Generation of Human Epidermal Constructs on a Collagen Layer Alone

Because engineered tissues are designed for clinical applications in humans, a major problem is the contamination
of cocultures and tissues by allogenic molecules used to grow stem cells in vitro. The protocols
that are commonly applied to generate epidermal equivalents in vitro require the use of irradiated murine
fibroblasts as a feeder layer for keratinocytes. In this study, we report a simple procedure for growing
human keratinocytes, isolated from adult skin, to generate an epidermal construct on a collagen layer alone.
In this model, no human or murine feeder layers were used to amplify cell growth, and isolated keratinocytes
were seeded directly at high cell density on the collagen-coated flasks or coverslips in an epithelial
growth medium containing low calcium concentration.
Morphological, immunochemical, and cytokinetic features of epithelial colonies grown on the collagen
layer were typical of keratinocytes and were comparable with those reported for keratinocytes grown on
a feeder layer. The stratification of keratinocytes generated 3-dimensional synthetic constructs displaying
a tissue architecture comparable with that of natural epidermis. Epithelial cells expressed specific markers
of keratinocyte terminal differentiation, including involucrin and filaggrin. Nevertheless, the number of cell
layers was lower than in natural skin, and electron microscopical analysis revealed that the overall organization
of these layers was poor compared with natural epidermis, including the formation of junctional
complexes, basement membrane, and keratinization. The lack of epithelial–mesenchymal interactions that
occur during skin histogenesis may account for such an incomplete maturation of epidermal constructs.