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Objective Irreversible electroporation (IRE) is a non thermal tissue ablation therapy which is induced by applying high voltage waveforms across electrode pairs. When multiple electrode pairs are sequentially used, the treatment volume (TV) is typically computed as the geometric union of the TVs of individual pairs. However, this method neglects that some regions are exposed to overlapping treatments. Recently, a model describing cell survival probability was introduced which effectively predicted TV with overlapping fields in vivo. However, treatment overlap has yet to be quantified. This study characterizes TV overlap in a controlled in vitro setup with the two existing methods which are compared to an adapted logistic model proposed here. Methods CHO cells were immobilized in agarose gel. Initially, we characterized the electric field threshold and the cell survival probability for overlapping treatments. Subsequently, we created a 2D setup where we compared and validated the accuracy of the different methods in predicting the TV. Results Overlap can reduce the electric field threshold required to induce cell death, particularly for treatments with low pulse number. However, it does not have a major impact on TV in the models assayed here, and all the studied methods predict TV with similar accuracy. Conclusion Treatment overlap has a minor influence in the TV for typical protocols found in IRE therapies. Significance This study provides evidence that the modeling method used in most pre-clinical and clinical studies seems adequate.

Background and objectives: Electroporation is the phenomenon by which cell membrane permeability to ions and macromolecules is increased when the cell is briefly exposed to high electric fields. In electroporation-based treatments, such exposure is typically performed by delivering high voltage pulses across needle electrodes in tissue. For a given tissue and pulsing protocol, an electric field magnitude threshold exists that must be overreached for treatment efficacy. However, it is hard to preoperatively infer the treatment volume because the electric field distribution intricately depends on the electrodes’ positioning and length, the applied voltage, and the electric conductivity of the treated tissues. For illustrating such dependencies, we have created EView (https://eview.upf.edu), a web platform that estimates the electric field distribution for arbitrary needle electrode locations and orientations and overlays it on 3D medical images. Methods: A client-server approach has been implemented to let the user set the electrode configuration easily on the web browser, whereas the simulation is computed on a dedicated server. By means of the finite element method, the electric field is solved in a 3D volume. For the sake of simplicity, only a homogeneous tissue is modeled, assuming the same properties for healthy and pathologic tissues. The non-linear dependence of tissue conductivity on the electric field due to the electroporation effect is modeled. The implemented model has been validated against a state of the art finite element solver, and the server has undergone a heavy load test to ensure reliability and to report execution times. Results: The electric field is rapidly computed for any electrode and tissue configuration, and alternative setups can be easily compared. The platform provides the same results as the state of the art finite element solver (Dice = 98.3 ± 0.4%). During the high load test, the server remained responsive. Simulations are computed in less than 2 min for simple cases consisting of two electrodes and take up to 40 min for complex scenarios consisting of 6 electrodes. Conclusions: With this free platform we provide expert and non-expert electroporation users a way to rapidly model the electric field distribution for arbitrary electrode configurations. This work was supported by the Ministry of Economy and Competitiveness of Spain (grant number TEC2014-52383-C3-2-R), the María de Maeztu Units of Excellence Programme (grant number MDM-2015-0502) and by the Spanish Ministry of Economy and Competitiveness under the Programme for the Formation of Doctors (grant number BES-2017-081164). Antoni Ivorra gratefully acknowledges the financial support by ICREA under the ICREA Academia programme.

International audience; Objective: The purpose of the present work is to assess the ability of sine waves to perform Electrochemotherapy (ECT) and to study the dependence of the frequency of the applied sine wave on the treatment efficacy. Methods: A subcutaneous tumor model in mice was used, and the electric field was delivered in combination with bleomycin. Sinusoidal electric fields of different frequencies, amplitudes, and durations were compared to square waves. Computer simulations were additionally performed. Results: The results confirmed the ability of a sinusoidal electric field to obtain successful ECT responses. A strong dependence with frequency was obtained. The efficacy of the treatment decreased when the frequency of the sine waves was increased. At low sinusoidal frequency, the efficacy of the treatment is very similar to that obtained with a square wave. The collateral effects such as skin burns and muscle contractions decreased for the highest frequency assayed. Conclusion: The use of sine wave burst represent a feasible option for the treatment of cancer by ECT. Significance: These results could have important implications for the treatment of cancer in the clinical world where ECT is performed with DC square pulses.

High-frequency irreversible electroporation (H-FIRE) has emerged as an alternative to conventional irreversible electroporation (IRE) to overcome the issues associated with neuromuscular electrical stimulation that appear in IRE treatments. In H-FIRE, the monopolar pulses typically used in IRE are replaced with bursts of short bipolar pulses. Currently, very little is known regarding how the use of a different waveform affects the cell death dynamics and mechanisms. In this study, human pancreatic adenocarcinoma cells were treated with a typical IRE protocol and various H-FIRE schemes with the same energized time. Cell viability, membrane integrity and Caspase 3/7 activity were assessed at different times after the treatment. In both treatments, we identified two different death dynamics (immediate and delayed) and we quantified the electric field ranges that lead to each of them. While in the typical IRE protocol, the electric field range leading to a delayed cell death is very narrow, this range is wider in H-FIRE and can be increased by reducing the pulse length. Membrane integrity in cells suffering a delayed cell death shows a similar time evolution in all treatments, however, Caspase 3/7 expression was only observed in cells treated with H-FIRE. Ministry of Economy and Competitiveness of Spain [TEC2014-52383-C3-2-R]; Pancreatic Cancer Action Network [16-65-IANN]; Cures Within Reach [PUJFSANY]; ICREA under the ICREA Academia programme The authors would like to thank Dr. Elisa M. Wasson for her help in the image analysis of 3D cell cultures. This work was supported by the Ministry of Economy and Competitiveness of Spain (Grant No. TEC2014-52383-C3-2-R), the Pancreatic Cancer Action Network (Grant No. 16-65-IANN) and Cures Within Reach (Grant No. PUJFSANY). Antoni Ivorra gratefully acknowledges financial support by ICREA under the ICREA Academia programme.

Pulsed radiofrequency (PRF) treatments for chronic pain consist in the delivery of a train of sinusoidal electric bursts to the targeted nerve. Despite numerous clinical evidence of its efficiency, the mechanism of action of PRF remains unclear. Since most of the reported biological effects of PRF can be initiated by a calcium influx into the neurons, we hypothesized that PRF may induce a mild electroporation effect causing a calcium uptake. To test this hypothesis, HEK-293 cells were exposed to PRF bursts and cytosolic calcium and Yo-Pro-1 uptake were monitored. After a single burst, calcium peaks were observed for electric fields above 480 V/cm while the uptake of Yo-pro-1 was insignificant. After a train of 120 bursts, the electric fields required to induce a calcium and Yo-pro-1 uptake decreased to 330 V/cm and 880 V/cm respectively. Calcium peaks were not detected when cells were treated in calcium free media. The temperature increase during the treatments was lower than 5 °C in all cases. Finally, the cell response for different burst frequencies and extracellular media conductivities correlated with the induced transmembrane voltage calculated with a numerical model. Our results support the hypothesis of an electroporation mediated calcium influx. This work was supported by the Ministry of Economy and Competitiveness of Spain through the grants TEC2014-52383-C3-2-R and SAF2014-52228-R. Antoni Ivorra gratefully acknowledges the financial support by ICREA under the ICREA Academia programme.

Electrolytic Electroporation (E2) is the combination of reversible electroporation and electrolysis. It has been proposed as a novel treatment option to ablate tissue percutaneously. The present in vitro study in cells in suspension was performed to investigate the underlying mechanisms of action of E2. Different types of experiments were performed to isolate the effects of the electrolysis and the electroporation components of the treatment. Additionally, thermal simulations were performed to determine whether significant temperature increase contributes to the effect. The results indicate that E2’s cell killing efficacy is due to a combinational effect of electrolysis and reversible electroporation that takes place within the first two minutes after E2 application. The results further show that cell death after E2 treatment is significantly delayed. These observations suggest that cell death is induced in permeabilized cells due to the uptake of electrolysis species. Thermal simulations revealed a significant but innocuous temperature increase.

Optimization of protocols is required for each specific type of biomass processed by electroporation of the cell membrane with high voltage pulsed electric fields (PEF). Such optimization requires convenient and adaptable laboratory systems, which will enable determination of both electrical and mechanical parameters for successful electroporation and fractionation. In this work, we report on a laboratory PEF system consisting of a high voltage generator with a novel asymmetric voltage multiplying architecture and a treatment chamber with sliding electrodes. The system allows applying pulses of up to 4 kV and 1 kA with a pulse duration between 1 μs and 100 μs. The allowable energy dissipated per pulse on electroporated biomass is determined by the conditions for cooling the biomass in the electroporation cell. The device was tested on highly conductive green macroalgae from Ulva sp., a promising but challenging feedstock for the biorefinery. Successful electroporation was confirmed with bioimpedance measurements. Industrial relevance Seaweed biomass is an emerging feedstock for biorefineries with already 30 million tons per year of global industrial production. However, most of the biomass produced today is lost. Pulsed electric field (PEF) extraction could allow saving energy on biomass drying, deashing and it could allow extracting various organic compounds. However, the parameters needed to seaweed biomass treatment with PEF are not known and will differ from species to species. Furthermore, very high salt content challenges most of the available laboratory PEF devices, limiting the ability for parameters optimization in the lab. The developed laboratory scale PEF system coupled to bioimpedance measurement provides a necessary set of tools and methods for PEF parameters optimization required for process scale-up.

Electroporation based treatments consist in applying one or multiple high voltage pulses to the tissues to be treated. As an undesired side effect, these pulses cause electrical stimulation of excitable tissues such as nerves and muscles. This increases the complexity of the treatments and may pose a risk to the patient. To minimize electrical stimulation during electroporation based treatments, it has been proposed to replace the commonly used monopolar pulses by bursts of short bipolar pulses. In the present study, we have numerically analyzed the rationale for such approach. We have compared different pulsing protocols in terms of their electroporation efficacy and their capability to trigger action potentials in nerves. For that, we have developed a modeling framework that combines numerical models of nerve fibers and experimental data on irreversible electroporation. Our results indicate that, by replacing the conventional relatively long monopolar pulses by bursts of short bipolar pulses, it is possible to ablate a large tissue region without triggering action potentials in a nearby nerve. Our models indicate that this is possible because, as the pulse length of these bipolar pulses is reduced, the stimulation thresholds raise faster than the irreversible electroporation thresholds. We propose that this different dependence on the pulse length is due to the fact that transmembrane charging for nerve fibers is much slower than that of cells treated by electroporation because of their geometrical differences. This work was supported by the Ministry of Economy and Competitiveness of Spain through the grant TEC2014-52383-C3-2-R. RVD’s research is supported by the National Institutes of Health under award number NIH 1R21 CA192041-01.

It is widely accepted that electroporation occurs when the cell transmembrane voltage induced by an external applied electric field reaches a threshold. Under this assumption, in order to trigger electroporation in a spherical cell, Schwan’s equation leads to an inversely proportional relationship between the cell radius and the minimum magnitude of the applied electric field. And, indeed, several publications report experimental evidences of an inverse relationship between the cell size and the field required to achieve electroporation. However, this dependence is not always observed or is not as steep as predicted by Schwan’s equation. The present numerical study attempts to explain these observations that do not fit Schwan’s equation on the basis of the interplay between cell membrane conductivity, permeability, and transmembrane voltage. For that, a single cell in suspension was modeled and the electric field necessary to achieve electroporation with a single pulse was determined according to two effectiveness criteria: a specific permeabilization level, understood as the relative area occupied by the pores during the pulse, and a final intracellular concentration of a molecule due to uptake by diffusion after the pulse, during membrane resealing. The results indicate that plausible model parameters can lead to divergent dependencies of the electric field threshold on the cell radius. These divergent dependencies were obtained through both criteria and using two different permeabilization models. This suggests that the interplay between cell membrane conductivity, permeability, and transmembrane voltage might be the cause of results which are noncompatible with the Schwan’s equation model. This work was supported by the Ministry of Economy and Competitiveness of Spain through Grant TEC2014-52383-C3-2-R. PTV received support from the Old Dominion University Frank Reidy Research Center for Bioelectrics and the Air Force Office of Scientific Research (FA9550-15-1-0517, FA9550-14-1-0123).

Electroporation based treatments typically consist in applying high voltage dc pulses. As an undesired side effect, these dc pulses cause electrical stimulation of excitable tissues such as motor nerves. In the present in vivo study, it was explored the use of bursts of sinusoidal voltage in the frequency range from 50 kHz to 2 MHz to induce irreversible electroporation (IRE) whilst avoiding neuromuscular stimulation. Series of 100 dc pulses or sinusoidal bursts, both with an individual duration of 100 μs, were delivered to rabbit liver through thin needles in a monopolar electrode configuration and thoracic movements were recorded with an accelerometer. Tissue samples were harvested three hours after treatment and later postprocessed to determine the dimensions of the IRE lesions. Thermal damage due to Joule heating was ruled out via computer simulations. Sinusoidal bursts with a frequency equal or above 100 kHz did not cause thoracic movements and induced lesions equivalent to those obtained with conventional dc pulses when the applied voltage amplitude was sufficiently high. IRE efficacy dropped with increasing frequency. For 100 kHz bursts, it was estimated that the electric field threshold for IRE is about 1.4 kV/cm whereas that of dc pulses is about 0.5 kV/cm. This work was supported by the Ministry of Economy and Competitiveness of Spain through the grant TEC2014-52383-C3-R (TEC2014-52383-C3-2-R and TEC2014-52383-C3-3-R).

[EN] Background: The thermal and electrical effects of pulsed radiofrequency (PRF) for pain relief can be controlled by modifying the characteristics of the RF pulses applied. Our goal was to evaluate the influence of such modifications on the thermal and electric performance in tissue. Methods: A computational model was developed to compare the temperature and electric field time courses in tissue between a standard clinical protocol (45 V pulses, 20ms duration, 2 Hz repetition frequency) and a new protocol (55 V pulses, 5ms duration, 5 Hz repetition frequency) with a higher applied electric field but a smaller impact on temperature alterations in tissue. The effect of including a temperature controller was assessed. Complementarily, an agar-based experimental model was developed to validate the methodology employed in the computer modelling. Results: The new protocol increased the electric field magnitude reached in the tissue by around +20%, without increasing the temperature. The temperature controller was found to be the fundamental factor in avoiding thermal damage to the tissue and reduced the total number of pulses delivered by around 67%. The experimental results matched moderately well with those obtained from a computer model built especially to mimic the experimental conditions. Conclusions: For the same delivered energy, the new protocol significantly increases the magnitude of the applied electric field, which may be the reason why it is clinically more effective in achieving pain relief., This work was supported by the Spanish "Plan Estatal de Investigacion, Desarrollo e Innovacion Orientada a los Retos de la Sociedad" under Grant TEC2014-52383-C3-R (TEC2014-52383-C3-1-R and TEC2014-52383-C3-2-R). Elzbieta Ewertowska has a Predoctoral Grant (BES-2015-073285) from the Ministry of Economy, Industry and Competitiveness (Government of Spain).