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Jason Miller, Qitao Zhang, Steve Rowe, Daniel Peisach, David N Zacks, Sami Barmada, Debra A Thompson; Evaluating Retinal Pigment Epithelium Cultures for Establishing Automated, Live-Cell Tracking of Outer Segment Degradation. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4225.
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© ARVO (1962-2015); The Authors (2016-present)
To study the degradation of outer segments (OS) by the retinal pigment epithelium (RPE), we seek to establish a live-cell, automated microscopy system to track OS as they traverse the RPE from apical engulfment to basolateral lysosomal degradation. As a first step in establishing this system, we evaluated primary RPE cultures for optimal culture conditions, expression of RPE-specific genes, capacity for chemical transfection, and phagocytic efficiency.
Porcine, bovine, and human fetal (hf) RPE cultures were grown on Transwell supports. The impact of media change frequency on long-term (> 6 weeks) cultures was evaluated by both trans-epithelial electrical resistance (TEER) and expression levels of specific RPE proteins. Approximately 30 different chemical reagents were evaluated for transfection efficiency and impact on TEER. Species-specific differences in phagocytic efficiency were evaluated by immunofluorescence and Western blot. Finally, an automated imaging platform for tracking OS degradation was established using a widefield microscope with Nikon PerfectFocus technology, driven by customized scripts.
Decreasing the frequency of media changes had no impact on TEER or levels of RPE-specific markers. Porcine and bovine RPE expressed significantly higher levels of RPE65 than hfRPE, but comparable levels of MERTK. OTX2 expression was higher in hfRPE than porcine or bovine RPE. Passage 1 hfRPE expressed higher levels of RPE65 than passage 2 hfRPE. Among transfection reagents tested, Viafect (Promega) at a 4:1 reagent:DNA ratio and 1.33ng/ul DNA concentration produced the highest efficiency with nominal toxicity in bovine and hfRPE. No chemical reagent performed adequately in porcine cultures. Phagocytic efficiency was higher in porcine RPE than hfRPE, but both cleared OS by 24-48 h. The automated imaging platform is capable of tracking hundreds of individual RPE cells in 3d over time while on Transwells, laying the groundwork for tracking OS degradation.
Given the capacity to transfect confluent, mature hfRPE with fluorescently-tagged proteins, we have chosen hfRPE cultures as our platform for live-cell tracking of OS degradation. We intend that this platform will provide insights into how OS degradation functions, how it can fail, and the impact of this failure on RPE atrophy in diseases such as macular degeneration.
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