Characterization of human gingival keratinocytes cultured in a serum-free medium

Primary cultures of keratinocytes were established from gingival tissue explanted on the surface of type I collagen gels and fed a serum-containing medium. Cells could be routinely subcultured for at least five passages in a basal nutrient medium (MCDB 153) containing low calcium (0.1 mM), and supplemented with ethanolamine, phosphoethanolamine, hydrocortisone, insulin, epidermal growth factor and protein of bovine pituitary extract. Cells seeded at low densities doubled exponentially in number every 24-30 h and formed a confluent monolayer within 10-14 days. Phase-contrast light and transmission electron microscopy showed that the keratinocyte cultures had features typical of epithelial cells, including desmosomes and perinuclear tonofilament bundles. Immunofluorescent microscopy showed the presence of specific keratin proteins in basal cells of proliferating cultures. Gel electrophoresis of the insoluble cytosolic proteins of gingival and skin keratinocytes showed several differences. Suspension of dividing gingival keratinocytes in 1.3% methylcellulose medium induced greater than 50% cross-linked envelopes, suggesting the existence of a terminal differentiation pathway in gingival basal cells. Clonal growth experiments showed that both insulin and epidermal growth factor were required for optimal clonal growth. The growth of subcultures was arrested and the unstratified epithelial monolayer induced to form a stratified sheet by replacing the growth medium with basal MCDB 153 medium depleted of growth factors and containing 2 mM calcium. Sheets of stratified gingival epithelium formed on and later released from the dish by enzymatic treatment may be suitable for a variety of experimental and clinical uses.