Abstract: Cultures of keratinocyte cells are provided which are free from nonautologous fibroblasts and organ extracts, and which have a high speed of cell amplification for a minimum seeding density. Preferably, a culture contains no more than about 10% autologous non-keratinocyte cells such as star-shaped, non-keratinocyte cells and no more than about 1% autologous fibroblasts. The cultures can be cryopreserved in a buffered isotonic medium containing serum and a cryoprotectant. The cultures are produced by a process that does not involve the use of a feeder layer and organ extracts. A culture medium used contains Medium 199, serum, epidermal growth factor, cholera toxin and/or hydrocortisone, and optionally insulin. A substance for wound healing and for cosmetic applications is derived from cultured human keratinocytes. Lyophilized keratinocyte cell cultures or an extract therefrom is used to provide a pharmaceutical composition. Confluent and cohesive keratinocyte sheets are prepared for use in wound healing.