Neural stem cells (NSCs) are valuable because of their ability to differentiate into neurons and glial cells with applications in neuroscience and clinical use for treatment of neurodegenerative disease and neurological disorders. NSCs are obtained by isolation from tissue or differentiated from pluripotent cells. This protocol describes methods for expanding human or rat NSCs in cell culture and their subsequent characterization.

StemPro NSC SFM complete medium consists of KnockOut D-MEM/F-12 with StemPro Neural Supplement, bFGF, EGF, and GlutaMAX Supplement. Complete medium is stable for 4 weeks when stored in the dark at 2–8°C.

To prepare 100 mL of complete medium:

1. Reconstitute bFGF and EGF with 0.1% BSA solution (in KnockOut D-MEM/F-12) at a concentration of 100 μg/mL. You will need 20 μL of each per 100 mL of complete medium. Freeze unused portions in aliquots.
2. Mix the following components under aseptic conditions. For larger volumes, increase the component amounts proportionally.

For culture of adherent cultures, use either fibronectin protein or CELLstart substrate to prepare a matrix for coating your plates.

1. Dilute CELLstart substrate 1:100 in D-PBS with calcium and magnesium (i.e., 50 μL of CELLstart substrate into 5 mL of D-PBS).

2. Coat the surface of the culture vessel with the working solution of CELLstart substrate (14 mL for T-75, 7 mL for T-25, 3.5 mL for 60-mm dish, 2 mL for 35-mm dish).
3. Incubate the culture vessel at 37°C in a humidified atmosphere of 5% CO₂ for 1 hour.
4. Remove the vessel from the incubator and store it until use. Remove all CELLstart substrate immediately before use, and fill the vessel with complete StemPro NSC SFM.

1. Dilute fibronectin in distilled water to make a 1 mg/mL stock solution.
2. Store working solution at –20°C until use.
3. Add stock solution to DPBS to make a working solution of 20 μg/mL.
4. Add enough working solution to cover the surface of the culture vessel (10 mL for T-75, 2.5 mL for 60-mm dish, 1.5 mL for 35-mm dish).
5. Incubate the culture vessel at 37°C in a humidified atmosphere of 5% CO₂ for 1 hour.
6. Remove the vessel from the incubator and store it until use. Remove fibronectin solution immediately before use and fill the vessel with complete StemPro NSC SFM.

NSC populations can be expanded from frozen stocks and grown in StemPro NSC SFM complete medium as adherent cultures, or as suspension cultures. In either environment, change the spent culture medium every other day. When the cells in adherent culture reach >90% confluency, they are ready to be passaged. When the neurospheres in suspension culture become >3.5 mm in diameter, they are ready to be passaged. Cells cultured during expansion can be frozen down to create additional frozen stocks of higher passage number.

Read more about cryopreservation and recovery of neural cells

1. Aspirate the medium and wash with D-PBS without Ca²⁺ and Mg²⁺.
2. Add 1 mL of TrypLE Enzyme or Accutase reagent to the culture vessel.
   Note: The monolayer lifts off from the culture dish within 30 seconds of application of TrypLE Enzyme or Accutase reagent.
3. Gently pipette to loosen monolayer into a single cell suspension. Neutralize the treatment by adding 4 mL of medium. Do not treat the cells for longer than 3 minutes after addition of TrypLE Enzyme or Accutase reagent.
4. Spin down the cells by centrifugation at 1,200 rpm for 4 minutes. Aspirate and discard the supernatant.
5. Resuspend the cells in StemPro NSC SFM complete medium.
6. Count the cell number using a hemacytometer.
7. Plate cells in fresh medium on a CELLstart substrate- or fibronectin-coated plate at a density of 1 × 10⁴-1 × 10⁵ cells/cm² or split the cells at a 1:4 ratio.

1. Transfer medium containing neurospheres into a 15- or 50-mL conical tube.
2. Leave the tube at room temperature and allow the neurosphere to settle to the bottom of tube. Alternatively, spin down the cells by centrifugation at 500 rpm (200 × g) for 2 minutes.
3. Aspirate the supernatant carefully and leave the neurospheres in a minimum volume of medium.
4. Wash the neurospheres with 10 mL D-PBS without Ca²⁺ and Mg²⁺, aspirate the D-PBS supernatant carefully, and leave the neurospheres in a minimum volume of D-PBS.
5. Add 1 mL of TrypLE Enzyme to the spheres and gently triturate neurospheres using a Pasteur pipette to create a single cell suspension.
6. Neutralize the treatment by adding 4 mL of medium.
7. Spin down the cells by centrifugation at 1,200 rpm for 4 minutes. Aspirate and discard the supernatant.
8. Resuspend the cells in StemPro NSC SFM complete medium.
9. Count cell number using hemacytometer.
10. Seed the cells in fresh medium in a suspension dish (a non-coated flask can be used) at a density of 200,000 cells/mL.

Antibodies for NSC characterization

Use the antibodies listed in the following table to characterize NSCs by immunocytochemistry.

Use the primer sets listed in the following table to characterize NSC by PCR.

StemPro NSC SFM complete medium consists of KnockOut D-MEM/F-12 with StemPro Neural Supplement, bFGF, EGF, and GlutaMAX-I. Complete medium is stable for 4 weeks when stored in the dark at 2–8°C.

To prepare 100 mL of complete medium:

1. Reconstitute bFGF and EGF with 0.1% BSA solution (in KnockOut D-MEM/F-12) at a concentration of 100 μg/mL. You will need 20 μL of each per 100 mL of complete medium. Freeze unused portions in aliquots.
2. Mix the following components under aseptic conditions. For larger volumes, increase the component amounts proportionally.

You may observe a white precipitate when thawing StemPro Neural Supplement; this precipitate will disappear when the supplement is completely thawed or dissolved.

For adherent cultures, prepare plates with CELLstart substrate as described below.

1. Dilute CELLstart substrate 1:100 in D-PBS with calcium and magnesium (e.g., 50 μL of CELLstart substrate into 5 mL of D-PBS).

2. Coat the surface of the culture vessel with the working solution of CELLstart substrate (14 mL for a T-75 flask, 7 mL for a T-25 flask, 3.5 mL for a 60-mm dish, 2 mL for a 35‑mm dish).
3. Incubate the culture vessel at 37°C in a humidified atmosphere of 5% CO₂ for 1 hour.
4. Remove the vessel from the incubator and store at 4°C until use. Remove all CELLstart substrate solution immediately before use and fill the vessel with complete StemPro NSC SFM.

1. Resuspend the rat fetal NSCs as follows:
   • For freshly prepared rat fetal NSCs, after rinsing with D-PBS, resuspend in warmed complete StemPro NSC SFM at a density of 1 × 10⁷ viable cells/mL.
   • For thawed rat fetal NSCs, after determining the viable cell count, resuspend in warmed complete StemPro NSC SFM at a cell density of 1 × 10⁷ viable cells/mL.
2. Plate rat fetal NSCs onto CELLstart substrate-coated culture vessels at a density of 5 × 10⁴cells/cm². See the following table for recommended seeding densities for common culture vessels.
   
   

Vessel size	Growth area	Volume of media	No. of cells
96-well plate	0.32 cm2/well	0.1 mL	1.6 × 104
24-well plate	1.9 cm2/well	0.5 mL	1.0 × 105
12-well plate	3.8 cm2/well	1 mL	1.9 × 105
35-mm dish	8 cm2/well	2 mL	4.0 × 105
6-well plate	9.6 cm2/well	2 mL	4.8 × 105
60-mm dish	19.5 cm2	5 mL	9.8 × 105
T-25 flask	25 cm2	5 mL	1.3 × 106
100-mm dish	55 cm2	10 mL	2.8 × 106
T-75 flask	75 cm2	15 mL	3.8 × 106

3. Add the appropriate volume of cells to each culture vessel and incubate at 37°C, 5% CO₂, and 90% humidity.
4. Re-feed the rat fetal NSC cultures every 2−3 days with fresh complete StemPro NSC SFM. The morphology of rat fetal NSCs should exhibit short stellate-like processes with uniform density (Figure 1).

5. When cells reach 75–90% confluency (3–4 days after seeding), the rat fetal NSC cultures are ready to be passaged.
6. Rinse the culture vessel once with D-PBS without calcium and magnesium, then remove the medium.
7. Add pre-warmed StemPro Accutase and let the cells detach from the culture surface (within approximately 30 seconds).
8. After detachment, gently pipet the cells up and down to break the clumps into a uniform cell suspension and add four volumes of complete StemPro NSC SFM to the culture vessel.
9. Disperse the cells by pipetting over the culture surface several times to generate a homogenous cell solution.
10. Transfer the cells to a sterile centrifuge tube and centrifuge at 300 × g for 4 minutes at room temperature. Aspirate and discard the medium.
11. Resuspend the cell pellet in a minimal volume of pre-warmed complete StemPro NSC SFM and remove a sample for counting.
12. Determine the total number of cells and percent viability using trypan blue stain or the LIVE/DEAD Cell Vitality Assay Kit
13. Add enough complete StemPro NSC SFM to tube for a final cell solution of 1 × 10⁶ viable cells/mL. Incubate at 37°C, 5% CO₂ and 90% humidity. Rat fetal NSC cultures should not be maintained for more than 3 passages.

Important: If you are re-feeding rat fetal NSC in a growth medium other than complete StemPro NSC SFM, ensure that the medium is supplemented with 10 ng/mL bFGF to maintain the undifferentiated state of the rat fetal NSCs.

1. Resuspend the rat fetal NSCs as follows:
   • For freshly prepared rat fetal NSCs, after rinsing with D-PBS, resuspend in warmed complete StemPro NSC SFM at a cell density of 1 × 10⁷ viable cells/mL.
   • For thawed rat fetal NSCs, after determining the viable cell count, resuspend in warmed complete StemPro NSC SFM at a cell density of 1 × 10⁷ viable cells/mL.
2. Plate the rat fetal NSCs onto uncoated or low-attachment culture vessels at a density of 2 × 10⁵ viable cells/cm². See the table below for recommended seeding densities.



Vessel size	Growth area	Volume of media	No. of cells
96-well plate	0.32 cm2/well	0.1 mL	6.4 × 104
24-well plate	1.9 cm2/well	0.5 mL	3.8 × 105
12-well plate	3.8 cm2/well	1 mL	7.6 × 105
35-mm dish	8 cm2/well	2 mL	1.6 × 106
6-well plate	9.6 cm2/well	2 mL	1.9 × 106
60-mm dish	19.5 cm2	5 mL	3.9 × 106
T-25 flask	25 cm2	5 mL	5.0 × 106
100-mm dish	55 cm2	10 mL	1.1 × 107



3. Add the appropriate volume of cells to each culture vessel and incubate at 37°C, 5% CO₂, and 90% humidity.
4. Carefully re-feed the neurosphere suspension of rat fetal NSCs every 2−3 days with fresh complete StemPro NSC SFM without removing any developing neurospheres. The morphology of the neurospheres should exhibit spherical and transparent multicellular complexes (Figure 2).

5. When the neurospheres reach a diameter of 3.5 mm or larger, the rat fetal NSCs are ready to be passaged.
6. Transfer the neurosphere suspension into a sterile centrifuge tube and let the neurospheres settle by gravity or centrifuge at 200 × g for 2 minutes. Aspirate the supernatant carefully to leave the neurospheres in a minimal volume of medium.
7. Rinse the neurospheres once with D-PBS without calcium and magnesium and leave a minimal volume of D-PBS.
8. Add 1 mL of pre-warmed Accutase reagent to the neurospheres and incubate for 10 minutes at room temperature.
9. After incubation, gently pipette the cells up and down to get a single-cell suspension and add 4 mL of complete StemPro NSC SFM to the tube.
10. Centrifuge at 300 × g for 4 minutes at room temperature, carefully aspirate the supernatant, resuspend in a minimal volume of pre-warmed complete StemPro NSC SFM, and remove a sample for counting on a hemacytometer or automated cell counter.
11. Determine the total number of cells and percent viability.
12. Add enough complete StemPro NSC SFM to the tube for a final cell solution of 1 × 10⁷ viable cells/mL. Incubate at 37°C, 5% CO₂, and 90% humidity. Neurosphere suspension cultures should not be maintained for more than 3 passages.