Pancreatic islet isolation by mechanical-enzymatic separation, stationary collagenase digestion and dextran discontinuous density gradient purification: experimental study in dogs

Abstract

The prospects for allotransplantation of pancreatic islets in man depend on the development of methods that provide sufficient quantities of pancreatic islets from a single donor, which are capable, when transplanted, of achieve the normalization of carbohydrate metabolism. Objective: Evaluate the efficacy of the isolation of Langerhans islets from dogs, by means of mechanical-enzymatic separation technique with stationary digestion using collagenase, and purification with a discontinuous dextran density gradient. Methods: The counting of islet numbers and evaluation of their sizes was accomplished by staining with diphenylthiocarbazone and using stereoscopic microscopes equipped with eyepiece reticule for the measurement of average diameters of stained islets. Results: The results disclosed that the average number of islets isolated was 81032.20 ± 24736.79 and the average number of islets isolated per kg of body weight was 6938.70 ± 1392.43. The average number of islets isolated per kg of body weight showed significant correlation with body weight and weight of the pancreas resected. Conclusion: The number of islets isolated, of a single donor, by mechanical-enzymatic separation, stationary collagenase digestion and discontinuous dextran density gradient purification can be sufficient to success of pancreatic islets transplant in dogs.

Pancreatic islet allograft in spleen with immunossuppression with cyclosporine. Experimental model in dogs

“…The main pancreatic duct was immediately isolated before its penetration of the duodenal wall. The preparation of the pancreatic tissue, the pancreatectomy, and the pancreatic islet isolation procedures were performed as previously described 9,12 . The degree of contamination was measured by comparison relative to the quantity of stained islets and of the stained acinar and non-acinar tissue (Figure 1).…”

“…The disadvantages of pancreatic islet transplant are the difficulty of isolating viable and functioning human islets in sufficient quantities to maintain a normoglycemic state, and the immunogenocide of the islets that requires considerable amounts of immunosuppressive drugs to prevent rejection 5 . However, the more compact pancreas of large mammals yields a low number of functionally viable and isolated pancreatic islets caused by the strong connections between their own islets and the pancreatic exocrine tissue that surrounds them [6][7][8][9] . For the clinical transplantation of pancreatic islets to be enhanced, it is imperative that certain conditions must be assured.…”