September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
Development of a high content imaging photoreceptor outer segment phagocytosis assay in inducible pluripotent stem cell (iPSc)-derived RPE cells
Author Affiliations & Notes
  • Megan Jabour
    Novartis Institutes for BioMedical Research, Cambridge, Massachusetts, United States
  • Heather MacLeod
    Novartis Institutes for BioMedical Research, Cambridge, Massachusetts, United States
  • Frada Berenshteyn
    Novartis Institutes for BioMedical Research, Cambridge, Massachusetts, United States
  • Arnaud Lacoste
    Novartis Institutes for BioMedical Research, Cambridge, Massachusetts, United States
  • Kathryn McAllister
    Novartis Institutes for BioMedical Research, Cambridge, Massachusetts, United States
  • Footnotes
    Commercial Relationships   Megan Jabour, Novartis (E); Heather MacLeod, Novartis (E); Frada Berenshteyn, Novartis (E); Arnaud Lacoste, Novartis (E); Kathryn McAllister, Novartis (E)
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 259. doi:
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      Megan Jabour, Heather MacLeod, Frada Berenshteyn, Arnaud Lacoste, Kathryn McAllister; Development of a high content imaging photoreceptor outer segment phagocytosis assay in inducible pluripotent stem cell (iPSc)-derived RPE cells. Invest. Ophthalmol. Vis. Sci. 2016;57(12):259.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Daily phagocytosis of shed photoreceptor outer segments (OS) by the retinal pigmented epithelium (RPE) is essential to maintaining the health of the retina, and dysfunction of this process has been implicated in retinal degeneration. Prior studies have focused on OS phagocytosis by transformed RPE cell lines or freshly isolated RPE cells. The aim of these studies is to use high content imaging to develop an OS phagocytosis assay with iPSc derived RPE cells.

Methods : iPSc derived RPE cells and the transformed RPE cell line, ARPE-19, were cultured in 384-well plates for 3-5 weeks prior to phagocytic challenge. Isolated bovine OS were labeled with FITC isomer or Qtracker 800 nanocrystals. OS were incubated with RPE cells for 2-24 hours at 37°C, washed, and fixed with 4% paraformaldehyde. Cells were then permeabilized with ice cold methanol and stained for rhodopsin or ZO-1, to evaluate tight junction formation. Images were acquired on the Molecular Devices ImageXpress MicroXL, and OS counts quantified using MetaXpress Transfluor HT analysis module.

Results : Both iPSc derived RPE and ARPE-19 cells form tight junctions when cultured in 384 well format, but only iPSc derived RPE develop the cobblestone morphology typical of RPE cells. Visualization of RPE phagocytosis was achieved through direct labeling of OS with either Qtracker nanocrystals or FITC isomer. Complete colocalization with rhodopsin staining was observed with FITC labeled OS. Further evaluation of serial diluted OS demonstrate that ARPE-19 and iPSc derived RPE cells phagocytose OS in a concentration-dependent manner with maximal phagocytosis occurring at approximately 4 hours.

Conclusions : We have developed a 384-well high content imaging assay allowing for visualization and quantification of OS phagocytosis by iPSc derived RPE cells. This in turn may enable high throughput evaluation and deconvolution of pathways involved in the phagocytic process.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

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