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Prevention of oxidative stress in porcine islet isolation

Journal of Artificial Organs volume 13, pages 38–47 (2010)Cite this article

Abstract

High yields of pure and viable porcine islet cells (PICs) to be used for microencapsulation are crucial for successful xenotransplantation. Mechanical disruption of the pancreas, enzymes used for digestion, digestion temperature and time are among the factors known to cause oxidative stress and to impact on the yield, purity and viability of PICs. The aim of our study was to optimize conventional procedures in order to minimize the oxidative stress that occurs during the isolation and purification of PICs. Porcine pancreatic tissue was harvested at a local slaughterhouse, and 15 consecutive isolations of PICs were performed with a modified automated Ricordi method (Graz method) using a shorter digestion time, a lower digestion temperature and minimal mechanical stress. PICs were purified with the Lymphoprep density gradient medium. Purity and viability were assessed immediately after the isolation process and after overnight culture. PIC function was tested in glucose stimulation experiments and insulin concentration was determined by ELISA. Oxidative stress was assessed by measuring isoprostanes (IP), malondialdehyde (MDA) and lipase levels using a HPLC-based, colorimetric liquid assay or ELISA, respectively. The mean yield of PICs was 3479 ± 542 IEQs/g pancreas, with 96.4% viability and 97.7% purity. There was no significant loss in PIC viability after overnight culture. Insulin secretion in response to glucose was not impaired after isolation and purification. IP, MDA and lipase levels did not change significantly during the isolation procedure. With our new Graz method we seem to have succeeded in preventing oxidative stress and achieving high yields of pure and viable PICs.

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Affiliations

  1. Division of Transplantation Surgery, Department of Surgery, Medical University of Graz, Auenbruggerplatz 29, 8036, Graz, Austria

    Philipp Stiegler, Florian Hackl, Silvia Schaffellner, Florian Iberer & Karlheinz Tscheliessnigg

  2. Division of Gastroenterology and Hepatology, Department of Internal Medicine, Medical University of Graz, Auenbruggerplatz 15, 8036, Graz, Austria

    Vanessa Stadlbauer

  3. Institute of Physiological Chemistry, Center for Physiological Medicine, Medical University of Graz, Harrachgasse 21, 8010, Graz, Austria

    Joachim Greilberger

  4. Division of Hematology, Department of Internal Medicine, Medical University of Graz, Auenbruggerplatz 38, 8036, Graz, Austria

    Dirk Strunk

  5. Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University of Graz, Auenbruggerplatz 29, 8036, Graz, Austria

    Sieglinde Zelzer

  6. Institute of Pathology, Medical University of Graz, Auenbruggerplatz 25, 8036, Graz, Austria

    Carolin Lackner

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  1. Philipp Stiegler
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  2. Vanessa Stadlbauer
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Correspondence to Philipp Stiegler.

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Stiegler, P., Stadlbauer, V., Hackl, F. et al. Prevention of oxidative stress in porcine islet isolation. J Artif Organs 13, 38–47 (2010). https://doi.org/10.1007/s10047-010-0488-x

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  • DOI: https://doi.org/10.1007/s10047-010-0488-x

Keywords

  • Islet transplantation
  • Islet isolation
  • Xenotransplantation
  • Oxidative stress
  • Isoprostanes