A reliable method for isolation of viable porcine islet cells

C. Denise Ching, Robert C. Harland, Bradley H. Collins, William Kendall, Hasan Hobbs, Emmanuel C. Opara

Research output: Contribution to journalArticle

24 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)276-279
Number of pages4
JournalArchives of Surgery
Volume136
Issue number3
DOIs
StatePublished - Jan 1 2001
Externally publishedYes

ASJC Scopus subject areas

  • Surgery

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    Denise Ching, C., Harland, R. C., Collins, B. H., Kendall, W., Hobbs, H., & Opara, E. C. (2001). A reliable method for isolation of viable porcine islet cells. Archives of Surgery, 136(3), 276-279. https://doi.org/10.1001/archsurg.136.3.276