All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.
1. Solutions and reagents
- 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.
- Prepare neurosphere medium: add 10 ng/mL Basic Fibroblast Growth Factor (bFGF); 10 ng/mL Epidermal Growth Factor (EGF) to standard medium.
- 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.
- 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.
- Prepare sucrose solution: Hanks' Balanced Salt Solution (HBSS), 0.3 g/mL sucrose.
- Prepare Bovine serum albumin (BSA) washing solution: Earle's Balanced Salt Solution (EBSS), 40 mg/mL BSA, 0.02 mL/l HEPES.
- 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).
- Adult NSC cultures
- Sacrifice animals by cervical dislocation.
- Collect brains from 4–5 mice in a 50 mL tube containing ice cold HBSS.
- 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.
- Place the brain ventral side downwards, in rostro-caudal direction, and remove the olfactory bulbs using a razor blade.
- Using a razor blade, cut 2–3 coronal slices of 1 mm thickness, from the cortex to the optical chiasma.
- Place the slices on the cold surface in a ventro-dorsal position and identify the corpus callosum and the two lateral ventricles.
- Using magnifying glasses or a stereoscope, isolate the walls of the lateral ventricles, taking care not to carry pieces of the corpus callosum.
- Put the isolated tissue in the enzymatic dissociation buffer (5–10 mL) and incubate at 37 °C for 15 min.
- Mix the solution, pipetting several times (at least 50), and incubate again at 37 °C for 10 min.
- Neutralize the trypsin by adding 5 mL of standard culture medium and filter the solution using a 70 µm filter.
- Centrifuge the filtered solution for 5 min at 400 x g.
- Resuspend the pellet in the sucrose solution and centrifuge for 10 min at 500 x g.
- Resuspend the pellet in BSA washing solution and centrifuge for 7 min at 400 x g.
- 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
- Add the growth factors (bFGF/EGF) every 2 days.
- Every 4–6 days (depending on cell density), change half of the medium as follows:
- Transfer the entire cell suspension to a 15 or 50 mL tube.
- Centrifuge for 5 min at 400 x g.
- Remove half of the volume.
- Add the same amount of fresh medium, gently mix by pipetting, and add growth factors.
4. Oligospheres
- 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.
- Rapidly evaluate the diameter by taking pictures of the spheres using an inverted transmitted light microscope and opening them by ImageJ software.
- Click on the Analyze menu and from the Tools window, select Scale bar.
- Set 150 µm as Width in microns and compare the scale bar with the spheres.
- 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.
- Add 810 µL of fresh standard culture medium, count the cells, and re-plate them as described for the neurospheres.
- Add bFGF/PDGF-AA 10 ng/mL every 2 days
- Every 4–6 days (depending on cell density), change half of the medium as follows:
- Transfer the entire cell suspension to a 15 or 50 mL tube.
- Centrifuge for 5 min at 400 x g.
- Remove half of the volume.
- Add the same amount of fresh medium, gently mix by pipetting, and add growth factors.
5. Plate coating
- 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.
- The following day, remove the liquid and wash three times with distilled sterile water.
- 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
- 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.
- 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.
- Add 810 µL of fresh standard culture medium and count the cells.
- 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
- After 3 days, remove the entire medium and add the same volume of oligodendrocyte differentiation medium.
- Change half of the medium every 4 days and add fresh differentiation mix (T3/CNTF/NAC) every 2 days.
