Challenges in the standardization of in vitro cytotoxicity assays for comparative risk assessment of cold atmospheric pressure plasma devices

Cold atmospheric pressure plasma (CAP) has emerged as a promising therapeutic modality in wound healing, with multiple devices now certified for clinical use. However, the constructive and functional diversity of CAP technologies poses significant challenges for cross-device comparison in preclinical in vitro studies. In this study, we evaluated how device-specific parameters and experimental conditions influence cytotoxic outcomes across different CAP technologies. Using L929, GM00637, and HaCaT cells, we compared direct treatment with a plasma jet (kINPen^® MED) and a surface micro-discharge device (plasma care^®), revealing significant differences in the reduction of metabolic activity under otherwise identical conditions. Assessment of treatment geometry-specifically the radius of circular motion of the plasma jet-significantly affects metabolic activity, even at identical exposure times. To standardize conditions across devices, we further investigated an indirect treatment approach using a metal grid to generate plasma-conditioned PBS. However, we found a non-linear relationship between liquid volume, treatment time, and biological outcome. Moreover, indirect treatment excludes short-lived reactive species and non-chemical plasma components, limiting its biological relevance. Our findings demonstrate that neither direct nor indirect treatment protocols reliably enable cross-device comparisons in vitro. We therefore advocate for transparent, comprehensive reporting of all device and experimental variables, rather than pursuing a single standardized protocol. This enables meaningful data integration and cross-study comparisons, even when protocols differ.