Cultivation of limbal stem cells‐derived corneal epithelium on different biologic materials for clinical transplantation

Abstract

Abstract Purpose To develop simple, reproducible, animal‐materials free method for cultivating limbal stem‐cells and differentiating them into corneal epithelium on different human biologic materials for clinical transplantation. Methods The limbal tissues (2x2mm) were harvested from cadavers not more than 8 hours after death and proliferated in vitro on cell culture tissue plates, human amniotic membranes (HAM) or human lens capsules in medium containing human AB serum. Cell viability was tested using the MTT assay and annexin‐FITC/Propidium Iodide positivity methods. Molecular gene and immunofluorescent marker studies for stemness, proliferation and differentiation were used for the analysis. Results Over a period of one year, 50 limbal tissue explants were cultivated. Emergence of cells at one edge of the explants occurred within 24 hours from culturing and formed monolayer within 14 days. Although the speed of cell growth varied among donors and types of media for growth, inadequate growth at two weeks was never recorded. The viability of the cells at 7 and 14 days of cultivation was higher than 96% except in case of HAM use where viability was below 80%. The growing cells were characterized for their positivity for stemness (P63, ABCG2), proliferation (ki67) and epithelial cell markers CK 3, 8, 12, 14, 18 and 19. Conclusion We demonstrate a simple, animal‐materials free technique for generating corneal epithelium from cadavers or alternatively from autologous donors for viable cell growth on different biologic materials for transplantation. The growth of corneal epithelium on lens capsules proved to be superior compared to the other cultivation techiques.