Transplantation and in vitro perifusion of rat islets of Langerhans after slow cooling and warming in the presence of either glycerol or dimethyl sulfoxide

Abstract

The cryoprotectants dimethyl sulfoxide (Me 2SO) and glycerol have been used for the cryopreservation of fetal rat pancreases but only Me 2SO has been reported for the cryopreservation of adult rat islets. Since glycerol may be preferred to Me 2SO for clinical use, this study was undertaken to compare the effectiveness of these cryoprotectants during the slow cooling of isolated adult rat islets. Islets of Langerhans prepared from the pancreases of WAG rats by collagenase digestion were stored at −196 °C after slow cooling (0.3 °C/min) to −70 °C in the presence of multimolar concentrations of either Me 2SO or glycerol. Samples were rewarmed slowly (~10 °C/min) and dilution of the cryoprotectant was achieved using medium containing sucrose. Function was assessed by determination of the time course of the glucose-induced insulin release during in vitro perifusion at 37 °C and also by isograft transplantation. Transplants were carried out by intraportal injection of a minimum of 1700 frozen and thawed islets into streptozotocin-induced diabetic recipients and tissue function was assessed by monitoring blood glucose levels and body weight changes. Without exception the islets frozen and thawed in the presence of glycerol failed to reduce high serum glucose levels of recipient rats and in vitro dynamic release curves showed that islets frozen with glycerol were unable to demonstrate a glucose-sensitive insulin release pattern. Reversal of the diabetic conditions was achieved in two of five animals receiving islets which had been frozen and thawed with 2 M Me 2SO; and in one of three animals receiving islets cryopreserved with 3 M Me 2SO. Nevertheless, perifusion studies showed that the pattern of insulin secretion from groups of cryopreserved islets which did show an ability to secrete insulin was atypical compared with that of untreated controls, suggesting that the tissue was altered or damaged in some way.