A Method for Generating Oligodendrocyte-Conditioned Medium

1. OL lineage (OL) cell harvesting and culture

NOTE: These steps should be performed in a laminar flow hood under sterile conditions.

1. On the day after shaking, prepare the Bottenstein-Sato (BS) medium according to Table 1.
2. Rinse coated Petri dishes 3 times with sterile distilled water.
3. Harvest flasks' supernatant containing mainly OL lineage cells but also some microglial cells and plate it on non-coated 100 mm Petri dishes.
   NOTE: This step allows the removal of microglial cells through differential fast adhesion on the dish surface.
4. Incubate the Petri dishes for 15 min in a humidified incubator at 37 °C under 5% CO₂.
5. Fill each T150 flask with 25 mL of warm, freshly prepared culture medium and incubate in a humidified incubator at 37 °C under 5% CO₂ until the second shaking.
6. Transfer the supernatant from the Petri dishes into new non-coated 100 mm Petri dishes to allow adhesion of residual microglial cells.
7. Incubate the Petri dishes for 15 min in a humidified incubator at 37 °C under 5% CO₂.
8. Remove the supernatant, which contains non-adherent OL lineage cells, and transfer it into 50 mL tubes (supernatant from 2 Petri dishes for a 50 mL tube). Discard Petri dishes plated with microglia.
9. Centrifuge the supernatant for 5 min at 423 x g. Carefully remove the supernatant and resuspend the cell pellet with 1 mL of BS medium. Pool all pellets in a common 50 mL tube and adjust the volume to 10 mL with BS medium.
10. Determine cell density by counting cells under a microscope.
    NOTE: A cell density between 3 x 10⁵ cells/mL and 5 x 10⁵ cells/mL should be obtained.
11. Add 20 mL of BS if cell density is higher than or equal to 4 x 10⁵ cells/mL to obtain a final volume of 30 mL, or add only 10 mL of BS if cell density is less than 4 x 10⁵ cells/mL to obtain a final volume of 20 mL.
12. Plate two or three pre-coated 100 mm Petri dishes with 10 mL of cell suspension. Incubate in a humidified incubator at 37 °C under 5% CO₂.
13. Clear the debris from the Petri dishes by refreshing all of the BS medium 2 h later.
    NOTE: Examine the culture under the microscope before and after clearing to verify cell density and efficiency of debris removal.
14. Incubate for 2 days in BS medium in a humidified incubator at 37 °C under 5% CO₂.
    NOTE: Examine the culture under the microscope. The confluence should be 70% to 80%.

2. OCM production

NOTE: Perform these steps in a laminar flow hood under sterile conditions.

1. Prepare NB-B27low medium according to Table 2.
2. Renew the culture medium with 10 mL of warm NB-B27low medium. Incubate for 2 days in a humidified incubator at 37 °C under 5% CO₂.
3. Harvest the OCM, i.e., the supernatant containing OL-secreted factors. Filter-sterilize OCM using a 0.22 µm filter.
   NOTE: Store OCM at 4 °C for a maximum of 2 months.

Table 1: Preparation of Bottenstein-Sato (BS) media.

Table 2: Preparation of NB-B27low and NB-B27 media.