Culturing BG01V Human Embryonic Stem Cells with MEF-conditioned Medium

Introduction

Human embryonic stem (hES) cells are pluripotent stem cells derived from pre-implantation embryos that can be maintained and expanded in an undifferentiated state or induced to differentiate along somatic or germ cell lineages. They can be maintained either on a layer of mitotically inactivated feeder cells (1) such as irradiated mouse embryonic fibroblasts (iMEF, R&D Systems, Catalog # PSC001) or using mouse or human feeder cells conditioned medium (2) (R&D Systems, Catalog # AR005 or AR007, respectively). The protocol below has been used with the BG01V line of hES cells (3, 4).
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.

Materials Required

• Recombinant human FGF basic (R&D Systems, Catalog # 233-FB, 4114-TC, or equivalent)
• Accutase (Innovative Cell Technologies, Catalog # AT104 or equivalent)
• Cultrex® Basement Membrane Extract (BME) (R&D Systems, Catalog # 3433-005-01 or equivalent)
• DMEM/F12 (Invitrogen, Catalog # 12500-096 or equivalent)

• BG01V human embryonic stem cells
• Tissue culture dishes (60 mm; Fisher, Catalog # 08-772B, 100 mm; Fisher, Catalog # 08-772E, or equivalent)
• 15 mL conical tubes (Corning Costar, Catalog # 430052 or equivalent)
• 0.2 micrometer filter unit (Nalgene, VWR Catalog # 28198-496 or equivalent)
• Pipettes and pipette tips

• 37° C and 5% CO₂ humidified incubator
• Centrifuge (low speed clinical or equivalent)
• Hemacytometer
• Inverted microscope

Procedure

1. Thawing and Expanding Cryopreserved Cells:
   1. Prepare the Cultrex BME coated plate.
      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 at 2 - 8° C overnight.
      4. Dilute Cultrex BME 1:40 in DMEM/F12. This can be stored for up to 2 weeks at 2 - 8° C.
      5. Coat the desired number of plates with diluted Cultrex BME (approximately 2.5 mL per 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. Thawing of BG01V hES Cells:
      1. Warm the MEF Conditioned Medium to 37° C.
      2. Thaw the vial of BG01V hES cells by warming until just thawed and then immediately transfer to a 15 mL conical tube containing at least 5 mL of pre-warmed MEF Conditioned Medium.
      3. Spin at 200 x g for 4 minutes.
      4. Remove the supernatant and gently flick the pellet. Resuspend the pellet in an appropriate amount of MEF Conditioned Medium 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 the 37° C, 5% CO₂ incubator. Change the medium daily and monitor the cells. Passage the cells at the desired confluency.
   3. Passaging of BG01V hES Cells:
      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 Conditioned Medium to 37° C.
      3. Remove the MEF Conditioned Medium 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 conical tube containing 5 mL of MEF Conditioned Medium and spin at 200 x g for 4 minutes.
      7. Resuspend the pellet in MEF Conditioned Medium and count the cells using a hemacytometer.
      8. Plate the desired number of cells (approximately 1.0 x 10⁶ cells/60 mm plate) on the Cultrex BME coated plate in MEF Conditioned Medium containing 4 ng/mL of rhFGF basic.
      9. Change the medium daily. Monitor the cells for the desired confluency.

   References

   1. Thomson, J.A. et al. (1998) Science 282:1145.
   2. Xu, C. et al. (2001) Nature Biotechnology 19:971.
   3. Zeng, X. et al. (2004) Restor. Neuro. Neurosci. 22:421.
   4. Plaia, T. et al. (2006) Stem Cells 24:531.