November 16 - December 4, 2020 Online Event, Ljubljana, Slovenia Proceedings and workbook of the Electroporation-based Technologies and Treatments International SCIENTIFIC WORKSHOP and POSTGRADUATE COURSE Edited by: Peter Kramar Damijan Miklavčič Organised by: University of Ljubljana Faculty of Electrical Engineering Institute of Oncology, Ljubljana Supported by: Educell Energie Pulse Systems IGEA Iskra Medical Laboratorij-um Medtronic Micro+Polo Omega Pulse Biosciences www.ebtt.org Proceedings and workbook of the Electroporation-based Technologies and Treatments International SCIENTIFIC WORKSHOP and POSTGRADUATE COURSE Online Event, Ljubljana, Slovenia November 16 - December 4, 2020 3 Welcome note 4 In memoriam: Justin Teissié (1947–2020) 5 Invited Lecturers 7 Antoni Ivorra: Nerve Impulses: Avoiding Them in and by Electroporation and Inducing Them with Injectable Micr ostimulators for Movement Restoration in Paralysis 8 Gale Louise Craviso: Characterization of the Effects of Nanosecond Electric Pulses on Neuroedocrine Adr enal Chromaffin Cells 9 Indrawati Oey: Effect of Pulsed Electric Fields processing on plant-based foods 10 Emanuela Signori: DNA Immunization Protocols by Electrotransfer 11 François H. Cornelis: The applications of IRE/ECT in clinical practice 15 Short presentations 29 Laboratory exercises 31 Saša Haberl Meglič, Mojca Pavlin: L1 The influence of Mg2+ ions on gene electrotransfer efficiency 35 Tjaša Potočnik, Alenka Maček Lebar: L3 Visualization of local ablation zone distribution between two needle electrodes 39 Peter Kramar, Aljaž Velikonja, Alenka Maček Lebar: L6 Electroporation of planar lipid bilayers 43 Saša Haberl Meglič, Karel Flisar: L7 E. coli inactivation by pulsed electric fields in a continuous flow system 47 Saša Haberl Meglič, Matej Reberšek: L8 Analysis of electric field orientations on gene electrotransfer efficiency 51 Matej Kranjc, Igor Serša: L10 Monitoring of electric field distribution in biological tissue by means of magnetic resonance electrical impedance tomography 55 Gorazd Pucihar: L11 Measur ements of the induced transmembrane voltage with fluorescent dye di-8-ANEPPS 59 Computer modeling 61 Anže Županič, Bor Kos: C1 Treatment planning for electrochemotherapy and irreversible electroporation: optimization of voltage and electrode position 63 Paulo A. Garcia, Bor Kos : C2 Numerical Modeling of Thermal Effects during Irreversible Electroporation Treatments 67 Mounir Tarek: C3 Molecular dynamics simulations of membrane electroporation 69 E-learning 71 Selma Čorović, Samo Mahnič-Kalamiza: E1 Electroporation of cells and tissues - interactive e-learning course 75 Faculty members ISBN 978-961-243-410-6 Proceedings is available in PDF format at 2020.ebtt.org/proceedings November 16 – December 4, 2020 Online Event, Ljubljana, Slovenia Proceedings and workbook of the Electroporation-Based Technologies and Treatments International SCIENTIFIC WORKSHOP and POSTGRADUATE COURSE Edited by: Peter Kramar Damijan Miklavčič Organised by: University of Ljubljana Faculty of Electrical Engineering Institute of Oncology, Ljubljana Organising committee: Chair: Peter Kramar Members: Matej Kranjc, Lea Vukanović, Duša Hodžič Supported by: Educell Energie Pulse Systems IGEA Iskra Medical Laboratorij-um Medtronic Micro+Polo Omega Pulse Biosciences www.ebtt.org _____________________________________________________ Kataložni zapis o publikaciji (CIP) pripravili v Narodni in univerzitetni knjižnici v Ljubljani COBISS.SI-ID=37042947 ISBN 978-961-243-410-6 (pdf) _____________________________________________________ URL: 2020.ebtt.org/proceedings Copyright © 2020 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 Welcome note Dear Colleagues, dear Students, The idea of organizing the Workshop and Postgraduate Course on Electroporation-Based Technologies and Treatments (EBTT) at the University of Ljubljana had been developing for several years. After preliminary discussions, the Workshop and Course was organised for the first time in 2003. In 2020 the Course is organised for the 14th time! In these fourteen years, the Course has been attended by 844 participants coming from 41 different countries. And this year again – despite pandemics - we can say with great pleasure: “with participation of many of the world leading experts in the field” – unfortunately, as an online only event. The goals and aims of the Workshop and Course however remain unchanged: to provide the participants with sufficient theoretical background and practical knowledge to allow them to use electroporation effectively in their working environments. It is a great pleasure to welcome you to the EBTT and in particular to the practical lab work taking place in the virtual space of University of Ljubljana, Faculty of Electrical Engineering organised as an integral part of the Interdisciplinary doctoral programme Biomedicine. From the very beginning we were aiming to prepare lab work for participants, which would complement the lectures. As preparing lab work takes more time than preparing and organizing lectures, we introduced lab work at the second workshop in 2005. Lab work covers different aspects of research: biological experiments taking place in the cell culture labs, microbiological lab, lab for tissue and planar lipid bilayer; numerical and molecular dynamics modelling, e-learning using computer classrooms, pulse generator development and electrical measurements using electronic laboratory workshop and magnetic resonance electrical impedance tomography. This year we were faced with a new challenge as the Course is organised fully online due to Covid-19 pandemics. We have prepared in advance for this situation by prerecording wet lab practials and adapting those that can be conducted as on-line ( e.g. modelling, computer simulations). The team here in Ljubljana (and Nancy) was working hard and will provide live webinars of the lab works so that you will be able to benefit most even if not actually being in the lab. We understand this is not perfect but we would nevertheless like to offer you the opportunity to learn as much as possible. The biological experiments were pre-recorded in the labs of the network of research and infrastructural centre MRIC, University of Ljubljana, at the Faculty of Electrical Engineering in the Laboratory of Biocybernetics. Lab work would not be possible without extensive involvement and commitment of numerous members of the Laboratory of Biocybernetics and Igor Serša from Jožef Stefan Institute for what I would like to thank them all cordially. It also needs to be emphasized that all written contributions collected in the proceedings have been reviewed and then thoroughly edited by Peter Kramar. We thank all authors and editors. Also, I would like to express our sincere thanks to the faculty members and invited lecturers for their lectures delivered during the course. Finally I would like to thank our sponsors who are making our EBTT possible: Educell (Slovenia), EnergyPulse Systems (Portugal), IGEA (Italy), Iskra Medical (Slovenia), Laboratorij-um (Slovenia), Medtronic (USA), Micro+Polo (Slovenia), Omega (Slovenia), and Pulse Bioscience (USA). Thank you for participating in our Workshop and Course. We sincerely hope that you will benefit from being with us. Sincerely Yours, Damijan Miklavčič In memoriam: Justin Teissié (1947–2020) Justin Teissié, was a devoted scientist and a founding faculty member and still an active faculty member of the EBTT. Justin Teissié has dedicated his life to the study of biological membranes and the fundamental processes leading to membrane permeabilisation, and has played a major role in the development of its applications. He has been equally aware of the need and importance of developing fundamental knowledge and understanding of cell membrane electropermeabilisation, as well as of developing and promoting use of electroporation in medicine and biotechnology. For his work he received many honours: winner of the Prize for Medical Applications of Electricity awarded by the Institut Electricité Santé in 1995, CNRS honorary medallist in 2013 and laureate of the American Institute for Medical and Biological Engineering (AIMBE) in 2017. He became an Honorary Senator of the University of Ljubljana in 2015, and was Emeritus CNRS Research Director since 2012. Although retired, he never ceased to be an extremely involved and active researcher; Justin Teissié lived until his last days for Science. His passing creates an immense void in our community. His power of work, his integrity and scientific rigor, but also his humour, his great generosity, and his availability were exemplary. He was always posing questions, continuously reminding us that our knowledge is still incomplete, and even though we sometimes had an impression that we have done something completely new and complete, the literature search revealed Justin had at least addressed that specific question before. We, his students, colleagues, and friends, will never forget him, and will keep him in our memories. We will remember how invigorating scientific discussions with him were, and how he was capable of influencing our research with a simple question. Marie-Pierre Rols and Damijan Miklavčič INVITED LECTURERS EBTT WORKSHOP 2020 INVITED LECTURERS Nerve Impulses: Avoiding Them in and by Electroporation and Inducing Them with Injectable Microstimulators for Movement Restoration in Paralysis Antoni Ivorra, Department of Information and Communication Technologies, Universitat Pompeu Fabra, Carrer Roc Boronat 138, 08018, Barcelona, SPAIN INTRODUCTION activates a whole muscle or a group of muscles. For being In this talk I will briefly present some recent studies able to selectively activate portions of a muscle, we are conducted by the Biomedical Electronics Research Group of developing a technology for the implementation of wireless Universitat Pompeu Fabra that are about electroporation and networks of thin and flexible intramuscular implants to be electrical stimulation. In particular, I will focus on aspects deployed by injection [4,5]. that lie in the boundary between both phenomena. Electrical stimulation of peripheral nerves is frequently ACKNOWLEDGEMENTS an unwanted consequence of the delivery of currents to body This research was financially supported by the Ministry tissues. This is, for instance, the case in conventional of Economy and Competitiveness of Spain through the electroporation treatments; the delivery of relatively long grants TEC2014-52383-C3-R and SAF2014-52228-R, and (>10 µs) monophasic pulses causes pain and muscle by the European Research Council (ERC) under the contractions. In [1] we numerically explained how the European Union’s Horizon 2020 research and innovation delivery of bursts of short (≤ 5 µs) biphasic square pulses, in programme (grant agreement No 724244). a technique referred to as H-FIRE, can cause irreversible I gratefully acknowledge the financial support by electroporation (IRE) whilst minimizing stimulation. We ICREA under the ICREA Academia programme. then in vivo demonstrated that sinusoidal waveforms can also perform ablation by IRE and avoid muscle contractions [2]. REFERENCES Dwelling further on the delivery of high-frequency currents [1] B. Mercadal, C. Arena, R. Davalos and A. Ivorra, for therapeutics, we learnt about the non-ablative use of "Avoiding nerve stimulation in irreversible bursts of radiofrequency current for chronic pain treatment electroporation: a numerical modeling study," Phys. and we hypothesized that electroporation could somehow be Med. Biol., vol. 62(20), pp. 8060-8079, 2017. related to their mechanism of action. In a recent publication [2] Q. Castellví, B. Mercadal, X. Moll, D. Fontdevila, A. we report multiple in vitro evidence of an electroporation Andaluz and A. Ivorra, "Avoiding neuromuscular mediated calcium uptake when high frequency (500 kHz) stimulation in liver irreversible electroporation using sinusoidal bursts are delivered and discuss how such uptake radiofrequency electric fields," Phys. Med. Biol, vol. could explain pain relief [3]. 63(3), pp. 035027, 2018. Electrical stimulation of peripheral nerves would also be [3] B. Mercadal, R. Vicente and A. Ivorra, "Pulsed an adverse consequence of performing wireless power radiofrequency for chronic pain: in vitro evidence of an transfer (WPT) to implants by means of volume conduction electroporation mediated calcium uptake," at low frequencies. However, as we have shown recently [5], Bioelectrochemistry, vol. 136, pp. 107624, 2020. WPT based on volume conduction of high frequency [4] L. Becerra-Fajardo, M. Schmidbauer and A. Ivorra, (>1 MHz) currents is doable: powers above 1 mW can be "Demonstration of 2 mm thick microcontrolled injectable obtained in very thin (diameter < 1 mm) and short (length stimulators based on rectification of high frequency < 15 mm) implants when ac fields that comply with safety current bursts," IEEE Trans. Neural Syst. Rehabilitation standards are present in the tissues where the implants are Eng. , vol. 25(8), pp. 1343-1352, 2017. located. This is one of the main operating principles of a [5] M. Tudela-Pi, L. Becerra-Fajardo, A. García-Moreno, J. technology we have proposed and in vitro demonstrated for Minguillon and A. Ivorra, "Power Transfer by Volume the development of injectable passive sensors [7]. Conduction: In Vitro Validated Analytical Models On the other hand, electrical stimulation of excitable Predict DC Powers above 1 mW in Injectable Implants," tissues is intentionally performed for different clinical IEEE Access, vol. 8(1), pp. 37808-3782 , 2020. purposes. For instance, electrical stimulation of motor nerves [6] A. Eladly, J. Del Valle, J. Minguillon, B. Mercadal, L. is performed to restore motor functions in paralysis patients. Becerra-Fajardo, X. Navarro and A. Ivorra, "Interleaved A major limitation of electrical stimulation for paralysis is intramuscular stimulation with minimally overlapping that it rapidly induces muscle fatigue. We recently in vivo electrodes evokes smooth and fatigue resistant forces," J. demonstrated that such fatigue can be minimized or avoided Neural Eng. , vol. 17, pp. 046037, 2020. if multiple portions of the muscles are stimulated [7] S. Malik, Q. Castellví, L. Becerra-Fajardo, M. Tudela- asynchronously [6]. Such functionality is not feasible with Pi, A. García-Moreno, M. Shojaei Baghini and A. Ivorra, existing technology for stimulation. Neuromuscular "Injectable Sensors Based on Passive Rectification of stimulation is typically performed with relatively large and Volume-Conducted Currents," IEEE Trans. Biomed. invasive implants which contain bulky components (e.g. Circuits Syst. , vol. 14(4), pp. 867-878, 2020. batteries or coils) for power. Each stimulation channel 7 EBTT WORKSHOP 2020 INVITED LECTURERS Characterization of the Effects of Nanosecond Electric Pulses on Neuroendocrine Adrenal Chromaffin Cells Gale Louise Craviso1, Normand Leblanc1, Thomas Gould2, Josette Zaklit3, Ji Hwan Yoon3, Indira Chatterjee3, 1 Department of Pharmacology and 2 Physiology and Cell Biology, University of Nevada, Reno School of Medicine, Reno, Nevada, USA and 3 Department of Electrical and Biomedical Engineering, University of Nevada, Reno, USA INTRODUCTION There is a great deal of interest in developing electrostimulation approaches that can selectively target specific regions of the body non-invasively and thus without the need for implanted electrodes. To work toward this goal, we have been conducting a comprehensive study of the interaction of electric pulses in the low nanosecond regime (sub-10 ns) with excitable chromaffin cells isolated from the adrenal medulla. Chromaffin cells, which mediate the “fight or flight” response by releasing catecholamines into the circulation, have served as a well-established model of neural-type/neuroendocrine cells. In our studies we employ numerical cell modelling and a variety of experimental approaches to elucidate the mechanisms by which neural cell Figure 1: Typical whole-cell current recorded in a patch excitability is altered at the cellular and molecular levels. clamped chromaffin cell following exposure to a 5 ns pulse (NEP). The inward current, which is carried mainly by Na+, RESULTS is recorded with a delay of 8 ms after the pulse is delivered Exposing chromaffin cells to a single 5 ns pulse is to the cell. From [2]. sufficient to stimulate the release of the catecholamines epinephrine and norepinephrine via Ca2+-dependent ACKNOWLEDGEMENTS exocytosis [1], the same process used by neurons to release This work was supported by AFOSR grants FA9550-14-1- neurotransmitters. The source of Ca2+ is extracellular, where 0018, FA9550-20-1-0061, and AFOSR MURI grant Ca2+ enters the cells via voltage-gated Ca2+ channels FA9550-15-1-0517. (VGCC). While much effort has gone into elucidating the mechanism underlying VGCC activation, which we found REFERENCES relies on Na+ influx that leads to membrane depolarization [1] G.L. Craviso et al., “Nanosecond electric pulse-induced [2,3], we have not yet established whether the plasma increase in intracellular calcium in adrenal chromaffin membrane pathway by which Na+ enters the cell (Figure 1) cells triggers calcium-dependent catecholamine release,” is indicative of electropermeabilization and/or activation of IEEE Trans. Dielectr. Electri. Insul., vol.16, pp. 1294-a non-selective cation channel. Those studies are ongoing. 1301, 2009. Regardless, the sequence of events that leads to [2] J. Yoon et al., “Enhanced monitoring of nanosecond catecholamine release in cells exposed to a 5 ns pulse is electric pulse-evoked membrane conductance in whole-similar to that evoked by acetylcholine, the physiological cell patch clamp experiments,” J. Membrane Biol., vol. stimulus of chromaffin cells. 249, pp. 633-644, 2016. In other studies we also found that a 5 ns pulse [3] J. Zaklit et al, “Adrenal chromaffin cells exposed to 5-ns differentially inhibits cell membrane macroscopic ion pulses require higher electric fields to porate intracellular currents, which in chromaffin cells comprise a mixture of membranes than the plasma membrane: an experimental Na+, Ca2+ and K+ currents [4]. Thus, we have identified and modeling study,” J. Membrane Biol. DOI another way in which chromaffin cell excitability is affected 10.1007/s00232-017-9983-9, 2017. by a single nanosecond pulse and are currently investigating [4] L. Yang et al., “Nanosecond electric pulses differentially the cellular processes by which this occurs. affect inward and outward currents in patch clamped While we will continue to use primary cultures of adrenal adrenal chromaffin cells,” PLOS ONE 12(7): e0181002. chromaffin cells for our studies, we recognize the need to https://doi.org/10.1371/journal. pone.0181002, 2017. investigate effects of nanosecond pulses on these cells in a more physiological setting. Consequently, our efforts now include exposing chromaffin cells in intact adrenal gland tissue, in particular, adrenal tissue from transgenic mice in which the cells are targeted to express specific genetically- encoded fluorescent reporters. 8 EBTT WORKSHOP 2020 INVITED LECTURERS Effect of Pulsed Electric Fields processing on plant-based foods Indrawati Oey 1, 1 Department of Food Science, University of Otago, Dunedin, NEW ZEALAND Pulsed electric field processing (PEF) technology has Unfortunately, fruits and vegetables are often been used in food industries. This technique applies considered as being ‘homogenous’ plant structures. short pulses (microsecond ranges) of high voltages to The potential for using PEF for industrial processing of samples placed between two electrodes. PEF solid plant produce has been underestimated due to a processing could result in pore formation of cell limited understanding of PEF effects on solid plant membranes, a phenomenon called electroporation materials. For industrial applications, it is important to which can be irreversible or reversible - dependent on understand the relationship between PEF induced the treatment intensity applied. Under low or mild PEF structure modification and the subsequent industrial processing intensities, the pores formed in the cell applications (i.e., cutting, frying, extraction etc.). This membrane can reseal after pulse application. lecture discusses different methodologies that have Reversible electroporation can be used to induce stress been used to assess the effect of PEF treatment on fruits reactions in plant tissues, stimulate metabolic activity and vegetables and the applications of PEF for fruits and increase the biosynthesis of secondary metabolites and vegetables processing. such as production of antioxidants. While, at high PEF processing intensities, irreversible electroporation can READING MATERIALS occur and this phenomenon has been utilised as a [1] Oey, I., Faridnia, F., Leong, S.Y., Burritt, D. J., Liu, T. processing aid by food industries to improve the mass 2017. Determination of pulsed electric fields effects on transfer of metabolites prior to dehydration, extraction, the structure of potato tubers. In: Handbook of Electroporation 2, pp. 1489-1507, D. Miklavcic (Ed.), or pressing of foods. Various studies have shown that Springer International Publishing, DOI 10.1007/978-3- PEF can enhance the extraction of juice, reduce the 319-26779-1_151-1. cutting force and improve the drying and freezing [2] Duque, S. M. M., Leong, S. Y., Agyei, D., Singh, J., processes of plant based foods. Larsen, N., Oey, I. 2020. Modifications in the physicochemical properties of flour “fractions” after Most PEF studies have used liquid, semi-solid or solid Pulsed Electric Fields treatment of thermally processed plant materials that have been mechanically oat. Innovative Food Science & Emerging Technologies, fragmented prior to PEF treatment. The damage caused 102406. by mechanical fragmentation (e.g. mashing, slicing, [3] Liu, T., Burritt, D.J., Oey, I. 2019. Understanding the dicing or other mechanical breakdown) prior to PEF is effect of Pulsed Electric Fields on multilayered solid plant foods: Bunching onions (Allium fistulosum) as a likely to affect the nature of any PEF induced changes, model system. Food Research International 120, 560- which limits the widespread of PEF technologies for 567. plant produce processing. Plant-based foods such as fruits and vegetables are made up of many cell and tissue types (e.g. vascular tissues and ground tissues) with varying electrical and topological properties, which determine their sensitivity to electroporation. Hence, their heterogeneous structures need to be taken into consideration when investigating PEF-induced structural changes in plant cells, tissues and organs. Additionally, when placing plant materials with heterogeneous structures in a homogeneous electric field, the uniformity of the PEF effect across the plant materials can be disrupted and the heterogeneity of PEF induced structural changes in intact plant based foods could affect the subsequent processing. 9 EBTT WORKSHOP 2020 INVITED LECTURERS DNA Immunization Protocols by Electrotransfer Emanuela Signori1,2, 1CNR, Institute of Translational Pharmacology, 2University Campus Bio-Medico of Rome, School of Medicine, Rome, ITALY INTRODUCTION present (lack of ?) knowledge”, Biochim. Biophys. Acta, Important advances in understanding the biology of the vol. 1724, pp. 270-80, 2005. immune system are leading to new therapeutic strategies for [4] L.M. Mir, “Electroporation-based gene therapy: recent the treatment of infectious and cancer diseases, such as those evolution in the mechanism description and technology based on gene therapy [1, http://www.abedia.com/wiley/]. developments”, Methods Mol Biol. , vol. 112, pp. 3-23, Gene Electro-Transfer (GET) is an efficient method that 2014. involves the use of Electroporation (EP) after the injection of [5] P. Chiarella, E. Massi, M. De Robertis, et al., nucleotide sequences coding antigens or immunomodulatory “Electroporation of skeletal muscle induces danger signal molecules, delivered by different viral or non-viral vectors release and antigen-presenting cell recruitment [1], to elicit or enhance an immune response into the host. independently of DNA vaccine administration”, Exp. Op. Biol. Ther., vol. 8, pp. 1645-57, 2008. ADVANTAGES OF GET IMMUNIZATION [6] P. Chiarella, E. Massi, M. De Robertis, et al. “Strategies Application of controlled electric pulses at the injection site for effective naked-DNA vaccination against infectious induces a transient perturbation of the cell membrane leading diseases”, Recent Patents on Anti-Infective Drug to a higher uptake of therapeutic nucleotides [2-4]. Mild Discovery, vol. 3, pp. 93-101, 2008. local tissue damage induced by EP plays a role in enhancing [7] R. Heller, L.C. Heller, “Gene electrotransfer clinical both arms of the immune response, making EP a good trials”, Adv. Genet. , vol. 89, pp. 235-62, 2015. approach for immunization protocols [5]. In the last two [8] U. Lampreht, L. Loiacono, M. Cemazar, et al. “Gene decades, GET strategy by employment of plasmid DNA Electrotransfer of Plasmid-Encoding IL-12 Recruits the demonstrated its efficacy and safety in pre-clinical and M1 Macrophages and Antigen-Presenting Cells Inducing clinical vaccination protocols for infectious and cancer the Eradication of Aggressive B16F10 Murine diseases [7]. Many anti-cancer GET therapies are based on Melanoma”, Mediators Inflamm, 2017:5285890, 2017. the delivery of interleukin-12 [8]. [9] K. Cepurniene, P. Ruzgys, R. Treinys et al. “Influence of plasmid concentration on DNA electrotransfer in vitro CRITICAL POINTS OF GET IMMUNIZATION using high-voltage and low-voltage pulses”, J Membr Different routes of plasmid DNA administration Biol. Vol. 236, pp. 81-5, 2010. (intradermal, intramuscular, intratumoral, peritumoral), [10] G. Sersa, J. Teissie, M. Cemazar, et al. different doses of plasmid DNA, combined treatments, as “Electrochemotherapy of tumors as in situ vaccination well as appropriate choice of electric parameters and boosted by immunogene electrotransfer”, Cancer electrodes, in vivo models and mathematical modeling for Immunol Immunother. vol. 64, pp. 1315-27, 2015. effective GET therapies [9-12], are essential steps of [11] S. Corovic, I. Lackovic, P. Sustaric, et al., “Modeling investigation for the translation of DNA EP pre-clinical of electric field distribution in tissues during protocols into clinical applications. electroporation”, Biomed Eng Online., vol. 12, 16, pp.1- 27, 2013. CONCLUSIONS [12] T. Kotnik, L. Rems, M. Tarek, and D. Miklavčič, Several clinical trials based on GET have been started [7,13]. “Membrane Electroporation and Due to the recent results in this field, we can be confident Electropermeabilization: Mechanisms and Models,” good improvements in electrogene immunotherapy will be Annu. Rev. Biophys., vol. 48, pp. 63-91, 2019. reached soon, so opening new perspectives for therapies able [13] S. Pierini, R. Perales-Linares, M. Uribe-Herranz, et al., to increase the patients’ quality of life in the next future [14]. “Trial watch: DNA-based vaccines for oncological indications”, Oncoimmunology, vol. 6, e1398878, 2017. REFERENCES [14] A. Lopes, G. Vandermeulen, V. Préat, “Cancer DNA [1] S.L. Ginn, A.K. Amaya, I.E. Alexander, et al., “Gene vaccines: current preclinical and clinical developments therapy clinical trials worldwide to 2017: An update”, and future perspectives”, J Exp Clin Cancer Res., vol. 38, The Journal of Gene Medicine, vol. 20 e3015, pp.1-16, 146, pp.1-24, 2019. 2018. [2] E. Neumann, M. Schaefer-Ridder, Y.Wang, et al., “Gene transfer into mouse lyoma cells by electroporation in high electric fields”, EMBO J., vol. 1, pp. 841-45, 1982. [3] J. Teissie, M. Golzio, M.P. Rols. “Mechanisms of cell membrane electropermeabilization: A minireview of our 10 EBTT WORKSHOP 2020 INVITED LECTURERS The applications of IRE/ECT in clinical practice L. Razakamanantsoa, MD1, 2; G. Srimathveeravalli, PhD2; O. Seror, MD, PhD3; F.H. Cornelis, MD, PhD1 ; 1Sorbonne University, Paris, France, 2University of Massachusetts, Amherst, USA, 3University Paris 13, Paris, FRANCE Percutaneous irreversible electroporation (IRE) and complications and with an improvement of pain ( >50%) in electrochemotherapy (ECT) have been introduced in the past 84% of the cases [24,25]. The Italian Society of Orthopedics few years in Interventional Radiology. Various successful and Traumatology (SIOT) has included now this therapy in clinical applications have been reported so far. the guidelines for the management of unresectable tumors of The liver was found to be a suitable target for the sacrum. percutaneous IRE. In the case of small tumors efficacy varies IRE was supposed to be effective in lung cancer widely (45.5-100%) among teams [1], but efficacy remains treatment but clinical translation failed to demonstrate it, limited in case of larger tumors > 5 cm [2]. Compared to probably explained by high differences in electric radiofrequency ablation (RFA) [3–5], IRE allows liver conductivity between lung parenchyma and tumor tissue regeneration in patients without severe cirrhosis or previous [26]. Animal studies demonstrated encouraging results for chemoembolization and can potentially be mooted in ECT [15][27]. selected patients unsuitable for other local treatments [6,7]. Several other clinical trials are investigating the use of IRE efficacy may be limited in case of metallic implants [8]. ECT for malignant melanoma (NCT03448666), vulvar ECT was also found to be effective to treat primary or carcinoma (NCT03142061), head and neck cancer (NCT secondary liver tumors that are unresectable because 02549742). Finally, the use of calcium combined to adjacent to portal vein or bile duct [9–12]. electroporation unveiled to be a new, safe and inexpensive Regarding pancreatic cancer, which was thought to be an antitumor treatment demonstrating a response rate excellent indication for IRE/ECT, results are controversial comparable to ECT with bleomycin (72% and 84%, p=0.5) with high morbidity and limited effectiveness, although with less ulceration (38% vs. 68%) in superficially cutaneous some studies reported an improvement in long-term metastatic melanoma or breast cancer tumors [28]. outcomes compared to other therapies [13]. A clinical trial in To improve the evidence on safety and efficacy phase I/II using ECT with bleomycin for the treatment of strengthening the use of IRE/ECT in clinical practice, metastatic unresectable pancreatic cancer is presently under prospective trials comparing them to other therapies such as investigation (NCT03225781). radiation therapy or thermal ablation are needed. Unlike IRE seems interesting for nephron, urinary-tract sparing surgery, the additional ability to stimulate the immunogenic treatment of renal tumors. It has been demonstrated in system to enhance tumor response, creating a potential animals to safeguard urine-collecting system by preserving immunogenic window remains a promise for IRE/ECT in normal morphology and function, allowing urothelial clinical research [29–31]. regeneration without urine leakage or stenosis after treatment [14–16]. Therefore, IRE was used in case of small renal REFERENCES masses up to 4 cm [14,17]. However, more clinical evidence [1] Geboers B, Scheffer HJ, Graybill PM, Ruarus AH, is still required to translate this technic to a standard option Nieuwenhuizen S, Puijk RS, et al. High-Voltage such as RFA. A case-report demonstrated tumor control after Electrical Pulses in Oncology: Irreversible ECT with no evidence of residual disease [18]. Electroporation, Electrochemotherapy, Gene Focal therapy for localized clinically significant prostate Electrotransfer, Electrofusion, and cancer (PCa) offers an alternative to radical treatment aiming Electroimmunotherapy. Radiology. Radiological to eliminate PCa while preserving benign prostatic tissue, Society of North America (RSNA); 2020;295:254–72. bladder neck, sphincter, and adjacent neurovascular [2] Thomson KR, Cheung W, Ellis SJ, Federman D, structures to minimize treatment-related toxicity [19]. A Kavnoudias H, Loader-Oliver D, et al. Investigation of study supported the safety and feasibility of IRE as a primary the safety of irreversible electroporation in humans. J treatment for localized PCa with effective short-term Vasc Interv Radiol. 2011;22:611–21. oncological control in carefully selected men [20]. ECT was [3] Shady W, Petre EN, Gonen M, Erinjeri JP, Brown KT, used in a patient presenting with a PCa and infiltration of the Covey AM, et al. Percutaneous Radiofrequency urethral sphincter with low toxicity, mild adverse events and Ablation of Colorectal Cancer Liver Metastases: Factors absence of tumor activity or impairment in the surrounding Affecting Outcomes--A 10-year Experience at a Single tissues or organs 6 months after treatment [21]. Center. Radiology. 2016;278:601–11. In bone, IRE might be an accurate treatment alternative [4] Cornelis FH, Durack JC, Kimm SY, Wimmer T, close to sensitive structures such as nerves [22]. However, Coleman JAJA, Solomon SB, et al. A Comparative contrary to some animal studies, permanent neural function Study of Ablation Boundary Sharpness After impairment can occur [23]. ECT seems more tolerate in well- Percutaneous Radiofrequency, Cryo-, Microwave, and selected patients with bone metastases of the spine, pelvis or Irreversible Electroporation Ablation in Normal Swine appendicular skeleton with no intra- or post-operative Liver and Kidneys. Cardiovasc Intervent Radiol. 11 EBTT WORKSHOP 2020 INVITED LECTURERS 2017;40:1600–8. [17] Buijs M, Zondervan PJ, de Bruin DM, van Lienden KP, [5] Vroomen LGPH, Petre EN, Cornelis FH, Solomon SB, Bex A, van Delden OM. Feasibility and safety of Srimathveeravalli G. Irreversible electroporation and irreversible electroporation (IRE) in patients with small thermal ablation of tumors in the liver, lung, kidney and renal masses: Results of a prospective study. Urol Oncol bone: What are the differences? 2017;98:609–17. Semin Orig Investig. Elsevier BV; 2019;37:183.e1- [6] Li S, Zeng Q, Zhong R, Mao S, Shen L, Wu P. [Liver 183.e8. regeneration after radiofrequency ablation versus [18] Andresciani F, Faiella E, Altomare C, Pacella G, irreversible electroporation]. Zhonghua Yi Xue Za Zhi. Beomonte Zobel B, Grasso RF. Reversible 2015;95:66–8. Electrochemotherapy (ECT) as a Treatment Option for [7] Silk MT, Wimmer T, Lee KS, Srimathveeravalli G, Local RCC Recurrence in Solitary Kidney. Cardiovasc Brown KT, Kingham PT, et al. Percutaneous ablation of Intervent Radiol. Springer Science and Business Media peribiliary tumors with irreversible electroporation. J LLC; 2020;43:1091–4. Vasc Interv Radiol. 2014;25:112–8. [19] Srimathveeravalli G, Cornelis F, Mashni J, Takaki H, [8] Cornelis FH, Cindrič H, Kos B, Fujimori M, Petre EN, Durack JCJC, Solomon SBSB, et al. Comparison of Miklavčič D, et al. Peri-tumoral Metallic Implants ablation defect on MR imaging with computer Reduce the Efficacy of Irreversible Electroporation for simulation estimated treatment zone following the Ablation of Colorectal Liver Metastases. Cardiovasc irreversible electroporation of patient prostate. Intervent Radiol. 2019; Springerplus. 2016;5:219. [9] Djokic M, Cemazar M, Popovic P, Kos B, Dezman R, [20] van den Bos W, Scheltema MJ, Siriwardana AR, Bosnjak M, et al. Electrochemotherapy as treatment Kalsbeek AMF, Thompson JE, Ting F, et al. Focal option for hepatocellular carcinoma, a prospective pilot irreversible electroporation as primary treatment for study. Eur J Surg Oncol. 2018;44:651–7. localized prostate cancer. BJU Int. Wiley; [10] Coletti L, Battaglia V, De Simone P, Turturici L, 2017;121:716–24. Bartolozzi C, Filipponi F. Safety and feasibility of [21] Klein N, Gunther E, Zapf S, El-Idrissi R, Atta J, Stehling electrochemotherapy in patients with unresectable M. Prostate cancer infiltrating the bladder sphincter colorectal liver metastases: A pilot study. Int J Surg. successfully treated with Electrochemotherapy: a case 2017;44:26–32. report. Clin case reports. John Wiley and Sons Inc.; [11] Edhemovic I, Brecelj E, Gasljevic G, Marolt Music M, 2017;5:2127–32. Gorjup V, Mali B, et al. Intraoperative [22] Coleman RE, Rubens RD. The clinical course of bone electrochemotherapy of colorectal liver metastases. J metastases from breast cancer. Br J Cancer. 1987;55:61– Surg Oncol. 2014;110:320–7. 6. [12] Cornelis FHH, Korenbaum C, Ben Ammar M, Tavolaro [23] Vroomen LGPH, Scheffer HJ, Melenhorst MCAM, van S, Nouri-Neuville M, Lotz JPP. Multimodal image- Grieken N, van den Tol MP, Meijerink MR. Irreversible guided electrochemotherapy of unresectable liver Electroporation to Treat Malignant Tumor Recurrences metastasis from renal cell cancer. Diagn Interv Imaging. Within the Pelvic Cavity: A Case Series. Cardiovasc 2019;100:309–11. Intervent Radiol. 2017;40:1631–40. [13] Moris D, Machairas N, Tsilimigras DI, Prodromidou A, [24] Bianchi G, Campanacci L, Ronchetti M, Donati D. Ejaz A, Weiss M, et al. Systematic Review of Surgical Electrochemotherapy in the Treatment of Bone and Percutaneous Irreversible Electroporation in the Metastases: A Phase II Trial. World J Surg. Treatment of Locally Advanced Pancreatic Cancer. Ann 2016;40:3088–94. Surg Oncol. Springer Science and Business Media LLC; [25] Cornelis FH, Ben Ammar M, Nouri-Neuville M, Matton 2019;26:1657–68. L, Benderra MA, Gligorov J, et al. Percutaneous Image- [14] Wendler JJ, Pech M, Köllermann J, Friebe B, Siedentopf Guided Electrochemotherapy of Spine Metastases: S, Blaschke S, et al. Upper-Urinary-Tract Effects After Initial Experience. Cardiovasc Intervent Radiol. Irreversible Electroporation (IRE) of Human Localised 2019;42:1806–9. Renal-Cell Carcinoma (RCC) in the IRENE Pilot Phase [26] Ricke J, Jürgens JHW, Deschamps F, Tselikas L, Uhde 2a Ablate-and-Resect Study. Cardiovasc Intervent K, Kosiek O, et al. Irreversible electroporation (IRE) Radiol. Springer Science and Business Media LLC; fails to demonstrate efficacy in a prospective multicenter 2017;41:466–76. phase II trial on lung malignancies: the ALICE trial. [15] Kodama H, Vroomen LG, Ueshima E, Reilly J, Brandt Cardiovasc Intervent Radiol. 2015;38:401–8. W, Paluch L-R, et al. Catheter-based endobronchial [27] Ahmad U, Machuzak M. Electric shock therapy for lung electroporation is feasible for the focal treatment of cancer: Taking palliation to the next level. J Thorac peribronchial tumors. J Thorac Cardiovasc Surg. Cardiovasc Surg. Elsevier BV; 2018;155:2160–1. 2017/12/20. 2018;155:2150-2159.e3. [28] Falk H, Forde PF, Bay ML, Mangalanathan UM, [16] Srimathveeravalli G, Cornelis F, Wimmer T, Monette S, Hojman P, Soden DM, et al. Calcium electroporation Kimm SYSY, Maybody M, et al. Normal Porcine Ureter induces tumor eradication, long-lasting immunity and Retains Lumen Wall Integrity but Not Patency cytokine responses in the CT26 colon cancer mouse Following Catheter-Directed Irreversible model. Oncoimmunology. Taylor & Francis; Electroporation: Imaging and Histologic Assessment 2017;6:e1301332–e1301332. over 28 Days. J Vasc Interv Radiol. 2017;28. [29] White SB, Zhang Z, Chen J, Gogineni VR, Larson AC. 12 EBTT WORKSHOP 2020 INVITED LECTURERS Early Immunologic Response of Irreversible 2019/04/24. American Physiological Society; Electroporation versus Cryoablation in a Rodent Model 2019;317:F52–64. of Pancreatic Cancer. J Vasc Interv Radiol. Elsevier BV; [31] Takaki H, Imai N, Thomas CT, Yamakado K, 2018;29:1764–9. Yarmohammadi H, Ziv E, et al. Changes in peripheral [30] Ueshima E, Fujimori M, Kodama H, Felsen D, Chen J, blood T-cell balance after percutaneous tumor ablation. Durack JC, et al. Macrophage-secreted TGF-β(1) Minim Invasive Ther Allied Technol. Informa UK contributes to fibroblast activation and ureteral stricture Limited; 2017;26:331–7. after ablation injury. Am J Physiol Renal Physiol. 13 EBTT WORKSHOP 2020 INVITED LECTURERS 14 SHORT PRESENTATIONS EBTT WORKSHOP 2020 SHORT PRESENTATIONS Irreversible electroporation and radiofrequency ablation: A computational modelling comparison of lesion morphology in cardiac tissue Mario G. Barea1, Tomás García-Sánchez1, and Antoni Ivorra1; 1 Department of Information and Communication Technologies, Universitat Pompeu Fabra, Carrer de Roc Boronat 138, 08018 Barcelona, SPAIN INTRODUCTION Radiofrequency (RF) ablation is the most extended non- pharmacological treatment for atrial fibrillation (AF). However, it can cause thermal damage in surrounding organs. Irreversible electroporation (IRE) is a new non- thermal ablation technique that preserves the extracellular matrix, which favors its recellularization, avoiding the negative effects of RF. IRE traditionally applies monophasic pulses of 100 µs. However, it requires the use of sedation or anesthesia because of the occurrence of muscle stimulation. Figure 1: (RF) Temperature solution of radiofrequency As an alternative to reduce it, high-frequency biphasic pulses model. White line shows the boundary at 50 ºC. have been proposed, allowing a monopolar catheter (IRE) Electric field solution of irreversible electroporation configuration. The objective is to compare the injury model. White line shows the boundary at 700 V/cm. morphology of both techniques by using a computational model. CONCLUSIONS To obtain a lesion depth of 2 mm (thickness of the METHODS atrium), a 17.5 V amplitude needs to be applied in RF and The 3D models follow the geometry described in [1]. 780 V in IRE. The blood flow generates an irregular lesion They consist of a fragment of a cardiac chamber filled with in RF. In IRE the influence of flow is minimal, obtaining a circulating blood, with a catheter inserted 0.5 mm deep and regular geometry of the lesion. Our results suggest that IRE perpendicular to the tissue, and a dispersive electrode at the may have lower spatial resolution than RF with wider lesions base of the tissue to simulate a monopolar catheter for equal depth. This may be translated in fewer application configuration. Materials are those used in the same study. sites for IRE compared to RF to cause a continuous lesion of The models are based on triple coupling of electric-thermal the same size. Further studies should be performed to verify problem and fluid dynamics. Electrical and thermal this statement. conductivity of cardiac tissue are considered temperature- dependent [2]. In IRE model, electrical conductivity with REFERENCES electric field dependence is included [3]. In RF model, constant voltage ablation method has been used, setting an [1] Ana González-Suárez and Enrique Berjano. “Compara- arbitrary voltage for 60 s. The IRE protocol uses 20 bursts of tive analysis of different methods of modelling the high-frequency biphasic pulses at 150 kHz. Duration of the thermal effect of circulating blood flow during RF burst is 100 µs. Amplitudes were adjusted to obtain a depth cardiac ablation” . In: IEEE Transactions on Biomedical of injured tissue of 2 mm from the catheter tip. Blood Engineering. 63.2 (2015), pp. 250–259 velocity is set to 3 cm/s. [2] Mudit K Jain and Patrick D Wolf. “Temperature- To evaluate lesion morphology, RF model considers controlled and constant-power radio-frequency ab-lation: tissue temperatures above 50 ºC. IRE model considers the what affects lesion growth?” In: IEEE Transactions on affected tissue by an electric field greater than 700 V/cm. Biomedical Engineering. 46.12 (1999), pp. 1405–1412. This value has been arbitrarily chosen due to the lack of [1] [3] David Schutt, Enrique J. Berjano, and Dieter Haem- experimental data that offer a threshold value for irreversible merich. “Effect of electrode thermal conductivity in electroporation in cardiac tissue. cardiac radiofrequency catheter ablation: a computa- tional modelling study” . In: International journal of RESULTS hyperthermia. 25.2 (2009), pp. 99–107. Figure 1 shows the volume within the white line as injured tissue in both models. The volume of damaged tissue in RF (9.74 mm3) is 2.6 times smaller than in IRE (25.33 mm3). With the settings established in this study, to cause a lesion 2 mm deep, the RF voltage obtained is 17.5 V while for IRE is 780 V. Regarding lesion geometry, the RF model predicts a more irregular geometry than in the IRE model due to the influence of blood flow. Interestingly, for this catheter geometry, the IRE lesion is wider than the RF lesion. 17 EBTT WORKSHOP 2020 SHORT PRESENTATIONS High-Voltage Generator for Delayed Bipolar and Paired Nanosecond Pulsed Electric Fields (nsPEF) Rosa Orlacchio1, Lynn Carr1,2, Cristiano Palego2, Delia Arnaud-Cormos1,3, Philippe Leveque1; 1 Univ. Limoges, CNRS, XLIM, UMR 7252, F-87000 Limoges, FRANCE, 2 School of Electronic Engineering, Bangor University, Bangor, UK, 3 Institut Universitaire de France (IUF), 75005, Paris FRANCE INTRODUCTION m cable connected to the output of the 2-port box. Note that Cancellation or increase of the classical bio-effects induced by varying the bias voltage from 4 to 8 kV, it is possible to by unipolar nanosecond pulsed electric fields (nsPEF) have vary the amplitude of the pulses from about 2 to 4 kV. been observed when bipolar –a negative polarity pulse follows a positive polarity pulse– [1] or paired –two identical pulses with positive polarity– [2] nsPEF are applied, respectively. However, the precise mechanism responsible for these phenomena remains unknown. The possibility to explore different pulse durations as well as different delays between a positive and a negative polarity or two positive polarities might be of interest to provide new insights for the understanding of the above-mentioned events. The aim of this work is to introduce a novel versatile high-voltage generator able to provide 10-ns bipolar or paired pulses with very short interphase intervals between 17 and 360 ns. METHODS The generator proposed in this study exploits the frozen- wave concept fully detailed in [3]. The main components Figure 1: 10-ns bipolar (solid lines) and paired (dashed are: 1) a high-voltage DC power supply (SR20kV-300W, lines) pulses with a delay of 17 ns. Bias voltage is set to 4 Technix, France), 2) two photoconductive switches (black lines) and 8 kV (gray lines). (PCSSs) embedded in a 2- and 3- port coaxial boxes filled with oil, 3) a high-energy 3-ns pulsed laser (PL2241A, CONCLUSIONS Ekspla, Lithuania), 4) a tap-off (Model 245-NMFFP-100, In this study, a new flexible generator for in vitro Barth Electronics, Inc, NV, USA), connected between the bioelectric experiments has been introduced. The main generator and the electrodes, for the real-time measurement novelty consists in the possibility to obtain bipolar and of the generated pulses, and 5) a 12-GHz oscilloscope paired pulses with different delays between the polarities in (DSO, TDS6124C, Tektronix, USA) for the visualization of the ns range (17 – 360 ns) by varying the length of the the pulses. The energy delivered by the high-voltage DC transmission line at the output of the 2-port box. power supply is stored into a transmission line (coaxial Complementary information can be found in [4]. cable) and conveyed to the load (biological cells) through a pair of electrodes when the laser optically triggers, REFERENCES simultaneously, the two PCSSs. The 3-port box is [1] B. L. Ibey et al. , "Bipolar nanosecond electric pulses are connected to the high-voltage DC power supply, the tap-less efficient at electropermeabilization and killing cells off, and the 2-port box. Depending on the termination at the than monopolar pulses", Biochem. Biophys. Res. output of the 2-port coaxial box it is possible to generate Commun. , vol. 443, pp. 568–573, 2014. unipolar, bipolar, or paired pulses by using a 50 Ω [2] E. C. Gianulis, M. Casciola, S. Xiao, O. Pakhomova, terminator, short circuit, or open circuit, respectively. The and A. Pakhomov, "Electropermeabilization by uni- or duration of the interphase interval is established by the bipolar nanosecond electric pulses: The impact of length of the transmission line at the output of the 2-port extracellular conductivity", Bioelectrochemistry, vol. coaxial box. In this study, interphase delays within the 17– 119, pp. 10–19, 2018. 360 ns range were generated using a coaxial cable whose [3] D. Arnaud-Cormos, V. Couderc, and P. Leveque, length went approximately from 0.5 up to 35 m. In addition, "Photoconductive switching for pulsed high-voltage the proposed generator allows to obtain pulses of different generators", Handbook of Electroporation, D. duration by varying the length of the coaxial cable Miklavcic, Cham: Springer International Publishing, connecting the 2- and 3- port boxes, i.e., for two 10 ns- 2017, pp. 1–21. duration pulses, a 2-m cable was used. [4] R. Orlacchio, L. Carr, C. Palego, D. Arnaud-Cormos, and P. Leveque, "High-voltage 10 ns delayed paired or RESULTS bipolar pulses for in vitro bioelectric experiments", Figure 1 shows an example of bipolar and paired pulses Bioelectrochemistry, (in press) . with the interphase interval of 17 ns, obtained with a ~0.5- 18 EBTT WORKSHOP 2020 SHORT PRESENTATIONS Detection of electroporation using bioluminescence of biological cells Vitalij Novickij1, Auksė Zinkevičienė2, Veronika Malyško1, Jurij Novickij1, Julita Kulbacka3, Nina Rembialkowska3 and Irutė Girkontaitė2; 1 Faculty of Electronics, Vilnius Gediminas Technical University, Vilnius, LITHUANIA 2 State Research Institute Centre for Innovative Medicine, Department of Immunology, Vilnius, LITHUANIA 3 Department of Molecular and Cellular Biology, Wroclaw Medical University, Wroclaw, POLAND INTRODUCTION Electroporation is the methodology which is applied to increase and control cell membranes permeability based on pulsed electric field (PEF) [1]. The combination of electroporation phenomenon with drugs, which is known as electrochemotherapy, is the highly effective method in cancer treatment [2][3]. The effectiveness and result of electrochemotherapy depends on electric field amplitude, pulse shape, duration, repetition rate and other physical factors. In this work, we propose a cell membrane permeabilization and electroporation efficiency in vitro detection model based on Sp2/0 bioluminescent myeloma cells. METHODS SP2/0 cells were maintained in RPMI 1640 supplemented with 2 mM glutamine, 100 U/ml penicillin, Figure 1: Changes in cell bioluminescence depending on 100 mg/ml streptomycin and 10% of fetal calf serum (FCS). pulsed electric field treatment. All cell culture reagents were obtained from Gibco, Thermo Fisher Scientific, USA. Initially the cell were electro- CONCLUSIONS transfected (4 x 100 µs x 1.2 kV/cm) with Luciferase- The bioluminescence response is dependent on the cell pcDNA3 plasmid (Adgene plasmid #18964, a kindly gift permeabilization state and can be effectively used to detect from William Kaelin) linearized with Bgl II. permeabilization. During saturated permeabilization the Up to 3 kV, 100 ns – 1 ms square wave high voltage and methodology accurately predicts the losses of cell viability high frequency pulse generator and electroporation cuvette due to irreversible electroporation. with 1 mm gap aluminum electrodes (Biorad, Hercules, USA) was used for electroporation. Electroporation has been ACKNOWLEDGEMENTS studied using the 100 µs x 1–8 pulsing protocols in 1–2.5 This work was supported by grant Nr. S-MIP-19-22 from kV/cm PEF range. Research Council of Lithuania. The study was also partly After electroporation 40 µL of the cells (each data point) supported by PL NCN Grant SONATA BIS 6 were transferred into the white plates with 96 wells. D- (2016/22/E/NZ5/00671; PI: J. Kulbacka). Luciferin (Promega, USA) was added to the cells at final concentration of 150 µg/ml. The luminescence of SP2/0 cells REFERENCES was evaluated using a Synergy 2 microplate reader and Gen5 [1] U. Kauscher, M.N. Holme, M. Björnmalm, M.M. software (BioTek, USA). Stevens, "Physical stimuli-responsive vesicles in drug delivery: Beyond liposomes and polymersomes," RESULTS Advanced Drug Delivery Reviews, 2018. The changes in bioluminescence were detected after [2] D. Miklavčič, G. Serša, E. Brecelj, J. Gehl, D. Soden, G. using microsecond range (100 µs x 1–8) PEF treatment and Bianchi, P. Ruggieri, C.R. Rossi, L.G. Campana, T. Jarm, the summary of the data is presented in Fig. 1. "Electrochemotherapy: Technological advancements for In all cases, (1–2.5 kV/cm) when the electroporation was efficient electroporation-based treatment of internal predominantly reversible a statistically significant increase tumors," Med. Biol. Eng. Comput., 2012. of RLU was observed. By increasing electric field to 1.5 [3] T. Kotnik, L. Rems, M. Tarek, D. Miklavčič, kV/cm (which corresponds to partly irreversible "Membrane Electroporation and electroporation) RLU significantly decreased after 4 pulses Electropermeabilization: Mechanisms and Models," indicating loss in cell viability. Annu. Rev. Biophys., 2019. 19 EBTT WORKSHOP 2020 SHORT PRESENTATIONS Bacterial inactivation with combination of electroporation and antibiotics with different targets Žana Lovšin, Tadej Kotnik, University of Ljubljana, Faculty of Electrical Engineering, Trzaska 25, SI-1000 Ljubljana, SLOVENIA INTRODUCTION inactivation rates without electroporation as ampicillin, but The growing number of antibiotic-resistant when combined with electroporation, the inactivation rates microorganisms has increased the demand for alternative were lower than the corresponding ones obtained with bacterial inactivation methods. One of the most efficient ampicillin. The reason could be in the location of the such methods is irreversible electroporation [1], however, antibiotics’ target, which is intracellular for ciprofloxacin, alone it often induces insufficient inactivation, motivating which must thus traverse the membrane fully in order to the investigation into synergistic treatments [2]. In such inhibit DNA replication. Tetracycline alone had very little treatments, electroporation is combined either with physical effect even at the highest concentration investigated. methods or antimicrobial agents [1], which become more Electroporation increased its effect, but with lower electric effective if the pathogens are previously sublethally fields this difference was small. Tetracycline must get across damaged by electroporation [2]. the membrane to inhibit protein synthesis and it is a bit larger Our aim was to assess the inactivation of a non-resistant than ciprofloxacin, so that could explain why lower electric strain of Escherichia coli using synergistic treatment of fields are not sufficient for its internalization. electroporation and antibiotics with different modes of action. Here we report our first results. Table 1: Inactivation rates (log+-sd) for E. coli with electroporation and three different antibiotics. E=electric METHODS field (5, 10, 15 or 20 kV/cm), MIC=minimal inhibitory Working concentrations were determined based on MIC concentration in overnight culture. (minimal inhibitory concentration in an overnight culture) Parameters Ampicillin Ciprofloxacin Tetracycline concentrations of antibiotics for our non-resistant E. coli strain (K12 ER1821, New England BioLabs). Antibiotics MIC -0.55 +-0.07 -0.50+-0.1 -0.07+-0.02 used in this study were ampicillin (#A9518), tetracycline E5-MIC -0.80+-0.28 -0.67+-0.25 -0.23+-0.03 (# T3383) and ciprofloxacin (#17850). E10-MIC -1.64+-0.22 -1.23+-0.41 -0.75+-0.06 For electroporation, a single rectangular electric pulse E15-MIC -2.20+-0.25 -1.90+-0.53 -1.28+-0.07 was used, with a duration of 1 ms and an amplitude (voltage- E20-MIC -2.65+-0.22 -2.18+-0.37 -1.78+-0.12 to-distance ratio, serving as an approximation of the electric field) of up to 20 kV/cm. For each antibiotic, 30xMIC -2.17+-0.14 -2.10+-0.3 -0.32+-0.01 25 different combinations were tested: five different E5-30xMIC -2.86+-0.13 -2.33+-0.24 -0.73+-0.03 concentrations of the antibiotic and five different pulse E10-30xMIC -3.31+-0.04 -3.08+-0.15 -2.05+-0.17 amplitudes. Samples were incubated for 2 hours 40 minutes E15-30xMIC -3.80+-0.16 -3.44+-0.13 -2.71+-0.23 at room temperature before plating. CFU counts were made the next day and inactivation rate was determined as log E20-30xMIC -4.02+-0.15 -3.47+-0 -3.45+-0.15 (N/N0). ACKNOWLEDGEMENTS RESULTS This work was supported by the Slovenian Research With electroporation alone, we achieved up to -1.8 log Agency (funding for Junior Researcher to ŽL). Experiments inactivation at 20 kV/cm. When only antibiotics were used, were performed within Infrastructure Programme: Network increasing the concentration from MIC to 30xMIC increased of research infrastructure centers at University of Ljubljana bacterial inactivation, but with tetracycline this difference (MRIC UL IP-0510). was small (0.25 log). When a combination of electroporation and antibiotic were used, bacterial inactivation increased REFERENCES with increasing electric field and antibiotic concentration. [1] A.L. Garner, Pulsed electric field inactivation of However, with tetracycline, this increase in inactivation was microorganisms: from fundamental biophysics to small for electric field below 10 kV/cm. Highest inactivation synergistic treatments, Applied Microbiology and was achieved with the highest concentration of ampicillin Biotechnology, vol. 103, pp. 7917–7929, 2019. and electric field of 20 kV/cm (-4.02 log). The results are [2] D. Berdejo, E. Pagan, D.G. Gonzalo and R. Pagan, summarized in Table 1. Exploiting the synergism among physical and chemical processes for improving food safety, Current Opinion in CONCLUSIONS Food Science, vol. 30, pp. 14-20, 2019. Highest inactivation was achieved with ampicillin which targets cell wall synthesis. Ciprofloxacin had similar 20 EBTT WORKSHOP 2020 SHORT PRESENTATIONS Advanced numerical model of a continuous PEF treatment chamber Peter Lombergar, Samo Mahnic-Kalamiza, Karel Flisar, Damijan Miklavcic; University of Ljubljana, Faculty of Electrical Engineering, Trzaska 25, SI-1000 Ljubljana, SLOVENIA INTRODUCTION RESULTS AND DISCUSSION Pulsed electric field (PEF) treatment is a promising novel The model was validated with experimentally measured technique in the field of food processing, biotechnology, and electric current and changes in temperature of the fluid environmental engineering. It is based on applying short, within the outlet channel of the treatment chambers during high-intensity electric pulses. One of the main components pulse application. Good agreement between measured and of a PEF treatment system is a treatment chamber, in which simulated values was obtained (Figure 1). the product is exposed to electric pulses. Numerical modelling provides a unique insight into the distribution of the electric field, fluid flow, and temperature inside the treatment chamber, which cannot be provided experimentally. Existing literature is reporting only on stationary state simulations that do not account for the influence of individual electric pulses, but rather employ a time- averaging of the voltage applied to electrodes. In the study we present a time-dependent numerical model of a continuous PEF treatment chamber where the influence of each individual pulse is accounted for. MATERIALS AND METHODS Experiments were performed using 0.18 % NaCl solution, since its electrical conductivity is similar to that of fruit juices. Saline solution was pumped with a syringe pump Figure 1: Comparison of simulated and measured Aladin-1000 (World Precision Instruments, USA) and temperature change within the outlet channel of large commercial 50 ml plastic syringe. Three different continuous parallel plate treatment chamber. Pulse protocol: U m = 1000 treatment chambers were used; a co-linear treatment V, τ = 100 µs, f = 1.1 Hz, F = 20 ml/min. chamber [1], a large parallel plate treatment chamber [2], and a small parallel plate treatment chamber (University of Presented numerical model allows for the analysis of Ljubljana, Slovenia). Unipolar square wave pulses were variables in the treatment chamber (temperature, electric applied using a prototype pulse generator [1]. Voltage and field, etc.) during each individual electrical pulse, between current were recorded with an oscilloscope, a high voltage pulses and after pulse application. It can be used as a differential probe, and a current probe (all Teledyne LeCroy, complement to experiments or as a substitution, to study the USA). During the application of electrical pulses, the effect of different values of PEF parameters that could not be temperature within the outlet channel of the chamber was performed due to experimental limitations. recorded using an optical thermometer (OpSens Solutions INC, Canada). ACKNOWLEDGEMENTS In the study protocols with fixed number of pulses We acknowledge the financial support of the Slovenian applied (e.g. N = 10) and continuous pulse application were Research Agency (ARRS), through research programme P2- used for each treatment chamber. Pulse lengths ( τ) of 10 µs 0249, postdoctoral project Z7-1886, and the Infrastructural and 100 µs were used in combination with repetition Centre “Cellular Electrical Engineering” (I0-0022). frequencies ( f) of 10.3 Hz and 1.1 Hz, respectively. Flow rates ( F) and peak voltage ( U REFERENCES m) for each chamber were chosen within limitations of experimental equipment. [1] K. Flisar et al. , ‘Testing a prototype pulse generator for a Time-dependent numerical model was built in COMSOL continuous flow system and its use for E. coli inactivation Multiphysics. In the numerical model, geometries of and microalgae lipid extraction’, Bioelectrochemistry, treatment chambers were constructed in 3D and constituent vol. 100, pp. 44–51, 2014. materials were chosen from the Material Library. A [2] G. Pataro et al. , ‘Pulsed electric fields assisted microbial stationary study step was used to solve the fluid flow profile. inactivation of S. cerevisiae cells by high pressure carbon Two separate time-dependent study steps were used to model dioxide’, The Journal of Supercritical Fluids, vol. 54, no. conditions in a chamber during and after pulse application. 1, pp. 120–128, 2010. Electrical pulses were described with experimentally recorded voltage and Events interface was used to accurately model each individual pulse. 21 EBTT WORKSHOP 2020 SHORT PRESENTATIONS Calcium electroporation as a potential ovarian cancer treatment modality Zofia Łapińska, Julita Kulbacka, Jolanta Saczko Department of Molecular and Cellular Biology, Wroclaw Medical University, Borowska 211A, 50- 556, Wroclaw POLAND INTRODUCTION production. The depletion of ATP resources leads the cell to Epithelial ovarian cancer is one of the most fatal necrosis. Moreover, it has been shown that this disturbance estrogen-dependent gynecological malignancies. of calcium homeostasis can induce the production The high mortality rate is associated with the fact, that of reactive oxygen species and the activation of proteases almost 75% of women are not diagnosed before the disease and lipases [3]. The mechanism of the CaEP procedure has progressed to stage III or IV [1]. This is because, in most is shown in Fig. 1. cases, cancer has no or very vague symptoms. So far, CaEP has been tested in vitro and in vivo in five The conventional treatment strategy is mainly based on the human tumors grown subcutaneously combination of the tumor resection surgery and in immunocompromised mice. CaEP induced tumor cell chemotherapy with cisplatin or its derivatives. death in all tested tumors with different sensitivity In the early stages of the disease, this approach is initially to treatment [5]. Unfortunately, there are no literature reports effective. Unfortunately, in a large number of patients, about the effectiveness of this treatment method in ovarian the disease relapse without responding to re-treatment. cancer. Therefore, there is a necessity to develop new therapeutic methods, that also have less toxicity to normal cells. AIM OF THE FUTURE WORK Calcium electroporation (CaEP) is a novel promising Our future work will focus on investigating the cancer treatment based on conventional effectiveness of calcium electroporation in ovarian cancer as electrochemotherapy (ECT), where the chemotherapeutic a treatment modality. agent is replaced by calcium ions (Ca2+) [2]. Intracellular calcium (Ca2+) is an essential second messenger involved in Funding: Subsidy of Department of Molecular and Cellular a wide range of cellular processes, from fertilization to cell Biology SUB.D260.20.009. death [3]. In the eukaryotic cell, there is a concentration gradient of calcium ions across the cell membrane. REFERENCES Depending on the location, this gradient is 10-20,000-fold [1] C. Stewart, C. Ralyea, and S. Lockwood, “Ovarian and it is tightly controlled using several mechanisms [5]. Cancer: An Integrated Review,” Seminars in Oncology Nursing, vol. 35, no. 2. W.B. Saunders, pp. 151–156, 2019. [2] S. K. Frandsen and J. Gehl, “A review on differences in effects on normal and malignant cells and tissues to electroporation-based therapies: A focus on calcium electroporation,” Technology in Cancer Research and Treatment, vol. 17. pp. 1–6, 2018. [3] B. Staresinic et al. , “Effect of calcium electroporation on tumour vasculature,” Sci. Rep. , vol. 8, no. 1, pp. 1– 14, 2018. [4] S. K. Frandsen et al. , “Normal and malignant cells exhibit differential responses to calcium Figure 1: Calcium electroporation (CaEP). (A) 5-225 mM electroporation,” Cancer Res. , vol. 77, no. 16, pp. CaCl 4389–4401, 2017. 2 injection into the tumor cell (B) electroporation and membrane permeabilization (C) influx of Ca2+ (D) cell [5] Y. Chen, et al. , “Chemical Enhancement of Irreversible necrosis [2][6][7]. Electroporation: A Review and Future Suggestions,” Technology in cancer research & treatment, vol. 18. pp. 1–13, 2019. The formation of pores in the cell membrane as a result [6] K. L. Hoejholt et al. , “Calcium electroporation and of electroporation increases its permeability, which allows electrochemotherapy for cancer treatment: Importance the influx of Ca2+ into the tumor cell and an increase of its of cell membrane composition investigated by intracellular concentration [4]. The cell extrudes the excess lipidomics, calorimetry and in vitro efficacy,” Sci. of Ca2+ outside through increased activity of plasma Rep. , vol. 9, no. 1, pp. 1–12, 2019. membrane calcium ATPase (PMCA). The energy needed for [7] C. C. Plaschke et al. , “Calcium Electroporation for its functioning comes from adenosine triphosphate (ATP) Recurrent Head and Neck Cancer: A Clinical Phase I hydrolysis. A high concentration of Ca2+ leads to the Study,” 2019. amplified activity of Na+/K+-ATPase [5]. The loss of the electrochemical gradient of mitochondrial membranes has also been noted, which results in the inhibition of ATP 22 EBTT WORKSHOP 2020 SHORT PRESENTATIONS Gene therapy of murine melanoma B16F10 using a combination of interleukin 2 and 12 gene electrotransfer T.Komel1,2, M. Bosnjak1, S. Kranjc Brezar1, E. Signori3,4, M. De Robertis5,6, G. Sersa1,7 and M. Cemazar1,8; 1Institute of Oncology Ljubljana, Department of Experimental Oncology, Zaloska 2, SI-1000 Ljubljana, SLOVENIA; 2University of Ljubljana, Faculty of Medicine, Vrazov trg 2, SI-1000 Ljubljana, SLOVENIA; 3National Research Council-Institute of Translational Pharmacology (CNR-IFT), Via Fosso del Cavaliere 100, Rome, ITALY; 4University Campus Bio-Medico of Rome, School of Medicine, Via Álvaro del Portillo 21, 00128 Rome, ITALY; 5Institute of Biomembranes, Bioenergetics and Molecular Biotechnologies, Consiglio Nazionale delle Ricerche (CNR), 70126 Bari, ITALY; 6Department of Biosciences, Biotechnology and Biopharmaceutics, University of Bari Aldo Moro, 70125 Bari, ITALY; 7University of Ljubljana, Faculty of Health sciences, Zdravstvena pot 5, SI – 1000 Ljubljana, SLOVENIA; 8University of Primorska, Faculty of Health sciences, Polje 42, SI – 6310 Izola, SLOVENIA INTRODUCTION complete response (Figure1). Additionally, no tumour Immunotherapy has become an important pillar of cancer growth after rechallenge was observed in mice from the GET treatment improving the outcome of many patients [1]. One combination group. Increased protein expression of IL-2 and approach is the use of interleukin 2 (IL-2) and 12 (IL-12) that IL-12 was detected only in the respective groups (GET pIL- are potent cytokines that exhibit antitumor effect [2]. These 2 or GET pIL-12) and in GET combination group. two cytokines use separate signaling pathways to induce complementary biological effects, thus the aim of our study CONCLUSIONS was to determine the anitumour effects of gene therapy of To conclude, we successfully transfected B16F10 cells murine melanoma B16F10 using a combination of IL-2 and with pIL-2 and pIL-12 in vitro. Treatment of cells with IL-12 gene electrotransfer (GET). plasmid DNA and thereafter with ECT pulses yielded higher cell viability but lower expression than treatment with GET METHODS pulses. The treatment with GET pulses coupled with pIL-12 The cytotoxic effect of GET coupled with both plasmids alone and in combination with pIL-2 resulted in complete individually and combined was determined in B16F10 responses of tumours. In addition, all mice in the murine melanoma cell line. Two pulse protocols were used, combination group developed also immune memory, as they called ECT (8 pulses with a voltage to distance ratio of 1300 remained tumours free 100 days after the rechallenge. V/cm, duration of 100 µs and frequency of 1 Hz) and GET (8 pulses with a voltage to distance ratio of 600 V/cm, Treatment groups duration of 5 ms and frequency of 1 Hz). Cell viability was 600 determined using PrestoBlue assay and the mRNA ) GET Comb 3 expression profile of Il2 and Il12 in cells was determined by m GET pIL-12 400 GET pIL-2 qRT-PCR. In vivo gene electrotransfer of pIL-2 and pIL-12 e (m using GET protocol was performed in C57BL/6 mice 200 bearing subcutaneous B16F10 melanoma tumours. Tumour or volum growth delay was calculated and the IL-2 and IL-12 protein Tum 0 expression in tumour and serum samples was determined 0 20 40 60 using ELISA. The experimental procedures were performed Time (days) in compliance with the guidelines for animal experiments of Figure 1: Growth curves of tumours in the therapeutic the EU directive (2010/63/EU) and the permission from the treatment groups. Veterinary Administration of the Ministry of Agriculture, Forestry and Food of the Republic of Slovenia (permission REFERENCES no. U34401–1/2015/38). [1] K. Esfahani, L. Roudaia, N. Buhlaiga, S. V. Del Rincon, RESULTS N. Papneja, and W. H. Miller, “A review of cancer The viability of cells ( in vitro) treated with both plasmids immunotherapy: From the past, to the present, to the individually and combined coupled with ECT pulses was future,” Curr. Oncol., vol. 27, no. S2, pp. 87–97, 2020. notably higher compared to the viability after treatment with [2] Gollob JA, Schnipper CP, Murphy EA, Ritz J, Frank DA. GET pulses (p<0.05). However, the expression of mRNA The functional synergy between IL-12 and IL-2 involves coding for Il2 and Il12 in cells treated with ECT pulses was p38 mitogen-activated protein kinase and is associated lower than in cells treated with GET pulses. In vivo, with the augmentation of STAT serine phosphorylation. prolonged tumour growth delay was observed in the GET J Immunol. 162(8): 4472-81, 1999. pIL-12 and GET combination group and in some mice also 23 EBTT WORKSHOP 2020 SHORT PRESENTATIONS Electroporation based modifications of plasma membrane organization in cancer cells for targeted therapy Urszula Szwedowicz, Jolanta Saczko, Anna Choromańska; Department of Molecular and Cellular Biology, Wroclaw Medical University, Borowska 211A, 50-556 POLAND INTRODUCTION Pharmacotherapy has always been the most frequently miRNAs MODULATE VAROIUS DRUG RESISTANS chosen therapeutic option to treat a patient. Currently, the MECHANISM biggest problem is not so much finding a drug for a specific Considering the significant role of proteins in multidrug disease, but looking for an effective method of its resistance, a method was developed that allows miRNA to administration, so that it has a justified clinical effect. Many regulate their expression. The prospect of influencing the active substances have been tested for pharmacological number of synthesized membrane transporters in the cell activity and have often obtained spectacular results in vitro, membrane has opened the possibility of using miRNAs in but their final effect in the subsequent stages of the tests was the pharmacotherapy of neoplastic diseases. Another far from the expected. One of the main reasons is the mechanism that can be modulated by miRNAs is autophagy. imperfection of the absorption and distribution of drugs in This process maintains homeostasis in cells by breaking the human body. Effective drug action is also related to the down damaged cellular elements that can later be reused as concentration at the site of action, which is often only a nutrients. Inhibiting this process with miRNA allows fraction of the starting concentration. Even if the pathway of disturbing the further development and proliferation of the the drug in the body is shortened, the question of the direct neoplastic cell, which loses the possibility of its repair and effect of the drug on target cells remains [1]. rearrangement [5]. Some miRNAs are designed to inhibit the apoptosis of cancer cells, so they indirectly contribute to MULTIDRUG RESISTANCE OF NEOPLASTIC their immortality. Therefore, scientists are interested in CELLS substances that will limit the action of such miRNAs [6]. Due to numerous abnormalities, neoplastic cells lose Physical and mechanical methods, i.e. electroporation and their self-control mechanisms, proliferate, are resistant to microgravity, that will be used in the research, are aimed at apoptosis, tend to expand and invade, and are characterized sensitizing cancer cells to the chemotherapeutic agent used. by specific metabolism. Due to a large number of divisions, Cells with an altered structure of the cell membrane and its they also quickly acquire new features that make it easier for protein elements will be assessed in terms of their ability to them to adapt to the prevailing conditions. One of them is the accumulate the drug after the applied treatments concerning development of multidrug resistance (MDR), which is the control sample. mainly based on the altered membrane composition. Such cells often contain more cholesterol in it, making it more REFERENCES rigid and less permeable to various substances, including [1] Wiela-Hojeńska A. Kinetyczna charakterystyka losów drugs [2]. leków w organizmie Internet: Another type of multi-drug resistance in neoplastic cells https://www.farmacja.umed.wroc.pl/sites/default/files/st is the overexpression of membrane transporters. These ruktura/farmacja/farmakologia/1_Farmakokinetyka.pdf proteins limit the supply of the drug inside the cell or even [2] Zalba S., L.M. ten Hagen T. Cell membrane modulation actively pump it out of the cell. The best-known membrane as adjuvant in cancer therapy Internet: transporters are ABC transporters, which, even against the https://www.sciencedirect.com/science/article/pii/S0305 concentration gradient, can excrete the chemotherapeutic 737216301153. agent toxic to the cell beyond the cell membrane, preventing [3] Popęda M., Płuciennik E., Bednarek A. Białka w its accumulation [3]. oporności wielolekowej nowotworów Postepy Hig Med Dosw (online), 2014; 68: 616-632 ELECTROCHEMOTHERAPY [4] Skołucka N., Saczko J., Kotulska M., Kulbacka J., One of the possibilities of modulating biological Choromańska A. Elektroporacja i jej zastosowanie Pol. membranes is the use of electroporation. This method is Merk. Lek., 2010, XXVIII, 168, 501 aimed at increasing the membrane permeability by [5] An X., Sarmiento C., Tan T., Zhu H. Regulation of disrupting its integrity under the influence of an multidrug resistance by microRNAs in anti-cancer electromagnetic field. This effect is reversible and, therapy Acta Pharmaceutica Sinica B Volume 7, Issue 1, depending on the pulses used, it may last several minutes. January 2017, Pages 38-51 The created pores allow free movement of particles of larger [6] Aung T., Qu Z., Kortschak D., Adelson D. Understanding size into the cell, which allows a better pharmacological the Effectiveness of Natural Compound Mixtures in effect to be obtained, without an overall increase in the Cancer through Their Molecular Mode of Action Int. J. concentration of the drug and surgical intervention. Mol. Sci. 2017, 18, 65. Temporary damage to the regular structure of the membrane will also disrupt the placement of drug transport proteins, thus their localization in the membrane and activity [4]. 24 EBTT WORKSHOP 2020 SHORT PRESENTATIONS Percutaneous Electrochemotherapy of Hepatocellular Carcinoma: A case report Miha Stabuc1 , Rok Dežman1, Maja Cemazar2, Mihajlo Djokic,3, Bor Kos4, Gregor Sersa2, Peter Popovic1; 1Universty Medical Centre Ljubljana, Clinical Institute of Radiology, Zaloška 7, Ljubljana, SLOVENIA 2Institute of Oncology Ljubljana, Department of Experimental Oncology, Zaloška 2, Ljubljana, SLOVENIA 3University Medical Centre Ljubljana, Clinical Department of Abdominal Surgery, Zaloška 7, Ljubljana, SLOVENIA 4University of Ljubljana, Faculty of Electrical Engineering, Tržaška cesta 25, Ljubljana, SLOVENIA INTRODUCTION and grant of University Clinical Center Ljubljana Electrochemotherapy (ECT) is a local nonthermal #20180061. treatment of cancer that combines chemotherapy and the application of electric pulses. ECT is already recognized as REFERENCES safe and effective treatment of cutaneous tumors and skin [1] Campana LG, Clover AJ, Valpione S, Quaglino P, Gehl metastases [1-3]. It has also proved to be safe and effective J, Kunte C. Recommendations for improving the quality treatment of deep-seated tumors during open surgery [4- of reporting clinical electrochemotherapy studies based 6]. Percutaneous ECT has also already been described, but on qualitative systematic review. Radiol not on HCC [7,8]. Oncol. 2016;50:1–13. [2] Gehl J, Sersa G, Matthiessen LW, Muir T, Soden D, CASE DESCRIPTION Occhini A. Updated standard operating procedures for We report a case of a 66-year old man with Child A liver electrochemotherapy of cutaneous tumours and skin cirrhosis with single HCC with diameter of 18mm in liver metastases. Acta Oncol. 2018;57:874–82. segment 3 and no signs of extrahepatic disease. His [3] Campana LG, Miklavčič D, Bertino G, Marconato R, documentation was reviewed on hepatopancreaticobiliary Valpione S, Imarisio I. Electrochemotherapy of (HPB) multidisciplinary team meeting, which recognized superficial tumors - current status: basic principles, this lesion to be suitable for minimally invasive percutaneous operating procedures, shared indications, and emerging electrochemotherapy. applications. Semin Oncol. 2019;46:173–91. The detailed technological procedure has already been [4] Edhemovic I, Brecelj E, Gasljevic G, Snoj M, Miklavcic reported in another article [9]. Treatment was performed D, Gadzijev E. Intraoperative electrochemotherapy of under general anesthesia and deep muscle relaxation. colorectal liver metastases. J Because the lesion was not visible on ultrasound the insertion SurgOncol. 2014;110:320–7. of four long needle electrodes was performed under contrast [5] Djokic M, Cemazar M, Popovic P, Kos B, Dezman R, enhanced cone-beam computed tomography (CBCT) Bosnjak M. Electrochemotherapy as treatment option guidance. Before the electric pulses were delivered, for hepatocellular carcinoma, a prospective pilot bleomycin was administrated intravenously. study. Eur J Surg Oncol. 2018;44:651–7. After electrode extraction, control CBCT with contrast [6] Edhemovic I, Brecelj E, Cemazar M, Boc N, Trotovsek injection through microcatheter showed no bleeding or B, Djokic M. Intraoperative electrochemotherapy of hematoma and also that the lesion was avital. colorectal liver metastases: a prospective phase II There were no complications after the procedure and the study. Eur J Surg Oncol. 2020. patient was discharged the day after the procedure with [7] Tarantino L, Busto G, Nasto A, Fristachi R, Cacace L, analgesics and antithrombotic prophylaxis. Talamo M. Percutaneous electrochemotherapy in the treatment of portal vein tumor thrombosis at hepatic RESULTS hilum in patients with hepatocellular carcinoma in In this case, percutaneous ECT showed to be an effective cirrhosis: a feasibility study. World J and safe treatment of single small HCC, considering the Gastroenterol. 2017;23:906–18. complete response immediately after the procedure, absence [8] Cornelis FH, Ben Ammar M, Nouri-Neuville M, Matton of disease relapse after 18 months and absence of adverse L, Benderra MA, Gligorov J. Percutaneous image- events. guided electrochemotherapy of spine metastases: initial experience. Cardiovasc Intervent CONCLUSION Radiol. 2019;42:18069. Percutaneous ECT could be an effective technique for the [9] Djokic M , Dezman R, Cemazar M, Stabuc M, Petric M, treatment of HCC, especially for tumors not suitable for Smid LM, Jansa R, Plesnik B, Bosnjak M, Lampreht surgery or percutaneous ablation. However, more studies are Tratar U, Trotovsek B, Kos B, Miklavcic D, Sersa G, needed to confirm our results. Popovic P Percutaneous image guided electrochemotherapy of hepatocellular carcinoma: ACKNOWLEDGEMENTS technological advancement Radiol Oncol. 2020 Jun This work was financially supported by the Slovenian 20;54(3):347-352. Research Agency (ARRS), grant No. P3-0003 and P2-0249 25 EBTT WORKSHOP 2020 SHORT PRESENTATIONS Treatment of lymph node metastases from colorectal cancer with percutaneous image guided electrochemotherapy: a case report Marko Orešnik1, Rok Dežman1, Miha Štabuc1, Maja Čemažar2, Maša Bošnjak2, Tanja Mesti2, Bor Kos3, Gregor Serša2, Peter Popovič1 1Institute of Radiology, University Medical Centre Ljubljana, Zaloška cesta 7, 1000 Ljubljana, Slovenia; 2Institute of Oncology Ljubljana, Zaloška cesta 2, 1000 Ljubljana, Slovenia; 3Faculty of Electrical Engineering, University of Ljubljana. INTRODUCTION intravenous contrast showed centrally necrotic treated lesion, Electrochemotherapy is effective in treatment of a variety however new metastasis occured in other locations. of tumors. Intraoperative electrochemotherapy of colorectal liver metastasis proved to be efficient treatment modality and could be translated into percutaneous treatment of colorectal lymph node metastasis. The aim of this case presentation is to demonstrate the feasibility, safety and effectiveness of electrochemotherapy with percutaneous approach on colorectal lymph node metastasis. PATIENT AND METHODS A 47-year-old man was presented at multidisciplinary team meeting with a diagnosis of lymph node metastasis from recurrent adenocarcinoma of the ascending colon in May 2020. FDG-PET/CT from two months earlier showed pathological FDG uptake form enlarged retroperitoneal lymph node, indicating metastasis (Figure 1). Subsequent abdominal CT scan demonstrated an enlarged right para- aortic lymph node measuring 3.5 cm in diameter. At the time the patient has just finished first cycle of FOLFOXIRI regimen in combination with bevacizumab. According to the multidisciplinary team meeting, the patient was eligible candidate for percutaneous electrochemotherapy. after the procedure (A, B). Position of the electrodes in relation to metastasis on cone-beam CT (C, D). Treatment was performed under general anesthesia and deep muscle relaxation. Figure 1: A 47-year old male with colorectal lymph node Five electrodes with 3 cm active length were placed metastasis. PET/CT demonstrating the lesion before and percutaneously around the tumor under stereotactic CBCT guidance. In keeping with European Standard Operating CONCLUSIONS Procedures on Electrochemotherapy (ESOPE) We describe the first case of percutaneous recommendations, 28.500 IU of bleomycin in 20 ml of electrochemotherapy of coloretal lymph node metastasis. physiological saline was administered intravenously in bolus This case shows that percoutaneous electrochemotherapy and two trains of 4 electric pulses (duration 100 µs, pulse could provide an effective treatment for a selected group of repetition frequency 1 kHz) of opposite polarity with patients with unresectable colorectal lymph node metastasis. voltage-to-distance ratio of 1000 V/cm were delivered A prospective cohort study is needed to confirm our findings. between all electrode pairs starting 8 minutes after the bleomycin injection (total number of pulses = 48). REFERENCES [1] I.Edhemovic, et al.,“Intraoperative electrochemotherapy RESULTS of colorectal liver metastases,” J Surg Oncol. , vol. Only a slight increase in inflammatory parameters was 110(3), pp.320-7, 2014. observed during hospitalization, which was attributed to the [2] M. Djokic, et al., “Percutaneous Image Guided inflammatory reaction of the treated tissue to the procedure. Electrochemotherapy of Hepatocellular Carcinoma: Six weeks after percutaneous electrochemotherapy, Technological Advancement”, Radiol Oncol., vol. 54(3), control PET/CT scan showed marked reduction of FDG pp. 347–352, 2020. uptake from lymph node metastasis, with only peripheral rim [3] Fennell and R. Hauptmann, Electroporation and PEG of enhancement remaining. The patient was feeling well, in delivery of DNA into maize microspores. Plant Cell good physical condition and pain-free. On the second follow- Reports, vol. 11, pp. 567-570, 1992. up three months after the procedure, a control CT scan with 26 EBTT WORKSHOP 2020 SHORT PRESENTATIONS Comparable effectiveness of electrochemotherapy with cisplatin and oxaliplatin, but not bleomycin in B16F10, CT26 and 4T1 murine cell lines in vitro Urša Kešar1,2, Katja Uršič1, Tanja Jesenko1,2, Maja Čemažar1,3, Primož Strojan2,4, Gregor Serša1,5; 1 Institute of Oncology Ljubljana, Department of Experimental Oncology, Zaloška cesta 2, SI-1000 Ljubljana, SLOVENIA 2 Faculty of Medicine, University of Ljubljana, Vrazov trg 2, SI-1104 Ljubljana, SLOVENIA 3 Faculty of Health Sciences, University of Primorska, Polje 42, SI-6310 Izola, SLOVENIA 4 Institute of Oncology Ljubljana, Department of Radiation Oncology, Zaloška cesta 2, SI-1000 Ljubljana, SLOVENIA 5 Faculty of Health Sciences, University of Ljubljana, Zdravstvena pot 5, SI-1000 Ljubljana, SLOVENIA INTRODUCTION In electrochemotherapy (ECT), electric pulses increase the uptake of cytostatics in the cells which causes stronger cytotoxic effect [1]. Furthermore, ECT can induce immunogenic cell death (ICD), apoptosis or necrosis in tumor cells [1–3]. The aim of the study was to compare the sensitivity of three immunologically different cell lines to ECT with cisplatin (CDDP), oxaliplatin (OXA), or bleomycin (BLM) in vitro in order to determine the IC30, IC50 and IC70 concentrations of cytostatics. These concentrations will later be used for the evaluation of immunological effects of ECT. METHODS We used three different murine tumor cell lines: B16F10, CT26 and 4T1, which are known to induce tumors with different immunological profiles in vivo. We treated them Figure 1: A Surviving of cells after electroporation (EP) either with electric pulses alone or with CDDP, OXA and alone (n = 9); B IC50 concentration for ECT with OXA (n = BLM alone, as well as in combination with electric pulses. 3); C IC50 concentration for ECT with CDDP (n = 3); D For electroporation, we used plate electrodes with 2 mm IC50 concentration for ECT with BLM (n = 3). distance and ECT electric pulses (1300 V/cm, 100 µs, 1 Hz, 8 pulses). We compared the cytotoxicity of the three ACKNOWLEDGEMENTS cytostatics among cell lines using a clonogenic assay. This work received funding from Slovenian Research Additionally, we determined the concentration of tested Agency (ARRS): research programme P3-0003. drugs that induce death of 30 %, 50 % and 70 % of cells (IC30, IC50, IC70 values). REFERENCES [1] G. Sersa et al. , “Electrochemotherapy of tumors as in situ RESULTS vaccination boosted by immunogene electrotransfer,” All three cell lines were comparably sensitive to electric Cancer Immunology, Immunotherapy, vol. 64, no. 10. pulses alone and to ECT with CDDP and OXA at IC30, IC50 Springer Science and Business Media Deutschland and IC70 (IC50 results shown in Figure 1: A, B and C). GmbH, pp. 1315–1327, 02-Oct-2015. However, a statistically significant difference (p < 0.05) [2] C. Y. Calvet, D. Famin, F. M. André, and L. M. Mir, between CT26 and 4T1 cell lines at IC50 (but not at IC30 “Electrochemotherapy with bleomycin induces and IC50) for ECT with BLM was demonstrated (Figure 1: hallmarks of immunogenic cell death in murine colon D). cancer cells,” Oncoimmunology, vol. 3, no. 4, 2014. [3] R. M. Brock, N. Beitel-White, R. V. Davalos, and I. C. CONCLUSIONS Allen, “Starting a Fire Without Flame: The Induction of We have shown that all three cell lines are equally Cell Death and Inflammation in Electroporation-Based sensitive to electric pulses. However, 4T1 cells were more Tumor Ablation Strategies,” Frontiers in Oncology, vol. sensitive to ECT with BLM (IC50) than CT26 cells. The 10. Frontiers Media S.A., p. 1235, 28-Jul-2020. determined IC30, IC50 and IC70 values for all cytostatics in all three cell lines will enable us further exploration of immunological effects of ECT. 27 EBTT WORKSHOP 2020 SHORT PRESENTATIONS Adjuvant electrochemotherapy in canine oral melanoma – Case Report Jean C. S. Luz1, Cláudia Russo2; 1Cancer Institute of São Paulo, Av. Dr. Arnaldo, 251, 01246-000, São Paulo - BRASIL. ² Convet, Av. Brasil, 393 - Zona 08, 87050-465, Maringá – PR – BRASIL. INTRODUCTION The oral cavity is a common location for the development of neoplasia in small animals [1]. As the most common malignant oral tumor in dogs, oral melanoma typically occurs on the gingiva and is characterized by rapid growth, local invasion and early metastasis. In general, oral tumors can be difficult to manage surgically because of their close association with local periodontal structures including oral bones [1,2] and frequently are resistant to chemotherapy and radiation therapy [1,3] being the electrochemotherapy (ECT) a valuable indication [4]. CASE REPORT Chokito, a 13-years old dachshund, attended under tutor's complaint of halitosis was referred for treatment of periodontal disease. During orotracheal intubation, the presence of a neoformation mass of 3cm in the soft palate was detected (Figure 1a). Through histopathological analysis it was confirmed as melanoma. The indicated treatments were tumor debulking + ECT (8 pulses/ 1300 V/cm/ 100 µs/ 5 kHz) with I.V. bleomycin (15 UI/m²) in the surgical site as well as right submandibular lymphadenectomy due to fine- needle aspiration cytology (FNAC) indicating lymph node metastasis. Six sessions of carboplatin 300 mg/m² and melphalan (2mg/m²) was also administered. Oral cavity assessments under anesthesia never revealed recurrence, yet a second ECT session was performed 20 days after the first ECT. A new biopsy three weeks after the 2nd ECT was performed and indicated the absence of tumor cells in the surgical site. The animal was discharged from treatment follow-up 135 days after treatment without signs of tumor Figure 1: A. Tumor before first treatment (white line), 30th relapse (Figure 1b), and after 1 year and 8 months it remained day after biopsy. B. Tumor site (white line), 135th day after healthy and without local tumor recurrence or metastasis. treatment, with visualization of the caudal region of the soft palate (light area) and no signals of tumor relapse. Arrow: DISCUSSION epiglottis. Asterisk: right molar. Oral tumors in companion animals often arrive at an advanced stage in the veterinary clinic, due to the difficulty REFERENCES or lack of habit of visualizing the oral cavity by the animal's [1] H. Smith, H. Goldschmidt, P.M. Mcmanus, "A tutors. Due to the impossibility of a surgical resection with Comparative review of melanocytic neoplasms". Vet. safety margins in this case, debulking of the macroscopic Pathol, vol. 39, pp. 651–678, 2002. mass and cleaning of residual tumor cells with ECT was [2] JF Modiano, MG Ritt, J Wojcieszyn J, "The Molecular performed. Due to the early metastasis profile of positivity Basis of Canine Melanoma: Pathogenesis and Trends in in lymph node FNAC, systemic therapy (conventional Diagnosis and Therapy". J Vet Intern Med, vol. 13, pp. chemotherapy) was applied. The disease-free survival of 1 163-74, 1999. year 8 months indicates successful treatment and highlights [3] B Hernandez, H Adissu, et al., "Naturally Occurring the role of ECT as adjuvant therapy in incompletely resected Canine Melanoma as a Predictive Comparative Oncology tumors. Model for Human Mucosal and Other Triple Wild-Type Melanomas". Int J Mol Sci, vol. 19 pp. 394, 2018. [4] CC Plaschke, G Bertino, JA McCaul, et al., European Research on Electrochemotherapy in Head and Neck Cancer (EURECA) project: Results from the treatment of mucosal cancers. Eur J Cancer, vol. 87 pp.172–81, 2017. 28 LABORATORY EXERCISES EBTT WORKSHOP 2020 THE INFLUENCE OF MG2+ IONS ON GENE ELECTROTRANSFER EFFICIENCY The influence of Mg2+ ions on gene electrotransfer efficiency L1 Saša Haberl Meglič, Mojca Pavlin University of Ljubljana, Faculty of Electrical Engineering Duration of the experiment: day 1: 60 min; day 2: 60 min Max. number of participants: 4 Location: Cell Culture Laboratory 3 Level: Basic PREREQUISITES Participants should be familiar with the Safety rules and Rules for sterile work in cell culture laboratory. No other specific knowledge is required for this laboratory practice. THEORETICAL BACKGROUND Gene electrotransfer is a non-viral method used to transfer genes into living cells by means of high-voltage electric pulses. An exposure of a cell to an adequate amplitude and duration of electric pulses leads to transient increase of cell membrane permeability for molecules which are otherwise deprived of membrane transport mechanisms. This allows various nonpermeant molecules, including DNA, to be transported across the membrane and enter the cell. Although mechanisms of the process are not yet fully elucidated, it was shown that several steps are crucial for gene electrotransfer: interaction of plasmid DNA (pDNA) with the cell membrane, translocation of pDNA across the membrane, migration of pDNA towards the nucleus, transfer of pDNA across the nuclear envelope and gene expression (Figure 1). Figure 1. Steps involved in gene electrotransfer. Many parameters have been described to influence the efficiency of gene electrotransfer. A few published reports have shown that the concentration of Mg2+ ions in electroporation media have important impact on forming a complex between DNA and the cell membrane during application of electrical pulses. Namely, DNA is negatively charged polyelectrolyte and Mg2+ ions can bridge the DNA with negatively charged cell membrane. But it was shown that Mg2+ ions at higher concentrations may bind DNA to the cell membrane strongly enough to prevent translocation of DNA into the cell during electroporation thus gene electrotransfer efficiency is decreased. The aim of this laboratory practice is to demonstrate how different Mg2+ concentrations in electroporation media affect the efficiency of gene electrotransfer and cell viability. 31 EBTT WORKSHOP 2020 THE INFLUENCE OF MG2+ IONS ON GENE ELECTROTRANSFER EFFICIENCY EXPERIMENT We will transfect Chinese hamster ovary cells (CHO-K1) with plasmid DNA (pEGFP-N1) that codes for GFP (green fluorescent protein) using two different electroporation media (see Protocol section). To generate electric pulses Jouan GHT 1287 electroporator (Jouan, St. Herblain, France) will be used. Pulses will be monitored on osciloscope (LeCroy 9310C). We will determine gene electrotransfer efficiency and cell viability for both electroporation media. Figure 2. Gene electrotransfer of plated CHO cell 24 h after pulse application in 1 mM Mg or 30 mM Mg media. 8 x 1 ms (stainless steel wire electrodes with inter-electrode distance d = 2 mm; applied voltage U = 140 V resulting in electric field strength E = 0.7 kV/cm) pulses were applied with repetition frequency of 1 Hz to deliver pEGFP-N1 (concentration of DNA in electroporation media was 10 µg/ml) into the cells. Phase contrast images of treated cells for (A) 1 mM Mg and (C) 30 mM Mg media and fluorescence images of treated cells for (B) 1 mM Mg and (D) 30 mM Mg media are presented. To visualize transfection 20x objective magnification was used. Protocol 1/2 (Gene electrotransfer with different electroporation media): CHO cells will be grown in multiwells as a monolayer culture in Ham’s tissue culture medium for mammalian cells with 10% fetal bovine serum (Sigma-Aldrich Chemie GmbH, Deisenhofen, Germany) at 37º C. Cells will be plated 24 h before the experiment in concentration 5 x 104 cells per well. Just before the experiment remove culture media and replace it with 150 µl of electroporation media containing plasmid DNA with concentration 10 µg/ml. Use 1 mM or 30 mM electroporation media. Sucrose molarity has also been changed in order to attain the molarity of the media: 32 EBTT WORKSHOP 2020 THE INFLUENCE OF MG2+ IONS ON GENE ELECTROTRANSFER EFFICIENCY a) 1 mM Mg media (10 mM phosphate buffer Na2HPO4/NaH2PO4, 1 mM MgCl2, 250 mM sucrose; pH = 7.2) b) 30 mM Mg media (10 mM phosphate buffer Na2HPO4/NaH2PO4, 30 mM MgCl2, 160 mM sucrose; pH = 7.2) Incubate cells with plasmid DNA for 2-3 minutes at room temperature. Then apply a train of eight rectangular pulses with duration of 1 ms, U = 140 V resulting in electric field strength E = 0.7 kV/cm and repetition frequency 1 Hz to deliver plasmid DNA into the cells. Use stainless steel wire electrodes with inter-electrode distance d = 2 mm. Cells in the control are not exposed to electric pulses. Immediately after exposure of cells to electric pulses add 37.5 µl of fetal calf serum (FCS-Sigma, USA). Incubate treated cells for 5 minutes at 37ºC and then add 1 ml of culture media. Protocol 2/2 (Determining gene electrotransfer efficiency and cell viability): After 24 h incubation at 37ºC determine the difference in gene electrotransfer efficiency and cell viability for both electroporation media by fluorescent microscopy (Leica, Wetzlar, Germany) at 20x magnification using GFP filter with excitation at 488 nm. You will determine gene electrotransfer efficiency from the ratio between the number of green fluorescent cells (successfully transfected) and the total number of viable cells counted under the phase contrast. You will obtain cell survival from phase contrast images as the ratio between the number of viable cells in the treated sample and the number of viable cells in the control sample. FURTHER READING: Haberl S., Pavlin M., Miklavčič D. Effect of Mg ions on efficiency of gene electrotransfer and on cell electropermeabilization. Bioelectrochemistry 79: 265-271, 2010 Haberl S., Kandušer M., Flisar K., Bregar V.B., Miklavčič D., Escoffre J.M., Rols M.P., Pavlin M. Effect of different parameters used for in vitro gene electrotransfer on gene expression efficiency, cell viability and visualization of plasmid DNA at the membrane level. J Gene Med 15: 169-181, 2013 Rosazza C., Haberl Meglič S., Zumbusch A., Rols M.P., Miklavčič D. Gene electrotransfer: a mechanistic perspective. Curr Gene Ther 16: 98-129, 2016 NOTES & RESULTS Electroporation Treated Viable Green Gene Viability [%] media viable cells in fluorescent electrotransfer cells control cells efficiency [%] 1 mM Mg media 30 mM Mg media 33 EBTT WORKSHOP 2020 THE INFLUENCE OF MG2+ IONS ON GENE ELECTROTRANSFER EFFICIENCY NOTES & RESULTS 34 EBTT WORKSHOP 2020 VISUALIZATION OF LOCAL ABLATION ZONE DISTRIBUTION BETWEEN TWO NEEDLE ELECTRODES Visualization of local ablation zone distribution between two L3 needle electrodes Tjaša Potočnik, Alenka Maček Lebar University of Ljubljana, Faculty of Electrical Engineering Duration of the experiments: 60 min Max. number of participants: 4 Location: Cell Culture Laboratory 3 Level: basic PREREQUISITES Participants should be familiar with Laboratory safety (S1) and Electroporation hardware safety (S2). No other specific knowledge is required for this laboratory practice. THEORETICAL BACKGROUND Electroporation is the method in which by applying external electric field of sufficient amplitude and duration membrane of exposed cells becomes permeabilized for molecules that otherwise cannot pass cell membrane. After reversible electroporation cell membrane reseals. With increasing amplitude of electric field the level of cell membrane permeabilization and the number of cells that are permeabilized increases. When pulses with sufficient magnitude and duration are applied, cell death is achieved and the process is defined as irreversible electroporation (IRE). IRE is an emerging ablation technique inducing apoptosis in successfully treated cells or tissues. Usually there is a sharp border between treated and untreated tissue regions because only the cells that are exposed to high enough electric field are ablated. Effective prediction of electric field can be obtained by numerical modeling, which includes the shape and position of the electrodes and parameters of electric pulses (amplitude, duration, number, frequency) used as well as electrical properties of the tissue. Using treatment planning, IRE offers benefits over other cancer therapies because it can be performed near large blood vessels, nerves, and ducts without causing damage to these structures, sparing extracellular matrix. Electroporation can be detected by measuring increased transport of molecules across the membrane. Cell uptake of dyes, either fluorescent molecules (lucifer yellow, yo-pro-1, propidium iodide) or colour stains (such as trypan blue), is most often used for in vitro electroporation detection. Trypan blue can be used as an indicator of plasma membrane integrity and of cell viability. Trypan blue is normally impermeant to healthy cells. When cell membrane integrity is compromised, the dye is able to enter the cell and stains cellular structures blue, especially nuclei, making the cell appear blue. Cells that take up this dye several hours after exposure to electrical pulses are usually considered dead or dying. The aim of this laboratory practice is to visualize local ablation zone distribution between two needle electrodes with increasing pulse amplitude using trypan blue. EXPERIMENT We will visualize local ablation zone distribution between two needle electrodes using trypan blue. The effect of the pulse amplitude on the local ablation zone distribution between two needle electrodes will be determined for a train of eight 100 µs rectangular pulses delivered with the repetition frequency 1 Hz. The area of blue cells that is a consequence of efficient ablation increases with increasing pulse amplitude is presented in Figure 1. 35 EBTT WORKSHOP 2020 VISUALIZATION OF LOCAL ABLATION ZONE DISTRIBUTION BETWEEN TWO NEEDLE ELECTRODES Protocol: You will use Chinese hamster ovary cells (CHO), plated 48 h before experiment in concentration 2.5 x 105 cells per tissue culture dish. Cells are attached to the culture dish surface. Immediately before electric pulses are applied replace the growth medium with electroporation buffer. As electroporation buffer you will use isotonic 10 mM K2HPO4/KH2PO4 containing 1 mM MgCl2 and 250 mM sucrose with pH 7.4. You will use needle electrodes 1 mm apart. For pulse delivery Gemini X2 electroporator (Hardvard apparatus BTX, USA) will be used. It can produce square and exponential pulses. During the experiment current will be monitored with an oscilloscope and a current probe. Electric field in the needle surrounding can be calculated numerically. Figure 1: The sequence of the images of local ablation zone after cells were exposed to electric pulses with increasing pulse amplitude. The images were obtained by light microscopy under 10 × objective magnifications (top row) and under 5 × objective magnifications (bottom row). Remove the tissue culture dish from the incubator and replace the growth medium with 500 µl of electroporation buffer. Carefully place needle electrodes on edge of tissue culture dish and apply electric pulses. Electric pulse parameters used are: 8 pulses, 100 µs duration and pulse repetition frequency 1 Hz, while pulse amplitude increases gradually. Increase the pulse amplitude from 0 V to 100 V, 300 V, 500 V and 700 V. After electroporation leave cells for 10 minutes at room temperature. Remove electroporation buffer and add 500 µl of trypan blue to tissue culture dish. Leave the cells for 5 minutes at room temperature then replace the trypan blue with 500 µl of fresh electroporation buffer. For visualization of local ablation zone, EVOS XL Core Imaging System (Invitrogen™) will be used. FURTHER READING: Batista Napotnik T, Miklavčič D. In vitro electroporation detection methods – An overview. Bioelectrochemistry 120: 166-182, 2018. Čemažar, M, Jarm T., Miklavčič D, Maček Lebar A., Ihan A., Kopitar N.A., Serša G. Effect of electric field intensity on electropermeabilization and electrosensitivity of various tumor cell lines in vitro. Electro and Magnetobiology 17: 263-272, 1998. Čorović S, Pavlin M, Miklavčič D. Analytical and numerical quantification and comparison of the local electric field in the tissue for different electrode configurations. Biomed. Eng. Online 6: 37, 2007. Davalos RV, Mir IL, Rubinsky B. Tissue ablation with irreversible electroporation. Ann Biomed Eng 33(2):223-31, 2005. Dermol J, Miklavčič D. Predicting electroporation of cells in an inhomogeneous electric field based on mathematical modeling and experimental CHO-cell permeabilization to propidium iodide determination. Bioelectrochemistry 100: 52-61, 2014. Puc M., Kotnik T., Mir L.M., Miklavčič D. Quantitative model of small molecules uptake after in vitro cell electropermeabilization. Bioelectrochemistry 60: 1 – 10, 2003. Rols M.P. Electropermeabilization, a physical method for the delivery of therapeutic molecules into cells. Biochim. Biophys Acta 1758: 423-428, 2006 36 EBTT WORKSHOP 2020 VISUALIZATION OF LOCAL ABLATION ZONE DISTRIBUTION BETWEEN TWO NEEDLE ELECTRODES NOTES & RESULT 37 EBTT WORKSHOP 2020 VISUALIZATION OF LOCAL ABLATION ZONE DISTRIBUTION BETWEEN TWO NEEDLE ELECTRODES NOTES & RESULTS 38 EBTT WORKSHOP 2020 ELECTROPORATION OF PLANAR LIPID BILAYERS Electroporation of planar lipid bilayers L6 Peter Kramar, Aljaž Velikonja, Alenka Maček Lebar University of Ljubljana, Faculty of Electrical Engineering, Slovenia Duration of the experiments: 120 min Max. number of participants: 4 Location: Laboratory for skin and planar lipid bilayers Level: Basic PREREQUISITES Participants should be familiar with Laboratory safety (S1). No other specific knowledge is required for this laboratory practice. THEORETICAL BACKGROUND A planar lipid bilayer can be considered as a small fraction of total cell membrane. As such has often been used to investigate basic aspects of electroporation; especially because of its geometric advantage allowing chemical and electrical access to both sides of the lipid bilayer. Usually a thin bi-molecular film composed of specific phospholipids and organic solvent is formed on a small aperture separating two aqueous compartments. Electrodes immersed in these two aqueous compartments allow to measure current and voltage across the lipid bilayer (Figure 1). Two different measurement principles of planar lipid bilayer’s properties can be used: voltage or current clamp method. Planar lipid bilayer from an electrical point of view can be considered as imperfect capacitor, therefore two electrical properties, capacitance and resistance, mostly determine its behaviour. Figure 1: Equivalent circuit of a planar lipid bilayer. The aim of this laboratory practice is to build a planar lipid bilayer by painting method (Muller - Rudin method) or/and foldig method (Montal – Mueller) and to determine capacitance and resistance of the planar lipid bilayer using LCR meter. Basic aspects of planar lipid bilayer electroporation will be given by observing formation of the pores and determining its breakdown voltage. EXPERIMENT Protocol: Muller-Rudin method Form a planar lipid bilayer by covering the surface of the aperture in a barrier separating two compartments of a measuring vessel with a lecithin solution (20 mg/ml of hexane). After evaporation of hexane, fill compartments with solution consisting of 0.1 mol KCl, 0.01 mol of HEPES, at pH=7.4. Connect the electrodes and apply a drop of lecithin dissolved in decane (20 mg/ml) to the aperture by 39 EBTT WORKSHOP 2020 ELECTROPORATION OF PLANAR LIPID BILAYERS the micropipette or paint it by a teflon brush. Measure capacitance and determine if the formation planar lipid bilayer is appropriate. Montal – Mueller method Cover the surface of the aperture in a barrier separating two compartments of a measuring vessel with 1 µl lecithin solution (10 mg/ml of hexane and ethanol absolute in ratio 9:1). After evaporation of hexane and ethanol, add on the aperture 1,5 µl solution of pentan and hexadecane in ratio 7:3. Fill compartments with solution consisting of 0.1 mol NaCl, 0.01 mol of HEPES, at pH=7.4. On the solution surface apply 2 µl of lecithin solution in each compartment. Wait approximately 15 minutes that lipid molecules are equally spread on the solution surface. Then rise the solution surface above aperture synchronously in both compartments by pumps. Measure capacitance and determine if the formation planar lipid bilayer is appropriate. Measuring methods: When the planar lipid layer is formed, we apply the current or voltage to the planar lipid bilayer. In the current clamp method the current is applied to the planar lipid bilayer and we measure voltage across the bilayer. Apply a linearly increasing current and record a voltage across the bilayer. During the experiment you will obtain the time course of the voltage across the bilayer and the plot of the programmed current flowing between two current electrodes. In the voltage clamp method the voltage across the planar lipid bilayer is applied and current, which flows through planar lipid bilayer, is measured. To the planar lipid bilayer apply a linearly increasing voltage and record a flowing current. Like at the current clamp method you will obtain the time course of the flowing current and the plot of the programmed voltage across the planar lipid bilayer. From collected data determine the breakdown voltage ( Ubr) and the lifetime ( tbr) of planar lipid bilayer. FURTHER READING: Kalinowski S., Figaszewski Z., A new system for bilayer lipid membrane capacitance measurements: method, apparatus and applications, Biochim. Biophys. Acta 1112:57-66, 1992. Pavlin M, Kotnik T, Miklavcic D, Kramar P, Macek-Lebar A. Electroporation of planar lipid bilayers and membranes. In Leitmanova Liu A (ed.), Advances in Planar Lipid Bilayers and Liposomes, Volume 6, Elsevier, Amsterdam, pp. 165-226, 2008. Koronkiewicz S., Kalinowski S., Bryl K., Programmable chronopotentiometry as a tool for the study of electroporation and resealing of pores in bilayer lipid membranes. Biochim. Biophys. Acta, 1561:222–229, 2002. Kotulska M., Natural fluctuationsof an electropore show fractional Lévy stable motion, Biophys. J. , 92:2412-21, 2007. Montal M., Mueller, P., Formation Of Bimolecular Membranes From Lipid Monolayers And A Study of their Electrical Properties, PNAS, 69:3561-3566, 1972. Kramar P, Miklavčič D, Maček-Lebar A. Determination of the lipid bilayer breakdown voltage by means of a linear rising signal. Bioelectrochemistry 70: 23-27, 2007. NOTES & RESULTS 40 EBTT WORKSHOP 2020 ELECTROPORATION OF PLANAR LIPID BILAYERS NOTES & RESULTS 41 EBTT WORKSHOP 2020 ELECTROPORATION OF PLANAR LIPID BILAYERS NOTES & RESULTS 42 EBTT WORKSHOP 2020 E. COLI INACTIVATION BY PULSED ELECTRIC FIELDS IN A CONTINUOUS FLOW SYSTEM E. coli inactivation by pulsed electric fields in a continuous flow L7 system Saša Haberl Meglič, Karel Flisar University of Ljubljana, Faculty of Electrical Engineering Duration of the experiment: day 1: 90 min; day 2: 60 min Max. number of participants: 4 Location: Microbiological laboratory Level: Basic PREREQUISITES Participants should be familiar with the Safety rules and Rules for sterile work in cell culture laboratory. No other specific knowledge is required for this laboratory practice. THEORETICAL BACKGROUND The first description of the profound effect of electrical pulses on the viability of a biological cell was given in 1958. If a cell is exposed to a sufficiently high electric field, its membrane becomes permanently permeable, resulting in leakage of cellular components, which leads to cell death. The method gained ground as a tool for microbial inactivation and the influence of different pulsed electric fields (PEF) on microbial viability was extensively studied on various microorganisms. Since PEF microbial inactivation in controlled laboratory conditions showed promise, the idea arose of also removing pathogenic microorganisms from various water sources, hospital wastewaters and liquid food, without destroying vitamins or affecting the food’s flavour, colour or texture. In order to facilitate PEF application on a large scale, the development of flow processes has been pursued. A standard PEF treatment system therefore consists of a pulse generator that enables continuous pulse treatment, flow chambers with electrodes and a fluid-handling system. Several parameters have been described, which can influence inactivation of microbial cells. Specifically in a continuous flow system the flow rate of a liquid must be adjusted in order for each bacterial cell to be exposed to appropriate pulse conditions. The aim of this laboratory practice is to demonstrate how different pulse parameters in a continuous flow system affect bacterial inactivation. EXPERIMENT We will inactivate Escherichia coli K12 TOP10 cells carrying plasmid pEGFP-N1, which encodes kanamycin resistance (Clontech Laboratories Inc., Mountain View, CA, USA) in a continuous flow system (see Figure 1) using different electric pulse parameters. To generate electric pulses square wave prototype pulse generator will be used. Pulses will be monitored on osciloscope (LeCroy 9310C). The inactivation level will be determined by plate count method. Bacterial cells will be grown prior experiment for 17 hours at 37°C in Luria Broth (LB) medium (Sigma-Aldrich Chemie GmbH, Deisenhofen, Germany) with shaking. 43 EBTT WORKSHOP 2020 E. COLI INACTIVATION BY PULSED ELECTRIC FIELDS IN A CONTINUOUS FLOW SYSTEM Figure 1. Continuous flow electroporation system. The circuit system includes a flow chamber with electrodes and a prototype square wave pulse generator. Voltage and current are both monitored throughout the experiment. Protocol 1/2 (Electroporation of bacteria in a continuous flow system): On the first day of experiment E. coli cells will be centrifuged (4248 g, 30 min, 4°C) and the pellet will be re-suspended in 10 ml of distilled water and 100x diluted. The total volume of prepared bacterial cells for the treatment will be 0.3 L. In order to determine the number of bacterial cells in our sample, you will prepare serial dilutions of bacterial sample ranging from 10-1 to 10-6 (in 900 µl of sterile distilled water you will dilute 100 µl of bacterial sample). You will pipette 100 µl of dilutions 10-5 and 10-6 on LB agar containing kanamycin antibiotic and evenly spread the liquid with sterile plastic rod. The exposure of cells to electric pulses in flow through chamber in a continuous flow system depends on the geometry of the chamber, the frequency of pulses at which electroporator operates. The number of pulses is given by equation 1. At that flow, the desired number of pulses are applied to the liquid and thus to the cells in the flow-through chamber. Because the volume of our cross-field chamber between the electrodes ( Q = 0.0005 L) and the frequency (10 Hz in our case) are constant, the flow through the chamber can be determined: " = $ ∙ ' (1) % where q (L/min) is the flow rate, Q (L) the volume between the two electrodes and n is the number of pulses received by the fluid in the chamber in residence time. For a frequency of 10 Hz, you will calculate the flow rate ( q) at which the whole liquid will be subjected to at least one pulse. For PEF flow through 44 EBTT WORKSHOP 2020 E. COLI INACTIVATION BY PULSED ELECTRIC FIELDS IN A CONTINUOUS FLOW SYSTEM treatment you will use 0.3 L (10-2 dilution) of prepared bacterial cells. Bacterial cells will be pumped through the system at the calculated flow rate and pulses will be applied by prototype pulse generator. After PEF treatment take 100 µl of treated sample and prepare dilutions ranging from 10-1 to 10-6. You will pipette 100 µl of dilutions 10-4, 10-5 and 10-6 on LB agar with kanamycin antibiotic and evenly spread the liquid with sterile plastic rod. Protocol 2/2 (Determining bacterial viability): After 24 h incubation at 37ºC count colony forming units. Express the viability as log (N/N0), where N represents the number of colony forming units per ml in a treated sample (bacterial cells exposed to electric pulses) and N0 the number of colony forming units per ml in an untreated sample (bacterial cells not exposed to electric pulses). Example of determining bacterial viability: You counted 70 CFU in a control sample (dilution 10-7) and 30 CFU in a treated sample (dilution 10-5). Number of bacterial cells per ml (control sample) = 70 x 107 (dilution factor of sample) x 10 (dilution factor of plating) = 7 x 109 bacterial cells/ml Number of bacterial cells per ml (treated sample) = 30 x 105 (dilution factor of sample) x 10 (dilution factor of plating) = 3 x 107 bacterial cells/ml log N/N0 = log (3 x 107/ 7 x 109) = -2.368 FURTHER READING: Flisar K., Haberl Meglic S., Morelj J., Golob J., Miklavčič D. Testing a prototype pulse generator for a continuous flow system and its use for E. coli inactivation and microalgae lipid extraction. Bioelectrochemistry doi: 10.1016/j.bioelechem.2014.03.008, 2014 Gerlach D., Alleborn N., Baars A., Delgado A., Moritz J., Knorr D. Numerical simulations of pulsed electric fields for food preservation: A review. Innov Food Sci Emerg Technol 9: 408-417, 2008 Gusbeth C., Frey W., Volkmann H., Schwartz T., Bluhm H. Pulsed electric field treatment for bacteria reduction and its impact on hospital wastewater. Chemosphere 75: 228-233, 2009 Pataro G., Senatore B., Donsi G., Ferrari G. Effect of electric and flow parameters on PEF treatment efficiency. J Food Eng 105: 79-88, 2011 NOTES & RESULTS 45 EBTT WORKSHOP 2020 E. COLI INACTIVATION BY PULSED ELECTRIC FIELDS IN A CONTINUOUS FLOW SYSTEM NOTES & RESULTS 46 EBTT WORKSHOP 2020 ANALYSIS OF ELECTRIC FIELD ORIENTATIONS ON GENE ELECTROTRANSFER EFFICIENCY Analysis of electric field orientations on gene electrotransfer L8 efficiency Saša Haberl Meglič, Matej Reberšek University of Ljubljana, Faculty of Electrical Engineering Duration of the experiment: day 1: 90 min; day 2: 60 min Max. number of participants: 4 Location: Cell Culture Laboratory 1 (1st day) and 3 (2nd day) Level: Advanced PREREQUISITES Participants should be familiar with the Safety rules and Rules for sterile work in cell culture laboratory. The basic knowledge of handling with cells is required for this laboratory practice. THEORETICAL BACKGROUND Gene electrotransfer is a non-viral method used to transfer genes into living cells by means of high-voltage electric pulses. An exposure of a cell to an adequate amplitude and duration of electric pulses leads to transient increase of cell membrane permeability for molecules which are otherwise deprived of membrane transport mechanisms. This allows various nonpermeant molecules, including DNA, to be transported across the membrane and enter the cell. Although mechanisms of the process are not yet fully elucidated, it was shown that several steps are crucial for gene electrotransfer: interaction of plasmid DNA (pDNA) with the cell membrane, translocation of pDNA across the membrane, migration of pDNA towards the nucleus, transfer of pDNA across the nuclear envelope and gene expression. Many parameters (such as electric pulse protocol) can influence the first step (interaction of DNA with the cell membrane) and by that gene electrotransfer efficiency. Therefore different electric pulse protocols are used in order to achieve maximum gene transfection, one of them is changing the electric field orientation during the pulse delivery. Since DNA is a negatively charged molecule and it is dragged towards the cell with the electrophoretic force in the opposite direction of the electric field, changing electric field orientation increases the membrane area competent for DNA entry into the cell. The aim of this laboratory practice is to demonstrate how different pulse polarity affects the efficiency of gene electrotransfer and cell viability. EXPERIMENT For the experiment we will use Chinese hamster ovary cells (CHO-K1) and plasmid DNA (pEGFP-N1) that codes for GFP (green fluorescent protein). To generate and deliver electric pulses a high-voltage prototype generator and electrodes with four cylindrical rods, which were developed at a Laboratory of Biocybernetics will be used. Pulses will be monitored on osciloscope (LeCroy 9310C). Pulse protocols (see also Figure 1): a) SP (single polarity): the direction of electric field is the same for all pulses b) OBP (orthogonal both polarities): the direction of the electric field is changed between the pulses 47 EBTT WORKSHOP 2020 ANALYSIS OF ELECTRIC FIELD ORIENTATIONS ON GENE ELECTROTRANSFER EFFICIENCY Figure 1: Two different pulse protocols will be used: single polarity (SP) and orthogonal both polarities (OBP) Protocol 1/2 (Gene electrotransfer with different pulse parameters): CHO cells will be grown in multiwells as a monolayer culture in Ham’s tissue culture medium for mammalian cells with 10% fetal bovine serum at 37º C. Cells will be plated 24h before the experiment in concentration 5 x 105 cells per well. Just before the experiment remove culture medium and replace it with 150 µl of electroporation buffer containing plasmid DNA with concentration 10 µg/ml. Incubate cells with plasmid for 2-3 minutes at room temperature. Then apply a train of eight pulses with amplitude of 225 V, duration of 1 ms and repetition frequency 1 Hz using single polarity and orthogonal both polarities (see Pulse protocols) to deliver plasmid DNA into the cells. Cells in the control are not exposed to electric pulses. Immediately after exposure of cells to electric pulses add 37 µl of fetal calf serum (FCS-Sigma, USA). Incubate treated cells for 5 minutes at 37º C and then add 1 ml of culture medium. Protocol 2/2 (Determining gene electrotransfer efficiency and cell viability): After 24 h incubation at 37º C determine the difference in gene electrotransfer efficiency and cell viability for both pulse protocols by fluorescent microscopy (Leica, Wetzlar, Germany) at 20x magnification using GFP filter with excitation at 488 nm. You will determine gene electrotransfer efficiency from the ratio between the number of green fluorescent cells (successfully transfected) and the total number of cells counted under the phase contrast. You will obtain cell survival from phase contrast images as the ratio between the number of viable cells in the treated sample and the number of viable cells in the control sample. 48 EBTT WORKSHOP 2020 ANALYSIS OF ELECTRIC FIELD ORIENTATIONS ON GENE ELECTROTRANSFER EFFICIENCY FURTHER READING: Faurie C., Reberšek M., Golzio M., Kandušer M., Escoffre J. M., Pavlin M., Teissie J., Miklavčič D., Rols M. P. Electro-mediated gene transfer and expression are controlled by the life-time of DNA/membrane complex formation. J Gene Med 12: 117-125, 2010 Golzio M., Teissié J., Rols M. P. Direct visualization at the single-cell level of electrically mediated gene delivery. PNAS 99: 1292-1297, 2002 Pavlin M., Haberl S., Reberšek M., Miklavčič D., Kandušer M. Changing the direction and orientation of electric field during electric pulses application improves plasmid gene transfer in vitro. J Vis Exp, 55: 1-3, 2011 Reberšek M., Faurie C., Kandušer M., Čorović S., Teissié J., Rols M.P., Miklavčič D. Electroporator with automatic change of electric field direction improves gene electrotransfer in vitro. Biomed Eng Online 6: 25, 2007 Reberšek M., Kandušer M., Miklavčič D. Pipette tip with integrated electrodes for gene electrotransfer of cells in suspension: a feasibility study in CHO cells. Radiol Oncol 45: 204-208, 2011 NOTES & RESULTS Gene Pulse parameters electrotransfer Cell viability efficiency [%] [%] Single polarity Orthogonal both polarities 49 EBTT WORKSHOP 2020 ANALYSIS OF ELECTRIC FIELD ORIENTATIONS ON GENE ELECTROTRANSFER EFFICIENCY NOTES & RESULTS 50 EBTT WORKSHOP 2020 MONITORING OF ELECTRIC FIELD DISTRIBUTION IN BIOLOGICAL TISSUE BY MEANS MREIT Monitoring of electric field distribution in biological tissue by L10 means of magnetic resonance electrical impedance tomography Matej Kranjc1, Igor Serša2 1University of Ljubljana, Faculty of Electrical Engineering 2Jozef Stefan Institute Duration of the experiment: day 1: 90 min Max. number of participants: 4 Location: MRI Laboratory (Jožef Stefan Institute) Level: Basic PREREQUISITES Participants should be familiar with Laboratory safety (S1). No other specific knowledge is required for this laboratory practice. THEORETICAL BACKGROUND A method capable of determining electric field distribution during the pulse delivery has a practical value as it can potentially enable monitoring of the outcome of electroporation which strongly depends on the local electric field. Measurement of electric field distribution enables detection of insufficient electric field coverage before the end of either reversible or irreversible electroporation treatment, thus enabling corrections of field coverage during the treatment and consequently increasing and assuring its effectiveness. As there are no available approaches for measurement of electric field distribution in situ, an indirect approach using magnetic resonance techniques was suggested. Magnetic resonance electrical impedance tomography (MREIT) enables reconstruction of electric field distribution by measurement of electric current density distribution, first, and calculation of electrical conductivity of the treated subject during application of electric pulses using MRI data as an input to numerical algorithms, second. This method enables determination of electric field distribution in situ also accounting for changes that occur in the tissue due to electroporation. MREIT is a relatively new medical imaging modality based on numerical reconstruction of electrical conductivity inside a tissue by means of current density distribution measured by current density imaging (CDI) sequence. The MREIT algorithm applied for reconstruction of electrical conductivity of the tissue is based on solving Laplace’s equation through iterative calculation. Electrical conductivity is updated after each iteration ( k+1): |- ()*+ = ./0|. |∇2)| where JCDI is current density obtained by CDI and u k is electric potential obtained as a solution of Laplace’s equation. When difference between two successive conductivities falls below certain value electric field distribution can be calculated using: - 4 = ./0. ( The aim of this laboratory practice is to demonstrate monitoring of electric field distribution in a biological tissue using MREIT. 51 EBTT WORKSHOP 2020 MONITORING OF ELECTRIC FIELD DISTRIBUTION IN BIOLOGICAL TISSUE BY MEANS MREIT EXPERIMENT We will monitor current density distribution and electric field distribution in biological tissue exposed to electric pulses by means of MREIT. We will then compare measured current density distribution and reconstructed electric field distribution with simulation results obtained by a numerical model of the tissue. Protocol The experiment will be performed on biological tissue (chicken liver) sliced in a disc-like sample measuring 21 mm in diameter and 2 mm in height (Fig. 1a). Electric pulses will be delivered via two cylindrically shaped electrodes inserted into the sample. After the insertion, the electrodes will be connected to an electric pulse generator connected to an MRI spectrometer. The sample will be placed in a 25 mm MR microscopy RF probe (Fig. 1b) inside a horizontal-bore superconducting MRI magnet (Fig. 1c). Electroporation treatment of the sample will be performed by applying two sequences of four high voltage electric pulses with a duration of 100 µs, a pulse repetition frequency of 5 kHz and with an amplitude of 500 V and 1000 V. Figure 1: Biological sample (a) placed in a MR microscopy probe (b) inside a horizontal MRI magnet (c). MR imaging will be performed on a MRI scanner consisting of a 2.35 T (100 MHz proton frequency) horizontal bore superconducting magnet (Oxford Instruments, Abingdon, United Kingdom) equipped with a Bruker micro-imaging system (Bruker, Ettlingen, Germany) for MR microscopy with a maximum imaging gradient of 300 mT/m and a Tecmag Apollo spectrometer (Tecmag, Houston TX, USA). Monitoring of electric field is enabled by CDI, which is an MRI method that enables imaging of current density distribution inside conductive sample. We will apply two-shot RARE version of the CDI sequence (Fig. 2). Figure 2: Two-shot RARE pulse sequence used for acquisition of current density distribution. The sequence consists of a current encoding part with a short (100 µs long) high-voltage electroporation pulse ( U el) delivered immediately after the nonselective 90° radiofrequency (RF) excitation pulse. In the second part of the sequence signal acquisition is performed using the single-shot RARE signal acquisition scheme that includes standard execution of readout ( G r), phase-encoding ( G p) and slice-selection ( G s) magnetic field 52 EBTT WORKSHOP 2020 MONITORING OF ELECTRIC FIELD DISTRIBUTION IN BIOLOGICAL TISSUE BY MEANS MREIT gradients. Due to auxiliary phase encoding induced by the electric pulse, the RARE sequence is repeated twice, each time with a different phase of the refocusing pulses (0°and 90°), and the corresponding signals are co-added. Electric field distribution in the sample will be reconstructed by iteratively solving Laplace's equation using J-substitution mathematical algorithm and finite element method with the numerical computational environment MATLAB on a desktop PC. We will compare measured current density distribution obtained by means of CDI and reconstructed electric field distribution obtained by means of MREIT in the sample with simulation results obtained by a numerical model of the sample. FURTHER READING Kranjc M., Bajd F, Sersa I., Miklavcic D., Magnetic resonance electrical impedance tomography for monitoring electric field distribution during tissue electroporation. IEEE Trans Med Imaging 30:1771–1778, 2011. Kranjc M., Bajd F., Serša I., Miklavčič D., Magnetic resonance electrical impedance tomography for measuring electrical conductivity during electroporation. Physiol Meas 35:985–96, 2014. Kranjc M, Markelc B, Bajd F, Čemažar M, Serša I, Blagus T, Miklavčič D. In situ monitoring of electric field distribution in mouse tumor during electroporation. Radiology 274: 115-123, 2015. Kranjc M., Bajd F., Serša I. de Boevere M., Miklavcic D., Electric field distribution in relation to cell membrane electroporation in potato tuber tissue studied by magnetic resonance techniques. Innov Food Sci Emerg Technol, 2016. Woo E. J. and Kranjc M. Principles and use of magnetic resonance electrical impedance tomography in tissue electroporation in Handbook of Electroporation (ed. Miklavcic, D.) 1–18 Springer, 2016. Seo J.K., Woo E.J., Magnetic Resonance Electrical Impedance Tomography (MREIT). SIAM Rev 53:40–68, 2011. Sersa I. Auxiliary phase encoding in multi spin-echo sequences: application to rapid current density imaging. J Magn Reson, 190(1):86– 94, 2008. NOTES & RESULTS 53 EBTT WORKSHOP 2020 MONITORING OF ELECTRIC FIELD DISTRIBUTION IN BIOLOGICAL TISSUE BY MEANS MREIT NOTES & RESULTS 54 EBTT WORKSHOP 2020 MEASUREMENTS OF THE INDUCED TRANSMEMBRANE VOLTAGE WITH FLUORESCENT DYE DI-8-ANEPPS Measurements of the induced transmembrane voltage with L11 fluorescent dye di-8-ANEPPS Gorazd Pucihar University of Ljubljana, Faculty of Electrical Engineering Duration of the experiments: 60 min Max. number of participants: 4 Location: Cell Culture Laboratory 1 Level: Advanced PREREQUISITES Participants should be familiar with Laboratory safety (S1). The basic knowledge of handling with cells is required for this laboratory practice. THEORETICAL BACKGROUND When a biological cell is placed into an external electric field the induced transmembrane voltage (ITV) forms on its membrane. The amplitude of the ITV is proportional to the amplitude of the applied electric field, and with a sufficiently strong field, this leads to an increase in membrane permeability - electroporation. Increased permeability is detected in the regions of the cell membrane where the ITV exceeds a sufficiently high value, in the range of 250 – 1000 mV, depending on the cell type. In order to obtain an efficient cell electroporation it is therefore important to determine the distribution of the ITV on the cell membrane. The ITV varies with the position on the cell membrane, is proportional to the electric field, and is influenced by cell geometry and physiological characteristics of the medium surrounding the cell. For simple geometric shapes the ITV can be calculated analytically (e.g. for a spherical cell, using Schwan's equation). For more complicated cell shapes experimental and numerical methods are the only feasible approach to determine the ITV. The aim of this laboratory practice is to measure the ITV on a spherical cell by means of a fluorescent potentiometric dye di-8-ANEPPS. EXPERIMENT Potentiometric fluorescent dyes allow observing the variations of the ITV on the membrane and measuring its value. Di-8-ANEPPS is a fast potentiometric fluorescent dye, which becomes fluorescent when it binds to the cell membrane, with its fluorescence intensity varying proportionally to the change of the ITV. The dye reacts to the variations in the ITV by changing the intramolecular charge distribution that produce corresponding changes in the spectral profile or intensity of the dye's fluorescence. Protocol: The experiments are performed on Chinese hamster ovary cells (CHO) grown in Lab-Tek chambers (Nunc, Germany) in culture medium HAM-F12 supplemented with 10% fetal bovine serum, L-glutamine (all three from Sigma-Aldrich) and antibiotics. When cells attach to the cover glass of a Lab-Tek chamber (usually after 2 to 3 hours to obtain attached cells of spherical shape), carefully replace the culture medium with 1 ml of SMEM medium (Spinner’s modification of the MEM, Sigma-Aldrich) containing 30 µM of di-8-ANEPPS and 0.05% of Pluronic (both Life Technologies). After staining for 12 min at 4°C, wash the cells thoroughly with pure SMEM to remove the excess dye. After washing leave 1.5 ml of SMEM in the chamber. Place the chamber under a fluorescence microscope (Zeiss AxioVert 200, Germany) and use ×63 oil immersion objective. Position two parallel Pt/Ir wire electrodes, with a 4 mm distance between them, to the bottom of the chamber. Set a single 40 V, 50 ms pulse on the programmable square wave electroporator TSS20 (Intracel). This will result in a voltage-55 EBTT WORKSHOP 2020 MEASUREMENTS OF THE INDUCED TRANSMEMBRANE VOLTAGE WITH FLUORESCENT DYE DI-8-ANEPPS to-distance ratio of ~100 V/cm. The pulse must be synchronized with the image acquisition. Set the excitation wavelength to 490 nm and use ANEPPS filter to detect fluorescence (emission 605 nm). Find the cells of interest. Acquire the control fluorescence image and subsequently the image with a pulse, using a cooled CCD camera (VisiCam 1280, Visitron) and MetaFluor 7.7.5 (Molecular Devices). Apply four pulses with a delay of 4 s between two consecutive pulses. For each pulse, acquire a pair of images, one immediately before (control image) and one during the pulse (pulse image) (Figures 1A and B). Open the images in MetaMorph 7.7.5 (Molecular Devices). To qualitatively display the ITV on the cell membrane, convert the acquired 12-bit images to 8-bit images. For each pulse, obtain the difference image by subtracting (on a pixel-by-pixel basis) the control image from the pulse image. Add 127, so that 127, i.e. mid-gray level, corresponds to 0 V, brighter levels to negative voltages, and darker levels to positive ones (Figure 1C). Average the three difference images to increase the signal-to-noise ratio. To quantitatively determine the ITV, open the acquired, unprocessed fluorescence images. Determine the region of interest at the site of the membrane and measure the fluorescence intensities along this region for the control and pulse image. Transform the values to the spreadsheet. Measure the background fluorescence in both images and subtract this value from the measured fluorescence. Calculate the relative changes in fluorescence (Δ F/ F C) by subtracting the fluorescence in the control image F C from the fluorescence in the pulse image F P and dividing the subtracted value by the fluorescence in the control F C; Δ F/FC = (F P – F C) / FC. Average the relative changes calculated for all four acquired pairs of images. Transform the fluorescence changes to the values of the ITV (ΔF/F = -6% / 100 mV), and plot them on a graph as a function of the arc length (Figure 1D). A B E C D Figure 1: Measurements of the induced transmembrane voltage (ITV) on an irregularly shaped CHO cell. (A) A control fluorescence image of a cell stained with di-8-ANEPPS. Bar represents 10 µm. (B) Fluorescence image acquired during the exposure to a 35 V (~88 V/cm), 50 ms rectangular pulse. (C) Changes in fluorescence of a cell obtained by subtracting the control image A from the image with pulse B and shifting the grayscale range by 50%. The brightness of the image was automatically enhanced. (D) ITV measured along the path shown in C. 56 EBTT WORKSHOP 2020 MEASUREMENTS OF THE INDUCED TRANSMEMBRANE VOLTAGE WITH FLUORESCENT DYE DI-8-ANEPPS FURTHER READING: Teissié J., and Rols M. P. An experimental evaluation of the critical potential difference inducing cell membrane electropermeabilization. Biophys. J. 65:409-413, 1993. Gross D., Loew L. M, and Webb W. Optical imaging of cell membrane potential changes induced by applied electric fields Biophys. J. 50:339-348, 1986. Montana V., Farkas D. L., and Loew L. M. Dual-wavelength ratiometric fluorescence measurements of membrane-potential. Biochemistry 28:4536-4539, 1989. Loew L. M. Voltage sensitive dyes: Measurement of membrane potentials induced by DC and AC electric fields. Bioelectromagnetics Suppl. 1:179-189, 1992. Hibino M., Itoh H., and Kinosita K. Time courses of cell electroporation as revealed by submicrosecond imaging of transmembrane potential. Biophys. J. 64:1789-1800, 1993. Kotnik T., Bobanović F., and Miklavčič D. Sensitivity of transmembrane voltage induced by applied electric fields – a theoretical analysis. Bioelectrochem. Bioenerg. 43:285-291, 1997. Pucihar G., Kotnik T., Valič B., Miklavčič D. Numerical determination of transmembrane voltage induced on irregularly shaped cells. Annals Biomed. Eng. 34: 642-652, 2006. Video Article: Pucihar G., Kotnik T., Miklavčič D. Measuring the induced membrane voltage with di-8-ANEPPS (Video Article). J. Visual Exp. 33: 1659, 2009. NOTES & RESULTS 57 EBTT WORKSHOP 2020 MEASUREMENTS OF THE INDUCED TRANSMEMBRANE VOLTAGE WITH FLUORESCENT DYE DI-8-ANEPPS NOTES & RESULTS 58 COMPUTER MODELING EBTT WORKSHOP 2020 TREATMENT PLANNING FOR ELECTROCHEMOTHERAPY AND IRREVERSIBLE ELECTROPORATION Treatment planning for electrochemotherapy and irreversible C1 electroporation: optimization of voltage and electrode position Anže Županič1, Bor Kos2 1Eawag - Swiss federal institute of aquatic science and technology, Switzerland 2University of Ljubljana, Faculty of Electrical Engineering Duration of the experiments: 90 min Max. number of participants: 6 Location: Laboratory of Biocybernetics Level: Basic PREREQUISITES No specific knowledge is required for this laboratory exercise. THEORETICAL BACKGROUND Electrochemotherapy (ECT) is an efficient local treatment of cutaneous and subcutaneous tumors, which combines the delivery of nonpermeant, cytotoxic chemotherapeutics (e.g. bleomycin, cisplatin) and short high-voltage electric pulses. The pulses induce electric fields inside the tissue, thereby increasing cell membrane permeability in tissue (electropermeabilization) to otherwise nonpermeant chemotherapeutics. ECT requires the electric field inside the tumor to be higher than the threshold value needed for reversible electroporation ( Erev) while irreversible electroporation ( Eirrev) in nearby critical structures should be limited. For IRE, the electric field in the entire tumor volume needs to be above the irreversible electroporation threshold. It is not necessary that the whole tumor is electropermeabilized by one pulse or pulse sequence - sometimes a combination of several pulse sequences or a combination of different electrodes is required. The aim of this laboratory practice is to learn how to use optimization techniques to achieve suitable electric field distribution for electrochemotherapy experimental planning and treatment planning. EXPERIMENT A finite element based numerical modeling program package COMSOL Multiphysics version 5.4 (COMSOL AB, Stockholm, Sweden) will be used to optimize voltage between the electrodes and position of the electrodes on a simple 3D model of a spherical subcutaneous tumor and surrounding tissue (Figure 1a). Electrode positions and the applied voltage should be chosen, so that the following objectives are fulfilled: • For electrochemotherapy: the tumor is permeabilized ( Etumor > Erev = 400 V/cm), • For irreversible electroporation: the tumor is permeabilized above the irreversible threshold ( Etumor > Eirrev = 600 V/cm), • the damage to healthy tissue is kept to a minimum. We will calculate the electric field distribution in the model after each change of the electrode placement or voltage. The final goal of this exercise is to achieve 100 % Etumor > Erev (or 100 % Etumor > Eirr when planning for IRE) and minimize Eirr in healthy tissue . Protocol: Build the 3-d model by following the lecturer’s instructions and take into account your tissue-specific electric properties. Solve the model and evaluate the initial solution. In case, the initial solution is inappropriate (see e.g., Figure 1b), try to improve on the solution by changing electrode positions and voltage between the electrodes. Calculate the electric field distribution in the model after changing the 61 EBTT WORKSHOP 2020 TREATMENT PLANNING FOR ELECTROCHEMOTHERAPY AND IRREVERSIBLE ELECTROPORATION electrode positions or voltage and then determine the coverage of tumor tissue with Etumor > (Erev or Eirrev) and determine damage to healthy tissue due to irreversible electroporation. Repeat the process, until the quality of your solution reaches the set goals. Compare the results with others, who have used different tissue properties. Use a parametric study to find the lowest voltage which achieves the objective for the selected electrode geometry. A B Figure 1: (A) Simple 3D model of tumor and needle electrodes in healthy tissue; (B) electric field over reversible threshold inside the healthy tissue and the tumor. FURTHER READING: Miklavčič D, Čorović S, Pucihar G, Pavšelj N. Importance of tumor coverage by sufficiently high local electric field for effective electrochemotherapy. EJC Supplements, 4: 45-51, 2006. Čorović S, Županič A, Miklavčič D. Numerical modeling and optimization of electric field distribution in subcutaneous tumor treated with electrochemotherapy using needle electrodes. IEEE Trans. Plasma Sci., 36: 1665-1672, 2008. Županič A, Čorović S, Miklavčič D. Optimization of electrode position and electric pulse amplitude in electrochemotherapy. Radiol. Oncol., 42: 93-101, 2008. Edd JF, Davalos RV. Mathematical modeling of irreversible electroporation for treatment planning, Technol. Cancer Res. Treat., 6: 275-286, 2007. Kos B, Zupanic A, Kotnik T, Snoj M, Sersa G, Miklavcic D. Robustness of Treatment Planning for Electrochemotherapy of Deep-Seated Tumors, Journal of Membrane Biology 236: 147-153, 2010. Cukjati, D, Batiuskaite D, Andre F, Miklavcic D, Mir L. Real Time Electroporation Control for Accurate and Safe in Vivo Non-viral Gene Therapy. Bioelectrochemistry 70: 501–507, 2007. NOTES & RESULTS 62 EBTT WORKSHOP 2020 NUMERICAL MODELING OF THERMAL EFFECTS DURING IRE TREATMENTS Numerical Modeling of Thermal Effects during Irreversible C2 Electroporation Treatments Paulo A. Garcia1 and Bor Kos2 1Virginia Tech – Wake Forest University 2University of Ljubljana, Faculty of Electrical Engineering Duration of the experiment: 90 min Max. number of participants: 6 Location: Laboratory of Biocybernetics Level: Advanced PREREQUISITES Basic to advanced knowledge of finite element modeling THEORETICAL BACKGROUND Irreversible electroporation (IRE) is a new, safe, and effective minimally invasive ablation modality with the potential to treat many currently unresectable and/or untreatable tumors. The non-thermal mode of cell death in IRE is unique in that it does not rely on thermal changes from Joule heating to kill tumor cells thus allowing for successful treatment even in close proximity to critical structures and without being affected by the heat sink effect. Accurate modeling of the electrical and thermal responses in tissue is important to achieve complete coverage of the tumor and ensure that the thermal changes during a procedure do not generate thermal damage, especially in critical structures (e.g. bile ducts, nerves and sensitive blood vessels). Figure 2: Electric Field distribution resulting from a bipolar electrode with an applied voltage of 1250 V. The temperature distribution ( T) within the tissue wil be obtained by transiently solving a modified heat conduction equation with the inclusion of the Joule heating source term ' = (|∇5|6 78 9: = ∇ ∙ (=∇>) + ' (1) 9; 63 EBTT WORKSHOP 2020 NUMERICAL MODELING OF THERMAL EFFECTS DURING IRE TREATMENTS where ( is the electrical conductivity, 5 the electric potential, = is the thermal conductivity, 8 is the specific heat capacity, and 7 is the density of the tissue. At each time step, the current density and electric field distribution are determined and updated in the Joule heating term to capture the electrical conductivity changes in liver tissue from electroporation and temperature. Figure 3: Temperature distribution after a ninety 100-µs pulse IRE treatment in liver tissue at 1 pulse per second. Thermal damage is a process that depends on temperature and time. If the exposure is long, damage can occur at temperatures as low as 42°C, while 50°C is general y chosen as the target temperature for instantaneous damage. The damage can be calculated based on the temperatures to assess whether a particular set of pulse parameters and electrode configuration wil induce thermal damage in superposition with IRE. The thermal damage wil be quantified using the Arrhenius rate equation given by: FGH Ω(B) = ∫;LM D ∙ EI∙J(K) OB (2) ;LN where P is the universal gas constant, 8.314 J/(mol·K); D is the pre-exponential factor, 7.39 × 1039 s-1, a measure of the effective collision frequency between reacting molecules in bimolecular reactions; QR the activation energy barrier that molecules overcome to transform from their "native state" to the "damaged state", 2.577 × 105 J/mol for liver tissue. It is important to note that the pre-exponential factor and activation energy are tissue specific parameters that describe different modes of thermal damage such as microvascular blood flow stasis, cel death, and protein coagulation. In terms of finite element modeling of thermal damage, an integral value Ω(B) = 1 corresponds to a 63% probability of cel death and an integral value Ω(B) = 4.6 corresponds to 99% probability of cel death due to thermal effects. In order to convert the damage integral to a probability of cell death, V(%), we wil use: V(%) = 100 ∙ Y1 − E[\(;)] 64 EBTT WORKSHOP 2020 NUMERICAL MODELING OF THERMAL EFFECTS DURING IRE TREATMENTS Figure 4: Thermal damage probability of cell death due to excessive thermal effects as a result of Joule heating. The aim of this laboratory practice is to get familiar with the numerical simulation tools needed for capturing the electrical and thermal responses during a ninety 100-µs pulse IRE. We will accomplish this by coupling the Laplace, Heat Conduction, and Arrhenius equations using COMSOL Multiphysics 5.4 (Comsol AB, Stockholm, Sweden) to determine the IRE zones of ablation and evaluate if the increase in temperature due to Joule heating due to the pulses generates any potential thermal damage. EXPERIMENT In this exercise we wil compare the effect of a static, (N, and dynamic, ((Q), electrical conductivity functions in the resulting electrical and thermal effects during an entire IRE protocol in liver tissue. Initial y we wil determine the volume of tissue affected by IRE from the electric field distributions. We wil then evaluate the temperature increase in liver tissue as a result of the Joule heating and determine if there was a probability of cell death due to thermal damage with the given IRE protocols employed. This exercise wil provide the participants with accurate predictions of al treatment associated effects which is a necessity toward the development and implementation of optimized treatment protocols. Specificaly: 1) Simulate the electric field distribution using a static conductivity and 1000 V, 1500 V, and 2000 V. 2) Simulate the electric field distribution using a dynamic conductivity and 1000 V and 1500 V. 3) Include the Heat Conduction Equation by coupling with the Laplace Equation via Joule Heating. 4) Explore the resulting temperature distributions as a function of pulse number and frequency. 5) Incorporate the Arrhenius equation to assess potential thermal damage from the Joule Heating. 6) Investigate the effect of pulse frequency (1 Hz, 10 Hz, and 100 Hz) for ninety 100-μs pulses. FURTHER READING: Davalos RV, Rubinsky B, Mir LM. Theoretical analysis of the thermal effects during in vivo tissue electroporation. Bioelectrochemistry 61(1-2): 99-107, 2003 Chang, IA and Nguyen, UD., Thermal modeling of lesion growth with radiofrequency ablation devices. Biomed Eng Online, 3(1): 27, 2004 Davalos, R.V. and B. Rubinsky, Temperature considerations during irreversible electroporation. International Journal of Heat and Mass Transfer, 51(23-24): 5617-5622, 2008 Pavšelj N and Miklavčič D, Numerical modeling in electroporation-based biomedical applications. Radiology and Oncology, 42(3): 159-168, 2008 Lacković I, Magjarević R, Miklavčič D. Three-dimensional finite-element analysis of joule heating in electrochemotherapy and in vivo gene electrotransfer. IEEE T. Diel. El. Insul. 15: 1338-1347, 2009 Garcia, PA, et al., A Parametric Study Delineating Irreversible Electroporation from Thermal Damage Based on a Minimally Invasive Intracranial Procedure. Biomed Eng Online, 10(1): 34, 2010 Pavšelj N, Miklavčič D. Resistive heating and electropermeabilization of skin tissue during in vivo electroporation: A coupled nonlinear finite element model. International Journal of Heat and Mass Transfer 54: 2294-2302, 2011 65 EBTT WORKSHOP 2020 NUMERICAL MODELING OF THERMAL EFFECTS DURING IRE TREATMENTS Garcia PA, Davalos RV, Miklavčič D. A numerical investigation of the electric and thermal cell kill distributions in electroporation-based therapies in tissue. PLOS One 9(8): e103083, 2014. NOTES & RESULTS 66 EBTT WORKSHOP 2020 MOLECULAR DYNAMICS SIMULATIONS OF MEMBRANE ELECTROPORATION Molecular dynamics simulations of membrane electroporation C3 Mounir Tarek CNRS- Université de Lorrains, Nancy France Europeen Laboratory EBAM Duration of the experiments: 90 min Max. number of participants: 18 Location: Computer room (CIT) Level: Basic PREREQUISITES No specific knowledge is required for this laboratory practice. THEORETICAL BACKGROUND The application of high electric fields to cells or tissues permeabilizes the cell membrane and is thought to produce aqueous-filled pores in the lipid bilayer. Electroporation is witnessed when the lipid membrane is subject to transmembrane voltages (TMV) of the order of few hundred millivolts, which results from the application of electrical pulses on a microsecond to millisecond time scale Figure 1: Configurations from the MD simulation for a large POPC subject to a transverse electric field (A) Bilayer at equilibrium. (B-C) Formation of water wires at the initial stage of the electroporation process (D-F) Formation at a later stage of large water pores that conduct ions across the membrane and that are stabilized by lipid head-group (yellow cyan). (Delemotte and Tarek. J. Membr. Biol. 2012). which are sufficient to produce a transient trans-membrane potential and an electrical field across the membrane of the order of ~ 108 V/m. This process is believed to involve (1) charging of the membrane due to ion flow, (2) rearrangement of the molecular structure of the membrane, (3) formation of pores, which perforate the membrane and are filled by water molecules (so-called aqueous, or hydrophilic, 67 EBTT WORKSHOP 2020 MOLECULAR DYNAMICS SIMULATIONS OF MEMBRANE ELECTROPORATION pores), (4) an increase in ionic and molecular transport through these pores, and, under appropriate conditions, membrane integrity recovery when the external field stress is removed. Molecular Dynamics (MD) simulations belong to a set of computational methods in which the dynamical behaviour of an ensemble of atoms or molecules, interacting via approximations of physical pair potentials, is determined from the resolution of the equation of motions. MD simulations enable ones to investigate the molecular processes affecting the atomic level organization of membranes when these are submitted to voltage gradient of magnitude similar to those applied during electropulsation. The aim of this practical exercise is to characterize from MD simulations trajectories the electrostatic properties of membranes subject to a transmembrane potential (0 to 2 V). Figure 2: Electrostatic potential maps generated from the MD simulations of a POPC lipid bilayer (acyl chains, green; head groups, white) surrounded by electrolyte baths at 1 M NaCl (Na+ yellow, Cl- green, water not shown) terminated by an air/water interface. Left: net charge imbalance Q = 0 e (TMV=0 mV). Right: Q = 6 e (TMV=2 V). The aim of this laboratory practice is to get familiar with the tools for molecular dynamics, possibilities to set on models and graphical presentation of atomistic models. EXPERIMENT Due to the limited time and large resources needed to generate MD trajectories of membranes, the latter will be provided to the students. The simulations concern pure planar phospholipid bilayers (membrane constituents) and water described at the atomic level. A set of long trajectories spanning few nanoseconds generated with or without a transmembrane voltage induced by unbalanced ionic concentrations in the extracellular and intracellular will be provided. The students will (1) determine the distribution of potential and electric field in model membrane bilayers (2) measure the membrane capacitance, (3) visualize at the molecular level the formation of membrane pores under the influence of a transmembrane voltage, and measure the intrinsic conductance of such pores. FURTHER READING: Tarek, M. Membrane Electroporation: A Molecular Dynamics Study Biophys. J. 88: 4045-4053, 2005. Dehez, F.; Tarek, M.; and Chipot, C. Energetics of Ion Transport in a Peptide Nanotube J. Phys. Chem. B 111: 10633-10635, 2007 Andrey A. Gurtovenko, Jamshed Anwar, and Ilpo Vattulainen, Defect-Mediated Trafficking across Cell Membranes: Insights from in Silico Modeling, Chem. Rev. 110: 6077-6103, 2010. Delemotte, L. and Tarek, M. Molecular Dynamics Simulations of Membrane Electroporation J. Membr. Biol. 245/9:531-543, 2012. NOTES & RESULTS 68 E-LEARNING EBTT WORKSHOP 2020 ELECTROPORATION OF CELLS AND TISSUES - INTERACTIVE E-LEARNING COURSE Electroporation of cells and tissues - interactive e-learning course E1 Selma Čorović and Samo Mahnič Kalamiza University of Ljubljana, Faculty of electrical engineering Duration of the experiment: app. 90 min Max. number of participants: 18 Location: Computer room (P5) Level: Basic PREREQUISITES No specific knowledge is required for this laboratory practice. The aim of this laboratory practice is to provide the participants with basic knowledge on local electric field distribution in cells and tissues exposed to high voltage electric pulses (i.e. electroporation pulses) by means of interactive e-learning course content. The e-learning content is based on the available knowledge from the scientific literature. PROTOCOL OF THE E-LEARNING COURSE The participants will be gathered in a computer-computer classroom providing each participant with a computer. A short test will be given to establish the baseline knowledge before the e-learning course. Within the first part of the e-learning course we will bring together the educational material on basic mechanisms underlying electroporation process on the levels of cell membrane, cell and tissues as a composite of cells (Figure 1). Figure 1: Introduction of small molecules (blue molecules) through a cell membrane (a) into an electroporated cell (b) and into the successfully electroporated cells within an exposed tissue (c) (Čorović et al., 2009). Within the second part of the course we will provide basic knowledge on important parameters of local electric field needed for efficient cells and tissue electroporation, such as: electrode geometry (needle or plate electrodes as illustrated in Figure 2, electrode position with respect to the target tissue and its surrounding the tissues (Figure 3), the contact surface between the electrode and the tissue, the voltage applied to the electrodes and electroporation threshold values. This part of the e-learning course content will be provided by an interactive module we developed in order to visualize the local electric field distribution in 2D and 3D dimensional tissue models. The objective of this module is to provide: - local electric field visualization in cutaneous (protruding tumors) and subcutaneous tumors (tumors more deeply seeded in the tissue); - guideline on how to overcome a highly resistive skin tissue in order to permeabilize more conductive underlying tissues and 71 EBTT WORKSHOP 2020 ELECTROPORATION OF CELLS AND TISSUES - INTERACTIVE E-LEARNING COURSE - visualization and calculation of successfully electroporated volume of the target tissue and its surrounding tissue (i.e. the treated tissue volume exposed to the electric field between reversible and irreversible electroporation threshold value Erev ≤ E < Eirrev) with respect to the selected parameters such as: number and position of electrodes, applied voltage on the electrodes. Figure 2: Plate electrodes vs. needle electrodes with respect to the target tissue (e.g. tumor tissue). Figure 3: Electric field distribution within the tumor (inside the circle) and within its surrounding tissue (outside the circle) obtained with three different selection of parameters (number and position of electrodes and voltage applied): (a) 4 electrodes, (b) 8 electrodes and (c) 8 electrodes with increased voltage on electrodes so that the entire volume of tumor is exposed to the Erev ≤ E < Eirrev. After the e-learning course the pedagogical efficiency of presented educational content and the e-learning application usability will be evaluated. FURTHER READING: Čorović S, Pavlin M, Miklavčič D. Analytical and numerical quantification and comparison of the local electric field in the tissue for different electrode configurations. Biomed. Eng. Online 6: 37, 2007. Serša G, Miklavcic D: Electrochemotherapy of tumours (Video Article). J. Visual Exp. 22: 1038, 2008. Čorović S, Županič A, Miklavčič D. Numerical modeling and optimization of electric field distribution in subcutaneous tumor treated with electrochemotherapy using needle electrodes. IEEE T. Plasma Sci. 36: 1665-1672, 2008. Čorović S, Bešter J, Miklavčič D. An e-learning application on electrochemotherapy. Biomed. Eng. Online 8: 26, 2009. Čorović S, Županič A, Kranjc S, Al Sakere B, Leroy-Willig A, Mir LM, Miklavčič D. The influence of skeletal muscle anisotropy on electroporation: in vivo study and numerical modeling. Med. Biol. Eng. Comput. 48: 637-648, 2010. Edhemovic I, Gadzijev EM, Brecelj E, Miklavcic D, Kos B, Zupanic A, Mali B, Jarm T, Pavliha D, Marcan M, Gasljevic G, Gorjup V, Music M, Pecnik Vavpotic T, Cemazar M, Snoj M, Sersa G. Electrochemotherapy: A new technological approach in treatment of metastases in the liver. Technol Cancer Res Treat 10:475-485, 2011. Bergues Pupo AE, Reyes JB, Bergues Cabrales LE, Bergues Cabrales JM. Analitical and numerical quantification of the potential and electric field in the tumor tissue for different conic sections. Biomed. Eng. Online 10:85, 2011. Neal RE II, Garcia PA, Robertson JL, Davalos RV. Experimental characterization and numerical modeling of tissue electrical conductivity during pulsed electric fields for irreversible electroporation treatment planning. IEEE T. Biomed. Eng. 59(4):1077-1085, 2012. 72 EBTT WORKSHOP 2020 ELECTROPORATION OF CELLS AND TISSUES - INTERACTIVE E-LEARNING COURSE Čorović S, Mir LM, Miklavčič D. In vivo muscle electroporation threshold determination: realistic numerical models and in vivo experiments. Journal of Membrane Biology 245: 509-520, 2012. Essone Mezeme M, Pucihar G, Pavlin M, Brosseau C, Miklavčič D. A numerical analysis of multicellular environment for modeling tissue electroporation. Appl. Phys. Lett. 100: 143701, 2012. Mahnič-Kalamiza S, Kotnik T, Miklavčič D. Educational application for visualization and analysis of electric field strength in multiple electrode electroporation. BMC Med. Educ. 12: 102, 2012. Čorović S, Lacković I, Šuštarič P, Šuštar T, Rodič T, Miklavčič D. Modeling of electric field distribution in tissues during electroporation. Biomed. Eng. Online 12: 16, 2013. NOTES & RESULTS 73 EBTT WORKSHOP 2020 ELECTROPORATION OF CELLS AND TISSUES - INTERACTIVE E-LEARNING COURSE NOTES & RESULTS 74 EBTT WORKSHOP 2020 FACULTY MEMBERS Faculty members Damijan Miklavčič University of Ljubljana, Faculty of Electrical Engineering, Tržaška 25, SI-1000 Ljubljana, Slovenia E-mail: damijan.miklavcic@fe.uni-lj.si Lluis M. Mir Université Paris-Saclay, CNRS, Institut Gustave Roussy, Metabolic and systemic aspects of oncogenesis (METSY), 114 rue Edouard Vaillant, F-94805 Villejuif Cédex, France E-mail: Luis.MIR@gustaveroussy.fr Marie-Pierre Rols Institute of Pharmacology and Structural Biology, CNRS - University of Toulouse III, IPBS UMR 5089, 205, route de Narbonne, 31077 Toulouse, France E-mail: marie-pierre.rols@ipbs.fr Gregor Serša Institute of Oncology, Zaloška 2, SI-1000 Ljubljana, Slovenia E-mail: gsersa@onko-i.si Mounir Tarek Theoretical Physics and Chemistry Laboratory, Université de Lorraine, CNRS, LPCT, F-54000 Nancy E-mail: mounir.tarek@univ-lorraine.fr P. Thomas Vernier Frank Reidy Research Center for Bioelectrics, Old Dominion University, 4211 Monarch Way, Norfolk, VA 23508, USA E-mail: pvernier@odu.edu Julie Gehl University of Copenhagen, Department of Clinical Medicine, Blegdamsvej 3, 2200 København N Denmark E-mail: julie.gehl@sund.ku.dk 75 See you next time in Copenhagen! We will all be there again!