NeuroPorator: An open-source, current-limited electroporator for safe in utero gene transfer.

BACKGROUND: Electroporation is an effective technique for genetic manipulation of cells, both in vitro and in vivo. In utero electroporation (IUE) is a special case, which represents a fine application of this technique to genetically modify specific tissues of embryos during prenatal development. Commercially available electroporators are expensive and not fully customizable. We have designed and produced an inexpensive, open-design, and customizable electroporator optimized for safe IUE. We introduce NeuroPorator. METHOD: We used off-the-shelf electrical parts, a single-board microcontroller, and a cheap data logger to build an open-design electroporator. We included a safety circuit to limit the applied electrical current to protect the embryos. We added full documentation, design files, and assembly instructions. RESULT: NeuroPorator output is on par with commercially available devices. Furthermore, the adjustable current limiter protects both the embryos and the uterus from overcurrent damage. A built-in data acquisition module provides real-time visualization and recordings of the actual voltage/current pulses applied to each embryo. Function of NeuroPorator has been demonstrated by inducing focal cortical dysplasia in mice. SIGNIFICANCE AND CONCLUSION: The simple and fully open design enables quick and cheap construction of the device and facilitates further customization. The features of NeuroPorator can accelerate the IUE technique implementation in any laboratory and speed up its learning curve.

Revisiting the role of pulsed electric fields in overcoming the barriers to in vivo gene electrotransfer.

Gene therapies are revolutionizing medicine by providing a way to cure hitherto incurable diseases. The scientific and technological advances have enabled the first gene therapies to become clinically approved. In addition, with the ongoing COVID-19 pandemic, we are witnessing record speeds in the development and distribution of gene-based vaccines. For gene therapy to take effect, the therapeutic nucleic acids (RNA or DNA) need to overcome several barriers before they can execute their function of producing a protein or silencing a defective or overexpressing gene. This includes the barriers of the interstitium, the cell membrane, the cytoplasmic barriers and (in case of DNA) the nuclear envelope. Gene electrotransfer (GET), i.e., transfection by means of pulsed electric fields, is a non-viral technique that can overcome these barriers in a safe and effective manner. GET has reached the clinical stage of investigations where it is currently being evaluated for its therapeutic benefits across a wide variety of indications. In this review, we formalize our current understanding of GET from a biophysical perspective and critically discuss the mechanisms by which electric field can aid in overcoming the barriers. We also identify the gaps in knowledge that are hindering optimization of GET in vivo.

An ultra-low-cost electroporator with microneedle electrodes (ePatch) for SARS-CoV-2 vaccination.

Vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other pathogens with pandemic potential requires safe, protective, inexpensive, and easily accessible vaccines that can be developed and manufactured rapidly at a large scale. DNA vaccines can achieve these criteria, but induction of strong immune responses has often required bulky, expensive electroporation devices. Here, we report an ultra-low-cost (<1 USD), handheld (<50 g) electroporation system utilizing a microneedle electrode array ("ePatch") for DNA vaccination against SARS-CoV-2. The low cost and small size are achieved by combining a thumb-operated piezoelectric pulser derived from a common household stove lighter that emits microsecond, bipolar, oscillatory electric pulses and a microneedle electrode array that targets delivery of high electric field strength pulses to the skin's epidermis. Antibody responses against SARS-CoV-2 induced by this electroporation system in mice were strong and enabled at least 10-fold dose sparing compared to conventional intramuscular or intradermal injection of the DNA vaccine. Vaccination was well tolerated with mild, transient effects on the skin. This ePatch system is easily portable, without any battery or other power source supply, offering an attractive, inexpensive approach for rapid and accessible DNA vaccination to combat COVID-19, as well as other epidemics.

Novel platinum bipolar electrode for irreversible electroporation in prostate cancer: preclinical study in the beagle prostate.

The exposure of the prostate to high electric field strength during irreversible electroporation (IRE) has been extensively investigated. Multiple monopolar electrodes, however, have risks of organ piercing and bleeding when placing electrodes. A novel bipolar electrode made of pure platinum and stainless steel was developed for prostate cancer ablation. Voltages of 500 and 700 V were applied to the beagle prostate with this electrode to evaluate ablated tissues and their characteristics. IRE procedures were technically successful in all dogs without procedure-related complications. The current that flowed through the anode and cathode while applying 500 and 700 V were 1.75 ± 0.25 A and 2.22 ± 0.35 A, respectively. TUNEL assays showed that the estimated ablated areas when applying 500 and 700 V were 0.78 cm2 and 1.21 cm2, respectively. The minimum electric field strength threshold required for induction of IRE was 800 V/cm. The platinum electrode was resistant to corrosion. The IRE procedure for beagle prostates using a single bipolar electrode was technically feasible and safe. The novel bipolar electrode has great potential for treating human prostate cancer with fewer IRE-related complications.

Peculiarities of Neurostimulation by Intense Nanosecond Pulsed Electric Fields: How to Avoid Firing in Peripheral Nerve Fibers.

Intense pulsed electric fields (PEF) are a novel modality for the efficient and targeted ablation of tumors by electroporation. The major adverse side effects of PEF therapies are strong involuntary muscle contractions and pain. Nanosecond-range PEF (nsPEF) are less efficient at neurostimulation and can be employed to minimize such side effects. We quantified the impact of the electrode configuration, PEF strength (up to 20 kV/cm), repetition rate (up to 3 MHz), bi- and triphasic pulse shapes, and pulse duration (down to 10 ns) on eliciting compound action potentials (CAPs) in nerve fibers. The excitation thresholds for single unipolar but not bipolar stimuli followed the classic strength-duration dependence. The addition of the opposite polarity phase for nsPEF increased the excitation threshold, with symmetrical bipolar nsPEF being the least efficient. Stimulation by nsPEF bursts decreased the excitation threshold as a power function above a critical duty cycle of 0.1%. The threshold reduction was much weaker for symmetrical bipolar nsPEF. Supramaximal stimulation by high-rate nsPEF bursts elicited only a single CAP as long as the burst duration did not exceed the nerve refractory period. Such brief bursts of bipolar nsPEF could be the best choice to minimize neuromuscular stimulation in ablation therapies.

Feasibility and effectiveness of endoscopic irreversible electroporation for the upper gastrointestinal tract: an experimental animal study.

Irreversible electroporation (IRE) is a local non-thermal ablative technique currently used to treat solid tumors. Here, we investigated the clinical potency and safety of IRE with an endoscope in the upper gastrointestinal tract. Pigs were electroporated with recently designed endoscopic IRE catheters in the esophagus, stomach, and duodenum. Two successive strategies were introduced to optimize the electrical energy for the digestive tract. First, each organ was electroporated and the energy upscaled to confirm the upper limit energy inducing improper tissue results, including bleeding and perforation. Excluding the unacceptable energy from the first step, consecutive electroporations were performed with stepwise reductions in energy to identify the energy that damaged each layer. Inceptive research into inappropriate electrical intensity contributed to extensive hemorrhage and bowel perforation for each tissue above a certain energy threshold. However, experiments performed below the precluded energy accompanying hematoxylin and eosin staining and terminal deoxynucleotidyl transferase dUTP nick-end labeling assays showed that damaged mucosal area and depth significantly decreased with decreased energy. Relevant histopathology showed infiltration of inflammatory cells with pyknotic nuclei at the electroporated lesion. This investigation demonstrated the possibility of endoscopic IRE in mucosal dysplasia or early malignant tumors of the hollow viscus.

A wideband picosecond pulsed electric fields (psPEF) exposure system for the nanoporation of biological cells.

The effects and mechanisms of ultrashort and intense pulsed electric fields on biological cells remain some unknown. Especially for picosecond pulsed electric fields (psPEF) with a high pulse repetition rate, electroporation or nanoporation effects could be induced on cell membranes and intracellular organelle membranes. In this work, the design, implementation, and experimental validation of a wideband psPEF exposure system (WPES) is reported, comprising picosecond pulser and wideband biochip, for the in vitro exposure of suspended cells to high-intensity psPEF. Excited by repetitive picosecond pulses (the duration of 200 ps and the amplitude of a few kilovolts), the proposed biochip adopts grounded coplanar waveguide (GCPW) for a wide working bandwidth, which was fabricated with 160 µm thick electrodes for uniform distribution of psPEF in the cross-section. To ensure that only psPEF is generated in the biological medium containing cells except for ionic current, this work proposes to install capillary tubes in the electrode gaps for electrical insulation and cells delivery. By electrical measurements in the time domain and frequency domain, the exposure system is adapted for local generation of extremely high-intensity psPEF with the 3 dB bandwidth up to 4.2 GHz. Furthermore, biological experiments conducted on the developed exposure system verified its capability to permeabilize biological cells under the exposure of high-intensity psPEF.

In Vitro Cell Selectivity of Reversible and Irreversible: Electroporation in Cardiac Tissue.

[Figure: see text].

Accuracy of Electrode Placement in IRE Treatment with Navigated Guidance.

PURPOSE: Evaluate the accuracy of multiple electrode placements in IRE treatment of liver tumours using a stereotactic CT-based navigation system. METHOD: Analysing data from all IRE treatments of liver tumours at one institution until 31 December 2018. Comparing planned with validated electrode placement. Analysing lateral and angular errors and parallelism between electrode pairs RESULTS: Eighty-four tumours were treated in 60 patients. Forty-six per cent were hepatocellular carcinoma, and 36% were colorectal liver metastases. The tumours were located in all segments of the liver. Data were complete from 51 treatments. Two hundred and six electrodes and 336 electrode pairs were analysed. The median lateral and angular error, comparing planned and validated electrode placement, was 3.6 mm (range 0.2-13.6 mm) and 3.1° (range 0°-16.1°). All electrodes with a lateral error >10 mm were either re-positioned or excluded before treatment. The median angle between the electrode pairs was 3.8° (range 0.3°-17.2°). There were no electrode placement-related complications. CONCLUSION: The use of a stereotactic CT-based system for navigation of electrode placement in IRE treatment of liver tumours is safe, accurate and user friendly.

Episomal Reprogramming of Human Peripheral Blood Mononuclear Cells into Pluripotency.

Peripheral blood is an easily accessible cell resource for reprogramming into pluripotency by episomal vectors. Here, we describe an approach for efficient generation of integration-free induced pluripotent stem cells (iPSCs) under feeder or feeder-free conditions. Additionally, in combination with the CRISPR-Cas9 genome-editing system, we can directly generate edited iPSCs from blood cells. With this protocol, one can easily generate either integration-free iPSCs or genetically edited iPSCs from peripheral blood at high efficiency.

Mediated amperometry as a prospective method for the investigation of electroporation.

Pulsed electric field effects induced in a membrane, as well as intracellular structures, depend on cell type, field and media parameters. To achieve desired outcomes, membranes should be permeabilized in a controlled manner, and thus efficiency of electroporation should be investigated in advance. Here, we present a framework for using mediated amperometry as a prospective method for the investigation of electroporation and its effects on cellular machinery. Whole-cell sensors with single mediator systems comprised of hydrophilic or lipophilic mediators were successfully employed to investigate membrane permeability as well as cellular responses. Exposure of yeast cells to single electric field pulse (τ = 300 µs, E = 16 kV/cm) resulted in up to tenfold increase of current strength mediated with hydrophilic mediators. Exposure to PEF resulted in decrease of menadione mediated current strength (from 138 ± 15 to 32 ± 15 nA), which could be completely compensated by supplementing electrolyte with NADH.

Irreversible Electroporation for De-epithelialization of Murine Small Intestine.

BACKGROUND: Nonthermal irreversible electroporation (NTIRE) has been shown to ablate the small intestinal epithelium while maintaining submucosal and muscularis propriae integrity. NTIRE is used here in a first-in-mouse study to eliminate the native intestinal stem cell population to understand optimal parameters and timeline of mucosal regeneration. METHODS: Adult C57 background mice underwent laparotomy and electroporation of 1.5 cm of jejunum using a BTX 830 ECM electroporator and electrode calipers. Parameters were varied by voltage, pulse number, interval, and duration to determine optimal de-epithelialization. Electroporated segments were extracted 1 to 3 d after intervention with same-animal control segment. Cross sections were preserved, and measurements were taken to compare effects of parameters on villi height, crypt depth, crypt obliteration, and serosal thickness. RESULTS: Morbidity was limited at a standard set of electroporation parameters (14%), and increased with higher voltage, longer interval, and shorter or longer pulses. Serosa/muscularis thickness was unaffected by varying interventions. Crypt depth and obliterated crypts were most affected by modulating pulse number, intervals, and duration. Villi height was most significantly shortened by altering pulse duration, with limited recovery by day 3, otherwise mucosal regeneration was observed in most cases by this point. CONCLUSIONS: NTIRE is an effective method of denuding small intestinal epithelium in mice and temporarily ablating crypts while sparing the support scaffold for native regeneration. This first-in-mouse study of electroporation suggests it is a practical tool that can be utilized in a small mammalian system.

Transformation of Escherichia coli by Electroporation.

Preparing electrocompetent bacteria is considerably easier than preparing cells for transformation by chemical methods. Bacteria are simply grown to mid-log phase, chilled, centrifuged, washed extensively with ice-cold buffer or H2O to reduce the ionic strength of the cell suspension, and then suspended in an ice-cold buffer containing 10% glycerol. DNA may be introduced immediately into the bacteria by exposing them to a short high-voltage electrical discharge. Alternatively, the cell suspension may be snap-frozen and stored at -70°C for up to 6 mo before electroporation, without loss of transforming efficiency.

A Digital Controlled Pulse Generator for a Possible Tumor Therapy Combining Irreversible Electroporation With Nanosecond Pulse Stimulation.

The irreversible electroporation with microsecond electric pulses is a new ablation technique adopted in the tumor therapy worldwide. On the other hand, the nsPEF (nanosecond pulsed electric field) has been proved to provide a means to induce immunogenic cell death and elicits antitumor immunity, which is under intensive in-vitro and in-vivo studies and in clinical trials. Normally, one needs two different types of electric pulse generators for producing the pulses in the ranges of nanosecond and microsecond, respectively. In order to realize these two types of tumor treatments in complementary and optimize electrical pulse parameters, we have developed a compact high-voltage pulse generator with a wide pulse width tuning range, based on a capacitor discharging configuration digitally controlled by a silicon carbide MOSFET switching array through a pair of optic-coupler drivers. The developed digital pulse generator is capable of adjusting: pulse width over 100-100 µs, voltage over 0-2 kV and repetition rate up to 1.2 kHz. The pulse generator is designed in simulation, implemented and verified in experiments. The pulse generator is shown to deliver a complementary treatment on Murine melanoma B16 cell lines, i.e., triggering the cell early apoptosis under the 300 ns pulse stimulation while a complete killing under the 100 ns pulses. The pulse generator is further demonstrated to induce antitumor immunity in a preliminary in vivo study on the mice model.

Microfluidic Electroporation Coupling Pulses of Nanoseconds and Milliseconds to Facilitate Rapid Uptake and Enhanced Expression of DNA in Cell Therapy.

Standard electroporation with pulses in milliseconds has been used as an effective tool to deliver drugs or genetic probes into cells, while irreversible electroporation with nanosecond pulses is explored to alter intracellular activities for pulse-induced apoptosis. A combination treatment, long nanosecond pulses followed by standard millisecond pulses, is adopted in this work to help facilitate DNA plasmids to cross both cell plasma membrane and nuclear membrane quickly to promote the transgene expression level and kinetics in both adherent and suspension cells. Nanosecond pulses with 400-800 ns duration are found effective on disrupting nuclear membrane to advance nuclear delivery of plasmid DNA. The additional microfluidic operation further helps suppress the negative impacts such as Joule heating and gas bubble evolution from common nanosecond pulse treatment that lead to high toxicity and/or ineffective transfection. Having appropriate order and little delay between the two types of treatment with different pulse duration is critical to guarantee the effectiveness: 2 folds or higher transfection efficiency enhancement and rapid transgene expression kinetics of GFP plasmids at no compromise of cell viability. The implementation of this new electroporation approach may benefit many biology studies and clinical practice that needs efficient delivery of exogenous probes.

A low-cost smartphone controlled portable system with accurately confined on-chip 3D electrodes for flow-through cell electroporation.

Microscale flow-through electroporation at DC voltage has advantages in delivering small molecules. Yet, electroporation based on constant voltage are liable to generate electrolysis products which limits the voltage-operating window. Parallel on-chip 3D electrodes with close and uniform spacing are required to cut down voltage as well as provide enough electric field for electroporation. Here we present a simple electrode fabrication method based on capillary restriction valves in Z-axis to achieve parallel 3D electrodes with controllable electrode spacing in PDMS chips. With electrodes accurately placed in close range, a low voltage of only 1.5 V can generate enough electric field (>400 V/cm) to make cell membrane permeable. Squeeze flow is introduced to produce higher electric field (>800 V/cm) at a fixed voltage for more efficient electroporation. Benefit from the electrode fabrication method and application of squeeze flow, we develop a smartphone controlled microfluidic electroporation system which integrate functions of sample injection, pressure regulating, real-time observation and constant DC power supply. The system is used to electroporate two cell lines, showing a permeabilization percentage of 63% for HEK-293 cells and 58% for CHO-K1 cells with optimal parameters. Thus, the portable microfluidic system provides a cost-effective and user-friendly flow-through cell electroporation platform.

In vitro study on the mechanisms of action of electrolytic electroporation (E2).

Electrolytic Electroporation (E2) is the combination of reversible electroporation and electrolysis. It has been proposed as a novel treatment option to ablate tissue percutaneously. The present in vitro study in cells in suspension was performed to investigate the underlying mechanisms of action of E2. Different types of experiments were performed to isolate the effects of the electrolysis and the electroporation components of the treatment. Additionally, thermal simulations were performed to determine whether significant temperature increase contributes to the effect. The results indicate that E2's cell killing efficacy is due to a combinational effect of electrolysis and reversible electroporation that takes place within the first two minutes after E2 application. The results further show that cell death after E2 treatment is significantly delayed. These observations suggest that cell death is induced in permeabilized cells due to the uptake of electrolysis species. Thermal simulations revealed a significant but innocuous temperature increase.

The application of point source electroporation and chemotherapy for the treatment of glioma: a randomized controlled rat study.

The prognosis of Glioblastoma Multiforme patients is poor despite aggressive therapy. Reasons include poor chemotherapy penetration across the blood-brain barrier and tumor infiltration into surrounding tissues. Here we studied the effects of combined point-source electroporation (EP) and systemic chemotherapy in glioma-bearing rats. 128 rats were studied. Treatment groups were administered systemic Cisplatin/Methotrexate before EP (either 90 or 180 pulses). Control groups were treated by EP, chemotherapy, or no treatment. Tumor volumes were determined by MRI. Tumors growth rates of the EP + Methotrexate group (1.02 ± 0.77) were significantly lower (p < 0.01) than the control (5.2 ± 1.0) 1-week post treatment. No significant difference was found compared to Methotrexate (1.7 ± 0.5). Objective response rates (ORR) were 40% and 57% for the Methotrexate and EP + Methotrexate groups respectively. Tumor growth rates and ORR of the EP + Cisplatin groups (90 pulses 0.98 ± 0.2, 57%, 180 pulses 1.2 ± 0.1, 33%) were significantly smaller than the control (6.4 ± 1.0, p < 0.01, p < 0.02, 0%) and Cisplatin (3.9 ± 1.0, p < 0.04, p < 0.05, 13%) groups. No significant differences were found between the control groups. Increased survival was found in the EP + Cisplatin group, Χ2 = 7.54, p < 0.006 (Log Rank). Point-source EP with systemic chemotherapy is a rapid, minimal-invasive treatment that was found to induce significant antineoplastic effects in a rat glioma model.

CMOS microcavity arrays for single-cell electroporation and lysis.

Transfection is a key function for many single-cell analyses. Reversible electroporation (EP) using high intensity electric fields is a simple means of transfection applicable to most cell types. For reversible EP, precise control over the electric field is critical to regulate the induced pore densities in the membrane and maintain cell viability. Individually accessible microelectrode arrays enabled by semiconductor fabrication methods have emerged as a viable technology for single-cell analyses but do not provide for effective electroporation capabilities due to the planar arrangement of electrodes. Towards the goal of a fully integrated single-cell analysis platform, we utilize a commercial complementary metal-oxide-semiconductor (CMOS) process to realize microcavities which allow for single-cell confinement with integrated three-dimensionally aligned electrodes for effective poration. The structure is formed using the inherent metal stack available within the CMOS process as a hard etch mask for deep-reactive ion etching. Using this structure, to our knowledge, we present the first on-CMOS demonstration of controlled electroporation with the goal of transfection using human embryonic kidney cells (HEK-293) stained with Calcein as a model. We report an increase in calcein leaching from the cells subject to increasing electric field intensities with subsequent reuptake confirming cell viability post electroporation. These results are supported by numerical simulation of theoretical pore density which are in good agreement with numerical simulation. Combined with simple optical or electrical feedback, the structure is suitable for precise electroporation control in single-cells.

ElectroPen: An ultra-low-cost, electricity-free, portable electroporator.

Electroporation is a basic yet powerful method for delivering small molecules (RNA, DNA, drugs) across cell membranes by application of an electrical field. It is used for many diverse applications, from genetically engineering cells to drug- and DNA-based vaccine delivery. Despite this broad utility, the high cost of electroporators can keep this approach out of reach for many budget-conscious laboratories. To address this need, we develop a simple, inexpensive, and handheld electroporator inspired by and derived from a common household piezoelectric stove lighter. The proposed "ElectroPen" device can cost as little as 23 cents (US dollars) to manufacture, is portable (weighs 13 g and requires no electricity), can be easily fabricated using 3D printing, and delivers repeatable exponentially decaying pulses of about 2,000 V in 5 ms. We provide a proof-of-concept demonstration by genetically transforming plasmids into Escherichia coli cells, showing transformation efficiency comparable to commercial devices, but at a fraction of the cost. We also demonstrate the potential for rapid dissemination of this approach, with multiple research groups across the globe validating the ease of construction and functionality of our device, supporting the potential for democratization of science through frugal tools. Thus, the simplicity, accessibility, and affordability of our device holds potential for making modern synthetic biology accessible in high school, community, and resource-poor laboratories.