Prednosti i nedostaci različitih pristupa generiranja impulsa za elektroporaciju

Electroporator is a generator of electric pulses that is used for permeabilization of cells. There are five major concepts of electroporation design. Capacitor discharge, square wave generator, and analog generator are used to generate classical electroporation pulses that are longer than microsecond and pulse forming network, and resonant charging generator that are used to generate nanosecond electroporation pulses. The choice of an electroporator design is always driven by the biotechnological or biomedical application. Electroporators can be used for introduction of small (electrochemotherapy) and large molecules (gene electrotransfer), cell fusion, insertion of proteins into cell membrane, electroporation of organelles, pasteurization, tissue ablation etc. Basic concepts and foreseeable future developments in electroporator design are presented in this article.Elektroporator je generator impulsa koji se koristi za permeabilizaciju stanica. Postoji pet glavnih izvedbi elektroporatora. Pražnjenje kondenzatora, generator pravokutnog valnog oblika i analogni generator se koriste za klasične elektroporacijske impulse koji su duži od mikrosekunde, a mreža za formiranje impulsa i generator s rezonantnim nabijanjem se primjenjuju za generiranje nanosekundnih elektroporacijskih impulsa. Izbor izvedebe elektroporatora vođen je uvijek biotehnološkom ili biomedicinskom primjenom. Elektroporatori se mogu koristiti za ubacivanje malih (elektrokemoterapija) i velikih molekula (elektro genski prijenos), fuziju stanica, umetanje proteina u staničnu membranu, elektroporaciju organela, pasterizaciju, ablaciju tkiva itd. U radu su prikazani temeljni pristupi u izvedbama elektroporatora i predvidivi budući razvoj

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: 1–10 ns, B: 11–100 ns and C: 101–999 ns. 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

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

<p>Abstract</p> <p>Background</p> <p>Gene 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.</p> <p>Methods</p> <p>For 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.</p> <p>Results</p> <p>The results of <it>in-vitro </it>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.</p> <p>Conclusion</p> <p>On the basis of our results we can conclude that the new system can be used in <it>in-vitro </it>gene electrotransfer to improve cell transfection by changing electric field direction between electrical pulses, without affecting cell survival.</p

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Electropermeabilization of endocytotic vesicles in B16 F1 mouse melanoma cells

It has been reported previously that electric pulses of sufficiently high voltage and short duration can permeabilize the membranes of various organelles inside living cells. In this article, we describe electropermeabilization of endocytotic vesicles in B16 F1 mouse melanoma cells. The cells were exposed to short, high-voltage electric pulses (from 1 to 20 pulses, 60 ns, 50 kV/cm, repetition frequency 1 kHz). We observed that 10 and 20 such pulses induced permeabilization of membranes of endocytotic vesicles, detected by release of lucifer yellow from the vesicles into the cytosol. Simultaneously, we detected uptake of propidium iodide through plasma membrane in the same cells. With higher number of pulses permeabilization of the membranes of endocytotic vesicles by pulses of given parameters is accompanied by permeabilization of plasma membrane. However, with lower number of pulses only permeabilization of the plasma membrane was detected