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T.R. Kinnick, J.B. Stanton, B. Bakall, B.S. McKay, A.D. Marmorstein; Accumulation of Lipofuscin in Response to Phagocytosis of Photoreceptor Outer Segments in Fetal Human RPE Cells . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2867.
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To determine the effects of photoreceptor outer segment phagocytosis on the accumulation of lipofuscin granules and melanolipofuscin granules in fetal human RPE (fhRPE).
RPE cells were harvested and maintained according to published methods (Hu and Bok, Mol Vis 2000; 7: 14–19). Outer segments (OS) were harvested from porcine eyes under dim red light and separated in 25 – 60% (w/v) continuous sucrose gradients. OS were fed to fhRPE at 5 X 107 cells/insert for 14 days, either in the presence of light (12 hours light by 12 hours dark) or in the dark. Subcellular fractionation was used to isolate lipofuscin, melanolysosomes, and melanosomes, and particles were counted under a microscope.
Feeding of OS to fhRPE resulted in the formation of melanolysosomes and lipofuscin granules in the cells and a diminished number of melanosomes. In contrast, control monolayers of fhRPE which were not exposed to OS did not have melanolysosomes or lipofuscin granules. Exposure of the cells to white light caused the accumulation of lipofuscin–like granules even in the absence of OS feeding. However, lipofuscin accumulation was significantly greater in light exposed fhRPE fed OS.
We have shown that fhRPE in vitro create lipofuscin granules and melanolysosomes in response to OS feeding regardless of exposure to light. Light exposure increases the number of lipofuscin granules formed and causes accumulation of other lipofuscin like material regardless of OS feeding. The fhRPE primary cell culture system is ideal for studying the OS degradation pathway in pigmented RPE cells in vitro.
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