Immediate and Delayed Side Effects of Irreversible Electroporation on Epidermal Keratinocytes

Irreversible electroporation (IRE) has been explored significantly for health care applications, especially cancer therapy and wound healing. It is associated with massive cell death via the bursting of cells caused by the creation of nanopores due to the rapid alteration of a voltage gradient across the cell membrane. It is reported to disinfect chronic wounds and burn injuries, minimize scar tissue formation, and regenerate accessory organs like hair follicles. However, several theories surround this therapy’s efficacy, such as the activation of immune pathways via paracrine secretion. The current study used a keratinocyte cell line (HaCaT) monolayer as an in vitro model to explore the significant immediate side effects of applying IRE. IRE was applied to cellular monolayers via needle electrodes as bursts of pulsed electric field (PEF). We observed via microscopic image analysis that there were input voltage-dependent alterations in cellular monolayer morphology visible in both phase contrast and using fluorescent live/dead staining. Similar changes were observed in reactive oxygen species-specific staining, mitochondrial membrane potential-specific staining, and cell membrane lipid peroxidation-specific staining. All the micrographic imaging showed that the affected area was highly correlated with the input voltage. Furthermore, a multiphysics simulation of the IRE applied to the cellular monolayer for an individual pulse was performed to characterize the electric field intensity spatial distribution. It exhibited similarities with the morphological alteration profiles in the cellular monolayer post-PEF application. We also found multinucleated or fused cells outside the IRE-induced immediate death zone, which could also be another side effect of this exposure.