This article details a protocol for culturing astrocytes from neonatal mouse brains, highlighting the importance of these cells in neuroscience research. The method emphasizes the steps necessary to ensure a pure astrocyte culture, avoiding contamination from other cell types.
The astrocyte in vitro culture helps investigate the biological role of astrocytes - the most abundant brain cells.
Begin with a neonatal mouse brain placed under a dissecting microscope.
Remove the olfactory bulbs and the cerebellum.
Perform a midline incision between the two hemispheres to separate the cortex.
Remove the meninges to avoid contamination in the astrocyte culture from fibroblasts and meningeal cells.
Transfer the cortex hemispheres into an ice-cold medium and mince into small pieces.
Collect the cortical pieces in a tube containing trypsin. Trypsin, a protease enzyme, digests the cell adhesion proteins, facilitating cell separation.
Centrifuge to pellet the digested tissue.
Remove the supernatant and add an astrocyte medium to the pellet. Pipette vigorously to release a mixed cell population of astrocytes, microglia, and oligodendrocyte precursor cells or OPCs.
Seed this cell suspension into a poly-lysine coated tissue culture flask and incubate.
The negatively charged cells attach to the positively charged poly-lysine and grow over the entire surface to form a confluent layer.
Shake the flask to detach the loosely bound microglia and OPCs. Remove the supernatant containing detached cells.
Add trypsin to the astrocyte layer. Collect the cells and centrifuge.
Resuspend the pellet and divide the astrocytes into multiple flasks.
Finally, assess the purity of the astrocyte culture by immunostaining cell-specific markers.
Transfer the dish containing the brains to a stereomicroscope. Next, to isolate the cortices, grab the posterior end of each brain with fine forceps and perform a midline incision between the hemispheres.
Then, insert a second set of forceps to the created groove and peel away the plate-like structure of the cortex from the rest of the brain.
Once all cortices have been isolated, carefully peel the meninges away from the cortex by pulling with the fine forceps. This step avoids contamination of the final astrocyte culture by meningeal cells and fibroblasts.
Transfer the prepared cortical hemispheres into a second dish filled with chilled HBSS and place on ice. Continue accordingly with all four cortices. Finally, cut each hemisphere into four to eight small pieces using sharp blades.
Under sterile conditions, transfer the pieces of cortex into one 50-milliliter Falcon tube and add HBSS to a final volume of 22.5 milliliters. Then, add 2.5 milliliters of 2.5% trypsin.
Replace the cap, mix, and incubate the tissue in the water bath at 37 degrees Celsius for 30 minutes. Mix by shaking every 10 minutes. Then, after centrifuging for 5 minutes at 300 x g to pellet the cortex tissue pieces, carefully decant the supernatant.
Add 10 milliliters of astrocyte plating medium to the pellet, and use a 10-milliliter pipette to vigorously pipette the medium containing the tissue up and down 20 to 30 times until a single cell suspension is obtained.
Next, add astrocyte plating medium to a final volume of 20 milliliters and count the cells using a hemocytometer or automated cell counter according to standard procedures.
One preparation of four mouse pup cortices should yield 10 to 15 times 10 to the sixth dissociated single cells. Finally, aspirate the poly-D-lysine from a previously prepared flask and transfer the dissociated cell suspension to the flask.
Incubate the culture at 37 degrees Celsius in the tissue culture incubator for 2 days and then change the media. The media should then be changed every 3 days thereafter.
The astrocyte should appear confluent with an overlying layer of microglia after 7 to 8 days in culture. At this point, set the T75 flask on an orbital shaker set at 180 rotations per minute for 30 minutes to remove microglia.
Discard the supernatant containing microglia or spin it down and plate for culture. Then, add 20 milliliters of fresh astrocyte culture medium and continue by shaking the flask at 240 rpm for 6 hours to remove oligodendrocyte precursor cells.
Since some OPCs will not completely detach from the astrocyte layer, continue to shake vigorously by hand for 1 minute in order to prevent OPC contamination. Again, discard the supernatant or spin it down on a plate to culture OPCs.
Rinse the remaining confluent astrocyte layer twice with PBS. Aspirate the PBS and add 5 milliliters of trypsin-EDTA. Incubate in the tissue culture incubator at 37 degrees Celsius.
Check for detachment of astrocytes every 5 minutes and enforce the detachment of astrocytes by hitting the flask against the palm of your hand two to three times.
Once the astrocytes have detached from the flask, add 5 milliliters of astrocyte culture medium. Spin the cells at 180 x g for 5 minutes, aspirate the supernatant, and add 40 milliliters of fresh astrocyte plating medium.
One T75 flask of mixed cortical cells should yield around 1 times 10 to the sixth cells enriched for astrocytes after the first passage.
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