Ward, R. K., P. N. Nation, M. Maxwell, C. L. Barker, and R. H. Clothier. Evaluation In Vitro of Epidermal Cell Keratinization. Toxic. in Vitro 1997. 11: 633-636. [Reprinted with permission from Elsevier Science]

1,25(OH)₂ vitamin D₃

Rhodamine B has been used as a histopathological stain for keratinization and cornification. Its ability as an in vitro indicator of the degree of epidermal keratinization was investigated in these preliminary studies. An immortalized human keratinocyte cell line, SVK-14, was evaluated as an alternatives to primary human keratinocytes. The influence of extracellular calcium levels was evaluated alongside the effect of exposure to 1,25 (OH)₂ vitamin D₃ in serum-free and serum-containing media. Alamar blue (AB) conversion was used to measure changes in cellular reductive potential, and the amount of bound Rhodamine B relative to total protein per well was taken as an indicator of keratinization. Exposure to 1,25 (OH)₂ vitamin D₃ for 7 or 10 days did not increase Rhodamine B binding to confluent SVK-14 cultures, regardless of calcium concentration. Variation in Rhodamine B dye-binding to cells made it difficult to interpret the data. In addition, concern regarding the ability of SVK-14 cells to differentiate suggests that further studies need to be performed using normal human keratinocytes to validate this in vitro endpoint, with epidermal growth factor, insulin and hydrocortisone removed from the media to enhance epidermal differentiation.