Cryopreservation and Thawing of ES/iPS Cells
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)
- Generate larger clumps (> 150 µm):
- Use 2 mL serological pipettes
- GCDR: Reduce incubation time ~1 - 2 minutes & minimize scraping
- ReLeSR™: Reduce incubation time ~1 - 2 minutes
- 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.