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Sterility control and long-term eye bank storage of cultured human limbal epithelial cells for transplantation
  1. Tor Paaske Utheim,
  2. Sten Raeder (sten.rader{at}medisin.uio.no),
  3. Øygunn Aass Utheim,
  4. Maria de la Paz,
  5. Borghild Roald,
  6. Edvard Messelt,
  7. Torstein Lyberg
  1. Center for Clinical Research, University of Oslo, Ullevaal University Hospital, Norway
  2. Center for Clinical Research, University of Oslo, Ullevaal University Hospital, Norway
  3. Department of Ophthalmology, University of Oslo, Ullevaal University Hospital, Norway
  4. Institut Universitari Barraquer/Universitat Autonoma de Barcelona, Barcelona, Spain
  5. Department of Pathology, Ulleval University Hospital, University of Oslo, Norway
  6. Department of Oral Biology, Faculty of Dentistry, University of Oslo, Norway
  7. Center for Clinical Research, University of Oslo, Ullevaal University Hospital, Norway

    Abstract

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

    Methods: HLEC cultures 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, MD, USA) 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 one week storage. Viability of cultured HLEC was 87.9 ± 6.4% and 52.7 ± 13.1% after two and three 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 two weeks of storage, but lost after three weeks. A less differentiated phenotype was maintained.

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

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