A Reliable Method for Isolation of Viable Porcine Islet Cells

Ching C, Harland RC, Collins BH, et al. — Thu, 01 Mar 2001 00:00:00 GMT

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°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.

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A Reliable Method for Isolation of Viable Porcine Islet Cells