Fetal Rat Keratinizing Epidermal Cells in Culture: Effects of Long-Term Treatment by Benzo[a]pyrene on Their Growth Characteristics23

Abstract

After serial 20-minute trypsinization, skin from near-term fetal MRC rats was separated into epidermis attached to basal lamina and dermis with trapped hair follicles. Fragmentation of the basal lamina by trypsinization caused the epidermal basal cell layer to form cell clumps of various sizes, whereas dermal mesenchymal cells dissociated into single cells, leaving a fibrous network with hair follicles. Clumps of epidermal basal cells were then isolated with few contaminating hair follicles. At 30–32.5° C, a cornification process caused the clumps to form thickened sheets of nearly pure keratinizing epidermal cells, which were isolated in petri dishes and grew to fill the dishes. The mode of cell differentiation in these sheets resembled an in vivo cornification process; the sheets showed maximum growth at 30° C and failed to survive at 35° C or above. Benzo[a]pyrene (BP) treatment accelerated the growth of cell sheets and enabled them to adapt to 35 and 37.5° C. Keratinizing epidermal cells treated with BP at 35 and 37.5° C were serially subcultured at these temperatures and lost their original dependence on lower temperatures for growth. The cell sheets were still capable of in vitro keratinization; however, some qualitative and quantitative changes were observed