Cryopreservation of mouse pancreatic islets: Effects of different glucose concentrations in the post-thaw culture medium on islet recovery

Abstract

It was the aim of this study to investigate the influence of the glucose concentration of the post-thaw culture medium on islet B-cell survival after Cryopreservation by the combined assessments of islet recovery, islet DNA and insulin contents, and insulin release. Collagenase isolated mouse islets were kept in culture for 3 days in the presence of 11.1 m M glucose and then transferred to freezing ampoules containing Hanks' solution supplemented with 10% calf serum and 2 M dimethyl sulfoxide. After a 20-min incubation at 0 °C the islets were cooled at a rate of 25 °C/min to −70 °C and subsequently plunged into liquid nitrogen. After 2 hr the frozen islets were rapidly thawed at 37 °C, transferred to culture dishes, and cultured for another 3 days in the presence of 2.8, 5.6, 11.1, 16.7, or 28 m M glucose. Nonfrozen control islets were treated identically after a preceding 3-day culture at 11.1 m M glucose. The percentage recovery of cryopreserved islets was decreased compared to that of nonfrozen islets, but was increased when higher glucose concentrations were used in the post-thaw culture medium. Since the DNA content of the cryopreserved islets was slightly decreased, the overall survival rate of the cryopreserved B-cells, when cultured at the higher glucose concentrations after thawing, was found to be about 75%. The insulin content of the cryopreserved islets was decreased but the glucose-stimulated insulin release was essentially the same as that of the nonfrozen islets. We have previously demonstrated that islets cryopreserved with the present technique have a preserved (pro)insulin biosynthesis and therefore the decreased insulin content is most likely to be due to minor intracellular damage leading to a reduced capacity for insulin storage. The present findings also suggest that a high glucose concentration is not deleterious to cryopreserved islets. Rather the islet recovery was enhanced by glucose, presumably because of its energy generation in the B cells, and this may allow repair of small injuries induced by the freeze-thawing.