Obtaining a Mixed Oligodendrocyte and Astrocyte Culture From Adult Mouse Neural Stem Cells

Take a coronal mouse brain slice.

Identify the lateral ventricles, the C-shaped cavities in the brain, and isolate the ventricle walls.

Place the tissue in a buffer containing trypsin to degrade the extracellular matrix, releasing the cells.

Centrifuge and remove the supernatant. Resuspend the cells in a medium.

Plate the cells and incubate them vertically to maintain them in suspension.

Add growth factors that induce neural stem cell proliferation and aggregation to form three-dimensional neurospheres.

Harvest the neurospheres, centrifuge, and discard the supernatant.

Add fresh medium and mechanically dissociate the neurospheres.

Plate the cells and add a medium containing growth factors that induce neural stem cell differentiation into oligodendrocyte precursors.

The precursors proliferate and form oligospheres.

Mechanically dissociate the oligospheres to release the precursors.

Seed the precursors in extracellular matrix protein-coated wells to facilitate cellular attachment.

Add a medium containing specific hormones and neurotrophic factors to induce precursor maturation into oligodendrocytes and astrocytes.

For neural stem cell isolation from the subventricular zone from 2.5-month-old adult mice, place brains from four to five adult mice into a 50-milliliter tube of ice-cold HBSS and place one brain ventral side down in the rostrocaudal direction on a sterile aluminum foil-covered flask filled with -20 degrees Celsius overnight cold water. Use a razor blade to remove the olfactory bulbs, and to cut two to three one-millimeter thick coronal slices 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 magnification, isolate the walls of the lateral ventricles, taking care not to include pieces of the corpus callosum, and place the isolated tissue in 5 to 10 milliliters of enzymatic dissociation buffer for a 15-minute incubation at 37 degrees Celsius.

At the end of the incubation, pipette the tissues at least 50 times before incubating the samples at 37 degrees Celsius for an additional 10 minutes. At the end of the incubation, neutralize the trypsin with 5 milliliters of standard culture medium and filter the tissue suspension through a 70-micron filter. Centrifuge the filtered solution for five minutes at 400 times g and resuspend the pellet in sucrose solution for a second centrifugation. At the end of the centrifugation, resuspend the pellet in bovine serum albumin washing solution for another centrifugation, then resuspend the pellet in standard culture medium for counting and plate the cells in an upright T25 or T45 flask.

For primary neurosphere differentiation, add basic fibroblast and endothelial growth factors to the neural stem cell culture every two days. When the neurospheres reach a 100 to 500-micron diameter, passage the cells by mechanical dissociation according to standard protocols.

For oligosphere differentiation, treat the cells with basic fibroblast growth factor and platelet-derived factor AA every two days. When the oligospheres reach a 100 to 150-micron diameter, dissociate the spheres by mechanical dissociation according to standard protocols, and seed the cell suspension at a 3,000 cell per square centimeter density onto Poly-D/L-Ornithine laminin coated plates. After three days, replace the supernatant of each culture with the same volume of oligodendrocyte differentiation medium.