Matej Reberšek

Role of pulse shape in cell membrane electropermeabilization

The role of the amplitude, number, and duration of unipolar rectangular electric pulses in cell membrane electropermeabilization in vitro has been the subject of several studies. With respect to unipolar rectangular pulses, an improved efficiency has been reported for several modifications of the pulse shape: separate bipolar pulses, continuous bipolar waveforms, and sine-modulated pulses. In this paper, we present the results of a systematic study of the role of pulse shape in permeabilization, cell death, and molecular uptake. We have first compared the efficiency of 1-ms unipolar pulses with rise- and falltimes ranging from 2 to 100 micros, observing no statistically significant difference. We then compared the efficiency of triangular, sine, and rectangular bipolar pulses, and finally the efficiency of sine-modulated unipolar pulses with different percentages of modulation. We show that the results of these experiments can be explained on the basis of the time during which the pulse amplitude exceeds a certain critical value.

Equivalent Pulse Parameters for Electroporation

Electroporation-based applications require the use of specific pulse parameters for a successful outcome. When recommended values of pulse parameters cannot be set, similar outcomes can be obtained by using equivalent pulse parameters. We determined the relations between the amplitude and duration/number of pulses resulting in the same fraction of electroporated cells. Pulse duration was varied from 150 ns to 100 ms, and the number of pulses from 1 to 128. Fura 2-AM was used to determine electroporation of cells to Ca2+. With longer pulses or higher number of pulses, lower amplitudes are needed for the same fraction of electroporated cells. The expression derived from the model of electroporation could describe the measured data on the whole interval of pulse durations. In a narrower range (0.1-100 ms), less complex, logarithmic or power functions could be used instead. The relation between amplitude and number of pulses could best be described with a power function or an exponential function. We show that relatively simple two-parameter power or logarithmic functions are useful when equivalent pulse parameters for electroporation are sought. Such mathematical relations between pulse parameters can be important in planning of electroporation-based treatments, such as electrochemotherapy and nonthermal irreversible electroporation.

ELECTRO-MEDIATED GENE TRANSFER AND EXPRESSION ARE CONTROLLED BY THE LIFE-TIME OF DNA/MEMBRANE COMPLEX FORMATION

Electroporation is a physical method used to transfer molecules into cells and tissues. Clinical applications have been developed for antitumor drug delivery. Clinical trials of gene electrotransfer are under investigation. However, knowledge about how DNA enters cells is not complete. By contrast to small molecules that have direct access to the cytoplasm, DNA forms a long lived complex with the plasma membrane and is transferred into the cytoplasm with a considerable delay.

Effects of high voltage nanosecond electric pulses on eukaryotic cells (in vitro): A systematic review

For this systematic review, 203 published reports on effects of electroporation using nanosecond high-voltage electric pulses (nsEP) on eukaryotic cells (human, animal, plant) in vitro were analyzed. A field synopsis summarizes current published data in the field with respect to publication year, cell types, exposure configuration, and pulse duration. Published data were analyzed for effects observed in eight main target areas (plasma membrane, intracellular, apoptosis, calcium level and distribution, survival, nucleus, mitochondria, stress) and an additional 107 detailed outcomes. We statistically analyzed effects of nsEP with respect to three pulse duration groups: A: 1-10ns, B: 11-100ns and C: 101-999ns. The analysis confirmed that the plasma membrane is more affected with longer pulses than with short pulses, seen best in uptake of dye molecules after applying single pulses. Additionally, we have reviewed measurements of nsEP and evaluations of the electric fields to which cells were exposed in these reports, and we provide recommendations for assessing nanosecond pulsed electric field effects in electroporation studies.

Cell electrofusion using nanosecond electric pulses

Electrofusion is an efficient method for fusing cells using short-duration high-voltage electric pulses. However, electrofusion yields are very low when fusion partner cells differ considerably in their size, since the extent of electroporation (consequently membrane fusogenic state) with conventionally used microsecond pulses depends proportionally on the cell radius. We here propose a new and innovative approach to fuse cells with shorter, nanosecond (ns) pulses. Using numerical calculations we demonstrate that ns pulses can induce selective electroporation of the contact areas between cells (i.e. the target areas), regardless of the cell size. We then confirm experimentally on B16-F1 and CHO cell lines that electrofusion of cells with either equal or different size by using ns pulses is indeed feasible. Based on our results we expect that ns pulses can improve fusion yields in electrofusion of cells with different size, such as myeloma cells and B lymphocytes in hybridoma technology.

Skin electroporation for transdermal drug delivery: The influence of the order of different square wave electric pulses

Electroporation can be used as an active enhancement method for intra- and transdermal drug delivery. Differences in response of skin to electric pulses depend on their amplitude, duration and number and have been a point of interest in the past. While protocols consisting of the same repetitive, mostly exponentially decaying pulses have been used before, this study is focused on comparing different combinations of square wave short high voltage (HV) and longer low voltage (LV) electroporation pulses. Our in vitro experimental results show that longer LV pulses significantly increase subsequent passive transport of calcein through dermatomed pig skin, while short HV pulses alone result in negligible calcein passive transdermal transport. Surprisingly, when the long LV pulses are preceded by short duration HV pulses, the total calcein transported is reduced significantly. This result is explained using a theoretical physics based model of individual local transport region (LTR) evolution during the applied LV pulse. The theoretical model shows that HV pulses alter the structure of the stratum corneum in such a way that when the LV pulses are applied, insufficient thermal energy is generated to initiate LTR expansion. Together, the experimental results and theoretical predictions show that the total pulse energy alone cannot account for total solute transport: that the order of the types of pulses administered must also be considered. Our findings open a direction for further improvement of the method using new protocols.

Electroporator with automatic change of electric field direction improves gene electrotransfer in-vitro

BackgroundGene electrotransfer is a non-viral method used to transfer genes into living cells by means of high-voltage electric pulses. An exposure of a cell to an adequate amplitude and duration of electric pulses leads to a temporary increase of cell membrane permeability. This phenomenon, termed electroporation or electropermeabilization, allows various otherwise non-permeant molecules, including DNA, to cross the membrane and enter the cell. The aim of our research was to develop and test a new system and protocol that would improve gene electrotransfer by automatic change of electric field direction between electrical pulses.MethodsFor this aim we used electroporator (EP-GMS 7.1) and developed new electrodes. We used finite-elements method to calculate and evaluate the electric field homogeneity between these new electrodes. Quick practical test was performed on confluent cell culture, to confirm and demonstrate electric field distribution. Then we experimentally evaluated the effectiveness of the new system and protocols on CHO cells. Gene transfection and cell survival were evaluated for different electric field protocols.ResultsThe results of in-vitro gene electrotransfer experiments show that the fraction of transfected cells increases by changing the electric field direction between electrical pulses. The fluorescence intensity of transfected cells and cell survival does not depend on electric field protocol. Moreover, a new effect a shading effect was observed during our research. Namely, shading effect is observed during gene electrotransfer when cells are in clusters, where only cells facing negative electro-potential in clusters become transfected and other ones which are hidden behind these cells do not become transfected.ConclusionOn the basis of our results we can conclude that the new system can be used in in-vitro gene electrotransfer to improve cell transfection by changing electric field direction between electrical pulses, without affecting cell survival.

Cell membrane electroporation-Part 3: the equipment

As described in Part 1, a cell membrane can be made permeable to various molecules by carrying out a procedure called electroporation [1]. This procedure is being successfully used in biology, biotechnology, and medicine [2], [3]. It requires electroporators and electrodes. An electroporator generates short HV pulses of specific shape, amplitude, duration, number, and repetition frequency [4], and the pulses are applied to the target cells or load through the electrodes [5]. The energy delivered to the load is governed by the number of pulses and the pulse voltage, current, and duration. In biomedical applications that energy can be several joules; in biotechnology, where electroporation is used for treatment of agricultural products and water, it can be several kilojoules.

Blumlein Configuration for High-Repetition-Rate Pulse Generation of Variable Duration and Polarity Using Synchronized Switch Control

Blumlein generators are used in different applications such as radars, lasers, and also recently in various biomedical studies, where the effects of high-voltage nanosecond pulses on biological cells are evaluated. In these studies, it was demonstrated that by applying high-voltage nanosecond pulses to cells, plasma membrane and cell organelles are permeabilized. As suggested in a recent publication, the repetition rate and polarity of nanosecond high-voltage pulses could have an important effect on the electropermeabilization process, and consequently, on the observed phenomena. Therefore, we designed a new Blumlein configuration that enables a higher repetition rate of variable duration of either bipolar or unipolar high-voltage pulses. We achieved a maximal pulse repetition rate of 1.1 MHz. However, theoretically, this rate could be even higher. We labeled endocytotic vesicles with lucifer yellow and added propidium iodide to a cell suspension for testing the cell plasma membrane integrity, so we were able to observe the permeabilization of endocytotic vesicles and the cell plasma membrane at the same time. The new design of pulse generator was built, verified, and also tested in experiments. The resulting flexibility and variability allow further in vitro experiments to determine the importance of the pulse repetition rate and pulse polarity on membrane permeabilization - both of the cell plasma membrane as well as of cell organelle membranes.

Optimization of bulk cell electrofusion in vitro for production of human-mouse heterohybridoma cells.

Cell electrofusion is a phenomenon that occurs, when cells are in close contact and exposed to short high-voltage electric pulses. The consequence of exposure to pulses is transient and nonselective permeabilization of cell membranes. Cell electrofusion and permeabilization depend on the values of electric field parameters including amplitude, duration and number of electric pulses and direction of the electric field. In our study, we first investigated the influence of the direction of the electric field on cell fusion in two cell lines. In both cell lines, applications of pulses in two directions perpendicular to each other were the most successful. Cell electrofusion was finally used for production of human-mouse heterohybridoma cells with modified Koehler and Milstein hybridoma technology, which was not done previously. The results, obtained by cell electrofusion, are comparable to usually used polyethylene glycol mediated fusion on the same type of cells.