June 2015
Volume 56, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2015
Evaluating Retinal Pigment Epithelium Cultures for Establishing Automated, Live-Cell Tracking of Outer Segment Degradation
Author Affiliations & Notes
  • Jason Miller
    Ophthalmology and Visual Sciences, Kellogg Eye Center, University of Michigan, Ann Arbor, MI
  • Qitao Zhang
    Ophthalmology and Visual Sciences, Kellogg Eye Center, University of Michigan, Ann Arbor, MI
  • Steve Rowe
    Cybernet Systems Corporation, Ann Arbor, MI
  • Daniel Peisach
    School of Medicine, Wayne State University, Detroit, MI
  • David N Zacks
    Ophthalmology and Visual Sciences, Kellogg Eye Center, University of Michigan, Ann Arbor, MI
  • Sami Barmada
    Department of Neurology, University of Michigan, Ann Arbor, MI
  • Debra A Thompson
    Ophthalmology and Visual Sciences, Kellogg Eye Center, University of Michigan, Ann Arbor, MI
  • Footnotes
    Commercial Relationships Jason Miller, None; Qitao Zhang, None; Steve Rowe, None; Daniel Peisach, None; David Zacks, None; Sami Barmada, None; Debra Thompson, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2015, Vol.56, 4225. doi:
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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)

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Abstract

Purpose: 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.

Methods: 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.

Results: 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.

Conclusions: 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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