Isolating and Culturing Oligodendrocyte Precursor Cells from Mouse Pup Brain Cortex

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

1. Tissue dissociation

NOTE: All the following procedures are carried out in a sterile tissue culture designated biosafety cabinet using aseptic technique and sterile materials.

1. Add 1 mL of 0.05% trypsin containing 0.53 mM EDTA to each 15 mL conical tube of 9 mL DMEM and tissue to begin tissue lysis.
   NOTE: DMEM contains a high amount of calcium chloride, which can act as an inhibitor of trypsin. If dissociation is not complete following the outlined steps, Hanks’ Balanced Salt Solution without calcium or magnesium can be used. After trypsinization, the enzyme can be neutralized by adding a trypsin inhibitor, although calcium should be added back to the solution as it is a cofactor for DNase I, which is used in subsequent steps.
2. Triturate with a 10 mL pipette approximately 20x.
3. Transfer the cell suspensions to empty 50 mL conical tubes.
4. Incubate the solution at 37 °C, 5% CO₂ for 15 min, gently agitating the lysates after 8 min.
5. Add 5 mL of MGM and 200 µL (5 mg/mL) DNase I to each tube for a final concentration of 50 µg/mL.
6. Triturate each lysate with a 10 mL pipette 10x.
7. Let the cell suspensions sit for 3 min at room temperature to allow non-dissociated tissue to settle at the bottom of the tubes.
8. Transfer the cell suspensions to new 50 mL conical tubes, leaving behind the non-dissociated tissue.
   NOTE: The lysis and trituration steps described above significantly limits the amount of non-dissociated tissue.
9. Centrifuge the tubes at 300 x g for 3 min at 4 °C without brake.
10. Aspirate the supernatant and resuspend the remaining cell pellets in 5 mL of MGM.
11. Triturate the pellet with a 5 mL pipette 20x.
12. Plate the 5 mL cell suspensions on coated T25 flasks.
13. Incubate the cells at 37 °C, 5% CO₂ and change media initially after 24 h to remove cell debris.
    NOTE: Some protocols recommend an initial media change after 72 h. Optimization of this step may be required.
14. Perform a 100% media change with MGM every 48-72 h until cells are 80% confluent (approximately 5-7 days).
    NOTE: All media must be warmed to 37 °C before media changes.

2. Oligodendrocyte precursor cell isolation

When plating OPCs following initial isolation, they must be plated on a poly-D-lysine-coated surface (sterile plate or coverslip). Prepare these materials before completing this section.

1. Following 15 h shake, remove the supernatant from the flasks and plate on a sterile 100 mm Petri dish.
2. Incubate the supernatant at 37 °C, 5% CO₂ for 30 min, swirling after 15 min to remove remaining microglia, as these will very quickly adhere to the dish. Non-tissue culture-treated Petri dishes may be used for this step.
3. Remove non-adherent cell supernatant, count, and plate on a poly-D-lysine-coated surface. Typically, 7,500-10,000 OPCs are plated/cm².
4. Incubate at 37 °C, 5% CO₂ for at least 1 h (up to 6 h), then gently aspirate 95% of media, and slowly add warm OPC Media, pipetting media against the wall of the well to minimize disruption of OPCs. Change media every 48 h until cells are ready for use.
   NOTE: It is critical that only one well is changed at a time. OPCs are sensitive and are especially intolerant to dry conditions. The addition of PDGF-AA in OPC media is to delay OPC maturation into oligodendrocytes. This factor may be excluded from culture media if the experimental focus is mature oligodendrocytes.

Table 1: Buffer and Media Recipes.

Mixed Glia Media (MGM)

OPC Media (50mL)