The purpose of this study was to investigate the effect of culture microenvironment on the cell death of isolated rat pancreatic islets. After isolation by the conventional collagenase digestion technique, the islets were cultured in a hydrogel of collagen type I mixed with collagen type III, type IV,and laminin. Irrespective of the type of mixture, islet cell death was significantly suppressed by their co-culture with the collagen hydrogel mixtures, although no change in islet morphology was observed. Co-culture with the collagen mixtures had no influence on the expressed mRNA level of insulin, glucagon, and somatostatin of the islets, or the islet secretion of hepatocyte growth factor (HGF), interleukin (IL)-1α, and IL-1β. These findings suggest that three-dimensional culture in the collagen hydrogel and the mixture of laminin is able to maintain the cell viability of islets.