Fabrication of functional rat pseudo-islets after cryopreservation of pancreatic islets or dispersed islet cells

摘要

Dispersed single cells from pancreatic islets can configure the three-dimensional islet-like architecture (pseudo-islets) with insulin secretion potential and controllable size through their aggregation property. The present study was designed to investigate whether cryopreservation of islets or islet cells can contribute to the efficient pseudo-islet fabrication in the rat model. In control group (CT), islet single cells were prepared by trypsin digestion of 50-400-mu m o fresh control islets, and then cultured for 3 days in the U-bottom microwell to fabricate pseudo-islets. In vitrification-warming group (VW), islet single cells were prepared from postwarm islets cryopreserved by vitrification on nylon mesh device, and then cultured for 3 days. In freezing group (FR), islet single cells originated from fresh islets were subjected to a conventional Bicell(R) freezing, and postthaw cells were cultured for 3 days. To generate 1 islet equivalent pseudo-islets (150 mu m o) by the sphere culture, 1250 CT cells, 1250 VW cells, and 1500 FR cells were seeded to each microwell. The viability of the pseudo-islets was comparable among the three groups (93.9%-96.9%). Furthermore, the insulin secretion assay showed that those pseudo-islets responded sufficiently to the high glucose stimulation. Immunostaining for insulin and glucagon showed that the endocrine cell arrangement of those pseudo-islets is similar to that of native and isolated islets. These islets/pseudo-islets had the beta-cells in core and the alpha-cells in mantle, which was typical characteristic of the rodent islets. However, some clusters of alpha-cells were observed inside the FR pseudo-islets. Interestingly, the VW pseudo-islets had significantly fewer alpha-cells than the CT or FR pseudo-islets. These results suggest that the sphere culture of islet cells is useful tool to generate the pseudo-islets with the customized size and normal functionality, even after islet cryopreservation.