| Objective: This experiment is to develop a simple and effective method for isolating and purifying rat pancreatic islet, and to assess the biological function of the purified islets in vitro and in vivo. The most important thing is to reduce islet exposition of warm collagenase and gel formation.Methods: (1), the incubation medium includes glycerol—a common gelatin solvent at 5% volume (V/V). (2), The minced tissue-to-total incubation volume ratio was ≥1:10. (3), Free-islet exposure to pancreatic digestion products was minimized by frequent separation of islets. (4), Incubation temperature at 38±1°C and (5), pH in the range 7.7-7.9. (6), The collagenase-injected arid sectioned pancreas was placed in a chamber containing 1mg/ml collagenase, and gently agitated in a 37°C water bath for 15-20min, until most of the pancreas was partially digested, and was cultured for 1 to 2d at 24°C in RMPI-1640 media supplemented with 10% fetal calf serum until most of the islets were isolated from the extrocrine parts of the pancreas. (7) , Purification of the isolated islets was performed by histopaque1077 gradients. (8), Purified islets were tested by glucose and transplanting into streptozotocin-diabetic ICR mice.Results: Gel formation was greatly reduced or eliminated, warm-ischemia was 13 ±3min, cold-ischemia time was 10±0.5min. there were 1680 +170 islets per SD rat's pancreas after digestion, and 870∽1300 islets per SD rat's pancreas after histopaque 1077 gradients with purity of(80±3.5)%. The...
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