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C. D'hondt, R. Ponsaerts, S. P. Srinivas, J. Vereecke, B. Himpens; Morphologic Appearance in Bovine Corneal Endothelial Cells Over Prolonged Time in Culture. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2707.
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© ARVO (1962-2015); The Authors (2016-present)
Aging is known to decrease endothelial cell density. Due to the absence of proliferative capacity of corneal endothelial cells, loss of cells is usually compensated by cell migration and cell enlargement, resulting in an age-related decrease in cell density. Primary cultured bovine corneal endothelial cells (BCEC) are frequently used as a model to study barrier integrity and intercellular communication. To date there have been no reports describing morphologic appearance of BCEC with time in culture. The purpose of this study was to investigate whether BCEC show morphological changes during prolonged time in culture.
Bovine corneal endothelial and epithelial cells were freshly isolated from the cornea (animal age < 2 years) and cultured for 8 to 30 days. Confocal microscopy and FACS analysis were performed to study cell size. Von Willebrand factor was used as an endothelial marker, changes in cytoskeleton morphology were studied by visualization of F-actin and α-tubulin. In order to examine cell morphology in fresh bovine corneas, we applied scanning electron microscopy (SEM).
Analysis of confocal images showed a significant increase in cell area from 710.6 ± 7.8 µm2 (N= 3350) on day 8 after isolation to 2517.4 ± 67.1 µm2 (N= 2454) on day 30. FACS-data are in line with such a cell size increase. Cultured corneal epithelial cells grown for 8 and 30 days did not show a difference in average cell size. Immunocytochemistry also showed a growing perijunctional ring of actin in BCEC with increasing days in culture. We also noted formation of interdigitations in cells cultured for 21 to 30 days. SEM images of corneas showed an increase in cell size and a decrease in cell density with age.
Cultured BCEC show differences in cell size and morphology with time in culture, while these changes are absent in cultured bovine corneal epithelial cells. Native BCEC show age-related changes in cell size. This is the first report of morphological changes with time in culture in BCEC and of age-related changes in native BCEC. These findings can be of importance when performing studies on barrier integrity, wound repair and intercellular communication in cultured BCEC.
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