We thank Drs. Daniel Hass and Jim Hurley for the idea of testing O2 solubility in new versus conditioned media as a control. We thank Dr. Magali Saint-Geniez for editorial input on the manuscript. We thank Scott Szalay at Instrument and Electronic Services Core, Kellogg Eye Center, for retrofitting the hypoxia chamber with the Resipher USB cable. No federal funds were used for HFT research. The Electronic Services Core is supported by P30 EY007003 from the National Eye Institute. This work is supported by an unrestricted departmental grant from Research to Prevent Blindness (RPB). J.M.L.M. is supported by the James Grosfeld Initiative for Dry Age-Related Macular Degeneration, the E. Matilda Ziegler Foundation for the Blind, an Eversight eye-bank grant, a K08EY033420 grant from the National Eye Institute, and support from Dee and Dickson Brown as well as the David and Lisa Drews Discovering Hope Foundation. D.Y.S. is supported by the UNSW Scientia Program. L.A.K. is supported by the Iraty Award, Monte J. Wallace, Michel Plantevin, an R01EY027739 grant from the National Eye institute, and the Department of Defense Army Medical Research Acquisition Activity VR220059.

For protocols to establish human primary or iPSC-RPE cultures, see the following references15,16,17,18. The acquisition and use of human tissue for these protocols were reviewed and permitted by the University of Michigan Institutional Review Board (HUM00105486).
1. General application of the system to RPE culture
<ol>
	<li>Plate human induced plur...

Novel OCR Monitoring System for Differentiated RPE Cultures

<ol>
	<li>Shu, D. Y., Butcher, E., Saint-Geniez, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EMT+and+EndMT:+emerging+roles+in+age-related+macular+degeneration.">EMT and EndMT: emerging roles in age-related macular degeneration.</a> Int J Mol Sci. 21 (12), 4271 (2020).</li><li>Shu, D. Y., Lovicu, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myofibroblast+transdifferentiation:+the+dark+force+in+ocular+wound+healing+and+fibrosis.">Myofibroblast transdifferentiation: the dark force in ocular wound healing and fibrosis.</a> Prog Retin Eye Res. 60, 44-65 (2017).</li><li>Hurley, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retina+metabolism+and+metabolism+in+the+pigmented+epithelium:+a+busy+intersection.">Retina metabolism and metabolism in the pigmented epithelium: a busy intersection.</a> Annu Rev Vis Sci. 7, 665-692 (2021).</li><li>Wu, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deficient+RPE+mitochondria+energetics+leads+to+subretinal+fibrosis+in+age-related+macular+degeneration.">Deficient RPE mitochondria energetics leads to subretinal fibrosis in age-related macular degeneration.</a> Invest Ophthalmol Vis Sci. 64 (8), 2420 (2023).</li><li>Hansman, D. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+reprogramming+of+the+retinal+pigment+epithelium+by+cytokines+associated+with+age-related+macular+degeneration.">Metabolic reprogramming of the retinal pigment epithelium by cytokines associated with age-related macular degeneration.</a> Biosci Rep. 44 (4), BSR20231904BSR20231904 (2024).</li><li>Li, K. X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+pyruvate+kinase+M2+activity+as+a+therapy+in+a+primary+RPE+cell+culture+model+of+proliferative+vitreoretinopathy.">Modulation of pyruvate kinase M2 activity as a therapy in a primary RPE cell culture model of proliferative vitreoretinopathy.</a> Invest Ophthalmol Vis Sci. 62 (8), 2213 (2021).</li><li>Gupta, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+in+stem+cells-based+replacement+therapy+for+retinal+pigment+epithelium:+in+vitro+differentiation+to+in+vivo+delivery.">Progress in stem cells-based replacement therapy for retinal pigment epithelium: in vitro differentiation to in vivo delivery.</a> Stem Cells Transl Med. 12 (8), 536-552 (2023).</li><li>Wilson, D. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen+distribution+and+vascular+injury+in+the+mouse+eye+measured+by+phosphorescence-lifetime+imaging.">Oxygen distribution and vascular injury in the mouse eye measured by phosphorescence-lifetime imaging.</a> Appl Opt. 44 (25), 5239-5248 (2005).</li><li>Hass, D. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Medium+depth+influences+O2+availability+and+metabolism+in+human+RPE+cultures.">Medium depth influences O2 availability and metabolism in human RPE cultures.</a> Invest Ophthalmol Vis Sci. 64 (14), 4 (2023).</li><li>Shu, D. Y., Butcher, E. R., Saint-Geniez, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suppression+of+PGC-1&#945;+drives+metabolic+dysfunction+in+TGF&#946;2-Induced+EMT+of+retinal+pigment+epithelial+cells.">Suppression of PGC-1&#945; drives metabolic dysfunction in TGF&#946;2-Induced EMT of retinal pigment epithelial cells.</a> Int J Mol Sci. 22 (9), 4701 (2021).</li><li>Shu, D. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dimethyl+fumarate+blocks+tumor+necrosis+factor-alpha-driven+inflammation+and+metabolic+rewiring+in+the+retinal+pigment+epithelium.">Dimethyl fumarate blocks tumor necrosis factor-alpha-driven inflammation and metabolic rewiring in the retinal pigment epithelium.</a> Front Mol Neurosci. 15, 896786 (2022).</li><li>Ng, P. Q., Saint-Geniez, M., Kim, L. A., Shu, D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Divergent+metabolomic+signatures+of+TGF&#946;2+and+TNF&#945;+in+the+induction+of+retinal+epithelial-mesenchymal+transition.">Divergent metabolomic signatures of TGF&#946;2 and TNF&#945; in the induction of retinal epithelial-mesenchymal transition.</a> Metabolites. 13 (2), 213 (2023).</li><li>Kar, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Choriocapillaris+impairment+is+associated+with+delayed+rod-mediated+dark+adaptation+in+age-related+macular+degeneration.">Choriocapillaris impairment is associated with delayed rod-mediated dark adaptation in age-related macular degeneration.</a> Invest Ophthalmol Vis Sci. 64 (12), 41 (2023).</li><li>Curcio, C. A., Kar, D., Owsley, C., Sloan, K. R., Ach, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+macular+degeneration,+a+mathematically+tractable+disease.">Age-related macular degeneration, a mathematically tractable disease.</a> Invest Ophthalmol Vis Sci. 65 (3), 4 (2024).</li><li>Maminishkis, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confluent+monolayers+of+cultured+human+fetal+retinal+pigment+epithelium+exhibit+morphology+and+physiology+of+native+tissue.">Confluent monolayers of cultured human fetal retinal pigment epithelium exhibit morphology and physiology of native tissue.</a> Invest Ophthalmol Vis Sci. 47 (8), 3612-3624 (2006).</li><li>Maminishkis, A., Miller, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+models+for+study+of+retinal+pigment+epithelial+physiology+and+pathophysiology.">Experimental models for study of retinal pigment epithelial physiology and pathophysiology.</a> J Vis Exp: JoVE. (45), e2032 (2010).</li><li>Zhang, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+platform+for+assessing+outer+segment+fate+in+primary+human+fetal+RPE+cultures.">A platform for assessing outer segment fate in primary human fetal RPE cultures.</a> Exp Eye Res. 178, 212-222 (2019).</li><li>Sharma, R., Bose, D., Montford, J., Ortolan, D., Bharti, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Triphasic+developmentally+guided+protocol+to+generate+retinal+pigment+epithelium+from+induced+pluripotent+stem+cells.">Triphasic developmentally guided protocol to generate retinal pigment epithelium from induced pluripotent stem cells.</a> STAR Protoc. 3 (3), 101582 (2022).</li><li>Marchetti, P., Fovez, Q., Germain, N., Khamari, R., Kluza, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+spare+respiratory+capacity:+Mechanisms,+regulation,+and+significance+in+non-transformed+and+cancer+cells.">Mitochondrial spare respiratory capacity: Mechanisms, regulation, and significance in non-transformed and cancer cells.</a> FASEB J. 34 (10), 13106-13124 (2020).</li><li>Rosenfeld, P. J., Trivizki, O., Gregori, G., Wang, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+update+on+the+hemodynamic+model+of+age-related+macular+degeneration.">An update on the hemodynamic model of age-related macular degeneration.</a> Am J Ophthalmol. 235, 291-299 (2022).</li><li>Fitch, T. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+analysis+of+bioenergetics+in+primary+human+retinal+pigment+epithelial+cells+using+high-resolution+respirometry.">Real-time analysis of bioenergetics in primary human retinal pigment epithelial cells using high-resolution respirometry.</a> J Vis Exp: JoVE. (192), (2023).</li><li>Calton, M. A., Beaulieu, M. O., Benchorin, G., Vollrath, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+measuring+extracellular+flux+from+intact+polarized+epithelial+monolayers.">Method for measuring extracellular flux from intact polarized epithelial monolayers.</a> Mol Vis. 24, 425-433 (2018).</li><li>Grumbine, M. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintaining+and+assessing+various+tissue+and+cell+types+of+the+eye+using+a+novel+pumpless+fluidics+system.">Maintaining and assessing various tissue and cell types of the eye using a novel pumpless fluidics system.</a> J Vis Exp: JoVE. (197), (2023).</li><li>Mu, C., Shearer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocol+for+measuring+respiratory+function+of+mitochondria+in+frozen+colon+tissue+from+rats.">Protocol for measuring respiratory function of mitochondria in frozen colon tissue from rats.</a> STAR Protoc. 4 (4), 102560 (2023).</li><li>Rocha, D. S., Manucci, A. C., Bruni-Cardoso, A., Kowaltowski, A. J., Vilas-Boas, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+practical+and+robust+method+to+evaluate+metabolic+fluxes+in+primary+pancreatic+islets.">A practical and robust method to evaluate metabolic fluxes in primary pancreatic islets.</a> Mol Metab. 83, 101922 (2024).</li><li>Kanaan, M. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cystine/glutamate+antiporter+xCT+controls+skeletal+muscle+glutathione+redox,+bioenergetics+and+differentiation.">Cystine/glutamate antiporter xCT controls skeletal muscle glutathione redox, bioenergetics and differentiation.</a> Redox Biol. 73, 103213 (2024).</li><li>da Veiga Moreira, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methylene+blue+metabolic+therapy+restrains+in+vivo+ovarian+tumor+growth.">Methylene blue metabolic therapy restrains in vivo ovarian tumor growth.</a> Cancers. 16 (2), 355 (2024).</li><li>McNally, L. A., Altamimi, T. R., Fulghum, K., Hill, B. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Considerations+for+using+isolated+cell+systems+to+understand+cardiac+metabolism+and+biology.">Considerations for using isolated cell systems to understand cardiac metabolism and biology.</a> J Mol Cell Cardiol. 153, 26-41 (2021).</li><li>Misare, K. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+consequences+of+tetraploidy+on+Caenorhabditis+elegans+physiology+and+sensitivity+to+chemotherapeutics.">The consequences of tetraploidy on Caenorhabditis elegans physiology and sensitivity to chemotherapeutics.</a> Sci Rep. 13 (1), 18125 (2023).</li></ol>

Richard A. Bryan and Kin Lo are employees of Lucid Scientific, which manufactures the Resipher system.

Mitochondrial metabolism of the RPE plays a critical role in the pathogenesis of common blinding retinal diseases, including AMD and PVR. Modeling RPE mitochondrial metabolism in vitro allows one to isolate its metabolic state from those of surrounding tissues, along with subjecting the tissue to different disease-simulating insults in a controlled manner. Such in vitro modeling of RPE mitochondrial metabolism has been facilitated by the advent of high-fidelity human primary and iPSC-RPE cultures that a...

The retinal pigment epithelium (RPE) is a monolayer of functionally postmitotic, highly polarized epithelial cells that form a barrier between light-sensitive photoreceptors in the retina and their blood circulation, a capillary bed termed the choriocapillaris. Like the role of glia-supporting neurons, the RPE carries out myriad functions to support photoreceptors, including phagocytosis of photoreceptor outer segments, transport of nutrients and metabolic support for photoreceptors, and secretion of essential growth factors, all critical for maintaining visual function.
Degeneration of the RPE underlies several common degenerative disorders of vision. In age-related macular degeneration (AMD), one of the most common causes of incurable vision loss in the world, the RPE dies, and overlying photoreceptors therefore suffer a secondary degeneration. In proliferative vitreoretinopathy (PVR), the RPE instead exits its normally quiescent postmitotic state, proliferating and dedifferentiating into a mesenchymal state (a so-called epithelial-to-mesenchymal transition [EMT]) with alterations in its metabolism. RPE dedifferentiation causes a loss of RPE support to photoreceptors while also triggering a more fibrotic state. This results in both photoreceptor degeneration and RPE-induced scarring, both of which trigger vision loss1,2.
A major part of the RPE&#39;s support to photoreceptors is metabolic, and metabolic dysregulation is a critical factor in numerous retinal diseases, including AMD and PVR. The RPE serves as a regulatory barrier between photoreceptors and their source of oxygen and nutrients, the choriocapillaris. Thus, what the RPE chooses to metabolize versus what the RPE chooses to pass through from the choriocapillaris to the photoreceptors strongly governs photoreceptor metabolism and survival. Numerous studies have shown that the RPE is heavily dependent on mitochondrial metabolism for its normal health, and that photoreceptors instead rely heavily on glycolysis3. This has introduced the concept of complementary, intertwined metabolic states between photoreceptors and the RPE. Specifically, the RPE reduces its metabolism of preferred photoreceptor metabolic substrates and instead utilizes the byproducts of photoreceptor metabolism combined with the metabolites that photoreceptors do not consume. In diseases such as PVR and AMD, evidence strongly suggests that the RPE becomes more glycolytic and less dependent on mitochondrial metabolism; this shift towards RPE glycolysis may starve photoreceptors of metabolites it needs, triggering degeneration4,5,6. Given how interdependent RPE and photoreceptor metabolism are and how much altered metabolism underlies retinal disease, there is strong interest in modeling and manipulating RPE metabolism for therapeutic purposes.
While studying RPE mitochondrial metabolism in vivo is ideal, many aspects of RPE mitochondrial metabolism can only be practically probed in an in vitro culture system. Significant progress towards high-fidelity RPE cultures has been made in the past several decades, to the point that the most carefully groomed RPE cultures are now being used for cell replacement therapy in human clinical trials7. To maintain such high-fidelity cultures, the RPE needs to be grown on particular substrates in particular media for months prior to experimentation. With these conditions, RPE cultures are maximally differentiated and polarized, approximating in vivo RPE. Unfortunately, there is no equipment currently available that can measure mitochondrial metabolism specifically from the RPE in vivo. While oxygen monitoring of the retinal capillary network has been achieved in vivo using electron paramagnetic resonance (EPR) oximetry8, this is not possible for RPE analysis. Differences between RPE metabolism in vivo and in vitro are not well-described, but RPE cultures have been shown to have high mitochondrial activity, similar to RPE in vivo3,9, suggesting significant insight into RPE mitochondrial metabolism can be gained using high-fidelity RPE cultures.
As all mitochondrial metabolism leads to oxygen consumption, measuring RPE oxygen consumption rates (OCR) is a faithful proxy for mitochondrial metabolism. Measuring OCR in RPE cultures has been difficult, as the conditions that promote maximal RPE polarization and differentiation often preclude long-term accurate OCR measurements with currently available techniques, such as the Seahorse Analyzer. In this methods paper, a novel device, termed the Resipher (hereafter referred to as &#34;the system&#34;), is introduced, which allows continuous measurement of OCR over weeks in RPE grown under conditions that maximally promote polarization and differentiation. The ease with which OCR can be measured by this system in RPE culture conditions that maximally promote RPE differentiation and polarization is unique among existing OCR-measuring devices.
This paper provides tips and tricks for using the system with RPE cultures, followed by a demonstration of two particular applications. First, RPE EMT, mimicking PVR, is triggered by exposure to transforming growth factor-beta (TGF&#946;)1,10,11,12. The system is used to monitor how RPE metabolism evolves during the EMT process. Second, the role of hypoxia in RPE metabolism is explored using this system. Hypoxia is an important contributor to the pathogenesis of AMD, as the choriocapillaris thins with age13,14. Combining this system with hypoxia chambers allows one to model altered RPE mitochondrial metabolism with the subtle hypoxia that accompanies aging. Finally, an online calculator using Resipher data is introduced to allow one to determine whether RPE cultures are in hypoxic, normoxic, or hyperoxic conditions. Such information is critical for drawing any conclusions about RPE metabolism from in vitro RPE culture studies.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.25% Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>#25-200-056</td>
			<td></td>
		</tr>
		<tr>
			<td>3,3&#39;,5-triiodo-L-thyronine sodium salt</td>
			<td>Sigma</td>
			<td>T5516</td>
			<td></td>
		</tr>
		<tr>
			<td>32-channel Resipher lid</td>
			<td>Lucid Scientific</td>
			<td>NS32-101A for Falcon</td>
			<td></td>
		</tr>
		<tr>
			<td>Antimycin A from streptomyces sp.</td>
			<td>Sigma</td>
			<td>A8674-25MG</td>
			<td>Inhibitor of Complex III of the electron transport chain</td>
		</tr>
		<tr>
			<td>BAM15</td>
			<td>Sigma</td>
			<td>SML1760-5MG</td>
			<td>Uncoupling agent to increase mitochondrial respiration</td>
		</tr>
		<tr>
			<td>DMSO, cell culture grade</td>
			<td>Sigma-aldrich</td>
			<td>D4540-100ML</td>
			<td>Vehicle for reconstituting mitochondrial drugs</td>
		</tr>
		<tr>
			<td>Extracellular matrix coating substrates: 
			Synthemax II-SC</td>
			<td>Corning</td>
			<td>#3535</td>
			<td>Extracellular matrix for hfRPE</td>
		</tr>
		<tr>
			<td>Extracellular matrix coating substrates: Vitronectin</td>
			<td>Gibco</td>
			<td>A14700</td>
			<td>Extracellular matrix for iPSC-RPE</td>
		</tr>
		<tr>
			<td>FCCP</td>
			<td>Sigma</td>
			<td>C2920-10MG</td>
			<td>Uncoupling agent to increase mitochondrial respiration</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (Bio-Techne S11550H)</td>
			<td>Bio-Techne</td>
			<td>S11550H</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrocortisone-Cyclodextrin</td>
			<td>Sigma</td>
			<td>H0396</td>
			<td></td>
		</tr>
		<tr>
			<td>Hypoxia chamber</td>
			<td>Embrient Inc.</td>
			<td>MIC-101</td>
			<td></td>
		</tr>
		<tr>
			<td>N1 Media Supplement</td>
			<td>Sigma</td>
			<td>N6530</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-Essential Amino Acids Solution</td>
			<td>Gibco</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr>
			<td>O2 sensor</td>
			<td>Sensit technology or Forensics Detectors</td>
			<td>P100 or FD-90A-O2</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human TGF&#946;2</td>
			<td>Peprotech</td>
			<td>100-35B</td>
			<td>Transforming growth factor beta-2 to induce epithelial-mesenchymal transition</td>
		</tr>
		<tr>
			<td>Rotenone</td>
			<td>Sigma</td>
			<td>R8875-1G</td>
			<td>Inhibitor of Complex I of the electron transport chain</td>
		</tr>
		<tr>
			<td>System-compatible plate</td>
			<td>Corning</td>
			<td>#353072</td>
			<td></td>
		</tr>
		<tr>
			<td>Taurine</td>
			<td>Sigma</td>
			<td>T8691</td>
			<td></td>
		</tr>
		<tr>
			<td>&#945;MEM (Alpha Modification of Eagle&#39;s Media)</td>
			<td>Corning</td>
			<td>15-012-CV</td>
			<td></td>
		</tr>
	</tbody>
</table>

long-term monitoring of oxygen consumption rates in highly differentiated and polarized retinal pigment epithelial cultures

Mitochondrial metabolism is critical for the normal function of the retinal pigment epithelium (RPE), a monolayer of cells in the retina important for photoreceptor survival. RPE mitochondrial dysfunction is a hallmark of age-related macular degeneration (AMD), the leading cause of irreversible blindness in the developed world, and proliferative vitreoretinopathy (PVR), a blinding complication of retinal detachments. RPE degenerative conditions have been well-modeled by RPE culture systems that are highly differentiated and polarized to mimic in vivo RPE. However, monitoring oxygen consumption rates (OCR), a proxy for mitochondrial function, has been difficult in such culture systems because the conditions that promote ideal RPE polarization and differentiation do not allow for easy OCR measurements. 
Here, we introduce a novel system, Resipher, to monitor OCR for weeks at a time in well-differentiated RPE cultures while maintaining the RPE on optimal growth substrates and physiologic culture media in a standard cell culture incubator. This system calculates OCR by measuring the oxygen concentration gradient present in the media above cells. We discuss the advantages of this system over other methods for detecting OCR and how to set up the system for measuring OCR in RPE cultures. We cover key tips and tricks for using the system, caution about interpreting the data, and guidelines for troubleshooting unexpected results. 
We also provide an online calculator for extrapolating the level of hypoxia, normoxia, or hyperoxia RPE cultures experience based on the oxygen gradient in the media above cells detected by the system. Finally, we review two applications of the system, measuring the metabolic state of RPE cells in a PVR model and understanding how the RPE metabolically adapts to hypoxia. We anticipate that the use of this system on highly polarized and differentiated RPE cultures will enhance our understanding of RPE mitochondrial metabolism both under physiologic and disease states.

Author Spotlight: Extended Oxygen Consumption Measurement in Retinal Pigment Epithelium Using Resipher

Long-term Monitoring of Oxygen Consumption Rates in Highly Differentiated and Polarized Retinal Pigment Epithelial Cultures

Degeneration of the RPE, or retinal pigment epithelium, underlies many causes of vision loss. Mitochondrial dysfunction underlies this RPE degeneration in many cases. We can measure mitochondrial function by looking at oxygen consumption rates, or OCR.Here we introduce a new method for measuring OCR in the RPE. Thanks to the work from multiple labs, it's now well-established, the RPE requires mitochondrial activity to maintain its health and function. Research is now more focused on what nutrients the RPE preferentially uses, and if that changes under stress.Prior methods for assessing OCR in RPE have limitations. Either the RPE was grown in non-ideal conditions, or it could not be assessed over long periods of time. So measuring OCR in high fidelity cultures that maintain features in vivo is one of the main challenges.The method for monitoring OCR introduced in this manuscript enables us to measure OCR over a week while manipulating the RPE under multiple experimental challenges. This is all done while the RPE is in ideal culture conditions that allows it to mimic ideal RPE in vivo. Recipher is a novel device for long-term monitoring of oxygen consumption in cell cultures, such as retinal pigment epithelium.Unlike short-term methods like seahorse analyzer, recipher supports extended monitoring under standard conditions, enabling detailed bioenergetic profiling over weeks while preserving cell health.

The &#34;sandwich&#34; setup for the Resipher experiment is demonstrated in Figure 2A. Sensing lids with 32 probes corresponding to columns 3, 4, 9, and 10 on the 96-well plate sit between the cell plate and the Device. After connecting to the Hub, the Device activates motors to move the sensing lid up and down, measuring O2 concentration in the media column at a range of heights above the cell monolayer (typically 1-1.5 mm). The O2 gradient is therefore continuously me...

We introduce a novel device for measuring oxygen consumption rates (OCR) in retinal pigment epithelial (RPE) cultures. The device can measure OCR for weeks at a time on RPE grown on standard cell culture plates with standard media while the plates are in a standard cell culture incubator.

Mitochondrial metabolism is critical for the normal function of the retinal pigment epithelium (RPE), a monolayer of cells in the retina important for ...

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JoVE Journal

Biology

Setup of Resipher System &#34;Sandwich&#34; to Monitor Oxygen Consumption Rate in Well-Differentiated RPE Cultures

To begin, place the sensing lid on an empty 96 well receiver plate and place it in the cell culture incubator. Align and mount the device on the sensing lid and connect it to the hub via the provided USB cable creating a Recypher sandwich. Now go to the Lucid Lab website and click the new experiment button on the top right corner to create a new experiment.Name the title of the experiment and input any relevant experimental notes for the study. Next, create well conditions in treatment groups. For example, if testing the effects of different serum concentrations in the media on oxygen consumption rate, select serum in group name intermediate serum concentrations to be used, and add more serum percent values by clicking the plus button.Then assign which wells will receive a particular serum percentage and select a color pattern for those wells. Click the add group button to add more groups as needed. Next, define the plate setup and select the corresponding device and plate style.To do so, click the add plate button, choose the device and select the plate type. Select treatment, and click on the corresponding well. Now, click the start now button to start the experiment.Check that the indicator on the website and the light emitting diode on the hub is solid green. Let the system run for 15 to 60 minutes to allow for testing that each sensor is well calibrated and detecting atmospheric oxygen. Afterward, remove the device from the sensing lid and place the device upside down in the incubator to allow the motor on the device to reset.Now, place the plate with the sensing lid back in the cell culture hood, along with a 96 well plate containing the RPE cultures. Change the media in the plate with RPE cultures to the same media and volume for all wells to obtain baseline oxygen consumption rate values for each well. Then transfer the sensing lid to the plate containing cell cultures and place the standard lid on the plate containing cell cultures to the empty 96 well receiver plate, to maintain the sterility of the standard lid.Place the plate with cell cultures and the sensing lid back in the cell culture incubator. Align and mount the device on the sensing lid and connect it to the hub via the provided USB cable. Check that the indicators on the website interface and the hub are all solid green.Let the device measure oxygen consumption rates or OCR on each well for at least 12 to 24 hours to capture a baseline OCR. After the experiment, immerse the sensing lid in 70%ethanol for 10 minutes in a cell culture hood to sterilize it. Once the ethanol and water totally evaporate, put the sensing lid on a new 96 well receiver plate.

Measuring Changes in Mitochondrial Metabolism in Hypoxic RPE Using Resipher System

To begin, assemble the sensing lid with the cell culture plate in the hood and transfer them into the hypoxia chamber. Set up the sandwich in the hypoxia chamber. After that, place a Petri dish with sterile water in the hypoxia chamber to help maintain humidity.Finally, place a portable oxygen sensor in the hypoxia chamber. Then, seal the hypoxia chamber. Ensure the USB cable is coming out of the hypoxia chamber, and the hole where the cable exits is sealed with putty or silicone grease.Connect the hypoxia chamber to a gas cylinder with a hypoxic concentration of oxygen and a standard cell culture concentration of carbon dioxide. Slowly exchange the air in the hypoxia chamber with the 1%oxygen and 5%carbon dioxide air from the cylinder until the oxygen sensor shows the desired oxygen concentration, usually between 1%and 10%Now, close the inlet and outlet valves of the hypoxia chamber and transfer the entire chamber into the incubator. Then set up and start the experiment.Media equilibrated with atmospheric oxygen took longer to reach equilibrium at higher volumes. The hypoxia chamber showed a slow leak, causing oxygen levels to approach atmospheric concentration by 30 hours. Oxygen solubility was identical between fresh and conditioned media, indicating that media composition changes over time did not affect oxygen solubility.

Calculating the O2 Concentration at the RPE Monolayer to Determine if Cells are in Hypoxic, Normoxic, or Hyperoxic Conditions

To begin, monitor oxygen consumption rates, or OCR, in well-differentiated RPE cultures in a standard cell culture incubator using Resipher system. After the experiment, open the calculator to estimate oxygen levels at the cell monolayer and enter the media volume in microliters. Enter the saturated oxygen concentration at the air liquid interface.In the flux input, enter the flux or OCR reported by the system. Based on these values, the steady state oxygen concentration at each height in the media column is calculated and shown in the plot. The X axis of the plot represents the height in millimeters above the bottom of the well.The Y axis represents the calculated oxygen concentration at that height. The oxygen concentration at the bottom of the well is reported in the line below the plot.

4.2K Views.  University of Michigan, Ann Arbor.  We introduce a novel device for measuring oxygen consumption rates (OCR) in retinal pigment epithelial (RPE) cultures. The device can measure OCR for weeks at a time on RPE grown on standard cell culture plates with standard media while the plates are in a standard cell culture incubator. 

Novel OCR Monitoring System for Differentiated RPE Cultures

Summary