Experiments on reversible and irreversible cell electroporation were carried out with an experimental setup based on a standard apparatus for horizontal electrophoresis, a syringe pump with regulated cell suspension flow velocity and a dcEF power supply. Cells in suspension flowing through an orifice in a barrier inserted into the electrophoresis apparatus were exposed to defined localized dcEFs in the range of 0–1000 V/cm for a selected duration in the range 10–1000 ms. This method permitted the determination of the viability of irreversibly electroperforated cells. It also showed that the uptake by reversibly electroperforated cells of fluorescent dyes (calcein, carboxyfluorescein, Alexa Fluor 488 Phalloidin), which otherwise do not penetrate cell membranes, was dependent upon the dcEF strength and duration in any given single electrical field exposure. The method yields reproducible results, makes it easy to load large volumes of cell suspensions with membrane non-penetrating substances, and permits the elimination of irreversibly electroporated cells of diameter greater than desired. The results concur with and elaborate on those in earlier reports on cell electroporation in commercially available electroporators. They proved once more that the observed cell perforation does not depend upon the thermal effects of the electric current upon cells. In addition, the method eliminates many of the limitations of commercial electroporators and disposable electroporation chambers. It permits the optimization of conditions in which reversible and irreversible electroporation are separated. Over 90% of reversibly electroporated cells remain viable after one short (less than 400 ms) exposure to the localized dcEF. Experiments were conducted with the AT-2 cancer prostate cell line, human skin fibroblasts and human red blood cells, but they could be run with suspensions of any cell type. It is postulated that the described method could be useful for many purposes in biotechnology and biomedicine and could help optimize conditions for in vivo use of both reversible and irreversible electroporation.
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