Isolation of Primary Mouse Retinal Pigmented Epithelium Cells
November 4th, 2022
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.
This protocol will outline an easy and efficient way to successfully isolate primary mouse retinal pigmented epithelium cells. Retinal pigmented epithelium cells play a very important role to maintain the photoreceptor cells and to ensure the normal function of the retina. Our lab has been isolating primary mouse pigmented epithelium cells as an important tool to study the outer blood-retinal barrier and related diseases such as age-related macular degeneration.
To extract the mouse eyes, ethically euthanize the mouse according to your institution's approved methods, then place the mouse on a sterile underpad and nucleate the eye by pressing a 5/45-style tweezer on the orbital area to induce proptosis, then move the tweezer to the posterior of the eye and gently pull to remove the eye ensuring that the optic nerve is still intact. While using forceps, submerge the eyes in a 2-milliliter microcentrifuge tube containing 5%povidone iodine. Incubate the eyes at room temperature for 2 to 3 minutes.
Remove the eyes from the povidone iodine and place them in a Petri dish. While using a sterile transfer pipette, wash the eyes with Hanks'Balanced Saline Solution until the orange color of the povidone iodine is gone. This takes approximately 2 to 3 washes.
Place the eyes in a new Petri dish and submerge them in cold complete RPE cell growth media. From there, you can begin dissecting under a dissecting microscope. Under the microscope, use tweezers and/or Vannas scissors to pull or cut away connective tissue in extraocular muscles.
Eyes can then be held for an hour in cold RPE cell culture media. However, it is preferred to proceed directly to the next step after cleaning the eyeball. Transfer the eyes to a 2-milliliter microcentrifuge tube containing the enzyme A solution, then incubate the eyes inside an incubator at 37 degrees Celsius for 40 minutes.
Remove the eyes from the enzyme A solution and place them in a new 2-milliliter microcentrifuge tube containing the enzyme B solution, then place them inside an incubator and incubate at 37 degrees Celsius for 50 minutes. Remove the eyes from enzyme B and place them in a 60-millimeter Petri dish and submerge them and complete RPE cell growth media, then incubate the eyes at 4 degrees Celsius for 30 minutes. Place the dish under the dissecting microscope and begin dissecting.
Use M5S forceps and a needle of a syringe to make an incision in the ora seratta section. Now with two forceps, grasp the inner part of the incision with one forcep and grasp the cornea with the other forcep. Begin to slowly tear the cornea from the retina by pulling the cornea.
Be sure to tear in small increments and move down the tear as you remove the cornea. This will ensure that the RPE will remain mostly intact and you will get a cleaner tear. Once the cornea is completely detached, you should be able to see the iris and lens.
Remove both the iris and lens with forceps to maintain the purity of the isolation. To remove the neural retina from the RPE layer, grasp the outer layer of the eyecup with one tweezer and position an arm of a second tweezer between both the RPE and neural layers. From there, gently pull or scoop the neural retina away from the RPE layer, then discard the neural retina.
Move the RPE layer to a different place on the dish free of debris. To separate the RPE from the choroid and sclera, gently scrape the inside of the eyecup with a 5/45-style tweezer to ensure the separation of the RPE cells. The RPE cells appear cloudy when suspended and complete RPE cell growth media.
Aspirate the cells using a 3.2-milliliter sterile transfer pipette and transfer to a new 15-milliliter centrifuge tube containing complete RPE cell growth media. The cells can be centrifuged at 300g for 10 minutes at room temperature. You can then aspirate out the supernatant and resuspend the pellet in warmed RPE cell growth media.
Plate the cells on a 100-millimeter Petri dish and incubate at 37 degrees Celsius and 5%CO2. These images show a successful isolation at passage 0 and passage 1, which is evident by the pigmentation of the RPE cells. At passage 4, the cells become less characteristic and exhibit less pigment, show a spindle-like appearance or become more robust with a larger surface area.
We validated the RPE cells with an RPE-specific marker, RPE65 and a cytoskeletal protein F-actin. We also checked for potential contamination with Muller cells by staining with a Muller cell-specific marker, vimentin. This protocol is a simplified adaptation of existing protocols.
The protocol uses materials and associated equipment that are available in most research labs, which will help isolating primary mouse RPE cells in an effective way.
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Overview
This study presents a simplified protocol for isolating retinal pigmented epithelium (RPE) cells from mouse eyes, which are crucial for maintaining photoreceptor function within the retina. The stepwise method includes eye enucleation, dissection, and the subsequent isolation and culturing of RPE cells, aimed at facilitating research on retinal conditions.
Key Study Components
Research Area
- Cell biology
- Retinal health and diseases
- Isolation techniques
Background
- RPE cells support photoreceptor maintenance and function.
- Primary RPE isolation is a critical tool for studying the outer blood-retinal barrier.
- Relevance to conditions such as age-related macular degeneration.
Methods Used
- Dissection and enzymatic treatment of mouse eyes.
- Mouse model system.
- Cell culture and validation techniques (RPE65 marker).
Main Results
- Successful isolation of RPE cells with maintained pigmentation and viability.
- Demonstrated integrity of RPE cells validated by specific markers.
- Transmission of a simplified adaptation of existing isolation protocols.
Conclusions
- This study outlines an effective method for RPE cell isolation.
- The protocol can enhance research regarding retinal diseases and cellular functions in biology.
Frequently Asked Questions
Executive Industry Relevance
Isolation of primary mouse retinal pigmented epithelium (RPE) cells enables direct investigation of disease mechanisms and therapeutic responses in ocular research. This protocol supports early-stage target validation and mechanistic de-risking for retinal disease models, providing a foundation for translational studies and preclinical evaluation. Reliable RPE isolation underpins portfolio decisions in ophthalmic drug discovery by enabling comparative studies across genetic backgrounds and treatment conditions.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables interrogation of molecular mechanisms underlying RPE pathology in disease-relevant mouse models.
- Supports functional target validation by allowing direct comparison of wild-type and genetically modified RPE cells.
- Facilitates mechanistic de-risking for candidate targets implicated in retinal disorders.
Screening & Assay Development
- Provides a validated primary cell system for downstream drug screening and efficacy testing.
- Ensures reproducibility and standardization through marker-based cell characterization (e.g., RPE65, F-actin).
- Enables quantitative assessment of drug effects on RPE morphology and pigmentation.
Translational & Preclinical Research
- Aligns with disease-relevant models for translational biomarker studies in retinal degeneration.
- Supports continuity from discovery through preclinical validation by enabling in vitro and ex vivo analyses.
- Reduces biological risk by providing a physiologically relevant system for early efficacy and toxicity assessment.
Pipeline & Workflow Integration
This RPE isolation protocol fits at the interface of early discovery and preclinical research, supporting workflows from hypothesis testing to lead identification in ocular drug development.
- Discovery Biology: Facilitates hypothesis-driven studies of RPE function and disease mechanisms.
- Screening: Delivers assay-ready primary cells for reproducible compound evaluation.
- Analytics: Enables quantitative readouts of cell morphology, pigmentation, and marker expression.
- Translational Research: Bridges in vitro findings with in vivo disease models for biomarker alignment.
- Enterprise Reuse: Establishes a reusable protocol adaptable to various mouse models and experimental designs.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence and reduces mechanistic ambiguity in retinal research.
- Operational Value: Promotes standardization, reproducibility, and scalability across research teams.
- Strategic Value: Informs go/no-go decisions and enhances capital efficiency in ophthalmic portfolios.
- Portfolio Impact: Supports risk-adjusted prioritization and advancement of retinal disease programs.
Implementation Considerations
- Requires expertise in mouse dissection and primary cell culture techniques.
- Needs access to standard cell culture and imaging instrumentation for validation.
- Demands cross-team standardization of isolation and characterization protocols.
- Adaptable to different mouse genotypes and ocular disease models.
- Limited passage number (up to 3-4) may constrain long-term studies.
