Reversible and irreversible electroporation of cell suspensions flowing through a localized DC electric field

Abstract

12 664 61 42Abbreviations used: DC -direct current; dcEF -direct current electric field; Eth BR 2 -ethidium bromide; FBS -fetal bovine serum; FDA -fluorescein diacetate; HSF -human skin fibroblasts; IRE -irreversible electroporation; PBS -buffered saline without or without calcium and magnesium ions; RBC -red blood cells; RE -reversible electroporation Research article REVERSIBLE AND IRREVERSIBLE ELECTROPORATION OF CELL SUSPENSIONS FLOWING THROUGH A LOCALIZED DC ELECTRIC FIELDWŁODZIMIERZ KOROHODA*, MACIEJ GRYS and ZBIGNIEW MADEJA* Department of Cell Biology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387, Cracow, Poland Abstract: 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.

Decreasing the thresholds for electroporation by sensitizing cells with local cationic anesthetics and substances that decrease the surface negative electric charge

“…We applied the recently described method of cell electroporation by flow of cell suspension through a localized electric field [10]. The experiments were carried out on a rat prostate cell line and human skin fibroblasts (HSF) using anesthetics at lower concentrations than commonly used for surgical anesthesia.…”

“…1A, 10 mM procaine HCl caused a decrease in the LD 50 dcEF from 784 to 426 V/cm. The two cell viability tests (the calcein [10,20,21] and FDA/ethidium bromide tests [18]) gave compatible results. We then studied the effects of the cationic local anesthetics tetracaine, procaine and lidocaine on cell viability using the calcein uptake test.…”

“…Cell electroporation was carried out with the setup described in [10], in a solution containing calcein, carboxyfluorescein or Lucifer yellow. The effectiveness of the method was established by scoring the number of fluorescent cells found 15-30 min following the procedure.…”

“…The effectiveness of the method was established by scoring the number of fluorescent cells found 15-30 min following the procedure. A fluorescent viability test using fluorescein diacetate (FDA) and ethidium bromide (EthBr 2 ) was carried out to determine the number of live cells [10,[16][17][18][19][20]. During the electroporation procedure, the electroporated cells remained in the electroporation medium with or without added substances for no more than one hour.…”

“…This approach was shown to facilitate the uptake of bleomycin and propidium iodide by the cells. We used the recently described the method of cell electroporation using a flow of cell suspension through a localized electric field [10] to examine the effects of substances known to modify cell membrane properties. This method allows the numbers of reversibly and irreversibly electroporated cells to be determined as a function of dcEF applied to the cells for a given period of time (time duration).…”