Culturing Mouse Cortical Stem Cells: Expansion using the Monolayer System: R&D Systems

This protocol must be read in its entirety before using Catalog # NSC002 and AR003.

Caution: The mouse cortical stem cells used in this protocol contain trace amounts of human transferrin and DMSO, and the media used in this protocol contains trace amounts of transferrin. The transferrin was tested at the donor level using an FDA licensed method and found to be non-reactive for anti HIV-1/2 and Hepatitis B surface antigen. Because no testing can offer complete assurance of freedom from infectious agents, these reagents should be handled as if capable of transmitting infection.

Supplies Required

Reagents

Mouse Cortical Stem Cells (R&D Systems, Catalog # NSC002)
N-2 Plus Media Supplement (R&D Systems, Catalog # AR003)
Recombinant Human Fibroblast Growth Factor basic (FGF basic) (R&D Systems, Catalog # 3718-FB or 4114-TC)
Recombinant Human Epidermal Growth Factor (EGF) (R&D Systems, Catalog # 236-EG)
Purified Bovine Fibronectin (R&D Systems, Catalog # 1030-FN)
PBS
DMEM/F12
Glucose
Glutamine
NaHCO₃
Penicillin-Streptomycin, 100X
Poly-L-ornithine
Hank’s Balanced Salt Solution (HBSS) (Ca²⁺ /Mg²⁺ -free), 10X
HEPES
BSA, very low endotoxin
Acetic acid
Trypan blue
Deionized (DI) water

Materials

10 cm tissue culture dishes
15 mL tubes
50 mL Falcon tubes
0.2 µm, 1000 mL filter unit
0.2 µm, 500 mL filter unit
Plastic cell scraper
Pipettes and pipette tips

Equipment:

37° C and 5% CO₂ incubator
Centrifuge
Hemocytometer
Microscope
Water bath

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 Hanks Balanced Salt Solution (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. 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.
EGF Stock (1000X) - Add sterile 0.1% BSA in 10 mM acetic acid to the Human EGF 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.

Poly-L-ornithine and Fibronectin Coated Plates

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 and 5% CO₂.
Discard the Poly-L-ornithine solution. Wash each dish 3 times with 10 mL of PBS each time.
Add 10 mL of PBS to each dish. Incubate overnight at 37° C and 5% CO₂.
Allow the vial of Bovine Fibronectin to warm to room temperature. Do not agitate. 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 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 and 5% CO₂ for 3 - 30 hours.
Discard the Bovine Fibronectin solution. Wash each dish once with 10 mL of PBS before use.

Thawing of Cryopreserved Cells

Review the following protocol in detail before thawing the cells.

Warm 30 mL of Completed NSC Base Media containing FGF basic and EGF in a 37° C water bath.
Add 20 mL of pre-warmed Completed NSC Base Media with mitogens to a 50 mL tube. Reserve the remaining 10 mL pre-warmed Completed NSC Base Media for step # 5.
Remove the cryovial containing frozen mouse cortical stem cells (Catalog # NSC002) 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 Trypan blue and count the live cells on a hemocytometer.
Seed cells at a density according to the appropriate expansion protocol.

Procedure

Use serological pipettes to transfer and remove solutions.

Expansion

Seed 2.0 x 10⁶ mouse cortical stem cells in 10 mL of Completed NSC Base Media supplemented with 20 ng/mL each of FGF basic and EGF on a Poly-L-ornithine and Fibronectin Coated Plate.
Incubate the cells at 37° C and 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 1000X FGF basic stock and 10 µL of 1000X EGF stock to the culture.
Every second day, replace the medium with fresh Completed NSC Base Media.
Supplement the medium with 10 µL of 1000X stock (20 ng/mL) of both FGF basic and EGF each day.
Passage the cells when they reach 70 - 80% confluency according to the procedure described below.

Passage

Warm the Buffered HBSS (1X) and Completed NSC Base Media supplemented with FGF basic and EGF to 37° C.
Remove the Medium from the cells, and 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 cells round up (check frequently).
Dislodge as many of the cells as possible from the plate by pipetting. Any remaining cells can be scraped with a hard plastic cell scraper. Transfer the cells to a 15 mL conical tube.
Centrifuge for 5 minutes at 200 x g. Remove the supernate.
Resuspend the cells with 5 mL of Completed Base Media containing FGF basic and EGF 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 Trypan blue and count the live cells on a hemocytometer.
Seed 2.0 x 10⁶ viable cells in 10 mL of Completed Base Media containing FGF basic and EGF on a Poly L ornithine/Fibronectin Coated Plate.
Incubate the cells at 37° C and 5% CO₂. Repeat steps 4 and 5 in the Expansion section (see above). Passage the cells after 3 days or when cells reach 70 - 80% confluence.

Sample Data



Figure 1: A. Undifferentiated mouse cortical stem cells cultured on poly-L-ornithine and fibronectin coated dishes. B - D. Mouse cortical stem cells differentiated for 7 days. Neuronal lineage cells (B) were detected with mouse anti-neuron specific beta III tubulin (R&D Systems, Catalog # MAB1195) followed by NorthernLights™ 557-conjugated donkey anti-mouse IgG (R&D Systems, Catalog # NL007); astrocyte lineage cells (C) were detected with sheep anti-human GFAP (R&D Systems, Catalog # AF2594) followed by NorthernLights 557-conjugated donkey anti-sheep IgG (R&D Systems, Catalog # NL010); and oligodendrocyte lineage cells (D) were detected with mouse anti-oligodendrocyte marker O4 (R&D Systems, Catalog # MAB1326) followed by Rhodamine Red®-conjugated goat anti-mouse IgM. Image A is shown at 100X. Images B, C, and D are shown at 200X. Rhodamine Red is a trademark of Invitrogen.