Definitive Endoderm Differentiation
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
Corning™ RPMI 1640 Medium (Mod.) 1X with L-Glutamine Fisher Scientific MT10041CV
mTesR Medium Stemcell Technologies 85850
Rock Inhibitor Stemcell Technologies 72302
Accutase Innovative Cell Technologies AT-104
CHIR99021 TOCRIS 4423
Activin A R&D Systems 338-AC-010
EDTA Various
FBS Various
Required Equipment / Consumables:
  • Biosafety Cabinet
  • CO2, O2, humidified, water jacketed incubator
  • Sterile, glass, borosilicate pipets (Fisherbrand)
  • Reagent Setup:
    CHIR99021 (15mM Stock):
    • Check the molecular weight.
    • Dissolve in DMSO until 15mM.
    • Divide the solution into 200-μl aliquots in 1.5-ml tubes.
    • Store it at −80 °C for up to 6 months.
    Activin A (10 μg/mL Stock):
    • Dissolve 50 μg of Activin A in 5 ml of 0.1% BSA/PBS.
    • Divide the solution into 500-μl aliquots in 1.5-ml tubes.
    • Store it at −80 °C for up to 6 months.
    Prepare Matrigel 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.
    Prepare EDTA Solution:
    1. Prepare a 0.5mM EDTA solution by diluting 0.5M EDTA 1:1000 in PBS
    Procedure:
    Culture of hPSCs
    1. Prepare a matrigel-coated 6-well plate.
    2. Quickly thaw 1 vial of hPSCs from liquid N2 in a 37°C water bath.
    3. Transfer the cells to a sterile 15mL conical tube.
    4. Add a few drops of mTeSR1 and gently swirl the conical tube.
    5. Add 2mL of mTeSR1 to the conical tube.
    6. Centrifuge the cells at 120g for 3 min at room temperature. Aspirate and discard the supernatant.
    7. Aspirate the liquid from the matrigel-coated plates.
    8. Resuspend the cell pellet in 2mL of mTeSR1 + Rock inhibitor. Add the cell suspension to 1 well of the matrigel-coated plate.
    9. 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.
    10. Incubate a 37°C incubator overnight.
    11. On the next day, aspirate the medium in each well. Add 2mL of fresh mTeSR1 media. Repeat daily with increasing volumes of mTeSR1 until cells are ready to be passaged (usually about 3-4 days).
    Definitive Endoderm Induction
    1. Prepare a matrigel-coated 24-well plate.
    2. When cells are 80-90% confluent, aspirate the old medium and add 1mL of Accutase to each well to be passaged.
    3. Incubate for 6-10 minutes at 37°C until cells detach.
    4. Once incubation is complete, add 3mL of mTeSR1 to neutralize the Accutase, and collect cells to be passaged using fresh media using a 5mL pipet.
      Note: Cells can be passaged with EDTA as well. If passaging with EDTA, passage the cells 1:8.
    5. Spin the cells at 120g for 3 minutes.
    6. Aspirate the supernatant, leaving a trace amount to prevent the pellet from drying.
    7. Resuspend the cell pellet in 1mL of mTeSR1 + Rock inhibitor.
    8. Aspirate the liquid from the matrigel-coated plates and add 500 μl of mTeSR1 +Rock inhibitor.
    9. Take 20 μl of the cell suspension and count the cells.
    10. Add the appropriate number of cells to the medium-containing wells.
      • For a 24-well plate, seed the wells with 100,000 cells.
    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. Change media daily with 1mL of mTeSR1.
    14. Day 0: On the next day, or when cells are fully confluent, aspirate the old medium and add 1mL of: RPMI + 100ng/mL Activin A + 3μm CHIR99021 to each well.
    15. Day 1: On the next day, aspirate the old medium and add 1mL of: RPMI + 100ng/mL Activin A + 0.2% FBS to each well. Repeat daily until day 3.