An improved cryopreservation procedure for human fetal pancreas tissues

Improved viability and function of insulin-producing beta (B) cells of frozen-stored human fetal pancreatic tissue was obtained by a two-step method utilizing high concentrations of dimethyl sulfoxide (DMSO). Human fetal pancreata (14-23-week gestation) obtained from pathologic abortions were teased and cultured overnight. Prior to freezing the tissues were immersed in 0.9% saline containing 0.5 M DMSO for 30 min (room temperature) and then placed in 2.1 M DMSO on ice for 5 min. The tissues were frozen by the method previously developed in our laboratory and stored at -196 degrees C. The frozen-stored tissues were subsequently thawed at 24 degrees C and cultured overnight before viability testing. Viability and function of the B cells were assessed by several specific assay methods; glucose plus theophylline-induced insulin release during static incubation and perifusion, 3H-leucine incorporation into insulin, and insulin content of the tissue grown in athymic mice for 7 days. The response to glucose plus theophylline stimulation, measured on the frozen-thawed tissue one day after thawing, was 80% of the level measured in control tissue maintained in organ culture. Frozen-thawed tissues maintained in organ culture for 1 week responded comparably in the in vitro assay systems. The insulin content of frozen-thawed pancreatic tissue removed from athymic mice 1 week after transplantation was approximately 60% of the amount measured in the control grafts. These results demonstrate the utility of our procedure in the maintenance of the viability and function of frozen-stored human B cells both in culture and after transplantation.