Primary culture of human oral epithelial cells. Growth requirements and expression of differentiated characteristics

Abstract

Epithelial cell cultures were initiated from explants of normal human oral mucosa. Growth parameters, cell type, and degree of maturation/cytodifferentiation were assessed by morphological and surface topographical criteria (light and scanning electron microscopy) together with immunofluorescence studies with a panel of antibodies to cytokeratins and extracellular matrix components. The effects of different media formulations were compared. Whereas stromal cell over-growth soon became apparent in media containing 10% serum, in low serum (0.5%) media containing insulin, hydrocortisone, epidermal growth factor (EGF), and/or cholera toxin (CT), epithelial growth was maintained with minimal or absent stromal cell contamination. Cell proliferation, maturation, and differentiation were modulated by EGF and CT: cultures maintained on EGF showed optimal growth but cells typically displayed only limited differentiation. By contrast, CT promoted considerably more cytodifferentiation but at the expense of proliferative capacity. Both factors together were complementary, resulting in maintenance of cells of a more mature phenotype of high proliferative capacity. Cytokeratins of normal oral epithelium in situ demonstrated characteristic changes in patterns of expression associated with differentiation. In culture, proliferative epithelial cells expressed keratins typical of the basal layer, whereas the most differentiated cells were identified by their strong reactivity with antibodies to epidermal keratins. Less mature cells showed expression of keratins associated with nonstratified epithelia. In cultures maintained with CT but no EGF, there was a tendency for weaker expression of basal type keratins, further suggesting that these cells were maintaining a more differentiated phenotype. Extracellular matrix components (fibronectin, laminin, collagen type IV) were not expressed by any epithelial cells in culture. Irrespective of medium composition, cultures did not survive beyond 100 days (5 or 6 subcultivations) before undergoing an irreversible 'crisis' of growth arrest and onset of degenerative changes.