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 growing 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 weeks 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. Resipher 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, Resipher supports extended monitoring under standard conditions, enabling detailed bioenergetic profiling over weeks while preserving cell health. 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 Resipher 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 and treatment groups.

For example, if testing the effects of different serum concentrations in the media on oxygen consumption rate, select Serum in group name. Enter media 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. 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 exit 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. 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.