Here we present a protocol for in vitro isolation of multiple glial cell populations from a mouse CNS. This method allows for the segregation of regional microglia, oligodendrocyte precursor cells, and astrocytes to study the phenotypes of each in a variety of culture systems.
The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine neonates for the isolation of glial subpopulations. The purpose of the procedure is to dissociate microglia, oligodendrocyte progenitor cells (OPCs), and astrocytes from cortical, cerebellar, brainstem, and spinal cord tissue to facilitate further in vitro analysis. The CNS region isolation procedures allow for the determination of regional heterogeneity among glia in multiple cell culture systems. Rapid CNS region isolation is performed, followed by the mechanical removal of meninges to prevent meningeal cell contamination of glia. This protocol combines gentle tissue dissociation and plating on a specified matrix designed to preserve cell integrity and adherence. Isolating mixed glia from multiple CNS regions provides a comprehensive analysis of potentially heterogenous glia while maximizing the use of individual experimental animals. Additionally, following dissociation of regional tissue, mixed glia are further divided into multiple cell types including microglia, OPCs, and astrocytes for use in either single cell type, cell culture plate inserts, or co-culture systems. Overall, the demonstrated techniques provide a comprehensive protocol of broad applicability for careful dissection of four individual CNS regions from murine neonates and includes methods for the isolation of three individual glia cell types to examine regional heterogeneity in any number of in vitro cell culture systems or assays.
Glia are necessary for proper neuronal function in the CNS. They are composed of three major subpopulations, astrocytes, oligodendrocytes, and microglia, each with a different, yet indispensable role1. Without the proper glial cell diversity and activity, neuronal function would be severely impacted, leading to CNS impairment. Glia are capable of influencing neurotransmission, and each cell type does so in a unique manner. Glial cells in the brain have the capacity to communicate amongst themselves, as well as with neuronal cells, in order to facilitate proper CNS function2. Oligodendrocytes increase the speed of electrical transmission through the formation of a myelin sheath, which facilitates the clustering of ion channels at the nodes of Ranvier, the sites of neuronal action potential generation3. Microglia are critical for the pruning of synapses by monitoring synaptic transmission and “rewiring” neuronal connections following injury4. In addition, microglia are the most abundant resident immune cell of the CNS, acting as the primary form of host defense against pathogens5. Astrocytes can regulate synaptic transmission between neurons by modifying the concentration of extracellular potassium6. They also have roles in controlling local blood flow7, releasing and taking up neuromodulatory elements8, and have a key role in blood-brain barrier maintenance9. Thus, each glial subtype is critical for CNS function, as defects in any type have long been associated with a wide variety of pathological states, including psychiatric diseases, epilepsy, and neurodegenerative conditions10.
The greatest obstacle in the study of CNS pathobiology is the inability to investigate human cells in the context of their microenvironmental niche. Human biopsy tissue is most collected post-mortem and cells can easily be damaged or lost during the extraction and processing. Furthermore, it is a challenge to keep human cells alive and viable in vitro for any length of time without deriving immortalized cell lines from tumors, at which point they no longer accurately reflect their normal physiological properties11,12. Additionally, there is a significant amount of regional heterogeneity among individual glia cell types13,14,15, and obtaining regional CNS samples from individual patients is nearly impossible. As such, it is necessary to develop alternative models to study the contribution of regional glia in specific CNS disorders.
Here, we describe an in vitro system using mouse CNS region-specific isolation of multiple glial subpopulations, allowing for the manipulation and quantification of microglia, oligodendrocyte precursor cells (OPCs), which give rise to mature oligodendrocytes, and astrocytes. Each population can be independently isolated and subjected to a wide variety of experimental techniques including drug or molecule treatment, immunocytochemistry, protein/RNA extraction and analysis, and other co-culture systems depending on experimental necessity. Additionally, this isolation technique yields high cell number, allowing for the characterization and investigation of each glial population in a high-throughput manner. It also enables the study of CNS cell differentiation, growth, and proliferation in response to a wide variety of microenvironmental stimuli in a controlled manner in order to avoid confounding factors which are typically present in an in vivo setting. Lastly, this cell isolation technique facilitates the manipulation of glial cell populations within different CNS regions to investigate how regional glia interact with each other and respond to varying stimuli, allowing for precision and reproducibility.
NOTE: All animal studies were authorized and approved by the Cleveland Clinic Lerner Research Institute Institutional Animal Care and Use Committee.
1. Prepare media and supplies for dissection
NOTE: All buffer and media recipes are provided in Table 1. This procedure is done under sterile conditions in a tissue culture designated biosafety cabinet.
2. Cortex, cerebellum, brainstem, and spinal cord dissection
NOTE: This procedure can be done on the benchtop and requires a dissection scope. Use strict aseptic technique for all steps of the procedure and minimize tissue exposure to the room air. Keep all media chilled on ice during dissection to ensure maximal tissue preservation. Alternatively, this procedure could be done in a hood that allows the use of an internal dissection scope.
3. Tissue dissociation
NOTE: All the following procedures are carried out in a sterile tissue culture designated biosafety cabinet using aseptic technique and sterile materials.
4. Microglia isolation
5. Oligodendrocyte precursor cell isolation
NOTE: When plating OPCs following initial isolation, they must be plated on a poly-D-lysine-coated surface (sterile plate or cover slip). Prepare these materials prior to the completion of this section.
6. Astrocyte isolation
7. Identification and isolation of microglia, OPCs, astrocytes, and mature oligodendrocytes using immunocytochemistry
Representative data shown below illustrates that IFNγ signaling influences OPC differentiation and maturation. Without the presence of IFNγ receptor (IFNγR), cortical OPCs do not differentiate into mature myelinating oligodendrocytes as readily, which is evidenced by the absence of MBP staining (Figure 1). Since oligodendrocytes and astrocytes are derived from a common progenitor, we analyzed GFAP expression, which labels astrocytes. We found that IFNγR-deficient cells strongly express GFAP suggesting that they may be adopting an astrocytic phenotype, corroborating earlier reports19.
Additional evidence for regional heterogeneity in CNS cells is evidenced by varying astrocyte morphology as seen in astrocytes from the cortex, cerebellum, brainstem, and spinal cord (Figure 2). Of note, astrocytes from the same region may also exhibit morphological heterogeneity, supporting the notion that this glial subtype is highly dynamic. The differences in cellular architecture is suggestive of functional diversity and thus the ability to isolate glial populations is necessary to study phenotypic responses in the absence and presence of microenvironmental stimuli.
Oligodendrocytes are critical for the myelination of neuronal axons and are necessary for proper CNS repair and function. OPCs give rise to their mature counterparts, making it critical to understand the biology behind their ability to differentiate. Cytokine signaling significantly influences stem and immune cell behavior. Thus, it is important to understand how regional responses of OPCs may vary to differential cytokine stimulation (Figure 3), which may impact their ability to differentiate into mature myelinating oligodendrocytes.
Figure 1: Representative data showing OPC differentiation in WT and IFNγR-/- mice in the presence of exogenous IFNγ. Cortical OPCs were isolated from (A) WT and (B) IFNγR-/- P4 mouse pups following the procedure outlined above. Cells were treated with 1 ng/mL IFNγ for 48 h, then fixed and stained to delineate cell differentiation. OPCs were labeled for NG2 and GFAP to identify those which were adopting an astrocytic phenotype. Likewise, OPCs were also labeled for NG2 and MBP to identify those that were differentiating into mature oligodendrocytes. Scale bar = 20 μM. Please click here to view a larger version of this figure.
Figure 2: Representative data demonstrating regional heterogeneity in astrocyte morphology. Astrocytes from cortex, cerebellum, brainstem, and spinal cord were isolated from P4 mouse pups using the protocol described above and labeled for GFAP (green) by immunocytochemistry following 48 h in culture. Scale bar = 20 μM. Please click here to view a larger version of this figure.
Figure 3: Representative data demonstrating differential responses of regional OPCs to cytokines. OPCs were isolated from the brainstem and spinal cord of P4 mouse pups using the protocol described above. Cells were treated with increasing concentrations (1-10 ng/mL) of (A) IFNγ or (B) interleukin (IL)-17 in order to investigate the differential influence of cytokine on the ability of regional OPCs to differentiate into myelinating oligodendrocytes. Following a 48 h incubation with specified cytokines, OPCs were fixed and labeled for NG2 (green) and MBP (red). Scale bar = 20 μM. Data represent means ± SEM. **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 by 2-way ANOVA. Please click here to view a larger version of this figure.
PBS/Antibiotic Solution (PAS): | ||
1.0 mL | 100X Antibiotic/Antimycotic containing 10,000 units/mL penicillin and 10,000 mg/mL streptomycin | |
25 mg/mL | Amphotericin B | |
99 mL | 1X PBS | |
Mixed Glia Media (MGM) | ||
88 mL | 1X DMEM (high glucose, w/L-glutamine, w/Na pyruvate) | |
10 mL | Heat-inactivated FBS | |
1.0 mL | L-Glutamine (100X) | |
1.0 mL | Antibiotic/Antimycotic | |
OPC Media (50mL) | ||
49 mL | Neurobasal media | |
1.0 mL | B27 supplement (50X) | |
10 ng/ml | PDGF-AA | |
NOTE: PDGF-AA is added fresh prior to each media change. | ||
Astrocyte Media (1 L) | ||
764 mL | MEM with Earle's salts containing glutamine | |
36 mL | Glucose (use 100 mg/mL stock for final concentation of 20 mM) | |
100 mL | Heat-inactivated FBS | |
100 mL | Heat-inactivated horse serum | |
10 mL | Glutamine (use 200 mM stock if not included in stock medium) | |
OPTIONAL: 10 ng/mL recombinant mouse epidermal growth factor | ||
NOTE: Sterile filter all media and store at 4°C until used. |
Table 1: Buffer and Media Recipes.
In this protocol, we describe the isolation of the three major glial cell subpopulations from mouse CNS: microglia, OPCs, and astrocytes. A major setback for the investigation of neurodegenerative and neuroinflammatory CNS diseases is the lack of primary human cells and tissues, particularly those that are regional and from the same patient. In most instances, human CNS cell lines are derived from transformed, immortalized cancer cells which may not be accurate representations of their normal physiological behavior20,21,22. Thus, alternative methods are necessary to study CNS cell phenotypes in a controlled manner. Furthermore, the diversity of neurological glial cell populations makes it necessary to investigate each subtype both independently of one another, as well as in co-culture conditions in order to recapitulate both their cell autonomous and non-autonomous functions. Glial cells have a wide variety of critical functions in the CNS ranging from neuronal support23, learning/cognition24,25, and CNS immunological responses26. As such, it is necessary to understand the molecular and cellular functions of each glial subpopulation in a physiological and pathological context. In order to do so, we provide here a reliable method for the extraction and isolation of viable glia subtypes. Due to practical and ethical constraints in human subject’s research, animal models are currently the most relevant surrogates for human glial cell biology. In particular, mice are ideal model animals as their genome can be manipulated and analyzed to further dissect particular molecular mechanisms underlying health and disease. Therefore, the successful removal and separation of murine microglia, OPCs, and astrocytes is a key tool to investigate the functions of glia during physiological, neurodegenerative, or neuroinflammatory conditions.
This protocol can be optimized to explore CNS cell regional heterogeneity. It is becoming increasingly clear that glia exhibit regional heterogeneity in form and function. Astrocytes are regionally diverse and display distinct morphology depending on their location within the CNS27. Furthermore, the density of astrocytes and their mitotic index can define anatomical regions, supporting the hypothesis that regional astrocyte heterogeneity may reflect molecular and functional differences based on their location within the CNS28. Microglial regional heterogeneity is also under active investigation, although the underlying mechanisms and functional consequences of microglia diversity in CNS development or behavior are currently unclear. However, it is known that adult microglia display diversity in cell number, cell and subcellular structures, and molecular signatures29. Moreover, recent advances in multiplexed mass cytometry have further defined the regional heterogeneity of microglia, analyzing cellular phenotype from five different CNS regions of nine human donors, allowing for large-scale immunophenotyping of human microglia30. Currently, such approaches are in their nascent stages, making animal studies a viable solution for the study of regional glia in CNS disease development. Finally, regional heterogeneity has also recently been described in oligodendrocytes. Single-cell RNA sequencing on 5072 individual cells from 10 regions of juvenile and adult CNS identified 13 distinct subpopulations across different stages of differentiation31. Importantly, it was also found that as oligodendrocytes matured from OPCs, their transcriptional profiles diverged and their functional phenotypes changed, highlighting oligodendrocyte heterogeneity within the CNS31.
Thus, understanding regional heterogeneity of the various resident CNS cells in the context of their diverse neighboring neurons and other glia may provide important rationale for the future development of novel therapies to treat neuroinflammatory and neurodegenerative disorders. While this protocol focuses on the extraction, isolation, and identification of glial subpopulations, it provides a convenient starting point for the examination of their function. Furthermore, it can be adapted and combined with transgenic mouse models in order to study genetic mechanisms associated with glial cell biology. It can also be used to examine the responses of glial cells to each other in co-culture assays. The outlined steps represent a cost-efficient and high-throughput method of extracting and isolating different CNS glial populations which can then be adapted to a wide variety of experimental parameters. It should be noted; however, that the method described here utilizes neonates due to the lower levels of myelination and high density of proliferating glia. For these reasons, it is technically more feasible to isolate viable glia from neonates compared to adult animals. The phenotypic differences in neonatal glia compared to adult glia should thus be considered during experimental design and data interpretation.
The authors have nothing to disclose.
We thank Morgan Psenicka for manuscript editing and discussion and Dr. Grahame Kidd for assistance in figure formatting. This work was supported by NIAID K22 AI125466 (JLW).
0.05% Trypsin and 0.53 mM EDTA | Gibco | 25300054 | Tissue dissociation |
12-Well Plates | Greiner Bio-One | 665 180 | Cell culture plate |
1X PBS pH 7.4 | Gibco | 10010031 | Standard reagent |
32% Paraformaldehyde | Electron Microscopy Sciences | 15714-S | Fixative |
50 mL, 25 cm2 cell culture flask | Greiner Bio-One | 690 175 | Cell culture (T25) flask |
Antibiotic-Antimycotic 100X | Gibco | 15240-096 | Media component |
B-27 Supplement 50X | Gibco | 17504-044 | Media component |
Bovine serum albumin | Sigma | A9647-50G | Antibody diluent |
Confocal Microscope | Zeiss | LSM 800 | Confocal for imaging |
DAPI | ThermoFisher | D1306 | Nuclear stain |
DMEM (1X), high glucose with Na pyruvate | Gibco | 11995040 | Media component |
Dnase I | Sigma | 10104159001 | Tissue dissociation |
Fetal bovine serum heat inactivated | Gibco | A3840001 | Media component |
Fibronectin from bovine plasma | Sigma | F1141-1MG | Cell adherent |
Fine stitch Scissors | Sklar | 64-3260 | Dissection tools |
Goat anti-rabbit IgG Alexa Fluor 488 | Invitrogen | A11008 | Secondary staining antibody |
Goat anti-rat IgG Alexa Fluor 555 | Invitrogen | A21434 | Secondary staining antibody |
Hanks' Balanced Salt Solution (w/o Ca or Mg) | ThermoFisher | 14170120 | Tissue dissociation |
L-glutamine, 200mM | Gibco | 20530081 | Media component |
Murine epidermal growth factor | ThermoFisher | PMG8044 | Media component |
Murine IFN-γ | Peprotech | 315-05-20UG | Media component |
Murine PDGF-AA | Peprotech | 315-17 | Media component |
Neurobasal | Gibco | 21103-049 | Media component |
Normal goat serum | Sigma | G9023 | Blocking solution component |
Operating Scissors | Surgi-OR | 95-272 | Dissection tools |
Poly-D-Lysine 12 mm #1 German Glass Coverslip | Corning Biocoatt | 354086 | Cell adherent |
Prolong Gold Antifade Reagent | Cell Signaling Technology | 9071S | Mounting Media |
Rabbit anti-Iba1 | Wako | 019-19741 | Primary antibody |
Rabbit anti-NG2 Chondroitin Proteoglycan | Millipore | ab5320 | Primary antibody |
Rat anti-GFAP | ThermoFisher | 13-0300 | Primary antibody |
Rat anti-myelin basic protein | Abcam | ab7349 | Primary antibody |
Sharp Tip Scissors | Surgi-OR | 95-104 | Dissection tools |
Stereo Microscope | Leica | S4 E Stereo Zoom Microscope | Microscope for dissection |
Tissue Forceps | Sklar | 66-7644 | Dissection tools |
Triton X-100 | Fisher Bioreagents | BP151-100 | Cell permabilization |
Trypsin Inhibitor (from chicken egg white) | Sigma | 10109878001 | Tissue dissociation |