Nancy L. Parenteau

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.

Serial cultivation of normal human keratinocytes: A defined system for studying the regulation of growth and differentiation

SummaryWe have developed a defined method for human epidermal keratinocyte culture. The minimally supplemented basal medium supported establishment of primary cultures from neonatal foreskin in a defined environment. It also supported serial cultivation and rapid expansion of cell number. Casein replaced serum for defined cryopreservation. Cells were serially cultivated in medium containing 0.08 mM calcium. The rate of cell division however remained high after addition of 1.8 mM calcium. The particulate transglutaminase activity of the cultures was low at confluence, even in the presence of 1.88 mM calcium, indicating an enrichment of the basal cell population. Culture with small amounts (0.3%) of chelated serum increased particulate transglutaminase activity approximately 2.2-fold in low calcium cultures and approximately 3.5-fold in high calcium cultures. A gradual reduction in growth rate of serum-treated cultures upon serial cultivation also indicated a depletion of cells with basal cell character. Bovine hypothalamic extract and cholera toxin were able to avert, in part, the differentiation-promoting effects of serum. Keratinocytes serially cultivated in the defined medium maintained the ability to develop normally into a morphologically differentiated epidermis.

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...

Progenitor Cell Properties and the Engineering of Tissues

The need for human tissue to aid in organ repair or provide a curative therapy is well known. In this review, we discuss the properties of the epidermal keratinocyte progenitor cell and the biology that underlies the methods that have helped deliver cell therapies to the clinic using this cell type. In addition, we review what the keratinocyte and the dermal fibroblast have taught us about the potential immunogenicity of allogeneic cells. The many observations made using the keratinocyte have broader biological implications and we discuss how this body of work parallels neural stem cell culture and might help us interpret cell behavior in the pancreas.