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1. OL lineage (OL) cell harvesting and culture
NOTE: These steps should be performed in a laminar flow hood under sterile conditions.
- On the day after shaking, prepare the Bottenstein-Sato (BS) medium according to Table 1.
- Rinse coated Petri dishes 3 times with sterile distilled water.
- 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.
- Incubate the Petri dishes for 15 min in a humidified incubator at 37 °C under 5% CO2.
- 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.
- Transfer the supernatant from the Petri dishes into new non-coated 100 mm Petri dishes to allow adhesion of residual microglial cells.
- Incubate the Petri dishes for 15 min in a humidified incubator at 37 °C under 5% CO2.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- Prepare NB-B27low medium according to Table 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.
- 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.
| Bottenstein-Sato (BS) media | Final concentration |
| Dulbecco's Modified Eagle Medium | |
| Penicillin-Streptomycin | 100 IU/mL |
| apo-Transferrin human | 100 µg/mL |
| BSA (Bovine Serum Albumin) | 100 µg/mL |
| Insulin | 5 µg/mL |
| PDGF | 10 ng/mL |
| Progesterone | 62 ng/mL |
| Putrescine dihydrochloride | 16 µg/mL |
| Sodium selenite | 40 ng/mL |
| T3 (3,3',5-Triiodo-L-thyronine sodium salt) | 30 ng/mL |
| T4 (L-Thyroxine) | 40 ng/mL |
Table 2: Preparation of NB-B27low and NB-B27 media.
| NB-B27 low media | Final concentration |
| Neurobasal | |
| B27 supplement | 0.5x |
| L-glutamine | 0.5 mM |
| Penicillin-Streptomycin | 100 IU/mL |
| NB-B27 media | Final concentration |
| Neurobasal | |
| B27 supplement | 1x |
| L-glutamine | 0.5 mM |
| Penicillin-Streptomycin | 100 IU/mL |