This article details a method for isolating and culturing neural stem cells from the cerebellum of newborn mice. The process involves using magnetic microbeads to selectively bind stem cells, followed by techniques to promote their proliferation and differentiation into neurons and glia.
Take cells isolated from a newborn mouse cerebellum containing stem cells, neurons, and glia.
Add antibody-coated magnetic microbeads that bind to a stem-cell-specific surface glycoprotein.
Centrifuge, remove the unbound beads, and resuspend the cells in a buffer.
Load the cells onto a separation column placed in a magnetic separator, which retains the microbead-bound stem cells while other cells flow through.
Add a neurosphere medium to elute the cells and transfer them into a microplate. Incubate to induce the formation of primary neurospheres, self-renewing colonies of stem cells.
Centrifuge and discard the medium. Resuspend the neurospheres in a medium containing enzymes to digest the cellular adhesion proteins.
Centrifuge and remove the enzymes. Add the neurosphere medium and mechanically dissociate the cells.
Incubate to allow stem cell proliferation and secondary neurosphere formation, while the differentiated cells lacking stem cell characteristics do not proliferate.
Transfer the neurospheres to a differentiation medium and incubate, promoting differentiation into neurons and glia.
Place the centrifuge tubes on ice, then, strain the dissociated cells through a 40-micrometer cell strainer into a 50-milliliter tube. Top the filter with 10 milliliters of ice-cold HBSS solution, to ensure that the cells pass through the mesh.
Transfer the filtered cells to a fresh 15-milliliter centrifuge tube, and centrifuge the suspension at 300 times g for 10 minutes at 4 degrees Celsius. Then, use a vacuum aspirator to remove the supernatant completely.
Resuspend the cell pellet in 160 microliters of ice-cold magnetic column buffer. Add 40 microliters of anti-prominin-1 microbeads to the cell suspension. Then, incubate the tubes in a refrigerator for 15 minutes, to allow the antibody to bind to the prominin-1-expressing cells.
Wash the cells with 1 to 2 milliliters of column buffer X, and centrifuge them at 300 times g for 10 minutes. Aspirate the supernatant and resuspend the pellet in 1 milliliter of column buffer X. Place the magnetic columns on the magnetic stand, and rinse them once with 500 microliters of buffer X. Apply the labeled cell suspension onto the column, and collect the flow-through in fresh 15-milliliter tubes.
Wash the column three times with 500 microliters of buffer X. Then, remove the columns from the magnetic field and place them in 1.5-milliliter tubes. Add 1 milliliter of culture medium, and push the plunger into the column to flush out the cells tagged with prominin-1 beads.
To passage the neurospheres, transfer them along with the culture medium into a sterile 15-milliliter centrifuge tube. Pellet the neurospheres by centrifugation at 300 times g for 5 minutes, and discard the supernatant.
Resuspend the pellet in 5 milliliters of dissociation media with papain or 0.05% trypsin solution, and incubate the cells at 37 degrees Celsius for 10 minutes. Centrifuge the cell suspension again. Then, resuspend the cells in 5 milliliters of neurosphere medium, and dissociate them by slowly pipetting them up and down 10 times with a pasture pipette. Plate the cells in neurosphere media as described in the text protocol, and enrich them with secondary neurospheres after 7 to 10 days in culture as previously described.
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