Method Article

A Method for Generating Oligodendrocyte-Conditioned Medium

July 8th, 2025

In This Article

Abstract

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Source: Mazuir, E., et al. Generation of Oligodendrocytes and Oligodendrocyte-Conditioned Medium for Co-Culture Experiments. J. Vis. Exp. (2020)

This video demonstrates the production of oligodendrocyte-conditioned medium (OCM) from oligodendrocyte lineage cells by stressing them to enhance their molecular release into the medium. This medium, which is rich in growth factors, cytokines, and other bioactive molecules, can be added to neuron cultures to gain insight into the impact of oligodendrocytes-secreted factors on neuronal physiology and connectivity.

Protocol

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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.

  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 ....

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Disclosures

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No conflicts of interest declared.

Materials

List of materials used in this article
NameCompanyCatalog NumberComments
B27 supplementThermoFisher17504044
D-(+)-Glucose solutionSigmaG8769
Dulbecco's Modified Eagle MediumThermoFisher31966021
Ethanol 100%Sigma32221-M
Ethanol 70%VWR Chemicals83801.36
Fetal Calf SerumThermoFisher10082147
NeurobasalThermoFisher21103049
Penicillin-StreptomycinThermoFisher15140122
Phosphate Buffered Saline without calcium and magnesiumThermoFisherA1285601
Polyethylenimine(PEI)SigmaP3143
Bottenstein-Sato (BS) media
apo-Transferrin humanSigmaT1147
BSA (Bovine Serum Albumin)SigmaA4161
Dulbecco's Modified Eagle MediumThermoFisher31966021
InsulinSigmaI5500
PDGFPeprotechAF-100-13A
Penicillin-StreptomycinThermoFisher15140122
ProgesteroneSigmaP8783
Putrescine dihydrochlorideSigmaP5780
Sodium seleniteSigmaS5261
T3 (3,3',5-Triiodo-L-thyronine sodium salt)SigmaT6397
T4 (L-Thyroxine)SigmaT1775
Tools
0.22 µm filterSartorius514-7010
1 mL syringeTerumo1611127
100 mm Petri dishDutscher193100
15 mL tubeCorning Life Science734-1867
50 mL tubeCorning Life Science734-1869
60 mm Petri dishDutscher67003

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Tags

Oligodendrocyte Conditioned MediumOligodendrocyte Lineage CellsCell Attachment EnhancementNutrient Poor Medium IncubationMedium Filtration SterilizationBioactive Molecule SecretionOCM Storage PreservationPolymer Coated Petri DishHumidified Incubator ConditionsLaminar Flow Hood Sterility

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