ABSTRACT
Various culture media are used to propagate keratinocytes (KCs) in vitro. The COVID-19 pandemic resulted in supply chain shortages necessitating substitutions to standard laboratory protocols, which resulted in many laboratories having to use culture media different from those they typically use. We screened available media on the KC line N/TERT2G and found that biological responses varied considerably across three culture media: KC serum-free media, KC growth medium 2, and defined media. We observed qualitative and quantitative differences in proliferation; KCs cultured in defined media had significantly lower proliferative capacity. KC differentiation was assessed by western blot for CLDN1, occludin, cytokeratin-10, and loricrin. Elevated expression of differentiation markers was observed in cells cultured in either KC growth medium 2 or defined media compared with those in cells cultured in KC serum-free media. KC barrier function was measured by transepithelial electrical resistance. KCs cultured in KC growth medium 2 and defined media developed significantly higher transepithelial electrical resistance than those cultured in KC serum-free media, and when treated with IL-4 and IL-13 or IL-17A, we observed variable responses. H&E staining on day 5 -post-differentiation showed greater epithelial thickness in KCs cultured in defined media and KC growth medium 2 than in those cultured in KC serum-free media. These findings show that the choice of culture media impacts the biological response of KCs in a manner that persists through differentiation in the same media.