Comparison of different digestion techniques and density gradient purification procedures to prepare viable pancreatic islets from SD rats

Objective:Improving collagenase digestion methods and purification procedures to prepare pancreatic islets from rats for transplantation studies.Methods:Isolation of rat islets were performed with the basic technique described by Lim and with our modification. Islet were purified by Dextran density gradient. For identification, islet samples were stained with DTZ. Islet viabikity was determined with fluorescence （AO/PI）. In vivo the rat islet function was assessed by transplantation of Kunmin mice with streptozocin-induced diabetes.Results:No great quantitative differences could be detected in islet shape, number and size between SD and Wistar rats by histologic analysis. To analyze the influence of the type, consistency and reaction time of the enzyme on islet viability and yields suggests that 0.1% collagenase 30 mins was the best and that between Collagenase Ⅴ and Ⅺ gave similar results. The islets were recovered between the 9% and 14% Dextran layers, and between the 14% and 16%Dextran layers. Purity ranged 85%—95% with 22%—16%—14%—9% gradient. The islets prepared restored normoglycemia with 2-3 days from implantation in the diabetes mice.Conclusion:Tese results show using the digestion methods and purification procedure that we have developed could provided islets from rats for transplantation studies.