This work was supported by Agence Nationale de la Recherche (Jeunes Chercheuses/Jeunes Chercheurs to EFN), Fondation Voir et Entendre and Fondation Bettencourt Schueller (Young Investigator Grants to EFN), Centre National de la Recherche Scientifique (CNRS, permanent position for EFN), and The National Eye Institute of the National Institutes of Health (R01-EY13295 to SCF). Additionally, the Institut de la Vision is funded by Institut National de la Sant&#233; et de la Recherche M&#233;dicale, Universit&#233; Pierre et Marie Curie-Paris 6, Centre National de la Recherche Scientifique and D&#233;partment de Paris.

This POS isolation experiment is time consuming and may require up to 12 hr to complete if POS are labeled before storage. The protocol has been adapted from a paper published by R.S. Molday and colleagues in 198731 and modified by S.C. Finnemann and colleagues in 199710.
Animals were handled according to the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research. Protocols were reviewed and approved by the Charles Darwin Ethics Committee from University Pierre and Marie Curie-Paris 06 and the Fordham University Institutional Animal Care and Use Committee.
1. General Set Up
<ol>
	<li>Obtain 80 fresh pig or cow eyes as fresh as soon after slaughtering as possible and keep them chilled in the dark. Provide instructions to the provider so the company proceeds the same way. This protocol is optimized for 80 eyes. To proceed with more eyes and get bigger stocks (e.g.,, from 160 eyes), double the solution quantities and perform 2 rounds of ultracentrifugation; pool samples later at the collection step (see step 2.2.2).</li>
	<li>Set up on the bench the red safelight lamp, absorbent pads, wipes (Materials table). Prepare 15 cm dishes and biohazard bags for waste collection (unused eye parts, vitreous&#8230;). Wear tight-fitting labcoat, gloves, sleeve protectors and goggles.</li>
	<li>Precautions:
	<ol>
		<li>Pre-chill all solutions then keep them cold during all steps. Keep eyes and gradients chilled on ice as much as possible.</li>
		<li>Keep eyes and derived tissues in the dark as much as possible until the collection step after the ultracentrifugation to avoid photobleaching of visual pigments and loss of the orange-pink color of the active form of the photopigments.</li>
	</ol>
	</li>
</ol>
2. Protocol Actions
<ol>
	<li>Prepare solutions and gradients:
	<ol>
		<li>Thaw taurine stock completely using a 37 &#176;C water bath.</li>
		<li>Prepare all solutions from stock solutions (Table 1, Stock solutions) and mix thoroughly the sucrose with other ingredients using a magnetic stirrer for each solution.
		<ol>
			<li>Prepare 15 ml of the homogenization solution to a final concentration of 20% sucrose, 20 mM tris acetate pH7.2, 2 mM MgCl2, 10 mM glucose and 5 mM taurine.</li>
			<li>Prepare the 25% sucrose solution to a final concentration of 25% sucrose (using 70% sucrose stock), 20 mM tris acetate pH 7.2, 10 mM glucose and 5 mM taurine.</li>
			<li>Prepare the 60% sucrose solution to a final concentration of 60% sucrose (using sucrose powder), 20 mM tris acetate pH7.2, 10 mM glucose and 5 mM taurine.</li>
			<li>Prepare the Wash 1 solution to a final concentration of 20 mM tris acetate pH 7.2 and 5 mM taurine.</li>
			<li>Prepare the Wash 2 solution to a final concentration of 10% sucrose, 20 mM tris acetate pH 7.2 and 5 mM taurine.</li>
			<li>Prepare the Wash 3 solution to a final concentration of 10% sucrose, 20 mM sodium phosphate pH 7.2 and 5 mM taurine.</li>
		</ol>
		</li>
		<li>Steady the gradient maker on a magnetic stirrer and insert a stir bar in the chamber closest to the exit where the 60% solution will be inserted. Use a cut-off P200 pipet tip placed at the exit of the tubing to achieve proper casting speed.</li>
		<li>Cast linear gradients delicately by diluting and stirring the 60% sucrose solution with the 25% one using 12 ml from each solution (i.e., total 6 tubes for 80 eyes) in pre-chilled ultracentrifuge tubes. Stir the gradually-diluting 60% sucrose solution well. Steady the speed of gradient flow, pouring the solution smoothly and not too fast. 
		NOTE: In order to get a continuous, linear gradient, keep the pipet tip opening right at the surface of the solution for the entire gradient formation.</li>
		<li>Let the gradients sit on ice for around 1 hr for stabilization. Keep the gradients chilled until loaded. Do not shake the tubes to prevent disruption of the gradient.</li>
	</ol>
	</li>
	<li>Tissue collection:
	<ol>
		<li>Collect tissues under dim red light. Take an eye into one hand and poke the front with edge of razor blade while holding the eye away (to avoid splashing; Figure 1). Use the razor blade to cut the anterior eyeball into 2 halves (cornea plus at least 5 mm into the sclera). Remove the lens and flip the eyecup inside out so that it can be held over the tip of one finger thus exposing the retina.</li>
		<li>Using the razor blade at an angle, gently scrape the retina, detaching easily and appearing as a pinkish layer, off the tapetum surface and cut at the optic nerve head (Figure 1). Collect all retinas in 2 x 50 ml tubes each containing 15 ml of homogenization solution on ice.</li>
		<li>Retina homogenization:
		<ol>
			<li>Shake the suspension vigorously by hand for 2 min in order to disrupt the different cell layers, break POS off the rest of the PR cell, fragment POS and homogenize the retina suspension.</li>
			<li>Filter 3x through a double-layer of clean gauze to remove large tissue fragments and collect the flow-through in clean 50 ml tubes (Figure 1). The retinal suspension being quite thick, to maximize the yield, gently press on the gauze to release remaining fragmented tissues after each filtration.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Photoreceptor outer segment fragment (POS) isolation:
	<ol>
		<li>Gently lay the crude retina prep, around 6 - 7 ml per tube, over 6 x 30 ml ultracentrifuge tubes each containing 24 ml of fresh chilled continuous 25 - 60% sucrose gradient (Figure 1). Balance the opposing tubes as appropriate for the rotor to be used. Ultracentrifuge at 106,000 x g for 50 min at 4 &#176;C.</li>
		<li>Remove most of the solution above the orange-pink band in the upper third of gradient by aspiration (Figure 2A). Collect the orange-pink band with a cut-off P1000 tip into a beaker. Discard the rest after neutralizing using bleach (biological waste).</li>
		<li>Dilute with ~4 - 5 volumes of ice-cold Wash 1 solution. Separate into as many 30 ml tubes as needed. Centrifuge at 3,000 x g for 10 min at 4 &#176;C. Carefully discard the supernatant.</li>
		<li>Resuspend pellets in 10 ml Wash 2 solution and spin at 3,000 x g for 10 min as detailed above. Resuspend pellets in 15 ml Wash 3 and spin again at 3,000 x g for 10 min. Combine suspensions from several pellets to reduce the number of tubes needed before centrifugation in Wash 2 and Wash 3 solutions.</li>
	</ol>
	</li>
	<li>POS aliquoting without labeling:
	<ol>
		<li>Resuspend POS in 10 - 20 ml DMEM. Predilute 10 &#181;l in 490 &#181;l DMEM (1:50) and count elongated as well as whirled POS in a cell counting chamber (Figure 2B). Calculate yield and concentration in POS particles per mL. 
		NOTE: The yield varies, depending mostly on the age and strain of pigs/cows and size of the eyes, e.g., a good preparation ranges from 5 - 8 x 109 POS from 80 pig eyes.</li>
		<li>Decide on the final DMEM volume (typically 10 - 20 ml) and add sucrose to yield a final concentration of 2.5% sucrose.</li>
		<li>Prepare aliquots of the desired size, e.g., 5 x 107 POS per tube for a 2 ml resuspension volume for a 96-well experiment at 40 &#181;l/well. Spin 1 aliquot for 5 min at 2,300 x g at RT&#160;and evaluate pellet size. Prepare various aliquot sizes for different application volumes as POS should NOT be frozen and thawed twice.</li>
		<li>Freeze POS aliquots at -80 &#176;C until further use. 
		NOTE: Aliquots are stable for many months. In our hands, unlabeled or labeled POS can be stored for at least one year.</li>
	</ol>
	</li>
	<li>POS labeling and aliquoting: 
	NOTE: We typically use FITC to label POS for quantification of phagocytosis assays, but other dyes may be used as well depending on needs.
	<ol>
		<li>Resuspend 1 10 mg FITC vial at a 2.5 mg/ml concentration in 0.1 M Na carbonate buffer pH 9.5 by mixing 2.6 ml 0.1 M NaHCO3 pH 8.4 with 1.4 ml 0.1 M Na2CO3 pH 11.5 (Table 2, Stock solutions). Rotate for 1 hr at RT while protecting from light and spin for 5 min at maximum speed to pellet non-resuspended FITC solids.</li>
		<li>Resuspend the POS pellet in 10 ml of Wash 3 solution (or DMEM) and add 2 ml of FITC solution for 80 eyes. Store leftover FITC frozen at -80 &#176;C. Rotate for at least 1.5 hr at RT. Protect from light using aluminum foil.</li>
		<li>Wash labeled POS twice using Wash 3 solution as described in section 2.3.4, then once or twice in DMEM until almost no free FITC is seen in the supernatant fraction. Resuspend POS in DMEM and proceed as for unlabeled POS for counting and aliquoting (section 2.4).</li>
	</ol>
	</li>
	<li>Using POS in experiments:
	<ol>
		<li>Thaw POS at RT, and keep them in the dark as much as possible if they are fluorescently-labeled. Spin for 5 min at 2,300 x g at RT. Aspirate the supernatant.</li>
		<li>Immediately resuspend in appropriate volume of assay solution as dictated by the experiment. In case of different times of POS challenge, store resuspended POS at RT in the dark between time-points for several hours.</li>
	</ol>
	</li>
</ol>

The authors declare that they have no competing financial interest.

Three steps or conditions are crucial for an optimized purification: quality of gradient casting and delicate gradient tubes manipulation, keeping tissues chilled and in the dark until the collection step, strength of shaking of retinal homogenates to obtain proper POS isolation from the rest of the PR cell. If some issues arise in seeing the orange band properly, they are most likely due to one of the three reasons above (see also the second paragraph of the Results section). Some modification of certain conditions is possible, like the use of a 27 - 50% sucrose gradient concentration32 or of Optiprep step gradients33,21.
Some caution needs to be taken regarding aliquots for experiments as POS should not be frozen and thawed twice. Therefore, it is useful to prepare various sizes for different application volumes.
POS pre-labeling should be used with caution. Covalent labeling generates very highly fluorescent POS that make co-localization studies difficult. Moreover, pre-labeling followed by freeze-thaw may not maintain POS surface characteristics and alter POS immunogenicity. Hence, for co-localization assays it is advisable to store unlabeled POS and label them after thaw immediately before using them for an experiment10,13-15,32.
Preparing large-scale POS stocks is economical: a one-day preparation yields reliable experimental material for numerous experiments. Moreover, using rodent retinas to prepare POS for the same number of assays would require very large numbers of animals. Like synthetic particles like beads, a frozen stock of POS will provide a highly consistent tool to test directly the phagocytic ability of RPE cells. Unlike synthetic beads targeting other particle ingestion mechanism such as endocytosis not reacted to POS phagocytosis43,44, POS isolated using our protocol are physiologically-relevant phagocytic particles: (1) Isolated POS expose phosphatidylserines, a critical &#8220;eat me&#8221; signal recognized by the RPE&#8217;s phagocytic machinery; (2) POS can be efficiently digested by RPE cells after uptake and they thus allow studying all phases of RPE phagocytosis. A possible drawback to the use of porcine or bovine POS could be any species difference between POS as most cell culture phagocytosis assays do not use porcine RPE cells. For instance, the overall cone to rod ratio varies between species &#8211; from approximately 2 - 3% in rodents to 5% in humans and 20% in pigs &#8211; and cones are concentrated in the macular region in humans. However, current data on the phagocytic machinery show that POS particles are recognized by RPE cell receptors due to their ability to bind soluble ligands, such as the integrin ligand MFG-E89,12 and MerTK ligands Gas6 and Protein S45. These ligand proteins bind to phosphatidylserines regardless of species7-9,21,22,31-33 but may bridge to RPE receptors in a species-specific manner. Potential pitfalls from using porcine/bovine POS for RPE uptake assays can just be avoided by supplementing soluble ligands during assays that are tailored to the origin of the RPE to be used.
More recently, generation of RPE cells derived from reprogrammed stem cells like ES cells or iPS have instigated a renewed interest for isolated POS27,29,30,34. These RPE cells may be derived from healthy donors or from patients carrying specific mutant genes. They can either be explored functionally to characterize the precise cellular phenotypic effect of the mutations present, or they require to be validated for their phagocytic function before potential transplantation therapies27,30,34. These new approaches open a new demand for POS purified on a large scale, as RPE phagocytosis constitutes one of RPE cell main functions and reproducible tests are recommended for reliable comparison between sets of cells.

In the retina, vision is triggered by isomerization of photosensitive molecules called opsins, before being transformed into a signal that can be transmitted between neurons up to the visual areas in the brain. These molecules are embedded in stacks of membranous disks resembling pancakes that constitute the outer segment portions of photoreceptor cells (PRs). Being subjected to constant exposure to light and therefore considerable levels of oxidative stress, PRs continuously renew their outer segments to limit potential oxidative damage. Photoreceptor outer segments are in close contact with apical microvilli of the neighboring retinal pigment epithelial (RPE) cells. RPE cells constitute the outer part of the blood-retinal barrier and ensure numerous tasks that are crucial for photoreceptors health and function1, such as quenching light rays via melanin pigments, re-isomerization of the photoreactive opsin component retinal, providing nutrients and growth factors, participating in PR metabolite disposal.
In addition, RPE cells eliminate spent POS and recycle their components, a daily occupation that is regulated by the circadian rhythm in mammalian retina2,3. The clearance of shed POS is absolutely necessary for PR survival. When it is completely abrogated, POS debris accumulate and PRs degenerate causing rapid vision loss4,5. If the rhythmic profile is lost and replaced by a constant activity, PR and RPE defects accumulate with age6. Therefore, it is very important to characterize the molecular regulation of RPE phagocytosis in vitro in order to understand phenotypes linked to its dysfunction. Interestingly, the molecular machinery in RPE cells is very similar to the one used by macrophages to clear apoptotic cells and both are dependent on recognition of exposed phosphatidylserine on phagocytic debris7-9. Still, RPE cells and macrophages regulate phagocytosis differently, as macrophages opt for immediate elimination of apoptotic cells at encounter time while RPE cells rhythmically engulf POS only once a day despite their permanent contact with outer segments. This suggests specific regulation mechanisms that are not yet fully understood.
Many of the molecules implicated in the RPE phagocytic machinery have been identified or validated thanks to the use of isolated POS and cell culture phagocytosis assays. The alphavbeta5 integrin receptor located at the RPE apical cell surface, in coordination with its ligand MFG-E8, binds specifically to POS10-12, which are then internalized via the MerTK tyrosine kinase receptor13-15. The CD36 scavenger receptor has been shown to participate in POS intake and influence its speed16,17, and might serve as a sensor of oxidized phospholipids at the POS surface18. Internalization needs the recruitment of F-actin cytoskeleton-associated proteins such as annexin 219, myosin II20 and myosin VIIA21,22. Native or oxidized POS in vitro are also utilized to understand aging phenotypes of RPE cells in vivo linked to accumulation of poorly digested oxidized POS23-28. The generation of RPE cells derived from stem cells has initiated a new application for isolated POS that are used to prove functionality of cells before they are transplanted to animals or patients29,30,27.
First described by Molday and colleagues in 198731, the protocol for isolation of bovine POS combines an ultracentrifugation step of retinal homogenates on continuous sucrose gradients with observation of the characteristic orange appearance of unbleached retinal photopigment (carrying 11-cis retinal). In the past 10 years, due to precautions taken in order to minimize risk of mad cow disease, use of porcine eyes has become increasingly prominent. The protocol described here shows how to obtain large amounts of POS from porcine or bovine eyes that can be aliquoted and stored for extended periods of time. This eliminates the need to prepare POS from rodent eyes, which requires using a large number of animals per POS preparation and assay32,33,21,22. In addition, details about POS labeling before storage using fluorescent molecules are given, to quantify and visualize POS in a simplified and comparable manner for some applications compared to labeling POS after the phagocytosis assay32,10. Therefore, these large stocks allow for reproducibility and ease of use in many different types of experiments.

<table><tbody><tr><td colspan="4">Specific Material/Equipment</td></tr><tr><td>2 Chamber gradient maker</td><td></td><td></td><td>Gradient maker with 30 ml chambers</td></tr><tr><td>3 mm diameter silicone tubing</td><td></td><td></td><td>tubing for gradient casting</td></tr><tr><td>Small size magnetic stir bar</td><td></td><td></td><td>Stir bar fitting the gradient maker chamber</td></tr><tr><td>Red safelight lamp</td><td></td><td></td><td>Inactinic lamp for dissection in the dark</td></tr><tr><td>Ultra Clear 25 x 89 cm tubes</td><td>Beckman</td><td>344058</td><td>Ultracentrifugation tubes</td></tr><tr><td>PP Oak Ridge tubes</td><td>Nalgene</td><td>3119-0050</td><td>30 ml centrifugation tubes</td></tr><tr><td>Optima LE-80K</td><td>Beckman Coulter</td><td>365668</td><td>Ultracentrifuge</td></tr><tr><td>SW 32Ti swing rotor</td><td>Beckman Coulter</td><td>369694</td><td>Swing rotor for ultracentrifuge</td></tr><tr><td>Avanti J-26 XP</td><td>Beckman Coulter</td><td>393124</td><td>Centrifuge</td></tr><tr><td>JA-25.50 rotor</td><td>Beckman Coulter</td><td>363058</td><td>Rotor for Avanti J-26 XP centrifuge</td></tr><tr><td>FITC Isomer I</td><td>Life Technologies</td><td>F-1906</td><td>Fluorescent dye</td></tr><tr><td colspan="4">Other Material/Equipment</td></tr><tr><td>Counting chamber (such as Neubauer or Malassez)</td><td></td><td></td><td></td></tr><tr><td>Dark ice buckets with lids</td><td></td><td></td><td></td></tr><tr><td>Scales</td><td></td><td></td><td></td></tr><tr><td>Magnetic stirrer and upholding pole</td><td></td><td></td><td></td></tr><tr><td>Refrigated microcentrifuge</td><td></td><td></td><td></td></tr><tr><td>37 &amp;deg;C water bath</td><td></td><td></td><td></td></tr><tr><td>-80 &amp;deg;C freezer</td><td></td><td></td><td></td></tr><tr><td colspan="4">Consumables</td></tr><tr><td>Labcoat</td><td></td><td></td><td>Health and safety</td></tr><tr><td>Gloves</td><td></td><td></td><td></td></tr><tr><td>Sleeve protectors</td><td></td><td></td><td></td></tr><tr><td>Goggles</td><td></td><td></td><td></td></tr><tr><td>Absorbent pads</td><td></td><td></td><td></td></tr><tr><td>Biohazard trash bags and bins</td><td></td><td></td><td></td></tr><tr><td>Weck-Prep blades</td><td></td><td>Dissection</td><td>60 mm/2.25 inch wide razor blades</td></tr><tr><td>15 cm plastic dish</td><td></td><td></td><td></td></tr><tr><td>Sterile gauze sheets</td><td></td><td></td><td></td></tr><tr><td>15 and 50 ml tubes</td><td></td><td></td><td>Common consumables</td></tr><tr><td>Microtubes</td><td></td><td></td><td></td></tr><tr><td>Aluminum foil</td><td></td><td></td><td></td></tr></tbody></table>

large-scale purification of porcine or bovine photoreceptor outer segments for phagocytosis assays on retinal pigment epithelial cells

Analysis of one of the vital functions of retinal pigment epithelial (RPE) cells, the phagocytosis of spent aged distal fragments of photoreceptor outer segments (POS) can be performed in vitro. Photoreceptor outer segments with stacks of membranous discs containing the phototransduction machinery are continuously renewed in the retina. Spent POS are eliminated daily by RPE cells. Rodent, porcine/bovine and human RPE cells recognize POS from various species in a similar manner. To facilitate performing large series of experiments with little variability, a large stock of POS can be isolated from porcine eyes and stored frozen in aliquots. This protocol takes advantage of the characteristic of photopigments that display an orange color when kept in the dark. Under dim red light, retinae are collected in a buffer from opened eyecups cut in halves. The retinal cell suspension is homogenized, filtered and loaded onto a continuous sucrose gradient. After centrifugation, POS are located in a discrete band in the upper part of the gradient that has a characteristic orange color. POS are then collected, spun, resuspended sequentially in wash buffers, counted and aliquoted. POS obtained this way can be used for phagocytosis assays and analysis of protein activation, localization or interaction at various times after POS challenge. Alternatively, POS can be labeled with fluorophores, e.g., FITC, before aliquoting for subsequent fluorescence quantification of POS binding or engulfment. Other possible applications include the use of modified POS or POS challenge combined with stress conditions to study the effect of oxidative stress or aging on RPE cells.

The combination of the linear sucrose gradient and the ultracentrifugation allows the separation of the different components of the retinal suspension by density. Heavy larger retinal debris and RPE cells sink to or near the bottom of the gradient (Figure 2A). Lighter POS and lighter individual cells or cell debris from the retina migrate as separate bands to reach the top half of the gradient by the end of the centrifugation period. By keeping the eyes and the samples in the dark until after the centrifugation step, the band containing POS can be immediately recognized by its bright orange color. The width and intensity of this band depends on gradient quality and tube height (89 mm height is preferred). Typically, when isolated properly POS should appear elongated, straight or bent when observed on a microscope slide (Figure 2B). Depending on the focus plane on the microscope, they can appear either dark or shiny. If not resuspended properly, they will stay in clumps. If they are damaged (see paragraph below), they will appear as much smaller pieces, a little like a &#8216;dusty&#8217; background.
Problems that can arise are mostly related to three issues. The first possible issue is that the shaking step of the retinal homogenates is not vigorous enough, which leads to POS remaining attached to PRs thus to decreased yield. The second possible issue is linked to poor gradient casting or gradient quality loss to insufficient stabilization time before loading or excessive tube shaking at any step before the collection. In this case, the bands cannot be properly distinguished and POS cannot be collected in the corresponding tube. Finally, the third potential issue arises if the pH of any of the solutions is wrong: POS membranes may disintegrate and only small POS debris appears on the microscope when visualizing them for yield assessment.
Purified POS can be used for a number of different applications related to studying the phagocytic function of RPE cells. RPE cells are usually used as cell lines or in primary culture from animal models 32,6,12,18-21. More recently, RPE cells derived from reprogrammed stem cells like iPSC or ESC have been used29,30,27,34. Isolated POS serve to test directly the phagocytic ability of mutant RPE cells; e.g., POS are readily phagocytosed by primary RPE cells from wild-type mice, whereas cells from beta5 integrin and MFG-E8 knockout mice phagocytose less POS in the same amount of time6,12. Quantification of POS phagocytosis can be done either by counting FITC-labeled POS manually on microscope pictures6 (Figure 3A), using equipment capable of reading fluorescence intensity10,24,15,35 or a flow cytometer after cell trypsinization36,27. More recently, POS intake and degradation has been evaluated on immunoblots probed for opsins37,38,27.
In vivo, the POS uptake process occurs sequentially as recognition/binding precedes synchronized internalization6. The different phases of uptake can also distinguished in vitro by using appropriate experimental procedures39. Pulse-chase experiments can be performed using temperature switch: POS binding occurs at 20 &#176;C while internalization needs a temperature of 37 &#176;C to proceed32,40,7. This distinction also exists in macrophages, which can recognize POS as well and in which the molecular machinery to eliminate apoptotic cells is very similar to the RPE&#39;s machinery7. After FITC-POS phagocytosis, binding and internalization can be quantified in separate samples by quenching the FITC-fluorescence of surface-bound POS during pre-incubation with trypan blue before fixing the cells10. In trypan blue-treated cells, only internal POS will be visualized, and bound POS can be quantified by subtracting internal POS from total POS fluorescence counts. When phagocytosis is evaluated on immunoblots obtained using lysates collected after POS challenge, an equivalent treatment with EDTA before lysis will allow the detachment of POS bound at the cell surface for internal versus total POS phagocytosis comparison37,38.
Tremendous advances have been made in the identification of the phagocytic machinery thanks to experiments employing purified POS. Protein recruitment can be assessed by immunofluorescence co-localization assays13-15,19-21,37,41(Figure 3B). Protein recruitment or activation can be validated on immunoblots after immunoprecipitation or by checking for phosphorylation7,10,12-15,17,19,20,27,35,37,41 (Figure 3C). More open-ended studies on overall gene expression changes after different times of POS challenge have also been performed42.
Isolated POS can also be used to study POS elimination by RPE cells. Indeed, during normal aging and/or when POS are not digested properly, POS degradation products accumulate gradually as lipofuscin deposits and can lead to pathologies due to oxidation mechanisms24,27. Hence, understanding the effects of light and related oxidative damage may require POS challenge of RPE cells18,26. Finally, repeated feeding with normal27 or oxidized25,28 POS can be used to induce cumulative effects in RPE cells in culture in order to understand pathological mechanisms that develop in vivo over longer periods of time.
<img alt="Figure 1" src="/files/ftp_upload/52100/52100fig1highres.jpg" /> 
Figure 1. Retina isolation from porcine eyes. Drawing showing the different steps of the eye dissection in order to collect and homogenize the retina before the ultracentrifugation step on continuous sucrose gradients, starting top left. A 60 mm wide blade is used to cut the eye sequentially on one side and then the other in order to be able to stretch it inside-out on a finger tip, thus exposing the retina. Next, the retina is collected by scrapping it from the tapetum with the blade. Pooled retinae are then shaken thoroughly in homogenization buffer, filtered 3 times through double layers of gauze and delicately poured on top of the linear 25 - 60% sucrose gradient.
<img alt="Figure 2" src="/files/ftp_upload/52100/52100fig2highres.jpg" /> 
Figure 2. POS purification from porcine eyes. (A) Photograph of the various layers observed on a 25 - 60% sucrose gradient after ultracentrifugation of retina homogenates. The orange band corresponds to POS to be collected. The white upper band corresponds to non-POS related materials, as well as larger debris migrating near or at the bottom of the tube. (B) Picture of isolated POS as observed on a bright field microscope on a counting cell for yield assessment. POS exist in elongated (a, a&#8217;, c, d) and whirled (a, a&#8217;, b, b&#8217;, d) forms. Some elongated POS can display a coiled extremity (c). When changing the plane of focus, POS appearance shifts from shiny to dark (compare a and a&#8217;, b and b&#8217;). Panel e illustrates POS that have not been resuspended properly and reside in clumps. Panel f shows POS that have been damaged and are not usable, only small pieces are detectable. Scale 10 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52100/52100fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52100/52100fig3highres.jpg" /> 
Figure 3. Analysis of RPE phagocytosis using purified POS. (A) Confocal microscope picture and corresponding quantification of the number of phagosomes per cell with time showing that FITC-labeled POS (green) have been less phagocytosed by RPE primary cells isolated from beta5 integrin knockout (&#946;5-/-) compared to wild-type (wt or &#946;5+/+) control mice as indicated. Nuclei (red) were labeled using DAPI and cell junctions (blue) using the tight junction marker ZO-1. Scale bars 10 &#181;m. Reproduced from &#169;Nandrot et al., 2004, originally published in The Journal of Experimental Medicine 200(12):1539-1545. (B) Confocal microscope pictures showing co-localization (yellow-magenta, bottom right panel) between &#946;5-GFP fusion proteins (green, top left panel), surface &#945;v&#946;5 integrin receptors (red, top right panel) and POS (blue, bottom left panel). Scale bar 10 &#181;m. Reproduced from Nandrot et al., 2012, originally published in Biology of the Cell 104(6):326-341, &#169;Portland Press Limited. (C) Immunoblots (WB) and corresponding quantification (quantif.) showing that POS challenge (POS) increases phosphorylation of FAK on the Tyr397 residue when compared without treatment (/) or challenged with medium alone (m) in control cells (c) while cells expressing an inactivated form of FAK (F) do not react, as indicated. Reproduced from Finnemann, 2003, originally published in EMBO Journal 22(16):4143-4154. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52100/52100fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Stock solution</td>
			<td>Storage temperature</td>
		</tr>
		<tr>
			<td>70% sucrose</td>
			<td>4 &#176;C</td>
		</tr>
		<tr>
			<td>100 mM glucose</td>
			<td>4 &#176;C</td>
		</tr>
		<tr>
			<td>1 M MgCl2</td>
			<td>RT</td>
		</tr>
		<tr>
			<td>0.5 M taurine</td>
			<td>-20 &#176;C</td>
		</tr>
		<tr>
			<td>200 mM tris acetate pH 7.2</td>
			<td>RT</td>
		</tr>
		<tr>
			<td>200 mM sodium phosphate pH 7.2</td>
			<td>RT</td>
		</tr>
		<tr>
			<td>0.1 M NaHCO3 pH 8.4</td>
			<td>RT</td>
		</tr>
		<tr>
			<td>0.1 M Na2CO3 pH 11.5</td>
			<td>RT</td>
		</tr>
	</tbody>
</table>
Table 1. Recipes of stock solutions and associated storage temperatures.

Watch this Scientific Journal Video about Large-Scale Purification of Porcine or Bovine Photoreceptor Outer Segments for Phagocytosis Assays on Retinal Pigment Epithelial Cells at JoVE.com

Large-Scale Purification of Porcine or Bovine Photoreceptor Outer Segments for Phagocytosis Assays on Retinal Pigment Epithelial Cells

This article describes the protocol for the purification of photoreceptor outer segment fragments (POS) via ultracentrifugation from porcine/bovine retinae using homogenization and sucrose gradient centrifugation. This protocol allows the preparation of large stocks of POS aliquots, labeled or unlabeled, that can then be stored at -80 &#176;C.

Analysis of one of the vital functions of retinal pigment epithelial (RPE) cells, the phagocytosis of spent aged distal fragments of photoreceptor outer ...

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Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

12.1K Views.  INSERM, U968. This article describes the protocol for the purification of photoreceptor outer segment fragments (POS) via ultracentrifugation from porcine/bovine retinae using homogenization and sucrose gradient centrifugation. This protocol allows the preparation of large stocks of POS aliquots, labeled or unlabeled, that can then be stored at -80 °C.

Video: Large-Scale Purification of Porcine or Bovine Photoreceptor Outer Segments for Phagocytosis Assays on Retinal Pigment Epithelial Cells

Two Peeling Methods for the Isolation of Photoreceptor Cell Compartments in the Mouse Retina for Protein Analysis

Rod photoreceptors are highly polarized sensory neurons with distinct compartments. Mouse rods are long (~80 &#181;m) and thin (~2 &#181;m) and are laterally packed in the outermost layer of the retina, the photoreceptor layer, resulting in alignment of analogous subcellular compartments. Traditionally, tangential sectioning of the frozen flat-mounted retina has been used to study the movement and localization of proteins within different rod compartments. However, the high curvature of the rod-dominant mouse retina makes tangential sectioning challenging. Motivated by the study of protein transport between compartments, we developed two peeling methods that reliably isolate the rod outer segment (ROS) and other subcellular compartments for western blots. Our relatively quick and simple techniques deliver enriched and subcellular-specific fractions to quantitatively measure the distribution and redistribution of important photoreceptor proteins in normal rods. Moreover, these isolation techniques can also be easily adapted to isolate and quantitatively investigate the protein composition of other cellular layers within both healthy and degenerating retinae.

This protocol presents two techniques to isolate the subcellular compartments of murine rod photoreceptors for protein analysis. The first method utilizes live retinae and cellulose filter paper to separate rod outer segments, while the second employs lyophilized retinae and adhesive tape to peel away rod inner and outer segment layers.

Rod photoreceptors are highly polarized sensory neurons with distinct compartments. Mouse rods are long (~80 &#181;m) and thin (~2 &#181;m) and are ...

Neuroscience

Characterization and Morphological Dynamics of Cultured Primary Murine RPE

A Simplified Method for Isolation and Culture of Retinal Pigment Epithelial Cells from Adult Mice

Retinal pigment epithelial cells (RPE) are critical for the proper function of the retina. RPE dysfunction is involved in the pathogenesis of important retinal diseases, such as age-related macular degeneration, retinitis pigmentosa, and diabetic retinopathy. We present a streamlined approach for the isolation of RPE from murine adult eyes. In contrast to previously reported methods, this approach enables the isolation and culture of highly pure RPE from adult mice. This simple and fast method does not require extensive technical skill and is achievable with basic scientific tools and reagents. Primary RPE are isolated from C57BL/6 background mice aged 3- to 14-weeks by enucleation of the eye followed by the removal of the anterior segment. Enzymatic trypsinization and centrifugation are used to dissociate and isolate the RPE from the eyecup. In conclusion, this approach offers a quick and effective protocol for the utilization of RPE in the study of retinal function and disease.

Author Spotlight: Efficient Isolation of Primary Retinal Pigment Epithelial Cells from Adult Mice for Retinopathy Research

Retinal pigment epithelium (RPE) acts as a crucial barrier between the choroid and retina, promoting the health and function of retinal cell types, such as photoreceptors. Herein, we describe a simple and effective protocol for isolating and culturing adult murine RPE.

Retinal pigment epithelial cells (RPE) are critical for the proper function of the retina. RPE dysfunction is involved in the pathogenesis of important ...

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 ...

Biology

Isolation, Culture, and Genetic Engineering of Mammalian Primary Pigment Epithelial Cells for Non-Viral Gene Therapy

Age-related macular degeneration (AMD) is the most frequent cause of blindness in patients &#62;60 years, affecting ~30 million people worldwide. AMD is a multifactorial disease influenced by environmental and genetic factors, which lead to functional impairment of the retina due to retinal pigment epithelial (RPE) cell degeneration followed by photoreceptor degradation. An ideal treatment would include the transplantation of healthy RPE cells secreting neuroprotective factors to prevent RPE cell death and photoreceptor degeneration. Due to the functional and genetic similarities and the possibility of a less invasive biopsy, the transplantation of iris pigment epithelial (IPE) cells was proposed as a substitute for the degenerated RPE. Secretion of neuroprotective factors by a low number of subretinally-transplanted cells can be achieved by Sleeping Beauty (SB100X) transposon-mediated transfection with genes coding for the pigment epithelium-derived factor (PEDF) and/or the granulocyte macrophage-colony stimulating factor (GM-CSF). We established the isolation, culture, and SB100X-mediated transfection of RPE and IPE cells from various species including rodents, pigs, and cattle. Globes are explanted and the cornea and lens are removed to access the iris and the retina. Using a custom-made spatula, IPE cells are removed from the isolated iris. To harvest RPE cells, a trypsin incubation may be required, depending on the species. Then, using RPE-customized spatula, cells are suspended in medium. After seeding, cells are monitored twice per week and, after reaching confluence, transfected by electroporation. Gene integration, expression, protein secretion, and function were confirmed by qPCR, WB, ELISA, immunofluorescence, and functional assays. Depending on the species, 30,000-5 million (RPE) and 10,000-1.5 million (IPE) cells can be isolated per eye. Genetically modified cells show significant PEDF/GM-CSF overexpression with the capacity to reduce oxidative stress and offers a flexible system for ex vivo&#160;analyses and in vivo studies transferable to humans to develop ocular gene therapy approaches.

Here, a protocol to isolate and transfect primary iris and retinal pigment epithelial cells from various mammals (mice, rat, rabbit, pig, and bovine) is presented. The method is ideally suited to study ocular gene therapy approaches in various set-ups for ex vivo&#160;analyses and&#160;in vivo studies transferable to humans.

Age-related macular degeneration (AMD) is the most frequent cause of blindness in patients &#62;60 years, affecting ~30 million people worldwide. AMD is a ...

Efficient Dissection and Culture of Primary Mouse Retinal Pigment Epithelial Cells

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to understand the pathophysiology of ocular diseases because of their similarities with human anatomy and physiology. Alterations in the retinal pigment epithelium (RPE), including changes in morphology and function, are common features shared by many ocular disorders. However, successful isolation and culture of primary mouse RPE cells is very challenging. This paper is an updated audiovisual version of the protocol previously published by Fernandez-Godino et al. in 2016 to efficiently isolate and culture primary mouse RPE cells. This method is highly reproducible and results in robust cultures of highly polarized and pigmented RPE monolayers that can be maintained for several weeks on Transwells. This model opens new avenues for the study of the molecular and cellular mechanisms underlying eye diseases. Moreover, it provides a platform to test therapeutic approaches that can be used to treat important eye diseases with unmet medical needs, including inherited retinal disorders and macular degenerations.

This protocol, which was originally reported by Fernandez-Godino et al. in 20161, describes a method to efficiently isolate and culture mouse RPE cells, which form a functional and polarized RPE monolayer within one week on Transwell plates. The procedure takes approximately 3 hours.

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to ...

Primary Culture of Porcine Retinal Pigment Epithelial Cells

The retinal pigment epithelium (RPE) is a monolayer of polarized pigmented epithelial cells, located between the choroid and neuroretina in the retina. Multiple functions, including phagocytosis, nutrient/metabolite transportation, vitamin A metabolism, etc., are conducted by the RPE on a daily basis. RPE cells are terminally differentiated epithelial cells with little or no regenerative capacity. Loss of RPE cells results in multiple eye diseases leading to visual impairment, such as age-related macular degeneration. Therefore, the establishment of an in vitro culture model of primary RPE cells, which more closely resembles the RPE in vivo than cell lines, is critical for the characteristic and mechanistic studies of RPE cells. Considering the fact that the source of human eyeballs is limited, we create a protocol to culture primary porcine RPE cells. By using this protocol, RPE cells can be easily dissociated from adult porcine eyeballs. Subsequently, these dissociated cells attach to culture dishes/inserts, proliferate to form a confluent monolayer, and quickly re-establish key features of epithelial tissue in vivo within 2 wks. By qRT-PCR, it is demonstrated that primary porcine RPE cells express multiple signature genes at comparable levels with native RPE tissue, while the expressions of most of these genes are lost/highly reduced in human RPE-like cells, ARPE-19. Moreover, the immunofluorescence staining shows the distribution of tight junction, tissue polarity, and cytoskeleton proteins, as well as the presence of RPE65, an isomerase critical for vitamin A metabolism, in cultured primary cells. Altogether, we have developed an easy-to-follow approach to culture primary porcine RPE cells with high purity and native RPE features, which could serve as a good model to understand RPE physiology, study cell toxicities, and facilitate drug screenings.

Here, an easy-to-follow method to culture primary porcine retinal pigment epithelial cells in vitro is presented.

The retinal pigment epithelium (RPE) is a monolayer of polarized pigmented epithelial cells, located between the choroid and neuroretina in the retina. ...

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 ...

Isolation of Retinal Pigment Epithelial Cells from Guinea Pig Eyes

This protocol describes the isolation of cells of the retinal pigment epithelium (RPE) from the eyes of young pigmented guinea pigs for potential application in molecular biology studies, including gene expression analyses. In the context of eye growth regulation and myopia, the RPE likely plays a role as a cellular relay for growth modulatory signals, as it is located between the retina and the two walls of the eye, such as the choroid and sclera. While protocols for isolating the RPE have been developed for both chicks and mice, these protocols have proven not to be directly translatable to the guinea pig, which has become an important and widely used mammalian myopia model. In this study, molecular biology tools were used to examine the expression of specific genes to confirm that the samples were free of contamination from the adjacent tissues. The value of this protocol has already been demonstrated in an RNA-Seq study of RPE from young pigmented guinea pigs exposed to myopia-inducing optical defocus. Beyond eye growth regulation, this protocol has other potential applications in studies of retinal diseases, including myopic maculopathy, one of the leading causes of blindness in myopes, in which the RPE has been implicated. The main advantage of this technique is that it is relatively simple and&#160;once perfected, yields high-quality RPE samples suitable for molecular biology studies, including RNA analysis.

We describe a simple and efficient method for isolating cells of the retinal pigment epithelium (RPE) cells from the eyes of young pigmented guinea pigs. This procedure allows for follow-up molecular biology studies on the isolated RPE, including gene expression analyses.

This protocol describes the isolation of cells of the retinal pigment epithelium (RPE) from the eyes of young pigmented guinea pigs for potential ...

A Cost-Effective Method for Isolating Primary RPE Cells from Porcine Eyes

Isolation of Primary Porcine Retinal Pigment Epithelial Cells for In Vitro Modeling

The retinal pigment epithelium (RPE) is a crucial monolayer in the outer retina responsible for supporting photoreceptors. RPE degeneration commonly occurs in diseases marked by progressive vision loss, such as age-related macular degeneration (AMD). Research on AMD often relies on human donor eyes or induced pluripotent stem cells (iPSCs) to represent the RPE. However, these RPE sources require extended differentiation periods and substantial expertise for culturing. Additionally, some research institutions, particularly those in rural areas, lack easy access to donor eyes. While a commercially available immortalized RPE cell line (ARPE-19) exists, it lacks essential in vivo RPE features and is not widely accepted in many ophthalmology research publications. There is a pressing need to obtain representative primary RPE cells from a more readily available and cost-effective source. This protocol elucidates the isolation and subculture of primary RPE cells obtained post-mortem from porcine eyes, which can be sourced locally from commercial or academic suppliers. This protocol necessitates common materials typically found in tissue culture labs. The result is a primary, differentiated, and cost-effective alternative to iPSCs, human donor eyes, and ARPE-19 cells.

Author Spotlight: Modeling Retinal Pathologies with Enhanced RPE Cell-Based Disease Models

This protocol outlines the procedure for obtaining and culturing primary retinal pigment epithelial (RPE) cells from locally sourced porcine eyes. These cells serve as a high-quality alternative to stem cells and are suitable for in vitro retinal research.

The retinal pigment epithelium (RPE) is a crucial monolayer in the outer retina responsible for supporting photoreceptors. RPE degeneration commonly ...

Isolating RPE and IPE Cells from Bovine Eye: A Procedure to  Harvest and Culture Bovine Primary Pigment Epithelial Cells

Source: Bascuas, T. et al., <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/62145">Isolation, Culture, and Genetic Engineering of Mammalian Primary Pigment Epithelial Cells for Non-Viral Gene Therapy.</a> J. Vis. Exp. (2021).
This video demonstrates the procedure to isolate the retinal pigment epithelial, or RPE, cells and iris pigment epithelial, or IPE, cells from harvested bovine eyes. The isolated cells can be cultured to study their role in the treatment of ocular diseases.

Source: Bascuas, T. et al., Isolation, Culture, and Genetic Engineering of Mammalian Primary Pigment Epithelial Cells for Non-Viral Gene Therapy. J. Vis. ...

Large-Scale Purification of Porcine or Bovine Photoreceptor Outer Segments for Phagocytosis Assays on Retinal Pigment Epithelial Cells

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