Abstract
Purpose: :
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.
Methods: :
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.
Results: :
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.
Conclusions: :
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.
Keywords: cornea: epithelium • cornea: storage • transplantation