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, 10000 U: and a water-soluble analogue of vitamin E (Trolox), 1 mmol/L. After 20-minute incubations on ice and at 37°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°C. Results: The mean±SEM yield of intact purified islet cells (50-200 μm 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.