NOVEL APPROACHES TO CRYOPRESERVATION OF HUMAN PANCREATIC ISLETS1

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Abstract
Optimized conditions for survival and function of human islets must be defined if sufficient islets are to be recovered from a single human donor pancreas to reverse type-1 diabetes after isolation, cryopreservation, and transplantation. The objective of this study was to compare the cryoprotective effect of ethylene glycol (EG) with the standard cryoprotectant, dimethyl sulfoxide (DMSO), on isolated human islet survival and function. Furthermore, the effect of different addition protocols and equilibrium concentrations of the cryoprotectants were studied. Islets were isolated from human pancreata by using standard techniques of collagenase digestion and discontinuous Ficoll gradient purification. Aliquots of freshly isolated human islets were cryopreserved in six groups by using DMSO or EG. Cryoprotectants were added stepwise to produce a final concentration of 1.5 or 2.0 M, or added in a single step to a concentration of 1.5 M. Islets were cryopreserved by using established protocols and cultured for 48 hr at 37°C before assessment of percentage of recovery and in vitro viability. After cryopreservation, percentage of recovery of islets was significantly higher in the group treated with 1.5 M of DMSO added in a stepwise protocol (74±3%, mean±SEM) compared with the standard 2.0 M of DMSO (62±4%) (P <0.05, unpaired t test, n=6). There was no difference between the recovery of islets cryopreserved with either 1.5 M of DMSO stepwise (74±3%) or 1.5 M of DMSO one-step (69±3%). Islet recovery was higher in groups treated with DMSO compared with EG, regardless of concentration of cryoprotectant or addition protocol, although the difference was significant only when comparing DMSO and EG 1.5 M one-step. Furthermore, islets treated with 1.5 M of DMSO, added either stepwise (6.0±0.4) or in one-step (6.5±0.8), had significantly higher stimulation indices compared with islets treated with the standard cryoprotectant for human islets, 2.0 M of DMSO (4.5±0.5) (P <0.05). These results demonstrate that a lower concentration of DMSO (1.5 M) allows for the cryopreservation of human islets with superior survival and preservation of function post-culture compared with 2.0 M of DMSO and various concentrations of EG.