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

1. Preparation of Materials and Culture Medium

1. For the dissection and dissociation of the SCZ (Subcallosal zone), wrap the brain matrix, double-edged razor blade, and forceps with aluminum foil, and then sterilize them by autoclaving.
2. Prepare 50 ml of cold PBS (Phosphate-buffered saline) buffer to wash the whole mouse brain.
3. Set up a dissection microscope and prepare the surgical tools required for the dissection of the brain (autoclaved scissors and forceps) and the isolation of the SCZ (1 ml syringe, 30 G needle, brain matrix, and fine forceps).
4. Preparation of N2 Medium:
   1. Prepare F-12/DMEM (+L-glutamin, +sodium bicarbonate) medium with 2% B27, 1% N2 supplements, and 1% Penicillin-Streptomycin.
      Note: The growth medium consists of N2 medium and growth factors (20 ng/mL purified epidermal growth factor (EGF) and 20 ng/mL basic fibroblast growth factor (bFGF2)).
5. Prepare a digestion buffer (40 unit/ml papain, 2.4 unit/ml dispase II, and 2% penicillin-streptomycin in PBS) for tissue digestion.
6. Preparation of Coating Plate and Coverslip:
   1. Prepare poly-L-ornithine (PLO; 0.01%) and laminin (10 µg/ml dissolved in DH₂O). To coat 6-well plates for the maintenance of SCZ-aNSCs (Adult neural stem cells) as a monolayer or 18 mm coverslips for immunostaining, incubate them with PLO overnight at 4°C. Then wash them 3 times with DH₂O. Allow the plates and coverslips to dry after the last washing.
   2. Next, incubate the plates with laminin overnight at 4 °C. Then, wash them 3 times with DH₂O.
      Caution: Do not dry the laminin, which will affect cell attachment.
      Note: The coating solutions can be reused 3 times.

2. Isolation and Dissociation of the Adult SCZ

1. Prior to culture preparation, place the brain matrix and double-edged razor on ice.
   Note: Do not freeze the brain matrix, because the brain could attach to it.
2. Sacrifice a mouse (8 weeks old) by CO₂ asphyxiation or cervical dislocation.
   1. Cut off the head with sharp scissors after spraying 70% ethanol. To immobilize the head, hold both sides of the head tightly. Cut the skin with scissors at the midline in a caudal-rostral direction. This promotes the complete removal of the skin from the skull.
   2. Remove the caudal part of the skull (posterior to lambda) first, and then insert the forceps between the skull and brain at the dorsal midline position. Grab the left (or right) parietal bone and carefully peel it off. Repeat this procedure for the removal of the other parietal bone.
      Note: Disconnection of the optic nerve and removal of the meninges from the brain make it easier to detach the brain from the skull.
   3. Transfer the brain to cold PBS buffer (25 ml) and rinse it twice to remove excess blood.
3. Place the brain into the brain matrix on ice and make coronal cuts to obtain 1 mm thick slices. Transfer the brain slices (1 mm) containing the SCZ regions that are located in the posterior part of the brain to a cold PBS in a 35 mm plastic petri dish.
   Caution: In order to obtain a parallel plane of coronal sections, ensure that the brain fissure is placed in the midline of the brain matrix; it is critical to avoid unnecessary variations in the sections.
   Note: Obtain brain slices at 2-3 mm posterior to bregma; this allows the separation of the SCZ from the SVZ(subventricular zone).
4. Under the dissecting microscope with a low magnification, micro-dissect the SCZ from the white matter area of the cortex and hippocampus with a bent 30G needle6,9. Then, remove the cortex regions above the SCZ. Place the dissected SCZ region from the slices into a 35 mm plastic petri dish on ice without cold PBS.
   Note: Contamination of extra regions containing mature neurons could affect the viability of aNSCs and neurosphere formation because they undergo cell death in aNSC culture conditions.
5. Using a bent 30 G needle, immediately chop the dissected tissues into small pieces.
   Note: If a longer time is required for dissecting the SCZ tissue, immerse the dissected tissues in cold PBS before chopping.
6. Re-suspend the chopped tissue with 1 ml of digestion buffer, transfer the tissue to a 15 ml tube containing 2 ml of digestion buffer, and incubate it for 30 min in a 37°C water bath.
   Note: Shake the tube every 10 min to mix well.

3. Subcallosal Zone-derived Adult Neural Stem Cell Culture

1. Tap the tube mildly to dissociate the digested tissue, and then centrifuge the tube at 145 x g for 5 min. Discard the supernatant, re-suspended the digested tissue with 1 mL of pre-warmed N2 medium to wash out the digestion buffer, and gently pipet the sample solution a maximum of 5 times using a P1000 pipette.
   Note: Over-triturating with a narrow pipet tip can diminish cell viability and subsequent growth.
2. Centrifuge the tube at 145 x g for 5 min. After discarding the supernatant, re-suspend the cell pellet in 1 ml of N2 medium.
3. Prepare 1 ml of N2 medium in a non-coated 6-well plate and add 1 ml of the suspended cells to make a final volume of 2 ml.
4. Add EGF (20 ng/ml) and bFGF (20 ng/ml) into each well. Gently shake the 6-well culture dish by hand to mix the added growth factors with the plated cells. Keep the 6-well plate in a 37°C and 5% CO₂ incubator.
5. Add EGF (20 ng/ml) and bFGF (20 ng/ml) to each well every day for 8 days. Every third day, add 200 µl of N2 media to maintain the approximate 2 ml volume of the medium.

4. Passaging of NSCs as Neurospheres and to Monolayer Cultures

1. Gather the neurospheres and transfer them to a new 15 ml conical tube.
   Note: The number of neurospheres (>50 µm diameter) per well from the SCZ of a single mouse brain was 64.3 ± 7.31, which was less than that of the SVZ (190.5 ± 6.33).
2. Incubate the neurospheres with 0.5 ml of dissociation buffer for 5 min in a 37°C water bath to dissociate the neurospheres into single cells. Primary neurospheres can be dissociated into single cells and maintained over several passages as neurospheres or monolayer cultures.
   Note: Expanding SCZ-aNSCs as a monolayer is superior to neurospheres because SCZ-aNSCs can be passaged >10 times in a monolayer culture format but <5 times in a neurosphere culture format.
   Caution: SCZ-aNSCs exhibit strong aggregation, and it is difficult to dissociate them into single cells. Thus, the neurosphere culture format is not recommended for routine cell expansion.
3. Gently pipet the sample solution up and down with a P1000 pipette less than 5 times and centrifuge the tube at 145 x g for 5 min. Discard the supernatant and re-suspend the neurospheres with 1 ml of N2 medium.
   Note: Over-titurating with a narrow pipette tip can diminish cell viability and subsequent growth.
4. To count the cells, make a 1:1 mixture of the cell suspension (10 µl) and the 0.4% trypan blue solution, and then count the number of live cells on a hematocytometer. After coating a 6-well plate with PLO/laminin, plate the cells at 2.5 x 105 cells/ml with 2 ml of N2 medium for each well.
   Note: Changes in cell density can affect their condition and differentiation potential.
5. Maintain the SCZ-aNSCs with a daily treatment of growth factors (2 ml, 20 ng/ml) for 5 days, and then passage them.

5. Differentiation of Subcallosal Zone-derived Adult Neural Stem Cells

1. Plate aNSCs onto a PLO/Laminin-coated 18 mm coverslip with 1 x 105 cells/ml in 1 ml N2 with growth factors (20 ng/ml) for differentiation of the SCZ-aNSCs.
2. The next day, when the cells are firmly attached to the coverslip, exchange the growth medium with N2 to remove the growth factors.
3. After 6 days, wash the differentiated cells with 1 ml of PBS to remove cell debris and fix them for immunostaining.
   Note: BrdU can be incorporated into the newly synthesized DNA of proliferating cells. Therefore, if desired, BrdU (10 µg/ml) can be added to live cells before the fixation of cells.