In vitro reconstruction of tissue-engineered human corneal endothelium and characterization of its morphology and structure

• AIM: To reconstruct tissue-engineered human corneal endothelia (TE-HCE) in vitro and characterize them in morphology and structure. • METHODS: Monoclonal HCE cells (mcHCE cells) were cloned from untransfected HCE cell line by limited dilution, and their karyotypes were analyzed by routine methods of chromosomal preparing and karyosystematics. Modified denuded amniotic membranes (mdAMs) were prepared from amniotic membrane by inverted trypsin denudation and coated with extracellular matrix proteins. TE-HCEs were in vitro reconstructed by using mcHCE cells at logarithmic phase as seed cells and mdAMs tiled on well bottoms of a 24-well culture plate as scaffold carriers, which were cultured in 200mL/L fetal bovine serum (FBS)-containing DMEM/F12 medium at 37°C in a 50mL/L CO₂ incubator. The morphology of seed cells, formation of cell junctions, integrality of endothelial monolayer and its integrated status to mdAM were investigated by Alizarin red staining, freeze-section's hematoxylin-eosin (HE) staining, inverted microscopy and scanning electron microscopy. The ultrastructure of seed cells on mdAM and formation of cell junctions were examined by transmission electron microscopy. The expression patterns of different cell junction proteins of TE-HCE seeder cells were detected by immunofluorescent techniques. • RESULTS: Seven mcHCE cell strains with normal karyotype (2n =46) were screened out from the untransfected HCE cell line. About 30 hours after reconstruction initiation, mcHCE seeder cells formed an integrated monolayer on mdAM with a cell density as high as 3 413/mm². Most of seed cells were in polygonal morphology, integral endothelial monolayer was reconstructed with various cell-cell and cell-mdAM junctions. And the ultrastructure of seed cells was similar to that of HCE cells in vivo, with a lot of mitochondria scattered in cytoplasm. Besides, the seed cells maintained positive expression of cell junction proteins such as zonula occludens protein 1, N-cadherin, connecxin-43 and integrin áv/β5. • CONCLSUION: The TE-HCEs, with similar morphology and structure to those of HCE in vivo, were successfully reconstructed, and can be used promisingly as HCE equivalents for clinical corneal endothelium transplantation.