Concepts and Capabilities of In-House Built Nanosecond Pulsed Electric Field (nsPEF) Generators for Electroporation: State of Art

Electroporation is a pulsed electric field triggered phenomenon of cell permeabilization, which is extensively used in biomedical and biotechnological context. There is a growing scientific demand for high-voltage and/or high-frequency pulse generators for electropermeabilization of cells (electroporators). In the scope of this article we have reviewed the basic topologies of nanosecond pulsed electric field (nsPEF) generators for electroporation and the parametric capabilities of various in-house built devices, which were introduced in the last two decades. Classification of more than 60 various nsPEF generators was performed and pulse forming characteristics (pulse shape, voltage, duration and repetition frequency) were listed and compared. Lastly, the trends in the development of the electroporation technology were discussed.This article belongs to the Special Issue Electroporation Systems and Application

High Frequency Bipolar Electroporator with Double-Crowbar Circuit for Load-Independent Forming of Nanosecond Pulses

In this work, a novel electroporation system (electroporator) is presented, which is capable of forming high frequency pulses in a broad range of parameters (65 ns–100 µs). The electroporator supports voltages up to 3 kV and currents up to 40 A and is based on H-bridge circuit topology. A synchronized double crowbar driving sequence is introduced to generate short nanosecond range pulses independently of the electroporator load. The resultant circuit generates pulses with repetition frequencies up to 5 MHz and supports unipolar, bipolar, and asymmetrical pulse sequences with arbitrary waveforms. The shortest pulse duration step is hardware limited to 33 ns. The electroporator was experimentally tested on the H69AR human lung cancer cell line using 20 kV/cm bipolar and unipolar 100 ns–1 μs pulses. Based on a YO-PRO-1 permeabilization assay, it was determined that the electroporator is suitable for applied research on electroporation. The system offers high flexibility in experimental design to trigger various electroporation-based phenomena

Dielectrophoretic Manipulation of Cell Transfection Efficiency during Electroporation Using a Center Needle Electrode

Long duration electric pulses are frequently used to facilitate DNA electrotransfer into cells and tissues, while electroporation pulses can be combined with electrophoresis to maximize the transfection efficiency. In this work, we present the dielectrophoresis (DEP)-assisted methodology for electrotransfer of plasmid DNA (3.5 kbp pmaxGFP) into mammalian cells (CHO-K1). A prototype of an electroporation cuvette with center needle electrode for DEP-assisted transfection is presented resulting in a 1.4-fold of transfection efficiency increase compared to the electroporation-only procedure (1.4 kV/cm × 100 µs × 8). The efficiency of transfection has been compared between three DEP frequencies of 1, 100, and 1 MHz. Lastly, the effects of exposure time (1, 3, and 5 min) during the DEP application step have been determined. It is concluded that the proposed methodology and exposure setup allow a significant improvement of transfection efficiency and could be used as an alternative to the currently popular electrotransfection techniques

Predicting electrotransfer in ultra-high frequency sub-microsecond square wave electric fields

Measurement of cell transmembrane potential (TMP) is a complex methodology involving patch-clamp methods or fluorescence-based potentiometric markers, which have limited to no applicability during ultrafast charging and relaxation phenomena. In such a case, analytical methods are applied for evaluation of the voltage potential changes in biological cells. In this work, the TMP-based electrotransfer mechanism during ultra-high frequency (>= 1 MHz) electric fields is studied and the phenomenon of rapid membrane charge accumulation, which is non-occurrent during conventional low-frequency electroporation is simulated using finite element method (FEM). The influence of extracellular medium conductivity (0.1, 1.5 S/m) and pulse rise/fall times (10-50 ns) TMP generation are presented. It is shown that the medium conductivity has a dramatic influence on the electroporation process in the high-frequency range of applied pulsed electric fields (PEF). The applied model allowed to grasp the differences in polarization between 100 and 900 ns PEF and enabled successful prediction of the experimental outcome of propidium iodide electrotransfer into CHO-K1 cells and the conductivity-dependent patterns of MHz range PEF-triggered electroporation were determined. The results of this study form recommendations for development and pre-evaluation of future PEF protocols and generators based on ultra-high frequency electroporation for anticancer and gene therapiesBiologijos katedraVilniaus Gedimino technikos universitetasVilnius Gediminas Technical University, LithuaniaVytauto Didžiojo universiteta