26. 0.5-M EDTA stock solution (1000Â): Dissolve 1.96-g EDTA
tetrasodium salt in ultrapure water (final volume: 10 mL) by
adjusting pH to 8 and vigorously stirring. Filter the resulting
solution using a 0.2-μm membrane filter, and store at 4
C for
up to 6 months.
27. 0.5-mM EDTA dissociation buffer: Add 100 μL of 0.5-M
EDTA (pH 8.0) and 0.18-g NaCl (to increase osmolarity to
340 mOsm) to 99.8-mL PBS (final volume: 100 mL), sterilize
using a 0.2-μm membrane filter, and store at 4
C for up to
6 months. Alternatively, commercially available Versene solution that has a similar composition but slightly lower osmolality
can be used instead of the 0.5-mM EDTA dissociation buffer.
28. Complete E8 flex medium: Slowly thaw the E8 flex supplement
(50Â, 10 mL) at room temperature or at 4
C overnight, mix
the content, and then add to 489-mL E8 flex basal medium.
Also add 1-mL primocin solution to obtain a working concentration of 100 μL/mL.
29. Undefined cryopreservation medium at 2Â concentration:
Add sterile 20% (v/v) DMSO, 40% (v/v) Knock-Out Serum
Replacement (KSR), and 20-μM ROCK inhibitor Y-27632 to
HEPES-buffered DMEM/F-12, and store at 4
C after
mixing.
30. Xeno-free defined cryopreservation medium at 2Â concentration: Add 10% DMSO, 10% EG, 1% methylcellulose, 6-mM
reduced glutathione, 10-mM ascorbic acid 2-phosphate, 2%
Glutamax, 2% Non-Essential Amino Acids, 4% Essential
Amino Acids, 2% RPMI 1640 Vitamins Solution, and 20 μM
ROCK inhibitor Y-27632 to HBSS- (see Note 2).
31. Undefined thawing medium at 1Â concentration: Add 0.1-M
sucrose, 20% KSR, and 10-μM ROCK inhibitor Y-27632 to
HEPES-buffered DMEM/F-12 medium, filter under the
hood using a 0.2-μm membrane filter, and store at 4
C (see
Note 3).
32. Xeno-free chemically defined thawing medium at 1Â concentration: Add 0.1-M sucrose, 0.5% methylcellulose, 3-mM
reduced glutathione, 5-mM ascorbic acid 2-phosphate, 1%
Glutamax, 1% Non-Essential Amino Acids, 2% Essential
Amino Acids, 1% RPMI 1640 Vitamins Solution, and 10-μM
ROCK inhibitor Y-27632 to HBSSÀ (see Note 4).
33. Xeno-free chemically defined thawing medium without sucrose
and methylcellulose: Add 3-mM reduced glutathione, 5-mM
ascorbic acid 2-phosphate, 1% Glutamax, 1% Non-Essential
Amino Acids, 2% Essential Amino Acids, 1% RPMI 1640 Vitamins Solution, and 10-μM ROCK inhibitor Y-27632 to
HBSSÀ.
Chemically Defined Cryopreservation of hiPSCs
573
tetrasodium salt in ultrapure water (final volume: 10 mL) by
adjusting pH to 8 and vigorously stirring. Filter the resulting
solution using a 0.2-μm membrane filter, and store at 4
C for
up to 6 months.
27. 0.5-mM EDTA dissociation buffer: Add 100 μL of 0.5-M
EDTA (pH 8.0) and 0.18-g NaCl (to increase osmolarity to
340 mOsm) to 99.8-mL PBS (final volume: 100 mL), sterilize
using a 0.2-μm membrane filter, and store at 4
C for up to
6 months. Alternatively, commercially available Versene solution that has a similar composition but slightly lower osmolality
can be used instead of the 0.5-mM EDTA dissociation buffer.
28. Complete E8 flex medium: Slowly thaw the E8 flex supplement
(50Â, 10 mL) at room temperature or at 4
C overnight, mix
the content, and then add to 489-mL E8 flex basal medium.
Also add 1-mL primocin solution to obtain a working concentration of 100 μL/mL.
29. Undefined cryopreservation medium at 2Â concentration:
Add sterile 20% (v/v) DMSO, 40% (v/v) Knock-Out Serum
Replacement (KSR), and 20-μM ROCK inhibitor Y-27632 to
HEPES-buffered DMEM/F-12, and store at 4
C after
mixing.
30. Xeno-free defined cryopreservation medium at 2Â concentration: Add 10% DMSO, 10% EG, 1% methylcellulose, 6-mM
reduced glutathione, 10-mM ascorbic acid 2-phosphate, 2%
Glutamax, 2% Non-Essential Amino Acids, 4% Essential
Amino Acids, 2% RPMI 1640 Vitamins Solution, and 20 μM
ROCK inhibitor Y-27632 to HBSS- (see Note 2).
31. Undefined thawing medium at 1Â concentration: Add 0.1-M
sucrose, 20% KSR, and 10-μM ROCK inhibitor Y-27632 to
HEPES-buffered DMEM/F-12 medium, filter under the
hood using a 0.2-μm membrane filter, and store at 4
C (see
Note 3).
32. Xeno-free chemically defined thawing medium at 1Â concentration: Add 0.1-M sucrose, 0.5% methylcellulose, 3-mM
reduced glutathione, 5-mM ascorbic acid 2-phosphate, 1%
Glutamax, 1% Non-Essential Amino Acids, 2% Essential
Amino Acids, 1% RPMI 1640 Vitamins Solution, and 10-μM
ROCK inhibitor Y-27632 to HBSSÀ (see Note 4).
33. Xeno-free chemically defined thawing medium without sucrose
and methylcellulose: Add 3-mM reduced glutathione, 5-mM
ascorbic acid 2-phosphate, 1% Glutamax, 1% Non-Essential
Amino Acids, 2% Essential Amino Acids, 1% RPMI 1640 Vitamins Solution, and 10-μM ROCK inhibitor Y-27632 to
HBSSÀ.
Chemically Defined Cryopreservation of hiPSCs
573