Obtaining a Mixed Oligodendrocyte and Astrocyte Culture From Adult Mouse Neural Stem Cells

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

1. Solutions and reagents

1. Prepare standard medium: DMEM/F12 GlutaMAX 1x; 8 mmol/L HEPES; 100 U/100 μg Penicillin/Streptomycin (1% P/S); 1x B27; 1x N-2.
2. Prepare neurosphere medium: add 10 ng/mL Basic Fibroblast Growth Factor (bFGF); 10 ng/mL Epidermal Growth Factor (EGF) to standard medium.
3. Prepare oligosphere/oligodendrocyte precursor cell (OPC) medium: add 10 ng/mL bFGF; 10 ng/mL Platelet Derived Growth Factor-AA (PDGF-AA) to standard medium.
4. Prepare oligodendrocyte differentiation medium: add 50 nM Triiodothyronine (T3); 10 ng/mL Ciliary Neurotropic Factor (CNTF); 1x N-acetyl-L-cysteine (NAC) to standard medium.
5. Prepare sucrose solution: Hanks' Balanced Salt Solution (HBSS), 0.3 g/mL sucrose.
6. Prepare Bovine serum albumin (BSA) washing solution: Earle's Balanced Salt Solution (EBSS), 40 mg/mL BSA, 0.02 mL/l HEPES.
7. Prepare enzymatic dissociation buffer: HBSS, 5.4 mg/mL D-glucose, 15 mmol/L HEPES, 1.33 mg/mL Trypsin, 0.7 mg/mL Hyaluronidase, 80 U/mL DNase.

2. Dissection and NSC isolation

NOTE: Adult neural stem cells (NSCs) were isolated from 2.5-month-old adult sub-ventricular zone (SVZ).

1. Adult NSC cultures
   1. Sacrifice animals by cervical dislocation.
   2. Collect brains from 4–5 mice in a 50 mL tube containing ice cold HBSS.
   3. Place the brain on a cold sterile surface. For this purpose, use a T-25 flask filled with water and placed at -20 °C overnight. At the time of the experiment, cover the flask with sterile aluminum foil.
   4. Place the brain ventral side downwards, in rostro-caudal direction, and remove the olfactory bulbs using a razor blade.
   5. Using a razor blade, cut 2–3 coronal slices of 1 mm thickness, from the cortex to the optical chiasma.
   6. Place the slices on the cold surface in a ventro-dorsal position and identify the corpus callosum and the two lateral ventricles.
   7. Using magnifying glasses or a stereoscope, isolate the walls of the lateral ventricles, taking care not to carry pieces of the corpus callosum.
   8. Put the isolated tissue in the enzymatic dissociation buffer (5–10 mL) and incubate at 37 °C for 15 min.
   9. Mix the solution, pipetting several times (at least 50), and incubate again at 37 °C for 10 min.
   10. Neutralize the trypsin by adding 5 mL of standard culture medium and filter the solution using a 70 µm filter.
   11. Centrifuge the filtered solution for 5 min at 400 x g.
   12. Resuspend the pellet in the sucrose solution and centrifuge for 10 min at   500 x g.
   13. Resuspend the pellet in BSA washing solution and centrifuge for 7 min at 400 x g.
   14. Resuspend the pellet in the standard culture medium. Count the cells and plate them in suspension at a density of 10–50 cells/μL in a T-25 or T-45 flask containing 10–30 mL of neurosphere medium, kept in a vertical position to avoid cell adhesion.

3. Primary neurospheres

1. Add the growth factors (bFGF/EGF) every 2 days.
2. Every 4–6 days (depending on cell density), change half of the medium as follows:
   1. Transfer the entire cell suspension to a 15 or 50 mL tube.
   2. Centrifuge for 5 min at 400 x g.
   3. Remove half of the volume.
   4. Add the same amount of fresh medium, gently mix by pipetting, and add growth factors.

4. Oligospheres

1. When the neurospheres reach a diameter of 100–150 µm, they are ready to be passed. To do so, transfer the entire cell suspension to a 15 or 50 mL tube, and centrifuge for 5 min at 400 x g.
   1. Rapidly evaluate the diameter by taking pictures of the spheres using an inverted transmitted light microscope and opening them by ImageJ software.
   2. Click on the Analyze menu and from the Tools window, select Scale bar.
   3. Set 150 µm as Width in microns and compare the scale bar with the spheres.
2. Remove the entire volume by inversion and resuspend the pellet in 180 µL of fresh standard culture medium. Pipette 50 times to allow disaggregation of the spheres.
3. Add 810 µL of fresh standard culture medium, count the cells, and re-plate them as described for the neurospheres.
4. Add bFGF/PDGF-AA 10 ng/mL every 2 days
5. Every 4–6 days (depending on cell density), change half of the medium as follows:
6. Transfer the entire cell suspension to a 15 or 50 mL tube.
7. Centrifuge for 5 min at 400 x g.
8. Remove half of the volume.
9. Add the same amount of fresh medium, gently mix by pipetting, and add growth factors.

5. Plate coating

1. Poly-D,L-ornithine/laminin coating: at least 2 days before plating the OPCs, add 50 µg/mL poly-D,L-ornithine solution, diluted in PBS, to each well (40 µL/well for 96- well plates) and incubate at RT overnight.
2. The following day, remove the liquid and wash three times with distilled sterile water.
3. Let the plates dry at RT overnight. The following day, add a laminin solution diluted in PBS (5 µg/mL; 40 µL/well for 96-well plates) and incubate for 2 h at 37°C.

6. Cell seeding

1. When the oligospheres reach a diameter of 100–150 µm, they are ready to be dissociated and seeded on the poly-D,L-ornithine/laminin coated plates. To do so, transfer the entire cell suspension to a 15 or 50 mL tube, and centrifuge for 5 min at 400 x g.
2. Remove the entire volume by inversion and resuspend the pellet in 180 µL of fresh standard culture medium. Pipette 50 times to allow disaggregation of the spheres.
3. Add 810 µL of fresh standard culture medium and count the cells.
4. Remove the laminin solution from the wells and plate the cells at 3,000 cell/cm² density (100 µL/well for 96-well plates)

7. OPC differentiation induction

1. After 3 days, remove the entire medium and add the same volume of oligodendrocyte differentiation medium.
2. Change half of the medium every 4 days and add fresh differentiation mix (T3/CNTF/NAC) every 2 days.