This protocol outlines the isolation of purified astrocytes and microglia from the adult mouse spinal cord, facilitating subsequent applications such as RNA analysis and cell culture. It includes detailed cell dissociation methods and procedures designed to enhance both the quality and yield of isolated cells.
Astrocytes and microglia play pivotal roles in central nervous system development, injury responses, and neurodegenerative diseases. These highly dynamic cells exhibit rapid responses to environmental changes and display significant heterogeneity in terms of morphology, transcriptional profiles, and functions. While our understanding of the functions of glial cells in health and disease has advanced substantially, there remains a need for in vitro, cell-specific analyses conducted in the context of insults or injuries to comprehensively characterize distinct cell populations. Isolating cells from the adult mouse offers several advantages over cell lines or neonatal animals, as it allows for the analysis of cells under pathological conditions and at specific time points. Furthermore, focusing on spinal cord-specific isolation, excluding brain involvement, enables research into spinal cord pathologies, including experimental autoimmune encephalomyelitis, spinal cord injury, and amyotrophic lateral sclerosis. This protocol presents an efficient method for isolating astrocytes and microglia from the adult mouse spinal cord, facilitating immediate or future analysis with potential applications in functional, molecular, or proteomic downstream studies.
Astrocytes and microglia are versatile glial cells that play vital roles in the central nervous system (CNS), encompassing responsibilities such as regulating neuronal function, contributing to CNS development, maintaining the blood-brain barrier, and participating in other critical processes1,2,3,4. Besides their role in maintaining homeostasis, these glial cells also play a pivotal part in injury and repair mechanisms. Microglia are well-known for their phagocytic, inflammatory, and migratory capabilities following insults or injuries5,6,7. Astrocyte responses in disease are equally diverse, encompassing contributions to inflammation, the formation of glial scars, and the compromise of the blood-brain barrier8,9. Although our understanding of the detrimental and reparative roles of microglia and astrocytes in the CNS has grown, the inherent heterogeneity in both their structure and function necessitates robust tools for studying them in various contexts.
Gaining further insight into the roles of microglia and astrocytes in health and disease requires a combined approach of in vivo and in vitro investigations. In vivo techniques leverage the intricate crosstalk between glial cells and neurons within the CNS, while in vitro methodologies prove valuable when assessing single-cell functions or responses under specific stimuli. Each method offers unique advantages; in vitro studies are essential for understanding the specific roles of these cell types without direct or indirect input from neighboring cells. Additionally, in vitro assays utilizing immortal cell lines present certain benefits, including the ability to proliferate indefinitely, cost-efficiency, and ease of maintenance. However, it's important to note that primary cells more closely mimic normal physiological responses compared to cell lines. This physiological relevance is crucial in functional assays and transcriptomic analyses.
One of the challenges in obtaining primary cells, particularly from the adult mouse spinal cord, lies in the quantity and viability of the samples. The adult spinal cord, being smaller than the brain and containing a significant amount of myelin, poses unique difficulties. While there are several published protocols detailing the isolation of pure, viable glial cells from neonatal animals or the adult mouse brain10,11,12,13, these methodologies may not be suitable for studying diseases and injuries specific to the spinal cord. In this protocol, we offer a comprehensive procedure to efficiently isolate pure, viable microglia and astrocytes from the adult mouse spinal cord, facilitating downstream applications in cell culture and transcriptomic analyses. This protocol has been successfully employed to isolate these cells from adult mice aged 10 weeks to 5 months, demonstrating its utility across various contexts, including studies involving conditional knockout mice, drug responses, developmental research, and age-related models.
All animal care and experimental procedures were conducted following the approval of the Animal Care and Use Committee at The George Washington University School of Medicine and Health Sciences (Washington, D.C., USA; IACUC#2021-004). The study utilized male and female C57BL/6J wild-type (WT) mice aged 10 weeks to 5 months, which were sourced from a commercial supplier (see Table of Materials) and housed at The George Washington University. An overview of the protocol workflow is presented in Figure 1.
1. Preparation of the spinal cord
2. Enzymatic cell dissociation
3. Mechanical cell dissociation
4. Myelin removal
5. Microglia and astrocyte isolation
6. Plating cells for in vitro assays
NOTE: If cells will not be plated and will be used immediately for RNA analysis, proceed to step 8.
7. Immunohistochemistry
NOTE: It is best to perform immunohistochemistry analyses after at least 3 days when the media has been replaced at least once. This ensures debris has been removed and cells have completely adhered to the coverslip.
8. RNA extraction
NOTE: Ideally, there should be at least 100,000 cells to extract sufficient RNA for analysis. If necessary, cells from 2-3 spinal cords may be combined.
The methods outlined in this protocol enable the isolation of pure and viable microglia and astrocytes from the adult mouse spinal cord, facilitating various downstream applications, including in vitro functional or histological assays and RNA analysis.
A successful isolation for in vitro studies will result in continuous cell proliferation over several days. Adult cells exhibit a slower proliferation rate compared to cells isolated from neonatal animals, and some debris may be present in the first few days. By 4 days in vitro (DIV), cells should be largely clear of debris, with most cells adhering to the flask bottom. Astrocytes will begin to form longer processes, while microglia will assume an oval shape with shorter spindles (Figure 2). By 7 DIV, astrocytes should form a connected confluent layer, and microglia should display fewer and shorter processes (Figure 3).
Purity can be confirmed by double labeling ACSA2-sorted cells with GFAP and O4 to assess oligodendrocyte contamination in astrocyte cultures and CD11b-sorted cells with Iba1 and GFAP to evaluate astrocyte contamination in microglia cultures (Table 1).
A successful protocol will also yield high-quality RNA with minimal degradation and sufficient quantity (Figure 4A). Electropherograms of RNA extracted from isolated cells should exhibit prominent 18S and 28S peaks. Overdissociation of cells or prolonged time between perfusion and cell sorting can lead to RNA degradation (Figure 4B). Insufficient enzymatic and/or mechanical dissociation or inadequate myelin removal can result in reduced cell yield and RNA (Figure 4C). Isolated astrocytes can be sequenced to identify inflammatory markers. Comparing healthy astrocytes to inflammation-activated astrocytes (e.g., from an experimental autoimmune encephalomyelitis animal) will reveal relative ininhibitions of inflammatory pathways in healthy versus inflammatory astrocytes (Figure 4D).
Figure 1: Overview of spinal cord preparation, tissue dissociation, and cell sorting. The figure provides an overview of the spinal cord preparation process, including tissue dissociation and cell sorting. After spinal cord dissection, tissues undergo enzymatic and mechanical dissociation. Myelin is removed, and cells are labeled with anti-ACSA2 or anti-CD11b antibodies to target astrocytes and microglia. The sorted cells can then be utilized for cell culture and RNA analysis. Please click here to view a larger version of this figure.
Figure 2: Phase contrast images of astrocytes and microglia at 4 days in vitro (4DIV). (A) Depicts a representative example of ACSA2+ sorted cells with extended processes. (B) CD11b+ cells display oval-shaped cell bodies and short processes. Scale bar = 50 µm. This figure is adapted from Ahn, J. J. et al.16. Please click here to view a larger version of this figure.
Figure 3: Fluorescent images of astrocytes and microglia at 8 days in vitro (8DIV). (A) ACSA2+ cells labeled with GFAP (green) and O4 (red) exhibit minimal O4 staining and form a connected confluent layer of astrocytes. (B) CD11b+ cells labeled with Iba1 (green) and GFAP (red) show minimal presence of GFAP. Scale bar = 50 µm. This figure is adapted from Ahn, J. J. et al.16. Please click here to view a larger version of this figure.
Figure 4: Representative electropherograms of RNA samples. (A) High yield, high-quality RNA is expected after a successful cell isolation. (B) Low yield, low-quality RNA or (C) low yield, high-quality RNA may be expected in cases of cell death, insufficient dissociation, or inadequate debris removal. (D) RNA sequencing analysis of select inflammatory pathways in healthy astrocytes versus inflammatory astrocytes reveals relative inhibition of inflammation in sorted healthy astrocytes compared to inflammatory astrocytes. Please click here to view a larger version of this figure.
Cell type | Total cell count | Sorted cells | % Sorted/Total | Average % viability |
ACSA2 | 6.3 x 105 | 1.7 x 105 | 27 | 92 |
CD11b | 6.3 x 105 | 8.0 x 104 | 12.7 | 93 |
Table 1: Cell yield, purity, and viability after sorting for ACSA-2 and CD11b cells. This table is adapted from Ahn, J. J. et al.16.
The isolation of pure, viable primary cells is paramount for investigating the structure and function of specific cell types. In the adult mouse, particularly in the spinal cord, this task poses significant challenges, as existing protocols are often not tailored to the adult spinal cord10,17. This protocol presents an efficient and cost-effective method applicable to various downstream applications, including cell culture, flow cytometry, histology, and transcriptomic studies.
The speed of spinal cord preparation plays a crucial role in ensuring optimal cell viability and yield. While it's imperative to achieve effective clearance of red blood cells during cardiac perfusion, the number of isolated cells can vary widely based on the time elapsed between cardiac perfusion and enzymatic dissociation. We observed that initiating tissue dissociation more than 10 min after cardiac perfusion led to decreased cell viability. Additionally, enzymatic dissociation durations of less than 30 min proved ineffective, leaving undigested tissue fragments and reducing cell yield.
Although complete elimination of mechanical dissociation (such as triturating or chopping) did not affect cell viability, it did result in fewer cells isolated due to the presence of undigested tissue fragments. A combination of mechanical methods with enzymatic dissociation proved to be the most effective approach for cell isolation. However, it's worth noting that some level of cellular stress inevitably occurs during tissue dissociation, potentially impacting transcriptomic studies18. This is a common challenge with CNS tissue dissociation procedures19. Nevertheless, gentle trituration methods have been shown to minimize cell death, extraneous transcript activation, and unwanted proteolysis16. Furthermore, although viable cells can be obtained by eliminating mechanical dissociation entirely, this might necessitate the use of additional animals. For the sake of reproducibility, complementing enzymatic dissociation with gentle chopping and trituration to maximize cell yield per spinal cord is recommended. However, researchers may choose to modify the protocol by eliminating trituration or chopping if their study's targets are highly sensitive to cellular stress and require minimal dissociation.
In summary, the integration of gentle dissociation steps and expeditious tissue preparation ensures optimal cell yield and viability. Protocol flexibility is enhanced by the option to replace cold media with warm media for maintaining cells in culture. This methodology has been optimized for application in animal models ranging from 10 weeks to at least 5 months old, including disease models.
The authors have nothing to disclose.
We thank Castle Raley at the George Washington University Genomics Core for RNA analyses and Q2 Lab Solutions for RNA sequencing analyses. This work was supported by the National Institute of Neurological Disorders and Stroke [grant number F31NS117085] and the Vivian Gill Research Endowment to Dr. Robert H. Miller. Figure 1 was created with BioRender.com.
2,2,2-Tribromoethanol | Sigma Aldrich | T48402 | |
24 well tissue culture plate | Avantor | 10861-558 | |
2-Methyl-2-butanol, 98% | Thermo Fisher | A18304-0F | |
4',6-Diamidino-2-Phenylindole, Dihydrochloride | Invitrogen | D1306 | 1:1000 |
45% glucose solution | Corning | 25-037-CI | |
5 mL capped tubes | Eppendorf | 30122305 | |
Acetic acid | Sigma-Adlrich | A6283 | |
Adult Brain Dissociation Kit | Miltenyi | 103-107-677 | |
Anti-ACSA2 Microbead Kit | Miltenyi | 130-097-679 | |
Anti-Iba1 | Wako | 019-1974 | |
Bioanalyzer | Agilent Technologies | G2939BA | |
C57BL/6J wild-type (WT) mice | Jackson Laboratories | ||
CD11b (Microglia) MicroBeads | Miltenyi | 130-093-634 | |
Celltrics 30 µm filter | Sysmex Partec | 04-004-2326 | |
Counting Chamber (Hemacytometer) | Hausser Scientific Co | 3200 | |
Deoxyribonuclease I from bovine pancreas | Sigma Aldrich | D4527-40KU | |
Distilled water | TMO | 15230001 | |
DMEM/F12 | Thermo Fisher | 11320074 | |
DNase for RNA purification | Qiagen | 79254 | |
Dulbecco's phosphate-buffered saline | Thermo Fisher | 14040117 | |
Fetal bovine serum | Thermo Fisher | A5209401 | |
GFAP antibody (mouse) | Santa Cruz | sc-33673 | 1:500 |
GFAP antibody (rabbit) | Dako | Z0334 | 1:500 |
Goat anti-mouse 594 IgG | Invitrogen | a11032 | 1:500 |
Goat anti-mouse 594 IgM | Invitrogen | a21044 | 1:300 |
Goat anti-Rabbit 488 IgG | Invitrogen | a11008 | 1:500 |
Iba1 antibody (rabbit) | Wako | 019-1974 | 1:500 |
MACS Separator | Miltenyi | 130-042-303 | |
Masterflex C/L Pump System | Thermo Fisher | 77122-22 | |
MEM | Corning | 15-015-CV | |
Methanol | Sigma-Adlrich | 439193 | |
Mounting Medium | Vector Laboratories | H-1000-10 | |
MS Columns | Miltenyi | 130-042-401 | |
O4 Antibody | R&D | MAB1326 | |
Penicillin-Streptomycin | Gibco | 15070063 | |
Plugged 9" glass pasteur pipette | VWR | 14672-412 | |
RNeasy Plus Micro Kit | Qiagen | 74034 | |
Royal-tek Surgical scalpel blade no. 10 | Fisher scientific | 22-079-683 | |
Small Vein Infusion Set, 23 G x 19 mm | Kawasumi | D3K2-23G |