Optimizing Conditions for Rat Pancreatic Islets Isolation

Objective:To optimize conditions for rat islets isolation and purification,and lay a foundation for islet transplantation experiment.Methods:Islets were isolated from the pancreata of Sprague-Dawley rats by intraductal collagenase P digestion,and purified by discontinuous Ficoll density gradient centrifugation.The protocol was carried out to investigate the influence of collagenase concentration,digestion time,and rat weight on the result of islet isohtion.Rat islets were identified by Dithizon staining,and islets viability was assessed by Acridine orange/Propidium iodide staining.Glucose-stimulated insulin secretion was detected using insulin radioimmunoassay kits and used to assess the function of rat islets.Results:The concentration of collagenase,the time of digestion and the weight of the rat were important factors for efficacious isolation of islets.Under the condition of digestion by 1mg/ml collagenase P for 45min,the effect of islet isolation was optimal (P＜0.05).Rat with a higher weight (＞300g) showed a higher islet yield than those with a lower weight (250g) (P＜0.05).The purity of islets isolated under optimizing conditions was above 90%,and the viability was above 90%.The insulin secretion upon 2.8mmol/L and 16.Tmmol/L glucose simulation was (5.40±1.75) mIU/L/30IEQ and (12.27±2.55) mIU/L/30IEQrespectively (P＜0.05).The simulation index was 2.33±0.29.Conclusions:The result of islet isolation was concerned with collagenase concentration,digestion time,and rat weight.Optimizing conditions for islets isolation can improve the result of rat islet isolation.