September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
Utilization of an extracellular flux assay to measure mitochondrial respiration in hPSC-RPE
Author Affiliations & Notes
  • Divya Sinha
    Waisman Center, University of Wisconsin-Madison, Madison, Wisconsin, United States
    McPherson Eye Research Institute, University of Wisconsin-Madison, Madison, Wisconsin, United States
  • Gyda C Beeson
    Department of Drug Discovery and Biomedical Sciences, Medical University of South Carolina, Charleston, South Carolina, United States
  • Molly M Wilson
    Waisman Center, University of Wisconsin-Madison, Madison, Wisconsin, United States
  • Janis T Eells
    Department of Biomedical Sciences, University of Wisconsin-Milwaukee, Milwaukee, Wisconsin, United States
  • Craig C Beeson
    Department of Drug Discovery and Biomedical Sciences, Medical University of South Carolina, Charleston, South Carolina, United States
  • David M Gamm
    Department of Ophthalmology and Visual Sciences, University of Wisconsin-Madison, Madison, Wisconsin, United States
    McPherson Eye Research Institute, University of Wisconsin-Madison, Madison, Wisconsin, United States
  • Footnotes
    Commercial Relationships   Divya Sinha, None; Gyda Beeson, None; Molly Wilson, None; Janis Eells, None; Craig Beeson, None; David Gamm, None
  • Footnotes
    Support  Research to Prevent Blindness Catalyst Award, International Retina Research Foundation, Foundation Fighting Blindness, NIH R01EY024588, Retinal Research Foundation Emmett A. Humble Distinguished Directorship, McPherson Eye Research Institute (Sandra Lemke Trout Chair), Carl and Mildred Reeves Foundation, NIH P30HD03352, Muskingum County Community Foundation, Choroideremia Research Foundation, SIG 1S10OD018039-01 for Seahorse XFe96 analyzer, Support Grant P30 CA014520 to SMSSF (core service of the University of Wisconsin Carbone Cancer Center)
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 1140. doi:
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    • Get Citation

      Divya Sinha, Gyda C Beeson, Molly M Wilson, Janis T Eells, Craig C Beeson, David M Gamm; Utilization of an extracellular flux assay to measure mitochondrial respiration in hPSC-RPE. Invest. Ophthalmol. Vis. Sci. 2016;57(12):1140.

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

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Abstract

Purpose : Human pluripotent stem cell-derived RPE (hPSC-RPE) cells are being utilized to model retinal disease in vitro, as well as to potentially treat patients with certain blinding disorders via cell transplantation. In vivo, RPE cells form a highly metabolically active monolayer that performs several important functions requiring efficient mitochondrial oxidative metabolism. Thus, efforts to monitor and optimize hPSC-RPE health should at least in part take into account mitochondrial function. Toward this end, we examined the behavior of hPSC-RPE in a well-established, rigorous extracellular flux (XF) assay after optimizing critical assay parameters.

Methods : RPE cells were differentiated from human embryonic stem cells (hESC) or human induced pluripotent stem cells (hiPSCs) using protocols established by our lab. Pure monolayer cultures of hPSC-RPE were dissociated and then re-seeded at densities of 5000, 10000 or 30000 cells per well on XFe96-well microplates, and cultured for time periods varying from 1 day to two weeks. An XFe96 extracellular flux analyzer (Seahorse Bioscience) was used to measure basal respiration, ATP-coupled and maximal mitochondrial respiration via oxygen consumption rates (OCR). Data was analyzed using XF Wave software (Seahorse Bioscience).

Results : A seeding density of 30,000 cells/well on Seahorse 96-well microplates works optimally to reproducibly measure OCR for hPSC-RPE. At lower seeding densities, confluent monolayers are not formed in 1-2 days resulting in variable OCRs. Basal OCR levels can reach greater than 100 pmol/min when seeded hPSC-RPE cells are maintained longer than 3 days in culture and maximal OCR can be 1.5 – 3-fold of basal.

Conclusions : Efforts to optimize the XF assay for hPSC-RPE succeeded in producing highly robust and reproducible results when seeding density and length of time in culture were taken into consideration. hPSC-RPE demonstrated active mitochondrial respiration in these assays, indicative of overall excellent cell health.

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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