Procedure for Culturing BG01V Human Embryonic Stem Cells on Irradiated Mouse Embryonic Fibroblasts

Stem Cell Protocol: Procedure for Culturing BG01V Human Embryonic Stem Cells on Irradiated Mouse Embryonic Fibroblasts (iMEF)

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 embryo fibroblasts (iMEF, R&D Systems, Catalog # PSC001) or using MEF conditioned medium (2) (R&D Systems, Catalog # AR005). 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

• Fetal bovine serum (Sigma, Catalog # 12103C or equivalent)
• Knockout™ serum replacer (Invitrogen, Catalog # 10828)
• Non-Essential Amino Acids, 100X (Irvine Scientific, Catalog # 9304 or equivalent)
• L-Glutamine, 200 mM solution (Irvine Scientific, Catalog # 9317 or equivalent)
• Penicillin/Streptomycin, 100X solution (Sigma, Catalog # P4333 or equivalent)
• Beta-mercaptoethanol (Invitrogen, Catalog # 21985-023 or equivalent)
• DMEM/F12 (Invitrogen, Catalog # 12500-096 or equivalent)
• High glucose DMEM (Invitrogen, Catalog # 12100-046 or equivalent)
• Recombinant human (rh) FGF basic (R&D Systems, Catalog # 4114-TC, 233-FB, or equivalent)
• Accutase (Innovative Cell Technologies, Catalog # AT104 or equivalent)
• 0.1% w/v solution of gelatin (Sigma, Catalog # G2500 or equivalent) in sterile dH2O

• 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, 5% CO₂ incubator
• Centrifuge (low speed clinical or equivalent)
• Hemacytometer
• Inverted microscope

Reagent Preparation

• 10% fetal bovine serum
• 90% high glucose DMEM
• 2 mM L-glutamine
• 1X penicillin/streptomycin
  
  Combine all of the above ingredients and filter sterilize.

• 15% fetal bovine serum
• 5% Knockout serum replacer
• 2 mM non-essential amino acids
• 2 mM L-glutamine
• 1X penicillin/streptomycin
• 0.1 mM beta-mercaptoethanol
• 80% DMEM/F12
  
  Combine all of the above ingredients except rhFGF basic and filter sterilize.

Procedure

1. Thawing and plating of the iMEF feeder cells:
   1. Gelatin coat the appropriate sized plate(s) for the desired number of cells by covering the surface of the dish with 0.1% sterile gelatin for 15 minutes. For example, one vial of 6 x 10⁶ iMEF can be plated onto two 100 mm dishes, six 60 mm dishes, or two 6 well plates.
   2. Warm MEF medium to 37° C.
   3. Thaw the desired number of vials of iMEF cells by quickly warming the cryotube(s) in a 37° C water bath until the cells are just thawed and then immediately transferring the contents of one vial to a 15 mL conical tube containing at least 5 mL of pre-warmed MEF medium. Rinse the vial with an additional 1 mL of medium to ensure the removal of all the cells.
   4. Spin at 200 x g in a clinical centrifuge for 5 minutes.
   5. Remove the supernatant and flick the pellet.
   6. Aspirate the 0.1% gelatin from the plate(s).
   7. Resuspend the iMEF cells from step "e" (above) in MEF medium and transfer to the gelatin-coated plates at a density of approximately 1 x 10⁶ cells/60 mm plate.
   8. Incubate overnight in a 37° C, 5% CO₂ incubator.
   
   
2. Thawing the BG01V hES cells:
   1. Warm the hES medium to 37° C.
   2. Thaw the cryovial of hES cells in a 37° C water bath until just thawed and then transfer immediately to a 15 mL conical tube containing at least 5 mL of pre-warmed hES medium. Rinse the vial with an additional 1 mL of medium to ensure the removal of all the cells.
   3. Spin at 200 x g in a clinical centrifuge for 4 minutes.
   4. Remove the supernatant and gently flick the pellet.
   5. Resuspend the pellet in an appropriate amount of hES medium freshly supplemented with 4 ng/mL of rhFGF basic (typical volume is 5 mL per 60 mm plate).
   6. Remove the MEF medium from the iMEF cells plated in step 1 and add hES cell suspension to the plate. Typically, 1 x 10⁶ cells are thawed to a 60 mm plate.
   7. Grow in a 37° C, 5% CO₂ incubator. Cells should be fed daily with hES medium freshly supplemented with rhFGF basic. Passage cells before the hES colonies touch at their edges.
3. Passaging of the BG01V hES cells:
   1. Plate the iMEF cells on the desired number of plates as described above, 1 day prior to passaging.
   2. Warm the hES medium to 37° C.
   3. Remove the hES 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 Accutase gently over the plate until all the cells have been detached.
   5. Gently pipette 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 hES medium and spin at 200 x g for 4 minutes.
   7. Remove the supernatant and gently flick the pellet.
   8. Resuspend the pellet in hES medium and count the cells using a hemacytometer.
   9. Plate the desired number of cells (approximately 0.5 - 1.0 x 10⁶ cells/60 mm plate) on the previously plated iMEF cells in hES medium containing 4 ng/mL of rhFGF basic.
   10. Feed the cells daily with hES medium freshly supplemented with rhFGF basic.

Typical Data

Sample data of BG01V hES cells cultured on R&D Systems iMEF.

Figure 1: BG01V colonies growing on R&D Systems iMEF feeder cells.
Figure 2: Flow cytometric analysis of pluripotency markers R&D Systems iMEF feeder cells. Oct4 (A) and SSEA-4 (B). BG01V cells were cultured on R&D Systems iMEF cells for two passages. At the third passage, cells were harvested for flow cytometry with markers of pluripotency. (A) Cells were fixed for intracellular flow cytometry with PE conjugated rat anti-human Oct4 (filled histogram in A; R&D Systems, Catalog # IC1759P). (B) Cells were used for surface flow cytometry with PE conjugated SSEA-4 (filled histogram in B; R&D Systems, Catalog # FAB1435P). Isotype controls (open histograms in A and B) used were PE conjugated rat IgG2B (R&D Systems, Catalog # IC013P) and PE-conjugated mouse IgG3, respectively
Figure 3: Immunocytochemical analysis of pluripotency markers Oct4 (A) and Nanog (B). BG01V cells were cultured on R&D Systems iMEF cells for two passages. At the third passage, iMEF and BG01V were plated onto coverslips and grown for 1 - 2 days. Coverslips were fixed and stained with (A) goat anti-human Oct4 (R&D Systems, Catalog # AF1759, followed by NorthernLights 557-conjugated anti-goat antibody (R&D Systems, Catalog # NL001) or (B) goat anti-human Nanog (R&D Systems, Catalog # AF1997) followed by NorthernLights 557-conjugated anti-goat antibody.

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.