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 karyosyste-matics.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℃ 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 expres-sion 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 untrans-fected HCE cell line.About 30 hours after reconstruction initiation,mcHCE seeder cells formed an integrated mono-layer on mdAM with a cell density as high as 3413/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 αv/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.