MELANOCYTES IN CULTURED EPITHELIAL GRAFTS ARE DEPLETED WITH SERIAL SUBCULTIVATION AND CRYOPRESERVATION - IMPLICATIONS FOR CLINICAL OUTCOME

Patchy hypopigmentation often occurs unpredictably in the skin regener ated from cultured epidermal autografts, especially when that skin is grown from frozen cells, serially passaged, or both. The impact of ser ial subcultivation and cryopreservation on melanocyte viability in the cultured epidermal autograft culture system was investigated. Serial subcultivation of human keratinocytes through as many as eight passage s was performed, and melanocyte densities in confluent cultures at eac h passage were determined after specific labeling of melanocytes. The experimental cells were frozen before cultivation and between passages to determine the effect of standard cryopreservation on melanocyte su rvival. freshly passaged cells that had not been frozen served as cont rols. Melanocytes were gradually depleted during fresh passage of epid ermal cells but persisted through as many as seven passages. Freezing before or after the first passage or between subsequent passages resul ted in a complete loss of melanocytes by the third or fourth passage. The findings suggest that cryopreservation should be avoided during cu ltured epidermal autograft production to optimize melanocyte survival and minimize pigmentation abnormalities that may occur after grafting.