A method for growing human epidermal cells in culture in the absence of dermal components has been modified in our laboratory to eliminate HLA-DR bearing Langerhans cells. These sheets have been used as allografts on burn patients. These cultured allografts increased the rate of healing of partial thickness burn wounds and did not cause a rejection reaction. A limitation of the procedure, however, is that only primary cultures are suitable for grafting so that a constant source of donor skin is required. Furthermore, current techniques require three weeks of incubation and coverage is needed in these burn patients prior to that time. In order to develop procedures for expanding epidermal cultures while maintaining their differentiated nature in vitro, it is necessary to characterize in detail the kinetics of growth and differentiation in these cultures. The ultimate aim is to produce cultured epidermal grafts which will be readily available to improve the healing of burn wounds. The experiments propose in this application are: I. To characterize the cell cycle kinetics of human keratinocytes grown according to our culture system. II. To assess the status of basal cells at various times during the lifespan in culture of human epidermal cells. III. To examine changes occurring in human keratinocytes as they differentiate in culture by looking for the presence of different molecular weight keratins and correlating their appearance with cell size, DNA content and RNA content. IV. To study the effect of cryopreservation of epidermal cell suspensions and sheets on the growth and differentiation of these cells. V. To examine the effect on growth and differentiation of cultured epidermal cells of various calcium ion concentrations, oxygen tensions and soluble factors elaborated by HLA-DR bearing Langerhans cells.
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