Insulin and hydrocortisone influences on cultured rat tongue epithelium

Abstract

Rat tongue epithelium was separated from underlying connective tissue by trypsinization, dispersed into single cells and small clusters and plated on cellulose acetate-nitrate filters, collagen gels or plastic substrate in serum-supplemented media. Morphologically, abundance of growth and stratification of epithelium was greatest on floating collagen gels, intermediate on plastic and least on the filters. Cultures on the plastic substrate were used to test the effects of insulin, hydrocortisone, dimethyl sulphoxide, glucose and soybean trypsin inhibitor upon epithelial outgrowth. Cultures were plated at the same density, grown to partial con-fluency, and analysed with a superimposed point lattice to determine the fraction of total subsurface covered. Cells cultured 1.25 − 5 × 10 5 cells/cm 2 in serum-supplemented αMEM or McCoy's 5A media produced the best outgrowth. Additional insulin supplementation at 40 μg/ml gave better outgrowth than 400 or 4 μg/ml within the first week. Insulin at 40 μg/ml also gave better outgrowth than a mixed insulin-hydrocortisone supplement at three different levels or than hydrocortisone supplement alone at three different levels. When 0.5 per cent DMSO was added to any of these supplementary formulas, epithelial outgrowth was reduced compared with the same formulae without DMSO. Addition of three times the formula level of glucose also reduced epithelial outgrowth. Addition of soybean trypsin inhibitor to αMEM supplemented with insulin, but not fetal calf serum, caused increased epithelial outgrowth. These findings helped define appropriate conditions for cell attachment, migration, proliferation and differentiation of primary rat tongue epithelial cultures.