CHARACTERIZATION OF GROWTH AND DIFFERENTIATION OF NORMAL HUMAN SUBMANDIBULAR-GLAND EPITHELIAL-CELLS IN A SERUM-FREE MEDIUM

Parenchymal tissue of human submandibular glands was cultured in a ser um-free medium consisting of a 1:9 mixture of Dulbecco's modified Eagl e's medium and MCDB 153 supplemented with 10 ng/ml epidermal growth fa ctor, 10 muM dexamethasone and 1 mug/ml insulin. Cultivation of the ti ssue in this medium resulted in propagation of loosely arranged epithe lioid cells on plastic, without the necessity of a matrix. Epidermal g rowth factor significantly enhanced mitogenesis of cultured cells, whi ch expressed specific high- and low-affinity receptors for epidermal g rowth factor. The epithelioid cells were found to represent the undiff erentiated ultrastructure of ductal cells. Immunocytochemically, cultu red epithelioid cells expressed antigens specific to basal cells of th e intra- and interlobular ducts in situ, including cytokeratins 3 and 6 and cytokeratins 13 and 16, vimentin, and alpha-smooth muscle actin. Moreover, cytoplasm of the cells was immunostained using antibody aga inst the basement membrane component, type IV collagen. These results suggested that cultured epithelioid cells are undifferentiated ductal cells, which have the characteristics of basal cells of the intra- and /or interlobular ducts. Cultured epithelioid cells maintained the char acteristics for serial passage until the time that the cultures were c onfluent. On the other hand, several stratified foci developed on the confluent monolayer. The stratified cells were strongly positive for c ytokeratins 3 and 6, but negative for vimentin, alpha-smooth muscle ac tin and type IV collagen. Moreover, the stratified cells were strongly stained with the antibody against epithelial membrane antigen. This a ntibody stained the luminal membrane domain of salivary epithelial cel ls. Electron micrograph of the vertical section through the foci revea led stratified cell layers with a gradual transition from basal cells to squamous epidermoid cells. This result suggests that cultured epith elioid cells, which have the characteristics of basal cells of the int ra- and/or interlobular duct, have the potential to differentiate into luminal duct cells.