Y-27632 (for xeno-free chemically defined cryopreservation)
or in cold E8 medium supplemented with 10-μM ROCK
inhibitor Y-27632 (for undefined cryopreservation).
6. Add dropwise an equal volume of either ice-cold 2Â chemically
defined cryomedium (for xeno-free chemically defined cryopreservation) or ice-cold 2Â undefined cryomedium (for undefined cryopreservation) while mixing by tapping the tube.
7. Quickly aliquot the resulting cell suspension in either 1Â
chemically defined cryomedium or 1Â undefined cryomedium
into cryovial as 200- to 300-μL aliquots. Small aliquots can be
thawed faster and more uniformly.
8. Transfer the cryovials to a precooled (4
C) freezing container
(e.g., Mr. Frosty), and immediately place the container in a
À80
C freezer. hiPSCs seem to quickly equilibrate with cryoprotectants. Thus, aliquoting and subsequent transferring to a
À80
C freezer can be completed within 5 min to minimize
cryoprotectant toxicity.
9. After slow cooling to À80
C overnight, the cryovials containing hiPSCs should be transferred to a liquid nitrogen tank for
long-term storage.
3.5 Thawing of
hiPSCs
1. Prepare vitronectin-coated plate(s) ready to seed thaw hiPSCs
(see Subheading 3.1).
2. Remove a cryovial containing hiPSCs from the liquid nitrogen
tank, and hold in air for 20 s.
Fig. 1 Overview of freezing and thawing steps. Shown are critical steps with relevant processing times. See
text for details
576
Juliette Seremak and Ali Eroglu
or in cold E8 medium supplemented with 10-μM ROCK
inhibitor Y-27632 (for undefined cryopreservation).
6. Add dropwise an equal volume of either ice-cold 2Â chemically
defined cryomedium (for xeno-free chemically defined cryopreservation) or ice-cold 2Â undefined cryomedium (for undefined cryopreservation) while mixing by tapping the tube.
7. Quickly aliquot the resulting cell suspension in either 1Â
chemically defined cryomedium or 1Â undefined cryomedium
into cryovial as 200- to 300-μL aliquots. Small aliquots can be
thawed faster and more uniformly.
8. Transfer the cryovials to a precooled (4
C) freezing container
(e.g., Mr. Frosty), and immediately place the container in a
À80
C freezer. hiPSCs seem to quickly equilibrate with cryoprotectants. Thus, aliquoting and subsequent transferring to a
À80
C freezer can be completed within 5 min to minimize
cryoprotectant toxicity.
9. After slow cooling to À80
C overnight, the cryovials containing hiPSCs should be transferred to a liquid nitrogen tank for
long-term storage.
3.5 Thawing of
hiPSCs
1. Prepare vitronectin-coated plate(s) ready to seed thaw hiPSCs
(see Subheading 3.1).
2. Remove a cryovial containing hiPSCs from the liquid nitrogen
tank, and hold in air for 20 s.
Fig. 1 Overview of freezing and thawing steps. Shown are critical steps with relevant processing times. See
text for details
576
Juliette Seremak and Ali Eroglu