Abstract
We provide our detailed, standardized in vitro protocol for the culture and differentiation of human retinal pigment epithelial (RPE) cells into a highly polarized and functional monolayer. Disruption of the polarized RPE function plays an important role in the pathogenesis of common blinding disorders of the retina. The availability of this polarized RPE monolayer allows for reproducible evaluation of RPE function, modeling of RPE dysfunction in retinal disease and in vitro evaluation of new therapies. The protocol, which takes approximately 6 weeks to complete, describes the culture of RPE from human fetal donor eyes, the differentiation of these cells into a polarized monolayer with high transepithelial resistance and morphologic characteristics that mimic the RPE monolayer in vivo. By modifying the procedure for initial isolation of pure RPE cells and the culture conditions used in existing protocols, we have established a standardized protocol that provides highly reproducible RPE monolayers from the same donor eye.
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Acknowledgements
Supported by funds from The Arnold and Mabel Beckman Foundation, National Institutes of Health Grants EY01545 and core grant EY03040, and the Research to Prevent Blindness Inc. The authors thank P.G. Sreekumar for his early contributions to the development of these methods.
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Sonoda, S., Spee, C., Barron, E. et al. A protocol for the culture and differentiation of highly polarized human retinal pigment epithelial cells. Nat Protoc 4, 662–673 (2009). https://doi.org/10.1038/nprot.2009.33
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DOI: https://doi.org/10.1038/nprot.2009.33
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