Sterility control and long-term eye-bank storage of cultured human limbal epithelial cells for transplantation

• Published in: British journal of ophthalmology Vol. 93; no. 7; pp. 980 - 983
• Main Authors: Utheim, T P, Raeder, S, Utheim, Ø A, de la Paz, M, Roald, B, Lyberg, T
• Format: Journal Article
• Language: English
• Published: BMA House, Tavistock Square, London, WC1H 9JR BMJ Publishing Group Ltd 01.07.2009; BMJ Publishing Group; BMJ Publishing Group LTD
• Subjects: Biological and medical sciences; Birth control; Cells, Cultured - cytology; Cells, Cultured - transplantation; Corneal Diseases - pathology; Culture Media; Epithelial Cells - cytology; Epithelial Cells - transplantation; Epithelium, Corneal - cytology; Eye Banks; Gynecology. Andrology. Obstetrics; Humans; Laboratories; Limbus Corneae - cytology; Medical sciences; Miscellaneous; Morphology; Ophthalmology; Organ Preservation; Staining and Labeling; Stem Cell Transplantation; Sterility. Assisted procreation; Sterilization; Studies; Surgery; Tissue engineering; Tissue Survival; Norway; Human; Cell culture; Rim of sclera; Conservation; Transplantation; Long term; Sterility; Eye; Storage; Treatment; Surgery; Graft; Epithelial cell; Ophthalmology
• ISSN: 0007-1161; 1468-2079
• DOI: 10.1136/bjo.2008.149591

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.