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S. Proulx, A.F. Laplante, P. Carrier, M. Talbot, A. Deschambeault, S.L. Guérin, F.A. Auger, L. Germain; Improvement of the Growth Conditions of Reconstructed Human Corneas . Invest. Ophthalmol. Vis. Sci. 2005;46(13):5004.
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Purpose: This study was conducted in order to improve the macroscopic and histologic features of our model of tissue–engineered corneas reconstructed from cultured human cells. Methods: Reconstructed human corneas were produced by culturing fibroblasts in presence of ascorbic acid to produce sheets on which epithelial cells were seeded and cultured. Limbal epithelial cells came from 2 different donors, and cells from the first donor were used following either one or two cryopreservations. The reconstructed corneas were cultured in growth medium supplemented with either 10%, 30% or 50% fetal bovine serum (FBS) or 10% human serum. The tissue was raised at the air/liquid interface to stimulate differentiation of epithelial cells. For the reconstructed corneas grown in the presence of 10% serum, the epithelium was either left dry or drops of serum (bovine or human) were added twice a day for 2 weeks. The reconstructed corneas were then photographed for macroscopic evaluation and processed for histological studies. Results: The macroscopic evaluation showed that in general, all reconstructed corneas had a nice appearance. Corneas reconstructed with the donor cells that were frozen once gave better results than those from the same donor that were frozen twice. The epithelium that received drops of serum had a better appearance than those that were left dry, they had fewer swirls and were more uniform. The tissues that were cultured in higher serum concentrations (30% and 50% FBS) gave better results than those at 10% serum. There was no evident amelioration for the tissues cultured in medium supplemented with human rather that bovine serum. Conclusions: This study allowed us to optimize the growth conditions for the production of our reconstructed cornea model. Higher serum concentrations in the growth medium improved the macroscopic aspect of the tissue. Adding drops of serum on the epithelium also enhanced the quality of the tissue. The improved reconstructed corneas will then be used for the studies of reepithelialization of the corneal epithelium following wound–healing.
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