Culturing Mouse Cortical Stem Cells: Expansion using the Neurosphere System

This protocol must be read in its entirety before using Catalog # NSC002 and AR003.

Caution: The mouse cortical stem cells used in this protocol contain trace amounts of human transferrin and DMSO, and the media used in this protocol contains trace amounts of transferrin. The transferrin was tested at the donor level using an FDA licensed method and found to be non-reactive for anti HIV-1/2 and Hepatitis B surface antigen. Because no testing can offer complete assurance of freedom from infectious agents, these reagents should be handled as if capable of transmitting infection.

Supplies Required

Reagents

  • Mouse Cortical Stem Cells (R&D Systems, Catalog # NSC002)
  • N-2 Plus Media Supplement (R&D Systems, Catalog # AR003)
  • Recombinant Human Fibroblast Growth Factor basic (FGF basic) (R&D Systems, Catalog # 233-FB or 4114-TC)
  • Recombinant Human Epidermal Growth Factor (EGF) (R&D Systems, 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

Reagent & Media Preparation

Note: Sterile technique is required when handling the reagents.

  • Completed NSC Base Media - Mix the components listed in the chart below with DI water to make 500 mL of Completed NSC Base Media. Adjust the pH to 7.2 ± 0.2. Sterile filter the solution using a 0.2 µm filter unit and store in the dark at 2-8° C for up to 2 weeks.
    Component Amount
    DMEM/F12 6 g
    Glucose 0.775 g
    Glutamine 0.0365 g
    NaHCO3 0.845 g
    N-2 Plus Media Supplement 5 mL
  • FGF basic Stock (1000X) - Add sterile 0.1% BSA in PBS to the Human FGF basic vial to make a 20 µg/mL stock. Aliquot and store at < -20° C in a manual defrost freezer for up to 6 months. Avoid repeated freeze-thaw cycles.
  • EGF Stock (1000X) - Add sterile 0.1% BSA in 10 mM acetic acid to the Human EGF vial to make a 20 µg/mL stock. Aliquot and store at < -20° C in a manual defrost freezer for up to 6 months. Avoid repeated freeze-thaw cycles.

Thawing of Cryopreserved Cells

Review the following protocol in detail before thawing the cells.

  1. Warm 30 mL of Completed NSC Base Media containing FGF basic and EGF in a 37° C water bath.
  2. Add 20 mL of pre-warmed Completed NSC Base Media with mitogens to a 50 mL tube. Reserve the remaining 10 mL pre-warmed Completed NSC Base Media for step # 5.
  3. Remove the cryovial containing frozen mouse cortical stem cells (Catalog # NSC002) from the liquid nitrogen. Using a 2 mL pipette, immediately add 1 mL of fresh pre-warmed media to the vial by gently pipetting up and down. As cells begin to thaw, transfer the thawed portion into the pre-warmed media in the 50 mL tube. Repeat this process with the warmed media until all of the cells have thawed.
    Note: Most of the frozen cells will be at the bottom of the cryovial.
  4. Centrifuge the cells at 200 x g for 5 minutes.
  5. Aspirate off 95% of the supernatant carefully and resuspend by gently pipetting the cell pellet up and down with 10 mL of Completed NSC Base Media with mitogens.
    Note: Rapid resuspension of frozen cells in warmed media during thawing is critical. Allowing cells to thaw slowly in the DMSO will dramatically reduce viability. Around 90% cell viability is expected from the freshly thawed cells when the appropriate thawing procedure is followed.
  6. Seed cells at a density according to the appropriate expansion protocol.

Procedure

Use serological pipettes to transfer and remove solutions.

Expansion

  1. Seed approximately 2 x 105 mouse cortical stem cells in 5 mL of Completed NSC Base Media supplemented with 20 ng/mL of EGF and 20 ng/mL of FGF basic per well in a 6-well plate.
  2. Incubate the cells at 37° C and 5% CO2.
  3. Add fresh EGF (20 ng/mL) and FGF basic (20 ng/mL) each day to the medium. Every fourth day, based on the number of neurospheres, replace the media according to the steps described below.
    1. a) 50 neurospheres or more - Transfer the medium containing the neurospheres to a 15 mL tube. Centrifuge for 5 minutes at 100 x g and remove the media (gently resuspend the pellet using a small quantity of fresh Completed NSC Base Media containing EGF (20 ng/mL) and FGF basic (20 ng/mL). Add the neurosphere suspension to 5 mL of fresh Completed Base Media containing EGF (20 ng/mL) and FGF basic (20 ng/mL) in one well of a 6-well plate.
    2. b) Less than 50 neurospheres - Transfer the neurospheres, using a Pasteur pipette, directly into 2.5 mL of Completed NSC Base Media containing EGF (20 ng/mL) and FGF basic (20 ng/mL) in one well of a 6-well plate. Do not discard the conditioned medium. Add 2.5 mL of this conditioned medium to the well. When there are fewer neurospheres, conditioned medium is required. Only half of the medium is replaced with fresh Completed NSC Base Media containing EGF (20 ng/mL) and FGF basic (20 ng/mL).
  4. Pass the cells at 5 - 7 days, or when the neurospheres have a dark clump inside or ruffling on the outside of the neurosphere, according to the procedure described below.

Passage

  1. Transfer the media containing the floating neurospheres to a 15 mL tube. Do not dislodge attached neurospheres for passage.
  2. Centrifuge for 5 minutes at 100 x g.
  3. Partially dissociate the neurospheres by pipetting up and down 20 times, being careful not to create bubbles in the suspension.
    Note: For optimal dissociation of the neurospheres, it is recommended that a P200 pipette be used.
  4. At passages 1 and 2, the cells should be split 1:1. After passage 2, the cells can be split 1:2.

Sample Data


Figure 1
Figure 2: Passage 3 mouse cortical stem cells cultures as neurospheres stained with PE-conjugated mouse anti-rat Nestin (Catalog # IC2736P; filled histogram) or with isotype control antibody (Catalog # IC003P; open histogram.

References

  1. Johe, K.K. et al. (1996) Genes and Development 10:3129.
  2. Kim, J.H. et al. (2003) Methods Enzymol. 365:303.
  3. Tropepe, V. et al. (1999) Dev. Biol. 208:166.
  4. T.J. Kilpatrick and P.F. Bartlett (1993) Neuron 10:255.