Summary

Generating and Co-culturing Murine Primary Microglia and Cortical Neurons

Published: July 26, 2024
doi:

Summary

This protocol describes a microglia-neuronal co-culture established from primary neuronal cells isolated from mouse embryos at embryonic days 15-16 and primary microglia generated from the brains of neonatal mice at post-natal days 1-2.

Abstract

Microglia are tissue-resident macrophages of the central nervous system (CNS), performing numerous functions that support neuronal health and CNS homeostasis. They are a major population of immune cells associated with CNS disease activity, adopting reactive phenotypes that potentially contribute to neuronal injury during chronic neurodegenerative diseases such as multiple sclerosis (MS). The distinct mechanisms by which microglia regulate neuronal function and survival during health and disease remain limited due to challenges in resolving the complex in vivo interactions between microglia, neurons, and other CNS environmental factors. Thus, the in vitro approach of co-culturing microglia and neurons remains a valuable tool for studying microglia-neuronal interactions. Here, we present a protocol to generate and co-culture primary microglia and neurons from mice. Specifically, microglia were isolated after 9-10 days in vitro from a mixed glia culture established from brain homogenates derived from neonatal mice between post-natal days 0-2. Neuronal cells were isolated from brain cortices of mouse embryos between embryonic days 16-18. After 4-5 days in vitro, neuronal cells were seeded in 96-well plates, followed by the addition of microglia to form the co-culture. Careful timing is critical for this protocol as both cell types need to reach experimental maturity to establish the co-culture. Overall, this co-culture can be useful for studying microglia-neuron interactions and can provide multiple readouts, including immunofluorescence microscopy, live imaging, as well as RNA and protein assays.

Introduction

Microglia are tissue-resident macrophages that facilitate immunosurveillance and homeostasis in the central nervous system (CNS)1,2,3. They originate from yolk sac erythromyeloid progenitor cells that colonize the brain during embryonic development4,5,6 and are maintained throughout the organism's life span through self-renewal, which involves proliferation and apoptosis7. At steady-state, resting microglia have ramified morphology and engage in tissue surveillance8,9,10.

Microglia express numerous cell-surface receptors, which enables them to rapidly respond to changes in the CNS11,12 and to promote inflammatory responses in the event of infections or tissue injury12,13,14, as well as during neurodegenerative diseases9,15, such as multiple sclerosis (MS)16,17. Microglia also express receptors to various neurotransmitters and neuropeptides18,19,20, which suggests they may also respond to and regulate neuronal activity21,22. Indeed, microglia and neurons interact in various forms of bidirectional communication8,23 such as direct interactions mediated by membrane proteins or indirect interactions through soluble factors or intermediate cells23,24.

For instance, various neurotransmitters secreted by neurons can modulate the neuroprotective or inflammatory activity of microglia25,26,27. Additionally, direct interactions between neurons and microglia help to maintain microglia in a homeostatic state28. Conversely, direct interactions of microglia with neurons can shape neuronal circuitry29 and influence neuronal signaling30,31,32. As disruptions of these interactions induce hyperexcitability of neurons30 and microglia reactivity33,34, dysregulated microglia-neuronal interactions are implicated as a contributing factor to neurological diseases33,35. Indeed, psychotic23,26 and neurodegenerative diseases have been described to exhibit dysfunctional microglia-neuronal interactions33. While these observations highlight the importance of microglia-neuronal communication in the CNS, specific mechanisms of how these interactions regulate microglial and neuronal functions in health and disease are relatively unknown.

Within a complex milieu such as the CNS, multiple environmental factors can influence microglia-neuronal interactions, which limits the ability to study transient cellular interactions in vivo. Here, we present an in vitro microglia-neuronal co-culture system that can be used to study direct cellular interactions between microglia and neurons. This protocol describes the generation of primary microglia and neurons from the cortices of neonatal mice between post-natal days 0-2 and embryonic mice days 16-18, respectively. Neurons and microglia are then co-cultured in 96-well plates for downstream high-throughput experiments. We previously used this approach to demonstrate that microglia phagocytosis protects neurons from oxidized phosphatidylcholine mediated cell death37, suggesting that this method can help to understand the roles of microglia in the context of neurodegeneration and MS. Similarly, microglia-neuronal co-cultures may also be useful for investigating the impact of microglia-neuronal crosstalk in other contexts such as viral infections38 or neuronal injury and repair39. Overall, in vitro microglia-neuronal co-culture systems enable researchers to study microglia-neuronal interactions in a manipulatable and controlled environment, which complements in vivo models.

Protocol

All animals used in this study were housed and handled with approval from the University Animal Care Committee (UACC) of the University of Saskatchewan and the Canadian Council on Animal Care (CCAC). Post-natal days 0-2 CD1 male and female mice and embryonic days 16-18 (E16-18) embryos from pregnant CD1 mice were used for this study. The details of the reagents and the equipment used are listed in the Table of Materials.

1. Primary microglia culture

NOTE: It is crucial to time the mixed glia and neuronal cultures so that microglia are mature and ready for harvest within 2 days after neurons are seeded into a 96-well plate.

  1. Preparation
    1. Pre-warm complete glia culture media, which includes Dulbecco's Modified Eagle Medium (DMEM) with high glucose supplemented with 10% heat-inactivated fetal bovine serum, 50 U/mL Penicillin/Streptomycin with 2.92 mg/mL of L-glutamine, 1mM sodium pyruvate, 1x non-essential amino acids, and 20 mM of HEPES in a 37 °C water bath.
    2. Add 5 mL of 10 µg/mL poly-L-ornithine (PO) hydrobromide to each T-75 flask. Gently swirl the flask to ensure that PO completely covers the bottom of the flask. Incubate at 37 °C in a 5% CO2 incubator for at least 1 h.
      NOTE: This coating step is required to facilitate cell adhesion to the T-75 flasks.
  2. Isolation of brains from P0-P2 neonatal mice
    NOTE: Sterilize all dissection tools and maintain the sterility of reagents. Ensure adherence to aseptic techniques throughout.
    1. Before starting dissection, spray down the countertop with 70% ethanol or disinfecting spray where dissection will take place. Set up the dissection microscope and place the Petri dish under the microscope. Pour 20 mL of Leibovitz's L-15 media into the petri dish. Keep the lid of the Petri dish on while the microscope is not in use to reduce contamination.
    2. Place down several layers of paper towel and thoroughly spray the paper towels with 70% ethanol. Prepare the dissection tools (dissection scissors, a pair of micro-forceps, and tissue forceps) by thoroughly spraying them with 70% ethanol and laying the tools down on the paper towels.
    3. Transfer post-natal days 0-2 mouse onto the paper towels and thoroughly spray the mouse with 70% ethanol. Decapitate the mouse using dissection scissors (following institutionally approved protocols).
    4. While gently holding the head, create a midline incision in the cranium starting from the neck to the nose. Peel the skull back with tissue forceps to reveal the brain.
    5. Using tissue forceps, gently scoop the brain out by inserting the forceps under the brain and lift the brain out from the head. Immediately place the brain into the Petri dish containing 20 mL of Leibovitz's L-15 media.
    6. Under a dissection microscope, carefully remove the brain stem and the meninges using a pair of micro-forceps. Collect the brains in a 50 mL conical tube containing 5 mL of Leibovitz's L-15 media.
  3. Brain dissociation, seeding and maintaining mixed glia culture
    NOTE: Perform all subsequent steps in a biosafety cabinet.
    1. Mince the brains into approximately 1 mm2 pieces with a sterile disposable scalpel blade. Carefully transfer the minced brains into a new sterile 50 mL tube.
    2. To the minced brains, add a final concentration of 0.25% trypsin and incubate the tissue in a 37 °C water bath for 20 min. Every 2-3 min, mix the solution by gently inverting the tube to aid tissue dissociation.
    3. From the 5% CO2 incubator, transfer the T-75 flasks containing 5 mL of PO into the biosafety cabinet. Discard the PO and rinse the flasks 3 times with sterile 1x phosphate buffer solution (PBS). Ensure to thoroughly rinse the flasks as excess PO is toxic to cells.
    4. Transfer the digested tissue from the 37 °C water bath into the biosafety cabinet. Add 5 mL of complete glia culture media to neutralize the trypsin. Gently mix the tissue suspension using a 10 mL pipette.
    5. Place a sterile 70 µm nylon mesh cell strainer onto a sterile 50 mL conical tube and wet the mesh by pouring roughly 5 mL of L-15. Carefully decant the cell suspension through the cell strainer. Remove the plunger from a sterile 1 mL syringe, grind the tissue into the mesh using the plunger to get rid of tissue clumps, and generate a homogenized cell suspension.
    6. Periodically, pour approximately 5 mL of Leibovitz's L-15 media into the 70 µm nylon mesh and continue grinding the tissue on the mesh with the 1 mL syringe plunger. When little to no visible tissue remains on the mesh, discard the mesh and set the cell suspension aside.
    7. Mix the cell suspension by gently pipetting up and down. To each flask, add equal volumes of cell suspension containing 1-2 brains with glia culture media to a final volume of 15 mL.
    8. Incubate the cells at 37 °C in a 5% CO2 incubator overnight. The following day, wash the flasks twice with sterile 1x PBS to remove unattached cells and debris.
    9. Add 15 mL of fresh complete glia culture media to the culture and incubate at 37 °C in a 5% CO2 incubator. After 3 days, change half the media with 10 mL of fresh glia culture media supplemented with 40 ng/mL of macrophage colony-stimulating factor (MCSF).
      NOTE: Thereafter, half the media is replaced with 10 mL of fresh glia culture media supplemented with 40 ng/mL of MCSF every 3 days. The culture is maintained for 8-10 days before microglia are harvested.

2. Primary neuron culture

  1. Preparation
    1. Thaw B-27 plus supplement from B-27 plus neuronal culture system. Pre-warm neurobasal plus medium from B-27 plus neuronal culture system, 1x Hank's Balanced Salt Solution (HBSS), and heat-inactivated fetal bovine serum (FBS) at 37 °C in a water bath.
    2. Coat T-25 flasks with 5 mL of 10 µg/mL PO and incubate the flasks for at least 1 hour at 37 °C. Prepare instruments and supplies for dissection.
      NOTE: This coating step facilitates cell adhesion to the T-25 flasks.
  2. Brain isolation from E16-E18 mouse embryos
    NOTE: Sterilize all dissection tools and maintain the sterility of reagents. Ensure adherence to aseptic techniques throughout.
    1. Before starting dissection, spray down the countertop with 70% ethanol or disinfecting spray where dissection will take place. Set up the dissection microscope and place the Petri dish under the microscope. Pour 20 mL of 1x HBSS into the Petri dish. Keep the lid of the Petri dish on while the microscope is not in use to reduce contamination.
    2. Place down several layers of paper towel and thoroughly spray the paper towels with 70% ethanol. Prepare the dissection tools (spring scissors, a pair of micro-forceps, and tissue forceps) by thoroughly spraying them with 70% ethanol and laying the tools down on the paper towels.
    3. Anesthetize a pregnant mouse between gestation days 16-18 using isoflurane and euthanize the mouse by cervical dislocation (following institutionally approved protocols).
    4. Lay the mouse on its back on a piece of paper towel soaked in 70% ethanol. Disinfect the abdominal area with 70% ethanol. Lift the skin of the lower abdomen with tissue forceps and perform a V-shaped incision from this point to the lower rib cage with dissection scissors.
    5. Grab the uterine horns containing the embryos with tissue forceps and gently remove the embryos from the abdominal cavity. Briefly disinfect the uterine horns containing embryos with 70% ethanol.
    6. Dissect one embryo from its individual sac at a time. Using spring scissors, create a midline incision at the top of the skull, starting from the back of the neck to the nose. Then, lift the skull away from the incision site to reveal the brain. Gently scoop out the brain with tissue forceps and transfer the brain to a 10 cm Petri dish containing 15 mL of 1x HBSS.
    7. Under a dissection microscope, carefully remove the brainstem and the meninges on both sides of the brain cortices with a pair of micro-forceps. Transfer the brain cortices into a 50 mL conical tube containing 10 mL of 1x HBSS and place on ice.
  3. Brain dissociation and seeding of cortical neurons in cell culture
    NOTE: Perform all subsequent steps in a biosafety cabinet.
    1. Mince the brains into approximately 1 mm2 pieces in the 50 mL conical tube with a sterile disposable scalpel blade. Transfer the minced brains into a new sterile 50 mL conical tube.
    2. Add trypsin to the tube with a final concentration of 0.25%. Immerse the tube in a 35-38 °C hot water bath for 15 min and gently mix the tube to homogenize the tissue every 2-3 min.
    3. Take the tube out of the hot water bath. Gently pipette the tissue suspension up and down to further dissociate cells, then add 0.5 mL of heat-inactivated FBS to the homogenized suspension to neutralize trypsin activity. Avoid air bubbles to minimize toxicity to neuronal cells.
    4. Place a sterile 70 µm nylon mesh cell strainer onto a sterile 50 mL conical tube and wet the mesh by adding roughly 5 mL of neurobasal media. Filter the cell suspension through the 70 µm nylon mesh cell strainer. Using a 1 mL syringe plunger, gently grind the tissue into the cell strainer. Periodically pour approximately 5 mL of 1x HBSS while continuing grinding to wash the cell strainer.
    5. Centrifuge the conical tube at 300 x g for 5 min at 4 °C. During this time, dilute B-27 plus supplement in neurobasal plus media to 1x final concentration to use as complete neurobasal media.
    6. Discard the PO in the T-25 flasks and rinse the flasks three times with 5 mL of 1x PBS.
    7. Carefully decant to remove the supernatant and resuspend the cell pellet by gently pipetting up and down 2-3 mL of complete neurobasal media. Determine the cell concentration and total cell numbers with a hemocytometer and trypan blue. Expect up to 1.0 x 107 cells per embryo.
    8. Plate the cells in the PO-coated T-25 flasks with approximately 2.0 x 107 cells per flask in 5 ml of complete neurobasal media. Incubate the cell culture at 37 °C in a 5% CO2 incubator.
    9. Perform a half media change every 2-3 days by replacing 2.5 mL of media from the culture flask with freshly made complete neurobasal media.

3. Co-culture of primary neurons and microglia

NOTE: All subsequent steps are to be done in a sterile biosafety cabinet.

  1. Seeding neurons in 96-well plates
    1. Add 100 µL of 10 µg/mL PO per well and incubate the flasks for at least 1 h at 37 °C to coat the wells with PO. Wash the plate with 1x PBS 3 times before seeding cells and aspirate any remaining liquid after the last wash.
    2. Neurons grown in T-25 flasks are harvested for co-culture after 2-5 days in culture. Replace the media with 5 mL of 1x Versene solution with 0.25% trypsin and 1 mg/mL DNase I to detach neurons from the flask. Place the flasks inside the incubator for 5-6 min for digestion and gently swirl the flask every 2-3 minutes to aid cell detachment.
    3. Take the flask out of the incubator and check if cells are detached under a microscope. Add 0.5 mL of heat-inactivated FBS to neutralize the trypsin and DNase I. Transfer cells to a 15 mL conical tube. Wash the flask with 5 mL of complete neurobasal media once to maximize collected neurons. Collect all cells in the same 15 mL conical tube.
    4. Centrifuge the cell suspension at 300 x g for 5 min at 4 °C. Discard the supernatant and gently resuspend the cell pellet in 2-3 mL of complete neurobasal media.
    5. Count cells with a hemocytometer and add complete neurobasal media to achieve a final concentration of 7.5 x 105 neurons/mL. Seed 100 µL of the cell suspension to each well in the PO-coated 96-well plate to obtain 7.5 x 104 neurons/well. A yield of 5-6 million neurons per flask is expected.
    6. Incubate the neurons in a 96-well plate overnight. Add 100 µL of complete neurobasal media to each well the next day.
      ​NOTE: Once neurons display appropriate neurite process growth (around day 3-4 after seeding in 96-well plates), they are ready for co-culture with microglia.
  2. Isolation and microglia-neuronal co-culture
    1. Between 8-10 days of mixed glia cultivation, collect microglia by gently washing the T-75 flasks with glia culture media in each flask with a 10 mL pipette, and transfer the cells and media into a sterile 50 mL conical tube. Wash the flasks again with 10 mL fresh glia culture media to detach additional microglia from the flasks and collect the media. A yield of 7.5 x 105 microglia per flask is expected.
    2. Add 20 mL of fresh glia culture media supplemented with 20 ng/mL MCSF to each flask. Microglia may be harvested once or twice more if the mixed glia culture is maintained.
    3. Centrifuge the cell collection at 300 x g for 5 min at 4 °C. Carefully remove the supernatant and gently resuspend the pellet in 2 mL of complete neurobasal media.
    4. Count the cells with a hemocytometer and trypan blue. Add complete neurobasal media to a final concentration of 2.5 x 105 cells/mL.
    5. From the 96-well neuronal culture plate, remove 100 µL of neurobasal media and add 100 µL of 2.5 x 105 cells/mL cell suspension to seed 2.5 x 104 microglia per well of 7.5 x 104 neurons. Incubate the culture overnight at 37 °C in a 5% CO2 incubator to allow microglia to settle. The co-culture may now be used for downstream experiments.

Representative Results

A flowchart showing the key steps of the mixed glia culture for microglia is shown in Figure 1A. Overall, sparse cells and excessive cellular debris are expected on day 1 (Figure 1B). By day 4, increased cell number should be observed, especially with the generation of adherent astrocytes, as indicated by their elongated morphology (Figure 1C). A few microglia may be observed on top of the astrocytes or as small round cells floating in media. By day 8, a confluent layer of astrocytes will form the adherent layers of cells, whereas microglia will appear as round and bright cells that are semi-attached to the astrocytes or floating in media (Figure 1D). If microglial density is low by day 8, the culture may be incubated for additional days before harvest.

A flow chart showing the key steps of the neuronal culture is shown in Figure 2A. In a healthy culture, neurons will attach and form neurite processes after 1-3 days in vitro (Figure 2B). Similarly, upon seeding neurons into a 96-well plate, they will appear round initially, but by day 3, neurons should become adherent and form neurite processes that connect with neighboring neurons (Figure 2C). Conversely, unhealthy neuron cultures will contain cell clumps and cell debris, which can result from deficient cell dissociation or mishandling of neurons (Figure 2D).

A flow chart showing the key steps of the microglia-neuronal co-culture protocol is shown in Figure 3A. After adding 2.5 x 104 microglia to 7.5 x 104 neurons, round and large microglia should appear on top or between the neurons and neurite processes (Figure 3B). To further visualize the microglia-neuronal co-culture, microglia and neurons alone or in co-culture were labeled with ionized calcium-binding adaptor molecule 1 (IBA-1, red) and tubulin βIII (TUJ1, green) antibodies37 for immunofluorescence microscopy (Figure 3CF).

Figure 1
Figure 1: Overview of primary microglia culture. (A) Flow chart of the key steps in generating a mixed glia culture. (BD) Representative 10x bright-field microscope images of mixed glia culture on day 1 (B), day 4 (C), and day 8 (D). Scale bars: 100 µm. Please click here to view a larger version of this figure.

Figure 2
Figure 2: Overview of primary neuron culture. (A) Flow chart of the key steps in generating neurons. (B) Representative 10x bright field microscope images showing neurons cultured in T-25 flasks. (C) Representative 10x bright field microscope of neurons cultured in a 96-well plate. (D) Representative 10x bright field microscope of an unhealthy neuronal cluster in a 96-well plate. Scale bars: 100 µm. Please click here to view a larger version of this figure.

Figure 3
Figure 3: Overview of microglia-neuron co-culture. (A) Flow chart of the key steps for the microglia-neuronal co-culture. (B) Representative 10x bright-field microscope images of microglia-neuronal co-culture in 96-well plates. Scale bars: 100 µm. (C,D) Representative immunofluorescence microscopy images of neurons alone (C), microglia alone (D), day 2 neuron and microglia co-culture (E), or 12-day neuron and microglia co-culture (F) in a 96-well plate. Microglia and neurons are labeled with IBA-1 (red) and tubulin βIII (green), respectively. Nuclei are stained with DAPI (blue). Immunofluorescence images were acquired using a 25x, 0.8 NA, water immersion objective with a widefield fluorescence microscope. Scale bars: 50 µm. Please click here to view a larger version of this figure.

Discussion

This article describes a protocol for isolating and culturing mouse primary neurons and primary microglia, which are subsequently used to establish a microglia-neuronal co-culture that can be used to study how microglia and neuron interactions regulate their cellular health and function. This relatively simple and accessible approach can provide critical insights into the mechanisms and functional outcomes of microglia neuron interactions in the CNS.

To achieve an optimal co-culture, several critical aspects in this protocol require extra care. During brain isolations (steps 1.2.6 and 2.2.7), meninges attached to the surface of both the embryonic and post-natal brains should be removed completely before collecting the tissues. Like other neuroglia protocols, incomplete removal of the meninges may result in cell culture becoming contaminated with endothelial cells or blood-derived cells40,41,42. Additionally, when culturing primary neurons (steps 2.3.7 and 3.1.4), it is crucial to generate a single-cell suspension and avoid cell clumps before seeding neurons into flasks or 96-well plates. Neuronal aggregates will not properly adhere in culture and will lead to excessive neuronal stress and cell death. In addition, extended treatment of neurons with trypsin when attempting to dissociate them from T-25 flasks may cause unwanted cell death (step 3.1.2). To mitigate this, neurons may be directly seeded in 96-well plates at the desired cell density following dissociation from embryonic brains (step 2.3.7). Moreover, in this protocol, a 3:1 neuron: microglia ratio was selected to partially represent an elevated amount of microglia that may be present in the CNS microenvironment during neuroinflammation. Alternatively, the ratio of cells in the co-culture may be adjusted per experimental need. In addition, the timepoint selected for co-culture neurons may be extended if one aims to assess more mature neuronal states.

Notably, when dissociating neurons from T-25 flasks to 96-well plates, using trypsin alone may result in incomplete dissociation and the formation of cell clumps instead of a monolayer culture in 96-well plates (Figure 2D). To address this issue, neurons can be dissociated from the T-25 flask using 1x Versene solution containing 1 mg/mL DNase I. Other factors contributing to neuronal aggregation and reduced viability include incomplete trituration of the cell suspension, inadequate neutralization of trypsin with heat-inactivated FBS, excessive centrifugation, or prolonged cell pellet formation. Therefore, ensuring proper tissue dissociation and the generation of a single-cell suspension are critical for establishing a healthy microglia and neuron co-culture.

This protocol aims to recapitulate microglia-neuronal interactions in vitro to better understand the functional importance of their interactions in the CNS. While other types of co-culture systems have been developed, such as culturing individual cell populations on different surfaces to allow for independent treatment of two cell populations43,44, this mixed culture system enables direct and close contact of microglia and neurons. In addition, when maintained properly through half media changes every 3 days, this mixed culture system can remain healthy from 12 days (Figure 3F) to potentially 3 weeks, allowing for long-term studies of neuronal-microglia interactions45.

While the relative simplicity of the co-culture system is advantageous for accessibility, a key limitation of this approach is that the two-dimensional cell culture does not fully represent the CNS tissue environment. Indeed, a tri-culture of microglia, neurons, and astrocytes is shown to mimic in vivo neuroinflammatory response more reliably than microglia or astrocyte-neuronal co-culture46,47. Furthermore, recent developments in brain organoid systems with microglia and other glial cells, including astrocytes and oligodendrocytes, are more suitable for studying three-dimensional (3D) tissue architecture and interactions of the CNS48. Similarly, a human 3D triculture system comprising neurons, microglia, and astrocytes is shown to be effective in modeling Alzheimer's disease condition49. Other human microglia-neuron co-cultures may be more physiologically relevant to neurodegenerative disease studies. For example, the co-culture of microglia and motor neurons derived from human induced pluripotent stem cells50 may be useful for studying diseases such as amyotrophic lateral sclerosis51. Nevertheless, given the relative abundance and low cost of generating primary neurons and microglia from mice, assessing microglia-neuronal co-culture in 96-well plates can be a high throughput approach to studying the mechanisms and functional implications of microglia-neuronal interactions. Downstream assays may include using live cell imaging to determine cellular functional changes in real-time, RNA sequencing to determine cellular transcriptional changes, immunofluorescence microscopy to determine cellular phenotypic changes, mass spectrometry to determine cellular and proteomic changes, or enzyme-linked immunosorbent assay and western blot to measure protein secreted into the co-culture supernatant.

As microglia are important mediators of CNS health and diseases, this co-culture can also be used to investigate the effect of disease-related molecules and compounds on microglia-neuronal bi-directional communication. In the context of MS, activated microglia may take on a destructive role, contributing to the worsening of pathology52,53. For example, they may secrete pro-inflammatory cytokines and reactive oxygen species, as well as promote astrocyte activation45. This protocol may help to further interrogate the harmful role of microglia by treating microglia in the co-culture with inflammatory cytokines found elevated in MS and assessing the outcomes of neuron health. Alternatively, this microglia-neuronal co-culture has been used to show the protective effect of microglia on neurons in response to oxidized phosphatidylcholine, an oxidative product discovered in MS54. Moreover, neuronal apoptosis remodels microglia gene expression during development by promoting microglia survival and phagocytosis55. Therefore, this protocol may also help to determine the impact of neuronal death on microglia phenotype and function.

In summary, this protocol describes the generation and co-culturing of primary mouse-derived neurons and microglia in vitro. The microglia-neuronal co-culture set up in 96-well plates can be a high throughput approach to interrogate the mechanisms and function of microglia interactions with neurons in the context of CNS health and disease, including MS.

Disclosures

The authors have nothing to disclose.

Acknowledgements

JP acknowledges funding support from the Natural Sciences and Engineering Research Council of Canada and the University of Saskatchewan College of Medicine. YD acknowledges funding support from the University of Saskatchewan College of Medicine Startup Fund, the Natural Sciences and Engineering Research Council of Canada Discovery Grant (RGPIN-2023-03659), MS Canada Catalyst Grant (1019973), Saskatchewan Health Research Foundation Establishment Grant (6368), and Brain Canada Foundation Future Leaders in Canadian Brain Research Grant. Figure 1A, Figure 2A, and Figure 3A were created with BioRender.com.

Materials

10 cm Petri dish  Fisher  07-202-011 Sterile
1x Versene Gibco 15040-066
B-27 Plus Neuronal Culture System  Gibco  A3653401
Dissection microscope VWR
DNase I Roche 11284932001
Dulbecco’s Modified Eagle Medium (DMEM) Gibco 11960-044
Fetal Bovine Serum  ThermoFisher Sci 12483-020
HBSS (10x) Gibco 14065-056
Hemacytometer Hausser Scientific 1475
HEPES  ThermoFisher Sci 15630080
Leibovitz’s L-15 Medium (1x) Fisher Scientific  21083027
Macrophage colony stimulating factor  Peprotech 315-02
Micro-Forceps RWD F11020-11 Autoclaved/Sterile
Non-essential amino acids Cytiva SH3023801
PBS (10x) ThermoFisher Sci AM9625
Penicillin Streptomycin Glutamine (100x) Gibco 103780-16
Poly-L-ornithine hydrobromide  Sigma P3655-100MG
Sodium pyruvate (100 mM) Gibco 11360-070
Spring scissors RWD S11008-42 Autoclaved/Sterile
Surgical blade Feather 08-916-5D Sterile
T-25 flasks Fisher 10-126-9
T-75 flasks  Fisher 13-680-65
Tissue forceps Codman 30-4218 Autoclaved/Sterile
Tissue scissors RWD S12052-10 Autoclaved/Sterile
Trypan Blue  Thermofisher Sci  15250-061
Trypsin (2.5%) ThermoFisher Sci 15090046
Widefield Immunofluorescence Microscope Zeiss

References

  1. Yin, J., Valin, K. L., Dixon, M. L., Leavenworth, J. W. The role of microglia and macrophages in CNS homeostasis, autoimmunity, and cancer. J Immunol Res. 2017, 1-12 (2017).
  2. Colonna, M., Butovsky, O. Microglia function in the central nervous system during health and neurodegeneration. Annu Rev Immunol. 35 (1), 441-468 (2017).
  3. Ginhoux, F., Prinz, M. Origin of microglia: Current concepts and past controversies. Cold Spring Harb Perspect Biol. 7 (8), a020537 (2015).
  4. Dermitzakis, I., et al. Origin and emergence of microglia in the CNS-an interesting (hi)story of an eccentric cell. Curr Issues Mol Biol. 45 (3), 2609-2628 (2023).
  5. Ransohoff, R. M., Cardona, A. E. The myeloid cells of the central nervous system parenchyma. Nature. 468 (7321), 253-262 (2010).
  6. Ginhoux, F., et al. Fate mapping analysis reveals that adult microglia derive from primitive macrophages. Science. 330 (6005), 841-845 (2010).
  7. Askew, K., et al. Coupled proliferation and apoptosis maintain the rapid turnover of microglia in the adult brain. Cell Rep. 18 (2), 391-405 (2017).
  8. Vidal-Itriago, A., et al. Microglia morphophysiological diversity and its implications for the CNS. Front Immunol. 13, 997786 (2022).
  9. Wendimu, M. Y., Hooks, S. B. Microglia phenotypes in aging and neurodegenerative diseases. Cells. 11 (13), 2091 (2022).
  10. Hanisch, U. K., Kettenmann, H. Microglia: Active sensor and versatile effector cells in the normal and pathologic brain. Nat Neurosci. 10 (11), 1387-1394 (2007).
  11. Colonna, M., Butovsky, O. Microglia function in the central nervous system during health and neurodegeneration. Annu Rev Immunol. 35 (1), 441-468 (2017).
  12. Zhao, J. F., et al. Research progress on the role of microglia membrane proteins or receptors in neuroinflammation and degeneration. Front Cell Neurosci. 16, 831977 (2022).
  13. Yang, I., Han, S. J., Kaur, G., Crane, C., Parsa, A. T. The role of microglia in central nervous system immunity and glioma immunology. J Clin Neurosci. 17 (1), 6-10 (2010).
  14. Jurga, A. M., Paleczna, M., Kuter, K. Z. Overview of general and discriminating markers of differential microglia phenotypes. Front Cell Neurosci. 14, 198 (2020).
  15. Doens, D., Fernández, P. L. Microglia receptors and their implications in the response to amyloid β for Alzheimer’s disease pathogenesis. J Neuroinflammation. 11 (1), 48 (2014).
  16. Block, M. L., Zecca, L., Hong, J. S. Microglia-mediated neurotoxicity: Uncovering the molecular mechanisms. Nat Rev Neurosci. 8 (1), 57-69 (2007).
  17. Fischer, M. T., et al. NADPH oxidase expression in active multiple sclerosis lesions in relation to oxidative tissue damage and mitochondrial injury. Brain. 135 (3), 886-899 (2012).
  18. Marinelli, S., Basilico, B., Marrone, M. C., Ragozzino, D. Microglia-neuron crosstalk: Signaling mechanism and control of synaptic transmission. Semin Cell Dev Biol. 94, 138-151 (2019).
  19. Pocock, J. M., Kettenmann, H. Neurotransmitter receptors on microglia. Trends Neurosci. 30 (10), 527-535 (2007).
  20. Carniglia, L., et al. Neuropeptides and microglial activation in inflammation, pain, and neurodegenerative diseases. Mediators Inflamm. 2017, 5048616 (2017).
  21. Zhao, S., Umpierre, A. D., Wu, L. J. Tuning neural circuits and behaviors by microglia in the adult brain. Trends Neurosci. 47 (3), 181-194 (2024).
  22. Kettenmann, H., Kirchhoff, F., Verkhratsky, A. Microglia: New roles for the synaptic stripper. Neuron. 77 (1), 10-18 (2013).
  23. Haidar, M. A., et al. Crosstalk between microglia and neurons in neurotrauma: An overview of the underlying mechanisms. Curr Neuropharmacol. 20 (11), 2050-2065 (2022).
  24. Cserép, C., Pósfai, B., Dénes, &. #. 1. 9. 3. ;. Shaping neuronal fate: Functional heterogeneity of direct microglia-neuron interactions. Neuron. 109 (2), 222-240 (2021).
  25. Pocock, J. M., Kettenmann, H. Neurotransmitter receptors on microglia. Trends Neurosci. 30 (10), 527-535 (2007).
  26. Eyo, U. B., Wu, L. J. Bidirectional microglia-neuron communication in the healthy brain. Neural Plast. 2013, 456857 (2013).
  27. Strosznajder, J. B., Czapski, G. A. Glutamate and GABA in microglia-neuron cross-talk in Alzheimer’s disease. Int J Mol Sci. 22 (21), 11677 (2021).
  28. Lyons, A., et al. CD200 ligand-receptor interaction modulates microglial activation in vivo and in vitro A role for IL-4. J Neurosci. 27 (31), 8309-8313 (2007).
  29. Wake, H., Moorhouse, A. J., Miyamoto, A., Nabekura, J. Microglia: Actively surveying and shaping neuronal circuit structure and function. Trends Neurosci. 36 (4), 209-217 (2013).
  30. Merlini, M., et al. Microglial Gi-dependent dynamics regulate brain network hyperexcitability. Nat Neurosci. 24 (1), 19-23 (2021).
  31. Chen, Z., et al. Microglial displacement of inhibitory synapses provides neuroprotection in the adult brain. Nat Commun. 5 (1), 4486 (2014).
  32. Cantaut-Belarif, Y., et al. Microglia control the glycinergic but not the GABAergic synapses via prostaglandin E2 in the spinal cord. J Cell Biol. 216 (9), 2979-2989 (2017).
  33. Szepesi, Z., Manouchehrian, O., Bachiller, S., Deierborg, T. Bidirectional microglia-neuron communication in health and disease. Front Cell Neurosci. 12, 323 (2018).
  34. Chamera, K., Trojan, E., Szuster-Głuszczak, M., Basta-Kaim, A. The potential role of dysfunctions in neuron-microglia communication in the pathogenesis of brain disorders. Curr Neuropharmacol. 18 (5), 408-430 (2020).
  35. Gao, C., Jiang, J., Tan, Y., Chen, S. Microglia in neurodegenerative diseases: Mechanism and potential therapeutic targets. Signal Transduct Target Ther. 8 (1), 359 (2023).
  36. Brisch, R., et al. The role of microglia in neuropsychiatric disorders and suicide. Eur Arch Psychiatry Clin Neurosci. 272 (6), 929-945 (2022).
  37. Dong, Y., et al. Oxidized phosphatidylcholines found in multiple sclerosis lesions mediate neurodegeneration and are neutralized by microglia. Nat Neurosci. 24 (4), 489-503 (2021).
  38. Alvarez-Carbonell, D., et al. Cross-talk between microglia and neurons regulates HIV latency. PLoS Pathog. 15 (12), e1008249 (2019).
  39. Lorenzen, K., et al. Microglia induce neurogenic protein expression in primary cortical cells by stimulating PI3K/AKT intracellular signaling in vitro. Mol Biol Rep. 48 (1), 563-584 (2021).
  40. Güler, B. E., Krzysko, J., Wolfrum, U. Isolation and culturing of primary mouse astrocytes for the analysis of focal adhesion dynamics. STAR Protoc. 2 (4), 100954 (2021).
  41. Tomassoni-Ardori, F., Hong, Z., Fulgenzi, G., Tessarollo, L. Generation of functional mouse hippocampal neurons. Bio Protoc. 10 (15), e3702 (2020).
  42. Viviani, B. Preparation and coculture of neurons and glial cells. Curr Protoc Cell Biol. Chapter 2 (Unit 2.7), (2006).
  43. Roqué, P. J., Costa, L. G. Co-culture of neurons and microglia. Curr Protoc Toxicol. 74, 11.24.1-11.24.17 (2017).
  44. Goshi, N., Morgan, R. K., Lein, P. J., Seker, E. A primary neural cell culture model to study neuron, astrocyte, and microglia interactions in neuroinflammation. J Neuroinflammation. 17 (1), 155 (2020).
  45. Carroll, J. A., Foliaki, S. T., Haigh, C. L. A 3D cell culture approach for studying neuroinflammation. J Neurosci Methods. 358, 109201 (2021).
  46. Baxter, P. S., et al. Microglial identity and inflammatory responses are controlled by the combined effects of neurons and astrocytes. Cell Rep. 34 (12), 108882 (2021).
  47. Luchena, C., et al. A neuron, microglia, and astrocyte triple co-culture model to study Alzheimer’s disease. Front Aging Neurosci. 14, 844534 (2022).
  48. Park, J., et al. A 3D human triculture system modeling neurodegeneration and neuroinflammation in Alzheimer’s disease. Nat Neurosci. 21 (7), 941-951 (2018).
  49. Vahsen, B. F., et al. Human iPSC co-culture model to investigate the interaction between microglia and motor neurons. Sci Rep. 12 (1), 12606 (2022).
  50. Giacomelli, E., et al. Human stem cell models of neurodegeneration: from basic science of amyotrophic lateral sclerosis to clinical translation. Cell Stem Cell. 29 (1), 11-35 (2022).
  51. Yong, V. W. Microglia in multiple sclerosis: protectors turn destroyers. Neuron. 110 (21), 3534-3548 (2022).
  52. Kamma, E., Lasisi, W., Libner, C., Ng, H. S., Plemel, J. R. Central nervous system macrophages in progressive multiple sclerosis: relationship to neurodegeneration and therapeutics. J Neuroinflammation. 19 (1), 45 (2022).
  53. Dong, Y., Lozinski, B. M., Silva, C., Yong, V. W. Studying the microglia response to oxidized phosphatidylcholine in primary mouse neuron culture and mouse spinal cord. STAR Protoc. 2 (4), 100853 (2021).
  54. Anderson, S. R., et al. Neuronal apoptosis drives remodeling states of microglia and shifts in survival pathway dependence. eLife. 11, e76564 (2022).
  55. Harry, G. J., McPherson, C. A. Microglia: Neuroprotective and neurodestructive properties. Handbook of Neurotoxicity. , 109-132 (2014).
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Park, J., Yu, R., Dong, Y. Generating and Co-culturing Murine Primary Microglia and Cortical Neurons. J. Vis. Exp. (209), e67078, doi:10.3791/67078 (2024).

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