[Methodologic aspects in cultivating human keratinocytes]

The cultivation of human keratinocytes is an appropriate experimental model for a variety of cell biological, pharmacological and biochemical investigations and the production of in vitro epidermis suitable for grafting since Rheinwald and Green (1975) have established this method. A procedure is described to isolate and culture human keratinocytes using different culture conditions. About 1-2 million epidermal cells could be prepared by trypsinization over night at 4 degrees C from 1-2 cm2 skin. Primary cell cultures seeded on mitomycin C treated NIH 3T3 fibroblasts reached subconfluence after 15-20 days when Eagle MEM was used as culture medium and after 10-15 days of culture using a mixture of Dulbecco's MEM and Ham F12 (3:1). In each case the media were supplemented with different growth factors. Subcultures were seeded on NIN 3T3 feeder cells as well as in collagen or fibronectin coated dish. Multilayered epidermal sheets developed with in 12-15 days of culture.