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Author Notes:

Correspondence to: Athanassios Sambanis, PhD, Telephone: (213) 612-2003, asambanis@wmkeck.org , Postal Address: 550 South Hope Street, Suite 2500, Los Angeles CA 90071, USA

Sudhakar Muthyla performed all experiments, cell culture, insulin ELISA assays, alamar blue metabolic assays, confocal microscopy, data analyses and graphing, and writing of the manuscript; Susan Safley assisted with writing the manuscript and reviewed the manuscript; Kereen Gordon performed API isolation and islet encapsulation; Graham Barer cultured islets and assisted with API isolation and islet encapsulation.

Collin Weber was PI of the laboratory at Emory University in which much of the work was performed; Athanassios Sambanis was PI of the laboratory at Georgia Institute of Technology, in which much of the work was performed, and he assisted with interpretation of the data and reviewing the manuscript.

We gratefully acknowledge Dr. Lawrence Gazda and Dr. Robert Holdcraft of the Rogosin Institute, Xenia Division, OH for providing adult pig pancreata from Bob Evans Farms for our API isolations.

We also thank Neil Anthony (The Emory Integrated Cellular Imaging core) for assistance with Imaris (3D Image analysis software).

Subjects:

Research Funding:

This work was funded by Juvenile Diabetes Research Foundation and National Institute of Health under award R90DK098981.

Keywords:

  • Science & Technology
  • Life Sciences & Biomedicine
  • Medicine, Research & Experimental
  • Transplantation
  • Research & Experimental Medicine
  • hypoxia
  • insulin secretion
  • islet viability
  • microencapsulation
  • porcine islets
  • HUMAN PANCREATIC-ISLETS
  • BETA-CELLS
  • INSULIN INDEPENDENCE
  • ALAMAR BLUE
  • TRANSPLANTATION
  • OXYGEN
  • REVERSAL
  • XENOTRANSPLANTATION
  • XENOGRAFTS
  • SECRETION

The effect of hypoxia on free and encapsulated adult porcine islets-an in vitro study

Tools:

Journal Title:

Xenotransplantation

Volume:

Volume 24, Number 1

Publisher:

, Pages e12275-e12275

Type of Work:

Article | Post-print: After Peer Review

Abstract:

Background: Adult porcine islets (APIs) constitute a promising alternative to human islets in treating type 1 diabetes. The intrahepatic site has been used in preclinical primate studies of API xenografts; however, an estimated two-thirds of donor islets are destroyed after intraportal infusion due to a number of factors, including the instant blood-mediated inflammatory reaction (IBMIR), immunosuppressant toxicity, and poor reestablishment of extracellular matrix connections. Intraperitoneal (ip) transplantation of non-vascularized encapsulated islets offers several advantages over intrahepatic transplantation of free islets, including avoidance of IBMIR, immunopro tection, accommodation of a larger graft volume, and reduced risk of hemorrhage. However, there exists evidence that the peritoneal site is hypoxic, which likely impedes islet function. Methods: We tested the effect of hypoxia (2%-5% oxygen or pO 2 : 15.2-38.0 mm Hg) on free and encapsulated APIs over a period of 6 days in culture. Free and encapsulated APIs under normoxia served as controls. Islet viability was evaluated with a viability/cytotoxicity assay using calcein AM and ethidium bromide on days 1, 3, and 6 of culture. Alamar blue assay was used to measure the metabolic activity on days 1 and 6. Insulin in spent medium was assayed by ELISA on days 1 and 6. Results: Viability staining indicated that free islet clusters lost their integrity and underwent severe necrosis under hypoxia; encapsulated islets remained intact, even when they began to undergo necrosis. Under hypoxia, the metabolic activity and insulin secretion (normalized to metabolic activity) of both free and encapsulated islets decreased relative to islets cultured under normoxic conditions. Conclusions: Hypoxia (2%-5% oxygen or pO 2 : 15.2-38.0 mm Hg) affects the viability, metabolic activity, and insulin secretion of both free and encapsulated APIs over a six-day culture period. Encapsulation augments islet integrity under hypoxia, but it does not prevent loss of viability, metabolic activity, or insulin secretion.

Copyright information:

© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

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