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  • Title: [Improved methods of isolation and purification of rat islets and its viability research].
    Author: Xu Y, Fu H, Wang Z, Zhou X, Xu Y, Wang D, Xue S, Li X.
    Journal: Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi; 2010 Apr; 24(4):406-9. PubMed ID: 20458999.
    Abstract:
    OBJECTIVE: The purity and activity of islets will greatly affect the outcome of xenotransplantation therapy of type 1 diabetes mellitus. To set up an improved method of the isolation and purification of rat islets, which can obtain high-purity, high-yield, and high-viability islets. METHODS: Ten healthy and adult male SD rats, weighing 250-300 g were used as organ donors. Collagenase V was perfused into pancreas via pancreatic duct. Pancreas was digested with collagenase in water bath at 38 degrees C about 15 minutes, islet purification was performed using two techniques: with Ficoll 400 density gradient (group A), and Ficoll-Paque PLUS (group B). Dithizone (DTZ) was utilized for identifying islets, counting islets equivalent quantity (IEQ) and islets' purity. Trypan blue staining was used to detect the viability of islets. Islets of group B was encapsulated with alginate/poly-L-lysine/alginate (APA). Islets function of microencapsulated and nonmicroencapsulated was evaluated by the insulin release test. RESULTS: DTZ staining showed that islets shape were round, ellipse and irregular with a clear edge and a diameter range of 50-300 microm. The IEQ values were 338.04 +/- 76.61 and 834.80 +/- 54.00 in groups A and B, respectively, showing significant difference (P < 0.05). The purities were 88.31% +/- 2.67% and 95.63% +/- 1.96% in groups A and B, respectively, showing no significant difference (P > 0.05). The activities of islets were 67.40% +/- 5.15% and 86.05% +/- 2.52% in groups A and B, showing significant difference (P < 0.05). Islet APA microcapsules had round shape, unified size, and its diameter was between 1.5 and 2.0 mm. Each microcapsule was encapsulated of 1 to 3 islets. The result of insulin release assay was that the concentrations of insulin secretion with islets of microencapsulated and nonmicroencapsulated were (5.53 +/- 1.64) ng/mL and (4.76 +/- 0.26) ng/mL in low glucose, and its concentrations of insulin secretion in high glucose were (11.95 +/- 2.07) ng/mL and (14.34 +/- 3.18) ng/mL. Stimulated insulin secretion in high glucose was 2 times more than that in low glucose (P < 0.05), but there was no significant difference (P > 0.05) in the stimulation index between group A (2.16 +/- 0.30) and group B (3.01 +/- 0.59). CONCLUSION: The method of islets isolation and purification using Ficoll-Paque PLUS own the virtues of more convenient, high islet yield, and high islet purity. Both microencapsulated and nonmicroencapsulated islets show high-viability while culture in vitro.
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