CRYOPRESERVATION OF RAT PANCREATIC ISLETS

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Abstract
Inasmuch as cryopreservation can facilitate clinical islet transplantation by providing a means of storing supplemental islets in order to augment marginally adequate grafts, protocols are needed to allow for a minimal loss in viable beta cells. By replacing the cryoprotectant dimethyl sulfoxide (DMSO) with ethylene glycol (EG), a more simplified cryopreservation protocol was developed, which resulted in improved survival and function of rat pancreatic islets. Nonfrozen islets, islets cryopreserved in DMSO, and EG-cryopreserved islets were compared for percent recovery, cellular composition, in vitro viability, and metabolic function after transplantation. After cryopreservation in DMSO or EG, islet yield was similar to that of nonfrozen controls; however, islets cryopreserved in DMSO exhibited lower cellular DNA, insulin, and glucagon content, as well as an impaired insulin secretory capacity in vitro than the nonfrozen controls. When compared with controls, islets cryopreserved in DMSO contained a higher proportion of beta cells but a lower number of glucagon-positive cells, whereas cryopreservation with EG resulted in similar DNA/hormone contents, in vitro viability, and cellular composition. Transplantation of islet grafts composed of comparable numbers of beta cells (2.1-2.3 million) corrected diabetes in 100% (6/6; nonfrozen controls), 92% (10/11; DMSO), and 100% (14/14; EG) of the recipients; however, those who received DMSO-treated islets took longer to achieve euglycemia and remained glucose-intolerant. These results demonstrate that EG allows for the successful cryopreservation of rat islet beta and a cells with the same yield and quality as nonfrozen islets. The observation that alpha-cell survival was better after cryopreservation with EG may explain the improved functional viability of these grafts. Further studies are needed to assess whether this protocol provides any advantage for cryopreserving large numbers of human islets.