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.

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. 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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><tbody><tr><td>0.25% Trypsin-EDTA</td><td>Gibco</td><td>#25-200-056</td><td></td></tr><tr><td>3,3&#039;,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:&lt;br/&gt; 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&amp;beta;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>&amp;alpha;MEM (Alpha Modification of Eagle&#039;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.

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...

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

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.

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 ...

long-term-monitoring-oxygen-consumption-rates-highly-differentiated

Research

JoVE Journal

Biology

4.8K Views.  University of Michigan, Ann Arbor. Degeneratie van het RPE, of retinaal pigmentepitheel, ligt ten grondslag aan vele oorzaken van verlies van het gezichtsvermogen. Mitochondriale disfunctie ligt in veel gevallen ten grondslag aan deze RPE-degeneratie. We kunnen de mitochondriale functie meten door te kijken naar zuurstofverbruikssnelheden of OCR.Hier introduceren we een nieuwe methode voor het meten van OCR in de RPE. Dankzij het werk van meerdere laboratoria is het nu goed ingeburgerd,...

Video: Auteur in de schijnwerpers: Uitgebreide meting van het zuurstofverbruik in retinaal pigmentepitheel met behulp van Resipher

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

Pumpless Multi-Channel Flow Culture to Maintain and Assess Tissues and Cells

Maintaining and Assessing Various Tissue and Cell Types of the Eye Using a Novel Pumpless Fluidics System

Many in vitro models used to investigate tissue function and cell biology require a flow of media to provide adequate oxygenation and optimal cell conditions required for the maintenance of function and viability. Toward this end, we have developed a multi-channel flow culture system to maintain tissue and cells in culture and continuously assess function and viability by either in-line sensors and/or collection of outflow fractions. The system combines 8-channel, continuous optical sensing of oxygen consumption rate with a built-in fraction collector to simultaneously measure production rates of metabolites and hormone secretion. Although it is able to maintain and assess a wide range of tissue and cell models, including islets, muscle, and hypothalamus, here we describe its operating principles and the experimental preparations/protocols that we have used to investigate bioenergetic regulation of isolated mouse retina, mouse retinal pigment epithelium (RPE)-choroid-sclera, and cultured human RPE cells. Innovations in the design of the system, such as pumpless fluid flow, have produced a greatly simplified operation of a multi-channel flow system. Videos and images are shown that illustrate how to assemble, prepare the instrument for an experiment, and load the different tissue/cell models into the perifusion chambers. In addition, guidelines for selecting conditions for protocol- and tissue-specific experiments are delineated and discussed, including setting the correct flow rate to tissue ratio to obtain consistent and stable culture conditions and accurate determinations of consumption and production rates. The combination of optimal tissue maintenance and real-time assessment of multiple parameters yields highly informative data sets that will have great utility for research in the physiology of the eye and drug discovery for the treatment of impaired vision.

Author Spotlight: Advancing Eye Physiology Research via a Multi-Channel Flow Culture for Optimal Tissue Maintenance and Real-Time Assessment 

Real-time analysis of live tissue yields important functional and mechanistic data. This paper describes the protocols and critical variables to ensure accurate and reproducible generation of data by a novel and pump-free multi-channel fluidics system that maintains and assesses a wide range of tissue and cell models.

Many in vitro models used to investigate tissue function and cell biology require a flow of media to provide adequate oxygenation and optimal cell ...

Bioengineering

MicroRNA Expression Profiles of Human iPS Cells, Retinal Pigment Epithelium Derived From iPS, and Fetal Retinal Pigment Epithelium

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human iPS cells (iPS-RPE), and fetal RPE. The protocols include collection of RNA for analysis by microarray, and the analysis of microarray data to identify miRNAs that are differentially expressed among three cell types. The methods for culture of iPS cells and fetal RPE are explained. The protocol used for differentiation of RPE from human iPS is also described. The RNA extraction technique we describe was selected to allow maximal recovery of very small RNA for use in a miRNA microarray. Finally, cellular pathway and network analysis of microarray data is explained. These techniques will facilitate the comparison of the miRNA profiles of three different cell types.

The microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human induced-pluripotent stem (iPS) cells (iPS-RPE), and fetal RPE, were compared.

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, ...

Determination of Mitochondrial Respiration and Glycolysis in Ex Vivo Retinal Tissue Samples

Mitochondrial respiration is a critical energy-generating pathway in all cells, especially retinal photoreceptors that possess a highly active metabolism. In addition, photoreceptors also exhibit high aerobic glycolysis like cancer cells. Precise measurements of these metabolic activities can provide valuable insights into cellular homeostasis under physiological conditions and in disease states. High throughput microplate-based assays have been developed to measure mitochondrial respiration and various metabolic activities in live cells. However, a vast majority of these are developed for cultured cells and have not been optimized for intact tissue samples and for application ex vivo. Described here is a detailed step-by-step protocol, using microplate-based fluorescence technology, to directly measure oxygen consumption rate (OCR) as an indicator of mitochondrial respiration, as well as extracellular acidification rate (ECAR) as an indicator of glycolysis, in intact ex vivo retinal tissue. This method has been used to successfully assess metabolic activities in adult mouse retina and demonstrate its application in investigating cellular mechanisms of aging and disease.

Determination of Mitochondrial Respiration and Glycolysis in Ex Vivo Retinal Tissue Samples

Described here is a detailed protocol for performing mitochondrial stress assay and glycolytic rate assay in ex vivo retinal tissue samples using a commercial bioanalyzer.

Mitochondrial respiration is a critical energy-generating pathway in all cells, especially retinal photoreceptors that possess a highly active metabolism. ...

Neuroscience

Rodent Working Heart Model for the Study of Myocardial Performance and Oxygen Consumption

Isolated working heart models have been used to understand the effects of loading conditions, heart rate and medications on myocardial performance in ways that cannot be accomplished in vivo. For example, inotropic medications commonly also affect preload and afterload, precluding load-independent assessments of their myocardial effects in vivo. Additionally, this model allows for sampling of coronary sinus effluent without contamination from systemic venous return, permitting assessment of myocardial oxygen consumption. Further, the advent of miniaturized pressure-volume catheters has allowed for the precise quantification of markers of both systolic and diastolic performance. We describe a model in which the left ventricle can be studied while performing both volume and pressure work under controlled conditions. 
In this technique, the heart and lungs of a Sprague-Dawley rat (weight 300-500 g) are removed en bloc under general anesthesia. The aorta is dissected free and cannulated for retrograde perfusion with oxygenated Krebs buffer. The pulmonary arteries and veins are ligated and the lungs removed from the preparation. The left atrium is then incised and cannulated using a separate venous cannula, attached to a preload block. Once this is determined to be leak-free, the left heart is loaded and retrograde perfusion stopped, creating the working heart model. The pulmonary artery is incised and cannulated for collection of coronary effluent and determination of myocardial oxygen consumption. A pressure-volume catheter is placed into the left ventricle either retrograde or through apical puncture. If desired, atrial pacing wires can be placed for more precise control of heart rate. This model allows for precise control of preload (using a left atrial pressure block), afterload (using an afterload block), heart rate (using pacing wires) and oxygen tension (using oxygen mixtures within the perfusate).

Isolated working heart models can be used to measure the effect of loading conditions, heart rate, and medications on myocardial performance and oxygen consumption. We describe methods for preparation of a rodent left heart working model that permits study of systolic and diastolic performance and oxygen consumption under various conditions.

Isolated working heart models have been used to understand the effects of loading conditions, heart rate and medications on myocardial performance in ways ...

Primary Cell Cultures from the Mouse Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) is a highly polarized multi-functional epithelium that is located between the neural retina and the choroid of the eye. It is a single sheet of pigmented cells that are hexagonally packed and connected by tight junctions. The main functions of the RPE include absorption of light, phagocytosis of the shed photoreceptor outer segments, spatial buffering of ions, transport of nutrients, ions and water as well as active involvement in the visual cycle. With such important and diverse functions, it is critically important to study the biology of RPE cells. A number of RPE cell lines have been established; however, passaged and immortalized cells are known to quickly lose some of the morphological and physiological characteristics of natural RPE cells. Thus, primary cells are more suitable for studying different aspects of RPE cell biology and function. Mouse primary RPE cell culture is very useful to researchers since mouse models are widely used in biological studies, however collecting RPE cells from mouse is also very challenging due to their small size. Here, we present a protocol for establishing primary mouse RPE cell cultures which includes enucleation and dissection of the eyes and isolation of the RPE sheets to yield the cells for culturing. This method enables efficient cell recovery. The RPE cells obtained from two mice&#160;can reach confluency on one 12 mm polyester membrane insert pre-loaded in culture plate after one week of culture and display some of the original properties of bona fide RPE cells such as hexagonal shape and pigmentation after two weeks of culture.

The retinal pigment epithelium (RPE) is a multi-functional epithelium of the eye. Here we present a protocol to establish primary cell cultures derived from the murine RPE.

The retinal pigment epithelium (RPE) is a highly polarized multi-functional epithelium that is located between the neural retina and the choroid of the ...

Measurement of Energy Metabolism in Explanted Retinal Tissue Using Extracellular Flux Analysis

High acuity vision is a heavily energy-consuming process, and the retina has developed several unique adaptations to precisely meet such demands while maintaining transparency of the visual axis. Perturbations to this delicate balance cause blinding illnesses, such as diabetic retinopathy. Therefore, the understanding of energy metabolism changes in the retina during disease is imperative to the development of rational therapies for various causes of vison loss. The recent advent of commercially-available extracellular flux analyzers has made the study of retinal energy metabolism more accessible. This protocol describes the use of such an analyzer to measure contributions to retinal energy supply through its two principle arms - oxidative phosphorylation and glycolysis - by quantifying changes in oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) as proxies for these pathways. This technique is readily performed in explanted retinal tissue, facilitating assessment of responses to multiple pharmacologic agents in a single experiment. Metabolic signatures in retinas from animals lacking rod photoreceptor signaling are compared to wild-type controls using this method. A major limitation in this technique is the lack of ability to discriminate between light-adapted and dark-adapted energy utilization, an important physiologic consideration in retinal tissue.

This technique describes real time recording of oxygen consumption and extracellular acidification rates in explanted mouse retinal tissues using an extracellular flux analyzer.

High acuity vision is a heavily energy-consuming process, and the retina has developed several unique adaptations to precisely meet such demands while ...

Monitoring Dynamic Growth of Retinal Vessels in Oxygen-Induced Retinopathy Mouse Model

Oxygen-induced retinopathy (OIR) is widely used to study abnormal vessel growth in ischemic retinal diseases, including retinopathy of prematurity (ROP), proliferative diabetic retinopathy (PDR), and retinal vein occlusion (RVO). Most OIR studies observe retinal neovascularization at specific time points; however, the dynamic vessel growth in live mice along a time course, which is essential for understanding the OIR-related vessel diseases, has been understudied. Here, we describe a step-by-step protocol for the induction of the OIR mouse model, highlighting the potential pitfalls, and providing an improved method to quickly quantify areas of vaso-obliteration (VO) and neovascularization (NV) using immunofluorescence staining. More importantly, we monitored vessel regrowth in live mice from P15 to P25 by performing fluorescein fundus angiography (FFA) in the OIR mouse model. The application of FFA to the OIR mouse model allows us to observe the remodeling process during vessel regrowth.

This protocol describes a detailed method for the preparation and immunofluorescence staining of mice retinal flat mounts and analysis. The use of fluorescein fundus angiography (FFA) for mice pups and image processing are described in detail as well.

Oxygen-induced retinopathy (OIR) is widely used to study abnormal vessel growth in ischemic retinal diseases, including retinopathy of prematurity (ROP), ...

Real-Time Analysis of Bioenergetics in Primary Human Retinal Pigment Epithelial Cells Using High-Resolution Respirometry

Metabolic dysfunction of retinal pigment epithelial cells (RPE) is a key pathogenic driver of retinal diseases such as age-related macular degeneration (AMD) and proliferative vitreoretinopathy (PVR). Since RPE are highly metabolically-active cells, alterations in their metabolic status reflect changes in their health and function. High-resolution respirometry allows for real-time kinetic analysis of the two major bioenergetic pathways, glycolysis and mitochondrial oxidative phosphorylation (OXPHOS), through quantification of the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), respectively. The following is an optimized protocol for conducting high-resolution respirometry on primary human retinal pigment epithelial cells (H-RPE). This protocol provides a detailed description of the steps involved in producing bioenergetic profiles of RPE to define their basal and maximal OXPHOS and glycolytic capacities. Exposing H-RPE to different drug injections targeting the mitochondrial and glycolytic machinery results in defined bioenergetic profiles, from which key metabolic parameters can be calculated. This protocol highlights the enhanced response of BAM15 as an uncoupling agent compared to carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) to induce the maximal respiration capacity in RPE. This protocol can be utilized to study the bioenergetic status of RPE under different disease conditions and test the efficacy of novel drugs in restoring the basal metabolic status of RPE.

The metabolic status of human retinal pigment epithelial cells (H-RPE) reflects their health and function. Presented here is an optimized protocol for examining the real-time metabolic flux of H-RPE using high-resolution respirometry.

Metabolic dysfunction of retinal pigment epithelial cells (RPE) is a key pathogenic driver of retinal diseases such as age-related macular degeneration ...

Quantifying Retinal Pigment Epithelial Phagocytosis and Lipofuscin Accumulation

Improved Lipofuscin Models and Quantification of Outer Segment Phagocytosis Capacity in Highly Polarized Human Retinal Pigment Epithelial Cultures

The daily phagocytosis of photoreceptor outer segments by the retinal pigment epithelium (RPE) contributes to the accumulation of an intracellular aging pigment termed lipofuscin. The toxicity of lipofuscin is well established in Stargardt's disease, the most common inherited retinal degeneration, but is more controversial in age-related macular degeneration (AMD), the leading cause of irreversible blindness in the developed world. Determining lipofuscin toxicity in humans has been difficult, and animal models of Stargardt's have limited toxicity. Thus, in vitro models that mimic human RPE in vivo are needed to better understand lipofuscin generation, clearance, and toxicity. The majority of cell culture lipofuscin models to date have been in cell lines or have involved feeding RPE a single component of the complex lipofuscin mixture rather than fragments/tips of the entire photoreceptor outer segment, which generates a more complete and physiologic lipofuscin model. Described here is a method to induce the accumulation of lipofuscin-like material (termed undigestible autofluorescence material, or UAM) in highly differentiated primary human pre-natal RPE (hfRPE) and induced pluripotent stem cell (iPSC) derived RPE. UAM accumulated in cultures by repeated feedings of ultraviolet light-treated OS fragments taken up by the RPE via phagocytosis. The key ways that UAM approximates and differs from lipofuscin in vivo are also discussed. Accompanying this model of lipofuscin-like accumulation, imaging methods to distinguish the broad autofluorescence spectrum of UAM granules from concurrent antibody staining are introduced. Finally, to assess the impact of UAM on RPE phagocytosis capacity, a new method for quantifying outer segment fragment/tips uptake and breakdown has been introduced. Termed "Total Consumptive Capacity", this method overcomes potential misinterpretations of RPE phagocytosis capacity inherent in classic outer segment "pulse-chase" assays. The models and techniques introduced here can be used to study lipofuscin generation and clearance pathways and putative toxicity.

Author Spotlight: Improved Lipofuscin Models and Quantification of Outer Segment Phagocytosis Capacity in Highly Polarized Human Retinal Pigment Epithelial Cultures  

This protocol describes a lipofuscin accumulation model in highly differentiated and polarized human retinal pigment epithelial (RPE) cultures and an improved outer segment (OS) phagocytosis assay to detect the total OS consumption/degradation capacity of the RPE. These methods overcome the limitations of previous lipofuscin models and classical pulse-chase outer segment phagocytosis assays.

The daily phagocytosis of photoreceptor outer segments by the retinal pigment epithelium (RPE) contributes to the accumulation of an intracellular aging ...

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

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