Isolation of Pure Astrocytes and Microglia from the Adult Mouse Spinal Cord For In Vitro Assays and Transcriptomic Studies

Summary

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.

Abstract

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.

Introduction

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 processes^1,2,3,4. 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 injuries^5,6,7. Astrocyte responses in disease are equally diverse, encompassing contributions to inflammation, the formation of glial scars, and the compromise of the blood-brain barrier^8,9. 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 brain^10,11,12,13, 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.

Protocol

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

1. Anesthetize the animals using Avertin (12.5 mg/mL of 2,2,2-Tribromoethanol and 2.5% 2-methyl-2-butanol in sterile water) (see Table of Materials). Confirm the absence of a response to toe and tail pinches in the mice.
2. Perform cardiac perfusion with cold 1x Dulbecco's phosphate-buffered saline (DPBS) to remove peripheral blood mononuclear cells.
   1. Place the animal on a shallow tray and make a lateral incision to expose the pleural cavity. Insert a 23 G perfusion needle connected to a peristaltic pump (see Table of Materials) into the left ventricle and activate the pump. Once cold DPBS flows through the heart, create a small incision in the right atrium.
      ​NOTE: A faster perfusion rate is preferred in this step to enhance cell viability. Blood should be flushed out in under 1 min. Clearing of the liver indicates successful perfusion.
3. Decapitate the animal using large scissors and remove the spinal column¹⁴. Submerge it in cold 1x DPBS with Ca²⁺/Mg²⁺/0.2% glucose in a Petri dish on ice for tissue rinsing.
4. Starting now, ensure that all steps are conducted in a sterile environment. Use hydraulic extrusion¹⁴ to isolate the entire spinal cord. Employ an 18 G needle and a 10 mL syringe filled with cold 1x DPBS with Ca²⁺/Mg²⁺/0.2% glucose. Transfer the spinal cord to a Petri dish placed on ice packs containing sufficient cold 1x DPBS with Ca²⁺/Mg²⁺/0.2% glucose to submerge the entire tissue.
5. Carefully remove the meninges under a microscope within a laminar flow hood. Transfer the spinal cord to an empty Petri dish on ice packs, and using a No. 10 blade surgical scalpel, cut the entire spinal cord into 2-3 mm sections.

2. Enzymatic cell dissociation

1. Add Enzyme mix 1 and Enzyme mix 2 from the commercially available Adult Brain Dissociation Kit following the manufacturer's protocol (see Table of Materials). Swirl the enzymes in the dish several times to ensure uniform coating of the spinal cord, then incubate at 37 °C for 30 min. Every 5 minutes, gently swirl the dish to prevent tissue clumping and facilitate even distribution of reagents.
2. Remove the dish from the incubator and add 350 µL of 0.46 mg/mL DNAse I in Minimal Essential Medium (MEM) (see Table of Materials). Mix by gently swirling.
3. Add 1 mL of cold DMEM/0.5% Fetal Bovine Serum (FBS) and mix by gently swirling. Immediately transfer the entire contents to a 5 mL tube.

3. Mechanical cell dissociation

1. Using a P1000 pipette, gently triturate the tissue three times, ensuring that tissue pieces are drawn up each time to further dissociate the tissue. Then, using a plugged 9" glass Pasteur pipette, gently triturate five more times, ensuring no bubbles are generated during the process.
2. Place the 5 mL tube upright and allow the tissue to settle at the bottom for 30 s. Once the tissue has settled, draw up the supernatant using a P1000 pipette and pass it through a 30 µm filter into a 15 mL conical tube.
3. To the same 5 mL tube with the remaining tissue, add 1 mL of cold DMEM/0.5% FBS. Using a Pasteur pipette, gently triturate three times.
4. Allow the tissue to settle at the bottom for 30 s, then pass the supernatant through a 30 µm filter and collect it into the same 15 mL tube as before. Repeat step 3.3 and step 3.4 once more.
5. Centrifuge the filtered cell suspension at 300 x g for 10 min at room temperature (RT). Carefully remove and discard the resulting supernatant using a vacuum aspirator.

4. Myelin removal

1. Remove myelin using the debris removal solution (DRS) provided in the Adult Brain Dissociation Kit following the manufacturer's protocol. After debris removal, add 5 mL of cold DMEM/0.5% FBS to the cell pellet and gently invert the tube three times. Centrifuge the cell suspension at 300 x g for 10 min at room temperature (RT).
   NOTE: If the cells will be used for in vitro studies and will remain in culture, pre-warmed DMEM/0.5% FBS should be used instead.
2. Discard the supernatant and resuspend the cell pellet in 1 mL of DPBS/0.5% FBS (cold for RNA studies and warm for in vitro assays). Count the cells and assess viability using Trypan Blue.
   NOTE: Cell suspensions from multiple animals may be combined to increase cell concentration.
3. Wash the cells by adding DPBS/0.5% FBS up to a total volume of 5 mL. Centrifuge the suspension at 300 x g for 10 min at room temperature and discard the supernatant using a pipette.

5. Microglia and astrocyte isolation

1. Label the cells with anti-CD11b or anti-ACSA-2 microbeads following the manufacturer's protocol (see Table of Materials).
2. Sort the cells by positive selection using the MS columns according to the manufacturer's instructions (see Table of Materials). After sorting, centrifuge the sorted cells at 300 x g for 10 min and discard the supernatant.

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.

1. Resuspend the cells in 1 mL of warm DMEM/0.5% FBS. Count the cells and assess their viability using a hemacytometer (see Table of Materials).
2. Adjust the cell concentration to 75,000 cells per 50 µL. Add 50 µL of the cell suspension to each Poly-L-lysine-coated coverslip¹¹ in a 24-well plate.
3. Incubate at 37 °C for 2 h to allow the cells to adhere to the coverslip.
4. Carefully add 450 µL of warm DMEM/5% FBS/Penicillin-Streptomycin (Pen-Strep, see Table of Materials) to each well.
5. After 3 days, replace half of the media with 250 µL of warm DMEM/5% FBS/Pen-Strep. For maintenance, completely replace the media with 500 µL of warm DMEM/5% FBS/Pen-Strep every 4-5 days.

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.

1. Remove the media and label the cells with anti-mouse mAb O4 (1:100) (see Table of Materials) in warm DMEM/5% FBS/10% normal goat serum (NGS) for 15 min at room temperature (RT) or at 37 °C.
   NOTE: Skip this step and proceed to step 7.3 if the cells will not be labeled with O4.
2. Gently rinse the coverslips by dipping them into warm DMEM/5% FBS three times. Add goat anti-mouse 594 IgM (1: 300) (see Table of Materials) in warm DMEM/5% FBS/10% NGS and incubate at RT for 15 min in the dark. Gently rinse three times in warm DMEM/5% FBS.
3. Fix the cells with cold 5% acetic acid/methanol for 15 min at 4 °C. Wash three times with 1x PBS, then label with the appropriate antibody: anti-rabbit GFAP (1: 500), anti-mouse GFAP (1: 500), or anti-rabbit Iba1 (1: 500) in 1% Triton-X100/10% NGS in 1x DPBS (see Table of Materials). Incubate for 1 h at RT.
   NOTE: Keep coverslips in the dark if they have already been stained with O4.
4. Gently rinse three times with PBS. Label with the appropriate secondary antibody: anti-mouse 594 IgG (1: 500), anti-rabbit 488 IgG (1: 500) (see Table of Materials). Incubate for 30 min at RT in the dark.
5. Gently rinse three times with PBS. Counterstain with DAPI (1: 1000) for 1 min at RT. Rinse three times with PBS, add mounting medium (see Table of Materials), and mount coverslips onto slides.

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.

1. Extract RNA using a commercially available RNA isolation kit following the manufacturer's protocol (see Table of Materials). For each wash step with the RW1 wash buffer provided in the kit, leave the cells in the wash buffer for an additional 2 min.
2. Remove any remaining genomic DNA using DNase I according to the manufacturer's instructions (see Table of Materials). Incubate with DNase at RT for 15 min, then wash with RW1 buffer.
3. To increase RNA yield, prior to elution, incubate the samples in RNAse-free water at RT for 10 min. Assess RNA integrity and quantity using a bioanalyzer¹⁵ (see Table of Materials).

Representative Results

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.

Discussion

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 cord^10,17. 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.

Materials

Name	Company	Catalog Number	Comments
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