The most common devices used for measuring oxygen consumption don't support the simultaneous collection of outflow fractions. It's critical to also assay outflow to correlate metabolism with tissue function. Technologies measuring both can better assess cellular mechanisms mediating tissue function and disease.
The key challenge in measuring oxygen consumption in live tissue is delivering adequate oxygen to maintain tissue viability and function. This is best accomplished with flow systems. Utilizing gas pressure instead of standard peristaltic pumps greatly simplifies the procedure and also eliminates bubble formation.
The utility of a metabolic measurement is only as good as its ability to quantify and reproduce the results. Our protocol leverages the combination of precisely controlled flow rate with stable lifetime detection of dissolved oxygen in both inflow and outflow. This approach can reproducibly resolve changes in oxygen consumption of even just a few percent.
The biochemical processes mediating retinal function are the result of the metabolic interplay between various tissues of the eye. The ability of our flow system to assess multiple tissue types with diverse geometries facilitates the study of the unique intrinsic properties of each cell type and their coordinated functions. Many diseases afflicting the eye are caused by impaired metabolic pathways and signaling.
We are focusing on measuring the oxygen consumption and the release of metabolic byproducts by eye tissues in order to characterize, understand, and treat the development of retinal diseases and degeneration. To set up the novel pump-free multi-channel flow culture system after assembling the media reservoir module or MRM, insert the tubing assemblies into it and place the MRM into the heater. After securing the heater in place, proceed to insert the tissue chamber assemblies or TCAs into the perfusion chamber module or PCM Using the TCA insertion tool, insert each of the eight TCAs one at a time into the PCM holes.
Ensure to press firmly on the adapter with the tool until the top of the tubing sleeve around each chamber touches the surface, circumscribing the holes in the PCM. Set the partially assembled PCM aside, along with the PCM brace and six screws. 30 minutes following the enclosure assembly and after the MRM reaches the desired temperature, using a 50 milliliter pipette, transfer the pre-equilibrated perfusate into the preheated MRM insert by gently dispersing the liquid down the sides.
Next to place the PCM onto the MRM, insert the TCA resistance tubes emanating from the bottom of the PCM into the MRM inserts, four on each side of the MRM divider. Orient the module so that the tissue chambers rest against the oxygen detector once positioned. Secure the PCM to the support brace using six screws and the electric screwdriver.
Then fix the TCAs within the PCM support fins using the provided elastic band, stretching it around the fins at the level of the rubber gaskets. Position the oxygen detector on the detector stand, ensuring its face rests against the PCM fins. Align the LED photodetector pairs with the oxygen-sensitive dye in the tissue chambers by loosening the screws set on the side of the detector holder and adjusting the lateral guides of the detector if necessary.
Finally, place the lid on top of the enclosure. To load the tissue into the chambers, wait until the media level is 0.5 inches from the top. For loading the harvested retina or RPE choroid sclera, using fine-point forceps, gently place the retina without folding it into each chamber.
Meanwhile, use a tissue wipe to prevent the tissue chamber liquid from dripping onto the oxygen sensor. Let the tissue sink towards and onto the FRET. To load RPE cells on Transwell membranes, after passaging the prepared cells with 0.25%trypsin EDTA, seed them on polyethylene terephthalate track-etched filters having 0.4 millimeter pore size.
On the day of the experiment, cut the membranes into three equal-width strips and load them into the tissue chambers using forceps. The oxygen consumption rate or OCR of the retina was constant during the time the test compounds were injected, indicating stable health and function of the tissue and supporting the validity of the method. Consistent with data obtained using the conventional perfusion methods for both retina and RPE choroid sclera, OCR decreased in response to oligomycin and increased in response to FCCP.
The changes in lactate production rate or LPR observed were the reverse of those observed for OCR. RPE cells, which have not been previously analyzed with flow systems, responded similarly to RPE choroid sclera using this method.
