A NEW 3-DIMENSIONAL CULTURE OF HUMAN KERATINOCYTES - OPTIMIZATION OF DIFFERENTIATION

Many attempts have been made to obtain reconstructed human epidermis c omprised of keratinocytes and extracellular-matrix constituents (essen tially collagen) in the presence or absence of fibroblasts. A simple m odel of cultured human keratinocytes, grown at the air-liquid interfac e of a noncoated artificial membrane, has been developed. This culture system offers many advantages: easy control of environmental factors and routine examination using optical or electronic microscopy, immuno histochemistry and indirect immunofluorescence techniques. This model enables the analysis of well-known differentiation markers and also in tregrins, a family of cell-surface molecules involved in cell-cell and cell-extracellular matrix interactions, whose receptors are expressed on all basal keratinocytes. In our culture system, the expression of the different integrin subunits (alpha 2, alpha 3, alpha 5, alpha 6, b eta 1) was studied as a function of the differentiation state in two d ifferent media (K-SFM or DMEM/Ham's F12) supplemented with 5% fetal ca lf serum and adjusted to 1.5 mmol/L calcium. The most significant data are the preponderant expression of the alpha 2 and alpha 3 subunits i n the basal and suprabasal layers, with membrane expression differing according to the culture medium; terminal differentiation was obtained in DMEM/Ham's F12. The use of membrane inserts represents a significa nt technological advance in culturing keratinocytes and is an easy-to- handle and valid model for determining the influence of physiological or pharmacological factors on cell proliferation or differentiation.