Article Text

Download PDFPDF
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}


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.

Statistics from

Request Permissions

If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.


  • 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.