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S. Raeder, T. Paaske Utheim, Ø. Aass Utheim, M. de la Paz, J. R. Eidet, T. Lyberg; Eye Bank Storage of Cultured Corneal Epithelium: Sterility Testing of Storage Media, Long-Term Preservation, and Deepithelialization of the Amniotic Membrane Substrate. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5717.
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In recent studies we have examined the feasibility of eye bank storage of cultured human limbal epithelial cells (HLEC) for transplantation. This study aimed at investigating microbiological sterility, long-term preservation, and effects of deepithelialization of the amniotic membrane substrate of cultured HLEC subject to eye bank storage.
HLEC were expanded from cadaver explants on intact amniotic membranes (iAM) or denuded amniotic membranes (dAM) for 3 weeks and stored in a closed container in organ culture medium at 23°C. Sterility of the storage medium was tested using the Bactec blood bottle method. A laser confocal microscope and digital imaging were used to distinguish live (calcein AM-positive) from dead (ethidium homodimer-1-positive) cells in HLEC cultures on iAM following 2-and 3-week OC storage. Quantitative transmission electron microscopy analysis and scanning electron microscopy was performed on HLEC cultures on iAM and dAM following 1-week storage.
None of the 46 blood culture bottles (Bactec Plus Aerobic/F bottles (n=23) and Bactec Lytic/10 Anaerobic/F bottles (n=23)) were contaminated, giving a contamination rate of 0%. Basal layer viability of cultured limbal epithelial cells was 85.6% ± 13.5% (range 0.25-97%) after 2-weeks storage versus 52.7% ± 13.1% (range 0.26-81%) after 3-weeks storage (P<0.001). Following 1-week storage, the number of desmosomes per µm in HLEC cultured on iAM (1.39 ± 0.77 (range 0.49 - 2.51)) was not significantly different from HLEC cultured on dAM (0.98 ± 0.45 (range 0.02-1.37)), and scanning electron microscopy demonstrated tightly apposed superficial cells in both groups.
These data demonstrates that cultured HLEC may be easily controlled for microbial contamination during eye bank storage and that cultured corneal epithelium demonstrates high cell survival rates for up to 2-weeks storage. With respect to desmosomal attachments denuding of the amniotic membrane did not have any significant impact on the structural integrity of cultured HLEC following storage.
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