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.
