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Sterility control and long-term eye-bank storage of cultured human limbal epithelial cells for transplantation
  1. T P Utheim1,2,
  2. S Raeder1,2,
  3. Ø A Utheim2,
  4. M de la Paz3,
  5. B Roald4,
  6. T Lyberg1
  1. 1
    Center for Clinical Research, Ulleval University Hospital, Oslo, Norway
  2. 2
    Department of Ophthalmology, University of Oslo, Ulleval University Hospital, Norway
  3. 3
    Institut Universitari Barraquer/Universitat Autonoma de Barcelona, Barcelona, Spain
  4. 4
    Department of Pathology, Ulleval University Hospital, University of Oslo, Norway
  1. Dr S Raeder, Center for Clinical Research, University of Oslo, Ulleval University Hospital, Kirkeveien 166, 0407 Oslo, Norway; sten.rader{at}medisin.uio.no

Abstract

Background/aims: To assess sterility of cultured human limbal epithelial cells (HLEC) and to investigate the viability, morphology and phenotype of cultured HLEC following 2 and 3 weeks of organ culture storage.

Methods: HLEC cultured on amniotic membranes were stored in organ culture medium in a closed container at 23°C. Sterility of storage media was tested using a Bactec 9240 blood culture instrument (Becton Dickinson, Maryland) for incubation and periodic reading. Viability was analysed by calcein-acetoxymethyl ester/ethidium homodimer-1 assay, morphology by light microscopy and cellular phenotype by immunohistochemistry.

Results: No microbial contamination was observed after 1 week’s storage. Viability of cultured HLEC was 87.9 (SD 6.4)% and 52.7 (13.1)% after 2 and 3 weeks of storage, respectively, compared with 98.8 (2.6)% before storage (p<0.001). The multilayered structure was preserved in 70% of cultures following 2 weeks of storage but lost after 3 weeks. A less differentiated phenotype was maintained.

Conclusion: This study is the first to verify the sterility of HLEC cultures prior to transplantation. Although a slight decrease in viability was observed following 2 weeks of storage, the HLEC sheets remain acceptable, whereas 3 week’s storage was unsatisfactory.

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Footnotes

  • Competing interests: TPU and SR have filed a patent application on storage of cultured limbal epithelial cells.

  • Funding: This work was supported by the South-Eastern Norway Regional Health Authority.

  • Ethics approval: Ethics approval was provided by Regional komite for etikk, Region Øst-Norge.

  • Patient consent: Obtained.

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