We have found previously that human neonatal foreskin keratinocyte air/liquid (A/L) cultures developed normal-appearing epidermis, and that water flux was only two to three times higher than that found with intact human skin. To understand further the barrier properties of these A/L cultures, we have analyzed the composition and the gross conformational structures of the cultured stratum corneum (SC) lipids, and compared them with those of human SC. Electron microscopic examination demonstrated that cultured SC has high intracellular lipid content, but that it lacks both the basic unit repetition (e.g., broad/narrow/broad/broad/narrow/ broad pattern of electronlucent bands) normally found in human SC intercellular lipids and the covalently bound lipid envelope. These results indicate that the cultures are hyperproliferative, with characteristics similar to those found in SC obtained from patients with atopic dermatitis or psoriasis, or from animals whose diets lack essential fatty acids. In addition, the high free sterol content and the altered fatty acid/ceramide composition of these cultures argue that the compromised barrier function is linked to hyperproliferation and lipid synthesis, or vice versa. Infrared spectroscopic analysis of the SC lipid thermal transitions confirm that there are major conformational differences between the lipids of cultured and human SC, which clearly have an impact on permeability. The hyperproliferative state of growth and the profound differences between cultured and human SC in their lipid structural, compositional, and conformational properties together attest that water permeability alone is not a sufficiently sensitive marker of keratinocyte terminal differentiation for in vitro culture systems. It is not completely obvious how these differences in the cultured SC may influence the utility of this surrogate in studies involving skin biochemistry, pharmacology, permeability, metabolism, and toxicology. Nevertheless, it is clear that one needs to characterize the end product fully before one can judiciously interpret any results obtained from this or other similar skin surrogates.
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