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Ana Fernández, Jennifer Ramos, Marta López, Patricia Pérez, Elizabeth Santín, Leire Llorente, Yolanda Diebold, F. Javier Iglesias; Proliferation of porcine conjunctival fibroblasts in fibrin-based scaffolds using alamar blue assay. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1387.
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© ARVO (1962-2015); The Authors (2016-present)
Cell proliferation rate is a common biocompatibility parameter analyzed in tissue engineering when a new biomaterial is tested. Our aim was to analyze the suitability of the AlamarBlue™ proliferation assay (AB) to measure cell proliferation in tridimensional (3D) constructs using porcine conjunctival fibroblasts grown in 3D fibrin-based scaffolds.
Porcine eyes, obtained from a local slaughterhouse, were enzimatically digested (collagenase type I solution for 24 h) in order to obtain conjunctival fibroblast. Fibroblasts were expanded until fifth passage in standard cell culture conditions. To determine optimum cell density to be used as our standard in the AB assay, eight cell seeding densities, ranging from 1x104cells/ml to 1,5x106cells/ml, were assayed in monolayer culture (n=5). Percentage reduction of AB by fibroblasts was calculated using fluorescence measurements at 530nm excitation and 590 emission wavelengths after 4 h incubation (AB assay recommendation). Measurements for each cell density were obtained as standard straight lines. Scaffolds (n=20) were prepared from human cryoprecipitate or plasma at low (LC, 1,5mg/ml) or high (HC, 3,5mg/ml) fibrinogen concentrations. The same 8 cell densities were seeded in fibrin scaffolds and AB measured every h up to 30 hs. A total of 240 samples were analyzed.
Fluorescence signal obtained from fibroblast seeded-scaffolds reached the standard straight line at different AB incubation times. Average incubation time for LC cryoprecipitate and LC plasma scaffolds was 26 hs, while HC cryoprecipitate and HC plasma scaffolds required 28 hs and 30 hs, respectively.
Current knowledge about AB assay indicates that cell proliferation can be measured after 4 hour incubation regardless the used culture surface. Our study demonstrates that there is no dependence between type of culture surface and the incubation time recommended by the AB standard protocol. Rather, the material used for cell culture and, in our case, fibrinogen concentration in it determines optimum measurement protocol. It is relevant to consider this fact when biocompatibility of new materials is tested.
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