Successful Cryopreservation of Fetal Porcine Proislets

Abstract

This study reports the cryopreservation of purified fetal pig proislets (FPP) which were isolated by a culture technique. The FPP were equilibrated with 2 M dimethyl sulfoxide (Me 2SO), cooled at a rate of 0.25°C/min to -40°C, transferred to liquid nitrogen ( - 196°C), stored for 2 h to 2 months at - 196°C, and thawed in a 37°C water bath. The morphology of frozen-thawed FPP was similar to that of noncryopreserved FPP. One hundred percent recovery of response capacity was achieved when tested with 16.7 m M glucose plus 10 m M theophylline. We have recently shown that streptozotocin is not toxic to FPP. Accordingly, we transplanted a standardized dose (10-15 mg/mouse) of either cultured or frozen-thawed FPP beneath the renal capsule of nude mice. Three to four weeks later the recipients of the FPP were rendered diabetic by iv streptozotocin. All of the mice transplanted with cultured FPP and seven of eight mice receiving cryopreserved FPP achieved normoglycemia, 74.8 ± 32.9 and 54.7 ± 8.1 days ( P > 0.05) after transplantation. An intraperitoneal glucose tolerance test in the mice of both groups showed a flatter response to glucose compared to those of normal controls. Grafts removed from the mice with normoglycemia for > 3 weeks, in both groups, had the capacity to secrete insulin in response to 16.7 m M glucose alone and 16.7 m M glucose plus 10 m M theophylline during in vitro perifusion. Histological examination revealed that the extent of differentiation and development, in vivo of cryopreserved FPP was comparable with that of cultured FPP. These data indicate that cryopreservation, with the protocol used here, is successful in maintaining functional viability of frozen-thawed FPP. This study is valuable for clinical islet transplantation research.