Culturing Rat Fetal Neural Stem Cells

Rat neural stem cells (NSCs) serve as a well-established model for investigating human brain development, disease processes, and treatment strategies for debilitating central nervous system (CNS) disorders. This protocol describes the in vitro expansion, passaging, and morphology of rat fetal NSCs in adherent or neurosphere suspension cultures.

Required materials

Cells

• Gibco Rat Fetal Neural Stem Cells (Cat. No. N7744-100) or homogenous cell preparation from 14−18 days post-coitum rat brain tissue

Media and reagents

• Dulbecco’s Phosphate-Buffered Saline (D-PBS) (Cat. No. 14040)
• Dulbecco’s Phosphate-Buffered Saline (D-PBS) without calcium or magnesium (Cat. No. 14190)
• StemPro NSC SFM (Cat. No. A10509-01)
• StemPro Accutase Cell Dissociation Reagent (Cat. No. A11105-01)
• CELLstart CTS (Cat. No. A10142-01)
• Trypan blue (Cat. No. 15250) (included with the Countess Automated Cell Counter) or the LIVE/DEAD Cell Vitality Assay Kit (Cat. No. L34951)

Special tools

• Countess Automated Cell Counter (Cat. No. C10227) or hemacytometer

Medium for expanding neural stem cells

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 CTS as described below.

1. Dilute CELLstart CTS 1:100 in D-PBS with calcium and magnesium (e.g., 50 μL of CELLstart CTSinto 5 mL of D-PBS).
   Note:  CELLstart CTS should not be frozen, vortexed, 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 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 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 using the coated plates. Make sure the plates do not dry out.

Adherent cultures

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 CTS-coated culture vessels at a density of 5 × 10⁴ cells/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 (see Figure 1 below).
   
   
   Figure 1.  Rat fetal NSCs at passage 3 in adherent culture using StemPro NSC SFM.
   
   
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 (see Figure 2).
   
   
   Figure 2. Rat fetal NSCs at passage 3 in neurosphere culture using StemPro NSC SFM.
   
   
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 StemPro® Accutase 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 Countess 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.
    

Important: If you are re-feeding rat fetal NSCs 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.