CulturingBG01VHumanEmbryonicStemCellswithMouseEmbryonicFibroblast

If culturing in the absence of a feeder cell layer is desired, human embryonic stem (hES) cells can be maintained using Mouse or Human-Conditioned Media (Catalog # AR005, AR007). The protocol below has been used with the BG01V line of hES cells. Please note that other hES cell lines may require modifications of this protocol. Optimal culture conditions must be determined by the investigator for each hES line.

Please read the protocol in its entirety before starting.

Supplies Required

Reagents

• MEF-Conditioned Media (R&D Systems, Catalog # AR005) or Human Feeder Cell-Conditioned Media (R&D Systems, Catalog # AR007)
  

• Recombinant human FGF basic (R&D Systems, Catalog # 233-FB) or tissue culture grade FGF basic (R&D Systems, Catalog # 4114-TC)
  

• Accutase (Innovative Cell Technologies, Catalog # AT104 or equivalent)
  

• Cultrex? Reduced Growth Factor Basement Membrane Extract (BME) (R&D Systems, Catalog # 3433-005-01)
  

• DMEM/F12

Materials

• BG01V human embryonic stem cells (ATCC, Catalog # SCRC-2002)
  

• 60 or 100 mm tissue culture plates 
  

• 15 mL centrifuge tubes
  

• Pipettes and pipette tips

Equipment

• 37° C, 5% CO₂ incubator
  

• Centrifuge (low speed clinical or equivalent)
  

• Hemocytometer
  

• Microscope

Figure 1

Procedures

Note: When handling biohazard materials such as human cells, safe laboratory procedures should be followed and protective clothing should be worn.

Note: Sterile technique is required when handling the reagents.

1. Prepare the Cultrex BME-coated plate. (Figure 1)

1. Thaw Cultrex BME on ice at 2 - 8° C overnight.

2. Aliquot thawed Cultrex BME into pre-cooled tubes and store at -20° C.

3. Thaw the aliquot on ice or at 2 - 8° C overnight.

4. Dilute Cultrex BME 1:40 in DMEM/F12. This can be stored at 4° C for up to 2 weeks.

5. Coat the desired number of plates with diluted Cultrex BME (approximately 2.5 mL/60 mm plate) and incubate for 1-2 hours at room temperature.

6. Remove the Cultrex BME solution immediately prior to plating the cells.

2. Preparation and plating of BG01V Cells. (Figure 2)

   Figure 2
   
   

1. Warm the MEF or Human Feeder Cell-Conditioned Media to 37° C.

2. Warm the vial of BG01V hES cells until just thawed and then immediately transfer to a 15 mL centrifuge tube containing at least 5 mL of pre-warmed Conditioned Media. Rinse the cryovial with an additional 1 mL of media to ensure the removal of all the cells.

3. Spin in a clinical centrifuge at 200 x g for 4 minutes.

4. Remove the supernatant and gently flick the pellet. Resuspend the pellet in an appropriate amount of Conditioned Media supplemented with 4 ng/mL of recombinant human FGF basic.

5. Add the BG01V hES cell suspension to the Cultrex BME-coated plate.

6. Grow in a 37° C, 5% CO₂ incubator. Change the media daily and monitor the cells. Passage the cells at the desired confluency.

3. Passaging BG01V Cells (Figure 3)

   Figure 3
   
   

1. Prepare the desired number of plates by coating with Cultrex BME, as described above, 1 - 2 hours prior to passaging the cells.

2. Warm the MEF- or Human Feeder Cell-Conditioned Media to 37° C.

3. Remove the Conditioned Media from cells. Add 1 mL of Accutase solution to each 60 mm plate. Incubate at room temperature for 5 - 10 minutes or until cells begin to slough off the plate.

4. Pipette gently over the plate until all the cells have been detached.

5. Pipette the cell suspension up and down to break up large cell clumps.

6. Remove the cell suspension to a 15 mL centrifuge tube containing 5 mL of Conditioned Media and spin at 200 x g for 4 minutes.

7. Resuspend the pellet in Conditioned Media and count the viable cells using a hemocytometer.

8. Plate the desired number of cells (approximately 1.0 x 10⁶ cells/60 mm plate) on the Cultrex BME coated plate in Conditioned Media containing 4 ng/mL of recombinant human FGF basic.

9. Change the media daily. Monitor the cells for the desired confluency.

BG01V cells are licensed from BresaGen, Inc.
Cultrex is a registered trademark of Trevigen, Inc.