Culturing Rat Cortical Stem Cells: Expansion using the Neurosphere System

Ex vivo expanded neural stem cells serve as excellent tools for researchers studying neural development and neurological disorders. Ready-to-use primary cortical stem cells, isolated from E14.5 Sprague-Dawley rats (Catalog # NSC001), can be grown in monolayer or as neurospheres as described here. These cells retain capacity for multi-lineage differentiation into astrocytes, neurons, and oligodendrocytes.
Please read the protocol in its entirety before starting.

Supplies Required

Reagents

• N-2 Plus Media Supplement (R&D Systems, Catalog # AR003)
• Recombinant Human 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 (DI) water

Materials

• 6-well plates
• 15 mL centrifuge tubes
• Pasteur pipettes
• Pipettes and pipette tips
• 0.2 µm, sterile filter unit

Equipment

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

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.

Procedure

1. Thawing Cryopreserved Rat Cortical Stem Cells (Review the following section in detail before thawing the cells)
   1. Warm 30 mL of Completed NSC Base Media in a 37° C water bath.
   2. Add 20 mL of pre-warmed Completed Base NSC Media supplemented with FGF basic (20 ng/mL) to a 50 mL tube. Reserve the remaining 10 mL pre-warmed Completed Base NSC Media for step #5.
   3. Remove the cryovial containing frozen rat cortical stem cells 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 Base Media with growth factors.
      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 described below.
2. Neurosphere Expansion (Figure 1)
   1. Seed approximately 1 x 10⁵ NSCs in 5 mL of Completed NSC Base Media supplemented with 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 FGF basic (20 ng/mL) each day to the media. Every fourth day, based on the number of neurospheres, replace the media according to the steps described below.
      1. Less than 50 neurospheres - Transfer the neurospheres, using a Pasteur pipette, directly into 2.5 mL of Completed NSC Base Media containing and FGF basic (20 ng/mL) in one well of a 6-well plate. DO NOT DISCARD THE CONDITIONED Media. Add 2.5 mL of this conditioned media to the well. When there are fewer neurospheres, conditioned media is required. Only half of the media is replaced with fresh Completed NSC Base Media containing FGF basic (20 ng/mL).
      2. More than 50 neurospheres - Transfer the media 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 FGF basic (20 ng/mL). Add the neurosphere suspension to 5 mL of fresh Completed Base Media containing and FGF basic (20 ng/mL) in one well of a 6-well plate.
   4. Pass the cells according to the procedure described below at 5-7 days, or when the neurospheres have a dark clump inside or ruffling on the outside of the neurosphere.

   Figure 1

   
   
    
3. Passaging Neurospheres
   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 with a P200 pipette, being careful not to create bubbles in the suspension.
   4. At passages 1 and 2 the cells should be split 1:1. After passage 2 the cells can be split 1:2.