Cornification and Basement Membrane Formation in a Bilayered Human Skin Equivalent Maintained at an Air-Liquid Interface

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Abstract
Human keratinocytes that were grown in a skin equivalent at an air-liquid interface were analyzed morphologically and biochemically to demonstrate differentiation approaching that of human skin. Within 3 weeks of growth at the interface, cuboidal basal cells, distinct spinous and granular zones, and a fully developed cornified layer of enucleated cells formed the multilayered epidermis. Ultrastructurally, the keratinocytes in the upper granular layer contain tonofilament bundles and membrane-coating granules. These cells form cornified squames that are resistant to degradation by sodium dodecyl sulfate/dithiothreitol. Basal cells are attached to a developing basement membrane with hemidesmosomes. Immunogold silver staining analysis with monoclonal antibodies demonstrated the expression of basement membrane collagens IV and VII. This level of differentiation might improve "take" of human grafts and provides a useful system with which to study topical carcinogens and tumor promoters in vitro.

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