Abstract
In vitro culture of murine Langerhans islets usually ends in islet death after 1-3 wk. Given contradictory published data, we studied the influence of glucose on the function and survival of islets from DBA/2 mice. Islets were cultured on plastic microwells, using 1, 2, or 11 g/l glucose concentrations. Using our routine technique, insulin secretion was evaluated after islet incubation for 15 min in basal medium [(bIS), 1 g/1 glucose], followed by 15 min in stimulating medium [(sIS), 3 g/l glucose, 20 mM/l arginine, 5 mM/l theophylline]. Insulin secretion of islets cultured in 1 g/l glucose remained stable and normal over a period of 2 mo [Day 7: bIS, 6.3 ± 3.1 pU/50 μl; sIS, 16.6 ± 6.8 μU/50 μl. Day 60: bIS, 6.0 ± 4.0 pU/50 μl; sIS, 21.3 ± 10.5 μU/50 μl]. Islet morphology also remained normal. Islets cultured in 2 g/l glucose showed elevated insulin response under basal and stimulating conditions during 2-3 wk, followed by a dramatic drop in insulin secretion [Day 7: bIS, 19.5 ± 5.7 μU/50 μl; sIS, 80.9 ± 10.7 μU/50 μl. Day 60: bIS, 5.4 ± 5.0 μU/50 μl; sIS, 2.7 ± 1.4 μU/50 μl]. Severe morphologic alterations appeared rapidly and islet destruction was nearly complete by 60 days. At 11 g/l glucose, functional and morphological islet alterations were accelerated [Day 7: bIS, 10.3 ± 2.7 μU/50 μl; sIS, 18.8 ± 4.9 μU/50 μl. Day 21: bIS and sIS almost undetectable]. On the basis of the results obtained in our experimental conditions, it may be concluded that the optimal glucose concentration for long-term murine islet cultures is 1 g/l. Based on the results of this study and testing an extracellular matrix, we thereafter cultured islets from DBA/2 mice at the 1 g/l glucose concentration. These islets remained functional for a period of time > 100 days. Abbreviations: bIS: insulin secretion in basal medium; sIS: insulin secretion in stimulating medium; MEM: minimal essential medium.
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