2. Pre-coat the 6-well culture plate with 1 mL of the diluted
Matrigel (see Note 3) and leave the plate at room temperature
for an hour or at 37
C for at least 30 min.
3. Quickly thaw cryopreserved iPSCs in a 37
C water bath until
only a small ice crystal remains.
4. Wipe the outside of the cryovial with 70% ethanol.
5. Gently pipette the cell suspension and transfer to a 15-mL
conical tube.
6. Add 5 mL mTeSR™1 medium dropwise to the cell suspension
while gently rocking the tube.
7. Centrifuge the cells at 500 Â g for 5 min at room temperature.
8. Aspirate the diluted Matrigel from the well and add 1 mL
mTeSR™1 medium supplemented with 10 μM Y-27632.
9. After centrifugation, aspirate the supernatant.
10. Gently resuspend the cells in 1 mL mTeSR™1 medium supplemented with 10 μM Y-27632. Take care to maintain the
cells as clumps for better attachment.
11. Transfer the cell suspension onto the Matrigel-coated well
(step 8) and incubate at 37
C, 5% CO 2 .
12. After 24 h, replace the medium with mTeSR™1 medium
without Y-27632 and change the medium daily.
3.1.2 Passaging iPSCs
Passage when the cells reach 70% confluence, usually every 4–5 days
(see Note 4).
1. Pre-coat the 6-well culture plate with 1 mL of the diluted
Matrigel (see Note 3) for an hour at room temperature. Aspirate the diluted Matrigel from the well and add 2 mL of
mTeSR™1 medium.
2. Discard the medium from the cultured well and wash the cells
with DMEM/F12.
3. Add 1 mL of 1 mg/mL Dispase and incubate for 7 min at 37
C or until the edge of the colony is rounded up.
4. Aspirate Dispase, wash the well twice with DMEM/F12.
5. Add 2 mL of mTeSR™1 medium, gently scrape off the colonies using cell scraper.
6. Use a 5-mL seropipette, gently break up large clumps into
small clumps.
7. Transfer 200 μL of the cell clumps onto the Matrigel-coated
well containing 2 mL of mTeSR™1 medium (step 1) and
incubate at 37
C, 5% CO 2 .
8. Change the medium daily.
200
Methichit Wattanapanitch
Matrigel (see Note 3) and leave the plate at room temperature
for an hour or at 37
C for at least 30 min.
3. Quickly thaw cryopreserved iPSCs in a 37
C water bath until
only a small ice crystal remains.
4. Wipe the outside of the cryovial with 70% ethanol.
5. Gently pipette the cell suspension and transfer to a 15-mL
conical tube.
6. Add 5 mL mTeSR™1 medium dropwise to the cell suspension
while gently rocking the tube.
7. Centrifuge the cells at 500 Â g for 5 min at room temperature.
8. Aspirate the diluted Matrigel from the well and add 1 mL
mTeSR™1 medium supplemented with 10 μM Y-27632.
9. After centrifugation, aspirate the supernatant.
10. Gently resuspend the cells in 1 mL mTeSR™1 medium supplemented with 10 μM Y-27632. Take care to maintain the
cells as clumps for better attachment.
11. Transfer the cell suspension onto the Matrigel-coated well
(step 8) and incubate at 37
C, 5% CO 2 .
12. After 24 h, replace the medium with mTeSR™1 medium
without Y-27632 and change the medium daily.
3.1.2 Passaging iPSCs
Passage when the cells reach 70% confluence, usually every 4–5 days
(see Note 4).
1. Pre-coat the 6-well culture plate with 1 mL of the diluted
Matrigel (see Note 3) for an hour at room temperature. Aspirate the diluted Matrigel from the well and add 2 mL of
mTeSR™1 medium.
2. Discard the medium from the cultured well and wash the cells
with DMEM/F12.
3. Add 1 mL of 1 mg/mL Dispase and incubate for 7 min at 37
C or until the edge of the colony is rounded up.
4. Aspirate Dispase, wash the well twice with DMEM/F12.
5. Add 2 mL of mTeSR™1 medium, gently scrape off the colonies using cell scraper.
6. Use a 5-mL seropipette, gently break up large clumps into
small clumps.
7. Transfer 200 μL of the cell clumps onto the Matrigel-coated
well containing 2 mL of mTeSR™1 medium (step 1) and
incubate at 37
C, 5% CO 2 .
8. Change the medium daily.
200
Methichit Wattanapanitch