An experimental and theoretical study on cell swelling for osmotic imbalance induced by electroporation.

The cellular membrane serves as a pivotal barrier in regulating intra- and extracellular matter exchange. Disruption of this barrier through pulsed electric fields (PEFs) induces the transmembrane transport of ions and molecules, creating a concentration gradient that subsequently results in the imbalance of cellular osmolality. In this study, a multiphysics model was developed to simulate the electromechanical response of cells exposed to microsecond pulsed electric fields (µsPEFs). Within the proposed model, the diffusion coefficient of the cellular membrane for various ions was adjusted based on electropore density. Cellular osmolality was governed and described using Van't Hoff theory, subsequently converted to loop stress to dynamically represent the cell swelling process. Validation of the model was conducted through a hypotonic experiment and simulation at 200 mOsm/kg, revealing a 14.2% increase in the cell's equivalent radius, thereby confirming the feasibility of the cell mechanical model. With the transmembrane transport of ions induced by the applied µsPEF, the hoop stress acting on the cellular membrane reached 179.95 Pa, and the cell equivalent radius increased by 11.0% when the extra-cellular medium was supplied with normal saline. The multiphysics model established in this study accurately predicts the dynamic changes in cell volume resulting from osmotic imbalance induced by PEF action. This model holds theoretical significance, offering valuable references for research on drug delivery and tumor microenvironment modulation.

Dynamics of Cell Death Due to Electroporation Using Different Pulse Parameters as Revealed by Different Viability Assays.

The mechanisms of cell death due to electroporation are still not well understood. Recent studies suggest that cell death due to electroporation is not an immediate all-or-nothing response but rather a dynamic process that occurs over a prolonged period of time. To investigate whether the dynamics of cell death depends on the pulse parameters or cell lines, we exposed different cell lines to different pulses [monopolar millisecond, microsecond, nanosecond, and high-frequency bipolar (HFIRE)] and then assessed viability at different times using different viability assays. The dynamics of cell death was observed by changes in metabolic activity and membrane integrity. In addition, regardless of pulse or cell line, the dynamics of cell death was observed only at high electroporation intensities, i.e., high pulse amplitudes and/or pulse number. Considering the dynamics of cell death, the clonogenic assay should remain the preferred viability assay for assessing viability after electroporation.

Optimal Irreversible Electroporation Combined with Nano-Enabled Immunomodulatory to Boost Systemic Antitumor Immunity.

In this work, we proposed nµPEF, a novel pulse configuration combining nanosecond and microsecond pulses (nµPEF), to enhance tumor ablation in irreversible electroporation (IRE) for oncological therapy. nµPEF demonstrated improved efficacy in inducing immunogenic cell death, positioning it as a potential candidate for next-generation ablative therapy. However, the immune response elicited by nµPEF alone was insufficient to effectively suppress distant tumors. To address this limitation, we developed PPR@CM-PD1, a genetically engineered nanovesicle. PPR@CM-PD1 employed a polyethylene glycol-polylactic acid-glycolic acid (PEG-PLGA) nanoparticle encapsulating the immune adjuvant imiquimod and coated with a genetically engineered cell membrane expressing programmed cell death protein 1 (PD1). This design allowed PPR@CM-PD1 to target both the innate immune system through toll-like receptor 7 (TLR7) agonism and the adaptive immune system through programmed cell death protein 1/programmed cell death-ligand 1 (PD1/PDL1) checkpoint blockade. In turn, nµPEF facilitated intratumoral infiltration of PPR@CM-PD1 by modulating the tumor stroma. The combination of nµPEF and PPR@CM-PD1 generated a potent and systemic antitumor immune response, resulting in remarkable suppression of both nµPEF-treated and untreated distant tumors (abscopal effects). This interdisciplinary approach presents a promising perspective for oncotherapy and holds great potential for future clinical applications.

The Enhancement of Tumor Ablation Effect by the Combination of High-Frequency and Low-Voltage Bipolar Electroporation Pulses.

The H-FIRE (high-frequency irreversible electroporation) protocol employs high-frequency bipolar pulses (HFBPs) with a width of ∼1 µs for tumor ablation with slight muscle contraction. However, H-FIRE pulses need a higher electric field to generate a sufficient ablation effect, which may cause undesirable thermal damage. OBJECTIVE: Recently, combining short high-voltage IRE monopolar pulses with long low-voltage IRE monopolar pulses was shown to enlarge the ablation region. This finding indicates that combining HFBPs with low-voltage bipolar pulses (LVBPs), which are called composited bipolar pulses (CBPs), may enhance the ablation effect. METHODS: This study designed a pulse generator by modifying a full-bridge inverter. The cell suspension and 3D tumor mimic experiments (U251 cells) were performed to examine the enhancement of the ablation effect. RESULTS: The generator outputs HFBPs with 0-±2.5 kV and LVBPs with 0-±0.3 kV in one period. The pulse parameters are adjustable by programming on a human-computer interface. The cell suspension experiments showed that CBPs could enhance cytotoxicity, as compared to HFBPs with no cell-killing effect. Even at lower electric energy, the cell viability by CBPs was significantly lower than that of the HFBPs protocol. The ablation experiments on the 3D tumor mimic showed that the CBPs could create a larger connected ablation area. In contrast, the HFBPs protocol with a similar dose generated a nonconnected ablation area. CONCLUSION: Results indicate that the CBPs protocol can enhance the ablation effect of HFBPs protocol. SIGNIFICANCE: This proposed generator that uses the CBPs principle may be a useful tool for tumor ablation.

Superior Surface-Insulated Polymers with Low Leakage Current Enabled by Tailored Coatings Deposited with Colliding Plasma Jets.

Insufficient surface insulation margin is the primary challenge for a 10 kV plus high-voltage semiconductor module. Surface charge accumulation and electric field distortion are the leading causes of surface insulation failure. Power modules restrict leakage loss, so only insulation dielectrics with low surface conductivity can be used. However, low conductivity, accumulated charge dissipation, and distorted electric field optimization have always been contradictory. A potential barrier increase and electron affinity decrease are both less coupled approaches with conductivity, which may have the potential for reducing surface charge accumulation. Here, surface charge accumulation inhibition and local electric field optimization were synchronously realized by tailored coating deposition with colliding plasma jets. This novelty approach leads to a finer interfacial modification of the triple junction and its nearby interfaces. The high-barrier and low-affinity coatings deposited by colliding plasma jets suppress charge injection (electrode-polymer interface) and promote charge dissipation (gas-polymer interface), respectively. At the same time, the small-area semiconductor deposited at the triple junction relieves the distortion of the electric field. In the end, while maintaining a low leakage current, the surface flashover voltages of polytetrafluoroethylene, polyimide, and epoxy packaging polymers are significantly increased by 69.7, 43.2, and 39.6%, respectively. Notably, the normalized leakage loss is less than 3/10,000 of the commercially available SiC module, which vastly differs from the surface insulation improvement strategy that blindly increases surface conductivity. This tailored coating modification strategy provides a new idea for dielectric research. It has reasonable practicability due to fast, cheap, and environmentally friendly colliding plasma jets.

Scheduled dosage regimen by irreversible electroporation of loaded erythrocytes for cancer treatment.

Precise control of cargo release is essential but still a great challenge for any drug delivery system. Irreversible electroporation (IRE), utilizing short high-voltage pulsed electric fields to destabilize the biological membrane, has been recently approved as a non-thermal technique for tumor ablation without destroying the integrity of adjacent collagenous structures. Due to the electro-permeating membrane ability, IRE might also have great potential to realize the controlled drug release in response to various input IRE parameters, which were tested in a red blood cell (RBC) model in this work. According to the mathematical simulation model of a round biconcave disc-like cell based on RBC shape and dielectric characteristics, the permeability and the pore density of the RBC membrane were found to quantitatively depend on the pulse parameters. To further provide solid experimental evidence, indocyanine green (ICG) and doxorubicin (DOX) were both loaded inside RBCs (RBC@DOX&ICG) and the drug release rates were found to be tailorable by microsecond pulsed electric field (µsPEF). In addition, µsPEF could effectively modulate the tumor stroma to augment therapy efficacy by increasing micro-vessel density and permeability, softening extracellular matrix, and alleviating tumor hypoxia. Benefiting from these advantages, this IRE-responsive RBC@DOX&ICG achieved a remarkably synergistic anti-cancer effect by the combination of µsPEF and chemotherapy in the tumor-bearing mice model, with the survival time increasing above 90 days without tumor burden. Given that IRE is easily adaptable to different plasma membrane-based vehicles for delivering diverse drugs, this approach could offer a general applicability for cancer treatment.

Study on B16 Cell Cytotoxicity by High Frequency Reversible Electroporation With Bleomycin That Induces Hallmarks of Immunogenic Death.

Hundreds of high frequency bipolar pulse bursts with ∼1 µs have been suggested to alleviate muscle contractions and pain during the irreversible electroporation (IRE) tumor treatment. This study is performed to verify whether eight bursts of high frequency reversible electroporation pulses (HFREs) with bleomycin could be used for electrochemotherapy (ECT) tumor treatment. Firstly, in vitro experiments on B16 cells are performed to determine the cytotoxicity of the HFREs with bleomycin. The result indicates that the protocol of HFREs with bleomycin has a significant killing effect compared with only bleomycin, in which the used HFRE pulses are set to induce high membrane permeabilization while maintaining high cell viability. The immunogenic cell death (ICD) that generates danger associated molecular patterns (DAMPs) could trigger an adaptive immune response against tumors. We demonstrated that HFREs with bleomycin could trigger the hallmarks of ICD with obvious up-regulation of DAMPs, including ATP, HMGB1, and CRT. The ICD process may begin at 3 h but perform at 6 h after HFREs with bleomycin stimulation. The in vivo experiment on mice tumor treatment also showed that the protocol of HFREs with bleomycin could inhibit tumor growth with more cytotoxic CD8+ T cells infiltration. The results obtained from in vitro and in vitro experiments preliminary confirmed that the HFREs with bleomycin could be used for ECT tumor treatment associated with the hallmarks of ICD and preliminary trigger the adaptive immune response.

The Enlargement of Ablation Area by Electrolytic Irreversible Electroporation (E-IRE) Using Pulsed Field with Bias DC Field.

Irreversible electroporation (IRE) by high-strength electric pulses is a biomedical technique that has been effectively used for minimally invasive tumor therapy while maintaining the functionality of adjacent important tissues, such as blood vessels and nerves. In general, pulse delivery using needle electrodes can create a reversible electroporation region beyond both the ablation area and the vicinity of the needle electrodes, limiting enlargement of the ablation area. Electrochemical therapy (EChT) can also be used to ablate a tumor near electrodes by electrolysis using a direct field with a constant current or voltage (DC field). Recently, reversible electroporated cells have been shown to be susceptible to electrolysis at relatively low doses. Reversible electroporation can also be combined with electrolysis for tissue ablation. Therefore, the objective of this study is to use electrolysis to remove the reversible electroporation area and thereby enlarge the ablation area in potato slices in vitro using a pulsed field with a bias DC field (constant voltage). We call this protocol electrolytic irreversible electroporation (E-IRE). The area over which the electrolytic effect induced a pH change was also measured. The results show that decreasing the pulse frequency using IRE alone is found to enlarge the ablation area. The ablation area generated by E-IRE is significantly larger than that generated by using IRE or EChT alone. The ablation area generated by E-IRE at 1 Hz is 109.5% larger than that generated by IRE, showing that the reversible electroporation region is transformed into an ablation region by electrolysis. The area with a pH change produced by E-IRE is larger than that produced by EChT alone. Decreasing the pulse frequency in the E-IRE protocol can further enlarge the ablation area. The results of this study are a preliminary indication that the E-IRE protocol can effectively enlarge the ablation area and enhance the efficacy of traditional IRE for use in ablating large tumors.

A fundamental theoretical study on the different effect of electroporation on tumor blood vessels and normal blood vessels.

Electroporation achieved by the application of pulsed electric field is successfully used for clinical tumor ablation. Electrochemotherapy (ECT) and irreversible electroporation (IRE), which are two protocols based on electroporation, have been shown to ablate only tumor cells while preserving the function of normal blood vessels. However, the mechanism of this unique advantage is still not fully understood. This study first built a multilayer dielectric model of both normal and tumor blood vessels to study the electroporation effect. Since endothelial cells are the main component of normal and tumor blood vessels, this study mainly analyzed the electroporation effect on endothelial cells. The rich vascular smooth muscle cells (VSMCs), could play a protective function, allowing endothelial cells to suffer less electroporation effect in normal blood vessels. Interestingly, the endothelial cells in tumor blood vessel sustained a stronger electroporation effect than those in normal blood vessels due to the lack of VSMCs. This study may provide a conceivable explanation for why the structure of endothelial cells in normal blood vessels is preserved during electroporation treatment. This study also demonstrates that ECT or IRE may also damage both tumor blood vessels and cells while preserving normal blood vessels, which benefits complete tumor ablation.

Differential effects of nanosecond pulsed electric fields on cells representing progressive ovarian cancer.

Nanosecond pulsed electric fields (nsPEFs) may induce differential effects on tumor cells from different disease stages and could be suitable for treating tumors by preferentially targeting the late-stage/highly aggressive tumor cells. In this study, we investigated the nsPEF responses of mouse ovarian surface epithelial (MOSE) cells representing progressive ovarian cancer from benign to malignant stages and highly aggressive tumor-initiating-like cells. We established the cell-seeded 3D collagen scaffolds cultured with or without Nocodazole (eliminating the influence of cell proliferation on ablation outcome) to observe the ablation effects at 3 h and 24 h after treatment and compared the corresponding thresholds obtained by numerically calculated electric field distribution. The results showed that nsPEFs induced larger ablation areas with lower thresholds as the cell progress from benign, malignant to a highly aggressive phenotype. This differential effect was not affected by the different doubling times of the cells, as apparent by similar ablation induction after a synergistic treatment of nsPEFs and Nocodazole. The result suggests that nsPEFs could induce preferential ablation effects on highly aggressive and malignant ovarian cancer cells than their benign counterparts. This study provides an experimental basis for the research on killing malignant tumor cells via electrical treatments and may have clinical implications for treating tumors and preventing tumor recurrence after treatment.

In Vitro Experimental and Numerical Studies on the Preferential Ablation of Chemo-Resistant Tumor Cells Induced by High-Voltage Nanosecond Pulsed Electric Fields.

Chemoresistance causes tumor recurrence and metastasis, resulting in poor clinical outcomes and low survival, and has been considered an obstacle to tumor therapy. The development of novel therapeutic approaches that can effectively kill chemoresistant tumor cells (CRTCs) is therefore critical to overcoming these obstacles. OBJECTIVE: Here, we introduce an emerging physical feature-based therapeutic approach based on nanosecond pulsed electric fields (nsPEFs). The goal of this study is to investigate the effect of nsPEFs on CRTCs. METHODS: The cell viability, ablation effects on a 3D-cultured scaffold, and lethal thresholds of nsPEFs were evaluated according to fluorescence staining assays. RESULTS: nsPEF treatment preferentially affected chemoresistant cells (A549/CDDP) with a higher cell viability inhibition ability/cell death rate, larger ablation area, and lower ablation threshold compared to their respective homologous tumor cells (A549). The experimental and theoretical studies suggested that nsPEFs displayed selective behavior toward intracellular structures. With this selective character, nsPEFs can induce higher electroporation effects (e.g., higher pore number, larger electroporation area, and faster fluorescence dissipation on the nuclear envelope) on CRTCs due to their larger nuclear size and cell membrane capacitance. CONCLUSION: These findings demonstrated that nsPEFs induced preferential ablation of CRTCs over their respective homologous tumor cells. SIGNIFICANCE: This study provides an experimental and theoretical basis for the study of killing CRTCs by electrical treatments and suggests potential applications in the optimization of novel anti-chemoresistance methods.

Picosecond Pulse Electrical Field Suppressing Spike Firing in Hippocampal CA1 in Rat In Vivo.

Picosecond pulse electrical fields (psPEFs), due to their high temporal-resolution accuracy and localization, were viewed as a potential targeted and noninvasive method for neuromodulation. However, few studies have reported psPEFs regulating neuronal activity in vivo. In this paper, a preliminary study on psPEFs regulating action potentials in hippocampus CA1 of rats in vivo was carried out. By analyzing the neuronal spike firing rate in hippocampus CA1 pre- and post-psPEF stimulation, effects of frequency, duration, and dosimetry of psPEFs were studied. The psPEF used in this study had a pulse width of 500 ps and a field strength of 1 kV/mm, established by 1 kV picosecond voltage pulses. Results showed that the psPEF suppressed spike firing in hippocampal CA1 neurons. The suppression effect was found to be significant except for 10 s, 10 Hz. For short-duration stimulation (10 s), the inhibition rate of spike firing increased with frequency. At longer stimulation durations (1 and 2 min), the inhibition rate increased and decreased alternately as the frequency increased. Despite this, the inhibition rate at high frequencies (5 and 10 kHz) was significantly larger than that at 10 and 100 Hz. A cumulative effect of psPEF on spike firing inhibition was found at low frequencies (10 and 100 Hz), which was saturated when frequency reached 500 Hz or higher. This paper conducts a study on psPEF regulating spike firing in hippocampal CA1 in vivo for the first time and guides subsequent study on psPEF achieving noninvasive neuromodulation. © 2020 Bioelectromagnetics Society.

Analysis on reversible/irreversible electroporation region in lung adenocarcinoma cell model in vitro with electric pulses delivered by needle electrodes.

Irreversible electroporation (IRE) is a minimally invasive tumor therapy using pulsed electric field with high intensity while the important tissues such as blood vessel, bile duct, and nerve are preserved. In addition to ablation area, reversible electroporation (RE) region is also generated using needle electrodes for pulse delivery. The goal of this work is to study the generation of RE region and ablation region on a 2D lung adenocarcinoma cell model in vitro. The tumor model is exposed to electric pulses with various number. The calcium AM and propidium iodide (PI) are examined to detect the ablation area and electroporation area, respectively. The results show that electroporation area firstly tends to plateau after approximately 50 pulses, while the ablation area continues to increase. The percentage of IRE area in total electroporation area increases with additional pulses, which means that RE region could be gradually turned into ablation area with increased pulse number. However, the percentage of IRE area only achieves to 54% for 200 pulses, which indicates that RE region still cannot be completely removed. RE and IRE thresholds appear to converge as the number of pulses increases. An equation between pulse number and the electric field threshold of ablation including the electric field threshold of RE is also provided for lung adenocarcinoma cell ablation. This work may have the value for the optimization of IRE protocols on tumor ablation.

Development of a Multi-Pulse Conductivity Model for Liver Tissue Treated With Pulsed Electric Fields.

Pulsed electric field treatment modalities typically utilize multiple pulses to permeabilize biological tissue. This electroporation process induces conductivity changes in the tissue, which are indicative of the extent of electroporation. In this study, we characterized the electroporation-induced conductivity changes using all treatment pulses instead of solely the first pulse as in conventional conductivity models. Rabbit liver tissue was employed to study the tissue conductivity changes caused by multiple, 100 µs pulses delivered through flat plate electrodes. Voltage and current data were recorded during treatment and used to calculate the tissue conductivity during the entire pulsing process. Temperature data were also recorded to quantify the contribution of Joule heating to the conductivity according to the tissue temperature coefficient. By fitting all these data to a modified Heaviside function, where the two turning points (E 0, E 1) and the increase factor (A) are the main parameters, we calculated the conductivity as a function of the electric field (E), where the parameters of the Heaviside function (A and E 0) were functions of pulse number (N). With the resulting multi-factor conductivity model, a numerical electroporation simulation can predict the electrical current for multiple pulses more accurately than existing conductivity models. Moreover, the saturating behavior caused by electroporation can be explained by the saturation trends of the increase factor A in this model. The conductivity change induced by electroporation has a significant increase at about the first 30 pulses, then tends to saturate at 0.465 S/m. The proposed conductivity model can simulate the electroporation process more accurately than the conventional conductivity model. The electric field distribution computed using this model is essential for treatment planning in biomedical applications utilizing multiple pulsed electric fields, and the method proposed here, relating the pulse number to the conductivity through the variables in the Heaviside function, may be adapted to investigate the effect of other parameters, like pulse frequency and pulse width, on electroporation.

Application of bioimpedance spectroscopy to characterize chemoresistant tumor cell selectivity of nanosecond pulse stimulation.

The discriminating effects of nanosecond pulsed electric fields (nsPEFs) between chemoresistant tumor cells (CRTCs) and their respective homologous chemosensitive tumor cells (CSTCs) were investigated based on bioimpedance spectroscopy (BIS). The electrical properties of individual untreated cells were determined by fitting the impedance spectra to an equivalent circuit model and then using aided simulations to calculate the nuclear envelope transmembrane potential (nTMP) and electroporation area on the nuclear envelope. Additionally, fluorescence staining assays of cell monolayers after nanopulse stimulation (80 pulses, 200 ns, 3 kV) were conducted to validate the simulation results. The staining results indicated that CRTCs showed a larger ablation area and lower lethal threshold compared to CSTCs after exposure to the same nsPEF energy, which was in accordance with the higher nTMP and larger electroporation area calculated for CRTCs. The increase in the lethal effects of nsPEFs on CRTCs compared to CSTCs mainly resulted from the superposition of the changes in the electrical properties and nuclear size. The work shows that BIS can distinguish CRTCs and CSTCs and the corresponding nsPEF effects, suggesting potential applications for the optimization of novel anti-chemoresistance methods, including nsPEF-treatments.

Nanosecond pulses targeting intracellular ablation increase destruction of tumor cells with irregular morphology.

The decrease in killing sensitivity of the cell membrane to microsecond pulse electric fields (µs-PEFs) is ascribed mainly to the aberrant morphology of cancer cells, with clear statistical correlations observed between cell size and shape defects and the worsening of the electrical response to the PEF. In this paper, nanosecond pulsed electric fields (ns-PEFs) inducing the nucleus effect and µs-PEFs targeting the cell membrane were combined to enhance destruction of irregular cells. The fluorescence dissipation levels of the nuclear membrane and cell membrane exposed to the µs, ns, and ns + µs pulse protocols were measured and compared, and a dynamic electroporation model of irregular cells was established by the finite element software COMSOL. The results suggest that the cell membrane disruption induced by µs-PEFs is worse for extremely irregular cells and depends strongly on cellular morphology. However, the nuclear membrane disruption induced by ns-PEFs does not scale with irregularity, suggesting the use of a combination of ns-PEFs with µs-PEFs to target the nuclear and cell membranes. We demonstrate that ns + µs pulses can significantly enhance the fluorescence dissipation of the cell and nuclear membranes. Overall, our findings indicate that ns + µs pulses may be useful in the effective killing of irregular cells.

A 2-D Cell Layer Study on Synergistic Combinations of High-Voltage and Low-Voltage Irreversible Electroporation Pulses.

Irreversible electroporation (IRE) employs brief, high-electric field pulses to ablate tumors while preserving the extracellular matrix. Recently, we showed that combining short high-voltage (SHV) IRE pulses and long low-voltage (LLV) IRE pulses can enlarge the tissue ablation region, presumably through a synergistic effect. OBJECTIVE: The goal of this study is to further investigate the effect of this combination on a 2-D cell layer tumor model. METHODS: 2-D layers of tumor cells are exposed to various SHV and LLV combinations, and the results of propidium iodide (PI) and fluorescein diacetate staining are examined to correlate treatment protocols with the ensuing irreversible and reversible electroporation areas. RESULTS: The combination of SHV+LLV pulses produces a larger area of electroporation and ablation than LLV+SHV pulses, LLV pulses alone, and SHV pulses alone. CONCLUSION: Judiciously combining SHV and LLV pulses can produce a synergistic effect that enlarges the electroporation-induced ablation area. A hypothetical explanation for this effect is that it involves a combination of pore expansion and electrolysis induced by LLV pulses in the area that had been reversibly permeabilized by the SHV pulses. SIGNIFICANCE: This paper is valuable for the design of improved IRE protocols and provides a hypothesis for the mechanisms involved.

A Conceivable Mechanism Responsible for the Synergy of High and Low Voltage Irreversible Electroporation Pulses.

Irreversible electroporation (IRE), is a new non-thermal tissue ablation technology in which brief high electric field pulses are delivered across the target tissue to induce cell death by irreversible permeabilization of the cell membrane. A deficiency of conventional IRE is that the ablation zone is relatively small, bounded by the irreversible electroporation isoelectric field margin. In the previous studies we have introduced a new treatment protocol that combines few short high voltage (SHV) pulses with long low-voltage (LLV) pulses. In the previous studies, we also have shown that the addition of few SHV pulses increases by almost a factor of two the area ablated by a protocol that employs only the LLV pulses. This study employs potato and gel phantom to generate a plausible explanation for the mechanism. The study provides circumstantial evidence that the mechanism involved is the production of electrolytic compounds by the LLV pulse sequence, which causes tissue ablation beyond the margin of the irreversible electroporation isoelectric field generated by the SHV pulses, presumable to the reversible electroporation isoelectric field margin generated by the SHV pulses.

Electrofusion by a bipolar pulsed electric field: Increased cell fusion efficiency for monoclonal antibody production.

The excessive cell death rate caused by electrofusion with unipolar pulses (UPs) has been a bottleneck to increasing cell fusion efficiency in monoclonal antibody technology. Several studies have confirmed that compared with UPs, bipolar pulses (BPs) with microsecond pulse widths can increase electropermeabilization while reducing cell death. Given these characteristics, BPs were used to increase cell fusion efficiency in this study. Cell staining and hybridoma culture experiments were performed using SP2/0 mouse myeloma cells and lymphocytes. Based on the equal energy principle, UPs and BPs were delivered to electrodes at a distance of 3.81 mm, with electric field intensities ranging from 2 kV/cm to 3 kV/cm and pulse duration of 40 µs for the UPs and 20-20 µs for the BPs. The results of cell staining experiments showed that cell fusion efficiency was 3-fold greater with BPs than with UPs. Similarly, the results of the hybridoma culture experiments showed that the hybridoma yields were 0.26‰ and 0.23‰ (2.5 kV/cm and 3 kV/cm, respectively) in the UP groups and increased to 0.46‰ and 0.35‰ in the BP groups. Taken together, the results show that the efficiency of heterologous cell fusion can be greatly increased if BPs are used instead of the commonly applied UPs. This study may provide a promising method for monoclonal antibody technology.