Abstract
Purpose The purpose of this study was to create organ culture medium for preservation human corneas without components of animal origin. We evaluated the efficiency in maintaining the quality of corneas during organ culture in medium supplemented with umbilical cord blood platelet lysate (UCB-PL).
Methods Human corneas were stored in standard organ culture medium (Eagle’s Minimum Essential Medium buffered with 25 mM HEPES, 2 mM L-glutamine, penicilin, streptomycin and amphotericin B) supplemented with 2% UCB-PL and in the standard medium supplemented with 2% FBS during 28 days at 31 °C. Fourteen corneas (7 pairs) with endothelial cell density (ECD) ≥ 2000 cells/mm2 were enrolled. The quality of the corneal endotelium was assessed by ECD, viability (trypan blue), and morphology of endothelial cells (EC) by light microscopy at 0.,7.,14., 21. and 28. day of organ culture. Following organ culture, immunostaining of flat-mounted corneas was preformed by analysis of expression and distribution of Zonula occludens-1 (ZO-1), (Na+/K+)-ATPases, Ki-67 and caspase 3. Metabolical changes during organ culture were observed by glucose and lactate concentration. Differences in numerical variables between two measurement points were tested with the Wilcoxon test.
Results In terms of preservation of EC, both medium produced similar results. ECD decreased in both media, morphology changes and viabiliy of EC remained comparable between two groups with no significant differences between observed parameters during 28 days. After 28 days of organ culture, ZO-1 was expressed and distributed following a zig-zag line in EC, while (Na+/K+)-ATPases was expressed on the lateral surface of epithelial membranes and EC on corneas stored in both media. Ki67 was detected in the epithelial layer, and was not detected in the endothelium. The expression of caspase 3, was shown by some corneal epithelial cells but it was not detected in EC. Metabolic changes during organ culture showed, decrease in glucose and increase in lactate concentration in both tested media.
Conclusion The present study showed comparable effect of FBS and UCB-PL on biological characteristics on EC during organ culture. The quality of human corneas stored for 28 days at 31 °C in a medium with UCB-PL was preserved. This qualifies UCB-PL as a safe, effective and readily available substitute for FBS and the possibility of its use in serum-free medium in corneal preservation and eye banking field.
