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Che John Connon, James W Foster, Ricardo M Gouveia; Low-glucose Enhances Keratocyte-characteristic Phenotype from Corneal Stromal Cells in Serum-free Conditions. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):1303.
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© ARVO (1962-2015); The Authors (2016-present)
The avascular cornea is a uniquely isolated organ, with its stroma constituting a nutrient-poor environment. Consequently, the availability of metabolites such as glucose to corneal stromal cells is considerably reduced compared with other tissues, or indeed with media commonly used to culture these cells in vitro. However, the role of glucose on the behaviour of human corneal keratocytes has been largely overlooked. As such, we sought to investigate the effects of low-glucose formulations on the phenotype of human corneal stromal cells.
Human corneal keratocytes were extracted from normal corneal limbal rings and expanded in DMEM:F12 containing 5% foetal bovine serum for 2 passages to form differentiated fibroblasts. These cells were then maintained in serum free media of different glucose concentrations (low, 2 or high, 4.5 g.L-1) for >3 weeks and their phenotype monitored through immunohistochemistry, quantitative PCR and immuno-blotting.
Human stromal cells cultured in low-glucose were able to survive for extended periods when compared to high-glucose, serum-free conditions (p≥0.01). Furthermore, low-glucose enhanced their reversal from fibroblast to a keratocyte-characteristic phenotype. Specifically, cells within low-glucose medium assumed dendritic morphologies, with bean-shaped condensed nuclei, absence of alpha-smooth muscle actin or stress fibres, and a corresponding significant reduction in migratory and contractile activities when compared with high-glucose, serum-free conditions (p≥0.01). Moreover, cells within low-glucose recovered the ability to significantly express keratocyte-characteristic markers, CD34 and ALDH1A1 (p≥0.05), above traditional high-glucose serum free media conditions, whereas keratocan, lumican and ALDH3A1 remained comparable.
Our results indicate that low-glucose enhances keratocyte-characteristic phenotype above and beyond established media formulations and thus has important implications for corneal biology in health and disease.
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