Electroporation Device Research Articles

Therapeutic vaccination for the elimination of HPV16+ high-grade dysplasia: A phase I clinical trial of the DNA vaccine pNGVL4aCRTE6E7L2, administered via the TriGrid™ electroporation device

Therapeutic vaccination for the elimination of HPV16+ high-grade dysplasia: A phase I clinical trial of the DNA vaccine pNGVL4aCRTE6E7L2, administered via the TriGrid™ electroporation device

Real-World Safety of Prostate Cancer Focal Therapy: MAUDE Database Analysis.

Objective: The aim of this study was to assess the real-world safety profile of medical devices used in focal prostate cancer treatment utilizing the Manufacturer and User Facility Device Experience (MAUDE) database. Methods: The MAUDE database was searched for reports on high-intensity focused ultrasound (HIFU), cryoablation, laser ablation, and irreversible electroporation (IRE) devices used in prostate cancer treatment from 1993 to 2023. Adverse events were identified and categorized. Results: We identified 175 reports for HIFU, 1362 for cryoablation, 615 for laser ablation, and 135 for IRE devices, with 28, 284, 126, and 2 respective reports, directly related to prostate cancer treatment. The aggregated data revealed the majority of complications were minor, with 82.5% (n = 363 out of 440 total complications) classified as Clavien-Dindo grade 1 or 2. Common minor complications included erectile dysfunction and urinary retention. Severe complications such as rectal fistulas were noted in HIFU and IRE treatments, along with singular mortality due to pulmonary embolism in cryoablation. Conclusions: Our analyses from MAUDE reveal that devices used in focal therapy for prostate cancer are predominantly associated with minor complications, underscoring their overall real-world safety profile. However, the occurrence of severe adverse events emphasizes the critical importance of rigorous patient selection and meticulous procedural planning. These findings provide valuable insights into the safety profile of focal therapy devices and contribute to the growing body of evidence on their use in prostate cancer treatment.

Well Plate-Based Localized Electroporation Workflow for Rapid Optimization of Intracellular Delivery.

Efficient and nontoxic delivery of foreign cargo into cells is a critical step in many biological studies and cell engineering workflows with applications in areas such as biomanufacturing and cell-based therapeutics. However, effective molecular delivery into cells involves optimizing several experimental parameters. In the case of electroporation-based intracellular delivery, there is a need to optimize parameters like pulse voltage, duration, buffer type, and cargo concentration for each unique application. Here, we present the protocol for fabricating and utilizing a high-throughput multi-well localized electroporation device (LEPD) assisted by deep learning-based image analysis to enable rapid optimization of experimental parameters for efficient and nontoxic molecular delivery into cells. The LEPD and the optimization workflow presented herein are relevant to both adherent and suspended cell types and different molecular cargo (DNA, RNA, and proteins). The workflow enables multiplexed combinatorial experiments and can be adapted to cell engineering applications requiring in vitro delivery. Key features • A high-throughput multi-well localized electroporation device (LEPD) that can be optimized for both adherent and suspended cell types. • Allows for multiplexed experiments combined with tailored pulse voltage, duration, buffer type, and cargo concentration. • Compatible with various molecular cargoes, including DNA, RNA, and proteins, enhancing its versatility for cell engineering applications. • Integration with deep learning-based image analysis enables rapid optimization of experimental parameters.

SiRNA-mediated downregulation of BATF3 diminished proliferation and induced apoptosis through downregulating c-Myc expression in chronic myelogenous leukemia cells.

Despite considerable improvement in therapeutic approaches to chronic myeloid leukemia (CML) treatment, this malignancy is considered incurable due to resistance. However, investigating the molecular mechanism of CML may give rise to the development of extremely efficient targeted therapies that improve the prognosis of patients. Basic leucine zipper transcription factor ATF-like3 (BATF3), as transcription factor, is considered a key regulator of cellular activities and its function has been evaluated in tumor development and growth in several cancer types. This study aimed to evaluate the potential of the cellular impact of siRNA-mediated downregulation of BATF3 on CML cancer cells through cell proliferation, induction of apoptosis, and cell cycle distribution. The transfection of BATF3 siRNA to K562 CML cells was performed by electroporation device. To measure cellular viability and apoptosis, MTT assay and Annexin V/PI staining were carried out, respectively. Also, cell cycle assay and flow cytometry instrument were applied to assess cell cycle distribution of K562 cells. For more validation, mRNA expression of correlated genes was relatively evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). The data indicated that siRNA-mediated BATF3 inactivating severely promoted the cell apoptosis. Also, the targeted therapy led to high expression of Caspase-3 gene and Bax/Bcl-2 ratio. Silenced BATF3 also induced cell cycle arrest in phase sub-G1 compared to control. Finally, a noticeable decrement was obtained in c-Myc gene expression through suppression of BATF3 in CML cells. The findings of this research illustrated the suppression of BATF3 as an effective targeted therapy strategy for CML.

Using Electroporation to Improve and Accelerate Zebrafish Embryo Toxicity Testing.

Zebrafish have emerged as a useful model for biomedical research and have been used in environmental toxicology studies. However, the presence of the chorion during the embryo stage limits cellular exposure to toxic elements and creates the possibility of a false-negative or reduced sensitivity in fish embryo toxicity testing (FET). This paper presents the use of electroporation as a technique to improve the delivery of toxic elements inside the chorion, increasing the exposure level of the toxins at an early embryo stage (<3 h post-fertilization). A custom-made electroporation device with the required electrical circuitry has been developed to position embryos between electrodes that provide electrical pulses to expedite the entry of molecules inside the chorion. The optimized parameters facilitate material entering into the chorion without affecting the survival rate of the embryos. The effectiveness of the electroporation system is demonstrated using Trypan blue dye and gold nanoparticles (AuNPs, 20-40 nm). Our results demonstrate the feasibility of controlling the concentration of dye and nanoparticles delivered inside the chorion by optimizing the electrical parameters, including pulse width, pulse number, and amplitude. Next, we tested silver nanoparticles (AgNPs, 10 nm), a commonly used toxin that can lower mortality, affect heart rate, and cause phenotypic defects. We found that electroporation of AgNPs reduces the exposure time required for toxicity testing from 4 days to hours. Electroporation for FET can provide rapid entry of potential toxins into zebrafish embryos, reducing the time required for toxicity testing and drug delivery experiments.

Nanopore Electroporation Device for DNA Transfection into Various Spreading and Nonadherent Cell Types.

Cell transfection plays a crucial role in the study of gene function and regulation of gene expression. The existing gene transfection methods, such as chemical carriers, viruses, electroporation, and microinjection, suffer from limitations, including cell type dependence, reliance on cellular endocytosis, low efficiency, safety concerns, and technical complexity. Nanopore-coupled electroporation offers a promising approach to localizing electric fields for efficient cell membrane perforation and nucleic acid transfection. However, the applicability of nanopore electroporation technology across different cell types lacks a systematic investigation. In this study, we explore the potential of nanopore electroporation for transfecting DNA plasmids into various cell types. Our nanopore electroporation device employs track-etched membranes as the core component. We find that nanopore electroporation efficiently transfects adherent cells, including well-spreading epithelial-like HeLa cells, cardiomyocyte-like HL-1 cells, and dendritic-cell-like DC2.4 cells. However, it shows a limited transfection efficiency in weakly spreading macrophages (RAW264.7) and suspension cells (Jurkat). To gain insights into these observations, we develop a COMSOL model, revealing that nanopore electroporation better localizes the electric field on adherent and well-spreading cells, promoting favorable membrane poration conditions. Our findings provide valuable references for advancing nanopore electroporation as a high-throughput, safe, and efficient gene transfection platform.

Suitability of a universal electroporation device for genome editing and production of transgenic rats.

Mammalian genome editing has utilized expensive and highly specialized electroporator devices. The "Gene Pulser XCell," a modular electroporation system for transfecting all cell types, has not been used extensively in mammalian embryo genome editing. The present experiment was undertaken to determine the usefulness of the Gene Pulser XCell for inserting the CRISPR/Cas9 system into intact zygotes in order to obtain the enhanced green fluorescent protein reporter rats (eGFP-R). An electroporation pulse response test using mCherry mRNA was performed to optimize the settings of the electroporator. Forty-five combinations of five pulse voltages (15, 25, 30, 35 and 40V), three pulse durations (5, 10 and 25ms), and three pulse frequencies (2, 5 and 6 pulses) applied at a constant 100-ms pulse interval and temperature of 37.5°C were evaluated. The test revealed that the 35V was the only voltage suitable for insertion of mCherry mRNA into intact rat zygotes and the only one that resulted in the production of embryos attaining the blastocyst stage. The incorporation of mCherry mRNA increased but the survival of the electroporated embryos declined with an increment in the number of pulses. Subsequent transfer of 1112 surviving Sprague Dawley rat embryos (after 8h of incubating 1800 zygotes electroporated with the CRISPR/Cas9) resulted in the production of 287 offspring (25.8%). Ensuing PCR and phenotypic evaluation confirmed that twenty animals (6.96%) expressed eGFP in all body organs/tissues except for blood and blood vessels. The mortality of males and females before the attainment of puberty was 2 and 3 pups, respectively, and the final number/ratio of male to female of offspring was 9:11. All the surviving rats mated naturally and successfully transmitted the GFP transgene to their progeny. The Gene Pulser XCell total system with the settings predetermined in the present experiment can effectively be used to produce transgenic rats through the CRISPR/Cas9-mediated genome editing of zygotes.

A Low-Cost Electroporator for Genetically Modifying Social Amoeba Dictyostelium Discoideum

The social amoeba Dictyostelium discoideum is a commonly used eukaryotic model organism for the study of cell division, chemotaxis, differentiation, phagocytosis, and other cellular processes. Electroporation is an effective and efficient method for delivering plasmid DNA into D. discoideum, an invaluable tool for studying intracellular processes. The technology is readily available but often prohibitively expensive. Although several custom-built electroporation devices have been developed, none deliver the specific 8.5kV/cm exponentially decaying waveform required for D. discoideum transformation. The present study examined whether a simple, inexpensive device can be built to produce this waveform through a simple resistor-capacitor (RC) circuit. A pulse generator RC circuit was built incorporating inexpensive electronic components and a 3D printed cuvette chamber. All four possible combinations of custom-built and commercial pulse generators and custom-built and commercial cuvette chambers were used to transform D. discoideum cells with a plasmid encoding green fluorescent protein (GFP). There were no significant differences in the number of surviving cells immediately following or 24 hours post-transformation between the systems. All combinations of custom-built and commercial systems achieved comparably high transformation efficiency shown by percent of cells expressing GFP six days after the transformation. Since the waveform-specific electroporation system we present here can be built by non-experts with easily obtainable materials and 3D printing, we envision this device to benefit investigators in areas with low research budgets and educators in multiple STEM fields.

High‐Throughput CRISPR/Cas9 Mediated Gene Editing of Primary Human T Cells in a Microfluidic Device for Cellular Therapy Manufacturing

AbstractAutologous cellular therapies have been highly successful in treating hematological cancers and have the potential to be used for a variety of indications. Manufacturing these therapies rapidly and at low cost remains a major challenge. A key bottleneck in cellular therapy manufacturing is genetic modification of target cells, which is often done using viral vectors. Because vectors are expensive to develop and produce, non‐viral gene transfer using electroporation is emerging as a preferred transfection method for next‐generation therapies. However, most commercial electroporation systems are built for research use rather than large‐scale clinical manufacturing. The microfluidic, continuous‐flow electroporation device presented here offers several advantages including large‐scale and high throughput processing, high performance, and the potential for automation. It transfects primary human T cells with Cas9‐guide ribonucleic acid (RNA) ribonucleoprotein complexes (RNP) and messenger RNA (mRNA) with up to 99–100% efficiency and minimal impact on viability. In addition, this device transfects 3.5 kbp plasmid deoxyribonucleic acid with up to 86% efficiency after preliminary optimization studies. A single microchannel can deliver a total cellular processing throughput of up to 9.6 billion per hour. The combination of high throughput and high performance enables the scale of processing required for future “off‐the‐shelf” allogeneic cellular therapies.

Plant-based model for the visual evaluation of electroporated area after irreversible electroporation and its comparison to in-vivo animal data.

Electroporation (EP) is widely used in medicine, such as cancer treatment, in form of electrochemotherapy or irreversible electroporation (IRE). For EP device testing, living cells or tissue inside a living organism (including animals) are needed. Plant-based models seem to be a promising alternative to substitute animal models in research. The aim of this study is to find a suitable plant-based model for visual evaluation of IRE, and to compare the geometry of electroporated areas with in-vivo animal data.For this purpose, a variety of fruit and vegetables were selected and visually evaluated after 0/1/2/4/6/8/12/16/24 h post-EP. Apple and potato were found to be suitable models as they enabled a visual evaluation of the electroporated area. For these models, the size of the electroporated area was determined after 0/1/2/4/6/8/12/16/24 h. For apples, a well-defined electroporated area was visual within two hours, while in potatoes it reached a plateau after eight hours only. The electroporated area of apple, which showed the fastest visual results was then compared to a retrospectively evaluated swine liver IRE dataset which had been obtained for similar conditions. The electroporated area of the apple and swine liver both showed a spherical geometry of comparable size. For all experiments, the standard protocol for human liver IRE was followed. To conclude, potato and apple were found to be suitable plant-based models for the visual evaluation of electroporated area after irreversible EP, with apple being the best choice for fast visual results. Given the comparable range, the size of the electroporated area of the apple may be promising as a quantitative predictor in animal tissue. Even if plant-based models cannot completely replace animal experiments, they can be used in the early stages of EP device development and testing, decreasing animal experiments to the necessary minimum.

Effect of Experimental Electrical and Biological Parameters on Gene Transfer by Electroporation: A Systematic Review and Meta-Analysis.

The exact mechanisms of nucleic acid (NA) delivery with gene electrotransfer (GET) are still unknown, which represents a limitation for its broader use. Further, not knowing the effects that different experimental electrical and biological parameters have on GET additionally hinders GET optimization, resulting in the majority of research being performed using a trial-and-error approach. To explore the current state of knowledge, we conducted a systematic literature review of GET papers in in vitro conditions and performed meta-analyses of the reported GET efficiency. For now, there is no universal GET strategy that would be appropriate for all experimental aims. Apart from the availability of the required electroporation device and electrodes, the choice of an optimal GET approach depends on parameters such as the electroporation medium; type and origin of cells; and the size, concentration, promoter, and type of the NA to be transfected. Equally important are appropriate controls and the measurement or evaluation of the output pulses to allow a fair and unbiased evaluation of the experimental results. Since many experimental electrical and biological parameters can affect GET, it is important that all used parameters are adequately reported to enable the comparison of results, as well as potentially faster and more efficient experiment planning and optimization.

High-throughput sequencing revealed low-efficacy genome editing using Cas9 RNPs electroporation and single-celled microinjection provided an alternative to deliver CRISPR reagents into Euglena gracilis.

High-throughput sequencing revealed low-efficacy genome editing using Cas9 RNPs electroporation and single-celled microinjection provided an alternative to deliver CRISPR reagents into Euglena gracilis.

M-TUBE enables large-volume bacterial gene delivery using a high-throughput microfluidic electroporation platform.

Conventional cuvette-based and microfluidics-based electroporation approaches for bacterial gene delivery have distinct advantages, but they are typically limited to relatively small sample volumes, reducing their utility for applications requiring high throughput such as the generation of mutant libraries. Here, we present a scalable, large-scale bacterial gene delivery approach enabled by a disposable, user-friendly microfluidic electroporation device requiring minimal device fabrication and straightforward operation. We demonstrate that the proposed device can outperform conventional cuvettes in a range of situations, including across Escherichia coli strains with a range of electroporation efficiencies, and we use its large-volume bacterial electroporation capability to generate a library of transposon mutants in the anaerobic gut commensal Bifidobacterium longum.

Multiplexed high-throughput localized electroporation workflow with deep learning-based analysis for cell engineering.

Manipulation of cells for applications such as biomanufacturing and cell-based therapeutics involves introducing biomolecular cargoes into cells. However, successful delivery is a function of multiple experimental factors requiring several rounds of optimization. Here, we present a high-throughput multiwell-format localized electroporation device (LEPD) assisted by deep learning image analysis that enables quick optimization of experimental factors for efficient delivery. We showcase the versatility of the LEPD platform by successfully delivering biomolecules into different types of adherent and suspension cells. We also demonstrate multicargo delivery with tight dosage distribution and precise ratiometric control. Furthermore, we used the platform to achieve functional gene knockdown in human induced pluripotent stem cells and used the deep learning framework to analyze protein expression along with changes in cell morphology. Overall, we present a workflow that enables combinatorial experiments and rapid analysis for the optimization of intracellular delivery protocols required for genetic manipulation.

Broadband Microwave Electroporation Device for the Analysis of the Influence of Frequency, Temperature and Electrical Field Strength.

In this paper, a broadband microwave device for cell poration is presented, that enables the analysis of the relation between frequency, electrical field strengths and temperature for a successful cell poration. Electromagnetic-thermal coupled simulations in the frequency range from 1 GHz to 10 GHz show that the device reaches electrical field strengths of 100 V/cm and temperatures lower then 40°C. Electroporation experiments with adherent C2C12 mouse myoblast cells show successful uptake of an anti-histone γ -H2A.X nanobody at a frequency of 10 GHz. This MWP device allows the fast electro-poration of adherent cells. After 15 min, the cells show uptake of γ -H2A.X-specific nanobody while most of them survived.

Microtrap array on a chip for localized electroporation and electro-gene transfection

We developed a localized single-cell electroporation chip to deliver exogenous biomolecules with high efficiency while maintaining high cell viability. In our microfluidic device, the cells are trapped in a microtrap array by flow, after which target molecules are supplied to the device and electrotransferred to the cells under electric pulses. The system provides the ability to monitor the electrotransfer of exogenous biomolecules in real time. We reveal through numerical simulations that localized electroporation is the mechanism of permeabilization in the microtrap array electroporation device. We demonstrate the simplicity and accuracy of this microtrap technology for electroporation by delivery of both small molecules using propidium iodide and large molecules using plasmid DNA for gene expression, illustrating the potential of this minimally invasive method to be widely used for precise intracellular delivery purposes (from bioprocess engineering to therapeutic applications).

Recent Advances in Microscale Electroporation.

Electroporation (EP) is a commonly used strategy to increase cell permeability for intracellular cargo delivery or irreversible cell membrane disruption using electric fields. In recent years, EP performance has been improved by shrinking electrodes and device structures to the microscale. Integration with microfluidics has led to the design of devices performing static EP, where cells are fixed in a defined region, or continuous EP, where cells constantly pass through the device. Each device type performs superior to conventional, macroscale EP devices while providing additional advantages in precision manipulation (static EP) and increased throughput (continuous EP). Microscale EP is gentle on cells and has enabled more sensitive assaying of cells with novel applications. In this Review, we present the physical principles of microscale EP devices and examine design trends in recent years. In addition, we discuss the use of reversible and irreversible EP in the development of therapeutics and analysis of intracellular contents, among other noteworthy applications. This Review aims to inform and encourage scientists and engineers to expand the use of efficient and versatile microscale EP technologies.

Modification of a Ready-Made High-Voltage Pulse Generator for Non-Thermal Irreversible Electroporation.

We present an inexpensive device for non-thermal irreversible electroporation (IRE) manufactured by upgrading an industrial high-voltage pulse generator. In addition to the generator, the experimental hardware-and-software complex (IRE system) contains a microprocessor and a temperature sensor, which enables automatic control the pulse frequency in accordance with the temperature of the object. The IRE system was tested in in vitro and in vivo experiments: effective and safe non-thermal IRE was demonstrated. The developed system is easily reproducible and can be especially useful for demonstration experiments, pioneering research, as well as for teaching the basics of IRE.

Laser Machined Fiber-based Microprobe: Application in Microscale Electroporation.

Microscale electroporation devices are mostly restricted to in vitro experiments (i.e., microchannel and microcapillary). Novel fiber-based microprobes can enable in vivo microscale electroporation and arbitrarily select the cell groups of interest to electroporate. We developed a flexible, fiber-based microscale electroporation device through a thermal drawing process and femtosecond laser micromachining techniques. The fiber consists of four copper electrodes (80 μm), one microfluidic channel (30 μm), and has an overall diameter of 400 μm. The dimensions of the exposed electrodes and channel were customizable through a delicate femtosecond laser setup. The feasibility of the fiber probe was validated through numerical simulations and in vitro experiments. Successful reversible and irreversible microscale electroporation was observed in a 3D collagen scaffold (seeded with U251 human glioma cells) using fluorescent staining. The ablation regions were estimated by performing the covariance error ellipse method and compared with the numerical simulations. The computational and experimental results of the working fiber-based microprobe suggest the feasibility of in vivo microscale electroporation in space-sensitive areas, such as the deep brain.

Processing of sugar beets assisted by pulsed electric fields

Pulsed electric fields (PEFs) are becoming more and more popular in different applications in processing agricultural products. The PEF technology is based on the application of high voltage short pulses that allow electroporation in the cell membranes. This review outlines the PEF applications used in processing sugar beets. New perspective technologies of the cold or warm aqueous extraction of sugar, cold pressing of sugar beet cossettes, and combined pressing-diffusion process are discussed. Electroporation devices for pilot and industrial applications in the sugar beet industry are also presented.