Recent Advancements in Electroporation Technologies: From Bench to Clinic.

Over the past decade, the increased adoption of electroporation-based technologies has led to an expansion of clinical research initiatives. Electroporation has been utilized in molecular biology for mammalian and bacterial transfection; for food sanitation; and in therapeutic settings to increase drug uptake, for gene therapy, and to eliminate cancerous tissues. We begin this article by discussing the biophysics required for understanding the concepts behind the cell permeation phenomenon that is electroporation. We then review nano- and microscale single-cell electroporation technologies before scaling up to emerging in vivo applications.

Experimental and Numerical Investigation of Parameters Affecting High-Frequency Irreversible Electroporation for Prostate Cancer Ablation.

While the primary goal of focal therapy for prostate cancer (PCa) is conserving patient quality of life by reducing oncological burden, available modalities use thermal energy or whole-gland radiation which can damage critical neurovascular structures within the prostate and increase risk of genitourinary dysfunction. High-frequency irreversible electroporation (H-FIRE) is a promising alternative ablation modality that utilizes bursts of pulsed electric fields (PEFs) to destroy aberrant cells via targeted membrane damage. Due to its nonthermal mechanism, H-FIRE offers several advantages over state-of-the-art treatments, but waveforms have not been optimized for treatment of PCa. In this study, we characterize lethal electric field thresholds (EFTs) for H-FIRE waveforms with three different pulse widths as well as three interpulse delays in vitro and compare them to conventional irreversible electroporation (IRE). Experiments were performed in non-neoplastic and malignant prostate cells to determine the effect of waveforms on both targeted (malignant) and adjacent (non-neoplastic) tissue. A numerical modeling approach was developed to estimate the clinical effects of each waveform including extent of nonthermal ablation, undesired thermal damage, and nerve excitation. Our findings indicate that H-FIRE waveforms with pulse durations of 5 and 10 µs provide large ablations comparable to IRE with tolerable levels of thermal damage and minimized muscle contractions. Lower duration (2 µs) H-FIRE waveforms exhibit the least amount of muscle contractions but require increased voltages which may be accompanied by unwanted thermal damage.

A Comparative Modeling Study of Thermal Mitigation Strategies in Irreversible Electroporation Treatments.

Irreversible electroporation (IRE), also referred to as nonthermal pulsed field ablation (PFA), is an attractive focal ablation modality for solid tumors and cardiac tissue due to its ability to destroy aberrant cells with limited disruption of the underlying tissue architecture. Despite its nonthermal cell death mechanism, application of electrical energy results in Joule heating that, if ignored, can cause undesired thermal injury. Engineered thermal mitigation (TM) technologies including phase change materials (PCMs) and active cooling (AC) have been reported and tested as a potential means to limit thermal damage. However, several variables affect TM performance including the pulsing paradigm, electrode geometry, PCM composition, and chosen active cooling parameters, meaning direct comparisons between approaches are lacking. In this study, we developed a computational model of conventional bipolar and monopolar probes with solid, PCM-filled, or actively cooled cores to simulate clinical IRE treatments in pancreatic tissue. This approach reveals that probes with integrated PCM cores can be tuned to drastically limit thermal damage compared to existing solid probes. Furthermore, actively cooled probes provide additional control over thermal effects within the probe vicinity and can altogether abrogate thermal damage. In practice, such differences in performance must be weighed against the increased time, expense, and effort required for modified probes compared to existing solid probes.

A review: Dielectrophoresis for characterizing and separating similar cell subpopulations based on bioelectric property changes due to disease progression and therapy assessment.

This paper reviews the use of dielectrophoresis for high-fidelity separations and characterizations of subpopulations to highlight the recent advances in the electrokinetic field as well as provide insight into its progress toward commercialization. The role of cell subpopulations in heterogeneous clinical samples has been studied to deduce their role in disease progression and therapy resistance for instances such as cancer, tissue regeneration, and bacterial infection. Dielectrophoresis (DEP), a label-free electrokinetic technique, has been used to characterize and separate target subpopulations from mixed samples to determine disease severity, cell stemness, and drug efficacy. Despite its high sensitivity to characterize similar or related cells based on their differing bioelectric signatures, DEP has been slowly adopted both commercially and clinically. This review addresses the use of dielectrophoresis for the identification of target cell subtypes in stem cells, cancer cells, blood cells, and bacterial cells dependent on cell state and therapy exposure and addresses commercialization efforts in light of its sensitivity and future perspectives of the technology, both commercially and academically.

A novel ultralow conductivity electromanipulation buffer improves cell viability and enhances dielectrophoretic consistency.

Cell separation has become a critical diagnostic, research, and treatment tool for personalized medicine. Despite significant advances in cell separation, most widely used applications require the use of multiple, expensive antibodies to known markers in order to identify subpopulations of cells for separation. Dielectrophoresis (DEP) provides a biophysical separation technique that can target cell subpopulations based on phenotype without labels and return native cells for downstream analysis. One challenge in employing any DEP device is the sample being separated must be transferred into an ultralow conductivity medium, which can be detrimental in retaining cells' native phenotypes for separation. Here, we measured properties of traditional DEP reagents and determined that after just 1-2 h of exposure and subsequent culture, cells' viability was significantly reduced below 50%. We developed and tested a novel buffer (Cyto Buffer) that achieved 6 weeks of stable shelf-life and demonstrated significantly improved viability and physiological properties. We then determined the impact of Cyto Buffer on cells' dielectric properties and morphology and found that cells retained properties more similar to that of their native media. Finally, we vetted Cyto Buffer's usability on a cell separation platform (Cyto R1) to determine combined efficacy for cell separations. Here, more than 80% of cells from different cell lines were recovered and were determined to be >70% viable following exposure to Cyto Buffer, flow stimulation, electromanipulation, and downstream collection and growth. The developed buffer demonstrated improved opportunities for electrical cell manipulation, enrichment, and recovery for next generation cell separations.

High-Voltage Electrical Pulses in Oncology: Irreversible Electroporation, Electrochemotherapy, Gene Electrotransfer, Electrofusion, and Electroimmunotherapy.

This review summarizes the use of high-voltage electrical pulses (HVEPs) in clinical oncology to treat solid tumors with irreversible electroporation (IRE) and electrochemotherapy (ECT). HVEPs increase the membrane permeability of cells, a phenomenon known as electroporation. Unlike alternative ablative therapies, electroporation does not affect the structural integrity of surrounding tissue, thereby enabling tumors in the vicinity of vital structures to be treated. IRE uses HVEPs to cause cell death by inducing membrane disruption, and it is primarily used as a radical ablative therapy in the treatment of soft-tissue tumors in the liver, kidney, prostate, and pancreas. ECT uses HVEPs to transiently increase membrane permeability, enhancing cellular cytotoxic drug uptake in tumors. IRE and ECT show immunogenic effects that could be augmented when combined with immunomodulatory drugs, a combination therapy the authors term electroimmunotherapy. Additional electroporation-based technologies that may reach clinical importance, such as gene electrotransfer, electrofusion, and electroimmunotherapy, are concisely reviewed. HVEPs represent a substantial advancement in cancer research, and continued improvement and implementation of these presented technologies will require close collaboration between engineers, interventional radiologists, medical oncologists, and immuno-oncologists.

High-Frequency Irreversible Electroporation for Treatment of Primary Liver Cancer: A Proof-of-Principle Study in Canine Hepatocellular Carcinoma.

PURPOSE: To determine the safety and feasibility of percutaneous high-frequency irreversible electroporation (HFIRE) for primary liver cancer and evaluate the HFIRE-induced local immune response. MATERIALS AND METHODS: HFIRE therapy was delivered percutaneously in 3 canine patients with resectable hepatocellular carcinoma (HCC) in the absence of intraoperative paralytic agents or cardiac synchronization. Pre- and post-HFIRE biopsy samples were processed with histopathology and immunohistochemistry for CD3, CD4, CD8, and CD79a. Blood was collected on days 0, 2, and 4 for complete blood count and chemistry. Numeric models were developed to determine the treatment-specific lethal thresholds for malignant canine liver tissue and healthy porcine liver tissue. RESULTS: HFIRE resulted in predictable ablation volumes as assessed by posttreatment CT. No detectable cardiac interference and minimal muscle contraction occurred during HFIRE. No clinically significant adverse events occurred secondary to HFIRE. Microscopically, a well-defined ablation zone surrounded by a reactive zone was evident in the majority of samples. This zone was composed primarily of maturing collagen interspersed with CD3+/CD4-/CD8- lymphocytes in a proinflammatory microenvironment. The average ablation volumes for the canine HCC patients and the healthy porcine tissue were 3.89 cm3 ± 0.74 and 1.56 cm3 ± 0.16, respectively (P = .03), and the respective average lethal thresholds were 710 V/cm ± 28.2 and 957 V/cm ± 24.4 V/cm (P = .0004). CONCLUSIONS: HFIRE can safely and effectively be delivered percutaneously, results in a predictable ablation volume, and is associated with lymphocytic tumor infiltration. This is the first step toward the use of HFIRE for treatment of unresectable liver tumors.

Self-aligned microfluidic contactless dielectrophoresis device fabricated by single-layer imprinting on cyclic olefin copolymer.

The trapping and deflection of biological cells by dielectrophoresis (DEP) at field non-uniformities in a microfluidic device is often conducted in a contactless dielectrophoresis (cDEP) mode, wherein the electrode channel is in a different layer than the sample channel, so that field penetration through the interceding barrier causes DEP above critical cut-off frequencies. In this manner, through physical separation of the electrode and sample channels, it is possible to spatially modulate electric fields with no electrode-induced damage to biological cells in the sample channel. However, since this device requires interlayer alignment of the electrode to sample channel and needs to maintain a thin interceding barrier (~ 15 µm) over the entire length over which DEP is needed (~ 1 cm), variations in alignment and microstructure fidelity cause wide variations in cDEP trapping level and frequency response across devices. We present a strategy to eliminate interlayer alignment by fabricating self-aligned electrode and sample channels, simultaneously with the interceding barrier layer (14-µm width and 50-µm depth), using a single-layer imprint and bond process on cyclic olefin copolymer. Specifically, by designing support structures, we preserve fidelity of the high aspect ratio insulating posts in the sample channel and the interceding barrier between the sample and electrode channels over the entire device footprint (~ 1 cm). The device operation is validated based on impedance measurements to quantify field penetration through the interceding barrier and by DEP trapping measurements. The presented fabrication strategy can eventually improve cDEP device manufacturing protocols to enable more reproducible DEP performance. Graphical abstract.

The feasibility of using dielectrophoresis for isolation of glioblastoma subpopulations with increased stemness.

Cancer stem cells (CSCs) are aggressive subpopulations with increased stem-like properties. CSCs are usually resistant to most standard therapies and are responsible for tumor repropagation. Similar to normal stem cells, isolation of CSCs is challenging due to the lack of reliable markers. Antigen-based sorting of CSCs usually requires staining with multiple markers, making the experiments complicated, expensive, and sometimes unreliable. Here, we study the feasibility of using dielectrophoresis (DEP) for isolation of glioblastoma cells with increased stemness. We culture a glioblastoma cell line in the form of neurospheres as an in vitro model for glioblastoma stem cells. We demonstrate that spheroid forming cells have higher expression of stem cell marker, nestin. Next, we show that dielectric properties of neurospheres change as a result of changing culture conditions. Our results indicate that spheroid forming cells need higher voltages to experience the same DEP force magnitude compared to normal monolayer cultures of glioblastoma cell line. This study confirms the possibility of using DEP to isolate glioblastoma stem cells.

Experimental High-Frequency Irreversible Electroporation Using a Single-Needle Delivery Approach for Nonthermal Pancreatic Ablation In Vivo.

PURPOSE: To investigate the feasibility of single-needle high-frequency irreversible electroporation (SN-HFIRE) to create reproducible tissue ablations in an in vivo pancreatic swine model. MATERIALS AND METHODS: SN-HFIRE was performed in swine pancreas in vivo in the absence of intraoperative paralytics or cardiac synchronization using 3 different voltage waveforms (1-5-1, 2-5-2, and 5-5-5 [on-off-on times (µs)], n = 6/setting) with a total energized time of 100 µs per burst. At necropsy, ablation size/shape was determined. Immunohistochemistry was performed to quantify apoptosis using an anticleaved caspase-3 antibody. A numerical model was developed to determine lethal thresholds for each waveform in pancreas. RESULTS: Mean tissue ablation time was 5.0 ± 0.2 minutes, and no cardiac abnormalities or muscle twitch was detected. Mean ablation area significantly increased with increasing pulse width (41.0 ± 5.1 mm2 [range 32-66 mm2] vs 44 ± 2.1 mm2 [range 38-56 mm2] vs 85.0 ± 7.0 mm2 [range 63-155 mm2]; 1-5-1, 2-5-2, 5-5-5, respectively; p < 0.0002 5-5-5 vs 1-5-1 and 2-5-2). The majority of the ablation zone did not stain positive for cleaved caspase-3 (6.1 ± 2.8% [range 1.8-9.1%], 8.8 ± 1.3% [range 5.5-14.0%], and 11.0 ± 1.4% [range 7.1-14.2%] cleaved caspase-3 positive 1-5-1, 2-5-2, 5-5-5, respectively), with significantly more positive staining at the 5-5-5 pulse setting compared with 1-5-1 (p < 0.03). Numerical modeling determined a lethal threshold of 1114 ± 123 V/cm (1-5-1 waveform), 1039 ± 103 V/cm (2-5-2 waveform), and 693 ± 81 V/cm (5-5-5 waveform). CONCLUSIONS: SN-HFIRE induces rapid, predictable ablations in pancreatic tissue in vivo without the need for intraoperative paralytics or cardiac synchronization.

Cycled pulsing to mitigate thermal damage for multi-electrode irreversible electroporation therapy.

Purpose: This study evaluates the effects of various pulsing paradigms, on the irreversible electroporation (IRE) lesion, induced electric current, and temperature changes using a perfused porcine liver model. Materials and methods: A 4-monopolar electrode array delivered IRE therapy varying the pulse length and inter-pulse delay to six porcine mechanically perfused livers. Pulse paradigms included six forms of cycled pulsing schemes and the conventional pulsing scheme. Finite element models provided further insight into the effects of cycled pulsing on the temperature and thermal injury distribution. Results: 'Single pulse cycle with no interpulse delay' deposited maximum average energy (2.34 ± 0.35 kJ) and produced the largest ratio of thermally damaged tissue area and IRE ablation area from all other pulse schemes (18.22% ± 8.11, p < .0001 all pairwise comparisons). These compared favorably to the conventional algorithm (2.09 ± 0.37 kJ, 3.49% ± 2.20, p < .0001, all comparisons). Though no statistical significance was found between groups, the '5 pulse cycle, 0 s delay' pulse paradigm produced the largest average IRE ablation cross sectional area (11.81 ± 1.97 cm2), while conventional paradigm yielded an average of 8.90 ± 0.91 cm2. Finite element modeling indicated a '10 pulse cycle, 10 s delay' generated the least thermal tissue damage and '1 pulse cycle, 0 s delay' pulse cycle sequence the most (0.47 vs. 3.76 cm2), over a lengthier treatment time (16.5 vs. 6.67 minutes). Conclusions: Subdividing IRE pulses and adding delays throughout the treatment can reduce white tissue coagulation and electric current, while maintaining IRE treatment sizes.

Ablation outcome of irreversible electroporation on potato monitored by impedance spectrum under multi-electrode system.

BACKGROUND: Irreversible electroporation (IRE) therapy relies on pulsed electric fields to non-thermally ablate cancerous tissue. Methods for evaluating IRE ablation in situ are critical to assessing treatment outcome. Analyzing changes in tissue impedance caused by electroporation has been proposed as a method for quantifying IRE ablation. In this paper, we assess the hypothesis that irreversible electroporation ablation outcome can be monitored using the impedance change measured by the electrode pairs not in use, getting more information about the ablation size in different directions. METHODS: Using a square four-electrode configuration, the two diagonal electrodes were used to electroporate potato tissue. Next, the impedance changes, before and after treatment, were measured from different electrode pairs and the impedance information was extracted by fitting the data to an equivalent circuit model. Finally, we correlated the change of impedance from various electrode pairs to the ablation geometry through the use of fitted functions; then these functions were used to predict the ablation size and compared to the numerical simulation results. RESULTS: The change in impedance from the electrodes used to apply pulses is larger and has higher deviation than the other electrode pairs. The ablation size and the change in resistance in the circuit model correlate with various linear functions. The coefficients of determination for the three functions are 0.8121, 0.8188 and 0.8691, respectively, showing satisfactory agreement. The functions can well predict the ablation size under different pulse numbers, and in some directions it did even better than the numerical simulation method, which used different electric field thresholds for different pulse numbers. CONCLUSIONS: The relative change in tissue impedance measured from the non-energized electrodes can be used to assess ablation size during treatment with IRE according to linear functions.

Effects of internal electrode cooling on irreversible electroporation using a perfused organ model.

PURPOSE: This study evaluates the effects of active electrode cooling, via internal fluid circulation, on the irreversible electroporation (IRE) lesion, deployed electric current and temperature changes using a perfused porcine liver model. MATERIALS AND METHODS: A bipolar electrode delivered IRE electric pulses with or without activation of internal cooling to nine porcine mechanically perfused livers. Pulse schemes included a constant voltage, and a preconditioned delivery combined with an arc-mitigation algorithm. After treatment, organs were dissected, and treatment zones were stained using triphenyl-tetrazolium chloride (TTC) to demonstrate viability. RESULTS: Thirty-nine treatments were performed with an internally cooled applicator and 21 with a non-cooled applicator. For the constant voltage scenario, the average final electrical current measured was 26.37 and 29.20 A for the cooled and uncooled electrodes respectively ([Formula: see text]). The average final temperature measured was 33.01 and 42.43 °C for the cooled and uncooled electrodes respectively ([Formula: see text]). The average measured ablations (fixed lesion) were 3.88-by-2.08 cm and 3.86-by-2.12 cm for the cooled and uncooled electrode respectively ([Formula: see text], [Formula: see text]). Similarly, the preconditioned/arc-mitigation scenario yielded an average final electrical current measurement of a 41.07 and 47.20 A for the cooled and uncooled electrodes respectively ([Formula: see text]). The average final temperature measured was 34.93 and 44.90 °C for the cooled and uncooled electrodes respectively ([Formula: see text]). The average measured ablations (fixed lesion) were 3.67-by-2.27 cm and 3.58-by-2.09 cm for the cooled and uncooled applicators ([Formula: see text]). CONCLUSIONS: The internally-cooled bipolar applicator offers advantages that could improve clinical outcomes. Thermally mitigating internal perfusion technology reduced tissue temperatures and electric current while maintaining similar lesion sizes.

Enhancing Irreversible Electroporation by Manipulating Cellular Biophysics with a Molecular Adjuvant.

Pulsed electric fields applied to cells have been used as an invaluable research tool to enhance delivery of genes or other intracellular cargo, as well as for tumor treatment via electrochemotherapy or tissue ablation. These processes involve the buildup of charge across the cell membrane, with subsequent alteration of transmembrane potential that is a function of cell biophysics and geometry. For traditional electroporation parameters, larger cells experience a greater degree of membrane potential alteration. However, we have recently demonstrated that the nuclear/cytoplasm ratio (NCR), rather than cell size, is a key predictor of response for cells treated with high-frequency irreversible electroporation (IRE). In this study, we leverage a targeted molecular therapy, ephrinA1, known to markedly collapse the cytoplasm of cells expressing the EphA2 receptor, to investigate how biophysical cellular changes resulting from NCR manipulation affect the response to IRE at varying frequencies. We present evidence that the increase in the NCR mitigates the cell death response to conventional electroporation pulsed-electric fields (∼100 µs), consistent with the previously noted size dependence. However, this same molecular treatment enhanced the cell death response to high-frequency electric fields (∼1 µs). This finding demonstrates the importance of considering cellular biophysics and frequency-dependent effects in developing electroporation protocols, and our approach provides, to our knowledge, a novel and direct experimental methodology to quantify the relationship between cell morphology, pulse frequency, and electroporation response. Finally, this novel, to our knowledge, combinatorial approach may provide a paradigm to enhance in vivo tumor ablation through a molecular manipulation of cellular morphology before IRE application.

Quantification of cell membrane permeability induced by monopolar and high-frequency bipolar bursts of electrical pulses.

High-frequency bipolar electric pulses have been shown to mitigate undesirable muscle contraction during irreversible electroporation (IRE) therapy. Here, we evaluate the potential applicability of such pulses for introducing exogenous molecules into cells, such as in electrochemotherapy (ECT). For this purpose we develop a method for calculating the time course of the effective permeability of an electroporated cell membrane based on real-time imaging of propidium transport into single cells that allows a quantitative comparison between different pulsing schemes. We calculate the effective permeability for several pulsed electric field treatments including trains of 100µs monopolar pulses, conventionally used in IRE and ECT, and pulse trains containing bursts or evenly-spaced 1µs bipolar pulses. We show that shorter bipolar pulses induce lower effective membrane permeability than longer monopolar pulses with equivalent treatment times. This lower efficiency can be attributed to incomplete membrane charging. Nevertheless, bipolar pulses could be used for increasing the uptake of small molecules into cells more symmetrically, but at the expense of higher applied voltages. These data indicate that high-frequency bipolar bursts of electrical pulses may be designed to electroporate cells as effectively as and more homogeneously than conventional monopolar pulses.

A feasibility study for enrichment of highly aggressive cancer subpopulations by their biophysical properties via dielectrophoresis enhanced with synergistic fluid flow.

A common problem with cancer treatment is the development of treatment resistance and tumor recurrence that result from treatments that kill most tumor cells yet leave behind aggressive cells to repopulate. Presented here is a microfluidic device that can be used to isolate tumor subpopulations to optimize treatment selection. Dielectrophoresis (DEP) is a phenomenon where particles are polarized by an electric field and move along the electric field gradient. Different cell subpopulations have different DEP responses depending on their bioelectrical phenotype, which, we hypothesize, correlate with aggressiveness. We have designed a microfluidic device in which a region containing posts locally distorts the electric field created by an AC voltage and forces cells toward the posts through DEP. This force is balanced with a simultaneous drag force from fluid motion that pulls cells away from the posts. We have shown that by adjusting the drag force, cells with aggressive phenotypes are influenced more by the DEP force and trap on posts while others flow through the chip unaffected. Utilizing single-cell trapping via cell-sized posts coupled with a drag-DEP force balance, we show that separation of similar cell subpopulations may be achieved, a result that was previously impossible with DEP alone. Separated subpopulations maintain high viability downstream, and remain in a native state, without fluorescent labeling. These cells can then be cultured to help select a therapy that kills aggressive subpopulations equally or better than the bulk of the tumor, mitigating resistance and recurrence.

Irreversible electroporation for the ablation of pancreatic malignancies: A patient-specific methodology.

BACKGROUND AND OBJECTIVES: Irreversible Electroporation (IRE) is a focal ablation technique highly attractive to surgical oncologists due to its non-thermal nature that allows for eradication of unresectable tumors in a minimally invasive procedure. In this study, our group sought to address the challenge of predicting the ablation volume with IRE for pancreatic procedures. METHODS: In compliance with HIPAA and hospital IRB approval, we established a pre-treatment planning methodology for IRE procedures in pancreas, which optimized treatment protocols for individual cases of locally advanced pancreatic cancer (LAPC). A new method for confirming treatment plans through intraoperative monitoring of tissue resistance was also proved feasible in three patients. RESULTS: Results from computational models showed good correlation with experimental data available in the literature. By implementing the proposed resistance measurement system 210 ± 26.1 (mean ± standard deviation) fewer pulses were delivered per electrode-pair. CONCLUSION: The proposed physics-based pre-treatment plan through finite element analysis and system for actively monitoring resistance changes can be paired to significantly reduce ablation times and risk of thermal effects during IRE procedures for LAPC.

High-Frequency Irreversible Electroporation: Safety and Efficacy of Next-Generation Irreversible Electroporation Adjacent to Critical Hepatic Structures.

Irreversible electroporation (IRE) is a nonthermal ablation modality employed to induce in situ tissue-cell death. This study sought to evaluate the efficacy of a novel high-frequency IRE (H-FIRE) system to perform hepatic ablations across, or adjacent to, critical vascular and biliary structures. Using ultrasound guidance H-FIRE electrodes were placed across, or adjacent to, portal pedicels, hepatic veins, or the gall bladder in a porcine model. H-FIRE pulses were delivered (2250 V, 2-5-2 pulse configuration) in the absence of cardiac synchronization or intraoperative paralytics. Six hours after H-FIRE the liver was resected and analyzed. Nine ablations were performed in 3 separate experimental groups (major vessels straddled by electrodes, electrodes placed adjacent to major vessels, electrodes placed adjacent to gall bladder). Average ablation time was 290 ± 63 seconds. No electrocardiogram abnormalities or changes in vital signs were observed during H-FIRE. At necropsy, no vascular damage, coagulated-thermally desiccated blood vessels, or perforated biliary structures were noted. Histologically, H-FIRE demonstrated effective tissue ablation and uniform induction of apoptotic cell death in the parenchyma independent of vascular or biliary structure location. Detailed microscopic analysis revealed minor endothelial damage within areas subjected to H-FIRE, particularly in regions proximal to electrode insertion. These data indicate H-FIRE is a novel means to perform rapid, reproducible IRE in liver tissue while preserving gross vascular/biliary architecture. These characteristics raise the potential for long-term survival studies to test the viability of this technology toward clinical use to target tumors not amenable to thermal ablation or resection.

Modeling of Transmembrane Potential in Realistic Multicellular Structures before Electroporation.

Many approaches for studying the transmembrane potential (TMP) induced during the treatment of biological cells with pulsed electric fields have been reported. From the simple analytical models to more complex numerical models requiring significant computational resources, a gamut of methods have been used to recapitulate multicellular environments in silico. Cells have been modeled as simple shapes in two dimensions as well as more complex geometries attempting to replicate realistic cell shapes. In this study, we describe a method for extracting realistic cell morphologies from fluorescence microscopy images to generate the piecewise continuous mesh used to develop a finite element model in two dimensions. The preelectroporation TMP induced in tightly packed cells is analyzed for two sets of pulse parameters inspired by clinical irreversible electroporation treatments. We show that high-frequency bipolar pulse trains are better, and more homogeneously raise the TMP of tightly packed cells to a simulated electroporation threshold than conventional irreversible electroporation pulse trains, at the expense of larger applied potentials. Our results demonstrate the viability of our method and emphasize the importance of considering multicellular effects in the numerical models used for studying the response of biological tissues exposed to electric fields.

Electroporation of Brain Endothelial Cells on Chip toward Permeabilizing the Blood-Brain Barrier.

The blood-brain barrier, mainly composed of brain microvascular endothelial cells, poses an obstacle to drug delivery to the brain. Controlled permeabilization of the constituent brain endothelial cells can result in overcoming this barrier and increasing transcellular transport across it. Electroporation is a biophysical phenomenon that has shown potential in permeabilizing and overcoming this barrier. In this study we developed a microengineered in vitro model to characterize the permeabilization of adhered brain endothelial cells to large molecules in response to applied pulsed electric fields. We found the distribution of affected cells by reversible and irreversible electroporation, and quantified the uptaken amount of naturally impermeable molecules into the cells as a result of applied pulse magnitude and number of pulses. We achieved 81 ± 1.7% (N = 6) electroporated cells with 17 ± 8% (N = 5) cell death using an electric-field magnitude of ∼580 V/cm and 10 pulses. Our results provide the proper range for applied electric-field intensity and number of pulses for safe permeabilization without significantly compromising cell viability. Our results demonstrate that it is possible to permeabilize the endothelial cells of the BBB in a controlled manner, therefore lending to the feasibility of using pulsed electric fields to increase drug transport across the BBB through the transcellular pathway.