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•We review four promising biotechnological applications of electroporation.•Reversible electroporation is an efficient method for genetic transformation of microorganisms.•Irreversible electroporation allows the inactivation of microbes and the extraction of biomolecules.•Water release due to electroporation increases the efficiency of biomass drying. Electroporation is already an established technique in several areas of medicine, but many of its biotechnological applications have only started to emerge; we review here some of the most promising. We outline electroporation as a phenomenon and then proceed to applications, first outlining the best established – the use of reversible electroporation for heritable genetic modification of microorganisms (electrotransformation), and then explore recent advances in applying electroporation for inactivation of microorganisms, extraction of biomolecules, and fast drying of biomass. Although these applications often aim to upscale to the industrial and/or clinical level, we also outline some important chip-scale applications of electroporation. We conclude our review with a discussion of the main challenges and future perspectives.

Several reports have recently been published on effects of very short and intense electric pulses on cellular organelles; in a number of cases, the cell plasma membrane appeared to be affected less than certain organelle membranes, whereas with longer and less intense pulses the opposite is the case. The effects are the consequence of the voltages induced on the membranes, and in this article we investigate the conditions under which the induced voltage on an organelle membrane could exceed its counterpart on the cell membrane. This would provide a possible explanation of the observed effects of very short pulses. Frequency-domain analysis yields an insight into the dependence of the voltage inducement on the electric and geometric parameters characterizing the cell and its vicinity. We show that at sufficiently high field frequencies, for a range of parameter values the voltage induced on the organelle membrane can indeed exceed the voltage induced on the cell membrane. Particularly, this can occur if the organelle interior is electrically more conductive than the cytosol, or if the organelle membrane has a lower dielectric permittivity than the cell membrane, and we discuss the plausibility of these conditions. Time-domain analysis is then used to determine the courses of the voltage induced on the membranes by pulses with risetimes and durations in the nanosecond range. The particularly high resting voltage in mitochondria, to which the induced voltage superimposes, could contribute to the explanation why these organelles are the primary target of many observed effects.

Proteins extracted from microalgae for food, personal care products and cosmetics must be of high purity, requiring solvent-free extraction techniques despite their generally considerably lower protein yield and higher energy consumption. Here, three such approaches for green extraction of proteins from Chlorella vulgaris were evaluated: ultrasound, freeze-thawing, and electroporation; chemical lysis was used as positive control (maximal achievable extraction), and no extraction treatment as negative control. Compared to chemical lysis, electroporation yielded the highest fraction of extracted protein mass in the supernatant (≤27%), ultrasound ≤24%, and freeze-thawing ≤15%. After a growth lag of several days, electroporated groups of algal cells started to exhibit growth dynamics similar to the negative control group, while no growth regeneration was detected in groups exposed to ultrasound, freeze-thawing, or chemical lysis. For electroporation as the most efficient and the only non-destructive among the considered solvent-free protein extraction techniques, simultaneous extraction of intracellular algal lipids into supernatant was then investigated by HPLC, proving relatively low-yield (≤7% of the total algal lipid mass), yet feasible for glycerides (tri-, di-, and mono-) as well as other fatty acid derivatives. Our results show that electroporation, though lower in extraction yields than chemical lysis or mechanical disintegration, is in contrast to them a technique for largely debris-free extraction of proteins from microalgae, with no need for prior concentration or drying, with feasible growth regeneration, and with potential for simultaneous extraction of intracellular algal lipids into the supernatant.

Some previous research showed that average daily exposure to extremely low frequency (ELF) magnetic fields (MF) of more than 0.3 or 0.4 μT could potentially increase risk of childhood leukaemia. To allow calculations of ELF MF around high voltage (HV) power lines (PL) for the whole Slovenia, a new three-dimensional method including precision terrain elevation data was developed to calculate the long-term average ELF MF. Data on population of Slovenian children and adolescents and on cancer patients with leukaemia's aged 0-19 years, brain tumours at age 0-29, and cancer in general at age 0-14 for a 12-year period 2005-2016 was obtained from the Slovenian Cancer Registry. According to the large-scale calculation for the whole country, only 0.5% of children and adolescents under the age of 19 in Slovenia lived in an area near HV PL with ELF MF density greater than 0.1 μT. The risk of cancer for children and adolescents living in areas with higher ELF MF was not significantly different from the risk of their peers. The new method enables relatively fast calculation of the value of low-frequency magnetic fields for arbitrary loads of the power distribution network, as the value of each source for arbitrary load is calculated by scaling the value for nominal load, which also enables significantly faster adjustment of calculated estimates in the power distribution network.

Electrochemotherapy and irreversible electroporation are gaining importance in clinical practice for the treatment of solid tumors. For successful treatment, it is extremely important that the coverage and exposure time of the treated tumor to the electric field are within the specified range. In order to ensure successful coverage of the entire target volume with sufficiently strong electric fields, numerical treatment planning has been proposed and its use has also been demonstrated in practice. Most of numerical models in treatment planning are based on charge conservation equation and are not able to provide time course of electric current, electrical conductivity, or electric field distribution changes established in the tissue during pulse delivery. Recently, a model based on inverse analysis of experimental data that delivers time course of tissue electroporation has been introduced. The aim of this study was to apply the previously reported time-dependent numerical model to a complex in vivo example of electroporation with different tissue types and with a long-term follow-up. The model, consisting of a tumor placed in the liver with 2 needle electrodes inserted in the center of the tumor and 4 around the tumor, was validated by comparison of measured and calculated time course of applied electric current. Results of simulations clearly indicated that proposed numerical model can successfully capture transient effects, such as evolution of electric current during each pulse, and effects of pulse frequency due to electroporation effects in the tissue. Additionally, the model can provide evolution of electric field amplitude and electrical conductivity in the tumor with consecutive pulse sequences.

An analytical description of transmembrane voltage induced on spherical cells was determined in the 1950s, and the tools for numerical assessment of transmembrane voltage induced on spheroidal cells were developed in the 1970s. However, it has often been claimed that an analytical description is unattainable for spheroidal cells, while others have asserted that even if attainable, it does not befit the reality due to the nonuniform membrane thickness, which is unrealistic but inevitable in spheroidal geometry. In this paper we show that for all spheroidal cells, membrane thickness is irrelevant to the induced transmembrane voltage under the assumption of a nonconductive membrane, which was also applied in the derivation of Schwan’s equation. We then derive the analytical description of transmembrane voltage induced on prolate and oblate spheroidal cells. The final result, which we cast from spheroidal into more familiar spherical coordinates, represents a generalization of Schwan’s equation to all spheroidal cells (of which spherical cells are a special case). The obtained expression is easy to apply, and we give a simple example of such application. We conclude the study by analyzing the variation of induced transmembrane voltage as a spheroidal cell is stretched by the field, performing one study at a constant membrane surface area, and another at a constant cell volume.

Growing diversity of protein-based technologies dictates further development of bio manufacturing to lower the cost of production and maximize yields. Intracellularly expressed recombinant proteins must be extracted from production host prior to purification. Use of electroporation to obtain proteins from bacteria and yeasts has been demonstrated in several studies for different modes of operation and formats. Here we tested various protocols for protein extraction from by means of electroporation. The tested protocols were compared to established extraction methods of ultrasonication and glass-bead milling in terms of protein yields and content of impurities such as host cell DNA and endotoxins in the lysate. Protein extraction yield was maximal when exponentially growing bacteria were treated at 37°C, regardless of the electroporation mode of operation (batch or flow). We were unable to eliminate co-extraction of host DNA and endotoxins, but with 8 × 1 ms, 5 kV/cm, 1 Hz pulses they were minimized. Yields with optimized electroporation (up to 86 g protein/kg dry weight) were inferior to those in ultrasonication (up to 144 g protein/kg dry weight) and glass-bead milling (up to 280 g protein/kg dry weight). Nevertheless, electroporation largely avoids cell lysis and disintegration with which the extract is a mix of extracted proteins with debris of the bacterial envelope and bacterial DNA, which necessitates further purification.

Treatment of cutaneous and subcutaneous tumors with electrochemotherapy has become a regular clinical method, while treatment of deep-seated tumors is still at an early stage of development. We present a method for preparing a dedicated patient-specific, computer-optimized treatment plan for electrochemotherapy of deep-seated tumors based on medical images. The treatment plan takes into account the patient's anatomy, tissue conductivity changes during electroporation and the constraints of the pulse generator. Analysis of the robustness of a treatment plan made with this method shows that the effectiveness of the treatment is not affected significantly by small single errors in electrode positioning. However, when many errors occur simultaneously, the resulting drop in effectiveness is larger, which means that it is necessary to be as accurate as possible in electrode positioning. The largest effect on treatment effectiveness stems from uncertainties in dielectric properties and electroporation thresholds of treated tumors and surrounding tissues, which emphasizes the need for more accurate measurements and more research. The presented methods for treatment planning and robustness analysis allow quantification of the treatment reproducibility and enable the setting of suitable safety margins to improve the likelihood of successful treatment of deep-seated tumors by electrochemotherapy.

Background Electrochemotherapy is a local treatment that utilizes electric pulses in order to achieve local increase in cytotoxicity of some anticancer drugs. The success of this treatment is highly dependent on parameters such as tissue electrical properties, applied voltages and spatial relations in placement of electrodes that are used to establish a cell-permeabilizing electric field in target tissue. Non-thermal irreversible electroporation techniques for ablation of tissue depend similarly on these parameters. In the treatment planning stage, if oversimplified approximations for evaluation of electric field are used, such as U/d (voltage-to-distance ratio), sufficient field strength may not be reached within the entire target (tumor) area, potentially resulting in treatment failure. Results In order to provide an aid in education of medical personnel performing electrochemotherapy and non-thermal irreversible electroporation for tissue ablation, assist in visualizing the electric field in needle electrode electroporation and the effects of changes in electrode placement, an application has been developed both as a desktop- and a web-based solution. It enables users to position up to twelve electrodes in a plane of adjustable dimensions representing a two-dimensional slice of tissue. By means of manipulation of electrode placement, i.e. repositioning, and the changes in electrical parameters, the users interact with the system and observe the resulting electrical field strength established by the inserted electrodes in real time. The field strength is calculated and visualized online and instantaneously reflects the desired changes, dramatically improving the user friendliness and educational value, especially compared to approaches utilizing general-purpose numerical modeling software, such as finite element modeling packages. Conclusion In this paper we outline the need and offer a solution in medical education in the field of electroporation-based treatments, e.g. primarily electrochemotherapy and non-thermal irreversible tissue ablation. We present the background, the means of implementation and the fully functional application, which is the first of its kind. While the initial feedback from students that have evaluated this application as part of an e-learning course is positive, a formal study is planned to thoroughly evaluate the current version and identify possible future improvements and modifications.

Exposure of a cell to an electric field results in inducement of a voltage across its membrane (induced transmembrane voltage, ΔΨ m) and, for sufficiently strong fields, in a transient increase of membrane permeability (electroporation). We review the analytical, numerical and experimental methods for determination of ΔΨ m and a method for monitoring of transmembrane transport. We then combine these methods to investigate the correlation between ΔΨ m and molecular transport through an electroporated membrane for isolated cells of regular and irregular shapes, for cells in dense suspensions as well as for cells in monolayer clusters. Our experiments on isolated cells of both regular and irregular shapes confirm the theoretical prediction that the highest absolute values of ΔΨ m are found in the membrane regions facing the electrodes and that electroporation-mediated transport is confined to these same regions. For cells in clusters, the location of transport regions implies that, at the field strengths sufficient for electroporation, the cells behave as electrically insulated (i.e., as individual) cells. In contrast, with substantially weaker, nonelectroporating fields, potentiometric measurements show that the cells in these same clusters behave as electrically interconnected cells (i.e., as one large cell). These results suggest that sufficiently high electric fields affect the intercellular pathways and thus alter the electric behavior of the cells with respect to their normal physiological state.