Complete Growth Medium
There are two options for the base medium for this cell line.
Option 1: NeuroCult NS-A Proliferation kit (Catalog No. 5751, Stem Cell Technologies)
Option 2: DMEM/F12 (1:1) (Catalog No. 30-2006, ATCC) with an additional 0.9% glucose, 4 mM L-glutamine (Catalog No. 30-2214, ATCC), 25 µg/mL insulin, 100 µg/mL transferrin, 20 nM progesterone, 15 µM putrescine and 30 nM selenite
To make the complete growth medium, add the following supplements to either options of the base medium (see above):
- 20ng/mL recombinant human Epidermal Growth Factor (EGF, Catalog No. 100-15, PeproTech)
- 100 ng/mL recombinant human Platelet-Derived Growth Factor-AA (PDGF-AA, Catalog No. 100-13A, PeproTech)
- 20 ng/mL recombinant human Fibroblast Growth Factor 2 (bFGF, Catalog No. ACS-3025, ATCC)
- 2 µg/mL heparan sulfate (Catalog No. H9902, Sigma)
The cells grow as phase-bright, smooth spheres. The neurospheres should not get too big, ragged or dark as this is a sign of unhealthy, dying cells. The cells should be passaged when the neurospheres are about 200-400 μm in size.
Volumes used in this protocol are for 75 cm2 flask.
- Harvest and collect the entire cell suspension from the culture flask into a 15 mL tube.
- Centrifuge at 200 x g for 10 minutes.
- Aspirate supernatant, leaving approximately 200 µL to cover the pellet.
- Add 1 mL of complete culture medium.
- Triturate cells with a P1000 micropipette set to 800 µL by pipetting up and down 40 times or until the cells appear to be in a single cell suspension.
- Add 8 mL of complete culture medium and centrifuge at 200 x g for 10 minutes.
- Aspirate supernatant and resuspend the cells in 2 mL of complete culture medium.
- Count viable cells using trypan blue exclusion assay on a hemacytometer.
- Seed single cells between ranges of 8 x 103 to 2 x 104 cells/cm2.
Note: If accurate cell count is necessary, Accumax (Catalog No. AM105, Innovative Cell Technologies) can be used; however, the cells may take some time to recover from an enzymatic dissociation.
Freeze medium: Complete growth medium (90%) supplemented with 10% (v/v) DM
Storage temperature: liquid nitrogen vapor phase