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

Summary

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.

Abstract

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.

Introduction

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 retina^2,3. 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 loss^4,5. 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 debris^7-9. 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 POS^10-12, which are then internalized via the MerTK tyrosine kinase receptor^13-15. The CD36 scavenger receptor has been shown to participate in POS intake and influence its speed^16,17, 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 VIIA^21,22. 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 POS^23-28. 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 patients^29,30,27.

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 assay^32,33,21,22. 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 assay^32,10. Therefore, these large stocks allow for reproducibility and ease of use in many different types of experiments.

Protocol

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

1. 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).
2. 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…). Wear tight-fitting labcoat, gloves, sleeve protectors and goggles.
3. Precautions:
   1. Pre-chill all solutions then keep them cold during all steps. Keep eyes and gradients chilled on ice as much as possible.
   2. 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.

2. Protocol Actions

1. Prepare solutions and gradients:
   1. Thaw taurine stock completely using a 37 °C water bath.
   2. 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.
      1. 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.
      2. 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.
      3. 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.
      4. Prepare the Wash 1 solution to a final concentration of 20 mM tris acetate pH 7.2 and 5 mM taurine.
      5. Prepare the Wash 2 solution to a final concentration of 10% sucrose, 20 mM tris acetate pH 7.2 and 5 mM taurine.
      6. Prepare the Wash 3 solution to a final concentration of 10% sucrose, 20 mM sodium phosphate pH 7.2 and 5 mM taurine.
   3. 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.
   4. 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.
   5. 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.
2. Tissue collection:
   1. 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.
   2. 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.
   3. Retina homogenization:
      1. 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.
      2. 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.
3. Photoreceptor outer segment fragment (POS) isolation:
   1. 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 °C.
   2. 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).
   3. 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 °C. Carefully discard the supernatant.
   4. 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.
4. POS aliquoting without labeling:
   1. Resuspend POS in 10 - 20 ml DMEM. Predilute 10 µl in 490 µl 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.
   2. Decide on the final DMEM volume (typically 10 - 20 ml) and add sucrose to yield a final concentration of 2.5% sucrose.
   3. 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 µl/well. Spin 1 aliquot for 5 min at 2,300 x g at RT and evaluate pellet size. Prepare various aliquot sizes for different application volumes as POS should NOT be frozen and thawed twice.
   4. Freeze POS aliquots at -80 °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.
5. 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.
   1. 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.
   2. 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 °C. Rotate for at least 1.5 hr at RT. Protect from light using aluminum foil.
   3. 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).
6. Using POS in experiments:
   1. 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.
   2. 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.

Results

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 centrif...

Discussion

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 certai...

Materials

List of materials used in this article

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