4 th World Congress on Electroporation and Pulsed Electric Fields in Biology, Medicine, and Food & Environmental Technologies Copenhagen, Denmark 9−13 October, 2022 Book of Abstracts Organised by: ISEBTT − The International Society for Electroporation-Based Technologies and Treatments Edited by: Julie Gehl, Stine Frandsen Dobbelaar, and Samo Mahnič-Kalamiza ____________________________________________________ Kataložni zapis o publikaciji (CIP) pripravili v Narodni in univerzitetni knjižnici v Ljubljani COBISS.SI-ID 127487747 ISBN 978-961-243-444-1 (PDF) ___________________________________________________ URL: https://wc2022.electroporation.net/wp-content/ uploads/2022/10/ World_Congress_Electroporation_2022_Copenhagen_- _Book_of_Abstracts.pdf Copyright © 2022 Založba FE. All rights reserved. Razmnoževanje (tudi fotokopiranje) dela v celoti ali po delih brez predhodnega dovoljenja Založbe FE prepovedano. Založnik: Založba FE, Ljubljana Izdajatelj: Fakuleta za elektrotehniko, Ljubljana Urednik: prof. dr. Sašo Tomažič 1. elektronska izdaja P R O G R A M M E Plenary Lectures Page Monday Plenary Talks, Monday, Oct 10 2022, 8:50-10:20 Location: Congress Hall Session: Plenary Talks 37 Chairs: Richard Heller and Emanuela Signori Organizers/Conveners: n/a 8:50 Applying in vivo electroporation to large scale vaccination and immuno- 37 PL-01 therapies: Is this a moon shot? Matti Sallberg 9:20 Pulsed electric fields for winemaking: from the test tube to glass of wine 37 PL-02 Javier Raso 9:50 Gene Electrotransfer: Better understanding for better utilization 37 PL-03 Marie-Pierre Rols Tuesday Plenary Talks, Tuesday, Oct 11 2022, 8:30-10:00 Location: Congress Hall Session: Plenary Talks 38 Chairs: Damijan Miklavčič and Kars Neven Organizers/Conveners: n/a 8:30 Achieving non-invasive therapy and drug/vaccine delivery: Challenges and 38 PL-09 prospects Hamid Hosano 9:00 Preclinical Insights into Electroporation and the Myocardium: What have 38 PL-10 we achieved and what do we hope to achieve? Preclinical insights Jacob Koruth 9:30 Irreversible electroporation for the treatment of brain cancer: History and 38 PL-04 Future Directions Rafael V. Davalos Wednesday Plenary Talks, Wednesday, Oct 12 2022, 8:30-10:00 Location: Congress Hall Session: Plenary Talks 38 Chairs: Lluis Mir and Muriel Golzio Organizers/Conveners: n/a 8:30 Electrochemotherapy and IL-12 gene electrotransfer in veterinary medi- 38 PL-05 cine Maja Čemažar 9:00 Local intratumoral electroporation delivery of interleukin 12 immunother- 39 PL-11 apy leads to systemic anti-cancer response Adil Daud 9:30 Back to basics: Electroporation of artificial cells and what we can learn 39 PL-06 from them Rumiana Dimova 5 Thursday Plenary Talks, Thursday, Oct 13 2022, 9:00-10:00 Location: Congress Hall Session: Plenary Talks 40 Chairs: Erika Gabriella Kis and Giulia Bertino Organizers/Conveners: n/a 9:00 A snapshot of clinical research on electrochemotherapy: progress and fu- 40 PL-07 ture directions Luca G. Campana 9:30 Tumor Treating Fields: A Novel Treatment Modality for Solid Tumors 40 PL-08 David D. Tran 6 Oral Presentations Page Sunday Educational Session Track, Sunday, Oct 09 2022, 13:00-17:00 Location: Harlequin and Columbine Rooms combined Session: Educational Session 45 Chairs: Caterina Merla and Claudia Muratori Organizers/Conveners: Caterina Merla and Claudia Muratori 13:15 Pulsed electric fields 45 OR-03 Antoni Ivorra 13:45 Applications of pulsed electric field in the food industry - Educational 45 OR-192 session Federico Gómez Galindo 14:15 Pulsed Electric Field (PEF) Technology for Environmental Applications 45 OR-01 Christian Gusbeth 15:30 Biomedical applications of electroporation 46 OR-02 Gregor Serša 16:00 Electrochemotherapy- from Bench to Bedside and Beyond 46 OR-126 A. James P. Clover 16:30 Pulsed electric fields and the cardiovascular system: cardiac ablation and 47 OR-223 beyond Elad Maor Monday morning Track A, Monday, Oct 10 2022, 10:50-12:10 Location: Pierrot Room Session: P1 - General Applications for Food Processing 47 Chair: Federico Gómez Galindo Organizers/Conveners: Federico Gomez and Francesco Marra 10:50 Dielectric analysis of Gram-positive and -negative bacteria subjected to 47 OR-84 pulsed electric fields Hirotaka Nuki, Sunao Katsuki, Atsushi Tanaka, Kaichi Miyazaki, Ryuya Kimura 11:05 Assessment of microbial inactivation and lipid oxidation using non-thermal 47 OR-85 plasma on mussels Cinzia Mannozzi, Roberta Foligni, Lama Ismaiel, Silvia Tappi, Romolo Laurita, Luca Belleggia, Cristiana Cesaro, Andrea Osimani, Massimo Mozzon 11:20 Application of moderate electric fields to improve mass transfer steps in 48 OR-87 fruit processing Anne Kathrin Baier, Justus Knappert, Cornelia Rauh 11:35 Enhancement and Reversion of Irreversible Electroporation via Osmotic 48 OR-88 Shock Treatment Greta Gančytė, Povilas Šimonis, Arūnas Stirkė Monday morning Track B, Monday, Oct 10 2022, 10:50-12:10 Location: Harlequin Room Session: P12 - Electroporation and Cellular Pathways 49 Chairs: Mihaela Moisescu and Olga Pakhomova Organizers/Conveners: TBA 7 10:50 Identification of the ion channels affecting membrane permeabilization by 49 OR-167 nanosecond electric pulses Giedre Silkuniene, Uma Mangalanathan, Alessandra Rossi, Andrei G. Pakhomov, Olga N. Pakhomova 11:05 Denaturation of proteins subjected to 1 ns-long MV/cm electrical pulses 49 OR-168 Shin Goto, Yuya Sato, Koki Tsurusaki, Sunao Katsuki 11:20 Pulsed electric fields as tool for human skin remodeling 50 OR-170 Sara Gouarderes, Camille Ober, Layal Doumard, Jany Dandurand, Patricia Vicendo, Isabelle Fourquaux, Alexander Golberg, Valérie Samouillan, Laure Gibot 11:35 Electropermeabilization and the packing of bilayer lipids: a problem ad- 50 OR-171 dressed by real-time fluorescence measurements Ioan Tivig, Mihaela G. Moisescu, Tudor Savopol 11:50 Microsecond pulsed electric fields: effects on U87-cell line in different cul- 51 OR-172 ture conditions Arianna Casciati, Mirella Tanori, Elena Rampazzo, Isabella Gianlorenzi, Luca Persano, Giampietro Viola, Alice Cani, Silvia Bresolin, Carmela Marino, Mariateresa Mancuso, Caterina Merla Monday morning Track C, Monday, Oct 10 2022, 10:50-12:10 Location: Congress Hall Session: P34 - Development of pulse generators and electrodes 51 Chairs: Saulius Šatkauskas and Mark Jaroszeski Organizers/Conveners: Saulius Šatkauskas and Mark Jaroszeski 10:50 Piezoelectric Transformer based High-Voltage Pulse Generator using 51 OR-47 Wide-bandgap Semiconductors for Electroporation Ajay Chole, Maeve Duffy 11:05 Four channel 12 kV, 50 A, 100 ns - 100 µs generator for biomedical and 52 OR-98 biotechnological applications Aleh Kandratsyeu, Uladzimir Sabaleuski, Luis Redondo, Andrei G. Pakhomov 11:20 Super boosting for sub-nanosecond switching of high voltage SiC MOSFET 52 OR-48 Alicia del Barrio Montañés, Viliam Senaj, Thomas Kramer, Martin Sack 11:35 Histopathology study of nsPEF from a novel electroporator 53 OR-97 Ilan Wyn Davies, Paul Sibbons, Chris Hancock 11:50 Microfluidic chip with multi-detection modules for treatment of viable cells 53 OR-114 by pulsed electric field Arūnas Stirkė, Neringa Bakute, Virginija Siauruseviciute, Paulina Kizinievic, Justina Kavaliauskaite Monday morning Track D, Monday, Oct 10 2022, 10:50-12:10 Location: Columbine Room Session: P8 - Mechanisms of Cell Death in Electroporation-based Therapies 54 Chairs: Boris Rubinsky and Lluis Mir Organizers/Conveners: Boris Rubinsky and Lluis Mir 10:50 Acute ATP Loss During Irreversible Electroporation Supports Caspase 54 OR-57 Independent Cell Death Leo Razakamanantsoa, Neeraj Raghuraman Rajagopalan, Francois Cornelis, Michele Sabbah, Isabelle Thomassin-Naggara, Yasushi Kimura, Govind Srimathveeravalli 11:05 Characterization of the immunogenic cell death for electroporation treat- 54 OR-58 ments Nastaran Alinezhadbalalami, Kailee David, Zaid S. Salameh, Kenneth N. Aycock, Rafael V. Davalos 8 11:20 The Dynamics of Extraction and Impact of Intracellular DAMPs Released 55 OR-59 from Irreversibly Electropermeabilized Cells on the Viability of Electro- porated Cells Baltramiejus Jakštys, Rūta Palepšienė, Salvijus Vykertas, Dovilė Uždavinytė, Ingrida Šatkauskienė, Saulius Šatkauskas 11:35 Nano- and microsecond electric field thresholds for killing epithelial and 55 OR-60 smooth muscle cells Emily Gudvangen 11:50 Spatial distribution of permeabilization, pH fronts and temperature in a 56 OR-61 2D tissue model induced by electrolytic electroporation: a numerical study Enaide Maine Calzado, Luis Enrique Bergues Cabrales, Nahuel Olaiz, Pablo Turjanski Monday afternoon Track A, Monday, Oct 10 2022, 13:30-14:45 Location: Pierrot Room Session: P13 - Biological Processes Induced by Electrotransfer of Molecular Cargo 56 Chair: Fan Yuan Organizers/Conveners: Fan Yuan 13:30 Palmitoylation of STING following DNA electroporation in mouse skeletal 56 OR-49 muscle Amanda E. Sales Conniff, Jared Tur, Kris Kohena, Min Zhang, Justin Gibbons, Loree Heller 13:45 Electroporation as an aid in obtaining extracellular vesicles from cancer 57 OR-51 cells. Increasing the efficiency of EVs isolation using differential centrifu- gation Urszula Szwedowicz, Anna Choromańska 14:00 Improving Electrotransfer with Nonreducing Sugars 57 OR-52 Fan Yuan 14:15 Gene electrotransfer: comparison of parameters used in electrogenother- 57 OR-53 apy and high frequency electroporation on gene expression Alexia DeCaro, Marie-Pierre Rols, Muriel Golzio, Elisabeth Bellard 14:30 Electrotransfer of molecules in the reconstructed skin model 57 OR-54 Geraldine Alberola, Marie-Pierre Rols, Elisabeth Bellard, Alexia DeCaro, Muriel Golzio Monday afternoon Track B, Monday, Oct 10 2022, 13:30-14:45 Location: Harlequin Room Session: P2 - Electric Fields in Fermentation and Wine Production 58 Chairs: Javier Raso and Eugène Vorobiev Organizers/Conveners: Javier Raso and Eugene Vorobiev 13:30 Inactivation of resistant Escherichia coli by combining antibiotics with 58 OR-50 electroporation Saša Haberl-Meglič, Dejan Slokar, Damijan Miklavčič 13:47 Microbial stabilization of wine by Pulsed electric fields (PEF) 58 OR-118 Carlota Delso, Alejandro Berzosa, Ignacio Alvarez-Lanzarote, Javier Raso 14:04 Improved Pulsed Electric Field processing units for decontamination of 59 OR-117 thermosensitive protein-rich foods Claudia Siemer, Christopher McHardy, Marc Schmidt, Justus Knappert, Stefan Toepfl, Cornelia Rauh 9 14:21 Effectiveness of Pulsed Electric Field Treatment on Spices on Quality Prop- 59 OR-05 erties, Aflatoxin Decomposition and Its Mutagenity: Red Pepper Case Gülsün Akdemir Evrendilek, Nurullah Bulut, Bahar Atmaca, Sibel Uzuner Monday afternoon Track C, Monday, Oct 10 2022, 13:30-14:45 Location: Congress Hall Session: P34 - Development of pulse generators and electrodes 59 Chairs: Saulius Šatkauskas and Mark Jaroszeski Organizers/Conveners: Saulius Šatkauskas and Mark Jaroszeski 13:30 A novel electrode for Transurethral Electroporation Based Treatments for 59 OR-99 Bladder Tumors Balthazar M. BAH Hoffmann, Juan JLV Luis Vásquez, Julie Gehl 13:45 Effect of electrically insulating the tip and/or a part of the circumference of 60 OR-111 the active needle length on the electric field line pattern and temperature gradient during irreversible electroporation Annemiek M. Hogenes, Cornelis H. Slump, Gerben A. te Riet o.g. Scholten, Martijn W. J. Stommel, Jurgen J. Fütterer, Rudolf M. Verdaasdonk 14:00 An intradermal, hand-held electroporation device for in vivo applications 60 OR-112 Emran O. Lallow, Nandita C. Jhumur, Christine Roberts, Jerry W. Shan, David I. Shreiber, Jonathan P. Singer, Jeffrey D. Zahn, Joel N. Maslow, Hao Lin 14:15 An in vivo Electrotransfer Instrument that Incorporates Tissue Heating 61 OR-113 and Impedance Feedback-Based Electropulsation Mark J. Jaroszeski, Alex Otten, Andrew M. Hoff, Timothy J. Fawcett, Richard Heller 14:30 Design of a versatile nanosecond pulsed electric field (nsPEF) system 61 OR-201 Ilan Wyn Davies, Paul Sibbons, Chris Hancock Monday afternoon Track D, Monday, Oct 10 2022, 13:30-14:45 Location: Columbine Room Session: P9 - Electroporation-based vaccines and immunogenic effects of electroporation 62 Chairs: Loree Heller and Maja Čemažar Organizers/Conveners: TBA 13:30 Impact of electrochemotherapy on immune cells in the context of cancer 62 OR-203 – In vitro study Maura B. Bendix, Aileen Houston, Beth Brint, Patrick F Ford 13:45 Immunological effects of electrochemotherapy in b16f10, 4t1 and ct26 mur- 62 OR-202 ine cell lines in vitro Ursa Kesar, Tanja Jesenko, Bostjan Markelc, Katja Ursic Valentinuzzi, Maja Čemažar, Primoz Strojan, Gregor Serša 14:00 Skin dendritic cell mobilization upon electrochemotherapy: a dynamic 63 OR-204 analysis by fluorescence microscopy and flow cytometry Elisabeth Bellard, Nathalie Joncker, Marie-Pierre Rols, Muriel Golzio 14:15 Release of damage-associated molecular pattern molecules in vitro 63 OR-205 Tamara Polajžer, Tomaž Jarm, Damijan Miklavčič 14:30 The Influence of New High-Frequency Nanosecond Electrochemotherapy 64 OR-10 on the Elimination of LLC1 Tumours, Prolonging C57BL/6J Mice Survival and Positively Affecting Changes in Immune Cell Subpopulations Eivina Radzevičiūtė, Austėja Balevičiūtė, Augustinas Želvys, Vitalij Novickij, Veronika Malyško-Ptašinskė, Jurij Novickij, Irutė Girkontaitė 10 Monday late afternoon Track A, Monday, Oct 10 2022, 16:00-17:40 Location: Pierrot Room Session: P6 - Microalgae Biorefinery 64 Chairs: Gianpiero Pataro and Nabil Grimi Organizers/Conveners: Nabil Grimi and Gianpietro Pataro 16:00 Integration of Pulsed Electric Field (PEF) Technology into the Microalgae 64 OR-41 Biorefinery Christian Gusbeth, Damaris Krust, Wolfgang W. Frey 16:25 Microfluidic devices for the optimization of pretreatments in the context 65 OR-40 of microalgae-based productions Solène Prudhomme, Sakina Bensalem 16:40 No downstream without upstream: Challenges for PEF treatment of Arth- 65 OR-42 rospira platensis and studies on the mechanism of phycocyanin release Justus Knappert, Christoph Lindenberger, Christopher McHardy, Cornelia Rauh 16:55 Nanosecond Pulsed Electric Fields selectively foster cell proliferation in 66 OR-43 heterotrophic microalgae Byron Perez Simba, Robert Axelrod, Iris Haberkorn, Alexander Mathys 17:10 Scaling microalgal biomass extraction for industrial application 66 OR-44 Katja Zocher, Martina Balazinski, Marie-Christine Sommer, Raphale Rataj, Michael Timm, Marco Kreische, Elke Kurth, Petra Sandau, Juergen F Kolb 17:25 Application of pulsed electric fields and mechanical cell disintegration tech- 67 OR-45 niques in biorefinery cascade of microalgae Daniele Carullo, Francesco Donsì, Giovanna Ferrari, Gianpiero Pataro Monday late afternoon Track B, Monday, Oct 10 2022, 16:00-17:30 Location: Harlequin Room Session: P10 - Gene Electrotransfer for Immunotherapy 67 Chairs: Richard Heller and Emanuela Signori Organizers/Conveners: Siqi Guo and Kevin Hollevoet 16:00 Transfection by electroporation using pulses from nano- to millisecond 67 OR-67 duration in vitro Tjaša Potočnik, Tamara Polajžer, Alenka Maček-Lebar, Damijan Miklavčič 16:15 Skin dendritic cell mobilization upon Gene electrotransfer (GET) 68 OR-68 Elisabeth Bellard, Lise Pasquet, Sophie Chabot, Marie-Pierre Rols, Muriel Golzio 16:30 Electrophoretic Infusion of DNA into Gels and Tissues: Experimental and 68 OR-69 Theoretical Analysis Shaurya Sachdev, Damijan Miklavčič 16:45 Immunomodulatory effects of radiotherapy and gene electrotransfer of 68 OR-70 plasmid DNA encoding chemokines CCL5 and CCL17 in murine tumors Tim Bozic, Lucija Kozjek Mencinger, Simona Kranjc Brezar, Gregor Serša, Iva Santek, Maja Čemažar, Bostjan Markelc 17:00 OncoSec’s 2nd Generation Therapy: Amplification of the CXCR3/CXCL9 69 OR-71 axis by intratumoral electroporation of plasmid CXCL9 synergizes with plasmid IL-12 therapy to elicit robust anti-tumor immunity Jun Zhang, Jack Lee, Erica Browning, Mia Han, Kurt Sakurado, Vincent Wu, Nygel Oglesby, Tarsicio Juarez, H. Kim Lyerly, Daniel Fisher, Adil Daud, Alain Algazi, Joseph Skitzki, Chris Twitty, David A. Canton 17:15 Differences in Nano-Electrotransfection Efficiency Between Cancer and 69 OR-139 Primary Murine Immune Cells Eivina Radzevičiūtė, Veronika Malyško-Ptašinskė, Vitalij Novickij, Jurij Novickij, Irutė Girkontaitė 11 Monday late afternoon Track C, Monday, Oct 10 2022, 16:00-17:30 Location: Congress Hall Session: P15 - Nanosecond Pulse Stimulation and Electropermeabilization (MURI AFOSR) 70 Chairs: Andrei Pakhomov and Bennett Ibey Organizers/Conveners: Andrei Pakhomov and Bennett Ibey 16:00 Focusing nanosecond pulse effects away from electrodes 70 OR-04 Andrei G. Pakhomov, Iurii Semenov, Vitalii Kim, Aleh Kandratsyeu, Luis Redondo, Emily Gudvangen, Shu Xiao, Allen Kiester, Joel N. Bixler, Bennet L. Ibey 16:30 Nanosecond electric pulses target the alpha-1 subunit of membrane Na,K- 70 OR-06 ATPase Giedre Silkuniene, Uma Mangalanathan, Andrei G. Pakhomov, Olga N. Pakhomova 16:45 Ultrafast Imaging enabling the direct observation of the charging dynamics 71 OR-07 caused by pulsed electric field effects Joel N. Bixler, Zachary Steelman, Allen Kiester, Bennett L. Ibey 17:00 Excess Efficacy of Nanosecond Stimulation in Cardiomyocytes 71 OR-08 Christian W. Zemlin, Federica Serra 17:15 Cellular Excitability and nsPEFs: Involvement of lipid oxidation in action 72 OR-09 potential activation Aurianne Rainot, Lea Rems, Mounir Tarek Monday late afternoon Track D, Monday, Oct 10 2022, 16:00-17:30 Location: Columbine Room Session: P3 - Pulsed Electric Fields (PEF) Strategies in Enhancing Health-promoting Properties of Plant-based Foods 72 Chairs: Giovanna Ferrari and Pedro Elez-Martínez Organizers/Conveners: Pedro Elez-Martínez and Gianpiero Pataro 16:00 Pulsed electric fields (PEF) for the preservation and bioproduction of 73 OR-220 health-related compounds and properties in plant-based foods Robert Soliva-Fortuny, Olga Martin-Belloso, Pedro Elez-Martinez 16:25 PEF for the extraction and recovery of health-related compounds from 73 OR-221 vegetable by-products and wastes Giovanna Ferrari, Serena Carpentieri, Farid Soltanipour, Gianpiero Pataro 16:45 PEF-assisted processes for improving the health-related properties of plant 73 OR-125 foods Ignacio Alvarez-Lanzarote, Javier Raso 17:00 Evaluation of the beneficial effects of PEF-treated artichoke residues ex- 74 OR-124 tract on THP-1 human cell lines Serena Carpentieri, Giuseppina Augimeri, Jessica Ceramella, Adele Vivacqua, Stefania Sinicropi, Gianpiero Pataro, Daniela Bonofiglio, Giovanna Ferrari 17:15 Effect of Pulsed Electric Fields (PEF) on the extraction of phenolic com- 74 OR-123 pounds in orange by-products María del Carmen Razola-Díaz, Robert Sevenich, Eduardo Jesús EJGH Guerra-Hernández, Vito Verardo, Oliver Schlüter Tuesday morning Track A, Tuesday, Oct 11 2022, 10:30-12:10 Location: Pierrot Room Session: P11 - Immunogenic Effects of Electroporation-based Treatments 75 Chairs: Loree Heller and Maja Čemažar Organizers/Conveners: Loree Heller and Maja Čemažar 12 10:30 Adjuvant immunogenicity of nanosecond pulsed electric field anti-cancer 75 OR-206 treatments Claudia Muratori, Flavia Mazzarda, Alexandra E. Chittams-Miles, Julia Pittaluga, Andrew Ojeda, P. Thomas Vernier 10:55 Induction of bystander and abscopal effects after electroporation-based 75 OR-200 treatments Paulius Ruzgys, Neringa Barauskaite, Rūta Palepšienė, Baltramiejus Jakstys, Dovilė Uždavinytė, Salvijus Vykertas, Martynas Maciulevicius, Saulius Šatkauskas 11:10 Tumor Treating Fields-based Vaccination in Solid Tumors 75 OR-207 Dongjiang Chen, Son Le, David D. Tran 11:25 Nanosecond Electric Pulses Overturn Immunosuppression in the Tumor 76 OR-208 Microenvironment Siqi Guo, Anthony Nanajian, Megan Scott, Stephen J. Beebe 11:40 Employing the Electrochemical Side Effects of Pulsed Electric Fields 76 OR-209 Within the Tissue Microenvironment Zaid S. Salameh, Kenneth N. Aycock, Rafael V. Davalos 11:55 Immunogenicity of nucleic acid delivery to skin 77 OR-210 Tanja Jesenko, Maša Bošnjak, Katarina Znidar, Amanda E. Sales Conniff, Bostjan Markelc, Nina Semenova, Jared Tur, Kris Kohena, Simona Kranjc Brezar, Maja Čemažar, Loree Heller Tuesday morning Track B, Tuesday, Oct 11 2022, 10:30-12:10 Location: Harlequin Room Session: P14 - Cytoskeletal Changes and their Implications in the Different Types ofPulsed Electric Fields (PEF) based Technologies and Treatments 77 Chairs: Michal Cifra and Olga Zeni Organizers/Conveners: Olga Zeni and Michal Cifra 10:30 Suspended Nanonets Enable Precise Quantitation of Forces and Cyto- 77 OR-191 skeletal Changes Post Electroporation Philip M. Graybill, Aniket Jana, Rakesh Kapania, Rafael V. Davalos, Amrinder Nain 10:55 Molecular dynamics simulation studies of intense electric field on cytoskel- 78 OR-31 eton components Jiří Průša, Saurabh K. Pandey, Michal Cifra 11:10 Chip platform for PEF delivery to microtubules on total internal reflection 78 OR-33 fluorescence microscope Daniel Havelka, Ilia Zhernov, Michal Teplan, Zdenek Lansky, Djamel E. Chafai, Michal Cifra 11:25 Unraveling the interplay between DNA transport and cytoskeletal ele- 78 OR-30 ments during/after electroporation Pouyan E. Boukany 11:40 Transient disruption of blood-brain barrier on rat brains at low-voltage 78 OR-82 high-pulse using non-invasive electrode approach Neeraj Raghuraman Rajagopalan, William Ray Vista, Masashi Fujimori, Laurien G. P. H. Vroomen, Niranjan Khadka, Marom Bikson, Govind Srimathveeravalli 11:55 Cell shape, orientation, and pulsation buffer affect electroporation lethality 79 OR-32 for elongated cells aligned on suspended nanofibers Edward Jacobs, Philip M. Graybill, Aniket Jana, Atharva Agasha, Amrinder Nain, Rafael V. Davalos 13 Tuesday morning Track C, Tuesday, Oct 11 2022, 10:30-12:10 Location: Congress Hall Session: P21 - Electroporation for Cardiac Ablation: Mechanisms and Scientific Advances 79 Chairs: Damijan Miklavčič and Elad Maor Organizers/Conveners: Damijan Miklavčič and Elad Maor 10:30 Pulsed field ablation cardio-selectivity: Differential effect of High- 79 OR-173 Frequency Electroporation on myocardium and other tissues Yonatan Moshkovits, Dvora Grynberg, Eyal Heller, Leonid Maizels, Elad Maor 10:45 A Novel Single-Shot Pulsed Field Ablation System Is Associated with 80 OR-174 Large and Durable Ventricular Lesions In Vivo: A Preclinical Assessment of Safety and Efficacy Steffen Holzinger, Arash Aryana, Dorin Panescu, Cary Hata, Alan de la Rama, Ken Nguyen, André d’Avila 11:00 Direct and alternating current pulse filed ablation lesion comparison 80 OR-175 Martin Pesl, Eva Odehnalova, Jakub Hejc, Veronika Novotna, Petra Stachova, Vita Zampachova, Anna Siruckova, Dalibor Cervinka, Zdenek Starek 11:15 Multiscale Modeling of Pulsed Field Ablation in Anisotropic Myocardium 81 OR-181 Quim Castellvi, Emma Aoustin, Antoni Ivorra 11:30 Preventing cardiac cell damage after electric injuries 81 OR-182 Pamela Sowa, Aleksander Kielbik, Andrei G. Pakhomov, Emily Gudvangen, Uma Mangalanathan, Volker V.A. Adams, Olga N. Pakhomova Tuesday morning Track D, Tuesday, Oct 11 2022, 10:30-12:10 Location: Columbine Room Session: P4 - Electric Fields for Ohmic Heating in Food Processing 82 Chairs: Felix Schottroff and Sudhir Sastry Organizers/Conveners: Felix Schottroff and Sudhir Sastry 10:30 Differentiation of Thermal and Electric Field Effects During Ohmic Heat- 82 OR-115 ing Felix Schottroff 10:50 Electric Fields and their Effects on Vegetative Microorganisms, Spores and 82 OR-166 Enzymes Jin Hong Mok, Taras Pyatkovskyy, Chaminda P. Samaranayake, Sudhir K. Sastry 11:10 Industrial Applications of Ohmic Heating 82 OR-178 Henry Jaeger 11:30 Ohmic heating of patatin enriched potato protein: Influence of moderate 83 OR-55 electric fields on thermal induced gel properties Eike Joeres, Stephan Drusch, Stefan Töpfl, Ute Bindrich, Andreas Juadjur, Thore Völker, Volker Heinz, Nino Terjung 11:40 Pulsed Electric Fields as a new ohmic heating system for vegetable blanch- 83 OR-56 ing Leire Astráin Redín, Javier Raso, Guillermo J. Cebrián, Ignacio Alvarez-Lanzarote Tuesday afternoon Track A, Tuesday, Oct 11 2022, 13:30-14:45 Location: Pierrot Room Session: P20 - Electroporation-based Treatments in Veterinary Medicine II 84 Chairs: Felipe Maglietti and Maja Čemažar Organizers/Conveners: Felipe Maglietti and Maja Čemažar 14 13:30 Linear DNA amplicons delivered by electro-gene-transfer as veterinary 84 OR-75 Covid-19 vaccine candidate Antonella Conforti, Erika Salvatori, Lucia Lione, Mirco Compagnone, Eleonora Pinto, Brian Viscount, James A. Hayward, Clay D. Shorrock, Diego G. Diel, Fabio Palombo, Joe A. Impellizeri, Luigi Aurisicchio 13:50 Interleukin 12 gene electrotransfer to skin: experience from studies on 84 OR-76 pigs Ursa Lampreht Tratar, Tanja Jesenko, Karolina Belingar, Tanya Birk, Anja Osep, Maša Bošnjak, Gregor Serša, Maja Čemažar 14:05 A combination of electrochemotherapy and gene electrotransfer in canine 85 OR-77 stage III melanoma: Initial experience from peru Sergio S. Salgado, Matias M. Tellado, Emanuela Signori, Felipe H. Maglietti 14:20 From Cancer to COVID-19: gene electrotransfer as a versatile tool to 85 OR-218 design innovative veterinary vaccines Luigi Aurisicchio Tuesday afternoon Track B, Tuesday, Oct 11 2022, 13:30-14:45 Location: Harlequin Room Session: P28 - Electroporation for Cardiac Ablation: Clinical Use and Development 86 Chairs: Kars Neven and Jacob Koruth Organizers/Conveners: Kars Neven and Jacob Koruth 13:30 Epicardial high-density electrogram mapping dynamics during Pulsed 86 OR-46 Field Ablation (PFA) Tomas Garcia-Sanchez, Gerard Amorós-Figueras, Sergi Casabella-Ramon, Zoraida Moreno-Weidmann, Jose M. Guerra, Antoni Ivorra 13:45 Characterization of the effects of cryoablation, RF ablation or pulsed field 86 OR-176 ablation on compound action potentials of porcine phrenic nerves David A. Ramirez, Lars M. Mattison, Paul A. Iaizzo 14:00 Utilizing Human Induced Pluripotent Stem Cells to Study Cardiac Pulsed- 87 OR-177 Field Ablation Leonid Maizels, Eyal Heller, Michal Lendesberg, Irit Huber, Gil Arbel, Amira Gepstein, Roy Beinart, Amit Segev, Lior Gepstein, Elad Maor 14:15 Open-Chest Pulsed Electric Field Ablation of Cardiac Ganglionated Plexi 87 OR-179 in Acute Canine Models Martin van Zyl, Mariam Khabsa, Jason Tri, Thomas Ladas, Adetola O. Ladejobi, Omar Yasin, John Reilly, Barry O’Brien, Kenneth Coffey, Samuel J. Asirvatham 14:30 Early clinical experience with cardiac pulsed field ablation 88 OR-28 Jim Hansen 15 Tuesday afternoon Track C, Tuesday, Oct 11 2022, 13:30-14:45 Location: Congress Hall Session: P37 - Models for in vitro electroporation 88 Chairs: Urška Kamenšek and Laure Gibot Organizers/Conveners: Urška Kamenšek and Laure Gibot 13:30 Chitosan-based breast cancer cell cultures: a promising tool for in vitro 88 OR-127 evaluation of anticancer treatments Bianca Bazzolo, Annj Zamuner, Monica Dettin, Elisabetta Sieni, Patrizia Lamberti, Maria Teresa Conconi 13:45 Skin electroporation for non-invasive drug delivery:Electrical properties of 88 OR-128 skin models and fluorescent molecule delivery Georgios Y. Kougkolos, Juliette Simon, Geraldine Alberola, Bastien Jouanmiqueou, Marie-Pierre Rols, Lionel Laudebat, Muriel Golzio, Zarel Valdez-Nava, Emmanuel Flahaut 14:00 High-throughput cell transfection in a microfluidic electroporation chip 89 OR-129 Neringa Bakute, Elinga Brazionyte, Arūnas Stirkė 14:15 Inorganic nanoparticles as physical aids for local thermal ablation and 89 OR-130 electroporation enhancement: efficacy assessment in 2D and 3D cellular models Nicolas Mattei, Sebastjan Nemec, Slavko Kralj, Vincent Gruszka, Muriel Golzio, Marie-Pierre Rols, Jelena Kološnjaj-Tabi 14:30 Scanning electrochemical microscope as a tool for the electroporation 90 OR-131 Inga Morkvėnaitė-Vilkončienė, Antanas Zinovičius, Baltramiejus Jakštys, Arūnas Ramanavičius Tuesday afternoon Track D, Tuesday, Oct 11 2022, 13:30-14:45 Location: Columbine Room Session: P39 - Electroporation for clinical use 90 Chair: Julie Gehl Organizers/Conveners: TBA 13:30 Electrochemotherapy of portal vein tumor thrombus as dowstaging to liver 90 OR-119 transplantation Luciano Tarantino, Emanuele Balzano, Giuseppina Busto, Aurelio Nasto, Sara Bortone, Paolo Tarantino, Riccardo Aurelio Nasto, Paolo De Simone 13:45 Response to Calcium Electroporation in Cancers Affecting the Skin – a 90 OR-121 Phase II Clinical Study Mille Vissing, Mascha Pervan, Kitt Vestergaard, John Pløen, Mazen Schnefeldt, Søren Rafael Rafaelsen, Christina Louise Lindhardt, Lars Henrik Jensen, Achim Rody, Julie Gehl 14:00 Electrochemotherapy of Posterior Resection Surface for Lowering Disease 91 OR-83 Recurrence Rate in Pancreatic Cancer (PanECT Study) Mihajlo Djokić 14:15 Burst Sine Wave Electroporation for Large Blood-Brain Barrier Disrup- 91 OR-22 tion for Efficient Drug Delivery—A Feasibility Study Sabrina N. Campelo, Kenneth N. Aycock, Zaid S. Salameh, Rafael V. Davalos, John H. Rossmeisl, Melvin F. Lorenzo 14:30 Pain sensation and muscle contractions during delivery of high-frequency 92 OR-24 electroporation pulses Aleksandra Cvetkoska, Alenka Maček-Lebar, Peter Trdina, Damijan Miklavčič, Matej Reberšek 16 Tuesday late afternoon Track A, Tuesday, Oct 11 2022, 16:00-17:30 Location: Pierrot Room Session: P19 - Electroporation-based Treatments in Veterinary Medicine I 92 Chairs: Nataša Tozon and Joe Impellizeri Organizers/Conveners: Nataša Tozon and Joe Impellizeri 16:00 The use of electrochemotherapy in combination with other oncological 92 OR-217 therapies Matias M. Tellado, Juan Manuel Fernandez, Juan Manuel Osacar, Felipe Maglietti 16:20 From Bench-to Kennel-to Bedside: Deploying Novel Preclinical Animal 93 OR-74 Models of Cancer in the Development of Irreversible Electroporation for Human Patient Applications Irving C. Allen, Kiho Lee, Sherrie Clark-Deener, Christopher Byron, Michael R. Edwards, John H. Rossmeisl, Sheryl Coutermarsh-Ott, Kristin Eden, Nikolaos Dervisis, Shawna Klahn, Joanne Tuohy, Rafael V. Davalos 16:35 Electrochemotherapy in a pancreatic neuroendocrine tumor in a dog: A 93 OR-73 case report Sergio S. Salgado, Felipe H. Maglietti 16:50 Longer duty cycle effects of irreversible electroporation on pig’s pancreatic 94 OR-138 tissues: a pilot study Hong Bae Kim 17:05 Veterinary Guidelines for Electrochemotherapy of superficial tumors 94 OR-213 Felipe H. Maglietti, Matias M. Tellado, Lluis M. Mir Tuesday late afternoon Track B, Tuesday, Oct 11 2022, 16:00-17:30 Location: Harlequin Room Session: P22 - Irreversible Electroporation (IRE) and Immunotherapy 94 Chairs: Rafael Davalos and Leo Razakamanantsoa Organizers/Conveners: Rafael Davalos and Léo Razakamanantsoa 16:00 A phase II-study of electroporation potentiated immunotherapy in liver 94 OR-186 metastatic pancreatic cancer (EPIC-1) Rasmus Virenfeldt Flak, Laurids Østergaard Poulsen, Mogens Tornby Stender, Gintare Naujokaite, Olga Tcacenco, Alkwin Wanders, Sönke Detlefsen, Ralf Agger, Emil Kofod-Olsen, Ole Thorlacius-Ussing, Morten Ladekarl 16:15 Irreversible Electroporation and Immune Checkpoint Inhibitor Immuno- 95 OR-187 therapy provides improved primary and systemic cancer control Qi Shao, Minhan Jiang, John C. Bischof, Yoji Shimizu, Brandon J. Burbach 16:30 Irreversible electroporation selectively lyses cancer cells while preserving 95 OR-188 function and promoting tumor infiltration of chimeric antigen receptor (CAR) engineered T cells William-Ray Vista, Mary Sheehan, Stephen B Solomon, Prasad Adusumilli, Govind Srimathveeravalli 16:45 Experimental study on enhancing the bioelectric effect of tumor cells using 96 OR-214 the combination of nanosecond and microsecond pulsed electric field Wencheng Peng, Junyi Ning, Hongmei Liu, Shoulong Dong, Chenguo Yao 17:00 Decellularized intestinal tissue as a potential graft for bladder reconstruc- 96 OR-215 tion therapies Neeraj Raghuraman Rajagopalan, Brian Simoes, Feiyu Yang, Yubing Sun, Govind Srimathveeravalli 17 Tuesday late afternoon Track C, Tuesday, Oct 11 2022, 16:00-17:30 Location: Congress Hall Session: P32 - Pulsed electric field effects on neural tissues and the brain 97 Chair: Caterina Merla Organizers/Conveners: TBA 16:00 Characterization of Ca2+ fluxes modulation by nanosecond pulsed electric 97 OR-17 fields in neuroblastoma and mesenchymal stem cells Francesca Camera, Tomas Garcia-Sanchez, Barbara B. Benassi, Adeline Muscat, Claudia Consales, Leslie Vallet, Franck M. André, Carmela Marino, Lluis M. Mir, Caterina Merla 16:15 RISEUP: Regeneration of Injured Spinal cord by Electro pUlsed bio- 97 OR-18 hybrid imPlant Claudia Consales, Manuel Monleón Pradas, Paolo Marracino, Micaela Liberti, Franck M. André, Victoria Moreno Manzano, Caterina Merla, Jorge Más Estellés, Marco Balucani, Micol Colella, Francesca Apollonio, Lluis M. Mir 16:30 Effects of electrical stimulation in neural stem cells and mesenchymal stem 98 OR-19 cells cell fate Marina M. Sanchez Petidier, Romain Fernandes, Leslie Vallet, Franck Andre, Lluis M. Mir 16:45 High-rate nsPEF bursts stimulate neurons at paradoxically low electric 98 OR-16 field thresholds and without electroporation Mantas Silkunas, Emily Gudvangen, Andrei G. Pakhomov 17:00 Microsecond electric pulses effects on induced neuronal stem cells for re- 99 OR-20 generation of spinal cord injuries Giorgia G. Innamorati, Caterina Merla, Leslie Vallet, Marina M. Sanchez Petidier, Franck M. André, Laura L. Caramazza, Sara S. Fontana, Noemi N. Dolciotti, Maria M. Pedraza, Nesus N. Torres, Victoria Moreno Manzano, Barbara B. Benassi, Paola P. Giardullo, Francesca Apollonio, Paolo Marracino, Lluis M. Mir, Claudia Consales 17:15 Low pulsed electrical fields for inducing transient BBB disruption in a 99 OR-21 mouse model Shirley Sharabi, Yael Mardor, David Last, Dianne Daniels, Sigal Liraz-Zaltsman, Itzik Cooper Tuesday late afternoon Track D, Tuesday, Oct 11 2022, 16:00-17:30 Location: Columbine Room Session: P5 - Mechanisms and Applications of PEF in the Food Industry 100 Chairs: Claudia Siemer and Artur Wiktor Organizers/Conveners: Claudia Siemer and Artur Wiktor 16:00 The role of post-electroporation recovery on the survival of Thai basil 100 OR-89 leaves during drying Grant Thamkaew, Lars Wadsö, Allan G. Rasmusson, Federico Gómez Galindo 16:15 Evaluation of the Extraction Yield of Phenolic Compounds in Olive Leaf 100 OR-90 Treated with Pulsed Electric Fields María del Carmen Razola-Díaz, Robert Sevenich, Ana María Gómez-Caravaca, Oliver Schlüter, Vito Verardo 16:30 Suitability of different Electrode Materials for Pulsed Electric Field (PEF) 100 OR-212 Application Milena Zdravkovic, Eva Kancirova, Artur Wiktor, Ute Bindrich, Andreas Juadjur, Volker Heinz, Kemal Aganovic 18 16:45 Physical and chemical characterization of freeze-dried strawberries and 101 OR-95 red bell peppers pretreated by pulsed electric fields (PEF) Marianna Giancaterino, Henry Jaeger 17:00 Electrically conductive biocomposite film for in-pack pulsed electric field 101 OR-91 food sterilization Ana Barra, Cláudia Nunes, Eduardo Ruiz-Hitzky, Paula Ferreira 17:15 Optimization through Response Surface Methodology of Pulsed Electric 102 OR-120 Fields-Assisted Extraction of bioactive compounds from red grape pomace Serena Carpentieri, Giovanna Ferrari, Gianpiero Pataro Wednesday morning Track A, Wednesday, Oct 12 2022, 10:30-12:15 Location: Pierrot Room Session: P16 - New Technologies for Cells and Tissues Electropermeabilization 102 Chairs: Caterina Merla and Matej Kranjc Organizers/Conveners: Caterina Merla and Matej Kranjc 10:30 (Elongated) gold nanoparticles: injectable antennas locally amplifying the 102 OR-132 electroporation or just a thorn in our flesh? Jelena Kološnjaj-Tabi, Muriel Golzio, Marie-Pierre Rols 10:45 Application of a new electroporation microsystem to the study of the im- 103 OR-137 pact of tumor microenvironment on electrochemotherapy efficiency Pauline Bregigeon, Théo Le Berre, Julien Marchalot, Laure Franqueville, Christian Vollaire, Charlotte Riviere, Marie Frénéa Robin, Fréderic Prat 11:00 Spatially resolved, high efficiency electrotransfection on a CMOS micro- 103 OR-133 electrode array Bastien Duckert, Dries Braeken, Liesbet Lagae, Maarten Fauvart 11:15 The fabrication and operation of a continuous-flow microfluidic device for 104 OR-136 single-cell electroporation Maria Atzampou, Hao Lin, Jerry W. Shan, David I. Shreiber, Christine Roberts, Joel N. Maslow, Jeffrey D. Zahn 11:30 Localized single-cell electroporation using U shaped microstructures in a 104 OR-134 microfluidic channel Aswin Muralidharan, Georg Pesch, Hendrik Hubbe, Lea Rems, Mahdiyeh Nouri-Goushki, Pouyan Boukany 11:45 Smart, solid-state, nanosecond pulsed power techniques for medical, agro 105 OR-135 and environmental applications Guus Pemen 12:00 Optimization of Ablation Region and Electrode Positioning in H-FIRE via 105 OR-66 Machine Learning Alfredo De Cillis, Caterina Merla, Giuseppina Monti, Luciano Tarricone, Marco Zappatore Wednesday morning Track B, Wednesday, Oct 12 2022, 10:30-12:10 Location: Harlequin Room Session: P24 - Calcium Electroporation 105 Chairs: Erika Gabriella Kis and Julita Kulbacka Organizers/Conveners: Erika Kis and Julita Kulbacka 10:30 Phase II Investigation of the Histopathologic Effect of Calcium Electro- 105 OR-110 poration on Cancer in the Skin – CaEP-B Mille Vissing, Sandra Kristiane Sinius Pouplier, Lars Munch, Stine Frandsen, Anne-Vibeke Lænkholm, Julie Gehl 19 10:45 Endoscopic calcium electroporation in patients with Barrett’s esophagus 106 OR-160 high-grade dysplasia: A first-in-man phase 1 study Laser Arif Bazancir, Charlotte Egeland, Rajendra Singh Garbyal, Julie Gehl, Michael Patrick Achiam 11:00 Effectiveness of calcium electroporation on human sensitive and resistant 106 OR-161 breast cancer cells Jolanta Saczko, Anna Choromańska, Julita Kulbacka, Katarzyna Bieżuńska-Kusiak 11:15 Endoscopic Calcium Electroporation for Colorectal Cancer: a phase I 107 OR-162 study Malene Broholm, Rasmus Vogelsang, Mustafa Bulut, Trine Stigaard, Hanne Falk Hansen, Stine Frandsen, Dorte Levin Pedersen, Trine Perner, Anne-Marie Kanstrup Fiehn, Andreas Weinberger Rosen, Christina Andersen, Niels Pallisgaard, Ismail Gö- genur, Julie Gehl 11:30 Impact of variety type of calcium electroporation protocols on selected 107 OR-163 cellular attributes in human colon cancer Anna Szewczyk, Nina Rembiałkowska, Anna Choromańska, Katarzyna Bieżuńska-Kusiak, Jolanta Saczko, Julita Kulbacka 11:45 Antitumor effects of nanosecond PEFs with calcium ions in colon cancer 108 OR-164 in vitro and in vivo Julita Kulbacka, Joanna Rossowska, Agnieszka Chwikowska, Anna Choromańska, Zofia Łapińska, Nina Rembiałkowska Wednesday morning Track C, Wednesday, Oct 12 2022, 10:30-12:10 Location: Congress Hall Session: P30 - Cancer Immunotherapy and Pulsed Electric Fields (PEF) 108 Chairs: Richard Heller and Emanuela Signori Organizers/Conveners: Emanuela Signori and Richard Heller 10:30 Local intratumoral electroporation delivery of potent anti-cancer inter- 108 OR-144 leukin 12 immunotherapy leads to systemic anti-cancer response Adil Daud, Alain Algazi, David A. Canton, Mia Han, Bridget O’Keeffe, Brandon Phung, Jeffrey Silverman 10:45 Nano-Pulse Stimulation™ (NPS™) in combination with the TLR7/8 im- 109 OR-145 mune adjuvant resiquimod eliminates murine Pan02 pancreatic tumors and inhibits the growth of rechallenge tumors Amanda H. McDaniel, Kristin Von Rothstein, Dacia Gonzalez, Richard Nuccitelli 11:00 Delivery of Immune Modulators Using Gene Electrotranser to Induce a 109 OR-143 Robust Immune Response Against Solid Tumors Richard Heller, Jody Synowiec, Samantha Mannarino, Julie Singh, Guilan Shi 11:15 Electroporation Efficacy in breast cancer cell line co-cultured with T 110 OR-141 lymphocytes Elisabetta Sieni, Annj Zamuner, Mariangela De Robertis, Ramona Marino, Daniela Rutigliano, Massimo Sanchez, Flavio Keller, Monica Dettin, Maria Teresa Conconi, Vito Michele Fazio, Mario Cioce, Emanuela Signori 11:30 Protein sampling with electroporation facilitates profiling of spatial differ- 110 OR-142 ential protein expression in breast tumors in vivo Alexander Golberg, Edward Vitkin, Julia Wise, Shay Ben-Elazar, Zohar Yakhini 11:45 Experimental and theoretical Brownian Dynamics analysis of Ion transport 110 OR-140 during cellular electroporation of E. coli bacteria Juan A. Gonzalez Cuevas, Ricardo Arguello, Marcos Florentin, Franck M. André, Lluis M. Mir 20 Wednesday morning Track D, Wednesday, Oct 12 2022, 10:30-12:10 Location: Columbine Room Session: P7 - Pulsed Electric Fields (PEF) for Recovery of Components from Microorganisms 111 Chairs: Wolfgang Frey and Javier Raso Organizers/Conveners: TBA 10:30 Combination of plasma-activated water with non-lethal pulsed electric field 111 OR-63 on bacteria inactivation Robin Mentheour, Nofel Merbahi, Marie-Pierre Rols, Zdenko Machala 10:45 Continuous Extraction of Proteins from Microbial Cells by Pulsed Electric 111 OR-64 Fields Felix Schottroff, Jens Kastenhofer, Oliver Spadiut, David J. Wurm, Henry Jaeger 11:00 Sequential extraction of different compounds of interest from yeast bio- 112 OR-65 mass assisted by Pulsed Electric Fields Alejandro Berzosa, Carlota Delso, Jorge J Sanz, Ignacio Alvarez-Lanzarote, Cristina C Sánchez-Gimeno , Javier Raso 11:15 The effect of nanosecond pulsed electric field on the production of meta- 112 OR-148 bolites from lactic acid bacteria Sumiyo Kanafusa, Elisabeth Uhlig, Kunihiko Uemura, Federico Gómez Galindo, Åsa Håkansson 11:30 PEF-Processing of Microbial Biomass at KIT-IHM 112 OR-159 Wolfgang W. Frey Wednesday afternoon Track A, Wednesday, Oct 12 2022, 14:00-15:00 Location: Pierrot Room Session: P18 - Electroporation and cellular processes 113 Chairs: Anna Bulysheva and Olga Zeni Organizers/Conveners: TBA 14:00 Urine protects urothelial cells against nanosecond pulsed electric fields 113 OR-149 damage Aleksander Kielbik, Pamela Sowa, Andrei G. Pakhomov, Emily Gudvangen, Uma Mangalanathan, Julita Kulbacka, Olga N. Pakhomova 14:15 Synergistic Gene Electrotransfer and 3D Bioprinted Implants for Improv- 113 OR-13 ing Biomanufactured Implant Biological Integration for Enhancing Mus- culoskeletal Tissue Regeneration Anna Bulysheva, Kyle Christensen, Aislin West, Michael Francis 14:30 Combinatorial treatment with nsPEF and antibiotics increases Methicillin- 114 OR-151 Resistant Staphylococcus aureus inactivation Alexandra E. Chittams-Miles, Areej Malik, Erin Purcell, Claudia Muratori 14:45 Calcium Oscillations And Mesenchymal Stem Cells Fate: Characterization 114 OR-165 and Control Through Electroporation Leslie Vallet, Franck M. André, Marina M. Sanchez Petidier, Romain Fernandes, Lluis M. Mir Wednesday afternoon Track B, Wednesday, Oct 12 2022, 14:00-15:00 Location: Harlequin Room Session: P23 - Irreversible Electroporation (IRE) 115 Chairs: Govind Srimathveeravalli and Robert Neal II Organizers/Conveners: Govind Srimathveeravalli and Robert E Neal II 21 14:00 Does imaging response after irreversible electroporation correlate with 115 OR-189 survival in localized pancreatic cancer? Rasmus Virenfeldt Flak, Rune Vincents Fisker, Niels Henrik Bruun, Mogens Tornby Stender, Louise Stenholt, Ole Thorlacius-Ussing, Lars Jelstrup Petersen 14:15 Toward large ablation volumes with single insertion high-frequency irre- 115 OR-190 versible electroporation Kenneth N. Aycock, Sabrina N Campelo, Zaid S. Salameh, Edward Jacobs, Kailee David, Iain H McKillop, Rafael V. Davalos 14:30 Optimization of Irreversible Electroporation Needle Electrode Placement 116 OR-211 Using Ultrasound-dependent Respiratory Motion Tracking Filters Radwan Qasrawi Wednesday afternoon Track C, Wednesday, Oct 12 2022, 14:00-15:00 Location: Congress Hall Session: P29 - Electroporation-based Therapies - Head and Neck Cancer 116 Chairs: Giulia Bertino and Christina Caroline Plaschke Organizers/Conveners: Giulia Bertino and Caroline Plaschke 14:00 Electrochemotherapy for the treatment of cutaneous squamous cell car- 116 OR-102 cinoma: the INSPECT experience (2008-2019) Giulia Bertino, The INSPECT Group 14:15 Treatment of Basal Cell Carcinoma with Electrochemotherapy: Insights 117 OR-100 from the InspECT Registry (2008-2019) Luca G. Campana, Giulia Bertino, Tobian Muir, Luca G. Campana 14:30 Calcium electroporation for low risk basal cell carcinoma – a proof of 117 OR-101 concept study Stine Regin Wiegell, Kristoffer Hendel, Christine Fuchs, Gregor Jemec, Julie Gehl, Mille Vissing, Merete Hædersdal 14:45 Effects of Pulse Repetition Frequency on Nanosecond Electrochemother- 118 OR-109 apy Veronika Malyško-Ptašinskė, Eivina Radzevičiūtė, Irutė Girkontaitė, Jurij Novickij, Julita Kulbacka, Nina Rembiałkowska, Vitalij Novickij Wednesday afternoon Track D, Wednesday, Oct 12 2022, 14:00-15:00 Location: Columbine Room Session: P38 - Electroporation for cardiac ablation 118 Chairs: Tomas Garcia-Sanchez and Damijan Miklavčič Organizers/Conveners: TBA 14:00 Swine Coronary Lumen Contractures Following Irreversible Electropora- 118 OR-180 tion as Observed by Optical Coherence Tomography Amanda N. DeVos, David A. Ramirez, Paul A. Iaizzo 14:15 Differences in endocardial lesion morphology between Radiofrequency Ab- 119 OR-183 lation (RFA) and Pulsed Field Ablation (PFA): a computational modelling study Mario Gómez, Tomas Garcia-Sanchez, Antoni Ivorra 14:30 Experimental and numerical evaluation of effect of tissue-electrode prox- 119 OR-184 imity during cardiac pulsed field ablation Bor Kos, Brian Howard, Atul Verma, Wendy S. Tzou, Lars M. Mattison, Damijan Miklavčič, Birce J. Onal, Mark T. Stewart, Daniel C. Sigg 22 14:45 Effect of Contact Force on Pulsed Field Ablation Lesions in Porcine Car- 120 OR-185 diac Tissue Lars M. Mattison, Atul Verma, Tobias Reichlin, Birce J. Onal, Kevin Sack, Megan M. Schmidt, Damijan Miklavčič, Daniel C. Sigg Wednesday late afternoon Track A, Wednesday, Oct 12 2022, 16:00-17:30 Location: Pierrot Room Session: P27 - In memoriam of Justin Teissié - N’espérez pas, mesurez (don’t expect, measure) 120 Chairs: Lluis Mir and Muriel Golzio Organizers/Conveners: Marie-Pierre Rols 16:00 N’ESPÉREZ PAS, MESUREZ (DON’T EXPECT, MEASURE) 121 OR-219 Marie-Pierre Rols 16:15 Fourty Years of Electroporation (1982-2022) – New View on the Poration- 121 OR-72 Resealing Hysteresis Eberhard Neumann 16:30 Information about the ISEBTT « Justin Teissié » Award 121 OR-222 Muriel Golzio 17:00 Non-canonical biological targets of intense pulsed electric field: proteins 122 OR-122 Michal Cifra Wednesday late afternoon Track B, Wednesday, Oct 12 2022, 16:00-17:30 Location: Harlequin Room Session: P33 - Imaging and treatment planning in clinical trials 122 Chairs: Aleš Grošelj and Rasmus Virenfeldt Flak Organizers/Conveners: Ales Grošelj and Rasmus Virenfeldt Flak 16:00 Finite Element evaluation of the electric field distribution in a non- 122 OR-39 homogeneous environment Elisabetta Sieni, Bianca Bazzolo, Monica Dettin, Maria Evelina Mognaschi, Paolo Di Barba, Michele Forzan, Annj Zamuner, Patrizia Lamberti, Maria Teresa Conconi 16:15 Monitoring of current density and electric field distribution during elec- 122 OR-34 troporation of heterogeneous tissues using MR techniques Matej Kranjc, Marko Strucic, Jessica Genovese, Rok Šmerc, Vitalij Novickij, Samo Mahnič-Kalamiza, Igor Serša, Damijan Miklavčič 16:30 Discrete Dual Finite Volumes (DDFV) for electroporation modeling 123 OR-35 Thomas Bonnafont, Delphine Bessières, Jean Paillol 16:45 Coarse-to-fine segmentation of needles on CBCT scans for the evaluation 123 OR-37 of electroporation ablation Eloise Inacio, Luc Lafitte, Olivier Sutter, Olivier Seror, Baudouin Denis de Senneville, Clair Poignard 17:00 PIRET: a Software Platform for Treatment Planning in Electroporation- 124 OR-38 based Therapies Enric Perera-Bel, Kenneth N. Aycock, Zaid S. Salameh, Mario Gómez, Rafael V. Davalos, Miguel A. González Ballester, Antoni Ivorra 17:15 Electric Field Assisted Volumetric Tumor Profiling 124 OR-36 Mary C. Sheehan, Yasushi Kimura, Neeraj Raghuraman Rajagopalan, Brian Simoes, Govind Srimathveeravalli 23 Wednesday late afternoon Track C, Wednesday, Oct 12 2022, 16:00-17:30 Location: Congress Hall Session: P36 - New Technologies for Cells and Tissues Electropermeabilization-II 125 Chairs: Vitalij Novickij and Caterina Merla Organizers/Conveners: TBA 16:00 Membrane permeabilization by high-intensity pulsed electromagnetic 125 OR-147 fields – a non-contact electroporation? Damijan Miklavčič 16:30 Ultrashort high-intensity pulse generators for simultaneous cellular per- 125 OR-23 meabilization and endoscopic imaging for future biomedical applications Rosa Orlacchio, Nour Tabcheh, Delia Arnaud-Cormos, Philippe Leveque 16:45 Effect of the electric field vector change on the efficacy of nanosecond pulse 126 OR-158 trains Vitalii Pavlovich Kim, Andrei G. Pakhomov 17:00 Targeted excitation of murine hippocampal neurons by spatiotemporal 126 OR-146 summation of nanosecond electric pulses Iurii Semenov, Tatiana Zvonareva, Vitalii Kim, Joel Bixler, Allen Kiestler, Bennet L. Ibey, Stephen J. Beebe, Andrei G. Pakhomov Thursday morning Track A, Thursday, Oct 13 2022, 10:30-12:10 Location: Pierrot Room Session: P12 - Electroporation and Cellular Pathways 127 Chairs: Claudia Muratori and Jelena Kolosnjaj-Tabi Organizers/Conveners: TBA 10:30 Cold atmospheric plasma does not stimulate dermal collagen remodeling 127 OR-216 at tissue scale Sara Gouarderes, Aurélie Marchès, Patricia Vicendo, Michel Simon, Nofel Merbahi, Laure Gibot 10:45 Necroptosis and Pyroptosis Contribute to Cell Death of NTIRE Treatment 128 OR-196 Yanfang Zhang, Peipei Mai, Fentao Liu, Yunlong Wang, Boris Rubinsky 11:00 Pulsed electric fields with calcium ions stimulate oxidative alternations 128 OR-197 and lipid peroxidation in human non-small cell lung cancer Vitalij Novickij, Nina Rembiałkowska, Paulina Kasperkiewicz-Wasilewska, Dagmara Baczyńska, Adam Rzechonek, Piotr Błasiak, Jolanta Saczko, Julita Kulbacka 11:15 Ultrastructural analysis on normal astrocytes and medulloblastoma cancer 129 OR-198 stem cells after microsecond pulsed electric field exposure to dissect the cell response specificity Mirella Tanori, Arianna Casciati, Anna Rita Taddei, Carmela Marino, Caterina Merla, Mariateresa Mancuso 11:30 Finding an effective MRI sequence to visualise the electroporated area in 129 OR-195 plant-based models by quantitative mapping Athul Thomas, Teresa Nolte, Andreas Ritter, Marco Baragona 11:45 Flexible electronics integrated into increasingly complex glioblastoma 130 OR-199 models for the study of pulsed electric fields effect in tumor and its mi- croenvironment Marie Lefevre, Attila Kaszas, Andrea Slezia, Gerwin Dijk, Loig Kergoat, David Moreau, Franck Debarbieux, Rodney P. O’Connor 24 Thursday morning Track B, Thursday, Oct 13 2022, 10:30-12:10 Location: Harlequin Room Session: P25 - Electrochemotherapy for Cutaneous Metastases 130 Chairs: Joy Odili and A. James Clover Organizers/Conveners: Joy Odili and Jim Clover 10:30 Efficacy of electrochemotherapy in breast cancer patients of different hor- 130 OR-103 monal status: The INSPECT experience Claudia Di Prata, Eva Maria Grischke, Giuseppe Azzarello, Julie Gehl, Francesca De Terlizzi 10:45 Health-related quality of life trajectories in melanoma patients after elec- 131 OR-104 trochemotherapy: real-world insights from the InspECT register A. James P. Clover, Joy Odili, The INSPECT Group 11:00 High Frequency Electroporation and Chemotherapy for the treatment of 131 OR-105 Cutaneous Malignancies; Evaluation of Early Clinical Utility and Response A. James P. Clover, Phoebe Lyons, Dana Polini, Alison Bracken 11:15 Outcomes of patients with metastatic melanoma treated with electro- 132 OR-106 chemotherapy, pembrolizumab or their combination: a retrospective matched cohort analysis from InspECT and Slovenian Cancer Registry Luca G. Campana, Barbara Perič, Maša Bošnjak, Francesca De Terlizzi, Gregor Serša 11:30 Electrochemotherapy with intravenous bleomycin for patients with cu- 132 OR-107 taneous malignancies, across tumour histology: A systematic review Freya Bastrup, Mille Vissing, Julie Gehl 11:45 Hybrid ECT – a different approach 133 OR-108 Michael Rice, Giulia Colavitti, Antonio Orlando Thursday morning Track C, Thursday, Oct 13 2022, 10:30-12:10 Location: Congress Hall Session: P35 - Modelling 133 Chairs: Mounir Tarek and Lea Rems Organizers/Conveners: Mounir Tarek and Lea Rems 10:30 Mean field model of single cell electroporation 133 OR-25 Pedro Jaramillo, Annabelle Collin, Clair Poignard 10:50 Water Pores in Planar Lipid Bilayers with an addition of cholesterol 134 OR-26 Alenka Maček Lebar, Damijan Miklavčič, Peter Kramar 11:10 Build me a skeletal muscle in silico: Insights into tissue electroporation 135 OR-29 from an experimentally-validated multiscale numerical model Rok Šmerc, David A. Ramirez, Samo Mahnič-Kalamiza, Janja Dermol-Černe, Daniel C. Sigg, Lars M. Mattison, Paul A. Iaizzo, Damijan Miklavčič 11:30 Real-Time Conductivity Distribution Characterization for Electroporation 135 OR-27 using Plant Tissue Borja López-Alonso, Pablo Briz, Héctor Sarnago, José Miguel Burdío, Óscar Lucía 11:50 Characterization of an experimental setup for recording fluorescence in 136 OR-96 real-time from a cell membrane exposed to electric pulses Ioan Tivig, Mihaela G. Moisescu, Eugenia Kovacs, Tudor Savopol 25 Thursday afternoon Track A, Thursday, Oct 13 2022, 13:30-15:00 Location: Pierrot Room Session: P17 - Using Nanosecond Pulsed Electric Fields (nsPEF) to Treat Cancer 136 Chairs: Richard Nuccitelli and Olga Pakhomova Organizers/Conveners: Richard Nuccitelli and Olga Pakhomova 13:30 Nanosecond Pulsed Electric Field in Tumor Ablation, From Lab Experi- 136 OR-116 ment to Clinical Practice Xinhua Chen 13:47 Tissue-specific clearance thresholds using high repetition rate nanosecond 136 OR-14 pulsed electric fields Richard Nuccitelli, Amanda McDaniel, Kristin Von Rothstein, Dacia Gonzalez, Esin Sozer 14:07 Multicellular spheroids as three-dimensional in vitro models for bipolar 137 OR-11 cancellation assessment Rosa Orlacchio, Muriel Golzio, Jelena Kolosnjaj-Tabi, Philippe Leveque, Delia Arnaud-Cormos, Marie-Pierre Rols 14:24 Negative Effects of Cancellation During Bipolar Nanosecond Electro- 137 OR-12 chemotherapy Vitalij Novickij, Nina Rembiałkowska, Wojciech Szlasa, Julita Kulbacka 14:41 Electrochemotherapy Using Anticancer Drug Cocktail for Treatment of 138 OR-15 Drug-resistant Cancer Cells Nina Rembiałkowska, Vitalij Novickij, Julita Kulbacka Thursday afternoon Track B, Thursday, Oct 13 2022, 13:30-15:00 Location: Harlequin Room Session: P26 - Electrochemotherapy – Internal Tumors 138 Chairs: Ismail Gögenur and Ibrahim Edhemović Organizers/Conveners: Ismail Gögenur and Ibrahim Edhemović 13:30 Intraoperative electrochemotherapy of colorectal liver metastases: Long 139 OR-152 term results of a prospective phase II study Ibrahim Edhemovic, Erik Brecelj, Maja Čemažar, Nina Boc, Blaž Trotovšek, Mihajlo Djokić, Arpad Ivanecz, Stojan Potrc, Maša Bošnjak, Bostjan Markelc, Bor Kos, Damijan Miklavčič, Gorana Gasljevic, Gregor Serša 13:45 Long term results of a prospective phase II study evaluating intraoperative 139 OR-153 electrochemotherapy of hepatocellular carcinoma Mihajlo Djokić, Blaž Trotovšek, Maja Čemažar, Maša Bošnjak, David Badovinac, Damijan Miklavčič, Bor Kos, Miha Štabuc, Borut Štabuc, Rado Janša, Lojze Šmid, Peter Popovič, Gregor Serša 14:00 Bleomycin based electrochemotherapy using variable electrode geometry 139 OR-154 electrodes for the treatment of deep-seated soft tissue sarcomas Aurel Ottlakan, Gyorgy Lazar, Renata Koszo, Katalin Hideghety, Andras Nagy, Gabor Vass, Judit Olah, Erika Gabriella Kis 14:15 Calcium electroporation, an experimental cancer treatment – results from 140 OR-155 a pilot trial within advanced esophageal cancer Charlotte Egeland, Lene Jensen, Julie Gehl, Ismail Gögenur, Michael Patrick Achiam 14:30 Electrochemotherapy of peri-hilar primary liver tumors 140 OR-156 Luciano Tarantino, Giancarlo Bizzarri, Aurelio Nasto, Giuseppina Busto, Sara Bortone, Emanuele Balzano, Paolo Tarantino, Riccardo Aurelio Nasto, Paolo De Simone 14:45 Histologic changes of porcine portal vein anostomosis after electrochemo- 140 OR-157 therapy with bleomycin Mihajlo Djokić 26 Thursday afternoon Track C, Thursday, Oct 13 2022, 13:30-15:00 Location: Congress Hall Session: P31 - Gene Electrotransfer for Antibody Production 141 Chairs: Kevin Hollevoet and Bostjan Markelc Organizers/Conveners: Kevin Hollevoet and Boštjan Markelc 13:30 Combined treatment of intratumoral DNA-based anti-CTLA4 antibody 141 OR-78 gene electrotransfer and irradiation for treatment of solid tumors Bostjan Markelc, Simona Kranjc Brezar, Tanja Jesenko, Tim Bozic, Paul J. Declerck, Liesl Jacobs, Maja Čemažar, Kevin Hollevoet, Gregor Serša 13:45 Building a genetic medicine platform for DNA-encoded antibody thera- 142 OR-79 peutics Kevin Hollevoet, Giles Vermeire, Liesl Jacobs, James A. Williams, Debby Thomas, Stéphanie De Vleeschauwer, Trevor Smith, Maya Imbrechts, Rana Abdelnabi, Johan Neyts, Nick Geukens, Paul J. Declerck 14:00 Advancing DNA-based antibody therapeutics through evaluation and 142 OR-80 characterization of in vivo expression Marie-Lynn Cuypers, Nick Geukens, Kevin Hollevoet, Paul J. Declerck, Maarten Dewilde 14:15 MYO Technology for DNA-Based Delivery of Next-Generation Antibody 143 OR-81 Therapeutics Andrew D. Cameron, Debnath Maji, Xin Yao, Veronica Miguela, Robert Miller, Marek M. Drozdz, Delcora A. Campbell, Mitchell Sitnick T. Sitnick, Rachel A. Liberatore 14:30 Mouse, canine and human interleukin-12 antibiotic resistance gene-free 143 OR-169 plasmids: bacterial maintenance and gene electrotransfer efficiency Urska Kamensek, Andrej Rencelj, Tanja Jesenko, Tinkara Remic, Gregor Serša, Maja Čemažar Thursday afternoon Track D, Thursday, Oct 13 2022, 13:30-15:00 Location: Columbine Room Session: P5 - Mechanisms and Applications of PEF in the Food Industry 144 Chairs: Stefan Toepfl and Milena Zdravkovic Organizers/Conveners: Claudia Siemer and Artur Wiktor 13:30 Modelling and validation of the electrochemical phenomena at the 144 OR-194 electrode-solution interface of a PEF treatment chamber Gianpiero Pataro, Giovanna Ferrari 13:45 Effect of post-electroporation recovery of Thai basil leaves prior convective 144 OR-94 and vacuum drying Grant Thamkaew, Allan G. Rasmusson, Dmytro Orlov, Federico Gómez Galindo 14:00 Effect of pulsed electric field (PEF) intensity on separated cream yield, 144 OR-92 physico-chemical properties and stability Markus Walkling-Ribeiro, Thomas Jacob, Lilia Ahrné 14:15 Pulse electric fields-assisted steam peeling of different fruits and vegetables 145 OR-193 Gianpiero Pataro, Oscar Mauricio Pulgarin Lopez, Giovanna Ferrari 14:30 Combination of different techniques for assessing PEF-treatment in plant 145 OR-93 and animal tissues Jessica Genovese, Matej Kranjc, Igor Serša, Pietro Rocculi, Damijan Miklavčič, Samo Mahnič-Kalamiza 27 Poster Presentations Page Monday Poster Session Track, Monday, Oct 10 2022, 14:45-16:00 Location: Congress Hall Session: Poster Session (and Coffee break) 149 Chairs: Alexandra Chittams-Miles and Maura Bendix Organizers/Conveners: n/a PO-001 A Review of Electronic Technology for Medical Applications of Electro- 149 poration Borja López-Alonso, Pablo Briz, Héctor Sarnago, José Miguel Burdío, Óscar O Lucía PO-016 Membrane extracellular vesicles released after electroporation as mediat- 149 ors for melanoma-fibroblasts communication Anna Choromańska, Urszula Szwedowicz, Jolanta Saczko, Julita Kulbacka PO-019 Non-invasive real time analysis of electroporation phenomenon of indi- 150 vidual cells Anne Calvel, Katia Grenier, David Dubuc, Marie-Pierre Rols PO-023 The efficacy of microsecond electric pulses with calcium ions is related to 150 the expression of drug resistance genes and proteins Nina Rembiałkowska, Vitalij Novickij, Dagmara Baczyńska, Magda Dubińska-Magiera, Wojciech Szlasa, Jolanta Saczko, Julita Kulbacka PO-026 Electroporation of excitable cells studied with genetically engineered HEK 150 cells Tina Batista Napotnik, Bor Kos, Tomaž Jarm, Lea Rems PO-030 Efficacy of shock waves and expansion waves generated by nanosecond 151 pulsed electric discharges for permeabilizing cells and delivering drugs Ryuichi Nakajo, Nushin Hosano, Ryosuke Inoue, Takashi Sakugawa, Hamid Hosano PO-007 New high frequency electroporation protocols for human and veterinary 151 medicine applications Alexia DeCaro, Jean-Baptiste Leroy, Muriel Golzio, Marie-Pierre Rols PO-038 Pulsed electric field pasteurisation of orange juice: Inactivation of Escheri- 151 chia coli and native microflora and impact on product quality Kate Waldert, Robert Sevenich, Oliver Schlüter, Monika Springer PO-041 Effects of pulsed electric fields on technological properties and dietary fiber 152 content of carrot pomace Harold Antonio Pájaro Escobar, Gloria López Gámez, Robert Soliva-Fortuny, Olga Martin-Belloso, Pedro Elez-Martinez PO-043 Comparison of extraction of bio-molecules assisted by pulsed electric en- 152 ergy and ultrasonication: Efficiencies for different microalgal species Rui Zhang, Nikolai Lebovka, Eugene Vorobiev, Luc Marchal, Nabil Grimi PO-046 Red wine vinification on a pilot-plant winery based on Pulsed Electric 153 Fields (PEF) Mafalda M. Santos, Luis Redondo, Marcos M. Pereira PO-010 IREC study- electroporation (IRE), calcium electroporation (CaEP) and 153 electrochemotherapy (ECT) in pancreatic cancer patient’s treatment: From science to practice Julia Rudno-Rudziska, Ewelina Frejlich, Maciej Guziński, Julita Kulbacka, Nina Rembiałkowska, Wojciech Kielan 28 PO-048 Biological characterization of pulsed electric field-obtained extra virgin 154 olive oil Roberto Martínez-Beamonte, Marina Ripalda, Tania Herrero-Continente, Cristina Barranquero, Alberto Dávalos, María del Carmen López de las Hazas, Ignacio Alvarez-Lanzarote, Javier Raso, Joaquín Surra, Carmen Arnal, Jesús Osada, María Ángeles Navarro-Ferrando PO-051 Effect of Pulse Electric Fields on the Growth Stimulation of Lactobacillus 154 plantarum CCDM 181 Gabriela Kuncová, Iveta Horsáková, Jakub Reitmeier, Anna Tobolková, Rudolf Ševčík PO-054 Influence of pulsed electric fields on carotenoids content of carrot purees 155 during storage and changes in their bioaccessibility Gloria López Gámez, Pedro Elez-Martinez, Olga Martin-Belloso, Robert Soliva-Fortuny PO-056 Visualization of local thermalization of conductive fluids in a continuous 155 flow PEF treatment cell Kenshi Kajiwara, Bingyu Yan, Ryosuke Kadoya, Sunao Katsuki PO-059 Automated Irreversible electroporated region prediction in different elec- 156 trode type with deep learning approach Amir Khorasani PO-061 Electrodeformation study of Giant Unilamellar vesicles (GUVs) and Multi 156 Vesicular Vesicles (MVVs) under DC and AC electric field Pulses Rupesh Kumar, Mohammad Maoyafikuddin, Rochish Thaokar PO-064 Engineering the tumor environments in vitro using peptide-enriched, hy- 156 aluronic acid-based hydrogels Annj Zamuner, Leonardo Cassari, Monica Dettin, Maria Teresa Conconi, Elisabetta Sieni PO-067 Time-dependent numerical model of electroporation comparing prolate 157 spheroid and real-shaped geometry of a cardiomyocyte Maria Scuderi, Damijan Miklavčič, Janja Dermol-Černe PO-070 Tumor location method based on multi-electrode structures and machine 157 learning Pablo Briz, Borja López-Alonso, Héctor Sarnago, Óscar Lucía, José Miguel Burdío PO-073 Modeling of cardiac electrophysiology of a tissue containing an electropor- 158 ated area Clair Poignard, Annabelle Collin, Simone Nati Poltri PO-033 The bystander effect after Ca electroporation, BLM electrotransfer and 158 irreversible electroporation in multiple cell lines Neringa Barauskaite, Paulius Ruzgys, Rūta Palepšienė, Salvijus Vykertas, Saulius Šatkauskas PO-035 Pulsed electric field assisted rehydration of dry-salted cod (Gadus morhua) 159 Jessica Genovese, Silvia Tappi, Urszula Tylewicz, Fabio D’Elia, Ana De Aguiar Saldanha Pinheiro, Pietro Rocculi PO-075 Low pulsed electrical fields for inducing transient BBB disruption - Mech- 159 anism of action Shirley Sharabi, Yael Bressler, Orly Ravid, David Last, Dianne Daniels, Daniel Rand, Yael Mardor, Itzik Cooper PO-080 Microelectrode array-based electroporation for use in multifunctional ret- 160 inal implants Andrea Kauth, Anne-Kathrin Mildner, Sandra Johnen, Joachim Wegener, Sven Ingebrandt PO-015 Electrical Impedance changes as a possible real-time indicator of Pulsed 160 Field Ablation (PFA) efficacy Tomas Garcia-Sanchez, Gerard Amorós-Figueras, Mario Gómez, Jose M. Guerra, Antoni Ivorra 29 Tuesday Poster Session Track, Tuesday, Oct 11 2022, 14:45-16:00 Location: Congress Hall Session: Poster Session (and Coffee break) 161 Chairs: Clair Poignard and Urša Lampreht Tratar Organizers/Conveners: n/a PO-002 Successful Treatment with Electrochemotherapy for Multiple Non- 161 Melanoma Skin Cancers in Kidney Transplant Recipients Petra Rózsa, Dóra Ágoston, Edit Szederkényi, Anita Varga, Henriette Ócsai, Eszter Baltás, Lajos Kemeny, Judit Oláh, Erika Kis PO-005 Calcium electroporation in cancer treatment – design, test and evaluation 161 of an evidence-based curriculum Christina Louise Lindhardt PO-008 Electroporation in liver cancer multiphysics simulation 162 Ali Jouni PO-011 Anti-inflammatory response characterization of microsecond electric 162 pulses stimulation for spinal cord injuries application Giorgia G. Innamorati, Paola P. Giardullo, Barbara B. Benassi, Caterina Merla, Maria M. Pedraza, Nesus N. Torres, Leslie Vallet, Marina M. Sanchez Petidier, Laura L. Caramazza, Sara S. Fontana, Noemi N. Dolciotti, Victoria Moreno Manzano, Franck M. André, Paolo Marracino, Micaela Liberti, Claudia Consales PO-014 Treatment Planning under Uncertainty for Electroporation-Based Cancer 163 Therapies Prashanth Lakshmi Narasimhan, Zoi Tokoutsi, Nada Cvetkovic, Marco Baragona, Karen Veroy PO-017 Construction of three-dimensional structure and research of folding- 163 analogues of human kinetochore-microtubule used in transmembrane drug delivery Olga Venger, Andrii Venger PO-020 Structure and folding-analogues of mutant form of human mitotic ser- 164 ine/threonine kinase B delivered into cells by electroporation Andrii Venger, Olga Venger PO-024 Development of 3D melanoma cultures on a hyaluronic acid-based scaffold 164 with synthetic self-assembling peptides Annj Zamuner, Leonardo Cassari, Monica Dettin, Luigi Dall’Olmo, Luca G. Campana, Maria Teresa Conconi, Elisabetta Sieni PO-027 CaCl2 influence on pDNA electrotransfer efficiency and cell viability 165 Rūta Palepšienė, Paulius Ruzgys, Martynas Maciulevicius, Baltramiejus Jakstys, Dovilė Uždavinytė, Salvijus Vykertas, Neringa Barauskaite, Saulius Šatkauskas PO-028 Effects of calcium electroporation and irreversible electroporation on 165 HPAF II cells in vitro study Agnieszka Gajewska Naryniecka, Nina Rembiałkowska, Vitalij Novickij, Dagmara Baczyńska, Julia Rudno-Rudzińska, Wojciech Kielan, Julita Kulbacka PO-031 Measuring cell membrane charging limits in current clamp mode 166 Mantas Silkunas, Andrei G. Pakhomov PO-034 Combination of cold plasma and pulsed electric field for microalgae treat- 166 ment Kamile Jonynaite, Skirmantas Kersulis, Rolandas Uscila, Zydrunas Kavaliauskas, Liutauras Marcinauskas, Voitech Stankevic PO-036 Mass transfer modulation during salting of PEF pre-treated salmon fillets 167 Ana De Aguiar Saldanha Pinheiro, Fabio D’Elia, Jessica Genovese, Silvia Tappi, Urszula Tylewicz, Pietro Rocculi 30 PO-039 Developing and optimizing ohmic heating for cake 167 Aberham Hailu Feyissa, Anastasia Mantza, Felix Rabeler PO-042 Bioactive properties of air-dried organic apples pre-treated by pulsed elec- 168 tric field Artur Wiktor, Katarzyna Rybak, Dorota Witrowa-Rajchert, Malgorzata Nowacka PO-044 The impact of matrix on pulsed electric field preceded food drying 168 Aleksandra Matys, Alicja Baranska, Katarzyna Rybak, Dorota Witrowa-Rajchert, Magdalena Dadan, Katarzyna Pawlaczyk, Artur Wiktor PO-047 Plant-based model for the optical evaluation of electroporated area after 169 irreversible electroporation and its comparison to in-vivo animal data Kim Lindelauf, Athul Thomas, Marco Baragona, Ralph Maessen, Andreas Ritter PO-049 Study of the impact of Pulsed electric fields pretreatment on yellow meal- 169 worm insects in a biorefinery concept Rachelle El Hajj, Houcine Mhemdi, Karim Allaf, Colette Besombes, Nabil Grimi, Eugene Vorobiev PO-052 Eradication of Saccharomyces cerevisiae by PulsedElectric Field Treat- 169 ments Efrat Emanuel, Rivka Cahan, Roman Pogreb PO-055 Influence of pulsed electric field-assisted dehydration on the volatile com- 170 pounds of Genovese basil (Ocinum basilicum L.) Sumiyo Kanafusa, Mikael Agerlin Petersen, Federico Gómez Galindo PO-057 Mathematical model of biliary metal stent occlusion treatment using irre- 170 versible electroporation Martin Hemzal, Veronika Novotna PO-060 An optimal dose-response in terms of pulse length in EP-based treatment 170 protocols Isaac Rodriguez, Nahuel Olaiz, Felipe H. Maglietti, Sebastián D. Michinski, Alejandro Soba, Cecilia Suárez, Guillermo R. Marshall PO-062 Electric Field Fabrication: A method for 3D printing electroporation mi- 171 crodevices Josie L. Duncan, Rafael V. Davalos, Jeff Schultz, Zeke Barlow PO-065 Electrical parameters for (ir)reversible electroporation on hepatocellular 171 carcinoma cells in vitro Kim Lindelauf, Marco Baragona, Ralph Maessen, Andreas Ritter PO-068 Potentiating the efficacy of clinical antibiotics by electroporation 172 Žana Lovšin, Tadej Kotnik PO-071 Efficient recycling of electronic-waste by nanosecond pulsed electric dis- 172 charge Ryo Kaida, Mitsuhiko Sato, Nushin Hosano, Mijanur Rahman, Hamid Hosano PO-074 Study of permittivity change in the cervical cell membrane 3D realistic 172 model due to application of subnanosecond electric field using SRD based pulse generator Mayank Kumar, Ashutosh Mishra Wednesday Poster Session Track, Wednesday, Oct 12 2022, 15:00-16:00 Location: Congress Hall Session: Poster Session (and Coffee break) 173 Chairs: Bor Kos and Anna Szewczyk Organizers/Conveners: n/a 31 PO-003 Electrochemotherapy in Oncodermatology, current uses in Argentina 173 Felipe H. Maglietti, Sebastián D. Michinski, Carolina Abal, Matias M. Tellado, Guillermo R. Marshall, Adrián Barceló PO-006 The best treatment and care for patients suffering from skin cancer - lifting 173 competencies for nurses working in advanced cancer care with calcium electroporation Christina Louise Lindhardt PO-009 Randomised controlled trial investigating the effect of reduced bleomy- 174 cin in electrochemotherapy on patients with cutaneous malignancies: A protocol Freya Bastrup, Mille Vissing, Volker-Jürgen Schmidt, Mohammad Farooq Nassari, Taiba Alrasheed, Anni Linnet Nielsen, Camilla Kjær Lønkvist, Lisbeth Rosenkrantz Hølmich, Michael Prangsgaard Møller, Biljana Mojsoska, Elizabeth Emilie Rosted, Julie Gehl PO-012 Electrochemotherapy combined with immunotherapy as a facial nerve pre- 174 serving treatment modality of the late intraparotideal metastasis of a non- melanoma skin cancer Gabor Vass, Zsolt Bella, Aurel Ottlakan, Eszter Baltas, Judit Olah, Lajos Kemeny, Erika Gabriella Kis PO-018 Evaluation of TNF alpha production due to electrochemotherapy applied 174 on glioblastoma spheroids co-cultured with monocytes Bogdan Mircea Matei PO-021 Electroporation-based modalities fused with 17 β-estradiol in ovarian can- 175 cer therapy in vitro Zofia Łapińska, Anna Szewczyk, Julita Kulbacka, Jolanta Saczko PO-022 Effect of electrochemotherapy on myogenesis of mouse skeletal muscle 175 C2C12 cells in vitro: “side-effects” Simona Kranjc Brezar, Urska Matkovic, Bostjan Markelc, Ajda Vrabic, Tim Bozic, Mihaela Jurdana, Maja Čemažar PO-025 Molecular insight into denaturation of plasma membrane ion channels by 176 pulsed electric fields Lea Rems, Lucie Delemotte PO-029 Nanoparticle targeted drug delivery with nanosecond pulsed electric dis- 176 charge induced shock waves Ryosuke Inoue, Nushin Hosano, Ryuichi Nakajo, Hamid Ghandehari, Hamid Hosano PO-032 Role of resting membrane potential in Ca2+ influx following exposure to 177 intense electrical pulse Kazuya Matsunaga, Sunao Katsuki PO-037 Pulsed electric field treatment application to improve product yield and 177 waste valorization in kiwifruit processing Corinna Stühmeier-Niehe, Martina Comiotto Alles, Ivan Shorstkii, Maxim Sosnin, Claudia Siemer PO-040 Pulsed Electric Field Treatment of Seeds with Improvement of Seed Vigour 178 Gülsün Akdemir Evrendilek, Bahar Atmaca, Nurullah Bulut, Sibel Uzuner PO-045 Application of pulse electric fields (PEF) on drying and frying processes 178 of vegetables Caiyun Liu, Eugene Vorobiev, Nabil Grimi PO-050 Frequency Domain Dielectric Spectroscopy of Gram-negative Bacteria Ex- 178 posed to Pulsed Electric Fields Atsushi Tanaka, Sunao Katsuki, Hirotaka Nuki, Ryuya Kimura, Kaichi Miyazaki PO-053 Effect of pulsed electric fields and ultrasound on protein extraction, yield 179 and techno-functionality from duckweed (L. minor and L. gibba) in an indoor farming cultivation system Patricia Maag, Özlem Özmutlu Karslioglu, Claudia Siemer, Alica Lammerskitten 32 PO-058 The induction of cell electrosensitization or electrodesensitization with ap- 179 plication microsecond pulsed electric fields Paulius Ruzgys, Vitalij Novickij, Neringa Barauskaite, DIana Navickaitė, Saulius Šatkauskas PO-063 Application catheter parameters affecting PFA outcome based on simula- 180 tion Roman Kafka, Veronika Novotna, Martin Hemzal PO-066 Study of surface oxides modifications on nitinol electrodes during electro- 180 poration protocols and possible consequences Théo Le Berre, Pauline Bregigeon, Marie Frénéa Robin, Andrei Sabac, Charlotte Riviere, Fréderic Prat, Julien Marchalot PO-069 Persistent membrane depolarization following conventional electropora- 181 tion depends on temperature and is influenced by ion channel blockers Anja Blažič, Lea Rems PO-072 Could the presence of a coronary stent provoke distortion of the electric 181 field and any thermal side effect during epicardial pulsed field ablation? Insights from an in-silico computational modelling Ana González-Suárez, Barry O’Brien, Martin O’Halloran, Adnan Elahi PO-077 Downstaging of portal vein tumor thrombus from Hepatocellular Car- 181 cinoma with Electrochemotherapy as bridge to liver transplantation Luciano Tarantino, Emanuele Balzano, Giuseppina Busto, Aurelio Nasto, Sara Bortone, Paolo Tarantino, Riccardo Aurelio Nasto PO-078 Intense electric field effects on microtubule systems 182 Daniela Guadalupe Blanco Campoy, Tomáš Zakar, Michal Cifra PO-076 Nanosecond Pulse Water Surface Discharge for Water and Wastewater 182 Treatments Mijanur Rahman, Nushin Hosano, Ryo Kaida, Mitsuhiko Sato, R. Ruma, Hamid Hosano PO-004 Endoscopic calcium electroporation as a palliative treatment for inoperable 182 colorectal cancer Malene Broholm, Mustafa Bulut, Rasmus Vogelsang, Mikail Gögenur, Ismail Gögenur 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 requirements in energy and manpower during red wine- making. The electroporation of the grape skins by PEF Monday Plenary Talks increases the extraction rate of phenolic compounds that are responsible for the sensory properties (colour, fla- Oct 10, 8:50 - 10:20 vour, astringency, and bitterness), aging performance, and beneficial effects on health attributed to the mod- PL-01 erate consumption of wine. The improvement in poly- Applying in vivo electroporation to large scale vac- phenolic release permits a reduction in the duration of cination and immunotherapies: Is this a moon the maceration-fermentation by 2 to 5 days resulting in shot? an increment of the production capacity of a winery and Matti Sallberg energy-saving. In the case of white wine, wineries can also Karolinska Institutet, Sweden take advantage of PEF to improve the extraction of vari- A major obstacle in the wider use DNA-based vaccines etal aroma precursors that are located in the skin of some is the poor uptake into human cells. The so far best tech- white grape berries. This attractive effect may prevent the nology to promote immunogenicity of DNA vaccines is in use of macerating enzymes and/or save energy by short- vivo electroporation (EP). However, this technology cur- ening the duration of cold maceration in the production rently has limitations for a wider use in vaccinology due of white wine. The capability of PEF to inactivate spoil- to the dependence on advanced devices, stable electricity, age microorganisms while preserving physicochemical and and that the treatment often is associated with a transient sensorial properties of must and wines may help enhance pain. These are issues that needs to be overcome before in wine quality by guaranteeing reproducible fermentations vivo EP is a widely used and accepted technology for DNA and reducing or replacing the use of SO2 for wine stabil- vaccine delivery. During the COVID-19 pandemic, several ization. It has been also demonstrated that PEF triggers new technologies have been put to a wider use in vaccine yeast autolysis thereby accelerating the release of man-development including in vivo EP. The mRNA has proven noproteins from cell walls and decreasing the duration of to be safe and effective in fighting COVID-19, although the aging on lees. longevity of the immune response are still not clear. Aden- The current availability of commercial PEF units capable oviral vectors were rapidly developed but have been found of responding to the processing capacity demanded by the to have limitations as a platform for multiple doses. Thus, wineries and the authorization by the European regula-as a complementary technology to mRNA, plasmid DNA tion of the PEF technology as a new oenological practice is highly attractive. The two platforms have different lim- for white and red winemaking constitute a definitive im- itations and different advantages, with the ease of delivery pulse for the implementation of the PEF technology in the with mRNA being and obvious significant advantage. We wineries. are currently developing DNA-based vaccines for homolog- ous and heterologous prime-boost strategies. For COVID- PL-03 19, we have developed a booster vaccine with a plasmid Gene Electrotransfer: Better understanding for expressing three receptor bonding domain loops corres- better utilization ponding to three SARS-CoV-2 variants (WH1, Alpha and Marie-Pierre Rols Beta), combined with the highly conserved membrane and CNRS, IPBS, France nucleoproteins (M/N). This will be delivered using a new Cell membranes can be transiently permeabilized by delivery device that allows for single-step intra-muscular application of electric pulses. This process, called elec-DNA injection and EP treatment. However, will this be tropermeabilization or electroporation, allows hydrophilic applicable to mass vaccination? In parallel we are devel- molecules, such as anticancer drugs and nucleic acids, to oping DNA vaccines to prevent infections with Crimean-enter into targeted cells and tissues. Electroporation has Congo Hemorrhagic Fever Virus, and to treat chronic hep- been successfully developed in human and veterinary clin- atitis B and D virus infections. For these indications the ics to treat cutaneous and subcutaneous cancers. It is also use of in vivo EP is mot likely and easier concept to intro- promising for gene therapy and vaccination. The know-duce and to use. Thus, what is needed from in vivo EP to ledge of the processes involved in membrane permeabiliz- be a widely accepted technology for DNA vaccine delivery. ation and in gene transfer is mandatory for the method This will be discussed in the current presentation. to be efficiently and safely used in clinics. As will be presented, our strategy to address these processes is to PL-02 use different biological models with increasing complexit- Pulsed electric fields for winemaking: from the test ies and implement different imaging tools. The descrip- tube to glass of wine tion of the full mechanisms takes benefit from studies per- Javier Raso formed on different biological models (lipid vesicles, cells University of Zaragoza, Food Technology, Spain in 2D and 3D culture, mice) and from different micro- The ability of pulsed electric fields (PEF) to electro- scopy tools that allow to visualize the processes. Single porate the membranes of grape skin and microbial cells cell imaging experiments revealed that the uptake of mo- can be used by wineries to improve winemaking. The re- lecules (antitumor drugs, nucleic acids) takes place in well- lease of polyphenol from the grape skins in the maceration- defined membrane regions and depends on their chemical fermentation stage represents the stage with the highest and physical properties (size, charge). Small molecules can 37 freely cross the electropermeabilised membrane and have PL-10 a free access to the cytoplasm. Heavier molecules, such as Preclinical Insights into Electroporation and the plasmid DNA, face physical barriers (plasma membrane, Myocardium: What have we achieved and what cytoplasm crowding, nuclear envelope) which engender a do we hope to achieve? Preclinical insights complex mechanism of transfer and is a multi-step process Jacob Koruth steps including the initial interaction with the electroper- Mt Sinai Medical Center, United States meabilised membrane, the crossing of the membrane, the The presentation will cover a state of the art review transport within the cell towards the nuclei and finally of electroporation and its application on myocardium as gene expression. In a tissue, other barriers are present it relates to cardiac ablation. We will discuss the effects such as cell-cell contact, junction, extracellular matrix…To on atria and ventricles and review what has been demon-better understand the limits and improve the transfer of strated, what has been translated to clinical practice and molecules, we have developed 3D spheroids models that what we hope to do in the near future (with a focus on more accurately mimic the in vivo complexity of tumors ventricular myocardium). We will also briefly touch upon and reconstructed human skin containing a differentiated the role of reversible electroporation. dermis and epidermis. Since microscopes have a limited penetration in tissue, we implemented a clearing technique PL-04 to accurately and completely analyze and quantify the Irreversible electroporation for the treatment of transfection rate in the whole spheroids. All these models brain cancer: History and Future Directions allow to optimize the pulse parameters to obtain a high Rafael V. Davalos gene expression rate with minimum side effect and there- Virginia Tech, United States fore improve the development and use of electroporation in clinics. Irreversible Electroporation (IRE) is a minimally in- vasive surgical therapy we invented to treat unresect- able tumors using low-energy microsecond pulses. IRE is Plenary Talks unique among tissue ablation techniques in affecting only the cell membrane while tissue molecules, everything en- Tuesday Plenary Talks compassing collagen structures to proteins; remain intact, Oct 11, 8:30 - 10:00 thereby making treatment near critical structures such as major blood vessels and nerves possible. We are develop- ing an advanced form of the technology, high frequency PL-09 irreversible electroporation (HFIRE) for the treatment of Achieving non-invasive therapy and drug/vaccine glioblastoma (GBM). This new therapy preferentially tar-delivery: Challenges and prospects gets cancer cells over healthy cells, transiently disrupts Hamid Hosano the blood-brain barrier for delivery of therapeutics, and Kumamoto University, Japan induces a positive immune response. Our preclinical work The use of non-invasive or less-invasive medical pro- focuses on helping canine patients with naturally occur- cedures has numerous advantages, including cost, side ef- ring GBM, which are excellent translational models of hu- fects, and post-procedure care, over conventional methods, man GBM. Results of our ongoing trials have been ex- particularly for the aging population. Recent advances tremely positive, supporting that HFIRE is effective for in the control of electromagnetic/sonic waves propaga-the treatment of GBM, including tumors refractory to tion/interaction in deep tissue, have opened a new ho- surgery, radio- and chemotherapies. Additionally, I will rizon for moving from non-invasive diagnosis to therapy, discuss our use of bioelectrics to develop technologies with or even combining the two. Since the 1980s, extracorpor-applications in rare cell isolation, medical device design, eal shock wave lithotripsy (ESWL) with focusing shock 3D printing, and focal cancer therapy. waves has been a long-standing clinical practice. Cur- rently, this technique is utilized in orthopaedics for bone formation/pain management and offers promising oppor- Plenary Talks tunities in cardiovascular medicine and cancer therapy as well. Image guided high-intensity-focused-ultrasound Wednesday Plenary Talks (HIFU) therapy has been successfully used to ablate be- Oct 12, 8:30 - 10:00 nign and malignant tumors. Focusing of electromagnetic waves has also attracted considerable attention in this re- PL-05 spect. In this talk, we will summarize our group’s experi- Electrochemotherapy and IL-12 gene electrotrans- ences with electromagnetic and ultrasound waves focusing fer in veterinary medicine (shock waves and HIFU) used for non-invasive therapies Maja Čemažar and needle-free pain-free transcutaneous vaccine/drug de- Institute of Oncology Ljubljana, Slovenia livery. The current challenges and future opportunities will be discussed. The first electrochemotherapy (ECT) in veterinary medicine was performed in cats in the late 1990s, and since then ECT has become gradually used and recognised as a standard treatment modality for different cutaneous and 38 subcutaneous tumors in various animal species, predomin-events has become the primary focus in development of antly in dogs. Some canine tumors, such as mast cell tu- novel anti-cancer therapies and combination treatment ap- mors and perianal tumors respond excellent to ECT, while proaches. Intratumoral delivery of plasmids through elec- others, such as oral tumors responds poorly and in these troporation (EP) represents a promising local treatment cases the main focus of ECT is on improvement of the approach that generates systemic immune responses while quality of life. In addition to dogs, various tumor types minimizing treatment related side effects. OncoSec Med- in cats and horses can be treated by ECT. Specifically, ical System (OMS) pairs a generator and an applicator squamous cell carcinoma which is a common malignancy to induce localized expression of recombinant proteins by in cats and requires invasive treatments, has a pronounced EP in palpable tumor lesions. Intratumoral delivery of in- response to ECT with bleomycin. In horses, ECT with terleukin 12 (IL-12) tavokinogene telseplasmid (tavo) has cisplatin has proven to be an effective treatment for sarc- demonstrated clinical utility to safely treat cancer patients oids, the most common neoplasm in horses. In the last across solid tumor disease states. IL-12 is a pleiotropic decade ECT has also been described in treating various cytokine that has been extensively studied as a poten-tumors in small exotic animals, such as sea turtles, green tial cancer immunotherapy candidate due to its ability turtles, ferrets, rats, hedgehog and cockatiel, where sur- to engage multiple immune effectors and reverse tumor- gical removal of tumor would be difficult due to the size induced immunosuppression. However, systemic admin- of the animals or location of the tumor. Therefore, ECT istration of IL-12 has proven to be exceedingly toxic in is an effective local treatment of tumors in various animal clinical trials, ultimately limiting its clinical utility. In- species, however it lacks the systemic component of treat- tratumoral delivery of tavo via OMS EP resulted in local ment, which is essential in combating the metastatic dis- and systemic anti-tumor effects while inducing only min- ease. The cancer progression and metastasis are strongly imal treatment-related adverse events in multiple clinical connected to the insufficiency of antitumor immune re- trials. Here, we summarize findings from two of our trials sponse. For this reason, the use of interleukin 12 (IL- designed to evaluate a novel anti-tumor treatment com- 12) – a promising candidate for eliciting immune response bination of pembrolizumab (immune checkpoint inhibitor) – in combination with electroporation, called IL-12 gene and intratumoral tavo-EP. electrotransfer (IL-12 GET) has been introduced to pre- KEYNOTE-695 study is a single-arm, phase 2, open-label, clinical and clinical research in veterinary medicine more multicenter study of tavo-EP plus pembrolizumab in pa- than 10 years ago. Several studies have been published tients with unresectable or metastatic melanoma progress- using IL-12 GET alone or in combination with ECT for ing on standard of care immune checkpoint inhibitor(s). the treatment of various tumors, predominantly in dogs. Patients had a 27.8% objective response rate (ORR, IL-12 GET has proven to be mostly effective in round n=54), with 47% of responding patients having durable cell tumor, specifically mast cell tumor. In other tumor response lasting over 1 year. Median overall survival (OS) types such as squamous cell carcinoma, oral melanoma, was 23.5 months (n=56). Grade 3 treatment-related ad- osteosarcoma, adenocarcinoma, fibrosarcoma, the success verse events (TRAEs) were reported for 6.7% patients. of IL-12 GET alone or in combination with ECT is lim- No grade 4 or 5 TRAEs were reported. KEYNOTE-890 ited. The underlying mechanisms of the combined ther- study cohort 1 is a phase 2, open-label, multicenter study apy are, besides direct cytotoxicity, antiangiogenic action assessed the safety and efficacy of tavo-EP in combination and antitumor immune response. Moreover, IL-12 GET with pembrolizumab as 2L+ treatment for advanced triple in combination with ECT showed to be a safe treatment negative breast cancer (TNBC). Patients had a 17.4% with no, or minimal side effects and with minimal plasmid ORR (n=26), with a median duration of response of 16.6 DNA shedding. The future of ECT alone and in combin- months. Median OS was 11 months (n=26). Grade 3 ation with IL-12 GET in veterinary medicine is focused TRAEs were reported for 23.1% patients. No grade 4 or towards the finding the appropriate biomarkers for selec- 5 TRAEs were reported. In comparision to nanoparticle tion of tumors/patients that will respond to these types of and mRNA delivery, it is associated with reduced side ef- treatment and to optimization of the treatment modality, fects. specifically the frequency of IL-12 GET and/or ECT to In summary, EP delivery of tavo allows for safe adminis- obtain the optimal tumor response. tration of IL-12 treatment in patients with solid tumors. Findings from OncoSec clinical trials support continued PL-11 development of an electroporation-based delivery of po- Local intratumoral electroporation delivery of in- tent medicines as a promising approach to local delivery terleukin 12 immunotherapy leads to systemic of treatment with systemic effects while minimizing the anti-cancer response severe toxicities invariably associated with systemic im- Adil Daud munotherapy. University of California, Melanoma and Cutaneous Oncology Department, United States PL-06 Systemic administration of potent anti-tumor immun- Back to basics: Electroporation of artificial cells otherapies is often associated with profound tumor re-and what we can learn from them sponses but also with serious and sometimes irreversible Rumiana Dimova treatment-related adverse events. Optimizing efficacy of Max Planck Institute of Colloids and Interfaces, Germany treatment approaches while reducing severity of adverse Giant vesicles are a fascinating model membrane sys- 39 tem, which has been initially established and used as a with the future directions of investigation will be dis-workbench for studying basic properties of simple lipid cussed. As reported by the National Institute for Health bilayers (The giant vesicle book, Eds. Dimova & Marques, and Care Excellence (NICE) and recent meta-analyses, the CRC Press, 2019). Nowadays, they are increasingly em- current level of evidence of ECT remains low in quantity ployed by biophysicists to unravel the mechanisms driving and quality and its place in guidelines marginal. Hence, various biological processes occurring at the level of the the need for standardisation and continuous rigorous eval- cell membrane. Furthermore, giant vesicles provide excep- uation through comprehensive data collection and monit- tional biomembrane models for systematic studies on the oring of indications, outcomes, and costs. To this aim, effect of electric fields because the membrane response, in large databases will be essential. The International Net- terms of deformation, poration and permeation can be dir- work for Sharing Practices of ECT (InspECT) registry ectly visualized under the microscope (Dimova et al., Soft (https://insp-ect.eu) was established in 2008 to assess the Matter 3:817, 2007; Dimova et al., Soft Matter 5:3201, outcome of patients treated with ECT. Since then, it 2009). Methodologies for assessing the membrane mater- has provided relevant contributions to the advancement ial properties and effects of membrane remodeling factors of clinical ECT and, recently, has been structured into as deduced from measurements on giant vesicles become seven dedicated working groups (melanoma; basal cell and increasingly important (Dimova, Annu. Rev. Biophys. squamous cell carcinoma; rare histotypes; quality of life; 48:93, 2019). In this talk, we will introduce such methods elderly patients; breast cancer; calcium electroporation). and showcase approaches for measuring properties such As the current InspECT chair, I will present the main find-as membrane capacitance (Salipante et al., Soft Matter ings from the most recent publications, with particular 8:3810, 2012; Vitkova et al. Colloid Surf. A-Physicochem. emphasis on basal cell carcinoma, immunotherapy-ECT Eng. 557:51, 2018; Faizi 2021), bending rigidity (Gracia et combination in melanoma and care of elderly patients. al., Soft Matter 6:1472, 2010; Faizi et al. Electrophoresis, Over the last few years, the improvements in ECT equip- 42:2027, 2021), pore edge tension (Portet and Dimova, ment and treatment delivery, along with the introduction Biophys. J. 99:3264, 2010; Leomil et al., Bioinformatics of novel systemic therapies, have opened new exciting av- Advances 1:vbab037, 2021), permeation and membrane enues for translational and clinical research. However, viscosity (Faizi et al., Biophys. J. 121:910, 2022); all these whereas the beneficial effects of ECT for superficial tu- approaches are solely based on observing the response of mours and, more recently, deep-seated malignancies are giant vesicles to electric fields. We will show that the pres- widely accepted, the variability in responses across histo- ence of charged lipids and gangliosides in the membrane, types needs to be addressed. Currently, patient selection lowers the edge tension, thus increasing pore lifetimes and relies on clinical factors (tumour size, histotype, and ex- rendering membranes less stable against electroporation posure to previous oncological treatments); however, pre- (Lira et al., Adv. Sci., 8:2004068, 2021; Aleksanyan et dictive biomarkers are lacking. To this aim, researchers al., Biophys. J. in press, 2022). Presence of calcium ions will need to re-explore the biological factors underpinning reverses the propensity of membrane disruption. tumour response to ECT, including cancer cells them- selves and the tumour microenvironment, in the frame of a robust research roadmap. Notably, identifying biomark- Plenary Talks ers of response may improve the ECT technique by cus- tomising treatment parameters, improve patient outcomes Thursday Plenary Talks by manipulating the tumour and its microenvironment, Oct 13, 9:00 - 10:00 and allow the exploration of novel rational therapeutic combinations. However, whether these opportunities will PL-07 be exploited to produce clinically meaningful impact and A snapshot of clinical research on electrochemo- practice-changing results remains to be seen. therapy: progress and future directions Luca G. Campana PL-08 Manchester University NHS Foundation Trust, United King- Tumor Treating Fields: A Novel Treatment Mod- dom ality for Solid Tumors David D. Tran In medicine, the use of electrochemotherapy (ECT) has University of Florida College of Medicine, United States been enabled by standardisation of the technique and con- tinuous collaborative efforts across specialities. In clin- Tumor Treating Fields (TTFields) is a novel approved ical practice, this is having an impact at four levels: (1) therapy for glioblastoma (GBM) and malignant mesothe- for clinicians, via expansion of the available locoregional lioma and presently under phase 3 studies in several other therapies; (2) for multidisciplinary teams, by increasing solid cancers. TTFields employ non-invasive, external ap- the chance of novel combined approaches; (3) for patients, plication of low-intensity, intermediate-frequency, altern-through the availability of a low-demanding, quality-of- ating electric fields to disrupt the mitotic spindle, leading life-friendly treatment; finally, (4) for health systems, im-to chromosome mis-segregation and apoptosis. TTFields proving workflow by application of a straightforward and also target several DNA damage repair mechanisms and safe procedure. trigger ER stress-dependent autophagy. More recently, Next, the current limitations (including bias, level of sup- TTFields has been shown to induce blood brain barrier porting evidence, and lack of shared indications) along permeability, plasma membrane perforation, and immun- 40 ogenic cell death thought to result in peritumor inflammation that is routinely observed in TTFields-treated pa- tients. A potential mechanism of this property centers on TTFields-induced focal disruption and perforation of the nuclear envelope, leading to cytosolic release of large naked micronuclei clusters that recruit and intensely ac- tivate major DNA sensors and type 1 interferon (T1IFN)- dependent anti-tumor innate and adaptive immune stim- ulation. In a study involving patients with newly dia- gnosed GBM (ndGBM) treated with TTFields, robust T cell activation was detected specifically via the T1IFN tra- jectory, which highly correlated with T cell receptor clonal expansion, a hallmark of antigen-specific adaptive immune reactions. The combination of TTFields and an immune checkpoint inhibitor created a potential therapeutic syn- ergy, demonstrating highly promising efficacy with accept- able toxicity in ndGBM. However, resistance to TTFields eventually occurs in many patients. The mechanism of resistance follows TTFields’ effect on cellular membranes leading to mis-localization and mis-association of key mas- ter regulators controlling cancer stem cells and pro-tumor inflammation. Thus, current TTFields-based cancer im- munotherapeutic strategy in solid tumors should be fo- cused on maximizing its T1IFN-stimulated adaptive im- munity while also disrupting its stemness and inflammat- ory dysregulation. 41 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 phenomena are sometimes neglected by the newcomer. On the other hand, the applications of electroporation will be Sunday Educational Session overlooked as these will be presented in other talks in the educational session. Track Oct 09, 13:00 - 17:00 OR-192 Applications of pulsed electric field in the food in- OR-03 dustry - Educational session Pulsed electric fields Federico Gómez Galindo 1 Antoni Ivorra 1Lund University, Department of Food Technology, Engineer- Universitat Pompeu Fabra, Department of Information and ing and Nutrition, Sweden Communication Technologies, Spain 2Lund University, Department of Food Technology, Engineer- If living organisms are exposed to long electric fields, ing and Nutrition, Sweden most of the observable physiological effects appear to be The modern food industry is facing crucial challenges of thermal origin. On the other hand, if the electric field amid an increased global population, and the demand of exposure is brief (i.e., pulsed), it is possible to apply high high quality, safe, healthy, and nutritious products. Food electric fields without causing significant heating and two production should also be sustainable and with low envir-remarkable biophysical phenomena can be observed: elec- onmental impact. These challenges can only be achieved trical stimulation and electroporation. through research and innovation on food systems that in- Electrical stimulation consists in nonphysiologically indu- clude novel and nutrition-driven technologies. cing action potentials by delivering electric fields. (Action Pulsed electric field (PEF) is a technology that is already potentials are sudden transitions in transmembrane rest-being implemented in the food industry with clear advant- ing voltage that propagate along the cell membrane and ages on low-energy consumption for processing, improved are the basis of nerve impulses.) Electrical stimulation retention of nutrients, flavour or texture in comparison only occurs in excitable cells such as neurons or muscle with traditional processing technologies. PEF applica-cells. On the other hand, electroporation is a universal tions in the food industry include “cold pasteurization” phenomenon that occurs in all living cells. Electropora- of juices, extraction of cellular compounds, meat tenderiz- tion consists in nonphysiologically increasing the plasma ation and optimization of fermentation processes as well as membrane permeability to ions and molecules by exposing pre-treatment of vegetables prior to unit operations such the cells to high electric fields. Such increase in permeabil- as slicing, frying, and drying. ity is, presumably, related to the initial formation of nano- metric pores in the cell membrane; from which the term OR-01 electro-“poration” stems. Remarkably, both phenomena Pulsed Electric Field (PEF) Technology for Envir- occur when the transmembrane voltage is artificially in- onmental Applications creased above a threshold due to the presence of the elec- Christian Gusbeth tric field. (However, as we will see in the talk, the concept Karlsruhe Institute of Technology (KIT), Institute for Pulsed of transmembrane voltage threshold to initiate electropor- Power and Microwave Technology (IHM), Germany ation is debatable.) The environment is the external conditions, includ- The degree of permeabilization caused by electroporation ing all the biotic and abiotic factors that interact and depends on the characteristics of the exposure (e.g., field affect the survival and development of an organism or magnitude and duration) and on the sort of cells or tis- population. The aim of environmental applications is the sues and their environment. It can result in viable cells reduction of hazardous waste, water contamination, air (reversible electroporation) or it can result in cell death and atmospheric pollution and soil degradation. The dra- (irreversible electroporation). Many biomedical and bio- matic increase in the world’s population, combined with technological treatments are based on electroporation. In industrialisation, urbanisation, intensification of agricul- vitro, reversible electroporation is now commonly used for ture and water-intensive lifestyles, is increasingly leading gene transfection of cells in culture whereas irreversible to shortages in water supply. Currently, about 20% of the electroporation is used for cold pasteurization of liquid me-world’s population has no access to safe drinking water. dia or for facilitating the extraction of cellular contents. While water is a renewable resource, it is also a finite one. In vivo, reversible electroporation is used in living tissues Global freshwater consumption has increased sixfold in the for gene therapy and to enhance the penetration of anti- last century. The discharge of industrial effluents, sewage cancer drugs or calcium into undesirable cells. Also in and sludge into water bodies is responsible for infection vivo, irreversible electroporation (IRE) is used in minim- risks and health effects from contaminated drinking water. ally invasive procedures to ablate undesirable tissues for Therefore, improved technologies need to be developed cancer treatments or for managing cardiac arrhythmias. to control chemical and microbial contamination and the After an overview of fundamental concepts on electricity high consumption of this valuable resource. This lecture and bioelectricity, the talk will be focused on biophysical will discuss the environmental impact of bacterial contam-aspects of electroporation. However, electrical stimulation ination of industrial water circuits and hospital wastewa-and other phenomena that may accompany electropora- ter and how the application of PEF technology can help tion will also be presented. In particular, Joule heating solve these environmental problems. In the last decays and electrochemical reactions will be introduced as these 45 PEF techniques gained increasing importance in cellular eradication of tumors and can be considered an in-situ biology, in gene technology, in medicine, in food produc- vaccination. tion and in biotechnology. The significant part of the To further improve the therapeutic effect, electrochemo- lecture will focus on the PEF treatment for bacterial de- therapy can be used in combination with an immunothera- contamination of hospital wastewater effluents. The strin- peutic approach, it can be used either in combination with gent necessity of decontamination of such wastewater efflu- immune checkpoint inhibitors or immunostimulants. One ents relies on the fact that these effluents are loaded with of the approaches that is being explored is electrochemo- pathogenic and increasingly with antibiotic-resistant bac- therapy in combination with gene electrotransfer with a teria. One important issue addressed during the lecture is plasmid coding for interleukin 12 (IL-12). Electrochemo- the safety of the PEF technology, related to mutagenicity therapy in combination with gene electrotransfer is be-and induced electro tolerance in reference bacteria. In the ing used in veterinary oncology for the treatment of dogs. second part of the presentation, a promising application These combinations are also being explored with irrevers- of PEF treatment in electrocoating systems of paint shops ible electroporation. will be presented. The aim of this application is to prevent Due to their effectiveness, these approaches are constantly the bacterial contamination in the pretreatment and coat-being explored and applied in many different ways. In ing process to eliminate the use of biocides and drive down addition to application in oncology, gene electrotransfer both freshwater consumption and wastewater generation. can also be used for the delivery of vaccines into tissues. In addition, by efficiently monitoring the bacterial load in The technology with new skin applicators and plasmids or process liquid, a high finish quality can be maintained, re- RNA vaccines coding for proteins that trigger an immune working is avoided, and less paint consumption make more response are being tested. One of the applications is also efficient use of resources and lower operating costs. We the SARS-CoV-2 vaccine. To make all these biomedical have found that PEF treatment with short bipolar pulses applications even more effective or to refine their use, we and even with a high specific treatment energy, which is need to learn more about the biological factors that gov- required in extreme cases to achieve maximum bacterial ern the treatment effects. These factors will open new inactivation, does not affect the quality of the coating. possibilities for selection of patients suitable for treatment PEF treatment is therefore a suitable method for automa- and help to refine specific treatment approaches or even tion and effective in bacterial inactivation, does not affect personalize electroporation-based treatments. the quality of the coating. PEF treatment is therefore a suitable method for automation and effective in bacterial OR-126 decontamination of paints. Electrochemotherapy- from Bench to Bedside and Beyond OR-02 A. James P. Clover Biomedical applications of electroporation Cancer Research @UCC., University College Cork, Ireland Gregor Serša Electrochemotherapy is the combination of electropor- Institute of Oncology Ljubljana, Department of Experimental ation and low dose cytotoxic drug to achieve localised tu- Oncology, Slovenia mour control. This treatment had risen to become a well- Exposure of cells or tissues to an electric field can in- established treatment for cutaneous malignancies both of duce structural changes in the cell membrane that allow skin and non-skin origins. It is now delivered in many inflow of molecules into the cells. At the same time, the Cancer Centres across Europe and has become part of na- outflow of molecules is induced. Under specific conditions, tional treatment guidelines. This educational session talk the changes in the membrane are reversible; therefore, we will explore the journey of this development. call that reversible electroporation. This condition is use- Early investigators on both sides of the Atlantic realised ful for the introduction of cytotoxic drugs with hampered the potential for electrochemotherapy to add an additional transport into the cells, such as bleomycin and cisplatin. option in the tool box for Cancer treatments by combining This is called electrochemotherapy. Additionally, DNA or electroporation and low dose cytotoxic treatments. Early RNA can be transported into the cells, so it is useful as a treatment successes lead to the development of standard delivery system for gene therapy. This approach is called operating procedures and a consortium of clinicians across gene electrotransfer. When multiple pulses are applied to Europe that aimed to produce the experience and evid- the cells or tissues, irreversible changes are induced in the ence base to validate this treatment. Now the efficacy and cells, which lead to cell death. This is called irreversible durability of treatment effect is increasingly established. electroporation. However, both bench side and clinical researchers continue Electrochemotherapy is used as tissue ablation therapy for to strive to develop and expand treatment options avail- either superficial tumors or deep-seated tumors. Based on able to patients. Current developments include many ex- the specific mechanism of action, electrochemotherapy is citing treatment variations. These include treating solid effective in tumors with different histologies. In addition, organs as well as skin, altering the cytotoxic agent to in- electrochemotherapy is effective also for treatment of vas- clude novel agents such as calcium and investigating pulse cular malformations. Currently, it is used in many EU parameters. cancer centers and also in veterinary oncology around the This educational talk will give a comprehensive introduc- world. Due to the induction of immunogenic cell death, tion to Electrochemotherapy by covering the principles of this locally induced immune response contributes to the treatment, the evidence base for effectiveness and explor- 46 ing where future developments are likely. ant to PEF than Gram-negative bacteria. However, there is little discussion about the physical effect of PEF and OR-223 resistance to PEF at the cellular level based on their struc- Pulsed electric fields and the cardiovascular sys- tures and physical properties. In this study, we measured tem: cardiac ablation and beyond the dielectric properties of bacteria subjected to PEF us- Elad Maor ing an impedance spectroscopy, and evaluated the degree Sheba Medical Center, Israel of the PEF-induced bacterial damage. Listeria innocua as Pulsed field ablation (PFA) with irreversible electro- Gram-positive and Klebsiella aerogenes as Gram-negative poration has emerged as a promising technique for cath- were compared for the resistance to PEF by considering eter ablation of cardiac arrhythmias. While commercial the change in their dielectric properties measured using devices are being used clinically for the treatment of at- an impedance spectroscopy, together with the survivabil- rial fibrillation, there are significant knowledge gaps and ity in our sterilization experiment. Besides, we discussed contemporary industry-independent basic data is needed. the recovery of both bacteria damaged by PEF by the time In addition, there is an increasing interest in other pos- course of their dielectric properties. Our sterilization ex- sible application of PFA in the cardiovascular field. These periment showed that Gram-positive bacteria were more applications include treatment of ventricular fibrillation, resistant to PEF, as was previously reported. The imped- septal ablation, targeting other vascular structures and ance spectroscopy indicated that Gram-positive bacteria, cardiovascular neuromodulation. PFA has appealing char- compared to Gram-negative one, had a smaller change in acteristics for cardiologists, including its ability to be tis-dielectric properties and a larger electric field threshold sue specific and its nonthermal nature. The waveform at which dielectric property changed. A numerical study details of commercially used PFA systems are not been of electric field distribution on Gram-positive or Gram- disclosed by the industry due to intellectual property con- negative bacteria subjected to PEF implies the micro- cerns. These waveform properties (e.g., pulse intensity, scopic effect of PEF on the bacterial membrane. waveform shape, number of pulses, electrode configura- tion and geometry) are critical for treatment planning, OR-85 and can affect cardioselectivity, safety and efficiency of Assessment of microbial inactivation and lipid ox- the treatment. In this talk we will provide information on idation using non-thermal plasma on mussels the fundamentals of electroporation relating to the car- Cinzia Mannozzi 1, Roberta Foligni1, Lama Ismaiel1, Silvia Tappi2, Romolo Laurita2, Luca Belleggia1, Cristiana Cesaro1, diovascular system, summarize key studies and applica- Andrea Osimani1, Massimo Mozzon1 tions to date, and provide insight into future applications. 1Università Politecnica delle Marche, Italy Specifically, we will discuss (1) electroporation cardiose- 2University of Bologna, Italy lectivity including its effect on blood vessels and concerns regarding coronary vasospasm, (2) importance of high- Mediterranean mussels (Mytilus galloprovincialis) frequency-based protocols, (3) how human induced pluri- farming has a great impact on the economy of many potent stem cells models are used to study PFA effect on European countries. However, this kind of shellfish is a cardiomyocytes, (4) review contemporary clinical data. filter-feeding organism that can accumulate bacteria in very high quantities, thus plasma-activated water (PAW), as a non-thermal technology, can be a potential tool able P1 - General Applications for Food to guarantee food decontamination. Processing However, considering that the food decontamination occurs through the activity of the highly reactive species Monday morning Track A (e.g. hydrogen peroxide, nitrates, nitrites), this one can be induced undesired chemical changes as well, thus leading Oct 10, 10:50 - 12:10 to a deterioration of quality and nutritional features, such as peroxidation of food lipids. OR-84 The aim of this work was thus to assess the micro- Dielectric analysis of Gram-positive and -negative bial inactivation and investigate the oxidation degree of bacteria subjected to pulsed electric fields mussels processed with PAW obtained by exposing 500 ml Hirotaka Nuki, Sunao Katsuki, Atsushi Tanaka, Kaichi of distilled water for 4 min to a pulsed corona discharge Miyazaki, Ryuya Kimura driven by a high voltage power generator (AlmaPulse, Kumamoto University, Japan AlmaPlasma s.r.l.) using peak voltage of 18 kV and a Pulsed electric field (PEF) is a promising method for pulse repetition frequency of 5 kHz. PAW dipping times sterilizing conductive liquids at low temperatures. Bac- of 5, 10, and 15 minutes were explored vs controls dipped, terial resistance to PEF depends on the type of bacteria, for the same times, in demineralized water. including the shape, size and membrane structure classi- Total lipids were extracted from PAW-treated samples fied by the Gram staining. Gram-positive bacteria have and controls by the Bligh and Dyer (1959) method and a thick cell wall (tens of nm) with a membrane, while analysed for non-volatile (peroxides, oxysterols) and volat- Gram-negative bacteria have a thin cell wall sandwiched ile lipid oxidation products. Twelve oxygenated deriv-between the inner and outer membranes. Several experi- atives from the cleavage of the fatty acid hydroperoxide mental studies for example H. Htilsheger and B. Mazurek, isomers were detected in the headspace of mollusc lipids have shown that Gram-positive bacteria are more resist- 47 (C5-C9 aldehydes, alcohols, and ketones) and six choles- during further extraction at temperatures of 42 °C or 85 terol oxidation products (COPs) were identified in the °C by 50 and 30 %, respectively. This indicates that im- unsaponifiable matter. Peroxide value, total fatty acid proved extractability is a result of altered cell and tissue composition, volatile levels, cholesterol and COPs content structure, not only of an increase in temperature. These were not significantly affected by PAW soaking. A total effects could be used to decrease extraction time or to amount of 110-140 µ g of COPs/g of total lipid were ob- partly replace extraction steps with organic solvents. The served in the analysed samples, corresponding to 10-20 µ g polyphenol contents of the extracts were in good correla-of COPs/110 g of fresh matter. However, the efficacy of tion with the antioxidant capacity. the microbial decontamination (total mesophilic aerobes, MEF application is possible as an additional pro- Enterobacteriaceae, Pseudomonadaceae, and Escherichia cessing step before pressing or extraction as well as by coli) was very limited as well. installing electrodes into existing concepts for extraction The present work is part of the project “PRIN 2017 units. Thorough optimization of MEF parameters as well -PLASMAFOOD - Study and optimization of cold atmo- as adaption of the previous and subsequent processing spheric plasma treatment for food safety and quality im- steps is required to fully benefit from MEF effects on mass provement” founded by MIUR - Ministero dell’Istruzione transfer. dell’Università e della Ricerca. OR-88 OR-87 Enhancement and Reversion of Irreversible Elec- Application of moderate electric fields to improve troporation via Osmotic Shock Treatment mass transfer steps in fruit processing Greta Gančytė, Povilas Šimonis, Arūnas Stirkė Anne Kathrin Baier, Justus Knappert, Cornelia Rauh Sustainable food processing demands for holistic us- Center for Physical Sciences and Technology, Department of age of raw materials and energy-efficient processing con- Functional Materials and Electronics, Lithuania cepts. Moderate electric fields (MEF) provide an innov- Saccharomyces cerevisiae yeast employs the HOG ative and sustainable way of food processing due to the pathway to recover after dangerous shape modifications direct and very efficient usage of electrical energy. The and intracellular water disbalance caused by environ- combination of electrical effects on cells and tissue as well mental osmotic pressure changes. Pulsed electric field as increased processing temperatures due to ohmic heating (PEF) treatment is known to cause plasma membrane per- have a high potential for the improvement of mass trans- meabilization, an effect known as electroporation. Yet, port steps. The research project MEFPROC aimed at there is no information on whether the HOG biochemical generating knowledge on the application of MEF in mass pathway has a role in yeast response to PEF treatment. transfer operations to facilitate MEF uptake in industrial It was investigated if post-PEF osmotic pressure change of food processing. the media affects yeast cells’ viability, size and if the HOG The chain of fruit processing provides several options pathway is involved in recovery. Experiments were per- for MEF integration and was taken as an example of pos- formed with wild type (WT) Y00000 yeast and a mutant sible application. Juice recovery via mechanical pressing strain derived from WT, Y02724, with no active HOG1 can be improved by previous MEF application due to cell gene. Yeast suspension was exposed to a single electric rupture and facilitated flow of liquid from the tissue. An field pulse with a duration of 150µs and field strength of increase in cell disintegration of fruit mash by MEF could up to 10 kV/cm. Electroporation buffer was used as a be determined dependent on the previous degree of mech- reference point to represent isoosmotic conditions. Swift anical milling. For coarse mashes, an increase by factor 2 osmotic pressure change is defined as osmotic shock. After was measured while for fine mashes with already high de- PEF treatment, cells were transferred to hyperosmotic, gree of disintegration no further cell opening was possible. isoosmotic or hypoosmotic solution. Viability was assesed Depending on the mash fineness an increase in juice yield by counting colony forming units. Cell size was evaluated by up to 20 % was found. Disintegration of apple mash by measuring the turbidity of the solution. Protein and could as well be demonstrated in pilot scale. Besides the DNA efflux was observed by measuring light absorbtion of MEF treatment itself, the design of the subsequent juicing the media at 260 and 280nm wavelengths. showed to be of high importance. We showed that post-pulsed electric field treatment by a For processing of red currants, marked increases in the sudden change of osmolarity of the media has a significant polyphenol content and the antioxidant capacity of the impact on the yeasts’ viability, cell size and leakage of in- obtained juices after MEF treatment were observed. This tracellular components into the media when compared to was traced back to the temperature dependent extractabil- isoosmotic conditions. We show that electroporation effi- ity of phenols from the plant matrix. The MEF treatment ciency depends not only on electric field strength, duration thus could potentially replace time consuming mash incub-and the number of impulses applied, but also on whether ation before pressing and /or improve nutritional quality post-PEF treatment was applied. While it is known that of the juices. electric fields of 8-10 kV/cm cause irreversible damage to MEF led to significant increases in extraction yield for the membrane, after incubation in hyperosmotic condi- polyphenols from red currant press cakes using aqueous tions after PEF, viability increased by ˜30% and the ra- solvents. The highest differences between treated and un- dius of the cell was reduced by ˜2 µm. After weaker field treated samples were found directly after treatment for strengths of 2 and 4 kV/cm PEF and hyperosmotic shock an extraction time of 0 min. MEF also increased yields treatment, the viability of WT yeasts was restored to 100 48 %, for �hog this result was achieved only after 2 kV/cm Advanced CellScoring package of MetaMorph, then treatment. Hipoosmotic shock caused the opposite effect: averaged and normalized to the control transduced with after exposure to pulse of 4 kV/cm viability decreased by scrambled gRNA. 12 % (WT) and 39 % (�hog); after exposure to 6 kV/cm In the first screening, all 328 KOs were exposed to pulse, by 16 % and 25 %. The size of the cells almost 20 pulses. Based on the average of four independent doubled, after 10 kV/cm treatment. Protein and DNA experiments, we selected KOs with lower- and higher-YP leakage evaluation revealed that amount of intracellular uptake than in the control. The selected KO cell lines compounds in the media decreased after hyperosmotic were generated de novo, and the new screening was shock and increased after hipoosmotic shock, supporting performed two weeks after transduction. Forty and the hypothesis that mechanical cell shape alteration influ- twenty pulses were applied for lower-YP and higher-YP ences cells’ reaction to PEF. Furthermore, �hog strain was groups, respectively. In the lower-YP group, thirteen more sensitive to treatments suggesting that HOG path- KOs responded to nsEP with at least 8% YP uptake way is of relevance to recovery. reduction, thus reproducing the first screening results. To summarise, yeast membrane recovery after exposure to For three gene KOs (CLCA1, KCND3, and SHROOM1), PEF can be altered by subsequent change in osmolarity of YP reductions were statistically significant (p<0.05), media. HOG pathway involvement was linked to recovery and the overall effect was 15-27% lower than in the after electroporation. control. Unusually, ATP1A1 gene KO initially showed a strong reduction of the YP effect but weakened with time after the transduction. Increased YP uptake (>8%) P12 - Electroporation and Cellular was detected for at least fifteen gene KOs. For five Pathways genes (ATP2C2, CACNA1G, CHRNA10, KCTD17, and SCNN1B), the effect was significant (p<0.05) and 15-26% Monday morning Track B higher than in the control. Oct 10, 10:50 - 12:10 The genes whose KO resulted in lower YP uptake are likely producing proteins predisposed to injury by nsEP, OR-167 either structural or through interactions with lipids and Identification of the ion channels affecting mem- other proteins. The KOs leading to higher YP uptake brane permeabilization by nanosecond electric point to genes involved in plasma membrane stabilization pulses or electric injury repair. Giedre Silkuniene, Uma Mangalanathan, Alessandra Rossi, Andrei G. Pakhomov, Olga N. Pakhomova Support: AFOSR MURI grant FA9550-15-1-0517. Old Dominion University, United States OR-168 Several studies reported that electric fields could Denaturation of proteins subjected to 1 ns-long suppress the function of ion channels, possibly by elec- MV/cm electrical pulses troconformational changes in the channel proteins. We Shin Goto, Yuya Sato, Koki Tsurusaki, Sunao Katsuki hypothesized that electrically induced alterations in ion Kumamoto University, Japan channel structure contribute to cell membrane permeab- ilization. This work aimed at identifying ion channels The electric field on the plasma membrane of a affecting electroporation with nanosecond electric pulses cell exposed to a PEF of several kV/cm exceeds 1 (nsEP). MV/cm, which permeabilizes the membrane and induces We used a LentiArray CRISPR library to knockout (KO) trans-membrane ion flow. Also, membrane proteins as 328 ion channel genes in U937 human monocytes stably dielectric materials with diverse structures are subjected expressing Cas9 nuclease. In the library, gene-specific to the huge fields. Molecular dynamics simulations have guiding RNAs (gRNAs) are packed into lentiviruses predicted that MV/cm-class PEF stimulates membrane and arrayed in a 96-well format. Each well contains proteins and activates their functions, and there is four gRNAs designed to recognize different regions of growing interest in the effects of PEF on proteins.[1] the same gene and facilitate a permanent gene KO. A However, due to the significant biochemical response total of 328 U937 cell derivatives were generated, each following the PEF induced Ca2+ influx, it is difficult with a KO for a single ion channel gene. Each KO was to explore the effect of PEF on proteins. Previously, treated in electroporation cuvettes with trains of 20 or we experimentally demonstrated that intense electrical 40 pulses (300 ns, 7 kV/cm, 20Hz) in the presence of a pulses on the order of 200 kV/cm destroy urease protein membrane-impermeable fluorescent dye YoPro-1 (YP). in its aqueous solution using the specially adjusted Then the cells were placed in a 96-well plate and, in 50 electrophoresis.[2] Moreover, we reported the MV/cm minutes, imaged to access membrane permeabilization class electrical pulses disintegrate transthyretin aggreg- by YP entry. Imaging was performed using an Olympus ate.[3] Here, we investigated the primary, secondary and IX83 microscope configured for high-throughput screening tertiary structures in three kinds of proteins, lysozyme, with automated stage repositioning and auto-focusing. albumin, and urease, characterized by structures and Nine fields of view in each well were scanned and stitched molecular weights, exposed to 10 pulses of 1.3 MV/cm. in one image, yielding 300-600 cells per sample. YP Electrophoresis indicates that the primary structure in all fluorescence in individual cells was quantified with the three kinds of proteins were altered. Circular dichroism 49 (CD) analysis, applied to albumin protein, indicated Scanning Calorimetry were used as complementary tools the number of α-helices was significantly decreased by to analysed collagen structure. We demonstrated3 that the exposure to the pulse. Through the experiment, we pulsed electric fields induced 1) a rapid modulation (4h carefully monitored the change in pH, temperature, and after electrostimulation) of mRNA’s genes composing hydrogen peroxide concentration, which could affect the the matrisome, particularly a down-regulation of pro- protein conformation, none of these factors affected any collagens and ECM maturation’s enzymes; 2) a transient of proteins. decrease in pro-collagens production and hydroxyproline tissue content within a week after electrostimulation; 3) a References: long-lasting ROS-dependent over-activation of MMPs and [1] Paolo Marracino, Daniel Havelka, Jiří Průša, Micaela especially collagenase’s family for at least 48h and 4) a Liberti, Jack Tuszynski, Ahmed T Ayoub, Francesca down-regulation of TGF- β 1, a key player in pathological Apollonio, Michal Cifra. Tubulin response to intense fibrosis. First results also indicate that pro-angiogenic nanosecond-scale electric field in molecular dynamics processes occur after pulsed electric field application. simulation. Sci Rep 9 10477, 2019 Taken together, our results open up realistic and relevant [2] Urabe, G., Toshiaki Katagiri, Sunao Katsuki. Et al. prospects for pulsed electric field technology as a local Intense Pulsed Electric Fields Denature Urease Protein. and effective treatment of skin abnormal ECM. BIOELECTRICITY, Volume 2, Number 1, 2020 [3] Urabe, G., Sato, T., Nakamura, G. et al. 1.2 MV/cm References pulsed electric fields promote transthyretin aggregate 1. Gouarderes, S. et al. Vascular and extracellular degradation. Sci Rep 10, 12003,2020 matrix remodeling by physical approaches to improve drug delivery at the tumor site. Expert Opinion on Drug OR-170 Delivery, 17:12, 1703-1726 (2020) Pulsed electric fields as tool for human skin re- 2. Gouarderes, S. et al. Electroporation does not affect modeling human dermal fibroblast proliferation and migration Sara Gouarderes1, Camille Ober2, Layal Doumard1, Jany properties directly but indirectly via the secretome. Dandurand2, Patricia Vicendo1, Isabelle Fourquaux3, Alexan- Bioelectrochemistry. Aug;134:107531 (2020) der Golberg4, Valérie Samouillan2, Laure Gibot 1 3. Gouarderes, S. et al. Pulsed electric fields induce ex- 1IMRCP, CNRS UMR 5623, Université Toulouse III - Paul tracellular matrix remodeling through MMPs activation Sabatier, France, France and decreased collagen production. J Invest Dermatol 2CIRIMAT UMR 5085, Université Toulouse III - Paul Sabatier, 20;S0022-202X(21)02352-6 (2022) France, France 3CMEAB, France 4Tel Aviv university, Israel Impairment of extracellular matrix (ECM) remodeling OR-171 is observed in tumour microenvironment or fibrosis and Electropermeabilization and the packing of bilayer results in excessive collagen production and/or its de- lipids: a problem addressed by real-time fluores- creased degradation by metalloproteinases (MMPs). Due cence measurements to its pivotal role in tissue architectures and functions, Ioan Tivig, Mihaela G. Moisescu, Tudor Savopol ECM components and the proteins that regulate ECM Carol Davila University of Medicine and Pharmacy, Romania remodeling are thus promising therapeutic targets for Although there are studies that link electropermeab- human healthcare. Physical stimuli appear as attractive ilization (EP) to phenomena like: increased production tools to remodel ECM owing to their local and space-time of reactive oxygen species (ROS), enhanced endocytosis, control effects1. lipid peroxidation, etc., the mechanisms are yet to be For that purpose, we assessed the potential of pulsed elucidated. The goal of our work was to obtain pertinent electric field technology, classically applied to drug and data regarding the molecular changes in the biophysical plasmid delivery, to modulate cutaneous cells behavior parameters of the cell membrane due to the application as well as remodel collagen at tissue scale. Two distinct of EP pulses, mainly related to the lipid bilayer packing calibrated electric protocols classically used for gene and peroxidation [1]. electrotransfer (GET) (10 square-wave pulses of 5 ms, We used generalized polarization (GP) which is a 1 Hz) and electrochemotherapy (ECT) (8 square-wave parameter sensitive to changes in membrane lipids ar-pulses of 100 µ s, 1 Hz) were applied in our studies rangement. Membrane GP is measured using liposoluble without addition of any drugs. It appeared that dermal fluorophores (like: laurdan) having a high spectral fibroblasts proliferation and migration properties were sensitivity to the presence of water molecules in their not affected when grown in monolayer, whatever the proximity. The emission spectra of laurdan shift their electric field intensity applied2. We assessed the ECM maxima towards longer wavelengths if the lipid envir- remodeling after electroporation was applied onto a onment becomes less packed, allowing water molecules tissue-engineered human dermal substitute model, by ex- to penetrate deeper in the hydrophobic core of the amining genes modulation by transcriptomic and proteins membrane. In a less packed membrane, GP decreases. synthesis, as well as MMPs activity. Fourier-Transform We used a specially designed system of electrodes which Infrared-Attenuated Total Reflectance and Differential allowed performing electropermeabilization of cells in 50 suspension simultaneously with time-dependent measure-previous investigation by our group demonstrated the abil- ments of fluorescence and of temperature. ity of a specific electric pulse protocol (named PEF-5: NIH 3T3 cells were electroporated with 1 to 50 bipolar 0.3 MV/m, 40 us, 5 pulses) to selectively affect medullo- pairs of rectangular pulses. GP, ROS production and blastoma CSCs preserving normal cells. temperature were monitored in real time before and Here, we tested the same exposure protocol (PEF-5) to after pulse delivery. The GP behavior was analyzed in investigate the response of U87 GBM cells. conjunction with the temperature variation of the cell According to the dynamic nature of CSCs, we evaluated suspension. In order to distinguish between thermal the effects of PEF-5 exposure on U87-MG cells maintained and non-thermal effects of EP pulses, various buffer in standard culture conditions (U87 ML) or as neuro- conductivities (0.01, 0.04, 0.14 S/m) have been used for spheres (U87 NS). various number of pulses. Concerning the correlation Our data, obtained by analyzing different endpoints (i.e., of peroxidation with the GP parameter evolution, ROS cell viability, cell permeabilization, reactive oxygen species production was inhibited by using N-Acetyl L-Cysteine (ROS), full transcriptomic analysis and mRNA expression as a ROS scavenger. Endocytosis inhibitors (Chlorpro- of different genes, as well as clonogenic and invasion poten- mazine and Filipin III) were used to check whether the tial), showed that PEF-5 exposure substantially influenced variation of GP was caused by a specific clathrin/caveolin the fate of GBM CSCs. The exposure seems respons-endocytosis process triggered by EP pulses. ible of a differential regulation of many genes involved Two categories of effects were observed: i/ a thermal in hypoxia, inflammation and P53/cell cycle checkpoints, effect, consisting in an increased bilayer disorder (a deeper with consequent reduction of the cell’s ability to form new penetration of water into the hydrophobic core), and ii/ neurospheres and to transmigrate in vitro. Due to the a nonthermal effect, leading to a higher degree of lipids well-recognized radio-resistance reported for GBM, in this packing, the latter being attributed to a peroxidation work, we assessed the PEF-5 capability to affect GBM process (if EP was conducted in the presence of the cells also in combination with ionizing radiations (IRs). ROS scavenger, a reduction of the GP deflection was Combined PEF-5 exposure and IRs showed an additive observed). A correlation between the kinetics of ROS effect of the two physical stimuli with a dose-dependent production and that of the unpacking of membrane lipids effect of IR on the cell clonogenic potential. Further, cell triggered by EP was demonstrated, and this correlation invasiveness was suppressed in both U87 ML and NS ex- showed not to be linear. The membrane lipids packing, posed cells. Of note, PEF-5 treatment alone was significas observed by GP, was not affected by the inhibition of antly more effective in decreasing cell migration and in- endocytosis processes. An analysis of the permeabilization vasion than radiation exposure in both U87 ML and NS conditions in which the changes in the packing of the cells independently of the delivered dose. bilayer lipids and their peroxidation occur, was performed. Globally, our results confirm ultra-short pulsed electric fields as a promising treatment to destabilize GBM, [1] Tivig I et al., Bioelectrochemistry 138 (2021) opening up the possibility of developing effective PEF- 107689 mediated therapies. OR-172 Microsecond pulsed electric fields: effects on U87- P34 - Development of pulse generators cell line in different culture conditions and electrodes Arianna Casciati 1, Mirella Tanori1, Elena Rampazzo2, Isabella Gianlorenzi1, Luca Persano2, Giampietro Viola2, Monday morning Track C Alice Cani2, Silvia Bresolin2, Carmela Marino1, Mariateresa Mancuso1, Caterina Merla1 Oct 10, 10:50 - 12:10 1ENEA, Division of Health Protection Technologies, Italy 2University of Padova, Department of Women’s and Children’s OR-47 Health, Italy Piezoelectric Transformer based High-Voltage Pulse Generator using Wide-bandgap Semicon- Glioblastoma multiforme (GBM) is the most common ductors for Electroporation brain cancer in adults. GBM starts from a small frac- Ajay Chole, Maeve Duffy tion of poorly differentiated and aggressive cancer stem National University of Ireland, Galway, Electrical and Elec- cells (CSCs) responsible for aberrant proliferation and in- tronic Engineering, Ireland vasion. Notably, the canonical CSC paradigm has been recently revised, and it is now widely accepted that the The demand for nano-second to micro-second range CSC phenotype is characterized by dynamic cellular state HV pulse generators for electroporation is growing as evid- rather than a fixed population. Due to this extreme tu- enced in the recent literature, where the application of mor heterogeneity, actual therapies provide poor positive electroporation is of significantly increasing interest in the outcomes, and cancers usually recur. field of medicine and biotechnology. The HV pulse amp- Therefore, alternative approaches, possibly targeting litude, pulse-width and repetition frequency are decided CSCs, are necessary against GBM. Among emerging ther- depending on the specific application. apies, high intensity ultra-short duration pulsed electric In this work, a new application of Piezoelectric Trans- fields (PEFs) are considered extremely promising and a former (PT) based power converters to generate high- voltage (HV) bipolar pulses for electroporation in can- 51 cer treatment applications is proposed. In the first stage sol-id-state technology. In the scope of this article, a new of power conversion, the PT power converter with GaN pulsed generator, which includes four inde-pendent MOS- FETs and SiC diodes accepts a relatively low-voltage in- FET based Marx modulators, operating individually or put of 24 Vdc and converts it to a HV output, charging combined, controlled from a computer user interface is a capacitor to 2 kV. In the second stage, the charged HV described. The generator is capable of applying differ- capacitor is used as a source for a SiC MOSFET based in- ent pulse shapes, from unipolar to bipolar pulses into bio- verter to generate bipolar pulses with pulse widths of 600 logical loads, in symmetric and asymmetric modes, with ns. The detailed PT based converter design and selection voltages up to 12 kV and currents up to 50 A, in pulse of wide bandgap semiconductor (WBG) switches such as widths from 100 ns to 100 µs, including short-circuit pro- GaN FETs, high-voltage SiC diodes and SiC MOSFETs, as tection, current and voltage monitoring. This new sci- well as simulation results to demonstrate proof-of-concept entific tool can open new re-search possibility due to the using LTSpice are presented. Work is ongoing to develop flexibility it provides in pulse generation, particularly in hardware prototype to determine the performance, size, adjusting pulse width, polarity, and amplitude from pulse- and weight of the proposed HV pulse generator experi- to-pulse. It also permits operating in burst mode up mentally, and to compare it against commercially avail- to 5 MHz in four independent channels, for example in able electroporation power supplies. the application of synchronized asymmetric bipolar pulses, PTs are a special type of transformer that do not make which will be shown together with other characteristics of use of electromagnetic energy transfer mechanisms. The the generator. principle is to use vibration as a coupling medium to trans- fer input electrical energy to mechanical energy and then OR-48 again back to electrical energy at the output with a dif- Super boosting for sub-nanosecond switching of ferent voltage amplitude. high voltage SiC MOSFET Compared to traditional electromagnetic transformers for Alicia del Barrio Montañés 1, Viliam Senaj1, Thomas high step-up and high-voltage applications, PTs have ad- Kramer1, Martin Sack2 vantages such as high power-density, low electromagnetic 1CERN, Spain interference, reduced weight, high galvanic-isolation, and 2KIT, Institute for Pulsed Power and Microwave Technology, comparable efficiency. Almost all power converters pro- Germany posed for HV electroporation pulse generation in the lit- Fast HV generators were traditionally based on gas erature make use of either an inductor and/or a trans- switches with all their associated limitations. Recent pro- former to boost the input voltage. For a high-gain step- gress in HV Si and furthermore SiC MOS transistors up conversion from a relatively low-voltage input, PTs opened the door to replacing the gas switches; this pro-have been demonstrated in the past as a better alternative gress lowered their turn-on times in the range of 10’s of ns to magnetics-based power conversion. Furthermore, they and with up to 6.5 kV per component (i.e. SiC MOS). Fur- may enable the removal of magnetic components from the ther switching performance improvement can be achieved system which is advantageous for medical applications. by so called gate super-boosting technique with commut- The objective of this work is to explore and extend the ap- ation time approaching nanosecond level. plication of PTs to generate HV pulses for electroporation This technique is based on applying of very short over- at a better performance, size, weight, and cost compared voltage pulse of up to 300V on the MOS gate to coun- to existing magnetics-based power converters. The poten- terbalance the effects of the leads inductance and internal tial for extending the pulse voltage and repetition rates parasitic capacitance. In this way super boosting a 1.7 kV will also be considered. The performance of such a PT rated SiC MOS allows to reduce the MOS rise time (tr) based HV pulse generator using WBG devices specifically by a factor of > 26 (datasheet tr = 20 ns vs. measured for electroporation cannot be found in the published liter- tr < 800 ps), resulting in an output voltage slew rate > 1 ature. kV/ns and an amplitude > 1 kV into a 50 Ohm load. Similarly, our test with super-boosting of a 3.3 kV rated OR-98 SiC MOS resulted in an accelerated rise time by a factor Four channel 12 kV, 50 A, 100 ns - 100 µs generator of 20 (datasheet tr = 24 ns vs. measured tr < 1.2 ns), for biomedical and biotechnological applications with an Aleh Kandratsyeu1, Uladzimir Sabaleuski1, Luis Redondo 2, output voltage slew rate > 1.77 ns and an amplitude > Andrei G. Pakhomov3 2.5 kV into a 50 Ohm load. 1EnergyPulse Systems, Portugal To achieve this, the SiC MOS gate driver needs to be ex- 2Instituto Superior de Engenharia de Lisboa, GIAAPP/ISEL, tremely compact, using state of the art components in Portugal low inductance packages and with good decoupling of its 3Old Dominion University, Frank Reidy Research Center for power Bioelectrics, United States supply. Pulsed electric fields in the sub-microsecond range are Further increasing of the output voltage is possible either being increasingly used in biomedical and biotechnology by multiple series connected MOS transistors or by adding applications, where the demand for high-voltage and high-a Marx generator with sufficient number of stages to meet frequency pulse generators with enhanced performance the desired output voltage. and pulse flexibility is pushing the limits of pulse power We propose a fast generator based on the boosted com-52 mutation of cheap commercially available HV SiC MOS and a Marx topology to trigger thyristors to increase the Acknowledgements: The authors would like to thank output Creo Medical and the Barcroft centre and the pre-clinical voltage. The whole prototype uses commercial off-the- team for their continued support in this project and shelf (COTS) components, and the printed circuit board for providing access to their equipment, facilities and (PCB) is planned in a very compact design. expertise OR-97 Histopathology study of nsPEF from a novel elec- OR-114 troporator Microfluidic chip with multi-detection modules for Ilan Wyn Davies 1, Paul Sibbons2, Chris Hancock1 treatment of viable cells by pulsed electric field 1Bangor University, School of Computer Science and Elec- Arūnas Stirkė, Neringa Bakute, Virginija Siauruseviciute, tronic Engineering, United Kingdom Paulina Kizinievic, Justina Kavaliauskaite 2Independent Pre-Clinical Consultant in Surgery and Patho- Center for Physical Sciences and Technology, Lithuania logy, United Kingdom The possibility to artificially adjust and fine-tune gene A novel nanosecond electroporation system was expression is one of the key milestones in bioengineer-developed for in-vivo investigation of nsPEF, in the ing, synthetic biology, and advanced medicine. The ef- nanosecond pulse duration regime with amplitudes in the fects of proteins or other transgene products depend on region of 1 kV and rise times less than 2ns. This system the dosage, therefore controlled gene expression is re- was used for the primary investigation of the effect of quired both for laboratory use and therapeutic applic- nsPEF on live porcine liver tissue. ations, where even slight fluctuations of the transgene The fast nsPEF electroporation system developed for product impact its function and cell viability. In this con- this work utilised relatively slow charging and rapid text, physical techniques demonstrate optimistic perspect- discharging of an open circuit coaxial transmission line ives and pulsed electric field technology is a potential can- through a stack of avalanche transistors operating as didate for a non-invasive, biophysical regulator. It is chal- a fast-switching element. The nsPEF duration was lenging to integrate the biological technology with mech-directly dependent on the charged line length whilst the anical and electric engineering. The main task of engin- maximum nsPEF amplitude and transition time were eers is to apply their scientific and engineering knowledge dependent upon the number of avalanche transistors that to the solution of technical problems. Fusion of biology, were stacked in series. Timing control was managed by mechatronics and physics should further address biocom-an external trigger signal from a pulse generator with patibility guidelines to ensure complete functionality and repetition frequency limited by the associated charging reliability. However shorter nanosecond pulse duration time of the charged line. pulsed electric field (nsPEF) has only been studied for the A range of nsPEF was applied to a porcine liver at a past two decades, but has unique properties not observed range of settings. High voltage nsPEF were applied from with the longer pulses already described. Millisecond the novel electroporator through a parallel plate system PEFs are commonly used in life sciences, especially for where both plates were positioned on parenchymal DNA transfection to generate pores on the cell membrane. surfaces approximately 10mm apart with liver tissue On the other hand, nsPEFs can directly reach intracellu- occupying the entire space between the plates. Samples lar components without cell membrane destruction, and from the experimental sites were received as stained thus nsPEFs have emerged as a unique therapeutic tool for histology sections of liver mounted one tissue per slide. intracellular manipulation without any chemical interven- One section of each tissue was stained with haematoxylin tion. Although nsPEFs are now recognized as a drug-free and eosin and an additional section was stained with and purely electrical cancer therapy, the molecular mech- picro-sirius red combined with Millers elastin stain. All anism of nsPEF action remains largely unclear. In the past sections were scanned in their entirety and comments on few decades advances in the field of microfluidics and mi- any features of interest noted and documented with a crofabrication have contributed to the development of dy- representative photomicrograph. namic cell culture systems and in vitro models recapitulat- In conclusion, the electroporation device as configured in ing the human organs substitutes. Aside from improving this study was able to effect irreversible (and possibly the cellular and biological contents of the microfluidic sys- reversible) electroporation associated cellular changes tems, monitoring the tissues with integrated sensors and within liver tissue. The electroporated cellular features improved microscopy techniques will help generate more were well demarcated from normal tissue, within close data to gain a better understanding of the cellular beha- tolerance borders and related directly to the instrument viour in the device. We exposed mammalian cell lines, application sites. Morphologically, the effect of treatment transfected with NF- κ B pathway-controlled transcription is limited to cells. Collagen within the treated areas is system, to a range of microsecond to nanosecond pulsed unchanged from normal. electric fields parameters. Some possible designs of poten- The design of the novel electroporator used in this tial microfluidic chip will be presented as well. pre-clinical investigation is detailed in another abstract titled ‘Design of a versatile nanosecond pulsed electric field (nsPEF) system. 53 P8 - Mechanisms of Cell Death in ing IRE compared to RE and the addition of z-VAD-fmk Electroporation-based Therapies did not alter intracellular ATP levels. Exogeneous ATP supplementation did not impact proliferation or viability. Western blots revealed reduced pro-caspase 3 cleavage in Monday morning Track D the presence of exogenous ATP. The addition of z-VAD- Oct 10, 10:50 - 12:10 fmk increased caspase 8 activity, but did not impact levels of cell death, suggesting initiation but not the completion OR-57 of apoptotic cascade during IRE. Acute ATP Loss During Irreversible Electropora- Conclusion: Rapid and acute ATP loss during IRE im- tion Supports Caspase Independent Cell Death pedes completion of the apoptosis cascade, resulting in Leo Razakamanantsoa 1, Neeraj Raghuraman necrosis predominant cell death. This effect could not be Rajagopalan2, Francois Cornelis3, Michele Sabbah4, Isa- rescued by ATP supplementation and was insensitive to belle Thomassin-Naggara1, Yasushi Kimura2, Govind pan-caspase inhibitors. Cell necrosis from IRE is expec- Srimathveeravalli2 ted to be immunostimulatory and effective in cancer cells 1Great Hospital of Paris Tenon Hospital, Sorbonne University, that carry mutated or defective apoptosis genes. France 2University of Massachusetts Amherst, United States OR-58 3Memorial Sloan Kettering Cancer Center, United States Characterization of the immunogenic cell death for 4Saint-Antoine Research Center (CRSA), INSERM, CNRS, electroporation treatments France Nastaran Alinezhadbalalami, Kailee David, Zaid S. Introduction: Mode of cell death following tumor ab- Salameh, Kenneth N. Aycock, Rafael V. Davalos lation influences the type and strength of the immune re- Virginia Tech, United States sponse, wherein IRE has been reported to variably cause Introduction: When used as a focal ablation treatment, apoptosis, necrosis, oncosis and pyroptosis. Intracellular irreversible electroporation (IRE) and high frequency ATP is a key substrate for apoptosis and is rapidly de-irreversible electroporation (HFIRE) induce cell death pleted during Irreversible electroporation (IRE). The ob- by membrane destabilization and loss of homeostasis. jective of this study was to understand whether intracellu- In IRE, monopolar pulses are delivered into the tissue lar level ATP determines the mode of cell death following of interest while HFIRE utilizes shorter bipolar pulses. IRE. Recent in vivo studies suggest that both IRE and HFIRE Material and methods: Mouse bladder cancer cell line treatments can stimulate an immune response in addition (MB49) was treated in vitro with an increasing number to tissue ablation[1,2]. The extent of immunogenicity is of pulses (0, 10, 30, 50 or 90) with a duration of 100 µs often a determining factor in patients’ post-treatment and a frequency of 1 Hz at a fixed field strength (1500 outcomes with potentially desirable consequences such as V/cm) in 4mm gap cuvette. Cell proliferation and viabil- abscopal effects. Yet, the level of electroporation-induced ity were assessed with Cell Counting Kit-8 (CCK-8) and inflammatory response is not well-understood in correla- trypan blue staining respectively at 4 and 24-hours fol- tion with the treatment protocol. lowing treatment. ATP levels in culture were measured Here, we aim to study whether the extent of inflammation using Cell-Titer-Glo® Assay at serial timepoints (30 min, resulted from IRE and HFIRE treatments can vary based 2 hours, and 24 hours). The mode of cell death was evalu- on the applied treatment protocol. ated by combined Annexin-V/Propidium Iodide/ staining. Materials and Methods: As the first step for characteriz- Caspase 3/7 activity was measured at 4 hours following ing the electroporation-induced inflammatory response, pulse application. Change in the expression of caspase-the level of released ATP was measured from human 3 and caspase-8 were investigated by Western blotting. brain tumor cells after treatment with IRE and HFIRE. For blocking condition, apoptosis was inhibited by a pan- ATP is one of the damage associate molecular pattern caspase inhibitor (z-VAD-fmk, 50 µM). IRE treated cells (DAMPs) proteins that plays a role in activation of innate were supplemented with exogenous ATP (1.5 µM) to de- and adaptive immune system. Cells were cultured in well termine whether it can alter or reverse the mode of cell plates and treated with the IRE and HFIRE pulses. To death. keep the levels of cell death constant among treatment Results: Cell proliferation and viability decreased at both groups, the applied voltage was normalized to the electric assessed timepoints proportional to the number of pulses field thresholds for each waveform. ATP concentration applied and were not rescued by treatment with the z- was measured in the supernatant of the treated cells using VAD-fmk. Caspase 3/7 activation and apoptosis were pre- a luminescence-based assay (Abcam). dominant when treating cells with 10 pulses, where treat- Results and Discussion: Our preliminary results indicate ment with z-VAD-fmk rescued cells 24 hours after treat- that applying IRE and HFIRE pulses lead to the release ment. Such findings were muted in the groups receiving of ATP from the treated cells. The ATP release increases higher number of pulses. Annexin/PI assay demonstrated when higher electric fields are applied. Furthermore, our most cells stained positive for both stains after IRE treat- results suggest a direct correlation between the applied ment, indicative of both apoptotic/necrosis process which pulse width with the levels of released ATP. The release was not rescued by the treatment with z-VAD-fmk. De-of DAMPs is the first line of triggering an inflammatory crease in intracellular ATP was observed in all treatment response. Hence, our results can provide a guideline for conditions and all timepoints. ATP loss was greater dur-54 choosing the most optimum pulse parameters based on ation induced pores and electrophoretic forces occurring the desired inflammatory outcomes. Our results can also during the pulses that facilitated extraction of ICs. provide perspective on the temporal dynamics of the We hypothesize, that our results contribute to the explan- inflammatory response after IRE and HFIRE treatments. ation of a bigger picture of processes that may occur in An important determining factor in the level of im-the tumor microenvironment after IRE. munogenicity after local tumor treatments is the cell death mechanism. Often a combination of accidental OR-60 and programed cell death can be observed in a single Nano- and microsecond electric field thresholds for IRE or HFIRE treatment [3]. The future perspective of killing epithelial and smooth muscle cells this study is to more precisely quantify the cell death Emily Gudvangen mechanism in response to IRE and HFIRE both in vitro Old Dominion University Research Foundation, Center for Bio- and in vivo. electrics, United States Pulsed electric field (PEF) treatments are advant- References: ageous in their ability to non-thermally target cellular 1. Ringel-Scaia, et al. EBioMedicine 2019, 44, 112-125 components while sparing structural and vascular com- 2. Partridge, et al. J Vasc Interv Radiol 2020, 31, 482-491 ponents. We compared the electric field thresholds for e4. cell death in three human esophageal cell lines: normal 3. Mercadal, et al. Annals of biomedical engineering epithelial (ATCC CRL-2692), pre-malignant epithelial 2020, 48.5, 1451-1462. (ATCC CP-52731), and smooth muscle (H-6089, Cell Biologics). Thresholds were measured by placing two OR-59 cylindrical electrodes orthogonal to a dense cell monolayer The Dynamics of Extraction and Impact of Intra- and delivering trains of 100 uni- or bipolar pulses, 200 cellular DAMPs Released from Irreversibly Elec- ns to 10 µs duration, at 10 Hz. Propidium iodide was tropermeabilized Cells on the Viability of Electro- added two hours post-exposure to label dead cells, allow- porated Cells ing time for transiently electroporated cells to recover Baltramiejus Jakštys, Rūta Palepšienė, Salvijus Vykertas, their membrane integrity. The electric field strength Dovilė Uždavinytė, Ingrida Šatkauskienė, Saulius Šatkauskas which killed 50% of cells (LD50) was considered the cell Vytautas Magnus University, Lithuania death threshold. Unipolar pulses were significantly more Cells undergoing exposure to electric fields can exper- effective at killing pre-malignant compared to normal ience no electroporation, reversible electroporation (EP), epithelial cells, with an average 20% lower LD50 for all or irreversible electroporation (IRE) resulting in imme- tested pulse durations. Conversely, bipolar pulses with diate cell death. The interplay of cell death and EP is a single phase of less than 1 µs were less efficient in modulated by pulse parameters such as pulse strength, killing pre-malignant compared to normal epithelium. duration, pulse number, and pulse repetition frequency. Smooth muscle cells were the most sensitive to both IRE uses brief pulsed electric fields to damage cell plasma unipolar and bipolar PEF, particularly in the nanosecond membrane irreversibly thus leading to instant cell death range, where the LD50 was over 1.5-fold lower in the and inflammation due to DAMPs released and is applied smooth muscle than in the normal epithelium . Unex- for tissue ablation. pectedly, the response of smooth muscle cells to PEF Whereas intracellular DAMPs are known to be released was not determined by the electric field intensity alone, from cells following IRE, the influence of IRE extracted a phenomenon that has not been previously reported or intracellular compounds (ICs) on cancer cell viability has expected. It suggested that other factors play a role in never been studied. We created an in vitro model to in- smooth muscle sensitivity to PEF, such as cell orientation vestigate an impact of IRE extracted ICs on viability of relative to the electric field, cell-to-cell communication, cancerous cells. We discovered that ICs generated from and/or calcium signaling between cells. To explore this, IRE cells significantly increased the viability of reversibly we delivered ten 300 ns pulses to individual cells on a electropermeabilized cancer cells. Furthermore, results re-stage of a confocal microscope in either perpendicular or vealed that our EP parameters and conditions caused no parallel orientation to the electric field and measured the cell lysis. We determined that no more than 60 % of intra- YO-PRO-1 dye uptake by a time-lapse imaging for the cellular proteins could be extracted due to IRE. The main next 5 min. Smooth muscle cells aligned perpendicular amounts of RNA extracted were 26S and 18S ribosomal to the electric field were 5-times more permeable than RNA subunits and some messenger RNA. Surprisingly, we those aligned parallel to it. Additionally, we found that did not detect any differences in the qualitative composi-just a single pulse caused a calcium response lasting up tion of proteins extracted in the EP supernatants probably to 200 seconds, which could be spread between cells and due to the low sensitivity and specificity of the methods cause a propagation of effects outside of the electric field used. Controversially, we were able to obtain only slight threshold. Overall, the higher killing efficiency of unipolar DNA amounts in EP supernatants. pulses in pre-malignant compared to normal epithelium In conclusion, the levels of extracted ICs as proteins and suggest PEF is a promising treatment modality for RNA corresponded to intensifying electrical pulses despite ablation of pre-malignant cells to prevent the progression the release of DNA. Secondly, the process of electric field to cancer. Further studies will focus on the anomalous assisted extraction of ICs we attributed to the electropor- sensitivity and mitigation of damages to smooth muscle 55 cells. States 2University of South Florida, USF Genomics Core, United Acknowledgements: This study was supported by a States grant from Pulse Biosciences (to A.G.P.) Skeletal muscle is ideally suited as a target for thera- peutic gene delivery due to its abundance, high vascu- OR-61 larization, and high levels of protein expression. Intra- Spatial distribution of permeabilization, pH fronts muscular plasmid DNA (pDNA) injection is an easy but and temperature in a 2D tissue model induced by not particularly efficient delivery method. In electropora-electrolytic electroporation: a numerical study tion or electrotransfer, defined electric pulses increase the Enaide Maine Calzado 1, Luis Enrique Bergues Cabrales2, permeability of the cell membrane, facilitating the uptake Nahuel Olaiz1, Pablo Turjanski1 1 of pDNA into cells. Although clinical trials using pDNA Universidad de Buenos Aires, Argentina 2 electrotransfer are increasing, the molecular mechanisms Universidad de Oriente, Cuba that underlie this delivery technology remain unclear. One Electrolytic electroporation is a new tissue ablation of the consequences of pDNA electrotransfer is the ac- technique that is giving excellent results in the treatment tivation of DNA-specific pattern recognition receptors or of tumors. This technique consists of coupling electropor-DNA sensors. These sensors may signal via the adaptor ation with electrolytic ablation. The goal of this study protein stimulator of interferon genes (STING), which is to simulate the spatial distribution of permeabilization, relies on the post-translational modification palmitoyla- pH fronts and temperature generated by different elec- tion for activation. We investigated STING palmitoyla- trode arrays and to analyze how these parameters im- tion in mouse skeletal muscle and C2C12 myocytes after pact tissue damage when electrolytic electroporation is ap- pDNA electroporation. Muscle gene expression by RNA plied. A two-dimensional bicarbonate buffer model is used sequencing and protein expression by bead array was as- to simulate the biological tissue. The numerical model sessed four hours after delivery. DNA sensing was re- solves the two-dimensional Nernst-Planck equations for vealed by the upregulation of specific pro-inflammatory ion transport in a nine-ion electrolyte. In addition, the cytokines and chemokines after pDNA injection alone, bioheat transfer equation of Pennes was used to calculate electroporation alone, or the combination. The mRNAs the temperature distribution in tissue. Numerical simula- of 14 putative DNA sensors were significantly upregu-tions show that extreme pH fronts reach values of pH < lated in the muscle. STING was not significantly reg- 3.8 (around the anodes) and pH > 11.6 (near the cath- ulated, confirming that other mechanisms are involved odes). These pH values affect cell permeabilization levels, in its activation. We performed bioinformatical predic-resulting in the entry of high concentrations of molecules tions and identified high confidence palmitoylation sites into the treated tissue and, consequently, its destruction. on STING as well as the putative DNA sensors cGAS Temperature gradients produced in the medium are not (cyclic GMP–AMP synthase), Ddx58 (DExD/H-box hel- significant to induce severe tissue damage (maximum val- icase 58), Dhx36 (DEAH-box helicase 36), Dhx9 (DExH-ues were < 41 °C). Spatial pattern of pH fronts, per- box helicase 9), Ifi204 (interferon-activable protein 204) meabilization and temperature depend on the exposure and Zbp1 (Z-DNA binding protein 1). STING conserved time, applied charge, shape electrodes array, polarity and cysteine residues, highlighted by a cross-species alignment, separation distance between electrodes. Results obtained suggest the evolutionary importance of protein palmitoyla- in this study were contrasted with those obtained in in tion. STING palmitoylation stoichiometry was evalu- vitro and in vivo experiments of electrolytic electropora- ated using the novel acyl-PEGyl exchange gel-shift assay. tion reported in the international literature. We conclude Two previously characterized palmitoylation sites repres- that acid-base pH fronts and action they exert on tissue ented by a mobility shift in the presence of PEG-5k and permeabilization play an important role in tissue damage hydroxylamine-treated samples in the immunoblot were when electrolytic electroporation is applied, being remark- detected in all experimental groups. Uniquely, one addi- able for arrays of four and five electrodes arrays with 2 cm tional shift band was detected in all pulsed groups, indic- separation between them. ating increased STING palmitoylation following electro- poration. Palmitoylation plays a role in STING signal- P13 - Biological Processes Induced by ing, which suggests that palmitoylation sites are potential Electrotransfer of Molecular therapeutic targets that may have differential effects on immune signaling and regulatory functions. Understand- Cargo ing the molecular mechanisms of skeletal muscle gene ther- apy and creating safety biomarkers will be made possible Monday afternoon Track A by identifying unique post-translational modifications in Oct 10, 13:30 - 14:45 the muscle. OR-49 Palmitoylation of STING following DNA electro- poration in mouse skeletal muscle Amanda E. Sales Conniff 1, Jared Tur1, Kris Kohena1, Min Zhang2, Justin Gibbons2, Loree Heller1 1University of South Florida, Medical Engineering, United 56 OR-51 OR-53 Electroporation as an aid in obtaining extracellular Gene electrotransfer: comparison of parameters vesicles from cancer cells. Increasing the efficiency used in electrogenotherapy and high frequency of EVs isolation using differential centrifugation electroporation on gene expression Urszula Szwedowicz, Anna Choromańska Alexia DeCaro, Marie-Pierre Rols, Muriel Golzio, Elisabeth Wroclaw Medical University, Poland Bellard IPBS-CNRS, Toulouse, France Electroporation in biomedical sciences is especially useful as a method that facilitates the introduction of vari- Studied for more than 30 years, transfer of molecules ous substances into the cells. By overcoming the cell mem- in cells in vivo or in in vitro models mediated by the ap- brane barrier, numerous possibilities of using this method plication of electric fields has been the subject of many in diagnostics and therapy have arisen. Yet most of the advances in the medical field. By electrical stimulation scientific reports focus on the combination of electropor- of cells or tissues, it is possible to introduce therapeutics ation with gene therapy or chemotherapy, a less explored molecules and thus use electroporation for the treatment topic seems to be the possibility of using this method to of cancers or diseases such as muscle dysfunction, a pro- obtain various of its components from the cell, including cess called electrogenotherapy (EGT). These treatments extracellular vesicles (EVs), important participants in in- are possible using a precise protocol of long pulses and tercellular communication. There are many methods of high-voltage: 10 pulses of 5 ms at 0.6 kV/cm. One of isolating EVs from cells, but can they be improved? On- the objectives of our work is to compare this protocol of going studies investigate the utility of using reversible elec- long pulses with high frequency protocols presenting short troporation as an additional step in the extracellular ves- pulses of less than 5 µs, that may decrease side effects such icles isolation protocol with the differential centrifugation as muscle contraction and pain. To proceed, a new design method. Using, inter alia, the IncuCyte imaging system, of electrodes coupled to a high frequency generator has we determined that the application of electrical impulses been developed. Another pulse sequence, High Voltage – enhances the outflow of cell contents. More detailed re- Low Voltage (HV-LV) protocols, has been also tested for search, including flow cytometry and dynamic light scat- comparison. We first used HCT-116 2D colorectal cancer tering (DLS), confirmed that electroporation facilitated cells in suspension and submitted them to electric pulses in the isolation of EVs from cells. We conducted the research the presence of propidium iodide or of a Tomato-labeled on two melanoma cell lines (A375 and Me45) and immor-plasmid to address the effects of these different electric tal keratinocytes (HaCaT) with the use of the BTX Square pulses protocols on electrotransfer efficiency. Ongoing ex- Wave Electroporator. periments show that the pulse duration plays a dramatic role on gene expression. Short-duration pulses can per- OR-52 meabilize the cells but do not lead to more than 1.5% Improving Electrotransfer with Nonreducing Sug- of transfection rate. HV-LV protocols lead to 6 to 15% ars transfection, while the EGT protocol lead to 40 to 50% Fan Yuan of transfection. We are now performing the same experi- Duke University, Biomedical Engineering, United States ments on 3D spheroids models that more accurately mimic Electric pulses have been widely used in basic research the in vivo complexity of tumors. In order to accurately and preclinical studies for transferring molecular cargo and completely analyze and quantify the transfection rate (plasmid DNA, mRNA, and ribonucleoprotein) into cells. in the whole spheroids, we are implementing clarification, To improve the efficiency of electrotransfer, we investig- allowing to observe all the cells in the spheroid whatever ated effects of non-reducing sugars (NRS), such as sucrose, their localization, from the surface to the core. Altogether, trehalose, and raffinose, on cargo transport in cells. In these experiments should be of interest to optimize the experiments, the cells were pretreated with an NRS at pulse parameters of gene electrotransfer to obtain a high different concentrations for various periods. Then, the gene expression rate with minimum side effect. cargo was electrotransferred into the treated cells. At 24 hours post electrotransfer, we measure the transgene ex- OR-54 pression and cell viability. Our data showed that the treat- Electrotransfer of molecules in the reconstructed ment could induce the formation of amphisome‐like bodies skin model Geraldine Alberola, Marie-Pierre Rols, Elisabeth Bellard, (ALBs) and enlarge lysosomes. As a result, cargo degrad-Alexia DeCaro, Muriel Golzio ation was reduced and the efficiency of electrotransfer was CNRS, Institut de Pharmacologie et Biologie Structurale increased, compared to the untreated controls. These data (IPBS), France suggest that the NRS can be used to improve electrotrans- fer of molecular cargo in various applications. Pulsed electric fields are used in medicine for elec- trochemotherapy and DNA electrotransfer. Their use is mainly localized in the skin, which is an organ of choice, due to its accessibility, large surface area, vascularization and immune system. To better understand the limits and improve the transfer of molecules, we have developed a 3D system of reconstructed human skin containing a dif- ferentiated dermis and epidermis. This model has been 57 validated by different methods (biological markers, elec-hours with plate count method on Luria broth agar plates trical parameters), enabling us to have access to a com- without antibiotic. plex model mimicking the in-vivo conditions. We showed that electroporation can potentiate the ef- In this project, we have used this model to study the trans- ficiency of all antibiotics. The most profound effect was fer of molecules of different sizes and charges by electro- observed at higher electric field strength (10 kV/cm and poration. Molecules by modulating EGT electrical para- higher) and antibiotic concentration (where at least 50- meters and employing different types of electrodes. The 60% of bacteria died when treated with antibiotics only). first results show that the corneal layer becomes permeable Our results also suggested that for tetracycline, this po- to small molecules (less than 500 Da) when applying elec- tentiation can be higher than for other antibiotics. Never- tric field pulses, which is not the case for large molecules theless, further studies are needed in order to understand such as plasmid DNA. the dependence of the antibiotic efficacy potentiation by This work will help answer questions such as: electroporation. Our findings provide the basis for devel- - Is there a change in the “barrier” function of the skin? opment of new antimicrobial treatments in which electro- - Is there a disorganization of the intercellular junctions poration could be combined with antimicrobials, for in- at the level of the epidermis? activation of bacteria, particularly in contaminated fluids (e.g. hospital wastewaters). (1) Klein EY, et al., 2019; doi: 10.1136/bmjgh-2018- P2 - Electric Fields in Fermentation 001315 and Wine Production (2) Juma A, et al., 2020; doi: 10.3389/fm- icb.2020.01477 Monday afternoon Track B (3) Kotnik T, et al., 2015; doi: Oct 10, 13:30 - 14:45 10.1016/j.tibtech.2015.06.002 OR-118 OR-50 Microbial stabilization of wine by Pulsed electric Inactivation of resistant Escherichia coli by com- fields (PEF) bining antibiotics with electroporation Carlota Delso, Alejandro Berzosa, Ignacio Alvarez- Saša Haberl-Meglič, Dejan Slokar, Damijan Miklavčič Lanzarote, Javier Raso University of Ljubljana, Faculty of Electrical Engineering, University of Zaragoza, Spain Slovenia Winemaking is a complex process in which the presence Several bacterial species has developed resistance to of spoilage microorganisms may represent an enormous antibiotics, which is increasingly becoming serious concern risk to the final quality of wine with dramatic econom- and a global health threat (1). Development of novel ap- ical losses. Current strategies implemented in wineries proaches for efficient fight against infectious bacterial dis- for microbial control are the use of sulphur dioxide (SO2) eases is therefore crucial (2). One of promising approaches and sterilizing filtration prior to bottling. However, the is electroporation, where exposure of bacteria to short extensive use of SO2 is being questioned due to their po- electric pulses of sufficient strength renders their mem- tential toxic effect and sterilizing filtration involves high branes permeable, thus significantly facilitating transport operational costs and it is detrimental to the wine colour. of otherwise impermeable molecules through the mem- The ability of Pulsed Electric Fields (PEF) to inactivate brane (3). microorganisms at lower temperatures than those used in The aim of our study was to investigate electroporation thermal processing could represent a suitable alternative as a potentiator of antimicrobial efficacy against bacteria for microbial control in wineries. Although the poten- E. coli ER2420, resistant to tetracycline and chloramphen- tial of PEF for wine microbial decontamination has been icol. In the study we compared bacterial inactivation po-reported, most of these studies have been conducted un- tentiated by electroporation for antibiotics with different der laboratory conditions that cannot be transferred to modes of action: tetracycline (inhibits protein synthesis the wineries. In this investigation, the potential of PEF by preventing the attachment of aminoacyl-tRNA to the for microbial stabilization of wine in different winemaking ribosomal acceptor site), chloramphenicol (inhibits pro- steps has been investigated under processing conditions tein synthesis by preventing transfer of amino acids to the simulating an industrial process. growing peptide chains) and ampicillin (inhibits cell wall The PEF-resistance (15-25 kV/cm; 25- 400 µs; 40-170 synthesis by attachment to penicillin-binding proteins). kJ/kg) of three target microorganisms (Saccharomyces Antibiotics were added before pulse application at concen- bayanus, Brettanomyces bruxellensis and Oenococus oeni) tration achieving three different inactivation rates, where suspended in wine without SO2 and with added SO2 (5 80-90%, 50-60% or 10-15% of bacterial cells survived. Bac- - 40 ppm) was characterized. A 4-month shelf-life study terial cells with antibiotics were transferred to plate elec- was performed comparing decontamination by PEF (15 trodes (d = 1 mm) and exposed to a train of eight pulses, kV/cm; 40 and 115 kJ/kg) with SO2 (5-40 ppm) addition. 100 µs of duration of different electric field strength (5, In this study, it was evaluated the microbial stability and 10, 15 or 20 kV/cm). After the treatment cells were in- the oenological parameters of white wine, rosé wine and cubated again with antibiotic for 3 h with shaking at 37 red wine after the alcoholic fermentation and red wine °C in order for antibiotic to enter permeabilized bacterial after the malolactic fermentation. Finally, physicochem- membrane. Bacterial viability was determined after 24 h 58 ical and sensory analysis was conducted comparing steril-signed parallel treatment chamber compared to the cur- izing filtration with PEF decontamination (15 kV/cm; 85, rent standard for industrial PEF systems at the same inac- 127 and 168 kJ/kg) using a red wine ready for bottling. tivation rate. Moreover, real food products, such as milk, Results showed that up to 4.0 log cycles of inactivation were tested to demonstrate the usage for PEF technology can be obtained by PEF for all the microorganisms stud-for thermosensitive food products. ied S. bayanus, B. bruxellensis and O. oeni being the lat- ter the most resistant. Furthermore, a synergetic effect in OR-05 the lethality was observed by combining moderate PEF Effectiveness of Pulsed Electric Field Treatment on treatments with small doses of SO2. The shelf-life studies Spices on Quality Properties, Aflatoxin Decompos- performed in wines revealed the capacity of PEF to elim- ition and Its Mutagenity: Red Pepper Case inate or reduce SO2 doses used for microbial control in Gülsün Akdemir Evrendilek 1, Nurullah Bulut1, Bahar wines with no impact on the quality parameters of wines. Atmaca1, Sibel Uzuner2 Finally, a triangle sensory test revealed that an expert 1Bolu Abant Izzet Baysal University, Turkey panel was not able to discriminate the wines treated by 2Izmir Institute of Technology, Turkey PEF at different intensities from the wine that was treated As one of the most popular and most consumed spices by sterilizing filtration. Therefore, these results reveal the all over the World, red pepper (Capsicum annuum L.) great potential of PEF as a physical process to be used in is commonly used as spice and flavoring agent providing the wineries as for replacing or reducing SO2 doses or as unique flavor and attractive red color to food products. an alternative for sterilizing filtration. One of the major issues associated with red pepper is aflatoxin (AF) contamination in almost ¼ of the veget- OR-117 ables that causes 0.5-1.5 billion dollars lost in trade each Improved Pulsed Electric Field processing units year. Aflatoxin degradation because of their high heat sta- for decontamination of thermosensitive protein- bility and resistance to most of the food processing tech- rich foods nologies is still a big challenge. Thus, efficacy of pulsed Claudia Siemer 1, Christopher McHardy2, Marc Schmidt1, electric fields by energies in the range of 0.97 and 17.28 J Justus Knappert2, Stefan Toepfl1, Cornelia Rauh2 was applied to red pepper flakes for the determination of 1Elea Technology GmbH, Germany changes in quality properties of red pepper flakes, inactiv- 2TU Berlin, Germany ation of aflatoxin producing Aspergillus parasiticus as well Pulsed Electric Fields is an emerging technology for as aflatoxin decomposition, and reduction in mutagenity. the pasteurization of liquid foods. The technology’s work- While 64.37% inactivation with 17.28 J was obtained in ing principle requires small electrode gaps to create elec-mean initial A. parasiticus number; 99.88, 99.47, 97.75, tric fields with magnitudes being high enough to inactivate and 99.58% reductions were obtained on the mean initial foodborne microorganisms. In typical treatment chambers AfG1, AfG2, AfB1, and AfB2 concentrations. Depending being applied in industry, inhomogeneous electric fields on the applied PEF energy and aflatoxin concentration, and local temperature peaks are found. When treating mutagenic effect of aflatoxin is completely eliminated. protein-rich foods, this leads to local denaturation and agglomeration of proteins and finally to the formation of a fouling layer, which affects the product flow, electric P34 - Development of pulse generators field and temperature distribution and can lead to the and electrodes total clogging of the treatment zone as well as an inef- fective inactivation. Consequently, Pulsed Electric Fields Monday afternoon Track C for pasteurizing protein-rich liquid foods at industrial scale Oct 10, 13:30 - 14:45 is currently limited. The present contribution presents the results of a system- OR-99 atic study on how to avoid protein fouling while ensuring A novel electrode for Transurethral Electropora- a sufficient pasteurization at the same time. Methodolo- tion Based Treatments for Bladder Tumors gically, numerical simulations and experiments were per- Balthazar M. BAH Hoffmann, Juan JLV Luis Vásquez, Ju- formed using the example of milk pasteurization. A model lie Gehl was developed to simulate the treatment conditions in a Zealand University Hospital, Denmark PEF treatment chamber, which can be used to identify the points where fouling occurs. To demonstrate the mi- The limitation of electroporation-based technologies crobial efficiency the model solution was inoculated with for the treatment of tumors is that specific electrodes are Lactobacillus plantarum. Different parallel configurated needed in order to be able to deliver the electric pulses treatment chambers, suitable for a continuously operat- to the particular organ. Our group has previously invest- ing pilot scale PEF system, were simulated and stepwise igated the potential use of Electrochemotherapy (ECT) optimized to avoid protein denaturation and the occur-and Calcium Electroporation (CaEP) for the treatment of rence of protein fouling. The efficiency of each treatment bladder tumors with promising results in vivo. Therefore, chamber was experimentally studied focusing on protein we have designed a novel electrode that can be used endo- denaturation and microbial reduction. The results showed scopically (through the urethra) in the urinary bladder. less fouling and protein denaturation using the newly de- Our electrode assembly comprises a bipolar array of 6 (3 x 2) needle shaped electrode elements, in which 59 both central electrodes are off-set from an imaginary line line density occurs might contribute to a better control of between the two external electrodes. The novelty of this the ablation volume and might suppress temperature ef- design is that it produces an optimal electric field distri- fects at the ‘backside’ and tip of the electrode. Therefore, bution and allows direct visualization of the areas to be the effect of additional electrical insulation applied to the treated. Linear electrode arrays produce butterfly shaped original IRE electrodes, tip and/or active needle length electric fields with some cold spots. In this case, in order (ANL), on the electric field line pattern and temperature to ensure exhaustive treatment of all interested areas sub- gradient during IRE pulse delivery was investigated. stantial overlapping of the treated areas may be required, Methods: IRE was performed in a polyacrylamide gel and leading to potential tissue damage. Our electrode provides in a semolina in castor oil model by two monopolar 19G a homogeneous square shaped electric field. Thus, large IRE electrodes (AngioDynamics). Five designs of elec- overlapping during treatment is avoided. This solution trical insulation were investigated, the original electrode leads to faster and more efficient application of the elec- and insulation of: the electrode tip, a part of the circum- troporation treatments, with potentially minimal tissue ference of the ANL or both the tip and a part of the cir- damage. cumference of the ANL. The ANL was insulated for half Bladder tumors are common and highly prevalent. or three quarters of the electrode circumference. All types Treatment requires transurethral surgery (TURBT) (often of shielding were compared to the original unshielded IRE multiple) and multiple adjuvant intravesical treatments electrode. The color Schlieren method, an optical method with instrumentation of the urinary ways, all these as-to visualize a temperature gradient, was used to image the sociated with considerable side effects and high costs. temperature development. The (pulse) protocol used con- TURBT is normally performed with a resectoscope. sisted of 20 pulses with 90 µsec pulse length, 10 pulses/s, These devices use different ex-changeable instruments 1.5 cm inter-electrode distance, 1.5 cm ANL and a po-such as a loop electrode for cutting or resection of the tu- tential difference of 1500 V (BTX Gemini X2) between mor, also a roller ball for coagulation. The design of our the electrodes. The resistance was measured by the BTX electrode fits standard resectoscopes. This way, we can system, before and after pulsing. A static electric field take biopsies, inject drugs directly to tumors, and apply of 5.4 kV (MPL 500-10.000 V, FuG Elektronik GmbH) in the electric pulses under direct visualization, anywhere in combination with cameras placed in three dimensions were the bladder. This procedure is similar TURBT, making used to visualize the electric field line pattern, appearing it easy for urologists to learn, facilitating the introduc- by alignment of semolina. tion of electroporation as a potential treatment of bladder Results: Around the tip, the electric field lines showed the tumors. We expect that ECT or CaEP as a single treat- highest density and most strongly fanned out. The semo- ment will be an effective ablative treatment for bladder lina and color Schlieren results were in line, the highest tumors, thus leading to significant reduction in costs and change in temperature gradient was present around the side effects. tip, at the transition between the insulation and ANL for In conclusion, our electrode shows a novel design that tip insulation and along the negative electrode. The tem- provides an optimal electric field distribution, with the perature gradient showed locations where the highest tem- possibility of direct visualization of the bladder during peratures were reached. These locations could be at risk endoscopic treatments, using standard urological resecto- for thermal damage. The more insulation was applied, the scopes, opening the possibility of electroporation based higher the measured resistance. This resulted in a more treatments in the bladder. However, the novelty of the intense and enlarged area that showed a change in tem- off-set electrode element, which provides a homogeneous perature gradient. An enhanced parallel electric field line square shaped electric field, can also be implemented in pattern was realized when both the electrode tip and part other types of electrodes for different uses. The electrode of the ANL were insulated in comparison with the original is under initial development and clinical trials are cur- electrode. rently under planning. Conclusion: Electrically insulating the electrode tip in combination with a part of the circumference of the ANL OR-111 gives rise to a better parallel electric field and predictable Effect of electrically insulating the tip and/or a ablation zone in between the electrode pairs. Since the part of the circumference of the active needle tip had the largest share in the electric field disturbance length on the electric field line pattern and tem- and showed the highest electric flux density in comparison perature gradient during irreversible electropora- to the ANL, tip insulation is preferred over both tip and tion ANL insulation. Annemiek M. Hogenes 1, Cornelis H. Slump2, Gerben A. te Riet o.g. Scholten2, Martijn W. J. Stommel1, Jurgen J. OR-112 Fütterer1, Rudolf M. Verdaasdonk2 An intradermal, hand-held electroporation device 1Radboud University Medical Center, Netherlands for in vivo applications 2University of Twente, Netherlands Emran O. Lallow1, Nandita C. Jhumur 1, Christine Purpose: If temperature effects occur during irrevers- Roberts2, Jerry W. Shan1, David I. Shreiber1, Jonathan P. ible electroporation (IRE) treatment, they are expected Singer1, Jeffrey D. Zahn1, Joel N. Maslow2, Hao Lin1 1 near the electrodes, especially near the tip. Electrical insu- Rutgers, The State University of New Jersey, United States 2 lation of the electrode sides where the highest electric field GeneOne Life Science, Inc., Korea, Republic of 60 The development of DNA-based vaccines has been the eral decades. Recently, however, localized moderate heat-focus of extensive research and development efforts. DNA ing (to ˜43°C) during GET has been shown to increase vaccines and DNA based therapeutics have significant ad- expression of a delivered foreign gene. Tissue impedance vantages over other platforms including avoidance of cold- measured during electrical treatment has been used in a chain requirements and a superior safety profile. feedback manner to adjust electrical treatment in real-time A major challenge for DNA vaccination platforms is the to optimize the electroporation process. This resulted in need for a secondary delivery mechanism to promote cellu- increased expression too. This study focused combining lar transfection. Several approaches have been developed the use of heat and impedance to improve GET. for such purpose, and electroporation (EP) is the lead- A device that could be used to locally heat the target ing platform and state-of-the-art. To this end we have tissue, apply electric pulses, and use impedance feedback developed a hand-held, battery powered, simple-to-use to guide pulsation was designed, constructed, and tested. electroporation device with an intradermal microneedle- This system included an electrode array that contained electrode (MNE) array attachment for in vivo electropor- up to 16 individually addressable electrodes, a heat source, ation. The initial implementation is an 8x8 configuration and a thermal camera (FLIR Lepton 3.1) to monitor tissue where the MNEs are spaced equidistant at 750 µm. This temperature. The camera and heat source were integrated configuration allows for the creation of many zones for into a handle that contained the electrodes. Infrared, mi- EP with each zone consisting of a 2x2 mini-array to fo- crowaves, and warm air were investigated as mechanisms cus transfection on immediate peri-electrode volumetric to transfer heat that could be integrated into the device. region. The prototype device can produce up to 50 kHz Warm air proved to be as efficient as the other two meth- programmable electrical pulses with a maximum output ods with respect to heating times in phantom tissue and voltage of 35 V at pulse intervals of 10 µs. The use of porcine skin. It proved to be much simpler to implement lower voltage and current that avoids tissue damage. and integrate into a handle that contained electrodes. Im- Electrical testing for the device has been performed on ages from the camera were used in a control algorithm rat carcass skin and compared to a function generator- to heat tissue safely and to maintain temperature during amplifier system, to ensure proper pulse generation in treatment. The final system was able to heat tissue 5 mm air and in skin. Further studies have been performed in below the skin surface to 43°C in under a minute. Electric vivo on a rat model, delivering a green fluorescent pro- pulses were created by electronically switching the output tein (GFP) encoding plasmid. FITC images, 24 hrs post of a commercial power supply (Magna-Power, Fleming- pulsation, show distinct marks of the electroporation chip ton, NJ, USA). Impedance measurements were made us-with GFP expression localized around the needle locations ing the same electrodes as were used for applying electro- signifying the application of the electric field. The pulsing poration pulses. Relays were used to disconnect the high sequence is also tracked during the experiment and asso- voltage supply immediately after a pulse was applied to ciated with the corresponding GFP intensity. Numerical and allow impedance measurements using an impedance simulation is pursued in synergy with experimental tests, analysis chip (AD5933, Analog Devices). Electroporation where we simulate the realistic MNE array and skin tissue pulses could be applied at a maximum rate of about 3 geometry for the prediction of intradermal field and cur- Hz while making impedance measurements between each rent distribution. These results are compared with imped- pulse. The system could reproducibly detect impedance ance spectroscopy measurements and are used to optimize reductions in porcine skin that result from electropulsa- device and protocol design. tion. The combined heating and impedance-based pulsing are currently being tested for the delivery of DNA to OR-113 Guinea pig skin. An in vivo Electrotransfer Instrument that Incor- porates Tissue Heating and Impedance Feedback- OR-201 Based Electropulsation Design of a versatile nanosecond pulsed electric Mark J. Jaroszeski, Alex Otten, Andrew M. Hoff, Timothy field (nsPEF) system J. Fawcett, Richard Heller Ilan Wyn Davies 1, Paul Sibbons2, Chris Hancock1 University of South Florida, United States 1Bangor University, School of Computer Science and Elec- Pulsed electric fields have broad potential for molecu- tronic Engineering, United Kingdom 2 lar delivery applications relative to gene therapy, protein Independent Pre-Clinical Consultant in Surgery and Patho- therapies, vaccination, and chemotherapy. When electric logy, United Kingdom fields are used for gene delivery (gene electrotransfer or The nsPEF system developed in this work is based on GET), pulsed fields are generally used to induce the up- the relatively slow charging and rapid discharging of an take of exogenous plasmid DNA by cells comprising a open circuit coaxial transmission line through a stack of tissue. Wide ranges of electrical parameters have been avalanche transistors operating as a fast-switching ele- used and are typically specific to the electrode(s) being ment. This methodology allows for the generation of high used and the target tissue. These electrical parameters voltage nsPEF with a wide variety of nsPEF parameters. are pulse width, voltage, number of pulses, and period. The duration of the nsPEF produced is determined by Historically, these pulse parameters have been empirically the parameters of the charged or transmission line’s determined to arrive at optimal electrical conditions for associated time delay. The duration of the nsPEF is delivery. This technology has not been improved in sev- twice the associated time delay of the transmission line 61 affiliated with the system. The amplitude of the nsPEF is locally very effective, leading to sustained reduction of is restricted by the number of avalanche transistors that the treated tumour with little side effects to the patients. are stacked in series and their collective collector-emitter In contrast, systemic effects of this treatment have been breakdown voltage. The extent of the amplitude and rarely observed. the reflection coefficient of the system is determined by A systemic and long-lasting immune response is neces-the impedance ratio between the load impedance and sary to generate immunological memory, thus targeting the characteristic impedance of the transmission lines. metastatic disease and disease reoccurrence. The tumour For a system with zero reflection coefficient, the load microenvironment consists of a variety of cells including and characteristic impedance should match and results cancer and immune cells, all of which get impacted by in a nsPEF amplitude that is half the voltage level the ECT treatment. Currently the direct effects of ECT on charged line is charged to. The transition times of the immune cells regarding their viability and functionality is nsPEF are determined by the avalanche transistors that still unknown. operate as the fast-switching elements. Published data The aim of this study is to investigate the impact of EP suggests that they can produce transition times of 300 ps. and ECT on different immune cell in vitro permeability, A trigger, or a control signal, with an amplitude less than survival, immune profile and functionality in the context the base-emitter breakdown voltage, can be applied to of lung cancer in order to identify a means of understand- the base terminal of the lowest stacked transistors, Q1, to ing and improving the response to ECT. dictate when a nsPEF is produced, the number of nsPEF Method: Several immune cell subtypes were treated with that is produced and the repetition frequency between various EP and ECT conditions and the effect of these the nsPEF that is produced. Through manipulating the treatments on cell permeability and survival as well as current loop within the circuit and placing a load, or metabolic activity were established. Further, immune loads, across various potential differences in respect to cell subtypes were treated with cisplatin-ECT (based on the ground plane of the system, a positive, negative, established lung cancer cells in vitro results) and changes or bipolar (generation of positive and negative pulses to their viability, metabolic activity, immune profiles and simultaneously) nsPEF can be generated. Changing the functionality were observed. location of the load or loads in relation to the trans- Results: T cell viability and metabolic activity was un- mission line allows the selection of the polarity of the affected by all tested EP and ECT conditions. However, nsPEF generated. The developed nsPEF electroporation the viability of both macrophages and dendritic cells system can generate well-defined and specific numbers of (DCs) was negatively impacted with increasing electric symmetrical high voltage nsPEF, of various polarities, field strength for both EP and ECT treatments. Addi- with rise and fall times less than a nanosecond, with a tionally, the metabolic activity of DCs and macrophages wide variety of durations and repetition frequencies. The decreased with increasing electric field strength. Data resulting histopathology study of nsPEF generated from from currently ongoing experiments for immune profiles this electroporator is highlighted in another abstract and functionality after treatment will also be presented. titled ‘Histopathology study of nsPEF from a novel Taken together, these findings suggest that failure to electroporator’. generate an effective systemic immune response following ECT administration is due, at least in part, to the Acknowledgements: The authors would like to thank detrimental effect of the voltage currently used to treat Creo Medical for their continued support in this project patients on the antigen presenting cells present in the and for providing access to their equipment and expertise. tumour microenvironment. P9 - Electroporation-based vaccines OR-202 and immunogenic effects Immunological effects of electrochemotherapy in of electroporation b16f10, 4t1 and ct26 murine cell lines in vitro Ursa Kesar, Tanja Jesenko, Bostjan Markelc, Katja Ursic Valentinuzzi, Maja Čemažar, Primoz Strojan, Gregor Serša Monday afternoon Track D Institute of Oncology Ljubljana, Slovenia Oct 10, 13:30 - 14:45 Electrochemotherapy (ECT) elicits an immune re- OR-203 sponse in the treated tumors, however the effects of dif- Impact of electrochemotherapy on immune cells in ferent cytostatics and time frame of changes in different the context of cancer – In vitro study tumors models are still unknown. Usually described cell Maura B. Bendix, Aileen Houston, Beth Brint, Patrick F deaths after ECT are apoptosis, necrosis and immunogenic Ford cell death (ICD). ICD effectively induces adaptive im- University College Cork, Ireland mune response against neo-antigenes, which are released by dying or dead cells. The inflammatory response is Introduction: Electrochemotherapy (ECT) is an promoted by DAMPs (damage-associated molecular pat- established cancer therapy that delivers cytotoxic drugs terns) released from the cells and serve as danger signals. with the help of electroporation (EP) into the cells. It is Some of the DAMPs that can activate ICD are expos- widely used for the treatment of melanoma and is under ure of calreticulin (CRT) on the cell surface, the release investigation for treatment of internal malignancies. ECT 62 of ATP and high mobility group box 1 (HMGB1) from which resorbs progressively until day 3-5. We found a the cells. Immunologically important changes in tumor substantial decrease in the frequency of tumor infiltrating cells like loss of antigens, defects in the process of antigen DC (TIDC) as early as 6h and during the first 72h in presentation (MHC I, MHC II) or changes in immunolo- ECT-treated tumors. This decrease correlates with an gically important cell markers such as PD-L1 and CD40 increase of activated DC expressing CCR7 frequency. also affect the immune response. The aim of the study A slight decrease in TIDC frequency could be detected was to determine the changes in expression of DAMPs during the first 24h upon EP only, while bleomycin only (CRT, HMBG1) and specific cell markers (MHC I, MHC had no effect. These results suggest that ECT induces an II, PD-L1 and CD40) after ECT in vitro. We used three efficient activation/maturation of TIDC with a departure murine cell lines that form immunologically distinct tu- of DC infiltrating the tumors through the upregulation of mor models: B16F10 (melanoma), 4T1 (mammary car- CCR7. cinoma) and CT26 (colorectal carcinoma). They were We then hypothesized that in ECT-treated tumors, new treated with ECT with three different cytostatic at IC50 infiltrating DC could be recruited mainly from the normal concentrations (concentration that induces death of 50 % blood vessels adjacent to the tumor. To address this of cells) – cisplatin (CDDP), oxaliplatin (OXA) and bleo- question, we had set up different strategies using CD11c- mycin (BLM). HMGB1 release was determined with an DTR-GFP or CD11C-YFP mice bearing melanoma. ELISA kit, and for the expression of cell markers we used First, we analyzed the DC mobilization in the tumor flow cytometry. All values were measured 4, 24 and 48 of CD11c-YFP mice in dorsal skin window chambers h after the treatment. We demonstrated that ECT with using a DSD-macroscope. This technology allows us to CDDP caused the most potent changes of measured im- quantify DC over time in each tumor region and to follow munological markers in all tested cell lines. An increase in their behavior during the first days after treatment. We HMGB1, CRT, MHC I, PD-L1 and CD40 in all three cell observed a decrease in DC density in the tumor and in lines within 48 h after the treatment was detected. ECT the periphery with ECT. Upon ECT, we also observed with OXA induced similar but less pronounced changes as an increase in DC speed in the tumor but also in the ECT with CDDP. ECT with BLM induced only a few of periphery when compared to control mice. These findings the tested markers and the response was the most potent are consistent with an activation of DC induced by the in B16F10 melanoma cell line, however more immunolo- treatment. gically important changes were detected compared to 4T1 In parallel, the analysis by fluorescence microscopy on and CT26 cell line. In conclusion, ECT induced changes tumor sections of CD11c-DTR-GFP mice injected with a in the expression of immunologically important cell mark-fluorescent anti-CD31 antibody that stains the endothelial ers. In general, CDDP and OXA induced more changes cells of blood vessels, confirmed the decrease of DC in than BLM. For exact evaluation of immunological effects tumor after ECT. Seven days later, the re-infiltration of of ECT, further in vitro and in vivo studies are needed. DCs in the tumor occurred through the blood vessels and took place mainly in the periphery of tumor. OR-204 Our data provide new insights in the induction of immune Skin dendritic cell mobilization upon electro- responses against solid tumors upon ECT. Defective chemotherapy: a dynamic analysis by fluorescence pathways could be overcome upon delivery of thera- microscopy and flow cytometry peutic/immunostimulatory molecules at proper timings Elisabeth Bellard, Nathalie Joncker, Marie-Pierre Rols, to induce a systemic protective response. Muriel Golzio IPBS-CNRS, France References: Electroporation (EP) of tissues in humans is feasible, 1. S. Roux et al. Cancer Immunology, Immunotherapy, efficient and tolerable. Its most advanced routine clinical 2008. use is electrochemotherapy (ECT). EP increases drug 2. C. Y. Calvet et al. Oncoimmunology, 2014. delivery into tumor cells that results in up to 80% of complete responses in different tumor types. OR-205 T cell contributes to transitory or complete clearance of Release of damage-associated molecular pattern tumors post-treatment (1). ECT with bleomycin induces molecules in vitro an immunological cell death of tumor cells (2), which is Tamara Polajžer, Tomaž Jarm, Damijan Miklavčič key for efficient tumor Antigen presentation by dendritic University of Ljubljana, Faculty of Electrical Engineering, cells (DC) to T cells. Poor systemic protective responses Slovenia may be due to an insufficient anti-tumoral polarization of Activation of the immune response appears to affect T cell responses by DC at the tumor site. Investigation the outcome of electroporation-based therapy. Activation of the impact of ECT on DC mobilization and activation of the immune system can be triggered by immunogenic in the tumor should provide keys to improve current cell death, in which damage / danger-associated molecu-treatments. lar pattern molecules (DAMPs) are actively or passively We assessed the impact of ECT with bleomycin on the released from cells. In the extracellular space, DAMPs growth of subcutaneous melanoma and on mobilization act as an endogenous damage signal that is recognized of DC in C57Bl/6. We found that ECT treated mice by cells of the immune system, promoting the release of display an oedema at the tumor site as early as 6 hours, pro-inflammatory mediators and recruiting other immune 63 cells. Along with inflammation, the adaptive immune High-frequency nanosecond electrochemotherapy with response is activated, resulting in long-term protection nsECT3 and nsECT4 parameters has shown to signi- against previously introduced molecules or cancer cells. ficantly reduce carcinoma tumour progression and pro- Since in both electrochemotherapy and irreversible elec- long the lifespan of C57BL/6J mice. After nsECT, the troporation the immune system response plays an import- level of anti-tumour antibodies doubled in treated mice ant role in the outcome of cancer therapy, we investigated compared to untreated tumour-bearing mice. The ex- if and when specific DAMPs are released in response to pression of suppressor and activation receptors on im- electroporation in vitro. Eight 100 µs long pulses with a mune cells was investigated. Changes in immune cell repetition frequency of 1 Hz, as commonly used in tumor populations were observed after nsECT3 and nsECT4. treatment and soft tissue ablation, were used in the ex- Changes in myeloid cells: increased expression of CD31 periments. We examined the release of ATP, high mobil- on splenic monocytes, relatively more M1 macrophages ity group box 1 protein (HMGB1), and externalization of and a higher percentage of CD11b� dendritic cells in tu- calreticulin after electroporation because they are the gold mours. Changes in lymphocytes: plasma, memory B and standard for predicting immunogenic cell death in cancer CD4�CD8� T cells were increased in the spleens of nsECT- cells. However, since other DAMPs are also known, we ex- treated mice. Newly identified CD3�CD4�CD8�B220�Gr- tended our study to the detection of released nucleic acids 1�CD11b�CD11c�DX5�CD1d�MHCII�CD86�CD80� “negat- and uric acid. In this study, different cell lines were used ive lymphocytes” were increased in spleens and tumours (CHO - hamster epithelial cell line, B16F1 - murine melan- after nsECT3 and nsECT4. These “negative lympho- oma and H9C2 - cardiomyocytes) to investigate whether cytes” had higher expression levels of antigen presentation the release of DAMPs in different cell types is different. markers (MHCII, CD80, CD86) and decreased expression Although detection of certain DAMPs remains uncertain of suppressor PD-L1. As opposed to nsECT3 and nsECT4 (i.e. uric acid), successfully detected DAMPs show a treatment, µ sECT treatment led to decrease in cytotoxic strong correlation with cell survival/irreversible electro- CD8� T cells and no increase in plasma cells. poration and a much weaker correlation with membrane It was shown that nanosecond electric pulses result in suc-permeabilization/reversible electroporation. The trends cessful nano-electrochemotherapy and an immunogenic re- of release of DAMPs appears to be similar in different cell sponse. Ultrashort pulses could be used to develop effect- lines. However, the exact amount of released DAMPs var- ive anticancer treatment strategies, gradually replacing ies, which may be attributed to different physiological and standard microsecond procedures. metabolic needs of the cell type. OR-10 P6 - Microalgae Biorefinery The Influence of New High-Frequency Nanosecond Electrochemotherapy on the Elimination of LLC1 Monday late afternoon Track A Tumours, Prolonging C57BL/6J Mice Survival Oct 10, 16:00 - 17:40 and Positively Affecting Changes in Immune Cell Subpopulations OR-41 Eivina Radzevičiūtė 1, Austėja Balevičiūtė1, Augustinas Integration of Pulsed Electric Field (PEF) Tech- Želvys1, Vitalij Novickij2, Veronika Malyško-Ptašinskė2, Jurij nology into the Microalgae Biorefinery Novickij2, Irutė Girkontaitė1 Christian Gusbeth, Damaris Krust, Wolfgang W. Frey 1Centre for Innovative Medicine, Lithuania Karlsruhe Institute of Technology (KIT), Institute for Pulsed 2Vilnius Gediminas Technical University, Lithuania Power and Microwave Technology (IHM), Germany This work focuses on anti-tumour immune response In recent years, many studies have shown that the after nanosecond electrochemotherapy (ECT) to provide use of pulsed electric field (PEF) technology to extract new data and further support to employ nanosecond ECT valuable compounds from microalgae is suitable for en- in clinics. Carcinoma tumours were induced by subcu- ergetic and nutritional purposes and meets economic re- taneously injecting C57BL/6J mice with LLC1 cells. For quirements. This was only possible through the imple-tumour ablation, bleomycin electrochemotherapy (ECT) mentation of the microalgae biorefinery concept, which was used with 4 different conditions of high-frequency involves the use of the residual biomass after the extrac- nanosecond ECT (nsECT1-4) in comparison with µ sECT tion of high and medium value products (e.g., pigments, procedure (1.3 kV/cm x 8 pulses, 100 µ s, 1 Hz). nsECT polysaccharides, and proteins) for biofuel production. The parameters (3.5 kV/cm × 200 pulses) – nsECT1 (200 ns, International Energy Agency, IEA, defines biorefinery as 1 kHz), nsECT2 (200 ns, 1 MHz), nsECT3 (700 ns, 1 kHz) “the sustainable processing of biomass into a spectrum of and nsECT4 (700 ns, 1 MHz). Afterwards, mice survival marketable products and energy”. A major advantage of and tumour growth dynamics were evaluated. For the PEF treatment is the bypassing of drying and the immedi- determination of anti-tumour antibody titers, mice blood ate processing of the wet concentrated biomass. Moreover, sera were collected 10 days after the treatment. Spleens, PEF treatment can be used as a mild and effective method lymph nodes and tumours were isolated from euthanized of cell disruption, facilitating recovery of unaltered con- mice, and cell suspensions were prepared. Immune cells stituents at low energy costs and allows cascade processing were identified in organ suspensions and antibody titers for multiple component recovery, since it does not destroy were assessed by multi-colour flow cytometry. cell shape and maintains gravimetrical biomass separab- 64 ility after single extraction step. However, cell compon-and mechanical treatments at the single-cell level, with ents are not immediately available since electroporation high throughput. Specific architectures of microfluidic does not conduct to cell disintegration. It was assumed devices devoted to electrical or mechanical solicitation on that irreversible electroporation of the cell membrane and cells will be presented. the subsequent increased permeabilization are the main As a single-cell level study, we will investigate the impact effects which allows proteins and other ingredients to pass of PEFs on the rigidity of the cell wall. Indeed, previous through the membrane. We found in our studies that ef- results have revealed the major role of the cell wall as a ficient protein extraction after PEF treatment requires an barrier to efficient lipid extraction. incubation step, and that the progress and kinetics of this Gaining knowledge at the single-cell level will further lead release depend on the biomass concentration and the in- to the development of optimized pretreatment extraction cubation temperature. In addition to diffusion in a chem- and culturing techniques, therefore economically viable ical gradient, a second biological, enzyme-driven process large-scale production. within the PEF-treated biomass was confirmed to facilit- ate the release of more than 40 % of total proteins from OR-42 the cells. This is mainly the water-soluble protein fraction, No downstream without upstream: Challenges for but proteins from the cell organelles such as nucleus, mito- PEF treatment of Arthrospira platensis and stud- chondria and chloroplast are also released. It is not exactly ies on the mechanism of phycocyanin release known which biological processes are triggered by PEF Justus Knappert 1, Christoph Lindenberger2, Christopher treatment in the cell, which lead to the release of valuable McHardy1, Cornelia Rauh1 components - apart from the enzymatic autolysis. It seems 1TU Berlin, Germany that the type of cell death (programmed cell death, nec- 2OTH Amberg-Weiden, Germany rosis, or apoptosis) induced by PEF treatment influences The cyanobacterium Arthrospira platensis is a prom- the efficiency of protein extraction. Therefore, a better ising sustainable source for edible proteins and other high understanding of cell death in response to PEF treatment valuable substances such as the blue pigment-protein com-could lead to possible improvements in protein extraction plex phycocyanin. The technology of Pulsed electric field efficiency. This presentation will give an overview about (PEF) was recently studied to permeabilize the cell mem- the various factors, such as the post PEF treatment incub- brane, enhancing the mass transfer of water-soluble cell ation parameters, which have an impact on protein release, metabolites. A major drawback for PEF is the high elec- as it could be possible to optimize the incubation to ob- trical conductivity of the cultivation medium, which pre- tain higher yields. In addition, the role of an important cludes a direct treatment. An exchange of the medium discovery is discussed, namely a water-soluble factor that on the other hand makes the process unnecessary com- is released by PEF-treated cells and triggers cell death in plex. Further, the question of the release mechanism for untreated cells. Based on the cell death inducing factor water- soluble substances is not sufficiently clarified in the released after treatment, a working model for the recovery published literature. The two stated problems represent a of compounds from microalgae is proposed. hurdle for the implementation of PEF for cell disruption of Arthrospira platensis on a commercial level. In the first OR-40 part of this study, the extent to which the cultivation of Microfluidic devices for the optimization of pre- Arthrospira platensis in salt-reduced medium is possible treatments in the context of microalgae-based pro- was investigated. In the second part, the relation between ductions cell permeabilization and phycocyanin release was ana- Solène Prudhomme, Sakina Bensalem lyzed. ENS Paris-Saclay, France It was shown that a comparable growth rate and pigment In the present context where sustainable and renew- composition can be achieved in modified salt-reduced me- able carbon-neutral biofuels are needed as an alternat- dium by controlling the pH with CO2 gassing. The elec- ive to fossil fuels to meet the rising energy demands and trical conductivity of 4.6 mS cm-1 of the modified medium while facing environmental issues, microalgae have gained significantly reduces the dilution factor before PEF, in increasing attention both in the scientific and industrial comparison to full medium (20 mS cm-1). To investigate communities. These unicellular photosynthetic organisms the mechanism of phycocyanin release, the degree of cell owe this particular attention to their capacity to use sun- permeabilization (cell disintegration index) was directly light, CO2 fixation, nutrients, and water to generate bio- measured by means of a new method using the fluorescent mass at a high rate with rich energy content. However, the dye propidium iodide (PI). The novel method allows con- production chain when using algae as feedstock is energy- clusions to be drawn about the effects of treatment time, intensive and provides a poor product quality since com- electric field strength and treatment temperature. Using a ponent fractions are mixed or emulsified. self-developed algorithm for image segmentation, the dis- As a first part, this lecture will demonstrate the poten- integration of trichomes was observed over a period of 3 tial of pretreatments, such as Pulsed Electric Fields and hours. This revealed a direct correlation between the cell mechanical stress to increase the extraction efficiency of disintegration index and the decay of the trichomes. This lipids produced by microalgae cells. decay, in turn, could be brought into a direct temporal We will show that microfluidic technology is a suitable ap- relationship with the release of phycocyanin. proach as it allows the analysis of the impact of electrical This work shows that it is necessary to consider the en- 65 tire process to implement PEF for cell disruption of Arth-to scrutinize underlying mechanisms and transferability to rospira platensis. The relation between permeabilization multiple organisms. and the kinetics of particle decay and phycocyanin extrac- tion is demonstrated for the first time. Thus, the results OR-44 presented contribute to a deeper understanding regarding Scaling microalgal biomass extraction for indus- the release of cell metabolites in response to PEF. This fa- trial application cilitates the design of new processes to produce sustainable Katja Zocher 1, Martina Balazinski1, Marie-Christine products from Arthrospira platensis. Sommer1, Raphale Rataj1, Michael Timm1, Marco Kreische2, Elke Kurth2, Petra Sandau2, Juergen F Kolb1 1 OR-43 Leibniz Institute for Plasma Science and Technology , Ger- Nanosecond Pulsed Electric Fields selectively many 2 foster cell proliferation in heterotrophic microal- IGV GmbH, Germany gae Downstreaming of microalgae and their industrial Byron Perez Simba 1, Robert Axelrod1, Iris Haberkorn2, application has become a growing field of interest. Mi- Alexander Mathys1 croalgae can serve, for instance, as supplier for biofuel, 1ETH Zurich, Switzerland fine chemicals, pharmaceuticals, cosmetics, or food ap- 2ETH Singapore Centre, Singapore plications. However, the extraction of these compounds is Nanosecond pulsed electric fields (nsPEF) are a prom- challenging due to their sturdy cell wall. Standard extrac- ising tool to increase upstream efficiency in cellular agri- tion methods, e.g. bead-milling, sonication, chemical, or culture. Several biological effects including gene expres- enzyme extraction suffer from several drawbacks. They sion and growth stimulation have been reported for pho- are often ineffective against the cell wall, inacceptable toautotrophic microalgae species. The process window heat development occurs, long treatment times are tailoring such effects is defined by several electrical and necessary, or environmental harmful solvents are used. biological parameters. Optimization protocols have been Moreover, scaling of these methods are often difficult or limited to photoautotrophic organisms; however, hetero- uneconomic [1]. trophic species currently have a higher relevance owing Based on our previous studies [2] [3], we developed a spark to improved consumer perception and industrial-relevant discharge treatment in industrial scale for the extraction productivity. of Cyanidium caldarium, which is an extremophile mi- This research highlights nsPEF-related advances in the croalga. The alga is used for the production of cosmetics process optimization of heterotrophic microalgae (Aux- due to its high amounts of polyphenols and amino acids, enochlorella protothecoides and Chlorella vulgaris), lever- which are postulated to have anti-aging properties [4]. aging biomass productivity. Advanced statistical mod- The developed system was created modular to meet re- eling using Plackett Burman’s design was established to quirements from the industry partner concerning varying identify optimal process parameter combinations (pulse treatment volumes. Extraction processes were conducted width, -repetition frequency, -number, electric field with a pulse amplitude of 35-40 kV at 11 Hz, and a strength). The screening study (P=0.0002, R2 =0.8) pulse length of 100 ns. The algae suspension was moved showed a significant effect of microalgae species (P= through the system by a peristaltic pump, which ensured 0.00002) and electric field strength (P= 0.00167) on uniformly treatment of the bulk liquid and made external the percentage of biomass difference compared with the cooling unnecessary. Treatment times depended on the pumped control. The pulse width, -number, and time volume of biomass (dry weight 100g/l), usually between between treatments did not have a significant effect on 30 and 300 minutes. During the extraction process, the final biomass yield. The best productivity resul- the bulk temperature did not exceed 25 °C, which is ted in 12.28±0.028 g/L of C. vulgaris biomass harvested preferable for thermolabile extractives, such as proteins 96 h after inoculation and 72 h after nsPEF treatment or pigments. (15 kV/cm, 5 Hz, 40 ns, 2 treatments after 5 minutes). Additionally, under the set treatment parameters, it It was 26.22±1.25% higher than the control 9.73 ±0.55 was possible to extract the blue pigment phycocyanin (P=0.009). The model maximization determined an in- without the addition of chemicals or freeze thawing crease of 22.77% with a 95% confidence interval [16.02%- cycles. The extraction success was compared to several 29.52%] for C. vulgaris by applying two treatments after 5 conventional techniques. For comparing studies, two minutes each with 15 kV/cm, 5 Hz, 100 ns pulses. Auxeno- sonication treatment schemes and pulsed electric fields chlorella protothecoides did not show a significant increase treatment with equal energy input in each case as for in biomass 95%-CI [-3.7%, 2.22%]. The treatment effect on spark discharges were conducted. Moreover, a protocol lipids and protein content was analyzed while pore forma- of freeze-thaw-cycles were performed as most common tion, calcium flux, and transcriptomics were used to study extraction method for phycocyanin. For PEF treatment underlying mechanisms. and freeze-thawing, no noteworthy extraction yields of This work lays the foundation for improved microalgae phycocyanin were achieved in comparison to spark dis- productivity and, therefore, affordability of this poten- charge treatment. For one sonication scheme, an elevated tially more sustainable resource. It develops further know- extraction yield (13mg/g dw) was achieved compared ledge on using experimental design for screening several to 3mg/g dw of purified extract after spark discharge combined factors in nsPEF-research and opens the door treatment t. However, proteomic analysis revealed that 66 especially freeze-thaw cycles and sonication cause the ated the subsequent separation/purification stages. most modifications on the protein level of phycocyanin, This work demonstrated the feasibility of coupling elec-whereat spark discharges showed only minor changes, trical and mechanical technologies in the frame of mi-indicating that this method is much gentler to sensitive croalgal biorefinery, with the aim to develop a tunable extractives, such as pigments. technological platform capable of efficiently valorizing dis- parate microalgal strains, thus increasing the availability References: of high-added value molecules to feed multiple industrial [1] Lee, A.K. et al Biomass and bioenergy, 2012. 46: sectors. p. 89-101.; [2] Zocher, K., et al., Algal Research, 2019. 39: p. 101416; [3] Zocher, K., et al Journal of Physics D-Applied Physics, 2020. 53(21); [4] Pflaumbaum, M., et P10 - Gene Electrotransfer for al. Personal Care, 2013. 4: p. 113-6.; [5] Sommer, M.-C., Immunotherapy et al. Microorganisms, 2021. 9(7): p. 1452. Monday late afternoon Track B OR-45 Oct 10, 16:00 - 17:30 Application of pulsed electric fields and mechanical cell disintegration techniques in biorefinery cas- OR-67 cade of microalgae Transfection by electroporation using pulses from Daniele Carullo, Francesco Donsì, Giovanna Ferrari, Gian- nano- to millisecond duration in vitro piero Pataro Tjaša Potočnik, Tamara Polajžer, Alenka Maček-Lebar, University of Salerno, Italy Damijan Miklavčič Owing to the wide variability in structural features University of Ljubljana, Faculty of Electrical Engineering, characterizing the plethora of existing microalgal strains, Slovenia this work investigated the applicability of pulsed electric Gene electrotransfer (GET) or gene transfection by field (PEF) technology to promote the release of intracel- electroporation is a widely used method in which elec-lular compounds from two model species (e.g., A. platen- tric pulses cause temporary increase in cell membrane per- sis, and C. vulgaris) in combination with different mech- meability and thus enable the entry of nucleic acids into anical cell disruption techniques, namely high-shear ho-cells. The purpose of this work was to explore, evaluate mogenization (HSH), and high-pressure homogenization and demonstrate potential use of different pulses for in- (HPH). troducing plasmid DNA (pDNA) into cells in vitro and For this purpose, two different routes were followed, based compare efficiency and dynamics of gene expression after on delivering PEF treatments of constant intensity (20 GET. kV/cm, 100 kJ/kgSUSP.) to i) A. platensis after a gentle We performed experiments on cell suspensions of immor-HSH step (20.000 rpm, 96 kJ/kgSUSP.), and ii) C.vulgaris talized human skin fibroblasts 1306 cell line and on C2C12 prior to an ultimate HPH process (5 passes at 150 MPa, murine myoblasts with four ranges of pulse durations 750 kJ/kgSUSP.). Cell disruption efficiencies associated (nanosecond, high frequency bipolar pulses (HF-BP), mi- with either single or combined techniques were properly cro and millisecond). Cells were exposed to electric pulses assessed through morphological (optical/scanning elec- in 2 mm cuvettes and transfected with pDNA encoding tron microscopy, and particle size distribution), quali- green fluorescent protein (GFP). Six pDNA concentra- quantitative (content of water-soluble carbohydrates, and tions, namely 40, 60, 80, 100, 250 and 500 µg/ml, were proteins in the achieved supernatant after aqueous extrac- tested. After 24 hours cell survival was detected with tion step), and economical (calculation of the specific en- MTS assay and percentage of GFP positive cells and their ergy consumption per unit mass of dry weight target com- median fluorescence intensity (MFI) with flow cytometry. pound) analyses. To determine time dynamics of GFP expression after GET As a general trend, single PEF treatments induced no with different duration of pulses percentage of GFP pos- measurable effect on the cell shape/structure, but only itive cells and their MFI were measured every 8 hours for a surface shrinkage could be detected, which was likely 6 days. attributed to the occurrence of intracellular compounds Overall GET, which represents the percentage of trans- leakage during water diffusion step. Interestingly, the ap- fected cells relative to the initial population and considers plication of PEF in a sequential mode with mechanical both the efficiency of transfection and cell survival, in-treatments boosted the yield and selectivity of extraction creased with increasing pDNA concentration for all pulse from A. platensis and C. vulgaris cells, with an outstand- durations and both cell lines. Using the highest pDNA ing additive effect detected towards low molecular weight concentration, i.e. 500 µg/ml, we achieved between 35% compounds (e.g., carbohydrates). of overall GET with millisecond pulses in C2C12 myo- The cascaded combination of PEF and HSH/HPH tech- blast. Overall GET with millisecond pulses was however niques led to comparable and, in some cases, lower specific significantly lower in 1306 fibroblast, 15%. Interestingly, energy requirements for proteins and carbohydrates recov- overall GET with microsecond pulses was comparable in ery than those granted by single HPH processing, with both cell lines, at approximately 30%, as well as for HF- the latter causing cell debris formation and an undifferen- BP pulse protocol, at 20%. On the contrary, with nano- tiated release of intracellular compounds, which complic- second pulses overall GET was higher in 1306 fibroblast 67 compared to C2C12 myoblast. Also, MFI of GFP posit-tissues has not been explicitly explored. The current ive cells was significantly higher in 1306 fibroblast with all method of pressure-based injection of DNA solution pulse durations except for millisecond pulses showing that using a syringe does not allow precise control over the GET efficiency depends on cell line. Our measurements macroscopic distribution of DNA in tissues [1,2], leads to also showed that time dynamics of the onset of GFP (both inhomogeneous distribution [2], results in tissue damage percentage of GFP positive cells and their MFI) are com- due to excess pressure [3] and presents a challenge in parable for nanosecond, micro- and millisecond pulses in scaling up from small animals (mice) to large animals 1306 fibroblasts. and humans [1]. In this work, we use electrophoretic infusion through a micro-pipette to transport DNA over OR-68 macroscopic distances in-vitro in tissues models (3D Skin dendritic cell mobilization upon Gene elec- gels). We show that long-duration low-intensity electric trotransfer (GET) currents allow control over the macroscopic spread of Elisabeth Bellard, Lise Pasquet, Sophie Chabot, Marie- DNA through electrophoresis, and theoretical models Pierre Rols, Muriel Golzio can be used to predict the macroscopic distribution. CNRS-IPBS (Institut of Pharmacology and Structural Bio- Further, permeabilizing electric field can be subsequently logy), France applied and its distribution can be modelled to provide Electropermeabilization/electroporation (EP) is one a maximum overlap with the macroscopic distribution of of the non-viral methods based on the native transmem- DNA, leading to efficient gene electro-transfer without brane electric potential modulation of the cell by applying excess or insufficient coverage of electric field. electric pulses. This physical method thus enables the efficient and local delivery of chemotherapeutic drugs References: into tumor cells in Electrochemotherapy (ECT) (1) or [1] Dupuis, Marc, Kimberly Denis-Mize, Carolyn Woo, nucleic acids for interleukin-12 gene-electro transfer in Cheryl Goldbeck, Mark J. Selby, Minchao Chen, Gillis R. Immuno-Gene Electro-Therapy (IGET) (2). For Gene Otten et al. ”Distribution of DNA vaccines determines Electro-Therapy (GET), we set up electric field para- their immunogenicity after intramuscular injection in meters to obtain a high expression of both fluorescent mice.” The Journal of Immunology 165, no. 5 (2000): reporter and therapeutic genes while showing full safety 2850-2858. in living animals (3). The combination of pIL-12 GET [2] Henshaw, Joshua W., and Fan Yuan. ”Field distribu- and partial irreversible electroporation (IRE) not only tion and DNA transport in solid tumors during electric enhanced survival but also bring a curative effect in wild field-mediated gene delivery.” Journal of pharmaceutical type mice (2). This two-step treatment, named Immuno- sciences 97, no. 2 (2008): 691-711. Gene Electro-Therapy (IGET), led to the activation of [3] Boye, Carly, Sezgi Arpag, Nina Burcus, Cathryn Lund- the adaptive immune system and the development of an berg, Scott DeClemente, Richard Heller, Michael Francis, anti-tumor immune memory (2). We are now using GFP and Anna Bulysheva. ”Cardioporation enhances myocar- transgenic mice to evaluate the effect of EP parameters dial gene expression in rat heart.” Bioelectrochemistry on the skin dendritic cells by intravital microscopy 142 (2021): 107892. (unpublished data). OR-70 References: Immunomodulatory effects of radiotherapy and [1] Josserand V, et al. J Control Release. 2016 May gene electrotransfer of plasmid DNA encoding 4;233:81-87. chemokines CCL5 and CCL17 in murine tumors [2] Pasquet L, et al. J Immuno ther Cancer. 2019 Jun Tim Bozic 1, Lucija Kozjek Mencinger2, Simona Kranjc 26;7(1):161. Brezar1, Gregor Serša1, Iva Santek1, Maja Čemažar1, Bostjan Markelc1 [3] Pasquet L et al. Sci Rep. 2018 Nov 15;8(1):16833. 1Institute of Oncology Ljubljana, Slovenia 2 OR-69 University of Ljubljana, Faculty of Health Sciences, Slovenia Electrophoretic Infusion of DNA into Gels and Tis- Chemokines are small signaling proteins belonging to sues: Experimental and Theoretical Analysis the superfamily of cytokines, which regulate immune cell Shaurya Sachdev, Damijan Miklavčič migration. The degree and the type of infiltrated im- University of Ljubljana, Faculty of Electrical Engineering, mune cells into the tumor microenvironment affects the Slovenia disease progression and correlates with the efficacy and Gene electrotransfer is a process in which pulsed outcome of immunotherapies. Similarly, beneficial im- electric field is used to increase cell membrane per- munomodulatory effects were also observed after irradi- meability and introduce foreign genetic material into ation. Therefore, we sought to investigate gene electro-cells for gene therapeutic and gene editing applications. transfer (GET) of proinflammatory chemokines CCL5 or While most research in gene electrotransfer is dedicated CCL17 in combination with irradiation, as a potential to electrophoresis of DNA at the microscopic length therapeutic strategy for cancer therapy. Specifically, the scale close to cell membrane and DNA translocation chemotactic properties of investigated chemokines were across the cell membrane, the issue of DNA mobility first examined on cell cultures in vitro in murine CT26 over macroscopic length scales and DNA distribution in colon and 4T1 breast cancer models. Next, extravasation 68 inducing ability of chemokines was determined using in-and CXCL11. Signaling of these chemokines through the travital microscopy of fluorescently labeled splenocytes in CXCR3 receptor is hypothesized to improve the clinical CT26 and 4T1 dorsal window chamber models in vivo. response to anti-PD-1 therapies, including pembrolizu-Murine tumor models were chosen based on their immun- mab (ref 1). Biopsies collected during a Phase 2 clinical ophenotype – CT26 corresponding to inflamed and 4T1 to trial evaluating IT-TAVO-EP plus pembrolizumab in immunosuppressive tumor model – allowing for a compar- melanoma patients predicted to be refractive to anti-PD-1 ison of different tumor predisposition to immune response. immune checkpoint inhibitors (NCT03132675) showed The antitumor effectiveness of combined therapy utilizing that clinical outcome was closely tied to intratumoral GET of chemokines and two irradiation regimes (single CXCR3 mRNA levels, emphasizing the mechanistic dose of 10 Gy and fractionated dose of 3x 5 Gy) was de- importance of tumor-infiltrating CXCR3+ immune termined in vivo. Lastly, qRT-PCR was used to evaluate cells. Therefore, we asked if maximizing intratumoral gene expression of several cytokines in tumors after ther- chemokine levels by electroporation of pCXCL9 could apies, while changes in the abundance of CD4+, CD8+ augment the anti-tumor immune responses driven by cells and vasculature (CD31+ cells) were determined with intratumoral IL-12. immunofluorescent staining. Both chemokines CCL5 and In this study (ref 2), we show that the IT-pIL12-EP CCL17 were able to induce the migration of mouse mac- leads to an increase in CXCR3+ lymphocytes in the rophages RAW264.7 in vitro. Similarly, both CT26 and local lymph node and that blocking the CXCR3 signaling 4T1 dorsal window chamber models showed increased re- axis abrogates IL-12 anti-tumor efficacy. Intratumoral tention of splenocytes after GET of chemokines compared electroporation of CXCL9 with IL-12 augments the to control. CT26 tumor growth delay after combined ther- anti-tumor responses seen with intratumoral IL-12 alone. apy of GET of chemokines and both irradiation regimes Consistent with these findings, transcriptomic analyses of was significantly longer compared to control and even led treated lesions demonstrated upregulation of signatures to tumor cures. In the case of 4T1 tumors, only GET for interleukins, interferons, GPCR activation, antigen of chemokines combined with fractionated irradiation led presentation machinery, and TCR signaling, suggesting to a pronounced tumor growth delay but without tumor that IL-12/CXCL9 reshapes the TME to promote dend- cures. Gene expression analysis showed increased expres- ritic cell licensing and CD8+ T cell activation. Finally, sion of both chemokines after corresponding therapies. we show that the combination of IL-12 and CXCL9 drives Moreover, increased expression of CXCL9 and CXCL10, anti-tumor responses in distant untreated lesions and two potent chemoattractants of cytotoxic CD8+ T lymph-significantly improves anti-PD-1 response. These findings ocytes, was determined in most combined therapies. Com- show that the CXCL9/CXCR3 axis synergizes with parison of the degree of infiltrated immune cells into the IT-pIL12-EP therapy. Maximizing CXCL9 expression tumors showed increased numbers of helper CD4+ and through intratumoral electroporation may enhance the cytotoxic CD8+ T lymphocytes after GET of chemokines, efficacy of intratumoral IL-12 therapies in the clinic. however their numbers decreased whenever irradiation was used. Reduced numbers of immune cells were recapitu- References: lated in tumors after combined therapy along with the 1. Chow et al. Immunity. 2019 Jun 18;50(6):1498-1512.e5 decreased area of tumor vessels. Our results show the po- 2. Lee et al. Accepted at Mol. Ther. – Oncolytics. April tential of chemokines in cancer immunotherapy, however 2022. additional optimization of combined therapy is needed be- fore translation into clinics. OR-139 Differences in Nano-Electrotransfection Efficiency OR-71 Between Cancer and Primary Murine Immune OncoSec’s 2nd Generation Therapy: Amplifica- Cells tion of the CXCR3/CXCL9 axis by intratumoral Eivina Radzevičiūtė 1, Veronika Malyško-Ptašinskė2, Vitalij electroporation of plasmid CXCL9 synergizes with Novickij2, Jurij Novickij2, Irutė Girkontaitė1 plasmid IL-12 therapy to elicit robust anti-tumor 1Centre for Innovative Medicine, Lithuania immunity 2Vilnius Gediminas Technical University, Lithuania Jun Zhang1, Jack Lee1, Erica Browning1, Mia Han1, Kurt In this work we compared nano and microsecond Sakurado1, Vincent Wu1, Nygel Oglesby1, Tarsicio Juarez1, range electroporation protocols for electrotransfection H. Kim Lyerly2, Daniel Fisher3, Adil Daud4, Alain Algazi4, of cancer cell lines (4T1) and primary murine dend- Joseph Skitzki3, Chris Twitty1, David A. Canton 1 ritic cells (DC). The efficiency of electrotransfection 1Oncosec Medical Incorporated, United States was evaluated using two different molecular sizes and 2Duke University, United States different detectable protein-encoding plasmids: green 3Roswell Park Cancer Institute, United States fluorescent protein (GFP) encoding plasmid (4.7 kbp; 4University of California, United States p-EGFP-N1) and a plasmid expressing firefly luci- Previous studies have shown that intratumoral elec- ferase and red fluorescent protein (tdTomato) (8.5 kbp; troporation of plasmid IL-12 (tavokinogene telseplasmid, pcDNA3.1(+)/Luc2=tdT)). or IT-TAVO-EP) leads to upregulation of IFN- γ, a First of all, the permeabilization of the cells was studied critical driver of tumor infiltrating lymphocytes (TIL) to ensure that both µ sPEF and nsPEF protocols trigger through the action of its target genes, CXCL9, CXCL10, saturated permeabilization. The susceptibility to PEF of 69 all the cell lines involved in the study was similar. Two Effects of nanosecond electric pulses (nsEP) are re-different protocols were selected: nanosecond pulsed elec- duced (“canceled”) when the pulses are made bipolar. tric fields (nsPEF) (7 kV/cm x 300 ns x 100, 1 MHz) and This phenomenon of bipolar cancellation enables interfer- microsecond ( µ sPEFs) (1.2 kV/cm x 100 µ s x 8 pulses), ence targeting of nsEP effects, when two bipolar wave-which trigger high permeabilization. After that we used forms combine into a unipolar nsEP remotely from stimu- CHO-K1 cell line, as a model cell line, to determine the lating electrodes. The change in pulse shape, from bipolar optimal concentration of used plasmids for further usage (inefficient) near the electrodes to unipolar (efficient) at in 4T1 cell line and primary murine DCs electrotrans- a distance, cancels the bipolar cancellation (CANCAN ef- fection protocols. The efficiency of electrotransfection of fect). We showed CANCAN efficiency for preferential per- CHO-K1 cells was similar for both plasmids even though meabilization and ablation of cell monolayers in the center the size of the plasmid is different. Also, nsPEF and of a quadrupole electrode array. CANCAN focusing was µ sPEF returned comparable results. It was not the case demonstrated in CHO, HEK, and BPAE cell lines, using for 4T1 and DCs. The transfection rate with bigger plas- different pulse parameters and permeabilization markers. mid was reduced several-fold and at the same time the A world’s first single-body nsEP generator designed nsPEF protocol ensured significantly better transfection specifically for CANCAN targeting, EPULSUS-FPM4- efficiency than µ sPEFs. Afterward, we evaluated primary 7, was built by EnergyPulse Systems in Lisbon, Por- DCs transfection efficiency using immature and mature tugal. This generator utilizes a second-generation CAN- DCs, where for maturation LPS and TNF- α were used. CAN concept, with four channels of unipolar nsEP repla- However, there were no significant differences in transfec- cing two channels of bipolar nsEP. Four electrodes, form- tion efficiency between immature and mature DCs. ing a quadrupole, are alternately energized by unipolar It was shown that the used nsPEFs protocol ensured equi- nsEP in a series which creates multiphasic, bipolar elec- valent or better transfection efficiency than µ sPEFs. How- tric field oscillations near and between the electrodes. The ever, the plasmid size, concentration and the cell type af-electric field in the center of the quadrupole stays unipolar fect the transfection efficiency even though the sensitivity and is more efficient at producing bioeffects. The second-to electroporation itself is similar in all used cells. In this generation CANCAN is more versatile and confines nsEP study, we demonstrate the applicability of nsPEFs range effects to smaller remote targets. protocols for electrotransfection of primary murine DCs. We coupled the EPULSUS generator with a one-of-a- The results of this study are appliable in gene therapy and kind strobe laser workstation for imaging cell membrane DNA vaccination studies, where ultrashort pulses method- potential with nanosecond time resolution. It is utilized to ologies can be used. The nsPEF can be an alternative to explore complex effects of CANCAN stimulation and the available gene electrotransfection methods and protocols. dependence of bipolar cancellation on the angle change Acknowledgement: The project was funded by the Re- of the electric field vector. Fast strobe imaging may also search Council of Lithuania (No. S-MIP-19-22). provide answers to vital questions that have been debated for decades, such as the kinetics of electropore formation and resealing. We also plan to use strobe imaging to es- P15 - Nanosecond Pulse Stimulation tablish how CANCAN activates Na+ and Ca2+ voltage- and Electropermeabilization gated ion channels, to support future CANCAN applica- (MURI AFOSR) tions such as non-invasive deep brain stimulation. Support: AFOSR MURI grant FA9550-15-1-0517 to Monday late afternoon Track C AGP. Oct 10, 16:00 - 17:30 OR-06 Nanosecond electric pulses target the alpha-1 sub- OR-04 unit of membrane Na,K-ATPase Focusing nanosecond pulse effects away from elec- Giedre Silkuniene, Uma Mangalanathan, Andrei G. Pak- trodes homov, Olga N. Pakhomova Andrei G. Pakhomov 1, Iurii Semenov1, Vitalii Kim1, Aleh Old Dominion University, United States Kandratsyeu2, Luis Redondo3, Emily Gudvangen1, Shu Xiao1, Allen Kiester4, Joel N. Bixler4, Bennet L. Ibey4 Experimental evidence suggests that electric pulses 1Old Dominion University , United States (EP) can alter ion channel conformation. The structural 2EnergyPulse Systems, Portugal alterations in channel proteins likely impact membrane 3Lisbon Engineering Superior Institute, GIAAPP/ISEL, Por- permeability, providing pathways for transporting ions tugal and molecules across the plasma membrane. We per- 4Air Force Research Laboratory, United States formed a high-throughput screening to find ion channels affecting membrane permeabilization by nanosecond EP There is a tremendous promise in focusing bioeffects (nsEP). of electric stimuli away from electrodes, with applications Using lentiviral CRISPR/Cas9 gene editing we generated such as non-invasive deep brain stimulation and uniform gene knock-outs (KOs) in cell human monocytes U937 tumor ablation. The challenge is avoiding bioeffects near and obtained a total of 328 derivatives with KO for electrodes, where the electric field is the strongest, while a single ion channel gene. Each KO was exposed in achieving them remotely with a (much) weaker electric cuvettes to 20 or 40 pulses (300ns, 7 kV/cm, at 20 field. 70 Hz). In 50 min, cells were imaged for Yo-Pro-1 (YP) using streak camera microscopy (SCM). More recently, uptake on an Olympus IX83 microscope configured for we constructed a strobe illumination microscopy system high-throughput screening. We found that KO of the for use in conjunction with FlouVolt™ dye for imaging ATP1A1 gene coding for the alpha-1 subunit (ATP1A1) variations in cell membrane potential during the applica-of Na,K-ATPase (NKA) reduced YP uptake by up to tion of electric pulses. The system allows flexible variation 30 % in two independent series of experiments. The of trigger timing in order to image any time point of a cell- subsequent work aimed to validate and further explore electric pulse interaction. The system has minimum time this findings by other methods. resolution of 10 ns. While comparable to SCM, strobe First, YP uptake within 5 min after nsEP was measured photography requires the delivery of multiple electric and in ATP1A1 KO cells by time lapse imaging on a scanning illumination pulses to visualize the full time course of an confocal microscope. Ten 300ns pulses (7 kV/cm, 5 Hz) exposure event, but affords the ability to acquire full 2 di- were delivered to cells in the field of vision with a pair mensional image sequences. These tools are compliment- of tungsten electrodes. YP fluorescence in ATP1A1 KO ary in nature, and provide the ability to directly measure cells was ˜20% lower than in control cells, confirming the responses never before possible. Potential applications for data obtained with the high-throughput screening. the system include resolving the charging and discharging Next, ATP1A1 was suppressed by transduaction of U937 times of a cell membrane and measuring the time a cell cells with shRNA. The ATP1A1 knock-down (KD) was takes to exhibit a physical response to an electric pulse. validated with qPCR, and KD cells were accessed for Method: We imaged CHO-K1 cells, loaded with Fluo- YP uptake. We measured a reduction of YP uptake in Volt™, exposed to pulsed electric fields. Imaging was ATP1A1 KD cells by 20% and 25% in high-throughput conducted with both strobe microscopy and SCM. Electric and confocal setups, respectively. pulse energies were chosen such that no apparent cell dam- Next, we tested if NKA inhibition with ouabain affects age was observed, enabling acquisition of multiple images the YP uptake. U937 cells were incubated with 1 µM from the same cell in order to resolve the onset, duration, ouabain for 15 min, then exposed to 10 pulses (300 ns, and passing of each pulse. Using these systems, we cap- 7 kV/cm, 5 Hz) and assayed for YP uptake using the tured data observing the membrane charging response to confocal microscope setup. We found that the presence both square wave and AC waveforms. of ouabain did not reduce YP uptake but increased it Discussion: We demonstrated the ability to resolve a by 21%. This result confirms the role of ATP1A1 in whole cell interaction with an electric pulse with temporal YP uptake after nEP and indicates that this role is not resolution of sub-microsecond. Our systems enables the related to NKA physiological function as an ion pump. capture of the full 2D spatial scene with strobe and a During the high-throughput screening, we noticed that single spatial dimension with SCM. Hypothetically, the the initially strong inhibition of YP uptake diminishes strobe system should be able to resolve cellular response over time. We hypothesized that the function of the to frequency up to 30 MHz, given sufficient signal to noise ATP1A1 gene was compensated by paralog genes typ- for the fluorescent dye. Previous work with streak camera ically present in U937 at low expression levels. Using microscopy demonstrated a similar limit of 5 MHz as is qPCR we checked the expression level of other NKA’s reported here, based on limitations of the fluorescent dye. alpha subunits and found that ATP1A4 expression was By obtaining a full 2D image, we are able to resolve the upregulated in ATP1A1 KD cells. Therefore, upregula-charging kinetics of individual cells across a wide field of tion of NKA paralog genes can compensate for inhibited view and directly correlate that response to electric field ATP1A1. modeling results. This enables the validation of modeling Overall, our data point to ATP1A1 as a potential protein results for cell size, orientation, cell position relative to the target (or one of protein targets) for cell membrane electric field and when combined with other techniques, permeabilization by nsEP. cell cycle, pharmaceutical treatment and other physical stressors (mechanical fields, temperature, environmental Support: AFOSR MURI grant FA9550-15-1-0517 to buffer). AGP. Conclusion: Ultrafast imaging tools such as SCM and strobe photography are capable of resolving membrane charging dynamics in a variety of cell types with sub- OR-07 microsecond temporal resolution. Combined, these tools Ultrafast Imaging enabling the direct observation can enable visualization of charging rate dynamics, estim-of the charging dynamics caused by pulsed electric ation of frequency dependent dielectric properties, among field effects others. Joel N. Bixler, Zachary Steelman, Allen Kiester, Bennett L. Ibey OR-08 Air Force Research Laboratory, United States Excess Efficacy of Nanosecond Stimulation in Car- Introduction: Ultrafast Imaging tools have the poten- diomyocytes tial to enable direct visualization of cellular response to Christian W. Zemlin, Federica Serra pulsed electric fields. Previously, we demonstrated the Washington University in St. Louis, United States ability to image changes in membrane potential during It is generally assumed that when an electric field is cellular exposure to unipolar and bipolar electric pulses applied to a cell or tissue, the induced drift of ions and 71 accumulation of charges at the membrane is the main involves activation of voltage-dependent (voltage-gated) mechanism by which the field changes the transmembrane ion channels (VGIC), ubiquitous transmembrane proteins, voltage. Since theory predicts a drift velocity proportional that engage in the propagation of the electrical signal. to the applied field, the charging current, too, should be Under normal conditions, this activation is induced by a proportional to the applied field. If a certain charge ac- change in transmembrane potential in the order of few cumulation is required, e.g. to excite a cell, the required tens of mV and takes place on time scales of the order of field strength should be proportional to the duration of ten to a hundred microseconds. Hence, the mechanism of the field application. In other words, the basic theory of excitable cells’ activation by nsPEFs, i.e. pulses of few the drift of charges in an electric field predicts that the nanoseconds duration, is still puzzling, because of the strength-duration curve for the stimulation of an excit- significant difference in the time scales involved. There is able cell has slope -1 in a log-log plot. no clear consensus to date on the underlying mechanism In experiments, this theoretical prediction has generally leading to electrostimulation when cells are subject to been confirmed for nerve stimulation, but not for cardi- nsPEFs. omyocytes, for which the log-log slope of the strength- In this study we focus on a mechanism that might duration curve has been reported to be between -0.75 and potentially be implicated in the activation of VGICs. -0.5. This implies that short, strong pulses are more ef- nsPEFs were shown experimentally to generate both fective at charging the membrane than predicted by basic intracellular and extracellular Radical Oxygen Species theory. (ROS). Moreover, it has been suggested over two decades To understand what mechanisms can cause this excess ago, that model as well as cell membranes can be oxidized efficacy, we developed a model of membrane charging when subject to PEFs. There is experimental evidence that considers all know mechanisms by which the field that pulsed electric fields can increase the extent at which affects the transmembrane membrane: 1) Propagation unsaturated lipid acyl chain peroxidation occurs. of the applied field itself; 2) dielectric displacement; 3) Here, we first present results from computational drift/electrodiffusion; 4) active membrane response (open- chemistry methods, in particular molecular dynamics ing of ion channels); 5) ballistic ions, i.e. ions that are simulations of model lipid membranes that show that sufficiently close to the membrane to be able to reach it lipid oxidation induces changes in the properties of the without collisions with water or other molecules. We also membranes that can be drastic and long-lived enough considered different criteria for whether a pulse achieved to trigger activation of an action potential. Our results excitation: a) reaching a certain transmembrane voltage, show indeed that under certain conditions, lipid oxidation e.g. -45 mV or -55 mV; b) maintaining a certain depolar-of a patch model membrane might lead to a spontaneous ization for a minimum time (11 µs) to allow for chan- membrane depolarization which could be large enough to nel opening; c) when considering active membrane re-activate voltage-gated sodium channels and generate an sponses, whether the sodium activation variable m reaches action potential. the value 0.5. We then resorted to modelling of the action potential We found that the combined mechanisms of field propaga- at the cell level with Hodgkin-Huxley models. We tion, dielectric displacement, and drift lead to strength- present here results from equivalent circuit models’ duration curves with a plateau for short pulse durations implementation in Comsol Multiphysics to gain better that transitions into a log-log slope -1 for longer dura- understanding of the macroscopic response to the physical tions, consistent with experimental data for nerve stim- changes induced by lipid oxidation. Interestingly enough, ulation. The inclusion of an active response caused a these models confirmed as well, the predictions from the variety of interesting effects, including a profound reduc- microscopic (MD simulations) investigation. tion of the stimulation threshold for microsecond duration pulses (by more than 50%). The inclusion of an active re- sponse did not, however, substantially change the slope of the strength-duration curve. Our analysis of ballistic ions suggests that they may help explain the excess efficacy of short, strong pulses. OR-09 P3 - Pulsed Electric Fields (PEF) Cellular Excitability and nsPEFs: Involvement of Strategies in Enhancing lipid oxidation in action potential activation Health-promoting Aurianne Rainot1, Lea Rems2, Mounir Tarek 1 Properties of Plant-based Foods 1Université de Lorraine, France 2University of Ljubljana, Faculty of Electrical Engineering, Slovenia Monday late afternoon Track D Oct 10, 16:00 - 17:30 Several experimental studies have shown that nano- second pulsed electric fields (nsPEFs) can stimulate neuronal cells. Excitable cells stimulation results in gen- eral in a depolarization - repolarization of the membrane of these excitable cells, called action potential. The latter 72 OR-220 also to increase the sustainability of the agriculture and Pulsed electric fields (PEF) for the preservation food processing industry allowing mitigating the environ-and bioproduction of health-related compounds mental burdens of these sectors. and properties in plant-based foods The recovery of bioactive compounds from agro-food Robert Soliva-Fortuny, Olga Martin-Belloso, Pedro Elez- biomasses with sufficiently high yields is typically carried Martinez out via conventional solid-liquid extraction using organic University of Lleida, Food Technology, Spain solvents, which are mostly toxic, hazardous and harmful. The concentration and biological activity of phyto- However, being these compounds embedded in the chemicals, as well as of several compounds closely re- plant cells envelop, to reduce the resistance to mass trans- lated to food quality, is dramatically affected when high fer of intracellular compounds the implementation of effi- temperatures are applied during food processing. Elec- cient cell membrane permeabilization steps complement- tropermeabilization, cell wall disruption and other struc- ing conventional solvent extraction has been proposed. tural changes caused by pulsed electric field (PEF) treat- Among the technologies successfully tested, namely mi- ments may lead to significant microbial inactivation and crowave, ultrasound or ohmic heating, pulsed electric field increased shelf-life extension while preserving quality char- (PEF) gained great attention being a gentle and scalable acteristics. In addition, PEF can also be applied to cell disruption method of plant biomasses. enhance the release of phytochemical compounds from The ability of PEF to intensify the selective recovery the food matrix and, consequently, can be considered as of target intracellular compounds from various vegetable a strategy to tune the characteristics of processed food matrices with reduced extraction costs, thanks to the re- products. duction of energy and solvent consumption, and processing Applied as a non-thermal preservation method, PEF al- time has been demonstrated and valuable results are re- lows a significant reduction of the impact of processing ported in many papers available in the literature. on the fresh-like characteristics of fruit juices. At the In this lecture the recent findings on the utilization of same time, recent applications of low intensity PEF stand PEF for solid liquid extraction process acceleration will be out as a way either of stimulating the production of sec- presented and the effect of different combinations of the ondary metabolites in fresh commodities, hence increasing PEF processing variables on extraction efficiency will be their antioxidant potential, or as a strategy to improve the discussed, with particular focus on the results obtained in bioaccessibility of desirable compounds. the frame of the European Project Accelwater. The eco- This presentation will review several of the latest advances nomic viability of the PEF assisted extraction process with regarding the application of PEF for the development of respect to conventional methods will be also demonstrated safe, nutritious and high-quality food products. Key ex- via process simulation. amples of the PEF application to liquid plant-based foods with the aim of improving stability while preserving qual- OR-125 ity and health-related value will be discussed/shared. In PEF-assisted processes for improving the health- addition, application of PEF treatments to solid matrices related properties of plant foods in order to obtain plant foods with enhanced and more Ignacio Alvarez-Lanzarote, Javier Raso bioaccessible phytochemical contents will be presented and University of Zaragoza, Spain discussed. Pulsed electric fields (PEF) is a non-thermal process characterized by the electroporation of cell membranes OR-221 which is used for several applications in the food industry PEF for the extraction and recovery of health- including improving the extraction of intracellular com- related compounds from vegetable by-products pounds of interest from plant-based foods. There are sev- and wastes eral processes in which the application of PEF has im- Giovanna Ferrari 1, Serena Carpentieri2, Farid proved not only the yield but also the content of com- Soltanipour2, Gianpiero Pataro2 pounds associated with health-related properties such as 1University of Salerno and ProdAl S.c.a.r.l., Italy polyphenols, pigments, vitamins, etc. when producing or 2University of Salerno, Italy obtaining fruit juices, olive oil, wine, etc. Agricultural residues as well as food by-products and In this work, examples of processes (juice extraction, dry- side steams represent a cheap and rich source of valuable ing, freeze-drying, etc.) in which PEF technology has been bioactive compounds, that, if properly recovered, can be implemented will be presented centering when PEF was utilized in different industrial sectors, such as the food, used as a pre-treatment of the product (olives, grapes, pharma and cosmetic, to respond to the increasing con- apples, tomatoes) and its consequence in the obtained fi- sumers demand for natural ingredients with no poten- nal product (oil, juice, wine, etc.) and the possible en- tial toxicity. The number of product with novel formula- hancing increment of health-related compounds. In some tions incorporating natural compounds, which are repla-cases, new opportunities or products can be produced, cing synthetic additives, available in the market is increas- such as grape or apple juices with enhanced concentra- ing making the plant extracts market constantly growing. tions of polyphenols when PEF-treating the fruits previ-The valorization of agro-food residues through the re- ously to process them. In other cases, the application of covery of high value added bioactive compounds is there- PEF for other purposes, such as peeling of tomatoes, can fore strategic not only from the economic standpoint but result in by-products rich in lycopene which can be the 73 base of recovery processes of health-related compounds. was found to be the most abundant phenolic compound But not always and in all circumstances, PEF increase the in AE, representing 53% of the total polyphenol content, extraction of compounds or the effects are clear. The in- followed by naringin, catechin, epicatechin, sinapic acid, troduction of PEF technology in a standardized process phlorizin, cynarine, and gallic acid. The potential bio- implies the adaptation of the connected processing steps logical effects of AE were also investigated using THP- which need to be adjusted, or also changes in properties of 1 macrophages stimulated by Lipopolysaccharide (LPS), the raw material treated by PEF could affect those steps. as an in vitro model system of oxidative stress. A re- Also in this work, some limitations of the PEF technology duced reactive oxygen species production upon treatment will be commented, i.e. when obtaining extra virgin olive with AE was found. Moreover, AE was able to reduce oil (EVOO) or juice extraction. the secretion of the potent pro-inflammatory mediators Interleukin-6 and Monocyte Chemoattractant Protein-1 in OR-124 LPS-stimulated macrophages. Evaluation of the beneficial effects of PEF-treated These results highlight the anti-inflammatory and anti- artichoke residues extract on THP-1 human cell oxidant properties of the extracts from PEF-treated ar- lines tichoke by-products, corroborating their potential applica- Serena Carpentieri 1, Giuseppina Augimeri2, Jessica tion as a source of functional ingredients obtained through Ceramella2, Adele Vivacqua2, Stefania Sinicropi2, Gianpiero a feasible and sustainable process. Pataro1, Daniela Bonofiglio2, Giovanna Ferrari1 1University of Salerno, Italy OR-123 2University of Calabria, Italy Effect of Pulsed Electric Fields (PEF) on the Artichoke (Cynara scolymus L.), a typical Mediter- extraction of phenolic compounds in orange by- ranean edible plant, and its processing by-products, in- products cluding external bracts, leaves, and stems, have been ex- María del Carmen Razola-Díaz 1, Robert Sevenich2, tensively studied for their nutritional values and thera- Eduardo Jesús EJGH Guerra-Hernández1, Vito Verardo1, peutic properties. Recovery and valorization of agri-food Oliver Schlüter2 1 residues, still rich in valuable intracellular compounds, University of Granada, Spain 2 have emerged as a strategy to obtain high value-added Leibniz-Institut für Agrartechnik und Bioökonomie e.V. compounds at low cost for the formulation of functional (ATB), Germany foods. Orange peel is the main by-product of the orange However, the recovery of bioactives from plant cells with juice industry. This by-product is a well-known source sufficiently high yields, requires the adoption of an efficient of bioactive compounds widely studied for its antioxid-cell membrane permeabilization step before conventional ant, anti-inflammatory, anticancer, antirheumatic, anti-solvent extraction, in order to reduce the mass transfer diabetic, and cardioprotective activities. Therefore, the resistance of intracellular compounds from the inner part revaluation of this by-product is of great importance. In of the cell. this context, this study focuses on establishing the op- To this purpose, pulsed electric fields (PEF) is gaining timal conditions for pulsed electric field (PEF) techno- great interest as a gentle and scalable cell disruption tech- logy as a pretreatment for the orange peel extraction pro-nique of plant biomass, showing great potential to in- cess to obtain extracts rich in phenolic compounds, an tensify the selective recovery of target intracellular com- objective framed in the European Project SHEALTHY pounds from various plant matrices, while reducing the (https://www.shealthy.eu/project/). For this, a Box- energy, the solvent consumption, and the treatment time. Behnken design of 15 experiments with 3 independent However, to date, no studies have been carried out on factors has been used: pulse width (µs), number of pulses the application of PEF-assisted extraction of phenolics and field energy (kV/cm). In each experiment the phenolic from artichoke stems, nor on the subsequent evaluation content was extracted by ultrasound technology and ana-of the biological effects of the extracts using human cell lyzed by HPLC-MS. The response variables analyzed were lines. Therefore, in this study, PEF-assisted aqueous ex- the content of total phenolic compounds, total flavonoids, traction was utilized to recover effectively and sustainably total phenolic acids, and the two major compounds, Hes- valuable intracellular compounds from artichoke stems. peridin and Narirutin. The validity of the experimental The extracts were then subjected to membrane separa- design was confirmed by ANOVA and the optimal condi- tion and subsequent stabilization by freeze-drying. The tions were established by response surface methodology. biological effects of the obtained artichoke extracts (AE) With the optimal conditions of a PEF of 1.4 kV/cm and on human Lipopolysaccharide-stimulated THP-1 macro- 30 pulses of 110 µs, a recovery of 41.80 mg/g dry weight of phages, with specific emphasis on their antioxidant and total phenolic compounds was obtained from the orange anti-inflammatory activities, were investigated. by-product, a content 20% higher than that obtained with Interestingly, AE from PEF pre-treated (E=1 kV/cm; ultrasonic extraction without CEP as pretreatment. WT=5 kJ/kg) artichoke stems resulted to possess signi- ficantly higher content of total polyphenols (+122% on average) and antioxidant power (+155% on average), as compared with the untreated samples. Moreover, from HPLC-PDA analysis the chlorogenic acid 74 P11 - Immunogenic Effects of antitumor responses using EP, anticancer drugs, such as Electroporation-based Treatments bleomycin or cisplatin, or calcium locally or systemically are administered prior to delivery of electric pulses. These antitumor therapies correspondingly are known as anti- Tuesday morning Track A tumor electrochemotherapy and calcium electroporation. Oct 11, 10:30 - 12:10 On the other hand, IRE causes dramatic changes in mem- brane and subsequently cellular homeostasis that leads to OR-206 cell death without the administration of any exogenous Adjuvant immunogenicity of nanosecond pulsed substances. electric field anti-cancer treatments We have demonstrated that following IRE cells release Claudia Muratori, Flavia Mazzarda, Alexandra E. various substances, like ATP, proteins, and RNA, but not Chittams-Miles, Julia Pittaluga, Andrew Ojeda, P. Thomas DNA. It seems that released molecules promote cell sur- Vernier vival in vitro and might have an immunoregulating ef- Old Dominion University, Frank Reidy Research Center for fect in vivo. On the other hand, a dramatic negative ef- Bioelectrics, United States fect on nonelectroporated cell viability was observed when Using different tumor models, we found that treatment the medium was collected from cells treated with bleo-with nanosecond pulsed electric fields (nsPEF) enhance mycin or calcium electrotransfer and applied to directly the host antitumor immune response. The strongest ef- unaffected cells. These in vitro studies were translated fect was measured in mice bearing CT-26 colon carcinoma into in vivo studies where we aimed to test antitumor re- tumors where the local treatment with nsPEF protected sponses of combined treatments. Experiments were per- 78% of the animals from a second tumor cell challenge and formed on BALB/c mice bearing two 4T1 tumors on both delayed the growth of established distant lesions. Protec- back flanks. To find out whether the combined treat- tion in nsPEF-treated mice correlated with an increased ments can induce an abscopal effect one of the tumors antitumor effector activity in the spleen. Notably mice was treated and another was left untreated. Tumor treat- cured by nsPEF remained protected even one year post ment was performed by i.v. injection of bleomycin (200 treatment. We hypothesize that nsPEF-treated tumors µl, 470 µM) or/and i.t. injection of calcium chloride (half act as an in-situ cancer vaccine by initiating innate im- tumor volume, 168 mM)) followed by tumor electropor- munity while molding and sustaining adaptive immunity. ation using eight 100 µs pulses at 1200 or 1500 V/cm Indeed, we provide evidence that the damage created by pulse strength. Some mice were additionally treated with these short electrical stimuli is sensed in tumors by the in-IL2-coding plasmid electrotransfer into tibialis cranialis nate immune platform known as inflammasome. Upon as- muscles for possible immune response and abscopal effect sembly, the inflammasome induces membrane pore form- enhancement. ation and proinflammatory cytokine processing, leading We have shown the feasibility to obtain various biological to a form of inflammatory cell death known as pyroptosis. effects on directly unaffected cells after calcium electropor-We found that nsPEF triggered pyroptosis in macrophages ation and/or bleomycin electrotransfer both in vitro and accompanied by caspase-1 activation and release of the in vivo. Possible interplays of these effects are discussed. highly proinflammatory cytokine IL-1 β. Concurrently, to sustain adaptive immunity, nsPEF in tumor cells activ- OR-207 ate persistent ER-stress accompanied by phosphorylation Tumor Treating Fields-based Vaccination in Solid of translation initiation factor 2 α (eIF2 α): a biomarker Tumors of immunogenic cell death. Tumor antigen-release from Dongjiang Chen, Son Le, David D. Tran nsPEF-treated tumor cells comes with the release of ATP, University of Florida College of Medicine, United States a chemotactic factor for dendritic cells (DCs), HMGB1, Tumor Treating Fields (TTFields), a novel approved the DC maturation signal, and externalization of the “eat therapy for glioblastoma (GBM) and malignant mesothe- me” signal calreticulin. Understanding and manipulating lioma, employ non-invasive application of low-intensity, the cross talk between innate and adaptive immunity after intermediate-frequency, alternating electric fields to dis- nsPEF is expected to improve the immunotherapeutic re- rupt the mitotic spindle, leading to chromosome mis- sponse triggered by these treatments. segregation and apoptosis. Emerging evidence suggest that TTFields may also induce plasma membrane perfor- OR-200 ation, blood brain barrier disruption and inflammation. Induction of bystander and abscopal effects after However, the mechanism of these properties and whether electroporation-based treatments they can be harnessed therapeutically are unclear. Re- Paulius Ruzgys, Neringa Barauskaite, Rūta Palepšienė, cently, we reported that TTFields induce focal disrup- Baltramiejus Jakstys, Dovilė Uždavinytė, Salvijus Vykertas, Martynas Maciulevicius, Saulius Šatkauskas tion of the nuclear envelope, leading to cytosolic release of Vytautas Magnus University, Lithuania large naked micronuclei clusters that recruit and intensely activate the 2 major DNA sensors – cGAS and AIM2, Local delivery of electric field pulses can lead to revers- and their cognate cGAS/STING and AIM2/Caspase-1 inible (EP) or irreversible electroporation (IRE) of tumor flammasomes, thereby releasing large quantities of pro- cells. Both types of electroporation have been thoroughly inflammatory cytokines and type-1 interferons (T1IFNs) investigated and applied for in vivo and clinical studies that promote development and maturation of DCs and as novel modalities of antitumor treatment. To achieve 75 cytotoxic T cells. In murine models of GBM, TTFields-resident CD8 T cells indicated the initiation of anti-tumor treated GBM cells provide both tumor immunogens and specific cytotoxic T cells. danger signals to induce anti-tumor memory immunity Our discoveries support nsEPs are a potent TME modi-both intratumorally and systemically, producing a cure fier reversing the immunosuppressive barrier in the TME, rate approaching 66% and partial immunity in an addi- thus, are a promising in situ vaccination approach for im- tional 25% in a STING- and AIM2-dependent manner. In munosuppressive cells dominated cancers. patients with newly diagnosed GBM patients, we detec- ted robust post-TTFields activation of adaptive immunity OR-209 specifically via the T1IFN trajectory anchored by plas- Employing the Electrochemical Side Effects of macytoid DC activity, which was strongly correlated with Pulsed Electric Fields Within the Tissue Microen- T cell receptor (TCR) clonal expansion. Importantly, vironment we also defined a T cell-based gene signature predictive Zaid S. Salameh, Kenneth N. Aycock, Rafael V. Davalos of TTFields effects on T cell activation and TCR clonal Virginia Tech, United States expansion. Collectively, these studies defined a thera- Introduction: Irreversible Electroporation (IRE) is peutic strategy using TTFields as cancer immunotherapy a promising focal ablation procedure used clinically for in GBM and potentially other solid tumors. the treatment of unresectable tumors. [1] IRE involves the application of short, high-voltage pulses across the OR-208 target area via locally advanced electrodes. While the Nanosecond Electric Pulses Overturn Immunosup- non-thermal cell death mechanism of IRE can mitigate pression in the Tumor Microenvironment the thermal side effects seen in other focal ablation Siqi Guo, Anthony Nanajian, Megan Scott, Stephen J. modalities, the pulsed electric fields can instead cause Beebe electrochemical side effects. This is primarily a result Old Dominion University, United States of electrolytic reactions occurring around the electrode- Immunosuppression in the tumor microenvironment medium interface. These reactions form basic and acidic (TME) not only promotes tumor growth but also plays by-products at the cathode and anode respectively, which a critical role in cancer resistance to immunotherapy. We can generate substantial pH change. Some groups have and other groups demonstrated that an electrical engineer-studied these effects to minimize the role of electrochem- ing technology, nanosecond electric pulses (nsEPs) could ical fronts in their desired treatment,[2] while others effectively ablate local tumor meanwhile result in a long- use the electrolytic effects to increase ablation area. term immune protection in several animal models. Further Understanding how to control these effects could prove characterization of local and systemic immune profiles in beneficial in improving electroporation treatments. [3] two cancer models treated with nsEPs with one leading to Methods: Agar (1% w/v) tissue phantoms containing a strong vaccine effect but the other resulting in no im- bromothymol blue (10% v/v) were used to investigate the mune protection suggests changes in immunosuppressive effects of various pulse parameters (pulse duration, pulse cells in the TME but not in blood are associated with an-number, and voltage) as well as electrical conductivity titumor immunity. on local pH changes during IRE. This specific dye was This study is to explore mechanisms how nsEPs mount chosen as it conveniently shows areas of an appreciable a potent immune response in a predominately immun- pH change (±1.0). Thereafter, we translate our pulse osuppressive breast tumor. An orthotopic 4T1-luc mouse parameters into an ex vivo porcine liver model to provide mammary tumor model was treated with nsEPs (100ns, a physiologically relevant environment. As we cannot 50kV/cm, 3Hz and 1000 pulses). Blood, spleen, draining accessibly visualize pH change within a liver, here we lymph node (dLN) and tumor were harvested at 4-hour, utilize a pH electrode to provide point measurements on 8-hour, 1-, 3- and 7-day posttreatment for the analysis of the surface of the liver after treatment. frequencies, death and activation markers of various im- Discussion: Our results show that pH change, an indic- mune cells and suppressor function of regulatory T cells ator of electrochemical effects, can be manipulated to (Tregs). desired magnitudes in contained areas using extended, We found nsEP treatment greatly reduced immunosup- low-voltage pulses. While previously known to be a side pressive cells including Tregs, tumor-associated macro-effect of pulsed electric fields, these effects, if controllable, phages (TAMs) and myeloid-derived suppressor cells (MD-could be utilized to intentionally manipulate pH and SCs) locally and systemically. Contrarily, effector T cells induce electrochemical reactions. This could affect the were spared. The suppressor function of Tregs was greatly body’s acid-base relationship, which is critical to homeo- impaired and this was in line with a significant reduction stasis as it governs a multitude of micro and macroscopic of activation markers 4-1BB and TGF β and the selective functions.[4] With proper parameter selection, the ability elimination of active Tregs by nsEPs. Although all three to control electrochemical reactions within the body could types of immunosuppressive cells were diminished, the cell be clinically applicable across electroporation treatments. death in tumor occurred only to TAMs whereas Treg death was observed in the dLN. Nevertheless, no death of MD- [1] Davalos, Mir, and Rubinsky, “Tissue Ablation SCs took place in both tumor and dLN. Furthermore, a with Irreversible Electroporation.” switch of TAMs from MHC-II- M2-like to MHC-II+ M1- [2] Maglietti et al., “The Role of Ph Fronts in Tissue like was observed. Noticeably, a continuing rise of tissue Electroporation Based Treatments.” 76 [3] Klein et al., “Single Exponential Decay Waveform; a P14 - Cytoskeletal Changes and their Synergistic Combination of Electroporation and Electro- Implications in the Different lysis (E2) for Tissue Ablation.” Types ofPulsed Electric Fields (PEF) [4] Hopkins, Sanvictores, and Sharma, “Physiology, Acid Base Balance.” based Technologies and Treatments OR-210 Tuesday morning Track B Immunogenicity of nucleic acid delivery to skin Oct 11, 10:30 - 12:10 Tanja Jesenko1, Maša Bošnjak1, Katarina Znidar1, Amanda E. Sales Conniff2, Bostjan Markelc1, Nina Semenova3, OR-191 Jared Tur2, Kris Kohena2, Simona Kranjc Brezar1, Maja Suspended Nanonets Enable Precise Quantitation Čemažar1, Loree Heller 2 of Forces and Cytoskeletal Changes Post Electro- 1Institute of Oncology Ljubljana, Slovenia poration 2University of South Florida, United States Philip M. Graybill, Aniket Jana, Rakesh Kapania, Rafael 3Old Dominion University, United States V. Davalos, Amrinder Nain Skin is the largest organ in the body. With its het- Virginia Tech, United States erogeneous population of cells, it provides a passive phys- Intracellular cargo delivery by applying electrical ical barrier against infection and also contains elements pulses was first demonstrated in 1982. Since then, elec-of the innate and adaptive immune systems. Hence, it troporation (EP) has seen a meteoric rise with the suc- is an active immune organ and attractive target for DNA cessful delivery of a variety of cargos inside cells of mul- vaccination. Various cells can be found in the skin, includ- tiple lineages, both in vivo and in vitro. With EP events, ing keratinocytes, fibroblasts, endothelial cells and several the loss of cytoskeleton integrity and contractility is well types of immune cells. This diversity of cell types in skin established. However, after decades of advancement in could be important for DNA vaccination or other gene EP, a description linking loss and recovery of contractility therapies because DNA transfection could elicit different with cytoskeleton is still incomplete. In this talk, I will immune responses in different cell types present in the describe how single cells attached to nanofiber nanonets skin. The experience with different cell types and tumor experience a loss and recovery of forces post EP event, models demonstrated that electrotransfer of plasmid DNA correlated with loss and recovery of the actin cytoskeleton with or without the therapeutic gene (pDNA) activated structure. I will describe our Nanonet Force Microscopy several cytosolic DNA sensing pathways and resulted in (NFM) method for measuring single cell forces. Small dia- induction of different cytokines. Based on this research meter fibers (˜ 200 nm) capable of deflecting and acting and the innate immunogenicity of skin, here we correlas force sensors are deposited orthogonally on larger dia- ated the effects of electrotransfer of gWizBlank pDNA in meter non-deformable fibers (˜ 2 µm) in a suspended two- mouse skin to fibroblasts and keratinocytes in vitro us- layer fiber system and fused at the intersections to achieve ing reverse transcription real-time PCR (RT-qPCR) and fixed-fixed boundary conditions. Cell forces are estimated several types of protein quantification. After electrotrans- inversely by applying force vectors that originate at fo- fer of pDNA, the mRNAs of the putative DNA sensors cal adhesion clusters on fibers and are directed along the DEAD (AspGlu-Ala-Asp) box polypeptide 60 (Ddx60), tension-bearing actin stress fibers inside the cells. We in- Absent in melanoma 2 (Aim2), DNA-dependent activ- tegrate nanonets within a microfluidic device and deliver ator of interferon regulatory factor/ Z-DNA binding pro- ten 100 µ s pulses at three voltage magnitudes (500, 1000, tein 1 (Dai/Zbp1), Interferon activated gene 202 (Ifi202); and 1500 V) and two directions (parallel and perpendic- Interferon-inducible protein 204 (Ifi204) were upregulated ular to cell orientation), exposing cells to electric fields in keratinocytes, while Ddx60, Zbp1 and Ifi204 were up- between 441 V cm−1 and 1366 V cm−1. Post electropora- regulated in fibroblasts. Increased levels of the mRNAs tion, cellular contractility is lost and recovered in three dis- and proteins of several cytokines were detected and var- tinct stages that coincide with actin-cytoskeleton remodelied based on cell type. Mouse skin experiments in vivo ing. In stage 1, cells round up, followed by an unusual and confirmed our in vitro results with increased expression of unexpected biphasic stage (stage 2) characterized by in- the mRNAs of putative DNA sensors along with several creased contractility tens of minutes post-electroporation, cytokines and chemokines. Finally, with immunofluores- followed by force relaxation. The biphasic force behavior cent staining, we demonstrated that skin keratinocytes, is concurrent with the remodeling of actin stress fibers fibroblasts and macrophages contribute to the immune re- and membrane blebbing. Finally, in stage 3, cells elong- sponse observed after electrotransfer of pDNA. In conclu- ate and regain their pre-electroporation morphology and sion, our results confirm the essential immune functions contractility in 1−3 hr. We observe a significant drop in of both immune and non-immune cells in the skin which cell viability at high voltages applied perpendicularly to are essential for vaccination and gene therapy using pDNA the cell long-axis. Our experiments with multiple healthy electrotransfer. and cancerous cell lines demonstrate that contractile force is a more dynamic and sensitive metric than convention- ally accepted cell shape to electroporation. A mechanobi- ological understanding of cellular cytoskeleton remodeling linked with contractility post-electroporation provides a 77 new twist to exploring the mechanisms that drive mem-ence Foundation p.no. 18-23597S and Scientific Grant brane recovery with potential implications for molecular Agency VEGA project 2/0124/22 and institutional medicine, genetic engineering, and cellular biophysics. support from CAS (RVO: 86652036). Authors are also participating in COST Action CA17115. OR-31 Molecular dynamics simulation studies of intense OR-30 electric field on cytoskeleton components Unraveling the interplay between DNA transport Jiří Průša, Saurabh K. Pandey, Michal Cifra and cytoskeletal elements during/after electropor- Institute of Photonics and Electronics of the Czech Academy ation of Sciences, Czech Republic Pouyan E. Boukany Cytoskeleton is an ensemble of self-assembled protein Delft University of Technology, Netherlands fibers which are essential for the life of a cell. We focus Translocation and transport of genetic cargoes into eu- on microtubules, one type of cytoskeleton fibers, which karyotic cells is an essential step for the manipulation and enable cell division and intracellular transport. Here we genetic engineering of cellular activities medicine and fun-demonstrate that molecular dynamics simulations enable damental biology. The cytoskeletal elements (e.g. actin predictions of the potential effects of nanosecond scale in- cortex, microtubules) play critical roles in the several tense electric field effect on tubulin (a building block of steps associated with gene-electro transfer processes (from microtubules), microtubules, or kinesin. translocation across the cell membrane to the transport in- Authors acknowledge support from Czech Science side the cytosol), but the underlying mechanisms remain Foundation GX20-06873X. unclear and vague. This talk will describe how genetic cargoes with different sizes form aggregates during and OR-33 after electric pulses. Next, this talk will also explain how Chip platform for PEF delivery to microtubules on to understand the chaotic and active transport of genetic total internal reflection fluorescence microscope cargoes (with different sizes) through the cell cytoplasm. Daniel Havelka1, Ilia Zhernov2, Michal Teplan3, Zdenek Different modes of cargo transport within the cytoskeletal Lansky2, Djamel E. Chafai1, Michal Cifra 1 meshwork have been demonstrated for various mammalian 1Institute of Photonics and Electronics of the Czech Academy cells with different activities. We demonstrate that the of Sciences, Czech Republic electrotransferred DNA cargo undergoes anomalous diffu- 2BIOCEV, Institute of Biotechnology of the Czech Academy sion for different DNA sizes and cell types (non-cancerous of Sciences, Czech Republic and cancerous cell lines). All of these new insights and 3Institute of Measurement Science of the Slovak Academy of information will allow us to provide and develop a more Sciences, Slovakia predictable theory for gene-electrotransfer by implement- Pulsed electric field (PEF) technology is promising ing the contribution of cytoskeletal elements during and for the manipulation of biomolecular components and after electro-permeabilization. has potential applications in biomedicine and bionano- technology. Microtubules, nanoscopic tubular structures OR-82 self-assembled from protein tubulin, serve as important Transient disruption of blood-brain barrier on rat components in basic cellular processes as well as in brains at low-voltage high-pulse using non-invasive engineered biomolecular nanosystems. Recent studies in electrode approach cell-based models have demonstrated that PEF affects Neeraj Raghuraman Rajagopalan 1, William Ray Vista2, the cytoskeleton, including microtubules. However, the Masashi Fujimori2, Laurien G. P. H. Vroomen2, Niranjan Khadka3, Marom Bikson3, Govind Srimathveeravalli1 direct effects of PEF on microtubules are not clear. 1University of Massachusetts Amherst, Mechanical and Indus- In this work, we developed a lab-on-a-chip platform trial Engineering, United States integrated with a total internal reflection fluorescence 2Memorial Sloan Kettering Cancer Center, United States microscope system to elucidate the PEF effects on a 3The City College of New York, Department of Biomedical En- microtubules network mimicking the cell-like density of gineering, United States microtubules. The designed platform enables the delivery of short (microsecond-scale), high-field-strength (< 25 Introduction: High voltage pulses used for reversible kV/cm) electric pulses far from the electrode/electrolyte (RE) and irreversible electroporation (IRE) are known to interface. We showed that microsecond PEF is capable of induce Blood Brain Barrier (BBB) disruption when de- overcoming the non-covalent microtubule bonding force livered using invasive needle electrodes. We investigated to the substrate and translocating the microtubules. This the feasibility of recapitulating pulsed electric field (PEF) microsecond PEF effect combined with macromolecular induced BBB disruption using non-invasive, scalp moun- crowding led to the aggregation of microtubules. Our res- ted electrodes conventionally used for brain stimulation in ults expand the toolbox of bioelectronics technologies and animal models and human patients. electromagnetic tools for the manipulation of biomolecular Methods: Non-invasive application of PEF between a skull nanoscopic systems and contribute to the understanding mounted electrode and distal grounding pad was tested in of microsecond PEF effects on a microtubule cytoskeleton. Sprague Dawley rats. Combinations of pulses were applied by varying the voltage (1000 or 1500 V), pulse numbers Authors acknowledge support from the Czech Sci- (10 or 100), pulse width (20 or 100 µs) and frequency (1 78 or 10 Hz). BBB disruption was quantified by extravasa- crofluidic device to reproducibly generate elongated cells tion of intravenously administered Evan’s Blue (EB) dye. (5-25x length over width) with precisely controlled orient-Histology was performed to ascertain injury to the brain ations with respect to an applied electric field. We show and compared to invasive pulse application with a needle that post-electroporation cell viability is highly depend- electrode. Correlative COMSOL simulation studies were ent on cell orientation with respect to the electric field. performed on a rat head model to estimate electric fields Cells oriented parallel to the electric field had signific- in the brain using our electrode configuration. 2D mono- antly higher viabilities than seen in suspension or on a layer of b.End3 cells in a 24 well plate were treated at 2D surface. Subsequently, cells perpendicular to the elec- electric field strength levels estimated in the brain by sim- tric field had significantly lower viabilities. Additionally, ulation models at different pulse numbers, and compared the conductivity of the electroporation buffer significantly with conventional RE pulse parameters (1000 V/cm, 10 impacts viability outcomes in elongated cells. When the pulses; positive control). Changes in cell viability (CCK- pulsation buffer conductivity (0.1 mS/cm) is an order of 8) was measured at 2- and 24-hours post-treatment, while magnitude lower than the internal conductivity of the cell disruption of barrier function was determined by quantify- (2.5 mS/cm) we saw a trend reversal, with perpendicular ing alteration in the actin cytoskeleton and zona occludin cells having a significantly higher viability. By individu- (ZO-1) expression at cell-cell junctions. Changes in trans- ally analyzing cells attached to 1- or 2- fibers, we show capillary transport of IgG isotype control FITC antibody that there are minimal differences in post-electroporation and 70 kDa Rhodamine dextran dye following RE and viability despite differences in their cell adhesion and cyto- the test pulse parameters were studied with a microfluidic skeletal structure. Further, we show that cell survival for model of the BBB. elongated cells is still supported by the predictions of the Results: The percentage area of the brain with EB dye standard pore model of electroporation, applied with ac-was sensitive to increasing voltage, pulse number, with cepted model properties. More pores formed at the tips of the greatest coverage (24.55 %) occurring when maximum the cells elongated with the electric field and more pores energy was delivered (1500 V/cm, 100 pulses, 100 µs). formed at the flank of cells oriented perpendicular to the Correlative simulations suggested that the electric field electric field. An improved understanding of cell shape strength demarcating the region of BBB disruption ranged and pulsation buffer conductivity may lead to improved between 43 – 62 V/cm, where the area of disruption cor- methods for enhancing cell viability post-electroporation related with the total electrical charge deposited into the by engineering the cell morphology, cytoskeleton, and elec- brain. Disruption of actin cytoskeleton with nuclear trans- troporation buffer conditions. location of ZO-1at RE pulse parameters was recapitu- lated at lower electric field strengths (250 V/cm) with 160 pulses, without overt loss of cell viability or evidence of P21 - Electroporation for Cardiac electroporation. These pulse parameters reproduced al- Ablation: Mechanisms and Scientific terations in permeability in the microfluidic setup as well Advances to both antibody and small molecule dyes. Conclusion: PEF delivery with non-invasive electrodes Tuesday morning Track C can produce sizeable BBB disruption in rat models at elec- Oct 11, 10:30 - 12:10 tric field strengths that are substantially lower than the required threshold for RE. Simulation models suggest that OR-173 similar outcomes can be achieved in human brain. Pulsed field ablation cardio-selectivity: Differen- OR-32 tial effect of High-Frequency Electroporation on Cell shape, orientation, and pulsation buffer affect myocardium and other tissues Yonatan Moshkovits1, Dvora Grynberg2, Eyal Heller1, Le- electroporation lethality for elongated cells aligned onid Maizels 1, Elad Maor1 on suspended nanofibers 1Sheba Medical Center, Israel Edward Jacobs, Philip M. Graybill, Aniket Jana, Atharva 2 Agasha, Amrinder Nain, Rafael V. Davalos Sackler School of Medicine, Israel Virginia Tech, United States Background: Pulsed field ablation (PFA) is an emer- Cell membrane permeabilization by pulsed electric ging non-thermal ablation method based on the biophys- fields, known as electroporation, has applications in a vari- ical phenomenon of electroporation. While PFA is asso-ety of fields, yet the impact of cell shape has not yet been ciated with less collateral damage compared to thermal fully understood. For many electroporation applications, ablation, data on its cardiac-selectivity nature and on its cell survival and recovery post-treatment is desirable, as tissue-specific thresholds is lacking. Therefore, this study in gene transfection, electrofusion, and electrochemother- aimed to compare the in-vivo differential effect of high apy. In other applications viability is undesirable, as in frequency electroporation protocols (HF-IRE) on various tumor and cardiac ablations. A complete understand- tissues. Methods: 23 Sprague-Dawley rodents were al- ing of how cell shape and cytoskeletal morphology affect located to 3 different HF-IRE protocols with amplitudes cell viability post-electroporation may lead to improved of 300-, 600- and 900-volts. All protocols used twenty electroporation methods and treatment selection. In this 100-µs bursts at a frequency of 150 kHz. The in-vivo study, we use suspended nanofiber networks within a mi- experiment included HF-IRE ablation of cardiac muscle, skeletal striated muscle, liver, carotid artery and sciatic 79 nerve. Animals were euthanized after 14 days. Lesions the absolute acceleration of muscular contractions using were evaluated quantitatively by histologic analysis us- a calibrated Galaxy S6 accelerometer sensors and an app ing staining for fibrosis and by morphometric evaluation (phyphox, Aachen, Germany). The phone was taped to of tissue damage extent. Results: There were 8, 7 and animal’s chest. Lesions were assessed by pre- versus post- 8 animals in the 300-, 600- and 900-volts protocols, re- EGM analysis, pacing threshold, 3D voltage mapping (En- spectively. HF-IRE protocols demonstrated a graded ef- Site, Abbott, Chicago, IL), necropsy and histology. The fect on myocardial tissue with larger lesions in the 900V swine rete mirabile model was used to investigate for em- protocol compared with the other two protocols. Lesion bolic events related to PFA. width was 428±315µM, 694±493 µM and 1348±892 µM All applications were single-shot without repositioning the for 300-, 600- and 900-volts respectively (p<.05), with sim- catheter. No to minor microbubbling, but no skeletal ilar trend in lesion length measurements (p = .01). No muscle stimulation were observed. Acceleration was meas- damage to the carotid artery was observed in all proto- ured at < 0.5 m/s² (noise level). No tachycardiac rhythms cols. Partial damage to the sciatic nerve was observed in were induced by PFA applications. There was marked re- only 2 samples (25%) in the 600-volts group and in one duction in post- versus pre-PFA EGMs (0.7 ± 0.4 mV vs. sample (14.3%) in the 900-volts group. Liver and skeletal 1.8 ± 1.5 mV; P<0.0001 – or 0.5±0.2 mV vs. 2.0 ± 0.9 mV muscle tissues demonstrated ablation-induced fibrosis in when catheter was sized to ventricular dimensions) and all protocol groups with no graded effect between group. pacing threshold (7.5 ± 2.9 mA vs. � 20 mA; P<0.0001). Conclusions: Electroporation effect is tissue-specific such All lesions were large and durable up to 7 – 28 days of that myocardium is more prone to electroporation dam-follow-up. The lesions measured: 32.1 ± 4.7 mm in length, age compared to neural and vascular tissues. Our results 26.6 ± 7.8 mm in width, 8.4 ± 3.1 mm in depth, 62.9 ± suggest no neural or vascular damage with using a low 2.1 mm in circumference (when catheter and ventricular amplitude HF-IRE protocol. Further investigation is war- axes aligned) and 13.9 ± 4.7 cm³ in volume. Despite the ranted to better identify other tissues specific thresholds. higher waveform voltage and prolonged applications used, no or minor superficial thermal effects were detected at OR-174 necropsy or histology. Moreover, gross and microscopic A Novel Single-Shot Pulsed Field Ablation System examinations of the rete mirabile and kidneys revealed no Is Associated with Large and Durable Ventricu- evidence of thromboembolism in any of the animals. lar Lesions In Vivo: A Preclinical Assessment of In conclusion, the novel, single-shot PFA catheter system Safety and Efficacy is capable of creating large and durable ventricular lesions Steffen Holzinger 1, Arash Aryana2, Dorin Panescu3, Cary using 56-sec applications in vivo. Despite the presence of Hata3, Alan de la Rama3, Ken Nguyen3, André d’Avila4 minor microbubbles, examination of the rete mirabile and 1BIOTRONIK SE and Co. KG, Germany kidneys revealed no thromboembolic events in any of the 2Mercy General Hospital and Dignity Health Heart and Vas- animals. cular Institute, Sacramento, United States 3CRC EP Inc., United States OR-175 4Harvard Thorndike Electrophysiology Institute, Beth Israel Direct and alternating current pulse filed ablation Deaconess Medical Center, Boston, United States lesion comparison Pulsed field ablation (PFA) is a non-thermal ablative Martin Pesl 1, Eva Odehnalova1, Jakub Hejc1, Ver- strategy that achieves cell death in cardiac tissue by irre- onika Novotna2, Petra Stachova1, Vita Zampachova1, Anna Siruckova1, Dalibor Cervinka2, Zdenek Starek1 versible electroporation. 1International Clinical Research Center, St. Anne’s University The objective of this study is to recognize that a novel Hospital Brno, Czech Republic single-shot PFA catheter system (CRC EP Inc., Tustin, 2Brno University of Technology, Czech Republic CA) is capable of creating large and durable ablation lesions, without catheter repositioning, in both the atria Background: Pulse field ablations (PFA) are intro- and the ventricles without any adverse effects on adja- duced into clinical practice. A number of generators and cent structure or thromboembolic events based on swine catheters are emerging. while there is little known about rete mirabile examinations. In this context, the safety lesion formation, maturation, and long-term effects on the and efficacy of PFA was investigated using a novel 8-Fr, myocardium. Gross pathology inspection and examina-16-electrode, bidirectional, 25-, 30- or 35-mm spiral with tion with 9.4 T magnetic resonance imaging (MRI) are the larger catheters containing 2 distal mapping electrode standards for morphological evaluation. We aim to com- pairs. pare lesions from alternating current (AC) PFA, direct In total, 14 pulsed field applications were delivered in 5 current (DC) PFA, and radiofrequency (RF) lesions in swine (55–92 kg) under general anesthesia, without para- swine heart tissue. lytic agents, including: 7 lesions in the right (RV) and 7 Methods: Animals underwent AC, DC, and RFA ablation in the left ventricle (LV). The PFA catheters were inser- in the left ventricle. After animal euthanasia, hearts were ted through commercially-available 8.5-Fr steerable intro- explanted, fixed, and scanned by 9.4 T MRI, then sliced. ducers. Bipolar PFA (2.5–4.0 kV) was performed using Slices were photographed and measured. The pathological 56±18 sec, single-shot, QRS-gated applications under in- evaluation included staining alcian blue and yellow Mas- tracardiac echocardiographic guidance. The intensity of son trichrome. Evaluated were depth, width, and lesion skeletal muscle activation was quantified by measuring estuary, the volumes of the lesions were calculated using a 80 formula and directly measured from MRI with the “point- recent PFA studies. The multiscale model and approach by-point” method. presented in this study have been successfully employed Measurements obtained from gross pathology inspection to identify relevant factors able to improve the current and MRI did not differ, while distinct changes o for each understanding and predictive tools and the overall PFA ablation energy could be traced and attributed to different clinical outcomes. current characteristics. OR-182 OR-181 Preventing cardiac cell damage after electric injur- Multiscale Modeling of Pulsed Field Ablation in ies Anisotropic Myocardium Pamela Sowa 1, Aleksander Kielbik2, Andrei G. Quim Castellvi 1, Emma Aoustin2, Antoni Ivorra1 Pakhomov3, Emily Gudvangen3, Uma Mangalanathan3, 1Universitat Pompeu Fabra, Spain Volker V.A. Adams1, Olga N. Pakhomova3 2 1 Université Paris-Saclay, France Technische Universität Dresden, Germany 2Wroclaw Medical University, Poland Pulsed Field Ablation (PFA) is an electroporation- 3Old Dominion University, United States based treatment modality to perform cardiac tissue ab- lations. In this technique, a catheter is typically placed in The only effective therapy for ventricular fibrillation, contact with the endocardium where the electric pulses are electric shock, is also known to cause electroporation of delivered to ablate a target area of the myocardium. The cell membranes, which may be injurious to the myocar- cardiac parenchyma is principally constituted by elong- dium. A higher defibrillation dose increases the severity of ated myocytes organized in fibers oriented, mainly, per- post-resuscitation myocardial dysfunction, contributing pendicular to the endocardial surface. This anisotropic to the high mortality of post-cardiac arrest syndrome. morphology results in a higher electric conductivity at the Understanding the membrane integrity and restoration fibers direction, becoming a preferential pathway for the process is crucial for defibrillation without adverse electric current to flow along. According to that, wide electric injuries. This motivates the ongoing research to and shallow lesion morphology could be expected when investigate mechanisms of electropore resealing and the monopolar PFA applications are delivered focally. Con- search for its possible refinement. trary to that, some recently published pre-clinical data Myocyte cell membrane repair can take minutes. During present a deep elongated lesions morphology not aligned cardiopulmonary resuscitation (CPR), this delay can to the expected distribution of the electric field. This be fatal due to the reduced cardiac output resulting in study presents a multiscale simulation approach able to cell hypoxia. Block copolymers can represent a straight- identify factors needed to be considered when electropor- forward solution to maintain membrane integrity and ation treatments are applied in a high anisotropic tissue preserve cardiac myocyte viability, thus yielding a new such as the myocardium. First, a model was implemen- opportunity for a treatment of high clinical impact. ted where multiple cylindric myocytes were arranged mim- Regarding cardiac injury during CPR, the intervention icking the microscopic conformation of the cardiac tissue. with Poloxamer 188 (P188) has been evaluated only as Using that geometry, longitudinal and transversal elec- part of a bundle therapy with sevoflurane and standard tric fields at different frequencies and magnitudes were advanced life support. Therefore, its role in preventing applied to assess the interactions between the electric cur-cardiac electric damage has not been established. Here, rents, the ionic solutions, and the cell membranes. The for the first time, we demonstrate that P188 can prevent results of the simulations were employed to describe the cardiac electric injury by reducing the death of human expected anisotropic behavior at tissue level in terms of cardiomyocytes caused by electroporation. electric conductivity and expected membrane disruption We used nanofiber multiwell plates to create monolayers for each of the fiber orientations. Second, a macroscopic of AC16 human cardiomyocytes. A 3D printer customized model of the heart cavity was implemented. In this model, with an electrode holder was employed to precisely posi- a focal ablation catheter in contact with the myocardial tion stimulation electrodes orthogonal to cell monolayers. tissue was defined to simulate the delivery monopolar This technique also ensured the maintenance of time PFA treatments. The microscopic simulations results con- intervals between exposure and agent administration. firmed the anisotropic properties of the myocardium and Contact electrodes produced the electric field gradually reveal a complex behavior of this high structured tissue. decaying with distance from them, allowing the compar- Specifically, when low frequencies and low electric field ison of cell killing by a range of electric field strengths in magnitudes are applied, the induced membrane disrup- a single sample. tions are predominantly appearing when field is applied We used both wide-field and scanning confocal fluores- parallelly to the cardiac fibers. However, intense high fre- cence microscopy for measuring sarcolemma damage (by quency pulse waveforms are commonly required to mitig- YO-PRO1 dye uptake) and cell viability (by propidium ate skeletal contractions during PFA treatments. In that staining at 2- to 24 h after exposure) in monolayers situation, the results of the microscopic simulations re- treated by micro- and nanosecond electric pulses (µs and veal a demarcated higher sensitivity to the electric fields ns EP). Further, we evaluated the impact of P188 on in perpendicular orientation. The results provided by the membrane resealing and, consequently, cell survival. macroscopic model and the microscopic behavior, can ex- Our study revealed different time dynamics of cardiac cell plain the elongated lesion morphology reported in some death evoked by ns and µs EP. After a train of 200, 10 81 kV/cm, 300-ns pulses, the cell death rose from 74 after 2 OR-166 h to 90 percent after 8 h. Exposure to µs EP resulted in Electric Fields and their Effects on Vegetative Mi- cell death delayed up to 24 h. croorganisms, Spores and Enzymes Application of 1% P188 solution seconds after ns pulses Jin Hong Mok1, Taras Pyatkovskyy2, Chaminda P. reduced cell death about 1.5 times. We plan to use Samaranayake3, Sudhir K. Sastry 3 confocal microscopy with a mounted chamber perfusion 1Keimyung University, Korea, Republic of system to establish how the incorporation of P188 in the 2Ternopil National Medical University, Ukraine cell membrane enhances its resealing. 3The Ohio State University, United States These novel findings broaden the possibilities for future Interest in the use of electrotechnologies for food pro- prevention of electric injuries caused by defibrillation. cessing was revived in the 1980s due to industry interest Subsequent in vivo studies can warrant a further clinical in delivering improved food quality while assuring safety. trial. A notable development has been the discovery that under the right temperature conditions, low-frequency (< kHz) Support: This research has been made possible by electric fields result in inactivation of bacterial spores at the Kosciuszko Foundation. a greater rate than purely thermal methods. The effic- acy of combined electrical and thermal (electrothermal) P4 - Electric Fields for Ohmic Heating approaches suggests that the electric field interacts with specific components within the spore to cause inactiva- in Food Processing tion. Studies on the effects of electric fields a number of enzymes, including pectin methylesterase, alpha amyl- Tuesday morning Track D ase, peroxidase and cellulase, have shown activation of the Oct 11, 10:30 - 12:10 enzyme at sub-optimal activity temperatures, and acceler- ated inactivation at above-optimal activity temperatures. OR-115 Further, studies on the effect of frequency have shown en- Differentiation of Thermal and Electric Field Ef- hanced activity below specific frequencies. fects During Ohmic Heating Felix Schottroff OR-178 University of Natural Resources and Life Sciences (BOKU), Industrial Applications of Ohmic Heating Austria Henry Jaeger University of Natural Resources and Life Sciences (BOKU), Electrotechnologies use the direct application of elec- Austria tric fields to an electrically conductive product, which is placed in-between at least two electrodes. Due to the Compared to conventional heating methods, ohmic occurrence of an electrical current flow and the dissipa- heating can achieve shorter heating times while avoiding tion of electrical energy into thermal energy, an associ- hot surfaces and can reduce temperature gradients. Be- ated temperature increase (Ohmic heating) will occur dur- nefits may result for the processing of high viscous and ing these treatments. Although the primary objective of particulate foods as well as for solid foods. Although the ohmic heating is a fast and uniform temperature increase, technology had its first industrial application almost a cen- additional effects of the electric field on enzymes and bio- tury ago, the current use cases are still scarce. logical cells and tissues are discussed as well. Especially Challenges are resulting from the electrical, thermophys- for the design of decontamination processes, i.e. pasteuriz- ical and rheological properties of the products that have ation and sterilization, by electrotechnologies, knowledge to be mastered in order to achieve uniform heating and of the individual contribution of heat and electric field tothe full benefit resulting from the technology. In addi- ward the inactivation of microorganisms is crucial. Both tion to the product parameters, process parameters such are directly interconnected and the ultimate aim of these as the current frequency used, the electrode material and processes is to find a balance between product quality loss the geometry of the treatment chamber are also relevant and microbiological safety, thus ideally leading to a min- and need to be considered for specific applications. Fur- imization of the thermal load. Also, design and optimiza- thermore, equipment availability still depends on a small tion of processes distinctly depend on knowledge of these number of providers. mechanisms, enabling the utilization of the full potential Nevertheless, successful applications have been achieved, of these technologies. However, in order to gather this e.g. for cooking and baking as well as for the pasteurisa- knowledge, pronounced efforts in terms of experimental tion and sterilisation of sensitive products. Above men-design and simulation have to be undertaken. Thus, this tioned limitations have been addressed in these cases and presentation will focus on these issues, taking into account have resulted in optimized concepts regarding product for-experimental designs and considerations in terms of sim- mulation as well as process setup. ulation, enabling the differentiation of thermal and non- The presentation will highlight existing and future applic- thermal effects of electrotechnologies. ation scenarios. Product and process variables will be ad- dressed in order to emphasize the need for a re-engineering of existing processes when being replaced or complemen- ted by ohmic heating. Furthermore, aspects related to process validation will be discussed in order to exemplify 82 particularities resulting from the different heating prin-OR-56 ciple. Pulsed Electric Fields as a new ohmic heating sys- tem for vegetable blanching OR-55 Leire Astráin Redín, Javier Raso, Guillermo J. Cebrián, Ohmic heating of patatin enriched potato protein: Ignacio Alvarez-Lanzarote Influence of moderate electric fields on thermal in- University of Zaragoza, Spain duced gel properties Blanching is a thermal process widely used in the food Eike Joeres 1, Stephan Drusch2, Stefan Töpfl3, Ute industry, which is applied to vegetables and some fruits Bindrich1, Andreas Juadjur1, Thore Völker1, Volker Heinz1, prior to freezing, heat sterilization or dehydration. Tradi- Nino Terjung1 tional food blanching technologies include the use of boil- 1German Institute of Food Technologies (DIL e.V.), Germany ing water or steam, which are very time-consuming and 2Technical University of Berlin, Food Technology and Material energy-intensive treatments, potentially affecting the sens- Science, Germany ory quality of the products. Moreover, in the case of large 3University of Applied Science Osnabrueck, Food Processen- vegetables pieces, the fact that heat transfer occurs by gineering, Germany conduction from the hot medium to its core results in a Heating of proteinacious foods is an elementary step slow heat penetration rate leading to a problem of thermal during food processing as it commonly determines struc- uniformity. tural properties of the product. Ohmic Heating (i.e. heat- Therefore, volumetric heating systems are being invest- ing by the use of an electric field) is an emerging heat- igated. Pulsed Electric Fields (PEF) is one of the most ing technology, which offers several advantages over con- recently proposed alternatives for applying ohmic heating, ventional heating processes, e.g. a higher primary energy especially in processes where mass transfer occurs in ad- conversion or faster and more uniform heating. However, dition to heating. Therefore, the aim of this work was to the influence of Ohmic heating during thermal gelation optimize the PEF-assisted blanching of carrots immersed of globular plant proteins and their functionality is not in calcium chloride solutions. fully understood yet. This study aims to fill this lack of PEF heating of electric field strengths and frequencies up knowledge and to investigate possibilities of Ohmic heat- to 2 kV/cm and 150 Hz where applied to carrot samples ing to affect protein functionality and thereby properties immersed in calcium chloride solution at electrical con- of thermal induced gels. ductivities and temperatures up to 3.5 mS/cm and 80 °C. Therefore, 9 wt % patatin enriched potato protein solu- Heating curves of both matrices were used to character- tions at neutral pH and an ionic strength of 25 mM NaCl ize the influence of PEF parameters in the process and were heated from 25-65°C via Ohmic heating at several to compare with the conventional heating. A commercial electric field strengths and conventional heating, result-kit was used to study the inactivation of the peroxidase ing in come-up times of 240 and 1200 s as well as holding enzyme and the final texture of the freeze-thawed carrots times of 30 and 600 s. Gels were then physically char- was measured. acterised (texture, moisture, water holding capacity) and The results showed that increasing the frequency and elec- the level of protein denaturation was determined. To re- tric field strength did not improve the heating uniform- veal deviances between both heating methods on micro ity resulting in higher heating rates in carrots. However, and molecular scale, an SDS-PAGE, gel permeation chro- increasing the initial electrical conductivity of the saline matography, gel solubility tests, FTIR-spectroscopy and medium improved heating uniformity, achieving 80 °C in scanning electron microscopy were performed. both matrices after 60s when starting at an electrical con- Results indicated less proteins denatured and participated ductivity of 6 mS/cm (equal to the conductivity of the in gel network structures when heated via Ohmic heating. electroporated carrot). Nevertheless, in order to find a Conversely, more proteins remained in a partially nat- more feasible industrial application, the influence of the ive state and less hydrophobic interactions were measured initial temperature of the saline medium (20-80 °C) was within ohmically treated gel network structures. Scan- evaluated, determining a better heating uniformity apply- ning electron microscopy revealed the protein network to ing PEF at high temperatures. The best results were ob- be more gap-like and frayed when an electric field was tained when applying PEF-heating of 1.33 kV/cm, 100 Hz, present. Gel properties showed ohmically heated gels to an initial medium temperature of 80 °C and 3.5 mS/cm have tendencies of lower gel rigidity, moisture content and (at 80 °C) reaching 85 °C in 90 s in the carrot and in the water holding capacity, but higher gel elasticity. saline medium. A holding heating phase (85 °C for 50 The present study demonstrated that Ohmic heating can s) was required for complete peroxidase inactivation after be used as a tool to target and control protein denat- the PEF heating-up phase. Blanching assisted by PEF uration and thereby functionality. Thermal induced gels shortened the heating time by 60 % compared to conven- by Ohmic heating possess different gel properties (com- tional blanching by immersion in hot water. In addition, pared to conventional heating) which can be of interest PEF-blanched samples in a medium containing calcium for novel food products or biomedical applications. Fur- chloride showed higher (18.5 %) hardness values (determ-ther, Ohmic heating is applicable in case less denatura- ined after being freeze-thawed) than those traditionally tion during thermal treatment of food products containing blanched (also with CaCl2 added). globular plant proteins is desired. Based on the obtained results, it can be concluded that PEF allowed reducing the blanching time processing of 83 carrots improving their texture after a freezing-thawing 2 variants and T cell immune response specific for RBD process. region of Spike protein showed the induction of B and T cell response in vaccinated cats and ferrets. Moreover, in ferret clinical study, the two doses vaccination regimen P20 - Electroporation-based showed not only to elicit B and T cell immunity, espe- Treatments in Veterinary Medicine cially in 0.25 mg prime/boost vaccinated group, but also II to protect from viral challenge, with no detectable infec- tious virus in nasal swabs three days post challenge. Tuesday afternoon Track A Conclusions. Here we demonstrate that Linear DNA COVID-19 vaccine delivered by EGT is safe and immun- Oct 11, 13:30 - 14:45 ogenic in cats and induces protective immune response from viral challenge in ferrets. These results warrant fur- OR-75 ther investigations and hold promise for the development Linear DNA amplicons delivered by electro-gene- of veterinary vaccines to fight SARS-CoV-2 in animals and transfer as veterinary Covid-19 vaccine candidate potential transmission to humans. Antonella Conforti 1, Erika Salvatori2, Lucia Lione2, Mirco Compagnone2, Eleonora Pinto2, Brian Viscount3, James A. OR-76 Hayward3, Clay D. Shorrock3, Diego G. Diel4, Fabio Palombo5, Joe A. Impellizeri6, Luigi Aurisicchio1 Interleukin 12 gene electrotransfer to skin: exper- 1Evvivax srl, Italy ience from studies on pigs 2Takis , Italy Ursa Lampreht Tratar, Tanja Jesenko, Karolina Belingar, Tanya Birk, Anja Osep, Maša Bošnjak, Gregor Serša, Maja 3Adnas, United States Čemažar 4Cornell University, United States Institute of Oncology Ljubljana, Slovenia 5Neomatrix, Italy 6Veterinary Oncology Services, United States Gene electrotransfer (GET) of plasmid encoding inter- leukin 12 (IL-12) has already been used in clinical trials in Introduction. As reported by the American Veterinary combination with electrochemotherapy for the treatment Medical Association, besides human-to-human transmis-of various spontaneous tumors in dogs. Although com- sion, human-to-animal transmission of SARS-CoV-2 has bination therapy proved to be effective, there is a need been observed in some wild animals and pets. With an- for the optimization of gene therapy in order to improve imal models as an invaluable tool in the study of infectious the treatment outcome. Specifically, the optimisation con- diseases combined with the fact that the intermediate an- cerning the different concentration of plasmid DNA, the imal source of SARS-CoV-2 is still unknown, researchers use of invasive or non-invasive electrodes and the use of have demonstrated that cats and ferrets are permissive to plasmid DNA lacking the antibiotic resistance gene. In COVID-19 and are susceptible to airborne infections. To recent years, we have developed a plasmid encoding hu-address this issue, we have developed a genetic vaccina- man interleukin-12 (phIL12) that is currently in a Phase I tion platform based on a linear DNA amplicon (Linear clinical study enrolling patients with basal cell carcinoma DNA COVID-19 vaccine) encoding RBD region of SARS- of the head and neck (Clinicaltrials.gov: NCT05077033). CoV-2 Spike protein and delivered by electro-gene-transfer Plasmid phIL12 is free of the antibiotic resistance gene (EGT). In clinical trials conducted in cats and ferrets in and has therefore been developed in accordance with the USA, we have investigated safety, immunogenicity and ef- EMA guidelines for advanced therapy medicinal products. ficacy of Linear DNA COVID-19 vaccine. The plasmid was already evaluated in a preclinical study Methods. In feline trial, 11 client-owned Covid-19 neg- in the mouse tumor model CT26, where a plasmid with a ative adult cats received 1 mg prime-boost vaccination transcript for the mouse ortholog IL-12 was used, which (at days 0-28), intramuscularly via EGT. To assess vac- demonstrated its biological activity, safety, efficacy, phar-cine immunogenicity, blood samples were collected before macokinetic and pharmacodynamic properties. The aim and one month after boost. In ferret trial, 25 Covid-19 of this study was to investigate the use of different IL12 negative adult ferrets received two different vaccine doses GET modalities (different plasmid DNA concentrations (0.25 and 1 mg), either with prime-boost or only prime, and use of invasive or non-invasive electrodes) on IL-12 and were challenged with 5x105 PFU of live SARS-CoV- expression in the skin as well as confirm the pharmacokin- 2 intranasally two weeks after boost. Immune response etic characteristics of plasmid phIL12 in another animal and vaccine protective efficacy were assessed before and species. The porcine model was selected due to the fact after challenge. In both trials, we exploited a clinically that human IL-12 is biologically active in pigs and the skin validated device for veterinary electroporation called Vet- characteristics are similar between the pigs, dogs and hu-ePorator, based on Cliniporator technology currently ap- mans. The study was approved by national Animal Ethics proved for ECT applications in humans, adapted to EGT committee (U34401-2/2021/5). Gene transfer of phIL12 in and already used in many animal studies. the skin was performed on 9 pigs. Different concentration Results. No severe adverse effects related to EGT pro- of plasmid phIL12 (0 mg/ml, 1 mg/mL and 2 mg/mL) cedure were revealed in cats and ferrets administered with and two different types of electrodes: plate (non-invasive) two doses vaccination regimen. Linear DNA COVID-19 and needle (invasive) were used. Animals were euthanized vaccine proved to be safe in both trials. Measurement of 7, 14, and 28 days after GET. The expression of IL-12 binding/neutralizing antibodies against many SARS-CoV- 84 in the skin was performed on mRNA level by RT-qPCR presented with a mass on the soft palate extending to the and on protein level by ELISA test. Next, the distribu-nasopharynx, presenting difficulty to breath and eat. A 3 tion of plasmid DNA was evaluated in several different cm pedunculated mass infiltrating the tonsil was observed. organs as well as in skin by RT-qPCR. The results of our The patient had lymph node involvement and a suspicious study showed that the phIL12 GET with invasive elec- nodule in Chest radiographs. trodes induced higher expression of IL-12 on protein level ECT combined with GET was performed. The patient de-as well as on mRNA level 7 days after GET compared to veloped a mild inflammation in the treated area during the non-invasive ones. The distribution of the plasmid DNA first week. After the second GET an oronasal fistula de- showed the presence of plasmid in all of the samples tested veloped with no long-term symptoms. Two months later, with the highest plasmid copy number in the treated skin in follow-up radiographs, the suspicious nodule was not 7 days after GET. However, plasmid copy number in all of present. The patient remains disease free for 469 days. samples decreased over time and was at the minimum 28 Case 3: A 10 years old, schnauzer, spayed female, presen- days after GET. The results of this study demonstrated ted for lameness of the right anterior leg. A 1cm pig- the expression of phIL12 in porcine skin and similar phar- mented tumor on the nailbed was found. The cytology macokinetics properties as with the mouse ortholog. suggested the diagnose of melanoma. Digit amputation with lymphadenectomy was performed. The lymph node OR-77 involvement was confirmed. GET treatment was recom- A combination of electrochemotherapy and gene mended to improve survival and disease-free times. After electrotransfer in canine stage III melanoma: Ini- the treatment the patient did not have any side effects tial experience from peru and recovered uneventfully. After 261 days the patient Sergio S. Salgado 1, Matias M. Tellado2, Emanuela Signori3, remains free of disease. Felipe H. Maglietti4 All the patients remain disease free up to the writing of 1Universidad Peruana Cayetano Heredia, Lima, Peru this work. 2VetOncologia, Veterinary Oncology Clinic, DC, INFIP, DF - In conclusion, ECT combined with GET is a promising UBA CONICET, Argentina tool for the treatment of metastatic melanoma in dogs. 3Institute of Translational Pharmacology, Biomedical Sciences, Italy OR-218 4Instituto Universitario de Ciencias de la Salud Fundación H.A. From Cancer to COVID-19: gene electrotransfer Barceló , Anatomia, Argentina as a versatile tool to design innovative veterinary Introduction: Electrochemotherapy (ECT) and Gene vaccines electrotransfer (GET) are valuable tools in the treatment Luigi Aurisicchio of cancer. The aim of this study is to combine ECT and Takis S.r.l., Italy GET in three dogs with metastatic melanoma. We have recently developed Tel-eVax, a vaccine target- Patients and methods: All patients presented a confirmed ing Telomerase which was shown efficacious for treatment stage III melanoma. They were treated using ECT com- of canine B-cell lymphoma and other tumor types. Te- bined with GET. For the ECT intravenous bleomycin was lomerase is overexpressed in >90% tumor types and is a used. The electric pulses (8 100µs long of 1,000 V/cm at potential universal antigen. 5,000Hz for ECT and at 1 Hz for GET) were delivered us- The current pandemic demonstrated the urgent need ing an EPV-100 electroporator (Biotex, Argentina). Dis- to develop versatile vaccination platforms that could be posable needles electrodes were used. For GET, a plasmid quickly implemented for infectious diseases. It is of utmost coding canine IL-2, and a plasmid coding canine IL-12 importance to provide a vaccine in a time-critical manner were used, and the procedure was repeated 28 days later. for such diseases, as the frequency of such epidemics and Results: Case 1: A 10 years old, cross breed, spayed fe- pandemics as we see them today, will be heavily increas- male, presented with a mass on the gingiva of the premolar ing due to rise in global travel, global warming, increase in area on the right mandibula. The patient had cytore- population density, penetration into previously uninhab- ductive surgery, where an amelanotic melanoma was dia- ited areas and animal trade. Nucleic acid vaccines, such gnosed. Within the first month a relapse was observed. as those based on mRNA are endowed of these features. The tumor measured 3 cm, was ulcerated and hemor- To address the urgent need to find solutions to the SARS- rhagic. The owners rejected surgery, and only accepted CoV-2 Pandemic, Takis has developed COVID-eVax, a ECT. A complete response was achieved presenting a re- vaccine approach based on genetic engineering and DNA duction of the mandibular grith and bone remodeling, con- electroporation as part of the X-eVax platform, previously firming bone involvement. Three months later, a follow-up developed. The project started in 2020 and consisted of ultrasound revealed a heterogeneous lymph node. Surgical the molecular design of the vaccine, the development of the resection of it combined with ECT on the tumoral bed and reagents and tests necessary to test its effectiveness and on the scar of the original tumor was performed. GET was the experiments in animal models. Subsequently, GMP-performed using IL-2 near the original tumor and IL-12 in grade material (Good Manufacturing Practices) was pro- a close muscle. duced, all regulatory studies were conducted (toxicology, No side effects were observed. The patient remains free of biodistribution, immune response) and finally a phase 1 disease, reaching 649 days. study in humans, which ended in December 2021, achiev- Case 2: An 11 years-old, cross breed, spayed female, was ing all the objectives set and providing the basis for eval-85 uations in Phase 2 and 3 studies. damaged areas are still unknown. Of particular interest DNA vaccines advantages are: (1) simple and quick pro- from a clinical perspective is to determine how and when duction of DNA encoding the antigens by PCR or syn- the electrical activity of the reversible areas recovers after thetic methods (potential game-changers for new variants PFA. Studying the dynamics of this process can help to especially vaccine resistant strains), (2) easy large-scale understand the optimal use of electroanatomical mapping production, (3) safety compared to inactivated virus vac- systems in the PFA framework. cines, and (4) higher thermostability (minimal cold-chain In this study we acquired high-density local electrograms requirements), which is an issue with some vaccines. The before and during 60 min after PFA application. The DNA-based platforms offer great flexibility in manipulat- measurements were performed in different points of the ing the encoded vaccine antigen and have a great potential right and left ventricular epicardial surface of swine using for accelerated development. Recently, the first DNA vac- an open chest approach following a protocol approved by cine against SARS-CoV-2 (ZyCov-D) has been registered the Sant Pau Hospital Animal Experimentation Ethical in India for human use; moreover, DNA vaccines have been Committee. Under general anesthesia, a median sterno- extensively tested in multiple clinical trials in the onco- tomy was made to create a surgical window. Unipolar logy field and are commonly used in veterinary medicine. electrograms were recorded using a 128-channel electrode These vaccines (as opposed to mRNA-based vaccines) are matrix (BIOSEMI) attached to the epicardial surface (in- stable, do not require cold-chain supply, and can easily be terlectrode distance=0.75 mm, total dimensions 9.5 × 9 produced in large amounts in bacteria. All these advant- mm). For PFA ablation, a monopolar catheter (Biosense ages make this platform technology an attractive tool, as it Webster Thermocool STSF) was positioned in the region overcomes several shortcomings of alternative approaches corresponding to the center of the electrode array and a (e.g., complex production processes, stability issues, pur-return electrode patch was attached to the back of the chase price). animals. After the procedure, and at least 3 hours from In this presentation, opportunities and challenges of DNA- the last application, tissue was processed and lesion areas based vaccines will be discussed and their potential use in were stained with Triphenyltetrazolium Chloride (TTC) Veterinary medicine. to assess acute lesion morphology. Our results show an immediate change from basal nar- row biphasic electrograms to wide monophasic electro- P28 - Electroporation for Cardiac grams with marked ST-segment elevation. Narrow QRS- Ablation: Clinical Use and complexes gradually reappear while ST-elevation recovers Development back to basal conditions from the periphery towards the center of the mapped area The extent and dynamics of Tuesday afternoon Track B the observed changes depend on the voltage and the po- sition, i. e., depend on the electric field intensity. After Oct 11, 13:30 - 14:45 tissue staining, we observe that the area where the electro- grams are modified is beyond the area of acute damage. OR-46 This confirms that the electrical activity of the revers- Epicardial high-density electrogram mapping dy- ible electroporation area is also acutely modified. The namics during Pulsed Field Ablation (PFA) preliminary analysis of the results suggest that the differ- Tomas Garcia-Sanchez 1, Gerard Amorós-Figueras2, Sergi ent recovery dynamics observed could help in differenti- Casabella-Ramon2, Zoraida Moreno-Weidmann2, Jose M. Guerra2, Antoni Ivorra3 ating between reversible and irreversible electroporation 1CNRS, Gustave Roussy, Université Paris-Saclay, and Biomed- areas. This study could also help in establishing the op- ical Electronics Research Group (BERG), Universitat Pompeu timal acute time for remapping after treatment. Fabra, France 2 OR-176 Hospital de la Santa Creu i Sant Pau, Universitat Autònoma Characterization of the effects of cryoablation, RF de Barcelona, Spain 3 ablation or pulsed field ablation on compound ac- Universitat Pompeu Fabra, Spain tion potentials of porcine phrenic nerves The use of irreversible electroporation (IRE) as a David A. Ramirez 1, Lars M. Mattison2, Paul A. Iaizzo1 method for cardiac ablation in the treatment of cardiac 1University of Minnesota, United States arrhythmias, known as Pulsed Field Ablation (PFA), has 2Medtronic, United States rapidly moved from preclinical studies to clinic. One of the Introduction: Atrial fibrillation is conventionally major advantages of this novel ablation technique over ra- treated by electrical isolation of the pulmonary veins. diofrequency or cryoablation is safety. Although efficacy Phrenic nerve injury (PNI) has been reported as a com- and long-term lesion durability have been demonstrated plication associated with ablations performed with cryo in limited groups of patients, studies in larger groups of and radiofrequency (RF) ablation. Electroporation has individuals are still under development. been increasingly explored as a potential substitute to The reference indicator of acute lesion formation dur- these modalities, and while early research has demon- ing cardiac ablation with thermal techniques consists in strated promising safety results, temporary phrenic nerve recording the local electrical activity changes with elec- stunning has been observed. The purpose of this study was troanatomical mapping systems. In the case of PFA, the to investigate the dose response of phrenic nerve damage modifications produced in the reversible and irreversible 86 from the direct application of cryoablation, RF ablation, cell recovery time-constants, and the mechanistic nature and electroporation ex vivo. of its cytolytic and anti-arrhythmic properties. Methods: Phrenic nerves (PN) were dissected by remov- Methods and Results: Healthy-control hiPSC-derived caring extraneous connective tissues, fat and associated vas- diomyocytes were enzymatically dissociated and seeded as culature from healthy anesthetized swine (n = 44). Sub-circular cell sheets (hiPSC-CCSs). Electroporation pulse- sequently, PN were placed in oxygenated buffer and main- field ablation (PFA) experiments were performed by deliv- tained at 37°C. Each PN was then stimulated proximally ering high-voltage electrical pulses via two needle-shaped with a 1 V, 0.1 ms square wave pulse. Biphasic com-electrodes. Detailed voltage-mapping studies were sub- pound action potentials (CAPs) were recorded both prox- sequently conducted. PFA application generated electric-imally and distally along the PN: i.e., on either side of ally isolated lesions within the hiPSC-CCSs. Further char- the site of applied ablations. After stable baseline record- acterization reveled that; (1) lesions persisted over pro- ings were obtained, the following ablative therapies were longed periods of time, demonstrating cell death and indic-administered: focal cryo at -80 C for 30 and 60 seconds ating irreversible electroporation, (2) cell death occurred (n=4); focal RF at 55, 60, and 65°C for 1 minute (n=4), within 24hrs following PFA, and was localized in areas and electroporation energies of 500, 600, 800, 1200, 1800, exposed to the highest field-intensities, (3) a reversible and 2000 V applied with 90, 100µs, monophasic pulses electroporation component was also documented with a with probes placed 10 mm apart (5 mm on either side of temporal decrease in lesion-dimensions in areas exposed the nerve, n=4). CAPs were then recorded at 15-minute to lower field-intensities, (4) most tissue recovery had oc-intervals for 3 hours post-therapy. Waveforms were recor- curred within the first ˜30 minutes following PFA, (5) per ded before and after ablation, then analyzed to determ- single pulse, high-frequency PFA was less efficacious than ine changes in latencies, amplitudes, and slopes of elicited standard monophasic PFA, (6) increasing pulse-number CAPs. had augmented lesion area, due to cumulative field intens- Results: All nerves subjected to cryo ablation or radiofre- ity amplification, and (7) electroporation sensitization was quency ablations at a temperature of 65° C elicited com- achieved by increasing extracellular Ca2+, indicating its plete loss of function. Amplitudes of CAPs were reduced role in PFA-mediated cytolysis. over 90% from pre-ablation levels. RF ablations at 55° C Finally, evaluating for potential anti-arrhythmic proper- and 60° C caused moderate damage in nerve conduction ties, we targeted arrhythmic rotors created in the hiPSC- after 30 minutes, with an average CAPs amplitude reduc- CCSs. PFA abolished arrhythmic rotors directly and al- tion of 47%. The two highest voltage levels of electropor- lowed the generation of sustained line-blocks isolating the ation (1800 V and 2000 V) showed a significant reduction remaining arrhythmogenic substrates within the hiPSC- of CAP amplitude of 35% and 30% respectively compared CCSs. to baseline (p < 0.05). The other doses of electroporation Conclusion: This new model for the study of cardiac showed no statistically significant changes from baseline PFA provides novel insights into its temporal and elec- (p > 0.05). Reductions in conduction velocities were not trophysiological characteristics, and facilitates electropor- significant with p values > 0.05 for all treatments includ- ation protocol optimization, screening for potential PFA- ing control group. sensitizers, and studying the mechanistic nature of its anti- Conclusion: This study demonstrates that depending on arrhythmic properties. ablative therapy applied, the impacts on nerve activities can vary widely in an ex vivo experiment. In this isolated OR-179 swine phrenic nerve preclinical research model, direct cryo- Open-Chest Pulsed Electric Field Ablation of Car-ablation induced the greatest losses of function, RF was diac Ganglionated Plexi in Acute Canine Models dose dependent still showing a potential complete loss of Martin van Zyl1, Mariam Khabsa1, Jason Tri1, Thomas function. Electroporation showed decreased phrenic nerve Ladas1, Adetola O. Ladejobi1, Omar Yasin1, John Reilly2, function only at high doses and was associated with pre-Barry O’Brien 2, Kenneth Coffey2, Samuel J. Asirvatham1 1 served conduction at all voltage levels tested. Mayo Clinic, United States 2Atrian Medical Ltd., Ireland OR-177 Objectives: This study aimed to evaluate the safety Utilizing Human Induced Pluripotent Stem Cells and acute effect on markers of cardiac autonomic tone fol- to Study Cardiac Pulsed-Field Ablation lowing pulsed electric fields (PEF) delivered to epicardial Leonid Maizels 1, Eyal Heller1, Michal Lendesberg2, Irit ganglionated plexi (GP) during a cardiac surgical proced- Huber2, Gil Arbel2, Amira Gepstein2, Roy Beinart1, Amit ure. Segev1, Lior Gepstein2, Elad Maor1 Background: Ablation of GP as a treatment for atrial fib- 1Sheba Medical Center, Israel rillation (AF) has shown promise but thermal ablation 2Israel Institute of Technology, Laboratory for Cardiac Elec- energy sources are limited by risk of inadvertent collateral trophysiology and Regenerative Medicine, Israel tissue injury. Background: Cardiac electroporation is a novel prom- Methods: In acute canine experiments, median sterno- ising non-thermal ablation method. Nevertheless, signific- tomy was performed to allow identification of 5 epicardial ant knowledge gaps exist regarding its electrophysiological GP regions using an anatomy-guided approach. Each site consequences in human cardiomyocytes, including; pro-was targeted with saline-irrigated PEF (1000 V, 100 µs, tocol and delivery optimization, irreversibility threshold, 10 ECG-synchronized pulse sequences). Atrial effective 87 refractory period (AERP) and local electrogram (EGM) lular matrix (ECM), may lead to unreliable results because amplitude were measured before and after each treatment. they do not reflect the tissue architecture. Thus, several Histology was performed post-treatment from samples of three-dimensional (3D) in vitro models, such as spheroids treatment adjacent structures. and hydrogel-based cultures, have been developed to re- Results: In 5 animals, 30 (n=2) and 60 (n=3) pulses were semble the tumour microenvironment and reduce the use successfully delivered to all 5 target sites). There was no of animals in testing according to the 3R rule (Reduction, difference in local atrial EGM amplitude before and after Replacement, Refinement). PEF at each site (1.83 ± 0.41 mV vs. 1.92 ± 0.53 mV; Herein, a 3D in vitro model of breast cancer has been ob-p = 0.72). Mean AERP increased from 97 ± 15 ms at tained by seeding HCC1954 cells on lyophilized scaffolds baseline to 115 ± 7 ms following treatment at all sites composed of chitosan that derives from partial deacetyla- (18.6% increase, 95% CI [1.9-35.2%]; p = 0.037). There tion of chitin and presents a structural similarity to the were no sustained ventricular arrhythmias or acute evid- glycosaminoglycans (GAG) of ECM. Cultures were char- ence of ischemia following delivery. Histology showed com- acterized at various time points (1, 3, and 7 days from plete preservation of adjacent atrial myocardium, phrenic seedings): cell morphology, cell growth, stemness, and nerves, pericardium, and esophagus. matrix deposition were evaluated by means of phase con- Conclusions: The use of PEF to target regions rich in car- trast microscopy, cell viability, histochemical staining, and diac GP in open-chest canine experiments was feasible and mRNA expression analysis. Then after, the responses of effective at acutely altering markers of cardiac autonomic breast cancer cells to anticancer drugs and electropora- tone. tion (EP) were evaluated. All results were compared to that obtained on 2D cell cultures. Finally, the electrical OR-28 properties of the hydrated with medium and non-hydrated Early clinical experience with cardiac pulsed field scaffolds were determined in order to compare them with ablation typical tissue electrical characteristics. Jim Hansen Our data have shown that chitosan hydrogels allowed the Gentofte Hospital, University of Copenhagen, Denmark formation of 3D cultures where breast cancer cells organ- Catheter ablation is used increasingly to prevent recur- ized themselves to form not only monolayer but also spher- rent atrial fibrillation (AF). The primary ablation strategi oids and produce ECM molecules, such as collagen1A1 is pulmonary vein isolation (PVI) but succesrates with and laminin B1. In 3D cultures cancer stem cell popula- this strategy has been limited in part by insufficient dur- tion was enhanced, as demonstrated by increases in the ability of ablative lesions formed by conventional thermal expression of stemness markers, such as Nanog, SOX2, ablation methods. These methods also carries a risk of OCT4, and in sphere-forming ability. Furthermore, the collateral damage to extracardiac tissue such as the eso- N-cadherin/E-cadherin ratio was significantly higher than phagus and the phrenic nerve. Pulsed field ablation (PFA) that detected in 2D cultures. Finally, chitosan-based culis emerging as an efficient and safe method to produce tures were less sensitive to doxorubicin and electroporation durable PVI. The presentation will focus on the imple- than adherent cell cultures. mentation in clinical practice of a commercially available Collectively, results suggest that chitosan-based breast PFA system for PVI, and review our clinical experience as cancer cel cultures may represent a promising model for well as early published data on safety and efficacy for AF in vitro evaluation of anticancer treatments. ablation. OR-128 Skin electroporation for non-invasive drug deliv- P37 - Models for in vitro ery:Electrical properties of skin models and fluor- electroporation escent molecule delivery Georgios Y. Kougkolos 1, Juliette Simon1, Geraldine Alberola2, Bastien Jouanmiqueou2, Marie-Pierre Rols3, Lionel Tuesday afternoon Track C Laudebat4, Muriel Golzio3, Zarel Valdez-Nava4, Emmanuel Oct 11, 13:30 - 14:45 Flahaut1 1CIRIMAT, Université de Toulouse, CNRS UMR 5085, INPT, OR-127 UPS, France Chitosan-based breast cancer cell cultures: a 2IPBS, Université de Toulouse, CNRS UMR 5089, UPS, France promising tool for in vitro evaluation of anticancer 3IPBS-CNRS, France treatments 4LAPLACE, Université de Toulouse, CNRS UMR 5213, INPT, Bianca Bazzolo1, Annj Zamuner1, Monica Dettin1, Elisa- UPS, France betta Sieni 2, Patrizia Lamberti3, Maria Teresa Conconi1 1University of Padova, Italy The skin presents an accessible and convenient ad- 2University of Insubria, Italy ministration route for non-invasive drug delivery. Trans- 3University of Salerno, Italy dermal delivery platforms, such as nicotine patches, can ef- fectively administer drugs through the epidermis in a con- In drug development, two-dimensional (2D) cell cul- trolled manner. Advantages include increased bioavailab- tures are widely used to choose the most effective drug ility, systemic delivery and painless administration, which candidates that are then tested in animal models. Never- in turn improve patient compliance and quality of life. theless, these cultures, almost completely lacking extracel- 88 However passive diffusion of drugs through the skin is only certain geometries, inlet/outlet, channel depth and other achieved for low MW (<400-500 Da), relatively lipophilic parameters to precisely regulate the required function. molecules (logP around 2 to 3). Reversible skin electro- Since our group is studying an effect of pulsed electric poration can temporarily increase the permeability of the field to the cells, we have manufactured a microfluidic epidermis allowing bigger or more hydrophilic molecules chip designated for high-throughput electroporation of to overcome the skin barrier. cells. In our microchip, a cell culture chamber is divided We have developed a drug delivery platform, consisting of into two parallel channels by a membrane, meanwhile a nanocomposite, electrically-conductive hydrogel, acting electrodes for electroporation are attached to the wall of as a drug reservoir and an electrode for the application the channels. Both microchannels have their own inlet of electrical pulses. It is based on an agarose hydrogel, and outlet, enabling injection of transfection material reinforced with carbon nanotubes to increase its electrical separately. Our perspective is to perform electroporation conductivity. The hydrogel can be dried and then res- of mammalian cells in two different ways: (1) plasmid wollen in a liquid solution with a molecule of interest (e.g. and cells are injected in the same microchannel and (2) a therapeutic macromolecule or a fluorescent dye). injected into separate microchannels. Two skin models are used: freshly-isolated extracts of hair- less mice skin and lab-grown, reconstructed human skin [1] Tabeling P. Oxford University Press (2005) layers. The conductive hydrogels are placed on the skin [2] Edmond W. K. Young and David J. Beebe. J Chem models and a pulsed electric field is applied through a gen- Soc Rev.; 39(3): 1036–1048 (2010) erator. A multi-sided approach is employed to evaluate electroporation effectiveness and drug delivery. We meas- OR-130 ure the real-time electrical response of the system through Inorganic nanoparticles as physical aids for local a digital oscilloscope. The dynamic electrical resistance is thermal ablation and electroporation enhance- calculated from the tension and current measurement. In ment: efficacy assessment in 2D and 3D cellular parallel, we evaluate the macromolecule delivery through models fluorescence microscopy of the skin surface and histolo- Nicolas Mattei1, Sebastjan Nemec2, Slavko Kralj2, Vin- gical observations of fluorescent molecule penetration. cent Gruszka1, Muriel Golzio1, Marie-Pierre Rols1, Jelena Our results show that the dynamic resistance of the skin Kološnjaj-Tabi 1 1 rapidly decreases during the application of the pulsed elec- CNRS, Institut de Pharmacologie et Biologie Structurale tric field, due to the electroporation of the lipid bilay- (IPBS), France 2 ers and the subsequent increase in ionic charge transport. Jozef Stefan Institute, Department for Materials Synthesis, Depending on the electrical parameters applied (intensity Slovenia and duration of electric field), this decrease in resistance Cancer cells and the tumor microenvironment play a may be temporary or permanent, indicating us the revers- key role in cancer development, progression, and resist-ibility of electroporation. The fluorescent microscopy con- ance to treatment. In order to tackle both components, firms that application of a pulsed electric field increases often referred to as “the seed” and “the soil”, we herein the delivery rate of hydrophilic molecules (MW of 450 Da propose the use of different inorganic nanoparticles as up to 4 kDa). The histological observations show that the physical effectors to improve cancer treatment. delivery of smaller molecules is greatly enhanced but, for Conducting (gold) and semiconducting (iron oxide) the time being, bigger molecules (4 kDa) tend to accumu- nanoparticles have both: an excellent heating yield upon late in the outermost skin layers. exposure to light [1], and a non-negligible potential to We are going to present our latest results regarding the ef- locally enhance the electric field [2]. fect of pulsed electric fields on the electrical properties of Herein we compare different classes of commercially our skin models, coupled with observations on fluorescent available spherical and rod-shaped gold nanoparticles, as molecule delivery. well as custom-made spherical and chain-like magnetic anisotropic iron oxide nanoclusters [3]. OR-129 In our study, we evaluated three types of pulsed electric High-throughput cell transfection in a microfluidic field protocols: the ones used for electrochemotherapy, for electroporation chip irreversible electroporation, and for gene electrotransfer. Neringa Bakute, Elinga Brazionyte, Arūnas Stirkė The effects of pulsed electric fields, combined with Center for Physical Sciences and Technology, Functional Ma- spherical or elongated nanoparticles, were compared: terials and Electronics, Lithuania 1) in different aqueous media, where we evaluated the A technology of microfluidics is an emerging tool in heating of the suspensions, 2) in a type IV collagen gel, the field of biology, medicine and chemistry. Microfluidic where we evaluated nanoparticles suspensions potential device is also known as ‘lab-on-a-chip’ technology [1]. In for collagen denaturation, and 3) in cells grown in 2D moving from macro- to microscale, there is unprecedented and multicellular spheroids (3D) made with murine control over spatial and temporal gradients and patterns hepatocellular carcinoma cells (HEPA 1-6). that cannot be captured in conventional Petri dishes and Compared to electric pulses alone, the combination of well plates [2]. However, there is not a single standard pulsed electric fields with nanoparticles can disrupt the microfluidic chip designated for all purposes – every collagen gel and has a more pronounced detrimental different field of studies needs a specific microchip with effect on cells. The comparison of iron oxide-based 89 nanomaterials with gold nanoparticles indicates that P39 - Electroporation for clinical use magnetic nanoparticles can, upon specific conditions, dramatically outperform gold nanoparticles in different Tuesday afternoon Track D electroporation protocols. By simultaneously acting as physical (magnetic, pho- Oct 11, 13:30 - 14:45 tothermal and electric) effectors, the nanochain-based platforms offer original multimodal possibilities for OR-119 prospective cancer treatment, affecting both, the cells Electrochemotherapy of portal vein tumor throm- and the extracellular matrix. bus as dowstaging to liver transplantation Luciano Tarantino 1, Emanuele Balzano2, Giuseppina Busto3, Aurelio Nasto4, Sara Bortone3, Paolo Tarantino5, Ric- [1] A. Espinosa, J. Kolosnjaj-Tabi, A. Abou-Hassan, cardo Aurelio Nasto2, Paolo De Simone2 et al. Adv. Funct. Mater. 37 (2018) 1803660. 1A.Tortora Cancer Hospital, Italy [2] Lekner, J. (2014). Physics in Medicine & Biology, 59 2Pisa University, Italy (20) 6031. 3PO Pagani - ASL Salerno, Italy [3] S. Kralj, D. Makovec. ACS Nano. 9 (2015) 9700. 4PO Polla - ASP Salerno, Italy 5Dana Farber Cancer Institute - Boston (MA), United States OR-131 Scanning electrochemical microscope as a tool for Liver transplantation (OLT) is contraindicate in Portal the electroporation Vein tumor Thrombosis (PVTT) from Hepatocellular Car- Inga Morkvėnaitė-Vilkončienė, Antanas Zinovičius, Bal- cinoma at hepatic hilum(pH-HCC) Surgery,Thermal ab- tramiejus Jakštys, Arūnas Ramanavičius lation and chemotherapy show poorer outcomes Electro- Vilnius Gediminas Technical University, Lithuania chemotherapy (ECT) has been successfully used in pa- The influence of pulsed electric fields (PEF) on living tients with pH-HCC with PVTT. We report the results of cells is associated with the phenomenon of electroporation ECT as downstaging aimed to definitive cure by OLT. when pores open in the plasma membrane of cells. F.P. 53 years HBV related Cirrhosis Child-Pugh B7 Recently it was shown that scanning electrochemical class; EGDS F2 aesophageal Varices. Diabetes. April microscopy (SECM) and electrochemical impedance 2016 : Enhanced Computed Tomography (CT) detected spectroscopy (EIS) can be used for the determination of HCC(n.3 nodules in VII-VIII-VI;diameter range=2–5 cm) quantitative electrochemical characteristics before and and PVTT of right portal vein. The patient was con- after the electroporation. The results show that it is sidered ineligible for OLT. May 2016: first ablation ses- possible to use SECM for targeted electroporation at the sion with percutaneous Radiofrequency-ablation(RFA) of selected area of tissue. The advantages of SECM over 3 HCC-nodules . August 2016: second ablation session other standard methods used in electroporation studies with ECT of PVTT. CT october 2016: disappearance of are that the SECM methodology allows not only the PVTT and patent right portal vein. No intraparenchymal study of cellular responses to PEF at the population level recurrence. CT march 2017: No recurrence in portal vein but also single cells and enzymatic reactions. Moreover, and in the left lobe. local recurrence in the VII-VIII due to the small diameter of the ultramicroelectrodes (less segments. May 2017 : transarterial chemoembolization than 25 µm), the electrochemical signal settles quickly, (TACE) of right lobe recurrences. CT October 2017: pat-within a few nanoseconds, so SECM allows recording ent right portal vein. No recurrence. The patient was signal changes at an extremely high frequency. Using reconsidered for OLT. He underwent OLT in April 2018. SECM micro and nanoelectrodes it will be possible to At 36-months follow-up , no recurrence of HCC occurred. measure the signal of intracellular molecules coming out March 2021: enhanced CT and PET/CT detected a single of electroporated cells at an extremely high frequency, small nodule (1.5 cm) uptaking tracer in the left upper and this will allow seeing the processes taking place in pulmonary lobe, no hepatic recurrence . CT-guided FNB the plasma membrane of cells immediately after electro- showed metastasis from HCC . June 2021: left lung upper poration. It is expected that after applying SECM, it will lobectomy . At the current time the patient is alive and be possible to observe the beginning of cell permeation recurrence-free at 44 monts follow-up. or the dynamics of the formation and closing of primary ECT Could be aneffective technique as pre-OLT dowsta- pores. Next, microelectrodes and SECM will be used to ging in HCC with PVTT. evaluate the dynamics of cell death after electroporation OR-121 over a period of hours and even days by evaluating the Response to Calcium Electroporation in Cancers electrochemical properties of the growth medium. Affecting the Skin – a Phase II Clinical Study Mille Vissing 1, Mascha Pervan2, Kitt Vestergaard3, John This project has received funding from the European Pløen4, Mazen Schnefeldt4, Søren Rafael Rafaelsen4, Christina Regional Development Fund (project No 01.2.2-LMT-K- Louise Lindhardt4, Lars Henrik Jensen4, Achim Rody2, Julie 718-03-0063) under a grant agreement with the Research Gehl1 Council of Lithuania (LMTLT). 1Zealand University Hospital, Denmark 2University Hospital Schleswig-Holstein, Campus Lübeck, Ger- 90 many OR-83 3Absalon University College, Denmark Electrochemotherapy of Posterior Resection Sur- 4University Hospital of Southern Denmark, Denmark face for Lowering Disease Recurrence Rate in Pan- Background: Up to one in five cancer patients present creatic Cancer (PanECT Study) Mihajlo Djokić cutaneous malignancy. When cancer is present in the University Medical Centre Ljubljana, Department of Abdom- skin, symptoms and distress can effect patients’ quality inal Surgery, Slovenia of life. Surgical and medical treatment can be challenging and local treatments are warranted. Calcium elec- Pancreatic cancer remains one of the deadliest cancers troporation (EP) uses intratumoral calcium injection and despite numerous research in the past decades. The only electrical pulses applied directly to target tissue. EP cre- curative option is radical surgical resection. To improve ates transient membrane pores that facilitate diffusion of survival an earlier diagnosis or a more efficient treatment otherwise non-permeant calcium, into target cells. The method is needed. targeted cancer cells may die due to toxic calcium levels Study was designed as a prospective pilot study to as-whilst normal cells restore homeostasis. The method is sess feasibility, safety, and efficacy of electrochemotherapy safe and effective with low cost. Furthermore, calcium EP (ECT) of the posterior resection surface after a surgical has low treatment toxicity without mutagens and spares resection of pancreatic head carcinoma. ECT will be per- healthy tissues. formed within 8-28 min after intravenous in bolus admin- Materials and methods: This ongoing study aims to istration of bleomycin (15 mg/m2). Plate electrodes will investigate response to calcium EP in a non-randomized be used for ECT treatment, the electrodes will be placed phase II trial in 30 patients with cutaneous or subcu- between choledochal cut-end, truncus celiacus, remaining taneous malignancy of any histology. Patients must have of the pancreas and aortal lymph nodes. A phase I clin-been without response in cutaneous metastases over a two- ical study is designed to include 20 patients that meet the month period, and are allowed systemic treatment. Pa- inclusion criteria. Every patient will be closely followed-tients are treated once and followed at month 1, 2, 3, 4, 5, up after operation, findings will be noted and reported in 6, and 12. The primary endpoint is response two months line with Clavien-Dindo classification of surgical complic-after treatment. The overall response rate will be defined ations. Treatment effectiveness will be evaluated by US as number of responding lesions (partial or complete re- or CT imaging, to detect early local recurrence of the dis- sponse) relative to treated lesions evaluated by changes in ease. size (mm) by clinical examination with caliper measure- At the present time 4 patients were already included in ment, further documented by clinical photography. The the study. Preliminary results will be presented at the trial is a collaboration between three cancer centres in Congress. Næstved, Vejle (Denmark) and Lübeck (Germany). In Further research is needed to improve early detection and one patient subset, magnetic resonance imaging is used to radical treatment of pancreatic cancer. After enrolment of verify treatment area. In another subset, qualitative inter- 20 patients the analysis of feasibility, safety and efficacy views have been performed to describe patient experience. will be performed. We expect the local recurrence rate Results: To date 16 patients with a total of 45 meta- to be lower after hybrid surgical and electrochemotherapy stases of different cancer types have been in enrolled treatment in comparison to surgical resection alone. in the study; breast cancer (n=10), lung cancer (n=3), pancreatic- (n=1), gastric- (n=1) and endometrial can- OR-22 cer (n=1). Median follow-up is 2.5 months (1-12). Few Burst Sine Wave Electroporation for Large Blood-side effects have been observed and healthy tissues have Brain Barrier Disruption for Efficient Drug been spared without sign of hyperpigmentation. Nine pa- Delivery—A Feasibility Study tients have been interviewed and the qualitative data of Sabrina N. Campelo 1, Kenneth N. Aycock1, Zaid S. this subgroup is being analysed. MRI has been used to Salameh1, Rafael V. Davalos1, John H. Rossmeisl2, Melvin F. verify treatment areas in three patients. Preliminary res- Lorenzo1 1 ults from the first eight patients show an ORR of 42% (CI Virginia Tech, United States 2 23-63 %,) across all treated tumours after two months (n Virginia Maryland College of Veterinary Medicine, United = 24). Whereas efficacy of calcium EP for breast can- States cer metastases has previously reported, this is the first Introduction: Glioblastoma multiforme (GBM) is a account on treatment for lung, pancreatic, gastric and en- difficult tumor to treat due to its highly invasive nature dometrial cancer. and its intratumoral heterogeneity. Additionally, the Conclusion: This study seeks to define response rates presence of the blood-brain barrier (BBB), responsible for different types of malignant skin tumours treated with for restricting the passage of systemic neurotoxins, calcium EP in order to uncover the potential of calcium macromolecules, and infectious particles from the brain EP as a standard treatment option. Future studies re- parenchyma, poses a unique challenge. This selectivity garding mechanisms of action will further aid in predicting often leads to essential drug molecules designed to responses. The study is part of the Changing Cancer Care target malignant cells in the brain to be screened out by consortium supported by the European Fund for Regional the epithelial-like tight junction proteins coupled with Development. molecular efflux transporters. Despite major progress in the development of 91 electroporation-based ablation therapies, several chal- potentially useful for some clinical applications of elec-lenges and gaps in knowledge exist. Up until this point, a troporation such as tissue ablation, electrochemotherapy, large focus of high-frequency irreversible electroporation and gene electrotransfer. In cardiac tissue ablation, where (H-FIRE) has been dedicated to inducing tumor cell irreversible electroporation is used, the treatment has be- death through the use of rectangular pulses, but a come known as Pulsed Field Ablation. While the reduc- protocol for maximizing BBB disruption has not yet been tion of muscle contractions has been confirmed in sev- developed. This study investigates the use of burst sine eral in vivo studies, the reduction of pain sensation has wave electroporation (BSWE), to induce larger volumes not yet been confirmed in humans, nor has the relation-of BBB disruption while maintaining controlled volumes ship between muscle contraction and pain sensation been of tissue ablation. investigated. The data obtained by cell/animal experi- Methods: Four male Fischer rats were treated with either ments, modeling, and theoretical considerations, although a standard rectangular wave H-FIRE protocol (n=2, of great value, do not allow evaluation of pain reduc- 5-5-5 µs, 480V), or an equivalent characteristic frequency tion during HF electroporation therapy. Therefore, in our sine wave BSWE protocol (n=2, 50 kHz, 680 Vpeak = study, we investigated the pain sensation elicited by short 480VRMS) with 200 bursts of 100 µs of energized time. biphasic HF pulses in healthy subjects using the short- Rodents were administered Evans Blue Dye (EBD) as well form McGill Pain Questionnaire. Twenty-five healthy subas Gadolinium (Gd) contrast agent immediately prior to jects were subjected to electrical stimulation of the tibialis treatment. The solutions were allowed to circulate for one anterior muscle with biphasic HF pulses in the range of hour at which the rodents were then sacrificed. Rodents a few microseconds and both symmetric and asymmetric underwent T1 weighted contrast-enhanced imaging to interphase (between positive and negative phase) and in- quantify volumes of BBB disruption as demonstrated terpulse delays (between pulses). We also examined the by hyperechoic areas. Additionally, a craniotomy was relationship between muscle contraction and pain sensa-performed, and the rodent brain was sectioned to measure tion while varying the pulse parameters (pulse width, in- areas of EBD uptake. terphase, and interpulse delays). In addition, we analyzed Results: Our results demonstrated larger volumes of which pain fibers were more likely to be excited based on BBB disruption produced by 50 kHz BSWE (MRI: 152.8 the pain descriptors selected by the subjects from the pain mm3 ± 11.17; EBD: 25.27 mm2) compared to equivalent questionnaires (A-delta or C fibers). Our results confirm 5-5-5µs H-FIRE protocols (103.2 mm3 ± 7.99; EBD: that biphasic high-frequency pulses (1 and 2 µs) reduce 10.36 mm2) as measured through both MRI and EBD muscle contraction and pain sensation in comparison to quantification respectively. the longer monophasic pulses currently used. In addition, Conclusions: We have demonstrated a potential to we established that interphase and interpulse delays play capitalize on the frequency-dependent nature of cell an important role in reducing muscle contraction and/or permeabilization by utilizing narrowband sinusoidal pain sensation. This study has shown that the range of electrical waveforms to induce larger volumes of BBB optimal pulse parameters can be increased depending on disruption. While standard H-FIRE utilizes traditional the prerequisites of the therapy. rectangular wave electrical pulses to induce cellular permeabilization, our preliminary results suggest that BSWE for nonthermal ablation of malignant gliomas P19 - Electroporation-based and large BBB disruption may allow for the passing of Treatments in Veterinary Medicine large therapeutic molecules into the brain parenchyma I for longer treatment windows and promote more diffuse regions of reversible electroporation to induce more Tuesday late afternoon Track A efficient drug uptake into malignant cells. Oct 11, 16:00 - 17:30 OR-24 Pain sensation and muscle contractions during de- OR-217 livery of high-frequency electroporation pulses The use of electrochemotherapy in combination Aleksandra Cvetkoska 1, Alenka Maček-Lebar1, Peter with other oncological therapies Trdina2, Damijan Miklavčič1, Matej Reberšek1 Matias M. Tellado 1, Juan Manuel Fernandez2, Juan 1University of Ljubljana, Faculty of Electrical Engineering, Manuel Osacar3, Felipe Maglietti4 1 Slovenia VetOncologia, Veterinary Oncology Clinic, DC, INFIP, DF - 2University Medical Centre Ljubljana, Institute of Clinical UBA CONICET, Argentina 2 Neurophysiology, Slovenia VetOncologia, Argentina 3Anoikis , Argentina To minimize neuromuscular electrical stimulation dur- 4GEM, Argentina ing pulse delivery in electroporation-based treatments, it was proposed to replace long monophasic pulses of 50- Cancer therapy seeks to improve the quality of life of 100 veterinary patients and prolong their survival. In general, µ s with bursts of biphasic high-frequency pulses in the range of microseconds to reduce muscle contraction this is achieved by an efficient local control of the dis-and pain sensation. These pulses, often referred to as ease. Surgical oncology plays an important role for this high-frequency (HF) electroporation pulses, proved to be aim, but when used alone it can’t provide the best res- ults. The combination of different treatment modalities, 92 with different mechanisms of action, is what allows to probe essential for providing mechanistic insight. However, long survival times, maintain a good quality of life, and while these small animal models have notable translational provide very good long-term control of the disease. Elec- value, there are significant limitations in terms of scale trochemotherapy, now widely available across the world, and anatomical relevance. To address these limitations, plays a fundamental role in veterinary oncology. It can-our research team and collaborators have developed a di- not replace surgery in most of the cases, but it can com- verse range of large animal models and spontaneous tu- plement it effectively. Allowing to achieve better results mor studies in veterinary patients to complement existing when combined than when used separately. The same goes rodent models. These preclinical models and veterinary for chemotherapy, cryosurgery, immunotherapy and radio- patients are excellent at providing realistic avenues for therapy, which are also found among the existing cancer testing electroporation-based tumor ablation modalities, treatments. There is very few scientific evidence regarding perfecting surgical and treatment techniques, and devel- the combination of electrochemotherapy with other ther- oping treatment strategies for future use in human pa- apies in in veterinary medicine, however, from the the- tients. Here, we provide an overview of results generated oretical point of view there is no impediment for it. In using IRE and H-FIRE across a broad spectrum of pre- this work we present results of treatments that include a clinical animal models and spontaneous tumors in veter- combination of therapies including electrochemotherapy, inary patients. Our findings demonstrate the effectiveness in order share some light in this matter. of electroporation-based therapeutics on local tumor ab- lation under clinically relevant conditions. We also define OR-74 mechanisms associated with systemic anti-tumor effects From Bench-to Kennel-to Bedside: Deploying and immune system activation following focal tumor ab-Novel Preclinical Animal Models of Cancer in the lation. Finally, we discuss findings related to combination Development of Irreversible Electroporation for therapeutic approaches that successfully enhance local tu- Human Patient Applications mor ablation and augment systemic anti-tumor immunity. Irving C. Allen 1, Kiho Lee2, Sherrie Clark-Deener3, Christopher Byron3, Michael R. Edwards3, John H. Rossmeisl3, OR-73 Sheryl Coutermarsh-Ott3, Kristin Eden4, Nikolaos Dervisis3, Electrochemotherapy in a pancreatic neuroendo- Shawna Klahn3, Joanne Tuohy3, Rafael V. Davalos1 crine tumor in a dog: A case report 1Virginia Tech, United States Sergio S. Salgado 1, Felipe H. Maglietti2 2University of Missouri, United States 1Universidad Peruana Cayetano Heredia, Lima, Peru 3Virgina Maryland College of Veterinary Medicine, United 2Instituto Universitario de Ciencias de la Salud Fundación H.A. States Barceló , Anatomia, Argentina 4Virginia Tech Carilion School of Medicine, United States Introduction: The aim of this study is to use ECT in a Over the last decade, a multitude of focal tumor abla- case of an insulinoma, to improve the hypoglycemic con- tion methods have emerged as potential treatment options dition of the patient. for cancer. Unfortunately, almost all of these modalities Case: A 10 years-old, Shih tzu, spayed female presented have high complication risks and many patients are not with a history of a sudden seizure, with plasma glucose candidates for these strategies due to tumor progression, of 30g/dl. An ultrasound revealed a 2.66 cm mass in the tumor size, and location near critical structures. The over- right pancreatic branch. An insulinoma was suspected and whelming majority of clinically available ablation modal-derived to our clinic. ities are either ionizing or thermal-based and have critical The patient was received in a depressed mental state, and limitations, such as limited control in precision, inabil- three more episodes of seizures occurred. Blood samples ity to treat large or multiple tumor nodules, and incom- were obtained, and a bolus of 33?xtrose was administered. plete ablation near major vessels. The risk of injury to Isotonic fluid therapy plus dextrose was started. The critical structures near the ablation zone is also a major owners reported that the patient was weak and listless concern, particularly damage to blood vessels, nerves, or during the past few months. The patient did not have ducts. To address these limitations, our research team a seizure again; however, her blood glucose did not rise has deployed irreversible electroporation (IRE) and High above 50g/dl. Frequency Irreversible Electroporation (H-FIRE) for can- Lab results were unremarkable. The differential diagnosis cer therapy. IRE and H-FIRE deliver a series of electric was an insulinoma or a IGF – II producing tumor. pulses through electrodes inserted directly into the tumor. Chest radiographs were normal. A CT-scan was per- The induced electric field distribution is nonthermal and formed for staging and treatment planning, revealing no produces structural defects in the target cell membrane metastasis. that causes cell death. Unfortunately, the lack of clinic- Prior to surgery, the patient showed clinical decompensa- ally and physiologically relevant pre-clinical cancer mod- tion. She presented a blood glucose level of 18g/dl, which els is often a significant limitation with the development of was treated with dextrose supplementation upon stabiliz- novel biomedical devices and implementation of focal tu- ation. The patient was admitted to surgery for a partial mor ablation strategies. To date, the majority of studies pancreatectomy. However, during surgery the tumor was testing electroporation-based modalities for cancer treat- firm, vascularized, with a semicircular shape located next ment have focused on rodent models, which have been to the duodenum, rendering surgery very risky. . Then, critical in moving this field forward and will continue to ECT was performed as a safer option. 93 For the ECT, intravenous Bleomycin was used. The elec-the 50% duty cycle nor 83% of IRE in all the animals. tric pulses (8 100µs long of 1,000 V/cm at 5,000Hz) were Together, these data suggest that a longer duty cycle of delivered using an EPV-100 electroporator (Biotex, Ar-pulsing for IRE ensures the feasibility and safety for its gentina) using gold plated plates electrode. . application in clinical trials. Results: After the intervention the patient was evaluated using Glasgow Composite Measure Pain Scale, showing OR-213 mild to moderate discomfort. Specific canine pancreatic Veterinary Guidelines for Electrochemotherapy of lipase at 24 and 48 hours; the former was suspicious for superficial tumors pancreatitis (225.34 ug/L) but normalizing at 48 hours Felipe H. Maglietti 1, Matias M. Tellado2, Lluis M. Mir3 (150.3 ug/L). The blood glucose values stabilized in the 1Instituto Universitario de Ciencias de la Salud Fundación H.A. first 48 hours, allowing the cessation of dextrose supple- Barceló, Argentina mentation. Blood glucose was stable between 75g/dl to 2VetOncologia, Veterinary Oncology Clinic, DC, INFIP, DF - 85g/dl. Upon discharge blood glucose presented values of UBA CONICET, Argentina 140g/dl. In subsequent evaluations, fasting blood glucose 3Université Paris-Saclay, CNRS, Gustave Roussy, Metabolic was 270g/dl, then NPH insulin was started at a dose of and Systemic Aspects of Oncogenesis (METSY), France 0.25U/kg every 12 hours. Electrochemotherapy (ECT) consists in the applica- The histopathological examination with immunohisto- tion of electric pulses to increase chemotherapeutic drug chemistry confirmed an insulinoma. intake (bleomycin, cisplatin, or calcium) into the tumor During follow-up, ultrasonography revealed a reduction of cells. It has become a very valuable treatment option in more than 50% of the tumor one month after the procedure veterinary oncology. It is an effective and safe treatment (measurement of 1.1cm), confirming a partial response. modality, which is not only beneficial as a palliative treat- The quality of life was improved due to the cessation of ment, but also for a curative approach. Performing the the hypoglycemia. treatment adequately will ensure the best results possible, At time of writing, the patient remains free of clinical signs in the minimum number of sessions, and reduce complic- or progression of the disease. This is the first case of an ations. Usually, only one session is enough to achieve ex- insulinoma treated with ECT in veterinary medicine. cellent results, but the treatment can be repeated. Sev- In conclusion, ECT was an effective approach for treating eral sessions can be necessary in the case of incompletely a patient with an insulinoma. treated or very extended lesions, as well as in the occur- rence of new lesions. ECT is effective for superficial or OR-138 oral tumors of any histology that are accessible to the Longer duty cycle effects of irreversible electropor- electrodes. Intravenous bleomycin is the preferred drug ation on pig’s pancreatic tissues: a pilot study and route of administration, leaving other ways of ad- Hong Bae Kim ministration and drugs for selected cases. The guidelines Seoul National University, Biosystems Engineering, Korea, Re- presented here are destined to veterinarians who want to public of develop their understanding of the basis of ECT and wish A conventional irreversible electroporation (IRE) uses to perform it adequately and effectively. In this paper, a pulse width of the duty cycle ranging 0.01–0.04% to we also discuss common problems and how to solve them, cause irreversible permeabilization on the membrane of and we include practical tips to improve the treatment res- a cancer cell. However, such a duty cycle of pulsing ults based on common questions and mistakes of beginner may leave residual liver cells within the ablated tissue. users. The relationship between the duty cycle of pulse width with the feasibility and safety of IRE remains unclear. This study demonstrated the effect of varying the duty P22 - Irreversible Electroporation cycle of 50–83% on the feasibility and safety of applying (IRE) and Immunotherapy IRE in the pig’s pancreas. Triphenyltetrazolium chlor- ide staining demonstrated that during the 50% duty cycle Tuesday late afternoon Track B of IRE, the ablation area was significantly dependent on Oct 11, 16:00 - 17:30 time after IRE. Furthermore, no ablation was observed 14 days after either duty cycle 50%-IRE or 83%-IRE. This OR-186 indicated that the ablated tissues had recovered. For his- A phase II-study of electroporation potentiated topatholocial evaluation, most parameters of interstitial immunotherapy in liver metastatic pancreatic can-edema, hemorrhage, necrosis, and infiltration decreased cer (EPIC-1) 14 days after IRE of 50% duty cycle, except for fibrosis. Rasmus Virenfeldt Flak 1, Laurids Østergaard Poulsen1, For hematological evaluation, levels of aspartate transam- Mogens Tornby Stender1, Gintare Naujokaite1, Olga inase, alanine transaminase, total bilirubin, amylase, and Tcacenco1, Alkwin Wanders1, Sönke Detlefsen2, Ralf Agger3, lipase were the same as those in the control group after Emil Kofod-Olsen3, Ole Thorlacius-Ussing1, Morten Ladekarl1 14 days of duty cycle 50%-IRE. The pathological values 1Aalborg University Hospital, Denmark were reportedly lower than that of the 50% duty cycle 14 2Odense University Hospital, Denmark days after IRE for the 83% duty cycle of IRE. Moreover, 3Aalborg University, Denmark the electrocardiogram did neither depict arrhythmia after 94 Introduction: Pancreatic cancer (PC) is the fourth creased SPAS-1+ tissue-resident CD8+ memory T cells leading cause of cancer death. This is largely due to (TRM) in non-lymphoid tissues including skin. Mice that late diagnosis, aggressive tumor biology and resistance to had previously achieved complete remission following dual chemotherapy. Immune checkpoint inhibitors (ICI) have IRE + anti-CTLA-4 therapy were 100% protected from shown impressive results in several tumor types, but not in secondary tumor challenge, suggesting that enhanced tu- PC. Animal trials have, however, shown that ablation of a mor antigen-specific TRM in the skin are associated with tumor lesion using irreversible electroporation (IRE) can tumor protection. IRE immediately followed by anti-PD- alleviate resistance to ICI in PC and other cancers. The 1 did not show an effect on tumor growth or SPAS-1+ T aim of this trial was to examine the efficacy and safety of cell levels in blood. However, the anti-PD-1 group showed combined pembrolizumab and IRE ablation in liver meta- sustainable tumor regression (compared to IgG control, static PC. p<0.05) when given 10 days later, after both IRE and Materials & Methods: Patients (PS 0-1) with progression anti-CTLA-4 treatment. on or intolerance to first or subsequent lines of chemother- In the MC-38 colon cancer model, primary tumor control apy was included in a prospective single-arm trial. Pa- was significantly improved by combining IRE with antitients were administered pembrolizumab (400mg every 6 PD-1 treatment (median survival days: control: 8, anti- weeks) and treated with IRE of a single liver metastasis PD-1: 9, IRE: 16, IRE+anti-PD-1: 26). This was correl- after 10 days. CT scans were performed at baseline, 14 ated with a higher frequency of CD8+ tumor infiltrating days after IRE and every two months during follow-up. lymphocytes. IRE + anti-PD-1 also achieved a rate of Blood and tissue samples were collected at baseline, on secondary tumor rejection rate of 95%. In addition, CD8 the day before IRE and on the first postoperative day. depletion after IRE + anti-PD-1 treatment led to 0% re- Results: Inclusion was stopped after eight patients due to jection upon rechallenge (n=10). In another colon cancer progression in all eight patients during interim analysis. model, CT26, IRE + anti-CTLA-4 led to complete tumor No serious adverse reactions to pembrolizumab or surgical regression in 25% of the mice (n=8), compared to contin- complications were recorded. Flowcytometric classifica- ued tumor growth with IRE or ICI monotherapy. tion of circulating immune cells and RNA-sequencing of In summary, our results show that IRE and ICI can lead tumor samples are currently ongoing. to a synergistic and durable antitumor response, prolong Conclusions: Combined IRE and pembrolizumab does not overall survival, and induce long-term immune protection. have a clinically relevant effect size in previously treated This success appears to rely heavily on CD8 T cells (i.e., metastatic PC. The cancer immunological impact of IRE population, location, and phenotype). Insufficient priming is currently being investigated. of CD8 T cells can be addressed by focal therapy and early intervention of anti-CTLA-4 such as shown in the TRAMP OR-187 and CT-26 models. On the other hand, T cell exhaustion Irreversible Electroporation and Immune Check- can be mitigated by engineering of focal therapy and anti-point Inhibitor Immunotherapy provides improved PD-1 immunotherapy as shown in the MC-38 model and primary and systemic cancer control delayed administration of anti-PD-1 in TRAMP. Qi Shao, Minhan Jiang, John C. Bischof, Yoji Shimizu, Brandon J. Burbach OR-188 University of Minnesota, United States Irreversible electroporation selectively lyses can- Accumulating evidence suggests that focal therapies cer cells while preserving function and promot- combined with immunotherapies can provide systemic ing tumor infiltration of chimeric antigen receptor anti-cancer immunity. Here, we assessed the ability of (CAR) engineered T cells combined irreversible electroporation (IRE) and Immune William-Ray Vista 1, Mary Sheehan2, Stephen B Solomon1, Checkpoint Inhibitor (ICI) to improve primary and sys- Prasad Adusumilli1, Govind Srimathveeravalli2 1 temic tumor control in various murine cancer models. Memorial Sloan Kettering Cancer Center, United States 2 Hind limb tumors (TRAMP-C2, MC-38 and CT26) were University of Massachusetts Amherst, United States treated with IRE (1500 V/cm, 50 µ s pulse width, 50 pulses Purpose: We investigated optimal IRE pulse paramet-at 1Hz) by a set of parallel electrodes, followed by ICI im- ers for the selective depletion of cancer cells while sparing munotherapy with anti-CTLA-4 (200 µ g on day 1, 100 µ g Chimeric Antigen Receptor (CAR) T cells in the tumor on day 4, 7 and 10) or anti-PD-1 (100 µ g on day 1, 3 and microenvironment. We also studied the impact of IRE on 5). CAR T cell function and its recruitment into tumors. In the TRAMP-C2 prostate cancer model, IRE followed Materials and Methods: Following IRE (at varying by anti-CTLA-4 demonstrated superior outcomes than voltage, pulse width and numbers) on mouse (AB12) and either monotherapy. Anti-CTLA-4 failed to control tu- human (MSTO-GM) mesothelioma cells, T cells (CAR mor growth and did not alter SPAS-1 T cell distribution and control) were co-cultured. Cell survival and prolifer- while IRE-treated tumors showed a brief delay of growth ation were quantified at 2-, and 24-hours post-treatment. but were unable to successfully control the tumor. In con- Pulse parameters with greatest differential cytotoxicity trast, the combinatorial regimen of IRE followed by anti- between cancer and T cells was termed selective IRE CTLA-4 therapy led to complete tumor regression in 46% (sIRE) and further tested in a 3D tumor mimic with Prop- of mice. In addition, combination IRE and anti-CTLA-4 idium Iodide/Calcein staining. Numerical models were therapy increased intratumoral SPAS-1+ T cells and in-constructed in COMSOL to estimate induced transmem- 95 brane voltage in different cells, the electric field gradi-showed that nano-microsecond pulse electric field could ent, and statistical probability of cell death in a 3D in simultaneously target the nuclear membrane and the vitro tumor mimic. Impact of repeated sIRE on CAR T cytomembrane on cells. Compared with nsPEF or µ sPEF cells was evaluated with a 51Cr release and biolumines- alone, the n µ PEF could significantly promote the fluorescent cytotoxicity assay at various effector-to-target (E:T) cence dissipation degree of single cell, produce increasing ratio. Chemokine release from cancer cells and its effect apoptotic and necrosis of cells, and achieve the wider-range on CAR T cell migration were measured with a multiplex and low-residue ablation area. Besides, when the apply-Luminex bead immunoassay and transwell co-culture, re- ing sequence of nanosecond pulses and microsecond pulses spectively. Outcomes of sIRE on intratumor T cell popu- was reversed, the enhanced bioelectric effect was weaker lation and cancer ablation was studied in BALB/cJ mice than that of n µ PEF. Therefore, the multi-target charac- bearing AB12 subcutaneous tumors using flow cytometry teristic and enhancement effect of n µ PEF could provide and immunohistochemistry. Trafficking of intravenously the theoretical support for greater killing effect of tumor administered CAR T cells from chemokine release by sIRE cells and complete ablation of tumor tissues, which is ex- was assessed in NOD scid gamma mice bearing MSTO- pected to become a novel therapy of pulsed electric field G/M tumors. for tumors. Result: Simulations suggested that cancer cells develop 1.5 – 2 fold higher transmembrane voltage when treated with OR-215 the same pulse parameters, increasing their susceptibil- Decellularized intestinal tissue as a potential graft ity to IRE. Cuvette model studies confirmed this, where for bladder reconstruction therapies sIRE (1000 V/cm, 10 µ s pulse width, 125 pulses) provided Neeraj Raghuraman Rajagopalan, Brian Simoes, Feiyu greatest differential cytotoxicity (MSTO-GM: 43.2% vs. Yang, Yubing Sun, Govind Srimathveeravalli CAR T cell: 84.6%, p < 0.01), with similar outcomes University of Massachusetts Amherst, United States in a 3D gel model. sIRE effectiveness in 3D gel mod- Introduction: Irreversible electroporation (IRE) has els containing mixed cell populations could also be pre- previously been reported to kill cells without disrupting dicted by simulation models. sIRE parameters slightly the extracellular matrix (ECM), with potential applica-affected the proliferation, but not function of CAR T cells tions in tissue engineering. Here, we developed a workflow as measured on bioluminescent cytotoxic assays (p not and investigated the application of IRE for ex vivo decel- significant). CAR T cells remained potent following re- lularization of the small intestine, a widely used scaffold peated treatment (3x doses at 2:1 E:T ratio cytotoxicity material for reconstructive surgery. for sIRE: 45% vs. IRE: 29% & Sham: 75%). sIRE of Methods: Wistar rats (16-20 weeks old) were sacrificed to murine mouse tumors resulted in greater preservation of extract the small intestine (SI, 1 inch length). IRE and T cells (CD3+: 0.7%) at 4 hours post-treatment when detergent wash (DT - positive control) were used for tis- compared to IRE (CD3+: 0.46%), with greater recruit- sue decellularization, and results compared to sham treat- ment at 24 hours post-treatment (CD3+: 6.7%). Robust ment. IRE of SI in cold PBS was performed in a 4mm release of chemokines by sIRE induced greater migration gap cuvette with following pulse parameters: 1500 V/cm, in a transwell assay, findings that were validated in mouse 100 µs, 50 pulses at 1 Hz. IRE treated SI was then trans- bearing MSTO-G/M tumors. ferred to Iscove’s Modified Dulbecco’s Medium (IMDM) Conclusion: sIRE selectively lyses cancer cells while pre- with supplements and incubated for 4h, followed by 4x serving function of CAR T cells; sIRE-induced chemokine PBS wash (12 h), 1x PBS wash (1 h) and DNase wash gradient promotes CAR T-cell tumor infiltration, thereby (24 h). DT was performed by washing the SI in tri-augmenting the E:T ratio and anti-tumor efficacy. ton X-100 solution (52 h), sodium deoxycholate (26 h), DNase (1 h), 1x PBS (1 h), saline solution (1 h) and DI OR-214 water (1 h). H&E and Masson’s Trichrome stains were Experimental study on enhancing the bioelectric used for assessing morphological status and collagen in effect of tumor cells using the combination of nano- SI samples. Colorimetric quantitative assays were used to second and microsecond pulsed electric field measure residual DNA (Quantifluor dsDNA system), glyc- Wencheng Peng, Junyi Ning, Hongmei Liu, Shoulong Dong, osaminoglycan (Blyscan) and collagen (Sircol) levels in Chenguo Yao the tissue. Ultimate tensile test was performed on control Chongqing University, China and treatment groups to determine changes in mechanical Based on the different targets on cells and bioelec- properties from treatment. IRE treated IS was lyophil- trochemical mechanisms of the nanosecond pulsed elec- ized and made into a slurry to form scaffolds and seeded tric field (nsPEF) and microsecond pulsed electric field with bladder smooth muscle cells. Cytotoxicity assay was ( µ sPEF), this paper studied the enhanced bioelectric ef- performed on the scaffolds and compared to Matrigel cell fect of tumor cells using the combination of nsPEF and encapsulation (positive control). µ sPEF. With human malignant melanoma cells (A375) as Results: Outcomes of IS decellularization with IRE (41 experimental subjects, single-cell fluorescence assay, flow hrs) was comparable to DT (82 hrs) with shorter pro-cytometry double staining assay and monolayer cell ab- cessing time. H&E and Masson’s trichrome stained IRE lation experiment were carried out to analyze the tar- samples demonstrated complete removal of cellular com- geting characteristic and enhancement effect of nano- ponents with preservation of collagen (ECM), equivalent microsecond pulse electric field (n µ PEF). The results to DT. Total residual DNA in IRE samples (183 ng/mg) 96 group was considerably lower than sham group (2990 tric pulses dose was split in a few fractions, but the mech- ng/mg of tissue) but higher than DT (10 ng/mg). IRE anism of this phenomenon is still debated and unclear. samples had significant preservation of ECM content (col- Our experiments were conducted using fluorescence micro- lagen – 3.5 µg/mg, GAG – 0.27 µg/mg), comparable to scopy and acquiring fluorescence variations in real-time us- native IS (sham: collagen – 3 µg/mg, GAG – 0.77 µg/mg) ing FURA-2 ratiometric dye for calcium detection. Cells and higher than DT (collagen – 1.3 µg/mg, GAG – 0.05 were pulsed using coplanar electrodes of a planar wave- µg/mg). Mechanical stiffness of IRE (13.2 MPa) and DT guide integrated in an inverted microscope stage. Electric treated IS (10.38 MPa) group was higher than native tis- pulses lasting 10 ns in different numbers and different re- sue (5.3 MPa) without statistically significant difference. petition frequencies were used. Fluorescence images were Cytotoxicity assay showed that the cells grown in IRE acquired every 10 seconds starting from 1 minute before scaffolds were healthy and viable, equivalent to Matrigel the exposure up to 10 minutes after the end of the expos- culture. ure to monitor complete calcium fluxes dynamics after the Conclusion: IRE is a novel and effective approach for ex exposure. Sham exposures were performed during each set vivo decellularization of bulk tissue with outcomes com- of experiments. parable to conventional detergent wash at considerably The experiments were performed in the presence and in shorter time requirement. IRE provides added advantage absence of external Ca2+ ions to exclude the implication of not using harsh chemicals and better preservation of of internal cell organelles in regulating the cytosolic Ca2+ ECM. levels. Moreover, in order to demonstrate that the Ca2+ levels modulation is mainly due to the pores formation in- duced by the electrical stimulations, two different Ca2+ P32 - Pulsed electric field effects on channels inhibitors were also used and the results were neural tissues and the compared with experiments without inhibitors. brain Our results demonstrate that Ca2+ modulation seems in- dependent on the frequency content of the different pulsed Tuesday late afternoon Track C electric fields patterns applied. Similarly, no additive ef- Oct 11, 16:00 - 17:30 fects on cytosolic Ca2+ increase were observed when the pulse number was spread in multiple fractions, if com- pared to the fluorescence reported when the same number OR-17 of pulses was applied consecutively. The SH-SY5Y cells Characterization of Ca2+ fluxes modulation by seemed more sensitive to pulses stimulation, may be due nanosecond pulsed electric fields in neuroblastoma to their neuronal and tumor nature because Ca2+ is a and mesenchymal stem cells Francesca Camera 1, Tomas Garcia-Sanchez2, Barbara B. strong regulator of the functionality of such cells. Our res- Benassi3, Adeline Muscat4, Claudia Consales3, Leslie Vallet4, ults are interesting to better understand permeabilization- Franck M. André4, Carmela Marino3, Lluis M. Mir4, Caterina mediated Ca2+ dynamics to possibly control associated Merla4 cell functions for new therapeutic applications in cancer 1University of Rome La Sapienza, Italy and non-cancer diseases. 2CNRS, Gustave Roussy, Université Paris-Saclay, Depart- ment of Communications and Information Technologies, and OR-18 Biomedical Electronics Research Group (BERG), Universitat RISEUP: Regeneration of Injured Spinal cord by Pompeu Fabra, France Electro pUlsed bio-hybrid imPlant 3ENEA, Division of Health Protection Technologies„ Italy Claudia Consales 1, Manuel Monleón Pradas2, Paolo 4CNRS, Gustave Roussy, Université Paris-Saclay, France Marracino3, Micaela Liberti4, Franck M. André5, Victoria Moreno Manzano6, Caterina Merla1, Jorge Más Estellés2, To assess if any correlation can be established between Marco Balucani3, Micol Colella4, Francesca Apollonio4, Lluis the frequency content of different pulsed electric stimu- M. Mir5 lations and the permeabilization to calcium ions, the re- 1ENEA, Division of Health Protection Technologies, Italy sponse of two different cell cultures to a set of different 2Universitat Politècnica deValència, Spain nanosecond electric pulses was analyzed. These observa- 3Rise Technology S.r.L, Italy tions seem particularly interesting, as calcium is a fun-4Sapienza University, Italy damental second messenger in cells. In particular, both 5CNRS, Gustave Roussy, Université Paris-Saclay, France SH-SY5Y neuroblastoma cell line and mesenchymal stem 6CIPF, Neuronal and Tissue Regeneration Laboratory, Spain cells (MSCs) were analyzed. The use of two cellular mod- 7ENEA, Division of Health Protection Technologies, Italy els allows us to observe if the results obtained are cell Spinal Cord Injury (SCI), a major cause of paralysis, type-dependent and so to deepen which specific biological currently has no effective therapies. Every year almost activity can be modulated with pulsed electric stimula- 500.000 people are diagnosed with SCI worldwide. In tions to better address different therapeutic applications. Europe, the average investment is up to 2 M€ per pa- We also investigated if spreading the pulses in multiple tient in health are [1]. The difficulty on the neuronal groups enabled the increase of SH-SY5Y and MSCs mem-restoration after SCI is based on the complex cascade brane permeabilization. In fact, some authors observed, of events that inexorably cause a degenerative chronic looking at the electric field-mediated uptake of specific stage mainly favored by the non-permissive environment fluorescent dyes, cells “sensitization” if the delivered elec- and limited capacity for axonal regrowth. Multifaceted 97 strategies are considered the unique solution for functional exogenous electrical stimulation consists the most often in restoration by including cell substitution, neuroprotection triggering action potentials with low electric fields. This and axonal growth promotion. RISEUP project proposes allows us to non-chemically alter the niche signals of NSCs to attain neuronal functional regeneration after SCI by an or MSCs to promote proliferation and differentiation unprecedented and unique bio-hybrid-compatible electro- towards specific lineages. activated and wireless rechargeable implantable techno- Activation of calcium signalling is essential in neur-logy. RISEUP introduces high voltage microsecond elec- onal development, including neuronal induction [3] and tric pulses (micropulses) stimulations and low amplitude regulates physiological functions in the cell such as pro- direct currents on a combination of stem cells (induced liferation and differentiation [4]. Therefore, in this work, neural stem cells and multipotent stromal cells), whose intracellular calcium oscillations pattern has been studied transplantation is facilitated by an innovative scaffold bio- under conditions of proliferation and neuron-induced material. The RISEUP concept is that micropulses, being differentiation in NSCs and MSCs. Using electroporation able to impose and control cytosolic Calcium oscillations to control calcium oscillations [4], we studied the effects [2, 3], will facilitate cell maturation, survival and neur- of the oscillations on proliferation and induced neural otrophic factors secretion. Because Calcium signaling is differentiation of NSCs and MSCs. Calcium oscillations essential for neuronal activity, endogenous neuronal re- were recorded in time lapse microscopy with the calcium connections will also be favored. RISEUP goal, even if dye Fluo-4 AM. Morphological changes during the dif- ambitious, is concrete due to the multidisciplinary part- ferentiation process were studied by immunofluorescence ners’ competences, initiating from TRL1 a radically new techniques and brightfield microscopy. line of technology (electro-activated, remotely controlled, Manipulation of calcium patterns through electroporation biocompatible, biodegradable cell-containing implants for might be a potential therapy to promote cell proliferation the repair of neuronal lesions) establishing its proof-of- as well as neuron-induced differentiation in a simple principle (TRL3). The long-term vision of RISEUP is the and easily controllable way. Electrical stimulation is a radical change in SCI treatment modality to assure the feasible and flexible methodology in vitro and it is easily cure delivery without any machinery connection, dramat- transferable to in vivo in order to rescue central nervous ically improving patients’ quality of life. systems injuries. OR-19 [1] Fischer I, et al. Nat Rev Neurosci. 21 (7) :366- Effects of electrical stimulation in neural stem cells 383 (2020). and mesenchymal stem cells cell fate [2] S. Thuret, et al. Nat. Rev. Neurosci. 7, 628–643 Marina M. Sanchez Petidier, Romain Fernandes, Leslie (2006). Vallet, Franck Andre, Lluis M. Mir [3] Toth AB, et al. Cell Calcium. 59(2-3):124-134 (2016). CNRS, Gustave Roussy, Metabolic and Systemic aspects of the [4] Hanna, H., et al. Stem Cell Res Ther. 8, 91 (2017). oncogenesis (METSY), France Spinal cord injuries remain a significant therapeutic OR-16 challenge due to the inability of the central nervous High-rate nsPEF bursts stimulate neurons at para-system to regenerate lost neurons and restore functional doxically low electric field thresholds and without connections. Because of the enormous potential in electroporation the treatment of central nervous system disorders and Mantas Silkunas, Emily Gudvangen, Andrei G. Pakhomov injuries, Neural Stem Cells (NSCs) transplantation is an Old Dominion University, United States attractive therapy that demonstrates its ability to replace Nanosecond Pulsed Electric Field (nsPEF) is a new lost cells or repair host neural circuits, as well as its modality to achieve bioeffects, including cell stimulation. ability to differentiate into any required neural cell type However, use of high electric field is required for neuron [1]. Mesenchymal Stem Cells (MSCs) are considered a stimulation using nsPEF, thus increasing a risk of plasma competent cell source for tissue engineering applications. membrane disruption. Here we demonstrate that the high Transplantation of MSCs into spinal cord injury has an pulsed electric field requirement can be bypassed by ex- immunoregulatory effect on the tissue that contributes ploiting temporal summation when multiple subthreshold to structuring regenerative microenvironments in areas stimulatory nsPEF are compressed to high-rate bursts. of injury and has the potential to generate multiple Threshold electric field and neuron’s ability to respond differentiated progenies [2]. to multiple stimulation attempts was evaluated in a wide Studies with NSCs and MSCs, either separately or in range of nsPEF burst parameters. combination, show improved functional recovery after nsPEF burst stimulation was tested for excitation of transplantation, providing therapeutic benefit in terms of dissociated rat hippocampal neurons. A stimuli-induced decreasing the inflammatory environment. However, the action potential (AP) was detected using optical mem- low survival rate and uncontrolled graft differentiation brane potential registration at standard FITC settings requires the use of combination therapies. In this context, and transmembrane potential-sensitive fluorescent probe cell-based therapies still need improvements such as the FluoVolt™. nsPEF bursts enabled neuron stimulation enhancement of the cell survival and the neuronal differ- at 0.1±0.01kV/cm (400ns/pulses at 2MHz for 500µs), entiation and maturation, by complementary treatments, which was a substantial reduction compared to a single for example electrical stimulation. For this purpose, 400ns pulse that required 3±0.2kV/cm. Furthermore, 98 for constant burst duration the efficacy of nsPEF bursts The hypothesis is that, by modulating the calcium fluxes (reduction of threshold electric field) increased with the of iNSCs through µsec stimulation, these cells will differen- duty cycle, which could be achieved by an increment in tiate in mature neurons acquiring the ability to regenerate pulse repetition rate or pulse duration. Also, all tested the lesioned area. Moreover, MSCs stimulation could in- nsPEF bursts stimulated neurons at a lower time-average duce their differentiation in neuronal-like cells, able to re-electric field than a single matching pulse (both stimuli lease neurotrophic factors necessary for neurons survival. had identical time-average electric field and duration), Here we present the first results obtained by applying dif- indicating an unknown specific nsPEF burst effect. ferent exposure protocols in terms of repetion frequency, nsPEF bursts exhibited the lowest time-average threshold pulses number, amplitude, duration, and polarity. values (indicating the most effective temporal summation) The purpose of these preliminary results is to evaluate when the duty cycle was <10%. At the same time, these the survival, homeostasis and differentiation of cells in re- nsPEF bursts required the highest electrical field/pulse sponse to the different protocols of stimulation. ( ≈ 1-3kV/cm) to induce AP. These circumstances suggest that AP generation could be mediated due to membrane OR-21 disruption and increased permeability-mediated ions Low pulsed electrical fields for inducing transient fluxes (depolarization). Furthermore, the ability of BBB disruption in a mouse model neurons to generate responding AP to every stimulation Shirley Sharabi, Yael Mardor, David Last, Dianne Daniels, attempt (up to 100) was investigated. Neurons stimulated Sigal Liraz-Zaltsman, Itzik Cooper using single µs-range pulses (conventional stimulation) Sheba Medical Center, Israel responded by producing AP to all applied stimuli, while Brain diseases are extremely hard to treat, mainly to a single 400ns pulse responded only once. This due to poor penetration of therapeutics across the blood- revealed, that single pulse nsPEF was unsuitable for brain barrier (BBB). It was previously demonstrated by us multiple stimulations. Although, in general, nsPEF burst and others that BBB disruption (BBBd) can be achieved could be used for successful multiple stimulations (e.g., by applying electroporation (EP). Unfortunately, apply- 250 x 400ns/pulse at 500kHz or 150 x 100ns/pulse at ing high voltage pulses can induce significant adverse ef- 250kHz) responded to all stimuli. fect and require invasive surgical procedures. In the past years, we have discovered that low pulsed electric fields (L- Support: AFOSR MURI grant FA9550-15-1-0517 to PEFs), an order of magnitude below the threshold of elec- AGP troporation, can induce transient BBBd non-invasively. Here, we treated mice with 25-400 pulses (100-300V, elec- OR-20 trode gap 1.2-1.3 cm, 50µs pulses at 4Hz) using 2 plate Microsecond electric pulses effects on induced electrodes pressed against the skull. BBBd was studied neuronal stem cells for regeneration of spinal cord by MRI-based BBB maps developed uniquely for L-PEFs, injuries and by quantifying the accumulation of different molecu- Giorgia G. Innamorati 1, Caterina Merla1, Leslie Vallet2, lar weight therapeutics to the brain. Marina M. Sanchez Petidier2, Franck M. André2, Laura L. MRI studies: Contrast agent (Gd-Dota) was administered Caramazza3, Sara S. Fontana3, Noemi N. Dolciotti3, Maria M. to the tail vein 5 min to 4 hours post L-PEFs. BBBd Pedraza4, Nesus N. Torres4, Victoria Moreno Manzano4, Bar- bara B. Benassi1, Paola P. Giardullo1, Francesca Apollonio3, volumes and intensities were calculated using BBB maps, Paolo Marracino5, Lluis M. Mir2, Claudia Consales1 based on delayed-contrast MRI. A pixel by pixel ana- 1ENEA, Division of Health Protection Technologies, Italy lysis of the contrast intensity was used to evaluate BBBd 2CNRS, Gustave Roussy, Université Paris-Saclay, Metabolic and the volume and intensity of pixels above a certain and Systemic aspects of the oncogenesis (METSY), France threshold were calculated based on a 3 exponential model. 3Sapienza University, DIET, BioEMLab Group, Italy Correlation between the results and a finite elements sim- 4CIPF, Neuronal and Tissue Regeneration Laboratory, Spain ulation of the electric fields distribution in the brain was 5Rise Technology S.r.L, Italy studied. BBBd was depicted in the BBB maps Following 100V/100 Spinal cord injury (SCI) is a neurological and patho- pulses. The simulations showed that the electric field in logical state characterized by motor, sensory and auto- the disrupted volume was 28-65 V/cm, significantly below nomic dysfunctions, which affects around 500,000 people known EP threshold. A significant correlation was found every year. So far, several therapeutic strategies based between BBBd volume and the number and amplitude also on stem cell transplantation have been studied, but of the pulses (r2=0.96/0.91 respectively). BBB fully re- the results are poor and an effective protocol for spinal covered at 1h for 100V and 4hrs at 400V. cord regeneration is still missing. In order to evaluate drug accumulation in brain tissues, RISEUP project (fully described in another abstract sub- Doxorubicin (580Da), a biologic drug (ICTK, 50 kDa), mitted to WC2022), funded by the European community and IgG (150 KDa) were administered to the tail vein in the H2020 FET-OPEN program, provides for the de-of sham and L-PEFs-treated (200V/100 pulses) mice in velopment of an innovative system for the regeneration 3 separate experiments, and were allowed to circulate for of SCI based on the transplantation and the microsecond 4hrs. The mice were then perfused and the brains were (µsec) electric pulses stimulation, through a biocompat- removed and processed for drug concentration. Doxoru- ible, biodegradable, and electrified support, of mesen- bicin was detected by a fluorimeter, ICTK was detected chymal (MSCs) and induced neuronal (iNSCs) stem cells. 99 by ELISA and IgG was detected by immunofluorescence activities. Therefore, the revaluation of this by-product staining of brain slices. would be of great importance for the food industry. For Drug accumulation studies showed increased concentra- this reason, this study focuses on establishing the optimal tions of all tested drugs in the brain. Doxorubicin concen-conditions for a technology based on the use of pulsed elec- tration, 4 hours post L-PEFs, was 690±259 nM, which is tric fields (PEF) as a pretreatment in the olive leaf in or- X431 its IC50 in GL261 glioma cells, while undetected in der to obtain an extract enriched in phenolic compounds, the sham group. ICTK showed x13 increase (p<0.0001) an objective framed in the Project European SHEALTHY compared to sham. IgG staining revealed significant extra- (https://www.shealthy.eu/project/). For this, a Box- vasation into brain tissue while undetected in sham mice Behnken design of 15 experiments with 3 independent brains. factors has been carried out: frequency (Hz), treatment Our results demonstrate the feasibility of applying L- time (s) and field energy (kv/cm). In each experiment, the PEFs, non-invasively, for inducing transient and safe phenolic compounds were extracted using ultrasonic tech- BBBd in a mouse model. We further showed that this nology. The response variables were the content of total rapid treatment provides efficient delivery of small/large phenolic compounds, and the two major compounds, hy- therapeutics into the brain, thus leading the way to effi- droxytyrosol and oleuropein, measured by HPLC-MS. The cient treatment of various CNS disorders. validity of the experimental design was confirmed by AN- OVA and the optimal conditions were established by using the response surface methodology. The optimal conditions P5 - Mechanisms and Applications of by CEP were 0.6 kv/cm at 110 Hz for 11 seconds to obtain PEF in the Food Industry the maximum content of total phenolic compounds, which was 24 mg/g of dry leaf. The final data confirmed that the Tuesday late afternoon Track D treatment with CEP under these optimal conditions has Oct 11, 16:00 - 17:30 proven to be an effective pretreatment in improving the extraction of phenolic compounds in olive leaves, prob- OR-89 ably due to the increase in the permeability of the cell The role of post-electroporation recovery on the membrane. survival of Thai basil leaves during drying Grant Thamkaew, Lars Wadsö, Allan G. Rasmusson, Fed- OR-212 erico Gómez Galindo Suitability of different Electrode Materials for Lund University, Sweden Pulsed Electric Field (PEF) Application Milena Zdravkovic 1, Eva Kancirova1, Artur Wiktor2, Ute Horticultural crops have a low tolerance to dehydra- Bindrich1, Andreas Juadjur1, Volker Heinz1, Kemal Aganovic1 tion. In this paper, we show that the reversible electro- 1German Institute of Food Technologies (DIL e.V.), Germany poration of Thai basil leaves followed by 24 h resting before 2Warsaw University of Life Sciences, Institute of Food Sciences, hot air drying at 40 °C enhanced the survivability of the Poland tissues at certain levels of dehydration (moisture ratio = 0.2 and 0.1). However, this increased survival was rather Pulsed Electric Fields (PEF) is industrially used tech- limited. Through measurements of metabolic heat produc- nology for preservation of liquid products. Due to the dir- tion during resting, rehydration kinetics, respiration and ect contact of electrode surface and products, safety and photosynthesis of the rehydrated leaves, we show that rest- quality concers related to metal particle migration have ing after the application of a reversible pulse-electric field been raised in case of stainless steel. This resulted in com- (PEF) may allow a phase of hardening that has a protect- mon use of titanium electrodes, recently also regarded as ive effect on the cells, thus decreasing damage during the critical by EFSA. In this study suitability of six different subsequent drying phase. Increased preservation of cell electrode materials for PEF application was assessed. The vitality would be associated with a more turgid and fresh- selected materials were: titanium, tantalum, super duplex like rehydrated product, as cells would have the capacity stainless steel, silicon doped with phosphorus, graphite, to retain the rehydration water. and glassy carbon. The main criteria for the selection of the materials were: electrical conductivity, electrical res- OR-90 istance, corrosion resistance, and possible toxicity. Evaluation of the Extraction Yield of Phenolic The continuous parallel plate PEF chamber for liquid ap- Compounds in Olive Leaf Treated with Pulsed plication with electrode distance of 10 mm was built, de- Electric Fields signed to be operated in a pilot-scale machine (PEFPi- María del Carmen Razola-Díaz 1, Robert Sevenich2, Ana lot™ Dual, Elea GmbH, Germany). All electrode pairs María Gómez-Caravaca1, Oliver Schlüter2, Vito Verardo1 were tested for a period of 40 hours (5 cycles of 8 hours), 1University of Granada, Spain at electric field strength of 15 kV/cm, specific energy in- 2Leibniz-Institut für Agrartechnik und Bioökonomie e.V. put of 120 kJ/kg, and 2 bar backpressure. The pulse form (ATB), Germany was rectangular with width of bipolar pulses set to 7 µs. The electrode behaviour was tested in a model solution The olive leaf is one of the main by-products from the (pH=4, 20L) recirculated continuously at a flow rate of olive oil industry. This by-product is a rich source of phen- 80 L/h. The model solution was changed between cycles, olic compounds that have been shown to have beneficial meaning that each cycle had 32 PEF runs. After each health activities, which are due in part to their antioxidant 100 cycle concentration of eroded material in the solution was and chemical analyses (ascorbic acid, polyphenols, antiox-determined by inductively coupled plasma – optic emission idant compounds and anthocyanin content). spectroscopy (ICP-OES), while electrode surface changes Results showed that the PEF treatment positively af-were examined by scanning electron microscopy (SEM) fected the physical characteristics of the samples better and energy-dispersive X-ray spectroscopy (EDX) after 8 preserving the original shape of the fresh fruits and ve- and 40 hours of operation. getables. Compared to the untreated samples, a signific- The utilization of titanium electrodes resulted in the low- ant reduction of the shrinkage phenomenon for both, bell est migration rate of metal into the medium. During the peppers and strawberries, was detected by a reduction of first cycle, around 7.14 µ g/L of titanium was released per the volume loss of 30% and 50%, respectively. Pre-treated PEF run. The titanium concentration seemed to increase samples showed a lower firmness and increased rehydration with increase in electrode operation time, being 17.05 capacity. Moreover, PEF treatment does not appear to µ g/L per PEF run in the fourth PEF cycle. A similar have effects on the analyzed nutritional compounds com- tendency was observed in stainless steel, with release of pared to untreated samples. Fe, Cr and Mo. On contrary, the concentration of silicon The improved shrinkage behavior of the PEF treated ve- was 67.86 µ g/L after the first and 55.80 µ g/L after the getables could represent an important feature for market-fifth cycle per PEF run. According to the obtained results ing purposes as, due to the less volume losses, they resulted graphite and tantalum seem unsuitable for PEF applica- in more similar to the corresponding fresh products and tion in the tested set-up. likely more desirable for customers. Overall, the results The changes on electrode surface observed under micro- of this study suggest that PEF could be an effective pre- scope were visible for all tested electrode materials, with treatment to improve the freeze-drying process and the exception of glassy carbon after the first cycle. The final quality of freeze-dried fruits and vegetables. changes of cathode surface compared to anode surface were more pronounced in case of titanium and stainless OR-91 steel, where already after 8 hours of operation the surface Electrically conductive biocomposite film for in- of electrode was completely altered. The surfaces of both pack pulsed electric field food sterilization silicon electrodes were altered after 8 hours, while more Ana Barra 1, Cláudia Nunes1, Eduardo Ruiz-Hitzky2, Paula pronounced changes on the surface of glassy carbon anode Ferreira1 were observed when compared to cathode. 1CICECO - University of Aveiro, Portugal The obtained results indicate that electrodes made from 2ICMM – Instituto de Ciencia de Materiales de Madrid, CSIC, silicon doped with phosphorus or glassy carbon could be a Spain possible alternative for titanium electrodes currently used Pulsed electric field (PEF) is an emerging non for PEF application in food and other similar areas. thermal food sterilization technology. The preservation of nutritional and organoleptic food properties turns OR-95 this technology very attractive to extend the shelf life of Physical and chemical characterization of freeze- minimally processed food. The microbial inactivation is dried strawberries and red bell peppers pretreated achieved through electroporation, due to the application by pulsed electric fields (PEF) of high voltage pulses to food during microseconds. Marianna Giancaterino, Henry Jaeger Currently, food is sterilized inside a treatment chamber University of Natural Resources and Life Sciences (BOKU), before packaging. However, this process has a risk of Austria recontamination, that is preventing the industrial PEF Freeze-dried is considered a gentle drying process and implementation.[1] The in-pack food sterilization would it is largely used for the production of foods such as dried overcome this obstacle. Nonetheless, suitable electric- soups, dried snacks, breakfast cereals and cereal bars. The ally conductive food packaging materials are lacking. freeze-drying process better maintains the physical charac- In this context, electrically conductive biocomposite teristics and the nutritional and sensory properties of the materials might be a customizable and sustainable solu- raw materials compared to hot drying. However, during tion.[2] Herein, biocomposite films were prepared with the freeze-drying, phenomena as collapse and shrinkage alginate/zein biopolymers and carbon-clay electrically are likely to happen in sugar-rich plant materials. conductive fillers. A caramel-sepiolite nanocomposite was In this study, the effects on chemical and physical proper- graphitized at 550/800 ºC under N2 flow during 1 h.[3] ties of pulsed electric fields (PEF) pre-treated freeze-dried Multiwalled carbon nanotubes were used to improve the strawberries and red bell peppers was investigated. PEF electrical conductivity. The fillers were characterized by treatments at fixed electric field strength and frequency X-ray Diffraction (XRD), Raman spectroscopy, Solid (E= 1.0 kV/cm and f = 1 Hz) were performed. Variable State 13C Nuclear Magnetic Resonance (NMR), and number of pulses (20, 50, 100 and 200) and thereby en-Scanning Electron Microscopy (SEM). The XRD revealed ergy inputs between 0.3 and 6.0 kJ/kg on fresh products the maintenance of sepiolite structure during pyrolysis have been applied. The effects on the process efficiency at 550 ºC and Raman and 13C NMR confirmed the and the quality of freeze-dried fruits and vegetables were conversion of caramel into a graphitic material. The SEM evaluated by cell disintegration index, freezing kinetics, showed a homogeneous dispersion of sepiolite clay and freeze-drying kinetics, shrinkage measurement, rehydra- caramel. The filler with the highest electrical conductivity tion capacity, mechanical properties, color determination was dispersed into the alginate/zein matrix and the films 101 obtained by solvent casting. The film containing 70 involved in the extraction process, made of a PEF pre- wt% filler reached a maximum electrical conductivity of treatment followed by a subsequent SLE, applied on grape 329 and 6 µS/cm in-plane and through-plane directions, pomace. respectively. However, the mechanical resistance of films Therefore this study was focused on the optimization decreased in comparison with the control due to the high of the PEF-assisted extraction process using a central loads of carbon-clay filler. The electrically conductive composite design for response surface methodology from films will be used as food packaging electrodes to validate response surface methodology (RSM) with the aim to the in-pack PEF sterilization concept. sustainably intensify the extractability of phenolic com- pounds from red grape pomace. The cell disintegration Acknowledgements: index (Zp) was used as a response variable to identify the This work was developed within the scope of optimal PEF pre-treatment conditions of grape pomace the project CICECO - Aveiro Institute of Ma- in terms of field strength (E = 0.5–5 kV/cm) and energy terials, UIDB/50011/2020, UIDP/50011/2020 & input (WT = 1–20 kJ/kg), to be applied prior to the sub- LA/P/0006/2020, financed by national funds through the sequent solid-liquid extraction (SLE) process. For both FCT/MEC (PIDDAC). AB and PF are thankful to FCT untreated and PEF-treated samples, SLE was optimized for grant SFRH/BD/148856/2019 and the Investigator to determine the most effective combination of extraction FCT (IF/00300/2015), respectively. CN is grateful to temperature (20–50 °C), extraction time (30–300 min), Portuguese national funds (OE) through FCT IP in the and solvent concentration (0–100% ethanol in water). scope of the framework contract foreseen in the numbers Total phenolic content, flavonoid content, antioxidant 4, 5, and 6 of the article 23 of the Decree-Law 57/2016 activity, anthocyanin content, and tannin content of the of August 29 changed by Law 57/2017 of July 19. ER-H red grape pomace extract, were determined. The extrac- acknowledges financial support from PID2019-105479RB- ted compounds from untreated and PEF-treated samples I00 (AEI, Spain) project. at the optimal conditions were analyzed via HPLC-PDA analysis. References: Results revealed that under the application of the optim- [1] R. N. Arshad, et al., Trends Food Sci. Technol. 2021, ized PEF-assisted extraction conditions the extracts ob- 111, 43. tained from PEF-treated red grape pomace showed higher [2] A. Barra, et al., Compos. Sci. Technol. 2019, 173, 53. TPC (6%), FC (25%), FRAP (12%), DPPH (14%), TAC [3] C. Ruiz-García, et al., Phys. Chem. Chem. Phys. (54%), and TC (15%), as compared to the control extrac-2013, 15, 18635. tion. HPLC analyses revealed that epicatechin was one of the main phenolic compounds extracted, and no degrada- OR-120 tion phenomena occurred due to PEF application. Optimization through Response Surface Methodo- These results strengthen the feasibility of the innovative logy of Pulsed Electric Fields-Assisted Extraction approach investigated in supporting the valorization and of bioactive compounds from red grape pomace potential reutilization of grape processing by-products in Serena Carpentieri 1, Giovanna Ferrari2, Gianpiero Pataro1 the food industry. 1University of Salerno, Department of Industrial Engineering, Italy 2University of Salerno, Department of Industrial Engineering P16 - New Technologies for Cells and and ProdAl S.c.a.r.l., Italy Tissues Electropermeabilization Grape pomace is the major by-product generated dur- ing the winemaking process, with a production of 20 kg Wednesday morning Track A for each hectoliter of wine. The recovery of high-added Oct 12, 10:30 - 12:15 value intracellular compounds from grape pomace might represent a strategy to improve the sustainability of wine OR-132 production and promote economic opportunities. How- (Elongated) gold nanoparticles: injectable anten-ever, in order to weaken the cell membrane, which consti- nas locally amplifying the electroporation or just tutes a physical barrier that hinders the recovery of these a thorn in our flesh? compounds from plant residues via conventional solvent Jelena Kološnjaj-Tabi, Muriel Golzio, Marie-Pierre Rols extraction (SLE), an efficient permeabilization of the cell CNRS, Institut de Pharmacologie et Biologie Structurale envelopes should be necessary. (IPBS), France Pulsed electric fields (PEF) is gaining great interest as a Gold nanoparticles have been described as promising mild and easily scalable cell disruption technique, enhan- materials, which could locally enhance the electric field cing the extractability of intracellular compounds from via the “lighting rod effect”. While theory predicts that various plant matrices, with reduced solvent, time, and individualized elongated conducting nanoparticles with energy consumption. an aspect ratio (length/width) of about 15.7 could locally However, to maximize the benefits of PEF-assisted extrac- enhance the electric field up to 100 times [1], experimental tion over the SLE, an optimization step of the whole PEF- results obtained by our colleagues and us indicate this assisted extraction process should be carried out. To date, goal still remains elusive. few studies addressed the optimization of all the factors In order to master gold nanoparticles behavior around 102 and within living cells, we have to understand their such as spheroids produced with several types of cells, behavior in space and time. Upon administration to including fibroblasts. We believe that the microsystem cells, gold nanoparticles quickly undergo aggregation and we recently developed [2], enabling culture, monitoring cellular confinement, hampering nanoparticles potential and electroporation (EPN) of a large number of spheroids for electroporation enhancement. sharing similar characteristics will be a key asset to ana- As soon as gold nanoparticles meet proteins, either in lyze the effect of a dense fibrotic TME on ECT efficiency, cells or within (bodily) fluids, their surface structure and using pancreatic cancer as a pathological model. properties rapidly change. Conversely, their gold-based A 2% agarose hydrogel is molded to form microwells [3] core is rather inert and can persist in vivo over years and and bonded to an ITO coated glass slide, corresponding to decades. an electrode for EPN. Spheroids are obtained by seeding In this work, we present gold nanoparticles potential for PANC-1 pancreatic cancer cells with MRC-5 fibroblasts electroporation enhancement, focusing on theory and (ratio 2:1) transduced using NucLightTM green lentivirus, practice, summarizing the current state of the art. In the supplemented with 0.1 mg/mL of type I collagen. To prospect of exploiting the potential of gold nanoparticles perform EPN, another electrode is placed 1 mm above for electropermeabilization enhancement, we herein the one with the hydrogel (Figure 1c) with tubing for present gold nanoparticles behavior in vivo over a period the injection of low conductive EPN buffer (300 µS/cm). of one year [2], and we provide an overview of gold’s fate This buffer will be supplemented with a standard drug in humans, treated with ionic gold salts [3]. These salts, for pancreatic cancer treatment, gemcitabine, for ECT used in the eighties to treat rheumatoid arthritis, have tests. EPN is performed by applying 2 sine wave bursts been shown to locally generate gold nanoparticles and (10 kHz, 600 V/cm, 5 ms) whose relevance has recently elongated gold nanoparticles microstructures. been pointed out [4]. While we do not provide the miraculous recipe for gold The developed microsystem allows obtaining cohesive nanoparticles application in electroporation, we provide co-cultured spheroids after 3 days. As fibroblasts appear a general review of the (lack of) knowledge in the use in green, it will enable to differentiate them from cancer of gold-based particular and molecular compounds for cells for further immunostaining analysis. First tests have future studies and potential field development. been performed to determine the EPN threshold using propidium iodide (PI, 15 µM) to label electroporated [1] Lekner, J. (2014). Electroporation in cancer therapy cells and then fluorescein di-acetate to label living cells. without insertion of electrodes. Physics in Medicine & They demonstrated an efficient and reversible EPN for Biology, 59(20), 6031. 600 V/cm. [2] Kolosnjaj-Tabi, J., Javed, Y., Lartigue, et al (2015). We plan to perform ECT test and analyze its effect on The one year fate of iron oxide coated gold nanoparticles the produced co-cultured spheroids thanks global growth in mice. ACS nano, 9(8), 7925-7939. follow-up and in-situ proliferation analysis, revealed by [3] Balfourier, A., Kolosnjaj-Tabi, J., Luciani, N., Carn, confocal microscopy. We will compare these results to F., & Gazeau, F. (2020). Gold-based therapy: From those obtained with spheroids made of cancer cells only. past to present. Proceedings of the National Academy of It will enable to evaluate the effect of TME on ECT Sciences, 117(37), 22639-22648. efficiency, which presence will be studied by labeling collagen in the co-cultured spheroids. We also intend to OR-137 optimize the microsystem design to enable the monitoring Application of a new electroporation microsystem of spheroid growth with bio-impedance measurement. to the study of the impact of tumor microenviron- ment on electrochemotherapy efficiency [1] R. Sarathi, et al., in: 2014 IEEE Conf. Electr. Pauline Bregigeon 1, Théo Le Berre1, Julien Marchalot1, Insul. Dielectr. Phenom. CEIDP, 2014, pp. 232–234. Laure Franqueville1, Christian Vollaire1, Charlotte Riviere2, [2] P. Bregigeon, et al., Lab Chip (2022) DOI: Marie Frénéa Robin1, Fréderic Prat3 10.1039/d2lc00074a. 1Univ Lyon, Ecole Centrale de Lyon, INSA Lyon, Université [3] C. Rivière, et al., Plaque de Micropuits En Hydrogel Claude Bernard Lyon 1, CNRS, Ampère, UMR5005, France Biocompatible, Patent FR3079524A1. 2Institut Lumière Matière, Claude Bernard Lyon 1 Université, [4] T. García-Sánchez, et al., Biochim. Biophys. Acta CNRS, France BBA - Biomembr. 1860 (2018) 1022–1034. 3University Paris-Cité, INSERM U1016 and AP-HP, Paris, France, France OR-133 Pancreatic cancer has a very poor prognosis, with a Spatially resolved, high efficiency electrotransfec- percentage of survival inferior to 7% at 5 years [1]. Its tion on a CMOS microelectrode array tumor microenvironment (TME) presents a high density Bastien Duckert, Dries Braeken, Liesbet Lagae, Maarten of fibrotic tissues, inducing a low penetration of drugs in Fauvart cells. Electrochemotherapy (ECT) appears as a promising IMEC, Belgium treatment to enhance anticancer drug efficiency, and Electroporation faces the same hurdles typically more investigations are needed to improve ECT protocols encountered by any intracellular delivery technology: and drug selection. However, there are only a few studies higher transfection efficiencies are often achieved by comparing the effect of ECT on relevant 3D cell models submitting cells to more aggressive treatments, which in 103 turn results in lower cell viability. In this work, we take Here, we report the fabrication and operation of a advantage of a CMOS microelectrode array chip (MEA) flow-through electroporation system, consisting of a mi- [1] to perform spatially resolved electroporation of the crofluidic channel along with coincident pulsing electrodes cells grown on its surface. Due to the limited cellular area placed along the channel. As a cell passes between an elec- affected, electroporation with microelectrodes inherently trode set, the applied field is controlled by the electrodes has low toxicity and a large range of pulse parameters bias and spacing, while the pulse length is determined by can be modulated without compromising cell viability. the cell velocity and electrode width. Two sets of elec-Thanks to the ability to test different electroporation trodes are used to perform dual-pulse High voltage/Low conditions on different areas of the MEA, large screening voltage (HV/LV) electroporation. The HV electrodes are experiments designed to yield optimal electroporation narrow and biased to allow a short permeabilizing pulse parameters can be performed in parallel. We used this while the LV electrodes are wider to enable longer term method to define parameters that maximize the delivery molecular transport into the permeabilized cells. Voltage efficiency of an mCherry-encoding mRNA in primary is applied as a pulse train at high frequencies in order to fibroblasts while keeping cell toxicity to a minimum. avoid solution electrolysis seen with continuous DC pulses. After electroporation with those optimized conditions, This system is able to electroporate single cells in a expression of the fluorescent protein could be detected serial fashion at a throughput of >200 cells/sec. HEK293 after one hour and, six hours after electroporation, cells, hydrodynamically focused in the middle of the chan- successful transfection was observed on almost all the nel, were successfully transfected by delivering a GFP en- electrodes used to electroporate the cells. coding plasmid DNA via electroporation. The number of By sequentially introducing different fluorescent mo- pulses experienced by a cell were increased from 22 to 113 lecules or nucleic acids in the extracellular medium, and pulses by reducing the cell velocity (increasing residence electroporating cells at different locations on the MEA, time), showing a linear increase in eTE from 30% to 44% different patterns of cell fluorescence could be created. when applying a 40 V (E=216 kV/m) pulse train of 10 Our results showcase the promising applications that us pulses at 10 kHz, 10% duty cycle. Applying a dual electroporation on MEAs can serve, such as screening pulse scheme of 52 pulses at 40 V (E=216 kV/m) of 10 of large libraries of molecules (e.g., mRNA delivery in us at 10 kHz, 10% duty cycle followed by 340 pulses at dendritic cells) or creation of engineered tissues on chip 12.3 V (E= 66.5 kV/m) of 200 us at 1 kHz, 20% duty for drug screening applications. cycle, increased the eTE from 36% to 50%. The highest eTE achieved to date was 68 ± 9% with cell viabilities 1. Lopez, C. M. et al. A multimodal CMOS MEA approaching 100% up to 120 hours post-electroporation for high-throughput intracellular action potential meas- with voltages less than 40 V. Future work includes the urements and impedance spectroscopy in drug-screening utilization of impedance cytometry electrodes to electric- applications. IEEE J. Solid-State Circuits 53, 3076–3086 ally monitor the degree of cell membrane permeabilization (2018). following the application of the electroporation pulses, as well as the use of closely spaced interdigitated electrodes OR-136 using lower voltages to avoid solution electrolysis. The fabrication and operation of a continuous-flow microfluidic device for single-cell electroporation OR-134 Maria Atzampou 1, Hao Lin1, Jerry W. Shan1, David I. Localized single-cell electroporation using U Shreiber1, Christine Roberts2, Joel N. Maslow2, Jeffrey D. shaped microstructures in a microfluidic channel Zahn1 Aswin Muralidharan 1, Georg Pesch1, Hendrik Hubbe1, Lea 1Rutgers, The State University of New Jersey, United States Rems2, Mahdiyeh Nouri-Goushki1, Pouyan Boukany1 2GeneOne Life Science, Inc., Korea, Republic of 1Delft University of Technology, Netherlands 2 The ability to transform cells via the introduction of University of Ljubljana, Faculty of Electrical Engineering, genetic regulatory elements is now central to nearly every Slovenia aspect of biomedical research and development. Current Localized single-cell electroporation allows non-toxic, therapeutic innovations, such as CAR T-cell therapy, have controlled and efficient delivery of biomolecules to living traditionally relied on viral-mediated gene delivery. Al-cells. To obtain localized electroporation, it is required to though efficient, viral transformation techniques limit the apply the electric pulses through miniaturized structures. payload size within the viral coat, may have mutagen- Since the concentrated electric fields are localized only to esis risks, and have high manufacturing costs. These the vicinity of the miniaturized structures, the target cell drawbacks have renewed interest in alternative cell trans- must be attached to the miniaturized structures. This duction methods such as electroporation. Electropora- means single cells should be micro-manipulated to these tion is an efficient electro-physical, non-viral approach for structures by an external force (by optical or magnetic the intracellular delivery of exogenous materials. Micro- tweezers) or adhered to these structures (limiting to ad- scale electroporation is being investigated because it al- herent cell lines) for successful localized electroporation. lows lower pulse voltages, better control over the uniform- A major barrier to the routine operation of localized elec- ity of electric fields, along with the ability to monitor cell troporation in biological laboratories is the cumbersome membrane permeabilization in real-time, leading to higher manipulation and loading of single cells for drug and gene cell viability and electro-Transfection Efficiency (eTE). delivery. 104 To overcome this limitation, we fabricated a localized In this abstract, we focus on the so-called high-frequency single-cell electroporation chip consisting of a scalable hy- irreversible electroporation (H-FIRE). The adoption of H-drodynamic U-shaped micro-post array integrated into a FIRE in oncologic care is more and more frequent due to microfluidic channel. In our microfluidics device, the cells the reduction of heavy side effects as muscle and nerve are trapped in a microtrap array by a gentle flow, after contraction. which target molecules are supplied to the device and H-FIRE effectiveness is the result of a complex and long electrotransferred to the cells under electric pulses with process, involving numerical modeling defining the so- perfect cell viability. The system provides the ability to called treatment planning protocol. However, a large mar- monitor the electrotransfer of exogenous biomolecules in gin of uncertainty remains between prediction by treat- real-time. We reveal through numerical simulations that ment planning and the experiments, and the practice and localized electroporation is the mechanism of permeabil- experience of the surgeon is the best guarantee for the safe ization in the microtrap array electroporation device. We and effective outcome of the treatment. demonstrate the simplicity and accuracy of this microtrap As experimental results are already largely available in technology for electroporation by delivery of both small the current literature, we selected a number of data for molecule electrotransfer using propidium iodide and large the creation of a knowledge base and their preparation to molecule electrotransfer using plasmid DNA for gene ex- accurately train an ANN with a well-controlled and solid pression, illustrating the potential of this minimally in- methodology. Specifically, the focus is on two groups of vasive method to be widely used for precise intracellular target (output) parameters: 1) expected ablation area, delivery purposes (from bioprocess engineering to thera-and 2) the expected electrode placement. Then after fea- peutic applications). ture (input) definition and data assumption, two different artificial neuronal network typologies were defined. The OR-135 first one was tasked to estimate the ablation area given Smart, solid-state, nanosecond pulsed power tech- a specific experiment setup. The second one is aimed niques for medical, agro and environmental applicat determining the needed electrode configuration given ations a specific experiment setup and it involves the edge-to- Guus Pemen edge electrode distance, the electrode application depth, Eindhoven University of Technology, Netherlands and the electrode diameter as target parameters. Rapid developments in solid-state pulsed power tech- Both the two ANNs were fed with a dataset including nology offer opportunities to precisely adjust HV pulse 152 experiments (“Ablation ANN”) and 192 experiments shapes, even on a pulse-by-pulse basis. In this paper we (“Electrode ANN”), respectively. After algorithm iter-provide an overview of our research on solid-state, repet- ations, the residuals of the Ablation ANN are close to itive nanosecond pulsed power techniques. We are able to zero, thus indicating that the estimated ablation areas ”program”, set and adjust HV waveforms even during a are very reliable. Similarly, the residual for the Elec- pulse cycle. We will review several realized topologies, like trode ANN, which reports the estimation errors in [mm] our implementation of a solid-state impedance matched for the diameter, depth, and edge-to-edge distance of the Marx generator, and show the capabilities of these real-electrodes, shows that the majority of the residuals con- izations to generated flexible and adjustable high-voltage centrates around zero providing a successful and reliable waveshapes. estimation of these parameters. To conclude, the building up of a complete, high quality OR-66 and robust knowledge base, combined with an adequate Optimization of Ablation Region and Electrode design strategy of ANN, led to the identification of the Positioning in H-FIRE via Machine Learning optimum choice of important H-FIRE parameters, inter- Alfredo De Cillis1, Caterina Merla 2, Giuseppina Monti1, esting for a rapid optimization of future H-FIRE protocols. Luciano Tarricone1, Marco Zappatore1 1University of Salento, Engineering for Innovation Department, Italy P24 - Calcium Electroporation 2ENEA, Division of Health Protection Technologies, Division of Health Protection Technologies, Italy Wednesday morning Track B Authors recently demonstrated that the use of Machine Oct 12, 10:30 - 12:10 Learning (ML), and more specifically Artificial Neural Networks (ANN), is an appropriate way to improve the ef- OR-110 fectiveness of irreversible electroporation applied to cancer Phase II Investigation of the Histopathologic Ef- ablation. Indeed, modern ML techniques propose them- fect of Calcium Electroporation on Cancer in the selves as a smart strategy to identify optimized sets of Skin – CaEP-B parameters to be used in medical applications based on Mille Vissing, Sandra Kristiane Sinius Pouplier, Lars irreversible electroporation. Investigations based on em- Munch, Stine Frandsen, Anne-Vibeke Lænkholm, Julie Gehl pirical trials of the different involved parameters are the Zealand University Hospital, Denmark rule so far, but they cannot represent a sustainable ap- Background: Many patients with cancer may present proach in terms of costs and therapy effectiveness towards with cutaneous or subcutaneous tumours that can be diffi-extensive clinical applications. cult to manage with surgery, systemic treatments or radi- 105 ation. Calcium electroporation (EP) is a novel local treat-2Zealand University Hospital, Denmark ment using intratumoral calcium and pulsed electric fields, Background and aims: Barrett’s esophagus high-grade with the first clinical trials displaying safety and efficacy dysplasia (BE HGD) can transform into esophageal adeno- as well as limited side effects. Reports of long-term local carcinoma (EAC) in 19-28% of the cases. Once resected, a disease control and systemic responses following treatment shift in diagnosis from HGD to EAC happens in 21-38%. in the initial small-cohort trials imply a positive effect on Calcium electroporation combines a local injection of cal- cancer immunity caused by calcium EP-induced cell death. cium with electrical pulses to increase the individual cells’ This study investigates the histopathological effect of cal- permeability and flux of ions leading to necrosis. This cium EP and immune responses of the tumour microen- study presents the first results with calcium electropora- vironment , ClinicalTrials.gov Identifier: NCT04259658. tion in the esophagus. Materials and methods: This non-randomized phase II Methods: Six patients with BE HGD scheduled for an en- study will include 24 patients with cutaneous or subcu- doscopic submucosal dissection (ESD) were treated with taneous malignancy. Tumours are treated once and re- Ca-EP six weeks before the planned ESD. All side effects treated after one month with sequential biopsies taken at and adverse events (AEs) were registered, and the patients baseline and day 2, 7, 28, 30, 60 and 90 after initial treat- were later evaluated with gastroscopy. ment, depending on the number of included tumours. The Results: The observed adverse events included ret- primary endpoint is the change in proportion of tumour in- rosternal pain, throat irritation, coughing, and headache. filtrating lymphocytes two days after treatment compared No serious adverse events were observed. A hyperemic to before treatment. The samples will be analysed for area was observed in four patients after Ca-EP correspond- immune markers of the innate and adaptive immune sys- ing to treated areas. A fibrinous coating (three patients) tem as well as tumour necrosis, changes in vasculature and ulcers (four patients) were observed up to a week after and inflammation. Circulating tumour DNA from blood treatment. One patient had to undergo two CTA scans samples (baseline and day 28, 60, 90) will be analysed in a after treatment due to pain and a visually large fibrinous subgroup of patients . Secondary endpoints include clin- clot, which showed no perforation. ical response rate, PD-L1 expression of different tumour Conclusion: Ca-EP is safe and feasible in patients with BE histological subtypes and importance of previous irradi- HGD. This study paves the way for more extensive studies ation. Patients are followed up to 12 months. to investigate the effect on dysplasia cells and the safety Results: This study is still ongoing. Fourteen patients and feasibility of treating esophageal adenocarcinoma. with metastases of different tumour histology have been treated including breast cancer (n=11), lung cancer (n=1), OR-161 malignant melanoma (n=1) and bladder cancer (n=1) Effectiveness of calcium electroporation on human - the first bladder and lung cancer cases treated with sensitive and resistant breast cancer cells calcium EP. Both formalin-fixated, paraffin-embedded Jolanta Saczko, Anna Choromańska, Julita Kulbacka, samples (n=83) and frozen biopsies (n=21) have been ob- Katarzyna Bieżuńska-Kusiak tained. Preliminary results of HE-stained samples show Wroclaw Medical University , Department of Molecular and examples of tumour necrosis, residual tumour, inflammat-Cellular Biology, Poland ory reaction and change in tumour infiltrating lympho- cytes, mitosis rate, nuclear size and elastoid degeneration Breast cancer ranks among the top three most com- from sequential biopsies. The patient with bladder can- mon malignant neoplasms in Poland. The use of calcium cer metastases had progressive disease on immunotherapy, ion-assisted electroporation is an alternative approach to but clinically showed remission when re-treated with im-the classic treatment of this disease. The studies conduc- munotherapy after calcium EP treatment. ted in recent years confirm the effectiveness of electropor- Conclusion: This is the most comprehensive clinical study ation with calcium ions. They are signal transducers in to date investigating the effect of calcium electropora- many intracellular pathways, therefore, disturbance of in- tion on malignant tumours and their microenvironment. tracellular calcium ion homeostasis can lead the cell to The results of this trial may illuminate mechanisms un-death. Electroporation is a method that uses short elec- derlying this promising new treatment for cancer in the trical pulses that create transitional pores in the cell mem- skin across different tumour histological subtypes. Under- brane. They allow the penetration of certain drugs that standing changes in the tumour microenvironment could are not normally able to penetrate the cell membrane. uncover synergistic effects with the immune system and The aim of this study was to investigate the antitu- determine whether calcium EP has the potential to turn mor effect of electroporation alone and calcium ion- immunogenic “cold” tumours into “hot” tumours to bene- assisted electroporation on human mammary adenocar- fit targeted therapies. cinoma cells, sensitive (MCF-7/WT) and resistant to dox- orubicin (MCF-7/DOX). Cell viability was assessed using OR-160 independent tests: MTT and SRB. The consumption of Endoscopic calcium electroporation in patients the intracellular ATP pool was tested with the ATP-lite with Barrett’s esophagus high-grade dysplasia: A assay. The type of cell death after the applied therapy was first-in-man phase 1 study determined by TUNEL and flow cytometry (FACS) meth- Laser Arif Bazancir 1, Charlotte Egeland1, Rajendra Singh ods. The expression of α-1G and α-1H proteins was as-Garbyal1, Julie Gehl2, Michael Patrick Achiam1 sessed by immunocytochemistry, and changes in the mor- 1Rigshospitalet, Denmark phology of CaEP-treated cells were visualized using a holo- 106 tomographic microscope. Biopsies were analyzed for changes in CD3/CD8 as well The obtained results confirmed the effectiveness of the in- as PD-L1 and no clear trend of the density appeared. vestigated therapeutic method. The results of the work Blood sample analysis found no trend towards changes in constitute a good basis for planning research at the in vivo circulating cell free DNA level, and in the future, perhaps, to develop a more effect-Conclusion: This first study on calcium electroporation ive, safer, and more acceptable method of breast cancer for colorectal tumors shows that calcium electroporation treatment for patients. is a safe and feasible treatment modality in colorectal cancer. The treatment can be performed as an out-patient OR-162 treatment and may potentially be of great value for the Endoscopic Calcium Electroporation for fragile patient with limited treatment options. Colorectal Cancer: a phase I study Malene Broholm 1, Rasmus Vogelsang2, Mustafa Bulut2, Trine Stigaard2, Hanne Falk Hansen3, Stine Frandsen2, OR-163 Dorte Levin Pedersen2, Trine Perner2, Anne-Marie Kanstrup Impact of variety type of calcium electroporation Fiehn2, Andreas Weinberger Rosen2, Christina Andersen2, protocols on selected cellular attributes in human Niels Pallisgaard2, Ismail Gögenur2, Julie Gehl2 colon cancer 1Koege Hospital, Denmark Anna Szewczyk, Nina Rembiałkowska, Anna Choromańska, 2Zealand University Hospital, Denmark Katarzyna Bieżuńska-Kusiak, Jolanta Saczko, Julita Kulbacka 3Herlev Hospital, Denmark Wroclaw Medical University, Poland Background: Colorectal cancer is one of the most Electroporation is now commonly accepted as method common malignancies worldwide, with approximately using wide-range electric field pulses to rapidly increase 20% of the patients having an advanced disease. Local of transmembrane voltage and thereby rise of cell mem- symptoms from the tumor, such as pain, bleeding and brane conductance, which supported molecular transport stenosis, remains a common issue and affects quality of through cell membranes. According to type of pulses life. Electroporation is a method to permeabilize cell (duration, amplitude, number of pulses) reversible electro- membranes with high voltage pulses, allowing increased poration is divided into: 1) millisecond-, 2) microsecond-, passage of otherwise poorly or non-permeating substances 3) nanosecond-electroporation. The promising approach such as calcium (calcium electroporation). Recent studies gives combination of calcium ions with electroporation have shown that calcium electroporation efficiently (CaEP). The aim of our studies was evaluation the effects eliminates cancer cells. Additionally, studies show that of different type of PEF pulses combined with Ca2+ cancer cells are vulnerable to the treatment, whereas on cell structure and homeostasis. We tested different normal cells are more resistant to calcium electroporation. protocols of EP (µs- and nsPEF) +/- Ca2+ and observed The aim of this study is to determine safety of calcium its impact on cellular stress response, tumour biomarker electroporation for advanced colorectal cancer. expression and viability for colon cancer cell lines. Method: This exploratory phase 1 study investigated The LoVo (sensitive human colorectal adenocarcinoma safety of endoscopic calcium electroporation for colorectal cell line) and LoVoDX (doxorubicin-resistant human cancer. The study included six patients with inoperable colorectal adenocarcinoma cell line) were tested. As con- rectal and sigmoid colon cancer, all presenting with local trol the Hs738st/int (normal human intestine fibroblast) symptoms from the primary tumor. Patients were offered were used. The following CaEP protocols were used: (1) endoscopic calcium electroporation and were followed µs: 1.2 kV/cm; (2) ns: 37.5-50 kV/cm; +/- 2nm of Ca2+. with follow-up endoscopy and CT/MR scans. Additional The YO-PRO-1 uptake was assessed by flow cytometry. treatments were performed when appropriate. Secondar- The viability was measured by MTS assay and ROS level ily, biopsies and blood samples were collected before and analyzed by bioluminescent assay. The expression of after treatment. Biopsies were examined for histological aspartate- β-hydroxylase (ASPH) protein and heat shock changes and CD3/CD8 and PD-L1. Additionally, blood proteins (HSPs) were visualized using confocal laser samples were examined for circulating cell free DNA. scanning microscope (CLSM). The expression of CD133 Results: A total of ten procedures were performed, and marker was evaluated by immunocytochemistry. no serious adverse events occurred. All treatments were The YO-PRO-1 uptake depended on protocols paramet-performed as an out-patient procedure, and patients ers. MTS assay shown a viability decrease of malignant were discharged within one hour after treatment. Prior cell caused by µs- and nsCaEP. Normal cells were sensit- to inclusion, patients reported local symptoms, such as ive only to µsCaEP parameters. We obtained the most bleeding, pain, and stenosis. After treatment, five of six promising survival ratio between normal and malignant patients reported symptom relief within a few days after cells for nsCaEP (2 mM of Ca2+ with 50 kV/cm). treatment. Moreover, in malignant cells the significant release of In one patient, calcium electroporation was offered ROS after nsCaEP was detected and this was linked with followed by systemic chemotherapy. Complete visual increase of HSP 27 and HSP 70 expression. ASPH signal response of primary tumor was seen. Follow-up biopsies was clearly observed in untreated LoVoDX and LoVo cells after 12 weeks showed granulation tissue and ulcerous what is correlated with the aggressive tumorigenesis. The tissue and no adenocarcinoma was described. In addition, ASPH signal decreased after each of CaEP parameters. complete response of lung- and partial response of liver The opposite effect occurred in Hs378st.int cells which metastasis. 107 presented a low level of ASPH in untreated cells and it Conclusions. The application of nsPEF with calcium was higher after the nsCaEP application. Expression of ions seems to be an auspicious method for efficacious CD133 is different between normal and malignant cells cancer elimination and significantly facilitates the action as well as is modulated by CaEP. The obtained results of conventional cytotoxic pharmacological agents. confirmed that nsCaEP originates significant reduction Financing: This work was supported by the Pol- of colorectal cells viability, increases ROS/HSPs and ish National Science Centre project SONATA BIS 6 reduces ASPH signal that possibly is associated with cell (2016/22/E/NZ5/00671). overloaded by calcium influx and cell death. References: Financing: This work was supported by the Pol- [1] S.T. Pan, et al. Molecular mechanisms for tumour ish National Science Centre project SONATA BIS 6 resistance to chemotherapy, Clin. Exp. Pharmacol. (2016/22/E/NZ5/00671, PI: J. Kulbacka). Physiol. (2016), 43: 723-737 [2] E.P. Spugnini, et al. Definition of novel electro- OR-164 chemotherapy parameters and validation of their in vitro Antitumor effects of nanosecond PEFs with cal- and in vivo effectiveness, J. Cell. Physiol. 229 (2014) cium ions in colon cancer in vitro and in vivo 1177–1181. Julita Kulbacka 1, Joanna Rossowska2, Agnieszka [3] W. Szlasa et al. Oxidative Effects during Irreversible Chwikowska1, Anna Choromańska1, Zofia Łapińska1, Electroporation of Melanoma Cells—In Vitro Study, Nina Rembiałkowska1 Molecules. 26 (2020) 154. 1Wroclaw Medical University, Department of Molecular and Cellular Biology, Poland 2Hirszfeld Institute of Immunology and Experimental Therapy, P30 - Cancer Immunotherapy and Polish Academy of Sciences, Poland Pulsed Electric Fields (PEF) Background. Colon cancers are still poorly diagnosed; thus, their therapy does not have satisfactory outcomes. Wednesday morning Track C The main problem is the primary and acquired resistance, Oct 12, 10:30 - 12:10 characteristic of these types of cancers [1]. As chemother- apy is not as effective alone, pulsed electric fields (PEFs) OR-144 seem to support conventional anticancer methods. Des- Local intratumoral electroporation delivery of po- pite enhanced drug delivery, varied parameters of PEF tent anti-cancer interleukin 12 immunotherapy such as duration and number of pulses and the intensity leads to systemic anti-cancer response of electric field can be responsible for regulating specific Adil Daud 1, Alain Algazi1, David A. Canton2, Mia Han2, cellular responses [2]. NsPEF may induce oxidative stress Bridget O’Keeffe2, Brandon Phung2, Jeffrey Silverman2 in cells by stimulating ROS production and disrupting the 1University of California, United States balance of oxygenases, and antioxidant enzymes, which in 2Oncosec Medical Incorporated, United States turn cause cell damage with increased oxidative markers in membrane lipid peroxidation [3]. Systemic administration of potent anti-tumor immuno- Material and methods. In this study, we have used cancer therapies is invariably associated with undesirable rates of cell lines (LoVo, LoVoDX, MC38/D1) and murine models. treatment-related adverse events. Optimizing efficacy of For the electroporation process, 200 pulses of 10ns and treatment approaches while reducing severity of adverse high voltage (12.5 – 50 kV/cm) were used. In the case of events has become the primary focus in development of in vivo experiments, 400 and 1200 pulses were applied. novel anti-cancer therapies and combination treatment ap- The efficacy of nsPEFs protocols with and w/o calcium proaches. Intratumoral delivery of expression plasmids by ions was determined by clonogenic and MTT assay. The electroporation (EP) represents a promising local treat- cell response was validated by analyzing morphology ment approach to reduce toxicities observed with systemic by SEM, AFM, and confocal microscopy. Proteasomal treatments. OncoSec Medical System (OMS) pairs a gen- activity, GSH/GSSG assay, and ROS production were erator and an applicator to induce localized expression of evaluated as markers of oxidative stress and protein recombinant proteins by EP in palpable tumor lesions. damage. Finally, an animal model (C57BL/6 mice) was Specifically, intratumoral delivery of interleukin 12 (ILused in the study. NsPEF with CaCl2 or bleomycin as 12) tavokinogene telseplasmid (tavo) has demonstrated an ECT standard was performed. The observations were clinical utility to safely treat cancer patients across solid carried up for four weeks. tumor disease states. IL-12 is a pleiotropic cytokine that Results. Our results revealed that PEFs protocols signi- has been extensively studied as a potential cancer immun- ficantly reduced cell viability, particularly with calcium otherapy candidate due to its ability to engage multiple ions. We have observed an increase in oxidative stress immune effectors and reverse tumor-induced immunosup- markers in colon cancer cells. NsPEF application in pression. However, systemic administration of IL-12 has colon cancer in vivo, caused tumor size to decrease and proven to be exceedingly toxic in clinical trials, ultimately stimulated an increase in heat shock proteins (HSP27, limiting its clinical utility. Intratumoral delivery of tavo HSP40, and HSP70) expression. Moreover, ns pulses via OMS EP resulted in local and systemic anti-tumor stimulated the growth of CD45+ cells in tumor tissue. effects while inducing only minimal treatment-related ad- verse events in multiple clinical trials. Here, we summar- 108 ize findings from two of our trials designed to evaluate in combination with resiquimod (50ug; i.t.) was the op-a novel anti-tumor treatment combination of pembroli- timal treatment regimen for both eliminating a primary zumab (immune checkpoint inhibitor) and intratumoral Pan02 tumor (93%) as well as inhibiting growth of a Pan02 tavo-EP.KEYNOTE-695 study is a single-arm, phase 2, cell rechallenge tumor (60% TGI). In addition, when we open-label, multicenter study of tavo-EP plus pembroli- depleted the CD8+ T cells with an anti-CD8 antibody de-zumab in patients with unresectable or metastatic melan- livered in vivo before rechallenge, it reduced inhibition of oma progressing on standard of care immune checkpoint rechallenge tumor growth by 35% (NPS™ (180 mJ/mm3) inhibitor(s). Patients had a 27.8% objective response rate + resiquimod + aCD8ab = 25% TGI). After assessing ef- (ORR, n=54), with 47% of responding patients having ficacy, we analyzed rechallenge tumors for the presence of durable response lasting over 1 year. Median overall sur-tumor infiltrating CD8+ T cells using immunohistochem- vival (OS) was 23.5 months (n=56). Grade 3 treatment- istry. We found that rechallenge tumors in mice that re-related adverse events (TRAEs) were reported for 6.7% ceived the NPS™ (180 mJ/mm3) + resiquimod treatment patients. No grade 4 or 5 TRAEs were reported. had, on average, 6X (avg.=219) the number of CD8+ T KEYNOTE-890 study cohort 1 is a phase 2, open-label, cells than in tumors rechallenged after surgical resection of multicenter study assessed the safety and efficacy of tavo- the primary tumor (avg.=27) and 3X more than in tumors EP in combination with pembrolizumab as 2L+ treatment whose efficacy was depleted using an anti-CD8 antibody for advanced triple negative breast cancer (TNBC). Pa- (avg.=67). This number was also inversely correlated with tients had a 17.4% ORR (n=26), with a median duration tumor size, suggesting that the infiltrating CD8+ T cells of response of 16.6 months. Median OS was 11 months were responsible for inhibiting tumor growth. Overall, (n=26). Grade 3 TRAEs were reported for 23.1% pa-NPS™ (180 mJ/mm3) + resiquimod was the treatment tients. No grade 4 or 5 TRAEs were reported. condition most effective at eliminating a primary tumor In summary, EP delivery of tavo allows for safe adminis- and inhibiting a rechallenge tumor. This observed effect tration of IL-12 treatment in patients with solid tumors. was likely due to CD8+ T cell priming events that resul- Findings from OncoSec clinical trials support continued ted from the synergy between NPS-induced regulated cell development of an electroporation-based delivery of po-death and the immune adjuvant properties of resiquimod. tent medicines as a promising approach to local delivery We plan to continue to optimize our treatment approach of treatment with systemic effects while minimizing the by exploring the use of different adjuvants in combination severe toxicities invariably associated with systemic im- with NPS™ treatment energies to maximize efficacy. Our munotherapy. hope is that the NPS™ may one day become a novel can- didate for the treatment of pancreatic and other hard to OR-145 treat cancers. Nano-Pulse Stimulation™ (NPS™) in com- bination with the TLR7/8 immune adjuvant OR-143 resiquimod eliminates murine Pan02 pancreatic Delivery of Immune Modulators Using Gene Elec- tumors and inhibits the growth of rechallenge tu- trotranser to Induce a Robust Immune Response mors Against Solid Tumors Amanda H. McDaniel, Kristin Von Rothstein, Dacia Richard Heller, Jody Synowiec, Samantha Mannarino, Ju- Gonzalez, Richard Nuccitelli lie Singh, Guilan Shi Pulse Biosciences, Biology, United States University of South Florida, United States Pancreatic cancer is associated with an extremely poor Immunotherapy has the potential to be an effective prognosis and immunotherapy alone has not demonstrated therapeutic approach for cancer. The utilization of im- sufficient efficacy in the treatment of nonresectable tu- mune checkpoint inhibitors (ICIs) has enhanced the anti- mors. However, when a treatment modality capable of tumor effectiveness of immunotherapy approaches. While inducing tumor cell death is given in combination with an encouraging, the therapy is not without shortcomings. immunotherapy, the two treatments may synergize, signi- The tumors of many patients are initially refractory to ficantly increasing the response rate. Nano-Pulse Stimu- checkpoint inhibitor therapy or acquire resistance. Eflation™ is a bioelectric modality that initiates a cascade fectiveness of ICIs may be related to the amount of CD3 of events within cells that leads to regulated cell death and CD8 cells within the tumor or tumor periphery. Tu- (RCD), exposing tumor antigen to the immune system. mors can be categorized as cold (low checkpoint expres- We conducted studies to determine if NPS™, with or sion and minimal immune cell infiltrate), altered (excluded without the addition of the immune adjuvant resiquimod, (lymphocytes at the margin not infiltrating) or immun- could eliminate a murine Pan02 pancreatic tumor, while osuppressed (low level of infiltrate but a suppressive en- also preventing the growth of a Pan02 rechallenge by in- vironment) and hot (significant infiltrate and not a sup- ducing a CD8+ T memory cell response. After tumor pressive environment. A cold or altered tumor would be antigen is released and taken up by dendritic cells (DCs), resistant to ICI therapy. One approach to overcoming res- resiquimod further promotes adaptive immune recognition istance is to modify the tumor microenvironment (TME) by binding to TLR7 and TLR8 on DCs, stimulating the from cold or altered state to hot in order to enhance the production of type 1 IFNs and helping to prime and ex- efficacy of ICI therapy. We have been evaluating a gene- pand the population of tumor-specific CD8+ T cells. We based approach to modify the TME to increase T-cell in-found that using a medium dose of NPS™ (180 mJ/mm3) filtration and change from a suppressor environment to a 109 pro-inflammatory environment. To accomplish this, we tially recreate a tumor microenvironment. Two different have used gene electrotransfer (GET) to deliver proin-EP conditions (600 V/CM and 1000 V/cm), commonly flammatory cytokines directly at the tumor site. Both employed in electro-chemotherapy protocols, were applied, interleukin-15 and interleukin-12 have been effective in followed by the addition of PHA-M- or mock-stimulated T modifying the TME and inducing a robust immune re- cells, to test whether the potential adjuvant effect of EP sponse. Utilizing two tumor models B16.F10 melanoma could be modulated by T lymphocyte activation. model in C57Bl/6 mice and 4T1 breast cancer model in Our results demonstrated that EP alone and EP of co- BalbC mice, we observed a significant reduction in T reg- cultures of HCC1954 cells with resting T cells affected ulatory cells and myeloid derived suppressor cells while T significantly number and size of cancer cell-containing 3D effector cells were significantly increased within the TME structures. This reduction in 3D cancer cell spheroids par- following intratumor delivery. This approach also resulted alleled the T-lymphocyte infiltration and the induction of in prolonged disease-free survival as well as long term im- cell death as evidenced by PI staining. This effect was mune memory in a single tumor model. We further tested overall, largely improved when PHA-M activated T cells this approach in combination with anti-PD-1 using a two- were added. These results prove that EP may exert an-tumor model consisting of a subcutaneous B16F10 tumor ticancer effects by increasing the cell killing by activated and B16F10 cells expressing luciferase injected via the in- T-lymphocytes. We suggest this may be facilitated by EP- traperitoneal route in a C57Bl/6 mouse. Intratumor deliv- mediated antigen exposure on cancer cells. ery of pIL-12 GET as a monotherapy resulted in reduction Further experiments to evaluate Cancer Stem Cells mark- or elimination of the subcutaneous tumor. However, the ers in such experimental setting are on-going and the res- monotherapy approach was only successful in reducing the ults will be presented and discussed. peritoneal spread in about 50% of the mice. When pIL- 12 GET was combined with anti-PD1 administered via OR-142 an intraperitoneal injection not only was there an elim- Protein sampling with electroporation facilitates ination of the subcutaneous tumor, but it resulted in the profiling of spatial differential protein expression elimination of intraperitoneal metastatic growth. In both in breast tumors in vivo tumor models, upregulation of MHC class 1 and PDL1 Alexander Golberg 1, Edward Vitkin2, Julia Wise1, Shay expression was observed. The results have this study sug- Ben-Elazar2, Zohar Yakhini2 gest that the combination of delivering plasmids encoding 1Tel Aviv University, Israel cytokines combined with ICIs could be utilized as an ef- 2Reichman University, Israel fective combination therapy for solid tumors. Excision tissue biopsy, while central to cancer treat- ment and precision medicine, presents risks to the patient OR-141 and does not provide a sufficiently broad and faithful rep- Electroporation Efficacy in breast cancer cell line resentation of the heterogeneity of solid tumors. Here we co-cultured with T lymphocytes introduce e-biopsy – a novel concept for molecular profil- Elisabetta Sieni 1, Annj Zamuner2, Mariangela De ing of solid tumors using molecular sampling with elec- Robertis3, Ramona Marino4, Daniela Rutigliano4, Massimo troporation. As e-biopsy provides access to the molecular Sanchez5, Flavio Keller4, Monica Dettin2, Maria Teresa composition of a solid tumor by permeabilization of cell Conconi2, Vito Michele Fazio4, Mario Cioce4, Emanuela Signori6 membrane, it facilitates tumor diagnostics without tissue 1University of Insubria, DiSTA, Italy resection. Furthermore, thanks to its non tissue destruct- 2University of Padova, Italy ive characteristics, e-biopsy enables probing the solid tu- 3University of Bari, Italy mor multiple times in several distinct locations in the same 4University Campus Bio-Medico of Rome, Italy procedure, thereby enabling the spatial profiling of tumor 5Istituto Superiore Sanità, Italy molecular heterogeneity.We demonstrate e-biopsy in vivo, 6Institute of Translational Pharmacology, Biomedical Sciences, using the 4T1 breast cancer model in mice to assess its Italy performance, as well as the inferred spatial differential protein expression. In particular, we show that proteo- 3D scaffolds composed of hyaluronic acid and ionic- mic profiles obtained via e-biopsy in vivo distinguish the complementary self-assembling peptides can allow het- tumors from healthy breast tissue and reflect spatial tu- erogeneous cell-cell contact and the appropriate support mor differential protein expression. E-biopsy provides a of spatially organized extracellular matrix, thus evoking completely new molecular sampling modality for solid tu-more clinically relevant experimental conditions for grow- mors molecular cartography, providing information that ing cancer cells, as compared to conventional cultures. potentially enables more rapid and sensitive detection, at These 3D cultures may therefore represent a valuable sys- lesser risk, as well as more precise personalized medicine. tem to study the anticancer effect of Electroporation (EP) protocols, with translational potential. OR-140 Our working hypothesis is that EP of cancer cells growing Experimental and theoretical Brownian Dynamics in 3D scaffolds may elicit increased antigen exposure, in analysis of Ion transport during cellular electro- turn improving T lymphocyte migration and targeting of poration of E. coli bacteria cancer cells for their elimination. Juan A. Gonzalez Cuevas 1, Ricardo Arguello1, Marcos To this aim, we cultured breast carcinoma cells Florentin1, Franck M. André2, Lluis M. Mir2 (HCC1954) with T cells (Jurkat) in 3D scaffolds, to par- 1National University of Asuncion, Paraguay 110 2CNRS, Gustave Roussy, Université Paris-Saclay, Metabolic of the lipids in PAW rich in H2O2 and NO2-. and Systemic aspects of the oncogenesis (METSY), France Furthermore, we analyzed Giant unilamellar vesicles The motion of ions through pores formed in the in- (GUV) which are a simple spheroid membrane model ner and outer plasma membranes of Escherichia coli cells composed of lipid bilayer only. Observed GUV de- during electroporation is simulated in 3-D space using a gradation consecutive to PAW, PEF, and PAW+PEF Brownian dynamics model, which is mostly deterministic treatments indicated potential bacterial membrane following Newtonian mechanics, but has some stochastic damage by lipid degradation. We observed a strong properties to account for elastic ionic scattering in wa- degradation of the GUV for PAW+H2O2+PEF by ter. The pore’s conductance, diffusion coefficient, mobility optical microscopy. The lipid peroxidation induced by and translation time of Ca2+, Mg2+, Na+, K+ and Cl-PAW chemistry seems to be an important parameter of ions are estimated from the numerical model and valid- bacteria inactivation by plasma and plasma-activated ated with experiments conducted at the Gustave Roussy. water combined with a pulsed electric field. The results from this work provide a better understanding of the electroporation process, aiding in the design of elec- Supported by Slovak Research and Development Agency trical pulses and waveforms for maximizing the through- APVV-17-0382 and the Comenius university grant put of DNA, drugs and gene materials into cells, primarily UK/411/2022_FMFI. for application in cancer treatment. OR-64 Continuous Extraction of Proteins from Microbial P7 - Pulsed Electric Fields (PEF) for Cells by Pulsed Electric Fields Recovery of Components Felix Schottroff 1, Jens Kastenhofer2, Oliver Spadiut2, David J. Wurm2, Henry Jaeger1 from Microorganisms 1University of Natural Resources and Life Sciences (BOKU), Department of Food Science and Technology, Austria Wednesday morning Track D 2TU Wien, Austria Oct 12, 10:30 - 12:10 In food and bio technology, proteins are increasingly produced in microbiological production systems, by means OR-63 of fermentation. In conventional bioprocesses, down- Combination of plasma-activated water with non- stream processing is usually initiated by cell disintegra- lethal pulsed electric field on bacteria inactivation tion, using high-pressure homogenization. This step is fol-Robin Mentheour 1, Nofel Merbahi2, Marie-Pierre Rols3, lowed by extensive purification steps due to the resulting Zdenko Machala1 1 high loads of host cell impurities. Comenius University, France 2 In order to reduce impurities of the resulting protein solu- CNRS, Laboratoire LAPLACE, France 3 tions, pulsed electric field (PEF) treatment and the result- CNRS, IPBS, France ing electroporation was investigated for permeabilization Pulsed electric fields (PEF) and Plasma-activated of cell membranes and the selective release of target pro-water (PAW) are known for their antibacterial effects. teins from E. coli. For this purpose, continuous electro- PEF effects are usually present in the plasma discharges poration was employed to selectively extract recombinant applied in biomedicine. Our mild PEF treatment of 200 Protein A from the periplasm of E. coli. For this purpose, ns, 12.5 kV/cm, at 100 Hz applied for 100s on E. coli in a specifically designed flow-through PEF treatment cham- planktonic form did not result in bacteria inactivation. ber was deployed, operated at 1.5 kg/h, using rectangular Two different types of Plasma-activated water made by pulses of 3 ms at specific energy input levels between 10.3 a transient spark in atmospheric air were prepared. The and 241.9 kJ/kg. Energy input was controlled by vari- first type of PAW prepared in a closed-air reactor is rich ation of the electric field strength (28.4–44.8 kV/cm) and in NO2- and poor in H2O2, while the second one prepared pulse repetition frequency (50–1000 Hz). The effects of in an open-air environment is richer in H2O2. NO2- the process parameters on cell viability, product release, and H2O2 in PAW react together to form peroxynitrites and host cell protein (HCP), DNA, as well as endotoxin (ONOO-/ONOOH), which further degrade into �OH and (ET) loads were investigated. �NO2 radicals. It was found that a maximum product release of 89 % was We tested the antibacterial effects of PAW, PEF, and achieved with increasing energy input levels. Cell death their coupled effect applied to the decontamination of E. also gradually increased, with a maximum inactivation of coli in water, PAW, and PAW + additional H2O2. The -0.9 log at 241.9 kJ/kg. The conditions resulting in high reinforcement in H2O2 is to simulate the production of release efficiencies while keeping impurities low were elec- radicals through peroxynitrite chemistry. The radicals tric field strengths � 30 kV/cm and frequencies � 825 Hz. are known to damage the cell membrane and induce In comparison with high-pressure homogenization, PEF phospholipid peroxidation fragilizing the membranes to treatment resulted in 40 % less HCP load, 96 % less DNA the PEF treatment. We observed an antibacterial synergy load, and 43 % less ET load. of PAW with the PEF treatment only in presence of both Therefore, PEF treatment can be an efficient alternat- H2O2 and NO2- in the PAW. ive to the cell disintegration processes commonly used in The TBARS method which measures the membrane lipid downstream processing. Ultimately, PEF can contribute peroxidation showed a significantly stronger peroxidation 111 to design continuous or circular, more efficient biopro-OR-148 cesses for future applications in food and bio technology. The effect of nanosecond pulsed electric field on the production of metabolites from lactic acid bac- OR-65 teria Sequential extraction of different compounds of in- Sumiyo Kanafusa1, Elisabeth Uhlig1, Kunihiko Uemura2, terest from yeast biomass assisted by Pulsed Elec- Federico Gómez Galindo 1, Åsa Håkansson1 tric Fields 1Lund University, Sweden Alejandro Berzosa, Carlota Delso, Jorge J Sanz, Ignacio 2National Agriculture and Food Research Organizaion, Food Alvarez-Lanzarote, Cristina C Sánchez-Gimeno , Javier Raso Research Institute, Japan University of Zaragoza, Spain Watermelon juice is sensitive to heat, oxygen, and light Yeast biomass generated during alcoholic fermentation which makes it difficult to preserve using processing opera-represents the second most important by-product of oen- tions that may degrade its functional ingredients and gen- ological and brewing industries. Although yeast biomass erate unpleasant flavors. However, through an optimized is a valuable product, it has traditionally been used for fermentation process, watermelon juice can be preserved applications that do not add economical value or are dis- at the same time as the nutritional value is improved. In carded, which represents an environmental problem due this study, nanosecond pulsed electric field (nsPEF) was to the high biological and chemical oxygen demand. Bio- applied to the probiotic strain Lactobacillus plantarum molecules with different applications in the food, phar- DSM 9843 used to ferment watermelon juice. The object maceutical and cosmetic industries can be obtained from of this research is improving efficiency of the fermentation the yeast cytoplasm (protein, nucleic acids, glutathione, process by nsPEF-induced stress. polyphenols) and from their envelopes (mannoproteins, β- Fermentation of watermelon juice with L. plantarum DSM glucans). 9843 provoked a pH reduction from pH 5.6 to pH 3.8 The objective of this study was to evaluate a strategy and the production of 2.34 g/L D-lactic acid, 1.88 g/L based on the application of Pulsed Electric Fields (PEF) L-lactic acid and 0.075 g/L acetic acid after 20 h incub- for the development of a cascade process, which allows ation without nsPEF treatment. nsPEF treatment was obtaining a spectrum of valuable products from yeast bio-performed during the logarithmic growth phase and the mass. nsPEF conditions (repetition frequency, voltages and total Biomass of Saccharomyces cerevisiae 3D Viniferm was number of pulses) were changed. After the frequency in-treated by PEF at 15 kV/cm for 50 µs (26.0 kJ/kg), 150 creased from 1 Hz to 50 Hz, an increase of 9.4% D-lactic µs (96.8 kJ/kg) and 200 µs (129.5 kJ/kg) to electroporate acid and 7.5?etic acid were observed. It is noteworthy that 20, 85 and 99% of the population. After 24 hours of in- nsPEF treatment (5.0 kV, 700 pulses) resulted in 19% in- cubation at 25 ºC, the concentration of extracted intracel- crease (p< 0.01) in L-lactic acid compared to the control. lular compounds (proteins, amino acids, polyphenols and Significant increases in D-lactic acid (6.8%, p< 0.05) and glutathione) and the antioxidant capacity of the super- acetic acid (15%, p< 0.01) were also observed after nsPEF natant were determined. The cells were incubated again treatment (D-lactic acid: 4.5 kV, 700 pulses and acetic for obtaining mannoproteins and β-glucans due to the cell acid:4.5 kV, 1000 pulses) compared to the control. The wall autolysis triggered by the PEF treatment. nsPEF treatment did not affect the viability of the cells After 24 h incubation, hardly any extraction of cyto- and sufficient numbers remained in the product after fer- plasmic compounds was detected in the untreated cells mentation. (5-19%). However, the electroporation caused by the These results suggest that the metabolism of lactic acid PEF treatment facilitated the release of intracellular com- bacteria was affected by the nsPEF treatment. This re- pounds. Between 33 to 100% of the total content of the search demonstrates the potential of this novel electro- analysed compounds were extracted. Significant differ- technology for the increased efficiency of fermentation pro- ences in the amount of extracted compounds were not cesses. detected between the two most intense PEF treatments applied. PEF-induced autolysis resulted in a mannopro- OR-159 tein concentration in the supernatant of the three PEF- PEF-Processing of Microbial Biomass at KIT-IHM treated suspensions approximately 8 times higher than in Wolfgang W. Frey untreated cells. After the separation of the mannoprotein- KIT, IHM, Germany rich extract, an insoluble fraction with a concentration in This contribution will give a brief overview of current β-glucans ranging from 269 to 300 mg/g of dry extract R&D activities at KIT-IHM: was obtained from the PEF-treated yeast cells. In the past, PEF-processing was shown to allow recovery The results obtained in this research have shown that the of multiple cell components from microalgae by cascade electroporation of yeast cells caused by the application of processing. In collaboration with European partners, cas- PEF technology could be a useful tool in the development cade processing of Scenedesmus was implemented for the of an efficient, economical and sustainable method for the production of biostimulants and biofertilizer for agricul- valorisation of yeast used in the winemaking and brewing tural applications and lipids as an aquafeed additive. process. At lab- and pilot-scale, lipid extraction from microalgae is under focus. For lipid extraction from microalgae it is ad- vantageous to include an incubation step after PEF treat- 112 ment. This allows to reduce the treatment energy from ablation site. 1.5 MJ/kgDW to 0.25 MJ/kgDW. Despite this reduction We prepared artificial urine (AU) with compounds in treatment energy, economic efficiency is dominated by commonly present in the healthy human urine at the solvent extraction step. A techno-economic analysis physiological concentrations. Parameters known to of PEF-assisted lipid recovery pathways revealed, that the influence the therapy outcome, such as the concentration major part of energy expenses is caused by solvent recyc- of Ca2+, conductivity, and pH, were the same in all ling and not by biomass processing. First results indicate tested solutions. that the application of biphasic solvent systems, which use Electric field thresholds for complete ablation were meas- a minimum content of azeotropic mixtures of water and ured in monolayers of cancer and healthy urothelial cells. Ethanol, are advantageous, even at slightly reduced lipid The monolayers were treated by electric pulses using two yields. needle electrodes placed orthogonal to the monolayer and Compared to lab-scale batch cultivation, continuous large- generating the electric field, which gradually decreased scale open pond cultivation of microalgae results in elev- with distance from the electrodes. The region of cell ated conductivity values of the microalgae suspension after death was measured by staining with propidium iodide harvest. Energy efficiency of PEF-treatment depends on added to the cell monolayer at 2 hours after the nsPEF suspension conductivity. To overcome this general hurdle treatment. Cell death thresholds were determined by in PEF-assisted microalgae processing, a new approach for matching the stained areas to the simulated electric field microalgae pretreatment by pulsed microwave treatment intensity. Our study compared the cell death thresholds (PM) was tested. A cavity-based applicator was manu- for nsPEF exposures in a standard physiological solution factured and tested. Lipid extraction efficiency was well and the AU. We also conducted a separate confocal mi-comparable to PEF-processing. croscopy study to explore the impact of AU on membrane Heterotrophic cultivation of microbial biomass offers ad- permeabilization. vantages over autotrophic cultivation of microalgae, i.e. AU had a significant protective effect, increasing the cell higher biomass density and local independence from sun- death threshold 1.4 times for healthy urothelial cells and light. In particular, oleaginous yeasts can utilize residues 1.8 times for cancer cells. We proposed and investigated from organic industries, such as glycerol or molasses, as a several explanations for this effect, including the high carbon source, producing considerable lipid contents. For content of urea resulting in higher osmolarity and the component recovery, yeasts also require a cell disruption higher concentrations of Mg2+ and K+. Omitting urea step prior to lipid extraction. First results indicate that from AU, thus decreasing its osmolarity, resulted in a PEF-assisted lipid extraction allows complete lipid recov- decline in cell killing threshold for healthy urothelial cells ery from oleaginous yeasts. almost to the level achieved in physiological solution, but for cancer cells the threshold remained 1.6 fold higher. We also found that the higher concentration of Mg2+ in AU P18 - Electroporation and cellular was not responsible for increased cancer cell survival. The processes impact of other AU components is under investigation. Novel results of our study may support future clinical Wednesday afternoon Track A applications of nsPEF for bladder cancer ablation. Oct 12, 14:00 - 15:00 Support: This research has been made possible by OR-149 the Kosciuszko Foundation. Urine protects urothelial cells against nanosecond OR-13 pulsed electric fields damage Synergistic Gene Electrotransfer and 3D Bioprin- Aleksander Kielbik 1, Pamela Sowa2, Andrei G. Pakhomov3, Emily Gudvangen3, Uma Mangalanathan3, Julita Kulbacka1, ted Implants for Improving Biomanufactured Im- Olga N. Pakhomova3 plant Biological Integration for Enhancing Muscu- 1Wroclaw Medical University, Poland loskeletal Tissue Regeneration 2Technische Universität Dresden, Germany Anna Bulysheva 1, Kyle Christensen2, Aislin West3, Michael 3 Francis4 Old Dominion University, United States 1University of South Florida, United States Recent advances in understanding bioeffects of nano- 2Embody Inc, United States second pulsed electric fields (nsPEF) suggest that they 3Old Dominion University, United States could be safer than longer pulses and more efficient 4Asante Bio Inc, United States in causing a cytotoxic effect in tumor cells. For ur- othelial cancer treatment, nsPEF might minimize the In vivo gene therapy approaches and biomanufactur- neuro-muscular excitation while preserving the benefit of ing, or 3D bioprinting, have independently shown promise complete tumor ablation. in regenerative medicine, yet their synergistic potentials Permeabilization of cells results in the loss of intracellular have not been well explored. Musculoskeletal (MSK) tis-K+ and ATP and the influx of extracellular Na+ and sue injuries—from tendon tears, volumetric muscle loss, Ca2+. Since ablation efficiency depends on the tumor and myotendinous junction injuries—are commonplace environment, and the bladder is constantly exposed to and often lead to permanent disability and deformation. urine, we evaluated the effect of urine presence at the While there are current clinical regenerative treatments 113 for many MSK injuries, they have relatively long recovery cell membrane causing membrane destabilization, ion flux, times and often do not restore native function. To pro-and membrane depolarization. In our experiments, we spectively progress MSK tissue regeneration, we explore compared the cytotoxic effects of these antibiotics admin- combining gene electrotransfer (GET) with novel fibrous istered either before or after nsPEF. While doxycycline 3D bioprinted collagen microfibers to promote advanced caused 2 log inactivation only when cells were pretreated healing and tissue integration. First, GET parameters with nsPEF, combining nsPEF with daptomycin greatly for delivery to the skin were optimized with monophasic potentiated the effects of each monotherapy regardless of and biphasic pulses with reporter and effector genes to- the treatment order. Altogether our results show that wards optimizing underlying MSK tissue healing. Tis- nsPEF impacts MRSA viability, potentiates the effect of sue twitching and damage, as well as gene expression specific classes of antibiotics and that the order of the com- and distribution, were evaluated, quantified, and optim- bined treatment (antibiotic/nsPEF or nsPEF/antibiotics) ized. Bioprinted collagen microfiber constructs, mimick- can have major impact on the cotreatment efficacy. Res-ing healthy tendon structures, were then implanted subcu- ults from ongoing experiments looking at the effects of taneously for biocompatibility and angiogenesis analyses other antibiotics (vancomycin), investigating the efficacy as implants alone or combined with GET for human fibro- of the cotreatment on MRSA biofilms, and in vivo treat- blast growth factor-2 (FGF2). GET of FGF2 significantly ments of SSTIs will also be discussed. increased angiogenesis and histological biocompatibility of the bioprinted implants when compared to bioprinted con- OR-165 struct implant-only sites. The combination of biomanufac- Calcium Oscillations And Mesenchymal Stem tured collagen-microfiber-based implants and angiogenic Cells Fate: Characterization and Control Through GET therapy may together lead to better graft biocom- Electroporation patibility and prospectively tissue regeneration in MSK Leslie Vallet, Franck M. André, Marina M. Sanchez Peti- repair. dier, Romain Fernandes, Lluis M. Mir CNRS, Gustave Roussy, Université Paris-Saclay, METSY, OR-151 France Combinatorial treatment with nsPEF and antibi- Mesenchymal Stem Cells (MSCs) are adult stem otics increases Methicillin-Resistant Staphylococ- cells whose multipotency was initially described to cover cus aureus inactivation various cell types constituting connective tissues, such as Alexandra E. Chittams-Miles 1, Areej Malik2, Erin Purcell2, osteoblasts, adipocytes, or chondrocytes [1]. Nevertheless, Claudia Muratori1 under specific conditions, more recent studies shed light 1Old Dominion University , Frank Reidy Research Center for on extended differentiation abilities to other types of Bioelectrics, United States specialized cells such as muscle [2] or neuron-like cells [3]. 2Old Dominion University, Department of Chemistry and Bio- Not only due to this promising multipotency, but also chemistry, United States to interesting secretory activities [4] as well as rescuing Staphylococcus aureus (S. aureus) is a virulent bac- functions exerted towards damaged cells within the body terium that is one of the primary causes of hospital [5], these cells have attracted more and more interest in and community-acquired skin and soft tissue infections the context of regenerative therapies these last decades. (SSTIs). S. aureus has become antibiotic-resistant over In another respect, it was observed that MSCs naturally time, leading to the evolution of Methicillin-Resistant S. display spontaneous calcium (Ca2+) oscillations whose aureus (MRSA). The increasing prevalence of MRSA has frequency can vary over the course of differentiation put further strain on medical centers, and patients infec- or proliferation processes. Ca2+ is known to be an ted with this type of bacteria have worse outcomes. important cellular second messenger, often signaling in In this study, we are investigating the potential synergistic an oscillatory fashion. Frequency and/or amplitude of effects of dual treatment with nanosecond pulsed electric these Ca2+ oscillations can embed important information fields (nsPEF) and antibiotics. While nsPEF has been subsequently decoded by some proteins in the cell whose extensively used to treat eukaryotic cells, the effects of activity is Ca2+-sensitive [6]. This signaling mechanism these shorter pulses on bacteria have been less investig- allow for specific stimuli to modulate precise cellular ated. MRSA planktonic cells in exponential growth phase response [6]. More precisely, in this work, we focused were exposed to increasing number of either 300 or 600 first on the changes in Ca2+ oscillations triggered by ns pulses (3 MV/m, 1 Hz) in Luria Broth in electropor- stimuli involved in cellular proliferation or differentiation ation cuvette at room temperature. The highest pulse events. In a second part, we aimed to assess whether dose tested, namely 120 pulses of 600 ns duration, caused controlling Ca2+ oscillations frequency and/or amplitude 0.5 log10 inactivation. While these nsPEF doses were not might mimic or potentiate the effects of these stimuli. enough to completely inactivate MRSA planktonic cells, To this end, our laboratory has developed a technique they undoubtedly caused a reasonable level of damage. to homogeneously control Ca2+ oscillations within a cell We anticipated that the perturbation created by nsPEF population by physical means. This technique implies the would increase antibiotics efficacy. To test this hypothesis, use of microsecond pulsed electric fields (µsPEFs) capable we used two antibiotics approved to treat SSTIs: doxy- of generating slight permeabilization of the cell membrane cycline, an inhibitor of bacterial protein synthesis, and to the Ca2+ present in the surrounding medium. This daptomycin, a lipophilic peptide that intercalates into the subsequently triggers Ca2+ oscillations similar to the 114 natural ones (in shape, amplitude and duration) due to PET/CT-scans acquired from a prospective cohort of loc-the Ca2+ induced - Ca2+ release response [7]. This alized PC patients treated with IRE, were reexamined by work carries both fundamental and applicated aspects: two nuclear medicine and radiology experts. Semiauto- on the one side, to assess the role and the importance mated registration of tumor size, 18-FDG-standard up- of the Ca2+ oscillations in various cellular processes in take values (SUV), metabolic tumor volume (MTV), total MSCs, and on the other side, to develop a suitable device lesion glycolysis (TLG) and remote metastases were per- to control them on the long-term, in the perspective of formed. Comparative imaging outcomes on the lesion- and therapeutic applications. patient-level were tested for correlated with mortality rate (MR) by Poisson regressions with the Huber/White sand- References wich estimator. 1. M. Dominici et al., Cytotherapy, 8, 4, 2006, pp. Results: Of the 34 studies included in the systematic re- 315-317. view, 14 used standardized response criteria, 14 used self- 2. E. J. Gang, et al., Stem cells, 22, 4, July 2004 pp. determined criteria or absolute size comparison and six did 617-624. not report the response evaluation method. One study 3. A. J. Cardozo, et al., Gene, 511, 2, September 2012, statistically tested CT imaging outcomes with survival pp. 427-436. using a self-determined categorization and found a sig- 4. P. Kumar, et al., Cytokine & growth factor reviews, nificant correlation. Data from the clinical trial revealed 46, april 2019, pp. 1-9. a significant correlation between patient-level progressive 5. S. Paliwal, et al., Journal of biomedical science, 25, 1, disease (PD) based on RECIST 1.1. and MR in all time March 2018, art. 31. intervals. Patient-level PD based on EORTC PET re- 6. E. Smedler, and P. Uhlén, Biochimica Biophysica sponse criteria was correlated with MR in the three to Acta (BBA)-General Subjects, 1840, 3, March 2014, pp. six-month post-IRE period but failed to correlate in the 964-969. other time intervals. Lesion-level PD based on changes 7. H. Hanna, et al., Stem cell research & therapy, 8, April in tumor size, SUVmax, MTV and TLG failed to cor- 2017, art. 91. relate with MR. However, non-dichotomized lesion-level changes of the same variables revealed a significant correl- ation between MR and changes in tumor size, MTV and P23 - Irreversible Electroporation TLG. (IRE) Conclusions: There was a notable variation in the use of imaging response evaluation methods across the pub- Wednesday afternoon Track B lished literature. Only sparse evidence exists showing Oct 12, 14:00 - 15:00 that comparative imaging findings correlates with sur- vival. Patient-level PD based on RECIST 1.1. is cor- OR-189 related with poorer survival in PC patients in the current Does imaging response after irreversible electro- study but results should be validated in an independent poration correlate with survival in localized pan- cohort. Lesion-level analysis is inappropriate for evalu- creatic cancer? ation of efficacy, at this point in time, but several prom- Rasmus Virenfeldt Flak, Rune Vincents Fisker, Niels Hen- ising parameters should be examined in future trials. rik Bruun, Mogens Tornby Stender, Louise Stenholt, Ole Thorlacius-Ussing, Lars Jelstrup Petersen OR-190 Aalborg University Hospital, Denmark Toward large ablation volumes with single inser- tion high-frequency irreversible electroporation Introduction: Irreversible electroporation (IRE) and Kenneth N. Aycock 1, Sabrina N Campelo1, Zaid S. other local ablative therapies are being increasingly stud- Salameh1, Edward Jacobs1, Kailee David1, Iain H McKillop2, ied for the treatment of pancreatic cancer (PC), when Rafael V. Davalos1 surgical resection is not possible. Most published stud- 1Virginia Tech, United States ies use imaging outcomes as the primary endpoint for 2Atrium Healh - Carolinas Medical Center, United States evaluating efficacy. However, there are no consensus on Introduction: Hepatocellular carcinoma (HCC) is the how to evaluate the response and the commonly applied fastest growing type of cancer diagnosed in the United techniques have not been scientifically validated for this States and the third leading cause of cancer-related use. The aims of the current study were to examine the mortality worldwide. Additionally, the liver is a common use of response evaluation in this setting, to identify the site of metastasis from primary tumors in other organs. knowledge-gaps in the published literature and to test the However, most patients are not candidates for surgical validity of the most used methods in a cohort of PC pa- resection or liver transplantation, the only two definitive tients. therapies available for liver cancer [1]. Methods: First, a systematic literature search was per- High-frequency irreversible electroporation (H-FIRE) formed in PubMed. Studies reporting comparative is an emerging technology that utilizes pulsed electric imaging outcomes in ablation-treated locally advanced fields to generate nanoscale defects in the membranes PC were included. Studies were excluded if the out- of targeted cells, disrupting homeostatic equilibrium comes could not be differentiated between different dis- and generating cell death over several hours following ease stages, histology or surgical approaches. Secondly, 115 pulse delivery. H-FIRE offers numerous advantages over to radiation. thermal ablation and its predecessor, conventional IRE, Method: We developed a needle insertion navigation sys- such as reduced muscle contractions and electrochemical tem that optimize the electrode configurations in terms effects, potentially more predictable ablation volumes, of location, insertion depth, distance between electrodes and more consistent electric field distributions. However, and number of electrodes. The navigation system de- H-FIRE generated ablation volumes have struggled to signed based on 3D slicer open-source medical images pro- reach clinical relevance (˜3 cm). In this study, we cessing and analysis software. The system utilizes Com-examine the ability of internally cooled applicators and puter Tomography (CT) medical images for needle loca- newly introduced waveform manipulations to generate tion identification, while the needle insertion and place- large ablations in vivo. ment are fully controlled by respiratory organs motion Methods: Six female Yorkshire pigs (50–55 kg) were used tracking using real-time ultrasound images. The needle in experiments (Palmetto Research Swine, Reevesville, location tracked by the Channel and Spatial Reliability South Carolina), all of which were approved by the tracking algorithm (CSRT) for respiratory organ motion Institutional Animal Care and Use Committee. In all tracking, while the insertion was controlled by real-time cases, a custom generator (Voltmed, Inc., Blacksburg, distance and orientation estimation. VA) was used to deliver H-FIRE waveforms via an Results: The navigation system was tested and validated 18-gauge single-insertion monopolar or bipolar applicator on CT and US images data of 10 patients with liver and inserted into the liver. In experiments using a monopolar renal cell carcinoma (RCC) diseases with an average can- applicator, a grounding pad was placed distally ˜30 cer diameter of 6 and 5 cm respectively. The results in-cm from the insertion site. An accelerometer (Analog dicated that the average insertion and tracking accuracy Devices, Inc., Norwood, MA) was affixed to the skin was 88.2% and 85.7% for liver and renal cancers, with an approximately 15 cm from the insertion site. In all cases, average location error of 1.6 ±0.65 mm and 1.83±0.4 mm 300 bursts were delivered at a rate of 1 Hz, and animals respectively. were recovered for 24 hr prior to sacrifice. Variables under Conclusion: The control of patient respiratory motion study included applied voltage, probe irrigation, H-FIRE during the electroporation needle electrode placement de- waveform, and probe type. creases the placement errors and increases the probe place- Results: In all cases, treatments were well tolerated ment accuracy which in turn will improve the treatment with no side effects; all bursts were delivered in the quality. Our finding can enhance the Irreversible elec- absence of cardiac synchronization, and no cardiac troporation clinical treatment planning by providing real- abnormalities were observed. Muscle contraction severity time needle placement tracking and monitoring system. was significantly stronger for the monopolar electrode configuration and was dependent upon grounding pad placement location. By manipulating the interpulse delay P29 - Electroporation-based Therapies (5-2-5-100), applied voltages were increased by ˜500 V - Head and Neck Cancer over the standard waveform (5-2-5-2), and with internal probe irrigation, large (˜ 5 cm3), completely non-thermal Wednesday afternoon Track C ablations were achieved with a maximum treatment Oct 12, 14:00 - 15:00 voltage up to 3,300 V. Conclusions: In this study, we optimized several paramet- OR-102 ers toward maximizing ablation volumes with H-FIRE Electrochemotherapy for the treatment of cu- while retaining its advantages over conventional IRE taneous squamous cell carcinoma: the INSPECT and thermal ablation. To our knowledge, the ablation experience (2008-2019) volumes reported in this work are the largest reported Giulia Bertino 1, The INSPECT Group2 with H-FIRE and were achieved with a single insertion 1University of Pavia, Department of Otolaryngology, Italy device. 2INSPECT, United Kingdom References: Introduction: Cutaneous squamous cell carcinoma 1. Venook, A. P., Papandreou, C., Furuse, J. & Ladrón (cSCC) is a frequent skin cancer with a high risk of de Guevara, L. Oncologist 15, 5–13 (2010). recurrence characterized by tumor infiltration and poor prognosis. ECT (electrochemotherapy) is an alternative OR-211 treatment option for locally advanced or recurrent cSCC Optimization of Irreversible Electroporation that is not suitable for surgical resection. The aim of this Needle Electrode Placement Using Ultrasound- study was to evaluate the data in InspECT (International dependent Respiratory Motion Tracking Filters Network for Sharing Practice on ECT) registry of the re- Radwan Qasrawi ferral centers and to clarify the indications for use of ECT Al-Quds University, Palestine, State of as treatment modality for cSCC. Materials and methods: Patients with primary, recurrent Background: Irreversible electroporation is a novel ab- or locally advanced cSCC from 18 European centers were lation technique that uses needle electrodes to improve included. They underwent at least one ECT session with percutaneous interventions. The optimal probe placement bleomycin between February 2008 and November 2020, increases the treatment efficacy and reduces the exposure which was performed following the European Standard 116 Operating Procedures. patients. Results: The analysis included 162 patients (mean age 80 years; median, 1 lesion/patient). Side effects were mainly OR-101 local and mild (hyperpigmentation, 11%; ulceration, 11%; Calcium electroporation for low risk basal cell car- suppuration, 4%). The response to treatment per patient cinoma – a proof of concept study was 62% complete and 21% partial. In the multivariate Stine Regin Wiegell 1, Kristoffer Hendel1, Christine Fuchs1, model, intravenous drug administration and small tumor Gregor Jemec2, Julie Gehl2, Mille Vissing2, Merete Hædersdal1 size showed a significant association with a positive out- 1Bispebjerg Hospital, University of Copenhagen, Denmark come (objective response). One-year local progression-free 2Zealand University Hospital, Denmark survival was significantly better (p<0.001) in patients with Introduction: Basal cell carcinoma (BCC) is the most primary tumors (80% (95% C.I. 70%-90%) than in patients common type of cancer with increasing incidence rates. with locally advanced disease (49% (95%C.I. 30%-68%). In electrochemotherapy (ECT) permeabilization of the Conclusion: In the present study, ECT showed antitu- cell membrane by electric pulses (electroporation) allows mor activity and a favourable safety profile in patients chemotherapeutics such as bleomycin to enter the cell in-with complex cSCC for whom there is no widely accepted creasing the anti-tumor effect. ECT appears to be an standard of care. Better results seem to be obtainable in effective treatment of BCC. In calcium electroporation primary and small tumors (<3 cm) and using intravenous (CaEP) chemotherapy is replaced by calcium chloride in-administration of bleomycin. jection resulting in severe ATP depletion leading to nec- rosis. The effect of CaEP has been shown to be compar- OR-100 able to ECT in the treatment of cutaneous breast can- Treatment of Basal Cell Carcinoma with Electro- cer and malignant melanoma metastases. The aim of the chemotherapy: Insights from the InspECT Re- study was to evaluate the effect of CaEP in the treatment gistry (2008-2019) of BCC using either high or low frequency electropora- Luca G. Campana 1, Giulia Bertino2, Tobian Muir3, Luca tion. G. Campana4 Materials and Method: Patients with low risk primary 1Manchester University NHS Foundation Trust, United King- BCCs were treated in local anesthesia with injection of dom calcium chloride (225 mM) directly into the tumor includ- 2IRCCS Policlinico San Matteo Foundation, Pavia, Italy ing a safety margin of 5 mm, followed by electroporation 3South Tees NHS Foundation Trust, Middlesbrough, United (ePORE, Mirai, Ireland) with pulse frequencies of either Kingdom 250 kHz (high) or 5 kHz (low). The higher frequency was 4InspECT Collaborative, United Kingdom designed to limit muscle contraction and discomfort dur- Background: This report describes electrochemother- ing treatment. Histology proven non-complete responders apy (ECT) modalities in basal cell carcinoma (BCC) pa-were retreated after 3 months. Tumor demarcation, tumor tients and evaluates the efficacy, safety, and predictive depth and efficacy were evaluated using dermoscopy and factors. optical coherence tomography OCT. Methods: This is a cohort study. The International Results: 25 patients with superficial (7) or nodular (18) Network for Sharing Practices of Electrochemotherapy BCCs with a diameter of 5-30 mm were included. Seven (InspECT) multicenter prospective database was queried patients (28%) were in complete response at 3-month for BCC cases treated with bleomycin-ECT between 2008 follow-up. Of the 13 patients treated with high frequency 3 and 2019. had complete response after one treatment with no recur- Results: The analysis included 330 patients. There rence within 12-month. Ten patients were retreated res- were 623 BCCs (median number: 1/patient, range: 1- ulting in 4 partial responses and 6 with no response. Nine 7; size: 13 mm, range: 5-350), 85% primary, and 80% out of 12 patients treated with low frequency received two located in the head and neck region. The procedure was CaEP treatments, 2 patients declined re-treatment due to carried out under local anaesthesia in 68% of cases, with pain during the first treatment and 1 patient dropped out the addition of mild sedation in the remaining 32%. Out due to internal cancer. 4 patients had complete response of 300 evaluable patients, 242 (81%) achieved complete at 3-month follow-up. At 12-month follow-up 2 patients response (CR). No previous therapies (OR 0.35, 95% C.I. had microscopic recurrences, one was lost to follow-up and 0.19-0.67, p = 0.001) and deep tumour margin coverage one have not yet reached 12-month follow-up. Of the re-with electric pulses (OR 5.55, 95% C.I. 1.37-21.69, p = maining 5 patients treated with low frequency 4 patients 0.016) predicted CR achievement. Toxicity included skin had partial response and 1 patient no response. High ulceration (16%; G3, 1%) and hyperpigmentation (8.1%; frequency CaEP was significantly less painful than low G3, 2.5%). After 17 months, 28 (9.3%) patients developed frequency with a median pain score of 2 compared to 5 local recurrence/progression. (p=0.03). One patient had severe necrosis resulting in Conclusion: Despite no convincing evidence that ECT scar formation. The cosmetic result in patients with com- confers improved outcomes compared with surgical ex- plete response was good in 2 patients, fair in 2 patients cision, still, it might be considered in patients who are and poor in 3 patients. not fit for invasive interventions. BCC treatment naivety Conclusion: Using CaEP with a new type of electroporator and ability to cover deep cancer margin with electrodes the previously described effect of ECT on BCCs could not predict tumour clearance and may help clinicians select be reproduced. Low frequency CaEP were more painful 117 than high frequency. Further studies should be initiated to vivo studies need to be performed. explore the mechanism of low efficacy of CaEP on BCCs Acknowledgment: The research was funded by the and to optimize treatment procedures to obtain efficacy Research Council of Lithuania within the DAINA 2 rates as reported for ECT of BCC using bleomycin. framework grant No.: S-LL-21-4 (PI: V. Novickij) and supported by National Science Centre (Poland) within a OR-109 framework of DAINA 2 (2020/38/L/NZ7/00342; PI: J. Effects of Pulse Repetition Frequency on Nano- Kulbacka). second Electrochemotherapy Veronika Malyško-Ptašinskė 1, Eivina Radzevičiūtė2, Irutė [1] V. Novickij et al., High-frequency submicrosecond Girkontaitė2, Jurij Novickij1, Julita Kulbacka3, Nina electroporator. Biotechnol. Biotechnol. Equip., vol. 30, Rembiałkowska3, Vitalij Novickij1 no. 3, pp. 607–613, May 2016. 1Vilnius Gediminas Technical University, Lithuania 2State Research Institute Centre for Innovative Medicine, Lithuania 3Wroclaw Medical University, Poland P38 - Electroporation for cardiac This work focuses on nano-electrochemotherapy with ablation bleomycin and doxorubicin and the electroporation dependence on pulse amplitude (6–10 kV/cm), duration Wednesday afternoon Track D (100–500 ns), and pulse repetition frequency (10 kHz, 100 Oct 12, 14:00 - 15:00 kHz, and 1 MHz) with bursts of 10 pulses on murine Lewis lung carcinoma (LLC1) cell line. For electroporation, OR-180 0–3 kV, 100 ns–1 ms square wave high voltage pulse Swine Coronary Lumen Contractures Following Ir- generator [1], and a commercially available 1 mm gap reversible Electroporation as Observed by Optical electroporation cuvette were applied. For detection of Coherence Tomography cell permeabilization, Yo-Pro-1 and flow cytometry were Amanda N. DeVos, David A. Ramirez, Paul A. Iaizzo employed. Cell viability was evaluated 24-, 48-, and University of Minnesota, United States 72-hours post-electroporation. As a reference, ESOPE Epicardial ablation is gaining popularity as a viable (1.3 kV/cm x 100 µs x 8) protocol was used, and optimal approach for treatment of ventricular tachycardia. In or- concentrations of bleomycin and doxorubicin were de- der to minimize collateral damage to surrounding tissues termined. irreversible electroporation has been explored as an emer- It was shown that the permeabilization rate of LLC1 cells ging modality for cardiac tissues. This study demonstrates using nsECT could be manipulated by changing pulsing the worst-case scenario effects that irreversible electropor- protocols. As expected, the cell permeabilization was ation has on the coronary arteries when exposed directly scaled with pulse amplitude and duration. For example, to the ablative field. 10 kV/cm x 200 ns x 10 pulses delivered at 10 kHz, Electroporation therapies were delivered across the left resulting in ˜50% is being permeabilized, while for 300 anterior descending arteries (LADs) of reanimated swine ns pulses, the same level of permeabilization is reached hearts on the Visible Heart® apparatus. The electropor-already with 7 kV/cm electric field. When 10 kHz pulses ation energies were delivered utilizing two needle probes were used, permeabilization higher than 75% was achiev- inserted 1cm into the myocardium. The electrodes are 1cm able only with 8 – 10 kV/cm pulses and pulse durations apart with the LAD vessel placed squarely between. For exceeding 300 ns. Similar response was acquired with 100 translational impact, the generator used was the clinically kHz pulses. However, a further increase of the repetition approved NanoKnife electroporation system. Three dif- frequency to 1 MHz significantly improved the efficiency ferent energy levels were selected, 500V, 700V, and 900V of electroporation. High permeabilization rates could be to observe a dose response of the tissue. For each en- already reached with 200 ns pulses or PEF amplitudes in ergy level, 70 pulses were delivered, each with 90µs pulse the 6–8 kV/cm range. The MHz protocols were the most width. In order to prevent cardiac fibrillation, IRE pulses effective, showing high cell death rates for both drugs. were triggered in sync with an ECG R-wave monitor. A The efficiency of nsECT was saturated already when Dragonfly (TM) OCT catheter was inserted into the LAD pulses longer than 200 ns were used in the whole range using a 0.014” coronary guidewire. Prior to ablation, the of evaluated PEF amplitudes. The 100 and 200 ns pulses baseline measurement of the lumen is obtained as the OCT returned a -dependent response, indicating a scaling of the catheter pulls back such that a cross-sectional OCT im- ECT efficiency with cell membrane permeabilization. The age is taken every 0.1 mm over 54 mm of the artery using identical pulses delivered at a lower frequency resulted in the OPTIS (TM) System. Up to three locations along a significantly lower cell viability inhibition rate. the LAD are chosen for ablation. The guidewire was kept Our study confirmed that high-frequency nano- in place during the ablation, but the OCT catheter was electrochemotherapy with bleomycin and doxorubicin pulled back to prevent damaging it. The OCT catheter could be an effective alternative for already established was re-advanced to the same location, and a measurement ESOPE procedures. The proposed MHz range pulse re- was taken immediately after each ablation. Additional petition protocols trigger better or equivalent to ESOPE measurements were taken 15, 30, and 60 minutes follow- electrochemotherapy efficiencies. Although, further in ing each ablation to evaluate the coronary recovery. 118 At every energy level of electroporation, there was an the model materials were set according to the frequency initial acute contraction event. The effective cross sec- of each technique. A temperature-controlled RFA strategy tion area of the LAD was reduced to an average of 60% was used maintaining a constant temperature (55 ºC) at from its baseline level regardless of voltage delivered. At the electrode sensor (application of 30 seconds). In PFA, 15 minutes the average lumen recovered to 70-80% from the delivery protocol included 20 bursts of biphasic pulses baseline and at subsequent time points there was full re- (150 kHz) of 100 µs per burst repeated at 1 Hz. The ap- covery of lumen size for both the 500V and 700V IRE plied PFA voltages were adjusted to get the same lesion treatments. The 900V treatment remained at an average depth than in RFA, using a reference blood velocity (6 of 75% lumen area when compared to its pre-ablation size. cm/s). In RFA tissues were considered ablated if above 50 Mean diameter of the LAD also observed a similar trend. ºC. In PFA tissues were considered ablated if the local elec- Optical Coherence Tomography is a useful tool to measure tric field was above 1000 V/cm. This PFA threshold was and indicate to what extent electroporation affects cardiac arbitrarily set as a wort-scenario case under some assump- coronary function. In this study, it was observed that tions from other studies. Also, the Arrhenius’ equation while there is an acute contractile response of the coron- was used to compute possible thermal damage in PFA. ary arteries to electroporation, recovery does take place Simulations predict lesion differences in terms of morpho- soon after treatment. Higher levels of energy delivered logy between RFA and PFA for both catheter configura-may increase the recovery period. However, even when tions. For the same lesion depth, PFA lesions are wider, the ablation is delivered directly on the coronary there is more symmetrical and with a bigger volume than those less than 15% reduction of vessel size after 15 minutes of obtained in RFA. Blood velocity has high impact only on IRE delivery. RFA: lesions being shallower, narrower and more asym- metric for low velocities. Perpendicular catheter orient- OR-183 ations with respect heart surface generate higher volume Differences in endocardial lesion morphology lesions than sideways angles in RFA. The opposite effect between Radiofrequency Ablation (RFA) and is predicted for PFA. Pulsed Field Ablation (PFA): a computational modelling study OR-184 Mario Gómez 1, Tomas Garcia-Sanchez2, Antoni Ivorra1 Experimental and numerical evaluation of effect 1Universitat Pompeu Fabra, Spain of tissue-electrode proximity during cardiac pulsed 2CNRS, Gustave Roussy, Université Paris-Saclay and Uni- field ablation versitat Pompeu Fabra, France Bor Kos 1, Brian Howard2, Atul Verma3, Wendy S. Tzou4, Lars M. Mattison2, Damijan Miklavčič1, Birce J. Onal2, Mark Radiofrequency ablation (RFA) is commonly used in T. Stewart2, Daniel C. Sigg2 the treatment of atrial fibrillation (AF). RFA applies al- 1University of Ljubljana, Faculty of Electrical Engineering, ternating currents to induce thermal damage by ohmic Slovenia heating in living tissues. It can cause blood clots and 2Medtronic, United States steam-pops increasing the risk of strokes or tamponade. 3McGill University, Division of Cardiology, Canada Besides, heating of adjacent structures increases the risk 4University of Colorado School of Medicine, United States of atrioesophagial fistulas or internal bleeding. To min- imize these risks, a novel technique, based on irrevers- Introduction: Pulsed field ablation (PFA) for the ible electroporation, has emerged: Pulsed Field Ablation treatment of cardiac arrhythmias is a method that has (PFA). This study offers a comparison of lesion morpho- recently been the subject of intense research. Thermal logy for monopolar and bipolar catheter configurations, catheter ablation is currently the cornerstone of treatment evaluating the impact of blood velocity and catheter ori- for drug-resistant AF [1], but these technologies rely on entation in the lesions inside the cardiac chamber. A com- electrode-tissue contact to achieve optimal results [2,3]. putational model was implemented for both techniques us- Because PFA relies on irreversible electroporation as a ing the same geometry: a slab of heart cavity; and three method to induce cell death, it has been hypothesized tissues: blood (in motion), cardiac and skeletal muscles. that PFA does not require direct contact with tissue to Non-irrigated catheters were considered. Both models in-maintain its efficacy. clude three physics (electric currents, thermal and fluid Methods: Isolated hearts were prepared from male dynamics) with a double-coupled problem between elec- Yorkshire pigs (n=8). PFA was applied to the epicardial tric and thermal solution to model tissue Joule heating, surface of the ventricle with a 4-electrode linear catheter and between thermal and fluid dynamics to model the immersed in heparinized blood, using an offset tool to cooling effect of blood in motion. The temperature de- precisely control the distance between the electrodes and pendence of the electrical conductivity of the living tissues the epicardium. After a 2-hour period, the tissue was was included to account for the changes in conductivity of stained with triphenyl tetrazolium chloride (TTC), and ionic solutions with temperature. In PFA, the electric field the cross sections were imaged and analyzed using Im- dependence of the electrical conductivity (modelling the ageJ. Two numerical models were created using COMSOL electroporation) was also included. The thermal problem Multiphysics: with the offset tool and with unlimited was modelled using the Pennes’ Bioheat equation and the blood domain. The numerical model was validated with blood flow was solved as a laminar flow using the Navier- the measured values of the current during the treatment. Stokes’ equation. The electrical and thermal properties of Different values of electric field thresholds were used to 119 extract measurements of lesion depth. These values were model. used to train an explicit numerical model of lesion depth Methods: Isolated porcine hearts (n=7) were perfused us- as a function of electrode-tissue distance. The model ing a modified Langendorff set-up using a Krebs-Henseleit was then used to determine the electric field strength buffer. The hearts were submerged in 0.45% saline to threshold that best fit the experimental data. mimic the conductivity of blood. Mechanical motion of Results: Average lesion depth was 4.3 ± 0.4 mm, 2.7 the heart was arrested by delivering blebbistatin (25 – ± 0.4 mm, and 1.3 ± 0.4 mm for the 0, 2, and 4-mm 50 g) retrograde to the aortic root. A prototype focal electrode-tissue distances, respectively. The resulting PFA catheter was held perpendicular to the epicardium slope of the linear regression was -0.74 (R2=0.91). The and lowered until contact was made while attached to a best fitting electric field strength in the numerical model force gauge (Mark-10, Copiague, NY). Once the catheter was 490 V/cm. The slope of the numerical model was was in place, 1500 V PFA waveforms were applied eight -0.72. The numerical model with unlimited blood domain times in a bipolar, biphasic manner. Contact force was had a slope of -0.95. automatically recorded during each waveform delivery Discussion: The numerical model agrees very well with using a LabView program and averaged. Multiple the experimental results and even manages to reproduce locations on the epicardium of the right and left ventricle some effects at the edges of the experimental offset tool were ablated, avoiding coronary arteries and epicardial setup. Since the offset tool had a limited extension fat. Lesions were allowed to mature for 90 minutes, around the electrodes, the electric current density at the cross sectioned, and stained with triphenyl tetrazolium edges of the offset tool was larger than in the model with chloride to evaluate tissue viability. The lesions were unlimited blood pool. This resulted in a lesion depth that photographed, and the widths and depths were measured decreased less than the distance between the electrodes using ImageJ (NIH, Bethesda, MD). and the tissue. However, in the model where the current Results: A total of 83 lesions were evaluated with contact was not restricted with the offset tool, the depth of the forces between 1.3 g and 48.6 g. Lesion depth ranged lesion decreased at approximately the same rate as the from 2.1 mm to 7.4 mm with a mean depth of 4.8 ± distance between the electrodes and tissue increased. 0.9 mm. Lesion width ranged from 5.3 mm to 13.5 These results indicate that PFA does not critically depend mm with an average depth of 9.1 ± 1.3 mm. A linear on contact between electrodes and tissue. model showed an increase of 0.02 mm in depth (Depth = 0.02*Contact Force + 4.37) and 0.03 mm in width (Width References: = 0.03*Contact Force + 8.32) for each additional gram 1 Calkins H et al. Heart Rhythm 2017. of contact force. When evaluating low (0-8g, n=13)), doi:10.1016/j.hrthm.2017.05.012 medium (8-30g, n=37), and high (>30g, n=32) contact 2 Reddy VY et al. Heart Rhythm 2012. force, lesion depths were 4.3 ± 1.1 mm, 4.7 ± 0.9 mm, doi:10.1016/j.hrthm.2012.07.016 and 5.0 ± 0.8 mm respectively. Lesion widths for low, 3 Natale A et al. J Am Coll Cardiol 2014. medium, and high contact forces were 8.5 ± 1.4 mm, 8.9 doi:10.1016/j.jacc.2014.04.072 ± 1.0 mm, and 9.6 ± 1.4 mm. No statistical difference was observed between lesion depth, but lesion width with OR-185 high contact force was significant (p<0.05) compared to Effect of Contact Force on Pulsed Field Ablation the low and medium contact force groups. Lesions in Porcine Cardiac Tissue Conclusion: Increasing contact force using a bipolar, Lars M. Mattison 1, Atul Verma2, Tobias Reichlin3, Birce J. biphasic focal PFA system has minimal effects on acute Onal1, Kevin Sack1, Megan M. Schmidt1, Damijan Miklavčič4, lesion depths or widths in an isolated porcine heart model. Daniel C. Sigg1 1Medtronic, United States 1. Nakagawa, H., Castellvi, Q., Neal, R., Girouard, 2McGill University, Canada S., Ikeda, A., Kuroda, S., Hussein, A.A., Saliba, W.I. and 3University Hospital Bern, Switzerland Wazni, O.M., 2021. B-PO03-131 effects of contact force 4University of Ljubljana, Faculty of Electrical Engineering, on lesion size during pulsed field ablation. Heart Rhythm, Slovenia 18(8), pp.S242-S243. Background: Thermal based cardiac ablation techno- logies require tissue contact to enable heat transfer to the tissue to form a lesion. Due to this, contact force has P27 - In memoriam of Justin Teissié - been used as a tool to ensure lesion formation clinically. N’espérez pas, mesurez Pulsed Field Ablation (PFA) is a field-based ablation (don’t expect, measure) technology for which limited evidence of the importance of contact force on lesion size is available. Initial work Wednesday late afternoon Track has been done to establish the role of contact force with a unipolar, biphasic system, but a bipolar, biphasic system A has yet to be evaluated.1 Oct 12, 16:00 - 17:30 Objective: Determine the relationship between contact force and lesion size using a focal PFA Catheter with a bipolar, biphasic PFA system in an isolated porcine heart 120 OR-219 resealing back to the closed membrane. It is the slow N’ESPÉREZ PAS, MESUREZ (DON’T EXPECT, post-field responses that indicate „structural longevity“ MEASURE) of the induced porous states causing permeability for Marie-Pierre Rols Justin Teissié, a biophysicist, pioneer in the study molecules (electropermeabilisation) that usually do not of biological membranes and the basic processes of elec- penetrate the membrane like DNA. Long-lasting porosity troporation, passed away on September 2020. After his underlies transient endocytotic encapsulation of plasmid post-doctorate in the laboratory of Prof. T.Y. Tsong, at DNA. Another long-lived post-field effect refers to elec- the Johns Hopkins University, USA, he created and de- troporated cells in close contact slowly fusing to large veloped his research team in Toulouse, France. He has multinuclear syncytia (electrofusion) [4]. Long-lived pore dedicated his life to the study of biological membranes states also rationalize the accumulation of the effect of and the fundamental processes leading to membrane per-consecutive pulses in a pulse train. Physical chemical meabilisation, and has played a major role in the devel- thermodynamics rationalizes both rapid in-field poration opment of its applications in the medical field for elec-and slow post-field pore annealing and chemical repairing trochemotherapy and in industry for sterilization. He has as the two branches of the hysteresis in the degree of been equally aware of the need and importance of devel- poration f(E) as a function of the field intensity (E). oping fundamental knowledge and understanding of cell Oscillations in small-ion conductivity and in global shape membrane electropermeabilisation as well as of develop- of vesicles at constant applied field both represent the ing and promoting use of electroporation in medicine and cycling around the hysteresis loop of structural changes biotechnology. One of his favorite sentence was « Go back [5,6]. to the bench ». While being a researcher at the CNRS, he has constantly paid great attention to the training of [1] E. Neumann et al. (1982) Gene transfer into young people, trainees, doctoral and post-doctoral stu- mouse lyoma cells by electroporation, EMBO J. 1, dents. He was one of the funding faculty member of the 841-845. Electroproation Based Technologies and Treatments Post- [2] E. Neumann and K. Rosenheck (1972) Permeability graduate Course and International Scientific Workshop in changes induced by electric impulses in vesicular mem- Ljubljana, Slovenia. branes. J. Membr. Biol. 10, 279-290. Justin Teissié has been always asking questions re- [3] T. Griese et al. (2002) Conductometric and electro- minding us that our knowledge is still incomplete. We, optic relaxation spectrometry of nano-sized lipid vesicles, his students, colleagues and friends will never forget him, Phys Chem Chem Phys 4, 1217-1227. and he will remain as a model capable of enlightening our [4] E. Neumann et al. (1980) Cell fusion induced by future choices. high electric impulses applied to Dictostelium, Naturwis- senschaften 67, 414-115. OR-72 [5] V. A. Dimitrov et al. (2013) Transient oscillation of Fourty Years of Electroporation (1982-2022) – shape and conductivity in nano-sized vesicles, Phys Chem New View on the Poration-Resealing Hysteresis Chem Phys 15, 6303-6332. Eberhard Neumann [6] E. Neumann and S. Kakorin (2018) Membrane elec- Bielefeld University, Faculty of Chemistry, Germany troporation: chemical thermodynamics and flux kinetics revisited, Eur Biophys J. 47, 373-387 The term „Electroporation“, short for „electric pore formation“, has been coined in 1982 in the context of OR-222 the first electro-reprogramming of biological cells with Information about the ISEBTT « Justin Teissié » added foreign DNA by trains of electric pulses [1]. Prior Award to functional electro-uptake, it had been found in 1972 Muriel Golzio that electric pulsing causes release of intra-organellular CNRS, Institut de Pharmacologie et Biologie Structurale components without impairing cell viability [2]. The (IPBS), France concept of electroporation (EP) qualifies the very struc- tural basis of this electric field effect. The applied field This award is endorsed by the ISEBTT, and the nom- (pulse) causes rapid nucleation of electropores at „weak inees have to be members of our Society in good stand-membrane sites“ (e.g., short-chain lipids). In particular ing. It is the first price of our Society and we expect that structural electro-optic and ion-conductivity relaxation other awards will enlarge the list of the ISEBTT mer- spectrometry reveal that „two types of electropores“ are itorious members that our Society will recognize though apparent [3]. At low field intensity, small (Type I) pores these awards. The « Justin Teissié » Award is funded are dominant; caused by changes in the (non-covalent) by our members (and non-members, they are also wel-hydrophobic lipid-water interactions. At higher field come) through donations to a specific account in the Soci- intensities the „new interpretation“ suggests that pore ety, which was initiated by Justin’s colleagues and that is nucleation is preceded by chemical bond breaking. Field- fully dedicated to perpetuate the attribution of the Award. induced radicals cause lipid fragments (evidenced by Mir Contributions are welcome! et al.) that, as a part of the pore wall, constitute the larger Type II-pores as the origin for release of intracel- lular components under Maxwell stress (cell elongation). After pulse termination, the kinetic data indicate pore 121 OR-122 tures where production of fibrous extracellular matrix oc- Non-canonical biological targets of intense pulsed curs. In particular, it was evaluated how an accurate electric field: proteins simulation of the extracellular environment can predict Michal Cifra more accurately the electric field around the cell mem- Institute of Photonics and Electronics of the Czech Academy brane. Since the electric field distribution is correlated to of Sciences, Czech Republic the cell permeabilization, the electric field intensity using Pulsed electric field research and technology primar- different suspension media (e.g. RPMI or electroporation ily focus on the membranes, and for good reasons. buffer with low conductivity) was assessed. The electro- However, the growing accessibility of technology, which poration of cell was verified by means of cell suspension provides higher electric field strength enables triggering experiments. the response in the nanoscopic electromechanical machines It is well known that the distribution of the electric field which power life: proteins. Huge diversity in the proteome intensity in a material with non-homogeneous electrical opens new horizons for PEF in both fundamental biophys- characteristics is different respect to the one obtained in ics research, bionanotechnology, and applications. In this homogeneous electrical properties. In particular, the elec- talk, I present an overview of electric field effects on pro- tric field in presence of cells was simulated by FE Model teins, focusing on proteins that build up and interact with considering a parallelepiped and applying a voltage to two the cellular skeleton – a crucial system enabling intracellu- parallel faces to obtain an electric field with intensity equal lar transport and cell division. On an example of proteins to 1000 V/cm. In both models, homogeneous and non- constituting one type of cytoskeleton fibers - microtubules, homogeneous, cells were represented as circular objects. I will explain the primary mechanisms of coupling of an In the non-homogeneous model, the simulated region in- electric field to proteins. Then I will highlight selected cluded fibrous collagen areas immersed in the studied me- technologies combining advanced microscopy techniques dia to generate a discontinuity in electric conductivity. and chips that enable us to explore the real-time effects The electrical properties were experimentally determined of PEF. These results deepen our understanding of mo- and compared with those in literature. The numerical lecular level effects of electric field on proteins and open model assessed the intensity of the field in the examined new possibilities in biomedical and bionanotechnological region containing areas of non-homogeneity and compared applications. it with the field obtained in the same areas under condi- MC acknowledges support from Czech Science Found- tions of homogeneity. ation GX20-06873X. The local discontinuity could improve the electroporation efficiency for the same applied electric field. The data obtained showed that the cellular organization and the P33 - Imaging and treatment planning presence of extracellular matrix can modulate the local in clinical trials electrical properties influencing the efficiency of electro- poration. Wednesday late afternoon Track Simulation results were validated by means of suitable ex- periments with HCC1954 breast cancer cells in suspen- B sion and cultured on a hyaluronic acid (HA) hydrogels en- Oct 12, 16:00 - 17:30 riched with self-assembling peptides carrying IKVAV mo- tifs. Cell culture and suspensions was electroporated in OR-39 a parallelepiped chamber slide using EPS-01 (Igea S.p.A, Finite Element evaluation of the electric field dis- Carpi, MO, Italy) and a plate electrode. Electroporation tribution in a non-homogeneous environment was evaluated using Propidium Iodide. Elisabetta Sieni 1, Bianca Bazzolo2, Monica Dettin2, Maria Evelina Mognaschi3, Paolo Di Barba3, Michele Forzan2, Annj OR-34 Zamuner2, Patrizia Lamberti4, Maria Teresa Conconi2 Monitoring of current density and electric field dis- 1University of Insubria, Italy tribution during electroporation of heterogeneous 2Padova University, Italy tissues using MR techniques 3Pavia University, Italy Matej Kranjc 1, Marko Strucic1, Jessica Genovese2, Rok 4University of Salerno, Italy Šmerc1, Vitalij Novickij3, Samo Mahnič-Kalamiza1, Igor In this work, the effect of the electrical characteristics Serša4, Damijan Miklavčič1 1 of the extracellular environment on the electric field dis- University of Ljubljana, Faculty of Electrical Engineering, tribution in the electroporation conditions was studied by Slovenia 2 means of the Finite Element (FE) simulation and exper- University of Bologna, DISTAL, Italy 3 iments. In particular, the simulation results, exploiting Vilnius Gediminas Technical University, Lithuania 4 experimentally acquired electromagnetic properties of the Institut “Jožef Stefan”, Slovenia materials, showed how using the same boundary condi- Electrical properties of biological tissues have been of tions the inhomogeneities of the electrical characteristic interest for over a century as they determine the path-can modify the electric field distribution around the cells ways of current flow through the body, and are therefore respect to that happens by using a homogeneous media. important in the analysis of a wide range of biomedical ap- This is the case of in vitro experiments that include cells plications. Numerous factors determine electrical proper- electroporated in suspension compared to cells in 3D cul- 122 ties of biological tissues, such as cellular structure, amount and the dispersion of the media composing the biological of intra- and extra-cellular fluids, concentration and mo-cell. bility of ions in these fluids, temperature of the tissue, and Most of the computational schemes rely on the finite- their pathological conditions, to name only a few. Tissue element method or the transport lattice method. A Finite is a heterogeneous material with important interfacial pro- Volume method is proposed here because it is expected, cesses and cells of uneven size and different functions, and first to ensure flux consistency at each mesh cell, second to so from the electrical point of view, it is impossible to con- preserve the basic physical properties of the Poisson equa- sider tissue as a homogeneous material. tion, and third to provide accuracy on a coarse mesh with Measuring and evaluating electrical changes in real time is high heterogeneities. The Discrete Dual Finite Volume important for electroporation-based treatments and tech- (DDFV) scheme is a numerical method that has been ori-nologies. Therefore, an efficient approach is needed to ginally developed to solve the Poisson equation on coarse monitor the electric field in studied tissue during the deliv- and conforming mesh without the orthogonality condi- ery of electroporation pulses. Electric field can be recon- tions. structed from current density distribution data by mag- A complete electromagnetic model of a biological cell is netic resonance electric impedance tomography (MREIT). proposed. The Poisson equation is solved by a DDFV MREIT is enabled by Current Density Imaging (CDI), an method. Time-varying fields are computed by assuming MRI modality designed to detect electric currents via the that the quasi-static approximation is valid. Conductive temporal change of magnetic field that is induced by the media are modeled by a metal-dielectric equivalence. The currents. Since monitoring is performed during pulse de- cell model is oversimplified. It consists of a sphere sep- livery, the determined electric field distribution considers arated from the biological solution by a dielectric layer. all heterogeneities and changes that occur in the treated The problem is clearly 2.5D (axisymmetric 2D). The cell tissue. is described by three media: the inner of the cell, the mem- This work will give an overview of monitoring of elec- brane, and the biological solution. The electric transition tric field distribution in tissues of different levels of struc- conditions between each of them are accounted for. All of tural complexity. Experiments were performed in apple this leads to a generalized Poisson equation resolution with fruit, potato tuber, and carrot tissue since these vegetable appropriate jump conditions. Note that the membrane is matrices are – in this order – of ever increasing complexity. meshed so that the transmembrane potential is calculated Additionally, muscle tissue samples were taken from the by a simple difference of potential. DDFV unknowns are neck and thigh region of a domestic pig (Sus domesticus) located at each center of the mesh cell and also on ver- in order to evaluate anisotropy. Electroporation protocol tices. A new discrete balance equation is then associated consisted of two sequences of 4 pulses with a duration with each vertex. Two dual meshes are then introduced. of 100 µs per pulse and with a repetition rate of 5 kHz. The trans-membrane potential is computed without any The voltage amplitude was adjusted to obtain a sufficient interpolation or refinement. Numerical experiments are signal-to-noise ratio of the electric field in the sample. Ap-proposed. The time variation of the membrane potential plication of electric pulses was triggered by an MRI control when a nanosecond voltage pulse is applied to the biolo-unit and synchronised with the CDI pulse sequence. gical solution is given as an example. The dispersion prop- MREIT demonstrated to be suitable for monitoring of the erties of each medium are modeled by the classical Debye electric field distribution inside structures of plant tissues, formulation. The results are compared to those obtained whereas in muscle tissues we were able to detect different in the literature. current density behaviour based on application of electric pulses relative to the muscle fibre orientation. These find- OR-37 ings provide useful insights into the evaluation of electro- Coarse-to-fine segmentation of needles on CBCT poration and suggest that magnetic resonance techniques scans for the evaluation of electroporation ablation could be used as an efficient tool to improve the effective-Eloise Inacio 1, Luc Lafitte1, Olivier Sutter2, Olivier Seror2, ness of electroporation-based treatments. Baudouin Denis de Senneville1, Clair Poignard1 1INRIA and Université de Bordeaux, MONC, France OR-35 2Hosp. Paris Seine Saint Denis Avicenne Hosp., APHP, Univ. Discrete Dual Finite Volumes (DDFV) for electro- Paris 13, France poration modeling Electroporation ablation is a promising technique for Thomas Bonnafont1, Delphine Bessières2, Jean Paillol 2 the removal of deep-seated tumours as it can be used near 1Lab-STICC, UMR CNRS 6285, ENSTA Bretagne, France vital structures without affecting them. Indeed, it is min- 2Laboratoire SIAME, Université de Pau et des Pays de imally invasive and produces little thermal effects unlike l’Adour/E2s, France more traditional techniques such as radio-frequency abla- Numerous experimental studies have been carried out. tion. However, it remains very complex and thus requires In this context, the trans-membrane voltage is a data of cautious planning and evaluation. Our goal is to enable importance, it is generally determined by computation. the surgeons to evaluate the electroporation ablation pro- However, few accurate computational models have been cedure online by providing a visualisation of the electric developed. Modeling is challenging because of the thin field distribution along with the tumour localisation. thickness of the membrane compared to the overall di- A crucial step in this matter is the segmentation of the mensions, the electric field jump conditions at interfaces, electric probes delivering the electric pulses that leads to 123 the irreversible electroporation of tumorous cells. From form seamlessly integrates tools to plan patient-specific the localisation of the probes, the electric field distribu-electroporation interventions. First, patient anatomy is tion can be modelled to determine the area where the segmented from medical images and 3D reconstruction treatment is effective. However, challenges arise from the aids the user in placing the electrodes and setting up nature of the scans (CBCT scans have low contrast and treatment parameters. The electric field is then simulated low signal-to-noise ratio but low radiation exposure) and considering tissue non-linearities typically observed during the nature of the objects to segment (the probes are metal- electroporation therapies, that is, the dependency of the lic and thin, so artefacts are produced during capture and conductivity on the electric field and the dependency of the dataset is highly unbalanced). the conductivity on the temperature. Finally, thanks to To tackle this task, we propose a coarse-to-fine seg- the included postprocessing tools, the user can easily eval- mentation algorithm combining deep learning for the uate treatment feasibility and fine-tune the parameters to coarse segmentation of the probes and the Hough trans- ensure an optimal outcome. To illustrate the use of the form for their precise localisation. A modified U-Net with platform, four canine patients that had been treated with a patch optimisation learning strategy and a loss based irreversible electroporation were retrospectively planned on the Dice coefficient and the cross entropy provides with PIRET and with a workflow commonly used in previ-a rough segmentation mask then converted into a point ous studies, which uses different general-purpose software cloud. The Hough transform, completed with a voting platforms for each of the planning steps. We found that procedure, is applied to provide an analytical represent- PIRET was the fastest, outperforming the other method ation of the needles. Finally, a standard linear model is by 65 minutes on average (×1.7 faster). Both approaches used to compute the electric field from the probes local- computed similarly accurate electric field distributions, isation and the pulses information. It is known to under- with Dice scores across a sweep of the electric field higher estimate the electric field and thus leads to a suitable first than 0.93. The platform here presented is, to the best of approximation from the medical point of view. our knowledge, the most complete software for treatment The algorithm is evaluated on 8 of the 16 patients from planning in electroporation-based therapies, not only for our database, the rest of the data being used in the train- solid tumor treatment, as it was illustrated here, but po- ing of the U-Net. The artificial neural network is assessed tentially for other electroporation applications. Remark- by comparing the predicted segmentation and the ground ably, PIRET is an offline platform. This avoids transfer- truth with the Dice coefficient. The overall approach is as- ring personal sensitive data regarding patients. PIRET sessed by computing the distance between the needle tips stands for ”Planning Irreversible and Reversible Electro- as segmented and their real coordinates. Our approach is poration Treatments”. shown to be more robust and precise than the previously used segmentation based on thresholding and the Hough OR-36 transform. It is also suitable for an online evaluation dur- Electric Field Assisted Volumetric Tumor Profiling ing the procedure as it provides the probes location under Mary C. Sheehan, Yasushi Kimura, Neeraj Raghuraman two minutes on a commodity hardware. Rajagopalan, Brian Simoes, Govind Srimathveeravalli University of Massachusetts Amherst, United States OR-38 Introduction: Tumor sampling by Fine Needle Aspir- PIRET: a Software Platform for Treatment Plan- ation (FNA), a common technique for molecular cancer ning in Electroporation-based Therapies diagnosis, is restricted to the immediate vicinity of the Enric Perera-Bel1, Kenneth N. Aycock2, Zaid S. Salameh2, needle, with considerable variability in contents. Revers- Mario Gómez1, Rafael V. Davalos2, Miguel A. González ible electroporation (RE) is known to release intracellu- Ballester1, Antoni Ivorra 1 lar materials from permeabilized cells. We investigated 1Universitat Pompeu Fabra, Spain RE for enhancing FNA sample extraction and whether 2Virginia Tech, United States computational models can map the tumor volume being Tissue electroporation is the basis of several therapies. sampled. Electroporation is performed by briefly exposing the tis- Materials & Methods: Mouse breast (4T1) and blad- sues to high electric fields. These fields are typically gen- der cancer (MB49) cell lines underwent EP in a 4 mm erated by delivering short high-voltage waveforms across gap cuvette or in a 3D agarose hydrogel tumor mimic (2 electrodes in tissue. It is generally accepted that elec- pin electrodes, 1cm gap). EP parameters were screened troporation is effective where the electric field magnitude (1000-2000 V/cm, 8-15 pulses, 80 µs length) to determ- threshold is overreached. In therapies based on electropor- ine RNA (Nanodrop) and protein (BCA Assay) levels in ation, it must be ensured that the whole target volume is the sample and were adjusted for cell viability and prolif- covered by suprathreshold fields. However, it is difficult eration (Trypan blue stain and CCK-8 assay). Agilent to preoperatively estimate the field distribution because it Bioanalyzer and Coomassie brilliant blue staining were is highly dependent on patient anatomy and several treat- used to compare the molecular weight spectrum of RNA ment parameters. Thus, there is a need for treatment and proteins eluted by EP treated cells and whole cell lys-planning tools for therapies based on electroporation. In ate. A Comsol model of the in-vitro tumor mimic was used response, we have created PIRET, a platform to preoper- to map the electric field strength and energy delivered with atively predict the treatment volume in electroporation- the volume sampled by using two different reporters (cells based therapies that is user-friendly and fast. The plat- with GFP and Cell Tracker dye). Fluorescent microscopy 124 with nucleus (DAPI) and viability stains (Propidium Iod-high-intensity pulsed electromagnetic fields (HI-PEMF) ide/Calcein AM) were used to establish spatial extent of to achieve membrane permeabilization. Even though EP. In-vivo validation was done in mice with bilateral sub- the induced electric fields in our experiments were cutaneous tumors, comparing FNA +/- EP for total RNA significantly lower (up to 20 V/cm and 0.23 V/cm in and protein yield. Immunohistochemistry was performed experiments in vitro and in vivo, respectively) we have to identify damage or alterations in the tumors from EP. observed membrane permeabilzation as detected by Results & Discussion: Increasing EP energy dose correl- fluorescent dyes such as Propidium Iodide and YO-PRO ated to the quantity of RNA and protein in samples and in vitro; we then compared and achieved successful a reduction in cell viability and proliferation. Pulse para- electrochemotherapy in vivo in experimental murine meters with optimal viability (1500 V/cm, 8 pulses at 80 tumor model which yielded similar tumor growth as µs) had over 70?ll viability. At this EP condition, protein obtained by classical electrode mediated electroporation levels were 5:1 and 3:1 and RNA levels were 3:1 and 2:1 for both with cisplatin and bleomycin. Using HI-PEMF 4T1 and MB49 cells respectively when compared to sham. we also achieved electrotransfer of siRNA and plasmid Bioanalyzer results showed comparable levels for small and DNA in vivo, and recently also successfully achieved gene medium sized RNA, with whole cell lysate having slightly electrotransfer in vitro by HI-PEMF. higher levels of large RNA. In the 3D tumor mimic gel, Although the results obtained by HI-PEMF are compar- levels of material extraction correlated to the region show- able (albeit sometimes inferior) to electroporation using ing PI staining and could be controlled to extract a pre-direct contact of electrodes with target cells and tissue, dictable ratio of GFP and cell track dye. Simulation mod- the benefits of using non-contact electroporation are els calibrated to the 3D tumor mimic could be used to plan appealing. In the presentation the evidence available in region of dye extraction. Mouse studies demonstrated that the literature will be presented and possible mechanisms EP+FNA yielded more protein and RNA from both 4T1 discussed. (4:1 and 10:1, respectively) and MB49 (17:1 and 6:1, re- spectively) tumors versus FNA alone. T cell/macrophage References: stains showed that EP did not cause inflammation and Novickij, V. et al. IEEE Trans. Magn. 56, 1–6 (2020) TUNEL stains revealed that regions of necrosis were no Kranjc, S. et al. Radiol. Oncol. 50, 39–48 (2016) different from samples that underwent FNA alone. Kranjc, M. et al. Bioelectrochemistry 141, 107847 (2021) Conclusion: EP can enhance FNA sampling without in- Hu, Q. et al. IEEE Trans. Plasma Sci. 48, 1088–1095 jury to the tissue being sampled. Computational models (2020) can assist pulse parameter optimization and estimate volu- metric region being sampled. OR-23 Ultrashort high-intensity pulse generators for sim- ultaneous cellular permeabilization and endoscopic P36 - New Technologies for Cells and imaging for future biomedical applications Tissues Electropermeabilization-II Rosa Orlacchio, Nour Tabcheh, Delia Arnaud-Cormos, Philippe Leveque Wednesday late afternoon Track University of Limoges, CNRS, XLIM, UMR 7252, F-87000 , France C Oct 12, 16:00 - 17:30 High-intensity (˜50–100 kV/cm) ultrashort (duration < 1 µs) pulses can be used as a powerful tool for intracellular manipulation. Emerging technologies use OR-147 ultrashort pulses, for example, for cardiac defibrillation, Membrane permeabilization by high-intensity neurostimulation, or cancer therapy [1]. Indeed, pulses pulsed electromagnetic fields – a non-contact elec-with a duration of the order of the ns (nsPEF) and troporation? Damijan Miklavčič field strengths of a tenth of kV/cm, may affect internal University of Ljubljana, Faculty of Electrical Engineering, cellular structures inducing numerous bioeffects such as Slovenia membrane permeabilization through the formation of nanopores, loss of mitochondrial activity, or modulation Electroporation of cell membrane leading to increased of ion channels [1]. However, the delivery of strong membrane permeability that is exploited in electrochemo- electric fields of very short duration complicates the therapy and gene electrotransfer is usually achieved by engineering of the devices that require specific advanced exposing cells in vitro or in vivo to high voltage electric technologies to produce pulses with controllable amp- pulses. Although the value of electric field achieving litude, energy, power, duration, and shape. In the last membrane peremabilization will vary and will depend on 20 years, numerous generators have been developed cell size, orientation, density in vitro and tissue types paving the way for bioelectric studies [2]. To contribute in vivo as well as on pulse duration and number of to the understanding of biological effects induced by pulses applied the electric fields to which cells need to be ultrashort pulses, we designed and characterized –in the exposed are in the range of hundreds of V/cm. In all these frequency and time domains– novel versatile generators applications electrodes in contact with the medium/tissue able to provide unipolar, bipolar, and paired pulses of are used. about 10 ns, 1 ns, and a few hundred ps. The main We have used electric field induced by time varying 125 novelty of our generators consists in obtaining a very base of the triangle changed by 180o, but this angle short delay between the pulse polarities in the ns range gradually decreased to 0o closer to the apex electrode. (˜5 to 350 ns) which seems to play an important role in As a result, cells situated near and between the two cellular response [3]. The proposed systems allow pulse alternately energized electrodes experience “true” bipolar generation and delivery through a mechanism based on a electric field oscillations, while cells close to the apex “slow charging and fast discharging” of the energy also see only a small change of the electric field vector. A known as the frozen wave principle. This means that the train of unipolar pulses applied alternately to the base energy of a high voltage power supply is stored into a electrodes generates a long unipolar pulse near the apex, pulse-forming line and then released to a load (e.g., cells) with duration equal to the train duration. through a specific delivery system (i.e., electrodes) using We have employed this 3-electrode configuration in two high-power ultrafast switching elements. By exploiting different experiments. In the 1st one, we studied the the propagation of the electromagnetic waves in the line, effect of the vector change angle on the efficiency of we generated pulses with different duration, amplitude, electroporation by 600-ns pulses in BPAE cell monolay- shape (unipolar, bipolar, and paired), and interphase ers. The maximum bipolar cancellation (13-fold vs a delays. In addition, the characterization of a generator unipolar pulse) was observed with 180o vector change. providing pulses of the order of a few hundred ps is Cancellation tapered out towards the apex electrode ongoing. An optoelectronic delivery system based on a because of the reduction of the vector change angle. At pair of electrodes embedded in a polyethersulfone (PES) angles less than 90-120o, cancellation was replaced by fiber that also encloses an optical fiber will be used for summation, i.e., the effects became stronger (3-fold at the simultaneous pulse application and acquisition of 22o). In the 2nd experiment, we utilized this effect to endoscopic images. Bioelectric in vitro investigations are evoke electroporation and excitation (measured by Ca2+ envisaged aiming to the future development of medical mobilization) in smooth muscle cell monolayers. We applications using ultrashort pulses that may selectively achieved the effect at the apex electrode while avoiding target specific intracellular structures. it at two base electrodes, despite the stronger electric field there. Indeed, bipolar nsEP oscillations between References alternately energized electrodes had low biological ef- [1] S. Beebe et al., ‘Ultrashort Electric Pulse Effects in ficiency (because of both the ns duration and bipolar Biology and Medicine’, Bioelectrics Books, 2021. pulse shape). The same pulses merged into a long [2] D. Arnaud-Cormos et al., ‘Photoconductive switching unipolar pulse near the apex electrode. The duration for pulsed high-voltage generators’, in Handbook of of this created unipolar pulse can be made in µs or ms Electroporation, 2017, pp. 1–21. range, making it far more efficient than the bipolar ns [3] A. G. Pakhomov et al., ‘Cancellation of cellular re- oscillations. This effect can be utilized for enhancing focal sponses to nanoelectroporation by reversing the stimulus stimulation or electroporation at one of the electrodes polarity’, Cell. Mol. Life Sci., 2014, vol. 71, no. 22, pp. while fully avoiding it at the other electrodes. We are 4431–4441. further exploring the utility of this method for targeted focal brain stimulation and for directed “unipolar” tumor ablation. OR-158 Support: AFOSR MURI grant FA9550-15-1-0517 to Effect of the electric field vector change on the AGP. efficacy of nanosecond pulse trains Vitalii Pavlovich Kim, Andrei G. Pakhomov OR-146 Old Dominion University, United States Targeted excitation of murine hippocampal neur- A phenomenon of bipolar cancellation stands for the ons by spatiotemporal summation of nanosecond suppression of bioeffects of nanosecond electric pulses electric pulses (nsEP) when the electric field polarity is reversed. In Iurii Semenov 1, Tatiana Zvonareva1, Vitalii Kim1, Joel Bixler2, Allen Kiestler2, Bennet L. Ibey2, Stephen J. Beebe1, most studies, bipolar electric field is generated by ap- Andrei G. Pakhomov1 plying bipolar voltage to one electrode, while the second 1Old Dominion University, United States electrode serves for current return (ground). Bipolar 2Air Force Research Laboratory, United States electric field can also be created by applying unipolar pulses to two electrodes in alternation, when the electrode Nanosecond electric pulses (nsEPs) are inherently less not energized becomes ground. In both cases, the electric efficient than conventional ”long” pulses for neurostimula- field vector turns by 180o. To change this angle, we tion without cell damage. However, nsEPs can be utilized added a third electrode forming an isosceles triangle. to create novel and unique stimulation protocols. We This electrode at the apex was always connected to the took advantage of the bipolar cancellation phenomenon ground, while the other two electrodes were energized when nsEP effects are inhibited by switching the pulse in alternation. This way, the electric field lines from polarity. We used the bipolar cancellation phenomenon either active electrode converged on the apex electrode, in a 3-electrode array to trigger action potentials (APs) merging into a single long pulse. In other words, the in dissociated hippocampal neurons by spatiotemporal electric field direction between the electrodes at the summation of nsEPs at a chosen location. 126 Neurons were enzymatically isolated from newborn mice Sabatier, France, France and incubated in glass-bottomed culture dishes for 7-10 2Laplace UMR CNRS 5213, INFINITY INSERM , Université days until they started producing APs. To visualize Toulouse III - Paul Sabatier, Infinity, Toulouse University, plasma membrane (PM) electric potential changes, neur- CNRS, Inserm, Toulouse III – Paul Sabatier University, France ons were loaded with a FluoVolt voltage-sensitive dye. , France Time-lapse image stacks (3048 frames/s) were acquired 3INFINITY INSERM , Université Toulouse III - Paul Sabatier, using a CCD Camera on an inverted microscope. France The electrode array for neuron excitation constituted 4CNRS, Laboratoire LAPLACE, France three needles in the isosceles right triangle positioned Under physiological conditions, reactive oxygen spe- 50 µm above a culture dish perpendicular to the bot- cies (ROS) are known to play a role in cell signaling, tom. A bipolar electric field was created by alternating regulating a wide variety of functions, especially those twenty-four 300 ns unipolar electric pulses between two implicated in extracellular matrix (ECM) remodeling electrodes where one electrode served as a ground at [1]. Thus, the use of ROS-modulating technologies is the moment when the other was energized. Both active becoming a relevant and fruitful strategy to locally electrodes were distanced 1mm apart from the third, modulate ECM [2]. Cold atmospheric plasma (CAP) is permanently a ground one where electric field lines an emerging technology that generates a unique cocktail converged, combining into a single long pulse. Such of short- and long-lasting reactive oxygen and nitrogen exposure aimed to use bipolar cancellation to suppress species (RONS). Main medical applications of CAP are neuron excitation anywhere within the array except for antitumor strategy via multiple mechanisms of action and the targeted region at the ground electrode. wound treatment [3]. In antitumor context, it is nowadays To provide experimental validation of electric field bipolar largely unknown how CAP therapy outcome is affected oscillations, we used a custom-made strobe imaging by tumor microenvironment and more particularly ECM system to observe changes in the PM electric potential of [4]. CHO-K1 cells reflecting the electric field vector alterations In our study [5], we assessed the potential of direct cold upon exposure. Closer to active electrodes, PM potential atmospheric helium plasma jet to remodel collagen at at the side of the cell facing either electrode alternated dermal tissue scale. Four distinct conditions were studied: from de- to hyperpolarization at 300 ns intervals. Ex- no exposure (control), 30 sec exposure to helium plasma posure of the cell in a targeted region resulted in a �7 jet, 2 min exposure to helium plasma jet and control µs long continuous PM depolarization at the side facing condition of 2 min exposure to helium gas alone. First, the ground. If above the excitation threshold, this PM we chemically quantified major RONS generated in our depolarization would be sufficient to excite neurons. experimental set up. Secondly, we checked the antitumor Neurons were exposed at various distances from the properties of this plasma treatment onto 3D tumor spher- ground toward active electrodes to analyze how their oid model composed of human colorectal tumor HCT-116 position within the electrode array affects AP initiation. cells. Finally, using an original tissue-engineered human The amplitude of nsEPs in the trains was increased dermal substitute model rich in endogenous extracellular until a threshold of AP initiation. We found that AP matrix, we analyzed collagen remodeling after CAP can be triggered at the same threshold within 100 µm treatment through quantification of pro-collagen I secre- from the ground. In the range from 100 to 300 µm, the tion (ELISA), dosage of global metalloproteases MMPs AP threshold increased twofold. Farther than 300 µm activity (fluorimetry), MMP1 quantification (ELISA), from the ground, there was no successful AP initiation and chemical quantification of hydroxyproline (amino regardless of pulse amplitude. If neurons were exposed to acid specific to collagens family) of the whole tissue. a 7 µs pulse instead, AP was initiated anywhere within Our results indicated that H2O2, NO3- and NO2- were the array. This result shows how the bipolar cancellation produced during exposure to helium plasma jet. In phenomenon in the 3-electrode array can be utilized for our experimental condition, we confirmed that 2 min targeted stimulation at a single electrode. exposure to helium plasma jet was the only condition to induce tumor cell apoptosis and spheroid growth delay. Support: AFOSR MURI At human dermal tissue scale, the conditions applied did not 1) alter cell viability after 24h, 2) alter pro-collagen P12 - Electroporation and Cellular I secretion over 48h after treatment, 3) modify global Pathways MMPs activity over 48h after treatment, and 4) change hydroxyproline content over 5 days after treatment. The only modification observed was a transient increase in Thursday morning Track A MMP-1 level 6h after treatment in all conditions, which Oct 13, 10:30 - 12:10 disappeared at 24 post-treatment. In conclusion, our results indicate that helium-based cold OR-216 atmospheric plasma revealed to be efficient in inhibiting Cold atmospheric plasma does not stimulate tumor growth in in vitro 3D spheroid model while not dermal collagen remodeling at tissue scale inducing dermal extracellular matrix remodeling in the Sara Gouarderes1, Aurélie Marchès2, Patricia Vicendo1, same exposure conditions. Michel Simon3, Nofel Merbahi4, Laure Gibot 1 1IMRCP, CNRS UMR 5623, Université Toulouse III - Paul 127 [1]Di Meo, et al. Oxid. Med. Cell. Longev. 2016 the cell membrane[10], the RIP1/RIP3 and MLKL are also [2]Dunnill, et al. Int. Wound J. 2017 activated 6 hours after NTIRE[3], which adds complexity [3]Braný, et al. Int. J. Mol. Sci. 2020 of understanding NTIRE cell death. The fact that caspase [4]Privat-Maldonado, et al. Cancers 2019 1 and RIP1/RIP 3 are activated and induce the activation [5] Gouarderes, et al. Bioelectrochemistry 2022 of GSDMD and MLKL, tentatively suggests that NTIRE cell death is associated with a molecular path which com- OR-196 bines both elements of pyroptosis and necroptosis. Necroptosis and Pyroptosis Contribute to Cell However, our findings are interesting and different from Death of NTIRE Treatment conventional point, substantially more research is needed Yanfang Zhang 1, Peipei Mai1, Fentao Liu2, Yunlong to elucidate the mechanism of cell death from NTIRE. The Wang3, Boris Rubinsky4 findings and the possibilities we raise may be of interest to 1Luoyang Central Hospital Affiliated to Zhengzhou University, researchers studying the interaction of electric fields with Department of Endocrinology, China biological cells. 2School of Basic Medical Sciences, Zhengzhou University, De- partment of Pharmacology, China OR-197 3Henan Bioengineering Research Center, China Pulsed electric fields with calcium ions stimulate 4University of California Berkeley, Department of Mechanical oxidative alternations and lipid peroxidation in hu- Engineering and Department of Bioengineering, United States man non-small cell lung cancer Recently, non-thermal irreversible electroporation Vitalij Novickij1, Nina Rembiałkowska2, Paulina (NTIRE) has been employed as a new medical modality to Kasperkiewicz-Wasilewska3, Dagmara Baczyńska2, Adam Rzechonek2, Piotr Błasiak2, Jolanta Saczko2, Julita Kulbacka 2 ablate cancer. While the cell death mechanism of NTIRE 1Vilnius Gediminas Technical University, Lithuania is yet unclear. Nowadays, according to TUNEL stain ex- 2Wroclaw Medical University, Poland periments apoptosis is widely recognized as a noninflam- 3Wroclaw University of Science and Technology, Poland matory reason of cell death after NTIRE[1, 2]. However, in our previous study, we found the TUNEL Background. Pulsed electric fields (PEFs) are com- stain after NTIRE treatment is spread throughout the monly used to facilitate the delivery of various molecules, treated region, which is different from typical TUNEL including pharmaceuticals, into living cells [1]. However, stains of apoptotic cells, in which the nucleus is stained[3]. the applied protocols still require optimization regarding Moreover, cleaved Caspase 3 was not upregulated at any the conditions of the permeabilization process, i.e., pulse time after NTIRE, which is a key marker of apoptosis. So, waveform, voltage, duration, and the number of pulses we presumed that apoptosis, a non-inflammatory mechan- in a burst. This study highlights the importance of the ism of cell death, did not contribute to the cell death from electroporation buffers in the electropermeabilization and NTIRE. anticancer effects in the lung cancer model. The study of the mechanisms that lead to cell death has Material and methods. This research investigated evolved well beyond the concepts of necrosis and apop- the effects of electroporation on human non-small cell tosis[4]. A variety of other molecular mechanisms can lead lung cancer cells (A549) in potassium (SKM) and to various modes of programed necrosis characterized by HEPES-based buffers (SHM) using sub-microsecond the breaching of the cell membrane, including pyroptosis and microsecond range pulses. The experiments were and necroptosis. Pyroptosis is an inflammatory caspase- performed using 100 ns – 100 µ s (0.6–15 kV/cm) bursts dependent form of programmed necrosis[5]. Morphologic- with 8 pulses in a sequence. Cell membrane permeab- ally, pyroptotic cells display cell swelling and rapid plasma ilization rate was measured by flow cytometry using an membrane lysis. This form of cell death is driven by the impermeant dye Yo-Pro-1. Cell viability was analyzed inflammatory caspases: caspase 1, 4, 5 and 11. Activ- after 24 and 72h by MTT and SRB assay. The confocal ation of caspase 1 and failure to activate caspase 3 are microscopy method was used for CellROX® and HCS considered a key marker of pyroptosis[6]. Once caspases CellMask™ imaging. ROS level after PEF alone and 1, 11, 4 or 5 have been activated by either the canon- with calcium ions was detected by ROS-Glo™ H2O2 ical or non-canonical inflammasome pathway, they trigger luminescent assay. The lipid peroxidation process and pyroptosis by cleaving gasdermin D between Asp276 and neutral lipids were assessed by confocal microscopy Gly277[7]. Necroptosis is a programmed form of necrosis (Click-iTTM Lipid Peroxidation Imaging Kit and HCS that is dependent on activation of receptor-interacting LipidTOX™ Deep Red Neutral Lipid Stain). kinase (RIPK3) and the mixed lineage kinase domain-like Results. It was shown that depending on the buffer (MLKL) psuedokinase[8, 9]. This form of cell death in- composition, the susceptibility of cells to PEF varies, volves membrane rupture and release of cytoplasmic con- while calcium enhances the cytotoxic effects of PEF, tents and is also distinct from apoptosis. if high cell membrane permeabilization is triggered. It In our study, we found that both caspase-1 and GSDMD was also determined that electroporation with calcium were up-regulated at 6 and 24 hours after NTIRE treat- ions induces oxidative stress in cells, including lipid ment, which confirmed that pyroptosis type signaling was peroxidation, generation of reactive oxygen species, and activated after NTIRE treatment. Execution of necrop-neutral lipid droplets. tosis, occurs through MLKL introducing ion channels in Conclusions. The model of lung cancer used in the study the plasma membrane, resulting in the loss of integrity of is not typical for the electroporation studies. Here, we 128 demonstrated that calcium ions and optimized pulse filipodia and extra-cellular budding formation related to parameters could potentiate PEF efficacy and oxidative plasma membrane damage repair. alternations in lung cancer cells. Thus, the anticancer These observations seem to not indicate a clear difference efficacy of PEF in lung cancers in combination with in morphological changes induced by the exposure in both standard cytostatic drugs or calcium ions should be cell lines. considered, but this issue still requires in-depth detailed Therefore, an infrastructural approach by immunogold studies with in vivo models. staining in pre-embedding was performed to evaluate the distribution of CD133 protein in exposed cells. CD133 is a Acknowledgments: Funding was provided by the trans-membrane protein involved in cell stemness process. Polish National Centre of Science of DAINA 2 It is highly expressed in D283 cells and absent in NHA (2020/38/L/NZ7/00342; PI: J. Kulbacka), and the so it could be the molecular mediator of the observed dif- Research Council of Lithuania grant (Nr. S-LL-21-4, PI: ferential reaction on the two analyzed cell types. First V. Novickij). immunogold results showed a qualitative reduction of this protein after PEF-5 exposure in D283. Indeed, CD133 References: proteins are mainly positioned on cell protrusions highly [1] J. Gehl, Electroporation: Theory and methods, affected by the pulses exposure. In D283, CD133 dysfunc- perspectives for drug delivery, gene therapy and re- tion could explain the evasion in reactive oxygen species search, Acta Physiol. Scand. 177 (2003) 437–447. defense, and the consequent radio-sensitivity induction. https://doi.org/10.1046/j.1365-201X.2003.01093.x. In conclusion, this study investigates differences in mor- phological structure of two different cell lines after pulses OR-198 exposure suggesting the role of membrane protrusions in Ultrastructural analysis on normal astrocytes and the activation of specific cell signaling and molecular re- medulloblastoma cancer stem cells after micro- sponses. second pulsed electric field exposure to dissect the cell response specificity OR-195 Mirella Tanori1, Arianna Casciati1, Anna Rita Taddei2, Finding an effective MRI sequence to visualise Carmela Marino1, Caterina Merla 1, Mariateresa Mancuso1 the electroporated area in plant-based models by 1ENEA, Division of Health Protection Technologies, Italy quantitative mapping 2Tuscia University, High Equipment Centre, Italy Athul Thomas 1, Teresa Nolte1, Andreas Ritter1, Marco Pulsed electric fields (PEFs) have become an important Baragona2 1 clinical tool for the treatment of tumors. In this contest, Uniklinik, RWTH Aachen, Germany 2 our preview study showed that a specific pulse protocol Philips Research, Netherlands (PEF-5: 0.3 MV/m, 40 us, 5 pulses) was able to induce ir- MRI provides a means of visualizing the electropor- reversible membrane permeabilization and activate apop- ated region in 3D. Identifying an effective MRI sequence tosis and senescence in D283, a model of medulloblastoma for visualising electroporated area plays an essential role (MB) cancer stem cells (CSCs). PEF-5 exposure further in the evaluation and follow-up of in vivo electroporation promoted a radiosensitizing process enabling the complete treatments. For research purposes, plant-based electro- inhibition of engrafted tumor growth. poration models have emerged as an alternative to animal To verify if PEF-5 exposure could be selective for CSCs, experiments. In the past, the FLAIR MRI sequence was Normal Human Astrocytes (NHA), representing cells loc- found to have high contrast between electroporated and ated in the immediate surroundings of the tumor space, non-electroporated areas in potatoes (Hjouj et al. 2010). were also exposed to the same protocol. NHA were charac- This study aims to find an optimised MRI sequence for terized by a higher threshold for irreversible electropora-analysing electroporated tissue of plant-based models by tion than MB CSCs resulting more resistant to the electric employing both qualitative and quantitative MRI tech- pulses application maintaining a high viability. niques. To attempt understanding the mechanism of such select- Potatoes and apples were electroporated with 800, 1000, ive response (at morphological, functional and molecular 1500 V/cm and 800, 1000 V/cm and imaged with T1- level) and considering that the cell membrane seems the weighted, T2- weighted and FLAIR MRI sequences at main PEFs target, in this work, we performed an electron three different time points (3, 24, 48 hours) after elec- microscopy analysis on both cell types to highlight cell troporation. The sequence for best visualization of the morphological changing in relation to the specific plasma electroporated area with high contrast in apple and potato membrane composition and complexity. To achieve this was identified. To better understand the contrast beha- aim, Transmission Electron Microscopy (TEM) analysis viour, quantitative T1 maps of the central ablated slice was performed to explore inner and outer ultrastructural were obtained based on five inversion recovery measure- cell changes occurring one hour after PEF-5 exposure. ments and voxel-wise fitting of the function M( TI ) = M0 Results showed alterations of the internal organelles and * ( 1 - 2 * exp(-TI/T1) ), where M0, TI and TR are the membrane protrusions in both cell lines. proton density, the inversion time and the T1 relaxation Scanning Electron Microscopy (SEM) analysis was used to time. study cell surface changes occurring at the same time point All T1 weighted images showed a well-defined contrast after PEF-5 exposure and results confirmed retraction of between the electroporated and non-electroporated re- 129 gions compared to the T2 weighted images of potatoes the embryo’s vasculature over time. Indeed, at early after electroporation, which did not visualize the ablation embryonic stage, tumors are not recognized as foreign zone. This implies that differences in the T1 relaxation bodies due to the lack of immune system [4]. Using this time between the electroporated and non-electroporated model, we demonstrated that PEFs induced intracellular regions explain the contrast behaviour. calcium elevation, electropermeabilization of cellular In conclusion, morphologic and quantitative imaging membranes and vasoconstriction of tumors’ vasculature. showed that T1 changes are the driver for contrast in Also, that implanted flexible electrode devices could be plant-based (potato and apple) models for electropora- used to dysregulate intracellular calcium homeostasis. tion. The potato was found to be the more suitable model This showed that flexible devices can be used as a less for MRI-based visualization of the ablation zone. Import- invasive alternative to rigid metal electrodes for the antly, knowledge of T1 values for ablated and non-ablated delivery of PEF and that our model is suitable for first regions will enable fine-tuning of the MRI sequence para- experiments under intravital conditions, before using in meters towards optimal visualisations of the electropor- vivo animal models. Finally, preclinical studies were ated zone. performed with the same devices in a syngenic, orthograft immunocompetent mouse model to study the effect of OR-199 PEFs on tumors and the triggered immune response. Flexible electronics integrated into increasingly complex glioblastoma models for the study of [1] Davis, M. E., Clin J Oncol Nurs 20, S2-8 (2016). pulsed electric fields effect in tumor and its mi- [2] Lee, H., Bellamkonda, R. V., Sun, W. & Levenston, croenvironment M. E., J. Neural Eng. 2, 81–89 (2005). Marie Lefevre 1, Attila Kaszas2, Andrea Slezia2, Gerwin [3] Lefevre, M. C., npj Flexible Electronics 9 (2021). Dijk1, Loig Kergoat3, David Moreau1, Franck Debarbieux2, [4] Ribatti, D., International Review of Cell and Molecular Rodney P. O’Connor1 Biology vol. 270 181–224 (Academic Press, 2008). 1CMP-EMSE, France 2INT, France 3Panaxium SAS, France P25 - Electrochemotherapy for Glioblastoma multiforme is a brain cancer that is Cutaneous Metastases highly resistant and show high recurrence probability [1]. For this reason, bioelectric therapies based on the delivery Thursday morning Track B of pulsed electric fields (PEF) are more and more invest- Oct 13, 10:30 - 12:10 igated as an alternative to standard therapies. Indeed, PEFs have a direct effect on membrane permeability of OR-103 tumors and their microenvironment. However, most of Efficacy of electrochemotherapy in breast cancer these treatments are delivered by stiff metal electrodes patients of different hormonal status: The IN- resulting in a mechanical mismatch with the brain and SPECT experience causing acute and chronic injuries [2]. Claudia Di Prata 1, Eva Maria Grischke2, Giuseppe Our approach is based on the development of flexible Azzarello3, Julie Gehl4, Francesca De Terlizzi5 and biocompatible devices as a means of delivering 1University of Padova, Italy PEFs, that can be implanted onto glioblastoma tumors 2Univ. Frauenklinik, Germany without damaging surrounding tissues. In this purpose, 3ULSS 3 Serenissima-Mirano, Italy gold interdigitated electrodes were patterned onto a 4Zealand University Hospital, Denmark thin layer of the transparent organic parylene-c and 5INSPECT, Italy coated with the conductive polymer PEDOT:PSS. The effect of PEFs on tumors and their microenvironment Introduction. Electrochemotherapy has proven to be was studied at different levels. First, experiments were an efficient treatment for cutaneous metastases of various performed on glioblastoma cells stably expressing a cancers including breast cancer (BC). The large number of genetically encoded calcium indicator directly cultured patients collected within INSPECT database provide the on our in vitro devices. Mechanistic studies demonstrated possibility of a differentiated analysis on BC with different that the delivery of electric pulses induces changes in receptor status (estrogen receptor and HER2 receptor). intracellular calcium and allowed to investigate on the Materials and methods. Data on Breast Cancer patients role of ATP signaling in this calcium response. Despite with cutaneous metastases have been retrieved from the being simple and inexpensive, 2D in vitro experiments are INSPECT database. Patients have been divided into 3 less robust than in vivo models as they fail to mimic the groups: HER2+ = patients with HER2 positive, Hormone microenvironment of the tumors. In order to minimize receptor positive (HR+) = patients with either ER or PG the use of animal models that raises ethical issues, we positive (HER2 negative), Triple negative (TN) = patients developed an intermediate model combining a living or-with ER, PG and HER2 negative. The groups comprised ganism, a quail embryo, and an in vitro three-dimensional of 43, 94 and 34 patients, respectively. (3D) model of vascularized tumor [3]. 3D engineered Results. Groups were similar for: histological subtype glioblastoma spheroids were grafted in the chorioallantoic (ductal carcinoma was present in 72% HER2+, 82% HR+ membrane of a quail embryo and were vascularized by and 91% TN), location of the cutaneous nodules at chest level (92%, 89% and 95% respectively). Most of patients 130 in all groups have been pre-treated with surgery/systemic v5.0), and patient-reported HRQoL at baseline, one, two, therapy/radiotherapy. Preirradiation of treated lesions four and ten months (EuroQol [EQ-5D-3L] questionnaire, was observed in 76% of lesions in the TN group, 45% in including 5-item utility score [EQ-5D] and visual analogue HER2+ and 52% in HR+ group. Some patients (70% in scale for self-reported health state [EQ-VAS]). Comparis- HER2+, 55% in HR+, 38% in TN) were under concomit- ons were made for statistical and minimal important dif- ant systemic treatment at ECT session. Half of patients ferences (MID). Scores and clinical covariates were ana-were treated with ECT for multiple lesions. Multiple le- lysed to identify predictors of response in multivariate ana- sions were significantly smaller than single lesions: mean lysis. size of single lesions was 12.4±12.9 cm, whilst mean size Results: Complete response rate, G3 toxicity and one- of multiple lesions was 2.6±3.6 cm (p=0.003). year LPFS in 378 patients (76% of the InspECT melan- Response to ECT has been evaluated in terms of objective oma cohort) were 47%, 5%, and 78%. At baseline, age-response (OR) per patient and per nodule. OR per patient paired HRQoL did not differ from the general European is 86% in HER2+, 80% in HR+, 76% in TN (p=0.8664); population. Following ECT, both EQ-5D and EQ-VAS OR per nodule is 89% in HER2+, 86% in HR+, 83% in TN scores remained within MID boundaries, particularly (p=0.3846). Factors affecting response to ECT are differ- among complete responders. A subanalysis of the EQ- ent among groups, except for lesions’ size which signific- 5D items revealed a statistically significant worsening in antly affects the response in all groups (higher response pain/discomfort and mobility (at one-two months), and rate for sizes < 3cm, p=0.0105, p=0.0001, p=0.0266 re- self-care and usual activities (throughout the follow-up). spectively). Furthermore, in HER2+ group response is Concomitant checkpoint inhibition was associated with affected by metastatic condition (higher response rate in better EQ-5D and EQ-VAS trajectories. Baseline EQ-5D non-metastatic patients p=0.0143); in HR+ is affected by was the exclusive independent predictor of response (RR concomitant systemic therapy (higher response rate under 14.76, p=0.001). concomitant treatment p=0.0044) and lesion numerosity Conclusion: HRQoL of melanoma patients candidates (higher response rate in multiple lesions p=0.0065); in TN to ECT is similar to the general population and pre- is affected by lesions numerosity (higher response rate in served in complete responders. Transient deterioration multiple lesions p=0.0011). Local tumor control is higher in pain/discomfort and mobility domains and persistent for HER2+ and HR+ groups (1 year local progression free decline in self-care and usual activities may warrant tar- survival or 78% and 81% respectively) with respect to TN geted support interventions. Combination with check- group (61%). point inhibitors is associated with superior QoL outcomes. ECT treatment is equally effective among groups, despite Baseline HRQoL provides predictive information which different conditions, age, time since, diagnosis, previous can help identify patients most likely to respond. or concomitant treatments, treatment characteristics. Re- sponse and local tumor control seems to be better in small OR-105 multiple lesions than in big armour-like lesions, suggest- High Frequency Electroporation and Chemother- ing that treating smaller, even multiple, lesions at the time apy for the treatment of Cutaneous Malignancies; of occurrence is much more effective than treating bigger Evaluation of Early Clinical Utility and Response long lasting armour-like cutaneous lesions. A. James P. Clover 1, Phoebe Lyons2, Dana Polini3, Alison Bracken1 OR-104 1Cancer Research@UCC, University College Cork, Ireland Health-related quality of life trajectories in melan- 2Cork University Hospital, Department of Plastic Surgery, Ire- oma patients after electrochemotherapy: real- land world insights from the InspECT register 3School of Medicine, University College Cork, Ireland A. James P. Clover 1, Joy Odili2, The INSPECT Group3 Introduction: There has been increasing interest 1Cancer Research @UCC (University College Cork), Ireland in manipulating the pulse parameters used to deliver 2St. Georges University Hospitals NHS Trust, Department of successful electroporation in order to reduce muscle Plastic Surgery, United Kingdom contractions and the pain associated with treatment 3INSPECT Group, United Kingdom as well as potentially ensuring a more uniform tumour Introduction: Electrochemotherapy (ECT) effectively exposure. The ePORE device, Mirai Medical, Galway is controls skin metastases from cutaneous melanoma. The utilising a higher frequency electroporation delivery to objective of this study was to evaluate health-related qual- successfully permeabilise the tumour cell membrane and ity of life (HRQoL) in melanoma patients pre-/post-ECT enable patients to be treated under local anaesthesia. and its influence on treatment outcome. This allows for an increased group of patients to be Materials and methods: The analysis included prospect- treated with the anaesthetic risk reduced allowing for ive data from the International Network for Sharing Prac- a safer treatment. The pre-clinical data on the efficacy tices of ECT (InspECT) register. Following the Stand- of the new high frequency electroporation parameters ard Operating Procedures, patients received intraven- has been published which reduces the pulse length from ous or intratumoural bleomycin (15,000 IU/m2; 1000 IU 100 microseconds unipolar to packets of 2 microsecond mL/cm3) followed by 100-microsecond, 1000-V/cm elec- bipolar pulses. These high frequency parameters have tric pulses. Endpoints included response (RECIST v3.0), been successfully validated in-vitro on melanoma cells local progression-free survival (LPFS), toxicity (CTCAE to induce cell death when combined with a chemothera- 131 peutic. This case series examines early clinical utility and group (n=41 patients) received only ECT (one or more outcomes. sessions); the second group (n=44) received only pembrol- Method: Between July 2019 and March 2021, 20 patients izumab; the third group (n=45) received ECT in concom-were treated at Cork University Hospital, Ireland with itance with pembrolizumab. The percentage of patients high frequency electroporation and chemotherapy. 10 who had previous systemic treatment was 54% in the ECT were treated as part of the initial evaluation of efficacy alone group 32% in the pembrolizumab alone group, 67% and a further 10 for therapeutic indications. Up to 11 in the ECT plus pembrolizumab group (p=0.004). Local lesions per patient were included in the analysis, evaluated objective response (OR) was evaluated on skin metastases for response (CR, PR, NR, DP) over time as well as any at six months. complications. Patients included those who had disease Results: The OR rate was higher in patients who under- progression on current treatment regimes with no suitable went ECT and ECT+pembrolizumab (80.5% and 77.8% alternative options and those who could not tolerate or respectively) than those who received pembrolizumab declined general anaesthesia thus removing the option of alone (38.6%), p<0.001. The percentage of patients lower frequency electroporation. who experienced local progression was 26.7% in the ECT Results: Of the 20 patients treated, 16 are available + pembrolizumab group (after a mean time of 20±12 for follow up. 2 were unavailable for follow up and 2 months) and 56.1% in the pembrolizumab group (after were recently treated and outcome data is pending. All a mean time of 7±8 months). Cox regression analysis cor- successfully underwent treatment with no procedural rected for previous systemic treatment revealed a signific- complications and objectively successful electroporation. antly higher risk of local progression in the pembrolizu- 6 patients underwent treatment under local anaesthesia mab compared with the ECT+pembrolizumab group (RR with no sedation. 5.76, C.I. [2.41-13.77], p<0.001). One-year local progres- In total 278 lesions were treated and 89 of these included sion free survival was 86% in the ECT+pembrolizumab in the analysis. Lesions analysed included a variety of group and 51% in pembrolizumab group (p=0.0002). histiotypes including malignant melanoma (52), Basal Systemic OR was similar between pembrolizumab and Cell Carcinoma (29), Breast Carcinoma (7) and Squam- ECT+pembrolizumab groups (25.5% vs 24.4%, p=1). In ous cell carcinoma (1). At 12 weeks post treatment, the pembrolizumab group 61.4% of patients experienced Complete response (CR) was observed in 62/78 (79%,) systemic progression and 53.3% in ECT + pembrolizu- Partial response in 13% (10/78), Disease progression in mab group (p=0.522); the time to progression was sig- 5/78(6.5%) and unable to assess in 1/78 (1 %). nificantly shorter in the pembrolizumab compared with Conclusions: High frequency electroporation and chemo- the ECT+pembrolizumab group (8±9 vs 17±15 months, therapy is showing optimistic early response rates p<0.001). Cox regression analysis corrected for previous comparable to the rates seen with electrochemotherapy systemic treatment showed a significantly higher risk of using the standard procedures. No procedural issues systemic progression in the pembrolizumab group com- were encountered. As such, treatment with these novel pared with ECT+pembrolizumab group (RR 1.96, C.I. parameters represent an alternative treatment modality [1.07-3.60], p=0.0305). One-year systemic progression free that may expand the treatment envelope for patients who survival was 63% in the ECT+pembrolizumab group and could benefit form electroporation based treatment for 39% in the pembrolizumab group (p=0.0344). cutaneous malignancies especially under local anaesthesia. One-year overall survival was 88% and 64% in the ECT+pembrolizumab and pembrolizumab group, re- spectively (p=0.0062). Cox regression analysis correc- OR-106 ted for previous systemic treatment showed a significantly Outcomes of patients with metastatic melan- higher risk of death in the pembrolizumab compared with oma treated with electrochemotherapy, pembrol- respect to the ECT+pembrolizumab group (RR 2.02, C.I. izumab or their combination: a retrospective [1.01-4.03], p=0.045). matched cohort analysis from InspECT and Slov- Conclusions: Our results suggest that the combination of enian Cancer Registry pembrolizumab and ECT provides durable local disease Luca G. Campana 1, Barbara Perič2, Maša Bošnjak2, control on superficial metastases, which may beneficially Francesca De Terlizzi1, Gregor Serša2 impacts on the systemic progression of the disease. 1INSPECT, Italy 2Institute of Oncology Ljubljana, Slovenia OR-107 Background: Pembrolizumab is the standard of care Electrochemotherapy with intravenous bleomycin for patients with metastatic melanoma. Electrochemo- for patients with cutaneous malignancies, across therapy has been shown to provide durable local control tumour histology: A systematic review on superficial metastases. The combination of systemic Freya Bastrup 1, Mille Vissing2, Julie Gehl2 1 immunotherapy with pembrolizumab and local treatment Copenhagen University, Denmark 2 with ECT has not been investigated. Zealand University Hospital, Denmark Methods: We compared three groups of melanoma pa- Background: Electrochemotherapy (ECT) is an estab- tients with stage IIIC-IV disease and skin metastases. The lished treatment for primary and secondary skin tumours. groups were matched for age, disease stage, performance The method combines chemotherapy with electroporation. status (ECOG) and size of skin metastases. The first Bleomycin is the drug of choice for ECT, as it is already 132 well established as a treatment for several cancer types and is more effective in small volume deposits proportionally has the largest increase in efficacy when combined with reducing its efficacy as the lesions increase in volume. electroporation, enhancing the toxic effect several hundred Keeping in mind the above considerations we have de-fold. The response rates of ECT have been high and con- veloped our own protocol which has shown to be improve sistent over the past 30 years. However, some patients are our results. excluded from treatment due to possible side effects. Case Technique: Patients are managed under General Anaes- based reports point out that the efficacy possibly can be thetic as Day case maintained even when the dose of bleomycin is reduced. 1- All patients receive systemic Bleomycin via intraven- Consequently, in 2018, studies began investigating redu- ous administration with dose in line with ESOPE ( 15,000 cing the bleomycin dose. ECT is often performed as a IU/Sqm in 100cc of N.Saline), palliative treatment, which is why quality of life is an im- 2- A small amount (5-10mls) is taken from the IV bolus portant parameter to include in evaluation. (leaving the total dose administered unchanged and it is Aim: The purpose of this review is to summarize all data used to infiltrate intra-lesionally the bigger cancer depos- published using intravenous bleomycin for cutaneous ma- its. (10-20 mm in diameter) lignancies and is to our knowledge the first review to ex- 3- If there are nodules bigger than 2 cm they are surgically amine the use of a reduced bleomycin dose in ECT. Meth- excised with needle cutting diathermy with 1mm margin ods: This study is a systematic review. Fifty-five clinical and after achieving full haemostasis the wound is treated studies investigating ECT with intravenous bleomycin for with electroporation patients with any cutaneous malignancies (basal cell car- 4- The area treated with electroporation is extended to cinoma, squamous cell carcinoma, Kaposi’s sarcoma, ma- at least 3 cm margin around the clinically visible nodules lignant melanoma and breast cancer metastases) were in- or/and to any area where we would expect development cluded. of further recurrences Results: Studies published from 1993-2021 investigating Our pre-ECT clinical experience, when multiple skin de-the effect of ECT include 3729 patients and indicate a posit from melanoma and breast cancer where treated with consistent and high response with a mean objective re- narrow margin surgical excision, was characterised by a sponse rate (ORR) of 81.5%. Interestingly, studies using rapid early relapse. We have noticed a much longer dis- lower doses of bleomycin observe a similar ORR (85.5%), ease free interval since we have started treating a much opening the possibility that a lower dose may not be in- larger area of skin around the clinically visible nodules ferior. Eight studies have performed a quality of life as- and we believe that this is due to clearance of microscopic sessment using EORTC questionnaires, concluding an im- disease which is very susceptible to ECT. proved quality of life after ECT. The treatment of the bigger lesions with a combination Conclusion: This study gives an overview of published of local and systemic therapy has improved our complete studies on ECT with intravenous bleomycin for patients response. The surgical excision of bulky disease provides a with cutaneous malignancies, including the use of a re-clean wound which is more manageable, less symptomatic duced bleomycin dose, as preparation for a randomised and heals quicker than the ones following partial or com- study. plete tumour necrosis improving quality of life. Conclusions: We believe that our algorithm improves re- OR-108 sponse rate, disease free interval and reduce post treat- Hybrid ECT – a different approach ment symptoms avoiding long term management of nec- Michael Rice, Giulia Colavitti, Antonio Orlando rotic wounds. It is simple to implement without increase North Bristol NHS Trust , United Kingdom of risks as respects ESOPE recommendations of maximum Electro-Chemo-Therapy(ECT) is a combination treat- doses. ment based on the simultaneous administration of chemo- therapy and the application of an electric field to the area P35 - Modelling to be treated in order to produce reversible electroporation to the cancer cells. The electroporation increases the intra- Thursday morning Track C cellular migration of the cytotoxic drug locally enhancing Oct 13, 10:30 - 12:10 its efficacy. This technique has been a valuable tool in the treatment of various tumours for the last 15 years since OR-25 the publication of the ESOPE (European Standard Oper- Mean field model of single cell electroporation ating Procedure for Electrochemotherapy)in 2006. Pedro Jaramillo 1, Annabelle Collin2, Clair Poignard2 According to the ESOPE the bleomycin can be admin- 1Bordeaux University, France istered intravenously (bolus of 15,000 IU/Sqm) or intrale- 2INRIA, France sionally (1000 IU/Ml 0.25-1 ml/cm3). In our practice, based mainly on skin deposits from MM, Electroporation is a phenomenon that takes place SCC and Breast cancer, we frequently encounter extensive when a cell is immersed in a strong enough electric field. skin involvement suitable only for IV treatment. The cell membrane which is mainly made up of a lipid Published data shows that intra-lesional treatment (IL) is bilayer behaves like a dielectric separating two conductive more effective than intra-venous (IV) but it is mainly used media (the interior and exterior of the cell). Charge for localised disease. There is also clear evidence that ECT accumulates on either side of the membrane creating a transmembrane voltage (TMV). When the TMV 133 surpasses a certain threshold the membrane permeability Slovenia to the exterior environment drastically increases. This A fundamental understanding of the barrier proper- induces an exchange between the cell and its exterior ties of biological membranes can be obtained by studying medium and in particular enables certain molecules to model systems, such as planar lipid bilayers. The planar enter the cell that could not do so before [4]. lipid bilayer mimics a fragment of the cell membrane when Various models have been proposed over the years to it separates two compartments filled with electrolytes, i. e. explain this phenomenon at the scale of a single cell. when it is formed on a small aperture that separates two By far the most successful one was presented by Neu compartments. Electrodes immersed in each compartment and Krassowska in [2]. More recently [1], Leguebe et allow measurements of electric parameters of the planar al have proposed a more accurate model however it is lipid bilayer, which can be considered electrically as a non- phenomenological in nature and so it cannot explain the perfect capacitor - parallel connection of an ideal capacitor phenomenon. On the other hand molecular dynamics and resistor. The changes in planar lipid bilayer electrical simulations have enabled us to represent in extreme detail parameters can reflect structural changes of planar lipid small patches of cell membrane under the effect of an bilayer. external electric field [3]. However these simulations seem Using linearly increasing transmembrane voltage we to contradict what one would expect according to the measured the capacitance, breakdown voltage, and time model from Neu and Krassowska. Moreover, the patches required for rupture of planar lipid bilayers composed of cell membrane that are computationally feasible to of 1-pamitoyl 2-oleoyl phosphatidylcholine (POPC), 1- simulate are too small to compare results with both pamitoyl 2-oleoyl phosphatidylserine (POPS), mixture of models. Lastly, even molecular dynamics seem to agree POPC and POPS lipids in a 1:1 ratio, and POPC lip- with experiments but only qualitatively. In practice ids with an addition of 20, 30, 50 and 80 mol% of cho- they consider electric pulse that are about two orders of lesterol. We evaluated the change in the capacitance of magnitude stronger than the ones used in experiments. the planar lipid bilayer corresponding to the formation Our goal is to present a physically based, single cell of water pores, the radius of water pores at membrane electroporation model which is also coherent when ap- rupture, and the fraction of the area of the planar lipid plied to a small patch of the cell membrane and which bilayer occupied by water pores. The estimated pore radii takes into account pore interactions and dynamics. This of the planar lipid bilayer are 0.101 nm, 0.110 nm, and is important as it enables us to make a link current 0.106 nm for membranes composed of POPC, POPS, and molecular dynamics simulations of a small patches of cell POPC:POPS, respectively. For POPC lipids with 20, 30, membrane and a global description of the cell membrane. 50, and 80 mol% cholesterol added, the estimated pore The main idea is to use a phase ordering kinetics model to radii are 0.097 nm, 0.071 nm, 0.067 nm, and 0.083 nm, re- describe the state of the cell membrane. The evolution of spectively. The fraction of the surface occupied by water the membrane will then be determined by its free energy pores at the time of rupture of the planar lipid bilayer is also known as the mean field Ginzburg-Landau excess in the range of 0.1–1.8% for POPC, POPS and their mix- free energy. ture. For POPC lipids with an addition of 20, 30, 50, and We conclude this work with some simulations in the same 80 mol% cholesterol, the fraction of the surface occupied setting as in the molecular dynamics simulations using by water pores is estimated to be 12.55%, 22.85%, 0.4%, physically relevant coefficients in our model and compare and 0.9%, respectively. our results with current dynamical simulations. Interactions between lipids are important determinants of membrane organization and physical properties. It is [1] M. Leguèbe, A. Silve, L. M. Mir, and C. Poignard. known that cholesterol and unsaturated PC molecules are Conducting and permeable states of cell membrane not easily miscible, leading to the formation of cholesterol-submitted to high voltage pulses: mathematical and rich domains. These domains are small and have short life- numerical studies validated by the experiments. J. Theor. time; 1 to 100 ns. It was stipulated that the incorporation Biol., 360:83–94, 2014. of cholesterol (30 mol%) increases the penetration of water [2] K. A. DeBruin and W. Krassowska. Modeling elec- into the headgroup region and into the near-surface region troporation in a single cell. I. Effects of field strength of the hydrocarbon chains. The results of our study indic- and rest potential. Biophysical Journal, 77(3):1213–1224, ate that smaller water pores form in planar lipid bilayers 1999. composed of POPC lipids with an addition of 30 mol% [3] M. Breton, L. Delemotte, A. Silve, L. M. Mir, and cholesterol molecules in an electric field than in planar M. Tarek. Transport of siRNA through lipid membranes lipid bilayers composed of POPC lipids alone. However, driven by nanosecond electric pulses: an experimental and it is possible that the water pores occupy a large part (ap- computational study. J. Am. Chem. Soc., 134(34):13938– prox. 23%) of the planar lipid bilayer, which contributes 13941, 2012. to its greater stability in the electric field. [4] A. Gothelf, L. M. Mir, and J. Gehl. ... OR-26 Water Pores in Planar Lipid Bilayers with an ad- dition of cholesterol Alenka Maček Lebar, Damijan Miklavčič, Peter Kramar University of Ljubljana, Faculty of Electrical Engineering, 134 OR-29 cell in situ properties to bulk tissue properties, illustrating Build me a skeletal muscle in silico: Insights into the power of such a multiscale approach in understanding tissue electroporation from an experimentally- of the electroporation phenomenon from the mechanistic validated multiscale numerical model perspective. Rok Šmerc 1, David A. Ramirez2, Samo Mahnič-Kalamiza1, Janja Dermol-Černe1, Daniel C. Sigg3, Lars M. Mattison3, OR-27 Paul A. Iaizzo2, Damijan Miklavčič1 Real-Time Conductivity Distribution Characteriz- 1University of Ljubljana, Faculty of Electrical Engineering, ation for Electroporation using Plant Tissue Slovenia Borja López-Alonso, Pablo Briz, Héctor Sarnago, José 2University of Minnesota, United States Miguel Burdío, Óscar Lucía 3Medtronic, Inc., United States University of Zaragoza, Electronic Engineering and Commu- Gene electrotransfer is an important application of nications, Spain electroporation, with skeletal muscle the most used tar- This paper is focused on electroporation for medical get tissue due to its ability to express genes and secrete applications. Classical application of electroporation is proteins into the bloodstream. The specific architecture of carried out using fixed protocols based on preplanning or skeletal muscle, consisting of long fibres, imposes an aniso- monitoring the treatment by means of global impedance tropic electrical conductivity, leading to fibre-orientation measurements. Consequently, it is often preferred to ap-dependent electric field distribution at pulse application. ply the protocol repeatedly or to ensure a low enough final The electrical conductivity of skeletal muscle in the dir- global impedance to ensure complete treatment and pre- ection of fibres is higher than in the direction perpendic- vent tumour relapses. These methods, although partially ular to the fibres. The anisotropic electrical conductivity effective, do not allow to focus and control the treatment was also observed in cardiac muscle, a tissue of interest in effects and therefore healthy tissue can be overheated and another electroporation application of increasing import- damaged. In this context, new multi-output electropor- ance: the treatment of cardiac arrhythmias, particularly of ation systems with real-time monitoring tools have been atrial fibrillation, by means of pulmonary vein tissue abla- proposed to improve control and targeting of current treat- tion using irreversible electroporation. Due to a variety of ments. tissue types in electroporation-based treatments, further In this work, a multi-output electroporation generator has research on muscle anisotropy and its significance is of been used together with a multi-electrode structure to great importance. The objective of our study was to gain propose a real-time conductivity distribution estimation better insight into muscle anisotropy, with the goal of de- method. The goal of this method is to estimate the elec- termining the importance of field orientation in an aniso- tric conductivity distribution inside the tissue before each tropic tissue to the extent of irreversible damage by means electroporation pulse, i.e. in real-time. This will allow of an experimentally validated mathematical model. the system to target the treatment effect, improve its con- Our study consisted of an experimental and a numerical trol, and increase the preserved healthy tissue. The pro-part. In the experimental part, we delivered electrical posed method combines a small-signal impedance monit- pulses into the skeletal muscle tissue of pigs in vivo, in- oring block with a multi-electrode structure. Firstly, the serting the needle electrodes in two different orientations monitoring block allows to study the conductivity dynam-with respect to the muscle fibres: the first, in which the ics by means of the generation of measurement constant- direction of the applied electric field was parallel to the voltage pulse trains and a subsequent accurate current direction of the muscle fibres, and the second, in which measurement. Then, the multi-output structure allows ap-the electric field was perpendicular to the muscle fibres. plying the measuring pulses in different areas of the tissue We then used triphenyl tetrazolium chloride (TTC) stain- which allows to analyze the impedance in diverse volumes ing to determine the shape of the lesion for every ap- of the tissue. Finally, up to 81 impedance measurements plication. In the numerical part of the study, we built are processed by means of Least-Mean-Square algorithm a multiscale numerical model of the skeletal muscle. We to estimate a conductivity map composed of 18 voxels. used a single-cell model to determine cell-level conductiv- The proposed method has been developed by means of a ity during electroporation, and then generalised the calcu- FEA model in COMSOL Multiphysics, which allows to lated conductivity changes to the bulk tissue. In this way, simulate the impedance measurements taken by the mon-we were able to determine the electric field strength dis- itoring system, which are then normalized and processed tribution in skeletal muscle tissue during electroporation. by MATLAB. The proposed approach has been tested on Finally, we compared the experimentally determined le- potato tissue combined with phantom gel. These elements sions with the calculated field strength distributions using are selected because the potato is a vegetal tissue with ho- the Sørensen-Dice similarity coefficient to find the con- mogeneous electrical properties that allows to study the tours of the electric field strength threshold beyond which electroporation effects, and phantom gel allows to create irreversible damage is believed to occur. volumes with controlled and constant properties. These The study leads to two conclusions important for our un- are easy-to-use tissues for intensive experimentation, and derstanding of muscle electroporation and guiding further they have been widely used for similar purposes in mul- research; firstly, muscle anisotropy is of significant im- tiple electroporation studies. The tissue samples have a portance when considering electric field application, and thickness of 1 cm, and square multi-electrode based on secondly, we were able to extend the electroporated single parallel-plates electrodes that it is composed of isolated 135 square cells of 1 cm of side were used. P17 - Using Nanosecond Pulsed In conclusion, a method to estimate the electric conduct- Electric Fields (nsPEF) to Treat ivity distribution in real-time was developed and valid- Cancer ated by means of vegetal tissue model and finite element analysis. The final version of this paper will include ex- perimental results and future insights demonstrating the Thursday afternoon Track A feasibility of this proposal. Oct 13, 13:30 - 15:00 OR-96 OR-116 Characterization of an experimental setup for re- Nanosecond Pulsed Electric Field in Tumor Abla- cording fluorescence in real-time from a cell mem- tion, From Lab Experiment to Clinical Practice brane exposed to electric pulses Xinhua Chen Ioan Tivig, Mihaela G. Moisescu, Eugenia Kovacs, Tudor Zhejiang University, China Savopol Nanosecond pulsed electric field (nsPEF) utilizes high- Carol Davila University of Medicine and Pharmacy, Biophysics power short electric pulses to disrupt cellular membranes and Cellular Biotechnology, Romania and intracellular structures, e.g. nucleus and mitochon- Understanding the molecular mechanisms of the main dria, and consequently induces the apoptosis of tumor processes involved in electroporation continues to be of cells. It was approved in the laboratory by the differ- significant interest and requires experimental systems ent solid tumor models, indicating it is an ideal option for designed to record in real time the evolution of pertinent treating tumor nodules close to aforementioned delicate cell parameters. Here we present a setup which can structures. A prospective clinical trial (clinicaltrials.gov be used to evaluate by fluorescence various membrane identifier: NCT04039747) was conducted to evaluate the properties before, during and after application of the safety and efficacy of nsPEF ablation in 195 HCC pa- electroporation pulses to cells in suspension. tients with unresectable liver cancer that were ineligible The system is based on the design of adequate elec- for thermal ablation. The initial results suggest nsPEF is troporation electrodes, compatible with a standard an effective loco-regional treatment modality for liver car- spectrofluorometer cuvette housing (to allow the ex- cinoma located near gallbladder, hepatic vessels, portal citation beam to travel into the sample a window in vessels or gastrointestinal tract. After a follow-up of 6 the electrode plate was done). Four different window months. nsPEF has shown promising efficacy. Notably, geometries and sizes were considered: two circular and the non-thermal ablation mechanism of nsPEF could spare two rectangular; these geometries were 3D modeled in vital structures from collateral thermal damage as ob- AutoDesk Fusion 360; for all geometries, the spatial served in conventional thermal ablation. The concomitant electric field distribution was simulated in COMSOL activation of anti-tumor immunity by nsPEF can also po- Multiphysics 5.3a. tentially prevent tumor recurrence. However, the specific The electrodes ensuring the greatest homogeneity of the mechanism of nsPEF ablation inhibited tumor recurrence field in combination with the best possible illumination of needs further immune activation investigation from a clin- the sample, were then built using a computer-controlled ical perspective. The long term follow up of the above 195 cutting machine from a non-magnetizing stainless steel unresectable HCC patients who had the nsPEF ablation plate (to allow magnetic bar stirring of cells in suspen- is still ongoing. sion). As an example of the setup reliability, fluorescence OR-14 spectra of laurdan molecules labelling cells in suspension Tissue-specific clearance thresholds using high re- are presented together with the kinetics of the parameter petition rate nanosecond pulsed electric fields called “generalized polarization” for a varying number of Richard Nuccitelli, Amanda McDaniel, Kristin Von Roth- electroporation pulses. This parameter is strongly cor- stein, Dacia Gonzalez, Esin Sozer related to water presence in the hydrophobic core of cell Pulse Biosciences, Biology, United States membrane. The system may be employed for many other The proprietary Nano-Pulse Stimulation™ (NPS™) fluorescence measurements (fluorescence depolarization, treatment triggers regulated cell death and subsequent Förster resonance energy transfer, etc.) useful to the clearance of tissues by applying nanosecond pulses at high characterization of the electroporation process. amplitude electric fields (˜ 30 kV/cm) with low repetition rates (<10 Hz). The short, nanosecond pulse duration [1] Tivig I. et al., European Biophysics Journal, DOI : is significantly shorter than the typical tissue capacitive 10.1007/s00249-019-01417-9 charging time constant, resulting in a clearance require- ment of a very high electric field amplitude and the linear charging evolution of capacitive tissue components simil- arly across many tissue types. Here we report that, in contrast to the proprietary NPS™ treatments, the high repetition rate (1 -3 MHz) NPS™ treatments can achieve tissue clearance in packets of nano- second pulses (high-repetition-rate packets of up to 200 136 nanosecond pulses) at a lower electric field amplitude posed to 10 ns (50 kV/cm) pulses. Different shapes (uni- (e.g. 8 kV/cm). Our results show successful clearance polar and bipolar), number of pulses (up to 500), and across multiple tumor types indicating a capacitive char- interphase delay of 100 ns between the pulse polarities ging/discharging that takes much longer than the pulse were investigated using a high-voltage nsPEF generator duration and the off-time between adjacent pulses in the that we have previously designed and characterized [4]. high repetition rate packet. The longer time constant of We showed that cell membrane permeabilization, as well charging/discharging together with the lower electric field as cellular death, occur when at least 100 pulses are ap- application results in additive charging of the capacitive plied. Both effects increased as a function of the number components of the tissue during the whole duration of the of pulses. Similarly, spheroid growth decreased with the packet. Moreover, we show that the consistently successful increase of the number of pulses, with a complete growth clearance with MHz repetition rate NPS™ (MHz-NPS™) inhibition observed following the application of 500 pulses. treatment can be achieved with tissue-specific packet sizes. The application of bipolar pulses resulted in a significant At the same and even higher energy levels, tissue clearance reduction of the effects induced by unipolar pulses. How- is not possible unless the packet size is above a tissue- ever, the introduction of a delay of 100 ns between the specific threshold in length. This unique ability of MHz- two pulses’ phases, resulted in a complete restoration of NPS™ therapy to be able to deliver a fine-tuned electrical the unipolar effect. The results of this study suggest that exposure that targets a specific tissue type can be helpful bipolar cancellation might occur also within in vivo mod- when a pathology creates a localized electrically-distinct els. Further investigations with different pulses’ shapes environment with respect to the surrounding healthy tis- and interphase delays are needed to understand the mech- sue. We compare MHz-NPS™ exposures to equivalent anism behind nsPEF bipolar cancellation. microsecond (µs)-long pulses that provide an electrically- References equivalent plasma membrane charging. We find the equi- [1] S. Beebe, ‘usEP Induce Regulated Cell Death Mech- valent µs-pulse exposures are not able to provide the same anisms’, in Ultrashort Electric Pulse Effects in Biology and tissue clearance effectiveness as MHz-NPS™ therapy. We Medicine, 2021. discuss the intracellular membrane charging potentially [2] S. Beebe, ‘Effects of usEPs on Plasma Membranes— contributing to the final biological effect since it is a result Pores, Channels, and Repair’, in Ultrashort Electric Pulse of the high-frequency components of the NPS exposures Effects in Biology and Medicine, 2021. that are absent in µs-pulse exposures. [3] A. G. Pakhomov et al., ‘Cancellation of cellular responses to nanoelectroporation by reversing the stimulus OR-11 polarity’, Cell. Mol. Life Sci., 71(22), pp. 4431–4441, Multicellular spheroids as three-dimensional in 2014. vitro models for bipolar cancellation assessment [4] R. Orlacchio et al., ‘High-voltage 10 ns delayed Rosa Orlacchio 1, Muriel Golzio2, Jelena Kolosnjaj-Tabi2, paired or bipolar pulses for in vitro bioelectric experi-Philippe Leveque1, Delia Arnaud-Cormos1, Marie-Pierre Rols2 ments’, Bioelectrochemistry, (137), p. 107648, 2021. 1University of Limoges, CNRS, XLIM, UMR 7252, F-87000 , France OR-12 2Institut de Pharmacologie et Biologie Structurale (IPBS), Negative Effects of Cancellation During Bipolar France Nanosecond Electrochemotherapy Unipolar high-intensity nanosecond pulsed electric Vitalij Novickij 1, Nina Rembiałkowska2, Wojciech Szlasa2, fields (nsPEF) induce numerous bioeffects including regu-Julita Kulbacka2 1 lated cell death [1] and cellular permeabilization through Vilnius Gediminas Technical University, Institute of High the formation of nm-sized pores [2]. However, when a Magnetic Fields, Lithuania 2 second phase of opposite polarity is applied immediately Wrocław Medical Uniwersity, Department of Molecular and after the first polarity (e.g., bipolar pulse), the effects in- Cellular Biology, Poland duced by unipolar pulses are highly reduced [3]. This phe- Electrochemotherapy (ECT) with nanosecond pulses nomenon, called bipolar cancellation, is typical for nsPEF seems to be a logical evolution of currently established and it has been reported on numerous two-dimensional procedures based on microsecond pulse bursts. In (2D) cellular models for several pulse durations between this work, we studied the feasibility of high frequency 2 to 900 ns. Despite several hypotheses formulated, the nanosecond bipolar pulses for bleomycin and calcium mechanism behind is still unknown. However, it is of great electrochemotherapy. interest to the bioelectric community because it would al- As a model, A549 (human adenocarcinoma alveolar basal low to potentially tailor electric pulses to modulate spe- epithelial cells) and a multidrug-resistant H69AR human cific biological responses. In order to contribute to the lung cancer cell lines were used. For electroporation, the understanding of this newly discovered phenomenon, we bursts of 200 and 400 ns pulses (5, 7, 9 kV/cm) were studied for the first time, the occurrence of bipolar can-delivered (n=50) with a repetition frequency of 1 MHz. cellation in realistic three-dimensional (3D) multicellular Alternatively, the pulses were accompanied by a bipolar spheroids. These in vitro models made of cancerous cells, component (200, 400 ns) with or without delay (200 ns) allowed us to evaluate cellular permeabilization and viab- between them. For detection of cell permeabilization, ility upon nsPEF exposure. Spheroid tumor models, made Yo-Pro-1 (YP) and flow cytometry were employed. Elec- with HCT-116 human colorectal carcinoma cells, were ex- trochemotherapy was performed with the same pulsing 137 conditions but with the addition of bleomycin (100 nM) 1 mm gap electroporation cuvette. Bursts of 100 pulses or 2 mM CaCl2. were delivered with a varied frequency of 1 kHz and 1 It was shown that the application of unipolar nanosecond MHz. For detection of cell permeabilization, Yo-Pro-1 bursts triggers low to high permeabilization depending on and flow cytometry were employed. Cell viability was the pulse duration and amplitude, which is an expected evaluated 48-, 72- and 96-hours post-electroporation. As result. The introduction of a bipolar component (i.e., a reference, ESOPE (1.25 kV/cm x 100 µs x 8, 1 Hz) symmetrical pulse 200 + 200 ns), results in the cancella- protocol was used. tion effect. Basically, even though the delivered energy Without electroporation, all three drugs involved in the is doubled, the cells become impermeable to YP due to study (or the drug cocktail) did not result in any signi- rapid membrane depolarization by the opposite polarity ficant cell viability changes. However, combination with pulse. If the pulse is asymmetrical (i.e., 400 ns + 200 electroporation triggered detectable electrochemotherapy. ns), the cell permeabilization is still detectable but is The ESOPE protocol resulted in 75%, 52% and 53% significantly lower (P<0.05) compared to the unipolar viability after 72 h for CIS, BLM and MET, respectively. procedure. A total of 13 unipolar/bipolar protocols with The drug cocktail induced higher cell death (P<0.05) with various combinations of pulse durations and delays were up to 10% improvement. Further, the ESOPE protocol tested with three different amplitudes (5, 7, 9 kV/cm). was compared with nanosecond protocols. MET was not In all cases when a bipolar pulse component was intro- effective as a chemotherapeutic agent when nanosecond duced, the same phenomena of bipolar cancellation were sequences were involved. Only the highest intensity 8 detectable. Further, the permeabilization results were kV/cm x 500 ns x 100, 1 MHz protocol showed statistically supported by electrochemotherapy data with bleomycin significant changes versus control. Bleomycin with the or calcium in vitro. It was shown that the effects of same protocol induced comparable cell death rate (43%) electrochemotherapy are also canceled out when a bipolar as the ESOPE protocol. Respectively, more than 60% component is present. The cell viability remains the same of cells remained viable after nano-electrochmotherapy as in control samples, which were not treated by the with CIS. The 8 kV/cm x 500 ns x 100 protocol but pulsed electric field. delivered with 1 kHz frequency significantly hindered the Our study shows that high-frequency unipolar nanosecond treatment even though the permeabilization rate was still pulses result in successful electrochemotherapy and can be above 90%. Similar results were observed for 4 kV/cm an excellent alternative to ESOPE procedures. However, protocols indicating a dose and a frequency dependent the symmetrical bipolar nanosecond pulses have limited tendency. Finally, the drug cocktail was used. With the to no applicability due to the cancellation effect. In order best protocol (8 kV/cm, 1 MHz) the viability of the cells to overcome the problem, the modulation of the time was 35%, which is a considerable improvement taking delay between the unipolar and bipolar pulse should be into account the multi-drug resistance of the selected cell performed. line. Our results show that application of drug cocktails in the Acknowledgment: The research was funded by the context of electrochemotherapy can improve the response Research Council of Lithuania within the DAINA 2 of the drug-resistant cancer lines. Also, high-frequency framework grant No.: S-LL-21-4 (PI: V. Novickij). nanosecond electrochemotherapy can be as effective as The research was also supported by National Science ESOPE protocols, however, it’s also dependent on the Centre (Poland) within a framework of DAINA 2 chemotherapeutic agent involved. (2020/38/L/NZ7/00342; PI: J. Kulbacka). Acknowledgment: The research was funded by the Polish National Centre of Science of DAINA 2 OR-15 (2020/38/L/NZ7/00342; PI: J. Kulbacka), and also Electrochemotherapy Using Anticancer Drug supported by the Research Council of Lithuania grant Cocktail for Treatment of Drug-resistant Cancer (Nr. S-LL-21-4, PI: V. Novickij). Cells Nina Rembiałkowska 1, Vitalij Novickij2, Julita Kulbacka1 1Wrocław Medical Uniwersity, Poland P26 - Electrochemotherapy – Internal 2Vilnius Gediminas Technical University, Lithuania Tumors Electrochemotherapy (ECT) is the combination of electroporation and drugs with inhibited or limited trans- Thursday afternoon Track B port. In this work, we study the electrochemotherapy Oct 13, 13:30 - 15:00 with an anticancer cocktail containing a combination of bleomycin (BLM) with other drugs such as cisplatin (CIS), metformin (MET). The electroporation phe- nomenon’s dependence on pulse amplitude and pulse repetition frequency was investigated. As a model, drug- resitant human lung adenocarcinoma cell line (H69AR) was used. For electroporation, 4 and 8 kV/cm, 500 ns square wave high voltage pulses were delivered to cells in 138 OR-152 HCC in patients not suitable for other treatment options. Intraoperative electrochemotherapy of colorectal A prospective phase II clinical study was conducted in pa- liver metastases: Long term results of a prospect- tients with primary HCC who were not suitable for other ive phase II study treatment options according to the Barcelona Clinic Liver Ibrahim Edhemovic 1, Erik Brecelj1, Maja Čemažar1, Nina Cancer classification. A total of 24 patients with 32 tu- Boc1, Blaž Trotovšek2, Mihajlo Djokić2, Arpad Ivanecz3, Sto- mors were treated by electrochemotherapy. The proced- jan Potrc3, Maša Bošnjak1, Bostjan Markelc1, Bor Kos4, Dam- ure was effective, feasible, and safe with some procedure- ijan Miklavčič4, Gorana Gasljevic1, Gregor Serša1 related side effects. The responses of the 32 treated nod- 1Institute of Oncology Ljubljana, Slovenia ules were: 84.4% complete response (CR), 12.5% partial 2University Medical Centre Ljubljana, Slovenia response (PR), and 3.1% stable disease (SD). The treat- 3University Medical Centre Maribor, Slovenia ment was equally effective for nodules located centrally 4University of Ljubljana, Faculty of Electrical Engineering, and peripherally. Electrochemotherapy provided a dur- Slovenia able response with local tumor control over 60 months of Background: A previous pilot study proved the feas- observation in 78.0% of nodules. The patient responses ibility, safety and efficacy of electrochemotherapy in the were: 79.2% CR and 16.6% PR. The median progression- treatment of colorectal liver metastases. The aim of free survival was 15 months (range 2.7–60), and the over- this study was to evaluate long-term results and safety all survival over 5 years of observation was 72.0%. This of electrochemotherapy in the treatment of unresectable prospective phase II clinical study showed that electro- colorectal liver metastases. chemotherapy was an effective, feasible, and safe option Patients and Methods: In this prospective phase II study, for treating HCC in patients not suitable for other treat-patients with metachronous colorectal liver metastases ment options. were included. In all patients, at least one metastasis was unresectable due to its central location or a too-small fu- OR-154 ture remnant liver volume. Patients were treated by elec- Bleomycin based electrochemotherapy using vari- trochemotherapy using intravenously administered bleo- able electrode geometry electrodes for the treat- mycin during open surgery. ment of deep-seated soft tissue sarcomas Results: 84 metastases from 39 patients were treated. The Aurel Ottlakan, Gyorgy Lazar, Renata Koszo, Katalin objective response was 75% (63% CR, 12% PR). The me- Hideghety, Andras Nagy, Gabor Vass, Judit Olah, Erika Gab- dian duration of the response was 20.8 months for meta- riella Kis stases in CR and 9.8 months for metastases in PR. The University of Szeged, Hungary therapy was significantly more effective for metastases Introduction: Bleomycin based electrochemotherapy smaller than 3 cm in diameter than for larger ones. There (ECT) poses as an emerging technique not only in was no difference in response according to the metastatic the treatment of superficial skin tumours, but also in location, i.e., metastases in central vs. peripheral loca- case of advanced, metastatic and surgically inoperable tions. Progression-free survival was better in patients who deep-seated soft tissue sarcomas. responded well to electrochemotherapy compared to those Patients and Methods: During a 2-year period (February metastases that had a partial response or progressive dis- 2019- February 2021) 7 patients (5 male/2 female) with ease. However, there was no difference in overall survival, median age of 54 years (49-88) were treated with deep with a median of 29.0 months. seated, inoperable soft tissue sarcomas (STS) through Conclusions: Electrochemotherapy has proven to be safe bleomycin based electrochemotherapy at the University and effective in the treatment of colorectal liver meta- of Szeged Department of Surgery. Tumour histology stases, with a durable response. It provides local tumor included fibromyxoid sarcoma (n=2), epitheloid sarcoma control that enables patients with unresectable metastases (n=3), liposarcoma (n=1) and myofibroblastic sarcoma to receive further treatments. (n=1). All treatments were performed under general anesthaesia, OR-153 with the use of long needle VEG (variable electrode Long term results of a prospective phase II study geometry) electrodes. Treatment planning for electrode evaluating intraoperative electrochemotherapy of placement was preoperatively carried out in each case by hepatocellular carcinoma Pulsar software and confirmed with intraoperative ultra- Mihajlo Djokić 1, Blaž Trotovšek1, Maja Čemažar2, Maša sound during the procedure. Each procedure was started Bošnjak2, David Badovinac1, Damijan Miklavčič3, Bor Kos3, 8 minutes after intravenous bleomycin administration Miha Štabuc1, Borut Štabuc1, Rado Janša1, Lojze Šmid1, (15000 IU/m2) and lasted for a maximum of 40 minutes. Peter Popovič1, Gregor Serša2 Prior to- and after treatment (1 week, 1-2-4-6 months) 1University Medical Centre Ljubljana, Slovenia prospective data collection was carried out. Patient 2Institute of Oncology Ljubljana, Slovenia health status and QoL was assessed at each follow-up 3University of Ljubljana, Faculty of Electrical Engineering, visit. Tumour response was evaluated through imaging Slovenia (CT/PET CT/MRI) 2 months after ECT treatment as The aim of this clinical study was to investigate the per RECIST 1.1 guidelines, adverse events were evaluated effectiveness and long-term safety as well as long-term res- and graded according to Common Terminology Criteria ults of electrochemotherapy as an emerging treatment for for Adverse Events (CTCAE) version 4.0 [3]. Previous 139 treatments included surgical resection (7/7 cases), chemo- mor progression during the follow-up period. radiation (4/7 cases), radiation therapy (2/7 cases) and Conclusion: CaEP in patients with advanced esophageal immunotherapy (1/7 case). cancer was conducted without major safety concerns. This Results: Median operative time was 75 min (35-180), study opens the way for larger studies evaluating tumor median hospital stay 2 days (2-20). Treatments were regression and symptom palliation. well tolerated with minimal side effects. Grade 2 ul- ceration was experienced in four cases, and a transient OR-156 left musculus quadriceps femoris plegia occured in one Electrochemotherapy of peri-hilar primary liver patient. Median tumour diameter, tumour volume and tumors tumour depth was 5.9 cm (3.7-22.5), 131.13 cm3 (35.6- Luciano Tarantino 1, Giancarlo Bizzarri2, Aurelio Nasto3, 2456.22) and 6.18 cm (3.74-18.18), respectively. Two Giuseppina Busto4, Sara Bortone4, Emanuele Balzano5, Paolo month follow-up confirmed partial response in 5 patients Tarantino6, Riccardo Aurelio Nasto5, Paolo De Simone5 1 (71.42%), while stable disease in 1 patient (14.28%), and A.Tortora Cancer Hospital, Italy 2 progressive disease in 1 case (14.28%) as per RECIST PO Regina Apostolorum - Albano Laziele (RM), Italy 3 v.1.1. PO Polla - ASP Salerno, Italy 4 Conclusion: Local control of deep-seated STSs with PO Pagani - ASL Salerno, Italy 5 BLM-based VEG ECT combined with other treatment Pisa University, Italy 6 options hold a promising perspective and our results Dana Farber Cancer Institute - Boston (MA), United States may serve as a useful guide for further investigation and Purpose: To evaluate long-term efficacy of Electroche- treatment planning. mopherapy (ECT), of Hepatocellular Carcinoma (HCC) or Cholangiocarcinoma (CCC) infiltrating hepatic hilum (pH). OR-155 Material and Methods: We retrospectively reviewed Calcium electroporation, an experimental cancer data of 30 consecutive patients (20 M, 10 F; 43-85 treatment – results from a pilot trial within ad- year, mean: 62 year)with pH-HCC (n=19) or pH-CCC vanced esophageal cancer (n=11)treated with ECT. Tumors’ diameter ranged: 2.5- Charlotte Egeland 1, Lene Jensen1, Julie Gehl2, Ismail 7.5 cm (mean:4.1 cm). 63% (n=12/19) HCC had portal Gögenur2, Michael Patrick Achiam1 vein tumor thrombosis (PVTT). 66% (n=6/9) CCC with 1Rigshospitalet, Denmark Klatskin tumor had preliminar percutaneous biliary drain- 2Zealand University Hospital, Denmark age. 25 patients received US-guided percutaneous ECT Background: Calcium electroporation (CaEP) is a ablation, 5 patients had ECT in open laparatomy, and novel anti-cancer treatment where a high influx of cal- were followed-up with contrast-enhanced-MDCT 4 weeks cium, induced by local injection of a calcium solution and after treatment and every 6 months thereafter. locally applied reversible electroporation, leads to tumor Results: No perioperative major complication occurred. necrosis. In this first-in-man trial, CaEP was administered 2/30(6.7 %) HCC patients dropped out follow-up because to patients with advanced esophageal cancer. The primary of experienced fatal hemorrhage from gastroaesophageal aim of this exploratory study was to establish safety and varices 4-5 weeks after ECT. 2 patients underwent Liver feasibility. transplantation after successful ablation of portal vein tu- Materials and Methods: Patients with advanced esopha- mor thrombus with ECT. Post-treatment CT showed an geal cancer, without other available treatment options, overall complete ablation in 17/28 cases (60%). Complete could be included. In an outpatient setting, all patients ablation was achieved in 14/17%(83%) HCC patients and were put in general anesthesia. For the endoscopic proin 3/11(27%) CCC patients. The follow-up ranged from cedure, we used a pulse generator (ePORE®, Mirai Med- 6 to 66 months (median: 24 months,). Follow-up CT ical) and a single-use probe with two parallel electrodes showed local tumor recurrence in 15/17(88%) HCC pa- (EndoVE®, Mirai Medical) connected to a gastroscope. tients within 6 - 18 months, and in 9/11 (81%) CCC pa- The calcium solution (Calcium gluconate, B.Braun 0.23 tients within 3–12 months. After 6–66 months follow-up, mmol/ml) was injected into the tumor followed by loc- 9/17 (53%) HCC patients and 2/11 (18%) CCC patients ally applied electrical pulses (bipolar pulses of 5000 kilo- are still alive. Patients died for tumor progression , liver hertz/1000 Volt). After treatment, adverse events, pain, failure, gastrointestinal hemorrhage or cardiovascular fail- and dysphagia were registered and all patients were fol- ure lowed with computed tomography scans and upper endo- Conclusion: In our retrospective cohort, ECT showed en- scopies for up to three months. couraging efficacy for the local control of pH-HCC and Results: Eight patients were treated. One serious adverse was moderately effective for pH-CCC. event (one-day hospitalization due to anemia, requiring a single blood transfusion) and four adverse events were OR-157 registered including anemia, local pain, and oral thrush. Histologic changes of porcine portal vein anosto- Initially, five patients suffered from dysphagia, two repor- mosis after electrochemotherapy with bleomycin ted dysphagia relief and three reported no change. From Mihajlo Djokić the imaging evaluation, one patient had a partial response, University Medical Centre Ljubljana, Department of Abdom- three patients had no response, and four patients had tu- inal Surgery, Slovenia 140 Introduction: Locally advanced pancreatic cancer fre-P31 - Gene Electrotransfer for quently invades portal vein and it has to be resected and Antibody Production anastomosed. We currently do not know the effect of ECT on vein anastomoses, therefore it cannot be used in this setting to potentially reduce local recidives. The aim of Thursday afternoon Track C this study is to assess histologic changes of portal vein Oct 13, 13:30 - 15:00 anastomosis after ECT. Methods: Porcine model was chosen for the study as they OR-78 have similar hepatobiliary anatomy and function as hu- Combined treatment of intratumoral DNA-based mans. In total 12 pigs were enrolled in the experiment. anti-CTLA4 antibody gene electrotransfer and ir-Study was approved by the national Ethics Commission radiation for treatment of solid tumors for experiments on animals. After induction to anaes- Bostjan Markelc 1, Simona Kranjc Brezar1, Tanja Jesenko1, thesia, laparotomy was performed and inferior v. cava, Tim Bozic1, Paul J. Declerck2, Liesl Jacobs2, Maja Čemažar1, Kevin Hollevoet2, Gregor Serša1 v. portae and v. lienalis were identified. Then the ana- 1Institute of Oncology Ljubljana, Slovenia stomosis of portal vein (treated with ECT) and lienal vein 2KU Leuven, Belgium (control vein) were made. Inferior v. cava was treated with ECT. We will observe histological changes over 7, 14 Recombinant monoclonal antibodies (mAbs) are and 28 days after ECT. among the most promising classes of cancer immuno- Pigs were divided in 4 groups of three. First group was therapeutics. Since FDA’s approval of the anti-CTLA-4 control group (electroporation only) and was sacrificed 28 (cytotoxic T-lymphocyte–associated protein 4) antibody days after ECT. Group 2, 3 and 4 were treated with ECT Ipilimumab for the treatment of melanoma, the field dra- with bleomycin and sacrificed at 7, 14 and 28 days respect- matically expanded and mAbs targeting several other im- ively. Electroporation was made with plate electrodes. We mune checkpoints were developed. Immune checkpoint inused 12 electric pulses with amplitude of 1040 V, 20 µs hibitors (ICIs) are now widely used for treatment of dif- long and 0,5 Hz in frequency (IGEA S.p.A, Carpi, Italia). ferent cancer types. However, mAbs have several draw- Eight minutes before electroporation, groups 2, 3 and 4 backs including high production costs, frequent high dos- recived 15.000 IE/m2 of bleomycin. ing, limited efficacy as single agents, and systemic tox- Results: Out of 12 pigs, 3 of them had to be euthanized icity following i.v. infusion. Systemic immune-related 1-3 days earlier because of laparotomy dehiscence and ma- toxicity can be circumvented with intratumoral injection jor postoperative hernias. It did not have impact on the of mAbs, which, while retaining similar efficacy, still re- results as 2 of the animals were in the 28 day group. His- quire frequent dosing. On the other hand, plasmid DNA tological results were exceedingly homogenous. In 7 day (pDNA) encoding CI antibodies presents a promising al-group, we observed endothelial loss with signs of regener- ternative to the conventional mAb proteins. Gene electro- ation. Also, severe muscle cell loss and fibrosis was seen in transfer (GET) which utilizes electroporation, a clinically- tunica media. Thrombosed minor blood vessels were seen established technique, can be used to efficiently deliver in tunica adventitia. In the 14 day group endothelium pDNA to cells and tissues, including tumors. ICIs are es- regenerated completely. More and more fibrosis organiza- pecially effective in treatment of tumors with pre-existing tion were seen in tunica media on 14 and 28 day groups. anti-tumor immunity (‘hot tumors’), with limited efficacy Histological changes were comparable on intact vena kava in tumors lacking anti-tumor immunity (‘cold tumors’). and anastomosis of v. lienalis which was not treated with Radiotherapy can be used to trigger the beginning of ECT. No thrombosis was evident in all animals. Histolo- transition from cold to hot tumors, thus facilitating ICI gical findings did not differ from electroporation only and action. To examine the therapeutic potential of combining ECT groups. intratumoral GET of ICIs and radiotherapy we performed Discussion: Our study showed that ECT of vein anastom- GET of plasmid DNA encoding murine anti-mouse CTLA- osis is safe and feasible. Although ECT significantly di- 4 mAb (p(aCTLA4)) and combine it with two irradiation minished the muscular layer of veins, findings were com- regimes, a single dose of 10 Gy and fractionated dose of parable with untreated anastomosis which are made on a 3x 5 Gy in murine MC38 colon cancer. We first compared daily basis in operating rooms for variable reasons. Veins two previously established sets of GET pulse parameters: are a low-pressure system, therefore, any pseudoaneurysm one for pDNA delivery to tumors (8 pulses, 600 V/cm, formation is not likely. On the other hand, loss of mus- 5 ms, 1 Hz) and one for electrochemotherapy ((ECT), 8 cular layer could possibly predispose in pseudoaneurysm pulses, 1300 V/cm, 100 µs, 1Hz). GET of p(aCTLA4) formation if treating arteries. In our opinion, the risk of to tumors resulted in similar anti-CTLA4 mAb plasma thrombosis should not be higher than in normal vein ana- levels regardless of the pulse parameters, however; due to stomoses, because of complete endothelial regeneration. a pronounced anti-tumor effect of control plasmid DNA Conclusion: ECT of vein anastomosis is safe and feasible. (pCtrl) when delivered with pulse parameters for tumors, Care should be taken if treating arteries. ECT pulse parameters were used in the rest of experi- ments. When GET of p(aCTLA4) was combined with ir- radiation, the combination with fractionated dose of 3x 5 Gy, which induces more immunostimulatory effects, had a more pronounced anti-tumor effect than the combina- tion with single dose irradiation of 10 Gy. Moreover, only 141 the combination of fractionated irradiation with GET of provements. In vitro, mAb levels consistently improved p(aCTLA-4) outperformed better than the combination with decreasing sizes of plasmid backbone. In vivo, follow-with pCtrl. ing intramuscular pDNA electrotransfer in mice, the cor- We confirmed that GET of p(aCTLA4) could be a po- relation was less consistent. While improving biosafety, a tential alternative to systemic infusion of ICIs and when reduction in size beyond a standard conventional plasmid combined with irradiation, an anti-tumor effect can be backbone did not improve mAb levels in vivo. Cassette achieved. Future research will focus on determining the modifications, such as swapping antibody chain order or optimal schedule for a “booster” GET of p(aCTLA4) after use of two versus a single encoding plasmid, significantly irradiation and also exploring the use of pDNA encoding increased antibody expression in vitro, but failed to trans- other ICIs, to achieve a better anti-tumor effect. late in vivo. Conversely, a significant improvement in vivo but not in vitro was found with a set of muscle-specific pro- OR-79 moters compared to the ubiquitous CAG promoter. Des- Building a genetic medicine platform for DNA- pite the limited translation between in vitro and in vivo, encoded antibody therapeutics improvements in potency and biosafety were identified. Kevin Hollevoet 1, Giles Vermeire1, Liesl Jacobs1, James A. In conclusion, our new findings contribute to a broadly and Williams2, Debby Thomas1, Stéphanie De Vleeschauwer1, Tre- clinically applicable genetic medicine platform for DNA- vor Smith3, Maya Imbrechts1, Rana Abdelnabi1, Johan Neyts1, based antibody therapeutics. Nick Geukens1, Paul J. Declerck1 1KU Leuven, Belgium OR-80 2Nature Technology Corporation, United States Advancing DNA-based antibody therapeutics 3Inovio Pharmaceuticals, United States through evaluation and characterization of in vivo DNA-based delivery aims to administer the antibody- expression encoding nucleotides using a non-viral plasmid DNA Marie-Lynn Cuypers 1, Nick Geukens2, Kevin Hollevoet2, (pDNA) vector. Such genetic shortcut allows for pro- Paul J. Declerck1, Maarten Dewilde1 1 longed antibody production in vivo, and addresses some KU Leuven, Pharmaceutical Sciences, Belgium 2 of the bottlenecks of conventional antibody protein thera- PharmAbs, Belgium peutics. This can allow for a broader antibody accessib- DNA-based antibody therapy seeks to administer the ility and e.g. facilitate more effective cocktails. We pre- encoding nucleotide sequence, rather than the antibody viously presented preclinical proof of concept for various protein. This innovative approach can allow the patient’s DNA-encoded full-length monoclonal antibodies (mAbs) body to produce its own medicine, and presents an al- and nanobodies, delivered to muscle or tumor. ternative to the complex production and frequent admin- To advance our DNA platform, we evaluated examples of istration of antibody protein. We previously obtained product development. First, we focused on COVID-19. therapeutic proof of concept in mice for several plasmid-Using B cell mining in a convalescent patient, we identi- based (pDNA) antibody formats delivered via intramuscu-fied a panel of human mAbs that potently neutralized the lar electroporation, and demonstrated clinical translation main SARS-CoV-2 variants of concern. The lead mAb in sheep. To further improve the in vivo monoclonal an- 3B8 also retains its activity against omicron. In hamsters, tibody (mAb) expression, a better understanding of what intramuscular electroporation of DNA-encoded 3B8 res- happens after the administration of the plasmids is re- ulted in median mAb serum levels up to 90 µ g/ml ten quired. The current PK profile of mAbs expressed in vivo days after pDNA delivery, and protected animals against in mice is typically characterized by a steady increase in SARS-CoV-2 infection. Second, we focused on immuno- the first week(s) reaching peak levels in plasma of 1-10 therapy by delivering a cocktail of DNA-encoded immun- µ g/mL. Thereafter though, in most cases, a steep decline omodulators directly into the tumor. In a murine cancer of around 5 to 10-fold is observed. Ideally, plasma levels model, intratumoral electroporation of plasmids encoding need to be maintained above 10 µ g/mL for a longer time, IL-12 and an anti-PD-1 and anti-CTLA-4 antibody sig- in line with trough levels for most therapeutic mAbs. We nificantly delayed tumor growth compared to IL-12 alone aim to gain insight in the factors that affect mAb expres- and the combination of anti-PD-1 and anti-CTLA-4 an- sion after intramuscular pDNA electroporation at DNA, tibodies. The triple combination also enabled significant mRNA, protein, and cellular level. abscopal effects, which was not the case for the other treat- To evaluate the pDNA and RNA expression levels over ments. time, BALB/c mice were treated with pDNA encoding To assess translational feasibility, we previously reported the murine 4D5 mAb targeting HER2, delivered via intra- on intramuscular DNA-based antibody delivery in 40-70 muscular injection followed by electroporation. Following kg sheep, a clinically relevant model. Here, we further pDNA electrotransfer, biopsies and blood samples were aligned and affirmed the electroporation setup to clinical taken at different timepoints (up to 3 months). pDNA and practice. In groups of eight sheep each, escalating doses RNA was quantified using (reverse transcription) quant- of pDNA (1 and 4 mg) gave serum mAb levels up to the itative PCR. The RNA expression levels were on average µ g/ml range, without inducing anti-drug-antibodies dur- stable, but there was a high inter-animal variation. In ing the six week follow-up. contrast, a fast decline of pDNA was observed, i.e. 24h While the currently attained mAb titers are effective in after treatment the pDNA levels were <1% of the injec- various rodent disease models, we explored further im- ted amount. However, the fast and substantial loss of 142 pDNA does not correspond with the typically observed (pDNA) and a proprietary medical device for the intra-continuing protein detection, up to one year after pDNA muscular injection of pDNA, followed by the delivery of injection. We hypothesize that only a very small amount very short electrical pulses to the muscle tissue surround-of the administered pDNA is responsible for the observed ing the injection site. These pulses promote the in vivo protein expression and that the pDNA that is lost within transfection of muscle cells, resulting in antibody produc- the first 24h did not enter the cell and/or nucleus. To tion and secretion, and ultimately uptake into peripheral test this hypothesis, muscles isolated from treated anim-circulation. Animal proof-of-concept studies demonstrate als were sliced and used in an RNA scope assay to localize that these in vivo-produced antibodies are functional and the pDNA in the muscle cells. Preliminary results indic- demonstrate efficacy in a diverse array of disease mod- ate that the pDNA is localized mainly at the injection site. els, including cancer, inflammatory disease, and infectious Therefore, only a small amount of muscle cells will be part disease. Through significant improvements in manufac- of the protein factory. turing, distribution and storage, administration time, and In conclusion, this study investigates some fundamental administration frequency, MYO technology has the poten-aspects providing more insights about the efficiency of the tial to dramatically improve the accessibility and usage of DNA-based antibody delivery and the factors that can antibody drugs. play a role in the obtained protein levels. All methods generated in this study can be used as a toolbox to evalu- OR-169 ate other plasmid constructs or delivery methods with the Mouse, canine and human interleukin-12 antibi- ultimate goal to achieve a robust and prolonged antibody otic resistance gene-free plasmids: bacterial main- expression. tenance and gene electrotransfer efficiency Urska Kamensek, Andrej Rencelj, Tanja Jesenko, Tinkara OR-81 Remic, Gregor Serša, Maja Čemažar MYO Technology for DNA-Based Delivery of Institute of Oncology Ljubljana, Slovenia Next-Generation Antibody Therapeutics Background: Gene electrotransfer (GET) of plasmids Andrew D. Cameron, Debnath Maji, Xin Yao, Veronica encoding the cytokine interleukin 12 (IL-12) is currently Miguela, Robert Miller, Marek M. Drozdz, Delcora A. Camp- approaching clinical use for treatment of various superfi- bell, Mitchell Sitnick T. Sitnick, Rachel A. Liberatore cial solid tumors. In this study, we present the prepara- RenBio, Inc., United States tion and testing of antibiotic resistance gene-free plasmids As a class of drugs, antibodies have soared in both encoding mouse, canine and human IL-12, which com- clinical development and use in the last decade. There ply with the EU regulatory requirements that recommend are now more than 100 approved antibodies in the United against the use of antibiotics during the production of clin- States and Europe, for use in diverse disease areas from ical grade plasmids. The mouse orthologue was prepared arthritis and breast cancer to osteoporosis and migraines. to facilitate preclinical evaluation in mouse tumor mod- In addition, though long considered to have great poten- els, canine for cancer treatment of client-owned dogs, and tial within the infectious disease research community, the human for translation into human clinical testing. Our larger world has now been exposed to the utility of anti- aim was to evaluate the maintenance of these plasmids bodies for the treatment and prevention of viral diseases in a bacterial culture and test their transfection efficiency as part of the efforts to combat the COVID-19 pandemic. after GET to melanoma cells. Despite the broad applicability of antibody-based drugs, Methods: Plasmids encoding mouse (pORF-mIL-12- their more widespread use is limited by high manufac- ORT), canine (pORF-caIL-12-ORT) and human IL-12 turing costs and administration hurdles. Many antibod- (pORF-hIL-12-ORT) were prepared using the antibiotic- ies are delivered via time-consuming intravenous infusions free selection strategy operator-repressor titration (ORT®, and others, regardless of administration route, need to be Cobra Biologics), and all have the same backbone without dosed at regular 2-to-4-week intervals. Another drawback an antibiotic resistance gene. A commercially available of many biologics, also highlighted by the distribution and plasmid encoding a mouse IL-12 (pORF-mIL-12 (p40p35), storage of antibodies and vaccines for COVID-19, is the Invivogen), with an ampicillin resistance gene in the back- requirement for maintenance in a low temperature envir- bone was used as a control. Plasmid maintenance was onment prior to use. Such cold-chain dependencies can evaluated by determining plasmid yields and topologies significantly hinder the ability to get therapeutics to large after sub-culturing of transformed bacteria. Transfec- numbers of people, a problem that is amplified in resource tion efficiency was evaluated by determining the plasmid poor settings. MYO Technology™, a DNA-based platform copy number, expression and cytotoxicity after GET to for the delivery of therapeutic proteins, was developed to mouse (B16-F10), canine (CMeC-1) and human (SK-Mel- overcome these barriers to increased antibody use. DNA 28) melanoma cell lines. is easier to manufacture than antibodies and lacks most Results: Plasmid isolation yields of antibiotic resistance cold chain requirements. Additionally, administration us- gene-free plasmids were on average lower than that of the ing MYO Technology takes just a few minutes, and thera- commercial IL-12 plasmid. However, the yields remained peutic levels of antibody can potentially be maintained constant after sub-culturing, confirming that these plas-for months, in contrast to the weeks-long durability when mids are stably maintained in transformed bacteria dur- administered by standard methods. The MYO Techno- ing cell division, and are not lost over generations. After logy platform consists of antibody-encoding plasmid DNA GET to melanoma cells, the number of plasmids detected 143 in transfected cell was relatively low independently of the steel (type AISI 316L) electrodes of a PEF chamber. Dif-plasmid used (from 2 to 36 copies per cell). Nevertheless, ferent processing conditions obtained by coupling different IL-12 expression from all four plasmids was detected in field strength (12–31 kV/cm) and total specific energy in- all three cell lines, confirming that even 2 copies per cell put (20–100 kJ/kg) were simulated with two model liquid are sufficient for transgene expression. The IL-12 mRNA food solutions with different values of pH (3.5 and 7) and expression levels did not correlate with the plasmid copy electrical conductivities � (2 and 3.5 mS/cm) and different number. halides concentration chloride concentration (0 - 0.0201 Conclusions: We showed that the tested antibiotic res- mol/L). A validation of the predicted results was achieved istance gene-free IL-12 plasmids are stably maintained in by using experimental data on metal release measured bacteria and support sufficient IL-12 expression after GET with ICP-MS technique. in vitro. Therefore, these plasmids have the potential to The results showed that the developed model was able proceed to in vivo evaluation and, ultimately, translation to accurately predict the extent of metal release from the to clinical studies in Europe enabled by the absence of the electrodes of a PEF chamber. The concentration of metals antibiotic resistance gene. However, before translation, released from the electrodes depends on the process para- plasmid production still needs to be optimized to ensure meters of PEF treatment (field strength, energy input) improved plasmid yield, quality and, above all, standard- with a key role being played by the pulse repetition fre- ization of the final antibiotic-free product in good manu- quency. Moreover, regarding the treatment medium char-facturing practice conditions. acteristics, the presence of halides, high conductivity, and low pH, significantly affect the release of metals. Specific- ally, it was shown that higher electrical conductivity lead P5 - Mechanisms and Applications of to higher current passing through the chamber promoting PEF in the Food Industry the metal release. On the other hand, lower pH values in- crease the faradaic current density, thus increasing build- Thursday afternoon Track D up of charge at the double layer, leading to higher release Oct 13, 13:30 - 15:00 of metals. OR-94 OR-194 Effect of post-electroporation recovery of Thai Modelling and validation of the electrochemical basil leaves prior convective and vacuum drying phenomena at the electrode-solution interface of Grant Thamkaew, Allan G. Rasmusson, Dmytro Orlov, Fe- a PEF treatment chamber derico Gómez Galindo Gianpiero Pataro 1, Giovanna Ferrari2 Lund University, Sweden 1University of Salerno, Italy 2University of Salerno and ProdAl S.c.a.r.l., Italy Pretreatment by reversible electroporation followed by resting (storage under saturated moisture at 21 ± 2 °C) In PEF processing the food matrix is typically placed was evaluated for modification of the properties of dried in direct contact with charged metal electrodes of either and rehydrated Thai basil leaves. The treated leaves were batch or continuous treatment chamber and exposed to dried by convection at 40 °C or in a vacuum at room tem- repetitive (up to kHz) short duration (�s-ms) electric field perature. The results showed that vacuum drying pro- pulses of low (0.1-1 kV/cm), moderate (1-5 kV/cm) or high voked more cell damage and tissue collapse than convect- intensity (10-40 kV/cm), supplied by a high voltage pulse ive air drying at a moisture ratio (MR) of 0.2 and 0.1. Un- generator. der this level of MR, the pulsed electric field (PEF) and However, when these typical conditions for PEF pro- resting pretreatment exerts a protective effect of the tissue cessing are applied, undesired electrochemical reactions, for both drying methods. However, under complete de- especially those involving metal release from the elec- hydration (water activity, aw = 0.05) damage seems to be trodes, unavoidably occur at the electrode-solution inter- similar for both drying methods despite the PEF pretreat-face of a PEF treatment chamber. The occurrence of ment. Remarkably, reversible electroporation followed by these electrode reactions is a very complex phenomena, resting resulted in higher trichome preservation. At MR which is affected by several factors, such as PEF chamber of 0.05, the area of trichomes on the surface of convective- design and electrode material, PEF electrical parameters, dried, PEF-rested and fresh samples were not statistically as well as composition and chemical-physical properties of different at 2267 ± 89 µ m2 and 2218 ± 65 µ m2, respect-the treated products. ively, showing that this pretreatment still exerts a pro- For this reason, numerical simulations could significantly tective effect on trichomes when complete dehydration is help to improve the understanding of the electrochemical achieved. phenomena occurring at the electrode-food interface of a PEF chamber, by clarifying the effects of the main elec- OR-92 trical parameters and food constituents on electrode cor- Effect of pulsed electric field (PEF) intensity on rosion or release of electrode’s materials. separated cream yield, physico-chemical proper- In this work, a Multiphysics model based on the Finite Ele- ties and stability ment Method (FEM), was developed to predict the effect Markus Walkling-Ribeiro, Thomas Jacob, Lilia Ahrné of different electrical parameters as well as treatment me- University of Copenhagen, Food Science, Denmark dium characteristics on the metal release from the stainless 144 Pulsed electric field (PEF) technology has the poten-tainable peeling alternatives that can effectively peel fruits tial to be an effective emerging technology for the modi- and vegetables with minimum peeling losses and a better fication of structural components in dairy food, thereby, quality of the product, while providing less environmental opening the door to new applications and, typically, re-problems and reducing water and energy consumption. In taining the native character of the latter. In this study the recent years, the application of unconventional techno- the impact of PEF intensity on fat separation from raw logies as a pre-treatment for the peeling, such as infrared milk after centrifugation was investigated in terms of yield, radiation heating, ohmic heating, ultrasounds-assisted lye physico-chemical properties and physical stability of the peeling, pulse electric fields-assisted steam peeling, as well produced cream. as the use of enzymes, is intensively investigated. Raw milk was kept at 4°C prior to PEF treatment. The The aim of this study, which was in part carried out in the latter was carried out with a continuous-flow co-current frame of the European project AccelWater (Project ID: treatment chamber using electric field strengths ranging 958266), was to assess the potential of PEF technology from 9 to 27 kV/cm for a treatment time of 66 µs. The to facilitate the steam peeling of different fruits, includ- PEF-treated milk was then heated to 40°C before being ing tomatoes, peaches and apples. Samples were exposed skimmed, allowing for subsequent cream analyses using to PEF treatment of different intensity in terms of field a Milkoscan (yield and fat content), Mastersizer (particle strength (0.25-1 kV/cm) and energy input (0.25-1 kJ/kg), size), and high-shear mixer (overrun after 20 and 60 s) and before steam peeling (1-3 bar). The impact of PEF on the centrifugation (stability after 20 and 60 s). peelability of fruits was assessed by measuring the change Yield of cream increased significantly (P<0.05), for milk in the textural properties (hardness, peel strength) of the PEF-treated at 21 kV/cm and below considering the total fruits, as well as evaluating the peeling index, peeling loss, amount of fat removed from the skimmed milk. How- and weight. ever, no significant differences were observed for the fat Results obtained from this study demonstrate the poten- content when comparing cream from untreated and PEF- tial of PEF pre-treatment to reduce the peel strength and treated milk and for the particle size of its milk fat globules to induce high score of peeling ability for of all the fruits (P�0.05). The PEF treatments also influenced the overrun that led to less product loss. In particular, regardless of the produced cream, with the most intense treatment the type of processed fruit, the best results were obtained at 27 kV/cm decreasing the overrun capacity, whereas the for intermediate values of PEF treatment intensity (0.5 mildest treatment at 9 kV/cm increased it by 30% com- kV/cm, 0.5 kJ/kg). Moreover, as compared to the con- pared to cream that was not treated with PEF (P<0.05) ventional steam peeling method, the application of PEF at 20 s. At 60 s an increased overrun was observed from 9 pre-treatment enabled to reduce the steam pressure during to 21 kV/cm, achieving an overrun up to 65% greater for the thermophysical peeling phase by about 20% for toma- the least intense PEF treatment conditions than for un- toes, 33% for apples, and 25% for peaches, thus reducing treated cream (P<0.05). Moreover, the stability analyses the consumption of energy and water. of the cream showed a stronger cohesion induced by the OR-93 most intense exposure to PEF. Combination of different techniques for assessing PEF processing of raw milk at different intensities and PEF-treatment in plant and animal tissues subsequent milk separation for cream production, led to Jessica Genovese 1, Matej Kranjc2, Igor Serša3, Pietro a higher yield in cream, while not impacting fat content Rocculi1, Damijan Miklavčič2, Samo Mahnič-Kalamiza2 and particle size. Based on the different PEF intensities 1University of Bologna, DISTAL, Italy applied, cream overrun and stability could be tailored to 2University of Ljubljana, Faculty of Electrical Engineering, industrial applications. Slovenia 3Institut “Jožef Stefan”, Slovenia OR-193 Pulse electric fields-assisted steam peeling of dif- In the field of food innovation, pulsed electric field ferent fruits and vegetables (PEF) technology has attracted increasing interest in re- Gianpiero Pataro, Oscar Mauricio Pulgarin Lopez, Gio- cent years and has become one of the most attractive vanna Ferrari new non-thermal technologies due to its lower energy con- University of Salerno, Italy sumption and short treatment times. Although there is Peeling is the first unit operation performed during the a considerable body of scientific work available, detailed industrial transformation of fruit and vegetables, whose information on detection and quantification of the effects performance is crucial for maximizing the efficiency of of electroporation in complex and inhomogeneous multi- overall process, while preserving the quality of the fresh cellular systems, such as real food systems, is still limited. product. The effectiveness of PEF treatment depends on both pro- Current industrial methods of tomato peeling involve the cess parameters and properties of the biological material, use of hot lye (sodium hydroxide) solution or pressurized and the appropriate choice of methods to detect and as- steam, which provide high quality peeled tomato fruits. sess changes caused by electroporation in food matrices is However, both these methods suffer from various disad- needed. A deeper insight into the changes that occur after vantages such as disposal of caustic, high pH waste solu- electroporation and their relation to the complexity of the tion, and excessive water and energy consumption. food matrices tested is of great value to develop new ideas For these reasons, research is focusing towards new sus- for electroporation-based treatments in the food industry. 145 Therefore, different characterization techniques were combined, namely electrical impedance spectroscopy, current-voltage measurements, and magnetic resonance imaging to evaluate the physical changes caused by PEF treatment in raw plant and skeletal muscles of interest for food and/or feed. Experiments were performed on potato tuber and apple fruit, as these plant tissues are most com- monly used for industrial PEF applications, and on the chicken broiler Pectoralis major that was selected as ref- erence skeletal muscle. Electrical impedance spectroscopy was used to measure the dielectric properties of the biolo- gical tissue before and after the application of pulses, as this is a common technique in food PEF applications to determine the degree of cell disruption. Another possible but rarely used technique is to analyse the voltage and current waveforms recorded during the application of elec- trical pulses. Analysis of electric current signals allowed us to detect changes in electrical properties of the cell mem- brane in real time. Advanced MR techniques were the third technique used to monitor the spatially-dependent effect of PEF treatment. In particular, the transverse re- laxation time T2 provided evidence of the redistribution of water and solutes in the tissue as a function of the applied electric field during PEF treatment. The results of this study contribute to a better understanding of the effect of electroporation in complex and inhomogeneous multi- cellular systems, and provide important insights and calls for critical choice of electroporation assessment methods to optimize PEF treatment conditions. 146 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 (and Coffee break) als are being proposed in this field. The final version of this paper will include the references that allow to study the current electronic technology for Monday Poster Session Track medical applications. Oct 10, 14:45 - 16:00 PO-016 PO-001 Membrane extracellular vesicles released after A Review of Electronic Technology for Medical electroporation as mediators for melanoma- Applications of Electroporation fibroblasts communication Borja López-Alonso, Pablo Briz, Héctor Sarnago, José Anna Choromańska, Urszula Szwedowicz, Jolanta Saczko, Miguel Burdío, Óscar O Lucía Julita Kulbacka University of Zaragoza, Electronic Engineering and Commu- Wroclaw Medical University, Department of Molecular and nications, Spain Cellular Biology, Poland Electroporation is based on the permeability increment Analyzing the electroporation (EP) phenomenon and of cell membranes by means of the application of elec- the objective complexity of the effects at the cell level tric field pulses to induce controlled potential in the cell associated with it, we came across a problem that has not membranes. For this purpose, it is necessary to develop yet been investigated to increase electrochemotherapy’s electronic technology capable of applying and controlling therapeutic effectiveness (ECT). In the context of ECT electric field pulses. In the area of electroporation for med- optimization, we aim to isolate the extracellular vesicles ical applications the requirements are challenging, since it (EVs) released during the different reversible EP proced- is necessary to apply electric field pulses in a wide range ures. Up to now, the exact nature of that EVs is unknown. of intensities, and these must be controlled precisely to It is also not clear how the profile of the released EVs induce the desired effect. depends on the pulse duration (nano-, micro-and milli- This paper presents the basic concepts and a review of the second pulses). Today, we know that EVs are essential state-of-the-art of electronic technologies involved in med- mediators of intercellular communication, enabling the ical applications of electroporation, including electric-field functional transfer of bioactive molecules from one cell to pulse generators for the delivery of high-voltage electric another. Melanoma-derived exosomes stimulated cancer pulses, electroporation electrodes to control electric field, cell proliferation, mediated the epithelial-mesenchymal and electroporation monitoring and control systems to op- transition, and induced pre-metastatic niche formation timize treatment delivery. [Hatanaka et al. 2014]. Firstly, in the field of electroporation generators, it is es- This study investigated the EVs-mediated transformation sential to design technology that allows to apply voltage of normal fibroblasts to tumor-associated fibroblasts, waves in the most controlled and precise way. Nowadays, focusing on the functional regulation of vascular cell the voltage pulse is the most widely used waveform in adhesion molecule-1 (VCAM-1) expression, cell viability, medical electroporation application and, there are already and migration capacity. The experiments were performed a few commercially certified available generators. Des- on human primary fibroblasts and two commercial pite this, requirements of electroporation treatments are malignant melanoma cell lines: A375 - primary human becoming increasingly challenging, and a wide range of ex- skin malignant melanoma cell line with endogenous perimental electroporation generators have been proposed. BRAFV600E mutation, and Me45 – human metastatic These are based on multilevel electronic structures, trans- malignant melanoma cell line. Electroporation with a former topologies, resonant circuits, and multi-output sys- low-intensity electric field (with a range of 800-1600 tems, among others. V/cm) was delivered by a BTX square wave generator. Secondly, the electrodes are key elements in the electro- The Presto Blue test was applied to assess cell viability. poration process to generate the required electric field. Cellular migration was analyzed with the wound healing They may also have built-in sensors to send information assay, and the VCAM-1 expression in fibroblast cells to the control system and optimized geometries to create was determined by Real-Time PCR after 72 hours of special electric field patterns. Currently the most widely incubation. used are those based on needles or parallel plates, but We observed that after incubation with melanoma-derived there are many novel proposals including mono-electrode EVs, the primary human fibroblasts presented differences devices and multi-electrode implementations. in cell viability, migration capacity, and VCAM-1 ex- Finally, medical applications of electroporation require pression, dependent on EP parameters and the degree of control systems that allow to carry out effective and safe melanoma cells malignancy. treatments. Ideally, such systems allow to control the ap- plied electric field to achieve the desired effects, depend- Financing: This study was supported by funding ing on the electroporation medical application. For these from the Department of Molecular and Cellular Biology, reasons, it is necessary to develop monitoring and control Wroclaw Medical University grant no.SUBZ.D260.22.016. technologies that allow to know and control the treatment status. Nowadays, the most widespread methods of con- References: Hatanaka M, et al. Cleaved CD147 shed trol are based on the preplanning of treatments by means from the surface of malignant melanoma cells activates of finite element models and on-line monitoring of imped- MMP2 produced by fibroblasts. Anticancer Res. 2014, ance and temperature, although more innovative propos- 149 34, 7091-7096. PO-023 The efficacy of microsecond electric pulses with PO-019 calcium ions is related to the expression of drug Non-invasive real time analysis of electroporation resistance genes and proteins phenomenon of individual cells Nina Rembiałkowska 1, Vitalij Novickij2, Dagmara Anne Calvel 1, Katia Grenier1, David Dubuc1, Marie-Pierre Baczyńska1, Magda Dubińska-Magiera3, Wojciech Szlasa1, Rols2 Jolanta Saczko1, Julita Kulbacka1 1Laboratoire d’analyse et d’architecture des systèmes, LAAS- 1Wrocław Medical Uniwersity, Poland CNRS, France 2Vilnius Gediminas Technical University, Lithuania 2CNRS, IPBS, France 3University of Wrocław, Poland Background: Electropermeabilization (EP) is a prom- Drug and multidrug resistance (MDR) in cancers ising approach for the targeted treatment of diseases is still the unsolved problem of anticancer protocols. such as cancer. This technique allows the transient pores Various MDR inhibitors, modulators, or nanocarriers formation in the cell membranes, facilitating the access are used to omit this phenomenon. Here we propose of therapeutic agents such as bleomycin and cisplatin in a physical method that employs short electric pulsed the case of electrochemotherapy (ECT). to increase the efficacy of cytostatics in drug resistant However, while the efficacy and safety of EP have been cancers. The main purpose of the study was to investigate demonstrated, questions remain and hinder its use. The whether various electroporation parameters can modulate differences in response between healthy and cancerous the expression of drug resistance-related genes. We tested cells and the non-response of certain cancerous cells are different parameters of electroporation (1 and 1.5 kV/cm, still unknown. 8 pulses, 60 and 100 us square wave voltage, 1Hz) without The current study aims at proposing an early detection and with Ca2+ (CaEP – calcium electroporation). The of responses of healthy and cancerous cells, untreated or human cancer cell lines: sensitive and resistant, were treated by ECT or by different types of EP (reversible, tested. For detection of cell permeabilization, Yo-Pro-1 irreversible, calcium), using the microwave dielectric and flow cytometry were employed. Cell viability was spectroscopy (MDS) technique for cellular and tissue evaluated 72-hours post-electroporation. The confocal analysis. laser scanning microscope (CLSM) method was used for Methods: To carry out this study, we perform both on- Cadherin® and CellMask™ Deep Red imaging. Western chip EP experiments and dielectric sensing in miniature blotting was used for the analysis of multidrug resistance devices. This method has the advantages of non-invasive (MDR) protein expressed after electroporation without testing, with fast test times and rapid diagnosis, while and with Ca2+. MDR genes were analyzed by RT-PCR requiring small amounts of reagents and limited applied method. The obtained results confirmed that electropor- voltages to reach high electric fields. ation permeabilizes the cell membrane and enables the Among the different existing analysis methods, MDS effective delivery of molecules. Moreover,CaEP originates proves to be an innovative approach to discriminate a significant reduction in cells viability, and changes different cell types and states, while meeting the on chip the expression of genes related to drug resistance. It requirements. Indeed, for the microwave range, the cell was also determined that electroporation with calcium membrane becomes transparent, allowing the waves to ions induces significant reorganization of cadherin after reach the cell contents. The MDS method, adaptable exposure to electroporation with Ca2+. from single cell to tissue scales, allows to obtain the Concluding, we can state that CaEP can be an alternat- dielectric properties of the sample in a non-invasive and ive tool to treat cancers demonstrating drug resistance. non-destructive way, in real time and directly in the However, further investigation is required. culture medium [1]. Results: Single cell analysis is of paramount importance Acknowledgment: The research was funded by the for a better understanding of the effects of EP at larger Polish National Centre of Science of DAINA 2 scales. We measure the electrical signature of single (2020/38/L/NZ7/00342; PI: J. Kulbacka), and also cells isolated using a microfluidic device allowing to supported by the Research Council of Lithuania grant co-integrate EP and MDS. First, we calibrate the applied (Nr. S-LL-21-4, PI: V. Novickij) field for in-situ EP at the single cell level. We evaluate the different types of EP in a static manner. Second, in PO-026 order to investigate the dynamics of EP within individual Electroporation of excitable cells studied with ge- cells, we carry out real-time analyses. netically engineered HEK cells Tina Batista Napotnik, Bor Kos, Tomaž Jarm, Lea Rems References: University of Ljubljana, Faculty of Electrical Engineering, [1] A. Tamra, “Spectroscopie diélectrique HyperFréquence Slovenia des cellules biologiques soumises à l’électroporation,” Most electroporation-based treatments directly or in- Theses, Université Toulouse 3 Paul Sabatier, Mar. 2017. directly target excitable cells such as muscle and nerve [Online]. Available: https://hal.laas.fr/tel-01499406 cells. This may lead to undesirable side effects (muscle twitching, adverse effects on tissues adjacent to the elec- troporation area). Therefore, it is of great importance to 150 study how excitable cells, especially their voltage-gated expansion waves, and cavitation behind expansion waves ion channels, respond to electric pulses and how this af-were visualized by schlieren and shadowgraph techniques fects membrane permeability, excitability and function of using an ultra-high-speed framing camera. The pressure excitable cells. We used a genetically engineered human of shock and expansion waves was measured by a fiber op-embryonic kidney cell line HEK-293T for the study. This tic probe hydrophone (FOPH) pressure transducer. U937 cell line is characterized by stable expression of voltage- cells, human histiocytic lymphoma cell line, were used in gated sodium channels (NaV1.5) and has a Tet-on system an in-vitro setup to evaluate the survival rate, number for doxycycline-induced expression of inwardly rectifying of viable cells, cell growth, cells diameter and morpho- potassium channels (Kir2.1). This makes these cells a logy, and propidium iodide uptake, after exposure to shock simple model for excitable cells with minimal complement waves and expansion waves. The results show enhanced of sodium and potassium channels required for cellular ex-permeabilization/poration effects of expansion compare to citability. We were able to trigger single or multiple action shock waves. potentials in excitable HEK cells with single 100 µs elec- tric pulses. We were also able to detect depolarization in PO-007 nonexcitable HEK cells (cells lacking Kir2.1 channels) as New high frequency electroporation protocols for a result of electroporation. human and veterinary medicine applications Depolarization and action potentials were monitored op- Alexia DeCaro 1, Jean-Baptiste Leroy2, Muriel Golzio1, tically under a fluorescence microscope using a fluores- Marie-Pierre Rols1 cent potentiometric probe Di-4-ANEQ(F)PTEA with a 1IPBS-CNRS, Toulouse, France sampling time of 36 ms. To quantify how pulses of in- 2Leroy Biotech, France creasing amplitude influence the transmembrane voltage For more than 20 years, electroporation has been devel- changes and the ability of cells to trigger action potentials, oping in the field of cancer and is becoming increasingly we analyzed the fluorescence images with a custom code in common in the treatment of skin cancers. By increas- Matlab. Most representative membrane regions were se- ing the cytotoxic effect of anti-tumor drugs (bleomycin, lected by thresholding the images, then the fluorescence cisplatin), electrochemotherapy has already proven its ef- signal was corrected for photobleaching. This allowed fectiveness on tumors in human medicine but also in veter-automatic extraction of several time-dependent paramet- inary practice. However, this treatment requires loco- ers from the experimental conditions, such as the time regional or even general anesthesia, as electrical pulses can from pulse delivery to action potential and the time to be painful and cause muscle contractions. Several public- recovery of the fluorescence signal toward baseline fluor- ations proved that application of high frequency pulses escence. Overall, we find excitable and nonexcitable en- (5,000 Hz) resulted in much less discomfort to the patient gineered HEK cells a valuable tool for studying the effects than the 1 Hz protocol used in the clinical practice in the of electric pulses on excitability and electroporation of ex- 90’s and 2000’s. citable cells as well as ion channels. In order to maintain the effectiveness of the treatment and reduce the pain associated with contractions, we are devel- PO-030 oping new protocols using a high-frequency generator, and Efficacy of shock waves and expansion waves gen- new multipolar electrodes. Our ongoing results obtained erated by nanosecond pulsed electric discharges for on a colorectal cancer cells line (HCT-116) cultured both permeabilizing cells and delivering drugs 2D and 3D (spheroids) showed a clear effect on cell per- Ryuichi Nakajo, Nushin Hosano, Ryosuke Inoue, Takashi meability assessed by propidium iodide uptake. Using our Sakugawa, Hamid Hosano new protocols, the permeability rate was around 90 % and Kumamoto University, Japan the viability rate of 95 ?tween 1 kV/cm and 1.5 kV/cm on Clinical application of shock waves, as a completely cell suspensions with isoenergy tests. These results were non-invasive procedure, has been expanded during the compared to ESOPE protocol (8 pulses of 1,000 V/cm last- past decades. Recent investigations have revealed fur- ing 100 µs at 1 to 5,000 Hz) showing a permeability rate of ther interesting therapeutic and diagnostic potentials of 97% and a cell viability around 90%. Therefore, our high shock waves. While the effects of shock waves on cells frequency electroporation protocols appear promising for and tissue have been extensively studied, little is known an effective cell permeabilization with a low mortality rate about the effects of pulse (short duration, high intens- with similar temperature increase than ESOPE protocol ity) expansion waves. Furthermore, it is imperative to and without noticeable muscular contraction. have knowledge about effects of pulse expansion waves on tissue, as focused shock waves are always followed by an PO-038 expansion wave, and shock wave reflects as an expansion Pulsed electric field pasteurisation of orange juice: wave from low acoustic impedance tissue, like fat. Mean- Inactivation of Escherichia coli and native micro- while, tensile stress is more effective than compression to flora and impact on product quality permeabilize/porate cells, which makes expansion waves Kate Waldert 1, Robert Sevenich2, Oliver Schlüter2, Monika further interesting. In this study, pulse expansion waves Springer3 were produced by reflection of shock waves from a thin air 1University of Natural Resources and Life Sciences Vienna, layer in an innovative setup. Shock waves were produced Austria by nanosecond pulsed electric discharge. Shock waves, 2Leibniz-Institut für Agrartechnik und Bioökonomie e.V. 151 (ATB), Germany Large amounts of vegetable wastes are generated in 3Berliner Hochschule für Technik, Germany juice production industry. Among them, carrot pomace In the last years, consumer preferences have shifted is an excellent source of dietary fiber (DF). However, towards high-quality minimally processed foods with an the high content of insoluble dietary fiber (IDF) of car- extended shelf life and prioritizing food products with a rot pomace makes difficult its incorporation into food clean label and without the addition of preservatives. In products. Therefore, there is a great interest in modi- order to obtain such products, the thermal stress on the fying its structure for increasing the content of soluble food has to be kept low, to preserve its nutrients and dietary fiber (SDF) and improving the technological prop- sensory attributes. For this purpose, pulsed electric field erties. Pulsed electric fields (PEF) is a non-thermal pro- (PEF) treatment has emerged as a non-thermal preserva- cessing technology, which is able to modify the structure tion method, especially due to its suitability for food pas-and properties of different biomolecules. The aim of this teurisation. Therefore, the aim of this study was to invest- study was to evaluate the effect of PEF on the technolo-igate the efficacy of PEF for orange juice pasteurisation gical properties and DF content (SDF and IDF) of carrot by assessing microbial inactivation and quality retention. pomace. For this purpose, the inactivation of Escherichia coli DSM PEF treatments with different electric field strengths (5- 1116 (E. coli) and the natural microflora (predominantly 15 kV cm-1), and different number of pulses (5-75) were different yeast strains) by PEF was evaluated. For the fur-applied on carrot pomace (Daucus carota cv. Nantes). ther trials, process parameters were selected that caused a The PEF-treated pomace was freeze-dried, crushed and 5 log reduction of E. coli (11.3 kV/cm, 250 Hz, 403 kJ/kg). sieved (0.3 µm-mesh sieve). Then, water retention ca- A second series of PEF experiments with lower pulse re- pacity (WRC), oil retention capacity (ORC), cation ex- petition frequencies and specific energy input levels (120 change capacity (CEC), water swelling capacity (WSC), Hz, 182 kJ/kg) was carried out, aiming to inactivate the stabilizing and emulsifying capacity, solubility and DF native microbial flora in the juice under more gentle pro- content (SDF and IDF) were evaluated. cess conditions. After the treatments, the orange juice The pomace treated with 5 pulses of 15 kV cm-1 (1.17 kJ was stored in plastic bottles at 4 °C and 20 °C for eight kg-1) showed a significant increase (p<0.05) in the SDF weeks. Additionally, thermal pasteurisation (80°C, 1 min) content (20.9%), WRC (8.0%), ORC (8.1%) and solubil- and high pressure processing (HPP; up to 600 MPa, 2 ity (59.8%), while the IDF content was lower (15.4%) than min) were chosen as reference processes, representing es- that of the untreated pomace. Stabilizing and emulsifying tablished thermal and non-thermal preservation methods. capacities, CEC and WSC were not affected, regardless Both PEF process designs resulted in an extension of shelf- the treatment applied. Obtained results demonstrate that life at 4 °C (up to 21 days) compared to the native juice (7 PEF treatments could induce modifications in the config- days) but did not remain microbiologically stable through- uration and structure of the DF molecular chains. These out the entire storage period. At storage conditions of 20 changes could cause an increase in the content of the sol- °C, both PEF treated juices and the native juice showed uble fraction and, consequently, an improvement in its a comparable shelf-life of less than 7 days. PEF treat-solubility and its ability to retain water and oil. The re- ment conditions featuring lower pulse repetition frequen- duction in IDF content by PEF treatment was likely due cies (120 Hz) resulted in a comparable product quality to modification of the cell-wall structure of pomace where (soluble solids, cloud stability, vitamin C, colour, pH) to degradation of the insoluble fraction generally occurs. conventional thermal pasteurisation and HPP. However, PEF could be considered a promising technology to en-higher pulse repetition frequencies (250 Hz) resulted in hance the technological properties and to increase the SDF higher energy input levels (up to 403 kJ/kg) and treat- content of carrot pomace, thus facilitating its incorpora- ment temperatures (up to 68 °C) which led to a signific- tion as a functional ingredient in processed foods. ant loss of juice quality, mainly degradation of vitamin C and changes in colour. Therefore, it can be stated that PO-043 the pasteurisation of orange juice by means of PEF has Comparison of extraction of bio-molecules assisted distinct potential as a non-thermal pasteurisation method by pulsed electric energy and ultrasonication: Ef- for orange juice. However, further research is still needed ficiencies for different microalgal species for the product-related optimization of the process design, Rui Zhang1, Nikolai Lebovka2, Eugene Vorobiev2, Luc in order to be able to guarantee microbial stability as well Marchal3, Nabil Grimi 2 as quality retention throughout the whole desired storage 1Wuhan Polytechnic University, China period. 2University of Technology of Compiegne, France 3Université de Nantes, France PO-041 The effects of three physical treatments (pulsed elec- Effects of pulsed electric fields on technological trical fields (PEF), high voltage electrical discharges properties and dietary fiber content of carrot (HVED) and ultrasonication (US)) on cell disruption and pomace release of intracellular bio-molecules from different mi- Harold Antonio Pájaro Escobar, Gloria López Gámez, croalgal species (P. tricornutum, Nannochloropsis sp. and Robert Soliva-Fortuny, Olga Martin-Belloso, Pedro Elez- P. kessleri) were investigated. The extraction kinetic be- Martinez haviours of hydrophilic (carbohydrates and proteins) and University of Lleida, Food Technology, Spain hydrophobic (chlorophyll a) bio-molecules and selectivity 152 indexes were evaluated. At equivalent energy consump-Plant Winery, assessing various physico-chemical para- tion, the extraction efficiency arranged in the rows of meters such as phenolic compounds, native microflora, HVED > US > PEF (for carbohydrates) and US > HVED fermentation, maceration dynamics and also to determ- > PEF (for proteins) was observed for all tested microalgal ine factors that can impact the applicability of PEF in a species. Among them, the PEF treatment demonstrated winery. This specific variety was selected considering its the smallest efficiency for extraction of carbohydrates and availability in the Dão wine region of Portugal and repres- proteins due to its mechanism only cause cell membrane entativity in the viticultural international panorama, e.g. damage. However, for all tested physical treatments, the in Spain. extraction degree of carbohydrates was � 40%, while the Two batches of 1 ton of grapes each were acquired and extraction degree of proteins was � 10%. They allowed se- separated in two similar fractions to allow the compar- lective extraction more carbohydrates than proteins. The ison of vinification resorting to PEF vs Control. It was relative mild pulsed electric energy treatments (PEF and also possible to assess the impact of grape sanity on PEF HVED) have the higher extraction selectivity than US effectiveness. Grapes were treated on a continuous treat- treatment. Moreover, three physical treatments presented ment co-axial chamber, working at a flow of 4 T/h, with a different extraction behaviours of chlorophyll a. The ex-specific energy of 2 to 2.8 kJ/kg and electric field strength traction of chlorophyll a using PEF and HVED treatments of 2.0 kV/cm. Positive results were most visible in grapes occurs in one stage (diffusion). By contrast, the extrac-with lower sanity conditions for which PEF ended the 4- tion using US treatment occurs in two stages (convection day maceration period with +18.69% TPI, +44.74% IFC, and diffusion), the first stage with a fast chlorophyll a +51.5?techins, and +73.1% Anthocyanins than Control. transfer from the inside of microalgal cell, and the second With PEF processing the ability to inactivate microor- stage corresponds to the prolonged chlorophyll a transfer ganisms, it was also important to assess the impact of by molecular diffusion from interior of the microalgal cell. low field energy treatments on native flora of musts since Furthermore, based on the observation of the different be- anecdotal evidence shows an increased interest of the pro- haviors between green microalgae and diatom, the cell wall ducers and consumers on the use of spontaneous alcoholic of P. tricornutum was more fragile than Nannochlorop- fermentation. Results show, also, that PEF did not have sissp. or P. kessleri. These obtained results will help for a significant impact on the native microflora of musts at understanding the correlations between selected methods the used treatment field intensity. and efficiency of extracted target bio-molecules. The ap- propriated cell disruption methods should be selected and PO-010 used on tune the desired target molecules. However, in or- IREC study- electroporation (IRE), calcium der to obtain higher extraction efficiencies of intracellular electroporation (CaEP) and electrochemotherapy molecules, combined process will be needed. (ECT) in pancreatic cancer patient’s treatment: From science to practice PO-046 Julia Rudno-Rudziska, Ewelina Frejlich, Maciej Guziński, Red wine vinification on a pilot-plant winery based Julita Kulbacka, Nina Rembiałkowska, Wojciech Kielan on Pulsed Electric Fields (PEF) Wroclaw Medical University, Poland Mafalda M. Santos1, Luis Redondo 2, Marcos M. Pereira1 Pancreatic cancer still has an inferior prognosis. 1EnergyPulse Systems, Portugal Despite the improvement in cancer biology knowledge 2Instituto Superior de Engenharia de Lisboa, GIAAPP/ISEL, and major developments in radiotherapy procedures, Portugal pancreatic cancer has the same high index for mortality The increasing concern of food industry regarding sus- to morbidity for many years. According to the estimates, tainability and the public demand for food with higher by 2030, pancreatic cancer will become the second cause nutritional value and quality led to the development of of death in the USA and Germany. In Poland morbidity several novel food processing techniques. One of these is is about 3000 patients/year and mortality is almost the the application of Pulsed Electric Fields (PEF) in order same. 80 % of the cases are non-resectable pancreatic to enhance the mass extraction of valued food components cancers. For this patients only chemotherapy is the from cells through the electroporation effect, limiting the way of the treatment. Electroporation, as non- thermal thermal effect of conventional techniques. In winemaking, technique, may be another possibility for better patient’s PEF treatment of red grape musts improves mass transfer overral survival (OS), progression free survival (PFS) or phenomenon, translating in the reduction of maceration quality of life (QQL). times and enhancing the economic value and logistic ca- IREC (Effect of electroporation, calcium electroporation, pacity of wineries. eletrochemotherapy (IRE, CaEP, ECT) on quality of Although there are numerous studies of PEF in laborat- life and progression free survival in pancreatic cancer ory and batch condition, the reduced number of studies patients) was created and developed in cooperation of regarding PEF application in industrial winemaking facil- scientists and surgeons in Medical University of Wrocław, ities limits the capability of obtaining robust results that Poland on the basis of pilot study “Personalization of can be extrapolated to the daily functioning of a winery. pancreatic cancer treatment”. Inclusion criteria are This work was performed with the objective of further non- resectable, histopathologically confirmed pancreatic understand the impact of PEF on Tinta Roriz (Tem- cancer adult patients (stage III), with lesions to 6 cm in pranillo), a red wine variety in a medium scale Pilot-CT not older than 30 days, patient in 0,1,2 in ECOG 153 scale. Exclusion criteria are severe cardiac arrythmia, which rose from 1267 to 1372 mg/kg and in total phenolic pacemaker, lung fibrosis, bleomycin allergy. compounds which increased from 115 to 121 expressed as Patients are randomized to three groups. Group A- mg/kg of tyrosol. The PEF EVOO also displayed a sig- receive only electroporation (IRE), B- calcium elec- nificant increase (log2 fold change of 5.43 between both troporation (CaEP), C- electrochemotherapy (ECT) EVOO) in the microRNA, oeu-mir-31 5p. Mice consum- based on bleomycin with intratumoral and intravenous ing both oils did not show significant differences in feed administration. Patients are operated with open or consumption, body weight, atherosclerotic lesion or hep- transcutaneous technique in general anesthesia. Time of atic fat content expressed as lipid droplet area. However, observation is 12 months. CT or MR, laboratory tests females consuming the PEF EVOO showed a decrease in and physical examination are done. Also tests of quality plasma total cholesterol (498 ± 47 mg/dL) compared to of life (EORTC QLQ- PAN 26, QQL-WHO). the group receiving STD EVOO diet (588 ± 68 mg/dL). The primary end- points are patient’s disease free survival In addition, in both sexes there were slight differences in (DFS) and quality of life (QQL). Secondary end-points the ROS content of the different isolated lipoproteins. are overral survival, the best “therapeutic moment” for Conclusions: Our results showed that the PEF applied to the procedure- personalization of treatment, safety of the EVOO extraction improves the oil yield percentage, and procedure, comparison of effectiveness between A, B, C reduces the malaxing time maintaining the same quality of groups, immunological effects. EVOO. The observed increase in phytosterols, total phen- Project started on march 2022 and till this moment olic compounds and oeu-MIR-31 5p with the use of PEF several patients were included. technology may be responsible for the reduction in total cholesterol observed in females, and a slight change in the Acknowledgments: This study was supported by the oxidation profile of the different lipoproteins. No changes Medical Research Agency, Poland, IREC project No. were observed on pathological outcomes such as athero- 2020/ABM/01/00098/P/02 (PI: Prof. Wojciech Kielan). sclerosis or development of fatty liver disease. PO-048 PO-051 Biological characterization of pulsed electric field- Effect of Pulse Electric Fields on the Growth Stim- obtained extra virgin olive oil ulation of Lactobacillus plantarum CCDM 181 Roberto Martínez-Beamonte1, Marina Ripalda1, Tania Gabriela Kuncová 1, Iveta Horsáková1, Jakub Reitmeier1, Herrero-Continente1, Cristina Barranquero1, Alberto Anna Tobolková2, Rudolf Ševčík1 Dávalos2, María del Carmen López de las Hazas2, Ignacio 1University of Chemistry and Technology Prague, Czech Re- Alvarez-Lanzarote 1, Javier Raso1, Joaquín Surra1, Carmen public Arnal1, Jesús Osada1, María Ángeles Navarro-Ferrando1 2VITAVE Tech s.r.o., Czech Republic 1University of Zaragoza, Spain 2Instituto Madrileño de Estudios Avanzados (IMDEA), Spain The pulsed electric fields (PEF) is a very promising method in the food industry. Nowadays, it is already used Background and aim: Pulsed electric fields (PEF) is in industrial scale for non-thermal pasteurization and to a non-thermal process characterized by the electropora- streamline other processes during food production such as tion of cell membranes which is used for several applica- extraction, drying, freezing, etc. In terms of its impact tions including improving extraction of intracellular com- on microorganisms, the inactivation effect of the PEF has pounds of interest from plant-based foods. PEF techno- been examined so far. In some cases, it is desirable to logy applied to extra virgin olive oil (EVOO) extraction stimulate microorganisms, for example, during fermenta- slightly improves the extraction efficiency, without appar- tion processes. By exposing microorganisms to the pulsed ently affecting physic-chemical characteristics of EVOO. electric field at the sublethal level, reparation and protec-However, it has never been studied possible effects at bio- tion mechanisms are triggered. Thus, the microorganism logical level. The objective of the work is to characterize is able to accumulate nutrients, increase biomass produc-EVOO biological properties regarding atherosclerosis and tion and adapt to stressful conditions. As part of this ex- fatty liver disease by using Apoe-deficient mice as spon- perimental work, the influence of the set parameters (elec- taneous model of both pathologies. tric field strength, pulse length) on the bacterium Lacto- Methods and results: EVOO oils from Empeltre variety bacillus plantarum CCDM181 was observed in order to were prepared by standard (STD) and PEF procedures (2 achieve reversible electroporation leading to stimulation kV/cm; 3.9 kJ/kg) and characterized. Male and female of the growth of this bacterium. The impact of the PEF Apoe-deficient mice were fed for 12 weeks with 2 purified on the ability of this bacterium to grow under anaerobic Western diets differing on the type of used EVOO. After and aerobic conditions was also examined. Within the the dietary intervention, blood, aorta, heart and liver monitored parameters, stimulation of growth of 0.8 log samples were taken for analysis. As expected, PEF tech- was achieved with a PEF in unipolar mode with an in-nology increased of the oil yield from 10.2% to 12.2% with tensity of 2 kV/cm, a frequency of 100 Hz, a pulse length a malaxing time of 30 minutes, and reduced the malaxing of 5 µ s (the specific energy input 2 kJ/kg) and subsequent time required to obtain the maximal yield to 30 minutes aerobic cultivation. During anaerobic cultivation, stimu-instead of 60 minutes for the STD EVOO. Compared with lation of 0.6 log was achieved by treatment with a PEF in the STD EVOO, PEF EVOO showed a slightly increase in unipolar mode with an intensity of 3 kV/cm, a frequency the peroxide index from 6.9 to 8.2 meq O2/kg of fat, in free of 100 Hz and a pulse length of 5 µ s (the specific energy in-fatty acids from 0.11 to 0.14, in the phytosterol content 154 put 3 kJ/kg). It has been shown that with increasing pulse PO-056 length, the stimulating effects of the pulse electric field in- Visualization of local thermalization of conductive crease while maintaining the same electric field strength (3 fluids in a continuous flow PEF treatment cell kV/cm) and pulse frequency (100 Hz), when the greatest Kenshi Kajiwara, Bingyu Yan, Ryosuke Kadoya, Sunao stimulation was achieved using unipolar pulses of 8 µ s. In Katsuki terms of growth properties in the aerobic environment, an Kumamoto University, Japan improvement in growth was achieved after Lactobacillus The use of pulsed electric fields (PEF) is a promising plantarum CCDM 181 was exposed to PEF. method to sterilize conductive liquid foods at a relatively low temperature that does not damage the ingredients PO-054 (1). Since Sale & Hamilton’s experiment (2), numerous Influence of pulsed electric fields on caroten- batch-type experiments and small flow-rate experiments oids content of carrot purees during storage and have been reported. However, at the large flow-rate treat- changes in their bioaccessibility ment of 10 L/h or more, several unfavored phenomena, Gloria López Gámez, Pedro Elez-Martinez, Olga Martin- such as reduced sterilization effectiveness, lower dielectric Belloso, Robert Soliva-Fortuny strength and the electrode contamination are obstacles University of Lleida, Spain to the practical use. Some steady-state multi-physics Introduction. Carrots are an excellent source of simulations (3) have indicated that a significant local carotenoids. However, within the food structure, these thermalization occurs near the wall of treatment cell health-related compounds are surrounded by cellular bar- because of an intensive energy deposition to the reduced riers that hinder their release during digestion. Pulsed velocity layer near the wall. Here, we discuss the local electric fields (PEF) is a nonthermal technology that may thermalization of the fluids by visualizing the flow state facilitate the release of carotenoids through the disruption and temperature distribution of conductive fluids exposed of cell membranes, thus leading to improve their bioac- to repetitive high-power pulses in using time-resolved cessibility. In general, carotenoids bioaccessibility is quite Schlieren imaging and interferogram methods. low. Therefore, developing functional products that main- The PEF exposure chamber used to observe the fluid tain a high content during storage is essential to preserve flowing between the parallel electrodes consists of their beneficial health-promoting properties. Objective. stainless-steel electrodes 2 mm thick and 30 mm high, 4 The aim of this work was to evaluate the effect of PEF on mm apart and facing each other, sandwiched between two the carotenoid content of carrot purees during refrigerated glass windows. The cross section of the flow channel is storage and to study their bioaccessibility just after treat- H-shaped. Fluid was circulated using a peristaltic pump ments. Methodology. Purees were produced by blend- (IWAKI, PST-550H) with an average velocity of 1 m/s ing carrot pieces with water [1:1 (w/w)] and adding olive between electrodes. The fluid temperature was adjusted oil [5% (w/w)]. The resulting purees were subjected to to 26°C at the inlet of the PEF exposure section. An a PEF pre-treatment (5 pulses of 3.5 kV/cm) and then Nd:YAG laser (532 ns, Minilite, Continuum) with a pulse thermally-treated (T) (70 ºC for 10 min) to achieve shelf- width of 7 ns was used as the light source for observation. stability. Carotenoid content was evaluated for 21 days at By synchronizing the pulsed laser and the pulsed power 4 ºC by HPLC-DAD. In addition, purees were submitted supply, the phenomena were observed at arbitrary timing to in vitro digestion to assess carotenoid bioaccessibility relative to the PEF exposure. just after treatments. Obtained results were compared High repetition pulses were applied to three different to untreated purees (without PEF and/or thermal treat- fluids with different viscosities. For the high viscosity ments). Results. During storage, a carotenoid reduction temperature increased by 25°C only near the electrode of 33 % in thermally-treated purees was observed, whereas surface and by only 2°C at the center of the flow. carotenoid content of PEF-treated purees just decreased This local thermalization is attributed to the velocity by 16?ter 21 days. The highest carotenoid bioaccessibil- distribution of the fluid in the tube. Fluid near the ity was obtained in purees subjected to PEF or PEF/T tube wall flows very slowly and therefore receives more (18.7-20.4 %). These results suggest that electropermeab- electrical energy from the repetitive pulses. This localized ilization may allow a better release of carotenoids from thermalization causes a variety of problems in high-speed cells and facilitated their micellarization. PEF-treated PEF pasteurization of temperature-sensitive liquid foods, purees had lower content than those thermally-treated. such as milk and liquid whole eggs. On the other hand, Electroporation likely allows the release of intracellular it was observed that local thermalization near the wall compounds, which will be more available to be degraded surface is suppressed in low viscosity fluids. The Schlieren by free radicals formed during PEF treatment application. image of the fluid in this case shows turbulent flow. It is Conclusions. Results suggest that combining PEF and considered that local thermalization near the wall surface heat application is the most effective treatment to pro- is suppressed by lateral thermal diffusion in turbulent flow. mote carotenoids bioaccessibility. Furthermore, it stands also as a suitable technology to better keep the health- References promoting properties of carrot puree during storage. (1) T. Kajiwara, S. Katsuki, et al. , IEEE Trans. Dielectr. Electr. Insulat. Vol.22, No. 4, pp.1849-1855 (2015) (2) A. J. H. Sale and W. A. Hamilton: Effects of high electric fields on microorganisms. Killing of bacteria and 155 yeasts. Biochimica et Biophysica Acta., 148�1967�781-788 applied field is AC or a DC field. Apart from this, factors (3) R. Buckow, S. Schroeder, et al. : Simulation and eval- such as ratio of the internal and outer conductivity of uation of pilot-scale pulsed electric field(PEF) processing, the solution ( α), electric pulse duration, electric pulse Journal of Food Engineering 101 (2010) 67-77 amplitude, types of GUVs (charged and uncharged) also affect the deformation. Our experiments on GUVs under pulsed DC fields indicates that when the value of the PO-059 conductivity ratio of the inner to outer fluid, α =1, the Automated Irreversible electroporated region pre-GUVs prefers the prolate shape and shapes deformation diction in different electrode type with deep learn- shown more at higher electric field amplitude. However, ing approach when α >1, the GUVs gets deformed more into prolate Amir Khorasani shape and almost spindle types shapes are also formed. Isfahan University of Medical sciences, Medical Physics , Iran, On the other hand, oblate shapes are seen when α<1, Islamic Republic of means. The oblate shapes formation is due to compressive The main purpose of cancer treatment with irrevers- electric forces applied at the equator of the GUVs. The ible electroporation (IRE) is maximize tumor damage and range of electric field amplitude and pulse duration is minimize surrounding healthy tissue damage. Finite ele- investigated to know not only the shape deformation but ment analysis is one of the popular ways to calculate elec- study the reversible and irreversible electroporation of tric field and cell kill probability in IRE. However, this the GUVs. The reduction in the volume of the GUVs method also has limitations. This paper will focus on use after PEF treatment shows loss of liquid from inside of deep neural network (DNN) in IRE with different elec- of the GUV, indicating reversible electroporation. On trode types for prediction of irreversible electroporated re- the other hand, GUVs that burst show irreversible gions for treatment planning purposes. electroporation. Fluorescence microscopy studies indicate COMSOL Multiphysics was used to simulate the IRE. To that tubules could be formed either on the inner or on create accurate data sets of electric field distribution and the outer side of the vesicle. Our analysis indicates that cell kill probability distributions, the electric conductivity the formation of microscopic structures in such vesicles, change during IRE was considered. We used eight pulses are long lived, even after the pulse is swtiched off, and with a pulse width of 100 µ s, frequency of 1 Hz, and pulse depend sensitively on the size and strength of the pulse. voltage of 2500 V. Different electrode type such as needle In this particular study, the pulse width was changed pairs (1, 2, 3), plate, and bipolar electrodes were used in from 100 microseconds to 5 ms and electric field strength the current study. To create masks for DNN training, 90?ll was altered from a value of 0.2 kV/cm to 2 kV/cm. The kill probability contour was used. After data set creation, amount and variety of microstructures generated in the U-Net architecture was trained for predicting of irrevers- vesicle increases when the pulse width and the strength ible electroporated regions. of the electric field is increased. In this study, average U-Net DICE coefficient on test data were 0.96, 0.94, 0.91, 0.89, 0.96 with 1 pair needle elec- References: trode, 2 pair needle electrode, 3 pair needle electrode, 1. Priti Sinha, K., Das, S., Karyappa, R. B. & Thaokar, plate electrode, and bipolar electrode. Also, the average R. M. Electrohydrodynamics of Vesicles and Capsules. accuracy of U-Net for predicting irreversible electropor- Langmuir 36, 4863–4886 (2020). ated region with all electrode types was 0.98. 2. Riske, K. A. & Dimova, R. Electro-Deformation and The present study provides important evidence for use of Poration of Giant Vesicles Viewed with High Temporal U-Net for predicting of irreversible electroporated region Resolution. Biophysical Journal 88, 1143–1155 (2005). in treatment planning with different electrode type. 3. Dimova, R. et al. Giant vesicles in electric fields. Soft Matter 3, 817–827 (2007). PO-061 4. Dimova, R. et al. Vesicles in electric fields: Some novel Electrodeformation study of Giant Unilamellar aspects of membrane behavior. Soft Matter 5, 3201–3212 vesicles (GUVs) and Multi Vesicular Vesicles (2009). (MVVs) under DC and AC electric field Pulses Rupesh Kumar, Mohammad Maoyafikuddin, Rochish PO-064 Thaokar Engineering the tumor environments in vitro using Indian Institute of Technology, Bombay , CRNTS, India peptide-enriched, hyaluronic acid-based hydrogels Annj Zamuner 1, Leonardo Cassari1, Monica Dettin1, The GUVs are micrometers sized vesicles made up Maria Teresa Conconi1, Elisabetta Sieni2 of lipid bilayers. The exterior and interior of the GUVs 1University of Padova, Italy are filled with the liquid solution. The micrometers size 2University of Insubria, Department of Theoretical and Ap- and formation with lipid bilayers, makes a GUVs right plied Sciences – DiSTA, Italy choice as a bio mimic cell. To explore the fundamentals mechanisms of electroporation and electrodeformation of Electrochemotherapy is a well-assessed therapy used cells membrane, the GUVs are best choice to know the for some skin-related tumors like melanoma or breast can- physics behind all these mechanisms. cer recurrences after mastectomy. In recent years it was When a GUV is subjected to electric field it deforms. The evaluated in the treatment of some other types of tumors, shape deformation of the GUVs depends on whether the such as liver, head and neck, colon carcinomas, and soft 156 tissue sarcomas. abling complete and durable isolation of pulmonary veins The electroporation (EP) efficiency of the cell membrane avoiding damage to critical nearby structures. In the case depends on cell size and cell density as well as on the con-of cardiac ablation, the elongated and oriented cardiac ductivity and composition of the medium where cells are cells might affect the distribution of the electric field and, suspended during the EP. It is well known that in the consequently, the PFA treatment efficiency. As several tissue the cells experience cell-cell connection and are sur- different treatment parameters can be varied, a quick and rounded by the extra-cellular matrix that is a component efficient option to determine the optimal pulse parameters of the tumor microenvironment. In recent years, several of the treatment (amplitude, duration, number, repetition three-dimensional (3D) in vitro models have been used frequency) are numerical models. in EP experiments: the cell-cell interactions have been The shape of a cardiomyocyte is complex, being rod- simulated using spheroids, whereas the cell-ECM interac- shaped with a non-smooth membrane surface and a com-tions have been considered in cultures grown in hydro- plex array of microtubules. However, it is mostly approx-gels. Herein, hyaluronic (HA) acid-based scaffolds showed imated to a simple prolate spheroid which was shown to higher potentiality with respect to just formulated solu-be a good-enough approximation in stationary conditions. tions. These scaffolds promote spheroids formation where The aim of our research work was to establish if the pro- cells experiment cell-cell interactions, but also improve the late spheroid geometry is a good-enough approximation of deposition of ECM increasing cell-ECM interactions. HA- a cardiomyocyte also in the time domain. based scaffolds were enriched with a self-assembly peptide We developed a time-dependent numerical model of pore named EAbuK. Liver cancer (HepG2) cell lines were cul- formation on a cardiomyocyte. To represent a cardiomyo- tured on the scaffolds above mentioned. Both the cultures cyte, we used: 1) commonly used prolate spheroid and 2) were characterized by cell viability assessment and hem- real-shaped cardiomyocyte. The electric field was applied atoxylin/eosin and Masson’s stainings. Then after, cul- parallel and perpendicular to the long axis of the cardi- tures were electroporated varying the electric field amp- omyocyte at different electric fields, from 100 V/cm to litude in order to find the optimal EP condition. 10000 V/cm using different pulse lengths from 100 ns to 1 The EP experiments were performed using EPS02 EP ms. The initial value of pore density when no electric field equipment (manufactured by Igea SpA Carpi (MO), Italy) is applied is 109 pores/m2. By increasing the electric field, varying the amplitude of the applied voltage in the range pore density increases. The value of pores density usually 0-1200 V/cm. In these conditions, the EP efficiency was used as threshold of electroporation is 1013 pores/m2. evaluated by propidium iodide staining. Three-D cultures The prolate spheroid geometry is a good approximation on HA without functionalization and cells in adhesion elec- of a cardiomyocyte for very high and very low electric troporated using culture medium or phosphate-based EP fields. 1) When low electric fields are applied and pores buffer were taken as controls. Three-D cultures were elec- just start forming with pore density between 109 - 1011 troporated using a cell medium. pores/m2, i.e., probably before electroporation occurs. 2) Our data showed that HepG2 cells cultured on HA-EAbuK When high electric fields are applied with pore density scaffolds were already completely electroporated at 800 above 1015 pores/m2, i.e. when probably most of the cells V/cm, whereas cells cultured on HA started to be electro- are irreversibly electroporated. The prolate spheroid is porated at 1200 V/cm. In 2D cultures, HEPG2 cells were a too simplified approximation of a cardiomyocyte when electroporated at 1000 V/cm and 1200 V/cm in EP buffer medium electric fields are applied and the density of the and in culture medium, respectively. pores is between 1011 - 1015 pores/m2, i.e. when cells are Collectively, our preliminary results suggest that HepG2 probably reversibly electroporated. cell cultures on HA-EAbuK hydrogels may represent a In conclusion, we suggest using realistic shapes of cardi- promising tool for in vitro evaluation of EP efficiency. omyocytes at cell level numerical calculations when study- ing the effects of the application of a medium electric field PO-067 on a cardiomyocyte. Time-dependent numerical model of electropora- tion comparing prolate spheroid and real-shaped PO-070 geometry of a cardiomyocyte Tumor location method based on multi-electrode Maria Scuderi, Damijan Miklavčič, Janja Dermol-Černe structures and machine learning University of Ljubljana, Faculty of Electrical Engineering, Pablo Briz, Borja López-Alonso, Héctor Sarnago, Óscar Slovenia Lucía, José Miguel Burdío University of Zaragoza, Electronic Engineering and Commu- Cardiac arrhythmias are currently treated using two nications, Spain thermal energy methods: radiofrequency and cryoablation to ablate the targeted heart tissue. However, the Electroporation is a promising technique for cancer two methods cause potential damage to the esophagus, treatment that involves the application of high intensity phrenic nerve injury, and pulmonary vein stenosis. Con- electric fields. To achieve the optimum results, it is im- sequently, a new promising non-thermal ablation method portant to focus the treatment on the tumor tissue. In to treat cardiac arrhythmias in particular for atrial fib- this work, a machine learning algorithm is proposed to rillation called pulsed-field ablation (PFA), is being de- locate tumoral tissue by means the impedance measure- veloped. The PFA treatment involves the application of ments taken by a multi-output electroporation generator electric pulses to arrhythmogenic regions in the heart en- in conjunction with a multi-electrode structure. This can 157 also be used to apply a controlled and targeted electric tissue to isolate or eliminate the cause of the rapid and field, consequently improving the effectiveness of the local irregular heartbeats. Most of the catheter ablations are treatment. It can also simplify the electrode placement thermal by delivering a radiofrequency electromagnetic procedure, often the most time-consuming task. field (RFA). In this numerical study, we focus on the Several studies have determined that tumor tissue is more study of a novel and mainly non-thermal ablation tech- conductive than healthy tissue, usually at least 3 times nique: the pulsed electric field ablation (PFA), which more. Consequently, the purpose of this system is to loc- takes advantage of irreversible electroporation, a complex ate the tumor tissue between electrodes by means of the phenomenon of cell membrane rupture that occurs when impedance measurements on every part of the tissue. To tissues are subjected to very intense electric pulses. detect the tumor tissue inside the volume between the elec- This technique has been used in oncology for more than trodes, 81 impedance measurements are taken applying a decade, but it is still in its infancy in cardiology. low electric field pulses in different locations and direc- Preclinical evaluations of PFA in atrial fibrillation [2] tions. and ventricular ablation in large animal studies [3] show To find the tumors in the tissue, the volume between the successful results with possible transmural lesions, sparing electrodes is discretized into 18 voxels, in two square 3-by- vulnerable adjacent structures. 3 parallel planes, so that a matrix of 18 neural networks In this context, the objective of this work is to derive a can determine if there is tumor tissue or not in each cell. model considering an electroporated area in the geometry The 81 impedance measurements compose the input layer to better understand how PFA works over the long term of the 18 feedforward neural networks, each of them with and in particular why it appears to be more stable than 10 neurons in a single hidden layer. The output of each RFA. We propose a specific model of electroporated neural network is a single true or false statement, indicat-cardiac tissue, in order to couple the classical bidomain ing if there is or not tumor tissue in each voxel. Having system in the healthy parts of the ventricles with the a neural network per cell allows to design a simpler archi- electroporated region. Our numerical study enables to tecture of the neural networks, which is faster and easier quantify on synthetic data the degree of electroporation to train. The neural networks have been designed and needed to isolate the ill region of the hearts which gener- trained in Matlab with data obtained from a model de- ate the arrhythmias. In order to accelerate the simulation veloped in COMSOL Multiphysics. we propose a well adapted Schwarz algorithm to transmit The multi-electrode electroporation system consists of by the electric wave from the heart to the electroporated two differential square parallel-plate electrodes, each one region. of them is divided into a 3 by 3 matrix of plate electrodes individually driven by an 18-output generator. It can de- [1] Tung L. A bi-domain model for describing ischemic liver up to 1250 V unipolar or bipolar pulses and apply myocardial dc potentials low voltage pulses to measure tissue impedance. Each [2] Koruth J, Kuroki K, Iwasawa J, et al. Preclinical electrode cell has a side length of 8.5 mm, and 1.5mm Evaluation of Pulsed Field Ablation. Circ Arrhythm between cells guarantees isolation. Electrophysiol 2019; 12: e007781. An experimental validation has been carried out using 10 [3] Caluori G, Odehnalova E, Jadczyk T, et al. AC mm thick phantom gel and potato tissue samples. The gel Pulsed Field Ablation Is Feasible and Safe in At- has inserts of other more conductive gel in several areas, rial and Ventricular Settings: A Proof-of-Concept with conductivity ratios of 1:2, 1:3 and 1:5, emulating tu- Chronic Animal Study. Front Bioeng Biotechnol; 8, ht- mors of known location, conductivity, and size. Processing tps://www.frontiersin.org/article/10.3389/fbioe.2020.552357 data with the neural networks shows the voxels with the (2020, accessed 2 March 2022). more conductive gel effectively. In conclusion a method to locate tumor tissue between PO-033 electrodes has been developed and validated. The final The bystander effect after Ca electroporation, version of this paper will include experimental results and BLM electrotransfer and irreversible electropor- future insights demonstrating the feasibility of this pro- ation in multiple cell lines posal. Neringa Barauskaite, Paulius Ruzgys, Rūta Palepšienė, Salvijus Vykertas, Saulius Šatkauskas PO-073 Vytautas Magnus University, Lithuania Modeling of cardiac electrophysiology of a tissue Introduction: Novel electroporation-based cancer containing an electroporated area treatments utilize the phenomena of increased cell mem- Clair Poignard, Annabelle Collin, Simone Nati Poltri brane permeability to the exogenous molecules because INRIA, France of increased transmembrane potential induced by the ap- In healthy hearts, the propagation of the electrical plication of external electric fields. Electrochemotherapy waves follows a predictable pattern described by a and calcium electroporation anticancer therapies rely on so-called bidomain model [1]. When people suffer from a higher specific death-causing molecule entrance to the cell cardiac rhythm disorder, the electrical wave can become (Calcium ions, or chemotherapeutical drugs: bleomycin, chaotic and directly affects the pumping function of the cisplatin). However, the utilization of electroablation is heart. One of the main treatments for these arrhythmias based on irreversible electroporation phenomena causing is catheter ablation, which destroys small areas of heart the irreversibly affected cell membrane permeability to in- 158 crease, hence cell death. (EC-meter Mod. Basic 30, Crison, Spain). Trials were In a previous study, we showed the presence of the conducted filling the treatment chamber with a product- bystander effect on CHO cells after bleomycin electro- to-water ratio of around 1:5 (w/w) and delivering n=1000 transfer. In this study, we aimed to extend the invest- pulses at fixed amplitude (10 ± 1 µs) and frequency (100 igation of the bystander effect using 3 different cell lines, Hz). The rehydration process was carried out under static namely CHO, A-549, and 4T1 cell lines. In addition, we conditions for 6 days, immersing dry-salted cod samples implemented not only bleomycin electrotransfer but also in tap water (5 ± 0.5 °C) using a ratio of cod:water of calcium electroporation and irreversible electroporation in 1:10 (w/v). Mass transfer parameters were determined at vitro. 0, 4, 6, 24, 48, 72, 96,120 and 144 h of the rehydration Methodology: Chinese hamster ovary (CHO), adenocar- process. The results show that the use of PEF technology cinomic human alveolar basal epithelial (A-549), and mice increases the rate of the rehydration process of dry-salted breast cancer (4T1) cell lines were used for experiments. cod and influences the redistribution of salt. In general, One electric pulse (1400 V/cm 100 us) was used for bleo-the samples pre-treated with PEF showed higher weight mycin (20 nM) and calcium (1 mM) electrotransfer into gain and lower salt loss than the control samples during cells. Irreversible electroporation was triggered with one the rehydration process. Nevertheless, the calculated salt 1400 V/cm 100 us pulse. We used laboratory-made phos- content in both pre-treated sample was found to be in phate buffer and sucrose-based electroporation media at the range of commercial rehydrated cod products, namely pH 7, and conductivity at 0.1 S/m. The bystander effect about 10- 20 g·kg-1 NaCl (10.0 ± 7.0 g·kg-1 NaCl con- was measured by taking the media from the affected cells trol; 20.0 ± 6.0 g·kg-1 NaCl PEF (1); 10.5 ± 6.0 g·kg-1 24-72 hours post electroporation and applying it to the NaCl PEF (2)). The application of PEF prior to rehyd- bystander cells for clonogenic assay or Alamar blue assay. ration of salted cod samples could be of interest to the Results and discussion: The presence of the bystander ef-food industry due to higher process yield (higher weight fect was confirmed in all cell lines. However, depending gain) and the possibility to reduce the water renewal, as on the collection medium time after cell electroporation, less NaCl is lost in the wastewater. the bystander effect after irreversible electroporation had a positive impact on cell viability. We have also obtained PO-075 the difference in viability changes over time when com- Low pulsed electrical fields for inducing transient paring cell lines. Similar results were obtained by using BBB disruption - Mechanism of action the Alamar blue assay (metabolic activity of the cells) and Shirley Sharabi, Yael Bressler, Orly Ravid, David Last, Di-clonogenic assay (ability to form colonies). anne Daniels, Daniel Rand, Yael Mardor, Itzik Cooper Sheba Medical Center, Israel PO-035 The blood-brain barrier (BBB) limits transcellular and Pulsed electric field assisted rehydration of dry- paracellular passage of molecules from the blood system salted cod (Gadus morhua) into brain tissues, turning it into a major hurdle for treat- Jessica Genovese, Silvia Tappi, Urszula Tylewicz, Fabio ing brain diseases. High pulsed electrical fields (PEFs) D’Elia, Ana De Aguiar Saldanha Pinheiro, Pietro Rocculi can disrupt the BBB by inducing electroporation (EP), University of Bologna, DISTAL, Italy resulting in permeability of the transcellular route. We Salted cod (G. morhua) is a highly appreciated have discovered that low PEFs (L-PEFs), well below EP product, traditionally imported by Mediterranean coun- threshold, can induce transient BBB disruption (BBBd). tries and commercialised with different moisture content Here we studied the mechanisms involved in L-PEFs independing on the extension of the dehydration process. duced BBBd. Dry-salted cod must be rehydrated before consumption 10 pulses (5-800 V, 0.68 cm electrodes gap, 50 µs, 1 Hz) and this step can take up to five days.Desalting of cod were applied to a human in vitro BBB model composed of on an industrial scale is usually carried out immersing the brain-like endothelial cells and brain pericytes. Changes in product in stagnant water, resulting not only in sample permeability to different size molecules were studied. Vi- rehydration but also in the loss of salt. It therefore poses ability and membrane permeabilization (EP) were evalu- many problems, mainly related to the long processing ated by Presto-Blue, Lactate dehydrogenase release assays times and the quality of the final product. For this reason, and Propidium iodide. The effects on cytoskeleton and many researchers have focused on finding new desalting tight junction/adherent junction (TJ/AJ) proteins were methods to improve mass transfer. The application of studied using immunohistochemistry. Finally, phosphop- pulsed electric field (PEF) has been proposed as an al- roteomic analysis was conducted to identify molecular sig- ternative method to improve mass transfer in many food naling pathways. processes. However, there is no previous literature on the EP was undetected below 100 V, though max membrane use of PEF to improve animal tissue rehydration. There- permeabilization occurred at 500 V. Cell death was de- fore, the aim of this work was to investigate the influence tected at �1400 V. Starting at 10 V, BBBd increased for of two PEF pre-treatments (PEF (1) -500 V·cm-1 and small (NaF 376 Da) and large (IgG, 150 kDa) molecules in PEF (2) -1000 V·cm-1) on mass transport kinetics dur- a dose dependent manner: At 20 V permeability increased ing the rehydration process of salted cod. Samples were by 28.9±4.9/56 ±11.9% for NaF and IgG, while at 50 V treated at room temperature in tap water, with an ini- it increased by 135±12/212.94±33%, and permeability to tial electrical conductivity of 396 ± 5 µS·cm-1 at 25 °C an 8 kDa negatively charged antisense oligonucleotide in- 159 creased by 420±126%. of a parallel plate capacitor with perpendicular electric There were no changes in expression of ZO-1 or Beta field lines. Therefore, the cell suspension experiences a Catenin following L-PEFs or EP. Decreased expression of reasonably homogeneous electric field at the center dis- VE-cadherin was observed 30 min after EP only. There tance between the electrodes. However, in an MEA all was no significant change in the appearance of the cells electrodes are embedded in a planar substrate. Therefore, following L-PEFs (50 V), while at 800 V the cells seemed the electric field distribution becomes more complex and more elongated, swollen and electrofusion was clearly seen is quite inhomogeneous in the region of the attached cells. as early as 1 min after EP. At 24 hrs giant cells with mul- In this study, we present first results of an MEA- tinuclei were observed only after EP. based electroporation, using a FITC-labeled dextran as a 1 min after L-PEFs at 50 V, the expression pattern of fluorescent probe (2 MDa, Sigma-Aldrich) and an MEA- F actin, a key cytoskeleton protein, changed extensively. based electrofection, using pMAX GFP (Cell Line Nucle-Decreased density of the stress fibers across the cell body ofectorTM Kit V, Lonza). For this, we applied electrical and increase in misaligned fibers were observed. At 800 V, pulses to adherent HEK293T cells via our custom-made honeycomb-like peripheral actin and fragmented filaments MEAs comprising 60 individually addressable electrodes. were seen in abundance. Stress fibers across the cell body In our experiments, we varied the electrical pulse paramet- disappeared, the actin cortex became thicker and many ers (square pulses, sinusoidal pulses with different voltage misaligned fibers were seen. 24 hrs after treatment at 50 levels, frequencies, and durations) and the electrode ma- V, full recovery of the cytoskeleton was observed, while for terial (gold or iridium oxide). We also compared two dif- 800 V recovery attempt was observed, reflected by longer ferent electrode configurations, in which we either applied fragments of stress fibers across the cell body. This was a checkerboard arrangement of stimulation and counter supported by phosphoproteomic analysis showing strong electrodes on the MEA or short-circuited all electrodes activation of Rho GTPases signaling cascade which is a against a platinum wire as the counter electrode. We major regulator of cytoskeleton. found successful transfection of HEK293T cells with both Our results suggest that L-PEFs induce transient BBBd electrode arrangements and both materials. The highest via the paracellular pathway and that cytoskeleton reor- transfection efficiency was observed when applying a si- ganization mediated by Rho GTPases signaling is a pos- nusoidal 5V voltage pulse at 40kHz for 200ms to gold sible mechanism of action. These findings may lead the electrodes in the electrode arrangement with the platinum way to a new/non-invasive approach for drug delivery into wire. the brain. In the future, we plan to further optimize the MEA fabrication process and pulse protocol to achieve high ef- PO-080 ficiency with the checkerboard stimulation arrangement Microelectrode array-based electroporation for use eliminating the need for a large counter electrode. Once in multifunctional retinal implants successful, this concept should be further optimized for Andrea Kauth 1, Anne-Kathrin Mildner2, Sandra Johnen3, the ex vivo electrofection of retina explants and later im- Joachim Wegener2, Sven Ingebrandt1 plemented into the next generation of epiretinal implants. 1RWTH Aachen University, Germany 2Universität Regensburg, Germany PO-015 3Universitätsklinikum Aachen, Germany Electrical Impedance changes as a possible real- Retinal implants are intended to enable artificial vis- time indicator of Pulsed Field Ablation (PFA) ef- ion in patients suffering from degenerative diseases. In the ficacy past, we developed several types of epiretinal implants us- Tomas Garcia-Sanchez 1, Gerard Amorós-Figueras2, Mario ing microelectrode arrays (MEAs) to evoke a phosphene- Gómez3, Jose M. Guerra2, Antoni Ivorra3 1 based vision. However, all former concepts did not take CNRS, Gustave Roussy, Université Paris-Saclay, and Biomed- the retinal pathology and remodeling into account. Upon ical Electronics Research Group (BERG), Universitat Pompeu disease progression, the photoreceptor cells degenerate Fabra, France 2 and a functional reorganization of the neural network oc- Hospital de la Santa Creu i Sant Pau, Universitat Autònoma curs. This reorganization directly affects the stimulation de Barcelona, Spain 3 efficiency of the implants. In our next-generation ap- Universitat Pompeu Fabra, Spain proaches, we aim to incorporate additional functionalit- In recent years, the use of irreversible electroporation ies into the retinal implants. One hypothesis is that the (IRE) as a method for ablation of cardiac tissue in the remaining retinal cells could be protected by genetic modi- treatment of cardiac arrhythmias, also known as Pulsed fication, which could improve the stimulation efficiency of Field Ablation (PFA), has rapidly moved from preclinical the implants. While viral transduction is considered the studies to clinic. Although safety, efficacy and lesion dur- gold standard in gene therapy, transfection by electropora- ability of the treatments have been recently demonstrated tion (electrofection) is an immune-friendly alternative. In in limited groups of patients, studies in larger groups of in- the presented work, we investigate the possibility of using dividuals are still under development to show comparative MEAs to generate efficient electric field strengths for cell evidence of its superiority against radiofrequency ablation electrofection. and cryoablation. In a conventional electroporator, the electric field One of the many open questions of this new technology is between the electrodes is typically approximated to that how the local electrical activity of electroporated myocar- 160 dial tissue is modified during treatment and the differences tumors tend to be aggressive, multiplex, rapidly grow-between the reversibly and irreversibly damaged areas. ing lesions. The standard treatment is surgery, which This leads to question if the measurement of local electro- can be annoying regarding the number of lesions, and the grams, which are usually registered during ablation with left scars, or RT with limited possibilities of repetition. thermal methods, can accurately reflect the treatment out- Our clinical case report aims to highlight the possibility come and motivates the research on new real-time lesion of electrochemotherapy (ECT) as a successful treatment assessment methods. of the multiple non-melanoma tumors of renal transplant In this in vivo study we performed a parametric study patients nevertheless that raising evidence shows that im- using bursts of high frequency biphasic square waveforms mune system crucially contributes to ECT efficiency. of different amplitudes and frequencies leading to differ- Case presentations: Two 70-year-old male patients were ent levels of ablation efficacy. They consisted on square referred to the Department of Dermatology, University of biphasic bursts (10 bursts of 100 µs separated 1 s) at three Szeged, with multiple non-melanoma skin tumors (n=15, different frequencies (90, 260 and 450 kHz) and two fixed n=11) located in the head-neck area (n=11, n=10), on voltages (500 and 800 Vp), additional a group of mono- the limbs (n=2, n=1) and trunk (n=2, n=0). Both pa- phasic 100 µs square pulses at 500 V was also studied. PFA tients underwent renal transplantation 7, and 4 years be-in vivo treatments were performed in Sprague-Dawley rats fore referral, and have been treated with immunosuppress- following a protocol approved by the Sant Pau Hospital ive medication since then. Over the years, they developed Animal Experimentation Ethical Committee. Under gen- multiple non-melanoma skin tumors, which were previ- eral anesthesia, a left thoracotomy was made to create a ously removed surgically (n=27, n=3). Our multidiscip- surgical window. A monopolar epicardial electrode was linary tumor board decided to perform ECT. The patients carefully positioned over the left ventricle epicardium and were treated according to the Standard Operating Proced- a return electrode patch was attached to the back of the ures, under general anesthesia, with intravenous adminis- animals. During the procedure we continuously recorded tration of Bleomycin, because of the high number of le- the temperature at the electrode tip, the ECG and muscle sions. The treated lesions were in complete remission in contractions. In-burst electrical impedance was calculated both patient thirty-one and three months after ECT. One from the acquired voltage and electric current waveforms. patient received another session of ECT due to novel tu-Once the catheter was in position, baseline electrical im- mor lesions outside the treated area. pedance was recorded using 4 low voltage (50 Vp) bursts Conclusion: Organ transplant recipients with immunosup- identical to the treatment waveforms, subsequently, high pressive therapy are a unique group regarding high-risk, voltage treatment bursts were applied. Twenty-one days multiple skin tumor development. ECT can be a suitable later, animals were euthanized and the hearts were fixed treatment for immunosuppressed patients because of its for histological analysis with Masson’s trichrome staining high response rate, good cosmetic result, and only min- to assess the extent of fibrotic lesions. imal, local, low-grade side-effects. Our results show a clear dependence of ablation lesion size with frequency and amplitude of the applied wave- PO-005 form. This demonstrates how the IRE lethal threshold Calcium electroporation in cancer treatment – value increases with the increase in the frequency of the design, test and evaluation of an evidence-based applied waveform. The analysis of the impedance changes curriculum suggests that the impedance magnitude drop measured Christina Louise Lindhardt during PFA applications correlates with the chronic abla- University of Southern Denmark, Clinical Institute, Denmark tion lesions observed. These preliminary results suggest Calcium-electroporation has proven to be effective but the possible use of electrical impedance as a real-time in- yet gentle in treatment of skin cancer. dicator of PFA efficacy. Following the new treatment oncology must be updated to support the quality of treatment in order for the patients Poster Session (and Coffee break) to achieve a professional, equal and gentle way through the treatment and care. It is noted that in oncology nurses take over more tasks and skilled, up-to-date qual- Tuesday Poster Session Track ified nurses are needed in order to provide the patients Oct 11, 14:45 - 16:00 with the best and less stressful experience during treat- ment. With participation from researchers in Denmark PO-002 and Germany, the EU-based Interreg programme funded Successful Treatment with Electrochemotherapy the project “Changing Cancer Care”. One of its object-for Multiple Non-Melanoma Skin Cancers in Kid- ive was to develop, pilot and evaluate an evidence-based ney Transplant Recipients curriculum for continuing education of nurses working in Petra Rózsa, Dóra Ágoston, Edit Szederkényi, Anita Varga, oncology. Henriette Ócsai, Eszter Baltás, Lajos Kemeny, Judit Oláh, Erika Kis Methods: A multiple step approach was used: (1) Mixed- University of Szeged, Hungary methods studies and evidence syntheses were performed (2) A curriculum was developed for a 6-week (10 ETCS) Introduction: The risk of cutaneous malignancies is course targeting nurses working in oncology in Denmark. significantly higher in immunosuppressed patients com- (3) This curriculum was tested in a training course. (4) pared to the general population. These high-risk skin 161 A similar curriculum is being developed in Germany and Discussion: The electroporation results of both Electrical tested. The final prototype of the curriculum and scope- and Thermal Simulations are in line with literature, an review will be part of the deliverance. increasing electrical field will cause more permanent nan- Curriculum content: The curriculum addresses: calcium opores in the cell membrane, leading to more irreversible electroporation treatment, geronto-oncology clinical lead- cell damage and thereby apoptotic cell death [2]. The ership in nursing, dermatology, pain management, body different values of electric field and Temperature was due image, family dialogue, and technology. The course was to the different geometry and scales that were used in finalised with an oral examen. both simulations. Results: Nurses working in oncological care have gained Conclusion: To conclude, this study found an increasing a robust theoretical background in caring for patients fraction of irreversible electroporated cells over reversible treated using calcium electroporation. The evaluation was electroporated cells at increasing field strengths alongside positive, however insights into nutrition may enhance the with an increase in Temperature through simulation.ir course. Perspectives: This research demonstrates that investing References in an evidence-based curriculum lift the competences. It 1. Mir LM, Orlowski S, Belehradek J, Paoletti C. will benefit the patients to experience a safer, more reli- Electrochemotherapy potentiation of antitumour effect able pathway through the hospital system and eventually of bleomycin by local electric pulses. Eur J Cancer a higher quality of life. Clin Oncol [Internet]. 1991;27(1):68–72. Available from: http://www.sciencedirect.com/science/article/pii/027753799190064K PO-008 2. Ritter A, Bruners P, Isfort P, Barabasch A, Pfeffer Electroporation in liver cancer multiphysics simu- J, Schmitz J, et al. Electroporation of the Liver: More lation Than 2 Concurrently Active, Curved Electrodes Allow Ali Jouni New Concepts for Irreversible Electroporation and Elec- Uniklinik RWTH Aachen, Germany trochemotherapy. Technol Cancer Res Treat. 2018 Nov Introduction: Minimally invasive cancer therapies are 9;17:153303381880999. a popular area of research. Among these minimally invas- ive cancer therapies, electroporation-based therapies are PO-011 promising new methods, mainly due to their non-thermal Anti-inflammatory response characterization of effect and tumor specific approach. Electroporation uses microsecond electric pulses stimulation for spinal short electrical field pulses (in current clinical therapies cord injuries application about 70-100 µs) to create so called nanopores in the Giorgia G. Innamorati 1, Paola P. Giardullo1, Barbara B. Benassi1, Caterina Merla1, Maria M. Pedraza2, Nesus N. plasma membrane. There are two types of therapies: Torres2, Leslie Vallet3, Marina M. Sanchez Petidier3, Laura In irreversible electroporation, the cell membrane can’t L. Caramazza4, Sara S. Fontana4, Noemi N. Dolciotti4, Vic- repair the induced nanopores because of their size and toria Moreno Manzano2, Franck M. André3, Paolo Marracino5, amount which causes the cell to undergo apoptosis, the Micaela Liberti4, Claudia Consales1 natural cell death. In reversible electroporation, the 1ENEA, Division of Health Protection Technologies, Italy cell can repair their phospholipid bilayer and continue 2CIPF, Neuronal and Tissue Regeneration Laboratory, Spain on with their normal cell functions. In tumor therapy, 3CNRS, Gustave Roussy, Université Paris-Saclay, France those hydrophilic pores are used to highly induce the 4Sapienza University, DIET, Italy diffusion of a chemotherapeutic drug, and is known as 5Rise Technology S.r.L, Italy Electrochemotherapy[1]. This study aimed to predict the electric field lines around Spinal cord injury (SCI) is one of the most devastating the hepatic cancer tissue after electroporation, as well as and debilitating conditions that an individual can sustain. define the distribution of Temperature on the probe and It leads to temporary or permanent changes in the spinal the Tumor using computer simulations. cord’s normal motor, sensory, and autonomic functions. SCI is composed by two different phases: a primary and Methods: In order to predict the results, different simu- secondary injury. The first is due to the initial traumatic lations were done in COMSOL-Multiphysics simulation software with different voltages to see how the electric insult which damages or destroys the tissue and is due field was spreading across the cells. In order to obtain the to the hemostatic response and acute cell death. This strength of the Electric Field and the rise in Temperature, first phase is followed by the progressive secondary injury the following fixed parameters (70 pulses / 100 µs pulse cascade characterized by ischemia, proapoptotic signal- ing, and peripheral inflammatory cell infiltration. Over length / 100 µs interval) and variable parameters (200 the subsequent hours, the release of proinflammatory cy- V/600 V/1500 V) were applied on the model for both Electric field and Temperature Simulations. tokines and cytotoxic debris (DNA, ATP, reactive oxygen Results: The simulation that were done on COMSOL species) cyclically adds to the harsh postinjury microen- software showed that by increasing the voltage, and vironment, making this environment totally hostile to any thereby the electric field strength, the fraction of irre- kind of therapeutic treatments. For this reason, it is ex- tremely important, in case of SCI, to act against the in- versible electroporated cells over reversible electroporated flammatory cascade, to have the possibility of intervening cells will increase also the rise in Temperature will increase. therapeutically. Here we present the results obtained by applying differ- 162 ent exposure protocols in terms of repetition frequency, random model parameters and inputs. Thus, we define pulses number, amplitude, duration, and polarity on hu- the treatment planning algorithm as a stochastic optimiz-man microglia and macrophages cell lines, HMC3 and ation problem where the optimal treatment plan accounts U937, to evaluate the anti-inflammatory effect of these for the uncertainties in the treatment outcome predicted stimulations. These experiments are a specific task of by the stochastic model. To ensure usage during the treat- RISEUP project (fully described in another abstract sub- ment intervention, we propose a computationally efficient mitted to WC2022), funded by the European community approach involving a surrogate estimator of the propaga-in the H2020 FET-OPEN program, whose aim is to set an tion of the uncertainties from the input parameters to innovative approach for SCI regeneration based on micro the success measure of the treatment. This surrogate is pulses electric stimulation. a probabilistic representation of the treatment outcome and is validated against the predictive model for accur- PO-014 acy. The surrogate is ultimately used in a global optimiza- Treatment Planning under Uncertainty for tion routine to come up with optimally planned treatment Electroporation-Based Cancer Therapies strategies that are robust against uncertainties. Prashanth Lakshmi Narasimhan 1, Zoi Tokoutsi2, Nada Cvetkovic3, Marco Baragona2, Karen Veroy3 PO-017 1Eindhoven University of Technology and Philips Research, Construction of three-dimensional structure Centre for Analysis, Scientific Computing and Applications and research of folding-analogues of human (CASA), Netherlands kinetochore-microtubule used in transmembrane 2Philips Research, Eindhoven, Netherlands drug delivery 3Eindhoven University of Technology, Netherlands Olga Venger 1, Andrii Venger2 1 Electroporation-based treatment and therapies (EBT) The state institution South Ukrainian National Pedagogical are a novel advancement in the fight against cancer. These University named after K. D. Ushynsky, Ukraine 2 methods leverage the phenomenon of electroporation to Odessa National Medical University, Ukraine ablate the tumor through a minimally invasive proced- Introduction: It is obvious, that cellular and organis- ure. The procedure involves inducing a voltage difference mal fitness requires proper partitioning of genetic material across the tumor through needle-like electrodes to create during cell division. Furthermore, failure to accurately se- pores in the cell membrane. The pores can be utilized as gregate chromosomes causes aneuploidy, the most preval- temporary gateways to deliver chemo drugs (called electro ent genetic alteration in tumor cells and a potential factor chemotherapy (ECT)) or to damage the structural stabil- in the evolution of cancer. Chromosome segregation is ity (called irreversible electroporation (IRE)). The deliv- driven by microtubule-based forces, which are generated ery of ECT and IRE treatments is primarily based on the at kinetochores. electric field strength in the tumor cells. Hence, optimizing Basically, kinetochore-microtubule of human has import- the positioning and driving parameters of the electrodes ant role in drug and protein delivery in human cells dur- is an essential component of treatment planning. ing electroporation. But the structure of Kinetochore- In this work, we investigate model-based treatment plan- microtubule has been unfortunately still undescribed. ning algorithms that identify the optimal position and The aim of this work was to modulate the three- voltage parameters of the electrodes for a successful treat- dimensional (3D) structure and to research the folding- ment outcome. The optimal parameters are calculated analogues of human kinetochore-microtubule. by solving an optimization problem constrained by a bio- Material and methods: Template search with Blast and physical model for the EBT. The numerical solution of HHBlits has been performed by the SWISS-MODEL tem- the model is used to predict the electric field distribu- plate library. Models are built based on the target- tion and consequently the tumor ablation. However, the template alignment using ProMod3. Coordinates which electric field can depend on various factors (like the elec- are conserved between the target and the template are trode position, electrical conductivity values, etc.) and copied from the template to the model. Insertions and this can present difficulties in the planning. Firstly, the deletions are remodelled using a fragment library. Side tissue properties are commonly patient specific (based on chains are then rebuilt. Finally, the geometry of the result- water-fat content) and are not routinely measured prior ing model is regularized by a force field. In case loop mod- to or during interventions. Secondly, even if the measure- elling with ProMod3 fails, an alternative model is built ments were available, the measurement errors should be with PROMOD-II. taken into account. Finally, the inaccuracies in the elec- Results and Discussion: Three-dimensional (3D) structure trode positioning due to the lack of precise guiding tool, of human kinetochore-microtubule has been built. resolution of the medical imaging, image registration er- As folding-analogues of human kinetochore-microtubule rors and breathing motion may also affect the treatment there have been found the anaphase-promoting com-outcome. Hence, it is important to consider the effect of plex/Cyclosome, in complex with the Mitotic checkpoint these uncertainties in the planning for EBT interventions. complex and Cryo-EM unit of the Anaphase-promoting To account for such stochastic effects, the uncertainties are complex/Cyclosome, in complex with the Mitotic check- incorporated into the predictive biophysical model. This point complex. results in a stochastic computational model of the EBT, Information about three-dimensional (3D) structure comprising of a set of partial differential equations with and partial analogy with anaphase-promoting com- 163 plex/Cyclosome may help to understand physical proper-PO-024 ties as well as spatial configuration of human kinetochore- Development of 3D melanoma cultures on a hy-microtubule, and to examine its ability in drug and protein aluronic acid-based scaffold with synthetic self- delivery in human cells during electroporation. assembling peptides Annj Zamuner 1, Leonardo Cassari1, Monica Dettin1, Luigi PO-020 Dall’Olmo1, Luca G. Campana2, Maria Teresa Conconi1, Elisa- Structure and folding-analogues of mutant form of betta Sieni3 human mitotic serine/threonine kinase B delivered 1University of Padova, Italy into cells by electroporation 2Manchester University NHS Foundation Trust, United King- Andrii Venger 1, Olga Venger2 dom 1Odessa National Medical University, Ukraine 3University of Insubria, Italy 2The state institution South Ukrainian National Pedagogical Electrochemotherapy (ECT) with bleomycin is an University named after K. D. Ushynsky, Ukraine established and effective treatment in the care of pa- Introduction: Initially, mitotic serine/threonine kinase tients with superficially metastatic tumors. Cutaneous B is located to the kinetochore and plays a role in the melanoma is among the most frequent indications, with inhibition of the anaphase-promoting complex/cyclosome a complete response rate close to 50%, according to the (APC/C), delaying the onset of anaphase and ensuring most recent meta-analyses. Nonetheless, novel drugs and proper chromosome segregation. The interesting fact is electroporation (EP) conditions are being investigated to that the impaired spindle checkpoint function has been improve patient outcome further. found in many forms of cancer. Mutant forms of mitotic In general, ECT efficacy is evaluated in vitro on cell serine/threonine kinase B are leading to cancer cell mitosis suspensions or using cells in adhesion, and in vivo on depression and may be delivered into the cell by electro- animal models. Nevertheless, the first two mentioned poration. But currently the structure of mutant form of approaches completely lack extracellular matrix com- mitotic serine/threonine kinase B is still uncharacterized. ponents, leading to unreliable results because they do The aim of this work was to modulate the three- not reflect the natural tissue architecture. To overcome dimensional (3D) structure and to study the folding- this problem, several three-dimensional (3D) in vitro analogues of mutant form of human mitotic ser- models, such as spheroids and hydrogel-based cultures, ine/threonine kinase B. have been proposed to mimic the complex tumour Material and methods: Template search with Blast and microenvironment. These in vitro models always require HHBlits has been performed by the SWISS-MODEL tem- a suitable low-conductive medium to allow EP of the cell plate library. Models are built based on the target- membranes through a sequence of voltage pulses. In this template alignment using ProMod3. Coordinates which frame, a new synthetic scaffold based on hyaluronic acid are conserved between the target and the template are (HA) and self-assembling peptides is proposed as an ad- copied from the template to the model. Insertions and de- vantageous alternative. Self-assembling peptides (SAPs) letions are remodeled using a fragment library. Side chains are a class of ionic-complementary peptides (EAbuK) able are then rebuilt. Finally, the geometry of the resulting to aggregate, forming a hydrogel scaffold with a fibrous model is regularized by a force field. In case loop model- structure. The self-assembling sequence was condensed ling with ProMod3 fails, an alternative model is built with with an adhesive motif mapped on Laminin (IKVAV). PROMOD-II. SKMEL28 cells, a malignant melanoma cell line, were Results and Discussion: Three-dimensional (3D) structure seeded on these two-components scaffolds and cultures of mutant form of human mitotic serine/threonine kinase were characterized at 3 and 5 days by cell viability B has been built. assessment and Masson’s trichrome staining. As folding-analogues of mutant form of human mitotic Electroporation was performed using EPS02 EP equip- serine/threonine kinase B there have been detected: Mi- ment (Igea SpA, Carpi, Modena, Italy). Several voltage totic serine/threonine-protein kinase BUB1, beta, Mitotic amplitudes and strengths of the applied electric field (0, serine/threonine-protein kinase BUB1 beta, and Fibro- 400, 600, 800, and 1000 V/cm) were tested. The EP blast growth factor receptor 2. efficiency was evaluated at different electric field strengths Information about three-dimensional (3D) structure and using propidium iodide. Three-D cultures seeded on HA partial analogy with the detected folding-analogues may without SAPs and cells in adhesion electroporated using help to understand physical properties, furthermore spa-the culture medium or the electroporation buffer were tial configuration of mutant form of human mitotic ser- taken as controls. The 3D cultures were electroporated in ine/threonine kinase B, and to research the role of elec- the culture medium. troporation in delivering of mutant form of mitotic ser- Our data demonstrated that cells cultured on HA- ine/threonine kinase B into human cells. EAbuK-IKVAV scaffolds start to be electroporated at 400 V/cm (50% of electroporated cells). In contrast, the cells cultured on HA are not efficiently electroporated at the same electric field strength. The 2D experiments showed that EP starts at 600 V/cm using electroporation buffer and at 800 V/cm in culture medium but with very low efficiency (<50% of electroporated cells). In conclusion, 164 the HA-SAP 3D cultures allowed the simulation of a more PO-028 tumor-tissue-like environment and may represent a tool Effects of calcium electroporation and irreversible to study cell electroporation conditions. EP is greatly electroporation on HPAF II cells in vitro study influenced by the local conductivity of the extracellular Agnieszka Gajewska Naryniecka 1, Nina Rembiałkowska1, medium and cell-cell connections. Vitalij Novickij2, Dagmara Baczyńska1, Julia Rudno- Rudzińska1, Wojciech Kielan1, Julita Kulbacka1 1Wrocław Medical University, Poland PO-027 2 Vilnius Gediminas Technical University, Lithuania CaCl2 influence on pDNA electrotransfer effi- Pancreatic cancer (PC) was the seventh leading cause ciency and cell viability of cancer death in 2020, and the 5-year survival rate is less Rūta Palepšienė, Paulius Ruzgys, Martynas Maciulevicius, than 10%. This fatal disease is predicted to be the third Baltramiejus Jakstys, Dovilė Uždavinytė, Salvijus Vykertas, leading cause of cancer death in 2025. Pancreatic cancer Neringa Barauskaite, Saulius Šatkauskas has no symptoms until the disease has advanced and is ag- Vytautas Magnus University, Lithuania gressive cancer with early metastasis. Up to now, the only Calcium (Ca2+) is one of the most important second curative treatment is surgical resection, and it is possible messengers that is associated with a variety of different in the early stages of the disease. Irreversible electropora-processes including development, cell proliferation, and tion treatment offers new hope for patients with unresect- cellular motility. Intracellular free Ca2+ concentration is able tumors. Our study aims to determine the efficacy of tightly regulated and varies depending on cellular location. calcium electroporation (CaEP), in treating patients with Experiments already revealed that using electroporation – pancreatic cancer, which is expected to improve treatment a physical delivery method that increases the permeability outcomes and bring measurable benefits to patients. This of cell membranes – in combination with calcium necrotic innovative method combines a non-pharmacological ap- cell death can be induced. proach (electroporation) with calcium ion administration Although gene electrotransfer has been researched extens- upon IRE procedure beyond medicine specifications in an- ively in both in vitro and in vivo systems, the exact mech- ticancer therapy in patients diagnosed ”de novo” or who anisms by which DNA enters and navigates through cells do not improve with standard treatment methods. are still not fully understood. It has been proposed that Here we used the HPAF II cell line as a the negative charge of DNA induces its electrophoretic model for pancreatic cancer. The following EP movement near the cell membrane after an electric field protocols were used: 3.5kV/cm×900ns×100, 1kHZ; is applied. DNA interaction with the permeabilized cell 5.7kV/cm×600ns×100, 1kHz; 5.7kV/cm×900ns×100, membrane, followed by endocytosis results in DNA trans- 1kHz; compared to clinical standard ESOPE (European fer across the cell membrane and nuclear import. Exper- Standard Operating Procedures of Electrochemotherapy) imental data also revealed that divalent ions like Ca2+ 1.3kV/cm×100µs×8. All protocols were combined with which abolish electrostatic repulsion between DNA and 2.5mM calcium chloride (CaCl2) in a HEPES-based buf- the cell membrane might enhance DNA absorption on the fer, and cell viability was determined using the MTT assay cell membrane. However, less is known about how ex- after 48 and 72 hours. The cell membrane permeabiliz- actly calcium can influence the electrotransfer efficacy of ation rate was measured by flow cytometry using a cell- pDNA or how this simultaneous electrotransfer of DNA impermeant dye Yo-Pro-1. Additionally, proinflammatory and Ca2+ affects cell viability patterns. Thereby, the aim cytokines (IL-6 - Hs00174131_m1, IL-8 -Hs00174103_m1) of this study was to evaluate the effect of different extra- and anti-inflammatory cytokine (IL-10 - Hs00174086_m1) cellular CaCl2 concentrations (0 – 1mM) on the pDNA gene expression was quantitatively assessed in HPAF II transfection efficiency and cell viability tendencies. Ex- cells, 72h after EP protocols using real-time RT-PCR. periments were performed using two sets of electropora- The obtained results indicate a significant anticancer ef- tion parameters: 1400V/cm, 100 µ s, and 1200V/cm, 250 fect after calcium electroporation. In relation to EP alone, µ s. pDNA transfection efficiency and fluorescence intensthe addition of calcium ions caused cell viability to de- ity were evaluated using flow cytometry 24h after electro- crease to 20% of control cells after 48h and about 30 per- poration. Fluorescence microscopy was used for the evalu- cent after 72h. The most effective protocol occurred at ation of labeled pDNA electrotransfer tendencies. For the 5.7kV/cm×900ns×100, which was comparable to ESOPE. evaluation of cell viability patterns, flow cytometry assay Cell permeabilization increased with the increasing electric and MTT methods were used. Results revealed that the field intensity. We observed changes in cytokine mRNA higher Ca2+ (> 0.25mM) concentrations are associated expression levels, but additional research is needed. with significantly decreased plasmid electrotransfer effi- Our research is entirely innovative, and according to ciency and fluorescence intensity. What is more, a com- our current knowledge, there are limited reports about bination of pDNA and calcium electroporation resulted in calcium ion electroporation (CaCl2) in pancreatic cancer. a significant decrease in cell viability. The reason for this Acknowledgments: This study was supported by the phenomenon is still under investigation. Medical Research Agency, Poland, IREC project No. 2020/ABM/01/00098/P/02 (PI: Prof. Wojciech Kielan). 165 PO-031 100mV. Measuring cell membrane charging limits in cur- Support: AFOSR MURI grant FA9550-15-1-0517 to rent clamp mode AGP Mantas Silkunas, Andrei G. Pakhomov Old Dominion University, United States PO-034 When electrical stimuli increase the transmembrane Combination of cold plasma and pulsed electric membrane potential (TMP) to 200-500 mV, they damage field for microalgae treatment Kamile Jonynaite 1, Skirmantas Kersulis1, Rolandas the membrane by an electroporation. The first and argu- Uscila2, Zydrunas Kavaliauskas2, Liutauras Marcinauskas2, ably the most sensitive manifestation of electroporation is Voitech Stankevic1 the decrease in the membrane electrical resistance (Rm), 1State research institute Center for Physical Sciences and Tech- suggesting that measuring the electric current across the nology, Lithuania membrane is the best way to detect electroporation. In- 2Lithuanian Energy Institute, Lithuania deed, several studies utilized the whole-cell voltage clamp configuration of patch clamp to explore the limits to which The potential of microalgae helps to tackle the chal- cell membrane can be charged safely without electropora- lenges such as the growing demand for energy resources, tion. The same approach was employed for calibration of food and bioactive compounds is well known. However, voltage-sensitive dyes such as FluoVolt. solutions are still being investigated to efficiently reduce The inherent problem of voltage clamp measurements the cost of algae cell disruption while increasing the quantis the impact of the series resistance (Rs) at the tip of itative and qualitative yield of the resulting products. In the measuring pipette. The command voltage (Vc) from contrast to conventional chemical and mechanical meth- the patch clamp amplifier is divided between Rs and Rm ods of algae treatment, pulsed electric field (PEF) and proportionally to their values. When Rm»Rs, TMP is non-thermal plasma treatment have the greatest potential approximately equal to Vc. However, electroporation de- to address the above-mentioned problems. creases Rm to values comparable with Rs. Hence, the Studies have already been conducted showing that voltage drop at Rs has to be calculated from the measured mammalian and bacterial membranes oxidised in plasma current (I) and subtracted from Vc to obtain the actual are more sensitive to the PEF treatment [1,2]. How- TMP value as: TMP=Vc - Rs x I. This calculation works ever, there are no studies on the treatment of microal-for slow Vc changes and for steady-state Vc. However, cell gae. Therefore, this work aims to investigate the effect of capacitance (Cm) slows membrane charging to the inten- a combination of PEF and plasma treatment on microal-ded TMP proportionally to the charging time constant τ gae. = Cm x Rm. When Rm is changing due to electropora- The gliding arc discharge and PEF technologies were tion, the value of τ is not known and also changes in time. applied on Chlorella vulgaris cultivated in the BG-11 me- As a result, the actual TMP reached by fast Vc steps or dium. The plasma treatment was performed using these ramps is difficult or impossible to calculate. parameters: compressed air flow rate ˜22.8 l/min, the dis- We propose to address this problem by measuring tance between the ”knife-edge” type electrodes and surface TMP in the current clamp mode, when we apply current of the algae suspension was 30 mm, treatment duration steps or ramps and measure the voltage induced. This 300 s, power generator voltage 50–250 V and frequency voltage V equals to TMP when Rm»Rs. If Rm is de- 270 kHz. PEF treatment consisted of 10 µ s long 1-10 creased by electroporation, TMP = V - Rs x I, where I pulses at a frequency of 1 Hz, where applied electric field is clamped current value. Most important, this calcula- strength varied from 23-25 kV/cm. Afterwards, changes tion does not require the knowledge of Rm or τ , and can in the electrical conductivity, extracted chlorophyll-a and be employed with the fastest current steps or ramps. For protein content of untreated and treated algae suspension example, current clamp can be employed to test if the were determined. breakdown voltage depends on how fast the membrane is First, the effects of treatment using PEF or plasma polarized. Current clamp can also be employed to calib- were evaluated separately. The highest concentrations of rate voltage-sensitive dyes in intact and already electro- excreted chlorophyll-a concentration were obtained after porated membranes, to compare if electroporation affects 10 pulses of PEF (180 µg/mL) or 210-250 V plasma treat- dye sensitivity. ment (190 µg/mL). The combination of plasma and PEF We employed fast current ramps (0 to +/-1nA in 50ms) treatment did not significantly increase pigment levels to measure membrane breakdown voltage in CHO-K1 (200 ± 10 µg/mL) compared to the PEF effect only. cells. De-and hyperpolarization caused a distinct break- From a protein extraction perspective, the PEF treat- down at 229±11mV and -230±16mV, respectively. Fur- ment yielded 700 µg/ml of protein and the plasma treat-ther increase in current beyond this voltage neither in- ment (50-130 V) was 600 ± 100 µg/ml. In contrast, creased nor increased TMP, indicating that Rm is chan- plasma treatment with a discharge voltage of 210 V or ging dynamically to maintain the TMP at the breakdown more resulted in a lower concentration of protein extracted value. We employed current steps (50ms/step, in 100 pA from C. vulgaris compared to untreated algae. Meanwhile, increments down to -1 nA or up to +1 nA) to calibrate the combined effect of plasma and PEF did not lead to an voltage sensitivity of the FluoVolt dye in intact and elec- increase in the amount of proteins released, but rather to troporated cells. Electroporation had no significant im- a significant decrease. pact on the dye sensitivity, which averaged 17.1±1.3% per This unexpected decrease in protein extraction effi- 166 ciency suggests that further studies are needed to under-results obtained were the consequence of a conformational stand the effects of combined plasma and PEF treatment. change in the proteins induced by the applied treatment, References which allowed greater NaCl absorption and less water loss [1] C.M. Wolff, A. Steuer, I. Stoffels, T. von from the samples. Regarding texture, color and lipid ox-Woedtke, K.-D. Weltmann, S. Bekeschus, J.F. Kolb, idation, the treatment did not provide any difference in Combination of cold plasma and pulsed electric fields the treated samples compared to the control. – A rationale for cancer patients in palliative care, The result obtained may be of great importance to the Clinical Plasma Medicine. 16 (2019) 100096. ht- salmon processing industry because the achievement of tps://doi.org/10.1016/j.cpme.2020.100096. higher salt levels, in a product that simultaneously loses [2] Q. Zhang, J. Zhuang, T. von Woedtke, J.F. Kolb, less water, provides a technological advantage. In fact, the J. Zhang, J. Fang, K.-D. Weltmann, Synergistic antibac- salting process is thus more efficient as processing times terial effects of treatments with low temperature plasma are significantly reduced (to only 3 hours) and higher pro- jet and pulsed electric fields, Appl. Phys. Lett. 105 (2014) cessing yields are achieved with attractive cost savings for 104103. https://doi.org/10.1063/1.4895731. companies, improving the performance of industrial pro- cesses. PO-036 Mass transfer modulation during salting of PEF PO-039 pre-treated salmon fillets Developing and optimizing ohmic heating for cake Ana De Aguiar Saldanha Pinheiro, Fabio D’Elia, Jessica Aberham Hailu Feyissa, Anastasia Mantza, Felix Rabeler Genovese, Silvia Tappi, Urszula Tylewicz, Pietro Rocculi Technical University of Denmark, Denmark University of Bologna, DISTAL, Italy Conventional baking process is time and energy- Salting is one of the oldest and simplest methods of consuming process mainly due to heat transfer is limited preserving large quantities of fish for long periods of time; by internal conduction within the product. Ohmic heat- it is often used by the industry in combination with other ing is potentially solving this problem by allowing volu- traditional processing techniques, such as smoking, drying metric heating of the product and thereby, uniform and and cooking. This results in safer products with a better rapid heating and consequently, leads to a more energy- sensory appearance, but the salting kinetics require long efficient and sustainable process, however, its potential is times for the salt to diffuse into the product and to work not utilized yet. Thus, the presentation will focus on re-properly. Among existing emerging technologies, pulsed cent development in our understanding of ohmic heating electric fields (PEF) are a non-thermal treatment that has for baking processes. been shown to be effective in increasing mass transfer in The aim of current work focused on the investigation of both plant and animal tissues, without affecting the nu- the potential use of ohmic heating for cake baking and the tritional value, flavour, colour and texture of products. influence of the process on cake quality changes. The aim of the present study was to study the applic- An experimental design based on a Central Composite ation of PEF to Atlantic salmon filets before subjecting Design carried out to evaluate the effects of four key pro- them to dry salting in order to improve the process ef- cess parameters: applied voltage, salt, butter and baking fectiveness. The experimental design included 5 salmon powder content. The studied responses are process time, (Salmo salar) sample groups for each salting time (3 and expansion, moisture content, hardness and springiness of 6 hours): control (NT) and 4 types of PEF pre-treatment the cake. The results showed that voltage is the parameter (PEF1, PEF2, PEF3, PEF 4). At the end of the salting that affects all the responses significantly. Combination of times, the samples were rinsed in running water, dried and high voltage and high salt content led to decreased baking then subjected to the following analytical determinations: times (up to 80%), increased moisture loss and increased weight change, water activity, NaCl content change, wa- hardness. Baking powder affected positively the expansion ter content change, texture, colour and the level of thio-and springiness of the cake, but negatively the hardness barbituric acid reactive substances (tBars). The results and the process time. Butter did not influence signific- shown that pulsed electric fields of 0.64 kV/cm (detected antly the textural properties; however, it enhanced the by PEF3), applied prior to the 3-hour salting of salmon, expansion and reduced the final moisture content. promote the diffusion of salt into the tissues, leading to in- A mathematical model was established and used to creased NaCl retention by the muscle, thanks to the PEF identify the optimum settings for ohmic baking process. permeabilization effect on cell membranes. These process The quality properties of two commercial cakes and a parameters in fact generated a reversible electroporation pound cake baked in the convection oven were used as capable of favoring a more homogeneous distribution of target values. The cake baked at obtained optimum con- salt within the product, also allowing for a lower percent- dition resulted in similar textural properties as commer-age weight variation compared to untreated samples. PEF cial products. An assessment of the energy requirements did not provide any advantages in terms of reducing the showed that energy losses increase slightly with higher water activity of the samples, especially during the shorter electric fields, but ohmic heating can use up to 47% less salting times, but it did improve the water retention prop- energy than the industrial ovens. The results are encour- erties of the salmon with 3-hour salting, probably because aging and ohmic heating is a promising technology for cake of a more permeable structure that allowed retention of baking. more liquid within the tissue. It is also possible that the 167 PO-042 SUSFOOD2 and CORE Organic Cofunds, under the Bioactive properties of air-dried organic apples Joint SUSFOOD2/CORE Organic Call 2019 (MILDSUS- pre-treated by pulsed electric field FRUIT) as well as National Centre for Research and De- Artur Wiktor, Katarzyna Rybak, Dorota Witrowa- velopment (POLAND, decision DWM/SF-CO/31/2021). Rajchert, Malgorzata Nowacka Warsaw University of Life Sciences – SGGW (WULS-SGGW), PO-044 Poland The impact of matrix on pulsed electric field pre- Pulsed electric field (PEF) was demonstrated to en- ceded food drying hance the drying kinetics, reduce drying time and specific Aleksandra Matys1, Alicja Baranska1, Katarzyna Rybak1, Dorota Witrowa-Rajchert1, Magdalena Dadan1, Katarzyna energy consumption of the process. However, the literat- Pawlaczyk2, Artur Wiktor 1 ure on the quality aspects of such processed materials and 1Warsaw University of Life Sciences – SGGW (WULS-SGGW), especially chemical properties, is not very abundant. Poland The aim of this research was to evaluate the effect of 2Cedrus Sp. z o.o., Poland PEF treatment and air temperature on selected chem- ical properties of convective dried organic apples. PEF Pulsed electric field is one of the most promising application was carried out using batch system (PEFPi- technologies used for drying enhancement. The literature lot™, ELEA, Germany) and apple slices (thickness of 5 of the subjects shows that the effect of PEF on kinetics mm) were hot air dried (Promis, Poland). Response Sur- and quality strongly depends not only on parameters of face Methodology with central composite face centred plan pre-treatment, drying technique but also on matrix. (CCF) was used to design the experiment. The energy in- This aim of this research was to investigate the effect of put of PEF (1 kV/cm) varied from 1 to 6 kJ/kg and hot air PEF pre-treatment on air drying kinetics and selected temperature ranged between 60-80°C. Following chemical properties of apple, strawberry, carrot, and mushrooms. properties were evaluated in dried apple slices: vitamin The experiment was designed using response surface C, total phenolics (TPC) and total flavonoids (TFC), an- methodology with central composite face cantered plan. tioxidant activity with DPPH radical (AA) and reducing Drying was performed at temperature of 55, 70 and power (RP). 85°C whereas energy input of PEF was selected based The vitamin C concentration of investigated samples on electroporation efficiency measurement and it ranged varied between 6.5 and 130.6 mg/100 g d.m. In all cases, from 1-6 kJ/kg depending on the raw material. Kinetics untreated dried apples exhibited higher concentration of of the process and drying time was evaluated based on the vitamin C – it was equal 130.6, 112.2 and 82.2 mg/100 water evaporation curves. Following quality parameters g d.m., when temperature of 60, 70 and 80°C was used, were evaluated: total phenolics, total colour difference respectively. In turn, PEF treated samples exhibited vit- and antioxidant activity (DPPH assay, EC50). amin C content of 6.5-12.7, 7.3-19.8 and 8.0-10.1 mg/100 Obtained results showed that the role of matrix is of g d.m, for the same temperatures, respectively. Such tre- paramount importance when it comes to the effect of mendous decrease may be related to liberation of both PEF on drying improvement. For instance, in the case vitamin C and enzymes (e.g. ascorbic acid oxidase) which of apple tissue, drying time was reduced (by 5-20%) no remained active during drying. Similar results were found matter the parameters of drying and PEF treatment for phenolics and flavonoids. In these cases, alike all PEF were whereas in the case of mushrooms the reduction was evaluated variants were characterized by lower TPC and found mainly when the highest drying temperature was TFC. Moreover, at temperature of 60 and 70°C the negat- used (1-12%). The ambiguous results were also found ive relationship between energy input and phenolics and when strawberry was used. In the case of carrot samples, flavonoids content was found. For instance, apples dried the reduction of 4-30% was found but only at 55 and at 70°C were characterized by TPC od 1482 mg/100 g 70°C while no reduction or even extension of drying time d.m whereas materials pre-treated by 1, 3.5 and 6 kJ/kg was stated when drying was performed at 85°C. PEF exhibited values of 1201, 796 and 720 mg/100 g d.m., re- pre-treatment reduced total phenolics concentration of spectively. Antioxidant activity of dried slices pre-treated most investigated variants of apples and strawberries. by PEF was smaller in comparison to control apples. The Similar observations were withdrawn for antioxidant smallest difference was found when energy input was 1 activity. In turn, total phenolics content of mushrooms kJ/kg and drying was carried out at 70°C. Similar obser- increased no matter the treatment parameters were. Total vations can be withdrawn for reducing power. colour difference of PEF pre-treated materials was usually Performed analysis showed that PEF treatment in the higher in comparison to untreated materials, however range of 1-6 kJ/kg applied before air-drying decreased con- the direction of colour change was in some cases rather centration of analysed bioactive compounds and antioxid- desirable – for instance, carrot tissue was characterized ant activity. In most cases the reduction stayed in relation by higher share of red (a*) and yellow (b*) than control with the energy input which indicates that PEF condi- material. tions aimed towards drying enhancement should be selec- Research that was carried out shows that the effect of PEF ted carefully, especially when chemical properties conser- depends strongly on matrix and no standard, universal, vation is required. optimal protocol can be generated for all raw materials. This project has received funding from transna- Thus, the process requires optimization considering the tional funding bodies, partners of the H2020 ERA-NETs technological aim of drying and pre-treatmetn. 168 that can meet the growing demand for proteins, presenting Acknowledgment: The research was carried out as a many advantages over conventional farming, such as min- part of the Horizon 2020 program financed by the imal resource needs, low environmental impact (less GHG European Union (contract no. 817683) “Innovative and NH3 emissions), low feed conversion ratio (around 1.7 down-scaled FOod processing in a boX”, acronym FOX. for insects compared with 10 for cattle) and above all their integration into a logic of circular economy. PO-047 This research work is part of the RAFINSECT project Plant-based model for the optical evaluation of funded by the HDF region (France) and aims to set up electroporated area after irreversible electropor- an insect biorefinery. The objective is to fractionate and ation and its comparison to in-vivo animal data valorize all biomass fractions such as proteins, lipids and Kim Lindelauf1, Athul Thomas 1, Marco Baragona2, Ralph chitin by incorporating them into a variety of food and Maessen2, Andreas Ritter1 feed matrices, and into cosmetic products and packaging 1Uniklinik RWTH Aachen, Germany as well. 2Philips Research, Netherlands PEF treatment was applied to living mealworms, with an Electroporation is widely used in medicine, such as electric field strength between 200 and 2000 V/cm and a cancer treatment, in form of electrochemotherapy or irre- treatment time between 20 and 200 ms, under different versible electroporation. For electroporation device test- treatment conditions (in distilled water, tap water, com- ing, living cells or tissue inside living organism (including pacted or pre-rinsed). Treated larvae were then pressed animals) are needed. Plant-based models seem to be a as an energy-efficient defatting and dewatering method or promising alternative to substitute animal models. The dried in a ventilated oven. The impacts of the PEF treat- aim of this study is to find a suitable plant-based model ment on cell membrane electroporation, larval mortality, for optical evaluation of IRE, and to compare the geo- pressing ability, drying kinetics and product quality were metry of electroporated area with in-vivo animal data. studied. The treatment processes were compared and op- For this purpose, a variety of fruit and vegetables were timized. selected and optically evaluated after 0/5/22 hours after PEF pretreatment successfully killed the larvae with the electroporation. Apple, fresh and old potato were found to mortality being linked to the treatment’s electric field be suitable models as they enabled an optical evaluation strength, and pre-rinsing was found to improve the killing of the electroporated area. For these models, the electro-efficiency. The SEM images show visible damage and porated area was calculated after 0/5/22 hours. The elec- changes in the larvae’s structure. The treatment has sig- troporated area of apple, which showed the fastest optical nificantly increased the hydraulic press extraction yield results, was then compared to a retrospectively evaluated from 65% in blanched larvae to 78% in PEF-treated lar-swine liver IRE dataset which had been obtained for sim- vae, which might be caused by the enhanced membrane ilar conditions. The electroporated area of the apple and permeability due to electroporation, as well as accelerated swine liver both showed a spherical geometry of compar- the drying kinetics and water diffusivity (an improvement able size. For all experiments, the standard protocol for of water diffusivity by 470%). human liver IRE was followed. To conclude, potato and apple were found to be suitable PO-052 plant-based models for the optical evaluation of electro- Eradication of Saccharomyces cerevisiae by porated area after irreversible electroporation, with apple PulsedElectric Field Treatments being the best choice for fast optical results. Given the Efrat Emanuel 1, Rivka Cahan2, Roman Pogreb2 1 comparable range, the size of the electroporated area of Bar Ilan University, Israel 2 the apple may be promising as a quantitative predictor Ariel University, Israel in animal tissue. Even if plant-based models cannot com- One of the promising technologies that can inactivate pletely replace animal experiments, they can be used in microorganisms without heat is pulsed electric field (PEF) the early stages of electroporation device development and treatment. The aim of this study was to examine the influ- testing, decreasing animal experiments to the necessary ence of PEF treatment (2.9 kV cm−1, 100 Hz, 5000 pulses minimum. in trains mode of 500 pulses with a pulse duration of 10 µs) on Saccharomyces cerevisiae eradication and resealing PO-049 in dierent conditions, such as current density (which is Study of the impact of Pulsed electric fields pre- influenced by the medium conductivity), the sort of me- treatment on yellow mealworm insects in a biore- dium (phosphate buered saline (PBS) vs. yeast malt broth finery concept (YMB) and a combined treatment of PEF with the addi- Rachelle El Hajj1, Houcine Mhemdi1, Karim Allaf2, Colette tion of preservatives. When the S. cerevisiae were suspen- Besombes2, Nabil Grimi 1, Eugene Vorobiev1 ded in PBS, increasing the current density from 0.02 to 1University of Technology of Compiegne, France 3.3 A cm−2 (corresponding to a total specific energy of 2University of La Rochelle, France 22.04 to 614.59 kJ kg − 1) led to an increase of S. cerevisiae With the world’s population predicted to reach 9 bil-eradication. At 3.3 A cm−2 , a total S. cerevisiae erad- lion by 2050, it is essential to find new sustainable agricul- ication was observed. However, when the S. cerevisiae in tural practices and protein sources for the feed and food PBS was treated with the highest current density of 3.3 A sectors. Edible insects are promising alternative biomass cm−2 , followed by dilution in a rich YMB medium, a phe- 169 nomenon of cell membrane resealing was observed by flow cleaning with balloon catheter are not effective in case of cytometry (FCM) and CFU analysis. The viability of S. hyperplasia or tumour ingrowth. Endoluminal radiofre- cerevisiae was also examined when the culture was exposed quency ablation could be used but there is a certain risk to repeating PEF treatments (up to four cycles) with and of thermal damage that could be caused by the stent be- without the addition of preservatives. This experiment coming an active electrode. These factors suggest that was performed when the S. cerevisiae were suspended in IRE could be a convenient choice. YMB containing tartaric acid (pH 3.4) and ethanol to a fi- In this study, we present the analysis of the electric field nal concentration of 10% (v/v), which mimics wine. It was and temperature distribution inside the occluding tissue shown that one PEF treatment cycle led to a reduction of that was modelled as liver parenchyma. Simulations are 1.35 log10, compared to 2.24 log10 when four cycles were based on a 3D FEM model. Electroporation is induced applied. However, no synergic eect was observed when using a specialised 3-electrode catheter where two plate the preservatives, free SO2, and sorbic acid were added. electrodes are in contact with the undesired tissue block- This study shows the important and necessary knowledge ing metal biliary stent. Boundary conditions were set to about yeast eradication and membrane recovery processes drive the electrodes to deliver 100 of one hundred micro- after PEF treatment, in particular for application in the seconds long pulses with a frequency of 1 Hz. The tissue liquid food industry. was exposed to four different amplitudes of voltage (300 V, 650 V, 1000 V and 1300 V). The model parameters PO-055 were based on real experiments. Influence of pulsed electric field-assisted dehydra- The simulations show that the distribution of the electric tion on the volatile compounds of Genovese basil field in the occluding tissue is enclosed in the metal stent (Ocinum basilicum L.) if the stent mesh is dense enough. This is caused by the Sumiyo Kanafusa1, Mikael Agerlin Petersen2, Federico faraday cage-like behaviour which works to our advantage Gómez Galindo 1 and protects the healthy surrounding tissue from the IRE 1Lund University, Sweden effect. The ablation volumes are affected by the geometry 2University of Copenhagen, Denmark and position of the electrodes and stent but generally in- Pulsed electric field (PEF) was applied to basil leaves crease with higher voltage amplitudes. In our model, it (Ocinum basilicum L.) prior air drying at 40 °C. The para-ranges approximately from 76 mm3 to 184 mm3. The meters of the electric treatment were designed in such a temperature rise is acceptable in most cases. Only when way that (i) electroporated the tissue reversibly, provok- using an amplitude of 1300 V does the temperature reach ing a permanent opening of the stomatal guard cells and values above 50 °C. (ii) electroporated the tissue irreversibly, damaging the Based on the results of FEM simulations and the gen- cells. erally accepted threshold for IRE of 700 V/cm the elec- Treated leaves lost some volatile compounds due to both troporation effect should occur in all simulated cases. Yet PEF treatments, probably related with the direct effect based on the pilot experiments it seems that the threshold of permeabilization on the secretory cells of glandular for the IRE is higher and corresponds with the intensity trichomes. Upon drying, the irreversible permeabilization of the electric field above 1000 V/cm. According to our treatment showed the highest influence on the profile of findings, the optimal voltage for IRE in occluded biliary volatiles in the dried leaves showing better retention of stents ranges between 650 – 1000 V. If we would increase some terpenoids than the control. The performed statist- the voltage, the ablation volume would be higher but we ical analysis allowed to select six compounds that can be could induce thermal damage. The results suggest that used as markers both for the effect of pre-treatments prior this IRE application is feasible with certain parameters dehydration and for the effects of dehydration itself on the of the procedure. It has great potential, and if validated, volatile compounds of basil leaves. this form of recanalization could be used in other metallic stent occlusions. PO-057 Mathematical model of biliary metal stent occlu- PO-060 sion treatment using irreversible electroporation An optimal dose-response in terms of pulse length Martin Hemzal, Veronika Novotna in EP-based treatment protocols Brno University of Technology„ Czech Republic Isaac Rodriguez1, Nahuel Olaiz1, Felipe H. Maglietti2, Sebastián D. Michinski1, Alejandro Soba3, Cecilia Suárez1, Electroporation finds its place in a wide range of sci- Guillermo R. Marshall 1 entific and industrial fields. This abstract focuses on a 1University of Buenos Aires, Instituto de Física del Plasma, novel application of irreversible electroporation (IRE) to Argentina recanalize occluded biliary metal stents used in cholan- 2Instituto Universitario de Ciencias de la Salud Fundación H.A. giocarcinoma treatment. The occlusion, or blockage, of Barceló, Argentina the stent, is usually caused by epithelial hyperplasia, tu- 3Comisión Nacional de Energía Atómica, Argentina mour ingrowth or overgrowth into the stent mesh, as well as clot accumulation. The survival of patients has im- An optimal EP-based treatment, i.e., a treatment proved to exceed 12 months however, the metal stent based on electroporation (EP), such as electrochemother-patency rate is reportedly between 14 and 321 days. apy (ECT), Gene Electrotransfer (GET), or irreversible Standard recanalization techniques such as mechanical electroporation (IRE), is a function of pulse amplitude, 170 length, number, and frequency, among other variables. used to form the liquid build material in the desired shape Finding an optimal EP-based treatment in a space of of each layer. This shape is defined by an interdigitated 4 parameters is a difficult task. In previous works [1-2] electrode array– combining the resolution of integrated optimal GET was studied in terms of pulse number, for circuit fabrication and soft lithography with the complex- a range of pulse intensities, with all other variables kept ity of AM in the third dimension. After a layer is shaped, constant; unwanted damage due to pH fronts and the it is subsequently cured; this process is repeated until a 3D concept of the dose-response relationship was discussed. structure is formed. Because EFF does not take place in a Here we extend those results by analyzing an optimal vat of resin, components such as electrodes or membranes EP-based treatment in terms of pulse length considering can be included mid-build to create lab-on-a-chip systems damage due to temperature and/or irreversible electro- that are biologically relevant for treatment planning and poration effects. An optimal dose-response relationship characterization. A unique advantage of EFF is also the in an EP-based treatment, such as IRE, for the range of material selection. Proof-of-concept parts printed on this pulse intensities with fixed pulse number and frequency, platform include microdevices made with uncommon AM tested here, is predicted as the critical pulse length dosage materials but very common microfluidic materials such yielding maximum irreversibly electroporated tissue with as polydimethylsiloxane (PDMS), a biocompatible poly- minimal damage due to temperature. mer with sufficient transparency for optical monitoring. The devices printed on this platform include a millimeter- [1] Luján, E. et al. Optimal dose-response relationship scaled gear, an enclosed microfluidic tapered channel, and in electrolytic ablation of tumors with a one-probe-two- a layer of PDMS for interfacing with copper electrodes. electrode device, Electrochim. Acta 186, 494–503 (2015). Electric field fabrication is an additive manufacturing [2] Marino M. et al, OpenEP: an open‑source simulator method that combines high resolution fabrication tech-for electroporation‑based tumor niques with the complexity of additive manufacturing for Treatments, Scientific Reports (2021) 11:1423 faster, easier, and more innovative designs for electropor- ation microdevices. PO-062 Electric Field Fabrication: A method for 3D print- PO-065 ing electroporation microdevices Electrical parameters for (ir)reversible electropor- Josie L. Duncan 1, Rafael V. Davalos1, Jeff Schultz2, Zeke ation on hepatocellular carcinoma cells in vitro Barlow2 Kim Lindelauf 1, Marco Baragona2, Ralph Maessen2, An- 1Virginia Tech, United States dreas Ritter1 2Phase, Inc. , United States 1Uniklinik RWTH Aachen, Germany 2 Microfluidic devices have been used extensively in the Philips Research, Netherlands field of electroporation for characterization, transfection, Hepatocellular carcinoma (HCC), which accounts for and pasteurization and also provide a variety of advant- >80% of primary liver cancers worldwide, has a heavy dis-ages when compared to macro-sized systems. In these ease burden and is a leading cause of cancer-related death devices, spacing between the electrodes is small, ulti- in many parts of the world. Traditional treatment options mately requiring low applied potentials to electroporate are not always efficient in eradicating the tumor, hence the cell for its intended application. In turn, these low the need for new treatment methods. During the prom-potentials allow for a wide range of generators and sig- ising minimally invasive electroporation-based therapies, nals, not adequate for traditional setups, to be used. A biological cell membranes are exposed to an external, suffi- microdevice offers features comparable to cell size for high- ciently high, pulsed electric field, which can lead to a rapid resolution characterization, small required sample size for and large increase in electric conductivity and permeab- rare cell-types, compatibility with other microfluidic pro- ility, creating so called nanopores into the lipid bilayer cesses upstream and downstream, and the ability to mon- of the cell membrane. During irreversible electroporation itor the process in real-time. The design and complexity (IRE), the cell membrane cannot repair the induced nanof these devices, while they offer unique advantages for opores because of their size and amount, which causes the in vitro studies, are limited by current soft-lithography cell to undergo apoptosis. During reversible electropora-techniques and have yet to extensively benefit from the tion (RE), the cells can repair their phospholipid bilayer possible geometries of additive manufacturing (AM). and continue with their normal cell functions. In tumor Few additive manufacturing processes are appropri- therapy, those hydrophilic pores are used to increase the ate for the fabrication of microfluidic electroporation diffusion of a chemotherapeutic drug which is known as devices. The available high-resolution methods have lim- electrochemotherapy (ECT). For both IRE and RE, the ited biocompatible material options and do not permit success of the treatment is dependent on application of component integration between layers; thus, requiring as- the appropriate electric field. Therefore, this study aims sembly and electrode integration after the part is com- to define the (electrical) parameters for IRE and RE on plete. Here, we introduce Electric Field Fabrication hepatocellular carcinoma (HepG2) cells in-vitro. (EFF), an additive manufacturing method that manipu- In a custom-made in-vitro setup, HepG2 cell viability (at lates liquid build material using dielectrophoresis (DEP). 0, 5, 10 and 15 minutes), and the peak temperature were Dielectrophoresis, or polarization of a dielectric particle measured after electroporation with the different IRE and or fluid in the presence of a non-uniform electric field is RE pulsing protocols, to determine the most successful 171 settings for IRE and RE. In addition, A CAM/PI flow activation achieved with it. Although considerable inac-cytometric assay was performed to confirm cell permeab- tivation (3-4 log) with combined treatment was achieved, ilization for the RE pulsing protocols with the highest cell this may not be achievable for antibiotic concentrations viability. usually present in wastewater, which are typically much The results indicated that an IRE pulsing protocol (70 lower. Also, to reach considerable potentiation, some in- pulses, 100 µs pulse length, 100 ms interval) with an elec- cubation with antibiotic is required even for ampicillin. tric field strength of 4000 V/cm was needed as threshold for almost complete cell death of HepG2 cells. A RE PO-071 pulsing protocol (8 pulses, 100 µs pulse length, 1000 ms Efficient recycling of electronic-waste by nano- interval) with an electric field strength of 1000 V/cm was second pulsed electric discharge needed as threshold for viable and permeabilized HepG2 Ryo Kaida, Mitsuhiko Sato, Nushin Hosano, Mijanur Rah- cells. The low peak temperatures (max 30.1°C for IRE, man, Hamid Hosano max 23.1°C for RE) within this study indicated that the Kumamoto University, Japan reduction in HepG2 cell viability was caused by the ap- Recycling of valuable materials such as rare metals plied electric field and was not a result of Joule heating. is becoming an imperative topic from the perspective of resource conservation and environmental sustainability. PO-068 The existing techniques for electronic waste recycling and Potentiating the efficacy of clinical antibiotics by metal separation, e.g., chemical or mechanical crashing electroporation methods, are not cost effective and can cause further en- Žana Lovšin, Tadej Kotnik vironmental burden. In this regard, pulsed power tech- University of Ljubljana, Faculty of Electrical Engineering, nology has attracted considerable attention for green re- Slovenia cycling. In this study, metal coated electronics were used Electroporation is a promising complementary tech- as a separation processing model. A magnetic pulse com-nique for bacterial inactivation. Exposing bacteria to pression pulsed power generator (MPC-PPG) was used for short electric pulses of sufficient strength permeabilizes providing positive nanosecond pulses. By applying under- their envelope, thereby facilitating the uptake of mo- water electric discharge, the metallic parts were separated. lecules, including antibiotics. Electroporation is effective To understand the separation mechanisms an ultra-high-against a broad range of bacteria and as it is based on pore speed framing camera with 5 ns exposure time, equipped formation, it is largely impervious to development of res- with schlieren and shadowgraph optical setups, were em- istance. So far, most studies investigating electroporation ployed. The whole process of plasma generation, shock to potentiate the efficacy of antibacterials used substances waves production, propagation, and interface interactions, permissible in food industry, and only few used clinical an- and afterward flow fields were observed. A fiber optic tibiotics, as addition of these is problematic, which limits probe hydrophone (FOPH) pressure transducer with 3 acceptable applications to treatment of wastewaters in- ns rise time was used for incident and transmitted shock herently contaminated with such antibiotics. Moreover, waves pressure measurements. The results clearly confirm even the studies utilizing clinical antibiotics have mostly that the proposed method is highly efficient for green re- focused on achieving the maximal effect, and less on un- cycling. The technique can be applied to electronic waste derlying mechanisms and on the possible dependence of recycling on a commercial scale. the potentiation on the antibiotic’s target site or mode of action. PO-074 Our aim was to determine the effect of antibiotics mode Study of permittivity change in the cervical cell of action and concentration, electric field amplitude and membrane 3D realistic model due to application incubation time after treatment on Escherichia coli inac- of subnanosecond electric field using SRD based tivation. pulse generator Three antibiotics with different modes of action were used: Mayank Kumar, Ashutosh Mishra ampicillin (inhibition of cell wall synthesis in the pep- Indian institute of Information Technology Allahabaad, India tidoglycan layer of the cell wall), tetracycline (intracel- The paper represents a change in permittivity of the lular inhibition of protein synthesis) and ciprofloxacin (in- dielectric layer in the cell membrane of the cervical cell tracellular inhibition of DNA replication). Concentrations using a high voltage output generated by the diffused step used in the study were determined based on their previ-recovery diode producing a Gaussian pulse of 30ns and an ously determined minimum inhibitory concentrations. For electric field of 10KV/cm . The high-frequency electric electroporation treatment, one pulse of 1 ms duration and field allows the permittivity of the cell membrane to lower electric field amplitude of 5, 10, 15, or 20 kV/cm was used. down efficiently which can be easily studied with help of Level of inactivation was determined after different post- realistic geometry of a single cervical cell model gener- treatment incubation times at room temperature. ated using image processing techniques and CAD tools Our results show that with increasing pulse amplitude, an- and introduced into a multi-physics tool. The realistic tibiotic concentration, and/or incubation time, the poten- pulse generated by the diffused step recovery diode-based tiation of inactivation consistently increases. Ampicillin generator is introduced in the multi-physics area. The only needs to permeate the outer membrane of bacterial Debye dispersion relation is used to model the dielectric cell wall, which can explain the highest potentiation of in- relaxation in the cell membrane which is a bi-lipid layer 172 of 5nm thickness. The study of dielectric in the cervical status. Due to previous radiotherapy treatment for a sar-cells is important as the second-order time-domain equa- coma in the armpit, he developed two angiosarcomas (the tion helps the trans-membrane potential to easily reach first one was surgically removed). For treating the second the potential of 1-1.5V. This transmembrane voltage gen-one, he rejected a new surgical procedure because of the erated can cause pores formation on the nano-meter thin slow recovery time. The size of the remaining tumor was layer which is important in the case of drug and dye deliv- 12x6.5 cm. For the ECT procedure, the patient received ery. The dye delivery can easily detect the morphological mild sedation with intravenous ketamine and midazolam. changes in the cell structure of the cervical cells which is No local anesthetics were used. important in cancer detection. Drug delivery can be used Results: Total treatment time ranged from 18 to 30 for electrochemotherapy applications. Thus the change minutes. No cardiac rhythm alterations occurred during in permittivity of the bi lipid layer considerably reduces or after the procedure. A progressive reduction of the size the amount of electric field required to facilitate the pore of the lesion until its complete disappearance was seen formation. with no necrosis. All cases obtained a complete response, free of relapse during the follow-up time. Tolerance was excellent, even with local anesthesia. No systemic side Poster Session (and Coffee break) effects or post-procedural pain were observed. Hyperpig- mentation due to bleomycin developed in one patient, but Wednesday Poster Session Track it started to fade away progressively. The cosmetic results Oct 12, 15:00 - 16:00 in the other patients were very good. PO-003 PO-006 Electrochemotherapy in Oncodermatology, cur- The best treatment and care for patients suffering rent uses in Argentina from skin cancer - lifting competencies for nurses Felipe H. Maglietti 1, Sebastián D. Michinski2, Carolina working in advanced cancer care with calcium elec- Abal3, Matias M. Tellado4, Guillermo R. Marshall2, Adrián troporation Barceló1 Christina Louise Lindhardt 1Instituto Universitario de Ciencias de la Salud Fundación H.A. University of Southern Denmark, Clinical Institute, Denmark Barceló, Argentina 2University of Buenos Aires, Instituto de Física del Plasma, Purpose: The patients come first and need the best Argentina treatment and care is the objective of the project ‘Chan- 3Grupo de Electroporación en Medicina, Argentina ging Cancer Care‘. We identified how a tailored evidence- 4VetOncologia, Veterinary Oncology Clinic, DC, INFIP, DF - based training course for oncology nurses could improve UBA CONICET, Argentina qualifications in the care and treatment of patients treated with calcium electroporation (EP) and, subsequently, Introduction: Electrochemotherapy (ECT) is a stand- quality of life. Initially, we describe the development test ard treatment modality in Europe since 2006, and now and evaluation of the intervention, and implementation also in Argentina since 2020. In this work, we present the strategy, followed by the evaluation. The research, test results of three patients treated. and evaluation were conducted in 2019-2020. Patients and methods: The procedures were performed in Methods: The intervention was developed using a liter- an operating theater with heart monitoring. The patients ature review and individual- and focus group interviews were treated using IV bleomycin 15,000 IU/m2 in bolus. with healthcare professionals experienced in the field and The electric pulses were delivered using the electropor- patients. A prototype eight-day training course for nurses ator OncoPore (BIOTEX, Buenos Aires, Argentina), a working in advanced oncology where patients treated with medical-grade device. Needle electrodes were used in all calcium EP was developed, tested, and evaluated with cases, as well as a single-dose of intramuscular NSAID and mixed methods, including oral, written evaluation and an corticosteroids for pain management after the procedure. online assessment tool concerning content and relevance. Squamous cell carcinoma in the forehead. The patient was Results: The participating nurses evaluated the training 80 years old and presented a tumor surrounded by satel-course as relevant and appropriate for their practice in lite lesions (total size was 3.5x2.8 cm). The mild cognitive advanced cancer care. Knowledge of skin cancer focused impairment of the patient precluded the use of general on body image, skin and pain management, family dia- anesthesia, rendering the surgical procedures impossible. logue, person-centred approaches, clinical leadership, and Only lidocaine without epinephrine was used as a local shared decision making was evaluated as indispensable. anesthetic. The participant felt confident in using the newly acquired Basal cell carcinoma at the back of the ear. The patient skills immediately after the course and being resourceful was 60 years old and in good clinical shape. Smoker, for healthcare professionals on their ward. with peripheral vascular disease. The tumor had a size Conclusion: This research demonstrates that investing of 1x1.5 cm. Mild sedation with propofol plus local lido- in an evidence-based curriculum shifts nursing skills and caine without epinephrine was used for the procedure. The care. These skills will ultimately benefit patients by patient reported having no pain during or after the pro- providing a safer, more reliable transition through the hos- cedure. pital system and improving patient quality of life. The Angiosarcoma in the trunk. The patient was 85 years old curriculum based on theory and novel knowledge regarding and in good clinical shape. He was in optimal cognitive 173 calcium EP treatment for patients with cancer enhanced PO-012 the nurses’ qualifications and supported them to provide Electrochemotherapy combined with immunother-optimal and effective oncological care. Further curriculum apy as a facial nerve preserving treatment modal- developments may include patient involvement and co- ity of the late intraparotideal metastasis of a non- operation, e.g., patient and relatives’ boards and organ- melanoma skin cancer isations. Gabor Vass, Zsolt Bella, Aurel Ottlakan, Eszter Baltas, Judit Olah, Lajos Kemeny, Erika Gabriella Kis PO-009 University of Szeged, Hungary Randomised controlled trial investigating the ef- Authors present a case of a late intraparotideal meta- fect of reduced bleomycin in electrochemotherapy stasis of a non-melanoma skin cancer treated successfully on patients with cutaneous malignancies: A pro-with a combined treatment of electrochemotherapy (ECT) tocol and immunotherapy. Freya Bastrup 1, Mille Vissing2, Volker-Jürgen Schmidt2, The 72-year-old male patient was operated with a pla- Mohammad Farooq Nassari2, Taiba Alrasheed2, Anni Lin- nocellular carcinoma of the skin in the right temporal re- net Nielsen3, Camilla Kjær Lønkvist3, Lisbeth Rosenkrantz gion. Due to R1 resection and ipsilateral lymph node Hølmich3, Michael Prangsgaard Møller3, Biljana Mojsoska4, Elizabeth Emilie Rosted2, Julie Gehl2 metastases in the parotid gland and on the neck, re- 1Copenhagen University, Denmark resection, partial parotidectomy and neck dissection was 2 Zealand University Hospital, Denmark carried out. After negative staging examinations the pa- 3Copenhagen University Hospital, Denmark tient received definitive dose postoperative radiotherapy 4Department of Science and Environment, Roskilde University, according to oncoteam decision. Denmark 4 years later the patient was presented with a grow- ing mass in the remnant of the right parotid gland. The Background: Response rates of electrochemotherapy MRI suspected malignancy, the recurrent planocellular (ECT) with bleomycin have been high and consistent carcinoma was proven by open biopsy. Following the neg-when applied for the treatment of skin cancer. However, ative staging examinations, the oncoteam decision was im-due to possible side effects e.g. lung fibrosis, some pa- munotherapy (cemiplimab) in combination with ECT, be- tient groups are excluded from treatment. Consequently, cause surgical removal of the tumor would have destroyed in 2018, studies began to investigate the possibilities of the facial nerve. reducing the dose, finding a similar objective overall re- After 2 regimens of cemiplimab ECT was carried out sponse (ORR), thus indicating that a lower dose may not according to the Standard Operating Procedure (SOP) be inferior. with intravenous bleomycin, standard adjustable linear Aim: This protocol for a randomized controlled trial electrodes, Cliniporator Vitae by Igea S.p.A. The third (RCT) aims to investigate whether the bleomycin dose cemiplimab was administered 2 weeks after ECT. 1 month can be reduced with 50% and retain the ORR in patients after ECT complete remission was visible, which was with cutaneous malignancies. proved by MRI as well, with intact facial nerve functions. Methods: The RCT will include 55 patients, as calculated The patient is tumor-free 8 months after ECT and is re- in a statistical power analysis for non-inferiority studies ceiving regular immunotherapies. using categorical data. The patients will be randomised Our case demonstrates the organ and nerve function 1:1 in two groups; one group receiving the standard bleo- sparing ability of ECT in combination with immunother- mycin dose of 15,000 IU/m2 and the other group receiv- apy. ing 7,500 IU/m2. In order to be included the patients must be over 18 years old, have a histologically verified PO-018 cutaneous or subcutaneous, primary or secondary cancer Evaluation of TNF alpha production due to elec- of any histology, with a life expectancy over 3 months and trochemotherapy applied on glioblastoma spher- have a creatinine within normal upper limit. Patients will oids co-cultured with monocytes be excluded if they are pregnant, allergic to bleomycin or Bogdan Mircea Matei with impaired lung function. The primary endpoint is to Carol Davila University of Medicine and Pharmacy, Romania evaluate the ORR after three months. The trial will also There is an increased interest in the evaluation of include qualitative interviews for analysing quality of life immune response involvement in the clinical outcome of before and after ECT, and biopsies and blood samples to electrochemotherapy (ECT)(1, 2). In the present study investigate bleomycin pharmacokinetics and drug distri- glioblastoma spheroids were obtained, treated with ECT bution. The trial is planned to start in January 2023. and co-cultured with monocytes. We evaluated the pro- Conclusions: Reducing the bleomycin dose appears to be inflammatory TNF- promising. After searching the literature of clinical trials, α and anti-inflammatory IL 10 levels as a response of naïve resident monocytes to the exposure no RCT study has to our knowledge, considered lowering of the spheroids to ECT with either temozolomide (TMZ) the bleomycin dose for ECT treatment. This is necessary or cisplatin (Cis). to properly investigate the issue. Spheroids were prepared from U87 human glioblastoma This project is funded by the Danish Cancer Society. cell line (ATCC) at a seeding of 10k cells/well, grown for 7 days in 96 well plates, U bottom type, agarose coated wells. Spheroid growth rate was monitored every 2 days 174 via optical microscopy at 5x. ECT was applied on spher-The presented research aimed to investigate the impact oids using an Electro Cell B10 ( β-Tech) generator ( fol- of ECT (with cisplatin or calcium chloride (CaCl2) fused lowing the ESOPE protocol (3). After treatment, the with a high dose of 17 β-estradiol (E2) preincubation on co-culture was done by pipetting the spheroids in new OC cells (OvBH-1). Furthermore, the influence of the wells, previously covered with a monocyte layer (seeding analyzed treatment on normal cells (CHO-K1) has been 5k cells/well). Monocytes were freshly separated from the investigated. In the study, standard ESOPE protocol same human healthy donor on the day of ECT, using neg- (100 µs × 100 Hz × 8 pulses) with variable voltage ative immunomagnetic selection assay (MojoSort Human [kV/cm] has been applied. Subsequently, the impact on Pan Monocyte isolation kit). Multiple control groups were cells’ viability was examined by MTT assay. For each considered as follows: U87 spheroids in culture medium; parameter, the influence of ECT on cells’ mobility was U87 spheroids with monocytes; U87 spheroids exposed to studied using a wound-healing assay. The correlation chemotherapy alone; U87 spheroids exposed to chemother- between preincubation with E2 and plasma membrane apy and monocytes (without electric pulses). permeabilization was examined by measuring the uptake TNF- α was evaluated from co-culture growth medium 5 of the Yo Pro™-1 dye using flow cytometry. After hours after ECT and again at 7 days post treatment us- treatment, morphological changes occurring in cells have ing ELISA (TNF alpha Abcam 181421). IL-10 was also been visualized using fluorescent staining of the actin measured (IL-10 Abcam 185986). cytoskeleton and observed by confocal laser scanning The measurements showed that after ECT with Cis (at and holotomographic microscopy. Furthermore, the 833uM) or TMZ (at 100uM) applied on spheroids, there authors determined the type of cell death that occurred was a significant increase of TNF alpha levels in the co- post-treatment using Annexin V Apoptosis Necrosis culture medium at both 5 hours and 7 days with respect Assay. Then, the BAX, Bcl-2, and Caspase-12 levels were to the co-cultures not exposed to the electric pulses. The analyzed on protein (Western-Blotting) and transcript levels of TNF alpha were generally lower in case of ECT levels (Real-Time qRT-PCR). Also, a sensitive lumines- – TMZ group than in that of ECT – Cis. The levels of cent assay was used to assess the Caspase-3/-7 activity. IL-10 were not significantly modified by ECT. The obtained results revealed that preincubation with The spheroids growth rate was computed as an area-based E2 enhanced the cytotoxic effect of CT and ECT on OC index using Organoseg software(4). The index was de- cells. Concurrently, preincubation decreased the plasma creased at 7 days in the case of ECT + monocytes exposure membrane’s permeabilization. It may suggest that the for both TMZ and Cis when compared to chemotherapy enhanced efficiency of ECT after preincubation is not alone. related to plasma membrane permeability. It is expected that after electroporation intracytoplas- Funding: Statutory Subsidy Funds of the Department mic molecules are released and they could play the role of Molecular and Cellular Biology, Wroclaw Medical of immunogenic signals as classical DAMPs (ATP, calre- University grant no. SUBZ.D260.22.016 ticulin). Considering that our study showed increased levels of pro-inflammatory TNF-alpha but not for anti- Literature: inflammatory IL-10, we may speculate that ECT recruits 1.Menon, U. et al. Ovarian cancer population screening naïve monocytes towards the M1 pro-inflammatory type, and mortality after long-term follow-up in the UK Collab- with known anti-tumoral activity, avoiding in the same orative Trial of Ovarian Cancer Screening ( UKCTOCS ): type their transformation into tumor associated macro- a randomised controlled trial. Lancet 6736, 1–12 (2021). phages (TAMs, or M2 anti-inflammatory type) known for 2. Santen, R.J. The oestrogen paradox: A hypothesis. their pro-tumoral activity(5). Breast Cancer Res. 2007, 9, 1–5. 3. Traphagen, N.A.; Hosford, S.R.; Jiang, A.; Marotti, PO-021 J.D.; Brauer, B.L.; Demidenko, E.; Miller, T.W. High Electroporation-based modalities fused with 17 β- estrogen receptor alpha activation confers resistance estradiol in ovarian cancer therapy in vitro to estrogen deprivation and is required for therapeutic Zofia Łapińska, Anna Szewczyk, Julita Kulbacka, Jolanta response to estrogen in breast cancer. Oncogene 2021, 40, Saczko 3408–3421. Wroclaw Medical University, Poland Ovarian cancer (OC) is an estrogen-dependent ma- PO-022 lignancy and the most fatality among all gynecological Effect of electrochemotherapy on myogenesis of carcinomas [1]. Estrogens stimulate the proliferation of mouse skeletal muscle C2C12 cells in vitro: “side- OC; however, depending on their concentration, they effects” also can have a cytotoxic effect on cancer cells [2,3]. So Simona Kranjc Brezar 1, Urska Matkovic1, Bostjan far, this has not been thoroughly investigated. Calcium Markelc1, Ajda Vrabic1, Tim Bozic1, Mihaela Jurdana2, Maja Čemažar1 electroporation (CaEP) is a modification of electro- 1Institute of Oncology Ljubljana, Slovenia chemotherapy (ECT), which enables the introduction of 2University of Primorska, Faculty of Health Sciences, Slovenia supraphysiological doses of calcium ions (Ca2+) into the cytosol using pulsed electric fields (PEFs). Unfortunately, Electrochemotherapy (ECT) is a local ablative ther- the available literature lacks data focusing on the effi- apy for the treatment of different skin and subcutaneous ciency of CaEP as the potential OC treatment method. tumors as well as for certain tumors in internal organs. 175 The use of electroporation enables the application of small nels. Specifically, we studied three ion channels, including doses of bleomycin or cisplatin with high therapeutic ef- a bacterial sodium channel NavMs, an eucaryotic (cock- ficiency, resulting in minimal systemic toxicity. Skeletal roach) channel NavPaS, and a human hyperpolarization-muscle, in addition to cutaneous, vascular, endothelial, activated cyclic nucleotide-gated channel HCN1. We have and neural tissue, represents a major tumor-surrounding demonstrated that pores form more easily in VSDs that tissue, which is exposed to side effects of ECT. The aim are more hydrated and are electrostatically more favour- of this study was to investigate the effects of ECT on the able for the entry of ions. In addition, we demonstrated myogenesis of C2C12 cells in vitro, as the effects of ECT that pores in VSDs can expand into so-called complex on the different stages of myogenesis are not known. pores, which become stabilized by lipid headgroups and The effect of ECT was determined in mouse skeletal can lead to severe unfolding of VSDs from the channel. muscle C2C12 cell line. First, the electroporation effi- Corroborated by previous electrophysiological measure- ciency of differentiated C2C12 myotubes was determined ments, we predicted that ion channels with such unfolded at increasing voltages from 200 to 1300 V/cm using prop- VSDs become dysfunctional. Here we extend out study idium iodide (PI). Viability was measured using the Presto to various other ion channels, including members of po- Blue® assay. Further, the effect of ECT with bleomy- tassium channels, voltage-gated sodium channels, voltage-cin or cisplatin on the survival of C2C12 myoblasts and gated calcium channels, chloride channels, and a member myotubes using optimized electric pulse protocol was eval- of the transient receptor potential channel. We show that uated. Increasing doses of bleomycin (from 14000 nM complex pores can form in VSDs of many different ion to 0.14 nM) and cisplatin (from 200 µM to 5 µM) were channels but not all channels. Interestingly, we show that tested and cell survival was determined at 3, 5 and 7 days complex pores in VSDs can form more easily than in a pure after ECT. In addition, the effect of ECT on myoblast dif-phosphatidylcholine lipid bilayer. We discuss our findings ferentiation including IL-6 secretion was evaluated using in the light of how electric field-induced alteration of mem- ELISA. brane ion channels could affect the biological outcome of Permeabilization of C2C12 membranes by the intake of PI electroporation. was voltage-dependent with app. 42?ficiency at 500 V/cm and 74% to almost 100?ficiency at higher voltages from 900 PO-029 V/cm to 1300 V/cm. At these tested voltages the high cell Nanoparticle targeted drug delivery with nano- survival rate and myotube integrity has been maintained second pulsed electric discharge induced shock until day 5 after electroporation. The decrease of cell vi-waves ability in myoblast or myotubes has been observed after Ryosuke Inoue 1, Nushin Hosano1, Ryuichi Nakajo1, Hamid ECT with high doses of bleomycin or cisplatin, whereas Ghandehari2, Hamid Hosano1 low doses (up to 1.4 nM for bleomycin and 15 µM for cis- 1Kumamoto University, Japan platin) have shown no effect on cell survival. However, 2The University of Utah, United States myotubes were less sensitive to ECT with bleomycin or Underwater shock wave, in medical range, causes dis- cisplatin compared to myoblast, resulting in lower IC50 continuous change in the tissue’s pressure and density. values for bleomycin and cisplatin, up to 11.9-times and Shock waves are produced when stored energy is instant- up to 7.3-times, respectively. Moreover, ECT affected my- aneously released in the fluid. Shock waves are applied as a oblast differentiation capability, indicated by a significant non-surgical treatment method, as converging shock waves reduction of myotube formation, and a decrease in IL-6 ex-can be generated outside the body, coupled with the skin, pression compared to control. These preliminary findings and focused on the targeted tumor/tissue, without dam- contribute to the safety profile of ECT for tumor treat- aging the intervening tissue. In the meantime, nanomedi- ment. cine is rapidly developing for diagnostic and therapy to increase the bioavailability of drugs to tissue and tu- PO-025 mors. Nanoparticles, with their small size and large sur- Molecular insight into denaturation of plasma face area to volume ratio, are effective to bind, absorb membrane ion channels by pulsed electric fields and carry drugs, DNA, RNA, and proteins, along with Lea Rems 1, Lucie Delemotte2 imaging agents, to tumors with high efficiency. In this 1University of Ljubljana, Faculty of Electrical Engineering, paper, we used a solid-state magnetic pulse compression Slovenia circuit (MPC) to generate nanosecond pulsed electric dis- 2KTH Royal Institute of Technology, Science for Life Laborat- charges and to produce reliable and controllable underwa- ory, Dept. Applied Physics, Sweden ter shock waves. Shock pressure histories were measured Formation of aqueous pores in the plasma membrane by a fiber optic probe hydrophone (FOPH) pressure trans-lipid bilayer is a well-known mechanism of increased mem- ducer. Variety of shock wave overpressures from 20 to 150 brane permeability associated with electroporation. How- MPa were applied and tested. Silica nanoparticles were ever, much less is known whether and how membrane used as drug carriers and shock waves exposures were used proteins are affected by intense pulsed electric fields. In to enhance the effectiveness of the targeted drug delivery. our previous study (Rems et al., Biophys. J. 119:190- Suspension of human lymphoma cell line U937 were util- 205, 2020) we have shown through molecular dynamics ized for in-vitro experiments. The results are promising simulations that electric fields can induce pores in the and will be further developed for effective target drug de- voltage-sensor domains (VSDs) of voltage gated ion chan- livery. 176 PO-032 PO-037 Role of resting membrane potential in Ca2+ influx Pulsed electric field treatment application to im- following exposure to intense electrical pulse prove product yield and waste valorization in Kazuya Matsunaga, Sunao Katsuki kiwifruit processing Kumamoto University, Japan Corinna Stühmeier-Niehe 1, Martina Comiotto Alles1, Ivan Shorstkii2, Maxim Sosnin2, Claudia Siemer1 Introduction: Applying pulsed electric field (PEF) to 1ELEA Technology, Research and Development, Germany a living cell permeabilizes the cell membrane and sub- 2Kuban State Technological University, Russian Federation sequently leads to the transmembrane substance trans- portation. This phenomenon, known as the electroper- The present contribution focuses on the effect of Pulsed meabilization (EPB), is used for the introduction of drugs Electric Field (PEF) and the related impact of differ- and genes into cells and the extraction of intracellular com- ent treatment levels on the peeling characteristics of ponents [1-2]. In particular, calcium ion (Ca2+) influx kiwifruit and consequently on the physicochemical prop- following the PEF exposure is about to be used for can- erties. Kiwifruit is considered as a very healthy product cer treatment [3]. While typical pulse width of PEF is due to its high concentration in vitamin C and high nat- hundreds of microseconds or less, the subsequent Ca2+ ural antioxidative capacity correlating with a favorable influx lasts for from milliseconds to minutes. Although aroma. Because of the health promoting ingredients, it there are some hypotheses for mechanism of the Ca2+ in- is of high interest to use kiwi as an ingredient in various flux, such as a diffusion, an electrophoresis driven by the fruit products, such as smoothies. An important process resting membrane potential (RMP), or biochemical reac- step required for the use in juice production is peeling. tions including ion transporters, it is not clear. We have Mostly, abrasive peeling is used, but as it is related to a studied the ion influx using artificial cells with RMP. Ar-high waste percentage and massive yield loss the industry tificial cells allow us to exclude the biochemical processes is looking for alternatives. Moreover, the ripening status associated to the ion influx and outflux for discussing the and harvesting time are very important parameters to con- mechanism of Ca2+ influx. This paper mainly describes sider, which makes the peeling process even more complex. the production of RMP in artificial cells and the role of A promising technology for peeling is the use of PEF tech- RMP in the Ca2+ influx. nology. Materials and Method: We used giant unilamellar vesicles In this work, the influence of different PEF settings, de- (GUVs) as artificial cells. The w/o emulsion method was termined as specific energy, were studied on peeling effect-used to prepare GUVs, which contains potassium (K+) iveness and chemical properties of the kiwi using statistical ions inside. The membrane potential was generated for analysis. Experimental results showed that the specific simulating RMP in living cells by using the K+ ionophore energy input has a significantly influence on kiwifruit spe- valinomycin to drain internal K+ by the concentration cific peeling force. For applied PEF energy Wspec about gradient. The membrane potential was controlled by the 0.5 kJ/kg, the kiwifruit skin can be removed with a low initial intracellular K+ concentration. Fluorescent probe force indicating an improved peeling. A further increase for Ca2+, Fluo-8, contained only inside GUVs enables us in PEF specific energy did not change the peeling ability to detect Ca2+ influx following the PEF exposure. A significantly. The results demonstrated that the proposed single PEF (10 µ s, 5 kV/cm) was applied to GUVs with PEF treatment of kiwifruit had less peeling loss compared or without the membrane potential, and the amount of to untreated (control) sample. Low energy level of PEF Ca2+ uptake was evaluated by the brightness of fluores- treatment (0.3 kJ/kg) did not show an effect on the peel- cent intensity of Fluo-8. ing. Increasing the specific PEF energy, thereby increas- Result and Discussion: The observed rate of change of ing the number of pulses, improved peelability and de- fluorescence brightness values in the GUVs before and crease mass loss for achieving the desired peeling perform- after pulse application showed that the rate of change ance and product quality. was 2% for the GUVs without membrane potential, while PEF treatment also influenced the physical properties of 18% was observed for the GUVs with membrane poten- the kiwifruit at a specific energy of 1 kJ/kg. The pH tial. Only a slight increase in the intracellular Ca2+ con- values of the kiwi flour were significantly different to the centration was detected in the GUVs without membrane untreated control, as the pH of the PEF kiwi skin and potential. This slight increase is considered being owing to PEF bagasse were lower. Moreover, the water activity the diffusion through the electropermeabilized membrane. of the PEF treated skin was significantly higher than On the other hands, significant Ca2+ increase occurred in the untreated control skin samples. No significant differ- the GUVs with membrane potential. Besides, the increase ences were observed in the L*a*b*values. The influence of in the intracellular Ca2+ concentration was proportional the PEF treatment on the phenolic compounds was ana- to the membrane potential. These results clearly indic- lyzed and the PEF treated samples tended to have higher ate the membrane potential plays a significant role in the amount of phenolic compound than the untreated samples. Ca2+ influx rather than the diffusion. The antioxidant capacity of the PEF treated bagasse was These results will provide with the knowledge to under- significantly higher compared to the not treated bagasse. stand better the substance mobilization through the cell PEF treatment can positively influence the peeling and membrane following the PEF exposure and to find the chemical properties of kiwifruit. Depending on the ap- optimum parameters for the present various PEF applic- plied energy, different benefits can be generated and can ations. have an influence on the peeling process and on the health 177 promoting substances in kiwifruit juices. perature has a significant advantage in the preservation of heat-sensitive compounds (carotene, polyphenol,etc.). PO-040 The dried sample pretreated by PEF could better retain Pulsed Electric Field Treatment of Seeds with Im- the initial product color and had a reduced color devi- provement of Seed Vigour ation after rehydration. In regards to the frying process, Gülsün Akdemir Evrendilek 1, Bahar Atmaca2, Nurullah the application of the PEF treatment showed not only an Bulut1, Sibel Uzuner3 advantage in terms of the frying time but also in terms 1Bolu Abant Izzet Baysal University, Turkey of oil content absorbed and acrylamide content produced. 2Mardin Artuklu University , Turkey The oil and acrylamide content of PEF treated sample was 3Izmir Institute of Technology, Turkey lower compared to untreated ones. Moreover, the combin- Crop productions with the use of chemical additives ation of the PEF pretreatment and hot air pre-drying (or have intensified in order to satisfy the rapidly growing vacuum pre-drying) showed a synergistic efficiency on fry-human population. Chemicals help to boost agricultural ing time and also in terms of oil content absorbed. yields; however they cause environmental pollution and health problems in agriculture workers as well as human PO-050 consume these products. Seeds not only pass on genetic Frequency Domain Dielectric Spectroscopy of materials but also transmit different pathogens such as Gram-negative Bacteria Exposed to Pulsed Elec- bacteria, fungi and viruses. Seed-borne pathogens have tric Fields been controlled by chemicals, or by such treatments such Atsushi Tanaka, Sunao Katsuki, Hirotaka Nuki, Ryuya as heat, electrons, natural antifungal products or biolo- Kimura, Kaichi Miyazaki gical control. Although these methods appear to be ef- Kumamoto University, Japan fective in controlling seed-borne fungal diseases, there are Pulsed high electric field (PEF) has been studied as drawbacks such as fungicide resistance and incomplete effi- a non-thermal sterilization method for liquid foods. PEF ciency. Technological improvements and alternative meth- causes significant morphological and functional changes in odologies for efficient disease control are therefore needed. cells and tissues. The effect of PEF on bacteria is depend- Efficacy of pulsed electric fields on inactivation of endo- ent on the conditions of PEF, such as field strength, pulse genous bacteria and mold and yeast in addition to ger- width, and frequency of application, as well as the envir- mination rate (GR), normal seedling rate (NSR), and res- onment, such as temperature and conductivity. istance to cold and salt stresses. It was revealed that in- Previously, the effectiveness of PEF was determined em- creased energy application provided more inactivation on pirically from the results of sterilization experiments and endogenous microflora of vegetable seeds and grains with experiments using fluorescence microscopes, making it increased GR and NSR. PEF treatment provided stronger very difficult to optimize conditions. Therefore, it is im- root formation with more developed seedling. Seedlings portant to analyze the physical properties of the effect of developed significant resistance to cold and salt stresses. PEF on bacteria and to investigate the optimal conditions It is concluded that applied electric fields improved seed for each application. We focused on a technique called im- vigour being as an viable alternative to chemical seed pedance spectroscopy, which measures the reflected signal treatment. against the incident signal and measures how the amp- litude and phase change on measured objects. In partic- PO-045 ular, frequency-domain analysis is characterized by high Application of pulse electric fields (PEF) on drying information accuracy and a wide range of frequencies that and frying processes of vegetables can be measured, since the information density is uniform Caiyun Liu 1, Eugene Vorobiev2, Nabil Grimi2 with respect to frequency. In this study, we have estab- 1University of Shanghai for Science and Technology, China lished a method for analyzing the physical properties of 2University of Technology of Compiegne, France PEF-applied bacteria using impedance spectroscopy. This work focuses on the effect of pulsed electric We proposed to use impedance spectroscopy to measure field (PEF) treatment on various drying and frying pro- the dielectric properties of bacteria, and investigated its cesses from vegetables (potatoes and carrots). Inter- effectiveness and applicable conditions. actions between different drying modes and pretreat- It was found that a measurement frequency of 1 MHz, a ment have been studied. The impact of PEF treat- bacterial concentration of 1010 CFU/mL or higher, and ment and pre-drying by hot air or pre-drying by va- a conductivity of 0.73 mS/cm or lower were required for cuum drying on frying kinetics and the quality of fried accurate measurements. products were also analyzed. PEF pretreatment results in Then, the impedance method was then used to measure electro-permeabilization of the cell membranes, which fa- the dielectric properties of the bacteria when the number vors the acceleration of mass transfer processes and phys- of times PEF was applied was changed. ical/chemical changes of the product during drying and Capacitance decreased and conductance increased before frying. The results showed that the drying time was signi- and after PEF application. This is thought to be due to ficantly reduced in all processes (hot air drying, microwave the decrease in the ability to store electric charge due to drying, vacuum drying). The advantage of the PEF treat-the formation of pores in the bacterial membrane and to ment was also manifested by a decrease of the internal the leakage of the bacterial contents. temperature of the product during drying. This lower tem- The electromagnetic field simulation was used to estimate 178 the physical properties of the bacterial membrane, and performed based on pre-defined optimum pre-extraction the conductivity increased significantly with the increase procedure. Isolation of the proteins will be carried out by in the number of applications, while the relative permit- mild heat and ultrafiltration/diafiltration. Isolation con- tivity did not change significantly. This is thought to be ditions will be defined according to digestibility and flavor because the change in conductivity was so dominant that profile of duckweed protein isolate. the change in permittivity was almost negligible. PO-058 PO-053 The induction of cell electrosensitization or elec- Effect of pulsed electric fields and ultrasound on trodesensitization with application microsecond protein extraction, yield and techno-functionality pulsed electric fields from duckweed (L. minor and L. gibba) in an in-Paulius Ruzgys 1, Vitalij Novickij2, Neringa Barauskaite1, door farming cultivation system DIana Navickaitė1, Saulius Šatkauskas1 Patricia Maag 1, Özlem Özmutlu Karslioglu1, Claudia 1Vytautas Magnus University, Lithuania Siemer2, Alica Lammerskitten2 2Vilnius Gediminas Technical University, Lithuania 1University of Applied Sciences Weihenstephan-Triesdorf, In- Introduction: Nowadays the phenomenon of electro- stitute of Food Technology, Germany poration is being widely applied. However, it still narrows 2Elea Technology GmbH, Germany down to the transient (reversible electroporation) or An interdisciplinary Team of scientists from food tech- permanent (irreversible electroporation) cell membrane nology, horticulture and biotechnology from HSWT are permeability increase as a result of electric field applic- establishing the basis for optimal cultivation conditions ation. This phenomenon is fundamentally researched for duckweed in an indoor farming cultivation system. for a few decades yet still some additionally triggered In corporation with industry partner Elea Technology phenomena are still not fully understood among research-GmbH, gentle extraction methods will be established for ers. One of such phenomena is electrosensitization. This optimal protein extraction assisted by physical processes phenomenon indicates the change of cell sensitivity to the like pulsed electric fields (PEF) and ultrasound to observe electric field when cells were priorly affected by electric their implications on cell disruption to release protein from field. Usually, the effect of electrosensitization is being re- duckweed (L. minor and L. gibba). The project aims to searched when the time lag in between nanosecond pulsed investigate the effect of cultivation parameters (light spec- electric fields is in the sub-millisecond range. However, tra/intensities and nutrient solutions), innovative extrac- the electrosensitization has been discussed in microsecond tion methods and their implications on techno-functional pulsed electric field range with a time lag ranging from properties of duckweed protein. As new alternative pro-seconds to minutes. However, the term of “cell memory” tein source for food applications, duckweed has an increas- phase was used rather than electrosensitization. Here ing potential with its protein content, protein density and we investigate the process of the electrosensitization amino acid composition. phenomena in the time lag range that could be considered Different light spectra and intensities as well as compos- as “cell memory”. We have found that depending on the itions of macro- and micronutrients of the nutrient solu- number of applied electric pulses one can gain electro- tion are tested for their effect on protein composition in sensitization as well as desensitization. Indeed, we have duckweed species L. minor and L. gibba, with the aim of found a statistically significant changes (negative and optimizing protein content, yield and characteristics of the positive) in the percentage of electroporated cells when final protein extract. minutes post-application of sensitizing electric fields. As the main component of ”leaf proteins” RuBisCo pro- Methodology: Electrosensitization experiments were done tein, more specifically, an enzyme that plays an import- with Chinese hamster ovary cells. Electroporation has ant role in carbon fixation in all green plants, the ex- been performed with laboratory-made stainless steel traction process is divided into three processing steps: plate electrodes with 2 cm gap. All used pulsed electric pre-extraction, separation of pigments/antioxidants and fields that were used here were in between 1200 and protein isolation. Pre-extraction of freeze-dried and sub- 1400 V/cm. Electroporation was done with sucrose sequently ground duckweed powder as well as fresh mater- and glucose-based phosphate electroporation media at ial is carried out using PEF to break down plant cell mem- osmolarity at 270 mOsm, pH 7.1 and conductivity at brane by gentle cell disintegration. Ultrasound is used al- 0,1 S/m. The electrosensitization treatment experiment ternatively to support pre-extraction. setup was to apply electroporating electric field to PEF will be applied directly on the cell system and com- incubate cells at a particular time and electroporate pared in terms of its performance on increased protein ex- them again. Electroporation was evaluated by propidium traction yield and effects on techno-functional properties. iodide electrotransfer (40 �M). While ultrasound subsequently assists in the disruption of Results and discussion: We have found that after sensit- the particles and in the case of ground duckweed, resulted izing electric pulse and incubation in a range of minutes in smaller particles than 100 µm for 97% of the particles one can change cell response to electric fields by changing size distribution. This indicates cell disruption of plant their size. Depending on the applied electric field cells cells, which are normally 100-300 µm in size and resulted shrink or increase in size. Under these conditions, in about 10-15% increased protein solubility compared to viability was not significantly affected. Nevertheless, the untreated reference. Isolation of proteins will then be such change in cell size greatly influenced electroporation 179 efficiency, hence cells get desensitized or sensitized to the Our interest is also the development and testing of new electroporation. PFA catheter geometries. Two new designs show that it is theoretically possible to create lesions of the same size Funding: This work was supported by grant Nr. but with lesser current flow thus more suitable for a safe S-MIP-19-13 from the Research Council of Lithuania. PFA procedure. Parametric analyses revealed the impact of electrode distance on lesion size and temperature dis- PO-063 tribution. Application catheter parameters affecting PFA outcome based on simulation PO-066 Roman Kafka, Veronika Novotna, Martin Hemzal Study of surface oxides modifications on nitinol Brno University of Technology, Czech Republic electrodes during electroporation protocols and This abstract focuses on pulsed-field ablation (PFA), possible consequences which is a form of irreversible electroporation (IRE). In Théo Le Berre 1, Pauline Bregigeon1, Marie Frénéa Robin1, Andrei Sabac1, Charlotte Riviere2, Fréderic Prat3, Julien contrast to other ablation techniques, e.g. radiofrequency Marchalot1 ablation (RFA), innovative PFA has a big potential for 1Univ Lyon, Ecole Centrale de Lyon, INSA Lyon, Université cardiac ablation since it is tissue-selective and with min- Claude Bernard Lyon 1, CNRS, France imal danger to patients. 2Institut Lumière Matière, Claude Bernard Lyon 1 Université, In this work, we present 3D FEM simulations of ablation CNRS, France inside the heart with a focus on various parametric ana- 3University Paris-Cité, INSERM U1016 and AP-HP, France lyses, including electric field and temperature distribution in the tissue. The simulation is based on the usage of The purpose of this study is to evaluate the efficiency standard RFA catheters because, at this time, the com- and the possible risk of the utilization of nitinol as an mercial availability of PFA cardiac catheters is very lim- active electrode for electroporation (EPN). ited. Nitinol is widely used in medical devices for its particular The 3D FEM model includes a simple cylindrical cath- ultra-elasticity and shape-memory properties. It is also eter with a tip electrode which is in contact with heart considered as bio-compatible due to the TiO_2 layer tissue in a form of a cube. The electroporation pulses generated on the surface during processing. Different were set to be a 100 us log with a frequency of 1 Hz. We surface modification procedures are known to improve the were changing blood velocity on a Y-axis, the angle of the thickness of this layer in order to ensure the long term catheter-tissue contact, catheter pressure (electrode sur- bio-compatibility of passive implants [1]. face in contact with blood), as well as pulse parameters However, as this alloy is now commonly used as active such as voltage, pulse length and the time gap between electrode for different purposes, knowledge of the beha-pulse bursts. vior of nitinol in the body must be updated. It has been The simulations show the biggest Joule losses near the proven safe and efficient to use as micro-electrodes in active electrode in the blood, where is the highest current neural prosthesis [2], but its suitability as an electrode for density. It shows that blood velocity in the heart is suffi-EPN needs to be ensured. Indeed, the delivery of high- cient for cooling the electrode if it is not surrounded by the intensity square pulses (typically 1ms and 1000V/cm) tissue when pressed downward (no contact with blood). causes electrochemical reactions at the surface of the elec- However, the higher the voltage higher the temperature trodes, in particular metal releases due to the oxidation and the contact area must be cooled down. of the anode. This phenomenon has been observed in On the other hand, we show that with larger contact with vitro with aluminum [3] and stainless steel electrodes [4], blood, the electric current flow is bigger. That means a with the release of aluminum and iron ions respectively. greater danger for patients. There is a need to find a good In this case, the possible release of nickel ions into the compromise between the value of electric current and tem- body might be a problem for clinical use. Furthermore, perature where both of them are sufficiently low. as the TiO_2 layer on the nitinol is less conductive than In real experiments, the position of the catheter may not stainless steel, a drop in efficiency could be expected. be exactly perpendicular to the tissue but maybe at some We have developed a device enabling us to test the use angle. We show that with the perpendicular catheter- of different electrodes on a spheroid population. The tissue position, there is the lowest temperature rise. Fur- experimental comparison between nitinol and stainless ther, when the electrode is tilted in the direction of blood steel did not exhibit any significant difference in terms flow the cooling is better than when tilted in the opposite of EPN efficiency in this configuration. We then studied direction. The temperature during experiments is meas- the surface condition of differently processed nitinol wires ured with a thermocouple on the tip of the catheter. The after their use in EPN protocols in a conductive solution. simulations show that this kind of measurement is not op- We compared qualitatively with SEM images a heat- timal and needs improvement. The size of created lesions treated nitinol wire and a nitinol electrode, the latter in the myocardium is also discussed and compared to those being previously cleaned of its surface oxide and then from real experiments. The results show that the final ab- oxidized through an electroporation protocol. This con- lation outcome is affected by many variables which need firmed that the surface oxide thus formed is significantly to be taken into account for a more precise and reliable different from the one formed through heat-treatment. solution. We are currently extending this comparison to wires 180 protected by an oxide layer and used for EPN. We are PO-072 also using EDX and Raman spectroscopy to compare Could the presence of a coronary stent provoke quantitatively the exact surface composition of the distortion of the electric field and any thermal side samples. The presence of nickel releases in the solution effect during epicardial pulsed field ablation? In- after EPN will be investigated with Hach Lang test kits. sights from an in-silico computational modelling In the future, different EPN protocols will be tested, as Ana González-Suárez 1, Barry O’Brien2, Martin the use of shorter pulses seems to reduce metal releases [3]. O’Halloran1, Adnan Elahi1 1Translational Medical Device Lab, National University of Ire- [1] Shabalovskaya et al. (2008) Acta Biomater, land Galway, Ireland 10.1016/j.actbio.2008.01.013 2AtriAN Medical Limited, Ireland [2] Wong et al. (2016) 38th Annual International Confer- Background and objectives: Pulsed Field Ablation ence of the IEEE EMBC, 10.1109/EMBC.2016.7591718 (PFA) has been proposed as a non-thermal energy to treat [3] Vižintin et al. (2021) Bioelectrochemestry, atrial fibrillation by an epicardial approach, for ablation of 10.1016/j.bioelechem.2021.107798. ganglionated plexi. This energy provokes permeabilization [4] Rodaitė-Riševičienė et al. (2014) IEEE Transactions of the cell membrane (creation of pores), leading to celon Plasma Science, 10.1109/TPS.2013.2287499 lular homeostasis disruption and cell death. We recently checked the safety of this energy, which is totally delivered PO-069 at the target epicardial sites without damaging the un- Persistent membrane depolarization following con- derlying myocardium and the adjacent organs (lungs and ventional electroporation depends on temperature oesophagus). However, there is still no assessment on how and is influenced by ion channel blockers the electric field in the target zone could be affected by the Anja Blažič, Lea Rems presence of pre-existing implants, such as coronary stents University of Ljubljana, Faculty of Electrical Engineering, – neither is there an understanding of possible undesirable Slovenia thermal effects. The aim of this work is to develop compu- All cells maintain an electric potential difference across tational models to assess the electric field and temperature their plasma membranes, which results from the differ- distribution on the epicardium (target), upon application ences in membrane permeabilities for potassium, sodium, of high-intensity electric field pulses (PEFs). calcium, and chloride ions. This potential difference is Methods: Coupled electrical-thermal computer models called the resting voltage or resting membrane potential based on the heart anatomy including epicardial fat with and is maintained by a system of ion channels and pumps. the left circumflex coronary artery, myocardium and blood By convention the resting voltage is negative, meaning were built. The stent was placed within the coronary that the cell interior is electrically more negative com- artery. Different positions of the stent with respect to the pared to its exterior, and the membrane is considered hy- ablation device were also assessed. A period of latency perpolarized. The value of the resting voltage changes (i.e. a period of time without applying PEF energy) was dynamically with the cell cycle and has an important bio-taken into account after the application of PEFs to assess logical function by controlling the activity of various mem- if there was any collateral heating in the target site. PEF brane proteins. When cells are electroporated, their mem- parameters such as voltage profile, pulse width, inter-pulse brane voltage becomes disrupted and remains depolarized interval and number of pulses were those already used pre- for several minutes after pulse exposure. In this study we clinically and clinically to ablate epicardial ganglionated aim to gain a better understanding on the mechanisms of plexi. prolonged membrane depolarization upon electroporation. Results: The electric field distribution on the epicardium By studying different cell lines, including Chinese hamster was practically the same with and without having a stent, ovary cells and human glioblastoma cells, stained with a even at a very close distance from the ablation device (0.25 potentiometric dye, we show that depolarization markedly mm). The presence of a stent has not provoked any independs on the ambient temperature (room temperature creased heating after the latency period, with the temper- vs. 37°C). Not only do the cells remain depolarized for a ature being 37.5°C in the zone around the coronary artery longer period of time when electroporated at room temper- with and without stent. ature, they are often slightly depolarized even before elec- Conclusions: Computational results showed that the pres- troporation compared to conditions under 37°C. Further- ence of a coronary stent near the ablation site does not more, by using different ion channel blockers we show how cause any distortion of the electric field nor any thermal membrane depolarization in not purely the consequence of side effect in the target. ion leakage through nonselective pores in the permeabil- ized membrane, but involves the opening and closing of PO-077 membrane ion channels. We discuss the potential biolo- Downstaging of portal vein tumor thrombus from gical consequences of these findings, from the aspect of Hepatocellular Carcinoma with Electrochemother- cell handling for basic studies of cell electroporation, to- apy as bridge to liver transplantation wards the possibility of controlling cells’ susceptibility to Luciano Tarantino 1, Emanuele Balzano2, Giuseppina electroporation by pharmacological agents. Busto3, Aurelio Nasto4, Sara Bortone3, Paolo Tarantino5, Ric- cardo Aurelio Nasto2 1A.Tortora Cancer Hospital, Italy 181 2Pisa University, Italy enabling measurement of MT stability via absorbance in 3PO Pagani - ASL Salerno, Italy real time. Moreover, we follow the morphological changes 4PO Polla - ASP Salerno, Italy of MTs after nsPEF treatment with several imaging 5Dana Farber Cancer Institute - Boston (MA), United States techniques such as phase contrast microscopy, differential S.R. 53 years. January 2009 : HCV-related cir- interference contrast microscopy, holographic microscopy rhosis, Child-Pugh A5 class, EGDS no aesophageal and atomic force microscopy. These techniques are Varices. No important comorbidities. Treated with PEG- applicable also for the exploration of the effects of intense IFN+Ribavirin (march-november 2009) with subsequent subTHz electric field on tubulin and MTs. Our results sustained virologic response . HCVRNA absent overtime contribute to development of novel electromagnetic . October 2016 :CT detected small HCC nodule in the methods for modulating function of biomolecular matter VIII segment (diam.=12 mm). Treated with US guided at nanoscale. RF-ablation. November 2016 CT: complete necrosis. Un- fortunately, the patient dropped out US and CT follow-up Authors acknowledge support from Czech Science controls.September 2018: asthenia and weight loss. CT Foundation GX20-06873X. showed a large tumor infiltrating V-VII-VI segments and PO-076 complete PVTT of right portal vein and its branches . Sur- Nanosecond Pulse Water Surface Discharge for gical Consultation excluded indication to Liver resection Water and Wastewater Treatments and OLT . 23 october 2018: ECT of a large peri-hilar area Mijanur Rahman 1, Nushin Hosano1, Ryo Kaida1, Mit- of the tumor including the PVTT. 1 and 3 months post- suhiko Sato1, R. Ruma2, Hamid Hosano1 treatment CT showed complete necrosis and retraction of 1Kumamoto University, Japan the thrombus and residual viable tumor in the peripheral 2Dhaka University of Engineering and Technology, Bangladesh portion of the right lobe . Therefor, the patient was ree- valuated for OLT and considered eligible in waiting list . Underwater discharge and its dynamics have been March 2019: CT showed no perihilar or portal vein recur- studied with great interest over the past few decades. In rence and distant progression in the right lobe . March this regard, one of their most noteworthy applications has 2019 : Trans-arterial-Radio-therapy (TARE) of the right been drinking and wastewater treatments, as they can dir-lobe. Post-treatment CT demonstraded no perihilar or ectly affect our health and environment. While underwa- portal vein recurrence and extensive necrosis of the resid- ter electric discharge has been extensively studied, the re- ual tumor . December 2019: CT demonstrated several quired knowledge regarding the phenomenon of water sur-recurrences of HCC infiltrating the VI and VII segment face discharge is inadequate yet. This paper reports on the . Howewer no recurrence was observed at hepatic hilum physical characteristics of water surface discharge, includ- and in portal vessels . Therefore, on February 2020 the pa- ing plasma production and propagation, and simultaneous tient received OLT. At 2 years follow-up, no complication generation of shock waves in water and air. Nanosecond or recurrence or liver disfunction have been observed. pulsed electric fields on the surface of water were gen- erated using a magnetic pulse compression (MPC) unit, PO-078 comprising of a charger and a control unit. A point to Intense electric field effects on microtubule sys- plane electrode technique along with positive pulse was tems employed for water surface discharge. The discharge phe- Daniela Guadalupe Blanco Campoy, Tomáš Zakar, Michal nomenon and shock waves propagations were visualized Cifra using a time-resolved shadowgraph scheme by employing Institute of Photonics and Electronics of the Czech Academy an ultra-high-speed framing camera. Different gap dis- of Sciences, Czech Republic tances between the positive electrode and the surface of Understanding electromagnetic field-biomatter inter- water as well as different solution conductivities were con-action is crucial for the development of novel diagnostic sidered for the experiments, to confirm and understand and therapeutic methods as well as for novel procedures in the physical mechanisms at different environmental con- bio-nanotechnology. Tubulin proteins, the building blocks ditions of water and wastewater treatments. The results of microtubules (MTs) have exceptionally high structural confirm that nanosecond water surface discharge is an ef- electric charges and dipole moments. Consequently, it is fective and highly efficient method for decontaminating probable that MTs could be susceptible targets to electric water. field through which one could modulate the function of PO-004 biological systems. Our molecular dynamics simulations Endoscopic calcium electroporation as a palliative showed that intense nanosecond pulsed electric field treatment for inoperable colorectal cancer (nsPEF) opens the MT lattice on the nanosecond time Malene Broholm 1, Mustafa Bulut2, Rasmus Vogelsang2, scale and this depends on the electric field strength and Mikail Gögenur2, Ismail Gögenur2 temperature. 1Koege Hospital, Denmark We present follow-up experimental work which aims 2Zealand University Hospital, Denmark to verify these computational predictions. The work is centered around protocol development and systematic Background: Colorectal cancer is one of the most com- testing of a new experimental system which delivers mon malignancies worldwide cancer. Despite advances ˜ MV/m field strength nsPEF to MTs in vitro while within the field of colorectal cancer, a number of patients 182 can only be offered palliative treatment due to metastatic or inoperable colorectal cancer. Health-related quality of life is of great importance in this fragile group. In some cases, treatment options are limited as the patient may not be suitable for chemotherapy or radiotherapy due to comorbidities, making tumor-related symptoms very challenging in these cases. Calcium electroporation is a promising anticancer treatment, which has been shown to induce tumor cell necrosis. Studies show that cancer cells are vulnerable to the treatment, whereas normal cells are more resistant to calcium electroporation. An advantage is the simplicity of the procedure, facilitating use in a palliative setting. Method: In this case report, patients were unfit for standard palliative treatment and were offered endoscopic calcium electroporation as an experimental treatment. Treatments were performed under propofol sedation and patients were discharged within a few hours after the procedure. Calcium chloride was injected intratumorally, followed by electrical pulses delivered through the En- doVE device. The device was connected to the ePORE generator. Results : Case 1 was an 80-year old male with non- metastatic rectal cancer. The patient rejected surgical resection and chemotherapy. The patient received CaEP as an experimental treatment in May 2020 and additional treatments were performed in August 2020 and February 2021. Endoscopic assessment showed visual tumor regression after CaEP. Case 2 was an 88-year old male with sigmoid colon cancer. The patient suffered from anemia due to bleeding from the tumor. The patient was offered CaEP in July 2020 with repeated treatments in February 2021 and August 2021. Currently, the patient has been followed for 20 month and has stable hemoglobin levels. Case 3 was a 70-year old women with recurrent rectal cancer. The patient rejected further surgical interven- tions. CaEP was performed in February 2022 followed by electrochemotherapy in March 2022. Follow-up MRI showed tumor regression. Case 4 was a 76-year old male with sigmoid colon cancer. The patient had limited treatment options due to several comorbidities. He was treated with CaEP to limit further progression of stenosis. The patient reported relief of symptoms. Conclusion: Endoscopic calcium electroporation is a safe and feasible procedure for colorectal cancer and may be a valuable treatment modality for patients with limited treatment options. 183 Authors’ Index A Balucani, Marco 97, Bregigeon, Pauline 103, 180, Abal, Carolina 173, Balzano, Emanuele 140, 90, 181, Bresolin, Silvia 51, Abdelnabi, Rana 142, Baragona, Marco 129, 171, 169, 163, Bressler, Yael 159, Achiam, Michael Patrick 140, 106, Baranska, Alicja 168, Brint, Beth 62, Adams, Volker V.A. 81, Barauskaite, Neringa 165, 158, 179, Briz, Pablo 135, 157, 149, Adusumilli, Prasad 95, 75, Broholm, Malene 182, 107, Aganovic, Kemal 100, Barceló, Adrián 173, Browning, Erica 69, Agasha, Atharva 79, Barlow, Zeke 171, Bruun, Niels Henrik 115, Agerlin Petersen, Mikael 170, Barra, Ana 101, Bulut, Nurullah 178, 59, Agger, Ralf 94, Barranquero, Cristina 154, Bulut, Mustafa 107, 182, Ahrné, Lilia 144, Bastrup, Freya 174, 132, Bulysheva, Anna 113, Akdemir Evrendilek, Gülsün 59, 178, Batista Napotnik, Tina 150, Burbach, Brandon J. 95, Alberola, Geraldine 57, 88, Bazancir, Laser Arif 106, Burdío, José Miguel 157, 135, 149, Algazi, Alain 108, 69, Bazzolo, Bianca 88, 122, Busto, Giuseppina 181, 140, 90, Alinezhadbalalami, Nastaran 54, Becker, Sid ?? , Byron, Christopher 93, Allaf, Karim 169, Beebe, Stephen J. 76, ?? , 126, Błasiak, Piotr 128, Allen, Irving C. 93, Beinart, Roy 87, Alrasheed, Taiba 174, Belingar, Karolina 84, C Alvarez-Lanzarote, Ignacio 73, 58, 83, Bella, Zsolt 174, Cahan, Rivka 169, 112, 154, Bellard, Elisabeth 57, 57, 63, Calvel, Anne 150, Amorós-Figueras, Gerard 86, 160, Bellard, Elisabeth 68, Camera, Francesca 97, Andersen, Christina 107, Belleggia, Luca 47, Cameron, Andrew D. 143, André, Franck M. 99, 110, 114, 97, Ben-Elazar, Shay 110, Campana, Luca G. 132, 117, 162, 97, Benassi, Barbara B. 162, 99, 97, Campana, Luca G. 117, 40, 164, Andre, Franck 98, Bendix, Maura B. 62, Campbell, Delcora A. 143, Aoustin, Emma 81, Bensalem, Sakina 65, Campelo, Sabrina N 115, Apollonio, Francesca 97, 99, Bergues Cabrales, Luis Enrique 56, Campelo, Sabrina N. 91, Arbel, Gil 87, Bertino, Giulia 117, Cani, Alice 51, Arguello, Ricardo 110, Bertino, Giulia 116, Canton, David A. 69, 108, Arnal, Carmen 154, Berzosa, Alejandro 112, 58, Caramazza, Laura L. 162, 99, Arnaud-Cormos, Delia 137, Besombes, Colette 169, Carpentieri, Serena 102, 74, 73, Arnaud-Cormos, Delia 125, Bessières, Delphine 123, Carullo, Daniele 67, Aryana, Arash 80, Bieżuńska-Kusiak, Katarzyna 107, Casabella-Ramon, Sergi 86, Asirvatham, Samuel J. 87, 106, Casciati, Arianna 51, 129, Astráin Redín, Leire 83, Bikson, Marom 78, Cassari, Leonardo 156, 164, Atmaca, Bahar 178, Bindrich, Ute 100, 83, Castellvi, Quim 81, Atmaca, Bahar 59, Birk, Tanya 84, Cebrián, Guillermo J. 83, Atzampou, Maria 104, Bischof, John C. 95, Ceramella, Jessica 74, Augimeri, Giuseppina 74, Bixler, Joel N. 70, 71, Cervinka, Dalibor 80, Aurisicchio, Luigi 85, Bixler, Joel 126, Cesaro, Cristiana 47, Aurisicchio, Luigi 84, Bizzarri, Giancarlo 140, Chabot, Sophie 68, Axelrod, Robert 66, Blanco Campoy, Daniela Guadalupe Chafai, Djamel E. 78, Aycock, Kenneth N. 76, 124, 115, 91, 182, Chen, Xinhua 136, 54, Blažič, Anja 181, Chen, Dongjiang 75, Azzarello, Giuseppe 130, Boc, Nina 139, Chittams-Miles, Alexandra E. 75, Bonnafont, Thomas 123, 114, Á Bonofiglio, Daniela 74, Chole, Ajay 51, Ágoston, Dóra 161, Bortone, Sara 90, 181, 140, Choromańska, Anna 57, 106, 107, B Bošnjak, Maša 139, 139, 132, 84, 77, 108, 149, Baczyńska, Dagmara 165, 128, 150, Boukany, Pouyan 104, Christensen, Kyle 113, Badovinac, David 139, Boukany, Pouyan E. 78, Chwikowska, Agnieszka 108, Bakute, Neringa 89, 53, Bozic, Tim 175, 141, 68, Cifra, Michal 78, 78, 122, 182, Balazinski, Martina 66, Bracken, Alison 131, Cioce, Mario 110, Balevičiūtė, Austėja 64, Braeken, Dries 103, Clark-Deener, Sherrie 93, Baltás, Eszter 161, Brazionyte, Elinga 89, Clover, A. James P. 46, 131, 131, Baltas, Eszter 174, Brecelj, Erik 139, Coffey, Kenneth 87, 185 Colavitti, Giulia 133, Djokić, Mihajlo 140, ?? , 139, 139, 91, Gehl, Julie 107, 59, 117, 106, 105, 90, Colella, Micol 97, Dolciotti, Noemi N. 99, 162, 130, 140, 174, 132, Collin, Annabelle 133, 158, Dong, Shoulong 96, Genovese, Jessica 159, 167, 122, 145, Comiotto Alles, Martina 177, Donsì, Francesco 67, Gepstein, Amira 87, Compagnone, Mirco 84, Doumard, Layal 50, Gepstein, Lior 87, Conconi, Maria Teresa 88, 122, 110, Drozdz, Marek M. 143, Geukens, Nick 142, 156, 164, Drusch, Stephan 83, Geukens, Nick 142, Conforti, Antonella 84, Dubińska-Magiera, Magda 150, Ghandehari, Hamid 176, Consales, Claudia 99, 97, 162, 97, Dubuc, David 150, Giancaterino, Marianna 101, Cooper, Itzik 99, 159, Duckert, Bastien 103, Gianlorenzi, Isabella 51, Cornelis, Francois 54, Duffy, Maeve 51, Giardullo, Paola P. 162, 99, Coutermarsh-Ott, Sheryl 93, Duncan, Josie L. 171, Gibbons, Justin 56, Craviso, Gale L. ?? , Gibot, Laure 127, 50, E Cuypers, Marie-Lynn 142, Girkontaitė, Irutė 64, 69, 118, Eden, Kristin 93, Cvetkoska, Aleksandra 92, Gögenur, Ismail 182, 140, 107, Edhemovic, Ibrahim 139, Cvetkovic, Nada 163, Gögenur, Mikail 182, Edwards, Michael R. 93, Golberg, Alexander 50, ?? , Č Egeland, Charlotte 106, 140, Golberg, Alexander 110, Čemažar, Maja 62, 84, 77, 139, 139, El Hajj, Rachelle 169, Golzio, Muriel 57, 63, 68, 151, 102, 88, 68, 143, 38, 141, 175, Elahi, Adnan 181, 57, 137, 121, 89, Elez-Martinez, Pedro 73, 152, 155, d Gómez, Mario 119, 124, 160, Emanuel, Efrat 169, d’Avila, André 80, Gómez Galindo, Federico 170, 144, 45, de la Rama, Alan 80, F 112, 100, del Barrio Montañés, Alicia 52, Falk Hansen, Hanne 107, Gómez-Caravaca, Ana María 100, Fauvart, Maarten 103, Gonzalez, Dacia 109, 136, D Fawcett, Timothy J. 61, González Ballester, Miguel A. 124, D’Elia, Fabio 167, 159, Fazio, Vito Michele 110, Gonzalez Cuevas, Juan A. 110, Dadan, Magdalena 168, Fernandes, Romain 114, 98, González-Suárez, Ana 181, Dall’Olmo, Luigi 164, Fernandez, Juan Manuel 92, Goto, Shin 49, Dandurand, Jany 50, Ferrari, Giovanna 102, 145, 73, 67, Gouarderes, Sara 127, 50, Daniels, Dianne 159, 99, 144, 74, Graybill, Philip M. 79, 77, Daud, Adil 108, 39, 69, Ferreira, Paula 101, Grenier, Katia 150, Davalos, Rafael V. 54, 38, 124, 171, Feyissa, Aberham Hailu 167, Grimi, Nabil 152, 169, 178, 76, 77, 91, 93, 79, 115, Fisher, Daniel 69, Grischke, Eva Maria 130, Dávalos, Alberto 154, Fisker, Rune Vincents 115, Group, The INSPECT 116, 131, David, Kailee 54, 115, Flahaut, Emmanuel 88, Gruszka, Vincent 89, Davies, Ilan Wyn 61, 53, Flak, Rasmus Virenfeldt 94, 115, Grynberg, Dvora 79, De Aguiar Saldanha Pinheiro, Ana Florentin, Marcos 110, Gudvangen, Emily 55, 81, 113, 70, 98, 167, 159, Foligni, Roberta 47, Guerra, Jose M. 86, 160, De Cillis, Alfredo 105, Fontana, Sara S. 162, 99, Guerra-Hernández, Eduardo Jesús De Robertis, Mariangela 110, Ford, Patrick F 62, EJGH 74, De Simone, Paolo 140, 90, Forzan, Michele 122, Guo, Siqi 76, ?? , De Terlizzi, Francesca 130, 132, Fourquaux, Isabelle 50, Gusbeth, Christian 64, 45, De Vleeschauwer, Stéphanie 142, Francis, Michael 113, Guziński, Maciej 153, Debarbieux, Franck 130, Frandsen, Stine 105, 107, DeCaro, Alexia 151, 57, 57, H Franqueville, Laure 103, Declerck, Paul J. 141, 142, 142, Haberkorn, Iris 66, Frejlich, Ewelina 153, Delemotte, Lucie 176, Haberl-Meglič, Saša 58, Frénéa Robin, Marie 180, 103, Delso, Carlota 112, 58, Håkansson, Åsa 112, Frey, Wolfgang W. 112, 64, Denis de Senneville, Baudouin 123, Han, Mia 108, 69, Fuchs, Christine 117, Dermol-Černe, Janja 135, 157, Hancock, Chris 61, 53, Fujimori, Masashi 78, Dervisis, Nikolaos 93, Hansen, Jim 88, Fütterer, Jurgen J. 60, Detlefsen, Sönke 94, Hata, Cary 80, Dettin, Monica 88, 110, 156, 122, 164, G Havelka, Daniel 78, DeVos, Amanda N. 118, Gabay, Batel ?? , Hayward, James A. 84, Dewilde, Maarten 142, Gajewska Naryniecka, Agnieszka 165, Heinz, Volker 83, 100, Di Barba, Paolo 122, Gančytė, Greta 48, Hejc, Jakub 80, Di Prata, Claudia 130, Garbyal, Rajendra Singh 106, Heller, Richard 61, 109, Diel, Diego G. 84, Garcia-Sanchez, Tomas 97, 86, 160, Heller, Eyal 87, 79, Dijk, Gerwin 130, 119, Heller, Loree 77, 56, Dimova, Rumiana 39, Gasljevic, Gorana 139, Hemzal, Martin 180, 170, 186 Hendel, Kristoffer 117, Juadjur, Andreas 100, 83, Kralj, Slavko 89, Herrero-Continente, Tania 154, Juarez, Tarsicio 69, Kramar, Peter 134, Hideghety, Katalin 139, Jurdana, Mihaela 175, Kramer, Thomas 52, Hoff, Andrew M. 61, Kranjc, Matej 145, 122, K Hoffmann, Balthazar M. BAH 59, Kranjc Brezar, Simona 77, 175, 141, Kadoya, Ryosuke 155, Hogenes, Annemiek M. 60, 68, Kafka, Roman 180, Hollevoet, Kevin 141, Kreische, Marco 66, Kaida, Ryo 182, 172, Hollevoet, Kevin 142, Krust, Damaris 64, Kajiwara, Kenshi 155, Hollevoet, Kevin 142, Kulbacka, Julita 128, 175, 118, 150, Kamensek, Urska 143, Holzinger, Steffen 80, 137, 138, 149, 107, 108, 165, Kanafusa, Sumiyo 112, 170, Horsáková, Iveta 154, Kulbacka, Julita 113, Kancirova, Eva 100, Hosano, Nushin ?? , 151, 176, 182, Kulbacka, Julita 153, Kandratsyeu, Aleh 52, 70, 172, Kulbacka, Julita 106, Kanstrup Fiehn, Anne-Marie 107, Hosano, Hamid 38, 176, 172, 182, 151, Kumar, Rupesh 156, Kapania, Rakesh 77, ?? , Kumar, Mayank 172, Houston, Aileen 62, Kasperkiewicz-Wasilewska, Paulina Kuncová, Gabriela 154, Howard, Brian 119, 128, Kurth, Elke 66, Hubbe, Hendrik 104, Kastenhofer, Jens 111, Huber, Irit 87, Kaszas, Attila 130, L Hædersdal, Merete 117, Katsuki, Sunao 155, Ladas, Thomas 87, Hølmich, Lisbeth Rosenkrantz 174, Katsuki, Sunao 49, 47, 177, 178, Ladejobi, Adetola O. 87, Kauth, Andrea 160, Ladekarl, Morten 94, I Kavaliauskaite, Justina 53, Lafitte, Luc 123, Iaizzo, Paul A. 135, 86, 118, Kavaliauskas, Zydrunas 166, Lagae, Liesbet 103, Ibey, Bennet L. 70, 126, Keller, Flavio 110, Lakshmi Narasimhan, Prashanth 163, Ibey, Bennett L. 71, Kemeny, Lajos 161, 174, Lallow, Emran O. 60, Imbrechts, Maya 142, Kergoat, Loig 130, Lamberti, Patrizia 122, 88, Impellizeri, Joe A. 84, Kersulis, Skirmantas 166, Lammerskitten, Alica 179, Inacio, Eloise 123, Kesar, Ursa 62, Lampreht Tratar, Ursa 84, Ingebrandt, Sven 160, Khabsa, Mariam 87, Lansky, Zdenek 78, Innamorati, Giorgia G. 99, 162, Khadka, Niranjan 78, Lara, Grace ?? , Inoue, Ryosuke 176, 151, Khorasani, Amir 156, Last, David 99, 159, Ismaiel, Lama 47, Kielan, Wojciech 165, Laudebat, Lionel 88, Ivanecz, Arpad 139, Kielan, Wojciech 153, Laurita, Romolo 47, Ivorra, Antoni 160, 45, 119, 124, 81, Kielbik, Aleksander 113, 81, Lazar, Gyorgy 139, 86, Kiester, Allen 70, 71, Le, Son 75, J Kiestler, Allen 126, Le Berre, Théo 103, 180, Jacob, Thomas 144, Kim, Hong Bae 94, Leblanc, Normand ?? , Jacobs, Liesl 141, 142, Kim, Vitalii Pavlovich 126, Lebovka, Nikolai 152, Jacobs, Edward 115, 79, Kim, Vitalii 126, 70, Lee, Kiho 93, Jaeger, Henry 82, 101, 111, Kimura, Ryuya 47, Lee, Jack 69, Jakštys, Baltramiejus 55, Kimura, Ryuya 178, Lefevre, Marie 130, Jakštys, Baltramiejus 90, Kimura, Yasushi 54, 124, Lendesberg, Michal 87, Jakstys, Baltramiejus 75, 165, Kis, Erika 161, Leroy, Jean-Baptiste 151, Jana, Aniket 79, 77, Kis, Erika Gabriella 139, Leveque, Philippe 137, 125, Janša, Rado 139, Kis, Erika Gabriella 174, Levin Pedersen, Dorte 107, Jaramillo, Pedro 133, Kizinievic, Paulina 53, Liberatore, Rachel A. 143, Jarm, Tomaž 150, 63, Klahn, Shawna 93, Liberti, Micaela 97, 162, Jaroszeski, Mark J. 61, Knappert, Justus 59, 65, Lin, Hao 60, 104, Jemec, Gregor 117, Kofod-Olsen, Emil 94, Lindelauf, Kim 169, 171, Jensen, Lars Henrik 90, Kohena, Kris 56, 77, Lindenberger, Christoph 65, Jensen, Lene 140, Kolb, Juergen F 66, Lindhardt, Christina Louise 90, 173, Jesenko, Tanja 84, 62, 143, 141, 77, Kološnjaj-Tabi, Jelena 89, 102, 161, Jhumur, Nandita C. 60, Kolosnjaj-Tabi, Jelena 137, Lione, Lucia 84, Jiang, Minhan 95, Koruth, Jacob 38, Liraz-Zaltsman, Sigal 99, Joeres, Eike 83, Kos, Bor 150, 139, 139, 119, Liu, Hongmei 96, Johnen, Sandra 160, Koszo, Renata 139, Liu, Caiyun 178, Joncker, Nathalie 63, Kotnik, Tadej 172, Liu, Fentao 128, Jonynaite, Kamile 166, Kougkolos, Georgios Y. 88, López de las Hazas, María del Carmen Jouanmiqueou, Bastien 88, Kovacs, Eugenia 136, 154, Jouni, Ali 162, Kozjek Mencinger, Lucija 68, López Gámez, Gloria 155, 152, 187 López-Alonso, Borja 157, 149, 135, Mazzarda, Flavia 75, Nouri-Goushki, Mahdiyeh 104, Lorenzo, Melvin F. 91, McDaniel, Amanda 136, Novickij, Vitalij 165, 137, Lovšin, Žana 172, McDaniel, Amanda H. 109, Novickij, Jurij 118, 69, 64, Lucía, Óscar O 149, McHardy, Christopher 59, 65, Novickij, Vitalij 69, 122, 128, 138, Lucía, Óscar 157, 135, McKillop, Iain H 115, 118, 150, 64, 179, Luis Vásquez, Juan JLV 59, Mentheour, Robin 111, Novotna, Veronika 170, 80, 180, Lyerly, H. Kim 69, Merbahi, Nofel 111, 127, Nowacka, Malgorzata 168, Lyons, Phoebe 131, Merla, Caterina 97, 162, 105, 97, 99, Nuccitelli, Richard 136, Lænkholm, Anne-Vibeke 105, 51, 129, Nuccitelli, Richard 109, Lønkvist, Camilla Kjær 174, Mhemdi, Houcine 169, Nuki, Hirotaka 178, 47, Michinski, Sebastián D. 173, 170, Nunes, Cláudia 101, M Miguela, Veronica 143, Maag, Patricia 179, O Miklavčič, Damijan 157, 125, 92, 63, Maček Lebar, Alenka 134, O’Brien, Barry 87, 181, 120, 135, 122, 68, 119, 145, 139, 139, Maček-Lebar, Alenka 67, 92, O’Connor, Rodney P. 130, 58, 67, 134, Machala, Zdenko 111, O’Halloran, Martin 181, Mildner, Anne-Kathrin 160, Maciulevicius, Martynas 75, 165, O’Keeffe, Bridget 108, Miller, Robert 143, Maessen, Ralph 171, 169, Ober, Camille 50, Mir, Lluis M. 97, 99, 110, 97, 114, 98, Maglietti, Felipe H. 173, 93, 170, 85, Odehnalova, Eva 80, 94, 94, Odili, Joy 131, Mishra, Ashutosh 172, Maglietti, Felipe 92, Oglesby, Nygel 69, Miyazaki, Kaichi 178, 47, Mahnič-Kalamiza, Samo 145, 122, Ojeda, Andrew 75, Mognaschi, Maria Evelina 122, 135, Olah, Judit 139, Moisescu, Mihaela G. 50, 136, Mai, Peipei 128, Olah, Judit 174, Mojsoska, Biljana 174, Maine Calzado, Enaide 56, Oláh, Judit 161, Mok, Jin Hong 82, Maizels, Leonid 79, 87, Olaiz, Nahuel 170, 56, Monleón Pradas, Manuel 97, Maji, Debnath 143, Onal, Birce J. 120, 119, Monti, Giuseppina 105, Malik, Areej 114, Orlacchio, Rosa 125, 137, Moreau, David 130, Malyško-Ptašinskė, Veronika 118, Orlando, Antonio 133, Moreno Manzano, Victoria 99, 162, Malyško-Ptašinskė, Veronika 64, 69, Orlov, Dmytro 144, 97, Mancuso, Mariateresa 129, 51, Osacar, Juan Manuel 92, Moreno-Weidmann, Zoraida 86, Mangalanathan, Uma 49, 70, 81, 113, Osada, Jesús 154, Morkvėnaitė-Vilkončienė, Inga 90, Mannarino, Samantha 109, Osep, Anja 84, Moshkovits, Yonatan 79, Mannozzi, Cinzia 47, Osimani, Andrea 47, Mozzon, Massimo 47, Mansurov, Vasilii ?? , Otten, Alex 61, Muir, Tobian 117, Mantza, Anastasia 167, Ottlakan, Aurel 174, 139, Munch, Lars 105, Maor, Elad 87, Muralidharan, Aswin 104, Ó Maor, Elad 47, 79, Muratori, Claudia 114, 75, Ócsai, Henriette 161, Maoyafikuddin, Mohammad 156, Muscat, Adeline 97, Marchal, Luc 152, Ö Møller, Michael Prangsgaard 174, Marchalot, Julien 103, 180, Özmutlu Karslioglu, Özlem 179, Marchès, Aurélie 127, N P Marcinauskas, Liutauras 166, Nagy, Andras 139, Paillol, Jean 123, Mardor, Yael 99, 159, Nain, Amrinder 79, 77, Pájaro Escobar, Harold Antonio 152, Marino, Carmela 129, 51, 97, Nakajo, Ryuichi 151, 176, Pakhomov, Andrei G. 98, 70, 81, 126, Marino, Ramona 110, Nanajian, Anthony ?? , 76, 113, 70, 166, 49, Markelc, Bostjan 141, 175, 139, 77, Nassari, Mohammad Farooq 174, Pakhomov, Andrei G. 52, 126, 62, 68, Nasto, Riccardo Aurelio 140, 90, 181, Pakhomova, Olga N. 81, 113, 70, 49, Marracino, Paolo 97, 162, 99, Nasto, Aurelio 181, 90, 140, Palepšienė, Rūta 75, 165, 55, 158, Marshall, Guillermo R. 170, 173, Nati Poltri, Simone 158, Pallisgaard, Niels 107, Martin-Belloso, Olga 152, 73, 155, Naujokaite, Gintare 94, Palombo, Fabio 84, Martínez-Beamonte, Roberto 154, Navarro-Ferrando, María Ángeles Pandey, Saurabh K. 78, Más Estellés, Jorge 97, 154, Panescu, Dorin 80, Maslow, Joel N. 60, 104, Navickaitė, DIana 179, Pasquet, Lise 68, Matei, Bogdan Mircea 174, Nemec, Sebastjan 89, Pataro, Gianpiero 74, 73, 67, 144, 102, Mathys, Alexander 66, Neumann, Eberhard 121, 145, Matkovic, Urska 175, Neyts, Johan 142, Pawlaczyk, Katarzyna 168, Matsunaga, Kazuya 177, Nguyen, Ken 80, Pedraza, Maria M. 162, 99, Mattei, Nicolas 89, Nielsen, Anni Linnet 174, Pemen, Guus 105, Mattison, Lars M. 119, 86, 120, 135, Ning, Junyi 96, Peng, Wencheng 96, Matys, Aleksandra 168, Nolte, Teresa 129, Pereira, Marcos M. 153, 188 Perera-Bel, Enric 124, Remic, Tinkara 143, Sato, Mitsuhiko 182, 172, Perez Simba, Byron 66, Rems, Lea 181, 72, 150, 176, 104, Savopol, Tudor 50, 136, Perič, Barbara 132, Rencelj, Andrej 143, Schlüter, Oliver 151, 100, 74, Perner, Trine 107, Rice, Michael 133, Schmidt, Marc 59, Persano, Luca 51, Ripalda, Marina 154, Schmidt, Megan M. 120, Pervan, Mascha 90, Ritter, Andreas 169, 129, 171, Schmidt, Volker-Jürgen 174, Pesch, Georg 104, Riviere, Charlotte 180, 103, Schnefeldt, Mazen 90, Pesl, Martin 80, Roberts, Christine 60, 104, Schottroff, Felix 111, 82, Petersen, Lars Jelstrup 115, Rocculi, Pietro 145, 167, 159, Schultz, Jeff 171, Phung, Brandon 108, Rodriguez, Isaac 170, Scott, Megan 76, Pinto, Eleonora 84, Rody, Achim 90, Scuderi, Maria 157, Pittaluga, Julia 75, Rols, Marie-Pierre 89, 150, 151, 111, Segev, Amit 87, Pløen, John 90, 88, 137, 63, 102, ?? , 57, 57, Semenov, Iurii 70, 126, Pogreb, Roman 169, Rols, Marie-Pierre 37, 68, Semenova, Nina 77, Poignard, Clair 133, 123, 158, Rossi, Alessandra 49, Senaj, Viliam 52, Polajžer, Tamara 67, 63, Rossmeisl, John H. 91, 93, Seror, Olivier 123, Polini, Dana 131, Rossowska, Joanna 108, Serra, Federica 71, Popovič, Peter 139, Rosted, Elizabeth Emilie 174, Serša, Gregor 139, 84, 143, 141, 68, Potočnik, Tjaša 67, Rózsa, Petra 161, 132, 62, 46, 139, Potrc, Stojan 139, Rubinsky, Boris 128, Serša, Igor 145, 122, Poulsen, Laurids Østergaard 94, Rudno-Rudzińska, Julia 165, Sevenich, Robert 151, 74, 100, Prat, Fréderic 103, 180, Rudno-Rudziska, Julia 153, Shalom, Avshalom ?? , Prudhomme, Solène 65, Ruedlinger, Britney ?? , Shan, Jerry W. 104, 60, Průša, Jiří 78, Ruiz-Hitzky, Eduardo 101, Shao, Qi 95, Pulgarin Lopez, Oscar Mauricio 145, Ruma, R. 182, Sharabi, Shirley 159, 99, Purcell, Erin 114, Rutigliano, Daniela 110, Sheehan, Mary C. 124, Pyatkovskyy, Taras 82, Ruzgys, Paulius 158, 179, 165, 75, Sheehan, Mary 95, Q Rybak, Katarzyna 168, Shi, Guilan 109, Qasrawi, Radwan 116, Rybak, Katarzyna 168, Shimizu, Yoji 95, Rzechonek, Adam 128, Shorrock, Clay D. 84, R Shorstkii, Ivan 177, Rabeler, Felix 167, S Shreiber, David I. 60, 104, Radzevičiūtė, Eivina 64, 69, Sabac, Andrei 180, Siauruseviciute, Virginija 53, Radzevičiūtė, Eivina 118, Sabaleuski, Uladzimir 52, Sibbons, Paul 61, 53, Rafael Rafaelsen, Søren 90, Sabbah, Michele 54, Raghuraman Rajagopalan, Neeraj Sachdev, Shaurya 68, Siemer, Claudia 177, 179, 59, 124, 78, 96, 54, Sack, Martin 52, Sieni, Elisabetta 156, 88, 164, 122, Rahman, Mijanur 172, 182, Sack, Kevin 120, 110, Rainot, Aurianne 72, Saczko, Jolanta 107, 150, 128, 175, Sigg, Daniel C. 119, 120, 135, Ramanavičius, Arūnas 90, 149, Signori, Emanuela 110, 85, Ramirez, David A. 86, 118, 135, Saczko, Jolanta 106, Silkunas, Mantas 98, 166, Rampazzo, Elena 51, Sakugawa, Takashi 151, Silkuniene, Giedre 70, 49, Rand, Daniel 159, Sakurado, Kurt 69, Silverman, Jeffrey 108, Rasmusson, Allan G. 100, 144, Salameh, Zaid S. 91, 115, 124, 54, 76, Simoes, Brian 96, 124, Raso, Javier 83, 37, 73, 58, 154, 112, Sales Conniff, Amanda E. 56, 77, Simon, Michel 127, Rataj, Raphale 66, Salgado, Sergio S. 93, 85, Simon, Juliette 88, Rauh, Cornelia 65, 59, Sallberg, Matti 37, Singer, Jonathan P. 60, Ravid, Orly 159, Salvatori, Erika 84, Singh, Julie 109, Ray Vista, William 78, Samaranayake, Chaminda P. 82, Sinicropi, Stefania 74, Razakamanantsoa, Leo 54, Samouillan, Valérie 50, Sinius Pouplier, Sandra Kristiane 105, Razola-Díaz, María del Carmen 100, Sanchez, Massimo 110, Siruckova, Anna 80, 74, Sanchez Petidier, Marina M. 98, 114, Sitnick, Mitchell Sitnick T. 143, Reberšek, Matej 92, 99, 162, Skitzki, Joseph 69, Redondo, Luis 52, 153, Sánchez-Gimeno , Cristina C 112, Slezia, Andrea 130, Redondo, Luis 70, Sandau, Petra 66, Slokar, Dejan 58, Reichlin, Tobias 120, Santek, Iva 68, Slump, Cornelis H. 60, Reilly, John 87, Santos, Mafalda M. 153, Smith, Trevor 142, Reitmeier, Jakub 154, Sanz, Jorge J 112, Soba, Alejandro 170, Rembiałkowska, Nina 138, 150, 118, Sarnago, Héctor 135, 149, 157, Soliva-Fortuny, Robert 152, 73, 155, 128, 107, 165, 108, 137, Sastry, Sudhir K. 82, Solomon, Stephen B 95, Rembiałkowska, Nina 153, Sato, Yuya 49, Soltanipour, Farid 73, 189 Sommer, Marie-Christine 66, Thomas, Athul 129, 169, Vrabic, Ajda 175, Sosnin, Maxim 177, Thomas, Debby 142, Vroomen, Laurien G. P. H. 78, Sowa, Pamela 113, 81, Thomassin-Naggara, Isabelle 54, Vykertas, Salvijus 165, 75, 55, 158, Sozer, Esin 136, Thorlacius-Ussing, Ole 94, 115, v Spadiut, Oliver 111, Timm, Michael 66, van Zyl, Martin 87, Springer, Monika 151, Tivig, Ioan 50, 136, Srimathveeravalli, Govind 78, 124, 95, Tobolková, Anna 154, W 54, 96, Toepfl, Stefan 59, Wadsö, Lars 100, Stachova, Petra 80, Tokoutsi, Zoi 163, Waldert, Kate 151, Stankevic, Voitech 166, Töpfl, Stefan 83, Walkling-Ribeiro, Markus 144, Starek, Zdenek 80, Torres, Nesus N. 162, 99, Wanders, Alkwin 94, Steelman, Zachary 71, Tran, David D. 75, 40, Wang, Yunlong 128, Stender, Mogens Tornby 115, 94, Trdina, Peter 92, Wegener, Joachim 160, Stenholt, Louise 115, Tri, Jason 87, Weinberger Rosen, Andreas 107, Stewart, Mark T. 119, Trotovšek, Blaž 139, 139, West, Aislin 113, Stigaard, Trine 107, Tsurusaki, Koki 49, Wiegell, Stine Regin 117, Stirkė, Arūnas 53, 89, 48, Tuohy, Joanne 93, Wiktor, Artur 100, Stommel, Martijn W. J. 60, Tur, Jared 56, 77, Wiktor, Artur 168, 168, Strojan, Primoz 62, Turjanski, Pablo 56, Williams, James A. 142, Strucic, Marko 122, Twitty, Chris 69, Wise, Julia 110, Stühmeier-Niehe, Corinna 177, Tylewicz, Urszula 167, 159, Witrowa-Rajchert, Dorota 168, Suárez, Cecilia 170, Tzou, Wendy S. 119, Witrowa-Rajchert, Dorota 168, Sun, Yubing 96, Wu, Vincent 69, t Surra, Joaquín 154, Wurm, David J. 111, te Riet o.g. Scholten, Gerben A. 60, Sutter, Olivier 123, X Synowiec, Jody 109, U Xiao, Shu 70, Szederkényi, Edit 161, Uemura, Kunihiko ?? , 112, Szewczyk, Anna 107, Uhlig, Elisabeth 112, Y Szewczyk, Anna 175, Ursic Valentinuzzi, Katja 62, Yakhini, Zohar 110, Szlasa, Wojciech 137, 150, Uscila, Rolandas 166, Yan, Bingyu 155, Szwedowicz, Urszula 57, 149, Uždavinytė, Dovilė 55, 165, 75, Yang, Feiyu 96, Uzuner, Sibel 59, 178, Yao, Xin 143, Š Yao, Chenguo 96, Šatkauskas, Saulius 165, 75, 179, 158, V Yasin, Omar 87, 55, Valdez-Nava, Zarel 88, Yuan, Fan 57, Šatkauskienė, Ingrida 55, Vallet, Leslie 114, 98, 97, 99, 162, Yun, Sung Hae ?? , Ševčík, Rudolf 154, Varga, Anita 161, Šimonis, Povilas 48, Vass, Gabor 139, Z Šmerc, Rok 135, 122, Vass, Gabor 174, Zahn, Jeffrey D. 104, 60, Šmid, Lojze 139, Venger, Olga 163, 164, Zakar, Tomáš 182, Štabuc, Miha 139, Venger, Andrii 163, 164, Zaklit, Josette ?? , Štabuc, Borut 139, Verardo, Vito 74, 100, Zampachova, Vita 80, Verdaasdonk, Rudolf M. 60, Zamuner, Annj 164, 122, 156, 110, 88, T Verma, Atul 119, 120, Zappatore, Marco 105, Tabcheh, Nour 125, Vermeire, Giles 142, Zdravkovic, Milena 100, Taddei, Anna Rita 129, Vernier, P. Thomas 75, Zemlin, Christian W. 71, Tanaka, Atsushi 47, Veroy, Karen 163, Zhang, Rui 152, Tanaka, Atsushi 178, Vestergaard, Kitt 90, Zhang, Yanfang 128, Tanori, Mirella 129, 51, Vicendo, Patricia 127, 50, Zhang, Min 56, Tappi, Silvia 159, 167, 47, Viola, Giampietro 51, Zhang, Jun 69, Tarantino, Paolo 90, 181, 140, Viscount, Brian 84, Zhernov, Ilia 78, Tarantino, Luciano 90, 181, 140, Vissing, Mille 132, 174, 117, 105, 90, Zinovičius, Antanas 90, Tarek, Mounir 72, Vista, William-Ray 95, Znidar, Katarina 77, Tarricone, Luciano 105, Vitkin, Edward 110, Zocher, Katja 66, Tcacenco, Olga 94, Vivacqua, Adele 74, Zvonareva, Tatiana 126, Tellado, Matias M. 173, 92, 85, 94, Vogelsang, Rasmus 107, 182, Ž Teplan, Michal 78, Völker, Thore 83, Želvys, Augustinas 64, Terjung, Nino 83, Vollaire, Christian 103, Thamkaew, Grant 144, 100, Von Rothstein, Kristin 109, 136, Ł Thaokar, Rochish 156, Vorobiev, Eugene 152, 169, 178, Łapińska, Zofia 175, 108, 190 Document Outline 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