Rat Neural Stem Cell Expansion Protocol

This protocol describes the methods used to expand rat neural stem cells and is based on the use of the Rat Neural Stem Cell Expansion Medium (Millipore cat. no. SCM009), a two-component system that is convenient and easy to use. The Neural Stem Cell Basal Medium is a defined serum-free medium that has been optimized for the growth and in vitro differentiation of neural stem cells derived from rodents. A solution of basic FGF-2 growth factor is also provided separately and which, when added to the Neural Stem Cell Basal Medium (SCM003), allows for the growth and proliferation of rat neural stem cells. Withdrawal of FGF-2 from the Neural Stem Cell Basal Medium will result in the spontaneous differentiation of rat neural stem cells.

Material

Rat Neural Stem Cell Expansion Medium — Millipore cat. no. SCM003
Cryopreserved rat hippocampal neural stem cells — Millipore cat. no. SCR022
Neural Stem Cell Characterization Kit — Millipore cat. no. SCR019
Poly-L-ornithine — Sigma cat. no. P3655
Laminin — Sigma cat. no. L-2020
Accutase — Millipore cat. no. SCR005

Protocol

Preparation of Coated Plates

Note: Coating tissue culture plastic (or glasswares) that are used to culture rat neural stem cells with poly-L-ornithine and laminin is recommended.

Prepare stock solutions of poly-L-ornithine (10 mg/mL) by dissolving poly-L-ornithine in sterile water. The stock solution should be stored at –20°C or –80°C.
Dilute poly-L-ornithine with water from the stock concentration (10 mg/mL) to yield:
10 µg/mL for polystyrene plates
50 µg/mL for glass plates
Add enough of the poly-L-ornithine solution to cover the whole surface of the tissue cultureware. Use 5 mL volume for 6 cm plates and 10 mL volume for 10 cm plates and T75 flasks. Incubate overnight at room temperature.
The next day, rinse the tissue culture-wares with sterile water. Aspirate after each rinse.
Using sterile 1x PBS, dilute laminin to a final concentration of 5–7 µg/mL. Note: The same laminin concentration is used for both glass and polystyrene tissue culture-ware.
Add enough laminin (5–7 µg/mL) solution to the tissue culture-ware to cover the surface. Use 5 mL volume for 6 cm plates and 10 mL volume for 10 cm plates and T75 flasks. Incubate overnight at room temperature.
Coated plates and flasks can be stored in the laminin solution at –20°C for 6–8 months. The plates should be wrapped in plastic wrap before storage at –20°C .
Just before use, aspirate the laminin solution in the coated plates and wash the plates once with 1x PBS.

Thawing of Cells

Do not thaw the cells until the recommended medium and appropriately coated poly-Lornithine and laminin plasticware and/or glassware are on hand.
Remove the vial of rat neural stem cells (Millipore cat. no. SCR022) from liquid nitrogen and incubate in a 37°C water bath. Closely monitor until the cells are completely thawed. Maximum cell viability is dependent on the rapid and complete thawing of frozen cells. Important: Do not vortex the cells.
As soon as the cells are completely thawed, disinfect the outside of the vial with 70% ethanol. Proceed immediately to the next step.
In a laminar flow hood, use a 1 or 2 mL pipette to transfer the cells to a sterile 15 mL conical 300 tube. Be careful to not introduce any bubbles during the transfer process.
Using a 10 mL pipette, slowly add dropwise 9 mL of Neural Stem Cell Basal Medium (prewarmed at 37°C) to the 15 mL conical tube. Important: Do not add the whole volume of medium at once to the cells. This may result in decreased cell viability due to osmotic shock.
Gently mix the cell suspension by slow pipeting up and down twice. Be careful to not introduce any bubbles. Important: Do not vortex the cells.
Centrifuge the tube at 300 xg for 2–3 minutes to pellet the cells.
Decant as much of the supernatant as possible. Steps 4–8 are necessary to remove residual cryopreservative (DMSO).
Resuspend the cells in a total volume of 10 mL of Neural Stem Cell Basal Medium (prewarmed to 37°C) containing freshly added 20 ng/mL FGF-2.

Note:FGF-2 should always be added fresh to the Neural Stem Cell Basal Medium. To obtain a final concentration of 20 ng/mL FGF-2, add 2 µL of the FGF-2 stock to 10 mL of Neural Stem Cell Basal Medium.
Plate the cell mixture onto a poly-L-ornithine and laminin-coated 10 cm tissue culture plate.
Incubate the cells at 37°C in a 5% CO2 humidified incubator.
The next day, exchange the medium with fresh Neural Stem Cell Basal Medium (pre-warmed to 37°C) containing 20 ng/mL FGF-2. Exchange with fresh medium containing FGF-2 every other day thereafter.
When the cells are approximately 80% confluent, they can be dissociated with Accutase solution and passaged or alternatively frozen for later use.

Subculturing

Carefully remove the medium from the poly-Lornithine and laminin-coated 10 cm tissue culture plate containing the confluent layer of rat neural stem cells.
Apply 3–5 mL of Accutase solution and incubate in a 37°C incubator for 3 minutes.
Inspect the plate and ensure the complete detachment of cells by gently tapping the side of the plate with the palm of your hand.
Apply 5 mL of Neural Stem Cell Basal Medium (pre-warmed to 37°C) to the plate.
Transfer the dissociated cells to a 15 mL conical tube.
Centrifuge the tube at 300 xg for 2–3 minutes to pellet the cells.
Discard the supernatant.
Apply 2 mL of Neural Stem Cell Basal Medium containing 20 ng/mL FGF-2 to the conical tube and resuspend the cells thoroughly.
Count the number of cells using a hemocytometer.
Plate the cells to the desired density into the appropriate poly-L-ornithine and laminin-coated flasks, plates or wells in Neural Stem Cell Basal Medium containing 20 ng/mL FGF-2. Typically the cells are plated at ~2 million cells on poly-L-ornithine and laminin coated 10 cm plates or T75 flasks.

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