setup of resipher system &#34;sandwich&#34; to monitor oxygen consumption rate in well-differentiated rpe cultures

Novel OCR Monitoring System for Differentiated RPE Cultures

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.

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.

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.

setup-resipher-system-sandwich-to-monitor-oxygen-consumption-rate

Research

JoVE Journal

Biology

33 visualizações. Para começar, coloque a tampa de detecção em uma placa receptora de 96 poços vazia e coloque-a na incubadora de cultura de células. Alinhe e monte o dispositivo na tampa de detecção e conecte-o ao hub por meio do cabo USB fornecido, criando um sanduíche Recypher. Agora acesse o site do Lucid Lab e clique no botão novo experimento no canto superior direito para criar um novo experimento.Nomeie o título do experimento e insira quaisquer notas e...