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.