Feeder-free hPSCs Culture using EDTA/Accutase
ReagentVendorCatalog Number
Corning™ Matrigel™ hESC-Qualified Matrix Fisher Scientific 354277
Falcon™ Polystyrene Microplates Fisher Scientific Various
Corning™ Cell Culture PBS (1X) Thermo Fisher / Life Technologies MT21040CV
DMEM-F12 Thermo Fisher / Life Technologies MT10092CV
Essential 8 Medium Thermo Fisher / Life Technologies A1517001
Stemflex Medium Thermo Fisher / Life Technologies A3349401
mTesR Medium Stemcell Technologies 85850
Rock Inhibitor Stemcell Technologies 72302
Accutase Innovative Cell Technologies AT104
EDTA Various
Required Equipment / Consumables:
  • Biosafety Cabinet
  • CO2, O2, humidified, water jacketed incubator
  • Sterile, glass, borosilicate pipets (Fisherbrand)
  • Prepare Matrigel Plates:
    • Aliquot Matrigel according to manufacturer’s instructions. Freeze aliquots at -80°C.
    • Add the thawed matrigel in cold DMEM-F12 and plate in the appropriate sized cell culture vessel.
      • For a 6-well plate, we use 1mL of matrigel/DMEM-F12 per well.
      • For a 24-well plate, we use 250μl of matrigel/DMEM-F12 per well.
      • For a 96-well plate, we use 50μl of matrigel/DMEM-F12 per well
    • Allow ECM to attach to plate by incubating at 37°C for at least an hour. Plates can be left overnight at 37°C.
    Passaging with Accutase:
    1. Remove plate to be passaged from incubator and place it in the biosafety cabinet. 

    2. Remove the spent medium from the wells to be passaged by aspirating with borosilicate pipet.
    3. Rinse each well to be passaged with 1ml room temperature PBS.
    4. Add Accutase to each well to be passaged.
      1. For a 6-well plate, add 1mL Accutase to each well.
      2. For a 24-well plate, add 300μl Accutase to each well.
    5. Incubate for 6-10 minutes at 37°C until cells detach.
    6. Once incubation is complete, add the desired medium to neutralize the Accutase, and collect cells to be passaged using fresh media using a 5mL pipet.
      1. For a 6-well plate, add 3mL media to each well
      2. For a 24-well plate, add 1mL media to each well.
    7. Spin the cells at 120g for 3 minutes.
    8. Aspirate the supernatant, leaving a trace amount to prevent the pellet from drying.
    9. Resuspend the cell pellet with an appropriate amount of media for passaging.
      Note: If cells are cultured in mTeSR originally, resuspend the cells in an mTeSR solution containing Rock inhibitor.
    10. Remove media from Matrigel plates and add the appropriate volume of resuspended cells to each well.
    11. Gently shake the plate back and forth and front to back to evenly distribute the cells—avoid circular motions to prevent concentrating cells in the middle of the well.
    12. Place culture vessel into incubator overnight.
    13. Continue to feed cells until needed for experiments, passaging or freezing.
    14. In our hands, robust pluripotent stem cells can be passaged every 3-5 days. It is important 
to not let cells become overconfluent for optimal cell health.
    hESC Thawing:
    1. Aspirate DMEM+10%FBS media used to plate MEFs, and wash with PBS to eliminate traces of serum. Aspirate PBS and add fresh hESC media. Place dish in the incubator to pre-warm and correctly pH the media.
    2. Remove hPSCs from liquid nitrogen tank and move it rapidly to a 37°C water bath.
    3. Transfer cells into 9 mL of hESC media in a 15 ml conical tube.
    4. Spin at 200xg for 5 minutes.
    5. Resuspend pellet with media pre-warmed from the MEF plate and add cells directly back to the same MEF plate/wells. Incubate 37°C
    6. Feed daily with hESC
    Passaging with EDTA:
    1. Remove plate to be passaged from incubator and place it in the biosafety cabinet.
    2. Remove the spent medium from the wells to be passaged by aspirating with borosilicate pipet.
    3. Rinse each well to be passaged with 1ml room temperature PBS.
    4. Add room temperature 0.5mM EDTA to each well to be passaged.
      1. For a 6-well plate, add 1mL 0.5mM EDTA to each well.
      2. For a 24-well plate, add 300μl 0.5mM EDTA to each well.
    5. Incubate for 5-10 minutes at room temperature.
      Note: Do not incubate until cells detach; they should be loosely adherent to the culture plate and easily able to slough off the plate with gentle washing. If cells detach, collect and centrifuge the cells, resuspend in an appropriate volume for the proper passage ratio, and add the resuspended cells to new Matrigel or VTN-N coated plates.
    6. Once incubation is complete, aspirate the EDTA and, add fresh media using a 5mL pipet, and pipet up and down along the bottom of the well. Cells should be loosely attached and come off easily.
      Note: If cells are cultured in mTeSR originally, the cells do not need to be resuspended in an mTeSR solution containing Rock inhibitor.
    7. Remove media from Matrigel plates and add the appropriate volume of resuspended cells to each well.
    8. Gently shake the plate back and forth and front to back to evenly distribute the cells—avoid circular motions to prevent concentrating cells in the middle of the well.
    9. Place culture vessel into incubator overnight.
    10. Continue to feed cells until needed for experiments, passaging or freezing.
    11. In our hands, robust pluripotent stem cells can be passaged every 3-5 days. It is important to not let cells become overconfluent for optimal cell health.