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C. Maltusch, S. Johnen, L. Wickert, S. Kaempf, A. K. Salz, P. Walter, G. Thumann; 3T3 Feeder Cells Highly Improve Efficiency of Primary Pigment Cell Cultures. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3869.
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© ARVO (1962-2015); The Authors (2016-present)
To develop an optimized culture system for in vitro expansion of iris pigment epithelial (IPE) and retinal epithelial (RPE) cells.
Freshly isolated porcine and human RPE and IPE (PKH26-labeled) cells were seeded on a mitomycin inhibited 3T3 cell monolayer (DiO-labeled). Adhesion, spreading and cell division were monitored by video microscopy. At confluence cell phagocytic activity and tight junction formation were determined as an indication of cell functionality. Differences in gene expression between cells cultured on 3T3 layers and on plastic and between freshly isolated cells and cultured cells were examined by SYBR Green-based quantitative real-time PCR.
At 50 µg/ml mitomycin completely inhibited mitosis of 3T3 cells. In the presence of mitotically inhibited 3T3 cells, freshly isolated RPE and IPE cells adhere, spread and acquire the typical hexagonal epithelial shape within 12 hours of seeding. In cells cultured on a plastic substratum attachment or spreading was observed significantly delayed. After 2 weeks cells cultured on a mitotically inhibited cell layer, exhibited clear tight junctions and phagocytosis of rod outer segments (ROS). Both cell types, RPE and IPE cells showed a similar phagocytic activity. RPE as well as IPE cells cultured on feeder layer matrix showed equal mRNA expression of critical genes, e.g. RPE65 and CRALBP compared to cells cultured on plastic.
RPE and IPE cells attach, spread and acquire epithelial characteristics in as little as 12 hours after seeding. Culture of pigment cells on 3T3 feeder layers is ideal for in vitro studies of cell function, differentiation and dedifferentiation, especially in studies that require large number of cells.
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