A reliable method for isolation of viable porcine islet cells.
HYPOTHESIS:Mechanical injury and oxidative stress caused by reoxygenation of isolated porcine islet cells result in their unresponsiveness to glucose stimulation. DESIGN:Adult pigs (weighing 25-30 kg) were anesthetized, and following intra-arterial infusion of ice-cold University of Wisconsin solution, a complete pancreatectomy was performed. The pancreatic duct was cannulated for infusion of digestion medium containing collagenase type P, 1.5 mg/mL; deoxyribonuclease I, 10 000 U; and a water-soluble analogue of vitamin E (Trolox), 1 mmol/L. After 20-minute incubations on ice, and at 37 degrees C, the pancreas was hand shaken for 1 minute, followed by filtration and separation on an automatic cell separator (COBE 2991). Islet cells, identified by dithizone staining, were perifused at 37 degrees C. RESULTS:The mean +/- SEM yield of intact purified islet cells (50-200 microm in diameter), and mostly present in clusters, was 2398 +/- 143 cells per gram (n = 12). Glucose stimulation caused a significant increase in biphasic insulin secretion in the perifusion experiments. CONCLUSION:We have developed a simple, reproducible, and reliable procedure for isolating intact and viable porcine islet cells suitable for xenotransplantation.
Ching, CD; Harland, RC; Collins, BH; Kendall, W; Hobbs, H; Opara, EC
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