Isolating and Culturing Oligodendrocyte Precursor Cells from Mouse Pup Brain Cortex

Abstract

Source: Sinyuk, M., et al. Dissection and Isolation of Murine Glia from Multiple Central Nervous System Regions. J. Vis. Exp. (2020)

This video demonstrates a technique for isolating and culturing oligodendrocyte precursor cells (OPCs) from the mouse pup brain cortex.

Protocol

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)

88 mL	1X DMEM (high glucose, w/L-glutamine, w/Na pyruvate)
10 mL	Heat-inactivated FBS
1.0 mL	L-Glutamine (100X)
1.0 mL	Antibiotic/Antimycotic

OPC Media (50mL)

49 mL	Neurobasal media
1.0 mL	B27 supplement (50X)
10 ng/ml	PDGF-AA
NOTE: PDGF-AA is added fresh prior to each media change.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.05% Trypsin and 0.53 mM EDTA	Gibco	25300054	Tissue dissociation
1X PBS pH 7.4	Gibco	10010031	Standard reagent
50 mL, 25 cm square cell culture flask	Greiner Bio-One	690 175	Cell culture (T25) flask
Antibiotic-Antimycotic 100X	Gibco	15240-096	Media component
B-27 Supplement 50X	Gibco	17504-044	Media component
Bovine serum albumin	Sigma	A9647-50G	Antibody diluent
DMEM (1X), high glucose with Na pyruvate	Gibco	11995040	Media component
Dnase I	Sigma	10104159001	Tissue dissociation
Fetal bovine serum heat inactivated	Gibco	A3840001	Media component
Hanks' Balanced Salt Solution (w/o Ca or Mg)	ThermoFisher	14170120	Tissue dissociation
L-glutamine, 200mM	Gibco	20530081	Media component
Neurobasal	Gibco	21103-049	Media component
Normal goat serum	Sigma	G9023	Blocking solution component
Poly-D-Lysine 12 mm #1 German Glass Coverslip	Corning Biocoatt	354086	Cell adherent