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Britta Nommiste, Amanda-Jayne Carr, Carlos Gias, Peter Coffey; EFFECTS OF CULTURE MEDIUM AND SURFACE COATINGS ON RPE CELL DIFFERENTIATION. Invest. Ophthalmol. Vis. Sci. 2013;54(15):3744.
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The human RPE cell line, ARPE-19 is used to study RPE cells in vitro. In culture, with increasing passage, RPE cells dedifferentiate, losing morphology, pigmentation, and proteins key to cell function. Previous studies suggest culture medium affects the differentiation state of ARPE-19 cells. Here, we investigate the effects of culture media and extracellular matrix coatings on ARPE-19 growth to optimize conditions for differentiated RPE cell growth.
ARPE-19 cells were cultured on laminin, collagen I, matrigel, vitronectin and fibronectin coated plasticware. We tested a variety of culture media including HESC media +/-bFGF, DMEM+pyruvate with 1% or 10% fetal calf serum (FCS) and DMEM/F12 with 1% or 10% FCS for up to 70 days. Cells were processed for western blot, light microscopy and immunocytochemistry (ICC). Data was analysed in ImageJ and fluorescence levels evaluated based on a grading system.
Grading results suggested that the culture of ARPE-19 cells in DMEM+pyruvate+1%FCS, HESC+bFGF resulted in differentiation of cells towards RPE, whilst cells cultured in DMEM/F12+1% FCS maintained their undifferentiated state. ICC staining for Pmel17 and ZO-1 correlated with the grading analysis. Based on the fluorescent image analysis culture on laminin, matrigel and vitronectin resulted in more differentiation. The culture of cells on HESC-bFGF resulted in loss of adherence of the cells to the plasticware. Our analysis suggest DMEM/F12 1% serum is not suitable for promoting RPE cell differentiation.
Correct culture conditions are essential to maintain a differentiated cell phenotype. Optimising culture conditions will result in more reliable data when using ARPE-19 cells as a model system. In addition, information gained in these experiments could be transferred to maintain HESC or iPS-derived RPE in optimum conditions over passage.
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