ISLET CELL-CULTURE IN DEFINED SERUM-FREE MEDIUM

A serum-free, hormone- and factor-supplemented, defined medium was developed which maintains functional activity of primary cultures of adult islet cells and continuous islet cell lines. Medium supplements examined included proteose peptone (PP), transferrin (TrFe), insulin-like growth factor-I (IGF-I), an insulinotropic fragment of human GH [hGH-(6–13)], ethanolamine (EA), phosphoethanolamine (PEA), and human serum albumin (HSA). Glucose-stimulated insulin secretion from islet monolayers was determined after culture in serum-free supplemented medium by either 2- to 3-h static incubations or in a superfusion system with either low (2.8–8.3 μM) or stimulatory (16.7–19.4 μM) glucose concentrations. Glucose-induced secretion was not sustained after 3–4 days of culture in medium supplemented with PP (0.5 mg/ml) and TrFe (10 μg/ml) alone. Addition of T3did not restore glucose-induced secretion, although a combination of T3and IGF-I or of T3, IGF-I, and PRL (10–1010–9M) maintained glucose-induced insulin secretion for 1 month. No beneficial effects were noted with hGH-(6-13). The β-cell lines HIT-T15 and RINr 1046–38 were used to screen for a potential replacement for PP, the undefined component of the serum-free medium. A combination of HSA (1 mg/ml), EA (50 μM), and PEA (50 μM) provided a replacement for PP. In fact, insulin secretion from HIT-T15 cells was significantly better after culture in medium supplemented with HSA, EA, PEA, TrFe, T3, IGF-I, and PRL than in medium with PP, TrFe, T3, IGF-I, and PRL. HSA (1 μg/ml), EA (50 μM), and PEA (50 μM) in combination with TrFe (10 μg/ml), T3(0.1 nM), IGF-I (0.65 nM), and PRL (1 nM) were used in studies with primary islet monolayers. After 3 weeks of culture islet monolayers were superfused, and the biphasic glucose-induced insulin secretion of cells maintained in defined medium was indistinguishable from the insulin secretion of cells maintained in medium with 5% fetal bovine serum. These studies indicate that adult rat β-cells retain biphasic glucose-induced insulin secretion after extended culture in defined serum-free medium. The defined medium was also useful for cultures of RINr 1046–38 and HIT-T15 cells and should provide a basis for formulating media for islet cells from higher mammals, including man. © 1990 by The Endocrine Society.