實驗概要
Neural stem cells (NSC) are valuable resources 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. NSC are obtained by isolation from tissue, or differentiated from pluripotent cells. This page describes methods for expanding human NSC in cell culture and their subsequent characterization.
主要試劑
StemPro? NSC SFM
β-Mercaptoethanol
GlutaMAX?-I
CELLstart? CTS?
Fibronectin
Water, distilled
Dulbecco’s Phosphate-Buffered Saline (D-PBS) without Ca2 and Mg2
Dulbecco’s Phosphate-Buffered Saline (D-PBS)
StemPro? Accutase? Cell Dissociation Reagent
TrypLE? Express Stable Trypsin Replacement Enzyme
實驗材料
Human neural stem cells
實驗步驟
1. Preparing Matrix
1) Coating Culture Vessels with CELLstart? CTS?
a. 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.
b. 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).
c. Incubate the culture vessel at 37°C in a humidified atmosphere of 5% CO2 for 1 hour.
d. 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.
2) Coating Culture Vessels with Fibronectin
a. Dilute Fibronectin in distilled water to make a 1-mg/mL stock solution.
b. Store working solution at -20°C until use.
c. Add stock solution to D-PBS to make a working solution of 20 μg/mL.
d. 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).
e. Incubate the culture vessel at 37°C in a humidified atmosphere of 5% CO2 for 1 hour.
f. 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.
2. Culturing Neural Stem Cells
1) Thawing Frozen Neural Stem Cells
a. Aspirate the medium and wash with D-PBS without Ca2 and Mg2 .
b. ? Express or StemPro? Accutase?.
c. 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?.
d. Spin down the cells by centrifugation at 1,200 rpm for 4 minutes. Aspirate and discard the supernatant.
e. Resuspend the cells in StemPro? NSC SFM complete medium.
f. Count the cell number using a hemacytometer.
g. 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.
2) Passaging Neural Stem Cells (Adherent Culture)
a. Transfer medium containing neurospheres into a 15- or 50-mL conical tube.
b. 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.
c. Aspirate the supernatant carefully, and leave the neurospheres in a minimum volume of medium.
d. 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.
e. Add 1 mL of TrypLE? Express to the spheres and gently triturate neurospheres using a Pasteur pipette to create a single cell suspension.
f. Neutralize the treatment by adding 4 mL of medium.
g. Spin down the cells by centrifugation at 1,200 rpm for 4 minutes. Aspirate and discard the supernatant.
h. Resuspend the cells in StemPro? NSC SFM complete medium.
i. Count cell number using hemacytometer.
j. 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.
3) Passaging Neural Stem Cells (Suspension Culture)Cryopreserving Neural Stem Cells
a. Aspirate the medium and wash with D-PBS without Ca2 and Mg2 .
b. Add 1 mL of TrypLE? Express to the culture vessel.
c. 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.
d. Spin down the cells by centrifugation at 1,200 rpm for 4 minutes. Aspirate the supernatant.
e. Resuspend the cells in StemPro? NSC SFM complete medium at a density of 2 × 106 cells/mL.
f. 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.
g. 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.
h. Prepare 1 mL aliquots (1 × 106 cells) in cryovials and place the vials in an isopropanol chamber.
i. Put the isopropanol chamber at -80°C and transfer the vials to liquid nitrogen storage the next day.
