Abstract
We have investigated the freezing tolerance of rat pancreatic islets. Freshly isolated rat pancreatic islets were divided into three groups based on their longest diameter (small; 100-200 μm, medium; 201-300 μm, large; >300 μm). They were then cryopreserved at a slow cooling rate (-0.3°C/min) in the presence of dimethyl sulfoxide (Me2SO) or ethylene glycol (EG). After storage at -196°C for 1-4weeks, they were thawed and their ability to secrete insulin in response to fluctuations in glucose concentration was examined during three consecutive static incubations in vitro (1st; 2.8 mM, 2nd; 16.7 mM, 3rd; 2.8 mM). Morphological examination of the beta-granule population was determined by image analysis, and correlation with islets size was analyzed. The amount of insulin released from large-sized islets was significantly suppressed in EG (p<0.05) and Me2SO (p<0.01) groups compared to unfrozen islets. However, the mean volume of the large-sized islets isolated from one rat accounted for 43.0% of the total volume. On the other hand, the amount of insulin released from small- and medium-sized islets did not differ from those of unfrozen islets, and their mean volumes were 13.2 and 43.8%, respectively. The percentage of cells with beta-granules was significantly correlated with size in both EG (r=-0.52) and Me2SO (r=-0.35) groups, but no significant correlation was observed in the unfrozen islets groups. These findings suggest that large-sized islets are more susceptible to freezing injury than small- or medium-sized islets. Moreover, the volume distribution of isolated islets indicated that it may be important to retain the ability of insulin secretion from the large-sized islets.
