Single-Cell Splitting
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
Required Equipment / Consumables:
  • Biosafety Cabinet
  • CO2, O2, humidified, water jacketed incubator
  • Sterile, glass, borosilicate pipets (Fisherbrand)
    • Prepare ECM Plates:
    1. Aliquot Matrigel according to manufacturer’s instructions. Freeze aliquots at -80°C.

    2. To prepare tissue culture plates, take 1 aliquot of matrigel and thaw on ice or overnight at 4°C.
    3. 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.
    4. 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:
    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 500μl 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 1mL of media.
    10. Take 20μl of resuspended cells for cell counting.
      • Note: If cells are cultured in mTeSR originally, dilute the cells in an mTeSR solution containing Rock inhibitor.
    11. Make the appropriate dilution for desired number of cells per well.
    12. Remove basal media from Matrigel plates and add the appropriate volume of resuspended cells to each well.
    13. 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.
    14. Place culture vessel into 10% CO2 incubator.
    15. Feed cells after 2 days, then after 4 days, then after another 4 days until cells are ready to be passaged.
      1. After 2 days (2 days total): Add 80μl media/well.
      2. After 4 days (6 days total): Add 100μl media/well.
      3. After 4 days (10 days total): Add 100μl media/well.
      • Note: If cells were diluted in mTeSR containing Rock inhibitor, feed the cells with mTeSR only without Rock inhibitor.