title: Sterility control and long-term eye-bank storage of cultured human limbal epithelial cells for transplantation.
abstract: OBJECTIVE
  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 degrees 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Center for Clinical Research, University of Oslo, Oslo, Norway.
authors.forename: T P
authors.initials: TP
authors.lastname: Utheim
authors.forename: S
authors.initials: S
authors.lastname: Raeder
authors.forename: Ø A
authors.initials: ØA
authors.lastname: Utheim
authors.forename: M
authors.initials: M
authors.lastname: de la Paz
authors.forename: B
authors.initials: B
authors.lastname: Roald
authors.forename: T
authors.initials: T
authors.lastname: Lyberg
mesh_terms: D002478:Cells, Cultured / Q000166:cytology* / Q000637:transplantation; D003316:Corneal Diseases / Q000473:pathology; D003470:Culture Media; D004847:Epithelial Cells / Q000166:cytology* / Q000637:transplantation; D019573:Epithelium, Corneal / Q000166:cytology*; D005125:Eye Banks; D006801:Humans; D016850:Limbus Corneae / Q000166:cytology*; D009926:Organ Preservation; D013194:Staining and Labeling; D033581:Stem Cell Transplantation; D013242:Sterilization; D014022:Tissue Survival
chemical_list: D003470:Culture Media