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Olivier Roy, Mathieu Theriault, Olivier Rochette-Drouin, Stephanie Proulx; Re-establishment of a corneal endothelial phenotype from fibroblastic-like cells.. Invest. Ophthalmol. Vis. Sci. 2014;55(13):2045.
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Corneal endothelial cells often adopt a fibroblastic-like morphology following successive passages. However, fibroblastic-like cells cannot be used for the tissue engineering of a corneal endothelium. The purpose of this study was to modify the culture medium in order to obtain a monolayer of polygonal endothelial cells using multi-passaged cells that have a fibroblastic-like morphology.
Human corneal endothelial cells were cultured in growth-promoting media (Opti-MEM I, 8% serum, EGF, NGF, BPE, ascorbic acid, chondroitin sulfate and antibiotics). Third to sixth passaged human corneal endothelial cells were used for this study. Cells were seeded at high density (300 000 cells/cm2) and cultured in DMEM or Opti-MEM media at different calcium concentrations (0.05, 0.9 and 2mM) containing 8% serum. Cells were photographed to assess morphology. Mean cell size was determined using a particle size analyzer (Beckman Coulter Z2). Immunofluorescence staining of alpha-SMA, ZO-1 and N-cadherin was also performed in order to evaluate endothelial-mesenchymal transformation and protein expression of intercellular junctions, respectively.
Corneal endothelial cells that acquired a fibroblastic-like morphology (overlapping elongated cells) were able to revert to an endothelial morphology (a monolayer of contact-inhibited polygonal cells) when cultured in DMEM with 2mM calcium and 8% serum, or when the culture media was switched after 24h from 0.05mM to 2mM calcium. Prolonged culture in the 0.05mM calcium media resulted in high cell death. Not every cell population responded to the optimized media. Though a change of phenotype was observed, morphologically reverted endothelial cells remained slightly larger than their fibroblastic-like counterparts with respectively 15,4±0,2 µm and 14,9±0,1 µm average cell size (resuspended cells). All cells were negative for alpha-SMA and the majority expressed ZO-1 and N-cadherin.
This study shows that it is possible to re-establish a monolayer of polygonal corneal endothelial cells using cells that had a fibroblastic-like phenotype. From now on, cells that were previously discarded because of their fibroblastic-like morphology could now be used for the tissue engineering of a corneal endothelium of high cell density.
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