Isolation of Primary Mouse Retinal Pigmented Epithelium Cells

Podsumowanie

This manuscript describes a simplified protocol for the isolation of retinal pigmented epithelium (RPE) cells from mouse eyes in a stepwise manner. The protocol includes the enucleation and dissection of mouse eyes, followed by the isolation, seeding, and culturing of RPE cells.

Streszczenie

The retinal pigmented epithelium (RPE) layer lies immediately behind the photoreceptors and harbors a complex metabolic system that plays several critical roles in maintaining the photoreceptors' function. Thus, the RPE structure and function are essential to sustain normal vision. This manuscript presents an established protocol for primary mouse RPE cell isolation. RPE isolation is a great tool to investigate the molecular mechanisms underlying RPE pathology in the different mouse models of ocular disorders. Furthermore, RPE isolation can help in comparing primary mouse RPE cells isolated from wild-type and genetically modified mice, as well as testing drugs that can accelerate the development of therapy for visual disorders. The manuscript presents a step-by-step RPE isolation protocol; the entire procedure, from enucleation to seeding, takes approximately 4 hours. The media shouldn't be changed for 5-7 days after seeding, to allow the growth of the isolated cells without disturbance. This process is followed by the characterization of morphology, pigmentation, and specific markers in the cells via immunofluorescence. Cells can be passaged a maximum of three or four times.

Wprowadzenie

Retinal pigmented epithelium (RPE) cells are located between the choroid and the neural retina, forming a simple monolayer of cuboidal cells that lies behind the photoreceptor (PR) cells¹. The RPE plays a critical role in maintaining a healthy environment for PR cells, mainly by reducing the excessive accumulation of reactive oxygen species (ROS) and consequent oxidative damage¹. RPE cells oversee many functions, such as the conversion and storage of retinoids, the absorption of scattered light, fluid and ion transport, and phagocytosis of the shed PR outer segment membrane^2,3. Alterations in the RPE (morphology/function) can impair their function leading to retinopathy and this is a common feature shared by many ocular disorders⁴. Many ocular diseases are associated with alterations in the morphology and function of RPE cells, including some genetic diseases such as retinitis pigmentosa, Leber congenital amaurosis, and albinism^4,5,6, as well as age-related ocular disorders such as diabetic retinopathy (DR) and age-related macular degeneration (AMD)^7,8. Human cells are the most desirable, thus it would be ideal to study RPE disorders in primary human RPE cells for forming RPE monolayers. However, ethical matters and the limited availability of human donors due to the fact that most of these disorders lead to morbidity⁹, but not necessarily mortality¹⁰, thereby prevent the isolation of primary human RPE cells. This makes culturing RPE cells from nonhuman animal donors a preferred alternative. Rodents, particularly mice, are considered a great model for studying different ocular diseases since transgenic technology is more extensively established in these species¹¹. Even though the use of cultured primary RPE cells offers many advantages, it has been difficult to maintain growing cells for many passages, or to store and reuse the cells. The main limitation of this protocol is the mice's age; mice that are used for RPE isolation should be of a very young age (18-21 days old is optimal) as it has been difficult to culture RPE cells from adult mice^11,12,13. RPE cells can be isolated from mouse eyes at any age, however up to four cell passages was only successful with young mice (18-21 days old). RPE isolation from mouse retinas, using both C57BL6 mice and transgenic mice with deletion of the N-methyl-D-aspartate receptors (NMDARs) at the RPE cells, was performed to study the effect of elevated amino acid homocysteine on the development and progression of AMD¹⁴. In addition, isolated primary RPE cells helped in proposing a therapeutic target for AMD by inhibition of the NMDARs at RPE cells¹⁴. There are some NMDAR blockers that are approved by the Food and Drug Administration (FDA) and are currently used to treat moderate to severe confusion (dementia) related to Alzheimer's disease (AD), such as memantine¹⁶, which could be a potential therapeutic target for AMD¹⁴. Furthermore, isolated primary mouse RPE cells were used for the detection of inflammatory markers and the proposed induction of inflammation as an underlying mechanism for homocysteine-induced features of AMD and AD using a genetically modified mouse (CBS), which presents a high level of homocysteine^16,17.

This protocol was used to isolate RPE cells from both wild-type C57BL/6 mice and transgenic mice developed in our lab as a simplified adaptation of other published isolation protocols^13,18,19 to reach an easily applicable and reliable protocol. There is no sex preference in this protocol. While mice ages are critical for the isolation process, young, aged mice (18-21 days old) and older mice at any age (up to 12 months) were used for RPE isolation. However, we noticed that the RPE cells isolated from the young-aged mice lived longer, and up to four passages could be performed. The RPE cells isolated from older mice could be passaged once or twice, then they would stop growing at a normal rate and change their shape to be more elongated (fibroblast-like cells). Loss of pigmentation and decreased adhesion to the tissue culture plate followed by detachment was also observed.

Protokół

Animals were used as per the guidelines of Oakland university IACUC animal protocol number 21063 and the guidelines of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

1. Solution preparation

1. Prepare the complete RPE cell culture media by supplementing Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12) with 25% Fetal Bovine Serum (FBS), 1.5% penicillin/streptomycin, and 0.02% gentamicin.
2. Prepare Enzyme A by supplementing 741 µL of Hank's Balanced Salt Solution (HBSS) with 127 µL of Collagenase stock solution and 132 µL of Hyaluronidase stock solution. This makes a final volume of 1 mL, which can treat four eyes.
   1. Prepare the Collagenase stock solution by dissolving 1 mg of Collagenase from Clostridium histolyticum in 41 mL of HBSS (19.5 units /mL).
   2. Prepare the Hyaluronidase stock solution by dissolving 1 mg of Hyaluronidase from bovine testes in 18.4 mL of HBSS (38 units /mL).
3. Prepare Enzyme B by adding two parts of 0.25% Trypsin-EDTA to three parts of HBSS. Note that the total volume is dependent on the number of eyes dissected; 1 mL of Enzyme B can be used to treat four eyes.

2. Enucleation

1. Ethically euthanize the mouse using carbon dioxide (CO₂) inhalation according to IACUC standards²¹.
   1. Briefly, place the animal(s) in the CO₂ chamber to introduce 100% CO₂ with a fill rate of 30%-70% of the chamber volume per minute with CO₂ to achieve unconsciousness within 2 to 3 min, with minimal distress to the mice.
   2. Confirm death (lack of respiration and faded eye color), then move the mouse from the cage to a clean sterilized surgical area (scrubbed with alcohol) equipped with sterilized surgical equipment.
2. Place the mouse on a sterile underpad.
3. Enucleate the eyes by pressing 5/45 style tweezers on the orbital area to induce proptosis, followed by moving the tweezers to the posterior of the eye. Extract the eyes, with the optic nerve intact, by pulling the eyes gently from the orbital cavity.
4. Using forceps, submerge the eyes in a 2 mL microcentrifuge tube containing 1mL of 5% Povidone-Iodine. Incubate the eyes at room temperature for 2-3 min.
5. Remove the eyes from the povidone-iodine solution and place them in a Petri dish. Using a sterile transfer pipette, wash the eyes with HBSS until the orange color of the povidone-iodine is gone. This takes around two or three washes.
6. Place the eyes in a new 60 mm Petri dish and submerge them in cold (2-8 °C) complete RPE cell culture media prepared in step 1.1.

3. Dissection

1. Under a surgical microscope, use M5S style tweezers and Vannas scissors to remove the connective tissue and extraocular muscles.
   NOTE: The dissection is done under a tissue hood in a completely sterile environment. However, for video purposes, the dissecting microscope was placed outside the hood to achieve a higher quality demonstration/filming. Placing a small piece of lint-free tissue paper under the eye improves stability during dissection. The eyes can then be held temporarily (not for more than 1 hour) in the cold RPE cell culture media. However, it is preferable to proceed directly to the next step immediately after cleaning the eyes.
2. Transfer the eyes to a 2 mL microcentrifuge tube containing 1 mL of Enzyme A solution for every four eyes. Then, incubate the eyes in an incubator at 37 °C for 40 min.
3. Remove the eyes from the incubator and microcentrifuge tube. Then, transfer them to a new microcentrifuge tube containing 1 mL of Enzyme B solution for every four eyes. Incubate the tube in an incubator at 37 °C for 50 min.
4. Place the eyes in a new 60 mm suspension culture dish containing 10 mL of complete RPE cell growth media. Then incubate the eyes at 4 °C for 30 min.
5. Place the dish under a surgical microscope and begin dissecting.
6. Remove the anterior part of the eye (cornea, iris, and lens) from the posterior part of the eyecup (posterior retina).
   1. Grasp the eye with M5S style forceps and make an incision in the ora serrata section (most anterior part of the retina where the light blue ends) with a #11 blade or the needle of a syringe.
   2. Grasp the inner part of the incision with one pair of forceps, and grasp the cornea with another pair of forceps. Begin to slowly pull or tear the cornea from the retina. Be sure to tear in small increments and move down the tear while removing the cornea, to ensure that the RPE remains mostly intact and results in a cleaner tear.
      NOTE: Once the cornea is completely detached, the iris and lens can be seen.
   3. Separate both the cornea and iris.
7. Separate the lens from the eyecup with gentle compression of the posterior half of the eye.
8. To remove the neural retina from the RPE layer, grasp the outer layer of the eyecup with one pair of tweezers and position the arm of a second pair of tweezers between both the RPE and the neural layers. From there, gently pull or scoop the neural retina away from the RPE layer. Discard the neural retina.
   NOTE: In the video, the neural retina and lens are removed together. However, for beginners, it will be easier to remove each separately. What remains is the RPE, choroid, and sclera.
9. Discard the cornea, iris, lens, and neural retina. Move the remaining eyecup to a new area on the dish free of debris.
10. To separate the RPE from the choroid and sclera, gently scrape the inside of the eyecup with 5/45 style tweezers to ensure the separation of the RPE cells. The RPE cells appear cloudy when suspended in the complete RPE cell growth media.
11. Aspirate the cells using a 3.2 mL sterile transfer pipette and transfer to a new 15 mL centrifuge tube containing complete RPE cell growth media.

4. Centrifugation

1. Place the 15 mL centrifuge tube containing primary RPE cells inside a centrifuge and centrifuge the cells at 300 x g for 10 min at room temperature.
2. Aspirate the supernatant and resuspend the pellet with 10 mL of complete RPE growth media. The complete RPE growth media is more specific for RPE growth than other contaminating cells, such as Müller cells or choroidal endothelial cells. Müller cells require high glucose media, while choroidal endothelial cells require specific growth factors. By changing the media and passaging the culture, only the RPE cells will continue to grow.

5. Cell culture

1. Plate the resuspended primary RPE cells onto a sterile 100 mm Petri dish and transfer them to an incubator (after verifying the purity by staining cells with specific RPE/Müller cell markers, as indicated in Figure 1E-N). Incubate the cells at 37 °C and 5% CO₂.
   NOTE: The markers used are mentioned in the Table of Materials. This step is performed after the culture is established.
2. Incubate the cells for about 5 days to grow. During this time, do not change the media, disturb the cells, or move the cell culture plate (this is a critical step in the protocol; after isolation, the cells should be incubated and not be disturbed for 5 days).
   NOTE: The number of cells depends on the number of eyes used for the isolation. A greater number of eyes isolated will yield a greater number of cells. RPE isolation from two eyes will yield ~ 440,000-880,000 cells, or 5%-10% of the 100 mm Petri dish, which can hold 8.8 million cells.

6. Passaging

1. Aspirate out the media and wash with 2 mL of PBS. Then aspirate out the PBS.
2. Add 4 mL of 0.25% Trypsin-EDTA (1x), or enough to cover the base of the dish. Incubate for 5-10 min at room temperature.
3. Add 8 mL of pre-warmed RPE media or twice the volume of how much trypsin was used in step 6.2. Then collect the cells and place them in a 15 mL centrifuge tube.
4. Centrifuge at 300 x g for 7 min at room temperature. Aspirate the supernatant and resuspend the pellet in 10 mL of complete RPE cell culture media. Plate the whole suspension in a sterile 100 mm Petri dish.
   NOTE: The cells can be passaged up to four or five times¹⁸. However, the most consistent experiment results are found after two or three passages. After two to four passages, the cells changed their shape (as shown in Figure 1D) to be more elongated, accumulated in the middle of the plate, stopped growing, and detached.
5. Use immunofluorescence protocols, as per the previously published methods^8,14,15,16, to stain and validate the RPE specificity.

7. Immunofluorescence

NOTE: Immunofluorescence was performed as per the previously published methods^{8,14,15,16,17,18} to stain and validate the RPE specificity. The staining of primary RPE cells was typically done at passages P1 and P2. Here, a brief overview of the immunofluorescent protocol is presented.

1. Seed the cells on a cell culture chamber slide (30,000 cells per well for the 8-well chamber slide).
2. Culture the cells in complete RPE cell culture media at 37 °C and 5% CO₂ for 24-48 h.
3. When the cells are 80%-90% confluent, aspirate out the media and wash the chamber slide with 1x PBS.
4. Fix the slide with 4% paraformaldehyde for 10-15 min.
5. Aspirate out the 4% paraformaldehyde and then block with blocking solution (see Table of Materials).
6. Aspirate out the blocking solution and add the primary antibody, RPE65, and incubate for three hours at 37 °C (with an antibody concentration ranging from 1:200-1:250 in blocking buffer, at a volume of 150-200 µL/slide). Then, wash three times with PBS/TX-100 (5-10 min each wash).
7. Aspirate out the primary antibody and add the secondary antibody (with an antibody concentration 1:1,000 in blocking buffer, at a volume of 150-200 µL/slide). Incubate for one hour at 37 °C.
8. Aspirate out the secondary antibody. Wash three times with PBS/Trtion X-100 (5-10 min each wash).
9. Add a drop of DAPI mounting medium to label the nuclei and place a coverslip over the slide. Ensure that there are no bubbles beneath the coverslip.
10. Then, take images with a fluorescent microscope, accompanied by a high-resolution microscope (HRM) camera and image processing software (see Table of Materials).

Wyniki

Validation of the specificity, purity, and barrier function/formation of isolated RPE cells
The isolated cells were examined under a light microscope to verify their viability, morphology, and pigmentation. Images from P0 and P1 (Figure 1A,B) and images from P0 and P4 were captured (Figure 1C,D) to show the changes in the cells; shape, size, and pigmentation as the passages proceeded to the fourth passage (black arrows...

Dyskusje

The current protocol is a reported, modified, and simplified detailed procedure for RPE isolation from mouse eyes. The protocol includes enucleation, dissection, collection, seeding, culture, and characterization of RPE cells isolated from mouse eyes.

There are some limitations and critical steps that must be fulfilled for successful RPE isolation, such as mice age, the number of eyes dissected, the size of the tissue culture plate or dish, and cautions after seeding, storage, and passaging. T...

Materiały

Name	Company	Catalog Number	Comments
Beaker : 100mL	KIMAX	14000
Collagenase from Clostridium histolyticum	Sigma-Aldrich	C7657-25MG	For working enzyme, A
Disposable Graduated Transfer Pipettes :3.2mL Sterile		13-711-20
DMEM/F12	gibco	11330	Media to grow RPE cells
Fetal Bovine Serum (FBS)	gibco	26140079	For complete RPE cell culture media
Gentamicin Reagent Solution	gibco	15750-060	For complete RPE cell culture media
Hanks' Balanced Salt Solution (HBSS)	Thermo Scientific	88284	For working enzymes (A&B)
Heracell VISO 160i CO2 Incubator	Thermo Scientific	50144906
Hyaluronidase from bovine testes	Sigma-Aldrich	H3506-500MG	For working enzyme A
Kimwipes	Kimberly-Clark	34155
Luer-Lok Syringe with attached needle 21 G x 1 1/2 in., sterile, single use, 3 mL	B-D	309577
Micro Centrifuge Tube: 2 mL	Grainger	11L819
Mouse monoclonal anti-RPE65 antibody	Abcam, Cambridge, MA, USA	ab78036	For IF staining
Pen Strep	gibco	15140-122	For complete RPE cell culture media
Positive Action Tweezers, Style 5/45	Dumont	72703-DZ
Scissors Iris Standard Straight 11.5cm	GARANA INDUSTRIES	2595
Sorvall St8 Centrifuge	ThermoScientific	75007200
Stemi 305 Microscope	Zeiss	n/a
Surgical Blade, #11, Stainless Steel	Bard-Parker	371211
Suspension Culture Dish 60mm x 15mm Style	Corning	430589
Tissue Culture Dish : 100x20mm style	Corning	353003
Tornado Tubes: 15mL	Midsci	C15B
Tornado Tubes: 50mL	Midsci	C50R
Trypsin EDTA (1x) 0.25%	gibco	2186962	For working enzyme B
Tweezers 5MS, 8.2cm, Straight, 0.09x0.05mm Tips	Dumont	501764
Tweezers Positive Action Style 5, Biological, Dumostar, Polished Finish, 110 mm OAL	Electron Microscopy Sciences Dumont	50-241-57
Underpads, Moderate : 23" X 36"	McKesson	4033
Vannas Spring Scissors - 2.5mm Cutting Edge	FST	15000-08
Zeiss AxioImager Z2	Zeiss	n/a
Zeiss Zen Blue 2.6	Zeiss	n/a