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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. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
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.
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).
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.
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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