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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机译：可逆和不可逆细胞电穿孔的实验是通过基于水平电泳标准设备，调节细胞悬浮液流速的注射泵和dcEF电源的实验装置进行的。将悬液中的细胞流过插入到电泳仪中的屏障中的孔口，使其暴露在0-1000 V / cm范围内的定义的局部dcEF中，持续10-1000 ms的选定持续时间。这种方法可以确定不可逆电穿孔细胞的活力。它还表明，可逆电穿孔细胞对荧光染料（钙黄绿素，羧基荧光素，Alexa Fluor 488 Phalloidin）的摄取，否则它们不会穿透细胞膜，取决于在任何给定的单一电场暴露下的dcEF强度和持续时间。该方法产生可重现的结果，易于将大量非膜穿透性物质装载到大量细胞悬液中，并允许消除直径大于所需直径的不可逆电穿孔细胞。该结果与市售电穿孔器中有关细胞电穿孔的较早报告中的结果一致并详述。他们再次证明观察到的细胞穿孔不取决于电流对细胞的热效应。另外，该方法消除了商业电穿孔器和一次性电穿孔室的许多限制。它可以优化分离可逆电穿孔和不可逆电穿孔的条件。短时间（少于400毫秒）暴露于局部dcEF后，超过90％的可逆电穿孔细胞仍保持活力。实验是对AT-2癌症前列腺细胞系，人皮肤成纤维细胞和人红细胞进行的，但它们可以与任何细胞类型的悬浮液一起运行。假定所描述的方法可用于生物技术和生物医学中的许多目的，并且可帮助优化可逆和不可逆电穿孔在体内使用的条件。
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