Propidium Iodide electrotransfer dependence on cell shape in Monolayer In Vitro

Abstract

One of the most frequently used method for gene and drug delivery to the cells is electroporation. Cell transmembrane potential is increased when effected with electrical fields. If transmembrane potential reaches threshold (around 0.4 - 0.9 V) cell membrane temporal permeability to various molecules is increased. It is believed that cell membrane permeability increases as a result of induced hydrophilic pores on membrane. After induction of the pores on the membrane, various hydrophilic molecules diffuse and/or electrophoretically migrate through the hydrophilic pores in the membrane. This process is termed molecule electrotransfer. As there is no possibility to visualize electropores, the only way to study electroporation phenomenon is by using molecule like propidium iodide (PI) electrotransfer measurements. Once inside the cell PI intercalate to genetic material of the cell, resulting in significantly increased quantum efficiency of PI fluorescence. We used stated PI electrotransfer model to investigate the dependency of cell shape on electroporation. Here we have showed that cell shape has major impact to cell electroporation when induced transmembrane potential is at the electroporation threshold levels. Chinese hamster ovary (CHO) cell line was used as an object for PI electrotransfer in monolayer. Electroporation was performed with one (720 V/cm, 950 V/cm or 1400 V/cm at duration of 100 μs) pulse. Used PI concentration was 40 μg/ml. Cell electroporation was performed in electroporation medium (pH 7.1, conductivity 0.1 S/m, osmolarity 270 mOsm). Electrodes were placed under the inverted fluorescent microscope and all the electrotransfer process was imaged under fluorescent light starting from 3 seconds before electroporation, during electroporation and 80 s after electroporation every second. Taken images were processed with open access software ImageJ. [...]