Differentiating Mouse Embryonic Stem Cells into Embryoid Bodies in AggreWell Plates

METHOD

• 1. Coat the microwells of the AggreWell 400 plate with Pluronic F127 solution by adding 1 mL to each of those wells that contain microwells (the middle eight of the 24 wells in the plate contain microwells: B2 to C5).

  • Coating the microwells with Pluronic F-127 is not essential but helps to prevent EBs from sticking to the plate.

• 2. Centrifuge the plate at 3000g for 5 min to remove bubbles, and incubate for 30 min at room temperature.

• 3. Aspirate the Pluronic F127 solution and wash with 1 mL of DMEM per well. Remove the DMEM. Add 1 mL of EB medium to each well and centrifuge the plate again at 3000g for 5 min. Store the plate in the incubator until use.

• 4. If ES cells were cultured on MEFs, follow Steps 1–4 in Protocol: Differentiating Mouse Embryonic Stem Cells into Embryoid Bodies by Hanging-Drop Cultures (Behringer et al. 2016). Otherwise, trypsinize the cells using trypsin/EDTA, centrifuge at 200g for 5 min, and aspirate the supernatant.

• 5. Resuspend the cell pellet in EB medium. Count the cells and dilute to a final 2.4 × 10⁶ cells/mL using EB media to get EBs initiated by 2000 cells. The cell density can be changed if a different initiating cell number is required.

• 6. Without removing the media from the previously prepared AggreWell 400 plate, add 1 mL of the cell suspension to each well containing microwells. The total volume in each well will now be 2 mL. Immediately centrifuge the plate at 100g for 5 min to distribute the cells evenly between the microwells.

  • After centrifuging ES cells into the microwells, check for clumps or floating aggregates. These should be removed using a P200 tip to avoid clumping of embryoid bodies.

  • Examine the wells under a microscope to ensure that the microwells are filled with similar amounts of ES cells.

• 7. Place the plate into the incubator and incubate for 2–6 d.

  • Embryoid bodies can be collected from the AggreWell 400 plates as early as 24 h after aggregation, but they can be cultured for several days in the plates without affecting growth. Remove the embryoid bodies when they fill the size of the microwell or start clumping.

• 8. Change the medium after 48 h by carefully aspirating 1 mL of the medium with a 1-mL pipette tip, and gently add 1 mL of fresh medium to the wall of the well, avoiding turbulences.

  • When changing the medium in the AggreWell 400 plates, always leave the embryoid bodies covered with liquid to avoid formation of turbulence when adding the fresh medium. Fresh medium should be added as slowly as possible by pipetting the liquid to the inner wall of the well using a 1-mL pipette tip.

• 9. When the EBs reach the required size, rinse them from the microwells by gently pipetting up and down using a 1-mL pipette tip.

• 10. To remove single cells and dead cell clumps, the EBs can at this point be gently pipetted into a 40-μm cell strainer. Discard the flowthrough and wash back the EBs by inverting the cell strainer and rinsing with EB media.

• 11. Resuspend the EBs in EB medium, and distribute them between the wells of a six-well ultra-low-adherence plate. Culture the EBs until they reach the required size optimal for subsequent differentiation protocols.

  • Embryoid bodies should be plated at low densities in low-adherence plates for further growth to avoid clumping. When clumping is observed, further dilute the density.