EVALUATION IN-VITRO OF EPIDERMAL-CELL KERATINIZATION

Rhodamine B has been used as a histopathological stain for keratinizat ion and cornification. Its ability as an in vitro indicator of the deg ree of epidermal keratinization was investigated in these preliminary studies. An immortalized human keratinocyte cell line, SVK-14, was eva luated as an alternative to primary human keratinocytes. The influence of extracellular calcium levels was evaluated alongside the effects o f exposure to 1,25 (OH)(2) vitamin D-3 in serum-free and serum-contain ing media. Alamar blue (AB) conversion was used to measure changes in cellular reductive potential, and the amount of bound Rhodamine B rela tive to total protein per well was taken as an indicator of keratiniza tion. Exposure to 1,25 (OH)(2) vitamin D-3 for 7 or 10 days did not in crease 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 regardin g the ability of SVK-14 cells to differentiate suggests that further s tudies need to be performed using normal human keratinocytes to valida te this in vitro endpoint, with epidermal growth factor, insulin and h ydrocortisone removed from the media to enhance epidermal differentiat ion. (C) 1997 Elsevier Science Ltd.