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A protocol for the culture and differentiation of highly polarized human retinal pigment epithelial cells.

Nature protocols (2009-04-18)
Shozo Sonoda, Christine Spee, Ernesto Barron, Stephen J Ryan, Ram Kannan, David R Hinton
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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
3,3′,5-Triiodo-L-thyronine sodium salt, γ-irradiated, powder, suitable for, suitable for cell culture
Sigma-Aldrich
Taurine, ≥99%
Sigma-Aldrich
Anti-Na+/K+ ATPase α-1 Antibody, clone C464.6, clone C464.6, Upstate®, from mouse