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.

While lipofuscin universally accumulates in the RPE with aging, discerning its toxicity has been difficult. Here, we develop protocols that allow us to develop lipofuscin-like accumulation in highly polarized and mature RPE cultures to help discern lipofuscin's effects on RPE physiology. Studying lipofuscin toxicity in humans is confounded by the fact that lipofuscin decreases as the RPE dies.

Animal models of lipofuscin toxicity have had widely disparate results. We have carefully created in vitro models of lipofuscin-like material accumulation to facilitate the study of lipofuscin toxicity. The key to the success of our models is maintaining highly differentiated cultures, while inducing lipofuscin genesis via the same process that triggers lipofuscin in vivo, namely phagocytosis of photoreceptor outer segments.

Our in vitro protocol is unique in that lipofuscin-like material accumulates in RPE cultures that are highly differentiated to truthfully mimic RP in vivo. In these circumstances, we find that RPE culture are highly resistant to the lipofuscin accumulation and to toxic effect from lipofuscin. Additionally, in the process of assaying for phenotypes induced by lipofuscin-like accumulation in our RP cultures, we created a new type of phagocytosis assay called total consumptive capacity.

This assay allows us to determine the entire capacity of the RPE to phagocytose outer segments while avoiding some of the confounding interpretations that come with classical pulse chase phagocytosis assays. We are excited to utilize this model of lipofuscin-like material accumulation to better understand what RPE stressors may take normally inert lipofuscin into a more pathological role in RPE cell deaths. Begin by sterilizing polytetrafluoroethylene or PTFE-coated slides by immersing them in 70%ethanol for 10 minutes in a biosafety cabinet.

Allow the slides to air dry in a sterile 100 millimeter cell culture dish. Place each slide in one new 100 millimeter cell culture dish and add 200 microliters of serum-containing cell culture medium into each rectangle. Then place a handheld, 254-nanometer UV light on the 100-millimeter cell culture dish with the bulb directly facing the slide for 20 minutes.

Thaw the frozen aliquots of the outer segments, or OS, in a 37 degrees Celsius water bath. Then spin the OS at 2, 400 g for five minutes at room temperature. Immediately aspirate the supernatant in the biosafety cabinet using a pipette and resuspend the OS in PBS.

Now aspirate the medium from the slides and place up to 500 microliters of the two times 10 to the eighth OS per milliliter PBS solution of the two times 10 to the eighth OS per milliliter PBS solution on each slide rectangle. Place a handheld, 254-nanometer UV light over the cell culture dish with the bulb directly facing the OS for 40 minutes. Collect OS PBS solution in a 1.5 milliliter tube.

Wash the rectangle of the coated slide with 200 to 500 microliter of PBS and collect each wash in the micro centrifuge tube. Then, spin down the OS PBS solution at 2, 400 g for five minutes at room temperature. Aspirate the PBS in the biosafety cabinet with a pipetter, and resuspend the pellet in 500 microliters of standard medium to be used for the retinal pigment epithelium or RPE cultures.

Place 10 microliters of the suspension in a new microcentrifuge tube. Dilute 50 to 100 times with more cell culture medium and count the OS with a hemocytometer. Dilute the photooxidized OS or OXOS suspension.

Add a 5.6 times 10 to the seventh OS per milliliter concentration into a standard RPE medium. Add phagocytosis-bridging ligands, which facilitate OS uptake and lipofuscin accumulation. Then aliquot OXOS into single-use stalks and snap freeze in liquid nitrogen.

Thaw OXOS aliquots by diluting the frozen aliquot in 45 microliters of cell culture media before adding it to RPE transwells to induce lipofuscin accumulation. Then, characterize the oxidized OS by measuring the OXOS emission spectrum using lambda scanning on a confocal microscope. Confocal imaging showed that treated OS had increased autofluorescence compared to untreated OS.To begin, take the fixed transwell with lipofuscin-loaded RPE cultures.

After washing the cultures with PBS five times, leave a small amount of PBS in the apical chamber. Place the transwell upside down under a dissecting microscope. Using a razor blade, cut the semi porous membrane from the transwell by applying the cutting force at the junction of the membrane and the transwell.

Once the transwell membrane is cut, place it onto a microscope slide using forceps. Wipe away excess PBS with a task wipe while avoiding touching the cells. Add mounting medium followed by a cover slip.

Ensure to keep track of which side of the transwell is right side up. Next, detect the antigen of interest using a fluorophore with peak excitation of around 488 nanometers and emission detection at 500 to 530 nanometers. Set up a separate channel for autofluorescence detection with 405 nanometers excitation and 585 to 635 nanometers emission.

Autofluorescent granules induced by OXOS showed more accumulation after 20 feedings compared to five feedings. The ratio metric imaging helped to decipher UAM auto florescence from LC3 labeled with Alexa 488. The red channel contains only undigestible autofluorescence material and help to separate the LC3 signal from the green channel.

To begin, calculate the number of wells needed for the experiment, then thaw an appropriate amount of regular OS sample. Add enough RPE media to achieve a final OS concentration of four times 10 to the sixth per milliliter. Remove the apical medium and add 50 microliters of four times 10 to the sixth OS per milliliter with appropriate concentrations of bridging ligands.

After OS addition, incubate the samples for various time points. At the end of the incubation, add 16.67 microliters of four times Laemmli sample buffer with protease inhibitors to lyse both the cells and the overlying OS-containing supernatant. Using a P200 pipette, scratch the transwell surface and collect the combined cell supernatant and cell lysate together.

Vortex and leave at room temperature for 30 minutes for thorough denaturation. Western blot of the rhodopsin showed that the intact rhodopsin band was indistinguishable in control and UAM-laden RPE cells. However, the cleavage products of rhodopsin were higher in the UAM group, suggesting some mild degradative dysfunction in the phagolysosomal system.

The equal levels of GAPDH indicate that the cell count between wells is equal. Different rhodopsin antibodies recognize different degradation fragments. 4D2 recognizes the end terminus of rhodopsin, which is intact until the last steps of lysosomal degradation.

In contrast, 1D4 recognizes the C terminus of Rhodopsin, which is degraded earlier in the phagolysosomal process.