Short-term primary culture of acinar-intercalated duct complexes from rat submandibular glands

Abstract

Acinar-intercalated duct complexes dissociated from rat submandibular glands have been shown to be an excellent model for studying secretory responses of salivary gland components. However, they are functionally normal for only a few hours. We undertook a systematic manipulation of primary culture conditions in an attempt to extend the useful life of the complexes. The major modifications that were tested were increased oxygenation in increments to 95%; substitution of norepinephrine or carbamylcholine or both for isoproterenol in the medium; different sources of collagen for and addition of laminin, fibronectin and/or type IV collagen to the matrix gel; and varying the thickness of the collagen gel, richness of the cell suspension inoculate, and sources and concentrations of sera in the medium. Progress was monitored by light microscopic evaluation of routine sections of specimens until improved maintenance of acinar and other cells warranted carrying parallel cultures for biochemical, histochemical, and ultrastructural analyses. Best results were obtained with 90% O2, laminin in rat tail collagen gel, 10% fetal bovine serum, and 3 microM isoproterenol. Morphologically, there was good survival of acini and intercalated ducts after 1 d, with decreased acinar size being correlated with secretory response to the isoproterenol. Reorganization and considerable mitotic activity were seen at 2, 3, and 4 d, with most clusters of cells becoming much larger than the original complexes. During this period acinar cells steadily became less differentiated and their numbers decreased in proportion to intercalated duct or undifferentiated cells. However, there was good overall survival through 7 d. Biochemical analysis indicated that the cells were able to maintain significant biosynthetic activity for 4 d, with DNA, RNA, protein, and glycoprotein synthetic rates increasing over the culture period, but the secretory capacity of the cells diminished during the primary culture period, with mucin biosynthesis and secretion decreasing significantly after 1 d in culture.