3. Next, partially immerse it in a 37
C water bath, and gently
swirl for fast thawing. Remove the cryovial from the water bath
just before ice has completely melted.
4. Before placing the cryovial under the biosafety hood, spray and
wipe down the outside of it with 70% ethanol.
5. Under the biosafety hood, gently transfer the cryovial content
to a 15-mL tube for dilution of cryoprotectants.
6. In the case of xeno-free chemically defined cryopreservation,
add 100 μL of 1Â chemically defined thawing medium every
minute for a total of six times while gently mixing the tube
content by tapping each time. For the final dilution step, add
excess amount (10 mL) of defined thawing medium without
sucrose and methylcellulose.
7. In the case of undefined cryopreservation, perform the dilution
steps described above using 1Â undefined thawing medium.
For the final dilution step, add 10 mL of HEPES-buffered
DMEM/F-12 medium containing 20% KSR and 10-μM
ROCK inhibitor.
8. Wait for 1 min, and then pellet hiPSCs at 300 Â g for 5 min.
9. Aspirate the supernatant without disturbing the cell pellet, and
resuspend the cells in complete E8 flex medium containing
10-μM ROCK inhibitor.
10. Take out 40 μL of cell suspension to determine cell viability,
and then plate hiPSCs accordingly (e.g., 5 Â 10
4 cells/cm
2
).
11. Leave the plate undisturbed in a humidified CO 2 incubator at
37
C for at least 1 day to ensure the attachment of viable cells.
Subsequently, 2/3 of the culture medium can be changed daily.
4 Notes
1. Y-27632 (dihydrochloride) is less soluble in water (up to
30 mM) than in DMSO (up to 100 mM). The solvent can be
added directly to the vial that should then be vortexed vigorously for complete solubilization. When using DMSO as a
solvent, final DMSO concentration should not exceed 0.1%
to avoid its toxicity. The stock solution should not undergo
multiple freeze-thaw cycles and be protected from light.
2. It is extremely difficult to sterilize the defined cryopreservation
medium by filtering due to presence of methylcellulose. Therefore, we recommend first dissolving methylcellulose in HBSSwith or without DMSO and EG and then autoclaving the
resulting solution using a liquid program with slow pressure
release to prevent boilover. The aqueous solutions of DMSO
and EG are heat stable and can be autoclaved using a proper
Chemically Defined Cryopreservation of hiPSCs
577
C water bath, and gently
swirl for fast thawing. Remove the cryovial from the water bath
just before ice has completely melted.
4. Before placing the cryovial under the biosafety hood, spray and
wipe down the outside of it with 70% ethanol.
5. Under the biosafety hood, gently transfer the cryovial content
to a 15-mL tube for dilution of cryoprotectants.
6. In the case of xeno-free chemically defined cryopreservation,
add 100 μL of 1Â chemically defined thawing medium every
minute for a total of six times while gently mixing the tube
content by tapping each time. For the final dilution step, add
excess amount (10 mL) of defined thawing medium without
sucrose and methylcellulose.
7. In the case of undefined cryopreservation, perform the dilution
steps described above using 1Â undefined thawing medium.
For the final dilution step, add 10 mL of HEPES-buffered
DMEM/F-12 medium containing 20% KSR and 10-μM
ROCK inhibitor.
8. Wait for 1 min, and then pellet hiPSCs at 300 Â g for 5 min.
9. Aspirate the supernatant without disturbing the cell pellet, and
resuspend the cells in complete E8 flex medium containing
10-μM ROCK inhibitor.
10. Take out 40 μL of cell suspension to determine cell viability,
and then plate hiPSCs accordingly (e.g., 5 Â 10
4 cells/cm
2
).
11. Leave the plate undisturbed in a humidified CO 2 incubator at
37
C for at least 1 day to ensure the attachment of viable cells.
Subsequently, 2/3 of the culture medium can be changed daily.
4 Notes
1. Y-27632 (dihydrochloride) is less soluble in water (up to
30 mM) than in DMSO (up to 100 mM). The solvent can be
added directly to the vial that should then be vortexed vigorously for complete solubilization. When using DMSO as a
solvent, final DMSO concentration should not exceed 0.1%
to avoid its toxicity. The stock solution should not undergo
multiple freeze-thaw cycles and be protected from light.
2. It is extremely difficult to sterilize the defined cryopreservation
medium by filtering due to presence of methylcellulose. Therefore, we recommend first dissolving methylcellulose in HBSSwith or without DMSO and EG and then autoclaving the
resulting solution using a liquid program with slow pressure
release to prevent boilover. The aqueous solutions of DMSO
and EG are heat stable and can be autoclaved using a proper
Chemically Defined Cryopreservation of hiPSCs
577