Approaches to enhance proliferation of human epidermal keratinocytes in mass culture

Because of interest in mechanisms of carcinogenesis in human epithelial cells, quantitative procedures were developed for the mass culture of human epidermal keratinocytes in the absence of feeder cells. Several approaches were used to enhance proliferation since target cells are considered most susceptible to transformation if they are treated with carcinogenic agents during DNA synthesis. Mass cultures of enzymatically dispersed human foreskin were initiated in collagen-coated flasks containing medium NCTC 168 with 10% Chelex 100-treated horse serum. Under these conditions, human keratinocytes required a higher calcium ion concentration ([Ca2+]) than that reported for suspensions plated at low cell density. Neither the cohesiveness of the epidermal sheet nor continued proliferation was maintained by 0.15 mM Ca2+; 0.3 mM Ca2+ maintained these properties in primary culture only. A concentration of 1.0 mM Ca2+ provided the highest cell yield for prolonged growth as determined by the enumeration of cell nuclei isolated by citric acid. Reproducibility of successful initiation was achieved by inoculation of cells into a medium designed for clonal growth followed by culture in medium NCTC 168. Thus the balance of nutrients and electrolytes must be adjusted to satisfy the requirements of a dynamically expanding keratinocyte population.