Home | Technical Information | Protocols | Stem Cell Protocols | Rat Cortical Stem Cell Culture Protocol

Rat Cortical Stem Cell Culture Protocol

This protocol must be read in its entirety before using this product.

Contents

  • Expansion Protocols using Monolayer System
  • Expansion Protocols using Neurosphere System
  • Thawing of Cryopreserved Cells
  • References
  • Sample Data

EXPANSION PROTOCOL USING MONOLAYER SYSTEM

Supplies Required

Materials

  • 10 cm tissue culture dishes (Falcon, Catalog # 353003 or equivalent)
  • 50 mL Falcon tubes (Falcon, Catalog # 2070, Fisher, Catalog # 05-539-7, or equivalent)
  • 0.2 µm, 1000 mL filter unit (Nalgene, VWR Catalog # 28198-496 or equivalent)
  • Plastic cell scraper (Costar cell lifter, Catalog # 3008 or equivalent)
  • Pipettes and pipette tips

Reagents

  • StemXVivo™ Serum-Free NSC Base Media (R&D Systems, Catalog # CCM002)
  • Recombinant Human Fibroblast Growth Factor basic (FGF basic) (R&D Systems, Catalog # 233 FB/CF)
  • Purified Bovine Fibronectin (R&D Systems, Catalog # 1030-FN)
  • PBS (Invitrogen, Catalog # 10010-023 or equivalent)
  • Penicillin-Streptomycin, 100X (Invitrogen, Catalog # 15140-148 or equivalent)
  • Poly-L-ornithine (Sigma, Catalog # P3655 or equivalent)
  • Hank's Balanced Salt Solution (HBSS) (Ca2+/Mg2+-free), 10X (Invitrogen, Catalog # 14185 052 or equivalent)
  • HEPES (Sigma, Catalog # H7006 or equivalent)
  • BSA, very low endotoxin (Serologicals Proteins Inc., Catalog # 81-068-3 or equivalent)
  • Trypan blue (Invitrogen, Catalog # 15250-061)
  • Deionized water

Equipment

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

Reagent and Material Preparation

NSC Base Media - Thaw the StemXVivo Serum-Free NSC Base Media at 2 - 8° C or room temperature. Aliquot any remaining thawed medium and store at -20° C. Use within 10 days when stored in the dark at 2 - 8° C.

Completed NSC Base Media - Add Penicillin-Streptomycin (100X) to the NSC Base Media at a 1:100 dilution.

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.

Procedure Preparation

Poly-L-ornithine and Fibronectin Coated Plates

  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 each time.
  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. 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.
  7. Discard the PBS from each 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.

Procedure

Use serological pipettes to transfer and remove solutions.

Expansion

  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 and Fibronectin coated plate.
  2. Incubate the cells at 37° C and 5% CO2. Cells should become adherent after 24 hours.
  3. After 24 hours, add 10 µL of 1000X FGF basic stock to the culture.
  4. Every second day, replace the medium with fresh Completed NSC Base Media.
  5. Supplement the medium with 20 ng/mL of FGF basic each day.
  6. Passage the cells when they reach 60 - 70% confluency (approximately 4 days after initial plating) according to the procedure described below.

Passage

  1. Warm the Buffered HBSS (1X) and Completed NSC Base Media supplemented with FGF basic to 37° C.
  2. Remove the medium 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 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 Falcon tube.
  5. Centrifuge for 5 minutes at 200 x g. Remove the supernate.
  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 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 Base Media containing FGF basic on a Poly L ornithine/Fibronectin coated plate.
  9. 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% confluence.

EXPANSION PROTOCOL USING NEUROSPHERE SYSTEM

Supplies Required

Materials

  • 6-well plates (Costar, Catalog # 3516 or equivalent)
  • 15 mL tubes (Corning, Catalog # 430052 or equivalent)
  • Pipettes and pipette tips

Reagents

  • StemXVivo Serum-Free NSC Base Media (R&D Systems, Catalog # CCM002)
  • Recombinant Human Fibroblast Growth Factor basic (FGF basic) (R&D Systems, Catalog # 233 FB)
  • Recombinant Human Epidermal Growth Factor (EGF) (R&D Systems, Catalog # 236 EG)
  • PBS (Invitrogen, Catalog # 10010-023 or equivalent)
  • Penicillin-Streptomycin, 100X (Invitrogen, Catalog # 15140-148 or equivalent)
  • BSA, very low endotoxin (Serologicals Proteins Inc., Catalog # 81-068-3 or equivalent)
  • Acetic acid (J.T. Baker, Catalog # 9508-03 or equivalent)
  • Trypan blue (Invitrogen, Catalog # 15250-061)
  • Deionized water

Equipment

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

Reagent and Material Preparation

NSC Base Media - Thaw the StemXVivo Serum-Free NSC Base Media at 2 - 8° C or room temperature. Aliquot any remaining thawed medium and store at -20° C. Use within 10 days when stored in the dark at 2 - 8° C.

Completed NSC Base Media - Add Penicillin-Streptomycin (100X) into the NSC Base Media at a 1:100 dilution.

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.

Procedure

Use serological pipettes to transfer and remove solutions.

Expansion
  1. Seed approximately 1 x 105 NSCs in 5 mL of Completed NSC Base Media supplemented with 20 ng/mL of EGF and 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 EGF (20 ng/mL) and FGF basic (20 ng/mL) each day to the medium. Every fourth day, based on the number of neurospheres, replace the media according to the steps described below.
    1. > 50 neurospheres - Transfer the medium 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 EGF (20 ng/mL) and FGF basic (20 ng/mL). Add the neurosphere suspension to 5 mL of fresh Completed Base Media containing EGF (20 ng/mL) and FGF basic (20 ng/mL) in one well of a 6-well plate.
    2. < 50 neurospheres - Transfer the neurospheres, using a Pasteur pipette, directly into 2.5 mL of Completed NSC Base Media containing EGF (20 ng/mL) and FGF basic (20 ng/mL) in one well of a 6-well plate. DO NOT DISCARD THE CONDITIONED MEDIUM. Add 2.5 mL of this conditioned medium to the well. When there are fewer neurospheres, conditioned medium is required. Only half of the medium is replaced with fresh Completed NSC Base Media containing EGF (20 ng/mL) and FGF basic (20 ng/mL).
  4. Pass the cells at 5 - 7 days, or when the neurospheres have a dark clump inside or ruffling on the outside of the neurosphere, according to the procedure described below.
Passage
  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.

THAWING OF CRYOPRESERVED CELLS

Review the following protocol in detail before thawing the cells.

  1. Warm 30 mL of Completed NSC Base Media (refer to page 5) containing the desired mitogen (e.g. FGF basic and/or EGF) in a 37° C water bath.
  2. Add 20 mL of pre-warmed Completed Base NSC Media with desired mitogen 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 the desired mitogen.
    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.

REFERENCES

  1. Johe, K.K. et al. (1996) Genes and Development 10:3129.
  2. Kim, J.H. et al. (2003) Methods Enzymol. 365:303

SAMPLE DATA

Figure 1: Rat cortical stem cells cultured using the monolayer system (A) or the neurosphere system (B).
Figure 2: Expansion of rat cortical stem cells using the monolayer system (A) or the neurosphere system (B).