Tissue-engineered pancreatic islets: Culturing rat islets in the Chitosan sponge

Subcutaneous islet transplantation has become an attractive modality. With development of tissue-engineering techniques, it is possible to rectify the disadvantage of poor blood supply in the subcutaneous site by reconstructi on of the capillary network. According to reports, the Chitosan sponge (CS) could be used for reconstruction of in vitro capillary-like network and co uld be used in artificial skin equivalent. In this study, we cultured the i slets in CS for future application. CSs, having 200-500 mum pore size, were prepared by freeze-drying method. Rat islets were isolated from the pancre as of Lewis rats (10 weeks old, 280-300 g, male) by collagenase digestion f ollowed by discontinuous dextran gradient centrifugation method. Each 20 is lets were seeded equally into the CSs and were cultured for 62 days with va rious culture media such as RPMI-1640, Dulbecco's modified Eagle's medium ( DMEM), and Eagle's MEM. They contained 10% fetal bovine serum (FBS) and 5 m l/L antibiotic-antimycotic mixed stock solution in the culture dishes. Insu lin concentration both inside and outside of the islet-seeded CS was measur ed during culture. Changes in the morphology of islets were also observed i n this study. Freshly isolated islets had a loose appearance with an irregu lar border, and most were seen as a single islet. Occasionally a cluster, c onsisting of 2-4 islets ranging mainly from 150 to 250 mum in diameter, was observed. Islets cultured in the CSs in different culture media retained i nitial morphology, which had well-delineated smooth borders for at least 53 days. The insulin release behavior of islets cultured in the CS showed con stant secretory capacities for 49 days. After that they exhibited a rapid a nd definitive decline from the initial insulin release. Until this stage, i nsulin concentration in the CS was well maintained. The properties were dep endent on culture medium used and insulin diffusion released from islets. T his experiment is a new study model for establishment of islet culture in a three-dimensional matrix. Also extension of this observation will provide new insights for islet transplantation at the subcutaneous site by a tissue -engineering approach.