This article details a protocol for isolating oligodendrocyte precursor cells (OPCs) from mouse pup cortex. The method involves enzymatic digestion, mechanical dissociation, and selective media to promote cell survival and adherence.
Take a mouse pup cortex.
Add a proteolytic enzyme that digests the tissue's extracellular matrix. Then, mechanically dissociate the tissue, loosening the cells.
Add glial media and DNase to degrade any contaminating DNA.
Mechanically dissociate the tissue to release neurons and glial cells.
Once the undigested tissue settles, collect the media containing the cells and centrifuge. Remove the supernatant.
Resuspend cells in glia media and plate them on an extracellular matrix protein-coated flask.
Incubate. Astrocytes adhere more strongly to the coated surface than other cells. Glial media promote only glial cell survival.
Replace the media to remove debris.
Incubate with shaking to detach the overlying oligodendrocyte precursor cells, OPCs, and microglia.
Transfer the media with the cells onto a culture plate.
Incubate with shaking to facilitate differential microglial adhesion.
Transfer the non-adherent OPC supernatant onto a poly-D-lysine-coated multi-well plate. Incubate for OPC adherence.
Replace the media with OPC media, allowing OPC growth.
Add 1 milliliter of 0.05% trypsin with 0.53 millimolar EDTA to each tube with tissue. Triturate the mixture with a 10-milliliter pipette approximately 20 times. Then, transfer the cell suspension to an empty 50-milliliter conical tube.
Incubate the solution at 37 degrees Celsius and 5% carbon dioxide for 15 minutes, gently agitating the lysates after eight minutes. After the incubation, add 5 milliliters of MGM, and 200 microliters of DNase-I to each tube for a final concentration of 50 micrograms per milliliter. Triturate each lysate 10 times with a 10-milliliter pipette.
Then, let the cell suspension sit for three minutes at room temperature to allow nondissociated tissue to settle at the bottom of the tube. Transfer the cell suspensions to new 50-milliliter conical tubes, leaving behind the non dissociated tissue. After centrifugation, resuspend the cell pellet in 5 milliliters of MGM and triturate it 20 times.
Plate the cell suspensions on coated T-25 flasks, and incubate the cells at 37 degrees Celsius and 5% carbon dioxide. To perform oligodendrocyte precursor cell isolation, shake the cells for 15 hours. Then, remove the supernatant from the flask and plate it on a sterile Petri dish.
Incubate the supernatant at 37 degrees Celsius and 5% carbon dioxide for 30 minutes, swirling after 15 minutes to remove remaining microglia. Remove non-adherent cell supernatant. Count the cells and plate them on a poly-D-lysine-coated surface at the desired concentration.
Return the cells to the incubator for at least one hour. Then, gently aspirate 95% of media and slowly add warm OPC media, pipetting it against the wall of the well to minimize disruption of OPCs.
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