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Gesa M. Grobe, Stephan Reichl; Comparison of Vitamin C Induced Collagen Synthesis in Primary and Immortalized Human Corneal Keratocytes. Invest. Ophthalmol. Vis. Sci. 2011;52(14):2523.
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The purpose of this study was to compare the ability of primary and immortalized human corneal keratocytes to synthesize their own extracellular matrix induced by vitamin C supplementation.
Human corneal keratocytes isolated from corneo scleral rings (HuFib) and immortalized human corneal keratocytes (HCK) were cultivated for several weeks either with or without vitamin C (ascorbic acid) supplementation. The amount of collagen secreted by the keratocytes was quantified using Picrosirius Red staining depending on the time of cultivation. Sirius Red is an anionic dye which is able to interact with repetitive side chains of the collagen molecule. The amount of bound dye can be quantified by photometric measurement at 550 nm. Simultaneously cell viability was measured via MTT assay and cells were counted after trypsinization using a coulter counter. Furthermore, at the end of cultivation the optical transmission of the formed cell sheets was calculated after measuring their optical density.
Picrosirius Red staining revealed that collagen synthesis can be increased by supplementation of ascorbic acid in HuFib cells and in HCK cells whereas the effect was stronger in the primary cells. The total amount of synthesized collagen was comparable in both cell types but the total cell number of HCK cells was considerably higher. Cell sheet formed by HuFib cells showed higher optical transmission between 400 and 800 nm than cell sheets produced by HCK cells.
Vitamin C supplementation increased the collagen synthesis in both primary human corneal keratocytes and immortalized human corneal keratocytes leading to detachable cell sheets. Further experiments will be conducted to compare the tensile strength of the formed cell sheets.
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