实验概要
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.
主要试剂
Dulbecco’s Phosphate-Buffered Saline (D-PBS)
Dulbecco’s Phosphate-Buffered Saline (D-PBS) without calcium or magnesium
StemPro® NSC SFM
StemPro® Accutase® Cell Dissociation Reagent
CELLstart™ CTS™
Trypan blue (included with the Countess®) or the LIVE/DEAD® Cell
Vitality Assay Kit
主要设备
Countess® Automated Cell Counter or hemacytometer
实验步骤
1. Preparing Media
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.
Table 1
Component | Final concentration | Amount |
KnockOut™ D-MEM/F-12 | 1X | 97 mL |
GlutaMAX™-I Supplement | 2 mM | 1 mL |
bFGF (prepared as 100 μg/mL stock) | 20 ng/mL | 20 μL |
EGF (prepared as 100 μg/mL stock) | 20 ng/mL | 20 μL |
StemPro® Neural Supplement | 2% | 2 mL |
2. Coating Culture Vessels with CELLstart™
1) Dilute CELLstart™ CTS™ 1:100 in D-PBS with calcium and magnesium (e.g., 50 μL of CELLstart™ CTS™ into 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.
3. Expanding and Passaging of Rat NSCs
1) Resuspend the rat fetal NSCs as follows:
a. 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.
b. 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.
Table 2
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.
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.
4. Neurosphere Suspension Cultures
1) Resuspend the rat fetal NSCs as follows:
a. 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.
b. 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.
Table 3
Vessel size | Growth area | Volume of media |
96-well plate | 0.32 cm2/well | 0.1 mL |
24-well plate | 1.9 cm2/well | 0.5 mL |
12-well plate | 3.8 cm2/well | 1 mL |
35-mm dish | 8 cm2/well | 2 mL |
6-well plate | 9.6 cm2/well | 2 mL |
60-mm dish | 19.5 cm2 | 5 mL |
T-25 flask | 25 cm2 | 5 mL |
100-mm dish | 55 cm2 | 10 mL |
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.
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.