Mouse submandibular salivary epithelial cell growth and differentiation in long-term culture: influence of the extracellular matrix.
The adult mouse submandibular salivary gland provides a good model system to study gene regulation during normal and abnormal cell behavior because it synthesizes functionally distinct products ranging from growth factors and digestive enzymes to factors of relevance to homeostatic mechanisms. The present study describes the long-term growth and differentiation of submandibular salivary epithelial cells from adult male mice as a function of the culture substratum. Using a two-step partial dissociation procedure, it was possible to enrich for ductal cells of the granular convoluted tubules, the site of epidermal growth factor synthesis. Long-term cell growth over a period of 2 to 3 mo. with at least 3 serial passages was obtained only within three-dimensional collagen gels. Cells grew as ductal-type structures, many of which generated lumens with time in culture. Electron microscopic analysis in reference to the submandibular gland in vivo revealed enrichment for and maintenance of morphologic features of granular convoluted tubule cells. Reactivity with a keratin-specific monoclonal antibody established the epithelial nature of the cells that grew within collagen. Maintenance of cell differentiation, using immunoreactivity for epidermal growth factor as criterion, was determined by both cytochemical and biochemical approaches and was found to be dependent on the collagen matrix and hormones. Greater than 50% of the cells in primary collagen cultures contained epidermal growth factor only in the presence of testosterone and triiodothyronine. In contrast, cells initially seeded on plastic or cycled to plastic from collagen gels were virtually negative for epidermal growth factor. Biochemical analysis confirmed the presence of a protein with an apparent molecular weight of 6000 which comigrated with purified mouse epidermal growth factor. Epidermal growth factor was also present in detectable levels in Passage 1 cells. This culture system should permit assessment of whether modulation of submandibular gland ductal cell growth can be exerted via a mechanism that in itself includes epidermal growth factor and its receptor and signal transduction pathway.
Department of Microbiology, University of Texas Health Science Center, Dental Branch, Houston 77225.
E M
EM
Durban
D000818:Animals; D015153:Blotting, Western; D002454:Cell Differentiation; D002455:Cell Division; D002478:Cells, Cultured; D003094:Collagen; D004815:Epidermal Growth Factor / Q000096:biosynthesis; D004847:Epithelial Cells; D005109:Extracellular Matrix / Q000502:physiology*; D005455:Fluorescent Antibody Technique; D005782:Gels; D007633:Keratins / Q000502:physiology; D008297:Male; D051379:Mice; D008854:Microscopy, Electron; D010969:Plastics; D013363:Submandibular Gland / Q000166:cytology*; D013997:Time Factors
D005782:Gels; D010969:Plastics; D004815:Epidermal Growth Factor; D007633:Keratins; D003094:Collagen