The aim of this study was to investigate the dependence of calcein extraction and cell viability on the parameters of pulsed electric field (PEF). Two different approaches concerning PEF parameters were investigated: (1) extraction efficiency and cell viability dependence on pulse number, exploiting 1200 V/cm 100 µs duration high voltage (HV) electric pulses and (2) extraction efficiency and cell viability dependence on the pulses with different duration (44-400 µs) and electric field strength (600-1800 V/cm) that result in the same amount of electric field energy delivered to Chinese hamster ovary cells. Extraction efficiency was evaluated as a percentage ratio of calcein fluorescence intensity prior and after PEF treatment. Cell viability was evaluated using PI test and cell clonogenic assay. Moreover, calcein release dynamics from cells after 600 V/cm 400 µs, 1200 V/cm 100 µs, and 1800 V/cm 44 µs was evaluated. Our results show that HV pulses induce instant calcein extraction due to reversible electroporation; however, subsequent calcein leakage over time was only observed when 9 HV pulses of 1800 V/cm 44 µs were used. The increased number of pulses resulted in more efficient total calcein extraction. With the same total energy delivered via electric pulses, the increase of calcein extraction efficiency was more dependent on pulse strength rather than pulse duration. The highest calcein extraction efficiency (84.5 ± 7.4%) was obtained using 9 electric field pulses of 1800 V/cm, 44 µs at 1 Hz. Furthermore, the extraction efficiency can be significantly enhanced if external mechanical stress (pipetting) is applied to cells. Cell viability was determined to be dependent on different PEF exposure parameters. It varied from 96.8 ± 4.8 to 31.2 ± 8.9%, implying the possibility to adjust PEF parameter combinations to maintain high cell viability.
Keywords: Calcein; Cell viability; Electroporation; Extraction; Flow cytometry; Pulsed electric field (PEF).