Isolation and Culturing of Adult Neural Stem Cells from the Mouse Subcallosal Zone

Begin with a mouse brain on a chilled brain matrix.

Obtain sections containing the subcallosal zone or SCZ. Transfer the section to a cold phosphate buffer.

Under a dissecting microscope, cut out the SCZ portion.

Chop the dissected SCZ into smaller pieces.

Transfer these pieces into a digestion buffer. Incubate to dissociate the cells from the tissue.

Centrifuge and discard the supernatant. Resuspend the cells in a suitable medium.

Transfer the cells to a culture plate and add specific growth factors.

The adult neural stem cells divide, forming clusters called neurospheres.

Improper nutrient distribution in cell clusters limits cell growth.

To disperse the clusters, centrifuge and discard the supernatant. Resuspend the clusters in a dissociation buffer and incubate.

Pipette the solution up and down to generate single cells.

Centrifuge and discard the supernatant.

Resuspend the cells and transfer them into a suitable medium to obtain a monolayer.

Once dissected, transfer the mouse brain to cold PBS buffer, and rinse it twice to remove excess blood. Next, place the brain in the brain matrix on ice, and make coronal cuts to obtain one-millimeter-thick slices. Subsequently, transfer the posterior brain slices containing SCZ regions to cold PBS.

Under a dissecting microscope with low magnification, microdissect the SCZ from the white matter area of the cortex and the hippocampus with a bent 30-gauge needle. Then, remove the cortex regions above the SCZ, and place the dissected SCZ into cold PBS in a 35-millimeter Petri dish on ice.

The region of subcallosal zone can be dissected precisely from the whole tissue by using brain matrix and bent 30-gauge needle.

Following that, remove the PBS, and then, immediately chop the dissected tissues into small pieces. Resuspend them with one milliliter of digestion buffer. Afterward, transfer the sample to a 15-milliliter tube containing two milliliters of digestion buffer. Then, incubate it for 30 minutes in a 37 degrees Celsius water bath.

In this step, tap the tube mildly to dissociate the digested tissue, and then centrifuge the tube at 145 times g for five minutes. After five minutes, discard the supernatant, resuspend the digested tissue with one milliliter of pre-warmed and two medium to wash out the digestion buffer, and gently pipette the sample solution a maximum of five times.

Next, centrifuge the sample again at 145 times g for five minutes. After discarding the supernatant, resuspend the cell pellet in one milliliter of N2 medium. Place one milliliter of N2 medium in a non-coated six-well plate, and add one milliliter of the suspended cells to make a final volume of two milliliters.

Next, add 20 nanograms per milliliter EGF and 20 nanograms per milliliter bFGF into each well. Gently shake the six-well culture dish by hand to mix the added growth factors with the plated cells. Then, keep the six-well plate in a 37 degrees Celsius and 5% carbon dioxide incubator.

Add 20 nanograms per milliliter EGF and 20 nanograms per milliliter bFGF to each well every day for eight days. Every third day, add 200 microliters of N2 media to maintain approximately two milliliters of the medium. Here is an image of the neurospheres after eight days.

In this procedure, gather the neurospheres, and transfer them into a new 15-millimeter conical tube. Centrifuge the tube at 145 times g for five minutes. Subsequently, discard the supernatant, and resuspend the neurospheres with 0.5 milliliters of dissociation buffer. Incubate them with 0.5 milliliters of dissociation buffer for five minutes in a 37 degrees Celsius water bath to dissociate the neurospheres into single cells.

Gently pipette the sample solution up and down less than five times, and centrifuge the tube at 145 times g for five minutes. Subsequently, discard the supernatant, and resuspend the neurospheres with one milliliter of N2 medium. After coating a six-well plate with PLL laminin, plate the cells at 2.5 x 10⁵ cells per milliliter with two milliliters of N2 medium per well. Maintain the SCZ adult neural stem cells with a daily treatment of growth factors for five days before passaging them.