In Vitro Imaging and Molecular Characterization of Ca2+ Flux Modulation by Nanosecond Pulsed Electric Fields.

In recent years, the application of pulsed electric fields with very short durations (nanoseconds) and extremely high amplitudes (MV/m) has been investigated for novel medical purposes. Various electric protocols have been explored for different objectives, including the utilization of fractionated pulse doses to enhance cell electrosensitization to the uptake of different markers or an increase in apoptosis. This study focused on the use of fluorescence imaging to examine molecular calcium fluxes induced by different fractionated protocols of short electric pulses in neuroblastoma (SH-SY5Y) and mesenchymal stem cells (HaMSCs) that were electroporated using nanosecond pulsed electric fields. In our experimental setup, we did not observe cell electrosensitization in terms of an increase in calcium flux following the administration of fractionated doses of nanosecond pulsed electric fields with respect to the non-fractionated dose. However, we observed the targeted activation of calcium-dependent genes (c-FOS, c-JUN, EGR1, NURR-1, ß3-TUBULIN) based on the duration of calcium flux, independent of the instantaneous levels achieved but solely dependent on the final plateau reached. This level of control may have potential applications in various medical and biological treatments that rely on calcium and the delivery of nanosecond pulsed electric fields.

Dynamics of High-Density Unipolar Epicardial Electrograms During PFA.

BACKGROUND: Pulsed field ablation (PFA) is a novel nonthermal cardiac ablation technology based on irreversible electroporation (IRE). While areas of IRE lead to durable lesions, the surrounding regions, where reversible electroporation occurs, recover. The behavior of local electrograms in areas of different electroporation levels remains unknown. The goal of this study is to characterize electrogram dynamics after PFA in IRE and reversible electroporation areas. METHODS: A total of 6 domestic swine were used. PFA was applied in the epicardium of the right and left ventricles using a focal monopolar catheter. Additional radiofrequency ablations were performed. Epicardial unipolar electrograms were acquired at baseline and for 60 minutes post PFA/radiofrequency ablation using a high-density electrode matrix attached to the epicardium. Electrogram dynamics were analyzed in areas corresponding to different levels of electroporation. Acute lesion formation was assessed after 3 to 5 hours by triphenyl tetrazolium chloride staining. RESULTS: Electrogram analysis demonstrated a clear association between electrogram changes and the level of electroporation. Immediately after PFA, electrograms displayed the following: a significant decrease in R/S-wave amplitude; a large elevation of the ST-segment; and a large decrease in their |(dV/dt)|max. Marked changes in electrograms were observed beyond the lesion area. Thereafter, a gradual recovery was observed. The evolution of all the electrogram parameters throughout the 60 minutes after PFA was significantly different (P<0.05) between the IRE and reversible electroporation areas. Acute lesion staining showed significantly larger depth for PFA lesions compared with radiofrequency ablation. CONCLUSIONS: This study shows that unipolar electrograms can differentiate between reversible electroporation and IRE areas during the first 30 minutes post ablation. Differences after the first 30 minutes are less evident. Our findings could result useful for immediate lesion assessment after PFA and warrant further investigation.

Parametric Study of Pulsed Field Ablation With Biphasic Waveforms in an In Vivo Heart Model: The Role of Frequency.

BACKGROUND: Pulsed field ablation (PFA) is a novel nonthermal cardiac ablation technology based on irreversible electroporation. Unfortunately, the characteristics of the electric field waveforms used in clinical and experimental PFA are not typically reported. This study examines the effect of the frequency of biphasic waveforms and compares biphasic to monophasic waveforms. METHODS: A total of 29 Sprague-Dawley rats were treated with PFA using an epicardial monopolar electrode. Biphasic waveforms with three different frequencies, 90, 260, and 450 kHz (10 bursts of 100 µs duration at 500 V or 800 V) and monophasic waveforms (10 pulses of 100 µs duration at 500 V) were studied. Collateral neuromuscular stimulation and temperature increase in the point of application were directly measured. Lesion formation was assessed 3 weeks after treatment by histopathologic analysis. Computer simulations were used to estimate the electric field lethal threshold for each condition. A previous in vitro study was performed to draw a complete characterization of the studied dependencies. RESULTS: Morphometric analysis demonstrated a significant association between chronic lesion size and waveform characteristics. For the same voltage level, monophasic waveforms yielded the largest lesions compared with any of the biphasic protocols (P<0.05). Increasing PFA frequency was associated with reduced neuromuscular stimulation but also with reduced ablation efficacy. Maximum absolute temperature increase recorded along a complete treatment was 3 °C. Vascular structures inside the lesions were preserved for all conditions. Computer simulation-based analysis showed that waveform frequency had a graded effect on the lethal electric field threshold, with threshold of 600 V/cm for monophasic waveforms versus 2000 V/cm for biphasic waveforms with a frequency of 450 kHz. CONCLUSIONS: Frequency is a major determinant of efficacy in biphasic PFA. Our results highlight the critical need of disclosing waveform characteristics when reporting the results of different PFA systems.

A computational comparison of radiofrequency and pulsed field ablation in terms of lesion morphology in the cardiac chamber.

Pulsed Field Ablation (PFA) has been developed over the last years as a novel electrical ablation technique for treating cardiac arrhythmias. It is based on irreversible electroporation which is a non-thermal phenomenon innocuous to the extracellular matrix and, because of that, PFA is considered to be safer than the reference technique, Radiofrequency Ablation (RFA). However, possible differences in lesion morphology between both techniques have been poorly studied. Simulations including electric, thermal and fluid physics were performed in a simplified model of the cardiac chamber which, in essence, consisted of a slab of myocardium with blood in motion on the top. Monopolar and bipolar catheter configurations were studied. Different blood velocities and catheter orientations were assayed. RFA was simulated assuming a conventional temperature-controlled approach. The PFA treatment was assumed to consist in a sequence of 20 biphasic bursts (100 µs duration). Simulations indicate that, for equivalent lesion depths, PFA lesions are wider, larger and more symmetrical than RFA lesions for both catheter configurations. RFA lesions display a great dependence on blood velocity while PFA lesions dependence is negligible on it. For the monopolar configuration, catheter angle with respect to the cardiac surface impacted both ablation techniques but in opposite sense. The orientation of the catheter with respect to blood flow direction only affected RFA lesions. In this study, substantial morphological differences between RFA and PFA lesions were predicted numerically. Negligible dependence of PFA on blood flow velocity and direction is a potential important advantage of this technique over RFA.

Modeling Methods for Treatment Planning in Overlapping Electroporation Treatments.

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.

Biological effects of ultrashort electric pulses in a neuroblastoma cell line: the energy density role.

BACKGROUND: Despite the numerous literature results about biological effects of electromagnetic field (EMF) exposure, the interaction mechanisms of these fields with organisms are still a matter of debate. Extremely low frequency (ELF) MFs can modulate redox homeostasis and we showed that 24 h exposure to 50 Hz-1 mT has a pro-oxidant effect and effects on the epigenome of SH-SY5Y cells, decreasing miR-34b/c expression through the hypermethylation of their promoter. METHODS: Here, we investigated the role of the electromagnetic deposited energy density (ED) during exposures lasting 24 h to 1 mT amplitude MFs at a frequency of 50 Hz in inducing the above mentioned effects. To this end, we delivered ultrashort electric pulses, in the range of microsecond and nanosecond duration, with the same ED of the previously performed magnetic exposure to SH-SY5Y cells. Furthermore, we explored the effect of higher deposited energy densities. Analysis of i) gene and microRNA expression, ii) cell morphology, iii) reactive oxygen species (ROS) generation, and iv) apoptosis were carried out. RESULTS: We observed significant changes in egr-1 and c-fos expression at very low deposited ED levels, but no change of the ROS production, miR-34b/c expression, nor the appearance of indicators of apoptosis. We thus sought investigating changes in egr-1 and c-fos expression caused by ultrashort electric pulses at increasing deposited ED levels. The pulses with the higher deposited ED caused cell electroporation and even other morphological changes such as cell fusion. The changes in egr-1 and c-fos expression were more intense, but, again, no change of the ROS production, miR-34b/c expression, nor apoptosis induction was observed. CONCLUSIONS: These results, showing that extremely low levels of electric stimulation (never investigated until now) can cause transcriptional changes, also reveal the safety of the electroporating pulses used in biomedical applications and open up the possibility to further therapeutic applications of this technology.

Comparing High-Frequency With Monophasic Electroporation Protocols in an In Vivo Beating Heart Model.

This study compared monophasic 100-µs pulses with high-frequency electroporation (HF-EP) bursts using an in vivo animal model. Myocardial damage was evaluated by histologic analysis. Compared with 10 monophasic pulses, 20 bursts of HF-EP at 100 and 150 kHz were associated with less damage. However, when the number of HF-EP bursts was increased to 60, myocardial damage was comparable to that of the monophasic group. HF-EP protocols were associated with attenuated collateral muscle contractions. This study shows that HF-EP is feasible and effective and that pulse frequency has a significant effect on extent of ablation.

Possible molecular and cellular mechanisms at the basis of atmospheric electromagnetic field bioeffects.

Mechanisms of how electromagnetic (EM) field acts on biological systems are governed by the same physics regardless of the origin of the EM field (technological, atmospheric...), given that EM parameters are the same. We draw from a large body of literature of bioeffects of a man-made electromagnetic field. In this paper, we performed a focused review on selected possible mechanisms of how atmospheric electromagnetic phenomena can act at the molecular and cellular level. We first briefly review the range of frequencies and field strengths for both electric and magnetic fields in the atmosphere. Then, we focused on a concise description of the current knowledge on weak electric and magnetic field bioeffects with possible molecular mechanisms at the basis of possible EM field bioeffects combined with modeling strategies to estimate reliable outcomes and speculate about the biological effects linked to lightning or pyroelectricity. Indeed, we bring pyroelectricity as a natural source of voltage gradients previously unexplored. While very different from lightning, it can result in similar bioeffects based on similar mechanisms, which can lead to close speculations on the importance of these atmospheric electric fields in the evolution.

Electrophoresis-assisted accumulation of conductive nanoparticles for the enhancement of cell electropermeabilization.

The use of conductive nanoparticles (NPs) was previously proposed as a way to locally amplify the electric field (EF) intensity at the cell membrane to enhance cell electroporation. To achieve this, a close distance between the NPs and the cell membrane is mandatory. Here, a new method to improve the contact between NPs and cell surface using the effects of electric pulses (electrophoretic forces) is explored. The effects of two types of electric pulses are analyzed alone or combined in a two-pulse-train protocol on Chinese hamster DC-3F cells. Particularly we used 100 µs duration pulses, low intensity-millisecond pulses and combinations of both. Finally, we studied the use of surface coated NPs (PEGylated) for this application. Our results demonstrate that the delivery of an electric field prior to the electroporation pulses increases the accumulation of NPs around the cell membrane suggesting that NPs are pushed towards the cell surface through electrophoretic forces. This allowed reducing the need for long incubations between cells and NPs to observe an enhancement of electroporation mediated by conductive NPs. Thus low intensity-millisecond pulses can be used to increase the accumulation of either aggregated or individual (i.e. PEGylated) NPs supporting the electrophoretic nature of the observed effects.

Physiological changes may dominate the electrical properties of liver during reversible electroporation: Measurements and modelling.

This study presents electrical measurements (both conductivity during the pulses and impedance spectroscopy before and after) performed in liver tissue of mice during electroporation with classical electrochemotherapy conditions (8 pulses of 100 µs duration). A four-needle electrode arrangement inserted in the tissue was used for the measurements. The undesirable effects of the four-electrode geometry, notably concerning its sensitivity, were quantified and discussed showing how the electrode geometry chosen for the measurements can impact the results. Numerical modelling was applied to the information collected during the pulse, and to the impedance spectra acquired before and after the pulses sequence. Our results show that the numerical results were not consistent, suggesting that other collateral phenomena not considered in the model are at work during electroporation in vivo. We show how the modification in the volume of the intra and extra cellular media, likely caused by the vascular lock effect, could at least partially explain the recorded impedance evolution. In the present study we demonstrate the significant impact that physiological effects have on impedance changes following electroporation at the tissue scale and the potential need of introducing them into the numerical models. The code for the numerical model is publicly available at https://gitlab.inria.fr/poignard/4-electrode-system.

Monitoring the molecular composition of live cells exposed to electric pulses via label-free optical methods.

The permeabilization of the live cells membrane by the delivery of electric pulses has fundamental interest in medicine, in particular in tumors treatment by electrochemotherapy. Since underlying mechanisms are still not fully understood, we studied the impact of electric pulses on the biochemical composition of live cells thanks to label-free optical methods: confocal Raman microspectroscopy and terahertz microscopy. A dose effect was observed after cells exposure to different field intensities and a major impact on cell peptide/protein content was found. Raman measurements reveal that protein structure and/or environment are modified by the electric pulses while terahertz measurements suggest a leakage of proteins and other intracellular compounds. We show that Raman and terahertz modalities are a particularly attractive complement to fluorescence microscopy which is the reference optical technique in the case of electropermeabilization. Finally, we propose an analytical model for the influx and efflux of non-permeant molecules through transiently (electro)permeabilized cell membranes.

Successful Tumor Electrochemotherapy Using Sine Waves.

OBJECTIVE: The purpose of this 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 on 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 represents 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.

Conductive nanoparticles improve cell electropermeabilization.

Conducive nanoparticles (NPs) were proposed to locally amplify the external electric field (EF) intensity at the cell surface to improve cell electroporation. To better understand the physical mechanisms behind this improvement, different types of NPs and several incubation conditions were applied to adherent cells in the present study. The enhancement of electroporation was observed in the presence of conductive NPs but not when non-conductive NPs were used. Experimental data demonstrate the influence of the incubation conditions between cells and NPs, which impact on the number and quality (aggregated or isolated) of the NPs surrounding the cells. While NPs can increase the number of electroporated cells, they have a more pronounced impact on the level permeabilization of each individual cell. Our results reveal the potential of conductive NPs to enhance the efficiency of electroporation via the amplification of the local EF at the cell surface as shown by numerical simulations.

Impact of the number of electric pulses on cell electrochemotherapy in vitro: Limits of linearity and saturation.

In this study the evolution in the efficiency of electrochemotherapy (reversible electroporation) with pulse number was assessed in vitro. Experiments were performed using 100 µs pulses at different electric field intensities and the chemotherapeutic agent bleomycin. Additionally, electrical impedance spectroscopy measurements were used as a different method to study in real time the changes produced on cells with pulse number during trains of consecutive pulses. Our results show that the relation between pulse number and the observed outcome is complex and difficult to fully characterize. This relation can display a highly linear behaviour up to a certain number of pulses and/or field intensity applied. However, the relation between the number of pulses and the observed outcome always evolves to a saturation or at least a reduction in the electric field effects that is displayed when either electric field intensity or pulse number are increased. An exponential model was found to best describe this relation within the range of experimental conditions considered. Electrical impedance measurements confirmed the results and gave a more precise quantification of this dependence. The study highlights the importance that pulse number has in the electrochemotherapy protocols and establishes some limits in the use of this parameter.

In vitro analysis of various cell lines responses to electroporative electric pulses by means of electrical impedance spectroscopy.

This paper reports the comparative analysis, by means of electric impedance spectroscopy measurements, of three different cell lines subjected to electroporative pulses. The multifrequency information is recorded simultaneously at 21 frequency values in the range between 5 kHz and 1.3 MHz using a multisine based measuring approach. The analysis of the pre-electroporation impedance spectra shows how the system is able to detect differences and similarities between the cell lines under analysis. Particularly, a good agreement is found between the average cell diameter and the characteristic frequency (the frequency corresponding to a maximum in the imaginary part of the impedance). The measurements performed during electroporation at three different electric field intensities show how the impedance spectra changes dynamically between the consecutive pulses of a train of 8,100 µs pulses delivered at 1 Hz repetition rate. There are clear differences between the changes in the impedance measured at low and high frequency. The multifrequency information has been fitted to an electrical equivalent model in order to understand the different contributions in the observed impedance changes (mainly separate between membrane permeabilization and the conductivity changes in the extracellular medium). Finally, a ratio of the low and high frequency impedance information is used to estimate the accumulated impedance decay and to compare it to the internalization of a fluorescent permeabilization reporter. The comparison between both techniques at the three electroporation electric field intensities assayed confirms the ability of impedance measurements to detect in a precise way the level of membrane permeabilization. Additionally, this study demonstrates how the real time information obtained thanks to impedance measurements can provide a more precise quantification of the membrane permeabilization extent.

Sine wave electropermeabilization reveals the frequency-dependent response of the biological membranes.

The permeabilization of biological membranes by electric fields, known as electroporation, has been traditionally performed with square electric pulses. These signals distribute the energy applied to cells in a wide frequency band. This paper investigates the use of sine waves, which are narrow band signals, to provoke electropermeabilization and the frequency dependence of this phenomenon. Single bursts of sine waves at different frequencies in the range from 8 kHz-130 kHz were applied to cells in vitro. Electroporation was studied in the plasma membrane and the internal organelles membrane using calcium as a permeabilization marker. Additionally, a double-shell electrical model was simulated to give a theoretical framework to our results. The electroporation efficiency shows a low pass filter frequency dependence for both the plasma membrane and the internal organelles membrane. The mismatch between the theoretical response and the observed behavior for the internal organelles membrane is explained by a two-step permeabilization process: first the permeabilization of the external membrane and afterwards that of the internal membranes. The simulations in the model confirm this two-step hypothesis when a variable plasma membrane conductivity is considered in the analysis. This study demonstrates how the use of narrow-band signals as sine waves is a suitable method to perform electroporation in a controlled manner. We suggest that the use of this type of signals could bring a simplification in the investigations of the very complex phenomenon of electroporation, thus representing an interesting option in future fundamental studies.

Pyroelectricity as a possible mechanism for cell membrane permeabilization.

The effects of pyroelectricity on cell membrane permeability had never been explored. Pyroelectricity consists in the generation of an electric field in the surface of some materials when a change in temperature is produced. In the present study, tourmaline microparticles, which are known to display pyroelectrical properties, were subjected to different changes in temperature upon exposure to cells in order to induce an electric field at their surface. Then, the changes in the permeability of the cell membrane to a cytotoxic agent (bleomycin) were assessed by a cloning efficacy test. An increase in the permeability of the cell membrane was only detected when tourmaline was subjected to a change in temperature. This suggests that the apparition of an induced pyroelectrical electric field on the material could actually be involved in the observed enhancement of the cell membrane permeability as a result of cell electropermeabilization.

Recognition of Fibrotic Infarct Density by the Pattern of Local Systolic-Diastolic Myocardial Electrical Impedance.

Myocardial electrical impedance is a biophysical property of the heart that is influenced by the intrinsic structural characteristics of the tissue. Therefore, the structural derangements elicited in a chronic myocardial infarction should cause specific changes in the local systolic-diastolic myocardial impedance, but this is not known. This study aimed to characterize the local changes of systolic-diastolic myocardial impedance in a healed myocardial infarction model. Six pigs were successfully submitted to 150 min of left anterior descending (LAD) coronary artery occlusion followed by reperfusion. 4 weeks later, myocardial impedance spectroscopy (1-1000 kHz) was measured at different infarction sites. The electrocardiogram, left ventricular (LV) pressure, LV dP/dt, and aortic blood flow (ABF) were also recorded. A total of 59 LV tissue samples were obtained and histopathological studies were performed to quantify the percentage of fibrosis. Samples were categorized as normal myocardium (<10% fibrosis), heterogeneous scar (10-50%) and dense scar (>50%). Resistivity of normal myocardium depicted phasic changes during the cardiac cycle and its amplitude markedly decreased in dense scar (18 ± 2 Ω·cm vs. 10 ± 1 Ω·cm, at 41 kHz; P < 0.001, respectively). The mean phasic resistivity decreased progressively from normal to heterogeneous and dense scar regions (285 ± 10 Ω·cm, 225 ± 25 Ω·cm, and 162 ± 6 Ω·cm, at 41 kHz; P < 0.001 respectively). Moreover, myocardial resistivity and phase angle correlated significantly with the degree of local fibrosis (resistivity: r = 0.86 at 1 kHz, P < 0.001; phase angle: r = 0.84 at 41 kHz, P < 0.001). Myocardial infarcted regions with greater fibrotic content show lower mean impedance values and more depressed systolic-diastolic dynamic impedance changes. In conclusion, this study reveals that differences in the degree of myocardial fibrosis can be detected in vivo by local measurement of phasic systolic-diastolic bioimpedance spectrum. Once this new bioimpedance method could be used via a catheter-based device, it would be of potential clinical applicability for the recognition of fibrotic tissue to guide the ablation of atrial or ventricular arrhythmias.

Early detection of acute transmural myocardial ischemia by the phasic systolic-diastolic changes of local tissue electrical impedance.

Myocardial electrical impedance is influenced by the mechanical activity of the heart. Therefore, the ischemia-induced mechanical dysfunction may cause specific changes in the systolic-diastolic pattern of myocardial impedance, but this is not known. This study aimed to analyze the phasic changes of myocardial resistivity in normal and ischemic conditions. Myocardial resistivity was measured continuously during the cardiac cycle using 26 different simultaneous excitation frequencies (1 kHz-1 MHz) in 7 anesthetized open-chest pigs. Animals were submitted to 30 min regional ischemia by acute left anterior descending coronary artery occlusion. The electrocardiogram, left ventricular (LV) pressure, LV dP/dt, and aortic blood flow were recorded simultaneously. Baseline myocardial resistivity depicted a phasic pattern during the cardiac cycle with higher values at the preejection period (4.19 ± 1.09% increase above the mean, P < 0.001) and lower values during relaxation phase (5.01 ± 0.85% below the mean, P < 0.001). Acute coronary occlusion induced two effects on the phasic resistivity curve: 1) a prompt (5 min ischemia) holosystolic resistivity rise leading to a bell-shaped waveform and to a reduction of the area under the LV pressure-impedance curve (1,427 ± 335 vs. 757 ± 266 Ω·cm·mmHg, P < 0.01, 41 kHz) and 2) a subsequent (5-10 min ischemia) progressive mean resistivity rise (325 ± 23 vs. 438 ± 37 Ω·cm at 30 min, P < 0.01, 1 kHz). The structural and mechanical myocardial dysfunction induced by acute coronary occlusion can be recognized by specific changes in the systolic-diastolic myocardial resistivity curve. Therefore these changes may become a new indicator (surrogate) of evolving acute myocardial ischemia.

Interpulse multifrequency electrical impedance measurements during electroporation of adherent differentiated myotubes.

In this study, electrical impedance spectroscopy measurements are performed during electroporation of monolayers of differentiated myotubes. The time resolution of the system (1 spectrum/ms) enable 860 full spectra (21 frequencies from 5 kHz to 1.3 MHz) to be acquired during the time gap between consecutive pulses (interpulse) of a classical electroporation treatment (8 pulses, 100 µs, 1 Hz). Additionally, the characteristics of the custom microelectrode assembly used allow the experiments to be performed directly in situ in standard 24 multi-well plates. The impedance response dynamics are studied for three different electric field intensities (400, 800 and 1200 V/cm). The multifrequency information, analysed with the Cole model, reveals a short-term impedance recovery after each pulse in accordance with the fast resealing of the cell membrane, and a long-term impedance decay over the complete treatment in accordance with an accumulated effect pulse after pulse. The analysis shows differences between the lowest electric field condition and the other two, suggesting that different mechanisms that may be related with the reversibility of the process are activated. As a result of the multifrequency information, the system is able to measure simultaneously the conductivity variations due to ion diffusion during electroporation. Finally, in order to reinforce the physical interpretation of the results, a complementary electrical equivalent model is used.