Isolation of Pure Astrocytes and Microglia from the Adult Mouse Spinal Cord For In Vitro Assays and Transcriptomic Studies
October 20th, 2023
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.
Our research aims to characterize specific cell populations in the central nervous system, specifically the spinal cord, using preclinical models of disease. And we are trying to determine how these cells are affected under pathological conditions, and at different time points. It can be challenging to analyze cells, especially in the central nervous system because the cells in the brain and spinal cord, form a highly complex matrix, and everything is very tightly connected, so it can be difficult to isolate these cells without damaging the system.
Our protocol addresses limitations in existing methods for studying spinal cord diseases by efficiently isolating viable microglia and astrocytes from the small myelin-rich adult mouse spinal cord. This enables in vitro research on spinal cord related diseases in downstream analysis, filling a crucial research gap. This protocol presents several advantages, such as analyzing astrocytes and microglia under pathological conditions and at specific time points.
It also uniquely focuses on isolating glial cells from this adult spinal cord alone, excluding brain involvement. This allows researchers to study spinal cord pathologies at the cellular level. To begin, remove the spinal cord of a decapitated mouse that has been perfused with PBS.
Submerge the spinal cord in cold DPBS in a Petri dish on ice. With an 18-gauge needle and a 10-milliliter syringe filled with cold DPBS, use hydraulic extrusion to isolate the entire spinal cord in a laminar flow hood. Transfer the spinal cord into Petri dish with cold DPBS placed on ice packs.
Using a microscope inside the laminar flow hood, carefully remove any visible meninges with sharp forceps. Then transfer the spinal cord to an empty Petri dish, and use a number 10 surgical scalpel blade to cut it into two to three-millimeter long sections. For enzymatic dissociation of the spinal cells, add enzyme mixes one and two to the spinal cord pieces.
Swirl the enzymes in the dish several times to ensure uniform coating of the spinal cord. Incubate the dish at 37 degrees celsius for 30 minutes, manually swirling the dish every 10 minutes. After incubation, add 350 microliters of DNAse I.Swirl the dish gently to mix.
Next, add one milliliter of cold DMEM supplemented with 0.5%FBS before gently mixing. Immediately transfer the entire contents to a five-milliliter tube. With a P1000 pipette, gently triturate the tissue three times.
Then use a plugged nine-inch glass Pasteur pipette to gently triturate the tissue five more times. Place the tube upright for 30 seconds to allow the tissue to settle. With a P1000 pipette, aspirate out the supernatant, and transfer it through a 30-micrometer filter into a 15-milliliter conical tube.
To the tube with the remaining tissues, add one milliliter of FBS supplemented DMEM. Then gently triturate three times with a Pasteur pipette. After letting the tissue settle at the bottom, pass the supernatant through a 30-micrometer filter, and collect it into the same 15-milliliter tube used earlier.
Centrifuge the filtered cell suspension at 300G for 10 minutes at room temperature. Use a vacuum aspirator to carefully discard the supernatant. Use the debris removal solution provided in the adult brain dissociation kit to remove the myelin from the cell pellet.
Then add five milliliters of FBS supplemented DMEM to the pellet, and gently invert the tube three times to mix. After centrifuging, discard the supernatant and re-suspend the cell pellet in one milliliter of FBS supplemented DPBS. Centrifuge again before discarding the supernatant.
Label the cells with Anti-ACSA-2 MicroBeads, following the manufacturer's protocol. Use the magnetic separation columns to separate the cells by positive selection. Then centrifuge the cells at 300G for 10 minutes, before discarding the supernatant.
Next, re-suspend the cell pellet in one milliliter of warm FBS supplemented DMEM. Transfer the cell suspension to a hemocytometer to count the cells and assess their viability. Adjust the cell concentration to 75, 000 cells per 50 microliters with FBS/DMEM.
Then transfer 50 microliters of the cell suspension to a poly-l-lysine-coated cover slip in a 24-well plate. Incubate the plate at 37 degrees celsius for two hours to allow the to adhere to the cover slip. Then carefully add 450 microliters of warm antibiotic supplemented FBS/DMEM to each well.
Successful isolation and proliferation of microglia and astrocytes were observed. By day four, astrocytes were observed to form longer processes, while oval microglial cells with shorter spindles were seen. The astrocytes formed a connected confluent layer by day seven, with the microglia displaying fewer and shorter processes.
Cell sorting confirmed 92 to 93%cell viability of the isolated cell culture.
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Overview
This study characterizes cell populations in the central nervous system, focusing on isolating astrocytes and microglia from the adult mouse spinal cord. Using a refined protocol, the research enables subsequent applications like RNA analysis and cell culture, addressing challenges in studying spinal cord pathologies.
Key Study Components
Area of Science
- Neuroscience
- Cell Biology
- Pathology
Background
- Isolation of cells from the spinal cord is challenging due to its complex matrix.
- A focus on viable microglia and astrocytes aids in understanding spinal cord diseases.
- Existing methods often struggle with yielding high-quality isolated cells.
- This protocol specifically tackles the isolation of glial cells without involving the brain.
Purpose of Study
- To explore how astrocytes and microglia are affected under pathological conditions.
- To facilitate in vitro research for spinal cord-related diseases and fill research gaps.
- To develop a method for isolating these cell types from the adult mouse spinal cord.
Methods Used
- The primary method is the enzymatic dissociation of spinal cord tissues, followed by cell sorting.
- The biological model used is the adult mouse spinal cord, specifically targeting astrocytes and microglia.
- No multiomics workflow was mentioned.
- Critical steps include careful tissue handling, use of specific enzyme mixes, and density centrifugation.
- Cell sorting achieved 92-93% viability of the isolated cultures.
Main Results
- Successfully isolated microglia and astrocytes exhibiting viability and cellular responses.
- By day four, distinct morphological changes were observed in both cell types.
- Astrocytes formed a connected confluent layer, with microglia showing fewer processes.
- Confirmation of high cell viability indicates the protocol's effectiveness.
Conclusions
- The study demonstrates a reliable method for isolating specific glial cell populations from the spinal cord.
- This advancement enables detailed studies of spinal cord pathology at a cellular level.
- The findings have implications for understanding disease mechanisms in the spinal cord.
