SUCCESSFUL BANKING OF PANCREATIC ENDOCRINE-CELLS FOR TRANSPLANTATION

To determine optimal freezing and thawing conditions for rat pancreatic endocrine cells (PEC) and insulinoma cells, five different cryopreservation protocols were compared in this study. PEC and insulinoma cells were cooled at rates of between -0.3°C/min and -5°C/min to -70°C in the presence of 10%, 15%, or 20% dimethylsulfoxide (DMSO) with a programmable temperature controller and then transferred to liquid nitrogen for storage. Frozen cells were thawed by either rapid (in 37°C water bath) or slow (in air) thawing procedure. One hour after the thawing process, cellular viability was determined by trypan blue dye exclusion. The viability results for PEC and insulinoma cells were similar and showed that a slow cooling rate at -0.3°C/min in combination with a rapid thawing in 37°C water bath gave the best results, with up to 80% cellular viability. Cryoprotectant DMSO used at 10% concentration was the most effective among the three concentrations tested. Later, transplantation studies were performed with PEC cryopreserved with the best protocol, which is -5°C/min to 4°C, held for 3 minutes, -0.3°C/min to -7°C, held for 3 minutes, -0.3°C/min to -40°C, and -5°C/min from -40°C to -70°C in 10% DMSO with a programmable temperature controller then transferred to liquid nitrogen for storage. Intraportal transplantation of cryopreserved Wistar (Wi) strain PEC into allogeneic ACI diabetic recipients normalized their blood glucose (BG) for 8.3 ± 1.9 days (mean ± SD), which was not significantly different from that of noncryopreserved preparation of 6.6 ± 1.5 days. Cytotoxic antibody titers in the ACI recipients of cryopreserved and noncryopreserved Wi PEC graft were not significantly different. Intrathecal transplantation of frozen-thawed Wi PEC into allogeneic ACI diabetic recipients resulted in prolonged amelioration of diabetic state in 10 of 10 animals, which is similar to that seen with freshly prepared PEC. This study confirmed that cryopreservation is an effective procedure for the banking of PEC before transplantation. The in vivo survival period of PEC in allogeneic recipients was not significantly altered by the cryopreservation process used in this study. © 1990.