2. At the end of the incubation period, aspirate the medium, and
gently rinse the wells twice with 1 mL per well of prewarmed
EDTA dissociation buffer to completely remove calcium and
magnesium. The EDTA dissociation buffer should slowly be
added to the wall of each well to avoid early dislodging hiPSC
colonies.
3. Add 1 mL per well of prewarmed EDTA dissociation buffer for
the third time, and hold the plate(s) within the biosafety hood
for 3–5 min (longer incubation periods result in tiny clumps of
a few cells).
4. At the end of the incubation period, aspirate the EDTA dissociation buffer from each well without disturbing hiPSC colonies, and then pipette 1 to 2 mL fresh E8 medium containing
10 μM ROCK inhibitor Y-27632 into each well at a fast speed
to dislodge hiPSC colonies. Further detachment and dissociation of the colonies can be achieved by repeating up-and-down
pipetting a few times; however, excess pipetting should be
avoided so as not to end up with a single cell suspension.
5. Aspirate the partially dissociated hiPSC clumps, transfer to a
centrifuge tube, pellet at a low speed (200–300 Â g), resuspend
in E8 medium containing 10-μM ROCK inhibitor, and then
distribute to the vitronectin-coated wells. When passaging,
split ratios range from 1:3 to 1:6 depending on density and
growth characteristics of the cells. Usually, hiPSCs reach
70–80% confluency within 3–5 days after passaging.
3.4 Freezing of
hiPSCs
It is important that hiPSC colonies are healthy and in good shape at
the time of cryopreservation. Independent of the dissociation
approach, the cryopreservation-associated processes lead to disruption of cell-to-cell contacts, resulting in almost completely dissociated hiPSCs. Therefore, it is advisable to include 10-μM ROCK
inhibitor in freezing and thawing media, as well as in culture
medium before freezing (for ~1 h) and after thawing (for up to
48 h). The freezing and thawing steps are summarized in Fig. 1.
1. Prepare cryopreservation media in advance.
2. Determine the number of cryovials needed for freezing, and
label them with cell line name, passage number, type of cryopreservation medium, and date.
3. Place both defined and undefined cryopreservation media and
related solutions on ice. Adding and removal of cryoprotectants at a low temperature seem to be helpful in terms of
reducing cryoprotectant toxicity to hiPSCs.
4. Harvest hiPSC colonies as described earlier (see Subheading
3.3), and determine cell density and viability using trypan
blue and a hemocytometer or by commercially available kits.
5. After pelleting, resuspend the partially dissociated cell clumps
either in cold HBSS-containing 10-μM ROCK inhibitor
Chemically Defined Cryopreservation of hiPSCs
575
gently rinse the wells twice with 1 mL per well of prewarmed
EDTA dissociation buffer to completely remove calcium and
magnesium. The EDTA dissociation buffer should slowly be
added to the wall of each well to avoid early dislodging hiPSC
colonies.
3. Add 1 mL per well of prewarmed EDTA dissociation buffer for
the third time, and hold the plate(s) within the biosafety hood
for 3–5 min (longer incubation periods result in tiny clumps of
a few cells).
4. At the end of the incubation period, aspirate the EDTA dissociation buffer from each well without disturbing hiPSC colonies, and then pipette 1 to 2 mL fresh E8 medium containing
10 μM ROCK inhibitor Y-27632 into each well at a fast speed
to dislodge hiPSC colonies. Further detachment and dissociation of the colonies can be achieved by repeating up-and-down
pipetting a few times; however, excess pipetting should be
avoided so as not to end up with a single cell suspension.
5. Aspirate the partially dissociated hiPSC clumps, transfer to a
centrifuge tube, pellet at a low speed (200–300 Â g), resuspend
in E8 medium containing 10-μM ROCK inhibitor, and then
distribute to the vitronectin-coated wells. When passaging,
split ratios range from 1:3 to 1:6 depending on density and
growth characteristics of the cells. Usually, hiPSCs reach
70–80% confluency within 3–5 days after passaging.
3.4 Freezing of
hiPSCs
It is important that hiPSC colonies are healthy and in good shape at
the time of cryopreservation. Independent of the dissociation
approach, the cryopreservation-associated processes lead to disruption of cell-to-cell contacts, resulting in almost completely dissociated hiPSCs. Therefore, it is advisable to include 10-μM ROCK
inhibitor in freezing and thawing media, as well as in culture
medium before freezing (for ~1 h) and after thawing (for up to
48 h). The freezing and thawing steps are summarized in Fig. 1.
1. Prepare cryopreservation media in advance.
2. Determine the number of cryovials needed for freezing, and
label them with cell line name, passage number, type of cryopreservation medium, and date.
3. Place both defined and undefined cryopreservation media and
related solutions on ice. Adding and removal of cryoprotectants at a low temperature seem to be helpful in terms of
reducing cryoprotectant toxicity to hiPSCs.
4. Harvest hiPSC colonies as described earlier (see Subheading
3.3), and determine cell density and viability using trypan
blue and a hemocytometer or by commercially available kits.
5. After pelleting, resuspend the partially dissociated cell clumps
either in cold HBSS-containing 10-μM ROCK inhibitor
Chemically Defined Cryopreservation of hiPSCs
575