Pancreatic islets cultured on extracellular matrix: an excellent preparation for microfluorometry

摘要

We have examined the use of extracellular matrix (ECM) as a substratum for mouse pancreatic islet cultures with special reference to microfluorometry work. We find the techniques for obtaining and maintaining ECM-producing cell cultures and coating of glass straightforward. The islets attached readily to ECM-coated glass and with time spread out. Cultures were probed for glucose responses after one day, one week and two weeks. Upon stimulation with 10 mM glucose, the well-established initial changes in [Ca~(2+)]_i, (monitored with Fura-2), as well as oscillations upon prolonged exposure, were observed. At 20 mM glucose only an elevated level without oscillations was seen. Similar responses were observed during the two weeks in culture. After one week in culture or more, single cell measurements of qualitative changes in [Ca~(2+)]_i and cell membrane potential (monitored with the dye bis-(1,3-dibutyl-barbituric acid) trimethine oxonol (DiBAC_4(3)) could be performed concurrently on single cells at the islet periphery with ordinary fluorescence microscopy equipment. Simultaneous measurements of NAD(P)H and flavine adenine dinucleotide (FAD) fluorescence as well as measurements of rhodamine 123 fluorescence for mitochondrial membrane potential showed stable metabolic responses to glucose over the entire culture period. The islets had clearly elevated insulin secretion at 10 and 20 mM glucose compared to the secretion at 3 mM. In conclusion, mouse pancreatic islets cultured for up to two weeks on ECM-coated glass coverslips provide a functionally well-preserved and reproducible preparation for microfluorometry. The largest benefit of the method is that whole islet responses as well as single cell responses at the islet periphery can be monitored, using the same preparation.