5 W th orld Congress on Electroporation and Pulsed Electric Fields in Biology, Medicine, and Food & Environmental Technologies Rome, Italy September 15-19, 2024 Organised by The International Society for Electroporation-Based Technologies and Treatments and Sapienza University of Rome, Faculty of Civil and Industrial Engineering Programme and Book of Abstracts 5 th World Congress on Electroporation and Pulsed Electric Fields in Biology, Medicine, and Food & Environmental Technologies Rome, Italy 15−19 September, 2024 Programme and Book of Abstracts Organised by: The International Society for Electroporation-Based Technologies and Treatments & Sapienza University of Rome, Faculty of Civil and Industrial Engineering Edited by: Caterina Merla, Francesca Apollonio and Samo Mahnič-Kalamiza BBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBB .DWDORåQL]DSLVRSXEOLNDFLML&,3SULSUDYLOLY1DURGQLLQXQLYHU]LWHWQLNQMLåQLFLY/MXEOMDQL &2%,666,,' ,6%13') BBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBB 85/KWWSVZFHOHFWURSRUDWLRQQHWZSFRQWHQWXSORDGV :&B3URJUDPPHBDQGB%RRNBRIB$EVWUDFWVSGI &RS\ULJKW�=DORåED)($OOULJKWVUHVHUYHG5D]PQRåHYDQMHWXGLIRWRNRSLUDQMHGHOD YFHORWLDOLSRGHOLKEUH]SUHGKRGQHJDGRYROMHQMD=DORåEH)(SUHSRYHGDQR =DORåQLN=DORåED)(/MXEOMDQD ,]GDMDWHOM)DNXOHWD]DHOHNWURWHKQLNR/MXEOMDQD 8UHGQLNSURIGU6DãR7RPDåLþ .UDMLQOHWRL]LGD/MXEOMDQD HOHNWURQVNDL]GDMD Organization International Organizing Committee Apollonio, Francesca (Italy) Davalos, Rafael (USA) Gehl, Julie (Denmark) Merla, Caterina (Italy) Mir, Lluis M. (France) Raso, Javier (Spain) Rols, Marie-Pierre (France) Signori, Emanuela (Italy) Honorary Advisory Board Barbosa-Canovas, Gustavo (USA) Cadossi, Ruggero (Italy) Heller, Richard (USA Knorr, Dietrich (Germany) Marshall, Guillermo (Argentina) Miklavčič, Damijan (Slovenia) Neumann, Eberhard (Germany) Rubinsky, Boris (USA) Schoenbach, Karl H. (USA) Local Organizing Committee Apollonio, Francesca (Sapienza University) Merla, Caterina (ENEA) Cadossi, Matteo (IGEA) Camera, Francesca (ENEA) Caramazza, Laura (Sapienza) Casciati, Arianna (ENEA) Colella, Micol (Sapienza University) Consales, Claudia (ENEA) De Robertis, Mariangela (University of Bari) Dolciotti, Noemi (Sapienza University) Fionda, Bruno (Policlinico Gemelli) Lamberti, Patrizia (University of Salerno) Liberti, Micaela (Sapienza University) Marino, Carmela (ENEA) Marracino, Paolo (Rise Technology) Paffi, Alessandra (Sapienza University)) Pisano, Carmen (Sapienza University)) Scarfì, Maria Rosaria (CNR-IREA) Sieni, Elisabetta (University of Insubria) Signori, Emanuela (CNR) Tagliaferri, Luca (Policlinico Gemelli) Tylewicz, Urszula (University of Bologna) Scientific Program Committee Alexander Golberg (Israel), Matias Tellado (Argentina), Alexander Mathys (Switzerland), Maura Casciola (USA), Andrei Pakhomov (USA), Michal Cifra (Czchia), Anna Szewczyk (Poland), Mihaela Moisescu (Romania), Anna Bulysheva (USA), Mike Kempkes (USA), Antoni Ivorra (Spain), Mirella Tanori (Italy), Bennett Ibey (USA), Mounir Tarek (France), Chenguo Yao (China), Muriel Golzio (France), Christian Gusbeth (Germany), Nabil Grimi (France), Claudia Muratori (USA), Nadia Boussetta (France), Claudia Siemer (Germany), Nataša Tozon (Slovenia), Cornelia Rauh (Germany), Nikolai Lebovka (Ukraine), D. C. Sigg (USA), Nor Nadiah Abdul Karim Shah (Malaysia), Damijan Miklavcic (Slovenia), Olga Martin Belloso (Spain), Delia Arnaud-Cormos (France), Olga Pakhomova (USA), Pedro Elez-Martinez (Spain), Olga Zeni (Italy), Eugene Vorobiev (France), Paulo Garcia (USA), Fan Yuan (USA), Petr Lukes (Czech Republic), Felipe Maglietti (Argentina), Philippe Leveque (France), Gennaro Ciliberto (Italy), Richard Heller (USA), Gianpiero Pataro (Italy), Richard Nuccitelli (USA), Giulia Bertino (Italy), Robert Neal (USA), Govind Srimathveeravalli (USA), Robert Soliva (Spain), Gregor Sersa (Slovenia), Saulis Gintautas (Lithuania), Guillermo Cebrián (Spain), Saulius Satkauskas (Lithuania), Guillermo Marshall (Argentina), Sergio Salgado (Peru), Guus Pemen (The Netherlands), Shirley Sharabi (Israel), Hamid Hosano (Japan), Shu Xiao (USA), Hao Lin (USA), Sid Becker (New Zealand), Henry Jäger (Austria), Simona Salati (Italy), Ignacio Álvarez (Spain), Siqi Guo (USA), Indrawati Oey (New Zealand), Stefan Töpfl (Germany), James Lyng (Ireland), Stefania Romeo (Italy), Jiali Bao (China), Sudhir Sastry (USA), Joe Impellizeri (USA), Sunao Katsuki (Japan), Julita Kulbacka (Poland), Tom Vernier (USA), Kevin Hollevoet (Belgium), Tomas Garcia-Sanchez (Spain) Lea Rems (Slovenia), Tony Jin (USA), Loree Heller (USA), Urska Kamensek (Slovenia), Luigi Aurisicchio (Italy), Vitalij Novickij (Lithuania), Luis Redondo (Portugal), Wiktor Artur (Poland) Maja Cemazar (Slovenia), Wolfgang Frey (Germany), Maor Elad (Israel), Xiaoyun Ding (USA), Marco Benazzo (Italy), Xin-An Zeng (China), Mark Jaroszeski (USA), Zeng Xinan (China), Matej Kranjc (Slovenia), Zhong Han (China) Welcome Notes Dear Colleagues and Friends, Welcome to the 5th World Congress on Electroporation and Pulsed Electric Fields in Biology, Medicine, Food, and Environmental Technologies! We are thrilled to be part of this unique platform that brings together experts from diverse disciplines—biologists, medical professionals, engineers, and physicists—all united by a common goal: advancing the science of electroporation. This multidisciplinary approach is essential as we tackle critical challenges in areas such as cancer therapy, cardiovascular interventions, angiological malformations, DNA-based vaccines, and the development of innovative treatments. Beyond healthcare, our discussions will extend to pioneering applications in food technology, biotechnology, and environmental sustainability. Electroporation’s versatile capabilities offer new ways to enhance food safety and processing, optimize biotechnological processes, and address environmental challenges, demonstrating its profound impact on creating a better, more sustainable future. By exploring these diverse applications, we aim to unlock the full potential of electroporation, advancing science and technology for the benefit of society and the planet. This 5th edition brings several exciting features, including: i) the Industry Panel bridging the gap between academia and industry, with a focus on cardiac ablation using Pulsed Field Ablation; ii) the 3-Minute Thesis competition, where Ph.D. students present their thesis in three minutes in a clear and engaging way with the goal to enhance communication and presentation skills for a non-specialist audience; iii) the Senior2YS Meeting Corner, offering a unique opportunity for emerging researchers to connect with senior experts and industry leaders in the field of electroporation. The Educational Session, now in its second edition, proves to be a valuable part of the conference; newcomers to the field may find prior to the Congress useful and worthwhile introduction to electroporation and pulsed field technology, given by great lecturers. None of this would have been possible without the invaluable support of our sponsors, exhibitors, foundations, local and international committees, and, finally, the dedicated work of the scientific program committee. Each of these groups has played a crucial role in making this event a success. We are profoundly grateful for their support, that have made possible to realize all the initiatives and in particular the 34 Travel Awards for students and prizes for the best oral and poster presentations, recognizing outstanding work from both students and young scientists. It goes without saying that we extend our gratitude to all participants for their contributions and enthusiasm. The future will undoubtedly benefit from the work done here, as well as the new ideas and collaborations that will emerge from this congress. Welcome to Rome! The Local Organizing Committee, The Congress Chairs: Francesca Apollonio and Caterina Merla General Information Congress website Information for speakers wc2024.electroporation.net Guidelines for oral and poster presentation are available to download from the congress web-site. Important: personal Congress venue laptops cannot be used for Faculty of Civil and Industrial Engineering presentations. Sapienza University of Rome At the end of the congress, all presentations Via Eudossiana 18, 00184 Rome, Italy will be deleted to ensure that no copyright issues will arise. Congress Secretariat Ega Worldwide congresses&events Wi-Fi Viale Tiziano 19,00196 Rome, Italy Free access to the Wi-Fi at the congress mail: secretariat.wce2024@ega.it venue is provided. Personal access code will be given to each participant. Badges The congress name badges must be worn Journal during the congress. Access to the We have made an agreement with congress will not be granted without a name Bioelectricity, the official ISEBTT journal, badge issued by the congress secretariat. for a special issue dedicated to the conference, more information will be provided on the congress web-site. Certificate of Attendance Further special issues are foreseen nearly 6 A certificate of attendance will be sent to months after the end of the conference and participants by email after the conference. info will be provided to the congress presenters by email and on the congress web-site. Social Events Welcome Ceremony YS Night Date: Sunday, 15 September 2024 Date: Monday, 16 September 2024 Time: 17:30-21:00 (see schedule below) Time: 18:30-22:30 Places: San Pietro in Vincoli Basilica and Place: The Sanctuary Faculty of Civil and Industrial Engineering, Via delle Terme di Traiano, 4 University Sapienza of Rome All the students post-docs, and early carrier All the congress participants are invited to researchers registered to the congress are join the Welcome Ceremony and the invited to reach the YS Night and enjoy a Welcome Reception. The Welcome drink and glass of wine with food altogether Ceremony will start with the welcome of in a very nice place in the heart of Rome. It authorities and will end with a vibrant string will be great occasion to meet each other concert by the “Kepos” quartet. and to make new friendships. After the concert, a welcome cocktail will be served in the Cloister of the Faculty. Senior2YS Meeting Corner Date: Tuesday 17 September 2024 Tours of Rome Time: 18:30-20:00 Dates: Monday 16 and Tuesday 17 Place: Faculty of Civil and Industrial September 2024 Engineering, University Sapienza of Rome Time: at the end of the Scientific Sessions Duration: 1h30” This event offers an exceptional opportunity Departure place: Faculty of Civil and for young scientists to connect with senior Industrial Engineering, University Sapienza researchers and industry leaders in the of Rome field, gaining advice from experts, while enjoying a fresh free beer! All the congress participant registered to this event are invited to reach the departure point a 18:00 and enjoy the visit of the most beautiful places of the ancient Rome and Congress Dinner the wonderful company of colleagues from Date: Wednesday, 18 September 2024 all over the world. Time: 19:30-24:00 Places: Palazzo Brancaccio Viale del Monte Oppio, 7 Join the Congress Dinner in the magnificent location of Palazzo Brancaccio. An Italian style menu will be served accompanied by excellent Italian wine. After the dinner there will be a DJ set, music and dancing. N.B. If you have not yet purchased a congress dinner ticket (25 euros), you can do it at the registration desk by Monday 16. (subject to availability). Floor Plan: First Floor FIRST FLOOR Registration Speaker Ready Room Catering Area Scientific Sessions Halls Stand Lift Board Meeting Room Branded Panel Toilet Faculty of Civil and Industrial Engineering Sapienza University of Rome CLOISTER HALL FRESCOES’ HALL IGEA 1 HIGH VOLTAGE PROD. 2 ELEA TECHNOLOGY 3 MPORE 7 MONTENA 8 AMBER CHARGE 9 AEI ADVANCED ENERGY 10 HALL TAKIS 11 17 KIMEA 12 IVY BIOMEDICAL 4 CLOISTER LEROY BIOTECH 5 VITAVE 6 H2020 RISEUP BIOELECTRICITY 13 14 ELSEVIER B.V. PULSE BIOSCIENSES HALL HALL HALL RISE TECHNOLOGY 1 2 HALL 7 3 GARDEN GARDEN HALL 5 MAIN ENTRANCE VIA EUDOSSIANA 18 Floor Plan: Second Floor SECOND FLOOR Scientific Sessions Rooms Social Space Lift Poster Area Catering Area Toilet Faculty of Civil and Industrial Engineering Sapienza University of Rome HALL Boaga 24 Library Programme Overview TM Transformative possibilities for treating AFib Learn more at www.bostonscientific.eu CAUTION: The law restricts these devices to sale by or on the order of a physician. Indications, contraindications, warnings, and instructions for use can be found in the product labeling suppled with each device. Products shown for INFORMATION purposes only and may not be approved or for sale in certain countries. This material not intended for use in France. ©2024 Boston Scientific Corporation and its affiliates. All rights reserved. EP-1987504-AA Setting a new standard in safety. Engineered for efficiency. PulseSelect™ Pulsed Field Ablation (PFA) System The first and only PFA system approved for PAF and PerAF. Scan to learn more UC202411240 EE ©2024 Medtronic. Pulsed Electric Field food processing solutions. Improve your product & production with Elea PEF Advantage systems Pulsed Electric Field (PEF) processing transforms food, improving quality and providing new, greater opportunities. Elea PEF Advantage systems increase output, reduce costs, save energy, streamline supply chain logistics and help to minimise retail waste. With nearly 300 installed systems, we are the world’s leading provider of PEF solutions to the food, beverage and scientific sectors. Book a 30 minute PEF talk elea-technology.com info@elea-technology.com +49 (0) 5431 92629 70 Institutional Sponsors Meritorious travel grants sponsored by Travel grants also provided by 15 Industrial Sponsors Platinum 16 Industrial Sponsors Silver 17 Industrial Sponsors Bronze 18 Exhibitors 19 P R O G R A M M E Plenary Lectures Page Monday morning plenaries, Monday, Sep 16 2024, 8:50-9:50 Location: Hall 1 Session: Plenary talks 71 Chairs: Rafael Davalos and Emanuela Signori 8:50 Visualizing Electropore Dynamics in Live Cells 71 PL-01 Mantas Silkunas, Olga N. Pakhomova, Giedre Silkuniene, Andrei G. Pakhomov 9:20 Ion ion/protein mobilization following PEF application 71 PL-02 Sunao Katsuki Tuesday morning plenaries, Tuesday, Sep 17 2024, 8:30-10:00 Location: Hall 1 Session: Plenary talks 71 Chairs: Marie-Pierre Rols and Ruggero Cadossi 8:30 Lethal and non-lethal perturbation of cells by electroporation 71 PL-03 Antoni Ivorra 9:00 Mechanistic insights into the effects of electroporation on excitable cells 72 PL-04 Lea Rems 9:30 EP for Cardiac ablation 72 PL-05 Vivek Reddy Wednesday morning plenaries, Wednesday, Sep 18 2024, 8:30-10:00 Location: Hall 1 Session: Plenary talks 72 Chairs: Javier Raso and Samo Mahnič-Kalamiza 8:30 Pioneering sustainable food production with pulsed electric field technology 73 PL-06 Indrawati Oey 9:00 Unleashing the potential of pulsed electric fields in food processing: from 73 PL-07 techno-functional improvement to health-boosting innovations Robert Soliva-Fortuny, Pedro Elez-Martinez, Olga Martín-Belloso 9:30 Electroporation - The U.S. Veterinary Experience with ECT and GET 73 PL-08 Joseph A. Impellizeri 23 Thursday morning plenaries, Thursday, Sep 19 2024, 10:40-12:10 Location: Hall 1 Session: Plenary talks 73 Chairs: Matteo Cadossi and Lluis Mir 10:40 Enhanced Delivery of Plasmid DNA Encoding Therapeutic Agents as a Means 73 PL-09 to Induce a Robust Anti-Tumor Immune Response Richard Heller, Loree Heller, Guilan Shi, Jody Synowiec, Julie Singh, Alex Otten, Mark J. Jaroszeski 11:10 Clinical application in bone metastases compressing the spinal cord 74 PL-10 Frederic Deschamps 11:40 Vascular malformation ablation 74 PL-11 Walter A. Wohlgemuth 24 Oral Presentations Page Sunday Educational Session part 1, Sunday, Sep 15 2024, 13:00-15:00 Location: Hall 1 Session: Educational Session 77 Chair: Delia Arnaud-Cormos Info: Presentations Schedule 13:00-13:30 Luis M. Mir Words, concepts and facts: from the delivery of pulsed electric fields to electroporation? electropermeabilisation? reversible? irreversible? or even electropulsation? 13:30-14:00 Muriel Golzio Visualization of the delivery of molecules by electroporation: a step towards understanding the mechanisms involved 14:00-14:30 Maja Čemažar Electrochemotehrapy in human and veterinary oncology 14:30-15:00 Philippe Leveque Electroporation technologies Sunday Educational Session part 2, Sunday, Sep 15 2024, 15:30-17:00 Location: Hall 1 Session: Educational Session 77 Chair: Delia Arnaud-Cormos Info: Presentations Schedule 15:30-16:00 Michal Cifra Biophysical bases of PEF effects on proteins: modeling and experiments 16:00-16:30 Rafael Davalos Modeling of tissue electroporation and recent advances in irreversible electroporation 16:30-17:00 Samo Mahnič-Kalamiza Non-biomedical applications of electroporation: Traps for young players? Monday morning Track A, Monday, Sep 16 2024, 10:30-12:00 Location: Hall 1 Session: S21 - Cardiac ablation by irreversible electroporation - pulsed field ablation (PFA) 77 Chairs: Damijan Miklavčič and Ana González-Suárez Organizers/Conveners: D. Miklavčič, A. Ivorra 25 10:30 In situ characterization of electroporation-dependent tissue properties for can- 77 OR-01 cer and cardiac ablation Edward Jacobs, Pedro Santos, Rafael Davalos 10:45 Characterization of Thermal Safety Profile of a Novel Balloon-In-Basket PFA 78 OR-02 System under Repeated PFA Therapy Applications: Insights from in vivo and ex vivo Studies Lakshya Mittal, Ben Niemiera, Jed Overmann, Catherine Pipenhagen, L. Boyce Moon, Jeffrey Fish, Matthew Miller, Autumn Myhand, Taylor Spangler 11:00 Isolated primary rat ventricular cardiomyocytes response to electroporation: 78 OR-03 action potential – Ca2+ release – contraction Vid Jan, Marko Stručić, Tina Turk, Jernej Jurič, Monika Kos, Matej Reberšek, Martina Perše, Lea Rems, Damijan Miklavčič 11:15 Electrogram loss is neither sensitive nor specific for durable lesion formation 79 OR-04 in PFA Harikrishna Tandri, Shunsuke Uetake, Saleheldin Alhawamy, Parag Karmarkar 11:30 Arrhythmogenicity of monophasic and biphasic PFA waveforms in a porcine 80 OR-05 model Tugba Kumru, Lars M. Mattison, Atul Verma, Khaldoun Tarakji, Daniel C. Sigg 11:45 Surgical Ablation of Cardiac Tissue with Nanosecond Pulsed Electric Fields 80 OR-06 Christian W. Zemlin, Jakraphan Yu, Ralph Damiano Monday morning Track B, Monday, Sep 16 2024, 10:30-12:00 Location: Hall 7 Session: P5 - Electroporation and cellular processes and pathways 81 Chairs: Andrei Pakhomov and Anna Bulysheva 10:30 Facilitation of gelonin cytotoxicity with electroporation and its prospects for 81 OR-07 electrochemotherapy Olga N. Pakhomova, Andrei G. Pakhomov 10:45 Concomitant Electrotransfer of Small and Large molecules 82 OR-08 Ruta Palepšiene, Salvijus Vykertas, Ernestas Urbanskas, Justinas Venckus, Martynas Maciulevičius, Paulius Ruzgys, Baltramiejus Jakštys, Saulius Šatkauskas 11:00 Extracellular DNA enhances cell membrane damage stimulated by electrical 82 OR-09 short-circuiting via an aqueous droplet in dielectric oil Hirofumi Kurita, Yoshino Tsurusaki, Rika Numano 11:15 The inhibition of electrotransfection caused by the simultaneous transfer of 83 OR-10 multiple types of plasmid DNA Ernestas Urbanskas, Baltramiejus Jakštys, Paulius Ruzgys, Salvijus Vykertas, Justinas Venckus, Saulius Šatkauskas 26 11:30 Role of the actin cortex in intracellular transport of electrotransferred DNA 84 OR-11 cargo Sophie de Boer, Aswin Muralidharan, Gijsje Koenderink, Bijoy Bera, Pouyan E. Boukany 11:45 Research on the behavior of annexin A4 protein after cell electroporation: in- 84 OR-12 sights into the active membrane repair mechanisms Baltramiejus Jakštys, Dominykas Makarovas, Saulius Šatkauskas Monday morning Track C, Monday, Sep 16 2024, 10:30-12:00 Location: Hall 17 Session: S05 - Intensification of fermentation processes by pulsed electric fields 85 Chairs: Felix Schottroff and Javier Raso Organizers/Conveners: F. Schottroff, J. Raso 10:30 The role of PEF in enabling biotechnological processes for sustainable food 85 OR-15 processing Claudia Siemer 11:00 Use of PEF for the extraction of oenological compounds and pigments from 86 OR-13 yeasts Alejandro Berzosa, Javier Marín-Sanchez, Juan Manuel Martínez, Ignacio Álvarez, Ana Cristina C. Sánchez Gimeno, Javier Raso 11:15 Technical implementation of nsPEF in industrial biotechnological cultures 86 OR-14 Marco Stefan Fluri, Katharina Übelhör, Lya Siegenthaler, Lukas Neutsch, Leandro Buchmann 11:30 Industrial application of nanosecond PEF 87 OR-16 Leandro Buchmann 11:45 Enhancement of protein extraction and growth stimulation of microbial cells 87 OR-17 by µs PEF Felix Schottroff Monday morning Track D, Monday, Sep 16 2024, 10:30-12:00 Location: Cloister Hall Session: S12 - Numerical modelling as an essential tool in electroporation research 88 Chairs: Bor Kos and Clair Poignard Organizers/Conveners: B. Kos, M. Casciola, C. Poignard 27 10:30 Estimation of spatial conductivity distribution within a tumor from impedance 88 OR-18 measurements and imaging following a simulated electroporation therapy pro- tocol Damien Voyer, Olivier Sutter, Clair Poignard 10:45 Finite Element evaluation of the electric field distribution in a cell-aggregates 89 OR-19 Patrizia Lamberti, Michele Forzan, Stefania Romeo, Elisabetta Sieni 11:00 Finite element analysis model to predict electroporation of adherent cells in 90 OR-20 round, flat bottom wells Patrizia Lamberti, Nicolas Mattei, Maria Evelina Mognaschi, Jelena Kolosnjaj-Tabi, Muriel Golzio, Marie-Pierre Rols, Elisabetta Sieni 11:15 Microdosimetric Study to Calibrate µsPEFs Application in Two Electrodes 90 OR-21 Technology for RISEUP Project Sara S. Fontana, Noemi Dolciotti, Amir Ghassabi, Laura L. Caramazza, Irene Cuenca Ortolá, Micol Colella, Alessandra Paffi, Victoria Moreno, Luis M. Mir, Franck M. Andre, Claudia Consales, Francesca Apollonio, Micaela Liberti 11:30 Quantifying uncertainty of the numerical model of irreversible electroporation 91 OR-22 in the liver Helena Cindrič, Damijan Miklavčič, Bor Kos 11:45 Simulation Investigation of Pulsed Magnetic Field-Induced Cell Permeabiliza- 92 OR-23 tion Using the Coupled Magnetic-Electric-Force Pore Energy Equation Chi Ma, Yan Mi Monday young initiative 3MT, Monday, Sep 16 2024, 13:00-13:40 Location: Hall 1 Session: Three Minute Thesis (3MT) 92 Chairs: Micol Colella and Mariangela De Robertis Info: Finalists: Author Affiliation Title Berzosa Alejandro University of Zaragoza “Valorization of Spent Yeast from the Brewing Industry Using Pulsed Electric Field (PEF) Processing” Fontana Sara Sapienza University of Rome “Electric Pulses to Awaken Stem Cells: A Breakthrough in Injured Spinal Cord Regeneration?” Gomez-Barea Mario Universitat Pompeu Fabra “Can you guess which electrical parameter can trigger different cell death mechanisms?” Innamorati Giorgia University of Rome ”Tor Vergata” “Effects of microsecond electrical pulses on the inflammatory response” Lanssens Lize Katholieke Universiteit Leuven “PEF sensory boost: steering vegetable aromas” Walder Kate BOKU University “Power up your plate: Electrifying ways to optimize vegetable cooking” 28 Monday afternoon Track A, Monday, Sep 16 2024, 13:40-14:40 Location: Hall 17 Session: S03 - Microalgae biorefinery 92 Chairs: Nabil Grimi and Urszula Tylewicz Organizers/Conveners: N. Grimi, U. Tylewicz 13:40 Microalgae Biocompound Extraction: Simulation and Experimental Based 93 OR-24 Analysis of Residence Time and Cell Suspension Characteristics for Consist- ent and Scalable Continuous Flow PEF Processing Byron Patricio Perez Simba, Paride Azzari, Reto Koller, Iris Haberkorn, Alexander Mathys 13:55 Plasma-based extraction of compounds from the extremophile microalgae Gal- 93 OR-25 dieria sulphuraria Katja Zocher, Martina Balazinski, Marie-Christine Sommer, Ulfilas Hoffmann, Ulfilas Timm, Jörg Ullmann, Tilo Mottschall, Juergen F. Kolb 14:10 Pulsed electric fields for efficient lipid droplet extraction from cell-wall deficient 94 OR-26 microalgae Julia Baumgartner, Sing Teng Chua, Fengzheng Gao, Maylin Blunier, Lorraine Archer, Robert Axelrod, Fabian Abiusi, Silvia Vignolini, Alison Smith, Michael Hans-Peter Studer, Alexander Mathys 14:25 Biological Signalling Supports Biotechnology: Cell Death Triggers Protein Re- 95 OR-27 lease from Chlorella vulgaris Christian Gusbeth, Alexander Müller, Wolfgang W. Frey Monday afternoon Track B, Monday, Sep 16 2024, 13:40-14:40 Location: Cloister Hall Session: S08 - Public health risks and pulsed electric fields in the food industry 95 Chairs: Ignacio Álvarez and Juan Manuel Martínez Organizers/Conveners: I. Alvarez-Lanzarote, J. M. Martinez 13:40 Microbiological characterization of almond-based milk alternative and decon- 96 OR-28 tamination using pulsed electric fields (PEF) Arisa Thamsuaidee, Claudia Siemer, Vasilis P. Valdramidis 13:55 Electrochemical reactions as a side effect of Pulsed electric field treatment 96 OR-29 Gianpiero Pataro 14:10 Investigation of microbial strain variability resistance under PEF treatments 97 OR-31 and identification of the underlying molecular mechanisms Fotios Lytras, Georgios Psakis, Ruben Gatt, Joerg Hummerjohann, Guillermo J. Cebrian, Javier Raso, Vasilis Valdramidis 29 Monday afternoon Track C, Monday, Sep 16 2024, 13:40-14:40 Location: Hall 5 Session: P3 - Irreversible electroporation 98 Chairs: Rafael Davalos and Tomas Garcia 13:40 Characterization of pulsed electric fields for in vitro tumor spheroids and meta- 98 OR-32 static invasion Julio Arroyo 13:55 Study of irreversible electroporation-induced cell death in a 3D spheroid hep- 98 OR-33 atocarcinoma model Nicolas Mattei, Alexia de Caro, Emma Leschiera, Clair Poignard, Jelena Kolosnjaj-Tabi, Marie-Pierre Rols, Muriel Golzio 14:10 Acute efficacy and durability of in vitro pulsed field ablation in relation to the 99 OR-34 delivered energy impulse Ivana Fišerová, Marek Novak, David Kvapil, Stanislava Martinkova, Jan Trnka, Petr Tousek, Pavel Osmančík, Marek Hozman, Dalibor Herman, Jan Vrba, David Vrba, Ondrej Fiser Monday afternoon Track D, Monday, Sep 16 2024, 13:40-14:40 Location: Hall 7 Session: S01 - Medical applications of nsPEFs 100 Chairs: Richard Nuccitelli and Olga Pakhomova Organizers/Conveners: R. Nuccitelli, O. Pakhomova 13:40 Nano-Pulse Stimulation initiates an immune response in several types of mur- 100 OR-36 ine tumors Richard Nuccitelli 13:55 Distinct Tumor Immune Responses to Nanosecond Pulsed Electric Fields 100 OR-37 (nsPEFs) Determine Immunity Stephen J. Beebe, Anthony Nanajian, Brrittney Ruedlinger, Kamal Asadipour, Siqi Guo 14:10 The effects of nanosecond pulses on cell growth and multi-drug resistance in 101 OR-38 pancreatic cancer cells Wojciech Szlasa, Olga Michel, Vitalij Novickij, Paulina Kasperkiewicz, Mounir Tarek, Jolanta Saczko, Julia Rudno-Rudzińska, Wojciech Kielan, Julita Kulbacka 14:25 Are high-voltage nanosecond pulsed electric fields selective for cardiomyo- 102 OR-35 cytes? Pamela Sowa, Vitalij Novickij, Aleksander Kiełbik, Ferdinand Kollotzek, David Heinzmann, Jürgen Schreieck, Oliver Borst, Meinrad Gawaz 30 Monday late afternoon Track A, Monday, Sep 16 2024, 16:10-17:40 Location: Cloister Hall Session: S22 - New waveforms and electric field management strategies for electroporation-based therapies 103 Chairs: Antoni Ivorra and Micaela Liberti Organizers/Conveners: A. Ivorra, M. Liberti 16:10 Fifty shades of Electroporation 103 OR-39 Damijan Miklavčič, Samo Mahnič-Kalamiza 16:25 Impact of pulse protocol parameters on the efficacy of electrochemotherapy in 103 OR-40 vitro Fabio Lepore, Simona Salati, Francesca De Terlizzi, Giulia Grisendi, Roberta Fusco, Massimo Dominici, Matteo Cadossi, Ruggero Cadossi 16:40 Voltage vs Current Control. Selecting the Best Delivery Strategy 104 OR-41 Quim Castellvi, Antoni Ivorra 16:55 Differentiating Electroporation Currents using Dynamic PEF Modeling 105 OR-42 Clara Teresa De Souza Ramos, Daniella Lourdes Luna Santana de Andrade, Guilherme Brasil Pintarelli, Raul Guedert, Daniela Ota Hisayasu Suzuki 17:10 Comparison of the Thresholds for Electroporation and Excitation for Pulses 105 OR-43 within Nanosecond–Millisecond Duration Range Gintautas Saulis, Mantas Šilkūnas, Rita Saulė 17:25 Study on Effect of Electroporation Using Amplitude Modulation Signal and Har- 106 OR-44 monic Addition Borja López-Alonso, Tamara Polajžer, Matej Reberšek, Héctor Sarnago, Óscar Lucía, Damijan Miklavčič 31 Monday late afternoon Track B, Monday, Sep 16 2024, 16:10-17:40 Location: Hall 17 Session: P10 - Electrofluidic, microfluidic and lab on a chip 107 Chairs: Mihaela Moisescu and Hamid Hosano 16:10 Microphysiological system for PEF treatment of mammalian cells with integ- 107 OR-45 rated oxygen and TEER sensors Neringa Bakute, Eivydas Andriukonis, Kamile Kasperaviciute, Jorunas Dobilas, Martynas Sapurov, Gatis Mozolevskis, Arūnas Stirkė 16:25 Study on the technique of inducing perforation of blood clots by pulsed elec- 108 OR-46 tromagnetic fields Yuan Lei, Lei Li, Biao Hu, Shoulong Dong, Dengfeng He, Chenguo Yao 16:40 Dielectrophoresis – tool for analysis of mesenchymal stem cells differentiation 108 OR-47 Ioan Tivig, Leslie A. Vallet, Romain Samiaa, Franck M. Andre, Luis M. Mir, Tudor Savopol, Mihaela G. Moisescu 16:55 OpenDEP: A free-access platform for collecting and analyzing dielectro- 109 OR-48 phoresis spectra Ioan Tivig, Tudor Savopol, Mihaela G. Moisescu 17:10 A Lab-On-Chip Based on Transferred Laser-Induced Graphene Electrodes and 110 OR-49 Machine Learning for Electroporation of Adhered Cells Gianni Antonelli, Francesca Camera, Arianna Mencattini, Arianna Casciati, Mirella Tanori, Alessandro Zambotti, Giorgia Curci, Joanna Filippi, Michele D’Orazio, Paola Casti, Caterina Merla, Eugenio Martinelli 17:25 Non-invasive vaccine/drug delivery and theranostics by microfluidics of shock 110 OR-50 waves Nushin Hosano, Hamid Hosano 32 Monday late afternoon Track C, Monday, Sep 16 2024, 16:10-17:40 Location: Hall 1 Session: P13 - General applications of electroporation for food processing 110 Chairs: Gianpiero Pataro and Indrawati Oey 16:10 Validating pulsed electric field pasteurization of protein rich plant-based milk 110 OR-51 alternatives: a novel challenge trial approach Nicholas Horlacher, Indrawati Oey, Sze Ying Leong, Dominic Agyei, Jessie King 16:25 Application of continuous pulsed electric field (PEF) treatment in human milk 111 OR-52 as an alternative pasteurisation technique Yiting Wang, Farzan Zare, Negareh Ghasemi, P. Nicholas Shaw, Nidhi Bansal 16:40 Comprehensive Analysis of Heat-Assisted Pulsed Electric Fields and Conven- 112 OR-53 tional Thermal Treatment for Orange Juice Pasteurization: Cost, Energy Effi- ciency, and Sustainability Assessment Giovanni Landi, Miriam Benedetti, Matteo Sforzini, Elham Eslami, Gianpiero Pataro 16:55 Effect of pulsed electric field processing on functional properties of plant pro- 113 OR-54 tein in yogurt alternative applications Nicholas Horlacher, Indrawati Oey, Sze Ying Leong, Dominic Agyei, Jessie King 17:10 Effect of pulsed electric fields on protein extraction of duckweed (L. minor and 113 OR-55 L. gibba) Patricia Maag, Özlem Özmutlu, Cornelia Rauh 17:25 Pulsed electric field-assisted preparation of dialdehyde starch and its effect on 114 OR-238 structure and physiochemical properties Zhong Han, Ying Li, Xin-Dong Xu, Xin-An Zeng Monday late afternoon Track D, Monday, Sep 16 2024, 16:10-17:40 Location: Hall 5 Session: P3 - Irreversible electroporation 114 Chairs: Saulius Šatkauskas and Muriel Golzio 16:10 Study on Segmented Model of Tissue Conductivity Recovery after High Voltage 114 OR-56 Pulsed Electric Field Treatment Yajun Zhao, Luhao Qi, Zhi Fang, Dong Xu 16:25 Development of a Single Needle Electroporation Device Towards More Spher- 115 OR-57 ical Ablations Zaid Salameh, Vinay J. Deshmukh, Rafael Davalos 16:40 Production of Large Spherical Ablations Using Pulsed Electric Fields Admin- 116 OR-58 istered Via a Single Applicator Jewels L. Darrow, Callie Fogle, Robert H. Williamson, Alexia Cash, Kyle Mathews, Nate Nelson, Christopher Fesmire, Matthew Dewitt, Michael Sano 33 16:55 Investigation of rabbit heart electrical activity changes after electroporation us- 116 OR-60 ing combined optical and transmural microelectrode technique Regina Mačianskienė, Jonas Juravičius, Antanas Navalinskas, Mantė Almanaitytė, Vilma Zigmantaitė, Dominyka Adamonė, Ieva Lankutyte, Mindaugas Visockis, Justinas Barakauskas, Ernestas Urbanskas, Aras Rafanavičius, Saulius Šatkauskas 17:10 Electrical conductivity in human liver tissue: In vivo Assessment on normal 117 OR-61 vs. tumor Fernando Burdio, Amirhossein Sarreshtehdari, Xavi Moll, Enrique Berjano, Tomas Garcia Tuesday morning Track A, Tuesday, Sep 17 2024, 10:40-12:10 Location: Hall 5 Session: S20 - PhD Students as important bricks in the wall of funded projects and basic research 117 Chairs: Sara Fontana and Giorgia Innamorati Organizers/Conveners: S. Fontana, G. Innamorati 10:40 Gene electrotransfer of plasmid encoding Interleukin-12: off-target effects in 118 OR-62 murine cancer cells in vitro Ajda Medved, Maja Čemažar, Tanja Jesenko 10:55 Randomised Controlled Clinical Trial Investigating the Effect of Reduced Bleo- 118 OR-63 mycin in Electrochemotherapy Treatment on Patients with Cutaneous Malig- nancies Marie Tolstrup, Julie Gehl 11:10 Validation of in situ electroporation performed within a single cell microwave 119 OR-64 biosensor Anne A. Calvel, Olivia Peytral-Rieu, David Dubuc, Katia Grenier, Marie-Pierre Rols 11:25 Empowered Cellular and Subcellular Modeling for Microdosimetric Investiga- 120 OR-65 tion of PEF Exposure Noemi Dolciotti, Laura L. Caramazza, Sara S. Fontana, Micol Colella, Alessandra Paffi, Victoria Moreno, Loris Mannino, Luis M. Mir, Franck M. Andre, Romain Samiaa, Claudia Consales, Francesca Apollonio, Micaela Liberti 11:40 Effects of Intense Electric field on TRPV4 ion channel: a Molecular Dynamic 121 OR-66 study Carmen Pisano, Laura L. Caramazza, Paolo Marracino, Federico Del Signore, Micaela Liberti, Francesca Apollonio 11:55 Anti-tumor efficacy of new high-frequency electrical protocols on in vitro three- 121 OR-67 dimension colorectal cancer model Alexia de Caro, Jean-Baptiste Leroy, Jelena Kolosnjaj-Tabi, Muriel Golzio, Marie-Pierre Rols 34 Tuesday morning Track B, Tuesday, Sep 17 2024, 10:40-12:10 Location: Cloister Hall Session: P1 - Electroporation and immune response 122 Chairs: Lluis Mir and Marco Benazzo 10:40 Immunological changes in murine tumor cell lines following electrochemother- 122 OR-68 apy in vitro Ursa Kesar, Tanja Jesenko, Boštjan Markelc, Katja Ursic Valentinuzzi, Maja Čemažar, Primoz Strojan, Gregor Serša 10:55 Electroporation of 3D-cultured breast cancer cells elicits T lymphocyte- 123 OR-69 mediated killing Mariangela De Robertis, Ramona Marino, Elisabetta Sieni, Mario Cioce, Andrea Marra, Vincenzo Maria Perriello, Nico Martarelli, Annj Zamuner, Sonia Perrelli, Monica Dettin, Maria Teresa Conconi, Vito Michele Fazio, Flavio Keller, Emanuela Signori 11:10 Electroporation alters the proteomic output in human ex vivo GI cancer explant 123 OR-70 models: boosting systemic anti-tumour immunity and polarizing immune cell populations Aisling Uí Mhaonaigh, Lorraine Smith, Matthew McElheron, Aoibhín Woods, Fiona O’Connell, Cosima Sagurna, Kirstan Murphy, Meghana Menon, Yasir Bashir, Vincent Varley, Niamh O’Connor, Cian Muldoon, Ciara Ryan, Brian Mehigan, Waqas Butt, Narayanasamy Ravi, Claire Donohoe, Noel Donlon, John Larkin, Paul McCormick, Dara Kavanagh, Michael Kelly, John Reynolds, Declan Soden, Jacintha O’Sullivan 11:25 Enhanced antitumor efficacy of bleomycin electrochemotherapy combined 124 OR-71 with anti-PD-1 in mouse tumor models Simona Kranjc Brezar, Maša Omerzel, Barbara Lisec, Urša Lampreht Tratar, Tanja Jesenko, Gregor Serša, Maja Čemažar 11:40 Immunotherapy in combination with electrochemotherapy (Immune-ECT) in 125 OR-72 head and neck cancer Marta Minuti, Giulia Bertino, Ilaria Imarisio, Marco Benazzo 35 Tuesday morning Track C, Tuesday, Sep 17 2024, 10:40-12:10 Location: Hall 7 Session: P5 - Electroporation and cellular processes and pathways 126 Chairs: Olga Pakhomova and Marie-Pierre Rols 10:40 Optimizing Electroporation: Efficiency and Cell Viability in the Simultaneous 126 OR-73 Transfer of Bovine Serum Albumin, Propidium Iodide and Nucleic Acids Justinas Venckus, Ernestas Urbanskas, Salvijus Vykertas, Baltramiejus Jakštys, Paulius Ruzgys, Neringa Barauskaite-Šarkinienė, Saulius Šatkauskas 10:55 Mitochondrial Depolarization and ATP Loss During High Frequency Nano- 126 OR-74 second Electroporation Paulina Malakauskaitė, Augustinas Želvys, Auksė Zinkevičienė, Eglė Mickevičiūtė, Eivina Radzevičiūtė-Valčiuke, Veronika Malyško-Ptašinskė, Barbora Lekešytė, Jurij Novickij, Vytautas Kašėta, Vitalij Novickij 11:10 Generation of hypochlorous acid by high-voltage pulses and its influence on 127 OR-75 the cell plasma membrane Gintautas Saulis, Raminta Rodaitė, Jurgita Sventoraitiene, Viktorija Dainauskaite, Danute Batiuskaite, Alexander Golberg, Rita Saulė 11:25 Application of Pulsed Electric Fields to Gating Blood-Brain Barrier for Drug 128 OR-76 Delivery Pavel A. Solopov, Siqi Guo, Shu Xiao, John D D. Catravas 11:40 Is irreversible electroporation immunologically superior to thermal ablation or 128 OR-77 cryoablation? A closer look at antigen presentation, T cell activation and syn- ergy with immune checkpoint blockades Qi Shao 11:55 Involvement of mitochondria in the selective response to microsecond pulsed 129 OR-78 electric fields on both healthy and cancer stem cells in the brain Arianna Casciati, Anna Rita Taddei, Elena Rampazzo, Luca Persano, Giampietro Viola, Alice Cani, Silvia Bresolin, Vincenzo Cesi, Francesca Antonelli, Mariateresa Mancuso, Caterina Merla, Mirella Tanori 36 Tuesday morning Track D, Tuesday, Sep 17 2024, 10:40-12:10 Location: Hall 1 Session: P13 - General applications of electroporation for food processing 130 Chairs: Federico Gómez Galindo and Luis Redondo 10:40 Nondestructive extraction of functional molecules in yeast using 100 kV/cm 130 OR-79 class electrical pulses Hiroto Hashisako, Koya Asada, Masamori Higuchi, Sunao Katsuki 10:55 Pulsed Electric Field Treatment for Preservation of Chlorella Suspensions 130 OR-80 Cora De Gol, Ailsa Moodycliffe, Heidy M. W. den Besten, Marcel Zwietering, Michael Beyrer 11:10 Correlation of PEF induced biological autochemiluminescence with yeast cell 131 OR-81 electroporation Martin Bereta, Michal Teplan, Tomáš Zakar, Hoang Vuviet, Michal Cifra, Djamel Eddine E. Chafai 11:25 Germination and stress tolerance of oats treated with pulsed electric field at 131 OR-82 different phases of seedling growth Alia Hussain Al-Khafaji, Stephen Kwao, Federico Gomez Galindo, Sajeevan Radha Sivarajan 11:40 Effects of different combinations of pulsed electric field and pH shifting treat- 132 OR-83 ment on the aggregation structure and functional properties of soybean protein isolates Rui Wang, Pei-Feng Guo, Jing Nie, Xin-An Zeng 11:55 Analysis of temperature dependent dielectric properties of bacteria for effect- 132 OR-84 ive PEF pasteurization Ryuya Kimura, Sunao Katsuki, Bingyu Yan, Misato Kikuchi, Shoko Ishikawa, Kazuhiro Inobe, Ryo Sasahara, Taiga Kajiwara, Naoya Masuda, Yoshiharu Shimizu PFA Industry Panel Track, Tuesday, Sep 17 2024, 13:20-14:10 Location: Hall 1 Session: PFA Industry Panel 133 Chair: Damijan Miklavčič Info: Panelists: Ruggero Cadossi, IGEA, Italy; Maura Casciola, FDA, USA; Brendan Koop, Boston Scientific, USA; Steve Miller, Abbott, USA; Vivek Reddy, Mount Sinai Hospital, New York, USA; Tushar Sharma, Biosense Webster, USA; Daniel C. Sigg, Medtronic, USA; Darrin Uecker, Pulse Biosciences, USA. 37 13:20 Pulsed Field Ablation – gaps in knowledge and future directions of develop- 133 OR-085 ment Damijan Miklavčič Tuesday afternoon Track A, Tuesday, Sep 17 2024, 14:20-15:20 Location: Hall 1 Session: S21 - Cardiac ablation by irreversible electroporation - pulsed field ablation (PFA) 134 Chairs: Tomás García-Sánchez and Tugba Kumru Organizers/Conveners: D. Miklavčič, A. Ivorra 14:20 Initial single centre experience with pulsed field ablation for treatment of car- 134 OR-86 diac arrhythmias Jernej Štublar, Tine Prolič Kalinšek, Jernej Iršič, Damijan Miklavčič, Matevž Jan 14:35 Intraoperative Assessment of Irreversible Lesion Formation During PFA 134 OR-87 Parag Karmarkar, Shunsuke Uetake, Sivanag Maddineni, Bhupendra Mahar, Zhi-Qu Xu, Saleheldin Alhawamy, Harikrishna Tandri 14:50 Lesion Durability Prediction based on Real-Time Impedance Analysis Al- 135 OR-88 gorithms: Validation with First-in-Human Clinical Data from the RESET-AF Trial Laura Boehmert, Steffen Holzinger, Dorin Panescu 15:05 Investigation of bubble formation in intracardiac pulsed field ablation 136 OR-89 Samo Mahnič-Kalamiza, Damijan Miklavčič, Peter Lombergar, Blaž Mikuž, Lars M. Mattison, Daniel C. Sigg, Bor Kos Tuesday afternoon Track B, Tuesday, Sep 17 2024, 14:20-15:20 Location: Cloister Hall Session: S08 - Public health risks and pulsed electric fields in the food industry 137 Chairs: Ignacio Álvarez and Juan Manuel Martínez Organizers/Conveners: I. Alvarez-Lanzarote, J. M. Martinez 14:20 A multivariate study on continuous-mode pulsed electric field treatment of E. 137 OR-90 coli in water Yiting Wang, Farzan Zare, Elisabeth Prabawati, Buddhi Dayananda, MirHojjat Seyedi, Mark Turner, Negareh Ghasemi, Nidhi Bansal 14:35 Inactivation of zoonotic parasites by PEF, beyond single-cell electroporation 137 OR-91 Juan Manuel Martínez, Ignacio Álvarez, Guillermo J. Cebrian, Vanesa Abad 38 14:50 Rapid Recovery of Bacterial Membrane Following Exposure to Pulsed Electric 138 OR-92 Fields Bingyu Yan, Ryuya Kimura, Misato Kikuchi, Shoko Ishikawa, Kazuhiro Inobe, Ryo Sasahara, Taiga Kajiwara, Sunao Katsuki 15:05 Limitations of PEF for Food Pasteurization: role of membrane resealing in the 139 OR-93 microbial inactivation kinetics Carlota Delso, Juan Manuel Martinez, Ignacio Álvarez, Javier Raso Tuesday afternoon Track C, Tuesday, Sep 17 2024, 14:20-15:20 Location: Hall 7 Session: S09 - Treatment of spinal cord injury: novel strategies and updates from the RISEUP project 140 Chairs: Claudia Consales and Micol Colella Organizers/Conveners: C. Consales, M. Colella 14:20 Sensorimotor contributions to human cognition and emotion: clinical neuros- 140 OR-94 cience clues for optimizing engineering approaches to functional restoration in people with spinal cord lesions Salvatore M. Aglioti, Valentina Moro 14:35 Mechanisms of spinal cord regeneration 140 OR-95 Mark Anderson, Jordan Squair, Alexandra de Coucy, Matthieu Gautier, Zhigang He, Bernard Schneider, Michael Sofroniew, Jocelyne Bloch, Gregoire Courtine 14:50 Towards neuronal reconnection after a spinal cord injury using graphene- 141 OR-96 based nanocomposites – The NeuroStimSpinal project Paula Marques 15:05 Neuroprotective effect of Pulsed Electromagnetic Fields after Acute Ischemic 141 OR-97 Stroke Simona Salati, Micol Colella, Micaela Liberti, Ruggero Cadossi 39 Tuesday afternoon Track D, Tuesday, Sep 17 2024, 14:20-15:20 Location: Hall 5 Session: P11 - Electroporation modeling and mechanisms 142 Chairs: Damien Voyer and Daniela O. H. Suzuki 14:20 Multi-stages pulse modulation strategy (MSPM) enhances electroporation- 142 OR-98 mediated intracellular delivery by regulating the distribution and accumulation of drugs Xiaonan Tao, Kefu Liu 14:35 Multi-scalar microscopic molecular dynamics, coarse-grained and macro- 143 OR-99 scopic study of voltage-gated protein interactions and complex lipid pore form- ation during cellular electropermeabilization Juan A. Gonzalez Cuevas, Diego Stalder, Santiago Ferreyra, Carolina Recalde, Antoni Ivorra, Luis M. Mir 14:50 Comparison of sharpness and electrical field distribution of different electrode 143 OR-100 needles for electrochemotherapy Ana Laura Campastri, Antonella María Cilio, Jesica Rodríguez Miranda, Ximena Manglano, Sebastian D. Michinski, Felipe H. Horacio Maglietti 15:05 Cell electropermeabilization with subnanosecond pulsed electric fields 144 OR-101 Leslie A. Vallet, Njomza Ibrahimi, Laurent Ariztia, Marc Rivaletto, Antoine Silvestre de Ferron, Bucur M. Novac, Alexey Zhabin, Clair Poignard, Anthony Ranchou-Peyruse, Laurent Pecastaing, Franck M. Andre, Luis M. Mir 40 Tuesday late afternoon Track A, Tuesday, Sep 17 2024, 16:50-18:20 Location: Cloister Hall Session: S23 - Electroporation-based treatments in veterinary medicine 145 Chairs: Maja Čemažar and Nataša Tozon Organizers/Conveners: M. Čemažar, N. Tozon 16:50 Electro-Chemo-Immuno Therapy: activating local and systemic immunity 145 OR-102 Joseph A. Impellizeri, Antonella Conforti, Erika Salvatori, Luicia Lione, Luigi Aurisicchio 17:05 Electrochemotherapy for bilateral limbal squamous cell carcinoma in a horse 145 OR-103 Majbritt M. E. Larsen 17:20 Predictive factors in electrochemotherapy with or without IL-12 gene electro- 146 OR-104 transfer in dogs and cats Nataša Tozon, Urša Lampreht Tratar, Nina Milevoj, Masa Vilfan, Gregor Serša, Maja Čemažar 17:35 Comparison of intratumoral or peritumoral IL-12 gene electrotransfer in com- 146 OR-105 bination with electrochemotherapy for the treatment of spontaneous mast cell tumors in dogs Urša Lampreht Tratar, Nina Milevoj, Maja Čemažar, Katarina Žnidar, Katja Ursic Valentinuzzi, Andreja Brozic, Katerina Tomsic, Gregor Serša, Nataša Tozon 17:50 A Veterinary Electrotransfer System that employs Heat and Impedance – Pro- 147 OR-106 gress Toward Commercialization Mark J. Jaroszeski, Alex Otten, Gary Strange, Richard Heller 18:05 Electrical characterization of VX2 tumor in rabbit model for electroporation pur- 148 OR-107 poses Borja López-Alonso, Jorge Sánchez, Pablo Briz, Eva Monleón, José Aramayona, María Dolores Arribas, Héctor Sarnago, José M. Burdío, Óscar Lucía, Antonio Güemes Tuesday late afternoon Track B, Tuesday, Sep 17 2024, 16:50-18:20 Location: Hall 5 Session: S07 - Potential applications of PEFs technology in vegetable and fruit processing 148 Chairs: Marianna Giancaterino and Claudia Siemer Organizers/Conveners: C. Siemer, M. Giancaterino 16:50 How does PEF impact membrane integrity and the volatile profile of leek? 148 OR-108 Lize Lanssens, Sophie Delbaere, Ann Van Loey 17:05 Practical application using Pulse Electric Field (PEF) approach in milking the 149 OR-109 roots from aeroponic system Sylwester Ślusarczyk, Kajetan Grzelka, Joanna Jaśpińska, Adam Matkowski 41 17:20 Seaweed processing with pulsed electric fields: from batch to continuous pro- 150 OR-110 cess development for functional ingredients production Alexander Golberg 17:35 Optimizing valuable compound recovery from food side streams and microbial 150 OR-111 Biosynthesis through PEF-Induced Extraction and Stress Strategies Robert Sevenich 17:50 Increasing the yield of juice and bioactive compounds extracted from blueber- 151 OR-112 ries using pulsed electric field Shao-Keng Tai, Farzan Zare, Joseph Nastasi, Nidhi Bansal Tuesday late afternoon Track C, Tuesday, Sep 17 2024, 16:50-18:20 Location: Hall 7 Session: S09 - Treatment of spinal cord injury: novel strategies and updates from the RISEUP project 151 Chairs: Micol Colella and Claudia Consales Organizers/Conveners: C. Consales, M. Colella 16:50 Boosting the development of Electro Pulsed Bio-hybrid implantable devices 151 OR-113 through advanced modelling in vitro and in vivo Micol Colella, Francesca Apollonio, Marco Balucani, Laura L. Caramazza, Noemi Dolciotti, Sara S. Fontana, Paolo Marracino, Alessandra Paffi, Micaela Liberti 17:05 Materials solutions for an electrostimulable device for use in spinal cord injury 152 OR-114 model in rat Fernando Gisbert Roca, Sergiy Ivashchenko, Francisco Navarro Páez, Cristina Martinez, Jorge Más Estellés, Manuel Monleon 17:20 Electromanipulation of calcium oscillations in Mesenchymal Stem Cells, a con- 153 OR-115 trol of cell fate? Leslie A. Vallet, Marina Sanchez Petidier, Romain Samiaa, Nataliia Naumova, Claudia Consales, Giorgia Innamorati, Caterina Merla, Franck M. Andre, Luis M. Mir 17:35 Effects of microsecond electrical pulses on the inflammatory response 154 OR-116 Giorgia G. Innamorati, Francesca Camera, Fernando Gisbert, Sergiy Ivashchenko, Romain Samiaa, Sara S. Fontana, Noemi Dolciotti, Micol Colella, Alessandro Zambotti, Caterina Merla, Franck M. Andre, Victoria Moreno, Paolo Marracino, Claudia Consales 17:50 Emerging Approaches to Neural Tissue Regeneration: Electrical Stimulation 154 OR-117 of Stem Cells Marina Sanchez Petidier, Romain Samiaa, Leslie A. Vallet, Giorgia Innamorati, Claudia Consales, Caterina Merla, Victoria Moreno, Fernando Gisbert, Sergiy Ivashchenko, Manuel Monleon, Micaela Liberti, Franck M. Andre, Luis M. Mir 42 18:05 Regeneration of Injured Spinal Cord by applying subdural electro pulsed stim- 155 OR-118 ulation and stem cell bio-hybrid approach Loris Mannino, Eric Lopez, Maria Pedraza, Paolo Marracino, Marco Balucani, Marina M. Sanchez, Franck M. Andre, Luis M. Mir, Romain Samiaa, Manuel Monleon, Cristina Martinez, Andres Alba, Sara S. Fontana, Micol Colella, Micaela Liberti, Francesca Apollonio, Caterina Merla, Giorgia G. Innamorati, Victoria Moreno Tuesday late afternoon Track D, Tuesday, Sep 17 2024, 16:50-18:20 Location: Hall 1 Session: P7 - Electroporation for clinical use 156 Chairs: Michael Sano and Julita Kulbacka 16:50 Optimization of Bipolar Microsecond Electric Pulses for DNA Vaccine Delivery 156 OR-119 Robert H. Williamson, Matthew Dewitt, Driss Elhanafi, David Zaharoff, Michael Sano 17:05 Keloid treatment with Electrochemotherapy 157 OR-120 Sebastian D. Michinski, Ana Dimitri, Ana Campastri, Antonella Cilio, Raquel Lertora, Felipe H. Horacio Maglietti 17:20 Development of a specific gel for skin cancer electrochemotherapy. 158 OR-121 Antonella María Cilio, Ana Campastri, Jesica Rodríguez Miranda, Ximena Manglano, Sebastian D. Michinski, Felipe Maglietti 17:35 The Synergy of Conductive Nanoparticles with Nanosecond and Microsecond 158 OR-122 Pulse Bursts for Bleomycin-based Electrochemotherapy Barbora Lekešytė, Paulina Malakauskaitė, Eglė Mickevičiūtė, Eivina Radzevičiūtė- Valčiuke, Veronika Malyško-Ptašinskė, Anna Szewczyk, Natalija German, Almira Ramanavičienė, Julita Kulbacka, Vitalij Novickij 17:50 Development of novel genetic vaccine platforms: from the idea to GMP pro- 159 OR-123 duction Luigi Aurisicchio 18:05 Low-Dose Electrochemotherapy Enhances DNA Damage and Overcome Res- 160 OR-124 istance through Synergistic Drug Delivery Vaishali Malik, Laurien G. P. H. Vroomen, Masashi Fujimori, Emma Gerace, Jaad Ismail, Govindarajan Srimathveeravalli Wednesday morning Track A, Wednesday, Sep 18 2024, 10:40-12:10 Location: Cloister Hall Session: S04 - Advanced applications of PEF for food quality enhancement, food component modification, and structural alterations 160 Chairs: Samo Mahnič-Kalamiza and Jessica Genovese Organizers/Conveners: S. Mahnic-Kalamiza, J. Genovese 43 10:40 Introduction to Advanced Applications of PEF for Food Quality Enhancement, 160 OR-236 Food Component Modification, and Structural Alterations Jessica Genovese 10:46 PEF for more sustainable, nutritious biomass and macromolecules for food 161 OR-125 applications with a case study on microalgae Iris Haberkorn, Byron Perez, Alexander Mathys 10:58 The influence of a pulsed electric field on the osmotic dehydration process 162 OR-126 and selected physical properties of orange fruits dehydrated in unconventional solutions Agnieszka Ciurzyńska, Katarzyna Rybak, Dorota Witrowa-Rajchert, Katarzyna Pobiega, Sabina Galus, Małgorzata Nowacka 11:10 The manifold manifestations of electroporation effects on plant tissue and how 162 OR-127 their quantification depends on the method of analysis Madita Anna-Maria Kirchner, Claudia Siemer, Damijan Miklavčič, Stefan Töpfl, Samo Mahnič-Kalamiza 11:22 Enhancing Chemical Reactions and Modification of Food Ingredients Using 163 OR-128 Pulsed Electric Fields: An Alternative Technique Xin-An Zeng 11:34 Opportunities for implementing pulsed electric fields for the enhanced pro- 164 OR-129 cessing of plant-based foods George Dimopoulos, Varvara Andreou, Athanasios Limnaios, Alexandros Katsimichas, Ioanna Thanou, Efimia Dermesonlouoglou, George Katsaros, Petros Taoukis 11:46 Comparison of nanosecond and microsecond PEF for physical property and 165 OR-130 substance mobilization in potato Yuji Takahashi, Kiyohira Hagimoto, Sunao Katsuki, Yuji Okada 11:58 Effect of PEF on ginger roots: Improving juice extraction yield or product qual- 165 OR-131 ity Rian A. H. Timmermans, Deniz Döner, Lijiao Kan, Joanne Siccama, Bert Dijkink, Martijntje Vollebregt 44 Wednesday morning Track B, Wednesday, Sep 18 2024, 10:40-12:10 Location: Hall 17 Session: S17 - Voltage control of biological membrane pores 166 Chairs: Federica Castellani and Manfred Lindau Organizers/Conveners: M. Lindau, F. Castellani 10:40 Voltage sensitivity of electropores limits the membrane potential 166 OR-132 Mantas Silkunas, Andrei G. Pakhomov 10:55 Using the same electrode to electroporate a chromaffin cell and measure the 166 OR-135 resulting exocytosis of catecholamine Jaya Ghosh, Xin Liu, Kevin Gillis 11:10 Molecular mechanisms of vesicle priming, fusion pore formation and transmit- 167 OR-136 ter release by electrodiffusion Manfred Lindau 11:25 Lipid protein interactions guide fusion pore opening and expansion during reg- 168 OR-137 ulated exocytosis Volker Kiessling 11:40 Voltage-activation mechanisms of ion channels with different electrical polar- 168 OR-134 ities Peter Larsson 11:55 Visualizing membrane fusion and budding in live cells 169 OR-133 Ling-Gang Wu Wednesday morning Track C, Wednesday, Sep 18 2024, 10:40-12:10 Location: Hall 5 Session: S18 - Bridging the gap between experimental and modeling studies in PEF electroporation: a Young Professional‘s perspective 169 Chairs: Laura Caramazza and Rosa Orlacchio Organizers/Conveners: L. Caramazza, R. Orlacchio 10:40 Evaluating biological membrane response to PEF: A multiscale computational 169 OR-138 approach Laura L. Caramazza, Paolo Marracino, Micaela Liberti, Francesca Apollonio 10:55 Effects of pulsed electric fields on collagen self-assembly and collagen secre- 170 OR-139 tion by dermal fibroblasts Emma Barrere, Nicolas Mattei, Ophelie Cordier, Marie-Pierre Rols, Muriel Golzio, Hermes Desgrez-Dautet, Matthieu Chavent, Jelena Kolosnjaj-Tabi 11:10 Exploring Vibrational and Electromagnetic Properties of Protein Tubulin using 171 OR-140 Normal Mode Analysis and Molecular Dynamics Simulations Saurabh Kumar Pandey, Michal Cifra 45 11:25 Nanosecond pulsed electric fields and gold nanoparticles for cancer treatment 171 OR-141 Rosa Orlacchio, Jelena Kolosnjaj-Tabi, Nicolas Mattei, Lionel Michard, Hafsa Tjiou, Léna Serradeil, Isabelle Lagroye, Florence Poulletier de Gannes, Yann Percherancier, Philippe Leveque, Marie-Pierre Rols, Delia Arnaud-Cormos, Muriel Golzio 11:40 Deciphering the behavior of multicellular 3D spheroids exposed to high- 172 OR-142 intensity pulsed electric fields by a mathematical modeling approach Annabelle Collin, Jelena Kolosnjaj-Tabi, Muriel Golzio, Marie-Pierre Rols, Clair Poignard 11:55 On the complementarity of modeling and experimentation in the study of bio- 173 OR-143 logical effects of subnanosecond pulsed electric fields Leslie A. Vallet, Njomza Ibrahimi, Laurent Ariztia, Marc Rivaletto, Antoine Silvestre de Ferron, Bucur M. Novac, Alexey Zhabin, Clair Poignard, Mounir Tarek, Laurent Pecastaing, Franck M. Andre, Luis M. Mir Wednesday morning Track D, Wednesday, Sep 18 2024, 10:40-12:10 Location: Hall 7 Session: S16 - Electroporation in veterinary and translational medicine 174 Chairs: Felipe Horacio Maglietti and Joseph Impellizeri Organizers/Conveners: F. Maglietti, J. Impellizeri 10:40 Chimeric DNA vaccination against the Chondroitin Sulfate Proteoglycan 4: a 174 OR-144 potential allied in combinatorial approaches for the treatment of melanoma and osteosarcoma Federica Riccardo, LIdia Tarone, Carlotta Montana, Davide Giacobino, Lorenza Parisi, Selina Iussich, Giuseppina Barutello, Laura Conti, Maddalena Arigoni, Paolo Buracco, Emanuela Morello, Federica Cavallo 10:55 Electrochemotherapy plus IL-2+IL-12 gene electrotransfer in spontaneous in- 175 OR-145 operable stage iii-iv canine oral malignant melanoma Sergio S. Salgado, Matias N. Tellado, Mariangela De Robertis, Daniela Montagna, Daniela Giovannini, Sebastian D. Michinski, Emanuela Signori, Felipe Maglietti 11:10 Adjuvant Electrochemotherapy and/or Radiotherapy in Feline Injection Site 175 OR-146 Sarcoma Matias N. Tellado, Franco Portillo, Vanda Guillen, Tadeo Sabella, Maura Diaz, Felipe Maglietti 11:25 Retrospective analysis of the outcome and survival time of dogs with mast cell 176 OR-147 tumors with different degrees of malignancy treated with electrochemotherapy Javier Ojeda, Paulina Sandoval 46 11:40 Evaluation of the safety and feasibility of electrochemotherapy with intraven- 177 OR-148 ous bleomycin as local treatment of bladder cancer in dogs Marcelo Monte Mor Rangel, Lais Calazans Menescal Linhares, Daniela Ota Hisayasu Suzuki, Krishna Duro de Oliveira, Felipe H. Horacio Maglietti, Andrigo Barbosa De Nardi Wednesday morning Track E, Wednesday, Sep 18 2024, 10:40-12:10 Location: Hall 1 Session: P7 - Electroporation for clinical use 177 Chairs: Yan Mi and Govindarajan Srimathveeravalli 10:40 Enhancing sensitivity to radiation therapy using electroporation in a radio- 177 OR-149 resistant model of oesophageal cancer Aoibhín Woods, Aisling Uí Mhaonaigh, Aisling Heeran, Lorraine Smith, Stephen Maher, Niamh Lynam-Lennon, Declan Soden, Jacintha O’Sullivan 10:55 Electroporation treatment alters the inflammatory tissue microenvironment in 178 OR-150 the human inflammatory condition, Barrett’s Oesophagus Lorraine Smith, Cian Gargan, Aisling Uí Mhaonaigh, Irene Narinda, Aoibhín Woods, Aoife Kilgallon, Matthew McElheron, Meghana Menon, Fiona O’Connell, James Phelan, Declan Soden, Jacintha O’Sullivan 11:10 Intraoperative electrochemotherapy of the posterior resection surface after 179 OR-151 pancreaticoduodenectomy: Preliminary results of a hybrid approach treatment of pancreatic cancer Žan Čebron, Mihajlo Djokic, Miha Petrič, Maja Čemažar, Maša Omerzel, Gregor Serša, Blaz Trotovsek 11:25 Novel Synergistic Electric Pulses and First Human Cancer Clinical Trials: To- 179 OR-152 wards the Balance between Negligible Muscle Contraction and Enhanced Ab- lation Hongmei Liu, Jianhao Ma, Shoulong Dong, Chenguo Yao 11:40 Bleomycin based electrochemotherapy with standard electrodes for advanced 180 OR-153 stage, recurring vulvar/cervix carcinomas Aurel Ottlakan, Marton Vas, Gyorgy Lazar, Judit Olah, Gabor Vass, Mario Vincze, Erika Gabriella Kis 11:55 Electrochemotherapy: from palliation to important player in the multidisciplin- 181 OR-154 ary management of the cancer patient Antonio Bonadies, Tiziano Pallara, Marinella Tedesco, Paola Parisi, Michela Battista, Flavio Andrea Govoni, Gennaro Ciliberto, Emilia Migliano 47 Wednesday afternoon Track A, Wednesday, Sep 18 2024, 14:20-15:20 Location: Hall 1 Session: S21 - Cardiac ablation by irreversible electroporation - pulsed field ablation (PFA) 181 Chairs: Lakshya Mittal and Antoni Ivorra Organizers/Conveners: D. Miklavčič, A. Ivorra 14:20 Protocol-specific modelling of cardiac pulsed field ablation 181 OR-155 Argyrios Petras, Aurel Neic, Edward Vigmond, Gernot Plank, Luca Gerardo-Giorda 14:35 Endocardial or Epicardial Delivery of Pulsed Field Ablation of Ganglionated 182 OR-156 Plexi? Assessment and Quantification from An In-Silico Modelling Study Francisco Estevez-Laborí, Barry O’Brien, Ana González-Suárez 14:50 Modeling the long-term effects of Pulsed-Field Ablation including comparison 183 OR-157 with Radio-Frequency Ablation Simone Nati Poltri, Annabelle Collin, Clair Poignard 15:05 Multiscale Simulation of Calcium-Mediated Cardiac Lesion and Stunning in 183 OR-158 Pulsed Field Ablation Quim Castellvi, Antoni Ivorra Wednesday afternoon Track B, Wednesday, Sep 18 2024, 14:20-15:20 Location: Hall 7 Session: S01 - Medical applications of nsPEFs 184 Chairs: Richard Nuccitelli and Olga Pakhomova Organizers/Conveners: R. Nuccitelli, O. Pakhomova 14:20 Investigating the mechanism and dynamics of Ca2+-mediated pore expansion 184 OR-159 after nsPEFs in healthy and cancerous urothelial cells Aleksander Kiełbik, Aleksandra Mariianats, Pamela Sowa, Wojciech Szlasa, Vitalij Novickij, Igor Tsaur, Julia Marzi, Bastian Amend 14:35 Synergistic effects and mechanisms of nanosecond pulsed electric fields and 185 OR-160 cold atmospheric plasma to treat pancreatic cancer Siqi Guo, Zobia Minhas, Edwin A. Oshin, Shanaya M. Haque, Yu Jing, Lifang Yang, Chunqi Jiang 14:50 Nanosecond Bursts of Ultra-High Frequency for Electrochemotherapy and 186 OR-161 Gene Delivery Vitalij Novickij 15:05 Characterizing the Immune Response Following High Frequency Nanosecond 186 OR-162 Bipolar and Unipolar Calcium Electrochemotherapy Eivina Radzevičiūtė-Valčiuke, Augustinas Želvys, Eglė Mickevičiūtė, Jovita Gečaitė, Paulina Malakauskaitė, Barbora Lekešytė, Veronika Malyško-Ptašinskė, Auksė Zinkevičienė, Vytautas Kašėta, Julita Kulbacka, Joanna Rossowska, Vitalij Novickij 48 Wednesday afternoon Track C, Wednesday, Sep 18 2024, 14:20-15:20 Location: Hall 5 Session: S13 - High voltage electrical discharges: principles and applications 187 Chairs: Nadia Boussetta and Eugene Vorobiev Organizers/Conveners: N. Boussetta, E. Vorobiev 14:20 High-Performance Solid-State Generator for nsPEF Applications 187 OR-59 Pablo Briz, Héctor Sarnago, Óscar Lucía 14:35 A Synergistic Bipolar Pulse Power Generator for Expanding Ablation Area and 188 OR-164 Inhibiting Muscle Contraction Shoulong Dong, Chenguo Yao, Jianhao Ma, Lisheng Zhao, Yancheng Wang, Hongmei Liu 14:50 Pulsed Atmospheric Pressure Plasma for the destruction of emerging contam- 188 OR-165 inants and the inactivation of bacteria in water Konstantia Papalexopoulou, Irene-Eva Triantaphyllidou, Christos Aggelopoulos 15:05 Degradation of pesticide atrazine in water by high voltage electrical discharges 189 OR-30 Junting Hong, Nadia Boussetta, Gérald Enderlin, Franck Merlier, Nabil Grimi Wednesday afternoon Track D, Wednesday, Sep 18 2024, 14:20-15:35 Location: Cloister Hall Session: S11 - In vivo delivery of genetic medicine through gene electrotransfer 190 Chairs: Kevin Hollevoet and Emanuela Signori Organizers/Conveners: K. Hollevoet, E. Signori 14:20 Exploring gene electrotransfer as a DNA vaccination strategy: insights from a 190 OR-166 COVID-19 vaccine study Urška Kamenšek, Simona Kranjc Brezar, Tanja Jesenko, Špela Kos, Katarina Žnidar, Boštjan Markelc, Živa Modic, Tilen Komel, Maja Čemažar, Gregor Serša 14:35 Enhancing molecular cargo electrotransfer by modulating vesicular transport 190 OR-167 in cells Fan Yuan, Chunxi Wang 14:50 LiveGT Enhances Skeletal Muscle Reprogramming and Physiological Levels of 191 OR-168 Insulin Production Michael Francis, Jacob Hensley, Alex Otten, Tina Gagliardo, Anna Bulysheva 15:05 Magnetoporation: A novel method of molecular delivery for cell and gene ther- 192 OR-169 apies Zachary Rapp 49 15:20 Development of in vivo-launched synthetic DNA-encoded antibodies employ- 192 OR-237 ing CELLECTRA® electroporation technology Trevor Smith, Paul Fisher, Ami Patel, Elizabeth Parzych, Kevin Hollevoet, David B. Weiner, Laurent Humeau Wednesday late afternoon Track A, Wednesday, Sep 18 2024, 16:50-17:50 Location: Hall 5 Session: P11 - Electroporation modeling and mechanisms 193 Chairs: Jelena Kolosnjaj-Tabi and Patrizia Lamberti 16:50 AC electrodeformation studies on Compound Giant Unilamellar Vesicle as a 193 OR-170 model of eukaryotic cell Rupesh Kumar, Rajarshi Chakrabarti, Rochish Thaokar 17:05 Suitability (and not) of Giant Unilamellar Vesicles in electroporation studies for 193 OR-171 biological applications Rochish Thaokar, Mohammad Maoyafikuddin 17:20 Correlation between numerical simulations and clinical outcomes of irrevers- 194 OR-172 ible electroporation for hepatocellular carcinoma Olivier Sutter, Luc Lafitte, Baudouin Denis de Senneville, Damien Voyer, Jean Pierre Tasu, Arthur Petit, Timothée Molango, Lorenzo-Carlo Pescatori, Olivier Seror, Clair Poignard 17:35 Simulation study on waveform characteristics of measuring bio-impedance us- 195 OR-173 ing pulse frequency response method Lisheng Zhao, Sizhe Xiang, Haobo Yang, Shoulong Dong, Chenguo Yao, Liang Yu Wednesday late afternoon Track B, Wednesday, Sep 18 2024, 16:50-17:50 Location: Hall 17 Session: S15 - Advanced imaging techniques for visualizing the mechanisms of pulsed electric field interactions 195 Chairs: Joel Bixler and Bennett Ibey Organizers/Conveners: J. Bixler, B. Ibey 16:50 PEFfect Illumination: Observing Protein Oxidation Effects of Pulsed Electric 196 OR-174 Field Through Monitoring (Bio)Chemiluminescence Kateřina Červinková, Petra Vahalová, Michaela Poplová, Tomáš Zakar, Daniel Havelka, Martin Paidar, Viliam Kolivoška, Michal Cifra 17:05 Electric field effects on human skeletal muscle-derived mesenchymal 196 OR-175 stem/stromal cells investigated by scanning electrochemical microscopy Inga Morkvenaite-Vilkonciene, Tomas Mockaitis 50 17:20 Effects of Nanosecond Pulsed Electric Field on Cancerous and Normal Cells 197 OR-176 — Fluorescence Microscopy and Autofluorescence Lifetime Imaging Nobuhiro Ohta Wednesday late afternoon Track C, Wednesday, Sep 18 2024, 16:50-18:05 Location: Cloister Hall Session: S11 - In vivo delivery of genetic medicine through gene electrotransfer 197 Chairs: Kevin Hollevoet and Emanuela Signori Organizers/Conveners: K. Hollevoet, E. Signori 16:50 Immunomodulatory effects of plasmid DNA following gene electrotransfer in 197 OR-177 colon cancer utilizing different electric pulse protocols Tim Bozic, Mariangela De Robertis, Iva Santek, Flaviana Marzano, Boštjan Markelc, Alessandro Silvestris, Apollonia Tullo, Graziano Pesole, Maja Čemažar, Emanuela Signori 17:05 Calcium electroporation and interleukin-12 gene electrotransfer 198 OR-178 Barbara Lisec, Boštjan Markelc, Katja Ursic Valentinuzzi, Gregor Serša, Maja Čemažar 17:20 Unraveling a multifactorial host immune response to intramuscular electro- 199 OR-179 transfer of dna-encoded antibody therapy Debby Thomas, Jenny Sprooten, Jannes Govaerts, Pascal Merchiers, Maarten Dewilde, Kevin Hollevoet, Abhishek Garg, Nick Geukens 17:35 Enhancing the Therapeutic Benefits of Proteins with Short Half-Lives: Delivery 200 OR-180 of G-CSF and GLP-1 with DNA-Based MYO Technology Debnath Maji, Andrew D. Cameron, Linda Sasset, Sayantani Sinha, Andy Thompson, Carleigh Sussman, Delcora A. Campbell, Robert Miller, Marek M. Drozdz, Rachel A. Liberatore 51 Wednesday late afternoon Track D, Wednesday, Sep 18 2024, 16:50-17:50 Location: Hall 7 Session: S19 - Pulsed electric fields in meat and fish and their by-products processing 200 Chairs: Urszula Tylewicz, Pietro Rocculi and Silvia Tappi Organizers/Conveners: U.Tylewicz, P. Rocculi, S. Tappi 16:50 New advancement on meat processing using Pulsed electric field technology 200 OR-182 Indrawati Oey 17:05 Inactivation by Pulsed Electric Fields of Anisakis in naturally infected hake 201 OR-183 meat. Vanesa Abad, Javier Raso, Juan Manuel Martínez, Guillermo J. Cebrian, Ignacio Álvarez 17:20 Valorization of shrimp by-products: Extraction of high value-added com- 202 OR-184 pounds by pulsed electric field (PEF) and accelerated solvent extraction (ASE) Ana Cristina De Aguiar Saldanha Pinheiro, Francisco J. Martí-Quijal, Francisco J. Barba, Urszula Tylewicz, Silvia Tappi, Santina Romani, Pietro Rocculi Wednesday late afternoon Track E, Wednesday, Sep 18 2024, 16:50-18:20 Location: Hall 1 Session: S10 - Electrochemotherapy of cutaneous tumors 202 Chairs: Giulia Bertino and Julie Gehl Organizers/Conveners: G. Bertino, J. Ghel 16:50 Electrochemotherapy for Kaposi Sarcoma and Merckel Cell Carcinoma: find- 202 OR-185 ings of the InspECT Rare Tumours Working Group Joy Odili, Pietro Quaglino, Matteo Brizio, Giulia Bertino, Erika Kis, Matteo Mascherini, Michela Battista, Christian Kunte, David Mowatt, Francesco Russano, Roberto Giorgione, James P. Clover, Hadrian Schepler, Gregor Serša, Marta Minuti 17:05 Electrochemotherapy in the treatment of cutaneous melanoma metastases – 203 OR-186 the InspECT experience Erika Kis, Barbara Perić, Matteo Brizio, Giuseppe Riva, Giulia Bertino, Nunzia Di Cristo, Barbara Silvestri, Hadrian Schepler, Joy Odili, Siva Kumar, Matteo Mascherini, Christian Kunte, Francesca Tauceri, Giulia Colavitti, Veronica Seccia 17:20 InspECT database and clinical results of electrochemotherapy 204 OR-187 Giulia Bertino, Ales Groselj, Christian Kunte, Hadrian Schepler, Julie Gehl, Tobian Muir, James P. Clover, Pietro Quaglino, Erika Kis, Matteo Mascherini, Brian Bisase, Giancarlo Pecorari, Falk Bechara, Paolo Matteucci, Joy Odili, Francesco Russano, Giulia Colavitti, Rowan Pritchard-Jones, David Mowatt, Barbara Silvestri, Veronica Seccia, Gregor Serša 52 17:35 Electrochemotherapy for the treatment of cutaneous metastases from breast 205 OR-188 cancer Julie Gehl 17:50 Differential expression analysis of cutaneous squamous cell carcinoma and 206 OR-189 basal cell carcinoma proteomic profiles sampled with electroporation-based molecular biopsy Alexander Golberg, Edward Vitkin, Ariel Berl, Julia Wise 18:05 Electrochemotherapy in the treatment of chronic suppurative benign skin con- 206 OR-190 ditions: The St George’s Hospital experience Joy Odili Thursday morning Track A, Thursday, Sep 19 2024, 8:30-10:00 Location: Hall 1 Session: S24 - Emerging role of Electrochemotherapy in the treatment of GastroIntestinal cancer 207 Chairs: Luca Tagliaferri and Roberto Iezzi Organizers/Conveners: L. Tagliaferri, R. Iezzi 8:30 The Interventional Oncology in the modern interdisciplinary scenario 207 OR-191 György Kovács 8:45 The role of Interventional Radiology 207 OR-192 Laura Crocetti 9:00 The role of Interventional Endoscopy 207 OR-193 Fabia Attili 9:15 The role of Interventional and External Beam Radiotherapy 207 OR-194 Bruno Fionda 9:30 The role of Electrochemotherapy 207 OR-195 Martina Ferioli 9:45 The synergistic effect of Electrochemotherapy in the modern Oncology scen- 208 OR-196 ario Attila Kovacs Thursday morning Track B, Thursday, Sep 19 2024, 8:30-10:00 Location: Hall 7 Session: P12 - Biomass transformation and biocompounds 208 Chairs: Samo Mahnič-Kalamiza and Wolfgang Frey 53 8:30 Non-lethal extraction of phytochemicals and growth promotion of Iris domest- 208 OR-197 ica (L.) DC roots enabled by electroporation Kajetan Grzelka, Joanna Jaśpińska, Adam Matkowski, Sylwester Ślusarczyk 8:45 Bioactive Potential of Yeast Proteins Extracted with HPH and PEF 208 OR-198 Javier Marín-Sanchez, Alejandro Berzosa, Ignacio Álvarez, Ana Cristina C. Sánchez Gimeno, Javier Raso 9:00 Influence of Pulsed Electric Fields in combination with other processes on the 209 OR-199 extraction of valuable compounds from brewer’s spent yeast cells Sofie Schröder, Jan-Michel Schulte, Corinna Stühmeier-Niehe, Claudia Siemer, Stefan Töpfl 9:15 Solvent Lipid Extraction from Oleaginous Yeast assisted by Pulsed Electric 210 OR-200 Fields (PEF) Carlota Delso, Nataljia Nazarova, Wolfgang W. Frey 9:30 PEF treatment for the enhancement of microalgae cultivation 210 OR-201 Iris Haberkorn, Byron Perez, Alexander Mathys Thursday morning Track C, Thursday, Sep 19 2024, 8:30-10:00 Location: Hall 5 Session: S07 - Potential applications of PEFs technology in vegetable and fruit processing 211 Chairs: Marianna Giancaterino and Claudia Siemer Organizers/Conveners: C. Siemer, M. Giancaterino 8:30 Understanding of the applicability and the mechanism behind pulsed electric 211 OR-202 fields (PEF) as an alternative peeling method Marianna Giancaterino, Henry Jäger 8:45 Effects of pulsed electric field pre-treatment on the heating uniformity and final 212 OR-203 product quality of ohmic cooked vegetables Kate Waldert, Sarah Elisabeth Prenner, Marianna Giancaterino, Henry Jäger 9:00 Germination and stress tolerance of oats treated with pulsed electric field at 213 OR-204 different phases of seedling growth Alia Hussain Al-Khafaji 9:15 Pulsed electric field, a possible strategy for mitigation of process contaminants 213 OR-205 in vegetable snacks. Stefan Toepfl 9:30 Sustainable extraction of plant-based food colorants with Pulsed Electric 213 OR-206 Fields Madita Anna-Maria Kirchner, Stephanie Wink, Claudia Siemer, Stefan Töpfl 54 9:45 Biospeckle activity: New electroporation assessment method for treated fruits 214 OR-207 and vegetables Aleksandra Matys, Piotr Pieczywek, Artur Zdunek, Dorota Witrowa-Rajchert, Artur Wiktor Thursday morning Track D, Thursday, Sep 19 2024, 8:30-10:00 Location: Cloister Hall Session: S12 - Numerical modelling as an essential tool in electroporation research 215 Chairs: Bor Kos and Clair Poignard Organizers/Conveners: B. Kos, M. Casciola, C. Poignard 8:30 Electrodissociation of cytoskeleton proteins by intense electric field: in silico 215 OR-208 Jiri Prusa, Saurabh Kumar Pandey, Michal Cifra 8:45 A coarse-grained lattice model of PEF Inactivation kinetics from percolation 215 OR-209 theory Feiyu Wu, Chenguo Yao 9:00 Quantum chemical simulations of the interaction of Fe2+ with glycerophos- 215 OR-210 pholipids Teresė Kondrotaitė, Alytis Gruodis, Gintautas Saulis 9:15 Modelling the impact of electroporation on spheroid growth and the release of 216 OR-211 damage-associated molecular pattern molecules Emma Leschiera, Nicolas Mattei, Muriel Golzio, Jelena Kolosnjaj-Tabi, Clair Poignard, Marie-Pierre Rols 9:30 Skeletal muscle anisotropy from the perspective of experimental and model- 216 OR-212 based electrical impedance spectroscopy Rok Šmerc, Damijan Miklavčič, Samo Mahnič-Kalamiza 55 Thursday afternoon Track A, Thursday, Sep 19 2024, 14:10-15:40 Location: Hall 7 Session: P6 - Calcium electroporation 217 Chairs: Boštjan Markelc and Luigi Aurisicchio 14:10 Calcium Assisted Irreversible Electroporation Treats Early-Stage Bladder Can- 217 OR-213 cer by Uniformly Ablating the Urothelial Layer Mary Chase Sheehan, Shengwei Wu, William Ray-Vista, Kimberly Crowley, Masashi Fujimori, Neeraj Raghuraman Rajagopalan, Brian Simoes, Yasushi Kimura, Govindarajan Srimathveeravalli 14:25 Characterization of two distinct immortalized endothelial cell lines, EA.hy926 218 OR-214 and HMEC-1: Exploring the impact of calcium electroporation, Ca2+ signaling and transcriptomic profiles Tim Bozic, Barbara Lisec, Iva Santek, Boštjan Markelc, Milka Vrecl, Robert Frangez, Maja Čemažar 14:40 Calcium Ascorbate delivered by Electroporation as a novel effective strategy 219 OR-215 for colorectal cancer treatment Erika Salvatori, Luicia Lione, Mirco Compagnone, Eleonora Pinto, Mariantonina Greco, Melanie Paccagnella, Valentina Frezza, Giuseppe Roscilli, Luigi Aurisicchio, Antonella Conforti 14:55 Modeling the Calcium Oscillations Response to Pulsed Electric Fields for 220 OR-216 Spinal Cord Regeneration Alessandra Paffi, Laura L. Caramazza, Micol Colella, Noemi Dolciotti, Sara S. Fontana, Francesca Apollonio, Micaela Liberti Thursday afternoon Track B, Thursday, Sep 19 2024, 14:10-15:40 Location: Cloister Hall Session: P8 - Electroporation in veterinary oncology 220 Chairs: Urša Lampreht Tratar and Maja Čemažar 14:10 Electrochemotherapy (ECT) with intratumoral and intravenous chemotherapy 220 OR-217 for the treatment of equine skin neoplasias Carolina Duran, Javier Ojeda 14:25 Safety of concurrent administration of electrochemotherapy with intravenous 221 OR-218 bleomycin and intravenous carboplatin or vinblastine in tumour-bearing dogs and cats: a case series Chiara Penzo, Jinjing He, Sarah Jayne Baker, Daisy Trewin, Stephen John Baines 14:40 Electrochemotherapy of Cutaneous Tumors in Exotic Pets 222 OR-219 Joško Račnik, Maja Čemažar, Gregor Serša, Urša Lampreht Tratar, Tanja Švara, Nina Kočar, Maruša Škrbec, Nataša Tozon 56 Thursday afternoon Track C, Thursday, Sep 19 2024, 14:10-15:40 Location: Hall 17 Session: S15 - Advanced imaging techniques for visualizing the mechanisms of pulsed electric field interactions 222 Chairs: Joel Bixler and Bennett Ibey Organizers/Conveners: J. Bixler, B. Ibey 14:10 Optical streaking microscopy enables visualization of ultra-fast response to 222 OR-220 charge accumulation from MHz bursts of nanosecond pulsed electric fields Mark Keppler, Sean O’Connor, Gleb Tolstykh, Benjamin Kasukonis, Vladislav V. Yakovlev, Joel N. Bixler 14:25 Visualization of Sub-microsecond Changes in Plasma Membrane Potential 223 OR-221 After Exposure to a Single Microsecond Electric Pulse, or 5 MHz Burst of Low Energy Nanosecond Electric Pulses Gleb Tolstykh, Mark Keppler, Roberto Rodriguez, Sean O’Connor, Joel N. Bixler, Benjamin Kasukonis 14:40 Changes in hydration of cell membranes exposed to pulsed electric fields de- 224 OR-222 tected by wide-field Coherent anti-Stokes Raman microspectroscopy Caterina Merla, Francesca Camera, Michael Scherman, Brigitte Attal-Tretout, Luis M. Mir 14:55 Identifying the Differences of Nanosecond Pulsed Electric Field Effects on In- 224 OR-235 tracellular Functions among Breast Cancerous and Normal Cells through Real- time Monitoring Kamlesh Awasthi, Nobuhiro Ohta Thursday afternoon Track D, Thursday, Sep 19 2024, 14:10-15:40 Location: Hall 5 Session: S14 - Electromagnetic modelling for pulsed electric fields 225 Chairs: Elisabetta Sieni, Raji Sundararajan, Stefania Romeo and Patrizia Lamberti Organizers/Conveners: E. Sieni, R. Sundararajan, S. Romeo, P. Lamberti 14:10 A coplanar waveguide picosecond pulsed electric fields (psPEF) delivery sys- 225 OR-224 tem for the electro-permeabilization of biological cells Hafsa Tjiou, Lionel Michard, Philippe Leveque, Claire Dalmay, Delia Arnaud-Cormos 14:25 A few hundred optoelectronic pulsed electric field generator with fully config- 226 OR-225 urable pulse shape, duration and amplitude Lionel Michard, Hafsa Tjiou, Vincent Couderc, Philippe Leveque, Delia Arnaud-Cormos 14:40 Complex Electrical Impedance and β-dispersion for Electroporation Sensing 227 OR-226 Guilherme Brasil Pintarelli, Raul Guedert, Jéssica Rodrigues da Silva, Lucas Bertinetti Lopes, Daniela Ota Hisayasu Suzuki 57 14:55 Interpulse bioimpedance reading during electroporation as a tool for monitor- 227 OR-227 ing ablation completeness Pedro Paulo Santos, Hee Chang Shin, Edward Jacobs, Rafael Davalos 15:10 Optimization of Electrode Arrangement in 96-Well Plates for In Vitro Electro- 228 OR-228 poration Experiments Ondrej Fiser, Ivana Fišerová, Jan Trnka, Pavel Osmančík, Marek Hozman, Jan Vrba, David Vrba, Marek Novak Thursday afternoon Track E, Thursday, Sep 19 2024, 14:10-15:40 Location: Hall 1 Session: S06 - Electrochemotherapy in treatment of vascular malformations 228 Chairs: Tobian Muir and Gregor Serša Organizers/Conveners: T. Muir, G. Sersa 14:10 Principles and mechanisms of bleomycin electrosclerotherapy in treatment of 228 OR-229 vascular malformations Gregor Serša, Maja Čemažar, Tobian Muir 14:25 Electrode for the Treatment of Atheromatous Plaques by means of a combina- 229 OR-230 tion of reversible and irreversible electroporation Ximena Manglano, Jesica Rodríguez Miranda, Aldo Perusso, Sebastian D. Michinski, Antonella María Cilio, Ana Laura Campastri, Felipe Maglietti 14:40 Development of in vitro and in vivo models of vascular malformations for de- 230 OR-231 termining bleomycin electrosclerotherapy (BEST) efficacy Boštjan Markelc, Barbara Lisec, Tanja Jesenko, Simona Kranjc Brezar, Maša Omerzel, Maja Čemažar, Gregor Serša 14:55 Use of bleomycin electroscleotherapy (best) in hereditary hemorrhagic tel- 230 OR-232 angiectasia patient (HHT): a case report Marta Minuti, Giulia Bertino, Fabio Pagella, Rebecca Gelli, Marco Benazzo 15:10 Clinical applications of BEST in treatment of vascular malformations 231 OR-233 Tobian Muir, Alan Batterham, Shaun Wellburn, Sri Murugan 15:25 Bleomycin Electrosclerotherapy (BEST): experience with lymphatic malforma- 231 OR-234 tions Giulia Bertino, Marta Minuti, Rebecca Gelli, Valentina Ravetta, Marco Benazzo 58 Poster Presentations Page Coffee Break and Poster Session, Monday, Sep 16 2024, 14:40-16:10 Location: Coffee break area (2nd floor) Session: Poster session 235 Chairs: Samo Mahnič-Kalamiza, Gennaro Ciliberto and Guus Pemen 14:40 Surgery and electrochemotherapy: an option for amputation of the posterior 235 PO-01 limb of a canine with an infiltrating sarcoma Oscar Pagoto 14:40 Outcome of one session of electrochemotherapy with bleomycin as single or 235 PO-02 adjuvant treatment in equine cutaneous sarcoids and melanoma Majbritt M. E. Larsen 14:40 Squamous cell carcinoma treated with electrochemotherapy 236 PO-03 Jesica Rodríguez Miranda, Antonella María Cilio, Ana Laura Campastri, Raquel Lertora, Sebastian D. Michinski, Ana Clara Acosta, Felipe Maglietti 14:40 Electrochemotherapy as salvage treatment of adrenal metastasis 236 PO-04 Barbara Perić, Nina Boc, Maja Čemažar 14:40 The Pancreatic Cancer Cellular Response to Calcium Irreversible Electropora- 237 PO-05 tion Agnieszka Gajewska- Naryniecka, Nina Rembiałkowska, Dagmara Baczyńska, Katarzyna Bieżuńska-Kusiak, Anna Szewczyk, Vitalij Novickij, Julia Rudno-Rudzińska, Wojciech Kielan, Julita Kulbacka 14:40 Tissue-ablation App for Electroporation-based Therapy 238 PO-06 Sudip Kumar Das 14:40 The Effects of High Frequency Nanosecond Pulsed Electric Fields with Calcium 238 PO-07 on 3D Spheroidal Model of Lung Cancer Wojciech Szlasa, Julia Kucharczyk, Eivina Radzevičiūtė-Valčiuke, Vitalij Novickij, Julita Kulbacka, Nina Rembiałkowska 14:40 Irreversible Electroporation: Impact of novel multiple train pulse field on non- 239 PO-84 thermal tumor ablation Ciara Nulty 14:40 Effects of calcium electroporation (CaEP), electrochemotherapy (ECT) and ir- 240 PO-08 reversible electroporation (IRE) in patients with locally advanced pancreatic adenocarcinoma. On-going clinical trial. (IREC) Julia Rudno-Rudziska, Ewelina Frejlich, Karolina Rodak-Mirocka, Wojciech Kielan, Julita Kulbacka 59 14:40 2D, 3D and in vivo osteosarcoma models for electrochemotherapy studies 240 PO-09 Sasa Kupcic, Urša Lampreht Tratar, Gregor Serša, Maja Čemažar 14:40 Cell death mechanisms detected in cardiomyoblasts after conventional IRE and 241 PO-10 after H FIRE Mario Gómez-Barea, Tomás García-Sánchez, Antoni Ivorra 14:40 Lesion Depth Analysis for Pulsed Field Ablation 242 PO-11 Kailee David 14:40 Evaluation of collagen role in electroporation: two cell lines compared 242 PO-12 Annj Zamuner, Monica Dettin, Serena Zaccara, Caterina Antognazza, Maria Teresa Conconi, Elisabetta Sieni 14:40 Electro-gene-transfer of a synthetic gene: a possible approach for treatment 243 PO-13 of Glycogen Storage Disease type III Doriana Triggiani, Caterina Merla, Emanuela Pasquali, Simona Salati, Rosella Franconi 14:40 Comparative Evaluation of Lipofectamine and Electroporation Side Effects on 243 PO-14 Cellular Functions: Emphasizing IMPDH Regulation Yusuf Elhaei, Samira Samadieh, Seyed Mojtaba Yazdanparast, Razieh Yazdanparast, Mohammad Abdolahad 14:40 New microsystem for gradual electroporation of a regular spheroid population 244 PO-15 and application for protocol comparison Théo Le Berre, Julien Marchalot, Guilhem Rival, Charlotte Rivière, Marie Frénéa-Robin 14:40 Z-Can modality for remote selective stimulation incorporating bipolar nano- 245 PO-16 second cancellation Shu Xiao, Pavel A. Solopov, Siqi Guo, Minglei Guo 14:40 Scanning electrochemical microscopy as a tool for microscale imaging after 245 PO-17 electroporation Gustas Tolvaisas, Inga Morkvenaite-Vilkonciene 14:40 Development of a high-voltage pulse generator with an integrated function of 246 PO-18 measuring tissue bio-impedance spectrum Yajun Zhao, Shiji Li, Shanshan Jin, Zhi Fang, Dong Xu 14:40 A numerical model of irreversible electroporation at tissue scale specific to 246 PO-19 cardiac pulsed field ablation Simon Bihoreau, Guido Caluori, Pierre Jaïs, Clair Poignard, Michael Leguèbe, Annabelle Collin 14:40 Electrical Equivalent Thermal Modelling of Tissues During High-Frequency Ir- 247 PO-20 reversible Electroporation Hamza Benchakroun, Cian Kerrigan, Jason McNamara, Maeve Duffy 60 14:40 Rate of pore formation by electroporation in black lipid membranes 248 PO-21 Burak Altun, Antoni Ivorra 14:40 Advancing Electroporation Studies with Automated SECM and Machine Learn- 248 PO-22 ing Kristijonas Klimaitis, Inga Morkvenaite-Vilkonciene 14:40 Characterization of Transferred Electrodes obtained with Laser Induced Car- 249 PO-23 bonization Process for Electroporation of Adhered Cells Francesca Camera, Gianni Antonelli, Alessandro Zambotti, Antonino Cataldo, Stefano Bellucci, Laura Micheli, Rocco Cancelliere, Mauro Falconieri, Serena Gagliardi, Eugenio Martinelli, Caterina Merla 14:40 Power Supply Chain with Series Insulation Structure for High Voltage Marx 249 PO-24 Pulse Generator Yue Chen, Chenguo Yao, Shoulong Dong 14:40 Hybrid Digital-Analogue Square Wave Generator for Bioelectronics Applica- 250 PO-116 tions Adam Parry, Cristiano Palego 14:40 Irreversible electroporation of tethered bilayer membranes by scanning elec- 250 PO-26 trochemical microscopy Tomas Sabirovas, Rūta Bagdonaitė 14:40 Scaling of continuous PEF processes by means of dimensionless numbers and 251 PO-25 computational fluid dynamics (CFD) Justus Knappert, Felix Schottroff, Claudia Siemer, Cornelia Rauh 14:40 Potential use of pulsed electric field (PEF) treatment to increase the concentra- 252 PO-27 tion of bioactive compounds during fermentation of Clementina peel pomace: conversion of waste into food additives Junior Bernardo Molina Hernandez, Beatrice Cellini, Emiliana Giordano, Pietro Rocculi, Francesca Patrignani, Urszula Tylewicz, Silvia Tappi, Rosalba Lanciotti, Marco Dalla Rosa, Lucia Vannini 14:40 Study on the functional properties of starch regulated by pulsed electric field 252 PO-28 assisted esterification boru Chen 14:40 Detection and differentiation of bacteria permeabilization induced by pulsed 253 PO-29 electric fields (PEF) using electrochemical admittance spectroscopy (EAS) Mindaugas Visockis, Paulius Ruzgys, Simona Gelažunaitė, Aras Rafanavičius, Saulius Šatkauskas 14:40 Extraction intensification of caffeoylquinic acids from Forced Chicory Roots 254 PO-30 by pulsed electrical field Etienne Diemer, Morad Chadni, Irina Ioannou, Nabil Grimi 61 14:40 Impact of Pulsed Electric Field Pretreatment on the Functional and Structural 254 PO-31 Characteristics of Rapeseed Protein isolate from Rapeseed Cake Busra Oktar, Ana Cristina De Aguiar Saldanha Pinheiro, Silvia Tappi, Urszula Tylewicz, Germana Barbieri, Andrea Brutti, Pietro Rocculi, Marco Dalla Rosa 14:40 Inactivation of Alicyclobacillus acidoterrestris vegetative cells and spores in- 255 PO-32 duced by atmospheric cold plasma: Efficacy and underlying memchanism Lang-Hong Wang, Xin-An Zeng 14:40 In vitro study of the antifungal activity of chloride species and peroxide hy- 255 PO-33 droxide generated during treatment with pulsed electric field - Potential use as sanitizing equipment and food handling art Junior Bernardo Molina Hernandez, Giulio Gannini, Lorenzo Siroli, Silvia Tappi, Urszula Tylewicz, Marco Dalla Rosa, Francesca Patrignani, Pietro Rocculi 14:40 Value-added compounds extraction from apple by-products using pulsed elec- 256 PO-34 tric fields Maite Gagneten, María de los Ángeles Saucedo, Irina Mailén Siniuk, Isaac A. Rodríguez Osuna, Guillermo R. Marshall, Carolina Schebor, Nahuel Olaiz 14:40 Modification of dietary fiber from apple bagasse by combining pulsed electric 257 PO-35 fields and enzymatic hydrolysis Alba Díaz Núñez, Pedro Elez-Martinez, Robert Soliva-Fortuny, Olga Martín-Belloso 14:40 A study for achieving a higher effectiveness at less irradiation number on ster- 257 PO-36 ilization using pulsed plasma for cut vegetables packaging low oxygen atmo- sphere Pengcheng Cui 14:40 Application of pulsed electric field (PEF) treatment before ultrasound-assisted 258 PO-37 convective drying of organic strawberries Katarzyna Rybak, Dorota Witrowa-Rajchert, Małgorzata Nowacka 14:40 Unveiling the interplay between gliding arc discharge (GAD) plasma pretreat- 259 PO-38 ment and pulsed electric field (PEF) on Chlorella vulgaris microalgae Kamilė Jonynaitė, Rolandas Uscila, Mindaugas Aikas, Skirmantas Keršulis, Žy-drūnas Kavaliauskas, Liutauras Marcinauskas, Arūnas Stirkė, Voitech Stankevic Coffee Break and Poster Session, Tuesday, Sep 17 2024, 15:20-16:50 Location: Coffee break area (2nd floor) Session: Poster session 259 Chairs: Olga Zeni, Alexander Mathys and Óscar Lucía 15:20 Minimally invasive electrochemotherapy for the treatment of hepatocellular 260 PO-39 carcinoma: single centre study Mihajlo Djokic, Blaz Trotovsek, Rok Dezman, Miha Stabuc, Maja Čemažar, Gregor Serša, Benjamin Hadzialjevic 62 15:20 Advancing Cancer Treatment: Automated Application of Electric Pulses and 260 PO-40 Radiation Targeting Stem Cells guides by artificial intelligence (AI) algorithm Arianna Casciati, Mirella Tanori, Francesca Camera, Nicolo’ Colistra, Marco Salvatore Zappatore, Luciano Tarricone, Raffaele Crusi, Alfredo De Cillis, Simona Salati, Giacomo Perazzolo Gallo, Caterina Merla 15:20 Optimal Interphase Delay to Mitigate Cancellation Phenomenon in Bipolar 261 PO-41 Pulse Electrochemotherapy with Cisplatin Veronika Malyško-Ptašinskė, Aušra Nemeikaitė-Čėnienė, Eivina Radzevičiūtė- Valčiuke, Eglė Mickevičiūtė, Paulina Malakauskaitė, Barbora Lekešytė, Vitalij Novickij 15:20 Analyzing Breast Cancer Cell Electroporation: Perspectives from Scanning 261 PO-42 Probe Microscopy Methods Terese Kondrotaite, Tomas Mockaitis, Antanas Zinovicius, Inga Morkvenaite-Vilkonciene 15:20 Synergistic Bipolar Irreversible Electroporation (SBIRE): A Novel Approach for 262 PO-43 Effective Tumor Removal without Inducing Muscle Contractions Yancheng Wang, Kun qian, Qiang Yang, Yizhen Lei, Shoulong Dong, Chenguo Yao 15:20 Cisplatin and bleomycin increase cell mortality during partial irreversible elec- 263 PO-44 troporation on hepatocellular carcinoma spheroids model Alexia de Caro, Nicolas Mattei, Mathilde Poutier, Marie-Pierre Rols, Jelena Kolosnjaj-Tabi, Muriel Golzio 15:20 Microplastic particles (MPs) delivery by electroporation (EP) and their effects 263 PO-45 on the development of breast cancer cells Katarzyna Bieżuńska-Kusiak, Agnieszka Gajewska- Naryniecka, Agnieszka Chwiłkowska, Urszula Szwedowicz, Dagmara Baczyńska, Nina Rembiałkowska, Anna Szewczyk, Julita Kulbacka 15:20 Enhanced Visualization and Control of Drug Distribution in Electrochemother- 264 PO-46 apy Using Indocyanine Green with Bleomycin in a Murine 4T1 Mammary Tumor Model Joanna Tunikowska, Urszula Bazylińska, Anna Szewczyk, Nina Rembiałkowska, Zdzisław Kiełbowicz, Justyna Mączyńska, Vitalij Novickij, Julita Kulbacka 15:20 Exploring Immune Stimulation for Cancer Treatment 265 PO-47 Anna Szewczyk, Nina Rembiałkowska, Katarzyna Bieżuńska-Kusiak, Vitalij Novickij, Julita Kulbacka 15:20 Electrochemoterapy in personalized medicine.A predictive in vitro model for 265 PO-48 electrochemotherapy in metastatic melanoma Nicolò Martinelli, Annj Zamuner, Monica Dettin, Luigi Dall’Olmo, Luca Menilli, Luca Giovanni G. Campana, Elisabetta Sieni, Maria Teresa Conconi 63 15:20 Modulating Electrochemotherapy Efficacy in Ovarian Carcinoma with Bipolar 266 PO-49 nsPEFs: Insights into Cell Membrane Permeabilization and Reactive Oxygen Species Levels Zofia Łapińska, Vitalij Novickij, Nina Rembiałkowska, Eivina Radzevičiūtė-Valčiuke, Anna Szewczyk, Magda Dubińska-Magiera, Julita Kulbacka, Jolanta Saczko, Dagmara Baczyńska 15:20 Curcumin-Electroporation downregulates key heat shock and heat stable pro- 267 PO-50 teins in Curcumin supplementation rats Praveen Sahu, Lakshya Mittal, Ignacio G. Camarillo, Raji Sundararajan 15:20 Gene electrotransfer of tumor and muscle tissue with clinically used electric 267 PO-51 pulse parameters Maša Omerzel, Simona Kranjc Brezar, Boštjan Markelc, Gregor Serša, Maja Čemažar 15:20 Optimisation and validation of electroporation protocols in 3D bioprinted tu- 268 PO-52 mour models of colorectal cancer Yordan Sbirkov, Tsvetomira Ivanova, Milena Draganova, Iva Ilieva, Stefan Hubenov, Victoria Sarafian 15:20 The bystander effect after electroporation with microsecond and nanosecond 269 PO-53 pulses Neringa Barauskaite-Šarkinienė, Ugne Borinskyte, Vitalij Novickij, Saulius Šatkauskas, Paulius Ruzgys 15:20 Calcium-mediated Inactivation of Drug-resistant Microorganisms Using Pulsed 269 PO-54 Electric Fields Gediminas Staigvila, Jurgita Švedienė, Svetlana Markovskaja, Paulina Malakauskaitė, Veronika Malyško-Ptašinskė, Jurij Novickij, Algimantas Paškevičius, Vitalij Novickij 15:20 The Effects of Bipolar Cancellation Phenomenon on Nano- 270 PO-55 Electrochemotherapy of Melanoma Tumors Eglė Mickevičiūtė, Eivina Radzevičiūtė-Valčiuke, Veronika Malyško-Ptašinskė, Paulina Malakauskaitė, Barbora Lekešytė, Nina Rembialkowska, Julita Kulbacka, Vitalij Novickij 15:20 Reversible and irreversible electroporation mechanisms: an in vitro study on 270 PO-56 two pancreatic cancer cell models Mariateresa Allocca, Luigi Sapio, Anna Sannino, Olga Zeni, Maria Rosaria Scarfì, Stefania Romeo 15:20 Delivery of Anticancer Drugs with Protein-Based Nanocarriers Using Nano- 271 PO-57 second Pulsed Electric Fields and Shock Waves Shirin Khakpour, Zahra Moosavi-Nejad, Nushin Hosano, Hamid Hosano 15:20 The effect of pulse duration on electrostimulation and electroporation of excit- 271 PO-58 able S-HEK cells Tina Batista Napotnik, Tina Cimperman, Lea Rems 64 15:20 Protective Effects of Iron Compounds on Controlled Membrane Damage In- 272 PO-61 duced by Varied Pulsed Electric Field Durations in Cardiac and Skeletal Myo- cytes Nina Rembiałkowska, Anna Szewczyk, Katarzyna Bieżuńska-Kusiak, Dawid Przystupski, Eivina Radzevičiūtė-Valčiuke, Vitalij Novickij, Julita Kulbacka 15:20 A comparison of small molecule intracellular electrotransfer in spheroids and 273 PO-62 cell suspension Neringa Barauskaite-Šarkinienė, Simona Gelažunaite, Aras Rafanavičius, Gabija Andreikė, Vitalij Novickij, Paulius Ruzgys 15:20 Investigation of the state of cell death by applying pulsed electric field under 273 PO-63 ROS suppression Yasushi Minamitani, Takayoshi Kowase, Koki Saito 15:20 Electroporation-generated extracellular vesicles in tumor and normal cells in- 274 PO-64 teractions Anna Choromańska, Urszula Szwedowicz, Anna Szewczyk, Dagmara Baczyńska, Roksana Kruszakin, Krzysztof J. Pawlik, Julita Kulbacka 15:20 Electroporation induced protein elution out to extracellular media and cyto- 274 PO-65 plasmic membrane blebbing Salvijus Vykertas, Baltramiejus Jakštys, Saulius Šatkauskas 15:20 Synthetic Cell Models to Understand the Impact of the Actin Cortex on Mem- 275 PO-66 brane Electroporation Nikki Nafar, Gijsje Koenderink 15:20 Deciphering the resealing of membranes after a pulse using impedance meas- 275 PO-111 urements by numerical modelling Audrey Gossard, Tomas Garcia, Luis M. Mir, Annabelle Collin, Clair Poignard 15:20 Electroporation in vesicles under ms-pulsed electric field 276 PO-67 Nalinikanta Behera, Rochish Thaokar 15:20 Unveiling Fusion Pore Dynamics: Integrating Fluorescence and Electrochem- 276 PO-68 ical Imaging on Supported Bilayers Federica Castellani, Weronika Tomaka, Daniella Lopera, Nadia Prasad, Juana Sefair, Volker Kiessling, Manfred Lindau 15:20 Efficient Lipid Extraction with Underwater Pulsed Electric Discharge Shock 277 PO-69 Waves Seyedmasih Hosseini, Md Mijanour Rahman, Nushin Hosano, Hamid Hosano 15:20 A novel approach for modelling membrane electroporation dynamics 278 PO-70 Rashid Ali Faridi, Rochish Thaokar 15:20 Effect of electroporation in combination with inorganic particles used in tattoo 278 PO-71 inks Ophelie Cordier, Emma Barrere, Nicolas Mattei, Marie-Pierre Rols, Muriel Golzio, Jelena Kolosnjaj-Tabi 65 15:20 A mechanistic numerical model of cell membrane electroporation that links 279 PO-72 electro-poration and electro-permeabilization Ting Shu, Antoni Ivorra 15:20 Electrical conductivity effect on Anisakis spp inactivation by PEF and impact 280 PO-73 on fish quality Vanesa Abad, Natalia Escursell, Teresa Peiro, Adrián Ruiz, Javier Raso, Guillermo J. Cebrian, Ignacio Álvarez 15:20 The dynamics of synergetic bacteriocidic effect of pulsed electric fields and 280 PO-74 antibiotics Simona Gelažunaite, Mindaugas Visockis, Aras Rafanavičius, Saulius Šatkauskas, Paulius Ruzgys 15:20 Comparative Study of the Effects of Nanosecond and Microsecond Pulsed 281 PO-75 Electric Fields on Saccharomyces cerevisiae Pablo Briz, Alejandro Berzosa, Javier Marín-Sanchez, Borja López-Alonso, Cristina Calvo, Héctor Sarnago, Óscar Lucía, Javier Raso 15:20 Stimulation of Saccharomyces cerevisiae metabolism and growth using pulsed 282 PO-76 electric fields Benjamin Schmiedl 15:20 Modification of corn starch using pulsed electric fields: effects on composi- 282 PO-117 tion, structure, and techno-functionality Núria Farràs-Moragues, Saqib Gulzar, Pedro Elez-Martinez, Olga Martín-Belloso, Robert Soliva-Fortuny 15:20 The impact of electroporation on the therapeutic efficiency of colorectal cancer 283 PO-59 3d printed cells under hypoxia Tsvetomira Ivanova, Yordan Sbirkov, Victoria Sarafian 66 Coffee Break and Poster Session, Wednesday, Sep 18 2024, 15:20-16:50 Location: Coffee break area (2nd floor) Session: Poster session 283 Chairs: Rosa Orlacchio, Anna Szewczyk and Olga Martín-Belloso 15:20 Surgery and electrochemotherapy: An option for a feline with recurring infilt- 283 PO-77 rating sarcoma Oscar Pagoto 15:20 Electrochemotherapy in combination with surgery and radiotherapy. The role 284 PO-78 of translational medicine Felipe H. Horacio Maglietti, Matias N. Tellado, Antonella Cilio, Ana Campastri, Sebastian D. Michinski, Ana Clara Acosta, Raquel Lertora 15:20 Impact of Reversible Electroporation on Melanoma Cell Viability and Extracel- 285 PO-79 lular Vesicle Function Urszula Szwedowicz, Anna Choromańska 15:20 Potential of Ultrashort Pulsed Electric Fields to Empower Traditional Cancer 285 PO-80 Treatment by Breaking Solid Tumor Barriers Kun Qian, Chenguo Yao 15:20 Novel Bipolar Pulses for Improved Co-transfection Outcomes: Implications for 286 PO-81 CRISPR Cas 9 Delivery Alexia Cash, Robert H. Williamson, Mike Sano 15:20 Study on the Effect of Microsecond Pulsed Electric Field in Promoting Wound 286 PO-82 Healing in Diabetic Mice Lei Li, Chenguo Yao 15:20 PEF effect on a 3D in vitro model: a breast cancer case 287 PO-83 Patrizia Lamberti, Donatella Fiore, Maria Chiara Proto, Annj Zamuner, Monica Dettin, Elisabetta Sieni, Raji Sundararajan, Maria Teresa Conconi, Patrizia Gazzerro 15:20 Rare non-malignant, locally aggressive lesions of the head and neck treated by 287 PO-85 electrochemotherapy Gabor Vass, Aurel Ottlakan, Ildiko Csanyi, Eszter Baltas, Rolland Gyulai, Judit Olah, Erika Gabriella Kis 15:20 Bleomycin electrosclerotherapy (BEST) to manage head and neck venous mal- 288 PO-86 formations: a new therapeutic option and a case series Rebecca Gelli, Giulia Bertino, Marta Minuti, Marco Benazzo 15:20 Nano-Electrochemotherapy (NEC) to enhance head and neck cancer treatment 288 PO-87 Silvia Pisani 15:20 Characterising and enhancing immunogenic cell death following reversible ion 289 PO-88 electroporation Megan McAuley, Ciara Nulty, Declan Soden, Vincent Kelly 67 15:20 The Effects of Buffer Composition on Gene Electrotransfer by Nanosecond 290 PO-89 Electric Field Pulses Eivina Radzevičiūtė-Valčiuke, Jovita Gečaitė, Anna Szewczyk, Barbora Lekešytė, Veronika Malyško-Ptašinskė, Eglė Mickevičiūtė, Paulina Malakauskaitė, Julita Kulbacka, Vitalij Novickij 15:20 Lentigo Maligna Melanoma and Acral Lentiginous Melanoma Treatment with 290 PO-107 Electrochemotherapy Petra Rozsa 15:20 The synergistic electrotransfection effect of low-amplitude continuous wave 291 PO-90 application and nanosecond electroporation Paulius Ruzgys, Neringa Barauskaite-Šarkinienė, Eivina Radzevičiūtė, Saulius Šatkauskas, Vitalij Novickij 15:20 Electroporation-Enhanced Resveratrol Delivery into 3D-Hyaluronic Acid- 292 PO-91 Peptide Scaffold Cells for Effective Triple-Negative Breast Cancer Treatments Pragatheiswar Giri, Praveen Sahu, Ignacio G. Camarillo, Monica Dettin, Annj Zamuner, Maria Teresa Conconi, Raji Sundararajan, Elisabetta Sieni 15:20 The search for an optimal IRE protocol in terms of pulse duration considering 292 PO-92 damage due to temperature effects Isaac Rodriguez, Nahuel Olaiz, Felipe H. Horacio Maglietti, Ezequiel Goldberg, Sebastian D. Michinski, Cecilia Suárez, Alejandro Soba, Guillermo R. Marshall 15:20 Effectiveness of a Novel Basket-Shaped Pulsed Field Ablation Catheter for Intra 293 PO-93 Pulmonary Vein Ablation Jason Tri 15:20 Comparison of high-frequency pulse train alternating form on endothelial cell 293 PO-94 electroporation and permeability Liang Yu, Lvheng Ren, Sicong Wang, Shoulong Dong, Chenguo Yao 15:20 Dynamics of plasma membrane charging and relaxation measured by strobe 294 PO-95 fluorescence microscopy Iurii Semenov, Joel Bixler, Allen Kiester, Bennet L. Ibey, Andrei G. Pakhomov 15:20 Stream pulsed electric fields integral (sPEFI) and energy properties of tissue 295 PO-96 ablation on irreversible electroporation Weiwei Huang, Jiali Bao 15:20 Impact of pulse parameters on the conductivity variations in Biological tissues, 295 PO-97 treated with electroporation Praveen Sahu, Marco Barozzi, Hala Mohamed Abd El Megeed, Patrizia Lamberti, Ignacio G. Camarillo, Elisabetta Sieni, Raji Sundararajan 15:20 Pulsed field ablation for cardiac arrythmias: parameters prediction via machine 296 PO-60 learning Raffaele Crusi, Nicolo’ Colistra, Francesca Camera, Marco Salvatore Zappatore, Giuseppina Monti, Caterina Merla, Luciano Tarricone 68 15:20 Optimization of pulsed electric field (PEF) processing conditions for wheat 296 PO-98 flour treatment using Response Surface Methodology (RSM) Dominique Larrea-Wachtendorff, Danela Silva-Ferrer, Mario Perez-Won, Gipsy Tabilo-Munizaga 15:20 Flyback Versus Piezo Transformer Based Converter Topologies for Bipolar 297 PO-99 Pulsed-Power Applications Ajay M. Chole, Jeya Shree Thulasidas, Maeve Duffy 15:20 A Smart and Portable Electroporation System for More Rigorous Experiments 297 PO-100 Junrui Zhang, Xingyou Zhou, Junyan Qian, Ren Wang, Federico Jara Crua, Zhixian Deng, Shulin Wu, Yumei Xue, Xin Chen, Sixiang Li, Yuehua You, Shiyu Cheng 15:20 High-Performance Modular Pulse Generator for Electroporation Applications 298 PO-101 Héctor Sarnago, Borja López-Alonso, Pablo Briz, José M. Burdío, Óscar Lucía 15:20 Simulation study on magnetoporation induced by pulsed magnetic field com- 299 PO-103 bined with magnetic nanoparticles based on pore energy Wei Zheng, Yan Mi 15:20 Rapid joule heating improves vitrification based cryopreservation 299 PO-102 Qi Shao 15:20 Experimental study on protein denaturation induced by MV/cm class electrical 300 PO-104 pulses Koki Tsurusaki, Yuya Sato, Keisuke Endo, Sunao Katsuki 15:20 Where exactly do pores form in the complex organization of the plasma mem- 301 PO-105 brane? Insights from molecular simulations Lea Rems 15:20 Effects of microsecond pulsed electric field on tubulin structure and self- 301 PO-106 assembly Michaela Poplová, Tomáš Zakar, Viliam Kolivoška, Michal Cifra 15:20 nsPEF-mediated productivity improvement in bioprocessing – A cross-species 302 PO-108 evaluation of bacterial and yeast expression platforms in bioreactor cultures Lukas Neutsch 15:20 Enhancing starch for 3D food printing: pulsed electric field modification and 302 PO-109 functional insights Saqib Gulzar, Pedro Elez-Martinez, Olga Martín-Belloso, Robert Soliva-Fortuny 15:20 Inhibition of Color Change for Long Term on Meat of Bonito during -18� Freez- 303 PO-110 ing by Applying Pulsed High Electric Field Koki Saito, Shoichiro Kosugi, Koshi Kawasaki, Yasushi Minamitani, Ryo Sawada 69 15:20 Development of pulsed electric field pasteurization system for protein-rich li- 304 PO-112 quid foods Tomohiro Nakamura, Bingyu Yan, Akira Moriyama, Ryousuke Kadoya, Sunao Katsuki, Naoya Masuda, Yoshiharu Shimizu, Ryo Sasahara, Taiga Kajiwara, Jiro Kurihara 15:20 Enhancement of bioactive properties of maillard reacted peptides by pulsed 304 PO-113 electric fields Carolina Herrera-Lavados, Mario Perez-Won, Luis Moreno-Osorio, Gipsy Tabilo-Munizaga 15:20 Polyphenolic content and antioxidant activity of pulsed electric field-assisted 305 PO-114 extracts of green rooibos. Lusani Vhangani 15:20 Enhanced Extraction of Cellulose and Lignin from Agro-Industrial Wastes Util- 305 PO-115 izing Alkali Treatment Assisted by High-Voltage Electrical Discharges (HVED) for Wood Adhesives Application Yassine El Khayat Driaa, Hafida Maarir, Nabil Grimi, Amine Moubarik, Nadia Boussetta P L E N A R Y L E C T U R E S ’ A B S T R A C T S Plenary talks Charging the membrane to -200 mV and beyond produced one or several focal fluorescence transi- Monday morning plenaries ents. Their brightness decreased sideways from the center, consistent with Ca2+ entry through a Sep 16, 8:50 - 9:50 pore followed by a radial diffusion. These transi- PL-01 ents disappeared within 10-20 ms after the voltage Visualizing Electropore Dynamics in Live Cells step and marked the formation of short-lived Mantas Silkunas, Olga N. Pakhomova, Giedre electropores with 50- to 200-pS conductance. Silkuniene, Andrei G. Pakhomov Larger voltage steps increased the brightness, Old Dominion University, United States the size, and the number of transients, which did not necessarily appear at the same locations Discerning individual membrane lesions in as with the smaller steps. This pattern indicates electroporated cells has been a challenge due to that pre-existing lesions, by increasing local their size, which falls below the resolution limit membrane permeability and reducing charging of optical microscopy. Nonetheless, we have efficiency, could make the membrane locally less succeeded in tracking individual electropores by vulnerable to charging. At -240 mV we observed imaging Ca2+ entry with total internal reflection high-conductance pores (> 1 nS) that persisted fluorescence (TIRF) microscopy. TIRF imaging for seconds after the voltage step. Attempts to is restricted to an extremely shallow (˜100-nm) charge the membrane beyond the limit of about subplasmalemmal layer, which facilitates the -260 mV increased pore conductance in an ad- observation of Ca2+ fluxes through individual aptive manner, “clamping” the induced membrane pores instead of seeing a diffuse fluorescence potential at this limit or even depolarizing it. These cloud. Measuring pore currents simultaneously observations suggest possible paradigm shift with imaging revealed different types of lesions in recognizing electroporation not merely as a and the dynamics of pore creation and resealing. membrane injury but also as a potential protective A whole-cell voltage clamp configuration was mechanism. established in human embryonic kidney cells placed on glass coverslips with an indium tin Supported in part by NIH NEI R21EY034258 oxide (ITO) layer as electrical ground. Voltage and R21EY034803 (to A.G.P). steps, 1 to 25 ms, applied between the pipette and the ITO, induced lesions in the membrane PL-02 portion adjacent to the ITO. Loading cells with the Ion ion/protein mobilization following PEF ap- Ca2+ sensitive dye CAL-520 enabled dynamic plication visualization of Ca2+ fluxes through these lesions. Sunao Katsuki These experiments provided the first evidence of Kumamoto University, Japan electropore persistence for at least a minute in the absence of an applied membrane potential, as well No abstract was provided. as direct measurements of single pore electrical conductance. Plenary talks Hyperpolarizations to about -100 mV induced a diffuse zonal electropermeabilization, manifested Tuesday morning plenaries by momentary and subtle Ca2+ fluorescence Sep 17, 8:30 - 10:00 upticks (scintillas) over large membrane areas. PL-03 The scintillas lacked definitive brighter spots Lethal and non-lethal perturbation of cells by which would indicate a pore opening, and were electroporation accompanied by a modest (<100 pS) increase in Antoni Ivorra the whole-cell conductance. Universitat Pomepu Fabra, Spain 73 markably, researchers in the field of tumor treat-It has often been stated that irreversible elec- ment fields (TTFields), using alternating fields (200 troporation kills cells by disrupting cellular homeo- kHz) with extremely low electric field magnitudes stasis. This vague statement, while true, impli- (4 V/cm) applied for days, observed an increase in citly acknowledges our ignorance regarding cellu- cell membrane permeability to relatively large mo- lar death mechanisms after non-thermal large elec- lecules (> 4 kDa) [Cell Death Discov, 2018, 4:113] tric field exposure. Since early observations, it is which might also be due to calcium uptake. understood that, unless field exposure is extreme, both in magnitude and in time, cell death does PL-04 not occur immediately via necrosis. Delayed cell Mechanistic insights into the effects of electro- death, via regulated cell death mechanisms such poration on excitable cells as necroptosis, pyroptosis, and even apoptosis, Lea Rems has been reported for more moderate exposures University of Ljubljana, Slovenia [Bioelectrochemistry, 2021, 141:107871]. We also now know that extracellular calcium (Ca2+) uptake Understanding electroporation of excitable cells probably has a major role in cell death by electro- is becoming increasingly important due to the rapid poration [Bioelectrochemistry, 2020, 131:107369; advancement of applications targeting excitable tis- Bioelectrochemistry, 2021, 142:107927]. In hind- sues, including but not limited to electroporation- sight, this is unsurprising given the large differ- based cardiac tissue ablation, gene electrotrans- ence in calcium concentration between the cell ex- fer of nucleic acids into muscle tissue, irrevers- terior (˜2 mM) and the cell interior (0.2 µM) and ible electroporation of brain tumors and epileptic that calcium participates as a second messenger zones, as well as electrostimulation using nano- in several cellular signaling processes. That is, it second electric pulses. Excitable cells (muscle, is reasonable to assume that, if the plasma mem- nerve, and neuroendocrine cells) are characterized brane is made much more permeable to calcium by by expression of voltage-gated ion channels that electroporation, the inflow of extracellular calcium enable the cells to trigger electrical signals called can severely interfere with cellular processes to the action potentials. This lecture will provide new in-point of triggering cell death. And it is also reas- sights from experiments, continuum modeling, and onable to hypothesize that the calcium uptake by molecular dynamics simulations on how electropor- electroporation may have a profound impact on cel- ation can affect the electrophysiological response lular processes such as gene expression and cell of excitable cells and discuss the open questions proliferation for non-lethal exposures; particularly if relevant to the above-listed medical applications. the insult is continuous or repeated over time. In PL-05 my view, non-lethal electroporation using low fields EP for Cardiac ablation and long exposures represents an opportunity for Vivek Reddy developing new therapeutic modalities. Electro- Icahn School of Medicine at Mount Sinai, United States poration is frequently depicted as a phenomenon that cannot be observed for low field magnitudes, No abstract was provided. even if the exposure is very long. However, recent experimental observations challenge such depic- Plenary talks tion. In the context of explaining the long-term neur-omodulatory capabilities of pulsed radio-frequency Wednesday morning currents (ac 500 kHz), it has been demonstrated plenaries that, albeit mildly, cell permeability does increase Sep 18, 8:30 - 10:00 for calcium when relatively long exposures (˜ 2 seconds) of low magnitude (<200 V/cm) are applied [Bioelectrochemistry, 2020, 136:107624]. And, re- 74 PL-06 Electroporation as a therapeutic modality for Pioneering sustainable food production with cancer treatment is still “novel” in veterinary on- pulsed electric field technology cology especially in the United States and is pre- Indrawati Oey dominantly dominated by ECT. This presentation University of Otago, New Zealand will discuss the limited history of usage, miscon- ceptions regarding the technology, the challenges No abstract was provided. faced with education of new and interested spe- PL-07 cialists and pet owners, the availability of generat- Unleashing the potential of pulsed electric ors and electrodes, the awareness and limitations fields in food processing: from techno- with immunotherapy delivered via GET vs. tradi- functional improvement to health-boosting in- tional methods and finally, discuss future concepts novations for solutions on improving these current limitations Robert Soliva-Fortuny, Pedro Elez-Martinez, Olga and challenges. Martín-Belloso University of Lleida, Spain Plenary talks Applications of pulsed electric fields (PEF) in food processing are continuously expanding, Thursday morning plenaries demonstrating its capability to enhance the sustain- Sep 19, 10:40 - 12:10 ability of food processes as well as the quality of the obtained products. Beyond its remarkable im- PL-09 pact on food structure and functionality, PEF can Enhanced Delivery of Plasmid DNA Encoding be strategically used to preserve, and even en- Therapeutic Agents as a Means to Induce a Ro- hance, the health-related content of plant-based bust Anti-Tumor Immune Response products. Recent applications of mild PEF treat- Richard Heller, Loree Heller, Guilan Shi, Jody Synowiec, Julie Singh, Alex Otten, Mark J. Jaroszeski ments emerge as a strategy to stimulate the pro-University of South Florida, United States duction of secondary metabolites in fresh commod- ities, thereby increasing their antioxidant potential. Gene electrotransfer (GET) has been used to PEF also stands as a potent tool to improve the successfully deliver plasmid DNA to multiple tis- bioaccessibility of desirable compounds, introdu- sue targets. While there have been successes with cing a health-centric dimension to food processing. GET, there is still additional hurdles that need to Recent studies unveil the ability of PEF in elev- be overcome to advance the technology and en- ating the techno-functional and health-related prop- hance results. A key to enhancing effective gene- erties of food macromolecules, such as polysac- based immunotherapy is controlling the reprodu- charides (e.g. dietary fiber, starch) and proteins. cibility and expression levels of the delivered transThis lecture will explore the multifaceted role of gene. To this end, our research group has mod- electroporation for producing enhanced food in- ified the electrotransfer approach to enhance its gredients, simultaneously addressing sustainable utilization in multiple applications. One area that valorization, technological innovation, and health- we are testing with this new approach is immuno- boosting applications in plant-based products. therapy of solid tumors. The discovery and utiliz- ation of immune checkpoint inhibitors (ICIs) have PL-08 been demonstrated to have positive clinical out- Electroporation - The U.S. Veterinary Experi- comes, but have been limited by both primary and ence with ECT and GET acquired resistance. A major issue is the absence Joseph A. Impellizeri of T cells within the tumor microenvironment (TME). Veterinary Oncology Services, United States We previously demonstrated that the intratumor de- livery of a plasmid encoding interleukin-12 (pIL- 75 12) using GET resulted in a significant increase Metastatic epidural spinal cord compression in T effector cells and a reduction in T regulatory (MESCC) frequently results in severe pain and cells and myeloid derived suppressor cells within neurologic impairment such as paraplegia. Con- the TME. Adding ICIs together with pIL-12 GET in- ventional external beam radiation therapy is the duced a robust immune response leading to both standard of care for frontline therapy. Unfortu- local and systemic responses in preclinical and clin- nately, recurrence is common and the option of re- ical studies. While these results were encouraging, irradiation is often limited out of concern for spinal there was still a need to enhance the delivery pro-cord damage from cumulative radiation dose. Elec- cess. For example, the established GET techno- trochemotherapy is now an option to radiotherapy- logy requires high applied voltage for plasmid de- resistant MESCC, providing rapid and durable pain livery and lacked a means to determine if delivery relief and neurological improvement in the ad- had occurred during the process. Our current re- vanced cancer setting. However, neurological search has focused on developing a GET system complications can occured, related to the inevit- that could overcome these issues. A new system able technological reality that hyperthermia and ir- that incorporates a heat source and a tissue imped- reversible electroporation occur in the immediate ance measuring device as well as an array that in- vicinity of the electrodes. Further research to tailor corporates independently addressable electrodes this technique is merited to improve the safety pro- was developed and tested. A moderate elevation file and risk/benefit balance. of the tissue temperature coupled with the elec- trode array enabled approximately a 60% reduc- PL-11 tion in the applied voltage. We tested the approach Vascular malformation ablation in the B16.F10 mouse melanoma model. Delivery Walter A. Wohlgemuth of pIL-12 with the new system resulted in >80% UMH - Halle University Clinic, Germany long-term complete regression. In addition, mon- No abstract was provided. itoring impedance within each independent section of the electrode array revealed different pulse num- bers were needed to achieve delivery in each sec- tion. The utilization of this enhanced approach en- ables a more controlled delivery and application of the pulse fields. Another aspect of the current re- search is to examine the ability to deliver plasmid DNA encoding checkpoint peptides, i.e., PD1 and combine that with pIL-12. In a multi-tumor model, the plasmids were delivered to a subcutaneous tu- mor; tumors generated with an intraperitoneal in- jection were left untreated. This new therapy in- duced complete regression of the subcutaneous tu- mor and blockage of peritoneal tumor growth as assessed via in vivo imaging. Work is ongoing to translate this approach to clinical evaluation. PL-10 Clinical application in bone metastases com- pressing the spinal cord Frederic Deschamps Institut Gustave Roussy, France 76 O R A L P R E S E N T A T I O N S ’ A B S T R A C T S Educational Session properties, which both differ between patients in healthy and malignant tissues and change in an Sunday Educational Session electric field- dependent manner from the electro- part 1 poration process itself. We use an in situ method , termed voltage ramp, Sep 15, 13:00 - 15:00 that applies a series of increasing voltages across Info: This session is not supported by abstract treatment electrodes and measures the resulting submissions. current . Due to the inherent non-linearity in the system, we develop a robust deep neural network, Educational Session trained on finite element model simulations, to un- ravel the relationship between V/I characteristics and tissue properties. We found minimal test er- Sunday Educational Session ror (p < 0.0001), and our model was validated to part 2 correctly predict the complete dynamic conductiv- Sep 15, 15:30 - 17:00 ity curve in a previously characterized ex vivo liver Info: This session is not supported by abstract model (p < 0.0001). submissions. We believe this platform can be incorporated prior to treatment to rapidly ascertain patient-specific tissue properties paramount in electroporation treat- S21 - Cardiac ablation by ment planning models and real-time treatment pre- irreversible electroporation - diction algorithms, and this method can be used pulsed field ablation (PFA) over current ex vivo methods for in situ tissue char- acterization . We characterized and validated the Monday morning Track A first reported electrical tissue properties of lung tu-Sep 16, 10:30 - 12:00 mors from five canine patients. Further, we have characterized in vivo pancreatic cancer tumors in OR-01 both mice and immunocompromised swine. Lastly, In situ characterization of electroporation- we have characterized the first-reported properties dependent tissue properties for cancer and car- for cardiac tissue from for pulse widths ranging from diac ablation 500 ns to 100 us, using ex vivo porcine tissue within Edward Jacobs, Pedro Santos, Rafael Davalos 5 minutes of removal from the animal and main- Georgia Tech, United States tained at body temperature. Pulsed electric field (PEF) therapies deliver The nonthermal mechanisms for IRE are para- high-voltage, short electric pulses directly into tis-mount for treating tissue near anatomically sens- sue to permeabilizes cells through the generation of itive structures. Numerous thermal mitigation pro- nano-scale pores (electroporation). The transitory tocols have been proposed to minimize temperat- formation of pores is called reversible electropora- ure rise, but intraoperative temperature monitoring tion and is used to deliver impermeable substances is still needed. We demonstrate here that an ac- into cells. Larger and longer PEFs may induce curate and robust temperature prediction machine cell death through loss of homeostasis, termed irre- learning model can be developed using estimated versible electroporation (IRE). IRE was considered tissue properties (bulk and dynamic conductivity), the upper limit of reversible electroporation but has known geometric properties (probe spacing), and been developed as a standalone method for tis- easily measurable treatment parameters (applied sue ablation. PEF treatment efficacy depends on voltage, current, and pulse number). We show that the application of a critical electric field over the the model can predict temperature rise within ex targeted tissue volume, but the electric field dis-vivo perfused porcine livers, with error <0.5 °C , and tribution depends on the tissue-specific electrical is shown to predict temperature rise in over 1000 79 unique computational test conditions with <1 °C er-croscopic) collagen denaturation was rarely noted ror and no observable outliers . on the endocardium only, possibly suggesting prox- imity to a device electrode. The interpretation of OR-02 contraction band morphology, lack of overt or pre- Characterization of Thermal Safety Profile of dominate thermal signatures and other features a Novel Balloon-In-Basket PFA System under such as artery and nerve sparing strongly suggests Repeated PFA Therapy Applications: Insights that the lesions created were almost exclusively a from in vivo and ex vivo Studies response to PFA rather than thermal coagulation Lakshya Mittal 1, Ben Niemiera1, Jed Overmann1, necrosis. Catherine Pipenhagen1, L. Boyce Moon1, Jeffrey Fish1, The Ex vivo studies indicated an initial temperat-Matthew Miller1, Autumn Myhand1, Taylor Spangler2 ure increase with 8× and 16× therapy applications, 1Abbott laboratory, United States which plateaued well below 50°C. Heating was 2Bayside Preclinical Research Services, Inc, United limited to electrode-tissue interface (8×=45.76°C; States 16×=45.96°C max. temperatures at 95/95 confid- Introduction: Pulsed field ablation (PFA) is an ence level) with minimal temperature rise at 3mm attractive alternative to thermal modalities for treat- (8×=42.11°C; 16×=42.38°C max. temperatures at ing cardiac arrhythmias using electrical pulses due 95/95 level) and 7 mm (8×=39.83°C; 16×=40.74°C to an improved safety profile and ease of use. Op- max. temperatures at 95/95 level) indicating an ab- timization of electrical pulse parameters is essen- sence of bulk heating. These temperatures were tial for effective irreversible electroporation without not sufficient to create thermal damage, as also in-a significant Joule heating. This study investig- dicated by an absence of thermal lesion and/or char ates the impact of repeated therapy application on formation on tissue. Despite using non-perfused thermal profile of a novel PFA system. tissue and no direct saline/blood flow for heat dis- Methods: The VoltTM PFA system, comprised sipation, favorable thermal observations were re- of novel balloon-in-basket catheter and generator, corded in this worst-case ex vivo model. was studied. Conclusions: The in vivo and ex vivo results indic- An acute swine (N=6) cohort was evaluated using ate that the Volt PFA system is thermally safe un- PFA applications applied 8 times, with rotation of der repeated therapy applications in these models. the catheter between each application, at each pul- The favorable thermal safety profile could be attrib- monary vein (RSPV, LSPV, CIPV). Gross and his- uted to the carefully optimized catheter design and topathological evaluations were performed to as- waveform selection. sess in vivo thermal safety. To study thermal profile in worst-case heating scen- OR-03 ario, an ex vivo bovine right-ventricular tissue abla-Isolated primary rat ventricular cardiomyocytes tion model was evaluated at 37°C. Multiple consec- response to electroporation: action potential – utive therapy applications were performed (8× and Ca2+ release – contraction 16×), without catheter rotation and as fast as pos- Vid Jan, Marko Stručić, Tina Turk, Jernej Jurič, Monika Kos, Matej Reberšek, Martina Perše, Lea Rems, sible. Temperatures were recorded using Fiber Op- Damijan Miklavčič tic probes at electrode-tissue interface and at 3mm University of Ljubljana, Slovenia and 7mm depths to characterize bulk tissue heat- ing. Atrial fibrillation (AF) is the predominant form of Results: Acute in vivo gross and histopathological arrhythmia encountered in clinical practice. Some findings at PFA treatment sites showed no morpho- research estimate that 1 in 4 adults older than logical features indicative of significant thermal in-40 years will experience a form of AF. Catheter jury in cardiac tissues. While key pathological fea- ablation with pulmonary vein isolation is reported tures of thermal injuries were absent, minimal (mi- to be the most effective treatment of paroxysmal 80 AF. Pulsed Field Ablation (PFA) is a promising AP-CaT-SS are important as they demonstrate that new ablative intervention which uses irreversible the disappearance of intracardiac electrograms, electroporation to isolate electric triggers in pul- observed in PFA, does not necessarily indicate the monary veins from the heart tissue and thus stops absence of contraction. the arrhythmic beating of the heart. Although reported to be comparably effective and safer than OR-04 conventional thermal ablation methods, questions Electrogram loss is neither sensitive nor spe- remain, how PFA/electroporation affects cardiac cific for durable lesion formation in PFA tissue on cellular level and why arrhythmias some- Harikrishna Tandri 1, Shunsuke Uetake1, Saleheldin Alhawamy1, Parag Karmarkar2 times reoccur in procedurally successfully isolated 1Vanderbilt University Medical Center, United States pulmonary veins. 2Johns Hopkins University, United States In muscle (and cardiac) cells contrac- tion/sarcomere shortening is preceded by Ca2+ Background: Loss of local electrograms occurs release, triggered by an action potential, i.e. cell frequently during pulsed field ablation and has been depolarization. In our study we used optical affirmed as a measure of success. Whether this electrophysiology to explore how electroporation correlates with durable lesion formation is unclear with different pulse parameters affects action at present. Furthermore, the determinants of local potentials (AP), calcium transients (CaT) and sar- electrogram loss have not been determined. comere shortenings (SS) in primary rat ventricular Objective: To investigate energy determinants of cardiomyocytes. During the regular/continuous electrogram loss during PFA and correlate that with pacing resembling physiological pacing the three durable lesion formation in an in-vivo porcine right monitored signals were well synchronized and atrium. consistent in amplitudes and dynamics. We have Methods: A 9 French focal ablation PFA cath- then exposed cells to either conventional eight eter with 5 different closely spaced bipoles was 100 µ s monopolar pulses, eight bursts of 25 × 2 designed and tested in vivo in a porcine model. µ s bipolar pulses with interphase and interpulse The ablation electrode was designed as an Omni delays of 2 µ s, or eight bursts of 40 × 200 ns directional antenna to assess for contact. High-monopolar pulses with interpulse delay of 100 µ s. frequency electrical properties were monitored and Bursts and 100 µs pulses were applied at 1 Hz. calibrated to confirm electrode tissue contact. Se- Irrespective of the waveform used, we observed quential PFA pulses were applied from 250 V to the uncoupling between AP-CaT-SS with pulses 1000 V to the right atrial myocardium and electro- that were below the lethal threshold. Uncoupling gram changes were carefully recorded. Lesions was reached at lower electric fields in cells that were carefully catalog using EnSite mapping sys- were oriented parallel to electric field, compared to tem. Lesions were assessed by cross histology us-perpendicular ones, when using the longest 100 ing TTC staining 12 to 14 hours post ablation. µ s pulses. Opposite was observed when cells Results: A total of 8 lesions were assessed and were exposed to either 2 µ s or 200 ns pulses. 40 bipolar electrograms were evaluated. All lesions The results further show that contractions of elec- were delivered with adequate contact. Electrogram troporated cardiomyocytes can occur even in the loss was voltage dependent. Voltage below 500 absence of APs, due to the AP-CaT-SS uncoup- V did not result in complete electrogram abolition. ling, since Ca2+ ions can enter the cardiomyocytes Voltages > 750 V resulted in electrogram dimuni- through the permeabilized sarcolemma instead tion within 2 bursts of PFA. Of the 8 lesions, only 3 of voltage-gated Ca2+ channels. The effects of were detectable on TTC staining (250 V 40 bursts, supraphysiological electric pulses on AP and CaT 750 V and 40 bursts, 1000 V 20 bursts). Although were described well with increased conductivity of 500 V lead to electrogram dimunition no lesion was sarcolemma in the Luo-Rudy model. Results that observed. sublethal electric pulses can lead to uncoupling of 81 Electrogram loss is neither sensitive nor specific in 10 ms increments. The energy administration for durable lesion formation in PFA. Electrogram began before the onset of the T wave, extending loss routinely occurs with PFA once the voltage is through the duration of the T wave or until the greater than 500 V, however, this may not ensure onset of ventricular fibrillation (VF), depending on durable lesion. Durable lesion was observed even which occurred first. If VF was induced, the animal with 250 V despite lack of complete loss of electro- was resuscitated while keeping the catheter in gram. Higher voltages i.e ≥ 1000 V can result in place, utilizing external defibrillation through patch durable lesions with good contact along with elec-electrodes. trogram loss. Alternate methods of lesion assess- Results: In two RV positions, VF was induced ment are need to ensure durable lesion delivery in- during monophasic pulse wave deliveries but not traoperatively. during biphasic pulse wave deliveries 270/300ms and 310/320ms after R-wave detection respect- OR-05 ively. And in 3 RV positions, VF was induced during Arrhythmogenicity of monophasic and monophasic but not biphasic PFA application. In biphasic PFA waveforms in a porcine model summary, VF was induced in 7/7 monophasic Tugba Kumru 1, Lars M. Mattison1, Atul Verma2, Khal-PFA deliveries during the vulnerable period of the doun Tarakji1, Daniel C. Sigg1 T-wave and 0/5 biphasic energy deliveries. Even 1Medtronic , United States though utilizing the consistent delivery parameters 2McGill University, Canada including identical voltage, pulse width, delivery Intorduction: Pulsed Field Ablation (PFA) location, and number of pulses for both mono- has emerged as a novel energy modality for ad- phasic and biphasic deliveries, delivering biphasic dressing cardiac arrhythmias through irreversible PFA applications did not result in VF, highlighting electroporation [1]. While PFA is widely used in the notable effectiveness of the biphasic approach pulmonary vein isolation and the treatment of atrial compared to monophasic delivery methods. fibrillation, its applicability and arrhythmogenic po-Conclusion: The objective of this study was to tential in ventricles remains less explored. There evaluate monophasic and biphasic PFA waveforms has not been a direct comparison of the arrhyth- for arrhythmogenic risk. The tested waveforms mogenic potential of biphasic and monophasic had identical amplitude, pulse widths, delivery pulse wave deliveries. The aim of the present location, and number of pulses. Monophasic PFA study is to compare the arrhythmogenic potential waveforms consistently induce VF when applied of an atrial loop catheter (PulseSelect™ pulse field in porcine ventricles during the T wave, while ablation (PFA) system , Medtronic, USA) delivering biphasic PFA waveforms do not. This emphasizes either biphasic, bipolar or monophasic, bipolar the distinct advantage of biphasic delivery over PFA energy to porcine ventricles. monophasic delivery, highlighting the significance Methods: Two swine (68.1 kg and 65.4 kg) were of waveform selection. anesthetized, and arterial pressures and ECG measurements were recorded after establishing REFERENCES: vascular access. No anti-arrhythmic medication [1] Verma, A. et al., Circ. Arrhythm. Electrophysiol. was given. After the PulseSelect™ catheter 15, e010168 (2022). was advanced into the right ventricle (RV), two OR-06 different matched PFA waveforms were tested: Surgical Ablation of Cardiac Tissue with Nano- a monophasic and a biphasic waveform. PFA second Pulsed Electric Fields energy with a delivery sequence of monophasic- Christian W. Zemlin, Jakraphan Yu, Ralph Damiano biphasic-monophasic (n=2 RV positions) or Washington University in Saint Louis, United States monophasic-biphasic (n=3 RV positions) was delivered relative after detection of the R-wave Ablation of cardiac tissue creates nonconduct- 82 ing lesions that either disrupt common pathways of mune cell infiltration of the treated tissue. The reentrant arrhythmias or electrically isolate known safety profile was excellent with no arrhythmias in- sources of arrhythmic activity, for example the pul- duced during or following PFA application. monary veins. In current clinical practice, car- In summary, acute and chronic porcine studies diac ablation is performed thermally, either by heat- have demonstrated the ability of nanosecond PFA ing tissue with radiofrequency (RF) currents or by to quickly and reliably create transmural lesions freezing it with a cryogen. A rapidly developing al- without inducing arrhythmias. This is a promising ternative approach is pulsed field ablation (PFA) technology which may allow for more efficacious that relies on strong electric fields to disrupt cell and less invasive surgical ablation. membranes in order to ablate tissue. We compared the results of several studies that P5 - Electroporation and cellular we performed using nanosecond PFA to create le- processes and pathways sions on porcine hearts (n=30 combined). All le- sions were created with clamp electrodes with an open chest on the beating heart, similar to clin- Monday morning Track B ical practice for surgical ablation of cardiac tissue. Sep 16, 10:30 - 12:00 In the atria, 5 to 8 lesions per animal were cre- OR-07 ated using pulse amplitudes up to 15 kV; some of Facilitation of gelonin cytotoxicity with electro- our acute studies also included ventricular lesions. poration and its prospects for electrochemo- Ablation time was 2.5- 6 s for atrial and 3-6 s for therapy ventricular lesions. The study durations ranged Olga N. Pakhomova, Andrei G. Pakhomov from acute to 6 months survival. Some lesions Old Dominion University, United States were placed across the mitral and tricuspid valves in order to assess the effect of nanosecond PFA on Gelonin, derived from the plant Gelonium mul- valve tissue and valve function (using echocardio- tiforum, is a ribosome-inactivating protein (RIP) graphy) as well as coronary arteries. Transmurality known for its potent cytotoxicity. It functions en- of lesions was assessed using triphenyl tetrazolium zymatically, requiring only a few molecules to kill a (TTC) stains as well as histological trichrome stains cell. At the same time, gelonin poorly permeates on multiple sections per lesion. In one acute study into intact cells. The combination of extreme in- (n=6) exit block testing was conducted to assess tracellular cell killing efficiency with a low systemic whether the lesions electrically isolated pulmonary toxicity makes gelonin a promising candidate for veins and atrial appendages as intended, both im- electrochemotherapy-type ablation treatments. mediately after lesion creation and following a delay We compared the cytotoxic efficiency of electropor-of 2 hours. ation with and without gelonin in the medium in cul- Nanosecond PFA achieved excellent transmurality, tured CT-26 cells (colon carcinoma) and T24 cells with more than 99% of the (add # of histologic sec- (urinary bladder cancer). The cells were suspen- tions examined) atrial sections transmural even for ded in the growth medium and exposed in elec- the shortest ablation times (2.5 s). Lesion width troporation cuvettes to different numbers of 9 us, was highly consistent across the myocardial wall. 2 kV/cm, 10 Hz electric pulses. Cell survival was Even ventricular tissue up to 12 mm thick was re- measured with the Presto blue metabolic assay at liably ablated. Exit block was confirmed for 96% 24 and 48 hours after the EP treatment. In CT-26 of lesions at lesion creation and 96?ter a delay of cells, 100 nM gelonin reduced the EP number to kill 2 hours. Our preliminary results suggested no ef- 50% of cells (LD50) at least 20- and 30-fold for the fect of PFA on valve function and no histological 24- and 48-hr timepoints, respectively. changes in the treated valves and coronary arter- Cytotoxic efficiency of different gelonin concentra- ies. Hearts harvested at 4 to12 days post-ablation tions was compared after electroporation with 10 showed a strong inflammatory response with im- EP (9 us, 2 kV/cm, 10 Hz), a low dose that had 83 no cytotoxic effect by itself. Gelonin concentration ciency of electrotransfer of bleomycin molecules. that reduced survival by 50% (IC50) was only 5 nM. On the other hand, we showed that plasmid DNA Without electroporation, even the highest gelonin inhibits the electrotransfer of labeled siRNA and oli-concentration we could test (1,000 nM) reduced cell gonucleotides. The mechanism of this inhibition is survival by only 10-15% (not statistically signific- not fully understood. Some hints can be extracted ant). from the findings showing that inhibition of electro- These promising in vitro data justify further studies transfer of oligonucleotides occurs on both sides of of electroporation-mediated gelonin delivery into the cells. i.e., facing the anode and the cathode. tissues. Compared to the conventional electro- Stronger inhibition occurs at the cell side facing the chemotherapy with bleomycin and cisplatin, the cathode. The analysis shows that this is related cytotoxicity of gelonin does not rely on cell divi- to the occlusion of the electroporated membrane sion. This could make it suitable for ablation of tis-with pDNA aggregates, which form predominantly sues and tumors which are poorly sensitive to the on the cell side facing the cathode. The inhibition conventional electrochemotherapy. It could have on the cell side facing the anode is presumably re- a niche for ablation of slow-growing cancers, pro- lated to the aggregation of small oligonucleotide state hyperplasia, as well as for the cardiac pulsed molecules and pDNA. In addition to these results, field ablation. Gelonin toxicity can also be utilized we performed an analysis of the concomitant elec-to reduce the electroporation dose, thereby minim- trotransfer of pDNA of different sizes as well as the izing heat production and adverse neuromuscular impact of proteins on the delivery of nucleic acids. side effects of EP treatments. OR-09 OR-08 Extracellular DNA enhances cell membrane Concomitant Electrotransfer of Small and damage stimulated by electrical short- Large molecules circuiting via an aqueous droplet in dielectric Ruta Palepšiene, Salvijus Vykertas, Ernestas oil Urbanskas, Justinas Venckus, Martynas Maciulevičius, Hirofumi Kurita, Yoshino Tsurusaki, Rika Numano Paulius Ruzgys, Baltramiejus Jakštys, Saulius Toyohashi University of Technology, Japan Šatkauskas Vytautas Magnus University, Lithuania We have been investigating an electroporation method using electrical short-circuiting via a cell It is well described that the electrotransfer of suspension droplet in dielectric oil. An aqueous small and large molecules, like nucleic acids, into droplet of a few microliters placed between a cells is governed by distinct mechanisms. In both pair of electrodes can be deformed by an intense cases, the critical steps are cell electroporation and DC electric field depending on the electric field electrophoretic forces that drag the molecules to or intensity. When a droplet containing suspended through the electroporated membrane. In the case cells and plasmid DNA elongates during deform- of small nucleic acids, it is believed that electro- ation and connects the electrodes, the resulting phoretic forces drag the molecules to and through short-circuiting can cause successful gene elec- the membrane directly into the cytosol, while in the trotransfection into various mammalian cells. We case of pDNA, electrophoretic forces drag the mo- also investigated the mechanisms of gene elec- lecules to the electroporated membrane, where the trotransfection using short-circuiting [1] and the molecules form pDNA aggregates. These aggreg- influence of the electroporation medium on mem- ates are then uptaken by the cell, employing en- brane permeabilization [2]. The short-circuiting docytotic pathways. What is impact of presence of using a low-conductivity electroporation medium other, especially large molecules on electrotransfer enhanced transient and irreversible membrane efficiency of other molecules remains unclear. Re- pore formation. Taking these investigations into cently we showed that presence of pDNA in elec- account, we aimed to investigate the influence of troporation medium has a positive effect on effi- 84 the conductivity of the electroporation medium on question of the most effective approach to achieve the gene electrotransfer. As a result, the short- this. Various methods for GET exist for different circuiting using a low-conductivity medium with genes, including using two pDNA simultaneously, plasmid DNA succeeded gene electrotransfer and using a single pDNA containing two different genes, resulted in a significant decrease in cell viability or transferring pDNA separately. The primary ob- compared to the high-conductivity medium with jective of this research was to evaluate the effect- plasmid DNA. However, short-circuiting using the iveness of gene electrotransfer (GET) by simultan- low-conductivity medium without plasmid DNA did eously transferring plasmid DNA (pDNA) of differ- not show a decrease in cell viability. Thus, we ent sizes and comparing it with separate transfec- demonstrated the influence of extracellular DNA tion using the same plasmids. on cell membrane damage stimulated by droplet Experiments were conducted utilizing the Chinese electroporation using a low-conductivity medium. hamster ovary (CHO-K1) cell line, employing 1.2 The electrical stimulation with the combination of kV/cm electric field strength, 100 µs and 1 – 5 plasmid DNA and the low-conductivity medium pulses to assess the GET, by using two sizes of resulted in tremendous membrane damage. Lin- pDNA. The efficiency of transfecting plasmids cod- earized plasmid DNA stimulated more significant ing for Green Fluorescent Protein (pEGFP-N1, 4.7 membrane damage than circular DNA. However, kb) and Red Fluorescent Protein (mCardinal, 6.2 the size of linear DNA did not influence the efflux kb) was examined by quantifying the count of trans- of small intracellular molecules. Although further fected cells through the employment of a flow cyto- investigations are required to prove the detailed meter (AccuriTM C6, USA) 24 hours after the ap- mechanism observed here, this study may have plication of high voltage (HV) pulses. Additionally, implications for understanding the transportation the viability of the cells after GET was determined mechanism of electroporation. using the MTS assay. Applying 1.2 kV/cm, 100 µs 5 pulses reached [1] H. Kurita, et al., PLOS ONE, vol. 15, e0243361, the highest transfection efficiency for both used 2020. pDNA’s. Although, pEGFP transfection was [2] Y. Watanabe, et al., Sensors, vol. 22, 2494, around 4 times higher than mCardinal. Using same 2022. pulse parameters for simultaneous transfer of both [3] Y. Tsurusaki, et al., PLOS ONE, vol. 18, pDNA’s resulted in drastic decrease of transfec- e0285444, 2023. tion efficiency. Upon the simultaneous cotrans- fer of both pDNA the pEGFP transfection reached OR-10 10 % (as compared to 62 % of transfection effi- The inhibition of electrotransfection caused by ciency when single pEGFP electrotransfer was per- the simultaneous transfer of multiple types of formed). mCardinal transfection dropped from 16 plasmid DNA % to 8 % respectfully. Ernestas Urbanskas, Baltramiejus Jakštys, Paulius Our findings indicate that transfection of smaller Ruzgys, Salvijus Vykertas, Justinas Venckus, Saulius pDNA is more effective. Transfection efficiency Šatkauskas of both plasmids is decreased by transferring both Vytautas Magnus University, Lithuania pDNAs at the same time; however, transfection ef- Over the past decade, there have been signific- ficiency of small plasmids decreased by 6 times, ant advancements in safely delivering therapeutic whereas transfection of mCardinal decreases only compounds to tissues and organs using electric by 2 times. pulses. Gene electrotransfer (GET) has emerged as an effective method for facilitating the trans- fer of naked plasmid DNA (pDNA). However, the need to transfer two different genes prompts the 85 OR-11 cell membrane after a unipolar electric field has Role of the actin cortex in intracellular transport been applied. The intracellular transport has been of electrotransferred DNA cargo analysed through single particle trajectories and Sophie de Boer, Aswin Muralidharan, Gijsje Koen-mean squared displacement, and gives insights derink, Bijoy Bera, Pouyan E. Boukany on the anomalous diffusion regimes involved. Delft University of Technology, Netherlands Thus far, we can conclude that at short lag times, most of the DNA cargoes show caged or subdif- Genome editing methods can precisely alter fusive behaviour [2]. mammalian cell genomes, driving cell biology Future experiments will internalize larger plasmid advancement and promising treatments for con- DNA cargoes into mammalian cells. Of these genital diseases. Efficient genome editing for cargoes, some will contain DNA segments en- therapy involves delivering cargo such as nuc- coding proteins with specific genetic functions. leic acids and editing enzymes to cell nuclei by These nucleic acids will be electrotransferred breaching the cell membranes. Electroporation into a range of cell types such as benign, ma- is a technique that can permeabilize and induce lignant, and metastatic breast carcinoma cell lines. pores in the cell membrane using high-voltage pulses. Electrotransfer describes the translocation [1] Rosazza et al. (2013). “Intracellular track- of cargo from outside the cell, into the cell after ing of single-plasmid DNA particles after delivery electroporation. However, clear understanding by electroporation.” Molecular therapy, 21(12), about the mechanism behind this transport is lack- 2217-2226. ing, which hinders efficacy in clinical applications. [2] Muralidharan et al. ”Intracellular transport While prior research studied the active and passive of electrotransferred DNA cargo is governed by intracellular transport of genetic cargoes [1], this coexisting ergodic and non ergodic anomalous research highlights the overlooked impact of the diffusion.” BioRxiv (2021): 2021-04. DNA cargo size during gene electrotransfer. [3] Perrier et al. (2019). Response of an actin There are multiple intracellular pathways for network in vesicles under electric pulses. Scientific DNA cargoes to reach the nucleus [1]. Previ- reports, 9(1), 81 ous research has shown that internalized DNA cargoes undergo anomalous diffusion [2]. This OR-12 work extends on testing the dependency of the Research on the behavior of annexin A4 protein diffusion coefficient and transport behaviour of after cell electroporation: insights into the act- electrotransferred DNA cargo on the DNA size for ive membrane repair mechanisms different clinically relevant cell types [2]. Baltramiejus Jakštys, Dominykas Makarovas, Saul-Additionally, this ongoing study aims to uncover ius Šatkauskas how the actin cortex (a network of filaments that Vytautas Magnus University, Lithuania strengthens the cell membrane) contributes to the transport of nucleic acids in the cytoplasm Increase in cell membrane permeability after after transfer across the cell membrane. This cell treatment with short, high-voltage electric is pertinent due to the actin cortex’s effect on pulses (electroporation) is related with the forma- the resealing dynamics of electropermeabilized tion of hydrophilic pores and ROS induced phos- membranes [3], and its unclear role in intracellular pholipid oxidation. In any case, cell plasma mem- transport after electroporation. brane is permeabilized and its recovery plays a cru- Using inverted fluorescence microscopy, time- cial part to sustain cell viability. It is known that lapse images are taken to track the movement of proteins of annexin family are responsible for cell fluorescently labelled pEGFP-C1 aggregates in plasma membrane reparation processes. Specific- Chinese Hamster Ovary cells [2]. Results show ally, one of the most abundant and most interest- DNA aggregate complexes interacting with the ing annexin proteins is the annexin A4 protein. The 86 activity of annexin A4 protein is ATP independent, S05 - Intensification of while Ca2+ is its ligand and precursor leading to fermentation processes by pulsed polymerization of the protein and fusion with the electric fields positive phospholipids in cell plasma membrane forcing the membrane to curve, bend, and seal. Back to electroporation, it is known, that Ca2+ wor- Monday morning Track C sens the effectiveness of electroporation by indu- Sep 16, 10:30 - 12:00 cing faster cell plasma membrane recovery. Con- OR-15 sidering all above, we intended to investigate the The role of PEF in enabling biotechnological involvement of annexin A4 protein in cell plasma processes for sustainable food processing membrane recovery after cell electroporation and Claudia Siemer correlate this data to cell viability, and pore reseal-Elea Technology GmbH, Germany ing. We used MCF7 wild-type cells with intact annexin These days, sustainable food production is in- A4 gene and MCF7-AnxA4-KO cells with blocked dispensable. All manufacturers strive to produce annexin A4 gene expression. To visualize the an- less waste, achieve higher yields through more ef- nexin A4 activity in cells, we restored annexin A4 ficient production methods, save energy, and re- protein by transfecting MCF7-AnxA4–KO cells with quire fewer additives such as water or extraction Anx-A4-GFP plasmid. The MTS assay was utilized agents. PEF, as an innovative process, is cur- to assess the vitality of the cells, and flow cytometry rently widely used in various food productions, such was employed to determine the dynamics of elec- as vegetable processing to make the production of tropermeabilization and plasma membrane repair products like French Fries or canned carrots more by quantifying the amount of propidium iodide per- sustainable. Many publications and inquiries from meable cells. Fluorescence microscopy was used the industry demonstrate the potential of PEF tech- to monitor the activity of ANX-A4-GFP protein. nology for biotechnological applications. For fer- Results revealed that MCF7-WT cells better with- mentation, PEF can be applied in both upstream stood the negative effects of electroporation in and downstream processing, making it a highly at- comparison to MCF7-AnxA4- KO cells. Secondly, tractive technology for industrial use. The present- the amount of permeable cells 35 mins after elec- ation provides an overview of the current state of troporation decreased in both cell lines signifying the art and offers insights into current industrial im-active cell plasma membrane restoring processes, plementation for the use of PEF in biotechnological which were more pronounced in MCF-WT cell line. applications. During upstream processing, PEF Microscopy image analysis indicated that calcium can be used to induce a stress response in cells. had a drastic impact on ANX-A4 activity causing The resulting effect varies depending on the strain much faster translocation from the cytosol to the and the goal. On the one hand, fermentation can plasma membrane. To continue, we observed be accelerated, and on the other hand, metabolic that annexin A4 translocation speed increased with processes can be accelerated to selectively enrich pulse intensity. More interestingly, visual data target components within the cell. One example is showed that annexin A4 translocated from cytosol yogurt cultures. After PEF treatment, the pH value to plasma membrane in Ca2+ absent medium, decreases significantly faster during the fermenta- while electroporation of cells in Ca2+ caused an- tion, resulting in improved yogurt quality, particu- nexing A4 translocation not only to plasma mem- larly regarding syneresis. But also, the treatment brane but also to nucleus membrane. of starter cultures for salami production shows pro- cess advantages due to a faster decrease in pH value. In microalgae, increased formation of fats was observed, leading to higher yields. In down- stream processing, PEF can be used as a cell dis- 87 ruption method. Current industrial efforts aim on tion from Rhodotorula species. extracting oil from yeast, which is intended as a Obtaining bioproducts from yeasts for various ap- substitute for palm oil. Traditional cell disruption plications requires a release step to remove them methods yield in low extraction rates and/or neg- from the cell, posing challenges due to the barrier atively affect the quality of the oil. In the field of effect of the cell wall and cytoplasmic membrane. microalgae as well, PEF can be used for cell dis- Implementing strategies that permit selective and ruption. Depending on the type of microalgae and efficient release of desired products to achieve high the composition of its membrane, high yields can recovery and reduced contaminants represents a be achieved. A classic example is the extraction of subject of considerable interest for exploiting the the pigment phycocyanin from the spirulina algae, bioactive compounds produced by yeasts. where the pigment can be extracted with high yield Pulsed Electric Fields (PEF) technology has and purity. In summary, PEF technology has great emerged as a promising technology for improving potential for biotechnological applications due to its the recovery of compounds from yeasts cells. Re-versatility. It can save materials such as solvents, cent studies have shown the potential of PEF for shorten fermentation times, and save energy. the development of a cascade extraction process that allows sequential extraction of compounds of OR-13 interest from yeasts biomass based on their mo- Use of PEF for the extraction of oenological lecular weight. Electroporation of the cytoplas- compounds and pigments from yeasts mic membrane enables the extraction of soluble Alejandro Berzosa, Javier Marín-Sanchez, Juan compounds present in the cytoplasm (glutathione, Manuel Martínez, Ignacio Álvarez, Ana Cristina C. amino acids, and proteins) in the first step. Sub- Sánchez Gimeno, Javier Raso sequently, cell wall compounds (mannoproteins Universidad de Zaragoza, Spain and β-glucans) can be extracted due to the hydro-Yeasts are eukaryotic microorganisms extens- lytic action of the yeast’s own enzymes. ively utilized in the production of a wide range of Triggering endogenous enzymatic activity through fermented products such as alcoholic beverages, PEF treatment has also been shown to be be- dairy products, and bread. Additionally, yeasts neficial in improving the extraction of lipophilic biomass generated during fermentation processes pigments from yeasts, such as carotenoids and or specifically cultivated in different industrial pro-terpenoids. A previous incubation of the yeasts bio- cesses is a rich source of valuable compounds mass after PEF treatment allows the extraction of such as amino acids, glutathione, proteins, nuc- these pigments from both fresh or dried biomass leic acids, vitamins, mannoproteins, or β-glucans. using green solvents such as ethanol or eutectic While these compounds have diverse applications mixtures. in pharmaceuticals, cosmetics, and the food in- The aim of this presentation is to demonstrate how dustry due to their antioxidant, nutritional, and func-PEF technology may represent a valuable tool for tional attributes, their significance in the oenolo- the biotechnological industry in designing efficient gical industry is particularly noteworthy. Gluta- processes for extracting bioactive compounds from thione has been proposed as a promising altern- yeasts biomass with minimal environmental im- ative to sulfur dioxide, while amino acids and pro- pact. teins could serve as nutrients for fermentations, and mannoproteins contribute to the sensory prop- OR-14 erties of wine. More recently, yeasts have been Technical implementation of nsPEF in indus- recognized as extraordinary alternative sources of trial biotechnological cultures natural carotenoids and pigmented terpenoids with Marco Stefan Fluri 1, Katharina Übelhör1, Lya Siegenthaler2, Lukas Neutsch1, Leandro Buchmann2 multiple applications, including astaxanthin produc- 1Zürcher Hochschule für angewandte Wissenschaften , tion from X. dendrorhous and β-carotene produc-88 Switzerland OR-16 2Bühler Group, Switzerland Industrial application of nanosecond PEF Leandro Buchmann When advancing beyond the proof-of-concept Bühler Group, Switzerland stage towards industrial use, typical challenges of new technologies in entering the bioprocessing Climate change, biodiversity loss and social in- sector involve the need for validated scale-up / equality are major challenges facing society today. scale-down systems, and multiple other aspects re- Ensuring food safety and security for a growing lated to standardization, data and IoT-integration, world population, while mitigating the negative im- or physical interfacing. Applicability to biotechno- pacts of the above, is a daunting challenge. Biopro- logical cultures calls for a design that meets strin- cessing solutions have great potential to reduce the gent hygiene requirements and is equally compat- land and CO2e footprint of food systems. How- ible with sensitive cells, as well as harsh cleaning ever, major step changes are required to sustain- and sterilization protocols. Moreover, the range ably reduce the unit cost of production. Nano- of environmental conditions encountered in biopro- second PEF has the potential to address some of cesses is broad, for example in regard to cell con- today’s challenges in industrial bioprocessing. In- centrations (viscosity), media pH and conductivity dustrial bioprocesses with higher cell concentration levels, aeration rates (air bubble presence) in fed- or total biomass and product titre yields will be crit-batch cultures with high oxygen demand, and a ical to the required transformation of the food sys- high general bioburden with dense organic matter tem in the future. Buhler AG, in close collaboration that renders tubing, connectors and valves prone with ETH Zurich, has developed Stellar™ techno- to fouling. logy based on nanosecond PEF, which, unlike con- This work presents field-proven implementation ventional pulsed electric field applications, facilit-concepts and application data from a nanosecond ates only the mass transfer of ions and small mo- Pulsed Electric Field (nsPEF) ecosystem tailored lecules. When applied precisely, Stellar enables to rapid optimization and scaling in industrial bi- non-invasive process optimisation without altering otech. Engineering aspects like electrical shielding the target organism, in addition to or as an altern- of sensitive in-process analytical equipment, sterile ative to other optimisation methods. Stellar™ tech-interfacing in bypass loop configuration, and ac- nology combines in-depth scientific knowledge with curate fluid processing of shear-sensitive cell sus- industrial process expertise to push the boundaries pensions have successfully been solved. Next of bioprocess efficiency. Applicable across biolo- to the comparability of technical parameters, bio- gical kingdoms, Stellar™ technology enables pro- logical key characteristics of diverse origin have cess intensification for yeast, bacteria, microalgae, been compared to prove the validity of scale-down cyanobacteria, fungi, mammalian and plant cell- setups. based bioprocesses at scale with minimal energy Our results underline the need for accurate requirements <0.5kWh/m3. Stellar™ technology pulse control and methods for compensation of offers flexible process integration from laboratory conductivity effects. Moreover, we illustrate how (<1mL) to industrial scale (>50m3). and why the seamless interfacing of industrial nsPEF systems with bioprocess control software is OR-17 an important asset for robust and efficient applicab- Enhancement of protein extraction and growth ility. In combination with appropriate PAT tools and stimulation of microbial cells by µs PEF Felix Schottroff analysis routines, a straightforward transfer from Boku University, Austria shake flask scale to industrial bioprocessing set- tings is possible in such settings. Foods and food constituents are increasingly produced through fermentation and biotechnolo- gical methods, owing to their significant potential in 89 the transformation process towards more sustain-S12 - Numerical modelling as an able food systems. In this context, the utilization essential tool in electroporation of pulsed electric fields (PEF) treatment is a prom- research ising approach for process intensification and op- timization. Hence, this talk will give an overview on the topic and provide selected examples from re-Monday morning Track D search, considering the enhancement of microbial Sep 16, 10:30 - 12:00 growth by PEF, as well as the selective recovery of OR-18 valuable target compounds from microbial cells. Estimation of spatial conductivity distribution Growth stimulation by mild PEF treatment (0.5-5 within a tumor from impedance measurements kV/cm, 5-50 kJ/kg, 3-5 µs pulses, batch and con- and imaging following a simulated electropora- tinuous: 1 L/h) was demonstrated to increase the tion therapy protocol rate of biomass formation as well as the production Damien Voyer 1, Olivier Sutter2, Clair Poignard3 of microbial target substances. Growth stimulation 1EIGSI La Rochelle, France was carried out using the yeasts Saccharomyces 2Hôpital Avicenne, France cerevisiae and Pichia pastoris. PEF treatment led 3Univ. Bordeaux, France to an acceleration of fermentation time of up to 14 %, whereas CO2 formation (S. cerevisiae) and pro- Electroporation-based therapies are becoming duction of recombinant food proteins (P. pastoris) increasingly popular for treating cancerous tumors. was increased by up to 8 %. Successful treatment is highly dependent on the Selective extraction of recombinant proteins by electric field generated by the electrodes. In this continuous PEF treatment (10-50 kV/cm, 50-300 context, it is essential to have a numerical tool for kJ/kg, 3-5 µs pulses, 1-1.5 L/h) was investigated predicting electric field distribution, particularly in using E. coli and P. pastoris. Product release, yield, the case of deep-seated tumors. Imaging tools as well as purity of the extract and viability of the make it possible to build a precise geometric cells were evaluated. It was shown that electric model of the patient, including the various tissues. field strengths on the lower end of the spectrum However, fixing the electrical properties of tissues, were sufficient to obtain protein release (up to 89 and in particular their conductivity, is much more %), whereas energy input levels in the higher range delicate. (corresponding to pulse repetition frequencies of up In a recently published paper [1], we have shown to 1 kHz) were beneficial. However, viability of the that it is possible to combine computed tomo- cells was increasingly maintained for lower energy graphy scans with electrical measurements made input levels. Therefore, if preservation of viabil- by clinical electroporators to estimate the electrical ity is targeted, several consecutive treatments with conductivity of patient tissues. The technique is lower energy input levels may be beneficial. based on the concepts of impedance tomography. In summary, PEF treatment can be considered The inverse problem was successfully solved with a promising building block for future sustainable clinical data, exploiting electrical measurements food production. The technology has the potential of pre-pulses performed prior to electroporation to contribute to exploit and optimize microbial fer- pulses. However, the conductivity of the different mentation processes, ultimately leading to higher tissues was assumed to be constant, which is a yields and purity of extracts. simplifying assumption. In this study, we propose to solve the inverse problem to calculate the spatial distribution of conductivity, considering that conductivity may not be constant in a tissue. The difficulty lies in the fact that the number of measurements is small (there is no more than six electrodes around the 90 tumor) whereas the description of a conductivity Electroporation of cells is obtained by applying distribution requires a thousand elements in 2D pulsed electric fields generated by a sequence of modeling, and even more in 3D. The key point voltage pulses. In the past, the influence of the is the choice of the penalty term, introduced as electrical characteristics of the medium and cell in traditional tomography problem [2]. Imaging density was studied experimentally and with Finite provides us with valuable information about tissues Element simulation. In particular, the simulation of that we can exploit to set the covariance matrix inhomogeneities in terms of electric conductivity in related to the penalty term. the treatment area showed that the electric field dis-In the numerical experiments we will present, we tribution around the cells is modified with respect to aim to reconstruct the conductivity in a tumor, the use of a homogeneous media. Moreover, cell considering that it is higher in the center due to aggregation evidenced a local modification of the necrosis. Six electrodes are placed close to the electric field and transmembrane potential. In par- tumor. The geometric model is known, along with ticular, accurate simulations of the extracellular enthe position and shape of the tumor and the exact vironment have been demonstrated to predict more position of the electrodes (this information would accurately the electric field around the cell mem- be obtained by imaging). The impedance meas- brane. Moreover, the electric field distribution is urements are calculated by solving the forward correlated also to cell permeabilization. This paper problem (this information would be provided by evaluates numerically the electroporation of cells the electroporator). Different strategies, including considering aggregates of 9 or 25 cells. The cells Tikhonov regularisation and Total Variation de- are modelized as circles, with a given diameter, noising, have been tried for calculating the spatial surrounded by a membrane with a thickness of 7 distribution of conductivity from the impedances nm. The electroporation effect was simulated mod- and the geometric model. Numerical results show elling the membrane layer with the Smoluchowski that the inhomogeneity of conductivity within the equation that estimates the number of pores as a tumor can be reproduced. function of the electric field intensity. The 2D geometry of the Finite Element Model in- [1] O. Sutter et al., “How Impedance Measure- cludes a square with a side of 1 mm where the ments and Imaging Can Be Used to Characterize circles that represent the cells are in the center of the Conductivity of Tissues During the Workflow of this area. The time varying electric field is applied an Electroporation-Based Therapy”, IEEE Trans-by a rectangular voltage to two parallel faces that actions on Biomedical Engineering, Vol. 17, No. 4, represent the electrodes. The pulse is 100 µ s long 2024 and it is characterized by a 10 µ s rise/fall time. The [2] M. Vauhkonen et al., “Tikhonov Regularization amplitude of the voltage is set to 100 V that is equi-and Prior Information in Electrical Impedance valent to 1000 V/cm in the center of the pulse in Tomography”, IEEE Transactions on Medical homogeneous conditions. A conduction problem Imaging, Vol. 17, No. 2, 1998 and an electromagnetic time transient problem was solved using COMSOL software. OR-19 By using the numerical model, it is possible to es- Finite Element evaluation of the electric field timate the intensity of the electric field and trans- distribution in a cell-aggregates membrane potential in the examined region with Patrizia Lamberti1, Michele Forzan2, Stefania cells. The effect of cell aggregates on electric field Romeo3, Elisabetta Sieni 4 distribution and transmembrane potential values is 1Università di Salerno, Italy compared to the effect obtained in the case of a 2Padova University, Italy single cell. Moreover, the transmembrane potential 3National Council of Research CNR, Italy and electric field distribution with and without the 4University of Insubria, Italy Smoluchowski equation in cell membrane is evalu- ated. Simulation results will be validated with a set 91 of experiments with cells in adhesion. etic tool capable to estimate the electroporation area under different electric field strength condi- OR-20 tions considering a rectangle englobing the area Finite element analysis model to predict elec- between the electrodes. The tool is validated by in troporation of adherent cells in round, flat bot- vitro experiments using the green fluorescent pro- tom wells tein (GFP) expressing cells (HCT-116 and HEPA 1- Patrizia Lamberti 1, Nicolas Mattei2, Maria Evelina 6), pulsed in presence of 50 µM propidium iodide Mognaschi4, Jelena Kolosnjaj-Tabi2, Muriel Golzio2, (PI) to evidence cell permeabilization. After elec- Marie-Pierre Rols2, Elisabetta Sieni3 troporation, the bright field micrographs as well as 1Università di Salerno, Italy the green (GFP, excitation of 480/40 nm, emission 2Université de Toulouse, France of 527/30 nm) and red (PI, excitation of 560/40 nm, 3University of Insubria, Italy emission of 630/75 nm) fluorescence micrographs 4Pavia University, Italy were visualized and quantified. Experimental data In laboratory settings, parallel plate electrodes are first used to build the FEA model. To each mi- are commonly used to electroporate adherent crograph, a field of interest is fitted, comprising of a cells grown in round, flat-bottom culture plates. rectangle representing the width of the electrodes This configuration selectively permeabilizes cells and the gap between them, and the PI positive cells between the electrodes, with the permeabiliza- are enumerated. Subsequently the analyzed area tion extent depending on electric field intensity. is used to build a predictive computing tool, which The electroporation yield is subsequently assessed predicts the percentage of permeabilized cells in re- visually by counting cells that uptake a fluorescent spect to a desired cell density. probe upon membrane destabilization, and this is This modeling strategy will allow reducing and re- a time-consuming process. An alternative strategy fining in vitro experiments, exploring broader para- to determine electroporated cells would be useful to meters and obtaining results in a faster and more experimenters. As fluorescent cell ratio correlates cost-efficient way. with applied field, finite element analysis (FEA) can predict electric field distribution and cell permeab-OR-21 ilization. This study thus aims to identify a method Microdosimetric Study to Calibrate µsPEFs Ap- predicting electroporation efficiency across varying plication in Two Electrodes Technology for cell densities and pulse intensities using parallel RISEUP Project plate electrodes. Sara S. Fontana 1, Noemi Dolciotti1, Amir Ghassabi1, Laura L. Caramazza1, Irene Cuenca Ortolá5, Micol We evaluated the effect of electric pulses, applied Colella1, Alessandra Paffi1, Victoria Moreno2, Luis M. to adherent cells in round flat 96-well plates us- Mir3, Franck M. Andre3, Claudia Consales4, Francesca ing stainless steel 4 mm wide plate electrodes with Apollonio1, Micaela Liberti1 a 3 mm fixed gap. The protocol consisted of ten 1Universita Degli Studi Di Roma La Sapienza, Italy 5 ms long electric pulses with variable amplitude 2Principe Felipe Research Centre, Spain up to 800 V/cm. Two different electroporation me- 3Centre National De La Recherche Scientifique, France dia were used to perform electroporation: cell cul-4ENEA, Italy ture medium, supplemented with 10 ?S, 1% peni- 5Universitat Politècnica de València, Spain cillin/streptomycin, or a low conductivity phosphate buffer. Cell culture medium is more conductive and The application of highly intense (kV/m) pulsed a higher electroporation yield is expected. How- electric fields with duration of µs (µsPEFs) can ever, electric field distribution between electrodes increase cellular membranes permeabilization, may be uneven, resulting in non-uniform cell per- extending also to the endoplasmic reticulum (ER) meabilization. and leading to the intracellular calcium fluxes The proposed work presents a technical theor- regulation [1]. This phenomenon is being lever- aged within the RISEUP project [2], to foster stem 92 cells’ differentiation in neuronal lineage, through second Pulsed Electric Fields: Quantitative Study an Electro Pulsed Bio-hybrid (EPB) device for with Calcium Ions,” Sci. Rep., vol. 7, no. 1, 2017. the regeneration of the Spinal Cord Injury (SCI). [2] https://www.riseup-project.eu/. Preliminary in vitro experiments on stem cells [3] S. Fontana et al., “Electric field bridging-effect have been carried out through an exposure system in electrified microfibrils’ scaffolds,” Front. Bioeng. comprising titanium faced electrodes, to fine-tune a Biotechnol., vol. 11, 2023. stimulation protocol suitable for the project targets. [4] A. De Angelis et al., “Confocal Microscopy Nonetheless, the final EPB device will employ Improves 3D Microdosimetry Applied to Nan- a technology of golden planar and interdigitated oporation Experiments Targeting Endoplasmic electrodes, that induces an heterogeneous electric Reticulum,”, Front. Bioeng. Biotechnol. 8:552261, (E-) field distribution [3], different from the homo- 2020. geneous one induced inside the faced electrodes. Therefore a calibration procedure, providing nu- OR-22 merical estimate of the voltage intensity to be Quantifying uncertainty of the numerical model applied on the final EPB, becomes mandatory to of irreversible electroporation in the liver achieve same poration effects on cells exposed Helena Cindrič, Damijan Miklavčič, Bor Kos inside the two systems. Both faced electrodes University of Ljubljana, Slovenia (with an inter electrodes distance of 2 mm) and The success of electroporation-based treat- a simulative core of interdigitated electrodes (as ments depends on the complete coverage of the proposed in [3]) are modeled in Comsol Multiphys- target tissue with a sufficiently high electric field, i.e. ics v. 6.0. A realistic 3D Mesenchymal stem cell above the threshold value. Numerical models are model, including cytoplasm, ER and nucleus, is an indispensable tool for investigating the electric placed inside both electrodes models and oriented field distribution in the tissue and form the basis for with its major axis parallel to the electrodes. The computer-aided treatment planning. However, nu- multiphysics problem is reproduced in Comsol merical models usually contain several parameters linking the electric current module, to simulate with varying degrees of uncertainty. All uncertain- the µsPEFs application on the active electrode, ties in the model parameters lead to uncertainties in and the Boundary ODEs and DAEs to implement the model results and thus influence the treatment numerically the pore formation dynamics [4]. Res- plan. Uncertainty quantification can be performed ults will show that, considering a homogeneous to assess the robustness of the numerical model E-field of 30 kV/m within the faced electrodes, a used for treatment planning. voltage of 4.5 V is required to be applied to the The aim of this study was to quantify the un- interdigitated electrodes to obtain a mean E-field certainty associated with parameters governing the of 30 kV/m on the electrodes’ plane, between the nonlinear conductivity function that is usually used electrodes. Furthermore, the stimulation results to model the phenomenon of electroporation. In are in line, in terms of transmembrane potential, particular, we sought to evaluate the robustness pore density and conductivity induced on plasma of a numerical model employed for the planning of and ER membranes. Different rotations of the irreversible electroporation (IRE) treatments in the same cell, as well as a different cell shape, were liver. considered, to confirm that the calibration is robust We used COMSOL Multiphysics to create a 3D to different cells dimension and dispositions. In numerical model of liver tissue undergoing IRE us- conclusion, this numerical study provided the EPB ing two stand-alone needle electrodes. In parallel, calibration in order to be used readily once its a series of in vivo experiments were performed on manufacturing process will be finalized. porcine livers mirroring the setup of the numerical model to obtain data for model development and [1] H. Hanna et al., “Electropermeabilization validation. The collected liver samples were his- of Inner and Outer Cell Membranes with Micro- 93 tologically analyzed to delineate the zones of cell simulation studies primarily rely on traditional elec-damage induced by IRE. We correlated the histo- troporation models, focusing solely on investigat- logic samples with the electric field distribution caling the transmembrane voltage generated by the culated with the 3D numerical model and identified induced electric field from pulsed magnetic fields. the electric field isocontours that best matched the As a result, it becomes challenging to compare sim- damaged cell zones, i.e., we determined the IRE ulation results with experimental findings. Build- threshold that can be used for treatment planning. ing upon this foundation, our study further incor- For the uncertainty analysis, we used the porates the deformation of the cell membrane in- variance-based Sobol method with a polynomial duced by the electromagnetic stress generated by chaos-expansion metamodel. We quantified the the electromagnetic field, thereby expanding upon relative contribution of each uncertain model para- the traditional electroporation model. Specifically, meter to the total variance of three key model out- we employed the Maxwell stress equations to com- puts – the electric current, the IRE threshold, and pute the magnetic and electric stress resulting from the extent of the IRE treated zone. the pulsed magnetic field and induced electric field The analysis showed that the most influential at the cell membrane surface. Subsequently, ac- parameter affecting the electric current was the counting for the impact of this stress on the cell baseline electrical conductivity of the tissue, while membrane’s surface tension, we modified the pore the size of the IRE treated zone was more influ- energy equation, thereby refining the traditional enced by the conductivity increase factor and the electroporation model. The conclusive simula- onset of the conductivity increase. tion results demonstrate that the electroporation model founded on the coupled magnetic-electric- OR-23 force pore energy equation provides a more ac- Simulation Investigation of Pulsed Magnetic curate depiction of pulsed magnetic field-induced Field-Induced Cell Permeabilization Using the cell permeabilization. This establishes a solid the- Coupled Magnetic-Electric-Force Pore Energy oretical groundwork for investigating the mechan- Equation ism behind pulsed magnetic field-induced cell per- Chi Ma, Yan Mi meabilization, signifying its profound scientific im- Chongqing University, China portance. Pulsed magnetic field treatment has the cap- ability to enhance cell membrane permeability, en- Three Minute Thesis (3MT) abling larger molecular substances that were pre- viously unable to traverse the cell membrane to Monday young initiative 3MT enter the cell. This non-invasive treatment holds Sep 16, 13:00 - 13:40 substantial promise for biomedical applications and represents a forward-looking medical approach. Info: Look for the abstracts of the finalists in their Nonetheless, the mechanism behind pulsed mag-respective sessions. netic field-induced cell permeabilization remains elusive. Given the intricate coupling of electro- S03 - Microalgae biorefinery magnetic fields in space, designing experiments solely to investigate the effects of magnetic fields and induced electric fields on cell membrane per- Monday afternoon Track A meability proves challenging. Consequently, there Sep 16, 13:40 - 14:40 is considerable importance in constructing simula- tion models through theoretical analysis to elucid- ate the mechanism behind pulsed magnetic field- induced cell permeabilization. However, current 94 OR-24 perature minimally impacted residence time, with Microalgae Biocompound Extraction: Simula- laminar flow being predominant. The research tion and Experimental Based Analysis of Resid- successfully achieved 90±5% of electropermeab- ence Time and Cell Suspension Characteristics ilization (PEF processing parameters: 25 kV/cm, for Consistent and Scalable Continuous Flow 6x15 µs square pulses, 80.54±3.5 kJ/kgsus) across PEF Processing the different flow rates and microalgae concentra- Byron Patricio Perez Simba 1, Paride Azzari2, Reto tions, enhancing flow processing capacity from 1 Koller1, Iris Haberkorn1, Alexander Mathys2 to 18 L/h. It also highlighted that while consistent 1Singapore ETH Centre, Singapore electropermeabilization is achievable across differ- 2ETH Zurich, Switzerland ent flow rates and microalgae concentrations with precise control of residence time, protein yields The increasing global population and arable are strongly influenced by cell concentration. The land degradation have intensified the search for highest protein extraction yield was observed at a more sustainable bioresources. Microalgae, such concentration of 10 g/L, with yields dropping 40- as Auxenochlorella protothecoides, have emerged 50% at microalgae concentrations of 50 and 90 g/L. as potential alternatives in the bio-based eco- To optimize extraction, further research on an act- nomy, towing their minimal land requirements, ive extraction strategy post-PEF treatment for high- rapid growth and rich biocompound content. How- concentration suspensions is required. In con- ever, traditional mechanical extraction methods of- clusion, this research established a solid founda- ten result in excessive cell debris, hindering ef- tion for further upscaling of PEF technology in mi- ficient biocompound recovery. Pulsed Electric croalgae biocompound extraction. It provides in- Fields (PEF) are a promising mild extraction al- sights and tools for designing consistent processes ternative due to their extraction selectivity mainly across various microalgae concentrations and flow through cell electropermeabilization. Neverthe- rates, paving the way for more sustainable biore- less, the understanding of PEF processing para- source utilization and more efficient biocompound meters’ influence on microalgal electropermeabiliz- extraction from microalgae. ation remains incomplete, and most of the data has been generated in small batch setups. This know- OR-25 ledge gap poses challenges in upscaling biocom- Plasma-based extraction of compounds from pound extraction by PEF in relevant continuous the extremophile microalgae Galdieria sulphur- flow systems owing to the dependence of the treat- aria ment outcome on the residence time distribution in Katja Zocher 1, Martina Balazinski1, Marie-Christine the treatment chamber. Additionally, the microal-Sommer1, Ulfilas Hoffmann1, Ulfilas Timm1, Jörg gae suspension rheological behavior is affected Ullmann2, Tilo Mottschall2, Juergen F. Kolb1 by the residence time determined by factors such 1Leibniz Institute for Plasma Science and Technology, as cell concentration and temperature. The aim Germany of this study was to achieve consistent microal- 2Algenfarm Klötze, Germany gae electropermeabilization and protein extraction across PEF processing parameters, cell concen- The utilisation of microalgae as biogenic re- trations and scaled flow rates in continuous flow source in the sense of a viable bioeconomy has systems. The study used a combination of exper- become a growing field of interest. Microalgal imental methodologies and COMSOL Multiphysics metabolites can serve e.g., as resource for bio- simulations to accurately determine the residence fuels, chemicals, pharmaceuticals, cosmetics, time based on the experimental microalgae rheolo- or nutraceuticals. Nevertheless, the extraction gical data, while flow cytometry was used to assess of these compounds is challenging due to their cell permeabilization. The findings indicated that robust cell wall. Standard extraction methods, variations in suspension concentration and tem- e.g., bead-milling, sonication, chemical, or enzyme 95 extraction suffer from several problems. They are 101416. often ineffective against the cell wall, inacceptable 3. Zocher, K., et al., Journal of Physics D-Applied heat development occurs, long treatment times are Physics, 2020. 53(21). necessary, or environmental harmful solvents are 4. Sommer, M.-C., et al., Microorganisms, 2021. used. Moreover, scaling of these methods for in- 9(7): p. 1452. dustrial application is often difficult or uneconomic, which is why microalgal compounds are still an OR-26 economical niche.[1] Pulsed electric fields for efficient lipid droplet Based on our previous studies [2] [3] [4], we extraction from cell-wall deficient microalgae developed a spark discharge treatment system for Julia Baumgartner 1, Sing Teng Chua2, Feng- zheng Gao1, Maylin Blunier1, Lorraine Archer2, Robert the extraction of Galdieria sulphuraria, which is Axelrod1, Fabian Abiusi1, Silvia Vignolini2, Alison Smith2, an extremophile microalga, to address the afore-Michael Hans-Peter Studer3, Alexander Mathys1 mentioned obstacles. A semi-automatic setup 1ETH Zürich, Switzerland was developed, which can treat volumes up to 1 2University of Cambridge, United Kingdom litre within 30 minutes. Extraction processes were 3Bern University of Applied Sciences, Switzerland conducted with a pulse amplitude of 35-40 kV at 11 Hz, and a pulse length of 100 ns. The algae Microalgae are considered an under-exploited suspension was moved through the system by a sustainable source of valuable macro-and micronu- peristaltic pump, which ensured uniformly treat- trients. Due to their fast, soilless growth and great ment of the bulk liquid and made external cooling metabolic diversity, they bear the potential to pro- unnecessary. The biomass with an average dry duce large quantities of valuable lipids and proteins weight of 0.8 g/L and volume of 500 ml was treated independent of season. Like seeds, microalgae for 30 minutes. During the extraction process, the can store neutral lipids in lipid droplets. Such pre- bulk temperature did not exceed 25 °C, which is emulsified oleosomes can be directly integrated preferable for thermolabile extractives, such as into food or cosmetic products without the need of proteins or pigments. solvent extraction. Whilst suitable oleosome ex- After plasma treatment, samples were prepared traction technologies have been developed for lipid for profiling flavonoids, fatty acids and the pigment droplets from seeds, they are lacking for microal-phycocyanin with LC-MS, GC-MS and UV/vis gae. This research focused on two technologies to measurements. Interestingly, the flavonoids aqueously extract oleosomes from a cell-wall de- compartment revealed a high number of lipophilic ficient Chlamydomonas reinhardtii strain: pulsed flavonoids, but only minor amounts of hydrophile electric fields (PEF) and NaCl treatment. Microal- flavonoids. Also, the fatty acid profile shows a gae were grown under photoautotrophic conditions wide range of interesting substances, which can and lipid droplet production was induced by nitro- be of industrial interest. The pigment phycocyanin, gen limitation. Continuous PEF treatments (4-15 in contrast, is only detectable in minor yields, kV/cm, 5-30 µ s, 3-9 Hz, up to 30 kJ/L) with square-which contradicts to our previous findings. It is wave pulses were employed, and the optimal elec- assumed that the cultivation conditions of Galdieria tric field strength, pulse number and pulse dura- sulphuraria with regard to phycocyanin yields is tion were chosen based on a parameter screening. highly sensitive towards light exposure and tem- Aqueous lipid droplet extraction was possible after perature.[4] both PEF and NaCl treatment. These two techno- lipids for biofuels: processes and specific energy logies were compared in terms of total lipid yield requirements. (measured by GC-FID), color (measured spectro- photometrically), and protein content (based on ni- 1. Lee, A.K., et al. Biomass and bioenergy, trogen content) of the extract. The NaCl treatment 2012. 46: p. 89-101. led to an extraction of up to 62 % of the total lip- 2. Zocher, K., et al., Algal Research, 2019. 39: p. ids, while with the PEF treatment up to 46 % of 96 the total lipids were extracted. The PEF treatment incubation of fresh algal suspensions with algal ex-led to whitish extracts, as opposed to greenish ex- tract containing CDIF, as well as with extract alone. tracts from the NaCl treatment. Both processes led While algal cell death induced by PEF treatment, to a co-extraction of proteins (up to 60 % of the even at low specific energies, resulted in a signi- total protein). The PEF-based process was fur- ficant increase in intracellular ROS, no alterations thermore scalable and cyclic lipid droplet extrac- in inter- and intracellular ROS were observed dur- tion was possible. Both PEF and NaCl treatment ing cell incubation with CDIF-containing extract. therefore showed great potential as oleosome ex- This discrepancy may be attributed to the extract’s traction techniques for cell-wall deficient microal- antioxidant-rich composition, which not only con- gae, allowing for a solvent-free microalgal biore- tains CDIF but also suppresses ROS levels. This finery approach. observation suggests that ROS depletion disrupts cell signaling pathways, ultimately leading to pro- OR-27 grammed cell death. To validate this hypothesis, Biological Signalling Supports Biotechnology: we evaluated the cytotoxicity of antioxidants such Cell Death Triggers Protein Release from as Trolox, glutathione, and ascorbic acid on algal Chlorella vulgaris cells at various concentrations. Surprisingly, ascor- Christian Gusbeth, Alexander Müller, Wolfgang W. bic acid increased algal mortality over a 24 h incub- Frey ation period and promoted the release of proteins, Karlsruhe Institute of Technology, Germany correlating with algal mortality. Based on these in- Multiple studies have demonstrated that follow- sights, we investigated the potential of enhancing ing Pulsed Electric Fields (PEF) treatment, an in- the extraction process by combining PEF treatment cubation period exceeding 6 h in buffer is neces- with a subsequent incubation step in the presence sary to enhance the bio-accessibility of cell com- of ascorbic acid. Our investigation unveiled a sig- ponents such as proteins and lipids. This enhance- nificant increase in protein yield compared to PEF ment is primarily attributed to enzymatic processes treatment alone. This finding bears significance activated during incubation, which facilitate the re- as the synergistic application of cell disruption and lease of proteins and promote the solvent extrac-ascorbic acid not only enhances the yield of ex- tion of lipids from various microalgal species. The tracted cell components from algae but also shields induction of this autolytic process can be triggered them from oxidative degradation. This would allow by various means, including dark anoxia, where mi- for the development of new downstream processes croalgae are incubated in darkness for 24 to 48 h, that align with environmentally conscious practices, or even by PEF treatment with very low specific marking a significant step towards a more sustain- energies (< 5 J/g). Notably, in C. vulgaris, there’s able and efficient approach in biotechnological ap-evidence suggesting the presence of a specific cell plications. death inducing factor (CDIF) of protein origin, which triggers cell death in intact cells. The prevailing S08 - Public health risks and concept suggests that CDIF plays a pivotal role pulsed electric fields in the food during the algae incubation period post-PEF treat- industry ment, prompting cell death and subsequent auto- lytic processes, thereby enhancing the extraction process. The search for the identity of the CDIF Monday afternoon Track B led to the assumption that radical oxygen species Sep 16, 13:40 - 14:40 (ROS) may play a decisive role as signalling mo- lecules involved in many cell signalling pathways. To investigate this mechanism, we examined the intra- and intercellular ROS production during the 97 OR-28 tions in terms of the electric field strength and spe-Microbiological characterization of almond- cific energy at a defined inlet temperature. based milk alternative and decontamination us- Conclusions: The study offered insights into the ing pulsed electric fields (PEF) type of microbial contaminants found in almond- Arisa Thamsuaidee 1, Claudia Siemer1, Vasilis P. based beverages. While PEF proved to be a prom- Valdramidis2 ising alternative to thermal pasteurization, it will be 1Elea Technology GmbH, Germany crucial to define the target pertinent microorgan- 2National and Kapodistrian University of Athens, Greece isms as part of the risk assessment and process optimization, considering the species variability in Background and objectives: While plant-based microbial resistance. milk alternatives continue to grow in popularity, their microbiological quality and safety risks re- OR-29 main poorly understood. Furthermore, many plant- Electrochemical reactions as a side effect of based beverages are processed at very high tem- Pulsed electric field treatment peratures which could result in organoleptically and Gianpiero Pataro nutritionally inferior products. Pulsed electric field University of Salerno, Italy (PEF) is proposed as a gentle preservation tech- nique that could help to retain heat-sensitive com- Pulsed Electric Fields (PEF) is an advanced ponents while extending the product durability. The non-thermal processing method for achieving study aimed to identify the microbial contaminants tailored cell disintegration of biological tissues or in unprocessed almond beverage and evaluate the microbes. Various applications have been identi- resistance of an indigenous lactic acid bacterial fied in several processes of food industry involving isolate in comparison to a laboratory-derived strain the inactivation of microbial cells in liquid foods against PEF inactivation. (e.g., pasteurization), or mass transport phenom- Materials and methods: Microbiological character- ena of juice, water, and bioactive compounds from ization was performed by plating the almond raw plant sources (e.g., extraction, expression, drying, materials and unprocessed beverages on plate- osmotic dehydration, freeze-drying, freezing), or count agar. After the isolation of phenotypically requiring structural modifications to facilitate some different colonies, the microbiota was identified unit operation of food processing (e.g., peeling, cutvia MALDI-TOF MS and/or 16S rRNA sequencing. ting). The first industrial commercial applications Separate challenge tests were performed for model have been achieved. However, several limitations Lactiplantibacillus plantarum WCFS1 and indigen- still remain and have to be addressed prior to the ous Pediococcus pentosaceus to quantify the mi- full exploitation of PEF technology in food industry. crobial resistance against PEF in almond bever- Most of these limitations are related to the unavoid- age. Inactivation kinetic parameters were identified able occurrence of electrochemical reactions at by using a Weibull type model. electrode-food interface of a PEF treatment cham- Results: Pediococcus pentosaceus was the most ber, especially when typical PEF conditions (15- frequently identified vegetative non-spore forming 40 kV/cm) for pasteurization of liquid foods are ap- microorganism in the almond raw materials and plied. These reactions, especially those leading to unprocessed beverage. The microbial load and corrosion and fouling of the electrodes, electrolysis contaminants in almond beverage were depend-of water, migration of electrode material compon- ent on the source materials, but also on the pro- ents, and chemical changes of food product, must duction practices prior to the decontamination step. be minimized since may affect PEF commercial- PEF processing of almond beverage resulted in ≥ ization through safety, quality, process efficiency, 5 log10 reductions of both model and indigenous equipment reliability and cost aspects. In this lactic acid bacteria, with survival curves displaying frame, it should be also considered that the occur-downward concavity at similar processing condi- rence of these reactions is a very complex phenom- 98 ena, which is affected by several factors, such as scriptomic analysis was employed specifically for L. circuit topology of the pulse generator, PEF cham- monocytogenes EGD-e with the primary objective ber design and electrode material, PEF electrical of the analysis to discern the transcriptional profiles parameters, as well as composition and chemical-by comparing untreated and PEF-treated samples, physical properties of the treated products. In this and with the focused aim to elucidate the underly- work, the basic concept of electrochemistry along ing mechanisms of microbial resistance. with a description of the electrochemical phenom- The findings revealed that depending on the spe- ena that occur at the electrode-food interface of a cies and strain, microorganisms exhibited various PEF treatment chamber are briefly described. Par- resistances. PEF microbial resistance and strain ticular attention is devoted to the main side effects variability data were correlated to the total spe-associated to the occurrence of electrochemical re- cific energy applied. In general, L. monocytogenes actions and chemical processes. Finally, the main strains identified more resistant compared to the causes for electrochemical reactions are identified, E.coli strains in all the different PEF parameters, and suggestions on how to limit the extent of elec- which may is related to the the thicker cell wall and trochemical reactions and their side effects are also stiffening lipoteichoic acids Gram positive bacteria reported. have and the size of the microorganism. E. coli strains exhibited significant Log inactivation differ-OR-31 ences between the strains, under both 88 and 136 Investigation of microbial strain variability res- kJ/kg treatments with Log reductions ranging from istance under PEF treatments and identification 1.85 to 4.24 and from 2.19 to 4.59, respectively. In of the underlying molecular mechanisms contrast, L. monocytogenes strains under both 88 Fotios Lytras 1, Georgios Psakis1, Ruben Gatt1, and 136 kJ/kg treatments displayed no significant Joerg Hummerjohann2, Guillermo J. Cebrian3, Javier statistical differences and a <1.05 Log reduction. Raso3, Vasilis Valdramidis1 Under the highest PEF treatment parameter (184 1University of Malta, Malta kJ/kg), E. coli strains showed no statistical differ- 2National Reference Laboratory for STEC, Switzerland ences and a Log reduction ≥ 4.04 for all the strains. 3Universidad de Zaragoza, Spain On the same condition, L. monocytogenes strains Pulsed Electric Field (PEF) stands as an emer- displayed significant statistical differences and L6 ging technology, providing a compelling alternative strain emerged as the most PEF-resistant strain. to traditional thermal treatments. Typically, regard- The transcriptomic analysis revealed a diverse ar- less of processing, microbial inactivation efficiency ray of genes as key players in the PEF stress envir-requires challenge tests employing selected target onment, showcasing the dynamic up-regulation of strains. In the realm of this study, our focus was glnR, lmo2419, tktB, and the synchronized down- twofold, firstly the identification of microbial targets regulation of rpmA and cspL. resistant to PEF and secondly revealing the resist- Unraveling the molecular mechanisms responsible ance molecular mechanisms for future optimization for the resistance under PEF treatments, particu- of the process. larly in the case of L. monocytogenes, can provide This study commenced with a comprehensive in- insightful information on developing customized vestigation into the effects of PEF treatments ap- Pulsed Electric Field (PEF) treatments. This could plied with an electric field strength of 20 kV/cm allow for better optimisation of processing paramet- across a range of total specific energies on micro- ers or identification of ideal hurdle combinations, bial samples suspended in McIlvaine buffer at pH whose synergistic effects can ultimately enhance 7.0. A selection of 20 strains, originating from two strategies for elevating food safety standards while pathogenic model microorganisms, Listeria mono- preserving the nutritional value of food products. cytogenes and Escherichia coli, were systemat- ically examined. Following this, RNA-Seq tran- 99 P3 - Irreversible electroporation mor spheroids was tracked daily using Brightfield imaging and analyzed using ImageJ. Monday afternoon Track C Tumor spheroids partially ablated during their ini- tial treatment were found to gradually recover after Sep 16, 13:40 - 14:40 5 days during the study. The XTT viability as- OR-32 say revealed that the longer pulse durations, such Characterization of pulsed electric fields for in as IRE and the 5-1-5, caused deeper penetra- vitro tumor spheroids and metastatic invasion tion within the tumor spheroids producing immedi- Julio Arroyo ate stark decreases in viability as the voltage in- Georgia Institute of Technology , United States creased. Shorter pulse durations such as the 2- 1-2 displayed marginal cell death until 2500 V/cm, Electroporation is a biophysical phenomenon while the 1-1-1 presented minimal cell death across where nanoscale hydrophilic pores are generated all PEFs applied. Only the longer pulse durations on a cell’s plasma membrane when exposed to an at the highest voltages applied were consistently applied external electric field. Irreversible electro-successful in tumor spheroid elimination and com- poration (IRE) produces protracted nanopore form- plete metastatic invasion elimination. Metastatic ation, causing cell death [1]. A second genera- elimination was consistently initially seen during tion of IRE, known as High-Frequency Irreversible treatment; however, metastatic inhibition varied de- Electroporation (H-FIRE), delivers shorter bipolar pending on the level of penetration within the spher- pulses in series of bursts to mitigate muscle con- oids. Due to the presence of cancerous cells post- tractions induced by IRE. Tumor spheroids, cancer electroporation, adjuvant therapies such as chemo- cells cultured into a sphere (.01-2cm), can mimic therapeutics or immunotherapies are optimal solu- in vivo conditions due to their spatial organiza- tions to eradicate remaining malignancies. tion and cell-ECM interactions. Previously, tumor spheroids have been treated with electroporation to OR-33 cause permeation for delivering chemotherapeut- Study of irreversible electroporation-induced ics [2] and high calcium concentrations [3] to as- cell death in a 3D spheroid hepatocarcinoma sess cell death in a physiologically relevant model. model Here, we aim to subject the tumor spheroids with Nicolas Mattei 1, Alexia de Caro1, Emma Leschiera2, lethal and sub-lethal pulse electric fields (PEFs) us-Clair Poignard3, Jelena Kolosnjaj-Tabi1, Marie-Pierre ing various voltages and pulse durations to identify Rols1, Muriel Golzio1 the extent of cell death and metastatic elimina- 1Université de Toulouse, France tion/inhibition. 2Léonard de Vinci Pôle Universitaire, France Human glioblastoma cells (U251) were used to 3Université de Bordeaux, France form spheroids, seeded onto collagen hydrogels, While radiofrequency ablation has been the and subjected to a uniform electric field. Day of main method to thermally destroy deep-seated treatment was denoted as day 0. A custom pulse solid tumors such as hepatocellular carcinoma, it generator was used to deliver 500-2000 V/cm of cannot be used when tumors are located close IRE or 1000-2500 V/cm of H-FIRE with 500 V/cm to vital vessels or bile ducts. Alternatively, irre- intervals. The biphasic PEFs had varying pulse versible electroporation (IRE), consisting of the ap- widths of 1, 2, and 5 µs, but maintained similar in- plication of about eighty 100-µs long, high intens- terphase delays of 1 µs. The burst on-time and ity pulsed electric fields (PEFs), has emerged as bursts delivered remained constant between IRE an athermal ablation method. However, anatom- and H-FIRE at 100 µs over 100 bursts, respect- ical barriers can hamper an optimal placement of ively. Viability was measured using live/dead stain- the electrodes, yielding to inefficient IRE and con- ing and an XTT viability assay at days 1, 3, and 5 sequent cancer relapse. In order to evaluate can- post-treatments. Metastatic invasion from the tu- 100 cer cell potential to trigger an immune response these experiments serve as basis to develop math-at sub-therapeutic IRE pulses, we herein study ematical models for prediction of spheroid growth the immunogenicity of IRE-induced cell death. If and DAMPs release after IRE treatment. immunogenic, therapeutic immunostimulants could therefore be used to prevent relapse. In this study OR-34 we assessed the growth of 3D multicellular spher- Acute efficacy and durability of in vitro pulsed oids after PEFs exposure. Then, we assessed field ablation in relation to the delivered energy the immunogenicity of the cell death by measur-impulse ing apoptosis (through caspase 3/7 activation) and Ivana Fišerová 1, Marek Novak2, David Kvapil1, Stanislava Martinkova1, Jan Trnka1, Petr Tousek1, Pavel the release of major Damage Associated Molecular Osmančík1, Marek Hozman1, Dalibor Herman1, Jan Patterns (DAMPs): ATP, HMGB1 and calreticulin Vrba2, David Vrba2, Ondrej Fiser2 (CRT). 1Charles University, Czech Republic Our IRE treatment consisted of eighty 100-µs 2Czech Technical University in Prague, Czech Republic long pulses with a repetition rate of 1000 Hz applied with two parallel plate electrodes, with PEF Background: Pulsed-field ablation (PFA) is a intensity varying between 0 and 2500 V/cm. Murine new method for catheter ablation of arrhythmias hepatoma-derived Hepa 1-6 cells (GFP-expressing based on the destruction of cardiomyocytes by or wild type) were used in this work. short, high-voltage electric pulses [1]. Electric Spheroids were treated with IRE and imaged pulse generators with different characteristics are with fluorescence microscopy for four days. Cas- now being developed for the PFA rate. The efficacy pase 3/7 activation over time was assessed with a in terms of complete and durable tissue destruction specific fluorescence kit. ATP, HMGB1 and CRT may vary according to the intensity of the applied release were assessed by a bioluminescent assay, energy and the length and frequency of the pulses. immunoblotting and immunostaining, respectively. Moreover, the durability of tissue destruction can- Partial IRE (transient loss of GFP and regrowth not be tested during in vivo human procedures. of residual cells) and complete IRE (full loss of GFP Purpose: The aim of this study was to determine 1) expression) were determined at 1500 V/cm and the efficiency of inducing cell death in HL-1 cardi- 2500 V/cm, respectively. An increased caspase omyocytes inn relation to different PFA electric field activation was observed after treatment. Interest-strengths and 2) the durability of tissue desctruciton ingly, the peak of activation occurred 6 hours after in 24 hours after the procedure. partial IRE treatment, and only 3 hours after com- Methods: A tumor line of murine cardiomyocytes plete IRE treatment. All DAMPs observed show an was used for this study. PFA was conducted using increased release correlating with the PEF intens- a commercial TONAPULSE electrical pulse gen- ity. HMGB1 was detected 6 hours but not 3 hours erator equipped with an electrode plate (TONA- after 1500 V/cm treatment. GENA, CZ). Cardiomyocytes were exposed to a When the PEF intensity does not reach a burst consisting of 216 bipolar pulses lasting 2 µ s, sufficient level, residual cells are capable of re-with 5 µ s pauses in between of the pulses. Each growth, which might account for relapse in patients. burst was repeated 20 times with a 1 second pause Moreover, we saw that IRE induces ICD, and there- between. Applied electric fields ranged from 250 fore has the potential to induce an immunogenic V/cm to 1500 V/cm. The ratio of viable to non- response. Notably, both caspase 3/7 activation viable cells was assessed at 1 hour and 24 hours and extracellular HMGB1 results highlight that the post-electroporation with fluorescent microscopy. cell death is not only PEF intensity-dependent, but Results: Electric fields ranged from 250 to 500 also time-dependent. Thus, the timing parameter V/cm did not result in a any acute and sufficient rate should be considered in immunotherapy. Taken tocell death. Electric field of 750 V/cm, 1000 V/cm, gether, this work is in favor of the combination of 1250 V/cm, and 1500 V/cm induced cell death in IRE and immunotherapeutic agents. Additionally, 27 ± 6 %, 44 ± 6 %, 56 ± 7 %, and 75 ± 18 % of 101 exposed cardiomyocytes. After 24 hours and in re-response as indicated by the rejection of injec- lation to the PFA energy, the rate of destroyed car- ted rechallenge tumor cells with melanoma and diomyocytes was 16.5 ± 2.9 %, 37.3 ± 10 %, 50.3 ± hepatocellular carcinoma exhibiting the strongest 2.4 %, and 62.3 ± 15.3 ?ter exposure of 750 V/cm, response and lung carcinoma the weakest re- 1000 V/cm, 1250 V/cm, and 1500 V/cm. sponse. Conclusion: The minimum electric filed associ- The rejection of the rechallenge tumor indicates ated with any cardiomyocyte damage is 750 V/cm, that an immune response specific to the tumor has but sufficient, acute myocardial damage is present been initiated, but ultimately, we want the immune after 1500 V/cm. The durability of cardiomyo- system to eliminate metastases of the primary tu- cyte death is very similar after 24 h confirmed the mor. While metastases are difficult to routinely gen- acute success rate, confirming minimal cell repar- erate in mice, injecting a second, abscopal tumor ation, and excellent durability of acutely achieved at the same time as the primary tumor mimics the lessions. The slight decrease of dead cells may be presence of a metastasis. We therefore studied the due to slight reparation of damaged cells or prolif- changes in an abscopal tumor following different eration of cardiomyocytes due to their tumorigenic treatments of the primary tumor. While NPS treat- nature. ment alone does not affect the growth of the ab- scopal tumor, when it is combined with the injection S01 - Medical applications of of an immune system stimulant, we observe signi- ficant inhibition of abscopal growth. Our best res- nsPEFs ult came from injecting the anti-OX40 antibody to agonize the function of the co-stimulatory T cell re- Monday afternoon Track D ceptor, OX40. When we inject aOX40 into a Pan02 Sep 16, 13:40 - 14:40 tumor following NPS treatment, up to 80% of un- treated abscopal tumors were eliminated, suggest- OR-36 ing that this immune response stimulation might be Nano-Pulse Stimulation initiates an immune re- strong enough to go after metastases of the primary sponse in several types of murine tumors tumor. Richard Nuccitelli Pulse Biosciences, United States OR-37 Nano-Pulse Stimulation therapy (NPS) applies Distinct Tumor Immune Responses to Nano- electric pulses in the nanosecond domain to initi- second Pulsed Electric Fields (nsPEFs) De- ate regulated cell death in the treated tissues. This termine Immunity non-thermal therapy has been used to treat a wide Stephen J. Beebe 1, Anthony Nanajian1, Brrittney range of murine tumors and has been shown to ac-Ruedlinger1, Kamal Asadipour1, Siqi Guo2 1 tivate the immune system to inhibit the growth of Old Dominion University, United States 2 rechallenge tumors as well as untreated, abscopal Frank Reidy Research Center for Bioelectrics, United tumors when accompanied by the injection of some States immune system stimulants into the treated tumors. Nanosecond pulsed electric field fields Murine tumor types treated include melanoma, (nsPEFs) is a pulsed power technology that squamous cell carcinoma, lung carcinoma, breast stores and then releases high-powered non- carcinoma and pancreatic carcinoma. The en- thermal electric pulses in nanosecond durations ergy required to ablate these tumors has been de- that induce in situ vaccination (ISV) after ablation termined with pancreatic carcinoma and lung car- of orthotopic rat N1-S1 liver (75-80%) and mouse cinoma exhibiting ablation with 240 mJ/mm3, lung 4T1-luc breast tumors (80-95%); however, not carcinoma and squamous cell carcinoma requir- in the mouse melanoma (10-20%) models The ing 360 mJ/mm3, and melanoma requiring 480 studies here are designed to determine immune mJ/mm3. NPS therapy initiated a variable immune 102 mechanisms for nsPEFs in cancer models that do OR-38 or do not readily induce immunity with ISV. The effects of nanosecond pulses on cell In 4T1-luc breast cancer, nsPEFs selectively tar- growth and multi-drug resistance in pancreatic geted apoptosis-induction in activated T-regulatory cancer cells cells (Tregs) and tumor-associated macrophages Wojciech Szlasa1, Olga Michel1, Vitalij Novickij2, (TAMs) and eliminating myeloid-derived sup- Paulina Kasperkiewicz1, Mounir Tarek3, Jolanta pressor cell (MDSC) levels, showing relief of Saczko1, Julia Rudno-Rudzińska1, Wojciech Kielan1, immunosuppression in local and systemic envir- Julita Kulbacka 1 1 onments. There was a reduction in functional Treg Wroclaw Medical University, Poland 2 suppression capacity, which is likely explained State Research Institute Centre for Innovative Medicine, by decreases in activation markers (4-1BB and Lithuania 3 TGF Université de Lorraine, France β) and a shift of Treg phenotype from pre- dominantly activated (CD44+CD62L-) to naïve Nanosecond pulsed electric fields (nsPEF) (CD44-CD62L+). This stronger nsPEF apoptotic have been shown to exert anticancer effects. How- bias for activated Tregs spared effector CD4+ ever, the mechanisms triggered in cancer cells by and CD8+ T cells leading to a concomitant rise nanosecond-length pulses are not well known, es- in effector CD4+ T cells and a 2.7-fold increase pecially when low sub-permeabilization voltage is in the ratio of resident memory CD8+ CD103+ used. In this study, various human pancreatic can- T-cells to CD4+ Tregs. These findings show cer cell lines were treated with nsPEF, and mo- nsPEFs effectively switch the TME and secondary lecular changes at the cellular level were analyzed. lymphatic systems from immunosuppressive to We have used paclitaxel chemotherapy following immunoactive allowing cytotoxic T cell function nsPEF treatment and determined the correlation and immune memory formation to eliminate cancer with the changes in the expression of multi-drug cells and account for the nsPEFs ISV. resistance (MDR) proteins. Finally, we examined Studies with nsPEF conditions that induce ISV the influence of nsPEF on the adhesive proper- in rat liver and mouse breast cancer models do ties of cancer cells and the formation and growth not readily induce ISV in mouse melanoma. In of pancreatic cancer spheroids. Cell line response the TME, there were increases in DCs express- differed when applying a 200 ns, 100 pulses, 8 ing costimulatory molecules indicating the first kV/cm, and 10 kHz PEF treatment. PEF treat- step in an immune response, but memory T-cell ment led to (1) the release of microvesicles (MV) in numbers were low and likely anergic. Unlike the EPP85-181RDB cells, (2) electropermeabilization 4T1-luc tumors, Tregs, MDSC, and TAMs were in EPP85-181RNOV cells, and (3) cell shrinkage in not significantly increase on post treatment days EPP85-181P cells. The release of MV’s in EPP85- 3 and 7. Overall, these responses allude to a 181RDB cells reduced the membrane content of P- narrow potential for immunity in the mouse melan- gp and LRP, leading to a transient increase in the oma model due primarily to immunosuppression. vulnerability of the cells towards paclitaxel. In all Studies indicate that nsPEF treated melanoma cell lines, we observed an initial reduction in the tumors co-incubated with carbon nanotubes (CNT) size of the cancer spheroids after the nsPEF treat- decreased the charging effect required for effective ment. Cell line EPP85-181RNOV exhibited a per- tumor elimination but had no effect on immunity. manent reduction in the spheroid size after nsPEF. We propose a mechanism in which the surface ten- sion of the membrane, regulated by the organiza- tion of actin fibers, modulates the response of can- cer cells toward nsPEF. When a membrane’s sur- face tension remains low, we observed some cells form protrusions and release MVs containing MDR 103 proteins. In contrast, the cell membrane is elec-response curves. Ca2+ transient imaging was per- troporated when cell surface tension remains high. formed to follow the permeabilization of internal cell The latter effect may be responsible for the reduced membranes after exposure to nsPEFs. Further- tumor growth following nsPEF treatment. Our find- more, we validated the effect of nsPEFs fraction- ings underscore the clinical relevance of nsPEF as ation in the ex-vivo murine model. a novel therapeutic approach for pancreatic cancer, Our study showed that the permeabilization of meriting further investigation into its role in modu- plasma membranes for YO-PRO-1 dye is more ef- lating drug resistance and enhancing chemother- ficient in endothelial cells than in cardiomyocytes. apy effectiveness. This study lays the groundwork The calculated ED50 (electric field intensity which for future clinical trials aimed at integrating nsPEF affects 50% of cells) for trains of 200, 300-ns into existing treatment regimens, aiming to improve pulses, 10 Hz was almost 50% higher for cardi- patient outcomes in pancreatic cancer care. omyocytes (8,05 kV/cm vs. 5.43kV/cm, p <0,0001). Funding: Funding: This study was supported by Differences in plasma membrane permeabilization the Medical Research Agency, Poland, IREC pro- did not reflect on ablation efficiency. ED50 for en- ject No. 2020/ABM/01/00098/P/02 (PI: Prof. Woj- dothelial cells was only 8% lower (5,51 kV/cm vs. ciech Kielan). 5,98 kV/cm, p< 0,0001). The latter can be ex- plained by the increase of cytoplasmatic Ca2+ after OR-35 exposure in a Ca2+-free medium which was 2 times Are high-voltage nanosecond pulsed electric higher in cardiac cells, suggesting a superior effi- fields selective for cardiomyocytes? ciency of nsPEFs in intracellular compartment per- Pamela Sowa 1, Vitalij Novickij2, Aleksander Kiełbik1, meabilization. Ferdinand Kollotzek1, David Heinzmann1, Jürgen Fractionation of nsPEFs (4 trains of 50 pulses, Schreieck1, Oliver Borst1, Meinrad Gawaz1 at 10 Hz, with 300-ns duration, each train separated 1University Hospital Tübingen, Germany by a 50-sec interval and 2 trains of 100 pulses with 2Vilnius Gediminas Technical University, Lithuania a 100-sec interval between trains) increased en- Nanosecond pulsed electric fields (nsPEFs) are dothelial cell permeabilization. The ED50 was 12- an emerging cardiac ablation method based on 13% lower after fractionated nsPEFs in comparison electroporation. The nearly inevitable endothelial to a single train of 200 pulses, (p<0,0001). Consist-injury during the procedure may contribute to tran- ently, the endothelial cell-killing effect increased by sient myocardial dysfunction, resulting in electrical 6-11% (p<0,0001). Conversely, fractionation had and structural remodeling of the atria, a potential neither an effect on the susceptibility of cardiomyo- substrate for early arrhythmias recurrence. cytes nor an effect on their plasma membranes per- Although studies suggest that cardiomyocytes meability. Also, ex vivo studies showed no signific- are more sensitive to electroporation than en- ant effect of fractionated exposure on the ablation dothelial cells, there is still a lack of data unequivoc-area of the myocardium. ally confirming it. This study aims to compare the We have shown that myocardium and endothe- efficacy of nsPEFs on both cell types and explore lium are similarly susceptible to nsPEFs, with a the effect of fractionated nsPEFs on the therapy’s greater electroporative effect on cardiomyocyte in- selectivity. tracellular substructures. Fractionation of exposure We applied monolayers of HL-1 murine cardi- results in increased endothelial injury without alter-omyocytes and MHEC 5-T murine endothelial cells ing cardiac ablation efficiency. and constructed a robotic system positioning stim- ulation electrodes orthogonal to the substrate. We measured cell viability and permeability using wide- field fluorescence microscopy and fitted the stained areas to a simulated electric field to obtain dose- 104 S22 - New waveforms and electric pulses is associated with less electrochemistry, nerve stimulation, and muscle contraction [7,8], al- field management strategies for though this has not been unequivocally demon- electroporation-based therapies strated. With shorter pulses, and use of bipolar pulses, higher amplitudes and/or more pulses are Monday late afternoon Track required to compensate for less efficient electropor- A ation by these new pulse waveforms. Higher amp- Sep 16, 16:10 - 17:40 litudes and a greater number of pulses can also sig- nificantly increase heating, so thermal damage can OR-39 become significant. Fifty shades of Electroporation To summarize, we can perform electroporation Damijan Miklavčič, Samo Mahnič-Kalamiza with the intention of treatment (ECT, IRE, GET) with University of Ljubljana, Slovenia nanosecond to millisecond pulses with monopolar The range of pulse durations used in and bipolar pulses, but with different pulse amp- electroporation-based applications extends from litude and number of pulses to achieve the desired a few nanoseconds (and even sub-nanoseconds) degree of electroporation. However, other effects to tens of milliseconds. Traditionally, these pulses (electrochemistry, temperature, pain, contraction, were used for specific applications: short, i.e. …) will be different and, more importantly, they may microseconds, to introduce small molecules (e.g. not depend on the pulse parameters in the same bleomycin and cisplatin) into cells in electrochemo- way as electroporation. The choice of paramet- therapy, and longer pulses in the millisecond range ers thus requires more comprehensive optimisation for gene electrotransfer. With the advent of nano- than just the amplitude. second pulses arose the ambition to manipulate [1] https://doi.org/10.2478/raon-2024-0005 cells by electroporation of membranes of internal [2] https://doi.org/10.3390/app12168237 organs without damaging the plasma membrane. [3] https://doi.org/10.1007/s10439-020-02462- Later, high-frequency bipolar pulses came on to 8 the stage with the promise of causing no muscle [4] https://doi.org/10.1016/j.bbamem.2016.06.024 contraction nor pain. [5] https://doi.org/10.3390/vaccines11061036 It was shown that we can introduce small and [6] https://doi.org/10.3389/fonc.2022.853779 large molecules in vitro with nano-, micro- (mono- [7] https://doi.org/10.1016/j.electacta.2020.137187 and bipolar), and millisecond pulses [1,2]. A com- [8] https://doi.org/10.1038/s41598–022-12112- bination of electroporation with bleomycin and cis- 9 platin was shown to be equally efficient by adapt- ing pulse amplitude and/or number of pulses, and OR-40 that the same number of molecules introduced by Impact of pulse protocol parameters on the ef- these various waveforms will result in a similar cell ficacy of electrochemotherapy in vitro death. Gene transfer was also successfully per- Fabio Lepore1, Simona Salati 2, Francesca De formed with these waveforms, albeit with shorter Terlizzi2, Giulia Grisendi1, Roberta Fusco2, Massimo Dominici1, Matteo Cadossi2, Ruggero Cadossi2 pulses requiring higher pDNA concentrations. Ir- 1University Hospital of Modena and Reggio Emilia, Italy reversible electroporation by the same waveforms 2IGEA, Italy has also been demonstrated [3,4]. Similarly, DAMP release was obtained with these waveforms, sug- Electrochemotherapy (ECT) is the combination gesting that a similar immune response may be of the electric field with a chemotherapeutic agent, triggered in vivo – which was then also confirmed usually bleomycin, or cisplatin, for the local treat- in vivo [5,6]. ment of cancer. The electrical protocol used in clin- At the same time, claims (and proofs) are be- ics (ESOPE) consists of the application of 8 mono- ing made that delivery of high-frequency bipolar 105 polar 100 µ s pulses at 1000 V/cm. This study will lead to the development of a new In clinical practice, the electric field amplitude fixed pulse generator that will integrate the READ forat 1000 V/cm could have some limitations, e.g., mula for the identification of appropriate pulse pro- when the tissue resistivity is particularly low, cur- tocols to obtain complete coverage of the target rent levels may exceed the limits of the device volume with sufficient energy to achieve effective and treatment is interrupted, or when large tumour electroporation. This technological advancement volumes are targeted, the electroporation (EP) can will increase ECT efficacy and flexibility of use, will be achieved only by multiple electrodes reposition-optimize the number of electrodes needed to cover ing. the tumour volume and will limit muscle contrac- In 2009, Ibey through in vitro experiments demon- tions and pain reducing the invasiveness of the pro- strated that EP of the cell membrane depended on cedure. Absorbed Dose (AD) i.e., electric field amplitude and duration/number of applied pulses. OR-41 AD = σ · E 2 · τ( n,t) Voltage vs Current Control. Selecting the Best ρ Where E is the local electric field, σ the conductivity Delivery Strategy of the tissue, ρ the density of the tissue, and τ the Quim Castellvi, Antoni Ivorra total duration of exposure to the electric field and a Universitat Pompeu Fabra, Spain function of n and t (n, number of pulses and t, the The outcomes of the electroporation-based duration of single pulse). therapies rely on the induction of the desired biolo- In this work, we aim at identifying the Reversible gical effect into the target area. For a defined elec-Electroporation Absorbed Dose (READ) value ne- tric waveform, the generated therapeutic effects cessary to induce reversible electroporation of the are mainly dependent on the electric field mag- cell membrane. The READ value could allow the nitudes present at the tissue area. Proper control use of different pulse protocols in which electric of the electric field magnitude distribution is crucial field amplitude, single pulse duration and number for the treatment successful, making the geometry, of pulses are combined to achieve reversible elec- location, and energization strategies of the elec- troporation of the cell membrane and allow the dif- trodes critical. fusion of the anticancer drug into the cytoplasm. Since homogeneous effects over the thera- The analysis of the impact that each component of peutic area are typically desired, confining the tar- the pulse protocol has on the effectiveness of ECT get tissue between parallel electrode plates at a was performed as follows: first of all, N50, V50 and constant voltage could be considered the optimal τ 50, respectively the number of pulses, the electric delivery approach. However, the inability to sur-field and the pulse duration resulting in a 50% re- round the target tissue between planar rigid elec- duction in the effectiveness of the ESOPE protocol, trodes limits this method to surface treatments. To were identified. Then, the individual parameters of overcome this limitation, alternative electrode geo- the pulse protocol were modulated to compensate metries such as needles or endoluminal catheters for the loss of ECT effectiveness due to the ap- have been used to place the therapeutic electrodes plication of V50, τ 50 and N50. The experimental close to the target in a minimally invasive man-data collected were used to identify and validate the ner These electrode configurations often result in a READ formula. Results from in vitro experiments non-homogeneous electric field distribution that re- will be further validated in relevant cancer animal quires additional consideration regarding delivery models. strategies. Specifically, the number of therapeutic Based on the READ, pulse parameters will be mod- electrodes (monopolar vs. bipolar) and the elec- ulated to identify new pulse protocols capable of tric magnitude delivery strategy (voltage vs. cur- covering larger tissue volumes and/or to reduce rent) are strategy approaches that plays a key role muscle contraction and pain associated with the in the electric field distribution and should be care-delivery of electrical pulses. 106 fully evaluated in relation to the different treatment OR-43 environments. Comparison of the Thresholds for Electro- Here, different electrode configurations and poration and Excitation for Pulses within treatment environments will be defined to assess Nanosecond–Millisecond Duration Range how various delivery strategies influence thera- Gintautas Saulis 1, Mantas Šilkūnas2, Rita Saulė1 peutic outcomes. This analysis will underscore 1Vytautas Magnus University, Lithuania both the advantages and disadvantages of the 2Old Dominion University, United States different electric field management approaches Exposure of cells with pulses of strong electric across different scenarios. Such variability high- field can electroporate the cell plasma membrane, lights the absence of a universally effective thera- stimulate of excitable cells, or cause both effects. peutic strategy, emphasizing the importance of In some applications, e.g. non-thermal ablation of tailored approaches to optimize treatment effect- cardiac or tumour tissue with irreversible electro- iveness. poration, excitation of muscle cells is not desirable, while in other ones, it would be better to avoid elec-OR-42 troporation. Differentiating Electroporation Currents using Theoretical analysis and experimental data avail- Dynamic PEF Modeling able show that, depending on the membrane char- Clara Teresa De Souza Ramos, Daniella Lourdes Luna Santana de Andrade, Guilherme Brasil Pintarelli, ging time constant, pulse strength and duration, the Raul Guedert, Daniela Ota Hisayasu Suzuki complex interplay of excitation and electroporation Universidade Federal de Santa Catarina, Brazil can be observed. Excitation with or without dam- aging of the cell plasma membrane due to electro- Distinguishing electroporation current from poration can be achieved. other macroscopic alterations is essential for However, while carrying theoretical analysis, some advancing the understanding and control of elec- minor simplifications were used. Meanwhile, these troporation in tissues. This study aims to identify simplifications might be important for the de- this differentiation by employing a dynamic model tailed comparison of electroporation and excita- of pulsed electric field (PEF). Tissue dielectric distion thresholds as for some pulse durations these persion is described by a multipole Debye function, thresholds are close to each other. The aim of implemented in the time domain through auxiliary this study was to analyse theoretically the de- differential equations method. Furthermore, the pendence of the threshold for electroporation in electroporation effects were elucidated by estab- more details within a wide range of pulse durations lishing an electric field-to-transmembrane potential (nanoseconds–milliseconds) and compare these (TMP) relation. The time-dependent increase in results with experimental data on excitation of neur- tissue conductivity was assessed through extra- ons obtained earlier. polation of a kinetic model of cell electroporation, Electroporation of mouse hepatoma MH-22A cells while the conductivity-temperature dependency was determined from the increase of the plasma is modeled as a linear function. Experimental membrane permeability to potassium ions. It was data collected from in vitro potato tuber (Solanum assumed that the threshold of electroporation cor- tuberosum) samples are utilized to implement the responds to the formation of one pore. Theoretical model using commercial finite element method dependence of the threshold of electroporation of a (FEM) software. The results demonstrate the neuron on pulse duration were calculated based of model’s efficacy in distinguishing the electropora- the mechanism of electroporation. Parameters re- tion currents from other macroscopic phenomena, quired were estimated from the experimental data thus offering a valuable tool for advancing the obtained in mouse hepatoma MH-22A and Chinese understanding and control of electroporation in hamster ovary cells. For the comparison with ex- tissue systems. perimental data, the dependence of the threshold 107 for excitation of dissociated E18 rat hippocampal the effects of combining radiofrequency signals neurons, determined for single square-wave elec- with traditional electroporation pulses. This is since tric pulses with the durations from 100 ns to 1 ms the radiofrequency thermal ablative treatments use obtained earlier was used. application systems, i.e., electrodes and generat- The experimental excitation-duration curve can be ors like those of electroporation, and it is feasible to approximated by the straight line in a log-log plot synthesize a combination of the voltage waves that with the slope equal to -0.5, which remains the are traditionally used in these treatments. In this same for all range of pulse durations studied (from way, these waveforms could improve the accurate 100 ns to 1 ms). Meanwhile, the slope of the de- application of treatments or to allow controlling the pendence of the electric field strength required to thermal distribution. electroporate the cell on the pulse duration varies In this work, a proof of concept has been carried out from -0.06 to -0.9 within the same range of pulse by developing a power electronic system to study durations. the effects of the combination of traditional radi- From this analysis, it can be concluded that the res- ofrequency and electroporation waveforms. For ults of the exposure of neurons by a single square- this purpose, printed electrodes have been used on wave pulse depend strongly on its duration: slides with a separation of 100 µm, and a high fre- 1) For pulses longer than 10–20 µs the threshold for quency digital-to-analog converter together with a neuron excitation is much lower than that for elec- high-power operational amplifier have been used to troporation. generate the voltage signals. With this testbench, 2) Pulses with the durations from 200–300 ns to two strategies have been studied to combine the 10–20 µs, which cause neuron excitation, should waves used in radiofrequency and electroporation: also electroporate them. harmonic addition and amplitude modulation. The 3) Threshold for excitation becomes close to or first is the direct addition of two pure harmonics, even lower than the threshold for electroporation one with frequency in the electroporation range, for pulses shorter than 200–300 ns. and the other one with the frequency of typical ra- diofrequency treatments. The purpose of this sig- OR-44 nal is to increase the applied power with respect Study on Effect of Electroporation Using Amp- to a traditional electroporation signal to generate a litude Modulation Signal and Harmonic Addi-controlled and homogeneous temperature increase tion that can improve the effectiveness of electropora- Borja López-Alonso 1, Tamara Polajžer2, Matej tion. The second strategy is the generation of an Reberšek2, Héctor Sarnago1, Óscar Lucía1, Damijan amplitude modulation wave by means of the inter- Miklavčič2 ference of two harmonics with frequency in the radi- 1Universidad de Zaragoza, Spain ofrequency range and amplitude in the electropora- 2University of Ljubljana, Slovenia tion range. In contrast to the previous strategy, this The electroporation effects are highly depend-wave does not have the electroporation harmonic; ent on the shape of the applied voltage signal. This however, it is thought that the membrane may de- shape can modify the transmitted power or the cur- modulate the signal leading to the desired effects. rent path, affecting the distribution of the electric To investigate the proposed strategies, membrane field or the temperature rise, among other effects. permeabilization experiments were performed on These effects, and their synergy with the phenom- CHO cells. Prior to the experiments, the cell sus- ena of electroporation and electropermeabilization, pension was mixed with propidium iodide (PI). 20 have been studied in several areas such as the µl of the mixed cell suspension was then trans- food industry, among others. However, in the medi- ferred to the electrodes, which were placed under cine field, these effects have not been studied in the fluorescence microscope. Three minutes after depth, although there has been hypothesis about electroporation, images of PI fluorescence were taken. The changes in PI fluorescence were stat- 108 istically analyzed to confirm or refute the initial hy-epithelial/endothelial electrical resistance (TEER) potheses. monitoring modules. A microfluidic channel is In conclusion a power electronics system has been divided into two subchannels by the porous PC proposed to perform a proof of concept to study membrane and performs several functions in the the feasibility of combining electroporation and ra- microchip: (i) separates two flows in subchannels, diofrequency treatments. (ii) serves as a substrate for cell adhesion, (iii) strengthens the electric field at the surface of the membrane. P10 - Electrofluidic, microfluidic We conducted the viability studies on rat glioma and lab on a chip C6 cell in suspension using PEF treatment with various electric field strengths and 100 µ s 1 Hz in Monday late afternoon Track our microchip. The PEF treatment in the microchip B revealed a minor impact to the cells up to 10 Sep 16, 16:10 - 17:40 kV/cm. For comparison, the viability after PEF treatment in a conventional cuvette decreased with OR-45 increasing electric field, and the 5 kV/cm electric Microphysiological system for PEF treatment field was lethal. We assume the difference of PEF of mammalian cells with integrated oxygen and impact arises from Joule heating which is known TEER sensors to depend on the dimension of the electrodes. Neringa Bakute 1, Eivydas Andriukonis1, Kamile We have measured the permeability in microchip Kasperaviciute1, Jorunas Dobilas1, Martynas Sapurov1, Gatis Mozolevskis2, Arūnas Stirkė1 to DAPI and the saturation of permeability with 1State Research Institute Center for Physical Science 16 pulses at 1.8 kV/cm was observed. These and Technology, Lithuania results proved that the cells undergo membrane 2University of Latvia, Latvia permeabilization during PEF treatment in the microchip with a very gentle impact on the viability Batch electroporation, a conventional method of cells. for PEF treatment, typically involves a cuvette The performance of integrated sensors was stud- with embedded electrodes. Although the use ied with the application of an electric field. We of commercially available cuvettes is a simple, monitored the oxygen concentration changes routine procedure, it does have certain drawbacks, immediately after PEF treatment, and the oxygen including distortions in the electric field, local difference between the two integrated sensors was pH variation, excessive heat generation, and evaluated. A slight change of oxygen concentra- the dissolution of metal ions leading to reduced tion was captured with and without PEF treatment electroporation efficiency and cell viability. with cell suspension and not with buffer. To address this, conventional batch pulsed electric To conclude, our MPS for PEF treatment has field (PEF) technology has shifted toward the mi- several advantages: (i) a relatively easy soft- croscale and integration with microfluidic devices lithography process, which circumvents the also known as ’lab-on-a-chip’, ‘organ-on-a-chip’ shortcoming of PDMS; (ii) the electroporation of and microphysiological system (MPS). In micro- the cells ensures higher viability ( (iii) monitoring of fluidics, most soft-lithography microchips are made physiological parameters can be performed simul- of polydimethylsiloxane (PDMS). An alternative taneously. (iv) the same setup of our microfluidic to PDMS, an off-stoichiometry thiol-ene (OSTE), chip can be used for electroporation of suspension. overcomes PDMS limitations while maintaining transparency, biocompatibility, and repeatability. We present an MPS with an OSTE-based microfluidic chip for the PEF treatment of mammalian cells with the oxygen and trans- 109 OR-46 application. Study on the technique of inducing perforation of blood clots by pulsed electromagnetic fields OR-47 Yuan Lei 1, Lei Li1, Biao Hu1, Shoulong Dong1, Deng-Dielectrophoresis – tool for analysis of mesen- feng He2, Chenguo Yao1 chymal stem cells differentiation 1Chongqing University, China Ioan Tivig1, Leslie A. Vallet2, Romain Samiaa3, 2The southwest hospital of Amu, China Franck M. Andre3, Luis M. Mir3, Tudor Savopol1, Mihaela G. Moisescu 1 While electroporation and magnetic perforation 1Carol Davila University of Medicine and Pharmacy, Ro-techniques are known for their ability to alter cell mania permeability, the current applications of this elec- 2Université Paris-Saclay, France tromagnetic perforation technique are mainly tar- 3Centre National De La Recherche Scientifique, France geted at biological cells or microorganisms, and few studies have applied this technique to throm- Electrical properties of mesenchymal stem cells bus therapy. In this study, we realized the perfora- (MSCs) along with their osteogenic and adipogenic tion of blood clots in vitro by driving magnetic nan- progenies were evaluated at different stages of oparticles through a high-intensity pulsed magnetic cells differentiation, using dielectrophoresis (DEP). field. First, we built a modular pulsed magnetic field The utilization of MSCs in therapeutic applications generator with an energy feedback function, which is increasingly significant due to their capabilities consists of front-end energy-feeding circuits and in cell replacement and their immunomodulatory discharge modules at various levels in cascade, effects. However, the differentiation protocols and is capable of realizing flexible adjustment of the are protracted and might lead to incomplete or coil magnetic field. By utilizing the state change of non-synchronous differentiation. It is essential the front-end feeder switch, the load coil energy can to provide procedures which segregate during be fed back into the capacitors at all levels, thus or post-differentiation, various cells for obtaining outputting various forms of pulsed magnetic field homogenous populations for clinical use. waveforms such as long exponential waveforms In practice, a DEP force is obtained when applying and short triangular waveforms. Secondly, we built a non-uniform AC electric field which manipulates a a three-stage modular pulsed magnetic field gen- cell based on the polarizability differences between erator platform and carried out in vitro thrombolysis the cell and medium. The AC field frequency de-experiments using this platform. By outputting a 1T pendence of a factor called Clausius-Mossotti high-intensity, high-gradient pulsed magnetic field, (which integrates geometrical and electrical prop- magnetic particles were rapidly directed in the dir- erties of the cell) represents a DEP spectrum. ection of the thrombus, thus generating magnetic Human adipose-derived MSCs were subject to force to induce perforation of the thrombus, which standard differentiation protocols towards osteo- in turn changed the permeability of the blood clot. genic and adipogenic progenies. The assessment We documented this clot perforation and disruption of differentiation was done weekly for 4 weeks, phenomenon by white light microscopy and fluor- using Alizarin Red and Bodipy staining and bio- escence imaging. Finally, pulsed magnetic force chemical tests (alkaline phosphatase and Oil Red in vitro thrombolysis experiments were carried out, O assays). and by comparing the thrombolysis results of mag- DEP spectra (20 frequencies between 10kHz and netic nanoparticles mixed with thrombolytic drugs 40MHz) were acquired weekly during the differen- under a pulsed magnetic field with those of a control tiation process, using a 3DEP system (LABtech, group such as drugs alone, it was shown that the UK). The DEP spectra were analyzed using method of pulsed magnetic force-assisted throm- an in-house developed program, OpenDEP (ht- bolysis can promote the thrombolysis efficiency, tps://github.com/IoanTivig/OpenDEP_Compute.git) and it has a promising prospect for research and and electrical parameters of MSCs and their 110 progenies have been calculated. To simulate sep-cies at which the DEP force is null, and a cell goes arations of MSCs from their progenies, Autodesk from negative to positive (or vice-versa) DEP beha- Fusion 360 was used to design a microfluidic vior, are called crossover frequencies (fCO). channel (2 inlets and 2 outlets) embedding DEP DEP leverages electric fields not only to analyze electrodes, and COMSOL Multiphysics to simulate but to sort cells based on their electrical proper- electric fields, fluid flow and cell tracking. ties. These properties together with the geomet- A notable reduction in both membrane permittivity rical ones are integrated into a Clausius-Mossotti and conductivity was observed after the initial factor, which is determined by cell and medium week of differentiation for both progenies. These permittivities and conductivities. This factor’s fre- changes preceded the microscopical morpholo- quency dependence represents a DEP spectrum, gical modifications specific to differentiation and crucial for identifying fCO. Cell sorting is enabled correlated with the biochemical tests results that by creating conditions for different cells to be simul-confirmed the cells undergoing transformation. taneously either at fCO or under a DEP movement. In subsequent weeks, the membrane permittivity We propose a platform for DEP spectra acquis- continued to slowly decrease. Changes in cells’ ition and processing, named OpenDEP. It has electrical parameters at the end of the first differen-two components: i/ a lab-on-a-chip (LoC) sys- tiation week indicate thus that the transformation tem integrating electrodes that allows the ac- process started. quisition of microscopic images of cells distri- Moreover, our simulations showed that these elec- bution under AC field exposure, ii/ a software trical changes enable the early-stage separation which, by images processing, computes DEP spec- of differentiating cells using the DEP microfluidic tra and calculates cells electric parameters (ht- channel, although those cells have sizes similar tps://github.com/IoanTivig/OpenDEP.git). The plat- to those of MSCs. DEP separation facilitates two form has been used for acquiring and processing applications: purifying differentiated cells from DEP spectra of NIH3T3 and DC3F cells, and res- mixed populations early in their differentiation pro- ults were validated by comparison with those ob- cess and isolating MSCs from other cells sources. tained with a commercially available device, 3DEP This method is advantageous as it requires no (LABtech, UK). cell labeling, utilizes low-cost lab equipment and OpenDEP requires image acquisitions for each fre- consumables, and can begin in the first week of quency to obtain a DEP spectrum. For the same treatment. number of AC frequencies, a spectrum acquisi- tion by OpenDEP typically takes 30 min, compared OR-48 to less than 1 min in the case of 3DEP. But the OpenDEP: A free-access platform for collecting OpenDEP platform offers several significant ad- and analyzing dielectrophoresis spectra vantages: a) Manufacturing Simplicity - the plat- Ioan Tivig, Tudor Savopol, Mihaela G. Moisescu form is produced using standard 3D printing tech- Carol Davila University of Medicine and Pharmacy, Ro- niques and features modular design, b) Optimized mania Chamber Design - allowing to focus under micro- Label-free living cell characterization proced- scope simultaneously cells and electrodes, c) Ro- ures based on dielectrophoresis (DEP) offer sig- bust Statistical Analysis - calculations are based on nificant advancements in cellular biology by pre-large cell populations, d) Effective Electrode Geo- serving cell integrity. DEP is the phenomenon by metry - the indium tin oxide electrodes are design which a dielectric polarizable object placed in an to generate a strong vertical DEP force, directing inhomogeneous AC field is experiencing a driving cells to the chamber’s lower plane (artifacts due force exerted on the direction or in opposition of the to gravitational deposition and light scattering are electric field gradient (situations called positive or reduced), d) Sterile and Versatile Lab Conditions - negative DEP, respectively). The AC field frequen- the LoC may operate in a sterile environment and is compatible with various microscopy techniques 111 (e.g., fluorescence microscopy), e) Open-Source Recent progresses in nanosecond duration Software - facilitating broader accessibility and con-ultra-high voltage pulses provides exciting possib- tinuous customization. ilities to produce direct intracellular effects. MeanThe platform features collectively enhance the while, we have been using ultrashort electrical functionality and application scope of OpenDEP, pulses and miniature shock drivers to generate fine making it a valuable tool for cellular experimenta- micro underwater shock waves, which can penet- tion. rate deep in soft tissue. Shock waves, as phys- OR-49 ical stimuli for cell manipulation, are of particular A Lab-On-Chip Based on Transferred Laser-interest, as they can transiently increase the per- Induced Graphene Electrodes and Machine meability of the cell membrane with less side ef- Learning for Electroporation of Adhered Cells fects. These unique characteristics make them Gianni Antonelli 1, Francesca Camera2, Arianna appropriate for delivering energy for vaccine/drug Mencattini1, Arianna Casciati2, Mirella Tanori2, Aless-delivery. Ultrasound and shock wave can be ap- andro Zambotti2, Giorgia Curci1, Joanna Filippi1, Michele plied through totally non-invasive procedures tak-D’Orazio1, Paola Casti1, Caterina Merla2, Eugenio ing advantage of focusing in soft tissue; appropri- Martinelli1 ate for clinical therapy. We utilized micro-streaming 1University of Tor Vergata, Italy of shock wave and cavitation to deliver liquid vac- 2ENEA, Italy cine/drug to the depth of dermis. The paper will cover our recent progresses with shock waves and Lab-on-Chips paved the way for great improve- pulse ultrasound for minimally invasive pain-free ments in biology research, thanks to integrating needleless microfluidic vaccine/drug delivery. multiple facilities in a small environment. In this scenario, Laser-Induced Graphene (LIG) has re- cently allowed the generation of conductive geo- P13 - General applications of metries, which has been demonstrated to be an al- electroporation for food ternative solution for integrated metal electrodes in processing lab-on-chip experiments. In this work, we present a new Lab-On-Chip platform based on LIG elec- Monday late afternoon Track trodes, which were transferred on a biocompatible C transparent polymer substrate (namely PMMA) for Sep 16, 16:10 - 17:40 the electroporation of adherent cells detected by fluorescence and brightfield microscopy. The pro- OR-51 posed platform was used to study the calcium in- Validating pulsed electric field pasteurization take and the pseudopods retraction on U-87 glio- of protein rich plant-based milk alternatives: a blastoma cell line after electroporation stimuli. Us- novel challenge trial approach ing a tailored machine-learning algorithm, the elec- Nicholas Horlacher, Indrawati Oey, Sze Ying Leong, trodes’ design and the intensity of the applied elec-Dominic Agyei, Jessie King tric field were adjusted to optimize the trends of cal-University of Otago, New Zealand cium flow inside the single cell and its motility after Plant based milk alternatives (PBMA) are an the electroporation stimulus, demonstrating the po- emerging food category that suffers from technical tentiality and reliability of the proposed technique. challenges due to a tendency of plant protein (such OR-50 as pea protein) to coagulate during thermal pro- Non-invasive vaccine/drug delivery and ther- cessing (e.g., UHT, HTST). Pulsed electric field anostics by microfluidics of shock waves treatment (PEF) with mild preheating has shown Nushin Hosano, Hamid Hosano promise as an alternative pasteurization method to Kumamoto University, Japan reduce these undesired thermal impacts. However, 112 the established procedures to validate this use of parameters for continuous PEF treatment com-PEF through microbial challenge testing typically bined with preheating to achieve a minimum 5-log require a thermal presterilization of the product. reduction of inoculated surrogate microorganisms PBMA containing heat-sensitive protein do not per- in PBMA containing heat-labile protein. mit the application of sterilization, presenting a challenge to food producers looking to conduct OR-52 such trials with PEF. As a solution, this work pro- Application of continuous pulsed electric field poses an alternative approach that permits a re- (PEF) treatment in human milk as an alternative liable enumeration of inoculated surrogate organ- pasteurisation technique isms during microbial challenge testing of a PBMA Yiting Wang, Farzan Zare, Negareh Ghasemi, P. Nicholas Shaw, Nidhi Bansal without the need for presterilization. The University of Queensland, Australia An oat-based PBMA enriched with commercial pea protein isolate was prepared as a model food sys- Thermal processing, typically holder pasteur- tem and inoculated with two surrogate organisms isation (HoP) has been used as a standard proced- (E. coli and Listeria innocua). Pasteurization was ure to ensure the microbiological safety of donor carried out by applying a preheating treatment (35- human milk in numerous countries worldwide. The 45 °C) followed by the continuous application of treatment involves heating the milk to 62.5°C for PEF at a constant field strength, pulse width, and 30 minutes and often compromises the nutritional flow rate (10 kV/cm, 20 µ s, and 14 L/h, respect-and antimicrobial components essential for new- ively). Pulse frequency was adjusted to modify the born immunity. Hence, this study aims to explore specific energy input to 97-245 kJ/L and the inactiv- the potential effects of pulsed electric field (PEF) ation of inoculated surrogates was assessed using treatment on donor human milk as an alternative a combination of selective and non-selective plat- pasteurisation technique. Donor human milk used ing techniques. Oxford and eosin methylene blue in this study was sourced from anonymous con- agar permitted a selective enumeration of E. coli senting donors and stored in an ultracold freezer and Listeria sp., respectively, despite the presence at -80°C. Before usage, milk samples from vari- of native microbial contaminants, while total plate ous donors were thawed in the refrigerator (4°C) and spore counts on brain heart infusion, Luria- overnight and pooled together. Escherichia coli Bertani, and plate count agar indicated overall mi- JM109 bacterial culture was incubated for 24h to crobial reductions. reach 109 colony-forming units per mL (CFU/mL) Using the selective enumeration approach, a min- and was then subjected to two washes with sterile imum 5-log reduction in inoculated surrogates, re- 0.85% (w/v) saline solution via centrifugation. The quired for treatment validation, was achieved via resulting E. coli suspension was then inoculated to preheating to 35 °C followed by PEF treatment at either MilliQ water or donor human milk to achieve 204 kJ/kg. L. innocua was more resistant than an initial count of approximately 8 log CFU/mL. E. coli towards the effects of PEF, which demon- Subsequently, 100 mL of raw human milk and E. strated the importance of appropriate surrogate or- coli inoculated samples were treated using a con- ganism selection. No change in total plate and se- tinuous PEF system with a flow rate of 100 mL lective bacterial counts during a subsequent brief per minute. This treatment involved the applica- storage period (48h, 4 °C) was detected and indic- tion of 240k pulses (60k pulses per step with a ated a limited occurrence of sub-lethal injury dur- 5 min rest interval time between steps) at 20kV ing PEF treatment. A significant presence of en- and 50Hz using a pulsed power generator cap- dospore forming bacteria was however detected able of delivering energy up to 0.8 J. pulse-1 (with and resisted PEF treatment, which will require at- a 500Ω resistive load) and peak amplitude up to tention during future investigations. Overall, our 30kV. Temperature measurements were conduc- study showed that this unconventional approach ted using an infrared thermal camera before and can be successfully used to identify processing 113 after each pulsing step. The bacterial enumera-For the HTST process (90°C for 15 s), used tion in the processed samples was performed us- as a benchmark, the study examined the influence ing tryptone soya agar (TSA), with plates incub- of Waste Heat Recovery (WHR) efficiency on pre- ated at 37°C for a minimum of 48h before enumer- heating temperature, thermal power, and steam ation. Following the PEF treatment, the MilliQ wa- flow rate. Results indicated that higher WHR ef- ter sample with an initial E. coli count of approx- ficiency resulted in elevated raw juice temperat- imately 8 log CFU/mL exhibited a reduction of ap- ures leaving the regeneration stage, thereby redu- proximately 4.4 log. In comparison, the E. coli in- cing the thermal power necessary to achieve the oculated human milk with a similar initial bacterial pasteurization target temperature of 90°C. Con- count, showed a reduction of approximately 5.3 sequently, this led to reduced specific energy con- log. The raw human milk sample initially contained sumption, cost savings, and enhanced energy effi- 101 CFU/mL and showed complete microbial inac- ciency. tivation post-PEF treatment. Throughout the pro- For the PEF treatment (E=36 kV/cm, WT= 40- cess, all samples were maintained at a temperat- 200 kJ/kg), findings revealed that in combination ure below 35°C. This study explored the potential with preheating at various inlet temperatures of of PEF treatment as a non-thermal alternative for the juice to the chamber, higher WHR efficiency donor human milk pasteurisation while retaining its was correlated with decreased electrical energy de- beneficial properties. The continuous PEF treat- mand. Specifically, PEF demonstrated 30% lower ment effectively pasteurised 100 mL of raw human electric consumption and less than 70% fuel gas milk sample and achieved up to 5 log reduction in usage compared to thermal processes, reducing donor human milk inoculated with 108 CFU/mL E. greenhouse gas emissions by less than 40%. PEF coli at a significantly lower temperature compared treatment, with its lower energy requirements and to thermal treatment, potentially preserving the in- reduced reliance on fossil fuels, exhibited lower tegrity of the essential milk. CO2 emissions and comparable water usage com- pared to HTST, showcasing its environmental ad- OR-53 vantages. Comprehensive Analysis of Heat-Assisted The economic viability was assessed through a Pulsed Electric Fields and Conventional comprehensive cost analysis, encompassing cap- Thermal Treatment for Orange Juice Pas- ital investments, utility expenses, labour costs, and teurization: Cost, Energy Efficiency, and facility-related charges. The results highlighted that Sustainability Assessment despite higher initial capital costs for PEF, its lower Giovanni Landi 1, Miriam Benedetti1, Matteo unit pasteurization cost and reduced annual ex- Sforzini1, Elham Eslami2, Gianpiero Pataro2 penses yielded significant cost savings compared 1ENEA, Italy to HTST. Additionally, the study explored pay- 2University of Salerno, Italy back periods under various energy cost scenarios, This study compared commercial heat-assisted identifying optimal configurations for PEF plants Pulsed Electric Fields (PEF) and High-Temperature with payback periods ≤ 5 years. Through the correl- Short-Time Treatment (HTST) technologies for ation of emissions and water consumption, the PEF pasteurizing orange juice, focusing on cost, energy plants with a WHR efficiency of 35% emerged as efficiency, and sustainability metrics. The invest- the most energy-efficient and sustainable solution, igation encompassed a broad range of thermal re- warranting its selection for optimal orange juice covery efficiencies ranging from 0 to 65% for both pasteurization capable of mitigating the impact of the PEF and HTST processes. Additionally, the adverse energy scenarios. The preheating temper- study examined the impact of varying inlet temper- ature of 45°C was chosen as the optimal solution atures of the juice to the PEF chamber, ranging due to its proven effectiveness in microbial inactiv- from 35°C to 55°C. ation compared to lower temperatures. In conclusion, this comprehensive analysis 114 provides invaluable insights for stakeholders in tion whereas untreated samples without PEF were the food processing industry, facilitating informed used as a negative control. decision-making towards cost-effective, energy- PEF at 88 kJ/kg with preheating to 45 °C permitted efficient, and sustainable juice pasteurization tech- the formation of a gel during subsequent fermenta- nologies. tion with similar hardness to the positive control. A further increase in gel hardness was observed with OR-54 increasing specific energy (up to 21% at 214 kJ/kg) Effect of pulsed electric field processing on regardless of pre-heating temperature. Protein sol- functional properties of plant protein in yogurt ubility increased with specific energy (up to 18% at alternative applications 181 kJ/kg, 45 °C) compared to the negative control Nicholas Horlacher, Indrawati Oey, Sze Ying Leong, prior to fermentation, whereas zeta potential and Dominic Agyei, Jessie King particle size decreased. An enhanced protein sol- University of Otago, New Zealand ubilisation by PEF, based on particle size reduction Plant proteins have gained popularity as a func- and changes in net surface charge, could induce tional ingredient in yoghurt alternatives to provide further cross-linking and stronger gel formation dur- texture by protein gelation while improving nutri- ing fermentation. This demonstrated the potential tional benefits of the product. Plant protein isolates of PEF as a suitable processing step to modify the are commercially produced from sources such as texture of yoghurt alternatives. PEF could therefore the yellow pea (Pisum sativum) but typically suffer play a significant role in the development of sus- from poor functionality. Thus, there is a need for tainable foods that satisfy ever growing consumer processing methods that can induce targeted struc- demands. tural changes to improve their function as food in- OR-55 gredients. Pulsed electric field (PEF) treatment has Effect of pulsed electric fields on protein extrac-shown promise in modifying plant protein function- tion of duckweed (L. minor and L. gibba) ality but has not been explored in yoghurt altern- Patricia Maag 1, Özlem Özmutlu1, Cornelia Rauh2 atives. Therefore, the aim of this study was to as- 1University of Applied Sciences, Germany sess the effect of PEF with preheating on the pro- 2Technical University of Berlin, Germany tein gelation of yoghurt alternatives. A model food system was prepared by hydrolys- The Department of Food Technology and Hor- ing oats (10% w/v) with α-amylase (60 °C, 60 min), ticulture at HSWT (University of Applied Sciences followed by filtration and mixing with a commercial Weihenstephan-Triesdorf) is establishing a mild ex- pea protein isolate (5% w/v). The resulting disper- traction process for optimized protein extraction sion was pre-heated to 35 - 45 °C and exposed using pulsed electric fields of moderate intensity to PEF of bipolar square waveform using an EL- (MIPEF), which is a research objective in the re- CRACK lab-scale PEF unit in continuous opera- search project Smart Indoor Farming (”Science and tion (14 L/h). A constant field strength and pulse the Arts in Bavaria”, STMWK), aiming to observe width (10 kV/cm, 20 µ s) were applied, while pulse the effects on cell disruption and mass transfer, frequency was adjusted to achieve targeted spe- with a focus on the release of proteins from the cific energies between 81 and 214 kJ/kg. Physi- plant cells of duckweeds (L. minor and L. gibba). cochemical properties (zeta potential, particle size, The equipment used is a bipolar, square wave, Brix) and protein solubility based on soluble nitro- pulse generator, model OmniPulser (Manufacturer: gen were measured after PEF, while yoghurt fer- VITAVE) with an attached paralell plate batch treat- mentation (35 °C, 10h) was applied to identify im- ment chamber. pacts on protein gelation via texture profile analysis In previous trials with duckweed, remarkably high (TPA). Thermal-treated samples (80 °C, 30 min) protein contents of 35-40% (dry matter) were in- were included as a positive control for protein gela- vestigated and other research data has found a bal- 115 anced amino acid composition as well as valuable OR-238 omega-3 fatty acids ( α-linolenic acid) and antioxid-Pulsed electric field-assisted preparation of di- ants (lutein), offering excellent potential as a new aldehyde starch and its effect on structure and healthy alternative protein source. Research into physiochemical properties duckweed has therefore been intensified since its Zhong Han 1, Ying Li1, Xin-Dong Xu1, Xin-An Zeng2 potential as a high-quality nutrient for food applica-1South China University of Technology, China tions was recognized. 2Foshan University, China To pursue sustainable and mild extraction pro- In this study, dialdehyde starch was prepared cesses in which chemical solvents can be reduced, using pulsed electric field (PEF)-assisted sodium pulsed electric field (PEF) technology is used for periodate oxidation of starch, and its structure and cell disruption to pre-extract proteins in freshly har-properties were studied. The results showed that vested duckweed. Utilizing a batch processing the aldehyde group content of DAS (12-DAS) pre- approach, PEF of unipolar and bipolar types are pared with the assistance of PEF of 12 kV/cm in- applied to disintegrate the plant cell membrane. creased by 11.6%, compared with the dialdehyde This method is directly applied to a pre-crushed starch (0-DAS) prepared without the assistance duckweed cell system and subsequently compared of pulsed electric field. And as the intensity of to untreated samples to evaluate its effective- PEF increased, the molecular structure of the pre- ness in enhancing protein extraction yield. PEF- pared DAS became more disordered. SEM res- Parameters such as electrical fields (1-5 kV/cm), ults showed that the particle surface of DAS pre- number of pulses, and pulse width are considered. pared with PEF assistance was rougher than that of In addition, the stirring time after PEF treatment is 0-DAS. The rheological properties results showed considered a parameter for the optimized protein that the viscosity of DAS decreases with increas- release, as well as having reversible or irrevers- ing PEF strength. The decrease in viscosity means ible effects on cell opening are observed. Within that the mobility of the molecular chains in the the scope of current research, it was determined solution increases and the resistance to solution that the protein release of PEF-treated fresh duck- flow decreases, which is beneficial to the chemical weed at 3-5 kV/cm was 10-15% higher compared to reaction of DAS. In addition, the solubility of 12- the untreated reference. Demonstrating the effect DAS was significantly increased to 89.21% com- of PEF-assisted protein release and mass trans- pared with the solubility of native starch (19.01%).In fer, the conductivity index before and after treat-summary, PEF-assisted preparation of DAS has a ment, fluorescence microscopy to visualize the pro- more disordered molecular structure, lower viscos- tein release from plant cells and protein measure- ity, and a higher degree of damage to the surface ment are presented. The effects of PEF on an- of starch granules. tioxidant capacities and polyphenolic content will also be evaluated through methodologies, includ- ing DPPH, FRAP, and Folin-Ciocalteu assays. P3 - Irreversible electroporation As a future objective, the study aims to isolate pro- teins using an optimized pre-extraction procedure. Monday late afternoon Track This will entail applying mild processing techniques D such as ultrafiltration or diafiltration, with a primary Sep 16, 16:10 - 17:40 focus on evaluating the techno-functional proper- ties and nutritional profile of the concentrated pro- OR-56 tein for potential food applications. Study on Segmented Model of Tissue Conduct- ivity Recovery after High Voltage Pulsed Elec- tric Field Treatment Yajun Zhao 1, Luhao Qi2, Zhi Fang2, Dong Xu1 1Zhejiang Cancer Hospital, China 116 2Nanjing Tech University, China OR-57 Development of a Single Needle Electropora- Irreversible electroporation, induced by high- tion Device Towards More Spherical Ablations voltage pulses electric field, has shown tremend- Zaid Salameh, Vinay J. Deshmukh, Rafael Davalos ous application prospects in the fields of tumor Georgia Institue of Technology, United States treatment and cardiac ablation. The tissue con- ductivity would have a significant increase dur- Irreversible Electroporation (IRE) is a non- ing treatment and recover to different extents after thermal focal ablation modality for the treatment treatment. In the present study, the rabbit liver of solid tumors by the application of high voltage tissue is employed to investigate the conductivity pulsed electric fields onto undesirable tissue. Clin- change after treatment. Following the electropor- ically, multiple monopolar 19-gauge needles (at ation process induced by high-voltage pulses, a least 2) are used to apply the necessary voltage series of low-voltage pulses to monitor the change across the target region. IRE treatment requires of tissue conductivity at different time points after precise spacing and minimal angular deflection treatment, finally, the conductivity model was built upon insertion of each probe for optimal electric to reflect the recovery process. The results showed field contours. Although IRE provides a viable that there is an exponential decay phase in tis- treatment option for tumors situated in regions pre- sue conductivity within 1 ms after pulse treatment, viously considered inoperable, the technical chal- which could be correlated to the recovery process lenges introduced limit widespread adoption. Util- of short-lived reversible electroporation. This is folizing a single insertion bipolar electrode can induce lowed by a slow recovery process corresponding clinically relevant lesions and simplifies the medical to the recovery process of long-lived electropora-procedure compared to a traditional multiple needle tion. The tissue conductivity eventually stabilized array. However, containing several electrodes to to a value that remains higher than the initial value a single probe amplifies preexisting limiting factors before treatment, indicating irreversible changes in such as electrical arcing and irregular electric field tissue electrical characteristics after high-voltage contours. Electrical arcing presents an issue in pulse treatment, and this is in agreement with the the single needle electrode configuration due to characteristics of irreversible electroporation. With the proximity of the cathode and anode to one an-the increase in the number of pulses, the final sta- other, encouraging a corona discharge across the bilized value of tissue conductivity would increase insulative spacer. Irregular electric field distribution first and then tend to saturate, indicating that the and the subsequent ablations are a product of the degree of irreversible electroporation caused by electrode geometry. Importantly, the non-spherical such conditions is saturating. Further increasing electric field contour is undesirable in the treatment the number of pulses may not increase the ablation of malignant tumors, as the irregular shape com- range but may instead cause tissue thermal dam- plicates treatment planning and incomplete abla- age. The above three stages of conductivity (rapid tions are susceptible to recurrence. The objective recovery, slow recovery, stability) are important ref-of this work was to optimize a single needle electro- erences for establishing a dynamic model of tis- poration device towards a more complete ablation sue conductivity under irreversible electroporation, region. By using several independent electrodes which would help us understand the dynamic de- of varying sizes on a single device, the electric velopment process of irreversible electroporation, field distribution can be controlled in multiple spatial and formulate dynamic treatment plans for irrevers-directions through cycled pulsing. Here, through ible electroporation, as well as predict the effect of numerical and experimental investigations, we de-irreversible electroporation ablation. velop strategies for a single needle electroporation device for more spherical ablations. 117 OR-58 markers of cardiac damage (troponin) were mon- Production of Large Spherical Ablations Us- itored and treatments were imaged via CT one ing Pulsed Electric Fields Administered Via a week following treatment prior to histological col- Single Applicator lection. Finite element analysis was used to model Jewels L. Darrow 1, Callie Fogle1, Robert H. cell death processes. Williamson1, Alexia Cash1, Kyle Mathews1, Nate The average treatment zone increased in size as Nelson1, Christopher Fesmire1, Matthew Dewitt2, Mi- pulse duration increased from 500ns (3.6cm3) to chael Sano1 2000ns (15.3 cm3). The temperature control al- 1North Carolina State University , United States gorithm successfully achieved and maintained tar- 2University of Virginia , United States get temperatures in vivo. Without cooling, the 65ºC Traditional IRE treatments (NK-IRE) have temperature set point resulted in faster delivery demonstrated significant promise for the treatment (19.5 ± 6.8 minutes) compared to the 45ºC un- of solid tumors; however, the monopolar pulses cooled treatments (67.6 ± 27.3 minutes). Active used induce intense muscle stimulation requiring cooling further reduced treatment times to 4.2 ± the use of intraoperative paralytics. Additionally 1.2 minutes and 7.1 ± 2.2 minutes for 65ºC and for pulses must be delivered during the absolute re- 45ºC set points, respectively. No evidence of car- fractory period of the heart to avoid potentially lethal diac damage was found following treatment and no cardiac complications. These challenges and in- other adverse events were observed during or after strumentation limitations effectively limit treatment treatment. zone sizes and can lead to long treatment times for This study indicates that INSPIRE can be safely large tumors. administered via a single applicator approach with Integrated nanosecond pulse irreversible electro- voltages up to 6000V. Active temperature con- poration (INSPIRE) continuously delivers ultrashort trol enabled rapid delivery of electroporation treat- (250 – 2000 ns) paired positive and negative elec- ments without a priori knowledge of tissue electrical trical pulses in conjunction with active temperature properties or local tissue perfusion conditions. Ad- control. The ultrashort pulses used in INSPIRE sig- ditionally, active cooling of the applicator signific-nificantly reduce muscle stimulation compared to antly reduced treatment times. traditional IRE protocols. This enables the use of OR-60 an applicator and grounding pad approach as well Investigation of rabbit heart electrical activ- as the use of greater voltages. This creates larger ity changes after electroporation using com- treatment zones than possible with a single NK-IRE bined optical and transmural microelectrode treatment. However, these greater voltages lead to technique increased Joule-heating requiring active temperat- Regina Mačianskienė1, Jonas Juravičius1, Ant- ure control to dynamically adjust the rate at which anas Navalinskas1, Mantė Almanaitytė1, Vilma pulses are delivered, which can be supplemen- Zigmantaitė1, Dominyka Adamonė1, Ieva Lankutyte1, ted with applicator cooling to enable faster delivery Mindaugas Visockis2, Justinas Barakauskas2, Ernestas rates. Urbanskas2, Aras Rafanavičius2, Saulius Šatkauskas 2 This study investigated the safety and reproducibil- 1Lithuanian University of Health Sciences, Lithuania ity of the INSPIRE approach in vivo. Healthy swine 2Vytautas Magnus University, Lithuania livers were accessed via an ultrasound-guided per- cutaneous approach with a temperature sensing Pulsed-field ablation (PFA) uses electrical internally cooled electrode applicator. Treatments pulses to induce nonthermal, irreversible electro- consisting of various waveforms were administered poration (EP), causing cardiac cell death in specific with an amplitude of 6000V, a total dose of 0.02s, tissue populations, with minimal damage to adja- and temperature set points of 45ºC or 65ºC, with cent structures. Compared to the thermal ablation or without active applicator cooling. Blood based methods, PFA is a safer technique, but some major drawbacks hinder further development and trans- 118 lation of PFA procedure into clinical settings. We impairs selective transhepatic IRE of tumour nod-aimed to identify PFA parameters capable of cre- ules. Even though there are several references ating controlled depth and size ablation zones and of ex vivo conductivity measurements of liver and investigate changes of functional-electrical activity healthy hepatic tissue, in vivo ones are scarce or induced by PFA in the rabbit heart left ventricular inexistent particularly in human tumors. tissue. We utilized an EP system with an elec- Methods: This study aimed to evaluate the elec- troporator and specialized electrodes (with 3 mm trical conductivity in human liver tissue, compar- gap) to examine the area and depth of tissue ab- ing normal versus tumor tissues and in-vivo versus lation. We varied the duration (25-100 µs) and ex-vivo conditions within a frequency range of 3 voltage (100-800 V) of applied pulses and used to 1000 kHz. Twenty patients were selected, and a series of 20 pulses in 900 ms intervals to in- with informed consent, the electrical conductivity of vestigate the effect of EP on electrical activity in their liver was measured during surgery and post- Langendorff-perfused whole-heart and ventricular dissection. wall of cut-opened hearts; a cut was performed Results: Our results showed higher values for the nearly through the center of the EP zone. Two ad- tumor compared to the normal, both under in-vivo ditional stimulating pulses between every EP pulse and ex-vivo conditions. At 3.08 kHz, in-vivo tu- were used to record action potential (AP) with 300 mor tissue showed a value of 0.44±0.12 S/m, while ms period, which allowed to follow changes of AP ex-vivo tumor tissue was of 0.31 ±0.04 S/m. In during the whole EP protocol. Optical action po- contrast, ex-vivo normal tissue showed the lowest tentials were recorded using voltage sensitive NIR value (0.11±0.04 S/m) followed by the in-vivo nor- fluorescent dye Di-4-ANBDQBS. In addition, trans- mal tissue (0.16±0.05 S/m). The electric properties mural AP were recorded using microelectrode tech- also demonstrated a promising potential for distin- nique. The results showed that EP slows down guishing between different types of tissues includ- velocity of propagation of AP and induces partial ing metastasis, cholangiocarcinoma (CCA), hep- or full conduction blocks. The depth of EP af- atocellular carcinoma (HCC), hepatic cirrhosis, and fected zones, with full inhibition of electrical activ-normal liver, in both in-vivo and ex-vivo conditions. ity, depending on EP parameters can reach up to At 3.08 kHz, electrical conductivity for cholan- 3 mm. In the zones close to EP, the resting po- giocarcinoma, HCC and metastasis (in-vivo) were tential, the velocity of upstroke and the duration of 0.35±0.00049, 0.49±0.25, and 0.45±0.12 S/m, re-AP were reduced. In overall, our study reveals that spectively. our developed system is fully suitable to evaluate Conclusions: These findings could potentially im- changes in functional activity of electrical signals in prove diagnostics of liver diseases by means of EP and proximal to EP zones. electrical measurements and treatment techniques involving electric fields particularly IRE applica- OR-61 tions. Future research should focus on expanding Electrical conductivity in human liver tissue: In the sample size to refine categorization and com- vivo Assessment on normal vs. tumor parison processes across diverse human liver tis- Fernando Burdio 1, Amirhossein Sarreshtehdari1, sues. Xavi Moll2, Enrique Berjano1, Tomas Garcia1 1Universitat Pompeu Fabra, Spain 2 S20 - PhD Students as important Universitat Autònoma de Barcelona, Spain bricks in the wall of funded Introduction: Spread hepatic tumours are not projects and basic research suitable to be treated with thermal ablation, con- ventional irreversible electroporation (IRE) or sur- Tuesday morning Track A gery. Unfortunately, tumour electrical conductivity Sep 17, 10:40 - 12:10 is usually higher than hepatic conductivity, which 119 OR-62 cascade of events resulting in cytokine production, Gene electrotransfer of plasmid encoding thus we also determined expression of 23 differ- Interleukin-12: off-target effects in murine ent mouse cytokines at 24, 48 and 72 hours after cancer cells in vitro GET in culture media of exposed cells. Transfec- Ajda Medved, Maja Čemažar, Tanja Jesenko tion efficacy was higher in B16-F10 cell line com- Institute of Oncology Ljubljana, Slovenia pared to CT26 cells. Cell viability was significantly reduced after pmIL-12 and pScramble GET in both During cancer gene therapy, therapeutic genes cell lines. GET of plasmid DNA led to upregulation are inserted into cells and recognized by cell de- of both DNA and RNA binding PRRs with higher fence mechanisms as exogenous nucleic acids basal levels of expression in CT26 cell line. In- and consequently as pathogen-associated molecu- triguingly, basal levels of these PRRs were inher- lar patterns (PAMPs). Exogenous nucleic acids ently higher in CT26 cells compared to B16F10 cell are then recognized by endosomal and cytosolic line, similarly as to the concentration of cytokines nucleic acid-specific pattern recognition receptors observed to be more elevated in the CT26 cell line (PRRs). Activation of PRRs induces a specific relative to the B16F10 cell line. Moving forward, fu- immune response that may lead to antitumor ef- ture research will explore the comprehensive mod- fects. One of the gene therapies that has already ulation of these sensors and cytokines following been proven to be safe and effective, having good mRNA GET, offering insights into the broader land- local tumor control and an abscopal effect in the scape of GET off-target effects. treatment of melanoma skin metastases, is gene electrotransfer (GET) of plasmid DNA coding for OR-63 cytokine interleukin-12 (IL-12). Plasmid DNA in- Randomised Controlled Clinical Trial Investig- troduced into cells by GET could activate differ- ating the Effect of Reduced Bleomycin in Elec- ent cytosolic sensors, therefore the aim of our trochemotherapy Treatment on Patients with study was to evaluate the effect of GET of plas- Cutaneous Malignancies mid DNA coding for IL-12 on PRRs. Cells (B16- Marie Tolstrup, Julie Gehl F10 melanoma and CT26 colon carcinoma cells) University of Copenhagen, Denmark in suspension (25x106 cells/ml) were prepared for GET using different plasmids: plasmid DNA en- Introduction: Skin tumours across cancer types coding murine IL-12 (1 mg/ml, pmIL12), noncod- have effectively been treated with electrochemo- ing backbone plasmid DNA (1 mg/ml, pScramble), therapy (ECT) for over two decades. This treat- and a plasmid coding for a green fluorescent pro- ment involves administering a standard dose of tein (1 mg /ml pEGFP). Cells were exposed to chemotherapy (15.000 IU/m2 bleomycin) intraven- electric pulses through parallel electrodes with a ously, followed by briefly applying electricity to 2-mm gap. A clinically used pulse protocol was the skin tumours. The cell membranes of malig- used for GET; 8 times 1300 V/cm pulses of 100- nant cells will briefly become permeable, facilitat- microsecond duration at a frequency of 5 kHz were ing easier entry of chemotherapy into the cells and applied with CLINIPORATOR®. Transfection ef- subsequent destruction. Literature has described ficiency was determined 2 days after the electro- studies where the chemotherapy dosage has been transfer of pEGFP using fluorescence microscopy. reduced during the execution of ECT for skin tu- Cell viability was determined 3 days after treat- mours, due to facts such as advanced age and re- ment using the PrestoBlue™ Cell Viability Reagent. duced renal function. Despite the reduced dose of The expression of 15 different PRRs (Dai, Ifi16, chemotherapy, positive results have been consist- Ifi204, Ddx60, Dhx9, Dhx36, Aim2, Cgas, Sting, ently achieved. Ddx41, Lrrfip1, Ku70, Mda5, Lgp2, Rig-I) was de- We aim to investigate whether halving the chemo- termined using qRT‒PCR 4, 24 and 48 hours after therapy dosage during ECT for skin tumours is non- treatment. Activation of cytosolic sensors leads to inferior to the standard ECT treatment. 120 Methods: We plan to conduct a double-blinded ran-OR-64 domized clinical trial, aiming to include 55 parti- Validation of in situ electroporation performed cipants with any kind of biopsy-verified cutaneous within a single cell microwave biosensor tumour. We anticipate that the patients will have Anne A. Calvel, Olivia Peytral-Rieu, David Dubuc, approximately 110 cutaneous tumours in total. Par-Katia Grenier, Marie-Pierre Rols ticipants will be randomized in a 1:1 ratio to re- Université de Toulouse, France ceive either full or half dose of chemotherapy. The Context: ElectroPoration (EP) is widely used primary endpoint will be the overall tumor response in clinical practice within ElectroChemoTherapy after three months assessed according to the modi- (ECT) treatments, that consist in the combination fied Response Evaluation Criteria in Solid Tumours of pulsed electric fields’ application and anti-cancer (RECIST). Follow-up will continue for up to a year. agents’ administration. The entry of the anti-cancer Additionally, we will collect biological samples to agents into cells is facilitated as the membranes measure bleomycin distribution in normal tissue, tu- are transiently permeabilized, thereby increasing mours and blood during the execution of ECT. Fur- drugs’ cytotoxicity. thermore, a biopsy counting percent of tumour cells If the efficacy of ECT has been demonstrated in in tissue will be compared to bleomycin concentra- the treatment of solid tumors, in ˜20% of cases, tion in a biopsy for the same tumour. The collected relapse occurs. Being able to predict treatment data will be uploaded to the database of the Inter- efficacy as early as possible constitutes a main national Network for Sharing Practices of Electro- challenge. Conducting studies at the single-cell chemotherapy registry (InspECT). level is essential for a better understanding of the Additionally, we aim to conduct qualitative inter- dynamics of the EP phenomenon and enables views with 16 of the patients, both before the treat- the study of inter-cellular differences. Among ment and after three months, to understand their the different existing techniques for electrical experiences with cutaneous tumours and ECT. characterization of cells, Microwave Dielectric Discussion: As we make advancements in treat- Spectroscopy (MDS) is a promising approach to ing various cancers and prolonging patient sur- obtain the dielectric properties of cells directly in vival, we are likely to see an increasing incidence their culture medium, in a non-invasive way. MDS of cutaneous malignancies. If halving the dose of can be performed at the single-cell level [1], and bleomycin proves to be noninferior to the stand- it has been effective in detecting cellular states ard dosage, it could create opportunities for treating following the application of EP and ECT treatments elderly and fragile patients as well as those with re- [2]. duced kidney function. Additionally more repeated In the current study, we aim to perform in-situ treatments can be performed if necessary. EP with conventional microwave probes, in a The patients participating in this double-blinded, biosensor initially designed for performing MDS randomized controlled trial are those who would analysis. Such probes are used here to apply DC receive ECT treatment regardless, meaning there voltages. is no additional risk associated with the treatment. Methods: We conduct this study on human cancer This setup closely resembles real-life scenarios, al- cells (THP-1), which we pre-label with Calcein AM. lowing us to apply the conclusions to everyday pa- After trapping a single-cell in the biosensor, we tient care. deliver electric pulses through microwave probes, Implications of this study will potentially be relevant placed on either side of the central strip of a not only to treatment of cutaneous metastases but coplanar waveguide, which has a 5 µm-capacitive also to treatment of deep seated tumours with use gap at its center. of ECT. The applied electric pulses are similar to those commonly used for ECT treatments (8 pulses of 100 µ s width, at a frequency of 1 Hz). We 121 calibrate the electric field by studying the impact context, microdosimetry emerges as crucial tool on permeabilization at different voltages, ranging for comprehending the interaction between biolo- from 0 to 15 V. gical matter and electric fields, offering insights at Electropulsation buffer containing propidium iodide the microscopic level. The European RiseUp pro- is used while applying the electric pulses. We ject [3] exemplifies innovative approaches, aim- assess the state of cellular permeabilization by ing to regenerate neuronal function post-spinal studying the entry of propidium iodide and leakage cord injury (SCI) using an innovative, wireless, of Calcein AM. rechargeable implantable device combined with Results: We validate in-situ EP using an existing Stem Cells (MSCs and iNSCs). Microdosimetric biosensor originally designed for single-cell MDS analyses on realistic cell models are needed for ac- analysis. This study allows us to 1) define the curately evaluating induced electrical quantities [4]. permeabilization threshold for the studied cell line; To this regard, a novel semi-automatic proced- 2) validate the use of microwave probes to apply ure for ad hoc reconstructing 3D cell models and DC voltages and perform EP; 3) pave the way their internal organelles from high-resolution con- for further MDS analysis of the EP phenomenon focal microscopy images was fine-tuned. The 3D performed in-situ. reconstruction process was composed of four main steps: (i) image acquisition of cells, (ii) image pre-References: processing to improve the quality of the image, [1] T. Chen et al., “Microwave biosensor dedicated (iii) segmentation and 3D reconstruction of numer- to the dielectric spectroscopy of a single alive ical cell, using 3D Slicer v. 4.11 software, and biological cell in its culture medium,” IEEE MTT-S (iv) model optimization in Blender v3.0 software. Int. Microw. Symp. Dig., 2013. The resulting advanced cell models were then com- [2] A. Tamra et al., “Single Cell Microwave Bi- bined with already available 3D mesenchymal stem osensor for Monitoring Cellular Response to cell (MSCs) models [4] in order to obtain a nu- Electrochemotherapy.,” IEEE Trans. Biomed. merical cell sample formed by a mixture of iNSCs Eng., 2022. and MSCs. To assess the local electrical quant- ities induced by µs PEF on a cells sample, the OR-65 planar device developed in RiseUP Project was Empowered Cellular and Subcellular Modeling considered and reconstruct in Comsol Multiphysics for Microdosimetric Investigation of PEF Ex- v. 6.0. posure Results reveal complex distributions of induced Noemi Dolciotti 1, Laura L. Caramazza1, Sara electric fields and transmembrane potentials (TMP) S. Fontana1, Micol Colella1, Alessandra Paffi1, Vic- within cell structures. The proximity of cells influ- toria Moreno2, Loris Mannino2, Luis M. Mir3, Franck ences field redistribution, affecting exposure and M. Andre3, Romain Samiaa3, Claudia Consales4, electroporation. TMP distributions on plasma and Francesca Apollonio1, Micaela Liberti1 1 internal membranes exhibit varying arrangements, Universita Degli Studi Di Roma La Sapienza, Italy 2 correlated with cell and organelle sizes. The study Principe Felipe Research Centre, Spain 3 emphasizes the importance of realistic cell and or- Centre National De La Recherche Scientifique, France 4 ganelle shapes in microdosimetric analyses, high- ENEA, Italy lighting the need for a comprehensive understand- Electromagnetic (EM) fields play pivotal roles ing of electroporation phenomena at the cellular in biomedicine, facilitating gene and drug deliv- and subcellular level. This study paves the way for ery, cellular manipulation, and differentiation [1]. numerically assisted experimental applications of For instance, microsecond pulsed electric fields µsPEF for the controlled manipulation of cells and (µsPEFs) induce cell electroporation, enhancing subcellular organelles, enhancing the efficacy and membrane permeability for applications like elec- safety of emerging therapies. trochemotherapy and gene transfer [2]. In this [1] W. Bai et al., “Comparison of effects of high- 122 and low-frequency electromagnetic fields on pro-different tissues, the interaction with physiological liferation and differentiation of neural stem cells,” pathways and distinctive structure makes them Neurosci Lett, 2021. promising targets for treating numerous diseases [2] T. Kotnik, et “Membrane Electroporation and [5]. Electropermeabilization: Mechanisms and Mod- In this study we will examine not only alterations in els,” Annu Rev Biophys, 2019. the protein’s configuration and functioning but also [3] https://www.riseup-project.eu/ its interaction with the surrounding environment. [4] De Angelis et al., “Confocal Microscopy Im- This investigation provides valuable insights into proves 3D Microdosimetry Applied to Nanopor- potential therapeutic applications through 100 ation Experiments Targeting Endoplasmic Retic- nanoseconds MD simulations [2]. The human(h ulum,” Front Bioeng Biotechnol 2020. )TRPV4 model was implemented using MOD- ELLER [6] starting from a model derived from OR-66 the open source PDB [7]. The simulation box Effects of Intense Electric field on TRPV4 ion is represented by the hTRPV4 embedded into a channel: a Molecular Dynamic study lipid bilayer membrane (POPC) and hydrated by Carmen Pisano 1, Laura L. Caramazza1, Paolo 178096 water molecules. The overall dimension Marracino2, Federico Del Signore1, Micaela Liberti1, of the simulation box is 18x18x21 nm³. The study Francesca Apollonio1 was carried out considering E-field intensity in 1Universita Degli Studi Di Roma La Sapienza, Italy line with ones usually adopted in literature [3]. 2Rise technology S.R.L., Italy Each MD simulation was performed in the NPT Pulsed electric fields are increasingly used in ensemble, with temperature and pressure kept medicine to stimulate cells, enhancing membrane at 310 K and 1 bar, respectively. Preliminary permeability via electroporation to deliver thera- results reveal different modification of the hTRPV4 peutic molecules into the cell [1]. One event that according to the applied E-field. contributes to the increase in membrane permeab- ility is the formation of pores in the membrane lipid [1] T. Kotnik et al., Annu Rev Biophys, 2019. bilayer [2]. Electrophysiological measurements [2] P. Marracino et al., Bioelectrochemistry, 2022. suggest that even membrane proteins as voltage- [3] L. Rems et al., Biophys J, 2020. gated ion channels are affected by the application [4] M. Zhang et al., Signal Transduction and of the E field, although the molecular mechanisms Targeted Therapy, 2023. by which the electric field could affects these [5] L. Yue et al., J Cell Sci, 2021. molecules remain unidentified [1]. To fill this gap, [6] B. Webb et al., Curr Protoc Bioinformatics, molecular dynamic (MD) simulations are used 2016. to unravel the molecular events that take place [7] Z. Deng et al., Nat Struct Mol Biol, 2018. in different ionic channels when exposed to an intense electric field [3]. OR-67 Thus, the aim of this study is to investigate, by Anti-tumor efficacy of new high-frequency elec- using a MD approach, the effects of high-intensity trical protocols on in vitro three-dimension E-field on non-selective calcium channels, as the colorectal cancer model transient receptor potential (TRP) channels, which Alexia de Caro 1, Jean-Baptiste Leroy2, Jelena Kolosnjaj-Tabi1, Muriel Golzio1, Marie-Pierre Rols1 besides sharing a high degree of similarity with 1Institut de Pharmacologie et de Biologie Structurale, the voltage-gated ones, are found in numerous France tissues and cell types [4]. These channels are 2Leroy Biotech, France responsible for various sensory like heat, cold, pain, stress and vision and can be activated by For more than 20 years, electrochemotherapy a variety of stimuli. The presence of the TRP in has been increasingly used in the treatment of can- 123 cer, as it increases the cellular uptake of hydro-P1 - Electroporation and immune philic antitumor drugs (bleomycin, cisplatin) and response has already proven its effectiveness on tumors in human medicine and in veterinary practice. How- Tuesday morning Track B ever, this treatment requires local or even general Sep 17, 10:40 - 12:10 anesthesia, as the electrical pulses can be pain- ful and cause muscle contractions. Several studies OR-68 have shown that the application of high-frequency Immunological changes in murine tumor cell pulses (above 5000 Hz) is much less uncomfort- lines following electrochemotherapy in vitro able for the patient than the 1 Hz protocol used in Ursa Kesar, Tanja Jesenko, Boštjan Markelc, Katja clinical practice, despite a slight increase in temper-Ursic Valentinuzzi, Maja Čemažar, Primoz Strojan, ature. The use of bipolar pulses can also reduce Gregor Serša muscle contractions, although the amplitude of the Institute of Oncology Ljubljana, Slovenia field and the number of pulses must be increased Electrochemotherapy (ECT) initiates an im- to achieve the same effectiveness. mune response within treated tumors, yet the im- In order to reduce the pain associated with the con- pact of various cytostatics and the temporal dynam- tractions, but also to maintain the efficacy of the ics of alterations across diverse tumor models re- treatment, we have developed new protocols that main uncertain. Commonly observed types of cell use a high-frequency generator associated with a death following ECT include apoptosis, necrosis, new set of multipolar electrodes. Cell death meas- and immunogenic cell death (ICD), which effect- urements were done on a colon cancer cell line ively induces adaptive immune responses against (HCT-116) cultured in both 2D and 3D (spheroids) neo-antigens released by dying or dead cells. This with different molecules (cisplatin, bleomycin, cal- inflammatory reaction is facilitated by damage- cium and carboplatin). Using our new optimized associated molecular patterns (DAMPs) released protocols on cell suspension, the viability rate were from cells, acting as danger signals. Key DAMPs around 10 % at 48h after treatment and less than capable of activating ICD include surface expos- 5 % at 10 days after treatment whatever the cyto- ure of calreticulin (CRT), ATP release, and high toxic molecules. The results on 3D spheroid mod- mobility group box 1 (HMGB1) release. Addition- els show a significant decrease in spheroid size, ally, immunologically significant changes in tumor suggesting growth impairment after a single treat- cells, such as defects in antigen presentation (e.g., ment . These results were similar to the ESOPE MHC I, MHC II), or alterations in immune-relevant (European standard operating procedures for elec- cell markers like PD-L1 influence the immune re- trochemotherapy) protocol currently used in the sponse. This study aimed to assess alterations in clinic. In addition, the mechanisms of cell death in- DAMP [removed]CRT, HMGB1 and ATP) and im- duced by these high-frequency protocols were in- mune relevant cell markers (MHC I, MHC II, PD-L1) vestigated, suggesting apoptosis death that does following ECT in vitro. Three murine cell lines form- not appear to occur via the mitochondrial degrad- ing immunologically distinct tumor models in mice ation pathway. Ongoing clinical trials on cats and (B16F10 melanoma, 4T1 mammary carcinoma and horses at the Veterinary School in Toulouse have CT26 colorectal carcinoma) were subjected to ECT shown that 80% of patients respond completely using three different cytostatic drugs at IC30, IC50 to treatment without noticeable pain. Therefore, and IC70 concentrations: cisplatin (CDDP), oxali- our painless high-frequency electroporation proto- platin (OXA), and bleomycin (BLM). Changes in ex- cols appear very promising for the widespreading of pression were determined at 4, 24, and 48 hours electrochemotherapy as an effective cancer treat- post-treatment. Our results demonstrated that ECT ment. with all three tested cytostatic drugs induced ICD- associated DAMPs, but the induced DAMP signa- 124 ture was cell line and drug concentration specific. different EP conditions (600 V/cm and 1000 V/cm) Moreover, we showed that ECT with CDDP, OXA commonly used in ECT protocols, to modulate T or BLM can modify the expression of MHC I, MHC II lymphocytes activation. Our results revealed that and PD-L1 cell surface markers that are important EP of co-cultures of HCC1954 cells with resting T in boosting the immunogenicity of the therapy. Sim- cells significantly influenced the number and size of ilarly, as for DAMPs, the potential of ECT to change cancer cell-associated 3D structures (spheroids). their expression was cell line and cytostatic drug T lymphocytes infiltration and cancer cells death concentration specific. Our results thus put the were associated to the reduction in 3D cancer cell ECT with clinically relevant cytostatic drugs CDDP, spheroids. The addition of PHA-M activated T cells OXA and BLM on the map of ICD inducing ther- significantly enhanced this overall effect. Follow- apies and provide the necessary mechanistic in- ing these results, we co-cultured HCC1954 cells sights needed to harness the potential of ICD to with human T lymphocytes to evoke a more clin- elicit a systemic anti-tumor immune response, es- ically relevant condition. Using flow cytometry, we pecially when combined with immunotherapies. confirmed the activation of T cells and their cyto- toxic activity, mediated by the ability of EP to induce OR-69 the release of IL-2, INF-gamma and TNF-alfa at the Electroporation of 3D-cultured breast cancer mRNA level. cells elicits T lymphocyte-mediated killing Our study demonstrates that EP alone can exert Mariangela De Robertis 1, Ramona Marino2, Elisa-anticancer effects by increasing tumor cell killing by betta Sieni3, Mario Cioce2, Andrea Marra2, Vincenzo activated T lymphocytes. We speculate that this is Maria Perriello4, Nico Martarelli4, Annj Zamuner5, So-facilitated by EP-mediated increase in antigen ex- nia Perrelli1, Monica Dettin5, Maria Teresa Conconi5, Vito posure on tumor cells. Michele Fazio2, Flavio Keller2, Emanuela Signori6 1University of Bari ’Aldo Moro’, Italy OR-70 2University Campus Bio-Medico of Rome, Italy Electroporation alters the proteomic output 3University of Insubria, Italy in human ex vivo GI cancer explant models: 4University of Perugia, Italy boosting systemic anti-tumour immunity and 5Padova University, Italy polarizing immune cell populations 6Institute of Translational Pharmacology, Italy Aisling Uí Mhaonaigh 1, Lorraine Smith1, Matthew Cancer cells destruction induced by electro-McElheron1, Aoibhín Woods1, Fiona O’Connell1, Co- chemotherapy (ECT) can trigger a local and sys- sima Sagurna1, Kirstan Murphy1, Meghana Menon1, Yasir Bashir1, Vincent Varley1, Niamh O’Connor1, Cian temic antitumor immune response as a result of re-Muldoon1, Ciara Ryan1, Brian Mehigan1, Waqas Butt1, leasing tumor antigens from electroporated cancer Narayanasamy Ravi1, Claire Donohoe1, Noel Donlon1, cells. 3D scaffolds composed of hyaluronic acid John Larkin1, Paul McCormick1, Dara Kavanagh1, Mi- and ionic-complementary self-assembling peptides chael Kelly1, John Reynolds1, Declan Soden2, Jacintha can enable extracellular matrix organization, thus O’Sullivan1 mimicking the complexity of the tumor microenvir- 1Trinity College Dublin, Ireland onment. They therefore represent a valid system to 2Mirai Medical, Ireland study the anticancer effect of Electroporation (EP) Electroporation-based antitumor therapies, cal- protocols. cium electroporation (CaEP) and irreversible elec- We propose that the use of EP alone on cancer troporation (IRE), have been shown to be very ef- cells embedded in 3D scaffolds may trigger high fective on directly treated tumours by altering the antigens exposure, inducing T lymphocytes migra- tumour microenvironment (TME) and triggering a tion and activation that could lead to cancer cells systemic immune response. We investigated the elimination. proteomic secretome following reversible electro- We co-cultured HCC1954 breast carcinoma cells poration (rEP), CaEP and IRE in upper and lower with Jurkat T cells in 3D scaffolds, applying two 125 Gastrointestinal (GI) cancer explants. We analysed highlighting the immunomodulatory potential of EP the proteomic secretome to further understand the treatments. immunological response to EP and investigated the Significance: Alterations in the immunomodulatory effect of electroporated tumour conditioned media secretome of tumour tissue after EP could affect (TCM) on immune cell polarization. immune cell function, alter the TME and illicit an ab-Hypothesis: rEP and IRE are reported to trigger an scopal response. Further interrogation is required abscopal effect, resulting in a systemic anti tumour to fully elucidate the effects of EP at different sites immune response. We hypothesise that different and could facilitate tailored regimens based on tu- EP treatments will differentially affect the proteome mour site whilst enhancing a systemic immune re-released from GI cancer explants and polarize im- sponse to clear distal metastatic tumours. mune cells in the TME enhancing an anti-tumour immunity. OR-71 Methodology: Following patient consent, ex- Enhanced antitumor efficacy of bleomycin elec- vivo tumour and matched normal tissue explants trochemotherapy combined with anti-PD-1 in from GI cancer patients were exposed to rEP mouse tumor models with/without 5.0mM CaCl2 and IRE using the Simona Kranjc Brezar, Maša Omerzel, Barbara Lisec, Urša Lampreht Tratar, Tanja Jesenko, Gregor ePORE electroporator (Mirai Medical, Galway). Serša, Maja Čemažar Treated explants were cultured at 37°C for 24 Institute of Oncology Ljubljana, Slovenia hours. The supernatants, termed tumour or normal conditioned media, TCM and NCM respectively, Electrochemotherapy (ECT) has emerged as were analysed via MSD 54plex ELISA to assess an effective treatment modality for various tumor secreted factors on angiogenic, chemokine, cy- types in both preclinical and clinical settings within tokine, inflammatory, TH17 or vascular injury pan-human and veterinary oncology. Recent studies els. The effects of TCM and NCM on dendritic have unveiled its potential to activate innate im- cell and macrophage polarisation was assessed by munity by triggering a signaling pathway through flow cytometry. damage-associated molecular patterns (DAMPs). Results: MSD profiling of TCM and NCM from EP This activation leads to specific antitumor immunity treated explants, showed a varied proteome that and subsequent immunogenic cell death. The was dependent on the treatment delivered and tu- observed immunogenic responses resulting from mour origin. In gastric cancer myeloid cell attract- ECT using bleomycin (BLM) suggest a potential ants such as IL-8 and MCP-4 were elevated on synergy with immunotherapy, a pivotal compon- EP treatment and not in colorectal cancer (CRC). ent of current treatment modalities, particularly PD- Normal tissue was less responsive to all treat- 1 inhibitors, known for their significant effective- ments than tumour tissue. Inflammatory markers ness in various cancer types. Therefore, our study such as IL-1 α (p=0.03), TNF- α (p=0.022) and IL-10 aimed to evaluate the antitumor efficacy of combin- (p=0.048) and macrophage released chemokines ing ECT with BLM treatment alongside anti-PD-1 in MIP-1 α (p=0.048) MIP1- β (p=0.018) and MIP-3 α mouse tumor models of various histological types. (p=0.0043) that attract lymphocytes were altered In vitro sensitivity of colorectal MC-38 and CT26 with CaEP. DCs and macrophages were differen- carcinoma, mammary 4T1 carcinoma, B16F10 tially affected by EP treatment at different GI sites, melanoma, and WEHI 164 fibrosarcoma to ECT this needs to be considered when designing treat- with BLM was assessed using the Presto Blue vi- ment regimens. In vitro conditioned media from ability assay. Subsequently, the antitumor effect tumour explants following EP treatment lower M1 of combined ECT with BLM and anti-PD-1 was in- macrophage marker expression in CRC but dif- vestigated in subcutaneous colorectal MC-38 car- ferentially affects in upper GI cancers. In vitro cinoma, B16F10 melanoma grown in C57Bl/6 mice, conditioned media from CRC tumour explants fol- and colorectal CT26 carcinoma, mammary 4T1 lowing EP treatment alter DC marker expression, 126 carcinoma, and WEHI-164 fibrosarcoma grown in effect of ECT that is the systemic effect induced Balb/c mice through tumor growth delay assay. by local treatment. Locally, cellular necrosis de- Our data revealed that WEHI-164 cells displayed termines the release and exposure of tumor an- heightened susceptibility to ECT with BLM com- tigens, triggering an inflammatory response with pared to the other cell lines examined. Specific- consequent activation of the systemic immune re- ally, a significant lower dose of BLM was required sponse. This process allows the antineoplastic ef- to achieve a 50% reduction in cell viability (IC50) of fect of immunotherapy to be supported and ampli-WEHI-164 cells following electroporation, whereas fied. B16F10 and 4T1 cells demonstrated comparable Based on these assumptions and the evidence of sensitivity to ECT with BLM in vitro. Correspond- the effectiveness of the treatment in other tumors, ingly, in vivo experiments demonstrated a com- we decided to apply this protocol for locally ad- plete remission rate of 100% in fibrosarcoma, a vanced or recurrent tumors of head and neck. 50% in mammary carcinoma, a 13% remission rate Materials & Methods: Since 2016, 11 patients (4 in colorectal carcinoma tumors and no remission M and 7 F, average age 74 years, range 43-88 in melanoma tumors. Adjuvant immunotherapy y.o.) with locally advanced or recurrent cancer was used in tumor models that demonstrated lower underwent Immune-ECT with palliative aim. Nine sensitivity to ECT with BLM. This led to complete patients were already submitted to surgery and/or remission in the 4T1 mammary model and a remis- chemo-radiotherapy; 7 patients had squamous cell sion rate of 78% in colorectal carcinoma MC-38, carcinoma, 3 melanoma and 1 Merkel cell car- while in melanoma, it reached up to 14%. Mono- cinoma. Immunotherapy was done before ECT in 2 therapies, on the other hand, displayed limited ef- patients, after ECT in 4 patients, concomitant with ficacy, with only up to an 8-day delay in tumor ECT in 5 cases. Tumor response was evaluated growth compared to controls in all tumor models. clinically and radiologically within 3 months. Overall, our study underscores the enhanced an- Results: No major or minor complications were ob- titumor effectiveness of combining ECT with im- served. Complete clinical and radiological resolu- munotherapy, particularly in less responsive tumor tion of the carcinoma was achieved in 3 patients types, such as colorectal carcinoma, and to some with a mean follow-up of 33 months. Three patients extent, melanoma. These results underscore the are alive with persistent/progressive disease. The potential of ECT to modify the tumor microenviron- remaining 5 patients had disease progression and ment synergistically with immunotherapy in cancer died after an average of 22 months. treatment. Nevertheless, further investigations are Conclusions: Immune-ECT is successfully used warranted to elucidate the underlying mechanisms in clinical practice for metastatic melanoma. The of this combined therapeutic approach. safety and efficacy of the procedure could lead to consider Immune-ECT a valid alternative in the OR-72 treatment options of locally advanced or recurrent Immunotherapy in combination with electro- tumors of the head and neck. Further studies chemotherapy (Immune-ECT) in head and neck are necessary to understand why some tumors cancer respond to Immune-ECT protocols and other do Marta Minuti, Giulia Bertino, Ilaria Imarisio, Marco not. Clinical trials comparing immunotherapy alone Benazzo versus Immune-ECT are needed. Further studies IRCCS Policlinico San Matteo Foundation, Italy are necessary to standardize this new therapeutic Introduction: The combination of immuno- procedure. therapy and electrochemotherapy with Bleomycin (Immune-ECT) is successfully used for the treat- ment of metastatic melanoma. The association of these two procedures is based on the “abscopal” 127 P5 - Electroporation and cellular ing the pulse number that resulted in the highest uptake for BSA and PI. Cell viability was assessed processes and pathways 24 hours post-electroporation using the MTT-PMS assay. Concurrently, the percentages of viable Tuesday morning Track C BSA+ and RFP+ cells were determined by flow Sep 17, 10:40 - 12:10 cytometry (FL1 and FL4). The corresponding con- OR-73 trol groups were treated with BSA for baseline up- Optimizing Electroporation: Efficiency and Cell take regardless of whether BSA was co-delivered Viability in the Simultaneous Transfer of Bovine with PI or pDNA; they were not exposed to elec- Serum Albumin, Propidium Iodide and Nucleic tric pulses. Additionally, fluorescent microscopy Acids will be used to confirm the presence of fluores- Justinas Venckus, Ernestas Urbanskas, Salvijus cence suggesting pRFP expression and the uptake Vykertas, Baltramiejus Jakštys, Paulius Ruzgys, Neringa of BSA and PI within cells, providing a visual eval-Barauskaite-Šarkinienė, Saulius Šatkauskas uation of the electrotransfer efficacy. The prelimin- Vytautas Magnus University, Lithuania ary results have shown that with BSA-Alexa Fluor 488 rising to 12.92% at 17 HV pulses, affirming The delivery of exogenous proteins into cells the method’s efficacy, co-electroporation of BSA via electroporation has been used for biological in- and PI slightly reduced PI uptake from 96.12% to quiries, such as CRISPR-Cas9 gene editing and 93.4% (p<0.05). The presence of BSA modulated intracellular signaling detection. However, there pRFP uptake post-electroporation, with a decrease currently exists no theoretical model for the entry in RFP-positive cells by 28.38%, while BSA uptake of proteins via electrotransfer. Their context- increased by 20%. Furthermore, MTT-PMS assay dependent behavior could be observed by reveal- revealed that pRFP and BSA co-electroporation ing its potential synergies or interferences with vari-decreased cell viability to 23.1% compared to 60% ous other migrating molecules, including small mo- for pRFP alone and 48% for BSA alone (p<0.05). lecules and nucleic acids, under electroporation. These findings might indicate the complex dynamic Since this approach may increase membrane dam- between BSA, PI, and pRFP where these cargoes age and/or endocytosis during their transfer across could affect each one’s uptake and overall cell vi- the membrane, the concerns about cell viability ability. should also be adressed. The aim of this study is to determine the effects of protein transfer on OR-74 the efficiency of simultaneously electroporating it Mitochondrial Depolarization and ATP Loss alongside propidium iodide and plasmid DNA into During High Frequency Nanosecond Electro- mammalian cells and vice versa, using varying poration pulse numbers to optimize multi-molecule delivery Paulina Malakauskaitė 1, Augustinas Želvys1, Auksė while maintaining cell viability. CHO cells were ex- Zinkevičienė1, Eglė Mickevičiūtė1, Eivina Radzevičiūtė- posed to 1, 9, or 17 high-voltage (HV) pulses of Valčiuke1, Veronika Malyško-Ptašinskė2, Barbora 60 µs at 1000 V/cm and 1 Hz to facilitate simul- Lekešytė1, Jurij Novickij2, Vytautas Kašėta1, Vitalij taneous and separate entry of bovine serum albu-Novickij1 1 min (BSA) Alexa Fluor 488 conjugate and prop- State Research Institute Centre for Innovative Medicine, idium iodide (PI). After 30 min., the percentage of Lithuania 2 viable BSA+ and PI+ CHO cells was measured by Vilnius Gediminas Technical University, Lithuania flow cytometry, analyzing 10000 events in the P1 It is predicted that ultra-short electric field pulses region (FSC-A/FSC-H) using FL1 and FL2 chan- (nanosecond) can selectively permeabilize intra- nels on an AccuriTM C6 cytometer. Another ex- cellular structures (e.g., mitochondria) without ma- perimental points were tested for the uptake of jor effects on the outer cell plasma membrane. BSA and mCardinal separately and together, us- Such a phenomenon would have high applicability 128 in cancer treatment context and could be employed of chloride ions. As a result of electrolysis, Cl2 gas for modulation of cell death type or immunogenic can be formed at the anode. Chloride ions react response. with the water molecules and form hypochlorous In this study, we compare the effects of 100 µs x acid (HOCl), which is a powerful oxidant – it can 8 pulses (ESOPE - European Standard Operating react with a wide variety of biomolecules including Procedures on Electrochemotherapy) and bursts DNA, RNA, cholesterol, and proteins. For prac- of 100 ns pulses for modulation of the mitochon- tical applications of electroporation, e.g, when us- dria membrane potential. We characterize the ef- ing electroporation to extract DNA, RNA or proteins ficacies of various protocols to trigger cell plasma (e-biopsy), it is important to avoid any contamina- permeabilization, depolarize mitochondria, evalu- tion of samples. ate the extent of ATP depletion and generation of The aim of this work was to study the formation of reactive oxygen species (ROS). Finally, we employ HOCl acid as a result of electrolysis during high- the most prominent protocols in the context of Ca2+ voltage pulses, as well as its influence on the vi- electrochemotherapy in vitro. ability of cells and the barrier function of the cell We provide experimental proof than 100 ns pulses plasma membrane. The formation of hypochlor- (7.5–12.5 kV/cm, n = 100–1000, 1 MHz) can ous acid was evaluated with fluorescent indicator be used for modulation of mitochondrial potential, of hypochlorite 3’-p-Aminophenyl fluorescein (APF) however, the permeabilization of the outer mem- along with the scavengers of various reactive oxy- brane is still a pre-requisite for depolarization. Sim-gen species (ROS). The viability of Chinese ham- ilar to 100 µs x 8 pulses, the higher is the permeab- ster ovary (CHO) cells was determined by a colony- ilization rate the higher is the mitochondrial depol- forming assay. The size of the pores created in arization. Nevertheless, 100 ns pulses result in human erythrocytes was estimated by studying the lesser ROS generation when compared to ESOPE, protective action of xylitol (152 Da), mannitol (182 even when the energy input is several-fold higher Da), and sucrose (342 Da) against colloid-osmotic than for the microsecond procedure. It was also lysis. shown, that 100 ns pulses can be successfully used It has been obtained, that during high–voltage elec- for Ca2+ electrochemotherapy ensuring excellent tric pulses, ROS are generated. In cell–free media, cytotoxic efficacy, while it is concluded, that even micro–millisecond pulsed electric field increased shorter pulses (i.e., sub-100 ns) are required to ob- fluorescence of hypochlorite indicator APF propor- serve the phenomenon of selective mitochondrial tionally to the pulse number and amplitude. APF depolarization. fluorescence was reduced by both vitamin C and mannitol. Also, it has been shown that ROS form- OR-75 ation was more intensive in the case of stainless– Generation of hypochlorous acid by high- steel electrodes comparing to the aluminum ones. voltage pulses and its influence on the cell The results of this work can be useful for optimizing plasma membrane the electroporation technology used in biotechno- Gintautas Saulis 1, Raminta Rodaitė1, Jurgita logy, medicine, and food industry. Sventoraitiene2, Viktorija Dainauskaite1, Danute The influence of hypochlorous acid on the viability Batiuskaite1, Alexander Golberg3, Rita Saulė1 of Chinese hamster ovary (CHO) cells in vitro was 1Vytautas Magnus University, Lithuania evaluated. HOCl caused the reduction of CHO vi- 2Lithuanian University of Health Sciences, Lithuania ability. Less than 50 % of CHO cells survived, when 3Tel Aviv University, Israel the concentration of hypochlorous acid in the cell Pulses of strong electric field utilized for cell growth medium was 0.8 mM. electropermeabilization, also cause electrolysis re- The influence of HOCl on the plasma membrane actions at the electrode–solution interfaces. Tis- of human erythrocytes was also studied. HOCl in- sues and solutions usually contain high amounts creased the permeability of the cell plasma mem- brane to ions and small molecules, what caused 129 haemolysis of erythrocytes. The estimated radius immunostained for visualization of VE-Cadherin. of permeable structures, which appeared in the Control groups included unpulsed cells, as well as plasma membrane of erythrocytes, was about 0.3– cells pulsed with 15 pulses of 200 V (100 µ sec 0.5 nm. This is close to the size of the pores gener-duration, frequency of 1 Hz); both control groups ated by the exposure of cells with pulses of strong exhibited physiologically normal expression of VE- electric field. cadherin. Endothelial cells, pulsed with 15 EP at Conclusion: hypochlorous acid can be formed due 500 and 1000 V (100 µ sec duration, frequency of 1 to electrolysis during high-voltage pulses. It can in-Hz) demonstrated a reduction in VE-Cadherin im- crease permeability of the cell plasma membrane munostaining, indicative of endothelial barrier dis- to ions and small molecules, which can cause the ruption. Additionally, 50 EP at 500 V completely reduction of the cell viability. disrupted the endothelial barrier. This data sug- gests that microsecond EP affects VE-Cadherin ex- OR-76 pression. Additional studies are needed to under- Application of Pulsed Electric Fields to Gating stand the mechanisms behind these observations. Blood-Brain Barrier for Drug Delivery Pavel A. Solopov, Siqi Guo, Shu Xiao, John D D. Ca-OR-77 travas Is irreversible electroporation immunologically Old Dominion University, United States superior to thermal ablation or cryoablation? A closer look at antigen presentation, T cell ac- The blood-brain barrier (BBB) serves as a tivation and synergy with immune checkpoint protective layer separating blood circulation from blockades neural tissue. It is crucial for preserving the delicate Qi Shao extracellular environment within the neuronal par- University of Minnesota, United States enchyma. Neuroinflammatory events can disrupt the BBB by affecting adherens junctions (AJs) and Minimally invasive tumor focal ablation tech- tight junctions (TJs). The BBB presents a signific- nique (commonly known as focal therapy, FT), in- ant obstacle to drug delivery to the central nervous cluding irreversible electroporation (IRE), thermal system (CNS). It is composed of a continuous layer therapy (Heat) and cryosurgery (Cryo) possesses of specialized endothelial cells connected by tight immunomodulatory properties and can synergize junctions, along with pericytes, a no-fenestrated with immunotherapy to engage the immune sys- basal lamina, and astrocytic foot processes. VE- tem for systemic and long-term disease control. In cadherin, a key component of the vascular system, particular, the combination treatment of FT and im- is particularly essential for the formation of AJs and mune checkpoint inhibitors (ICIs) holds great prom-the overall structure of the BBB. This intricate bar- ise in eliminating established tumors and prevent- rier regulates and restricts the entry of therapeutics ing tumor recurrence, as demonstrated by encour-into the CNS. Various innovative approaches have aging results in preclinical studies (since 2004) and been investigated to improve the transport of thera-clinical trials (since 2011) across a variety of can- peutics across the BBB, each offering distinct be- cer types. Although comprehensive reviews have nefits and drawbacks. We hypothesized that the compared the clinical performances among distinct application of pulsed electric fields could open the tumor ablation approaches, the immunological ef- BBB for drug delivery. fects specific to FT modalities have not been fully Human microvascular endothelial cells were grown assessed or compared, the underlying mechan- as monolayers to 90% confluence on 8-well plates. isms are not fully understood. They were pulsed with 15-50 electrical pulses Utilizing the in vitro assessment platform with the (EP) of 100 µ sec duration, frequency of 1 Hz, OVA model, we found that T cell proliferation and and applied an electric field of 200-1000 V. Four DC activation following IRE treatment of B16-OVA hours later the cells were fixed, permeabilized, and cells were significantly more robust than with Heat 130 or Cryo treatments, even though Cryo resulted in OR-78 the highest release of protein, antigen, ATP and Involvement of mitochondria in the selective re- HMGB1, followed by IRE and Heat. IRE treated sponse to microsecond pulsed electric fields B16 cell lysates induced the most significant im- on both healthy and cancer stem cells in the provement in DC activation and T cell proliferation brain compared to Heat and Cryo treatments when the Arianna Casciati1, Anna Rita Taddei2, Elena same quantity of antigen (naive OVA) was present. Rampazzo3, Luca Persano3, Giampietro Viola3, Alice In a murine colorectal cancer model (MC-38), the Cani3, Silvia Bresolin3, Vincenzo Cesi1, Francesca therapeutic efficacy of anti-PD-1 in combination Antonelli1, Mariateresa Mancuso1, Caterina Merla1, Mirella Tanori 1 with FT is evaluated, utilizing well-characterized 1Italian National Agency for Energy New Technologies miniature probes. IRE exhibits the most favorable and Sustainable Economic Development, Italy syngenetic effect with anti-PD-1 immunotherapy 2University of Tuscia, Italy than Heat or Cryo, leading to greatest primary tu- 3University of Padova, Italy mor growth delay, longest tumor-free survival, and highest protection against secondary tumor chal- In the last few years, pulsed electric fields have lenge. Furthermore, the co-administration of IRE emerged as promising clinical tools for tumor treat- and anti-PD-1 significantly fosters the infiltration of ments. This study highlights the distinct impact CD8+ T cells into the tumor coupled with a remark- of a specific pulsed electric field protocol named able stem-like progenitor phenotype. PEF-5 (0.3 MV/m, 40 µs, 5 pulses) on astrocytes Our in vitro study suggests that IRE is a promising (NHA), medulloblastoma (D283) and glioblastoma approach to induce immunogenic cell death, ac- (U87 NS) cancer stem-like cells (CSCs). We pur- tivate APCs, enhance antigen presentation and T sued this goal performing ultrastructural analyses cell activation, and stimulate anti-tumor immune re- corroborated by molecular/omics approaches to sponse. IRE, emerged as the most immunogenic understand the susceptibility or resistance mech- treatment, is largely attributed to its immunologic- anisms triggered by PEF-5 exposure in the differ- ally favorable “antigenicity” and “adjuvanticity”. ent cell types. Scanning and Transmission Elec- Our in vivo study demonstrates that IRE stands as tron Microscopies analysis highlighted membrane a promising modality that can potentiate the antitu- filopodium-like protrusions disappearance on the mor efficacy when the tumor is poorly responding to surface of all pulsed cells accompanied by a rapid the ICI monotherapy. In addition, IRE has a super- cell swelling. Moreover, morphological alterations ior capacity to activate immunity and enhance the in the cytoskeleton and internal compartments, in- immunogenicity of MC-38 tumors, in comparison to cluding organelles, were observed after the expos- cryoablation and thermal ablation at the given set- ure. Intracellular ATP evaluation and mitochondria tings. membrane depolarization results confirmed that It is important to acknowledge that the immunolo- mitochondrial organelles were the elective target gical outcomes of each ablation modality can vary of PEF-5. Overall, our results suggested that the depending on the energy field distribution, the tar- mitochondrial perturbation was proportionally cor- geted tissue and the extent of energy-tissue inter- related with CD133 content on the cell surface of action. These variations are heavily influenced by analyzed cells. Where a high CD133 content was the specifics of cell death, tissue damage, inflam- present we observed its decrease due to the pro- mation induction, and subsequent wound healing tein localization on the lost membrane protrusions. processes. This decrease contributed to mitochondria dysfunc- tion and ATP depletion which further conferred to cytoskeleton alterations. The medulloblastoma CSCs showed a significant decrease of CD133- positive cell proportion and severe damages on 131 mitochondria leading to massive cell death, glio-controlled flow system. Phosphate-buffered saline blastoma CSCs maintained the principal vital pro- solution including Saccharomyces cerevisiae was cesses, suggesting that the PEF-5 perturbation constantly flown with 100 mL/h for the treatment. was transient and reversible and finally NHA activ- Pulsed voltages of 400 ns duration and up to 30 ated multivesicular bodies formation as a protective kV were supplied between electrodes at 1 Hz us- mechanism. ing a spark-gap driven Blumlein pulse formation Altogether, these findings suggest the possib- network. Inlet temperature of the solution was ad- ility to use PEF-based technology for developing justed so that the outlet temperature did not ex- potential therapeutic strategies to target selectively ceed 50�. To analyze yeast morphology, cell mem-mitochondria of brain CSCs, in correlation with brane permeability was observed using propidium CD133 content, preserving healthy cells. iodide and cell wall morphology using concanavalin A-FITC in addition to brightfield imaging. Also, the cell surface was observed using an electron micro- P13 - General applications of scopy. To analyze the supernatant after centrifu- electroporation for food gation of the treated solution, protein concentration processing was quantified using the BCA method whereas the qualitative analysis was conducted using Native- Tuesday morning Track D PAGE. Furthermore, functional activity of invertase, Sep 17, 10:40 - 12:10 a hydrolytic enzyme, was examined using an en- zyme function assay kit. PEF up to 150 kV/cm was OR-79 continuously applied under 0.3 MPa. Cell morpho- Nondestructive extraction of functional mo- logy clearly showed that the cell wall of the yeast lecules in yeast using 100 kV/cm class electrical subjected to 100 kV/cm PEF was damaged. The pulses amount of protein increased with an electric field Hiroto Hashisako, Koya Asada, Masamori Higuchi, above 70 kV/cm. Our experiment shows the ultra-Sunao Katsuki high PEF is capable of destroying the yeast cell wall Kumamoto University, Japan to extract intracellular functional molecules. Proteins, amino acids, and lipids contained in yeasts are widely used for foods, feeds, cosmet- OR-80 ics, and pharmaceuticals because yeasts are inex- Pulsed Electric Field Treatment for Preserva- pensive and mass-producible nature. Also, gene tion of Chlorella Suspensions transfer enables us to make yeasts produce any Cora De Gol 2, Ailsa Moodycliffe2, Heidy M. W. den Besten1, Marcel Zwietering1, Michael Beyrer2 designed proteins. Thermal or chemical methods 1Wageningen University, Netherlands are used to extract the yeast components for the 2HES-SO Valais , Switzerland mass production, whereas non-thermal and phys- ical methods are preferred to extract functional mo- Pulsed electric field (PEF) processing has lecules without destroying them. Although pulsed emerged as an alternative to thermal pasteuriz- electric fields (PEFs) are known to permeabilize the ation for the shelf-life extension of heat-sensitive yeast cell membrane, the thick cell wall remains liquids at industrial scale. It offers the advantage unchanged. The wall interrupts the intracellular of minimal alteration in physicochemical charac- large molecules to go outside. We have tried to teristics and functional properties. destroy the yeast cell wall using ultra-high PEFs In this study, a pilot-scale continuous PEF more than 100 kV/cm and to extract the intracel- processing (T < 55 °C) was applied for the pre- lular molecules such as enzymes without destroy- servation of microalgae Chlorella vulgaris (Cv) ing them. The PEF treatment cell consisting of suspensions (pH = 6.5). Cv suspensions have stainless-steel parallel plate electrodes with a 2 mm been proposed as minimally processed, nutritious gap was installed in a pressurized, temperature- 132 and functional ingredient for manufacturing meat 2Institute of Measurement Science, Slovakia analogues via high moisture extrusion. Various 3Institute of Photonics and Electronics of the Czech PEF conditions (electric field strength E = 16, 19, Academy of Sciences, Czech Republic 22, 25 and 28 kV/cm and pulse repetition rate = 4Kanazawa University, Japan 100, 120 and 140 Hz, with a pulse width of 20 µ s Pulsed electric fields (PEFs) show promise and an inlet product temperature T of 30 °C, n in biotechnology and food industries. However, = 3) were tested on Cv inoculated with potential real-time monitoring of PEF treatment efficiency, food spoilage microorganisms (Pseudomonas especially at industrial and continuous produc- guariconensis, Enterobacter soli and Lactococcus tion scales, is challenging. To address this, we lactis). The aim was to evaluate the PEF induced developed a simple setup for real-time detection microbial reduction and monitor the microbial of yeast biological autochemiluminescence (BAL) outgrowth during a 10-day cold storage period during pulsing. We exposed Saccharomyces (10 °C). Additionally, selective media were used cerevisiae to 8 pulses of 100 µs width with elec- to differentiate between injured and intact cells, tric field strengths of 2-7 kV/cm. Our method’s and image processing techniques were employed sensitivity in detecting yeast electroporation was to study the impact of PEF on colony size during compared to established methods such as imped- growth. ance measurements, propidium iodide uptake, Maximum inactivation of 4.1 ± 0.2, 3.7 ± 0.1 and 3.6 cell growth assay, and fluorescence microscopy. ± 0.2 logs was achieved (28 kV/cm and 120 Hz) for Results demonstrate instantaneous monitoring the isolates mentioned above, respectively. Under of yeast electroporation during pulsing, making these conditions, the critical electric field strengths it ideal for industrial applications. The setup’s E above which inactivation was observed, ranged simplicity allows integration into continuous liquid from 22.6 to 24.6 kV/cm. The significant increase flow systems. We also established quantitative in injured cells compared to intact cells (approx- BAL indicators via statistical analysis. imately 100x) suggests considerable potential for further inactivation. The observed inactivation res- Acknowledgments: The authors thank the Czech ulted from a synergy between thermal and electric Science Foundation project no. 20-06873X for the effects. A second PEF cycle demonstrated similar support. inactivation efficiency, indicating its potential to enhance shelf-life further. OR-82 These findings are of industrial relevance and Germination and stress tolerance of oats offer valuable insights into innovative technologies treated with pulsed electric field at different that not only ensure the quality and longevity of phases of seedling growth plant based food ingredients but also contribute Alia Hussain Al-Khafaji1, Stephen Kwao2, Federico to minimizing the risks associated with traditional Gomez Galindo 1, Sajeevan Radha Sivarajan3 thermal processing, thereby promoting healthier 1Lund Univesity, Sweden and more sustainable food options. 2OptiCept Technologies AB, Sweden 3Swedish University of Agricultural Sciences, Sweden This study explores the impact of pulsed elec- OR-81 tric field (PEF) application on oat seedling growth Correlation of PEF induced biological auto- and stress tolerance. PEF treatment (99 mono- chemiluminescence with yeast cell electropor- polar, rectangular pulses lasting 10 µs each, with ation a frequency of 13 Hz and a nominal electric field Martin Bereta 1, Michal Teplan2, Tomáš Zakar3, Ho-strength of 2250 V/cm) was applied at two growth ang Vuviet2, Michal Cifra3, Djamel Eddine E. Chafai4 stages: (i) when the seedlings had 0.2 cm roots 1Catholic University in Ruzomberok, Slovakia emerging from the kernel, and (ii) when they 133 had a 0.4 cm shoot emerging from the kernel. kV/cm combined with pH 11 shifting has the highest Post-treatment, the seedlings were hydroponically solubility (90.23%), the smallest turbidity (0.072), grown for 8 days. To induce stress, the hydroponic the largest absolute zeta-potential value (44.4mV) medium was augmented with PEG (15%) to induce and the smallest particle size (63.5nm). The com- drought stress and NaCl (150 mM) to induce salin- bination of PEF with pH shifting treatment can in- ity stress. Results demonstrate that applying PEF duce the secondary and tertiary structure of SPI improved the growth of the root and shoot of oat to unfold the folded part, resulting in the trans- seedlings. This effect was more pronounced when formation of SPI from the spherical packing state applied to more developed seedlings. When PEF to a molten sphere state with lower levels of α- was applied during the later stage of germination, helix and β-sheet and higher levels of random coil-seedlings exposed to salinity stress showed en- ing. The SPI modified by different methods had hanced shoot growth compared to the control. Un- better emulsifying, foaming and binding properties der the studied conditions, the application of PEF with small molecules. The improvement effect of had no impact on the growth of seedlings under the three modification methods on SPI functional drought stress. characteristics was ranked as follows: PEF com- bined with pH shifting > pH shifting followed by OR-83 PEF > PEF followed by pH shifting. Compared Effects of different combinations of pulsed with the untreated group, the emulsification activity electric field and pH shifting treatment on the (EAI), emulsification stability (ESI), foaming activ- aggregation structure and functional properties ity (FA) and binding constant to lutein of SPI after of soybean protein isolates PEF combined with pH shifting were increased by Rui Wang, Pei-Feng Guo, Jing Nie, Xin-An Zeng 119.24%, 39.33%, 59.03% and 245.81%, respect-Foshan University, China ively. The processing conditions of this method Soybean protein Isolate (SPI), a kind of a pro- are mild, which is conducive to improving the func- tein product made from defatted soybean meal, has tional properties of SPI without impairing the sens- been widely used in food, materials and cosmet- ory properties and biological activity of the pro- ics industries for its high nutritional value and func-tein. It is compatible with the processing concepts tional properties. However, when SPI has been ex- of “green processing” and “minimal processing” of tracted and produced, the treatments such as alkali food, and has the potential to become the main- dissolution and acid deposition, heat sterilisation stream technology in the modification of plant pro- and freeze/spray drying would cause protein ag- teins in the future. gregation and generate protein aggregates, which OR-84 results in decreasing solubility of SPI and blocking Analysis of temperature dependent dielectric its functional properties, thus restricting its applica-properties of bacteria for effective PEF pasteur- tion in the food industry. Therefore, we attempted ization to modify SPI using pulsed electric field (PEF) and Ryuya Kimura 1, Sunao Katsuki1, Bingyu Yan1, pH shifting techniques to reduce the degree of ag- Misato Kikuchi2, Shoko Ishikawa2, Kazuhiro Inobe2, Ryo gregation of SPI and improve its functional proper-Sasahara2, Taiga Kajiwara2, Naoya Masuda3, Yoshiharu ties. The effects of three different combined treat- Shimizu3 ment methods of PEF and pH shifting (PEF be- 1Kumamoto University, Japan fore pH shifting, PEF after pH shifting, PEF com- 2Kewpie Corporation, Japan bined with pH shifting) on the aggregation struc- 3Iwai Kikai Kogyo Co., Ltd., Japan ture and functional characteristics were investig- ated. The results show that PEF combined with Pulsed electric field (PEF) is the most promising pH shifting was the best method for depolymer- low-temperature pasteurization method for protein- ization of SPI aggregates. SPI treated with 10 rich liquid foods, such as milk, liquid egg, etc. Target liquids are often preheated for the PEF applic- 134 ation since bactericidal effect of PEF is known to OR-085 be dependent on the temperature at which PEF is Pulsed Field Ablation – gaps in knowledge and applied. On the other hand, higher preheating tem- future directions of development peratures compel us to lessen the electrical input Damijan Miklavčič of PEF for preserving the ingredients. In this pa- University of Ljubljana, Slovenia per, we discuss the optimal preheating temperat- Pulsed Field Ablation is a new ablation mod- ure for effective PEF pasteurization based on the ality based on irreversible electroporation. Since temperature dependence of bacterial membrane first proposed (and soon abandoned in 1980s due properties measured using an impedance spectro- to major risks and side effects) it has gained trac- scopy. The impedance analyzer (PSM1735-IAI, tion following the development of irreversible elec- Newtons4th) was used to measure the capacitance troporation in 2000s. PFA is now approved for atrial and conductance of bacterial suspension of En- fibrillation treatment in Europe and by the FDA. The terobacter hormaechei (1010 CFU/ml) in a 2 mm drive propelling the rapid uptake by cardiac electro- gap electroporation cuvette. The capacitance and physiologists is its superior safety and speed with the conductance reflect state of the bacterial mem- respect to cryo- and radiofrequency ablation and its brane and the leakage of intracellular ionic sub- comparable efficacy as evidenced by available clin- stances, respectively. The temperature of the bac- ical studies. It seems that we have “tamed the lion” terial suspension was monitored using a fluores- in the last two decades. cent fiber thermometer (FL-2400, Anritsu) and pre- Preclinical and clinical results indeed provide ex- cisely controlled by our homemade system. When pected results with respect to safety by almost the temperature of the bacterial suspension was complete absence of “standard” complications of slowly increased from 30�, the capacitance was cryo- and RF-ablations: phrenic nerves, pulmonary decreased linearly with increasing temperature up veins, and esophagus damage and most rare but to 43�. The decrease rate of the capacitance was feared atrio-esophageal fistula. Somewhat unex- increased between 43� and 55 �, which implies pectedly, other complications have been reported: to be a phase transition of the membrane. On the most notably cardiovascular spasm and hemolysis, other hand, the conductance was increased with but also silent cerebral emboli and phrenic nerve increasing the temperature, and the increase rate palsy have been reported. Can we expect more was increased between 43 � and 55 �. This in- and others when numbers of treated patients will dicates the leakage of the intracellular ionic sub- increase, and new PFA systems will be introduced stances. The survival rate of the bacteria to a into the market? single 2 µs-long 20 kV/cm PEF at the temperature Somewhat disappointing was relatively low effic- between 35� and 50� was investigated. The tem- acy, i.e. comparable to cryo- and RFA (but still perature dependence of the bactericidal effect was long-term follow-up results are missing). It needs correlated with the temperature dependence of the to be emphasized that any new technology requires capacitance and the conductance determined by optimization (also from a procedural standpoint) It impedance analysis. also seems fair to note that results obtained by PFA first generation devices are unfair to be compared PFA Industry Panel to those obtained by RFA systems that benefited of decades of perfecting them. Much so also for cryo- PFA Industry Panel Track ablation. Sep 17, 13:20 - 14:10 How can we address the new safety and/or ther- apy related concerns such as hemolysis, coronary spasm, cough, neuromuscular stimulation? Can we improve the efficacy? Is there a magic wave- form that balances off safety concerns while it im- 135 proves efficacy? Is there a way to compare differ-the whole cohort and follow up procedural efficacy ent PFA systems? How will regulators enable new was 60% and 42% for paroxysmal and persistent and improved waveforms and devices to get on the AF respectively. We observed 3 PFA related com- market for the benefit of the patients? How can im- plications. proved understanding of electroporation and basic Discussion: As PFA has proven higher safety pro- research help address these questions. file, which is believed to be due to non-thermal nature, LA posterior wall isolation was done as per our clinical experience and expert consensus, S21 - Cardiac ablation by nevertheless one patient had postprocedural eso- irreversible electroporation - phagitis, that resolved 2 weeks later. Follow up pulsed field ablation (PFA) analysis showed, that for this patient higher 25A dose (intended for anterior aspect of LA) was used Tuesday afternoon Track A in LA posterior wall. One patient had acute peri- Sep 17, 14:20 - 15:20 carditis. Coronary spasm presenting with acute ST elevation was noticed in one procedure, while ab- OR-86 lating at the ostium of the left inferior pulmonary Initial single centre experience with pulsed field vein. ablation for treatment of cardiac arrhythmias Additionally, we used PFA in 4 patients after failed Jernej Štublar 1, Tine Prolič Kalinšek2, Jernej Iršič2, radiofrequency ablation of ventricular tachycardia. Damijan Miklavčič1, Matevž Jan2 Despite highest dose (25A) and multiple applica- 1University of Ljubljana, Slovenia tions delivered in the same location, only transi- 2University Medical Centre Ljubljana, Slovenia ent effect was achieved in all cases. This could Background: Pulsed field ablation (PFA) is an be because CentauriTM system was developed for emerging ablation modality for treatment of cardiac ablation of thin atrial wall, thus reaching deeper arrhythmias, based on irreversible electroporation. midmyocardial tachycardia isthmuses only with re- Methods: We investigated procedural parameters versible electroporation field. and clinical outcomes from our PFA registry. Cen- Conclusion: Based on small and heterogeneous tauriTM (Galvanize Therapeutics, Inc., USA) PFA patient cohort clinical efficacy comparison to the system was used. It is connected instead of a ra- established clinical practice is difficult. Point-by- diofrequency generator and thus easily adopted in point PFA seems moderately effective for treatment routine clinical procedures, which were performed of AF and ineffective for intramural ventricular sub- in deep sedation between February 21st, 2023 and strates. We believe that the biggest advantage of March 5th, 2024. Follow up included scheduled the PFA system presented here is its possibility of outpatient clinic visits and ECG recordings guided toggling between different energy sources espe- by symptomatic arrhythmia episodes. cially in the vicinity of neighbouring tissues (oeso- Results: We included 43 consecutive patients phagus) during extensive ablation of the LA pos- where at least one PFA application was delivered. terior wall. Mainly persistent (53%) and paroxysmal (33%) at- rial fibrillation (AF) were treated with pulmonary OR-87 vein isolation lesion set (81%) as recommended Intraoperative Assessment of Irreversible Le- by the manufacturer. For persistent AF isolation sion Formation During PFA of left atrial (LA) posterior wall (47%) was added. Parag Karmarkar 2, Shunsuke Uetake1, Sivanag Maddineni2, Bhupendra Mahar2, Zhi-Qu Xu1, Saleheldin Patients were male (65%), mean age 60,6 years, Alhawamy1, Harikrishna Tandri1 overweight (29,3 BMI). Totally, 92 ± 60 (mean ± 1Vanderbilt University Medical Center, United States SD) PFA applications were delivered per patient, 2SIGT LLC , United States with skin-skin procedural time 171 ± 64 (mean ± SD) min. Acute procedural efficacy was 81% for 136 Background: Currently, there are no methods to Conclusion: HFDS can potentially assess pro- assess and confirm irreversible lesion formation in cedure parameters and confirm irreversible lesion real-time during Pulsed Field Ablation (PFA). Loss formation in real-time during PFA. This method of local electrograms is a poor guide to confirm ir- does not rely on local electrogram detection and reversible lesion formation. can be used to assess completeness of ablation Objective: To evaluate the feasibility of assessing during PFA. irreversible lesion formation in vivo (in swine) using High Frequency Dielectric Sensing (HFDS) using OR-88 a focal PFA catheter with its ablation electrode de- Lesion Durability Prediction based on Real- signed as an antenna-sensor at MHz-GHz frequen- Time Impedance Analysis Algorithms: Valida- cies in real-time during PFA. tion with First-in-Human Clinical Data from the Methods: A 9F focal ablation catheter with its ab- RESET-AF Trial lation electrode redesigned as an onmidirectional Laura Boehmert 1, Steffen Holzinger1, Dorin Panescu2 antenna-sensor electrode was used in vivo (swine) 1Biotronik, Germany to perform PFA. High Frequency Electrical Proper- 2CRC EP, United States ties (HFEPs), namely, resonant frequencies (Fr), of the antenna sensor-electrode were simultaneously Pulsed field ablation (PFA) is a non-thermal ab- monitored in 150MHz-to-2.5GHz frequency sweep lative strategy that achieves cell death in cardiac measurement. Changes to HFEPs, namely Fr, tissue by irreversible electroporation. PFA is be- were monitored to confirm electrode-tissue contact, coming widely adopted as a technique for the treat- e-field deposition in tissue and durable lesion form- ment of atrial fibrillation (AF). A catheter is placed ation. PFA was carried out by applying trains of in patient’s left atrium (LA) and navigated to each biphasic pulses, in a monopolar application, at dif- of the four pulmonary veins (PVs). Pulsed-field en- ferent voltages ranging from 250 V to 1800V, 2 µs ergy is applied to achieve isolation of all PVs from pulse widths 1000 pulses per application. Abla- the body of the LA. Determining the quality and tion locations were cataloged using Ensite mapping uniformity of the ablation catheter contact with the system and lesions were assessed by gross histo- tissue is essential for the success of PFA proced- logy using TTC staining 12-14 hr post ablation. ures. Poor electrode-tissue contact is detrimental Results: A total of 29 lesions were assessed in 2 to achieving circumferential and transmural PV le- animals (farm pigs, RA and RV lesions). Distinctly sions. Additionally, patient movement may also different HFEPs i.e. Fr, are observed when elec- lead to catheter displacement which can lead to trode is in blood, when contacting myocardium, and lower efficacy. Therefore, it is important to detect during PFA pulse application. Baseline Fr in blood such catheter movements, as well as poor con- 450±15 MHz which increased to 500±15 MHz con- tact, and to alert users accordingly. These alerts firming stable electrode-tissue contact (p<0.001). may result in stopping treatment or in reposition- Following stable electrode-tissue contact confirm- ing the catheter. Data for this abstract was ob- ation, PEF pulses were applied and Fr increase in tained from the RESET-AF clinical trial. RESET-AF the range of 545MHz-to-630 MHz was observed is a prospective, nonrandomized, premarket first- (for voltages >1400V). An increase in Fr i.e. ∆Fr>30 in-human (FIH) study. MHz correlated with irreversible lesion formation on The aim of this study was to evaluate a PFA sys- histology. PFA in blood resulted in ∆ Fr <10MHz. tem that utilized real-time impedance measure- 21 out of 29 intracardiac lesions which were con- ments to predict treatment efficacy based on con- firmed in real-time by HFDS during PFA were con- tact uniformity and movement detection algorithms. firmed by histology and were transmural or >2.5 We used impedance data from 18 patients of the mm deep. 6 lesions where ∆Fr was <10 did not res- RESET-AF. A single PFA application was delivered ult in a discernable lesion, whereas 2 lesions where to each PV followed by a second ‘insurance’ applic- ∆Fr <30 resulted in superficial lesion ˜1 mm deep. 137 ation without repositioning the catheter. PV isola-medium (we call this degassing), b.) release of tion was confirmed by 3D-mapping and exit block. gas as water vapor (boiling) and c.) release of gas Additional applications were only allowed if acute as a product of hydrolysis. We performed an in PV isolation was not achieved. All patients under- vitro study with pulsed fields in saline using sev- went mandatory invasive remapping at 3-months. eral different treatment protocols and a modified RF The remapping data evaluated the lesion durabil- catheter for bipolar delivery and high-speed cam- ity and were used to define the prediction algorithm era imaging. Moreover, numerical simulations to accuracy. investigate thermal effects near the electrodes and Acute isolation was achieved in 100% of the PVs predict boiling. (n=72) using 2.1±0.4 applications/PV. PV isolation Materials and methods: We used monophasic durability at 3 months was confirmed in 91.7% of (100 µ s) and biphasic protocols and with varying the PVs and in 77.8% of the patients. For the duty factor to deliver pulses to 0.45% NaCl solu- isolated veins, the contact uniformity algorithm, in tion. Pulses were delivered bipolar via a modi- conjunction with the average electrode impedance fied 5 mm tip catheter (ConductR, Medtronic, Min- value, were able to predict durable lesions with a neapolis, MN, USA) between the tip and the ring positive predicted value of 94.7%. The movement electrodes for intracardiac electrogram recording. detection algorithm was able to detect all move- The voltage and current during the pulses were ment instances of single electrodes, as confirmed measured, and the area between the tip and the by fluoroscopy or 3D navigation. most distal ring electrode was imaged at 10 kfps In this FIH clinical trial, PV isolation was achieved using a Phantom v1212 high-speed camera (Vis-safely and effectively in patients with AF using a ion Research, Wayne, NJ, USA). Numerical mod- novel, single-shot PFA system without reposition- eling was performed with COMSOL Multiphyiscs ing the catheter. Real-time impedance algorithms (COMSOL AB, Stockholm, Sweden). An axisym- were shown to add an additional layer of efficacy metric 2D model of the catheter was created. The as means to predict lesion outcomes. model takes into account the heating due to pulse delivery, the increase in conductivity of the saline OR-89 solution due to heating and the phase transition Investigation of bubble formation in intracar- between the liquid and gaseous phase of the wa- diac pulsed field ablation ter, i.e. boiling. Samo Mahnič-Kalamiza1, Damijan Miklavčič1, Peter Results: High-speed video recordings show that Lombergar1, Blaž Mikuž3, Lars M. Mattison2, Daniel C. bubbles form in an even layer on both electrodes Sigg2, Bor Kos 1 (anode and cathode) during a monophasic 100 µ s 1University of Ljubljana, Slovenia pulse protocol. These bubbles are of electrochem- 2Medtronic, United States ical origin. Above a certain voltage threshold, an 3Jožef Stefan Institute, Slovenia additional effect is observed near the leading edges Introduction: Pulsed field ablation is a treat- of the ring electrodes: rapid appearance and disap- ment method for cardiac ablation based on electro- pearance of bubbles, which is consistent with the poration. Electroporation requires charge transfer boiling process. In biphasic protocols, the electro- between the electrodes and the cell suspension or chemical bubbles are not present, but boiling also tissue, which leads to redox reactions at the elec- occurs at sufficiently high voltages. The numerical trodes, which also produce gasses. The energy in- model was also used to determine the maximum put is so intensive that it leads to considerable heat-volume of degassed air due to the reduced solu- ing and can reach the boiling point of water. bility of nitrogen and oxygen at elevated water tem- There are three possible mechanisms of bubble peratures. The volume of degassed air was always formation and growth: a.) release of gas due to less than the volume of steam. lower gas solubility at higher temperatures of the Conclusions: The choice of treatment protocol for energy delivery and electrode design critically af- 138 fect the generation of bubbles near the electrodes. The sample media used in this treatment was Careful electrode design and numerical modelling water. The peak temperature of the pure water can aid in preventing bubbles. The results obtained samples only increased by a few degrees as the in saline may differ from results in blood. sample’s electrical impedance is very large. How- ever, when a small amount of salt was added the peak temperature was below 37 °C which will not S08 - Public health risks and influence/inactivate E. coli independently. pulsed electric fields in the food The bacterial reduction increased linearly for most industry of the treatments applied with some showing satur- ation as the pulse number (or specific energy input) Tuesday afternoon Track B is increased. The log reduction ranged between Sep 17, 14:20 - 15:20 1.1 to 6.6 across all PEF treatments. All three variables (voltage, pulse number, and initial bac- OR-90 terial concentration) were found to be independ- A multivariate study on continuous-mode ently statistically significant predictors of bacterial pulsed electric field treatment of E. coli in reduction, however no interaction between these water variables were identified to also be statistically sig-Yiting Wang, Farzan Zare, Elisabeth Prabawati, nificant. Buddhi Dayananda, MirHojjat Seyedi, Mark Turner, Negareh Ghasemi, Nidhi Bansal OR-91 The University of Queensland, Australia Inactivation of zoonotic parasites by PEF, bey- Pulsed electric field (PEF) technology in the li- ond single-cell electroporation quid food processing sector can be operated in Juan Manuel Martínez, Ignacio Álvarez, Guillermo J. batch- or continuous-mode. For industrial integ- Cebrian, Vanesa Abad ration it is necessary to move from a research- Universidad de Zaragoza, Spain focused batch-mode treatment to the more industri- Parasites are concerning food-borne patho- ally feasible continuous-mode. Although it is more gens. Some of them are currently not being complex to integrate a PEF system in continuous- routinely controlled in food, probably because mode, it is more advantageous as the transient their burden on public health is underestimated. temperature can be controlled because of the li- Parasitic incidences could be avoided if preventive quid flow. Whereas, in batch-mode heating of technologies were applied during food processing. the liquid food is more prevalent and should be Effective inactivation treatments are currently closely monitored. In this work the role of the based on heat or freezing, but their side effects voltage, pulse number, and initial bacterial concen- collide head-on with current consumer trends and tration as predictors of bacterial reduction of Es- new culinary habits. cherichia coli JM109 were investigated. A closed- Recent research supports the potential application loop continuous-flow system was designed with a of Pulsed Electric Field (PEF) technology in the peristaltic pump, reservoir containing 100mL of the control of food-borne parasites, to reduce the inoculated water, and custom-made cuvette. viability and infectivity of parasite transmission E. coli, at three concentrations (5, 7, and 8 log stages without affecting food quality. CFU.mL-1), was inoculated in water and treated in The feasibility of PEF as an alternative to traditional continuous-mode PEF at two voltage levels. The freezing processes for the inactivation of Anisakis pulse protocol was applied in four ‘steps’, each step in fish has been demonstrated. The viability of of 60k pulses was applied with a 5-minute resting Anisakis larvae was highly dependent on field time between steps. The resting time was used to strength and specific energy, while pulse width allow the liquid to cool down but also to extract a only exerted a notable influence at the lowest field sample to investigate the role of pulse number. 139 strengths tested (1 kV/cm). Central composite OR-92 design helped to define a PEF treatment of 3 kV/cm Rapid Recovery of Bacterial Membrane Follow- and 50 kJ/kg as the one capable of inactivating ing Exposure to Pulsed Electric Fields almost 100% of Anisakis larvae in pieces of hake, Bingyu Yan 1, Ryuya Kimura1, Misato Kikuchi2, while affecting the investigated quality parameters Shoko Ishikawa2, Kazuhiro Inobe2, Ryo Sasahara2, (moisture, water holding capacity, and cooking Taiga Kajiwara2, Sunao Katsuki1 1 loss) to a lesser extent than freezing and thawing. Kumamoto University, Japan 2 The inactivation of larvae of the nematode Kewpie Corporation, Japan Trichinella, causing parasitic infections after the PEF is a potential non-thermal sterilization tech- consumption of uncooked meat or meat products, nology, which has good application prospects for also has been studied. Meat inspection is costly liquid food sterilization. he principle of PEF steriliz-and tends to progressively be derogated in pig ation is to apply a high potential difference across holdings applying controlled housing conditions the cell membrane, disrupting its structure (forma- in certain EU regions. PEF has been evaluated tion of pores) and thereby sterilizing the cell– a phe-in the inactivation of Trichinella sp., including nomenon called electroporation. However, there excysted larvae isolated by artificial digestion and are two states for these pores: recoverable and encapsulated larvae found in meat from naturally non-recoverable, depending on their size. Recov- infected wild boars. The excysted larvae were erable small pores can quickly heal in a suitable inactivated ten minutes after an intermediate PEF environment (rich in nutrients, suitable temperat- treatment (1 kV/cm, 0.41 kJ/kg). Treating excysted ure) within a few minutes, which may weaken the larvae with the mildest PEF treatment (0.5 kV/cm, bactericidal effect. However, there are two states 0.05 kJ/kg) combined with a 3% NaCl incubation for these pores: recoverable and non-recoverable, resulted in synergistic inactivation. The application depending on their size. Recoverable small pores of 3 kV/cm (20 kJ/kg) to wild boar meat resulted can quickly heal in a suitable environment (rich in the inactivation of over 90% of encapsulated in nutrients, suitable temperature) within a few Trichinella larvae. The viability of Trichinella in minutes, which may weaken the bactericidal ef- meat was inversely correlated to the field strength fect. The recovery process is anticipated to pro- applied (1-6 kV/cm) for equal energy input (20 gress in seconds or less from the viewpoint of the kJ/kg). thermal motion of lipids in the membrane, however, The development of new PEF equipment is advan- there are few studies reporting the recovery pro- cing at a rapid pace, allowing for food treatment at cess within minutes following PEF treatment. a scale that would have been unimaginable some The purpose of this study was to utilize fast dielec- years ago. A review of more basic-science studies tric spectroscopy to investigate the time scale of carried out on buffer media would contribute to recovery of injured bacteria from electric field ex- progress in addressing the underlying drawbacks posure at different field strengths. The advantages that remain to be solved. Thoroughly different of dielectric spectroscopy are that it can meas- fields (parasitology, physics, food engineering, ure phenomena within a few seconds after PEF water sanitation, etc.) should converge to achieve application and that it eliminates arbitrariness be- the industrial implementation of PEF for the cause the measured values are averages of all inactivation of food-borne parasites. bacteria contained in the target solution. En- terobacter hormaechei (Gram-negative bacteria) suspended in buffer solution containing calcium ions were irradiated with a single PEF 2 µs, 5-20 kV/cm) and measured using an impedance ana- lyzer (PSM1735-IAI, Newtons4th) at 1 MHz, a fre- quency that is less affected by the electric double 140 layer. Saccharomyces cerevisiae were evaluated as Dielectric measurements revealed that exposure Gram-negative and Gram-positive bacteria and to PEF with field strengths of 10 kV/cm or higher yeast strains respectively. PEF-treatments (12-30 led to a significant decrease in capacitance and an kV/cm) were applied in static conditions at low fre- increase in conductance of the bacterial solution. quencies (1 Hz), resulting in a negligible temper- The decrease in capacitance suggests an increase ature increase (< 5 °C). The study compared the in the permeability of the bacterial membrane, while lethality of PEF-treatments applied in two trains of the increase in conductance indicates permeability pulses (n/2 + n/2) separated by a period of time and leakage of intracellular ionic material into the ranging from 1 minute to 24 hours with the same solution. At 12 kV/cm, slightly above the critical treatment applied in a single train (n). Microbial field strength, the microscopic membrane dielectric viability and cytoplasmic membrane permeability constant decreased once and then recovered to the were monitored over experimental time by the plate value before PEF exposure. This provides evid- count method and propidium iodide staining proto- ence of recovery of the electro-permeable mem- cols. brane. However, for E > 15 kV/cm, no recovery was When a single train of pulses was applied, all the detected. These results indicate that the cell mem- strains exhibited a rapid inactivation in the first mo-brane recovers at different speeds depending on ments but then the inactivation barely increased by the electric field intensity. Additionally, the length of augmenting the number of pules (tailing). How-the gap time after the application of PEF will be one ever, for the three microorganisms, this tendency of the important parameters to consider in industrial was mitigated when pulses were applied in two applications of PEF combinatorial sterilization. separate trains, being more evident with a longer resting time in between. Therefore, the tail of the OR-93 survival curves was a consequence of a transi- Limitations of PEF for Food Pasteurization: role ent increment of the resistance of a proportion of of membrane resealing in the microbial inactiv- the population to the external electric field. Mem- ation kinetics brane permeabilization studies indicated that, dur- Carlota Delso 1, Juan Manuel Martinez2, Ignacio ing the time between trains, a certain percentage Álvarez2, Javier Raso2 of cells progressively recovered their membrane in- 1Karlsruhe Institute of Technology, Germany tegrity. Therefore, it seems that an intact cytoplas- 2Universidad de Zaragoza, Spain mic membrane is required to be electroporated by The capacity of Pulsed Electric Fields to inactiv- PEF. This observation might be related to the form- ate vegetative forms of pathogenic microorganisms ation of reversible local defects that increases the has led to its extensive study as a non-thermal tech- conductivity of the cytoplasmic membrane and pre- nology for pasteurizing liquid food. Efforts have vents the increment of the induced transmembrane been made to define PEF processing parameters voltage (ITV) during subsequent pulses. After a to ensure food safety. However, some studies resting period of time, the reversibly electroporated revealed limitations in PEF treatments that could cells can recover their membrane integrity and be- hinder its implementation at an industrial scale. come sensitive to subsequent PEF treatments. In particular, the kinetics of PEF-inactivation often In conclusion, splitting the delivered pulse train with exhibit a tail, which may require extremely enera delay period between them may contribute to re- getic protocols for cold-pasteurization. The present ducing the energy requirements of PEF protocols study explored the responses of various microor- to achieve the required inactivation levels to guar- ganisms subjected to electric fields to comprehend antee food safety and stability. the reason behind the increment of microbial res- istant to PEF during processing. Salmonella Typhimurium, Bacillus pumillus and 141 S09 - Treatment of spinal cord spinal cord injury. injury: novel strategies and OR-95 updates from the RISEUP project Mechanisms of spinal cord regeneration Mark Anderson 1, Jordan Squair1, Alexandra de Tuesday afternoon Track C Coucy1, Matthieu Gautier1, Zhigang He2, Bernard Sep 17, 14:20 - 15:20 Schneider1, Michael Sofroniew3, Jocelyne Bloch4, Gregoire Courtine1 OR-94 1Swiss Federal Institute of Technology, Switzerland Sensorimotor contributions to human cogni- 2Harvard University, United States tion and emotion: clinical neuroscience clues 3University of California, United States for optimizing engineering approaches to func- 4University Hospital Lausanne, Switzerland tional restoration in people with spinal cord le- Understanding why axons fail to regenerate sions has been one of the longest standing problems Salvatore M. Aglioti 1, Valentina Moro2 in contemporary neuroscience. For over eighty 1Sapienza University of Rome, Italy years, it was believed that the primary obstacle en- 2University of Verona, Italy countered by regenerating axons was the forma- The massive body-brain disconnection con- tion of an astrocyte border, which many believed sequent to spinal cord injuries implies a loss of acted as a physical and chemical barrier. My re- sensory and motor bodily functions below the le- search overturned this longstanding and incorrect sion but no direct influence on higher-order brain dogma, and demonstrated that the formation of an regions. Based on this, dichotomic views of mind- astrocytic border aids, rather than prevents, axon body interactions posit that basic perceptual and regeneration (Anderson et al, Nature, 2016). Fol- motor functions are separated from higher order lowing this discovery, I went on to expose three fun- ones that in turn are exclusively based on the ma- damental biological requirements to achieve axon nipulation of abstract, amodal symbolic systems. regeneration across severe SCI: i) upregulation of Telling, however, Embodied Cognition Theories dormant developmental growth programs in neur- postulate that creating and maintaining cognition ons above the injury, ii) expression of axon growth and emotion may be shaped by somatosensory permissive molecules within the core of the injury, and motor representations. In this vein, somato- and iii) chemoattraction to guide axons to relevant sensory and motor bodily representations are not targets below the injury. This strategy, based on purely instrumental, ancillary components of men- gene therapies, propelled severed axons through tal functions but are fundamentally important for and past anatomically complete SCI in mice, but their optimal functioning. Here, we review clin- they failed to reestablish movements (Anderson et ical neuroscience studies suggesting not only that al, Nature, 2018). My team and I recently dis- space and action representations but also higher covered additional mechanisms that are required order functions like fluid mental abilities (e.g. at- for such axon regeneration to mediate the recov- tention, executive functions) and emotional reactiv- ery of neurological function: one must reestab- ity and regulation are impaired in people with SCI. lish the projections of specific neuronal subpopu- While the relevance of factors like completeness, lations to their natural target region (Squair et al, severity and level of the lesion needs to be cla-Science, 2023). When all these mechanisms are rified, the existing data hint at the crucial role of targeted with the appropriate spatial and temporal bodily functions in modulating cognition and emo- sequence, paralysis can be reversed despite the tion. Thus, taking into account the bi-rectional link complete interruption of nerve fibers in the spinal between bodily and higher-order mental functions cord. Finally, this understanding also informed ex- is of key importance for any engineering-based at- periments that exposed the efficacy of targeting in- tempt to restore functions in people affected by dividual requirements to improve neurological func- 142 tions following incomplete spinal cord damage, or electrical stimulation and its fabrication method,” when spontaneous capacities decline due to age was developed, and an international patent applic- (Skinnider, Gautier, et al, Nature, in press). ation was published (WO 2023/209676 AI). The adECM and adECM-rGO scaffolds were OR-96 shown to be safe in vivo studies, with no systemic Towards neuronal reconnection after a spinal reactions or toxicity seen after implantation. The cord injury using graphene-based nanocom- results of histological studies showed that the posites – The NeuroStimSpinal project scaffolds allowed cells to successfully invade and Paula Marques integrate into the host tissue. This supports the Universy of Aveiro, Portugal idea that they could be used for neural tissue NeuroStimSpinal (NSS) was a collaborative engineering. The research further delved into the project funded through the H2020 FET Open implantation of scaffolds in SCI models, noting the program (No. 829060). The mission of NSS was significant tissue integration and limited fibrous to contribute to the development of a treatment encapsulation, especially in adECM-rGO foams. for spinal cord injuries (SCIs). SCIs result in Moreover, the macrophage-mediated uptake of para- and tetraplegia due to the disruption of rGO suggests a favourable biodegradation profile descending motor and ascending sensory neurons for these materials. Following these results, the on a partial or complete basis. The objective was hemisection in rats at the 10th thoracic vertebrae to produce an innovative, stimulus-responsive SCI implantation without electrical stimulation scaffold capable of stimulating neural tissue repair (ES) was performed on 46 animals divided into following SCI. The scaffold was composed of different groups. The experiment combining the graphene-based materials (GBM) and decellu- scaffold with the ES device was conducted on a larized human adipose tissue (adECM) and was limited number of animals; however, it allowed us intended to be implanted at the site of a traumatic to demonstrate the proof of concept. injury. By combining the scaffold with an electrical stimulation device, it was aimed to promote neuron Acknowledgments: This work was supported outgrowth and reconnection. by the European Union’s Horizon 2020 research A series of innovative scaffolds based on adECM and innovation programme under grant agreement and GBM, showing promising applications for No 829060 (NeuroStimSpinal project). neural tissue regeneration, were developed. The OR-97 scaffolds encompassed 3D foams, nanofibrous Neuroprotective effect of Pulsed Electromag- scaffolds, hydrogels, and bioinks. The NSS project netic Fields after Acute Ischemic Stroke advanced the understanding of cellular interac- Simona Salati 1, Micol Colella2, Micaela Liberti2, Rug-tions with 3D adECM/rGO foams. The unique gero Cadossi1 biochemistry of adECM allows neural stem cells to 1IGEA, Italy adhere and grow. It is important to note that high 2Sapienza University of Rome, Italy levels of rGO directly control cell fate by turning NE-4C cells and embryonic neural progenitor Acute cerebral ischemia is characterized by cells into neurons. Furthermore, increasing rGO several pathological processes evolving during modulates primary astrocyte fate by boosting the time, which contribute to the final tissue damage. expression of reactivity markers, while unaltering Secondary processes, such as prolonged inflam- the expression of scar-forming ones. matory response, impaired mitochondrial function To explore and optimise the electrostimulation and oxidative stress, are responsible for the pro- parameters delivered to the scaffold during the gression of brain injury to the peri-infarct area, in vitro cell cultures, a new device, “A multi-well called “penumbra.” Adenosine has been shown to graphene-multielectrode array device for in vitro 3D play a crucial role in regulating the inflammatory 143 cascade following brain ischemia. OR-98 Pulsed electromagnetic fields (PEMFs) act as mod- Multi-stages pulse modulation strategy (MSPM) ulators of adenosine receptors, increasing the func- enhances electroporation-mediated intracellu- tionality of the endogenous adenosine. In particu- lar delivery by regulating the distribution and lar, PEMF exposure induces a significant upregula- accumulation of drugs tion of A2A and A3 adenosine receptors in different Xiaonan Tao, Kefu Liu neuronal cell types. Fudan University, China Several lines of evidence suggest that PEMF ex- Electroporation-mediated intracellular drug de- posure might play a neuroprotective role after livery provides an efficient and universal method in ischemic damage. biological and biomedical applications [1] such as PEMFs counteract hypoxia-induced apoptosis and cellular monitoring [2] or cell therapy [3]. Reversible ROS production in neuronal-like cells and ex-membrane permeabilization is preferred in this pro- ert a strong anti-inflammatory effect on microglial cess which is crucial for the reconstruction of cel- cells. Data from stroke animal models showed lular homeostasis and cell survival [4]. Pulse mod- that PEMFs exposure is able to reduce the size ulation matters as it plays various roles at different of the infarct area and decrease the levels of pro-stages of membrane pore formation, development, inflammatory mediators. In clinical studies, PEMFs and resealing [5]. Here, we propose a multi-stages stimulation proved to be safe and well tolerated. pulse modulation (MSPM) strategy that uses the A randomized, placebo-controlled, double-blind microsecond high-voltage pulses (HSPs) and the study aimed at evaluating whether PEMFs expos- millisecond low-voltage pulse (LLP) in a progress- ure is able to promote recovery in acute ischemic ive manner to realize effective perforation and stroke patients (NCT02767778, I-NIC study) has enhanced delivery efficiency, respectively. This been recently completed. The MRI evaluation methodology is effective for the delivery of both performed at 45 days after stroke showed PEMF small charged molecules and nucleic acids. treatment induces a significant reduction of brain For small charged molecules, MSPM lever- ischemia accompanied by a large clinical recovery ages conventional HSPs (eight pulses, 1 Hz, 100 compared to controls. µ s, 1000 V/cm) for electrochemotherapy and LLP Altogether, our data demonstrate the efficacy of (100 ms, 75 V/cm) with individually optimized PEMFs against several mechanisms underlying field strength for HeLa cells. Various time se- ischemic damage and suggest that PEMF treat- quences of pulses are tested with LLP before ment should be considered to reduce the neuronal or/and after HSPs with the same overall elec- damage occurring in the “penumbra”, offering to tric dose (LLP-HSPs, LLP-HSPs-LLP, HSPs-LLP). clinicians the opportunity to extend the time for in- Of which, HSPs-LLP achieves the best balance tervention in patients suffering from ischemic stroke between cell viability and delivery efficiency at after the narrow time window for reperfusion treat- selected parameters. To better understand this ments. process, a numerical model integrating molecule transport into traditional electroporation theoretical P11 - Electroporation modeling and framework is built by introducing the dynamic pore mechanisms conductivity to simulate the delivery process. Since pore evolution process is highly dynamic, there Tuesday afternoon Track D is a necessity to couple molecule transmembrane Sep 17, 14:20 - 15:20 transfer with the fluctuation in pore current [6]. The modified model aligns well with the experimental results, where propidium iodide (PI) is utilized as a fluorescent indicator to track the delivery pro- cess. MSPM can augment rapid entry and distri- 144 bution of PI during pulse implement and ultimately OR-99 increase intracellular accumulation by about 30% Multi-scalar microscopic molecular dynam- compared to using HSPs alone, which indicates ics, coarse-grained and macroscopic study of the involvement of diffusion and enhanced elec- voltage-gated protein interactions and complex trophoresis. Besides, we evaluate the short-term lipid pore formation during cellular electroper- and long-term disturbances of MSPM alone on cells meabilization by transcriptome analysis, flow cytometry and MTT Juan A. Gonzalez Cuevas 1, Diego Stalder1, Santi-assay, which shows tolerable influences on cellular ago Ferreyra1, Carolina Recalde1, Antoni Ivorra2, Luis activities upon parameter optimization. Further ex-M. Mir3 1 periments confirm that MSPM effectively enhances National University of Asuncion, Paraguay 2 the delivery of small charged chemotherapy drugs Universitat Pomepu Fabra, Spain 3 (bleomycin and cisplatin), with approximately 10% Centre National De La Recherche Scientifique, France of decrease in IC50 for 24 h compared to using Increasing experimental evidence shows that HSPs alone. Though at reduced bleomycin con- application of pulsed electric fields on cells dur- centration, MSPM still increases the proportion of ing electropermeabilization affects voltage-gated apoptotic cells and effectively triggers apoptosis- ion channels, produces cell membrane peroxida- related pathways within 12 hours. tion and alters the action potential. However, few Additionally, since nucleic acids-related drug studies have been conducted on these effects, with delivery has gained broad popularity in cancer ther- challenges remaining due to the wide range of tem- apy, we also investigate the effectiveness of MSPM poral and spatial scales involved in experiments in delivering siRNA. MSPM outperforms traditional and applications. In this regard, we undertake a RNAiMAX Lipofection for in-vitro siRNA delivery multi-scalar approach combining molecular dynam- with higher viability (69.89% vs 48.51%) though ics, coarse-grained simulations, continuum mod- knock-down efficiency (56.58% vs 55.10%) is sim- els and artificial intelligence to simulate and ana- ilar at further optimized parameters for MDA-MB- lyze the formation of complex pores and increased 231-Luciferase cells (fifty pulses, 10 Hz, 20 µ s, permeability of human Na+, K+ and Ca2+ ion-750 V/cm for HSPs and 100 ms, 75 V/cm for LLP). channels during electropermeabilization. We ex- Besides, we find it also shows promise for in-vivo plore the effect of different electrical pulse para- siRNA delivery with minimal side effects. meters on the electroconformational changes of in- In summary, MSPM provides a feasible solu- dividual channels, protein-protein and lipid-protein tion for safe, efficient and rapid intracellular de- interactions. The results from this work provide a livery, further improving the delivery efficiency of better understanding of pore formation and mem- charged chemotherapy drugs and reducing the brane protein function modulation by external elec- dose demand by time-scaled regulation of molecu- tric fields, with potential biomedical applications tar-lar entry, distribution and intracellular accumulation geting excitable and non-excitable cells, such as compared to using HSPs alone. It also shows signi- gene therapy, DNA vaccination, cardiac ablation, ficant potential for nucleic acid drug delivery, mak- and electrochemotherapy for cancer treatment. ing it a valuable tool in biomedical research and therapy. OR-100 Comparison of sharpness and electrical field distribution of different electrode needles for electrochemotherapy Ana Laura Campastri 1, Antonella María Cilio1, Jesica Rodríguez Miranda1, Ximena Manglano1, Sebastian D. Michinski2, Felipe H. Horacio Maglietti1 1Fundación H.A. Barceló, Argentina 2Universidad de Buenos Aires, Argentina 145 Conclusions: An electrode using triangular needles Introduction: Electrochemotherapy (ECT) is an could provide an adequate electric field distribution accepted treatment used for human and veterinary and optimal sharpness after several applications cutaneous primary and secondary tumors of varied for the treatment of cutaneous and subcutaneous histologies. Several aspects of ECT remain to be lesions with electrochemotherapy. elucidated. The aim of this work is to develop a new needle for OR-101 electrochemotherapy electrodes that resists mech- Cell electropermeabilization with subnano- anical wear providing adequate sharpness and second pulsed electric fields electric field distribution throughout the treatment. Leslie A. Vallet 1, Njomza Ibrahimi2, Laurent Ariztia2, Materials and Methods Three types of needles Marc Rivaletto2, Antoine Silvestre de Ferron2, Bucur were evaluated; conical, beveled, and triangu- M. Novac5, Alexey Zhabin2, Clair Poignard3, Anthony lar shaped. Electric field simulations were per- Ranchou-Peyruse2, Laurent Pecastaing2, Franck M. formed using COMSOL Multiphysics () to address Andre4, Luis M. Mir4 the best orientation of the different shapes in the 1Université Paris-Saclay, France conformation of a six-needle electrode. To evalu- 2Université de Pau et des pays de l’Adour, France ate the difference in the insertion force required to 3Univ. Bordeaux, France pass through the tissue with the different types of 4Centre National De La Recherche Scientifique, France needles, several insertions were performed on an 5Loughborough University, United Kingdom ex vivo tissue model. The insertion force was es- The ability of short pulsed electric fields (PEFs) timated using a scale, and three different operators to generate cell electropermeabilization has been performed several applications in the tissue. The extensively studied for decades. This knowledge average weights were calculated, as well as the has led to the development of a wide scope of maximum and minimum values. To evaluate the applications in various fields, ranging from the durability of the sharpness, the insertion force was biomedical field to the food processing industry. measured in over 300 insertions. Curves for the To optimize these applications and to develop strength required were performed on each type of new ones, it is important to investigate all the needle. The new electrodes developed were val- mechanisms involved in this phenomenon, taking idated in veterinary patients with spontaneous tu- advantage of the progress in the generation of mors. transient voltage pulses of ultrashort durations. Results: Triangular shaped electrodes required The ultrashort pulses might display distinct prop- less force for the insertion in the tissue followed erties on biological membranes and might cope by beveled and conical needles respectively. The with some technical limitations associated with electric field distribution was more homogeneous in the exposure to longer pulses. If the action of conical shaped needles, followed by triangular and µs-PEFs and ns-PEFs on the cell membranes beveled shaped electrodes respectively. Great in- have been already well characterized, studies homogeneities are seen close to the tip of all the investigating the ability of subnanosecond-PEFs tested electrodes. Conical shaped electrodes lose to electropermeabilize cells are still relatively sharpness before 50 insertions. Beveled needles scarce, mainly as a consequence of the scarcity show adequate insertion force, but great electric in- of subnanosecond pulsed power exposure sys- homogeneities in the surroundings of the tip, and tems itself [1]. The present work focuses on greater tolerance to sharpness wear, resisting al- the effects of ca. 910 ps duration PEFs on the most 150 insertions. Triangular needles provide level of electropermeabilization of E. coli DH5 α to adequate sharpness for more than 200 insertions, YO-PRO-1, a cell-impermeant DNA binding dye. with an acceptable homogeneous electric field in The influences of several parameters have been the proximity of the tip, provided the flat faces of investigated, such as the effect of the number of the needles are facing each other. pulses applied, the pulse repetition frequency, the 146 electric field amplitude or the temperature of the trol of cutaneous and subcutaneous tumor nod-sample during the exposure to the pulses. While ules. Nonetheless, ECT results in no impact on the effects of some parameters are in line with inaccessible, distant metastases, thus resulting in those observed with pulses of longer duration (for no significant effects on patients’ overall survival. example, the electropermeabilization as a function Preliminary evidences suggest that ECT can ac- of the number of pulses applied), the effects of tivate the Immune System. However, this issue other parameters surprisingly differ. This has led has never been investigated in details. Based on to the elaboration of new mechanistic hypotheses, a similar technology, we have developed a genetic consistent with the fact that events commonly vaccination platform based on the use of muscle leading to electroporation with pulses of longer DNA electrogenetransfer (DNA-EGT). Erb-eVax is duration do not have the time to occur within hun- a vaccine targeting the oncogene HER2/neu, over- dreds of picoseconds. Importantly, the electric field expressed in breast cancer and canine osteosar-amplitudes reported here to generate an efficient coma. Here, we hypothesize that standard ECT electropermeabilization of E. coli to YO-PRO-1 can induce a low, but detectable immune response are considerably lower than those typically used against Tumor Associated Antigens (TAA) and that in studies involving pulses of subnanosecond ECT and DNA-EGT can achieve synergic effects. duration [2,3]. The achievement of efficient elec- Methods: In this study, we have utilized a clinic- troporation of E. coli using subnanosecond pulses ally validated device for Veterinary electroporation of relatively low electric field amplitude is a step called Vet-ePorator, based on Cliniporator techno- forward in the perspective of developing novel logy currently utilized and approved in Europe for technologies for PEF delivery. ECT applications and adapted to EGT. Mice chal- lenged with a breast cancer cell line overexpress- [1] S. Xiao et al., Med Biol Eng Comput, 55, ing HER2 were treated with ECT, DNA-EGT or their 7, 2017, pp1063 combination. Dogs with metastatic breast cancer [2] K. H Schoenbach et al., IEEE Trans. Plasma or Osteosarcoma are currently being treated with Sci, 36, 2, 2008, pp414 ECT and/or Erb-eVax. [3] I. Semenov et al., Biochem Biophys Rep, 6, Results: ECT induced a detectable cell-mediated 2016, pp253 immune response against HER2/neu and transi- ent control of tumor growth. DNA-EGT was able to delay tumor growth over time whereas the com- S23 - Electroporation-based bination ECT/DNA-EGT resulted in complete tumor treatments in veterinary medicine rejection. ECT reduced tumor masses and Erb- eVax induced cell mediated and antibody immune Tuesday late afternoon Track responses against HER2/neu in treated dogs. A Conclusions: Our findings provide new data on tu- Sep 17, 16:50 - 18:20 mor immunobiology and most importantly improve the efficacy of ECT and may open up novel immun- OR-102 otherapeutic approaches, thus having a significant Electro-Chemo-Immuno Therapy: activating impact on patient’s survival and quality of life. local and systemic immunity Joseph A. Impellizeri1, Antonella Conforti2, Erika OR-103 Salvatori3, Luicia Lione3, Luigi Aurisicchio 3 Electrochemotherapy for bilateral limbal 1Veterinary Oncology Services, United States squamous cell carcinoma in a horse 2Evvivax, Italy Majbritt M. E. Larsen 3Takis, Italy Evidensia Specialist Animal Hospital in Helsingborg, Introduction: Electrochemotherapy (ECT) is an Sweden approved and efficacious treatment for local con- 147 Objective: To describe the procedure and out-and systemic response to treatment. Therefore, it come of electrochemotherapy (ECT) with bleomy- is important to identify other types of biomarkers cin as a first-line treatment for bilateral squamous that could serve as predictive factors for treatment cell carcinoma in the eye of a horse. success in order to contribute to the increasing re- Animal Studied: A client-owned 5-year old cognition of ECT for tumour treatment in veterinary Haflinger gelding with limbal-conjunctival squam- oncology. ous cell carcinoma. Previous studies have shown that the expected ob- Procedures: During general and local anaesthesia, jective response (OR) depends on the type of tu- injection of bleomycin in the ocular tumour was fol- mour: epithelial tumours (perianal adenomas and lowed by electroporation, applied with a 15 mm adenocarcinomas) respond in 100%, round cell tu- needle electrode perpendicular to the ocular sur- mours (MCT) in 85%, mesenchymal tumours (STS) face. in 75% and oral tumours (oral melanoma (OM), oral Results: Treatment with ECT resulted in thera- SCC) in 40%. The OR in ECT as adjuvant therapy peutic electric pulses, and complete tumour re- for unclean margins can be expected to be 90% for sponse with no recurrence during follow up. Tu- both STS and MCT. The OR also decreases with mour toxicity as a result of treatment was mild, with advancing clinical stage. no adverse effect to normal tissue. In MCT, the combined treatment of ECT and gene Conclusions: In this case of bilateral ocular tu- therapy with cytokine interleukin 12 (IL-12 GET) mours, staged ECT with bleomycin was shown to resulted in better efficacy due to systemic support be a safe and effective treatment with complete tu- of the antitumor immune response. Similar to stud- mour remission. The result suggests ECT as a ies using only ECT, the response was worse in tu- competitive treatment in both human and veterinary mours > 2 cm3. In addition, the poorer response ocular tumours, with further research recommen- is also associated with a lower proliferation index ded. (Ki67) and increased PD-1 and PDL-1 expression in tumour tissue prior to treatment. In contrast, the OR-104 response was better in tumours with a higher infilt- Predictive factors in electrochemotherapy with ration of CD3+ cells. Serum/plasma levels of IL-12 or without IL-12 gene electrotransfer in dogs and IFN γ, ferritin and nucleosomes were also not and cats related to clinical response. In contrast, we have Nataša Tozon 1, Urša Lampreht Tratar2, Nina shown that elevated LDH levels may be helpful in Milevoj1, Masa Vilfan1, Gregor Serša2, Maja Čemažar2 predicting treatment response. 1University of Ljubljana, Slovenia The results of our large study on MCT in dogs sug- 2Institute of Oncology Ljubljana, Slovenia gest that the combination of ECT and IL-12 GET is Electrochemotherapy (ECT) is a relatively new successful, but additional biomarkers are needed method of tumour treatment in both human and for a more accurate disease prognosis. veterinary oncology. In veterinary oncology, ECT OR-105 is used to treat various histological tumour types, Comparison of intratumoral or peritumoral IL- most commonly perianal tumours, soft tissue sar- 12 gene electrotransfer in combination with comas (STS) and mast cell tumours (MCT) in dogs electrochemotherapy for the treatment of spon- and squamous cell carcinomas (SCC) in cats. Al- taneous mast cell tumors in dogs though the success of therapy varies depending on Urša Lampreht Tratar 1, Nina Milevoj2, Maja the type of tumour, considerable differences can Čemažar1, Katarina Žnidar1, Katja Ursic Valentinuzzi1, be observed even within the same histological tu-Andreja Brozic1, Katerina Tomsic2, Gregor Serša1, mour types. A good knowledge of biomarkers, such Nataša Tozon2 as histologic tumour type, is an indispensable tool 1Institute of Oncology Ljubljana, Slovenia for tailoring treatment regimens and predicting local 2University of Ljubljana, Slovenia 148 OR-106 The use of electrochemotherapy (ECT) and A Veterinary Electrotransfer System that em- interleukin-12 (IL-12) gene electrotransfer (GET) ploys Heat and Impedance – Progress Toward for the treatment of various histological types of Commercialization spontaneous tumors in dogs has been described Mark J. Jaroszeski 1, Alex Otten1, Gary Strange2, in several clinical studies. Although the results of Richard Heller1 these studies show that the treatment is successful 1University of South Florida, United States and safe, the routes of administration of IL-12 GET 2LifePulse Bioscience, United States have been either intratumoral (i.t.) or peritumoral (peri.t.). Therefore, the aim of this study was to Pulsed electric fields have broad potential for compare the two routes of administration of IL-12 molecular delivery applications for applications GET in combination with ECT and their contribution such as gene therapy, protein therapies, vac- to the enhanced response to ECT. Seventy-seven cination, and chemotherapy. Countless studies dogs diagnosed with spontaneous mast cell tumors have examined different electrode configurations (MCT) were divided into three groups: one group as well as wide ranges of electrical parameters was treated with a combination of ECT + GET peri.t. such as pulse width, voltage applied, number of (29 dogs), the second with the combination of ECT pulses, and period all generally focused on maxim- + GET i.t. (30 dogs) and the third with ECT alone izing/optimizing delivery or tissue response. More (18 dogs). In addition, immunohistochemical ana- recently, localized moderate heating (to ˜43°C) lysis of tumor samples before treatment and flow during the delivery of plasmid DNA using pulses cytometric analysis of peripheral blood mononuc- electric fields has been shown to increase expres- lear cells (PBMCs) before and after treatment were sion of a delivered foreign gene. Furthermore, tis- performed to determine any immunological effects sue impedance measured during electrical treat- of treatment. The results showed significantly im- ment has been used in a feedback manner to ad- proved local tumor control in the ECT + GET i.t. just electrical treatment in real-time to customize group (p < 0.050) compared to the ECT + GET the electroporation process during delivery. Based peri.t. and ECT alone groups. In addition, a re- upon lab prototype devices and results in small an- markable prolongation of the disease-free interval imal models, it was clear that there could be many (DFI) and progression-free survival (PFS) was ob- applications in human and veterinary clinics. As a served in the ECT + GET i.t. group compared to first step, a commercial device for providing electrothe other two groups (p < 0.050). The results in chemotherapy to veterinary tumors was developed, terms of local tumor response, disease-free inter- designed, and produced in conjunction with Life- val (DFI) and progression-free survival (PFS) were Pulse™, Inc. The device consisted of a handle consistent with the immunological assessments, as that had a replaceable array of needle electrodes. evidenced by the increased presence of anti-tumor After placement of the array into a tumor, the tu- immune cells in the circulation after treatment in the mor was heated using warmed air. Temperature ECT + GET i.t. group. This observation suggests was maintained using a FLIR camera that was also that a systemic immune response was induced. contained in the handle. Pulses were applied us- Moreover, no adverse severe side effects were ob- ing a custom pulse generator, and real-time imped- served. Finally, due to the more pronounced local ance measurements were used to guide pulsation. response after ECT + GET i.t., we suggest that The entire system was automatic and controlled us- the treatment response assessment should be per- ing a graphical user interface and software on a formed at least two months after treatment, which laptop. Ten of these first commercial devices have is in line with the iRECIST criteria. been circulated among about 20 veterinary onco- logy practices across the United States during the past two years. A multitude of different tumor types have been treated mostly in dogs and horses. De- 149 tails of the system operation and features will be characterize the model at different time points of provided along with features of the final commer- development. To perform the impedance measure- cial designs. 4 case studies will also be presented. ments with the developed system, tumors with a These will be from the treatment of an equine sarc- diameter between 0.5 and 2 cm were considered, oid, equine melanoma, canine soft-tissue sarcoma, so the time points studied were: 14, 17, and 21 and canine nasal squamous cell carcinoma. days. It is remarkable that, this last time point already presented a significant internal volume of OR-107 necrosis. The implementation procedure consisted Electrical characterization of VX2 tumor in rab- of a laparotomy implanting 1 mm3 portions of tumor bit model for electroporation purposes in a small incision in a left hepatic lobe. The meas- Borja López-Alonso 1, Jorge Sánchez1, Pablo Briz1, urements were taken in vivo and 10 minutes after Eva Monleón1, José Aramayona1, María Dolores the sacrifice of the animals. Arribas2, Héctor Sarnago1, José M. Burdío1, Óscar In conclusion, an impedance measurement sys- Lucía1, Antonio Güemes2 tem has been developed to carry out an electrical 1Universidad de Zaragoza, Spain characterization of a very interesting tumor model 2Hospital Clínico Universitario Lozano Blesa, Spain for the study of electroporation. The data have Within the diverse fields of application of elec- been statistically analyzed to obtain representative troporation, oncological treatments have a great in- conclusions and models. The final version of this terest. Although several treatments such as tumor paper will include experimental results and future ablation by irreversible electroporation or electro- insights demonstrating the feasibility of this pro- chemotherapy do already have clinical application, posal. there is still a need to improve the safety and control of these therapies. To carry out this research it is S07 - Potential applications of necessary to develop tools such as simulation and PEFs technology in vegetable and experimental models, which are complex due to the great variability of biological systems. Specifically, fruit processing within this branch it is necessary to study and model biological tissues in order to adequately estimate Tuesday late afternoon Track the distribution of the electric field, the feasibility B and efficiency of the treatments, and the possibil- Sep 17, 16:50 - 18:20 ity of thermal side effects. OR-108 The aim of this work is to present the electrical How does PEF impact membrane integrity and characterization of an animal tumor model widely the volatile profile of leek? used in this field. The rabbit VX2 is a large an- Lize Lanssens, Sophie Delbaere, Ann Van Loey imal model of cancer used for studying and devel- KU Leuven, Belgium oping oncological treatments and consists of one implantation in the hepatic lobe of a rabbit of VX2 In the past few decades, pulsed electric fields carcinoma, that is an anaplastic squamous cell car- (PEF) technology emerged as an innovative non- cinoma derived from a virus-induced papilloma in thermal processing technique for several types rabbits. For this purpose, an impedance meas- of food. At low electric field strengths (< 4 urement system has been designed to carry out in kV/cm), PEF has the potential to induce irrevers- vivo and ex vivo measurements in real time. With ible cell membrane permeabilization in plant-based this system and the acquired measurements, FEA matrices, which is currently mainly been investig- models have been developed to accurately estim- ated in the context of enhanced drying and en- ate the electrical properties of the tissue. hancement of extraction and cutting effectiveness. The experimental process consisted of the im- However, PEF at low electrical field strength can plantation of 9 animals divided into 3 groups to also impact enzyme-substrate interactions as a 150 result of membrane disintegration (i.e., electropor-OR-109 ation), thereby imparting their volatile profile, es- Practical application using Pulse Electric Field pecially in matrices where enzyme and its corres- (PEF) approach in milking the roots from aero- ponding substrate are present in different cells or ponic system different cell compartments, physically separated Sylwester Ślusarczyk, Kajetan Grzelka, Joanna by membranes. There are only limited studies that Jaśpińska, Adam Matkowski focus on the potential of PEF in this context. Due to Wroclaw Medical University, Poland the inhomogeneous microstructural characteristics The aim of this work was to develop and of many vegetables (e.g., different cell types and optimize PEF treatment for the reversible electro- cell sizes), it is hypothesized that the spatial ori- poration from the roots and rhizome of 3 model entation of the product in the treatment chamber plants Iris domestica (syn. Belamcanda chinensis might influence the PEF-induced effect. The latter L. DC. Iridaceae) Scutellaria baicalensis L and aspect has not been fully elucidated, but is interest-Cicerbita alpina (L.), all cultivated in aeroponic ing to consider as quality characteristics of similar system. Electroporation can be successfully used food products could be affected to different extents in extraction of compounds from plant tissue what if orientation in the treatment chamber is modified. is very well known.[ 1,2] but is it possible such In this research, PEF treatments at low electric extraction for living plants? field strengths were applied to leeks (Allium am- In our work we expand on these findings and peloprasum var. porrum), a vegetable that is further optimize the treatment parameters. 3 characterized by prominent compartmentalized en- months old aeroponic cultivar of model plants were zymes and substrates which can determine the electroporated using different pulsed electric field volatile profile of the vegetable. As a first re- strength from 0,2 to 7.5 kV/cm with constant 50 or search question, the effect of varying the total spe- 100 µ s duration of pulse and repetition N 50, 100 cific energy input of PEF treatments was investig- and 200 pulses. We successful milking the roots ( ated on the membrane integrity and volatile profile. extracted) of secondary metabolites after PEF in A second research question aimed to investigate different solvents system. We developed special the effect of the spatial orientation of leeks dur- chamber for this purpose. Treated plants survived ing PEF treatment, relative to the applied electric this process and it can be regularly repeated on field, on both the vegetable’s membrane integrity the same individual plants as a continuation of and volatile profile. To assess the membrane integ- milking the roots. The most promising variant was rity, measurements of tissue and medium conduct- when we applied: E = 0.5 kV/cm N = 100 f = 1 Hz ivity were performed. The volatile profile was ana- t = 100 us (A) and E = 0.5 kV/cm N = 200 f = 10 lyzed using an untargeted headspace-solid phase Hz t = 50 us (B) For extraction following solvents microextraction-gas chromatography-mass spec- were used: choline chloride : glucose (1:2) + 30% trometry (HS-SPME-GC-MS) approach. This work water; choline chloride : ethylene glycol (1:2); showed that PEF at low field strengths is able to choline chloride : fructose (1:2) + 30% water; induce membrane disintegration in leeks. A sub- choline chloride : saccharose (1:2) + 40% water stantial effect of applying different total specific en-and tap water. After PEF plants were cultivated in ergy inputs between 1.50 and 22.50 kJ/kg at a con- aeroponic systems and observed throughout the stant low electric field strength of 1.01 kV/cm on the next 4 months, compare to control. For HPLC-MS resulting cell membrane disintegration and volatile analysis all collected sample (directly after PEF profiles of leeks was observed. Furthermore, this and after week) were first purified on SPE system study demonstrated that the spatial orientation of to 10 mg/ml concentration. Main disadvantage of leeks during PEF treatment has an influential role this approach is frequent infection of aeroponic in inducing membrane electroporation and shaping cultivars with typical pests and frequent fungal and the volatile profile. bacterial overfeeding, which must be combated by 151 biological protection and keeping sterility regime. followed by aqueous extraction removed 69.11% of the ash from the Gracilaria sp. biomass. The Acknowledgements: The research was funded in in vitro digestibility of the Gracilaria sp. proteins in whole of the National Science Center POLAND the water-soluble extract from the PEF process was basis of the decision number National Science 70% compared to that of casein. These findings Centre Poland; UMO-2020/39/D/NZ9/01402 suggest that employing a continuous PEF process could position marine macroalgae as an important 1. Mahnič-Kalamiza, S., Vorobiev, E. & Mik- potential source for novel proteins. lavčič, D. Electroporation in Food Processing and Biorefinery. J. Membr. Biol. 247, 1279–1304 OR-111 (2014). Optimizing valuable compound recovery from 2. Liu, Z. W., Zeng, X. A. & Ngadi, M. Enhanced food side streams and microbial Biosynthesis extraction of phenolic compounds from onion through PEF-Induced Extraction and Stress by pulsed electric field (PEF). J. Food Process. Strategies Preserv. 42, 4–11 (2018). Robert Sevenich Leibniz-Institut für Agrartechnik und Bioökonomie e.V. (ATB), Germany Aim: In the last years several studies have OR-110 demonstrated the feasibility of pulsed electric fields Seaweed processing with pulsed electric fields: (PEF) as a good pre-treatment technology able from batch to continuous process development to enhance the bioactive compounds extraction. for functional ingredients production Within the EU founded project SHEALTHY it was Alexander Golberg investigated to what extend the extraction yield of Tel Aviv University, Israel essential oils as well as polyphenols could be in- Marine macroalgae Ulva sp. and Gracilaria sp. creased with PEF in the range of 1-5 kJ/kg in com- can reach a high protein content and a high com- parison to an untreated sample. position of essential amino acids. We have de- Methods: Based on the literature, higher electric veloped a novel device and investigated the para- field strength are necessary to extract valuable meters for a continuous Pulsed Electric Field (PEF) compounds from side streams, especially from or- process, coupled with enzyme treatment, to facilit- ange peels. The HVP 5 PEF (30 kV) systems with ate the aqueous fractionation of those seaweeds. a 2 cm treatment cell was used to aplly an electric The process demonstrated 8.79±0.58 (% w/w) Ulva field strength up to 10 kV/cm. The experimental protein extraction yield. Furthermore, the aqueous parameter setup for orange peels was based on fractionation of Ulva protein exhibited a substan- literature, where different electric field strength for tial 41.45% essential amino acid content and a the extraction of polyphenols from different side branched amino acids composition of 17.58%. Ad- streams were applied. For the PEF-treatment of ditionally, it displayed an in vitro relative digestibility olive leaves, parameters from a study conducted of 87.4±1.36% compared to soy protein, along with by UGR were used. The knowledge of the possible a water holding capacity of 7.15 ± 0.17 g water/g effective process conditions was translated into a sample and an oil holding capacity of 1.76 ± 0.11 g Design of Experiments (DoE). After the treatment oil/g sample. For Gracilaria sp., the water extracts the samples were dried and send to the analytic from the PEF-treated biomass showed higher pro- partner for analyses. tein yield (37.82 ± 7.96 to 47.56 ± 10.54 mg/g Results: For orange peels the electric field strength initial dry weight (DW) of biomass, depending on was between 1.0 – 2 kV/cm. Based on the DoE the PEF conditions) compared to extracts without PEF optimal treatment conditions for the increase of ex- (33.82 ± 6.01 mg/g initial DW of biomass). PEF traction in comparison to the control 50 % (Flavon- 152 oids), 26 % (Polyphenols), 32 %(Hesperidin), 68 % the pH at a near constant value; similarly pure wa- (Narirutin) was 1.5 kV/cm, 30 pulses and a pulse ter can be used to reduce volatility in the transient width of 30 us. In comparison to ultrasound optimal temperature during PEF excitation. This method conditions for TPC 22 % more was extracted. The can be used to treat any solid food product placed optimal treatment conditions for olive leaves were between electrodes surrounded by pure water. 0.8 kV/cm, 110 Hz, 11 s of treatment time and a The pH, sugar content, total solids, colour, total pulse width of 15 us. This lead to an increase of phenolic content, anthocyanin content, and DPPH TPC by 9% and of DPPH by 22 % in comparison antioxidant activity were investigated after pulsed to the control. Ultrasound and PEF extraction had electric stimuli applied to blueberries. No sig- similar results. nificant differences between PEF and non-PEF Conclusion: PEF extraction shows high potential samples were identified for pH, sugar content, nor for the extraction of valuable compounds from side total solids. The colour of the ‘pure water’ used for streams in comparison to untreated as well as other PEF treatment changed significantly as the specific green extraction technologies like ultrasound. The energy input increased. validation of the optimal treatment conditions as The phenolic content of both the blueberry juice well as the scale up are ongoing. and skin was significantly different to the control (no PEF). The anthocyanin content in the blueberry OR-112 skin was found to be significantly different to the Increasing the yield of juice and bioactive control. The DPPH antioxidant activity was statist- compounds extracted from blueberries using ically significant in the blueberry skin, however not pulsed electric field in the extracted juice. Shao-Keng Tai, Farzan Zare, Joseph Nastasi, Nidhi PEF through the electroporation and electroper-Bansal meabilization processes have shown to signific- The University of Queensland, Australia antly facilitate and support the extraction process Pulsed electric fields (PEF) is a widely used from blueberries. The method used in this work can processing technique in the food and beverage in- be further used in future work targeting any solid dustries for extraction, safety and/or quality of li- foods. quids, semi-solid, and solid foods. One of the main disadvantages of PEFs is that the generat- S09 - Treatment of spinal cord ors used to create them are dependent voltage injury: novel strategies and sources. The output pulse characteristics such as updates from the RISEUP project the voltage and pulse width are highly dependent on the sample’s electrical properties. In this work Tuesday late afternoon Track we have developed a method to treat high-value C solid food products without voltage dependence on Sep 17, 16:50 - 18:20 the sample. Blueberries were chosen as a tar- get high-value food product to show the proof-of- OR-113 concept which can be applied to any other solid Boosting the development of Electro Pulsed food. Bio-hybrid implantable devices through ad- The method uses pure water to set the electrical im- vanced modelling in vitro and in vivo pedance of the sample which is not influenced by Micol Colella 1, Francesca Apollonio1, Marco the target food product (in this case blueberries). Balucani2, Laura L. Caramazza1, Noemi Dolciotti1, Pure water has a large impedance (low conductiv- Sara S. Fontana1, Paolo Marracino2, Alessandra Paffi1, ity), whereas the blueberry itself cannot influence Micaela Liberti1 the electrical impedance until the PEF excitation 1Universita Degli Studi Di Roma La Sapienza, Italy has begun. In biochemistry buffers are used to fix 2Rise technology S.R.L., Italy 153 Spinal cord injury (SCI) is one of the most approach is applied [3], by manipulating licensed debilitating conditions existing [1]. Researchers detailed rat computational phantoms to host the are working worldwide on innovative treatments model of a SC lesion obtained from microscopy to restore the lost functions [2]. Particularly, the images. The EPB model is placed over the lesion, project “Regeneration of injured spinal cord by as done during rat surgery: dosimetric results electro pulsed bio-hybrid approach” (RISEUP, FET assess whether the optimal stimulation conditions Open of Horizon Europe Program 2020) aims at can be achieved in the lesion environment, and developing an innovative Electro Pulsed Bio-hybrid allow the design of additional stimulation devices (EPB) device for spinal cord regeneration based on when needed. stem cells transplantation and electric stimulation via µs-Pulsed Electric Fields (µsPEF) and direct References: current (DC) stimulation. [1] Barbiellini Amidei, et al., Spinal Cord 60, 812– In this context 3D advanced computational mod- 819 (2022) elling represents a fundamental step to potentiate [2] Hu, et al., Sig Transduct Target Ther 8, 245 the engineering work of design and production (2023) of the device able to generate field necessary to [3] Colella, et al., Sci Rep 10, 2945 (2020). induce the neuroregenerative processes on cells. Herein, we present the workflow to attain the 3D OR-114 realistic model of the EPB working in its intended Materials solutions for an electrostimulable environments of applications, i.e., in vitro and device for use in spinal cord injury model in rat in vivo. The computational approach to this full Fernando Gisbert Roca, Sergiy Ivashchenko, Francisco Navarro Páez, Cristina Martinez, Jorge Más Es- characterization of the EPB starts from modelling tellés, Manuel Monleon the initial device and subsequently adding details Univeristat Politècnica de València, Spain that increase complexity and level of realistic representation. The use of natural and synthetic biomaterials As a first step, bare electrodes are simulated forming hydrogels or scaffolds has served as a suc- to define and optimize their configuration, so cessful interface for physical, biological, and chem- obtaining the electrified part of the EPB. Secondly, ical support to endogenous and transplanted cells. a reliable and fast reconstruction procedure to This is a very promising field with great potential obtain realistic 3D models of the microfibril lanes to overcome the low efficacy of cell transplantation is carried out, starting from microscopy images, so in spinal cord injury (SCI). In this work, an Electro-reproducing the bio-hybrid scaffold, in tight contact Pulsed Biohybrid (EPB) device is developed as part with the electrodes. of the FET-OPEN project “Regeneration of injured Once that the EPB is numerically characterized, its spinal cord by electro pulsed bio-hybrid approach” behavior during in vitro experiments must be repro- (RISEUP, n.964562). Due to the characteristics of duced. An additional degree of detail is added, by the implant for SCI in rat model, the device had to simulating the presence of the stem cells over the meet a series of specific properties. Biocompatible microfibril lanes, with highly realistic cells models, and biodegradable materials (polylactic acid (PLA) obtained from high-resolution confocal microscopy and hyaluronic acid (HA)) were selected with the images: 3D-microdosimetry simulations allow to aim of achieving a slow degradation rate that would identify conditions able to guarantee threshold provide integrity to the device throughout the entire levels needed to attain the desired effect. Finally, regeneration process of the SCI. The device had these optimal conditions identified in vitro must to be able to deliver cells to the injured area using be ensured in the in vivo environment, where the smallest possible volume of material. There- the concept of neuronal regeneration should be fore, the scaffold had to have a high specific surface proved. Hence, an accurate 3D model of a spinal area and, at the same time, present a high flex- cord (SC) injured rat is needed. The semi-specific 154 ibility. This was achieved by using polylactic acid in the context of regenerative therapies in last microfibers of 10 microns in diameter arranged in decades. In another respect, calcium (Ca2+) is a monolayer, forming a microfiber lane. This as- an important second messenger in cells. Ca2+ sembly had to be subjected to electrical stimula- signaling often occurs in the form of oscillatory tion. For this purpose, a PLA membrane was de- patterns, referred to as Ca2+ oscillations, whose signed to place the electrodes. Since the conduct- frequency and/or amplitude embeds information ivity levels required for the electrodes were very that is subsequently decoded by effector proteins high, it was necessary to use metal electrodes in- whose activity is Ca2+-sensitive 5. Spontaneous stead of electroconductive polymers, and gold elec- Ca2+ oscillations have been observed in MSCs, trode tracks were incorporated on the PLA support- the frequency of which varies over the course of ive membrane. Tissue adhesions after surgery can differentiation or proliferation events. The question compromise the effectiveness of cell transplanta-subsequently arises to wonder whether taking tion. In order to minimize this danger, a hydro- the control over Ca2+ oscillations could be used gel coating of HA was produced on the face of the as a mean to influence the cell fate. As a first device opposite to that of the electrodes. HA is step, we focused on assessing the changes in a natural hydrogel that prevents these processes. Ca2+ oscillation patterns along proliferation or With all these premises, a device was designed differentiation events by developing time-lapse and manufactured, consisting in an electrified sup- microscopy with fluorescent Ca2+ probes and Fast portive membrane (PLA) with a monolayer of mi- Fourier Transform analysis, allowing the analysis crofibers (PLA) on one face and a hydrogel coat- of Ca2+ oscillations in hundreds of cells in a single ing (HA) on the other. Two adapted designs of the experiment. In a second time, we took the control device were developed taking account of the cir- of Ca2+ oscillations using microsecond pulsed cumstances of in vitro experiments and of in vivo electric fields generating a slight permeabilization surgery in a rat SCI model. of the cell membrane to Ca2+. This limited and controlled permeabilization to Ca2+ subsequently OR-115 triggers Ca2+ oscillations similar to natural ones Electromanipulation of calcium oscillations in (in shape, amplitude and duration) due to the Mesenchymal Stem Cells, a control of cell fate? Ca2+-induced-Ca2+-release response 6. The Leslie A. Vallet 1, Marina Sanchez Petidier2, Romain evolution of Ca2+ oscillation patterns in prolif-Samiaa3, Nataliia Naumova1, Claudia Consales4, Gior- eration and in various differentiations led to the gia Innamorati4, Caterina Merla4, Franck M. Andre3, Luis hypothesis of a link between Ca2+ oscillations and M. Mir3 cell cycle progression in MSCs. In this context, 1Université Paris-Saclay, France the effect of the frequency of Ca2+ oscillations 2Fundación Hospital Nacional de Parapléjicos, Spain on specific signaling pathways associated with 3Centre National De La Recherche Scientifique, France the cell cycle progression and the evolution of the 4ENEA, Italy Ca2+ oscillation patterns within a cell cycle are Mesenchymal stem cells (MSCs) are adult currently under investigation. This work carries multipotent stem cells which can differentiate both fundamental and applicated aspects: to into various connective tissue cell types such as assess the role and the importance of the Ca2+ osteoblasts, adipocytes… 1 In addition, under oscillations in various cellular processes in MSCs, specific conditions, their differentiation abilities and to develop a suitable device to control them can extend to other specialized cell types, such on the long-term, in the perspective of therapeutic as muscle or neuron-like cells 2. Not only due to applications. this promising multipotency, but also to interesting secretory activities 3 as well as rescue functions 1 M.Dominici et al., Cytotherapy, 8, 2006, pp315 towards damaged cells in their environment 4, 2 A.Halim et al., Stem Cell Rev and Rep, 16, 2020, these cells have attracted increasing interest pp661 155 3 P.Kumar, et al., Cytokine Growth Factor Rev, 46, of SCI based on the transplantation and the mi-2019, pp1 crosecond electric pulses (µsPEFs) stimulation of 4 S.Paliwal, et al., J Biomed Sci, 25, 2018, 31 mesenchymal (MSCs) and induced neuronal (iN- 5 E.Smedler, and P. Uhlén, Biochim Biophys Acta, SCs) stem cells, through a biocompatible, biode- 1840, 2014, pp964 gradable, and electrified support. 6 H.Hanna, et al., Stem Cell Res Ther, 2017, 8, 91 The hypothesis is to modulate through µsPEFs the intracellular calcium fluxes resulting in the con-OR-116 trol of proliferation and differentiation of these cells; Effects of microsecond electrical pulses on the iNSCs could differentiate in mature neurons to re- inflammatory response generate the lesioned area, and MSCs in neuronal- Giorgia G. Innamorati 2, Francesca Camera2, like cells to release neurotrophic factors. Fernando Gisbert1, Sergiy Ivashchenko1, Romain To better understand the effect of this device on Samiaa3, Sara S. Fontana4, Noemi Dolciotti4, Micol the pro-inflammatory environment of SCI, the two Colella4, Alessandro Zambotti2, Caterina Merla2, Franck µsPEFs protocols (proliferation and differentiation), M. Andre3, Victoria Moreno5, Paolo Marracino6, Claudia identified in the context of the project, have been Consales2 1 tested on a human cell line of monocytes (THP-1), Universitat Politècnica de València, Spain 2 macrophages (obtained by treating monocytes with ENEA, Italy 3 PMA) and microglia (HMC3). Centre National De La Recherche Scientifique, France 4 Here we present the first results obtained by ap- Universita Degli Studi Di Roma La Sapienza, Italy 5 plying these stimulation protocols to the cells men- Principe Felipe Research Centre, Spain 6 tioned above in terms of proliferation, gene expres- Rise technology S.R.L., Italy sion, and inflammatory response. Spinal cord injury (SCI) is a neurological and The purpose of these analyses is to assess the pathological state caused by direct damage of the effect of the final device on the pro-inflammatory spinal cord or of the tissue and vertebrae sur- environment of SCI, to avoid any side effects, rounding it, characterized by permanent or tempor- hoping for the induction of a beneficial anti- ary changes in the motor, sensory, and autonomic inflammatory effect. function. Two different phases characterize SCI. The OR-117 primary injury occurs immediately after the trau- Emerging Approaches to Neural Tissue Regen- matic insult which damages or destroys the tissue eration: Electrical Stimulation of Stem Cells and is due to the hemostatic response and acute Marina Sanchez Petidier1, Romain Samiaa 2, Leslie cell death. This first phase is followed by the proA. Vallet3, Giorgia Innamorati4, Claudia Consales4, Ca-gressive secondary injury cascade, characterized terina Merla4, Victoria Moreno5, Fernando Gisbert6, Ser-by ischemia, proapoptotic signaling, and peripheral giy Ivashchenko6, Manuel Monleon6, Micaela Liberti7, Franck M. Andre2, Luis M. Mir2 inflammatory cell infiltration. Over the subsequent 1Fundación Hospital Nacional de Parapléjicos, Spain hours, the strong inflammatory response, mediated 2Centre National De La Recherche Scientifique, France by cell activation and release of proinflammatory 3Université Paris-Saclay, France cytokines, makes the SCI environment hostile to 4ENEA, Italy any kind of therapeutic treatment. Indeed, so far, 5Principe Felipe Research Centre, Spain several therapeutic strategies for SCI have been 6Universitat Politècnica de València, Spain studied, but an effective protocol for spinal cord re-7Sapienza University of Rome, Italy generation is still missing. RISEUP project, funded by the European com- Spinal cord injury (SCI) treatment remains a munity in the H2020 FET-OPEN program (grant significant challenge due to inability to regenerate agreement N° 964562), provides for the develop- lost neurons and restore functional connections ment of an innovative system for the regeneration after an SCI. The complex cascade of events that 156 follow an SCI, including a non-permissive envir-lation, which could promote cellular proliferation or onment and limited capacity for axonal regrowth, differentiation events. often leads to a degenerative chronic stage In conclusion, this work aims at defining the elec- [1]. This complexity necessitates multifaceted trical stimulation protocols that are able to induce strategies for functional restoration, including cell specific changes in the Ca2+ oscillations of MSC substitution, neuroprotection and axonal growth which may promote proliferation or neural differ- promotion. entiation. This could potentially lead to significant Mesenchymal Stem Cells (MSC) have the potential advancements in the treatment of SCI, paving the to generate multiple differentiated progenies and way for more effective recovery strategies. provide a suitable microenvironment for tissue regeneration [1]. MSC are present in several References tissues of the human body, making them a readily [1]Thuret S et al. doi:10.1038/nrn1955 available source of stem cells for therapeutic use. [2]Carafoli, E. et al. doi:10.1074/jbc.R116.735894 Also, it is reported that MSC transplantation into [3]Berridge, M.J. et al. doi:10.1038/nrm1155 SCI provides immunoregulatory effects. However, [4]Kawano S. et al. the transplantation process is challenged by a doi:10.1016/S0143416002001240 number of issues such as a low survival rate and [5]Hanna H. et al. doi:10.1186/s13287-017-0536-z the lack of neural differentiation and maturation. Therefore, in order to improve cell-based ther- OR-118 apies it is important to develop complementary Regeneration of Injured Spinal Cord by apply- treatments for example electrical stimulation par- ing subdural electro pulsed stimulation and ticularly if it may avoid using exogenous chemical stem cell bio-hybrid approach compounds. Loris Mannino 1, Eric Lopez1, Maria Pedraza1, Paolo Marracino2, Marco Balucani2, Marina M. Sanchez3, Our work explores a new combinatory therapy Franck M. Andre3, Luis M. Mir3, Romain Samiaa3, for SCI treatment that employs high voltage mi- Manuel Monleon4, Cristina Martinez4, Andres Alba4, crosecond electric pulses ( µ sPEF) stimulations Sara S. Fontana5, Micol Colella5, Micaela Liberti5, and low amplitude direct currents in conjunction Francesca Apollonio5, Caterina Merla6, Giorgia G. with stem cell transplantation on a biocompatible Innamorati6, Victoria Moreno1 device. The purpose of using µ sPEF stimulation is 1Principe Felipe Research Centre (CIPF), Spain to generate cell electropermeabilisation to calcium 2Rise technology S.R.L., Italy (Ca2+), a simple molecule which performs a 3Centre National De La Recherche Scientifique, France multitude of functions within cells [2]. It serves as 4Universitat Politécnica de València, Spain universal second messenger in many biological 5Universita Degli Studi Di Roma La Sapienza, Italy process [3]. Minor changes in Ca2+ concentration, 6ENEA, Italy known as Ca2+ oscillations, are crucial for cellular differentiation and function. Thus, the control of Spinal cord injury (SCI) is a dramatic event Ca2+ oscillations could potentially optimize the that leads to an irreversible disabling condition process of cell transplantation. with loss of motor and sensory functions (1). The This objective is thus the development of electrical main symptoms are the temporary or permanent protocols that can induce differentiation or prolif- loss of sensations, muscle strength, bowel and eration. We started using a 2D model and we are bladder function. SCI condition affects around now performing experiments in a more realistic 3D 300.000 people each year with different incid- system. Indeed, MSC exhibit spontaneous Ca2+ ences in the different countries and the severity oscillations, which vary if MSC are in differentiation of the SCI mainly depends on two aspects: i) or proliferation processes [4]. This study is the the anatomical structure involved and ii) the site continuation of our previous work [5], exploring the of the lesion (closer the trauma is to the cer- changes that might be triggered by µ sPEF stimu-vical vertebrae and greater will be the area af- 157 fected by the consequences of the trauma), (1). 1 Alizadeh, Dyck et al. 2019 SCI could be subdivided in two pathophysiological 2 Waxman 1989 phases: the primary injury, induced directly by the 3 Anjum, Yazid et al. 2020 trauma, leading to neural cell death and axonal 4 Alizadeh and Karimi-Abdolrezaee 2016 network damage and the delayed secondary in- 5 Varma, Das et al. 2013 jury characterized mainly by further dead of neur- 6 Hanna, Andre et al. 2017 ons and oligodendrocytes, increased astrocytes and immune cell activation and free radical form- P7 - Electroporation for clinical use ation (2,3). Among all, the loss of oligodendro- cytes and the consequent progressive demyelin- Tuesday late afternoon Track ation result in axonal dysfunction, degeneration D and loss of sensory and motor functions (4). Al- Sep 17, 16:50 - 18:20 though many progresses have been done to minim- ize the spinal cord damage and optimize the func- OR-119 tionality of spared connections, nowadays there is Optimization of Bipolar Microsecond Electric no effective therapy for the treatment of SCI (5). Pulses for DNA Vaccine Delivery Multifaceted strategies are considered the unique Robert H. Williamson 1, Matthew Dewitt2, Driss solution for functional restoration by including cell Elhanafi1, David Zaharoff1, Michael Sano1 substitution, neuroprotection and axonal growth 1North Carolina State University , United States promotion. RISEUP project (https://www.riseup- 2University of Virginia , United States project.eu/) proposes to achieve neuronal func- Gene-related therapeutics such as DNA vac- tional regeneration after SCI by an unprecedented cines are currently hindered by poor cellular up- and unique bio-hybrid-compatible electro-activated take. Reversible electroporation (RE) has been and potentially be wireless rechargeable implant- used to significantly improve DNA delivery, but tra- able technology. RISEUP introduces high voltage ditional monopolar protocols induce severe muscle microsecond electric pulses (micro pulses) stimu- stimulation and pain in vivo. Novel bipolar micro- lations and low amplitude direct currents on a com- second and sub-microsecond pulses have been bination of stem cells (induced neural stem cells shown to alleviate this stimulation when used for and multipotent stromal cells), whose transplanta- irreversible electroporation (IRE); however, these tion is facilitated by an innovative scaffold bioma- pulses introduce several parameters which have terial. The RISEUP concept is that micro pulses, yet to be explored in the context of RE. In this being able to impose and control cytosolic Calcium study, the parameters of waveform, delivery rate, oscillations (6), will facilitate cell maturation, sur-and dose were explored to begin optimizing bipolar vival and neurotrophic factors secretion. Because microsecond pulses for clinical use as a DNA de- Calcium signaling is essential for neuronal activity, livery vehicle. endogenous neuronal re-connections will also be To accomplish this, cells were embedded in 3D col- favored. RISEUP goal, even if ambitious, is con- lagen tissue mimics and allowed to achieve nat- crete due to the multidisciplinary partners’ compet- ive morphologies. A coaxial ring-and-pin electrode ences, initiating from TRLI a radically new line of was used to deliver treatments with varied wave- technology (electro-activated, remotely controlled, forms, delivery rates, and doses while exploring biocompatible, biodegradable cell-containing im- a wide range of electric field intensities. Samples plants for the repair of neuronal lesions) establish- were maintained at 37°C to replicate physiological ing its proof-of-principle (TRL3). The long-term vis- conditions and active temperature control was used ion of RISEUP is the radical change in SCI treat- during treatments to ensure peak temperatures did ment modality to assure the cure delivery without not exceed 42°C, eliminating thermal effects as a any machinery connection, dramatically improving confounding factor. RE thresholds were assessed patients’ quality of life. 158 via addition of propidium iodide prior to pulse de-injuries deep enough to affect the reticular layer of livery. IRE thresholds were assessed 24 hours the dermis (burns, insect bites, surgeries, tattoos post treatment in matched experiments using PI and piercings) and are the result of abnormal and Calcein AM to indicate live and dead cells, re- healing responses, producing excess fibrinogen spectively. and collagen (hyperactivation of keloid fibroblasts). A combination of finite element models and a MAT- Keloid scars are often disfiguring, profoundly LAB algorithm were then used to determine the impair quality of life, and cause immense physical electric field intensities associated with RE and IRE and mental discomfort in affected individuals, outcomes with optimal parameter sets being those especially those who have symptomatic (pruritic, which maximized the difference between these painful) and/or hyperpigmented scars. Surgery, thresholds. DNA transfection with a plasmid en- corticosteroids, and even intralesional bleomycin, coding for green fluorescent protein was then eval- among many others, are the treatments proposed uated via microscopy in a subset of best-performing for this pathology. Despite this, many of these groups and compared to traditional electroporation scars do not improve or if they do, they recur in protocols. the vast majority of cases. It is proposed that Of the parameters tested, dose had the most signi- electrochemotherapy (ECT) could be effective ficant impact on RE and IRE thresholds, with higher in treating of recurrent keloids, even when other doses reducing the magnitude of these thresholds, treatments have failed. Here we present the but reducing the safety margin between reversible preliminary results of 4 patients (5 keloids). and irreversible effects. The rate of energy delivery Materials and Methods: Four patients with five also influenced these thresholds, with higher initial keloids were treated with one session of ECT, rates resulting in higher RE and IRE thresholds and three patients with one keloid (two on the chest a larger safety margin between thresholds. Accord- and one on the back) and one patient with two ingly, when an optimal parameter set was used in keloids on the ear. For the ECT procedures, a final transfection validation study, we achieved a Oncopore electroporator (Biotex SRL, Buenos 7730% increase in gene expression over traditional Aires, Argentina) and a 6-needle electrode were monopolar pulse protocols. used. Bleomycin (Bleocris 15, LKM) was applied These results indicate that RE offers a promising intralesionally. The concentration of the solution avenue for DNA delivery and that bipolar micro- used was 1,000IU/cm3. The volume used was second pulses offer significant improvements over calculated by using the formula V = ab2 π /6, in current technology, which warrants more research each case. Patient follow-up was performed in to further explore the effects of these and other bi- a weekly basis for the first month, and monthly polar pulse parameters on RE outcomes. for the following 2 months. The lesions were measures on each follow-up visit. OR-120 Results: Three months after treatment, the overall Keloid treatment with Electrochemotherapy average reduction in the volume of the keloids Sebastian D. Michinski 1, Ana Dimitri3, Ana was 73% (range, 41.8 to 100%). In all cases Campastri2, Antonella Cilio2, Raquel Lertora3, Fe- the symptoms were reduced (itching, pain), but lipe H. Horacio Maglietti2 the hyperpigmentation remains. Treatment was 1Universidad de Buenos Aires, Argentina well tolerated by patients, and no serious adverse 2Fundación Barceló - CONICET, Argentina events were observed. 3Hospital Zonal General de Agudos Petrona V. de Cord- Discussion and Conclusion: There is little ex- ero, Argentina perience in using ECT to treat keloids, but the Background: Keloids are characterized by results have been encouraging (1,2). Here we the growth of a firm, extensive scar beyond the present only the preliminary results of 4 patients (5 edges of the original wound. These occur due to keloids). We conclude that ECT is safe and could be effective in recurrent keloids, even when other 159 treatments have failed. creasing concentrations in the agar-agar gels, un- til the application of the pulses did not induce the 1- Electrochemotherapy treatment of a re- formation of pH fronts or they were quickly neutral- calcitrant earlobe keloid scar with chronic ized.The final gel was prepared with carbopol 940 + lymphocytic leukaemia infiltration. ht- established buffer concentration (sodium bicarbon- tps://doi.org/10.1016/j.bjps.2010.05.008 ate) + an adjusted conductivity by addition of 0.5% 2- Treatment of Keloids and Hypertrophic Scars sodium chloride with Bleomycin and Electroporation. DOI: Results: Pulse delivery induces extreme pH 10.1097/PRS.0b013e3182a053c8 changes, which deteriorate the surface of the needles demonstrated by the emerging of irreg- ular fronts in heavily used needles. This irreg- OR-121 ular electrical conduction leads to irregular elec- Development of a specific gel for skin cancer tric fields, which lowers treatment efficiency. The electrochemotherapy. gel developed effectively neutralizes extreme pH Antonella María Cilio 1, Ana Campastri1, Jesica changes, preventing electrode damage. Rodríguez Miranda1, Ximena Manglano1, Sebastian D. Conclusion: The specific gel developed for electro- Michinski2, Felipe Maglietti1 poration reduces electrode wear increasing treat- 1Fundación H.A. Barceló, Argentina ment efficiency. In addition, it may ameliorate skin 2Universidad de Buenos Aires, Argentina side effects provoked by extreme pH changes. Introduction: Electrochemotherapy (ECT) is an accepted treatment used for cutaneous primary OR-122 and secondary tumors of varied histologies. Sev- The Synergy of Conductive Nanoparticles with eral aspects of ECT remain to be elucidated, for Nanosecond and Microsecond Pulse Bursts for example how to mitigate the side effects of elec- Bleomycin-based Electrochemotherapy trolysis during treatment. Electrolysis byproducts; Barbora Lekešytė 1, Paulina Malakauskaitė1, Eglė Mickevičiūtė1, Eivina Radzevičiūtė-Valčiuke1, Ver- acid at the anode and base at the cathode, can onika Malyško-Ptašinskė2, Anna Szewczyk3, Natalija cause needle deterioration, tissue damage, and German1, Almira Ramanavičienė1, Julita Kulbacka3, skin irritation. Vitalij Novickij1 Aim: To study the magnitude of pH changes pro- 1State Research Institute Centre for Innovative Medicine, duced by the electrolysis and to develop a gel that Lithuania neutralizes these changes. 2Vilnius Gediminas Technical University, Lithuania Materials and Methods: We used agar-agar gels 3Wroclaw Medical University, Poland with a conductivity similar to tissues, adjusted by the addition of 0.5% sodium chloride. Tissue con- The application of nanotechnology and nan- ductivity was calculated using current data ob- oparticles in the area of drug and gene delivery tained from ECT treatments previously performed is heavily exploited. Recently, it was shown that in veterinary and human medicine. pH fronts were conductive nanoparticles can be used for electric stained using methyl red for the acidic front and field amplification in a close proximity with the cell phenolphthalein for the basic front. The formation membrane, introducing the capability to improve of the fronts were captured by means of a high- available electrochemotherapy methods and solve speed camera (SAMSUNG 108 MP, 920 FPS at problems associated with field non-homogeneity. a resolution of 1280x720). The images were pro- However, the effects of conductive nanoparticles cessed and analyzed with the NIH ImageJ soft- were not yet characterized in the context of ware implemented in an image processing station nanosecond pulse bursts or in the context of drug for quantifying pH front extension. delivery, i.e., electrochemotherapy. Therefore, in We added sodium bicarbonate (as a buffer) at in- this work, we characterize the potential use of gold nanoparticles (AuNPs: 13 nm) in combination with 160 microsecond: 0.6–1.5 kV/cm × 100 µs × 8 (1 Hz) trade. Nucleic acid vaccines, such as those based and nanosecond: 6 kV/cm × 300–700 ns × 100 (1, on mRNA are endowed of these features. 10, 100 kHz and 1 MHz) electric field pulses. We To address the urgent need to find solutions to show that in the nano-pulse range, the pulse re- the SARS-CoV-2 Pandemic, Takis has developed petition frequency significantly affects the synergy COVID-eVax, a vaccine approach based on ge- between AuNPs and electroporation. Basically, netic engineering and DNA electroporation as part the synergistic effects (improved permeabiliza- of the X-eVax platform, previously developed. The tion and electrotransfer) are profound only with project started in 2020 and consisted of the molecu- increased burst frequency and are non-existent lar design of the vaccine, the development of the with low-frequency bursts. Additionally, AuNPs reagents and tests necessary to test its effective- not only reduce the permeabilization thresholds ness and the experiments in animal models. Sub- but also affect pore resealing, i.e., irreversible sequently, GMP-grade material (Good Manufactur- electroporation is triggered in case the field is not ing Practices) was produced, all regulatory stud-adjusted. ies were conducted (toxicology, biodistribution, im- Finally, we have tested the most prominent pro- mune response) and finally a phase 1 study in hu- tocols (microsecond and nanosecond) in the mans, which ended in December 2021, achieving context of bleomycin-based electrochemotherapy all the objectives set and providing the basis for (4T1 cell line). It was shown that a saturated evaluations in other applications. cytotoxic response with AuNPs can be triggered DNA vaccines advantages are: (1) simple and at significantly lower electric fields, which are quick production of DNA encoding the antigens considered sub-threshold and/or not applicable for by PCR or synthetic methods (potential game- electrochemotherapy otherwise. At the same time, changers for new variants especially vaccine res- the nanoparticles themselves are non-toxic for the istant strains), (2) easy large-scale production, (3) cells either separately or in combination with bleo-safety compared to inactivated virus vaccines, and mycin, indicating the methodology’s prospective (4) higher thermostability (minimal cold-chain re- applicability in the context of electrochemotherapy. quirements), which is an issue with some vac- cines. The DNA-based platforms offer great flex- Acknowledgments: The research was suppor- ibility in manipulating the encoded vaccine antigen ted by the Research Council of Lithuania, Grant and have a great potential for accelerated devel- Nr. S-PD-24-5. opment. Recently, the first DNA vaccine against SARS-CoV-2 (ZyCov-D) has been registered in In- OR-123 dia for human use; moreover, DNA vaccines have Development of novel genetic vaccine plat- been extensively tested in multiple clinical trials in forms: from the idea to GMP production the oncology field and are commonly used in veter- Luigi Aurisicchio inary medicine. These vaccines (as opposed to Takis, Italy mRNA-based vaccines) are stable, do not require The COVID-19 pandemic demonstrated the ur- cold-chain supply, and can easily be produced in gent need to develop versatile vaccination plat- large amounts in bacteria. All these advantages forms that could be quickly implemented for infec- make this platform technology an attractive tool, as tious diseases. It is of utmost importance to provide it overcomes several shortcomings of alternative a vaccine in a time-critical manner for such dis- approaches (e.g., complex production processes, eases, as the frequency of such epidemics and stability issues, purchase price). pandemics as we see them today, will be heav- In this presentation, opportunities and challenges ily increasing due to rise in global travel, global of DNA-based vaccines and Takis biotech experi- warming, increase in population density, penetra- ence will be discussed. tion into previously uninhabited areas and animal 161 OR-124 pared to free drug BLM without compromising vi- Low-Dose Electrochemotherapy Enhances ability significantly. However, when combined DNA Damage and Overcome Resistance with DNA repair pathway-targeting chemothera- through Synergistic Drug Delivery peutic drugs (TMZ or OLA), viability significantly de-Vaishali Malik 1, Laurien G. P. H. Vroomen2, Masashi creased from 95% (TMZ free drug) to < 40% (ECT Fujimori3, Emma Gerace1, Jaad Ismail1, Govindarajan 1:1 TMZ, ****p < 0.0001) in T98G and further re- Srimathveeravalli1 duction <15% viability (ECT 1:2 TMZ **p<0.005) 1University of Massachusetts Amherst, United States U87 cell lines. Similarly, viability decreased from 2Amsterdam University Medical Center, Netherlands 90% (OLA free drug) to <50% (ECT 1:1 OLA, 3Mie University, Japan ****p<0.0001) in both cell lines. Clonogenic assay Introduction: A lethal dose of bleomycin (BLM) showed comparable cell toxicity to high dose irra- is delivered into cells during electrochemotherapy diation (10Gy) with no significant difference com- (ECT) to induce acute cell death. Given BLM pro- pared to ECT+TMZ or ECT+OLA. In vivo results motes double-strand breaks (DSBs) in DNA, we demonstrated complete tumor eradication in sub- investigated whether the dose could be titrated to cutaneous mouse model (**p<0.01) when com- achieve DNA damage equivalent to external beam pared to untreated control at week 4, with doubled radiation therapy. Subsequently, we tested our survival from 4 weeks to 8 weeks untreated and strategy of low-dose ECT (LD-ECT) to treat radi- ECT+TMZ or ECT+OLA treatment respectively. In- ation sensitive glioblastoma (GBM), studying syn- tracranial GBM model showed reduction of biolu- ergy with standard of therapeutics; temozolomide minescence as early as 1-week post treatment, (TMZ) and olaprib (OLA), and illuminating novel av- supported by histological data which showed no enues for enhanced therapeutic efficacy in GBM signs of tumor cells at 8-week sacrifice. Conclu- treatment. sion: Non-ablative LD-ECT mimics high-dose ra- Methodology: TMZ-sensitive (U87) and - diation, enhancing standard-of-care therapeutics’ insensitive (T98G) GMB cell lines were screened efficacy and overcoming resistance, with potential for optimal reversible electroporation parameters, superior healthy brain sparing. and BLM dosing was titrated to maximize DNA damage as measured by ɣ-H2AX analyzed by flow S04 - Advanced applications of cytometry. Synergy with TMZ and OLA was tested PEF for food quality enhancement, at different dose ratios (0:3, 1:1, 1:2, 1:3) with LD-food component modification, and ECT, and outcomes evaluated with clonogenic as- structural alterations say for survival and proliferation. Radiation ther- apy at 2, 5 and 10Gy served as positive control. In Wednesday morning Track A Murine subcutaneous tumor model, TMZ or OLA was delivered for 10 consecutive days after LD- Sep 18, 10:40 - 12:10 ECT, then discontinued. Tumor volume and sur- OR-236 vival were recorded. Further evaluations conduc- Introduction to Advanced Applications of PEF ted in an orthotopic GBM nude mice model, U87 for Food Quality Enhancement, Food Compon- cells injected into left frontal cortex and treated ent Modification, and Structural Alterations with TMZ or TMZ + LD-ECT. Mice were mon- Jessica Genovese itored with bioluminescence imaging, and histo- Università degli Studi di Milano, Italy logical evaluation was performed at euthanasia. Results: In vitro studies revealed that delivery Pulsed electric field (PEF), also known as elec- of nanomolar concentration (0.625µg/mL) of BLM troporation, is a biophysical process in which an ex- (ECT) increased DNA damage >85% in T98G ternal electric field is applied to conductive materi- (****p<0.0001) and >65% in U87 (**p<0.01) comals, such as cells and tissues, increasing cell mem- brane permeability and inducing physicochemical 162 modifications of the cell membrane. The widely ity – Working ON Foods”. accepted mechanism underlying electroporation is the formation of hydrophilic pores in the cell’s phos-OR-125 pholipid bilayer. This process also causes mech- PEF for more sustainable, nutritious biomass anical, osmotic, viscoelastic, and hydrodynamic in- and macromolecules for food applications with stabilities in biological materials. a case study on microalgae PEF treatment has been found to induce Iris Haberkorn 1, Byron Perez1, Alexander Mathys2 1 the production of highly reactive oxygen species ETH Singapore Centre, Singapore 2 (ROS), such as superoxide anion and hydroxyl ETH Zurich, Switzerland radical, partly generated by electrochemical reac- Combining emerging processing technolo- tions at the electrode-electrolyte interface, but are gies with new raw materials and the inclusion of primarily the result of the cell’s response to oxidat-sustainability assessment, covering nutritional, ive stress (only present when the electroporation is social, and economic aspects, could form a holistic reversible). Chemical reactions at the electrode in- approach for developing sustainable food systems. terface, due to the passage of the electric current New materials like microalgae offer alternatives to through an aqueous solution, generate hydroxide creating more sustainable, nutritious, protein-rich ions at the cathode and protons at the anode, res- foods. Technologies including pulsed electric ulting in localized pH alterations. These changes fields (PEF) can facilitate their adoption by en- can influence the charge, reactivity, and stability hancing the efficiency of associated value chains. of various biomacromolecules, including polysac- This presentation highlights PEF applications for charides, proteins, nucleic acids, and lipids. sustainable, nutritious food development, using In summary, the effects of PEF treatment microalgae as a case study. are exerted through a complex interplay between Upstream, nanosecond PEF (nsPEF) (100 ns electrophysical and electrochemical mechanisms. pulses, 10 kV cm−1) maximizes the efficiency Electrophysical mechanisms include the induction of single-cell bioprocesses up to 17.53 ± 10.46 of transmembrane voltage (TMV), electrophoresis, percent across a range of diverse organism do- rotation of molecule dipoles, potential heating, and mains, including Arthrospira platensis (3×256±67 the generation of mechanical waves, directly alter- J kgsus−1) and heterotrophic Chlorella vulgaris ing cellular structures and functions. Electrochem- (3×227±60 J kgsus−1). ical mechanisms, such as pH changes, water elec- Downstream, combining microsecond PEF trolysis, metal cations release from electrodes, and (µsPEF) with an incubation step can modify the the generation of ROS, further impact cellular re- cell wall structure, potentially increasing nutrient sponses and can induce alterations in the micro- bioaccessibility. Applying 10×5 µs pulses at 20 structure and macromolecular interactions of bio- kV cm−1 (31.8 kJ kgsus−1) enhances C. vulgaris macromolecules. lipid bioaccessibility 3-fold while preserving oxid- Overall, the diverse applications of PEF treat- ative stability, therefore enhancing raw material ment have the potential to revolutionize various as- nutritional value. Microalgae protein extraction pects of food production and preservation, waste via µsPEF combined with incubation (80.7 ± 1.9 management, and food ingredient modifications. kJ kgsus-1) is more energy-efficient compared to Acknowledgement: Project funded under the conventional methods like high-pressure homo- National Recovery and Resilience Plan (NRRP), genization. Additionally, PEF facilitates protein Mission 4 Component 2 Investment 1.3 - Call for separation by microfiltration by preserving the proposals No. 341 of 15 March 2022 of Italian cell structure, reducing fouling and improving flux Ministry of University and Research funded by the rates. PEF yields small soluble proteins with European Union – NextGenerationEU. Project title specific functional properties for food applications. “ON Foods - Research and innovation network on This talk showcases the effect of PEF on the food and nutrition Sustainability, Safety and Secur- 163 quality and functional properties of microalgae and electric field of 1.5 kJ/kg resulted in the highest extracted biocompound fractions, highlighting the increase in extract, increase in dry matter content potential of advanced PEF applications for novel and loss of water. In most cases, the influence of food raw material components modifications and the type of osmotic solution on the kinetics of the structural alterations. process was insignificant. The highest dry matter PEF could emerge as high-impact technology, content was observed in fruits dehydrated in a addressing challenges in single-cell-based value rosehip juice solution with the addition of sucrose, chains by enhancing upstream efficiency while and the lowest water activity was observed in improving the downstream recovery of biocom- fruits dehydrated using a sucrose solution. The pounds. PEF supports the provision of nutritious texture parameters of oranges depended on the foods by facilitating the adoption of new raw type of osmotic solution used and the duration materials and, thus, contributes to establishing of the dehydration process, while the use of a more sustainable food systems. pulsed electric field before the osmotic dehydration process did not significantly change the texture OR-126 of dehydrated orange fruits, but the structure did The influence of a pulsed electric field on change. The use of PEF pre-treatment and dehyd- the osmotic dehydration process and selected ration in a 50% sucrose solution and a 50% rosehip physical properties of orange fruits dehydrated solution with the addition of sucrose increased in unconventional solutions brightness, color saturation, and the share of Agnieszka Ciurzyńska, Katarzyna Rybak, Dorota yellow in the osmodehydrated oranges. The visual Witrowa-Rajchert, Katarzyna Pobiega, Sabina Galus, assessment showed a darkening of the tissue Małgorzata Nowacka of fruit dehydrated in an unconventional rosehip Warsaw University of Life Sciences, Poland solution with the addition of sucrose compared to Aim: The aim of the work was to investigate the samples dehydrated in a sucrose solution. PEF effect of pre-treatment with a pulsed electric field significantly increased the ΔE coefficient, which (PEF) before osmotic dehydration (OD) treatment indicates a visible difference in color between on the properties of osmodehydrated orange fruit. osmotically dehydrated and PEF-treated tissue Method: The samples were treated using a pulsed and fresh tissue. electric field with specific energy input of 1.5 and 6.5 kJ/kg, and then subjected to the osmotic Acknowledgment: This project received fund- dehydration using two solutions: 50% sucrose ing from transnational funding bodies, partners of solution and rosehip juice solution with the addition the H2020 ERA-NETs SUSFOOD2 and CORE Or-of sucrose. The process was carried out at a tem- ganic Cofunds, under the Joint SUSFOOD2/CORE perature of 30°C for 3 hours, with the evaluation Organic Call 2019 (MILDSUSFRUIT), as well as of fruits after 0.5, 1, 2, and 3 hours. The ratio of the National Centre for Research and Development the mass of the solution to the mass of the raw (POLAND, decision DWM/SF-CO/31/2021). material was 1:4. The osmotic kinetics based on changes in total mass, water mass, and solids OR-127 mass were evaluated. Additionally, the chosen The manifold manifestations of electroporation properties of the orange fruit were analyzed (color, effects on plant tissue and how their quantific- dry substance content and water activity, structure ation depends on the method of analysis and texture). Madita Anna-Maria Kirchner 1, Claudia Siemer1, Damijan Miklavčič1, Stefan Töpfl1, Samo Mahnič- Results: The results showed that PEF treatment Kalamiza2 had the greatest effect on tissue diffusivity during 1Elea Technology GmbH, Germany short osmotic dehydration. In the case of dehyd- 2University of Ljubljana, Slovenia ration for 0.5 and 1 h, treatment with a pulsed 164 The use of Pulsed Electric Fields (PEF) has efficient production process. been successfully implemented in the potato in- dustry, but its application in the vegetable industry OR-128 remains limited due to the lack of knowledge on Enhancing Chemical Reactions and Modifica- the behavior of various plant tissue types in sub- tion of Food Ingredients Using Pulsed Electric sequent processing steps. The exposure to PEF Fields: An Alternative Technique reduces the needed blanching time as the elec- Xin-An Zeng troporation increases the rate of heat and mass Foshan University, China transfer in tissue, allowing lethal temperatures for Pulsed Electric Fields (PEF) represent a prom- browning enzymes like peroxidase to be reached ising non-thermal technology in food processing, more quickly in PEF treated plant tissues. How- enhancing chemical reactions and modifying the ever, this effect has not been consistently con- physical properties of food ingredients without com- firmed across all tissue types and in some cases promising their nutritional and sensory qualities. an increased enzyme activity was measured. Our Our investigations explored the application of PEF work aimed to investigate the non-uniform effects across diverse food matrices, including flavonoids, of PEF treatment due to the structural diversity of starches, and soy protein isolates. We found that plant tissues commonly used in industrial applic- PEF effectively augments the antiglycation activ- ations. These include mostly homogeneous tis- ity of Noni flavonoids by modulating flavone glyc- sues (red beet, sweet potato), tissues with lignified osides into aglycones. Specifically, PEF treat- parts (broccoli), and tissues with varying character- ment resulted in a substantial increase in the quer- istics (inhomogeneities) within the same raw ma- cetin proportion from 0.08 to 0.91 (molar ratio terial (parsnip, carrot). The applied PEF treatment with rutin) after 4000 pulses. This elevation in ranges from a field strength of 0.4 to 3 kV/cm with quercetin content significantly enhanced the NF specific energy inputs up to 6.5 kJ/kg. Electrical sample’s antiglycation potential, demonstrating up impedance measurements were conducted before to 2.98 times higher inhibitory effects on fructosam- and immediately after the PEF treatment as well as ine, α-dicarbonyl compounds, and advanced glyc-after a holding time of 10 minutes to monitor effects ation end-products (AGEs) in bovine serum albu-of water redistribution. Texture analysis was per- min (BSA)-fructose model. The enhanced antiglyc- formed using compression and cutting force meas- ation potential suggests significant health benefits, urements to determine the responsiveness of PEF particularly in preventing oxidative stress-related treated tissue to different force applications by com-diseases by inhibiting the formation of AGEs. In paring thresholds. The results show that the more the starch modification, PEF treatments were ap- complex the tissue structure, the less homogen- plied to corn and potato starches, achieving notable eous the PEF treatment. This can be attributed to improvements in freeze-thaw stability and pasting factors such as air pockets, low-conductivity struc- properties. After five cycles, PEF-assisted esteri- tures (e.g. lignin), and variations in water content fication reduced the syneresis of starch by 29.5%, and distribution. Due to these, the orientation of the compared to 10.17% without PEF, and lowered the tissue in the electric field also plays a crucial role pasting temperature by 7.6–15.1°C at 2–6 kV/cm. when treating inhomogeneous materials. Varying This PEF-assisted method also reduced reaction temporal changes in the average impedance ratio time and improved efficiency by 6.1–39.1% over (after-to-before the treatment) were observed for the control. These enhancements are critical for different treatment protocols and need to be con- applications requiring robust performance during sidered for industrial processes when holding times storage and processing, such as in frozen foods are part of the production line. Based on our find- and ready-to-eat meals. Additionally, the function- ings, appropriate PEF assessments and treatment ality of commercial soy protein isolate (SPI) was conditions can be defined for different groups of substantially improved by targeted PEF treatments plant raw materials, ensuring a gentler and more 165 and pH shifts. Structural unfolding and promo- field strengths ranging from 0.5 up to 2.0 kV/cm with tion of soluble aggregate formation were induced specific energy inputs up to 30 kJ/kg. The effects under moderate PEF intensity (10 kV/cm) and al- of the pretreatment were assessed based on out- kaline conditions (pH 11), resulting in a significant comes such as extraction yield, moisture transfer, increase in the solubility of commercial SPI from extraction of quality-enhancing compounds or re- 26.06% to 70.34%. This combination of treatments moval of undesired compounds. also greatly improved the emulsification and foam- In terms of juicing, PEF was successfully imple- ing properties of commercial SPI, crucial for ex- mented as a pretreatment in carrot and peach, panding its use in a wider range of food products which pose challenges during comminution and where textural integrity and stability are paramount. juicing. PEF also modified the textural properties The outcomes of our studies not only demonstrate of the ligneous tissues thereby facilitating cutting, the efficacy of PEF in enhancing the functional destoning, and juicing, while saving energy com- properties of food components but also its potential pared to thermal treatments (up to 99 %). Juice to contribute to the innovation and sustainability of extraction yield was increased by up to 17% for car-food manufacturing processes. rot and 5% for peach. Olive processing also exhib- ited promising results, increasing oil yields by up OR-129 to 10%. The enhanced extractability also led to in- Opportunities for implementing pulsed electric creased bioactive compound concentrations in the fields for the enhanced processing of plant- oil; 7% more total phenolic compounds and 57% based foods tocopherols, leading to increased oxidative stabil- George Dimopoulos 1, Varvara Andreou2, Athanasios ity during storage. The treatment of fruits and ve-Limnaios3, Alexandros Katsimichas3, Ioanna Thanou3, getables such as kiwi, cherry tomato, spinach, and Efimia Dermesonlouoglou3, George Katsaros2, Petros goji berry benefited from the increased tissue per- Taoukis3 meability during osmotic dehydration and drying, 1University of Natural Resources and Life Sciences, Aus-exhibiting increased dehydration rates. The effect- tria ive moisture removal also led to significant shelf- 2Institute of Technology of Agricultural Products, Greece life extension, adding up to 34 d to the shelf life of 3National Technical University of Athens, Greece osmotically dehydrated spinach leaves. PEF pro- The increasing demand in sustainable plant- cessing of zucchini also led to the reduction of oil based foodstuffs necessitates the development of uptake during deep frying by up to 36%, underlining energy efficient techniques as pretreatments, tar- the potential of PEF for healthier food production. geting to milder and improved processing, thus re- PEF processing of chickpeas improved their rehyd- taining quality. Pulsed Electric Fields (PEF) select- ration characteristics and led to significant reduc- ively increase plant cell permeability at low energy tion of undesirable oligosaccharides during soak- inputs through electroporation, offering significant ing, up to 87%. benefits in mass transfer operations to and from The presented results highlight the potential and ef- plant tissues such as drying, osmotic dehydration, ficiency of PEF pretreatment at improving the yield, extraction, and juicing, compared to conventional quality and stability of plant-based foods with min- processes. Enhanced mass transfer rates during imal energy consumption. The availability of indus- such processes lead to significant quality preserva- trial scale continuous equipment makes the applic- tion and textural modification. This work provides ation of the process a highly realistic prospect after an overview of successful case studies of the im-economic and environmental factors have been plementation of PEF processing as a pretreatment considered. to core food processes for a diverse range of plant tissues, with the overarching principle being the im- provement of further processing outcomes. Plant materials were treated with PEF at electric 166 OR-130 OR-131 Comparison of nanosecond and microsecond Effect of PEF on ginger roots: Improving juice PEF for physical property and substance mo- extraction yield or product quality bilization in potato Rian A. H. Timmermans, Deniz Döner, Lijiao Kan, Yuji Takahashi 1, Kiyohira Hagimoto1, Sunao Joanne Siccama, Bert Dijkink, Martijntje Vollebregt Katsuki1, Yuji Okada2 Wageningen University & Research, Netherlands 1Kumamoto University, Japan This study investigated the combined effect of 2Aohata Corporation, Japan Pulsed Electric Field (PEF) treatment and (mild) Pulsed electric fields (PEFs) are gaining atten- heat treatment on the juice extraction yield and tion in the food industry as a non-thermal food juice quality of ginger roots. Treatments were com- processing technology, for sterilization, extraction, pared to a thermal treatment (30 min 80°C) which drying and texture control of agricultural and fish- is an industrial standard. ery products. The key factors of PEF effect on Fresh ginger roots were subjected to PEF treat- agricultural crops are field intensity, pulse duration ment (E = 2 kV/cm, τ = 8 µs, w = 6 kJ/kg: 5/7 ginger-and number of pulses. Much of what is expected water ratio, w/w) combined with different heat treat- from PEF in food applications is to increase the ments (30 min 80°C, 2 min 90°C, 10 min 80°C, 10 permeability of cell membranes. Generally longer min 50°C). After PEF treatment, the ginger roots pulses are more effective in increasing cell mem- were processed, which includes an initial juicing brane permeability than larger electric fields. For step using an Angel juicer to separate pulp and this reason, microsecond or millisecond PEFs are juice. The separated pulp and juice were then often used in much of food processing, as it allows recombined and heated. This mixture was pro- treatment with small electric fields. Nanosecond cessed again using an Angel juicer to further ex- PEF has rarely been used. It is easy to imagine tract juice. Control samples without PEF treatment that the morphology of cell membranes subjected were also prepared for comparison. The juice yield to nanosecond PEF is different from that of micro- was measured by weighing the extracted juice. second PEF [1]. Relatively high field, nanosecond Quality parameters such as dry matter, soluble and pulses are expected to form a number of small non-soluble solids, color, and the gingerol content pores on the membrane, whereas the microsecond were analyzed. pulses produce a few large pores. The variation The results indicated that a combination of PEF of the membrane morphology dependent on the with heat treatment (30 min 80°C) improves the pulse waveform can be unitized to control the sub- extraction yield of juice with 3% compared to the stances mobilizing through the membrane. In this control sample using only heat. Combinations of study, 100’s nanosecond PEFs was compared to PEF and milder heat had the same extraction yield 10 microseconds PEFs for the dielectric properties as the control. For both PEF treated and control of potatoes and for the substances in the soaked samples, the most heat intensive treatment (30 min juice, and for the drying rate. The capacitance and 80°C) tend to darken the juice and reduce the yel- conductance of potatoes were measured using the low intensity. The other combined treatments of impedance analyzer (IM3570, Hioki) to discuss the PEF with milder heat treatments retain the natural cell membrane permeability in the tissue. The sub- color. stances in the soaked juice will also be analyzed Combination of PEF with heat can lead to inter- using the high-performance liquid chromatograph esting alternatives for conventional thermal treat- (EXTREMA, Jasco) to discuss the substances mo- ment. Either one can add PEF to the current ex- bilizing through the membrane in the tissue. Fur- isting thermal treatment to increase juice extraction thermore, the drying rate of potatoes was meas-yield, while keeping the same quality. Alternatively, ured to discuss the fluidity of water in the tissue. one can combine PEF with a milder heat treatment, leading to the same extraction yield as is the current 167 standard practice, but with a better quality. Reduc-reached the critical voltage. tion of the heating temperature or time could also At command voltages below -200 mV, the opening contribute to a reduction of energy use. of high-conductance electropores reduced the efficiency of membrane charging. As a result, the induced transmembrane potential (TMP) became S17 - Voltage control of biological progressively smaller than the applied command membrane pores voltage. In the range between -240 and -300 mV, the induced TMP reached a ceiling and could not Wednesday morning Track B be hyperpolarized any further. Larger command Sep 18, 10:40 - 12:10 voltage steps increased the electropore conduct- ance in an adaptive manner so that the TMP OR-132 remained unchanged or even depolarized. This Voltage sensitivity of electropores limits the effect was reproducibly demonstrated in different membrane potential cell types using different intra- and extracellular Mantas Silkunas, Andrei G. Pakhomov solutions, and with voltage step, voltage ramp, and Old Dominion University, United States current ramp protocols. For example, the most Charging cells beyond physiological limits hyperpolarized TMPs that could be reached with increases membrane conductance in a nonlinear voltage ramps averaged -270 ± 6 mV in HEK cells, manner. This effect does not depend on voltage- -284 ± 5 mV in CHO cells, and -243 ± 9 mV in gated ion channels. The additional conductance hippocampal neurons. The cell plasma membrane pathway created by membrane overcharging is becomes essentially transparent to the electric commonly associated with electropore formation. field in excess of what is needed to reach this limit, It remains to be established whether electropores thus allowing it to reach intracellular structures. are perforations in the lipid bilayer or in membrane The adaptive increase in pore conductance was proteins or if they are complex multicomponent reversible and protected cells from reaching still structures. It is likely that several types of electro-higher TMP that could disintegrate the membrane. pores can be generated, and their yield depends It remains to be studied whether faster charging, on the electric field parameters. e.g., with high-intensity nanosecond electric Voltage-clamp measurements in diverse mam- pulses, could override the increase in adaptive malian cells electroporated by nanosecond-range pore conductance and drive cells to an even higher electric pulses established that small, nanometer- TMP. sized electropores (“nanopores”) are voltage sensitive and selective to cations. These pores are supported in part by NIH NEI R21EY034258 also inwardly rectifying, meaning that they conduct and R21EY034803 (to A.G.P.) more current when the cell is hyperpolarized than when it is depolarized to the same potential. OR-135 The formation of voltage-sensitive electropores can Using the same electrode to electroporate a also be observed with ms-range pulses. By com- chromaffin cell and measure the resulting exo- bining a whole-cell patch clamp with total internal cytosis of catecholamine reflection fluorescence (TIRF) microscopy, we Jaya Ghosh, Xin Liu, Kevin Gillis were able to observe the formation of electropores University of Missouri, United States concurrently with monitoring their conductance. Adrenal chromaffin cells release the cat- The opening of large, high-conductance (> 1 nS) echolamines adrenaline and noradrenaline as part pores, manifested as an abrupt increase in the of the fight-or-flight response. These hormones whole-cell current and a bright focal Ca2+ entry are stored in vesicles within cells, and, upon an fluorescence transient, was typically delayed by appropriate stimulus, a vesicle will fuse with the milliseconds after the membrane had already 168 cell membrane and release its contents as a dis-vesicular membrane and the plasma membrane. crete packet via “quantal” exocytosis. Since cat- This process of membrane fusion is mediated by echolamines are readily oxidized, quantal exocyt- the Soluble N-ethylmaleimide-Sensitive Factor osis can be detected by an electrochemical elec- Attachment Proteins REceptor (SNARE) protein trode located immediately adjacent to the cell as complex, which in neurons and neuroendocrine a spike of amperometric current for each vesicle cells is composed of the vesicular SNARE pro- released. Here we report using a microelectrode tein Synaptobrevin 2 and the plasma membrane to both stimulate and measure the release of cat- proteins Syntaxin 1 and SNAP25 (Synaptosomal- echolamines from individual vesicles in individual Associated Protein of 25kDa). Before a vesicle cells. Stimulation occurs by either eliciting action can undergo fusion and release of its contents, it potentials in the cell or by electropermeabilizing the must dock at the plasma membrane and undergo cell to allow entry of stimulating substances into the a priming process that makes it ready for release. cell. Gold electrodes were fabricated on glass sub- Experiments using a SNAP25 based FRET con- strates using photolithography and insulated with a struct indicate the incorporation of SNAP25 into thick photoresist with openings to define the work- the SNARE complex, which appears to be the final ing electrodes at the bottom of cell-sized microw- step and it is this step, which defines the formation ells to trap single cells. A customized potentiostat of the primed state [1]. This step requires the was used to allow large transient currents to be ap- presence of the vesicular SNARE Synaptobrevin plied to stimulate cells followed by low-noise cur- 2. The assembly of the SNARE complex is fol- rent recordings to resolve picoamp current spikes lowed by its disassembly, reversing the FRET ratio resulting from quantal exocytosis. Trains of voltage increase. The changes in the SNARE complex pulses triggered release with spike characteristics leading to fusion are stimulated by activation of similar to those found with more physiological stim- voltage gated Ca2+ channels, increasing the uli, therefore release occurs from individual ves- Ca2+ concentration near the fusion site, which icles. Uptake of trypan blue into cells demonstrated is sensed primarily by the vesicular Ca2+ sensor that cell permeabilization occurred with strong stim- Synaptotagmin. However, Ca2+-independent but uli. Surprisingly, quantal exocytosis was depend- voltage-dependent secretion (CiVDS) has also ent on the chloride concentration in the bath solu- been reported in primary sensory neurons and in tion, but not the concentration of calcium. Using the sympathetic nervous system [2, 3]. Fusion the same electrode to stimulate and record release and transmitter release begins with formation of a of catecholamines from an individual cell allows de- narrow fusion pore connecting the lumen of secret- tailed information about exocytosis to be obtained ory vesicles to the extracellular space. Release and can be used to load membrane-impermeant of charged neurotransmitter molecules through a substances into the cell such as DNA or fluorescent narrow fusion pore requires charge compensation markers. and is therefore a process of electrodiffusion. The charge compensation for release of positively OR-136 charged catecholamines from chromaffin cells is Molecular mechanisms of vesicle priming, fu- coupled to Na+ influx through the fusion pore and sion pore formation and transmitter release by not to co-release of anions [4]. Anion substitution electrodiffusion experiments and molecular dynamics simulations Manfred Lindau indicate that very narrow fusion pores are cation University of Miami, United States selective but more dilated fusion pores become Various cell types release neurotransmitters, anion permeable. The transition occurs around a hormones and many other compounds that are fusion pore conductance of ˜300 pS [5]. The cation stored in secretory vesicles by exocytosis via selectivity of a narrow fusion pore accelerates the the formation of a fusion pore traversing the release of positively charged transmitters such as dopamine, noradrenaline, adrenaline, serotonin 169 and acetylcholine, while release of negatively brane region becomes more rigid, force is exerted charged transmitters such as ATP or glutamate on the SNARE transmembrane domains to drive may require a more dilated fusion pore. the merger of the two bilayers while the trans- SNARE complex completes folding into the cis- Supported by NIH Grants R01NS38200, SNARE complex. The flexibility of syntaxin’s jux- R01GM121787, R01GM085808 and tamembrane region can be modulated by the order R35GM139608. of the lipid bilayer, and the order of the bilayer is changed by Ca2+ dependent binding of the two C2 1. Zhao, Y., et al., PNAS, 2024. 121: domains of Syt1 to the membrane. The strong de- e2309161121. pendency of vesicle fusion efficiency on membrane 2. Huang, R., et al., PNAS, 2019. 116: 20201- order is further confirmed in live INS1 cell experi- 20209. ments where the plasma membrane is acutely en- 3. Zhang, C. and Z. Zhou, Nature Neuroscience, riched with lipids of defined acyl-chain saturation. 2002. 5: 425-430. In addition to fusion efficiencies and fusion kinetics, 4. Gong, L.W., G.A. de Toledo, and M. Lindau, Nat TIRF data from single vesicle fusion events con- Cell Biol, 2007. 9: 915-922. tains information about how the fluorescent content 5. Delacruz, J.B., et al., Cell Rep, 2021. 36: is released. Of particular interest is, how fast the 109580. content is released. We present data that show how interactions between PIP2 and conserved ar- OR-137 ginine residues of Syt1’s C2B domains regulate fu- Lipid protein interactions guide fusion pore sion pore expansion. opening and expansion during regulated exo- cytosis OR-134 Volker Kiessling Voltage-activation mechanisms of ion channels University of Virginia, United States with different electrical polarities Peter Larsson It has long been known that synatpotagmin- Linköping University, Sweden 1 (Syt1) is the sensor that triggers fast, evoked release of neurotransmitter by the fusion of syn- Hyperpolarization-activated cyclic nucleotide- aptic vesicles to the presynaptic membrane in gated (HCN) channels are essential for rhythmic response to calcium. It is also well estab- activity in the heart and brain. Mutations in lished that the SNAREs syntaxin-1a, SNAP-25, HCN channels are linked to heart arrhythmia and synaptobrevin-2/VAMP-2 form the core of the and epilepsy. HCN channels belong to the membrane fusion machinery that drives calcium- family of voltage-gated K+ (Kv) channels. triggered exocytosis not only in neurons but also Hyperpolarization-activated HCN channels and in secretory cells like insulin releasing beta-cells. the related depolarization-activated Kv10.1 (EAG) We recently proposed a mechanism where the and Kv11.1 (hERG) channels have very similar lipid bilayer is intimately involved in coupling cal- tetrameric structures with six transmembrane cium sensing to fusion. Using TIRF- (total in- segments (S1-S6) per subunit: S1-S4 form the ternal reflection fluorescence) and sd-FLIC (site- voltage-sensing domain (VSD) and S5-S6 form the directed fluorescence interference contrast) micro- pore domain (PD). In both Kv and HCN channels, scopy, we demonstrate that fusion of purified dense S4 is the positively charged voltage sensor and core vesicles and insulin granules with supported the C-terminal part of S6 forms the gate. However, membranes containing syntaxin-1a and SNAP-25 why HCN channels are activated by hyperpol- is strongly linked to the flexibility of the juxtamemarization whereas Kv channels are activated by brane region between SNARE domain and trans- depolarization is not clear. Using voltage clamp membrane domain of syntaxin. As the juxtamem- fluorometry, FRET, cysteine accessibility, and 170 cysteine crosslinking, we have measured the S18 - Bridging the gap between movement of S4 and the gate in HCN channels experimental and modeling studies to determine the mechanism of activation by hy- in PEF electroporation: a Young perpolarization. Our main hypothesis is that small differences in free energy between the closed and Professional‘s perspective open states, due to different interactions between S4 and the pore in different channels determines Wednesday morning Track C whether HCN channels or HCN-related channels Sep 18, 10:40 - 12:10 open by hyperpolarizations or depolarizations. OR-138 OR-133 Evaluating biological membrane response to Visualizing membrane fusion and budding in PEF: A multiscale computational approach live cells Laura L. Caramazza 1, Paolo Marracino2, Micaela Ling-Gang Wu Liberti1, Francesca Apollonio1 1 NINDS, NIH, United States Universita Degli Studi Di Roma La Sapienza, Italy 2Rise technology S.R.L., Italy Membrane fusion and budding mediate fun- damental processes like intracellular trafficking, In recent decades, there has been a surge in exocytosis, and endocytosis. Fusion is thought the study and application of pulsed electric fields to open a nanometer-range pore that may sub- (PEFs) in biomedical research. Characterized by sequently close or dilate irreversibly, whereas short durations (ranging from milliseconds to nano- budding transforms flat membranes into vesicles. seconds) and high intensities up to megavolts per Here I will describe a model of membrane fusion meter (MV/m), PEFs are utilized in established and budding obtained from direct visualization of techniques like electrochemotherapy, as well as vesicle fusion and endocytosis in live chromaffin emerging methods such as gene therapy and con- cells. Fusion involves hemi-to-full fusion, pore trolled drug delivery [1]. The investigation of the expansion, constriction and/or closure while fus- biophysical mechanisms underlying the interac- ing vesicles may shrink, enlarge, or receive an- tion of electromagnetic fields (EMFs) has led to other vesicle fusion; endocytosis follows exocyt- a consensus that molecular-level alterations can osis primarily by closing Ω-shaped profiles pre- trigger subsequent responses as biological com- formed through the flat-to-Λ-to-Ω-shape transition plexity increases, ultimately yielding macroscopic or formed via fusion. Calcium/SNARE-dependent consequences for the organism [2]. The chal- fusion machinery, cytoskeleton-dependent mem- lenges associated with studying these mechan- brane tension, osmotic pressure, calcium/dynamin- isms through traditional experimental approaches dependent fission machinery, and actin/dynamin- underscore the necessity of numerical investiga- dependent force machinery work together to gen- tions. Consequently, a multiscale computational erate fusion and budding modes differing in approach is proposed here, and it could be the key pore status, vesicle size, speed and quant- to planning and monitoring EMFs-based biomed- ity, controls release probability, synchronization ical applications. and content release rates/amounts, and underlies Such a multiscale approach endeavors to link exo-endocytosis coupling to maintain membrane the outcome of discrete modeling conducted on a homeostasis. molecular level with the inputs needed to provide a continuum simulation on a microscopic scale level. In fact for example Molecular dynamics (MD) sim- ulations have elucidated that electropermeabiliza- tion arises from phospholipid rearrangements and the formation of aqueous pores within the mem- brane [3-4]. However, MD simulations are typ- 171 ically performed on reduced models (with dimen-2Université Paul Sabatier, France sions up to tens of nm) and timescales (on the order Electroporation is a physical method that is of hundreds of ns) to mitigate computational costs gaining momentum in cancer treatment, to erad- while maintaining atomistic precision. On the con- icate cancerous cells, while sparing surrounding trary, whole cell exposure to PEFs induces various healthy tissues. Pulsed electric fields act on cellular signaling pathways, primarily through the cell membranes and can induce the occurrence well-known process of electropermeabilization or of cell membrane “pores”. Nevertheless, more electroporation of cellular membranes. These phe- knowledge is needed on electroporation effects nomena usually can be represented via continuum on the surrounding healthy tissues and on the models with dimensions up to �m and timescales extracellular matrix (ECM). Adjacent tissues are on the order of hundreds of �s. essential for structure and nutrition and include Thus, besides molecular modeling of mem- supporting/connective tissues and blood vessels. branes, microdosimetric simulations have recently Within them, the ECM contains the collagen, garnered significant attention [5]. These simula- which is one of the most abundant bodily proteins. tions estimate the electrical quantities induced at Omnipresent in our body, this protein also plays subcellular and cellular levels, solving the quasi- an important role in diseases such as fibrosis or static EM problem and the pore evolution over time cancer. Thus, the study of collagen is primordial in a multiphysics manner. Recent advancements in cancer research and treatment, because the focus on employing advanced 3D cellular mod- ECM is involved in tumor development, dissemin- els reconstructed from microscope images. These ation, and metastasis progression. Furthermore, models accurately capture the irregular shape and type I collagen is among the most frequently dimensions of intracellular compartments, provid- up-regulated collagens in solid cancers, yet its ing spatial distribution of electrical quantities that implication in disease progression is still unclear can be correlated with physicochemical measure- [1]. ments obtained from experiments [6]. While mi- To determine if square wave electric pulses, such crodosimetric investigations offer valuable insights, as the ones used for electroporation, can contrib-their accuracy remains confined to the microscopic ute to the remodeling of the ECM, we evaluate level. if and to what extent electroporation parameters [1] D. Miklavčič and T. Kotnik, Bioelectromag- might contribute to the alteration of the collagen netic Medicine, 2021. structure. This is also of particular importance in [2] F. Apollonio et al., IEEE Trans Microw Theory the context of lower intensity electric fields, which Tech, 2013. can stimulate wound healing and could increase [3] M. Tokman et al., PLoS ONE, 2013 collagen secretion by fibroblasts [2]. [4] P. Marracino et al., Bioelectrochemistry, To shed light on biological effects on collagen itself 2022. and on collagen building blocks (tropocollagen) [5] M. Scuderi et al., Bioelectrochemistry, 2022. upon pulsed electric field exposure, dermal sheets, [6] A. De Angelis et al., Front Bioeng Biotechnol, produced with primary dermal fibroblasts, and 2020. tropocollagen monomers are exposed to square OR-139 wave micro- and millisecond electric pulses at Effects of pulsed electric fields on collagen self-different electric field intensities. The collagen assembly and collagen secretion by dermal is analyzed with second harmonic generation fibroblasts microscopy and its amount and distribution is Emma Barrere 1, Nicolas Mattei1, Ophelie Cordier1, assessed. The obtained data will be subsequently Marie-Pierre Rols1, Muriel Golzio1, Hermes Desgrez- used to model structural alterations with Coarse Dautet2, Matthieu Chavent1, Jelena Kolosnjaj-Tabi1 Grain Molecular Dynamics (CG-MD) simulations 1Université de Toulouse, France using the MARTINI model. 172 This work highlights the implication of pulsed elec-to 140 GHz for our model. Introduction of water tric fields in collagen organization within dermal layers into the model modulates the frequencies, sheets. Moreover, it also address the question if resulting in reduced variations among snapshots. collagen formation from tropocollagen monomers Notably, we discern a distinct cluster of the first can be impacted, or if collagen secretion can be three modes within the subTHz frequency domain. amplified by fibroblasts stimulation with pulsed Leveraging our normal mode ensemble analysis, electric field. we predict the electromagnetic absorption of tubulin’s vibration modes, incorporating vibrational [1] Hsu, K., et al. Nat Commun., 2022, 13(1):7078. damping as a variable. [2] Nguyen, E.B., et al. J Electroanal Chem This investigation advances our understanding (Lausanne), 2018, 812:265–272. of the vibrational and electromagnetic properties of tubulin, establishing a groundwork for future OR-140 explorations into manipulating protein dynamics Exploring Vibrational and Electromagnetic through external electromagnetic fields. Properties of Protein Tubulin using Normal Furthermore, we delve into the complex and Mode Analysis and Molecular Dynamics dynamic post-translational modification of tubulin Simulations termed polyglutamylation. Multiple molecular dy- Saurabh Kumar Pandey, Michal Cifra namics simulations, incorporating various lengths Institute of Photonics and Electronics of the Czech and positions of polyglutamate chains on the Academy of Sciences, Czech Republic C-terminal tails of tubulin- αβ dimers, are currently Proteins, serving as nature’s intricate nanoma-in progress. Our objective is to elucidate the chines, orchestrate essential biological processes influence of electromagnetic fields on the structural by communicating motions across their complex characteristics of tubulin and how the presence structures. Understanding the elusive mechan- of polyglutamylated side chains influences the ism underlying the connection of distant sites structure and dynamics of tubulin dimers. within proteins often necessitates computational methods due to the challenging nature of direct Acknowledgments: The authors thank the Czech experimental spectroscopic detection of these Science Foundation project no. 20-06873X for the motions. In this investigation, we utilize normal support. mode analysis to explore the dominant vibration OR-141 modes of an all-atom model of tubulin protein and Nanosecond pulsed electric fields and gold scrutinize their interaction with electromagnetic nanoparticles for cancer treatment fields. Our primary aim is to discern the first few Rosa Orlacchio 1, Jelena Kolosnjaj-Tabi2, Nicolas vibrational modes and their frequencies, which Mattei2, Lionel Michard3, Hafsa Tjiou3, Léna Serradeil1, correlate with specific conformations of tubulin Isabelle Lagroye1, Florence Poulletier de Gannes1, Yann dimers implicated in the dynamic assembly and Percherancier1, Philippe Leveque3, Marie-Pierre Rols2, disassembly of microtubules. Delia Arnaud-Cormos3, Muriel Golzio2 Our inquiry focuses on a model of the tubulin- αβ 1Univ. Bordeaux, France dimer complexed with two GTP molecules, subjec- 2Université de Toulouse, France ted to molecular dynamics (MD) simulations. We 3University of Limoges, France extract multiple snapshots from these simulations The considerable progress in pulsed power al- to enhance sampling and employ normal mode lowed the generation and delivery of high-intensity analysis to ascertain the vibrational frequencies. (MV/m) ultrashort pulsed electric field (usPEF) of Our findings demonstrate that these frequencies hundreds nanosecond and picosecond paving the are situated in the sub-terahertz (subTHz) band, way toward novel targeted cancer therapies. The particularly within the range of approximately 40 unique capability of usPEF to trigger regulated cell 173 death or initiate an adaptive immune response pulses and ion channels. To achieve this, we are showed high efficacy in treating cancers in pilot implementing an innovative biophysical spectro- studies in vivo and clinical trials. The main advant- scopic methodology based on bioluminescence en- age of this technique is achieving highly localized ergy transfer, i.e., BRET. Detailed results will be tumor destruction without thermal ablation. While presented and discussed at the conference. most of published studies focus on pulses ≥ 100 ns, our recent research reveals that, under specific OR-142 exposure conditions, pulses from 2 to 10 ns (up Deciphering the behavior of multicellular 3D to 100 kV/cm) can elicit cellular death and inhibit spheroids exposed to high-intensity pulsed the growth of 3D multicellular spheroids (MCSs) de- electric fields by a mathematical modeling ap- rived from HCT-116 colorectal carcinoma cells. In proach parallel, theory suggests that PEF can be locally Annabelle Collin 2, Jelena Kolosnjaj-Tabi1, Muriel Golzio1, Marie-Pierre Rols1, Clair Poignard2 amplified by conducting nanoparticles (NP), such 1Université de Toulouse, France as gold nanoparticles (AuNPs). Spherical AuNPs 2Université de Bordeaux, France could theoretically enhance the electric field up to 3-fold and elongated NP may theoretically locally While irreversible electroporation (IRE) is enhance the electric field up to 100 times. While at- efficient in most cases, with a complete ablation tractive, this effect has not been extensively studied rate of about 75% of treated tumor nodules after experimentally and has never been studied in com- an IRE procedure [1], it is known that in some bination with usPEF. While some studies aimed patients ablation is not successful and the event to assess the combined effect of electric pulses may lead to accelerated tumor growth. To better and spherical AuNPs on individual cells, the com- understand the effects of pulsed electric fields bined effect of spherical or elongated AuNPs on on tumor response, we used multicellular tumor 3D MCSs remains to be investigated. This work spheroids as an in vitro microtumor model. Multi- aims to combine usPEF with AuNPs to develop a cellular tumor spheroids are an important tool in novel tool that could improve cancer therapy. Pre- cancer research as they bridge the gap between liminary results have been performed on two types 2D cell models and in vivo tumors by mimicking of spheroids derived from colorectal cancer (HCT- the characteristics of early avascular tumors. In 116) and hepatocellular carcinoma cells (Hepa 1- this work, multicellular HCT-116-GFP spheroids 6). MCSs with or without AuNPs, located outside were exposed to different electric field intensities MCSs in the pulsing buffer or embedded within can- and their volumes were monitored by fluorescence cerous cells, were exposed in HBSS (Hanks’ Bal- and bright field microscopy. To characterize their anced Salt Solution) buffer to trains of 500 unipolar regrowth after pulse delivery with respect to pulse pulses (20 Hz) with field intensity of approximately magnitude, we developed a two-step approach 50, 75, and 100 kV/cm. These two NP localizations based on mathematical modeling and data assim- (at the external membrane or within the cells) have ilation. a twofold interest. Outside the cells, the NPs could First, we consider a reduced Gompertz model - serve as antennas amplifying the electric field. In- a very classical model for tumor growth [2] - for side the cells, NPs could serve as internal local volume evolution. This volume approach showed agents, destabilizing cells homeostasis. Cellular vi- encouraging preliminary results. However, it did ability, spheroids growth, and local NPs’s effect de- not allow to properly quantify the effects of electro-pending on their peri- or intra-cellular position were poration and to account for tumor heterogeneity. evaluated. In parallel, we are exploring the possib- These two limitations motivated the introduction ility to monitor in real-time, non -invasively and on of a system of partial differential equations in a living cells, the activity of ion channels possibly im-second part. Three densities of tumor cells were plicated in cancer to unravel the molecular mech- considered: the proliferative cells, the quiescent anism underlying the interaction between electric 174 cells and the cells whose functioning was altered ies can be very useful to prepare the experiments by the effect of the electric field. To fit the biological or to explain the results already obtained experi-data with our models, we considered a para- mentally. In turn, experimental studies are the ulti- meter estimation strategy based on a population mate confirmation of modeling results. In the field approach that allows to compensate for sparse of electroporation research, some research sub- sampling times and measurement uncertainties by topics may particularly highlight this complement- constraining the variability of the parameters in the arity. For example, in the context of studying the ef-population. With this approach, it was possible to fects of subnanosecond pulsed electric fields (sub- determine the percentage of cells destroyed and nsPEFs) on the electropermeabilization of cells, it the percentage of cells whose functioning was was a prerequisite from the beginning to know pre- altered by the effect of electroporation for each cisely the electric field to which the cells would be value of the electric field. exposed. Therefore, it was necessary to perform Interestingly, our results indicate that the partially 3-D electromagnetic modeling of the electric field irreversible electrical pulses may lead to acceler- distribution generated by voltage pulses inside the ated cell regrowth. system. This, together with experimental measure- The advantage of our approach, is that soon after ments of the output voltage of the generator and the electroporation, we can detect the trend of the voltage recorded closest to the sample by a tumor growth. In practice, this model can thus Vdot probe system, allowed a correct interpreta- allow to assess which tumors were inefficiently tion of the experimental results. This part of the treated, and that the patient is on his or her way to study was performed in the SIAME laboratory spe-relapse. cialized in high power pulsed systems. For experi- mental convenience, the exposure system was de- [1] R. Cannon et al. Safety and early efficacy signed to allow the use of commercial electropor- of irreversible electroporation for hepatic tumors ation cuvettes in which cells were placed in sus- in proximity to vital structures. Journal of surgical pension. We needed to ensure the homogeneity of oncology, 2013. the electric field within the cuvette, the value of the [2] C. Vaghi et al. Population modeling of tumor electric field achieved, as well as the shape of the growth curves and the reduced Gompertz model waveform. In particular, we now hypothesize that improve prediction of the age of experimental both the value of the electric field reached and the tumors. PLoS computational biology, 2020. rise and fall times of the impulse are of paramount importance in the electropermeabilization obtained OR-143 with such transient PEFs. Experimentally, we have On the complementarity of modeling and exper- carried out a parametric study in our laboratory to imentation in the study of biological effects of evaluate the influence of the number of pulses ap- subnanosecond pulsed electric fields plied, the pulse repetition frequency, the electric Leslie A. Vallet 1, Njomza Ibrahimi2, Laurent Ariztia2, field amplitude, as well as the temperature of the Marc Rivaletto2, Antoine Silvestre de Ferron2, Bucur M. sample during exposure to PEFs. In the process of Novac3, Alexey Zhabin2, Clair Poignard4, Mounir Tarek5, interpreting the experimental results obtained and Laurent Pecastaing2, Franck M. Andre6, Luis M. Mir6 further investigating the mechanisms behind the 1Université Paris-Saclay, France observed electropermeabilization, we resorted to 2Université de Pau et des pays de l’Adour, France other types of modeling studies. These included 3Loughborough University, United Kingdom mathematical modeling performed at the Institut de 4Univ. Bordeaux, France Mathématiques de Bordeaux as well as molecu- 5Université de Lorraine, France lar dynamics modeling studies. With regard to our 6Centre National De La Recherche Scientifique, France question, all these elements have been comple-The complementarity between modeling and mentary pieces of a puzzle, allowing us to consol- experimental studies is undeniable. Modeling stud- idate our mechanistic hypotheses. This also high- 175 lights how the collaboration within the electropora-OSA mouse models. In canine MM and OSA pa- tion research community has been incredibly useful tients the electrovaccination procedure was safe, in our research on sub-nsPEFs. immunogenic and with a clinical benefit, signific- antly increasing the survival of vaccinated dogs as S16 - Electroporation in veterinary compared to controls. Induced-cellular and anti- body responses, encompassing multiple mechan- and translational medicine isms of action, correlated with patients’ survival. As a step forward, we have initiated to evalu- Wednesday morning Track D ate the potential combination of CSPG4-targeting Sep 18, 10:40 - 12:10 with front-line therapies for MM (BRAF inhibitors, BRAFi, Vemurafenib) and OSA (chemotherapy). OR-144 Ongoing studies suggests that CSPG4 could be re- Chimeric DNA vaccination against the Chon- lated to the emergence of BRAFi-resistance: pre- droitin Sulfate Proteoglycan 4: a potential allied liminary data show that in MM cells, CSPG4 ex-in combinatorial approaches for the treatment pression drops in the early response to BRAFi, to of melanoma and osteosarcoma reemerge again in resistant cells. Moreover, pre- Federica Riccardo, LIdia Tarone, Carlotta Montana, Davide Giacobino, Lorenza Parisi, Selina Iussich, Gi-liminary bioinformatic analysis of public datasets useppina Barutello, Laura Conti, Maddalena Arigoni, from melanoma patients’ biopsies collected before Paolo Buracco, Emanuela Morello, Federica Cavallo and after targeted-therapy, revealed similar trend University of Turin, Italy with an early drop in CSGP4 mRNA followed by a raise in therapy-resistant metastases. Our in Discovering effective therapies to combat tu- vitro results suggest that the combination between mor relapse and progression remains an unmet HuDo-CSPG4 antibodies induced by electrovac- need due to resistance to conventional treatments. cination and BRAFi could have a synergistic effect Rational combinatorial approaches to attack tu- against the malignant behavior of MM cells. mor cells from multiple sides are needed for dur- Moreover, our in vitro studies have shown that able clinical response. Combining standard ther- when treated with chemotherapeutic agents, an in- apies with cancer vaccines against tumor associ- crease in CSPG4 expression in OSA surviving cells ated antigens, such as the chondroitin sulfate pro- is observed, suggesting a possible involvement of teoglycan (CSPG)4, which is barely expressed in CSPG4 in resistance development. In line, com- normal tissues, but overexpressed by different tu- bining vaccine induced anti-CSPG4 antibodies with mors, could be pivotal. We have characterized chemotherapy enhance its effects on human and the relevant CSPG4 role in malignant melanoma canine OSA cells and spheres. (MM) and osteosarcoma (OSA), leading to invest- In conclusion, these results highlight the rel- igations of the potential of anti-CSPG4 DNA vac- evant CSPG4 role in MM and OSA and provide cination for their treatment alone or in combinatorial the bases to propose HuDo-CSPG4 electrovaccin-approaches with the standard of care. ation to effectively break immune tolerance and Since the CSPG4 is a non-mutated, poorly treat CSPG4+ MM and OSA patients in rational- immunogenic, self-antigen, we generated a hu- based combinatorial approaches, including BRAF man (Hu)-dog (Do)-CSPG4 hybrid plasmid, to over- targeted-therapy for MM, and chemotherapy for come host’s unresponsiveness. We tested HuDo- OSA, to finally improve patients’ survival. CSPG4 plasmid intramuscular injection followed by in vivo electroporation (electrovaccination) in mice challenged with transplantable tumors and in dogs with surgically resected spontaneous CSPG4+ MM and OSA. HuDo-CSPG4 was immunogenic and endowed with an anti-tumor potential in MM and 176 OR-145 GET were performed using an EPV-100 electropor- Electrochemotherapy plus IL-2+IL-12 gene ator (BIOTEX, Argentina), delivering 8 100 us long electrotransfer in spontaneous inoperable pulses of 1,000 V/cm at a repetition frequency of stage iii-iv canine oral malignant melanoma 5,000 Hz for ECT and 1 Hz for GET. Sergio S. Salgado 1, Matias N. Tellado2, Mariangela Results: An objective response (OR) for the local De Robertis3, Daniela Montagna4, Daniela Giovannini5, treatment was achieved in 80% of ECT+GET group Sebastian D. Michinski2, Emanuela Signori7, Felipe with a complete response (CR) in 30% of patients Maglietti6 and 50% of partial response (PR). In the control 1Universidad Peruana Cayetano Heredia, Peru group the OR was of 65% (CR of 5% and PR 60%). 2Universidad de Buenos Aires, Argentina The results showed a peak of IL-2 and IL-12 in 3University of Bari ‘A. Moro’, Italy blood levels after 7-14 days of transfection The me- 4Academia Nacional de Medicina, Argentina dian survival time (MST) was of 5.5 months (3- 5ENEA, Italy 32months) for the ECT+GET group. MST for the 6Fundación H.A Barceló, Argentina control group was 6 months (2-17months). The 7Institute of Translational Pharmacology, Italy median progression free survival (PFS) was 5.5 Introduction: Electrochemotherapy (ECT) and moths (3-32months) for the GET+ECT group, while Gene electrotransfer (GET) are valuable tools in for the ECT group was 4 months (1-16months). A the treatment of cancer. Both have the ability significantly difference was noted for the PFS of the to enhance an immune response; nevertheless, patients in the treatment group (p=0.0284). The ECT mainly induces a local immune response but treatment was well tolerated with only grade 1-2 fails to induce an effective systemic immune re- side effects presents for all groups. sponse, mainly because the stimulation of im- Conclusion: The addition of GET with peritumoral mune response induced by ETC is due to the re- canine IL-2 and intramuscular canine IL-12 can im- lease of damage-associated signal which triggers prove treatment outcomes by increasing the PFS a humoral immune response that lack the strength without adding discomfort or side effects. to affect distant metastasis. Switching this humoral OR-146 response to a cellular immune response would in- Adjuvant Electrochemotherapy and/or Radio- crease the possibility of an abscopal effect. This therapy in Feline Injection Site Sarcoma possibility could be enhanced by the transfection of Matias N. Tellado 2, Franco Portillo2, Vanda Guillen2, canine IL-2 and IL-12 plasmids by GET. The aim of Tadeo Sabella2, Maura Diaz2, Felipe Maglietti1 this retrospective study is to evaluate the addition 1Fundación H.A Barceló, Argentina of gene electrotransfer (GET) of canine IL-2 per- 2VetOncologia, Argentina itumorally and IL-12 intramuscularly for the treat- ment of inoperable stage III-IV canine oral melan- A retrospective analysis of cats with confirmed oma. diagnosis of Feline Injection Site Sarcoma (FISS) Materials and methods: Thirty patients were in- was conducted in our clinic between 2020 and cluded, of which twenty received only ECT (control 2022. The aim of this work was to evaluate the group), and ten a combination of ECT+GET (treat- impact of surgery combined with electrochemother- ment group). For the ECT treatment, intravenous apy (ECT), or radiotherapy(RTP), or both in the dis- bleomycin was used. Eight minutes after the ad- ease free survival (DFS). ministration of the drug, the electric pulses were The analysis included 85 feline patients with an av- delivered. For the treatment group. after the ECT erage age of 10.4 years (3-20). Fourty-one percent session, the GET session was performed transfect- (36/85) of the included patients had a recurrence ing canine IL-2 to the periphery of the tumor, and from previous surgery. The analysis included the canine IL-12 to the quadriceps muscle. The GET histological grade (G1, G2, and G3), histological procedure was repeated 28 days later. ECT and margins (compromised, narrow, and clean), and 177 anatomical location of the sarcoma (Flank/thoracic OR-147 and Dorsal/interscapular/spine). Retrospective analysis of the outcome and sur- Four treatment groups were defined: 1. Sur- vival time of dogs with mast cell tumors with dif- gery alone (n=21), 2. Surgery+ECT (n=25), 3. ferent degrees of malignancy treated with elec- Surgery+RTP (n=27), and 4. Surgery+ECT+RTP trochemotherapy (n=12). Javier Ojeda, Paulina Sandoval ECT treatment was performed intraoperatively in a Universidad Austral de Chile, Chile single session using plate or needle electrodes de- Electrochemotherapy (ECT) has shown good pending on the depth of the margins required. In- response in low-grade mastocitomas (MCT), either travenous blemycin was administered followed by as the main or adjuvant therapy after surgery. the application of electric pulses using an EPV-300 A retrospective analysis from data of the medical Electroporator (BIOTEX SRL, Argentina). Pulse records of patients with MCT without metastasis trains consisted of 8 square wave pulses of 100us and treated with ECT was performed (2018-2023). long 1000V/cm at 5KHz. In the case of RTP, it was In total, 29 patients and 39 tumors were analyzed. performed after the surgical wound healed, using According to the histopathological analysis (Kiupel an orthovoltage unit in 12 fractions of 3.3-4Gy, with grading) 28 tumors had low grade and 11 tumors a total accumulated dose of 40-48 Gy. had high grade. For all patients, ECT with bleomy- Results showed that when considering the histo- cin was used at a dose of 15IU/m2, using a Veterin- logical grade, no differences was found between ary Cellporator VetCP 125 device with pulse needle grades G1, G2, and G3, regarding DFI in any of electrode: 8 monophasic square; pulses duration the treatment’s groups. When considering patients of 100 µ s ; frequency of 5 Hz and pulse amplitude with clean surgical margins, none of the treatment was 800-1200 volts/cm. The evaluation time for re- groups differ significantly in terms of DFI p>0.05. sponse to treatment for each dog was at least 6 However, patients with compromised and narrow months with routine monthly controls. The size of surgical margins that received additional treatment the tumors was recorded prior to ECT. The average after surfery had a higher DFI when compared size of the tumors was 4.6 cm3. According to the with patients that received surgery alone (tp=0.05, location, 10 were located in the head (25.6%), 12 in p=0.0063, and p=0.05). the limbs (30.7%), 5 in the thoracic area (12.8%), 4 Side-effects in group 1 were one case of delayed in the pectoral area (10.2%). To a lesser extent, the healing related to the polypropylene mesh used. tumors were located in zone 3 of mammary glands In group 2, two patients presented partial dehis- (7.6%), 1 in the neck (2.5%), 2 periscrotal (5.1%) cence of the surgical wound in the flap that re- and 2 perivulvar (5.1%). solved without problems. In group 3, we observed The evaluation during the period it was determined two cases of dry radiodermitis that resolved quickly that 23 patients (79%) had complete remission. Of after 7 days. All the patients in groups 3 and 4 them, 18 patients had low-grade, and 5 patients presented alopecia and change in hair color of the had a high degree of malignancy. It was determ- irradiated area. All side-effects were categorized ined that 6 patients (21%) had disease progression, as mild and were well tolarated by the patient. all of them had high-grade mast cell tumors. Com- In conclusion, additional treatment with ECT, RTP plete remission was achieved in 27 low-grade tu- or both in patients with compromised or narrow sur- mors and 6 high-grade tumors. The remaining 6 gical margins improved DFI with acceptable side- tumors had disease progression, having a high de- effects. gree of malignancy. According to the survival time analysis of the treated patients and their histopathological grade, it was determined that low-grade MCT patients had a survival of 1101 days on average compared to 178 high-grade MCT patients where the average sur-osis. Complete response (CR) was achieved in vival was 210 days (p <0.05). Considering the av- 62% of the cases (13/21), while partial response erage tumors size of 4.6 cm³, the survival curve for (PR) was achieved in 24% (5/21). The median sur- those patients with tumors larger than 4.6 cm³ was vival and disease-free survival times were 284 and 124 days with a significant difference with the sur- 270 days, respectively. Overall survival was sig- vival of patients with tumors < 4.6 cm³, which was nificantly better in the dogs who achieved a CR. In 900 days (p<0.05). conclusion, ECT was welltolerated in dogs with UC, The results concluded that ECT is a method that demonstrating its safety and feasibility. These data can achieve complete remission in low-grade MCT. pave the way for new Positive outcome were achieved in neoplasia that studies aimed at evaluating the effectiveness of had a size less than 4.6 cm³ . On the other hand, ECT in canine bladder UC as a translational model with high degree of malignancy of MCT, therapeutic for human disease. responses will depend on the size of the tumor. This is the first study that evaluates the ECT for P7 - Electroporation for clinical use the treatment of low and high histopatological ma- lignancy MCT. Wednesday morning Track E Sep 18, 10:40 - 12:10 OR-148 Evaluation of the safety and feasibility of elec- OR-149 trochemotherapy with intravenous bleomycin Enhancing sensitivity to radiation therapy us- as local treatment of bladder cancer in dogs ing electroporation in a radio-resistant model of Marcelo Monte Mor Rangel 1, Lais Calazans Menes-oesophageal cancer cal Linhares2, Daniela Ota Hisayasu Suzuki3, Krishna Aoibhín Woods 1, Aisling Uí Mhaonaigh1, Aisling Duro de Oliveira4, Felipe H. Horacio Maglietti5, Andrigo Heeran1, Lorraine Smith1, Stephen Maher1, Niamh Barbosa De Nardi2 Lynam-Lennon1, Declan Soden2, Jacintha O’Sullivan1 1Vet Câncer Surgical Oncology, Brazil 1Trinity College Dublin, Ireland 2UNESP JABOTICABAL, Brazil 2Mirai Medical, Ireland 3IEB - UFSC, Brazil Oesophageal cancer is a poor prognostic can- 4VC LAB PATHOLOGY, Brazil cer with a five-year survival of less than 25%, and 5Fundación Barceló-CONICET, Anatomia, Argentina accounts for over 500,000 cancer-related deaths Local treatment of canine urothelial carcinoma annually. A significant number of patients fail to (UC) of the bladder is a challenge. More than respond to conventional treatments, chemotherapy 90% of the cases invade the muscular layer, more and radiation therapy, where only approximately than 50?velop on bladder sites with a difficult sur- 30% of patients achieve a pathological response. gical approach and often requiring radical sur- Despite advancements in the multi-armed treat- gical procedures. This study aims to evaluate ment approach of oesophageal cancer, numerous the safety and feasibility of electrochemotherapy initial responders to conventional therapies acquire (ECT) with intravenous bleomycin (BLM) as a local treatment resistance, which poses a particular clin- therapy for bladder UC. This prospective study in- ical challenge as their tumours become increas- cluded 21 dogs with spontaneous bladder UC. Re- ingly refractory to treatment. gional/distant As such there is a pressing need for new therapies metastases and neoplastic infiltration of the to improve the survival and quality of life of these serosa was considered the main exclusion criteria. patients and technologies to enhance the efficacy We had no deaths during ECT or in the imme- of current standard of care treatment regimens. diate postoperative period, and no suture dehis- As an anti-tumour strategy, electroporation has cence. Most dogs (19/21) developed mild adverse shown promise clinically in the treatment of cu- effects, whereas two dogs developed ureteral sten- taneous malignancies, but it’s therapeutic benefit 179 could extend to several solid malignancies. Tech-OR-150 nological advancements in the endoscopic deliv- Electroporation treatment alters the inflammat- ery of electroporation will facilitate the treatment of ory tissue microenvironment in the human in-gastrointestinal malignancies (EndoVE, Mirai Med- flammatory condition, Barrett’s Oesophagus ical, Galway). Lorraine Smith 1, Cian Gargan1, Aisling Uí We hypothesise that electroporation could function Mhaonaigh1, Irene Narinda1, Aoibhín Woods1, Aoife as a treatment-sensitizer to enhance the response Kilgallon1, Matthew McElheron1, Meghana Menon1, of resistant oesophageal tumour cells to ionizing ra- Fiona O’Connell1, James Phelan1, Declan Soden2, Jacintha O’Sullivan1 diation. 1Trinity College Dublin, Ireland Using an isogenic model of radio-resistant oe- 2Mirai Medical, Ireland sophageal adenocarcinoma developed within our lab, we investigated if electroporation, using the Barrett’s Oesophagus (BO) is an inflammatory ePORE electroporator (Mirai Medical, Galway), condition thought to be caused by chronic gast- could enhance treatment efficacy in both radio- ric acid reflux. It is the only known precursor sensitive and radio-resistant cells. We examined for oesophageal adenocarcinoma (OAC) increas- the surviving fraction of cells using a clonogenic asing the risk by 30-125 fold. Approximately 12% of say, as a measure of the cell’s reproductive capa- OAC patients when diagnosed had a previous dia- city. gnosis of BO but approximately 57% have concur- We identified that electroporation treatment prior to rent BO. Current treatments for BO include Radio exposure to 2Gy irradiation significantly reduced tu- Frequency Ablation (RFA) and cryotherapy for pa- mour cell viability, in both the radio-sensitive, and tients who have progressed to dysplasia. However, radio-resistant cell lines, p = 0.0333 (*) and p = these treatments are not without side effects, RFA 0.0033 (**) respectively, compared to irradiation is a thermal treatment while accuracy with cryother- alone, with the effects more pronounced in the apy is difficult. Thus, we hypothesise that electro- radio-resistant cells. poration is a feasible new treatment option. This work provides novel insight to the potential Using a novel human ex vivo Barrett’s explant of reversible electroporation as a radio-sensitizer, model, we treated 10 matched Barrett’s and adja- using an isogenic model of radio-resistant oeso- cent normal tissue ex vivo biopsies (five short and phageal cancer. To interpret this response, we five long segment patients) with various electropor- are validating these findings at alternative voltages ation (EP) conditions- EP only, calcium EP (5 mM and testing DNA damage and repair mechanisms CaCl2) and Irreversible Electroporation (IRE) using to help explain mechanistically this significant ra- the ePORE electroporator (Mirai Medical). After, diosensitizing effect by electroporation. Currently untreated and treated biopsies were cultured for 24 we are also examining the potential of electropor- hours. The tissue cultured media was then used ation to enhance chemosensitivity using a novel to analyse inflammatory secretions using the MSD isogenic model of cisplatin-resistant oesophageal multiplex platform (54-plex). Biopsies were histolo- cancer treated with an IC50 dose of cisplatin. gically categorised according to the Vienna grading system. Results confirm at baseline the ex vivo Bar- rett’s tissue explants were indeed more inflammat- ory compared to their matched normal tissue, with significantly higher levels of IFN- γ, IL-6 and IL-8 (p < 0.01). Compared to untreated Barrett’s tis- sue, EP only and CaEP did not significantly alter the levels of any cytokines apart from CaEP signi- ficantly reducing levels of VEGF (p<0.05). In con- 180 trast, IRE significantly reduced the levels of differ-erative electrochemotherapy of the posterior resec- ent cytokines compared to untreated tissue ((GM- tion surface. We outline the study protocols and CSF, IL-8, MCP-1, IL-10, IL-4, IL-6, TNF- α, IL-2, initial findings from a prospective pilot study invest-VEGF and IFN- γ) (p<0.01)). This may be a con-igating this approach. sequence of IRE directly killing the tissue. Then, Methods: Patients meeting the inclusion criteria clinical variables such as Vienna Grade and length and consenting to participate, with resectable pan- of disease segment were correlated with cytokine creatic head ductal adenocarcinoma, were enrolled profiles. With increasing disease severity, Bar- in the study. Following surgical resection, electro- rett’s tissue treated with CaEP negatively correl- chemotherapy with bleomycin was performed us- ated with most cytokines while IL-1B, TNF-a, IL- ing plate electrodes to cover the area between ana- 8, MCP-1 and MIP-1a were statistically significant tomical landmarks. (p<0.05). A similar trend was observed for the Results: Electrochemotherapy of the posterior re- same cytokines with increasing segment length. section surface was feasible in all 7 patients. Pan- Contrastingly, Barrett’s tissue treated with IRE with creatic fistula grade B occurred in only one patient; increasing disease severity positively correlated other observed complications were predominantly with most cytokines with IFN- γ statistically signific-Clavien-Dindo grade 2 or lower. Hospital mortality ant (p<0.05). This trend was also observed with was at 0%. longer Barrett’s disease segment length with IL-22, Conclusions: Our preliminary findings indicate that IL-15 and IL-23 statistically significant (p<0.05). the hybrid approach combining surgery with intra- Future work will analyse how these treatments operative electrochemotherapy appears to be both affect immune cell activity namely T cells and safe and feasible. Dendritic cells. We will also incorporate more pa- tients, especially those with advanced disease and OR-152 include a HFIRE treatment condition to further test Novel Synergistic Electric Pulses and First Hu- the validity in using electroporation as a treatment man Cancer Clinical Trials: Towards the Bal- for BO. ance between Negligible Muscle Contraction and Enhanced Ablation OR-151 Hongmei Liu, Jianhao Ma, Shoulong Dong, Chenguo Intraoperative electrochemotherapy of the Yao posterior resection surface after pancre- Chongqing University, China aticoduodenectomy: Preliminary results of Irreversible electroporation (IRE), a nonthermal a hybrid approach treatment of pancreatic therapy for solid tumors, has been implemented in cancer clinical applications and made significant advance- Žan Čebron 2, Mihajlo Djokic2, Miha Petrič2, Maja ments in recent years. However, there is still an Čemažar1, Maša Omerzel1, Gregor Serša1, Blaz extremely urgent for novel pulses that strike a bal- Trotovsek2 ance between effective tumor ablation and min- 1Institute of Oncology Ljubljana, Slovenia imizing side effects, especially the occurrence of 2University Medical Center Ljubljana, Slovenia muscle contractions during this treatment, neces- Background: Despite extensive research in re- sitating the use of deep muscle anesthesia and ad- cent decades, pancreatic cancer remains one of equate muscle relaxants. This study focuses on the deadliest forms of cancer, with survival rates the development and clinical evaluation of a new showing little improvement. Local recurrences ac- IRE protocol modality involving synergistic electric count for approximately 30 per cent of all disease pulses for cancer therapy, aiming to achieve both recurrences. With the intent to improve survival, we enhanced tumor ablation and negligible muscle designed a novel, hybrid treatment strategy con- contraction. sisting of surgical resection and additional intraop- Here, we introduced a novel IRE strategy combin- 181 ing optimized high and low-voltage pulsed protocol OR-153 (S-IRE) to study the ablation effect and muscle con- Bleomycin based electrochemotherapy with tractions with monopolar and bipolar electrodes. standard electrodes for advanced stage, recur- Through numerous experiments on cells and an- ring vulvar/cervix carcinomas imals, we validated the benefits of S-IRE in inhibit- Aurel Ottlakan, Marton Vas, Gyorgy Lazar, Judit ing nerve stimulation-induced muscle contractions Olah, Gabor Vass, Mario Vincze, Erika Gabriella Kis and killing tumor cells compared to traditional IRE, University of Szeged, Hungary allowing applied under local anesthesia during ab- Recurrent vulvar cancer occurs in an average lation. A device Wknife based on the S-IRE was of 24% of cases after primary treatment after sur- developed and underwent successful safety testing gery with, or without radiation. It is often treated in preclinical trials involving various tissues (liver, with concurrent or sequential multimodal therapies, thyroid, prostate, and lung tissue), showing prom- however, despite the use of multimodal treatments, ising outcomes and providing a strong rationale for relapses are recorded in approximately one third of exploring the efficacy of this technique in human patients with limited salvage options. subjects. Following successful electrical & EMC In a 2-year period (Dec 2020- Dec 2022), five sur- testing, the Wknife device received approval from gically inoperable V/C cases were treated during the NMPA (National Medical Products Administra- six sessions of ECT at the University of Szeged, tion) for clinical trials. The first human clinical trials Department of Surgery, Hungary. After preoper-on liver, thyroid, prostate, and lung cancers, had ative MRI, each ECT was carried out under gen- been conducted in collaboration with renowned on- eral anesthesia, using standard finger electrodes cologists and medical centers respectively. Pre- and intravenous bleomycin (15.000 IU/m2) admin- liminary clinical data has supported the feasibil- istered 8 min before first pulse generation. Pro- ity of the S-IRE in reducing muscle contractions spective data collection was carried outat 1 week, and local anesthesia perforation, it also primarily 1-2-4-6 months. Patient health status and QoL was demonstrated the safety and efficacy of Wknife in assessed at each follow-up. Tumor response was tumor ablation. Now, a multi-center randomized evaluated through MRI, and gynaecological exam- controlled clinical trial is being conducted for com- ination 2 months after ECT as per RECIST 1.1, prehensive assessment and monitoring of patient adverse events were evaluated according to Com- outcomes, along with long-term follow-up, which mon Terminology Criteria for Adverse Events (CT- is expected to provide valuable clinical evidence CAE) version 4.0. Quality of life was assessed on the feasibility and potential of this novel cancer using the EQ-5D (Euro Quality of Life - 5 dimen-treatment approach. sions) questionnaire.Three squamous cell vulva-, In conclusion, this study offers valuable insights 1 squamous cell cervix- and 1 columnal cell cervix into the novel S-IRE ablation technique, which ef- carcinoma were treated, with median age of 70 (64- fectively balances minimal muscle contraction with 85) years. Median operative time was 23 min (7- enhanced tumor ablation. It has the potential to 32), median hospital stay 2 days (2–6). Mean BMI greatly improve the operability and safety of IRE (body mass index) was 29.7, mean ASA 2, mean treatment, offering a less invasive/side effect and CCI (Charlson Comorbidity Index) 5. Two tumors effective treatment option for cancer patients. This were located in the cervical stump (after hysterec- advancement is expected to build an important tomy), 3 at the vulva. Previous treatments included basis for its integration into cancer treatment pro- surgery (5 cases: 2 hysterectomies+ 3 vulvectom- tocols in the future. ies), preop radiotherapy (RT) in 3, postop RT in 2 cases, postop chemotherapy in 1 case. Follow-up MRI at two months confirmed 1 CR (complete response), 1 PR (partial response), 2 SD (stable disease) and 1 PD (progressive disease). 182 No major adverse events were observed, novum organ preservation and aesthetic outcome in the atrial fibrillation occurred in 1 case median postop management of skin metastases of HN cancers. pain level (VAS) was 2. Previous odour. A retrospective outcomes analysis of HN skin can- cer patients who underwent ECT at San Gallicano OR-154 Dermatological Institute IRCCS was performed. Electrochemotherapy: from palliation to im- Treatment consisted of intravenous bleomycin in- portant player in the multidisciplinary manage- fusion followed by locally delivered electric pulses. ment of the cancer patient Loco-regional tumour response and side effects Antonio Bonadies 1, Tiziano Pallara1, Marinella were evaluated. Post ECT evaluation of scars’ ap- Tedesco1, Paola Parisi1, Michela Battista2, Flavio An-pearance and aesthetics were assessed using the drea Govoni3, Gennaro Ciliberto4, Emilia Migliano1 Vancouver Scar Scale (VSS) and the Manchester 1San Gallicano Dermatological Institute, Italy Scar Scale (MSS) in complete response patients. 2IGEA S.p.A, Italy Among 33 selected patients, 27 (82%) reached 3ACO San Camillo - Forlanini, Italy a complete response (CR) and 6 (18%) a partial 4Regina Elena National Cancer Institute, Italy response (PR). Local control of the disease was In these past 15 years we have seen the evolu- maintained for a median time of 9.5 months (range tion of electrochemotherapy (ECT) from palliation 0.3 - 52.7). All side effects were easily managed of inoperable tumours to treatment of locally ad- and resolved spontaneously. Among 26 CR pa- vanced non-melanoma skin cancers, multiple unre- tients evaluated for cosmetic outcome, at a median sectable in transit melanoma metastases and ad- time of 7 months (range 4 - 16), the VSS and the vanced skin recurrences of breast cancer. From MSS total scores were 4 or less and 10 or less, re- the beginning, we performed ECT also as a cur- spectively, in 25 patients (96%). ative treatment of a skin cancer with a multi- In conclusion, for the treatment of skin cancers of focal/multidistrict primary presentation such as cu- HN, ECT represents a favourable local anticancer taneous Kaposi’s sarcoma. The effective results procedure with minimal side effects and excellent achieved in this type of cancer patient led to the inaesthetic and functional results. Overall, ECT can clusion of ECT in the European Guidelines for its be regarded as an effective and safe adjunct to the treatment. Today, we use ECT as a part of a mul- armamentarium of the interdisciplinary oncological tidisciplinary skin cancer management providing a team and a feasible curative tool for uresectable spot treatment while continuing ongoing systemic primary skin cancer lesions. therapies, in case of disease progression or local cutaneous or subcutaneous recurrences. Regard- S21 - Cardiac ablation by ing the management of cutaneous tumours of the irreversible electroporation - head and neck (HN) region, the surgical treatment requires achieving safety margins. This endpoint pulsed field ablation (PFA) often demands a large demolition and a complex reconstruction with long operating times that are Wednesday afternoon Track sometimes not compatible with the frequent comor- A bidities of elderly and frail patients. Furthermore, Sep 18, 14:20 - 15:20 some functional and aesthetic disfiguring results OR-155 are not easily accepted by the patient. In this set- Protocol-specific modelling of cardiac pulsed ting, ECT offers several advantages over surgery field ablation including higher preservation of normal surround- Argyrios Petras 1, Aurel Neic2, Edward Vigmond3, ing tissues, decreased morbidity, and shorter hos-Gernot Plank4, Luca Gerardo-Giorda5 pitalization. The purpose of our clinical investig- 1Johann Radon Institute for Computational and Applied ation was to address the ECT antitumor efficacy, 183 Mathematics, Austria endocardial vs. epicardial approach is nearly im- 2NumeriCor GmbH, Austria possible in experimental terms. Therefore, the aim 3Heart Rhythm Disease Institute, France of our study was to compare the electric field dis- 4Medical University of Graz, Austria tribution and the thermal-side effects of both ap- 5Johannes Kepler University, Austria proaches under equal conditions in terms of elec- trode design, pulse protocol, and anatomical char- Pulsed field ablation (PFA) is a recent proced- acteristics of the tissues involved (epicardial fat and ure for the treatment of cardiac arrhythmias. The myocardium). procedure selectively targets cardiac tissue, creat- Methods: Two-dimensional computational ing nonthermal damage to cardiomyocytes through models with axial symmetry were built for en- electroporation, while sparing other types of ad- docardial and epicardial approaches using a jacent tissue structures. Despite the promising conventional cardiac ablation catheter (7 Fr – 4 clinical and experimental results, no mathemat- mm). Atrial (1.5−2.5 mm) and fat (1−5 mm) thick- ical model is capable of simulating all the differ- nesses were varied to simulate a representative ent ablation protocols used. This work introduces patient cohort treated with PFA using different ap- a protocol-specific framework for the modelling of plied voltage values (1000, 1500 and 2000 V) and PFA procedure. The cardiac bidomain equations number of pulses (30 and 50). The sequence of are coupled with an electroporation term which ac- pulses was monophasic with a width of 100 µ s and counts for the number of membrane pores per unit 1 Hz frequency. The PFA-induced electric effects area. A physiological model tracks pore generation were assessed by the volume of fat above 1000 at the cellular membrane and allows for the simula- V/cm, as well as the collateral volume of electric tion of pore resealing. After model calibration and field damage in the myocardium. Thermal-side comparison against experimental results available effect provoked by PFA was evaluated by the in the literature, the impact of two different proto- evolution of maximum temperature in the fat and cols, the bipolar interlace and the circular bipolar the myocardium and the quantification of thermal methods, is explored in terms of the acute lesion damage lesion by using the Arrhenius Equation. characteristics. The normality of the data was checked using OR-156 Shapiro-Wilk Test. The comparison of outcomes Endocardial or Epicardial Delivery of Pulsed between both approaches was performed using the Field Ablation of Ganglionated Plexi? Assess- paired t-test for normally distributed data. In case ment and Quantification from An In-Silico Mod- of non-normal distribution, the comparison was elling Study carried out using the Wilcoxon Signed-Rank-test. Francisco Estevez-Laborí1, Barry O’Brien2, Ana Results: The epicardial approach was superior González-Suárez 3 for capturing greater volumes of fat when the ap- 1Universidad Internacional de Valencia, Spain plied voltage was increased: 231 mm3/kV with the 2AtriAN Medical Ltd., Ireland epicardial approach vs. 182 mm3/kV with the en- 3University of Galway, Ireland docardial approach. In relation to collateral dam- age to the myocardium, the epicardial approach Background: Pulsed Field Ablation (PFA) has considerably spared the myocardium, unlike what recently been proposed as a non-thermal energy to occurred with the endocardial approach. Although treat atrial fibrillation by selective ablation of gan-the epicardial approach caused much more thermal glionated plexi (GP) embedded in epicardial fat. damage in the fat, there was not a significant differ-While most PFA-technologies use an endocardial ence between the approaches in terms of the size approach, the use of epicardial access has given of thermal damage in the myocardium. promising pre-clinical results. However, as each Conclusions: Our results suggest that the epi- technology employs a different and sometimes pro- cardial PFA ablation of GPs is more effective than prietary pulse application protocol, comparing the the endocardial approach. The proximity and direc- 184 tionality of the electric field deposited using an epithe ablation, over the long term. Both types of cardial approach are key to ensuring higher electric ablation involve isolation of a pathological area, but field strengths and increased temperatures within we describe them differently by using appropriate the epicardial fat, thus contributing to selective ab-transmission conditions at the interface between lation of the GPs with minimal myocardial damage. the ablated area and the healthy heart. We propose numerical simulations in the context OR-157 of AF and we consider the isolation of one PV of Modeling the long-term effects of Pulsed-Field a synthetic geometry of the left atrium. Both the Ablation including comparison with Radio- RFA and PFA models lead to the isolation of the Frequency Ablation PV. The main difference between the two models Simone Nati Poltri, Annabelle Collin, Clair Poignard results in the isolation of the extracellular potential Univ. Bordeaux, France in the case of RFA being continuous for PFA. Our In healthy hearts, the propagation of electrical modeling also enables to propose a mathematical waves follows a predictable pattern, whereas in explanation for the lower recurrence of AF after people suffering from a cardiac rhythm disorder, PFA compared with RFA. Partial disconnection of the electrical wave can become chaotic and the PV can be seen as the emergence of electrical directly affect the pumping function of the heart. pathways between the atrium and the PV. It could Isolation of the pulmonary veins (PVs) by catheter be caused by the development of necrotic fibrosis ablation has become the treatment of choice for after RFA leading to conduction pathways, which atrial fibrillation (AF). The main goal is to isolate is not the case in PFA. the PVs from which the fibrillation is supposed to originate by physical procedures such as the [1] J. Joseph and K. Rajappan. Radiofrequency RadioFrequency Ablation (RFA) [1] - a thermal ablation of cardiac arrhythmias: past, present and ablation that is currently the most commonly used future. QJM, 2012. technique - and the novel Pulsed electric Field [2] G. Caluori et al. AC pulsed field ablation is Ablation (PFA) [2], which is based on non-thermal feasible and safe in atrial and ventricular settings: irreversible electroporation. a proof-of-concept Despite the great interest that RFA and PFA have OR-158 generated in the treatment of AF, there is still a Multiscale Simulation of Calcium-Mediated Car- lack of understanding – and thus modelling – of diac Lesion and Stunning in Pulsed Field Abla- the underlying biophysical phenomena of these tion different therapies. On one hand, it is well known Quim Castellvi, Antoni Ivorra that RFA ablation leads to coagulation necrosis Universitat Pompeu Fabra, Spain with complete loss of cellular and vascular archi- tecture by leaving a scar composed of a fibrotic Electroporation is the basis of cardiac Pulsed tissue. On the other hand, PFA is known to destroy Field Ablation (PFA). Although it has been proven mainly the cardiomyocytes, but the tissue scaffold as an effective and safe technique clinically, the is preserved. Therefore, the physical properties cell death mechanisms and physiological effects of the cardiac tissue after RFA and PFA are very remain not fully understood. Computational model- different, although they have the same goal, which ing has become a powerful tool in the field of elec- is to isolate the PVs. trophysiology and can also be used to elucidate the This work aims to modify the classical bidomain alterations induced by electroporation pulses. model – which describes the propagation of intra- In this study, the permeabilization of the cell cellular and extracellular potentials in the heart – membrane in myocytes has been simulated at to introduce a region ablated by RFA or PFA, and different electric fields using Krassowska’s model study the propagation of the electric signal after (Biophys J. 2007 Jan 15;92(2):404-17). According 185 to that, the induced flow of ions (Na+, K+, Ca2+ electrophysiologic and structural effects induced by and Cl-) through the permeabilized membrane electroporation in myocardial tissue. have been incorporated into an electrophysiolo- gical model (Biophys J. 2000 May;78(5):2392-404.) S01 - Medical applications of which considers key ionic channels and pumps nsPEFs (e.g. RyR, SERCA and Na+/K+). Considering the solute changes induced by electroporation, the os- motic pressures and water flow have also been in- Wednesday afternoon Track corporated. The resulting model can estimate the B temporal distribution ionic concentrations and pre- Sep 18, 14:20 - 15:20 dict how the electrophysiologic behavior of the cell OR-159 is affected. Considering the critical role of calcium Investigating the mechanism and dynamics of in the regulation of cardiac myocytes, its intracellu-Ca2+-mediated pore expansion after nsPEFs in lar concentration has been linked to the different ef-healthy and cancerous urothelial cells fects observed in the cardiac cells after PFA pulses. Aleksander Kiełbik 1, Aleksandra Mariianats1, The results at cellular level show that for low Pamela Sowa1, Wojciech Szlasa2, Vitalij Novickij3, Igor concentration changes, the cardiomyocytes inter-Tsaur1, Julia Marzi1, Bastian Amend1 rupt their conduction capability showing a transi- 1University of Tübingen, Germany ent stunning effect. For larger ionic disturbances, 2Medical University Hospital, Poland the cell is not able to rapidly restore the physiolo- 3Vilnius Gediminas Technical University, Lithuania gical levels, and this will result in a long-term cell death. For higher concentrations, the calpain proA major effect on the cell-killing efficiency of teases are activated, disrupting large proteins and pulsed electric fields (PEFs) has been attributed cardiomyocyte structure which will lead a rapid cell to an excessive influx of extracellular Ca2+, with death. some studies suggesting a greater effect against The microscopic simulations of ionic distribu- cancer cells. Earlier in vitro research has shown tions and the triggered pathways have been em- that Ca2+ triggers abrupt pore expansion leading ployed to predict the spatio-temporal effects at the to early cell death. Our studies investigate the link macroscopic tissue level. A 3D numerical model between pore expansion dynamics and susceptib- of a clinical PFA procedure with a focal catheter in ility to PEFs, as well as the underlying mechanism, contact with the myocardium has been employed focusing on urothelial cancer cells. to deliver the therapeutic pulses and evaluate the We compared the response to an electric field extension of the induced effects. measured in monolayers and 3D cultures of nor- The results of this study predict the appear- mal and malignant human urothelial cell lines (SV- ance of a large area around the treatment loca- HUC-1, T24, UC3, RT4). We used a robotic system tion where the cardiomyocytes are stunned and un- to precisely place needle electrodes orthogonal to able to perform their physiologic electric conduc- the monolayer and plate electrodes on both sides of tion. Within the ablated section, the zone closer the spheroid. Imaging was performed using an in- to the catheter electrode, with higher homeostatic verted fluorescence microscope configured for high disturbances, presents a fast structural change in- throughput screening with automatic stage shift duced by calcium-activated calpain. These results and autofocus. The area of cell death and per- are in agreement with preclinical and clinical ob- meabilization after application of 300ns PEFs at 10 servations, including the recent observation of fast Hz was measured by staining with propidium iodide structural changes after PFA (Circ Arrhythm Elec- (PI) and YO-PRO-1 dye. Dose-response curves trophysiol, 2024, 17(3):e012255). The presented were obtained by fitting the stained areas of cell model with a multiscale approach is able to es- monolayers to the simulated electric field strength. timate the spatial and temporal evolution of both We showed that the presence of Ca2+ in the extra- 186 cellular medium resulted in a decrease in the im-present data to demonstrate synergistic effects of mediate membrane permeabilization of various ur- nsPEFs and cold atmospheric plasma (CAP) for othelial cancer cell lines measured with YO-PRO-1 pancreatic cancer and further explore the mechan- Uptake during PEFs delivery. Subsequently start- isms behind this synergy. ing from 20 min after exposure, Ca2+ caused an Mouse Pan02 pancreatic cancer cells were treated abrupt increase in cell permeability to PI and YO- with nsPEFs (60 ns 50 kV/cm, 1 Hz and pulse num- PRO-1 dye suggesting the expansion of cell mem- bers varied), CAP (9 kV, 200 ns pulses, 2 kHz and brane pores. treatment times varied), or the combination of these Interestingly, healthy SV-HUC cells, which demon- two treatments. Cell viability was examined with strated weaker YO-PRO-1 uptake at the time of WST-1 assays. Cell membrane permeabilization PEFs delivery, exhibited an earlier Ca2+ mediated was monitored with Electric Cell-substrate Imped- abrupt permeabilization compared to other cancer ance Sensing (ECIS). Generation of reactive oxy- cells. The time course of pore expansion does gen species (ROS) was quantified with H2DCFDA, not correlate with the sensitivity of the cells to the MitoSox, and Amplex red for cytosol ROS, mito-nsPEFs. Due to calcium-mediated pore expansion, chondrial superoxide, and H2O2, respectively. Mi- early cell death can be achieved at lower electric tochondrial membrane potential was determined field intensities. The LD50 (electric field dose that with TMRE. Cell death pathways including apop-kills 50% of the cells) for SV-HUC cell death was tosis and pyroptosis were examined with caspase 1.2 times lower in 2mM Ca2+ and 1.5 times lower activity assays and/or Western Blot. Additionally, in 5mM Ca2+ compared to the Ca2+-free medium the changes of signaling pathways and up-/down- (p<0.001). For the UC3 cancer cell line the LD50 regulation of gene expression were analyzed with was 1.3 and 1.5 times lower and for T24 1.4 and proteomic analysis as well. 1.9 times lower in 2 mM and 5 mM Ca2+ respect- We have found that nsPEFs at a low dose sensit- ively (p<0.001). The underlying mechanism of pore ize cancer cells for CAP treatments in terms of cell expansion remain to be explained using Raman membrane permeabilization and cytotoxicity. Both microspectroscopy and fluorescence lifetime ima- nsPEFs and CAP induce ROS generation including ging. cytosol ROS, mitochondrial superoxide and H2O2. However, it seems that nsPEFs are more effective OR-160 to elicit the drop of mitochondrial membrane poten- Synergistic effects and mechanisms of nano- tial than CAP does. CAP leads to apoptotic cell second pulsed electric fields and cold atmo- death with the upregulation of Caspase3/7 activ- spheric plasma to treat pancreatic cancer ity whereas nsPEFs induce non-apoptotic, non- Siqi Guo 1, Zobia Minhas1, Edwin A. Oshin1, pyroptotic cell death with a negative Caspase3/7 Shanaya M. Haque2, Yu Jing1, Lifang Yang2, Chunqi and Caspase1 activity. Notably, a mild dose of Jiang1 nsPEFs can significantly augment apoptotic cell 1Old Dominion University, United States death induced by CAP. Proteomic analysis shows 2Eastern Virginia Medical School, United States remarkable differences between nsPEFs and CAP A pulsed power technology generating nano- treatments in terms of gene expression changes second pulsed electric fields (nsPEFs) has been and signaling pathways involved. demonstrated to effectively treat many types of Our discoveries suggest distinctive cell death cancer in animal models. However, there are some mechanisms involved in nsPEFs and CAP, which limitations of nsPEF cancer treatments. It requires contribute to the synergistic effects of the combin- high-electric fields for cancer ablation which limit ation treatment. its application and effectiveness. Our group and others have proposed and investigated several ap- proaches to overcome this limitation. Here, we 187 OR-161 Acknowledgement: The research was sup- Nanosecond Bursts of Ultra-High Frequency for ported by the Research Council of Lithuania (Grant Electrochemotherapy and Gene Delivery nr. S-MIP-19-13, S-LL-21-4, S-MIP-23-124). Vitalij Novickij State Research Institute Centre for Innovative Medicine, OR-162 Lithuania Characterizing the Immune Response Follow- ing High Frequency Nanosecond Bipolar and Currently, the field of reversible electroporation Unipolar Calcium Electrochemotherapy (associated with molecular delivery) is dominated Eivina Radzevičiūtė-Valčiuke 1, Augustinas by the microsecond pulses, while the irreversible Želvys1, Eglė Mickevičiūtė1, Jovita Gečaitė1, Paulina electroporation (associated with tissue ablation in Malakauskaitė1, Barbora Lekešytė1, Veronika Malyško- the biomedical context) is far more flexible with Ptašinskė2, Auksė Zinkevičienė1, Vytautas Kašėta1, nanosecond protocols being present on the market Julita Kulbacka3, Joanna Rossowska4, Vitalij Novickij1 for several decades. One of the reasons could be 1State Research Institute Centre for Innovative Medicine, the lack of sufficient electrophoretic component Lithuania and inferior pore size following nano protocols, 2Vilnius Gediminas Technical University, Lithuania which negatively affects molecular transfer. As 3Wroclaw Medical University, Poland a result, the gene delivery or drug delivery was 4Hirszfeld Institute of Immunology and Experimental not covered at all in the sub-microsecond pulse Therapy, Poland range with the first works starting to appear only Calcium electrochemotherapy (CaECT) is a recently. In both application cases, if the bursts are new and effective cancer treatment approach delivered with common 1 Hz – 10 kHz frequencies, enabling the replacement of standard chemothera- the nano-pulses have significant limitations in peutic drugs with non-toxic calcium. Recently, it terms of electrotransfer efficiency. However, with was shown that the application of sub-microsecond the development of silicon-carbide MOSFET tech- pulses, which are compressed into a high fre- nology, high power and high frequency generation quency (MHz) burst, significantly boosts the of pulses became possible. It was shown that there efficacy of the CaECT methodology. However, this is a threshold pulse repetition frequency (500+ is true only for monophasic procedures, while in kHz), when the depolarization of the cell mem- the case of bipolar nanosecond pulses, a bipolar brane is longer than the delay between the pulses, cancellation (BPC) phenomenon can be triggered. which enables sustaining the cells in a polarized Most of the effects of BPC are reported in the state throughout the whole burst without triggering context of tissue ablation, while data on bipolar irreversible electroporation. This polarization ECT are hardly available. At the same time, the phenomenon positively affects electrotransfer application of bipolar pulses potentially reduces and efficacy of nano-electroporation, enabling muscle excitation and associated pain, ensures cell plasma permeabilization at significantly lower a more homogeneous spatial electric field distri- thresholds. As a result, the nanosecond protocols, bution within the tumor, and makes the treatment but delivered at ultra-high frequency (1+ MHz) less likely to induce electrolysis and pH changes. are competitive and in many cases superior to Therefore, in this work, we have compared the conventional microsecond pulses for drug & gene feasibility of unipolar and bipolar sub-microsecond delivery, which was confirmed both in vitro and in pulses (7 kV/cm x 300 ns x 250, 1 MHz) in the vivo. context of CaECT and compared them to ESOPE: This presentation is dedicated to provide a quick European Standard Operating Procedures for critical overview of the current status of the tech- Electrochemotherapy (1.5 kV/cm x 100 µ s x 8, 1 nology, benefits and limitations incl. a vision for Hz). Characterization of the immune response further development. following the treatments was of particular interest. A luminescent murine breast cancer (4T1-Luc) 188 cell line was used to induce tumors in Balb/C ranging from many scientific disciplines to industrial mice (n=60). When tumors reached the needed fields. Although all these applications are based volume (˜50 mm3), the CaECT (250 mM) treat- on the same basics, pulse shape, frequency and ment was performed with various electric field patterns, voltage, and current requirements can be protocols. Afterward, volumetric measurements very different for each specific scenario. These re- and bioluminescence tumor imaging were used to quirements can range from a few V and mA up to characterize the tumor response. Also, at the end hundreds of kV and A. Furthermore, the effects of of the experiment, mice spleens, lymph nodes and shorter pulses are currently being studied due to blood samples were isolated and used for further their potential advantages over longer and lower analysis with flow cytometry. voltage pulses. In this context, there is a cur- Our study demonstrated that both uni- and bipolar rent challenge to develop generators for scenarios nanosecond pulses can be used in CaECT context where pulses with widths in the range of tens of resulting in partial or complete response of the tu- ns, several kV and hundreds of A are needed. In mors. The treatments also triggered an increased this context, the impedance of the biological target percentage of CD4� memory T cell subsets in plays a key role in the design of such pulse gen- spleens and tumor-associated lymph nodes. Also, erators. There are cases where the impedance of increased helper T cells and decreased CD4� reg- the biological target can be well characterized, but ulatory T cells were detected in the lymph nodes it is also common to cope with unknown impedance after CaECT treatment. Moreover, modulation of targets. Moreover, the impedance of biological tar- humoral immune response (levels of antitumor gets varies over time during the electroporation pro- IgG antibodies) post calcium ECT treatment was cess. For those reasons, achieving low output im- detected. pedance is critical in these devices. Interestingly, the effects of BPC were not profound Considering this challenging context, this paper for the bipolar 7 kV/cm x 300 ns x 250, 1 MHz pro- proposes a versatile modular high-performance tocol, which was predicted by the in vitro viability pulse generator intended for nsPEF based treat- and permeabilization data supporting the research. ments. This paper focuses on detailing the design and its implementation, as well as the main exper- Acknowledgment: The research was suppor- imental results. The design is based on series- ted by the Research Council of Lithuania (Grant connected half-bridge inverters featuring 650-V Nr. S-MIP-23-124). Gallium Nitride (GaN) transistors. These are wide bandgap devices with fast switching capabilities, S13 - High voltage electrical i.e., with rising and falling times even under 1 ns. In the proposed topology, each module conducts all discharges: principles and the current delivered to the load but only needs to applications withstand a fraction of the applied voltage, depend- ing on the number of modules employed. Besides, Wednesday afternoon Track very low output impedance is achieved, making this C pulse generator suitable for working with a broad Sep 18, 14:20 - 15:20 range of biological targets, independently of their impedance. OR-59 By using this approach, a test prototype consist- High-Performance Solid-State Generator for ing of 4 modules has been developed, capable of nsPEF Applications generating pulses down to 5 ns long, and up to 2 Pablo Briz, Héctor Sarnago, Óscar Lucía kV and 200 A output voltage and current, respect- Universidad de Zaragoza, Spain ively. Moreover, the output voltage can be regu- Nowadays, nanosecond pulsed electric fields lated adjusting the number of active modules and (nsPEF) have an extensive scope of applications, DC bus voltage. Finally, there is an optically isol- 189 ated control board to manage the device. The ap-ate high-voltage short pulses with a maximum out- plied pulse pattern can be arbitrarily configurable to put voltage of ±12 kV, adjustable pulsewidth from match the requirements of the application, and the 100 ns to 1 µ s. Additionally, it can generate low-dv/dt is also controllable. voltage long pulses with a maximum output voltage The final version of this paper will include detailed of ±3 kV, adjustable pulsewidth from 10 to 100 design, implementation and experimental meas- µ s. The combination of high-voltage short pulses urements, including key waveforms. and low-voltage long pulses is adjustable in various forms. In addition, we conducted further clinical tri-OR-164 als. A Synergistic Bipolar Pulse Power Generator for Expanding Ablation Area and Inhibiting OR-165 Muscle Contraction Pulsed Atmospheric Pressure Plasma for the Shoulong Dong, Chenguo Yao, Jianhao Ma, Lisheng destruction of emerging contaminants and the Zhao, Yancheng Wang, Hongmei Liu inactivation of bacteria in water Chongqing University, China Konstantia Papalexopoulou, Irene-Eva Triantaphyl- lidou, Christos Aggelopoulos Pulse power technology has a wide range of Foundation for Research and Technology, Greece applications in the field of biomedicine, especially in tumor ablation scenarios based on irreversible Environmental pollution of aquatic systems is electroporation technology. Previous studies have a persistent concern for societies, with detrimental shown that exponential decay pulses result in their effects on human health, biodiversity, and socio- energy utilization rate being too low to completely economics. Various chemical compounds, ori- kill tumor cells. ginating from human activities, are extensively The exponential decay pulse causes its energy used and enter the aquatic environment through utilization to be too low to kill tumor cells com- wastewater. These compounds ultimately contam- pletely. The ablation area of the tumor is large inate surface water bodies, groundwater and drink- when unipolar rectangular pulses, are applied to ing water reservoirs. Additionally, a significant is- the target, but the DC component contained in uni- sue regarding water quality is sanitation, as patho- polar rectangular pulses will induce strong muscle gens such as bacteria, viruses, and protozoa are contractions for the patients. Muscle contraction introduced into water bodies, resulting in the pro- can cause discomfort to the patient and even dis- liferation of numerous waterborne infectious dis- place the electrode needle, which affects the ac- eases. Water bodies harbor a multitude of pol- curacy of the treatment. The bipolar rectangular lutants, including pharmaceuticals and, more re- pulses have no DC component and can suppress cently, so-called ”forever chemicals” known as per- the intensity of muscle contraction. However, the and polyfluoroalkyl substances (PFAS). Undiges- ablation area of bipolar rectangular pulses is smal- ted pharmaceuticals and their metabolites pose ler compared to unipolar rectangular pulses. A a threat to food chains and must be addressed. two-stage combined pulse, also called synergistic PFAS, extensively utilized in various products, are pulse, can improve the ablation efficacy. of significant concern due to their persistence and It utilizes a dual Marx structure with nested high- propensity for bioaccumulation in the environment, voltage short pulse modules and low-voltage long rendering them emerging contaminants of high pulse modules. This structure uses two different priority. Numerous conventional methods have sets of storage capacitors, charging sources and been employed for water remediation. However, solid-state switches that can respectively control many of these methods suffer from drawbacks such the parameters of the high-voltage long pulse and as limited pollutant/pathogens removal, high en- the low-voltage short pulse. An experimental proto- ergy consumption, prolonged treatment times and type is built to verify the scheme which can gener- the risk of secondary pollution. In recent years, 190 cold atmospheric plasma has emerged as a highly ter. Atrazine is also found in underground waters, promising water treatment method due to its cost- due to its low absorption from the soil. The chronic effectiveness and environmentally friendly nature. toxicity of atrazine is of concern. Atrazine inter- Cold plasma operates by generating highly reactive feres with the endocrine system, causing a series oxygen and nitrogen species, UV photons, and hy- of pathological changes and reproductive abnor- drated electrons, which effectively degrade organic malities. Atrazine causes breast, ovarian and uter- pollutants and inactivate pathogens in water. ine cancers as well as leukemia and lymphoma. In the present study, a comprehensive investiga- Therefore, it is very important to find clean and ecotion was conducted to assess the efficacy of pulsed nomical processes to degrade atrazine. atmospheric plasma in the degradation of vari- In this study, high-voltage electrical discharges ous organic pollutants, namely valsartan (a phar- (HVED) were used for the degradation of atrazine. maceutical compound) and perfluorooctanoic acid This process was compared to chemical treatment (PFOA), a member of the ’forever chemicals’ fam- with oxidation Fenton as well as physical treat- ily. Additionally, the study examined the inactiv- ment with high-frequency sonication. An analytical ation of Escherichia coli, a bacterium commonly monitoring of the degradation process was carried found in contaminated water. Two plasma react- out, by High-performance liquid chromatography ors were designed, constructed, and compared: (i) (HPLC) and Liquid chromatography–mass spec- a gas-liquid dielectric barrier discharge and (ii) a trometry (LC-MS). Degradation metabolites were plasma bubbles reactor. Both reactors were en- identified and compared for each process. ergized by high-voltage microsecond and/or nano- The work carried out as part of this study has second pulses to investigate and compare their ef- shown that the degradation rate of atrazine by fectiveness and energy requirements for the de- Fenton oxidation is the highest (around 95% of at- struction of pollutants and pathogens in water. Vari- razine degradation was observed after 2h of treat- ous critical parameters such as treatment time, ment). The use of low-frequency ultrasound at 50 pulse voltage, plasma gas, water matrix, and ini- kHz slightly degraded atrazine (around 23% of at- tial pollutant concentration, were also investigated. razine degradation was observed after 2h of treat- Optimal operational conditions with the highest en- ment). The use of high-frequency ultrasound at 525 ergy efficiency were determined. Additionally, de- kHz degraded 87% of atrazine after 2 h. The HVED tailed physicochemical characterization and ana- treatment was much more efficient, as 10-ms elec- lysis of plasma species formation under different trical shocks degraded atrazine by 83%. During the conditions were performed. The results of this degradation of atrazine, eleven compounds were study suggest that pulsed plasmas offer a prom- identified. The formation kinetics of these com- ising, environmentally friendly, and cost-effective pounds were studied for all the treatments applied. solution for the destruction of pollutants and patho- The result showed that the degradation products of gens in water. atrazine are related to the nature of the treatment applied. OR-30 The study showed the feasibility of high voltage Degradation of pesticide atrazine in water by electrical discharge (HVED) treatment, as an al- high voltage electrical discharges ternative technology, for the degradation of at- Junting Hong, Nadia Boussetta, Gérald Enderlin, razine. Hydroxyatrazine (HA) is the main degrada-Franck Merlier, Nabil Grimi tion product in HVED treatment, and it has a lower Université de Technologie de Compiègne, China toxicity than other degradation metabolites of at- Atrazine is an herbicide used primarily for the razine. This research paves a way for the devel- control of grassy and broadleaf weeds such as opment of a new clean and economical technology maize, sugar cane and sorghum. Atrazine pollutes for wastewater treatment. surface water because it is relatively soluble in wa- 191 S11 - In vivo delivery of genetic tems) in blood serum, and cell-mediated immunity with a tetramer assay (Tetramer Shop) on isolated medicine through gene splenocytes. electrotransfer The expression of S and N antigens was con- firmed both in vitro and in vivo by qRT-PCR. Wednesday afternoon Track The N antigen was also confirmed at the protein D level, while S was undetectable with the avail- Sep 18, 14:20 - 15:35 able ELISAs, indicating issues with its correct ex- pression. Consequently, vaccination with S did OR-166 not result in a significant induction of anti-S an- Exploring gene electrotransfer as a DNA vac- tibodies. Nevertheless, a significant induction of cination strategy: insights from a COVID-19 S-specific cytotoxic T cells was observed. Both vaccine study skin and muscle vaccination were well tolerated. Urška Kamenšek, Simona Kranjc Brezar, Tanja However, muscle vaccination led to a significantly Jesenko, Špela Kos, Katarina Žnidar, Boštjan Markelc, Živa Modic, Tilen Komel, Maja Čemažar, Gregor Serša higher expression of antigens and a stronger induc- Institute of Oncology Ljubljana, Slovenia tion of antigen-specific humoral and cell-mediated immune responses. Interestingly, the adjuvant and DNA vaccination is a promising strategy for the booster did not significantly enhance the immune treatment and prevention of cancer and infectious responses. diseases. The aim of this study was to evaluate the Our study reaffirmed the feasibility of GET for feasibility of gene electrotransfer (GET)-mediated DNA vaccination. It showed that muscle deliv- DNA vaccination on a model of COVID-19 vaccine. ery outperforms skin delivery in terms of anti- Plasmids encoding the SARS-CoV-2 spike (S) gen expression and induction of humoral and cell- or nucleocapsid (N) protein (pUNO1-SARS2-N, mediated immunity. The study also provided valu- pUNO1-SARS2-S-d19, InvivoGen) were used as able insights into the role of presentation pathways antigen source and an interleukin 12 (IL-12)- for inducing cell-mediated immune responses and encoding plasmid (pORFmIL-12(p40:p35), Invivo- demonstrated that both immunological adjuvant Gen) as an immunological adjuvant. The expres- and booster may be redundant after vaccination sion of the S and N antigens was first assessed with GET. Overall, the results support the use of after in vitro GET (8 × 1300 V/cm, 100 µ s, 5 GET for vaccination against infectious diseases kHz) to murine myoblasts (C2C12) and fibroblasts and solidify its role in therapeutic vaccinations for (L929) using qRT-PCR and ELISA assays (Abcam, cancer treatment. Krishgen Biosystems). Vaccination was performed in the skin (right flank) or muscle (right anterior OR-167 tibialis muscle) of female C57BL/6J mice on days 0 Enhancing molecular cargo electrotransfer by and 14 (booster dose) using previously optimized modulating vesicular transport in cells transfection protocols for skin (MultiElectrode Ar- Fan Yuan, Chunxi Wang ray, 24×170 V/cm, 150 ms, 2.82 Hz) and muscle Duke University, United States (plate electrodes, 1×HV: 600 V/cm, 100 µs, 4×LV: We have demonstrated previously that treat- 80 V/cm, 100 ms, 1 Hz). Two weeks after vaccina- ment of cells with non-reducing sugars (NRSs), tion, blood, spleens, and transfected tissues were such as sucrose, trehalose, and raffinose, can sig- collected from vaccinated mice. The expression of nificantly enhance the efficiency of electrotransfer S, N, IL-12, and interferon γ (Ifn- γ) in the transfec- (ET) for delivery of various molecular cargo (plas- ted tissue was determined by qRT-PCR, and serum mid DNA, mRNA, and ribonucleoprotein) into cells. IFN- γ by ELISA (R&D Systems). The induction of The enhancement has been observed in more than humoral immunity was evaluated with ELISAs for 20 cell lines and primary human cells. To achieve S- and N-specific IgG antibodies (Krishgen Biosys- 192 the enhancement, the cells are pretreated with an OR-168 NRS. After a certain period (6-24 hours), the cargo LiveGT Enhances Skeletal Muscle Reprogram- is electrotransferred into the treated cells. At 24- ming and Physiological Levels of Insulin Pro- 48 hours post ET, the gene delivery efficiency and duction cell viability are quantified. However, the same Michael Francis, Jacob Hensley, Alex Otten, Tina treatment led to minimal improvement in the ET for Gagliardo, Anna Bulysheva cargo delivery in mouse muscle. We hypothesized University of South Florida, United States that the lack of improvement was due to short half- Introduction: Gene electrotransfer is an estab- lives of NRSs in tissues because they are small, lished physical method for delivery to various tis- hydrophilic molecules that can be quickly cleared sue and organs in vivo including melanoma tumors, through vascular systems and diffusion into sur- skin, cardiac muscle, and skeletal muscle. GET rounding tissues. As a result, the treatment of cells advantages include nonimmunogenic and nonin-was reduced. To test the hypothesize, we encap- tegrative gene delivery to targeted tissues. Dis- sulated sucrose in biomimetic lipid nanoparticles advantages include low expression levels, short- (sucrose-LNPs) for targeted delivery into muscle lived/transient expression, and muscle stimulation. cells, and used it to treat the mouse muscle of Skeletal muscle, localize in vivo electro gene ther- the hind limb prior to the electrotransfer of plas- apy (liveGT) as a next generation GET, was utilized mid DNA (pDNA) into the same tissue. At 1-7 days with a monopolar electrode and optimized pulse se- post ET, the gene delivery efficiency was quanti- quences to determine longevity of expression and fied. Our data showed that the pretreatment with feasibility of reaching and maintain physiological sucrose substantially increased and prolonged the levels of blood circulating protein replacement ther- transgene expression in mice. The amount of in- apy. Maintain expression at physiological serum crease at 2 days post ET was equivalent to increas- levels for extended periods of time is an important ing the dose of pDNA by approximately 3000 folds. step in moving liveGT toward clinical translation as Mechanisms of the enhancement were associated a protein replacement alternative. Gene delivery of with the formation of large vesicles elicited by the plasmid DNA encoding human insulin was used to sucrose treatment. These vesicles included amphi- correlate pulsing parameters to serum levels in the some�like bodies (ALBs) and enlarged lysosomes. physiological range, as cannot be done with report- The ALB formation hindered vesicular transport of ers. pDNA to lysosomes, while the lysosome enlarge- Methods: Various LiveGT pulsing parameters were ment was correlated to the reduction of its acid- administered directly to the skeletal muscle of ity, which in turn inactivated nucleases. The ob- Sprague Dawley rats, with plasmid DNA encod- servation of large vesicle formation and alterations ing luciferase or human insulin and glucokinase co- in vesicle trafficking could be explained by the data delivery. Expression levels were monitored with from analysis of transcriptomic profiles in sucrose bioluminescence imaging as well as directly from treated cells. The changes in vesicular transport serum samples via ELISA’s over nine months post and inhibition of lysosomal function collectively de- initial liveGT. Additionally, to blood glucose meas- creased pDNA degradation, thereby increasing the urements were also monitored. Two-way ANOVA efficiency of ET. The study concluded that the NRS and Tukey multiple comparison tests were used to could be used to improve electrotransfer of molecu- determine statistically significant differences, with lar cargo in vitro and in vivo. p<0.05 considered significant. Control groups re- ceived vehicle, or no delivery at all. Results: Reporter expression was observed to be maintained for over six months with signific- antly higher (over 100-fold) than respective con- trols. Exogenous human insulin expression and 193 blood glucose modulation were maintained over increase cellular stress. Further, magnetoporation three months, with significant reduction in mean cir- eliminates electrical arcing within the sample and culating glucose compared untreated animals. the need for special conductive buffers. The pro- Conclusions: Our preliminary findings implicate cess can be fine-tuned to the magnetic field re- liveGT skeletal muscle reprogramming as a viable quirements of a particular cell, minimizing manip- target for secretory protein therapies. Physiological ulation and physical stressors to the cell, and has levels of insulin as a surrogate for other blood cir- shown high efficiency of intracellular transfer into culating proteins can be achieved and maintained a variety of immortalized and primary cell types in- in serum for several months without risks of integ- cluding CD4 T cells and red blood cells. It has also ration and immunogenicity. proven effective for various sizes and types of pay- loads, including DNA, RNA, and carbohydrates. ‘ OR-169 The hardware is composed of commercial off-the- Magnetoporation: A novel method of molecular shelf, custom-order, and internally manufactured delivery for cell and gene therapies components in the categories of hardware, elec- Zachary Rapp tronic components, and electronic circuitry. The Sigma Genetics, United States software and user interface are exclusively propri- In the field of gene-modified cell therapies, suc- etary. The company is presently working towards cessful intracellular gene delivery remains a signi- a cGMP-compliant flow-through instrument, while ficant challenge. Sigma Genetics offers an innov- also exploring other potential applications for the ative solution to this critical issue using propriet- technology including in the rapidly advancing gene- ary magnetoporation technology. Magnetoporation modified cell therapy space. Our technology is the uses external, pulsed magnetic fields to efficiently first to apply pulsed power at the cellular level and introduce foreign molecules and genes into cells. improve the manufacturing of clinical cell-based Magnetoporation induces an electric current across therapeutics; our first commercial application. each individual cell leading to transient poration of OR-237 the cell membrane. Cells are placed within a cu- Development of in vivo-launched synthetic vette inside a fixed and cooled coil. Magnetic fields DNA-encoded antibodies employing CELLEC-generated by pulsed power transmit through the TRA® electroporation technology coil which amplifies the magnetic field and also acts Trevor Smith 1, Paul Fisher1, Ami Patel2, Elizabeth as a reservoir when saturated. The core’s passage Parzych2, Kevin Hollevoet3, David B. Weiner2, Laurent into and out of saturation causes the magnetic per-Humeau1 meability of the sample volume to change rapidly, 1Inovio Pharmaceuticals, United States which in turn causes a higher rate of change in the 2The Wistar Institute, United States local magnetic field. The electric current induces 3University of Leuven, Belgium transient poration of the cell membranes and entry of extracellular material. The higher the rate of Monoclonal antibodies (mAb) have proved a change of the magnetic field, the higher the trans- simple passive immunization strategy to provide fer efficiency. Importantly, the electromagnetic cir- protection against infectious disease. How- cuitry dynamics of the magnetoporation process ever, access to potentially life-saving mAb biolo- are uniquely suited to scalability; as sample volume gics faces multiple challenges for broader pop- increases, the uniformity of the magnetic field and ulation coverage including high doses (in milli- the efficiency by which it interacts with the sample grams/kilogram), shelf-stability, temperature stabil- both increase. The result is an increase in transfer ity and distribution barriers for low-/middle-income efficiency with higher processing volumes without countries and resource-limited settings. Therefore, additional stress on cells, unlike electroporation additional strategies that can further facilitate mAb and sonoporation, where higher volume processing uptake and global availability would be valuable for 194 infection control. DNA-encoded monoclonal anti-cell electroporation. The deformation of single Gi- bodies (dMAb™) offer a possible alternative tech- ant Unilamellar Vesicles (sGUV) under alternat- nology to traditional recombinant mAb. Synthetic ing current (AC) electric fields has been extens- DNA constructs are delivered to muscle tissues ively investigated through a combination of ex- with in vivo electroporation to permit local tissue perimental and theoretical approaches in literat- expression of the antibody transgene and transi- ure. Both analytical and numerical models have ent production and secretion of mAb into circula- been established to understand the membrane’s tion. dMAbs have the potential to be a transform- electro-mechanical properties, including paramet- ative technology, allowing sustained trough levels ers such as bending rigidity, tension, and capacit- of bioavailable mAb, flexibility for administration of ance. These studies have provided an understand-multiple constructs, rapid production at dramatic- ing of the electromechanical response of sGUVs, ally lower costs, and long-term drug product stabil- as well as anucleate cells such as RBC , to electric ity. Through a combination of in vivo electropora- fields. tion and drug formulation circulating levels of mAbs However, eukaryotic cells have a nucleus whereby providing disease protection can be achieved by Compound Giant Unilamellar Vesicles (cGUVs) this technology platform. Here we discuss the emerge as an appropriate biomimetic model of bio- nonclinical path for development of DNA-encoded logical cells. cGUVs have a vesicle-in-vesicles in monoclonal antibodies. Through case studies we structure, where outer and inner vesicles represent investigate the delivery conditions, modeling and a biomimic, eukaryotic cell and nuclear membrane, understanding the pharmacokinetics of in vivo mAb respectively. In this work, a method for cGUV syn- expression, and identify the requirements for scal- thesis is improved for forming well formed cGUVs, ing up the technology into clinically relevant mod- allowing for modification of the electrical conduct- els. This process has resulted in the successful ivities in their inner, annular, and outer regions. A translation of multiple dMAb candidates as medical thorough experimental study on the EHD of cGUVs countermeasures into clinical testing. demonstrates promising experimental findings with an agreement with theoretical work. In response to weak externally applied AC fields, the observed P11 - Electroporation modeling and deformations of cGUVs—such as spherical, pro- mechanisms late, or oblate spheroidal—depend on the mem- brane’s electromechanical properties and the Max- Wednesday late afternoon well stress, which vary with the relative times- Track A cales associated with the frequency of the applied Sep 18, 16:50 - 17:50 AC field, membrane charging time, and Maxwell- Wagner relaxation time. This work firmly estab- OR-170 lishes cGUVs as suitable biomimetic models for AC electrodeformation studies on Compound conducting EHD studies on eukaryotic cells. Giant Unilamellar Vesicle as a model of euka- ryotic cell OR-171 Rupesh Kumar, Rajarshi Chakrabarti, Rochish Suitability (and not) of Giant Unilamellar Ves- Thaokar icles in electroporation studies for biological Indian Institute of Technology Bombay, India applications Insights gained from studies on the electro- Rochish Thaokar 1, Mohammad Maoyafikuddin2 1 hydrodynamics (EHD) of Single Giant Unilamel- IIT Bombay, India 2 lar Vesicles (sGUVs) have significantly contributed Raman Research Center, India to our understanding of how biological cells re- Giant Unilamellar vesicles have emerged as act to electric fields, leading to the advancement very promising systems to investigate biological in the understanding of cell dielectrophoresis and 195 processes, including the mechanism of electropor-ctric field (SSPEF) and a square wave pulsed elec- ation. The choice of GUVs is attractive because tric field (SWPEF) of same (rms) amplitude electric of simplicity and control of the biomembrane com- field. The occurrence of cylindrical deformations position, absence and choice of internal objects, in both SSPEF and SWPEF indicates the depend- and ability to manipulate electro mechanical prop- ence of deformation on TMP. In both SSPEF and erties of the membrane as well as electrical con- SWPEF, the vesicles deformed into prolate cylin- ductivities of the inner and outer solutions. In this ders as a result of Maxwell stress for both similar work, I will discuss the reliability and limitations of electrical conductivity β = 1 and greater inner con-Giant Unilamellar vesicles as biomemetic electro- ductivity β > 1, whereas they were compressed into porating systems, and highlight the similarities and oblate cylinders for greater outer conductivity β < differences between GUV electroporation and cell 1. Vesicles subjected to a SSPEF relaxed follow- electroporation. ing the pore closure mechanism for β= 1 and β < Unlike biological cells, GUVs show huge electrode-1. formation over millisecond time scales when sub- jected to electroporating DC fields, We used a high- OR-172 speed imaging camera to characterize the elec- Correlation between numerical simulations and trodeformation of GUVs subjected to electroporat- clinical outcomes of irreversible electropora- ing pulsed DC fields, measuring their aspect ratio tion for hepatocellular carcinoma and shape deformations. The results are presen- Olivier Sutter 1, Luc Lafitte2, Baudouin Denis de Senneville2, Damien Voyer3, Jean Pierre Tasu4, Arthur ted as a function of amplitude of electric field and Petit1, Timothée Molango1, Lorenzo-Carlo Pescatori1, conductivity ratio ( β) of the enclosed medium to the Olivier Seror1, Clair Poignard2 suspending medium of vesicles. 1Hôpital Avicenne, France In our first study, we examined the responses of 2Université de Bordeaux, France GUVs synthesized using the gel-assisted method 3EIGSI La Rochelle, France to a dc pulsed electric field. For the low salt case, 4University Hospital of Poitiers, France where the ion concentration in the enclosed me- dium (Cen) was less than or equal to 0.3 mM, and Objectives: To assess the clinical relevance of for the high salt case, where Cen was greater than retrospective numerical simulations of irreversible or equal to 25 mM, we observed that the shape electroporation (IRE) for hepatocellular carcinoma deformation and degree of deformation of the ves- (HCC). icles remained unchanged for both similar electrical Material and Methods: IRE procedures conducted conductivities, β=1 and greater inner conductivity at our center in 2022 were used for retrospective β>1, even with an increase in Cen. However, for numerical modeling and treatment evaluation stud-greater outer conductivity β<1 and Cen=25 mM, we ies. Per-procedural imaging (Cone Beam CT be-observed a transition from oblate to prolate shape fore and after electrodes insertion) was used to cre- in the GUVs due to a higher differential ionic con- ate a 3D geometric model of the tumor and its en- centration across the bilayer membrane. Interest- vironment using software developed by our team. ingly, the relaxations of the vesicles were not af- The spatial coordinates of the electrodes, as well fected by Cen for both β=1 and β<1. Conversely, as treatment parameters from the Nanoknife® IRE in the case of greater outer conductivity β>1 and generator, were used to retrospectively simulate Cen=100 mM, a higher edge tension resulted in in- the extent of the electric field (EF). The overall EF at hibited pore growth and faster relaxation of the ves-the maximum voltage administered between each icles. pair of electrodes was simulated using a static lin- In our second work, we have studied the effect of ear model by the finite element method. The rates waveform on the deformation and relaxations of of tumor coverage by 3D EF mappings were com- GUVs where vesicles were subjected to a high- pared to clinical and imaging follow-up of the pa- intensity, single cycle of a sinusoidal pulsed ele tient in terms of primary efficacy (1 month MRI) and 196 local recurrence (after one month). immediately during the procedure, some scholars Results: Among the 40 IRE procedures, 23 were have shown that the impedance characteristics of included as they involved the treatment of one (15 biological tissues can reflect the ablation effect of procedures) or two (8 procedures) nodular HCCs tumors. Impedance measurement technology of <5cm with all parameters available for numerical biological tissues is a key technology to assess treatment simulations. The 31 treated HCCs had ablation efficacy. Pulse waveform has rich fre- a mean diameter of 21mm (range: 7mm to 45mm) quency characteristics, and different pulse wave- and 3 to 6 electrodes were employed. The clinical forms have different amplitude-frequency charac- treatment protocol consisted of applying at least teristics. In this paper, pulse frequency response 100 pulses of 90µsec between each electrode pair. method is used to measure the impedance char- Numerical simulations revealed a median percent- acteristics of biological tissues. Firstly, the prin- age of tumor segmentation coverage by simulated ciple of measuring biological impedance from pulse EF of 100% at 300V/cm, 99,1% at 400V/cm, 94,1% voltage and current waveforms is theoretically ana- at 500V/cm and 80,9% at 600V/cm. lyzed. Secondly, simulation methods are used to At 1 month, 29/31 tumors (93,5%) exhibited com- set different repetition frequencies, pulse widths, plete ablation on MRI. Eight out of these 29 initially and pulse waveform types to explore the measure-ablated tumors recurred during follow-up (median ment results of biological impedance under differ- delay: 7 months). Overall, 21/31 tumors (68%) re- ent combinations of pulse characteristic paramet- mained inactivated after a median follow-up of 17 ers. The measurement pulse waveforms in this months. The median percentages of tumor cover- article include square-wave pulse current, square- age by different EF isolines were significantly lower wave pulse voltage, bipolar pulse waveform, syn-for the 10 cases of local IRE failure than for the 21 ergistic pulse voltage waveform, and synergistic cases of IRE success: 95,1% vs. 100% at 300V/cm pulse voltage and current waveform. By compar- (p<0.001); 83% vs. 100% at 400V/cm (p<0.0001); ing the above results, the pulse current square 72,3% vs. 98,9% at 500V/cm (p<0.001) and 61,4% wave (at burst 10Hz) measurement of biological vs. 89,9% at 600V/cm (p<0.01). A tumor coverage impedance has the smallest error, with a capacit- <95% by the 400V/cm EF isoline predicted a local ance measurement error of less than 3%. This IRE failure with a positive predictive value >80%. simulation lays the foundation for measuring bio- Conclusion: The results of numerical simulations logical impedance using the pulse frequency re- of IRE conducted on a retrospective cohort of pa- sponse method. tients are correlated with the local effectiveness of the treatment. This study suggests that, using a S15 - Advanced imaging static linear model of IRE, the isoline of the calcu- techniques for visualizing the lated EF providing the best prediction of the clin- ical outcome for the treatment of HCC is around mechanisms of pulsed electric 400V/cm. field interactions OR-173 Wednesday late afternoon Simulation study on waveform characteristics Track B of measuring bio-impedance using pulse fre- Sep 18, 16:50 - 17:50 quency response method Lisheng Zhao, Sizhe Xiang, Haobo Yang, Shoulong Dong, Chenguo Yao, Liang Yu Chongqing University, China Due to the difficulty in assessing the efficacy of pulse electric field tumor ablation technology 197 OR-174 support. PEFfect Illumination: Observing Protein Oxid- ation Effects of Pulsed Electric Field Through OR-175 Monitoring (Bio)Chemiluminescence Electric field effects on human skeletal muscle- Kateřina Červinková 1, Petra Vahalová1, Michaela derived mesenchymal stem/stromal cells in-Poplová1, Tomáš Zakar1, Daniel Havelka1, Martin vestigated by scanning electrochemical micro- Paidar2, Viliam Kolivoška3, Michal Cifra1 scopy 1Institute of Photonics and Electronics of the Czech Inga Morkvenaite-Vilkonciene, Tomas Mockaitis Academy of Sciences, Czech Republic State research institute Center for Physical Sciences and 2University of Chemistry and Technology, Czech Repub-Technology, Lithuania lic The human skeletal muscle-derived mesen- 3J. Heyrovský Institute of Physical Chemistry of the chymal stem/stromal cells (SM-MSCs) possess Czech Academy of Sciences, Czech Republic myogenic differentiation potential and participate Pulsed electric field (PEF) technology has in muscle regeneration. However, the question of become adaptable and useful across a range of how to improve human skeletal muscle regenera- research and industrial fields. While its primary tion remains actual. In this study, the total SM- effects have traditionally focused on lipid mem- MSCs population was separated into subpopula- branes, growing interest surrounds its impact on tions according to the neural cell adhesion mo- proteins, essential biomolecules governing cellular lecule (NCAM, CD56), stimulated with an alternat- processes. ing electric field (AC) using scanning electrochem- In this study, we utilized a novel and non- ical microscopy (AC-SECM) and the intracellular destructive endogenous chemiluminescence- redox changes, and myogenic differentiation mark- based sensing platform to investigate the effects ers were evaluated. The myogenic differentiation of PEF-generated ROS on a model protein, bovine of the total SM-MSCs population before and after serum albumin (BSA). Our technique exhibited AC stimulation was evaluated immunohistochem- superior sensitivity compared to traditional assays, ically by the levels of desmin and myogenin. The enabling the detection of oxidative effects induced effect of AC stimulation on the redox capacity of by PEF. Additionally, we assessed the influence CD56(+) and CD56(-) cell subpopulations, as well of prooxidants (such as hydrogen peroxide) and as on the level of myogenin on the indium tin oxide antioxidants (catalase and superoxide dismutase) (ITO) surface, were also investigated. Results: The on PEF-induced protein oxidation, elucidating their total SM-MSCs population grown on a glass cover- roles in modulating oxidative processes. slip weakly responded to AC stimulus, i.e. the level We proposed a comprehensive reaction scheme of desmin was slightly increased by the 3rd day of describing the pathways through which PEF- differentiation, while in the not stimulated cells, it in-generated ROS induce protein oxidation and the creased only by the 7th day. The level of myogenin modulation by pro- and antioxidants. Our findings did not change after AC stimulation. However, the provide valuable insights into the mechanisms CD56(+) and CD56(-) subpopulations had different underlying PEF-induced protein oxidation and redox activities and myogenic differentiation poten- highlight the importance of considering pro- and tial: the CD56(+) cells had stronger natural diffu- antioxidants in PEF-treated proteins. Understand- sion and were more redox active compared to the ing these processes holds significant implications CD56(-) cells; an alternating electric field more actfor diverse applications, including biomedicine, ively stimulated the redox activity of CD56(+) cells food processing, and biotechnology. than in CD56(-) cells; at control level, the CD56(+) cells had more myogenic differentiation-regulating Acknowledgments: The authors thank the Czech transcription factor myogenin, which AC more in- Science Foundation project no. 20-06873X for the tensively stimulated than in CD56(-) cells. Data 198 show that the total population of human SM-MSCs urements of intensity and lifetime images of fluores-is heterogeneous, with different regenerating po- cence not only of exogenous fluorophores stained tential cells that do not equally respond to extra- in cells but also endogenous fluorophores such cellular stimuli. The SECM can be used in both as nicotinamide adenine dinucleotide (NADH) or ways: (i) for extracellular stimulation and (ii) for the flavin adenine dinucleotide (FAD) in cultured cells investigation of intracellular redox changes of the were carried out before, during and after applica- human SM-MSCs or their subpopulations, allowing tion of nsPEF by using inverted microscope, time- a deeper understanding of the mechanisms medi- correlated single photon counting system, and ating skeletal tissue regeneration both in vitro and steady-state excitation light and femtosecond pulse in vivo. laser system. Results and Discussion: The effects of applied OR-176 electric field having a pulse-width of 50 ns and Effects of Nanosecond Pulsed Electric Field field strength 20 kV/cm (nsPEF(50)) have been ex- on Cancerous and Normal Cells — Fluores- amined for lung cancerous cells H661 and A549 cence Microscopy and Autofluorescence Life- and lung normal cells MCR-5 attached on the glass time Imaging substrate of the microelectrodes with the following Nobuhiro Ohta measurements: 1) change in cell morphology; 2) National Yang Ming Chiao Tung University, Taiwan change in cell viability; 3) phosphatidylserine (PS) Introduction: Electric field effects on dynamics externalization; 4) AFLIM of NADH and FAD; 5) and function of biological systems may depend on generation of superoxide anion; 6) generation of the cell membrane capacitance and on the pulse singlet oxygen; 7) change in mitochondrial mem- duration of the applied field, which enables select- brane potential; 8) caspase activation: 9) change ive applications of pulsed electric field in specific or-in calcium ion concentration. The obtained results ganelles and specific living cells. If pulses shorter show that caspase-dependent apoptosis is induced than the charging time of the outer membrane are by nsPEF in H661 and A549, whereas such field applied to living systems, the pulsed electric field effect was less or not induced in MCR-5, indicating may induce changes in subcellular organelles, sig- that cancerous and normal cells show very different nal proteins, and biochemical processes, without electric field effects from each other. affecting the outer plasma membrane. Consider- able advances have been made regarding the un- S11 - In vivo delivery of genetic derstanding of the pulsed electric field effects on medicine through gene the cellular functions in different cell types. Never-electrotransfer theless, it is still elusive how the effects of nsPEF on human cancerous cells differ from that on the nor-Wednesday late afternoon mal cells of the same origins. This is very important Track C from the clinical point of view because the damage Sep 18, 16:50 - 18:05 of the healthy cells limits the effectiveness of the cancer treatments. In the present study, effects OR-177 of nanosecond pulsed electric field (nsPEF) on dy- Immunomodulatory effects of plasmid DNA fol- namics and function of live cells have been ex- lowing gene electrotransfer in colon cancer util- amined by using fluorescence microscopy includ- izing different electric pulse protocols ing autofluorescence lifetime imaging (AFLIM), with Tim Bozic 1, Mariangela De Robertis2, Iva Santek1, a special attention to the difference of the electric Flaviana Marzano3, Boštjan Markelc1, Alessandro field effects between cancer and normal cells. Silvestris2, Apollonia Tullo3, Graziano Pesole2, Maja Methods: An electrode microchamber has been Čemažar1, Emanuela Signori3 constructed for applying nsPEF to live cells. Meas- 1Institute of Oncology Ljubljana, Slovenia 199 2University of Bari ‘A. Moro’, Italy death signaling represented by immunogenic 3Consiglio Nazionale delle Ricerche, Italy necrotic pathways, as well as the innate and adaptive immune response. These results sug- Gene electrotransfer (GET) has recently gest that specific pulse parameters can induce emerged as a promising non-viral approach for distinct immunomodulatory profiles in the tumor delivering plasmid DNA in gene therapy strategies microenvironment, highlighting the importance of for various pathologies, including cancer, autoim- pulse parameter selection in EP-based treatments. mune, or inflammatory diseases. Exposure of Therefore, this analysis could pave the way to cells to electric pulses in reversible electroporation identify information useful for selecting the best EP (RE) approaches induces a transient increase in method or developing new EP-based techniques cell membrane permeability, leading to diverse for an immunization approach. biological responses and even cell death. Despite multiple mechanisms influencing cell biology OR-178 impacting the outcome of GET treatments, the Calcium electroporation and interleukin-12 molecular effects of in vivo DNA delivery using gene electrotransfer different electric pulse parameters remain poorly Barbara Lisec, Boštjan Markelc, Katja Ursic characterized. Therefore, in this study, we charac- Valentinuzzi, Gregor Serša, Maja Čemažar terized, for the first time in vivo, two GET protocols Institute of Oncology Ljubljana, Slovenia for intratumoral delivery of plasmid DNA, via Calcium electroporation (CaEP) utilizes elec- transcriptomic profiling using RNA sequencing of troporation to facilitate cellular uptake of elevated treated murine colorectal tumors. An electropora-levels of Ca2+, leading to cell death induction. tion (EP) pulsing protocol based on Short and High While the efficacy of CaEP has been assessed Voltage (SH-V) electric pulses and an adapted in clinical trials, additional preclinical investigations High Voltage – Low Voltage (HV-LV) pulse protocol are necessary to fully understand its effectiveness were investigated, and the effects compared at and underlying mechanisms. This study assesses three and seven days post-therapy. Although there the effectiveness of CaEP in two tumor models and were no significant effects on mice body weight and compares it with electrochemotherapy (ECT). In tumor growth delay between treatment groups, the addition, the CaEP was combined with gene elec- SH-V pulse protocol showed better overall survival, trotransfer (GET) of plasmid encoding interleukin- with two mice exhibiting a partial response and a 12 (IL-12), aiming to elucidate potential synergistic steady disease response alongside a prolonged effects. Our hypothesis was that IL-12 enhances tumor growth delay. Immunofluorescence analysis the antitumor efficacy of local ablative therapies revealed increased CD4+ T cells and even higher such as CaEP and ECT. CD8+ T cells recruitment on day seven after both The impact of CaEP was evaluated in vitro and in treatments compared to day three, with statistical vivo using murine melanoma B16-F10 and murine significance for CD8+ T cells in the SH-V pulse mammary carcinoma 4T1 models, in comparison to protocol. Notably, the most significant changes ECT with bleomycin. Various treatment protocols were observed in the number of macrophages, involving CaEP with escalating calcium concentra- which significantly decreased on day three after tions alone or combined with IL-12 GET were ex- the SH-V pulse protocol and was restored on day amined. The tumor microenvironment was extens- seven, while GET using the HV-LV pulse protocol ively analyzed through immunofluorescence stain- resulted in the opposite trend. Consistent with ing of immune cells, blood vessels, and proliferat- these data, RNA sequencing analysis showed ing cells. that, if apoptosis is the predominant mode of cell In vitro, both CaEP and ECT with bleomycin death due to the application of SH-V pulses, the demonstrated dose-dependent decrease in cell vi- altered gene profile following the HV-LV pulse ability, with no differences in sensitivity between protocol is associated with the activation of cell 200 the two cell lines. In vivo, a dose-dependent re-response in mouse muscle following (IM) intra- sponse was also observed, with greater efficacy muscular pDNA EP and identify factors directly noted in 4T1 tumors compared to B16-F10 tu- linked to expressed mAb PK. To achieve this, mors. CaEP with 250 mM Ca resulted in significant knockout (KO) models and immune phenotyping growth delay in 4T1 tumors, which was compar- are employed, utilizing a validated pDNA encoding able to ECT with bleomycin. Additionally, adjunct a murine 4D5 anti-HER2 mAb. Earlier experiments peritumoral IL-12 GET following CaEP prolonged demonstrated that mAb PK in athymic nude and survival in B16-F10-bearing mice, but not in 4T1- RAG1 KO mice did not significantly differ from bearing mice. Furthermore, CaEP with peritumoral wild-type (WT) mice, suggesting that T and B cells, IL-12 GET altered tumor immune cell populations, including antigen-presenting pathways, do not cell proliferation and vasculature. drive the decline in mAb expression. To assess In conclusion, mice bearing 4T1 tumors exhibited the impact of the innate immune response, we superior response to CaEP compared to those with evaluated 4D5 PK in IFNAR1 KO mice, which lack B16-F10 tumors, suggesting potential immune sys- type-I interferon receptor function. After IM p4D5 tem involvement, since the difference in sensitiv- EP, plasma mAb levels showed no significant ity was not confirmed in vitro. The involvement of difference between IFNAR1 KO and WT mice immune response in antitumor effectiveness was throughout the 12-week follow-up. This finding further supported by enhanced antitumor efficacy diminishes the likelihood that DNA sensing, includ- observed with CaEP or ECT combined with IL-12 ing cGAS/STING, affects mAb PK. GET. However, the augmentation of CaEP efficacy Concurrently, we ran flow cytometry panels on WT varied depending on tumor type, with greater en- mice muscle, harvested one week post-IM EP. hancement seen in poorly immunogenic B16-F10 Compared to control muscle (including untreated, tumors compared to moderately immunogenic 4T1 vehicle-injected, and EP only), infiltrated T cells tumors. showed more of the effector phenotype and dend- ritic cell (DC) muscle infiltration was increased. OR-179 NK cell-mediated cytotoxicity was considered, Unraveling a multifactorial host immune re- but muscle NK cell infiltration was limited and did sponse to intramuscular electrotransfer of dna- not increase after pDNA EP. This suggests that encoded antibody therapy cytotoxic T-cell responses, facilitated by DC-based Debby Thomas1, Jenny Sprooten 1, Jannes antigen presentation, may target mAb-producing Govaerts1, Pascal Merchiers2, Maarten Dewilde1, cells. However, this does not appear to impact Kevin Hollevoet1, Abhishek Garg1, Nick Geukens1 mAb kinetics based on earlier observations in 1University of Leuven, Belgium RAG1 KO mouse model. 2Biomerpa BV, Belgium In addition, while assessing the IM macrophage DNA-based antibody gene electrotransfer population upon treatment, we found that the aims to administer antibody-encoding nucleotides, percentage of pro-inflammatory macrophages rather than the antibody protein, utilizing a non-viral (M1) increased, while the anti-inflammatory mac-plasmid DNA (pDNA) vector. This strategy aims rophages (M2) population remained unchanged. to achieve sustained in vivo antibody production, This data, supported by an increased DC infiltra- addressing challenges inherent to conventional tion, implies that phagocytic antigen-presenting antibody production and delivery. Previously, cells play a role in the decreased mAb production. we demonstrated preclinical proof of concept Potentially, cells transfected with mAb-encoding for diverse DNA-encoded monoclonal antibodies pDNA increase their “eat-me signals” due to cell (mAbs) and nanobodies delivered in muscle or stress (EP, protein overproduction, ER stress, tumor using electroporation (EP). etc.), attracting phagocytic cells. Ongoing work In this study, we aim to delineate the host immune investigates whether modulating phagocytosis impacts mAb kinetics. 201 In conclusion, this work started to unravel a mul-ments. tifactorial host immune response triggered by IM Here, we report MYO Technology delivery of two electrotransfer of a DNA-encoded mAb. As we molecules from a class of biologics that have a elaborate on these observations, factors demon- short half-life (about 4 hours): G-CSF (granulocyte- strably associated with mAb kinetics will be of colony stimulating factor) and GLP-1 (glucagon-like particular interest, as modulating these responses peptide-1). We demonstrate the long-term thera- could allow for a more robust and prolonged in peutic effect of G-CSF, used to treat severe chronic vivo mAb expression. neutropenia, when delivered via MYO Technology in both mice and rabbits. Mice were found to main- OR-180 tain durable protein expression and elevated neut- Enhancing the Therapeutic Benefits of Proteins rophil counts for over a year. Similarly, proof-of- with Short Half-Lives: Delivery of G-CSF and concept studies in mice demonstrate that expres- GLP-1 with DNA-Based MYO Technology sion of MYO Technology-delivered GLP-1/GIP re- Debnath Maji, Andrew D. Cameron, Linda Sasset, ceptor agonists remains stable for over six months Sayantani Sinha, Andy Thompson, Carleigh Sussman, and is efficacious in stimulating weight loss in Delcora A. Campbell, Robert Miller, Marek M. Drozdz, mouse models of diet-induced obesity. Rachel A. Liberatore Although initial animal studies have demonstrated RenBio, United States the potential of DNA-based platforms for delivering Numerous recombinant proteins have received therapeutic proteins, a notable challenge persists clinical approval, drastically transforming the stand-in enhancing protein yield to within a therapeutic ard of care landscape. However, the therapeutic range in larger animals. The proteins produced efficacy of many small biologics is hindered by and released systemically encounter a dilutional ef- their relatively short half-life, often due to phar- fect influenced by the size and blood volume of macokinetic properties, receptor binding kinetics, each species. To address this, extensive optimiz- and disease characteristics that necessitates fre- ations of the MYO Technology platform have been quent dosing to achieve and maintain therapeutic made that address the disparity in therapeutic pro- effectiveness, thereby causing significant patient tein concentrations achieved between smaller and discomfort and potentiating nonadherence to the larger animal models. therapy. Additionally, significant challenges like Thus, by enabling durable, scalable expression of high manufacturing costs and cold chain require- small therapeutic proteins, MYO Technology has ments continue to limit broader access to these life- the potential to expand the therapeutic advantages changing therapeutics, a problem amplified in low- of proteins with short half-lives. and middle-income countries. MYO Technology, a platform for the delivery of S19 - Pulsed electric fields in meat plasmid DNA (pDNA)-based therapeutics via in- and fish and their by-products tramuscular electroporation, offers several advant- ages over standard delivery of therapeutic proteins. processing A single administration of pDNA medicine using MYO Technology takes just a few minutes, and the Wednesday late afternoon serum level of the expressed therapeutic protein is Track D maintained for many months to years, without the Sep 18, 16:50 - 17:50 need for redosing, thus alleviating the dependence on patient compliance for durable therapeutic ef- OR-182 fect. Furthermore, pDNA manufacturing is a sim- New advancement on meat processing using pler and less specialized process as compared to Pulsed electric field technology Indrawati Oey most protein manufacturing processes, and pDNA University of Otago, New Zealand is very stable and lacks most cold chain require- 202 activation by PEF in naturally infected hake belly fil-Pulsed Electric Fields (PEF) is an innovative lets and the evaluation of the quality of fish samples nonthermal technique where high-voltage electrical during their shelf life after PEF treatments. energy is briefly applied to food in pulses lasting mi-Method: Application of PEF technology (evaluation croseconds to milliseconds. This energy induces of field strength – 3 to 5 kV/cm-, pulse width - 10 to various physicochemical changes in meat, particu-30 µs -, and specific energy - 10 to 30 kJ/kg -) for larly enhancing tenderness while preserving attrib- inactivation of Anisakis spp. from hake (Merluccius utes such as color, flavor, water retention, volatile merluccius) in naturally parasitized hake belly. The profile, and mineral content, and without promot- effect of MAP storage on the survivability of Anisa- ing lipid oxidation. Furthermore, PEF pre-treatment kis spp was also evaluated after applying the PEF enhances the efficiency, cost-effectiveness, and treatment. sustainability of meat processing methods like ac- The fish microbiota of control and PEF samples celerated ageing, salting, brining, drying, and sous was evaluated during shelf life in MAP (50%CO2- vide (SV) cooking. Particularly in sous vide pro- 50%N2). Quality tests (drip loss, moisture, water cessing, PEF pre-treatment has been found to re- holding capacity, and cooking loss) were also car- duce electrochemical variability in meat, signific- ried out on control, PEF, and freeze/thawed hake antly shorten SV cooking times, and consistently pieces to determine the impact of the technology improve tenderness, even in tougher cuts such on quality. as brisket and short ribs with bone. Additionally, Results: Inactivation of Anisakis spp in naturally the subsequent PEF followed by sous vide pro- parasitized hake bellies was highly dependent on cessed short ribs have shown increased in vitro PEF parameters, among which the two most im- protein digestibility without compromising the mi- portant were field strength and specific energy. In crobial safety of meat during refrigerated storage. addition, Anisakis survivability depended on the In this presentation, a recently built new continuous parasite’s location, as it was more resistant to PEF PEF chamber design will be introduced. when located inside the fish meat than when loc- OR-183 ated on the most superficial part of the belly. Al- Inactivation by Pulsed Electric Fields of Anisa- most complete inactivation of the parasite in hake kis in naturally infected hake meat. belly was achieved with a treatment of 5 kV/cm and Vanesa Abad, Javier Raso, Juan Manuel Martínez, 30 kJ/kg, applying pulses of 30 µs. The degree of Guillermo J. Cebrian, Ignacio Álvarez inactivation increased over time when the samples Universidad de Zaragoza, Spain were stored after PEF treatments in a modified at- mosphere containing 50% CO2. Introduction: Anisakis is a zoonotic parasite After PEF treatments, quality analyses during shelf found in the stomach of marine mammals. Humans life indicated that fish microbiota evolved simil- become accidental hosts when they consume raw arly to untreated samples; however, the MAP lim- or undercooked fish, or cephalopods, leading them ited the growth of the microbiota. In PEF-treated to suffer from intestinal syndromes and allergic re- samples, quality parameters (drip loss, moisture, actions. One of the most parasitized species in water holding capacity, and cooking loss) were Europe is hake. In Europe, the officially prescribed closer to those of fresh hake and superior, in terms methods for inactivation of Anisakis are heat treat- of quality, to the values obtained in frozen/thawed ment or freezing, both of which can affect fish qual- samples during their entire shelf life. ity. Conclusion: These results suggest that PEF could Several studies have demonstrated the effective- represent a promising alternative to freezing as a ness of Pulsed Electric Fields (PEF) for the inac- strategy for the elimination of Anisakis spp in fish tivation of Anisakis; however, none of them has without affecting fish quality, specifically in hake. featured naturally infected samples. Therefore, the objective of this study focuses on Anisakis spp in- 203 OR-184 streams from astaxanthin extraction were used for Valorization of shrimp by-products: Extrac- the extraction of chitosan biopolymer. The results tion of high value-added compounds by pulsed showed that PEF and ASE increased the astax- electric field (PEF) and accelerated solvent ex- anthin content in the extracts for both shrimp spe- traction (ASE) cies and solvent used, and the higher recovery was Ana Cristina De Aguiar Saldanha Pinheiro 1, Fran-obtained using their combination. However, the in- cisco J. Martí-Quijal2, Francisco J. Barba2, Urszula crease in antioxidant capacity varied depending on Tylewicz1, Silvia Tappi1, Santina Romani1, Pietro the solvent type. Both technologies seem to be an Rocculi1 effective tool to recover astaxanthin and antioxidant 1University of Bologna, Italy extracts from shrimp by-products. The techniques 2University of Valencia, Spain for recycling shrimp by-products are not yet well es- Shrimp and prawns are one of the most import- tablished for industrial use due to a lack of stand- ant internationally traded seafood products. Crus- ardization. Further research is needed to confirm tacean by-products are important natural sources these promising results and to explore other valu- of high-value compounds. Recently, pulsed elec- able compounds in crustacean by-products. This tric field (PEF) treatment has emerged as a prom- research was funded by the National Recovery and ising method for the isolation and extraction of vari-Resilience Plan (NRRP), Mission 4 Com-ponent 2 ous components from seafood by-products such Investment 1.3 - Call for tender No. 341 of 15 as calcium, chondroitin sulfate, collagen, chitosan March 2022 of Italian Ministry of University and Re- and protein. Accelerated solvent extraction (ASE) search funded by the European Union - NextGen- is considered an environmentally friendly method erationEU; Project code PE00000003, Concession for the extraction of bioactive and nutrient-rich com-Decree No. 1550 of 11 October 2022 adopted pounds from plants and food matrices. The main by the Italian Ministry of University and Research, objective of the present study was to apply PEF CUP D93C22000890001, Project title ”ON Foods - and ASE to recovery astaxanthin from shrimp by- Research and innovation network on food and nu- products and evaluate the effects of these tech- trition Sustainability, Safety and Security - Working nologies used independently or in combination on ON Foods”. the astaxanthin content and antioxidant activities of the extracts. In this study, fresh samples of red S10 - Electrochemotherapy of shrimp (Aristeus antennatus) and camarote shrimp cutaneous tumors (Melicertus kerathurus) were obtained from a local market in Valencia, Spain. PEF (3 kV/cm, 100 Wednesday late afternoon kJ/kg, 74 pulses) was used as a pretreatment be- Track E fore traditional solvent extraction and before the in-Sep 18, 16:50 - 18:20 novative extraction procedure ASE (50 °C, 15 min, 103.4 bar). The combined and independent effects OR-185 of the emerging technologies PEF and ASE using Electrochemotherapy for Kaposi Sarcoma and different solvents (EtOH and DMSO) on the extrac- Merckel Cell Carcinoma: findings of the In- tion of astaxanthin were evaluated for each shrimp spECT Rare Tumours Working Group species (M. keranthurus and A. antennatus). The Joy Odili 1, Pietro Quaglino2, Matteo Brizio2, Gi-astaxanthin content in extracts from shrimp by- ulia Bertino3, Erika Kis4, Matteo Mascherini5, Michela products was analyzed using spectrophotometric Battista6, Christian Kunte7, David Mowatt13, Francesco and HPLC methods. The antioxidant capacity of Russano8, Roberto Giorgione9, James P. Clover10, Had- the extracts was evaluated by Trolox equivalent rian Schepler11, Gregor Serša12, Marta Minuti3 1 antioxidant capacity (TEAC) and oxygen radical St. Georges University Hospital, United Kingdom absorbance capacity (ORAC) assays. Solid side 204 2University of Turin, Italy complete response. One-year local progression- 3IRCCS Policlinico San Matteo Foundation, Italy free survival in the whole population was 91%, 2 4University of Szeged, Hungary, Hungary years LPFS was 87%. 5IRCCS Ospedale Policlinico San Martino, Italy Conclusion: In the present study, ECT showed an- 6IGEA S.p.A, Italy titumor activity and a favorable safety profile in pa-7Artemed Hospital Munich, Germany tients with cutaneous Kaposi Sarcoma and Meckel 8IRCCS Veneto Institute of Oncology IOV, Italy Cell Carcinoma, where conventional treatments 9University of Eastern Piedmont, Italy were unsuitable or refused. Best results were ob- 10Cork University Hospital, Ireland tained in small tumors (<3 cm) using hexagonal 11Johannes Gutenberg University, Germany electrodes. 12Institute of Oncology Ljubljana, Slovenia 13The Christie Hospital, United Kingdom OR-186 Electrochemotherapy in the treatment of cu- Introduction: The rare tumors working group of taneous melanoma metastases – the InspECT the International Network for sharing practices on experience electrochemotherapy (InspECT) group has been Erika Kis 1, Barbara Perić2, Matteo Brizio3, Giuseppe tasked with reviewing the registry and analysing the Riva3, Giulia Bertino4, Nunzia Di Cristo5, Barbara information on rare tumors collected by InsPECT Silvestri6, Hadrian Schepler7, Joy Odili8, Siva Kumar9, centres. Kaposi Sarcoma and Merkel Cell Car- Matteo Mascherini10, Christian Kunte11, Francesca cinoma were found in this group of rare tumours. Tauceri12, Giulia Colavitti13, Veronica Seccia14 ECT is an alternative treatment option for patients 1University of Szeged, Hungary with cutaneous malignancies of skin and non-skin 2Institute of Oncology Ljubljana, Slovenia origin, not suitable for conventional treatments. In 3University of Turin, Italy this study, we aimed to assess outcomes of the use 4IRCCS Policlinico San Matteo Foundation, Italy of ECT as a treatment modality for Kaposi Sarcoma 5University of Eastern Piedmont, Italy and Merckel Cell Carcinoma within the InspECT re- 6ULSS 3 Serenissima, Italy gistry. 7Johannes Gutenberg University, Germany Materials and methods: Patients with superficial le- 8St. George’s University Hospital, United Kingdom sions of Kaposi Sarcoma and cutaneous Merkel 9Queen Victoria Hospital, United Kingdom cell carcinoma. Data from 15 European centres 10IRCCS Ospedale Policlinico San Martino, Italy was included. Patients underwent at least one 11Artemed Hospital Munich, Germany ECT session with bleomycin, performed follow- 12Morgagni-Pierantoni Hospital, Italy ing the European Standard Operating Procedures, 13Southmead Hospital, United Kingdom between March 2011 and October 2021. 14University Hospital of Pisa, Italy Results: The analysis included 61 Kaposi Sarcoma Melanoma is the most dangerous type of skin patients (mean age 73 years; median number of le- cancer. The treatment of melanoma skin meta- sions per patient 3) and 18 Merkel cell carcinoma stases are challenging despite multimodal avail- patients (mean age 74 years; median number of able systemic and local therapies. The melanoma lesions per patient 1.5). Side effects were repor- working group of the International Network for shar- ted as mild and easily manageable (hyperpigment- ing practices on ECT (InspECT) group investigates ation, 16%; ulceration, 5%; suppuration, 2%). The treatment outcomes after ECT using a common response to treatment per patient was 85% com- database with defined parameters. plete and 11% partial for Kaposi Sarcoma. In pa- Objectives: In our four studies the aim was to in- tients with Merkel cell carcinoma the response was vestigate the effectiveness of ECT in cutaneous 45% complete and 45% partial. In the multivari- metastases of melanoma, the effectiveness of ECT ate model, time since diagnosis to ECT and small in combination with pembrolizumab and the optimal tumor size showed a significant association with a timing of ECT in combination with immunotherapies 205 as well as to evaluate health-related quality of life group than in the pembrolizumab group. Our res- (HRQoL) changes in melanoma patients. ults suggest that in combined application the best Methods: All analysis included prospective data tumor response can be achieved with ECT during from the InspECT register. Endpoints included re- concomitant immunotherapy. sponse (RECIST v3.0), local progression-free sur- vival (LPFS), systemic PFS, overall survival (OS) OR-187 rates, toxicity (CTCAE v5.0), patient-reported HR- InspECT database and clinical results of elec- QoL outcomes ([EQ-5D] and [EQ-VAS]). We also trochemotherapy undertook a retrospective matched cohort analysis Giulia Bertino 1, Ales Groselj2, Christian Kunte3, Hadrian Schepler4, Julie Gehl5, Tobian Muir6, James on the effectiveness of ECT in combination with P. Clover7, Pietro Quaglino8, Erika Kis9, Matteo pembrolizumab in stage IIIC–IV melanoma patients Mascherini14, Brian Bisase10, Giancarlo Pecorari8, Falk included in the InspECT and the Slovenian Can-Bechara12, Paolo Matteucci20, Joy Odili13, Francesco cer Registry, and compared patient outcomes after Russano14, Giulia Colavitti15, Rowan Pritchard-Jones16, the following treatments: (a) pembrolizumab, (b) David Mowatt21, Barbara Silvestri17, Veronica Seccia18, pembrolizumab and ECT, and (c) ECT alone. Re-Gregor Serša19 sponse rates were checked in groups of patients 1University of Pavia, Italy where ECT was performed before, during or after 2University Medical Centre Ljubljana, Slovenia immunotherapy. 3Artemed Hospital Munich, Germany Results: The response for MM was with a CR of 4Johannes Gutenberg University, Germany 64% (600/932) and OR of 82% (768/ 932) and no 5Zealand University Hospital, Denmark serious adverse events were observed. The local 6James Cook University Hospital, United Kingdom objective response rate (ORR) was higher in the 7Cork University Hospital, Ireland pembrolizumab-ECT group than in the pembroli- 8University of Turin, Italy zumab group (78% and 39%, p < 0.001) amongst 9University of Szeged, Hungary stage IIIC–IV melanoma patients.The 1 year LPFS 10Queen Victoria Hospital, United Kingdom rates were 86% and 51% (p < 0.001), and the 1 11IRCCS Ospedale Policlinico San Martino, Italy year systemic PFS rates were 64% and 39%, re- 12Ruhr-University Bochum, Germany spectively (p = 0.034). The 1 year overall sur- 13St. Georges University Hospital, United Kingdom vival (OS) rates were 88% and 64%, respect- 14IRCCS Veneto Institute of Oncology IOV, Italy ively (p = 0.006). The further investigation of 15Southmead Hospital, United Kingdom the optimal timing of ECT in combination with im- 16St. Helens & Knowsley Teaching Hospital, United munotherapies showed the best tumor response Kingdom (CR:80% OR:93%) in small lesions (<3 cm) when 17ULSS 3 Serenissima, Italy ECT was performed during concomitant immuno- 18University Hospital of Pisa, Italy therapy.Following ECT, both EQ-5D and EQ-VAS 19Institute of Oncology Ljubljana, Slovenia scores remained within MID boundaries, particu- 20Hull University Teaching Hospital, United Kingdom larly among complete responders. Combination 21The Christie Hospital, United Kingdom with checkpoint inhibitors was associated with bet- The International Network for Sharing Practices ter QoL outcomes. on ElectroChemoTherapy (InspECT) is a group of Conclusion: ECT is a highly effective local treat- expert clinicians working with ECT formed in 2008 ment for melanoma metastases in the skin, with to meet and discuss issues related to the use of no severe adverse effects and preserves patient ECT. The aim is to bring faster and better treat- QOL. The combined application of pembrolizumab ment to patients by publishing high quality research and ECT was safe and more efficacious in prevent- and by networking for continuous training in the ing further growth of cutaneous metastases than field. The major priority is a web-database, where pembrolizumab alone. Interestingly, we observed treatment data from the centres can be collected. longer PFS and OS in the pembrolizumab-ECT 206 The group is composed by 43 centres and has suitable for standard treatments because of severe collected more than 2200 patients over the last general comorbidities. 15 years of activity, publishing 20 papers in peer reviewed journals. The most treated hystological OR-188 types are: malignant melanoma, basal cell car- Electrochemotherapy for the treatment of cu- cinoma, squamous cell carcinoma, and breast can- taneous metastases from breast cancer cer. Julie Gehl In this lecture we focused on head & neck (HN) Zealand University Hospital, Denmark cutaneous squamous cell carcinoma (cSCC) and This special session presentation aims to dis- long-term basal cell carcinoma (BCC) data extrac- cuss clinical indications, results and future direc- ted from the database. tions of the treatment of breast cancer metastases A total of 162 patients with HN cSCC and a mean using electrochemotherapy. age of 80 years were analyzed: side effects were Breast Cancer is the most common cancer in mainly local and mild (hyperpigmentation, 11%; ul- women, with around 1 of 8 women receiving a ceration, 11%; suppuration, 4%). The response breast cancer diagnosis during their lifetime. to treatment per patient was 62% complete and Breast cancer is treated with respectively endo- 21% partial. In the multivariate model, intravenous crine treatment with our without cycline kinase in- drug administration and small tumor size showed hibitors (for estrogen positive (ER+) cancer only), a significant association with a positive outcome. HER2 inhibitors (for HER2positive disease only), One-year local progression-free survival was signi- chemotherapy, in some cases immunotherapy and ficantly better in patients with primary tumors (80%, in subset of patients targeted treatments. Surgery 95% C.I. 70%-90%) than in patients with locally remains a very impactful treatment, but is not pos- advanced disease (49%, 95% C.I. 30%-68%). In- sible in advanced cancer. Radiotherapy is an im- terestingly, ECT showed antitumor activity and a portant tool but can only be used regionally and with favorable safety profile in patients with complex a upper dose limit. cSCC for whom there was no widely accepted In numbers, cutaneous metastasis in breast standard of care. cancer patients is one of the most frequent patient Among long term follow-up patients in the InspECT groups with cutaneous metastases, due to the fre- registry, a relevant number of patients affected by quency of cutaneous metastases multiplied by the HN BCC could be retrieved: 129 patients have a population at risk. Tumors may present as smal- follow-up longer than 5 years with a median follow- ler lesions, often multiple small lesions, but may up time of 7.2 yrs (range 5.0-11.6 yrs). Median also by large confluent areas. Patients are often age is 74 years (range 41-93 yrs). Most of them quite affected by cutaneous metastases, as this were treated for a BCC lesion on the nose (40%), in may lead to changes in body image, oozing, bleed- the auricular region (12%), at the forehead/temple ing and pain. (10%) or in the eye region (10%). Overall disease- Electrochemotherapy has a high response rate free survival at 5 years was 94%, with some slight of 77%, with 62% complete response (Clover et al, non-significant differences in primary vs recurrent EJC, 2020). No difference between breast can- lesions (95% vs 92%), non-ulcerated vs ulcerated cer subtypes (ER+, HER2+, or negative) was ob- lesions (97% vs 86%) and small (< 3 cm) vs large served (Di Prata et al, Cancers 2023). Responses (> 3 cm) lesions (98% vs 90%). are lower for larger lesions, yet even very large le- In conclusion, ECT is a safe and effective treat- sions do respond to treatment. Electrochemother- ment for cSCC and BCC with high percentages of apy may be combined with other treatment modal- complete response lasting over time. It is partic- ities, e.g. a patient responding well to endocrine ularly indicated for lesions located in anatomical treatment in other disease site, but with progres- sites where surgery could cause aesthetical disfig- sion in skin metastases, may be treated with elec- urement or loss of function and in old people not 207 trochemotherapy in addition to systemic treatment. teins observed with non-zero intensity in at least Many breast cancer patients have experienced pre- one dataset samples. The observed intra-patient vious surgery and/or radiotherapy to the area af- Pearson correlation of 0.923 ± 0.053 for BCC fected by breast cancer metastases. A slightly patients and 0.901 ± 0.060 for SCC patients in- lower response rate is observed after radiotherapy, dicates high consistency of the e-biopsy sampling but still high. technique. The obtained mass spectra presented A novel treatment using calcium electropora- significantly different proteome profiles for cSCC tion is now tested for breast cancer, also show- and BCC with several hundreds of proteins sig- ing high response rates. Interestingly, preliminary nificantly differentially expressed in each tumor data indicate that calcium electroporation and elec- in comparison to the other. Notably, our study trochemotherapy may be used interchangeably in showed that proteomes sampled with e-biopsy case of resistance to treatment. from cSCC and BCC lesions are different, and Many patients are referred when tumors are that 7 proteins were significantly overexpressed reached a considerable size, so one future goal is in BCC in comparison to cSCC even after the to work for earlier referral in order to use electro- Bonferroni correction (FDR=7.1e-03). Our results chemotherapy when the disease burden is more provide evidence that the e-biopsy approach could limited. potentially be used as a tool to support cutaneous As electrochemotherapy is increasingly being tumors classification with rapid molecular profiling. used across Europe, access to treatment for pa- tients is becoming easier. OR-190 Electrochemotherapy in the treatment of OR-189 chronic suppurative benign skin conditions: Differential expression analysis of cutaneous The St George’s Hospital experience squamous cell carcinoma and basal cell Joy Odili carcinoma proteomic profiles sampled with St. George’s University Hospital, United Kingdom electroporation-based molecular biopsy Introduction: Electrochemotherapy (ECT) with Alexander Golberg 1, Edward Vitkin1, Ariel Berl2, Ju-Bleomycin is a highly effective local treatment for lia Wise1 cancer in the skin, regardless of histology, with no 1Tel Aviv University, Israel severe adverse effects. It has also been shown to 2Meir Medical Center, Israel be effective in treating benign skin conditions such Clinical misdiagnosis between cutaneous as keloid scarring. Bleomycin in isolation is also squamous cell carcinoma (cSCC) and basal cell used to treat benign conditions such as keloid scar- carcinoma (BCC) affects treatment plans and ring and vascular malformations and is known for carries risks of potential for recurrence, meta- its properties as an antibiotic. Acne keloids, follicu-stases, morbidity and mortality. We report the litis decalvans, and hidradenitis suppurativa belong development of a novel tissue sampling approach to a group of chronic inflammatory skin conditions with molecular biopsy using electroporation. This with limited treatments. Treatment goals include method, coined e-biopsy, enables non-destructive reducing inflammation, improving keloidal lesions, non-thermal permeabilization of cells in the skin and limiting exacerbating factors. We wondered for efficient vacuum-assisted extraction of inform- whether the addition of electroporation would po- ative biomolecules for rapid diagnosis. We used tentiate the action of Bleomycin in chronic suppur- e-biopsy for ex vivo proteome extraction from 3 ative benign skin conditions where all traditional locations per patient in 21 cSCC and 20 BCC treatments including surgery had failed. pathologically validated human tissue samples. Methods: We identified 9 patients with the follow- The total 123 extracted proteomes were profiled ing benign conditions were treated: acne keloidalis, using LC/MS/MS. In total, we identified 7087 pro- folliculitis decalvans, and hidradenitis suppurative 208 (6 scalp, 2 jaw/ chin, 1 buttock). All were male. Interventional Oncology is an umbrella term for All presented with suppuration and pain. ECT was the practical, minimally invasive treatment of cer- delivered under General Anaesthetic. Bleomycin tain types of cancer. The roadmap of Interventional was administered both Intratumourally and intra- Oncology was first published in 2018. The cooper- venously (combined approach). A linear probe was ation of interventional radiotherapy, chemotherapy, used in all cases, using the Cliniporator (Igea). endoscopy, and radiology within a dedicated Inter- Results of the treatments were stored on a shared ventional Oncology Center is the best choice for fo- database (International Network for sharing prac- cal approaches. tices on ECT, InspECT). By combining these focal expert treatments, we Results: all patients reported a significant improve- not only enhance local control but also have the po- ment is suppuration, pain, and itch in the treated tential to significantly reduce toxicity. A dedicated areas. Flattening and softening of the scarred multidisciplinary tumor board further enhances the areas was a secondary effect which continued to success of these treatments, offering a truly per- improve several months after the treatment. No sonalized approach. Moreover, the integration of serious adverse effects to the treatment were re- interventional oncology and immune oncology her- ported. Most of the patients required 2 treatments 6 alds a new era in minimally invasive personalized months apart. None of the treated patients required treatments. oral antibiotics or steroid injections after ECT. Recent large cohort clinical trials create the Conclusion: Electrochemotherapy is an estab- basis for future well-designed and objective mul- lished treatment for the management of cancers in tidisciplinary trials in personalized cancer patient the skin and deeper tissues. The exact mechan- care. ism for the effectiveness of ECT in treating chronic suppurative conditions such as acne keloids, fol- OR-192 liculitis decalvans and hidradenitis is unclear. One The role of Interventional Radiology of the factors may be the role of Bleomycin as a gly- Laura Crocetti coprotein antibiotic. Enhanced extracellular matrix University of Pisa, Italy turnover, and the effect on vascularity at the treated No abstract was provided. areas are also factors which may limit inflamma- tion, making this treatment effective for this group of OR-193 patients. The preliminary results from St George’s The role of Interventional Endoscopy Hospital are promising and are presented here. Fabia Attili Policlinico Gemelli, Italy S24 - Emerging role of No abstract was provided. Electrochemotherapy in the OR-194 treatment of GastroIntestinal The role of Interventional and External Beam cancer Radiotherapy Bruno Fionda Thursday morning Track A Policlinico Gemelli, Italy Sep 19, 8:30 - 10:00 No abstract was provided. OR-191 OR-195 The Interventional Oncology in the modern in- The role of Electrochemotherapy terdisciplinary scenario Martina Ferioli György Kovács University of Bologna, Italy Università Cattolica Sacro Cuore, Italy No abstract was provided. 209 OR-196 conclude that PEF-assisted extraction of phyto- The synergistic effect of Electrochemotherapy chemicals from living roots was a success. Plant in the modern Oncology scenario survival rate was 92,4% and the acquired extracts Attila Kovacs were largely synonymous with control quality-wise. Klinik für Diagnostische und Interventionelle Radiologie Six-month-old plants cultivated in aeroponic sys-und Neuroradiologie, Germany tems were separated into control (sham treatment) and study groups, paired with a specific Natural No abstract was provided. Eutectic Solvent (NES): choline chloride : xylose (1:2) + 30% water, choline chloride : glucose P12 - Biomass transformation and (1:2) + 30% water, choline chloride : ethylene biocompounds glycol (1:4) or tap water. Each specimen was put into a cuvette filled with designated solvent Thursday morning Track B for 5 min and afterwards subjected to PEF of Sep 19, 8:30 - 10:00 the following parameters: E = 0.3 kV/cm, t = 50 µ s, f = 1 Hz, N = 33. Such treatment was then OR-197 repeated once (Ntotal = 66) or twice (Ntotal = 99) Non-lethal extraction of phytochemicals and in 5 min intervals. Solvents were then collected, growth promotion of Iris domestica (L.) DC purified and analyzed using HPLC-MS technique. roots enabled by electroporation Conductivity, temperature and mass of the media Kajetan Grzelka, Joanna Jaśpińska, Adam were measured before and after treatment. The Matkowski, Sylwester Ślusarczyk current and pulse parameters were recorded on an Wroclaw Medical University, Poland oscilloscope. Based on these data, specific energy Iris domestica is a perennial herb from Iridaceae input (Wspec) was calculated and correlated to family. It has been used for centuries in Traditional root growth (measured weekly over 6 weeks) and Chinese Medicine as an expectorant, antipyretic concentrations of compounds found in the NES and anti-inflammatory agent, however active extract (calculated based on peak intensity and compounds isolated from its rhizome were found standard curve). Finally, the plants were harves- to possess antimutagenic, antioxidant and antidia- ted and methanolic root extracts were prepared betic properties. These substances are classified and analyzed in the same way in order to eval- as polyphenols, and among them isoflavones uate changes in I. domestica phytochemical profile. such as tectoridin, tectorigenin, iridin and irigenin are the most abundant and also show promise in This research was funded in whole of the Na- developing new phytoestrogenic drugs. Our aim tional Science Center POLAND basis of the was to extract these compounds without killing decision number National Science Centre Poland; the plants, which is an innovative and much more UMO-2020/39/D/NZ9/01402 and WMU grant to K. eco-friendly approach when compared to methan- Grzelka no. SUBK.D030.24.011 olic extraction of dried material. In our previous study we found that applying PEF to plant roots OR-198 can promote plant growth due to induced abiotic Bioactive Potential of Yeast Proteins Extracted stress and modulate its phytochemical profile. This with HPH and PEF observation paired with low mortality rate of plants Javier Marín-Sanchez, Alejandro Berzosa, Ignacio Álvarez, Ana Cristina C. Sánchez Gimeno, Javier Raso suggested that after providing them with optimal Universidad de Zaragoza, Spain growth conditions it is highly probable to increase the yield of compounds of interest by applying PEF Yeast is a valued source of proteins and pep- several times throughout the plants’ life. tides with bioactive potential, garnering interest in After 2 years of empirically adjusting experimental the food industry and nutraceuticals. Extraction conditions and refining our methodology, we can 210 methods from microorganisms mainly involve cell ABTS method showed the highest antioxidant ca-disruption and cellular permeability modification, pacity in extracts from yeast treated with PEF after yielding products with unique properties. 48 hours of incubation. This phenomenon may be This study aimed to compare protein and pep- attributed to the formation of peptides during incub- tide extraction from yeast using High-Pressure Ho- ation that exerting the antioxidant capacity through mogenization (HPH) and Pulsed Electric Fields a different mechanism than those involved in the (PEF) and examining their antihypertensive and DPPH method. antioxidant capacities. The extraction kinetics and properties of yeast- S. cerevisiae brewing strain underwent HPH derived proteins and peptides varied depending on treatment (2 passes at 100 MPa) disrupting over the treatment applied to improve the extraction. 90% of the population and PEF treatment (15 Proteins and peptides extracted demonstrated sig- kV/cm for 100 µs) electroporating over 90%. After nificant bioactive potential, making them an in- treatment, samples were incubated at 37°C and triguing source of bioactive compounds for the in- filtered using 3kDa and 100kDa Amicon® Ultra dustry. Centrifugal Filters. Amino acids, glutathione, pro- teins, and antihypertensive and antioxidant capa- OR-199 cities were monitored over time. Influence of Pulsed Electric Fields in combin- HPH treatment resulted in the extraction of ation with other processes on the extraction of 610.29 mg/g of proteins after 1 hour, accounting valuable compounds from brewer’s spent yeast for 85% of those proteins over 100 kDa. After the cells same incubation time, the extraction of proteins Sofie Schröder 1, Jan-Michel Schulte2, Corinna Stühmeier-Niehe1, Claudia Siemer1, Stefan Töpfl1 from the electroporated yeast was lower (30.4% 1Elea Technology GmbH, Germany of the total amount of proteins). In this case, the 2Hochschule Osnabrück, Germany extracted proteins corresponded to proteins with a molecular weight lower than 3 kDa. As incub- In this study, the influence of pulsed electric ation time increased, proteolytic phenomena led fields (PEF) on the extraction of valuable compon- to decreased large protein concentrations in HPH- ents, such as proteins and minerals from brewer’s treated samples, resulting in peptide and amino spent yeast cells was investigated. In addition, the acid formation. Conversely, proteolysis in electro- influence of PEF in combination with high pressure porated cells facilitated protein extraction over time. homogenization (HPH) and an incubation time after The highest anti-hypertensive capacity in yeast treatment was also studied as a next step. For extracts obtained after HPH or PEF was observed this purpose, the yeast cells were treated with PEF, in the unfiltered fractions after 1 hour of incubation HPH, an incubation time or a combination of these (IC50 of 0.55 and 0.89 mg/mL for HPH and PEF, methods to increase the permeability of the cell respectively). Extending the incubation time led to membrane and extract valuable ingredients. Dif- a decline in this bioactive property, suggesting de- ferent electric field strengths (5 to 18 kV/cm), spe- gradation of bioactive compounds over time. Anti- cific energies (80 and 90 kJ/kg), pressures (50 to oxidant capacity varied significantly depending on 800 bar) and incubation times (one or two hours, the method of analysis. The DPPH method showed each at 30 °C and 60 °C) were set in order to in- also maximum efficacy in both extracts within the vestigate the effects on serum released, degree of first hour, decreasing by approximately half after 48 disintegration, soluble extract content and protein hours. No differences between fractions of different content. The comparison of PEF treated and un- molecular sizes were detected concerning the anti- treated yeast cells showed an increased serum re- oxidant activity determined by the DPPH method leased percentage from approx. 42 % to up to 65 and antioxidant capacity correlated with concen- %, higher degree of disintegration from 55 % to up tration of glutathione, a widely studied tripeptide to 100 %, and an increased soluble extract content, known for its antioxidant capacity. Conversely, the 211 which rose from 17 % to 30 %, after treatment with tions achieving cell dry weight (CDW) concentra-electric pulses. In addition, analysis of the extract tions of 20 to 35 g/L and lipid concentrations of after PEF treatment revealed higher protein con- 30 to 50 % of their CDW. The biomass harvested tent, compared to the untreated one. The study was pretreated by PEF (40 kV/cm, 150 kJ/kg) be- also shows a further increase in the yield of ex- fore undergoing solvent extraction (20 h of solvent- tracted ingredients with a combination of PEF and mixing). Various solvents or solvent systems HPH or an incubation time. HPH and incubation including ethanol-hexane, methyl terbutyl ether at 30 °C showed a synergistic effect in combina- (MTBE), ethyl-acetate or 2-methyltetrahydrofuran tion with PEF treatment. While incubation at 60 °C (MeTHF) were tested. Lipid yields were calculated alone also resulted in an increase and thus more gravimetrically after solvent evaporation. Whether of an additive effect was seen in the increase in an incubation time after PEF (24 h) and medium os- protein yield. The cell disintegration itself seems to molarity during this incubation could contribute to be mainly attributable to PEF, while 60 °C retention increasing lipids yield was also studied. Addition- time also tends to show an effect on permeabiliz- ally, the integrity of plasma membrane was evalu- ation. The results show that PEF in combination ated by staining with the fluorescent dye YoPro and with other procedures has great potential with re- flow cytometry techniques. gard to the extraction of valuable components from Results showed that great extraction yields of brewer’s spent yeast cells. 80-99 % of the total lipids were reached in all the strains evaluated, although by different strategies. OR-200 The successful lipid extraction on S. podzolica Solvent Lipid Extraction from Oleaginous Yeast and A. porosum required PEF plus an incubation assisted by Pulsed Electric Fields (PEF) period, while no pre-treatment was necessary for Carlota Delso, Nataljia Nazarova, Wolfgang W. Frey C. oleaginosum. This observation pointed out that Karlsruhe Institute of Technology, Germany solvent lipid extraction did not require plasma mem- Oleaginous yeasts are considered a promising brane permeabilization for C. oleaginosum- what renewable source of lipids that can be transformed was confirmed by neglectable Yopro uptake (< 10 into various oleochemicals, such as biodiesel or % of permeable cells). Incubation after PEF pro- biolubricants. Yeasts’ ability to use low-cost carbon cessing of S. podzolica in the same treatment me-sources and produce lipids at high rates, combined dia increased the percentage of permeable cells with their short cultivation times and independence (from 10 to 99 %) and the lipid yields (from 23 to from arable lands and climate conditions, make 62.8 % of total lipids). However, the lipid yields im-them strong competitors to vegetable oils. How- proved even further (up to 99 %) when the incub- ever, the downstream processing of oleaginous ation after PEF was performed in a low osmolar- yeast remains expensive and unfeasible. The high ity medium. In the case of A. porosum, only PEF energy demand of the pretreatments (for cell dis- plus incubation in a low osmolarity medium was ef- ruption) and the recycling costs and sustainability ficient for lipid extraction (83 % of total lipids). The concerns of the organic solvents represent the ma-successful of MeTHF, considered a green solvent jor drawbacks. The present study aimed to address and easy to recycle, in extracting lipids from C. these challenges by exploring the use of PEF as a oleaginosum and S. podzolica opens a promising low-energy pretreatment and identifying more sus- approach for further optimization and study for in- tainable solvents that are effective in lipid extraction dustrial application. from yeasts. OR-201 Three strains of oleaginous yeasts (Cutaneo- PEF treatment for the enhancement of microal- trichosporon oleaginosum, Saitozyma podzolica gae cultivation and Apiotrichum porosum) were grown in a biore- Iris Haberkorn 1, Byron Perez1, Alexander Mathys2 actor under nitrogen limitation and aerobic condi- 1ETH Singapore Centre, Singapore 212 2ETH Zurich, Switzerland kJ kgsus-1) enhanced mild biocompound extrac- tion from microalgae while preserving cell morpho- Today’s society is urged to find solutions that en- logy. Under the condition applied, PEF was 1.67x sure global food security while maintaining planet- more energy-effective than traditional unit opera- ary health. Technological advancement, coupled tions such as high pressure homogenization for the with the exploration of innovative raw materials, same extracted biocompound. holds promise for establishing more resilient food PEF has the potential to emerge as a high- systems. Value chains based on single-cell organ- impact technology that addresses challenges im- isms are experiencing increased traction. How- pairing the economic viability of single-cell-based ever, the economic viability and sustainability of value chains by tackling primary bottlenecks re- these value chains encounter obstacles owing in- lated to enhancing upstream efficiency and recov- efficiencies routed in the up- and downstream pro- ering downstream biocompounds. It underpins cessing. This talk highlights the potential of emer- the development of future food systems rooted in ging pulsed electric fields (PEF) for establishing more sustainable paradigms capable of supporting more sustainable multi-product biorefineries aiming a growing population. to foster continuous and circular bioprocessing with microalgae as a case study. Upstream, nsPEF processing is a technology- S07 - Potential applications of driven and resource efficient approach for single- PEFs technology in vegetable and cell bioprocess optimization. Applying treatments fruit processing during the early exponential growth phase signific- antly (p < 0.05) increased the biomass yields of in-Thursday morning Track C dustrially relevant Chlorella vulgaris (360 ± 114 J Sep 19, 8:30 - 10:00 kgsus−1). The biomass yield was increased in ax- enic (100 ns, 5 Hz, 10 kV cm-1) and non-axenic OR-202 (100 ns, 7 Hz, 10 kV cm-1) cultures by up to 17.5 Understanding of the applicability and the ± 10.5% and 50.1 ± 12.2%, respectively. In non- mechanism behind pulsed electric fields (PEF) axenic cultures, nsPEF-treatments posed a power- as an alternative peeling method ful tool for triggering cell-type specific differentiation Marianna Giancaterino, Henry Jäger and selective inactivation. A treatment window of University of Natural Resources and Life Sciences Vi- 100 ns, 7 Hz, 10 kV cm-1 decreased microalgae enna, Austria counts (-17.1 ± 13.8%) but had a stronger effect on Over the last decades, numerous strategies prokaryotes (-82.7 ± 14.6%). Quantitative assess- have been implemented in the food industry to pre- ment of manually obtained data from FCM-based vent soil and water contamination and limit the ex- analyses showed that nsPEF treatments enhanced ploitation of water and energy resources. One ap- cell proliferation of C. vulgaris. Growth stimulation proach was using electrotechnologies to improve was also obtained across other organism domains or replace existing industrial processes. However, repeatedly treated with 100 ns pulses at 10 kV several applications rely on empirical process con- cm−1: Cyanobacteria (Arthrospira platensis SAG ditions, and systematic approaches are still re- 21.99, 256 ± 67 J kgsus−1), heterotrophic C. vul- stricted due to the unclear mechanisms generated garis CCALA 256 (227 ± 60 J kgsus−1), and yeast by PEF at the intracellular level. Although PEF- (Saccharomyces cerevisiae DSM 70449, 173 ± 55 assisted peeling is of great interest for industrial J kgsus−1). Evidence suggests that this enhance- applications, the mechanism that drives the peel- ment is based on the induction of intracellular abi- ing process is still unclear. A study on the impact otic, sub-lethal stress related to transient, cytosolic of PEF treatment on the peeling ability of toma-surges in Ca2+, and effects on plasma membrane toes and kiwi fruits has been performed to invest- level. Downstream, microsecond PEF (80.72±1.90 igate the underlying mechanisms. This study used 213 monopolar exponential decay pulses with an elec-of vegetables in particular, thermal processing can tric field intensity of 1.0 kV/cm, resulting in total en-lead to loss of nutrients and product quality. Ohmic ergy inputs ranging from 0.6 to 5.0 kJ/kg for toma- heating (OH) provides the potential to optimize con- toes and 1.2 to 12.6 kJ/kg for kiwi fruits. Two pro- ventional thermal processes in terms of resource cedures were utilized for comparing the efficacy of efficiency, process efficacy and product quality re- PEF treatments to typical peeling methods: hot- tention. However, inhomogeneities in the heating water blanching (98 °C for 60 s) and lye peeling behavior of vegetables, caused by different tissue (98 °C for 45 s in 2% NaOH solution). The peeling structures, can lead to overprocessing of product efficiency was assessed using manual and mech- parts. Therefore, this study investigated the use anical methods. Texture, colour (L*, a*, b* scale), of pulsed electric fields (PEF) as pre-treatment to ascorbic acid concentration, chlorophyll a and b, disintegrate the plant cells and to minimize non- carotenoids, total polyphenol content, and antiox- uniformities in the electrical conductivity of the tis-idant activity were used to evaluate the quality of sue that affect the heating homogeneity. PEF treat- the final peeled product. Compared to traditional ment at a pulse repetition frequency of 2 Hz was techniques, the PEF treatment revealed similar or performed on peeled and unpeeled whole beet root superior peeling capabilities while dramatically re- and turnip cabbage with an electric field strength ducing product loss. Additionally, to investigate the of 0.5 – 4.0 kV/cm and a number of 25 – 500 mechanisms of PEF-induced peeling ability, the mi- pulses, resulting in a specific energy input of 0.5 croscopic structure of tomato pectin has been fur- – 150 kJ/kg. The OH was then carried out with ther studied. Atomic force microscopy (AFM) and 3 kW/kg at two different pulse repetition frequen- high-performance liquid chromatography (HPLC) cies (12 kHz and 300 kHz) until a temperature of analysis demonstrated that the ripening stage and 90 °C was reached with a subsequent holding time the intensity of PEF treatments affect the phys- of 5 min. Temperature kinetics and thermal ima- ical properties (fiber length and aggregate area) of ging were used to characterize the heating rate water-soluble pectin, chelator-soluble pectin, and and uniformity. The product quality and nutritional alkali-soluble pectin. The study has demonstrated value were evaluated by analyzing weight loss, tex- that electroporation-induced changes in plant tis- ture, color and vitamin C content. The results of sue may be used to improve or replace traditional the thermal analyses showed a significant reduc- food processes. Despite the promising findings, tion (up to 46 %) of the required heating time to this study provides awareness of how the advant- reach 90 °C for both unpeeled and peeled samples ages can only be converted to effective industrial pre-treated by PEF. In addition, thermal imaging re- strategy when the specifics of raw material struc- vealed an improved heating homogeneity within the ture are considered. different tissue regions due to prior cell disintegration of up to 85 % of the vegetable tissue. The faster OR-203 and more uniform cooking performance resulted in Effects of pulsed electric field pre-treatment on enhancement of the final product quality in terms the heating uniformity and final product quality of improved texture uniformity and better retention of ohmic cooked vegetables of vitamin C. This study demonstrated the potential Kate Waldert, Sarah Elisabeth Prenner, Marianna Gi-of using PEF as a pre-treatment to improve thermal ancaterino, Henry Jäger processing of vegetables by OH. The synergistic ef- University of Natural Resources and Life Sciences Vi- fects of combining both electrotechnologies lead to enna, Austria a rapid, homogeneous and energy-efficient cook- Healthy and sustainable food is an important ing performance with improved quality retention of consumer trend these days. In order to provide the final product. nutrient-rich and safe food, innovative techniques for gentle processing are required. In the case 214 OR-204 possibility to extract (reducing) sugars as well as Germination and stress tolerance of oats anti-nutritive substances has been observed. Sub- treated with pulsed electric field at different sequent washing has shown to reduce levels of phases of seedling growth precursors for process contaminants such as acryl- Alia Hussain Al-Khafaji amide, the cell opening induced allows lower frying Lund Univesity, Sweden time and temperature and hence reduced forma- tion of thermal reaction products. Dependent on This study explores the impact of pulsed elec- product type and raw material quality a reduction tric field (PEF) application on oat seedling growth potential of up to 50 % has been observed. The and stress tolerance. PEF treatment (99 mono- presentation will discuss mitigation strategies con- polar, rectangular pulses lasting 10 µs each, with sidering raw material composition and PEF impact a frequency of 13 Hz and a nominal electric field on product quality during various processing steps. strength of 2250 V/cm) was applied at two growth stages: (i) when the seedlings had 0.2 cm roots OR-206 emerging from the kernel, and (ii) when they Sustainable extraction of plant-based food col- had a 0.4 cm shoot emerging from the kernel. orants with Pulsed Electric Fields Post-treatment, the seedlings were hydroponically Madita Anna-Maria Kirchner 1, Stephanie Wink2, grown for 8 days. To induce stress, the hydroponic Claudia Siemer1, Stefan Töpfl1 medium was augmented with PEG (15 %) to induce 1Elea Technology GmbH, Germany drought stress and NaCl (150 mM) to induce salin- 2Universidade do Vale do Rio dos Sinos, Brazil ity stress. Results demonstrate that applying PEF improved the growth of the root and shoot of oat Food colorants are widely used for all types of seedlings. This effect was more pronounced when products in the food industry. Due to an increased applied to more developed seedlings. When PEF consumer demand for natural ingredients, artificial was applied during the later stage of germination, colorants are being replaced more and more with seedlings exposed to salinity stress showed en- extracts from plants like carrots, red beet, grapes, hanced shoot growth compared to the control. Un- or spinach. For the production of natural color- der the studied conditions, the application of PEF ants, rough methods, such as enzyme addition or had no impact on the growth of seedlings under massive disruption of the plant material, are used drought stress. to obtain high yields. The enzymes break down the cell walls und facilitate the extraction of intra-OR-205 cellular components, including pigments, but pro- Pulsed electric field, a possible strategy for mit-longed exposure times at enzyme specific tem- igation of process contaminants in vegetable peratures are needed to reach the desired effect. snacks. The physical process of electroporation by Pulsed Stefan Toepfl Electric Field (PEF) is an alternative to replace Elea Technology GmbH, Germany elaborate methods currently used in the industry. The increased color extraction after the PEF treat- Pulsed Electric Field induced electroporation of ment is caused by pore formation in the cell mem- vegetable tissue results in release of intracellular brane. Optimal PEF parameters, such as electric liquid and a reduced turgor pressure. This af- field strength and energy input, are crucial for max- fects subsequent processing steps such as cutting, imizing dye yield while minimizing resource con- washing, cooking or frying and allows mitigation of sumption. As a non-thermal alternative, the ex- process contaminants. The effect of PEF on fi- traction with PEF is particularly advantageous for nal product quality has been evaluated for sweet heat-sensitive compounds. In this work, colors potato, carrot, red beetroot and cassava. After a from the four main groups (anthocyanins, betalains, PEF treatment with a field strength of 0.5 to 1.5 carotenoids, chlorophylls) were extracted with PEF kV/cm and a specific energy input of 1 to 3 kJ/kg the 215 parameters relevant for industrial applications. The particles within cells, e.g., the movement of organ-color yields, quality and stability are compared to elles, cytoplasmic flow, and biochemical reactions. those of control samples and of enzymatic treated Therefore, this method makes it possible to obtain samples, allowing to focus on the cost and resource information about the life processes occurring in- efficiency of different extraction processes. Com- side a given cell. This research aimed to evalu- parative analyses highlight the advantages of PEF ate the impact of PEF treatment on the biospeckle over enzymatic methods in terms of higher extrac- activity of apple and carrot tissues. tion efficiency, reduced processing time, and lower Both studied materials were treated with PEF resource utilization. Further research is warran- (electric field strength 1 kV/cm, electrode voltage ted to optimize PEF conditions for each color com- 24 kV, pulse duration 7 µs, pulse frequency 20 Hz). pound and further types of raw materials. Har- For each material three different specific energy in- nessing the potential of PEF for extracting dyes puts were analyzed. In addition, untreated apples from food sources could offer sustainable and cost- and carrots were also evaluated. Right after PEF effective solutions for the food industry’s colorant treatment, both plant materials were subjected to needs. biospeckle activity analysis. Measurements were performed with 10-minutes intervals throughout 1 OR-207 hour. Biospeckle activity: New electroporation as- It was observed that treatment of the tested ma- sessment method for treated fruits and veget- terials (i.e., apples and carrots) with a PEF caused ables changes in the metabolism of these tissues. These Aleksandra Matys 2, Piotr Pieczywek1, Artur changes were evaluated on the basis of Tau coef- Zdunek1, Dorota Witrowa-Rajchert2, Artur Wiktor2 ficient - parameter determined from the autocorrel- 1Polish Academy of Sciences, Poland ation graph of frames from a video sequence. The 2Warsaw University of Life Sciences, Poland slower the observed process, the higher value of Each applied process causes stress to the this parameter. For 30 min after PEF treatment, all plants, which as a consequence induces physiolo- samples exhibited similar values of the Tau coeffi- gical and metabolic changes. A full understanding cient, which constitutes a similar rate of processes of the mechanism of stress-induced changes would occurring in the treated tissues. After 40 and 50 enable developing a method that allows to reduce minutes, there was a clear decrease in the Tau its effects. The pulsed electric field (PEF) may act coefficient value for the most processed samples as an abiotic stressor, influencing the metabolism (the highest specific energy input), which indicates of the treated tissue. Considering that the treat- a significant slowdown in their metabolism. ment with PEF, as well as all the methods of as- Measurement of biospeckle activity can be used sessing the effects of electroporation, are based as a new non-invasive method for assessing the on the necessity to interfere with the treated tis- level of electroporation of treated fruits and veget- sue, a method of measuring the obtained effects ables. in a non-invasive manner is essential, so that the This project has received funding from the Na- obtained result actually relates to only one, specific tional Science Centre (Poland) under the PRELU-process. Measurement of biospeckle activity is a DIUM grant agreement No 2022/45/N/NZ9/02859. technique used to analyze the life processes tak- ing place in the cell. Its non-invasiveness results from illuminating the tested sample with coherent light. An interference pattern is created on the de- tector, the dynamics of which depend on the rate of metabolic processes. Apparent activity of bio- speckle is the result of the physical movement of 216 S12 - Numerical modelling as an parameters can simultaneously describe the dis- order distribution of cell collectives, the random- essential tool in electroporation ness of discharge in liquid, and membrane rupture research of a single cell. We uncover the typical features of three-staged inactivation kinetics and study the uni- Thursday morning Track D versal scaling law of PEF settings on the survival Sep 19, 8:30 - 10:00 rate based on extensive simulations. This model helps us to quantitatively predict inactivation kinet-OR-208 ics in complex settings without resorting to empir- Electrodissociation of cytoskeleton proteins by ical data or FEM analysis with huge computational intense electric field: in silico burdens. Jiri Prusa, Saurabh Kumar Pandey, Michal Cifra Institute of Photonics and Electronics of the Czech OR-210 Academy of Sciences, Czech Republic Quantum chemical simulations of the interac- Although the effects of pulsed electric field tion of Fe2+ with glycerophospholipids (PEF) on biological membranes are being widely Teresė Kondrotaitė1, Alytis Gruodis 2, Gintautas explored, the effects of PEF on proteins are still Saulis1 1 understudied. Existing works on PEF effects on Vytautas Magnus University, Lithuania 2 proteins often focus on the effects on protein Vilnius University, Lithuania secondary or tertiary structures. Here we present Glycerophospholipids are the main compon- our molecular dynamics simulation-based per- ent of any cell plasma membrane. Under normal spective on the PEF effects on protein interactions, physiological conditions, most molecules cannot focusing on selected cytoskeletal proteins such directly permeate through the membrane without as microtubule-forming tubulin and kinesin. We physical or chemical perturbation of the bilayer. demonstrate in these examples that intense PEF Pulses of electric field, acidic and/or alkaline media, can lead to protein electrodissociation, i.e., electric and localization of aggressive metal ions cause cell field-catalyzed disconnection of interacting pro- membrane damage through lipid conformational teins. and destructive processes. Since the membrane structure is a layered lipid, it is necessary to evalu-Acknowledgments: The authors thank the Czech ate not only the physical but also the chemical as- Science Foundation project no. 20-06873X for the pects: the redox reactions taking place at the mem- support. brane create many ionized components that essen- tially catalyze the above-mentioned factors. Metal OR-209 ions belong to these factors. A coarse-grained lattice model of PEF Inactiva- To understand the dynamics of the formation of tion kinetics from percolation theory the pore in the lipid bilaer and its subsequent Feiyu Wu, Chenguo Yao closure at the molecular level, modeling of the Chongqing University, China structure of phospholipids and iron ion complexes Inactivation kinetics not only reflects the biolo- by quantum molecular theory methods was per- gical lethality of PEF but guides dosage settings formed. Quantum chemical simulations were run in clinic. Despite our insight in single cell death, using Gaussian16 package. unfortunately, inactivation kinetics of a multicellu- Optimization of associate geometry was performed lar system depends on a myriad of factors which by Gaussian16 program, using the semi-empirical has so far lacked a theoretical framework. Inspired density function method B3LYP by means of Gaus- by percolation theory from statistical physics, we sian basis set 6-31G. Influence of the solvent me- propose a lattice model to reconstruct the inactiv- dia (water) was evaluated by Polarized Continuum ation kinetics of cell suspension. Coarse-grained 217 Model (PCM). pulses are applied to cells in order to increase the It has been found that metal ion fixation to the lipid permeability of cell membrane. In reversible elec-chain is insignificant to cause lipid conformational troporation (RE), the pulse duration is sufficiently movement. Similarly, metal ion fixation in the case short to ensure that the cell membrane reseals of the –N–3(CH3)3 head in the lipid head group within several minutes. In irreversible electropora- was not observed. The iron ion binds two lipid tion (IRE), however, the pulses are more numerous molecules in the orthophosphoric region, forming or their amplitude is higher so that the cell mem- an energetically stable bridge between orthophos- brane is irreversibly destroyed, and the cells are phoric fragments. Both saturated and unsaturated killed. While the cells are destroyed, the integ- phosphatidylserine (PS) have been reported to be rity of adjacent tissue remains preserved, making similarly resistant to iron-dependent lipid peroxida- IRE very appealing for ablation of tumours. Recent tion. It is believed that the PS head group is re- studies have shown that IRE used for cancer treat- sponsible for this effect, as it binds the iron ion, ment also induces immunogenic cell death (ICD), a reducing the concentration of free iron ions. As form of cell death resulting in a regulated activation a result of this process, the lipid aliphatic chains of the immune response. In particular, damaged change their conformation - a curved chain around or dying tumour cells release damage-associated the metal ion centre is formed from a straight struc- molecular pattern molecules (DAMPs) which may ture. ultimately trigger an immunological response. A typical molecular charge redistribution during ex- In this talk, we present a hybrid model to investigate citation was determined and described. It is stated the ICD and regrowth of tumour spheroids exposed that due to the energetically favorable Fe2+ ion to IRE. In this model, a stochastic individual-based position, one lipid becomes a charge donor and model tracking the dynamics of single tumour cells the other – a charge acceptor. A typical associ- is coupled with a partial differential equation de- ate contains two phospholipids bridged by Fe2+ scribing IRE dynamics. Here, the death of tumour ion in the orthophosphoric region. Molecular orbit- cells and the release of DAMPs correlates with the als correspond to a forbidden electronic transition intensity of the IRE electric pulses. The model is of 0.50 eV from the ground state (MO 210,211) to confronted to biological measures of DAMPs re- the first excited state (MO 226). The iron ion binds lease and volume evolution of tumour spheroids two lipid molecules in the orthophosphoric region, submitted to electric pulses with different intensit- forming an energetically stable bridge between the ies. The results of computational simulations ob- orthophosphoric fragments. As a result, the lipid tained from the proposed model shed light on the aliphatic chains change the conformation - a curved way in which the intensity of the IRE electric pulses chain about the centre of the metal is formed from may affect the regrowth of tumour spheroids, as the straight structure. well as their release of DAMPs. OR-211 OR-212 Modelling the impact of electroporation on Skeletal muscle anisotropy from the perspect- spheroid growth and the release of damage- ive of experimental and model-based electrical associated molecular pattern molecules impedance spectroscopy Emma Leschiera 1, Nicolas Mattei3, Muriel Golzio3, Rok Šmerc, Damijan Miklavčič, Samo Mahnič- Jelena Kolosnjaj-Tabi3, Clair Poignard2, Marie-Pierre Kalamiza Rols3 University of Ljubljana, Slovenia 1Léonard de Vinci Pôle Universitaire, France Muscles are frequently the target tissue in 2Université de Bordeaux, France electroporation-based therapies. In particular, 3Université de Toulouse, France skeletal muscles are often chosen as the target Electroporation is a technique in which electric tissue for gene electrotransfer therapies, owing to 218 their capability to both express transgenes and re-of the model support and confirm the experimental lease proteins into the bloodstream. In addition, findings and demonstrate the importance of consid- the cardiac muscle is a tissue of interest in a novel ering the anisotropic electrical properties of muscle therapeutic approach for the treatment of atrial fib-tissue in electroporation-based therapies. rillation that utilises the phenomenon of irrevers- ible electroporation, known as pulse field ablation. P6 - Calcium electroporation The anatomy of the skeletal muscle, which com- prises elongated fibres, results in anisotropic elec- Thursday afternoon Track A trical properties of muscle tissue. This means that Sep 19, 14:10 - 15:40 the tissue properties (e.g. electrical, mass trans- fer, etc.) parallel to the fibres differ from those per-OR-213 pendicular to them. The aim of our study was to Calcium Assisted Irreversible Electroporation investigate the frequency-dependent properties of Treats Early-Stage Bladder Cancer by Uni- skeletal muscle tissue using electrical impedance formly Ablating the Urothelial Layer spectroscopy, complemented by numerical model- Mary Chase Sheehan 1, Shengwei Wu1, William Ray-ling to substantiate our findings. Vista2, Kimberly Crowley3, Masashi Fujimori4, Neeraj Raghuraman Rajagopalan1, Brian Simoes1, Yasushi Four electrode electrical impedance spectroscopy Kimura5, Govindarajan Srimathveeravalli1 experiments were conducted ex vivo in porcine bi- 1University of Massachusetts Amherst, United States ceps femoris skeletal muscle tissue. These exper- 2Memorial Sloan Kettering Cancer Center, United States iments were performed between an hour and sev-3Food and Drug Administration, United States eral hours post-mortem, ensuring that tissue prop- 4Mie University, Japan erties were assessed before as well as after signi- 5Osaka University Graduate School of Medicine, Japan ficant changes due to cell death occurred. To in- vestigate the effects of electroporation pulse deliv- Non-muscle invasive bladder cancer (NMIBC) ery on tissue properties, we measured the imped- has a high rate of local recurrence despite treat- ance both before and after pulse delivery. Our ex- ment with standard of care tumor resection and ad- perimental setup involved the delivery of 8 pulses juvant intravesical chemo or immunotherapy. The with a duration of 100 µs and a pulse repetition objective of our work was to develop technology for rate of 1/s. We cut and divided the tissue samples whole bladder urothelial cell ablation using calcium in two orientations, firstly such that the fibres were assisted irreversible electroporation (CAIR) as a oriented in the same direction as the applied elec- novel form of adjuvant therapy for NMIBC. tric field, and secondly such that the fibres were Contrast enhanced computed tomography images perpendicular to the direction of the applied elec- of a rat bladder were used to construct finite ele- tric field. Voltage and current measurements were ment models in Comsol Multiphysics that guided also carried out and the pulse shape was analysed the design of a novel catheter electrode (config- based on the orientation of the fibres. uration and sizing) and electric pulse parameters The numerical model was built using the Electric (intravesical conductivity and voltage) required for Currents interface and a Frequency Domain study whole-bladder CAIR of the urothelial layer. Sim- within the COMSOL Multiphysics software. We ulation results were validated in-vitro by perform- developed a detailed numerical representation to ing IRE with prototype catheters in a 3D rat blad- simulate the muscle tissue, incorporating individual der gel phantom embedded with murine bladder fibres in a three-dimensional geometry to accur- cancer (MB49) cells, using PI staining and micro- ately capture the anatomical complexity of the tis- scopy to map the penetration and distribution of sue. The model was used to determine the elec- ablation. Efficacy and safety of CAIR was tested trical impedance values both parallel and perpen- with healthy Sprague Dawley (n = 20) and can- dicular to the fibres, at the same frequencies used cer (n=22) bearing Wistar rats (by exposure to in our experimental setup. The calculated results N-butyl-N-(2-hydroxylbutyl) nitrosamine in water). 219 The bladder was surgically accessed for placement 2University of Ljubljana, Slovenia of the electrode catheter and was filled with 5mmol Disruption of Ca2+ homeostasis after calcium CaCl2 + 0.9% saline solution prior to treatment electroporation (CaEP) in tumors has been shown (1000 V/cm, 100 µ s pulse length, 100 pulses, 1 to elicit an enhanced antitumor effect with vary- Hz, 2x pulsing with repositioning of the electrodes). ing impacts on healthy tissue, such as endothe- Rats were clinically monitored prior to sacrifice at 1, lium. Therefore, our study aimed to determine 7, and 10 days post-treatment. Bladders were pro- differences in Ca2+ kinetics and gene expression cessed for histology with Hematoxylin and Eosin, involved in the regulation of Ca2+ signaling and Masson’s Trichrome, and TUNEL staining to as- homeostasis, as well as effects of CaEP on cyto- sess morphology, extracellular matrix status, and skeleton and adherens junctions of the established ablation status respectively. endothelial cell lines EA.hy926 and HMEC-1. Simulations showed that intravesical conductiv- CaEP was performed on EA.hy926 and HMEC- ity (.1 – 1.75 S/m) was inversely proportional to 1 cells with increasing Ca2+ concentrations. Viab- penetration and intensity of electric field strength ility after CaEP was assessed using Presto Blue, (EFS) in the urothelial layer and the EFS gradi- while the effect on cytoskeleton and adherens junc- ent between electrodes (17% and 100% reduc- tions was evaluated via immunofluorescence stain- tion, respectively), and directly proportional to curing (F-actin, α-tubulin, VE-cadherin). Differences rent drawn (35%). In-vitro results demonstrated in intracellular Ca2+ regulation ([Ca2+]i) were de- that depth and penetration of ablation increased termined with spectrofluorometric measurements with PEF dose and intravesical conductivity (2.4:1) using Fura-2-AM, exposing cells to DPBS, ionomy- was linked to heterogeneity in the depth of pen- cin, thapsigargin, ATP, bradykinin, angiotensin II, etration. Ultrasound measurements demonstrated acetylcholine, LaCl3, and GdCl3. Molecular dis- CAIR treatment did not impact bladder volume or tinctions were identified by analyzing differentially voiding in healthy or cancer bearing rats. In healthy expressed genes and pathways related to the cyto-rats, acute samples showed complete ablation of skeleton and Ca2+ signaling through RNA sequen- the urothelial layer on Day 1 followed by recovery cing. to baseline status by Day 10. In rats with bladder EA.hy926 cells, at increasing Ca2+ concen- cancer, the use of CAIR resulted in rapid, predict- trations, displayed higher CaEP susceptibility and able debulking of the urothelial layer within the en- lower survival than HMEC-1. Immunofluores- tire bladder without injury to the underlying muscu- cence confirmed CaEP-induced, time- and Ca2+- laris and submucosa. CAIR treatment of rats with dependent morphological changes in EA.hy926’s bladder cancer elicited regeneration of a healthy ur- actin filaments, microtubules, and cell‒cell junc- othelial layer by Day 10 post-treatment. tions. Spectrofluorometric Ca2+ kinetics showed Rapid, whole-bladder focal debulking of urothelial higher amplitudes in Ca2+ responses in EA.hy926 cancer with CAIR is feasible and safe. This may exposed to buffer, G protein coupled receptor ag- create a new adjuvant therapy for the treatment of onists, bradykinin, and angiotensin II compared to NMIBC in patients. HMEC-1. HMEC-1 exhibited significantly higher OR-214 [Ca2+]i changes after ionomycin exposure, while Characterization of two distinct immortalized responses to thapsigargin, ATP, and acetylcholine endothelial cell lines, EA.hy926 and HMEC-1: were similar in both cell lines. In a Ca2+-free me- Exploring the impact of calcium electropora- dium, ATP induced a significantly higher [Ca2+]i tion, Ca2+ signaling and transcriptomic profiles rise in EA.hy926, suggesting metabotropic P2Y re- Tim Bozic 1, Barbara Lisec1, Iva Santek1, Bošt-ceptor activation and Ca2+release from intracel- jan Markelc1, Milka Vrecl2, Robert Frangez2, Maja lular stores. RNA-sequencing analysis showed Čemažar1 significant differences in cytoskeleton- and Ca2+- 1Institute of Oncology Ljubljana, Slovenia related gene expression, highlighting upregulation 220 of ORAI2, TRPC1, TRPM2, CNGA3, TRPM6, re-fects of ascorbic acid combined with calcium, both sponsible for Ca2+ import in EA.hy926 compared in vitro and in vivo. Methods. MC38 or CT26 colon to HMEC-1. Moreover, KEGG analysis showed up- cancer cell lines were in vitro electroporated with regulated Ca2+ import and downregulated export a solution of either CaCl2 or CaAsc2 5 mM in a pathways in EA.hy926. 0.4 cm cuvette with a BTX square wave techno- Our finding show that significant differences logy. Post-treatment cell viability was assessed us- in CaEP response and [Ca2+]i regulation ex- ing the MTT assay. Apoptosis and cell cycle assays ist between EA.hy926 and HMEC-1, which may were also performed, and results were analyzed by be attributed to distinct transcriptomic profiles. flow cytometry. For in vivo studies, C57Bl/6 and EA.hy926, compared to HMEC-1, displayed higher Balb/c mice were challenged with MC38 or CT26 susceptibility and sensitivity to [Ca2+]i changes, colon cancer cell lines, respectively. A solution of which may be linked to overexpression of Ca2+- either CaCl2 or CaAsc2 168 mM was administered related genes and an inability to mitigate changes in the tumor via electroporation, as tumors reached in [Ca2+]i. The study offers a bioinformatic basis 4-8mm diameter, by means of Cliniporator techno- for selecting EC models based on research object- logy with the following electric conditions: 8 pulses ives. with a duration of 100 µs – 1000 V/cm. Results. Our study showed that both CaCl2 OR-215 and CaAsc2 had comparable effects on cell vi- Calcium Ascorbate delivered by Electropora- ability, inducing early and late apoptosis as well tion as a novel effective strategy for colorectal as necrosis in MC38 cell line. Interestingly, treat- cancer treatment ment with CaAsc2, with or without electroporation Erika Salvatori 1, Luicia Lione1, Mirco Compagnone2, (EP), resulted in a slight increase in the number of Eleonora Pinto1, Mariantonina Greco1, Melanie cells arrested in the G2 phase compared to CaCl2- Paccagnella3, Valentina Frezza1, Giuseppe Roscilli1, treated cells, thus preventing cells from replicat- Luigi Aurisicchio1, Antonella Conforti2 ing. Additionally, both treatments led to a slight 1Takis, Italy increase in HMGB1 levels, a marker of immun- 2Evvivax, Italy ogenic apoptosis. Treatment with either CaCl2 3Vitares, Italy or CaAsc2 significantly delayed tumor growth in Introduction. Over the last two decades, elec- MC38 mouse model and induced partial tumor re- troporation of the tumor has emerged as a valid gression in CT26 mouse model. In MC38 model, approach to deliver chemotherapy or other thera- treatment with CaAsc2 led to increased survival peutic agents in areas where surgical resection is rates compared to CaCl2 by the end of the study. not possible or chemotherapy proves ineffective. Finally, the analysis of cytokines in sera of CT26 Calcium chloride (CaCl2) is a very promising treat- mouse model revealed a slight increase in the ment because, unlike other chemotherapy agents levels of MIG and IL17A, responsible for T cell ac- such as bleomycin, is not genotoxic and is associ- tivation, after treatment with CaAsc2. Conclusions. ated with less severe side effects. The main mech- In summary, our findings demonstrate that both cal- anisms of calcium toxicity include ATP depletion, cium formulations exhibit similar effects on tumor production of ROS, and activation of hydrolytic en- growth both in vitro and in vivo and indicate cal- zymes. Many in vitro studies indicate the poten- cium ascorbate as a promising alternative to cal- tial antitumor properties of ascorbic acid, or vit- cium chloride for cancer electrochemotherapy. amin C, including among others ATP depletion and ROS production. Furthermore, preliminary clinical studies have demonstrated the efficacy of ascorbic acid, when combined with first-line therapies. Here we describe the use of a novel calcium formulation, calcium ascorbate (CaAsc2) and the synergistic ef- 221 OR-216 calcium dynamics exhibits a Hopf bifurcation, Modeling the Calcium Oscillations Response to showing periodic oscillations [4]. Calcium flux due Pulsed Electric Fields for Spinal Cord Regener- to the membrane permeabilization induced by the ation applied µsPEF represents an additive term acting Alessandra Paffi, Laura L. Caramazza, Micol Colella, on the calcium equilibrium. Noemi Dolciotti, Sara S. Fontana, Francesca Apollonio, Preliminary results on a simplified elliptical MSC Micaela Liberti exposed to a classical biphasic pulse of 100+100 Universita Degli Studi Di Roma La Sapienza, Italy µs duration and 30 kV/m intensity showed that, Recently, a growing number of biomedical despite the short duration of the µsPEF with applications focuses the attention on the possib- respect to the typical calcium dynamics (order ility of using pulsed electric (E) fields to obtain of a few minutes), its application can modulate cell regeneration and differentiation [1], [2], on the calcium oscillation period, accordingly with the bases of the well-established effect of cell experimental results [2]. membrane electropermeabilization. In particular, the European RISEUP project [3] explores the References: possibility of achieving neuronal regeneration [1] E.M. Kawamoto, C. Vivar, S. Camandola, after Spinal Cord Injury by delivering microsecond “Physiologyandpathology of calcium signaling in pulsed E fields (µsPEF) to induce and control stem the brain,” Front. Pharmacol., vol. 3, pp. 1–17, cells differentiation. Indeed, the change in ionic 2012. fluxes across the electroporated cell membranes [2] H. Hanna, F. M. Andre, L. M. Mir, “Electrical may alter the intracellular calcium concentration control of calcium oscillations in mesenchymal which plays an important role in proliferation and stem cells using microsecond pulsed electric differentiation of mesenchymal stem cells (MSCs) fields,” Stem Cell Research & Therapy, vol. 8, n. [2]. 91, 2017. In this study, a multiphysic and multiscale compu- [3] https://www.riseup-project.eu/ tational model was developed to understand and [4] J. Sneyd, J.M. Han, et al., “On the dynamical predict changes in calcium oscillations observed in structure of calcium oscillations”, Proc Natl Acad in vitro experiments on MSCs exposed to µsPEF. Sci U S A., vol. 114, n. 7, pp. 1456-1461, 2017. The computational model was developed in Comsol Multiphysics v. 6.1 by coupling four P8 - Electroporation in veterinary different physics: (i) the quasi-static EM problem oncology to calculate the induced transmembrane potential (TMP); (ii) the pore density equation, implemented Thursday afternoon Track B as a PDE applied to the cell membrane; (iii) the Sep 19, 14:10 - 15:40 transport of diluted species module to calculate the calcium leakage flux through the cell membrane as OR-217 a function of the pore density; (iv) the dynamics of Electrochemotherapy (ECT) with intratumoral the cytoplasmic calcium concentration described and intravenous chemotherapy for the treat- as PDEs [4] applied to the cell domain. This ment of equine skin neoplasias latter dynamics depends on the equilibrium of Carolina Duran, Javier Ojeda the incoming calcium flux through the inositol Universidad Austral de Chile, Chile triphosphate (IP3) receptors on the endoplasmic Skin neoplasias are the most common neo- reticulum, the outgoing fluxes through the pumps plasias in horses. Surgical excision is generally on the reticulum and on the plasma membrane successful in treating small and less invasive neo- and the leakage flux from the extracellular medium plasias (e.g. sarcoids). Nevertheless, in advanced [4]. Depending on the concentration of IP3, the stages or with aggressive tumors (e.g. squam- 222 ous cell carcinoma, malignant sarcoids, advanced OR-218 melanomas) ECT has been lately used for int- Safety of concurrent administration of elec- ralesional or margin treatments. Typically, 1-3 trochemotherapy with intravenous bleomycin treatments, 2-4 weeks apart are required. More and intravenous carboplatin or vinblastine in treatments (6-12) are needed with chronic/severe tumour-bearing dogs and cats: a case series presentations [personal-experience]. Thus, the Chiara Penzo, Jinjing He, Sarah Jayne Baker, Daisy use of ECT with intratumoral and total intravenous Trewin, Stephen John Baines chemotherapy was used to increase treatment suc- Willows Referral Service, United Kingdom cess. Introduction: Electrochemotherapy (ECT) with Medical records from horses treated in 2023 with intravenous (IV) bleomycin (BLEO) is an effective ECT using intratumoral and intravenous chemo- treatment (TX) for local control of neoplasia in dogs therapy were collected and analyzed. Cases were and cats with some patients requiring additional admitted and treated at the Veterinary Teaching systemic chemotherapy. We wanted to establish Hospital of Universidad Austral de Chile. Data in- the safety of such a combination. cluded were: age, sex, weight, breed, tumor loc- Methods: Client-owned dogs and cats with ma- ation, tumor size, diagnosis, prior and concurrent lignant tumours that underwent ECT with concur- therapies, number of ECT sessions, clinical signs, rent IV BLEO and IV carboplatin (Group CARBO) physical exam findings, follow-up, and disease pro- or vinblastine (Group VINBL) at our institution from gression. March 2021 to March 2024 were included. Signal- Four horses and 10 neoplasias were included. ment, tumour type, location, histological grade and Three horses were diagnosed with malignant sarc- margins, clinical stage, presence of microscopic or oids and 1 with equine melanoma. Age ranged gross disease, ECT modality and parameters, ad- from 5 to 15 years, 54% were males and 46?males, junctive medications, clinical history and results of all mix breeds. haematology, biochemistry and urinalysis were ret- In each patient chemotherapy with 90UI of bleomy- rospectively reviewed. Reversible electroporation cin was administered IV, 8-10min later, intratumor- (EP) was performed with one train of 8 biphasic ally cisplatin (0.5mg/cm2) and ECT with biphasic- electric pulses per site at 800-1150 V/cm lasting electrical-pulses was applied (700volts, 200ms, 50+50 µs each (interpulse 1ms) delivered with a 5KHz; VETCP 125-electroporator). veterinary electroporator (Onkodisruptor EXP-Vet; Surgical resection of ulcerated-bulging-masses Biopulse) 5 minutes after IV BLEO using double- was performed before ECT. plate (60mm length) and double-needle (45mm All cases remained clinically healthy, after each length) stainless-steel electrodes. Irreversible EP ECT session treated areas showed local inflamma- was performed with 5 trains of pulses per site at tion which resolved within 10-14d. 1000 V/cm. Pets were treated under general an- CBCs performed for 7d after treatment showed mild aesthesia or deep sedation. Blood/bone marrow, leucopenia and neutropenia 3-4d after treatment. constitutional and gastrointestinal Adverse Events Number of treatments ranged among 2 to 10 ses- (AEs) were graded according to the Veterinary Co- sions, depending on case severity. All horses operative Oncology Group’s common terminology were discharged, 100% of the sarcoids and 70% criteria for AEs (VCOG-CTCAE). Local toxicity was of melanomas showed complete remission. assessed by a published 5-point scale grading Intravenous-chemotherapy in conjunction with in- score for tissue necrosis. tratumoral ECT was well tolerated and successful Results: Five dogs and 6 cats met the inclusion in treating aggressive-chronic-sarcoids and melan- criteria (total 21 TXs). Group CARBO (1 dog, 6 oma in horses. This is the first report of total intra-cats, all carcinomas) received IV carboplatin at 200 venous chemotherapy with ECT in horses, improv- mg/m2 (total 15 TXs). Group VINBL (5 dogs, all ing treatment-efficacy when compared to the use mast cell tumours) received IV vinblastine at 1.8- intratumoral ECT alone. 223 2 mg/m2 (total 6 TXs). Intralesional (IT) medica-a squamous cell carcinoma and a hemangioma tions included cisplatin (1 cat, 1 TX), BLEO (1 cat, in a leopard gecko and a chomatophoroma in a 1 dog, 3 TXs) and calcium chloride (7 pets, 12 bearded dragon. A complete response of the tu- TXs) in the Group CARBO and IT calcium chlor- mors was achieved in almost all patients, with the ide (2 dogs, 3 TXs) in the Group VINBL. Adjunctive exception of a partial response of the chromato- medications for all pets included meloxicam (Group phoroma in a bearded dragon. No clinically vis- CARBO), prednisolone and paracetamol (Group ible side effects were observed in any of the treated VINBL) and maropitant and metoclopramide (both patients. In conclusion, ECT with cisplatin or bleo- Groups). Some pets received additional analgesia mycin has been shown in our cases to be a safe, or antibiotic prophylaxis (both Groups). Irrevers- effective and appropriate treatment option for the ible EP was performed in 5 patients in the Group treatment of the most common skin tumors in se- CARBO (10 TXs). AEs included Grade 1 neut- lected species of exotic pets. However, our study ropenia (2 cats, 2 TXs, Group CARBO), Grade 1-2 was conducted on only a few cases. Therefore, fur- gastrointestinal signs (9 pets, 17 TX, both Groups) ther studies with more patients and a larger number and Grade 2 skin toxicity (pyoderma)(1 dog, Group of different tumors are needed for a wider accept- VINBL). Local toxicity score was 1-2 (5 pets, 10 ance of ECT in the treatment of skin tumors in this TXs), 3 (2 pets, 2 TXs) and 4 (1 TX) for the Group animal species. CARBO and 0 in Group VINBL. Conclusions: Combining ECT with IV BLEO with S15 - Advanced imaging IV carboplatin or IV vinblastine was well tolerated. techniques for visualizing the Larger prospective studies are needed to confirm the safety and efficacy of these combinations. mechanisms of pulsed electric field interactions OR-219 Electrochemotherapy of Cutaneous Tumors in Thursday afternoon Track C Exotic Pets Sep 19, 14:10 - 15:40 Joško Račnik 1, Maja Čemažar2, Gregor Serša2, Urša Lampreht Tratar2, Tanja Švara1, Nina Kočar1, OR-220 Maruša Škrbec1, Nataša Tozon1 Optical streaking microscopy enables visualiz- 1University of Ljubljana, Slovenia ation of ultra-fast response to charge accumu- 2Institute of Oncology Ljubljana, Slovenia lation from MHz bursts of nanosecond pulsed Electrochemotherapy (ECT), a new antineo- electric fields Mark Keppler 1, Sean O’Connor1, Gleb Tolstykh2, plastic treatment for locally accessible tumors, has Benjamin Kasukonis3, Vladislav V. Yakovlev4, Joel N. been introduced with promising results in veterinary Bixler3 medicine for exotic pets. The aim of this study was 1Science Applications International Corporation, United firstly to clinically observe the safety aspect of us-States ing ECT in exotic animals and secondly to evaluate 2General Dynamics Information Technology, United the efficacy of ECT treatment based on histological States tumor type. In our study, ECT with bleomycin was 3Air Force Research Laboratory, United States used in eight patients: seven ferrets and one rabbit. 4Texas A&M University, United States Four cutaneous mast cell tumors, one squamous papilloma, one sebaceous gland adenoma, two se- Nanosecond electric pulses (nsEP) have been baceus epitheliomas and one basal cell tumor were recognized as a versatile tool due to their ability to treated in ferrets and papilloma in one rabbit. On elicit subtle changes in cellular permeabilization1. the other hand, ECT with cisplatin was used in It has been suggested that MHz nsEP trains could four patients: a cutaneous fibroma in a cockatiel, offer an efficient way to regulate the delivery of energy such that control can be established between 224 excitation and electroporation2. Testing pulse pro-threshold. On the contrary, nanosecond electric tocols at the MHz burst frequencies requires meas- pulses (NSEPs) are too brief to charge the PM urement equipment that can validate membrane re- of cells to initiate depolarization. It is believed sponses at time scales ranging from microseconds that a single NSEP depolarizes the cell via PM to milliseconds depending on the length of the electroporation after the application of high-power burst. Previously, our team used streak camera mi- EPs. Pakhomov et al. proposed using low-energy croscopy to capture sub-microsecond changes in (0.01–0.2 kV/cm) NSEPs combined with a very the membrane potential. While streak cameras can high pulse repetition rate (PRR) to prevent elec- capture sub-microsecond biophysics3, they are ex- troporation. This approach allows temporal sum- ceedingly expensive and cannot capture slow bio- mation of NSEPs and consequent PM charging physical responses beyond a few milliseconds. without damage. However, the exact mechanisms We developed an optical streaking microscope4 of this stimulation remain to be elucidated. It is that can acquire single-shot membrane voltage re- challenging to assess PM charging kinetics directly sponse down to microsecond temporal resolution, due to the rapid time scale of microsecond and with an unrestricted sequence depth. We used nanosecond EPs. Recently, we conducted a series this system to capture the membrane potential from of electrophysiological patch-clamp experiments to CHO-K1 cells labeled with FluoVolt voltage sensit- measure the degree of cellular PM depolarization ive dye exposed to MHz nsEP trains using bipolar directly after exposure to 5 MHz trains of NSEPs of electrodes. Optical streaking microscopy was val- 100 ns duration compared to single microsecond idated by visualizing the membrane charge accu- and ms pulses. However, the electrical stimulation mulation as demonstrated in Figure 1, which shows artifact prevented direct measurements of PM de- that the membrane response can be affected by polarization during exposures. To overcome these the repetition rate of the 100-pulse train (500 ns limitations, we employed optical measurements of in duration and 1.2 kV/cm in electric field amp- PM depolarization using an organic fluorescent re- litude). Optical streaking microscopy’s unrestric- porter of membrane potential (MP) (Fluovolt) and ted sequence depth enables analysis with micro- an in-house custom-made streak imaging system. second to millisecond temporal resolution, thereby Ultra-fast (2 ms long) streak kymographs of the offering a method for studying both microsecond MP changes were obtained after exposure to a biophysical and millisecond physiological effects. single 200 microsecond EP and 5 MHz trains of 1000 and 2000 NSEPs of 100 ns duration. The OR-221 electric field was constant between all exposures Visualization of Sub-microsecond Changes in (˜0.01 kV/cm). Immediately after exposures, a Plasma Membrane Potential After Exposure Fluovolt response (% fluorescence change) was to a Single Microsecond Electric Pulse, or 5 observed in the PM areas facing electrodes. The MHz Burst of Low Energy Nanosecond Electric duration of the response is directly correlated with Pulses the pulse width (PW) or duration of the NSEPs Gleb Tolstykh 1, Mark Keppler2, Roberto Rodriguez1, burst. During exposure, the PM near the anode Sean O’Connor2, Joel N. Bixler3, Benjamin Kasukonis3 electrode experienced hyperpolarization, while the 1General Dynamics Information Technology, United PM near the cathode electrode became depolar- States ized. The MP changes were as follows: 6.7±0.04% 2Science Applications International Corporation, United and -3.4±0.03% (n=8) after 200 microsecond EP; States 3.7±0.04% and -1.4±0.04% (n=9) after 5 MHz train 3Air Force Research Laboratory, United States of 1000 NSEPs of 100 ns duration; 4.1±0.05% and Low energy micro- and millisecond electric -1.6±0.05% (n=9) after 5 MHz train of 2000 NSEPs pulses (EPs) charge and depolarize the cellular of 100 ns duration. These results demonstrate that plasma membrane (PM) below the electroporation a single 200 microsecond EP at ˜0.01 kV/cm is more effective in charging PM than an equivalent 225 energy 5 MHz burst of 2000 NSEPs of 100 ns dur-of µm for HuMSC to a few µm for the erythrocytes. ation. Additionally, close amplitudes of PM fluor- Electro-CARS micro-spectroscopy enables the escence changes between ˜0.01 kV/cm bursts of recording of precise spatial and temporal signals 1000 and 2000 NSEPs suggest that an increase in originating from chemical vibrational modes of the either PW or applied voltage is required to increase molecules constituting real cell membranes which the extent of PM depolarization. we expected they would change in response to electropulsation. We paid attention to the phos- OR-222 pholipids C-H and water O-H vibrational modes Changes in hydration of cell membranes ex- located in the 2900-3480 cm-1 range. Thanks posed to pulsed electric fields detected by to the special geometrical arrangement of the wide-field Coherent anti-Stokes Raman micro- illuminating laser beams in our set-up, the probed spectroscopy water vibrations corresponded to the molecules of Caterina Merla 2, Francesca Camera2, Michael water located at the cell membrane surface (iden- Scherman1, Brigitte Attal-Tretout1, Luis M. Mir3 tified as interfacial water molecules) or confined 1Université Paris-Saclay, France in proximity of the phospholipid heads (identified 2ENEA, Italy as interstitial water molecules). We thus report 3Centre National De La Recherche Scientifique, France novel observations of spectral features originating The application of short (from hundreds of from cell phospholipids, interfacial and interstitial milliseconds down to few nanoseconds) and water in the CARS spectra of cells after their intense (from few kV/m up to tens of MV/m) electric exposure to µs and ns EP of high amplitude (few pulses (EP), known also as electropulsation [1], MV/m). To link these changes to the membrane facilitates biomolecules, peptides, proteins and/or permeabilization levels, we also measured them nucleic acids to cross the cell membrane. So far using pulses of different durations and fluorescent electropulsation initiation mechanisms have been markers such as YOPRO-1 and Fluo-4. Thanks studied only with molecular dynamics simulations to the capabilities of our ElectroCARS platform, or with indirect experimental approaches in living CARS signatures of single relevant Raman shifts cells or artificial models. In a recent paper [1], we associated to water molecules and lipids were also demonstrated the interest of wide-field Coherent followed in real-time during the exposure to the anti-Stokes Raman Scattering micro-spectroscopy high amplitude EP lasting µs and ns. (CARS) to study directly phospholipid and water This work is thus a valuable contribution to molecular responses after and during electropulsa- decipher the mechanisms of the membranes tion. This work [1] was carried on artificial lipid electroporation/electropermeabilization. vesicles, in the absence of fluorescent or molecular markers. Indeed, CARS is a non-linear optical Reference: technique enabling the label-free and fast time 1. C. Merla et al., Bioelectrochem. 147(2022) monitoring of the vibration modes belonging to 108218. specific chemical bonds in bio-samples. OR-235 In the present communication, we will report the Identifying the Differences of Nanosecond Electro-CARS platform used in [1]. It integrates a Pulsed Electric Field Effects on Intracellular CARS setup with microwave-based engineering Functions among Breast Cancerous and Nor- techniques to deliver microsecond (µs) and nano- mal Cells through Real-time Monitoring second (ns) duration EP. We exposed living cells: Kamlesh Awasthi, Nobuhiro Ohta human mesenchymal stem cells HuMSC, murine National Yang Ming Chiao Tung University, Taiwan lung fibroblasts DC-3F, and murine erythrocytes. These cells present different levels of biological Nanosecond pulsed electric fields (nsPEFs) complexity and very different diameters, from tens have garnered significant interest in biomedical re- 226 search due to their ability to modulate cellular func-S14 - Electromagnetic modelling tion by electrostimulating intracellular organelles, for pulsed electric fields functions, and signaling processes without interact- ing with the plasma membrane. However, nsPEF Thursday afternoon Track D faces significant challenges in clinical application Sep 19, 14:10 - 15:40 because the effects of nsPEFs on living cells de- pend on experimental conditions, such as field OR-224 strength and pulse duration, pulse rise and decay A coplanar waveguide picosecond pulsed elec- time, and cell type. Here, another challenge is to tric fields (psPEF) delivery system for the identify the nsPEF-induced changes in intracellular electro-permeabilization of biological cells space and interpret their relation to cell survivabil-Hafsa Tjiou, Lionel Michard, Philippe Leveque, Claire ity and death. Therefore, in-depth studies are re-Dalmay, Delia Arnaud-Cormos quired for the future development of nsPEF-based University of Limoges, France modalities for biomedical applications. From this Over the past few decades, the bio-electrics point of view, we have developed a real-time mon- field has been deeply engrossed in studying the itoring system by fabricating a 3D microelectrode effects of high-voltage, ultra-short pulsed elec- chip with a 200 µm electrode distance on the micro- tric fields (PEF) on biological cells and their po- scopic cover glass and incorporated it with a fluor- tential applications. When cells are exposed to escence microscope. high-voltage PEF of short duration (ranging from The differences in nsPEF effects, with the same microseconds to milliseconds) within an intens- pulse duration, field strength, and frequency, ity range of kV/cm to MV/m, one main outcome on intracellular functions including cell death, emerges, termed ”electroporation” or “electro- among breast cancerous (MCF7 and MDA-MB- permeabilization”. This phenomenon is defined by 231) and normal (MCF10A) cells have been iden- the formation of nano-pores and the permeability tified through real-time microscopic monitoring of increase in the cell’s membrane. This leads to nsPEF-induced changes in mitochondrial mem- an exchange of molecules between the cytoplasm brane potential, superoxide anion concentration, and the extra-cellular environment, either tempor- intracellular calcium ion concentration and mobility, arily (reversible electroporation) or permanently (ir-autofluorescence lifetime of NADH, caspase-3/7 reversible electroporation) if higher intensity PEF activity, phosphatidylserine externalization and the are applied, which may cause cell death or cell cell viability, without changing the cell culture con-apoptosis. Both reversible and irreversible elec- dition. The results show that the nsPEF-induced troporation have demonstrated utility across vari- changes in intracellular function and dynamics de- ous therapeutic and biological applications, includ- pend on the nature of the cells. The nsPEF applic- ing electrochemotherapy and electrogene therapy. ation to subtypes of breast cancerous and normal As for shorter duration of pulsed electric fields, e.g., cells shows different natures of cell death. The sur-nanosecond (nsPEF) and picosecond (psPEF), vivability of MCF10A cells was much higher than they were proven to electroporate or permeabil- that of MCF7 and MDA-MB-231 cells. We have ize intra-cellular organelles membranes. The ef- also identified that the mode of cell death was dif- ficient delivery of short, picosecond-duration, in- ferent among MCF7, MDA-MB-231, and MCF10A tense PEF requires generators designed to meet cells. The MCF7 cells died in a caspase-dependent specific pulse characteristics like shape, duration, manner, whereas MDA-MB-231 and MCF10A cells and amplitude. Additionally, there is ongoing in- died in a caspase-independent manner under the vestigation into delivery devices with reduced elec- application of nsPEF. trode gap distances, directly in contact with biolo- gical samples, to enhance the generation of high electric field intensity. In this paper, we propose 227 a miniaturized coplanar waveguide (CPW) trans-ator allows precise shaping of the pulses by using mission line designed as a delivery system for optical delay lines allowing to delay PCSSs trig- high intensity picosecond-PEF. The delivery sys- gering. The duration of single pulses can be ar- tem main characteristics are: i) a wide frequency bitrary varied as well as the temporal asymmetry band performance allowing the delivery of pico- of bipolar pulses. The inter-pulse delay of paired second pulses, ii) a miniaturized structure, espe- pulses can also be adjusted by varying the lengths cially the gap distance between electrodes which of transmission lines. Furthermore, the generator is equal to 130 µm insuring high intensity electric can absorb reflected pulses due to nonmatched fields (˜MV/m), and iii) a transparent substrate to load impedances and facilitates the use of any type enable real-time visualization of bio-effects on the of constant-impedance applicator. The entire sys- samples. The numerical assessment of the psPEF tem has been numerically studied by means of delivery system is ongoing, with plans for exper- software simulations with the aim to fit the exper- imental validation aimed at future utilization in in imental measurements. The presented state-of- vitro experiments to explore the impacts of high- the-art performances associated with the high con- intensity psPEF on the intra-cellular organelles. figurability of the pulse shapes enable further ex- ploration of the effects of picosecond pulses on bio- OR-225 logical cells and could lead to the discovery of new A few hundred optoelectronic pulsed electric bioelectrical phenomena. field generator with fully configurable pulse [1] A. G. Pakhomov and O. N. Pakhomova, shape, duration and amplitude “The interplay of excitation and electroporation in Lionel Michard, Hafsa Tjiou, Vincent Couderc, Phil-nanosecond pulse stimulation,” Bioelectrochem- ippe Leveque, Delia Arnaud-Cormos istry, vol. 136, p. 107598, Dec. 2020, doi: University of Limoges, France 10.1016/j.bioelechem.2020.107598. Recent research has shown growing interest in [2] Z. Mao, L. Liu, Y. Zhang, J. Zhang, N. Liu, high voltage, subnanosecond (sub-nsPEF) and pi- and Q. H. Liu, “Selective Electroporation of Organ- cosecond (psPEF) pulsed electric fields. For the in- elles Under an Intense Picosecond Pulsed Electric stance, picosecond pulses might be used to induce Field,” IEEE Journal on Multiscale and Multiphysics cell electroporation without excitation [1], electro- Computational Techniques, vol. 3, pp. 235–245, porate cell organelles [2] or in neurodegeneration 2018, doi: 10.1109/JMMCT.2018.2887000. research [3]. Nevertheless, the effects of subn- [3] M. Zamponi, R. Petrella, and P. A. Mol- anosecond and picosecond pulses on biological lica, “Picosecond Pulsed Electric Fields and Prom- cells, and the underlying interaction mechanisms ise in Neurodegeneration Research,” Bioelectricity, are yet to be explored to their full extent [4]. Gen- vol. 3, no. 3, pp. 176–185, Sep. 2021, doi: erating subnanosecond pulses is technologically 10.1089/bioe.2021.0005. challenging as at such high voltages, the switch- [4] I. Semenov and S. Xiao, “Electropermeab- ing element must be able to sustain high voltages ilization and Electrostimulation by Picosecond while allowing ultrafast switching. The generator Pulses,” in Handbook of Electroporation, D. Mik- we propose is based on photoconductive semicon- lavčič, Ed., Cham: Springer International Publish- ductor switches (PCSS) in conjunction with a pulse- ing, 2017, pp. 171–186. doi: 10.1007/978-3-319- forming network (PFN) constituted by coaxial trans- 32886-7_89. mission lines and optical delay lines (ODL). The PCSS are triggered using an optical pulse delivered by a 35 ps 1064 nm Nd:YAG laser. The proposed architecture can generate single, paired and bi- polar pulses with durations of a few hundred pico- seconds and amplitudes up to 15 kV. The gener- 228 OR-226 Introduction: The application of short, high- Complex Electrical Impedance and β- voltage electrical pulses induce pore formation in dispersion for Electroporation Sensing the cell membrane, ultimately leading to cell death; Guilherme Brasil Pintarelli, Raul Guedert, Jéssica this procedure is called Irreversible Electroporation Rodrigues da Silva, Lucas Bertinetti Lopes, Daniela Ota Hisayasu Suzuki (IRE) [1]. IRE is typically used in the clinic for Federal University of Santa Catarina, Brazil the treatment of unresectable tumors. Most IRE protocols involve the application of a fixed number Electroporation holds applications in various of pulses, disregarding the ablation completeness. fields such as gene transfer, cancer treatment, and This could lead to overtreatment, resulting in a industrial pasteurization. However, understand- higher temperature increase, or to undertreatment, ing the phenomenon is challenging due to the resulting in a higher tumor recurrence [2]. While nanoscale dimensions and dynamics of ‘electro’- the impact of electroporation on tissue bioimped- pores. Impedance spectroscopy, typically between ance is well known [3], a fixed way to monitor 1 kHz and 100 kHz, has been used to detect cell ablation completeness by means of bioimpedance membrane alterations. Yet, traditional impedance monitoring has yet to be implemented clinically. spectroscopy is not fast enough to analyze elec- Methods: To investigate the relationship between troporation dynamics and is prone to errors due increasing pulse number and interpulse bioimped- to electrode polarization (up to 10 kHz). Cur- ance measurements, 500 V/cm IRE pulses (100 rently, PEF equipment lacks feedback in their con- µ s) were delivered to vegetable tissue samples trol systems. This work investigates the altera- and stained with a metabolic dye to reveal ablation tion of complex electrical current during the ap- areas. A 2 ms low voltage pulse (LVP) was used plication of pulsed electric fields (PEF). The tested for the bioimpedance reading and was delivered hypothesis proposes utilizing information from β- 499 ms before each IRE pulse at 1 Hz. The Fast dispersion changes (intrinsic to biological tissue) to Fourier Transform was used to analyze the voltage study electroporation during PEF, considering that and current data obtained from the LVP. The the sample itself collaborates with the transduc- resulting interpulse bioimpedance was used to cal- tion of the effect, i.e., bioelectronic sensing. The culate metrics for shape and mean value, to reflect method uses the same electrodes as the PEF ap- the ablation and the temperature changes caused plication for sensing. Two signals are mixed: main by electroporation. Discussion: Our results show PEF protocol and a small signal centered at β- that the LVP doesn’t induce electroporation effects dispersion. By treating electrical current as a com- and that the LVP method for reading bioimpedance plex number, the analysis introduces an additional shows comparable accuracy to a commercial dimension for studying electroporation and voltage- impedance analyzer. Our results also suggest dependent phenomena. Experimental results us- that the ablation area saturates concurrently with ing vegetal tissue reveal significant changes in im- the attenuation of the beta dispersion, and this pedance and phase during PEF exposure. The attenuation can be measured by means of bioim- phase is reduced to 0º at the direct current PEF. pedance. This finding can be integrated into a The project contributes to the development of fast real-time monitoring system utilizing bioimpedance sensors for feedback and control of PEF. to assess the completeness of ablation. OR-227 [1] Davalos, Mir, and Rubinsky, “Tissue Ablation Interpulse bioimpedance reading during elec- with Irreversible Electroporation.” [2] Kiełbik et al. troporation as a tool for monitoring ablation ”Electroporation-Based Treatments in Urology” [3] completeness Ivorra and Rubinsky. ”In vivo electrical impedance Pedro Paulo Santos, Hee Chang Shin, Edward Jac-measurements during and after electroporation of obs, Rafael Davalos rat liver” Georgia Institute of Technology, United States 229 OR-228 that the configuration employing six needle elec- Optimization of Electrode Arrangement in 96- trodes, each with a radius of 0.2 mm and positioned Well Plates for In Vitro Electroporation Exper- at a distance of 1.25 mm from each other, achieved iments the highest levels of homogeneity in the electric Ondrej Fiser 1, Ivana Fišerová2, Jan Trnka2, Pavel field distribution within the well. This arrangement Osmančík2, Marek Hozman2, Jan Vrba1, David Vrba1, exhibited superior performance compared to other Marek Novak1 configurations tested, suggesting its potential for 1Czech Technical University in Prague, Czech Republic optimizing the electroporation process. 2Charles University, Czech Republic Conclusion: In summary, our study demonstrates Background: Pulsed-field ablation (PFA), a the significance of electrode arrangement in en- technique based on the application of electric hancing the efficiency of in vitro electroporation ex-pulses to cells, has emerged as a powerful tool periments. Through numerical simulations and rig- in molecular biology, biotechnology, and medicine. orous analysis, we identified a configuration with In vitro electroporation experiments, particularly six needle electrodes that achieved superior ho- those conducted in multi-well plates are becom- mogeneity in electric field distribution within the ing essential that lead to a better understanding well. This finding highlights the potential for op- of electroporation effects on cell and tissues. The timizing electroporation processes and improving electrodes are crucial part of the electroporation experimental consistency. Moving forward, these system and are submerged directly into the solu- insights may lead to advancements in molecular tion within the well. The shape and placement of biology, biotechnology, and medical research. Ul- these electrodes significantly impact the distribu- timately, our work contributes to the development tion of the electric field, posing a persistent chal- of more effective electroporation techniques with lenge. broad applications. Purpose: This study aims on the task of optimizing the arrangement of the electrodes within the well. S06 - Electrochemotherapy in Our primary objectives are twofold: firstly, to en- treatment of vascular hance the uniformity of the electric field throughout malformations the solution, and secondly, to maximize the volume exposed to electroporation. By refining the elec- Thursday afternoon Track E trode layout, we aim to achieve a more consistent Sep 19, 14:10 - 15:40 and efficient electroporation process. Methods: The impossibility of measuring the elec- OR-229 tric field distribution of short pulses within the well Principles and mechanisms of bleomycin elec-using conventional methods, make it only feasible trosclerotherapy in treatment of vascular mal- through analytical solutions or numerical simula- formations tions. In our study we used numerical simulator Gregor Serša 1, Maja Čemažar1, Tobian Muir2 COMSOL Multiphysics. We evaluate the field ho- 1Institute of Oncology, Ljubljana, Slovenia mogeneity, employing methods such as spatial av- 2James Cook University Hospital, United Kingdom eraging and statistical analysis of 2D images to assess uniformity, while also quantifying the elec- Vascular malformations are specific vascular troporated volume by analysing the isocontour of anomalies, either of the venous or arterial vascu- the electric field. Our investigation involved test- lar system. They often occur in children and young ing over 50 different arrangements of electrodes to adults. Treatment consists of either surgical inter- comprehensively explore the optimization possibil- vention or sclerotization of the vessels with vari- ities. ous agents, including bleomycin injections. Treat- Results: From our set of models, we discovered ment with bleomycin sclerotherapy is currently the most commonly used treatment approach. The 230 efficacy is usually good, but recurrence of the a common problem among high risk cardiovascular malformations is common. The first report on patients. It can be successfully treated by femoral the use of electrochemotherapy in the treatment angioplasty. However, restenosis is a problem that of capillary malformations dates back to 2019 by has still to be solved. The use of electroporation Sophie E.R. Horbach, who provided the proof of for cardiac ablation has been increasing its applic- concept for electrosclerotherapy. Later, this treat- ations worldwide. We present a novel electrode ment approach was used by several clinical groups for treating an atheromatous plaque in the femoral and was named Bleomycin ElectroScleroTherapy arthery with a combination of reversible and irre- – BEST. The review of clinical results has shown versible electroporation. very good efficacy and no side effects. Several Objective: Develop and validate a new electrode clinical trials are also currently underway. How- to treat atheromatous plaques in patients with peri- ever, standardization of the treatment is required pheral vascular disease by means of reversible and in order to standardize drug dosage and adminis- irreversible electroporation. tration as well as the application of electric pulses. Materials and Methods: The electrode was de- Electrochemotherapy has been used in the treat- signed and modeled in COMSOL Multyphisics soft- ment of tumors for three decades. Its effective- ware (Comsol, INC). It consists of a probe with 4 ness depends on the administration of drug to the rings of conductive material separated 4 mm from tumor cells, the induction of an immune response each other on the tip. Several pulse parameters and also on the vascular disrupting effect of this ap-were tested to achieve an effective electric field that proach. It has been shown that the vascular dis-could reach the vessel wall. The prototype elec- rupting effect is an important component of the an- trode was built in plastic and stainless steel. Dif- titumor effect of electrochemotherapy. Therefore, ferent pulse parameters were tested in a vegetable this mechanism must be involved in the efficacy of model using an EPV-LAB electroporator (BIOTEX BEST in the treatment of venous or arterial vascu- SRL, Buenos Aires, Argentina). lar malformations. The effect is limited to the tu- Results: The in-silico model showed that when mor vessels in the tumors, indicating selectivity of 600V were applied an adequate circumferential treatment to physiologically different tumor vessels electric field was generated. This field theoretically and possibly to altered vessels in the malforma-can reach the vessel wall and treat it completely. tions. Some molecular features of the vessels in The prototype developed showed promising results malformations have already been identified. How- in the in-vitro models when 32 pulses of 600 V were ever, further studies are needed on the character- delivered. The effective electric field reached a dis-istics of malformations and the potential of BEST tance of 2 cm away from each side of the electrode. for their treatment. To this end, we need to develop This spatial distribution is enough for ensuring suf- models and perform preclinical tests with BEST to ficient coverage of the affected arterial segment. optimize this treatment in humans. Conclusions: These results highlight the promise of the designed electrode for interventions targeting OR-230 peripheral artery disease associated with athero- Electrode for the Treatment of Atheromatous matous plaque. The ability to deliver targeted elec- Plaques by means of a combination of revers- trical pulses with sufficient spatial coverage holds ible and irreversible electroporation significant implications for enhancing blood flow, Ximena Manglano 1, Jesica Rodríguez Miranda1, reducing stenosis, and promoting vascular remod-Aldo Perusso1, Sebastian D. Michinski2, Antonella María eling in affected arteries. The developed elec- Cilio1, Ana Laura Campastri1, Felipe Maglietti1 trode could be an effective tool for the treatment 1Fundación H.A. Barceló, Argentina of peripheral atherosclerotic disease, with transla- 2Universidad de Buenos Aires, Argentina tional potential to human medicine. Further experi- Introduction: The peripheral vascular disease is mental validation, including in vivo studies in a suitable translational model is warranted to corroborate 231 these in silico and in vitro findings and assess the vasodilatory effect on the tumor’s blood vessels, safety and efficacy of the electrode. without damaging the normal vasculature around the tumor. Therefore, combining bleomycin sclero- OR-231 therapy and electroporation, i.e. bleomycin elec- Development of in vitro and in vivo models of trosclerotherapy (BEST) for the treatment of VMs, vascular malformations for determining bleo- could potentially improve the efficacy of bleomy- mycin electrosclerotherapy (BEST) efficacy cin sclerotherapy. Bleomycin is already one of the Boštjan Markelc, Barbara Lisec, Tanja Jesenko, Si-most commonly used sclerosing agents in the treat- mona Kranjc Brezar, Maša Omerzel, Maja Čemažar, ment of VMs. Therefore, the combination with elec- Gregor Serša trical pulses could only further enhance the efficacy Institute of Oncology LJubljana, Slovenia of bleomycin, as it would increase the uptake of the Vascular malformations (VM), a rare condition drug into the endothelial cells of the affected blood characterized by abnormally developed blood ves-vessels. In early clinical studies, BEST has already sels, can occur anywhere in the body, present- proven to be an effective and safe method for the ing a spectrum from simple and benign to com- treatment of VM, but its mechanisms of action are plex conditions. VM develop due to inherited or still not yet understood. Therefore, we have de- somatic mutations that disrupt the major intracel- veloped in vitro murine models of VMs based on lular endothelial receptor signaling pathways, i.e. the most common somatic mutations found in VMs the PIK3CA-AKT-mTOR, RAS - MAPK - ERK and and are now using them to determine the optimal SMAD signaling pathways. The resulting vascu- treatment parameters for BEST in vitro and to eval- lature in the VM is abnormal, often leaky and highly uate its therapeutic potential in treatment of VMs proliferative, i.e. very similar to tumor vasculature. in vivo. Our research will fill the gap in knowledge Vascular abnormalities are classified into two main of the mechanisms governing the response of VMs groups: tumors (true proliferative neoplasms) and to BEST treatment and provide standardized treat- malformations (morphological defects). Tumors ment parameters that could help guide the use of are further subdivided into benign, locally aggress- BEST in clinics. ive/marginal or malignant, while malformations are subdivided into simple, combined or associated OR-232 with other abnormalities. The selection of optimal Use of bleomycin electroscleotherapy (best) in treatment strategies for vascular malformations de- hereditary hemorrhagic telangiectasia patient pends on the specific type and anatomical location (HHT): a case report of the anomaly. Several different therapeutic op- Marta Minuti, Giulia Bertino, Fabio Pagella, Rebecca Gelli, Marco Benazzo tions are available, including observation, sclero- University of Pavia, Italy therapy, laser treatment, embolization and surgical interventions. Although current therapies for vas- Introduction: Hereditary Hemorrhagic Tel- cular malformations have shown efficacy in many angiectasia (HHT) is a bleeding disorder occurring cases, challenges and limitations still exist. In the in 1/5000 person. It is an invalidant disease be-case of ethanol, one of the most potent and effect- cause of frequent and recurrent bleeding episodes ive sclerosing agents, a high complication rate oc- of the arterio-venous malformations. Standard curs, with skin complications being the most com- treatments (sclerosant agents, Agon-laser, etc.) mon side effect, occurring in 8% of patients. are burdened by high recurrence rates. Electrochemotherapy is a local treatment that en- Recently, Bleomycin Electrosclerotherapy (BEST) hances the cytotoxicity of a drug exclusively at the has shown promising results in the treatment of site of application of the electrical pulses. In ad- vascular malformations. We present a case of oral dition to its direct cytotoxic effect on tumor cells, telangiectasia successfully treated with BEST. bleomycin electrochemotherapy also has a specific Materials & Methods: A 70 year-old man affected 232 by HHT with multiple telangiectasias of the tongue ily cross into the target cell. The utilisation of the was treated with BEST in August 2023. technique of electroporation can however address The procedure was performed under general an- this issue. In a number of interesting observa- aesthesia; reversible electroporation was applied tions, case reports and case series, the benefit with Cliniporator™ immediately after intralesional of adding electroporation to bleomycin sclerother- administration of 1,5 ml of Bleomycin (at concen- apy, coined as BEST (Bleomycin electrosclerother- tration of 0,25 mg/ml) according to the ESOPE pro- apy) has induced a significant improvement in re- tocol. sponses achieved and a reduction in the number Treatment response was assessed clinically and of repeat treatment sessions needed. Achieving a with Patient and Observer Scar Assessment Score quicker treatment response with less repeat ses- (POSAS) and Global Assessment of Change sions transfers significant benefits to patients, clini- (GAC) questionnaires. cians and institutions. Further exploration has led Results: At the end of BEST, the “vascular to the establishment of the BEST multi centre study lock” of electroporation effect was immediately vis- to assess treatment response and quality of life in ible. In the following days, a mild oedema of a patient and clinician reported outcome study. We the tongue was observed which resolved spon- will present our current understanding, and up-to- taneously. Moderate pain was controlled with date experience of this exciting novel treatment op- paracetamol. At the two-months follow-up, com- tion. plete remission of the lesion was observed. The scores on the evaluation questionnaires improved OR-234 significantly with a relative increase in the patient’s Bleomycin Electrosclerotherapy (BEST): exper-quality of life. At 7 months follow-up patient is still ience with lymphatic malformations NED. Giulia Bertino, Marta Minuti, Rebecca Gelli, Valentina Ravetta, Marco Benazzo Conclusions: BEST is a procedure that significantly IRCCS Policlinico San Matteo Foundation, Italy increases sclerosis of the vascular malformations. This is the first case of application of BEST in a Introduction: Congenital lymphatic malforma- HHT patient. The safety and efficacy of the proced- tions represent 12% of all the vascular anomalies ure could lead to consider BEST a valid alternative and two thirds of all the reported cases are found in the treatment options of oral vascular lesions in in the head and neck, especially in the oral mucosa HHT patients. or the neck. They are usually categorized as mac- rocystic or microcystic, but both can be found in the OR-233 same lesion. Intralesional injection of various scler-Clinical applications of BEST in treatment of osing agents, particularly bleomycin and picibanil, vascular malformations is the main treatment option, but multiple sessions Tobian Muir 1, Alan Batterham2, Shaun Wellburn2, Sri can be necessary to resolve or remarkably reduce Murugan1 the lesions. Recently, the association of reversible 1James Cook University Hospital, United Kingdom electroporation to bleomycin sclerotherapy (BEST) 2Teesside University, United Kingdom has been shown to increase the sclerosing proper- Nonsurgical Bleomycin injection (sclerother- ties of the drug with very high percentages of com- apy) of vascular anomalies is an effective and plete resolution of the lesions and without severe widely used treatment modality. Although this side effects; but up to now no dedicated stand- treatment is safe and efficacious, a response is ard operating procedures (SOP) exist. The Inter- usually only achieved after a series of repeat treat- national Network for Sharing Practices on Electro- ments. (An average of 4 treatment sessions were chemotherapy (InspECT) is the largest group of ex- needed in our series of 890 patients). Bleomycin perts (42 all over Europe) on electroporation-based is a large, charged molecule that does not eas- treatment and it is working on the definition of these 233 SOP. Here we report the experience with BEST of one of these Centers. Materials and Methods: We tested the efficacy of BEST in three patients with recurrent or non- responsive lymphangiomas, 1 child with intraoral lesions, and 2 adults with neck lesions. Results: No major or minor side effects were ob- served. All the patients underwent significant re- duction or resolution of the cysts with only one ses- sion, and the results are stable after more than 1 year of follow-up. Conclusions: The association of reversible elec- troporation to the standard application of intrale- sional bleomycin enhances the sclerosing effects of the drug because it increases the permeability of the cell membranes to the drug, and the result is acquired most of the time in one single session. Moreover, the treatment is safe, with organ and function sparing. It can be applied both in adults and in children and it can be considered a valid alternative in patients resistant to surgical or other sclerosing treatments. 234 P O S T E R P R E S E N T A T I O N S ’ A B S T R A C T S Poster session ans suggested amputation. But given the ampu- tation alternative, tumor resection, electrochemo- Coffee Break and Poster therapy and subsequent reconstruction with axial Session caudal superficial epigastric flap technique are pro- posed. The procedure was accepted by the owner Sep 16, 14:40 - 16:10 and due to the high recurrence rate of these types PO-01 of tumors, the pet was monitored every 15 days for Surgery and electrochemotherapy: an option 4 months and then every 2 months to evaluate the for amputation of the posterior limb of a canine evolution of the surgical suture. So far 24 months with an infiltrating sarcoma have passed since the procedure was performed Oscar Pagoto and there is no evidence of tumor recurrence and Sociedad Argentina de Oncología Veterinaria, Argentina no evidence of metastasis. In conclusion, the procedure performed avoiding Skin and subcutaneous tissue tumors are the amputation was satisfactory, without recurrences most common type of tumor in dogs (approximately or metastasis. 30%). Soft tissue sarcomas are 15%. All Sar- comas have a common mesenchymal origin and PO-02 similar behavior, but their differentiation is difficult Outcome of one session of electrochemother-diagnostic. They can present a pseudo capsule apy with bleomycin as single or adjuvant treat- or be very infiltrating and manifest a very aggress- ment in equine cutaneous sarcoids and melan- ive local growth, although those of high grade can oma also present metastasis rates of between 8-20% in Majbritt M. E. Larsen canines. Surgery is the main therapeutic tool. But Evidensia Specialist Animal Hospital in Helsingborg, if the surgery is performed with a narrow margin, Sweden the chances of recurrence increase significantly, The aim of this retrospective study was to eval- so other alternatives must be implemented: new uate the safety and efficacy of electrochemother- surgery, chemotherapy, radiotherapy, cryosurgery apy (ECT) with bleomycin in cutaneous tumours and electrochemotherapy. of the horse. All horses underwent one session In the Electrochemotherapy an electric field is gen- of treatment only. Fifteen horses with 49 sarcoids erated with predetermined frequency and time, a were treated with ECT and bleomycin as single chemotherapeutic (Bleomycin, Cisplatin) is applied treatment, and two horses with 5 sarcoids were locally and / or intravenously before. The chemo- treated with ECT and bleomycin as adjuvant treat- therapeutic enters the cell in greater percentage ment with marginal excision. One horse with 4 and induces the apoptosis. melanomas was treated with ECT and bleomycin It is possible to apply it on the tumor directly or as as single treatment. an adjuvant after surgery, inside the surgical bed For four horses with 18 sarcoids, information for foland treat possible cancer cells that could not be low up was not available. Of the remaining sarc- treated. It is very useful in those tumors that are oids, complete remission was observed in 21/31 very difficult to resects or resolved with little mar- (67,7%) tumours, partial remission in 5/31 (16,1%) gin or in those cases in the surgical intervention it tumours, and stable or progressive disease in 5/31 is very aggressive and may not be accepted by the (16,1%) tumours. In one horse with 4 melano- owner (Mandibulectomies, amputations, rhinotom- mas, complete remission was seen in 3/4 (75%) tu- ies). mours, and stable disease in 1 tumour. The case of a 15-year-old female canine with a For two horses with 5 sarcoids, after 1 session of soft-tissue sarcoma infiltrating the left limb knee of adjuvant treatment with ECT and bleomycin intra-rapid evolution, of very difficult surgical resolution operatively in combination with surgical/laser ex-with good margin, is presented. Other veterinari- 237 cision, complete remission was observed in 4/5 tu-treated with electrochemotherapy. They were not mours and recurrence in 1/5 tumours. candidates for first line therapies or they rejected Four horses with 5 sarcoids were treated with sed- them. Intravenous bleomycin was administered at ated, of which one was periocular. a dose of 15,000 IU/m2 and 8 minutes later the The results of the study show that ECT with bleo- electric pulses were delivered. mycin was safe, and effective in the large majority They consisted of 8 100 us long 1,000 V/cm pulses of tumours, with only one session. Location rather at a repetition frequency of 5 kHz by means of the than size was correlated to outcome, with periocu- OncoPore (BIOTEX SRL, Buenos Aires, Argentina) lar location having a worse outcome. using a six-needle electrode. Prophylactic antibiotics were administered after the PO-03 treatment. Squamous cell carcinoma treated with electro- The patients were followed-up weekly until the final chemotherapy response was obtained. Jesica Rodríguez Miranda 1, Antonella María Cilio1, Results: All the patients presented edema which Ana Laura Campastri1, Raquel Lertora2, Sebastian D. resolved after the first week. Patients with pain Michinski3, Ana Clara Acosta4, Felipe Maglietti1 prior to the treatment improved considerably dur- 1Fundación H.A. Barceló, Argentina ing the first days of follow-up. 2Hospital Zonal General de Agudos Petrona V. de Cord- According to RECIST criteria 86 % OR (67 % CR + ero, Argentina 29% PR) and 14% PD were obtained. 3Universidad de Buenos Aires, Argentina All but one patient required one treatment session. 4Hospital de Agudos JM Ramos Mejía, Argentina The patient with an ulcerated lesion in the scalp Introduction: Squamous cell carcinoma is a (initially without bone involvement determined by ct malignant proliferation of keratinocytes of the epi- scan), required two sessions. Despite having an dermis, is the second tumor in frequency after the initial good response, this patient progressed and Basal cell carcinoma and represents 20% of the cu- developed bone involvement requiring additional taneous tumors. treatment. The treatment was very well tolerated Risk factors that increase the probability of devel- in all cases, and the patients were willing to repeat oping Squamous cell carcinoma include unprotec-it if needed. ted UV exposure, as well as specific patient charac- Keywords: Electroporation, Cancer, Head and teristics such as age, skin phototype, immunosup- neck, epidermoid carcinoma pression, chronic inflammatory processes, among Conclusion: In advanced stages of squamous cell others. carcinoma where first line therapies provide poor In early stages, surgery is the treatment of choice. response, electrochemotherapy currently repres- Most cases of squamous cell cancer are limited to ents an effective and safe treatment for patients, the skin and are successfully treated with surgical being a valuable new tool for the oncologist. excision, local ablation, or radiotherapy. When these treatments fail or are rejected by the pa- PO-04 tients, new therapeutics options are needed. Elec- Electrochemotherapy as salvage treatment of trochemotherapy has been used in Europe since adrenal metastasis 2006 and in Argentina since 2020 as a standard of Barbara Perić, Nina Boc, Maja Čemažar care, being an effective treatment with few adverse Institute of Oncology Ljubljana, Slovenia effects in patients where other therapeutic options Introduction: Electrochemotherapy (ECT) is ef- have not been successful. fective and safe method of treatment for cutaneous Patients and Methods: These are the first seven melanoma (CM) skin metastases. ECT induces im- patients recruited from this prospective study with munogenic cell death and release of tumour anti- squamous cell carcinoma in the head that were gens. In the era of novel systemic CM treatment, 238 role of ECT in distant metastases treatment is less PO-05 clear. Approximately half of the patients don’t re- The Pancreatic Cancer Cellular Response to spond to the systemic treatment or relapse after ini- Calcium Irreversible Electroporation tial success. Studies have shown, that ECT can act Agnieszka Gajewska- Naryniecka 1, Nina in synergy with systemic immunotherapy. We are Rembiałkowska1, Dagmara Baczyńska1, Katarzyna presenting immunotherapy and ECT treatment of Bieżuńska-Kusiak1, Anna Szewczyk1, Vitalij Novickij2, resistant CM adrenal gland metastases. Julia Rudno-Rudzińska1, Wojciech Kielan1, Julita Kulbacka1 Case report: 59-years old patient was diagnosed 1Wroclaw Medical University, Poland with systemic, BRAF negative CM metastases in 2Vilnius Gediminas Technical University, Lithuania January 2020. Pembrolizumab was introduced combined with RT of the spleen 2021. Upon pro- Pancreatic cancer is an aggressive disease gression, 4 cycles of ipilimumab + nivolumab were that spreads early and has a 5-year survival rate given. In 2022 nivolumab monotherapy was intro- of only 10%. It often shows no symptoms until it duced, it is still ongoing. In December 2022 inop- has progressed significantly. Studies have shown erable 4.5 cm tumour in left adrenal gland (LAG) that cancer progression from stage I to IV takes an was described among the others. RT (TD 20 Gy) average of one year. The only curative treatment of LAG was initiated. is surgical resection, which is possible in the early In September 2023, CT showed 7.8 x 9.2 cm big stages of the disease. Irreversible Electroporation LAG metastases, new metastatic lymph node in left (IRE) is an innovative localized treatment that of- axilla (3.1 cm) and regression of other lesions. In fers new hope for those with unresectable tumors. November 2023 unsuccessful attempt of surgical IRE is a non-thermal ablative therapy that causes removal of LAG was performed, followed by open cell death by increasing cell membrane permeabil- LAG ECT. ity and activating apoptosis. It is well-established Eight minutes after i.v. injection of 34 mg of bleomy-that alterations in the mitochondria are the primary cin (BMI 30.8, 2 minutes bolus), hexagonal needle drivers of apoptosis activation, and calcium plays electrodes (IGEA, H-30-ST) were inserted in LAG. a crucial role in this mechanism. In our approach, All together 22 applications of electric pulses were the IRE technique involves the administration of delivered to the LAG, covering 2/3 of the tumour. calcium ions during an IRE procedure. CaEP Upon wound closure patient was moved to surgical is intended for patients who do not respond to department. Patient was released on the second standard treatment methods and holds promise postoperative day with no pain or side effects. for enhancing treatment outcomes. Our research Computed tomography (CT) 2 months after ECT investigated the efficacy of CaEP combined with showed partial response with extensive necrosis IRE and cellular responses of pancreatic cancer in the LAG and regression of ECT-untreated meta- cells. static lymph node, S100 and LDH were decreased. We have studied three immortalized cell lines, 5 months after ECT necrotic post-therapeutic area PL45, HPAFII, and BxPC3, as models for pan- was smaller on CT with slight increase of S100 and creatic cancer. The cells were trypsinized and LDH. Patient remains asymptomatic. exposed to calcium (2,5 mM CaCl2) and IRE Conclusion: ECT can be effectively and safely monotherapy in combination with a HEPES-based used as salvage therapy in case of inoperable, im- buffer (SHM). All protocols were compared to munotherapy resistant CM LAG metastases. clinical standard ESOPE (European Standard Operating Procedures of Electrochemotherapy) 1.3kV/cm×100µs×8. The viability of the cells was assessed at 24, 48, and 72 hours after exposure to the electric field. The cell membrane permeabiliz- ation rate was measured by flow cytometry using a 239 cell-impermeant dye Yo-Pro-1. Cellular responses presents the relationship between electrode conto EP protocols were assessed on gene and figurations, electrode geometry and the achievable protein levels at different time points depending ablation volume using the developed PF-IRE. Addi- on assay type (RT-qPCR, Western Blot, Miliplex® tionally, monitor the tissue temperature, which aids assay). in selecting right IRE parameters in clinical treat- Our study on electroporation revealed that the ment planning, avoiding thermal damages. The addition of calcium ions to electroporated cells led computational model of classical electrodynamics to a significant decrease in cell viability in all three for the solution of the electric field, coupled with cell lines compared to IRE alone. As the electric the statistical model for cell viability, can be very field intensity increased, we observed a corres-effective in planning electropermeabilization-based ponding increase in cell permeabilization. There tissue ablation. For the numerical implementation were noticed alternations in the RNA and protein of the mathematical model for the IRE, we followed expression levels of inflammatory and migratory the Finite Difference (FD) formulation in two di- markers (IL1a, IL2, IL4, IL6, IL8, IL10, IL17A, TNFa mensions, 2D, and three dimensions, 3D, and the MMP9, Cadherins, F-actin, Integrins, and TGFß. model is implemented in an app. The developed Calcium ions potentiated cell death combined mathematical model in our work aims to provide the with irreversible electroporation. According to our tissue-specific effective operation parameters with study, we state that CaEP may potentially alter the visual outputs through the app-based user in- cellular behavior and immune responses. Our terface of the solver for a better understanding of study is particularly innovative since few reports the clinicians. The tissue-ablation app includes the exist on applying calcium ion electroporation following concepts/features. (CaEP) in treating pancreatic cancer. > A mathematical model capable of predicting the tissue ablation volume in IRE by solving the electric Funding: This study was supported by the field and using the Peleg-Fermi model. Medical Research Agency, Poland, IREC project > Estimating the temperature is a necessary part of No. 2020/ABM/01/00098/P/02 (PI: Prof. Wojciech the treatment procedure; therefore, a module solv- Kielan), and SUBK.D260.24.024 . ing Pennes bioheat equation determines the tem- perature. > The model can consider electric field- or temperature-dependent electrical conductivity De- PO-06 pending on the treatment parameter set. Tissue-ablation App for Electroporation-based > Several treatment options include pulse type, Therapy pulse shape, pulse length, and the number of elec- Sudip Kumar Das trodes. TATA Consultancy Services, India > The GUI of the developed app provides easy ac- Pulse field irreversible electroporation (IRE)- cess to the selection of treatment parameters, the based minimally invasive tissue ablation technique provision of computation, and the visualization of uses high-intensity electric pulses of short duration results. to cause permanent damage to the cells in the tis- PO-07 sue. However, achieving safe and effective tissue The Effects of High Frequency Nanosecond ablation depends upon multiple factors, including Pulsed Electric Fields with Calcium on 3D the tissue properties, applied electric pulse poten- Spheroidal Model of Lung Cancer tial, and the electrode configuration. Thus, it de- Wojciech Szlasa1, Julia Kucharczyk1, Eiv- mands understanding the insights and optimizing ina Radzevičiūtė-Valčiuke2, Vitalij Novickij2, Julita the PF ablation parameters for the target region Kulbacka1, Nina Rembiałkowska 1 of interest. In this line of contribution, this work 1Wroclaw Medical University, Poland 240 2State Research Institute Centre for Innovative Medicine, PO-84 Lithuania Irreversible Electroporation: Impact of novel multiple train pulse field on non-thermal tumor Pulsed electric fields (PEFs) serve as a pre- ablation valent method for enhancing the intracellular deliv- Ciara Nulty ery of diverse molecules, including pharmaceutical Trinity College Dublin, Ireland agents. Despite widespread use, the precise para- meters governing the permeabilization process ne- Electroporation (EP) therapy, achieved by ap- cessitate further optimization. plication of electric pulses, can either be categor- Our study aimed to investigate how nano- ised as reversible or irreversible dependent on second pulses burst of varying frequency and field electric field strength and duration of treatment. intensity affect CaECT and cellular response (100 IRE is defined by at least 80-100pulses and a pulses of 8, 10, 12 kV/cm field intensity, 200 ns dur-high electric field amplitude of up to 3000V. This ation time, and high frequency pulses 10 kHz, 100 high magnitude and pulse duration overwhelm the kHz and 1 MHz). Also, the study seeks to identify cell capacity to regain homeostasis leading to cell the effects of nsPEF on the 3D spheroidal model death. of lung cancer (A549 cell line). ESOPE (European There is an existing clinical need for IRE protocol Standard Operating Procedures for Electrochemo- which is predictable with a larger ablation zone, therapy) protocols were used as a reference. while constraining the muscular contractions and We have shown that the increase of pulse repe- reducing temperature fluctuations near the elec- tition frequency nsPEFs significantly reduces cell trode site. Clinically, IRE requires an anesthetic viability and promotes membrane permeabilization. regime with paralytic agents to prevent severe As expected, the addition of calcium ions (5 mM muscle contractions associated with strong elec- CaCl2) to the nsPEF protocol further amplifies trical pulses, and analgesia management during re- these cytotoxic effects. Morphological analysis of covery. An electrocardiogram synchronizer should spheroids revealed inhibited tumor growth from 8 be used with IRE therapy to minimise risk of dys- kV/cm and 100 kHz pulses and higher paramet- rhythmia. Additionally, although the mechanism of ers with CaCl2. The control group’s tumor growth cell death by IRE is non-thermal, it can still create served as a reference. The nanosecond pulses ohmics heating with structures in direct contact with were as effective as the ESOPE sequence. Control the electrodes where the electrical field line dens- cells exhibited normal cadherin distribution on cell ity is highest, resulting in thermal ablation. Here membranes, while exposure to different PEF fre- we investigate different IRE parameters to spare quencies and calcium altered cadherin localization, surrounding healthy tissue, nerves and blood ves- impacting cell adhesion dynamics and responses sels, leading to tissue regeneration in the treatment to electrical stimuli. Exposure to PEF at different area from thermal ablation, while preventing severe frequencies shows an increasing p53 fluorescent, muscular contractions. indicating a frequency-dependent cellular stress re- In this study, development of Mirai Medical’s elec- sponse or activation of the tumor suppressor path- troporation technology, including electrode design way, modulated by calcium supplementation, as and IRE protocol, aimed to increase the efficacy evidenced by increased nuclear fluorescence of of IRE treatment, while overcoming the necessity p53. of anesthesia and muscle relaxant. Mirai Medical Financing: The research was supported by designed a novel IRE protocol with varied voltage the Statutory Subsidy Funds of the Department trains, duration and number of electrical pulses of Molecular and Cellular Biology (UMW) no. to overcome current limitations including severe SUBZ.D260.24.076. muscle contractions and thermal ablation. Our analysis showed that high frequency, high and low voltage trains can reduced skeletal muscle con- 241 tractions. Temperature monitoring during pulsing WHO scale below 2> .Pancreatic adenocarcinoma showed less heating around the electrode sites and may be diagnosed as primary tumor or recurrence. we also found alterations in probe design from the Patients excluded from IREC study are patients traditional needles was more user friendly and re- with severe cardiac arrythmia, WHO above 2 and duced superficial damage to tissue of pentretrating metastatic disease. needle electrodes. Until the date 10.04.2024 47 patients with stage Therefore, pain reduction in IRE can be achieved III of pancreatic adenocarcinoma (unresectable) by appropriately defining the protocol parameters were included to IREC study and underwent IRE or and electrode design. However, the further de- CaEP or ECT therapy with bleomycin. Open or per- velopment of these alternative protocols remains a cutaneous procedure was performed. During one- crucial point to be assessed for translation into clin-week stay in hospital the safety of the procedure ically setting as a standardised treatment. While was examined. Overal survival (OS), progression reducing associated IRE-morbidities, patients can free survival (PFS) were analysed as well as qual- undergo IRE therapy without anesthesia, while re- ity of life (WHOQOL-BREF, EORTC QLQ- PAN 26) taining non-thermal cell death of pulse treated tis- Patients were examined one, three, six and twelve sue. We suggest these new IRE operating proced- months after procedure. ures can be used in pre-malignant and malignant IREC is on-going study and the questions we need patients during a day procedure, reducing hospital to answer is weather this methods are effective, if wait times and reducing patients’ morbidities post any of these three methods is more suitable or safe, treatment and improving ability to withstand treat- is there the best moment for the patient to under- ment. went this procedure or is there any specific group of patients who will benefit most from the treatment. PO-08 Effects of calcium electroporation (CaEP), elec- PO-09 trochemotherapy (ECT) and irreversible elec- 2D, 3D and in vivo osteosarcoma models for troporation (IRE) in patients with locally ad- electrochemotherapy studies vanced pancreatic adenocarcinoma. On-going Sasa Kupcic, Urša Lampreht Tratar, Gregor Serša, clinical trial. (IREC) Maja Čemažar Julia Rudno-Rudziska, Ewelina Frejlich, Karolina Institute of Oncology Ljubljana, Slovenia Rodak-Mirocka, Wojciech Kielan, Julita Kulbacka Osteosarcoma (OS) is one of the most com- Wroclaw Medical University, Poland mon primary bone cancers in humans and dogs, Pancreatic cancer is 3% of cancers in Poland. which are a good model for the human disease. In It is located on the 10- th place in man and 12- th both humans and dogs, amputation with standard place in woman mortality. With it’s poor prognosis it adjuvant chemotherapy has been the established is on the 6- th and 7—th place in mortality. The mor- primary treatment for decades. Electrochemother- bidity increases in Poland. Because lack of symp- apy has already proven to be a good treatment op- toms pancreatic cancer is diagnosed late and in tion for various tumor types and could also be a 80% of the patients is unresectable. For those pa- good option for OS patients. To evaluate electro- tient only palliative chemotherapy and radiotherapy chemotherapy as a potential therapy for OS pa- is available and the overall survival with palliative tients, human and canine, we first had to create a systemic treatment is 8-10 months. predictive model of the disease. IREC study aimed to present and compare the Therefore, we established 2D and 3D cell mod- findings and outcomes of patients with locally els from the murine OS cell line K7M2 and an or- advanced, unresectable, histopathologically con- thotopic in vivo mouse model using the luciferase- firmed pancreatic adenocarcinoma (stage III). An- expressing cell line K7M2-luc. First, the 2D cell other inclusion criteria are age above 18 years old, model was used for initial cytotoxicity and prolifer- 242 ation assays. However, 2D models cannot reproPO-10 duce important properties of solid tumors in vivo. Cell death mechanisms detected in cardiomyo- Therefore, 3D multicellular tumor spheroids were blasts after conventional IRE and after H FIRE used as their architecture promotes cellular organ- Mario Gómez-Barea, Tomás García-Sánchez, Antoni ization, differentiation and communication. These Ivorra processes lead to the development of a spher- Universitat Pompeu Fabra, Spain oidal microenvironment that recapitulates the in Cell death mechanisms underlying the basis of vivo properties of solid, non-vascularised tumors; IRE protocols are still poorly understood. Signs growth kinetics, metabolic processes and resist- of accidental death (necrosis), regulated cell ance to cytostatic agents. We cultured spheroids death (e.g., necroptosis and pyroptosis), and pro- with different numbers of seeded cells, incubated grammed cell death (apoptosis) have been ob- them in a Clinostar® rotary incubator and harves- served. For protocols consisting of high�frequency ted them at selected time points. The percent- biphasic pulses (i.e., H�FIRE), our lab reported in age of apoptotic/necrotic cells was then determ- vitro data indicating that the portion of the total leined. Spheroids formed from 5000 cells and cul- sion due to delayed (i.e., non�necrotic) cell death tured for 1 week were selected as a suitable model mechanisms increased with frequency and that, for further studies. among that portion, apoptosis was less prevalent Since OS tumors usually develop at sites of bone as frequency increased (Ann Biomed Eng. 2020 growth and involve specific components of the May;48(5):1451-1462). That situation suggests bone microenvironment, the use of an orthotopic that other delayed cell death mechanisms must be mouse model is required. The implantation of cells involved in H�FIRE. The main aim of the present at the same anatomical site as the corresponding study was to understand which delayed cell death cancer in humans and dogs (in this case the prox- mechanisms are involved, apart from apoptosis, imal end of the tibia) better mimics the microenvir- when H�FIRE pulses are applied. H9C2 cells were onment and cellular interactions found in human 2D cultured and treated with a non�uniform field and canine tumors than the commonly used ec- similarly as in (IEEE Transactions on Biomedical topic (subcutaneous) models. Therefore, for the in Eng. 2022 ;69(4):1318-1327). A thin layer of low vivo studies, we first transduced the K7M2 cell line gelling agarose with 1 % v/v in DMEM was care- with the gene for luciferase. Next, the cells were fully placed over cells to immobilize the cells after inoculated into the proximal part of the tibia using treatment. The treatment consisted of either 60 a 29G needle. With a drilling action the needle bursts of biphasic pulses (internal frequencies of passed through the periosteum and once it reached 90, 150, 260 and 450 kHz) or 60 conventional IRE the medullar area, the cell suspension was injec- pulses, all with a duration of 100 µs repeated at 1 ted. The growth of the tumors was monitored once Hz. They were applied using a custom�made elec- weakly with IVIS. The mice were euthanized as trode in a monopolar configuration. Voltage amp- soon as bone lysis was pronounced and clinical litudes were adjusted to obtain similar areas free of signs of distress were detected, approximately 6 surviving cells after 24 h. Pyroptosis was assessed weeks after inoculation. In addition to the primary at 4 h and necroptosis at 18 h using indirect im- tumors, we also detected lung metastases, which munofluorescence detection methods. These time are an indicator of disease progression. In conclu- points were established in a preliminary study in sion, we have developed a suitable 2D, 3D and or- which it was determined when these two cell death thotopic in vivo model for further studies on electro-mechanisms were predominantly activated. Mem- chemotherapy of OS tumors. brane permeabilization by PI uptake was assessed at 4, 18 and 24 h. Electric field magnitude across the setup was computed by simulation with COM- SOL Multiphysics. An additional simulation study 243 was computed to neglect possible temperature ef-experiments for the generation of comparable data fects. The areas where pyroptosis and necroptosis between the two treatment platforms. A COMSOL were manifested were smaller than the total area finite element model will incorporate hydrogel ex- of the lesions as assessed by PI uptake at 24 h. perimental data to calculate electric field thresholds We reached a few conclusions. First, by using lar-to determine the necessary applied pulse paramet- ger voltage amplitudes, H�FIRE produces similar ers for a desired lesion area. Ex vivo tissue char- lesion sizes to those achieved by conventional IRE. acterization will provide us with realistic conduct- Second, H�FIRE lesion size cannot be accurately ivity and temperature data to incorporate into our estimated by PI uptake at 4 h because the final finite element model, further improving the accur- lesion, at 24 h, is substantially larger; as anticip- acy of the modeled lesion dimensions. By estab- ated, there are retarded mechanisms of cell death lishing mathematical relations between our in vitro when conventional IRE, and in particular H-FIRE, and ex vivo data, treatment plans can be optim- is applied. Third, signs of pyroptosis are maximal ized, allowing for the rapid refinement of pulsing after 4 h of treatment and of necroptosis after 18 h. protocols to minimize in vivo animal experiment- Fourth, both pyroptosis and necroptosis are highly ation while focusing on investigating lesion depth frequency�dependent, increasing their importance in porcine cardiac tissue. This comprehensive ap- with frequency. Fifth, in addition to necrosis, nec- proach presents a promising strategy for optimiz- roptosis, pyroptosis, and apoptosis, other undeter- ing cardiac ablation protocols to maximize lesion mined cell death mechanisms must be involved in depth, ultimately providing prolonged relief from at- ablation by conventional IRE and H�FIRE. rial fibrillation symptoms. PO-11 PO-12 Lesion Depth Analysis for Pulsed Field Ablation Evaluation of collagen role in electroporation: Kailee David two cell lines compared Georgia Tech, United States Annj Zamuner1, Monica Dettin1, Serena Zaccara2, Caterina Antognazza2, Maria Teresa Conconi1, Elisa-Pulsed field ablation (PFA) is a promising al- betta Sieni 2 ternative therapy to current thermally induced car- 1Padova University, Italy diac ablation techniques such as radiofrequency 2University of Insubria, Italy ablation and cryoablation for the treatment of atrial fibrillation (AF). PFA employs irreversible electro-Electrochemotherapy (ECT) uses electropora- poration to induce cell death in specified regions tion to improve drug uptake into cells. This is an es-of tissue with short duration (us), high amplitude tablished and effective treatment in the care of pa- (kV) electric pulses. Lesion depth, a critical factor tients with several types of superficially metastatic influencing arrhythmia recurrence, presents an is-tumors like breast cancer recurrences or melan- sue as the lack of deep, transmural lesions allows oma. Nevertheless, is applied to other types of tu- for irregular cell regrowth. A more efficient set mors. of protocols must be established to create robust In general, ECT efficacy is evaluated in vitro on cell lesions, ensuring long-term freedom from fibrillat-suspensions with liquid of low conductivity or us- ory symptoms. This problem can be addressed ing cells in adhesion. In recent years the use of through the use of 3D hydrogels and ex vivo tissue 3D culture models diffused. Cell suspension and characterization to gain insights into the efficacy adhesion cultures are very easy methods, never- of various IRE treatment protocols. Using a high- theless, they lack extracellular matrix components, throughput hydrogel model will efficiently narrow that are found in 3D cultures or in vivo. Previously, down optimal pulse parameters for ex vivo cardiac the authors studied the effect of inhomogeneity of tissue experiments. Two-needle electrode config- the scaffold materials and their electrical properties urations will be used for both in vitro and ex vivo in the distribution of the electric field when voltage 244 pulses are applied. When seeded on 3D scaffolds, ficiency of the ”Glycogen Debranching Enzyme” the breast cancer cells or melanoma cells produced (GDE). Mutations within the Agl gene, encoding their own extracellular matrix (ECM) that induced human GDE, result in the loss of its enzymatic an improvement in electroporation. In these cul- activity. Consequently, abnormal glycogen accu- tures, new collagen deposition was found, and au- mulates in skeletal and cardiac muscles, as well thors hypothesized that it played a role in the elec- as the liver, causing a spectrum of organ dysfunc- troporation process. tions with considerable heterogeneity. Currently, To investigate the role of collagen in the electro- no definitive cure exists, with therapeutic treat- poration process, HELA and SCOV-3 cells were ments relying solely on stringent dietary interven- cultured in two different scaffolds. The first scaf- tions. fold consisted of hyaluronic acid (HA) and 5% w/w Our investigation focused on the development of self-assembling peptides (EAbuK) condensed with a novel approach utilizing electroporation for ef- an adhesive motif mapped on Laminin (IKVAV), re- ficiently delivering genetic material into cells and ferred as HA-EAbuK-IKVAV. The second scaffold restoring missing enzymatic function. was HA enriched with 5% w/w calf Type I collagen Initially, we employed electro-gene-transfer to intro- (referred as HA-Collagen). duce of the human Agl gene into GSDIII fibroblasts The cell cultures were seeded on the scaffolds and using the syntethic plasmid pVAX-sGDE, which the cultures were characterized at 3 days by cell encodes the human GDE, and optimized electric viability assessment, hematoxylin-eosin staining, parameters to enhance transfection efficiency and and Masson’s trichrome staining. Moreover, the ensure targeted gene delivery. gene expression for collagen was investigated. Subsequently, we advanced our study obtaining Electroporation of the cells seeded on the two types GDE overexpression in vivo in the tibialis cranialis of scaffolds was performed using EPS02 EP equip- muscle of C57Bl/6 mice using the plasmid pVAX- ment (Igea SpA, Carpi, Modena, Italy). Different sGDE and two different electric pulse protocols. voltage amplitudes, generating different strengths The long-term goal of our work is to contribute to of the applied electric field (0, 400, 600, 800, and an alternative non-viral gene therapy approach for 1000 V/cm), were tested. The EP efficiency was GSDIII. evaluated using propidium iodide. Both the 3D cul- tures, HA-EAbuK IKVAV and HA-Collagen, were PO-14 electroporated in the culture medium. The cells cul- Comparative Evaluation of Lipofectamine and tured in the two scaffolds showed a different grade Electroporation Side Effects on Cellular Func- of electroporation improvement with respect to ad- tions: Emphasizing IMPDH Regulation herent cells. Yusuf Elhaei, Samira Samadieh, Seyed Mojtaba Yazdanparast, Razieh Yazdanparast, Mohammad Abdo- PO-13 lahad Electro-gene-transfer of a synthetic gene: a University of Tehran, Iran, Islamic Republic of possible approach for treatment of Glycogen Lipofectamine and electroporation are widely Storage Disease type III used for nucleic acid transfection, but their poten- Doriana Triggiani 1, Caterina Merla1, Emanuela tial side effects on cellular regulatory mechanisms Pasquali1, Simona Salati2, Rosella Franconi1 are not fully understood. Considering the crucial 1National Agency for New Technologies, Energy and roles of inosine 5′-monophosphate dehydrogenase Sustainable Economic Development, Italy (IMPDH) and its main regulatory form, cytoophidia, 2IGEA S.p.A., Italy in cell proliferation and fate, we examined the im- Glycogen Storage Disease Type III (GSDIII) is pact of these methods on IMPDH cytoophidia in a rare hereditary disease (with an approximate in- K562 cells. Utilizing a home-made electropor- cidence of 1 in 100,000 births) caused by the de- ation instrument, our results showed that Lipo- 245 fectamine treatment significantly decreased K562 above the spheroids, following the alignment of the cell viability compared to electroporation. Despite spheroid columns. 8 variable resistors (0-5kΩ) are both methods demonstrating similar siRNA trans- added to control the electrical potential on each fection efficiency with optimized parameters, Lipo- electrode. A PMMA structure is used to host the fectamine induced more pronounced changes in PCB and ensure watertightness of the chamber cell cycle distribution and erythroid differentiation and electrical contacts. The spheroids can thus in K562 cells than electroporation. Additionally, be exposed to an electric field by applying voltage Lipofectamine affected both the formation and size between the bottom electrode and the resistors of cytoophidia, whereas electroporation only influ- shared node. The actual electric field in the cham- enced cytoophidia size. The localization pattern of ber depends on the resistor values. The latter was cytoophidia remained consistent under both treat- adjusted using COMSOL software (Livelink with ments. These findings support the lack of ad- Matlab). verse effects of electroporation technique, relative EPN was performed on a population of HT-29 to the Lipofectamine approach, concerning the reg- spheroids after 3 days of culture, in presence of ulatory function of IMPDH-based cytoophidia and Propidium Iodide (PI), using eight 100 µ s large thus, it is a suitable replacement technique for monopolar pulses, with a 1 Hz repetition frequency. Lipofectamine-based investigations. The spheroids were exposed to a solution contain- ing Fluorescein Diacetate (FDA) after the proced- PO-15 ure as a measure of cell viability. New microsystem for gradual electroporation As IP dyes enter cells that have lost, temporary of a regular spheroid population and applica- or not, their membrane integrity, IP stains cells that tion for protocol comparison have been reversibly or irreversibly electroporated, Théo Le Berre 1, Julien Marchalot1, Guilhem Rival2, as well as dead cells. The IP intake thus correlates Charlotte Rivière1, Marie Frénéa-Robin1 to the permeabilization of the spheroids. 1Universite Claude Bernard Lyon, France On the other hand, FDA is converted into green 2University Lyon, France fluorescent fluorescein by living cells, thus assess- A microfluidic platform specifically designed by ing the reversibility of EPN at a given intensity. our team for culture, electroporation, and observa- The plot of IP and FDA intensity along the cham- tion of spheroids with regular size and shape has ber thus shows the permeabilization as a function been recently described (WCE22). of the electric field, as well as the statistical distri-We present in this study a microfluidic device bution per column (15 to 20 spheroids). A sigmoid derived from this work designed to apply a gradi- function was fitted to those data. ent of electric field to a spheroid population, in or-As the protocol used in this experiment is con- der to investigate effect of electroporation (EPN) sidered a standard for in vivo ECT procedures, we parameters on a spheroid model. Fluorescence can consider that this experimentation is a solid microscopy is used to estimate the efficiency of way to evaluate the potential of other protocols or EPN, it allows for the direct plot of permeabilization-waveforms in comparison to this standard. intensity relationship in a single experiment. We intend to use the device to compare the The system is centred around a microfluidic permeabilization-intensity relationship in different chamber, composed of a 1 mm silicone seal EPN protocols, in particular to investigate other between two electrodes, the chamber contains a waveforms. hydrogel scaffold hosting each spheroid at a spe- cific location, arranged into 16 columns. The bot- tom electrode is fully coated with ITO (Indium Tin Oxide) while the upper is gold-coated and wet- etched to set up 8 independent linear electrodes 246 PO-16 Z-Can modality for remote selective stimulation References: incorporating bipolar nanosecond cancellation [1] Pakhomov et al., Targeted Remote Electrostim- Shu Xiao 1, Pavel A. Solopov1, Siqi Guo1, Minglei ulation by Interference of Bipolar Nanosecond Guo2 Pulses, US Patent, 11,020,590 B2, Jun. 1, 2021 1Old Dominion University, United States [2] Xiao et al., Quadrupoles for Remote Elec- 2Eastern Virginia Medical School, United States trostimulation Incorporating Bipolar Cancellation. Bioelectricity. 2020 Dec 1;2(4):382-390. The feasibility of using nsEPs alone as a 3] Ibey et al. Bipolar nanosecond electric pulses targeted, receptor-independent, non-chemical are less efficient at electropermeabilization and stimulus has been demonstrated previously. killing cells than monopolar pulses. Biochem nsEPs have been shown to cause diverse ef- Biophys Res Commun 443, 568-573, 2014. fects, including electroporation, calcium influx, [4] Kim et al. Peculiarities of Neurostimulation by voltage-gated channel activation, and action Intense Nanosecond Pulsed Electric Fields: How potential activation. Extending the capability of to Avoid Firing in Peripheral Nerve Fibers. Int J nsEPs to deep-lying tissues will enable many more Mol Sci 22, doi:10.3390/ijms22137051, 202). therapeutic applications. We propose a modality that can potentially target deep responses while PO-17 suppressing superficial cell responses (Z-CAN). Scanning electrochemical microscopy as a tool This concept is derived from CANCAN stimulation for microscale imaging after electroporation [1, 2], exploiting the fact that tissue sensitivity to Gustas Tolvaisas, Inga Morkvenaite-Vilkonciene unipolar pulses (UP) is greater than to bipolar State research institute Center for Physical Sciences and pulses (BP) [3]. The difference could be as high Technology, Lithuania as 10-fold in the nanosecond range [4]. In Z-CAN, nsEPs of opposite polarity are delivered A scanning electrochemical microscope was to the tissues by two pairs of electrodes: P1 (with employed to investigate S. cerevisiae yeast-based a longer gap distance for deeper penetration) biofuel cells. One- and two-electron transfer me- and P2 (with a shorter gap distance for shallower diators and Nystatin were chosen to be tested to penetration). P1 electrodes are fired ahead of P2 create a convenient system and an efficient biofuel electrodes with a controllable delay to maximize element. The potential application in small con- the electrode isolation and cancellation effect. At centrations of Nystatin to improve electron trans- the tissue surface, the effect of P1 electrodes is port across yeast cell membranes by pore-forming, canceled by P2 electrodes with opposite polarity. thereby increasing the efficiency of the biofuel cell. However, this cancellation diminishes as depth in- Scanning electrochemical microscopy (SECM) has creases, allowing the cell response to be retained. been chosen to capture minimal electrochemical Specifically, the pulse waveform in the superficial activity in the yeast. Studies on the efficiency of region is controlled to be bipolar, while in the deep the developed biofuel cell by changing the external layer, it is unipolar. Only the tissue in the deep loads were also performed. region above the threshold will be activated. Other We compare results with varying Nystatin and volumes will either be exposed to subthreshold non-modified yeast modifications using the same BP or subthreshold UP and will be spared from hydrophilic and lipophilic mediators concentrations stimulation. with different Nystatin concentrations from 0 to 6 The Z-CAN concept will be simulated with a 3D µ g/ml. The research discovered that the power electromagnetic simulation (Computer Simulation generated by the microbial biofuel cell, based on Technology, CST) and tested on 3D cell tissue. Nystatin-modified yeast, was higher than that gen- Challenges regarding the pulse generator design erated by a non-modified yeast-based biofuel cell. and pulsing sequence will be discussed. The topographical redox activity maps produced 247 by the scanning electrochemical microscope have the circuit of the high-voltage pulse generator, the shown that larger electric currents are generated irreversible electroporation and impedance meas- from Nystatin-modified yeast during their cultiva- urement could be achieved simultaneously, which tion. provides the hardware support for real-time as- sessment of irreversible electroporation outcomes PO-18 and has significant implications for advancing the Development of a high-voltage pulse generator clinical application of IRE. with an integrated function of measuring tissue bio-impedance spectrum PO-19 Yajun Zhao 1, Shiji Li2, Shanshan Jin2, Zhi Fang2, A numerical model of irreversible electropora-Dong Xu1 tion at tissue scale specific to cardiac pulsed 1Zhejiang Cancer Hospital, China field ablation 2Nanjing Tech University, China Simon Bihoreau, Guido Caluori, Pierre Jaïs, Clair Poignard, Michael Leguèbe, Annabelle Collin Irreversible electroporation (IRE) as a novel tis- Univ. Bordeaux, France sue ablation technique has found widespread ap- plications in areas such as tumor therapy and car- Cardiac ablation is a technique used to address diac ablation. IRE employs high-voltage pulses to heart arrhythmias, a leading cause of mortality create nanoscale defects inducing cell death due worldwide. It involves the destruction of dys- to the loss of cell homeostasis. The cell death functional cardiac tissue in targeted areas using caused by IRE has a certain time lag in evalu- a catheter inserted into the heart. Irreversible ating therapeutic outcomes when using the cur- electroporation ablation is increasingly used in the rent medical imaging techniques, presenting chal- cardiac atrium, under the name of Pulsed Field lenges for surgeons. To address these issues, in Ablation (PFA) [1]. However, existing electro- the present study, an optimized topology of a high- poration models, used in oncology for instance, voltage pulse generator with impedance measure- are inadequate to describe PFA as they do not ment was proposed based on the previous stud- account for the complex geometry of cardiac cells ies assessing the outcome of irreversible electro- and their excitability. It is therefore a challenge poration using bioimpedance spectroscopy. The to describe both the pulses at tissue scale and impedance measurement was realized by relat- the microscopic electrical phenomena. Some ively lower voltage pulses extracted from a single- first simulations were presented in this direction, stage charging circuit of the Marx circuit. By doing combining the electroporation models and the this, the generator could simultaneously generate specific cardiac cells geometry [2]. To go further high-voltage pulses for irreversible electroporation in terms of modeling, we propose in this work a therapy and produce low-voltage pulse signals for new model of electroporation at tissue scale that measuring the impedance spectra of biological tis- is adapted to cardiac ablation, and the resulting sues during high-voltage pulse intervals. By theor- numerical simulations. etical analysis, simulation, and experimental veri- We obtain a model, using mathematical homo- fication, the prototype of the pulse generator could genization, by considering the cardiac electro- output pulses with a voltage of ±5kV, a maximum physiology cell-scale equations at the time and repetition frequency within a burst up to 1MHz, and length scales of PFA, together with the non-linear a pulse duration of 500 ns to 100 µ s. For the low-resistive term modeling membrane electroporation. voltage pulses, the pulse width and amplitude could Interestingly, a non-linear transport term depend- be adjusted from 500 ns to 100 µ s, and 10V to ing on the electric field appears, which is in 200V, respectively, and the maximum frequency for accordance with the experimental observations burst mode is 1MHz, the pulse number could be and with the phenomenological models used in adjusted as required. In conclusion, by optimizing oncology [3], and links the electric field with the 248 transmembrane potential at tissue scale. As a factors like pulsed electric field parameters, cell specificity of the homogenization technique, some membrane properties, and electrical conductivity. terms carry microscopic information, namely the Although application of H-FIRE pulses produces geometry of the cells. non-thermal cell death application of any pulse will We give some numerical illustrations in a realistic induce some tissue heating and in evaluating pro- context, including the geometry of catheters used posed H-FIRE pulse candidates it would be useful in clinical practice. Both bipolar (two electrodes on to be able to quantify the tissue heating expected the catheter) and unipolar (one electrode on the from a given pulse candidate before carrying out catheter and one on a distant patch) configurations experimental validation, for this reason a new ap- are studied. In the latter, an electrical current is set proach for thermal effect modelling has been de-instead of a voltage, in accordance with the clinical veloped. procedure. We investigate the influence of the In this work, we propose a novel method to contact force between the catheter and the tissue model the thermal response of tissues treated and of the cells geometry on the computed electric with H-FIRE: Electrical Equivalent Thermal Models field and thus on the estimated ablated zone. As (EETM). EETM represents the tissue geometry and our model involves time and has a cardiac-specific its surroundings as an equivalent electrical circuit, structure, the simulations are different from the enabling simplification of the thermal analysis com- quasi-static models. pared to Finite Element Methods for different com- binations of electroporation pulse parameters typic- [1] G. Caluori et al. AC Pulsed Field Ablation ally applied during treatment. The circuit elements Is Feasible and Safe in Atrial and Ventricular Set- signify the thermal resistance and specific heat ca- tings: A Proof-of-Concept Chronic Animal Study. pacity properties of targeted tissues. EETM is help- Front Bioeng Biotechnol; 8, 2020. ful for understanding how cells respond to pulsed [2] Q. Castellvi. In silico comparison between RFA electric fields, providing a simple way to analyse and PFA. Presentation at RICAM Modeling and both electrical and thermal changes during electro- simulation of ablation treatments Workshop, 2023. poration. [3] M. Leguebe et al. Conducting and permeable The proposed model includes the EndoVE device states of cell membrane submitted to high voltage (Mirai Medical, Ireland) and utilizing tissue elec- pulses. Mathematical and numerical studies val- trical properties from the IT’IS Foundation data- idated by the experiments. Journal of Theoretical base. It is implemented in LTspice—a free software Biology, 2014. for electrical circuit modelling. The equivalent cir- cuit assesses the thermal effect induced by H-FIRE PO-20 treatment with various amplitudes and frequencies. Electrical Equivalent Thermal Modelling of Tis- Temperature analysis of Mirai Medicals proprietary sues During High-Frequency Irreversible Elec- parameters reveals a rise from 37°C to 40.6°C, with troporation a 3.6°C increase, validated through experiments Hamza Benchakroun 1, Cian Kerrigan2, Jason using the EndoVE device on perfused ex-vivo ovine McNamara2, Maeve Duffy1 liver. 1University of Galway, Ireland The alignment of predicted EETM outcomes with 2Mirai Medical, Ireland experimental results highlights the value of our High-frequency irreversible electroporation (H- work, demonstrating the EETM’s potential as a re- FIRE) is a focal ablation therapy that uses high liable predictor for thermal assessment in H-FIRE voltage, short electrical pulses that has been pro- treatments with variable pulse parameters. This posed as an ablation technique for colorectal can- study contributes to the understanding and optimiz- cer. H-FIRE disrupts cell membranes, leading to ation of electroporation techniques, specifically H- cancer cell death, with outcomes influenced by FIRE, with implications for enhanced cancer treat- ment strategies. 249 PO-21 peaks of current due to the charging and dischar- Rate of pore formation by electroporation in ging of the membrane at the beginning and end of black lipid membranes the voltage pulses. These peaks can be attributed Burak Altun, Antoni Ivorra to the formation of short-lifetime pores by electro- Universitat Pompeu Fabra, Spain poration. Under this assumption, our preliminary quantitative analyses of the results seem to con- In a recent study on black lipid membranes firm that the rate of pore creation seems be pro- (BLM) by C.M. Marques and colleagues (Proc. portional to |V|exp( α|V|) rather than to �exp( α|V2|), Natl. Acad. Sci. USA, 2023 Mar 14, where V is the transmembrane potential as α is a 120(11):e2213112120) it has been observed that constant. Currently, we are conducting further as- the frequency of pores appearance by electropor- says and analyses to validate this conclusion and ation has a milder dependence on the transmem- to determine whether it is possible to observe elec- brane voltage than previously anticipated by stand- troporation for very low transmembrane voltages ard model of electroporation. We have carried (<200 mV) when the membrane is partially hydrop- out an experimental study to confirm such obser- eroxidized. vation and, in particular, to explore pore forma- tion when relatively low amplitude voltages are PO-22 applied. For that, we have acquired a commer- Advancing Electroporation Studies with Auto- cial BLM kit (ELEMENTS srl) consisting of a stim- mated SECM and Machine Learning ulator/current amplifier and a recording chamber Kristijonas Klimaitis, Inga Morkvenaite-Vilkonciene where two electrolyte-filled cuvettes are separated State research institute Center for Physical Sciences and by either polytetrafluoroethylene (i.e., Teflon) or Technology, Lithuania polyimide septa with a micrometer hole on which the lipid membrane is created. Voltage waveforms Scanning electrochemical microscopy (SECM) are applied across chlorinated silver electrodes employs an ultramicroelectrode (UME) as its probe (i.e., Ag-AgCl electrodes) immersed in the cuvettes in this scanning probe microscopy technique. It is so that transmembrane voltages are induced, and particularly effective in characterizing the electro- the resulting currents can be measured. We have chemical properties of surfaces and applying local- tried 80, 100, 110, 150 µm sized micro-holes. For ized electric fields to induce electroporation in cells. creating the membranes, we have either applied However, limitations such as slow imaging speeds the painting method (Muller - Rudin) or the fold- and dependence on user input restrict its full po- ing method (Montal – Mueller). The assayed mem- tential. To enhance SECM’s capabilities, we in- branes were composed of DPhPC or POPC phos- corporated visual recognition and machine learn- pholipids. Two electrolyte solutions were tried: 100 ing to detect micro-objects within images and de- mM KCl, 10 mM HEPES in double distilled water, termine their electrochemical activity. By recon- and 20 µL 1 M NaOH solution to adjust pH 7.4; and structing images from multiple approach curves, we a 0.1 M HCl solution. Most of the assays have con- achieved faster scanning and accurate detection sisted of delivering a series of rectangular pulses of active areas, including sites of electroporation. with varying amplitudes, alternating between pos- This approach reduces both scanning time and the itive and negative polarities. The applied voltages need for constant user presence. We developed an ranged from 0 mV to 500 mV. The time between automated SECM system with visual recognition, consecutive pulses and pulse durations were in the utilizing commercially available modules, low-cost order of seconds. As expected, the implemented components, proven design and software solutions membranes acted as a capacitance in the order from other fields, and a custom control and data of 0.1 µF/cm2. For voltage amplitudes of a few fusion algorithm to study electroporation and other hundreds of millivolts, we have observed sudden electrochemical phenomena efficiently. peaks in current that randomly occur in between the 250 PO-23 stantial differences at the molecular or bond level. Characterization of Transferred Electrodes ob- These analyses serve a crucial role in discerning tained with Laser Induced Carbonization Pro- whether the same chip can be reused with con- cess for Electroporation of Adhered Cells sistent performance or to what extent its function- Francesca Camera 1, Gianni Antonelli2, Alessandro ality persists over subsequent applications. Such Zambotti1, Antonino Cataldo1, Stefano Bellucci3, Laura insights are paramount for assessing the chip’s re-Micheli2, Rocco Cancelliere2, Mauro Falconieri1, Serena liability and longevity in practical usage scenarios. Gagliardi1, Eugenio Martinelli2, Caterina Merla1 1ENEA, Italy PO-24 2University of Tor Vergata, Italy Power Supply Chain with Series Insulation 3National Laboratory of Frascati INFN, Italy Structure for High Voltage Marx Pulse Gener- ator Lab-on-Chips revolutionized biology research Yue Chen, Chenguo Yao, Shoulong Dong by consolidating various functionalities into a com- Chongqing University, China pact platform. In particular, a lab-on-a-chip device can be a powerful tool for electroporation since Nanosecond pulse shows a wealth of physical with a small distance between electrodes, (hun- effects in cell electroporation and tumor ablation. dreds of micrometers), it can generate a high elec- For solid-state pulse generators (SSPG), insulation tric field providing a relatively low voltage (hundreds power supply (IPS) for multiple switches is a key of volts). issue for solid-state pulse generators (SSPG). The In this work, we reported a systematic characteriz- existing parallel structure for IPS is short of insula-ation of a lab-on-chip platform based on electrodes tion similarity, which results in low insulation utiliz-obtained with laser-induced carbonization process, ation ratio (IUR) and limited adjustability. Here we which were transferred on a biocompatible trans- propose a power supply chain (PSC) with series parent polymer substrate (namely PMMA) for the insulation structure. The insulation circumstance electroporation of adherent cells. The device com- of PSC maintains the same for each stage, which prises multiple cells featuring interdigitated elec- increases the minimum IUR to almost 90%, com- trodes positioned at varying distances ranging from pared to the existing parallel structures. The pro- 200 µm to 2 mm. These electrodes were utilized to posed PSC requires purely diodes, capacitors and generate an electric field up to 6 kV/cm. inductors to achieve IPS for switches, thus avoiding The device has been shown to be effective in elec- the customized cost and enhancing insulation ad- troporating U87 glioblastoma cell line looking at justability. After analyzing the working principle of calcium intake. So, we thoroughly characterized PSC, we evaluate the performance of PSC from the the electrode from multiple perspectives both pre- perspective of power supply and insulation. Finally, and post-pulse delivery. This comprehensive ana- an 8-staged Marx SSPG prototype is developed to lysis involved scanning electron microscopy (SEM) examine the compatibility of PSC and the protec- to examine surface morphology, Energy Dispers- tion circuit for PSC is implemented. The withstand ive X-ray Spectroscopy (EDS) for elemental ana- voltage of PSC can be easily adjusted within 14kV lysis at SEM, cyclic voltammetry to assess elec- according to the number of diodes. The PSC does trochemical characterization, Raman spectroscopy not require external control signals and does not to investigate carbon bond alterations and Fourier- interfere with gate driving synchronization or load Transform Infrared Spectroscopy (FTIR) for addi- waveforms, which offers a new solution of IPS for tional molecular characterization. compact Marx generators designed at nanosecond Preliminary results indicate that the morphology scale. undergoes slight modifications between pre- and post-pulse applications, even though the elec- trodes do not undergo oxidation processes or sub- 251 PO-116 like port manipulation, faster processing times Hybrid Digital-Analogue Square Wave Gener- were possible [1], [2] allowing for pulses to be ator for Bioelectronics Applications as short as 2 µ s. Realistic developments could Adam Parry, Cristiano Palego additionally target Field Programmable Gate Array Bangor University, United Kingdom (FPGA)-based digital synthesis to achieve much shorter pulses. Overall, the hybrid concept herein Short-pulse high-voltage waves are used for demonstrates a viable and cost-effective method a wide range of treatments and research, such for flexible waveform generation in bioelectronics as dielectrophoretic (DEP) control and Electropor- settings. The output waveform parameter range ation. Wave generators that create such waves achievable through the present approach com- tend to be expensive, bulky and highly application fortably lay in the realm of on-chip DEP trapping, specialised in terms of delivered wave parameters. sorting and High-Frequency Irreversible Electro- By contrast, a low-cost and flexible solution, poration (H-FIRE). allowing its output to be adapted for a range of different applications, could be of great value for [1] (2016) Arduino zero read in registers bioelectronics research teams while democratizing & speed question. [Online]. Available: treatment protocols and options. https://forum.arduino.cc/t/arduino-zero-read-in- This report demonstrates a hybrid concept relying registers-speed-question/378769 on digital synthesis to create square high-low [2] (2015) Ws2812b success on the esp-12. [On- portions of the wave along with analogue summing line]. Available: https://tech.scargill.net/ws2812b- and inversion to combine them together. This success-on-the-esp-12/ approach allows for a great variety of waveforms to be produced while ensuring fast/clean pulse PO-26 transitions owing to the inherently high slew rate Irreversible electroporation of tethered bilayer in digital signals. The design core concept and membranes by scanning electrochemical mi- a packaged prototype which was 3D printed and croscopy include an integrated heat sink, appear in Fig. 1 Tomas Sabirovas, Rūta Bagdonaitė (a) and (b), respectively. The present footprint, Vilnius University, Lithuania height and weight are 140mm x 130mm, 70mm and 1070g (with batteries) respectively. This study investigates the electroporation pro- The design concept consists of synthesizing two cess of tethered bilayer lipid membranes (tBLMs) digital signals through a microcontroller or equival- using ultramicroelectrodes and scanning elec- ent digital circuitry that implement the two phases trochemical microscopy (SECM). The effects of of a bipolar pulse, while also affording phase delay varying the potential applied to the ultramic- control. The digital signals can be programmed roelectrodes, relative to a silver/silver chloride allowing the user to synthesize a unique waveform (Ag/AgCl(sat)) reference electrode, on the form- according to the application. The achievable peak ation of pores within the tBLMs are elucidated to peak amplitude is 100 V for a 50 ohm load which through the experimental findings. In particular, it is dictated by the analogue inverter and amplifier. has been observed that applying relatively low po- The pulse width and delay can be controlled down tentials, ranging from 1 to 2 V, results in the form- to 2 µ s and up to over a second when a basic ation of pores of varying sizes within the tethered Arduino Uno is used for digital signal synthesis. bilayer lipid membrane. For ultramicroelectrodes The prototype here presented demonstrated easy with a diameter of 10 µm, the resulting electric fields reconfigurability through the Arduino IDE, including are estimated to be extremely high, approximately for bursts with an arbitrary number of pulses, a 100 kV/m at 1 V and 200 kV/m at 2 V. These high sample of which is reported in Fig. 1(c). field strengths are responsible for inducing signific-By utilising lower-level programming techniques ant variations in pore diameters within the tBLMs, 252 ranging from 5 µm to 140 µm. dependency of electric field distribution on insulator The study examined how pulse duration and the geometry. Existing models cannot predict energy number of impulses affect pore formation. Pulse input and field distribution for different chamber duration’s impact on pore formation was studied us- geometries, impeding process optimization. This ing durations of 120, 60, 30, and 5 seconds (one knowledge gap hinders the full potential of PEF in impulse). Furthermore, the impact of varying the the food industry, as safe processes can only be number of impulses from one to eleven, with dur- designed with extensive experiments. Computa- ations ranging from 0.1 seconds to milliseconds, tional fluid dynamics (CFD) simulation has been was investigated. It was observed that reducing proven to be a suitable tool for visualizing local the impulse duration resulted in a decrease in pore physical phenomena and therefore, to investig- size. Other factors affecting the size of the pores ate the influence of the geometry of the treatment in tBLMs after electroporation include the distance chamber. This helps to reduce the number of ex- between the ultramicroelectrodes and the surface periments, necessary to implement a new process. of the tBLM (from 1 to 30 µm), which are critical However, a mechanistic approach to transfer the determinants in modulating the pore size within the PEF process to other scales by means of CFD sim- lipid membrane. ulation is still missing. In conclusion, the use of tBLMs in experimental The presented work aims to fill this gap by deriving techniques holds great promise for advancing our dimensionless numbers based on the relevant bal- understanding of the mechanisms of electropora- ance equations. These numbers include the Reyn- tion and the factors that influence the properties olds and the Prandtl numbers as well as a newly of pores and their ensembles under electric field developed dimensionless factor which scales the conditions. The research demonstrates that the energy source term of the heat transport equation. combination of tBLM with scanning electrochemical CFD-Simulations on five different scales with five microscopy has the potential to greatly enhance different insulator geometries were performed us- the applicability of electroporation processes in bi- ing Ansys CFD. A relevant parameter space with otechnology and biosensing. Reynolds numbers from 200 to 3000, average field strengths from 5 to 26 kV/cm and specific energy PO-25 inputs from 0,7 to 200 kJ/kg was investigated. The Scaling of continuous PEF processes by means obtained data were used to investigate the depend- of dimensionless numbers and computational ency of relevant process parameters like the spe- fluid dynamics (CFD) cific energy input and the average electric field Justus Knappert 3, Felix Schottroff1, Claudia strength on the derived dimensionless numbers. Siemer2, Cornelia Rauh3 The results reveal that the specific energy input 1Boku University, Austria can be adequately described by the newly defined 2Elea Technology GmbH, Germany factor (R2=0.995). However, to determine the av- 3Technical University of Berlin, Germany erage electric field strength, more factors like the The colinear arrangement of electrodes and in- Reynolds and Prandtl numbers as well as dimen- sulators for continuous PEF treatment chambers sionless geometry factors for the insulators need is currently state of the art. This configuration en- to be considered. To ensure the correctness of ables higher field strengths to be generated at re- the simulations, validation experiments were per- latively low power and current. This design has formed on two of the five investigated scales with already been implemented in the industry for con- insulators of at least two different length to diameter tinuous treatment of liquid foods, e.g. to inactivate ratios. microorganisms or enhance mass transfer of valu- In conclusion, the study shows for the first time how able metabolites. the specific energy input and the field strength can However, scaling up poses challenges due to the be described independently of the treatment cham- ber geometry. This is an important contribution to 253 simplifying process design and enabling safe pro-6.3 hours without significantly (p≥0.05) affecting the cesses. maximum growth rate. These results suggest that PEF is a promising mild pretreatment technique PO-27 which can increase the efficiency of the industrial Potential use of pulsed electric field (PEF) treat-process by promoting the reduction of fermenta- ment to increase the concentration of bioactive tion time and increasing the amount of recovered compounds during fermentation of Clementina bioactive compounds. Clementine peels, a waste peel pomace: conversion of waste into food ad- product of the citrus processing industry, thus of- ditives fer potential economic benefits by returning to the Junior Bernardo Molina Hernandez, Beatrice Cel-food processing chain as an additive and providing lini, Emiliana Giordano, Pietro Rocculi, Francesca Pat-sustainable and creative approaches to food waste rignani, Urszula Tylewicz, Silvia Tappi, Rosalba Lanciotti, reduction. Marco Dalla Rosa, Lucia Vannini University of Bologna, Italy PO-28 The effects of pretreatment of Clementina peel, Study on the functional properties of starch inoculated with a yeast strain as starter for its ferregulated by pulsed electric field assisted es- mentation, with pulsed electric field (PEF) at 0.75 terification to 1 kV/cm on sucrose content, antioxidant activity boru Chen and yeast growth during fermentation were studied. School of Food Science and Engineering, China The results showed that permeabilization of Clem- Starch plays an important role in human life, it entina peel by PEF treatment at 22°C caused an is the main source of human energy and one of the increase in sucrose content and antioxidant activ- important industrial raw materials. However, due to ity compared with the control during the incuba- some undesirable characteristics of natural starch, tion period. Obtained results showed that the pre- such as high pasting temperature, poor emulsific- vious trituration of Clementina peel promoted the ation, easy retrogradation, and high glycemic in- highest conductivity when the electric field strength dex, the application of its original form in the food increased from 0.75 to 1.25 kV/cm. Treatments at 1 industry is subject to many restrictions. The func- kV/cm resulted in an immediate reduction of yeast tional properties of starch can be improved by phys- viability which however recovered over 3 days fer- ical, chemical or enzymatic modification to improve mentation attaining final cell loads similar (around the processing performance of starch. Octenyl suc- 6 Log CFU/g) to the control and PEF-treated ones cinic anhydride modified starches (OSA starch) is at 0.25-0.75 kV/cm. During fermentation total obtained by introducing OSA groups to the starch polyphenol, soluble solids (°Brix) and β-carotene molecular chain. It exhibits excellent pasting, emul-content significantly (p≤0.05) increased when PEF sification, water retention and anti-digestion prop- treatment was applied at 1 kV/cm after 1 hour erties, thus receiving wide attention from the starch of treatment. However, the DPPH index signific-further processing industry. At present, in prac- antly (p≤0.05) increased after 1 hour of PEF treat- tical applications, the traditional aqueous phase ment at 0.75 kV/cm. Further increase in electric method is usually used to produce octenyl succin- field strength did not significantly (p≥0.05) modify ate starch esters. However, the semi-rigid struc- this value. Thermograms obtained by using iso- ture of starch is difficult to be destroyed, which limits thermal calorimeter (TAM-Air) were analyzed ac-the attack of chemical/enzyme reagents on starch cording to the modified Gompertz equation to ob- molecular chains. Therefore, traditional aqueous tain the growth parameters (lag phase ( λ); max-phase method has the limitations of high reagent imum growth rate ( µ max) and maximum density consumption and high energy consumption. This (Amax) of the cells). The predicted curves well made an urgent requirement of development of fitted experimental points (R2=0.996). Increasing rapid, efficient and economical modification tech- PEF intensity, the lag phase decreased by about 254 niques. The pulsed electric fields (PEF) treat- properties. The measurement of the electrical im- ment can destroy starch structure and promote pedance of cells suspended in a liquid medium the chemical reaction time. At present, the effect before and after electroporation has been widely of PEF-assisted esterification on starch functional used for decades, offering the possibility to perform properties has rarely been reported, and its mech-label-free, rapid, real-time, and non-invasive as- anism is still unclear. Based on this, this study took sessment of electroporation efficacy. However, the green and renewable starch as the research ob- methodology for this technique is still considered ject, constructed a processing technology based on inadequate in predicting the dynamics of cell mem- PEF physical field to enhance the efficient modi- brane permeabilization and requires more research fication of starch. The study explores the mech- to improve its specificity for broader application in anism of PEF-assisted esterification in regulating the food and biotechnology industries. This study starch functionality under varying electric field in- aimed to improve the assessment of gram-positive tensities: (1) Low-intensity PEF (1.5-3 kV/cm) en- and gram-negative bacteria membrane permeabil- hances starch crystallinity, increases OSA grafting ization induced by pulsed electric fields by employ- degree, and improves enzyme resistance, leading ing the electrochemical admittance spectroscopy to reduced starch digestibility. The increased ra- (EAS) method. tio of short to long amylopectin branches inhibits Bacterial cells (E. coli and L. d. bulgaricus) cul- starch retrogradation, improving freeze-thaw sta- tivated in the exponential growth phase were sus- bility. (2) Medium-intensity PEF (3-4.5 kV/cm) dis- pended in deionized water (1.4µS/cm; pH - 6.5) rupts starch crystal structure, promoting OSA graft- to achieve a concentration of 10 OD and trans- ing on starch particle surfaces, enhancing inter- ferred into a 1 mm standard electroporation cu- facial activity and emulsification properties. (3) vette. Sets of 10 electrical pulses with pulse dur- High-intensity PEF (5-6 kV/cm) severely disrupts ations of 10 or 100 µs and pulsed electric field or even dissociates starch crystal structure, further strength ranging from 2.0 to 24 kV/cm were ap-weakening intermolecular bonds, improving water plied to the samples of 100 µl. EAS measurements accessibility, and lowering gelatinization temperat- were performed in a separate 1 mm electropora- ure.This research not only provides a novel meth- tion cuvette to ensure the accuracy of the results 10 odology for starch modification but also offers in- seconds after PEF treatment. The preassessment sights into the mechanism of PEF-assisted ester- of the release of metal ions from the aluminium ification, paving the way for the development of electrodes of the electroporation cuvette into the high-performance starch materials in the food and bacterial sample and temperature rise due to the industrial sectors. ohmic heating has not revealed any significant ef- fect on the admittance magnitude and phase angle PO-29 shift on the measurement results. Detection and differentiation of bacteria per- We found that L. d. bulgaricus cells demonstrated a meabilization induced by pulsed electric fields more significant increase in admittance magnitude (PEF) using electrochemical admittance spec- and a larger reduction in admittance phase after the troscopy (EAS) application of PEF strengths from 2 to 24 kV/cm Mindaugas Visockis, Paulius Ruzgys, Simona compared to E. coli, suggesting a higher suscept- Gelažunaitė, Aras Rafanavičius, Saulius Šatkauskas ibility to PEF. In contrast, results from metabolic Vytautas Magnus University, Lithuania activity, colony-forming units, and changes in op- The application of pulsed electric fields (PEF) tical density dynamics showed higher inhibition of on bacterial cells in suspension results in increased bacteria growth rate for E. coli cells than L. d. bul-cell membrane permeability and a change in the garicus, regardless of PEF strength. However, we admittance or impedance magnitude of the sample, determined that the specificity of EAS in evaluat- depending on the pulsing parameters and medium ing cell membrane permeabilization degree is sig- nificantly higher and correlates with results from 255 other methods when comparing differences in ad-PO-31 mittance and phase shift magnitude instead of ab- Impact of Pulsed Electric Field Pretreatment on solute values. the Functional and Structural Characteristics of Rapeseed Protein isolate from Rapeseed Cake PO-30 Busra Oktar1, Ana Cristina De Aguiar Saldanha Extraction intensification of caffeoylquinic Pinheiro 1, Silvia Tappi1, Urszula Tylewicz1, Germana acids from Forced Chicory Roots by pulsed Barbieri2, Andrea Brutti2, Pietro Rocculi1, Marco Dalla electrical field Rosa1 Etienne Diemer 1, Morad Chadni2, Irina Ioannou2, 1University of Bologna, Italy Nabil Grimi1 2Stazione Sperimentale per l’Industria delle Conserve Al-1Université Technologique de Compiègne , France imentari, Italy 2Urd abi, France The by-product of rapeseed after oil extraction, The aim of this study was to develop a new rapeseed meal, contains about 35–40% protein, method for extracting caffeoylquinic acids (CQAs) mainly consisting of storage proteins such as napin from forced chicory roots, which are a low-value by- and cruciferin. Rapeseed protein isolate (RPI) can product of Belgian endive cultivation. The extrac- be considered for various markets and applica- tion process was designed to be both environment- tions, e.g., for the fortification of dairy products and ally friendly and cost-effective. The two primary cereals, for the enrichment of fruit and vegetable CQAs under investigation are chlorogenic acid (5- juices and salad dressings. Its multifunctionality CQA) and dicaffeoylquinic acids (diCQAs), known not only expands product formulations, but also for their valuable biological activities such as anti-meets the growing demand for plant-based protein oxidant, antibacterial, anti-inflammatory, and anti- alternatives. As consumer interest in a plant- UV properties. However, the challenge lies in the based diet continues to grow, RPI is proving to low concentrations of these compounds in plants, be a sustainable and environmentally conscious making biomass valorization difficult. The primary choice and promises to fill a significant niche in the objective of this study was to produce a highly con-global protein market. Therefore, RPI is poised centrated solution of 5-CQA and diCQAs for sub- for broad application across multiple industries sequent purification and functionalization of the de- to drive innovation and meet the evolving needs sired products. of health-conscious consumers worldwide. The Innovative technologies, such as pulsed electrical aim of this study was to investigate the effects field (PEF), known to enhance extraction of poly- of pulsed electric field (PEF) pretreatment on the phenols have been investigated as pretreatments extraction yield of rapeseed protein isolates by to enhance the extraction of CQAs by forced evaluating their physicochemical and structural chicory roots pressing. The electrical-field strength, properties. Rapeseed cake was treated under temperature, and energy consumption were optim- three different conditions: PEF1 (1.7 kV/cm and 28 ized. Subsequently, the effect of PEF application kJ/kg at pH=11.5), PEF2 (1.7 kV/cm and 24 kJ/kg on the extraction yield of 5-CQA and diCQAs, com- at pH=7.0) and PEF3 (1.7 kV/cm and 40 kJ/kg at pared to conventional extraction, was conducted. pH=11.5). After PEF treatment, protein isolation Furthermore, the degradation of CQAs due to oxid- was performed using an alkaline extraction method ation was investigated and successfully prevented (pH 11.5), and subsequent protein recovery was using a tailor-made solution. The results demon- performed by isoelectric precipitation (pH 4.5). strated that optimized pressing with PEF led to The RPI was analyzed for water holding capacity, a four-fold increase in the CQAs content. Addi- oil holding capacity, Fourier transform infrared tionally, a recovery rate of 70-80% for the CQAs (FT-IR), protein solubility at different pH values, present in the biomass was attained. emulsifying activity and emulsifying stability. Ac- cording to FT-IR analysis, the samples pretreated 256 with PEF1 and PEF3 showed a very different apple juice and saline solution, respectively. Mor-protein structure, such as alfa-helix, beta-turn, phological analyses and intracellular substance re- beta-sheet based on the first amide region. In lease studies indicated severe structural damage addition, the results of solubility analysis showed to both vegetative cells and spores, with a notable that the solubility of the protein at pH 4 improved disruption in spore cortex and membrane integ- in the sample pretreated with PEF1. The results rity. Further investigations into the effects of culture of the study suggest that the PEF technology is temperature on microbial resistance showed that promising for the reuse of rapeseed waste. Nev- cells cultured at lower temperatures (25°C) exhib- ertheless, optimization of PEF pretreatment and ited reduced resistance to DBD-ACP. This suscept- extraction conditions is essential for generating ibility correlated with changes in fatty acid composi-protein isolates with crucial functional properties tion, notably a decrease in cyclohexaneundecanoic for the food industry. acid and an increase in more rigid fatty acids like cyclopentaneundecanoic acid, palmitic acid, and This research was funded by ProMixQual project stearic acid, as confirmed by Fourier Transform (CUP: E97G2200062000) Emilia-Romagna ERDF Infrared Spectroscopy and Gas Chromatography- Regional Program (ERDF RP) 2021-2027. Mass Spectrometry analyses. Proteomic analyses provided deeper insights, revealing that spores cul- PO-32 tivated at 25°C had diminished expression of pro- Inactivation of Alicyclobacillus acidoterrestris teins associated with sporulation, energy metabol- vegetative cells and spores induced by atmo- ism, and membrane transport, alongside a signi- spheric cold plasma: Efficacy and underlying ficant down-regulation in peptidoglycan and spore memchanism coat proteins compared to those at 45°C. These Lang-Hong Wang, Xin-An Zeng protein expression profiles suggest a structurally Foshan University, China weaker spore formation at lower temperatures, en- Alicyclobacillus acidoterrestris (A. acidoter- hancing susceptibility to DBD-ACP. These results restris), a thermophilic, spore-forming bacterium, highlight DBD-ACP’s potential as a viable non- poses substantial challenges in the fruit juice in- thermal technology for reducing A. acidoterrestris dustry due to its resilience against pasteuriza- in apple juices, with implications for improving food tion, leading to product spoilage. The applica- safety protocols and extending product shelf life. tion of dielectric barrier discharge-atmospheric cold Future studies will aim to optimize DBD-ACP para-plasma (DBD-ACP) as a non-thermal alternative meters for industrial applications and explore its effor inactivating vegetative cells and spores of A. ficacy against other resistant microbial strains. acidoterrestris have been investigated. Our re- PO-33 search collectively demonstrates the potential of In vitro study of the antifungal activity of chlor-DBD-ACP to significantly reduce microbial load ide species and peroxide hydroxide generated without compromising juice quality, as a comple- during treatment with pulsed electric field - Po- ment to the traditional heat-dependent techniques. tential use as sanitizing equipment and food For example, A. acidoterrestris vegetative cells de- handling art creased by 6.63 and 6.47 log under only DBD-ACP Junior Bernardo Molina Hernandez, Giulio Gannini, treatment of 30 kV for 6 min in saline solution and Lorenzo Siroli, Silvia Tappi, Urszula Tylewicz, Marco apple juice, respectively. However, DBD-ACP was Dalla Rosa, Francesca Patrignani, Pietro Rocculi less effective against spores, resulting in 2.64 and University of Bologna, Italy 2.43 log reductions at the same treatment para- meter. Enhanced sporicidal activity was observed In systems with pulsed electric field (PEF), elec- when DBD-ACP was combined with mild heat trochemical reactions can occur in the treatment (85°C), resulting in 4.03 and 3.95 log reductions in chamber. This can lead to partial electrolysis of 257 the solutes in the solution and subsequently gen-membranes, leading to cell death. In this frame- erate long-lived oxygenic reactive species such work, the extracellular release of DNA induced by as hydrogen peroxide (H2O2) and, in the case of electroporation can be considered a marker of cell solutions containing chloride compounds, lead to damage. These results provide new guidelines for electrolytic production. In this context, the anti- the use of saline solution treated with PEF as a dis- fungal activity of PEF-treated solutions was tested infectant to sanitise food processing equipment and against Penicillium spp, an opportunistic filament- items. ous fungus that has previously been reported to cause infections in immunocompromised patients PO-34 and to proliferate well in absorbent building materi- Value-added compounds extraction from apple als and able to contaminate and deteriorate various by-products using pulsed electric fields food products, with special attention given to po- Maite Gagneten, María de los Ángeles Saucedo, Ir- ina Mailén Siniuk, Isaac A. Rodríguez Osuna, Guillermo tential mycotoxin producers. Thus, in the present R. Marshall, Carolina Schebor, Nahuel Olaiz study Penicillium spp. conidial suspensions (105 Universidad de Buenos Aires, Argentina CFU/ml) were treated with PEF at 0.5 kV/cm for 15 seconds and the effects on conidial germination Apple production is one of the main economic and membrane damage were determined. The res- activities in Argentina’s Río Negro and Neuquén ults showed that conidia germination was reduced provinces. Apples not meeting the specifica- by almost 80 % (p ≤ 0.05). To better understand tions for fresh commercialization are mainly used the antifungal effect induced by the PEF treatment, to produce juice and cider, generating significant the saline solution was characterised after treat- volumes of waste. This residual biomass, a source ment. Chloride species (CS), oxidation-reduction of bioactive compounds of industrial and nutritional potential (ORP) (mV), pH and hydrogen peroxide interest, can be recovered to obtain high-value (H2O2) were measured immediately (0h), and at products. However, these compounds are difficult 1, 2, 3, 4, and 24 h after the treatment. Surpris- to isolate being usually associated with other con- ingly, a significant increase in the concentration of stituents of the food matrix like lignin, pectin, and CS (0.17 % ± 0.02 g/100 ml solution), ORP (245 proteins. The goal of this work was to study dif- ± 0.7 mV), and H2O2 (0.5 ug/ml) were found im- ferent PEF treatments to enhance the extraction mediately after the PEF treatment and it remained of polyphenols and antioxidant activity from apple relatively constant during 24 h. These compon- peels. Samples obtained after sanitizing and peel- ents can cause oxidative stress and, thus, the loss ing Red Delicious apples were immersed in dis- of viability of the conidia. To confirm the electro- tilled water in a custom-made chamber (parallel poration of conidial cells after PEF treatment, DNA plate electrodes with a 2 mm gap) and subjected to release was simultaneously analysed using prop- different PEF treatments with electric field intens- idium iodide dyes. After 72 h, DNA leakage in the ities of 9 and 15 kV/cm, 1 Hz and various treat- PEF-treated conidia was found to be 5-fold higher ment times. During the treatments, the electric cur- in the saline solution than in the control. To check rent was recorded and the total energy per mass whether the conidia recovered after PEF treatment, unit was computed. Immediately after PEF treat- the conidia were incubated for 24 h in a saline solu- ment, the samples were subjected to a water ex- tion treated with PEF. The result showed a signific- traction to perform the analytical measurements. ant reduction (p ≤ 0.05) of 2.47 ±0.02 log compared The effect of the PEF pretreatment was studied to the control sample. We hypothesized that the in terms of Total Phenolic Content (TPC) following above-mentioned damage by electroporation was the Folin-Ciocalteu method and Antioxidant Capa- due to the accumulation of CS and H2O2 leading to city (AC) by the bleaching method of the radical the leakage of intracellular fluids through cell dam- cation ABTS+˙. A control sample prepared follow- age. CL and H2O2 can accumulate on the surface ing the same procedure but without applying the of the conidia and easily diffuse through the cell 258 PEF pretreatment was conducted. Also, since the content of apple bagasse. Apple (Malus domestica PEF treatment caused a temperature rise of up to cv Golden Delicious) bagasse was subjected to en- 29 °C, an additional control was performed, sub- zymatic hydrolysis with Viscozyme (0.05-0.5%) for jecting the samples to that temperature. The stat- 1-4 h at 50 °C and/or PEF treatment (10 pulses,2 istical analyses of the results showed that the tem- kV cm-1). Water retention capacity (WRC), oil re- perature rise caused a 35 to 40% increase in AC tention capacity (ORC), solubility, soluble uronic and TPC. For the treatment at 9 kV/cm, although acid (UA), and soluble neutral sugar concentrations increments were observed compared to the con- (NS) of apple bagasse were determined. Over- trol, no significant differences with the control at all, all treatment conditions led to increased sol- 29 °C were found, suggesting that this electric field ubility, soluble UA, and NS content. PEF raised intensity was not strong enough to induce electro- WRC (27.24 +0.86 g/g) and ORC (6.68 + 0.79), re- poration and/or release the compounds of interest garding untreated bagasse (WRC = 25.97 + 0.63; from the food matrix. In contrast, treatments of 15 ORC = 6.10 + 0.30). This may have been a res- kV/cm yielded important increases in the TPC and ult of an elevated level of exposure of both polar AC even when compared to the temperature con- and non-polar groups. The highest content of sol- trol. A PEF treatment of 15 kV/cm and 180 J/g uble UA (10312.20 + 144 ppm), soluble NS (25.90 led to 35 and 50% increments of TPC and AC re- + 3 ppm), and solubility (69.93 + 0.9%) was ob- spectively, while the biggest improvements were tained after applying PEF and 0.5% of Viscozyme reached with 15 kV/cm and 750 J/g; this enabled during 4 h. The combination of treatments could doubling the amount of extracted phenols and trip- break down cell walls, as well as different veget- licating the AC. In conclusion, the results of this able membranes and its action can cause an in- work show that PEF technology can be applied crease in soluble polysaccharides and solubility of to recover value-added compounds with antioxid- DF. These results suggested that the combination ant capacity and potential health functional prop- of Viscozyme and PEF could be a potential strategy erties from a food by-product, contributing also to to obtain natural ingredients with improved techno- advance towards a more circular economy. logical properties and high soluble DF content from plant by-products. PO-35 Modification of dietary fiber from apple bagasse PO-36 by combining pulsed electric fields and en- A study for achieving a higher effectiveness zymatic hydrolysis at less irradiation number on sterilization us- Alba Díaz Núñez, Pedro Elez-Martinez, Robert ing pulsed plasma for cut vegetables packaging Soliva-Fortuny, Olga Martín-Belloso low oxygen atmosphere Universitat de Lleida, Spain Pengcheng Cui Yamagata University, Japan Large amounts of bagasse are discarded dur- ing apple juice production. These by-products, Pulsed plasma can be produced in a bag, even rich in dietary fiber (DF) and bioactive compounds, after packaging cut vegetables, by applying pulsed hold the potential for incorporation into foods as voltage to the package, sterilize the cut vegetables natural ingredients. However, their direct incor- by generated active species, and reduce the risk poration is limited by their insoluble DF content. of bacterial contamination. Currently, when the Pre-modification of DF to improve solubility is ne- cut vegetables are packaged in the bag, in many cessary, although commonly employed chemical cases, the inside of the bag is kept hypoxic at- methods carry significant environmental implica- mosphere by replacing some oxygen gas to nitro- tions. This study aimed to investigate the combined gen gas to keep the vegetables freshness. How- effect of enzymatic treatment and pulsed electric ever, under hypoxic atmosphere, both the con- fields (PEF) on technological properties and DF centration of generated ozone and direct inactiva- 259 tion efficacy by pulsed plasma are concurrently re-study aimed to investigate the impact of pulsed duced. Though increasing the number of pulses electric field (PEF) treatment and ultrasound- improves the inactivation effect, it concurrently assisted convective drying on the course of the amplifies damage to the vegetables. drying process and the properties of dried organic Therefore, this study is making full use of the strawberries. active species generated by pulsed plasma to PEF energy with the values of 0.065, 0.433, and achieve higher inactivation effect of packaged cut 0.800 kJ/kg was used as a pretreatment and the vegetables at fewer pulsed plasma irradiation num- power of ultrasonic waves: 120, 160, 200 W during ber under the controlled atmosphere (CA) condi- convective drying (temperature 70 °C, airflow 1.2 tions. According to our previous research and ana- m/s) were used to evaluate its effects on drying lysis, if we can further use the long-lived active spe-kinetics and properties of dried organic strawber- cies (such as ozone) generated by plasma at post- ries of the Roxana variety. The scope of the work treatment, there is still space for improvement in in-included the analysis of drying kinetics, color, and activation effect. In this presentation, we provide an selected chemical properties: total polyphenols, efficient solution to take the pulse plasma steriliza-total anthocyanins, vitamin C, sugars content, the tion of packaged sliced vegetables to a new level, ability to inactivate DPPH and ABTS radicals, and especially under the condition of 10? or lower (5%) ferric-reducing power. condition. The shortest drying time was recorded for samples By the experiment, we found that applying 50 PEF0.43_US160 and PEF 0.065 (125 minutes), shots (50 shots × 2) on each side with alternating while the longest drying time was achieved for flips resulted in higher inactivation efficacy than no-PEF0.43_US120 (175 minutes). With the use of flips with same shots. This innovative approach al- higher energies and higher US powers, a material lowed us to achieve superior inactivation results in with a brighter color, lower color saturation, and normal, 10?and 5?conditions. We successfully re- a lower redness was obtained, with a smaller solved the issue about low killing rate by reduced color deviation compared to fresh tissue, which ozone generation observed in low oxygen condi- was related to better preservation of the original tions, achieving almost same inactivation efficacy characteristics of the material. Application of as the normal condition. specific parameters - PEF with the highest energy and hybrid drying with the use of the lowest power PO-37 of ultrasound, as well as those pre-treated with the Application of pulsed electric field (PEF) treat- lowest PEF energy and dried using the highest ment before ultrasound-assisted convective power of ultrasound, were characterized by a drying of organic strawberries higher content of total polyphenols and anti-radical Katarzyna Rybak, Dorota Witrowa-Rajchert, activity. However, the use of non-thermal methods Małgorzata Nowacka resulted in a decrease in the content of vitamin C. Warsaw University of Life Sciences, Poland Furthermore, the highest loss of total sugars was Drying is one of the oldest and most important observed in the material treated with PEF_0.065, processing methods in food technology, with the PEF0.043_US160, and PEF0.043_US200. primary objective of reducing moisture content in Using PEF as a pre-treatment and US during food items to enhance their shelf life. However, this the hybrid drying process led to changes in the process is known to be highly energy-consumed structure of strawberry tissue and, consequently, and could potentially compromise product quality. with appropriate process parameters, a shortened Consequently, extensive research has been con- drying time and better physicochemical properties ducted in this domain, exploring advancements of organic strawberries can be obtained. such as alternative heat sources during drying and diverse pre-treatment methodologies. Thus, the Acknowledgement: This project has received fund- ing from transnational funding bodies, partners of 260 the H2020 ERA-NETs SUSFOOD2 and CORE Or-algal suspensions were evaluated. ganic Cofunds, under the Joint SUSFOOD2/CORE The results obtained show that the induced ef- Organic Call 2019 (MILDSUSFRUIT) as well as fects of the combined treatment depend on the National Centre for Research and Development plasma voltage parameters. The application of (POLAND, decision DWM/SF-CO/31/2021). a lower plasma voltage (Vout= 130 V) along- side PEF pulse increased algal cell permeability PO-38 comparable to that of PEF treatment alone. At Unveiling the interplay between gliding arc dis- 24 h post-treatment, nucleic acid and protein charge (GAD) plasma pretreatment and pulsed release was observed at levels comparable to electric field (PEF) on Chlorella vulgaris mi- ultrasound-treated control. These observations croalgae were accompanied by a loss of cell morphology, Kamilė Jonynaitė1, Rolandas Uscila2, Mindaugas suggesting that programmed cell death may have Aikas2, Skirmantas Keršulis1, Žydrūnas Kavaliauskas2, been triggered. In contrast, increasing Vout above Liutauras Marcinauskas2, Arūnas Stirkė1, Voitech 170 V resulted in a variable response of algae Stankevic 1 to the combined treatment. While the combined 1State research institute Center for Physical Sciences treatment induced cell permeability, DNA leakage and Technology, Lithuania tended to decrease with increasing Vout. This 2Lithuanian Energy Institute, Lithuania effect was particularly pronounced after exposure The combination of plasma and PEF is of to Vout of 210-250 V in combination with PEF, interest to enhance the treatment efficacy of where DNA release was similar to that of untreated PEF-induced effects. The mechanism is proposed algae. A similar trend was observed for protein to rely on plasma-induced radicals to induce release, with a significant decrease after exposure lipid oxidation, compromising membrane integrity to high-voltage plasma and PEF. Furthermore, and increasing combined treatment efficacy over microscopic examination showed that algae, al- plasma or PEF alone. In addition, the combined though metabolically inactive, retained a structure treatment has been shown to increase intracellular similar to intact cells. In conclusion, different ROS generation, affecting cytotoxicity and cell plasma output voltages induce variable changes death pathways. However, how the combined in algal cells after combined plasma and PEF treatment affects microalgal is poorly understood. treatment. These changes affect the efficiency of Therefore, this study was designed to investigate PEF for protein extraction and alter the mechanism the effects of GAD plasma in combination with of death of C. vulgaris. PEF on Chlorella vulgaris. To achieve this, the GAD reactor was powered by an AC power supply Acknowledgement: This work was supported consisting of a 270 kHz AC generator and a high by the Research Council of Lithuania under Grant voltage transformer (1:33) . Specifically, algal P-MIP-22-257. biomass was treated with plasma using: air flow of 22.8 l/min, electrode-to-suspension distance of Poster session 30 mm, and treatment duration of 300 s. The dis- charge voltage of the GAD plasma was controlled Coffee Break and Poster by adjusting the output voltage of the AC generator Session (Vout) within the range of 50-250 V. The plasma- Sep 17, 15:20 - 16:50 treated algal suspension was then subjected to PEF treatment, with parameters including a pulse duration of 7 µ s, 1-10 exponential pulses with a frequency of 1 Hz and a resulting electric field strength of 24-25 kV/cm. Changes in suspension and cell characteristics of the untreated and treated 261 PO-39 PO-40 Minimally invasive electrochemotherapy for the Advancing Cancer Treatment: Automated Ap- treatment of hepatocellular carcinoma: single plication of Electric Pulses and Radiation Tar- centre study geting Stem Cells guides by artificial intelli- Mihajlo Djokic 1, Blaz Trotovsek1, Rok Dezman1, gence (AI) algorithm Miha Stabuc1, Maja Čemažar2, Gregor Serša2, Ben- Arianna Casciati 1, Mirella Tanori1, Francesca jamin Hadzialjevic1 Camera1, Nicolo’ Colistra1, Marco Salvatore Zappatore2, 1University Medical Centre Ljubljana, Slovenia Luciano Tarricone2, Raffaele Crusi2, Alfredo De Cillis2, 2Institute of Oncology Ljubljana, Slovenia Simona Salati3, Giacomo Perazzolo Gallo3, Caterina Merla1 Introduction: Electrochemotherapy is a safe 1ENEA, Italy and effective method for the treatment of hepato- 2University of Salento, Italy cellular carcinoma (HCC). Initially, electrochemo- 3IGEA S.p.A., Italy therapy was used for HCC in the setting of open surgery. However, in recent years, with the devel- Tumors frequently show resistance to traditional opment of newer electrodes, the minimally invas- therapies due their extreme heterogeneity. Can- ive approach (i.e. percutaneous or laparoscopic) cer Stem Cells (CSCs) play a crucial role in tumor of electrochemotherapy has also been described in maintenance and recurrence. Therefore, alternat- the literature. ive approaches, possibly targeting CSCs, are ne- Methods: We retrospectively analyzed the cessary. Among emerging therapies, high intens- medical records of all patients with HCC treated ity ultra-short pulsed electric fields (PEFs) are con-with electrochemotherapy using a minimally invas- sidered extremely promising. Our previous res- ive approach at University Medical Centre Ljubljana ults demonstrated the ability of a specific elec- between January 2018 and March 2024. Thus, we tric pulse protocol to selectively affect medullo- only included patients who were treated with either blastoma CSCs by inducing irreversible electro- a percutaneous or laparoscopic approach. poration and radio-sensitization while sparing nor- Results: We identified 7 patients with HCC mal cells. In contrast, the same PEF-exposure treated with electrochemotherapy via a minimally protocols, in a glioblastoma model, exhibited re- invasive approach (3 patients via a percutaneous versible electroporation, leading to lower levels of approach and 4 patients via a laparoscopic ap- cell death and induction of neuronal differentiation, proach). In total, electrochemotherapy with bleo-which conferred radio-resistance. mycin was performed on 8 HCCs (1 patient had two The aim of our project is to develop an automated HCCs). Electrochemotherapy was feasible in all 8 device for the application of electric pulses and sublesions and none of the patients experienced ad- sequent doses of ionizing radiation suitable for dif- verse effects. During the median follow-up period ferent types of tumors, allowing practical, efficient, of 9 months, a complete response to treatment was and personalized therapy for each type of tumor. observed in all treated lesions. Our study aims to automate the application of PEFs Conclusion: Minimally invasive electrochemo- exposure and ionizing radiation using an artificial therapy for HCC proved to be safe, feasible and intelligence (AI) algorithm. AI approach will help effective in all 7 patients. The percutaneous ap- the operator to select an optimal PEF/radiation ex- proach should be considered as the first method posure protocol to eradicate CSCs of specific tu- as it is the least invasive. However, in cases mor in treatment. Different human cancer model where percutaneous approach is contraindicated will be used to provide data to train the AI, achieve (e.g. close proximity of tumor to vital organs), the maximum therapeutic benefit and minimize side ef- laparoscopic approach should be used. fects. The “trained” AI can play a key role in auto- mated delivery system, starting by the selection of PEF parameters administer the personalized dose 262 of ionizing radiation in a precise time lapse for each delay exists when the phenomenon is minimized, specific tumor to treat. enabling treatment efficiency comparable to mono- Results of this study will provide new opportunit- phasic pulses with identical parameters. ies to concretely improve existing therapeutic op- tions and develop more targeted and effective treat- Acknowledgement: The research was funded ments for cancer. by Research Council of Lithuania, Grant Nr. S-MIP-23-124. PO-41 Optimal Interphase Delay to Mitigate Cancel- PO-42 lation Phenomenon in Bipolar Pulse Electro- Analyzing Breast Cancer Cell Electroporation: chemotherapy with Cisplatin Perspectives from Scanning Probe Microscopy Veronika Malyško-Ptašinskė 1, Aušra Nemeikaitė- Methods Čėnienė2, Eivina Radzevičiūtė-Valčiuke2, Eglė Terese Kondrotaite1, Tomas Mockaitis2, Antanas Mickevičiūtė2, Paulina Malakauskaitė2, Barbora Zinovicius2, Inga Morkvenaite-Vilkonciene 2 Lekešytė2, Vitalij Novickij1 1Vilnius Gediminas technical University, Lithuania 1Vilnius Gediminas Technical University, Lithuania 2State research institute Center for Physical Sciences 2State Research Institute Centre of Innovative Medicine, and Technology, Lithuania Lithuania Cancer cells exhibit unique metabolic char- Nanosecond bipolar pulses trigger the can- acteristics, e.g., high ROS levels, including O2-, cellation phenomenon when the second pulse OH-, and H2O2. These ROS are crucial in cellular either partially or entirely nullifies the impact of physiology; cancer cells display elevated ROS the first pulse, thereby reducing the permeabiliz- levels compared to their healthy counterparts. ation of cells’ plasma membrane and the overall This distinction is pivotal for early identification efficacy of electroporation. To address this issue, and understanding of cancer cells’ physiological creating a time delay between the positive and and pathological activities at a single-cell level. negative phases of the bipolar pulses during Scanning electrochemical microscopy (SECM) electroporation procedures has been suggested is useful for investigating biologically-modified as a potential solution, although the specific surfaces, including living cells [1]. Several modes thresholds remain unclear. Our study examines of the SECM can be applied to investigate redox the impact of various interphase delay durations processes in living cells, such as feedback, (ranging from 0ms to 95ms) on symmetric bipolar generation-collection (GC-SECM), and redox nanosecond electrochemotherapy (300- and 500 competition (RC-SECM). GC-SECM mode is the ns pulses) with cisplatin, using 10 Hz, 100 Hz, most sensitive for detecting redox species. SECM and 1 kHz repetition frequency protocols in vitro. allows the investigation of substrate topography Mouse hepatoma MH-22a cell line is used as and its local reactivity with high resolution, as well a model. We evaluate the dependence of cell as the registering of electrically active materials plasma membrane permeabilization and viability secreted by the cells. The redox activity of human on different bipolar pulsed electric field protocols myocardium-derived mesenchymal stem cells and the resultant dependence of cisplatin-based (hmMSC) was investigated by GC-SECM mode, electrochemotherapy efficacy in the context of using 2-methylnaphthalene-1,4-dione (menadione, bipolar cancellation. It was demonstrated that MD) as a redox mediator [2]. SECM measure- the cancellation phenomenon is triggered when ments showed that healthy 2D-cultivated hmMSCs symmetrical 300, 500 ns bipolar pulses of 4–13 had much higher redox potential to reduce the MD kV/cm are employed, which significantly hinders compared to that of the 2D-cultivated pathological the efficacy of permeabilization. These effects are hmMSC. In addition, investigation of 2D- and 3D- more profound when there is no delay between cultivated hmMSCs showed that 3D cell cultivation separate phases. At the same time, a threshold 263 conditions positively affected the redox potential PO-43 of dilated myocardium-derived hmMSCs and Synergistic Bipolar Irreversible Electroporation improved resistance of healthy hmMSCs to MD. (SBIRE): A Novel Approach for Effective Tumor SECM’s non-invasive nature allows for measuring Removal without Inducing Muscle Contractions membrane permeability, cell respiratory activities, Yancheng Wang, Kun qian, Qiang Yang, Yizhen Lei, and other intra- and extra-cellular processes in Shoulong Dong, Chenguo Yao cancer and other types of cells without touching Chongqing University, China them [3]. The human skeletal muscle-derived Irreversible electroporation (IRE) has emerged mesenchymal stem/stromal cells (SM-MSCs) and as a promising modality for tumor treatment, lever- their subpopulations by Alternating Current SECM aging the controlled application of electrical pulses (AC-SECM) were stimulated, and the efficiency of to induce cell death. However, the associated myogenic differentiation was evaluated. CD56(+) muscle contractions during the procedure pose showed higher redox activity than CD56(-) cells, challenges. This study introduces a novel ap- their better response to AC electric field-based proach, termed Synergistic Bipolar Irreversible stimulation. These results encouraged us to Electroporation (SBIRE), aimed at achieving tu- apply SECM for electroporation of human breast mor ablation without the undesirable side effect cancer cells. Several tests were performed, of muscle contraction. SBIRE involves the simul- including redox mediators, electric pulses, and taneous application of ns (±1600 V between elec- SECM modes. Cells’ mechanical properties were trodes with a spacing of 0.2 cm or ±8000 V between measured by an atomic force microscope (AFM), electrodes with a spacing of 1 cm, ±500 ns, “+” to and there was a good correlation between SECM “-” delay 1 µs, “-” to “+” delay 200 µs, 5 cycles, and AFM results. 1 Hz) and µs (±300 V between electrodes with a spacing of 0.2 cm or ±1500 V between electrodes Funding: This research was funded by the with a spacing of 1 cm, ±2 µs, “+” to “-” delay 2 µs, Research Council of Lithuania (LMTLT), agree- “-” to “+” delay 1000 µs, 25 cycles, 1 Hz) bipolar ment No S-MIP-22-87. electrical pulses, strategically designed to syner- gistically target tumor cells while minimizing im- 1. Petroniene, J.; Morkvenaite-vilkonciene, I.; pact on adjacent muscle tissue. The experimental Miksiunas, R.; Bironaite, D.; Ramanaviciene, A.; setup includes in vitro and in vivo studies utilizing Rucinskas, K.; Janusauskas, V.; Ramanavicius, A. tumor cells and animal models to assess the ef- Electrochim. Acta 2020, 360. ficacy of SBIRE. Preliminary results demonstrate 2. Petroniene, J.; Morkvenaite-Vilkonciene, I.; the effectiveness of SBIRE in inducing irrevers- Miksiunas, R.; Bironaite, D.; Ramanaviciene, A.; ible electroporation within the tumor microenviron- Mikoliunaite, L.; Kisieliute, A.; Rucinskas, K.; Janument, leading to cell death, and the ablation effect sauskas, V.; Plikusiene, I.; et al. Electroanalysis is better than other parameter forms (24.41±0.23 2020, 32, elan.201900723. mm3 vs 19.54±0.25 mm3, p<0.0001). Importantly, 3. Bironaite, D.; Petroniene, J.; Miksiunas, muscle contraction is significantly reduced com- R.; Zinovicius, A.; Morkvenaite-Vilkonciene, I.; pared to traditional IRE procedures, highlighting the Ramanavicius, A. Electrochim. Acta 2023, 455, potential of SBIRE to enhance patient comfort and 142389. procedural success. To examine the immune re- sponse elicited by SBIRE and its potential in mitig- ating the advancement of post-ablation melanoma, a subcutaneous B16-OVA melanoma model was employed in C57BL/6J mice. The administration of IRE effectively suppressed the progression of melanoma, resulting in tumor-free mice following a 264 subsequent injection of the therapy. Furthermore, bution of the electric field is not homogeneous and an increased number of CD8+ T and dendritic cells, leads to partial ablation. To overcome this limita- but not CD4+ T, B, or NK cells, invaded the re- tion, we propose to couple the application of an ir- gion surrounding the ablation site on the seventh reversible electric field with low concentrations of day following IRE. Tumorigenesis was inhibited in cytotoxic drugs that are internalized in the revers- the SBIRE-treated group of mice through second- ibly permeabilized cells and cause their death. ary inoculation at the distal end, indicating the exist-Specifically, cells from murine hepatocarcinomas in ence of a protective immune response. Ultimately, suspension and in spheroids were treated accord- IRE has great potential as a technique to stimulate ing to the irreversible electroporation protocol with CD8+ T cell immunity and hinder the advancement 80 pulses of 100 µs at 1 Hz with field strengths of of melanoma following ablation. The development 1000, 1500, 2000 and 2500 V/cm with or without of SBIRE represents a significant advancement in cisplatin or bleomycin. After comparing cell viabil- the field of tumor ablation, addressing a key limita- ity with or without cytotoxic drugs, our preliminary tion associated with muscle contraction during IRE. results showed that the addition of drugs increased This technique not only offers a more targeted and cell mortality when ablation is partial, i.e. at 1000 precise approach to tumor treatment but also holds and 1500 V/cm. In the 2000 and 2500 V/cm proto- promise for minimizing procedural side effects. cols, the addition of drugs had no effect, as ablation is already complete without drugs. Similar results PO-44 were observed in cells suspension and spheroid Cisplatin and bleomycin increase cell mortal- models. In addition, studies of the cell death pro- ity during partial irreversible electroporation on cess have shown that the addition of drugs does not hepatocellular carcinoma spheroids model allow an increased release of ATP or increase the Alexia de Caro 1, Nicolas Mattei1, Mathilde Poutier2, percentage of apoptotic cells. Thus, coupling IRE Marie-Pierre Rols1, Jelena Kolosnjaj-Tabi1, Muriel as an ablation method with small amounts of cyto- Golzio1 toxic drugs could enable the eradication of cells in 1Institut de Pharmacologie et de Biologie Structurale, tumor areas where irreversible electropermeabiliz-France ation occurs only partially. 2Université Claude Bernard Lyon 1, France Irreversible electroporation (IRE) is a non- PO-45 thermal method of tissue ablation in which high field Microplastic particles (MPs) delivery by electro-strength electrical pulses are applied to unwanted poration (EP) and their effects on the develop- tissue, such as cancerous tissue, to cause cell ment of breast cancer cells death through irreversible membrane permeabiliz- Katarzyna Bieżuńska-Kusiak, Agnieszka Gajewska-Naryniecka, Agnieszka Chwiłkowska, Urszula Szwedow- ation. This technique has the advantage of be- icz, Dagmara Baczyńska, Nina Rembiałkowska, Anna ing selective because, unlike other thermal ablation Szewczyk, Julita Kulbacka methods, malignant tissue is targeted with the elec- Wroclaw Medical University, Poland trodes without affecting the surrounding tissue and vessels. In the field of oncology, numerous clinical Microplastic particles (MPs) (diameter from 0.1 trials have been conducted with irreversible elec- µ m to 5 mm) pose a huge threat to the natural troporation, giving encouraging results in tumors environment. Polystyrene (PS) and polyvinyl chlor- and cancers of the liver, pancreas, kidney and pro- ide (PVC) are commonly present in microplastic state. In most cases, however, the treatment ap- particles. They get into the body through food, pears to be effective only for small tumors and at inhalation and skin. They can cause cytotoxicity, an early stage. Relapses are observed in particu- oxidative stress, metabolism disorders, DNA dam- lar because certain areas of the tumors are only re- age, inflammation, immunological reactions, and versibly permeabilized, which means that the distri- even carcinogenesis. Breast cancer is the most common form of cancer and the second leading 265 cause of death due to cancer in women. The link may contribute to the development of protocols between microplastic particles and breast cancer for further in vivo studies and, in the future, the is a topic that requires further scientific research. application of new therapeutic methods of breast Our study aimed to determine the cytotoxicity of cancer treatment. microplastic particles delivered by electroporation on viability of breast cancer cells, and the level of Financing: SUBZ.D260.24.076 stress oxidative markers. For the study, we used human breast cell lines: PO-46 MDA-MB-453 and MCF-7. A solution with poly- Enhanced Visualization and Control of Drug styrene was used as microplastic particles for Distribution in Electrochemotherapy Using In- testing. Electroporation conditions in the target docyanine Green with Bleomycin in a Murine delivery of MPs, were selected based on the 4T1 Mammary Tumor Model protocols used in electrotransfer and drug carrier Joanna Tunikowska 1, Urszula Bazylińska5, Anna Szewczyk2, Nina Rembiałkowska6, Zdzisław delivery. Single milli- and microsecond pulses with Kiełbowicz1, Justyna Mączyńska4, Vitalij Novickij2, Julita low electric field strength were applied. The impact Kulbacka6 of MPs on cell viability after applying different re- 1Wroclaw University of Environmental and Life Sciences, versible electroporation parameters was assessed Poland using two independent tests (MTT and SRB). The 2State Research Institute Centre for Innovative Medicine, phenomenon of oxidative stress was determined Lithuania by examining the degree of lipid peroxidation. 3Wroclaw University of Environmental and Life Sciences, Immunocytochemical and -fluorescent methods Poland were used to assess the expression of antioxidant 4Łukasiewicz Research Network, Poland defense markers SOD1-3, GLRX and NOS1-3. 5Wroclaw University of Science and Technology, Poland The results showed that microplastic particles 6Wroclaw Medical University, Poland (MPs) cause cytotoxicity to both cancer and nor- mal cell lines. A significant decrease in cell viability Electrochemotherapy (ECT) is a promising was observed, especially in the case of cancer cell treatment modality in veterinary that combines lines. The synergistic effect of electroporation and short electric pulses with chemotherapeutic agents MPs was a significant decrease in cell viability, to enhance drug uptake and antitumor efficacy. especially in cancer cell lines. The additional use Our study introduces a novel application of ECT of EP increased the expression of oxidative stress using a combination of indocyanine green (ICG) markers compared to the use of MPs alone during and bleomycin (BLM), validated in the 4T1 murine incubation in solution with polystyrene. After the mammary tumor model. This approach is based use of EP and MPs, an increase in the degree of on the established efficacy of ECT with bleomycin lipid peroxidation was observed in breast cancer in treating various cancers and explores the use of cell lines. ICG to monitor and control drug distribution during The investigations showed that MPs can induce treatment. inflammation and oxidative stress in tissues, which We employed the 4T1 cell line to evaluate the may be associated with an increased risk of viability post-ECT using the MTT assay at 24 developing cancer, including breast cancer. In our and 72 hours. The experimental setup included research, the best anticancer effect was observed the administration of drugs both as free agents after applying EP with MPs. The cell survival and encapsulated within bilosomes, under the rate was lower than that of the method using conditions of the ESOPE protocol. Additionally, only one (EP or incubation in MPs solution). The fluorescent microscopy was utilized to visualize proposed research enabled us to determine the the uptake dynamics of ICG and the ICG-BLM impact of microparticles on human cells in normal combination facilitated by electroporation. and cancerous breasts. The obtained results Our preliminary findings indicate that ICG does not 266 adversely affect the ECT protocols in vitro, main-pulses (5-9 kV/cm, 10ns, 200; +/- Ca2+) were taining cell viability without significant detriment. used to stimulate P388 cells. Viability both cell Importantly, our in vivo trials demonstrated that lines were checked by MTT assay. Interactions ICG can effectively aid in the real-time visualization between various cells were observed using 3D of drug distribution, ensuring precise delivery and Cell Explorer Fluo holotomographic microscope aiding in the identification of sentinel lymph nodes and a confocal microscope. Immunofluorescence during surgical interventions. was utilized to determine activation markers of the This study not only supports the safety and effic- IS (IL-10, INF-ɤ). acy of ICG-enhanced ECT in vitro and in vivo but Results: Analysis revealed changes in markers also underscores the potential of this method in expression on P388 cells after exposure to EP improving the precision of drug delivery in cancer or CaEP. Initial observations hinted at increased therapy, particularly in breast cancer models. Our cancer cells (Ct26Wt) cytotoxicity post-CaEP results advocate for further investigation into the compared to P388 cells. Microscopic visualization clinical applicability of this combined approach in revealed interactions between macrophages and managing mammary tumors with the treatment tumor cells, as well as the death of Ct26Wt cells. combining ECT with fluorescent guided surgery. However, further exploration and wider parameter testing are required. This work was supported by the Wrocław Univer- Conclusions: Immunooncology, though nascent, sity of Environmental and Life Sciences (Poland) displays promising potential in cancer therapy. as part of the research project no N090/0015/23. Continued research and technological advance- ments hold the promise of refining treatment PO-47 strategies. Immunooncology might emerge as a Exploring Immune Stimulation for Cancer pivotal approach in combating not only cancer but Treatment also various other diseases in the future. Anna Szewczyk 1, Nina Rembiałkowska1, Katarzyna Bieżuńska-Kusiak1, Vitalij Novickij2, Julita Kulbacka1 Financing: Daina 2 (UMO-2020/38/L/NZ7/00342) 1Wroclaw Medical University, Poland and Grant Nr. S-PD-24-5 2State Research Institute Centre for Innovative Medicine, Lithuania PO-48 Electrochemoterapy in personalized medi- Background: Immunooncology, an emerging cine.A predictive in vitro model for electro- field bridging medicine and cell biology, aims to chemotherapy in metastatic melanoma activate the immune system (IS) against cancer Nicolò Martinelli 1, Annj Zamuner1, Monica Dettin1, cells. Despite the immune system’s ability to re-Luigi Dall’Olmo1, Luca Menilli1, Luca Giovanni G. cognize and eliminate cancerous cells, they often Campana2, Elisabetta Sieni3, Maria Teresa Conconi1 evade detection by mimicking normal cells. This 1Padova University, Italy study focuses on utilizing electroporation (EP), 2Manchester University, United Kingdom which involves short electrical impulses to alter 3University of Insubria, Italy cell membrane structure and enhance IS response against cancer. Personalized medicine is the new frontier in the Objectives: The hypothesis posits that EP can ac- treatment of cancer. This study aims at setting up tivate surface molecules on IS cells and augment a predictive in vitro model of the clinical response the secretion of factors, thereby improving the of cancer patients to recently introduced treatments elimination of cancer cells by the immune system. (target therapy, electrochemotherapy) and to new Materials and Methods: Murine colon cancer cells potential therapies. (Ct26Wt) and murine macrophages (P388) were The necessity of personalized therapy tailored on employed in the study. Nanosecond electrical the necessities of the patients is of paramount im- 267 portance; to obtain such a degree of personaliza-fields (nsPEFs) represents an emerging area tion is fundamental to use an experimental model within electroporation (EP)-based therapies, with that resemble the characteristics of the tumor in the potential to replace conventional European vivo such as tridimensionality and cell-cell and cell-protocols for electrochemotherapy (ECT). Short- matrix interactions. One of the most common and ening pulses’ duration reduces the frequency of lethal tumor type is metastatic melanoma. This painful muscle contractions, minimizes oxidation work aims at setting up a platform for the screening damage, and enhances treatment homogeneity of novel drugs against metastatic melanoma (tar- by incorporating higher-frequency elements. geted therapy) in combination with electrochemo- Moreover, the last decade brought the discovery therapy in melanoma spheroids. During the first of the bipolar cancellation (BPC) phenomenon. It phase of the study cells from a human melanoma was defined as the ability of electrical pulses with cell line (A375) will be used to generate spher- negative polarity (↓) to reduce or even eliminate oids, a 3D culture model, or will be seeded in hya- the effects of the preceding positive polarity pulses luronic acid scaffolds and/or self-aggregating pep- (↑) treatment. However, the exact molecular tide matrices, to resemble the microenvironment of mechanism remains unknown. the tumor and to allow the tumor cells to gener- The aim of our study was to analyse the influence ate their own extracellular matrix. Once the in vitro of the bipolar (BP) PEFs on the effectiveness 3D model of tumor tissue is characterized, these of BLM-based ECT of ovarian carcinoma (OC) scaffolds will be used to test different drugs and cell lines (MDAH-2774 and SKOV-3). Cells were their dosages in matrices seeded with cells derived exposed to pulses delivered in bursts but as uni- or from melanoma patients. Patients with in-transit bipolar, symmetrical, or asymmetrical sequences melanoma metastases will be the source of cancer with a duration of 500 ns (14 kV/cm) or 50 µs (4 cells. After informed consent, cells from human in- kV/cm). The effectiveness of cell membrane per- transit nodules will be separated, sorted, and cul- meabilization was investigated using Yo-ProTM-1 tured. Electrochemotherapy will be then applied uptake analysis. Cell viability was determined by to 3D cell cultures to increase the uptake of drug MTT assay. Moreover, changes in ROS levels molecules by the cancer cells, decreasing the drug were observed through DCFDA assay and IF dosage. The effects of the treatment will be as- staining. sessed by evaluating the spheroid growth and mor- The experiments that were performed revealed phology, extracellular matrix production, gene ex- reduced membrane permeabilization after cells’ pression, expression of membrane receptors (e.g. exposure to bipolar nsPEF and increased cell MC1R) and secondary messengers (cAMP). survival. Those effects were enhanced for sym- metrical bipolar nsPEF protocol. Interestingly, PO-49 using asymmetrical BP protocol caused increased Modulating Electrochemotherapy Efficacy in ROS levels compared to symmetrical PEF. Ovarian Carcinoma with Bipolar nsPEFs: In- In conclusion, the results indicate that modulation sights into Cell Membrane Permeabilization of the BPC phenomenon and ROS level might be and Reactive Oxygen Species Levels possible, depending on the symmetry of the used Zofia Łapińska1, Vitalij Novickij2, Nina BP PEF protocol. Rembiałkowska1, Eivina Radzevičiūtė-Valčiuke2, Anna Szewczyk2, Magda Dubińska-Magiera1, Julita Financing: The research was supported by Kulbacka 1, Jolanta Saczko1, Dagmara Baczyńska1 the Statutory Subsidy Funds of the Department 1Wroclaw Medical University, Poland of Molecular and Cellular Biology (UMW) no. 2State Research Institute Centre for Innovative Medicine, SUBZ.D260.24.076. Lithuania Drug delivery (i.e., cisplatin; CDDP or bleo- mycin, BLM) through nanosecond pulsed electric 268 PO-50 a distinct downregulation of CARHSP1-calcium- Curcumin-Electroporation downregulates regulated heat stable protein 1 and HSPB1, also key heat shock and heat stable proteins in known as heat shock protein 27. The protein- Curcumin supplementation rats protein interaction shows that proteins like CAR- Praveen Sahu, Lakshya Mittal, Ignacio G. Camarillo, HSP1 that respond to calcium levels typically have Raji Sundararajan roles in various cellular processes. Calcium is a Purdue University, United States crucial second messenger in signaling pathways, and proteins that are regulated by calcium are of- Breast cancer is a complex and heterogeneous ten involved in processes such as cell growth, ap- disease. In 2024, 310,720 women will be dia- optosis, and the response to cellular stress. gnosed with breast cancer in the U.S. It is alarming Likewise, Hsp27 has been shown to modulate sig- as every two minutes, a woman is diagnosed with naling pathways that contribute to cell growth, sur- breast disease in the U.S. alone. Various factors vival, and resistance to chemotherapy. It can have been identified as potential contributors to the promote cancer cell survival against chemothera-development and progression of breast cancer, in- peutic agents by upregulating the Akt/mTOR sig- cluding genetic mutations, hormonal changes, ex- naling cascade and inactivating p53, thus inhib- posure to chemicals and drugs, as well as life- iting chemotherapy-induced apoptosis. Overex- style factors such as diet. One important aspect pression of Hsp27 in cancer cell lines is associ- of lifestyle that has been implicated in breast can- ated with poor prognosis and treatment resistance. cer is the Western diet, which is characterized by a However, EP+Cur treatment shows downregula- high intake of processed foods, saturated fats, and tion and is favorable to the treatment of breast can- sugar. cer. Overall, the EP+Cur treatment approach and The Western diet has been shown to increase the alteration of protein/gene can be explored as poten- risk of breast cancer by 14%. Furthermore, con- tial targets for breast cancer and improving patient ventional therapies, including medications, surger- outcomes. ies, and radiation treatments, have high side effects and toxicity. However, studies have demonstrated PO-51 that curcumin (Cur) possesses anti-inflammatory, Gene electrotransfer of tumor and muscle tis- antioxidant, and anticancer properties, which are sue with clinically used electric pulse paramet- inexpensive natural compounds. Similarly, our pre- ers vious in vitro studies have demonstrated that the Maša Omerzel, Simona Kranjc Brezar, Boštjan electrical pulse (EP) application can further en- Markelc, Gregor Serša, Maja Čemažar hance the effectiveness of curcumin against breast Institute of Oncology Ljubljana, Slovenia cancer cells in electrochemotherapy (ECT). How- ever, the working mechanism is not well known. Gene electrotransfer, a technology for deliver- Towards this, we present a high-throughput, label- ing genetic material into cells and tissues, strongly free quantitative proteomic study of the anticancer relies on electric pulse parameters to achieve op- effects of EP with intratumoral curcumin adminis- timal transfection efficiency. Various parameters tration (EP+100µL Cur) on induced mammary tu- such as pulse duration, amplitude, and number mors in female rats. The rats were fed either a of pulses play crucial roles in determining the ef- Western diet (W) or a Western diet supplemen- ficiency. Therefore, in the study we first tested ted with 1% curcumin (W+Cur). Here, multiple different electric pulse protocols in vitro in mur- EP (1000V/cm,100µs,8 pulses at 100ms intervals ine B16F10 melanoma, L929 fibroblast and C2C12 between pulses) were administered using needle myoblast cells. One million cells were mixed array electrodes. with 10 µg of plasmid encoding GFP and placed We identified over 1000 proteins with differential between electrodes with 2.5 mm gap. Electric expressions in the W diet and W+Cur diet. Further, pulse protocols, delivered by Genedrive electropor- 269 ator (IGEA, Carpi, Italy) were as follows; GET1: Background: Electroporation offers the possib-250 V, 100 µs, 8 pulses, 1 Hz; GET2: 300 V, ility for gene transfer or enhanced chemotherapy 100 µs, 8 pulses, 1 Hz; GET3: 300 V, 100 µs delivery. While an ultimate goal of this method is bipolar pulse, 3 pulses, 1 Hz and GET4: 300 V, its wider application in the clinic and improvement 100 µs, 8 pulses, 5 kHz. Cells were seeded into of therapeutic strategies in oncology, the in vitro 96-well plate and placed into Cytation1 microplate experimental settings are not necessarily repres- reader, where fraction of transfected cells and me- entative of in vivo electroporation. Therefore, new dian fluorescent intensity was monitored every 2 h preclinical model systems should be established. for 24 h. Further, two most clinically relevant elec- Three-dimensional (3D) bioprinting allows for tric pulse protocols (GET2 and GET4) were used much better mimicking of the tumour architecture for transfection of 20 µg of plasmid with Cliniporator and microenvironment compared to standard (IGEA) in B16F10 murine melanoma tumors and monolayer cell culturing techniques. However, muscle using electrodes with 6 mm gap. After 48 h 3D biofabrication strategies have not been im- tissue was excised, frozen sections were stained plemented in vitro electroporation experiments with Hoechst, images were captured with micro- yet. Therefore, the aim of this pilot study was to scope and analyzed. In vitro fraction of transfec- establish protocols for the successful transfer of ted cells was higher in B16F10 ( ≈ 35%), when us- chemicals into 3D biorprinted tumour models of ing GET1 or GET2 protocol, compared to L929 or colorectal cancer (CRC). C2C12 cells ( ≈ 15-20%). When using GET3 ( ≈ 5- Materials and methods: A Clinivet electroporator 10%) or GET4 ( ≈ 20%), the fraction of transfected (IGEA, Italy) and a BioX extrusion-based bioprinter cells was similar regardless of cell type. However, (Cellink, Sweden) were used to carry out the initial median fluorescence intensity was similar for all cell experiments. Caco-2 CRC cells were bioprinted at lines when using GET1, 2 or 4 protocols and signi- density of 30 million cells/ml in a collagen-based ficantly lower (compared to other pulse protocols) bioink (MatriChem, Bulgaria). Electroporation pro- in C2C12 and B16F10 cells, but not in L929 cells tocols were optimized for 1) the buffer composition when using GET3 protocol. In tumor tissue both, and 2) the duration, strength and number of pulses. GET2 and GET4, protocols resulted in similar per- Assessment of the percentage electroporated cells cent of transfected cells in B16F10 melanoma tu- was carried out with propidium iodide (PI) and mors, approximately 2%, which is in line with previ- Calcein AM staining of the 3D bioprints. ously published research. However, only the GET Results: We have managed to successfully deliver 4 pulse protocol was successful in transfecting mur- PI inside 3D bioprinted CRC tumor models with ef- ine muscle. The findings of the study are import- ficiency of approaching 80%. Cell viability was not ant, since GET4 pulses, which are routinely used significantly affected by the protocol, allowing the in clinical practice for electrochemotherapy, suc- assessment of the effect of chemotherapeutics. cessfully delivered genetic material into the tumors Conclusion: This is the first study using electropor- and muscle. This indicates that concomitant elec- ation in a 3D bioprinted tumour model of CRC. This trochemotherapy and gene electrotransfer could be opens the possibility for more predictive pre-clinical feasible, using only one electric pulse protocol. studies related both to chemoelectroporation and gene (or miRNA) transfer. PO-52 Optimisation and validation of electroporation Acknowledgement: This study is financed by protocols in 3D bioprinted tumour models of the European Union-NextGenerationEU, through colorectal cancer the National Recovery and Resilience Plan of the Yordan Sbirkov, Tsvetomira Ivanova, Milena Dragan-Republic of Bulgaria, project No. BG-RRP-2.004- ova, Iva Ilieva, Stefan Hubenov, Victoria Sarafian 0007-C01. Medical University of Plovdiv, Bulgaria 270 PO-53 untreated cells 48 hours after exposure to electric The bystander effect after electroporation with fields. Cell viability was determined 30 minutes, 48 microsecond and nanosecond pulses hours (flow cytometry), and 6 days (clonogenic as- Neringa Barauskaite-Šarkinienė 1, Ugne Borinskyte1, say) after transferring the bystander medium. Vitalij Novickij2, Saulius Šatkauskas1, Paulius Ruzgys1 A significant difference in the use of nano- and 1Vytautas Magnus University, Lithuania micro-pulses with CHO-K1, 4T1, and A-549 cell 2State Research Institute Centre for Innovative Medicine, cultures was observed when performing the clono-Lithuania genic assay. It was found that the strongest bystander effect was observed in A-549 cells. Sim- Anti-cancer treatment by electrochemotherapy ilarly to microsecond pulses, nanosecond pulses is based on the phenomenon of electroporation, contributed to the induction of the bystander effect. where tumor tissue cells are exposed to high- Contrary to that, the bystander effect was observed intensity, short external electric fields. When the in CHO-K1 and 4T1 cell cultures after microsecond transmembrane potential exceeds a critical value, electroporation but not after nanosecond electro- cytotoxic drugs (such as bleomycin) can be intro- poration. duced into the cell cytosol during electrochemo- therapy or calcium electroporation. Cell elec- PO-54 troporation can be performed using microsecond Calcium-mediated Inactivation of Drug- and nanosecond-duration electrical pulses. Con- resistant Microorganisms Using Pulsed ventional microsecond electroporation typically re- Electric Fields quires about 100 µs (intensity 1–1.6 kV/cm), nano- Gediminas Staigvila 1, Jurgita Švedienė2, Svet- second electroporation pulse duration decreases to lana Markovskaja2, Paulina Malakauskaitė3, Ver- 100–300 ns, and the amplitude of used pulses in- onika Malyško-Ptašinskė1, Jurij Novickij1, Algimantas creases to 10–19 kV/cm. It is believed that the ef-Paškevičius2, Vitalij Novickij3 fect on the cell induced by nanosecond electropor- 1Vilnius Gediminas Technical University, Lithuania ation may significantly differ from that of conven- 2Nature Research Centre, Lithuania tional microsecond electroporation. 3State Research Institute Centre for Innovative Medicine, Recently, we demonstrated that a bystander ef-Lithuania fect is observed after microsecond electric field- Ca2+ ions are universal signal mediators that induced electrochemotherapy. Cells indirectly af- regulate many cellular functions, however, recently fected by electroporation and anticancer agents in- it was discovered that when calcium is combined fluence neighboring cells (Ruzgys et al., 2021). In with pulsed electric fields it shows high applicability this study, we aimed to investigate the bystander as anticancer treatment. During the treatment effect after nanosecond electroporation and com- pulsed electric fields are used to permeabilize pare it with microsecond electroporation. cell membrane (cell electroporation) allowing high Three cell lines are used in the study: CHO-K1, A- concentrations of Ca2+ ions enter the cell resulting 549, and 4T1. The efficiency of electrotransfer is in ATP depletion and rapid cell death. In this evaluated by changing the pulse amplitude, while work, we show that calcium electroporation can cell viability and bystander effect after exposure be successfully used to kill even drug-resistant or are assessed using BLM and calcium ion electro- gram-negative bacteria, while other microorgan- transfer. During electroporation using nanosecond isms are expected to have even higher sensitivity pulses, experiments were conducted using a nano- to the treatment due to thinner cell wall and higher electroporator (developed by Prof. Vitalij Novickij susceptibility to PEF. We have characterized the of VilniusTech), and microsecond pulses were ap- effects of 7.5–20 kV/cm microsecond (1–100 µ s) plied using the Amber Charge electroporator (Amand nanosecond pulse sequences (500 ns, n = ber Charge, Lithuania). The bystander effect was 200–2000) for inactivation of S. aureus, methicillin- induced by transferring cell growth medium onto resistant S. aureus (MRSA), P. aeruginosa and 271 have shown that it is possible to ensure revers-frequency), which results in lack of cell membrane ible electroporation, but to use Ca2+ ions (2, 5 permeabilization and consequent failure of electro- mM) for induction of a significant cytotoxic effect chemotherapy in vitro. Finally, we have performed similar to the Ca2+ electrochemotherapy, which a pilot in vivo study, where we compared the effic- is used to treat cancer. It is also shown that acy of monophasic (5 kV/cm, 500 ns, n=100) and microsecond pulses are more effective than bursts biphasic sequences (5 kV/cm, 500 ns + 500 ns, n of nanosecond pulses even when the energy = 100) in the context of Ca2+ electrochemotherapy input is equivalent. Importantly, that the both the (B16 F10 cell line, C57BL/6 mice, n = 28). The data drug-resistant or gram-negative bacteria are sus- indicated, that mice treated with bipolar pulses did ceptible to the treatment, which potentially shows not exhibit prolonged survival when compared to good applicability both in food processing related untreated control (tumor-bearing mice), therefore, context and for wound sterilization procedures. It the bipolar cancellation phenomenon was also oc- may allow to partially counter the arising problem current in vivo significantly impairing electrochemo- of antibiotic resistance. therapy. At the same time, monophasic nano- second pulses were as effective as the ESOPE se- Acknowledgments: The research was suppor- quence resulting in tumor reduction following the ted by Research Council of Lithuania, Grant Nr. treatment and prolonged survival of the animals. S-MIP-23-130. PO-56 PO-55 Reversible and irreversible electroporation The Effects of Bipolar Cancellation Phe- mechanisms: an in vitro study on two pancre- nomenon on Nano-Electrochemotherapy of atic cancer cell models Melanoma Tumors Mariateresa Allocca 1, Luigi Sapio2, Anna Sannino1, Eglė Mickevičiūtė 1, Eivina Radzevičiūtė-Valčiuke1, Olga Zeni1, Maria Rosaria Scarfì1, Stefania Romeo1 Veronika Malyško-Ptašinskė2, Paulina Malakauskaitė1, 1National Council of Research, Italy Barbora Lekešytė1, Nina Rembialkowska3, Julita 2University of Campania Luigi Vanvitelli, Italy Kulbacka3, Vitalij Novickij1 1 Electroporation (EP) is the increase of per- State Research Institute Centre for Innovative Medicine, meability of plasma membranes to ions and mac-Lithuania 2 romolecules, by intense and short pulsed electric Vilnius Gediminas Technical University, Lithuania 3 fields (PEF), that induce an over-threshold increase Wroclaw Medical University, Poland of the physiological transmembrane voltage. Be- The phenomenon known as the cancellation is low a certain electric field threshold, the cell can observed when bipolar nanosecond electric field restore the plasma membrane integrity (reversible pulses are used, which results in reduced elec- EP, REP). Above the threshold, irreversible EP troporation efficiency when compared to unipolar (IRE) occurs, leading to cell death due to extensive, pulses of the same parameters. Basically, the neg-non-repairable damage. The advantages of REP, ative phase of the bipolar pulse diminishes the ef- and IRE are widely recognized in biotechnology fect of the positive phase. Our study aimed to and medicine. Both REP and IRE applications rely investigate how bipolar cancellation affects Ca2+ on pulses with duration in the microsecond to mil- electrochemotherapy and cellular response under lisecond time scale, whereas the main differences varying electric field intensities and pulse durations are in the number of applied pulses, and/or in the (3–7 kV/cm, 100, 300, and 500 ns bipolar 1 MHz electric field strength. Despite the established ap- repetition frequency pulse bursts, n = 100). As a plications, physical and biological mechanisms of reference, standard microsecond range paramet- EP have not been fully elucidated, which implies ric protocols were used (100 µs x 8 pulses). We a poor control of PEF parameters, and definition have shown that the cancellation effect is extremely of pulsing protocols by trials and errors. Indeed, strong when the pulses are closely spaced (1 MHz 272 a better insight into the role of pulse parameters 2Alzahra University, Islamic Republic of Iran in determining membrane permeabilization or cell Protein-based nano-carriers show promise in death is crucial to control and optimize therapeutic adjusting drug pharmacokinetics, addressing chal- protocols. lenges like low solubility and non-specific distri- Here we report on the preliminary results of bution of anti-cancer drugs. They offer a poten- the activities carried out in the framework of a na- tial solution for precise targeting in drug delivery, tional project (Digging into rEversible and irrevers-crucial for reducing side effects and enhancing ef- ible ElectroPoration: in vitro and in silico multiphys-fectiveness. However, further research is needed ical analyses on cEll modelS for cancer Treatment due to limited understanding and development of – DEEPEST, PRIN 2022, Italian Ministry of Uni- these carriers, highlighting the ongoing need for versity and Research) which aims to develop and innovative, safe, and cost-effective drug delivery standardize a robust and sensitive methodology for systems. This research focuses on developing a multi-physics (electromagnetic, thermal and bio- an environmentally friendly, biodegradable protein- logical), multi-level (single cells, 2D and 3D cell sys-based nanoparticle using a cost-effective method. tems) analysis of EP mechanisms, under REP and The nanoparticles are served as carriers for drugs IRE pulsing conditions, that can be tailored to dif- and natural antioxidants. Nanosecond pulsed elec- ferent cell models, and used to improve outcome tric fields (nsPEFs) and their induced shock waves and optimization of pulsing protocols. are chosen for their effectiveness in cell transfec- First experiments were carried out on PANC- tion to facilitate the cellular uptake of the nano- 1 and MIA PaCa-2 pancreatic cancer cell lines to particle/drug complexes, efficiently delivering sub- identify REP and IRE conditions to be further in- stances ranging from small molecules to large pro- vestigated for molecular characterization of the cel- teins. The physical and chemical properties of the lular stress mechanisms leading to damage and nanoparticles are analyzed using techniques such death. Cells in a low-conductivity pulsing buffer as scanning electron microscopy (SEM), ultraviolet were exposed in 4 mm EP cuvettes by means of (UV)-visible Spectroscopy, dynamic light scatter- an ELECTROcell-B15 high voltage pulse gener- ing (DLS), and x-ray diffraction (XRD). The study ator (Leroy Biotech). Long (100 µ s) voltage pulses, investigates the delivery of the nanoparticles by 1 Hz repetition rate, were applied with variable physical drug delivery methods, including nsPEFs, pulse number (1-50) and voltage-to-distance ratio shock waves, and ultrasound. The effectiveness of (1 kV/cm – 2.5 kV/cm). A flow-cytometric method these delivery methods, along with their impact on based on double-staining of samples with the fluor- cellular models when subjected to physical stimula- escent dyes calcein acetoxymethyl ester (CAM) tion, are thoroughly assessed. The findings of this and propidium iodide (PI) was used to quantify EP study have the potential to pave the way for a safer efficiency and cell death, and to identify REP and and more straightforward drug administration sys- IRE pulsing conditions. Cell viability was assessed tem, proving beneficial for healthcare practitioners by the ability of cells to grow over a 24h and 48h and the pharmaceutical sector. period by means of MTT assay. The results of the ongoing activities will be presented at the confer- PO-58 ence. The effect of pulse duration on electrostimu- lation and electroporation of excitable S-HEK PO-57 cells Delivery of Anticancer Drugs with Protein- Tina Batista Napotnik, Tina Cimperman, Lea Rems Based Nanocarriers Using Nanosecond Pulsed University of Ljubljana, Slovenia Electric Fields and Shock Waves Shirin Khakpour 1, Zahra Moosavi-Nejad2, Nushin Electroporation-based treatments have been Hosano1, Hamid Hosano1 shown to affect the function of excitable cells such 1Kumamoto University, Japan as muscle cells and neurons. Electroporation and 273 excitability both depend on induced transmem-PO-61 brane voltage (TMV). Electric pulses used in rap- Protective Effects of Iron Compounds on Con- idly developing cardiac pulsed field ablation as well trolled Membrane Damage Induced by Varied as other electroporation-based treatments of excit- Pulsed Electric Field Durations in Cardiac and able tissues are of different parameters (duration, Skeletal Myocytes amplitude, number and repetition rate). Therefore, Nina Rembiałkowska 1, Anna Szewczyk2, Katar- in the context of the treatments, it is important to un-zyna Bieżuńska-Kusiak1, Dawid Przystupski1, Eiv- derstand how pulses of different parameters affect ina Radzevičiūtė-Valčiuke2, Vitalij Novickij2, Julita permeability, excitability and function of excitable Kulbacka1 1 cells. Wroclaw Medical University, Poland 2 We studied the effect of electric pulses of differ- State Research Institute Centre for Innovative Medicine, ent durations (0.5, 1, 10 and 100 µs) on the TMV Lithuania and intracellular calcium of genetically engineered Cardiovascular diseases are one of the leading excitable S-HEK cells in vitro. We have demon- causes of human death worldwide. Iron deficiency strated in our previous study that S-HEK are a valu- is prevalent among patients with cardiovascular able minimal in vitro experimental system for study- disease and is associated with poorer clinical out- ing electroporation in excitable cells. Changes in comes. Therefore, our study aims to investigate TMV (also action potentials) were monitored op- whether iron compounds, specifically iron citrate tically with potentiometric dye ElectroFluor630 and (III) and sodium EDTA iron (III) salt, exhibit pro- fluorescence microscopy, the signal of the whole tective effects following simulated damage to rat field of view was analysed. We also monitored cardiac muscle cells (H9C2) and skeletal muscle changes in intracellular calcium using a fluores- cells (L6). The varied sequences of pulsed electric cent calcium indicator Fura-2. With pulses of all field (PEF) were employed to induce different selected durations, one or more action potentials degrees of damage to the cells (3 pulses of 0.5, were triggered in S-HEK cells when the electric field 1 and 1.5 kV/cm field intensity, from 0.1 - 10 ms strength exceeded a certain threshold. Interest- duration, 1 Hz). ingly, we found that the characteristics of the ac- Our approach involved evaluating the cellular tion potential (shape, time of occurrence) triggered bioeffects affected by PEF and iron compounds, by the threshold electric field are influenced by including proliferation (colony formation assay), the pulse duration. By increasing the electric field viability, metabolism, the expression of heat shock above the threshold, we achieved prolonged depol- proteins, cell death, and oxidative stress. Initial arization, which indicates electroporation. All the studies allowed us to determine the non-toxic con- pulses used in the study also provoked a complex centrations of iron compounds for further research response in intracellular calcium. These results (10 - 50 µM for iron citrate (III) and 0.5 - 1 µM for contribute to understanding of basic mechanisms sodium EDTA iron (III) salt). Then, the degree of how the interplay between electroporation and of protection provided by the iron compounds excitation depends on the pulse parameters. following simulated cell damage was evaluated. This stage was conducted in two variants. In the first, cells were subjected to PEF directly in the presence of the iron compounds, while in the second variant, prior to the application of electric pulses, cells were incubated for 72 hours with the iron compounds. It was observed that cells subjected to PEF directly in the presence of the iron compounds showed no differences in proliferation when compared to PEF alone. How- 274 ever, in the second variant, iron citrate exhibited small molecule electrotransfer to cells in spheroids superior protective action, indicating its potential versus those in suspension. efficacy in mitigating cellular damage. This study 2D cultures (adherent cells) of three different cell demonstrated that particularly iron citrate (III) types were selected for the experiments. Various substantially enhances cardiac or skeletal muscle parameters of electric fields were applied to exam- functioning, cell damage-dependent pattern. In ine the electrotransfer differences. Bleomycin and summary, our findings suggest that pre-incubating cisplatin were chosen as anticancer drugs for the cells with iron compounds, particularly iron citrate, evaluation of intracellular electrotransfer efficiency. may enhance their resistance to damage induced The size of electroporated spheroids was evalu- by pulsed electric fields, thereby holding promise ated daily. Additionally, propidium iodide electro- for protective or regenerative therapy for cardiac transfer was performed on cells in spheroids and or skeletal muscle injuries. cells in suspension. To evaluate electrotransfer efficiency into cells in spheroids, a methodology Financing: The research was supported by the was developed to disaggregate spheroids into in- Subvention Funds (UMW) no. SUBK.D260.23.029. dividual cells, enabling microscopic and flow cyto- metry analysis. PO-62 The results showed that the electrotransfer effi- A comparison of small molecule intracellular ciency of small molecules into cells in a suspension electrotransfer in spheroids and cell suspen- or 3D setting was significantly different. The elec- sion troporation threshold of the applied electric field re-Neringa Barauskaite-Šarkinienė 1, Simona quired to achieve electroporation was also differ- Gelažunaite1, Aras Rafanavičius1, Gabija Andreikė1, ent. Spheroids require a higher electric field to Vitalij Novickij2, Paulius Ruzgys1 achieve electroporation compared to cell suspen- 1Vutautas Magnus University, Lithuania sion. Experiments using different cell lines showed 2State Research Institute Centre for Innovative Medicine, that when anticancer drugs were used, the same Lithuania cytotoxic effect, using the same electroporation Although electroporation has been a known conditions, was achieved when the concentration technique for quite some time, ongoing research of the drugs increased at least 10-fold in 3D set- has raised new unanswered questions. One such tings. Also, as the size of the spheroids increased, question is the transfer of established electropora- stronger electric fields and higher anticancer drug tion techniques from in vitro to in vivo settings. One concentrations were needed to achieve the same of the main challenges in this transition is the in- cytotoxic effect. creased complexity of tissues that are composed of various cells, and consequently, the responses PO-63 of the particular cell in the tissue to the applied elec-Investigation of the state of cell death by ap- tric field are different compared to in vitro settings. plying pulsed electric field under ROS suppres- This gap, to some extent, can be filled by employing sion cells in 3D cultures. These 3D cell cultures can be Yasushi Minamitani, Takayoshi Kowase, Koki Saito derived from adherent cells (2D cultures) by simply Yamagata University, Japan altering the surface of the plate while utilizing the It has been believed that the application of same growth medium. There are a small num- pulsed electric field with frequency components of ber of publications utilizing spheroids for small mo- a few MHz or lower to cells makes pores in a cell lecule electrotransfer; therefore, comparison of the membrane by dielectric breakdown and cell death efficiency of electrotransfer of small molecules into through shedding of internal tissues. However, cells in suspension and in 3D settings is still miss- when we sterilized green juice with anti-oxidation ing. The primary aim of this study was to compare using Pulsed Electric Field (PEF), the sterilization 275 ratio did not improve even when the application (HPFs). Results revealed differences in HPF viab-time of the pulses was extended. From this result, ility, migration capacity, VCAM-1, phospho-histone we have considered that the electrical membrane H3, focal adhesion kinase, and vezatin expression, breakdown may generate plasma, and it gener- depending on EP parameters and melanoma cell ate Reactive Oxygen Species (ROS), and con- grade. These findings underscore the significance sequently, ROS involved in the cell death. There- of further investigations into the effects of reversible fore, we suppressed ROS generated by applying EP on tumor cell-derived EV properties. the pulsed electric field using antioxidants and in- vestigated the sterilization ratio. In this study, we PO-65 compared survival rates and ROS generation de- Electroporation induced protein elution out to gree in Saccharomyces cerevisiae with and without extracellular media and cytoplasmic membrane adding dimethyl sulfoxide (DMSO) which is a scav- blebbing enger for OH radical when a PEF with a field Salvijus Vykertas, Baltramiejus Jakštys, Saulius Šatkauskas strength of 7.5 kV/cm was applied. As a res- Vytautas Magnus University, Lithuania ult, there were few killed cells on applying pulsed electric field under suppressing generated ROS by Electroporation, a method that uses elec- adding DMSO. trical pulses to temporarily permeabilize cell mem- branes, has emerged as a useful tool in cell biology PO-64 and biotechnology. Beyond its primary applications Electroporation-generated extracellular ves- in gene delivery and cell manipulation, electropora- icles in tumor and normal cells interactions tion facilitates interesting cellular phenomena, such Anna Choromańska 1, Urszula Szwedowicz1, Anna as membrane blebbing. Such stress response, Szewczyk2, Dagmara Baczyńska1, Roksana Kruszakin3, alongside the electroporation-assisted release of Krzysztof J. Pawlik3, Julita Kulbacka1 intracellular compounds like proteins and other mo- 1Wroclaw Medical University, Poland lecules that together produce Damage-Associated 2State Research Institute Centre for Innovative Medicine, Molecular Patterns (DAMPs), offers a window into Lithuania the complex interactions within the cellular and ex- 3Polish Academy of Sciences, Poland tracellular environment. Specifically, the release of While electroporation (EP) has long been util-DAMPs into the tumour microenvironment through ized in electrochemotherapy (ECT), the dynamics electroporation might have profound implications and composition of extracellular vesicles (EVs) re- for understanding immune responses and their im- leased through reversible electroporation remain pact on cancer progression. largely unexplored. Understanding whether and The primary aim of this study is to explore the how EP parameters influence the profile of re- dynamics of electroporation-induced membrane leased EVs is crucial. This study delves into the blebbing, protein release and possible effects in impact of various EP parameters on EV release modulating the tumour microenvironment. from human melanoma cells and their subsequent Experiments were conducted with Chinese Ham- effects on normal fibroblasts. Specifically, markers ster Ovary cell lines (CHO-K1 and stable eGFP indicative of EV-mediated transformation of fibro- transfected CHO-K1). For electroporation 1HV, blasts into tumor-associated fibroblasts were ex- 5HV and 9HV 100µs length pulses were used with amined, including the expression levels of vascu- electric field strengths from 0 kV/cm to 3.6 kV/cm lar cell adhesion molecule-1 (VCAM-1), changes in 0.6 kV/cm increments. Total protein quantitat- in phosphor-histone H3 expression, cell viability, ive and qualitative measurements were performed and migration capacity. EVs isolated from two ma- by colorimetric Pierce™ BCA Protein Assay Kit lignant melanoma cell lines subjected to revers- and SDS-Page electrophoresis. Intracellular eGFP ible EP were exposed to human primary fibroblasts fluorescence measurements were made by flow- 276 cytometry and fluorescent microscopy. cesses involved in electroporation. Specifically, the Our study on electroporation-induced protein re-actin cortex has been shown to influence mem- lease reveals that protein yield depends on pulse brane permeability, pore size, and pore resealing number and intensity. A comparison showed a time after applying electric pulses [D. L. Perrier et single high-voltage (HV) pulse was less effective al., Scientific Reports, 2019]. than multiple (5 or 9) pulses, with no yield differ- Our aim is to develop synthetic cell model sys- ence between the latter, indicating a yield plateau tems based on tunable actin cortices within giant beyond certain parameters. At 3.0 kV/cm for 5 and unilamellar vesicles (GUVs) [L. Baldauf et al., Bio- 9 pulses, protein elution reached 60% of total cel- physical Journal, 2023] to elucidate the mechan- lular protein, suggesting a maximum release limit. obiological mechanisms by which the actin cortex SDS-PAGE analysis showed no qualitative differ- modulates electroporation. We have successfully ences in protein bands between treated and con- developed GUVs with a branched actin cortex, and trol samples, suggesting cell lysis isn’t a significant our imaging data shows a strong impact of the cor-factor. We also measured intracellular eGFP fluor- tex on electroporation. By creating a more realistic escence over 32 minutes following the electropora- model system that incorporates the actin cytoskel- tion, which revealed complex elution dynamics con- eton, we hope to gain deeper insights compared to trary to expected rapid action. Additionally, electrothe inherent complexities of live cells or the over- poration caused cellular blebbing, with eGFP fluor- simplified GUV models typically used in previous escence diminishing in the cell body but remain- studies. ing constant in blebs, indicating unique protein re- tention dynamics. These findings highlight the nu- PO-111 anced effects of electroporation parameters on pro- Deciphering the resealing of membranes after tein elution and distribution. a pulse using impedance measurements by nu- merical modelling PO-66 Audrey Gossard 1, Tomas Garcia2, Luis M. Mir3, An-Synthetic Cell Models to Understand the Impact nabelle Collin1, Clair Poignard1 1 of the Actin Cortex on Membrane Electropora- Univ. Bordeaux, France 2 tion Universitat Pompeu Fabra, Spain Nikki Nafar, Gijsje Koenderink 3Centre National De La Recherche Scientifique, France Delft University of Technology, Netherlands Despite numerous experimental evidences Advances in genome editing technologies re- for the increase of membrane permeability after quire a comprehensive understanding of the bio- electrical pulses, there is still a lack of knowledge physical mechanisms underlying the effective intra-about the electrical properties of the membrane in cellular delivery of molecular cargo, such as nucleic the minutes after the pulse. Bioimpedancemetry is acids or proteins. While electroporation has proven a common technique to observe these changes. to be an effective delivery approach, the underlying In this work, we focus on data obtained with a biophysical mechanisms are poorly understood [T. fast electrical impedance spectroscopy setup for Kotnik, L. Rems, M. Tarek, and D. Miklavčič, Annu rapid measurement of cell properties after electro- Rev Biophys, 2019]. This research aims to bridge poration [1]. It consists of a disk with a diameter this gap in our knowledge. of 15 mm and six microelectrodes arranged in The actin cortex, a dense network of actin fila- parallel and spiralling from the center. The micro- ments located directly beneath the plasma mem- electrodes are placed on a cell layer of C2C12 brane, plays a crucial role in maintaining the cell’s myoblasts, avoiding direct contact with the cells shape and mechanical stability. Emerging evid- thanks to microseparators. The cell monolayer is ence suggests that this actin cortex is a critical, submitted to one biphasic microsecond pulse with yet often overlooked, factor in the biophysical pro- two different voltages. The measurements consist 277 of impedance data at different frequencies and PO-67 different buffer concentrations before the pulse Electroporation in vesicles under ms-pulsed and 5 minutes after. electric field Our goal is to develop numerical strategies to Nalinikanta Behera, Rochish Thaokar decipher the dynamics of cell membranes using Indian Institute of Technology Bombay, India these experiments. We first developed a robust Under electric field, vesicles may exhibit elec- calibration strategy to deal with the system dis- troporation, a fascinating process that augments tortion and to generate true sample impedances. the permeability of the vesicle membrane. While Then, we have developed two methods to analyze the experimental studies have demonstrated tre- the data. mendous controllability over electroporation under The first is based on an equivalent circuit model various electric pulses, reported theoretical mod- and a well-designed numerical optimization pro- els largely fail to provide mechanistic insights into cedure. It provided first very encouraging results the experimental results. In the present study, we and allowed to quantify the short-term behaviour propose an improved numerical formalism that can – within a few seconds after the pulse – of the predict pore evolution and consequent pore area membrane conductance. In particular, we were under millisecond-long electric pulses. The novelty able to distinguish between the release of [KCl] of our study lies in modeling the growth of large and the dynamics of the membrane resistance [2]. pores and the current passing through them. Im- However, the equivalent circuit does not explain plementing the above, we illustrate that the nature some long-term behaviours of the membrane, of electric pulse and its strength play a vital role in such as the incomplete recovery of the membrane governing the pore evolution. The polar regions resistance within five minutes. For this reason, are observed to admit maximum pore formation, we developed a second approach based on the whereas the large pores form near the angles of simulation of the entire experimental setup using minimum pore formation. The contribution of large a partial differential equation (PDE) model. The pores towards the total pore area is significantly whole 3D geometry (a disk with a diameter of more. The effect of charging time is also analyzed, 15 mm, a thickness of 15 microns and six spiral- showing that it can largely affect pore numbers and shaped microelectrodes on the top surface) is their size. Importantly, a higher charging time can considered. Our modelling takes properly into cease pore growth, and, in some cases, even their account the passive electrodes. When cells are formation. The transients in surface area variation, present in the buffer, transmission conditions captured by our model corroborate well with earlier on the electric potentials between intracellular experiment findings. The present work serves as and extracellular domains were described. Our a precursor for modeling efficient medical proced- strategy was validated first using impedance ures in electrochemotherapy. measurements without cells. Preliminary results with cells are then compared with impedance PO-68 measurements confirming the accuracy of our Unveiling Fusion Pore Dynamics: Integrating model. Fluorescence and Electrochemical Imaging on Supported Bilayers [1] T. García-Sánchez et al. A new spiral mi- Federica Castellani 1, Weronika Tomaka2, Danicroelectrode assembly for electroporation and ella Lopera1, Nadia Prasad3, Juana Sefair1, Volker impedance measurements of adherent cell mono- Kiessling2, Manfred Lindau1 layers. Biomedical microdevices, 2014. 1University of Miami, United States [2] A. Collin et al. Deciphering immediate post- 2University of Virginia, United States pulse membrane resealing from 4–electrode 3Cornell University, United States impedance measurements by numerical modeling. The release of neurotransmitters and hormones Bioelectricity, 2023. 278 occurs from the interior of secretory vesicles stor-of the individual fusion events. The amperometric ing these molecules, to the outside of the cell via spikes indicate the time of fusion with <1ms pre- formation of a fusion pore, which traverses two cision and the location of the release event with different membranes, the vesicle membrane and ˜300 nm precision. the plasma membrane. The process of fusion We present all the steps of the approach and pre- pore formation constitutes a fundamental step liminary experiments and results of CG-supported in various biological phenomena, including not membrane fusion events, observed under the only vesicular release, but also viral entry, and TIRF microscope. The approach will help to reveal intracellular membrane trafficking. the relation between molecular events of proteins Syntaxin 1 (Stx1) and SNAP25, together with Syn- and lipids with the phases of fusion pore formation aptobrevin 2 (Syb2), form the neuronal SNARE and dilation. (Soluble NSF Attachment Receptor) complex, which drives membrane fusion events such as PO-69 synaptic or neuroendocrine dense core ves- Efficient Lipid Extraction with Underwater icle exocytosis. Stx1 and SNAP25 are plasma Pulsed Electric Discharge Shock Waves membrane-associated proteins, while Syb2 is Seyedmasih Hosseini, Md Mijanour Rahman, Nushin Hosano, Hamid Hosano a vesicle membrane-associated protein (aka Kumamoto University, Japan VAMP2). SNARE proteins are recognized as the primary constituents of the molecular fusion Due to the many hazards and inconveniences of machine, they play a crucial role in bringing the fossil fuels, the ever-growing search for a suitable vesicle and the target membranes close together, alternative has been on the rise for nearly a cen- and in the formation of fusion pores mediating the tury. Plant-based bio-fuels taken from microalga release of vesicle contents, such as catecholam- offer numerous promising advantages which in- ines. clude, but are not limited to: renewability, a neut- To understand the relation between molecular ral carbon footprint, less consumption of land, and transitions measured by fluorescence imaging less flammability. Chlorella vulgaris is a green spe- and catecholamine release measured by electro- cies of single-celled microalgae of the Chlorophyta chemical detection of individual release events, we division, which is commonly used as a food sup- form a supported bilayer on top of a four-electrode plement, and due to its fast growth rate is industri- microfabricated electrochemical detector (ECD) ally produced for lipids. The application of pulsed array patterned on a microscope coverslip. This electric discharge is proven to be an effective way bilayer includes purified recombinant SNAP25 to create a breach in extra-cellular matrices of mi- and Stx1 proteins, incorporated into the bilayer croalgae, which causes the lipid cells to float freely, from proteoliposomes, simulating in this way which in turn enables more lipids to be extracted the cell plasma membrane composition. Natural as a result. This study investigates the effects of chromaffin granules (CGs) prepared from bovine the application of nanosecond pulsed electric un- adrenal glands carrying endogenous Syb2 and derwater discharge on its potential to extract lipids labeled with a fluorescent lipid are applied to the from the C. vulgaris. For the purpose of increasing supported bilayer in the space between the ECD the shock effect created by the discharge, an un- electrodes, leading to fusion events. In this way, derwater discharge apparatus was designed and the vesicle fluorescence and its changes can be constructed. Various numbers of pulses ranging imaged in total internal reflection fluorescence from 100 to 500 pulses with different frequencies (TIRF) excitation mode while simultaneously the were applied to the algae samples. Subsequently, release of catecholamine molecules occurs. The the lipids were extracted from the samples using released catecholamine molecules diffuse to the a variation of the Bligh-Dyer method, and the res- different ECD electrodes, generating amperomet- ults were analyzed and compared with the control ric currents that depend on the time and location 279 sample, which indicated a near 50% increase in the PO-71 quantity of the extracted lipids of treated samples. Effect of electroporation in combination with in- The results clearly prove the method to be effective, organic particles used in tattoo inks profitable, energy-saving, and applicable for large- Ophelie Cordier, Emma Barrere, Nicolas Mattei, scale usage. Marie-Pierre Rols, Muriel Golzio, Jelena Kolosnjaj-Tabi Université de Toulouse, France PO-70 Electroporation is a technique that exploits elec- A novel approach for modelling membrane trical pulses to increase cell membrane permeabil- electroporation dynamics ity. In order to kill tumor cells, electroporation is Rashid Ali Faridi, Rochish Thaokar combined with low systemic or local concentrations IIT Bombay, India of anticancer agents, which thus massively penet- Electroporation of biological cells using pulse rate into permeabilized tumor cells. This modality electric field, in the last few decades, brought sig- is also known as electrochemotherapy (ECT). ECT nificant attention towards itself owing to its grow- can be used to treat several types of cancers, in- ing applications in healthcare, food technology and cluding deep-seated and superficial tumors, such environment. While technological advancements as melanoma (1). Multiple modalities are avail- in the hardware is catering to its growing applica- able to treat melanoma, including surgery, immun- tions, a parallel effort is also there at unmasking otherapy, targeted therapy, radiotherapy, but ECT the mechanism behind electroporation when ex- appears as the best treatment, with an overall re- posed with such ultrashort electric pulses. Model- sponse rate of about 80%. ling attempts including full-blown simulations were In parallel, an increasing number of individuals are being performed in the past for cells assuming opting to wear one or more tattoos for aesthetic or spherical geometry, it is well-known that cells are reparative purposes. Tattoo inks contain micro- or hardly spherical. Biological cells tends to explore nano- particles, such as titanium dioxide and iron different morphological space under different stim- oxides, which are used as colorants. In case of uli, highlighting their plasticity and the limitations of ECT treatment, the presence of these particles in such spherical studies. This study aims at decoup- the skin could alter or amplify the effects of electroling geometrical considerations from mechanistic poration (2), if a tattoo is present in the zone that is understanding of electroporation. A simple patch being electroporated. of a cell where bi-layers at both end is coupled As to the best of our knowledge, there are little via cytoplasmic resistance is studied when micro- if any reports addressing the outcomes of elec- second pulse electric field is applied. When incor- troporation in combination with tattoo-ink-derived porated with realistic values of resting membrane particles and no ESOPE guidelines, our study fo- potential, the bilayers at both ends shows different cuses on the evaluation of the biological effects early-phase and long-term electroporation dynam- of such co-exposure. One of possible outcomes ics. Number of pores formed at the hyper-polarised can be an increased local tissue heating in vicin- end is almost three times than the pores formed ity of tattoo inks, local generation of reactive oxy- at the depolarised end at longer times. It has im- gen species (ROS) or local tissue damage due to plications on the respective conductance values irreversible electroporation. Cellular models, which and other parameters. Physical insights are also involve the use of skin-derived cells: malignant presented at the early-phase dynamics of electro- melanoma (A375 cell line) and normal dermal fibro- poration at both the ends. This study holds rel- blasts, isolated from a healthy skin biopsy, are used evance when cells with more realistic features will to obtain 2D and 3D models (multicellular spher- be studied having ion channels and other protein oids and dermal sheets). Different conditions are structures. compared: presence or absence of metallic/metal oxide particles derived from tattoo ink in combin- 280 ation with different electrochemotherapy-like elec-147:108216), and do not reproduce substantial troporation parameters (8 pulses, 100 µs duration, electroporation features such as the increased per- 1 Hz pulse repetition rate), applied at varying elec- meabilization obtained by splitting in time the elec- tric field intensity). tric field exposure. Ten years ago, it was proposed In the present study, we mainly focus on local tem- a semi-mechanistic numerical model of cell mem- perature measurement upon electroporation in ab- brane electroporation in which are distinguished a sence/presence of different tattoo inks, as well as conducting state and a permeable state (J Theor cell permeabilization and cell viability assessment Biol, 2014, 360:83-94). The conducting state following propidium iodide uptake and generalized was attributed to the formation of pores (“electro- oxidative stress assessment, which is determined poration”) and, although the authors did not expli- with cell oxidation detection kit. In addition, mor- citly indicate the mechanism behind the permeable phological alterations of the tissues are assessed state, later communications by them, and in par- with different microscopy techniques (optical, elec- ticular by Prof. Lluís M. Mir, suggest that the per- tron and second harmonic generation microscop- meable state would be caused by a chemical al- ies). teration in the composition of the membrane lipids, Taken together our study will allow to determine if a peroxidation, which causes a higher permeability tattoo inks do or do not have a harmful role upon (“electro-permeabilization”). Remarkably, a recent electroporation. study on artificial membranes characterizes the im- pact of the membrane hydroperoxidation degree PO-72 on the frequency of pore opening (Proc Natl Acad A mechanistic numerical model of cell mem- Sci USA, 2023, 120(11):e2213112120). Departing brane electroporation that links electro- from these studies, we have conceived and a new poration and electro-permeabilization mechanistic numerical model of cell membrane Ting Shu, Antoni Ivorra electroporation that considers electro-poration and Universitat Pompeu Fabra, Spain electro-permeabilization as distinguished but linked Electroporation can be defined as the biophys- mechanisms. The model assumes that short life- ical phenomenon in which cell membrane per- time pores are created at a rate determined by meability increases non-selectively to ions and mo- the transmembrane voltage and the oxidative state lecules due to exposure to an electric field. The of the membrane. In turn, the oxidative state of most widely accepted electroporation mechanism the membrane increases in the presence of pores. is the formation of pores, from which the term During the delivery of the electric field, both the “electro-poration” stems. Experiments on artificial formation of pores (electro-poration) and the perox- membranes and molecular dynamics (MD) simu- idation (electro-permeabilization) contribute to the lations indeed corroborate the creation of pores in permeabilization of the membrane. After the deliv- bilipid membranes when a transmembrane voltage ery of the electric field, only oxidation contributes to is stablished. However, the experimentally and the permeabilization of the membrane. However, computationally observed pores are short-lived and oxidated areas in the membrane laterally diffuse collapse immediately after cessation of the trans- and this maximizes pores creation in subsequent membrane voltage induced by the applied field. field exposures. Eventually, the oxidative damage This disagrees with the fact that the state of is repaired, and the physiological permeability is increased permeability can last for seconds or reestablished. minutes after field exposure. In addition, numer- ical models of cell membrane electroporation that only assume the formation of pores fail to ac- curately reproduce the uptake of small molecules in diverse scenarios (Bioelectrochemistry, 2022, 281 PO-73 whereas at 0.4 mS/cm, a field strength of 5 kV/cm Electrical conductivity effect on Anisakis spp and 20 kJ/kg were required for the same level of inactivation by PEF and impact on fish quality inactivation. Vanesa Abad 1, Natalia Escursell2, Teresa Peiro2, Fish microbiota was not affected by electrical con-Adrián Ruiz1, Javier Raso1, Guillermo J. Cebrian1, Ig-ductivity. Control and PEF samples showed no dif- nacio Álvarez1 ference in microbiological counts; it was the effect 1Universidad de Zaragoza, Spain of MAP that controlled microorganism growth. Pre- 2Scanfisk Seafood S.L., Spain liminary organoleptic analysis detected no differ- Larvae of the nematode family Anisakidae can ences in quality between control and PEF samples, cause parasitic infections in humans through the regardless of conductivity. consumption of fishery products. These larvae The lethality of Anisakis spp. in hake belly and blue are widely distributed geographically, with rates of whiting increased with higher electrical conductivity parasitism close to 100% in certain fish species. of the treatment medium. PEF treatments at higher Current legislation mandates freezing fish products electrical conductivity did not affect the microbiolo-that may contain Anisakis spp. if they are to be gical quality of hake, and no differences in quality eaten raw or undercooked. However, this method between control and PEF samples were observed. affects fish quality. Recently, Pulsed Electric Fields PO-74 (PEF) technology has been shown to effectively in- The dynamics of synergetic bacteriocidic effect activate Anisakis. The electrical conductivity of the of pulsed electric fields and antibiotics treatment medium significantly influences its lethal- Simona Gelažunaite, Mindaugas Visockis, Aras ity, but there is limited information on its effect on Rafanavičius, Saulius Šatkauskas, Paulius Ruzgys Anisakis inactivation. Vutautas Magnus University, Lithuania The objective of this work was to evaluate the influ- ence of the electrical conductivity of the treatment Currently, antibiotics are the primary defense medium on Anisakis inactivation in hake bellies and against various bacteria. However, their effective- blue whiting, and to assess its effect on hake micro- ness in killing bacteria has decreased due to the biota quality during shelf life. development of antibiotic resistance. To address The lethality of Anisakis spp. to PEF technology this issue, new antibiotics are being developed, (field strength: 1.2 to 3 kV/cm, pulse width: 7 µs, and efforts are underway to reduce antibiotic con- specific energy: 20 to 40 kJ/kg, and conductivity: sumption. Overuse of antibiotics in agriculture and 0.4 to 8 mS/cm) in hake (Merluccius merluccius) medicine contributes significantly to antibiotic res- and blue whiting (Micromesistius poutassou) was istance. Consequently, reducing antibiotic usage in determined by the mobility test described by EFSA. both fields is crucial. As a result, alternative meth-Fish microbiota of control and PEF (3 kV/cm; 7 µs; ods with the potential to substitute for antibiotics 20 kJ/kg; 0.4 and 8 mS/cm) samples of hake fil- are being heavily researched. One such method lets were evaluated during shelf life, both without is electroporation, which involves applying pulsed MAP and with MAP (50% CO2 - 50% N2) at 4ºC. electric fields (PEF) to bacterial suspensions. Elec- Additionally, an expert panel from a fish company troporation effectively deactivates bacteria, redu- (Scanfisk Seafood S.L., Zaragoza, Spain) conduc- cing their numbers by 4-5 logs and making it one ted a preliminary organoleptic analysis to detect dif-of the most efficient tools for bacterial inactivation. ferences between control and PEF samples. However, PEF alone can only temporarily decrease Inactivation of Anisakis increased when PEF treat- bacterial numbers, as bacteria tend to regain their ments were applied at higher electrical conductiv- numbers after a certain period following the PEF ities in both hake and blue whiting. A PEF treat- treatment. In contrast, antibiotics work more slowly ment of 3 kV/cm, 20 kJ/kg, and 8 mS/cm inactiv- but have a longer-lasting effect as long as they re- ated 85-95% of the Anisakis larvae in both species, main in the bacterial suspension. Combining PEF 282 with antibiotics offers the possibility of rapid initial for microbial inactivation, enhances the extraction bacterial reduction by PEF treatment, followed by of valuable intracellular compounds, increases fer- sustained inhibition using. Therefore, we investig- mentation rates and yields by improving substrate ated the simultaneous use of PEF and antibiotics uptake and metabolic activity, and induces cell on both gram-positive and gram-negative bacteria autolysis. While some applications require irre- to prolong the inhibitory effect of PEF. versible electroporation of the cell membrane, oth- Two types of bacteria were used in the experi- ers benefit from a reversible effect. The reversibil- ment: the gram-positive Lactobacillus bulgaricus ity or irreversibility of electroporation depends on delbrueckii and the gram-negative Escherichia coli processing parameters such as pulse and treat- DH5 α strain. PEF was applied using a BTX T820 ment duration, and electric field intensity. Most pulse generator with amplitudes up to 24000 V/cm current research on microbial electroporation has and pulse durations between 10 and 100 µs, deliv- been conducted using pulses with durations in the ering up to 10 pulses at 1 Hz. Cells at the concen- microsecond (µsPEF) range. However, the effects tration corresponding to 10 OD were electroporated of nanosecond pulses (nsPEF), characterized by in deionized water with a conductivity of 1 µS/m. extremely short durations coupled with high field Ampicillin, neomycin, and kanamycin were used strengths, remain less understood. as antibiotics at their respective IC50 concentra- This study addresses this gap by comparing the tions. Cell deactivation was assessed using meta- effects of nsPEF using a novel high-performance, bolic activity measurements at various time points solid-state multilevel inverter, against µsPEF treat- up to 24 hours post-PEF application. Changes ments, employing Saccharomyces cerevisiae as a in OD were monitored every 10 minutes up to 10 model microorganism. hours post-PEF. In addition, treatment efficiency Saccharomyces cerevisiae SafAle™ S-04 cells was evaluated using a colony formation assay. were collected in the stationary growth phase and The results demonstrate that PEF is a rapid and suspended in McIlvaine buffer (pH 7, 1 mS/cm). effective method for bacterial inactivation. How- The yeast suspensions were subjected to PEF ever, bacterial regrowth typically begins approxim- treatments at a constant electric field strength of ately 3 hours post-electroporation. In contrast, the 30 kV/cm. Unipolar pulses varied in pulse width: full effect of antibiotics is observed approximately nanoseconds (500 ns, 100 ns, 50 ns, 10 ns) and 10 hours after incubation. Our findings suggest microseconds (1 µs), and in treatment durations (1 that simultaneous application of PEF and antibiot- µs, 5 µs, 50 µs). Reversible and irreversible elec- ics results in rapid bacterial killing by PEF, with an-troporation, as well as yeast inactivation, were eval-tibiotics preventing bacterial regrowth. uated after 30 minutes and 24 hours of incubation. Pulses applied in the range from 1 µs to 100 PO-75 ns demonstrated similar performance. Treatments Comparative Study of the Effects of Nano- with a total duration of 50 µs irreversibly electropor-second and Microsecond Pulsed Electric Fields ated more than 50% of the population. When the on Saccharomyces cerevisiae total treatment duration decreased to 5 µs, revers- Pablo Briz, Alejandro Berzosa, Javier Marín- ible electroporation predominated, being observed Sanchez, Borja López-Alonso, Cristina Calvo, Héctor in up to 50% of the population. For a total treatment Sarnago, Óscar Lucía, Javier Raso time of 1 µs, only reversible electroporation was ob- Universidad de Zaragoza, Spain served, ranging from 10% of the cells (100 ns) to Pulsed Electric Field (PEF) technology has a 40% (1 µs). Moreover, under these treatments ap- wide range of applications in food processing and plied with pulses of 100 ns or higher, irreversible biotechnology due to its ability to modify the per- electroporation aligns with microbial inactivation. meability of cell membranes through electropora- In the case of shorter pulses, irreversible elec- tion. PEF provides an efficient non-thermal method troporation was not detected with 50 ns long pulses for treatment durations of 1 and 5 µs, and with 283 pulses of 10 ns for all the treatment durations can be viewed as basis for further studies. An ad-tested. However, these treatments inactivated up apted method for potential further studies, focusing to 40% of the population, suggesting a possible in- on microbial growth, glucose depletion and ethanol tracellular effect of PEF without affecting cytoplas- production was also devised. mic membrane permeability. The findings underscore the critical role of pulse PO-117 width in PEF treatments, indicating a need for fur- Modification of corn starch using pulsed elec- ther research to fully understand its effects on electric fields: effects on composition, structure, troporation efficiency and cell viability. This study and techno-functionality lays the groundwork for optimizing PEF parameters Núria Farràs-Moragues, Saqib Gulzar, Pedro Elez- Martinez, Olga Martín-Belloso, Robert Soliva-Fortuny for various applications in the food and biotechno-University of Lleida, Spain logical industries. Background: Starch, a ubiquitous carbohydrate PO-76 is widely used in the food industry due to its film- Stimulation of Saccharomyces cerevisiae meta- forming, gelling and viscosity properties. Neverthe- bolism and growth using pulsed electric fields less, native starches suffer from limitations such as Benjamin Schmiedl slow gelatinization kinetics and low digestion rates. University of Natural Resources and Life Sciences, Aus-To overcome these limitations, structural modifica- tria tions are required, but traditional methods such as The yeast Saccharomyces cerevisiae is a heating and chemical modifications may comprom- widely used microorganism in biotechnology and ise its nutritional quality. Non-thermal techniques food industry e.g. for production of ethanol, bio- such as pulsed electric fields (PEF) offer prom- pharmaceuticals or as a leavening agent for baked ising advantages, improving energy efficiency, and goods. Sublethal treatment of Saccharomyces preserving nutritional quality compared to conven- cerevisiae with Pulsed Electric Fields (PEF) has tional methods. This work aimed to evaluate the been reported in literature as stimulating the growth effect of PEF on the physicochemical properties and metabolism of Saccharomyces cerevisiae. of corn starch through the evaluation of alterations During my master thesis the inoculum for a fer- to granule structure, molecular organization, and mentation experiment was treated by PEF with an composition. electric field strength (E) of 0.5 – 5 kV/cm and a Materials and Methods: Corn starch was suspen- specific energy input (Wspec) of 0.5 – 25 kJ/kg. ded in distilled water (5% w/v) and subjected to The effect of the PEF treatment on the CO2 produc- bipolar 20- µ s pulses at 40 Hz with electric fields tion (measured as percent CO2 in the fermentation strengths ranging from 1.25 to 8 kV/cm and treat- vessel headspace) and the microbial growth rate ment times varying from 200 to 2000 pulses. Fol- (measured as colony forming units per mL) during lowing the PEF treatments, corn starch suspen- the following fermentation was investigated. It was sions were freeze-dried and sieved. The starch possible to show an increase of the rate of CO2 powder was characterized considering microstruc- production by a factor of 1.08 for three treatments ture (scanning electron microscopy) and techno- with E = 0.5 kV/cm and Wspec = 0.5/2.5/25 kJ/kg functional properties, including swelling power, wa- respectively and by a factor of 1.05 for a one treat- ter absorption capacity (WAC), and oil absorption ment with E = 5 kV/cm and Wspec = 25 kJ/kg. The capacity (OAC). The amylose/amylopectin content microbial growth rate was increased by a factor of and syneresis of the starch gel were also evalu- up to 1.14, although the results for microbial growth ated. rate were not statistically significant. These results Results: All PEF treatments disrupted the nat-show the potential of PEF treatment of Saccharo- ive granular structure and organization in a dose- myces cerevisiae in a fermentation inoculum and dependent manner. The SEM images proved 284 that PEF induced pores in the starch granules by current study aimed to investigate and evaluate electroporation, which resulted in the leaching of the electroporation efficiency of the therapeutic amylose from the granules. Generally, the amyl- scheme - standard chemotherapeutic (5-FU) and ose content of PEF-treated starch was reduced autophagy inhibitor (chloroquine), alone and in from 25.81% (control) to as low as 21.88% apply- combination, on 3D bioprinted HCT-116 3D cells ing 1.25 kV/cm and 2000 pulses. PEF treatment under normoxic and hypoxic conditions. The study significantly increased the swelling power (from showed that when used in IC50 values in combina- 4.38 to 4.76 g water/g starch) however, the oil tion, CQ enhances the cell growth inhibitory effect retention was reduced from 1.87 to 1.77 g oil/g of 5-FU in normal oxygen levels. However, this starch. Moreover, the PEF treatment of starch additive effect is lost when cancer cell 3D-printed appears to lower the syneresis of starch gels, aggregates are treated in an extremely hypoxic plausibly by inducing more H-bonding in the amyl- state. Interestingly, electroporation reverses the ose/amylopectin network. drug delivery efficiency and even enhances cell Conclusions: PEF treatments led to significant death under hypoxia. In conclusion, our data modulation of corn starch physicochemical charac- demonstrate better drug delivery with electropora- teristics, notably enhancing WAC while decreasing tion and suggest that electrochemotherapy could OAC. These changes are attributed to structural lead to more optimal treatment of human CRC modifications, including increased porosity and mi- cancer 3D cell models. This evidence indicates croscopic pore development within starch gran- their potential implementation as a more realistic ules, along with increased H-bonding and suggest platform for studying the effect of therapeutics and the potential of PEF to modify starch for various ap- subsequent application in personalized medicine. plications, thereby contributing to advancements in development of novel starch-based ingredients. Acknowledgments: This study was supported by the Next Generation EU, through the National PO-59 Recovery and Resilience Plan of the Republic of The impact of electroporation on the thera- Bulgaria, project № BG-RRP-2.004-0007-C01. peutic efficiency of colorectal cancer 3d printed cells under hypoxia Tsvetomira Ivanova, Yordan Sbirkov, Victoria Sarafian Poster session Medical University of Plvovdiv, Bulgaria Colorectal cancer (CRC) is the third leading Coffee Break and Poster malignant pathology in the world. The poor pro- Session gnosis is due not only to the high percentage of Sep 18, 15:20 - 16:50 late diagnosed cases, but also to the resistance acquired by tumor cells to chemo- and radio- PO-77 therapy. Therefore, new therapeutic approaches Surgery and electrochemotherapy: An option are urgently required. The reduced amount of for a feline with recurring infiltrating sarcoma oxygen in the tissue microenvironment leads to Oscar Pagoto autophagy, increases the self-renewal of cells, and SAOV Argentinian Society of Veterinary Oncology, Ar- CRC progression. Hypoxia is a major constituent gentina of the tumor microenvironment and is also a proven Soft tissue sarcomas are 7-15% of skin and driver of chemotherapy resistance. 3D bioprinted subcutaneous tumors in cats. Sarcomas have a tumors closely resemble primary carcinomas mor- common mesenchymal origin and similar behavior, phologically and biochemically, with shown higher but their differentiation is difficult diagnostic. They resistance to standard chemotherapeutics. The can present a pseudo capsule or be very infiltrat- 285 ing and manifest a very aggressive local growth, al-PO-78 though those of high grade can also present meta- Electrochemotherapy in combination with sur- stasis (5-15% in felines). Surgery is the main thera- gery and radiotherapy. The role of translational peutic tool, but if the surgery is performed with a medicine narrow margin, the chances of recurrence increase Felipe H. Horacio Maglietti 1, Matias N. Tellado2, Ant-significantly. So other alternatives must be imple- onella Cilio1, Ana Campastri1, Sebastian D. Michinski2, mented: new surgery, chemotherapy, radiother-Ana Clara Acosta3, Raquel Lertora4 1 apy, cryosurgery. In the Electrochemotherapy an Fundación Barceló, Argentina 2 electric field is generated with predetermined fre- Universidad de Buenos Aires, Argentina 3 quency and time, a chemotherapeutic (Bleomycin, Hospital de Agudos JM Ramos Mejía, Argentina 4 Cisplatin) is applied locally and / or intravenously Hospital Zonal General de Agudos Petrona V. de Cord- before. The chemotherapeutic enters the cell in ero, Argentina greater percentage and induces the apoptosis. Introduction. Electrochemotherapy is a new I possible to apply it on the tumor directly or as an treatment modality for skin tumors. It consists of adjuvant after surgery, inside the surgical bed and the administration of intravenous Bleomycin in a treat possible cancer cells that could not be treated. low dose followed by the application of an electric It is very useful in those tumors that are very difficult field that forms pores in the cell membrane. This to resects or resolved with little margin or in those allows the cytotoxic potency to be increased more cases in the surgical intervention it is very aggress-than 1,000 times, with very few adverse effects. It ive. produces tumor destruction regardless of the histo- February 2019, a 6-year-old male feline with a re- logy. It is used both in human and veterinary medi- current high-grade soft-tissue sarcoma infiltrative cine with very good results. with 2 previous surgeries, measuring 5 cm x 6 cm Objective. To describe the role of electrochemo- x 4 cm located between the back of the neck and therapy as a new step for treating cancer patients the inter scapular area, showed up at the oncology combined with surgery or radiotherapy, comparing consultation. Other veterinarians suggested eu- results in human and veterinary medicine. thanasia for the rapid evolution and recurrence and Methodology. In this work, we propose the indica- the location was difficult to resolve surgically with tions for electrochemotherapy in combination with margin. But given the others alternatives; tumor re- surgery or radiotherapy. The experience both in section, electrochemotherapy and subsequent re- human and veterinary patients is compared and construction with flap technique are proposed. The presented. Ten canine and ten human patients procedure was accepted by the owner and due with skin cancer in similar locations were treated to the high recurrence rate of these types of tu- (5 with surgery and ECT and 5 with radiotherapy mors, the cat was monitored every 15 days for 3 and ECT). The treatments in vet medicine were months and then every 1 months for 6 months and performed using the EPV-200 (BIOTEX SRL, Ar- every 2 months for 12 months to evaluate the evol- gentina) electroporator, and in human medicine the ution of the surgical suture. So far 48 months have OncoPore (BIOTEX SRL, Argentina) electropor- passed since the procedure was performed and ator. In both cases intravenous bleomycin was today, January 2024 there is no evidence of tumor used, and the procedures were conducted in the recurrence and no evidence of metastasis. In con- same manner. clusion, the procedure performed was very satis- Results. Electrochemotherapy is a very useful factory, without recurrences or metastasis. treatment modality that can be used in combina- tion with surgery, as a cytoreductive procedure to allow a less aggressive surgery. It can be used as a recue procedure when a relapse in the scar is seen, avoiding a new resection. When combining 286 with radiotherapy, it can be used as a rescue tool tion pathways and adhesion dynamics. These in-in irradiated areas. However, depending on the ex- sights suggest a promising role for reversible elec- tension of the relapse, a great tissue defect can be troporation in the modulation of EV-mediated cell produced, and this must be considered when plan- communication, with significant implications for the ning the treatment. development of targeted cancer therapies. Conclusion. Electrochemotherapy is a therapeutic alternative with a high local response rate and few PO-80 adverse effects for those patients who are not can- Potential of Ultrashort Pulsed Electric Fields didates for surgery or present a relapse after sur- to Empower Traditional Cancer Treatment by gery or radiotherapy. Human and veterinary treat- Breaking Solid Tumor Barriers ment outcomes are similar, and experience from Kun Qian, Chenguo Yao both can be summed to increase the knowledge in Chongqing University, China the field. Although cancer immunotherapy is deemed to shed light on the long-lasting fight against cancers, PO-79 its efficiency on solid tumors is barely satisfact- Impact of Reversible Electroporation on Melan- ory due to the compactness blocking antibodies. oma Cell Viability and Extracellular Vesicle Pulsed electric fields has been a powerful tool for Function ablating solid tumors, and narrowing pulse dura- Urszula Szwedowicz, Anna Choromańska tion can improve the field homogeneity penetrat- Wroclaw Medical University, Poland ing into the tumor. In this study, we comprom- Our study investigates the impact of revers- ised the integrity of multicellular tumor spheroids ible electroporation on melanoma cell viability and (MTSs), downgrading their compactness with ul- growth, along with the functional properties of isol- trashort pulsed electric fields. The 3D multicellu- ated extracellular vesicles (EVs). Preliminary ex- lar tumor spheroids were cultured in 96-well micro- periments established an electrical field strength plates coated with 1.5% (w/v) agarose. After 3-5 of 800-1600 V/cm for 100 µs at 1 Hz frequency days of culture, the consistency of size of C6 and and 8 pulses (ESOPE) as an optimal range that U-87 MG spheroids was confirmed. After being ex- minimizes cell death, thereby validating the revers- posed to ultrashort pulsed electric fields, the viabilible electroporation approach. EVs from untreated ity of spheroids was measured with Calcein-AM/PI melanoma cells were observed to promote growth; fluorescent images and CellTitler luminescent as- however, their stimulatory effect was compromised say. Immunofluorescence analysis confirmed that when melanoma cells were treated with EVs from ultrashort pulsed electric fields can suppress the electroporated cells, impeding tumor development. proliferation capacity with lower Ki-67 signal. Ki-67 Significantly, this study also recovers and evalu- protein is highly expressed during he G1, S, G2, M ates medium and large EVs—often overlooked in phase of the cell division, while cell in G0 phase isolation protocols—for their potential as carriers cannot express Ki-67. Thus Ki-67 index is posit- of biomolecules essential for EV biogenesis and ively related to cancer cell division, differentiation, function. Techniques such as impedance meas-infiltration and metastasis. In addition, the cell-cell urement along with wound healing and cell adhe-junction was broken by ultrashort pulsed electric sion molecule analysis revealed a ’bystander ef- fields with lower expression of adherens junction fect’ following electroporation, which resulted in protein N-cadherin and tight junction protein ZO- decreased proliferation and migration, particularly 1. It is evidenced that the capability of ultrashort evident in the A375 melanoma cell line. Further- pulsed electric fields to downregulate the intercel- more, the application of EVs influenced intracel- lular adherence, as well as suppress the epithelial- lular junctions and the adherence of melanoma mesenchymal transition, a key process of meta- cells, potentially altering intercellular communica- stasis of cancer cells. At last, aqueous fluorescent 287 nanoparticles were applied to simulate the antican-novel pulses introduce several new parameters cer drug or therapeutic antibodies. Under the su- such as shape, balance, pulse duration, and pulse pervision of fluorescence microscopy, the degree delay. After identifying an electric field strength of nanoparticles penetrating into the spheroids was which preserved cell viability, a variety of wave- positively related to the number of ultrashort pulsed forms and delivery rates were used to transfect electric fields, marked with a higher fluorescent sig-a green fluorescent protein (GFP)-encoding plas- nal from the inner quiescent zone or a necrotic core. mid. Transfection efficiency and viability were then In conclusion, we emphasize that ultrashort pulsed evaluated using microscopy and flow cytometry. electric fields could be promising for downgrading Optimal protocols were then used to assess co- the compactness of solid tumors, being a power- transfection using a red fluorescent protein (RFP)- ful assisted therapy for the delivery of anticancer encoding plasmid in addition to GFP plasmid to en- drugs and therapeutic antibodies. sure the feasibility of simultaneously delivering multiple molecular components (DNA, RNA, and Cas PO-81 9) required for CRISPR Cas 9 editing. Novel Bipolar Pulses for Improved Co- Cell viabilities similar to current electroporation pro-transfection Outcomes: Implications for tocols, that have been identified as optimal, were CRISPR Cas 9 Delivery achieved by utilizing the novel waveforms at an Alexia Cash, Robert H. Williamson, Mike Sano electric field intensity of 1500V/cm. However, the North Carolina State University , United States novel waveforms resulted in significantly higher CRISPR Cas 9 technology has been instru- transfection rates with the best performing treat- mental in genetic research since its first implement- ments resulting in a 94-fold increase compared to ation. Though the technology continues to show transfection rates seen in the traditional protocols. significant promise, there are challenges with clin- Interestingly, while trends in transfection rates were ical implementation. Notably, simultaneously deliv-preserved during co-transfection, the overall trans- ering requisite molecular components (donor DNA, fection rates increased with an average of 79% of guide RNA, and the Cas 9 protein) across the highly transfected cells expressing both plasmids. selective cell membrane is a challenge in vivo. The novel bipolar pulses used in this study demon- Electroporation has been proposed as a potential strate significant improvement over traditional elec- solution to this challenge due to its ability to induce troporation in relation to transfection and cotrans-transient pores in the cell membrane enabling sim- fection efficiency, however, these results and the ultaneous transport of all three molecular compon- assumed non-stimulatory nature of these specific ents of CRISPR systems. bipolar waveforms requires further in vivo valida- For clinical applications, traditional electroporation tion. protocols require substantial optimization to ensure PO-82 meaningful viability in vivo and issues surround- Study on the Effect of Microsecond Pulsed ing intense muscle contractions are associated with Electric Field in Promoting Wound Healing in monopolar pulses. Bipolar pulses have shown Diabetic Mice to be less stimulatory than traditional monopolar Lei Li, Chenguo Yao pulses, indicating that these bipolar pulses may be Chongqing University, China able to overcome current issues with clinical trans- lation. However, the transfection capabilities of bi- Effective treatment of diabetic wounds, which polar pulses have yet to be fully investigated. are chronic wounds that are difficult to heal be- In this study, transfection outcomes of a novel class cause of microenvironmental factors such as in-of bipolar pulse waveforms were compared to tra- flammation, immunodeficiency, infection, and hyp- ditional electroporation protocols using 2D suspen- oxia, remains challenging worldwide. Here, we in- sion models and 3D collagen tissue mimics. These troduce an emerging physical therapy that chooses 288 microsecond pulsed electric fields ( µ sPEFs) to can activate the immunological response of the cause reversible electroporation of fibroblasts and cells. The 3D cultures realized by Hyaluronic acid selectively kill senescent cells, providing new ideas scaffold functionalized with an adhesion sequence, for diabetic wound healing.Firstly, electroporation allow the generation of the extracellular matrix, experiments were conducted on the more import- ECM, typical of the cell lines and enhance the cell- ant fibroblasts in wounds, and by regulating the cell and cell-ECM interactions. From previous stud- experimental parameters of µ sPEF, it was found ies, a different response in terms of electroporation that µ sPEF could regulate the signaling pathways intensity of the 3D and 2D cell cultures to the same within the fibroblasts, and activate or inhibit the protocol conditions was evidenced. function of the fibroblasts; then, during wound heal- Therefore, here we evaluated two breast cancer ing in diabetic mice, µ sPEF improves the inflam-cell lines (MCF7 and MDA-MB-231) and estab- matory microenvironment during the inflammatory lished and validated their growth as 3D cell cul- phase, promotes cell proliferation and differenti- ture in Hyaluronic acid scaffolds. In these exper- ation, facilitates neovascularization and improves imental condition, the cells were able to generate wound blood flow during the repair phase, and reg- ECM with collagen and demonstrated an electro- ulates collagen synthesis ability during the remod- poration enhancement. In these models, the mark- eling phase to reduce the formation of scar tissue. ers of reversible and irreversible electroporation In summary, µ sPEF can provide favorable condi-will be evaluated and the results will be compared tions for wound healing. This study confirms the with 2D growth conditions. The 3D cultures will mechanism of action of µ sPEF in promoting the be exposed to different electroporation protocols, diabetic wound process and provides a new thera- one typical of RE and IRE, in order to evaluate the peutic strategy for clinical practice. stress response through PCR analyses of cytokine and inflammatory pathways. PO-83 PEF effect on a 3D in vitro model: a breast can- PO-85 cer case Rare non-malignant, locally aggressive lesions Patrizia Lamberti 1, Donatella Fiore1, Maria Chiara of the head and neck treated by electrochemo-Proto1, Annj Zamuner2, Monica Dettin2, Elisabetta therapy Sieni3, Raji Sundararajan4, Maria Teresa Conconi2, Pat-Gabor Vass, Aurel Ottlakan, Ildiko Csanyi, Eszter rizia Gazzerro1 Baltas, Rolland Gyulai, Judit Olah, Erika Gabriella Kis 1Università di Salerno, Italy University of Szeged, Hungary 2Padova University, Italy 3 Certain benign lesions in the head and neck University of Insubria, Italy 4 area can be locally aggressive causing serious Purdue University, United States symptoms. Such non-malignant tumors are Pulsed electric fields are used to perform cell fibromatosis and multiple adnexal neoplasia membrane electroporation. They are conveniently related to Brooke-Spiegler syndrome (BSS). adopted in cancer therapy associated with chemo- Fibromatosis is a slow-growing and locally infiltrat- therapy, in order to improve the drug uptake main- ive condition caused by uncontrolled proliferation of taining the cell integrity (i.e. reversible electropor-fibrous tissue arising from deep musculoaponeur- ation RE), or alone, in order to destroy cells with otic structures, while in BSS the numerous an irreversible process (i.e. irreversible electro- cylindromas, trichoepitheliomas, spiradenomas poration IRE). The RE therapy is currently used in are at a high risk of malignant transformation. In Europe as an effective treatment of several types both diseases surgery is usually the choice of of superficially metastatic tumors like breast cancer treatment. In fibromatosis it is difficult to achieve recurrences. IRE is used in USA for cancer treat-clear resection margin due to the complex ana- ment. tomy and frequent entrapment of neurovascular Recent studies put in evidence that electric stress 289 structures in the head and neck, while in BSS the Background & Aim: Venous malformations numerous lesions limit the surgical management. (VMs) are congenital vascular anomalies with a Between 2015-2022 five BSS patients (3 fe- prevalence of 1%. More than 40% occur in head male, 2 male, mean age of 47 years), and one and neck region. Diagnostic workup consists in 37-year-old female patient with severe symptoms clinical assessment of soft, dark-red, or bluish caused by compression of a bifocal histologically masses accompanied by Doppler Ultrasound and confirmed fibromatosis in the left upper neck and Magnetic Resonance. Only symptomatic or en- in the supraclavicular region were treated in our in- larged VMs are treated. Recent application of stitute with ECT. All BSS patients had multiple ad- intralesional Bleomycin with reversible electropor- nexal neoplasia located on the head and neck re- ation (electrosclerotherapy-BEST) demonstrates gion and had previous surgeries resulting in numer- promising results in VMs’ regression. ous scars and extensive areas of alopecia. Our pa- Material & Methods: Eight adults, 7 females and 2 tient with fibromatosis after incomplete resections males (median age 39 years, range 21-78) and a of the tumor, received immunomodulatory therapy total of 12 VMs (5 mobile tongues, 2 oral pelvis, 2 (thalidomide) which was suspended because of its submandibular spaces, 2 cheeks, 1 palatine tonsil) side effects. In the case of fibromatosis due to were treated. All patients were submitted to BEST severe symptoms caused by compression from the with Cliniporator ™ according to the standard op- tumor mass, and in the cases of BSS in order to erative procedures of the ESOPE Study. provide tumor removal and disease control, ECT Results: No major complications (loss of tissue or was decided by our multidisciplinary tumor board. functional impairment) were observed. Everyone In all cases ECT was performed according to had mild swelling and pain for some days. The 4 the ESOPE criteria with standard needle elec- tongues, 1 submandibular space and 1 oral pelvis trodes, and was repeated if necessary (in one case VMs underwent complete response and patients 8, in another case 2 settings of ECT were carried are still free of disease at 11 median months of fol- out) Tumor response was evaluated according to a low up (range 33-1 months). The patient with com- Recist 3.0, and iRecist criteria. plex tongue, cheek and oral pelvis VM and the other After 6 months all treated BSS lesions one with cheek VM underwent partial response at flattened, showing partial regression; in the 3 months after BEST and the other 2 VMs (1 sub- case of fibromatosis follow up was carried out by mandibular space and 1 tonsil) remained stable MRI, which confirmed 3 months after ECT the at 15 and 6 months, respectively; but patients no clinically significant regression: tumor volume was longer had pain or swelling. decreased by 77% for the bigger lesion and by Conclusions: BEST increases cell membranes’ 45% for the smaller one. We did not experience permeability to Bleomicyn, enhancing its sclerotiz- any serious side effects. ing effect.8 It can be applied in adults and children. With the ECT treatment of those nonmalignant Results are often acquired in a single session. Mul- tumors our aim, the disease control was achieved. tiple sessions are recommended in case of wide le- These cases demonstrate, that ECT has its role in sions or symptomatic recurrences. The treatment the treatment of selected benign lesions, which are is safe, with organ and functional sparing. surgically not manageable. PO-87 PO-86 Nano-Electrochemotherapy (NEC) to enhance Bleomycin electrosclerotherapy (BEST) to head and neck cancer treatment manage head and neck venous malformations: Silvia Pisani a new therapeutic option and a case series University of Pavia, Italy Rebecca Gelli, Giulia Bertino, Marta Minuti, Marco Benazzo Electroporation (EP) is the application of a loc- IRCCS Policlinico San Matteo Foundation, Italy alized electric field able to increase the permeal- 290 ization of molecules into cell membranes inducing Lps, with 70:30 DSPC:Chol molar ratio, showed the temporary depolarization of the voltage-gated greater values of EE% (about 30%) compared to channels. Electrochemotherapy (ECT), a combin- 50:50 DSPC:Chol (about 10 %). Single cycle EP ation of EP and chemotherapy, is used as standard (Volt:160V, n° pulse:8, length:10�s, RT) performed operating procedure (SOP) for cutaneous meta- on GS loading Lps allowed to achieve a 46% GS stases, primary skin and mucosal cancers that release from liposomes. The cellular uptake of Lps are not amenable to surgery. To increase the ef- undergoing EP, was faster compared to 0V stand- ficacy of ECT treatment, the combination of EP ard conditions. with nanomedicines is starting to present a valid The preliminary results are encouraging in be- adjuvant strategy for the treatment of head and ing able to use Lps nanosystems as drug carriers neck cancers. The designed nanocarriers (e.g., to obtain better cellular penetration and on-demand liposomes-Lps) should be able to guarantee an in- drug release by exploiting electroporation stimula- tracellular drug-controlled release by application of tion. Trials are ongoing on head and neck squam-external electric field. Moreover, thanks to the sim- ous cell carcinoma to exploit the nanocarrier-EP ilarity between cellular and liposomal membranes, platform. EP could be used as external trigger to obtain sim- ultaneous and reversible electro-permeabilization PO-88 that permits: i) easier uptake of the encapsulated Characterising and enhancing immunogenic drugs by the electroporated cells and ii) enhanced cell death following reversible ion electropora- drug release into cytosol. The proof of concept to tion evaluate the Lps response when subjected to elec- Megan McAuley 1, Ciara Nulty1, Declan Soden2, Vincent Kelly1 troporation both from a morphological and func- 1Trinity College Dublin, Ireland tional (drug release) point of view. Lps were pro- 2Mirai Medical, Ireland duced using microfluidic technique platform (Pre- cision NanoSystems Inc.). DSPC:Chol 50:50 and Calcium electroporation was first proposed 70:30 molar ratio in ethanol solutions [10mM] were as an alternative to electrochemotherapy due used for Lps production. The flow rates ratio (FRR) to the known safety, lower cost, and ease of and total flow rate (TFR) were optimized using a administration of calcium ions. The mechanism Design of Experiment (DOE) approach. Charged of CaEP-induced cell death is widely accepted to lipids DOTAP (cationic lipid) or DOPS (anionic lipid) involve ATP depletion and loss of homeostasis, were added until 5% molar ratio. Electroporator leading to either apoptosis or necrosis in tumours Gendrive IGEA was used. Gentamicin sulfate (GS depending on the severity of ATP depletion (1). 1mg/mL in PBS, pH 7.4) was used as model drug However, several studies have shown the ability of for liposomes loading. CaEP to induce the release of damage-associated Morphological and dimensional characteriza- molecular patterns (DAMPs) from cancer cells, tion was performed. Encapsulation efficiency including ATP and HMGB1 (2,3). It has also been (EE%) and effect of electroporation on drug release shown that CaEP is equally effective as electro- were tested. Preliminary cell uptake on Mesen- chemotherapy to induce immunogenic cancer cell chymal Stem cells (MSCs) was performed using death and long-term immunological memory in fluorescent labeled Lps. animal models (3) and is equally effective in clinical The results showed that more suitable Lps com-settings (4). This suggests that calcium may not position is DSPC:Chol 70:30 with DOTAP. Dimen- only act as a homeostatic disruptor but also as sional range was lower than 250nm with dispersion an adjuvant to drive anti-cancer responses within values between 0.2-0.3. EP, performed at increas- electroporated tumours. ing voltage values (160V, 200V and 250V) did not This project will investigate the mechanistic path- alter the dimensions of the Lps not causing irrevers- ways by which electroporation of calcium and ible poration of the lipidic membranes. Charged 291 other ionic species function to induce tumour cell therapy but also as a biotechnological step for the death. This study aims to elucidate the cellular development of long-term transfected cell lines. pathways underpinning cell death induced by ionic In this study, we evaluated the transfection ef- electroporation, and to enhance immunogenic ficiency of reporter genes (green fluorescent cell death and systemic anti-cancer responses by protein and luciferase) in Chinese hamster ovary delivering various ionic species. (CHO-K1) and murine breast cancer (4T1) cell lines using different composition of electroporation References: buffers (a total of 8 buffers). We have compared 1. Frandsen SK, Vissing M, Gehl J. A Compre- the microsecond protocols (1.2/1.5 kV/cm × 100 hensive Review of Calcium Electroporation—A µs, n = 2–8) with high frequency (1 MHz) bursts Novel Cancer Treatment Modality. Cancers. 2020 of nanosecond pulses (4/5 kV/cm × 300 ns, n = Jan 25;12(2):290. 250, 500). It was shown that buffers highly affect 2. Frandsen SK, Gissel H, Hojman P, Tramm T, transfection efficacy as well as cell viability post- Eriksen J, Gehl J. Direct Therapeutic Applications treatment. High-frequency nanosecond protocols of Calcium Electroporation to Effectively Induce ensured a better transfection efficacy than µsPEFs Tumor Necrosis. Cancer Res. 2012;72(6):1336– regardless of the plasmid used. 41. Finally, the most prominent high-frequency sub- 3. Falk H, Forde PF, Bay ML, Mangalanathan microsecond range protocol was successfully UM, Hojman P, Soden DM, et al. Calcium elec- applied for the long-term electrotransfection of troporation induces tumor eradication, long-lasting the murine 4T1 cell line with linearized luciferase- immunity and cytokine responses in the CT26 pcDNA3 plasmid. Later, as a proof of concept, colon cancer mouse model. OncoImmunology. 4T1-Luc tumors were induced in mice (BALB/C) 2017 May 4;6(5):e1301332. and tumor luminescence was visualized in vivo. 4. Falk H, Matthiessen LW, Wooler G, Gehl J. It is concluded that high-frequency nanosecond Calcium electroporation for treatment of cutaneous range protocols are suitable for transient and metastases; a randomized double-blinded phase II long-term transfection, while the efficacy of the study, comparing the effect of calcium electropor- procedure can be also modulated by buffer com- ation with electrochemotherapy. Acta Oncol. 2018 position. Mar 4;57(3):311–9. Acknowledgement: The research was sup- PO-89 ported by Research Council of Lithuania, Grant Nr. The Effects of Buffer Composition on Gene S-PD-24-5. Electrotransfer by Nanosecond Electric Field Pulses PO-107 Eivina Radzevičiūtė-Valčiuke 1, Jovita Gečaitė1, Lentigo Maligna Melanoma and Acral Lentigin- Anna Szewczyk1, Barbora Lekešytė1, Veronika ous Melanoma Treatment with Electrochemo- Malyško-Ptašinskė2, Eglė Mickevičiūtė1, Paulina therapy Malakauskaitė1, Julita Kulbacka3, Vitalij Novickij1 Petra Rozsa 1State Research Institute Centre for Innovative Medicine, University of Szeged, Hungary Lithuania 2Vilnius Gediminas Technical University, Lithuania Introduction: lentigo maligna melanoma (LMM) 3Wroclaw Medical University, Poland and acral lentiginous melanoma (ALM) are charac- terized by their ill-shaped borders and commonly The introduction of foreign genetic material into manifest on highly visible areas such as the face cells through the pores formed due to electropor- or the soles of the patients. The challenge in ation is a technique called gene electrotransfer treating these melanomas lies in preserving func- (GET) or electrotransfection. This non-viral gene tion and achieving satisfactory aesthetic outcomes delivery method is very promising not only for gene 292 while ensuring complete surgical excision with an Lithuania appropriate safety margin. Electrochemotherapy 3VilniusTech, Lithuania (ECT) can be an efficient treatment for both LMM Electroporation is a phenomenon in which and ALM. the cellular membrane temporarily becomes per- Case presentation: two patients with histologically meable to various hydrophilic molecules, including confirmed LMM and one patients with ALM presen- pDNA. It has been shown that plasmid DNA, fol- ted at the Department of Dermatology and Allergo- lowing cell treatment with electric pulses, interacts logy in Szeged, Hungary. A 79-year old female pa-with the affected membrane and forms membrane- tient presented with LMM on the left cheek, which pDNA complexes. Subsequently, the pDNA is in- was first treated with radiotherapy resulting in par- ternalized into the cell. tial remission. Following the recommendation of Recently, electrotransfection using nanosecond our multidisciplinary tumor board, ECT was per- electric pulses was demonstrated. However, the formed, leading to a clinically complete response transfection efficiency was not as high as compared after six months. A 83-year-old female patient had to that using microsecond electric pulses. One LMM on the forehead and right upper eyelid. After reason for this is the short duration of the pulses, ECT, she was prescribed topical imiquimod and which induce limited electrophoretic forces to drag showed partial response four months later. An pDNA to the affected membranes. Several publica- 82-year-old male patient diagnosed with ALM on tions suggest that additional low-voltage pulses can the right sole underwent two rounds of ECT. Dur- increase the transfection efficiency of microsecond ing the second treatment, two biopsies were ob- pulses. Here, we employed low-voltage continuous tained, neither confirming the presence of melan- waves after the application of nanosecond pulses oma. Only minimal, local side effects were ob- to enhance the transfection efficiency of the cells. served. All patients were treated with intraven- The CHO cell line was utilized for the experiments. ous bleomycin according to the ESOPE (European A concentration of 2 million cells per milliliter was Standard Operating Procedures of Electrochemo-prepared in a laboratory-made electroporation me- therapy) criteria. dium with a conductivity of 0.1 S/m, pH of 7.1, and Conclusions: scientific literature underscores the an osmolarity of 270 mOsm. Cell electroporation complexity of managing LMM and ALM due to their was performed using a 1 mm electroporation cu- propensity for irregular borders and localization on vette. Each treated sample contained 50 µl of cell cosmetically sensitive areas. Our cases support suspension. GFP-coding plasmid DNA was used the role of ECT as a promising treatment modal- at various concentrations ranging from 50 to 300 ity for LMM and ALM. Further clinical studies and µg/ml. Nanosecond electroporation was conducted refinement of treatment protocols are warranted to using a nanosecond pulse generator developed at elucidate the optimal utilization of ECT in the man- VilniusTech University (Lithuania). Various forms of agement of these melanomas and to expand its low-voltage waves were generated using a continu- clinical applicability. ous wave generator. Transfection efficiency was PO-90 assessed using a flow cytometer (BD Accuri C6). The synergistic electrotransfection effect of We utilized nanosecond electroporation paramet- low-amplitude continuous wave application ers to achieve approximately 20 percent transfec- and nanosecond electroporation tion efficiency. When an additional low-voltage Paulius Ruzgys 1, Neringa Barauskaite-Šarkinienė1, continuous wave was applied to the electroporated Eivina Radzevičiūtė3, Saulius Šatkauskas1, Vitalij cells, the transfection efficiency increased dramat- Novickij2 ically to 60 to 70%. Furthermore, the cells did not 1Vytautas Magnus University, Lithuania experience cell death, maintaining around 90 per- 2State Research Institute Centre for Innovative Medicine, cent cell viability. The results will give insights in the effectiveness of the waveform and the duration 293 of the continuous wave applications alongside the from a Caucasian woman was seeded within the application of nanosecond electric pulses. custom-fabricated HA-EAbuK-IKVAV scaffold that simulates the three-dimensional architecture of bio- PO-91 logical tissue environments. The experimental pro- Electroporation-Enhanced Resveratrol Deliv- tocol involves the administration of Resv with the ery into 3D-Hyaluronic Acid-Peptide Scaffold application of electric pulses with parameters set Cells for Effective Triple-Negative Breast Can- at 800–1,000 V/cm and 100 µ s duration, at a fre-cer Treatments quency of 1Hz, followed by a comprehensive as- Pragatheiswar Giri 1, Praveen Sahu1, Ignacio G. sessment of cell viability and reactive oxygen spe- Camarillo1, Monica Dettin2, Annj Zamuner2, Maria cies (ROS) production. The results of this study Teresa Conconi2, Raji Sundararajan1, Elisabetta Sieni3 are compelling, revealing a cell viability, as low as 1Purdue University, United States 24% at 24 hours of treatment with EP and Resv, 2Padova University, Italy underscoring the therapeutic potential of this ap- 3University of Insubria, Italy proach. Moreover, the concomitant use of Elec- Triple-Negative Breast Cancer (TNBC) is the troporation and Resveratrol at specified concentra- most aggressive sub-set of breast cancer, that tions significantly augmented ROS generation (4x) is also metastatic. TNBC is characterized by its compared to singular treatments, indicative of a po- clinical absence of the three most common re- tent synergistic interaction that exacerbates TNBC ceptors, Estrogen, Progesterone, and Human Epi- cell mortality via enhanced ROS-mediated apop- dermal Growth Factor Receptor 2 (Her2) and thera- tosis. These robust findings underscore the poten- peutic modalities, including the prevalent chemo- tial utility of Resveratrol as an anti-TNBC agent and therapeutic agent Cisplatin. It has the worst sur-highlight the pivotal role of electroporation in aug- vival and a lower 5-year survival rate compared to menting its cellular uptake and therapeutic impact. other subtypes, such as luminal A, luminal B, and HER2-positive breast cancers. Thus, there is an PO-92 unmet need for alternative, effective and affordable The search for an optimal IRE protocol in terms therapies that are gentle on both the body and the of pulse duration considering damage due to purse. temperature effects Towards this, in this research, we explored the ef- Isaac Rodriguez1, Nahuel Olaiz1, Felipe H. Horacio Maglietti2, Ezequiel Goldberg3, Sebastian D. Michinski1, ficacy of Resveratrol (Resv), a natural polyphenolic Cecilia Suárez1, Alejandro Soba3, Guillermo R. Mar-compound that is widely available in red grapes and shall 1 other dark colored fruits and vegetables, on MDA- 1Universidad de Buenos Aires, Argentina MB-231, human, TNBC cell line. For this purpose, 2Fundación Barceló-CONICET, Argentina a 3D scaffold matrix, composed of hyaluronic acid 3Comisión Nacional de Energía Atómica, Argentina (HA) and self-assembling peptides (SAPs; specific- ally, EAbuK), which are further condensed with a Searching for an optimal dose response in Laminin-derived adhesive motif (IKVAV). The major terms of pulse number or pulse length in an advantage of 3D cell culture is that it mimics more EP-based protocol requires the measurement closely the in vivo model, meeting the 3R principles of the electroporated tissue area and threshold (Replacement, Reduction, and Refinement). Given trajectories, a major task. Recently it was shown the known limitations surrounding the bioavailabil- that these trajectories could be determined by ity of Resveratrol, this study integrates Electropor- the time gradient of the associated electric field ation (EP) to enhance the uptake of the intracellular [1]. These results are extended to search for translocation of Resv, employing brief electric fields an optimal dose-response for an IRE protocol in to transiently permeabilize cellular membranes. terms of pulse duration considering damage due to MDA-MB-231 human TNBC cell line, derived temperature effects. The methodology introduced here is illustrated with partial data taken from [2] 294 who studied the effect of local temperatures on ab-The device is an expandable basket-shaped cath- lation size from an IRE protocol using constitutive eter (maximum diameter 20mm), equipped with pulses in the range 1-100 µs. Preliminary results 27 electrodes with pacing and sensing capabil- show that for constitutive pulse lengths in the range ity (panel A, Access PoinF-spline PentaRay cath-of 1-100 µs, without considering damage, ablation eter and Carto system (Biosense Webster, Dia- increases logarithmically with pulse duration, while mond Bar, CA). Total Joule delivery per vein was the threshold decreases exponentially. Moreover, 11.25, over 8 deliveries. Ablation was performed all pulse duration trajectories have common ab- in the superior vena cava (SVC), right inferior PV lation and threshold curves. Damage increases (RIPV), left inferior PV (LIPV), and right superior with pulse number and pulse duration. An optimal PV (RSPV); ablation was first performed within the dose-response in an IRE treatment, such as in respective vein (distal bipole delivery), then in the [2] but with a fixed pulse number and frequency, ostial aspect (proximal bipole delivery). PVs were is predicted as the critical pulse duration dosage identified by fluoroscopic, intracardiac echographic yielding maximum ablated tissue area with minimal and 3D EAM guidance (panel B). We assessed the damage due to temperature. local electrogram voltage amplitude and capture thresholds pre and post-ablation. [1] Marshall G. and Soba A., (2024) Predict- Results: In total, PFA was delivered to 11 PVs ing the electroporated tissue area trajectory in and 5 SVCs. The sharp (near-field) potential was Electroporation-based protocol Optimization, consistently eliminated by PFA application utiliz- http://arxiv.org/abs/2403.14022. ing the distal bipoles, consistent with PV abla- [2] Fesmire, C. et al. Irreversible electroporation is tion (panel C). Commensurate with this, PV sleeve a thermally mediated ablation modality for pulses capture was eliminated; however, ostial atrial po- on the order of one microsecond. Bioelechemistry tentials and capture threshold were not signific- 135: 107544 (2020). antly changed (7.1±3.2mA). Following ostial abla- tion, ostial signals were eliminated, and capture PO-93 threshold increased further. 11 veins were evalu- Effectiveness of a Novel Basket-Shaped Pulsed ated chronically at 30 days – of these, 9 remained Field Ablation Catheter for Intra Pulmonary Vein without PV potentials (panel D). Ablation Conclusions: This study demonstrates the effect- Jason Tri iveness of a prototype PFA device with ability to Mayo Clinic, United States achieve pulmonary vein sleeve and ostium ablation Background: Pulsed field ablation (PFA) has without need for catheter repositioning and sten- emerged as an alternative energy source for cath- osis. eter ablation for atrial fibrillation. The safety and PO-94 efficacy of this novel energy source have been Comparison of high-frequency pulse train al- demonstrated in previous reports. However, aim- ternating form on endothelial cell electropora- ing for pulmonary vein (PV) isolation may lead to tion and permeability recurrence due to one small arrhythmogenic re- Liang Yu, Lvheng Ren, Sicong Wang, Shoulong connection. We have previously demonstrated the Dong, Chenguo Yao safety of ablation within the PVs. This study aimed Chongqing University, China to elucidate the efficacy of a new PFA device for true “single-shot” PV ablation and isolation (PVI) Brain endothelial cell plays a key role in the through targeting of the intrapulmonary and ostial structure and function of the blood-brain barrier. in a single catheter position in both the acute and When high pulsed electric field is applied to cells, it chronic periods. can induce cell electroporation and increase intra- Methods: Five swine were utilized in this study. cellular pathway permeability. Two electrode pins 295 (1 mm diameter and 2.3 mm spacing) are used charge and cell diameter. The obtained curve had a to treat the adherent endothelial cells. The ap- sigmoidal shape with linearity in the range of ±45% plied electric pulse: the amplitude 1 kV, the pos- fluorescence change from the resting level. The itive/negative width 100 ns, 200 pulse burst with data correlated with the theoretical values with a lin-total energized time of 2 µs per burst at 4 Hz, ear range corresponding to ±400 mV TMP change. and We use change in fluorescence of YO-PRO- Beyond this range, FluoVolt signal did not change 1 dye to quantify membrane permeability by elec- despite applying a stronger electric field, presum- tric pulses. We investigate the alternating form the ably because the electropermeabilized membrane pulse interval between positive and negative the ef- did not charge further. However, electropermeab- fect of multiple alternating bipolar and single altern-ilization could not be detected by standard meth- ating bipolar pulsed electric fields on the permeab- ods (such as electroporative Ca2+ uptake) at field ility of endothelial cells. In multiple alternating bi-strengths below 1 kV/cm. polar mode, the electroporation effect area both in- The induced TMP was maximum at cell poles fa- creases first and then decreases with the increase cing the electrodes and gradually decreased to- of the period. we first fix the interval at 500 ns (peek wards the cell equator. At 70� angle, TMP change occurs when the period is around 5 µs), and then decreased on average by 62%. This depend- we adjusted the interval to half of the period (peek ence matched theoretical values in most cells ex- occurs when the period is around 15 µs). Under posed at 0.09-0.15kV/cm. Large cells exposed the same period, electroporation of the fixed inter- at �0.25kV/cm displayed a smaller induced TMP val one is weaker than that of half of the period. at the poles than predicted. The charging time In single alternating bipolar mode, the electropora- constant equaled the relaxation time constant for tion effect area decreases with the increase of the electric field strengths �0.15kV/cm. More intense period, but always stronger than multiple one. pulses reduced the charging time constant but did not affect the discharging one, and this difference PO-95 was more pronounced in larger cells. Dynamics of plasma membrane charging and Analysis of PM time constants at the different relaxation measured by strobe fluorescence angles to the electric field showed no difference microscopy between charging and discharging for the cells ex- Iurii Semenov 1, Joel Bixler2, Allen Kiester2, Bennet posed at �0.15kV/cm. In cells exposed at �0.25 L. Ibey2, Andrei G. Pakhomov1 kV/cm charging and discharging did not differ sig- 1Old Dominion University, United States nificantly at the 70� angle, where minimal polariz- 2Air Force Research Laboratory, United States ation was observed. However, the charging time Chinese Hamster Ovary (CHO) loaded with a constant decreased with the angle to the electric voltage-sensitive FluoVolt dye were exposed to 1- field, reaching the smallest value at the poles, while µs electric pulses at field strengths above and be-the discharging time constant remained nearly un- low electropermeabilization threshold. The cells changed. were exposed early after their attachment to the Our results suggest that more intense pulses briefly substrate when they still maintained a nearly round increase the membrane conductance, prevent- shape. The dye was excited by ˜8-ns laser flashes ing further charging. This electropermeabilization delivered at varied delays with respect to the elec- event is short-lived and apparently beyond the de- tric pulse. The delays were changed in 50-ns in- tection limits of common electroporation detection crements or decrements, to collect cell images at methods such as a dye or Ca2+ uptake. different times during cell charging and relaxation of the induced transmembrane potential (TMP). Cells were exposed at 0.09-0.48 kV/cm to build the dependence of FluoVolt fluorescence on PM 296 PO-96 PO-97 Stream pulsed electric fields integral (sPEFI) Impact of pulse parameters on the conductiv- and energy properties of tissue ablation on ir- ity variations in Biological tissues, treated with reversible electroporation electroporation Weiwei Huang 1, Jiali Bao2 Praveen Sahu1, Marco Barozzi2, Hala Mohamed 1Zhejiang University, China Abd El Megeed3, Patrizia Lamberti4, Ignacio G. 2Silveridge Institute of Technology, China Camarillo1, Elisabetta Sieni 1, Raji Sundararajan1 1Purdue University, United States The pulsed electric fields ablation (PEFA) based 2University of Insubria, Italy on irreversible electroporation (IRE) is a technique 3National Institute of Standards (NIS), Egypt that induces cell apoptosis or tissue ablation by cre-4University of Salerno, Italy ating irreversible pores in cell membranes under pulsed electric field (PEF), and has been widely High intensity, short duration voltage pulses in tumor therapy. The optimal combination of used for cell electroporation could also be used pulse electric field intensity, pulse length and pulse to determine the conductivity of the biological tis-number can obtain good tissue ablation effect in sues using different pulse protocols. Given the micro- and nanosecond PEF. In the paper aims are shape and the size of the sample, such as a paral- proposed a stream Pulsed Electric Fields Integral lelepiped, the pulses are applied between two par- (sPEFI) as an index of the combination, and es- allel faces, and voltage and are measured, and tablished a functional relationship between sPEFI the resistance of the parallelepiped sample is de- and electric field energy and tissue ablation by an- termined. From the resistance and sample size, imal experiments, and revealed the laws of PEFA. the conductivity is computed. In this research, Methods are to design some experiments of the the variation in the conductivity of the tissue, in pulse duration, pulse energy, pulse number and this case, potato tubers were measured, when the electrode spacing using Sprague-Dawley rat liver, pulse length, pulse number and pulse amplitude to setup the empirical equation of sPEFI and elec- were modified. The pulse number was varied from tric field energy properties of ablation area from 1 to 16 (1, 4, 8, 16) and the pulse lengths chosen experimental data, and no effect and thermal ef- were 10 µs, 100µs and 1 ms. The applied voltage fect threshold line in the Td-E0 map. Results were amplitude was varied from 5 to 700 V. Given an shown that sPEFI and energy curves exhibit ex- electrode gap of 7 mm the electric field intens- ponential increases and appear saturation at the ity varies between 3.5 V/cm to 1000 V/cm. The thresholds, the pulse initial voltage could control color change, which indicates the degree of elec- the saturation energy, and the no effect threshold troporation (electroporated sample becomes dark) of sPEFI is approximately 0.56 (kV/m)2s while the was verified after 24 h in order to determine the thermal effect is approximately 219.6 (kV/m)2s. electroporation threshold corresponding to the pro- Conclusions are that sPEFI depends on the pulse tocol. For each protocol at least three measure- waveform and is a controlled parameter, which can ments were considered and for each set of meas- be used as the index of the optimal combination urement the corresponding uncertainty was evalu- of pulse intensity, width and number. Electric field ated. energy of tissue consumption depends on pulse The results indicated that using 6 to 830 V/cm, 100 waveform and tissue resistance and is an uncon- µs pulses, the conductivity varied from 0.04 to 0.42 trolled parameter. S/m for one pulse, while it varied from 0.04-0.7 S/m for 16 pulses. The conductivity varied from 0.04 to 0.4 S/m for one pulse and to 0.04-0.8 S/m for 16 pulses, using 1 ms, 6 to 520 V/cm pulses. These mimic the biological tissue variations, which could be used for further studies. 297 PO-60 can potentially modify starches’ physicochemical Pulsed field ablation for cardiac arrythmias: and structural characteristics, which may influence parameters prediction via machine learning glucose release during gastrointestinal digestion Raffaele Crusi 1, Nicolo’ Colistra2, Francesca along with detrimental effects on the flour quality Camera2, Marco Salvatore Zappatore1, Giuseppina characteristics due to starch damage increase. Monti1, Caterina Merla2, Luciano Tarricone1 This work was focused on the optimization of the 1University of Salento, Italy pulsed electric fields (PEF) processing conditions 2ENEA, Italy for wheat flour using response surface method- Pulsed-field ablation (PFA) is a promising non- ology (RSM) with the aim to induce digestibility thermal tissue ablation technique that utilizes high- changes avoiding or minimizing the level of dam- amplitude ultra-short pulsed electrical fields (PEF) aged starch (DS). DS content was determined by to induce cell death through irreversible electropor- enzymatic analysis and used as a response vari- ation (IRE) in tissues. PFA can effectively treat able to identify the optimal treatment conditions in various medical conditions, including cancer and terms of flour-water concentration (5, 10, and 20% cardiac arrhythmias such as paroxysmal and per- w/w) and PEF processing conditions namely field sistent atrial fibrillation. However, the selection the strength of 3 and 5 kV/cm for 20-284 pulses, corres-PEF protocols, the electrode parameters and char- ponding to a total specific energy input of 3, 5 and acteristics to get specific and well defined ablat- 15 kJ/kg using 10 µs rectangular wave monopolar ive lesion is a challenging task which hinder IRE pulse at 50 Hz. The physicochemical properties cardiac ablation treatment standardization as well and digestibility of PEF-treated samples at optimal as optimal device development, as well as regu- conditions compared to untreated samples were latory decision-making for PFA protocols and pro- analyzed by SEM and an in vitro semi-dynamic di- cedures. In this context, artificial intelligence and gestion model, respectively. machine learning algorithms could potentially im- Results demonstrated that, the application of PEF prove the accuracy and effectiveness of PFA pro- at processing conditions of 10% of flour-water con- cedures by identifying the most suitable targets and centration (w/w), 34 pulses, and 5 kJ/kg induced a predicting optimal PEF parameters. Toward such lower level of DS (3.7%), which is within the accept- a direction, our study provides, for the first time, a able limit for high-quality flours (up to 8%) and sim-robust and effective prediction and optimization of ilar with those obtained for untreated samples. Un- the IRE parameters for human cardiomyocyte abla- der optimized conditions, PEF treatments on wheat tion treatment in vitro. To this aim, a novel artificial flour samples caused a significant decrease in the intelligence model has been developed and valid- rate of starch digestibility during the gastric and ated through esperimental data extracted from di- intestinal phase. For instance, a significant de- gital libraries and literature to estimate the ablation crease (from 24.4% to 18.1%) in the rapidly digest-area, electrode configuration, number of pulses, ible fraction (RDS) and a significant increase in the amplitude, period, and repetition rate of the applied resistant starch fraction (from 70.1 to 78%) were signals. observed. SEM micrographs exhibited unaffected size, shape and integrity of starch granules treated PO-98 by PEF optimal conditions. Optimization of pulsed electric field (PEF) pro- This preliminary study suggests that PEF treat- cessing conditions for wheat flour treatment ments may modulate the starch digestibility of using Response Surface Methodology (RSM) wheat flour by slowing the rate of digestion with Dominique Larrea-Wachtendorff, Danela Silva- minimal flour quality damage. However, further ex- Ferrer, Mario Perez-Won, Gipsy Tabilo-Munizaga periments should be performed in order to determ- Universidad del Bío-Bío, Chile ine the effects of PEF treatments on the functional and mechanical properties of wheat flour. Processing wheat flour with PEF technology 298 PO-99 while PZT utilizes the direct and indirect piezoelec- Flyback Versus Piezo Transformer Based Con- tric effect. verter Topologies for Bipolar Pulsed-Power Ap- Initial simulations show that in addition to re- plications moving magnetic components, the PZT solution Ajay M. Chole, Jeya Shree Thulasidas, Maeve Duffy demonstrates higher efficiency (92%) compared to University of Galway, Ireland FBT (80%). Furthermore, the PZT has a smal- ler size than the FBT electromagnetic transformer. Traditionally, magnetics-based power converter More detailed results of circuits operation over a topologies are the most prevalent in medical complete capacitor charging cycle will be provided devices, but with the emergence of piezoelectric during the conference, along with the complete cir- transformers (PZT), the possibility of magnetics- cuit designs. free solutions presents itself to address issues with Our findings offer valuable insights for demon-electromagnetic interference and compatibility with strating the potential of a magnetics-free PZT MRI. Our study presents a comparative analysis based solution for high voltage (HV) medical device between two topologies, flyback transformer (FBT) application requirements, balancing factors such and piezoelectric transformer (PZT) based convert- as efficiency, power consumption, and component ers, for use in high-voltage (HV) bipolar pulse gen- count. erators for medical applications such as electropor- ation. With stringent design requirements such as PO-100 high voltage and current ratings, rapid pulse repe- A Smart and Portable Electroporation System tition frequencies, fast rise times, high power dens- for More Rigorous Experiments ities, reliability, galvanic isolation, flexibility, and Junrui Zhang1, Xingyou Zhou2, Junyan Qian2, Ren controllability, the choice of pulse generation to- Wang2, Federico Jara Crua 2, Zhixian Deng2, Shulin pology is critical. In this study, performance met-Wu4, Yumei Xue4, Xin Chen4, Sixiang Li4, Yuehua You3, rics such as efficiency, charging time for capacit-Shiyu Cheng2 ors, input power requirements, component count 1Southern University of Science and Technology, China and capacitor configurations are thoroughly com-2Roumai Medical SA, Switzerland pared between the two topologies using LTspice 3People’s Hospital of Shenzhen Longhua District, China software simulation. Work is ongoing to compare 4Southern Medical University, China the solutions experimentally. Electroporators are prevalently applied in vari- Both topologies are designed to achieve the ous scenarios of cell and tissue engineering. An same circuit specifications, converting a 24 V in- electroporator delivers high intensity electric pulse put DC voltage, to a 2 kV output DC voltage and up waves to temporarily puncture reversible micro- to 40 A output current pulses. The PZT based to- holes in a cell membrane or a bacterial shell. pology requires four MOSFETs on the inverter side, It enables delivering of much larger gene seg- four diodes on the rectifier side, along with the com-ments across cell membranes and bacterial shells mercially available piezoelectric transformer. On compared to conventional transfection methods. the other hand, the FBT based solution employs However, electroporators of today are usually one MOSFET, one diode and a purpose designed heavy and large, in combination with old-fashioned electromagnetic transformer. “keypad + LCD display” operation, resulting in Both FBT and PZT topologies offer isolation, a steep learning curve and cumbersome to use. but they employ different mechanisms to achieve In addition, none of the existing electroporators it. In FBT, isolation is primarily achieved through provide visual feedback to confirm the correct gen- the transformer itself. In PZT topologies, isolation eration of the desired waveform. is typically achieved through the physical separa- We report an ultra-small electroporation system, tion of the piezoelectric elements. The FBT relies that includes ultra-small (155 x 155 x 33 mm, 0.7 on electromagnetic induction in a magnetic core, kg) host station and a set of accessories that facilit-299 ates experiments and provides users with scientific PO-101 research level data and plots to compare between High-Performance Modular Pulse Generator for the designed waveform and the actual generated Electroporation Applications one. Héctor Sarnago, Borja López-Alonso, Pablo Briz, The host station is a battery powered device that José M. Burdío, Óscar Lucía can be wirelessly controlled by a smart phone APP, Universidad de Zaragoza, Spain facilitating saving and retrieving of setting history, The application of pulsed electric fields (PEF) to measurement of resistance at the terminal, user- different biological targets in so-called electropora-defined waveform setting, and visualization of the tion processes causes a series of relevant micro- actual waveform to help users ensure correct ex- scopic and macroscopic effects that are of interest perimental setup. In detail, the host station is in a broad range of scientific and industrial fields. powered by a rechargeable battery with 12 V nom- This has created a wide range of electroporation inal voltage. Its output is in the form of high voltage applications ranging from in vitro cellular-level ap-pulses, whose parameters can be fine-tuned ac- plications to clinical macroscopic applications, or cording to needs. For example, voltage amplitude industrial intensive applications including. In all is tunable from 20 V to 200 V with 1 V step size, these applications, pulse generators play a key role voltage sign (+, -) is exchangeable by pre-setting generating the desired voltage/current levels with in the APP, center distance between two consecut- specific features for each application. ive pulses is settable from 8 µ s to 2 s, the number of In the current context, PEF applications require pulses to be released is controlled from 1 to 100. All generators able to generate pulses ranging from of these parameters are monitored by an internal few V and mA in in-vitro applications, to tens of kVs microprocessor and an analog-to-digital converter and hundreds of As in industrial applications. Fre- (ADC), and displayed on the APP interface together quencies and pulse widths also ranges from kHz with the set waveform, in order to visualize the dif- and hundreds of µs, to MHz and ns in other applic- ference between set value and the actual value. ations, pushing towards ps generators. The same In addition to the host device, the authors have de- applies to the generated waveform, including expo- veloped a series of accessories that are extremely nential decay or square waveforms, or even more convenient for researchers and their experiments. complex variable waveform. This context requires The first accessory is called Unity Module. It en- different technological solutions that leads often to ables connection of one electroporation cuvette suboptimal implementations and generator redund- to the high voltage pulses generated by the host ancies. device. The second is an Octuple Module that en- In this context, this paper details the design and imables parallel processing of multiple sample cell plementation of a scalable high-performance mod- suspensions loaded in multiple electroporation cu- ular pulse generator intended to be used in a wide vettes. Last but not least, a Clamp Module, which range of PEF applications. The proposed con- directs the high voltage pulses through to a pair of verter is based on the use of wide bandgap power crocodile clips. The clips can then be connected to devices, i.e. silicon carbide devices, that allows for any two electrodes in contact with cell. fast operation achieving rising times of a few ns, In conclusion, the authors developed an electropor- i.e., enabling frequencies beyond the MHz range. ator system with intelligent user interface, versatile The proposed generator is implemented following pulse generation and scientific level data record- a modular approach, where each module (Fig. 1(a- ing characteristics. The system is dedicated as a b)) includes two 750-V full-bridge inverters, allow- next-generation tool for research in cell and tissue ing 3000 Vpp bipolar output voltage with a current engineering and beyond. capability exceeding 100 A per module. Finally, the global architecture includes an isolated control and power module that enables building as many par- 300 allel or series modules as required to achieve the pulsed magnetic field parameters on the cell per-desired voltage and current levels. As a result, a foration characteristics was analyzed through the high-performance generator that can generate ar- calculation results. The results show that when the bitrary waveforms exceeding tens of kV and hun- magnetic induction intensity B≥0.8T, the pore dens- dreds of As in the MHz range is obtained (Fig. 1(c). ity reaches the order of magnitude of 1013 (perfor- Moreover, the scalable approach allows to cover ation threshold), leading to the perforation of the a wide range of applications using an efficient im- cell membrane. Once perforation initiates, the pore plementation. The final version of this paper will radius continues to increase until the pulse con- include detailed implementation and experimental cludes. Under the action of square wave, triangular results, including the main waveforms of the gen- wave and sinusoidal half-wave, the square wave erator already implemented. has the greatest influence on the pore radius, res- ulting in the strongest magnetoporation effect. With PO-103 the increase of pulsed amplitude and pulsed width, Simulation study on magnetoporation induced the perforation characteristics such as pore dens- by pulsed magnetic field combined with mag- ity, pore radius and pore area satisfy the change netic nanoparticles based on pore energy trend of exponential function. This study reveals Wei Zheng, Yan Mi the perforation characteristics of magnetic perfor- Chongqing University, China ation of cell membranes through simulation, offer- Compared with traditional therapy, magnetic ing a theoretical foundation and practical guidance field combined with magnetic nanoparticles ther- for the combined treatment of pulsed magnetic field apy is a promising development direction in tumor with magnetic nanoparticles. treatment due to its non-contact, small side effects PO-102 and wide adaptability. However, the specific theory Rapid joule heating improves vitrification and mechanism of this combination therapy have based cryopreservation not been fully elucidated, hampering its develop- Qi Shao ment and promotion process. Therefore, this pa- University of Minnesota, United States per takes as a clue the dynamic development re- lationship between pore energy and pore state in Biological time can be effectively stopped when cell membranes. Firstly, the magnetic stress of living biological systems are successfully cooled magnetic nanoparticles and tumor cells under mag- and stored at cryogenic temperatures (i.e., cryo- netic field was determined using the Maxwell stress preservation). Vitrification, an attractive ice-free tensor. Based on the function-energy relationship, alternative to conventional convective slow cool- the expression of the pore energy under the ac- ing methods, turns liquid water to a glassy phase tion of the magnetic field is obtained. Combined by cooling rapidly enough to avoid ice formation with the effects of line tension, surface tension and and chilling injury. However, rewarming techniques steric repulsion on the pore energy, the pore energy that are rapid and scalable (both in throughput and equation containing the above four contributions is biosystem size) for low concentrations of cryopro- formulated. Furthermore, tumor cells were selec- tective agent (CPA) for reduced toxicity are lacking, ted as the focus of investigation, and a coupling limiting the potential for translation. model was established in COMSOL to simulate the Here, we introduce a joule heating–based platform random distribution of magnetic nanoparticles in- technology, whereby biosystems are rapidly re- teracting with a monolayer of cells. The parameters warmed by contact with an electrical conductor that of the pulsed magnetic field were set by the mag- is fed a voltage pulse. A format of rapid rewarming netic field module. The pore energy and the perfor- using joule heating can achieve rapid and scalable ation characteristic were added to the partial differ-rewarming with low CPA concentrations for cryop- ential equation module. The influence of different reservation of biosystems at different scales. We 301 demonstrate successful cryopreservation of three diverse structures [1]. There is growing interest model biosystems with different thicknesses, in- in the effects of PEF on proteins, not only from a cluding adherent cells (˜4 µ m), Drosophila em-scientific perspective, but also in terms of its ap- bryos (˜50 µ m), and rat kidney slices (˜1.2mm), plication to the physical control of protein function using low CPA concentrations (2-4M). For all 3 ap-and structure, and the denaturation of proteins plications, a single monopolar voltage pulse was that occurs during the field sterilization of food deliver using a voltage pulse generator (ECM 830) products. However, it is not easy to identify PEF for rapid heating. We perform scaling analysis and effects on proteins in living cells because other numerical modeling to rationally design the joule biological events such as Ca2+ ion influx and heating to achieve rapid rewarming, followed by subsequent biochemical reactions make it difficult experimental validation. In short, the pulse width to identify PEF effects on proteins. Previously, we should be selected based on biosystem heat dif- experimentally demonstrated that electrical pulses fusion length, ranging from 10-100 µ s for single with 0.2 MV/cm or more denature urease proteins cell layer up to 100 ms for thin (˜ 1.2 mm) tissue in aqueous solution [2]. Furthermore, we reported slices. The optimal voltage is calculated by heat that electrical pulses on the order of 1 MV/cm capacity, the targeted heating rate (critical warm- class disassembled transthyretin aggregates [3]. ing rate, therefore heating power) and the size, ran- Based on these studies, we have experimentally ging from. For a targeted SAR in the range of 3– investigated the effects of 1 MV/cm class electric 600 × 1011 W/(m3) and system between 1-15 cm2, fields on the hierarchical structures of proteins. the voltage between 60-450 V (given internal ca- Two proteins, egg white ovalbumin (OVA) and pacitor of 4000 µ F and current limit 500 A) will be bovine serum albumin (BSA), with similar molecu-needed. Numerical simulation predicts that warm- lar weights and different structures, exposed to 10 ing rates from 5 × 104 to 6 × 108 °C/min can be times of 1.3 MV/cm, sub-nanosecond pulses, 1 achieved. ns, were analyzed and compared in the respective Comparison with conventional convective warming hierarchical structures. Native-PAGE showed that methods demonstrates that joule heating greatly PEFs with 0.5 MV/cm or more altered the tertiary improved viability at lower CPA concentrations for structure of OVA but not of BSE. Native-PAGE all the model biosystems studied. Our results showed that PEFs with 0.5 MV/cm or more altered present a general solution to the cryopreservation the tertiary structure of OVA but not of BSE. Thiol of a broad spectrum of cellular, organismal, and group (TG) measurements and dynamic light tissue-based biosystems. scattering (DLS) indicate that the tertiary structure of OVA was loosen by the cleavage of the sole PO-104 disulfide bond, whereas no significant change was Experimental study on protein denaturation in- observed in BSE. Circular dichroism (CD) analysis duced by MV/cm class electrical pulses indicates significant changes in the secondary Koki Tsurusaki, Yuya Sato, Keisuke Endo, Sunao structure of neither OVA nor BSA. Throughout Katsuki the experiment, pH and temperature remained Kumamoto University , Japan unchanged. Hydrogen peroxide concentration The electric field strength on the plasma mem- was not changed significantly to affect the three- brane of a living cell exposed to a pulsed electric dimensional structure of proteins. field (PEF) of several kV/cm is automatically enhanced and exceeds 1 MV/cm. While electric References: fields of 1 MV/cm are known to permeabilize [1] Jiří Průša, Michal Cifra, Molecular dynamics phospholipid membranes, molecular dynamics simulation of the nanosecond pulsed electric feld (MD) simulations predict that they affect the efect on kinesin nanomotor, Scientific Reports, function and structure of membrane proteins with 9:19721, 2019. [2] G. Urabe, T. Katagiri, S. Katsuki, “In- 302 tense pulsed electric fields denature urease voltage-gated ion channels. As such, these studies proteins”, Bioelectricity, 2, 1, March 2020, provide new insights into molecular mechanisms of doi.org/10.1089/bioe.2019.0021. increased membrane permeability associated with [3] G. Urabe, T. Sato, G. Nakamura, Y. Ko- electroporation. bashigawa, H. Morioka, S. Katsuki, “1.2 MV/cm Pulsed Electric Fields Promote Transthyretin PO-106 Aggregate Degradation”, Scientific Reports, Effects of microsecond pulsed electric field on 10:12003, July 2020, doi.org/10.1038/s41598- tubulin structure and self-assembly 020-68681-0. Michaela Poplová 1, Tomáš Zakar1, Viliam Kolivoška2, Michal Cifra1 1 PO-105 Institute of Photonics and Electronics of the Czech Where exactly do pores form in the complex or- Academy of Sciences, Czech Republic 2 ganization of the plasma membrane? Insights J. Heyrovský Institute of Physical Chemistry of the from molecular simulations Czech Academy of Sciences, Czech Republic Lea Rems Tubulins form the essential building blocks of University of Ljubljana, Slovenia microtubules, the largest type of filaments crucial Previous studies have shown that intense elec- for various cellular processes. Notably, tubulin tric field promotes formation of pores in a lipid proteins possess remarkably high structural elec- bilayer, where some types of lipids are more and tric charge and dipole moment. The functionality some less prone to pore formation. These studies and interactions of tubulin proteins are signific- typically investigated model lipid bilayers compris- antly influenced by the electric charge present ing up to three different types of lipids. However, on atomic groups in their amino acid residues. the cell plasma membrane is a complex assembly Consequently, applying an external electric field of various types of lipids and membrane proteins. presents a promising avenue for manipulating the The lipids are furthermore asymmetrically distrib- behavior of cytoskeletal proteins like tubulin. uted in the two bilayer leaflets and are organized In this study, we illustrate several alterations in in domains. Thus, it is important to understand tubulin structure and self-assembly polymerization how pores form in the complex lipid organization resulting from Pulsed Electric Field (PEF) treat- of the plasma membrane. This lecture will present ment (using pulses of 100 microseconds width, at results from molecular dynamics simulations using 1.5 MV/m, with 20-60 pulses fired at a repetition coarse-grained membranes containing more than frequency of 1 Hz). Dynamic Light Scattering 60 different lipid types, mimicking the realistic com-analysis revealed an increase in the effective size position of plasma membranes. Simulations using of tubulin molecule and the amount of aggregates such membranes demonstrate that pores do not following PEF treatment. Subsequently, we ob- form homogenously along the membrane surface, served a notable decrease in tubulin concentration but colocalize with domains that have specific fea- across all PEF-treated samples, particularly after tures, the most important being high density of poly- the removal of the majority of aggregates through unsaturated lipids. Furthermore, the simulations ultracentrifugation. Polymerization assays (UV-Vis show that poration does not depend solely on local turbidimetry measurements) demonstrated the lipid arrangement, but also on membrane mechan- loss of self-assembly capability in PEF-treated ical properties and the polarity of the electric field. tubulin, which was further confirmed through phase Combining results from these coarse-grained sim- contrast microscopy. Additionally, fluorescence ulations with atomistic simulations can help us un- spectroscopy employing 8-anilinonaphthalene-1- derstand how the rate of poration in lipid membrane sulfonic acid (ANS) probe indicated an upward domains compares with the rate of electric-field trend in fluorescence intensity in treated tubulin induced damage to membrane proteins such as samples, suggesting PEF might induce structural 303 alterations in tubulin, exposing more hydrophobic forms grown under strictly defined conditions in groups to the molecule surface. fully PAT-controlled bioreactor setups. Recombin- Our findings contribute to the advancement of ant E. coli and P. pastoris strains, containing GFP- novel electromagnetic techniques for influencing tagged proteins, served as models to investigate biomolecular function at the nanoscale. PEF the interdependence and cross-species comparab- treatment induces structural changes in tubulin ility of nsPEF treatment and biological response. molecules while retaining its fundamental self- A standardized experimental approach was taken, assembly capability. Furthermore, we identified with industry-near batch and fed-batch cultures ex- the PEF parameters threshold for irreversible ecuted in parallel twin runs interfaced to an nsPEF tubulin changes and observed the aggregating system in bypass loop configuration. On global cul- effect of PEF treatment on tubulin. ture and individual cell population level, physiolo- gical key parameters were followed via real-time We acknowledge support from the Czech Sci- monitoring and off-line analyses. ence Foundation, project no. GX20-06873X. Focussing on growth kinetics, morphological fea- tures and protein expression, positive overall im- PO-108 pact was observed across species borders, yet tra- nsPEF-mediated productivity improvement in cing back to differential effects depending on cul- bioprocessing – A cross-species evaluation of ture type and environment. Careful adjustment bacterial and yeast expression platforms in of nsPEF treatment conditions emerged as a pre- bioreactor cultures requisite for optimal outcome, and caution is due in Lukas Neutsch the selection and interpretation of state-of-art PAT Zürcher Hochschule für angewandte Wissenschaften, tools. Next to a holistic combination of multiple in- Switzerland dicators and data sources, our results recommend The technological advancement of pulsed elec- a closer look on the individual cells and popula- tric field treatments into the nanosecond (nsPEF) tion dynamics to leverage the full potential nsPEF- time scale has opened an exciting new field of ap- based stimulation in biotech production settings. plications in the stimulation of biotechnological cul-PO-109 tures. The underlying mechanisms and effects are Enhancing starch for 3D food printing: pulsed still largely unknown, but thought to combine into a electric field modification and functional in- milder and only temporary cellular response, which sights however can have long-lasting impact on the cul- Saqib Gulzar, Pedro Elez-Martinez, Olga Martín- ture. In bioprocess engineering, hope is that nsPEF Belloso, Robert Soliva-Fortuny treatment can be exploited as a versatile productiv- University of Lleida, Spain ity booster via its generically applicable, physical principle. Modifying starch for 3D printing is necessary Owing to the wide range of structural elements and to enhance printability, structural integrity, custom-metabolic pathways in cells that may be suscept- ization of properties, and compatibility with addit- ible to nsPEF-induced changes, factors related to ives, fostering innovation in food manufacturing biomass formation (e.g. cell division rate and sub- while meeting regulatory standards. This research strate utilization efficiency), as well as factors re-delves into the exploration of pulsed electric fields lated to the biosynthesis of compounds (e.g. gene (PEF) as a way of tailoring the physicochemical at- expression, intracellular accumulation or secretion) tributes and processing characteristics of starch. come into play as potentially fruitful optimization PEF is seen as a promising technique for modify- targets. ing food materials, such as starch, to enhance their In this study, we report on the in-process ap- functional properties and suitability for novel applic-plication of nsPEF to microbial expression plat- ations like 3D bioprinting. The study investigates 304 the impact of PEF on the physicochemical proper-PO-110 ties of pregelatinized corn starch (PGCS), and its Inhibition of Color Change for Long Term on potential for 3D printing. Meat of Bonito during -18� Freezing by Apply- PGCS was subjected to PEF treatments at an in- ing Pulsed High Electric Field tensity of 15 kV/cm, a 20 µ s pulse width, and op-Koki Saito 1, Shoichiro Kosugi1, Koshi Kawasaki1, erated at a frequency of 20 Hz for a range of 100-Yasushi Minamitani1, Ryo Sawada2 1 400 pulses. The study sought to observe the effect Yamagata University, Japan 2 of this treatment on the starch’s inherent granular Kyokuyo Co., LTD, Japan structure, composition, structural order changes, The temperature has to keep at -50°C or less tecno-functional properties, as well as the ability to by using ultra a low temperature freezer for long-form 3D printed constructs. term storage of meats and also at cutting and trans- The findings revealed that PEF disrupted the nat- porting, because the temperature below -50°C sup- ive architecture of the starch granules, leading to presses a color change of the meats. Therefore, changes in the structural organization. The treat- meat prices become high. The color change of the ment also improved the starch’s water and oil ab- meats is caused by that myoglobin converts to met- sorption capacities, with results showing an in- myoglobin by the decomposition of ATP. The de- crease of up to 56.8% and 104.5%, respectively. composition of ATP, which is a freshness indicator While PEF had a minimal impact on the starch’s of meats, causes by ATPase that is a decompos- composition, it significantly improved pasting vis- ition enzyme of ATP. If ATPase could be deactiv- cosities and the texture of the starch gel. X-ray dif-ated, the long-term storage would be possible in fraction (XRD) and differential scanning calorimetry a general-purpose refrigerator because the meat (DSC) analyses suggested that the PEF treatment color does not change at -18°C. Therefore, we con- resulted in reduced crystallinity and elevated trans- sidered the application of pulsed power technology ition temperatures in the modified PGCS. When to deactivation of ATPase. Pulsed high electric field used to make hydrogels, the PEF-treated PGCS has been reported to be able to suppress enzyme exhibited improved gel hardness. This character- activity without thermally impact to targets depend- istic was correlated with the creation of superior 3D ing on pulse conditions. printed constructs, which displayed sharper edges In this presentation, meat of bonito was used compared to those made from less modified coun- for a sample applying the pulsed high electric field, terparts. because it undergoes a noticeable color change Overall, the PEF-induced modifications enhanced from fresh red to red brown over a short period the functionalities of the starch, conferring desir- of time. Furthermore, since it has been reported able rheological attributes for 3D bioprinting PGCS- that myoglobin is easier to become metmyoglobin based foods. These findings underline the potential at between -5°C and -10°C, pieces of the meat of of non-thermal technologies, like PEF, in rationally bonito were stored in a freezer set at -6� and -18� manipulating the physicochemical and processing after applying the pulsed high electric field. Then, behavior of starch. Such advancements could the change in color of the meat of bonito and the open new avenues in food science and technology, rate of conversion from myoglobin to metmyoglobin particularly in the development of 3D-printed food for the elapsed time were investigated. As a result, products. it is found that application of the pulse high electric field can suppresses the conversion myoglobin to metmyoglobin in the meat of bonito. 305 PO-112 eral points of the system including inlet and outlet Development of pulsed electric field pasteuriz- of the PEF treatment cell. Long-life milk with Lis- ation system for protein-rich liquid foods teria innocua (2 x 107 /ml) was completely sterilized Tomohiro Nakamura 1, Bingyu Yan1, Akira by 26 kV/cm PEF and the thermal energy at 60�. Moriyama1, Ryousuke Kadoya1, Sunao Katsuki1, Liquid egg with Enterobacter hormaechei (2 x 107 Naoya Masuda2, Yoshiharu Shimizu2, Ryo Sasahara3, /ml) was treated by 19 kV/cm PEF and the thermal Taiga Kajiwara3, Jiro Kurihara4 energy at 55�, resulting in a 2.3 log reduction in 1Kumamoto University, Japan bacterial number. Both milk and liquid whole egg 2Iwai Kikai Kogyo Co., Ltd., Japan were continuously operated for more than 1 hour 3Kewpie Corporation, Japan without trouble under the same conditions as for the 4Rakunoh Mother’s, Japan pasteurization. No electrode damage or adhesion Pulsed electric field (PEF) is a promising non- was observed. thermal pasteurization method for protein-rich li- PO-113 quid foods. There are many reports showing the Enhancement of bioactive properties of mail- usefulness of PEF method in batch processing and lard reacted peptides by pulsed electric fields low flow rate experiments. However, in high-flow Carolina Herrera-Lavados, Mario Perez-Won, Luis rate processing of 100 L/h or more for industrializa-Moreno-Osorio, Gipsy Tabilo-Munizaga tion, serious problems such as reduced bactericidal Universidad del Bío-Bío, Chile effect, spark discharges, and ingredient adhesion to the electrodes occur. These problems are mainly Background: Maillard reaction (MR), which caused by the velocity distribution in the perpen- refers to a series of complex chemical reactions dicular to the flow. The flow velocity near the tube between amino acids, peptides or proteins with a walls is much lower than that in the central flow and reducing carbonyl compound, is commonly used in thus receives more electrical energy from the repet- food processing to improve the flavour and bioactiv- itive high-power pulses, resulting in the local heat- ity of hydrolysates and peptides. Emerging pro- ing. This not only degrades food components, but cessing technologies, such as pulsed electric field also promotes coagulation and deposition of com- (PEF), could be used to improve the extent of the ponents on the wall, triggering spark discharge. We MR, reducing the reaction temperature achieving have developed a continuously operating pasteur- similar flavour enhancement and bioactivity. This ization system combining a PEF and a moderate study aimed to investigate the effect of PEF on the thermal energy for protein-rich liquid foods such as progression of the MR and the bioactive and sens- milk and liquid whole eggs. This paper describes ory properties of Maillard-reacted peptides (MRPs) the ability of our system and the durability of the derived from salmon peptides. PEF treatment cell, which is basically co-linear type Methods: Salmon peptides obtained by enzymatic and can be stacked in series to the flow. The shape hydrolysis were subjected to wet PEF-assisted and geometry of the cell was designed to optimize Maillard reaction. The PEF treatment was applied in terms of the electric field distribution, the velocity in continuous mode at electric fields of 0, 10, 15, distribution of the flow, the temperature distribution and 20 kV/cm to a solution containing 4 mg/mL using COMSOL Multiphysics. The flow system was of salmon peptides (molecular weight <10 kDa), 1 pressurized to 0.5 MPa and the flow rate was adjus- mg/mL of xylose and 0.5 mg/mL of cysteine, at pH ted to 10 L/h by a needle valve located at the exit of 7.1. MR was then immediately performed at 70, 80, the flow system. 2 µs-long voltage pulses were re- and 90 °C for 150 min. The resulting MRPs were petitively delivered to the electrodes and the pulse analysed for browning index (at 420 nm), genera- repetition rate was set so that the average number tion of intermediates (at 294 nm), antioxidant activ- of pulses to bacteria for one stage was more than ity, Angiotensin I-Converting Enzyme (ACE) inhib- 1. Temperature of the flow was monitored at sev- itory activity, FTIR spectra, and sensory evaluation. 306 Results: Absorbance at 294 and 420 nm increased Regarding antioxidant activity, no significant dif-with reaction time, indicating the production of ferences (p > 0.05) were observed in DPPH-RS MR intermediates and browning compounds, which between the control and PEF-treated sample (1 was supported by FTIR results. At temperatures kv/cm) at 49 and 53%; however, an increase in of 70-80 °C the treatment at 20 kV/cm increased PEF intensity (2 kv/cm) resulted in an increase the browning index, but at 90 °C this was achieved in DPPH-RS of 83%. A similar phenomenon by the treatment at 10 kV/cm. PEF treatment also was observed where the PRS for the control, 1 decreased the final content of MR intermediate and 2 kv/cm were reported as 37, 39 and 42%, products. By applying electric fields of 15 kV/cm respectively. On the other hand, PEF had no effect the antioxidant and ACE Inhibitory activity of MRPs on the RP of CGRE since no significant differences was improved, but higher electric fields had no ef- (p > 0.05) were reported between treated and fect or even decreased the bioactivity of the MRPs, untreated samples. Consequently, the RP as possibly caused by the changes in final and in- depicted by an increase in absorbance at 700nm termediate MR products. All MRPs possessed a ranged from 1.56 – 1.70. meaty flavour. The results of this study demonstrated the effect Conclusion: The use of PEF-assisted MR on sal- of PEF as a suitable pre-treatment to enhance the mon peptides was effective to produce tasty MRPs extraction of polyphenols from green rooibos, even with improved bioactivity. at lower temperatures. Furthermore, varying the electric field strength was an important factor in PO-114 polyphenol extraction. Polyphenolic content and antioxidant activity of pulsed electric field-assisted extracts of green PO-115 rooibos. Enhanced Extraction of Cellulose and Lignin Lusani Vhangani from Agro-Industrial Wastes Utilizing Alkali Cape Peninsula University of Technology, South Africa Treatment Assisted by High-Voltage Electrical Discharges (HVED) for Wood Adhesives Applic- Antioxidant activity associated with green ation rooibos infusions is attributed to the activity of Yassine El Khayat Driaa 1, Hafida Maarir2, Nabil polyphenols, particularly aspalathin and nothof-Grimi3, Amine Moubarik2, Nadia Boussetta3 agin. This study investigated the polyphenolic 1Sorbonne Université, France content and antioxidant activity of pulsed electric 2Sultan Moulay Slimane University, Morocco field-assisted extraction of green rooibos. Aqueous 3Université de Technologie de Compiègne, France green rooibos was treated at a PEF intensity of (0 - 2kv/cm), generating specific energy of (0 – This study investigates a novel approach for 10kj/kg). After PEF treatment, crude green rooibos the extraction of cellulose and lignin from almond extracts (CGRE) were extracted at 40°C for 30 and walnut shells, agro-industrial wastes, specific- min. The resulting freeze-dried extracts were ally focusing on its applicability in the production analysed for total phenolic (TPC) and flavonoid of wood adhesives. In this research, we propose content (TFC), reducing power (RP), 2,2-diphenyl- a more sustainable and efficient technique by em- 1-picrylhydrazyl (DPPH-RS) and peroxyl radical ploying alkali treatment coupled with High-Voltage scavenging (PRS). Electrical Discharges (HVED). The synergistic ef- The TPC and TFC increased with an increase in fect of alkali and electrical discharges enhances PEF intensity, from 357.59 – 684.04 mg GAE/g the breakdown of biomass components, facilitating and 117.00 – 195.67 mg CE/g, respectively. the extraction of cellulose and lignin with improved Extraction of polyphenols occurred as a result of yield and purity. The employment of almond and pore formation and disintegration of the cellular walnut shells as a raw material not only addresses material. environmental issues linked to waste management 307 but also offers an economically viable and sustainable option for extracting cellulose and lignin. The application of HVED in the extraction process significantly enhanced the yield of cellulose and lignin from almond and walnut shells compared to conventional methods. Analysis of the structure was conducted using techniques such as Fourier Transform Infrared Spectroscopy (FTIR), X-ray Dif- fraction (XRD), and Two-Dimensional Heteronuc- lear Single Quantum Coherence Nuclear Magnetic Resonance (2D HSQC NMR) spectroscopy. These methods unveiled changes in the functional groups, crystalline structure, and chemical composition of the cellulose and lignin that were extracted. Ana- lysis of morphological changes was conducted us- ing Scanning Electron Microscopy (SEM) visualiza- tion, which unveiled advancements in morphology. Moreover, Differential Scanning Calorimetry (DSC) and Thermogravimetric Analysis (TGA) demon- strated the enhanced thermal stability of the isol- ated cellulose and lignin. The findings presented in this study illustrate the effectiveness of HVED- assisted alkali treatment in augmenting the extrac- tion efficiency and enhancing the characteristics of cellulose and lignin obtained from almond and walnut shells. Consequently, this approach shows great potential for the utilization of these materials in wood adhesive applications. 308 Authors’ Index A Aurisicchio, Luigi 219, 159, 145, Benedetti, Miriam 112, Abad, Vanesa 201, 137, 280, Awasthi, Kamlesh 224, Bera, Bijoy 84, Abd El Megeed, Hala Mohamed Axelrod, Robert 94, Bereta, Martin 131, 295, Azzari, Paride 93, Berjano, Enrique 117, Abdolahad, Mohammad 243, Berl, Ariel 206, Á Abiusi, Fabian 94, Bertinetti Lopes, Lucas 227, Álvarez, Ignacio 137, 280, 139, Acosta, Ana Clara 236, 284, Bertino, Giulia 202, 204, 231, 201, 208, 86, Adamonė, Dominyka 116, Bertino, Giulia 288, 203, B Aggelopoulos, Christos 188, Bertino, Giulia 125, 230, Baczyńska, Dagmara 237, 274, Aglioti, Salvatore M. 140, Berzosa, Alejandro 86, 208, 281, 263, 266, Agyei, Dominic 110, 113, Beyrer, Michael 130, Bagdonaitė, Rūta 250, Aikas, Mindaugas 259, Bieżuńska-Kusiak, Katarzyna Baines, Stephen John 221, Alba, Andres 155, 263, 272, 265, 237, Baker, Sarah Jayne 221, Alhawamy, Saleheldin 79, 134, Bihoreau, Simon 246, Bakute, Neringa 107, Ali Faridi, Rashid 278, Bisase, Brian 204, Balazinski, Martina 93, Allocca, Mariateresa 270, Bixler, Joel N. 223, 222, Baltas, Eszter 287, Almanaitytė, Mantė 116, Bixler, Joel 294, Balucani, Marco 151, 155, Altun, Burak 248, Bloch, Jocelyne 140, Bansal, Nidhi 151, 111, 137, Amend, Bastian 184, Blunier, Maylin 94, Bao, Jiali 295, Anderson, Mark 140, Boc, Nina 236, Barakauskas, Justinas 116, Andre, Franck M. 108, 154, 155, Boehmert, Laura 135, Barauskaite-Šarkinienė, Neringa 144, 154, 173, 120, 153, 90, Bonadies, Antonio 181, 126, 291, 269, 273, Andreikė, Gabija 273, Borinskyte, Ugne 269, Barba, Francisco J. 202, Andreou, Varvara 164, Borst, Oliver 102, Barbieri, Germana 254, Andriukonis, Eivydas 107, Boukany, Pouyan E. 84, Barbosa De Nardi, Andrigo 177, Antognazza, Caterina 242, Boussetta, Nadia 305, 189, Barozzi, Marco 295, Antonelli, Gianni 249, 110, Bozic, Tim 218, 197, Barrere, Emma 170, 278, Antonelli, Francesca 129, Brasil Pintarelli, Guilherme 227, Barutello, Giuseppina 174, Apollonio, Francesca 120, 169, Brasil Pintarelli, Guilherme 105, Bashir, Yasir 123, 90, 155, 121, 220, 151, Bresolin, Silvia 129, Batista Napotnik, Tina 271, Aramayona, José 148, Briz, Pablo 298, 148, 187, 281, Batiuskaite, Danute 127, Archer, Lorraine 94, Brizio, Matteo 202, 203, Batterham, Alan 231, Arigoni, Maddalena 174, Brozic, Andreja 146, Battista, Michela 202, 181, Ariztia, Laurent 144, 173, Brutti, Andrea 254, Baumgartner, Julia 94, Arnaud-Cormos, Delia 171, 226, Buchmann, Leandro 86, 87, Bazylińska, Urszula 264, Bulysheva, Anna 191, Arnaud-Cormos, Delia 225, Bechara, Falk 204, Buracco, Paolo 174, Arribas, María Dolores 148, Beebe, Stephen J. 100, Burdío, José M. 298, 148, Arroyo, Julio 98, Behera, Nalinikanta 276, Burdio, Fernando 117, Asada, Koya 130, Bellucci, Stefano 249, Butt, Waqas 123, Asadipour, Kamal 100, Benazzo, Marco 288, 231, Attal-Tretout, Brigitte 224, Benazzo, Marco 125, 230, C Attili, Fabia 207, Benchakroun, Hamza 247, Cadossi, Matteo 103, Cadossi, Ruggero 103, 141, Cifra, Michal 171, 131, 301, 215, Dall’Olmo, Luigi 265, Calazans Menescal Linhares, 196, Dalmay, Claire 225, Lais 177, Ciliberto, Gennaro 181, Damiano, Ralph 80, Caluori, Guido 246, Cilio, Antonella 284, 157, Darrow, Jewels L. 116, Calvel, Anne A. 119, Cilio, Antonella María 158, 236, Das, Sudip Kumar 238, Calvo, Cristina 281, 229, 143, Davalos, Rafael 77, 227, 115, Camarillo, Ignacio G. 292, 295, Cimperman, Tina 271, David, Kailee 242, 267, Cindrič, Helena 91, Dayananda, Buddhi 137, Camera, Francesca 110, 224, Cioce, Mario 123, De Aguiar Saldanha Pinheiro, 154, 249, 296, 260, Ciurzyńska, Agnieszka 162, Ana Cristina 202, 254, Cameron, Andrew D. 200, Clover, James P. 202, 204, De Cillis, Alfredo 260, Campana, Luca Giovanni G. 265, Colavitti, Giulia 203, 204, De Gol, Cora 130, Campastri, Ana Laura 229, 143, Colella, Micol 90, 155, 141, 151, De Robertis, Mariangela 197, 236, 120, 154, 220, 175, Campastri, Ana 157, 284, Colistra, Nicolo’ 260, 296, De Robertis, Mariangela 123, Campastri, Ana 158, Collin, Annabelle 246, 275, 172, De Souza Ramos, Clara Teresa Campbell, Delcora A. 200, 183, 105, Cancelliere, Rocco 249, Compagnone, Mirco 219, De Terlizzi, Francesca 103, Cani, Alice 129, Conconi, Maria Teresa 242, 123, Del Signore, Federico 121, Caramazza, Laura L. 151, 120, 287, 292, 265, Delbaere, Sophie 148, 220, 169, 121, 90, Conforti, Antonella 145, 219, Delso, Carlota 210, 139, Casciati, Arianna 129, 110, 260, Consales, Claudia 154, 90, 153, Deng, Zhixian 297, 120, 154, Cash, Alexia 286, 116, Denis de Senneville, Baudouin Conti, Laura 174, Castellani, Federica 276, 194, Cordier, Ophelie 278, 170, Castellvi, Quim 104, 183, Dermesonlouoglou, Efimia 164, Couderc, Vincent 226, Casti, Paola 110, Deschamps, Frederic 74, Courtine, Gregoire 140, Cataldo, Antonino 249, Desgrez-Dautet, Hermes 170, Crocetti, Laura 207, Catravas, John D D. 128, Deshmukh, Vinay J. 115, Crowley, Kimberly 217, Cavallo, Federica 174, Dettin, Monica 123, 292, 287, Crua, Federico Jara 297, Cebrian, Guillermo J. 137, 97, 242, 265, Crusi, Raffaele 296, 260, ?? , 280, 201, Dewilde, Maarten 199, Csanyi, Ildiko 287, Cellini, Beatrice 252, Dewitt, Matthew 156, 116, Cuenca Ortolá, Irene 90, Cesi, Vincenzo 129, Dezman, Rok 260, Cui, Pengcheng 257, Chadni, Morad 254, Di Cristo, Nunzia 203, Curci, Giorgia 110, Chafai, Djamel Eddine E. 131, Diaz, Maura 175, Chakrabarti, Rajarshi 193, Č Díaz Núñez, Alba 257, Chase Sheehan, Mary 217, Čebron, Žan 179, Diemer, Etienne 254, Chavent, Matthieu 170, Čemažar, Maja 146, 218, 240, Dijkink, Bert 165, Chen, Xin 297, 122, 260, 190, 228, 198, 124, Dimitri, Ana 157, Chen, Yue 249, 236, 146, 118, 222, 267, 230, Dimopoulos, George 164, Chen, boru 252, 197, 179, Djokic, Mihajlo 179, 260, Cheng, Shiyu 297, Červinková, Kateřina 196, Dobilas, Jorunas 107, Chole, Ajay M. 297, D Dolciotti, Noemi 120, 151, 220, Choromańska, Anna 285, 274, D’Orazio, Michele 110, 154, 90, Chua, Sing Teng 94, Dainauskaite, Viktorija 127, Dominici, Massimo 103, Chwiłkowska, Agnieszka 263, Dalla Rosa, Marco 255, 254, 252, Döner, Deniz 165, 310 Dong, Shoulong 249, 108, 179, Fogle, Callie 116, Giancaterino, Marianna 212, 293, Fontana, Sara S. 220, 155, 154, 211, Dong, Shoulong 188, 90, 120, 151, Gillis, Kevin 166, Dong, Shoulong 262, Forzan, Michele 89, Giordano, Emiliana 252, Donlon, Noel 123, Francis, Michael 191, Giorgione, Roberto 202, Donohoe, Claire 123, Franconi, Rosella 243, Giovannini, Daniela 175, Draganova, Milena 268, Frangez, Robert 218, Giri, Pragatheiswar 292, Drozdz, Marek M. 200, Frejlich, Ewelina 240, Gisbert, Fernando 154, 154, Dubińska-Magiera, Magda 266, Frénéa-Robin, Marie 244, Gisbert Roca, Fernando 152, Dubuc, David 119, Frey, Wolfgang W. 95, 210, Golberg, Alexander 127, Duffy, Maeve 297, 247, Frezza, Valentina 219, Golberg, Alexander 150, 206, Duran, Carolina 220, Fujimori, Masashi 160, 217, Goldberg, Ezequiel 292, Duro de Oliveira, Krishna 177, Fusco, Roberta 103, Golzio, Muriel 90, 263, 216, 172, 171, 98, 278, 170, 121, d G Gomez Galindo, Federico 131, de Boer, Sophie 84, Gagliardi, Serena 249, Gómez-Barea, Mario 241, de Caro, Alexia 263, 98, 121, Gagliardo, Tina 191, Gonzalez Cuevas, Juan A. 143, de Coucy, Alexandra 140, Gagneten, Maite 256, González-Suárez, Ana 182, den Besten, Heidy M. W. 130, Gajewska- Naryniecka, Ag- Gossard, Audrey 275, E nieszka 263, 237, Govaerts, Jannes 199, El Khayat Driaa, Yassine 305, Gallo, Giacomo Perazzolo 260, Govoni, Flavio Andrea 181, Elez-Martinez, Pedro 73, 282, Galus, Sabina 162, Greco, Mariantonina 219, 302, 257, Gannini, Giulio 255, Grenier, Katia 119, Elhaei, Yusuf 243, Gao, Fengzheng 94, Grimi, Nabil 189, 254, 305, Elhanafi, Driss 156, Garcia, Tomas 117, 275, Grisendi, Giulia 103, Enderlin, Gérald 189, García-Sánchez, Tomás 241, Groselj, Ales 204, Endo, Keisuke 300, Garg, Abhishek 199, Gruodis, Alytis 215, Escursell, Natalia 280, Gargan, Cian 178, Grzelka, Kajetan 149, Eslami, Elham 112, Gatt, Ruben 97, Grzelka, Kajetan 208, Estevez-Laborí, Francisco 182, Gautier, Matthieu 140, Guedert, Raul 105, F Gawaz, Meinrad 102, Guedert, Raul 227, Falconieri, Mauro 249, Gazzerro, Patrizia 287, Güemes, Antonio 148, Guillen, Vanda 175, Fang, Zhi 246, 114, Gečaitė, Jovita 290, 186, Guillén, Silvia ?? , Farràs-Moragues, Núria 282, Gehl, Julie 205, 204, 118, Gulzar, Saqib 282, 302, Fazio, Vito Michele 123, Gelažunaite, Simona 280, 273, Guo, Siqi 100, 245, 185, 128, Ferioli, Martina 207, Gelažunaitė, Simona 253, Guo, Pei-Feng 132, Ferreyra, Santiago 143, Gelli, Rebecca 230, 231, 288, Guo, Minglei 245, Fesmire, Christopher 116, Genovese, Jessica 160, Gusbeth, Christian 95, Filippi, Joanna 110, Gerace, Emma 160, Gutierrez, María ?? , Fionda, Bruno 207, Gerardo-Giorda, Luca 181, Gyulai, Rolland 287, Fiore, Donatella 287, German, Natalija 158, Fiser, Ondrej 99, 228, Geukens, Nick 199, H Fišerová, Ivana 99, 228, Ghasemi, Negareh 111, 137, Haberkorn, Iris 210, 161, 93, Fish, Jeffrey 78, Ghassabi, Amir 90, Hadzialjevic, Benjamin 260, Fisher, Paul 192, Ghosh, Jaya 166, Hagimoto, Kiyohira 165, Fluri, Marco Stefan 86, Giacobino, Davide 174, Han, Zhong 114, 311 Haque, Shanaya M. 185, Iussich, Selina 174, Kelly, Vincent 289, Hashisako, Hiroto 130, Ivanova, Tsvetomira 268, Kelly, Michael 123, Havelka, Daniel 196, Ivanova, Tsvetomira 283, Keppler, Mark 223, He, Zhigang 140, Ivashchenko, Sergiy 154, Keppler, Mark 222, He, Dengfeng 108, Ivashchenko, Sergiy 152, Kerrigan, Cian 247, He, Jinjing 221, Ivashchenko, Sergiy 154, Keršulis, Skirmantas 259, Heeran, Aisling 177, Ivorra, Antoni 143, 241, 279, 248, Kesar, Ursa 122, Heinzmann, David 102, 104, 183, 71, Khakpour, Shirin 271, Heller, Richard 147, 73, J Kielan, Wojciech 101, 237, Heller, Loree 73, Jacobs, Edward 77, 227, Kielan, Wojciech 240, Hensley, Jacob 191, Jäger, Henry 212, 211, Kiessling, Volker 276, 168, Herman, Dalibor 99, Jaïs, Pierre 246, Kiester, Allen 294, Herrera-Lavados, Carolina 304, Jakštys, Baltramiejus 84, Kiełbik, Aleksander 184, 102, Higuchi, Masamori 130, Jakštys, Baltramiejus 82, 83, Kiełbowicz, Zdzisław 264, Hoffmann, Ulfilas 93, 274, 126, Kikuchi, Misato 132, 138, Hollevoet, Kevin 199, Jan, Vid 78, Kilgallon, Aoife 178, Hollevoet, Kevin 192, Jan, Matevž 134, Kimura, Yasushi 217, Holzinger, Steffen 135, Jaroszeski, Mark J. 73, 147, Kimura, Ryuya 138, 132, Hong, Junting 189, Jaśpińska, Joanna 149, 208, King, Jessie 110, 113, Horacio Maglietti, Felipe H. 177, Jesenko, Tanja 124, 230, 122, Kirchner, Madita Anna-Maria 292, 284, 157, 143, 190, 118, 213, 162, Horlacher, Nicholas 113, 110, Jiang, Chunqi 185, Kis, Erika 204, 203, 202, Hosano, Nushin 271, 110, 277, Jin, Shanshan 246, Kis, Erika Gabriella 287, 180, Hosano, Hamid 271, 277, 110, Jing, Yu 185, Knappert, Justus 251, Hosseini, Seyedmasih 277, Jonynaitė, Kamilė 259, Kočar, Nina 222, Hozman, Marek 228, 99, Juravičius, Jonas 116, Koenderink, Gijsje 84, 275, Hu, Biao 108, Jurič, Jernej 78, Kolb, Juergen F. 93, Huang, Weiwei 295, K Kolivoška, Viliam 301, 196, Hubenov, Stefan 268, Kadoya, Ryousuke 304, Koller, Reto 93, Humeau, Laurent 192, Kajiwara, Taiga 304, 138, 132, Kollotzek, Ferdinand 102, Hummerjohann, Joerg 97, Kamenšek, Urška 190, Kolosnjaj-Tabi, Jelena 170, 172, Hussain Al-Khafaji, Alia 213, Kan, Lijiao 165, 121, 216, 171, 278, 263, 90, 98, 131, Karmarkar, Parag 134, Komel, Tilen 190, I Karmarkar, Parag 79, Kondrotaite, Terese 261, Ibey, Bennet L. 294, Kašėta, Vytautas 186, 126, Kondrotaitė, Teresė 215, Ibrahimi, Njomza 173, 144, Kasperaviciute, Kamile 107, Kos, Bor 136, 91, Ilieva, Iva 268, Kasperkiewicz, Paulina 101, Kos, Monika 78, Imarisio, Ilaria 125, Kasukonis, Benjamin 222, 223, Kos, Špela 190, Impellizeri, Joseph A. 145, 73, Katsaros, George 164, Kosugi, Shoichiro 303, Innamorati, Giorgia 153, 154, Katsimichas, Alexandros 164, Kovacs, Attila 208, Innamorati, Giorgia G. 154, 155, Katsuki, Sunao 304, 165, 130, Kovács, György 207, Inobe, Kazuhiro 132, 138, 71, 132, 138, 300, Kowase, Takayoshi 273, Ioannou, Irina 254, Kavaliauskas, Žydrūnas 259, Kranjc Brezar, Simona 230, 190, Iršič, Jernej 134, Kavanagh, Dara 123, 124, 267, Ishikawa, Shoko 138, 132, Kawasaki, Koshi 303, Kruszakin, Roksana 274, Ismail, Jaad 160, Keller, Flavio 123, Kucharczyk, Julia 238, 312 Kulbacka, Julita 290, 274, 237, Liberatore, Rachel A. 200, Malyško-Ptašinskė, Veronika 265, 264, 158, 186, 263, 238, Liberti, Micaela 154, 120, 90, 269, 290, 261, 158, 126, 270, 240, 101, 266, 272, 220, 155, 121, 141, 151, 169, 186, Kulbacka, Julita 270, Limnaios, Athanasios 164, Mancuso, Mariateresa 129, Kumar, Rupesh 193, Lindau, Manfred 167, 276, Manglano, Ximena 158, 229, Kumar, Siva 203, Lione, Luicia 219, 145, 143, Kumru, Tugba 80, Mannino, Loris 155, 120, Lisec, Barbara 230, 218, 198, Kunte, Christian 203, 204, 202, Maoyafikuddin, Mohammad 193, 124, Kupcic, Sasa 240, Marchalot, Julien 244, Liu, Hongmei 188, Kurihara, Jiro 304, Marcinauskas, Liutauras 259, Liu, Hongmei 179, Kurita, Hirofumi 82, Mariianats, Aleksandra 184, Liu, Xin 166, Kvapil, David 99, Marín-Sanchez, Javier 208, 86, Liu, Kefu 142, Kwao, Stephen 131, 281, Lombergar, Peter 136, Marino, Ramona 123, L Lopera, Daniella 276, Markelc, Boštjan 197, 190, 267, Lafitte, Luc 194, Lopez, Eric 155, 218, 198, 122, 230, Lagroye, Isabelle 171, López-Alonso, Borja 148, 298, Markovskaja, Svetlana 269, Lamberti, Patrizia 89, 287, 295, 281, 106, Marques, Paula 141, 90, Lourdes Luna Santana de An- Marra, Andrea 123, Lampreht Tratar, Urša 146, 124, drade, Daniella 105, Marracino, Paolo 121, 151, 155, 240, 146, 222, Lucía, Óscar 106, 281, 298, 187, 169, 154, Lanciotti, Rosalba 252, 148, Marshall, Guillermo R. 292, Landi, Giovanni 112, Lynam-Lennon, Niamh 177, Marshall, Guillermo R. 256, Lankutyte, Ieva 116, Lytras, Fotios 97, Martarelli, Nico 123, Lanssens, Lize 148, Martí-Quijal, Francisco J. 202, Larkin, John 123, M Martín-Belloso, Olga 257, 73, Larrea-Wachtendorff, Dominique Ma, Chi 92, 302, 282, 296, Ma, Jianhao 188, Martinelli, Eugenio 249, 110, Larsen, Majbritt M. E. 235, 145, Ma, Jianhao 179, Martinelli, Nicolò 265, Larsson, Peter 168, Maag, Patricia 113, Martinez, Juan Manuel 139, Lazar, Gyorgy 180, Maarir, Hafida 305, Martínez, Juan Manuel 201, 86, Le Berre, Théo 244, Mačianskienė, Regina 116, Martinez, Cristina 152, 155, Leguèbe, Michael 246, Maciulevičius, Martynas 82, Martínez, Juan Manuel 137, Lei, Yuan 108, Martinkova, Stanislava 99, Mączyńska, Justyna 264, Lei, Yizhen 262, Marzano, Flaviana 197, Maddineni, Sivanag 134, Lekešytė, Barbora 126, 261, 270, Marzi, Julia 184, Maglietti, Felipe 229, 158, 236, 158, 186, 290, Más Estellés, Jorge 152, 175, 175, Lepore, Fabio 103, Mascherini, Matteo 202, 204, Leroy, Jean-Baptiste 121, Mahar, Bhupendra 134, 203, Lertora, Raquel 236, 157, 284, Maher, Stephen 177, Masuda, Naoya 132, 304, Leschiera, Emma 216, 98, Mahnič-Kalamiza, Samo 162, Mathews, Kyle 116, Leveque, Philippe 226, 171, 216, 136, 103, Mathys, Alexander 210, 161, 94, Leveque, Philippe 225, Maji, Debnath 200, 93, Li, Lei 108, 286, Makarovas, Dominykas 84, Matkowski, Adam 149, 208, Li, Sixiang 297, Malakauskaitė, Paulina 158, 126, Mattei, Nicolas 170, 278, 90, 98, Li, Shiji 246, 270, 261, 186, 290, 269, 216, 263, 171, Li, Ying 114, Malik, Vaishali 160, Matteucci, Paolo 204, 313 Mattison, Lars M. 136, 80, Molina Hernandez, Junior Bern- Neutsch, Lukas 86, 302, Matys, Aleksandra 214, ardo 255, 252, Nie, Jing 132, McAuley, Megan 289, Moll, Xavi 117, Niemiera, Ben 78, McCormick, Paul 123, Monleón, Eva 148, Novac, Bucur M. 173, 144, McElheron, Matthew 123, 178, Monleon, Manuel 155, 154, 152, Novak, Marek 228, 99, McNamara, Jason 247, Montagna, Daniela 175, Novickij, Vitalij 269, 265, 264, Medved, Ajda 118, Montana, Carlotta 174, 238, 126, 101, 291, 266, 269, Mehigan, Brian 123, Monte Mor Rangel, Marcelo 177, 272, 273, Mencattini, Arianna 110, Monti, Giuseppina 296, Novickij, Vitalij 290, 158, 261, Menilli, Luca 265, Moodycliffe, Ailsa 130, 186, 186, 270, Menon, Meghana 178, 123, Moon, L. Boyce 78, Novickij, Jurij 126, 269, Merchiers, Pascal 199, Moosavi-Nejad, Zahra 271, Novickij, Vitalij 184, 102, Merla, Caterina 154, 154, 260, Morello, Emanuela 174, Novickij, Vitalij 237, 296, 249, 153, 243, 224, 110, Moreno, Victoria 155, 120, 154, Nowacka, Małgorzata 162, 258, 155, 129, 90, 154, Nuccitelli, Richard 100, Merlier, Franck 189, Moreno-Osorio, Luis 304, Nulty, Ciara 289, 239, Mi, Yan 92, 299, Moriyama, Akira 304, Numano, Rika 82, Michard, Lionel 171, 225, Morkvenaite-Vilkonciene, Inga O Michard, Lionel 226, 261, O’Brien, Barry 182, Michel, Olga 101, Moro, Valentina 140, O’Connell, Fiona 178, 123, Micheli, Laura 249, Mottschall, Tilo 93, O’Connor, Niamh 123, Michinski, Sebastian D. 157, 158, Moubarik, Amine 305, O’Sullivan, Jacintha 123, 177, 229, 175, 284, 143, 292, 236, Mowatt, David 202, 204, 178, Mickevičiūtė, Eglė 186, 158, 290, Mozolevskis, Gatis 107, Odili, Joy 204, 206, 203, 202, 126, 261, Muir, Tobian 228, 204, Oey, Indrawati 200, 113, 73, 110, Mickevičiūtė, Eglė 270, Muir, Tobian 231, Ohta, Nobuhiro 197, 224, Migliano, Emilia 181, Muldoon, Cian 123, Ojeda, Javier 220, 176, Miklavčič, Damijan 216, 134, Müller, Alexander 95, Okada, Yuji 165, 106, 103, 133, 136, 91, 78, 162, Muralidharan, Aswin 84, Oktar, Busra 254, Mikuž, Blaž 136, Murphy, Kirstan 123, Olah, Judit 287, Milevoj, Nina 146, 146, Murugan, Sri 231, Olah, Judit 180, Miller, Robert 200, Myhand, Autumn 78, Olaiz, Nahuel 256, 292, Miller, Matthew 78, N Omerzel, Maša 179, 230, 124, Minamitani, Yasushi 273, 303, Nafar, Nikki 275, 267, Minhas, Zobia 185, Nakamura, Tomohiro 304, Orlacchio, Rosa 171, Minuti, Marta 231, 125, 202, 230, Nanajian, Anthony 100, Oshin, Edwin A. 185, 288, Narinda, Irene 178, Osmančík, Pavel 99, 228, Mir, Luis M. 143, 224, 108, 154, Nastasi, Joseph 151, Ota Hisayasu Suzuki, Daniela 155, 144, 120, 275, 90, 153, 173, Nati Poltri, Simone 183, 227, 177, 105, Mittal, Lakshya 267, Naumova, Nataliia 153, Otten, Alex 73, 191, 147, Mittal, Lakshya 78, Navalinskas, Antanas 116, Ottlakan, Aurel 180, 287, Mockaitis, Tomas 261, Navarro Páez, Francisco 152, Overmann, Jed 78, Modic, Živa 190, Nazarova, Nataljia 210, O’Connor, Sean 223, 222, Mognaschi, Maria Evelina 90, Neic, Aurel 181, Moisescu, Mihaela G. 108, 109, Nelson, Nate 116, Ö Molango, Timothée 194, Nemeikaitė-Čėnienė, Aušra 261, Özmutlu, Özlem 113, 314 P Petras, Argyrios 181, Rafanavičius, Aras 253, Paccagnella, Melanie 219, Petrič, Miha 179, Rahman, Md Mijanour 277, Paffi, Alessandra 220, 90, 120, Peytral-Rieu, Olivia 119, Rajagopalan, Neeraj Raghura- 151, Phelan, James 178, man 217, Pagella, Fabio 230, Pieczywek, Piotr 214, Ramanavičienė, Almira 158, Pagoto, Oscar 235, 283, Pinto, Eleonora 219, Rampazzo, Elena 129, Paidar, Martin 196, Pipenhagen, Catherine 78, Ranchou-Peyruse, Anthony 144, Pakhomov, Andrei G. 71, 166, Pisani, Silvia 288, Rapp, Zachary 192, 81, Pisano, Carmen 121, Raso, Javier 208, 86, 97, 280, Pakhomov, Andrei G. 294, Plank, Gernot 181, 281, 139, 201, Pakhomova, Olga N. 81, 71, Pobiega, Katarzyna 162, Rauh, Cornelia 251, 113, Palego, Cristiano 250, Poignard, Clair 246, 183, 98, Ravetta, Valentina 231, Palepšiene, Ruta 82, 194, 216, 88, 275, 173, 144, 172, Ravi, Narayanasamy 123, Pallara, Tiziano 181, Polajžer, Tamara 106, Ray-Vista, William 217, Pandey, Saurabh Kumar 171, Poplová, Michaela 196, 301, Reberšek, Matej 106, 78, 215, Portillo, Franco 175, Recalde, Carolina 143, Panescu, Dorin 135, Poulletier de Gannes, Florence Reddy, Vivek 72, Papalexopoulou, Konstantia 171, Rembialkowska, Nina 270, 188, Rembiałkowska, Nina 263, 237, Poutier, Mathilde 263, Parisi, Paola 181, 238, 272, 265, 266, 264, Prabawati, Elisabeth 137, Parisi, Lorenza 174, Rems, Lea 72, 78, 301, 271, Prasad, Nadia 276, Parry, Adam 250, Ren, Lvheng 293, Prenner, Sarah Elisabeth 212, Parzych, Elizabeth 192, Reynolds, John 123, Pritchard-Jones, Rowan 204, Paškevičius, Algimantas 269, Riccardo, Federica 174, Prolič Kalinšek, Tine 134, Pasquali, Emanuela 243, Riva, Giuseppe 203, Proto, Maria Chiara 287, Pataro, Gianpiero 96, 112, Rival, Guilhem 244, Prusa, Jiri 215, Patel, Ami 192, Rivaletto, Marc 144, 173, Przystupski, Dawid 272, Patrignani, Francesca 252, 255, Rivière, Charlotte 244, Psakis, Georgios 97, Pawlik, Krzysztof J. 274, Rocculi, Pietro 255, 254, 202, Pecastaing, Laurent 144, 173, Q 252, Pecorari, Giancarlo 204, Qi, Luhao 114, Rodaitė, Raminta 127, Pedraza, Maria 155, Qian, Junyan 297, Rodak-Mirocka, Karolina 240, Peiro, Teresa 280, Qian, Kun 285, Rodrigues da Silva, Jéssica 227, Penzo, Chiara 221, Quaglino, Pietro 202, 204, Rodriguez, Isaac 292, Percherancier, Yann 171, Rodriguez, Roberto 223, q Perez, Byron 210, 161, Rodríguez Miranda, Jesica 143, qian, Kun 262, Perez Simba, Byron Patricio 93, 236, 229, 158, Perez-Won, Mario 296, 304, R Rodríguez Osuna, Isaac A. 256, Perić, Barbara 236, 203, Račnik, Joško 222, Rols, Marie-Pierre 90, 171, 263, Perrelli, Sonia 123, Radha Sivarajan, Sajeevan 131, 172, 278, 170, 119, 121, 98, 216, Perriello, Vincenzo Maria 123, Radzevičiūtė, Eivina 291, Romani, Santina 202, Persano, Luca 129, Radzevičiūtė-Valčiuke, Eivina Romeo, Stefania 89, 270, Perše, Martina 78, 266, 290, 186, Roscilli, Giuseppe 219, Perusso, Aldo 229, Radzevičiūtė-Valčiuke, Eivina Rossowska, Joanna 186, Pescatori, Lorenzo-Carlo 194, 158, 126, 270, 261, 238, 272, Rozsa, Petra 290, Pesole, Graziano 197, Rafanavičius, Aras 116, 273, Rudno-Rudzińska, Julia 237, Petit, Arthur 194, 280, 101, 315 Rudno-Rudziska, Julia 240, Sato, Yuya 300, Silvestre de Ferron, Antoine 173, Ruedlinger, Brrittney 100, Saucedo, María de los Ángeles 144, Ruiz, Adrián 280, 256, Silvestri, Barbara 203, 204, Russano, Francesco 204, 202, Saulė, Rita 127, 105, Silvestris, Alessandro 197, Ruzgys, Paulius 273, 126, 291, Saulis, Gintautas 105, 127, 215, Simoes, Brian 217, 82, 269, 83, 280, 253, Savopol, Tudor 108, 109, Singh, Julie 73, Ryan, Ciara 123, Sawada, Ryo 303, Sinha, Sayantani 200, Rybak, Katarzyna 162, 258, Sbirkov, Yordan 283, 268, Siniuk, Irina Mailén 256, Scarfì, Maria Rosaria 270, Siroli, Lorenzo 255, S Schebor, Carolina 256, Sabella, Tadeo 175, Smith, Lorraine 123, 178, 177, Schepler, Hadrian 202, 204, 203, Sabirovas, Tomas 250, Smith, Alison 94, Scherman, Michael 224, Saczko, Jolanta 101, 266, Smith, Trevor 192, Schmiedl, Benjamin 282, Sagurna, Cosima 123, Soba, Alejandro 292, Schneider, Bernard 140, Sahu, Praveen 292, 267, 295, Soden, Declan 123, 289, 178, Schottroff, Felix 251, 87, Saito, Koki 273, 303, 177, Schreieck, Jürgen 102, Salameh, Zaid 115, Sofroniew, Michael 140, Schröder, Sofie 209, Salati, Simona 260, 103, 243, Soliva-Fortuny, Robert 302, 282, Schulte, Jan-Michel 209, 141, 73, 257, Seccia, Veronica 203, 204, Salgado, Sergio S. 175, Solopov, Pavel A. 245, 128, Sefair, Juana 276, Salvatori, Erika 219, 145, Sommer, Marie-Christine 93, Semenov, Iurii 294, Samadieh, Samira 243, Sowa, Pamela 184, 102, Seror, Olivier 194, Samiaa, Romain 154, 155, 108, Spangler, Taylor 78, Serradeil, Léna 171, 120, 153, 154, Sprooten, Jenny 199, Serša, Gregor 222, 204, 124, Sanchez, Marina M. 155, Squair, Jordan 140, 179, 267, 190, 122, 146, 202, Sánchez, Jorge 148, Srimathveeravalli, Govindarajan 240, 228, 230, 146, 198, 260, Sánchez Gimeno, Ana Cristina 217, 160, Sevenich, Robert 150, C. 208, 86, Stabuc, Miha 260, Seyedi, MirHojjat 137, Sanchez Petidier, Marina 154, Staigvila, Gediminas 269, Sforzini, Matteo 112, Sanchez Petidier, Marina 153, Stalder, Diego 143, Shao, Qi 299, 128, Sandoval, Paulina 176, Stankevic, Voitech 259, Shaw, P. Nicholas 111, Sannino, Anna 270, Stirkė, Arūnas 107, 259, Shi, Guilan 73, Sano, Michael 116, 156, Strange, Gary 147, Shimizu, Yoshiharu 132, 304, Sano, Mike 286, Shin, Hee Chang 227, Strojan, Primoz 122, Santek, Iva 218, 197, Shu, Ting 279, Stručić, Marko 78, Santos, Pedro Paulo 227, Siccama, Joanne 165, Studer, Michael Hans-Peter 94, Santos, Pedro 77, Siegenthaler, Lya 86, Stühmeier-Niehe, Corinna 209, Sapio, Luigi 270, Siemer, Claudia 213, 162, 251, Suárez, Cecilia 292, Sapurov, Martynas 107, 96, 209, 85, Sundararajan, Raji 292, 295, Sarafian, Victoria 268, Sieni, Elisabetta 292, 287, 123, 267, 287, Sarafian, Victoria 283, 90, 242, 265, 295, 89, Sussman, Carleigh 200, Sarnago, Héctor 281, 148, 298, Sigg, Daniel C. 136, 80, Sutter, Olivier 88, 194, 187, 106, Signori, Emanuela 123, 175, Sventoraitiene, Jurgita 127, Sarreshtehdari, Amirhossein 197, Synowiec, Jody 73, 117, Silkunas, Mantas 71, 166, Szewczyk, Anna 265, Sasahara, Ryo 138, 304, 132, Silkuniene, Giedre 71, Szewczyk, Anna 290, 158, Sasset, Linda 200, Silva-Ferrer, Danela 296, Szewczyk, Anna 263, 237, 316 Szewczyk, Anna 274, 272, 266, Timm, Ulfilas 93, Vallet, Leslie A. 108, 173, 153, 264, Timmermans, Rian A. 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H. 160, Tanori, Mirella 129, 110, 260, Turk, Tina 78, Vuviet, Hoang 131, Tao, Xiaonan 142, Turner, Mark 137, Vykertas, Salvijus 274, 126, 82, Taoukis, Petros 164, Tylewicz, Urszula 254, 202, 252, 83, Tappi, Silvia 202, 252, 255, 254, 255, Tarakji, Khaldoun 80, W Ü Tarek, Mounir 173, 101, Waldert, Kate 212, Übelhör, Katharina 86, Tarone, LIdia 174, Wang, Lang-Hong 255, Tarricone, Luciano 296, 260, U Wang, Yancheng 262, Tasu, Jean Pierre 194, Uetake, Shunsuke 134, 79, Wang, Rui 132, Uí Mhaonaigh, Aisling 178, 123, Tauceri, Francesca 203, Wang, Chunxi 190, 177, Tedesco, Marinella 181, Wang, Sicong 293, Ullmann, Jörg 93, Tellado, Matias N. 284, 175, 175, Wang, Ren 297, Urbanskas, Ernestas 83, 126, Teplan, Michal 131, Wang, Yiting 137, 111, 116, 82, Thamsuaidee, Arisa 96, Wang, Yancheng 188, Ursic Valentinuzzi, Katja 198, Thanou, Ioanna 164, Weiner, David B. 192, 146, 122, Thaokar, Rochish 276, 278, Wellburn, Shaun 231, Uscila, Rolandas 259, Thaokar, Rochish 193, Wiktor, Artur 214, Thaokar, Rochish 193, V Williamson, Robert H. 286, 116, Thomas, Debby 199, Vahalová, Petra 196, 156, Thompson, Andy 200, Valdramidis, Vasilis P. 96, Wink, Stephanie 213, Thulasidas, Jeya Shree 297, Valdramidis, Vasilis 97, Wise, Julia 206, 317 Witrowa-Rajchert, Dorota 214, Yao, Chenguo 108, 215, 293, Zdunek, Artur 214, 162, 286, 262, 188, 249, 179, 285, Zemlin, Christian W. 80, Witrowa-Rajchert, Dorota 258, Yazdanparast, Seyed Mojtaba Zeng, Xin-An 132, 114, 163, 255, Wohlgemuth, Walter A. 74, 243, Zeni, Olga 270, Woods, Aoibhín 123, 178, 177, Yazdanparast, Razieh 243, Zhabin, Alexey 173, 144, Wu, Feiyu 215, Ying Leong, Sze 113, 110, Zhang, Junrui 297, Wu, Shengwei 217, You, Yuehua 297, Zhao, Yajun 246, 114, Wu, Ling-Gang 169, Yu, Jakraphan 80, Zhao, Lisheng 188, Wu, Shulin 297, Yu, Liang 293, Zheng, Wei 299, Yuan, Fan 190, Zhou, Xingyou 297, X Zigmantaitė, Vilma 116, Xiao, Shu 128, 245, Z Zaccara, Serena 242, Zinkevičienė, Auksė 186, 126, Xu, Xin-Dong 114, Zaharoff, David 156, Zinovicius, Antanas 261, Xu, Zhi-Qu 134, Zakar, Tomáš 196, 301, 131, Zocher, Katja 93, Xu, Dong 114, 246, Zambotti, Alessandro 110, 249, Zwietering, Marcel 130, Xue, Yumei 297, 154, Ž Y Zamuner, Annj 123, 265, 287, Želvys, Augustinas 186, 126, Yakovlev, Vladislav V. 222, 292, 242, Žnidar, Katarina 146, 190, Yan, Bingyu 138, 132, 304, Zappatore, Marco Salvatore 296, Yang, Qiang 262, 260, Ł Yang, Lifang 185, Zare, Farzan 137, 151, 111, Łapińska, Zofia 266, 318 Document Outline WC2024_BoA_Cover WC2024_BoA P R O G R A M M E P L E N A R Y L E C T U R E S ' A B S T R A C T S O R A L P R E S E N T A T I O N S ' A B S T R A C T S P O S T E R P R E S E N T A T I O N S ' A B S T R A C T S Authors' Index