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Current methods of islet isolation are limited, thus requiring islets to be pooled from multiple donors to provide sufficient islet mass to permit insulin independence following islet transplantation. Low temperature banking is one approach used to pool islet preparations. Recently, we developed a method for bulk cryopreservation of islets in a single freezer bag system that is less labor-intensive and more readily kept sterile. As a further improvement to this bulk cryopreservation protocol we examined islet survival following slow-step dilution or our standard sucrose dilution protocol. Known numbers of canine islets were cryopreserved in DMSO by slow cooling to -40°C, storing at -196°C, and rapid thawing. When islets were frozen and thawed in glass tubes the recovery of islets after 48 h of tissue culture was significantly higher when the DMSO was removed using either a slow step (71.7 + 2.7%) or a modified slow step (75.7 + 3.9%) protocol as compared with the standard sucrose dilution protocol (65.7 + 3.0%) (p < 0.05, unpaired t-test). Insulin secretion in vitro and in vivo graft function was similar between the experimental groups. Similarly, when islets were frozen then thawed in freezer bags, islet recovery following 48 h postcryopreser-vation tissue culture at 37° C was 74.8 + 2.4% for slow-step dilution compared with 66.2 + 2.7% for the standard sucrose dilution group (p < 0.05, unpaired t-test). Islets thawed in the freezer bag using the modified slow-step dilution protocol showed equivalent functional viability during static incubation to nonfrozen controls. Bulk cryopreservation of isolated islets in single blood freezer bags is a practical alternative to cryopreservation in glass tubes. Development of an automated protocol for the slow stepwise removal of the cryoprotectant from islets in freezer bags will facilitate low temperature tissue banking of islets.