Novel approaches to cryopreservation of human pancreatic islets

Background. Optimized conditions for survival and function of human islets must be defined if sufficient islets are to be recovered from a single huma n donor pancreas to reverse type-1 diabetes after isolation, cryopreservati on, and transplantation. The objective of this study was to compare the cry oprotective 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 co ncentrations of the cryoprotectants were studied. Methods. Islets were isolated from human pancreata by using standard techni ques of collagenase digestion and discontinuous Ficoll gradient purificatio n. Aliquots of freshly isolated human islets were cryopreserved in six grou ps by using DMSO or EG. Cryoprotectants were added stepwise to produce a fi nal concentration of 1.5 or 2.0 M, or added in a single step to a concentra tion of 1.5 M. Islets were cryopreserved by using established protocols and cultured for 48 hr at 37 degreesC before assessment of percentage of recov ery and in vitro viability. Results. After cryopreservation, percentage of recovery of islets was signi ficantly 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 betwee n 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 i n groups treated with DMSO compared with EG, regardless of concentration of cryoprotectant or addition protocol, although the difference was significa nt only when comparing DMSO and EG 1.5 Al one-step. Furthermore, islets tre ated with 1.5 Al of DMSO, added either stepwise (6.0 +/-0.4) or in one-step (6.5 +/-0.8), had significantly higher stimulation indices compared with i slets treated with the standard cryoprotectant for human islets, 2.0 M of D MSO (4.5 +/-0.5) (P <0.05). Conclusions. These results demonstrate that a lower concentration of DMSO ( 1.5 M) allows for the cryopreservation of human islets with superior surviv al and preservation of function post-culture compared with 2.0 M of DMSO an d various concentrations of EG.