Culturing Human Neural Stem Cells

StemPro® NSC SFM Complete Medium

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.

Component	Final concentration	Amount
KnockOut™ D-MEM/F-12	1X	97 mL
GlutaMAX™-I Supplement	2 mM	2 mM
bFGF (prepared as 100 μg/mL stock)	20 ng/mL	20 ng/mL
EGF (prepared as 100 μg/mL stock)	20 ng/mL	20 μL
StemPro® Neural Supplement	2%	2 mL

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

For culture of adherent cultures, use either Fibronectin or CELLstart™ CTS™ to prepare a matrix for coating your plates.

Coating Culture Vessels with CELLstart™ CTS™

1. Dilute CELLstart™ CTS™ 1:100 in D-PBS with calcium and magnesium (i.e., 50 μL of CELLstart™ CTS™ into 5 mL of D-PBS). Note: CELLstart™ CTS™ should not be frozen, vortex or exposed to vigorous agitation due to potential gel formation.


2. Coat the surface of the culture vessel with the working solution of CELLstart™ CTS™ (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™ CTS™ solution immediately before use, and fill the vessel with complete StemPro® NSC SFM. Note: You may coat the plates in advance and store them at 4°C, wrapped tightly with Parafilm®, for up to 2 weeks. Do not remove CELLstart™ CTS™ solution until just prior to use. Make sure the plates do not dry out.

Coating Culture Vessels with Fibronectin

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 D-PBS 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.
   

Note: You may store the Fibronectin-treated plates at 4°C, wrapped tightly with Parafilm®, for up to 2 weeks. Ensure that plates do not dry out. There is no washing step needed and use the dish directly after aspiration.

Neural stem cell (NSCs) 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.

Thawing Frozen Neural Stem Cells

1. Prepare 10 mL of 1X KnockOut™ D-MEM/F-12 and warm to 37°C.


2. Transfer vial of frozen NSC from nitrogen tank to water bath. It is important to make the transfer immediately to prevent crystal formation.


3. Transfer thawed cells into a 15-mL tube and add warmed 1X KnockOut™ D-MEM/F-12 to 10 mL.


4. Spin down the thawed cells by centrifugation at 1,000 rpm for 4 minutes. Aspirate and discard the supernatant.


5. Resuspend the cells in StemPro® NSC SFM complete medium and plate on a CELLstart™ CTS™ or Fibronectin-coated plate at high density (1 × 10⁵ cells/cm²).

Note: Viability of thawed cells should be ~80% if they were frozen following the cryopreservation protocol described here.

Passaging Neural Stem Cells (Adherent Culture)

1. Aspirate the medium and wash with D-PBS without Ca²⁺ and Mg²⁺.


2. Add 1 mL of TrypLE™ Express or StemPro® Accutase® to the culture vessel. Note: The monolayer lifts off from the culture dish within 30 seconds of application of TrypLE™ Express or StemPro® Accutase®.


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™ Express or StemPro® Accutase®.


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™ CTS™- or Fibronectin-coated plate at a density of 1 × 10⁴-1 × 10⁵ cells/cm², or split the cells at a 1:4 ratio.

Passaging Neural Stem Cells (Suspension Culture)

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™ Express 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.

Cryopreserving Neural Stem Cells

1. Aspirate the medium and wash with D-PBS without Ca²⁺ and Mg²⁺.


2. Add 1 mL of TrypLE™ Express to the culture vessel.


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™ Express.


4. Spin down the cells by centrifugation at 1,200 rpm for 4 minutes. Aspirate the supernatant.


5. Resuspend the cells in StemPro® NSC SFM complete medium at a density of 2 × 10⁶ cells/mL.


6. Prepare freezing medium consisting of 20% DMSO and 80% medium. Note: Freezing medium (2X) can be prepared on the day of use and stored at 4°C until use.


7. Add a volume of freezing medium equal to the amount of StemPro® NSC SFM complete medium used to resuspend the cells in a drop-wise manner.


8. Prepare 1 mL aliquots (1 × 10⁶ cells) in cryovials and place the vials in an isopropanol chamber.


9. Put the isopropanol chamber at -80°C and transfer the vials to liquid nitrogen storage the next day.

Antibodies for NSC Characterization

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

Category	Antigen	Type	Working concentration
Neural Stem Cells	Sox1	Mouse IgG	1:200
	Sox2	Mouse IgG	2 μg/mL
	CD133	Rabbit IgG	1:100
Proliferation	Ki67	Rabbit IgG	1:50
	EdU	Chemical	1:1,000
Isotype Control	Mouse	IgM and IgG	Do not dilute
	Rabbit	IgG	Do not dilute
	Rat	IgM and IgG	1:50

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

Target	Primer	Sequence	Tm	Amplicon size	Intron size
Neural Stem Cells	Sox1-F	GCGGAAAGCGTTTTCTTG	53.0	406	No Intron
	Sox1-R	TGACTTCTCCTCCC	50.2	406	No Intron
	Sox2-F	ATGCACCGCTACGACGTGA	59.3	437	No Intron
	Sox2-R	CTTTTGCACCCCTCCCATTT	56.0	437	No Intron
	Nestin-F	CAGCGTTGGAACAGAGGTTGG	58.6	389	1142
	Nestin-R	TGGCACAGGTGTCTCAAGGGTAG	60.7	389	1142
Endogenous Control	ACTB-F	ACCATGGATGATGATATCGC	58.2	281	135
	ACTB-R	TCATTGTAGAAGGTGTGGTG	54.4	281	135