THE ORGANOTYPIC CULTURE OF HUMAN SKIN KERATINOCYTES AND FIBROBLASTS TO ACHIEVE FORM AND FUNCTION

We describe an organotypic model of human skin comprised of a stratified layer of human epidermal keratinocytes and dermal fibroblasts within a contracted collagen lattice. Feasible and reproducible production of the skin construct has required the use of traditional as well as specialized culture techniques. The configuration of the construct has been engineered to maintain polarity and permit extended culture at the air-liquid interface. Morphological, biochemical and kinetic parameters were assessed and functional assays were performed to determine the degree of similarity to human skin. Light and ultrastructural morphology of the epidermis closely resembled human skin. The immunocytochemical localization of a number of differentiation markers and extracellular matrix proteins was also similar to human skin. Kinetic data showed a transition of the epidermal layer to a more in vivo-like growth rate during the development of the construct at the air-liquid interface. The barrier properties of the construct also increased with time reaching a permeability to water of less than 2%-h after approximately 2 weeks at the air-liquid interface which is still on average 30-fold more water-permeable than normal human skin. The construct is currently used for in vitro research and testing and is also being tested in clinical applications.