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J. K. Slettedal, K. Beraki, K. Haug, A. Thorsrud, B. Nicolaissen; Metal Ions Increase Cell Density in a Corneal Endothelial Cell Monolayer. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1796.
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© ARVO (1962-2015); The Authors (2016-present)
To examine the effect of various metal ions on corneal endothelium in cell culture. A too low endothelial cell density (ECD) leads to bullous keratopathy. In eye banks donor corneas are discarded due to too low ECD and after transplantation a decrease in ECD may result in graft failure.
Corneal endothelial cells were obtained from bovine eyes and primary culture was established. After trypsination cells were cultured for 48 hours in plastic dishes. The culture medium consisted of Minimal essential medium with 8% fetal bovine serum with the addition of aluminium, copper, lithium, magnesium, or zinc metal salts in concentration ranging from 1 to 300 mM. Three different concentrations were used for each metal salt. Cell density was measured by counting cells. BrdU was added to the medium 16 hours before fixation and incorporation into cells was assessed by immunostaining.
A confluent monolayer was observed in all cultures. In five similar control cultures without any addition of metal ions the ECD in the central area ranged from 1683 to 2525 (mean 2156) cells per mm². Cultures with added aluminium showed ECD from 2018 to 2224, copper 1614 to 2190, lithium 2452 to 3078, magnesium 3523 to 3986 and zinc 2213 to 3525 cells per mm ². BrdU-staining was largely negative in the central area of confluent cells and mostly positive at the edge of the confluent area in all cultures. The cells showed an endothelial phenotype in all cultures. In the cultures with added aluminium and copper, in which the highest concentrations of metal salts were used, the morphology showed great variability in cell size and shape as well as incorporation of small dark particles in the cytoplasm.
Our results indicate that addition of lithium, magnesium and zinc metal ions increases the maximum cell density in a corneal endothelial monolayer in cell culture. Cultures with added aluminium and copper did not show any increase in ECD, but likely toxic concentrations of metal salts were used. For neither of the metal ions could a dose-dependent increase in ECD be proved. However, this might be showed by using more optimal concentrations of metal ions. The mechanism might be interference with adhesion molecules, which in turn reduces contact inhibition, allowing the cells to proliferate more rapidly and thus to establish a more cell dense monolayer. The human corneal endothelium is regarded as a non-replicative cell layer in vivo. Manipulation of the ECD is of interest in relation to corneal eye banking and ultimately as in vivo therapy in cases with low ECD.
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