Growing human iPSC in feeder-free culture with mTeSR medium

Timing: ∼ 7 days

Note: The timing for human iPSC thawing is around 30 min.

Prepare Geltrex or Matrigel coated plates (see materials and equipment).

Transfer 5 mL of warmed mTeSR media (see materials and equipment) into a 15 mL tube.

Thaw a frozen vial of hiPSC containing at least 1.5–2.0 × 10⁶ cells in a 37°C water bath with gentle shaking until the ice starts to melt.

Transfer hiPSCs in a drop-wise manner into the 15 mL conical tube containing 5 ml prewarmed media (iPSC passage media). Gently rotate the tube to mix the cells with the media. Centrifuge the cells for 5 min at 300 × g at room temperature.

Remove the supernatant and add 3 mL of fresh iPSC passage media (see materials and equipment).

Seed the cells onto plates coated with Geltrex or Matrigel i.e., 1.5 × 10⁶ into one well of a 6-well plate. Make sure that cells are distributed evenly by rocking back and forth gently to distribute the cells and incubate at 37°C in a 5% CO₂ incubator.

After 24 h, change the iPSC passage media to regular mTeSR media.

Change the media every day.

CRITICAL: Changing media every day is very crucial since iPSCs proliferate quickly. Lack of nutrients in the exhausted media will lead to cell detachment and death.

Note: Different iPS cell lines are grown in different starting media such as StemFlex or Essential 8. We have used StemFlex and Essential 8 in substitution for mTeSR for cell lines requiring these media types. Cells should be approximately 70%–80% confluent after 7 days of culture. If cells do not reach this confluency, allow them to grow until they reach confluency. The cells can be cryopreserved or differentiated when they reach confluency. Follow the instructions below for cryopreservation or differentiation.