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T. Mimura, S. Amano, T. Usui, N. Honda, S. Yokoo, S. Yamagami; Cell Sheet Using Cultured Human Corneal Endothelial Cell Precursors. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5718.
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© ARVO (1962-2015); The Authors (2016-present)
To construct cell sheet from cultured human corneal endothelial cell (HCEC) precursors and to evaluate the sheet compared with cultured HCEC sheet.
Cultured fifth passage HCEC was used. Sphere-forming assay was performed to produce precursors from cultured HCEC. Precursors dissociated from spheres with EDTA, were seeded onto the denuded human amniotic membranes and were cultured for 48 hours. Cultured HCECs on amniotic membrane were used as a control. The expressions of 5-bromo-2'-deoxyuridine (BrdU), and a tight junction protein ZO-1 were examined in cells of HCEC precursors and cultured HCEC on amniotic membrane by immunocytochemistry. The transport activity of the HCEC precursor and cultured HCEC sheets were evaluated by Ussing chamber system.
The density of cells derived from HCEC precursors on amniotic membrane was significantly higher than that of cells from cultured HCECs (p<0.05). BrdU- and nestin-positive cell number in HCEC precursor sheet was higher than that in cultured HCEC sheet. Cells derived from HCEC precursors showed regular hexagonal morphology by ZO-1 staining, compared with those from cultured HCECs. HCEC precursor sheets, compared with human donor corneas, showed adequate pump function parameters by Ussing chamber system.
These findings indicate that sphere-forming assay using cultured HCEC provides high density and regular hexagonal morphology of cells for construction of HCEC sheet and may contribute to regeneration and transplantation of HCEC.
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