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Thomas Vo, Sina Abedi, Marilyn Chwa, Cristina M Kenney; Phagocytosis Assay to Measure Function of Human RPE Cells with AMD vs Normal Mitochondria. Invest. Ophthalmol. Vis. Sci. 2017;58(8):5242.
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© ARVO (1962-2015); The Authors (2016-present)
A critical biological function of RPE cells is phagocytosis of photoreceptor outer segments (POS) disc membranes. Each RPE cell ingests thousands of discs per day and incomplete digestion of POS leads to accumulation of lipofuscin granules, which are photo-inducible generators of reactive oxygen species. Mitochondrial damage and dysfunction are associated with RPE cells of AMD retinas. We hypothesize that RPE cells that contain AMD mitochondria will demonstrate different phagocytosis properties when compared to RPE cells with normal mitochondria.
Our lab created cytoplasmic hybrids (cybrids) by first isolating platelets from the blood of AMD and age-matched non-AMD patients. ARPE-19 cells, a cell line derived from human retinal epithelia (ATCC, Manassa, VA, USA), are converted to Rho0 (deficient of mtDNA) by serial passage in ethidium bromide. The cybrids are produced by fusion of platelets with Rho0 ARPE-19 cells. The resulting cells have unique mtDNA, but shared nuclear DNA. For experimentation, the cells were plated onto 6-well plates at a density of 800,000 cells/well. A bead solution was created by adding 10µL of yellow-green fluorescent beads (Polysciences Inc, diameter 1-µm) to 30mL media; reducing the initial concentration of 4.55 x 1010 beads/mL to 1.52 x 107 beads/mL. 2 mL of the solution was added to each well. Following 16 hours of incubation at 37o C, the cells were washed to remove unbound beads. Flow cytometry was performed with the ImageStreamX Mark II Imaging Flow Cytometer (EMD Millapore). Acquisition was set to a 5000 cell count per sample. Data analysis and image capture were additionally performed with the ImageStreamX software. Triplicate samples of the AMD and normal cybrids were evaluated for mean and standard deviation of the mean.
37.3% of the AMD cybrids versus 44% of the normal cybrids demonstrated phagocytosis of the beads at 16 hours. The standard deviation was 5.97 and 5.03, respectively. The results for this unpaired t-test were not statistically significant with a p-value of 0.2181.
Our initial findings suggest that the cybrids possessing AMD mitochondria have equivalent phagocytosis capacity to those with age-matched normal mitochondria. However, further studies will be undertaken to vary time exposures and concentration of fluorescent beads to characterize the role of mitochondria in the phagocytosis functions.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
Cybrids showing bead intake
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