Diverse effects of nanosecond pulsed electric fields on cells and tissues

The application of pulsed electric fields to cells is extended to include nonthermal pulses with shorter durations (10-300 ns), higher electric fields (< or =350 kV/cm), higher power (gigawatts), and distinct effects (nsPEF) compared to classical electroporation. Here we define effects and explore potential application for nsPEF in biology and medicine. As the pulse duration is decreased below the plasma membrane charging time constant, plasma membrane effects decrease and intracellular effects predominate. NsPEFs induced apoptosis and caspase activation that was calcium-dependent (Jurkat cells) and calcium-independent (HL-60 and Jurkat cells). In mouse B10-2 fibrosarcoma tumors, nsPEFs induced caspase activation and DNA fragmentation ex vivo, and reduced tumor size in vivo. With conditions below thresholds for classical electroporation and apoptosis, nsPEF induced calcium release from intracellular stores and subsequent calcium influx through store-operated channels in the plasma membrane that mimicked purinergic receptor-mediated calcium mobilization. When nsPEF were applied after classical electroporation pulses, GFP reporter gene expression was enhanced above that observed for classical electroporation. These findings indicate that nsPEF extend classical electroporation to include events that primarily affect intracellular structures and functions. Potential applications for nsPEF include inducing apoptosis in cells and tumors, probing signal transduction mechanisms that determine cell fate, and enhancing gene expression.     

Nanosecond pulsed electric fields act as a novel cellular stress that induces translational suppression accompanied by eIF2α phosphorylation and 4E-BP1 dephosphorylation

Recent advances in electrical engineering enable the generation of ultrashort electric fields, namely nanosecond pulsed electric fields (nsPEFs). Contrary to conventional electric fields used for DNA electroporation, nsPEFs can directly reach intracellular components without membrane destruction. Although nsPEFs are now recognized as a unique tool in life sciences, the molecular mechanism of nsPEF action remains largely unclear. Here, we present evidence that nsPEFs act as a novel cellular stress. Exposure of HeLa S3 cells to nsPEFs quickly induced phosphorylation of eIF2α, activation of its upstream stress-responsive kinases, PERK and GCN2, and translational suppression. Experiments using PERK- and GCN2-knockout cells demonstrated dual contribution of PERK and GCN2 to nsPEF-induced eIF2α phosphorylation. Moreover, nsPEF exposure yielded the elevated GADD34 expression, which is known to downregulate the phosphorylated eIF2α. In addition, nsPEF exposure caused a rapid decrease in 4E-BP1 phosphorylation irrespective of the PERK/GCN2 status, suggesting participation of both eIF2α and 4E-BP1 in nsPEF-induced translational suppression. RT-PCR analysis of stress-inducible genes demonstrated that cellular responses to nsPEFs are distinct from those induced by previously known forms of cellular stress. These results provide new mechanistic insights into nsPEF action and implicate the therapeutic potential of nsPEFs for stress response-associated diseases.     

Tumor ablation with irreversible electroporation

We report the first successful use of irreversible electroporation for the minimally invasive treatment of aggressive cutaneous tumors implanted in mice. Irreversible electroporation is a newly developed non-thermal tissue ablation technique in which certain short duration electrical fields are used to permanently permeabilize the cell membrane, presumably through the formation of nanoscale defects in the cell membrane. Mathematical models of the electrical and thermal fields that develop during the application of the pulses were used to design an efficient treatment protocol with minimal heating of the tissue. Tumor regression was confirmed by histological studies which also revealed that it occurred as a direct result of irreversible cell membrane permeabilization. Parametric studies show that the successful outcome of the procedure is related to the applied electric field strength, the total pulse duration as well as the temporal mode of delivery of the pulses. Our best results were obtained using plate electrodes to deliver across the tumor 80 pulses of 100 micros at 0.3 Hz with an electrical field magnitude of 2500 V/cm. These conditions induced complete regression in 12 out of 13 treated tumors, (92%), in the absence of tissue heating. Irreversible electroporation is thus a new effective modality for non-thermal tumor ablation.     

Stimulation of capacitative calcium entry in HL-60 cells by nanosecond pulsed electric fields

Nanosecond pulsed electric fields (nsPEFs) are hypothesized to affect intracellular structures in living cells providing a new means to modulate cell signal transduction mechanisms. The effects of nsPEFs on the release of internal calcium and activation of calcium influx in HL-60 cells were investigated by using real time fluorescent microscopy with Fluo-3 and fluorometry with Fura-2. nsPEFs induced an increase in intracellular calcium levels that was seen in all cells. With pulses of 60 ns duration and electric fields between 4 and 15 kV/cm, intracellular calcium increased 200-700 nM, respectively, above basal levels (approximately 100 nM), while the uptake of propidium iodide was absent. This suggests that increases in intracellular calcium were not because of plasma membrane electroporation. nsPEF and the purinergic agonist UTP induced calcium mobilization in the presence and absence of extracellular calcium with similar kinetics and appeared to target the same inositol 1,4,5-trisphosphate- and thapsigargin-sensitive calcium pools in the endoplasmic reticulum. For cells exposed to either nsPEF or UTP in the absence of extracellular calcium, there was an electric field-dependent or UTP dose-dependent increase in capacitative calcium entry when calcium was added to the extracellular media. These findings suggest that nsPEFs, like ligand-mediated responses, release calcium from similar internal calcium pools and thus activate plasma membrane calcium influx channels or capacitative calcium entry.     

DNA electrophoretic migration patterns change after exposure of Jurkat cells to a single intense nanosecond electric pulse

Intense nanosecond pulsed electric fields (nsPEFs) interact with cellular membranes and intracellular structures. Investigating how cells respond to nanosecond pulses is essential for a) development of biomedical applications of nsPEFs, including cancer therapy, and b) better understanding of the mechanisms underlying such bioelectrical effects. In this work, we explored relatively mild exposure conditions to provide insight into weak, reversible effects, laying a foundation for a better understanding of the interaction mechanisms and kinetics underlying nsPEF bio-effects. In particular, we report changes in the nucleus of Jurkat cells (human lymphoblastoid T cells) exposed to single pulses of 60 ns duration and 1.0, 1.5 and 2.5 MV/m amplitudes, which do not affect cell growth and viability. A dose-dependent reduction in alkaline comet-assayed DNA migration is observed immediately after nsPEF exposure, accompanied by permeabilization of the plasma membrane (YO-PRO-1 uptake). Comet assay profiles return to normal within 60 minutes after pulse delivery at the highest pulse amplitude tested, indicating that our exposure protocol affects the nucleus, modifying DNA electrophoretic migration patterns.     

Nanosecond pulsed electric fields have differential effects on cells in the S-phase

Nanosecond pulsed electric fields (nsPEFs) are a type of nonthermal, nonionizing radiation that exhibit intense electric fields with high power, but low energy. NsPEFs extend conventional electroporation (EP) to affect intracellular structures and functions and depending on the intensity, can induce lethal and nonlethal cell signaling. In this study, HCT116 human colon carcinoma cells were synchronized to the S-phase or remained unsynchronized, exposed to electric fields of 60 kV/cm with either 60-ns or 300-ns durations, and analyzed for apoptosis and proliferative markers. Several nsPEF structural and functional targets were identified. Unlike unsynchronized cells, S-phase cells under limiting conditions exhibited greater membrane integrity and caspase activation and maintained cytoskeletal structure. Regardless of synchronization, cells exposed to nsPEFs under these conditions primarily survived, but exhibited some turnover and delayed proliferation in cell populations, as well as reversible increases in phosphatidylserine externalization, membrane integrity, and nuclei size. These results show that nsPEFs can act as a nonligand agonist to modulate plasma membrane (PM) and intracellular structures and functions, as well as differentially affect cells in the S-phase, but without effect on cell survival. Furthermore, nsPEF effects on the nucleus and cytoskeleton may provide synergistic therapeutic actions with other agents, such as ionizing radiation or chemotherapeutics that affect these same structures.     

Activation of the JNK pathway by nanosecond pulsed electric fields

Nanosecond pulsed electric fields (nsPEFs) are increasingly recognized as a novel and unique tool in various life science fields, including electroporation and cancer therapy, although their mode of action in cells remains largely unclear. Here, we show that nsPEFs induce strong and transient activation of a signaling pathway involving c-Jun N-terminal kinase (JNK). Application of nsPEFs to HeLa S3 cells rapidly induced phosphorylation of JNK1 and MKK4, which is located immediately upstream of JNK in this signaling pathway. nsPEF application also elicited increased phosphorylation of c-Jun protein and dramatically elevated c-jun and c-fos mRNA levels. nsPEF-inducible events downstream of JNK were markedly suppressed by the JNK inhibitor SP600125, which confirmed JNK-dependency of these events in this pathway. Our results provide novel mechanistic insights into the mode of nsPEF action in human cells.     

A statistical model for multidimensional irreversible electroporation cell death in tissue

Background  

Irreversible electroporation (IRE) is a minimally invasive tissue ablation technique which utilizes electric pulses delivered by electrodes to a targeted area of tissue to produce high amplitude electric fields, thus inducing irreversible damage to the cell membrane lipid bilayer. An important application of this technique is for cancer tissue ablation. Mathematical modelling is considered important in IRE treatment planning. In the past, IRE mathematical modelling used a deterministic single value for the amplitude of the electric field required for causing cell death. However, tissue, particularly cancerous tissue, is comprised of a population of different cells of different sizes and orientations, which in conventional IRE are exposed to complex electric fields; therefore, using a deterministic single value is overly simplistic.     

Facilitation of electroporative drug uptake and cell killing by electrosensitization

Cell permeabilization by electric pulses (EP), or electroporation, is widely used for intracellular delivery of drugs and plasmids, as well as for tumour and tissue ablation. We found that cells pre‐treated with 100‐μs EP develop delayed hypersensitivity to subsequent EP applications. Sensitizing B16 and CHO cells by splitting a single train of eight 100‐μs EP into two trains of four EP each (with 5‐min. interval) decreased the LD₅₀ 1.5–2 times. Sensitization profoundly enhanced the electroporation‐assisted uptake of bleomycin, a cell‐impermeable cytotoxic agent accepted for killing tumours by electrochemotherapy. EP exposures that were not lethal per se caused cell death in the presence of bleomycin and proportionally to its concentration. Sensitizing cells by a split‐dose EP exposure increased bleomycin‐mediated lethality to the same extent as a 10‐fold increase in bleomycin concentration when using a single EP dose. Likewise, sensitization by a split‐dose EP exposure (without changing the overall dose, pulse number, or amplitude) enhanced the electroporative uptake of propidium up to fivefold. Enhancement of the electroporative uptake appears a key mechanism of electrosensitization and may benefit electrochemotherapy and numerous applications that employ EP for cell permeabilization.     

Bipolar nanosecond electric pulses are less efficient at electropermeabilization and killing cells than monopolar pulses

Multiple studies have shown that bipolar (BP) electric pulses in the microsecond range are more effective at permeabilizing cells while maintaining similar cell survival rates as compared to monopolar (MP) pulse equivalents. In this paper, we investigated whether the same advantage existed for BP nanosecond-pulsed electric fields (nsPEF) as compared to MP nsPEF. To study permeabilization effectiveness, MP or BP pulses were delivered to single Chinese hamster ovary (CHO) cells and the response of three dyes, Calcium Green-1, propidium iodide (PI), and FM1-43, was measured by confocal microscopy. Results show that BP pulses were less effective at increasing intracellular calcium concentration or PI uptake and cause less membrane reorganization (FM1-43) than MP pulses. Twenty-four hour survival was measured in three cell lines (Jurkat, U937, CHO) and over ten times more BP pulses were required to induce death as compared to MP pulses of similar magnitude and duration. Flow cytometry analysis of CHO cells after exposure (at 15 min) revealed that to achieve positive FITC-Annexin V and PI expression, ten times more BP pulses were required than MP pulses. Overall, unlike longer pulse exposures, BP nsPEF exposures proved far less effective at both membrane permeabilization and cell killing than MP nsPEF.     

A Numerical Investigation of the Electric and Thermal Cell Kill Distributions in Electroporation-Based Therapies in Tissue

Electroporation-based therapies are powerful biotechnological tools for enhancing the delivery of exogeneous agents or killing tissue with pulsed electric fields (PEFs). Electrochemotherapy (ECT) and gene therapy based on gene electrotransfer (EGT) both use reversible electroporation to deliver chemotherapeutics or plasmid DNA into cells, respectively. In both ECT and EGT, the goal is to permeabilize the cell membrane while maintaining high cell viability in order to facilitate drug or gene transport into the cell cytoplasm and induce a therapeutic response. Irreversible electroporation (IRE) results in cell kill due to exposure to PEFs without drugs and is under clinical evaluation for treating otherwise unresectable tumors. These PEF therapies rely mainly on the electric field distributions and do not require changes in tissue temperature for their effectiveness. However, in immediate vicinity of the electrodes the treatment may results in cell kill due to thermal damage because of the inhomogeneous electric field distribution and high current density during the electroporation-based therapies. Therefore, the main objective of this numerical study is to evaluate the influence of pulse number and electrical conductivity in the predicted cell kill zone due to irreversible electroporation and thermal damage. Specifically, we simulated a typical IRE protocol that employs ninety 100-µs PEFs. Our results confirm that it is possible to achieve predominant cell kill due to electroporation if the PEF parameters are chosen carefully. However, if either the pulse number and/or the tissue conductivity are too high, there is also potential to achieve cell kill due to thermal damage in the immediate vicinity of the electrodes. Therefore, it is critical for physicians to be mindful of placement of electrodes with respect to critical tissue structures and treatment parameters in order to maintain the non-thermal benefits of electroporation and prevent unnecessary damage to surrounding healthy tissue, critical vascular structures, and/or adjacent organs.     

High-frequency irreversible electroporation (H-FIRE) for non-thermal ablation without muscle contraction

Background

Therapeutic irreversible electroporation (IRE) is an emerging technology for the non-thermal ablation of tumors. The technique involves delivering a series of unipolar electric pulses to permanently destabilize the plasma membrane of cancer cells through an increase in transmembrane potential, which leads to the development of a tissue lesion. Clinically, IRE requires the administration of paralytic agents to prevent muscle contractions during treatment that are associated with the delivery of electric pulses. This study shows that by applying high-frequency, bipolar bursts, muscle contractions can be eliminated during IRE without compromising the non-thermal mechanism of cell death.

Methods

A combination of analytical, numerical, and experimental techniques were performed to investigate high-frequency irreversible electroporation (H-FIRE). A theoretical model for determining transmembrane potential in response to arbitrary electric fields was used to identify optimal burst frequencies and amplitudes for in vivo treatments. A finite element model for predicting thermal damage based on the electric field distribution was used to design non-thermal protocols for in vivo experiments. H-FIRE was applied to the brain of rats, and muscle contractions were quantified via accelerometers placed at the cervicothoracic junction. MRI and histological evaluation was performed post-operatively to assess ablation.

Results

No visual or tactile evidence of muscle contraction was seen during H-FIRE at 250 kHz or 500 kHz, while all IRE protocols resulted in detectable muscle contractions at the cervicothoracic junction. H-FIRE produced ablative lesions in brain tissue that were characteristic in cellular morphology of non-thermal IRE treatments. Specifically, there was complete uniformity of tissue death within targeted areas, and a sharp transition zone was present between lesioned and normal brain.

Conclusions

H-FIRE is a feasible technique for non-thermal tissue ablation that eliminates muscle contractions seen in IRE treatments performed with unipolar electric pulses. Therefore, it has the potential to be performed clinically without the administration of paralytic agents.     

Nanosecond pulsed electric fields (nsPEF) induce direct electric field effects and biological effects on human colon carcinoma cells

Nanosecond pulsed electric fields (nsPEFs) are ultrashort pulses with high electric field intensity (kV/cm) and high power (megawatts), but low energy density (mJ/cc). To determine roles for p53 in response to nsPEFs, HCT116 cells (p53+/+ and p53-/-) were exposed to nsPEF and analyzed for membrane integrity, phosphatidylserine externalization, caspase activation, and cell survival. Decreasing plasma membrane effects were observed in both HCT116p53+/+ and p53-/- cells with decreasing pulse durations and/or decreasing electric fields. However, addition of ethidium homodimer-1 and Annexin-V-FITC post-pulse demonstrated greater fluorescence in p53-/- versus p53+/+ cells, suggesting a postpulse p53-dependent biological effect at the plasma membrane. Caspase activity was significantly higher than nonpulsed cells only in the p53-/- cells. HCT116 cells exhibited greater survival in response to nsPEFs than HL-60 and Jurkat cells, but survival was more evident for HCT116p53+/+ cells than for HCT116p53-/- cells. These results indicate that nsPEF effects on HCT116 cells include (1) apparent direct electric field effects, (2) biological effects that are p53-dependent and p53-independent, (3) actions on mechanisms that originate at the plasma membranes and at intracellular structures, and (4) an apparent p53 protective effect. NsPEF applications provide a means to explore intracellular structures and functions that can reveal mechanisms in health and disease.     

Long-lasting plasma membrane permeabilization in mammalian cells by nanosecond pulsed electric field (nsPEF)

The barrier function of plasma membrane in nsPEF-exposed mammalian cells was examined using whole-cell patch-clamp techniques. A specialized setup for nsPEF exposure of individual cells in culture was developed and characterized for artifact-free compatibility with the patch-clamp method. For the first time, our study provides experimental evidence that even a single 60-ns pulse at 12 kV/cm can cause a profound and long-lasting (minutes) reduction of the cell membrane resistance (R(m)), accompanied by the loss of the membrane potential. R(m) measured in GH3, PC-12, and Jurkat cells (but not in HeLa cells) in 80-120 s after nsPEF exposure was decreased about threefold, and its gradual recovery could take 15 min. Multiple pulses enhanced permeabilization, for example, R(m) in GH3 cells fell about 10-fold after a train of five pulses. Within studied limits, permeabilization did not depend on the presence of Ca(2+), Mg(2+), K(+), Cs(+), Cd(2+), EGTA, tetraethylammonium, or 4-aminopyridine in the pipette or bath solutions. Our results supported theoretical model predictions of plasma membrane poration by nsPEF. However, the extended decrease in R(m), assumed to be related to the life span of the pores, and different nsPEF sensitivity of individual cell lines have yet to be explained. The phenomenon of long-lived membrane permeabilization provides new insights on the nature of nsPEF-opened conductance pores and on molecular mechanisms that underlie nsPEF bioeffects.     

Listeria monocytogenes cell wall constituents exert a charge effect on electroporation threshold

Genetically engineered cells with mutations of relevance to electroporation, cell membrane permeabilization by electric pulses, can become a promising new tool for fundamental research on this important biotechnology. Listeria monocytogenes mutants lacking DltA or MprF and assayed for sensitivity to the cathelicidin like anti-microbial cationic peptide (mCRAMP), were developed to study the effect of cell wall charge on electroporation. Working in the irreversible electroporation regime (IRE), we found that application of a sequence of 50 pulses, each 50μs duration, 12.5kV/cm field, delivered at 2Hz led to 2.67±0.29 log reduction in wild-type L. monocytogenes, log 2.60±0.19 in the MprF-minus mutant, and log 1.33±0.13 in the DltA-minus mutant. The experimental observation that the DltA-minus mutant was highly susceptible to cationic mCRAMP and resistant to IRE suggests that the charge on the bacterial cell wall affects electroporation and shows that this approach may be promising for fundamental studies on electroporation.     

Synergistic effects of nanosecond pulsed electric fields combined with low concentration of gemcitabine on human oral squamous cell carcinoma in vitro

Treatment of cancer often involves uses of multiple therapeutic strategies with different mechanisms of action. In this study we investigated combinations of nanosecond pulsed electric fields (nsPEF) with low concentrations of gemcitabine on human oral cancer cells. Cells (Cal-27) were treated with pulse parameters (20 pulses, 100 ns in duration, intensities of 10, 30 and 60 kV/cm) and then cultured in medium with 0.01 μg/ml gemcitabine. Proliferation, apoptosis/necrosis, invasion and morphology of those cells were examined using MTT, flow cytometry, clonogenics, transwell migration and TEM assay. Results show that combination treatments of gemcitabine and nsPEFs exhibited significant synergistic activities versus individual treatments for inhibiting oral cancer cell proliferation and inducing apoptosis and necrosis. However, there was no apparent synergism for cell invasion. By this we demonstrated synergistic inhibition of Cal-27 cells in vitro by nsPEFs and gemcitabine. Synergistic behavior indicates that these two treatments have different sites of action and combination treatment allows reduced doses of gemcitabine and lower nsPEF conditions, which may provide better treatment for patients than either treatment alone while reducing systemic toxicities.     

Plant actin controls membrane permeability

The biological effects of electric pulses with low rise time, high field strength, and durations in the nanosecond range (nsPEFs) have attracted considerable biotechnological and medical interest. However, the cellular mechanisms causing membrane permeabilization by nanosecond pulsed electric fields are still far from being understood. We investigated the role of actin filaments for membrane permeability in plant cells using cell lines where different degrees of actin bundling had been introduced by genetic engineering. We demonstrate that stabilization of actin increases the stability of the plasma membrane against electric permeabilization recorded by penetration of Trypan Blue into the cytoplasm. By use of a cell line expressing the actin bundling WLIM domain under control of an inducible promotor we can activate membrane stabilization by the glucocorticoid analog dexamethasone. By total internal reflection fluorescence microscopy we can visualize a subset of the cytoskeleton that is directly adjacent to the plasma membrane. We conclude that this submembrane cytoskeleton stabilizes the plasma membrane against permeabilization through electric pulses.     

Ablation of Myocardial Tissue With Nanosecond Pulsed Electric Fields

Background

Ablation of cardiac tissue is an essential tool for the treatment of arrhythmias, particularly of atrial fibrillation, atrial flutter, and ventricular tachycardia. Current ablation technologies suffer from substantial recurrence rates, thermal side effects, and long procedure times. We demonstrate that ablation with nanosecond pulsed electric fields (nsPEFs) can potentially overcome these limitations.

Methods

We used optical mapping to monitor electrical activity in Langendorff-perfused New Zealand rabbit hearts (n = 12). We repeatedly inserted two shock electrodes, spaced 2–4 mm apart, into the ventricles (through the entire wall) and applied nanosecond pulsed electric fields (nsPEF) (5–20 kV/cm, 350 ns duration, at varying pulse numbers and frequencies) to create linear lesions of 12–18 mm length. Hearts were stained either with tetrazolium chloride (TTC) or propidium iodide (PI) to determine the extent of ablation. Some stained lesions were sectioned to obtain the three-dimensional geometry of the ablated volume.

Results

In all animals (12/12), we were able to create nonconducting lesions with less than 2 seconds of nsPEF application per site and minimal heating (< 0.2°C) of the tissue. The geometry of the ablated volume was smoother and more uniform throughout the wall than typical for RF ablation. The width of the lesions could be controlled up to 6 mm via the electrode spacing and the shock parameters.

Conclusions

Ablation with nsPEFs is a promising alternative to radiofrequency (RF) ablation of AF. It may dramatically reduce procedure times and produce more consistent lesion thickness than RF ablation.     

Apoptosis initiation and angiogenesis inhibition: melanoma targets for nanosecond pulsed electric fields

Many effective anti-cancer strategies target apoptosis and angiogenesis mechanisms. Applications of non-ionizing, nanosecond pulsed electric fields (nsPEFs) induce apoptosis in vitro and eliminate cancer in vivo; however in vivo mechanisms require closer analysis. These studies investigate nsPEF-induced apoptosis and anti-angiogenesis examined by fluorescent microscopy, immunoblots, and morphology. Six hours after treatment with one hundred 300 ns pulses at 40 kV/cm, cells transiently expressed active caspases indicating that caspase-mediated mechanisms. Three hours after treatment transient peaks in Histone 2AX phosphorylation coincided with terminal deoxynucleotidyl transferase dUTP nick end labeling positive cells and pyknotic nuclei, suggesting caspase-independent mechanisms on nuclei/DNA. Large DNA fragments, but not 180 bp fragmentation ladders, were observed, suggesting incomplete apoptosis. Nevertheless, tumor weight and volume decreased and tumors disappeared. One week after treatment, vessel numbers, vascular endothelial growth factor (VEGF), platelet derived endothelial cell growth factor (PD-ECGF), CD31, CD35 and CD105 were decreased, indicating anti-angiogenesis. The nsPEFs activate multiple melanoma therapeutic targets, which is consistent with successes of nsPEF applications for tumor treatment in vivo as a new cancer therapeutic modality.     

Electroporation of DC-3F Cells Is a Dual Process

Treatment of biological material by pulsed electric fields is a versatile technique in biotechnology and biomedicine used, for example, in delivering DNA into cells (transfection), ablation of tumors, and food processing. Field exposure is associated with a membrane permeability increase usually ascribed to electroporation, i.e., formation of aqueous membrane pores. Knowledge of the underlying processes at the membrane level is predominantly built on theoretical considerations and molecular dynamics (MD) simulations. However, experimental data needed to monitor these processes with sufficient temporal resolution are scarce. The whole-cell patch-clamp technique was employed to investigate the effect of millisecond pulsed electric fields on DC-3F cells. Cellular membrane permeabilization was monitored by a conductance increase. For the first time, to our knowledge, it could be established experimentally that electroporation consists of two clearly separate processes: a rapid membrane poration (transient electroporation) that occurs while the membrane is depolarized or hyperpolarized to voltages beyond so-called threshold potentials (here, +201 mV and −231 mV, respectively) and is reversible within ∼100 ms after the pulse, and a long-term, or persistent, permeabilization covering the whole voltage range. The latter prevailed after the pulse for at least 40 min, the postpulse time span tested experimentally. With mildly depolarizing or hyperpolarizing pulses just above threshold potentials, the two processes could be separated, since persistent (but not transient) permeabilization required repetitive pulse exposure. Conductance increased stepwise and gradually with depolarizing and hyperpolarizing pulses, respectively. Persistent permeabilization could also be elicited by single depolarizing/hyperpolarizing pulses of very high field strength. Experimental measurements of propidium iodide uptake provided evidence of a real membrane phenomenon, rather than a mere patch-clamp artifact. In short, the response of DC-3F cells to strong pulsed electric fields was separated into a transient electroporation and a persistent permeabilization. The latter dominates postpulse membrane properties but to date has not been addressed by electroporation theory or MD simulations.