Culturing Rat Cortical Stem Cells: Expansion using the Monolayer 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, as described here, or as neurospheres. 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 FGF basic (R&D Systems, Catalog # 233-FB)
  • Bovine Fibronectin (R&D Systems, Catalog # 1030-FN)
  • PBS
  • DMEM/F12
  • Glucose
  • Glutamine
  • NaHCO3
  • Penicillin-Streptomycin 100x
  • Poly-L-Ornithine
  • Ca2+/Mg2+-free Hank’s Balanced Salt Solution (HBSS) (10x)
  • HEPES
  • BSA, very low endotoxin
  • Trypan blue, 0.4%
  • Deionized (DI) water

Materials

  • 10 cm tissue culture plates
  • 50 mL centrifuge tubes
  • 0.2 µm, sterile filter units
  • Plastic cell scraper
  • Pipettes and pipette tips

Equipment

  • 37° C, 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
  • Buffered HBSS (1x) - Add 100 mL of HBSS (10x) and 3.9 g HEPES to 900 mL of deionized water to make 1000 mL of buffered HBSS 1x. Adjust the pH to 7.2 ± 0.2. Sterile filter the solution using a 0.2 µm filter unit and store at room temperature for up to 6 months.
  • 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. Culture Dish Preparation
    1. Dissolve Poly-L-Ornithine in sterile PBS to make a 15 mg/mL stock (1000x). Aliquot and store at -20° C in a manual de­frost freezer for up to 6 months. Avoid repeated freeze-thaw cycles.
    2. Dilute the 1000x Poly-L-Ornithine Stock 1000-fold in sterile PBS to make a 15 µg/mL (1x) solution. Prepare fresh as needed.
    3. Add 10 mL of the (1x) Poly-L-Ornithine solution to each 10 cm tissue culture dish. Incubate overnight at 37° C.
    4. Discard the Poly-L-Ornithine solution. Wash each dish 3 times with 10 mL of PBS.
    5. Add 10 mL of PBS to each dish. Incubate overnight at 37 °C.
    6. Allow the vial of bovine Fibronectin to warm to room temperature without agitation. Make a 1 µg/mL solution by pipetting the bovine Fibronectin into sterile PBS and gently inverting the tubes. Prepare fresh as needed.
    7. Discard the PBS from each Poly-L-Ornithine-coated dish. Wash each dish once with 10 mL of PBS.
    8. Add 10 mL of 1 µg/mL bovine Fibronectin solution to each dish. Incubate at 37 °C for 3 - 30 hours.
    9. Discard the bovine Fibronectin solution. Wash each dish once with 10 mL of PBS before use.
  2. 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 supplemented with 20 ng/mL FGF basic in a 37 °C water bath.
    2. Add 20 mL of pre-warmed Completed Base NSC Media and FGF basic 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. Mix 10 µL of the cell suspension with 10 µL of 0.4% Trypan blue and count the live cells on a hemocytometer.
    5. Seed cells at a density according to the expansion protocol described below.
  3. Cell Expansion (Figure 1)
    1. Seed 1 - 1.5 x 106 NSCs in 10 mL of Completed NSC Base Media supplemented with 20 ng/mL of FGF basic on a Poly-L-Ornithine/Fibronectin-coated 10 cm plate.
    2. Incubate the cells at 37 °C, 5% CO2. After cells become adherent (3 hours to overnight), replace the medium with fresh completed NSC Base Media supplemented with FGF basic (20 ng/mL).
    3. After 24 hours, add 10 µL of FGF basic stock (1000x) to the culture.
    4. Every second day, replace the media with fresh Completed NSC Base Media.
    5. Supplement the media with FGF basic (to 20 ng/mL) each day.
    6. Passage the cells according to the procedure described below when they reach 60 - 70% confluency (approximately 4 days after initial plating).

    Figure 1.

  4. Passaging Cells (Figure 2)
    1. Warm the buffered HBSS (1x) and Completed NSC Base Media supplemented with FGF basic (20 ng/mL) to 37° C.
    2. Remove the media from the cells. Wash once in 10 mL of buffered HBSS (1x).
    3. Add 5 mL of buffered HBSS (1x). Incubate at room temp­erature for 15 - 45 minutes until the cells round up (check frequently).
    4. Scrape the cells from the plate with a hard plastic cell scraper. Transfer the cells to a 50 mL centrifuge tube.
    5. Centrifuge for 5 minutes at 200 x g and remove the super­natant.
    6. Resuspend the cells with 5 mL of Completed Base Media containing FGF basic by slowly pipetting up and down approximately 5 times with a 5 mL pipette.
    7. Mix 10 µL of the cell suspension with 10 µL of 0.4% Trypan blue and count the live cells on a hemocytometer.
    8. Seed 0.8 - 1.0 x 106 viable cells in 10 mL of Completed NSC Base Media containing FGF basic on a Poly-L-Ornithine/Fibronectin-coated plate.
    9. Incubate the cells at 37 °C, 5% CO2. Repeat steps 4 and 5 in the Expansion section (see above). Passage the cells after 3 days or when cells reach 70% confluence.

    Figure 2