Rat Cortical Stem Cells (3 x 10e6 cells/vial)

• 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 FGF basic.
• Centrifuge the cells at 200 x g for 5 minutes.

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

Neurosphere Expansion

• Perform a cell count.

• Plate cells at approximately 1.0 x 10⁶ NSCs in 5 mL of Completed NSC Base Media supplemented with FGF basic per well in a 6-well plate.
• Incubate the cells at 37 °C and 5% CO₂.
• Add fresh FGF basic to the media daily.

• 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 FGF basic.
     4. • Add the neurosphere suspension to 5 mL of fresh Completed Base Media containing FGF basic in one well of a 6-well plate.
• Passage the cells at 5-6 days or when the neurospheres have a dark clump inside or ruffling on the outside.

Passing Neurosphere

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

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