The following technical tip is for cryopreservation and thawing of human ES or iPS cells cultured in mTeSR™1 (Catalog #85850), mTeSR™ Plus (Catalog #05825), or TeSR™-E8™ (Catalog #05990) in 6-well plates using either mFreSR™ (mTeSR™1/mTeSR™ Plus only) or CryoStor® CS10 for cell aggregates, and FreSR™-S for single cells. For complete instructions, refer to the Technical Manual: Maintenance of Human Pluripotent Stem Cells in mTeSR™1 (Document #10000005505), mTeSR™ Plus (Document #10000005507), or TeSR™-E8™ (Document #10000005516), or contact us to request a copy.
Cultures should be harvested and cryopreserved at the time they would normally be ready for passaging. Each vial should contain the cell aggregates from one well of a 6-well plate. If using other cultureware, adjust volumes accordingly.
Should I Freeze Aggregates or Single Cells?
Faster recovery as no need to transition from single cells to clumps
Ease of use (no need for ROCK inhibitor)
Consistency between vials
More accurate cell count
Different number of cells (aggregates) per vial
Time to first passage less predictable
Requires ROCK inhibitor for first 24 hours
CryoStor™CS10 or mFreSR™ (for mTeSR™1 /mTeSR™ Plus only)
When thawing, lightly triturate larger clumps prior to seeding to generate 50 µm aggregates
If only a few undifferentiated colonies are observed after thawing, it may be necessary to select only these colonies for passaging and replate them in the same size well (i.e. without splitting) on a newly coated plate
FreSR™-S is the recommended cryopreservation medium
Use ACCUTASE™ or GCDR to generate single cells
Freeze 1 x 10^6 cells/cryovial, using a controlled rate freezing protocol
Passage human ES/iPS cells as aggregates; serial single-cell passaging can increase risk of karyotype abnormalities1,2
Chill cryopreservation medium before starting dissociation
Cryopreserve the contents of one well of a 6-well plate per cryovial at time of passage (can vary depending on density of cultures)
Freeze 1 x 10^6 cells/cryovial
Passage single cells as aggregates; serial single-cell passaging can increase risk of karyotype abnormalities1,2
Quickly warm cells to thaw, and before opening wipe down the outside of the bottles/vials with 70% ethanol or isopropanol
When only a small ice pellet remains, transfer the cells to an empty conical tube using a 2 ml serological pipette. Add the maintenance media dropwise to cells to avoid osmotic shock and improve recovery
Thaw into the same system the cells were cryopreserved from for one passage prior to transition into mTeSR™1/mTeSR™ Plus or TeSR™-E8™
Seed the equivalent of one cryovial into 1 - 2 wells of a coated 6-well plate (depending on number of aggregates or single cells cryopreserved)
First passage post-thaw may be required sooner than expected. The cultures tend to be very confluent and may merge into each other. After one passage with adjustment to a proper seeding density, the morphology should recover.
ROCK Inhibitor (Y-27632)
Add to single-cell cryopreserved cultures for 24 hours post-thaw
May be added to cultures frozen as aggregates post-thaw for 24 hours. We don't recommend it, as this may increase the seeding density and may force passage sooner than optimal or result in overgrowth or other issues
Will help attachment of human ES/iPS aggregates, but it is not necessary
Draper JS et al. (2004) Recurrent gain of chromosomes 17q and 12 in cultured human embryonic stem cells. Nat Biotechnol 22(1): 53–4.
Buzzard JJ et al. (2004) Karyotype of human ES cells during extended culture. Nat Biotechnol 22(4): 381–2.
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Cryopreservation and Thawing of ES/iPS Cells
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