Establishment of a Continuous Untransfected Human Corneal Endothelial Cell Line and Its Biocompatibility to Denuded Amniotic Membrane

Purpose: To establish an untransfected human corneal endothelial (HCE) cell line and characterize its biocompatibility to denuded amniotic membrane (dAM).

Methods: Primary culture was initiated with a pure population of HCE cells in DMEM/F12 media (pH 7.2) containing 20% fetal bovine serum and various supplements. The established cell line was characterized by growth property, chromosome analysis, morphology recovery, tumorigenicity assay, and expression of marker proteins, cell-junction proteins, and membrane transport proteins. The biocompatibility of HCE cells to dAM was evaluated by light microscopy, alizarin red staining, immunofluorescence assay, and electron microscopy.

Results: HCE cells proliferated to confluence 6 weeks later in primary culture and have been subcultured to passage 224 so far. A continuous untransfected HCE cell line with a population doubling time of 26.20 h at passage 101 has been established. Results of chromosome analysis, morphology, combined with the results of expression of marker protein, cell-junction protein and membrane transport protein, suggested that the cells retained HCE cell properties and potencies to form cell junctions and perform membrane transport. Furthermore, HCE cells, without any tumorigenicity, could form confluent cell sheets on dAMs. The single layer sheets that attached tightly to dAMs had similar morphology and structure to those of HCE in situ and had an average cell density of 3,413±111 cells/mm².

Conclusions: An untransfected and non-tumorigenic HCE cell line has been established, and the cells maintained positive expression of marker proteins, cell-junction proteins and membrane transport proteins. The cell line, with excellent biocompatibility to dAM, might be used for in vitro reconstruction of HCE and provides a promising method for the treatment of diseases caused by corneal endothelial disorders.