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T. Yasukawa, A. Nishiwaki, P. Wiedemann, A. Takase, Y. Yafai, W. Eichler, A. Bringmann, Y. Ogura; Three-Dimensional Spheroid Culture System Redifferentiates Retinal Pigment Epithelial Cells Independent of Number of Passages. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5548.
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The culture of retinal pigment epithelial (RPE) cells isolated from human eyes has a critical limitation: dedifferentiation and transdifferentiation into myofibroblasts with repeated passages. Previously, we reported a three-dimensional multicellular spheroid culture system of human RPE cells. The spheroids composed of RPE cells form a monolayer of RPE with tight junction and Bruch's membrane-like collagenous basal membrane. The object of this study is to evaluate the effect of the number of passages in cultured cells on redifferentiation of RPE.
Isolated human RPE cells underwent different number of passages in conventional culture. Then RPE cells at each passage were seeded onto 96-well U-bottom culture plates to allow spheroid formation. As a control, RPE cells were cultured on a flat culture plate. One and four weeks later, RPE cells were sampled for western blotting. The expressions of cytokeratin and smooth muscle actin were determined using corresponding 1st antibodies.
Spheroids composed of RPE cells showed intense expression of both cytokeratin and smooth muscle actin at week 1. Then cytokeratin became preferentially expressed at week 4, while smooth muscle actin was reduced in expression. The number of passages did not affect this characteristic of redifferentiation in the spheroid model. On the other hand, RPE cells in conventional culture tended to decrease the expression of cytokeratin and inversely increase that of smooth muscle actin with repeated passages, which suggested that RPE cells were dedifferentiated and transdifferentiated into myofibroblasts.
Our results demonstrated that RPE cells tended to lose original features as the epithelium in conventional culture and that the multicellular spheroid culture system redifferentiated RPE cells even after repeated passages. The 3-dimensional culture of RPE cells may be beneficial to elucidate functions of RPE.
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