CRYOPRESERVATION OF MOUSE PANCREATIC ISLETS

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Abstract
In most previous studies of cryopreserved isolated pancreatic islets, a slow cooling rate has been employed. We recently observed that faster cooling (5°C/min) resulted in better functional islet preservation than cooling at 0.5°C/min. We found that a culture period after the collagenase isolation of the islets, but prior to freezing, is crucial for the preservation of the islet B cell function. In the present investigation the function of isolated mouse pancreatic islets cooled in Hanks' solution supplemented with 2 M dimethylsulphoxide was compared with that of nonfrozen, cultured islets prepared from the same donors. The islets were cultured in RPMI 1640 + 10% calf serum for 3 days before freezing, and for 3 days after rapid thawing at 37°C. Islets were cooled at rates of 5, 15, or 25°C/min to 70°C and then plunged into liquid nitrogen. All three groups of cryopreserved islets responded with insulin secretion when challenged with high glucose concentrations in batch-type incubations. In further experiments it was found that glucose-stimulated (pro)insulin biosynthesis in islets frozen at 25°C/min was the same as that in the controls. Similar observations were made with respect to glucose-stimulated insulin release in perifusion experiments. However, a 30% reduction in insulin content was observed in the rapidly frozen islets. There was no difference in the replicatory capacity of the islets cells in vitro, as determined by an autoradiographic technique, between control islets and islets cooled at 5°C or 25°C/min. Intrasplenic implantation of 600–800 cryopreserved syngeneic islets into alloxan-diabetic mice led to complete or partial normalization of the hyperglycemia in seven of nine mice. When splenectomy was performed in five animals the serum glucose concentrations increased promptly. We conclude that relatively rapid cooling rates may be useful for cryopreservation of isolated pancreatic islets.