Abstract written by Anders Kvalheim, on the report:
Effects of Organ Culture and Optisol-GS Storage on Structural Integrity, Phenotypes, and Apoptosis in Cultured Corneal Epithelium.
Purpose of report: In this study, conventional organ culture (OC) storage and Optisol-GS storage of cultured human limbal epithelial cells (HLEC) are compared at different temperatures to find optimal conditions to store cultured tissue prior to transplantation to treat limbal stem cell deficiency. Finding the best possible way to store the HLECs, will benefit: Flexibility when planning transplantations, making it possible to perform microbiological testing, and safe transportation that will make operations possible not only at the sites of the few laboratories and eye-banks that perform the ex-vivo expansion of the HLECs.
Methods: HLECs cultured for three weeks, were stored in OC at 31°C or at 23°C, and in Optisol-GS at 5°C. All were stored in a closed container for 1 week. Morphology was studied by light microscopy and transmission electron microscopy. Phenotypic characterization was done by immunohistochemistry. Apoptosis was evaluated by three different methods: 1) Real-time RT-PCR microarray analysis. 2) Caspase-3 immunohistochemistry. 3) Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL).
Results: At 23°C the ultrastructure was maintained, but at both 5°C and 31°C the structure was changed. Detachment of epithelial cells, enlarged intercellular spaces and intracellular vacuoles, and separation of desmosomes were all seen at these temperatures. All cultured HLECs remained undifferentiated throughout all storage conditions. Caspase-3 and TUNEL staining did not show any significant increase under storage. Downregulation of BCL2A1, BIRC1, and TNF and upregulation of CARD6 under 23ºC and 5ºC storage conditions suggested a reduction in nuclear factor- B activity. This finding is particular interesting as it may lessen the need for immunosuppression subsequent to storage.
Conclusion: Apoptosis is minimal after OC storage. The article shows that storage of HLECs at the unconventional storage temperature of 23°C is superior to 31°C OC and at 5°C Optisol-GS.