Coculturing of Retinal Ganglion Neurons with Olfactory Ensheathing Glia
JoVE Encyclopedia of Experiments
Neuroscience
0 views • 3:24 min • July 8th, 2025
Take a rat's retina, a light-sensitive layer of the eye containing retinal ganglion neurons, or RGNs.
Place the retina in a cold-balanced salt solution to prevent tissue damage.
Transfer the retina to a solution containing protease and DNase and cut it into smaller pieces.
Transfer the contents to a tube and incubate.
The protease breaks down the extracellular matrix and loosens the cells, while DNase degrades any contaminating DNA.
Pipette up and down to disperse cell clumps.
Add an inhibitor solution to stop the protease action.
Centrifuge the cells to settle them. Remove the supernatant and resuspend the cells in a suitable growth medium.
Add this cell suspension to the monolayer of olfactory ensheathing glia or OEG cells, a type of glial cell, and incubate.
The RGNs integrate into the OEG monolayer, establishing the coculture model for assessing the neuroregenerative potential of OEGs.
Place the retina in a previously prepared P-60 cell culture dish with 5 milliliters of cold EBSS. Then, transfer it to P-60 cell culture dish with reconstituted papain plus 50 microliters of APV and 250 microliters of DNase plus 5 microliters of APV.
Cut the retina into small pieces with a scalpel. Transfer the pieces to a 15-milliliter plastic tube, and incubate them for 30 minutes in a humidified incubator at 37 degrees Celsius under 5% carbon dioxide agitating every 10 minutes. Dissociate cell clumps by pipetting up and down with a glass Pasteur pipette. Then, centrifuge the cell suspension at 200 times g for 5 minutes.
Discard supernatant and resuspend the cell pellet in albumin ovomucoid protease inhibitor with 150 microliters of DNase and 30 microliters of APV.
Carefully, add the cell suspension on 5 milliliters of albumin ovomucoid protease inhibitor, and repeat the centrifugation. While centrifuging, completely remove the ME-10 medium from an OAG 24-well plate, and replace it with 500 microliters of NBB-27 medium per well.
Discard the supernatant, and resuspend the cells in 2 milliliters of NBB-27 medium. Plate 100 microliters of retinal cell suspension into each well of the M24 plate onto PLL-treated or OEG monolayer coverslips.
Maintain cultures at 37 degrees Celsius with 5% carbon dioxide for 96 hours in NBB-27 medium.
Overview
This article describes a method for isolating retinal ganglion neurons (RGNs) from rat retinas and co-culturing them with olfactory ensheathing glia (OEG) cells. The process involves enzymatic dissociation of the retina, followed by cell culture techniques to assess the neuroregenerative potential of OEGs.
Key Study Components
Area of Science
- Neuroscience
- Cell Biology
- Regenerative Medicine
Background
- Retinal ganglion neurons are crucial for visual signal transmission.
- Olfactory ensheathing glia have shown potential in promoting neuronal regeneration.
- Understanding the interaction between RGNs and OEGs can provide insights into neuroregenerative strategies.
- Isolation and culture techniques are essential for studying these cells in vitro.
Purpose of Study
- To develop a coculture model for assessing the neuroregenerative potential of OEGs.
- To isolate RGNs from rat retinas effectively.
- To evaluate the integration of RGNs into OEG monolayers.
Methods Used
- Isolation of RGNs using protease and DNase treatments.
- Centrifugation and resuspension of cells in growth medium.
- Co-culturing RGNs with OEG cells on PLL-treated coverslips.
- Maintaining cultures under specific temperature and CO2 conditions.
Main Results
- Successful isolation of viable RGNs from rat retinas.
- Integration of RGNs into OEG monolayers observed.
- Establishment of a reliable coculture model for further studies.
- Potential for assessing neuroregenerative capabilities of OEGs demonstrated.
Conclusions
- The method provides a viable approach for studying RGNs and OEG interactions.
- Insights gained may contribute to developing therapies for retinal injuries.
- Future studies can build on this model to explore neuroregeneration.
Frequently Asked Questions
Executive Industry Relevance
Coculturing retinal ganglion neurons with olfactory ensheathing glia provides a controlled in vitro system to interrogate neuroregenerative mechanisms relevant to CNS injury. This model enables early-stage target validation and mechanistic de-risking for neuroregeneration-focused discovery portfolios. Its reproducibility and quantitative outputs support predictive confidence in translational research pipelines.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables direct assessment of neuroregenerative interactions between neuronal and glial cell types.
- Supports functional validation of glial-mediated axonal regeneration mechanisms.
- Facilitates mechanistic de-risking for neuroregeneration targets in preclinical pipelines.
Screening & Assay Development
- Provides a standardized coculture system for evaluating candidate molecules or interventions.
- Delivers reproducible, quantitative readouts of neuronal integration and survival.
- Enables assay scalability and platform reuse for compound screening.
Translational & Preclinical Research
- Aligns with disease-relevant models of CNS injury and regeneration.
- Supports continuity from discovery through preclinical validation of neuroregenerative strategies.
- Reduces translational risk by modeling cell-cell interactions critical for therapeutic development.
Pipeline & Workflow Integration
This coculture model fits within the early discovery to preclinical continuum, supporting hypothesis testing and target validation for neuroregeneration programs.
- Discovery Biology: Enables hypothesis-driven interrogation of glial-neuronal regenerative pathways.
- Screening: Provides a reproducible assay platform for quantitative evaluation of neuroregenerative interventions.
- Analytics: Supports measurement of neuronal integration and survival as key readouts.
- Translational Research: Bridges in vitro findings to preclinical models of CNS repair.
- Enterprise Reuse: Offers a reusable, standardized system for cross-program neuroregeneration studies.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence in neuroregeneration target validation.
- Operational Value: Standardizes coculture workflows for reproducibility and scalability.
- Strategic Value: Informs go/no-go decisions for neuroregeneration assets early in the pipeline.
- Portfolio Impact: Enables risk-adjusted prioritization of CNS repair programs.
Implementation Considerations
- Requires expertise in neuronal and glial cell isolation and culture.
- Demands access to cell culture infrastructure and analytical imaging tools.
- Necessitates cross-team standardization of coculture protocols for reproducibility.
- May require adaptation for different neuronal or glial subtypes depending on indication.
- Dependent on robust quantitative endpoints for meaningful interpretation.
Biopharma FAQs
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Last updated: 29 August 2026