Investigation of ATP influence on cells viability after electroporation

Abstract

Using high-voltage (HV) short (ns, μs) electrical pulses induce alterations in the cells plasma membrane that facilitates nonselective molecular transfer into the cells. Such a phenomenon was called electroporation (EP). Using very high electric field pulses cause instant cell death by necrosis and was called irreversible electroporation (IrEP). IrEP has already demonstrated a remarkable effect on treating tumors by induction of immune response. However, it is still not well understood why and how cells after EP and IrEP die. Moreover, it was proved that molecules such as ATP, proteins either DNA can move out of the cells through the electroporated membrane. The loss of intracellular biomolecules is thought to be one of the reasons that the viability of electroporated cells decreases. The aim of this study was to find out whether compensating the loss of ATP with extracellular ATP can help to preserve cell viability after EP. For experiments, we used Chinese hamster ovary (CHO) cell line. Electroporation was performed by using 9 HV 1200 V/cm electric field pulses with a duration of 100 μs repeating at 1 Hz. ATP was weighed before each experiment to avoid its degradation. The final concentration of ATP used was 20 μM/ml. Cells were electroporated in the presence or absence of ATP and kept for different periods in different temperatures again in the presence or absence of ATP. The viability of cells was assayed 24 hours after the experiment by Flow cytometry and after 6 days by colony formation test. Results have revealed that addition of ATP before EP has slightly increased the viability of the cells suggesting that loss of energetic resources during the pulses has a low impact on cell death in the short term. However, keeping the cells in growth medium supplemented with ATP for additional 2 hours in an incubator (37° C, 5 % CO2) increased viability of the cells for almost 30 % compared to the viability of cells that were electroporated without ATP. [...]