Effects of oxygen, insulin, and glucagon concentrations on rat submandibular acini in serum-free primary culture

Abstract

The objective of these studies was to develop serum-free culture conditions for dissociated acini from rat submandibular glands. Acini were isolated from the submandibular glands of 42-46 d old rats and cultured on reconstituted rat tail collagen containing laminin in 1:1 Ham's F12 and Dulbecco's media, supplemented with BSA, transferrin, insulin, T3, EGF, dexamethasone, retinoic acid, carbamylcholine, and trace elements, and gassed with 50% O2. The acini became partly embedded in the collagen gel and rapidly enlarged throughout the first 22 d of culture, maintaining modest seromucous acinar differentiation, as judged morphologically and by mucin secretion. Parallel cultures then were grown under 20, 35, 50, and 65% O2, and evaluated morphologically and by DNA content. Growth and retention of seromucous acinar characteristics were best with 35% O2, but lipid accumulation and cell death were unacceptably high. A spectrum of concentrations of insulin and glucagon then were tried. With 0.05 micrograms/ml insulin, cellular growth and organization were orderly, lipid accumulations were not excessive, and moderate differentiation was retained through 15 d of culture. With more than 0.1 microgram/ml insulin added to or subtracted from the optimum, the detrimental effects recurred. Addition of sufficient glucagon counteracted the effects of both optimum and excessive concentrations of insulin. We now have achieved an orderly growth of moderately differentiated rat submandibular acini for 15 d in serum-free primary culture.

Salivary Glands: A Paradigm for Diversity of Gland Development

“…In a short-term study (five days following ligation), granular duct and basal cells of the excretory ducts had the highest proliferative activity, as demonstrated by both 3H-thymidine uptake and proliferating cell nuclear antigen (PCNA) immunohistochemistry (Sumitomo et al, 1995). Acinar dedifferentiation also has been shown to occur after main duct ligation in the rat parotid gland (Donath et al, 1973;Leake et al, 1974), although in another study cell death reportedly destroyed the acini after five days in this model (Walker and Gobe, 1987 (Johnson, 1987;Arias and Bendayan,1991), but also to be relatively intolerant of decreased oxygen and nutrients (Rye et al, 1980;Quissell et al, 1994). Therefore, whether they atrophied or died in the cited duct ligature experiments might well have depended on the degree to which the blood and nerve supplies were obstructed by the ligature.…”

Development of Poly(Ethylene Glycol) Hydrogels for Salivary Gland Tissue Engineering Applications

“…As in vitro culture of salivary gland cells under adherent conditions leads to loss of secretory cell characteristics and reduced proliferation, [101][102][103] we have adopted the use of microspheres, which have been shown to contribute to gland function after transplantation. 10,13 It is generally acknowledged that the maintenance and expansion of functional salivary gland cells requires a biomaterial scaffold.…”

Development and characterization of SV40 immortalized rat submandibular acinar cell lines

“…Sections were cut and prepared for light and transmission electron microscopy as previously described (Redman and Quissell, 1993;Quissell et al, 1994). As shown in Fig.…”

“…Rather than utilizing antibiotic selection for the identification of individual cell lines, we established a selective screening procedure for the identification of well-differentiated clonal cell lines in order to prevent the potential loss following transfection of the more highly differentiated cells that are more susceptible to the toxicity of the selective antibiotics. Nontransformed adult rat submandibular acinar cells cannot be sustained in vitro without an extracellular matrix as substrate and they will not survive on plastic (Quissell et al, 1994). Therefore, the transfected cells were maintained on an extracellular matrix for the first few passages posttransfection to allow the cells time to adapt prior to their growth on plastic.…”