Generation of Oligodendrocytes and Oligodendrocyte-Conditioned Medium for Co-Culture Experiments

We describe an efficient method of oligodendroglial lineage cells purification and culture. This allows to address the molecular mechanisms controlling oligodendrocytes differentiation and their interactions with neurons during the myelination process. This shaking technique is not expensive and is optimal to obtain high quantity of oligodendrocytes.

Such cultures allow the production of oligodendrocyte-conditioned medium and co-cultures of oligodendrocytes with neurons. Multiple sclerosis is a disease caused by focal de-myelination in the central nervous system, secondary to oligodendrocytes loss. Oligodendrocytes culture provide a tool for better understanding how to promote re-myelination.

Only oligodendroglial cell culture could provide insights into intrinsic mechanisms regulating developments and biology of oligodendrocytes. Co-cultures with neurons allow to gain insight into their impacts on neuronal physiology. To begin, use curved forceps to maintain the head of the animal at eye level.

Use small surgical scissors to make a small incision at the base of the skull and cut the skull following the brain midline. Use forceps to gently peel off the two parts of the skull from the midline. Use a small surgical spoon to remove the brain from the head cavity.

Put the brain in a 60-millimeter Petri dish containing ice cold PBS glucose on ice. Viewing under a stereo microscope, use fine forceps to remove the cerebellum, the brain stem, and the olfactory bulbs from the cerebral hemispheres. Use fine forceps to separate the two cerebral hemispheres.

Use fine forceps to peel off the meninges. Put the cerebral cortices in a 60-millimeter Petri dish on ice. In a laminar flow hood, use a sharp scalpel to finely chop the cerebral cortices.

Transfer the minced tissue into a 50-milliliter tube containing enzyme digestion medium. Incubate for 30 minutes in a humidified incubator at 37 degrees Celsius under 5%carbon dioxide. After that, use a pipette to gently remove the enzyme digestion medium while making sure that the cortical tissue remains at the bottom of the tube.

Use a P1000 micropipette to add one milliliter of DMEM 10%fetal calf serum and gently triturate the tissue. Use a 70-micron filter and the piston of a one-milliliter syringe to filter the cortical tissue into a 50-milliliter tube. Rinse the residual tissue on the inner tube wall of the 50-milliliter tube several times with DMEM 10%fetal calf serum.

Fill the 50-milliliter tube with DMEM 10%fetal calf serum. Centrifuge at 423 times g for five minutes at room temperature. Carefully remove the supernatant and resuspend the cell pellet with two milliliters of DMEM 10%fetal calf serum.

Gently triturate the cell pellet with the P1000 micropipette and then, with the P200 micropipette. Dilute the cell suspension with the appropriate volume of DMEM 10%fetal calf serum. Plate five milliliters of the cell suspension on a T-150 flask at a density of one times 10 to the fifth cells per square centimeter.

Add 20 milliliters of warm DMEM 10%fetal calf serum to each T-150 flask. Incubate in a humidified incubator at 37 degrees Celsius under 5%carbon dioxide. After shaking the sample overnight at 250 rpm and 37 degrees Celsius, harvest the supernatant in the flask containing mainly oligodendrocyte lineage cells, but also, some microglial cells, and plate it on non-coated 100-millimeter Petri dishes.

Incubate the Petri dishes for 15 minutes in a humidified incubator at 37 degrees Celsius and 5%carbon dioxide. This allows removal of microglial cells through differential fast adhesion on the dish surface. In the meantime, fill each T-150 flask with 25 milliliters of warm, freshly prepared culture medium and incubate in a humidified incubator at 37 degrees Celsius under 5%carbon dioxide until the second shaking.

Next, transfer the supernatant from the Petri dishes into new non-coated 100-millimeter Petri dishes to allow adhesion of residual microglial cells. Incubate the Petri dishes for 15 minutes in a humidified incubator at 37 Celsius under 5%carbon dioxide. Remove the supernatant from every two Petri dishes, which contain non-adherent oligodendrocyte lineage cells, and transfer it into a 50-milliliter tube.

Discard the Petri dishes plated with microglia. Centrifuge the tubes for five minutes at 423 times g. Carefully remove the supernatant and resuspend the cell pellet with one milliliter of Bottenstein-Sato medium.

Pool all the pellets in a common 50-milliliter tube and adjust the volume to 20 or 30 milliliters depending on cell density with Bottenstein-Sato medium. Plate two or three precoated 100-millimeter Petri dishes with 10 milliliters of the cell suspension. Incubate in a humidified incubator at 37 degrees Celsius under 5%carbon dioxide.

Two hours later, clear the debris from the Petri dishes by refreshing all of the Bottenstein-Sato medium. Incubate for two days in the medium in a humidified incubator at 37 degrees Celsius under 5%carbon dioxide. After two days, examine the culture under a microscope.

In a laminar flow hood, under sterile conditions, renew the culture medium with 10 milliliters of warm MPB-27 low medium. Incubate for two days in a humidified incubator at 37 degrees Celsius under 5%carbon dioxide. To harvest the OCM, collect the supernatant containing oligodendrocytes secreted factors.

Filter sterilize the OCM using a 0.22-micron filter. In this protocol, oligodendrocyte lineage cells were purified from glial cultures by shaking off astrocytes and microglia. Analysis of the expression of different markers indicated that oligodendrocyte cultures were mostly pre-oligodendrocytes with 90 plus or minus 4%of O4 positive cells, 85 plus or minus 7%NG2 positive cells, and 4.7 plus or minus 2.1%of PLP positive cells, while 7.2 plus or minus 2.5%of cells were GFAP positive astrocytes.

OCM produced from such cultures were added at three days in vitro to purified hippocampal neuron cultures. This treatment promotes the clustering of notal proteins. Myelination of hippocampal neurons were studied through addition of oligodendroctyes at 14 days in vitro.

From 20 to 24 days in vitro, immuno staining of myelin markers, such as myelin basic protein, allowed visualization of the myelin segments and nodes of Ranvier. Core PBS is important for meninges removal. The brisk clearing is critical for oligodendroctyes viability and realizing the strengths of the flow applied with the pipette.

Oligodendrocyte-conditioned medium can be added to neuronal cultures to gain insight into the impact of oligodendroctyes secreted factors on neuronal physiology. Co-cultures of oligodendroctyes with neurons can also be performed to study the myelination process.