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 # 3718-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
- Culture Dish Preparation
- 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.
- Dilute the 1000x Poly-L-Ornithine Stock 1000-fold in sterile PBS to make a 15 µg/mL (1x) solution. Prepare fresh as needed.
- Add 10 mL of the (1x) Poly-L-Ornithine solution to each 10 cm tissue culture dish. Incubate overnight at 37° C.
- Discard the Poly-L-Ornithine solution. Wash each dish 3 times with 10 mL of PBS.
- Add 10 mL of PBS to each dish. Incubate overnight at 37 °C.
- 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.
- Discard the PBS from each Poly-L-Ornithine-coated dish. Wash each dish once with 10 mL of PBS.
- Add 10 mL of 1 µg/mL bovine Fibronectin solution to each dish. Incubate at 37 °C for 3 - 30 hours.
- Discard the bovine Fibronectin solution. Wash each dish once with 10 mL of PBS before use.
- Thawing Cryopreserved Rat Cortical Stem Cells (Review the following section in detail before thawing the cells)
- Warm 30 mL of Completed NSC Base Media supplemented with 20 ng/mL FGF basic in a 37 °C water bath.
- 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.
- 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. - Mix 10 µL of the cell suspension with 10 µL of 0.4% Trypan blue and count the live cells on a hemocytometer.
- Seed cells at a density according to the expansion protocol described below.
- Cell Expansion (Figure 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.
- 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).
- After 24 hours, add 10 µL of FGF basic stock (1000x) to the culture.
- Every second day, replace the media with fresh Completed NSC Base Media.
- Supplement the media with FGF basic (to 20 ng/mL) each day.
- Passage the cells according to the procedure described below when they reach 60 - 70% confluency (approximately 4 days after initial plating).
Figure 1.
- Passaging Cells (Figure 2)
- Warm the buffered HBSS (1x) and Completed NSC Base Media supplemented with FGF basic (20 ng/mL) to 37° C.
- Remove the media from the cells. Wash once in 10 mL of buffered HBSS (1x).
- Add 5 mL of buffered HBSS (1x). Incubate at room temperature for 15 - 45 minutes until the cells round up (check frequently).
- Scrape the cells from the plate with a hard plastic cell scraper. Transfer the cells to a 50 mL centrifuge tube.
- Centrifuge for 5 minutes at 200 x g and remove the supernatant.
- 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.
- Mix 10 µL of the cell suspension with 10 µL of 0.4% Trypan blue and count the live cells on a hemocytometer.
- 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.
- 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
