Mouse Cortical Stem Cells (2 x 10e6 cells/vial)

Refer to the product datasheet for complete product details.

Briefly, Mouse Cortical Stem Cells can be thawed and expanded by the neurosphere or monolayer system using the following procedure:

• Thaw the Mouse Cortical Stem Cells
• Plate the cells in Completed NSC Base Media containing EGF and FGF basic
• Expand the cells using the monolayer or neurosphere system

View Graphic
Procedure

View Graphic
Procedure

 

Reagents Supplied in Mouse Cortical Stem Cells (Catalog # NSC002):

• 2 vials of Mouse Cortical Stem Cells containing 2 x 10⁶ cells

Expanding Mouse Cortical Stem Cells Using the Neurosphere System

Reagents

• Mouse Cortical Stem Cells (Catalog # NSC002)
• N-2 Plus Media Supplement (Catalog # AR003)
• Recombinant Human Fibroblast Growth Factor basic (FGF basic) (Catalog # 233-FB or 4114-TC)
• Recombinant Human Epidermal Growth Factor (EGF) (Catalog # 236-EG)
• PBS
• DMEM/F12

• Glucose
• Glutamine
• NaHCO₃
• Penicillin-Streptomycin, 100X
• BSA, very low endotoxin
• Acetic acid
• Trypan Blue
• Deionized water

Materials

• 10 cm tissue culture dishes
• 15 mL tubes
• 50 mL Falcon tubes
• 0.2 µm, 1000 mL filter unit

• 0.2 µm, 500 mL filter unit
• Plastic cell scraper
• Pipettes and pipette tips

Equipment

• 37 °C and 5% CO₂ incubator
• Centrifuge
• Hemocytometer

• Microscope
• Water bath

Expanding Mouse Cortical Stem Cells Using the Monolayer System

Reagents

• Mouse Cortical Stem Cells (Catalog # NSC002)
• N-2 Plus Media Supplement (Catalog # AR003)
• Recombinant Human Fibroblast Growth Factor basic (FGF basic) (Catalog # 233-FB or 4114-TC)
• Recombinant Human Epidermal Growth Factor (EGF) (Catalog # 236-EG)
• Purified Bovine Fibronectin (Catalog # 1030-FN)
• PBS
• DMEM/F12
• Glucose

• Glutamine
• NaHCO₃
• Poly-L-ornithine
• Hank’s Balanced Salt Solution (HBSS) (Ca²⁺/Mg²⁺-free), 10X
• HEPES
• BSA, very low endotoxin
• Acetic acid
• Trypan Blue
• Deionized (DI) water

Materials

• 10 cm tissue culture dishes
• 15 mL tubes
• 50 mL Falcon tubes
• Pipettes and pipette tips

• 0.2 µm, 1000 mL filter unit
• 0.2 µm, 500 mL filter unit
• Plastic cell scraper

Equipment

• 37 °C and 5% CO₂ incubator
• Centrifuge
• Hemocytometer

• Microscope
• Water bath

Thawing Cryopreserved Mouse Cortical Stem Cells

• Pipette up and down and as cells thaw, transfer the thawed portion into a 50 mL tube containing pre-warmed Completed Base NSC Media supplemented with 20 ng/mL EGF and 20 ng/mL FGF basic.
• Centrifuge the cells at 200 x g for 5 minutes.

 

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• Resuspend the cell pellet in 10 mL of Completed NSC Base Media containing EGF and FGF basic.

 

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Neurosphere Expansion

• Perform a cell count.

 

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• Plate cells at approximately 2.0 x 10⁵ NSCs in 5 mL of Completed NSC Base Media supplemented with EGF and FGF basic per well in a 6-well plate.
• Incubate the cells at 37 °C and 5% CO₂.
• Add fresh EGF and FGF basic daily to the media.

 

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• Replace the media every 4 days according to the number of neurospheres present:
  1. a. Less than 50 neurospheres:
     1. • Transfer the neurospheres into one well of a 6-well plate using 2.5 mL of fresh media and 2.5 mL of spent/conditioned media.
  2. b. More than 50 neurospheres:
     1. • Transfer the media containing the neurospheres to a 15 mL tube.
     2. • Centrifuge for 5 minutes at 100 x g and remove the media.
     3. • Resuspend the pellet using a small quantity of fresh Completed NSC Base Media containing EGF and FGF basic.
     4. • Add the neurosphere suspension to 5 mL of fresh Completed Base Media containing EGF and FGF basic in one well of a 6-well plate.
• Passage the cells at 5-7 days or when the neurospheres have a dark clump inside or ruffling on the outside.

 

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Passing Neurosphere

• Transfer the media containing the floating neurospheres to a 15 mL tube.
• Centrifuge for 5 minutes at 100 x g.

 

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• Partially dissociate the neurospheres by pipetting up and down 20 times with a P200 pipette.
• At passages 1 and 2 the cells should be split 1:1. After passage 2 the cells can be split 1:2.

 

1. Add 1 mL of fresh pre-warmed media to the vial of frozen mouse cortical stem cells
2. See Details

Thawing Cryopreserved Mouse Cortical Stem Cells

• Add 1 mL of fresh pre-warmed media to the vial of frozen mouse cortical stem cells.
• Pipette up and down and as cells thaw, transfer the thawed portion into a 50 mL tube containing pre-warmed Completed Base NSC Media supplemented with 20 ng/mL EFG and 20 ng/mL FGF basic.

 

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• Mix 10 μL of the cell suspension with 10 μL of 0.4% Trypan Blue
• Perform a cell count.

 

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Monolayer Expansion

• Plate 2.0 x 10⁶ NSCs in 10 mL of Completed NSC Base Media supplemented with EGF and FGF basic onto a Poly-L-ornithine/Fibronectin-coated 10 cm plate.
• Incubate the cells at 37 °C and 5% CO₂.

 

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• Replace the media once cells become adherent. After 24 hours, add 10 μL of FGF basic stock (1000X) and 10 μL of EGF stock (1000X) to the culture.
• Replace the media with fresh Completed NSC Base Media every second day.
• Supplement the media daily with EGF and FGF basic
• Passage the cells when they reach 70-80% confluency.

 

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Passaging Cells

• Wash the cells once with pre-warmed HBSS.
• Add 5 mL of HBSS.
• Incubate at room temperature until the cells round up.

 

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• Scrape the cells from the plate.
• Transfer the cells to a 15 mL centrifuge tube.
• Centrifuge for 5 minutes at 200 x g.
• Remove the supernatant.

 

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• Resuspend the cells in 5 mL of Completed NSC Base Media containing EGF and FGF basic

 

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• Mix 10 μL of the cell suspension with 10 μL of 0.4% Trypan Blue
• Perform a cell count.

 

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• Plate 2.0 x 10⁶ viable cells in 10 mL of Completed NSC Base Media containing EGF and FGF basic onto a Poly-L-ornithine/Fibronectin-coated plate
• Incubate the cells at 37 °C and 5% CO₂.

 

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• Replace the media with fresh Completed NSC Base Media every second day
• Supplement the media with EGF and FGF basic daily
• Passage the cells after 3 days or when the cells reach 70-80% confluency.

 

1. Coat cell culture plates with Poly-L-ornithine and Fibronectin.
2. See Details