Abstract:

To reconstruct tissue-engineered human corneal endothelium (TE-HCE) suitable for corneal transplantation, this study was intended to screen out monoclonal cell strains from a untransfected human corneal endothelial (HCE) cell line, and reconstruct TE-HCE in vitro by using these cells as seeder cells. Monoclonal cell strains were screened out from the HCE cell line by limit dilution. Results of morphology, structure and chromosome analysis, combined with the results of expression of cell-junction proteins and membrane transport proteins, suggested that the cells from mcHCE2401 monoclonal cell strains had steady and normal morphology, structure, karyotype, and positive expression of cell junction proteins and membrane transport proteins as well. All these imply that mcHCE2401 cells have ideal characteristics of seeder cells for TE-HCE reconstruction. By using mcHCE2401 cells as seeder cells, modified denuded amniotic membrane (mdAM) as scaffold carrier, TE-HCE was reconstructed. Results of morphology and structure examination showed that polygonal mcHCE2401 cells formed a continuous and intact monolayer on mdAM with extensive cellcell and cellmdAM cell junctions. The average cell density of the monolyer was as high as 3602.22±45.22cell／mm² (equivalent to HCE cell density of a 0～3 years old baby). All these indicate that the reconstructed TE-HCE had very similar morphology and structure to those of HCE in situ. In conclusion, a monoclonal cell strain with normal morphology, structure, karyotype and celljunction and membrane transport protein expression has been established, and “youngest”TEHCE with similar morphology and structure to those of HCE in situ has been reconstructed successfully in vitro and provides a promising equivalent of donated HCE for clinical treatment of diseases caused by corneal endothelial disorders.

Key words: human corneal endothelial cell; monoclonal cell strain; modified denuded amniotic membrane; tissue-engineered human corneal endothelia; reconstruction