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Diabetologia

, Volume 45, Issue 6, pp 841–850 | Cite as

Impact of integrin-matrix matching and inhibition of apoptosis on the survival of purified human beta-cells in vitro

  • F. Ris
  • E. Hammar
  • D. Bosco
  • C. Pilloud
  • K. Maedler
  • M. Donath
  • J. Oberholzer
  • E. Zeender
  • P. Morel
  • D. Rouiller
  • P. Halban
Article

Abstract

Aims/hypothesis. Human islet cells survive poorly in culture and are overgrown by non-endocrine cells. The aims of this study were to sort human beta cells and to develop approaches for their improved survival in culture.

Methods. Human islets were infected with recombinant adenovirus expressing green fluorescent protein (GFP) under the control of the rat insulin promoter such that only beta cells expressed GFP. GFP-positive beta cells were sorted by flow cytometry, and expression of select integrins evaluated by RT-PCR. Beta cells were cultured on different extracellular matrices for up to 15 days. Apoptosis was measured by annexin V binding and ELISA. Insulin secretion was measured by ELISA.

Results. Sorted beta cells survived less well in culture than unsorted islet cells. This did not appear to be due to adenoviral infection and/or GFP expression. Purified beta cells expressed the integrins α3, α5, α6, αV, β1, but not β4. Of the various matrices tested, sorted beta cells attached and spread best on a lawn of lysed human bladder carcinoma cells (5637 cells). However, survival remained poor. Cell death was decreased but not prevented by continued presence of 10 mmol/l nicotinamide and apoptosis decreased by 24 h incubation with 20 µmol/l Z-VAD. Insulin secretion was maintained over 6 days following treatment with both agents.

Conclusions/interpretation. Purification of human beta cells induces marked apoptosis limiting their function and survival in vitro. This was improved by matching the extracellular matrix to the specific expression of integrins and by addition of nicotinamide and Z-VAD.

Integrins human beta cells apoptosis extracellular matrix insulin secretion GFP islet 

Copyright information

© Springer-Verlag 2002

Authors and Affiliations

  • F. Ris
    • 1
  • E. Hammar
    • 1
  • D. Bosco
    • 1
  • C. Pilloud
    • 1
  • K. Maedler
    • 3
  • M. Donath
    • 3
  • J. Oberholzer
    • 2
  • E. Zeender
    • 1
  • P. Morel
    • 2
  • D. Rouiller
    • 1
  • P. Halban
    • 1
  1. 1.Louis-Jeantet Research Laboratories, University of Geneva Medical Center, 1 rue Michel-Servet, 1211 Geneva 4Switzerland
  2. 2.Division of Surgical Research, Department of Surgery, University Hospital, GenevaSwitzerland
  3. 3.Division of Endocrinology and Diabetes, University Hospital, ZurichSwitzerland

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