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Olivier Roy, Isabelle Brunette, Stephanie Proulx; Influence Of Hydrodynamic Culture Conditions On The Expression Of Cell Junctions Of Tissue-Engineered Human Corneal Endothelium. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1663.
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This study was undertaken in order to evaluate the influence of fluid flow and pressure on the expression of cell junctions of tissue-engineered corneal endothelium in vitro.
Cultured human corneal endothelial cells were seeded and cultured on a previously devitalized corneal stroma, then either left in standard culture conditions (static cultures) or placed in an artificial anterior chamber with a perfusion rate of of 5 ul/min and a hydrostatic pressure of 18 mm Hg (hydrodynamic cultures). A higher perfusion rate than normal (2.6 ul/min) was chosen in order to maintain a constant perfusion pressure and allow for the additional leak of perfusate through the trabecular meshwork into the dish. After 4-6 days, corneas were photographed then fixed in 3.7% formaldehyde for histology, 2.5% glutaraldehyde for transmission electron microscopy or 4% paraformaldehyde for immunofluorescence staining of F-actin and the tight junction protein ZO-1. Controls consisted of devitalized corneas that were not seeded with corneal endothelial cells, and were processed in the same manner.
Macroscopically, corneas maintained in hydrodynamic cultures were more transparent than the static-cultured corneas. The corneal stromas in the histology cross-sections were thinner in the hydrodynamic-cultured corneas than in the static-cultured corneas. The mean (±SD) collagen spacing, calculated using transmission electron microscopy images, was 27 ±4 nm when corneas were under hydrodynamic culture conditions whereas spacing was 46 ±9 nm when corneas were left in static culture conditions. Control devitalized corneas with no endothelium had a mean collagen spacing of 32 ±6 nm when cultured under the same hydrodynamic conditions. Hydrodynamic-cultured corneal endothelium expressed higher amounts of ZO-1 protein, as assessed by immunostaining.
This study shows that corneal endothelial cells respond to an anterior chamber flow and pressure by improving cell junction expression in vitro. Ultimately, studying the effect of hydrodynamic culture will enable an improved understanding of morphogenesis and cell junction formation of the corneal endothelium.
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