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A Reliable Method for Isolation of Viable Porcine Islet Cells
C. Denise Ching, MD;
Robert C. Harland, MD;
Bradley H. Collins, MD;
William Kendall, MD;
Hasan Hobbs, BS;
Emmanuel C. Opara, PhD
Arch Surg. 2001;136:276-279.
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.
From the Department of Surgery, Duke University Medical Center, Durham, NC. Dr Harland is now with the Division of Transplantation Surgery, UMASS Memorial Health Care, Worcester, Mass.
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