Grys Maciej, Madeja Zbigniew, Korohoda Włodzimierz
Department of Cell Biology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Cracow, Poland.
Cell Mol Biol Lett. 2017 Jan 13;22:1. doi: 10.1186/s11658-016-0030-0. eCollection 2017.
The harmful side effects of electroporation to cells due to local changes in pH, the appearance of toxic electrode products, temperature increase, and the heterogeneity of the electric field acting on cells in the cuvettes used for electroporation were observed and discussed in several laboratories. If cells are subjected to weak electric fields for prolonged periods, for example in experiments on cell electrophoresis or galvanotaxis the same effects are seen. In these experiments investigators managed to reduce or eliminate the harmful side effects of electric current application.
For the experiments, disposable 20 μl cuvettes with two walls made of dialysis membranes were constructed and placed in a locally focused electric field at a considerable distance from the electrodes. Cuvettes were mounted into an apparatus for horizontal electrophoresis and the cells were subjected to direct current electric field (dcEF) pulses from a commercial pulse generator of exponentially declining pulses and from a custom-made generator of double and single rectangular pulses.
More than 80% of the electroporated cells survived the dcEF pulses in both systems. Side effects related to electrodes were eliminated in both the flow through the dcEF and in the disposable cuvettes placed in the focused dcEFs. With a disposable cuvette system, we also confirmed the sensitization of cells to a dcEF using procaine by observing the loading of AT2 cells with calceine and using a square pulse generator, applying 50 ms single rectangular pulses.
We suggest that the same methods of avoiding the side effects of electric current pulse application as in cell electrophoresis and galvanotaxis should also be used for electroporation. This conclusion was confirmed in our electroporation experiments performed in conditions assuring survival of over 80% of the electroporated cells. If the amplitude, duration, and shape of the dcEF pulse are known, then electroporation does not depend on the type of pulse generator. This knowledge of the characteristics of the pulse assures reproducibility of electroporation experiments using different equipment.
几个实验室观察并讨论了电穿孔过程中由于局部pH值变化、有毒电极产物的出现、温度升高以及作用于用于电穿孔的比色皿中细胞的电场不均匀性而对细胞产生的有害副作用。如果细胞长时间受到弱电场作用，例如在细胞电泳或趋电性实验中，也会出现同样的效应。在这些实验中，研究人员成功减少或消除了施加电流的有害副作用。
为进行实验，构建了具有由透析膜制成的两壁的一次性20微升比色皿，并将其置于距电极相当远的局部聚焦电场中。将比色皿安装到水平电泳装置中，细胞受到来自指数衰减脉冲的商用脉冲发生器以及定制的双矩形和单矩形脉冲发生器的直流电场（dcEF）脉冲作用。
在两个系统中，超过80%的电穿孔细胞在dcEF脉冲后存活。在直流电场中的流动以及置于聚焦直流电场中的一次性比色皿中，与电极相关的副作用均被消除。使用一次性比色皿系统，我们还通过观察钙黄绿素对AT2细胞的加载情况并使用方波脉冲发生器施加50毫秒的单矩形脉冲，证实了普鲁卡因使细胞对直流电场敏感。
我们建议，与细胞电泳和趋电性实验中避免电流脉冲施加副作用相同的方法也应应用于电穿孔。在确保超过80%的电穿孔细胞存活的条件下进行的电穿孔实验中证实了这一结论。如果已知直流电场脉冲的幅度、持续时间和形状，那么电穿孔不取决于脉冲发生器的类型。对脉冲特性的了解确保了使用不同设备进行电穿孔实验的可重复性。
