Valerie Mason

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 morein 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 forin vitro research and testing and is also being tested in clinical applications.

Skin in Complex Culture: The Transition from “Culture” Phenotype to Organotypic Phenotype

AbstractA model of human skin has been developed in vitro using epidermal keratinocytes, dermal fibroblasts, and type I collagen as starting materials. The bilayered model (Testskin, LSE) consists of a contracted collagen lattice populated with dermal fibroblasts overlaid with keratinocytes that form a multilayered epidermis at the air-liquid interface. Structural, kinetic, biochemical, and functional data suggest that during cultivation the construct undergoes a gradual transition from a “culture” phenotype to an organotypic pheno-type marked by changes in keratin content, cell kinetics, corneocyte shape and size, lipid biosynthesis, morphologic organization, and function. Timing of this transition and final functional characteristics can be manipulated somewhat by culture conditions. Metabolism and biological response are similar to in vivo response at this stage. However, the organotypic culture is as yet not completely developed with respect to a fully functional stratum corneum and continuous basemen...