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JoVE Journal Biology

Biology

Isolation of Primary Mouse Retinal Pigmented Epithelium Cells

Published: November 4, 2022 doi: 10.3791/63543
Automatic Translation
1,2, 3, 1,2, 1,2,3
1Eye Research Institute, Oakland University, 2Eye Research Center (OUWB)/ERC, William Beaumont School of Medicine, 3Oral Biology and Diagnostic Sciences, Dental College of Georgia, Augusta University

ERRATUM NOTICE

Summary

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.

Abstract

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.

Introduction

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 membrane2,3. 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 albinism4,5,6, 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 mice11,12,13. 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 homocysteine16,17.

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 protocols13,18,19 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.

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Protocol

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 methods8,14,15,16, to stain and validate the RPE specificity.

7. Immunofluorescence

NOTE: Immunofluorescence was performed as per the previously published methods8,14,15,16,17,18 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).

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Representative Results

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 are pointing to the isolated RPE cells in P4). An antibody for the RPE65 protein was used to verify the identity of the isolated cells. Immunofluorescence staining of the isolated cells was performed with an antibody against RPE65, followed by examination under the fluorescent microscope (Figure 1E,F: low magnification, and Figure 1G,H: high magnification, showing positively stained RPE cells in green and a nuclear marker, DAPI, in blue). RPE65 is an isomerohydrolase expressed in the RPE. It is essential for the regeneration of the visual pigment required for rods- and cone-mediated vision21 .To validate the barrier function of the isolated
cells, both cytoskeletal protein F-actin and tight junction protein ZO-1 immunofluorescence staining8 for the isolated cells were used (Figure 1I,J: green ZO-1 and blue nuclear staining). and Figure 1K,L: red F-actin and blue nuclear staining). However, the typical cobblestone shape of the RPE is very evident when cells are fully confluent.

Negative control was used in the experiments where the secondary antibody was added to the RPE cells without adding the primary antibody, to make sure the specificity of the antibody and the resultant color is specific to the antibodies that were used (not shown).

To validate the purity and contamination of the isolated RPE by Müller cells, immunofluorescence staining of the isolated cells with a specific cell marker for Müller cells, vimentin, was used (green and blue for nuclear staining as shown in Figure 1M,N) using Müller cells as a positive control. Isolated RPE cells showed negative staining for vimentin.

The isolated cells were examined under a light microscope to verify their viability, morphology, and pigmentation. Images from P0 and P1 (Figure 1K,L) and images from P0 and P4 were captured (Figure 1M,N) to show the changes in the cells' shape, size, and pigmentation as the passages proceeded to the fourth passage (black arrows are pointing to the isolated RPE cells in P4).

Figure 1
Figure 1: RPE isolation validation. (A) Light microscope for RPE passage zero (P0). (B) Light microscope for RPE passage one (P1). (C) Morphology characterization at P0. (D) Morphology characterization at P4. (E) Immunostaining for RPE65 at low magnification. (F) Immunostaining for RPE65 with nuclear staining with DAPI. (G) Immunostaining for RPE65 at high magnification. (H) Immunostaining for RPE65 with nuclear staining with DAPI at high magnification. (I) ZO-1 staining for RPE cells. (G) ZO-1 staining for RPE cells with nuclear staining with DAPI. (K) F-actin staining for RPE cells. (L) F-actin staining for RPE cells with nuclear staining with DAPI. (M) Vimentin staining for RPE cells. (N) Vimentin staining for Müller cells as a positive control (RMc-1) with nuclear staining with DAPI. Scale bars are equal to 300 µm, 20 µm, 300 µm, 300 µm, 50 µm, 10 µm, 50 µm, 50 µm, and 50 µm, respectively. Please click here to view a larger version of this figure.

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Discussion

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. To be able to passage the isolated cells up to three or four times, the best mice ages are between 18-21 days. To have a reasonable number of isolated cells that are able to grow and multiply, at least two eyes are needed for single isolation. The number of cells resulting in isolation is proportional to the number of eyes dissected (~220,000 cells/eye). A sterile 100 mm Petri dish/flask is recommended to obtain a larger number of cells in an early passage (P0). After seeding, cells should not be disturbed by moving the tissue culture plate from the incubator or by changing the media for at least 5 days. Also, one of the limitations ofthis technique is the storage and passaging of the isolated cells. Cells cannot be stored (freezed and rethawed) and can only be passaged three or four times. After passage 4, cells start to change their size and shape to become elongated (spindle shape) with a marked decrease in cell pigmentation (Figure 1D).

This protocol is different from other valuable and reliable published protocols for primary mouse RPE isolation13,19,20,24 in that it is simplified with a few steps that are easy to follow. RPE cells are able to be identified by their morphology, pigmentation, and specific RPE markers such as RPE654,5,21. Validation of the purity and contamination of Müller cells was achieved by staining isolated cells with a specific cell marker for Müller cells (vimentin)22. Many techniques have been used to evaluate the integrity of the blood-retinal barrier (BRB) in vitro, such as electron microscope (EM) examination, assessment of tight junction proteins (TJPs), FITIC dextran leakage assay, and transepithelial electrical resistance (TER) measurement that evaluates the physiological function of RPE as a monolayer8,23. RPE cells play an important role in maintaining the outer BRB, and to validate this function, we evaluated tight junction proteins (TJPs) such as Zona ocludin-1 (ZO-1) and cytoskeletal proteins like F-actin as previously published8. TJPs evaluation is a more convenient method for the evaluation of BRB integrity and barrier function which doesn't require expensive equipment that may not be available in all research labs, as opposed to EM evaluation or TER.

Primary RPE isolation is a technique that could be an important tool to study underlying mechanisms and pathogenesis, and for proposing new therapeutic applications for different eye diseases. The current technique enabled studying the changes in RPE cell structure and function and their impact on the outer BRB in age-related macular degeneration8. Moreover, it helped in studying the changes in RPE cells isolated from wild-type C57BL6 mice and different genetically modified mice, as well as testing the different pharmacological compounds seeking a therapeutic target for AMD14,16.

In conclusion, many available published protocols demonstrated primary mouse RPE isolation. The presented protocol is a simplified procedure resulting from modifying some existing protocols, using material, methods, and equipment that are available in most research labs to isolate primary RPE cells from mouse eyes.

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Disclosures

The authors have no conflicts of interest to declare that are relevant to the content of this article.

Acknowledgments

This work was supported by the National Eye Institute (NEI), and National Eye Institute (NEI) fund R01 EY029751-04. We would like to thank Dr. Pamela Martin for her help in our initial stages of RPE isolation.

Materials

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

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References

  1. Young, R. W., Droz, B. The renewal of protein in retinal rods and cones. The Journal of Cell Biology. 39 (1), 169-184 (1968).
  2. Sparrow, J. R., Hicks, D., Hamel, C. P. The retinal pigment epithelium in health and disease. Current Molecular Medicine. 10 (9), 802-823 (2010).
  3. Strauss, O. The retinal pigment epithelium in visual function. Physiological Reviews. 85 (3), 845-881 (2005).
  4. Marlhens, F., et al. Autosomal recessive retinal dystrophy associated with two novel mutations in the RPE65 gene. European Journal of Human Genetics. 6 (5), 527-531 (1998).
  5. Morimura, H., et al. Mutations in the RPE65 gene in patients with autosomal recessive retinitis pigmentosa or leber congenital amaurosis. Proceedings of the National Academy of Sciences. 95 (6), 3088-3093 (1998).
  6. Weiter, J. J., Delori, F. C., Wing, G. L., Fitch, K. A. Retinal pigment epithelial lipofuscin and melanin and choroidal melanin in human eyes. Investigative Ophthalmology & Visual Science. 27 (2), 145-152 (1986).
  7. Feeney-Burns, L., Hilderbrand, E. S., Eldridge, S. Aging human RPE: morphometric analysis of macular, equatorial, and peripheral cells. Investigative Ophthalmology & Visual Science. 25 (2), 195-200 (1984).
  8. Ibrahim, A. S., et al. Hyperhomocysteinemia disrupts retinal pigment epithelial structure and function with features of age-related macular degeneration. Oncotarget. 7 (8), 8532-8545 (2016).
  9. Zarbin, M. A. Age-related macular degeneration: review of pathogenesis. European Journal of Ophthalmology. 8 (4), 199-206 (1998).
  10. Dunn, K. C., Aotaki-Keen, A. E., Putkey, F. R., Hjelmeland, L. M. ARPE-19, a human retinal pigment epithelial cell line with differentiated properties. Experimental Eye Research. 62 (2), 155-170 (1996).
  11. Flannery, J. G. Transgenic animal models for the study of inherited retinal dystrophies. ILAR Journal. 40 (2), 51-58 (1999).
  12. Gibbs, D., Williams, D. S. Isolation and culture of primary mouse retinal pigmented epithelial cells. Advances in Experimental Medicine and Biology. 533, 347-352 (2003).
  13. Fernandez-Godino, R., Garland, D. L., Pierce, E. A. Isolation, culture, and characterization of primary mouse RPE cells. Nature Protocols. 11 (7), 1206-1218 (2016).
  14. Samra, Y. A., et al. Implication of N-Methyl-d-Aspartate receptor in homocysteine-induced age-related macular degeneration. International Journal of Molecular Sciences. 22 (17), 9356 (2021).
  15. van Marum, R. J. Update on the use of memantine in Alzheimer's disease. Neuropsychiatric Disease and Treatment. 5, 237-247 (2009).
  16. Elsherbiny, N. M., et al. Homocysteine induces inflammation in retina and brain. Biomolecules. 10 (3), 393 (2020).
  17. Tawfik, A., Elsherbiny, N. M., Zaidi, Y., Rajpurohit, P. Homocysteine and age-related central nervous system diseases: role of inflammation. International Journal of Molecular Sciences. 22 (12), 6259 (2021).
  18. Shang, P., Stepicheva, N. A., Hose, S., Zigler Jr, J. S., Sinha, D. Primary cell cultures from the mouse retinal pigment epithelium. Journal of Visualized Experiments. (133), e56997 (2018).
  19. Chen, M., et al. Characterization of a spontaneous mouse retinal pigment epithelial cell line B6-RPE07. Investigative Ophthalmology & Visual Science. 49 (8), 3699-3706 (2008).
  20. AVMA Guidelines for the Euthanasia of Animals: 2020 Edition. , Available from: https://www.avma.org/KB/Policies/Documents/euthanasia.pdf (2020).
  21. Cai, X., Conley, S. M., Naash, M. I. RPE65: role in the visual cycle, human retinal disease, and gene therapy. Ophthalmic Genetics. 30 (2), 57-62 (2009).
  22. Pérez-Álvarez, M. J., et al. Vimentin isoform expression in the human retina characterized with the monoclonal antibody 3CB2. Journal of Neuroscience Research. 86 (8), 1871-1883 (2008).
  23. Tawfik, A., Samra, Y. A., Elsherbiny, N. M., Al-Shabrawey, M. Implication of hyperhomocysteinemia in blood retinal barrier (BRB) dysfunction. Biomolecules. 10 (8), 1119 (2020).
  24. Promsote, W., Makala, L., Li, B., Smith, S. B., Singh, N., Ganapathy, V., Pace, B. S., Martin, P. Monomethylfumarate induces γ-globin expression and fetal hemoglobin production in cultured human retinal pigment epithelial (RPE) and erythroid cells, and in intact retina. Invest Ophthalmol Vis Sci. 55 (8), 5382-5393 (2014).

Tags

Isolation Primary Mouse Retinal Pigmented Epithelium Cells Protocol Photoreceptor Cells Retina Outer Blood-retinal Barrier Age-related Macular Degeneration Euthanize Tweezer Proptosis Optic Nerve Povidone Iodine Hanks' Balanced Saline Solution Petri Dish RPE Cell Growth Media Dissecting Microscope

Erratum

Formal Correction: Erratum: Isolation of Primary Mouse Retinal Pigmented Epithelium Cells
Posted by JoVE Editors on 12/07/2022. Citeable Link.

An erratum was issued for: Isolation of Primary Mouse Retinal Pigmented Epithelium Cells. The Discussion and References sections were updated.

The third paragraph of the Discussion section was updated from:

This protocol is different from other valuable and reliable published protocols for primary mouse RPE isolation13,19,20 in that it is simplified with a few steps that are easy to follow. RPE cells are able to be identified by their morphology, pigmentation, and specific RPE markers such as RPE654,5,21. Validation of the purity and contamination of Müller cells was achieved by staining isolated cells with a specific cell marker for Müller cells (vimentin)22. Many techniques have been used to evaluate the integrity of the blood-retinal barrier (BRB) in vitro, such as electron microscope (EM) examination, assessment of tight junction proteins (TJPs), FITIC dextran leakage assay, and transepithelial electrical resistance (TER) measurement that evaluates the physiological function of RPE as a monolayer8,23. RPE cells play an important role in maintaining the outer BRB, and to validate this function, we evaluated tight junction proteins (TJPs) such as Zona ocludin-1 (ZO-1) and cytoskeletal proteins like F-actin as previously published8. TJPs evaluation is a more convenient method for the evaluation of BRB integrity and barrier function which doesn't require expensive equipment that may not be available in all research labs, as opposed to EM evaluation or TER.

to:

This protocol is different from other valuable and reliable published protocols for primary mouse RPE isolation13,19,20,24 in that it is simplified with a few steps that are easy to follow. RPE cells are able to be identified by their morphology, pigmentation, and specific RPE markers such as RPE654,5,21. Validation of the purity and contamination of Müller cells was achieved by staining isolated cells with a specific cell marker for Müller cells (vimentin)22. Many techniques have been used to evaluate the integrity of the blood-retinal barrier (BRB) in vitro, such as electron microscope (EM) examination, assessment of tight junction proteins (TJPs), FITIC dextran leakage assay, and transepithelial electrical resistance (TER) measurement that evaluates the physiological function of RPE as a monolayer8,23. RPE cells play an important role in maintaining the outer BRB, and to validate this function, we evaluated tight junction proteins (TJPs) such as Zona ocludin-1 (ZO-1) and cytoskeletal proteins like F-actin as previously published8. TJPs evaluation is a more convenient method for the evaluation of BRB integrity and barrier function which doesn't require expensive equipment that may not be available in all research labs, as opposed to EM evaluation or TER.

In the References section, a 24th reference was added:

  1. Promsote, W., Makala, L., Li, B., Smith, SB., Singh, N., Ganapathy, V., Pace, B.S., Martin, P. Monomethylfumarate induces γ-globin expression and fetal hemoglobin production in cultured human retinal pigment epithelial (RPE) and erythroid cells, and in intact retina. Invest Ophthalmol Vis Sci. 55(8):5382-93 (2014).
Isolation of Primary Mouse Retinal Pigmented Epithelium Cells
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Tomaszewski, R., Rajpurohit, P.,… More

Tomaszewski, R., Rajpurohit, P., Cheng, M., Tawfik, A. Isolation of Primary Mouse Retinal Pigmented Epithelium Cells. J. Vis. Exp. (189), e63543, doi:10.3791/63543 (2022).

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