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?

 
Aggregates
Single Cells
Advantages
  • 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
Disadvantages
  • Different number of cells (aggregates) per vial
  • Time to first passage less predictable
  • Requires ROCK inhibitor for first 24 hours
Aggregates
Single Cells
  • 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

General Tips

Cryopreservation

  • 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

Thawing

  • 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)

Single Cells

  • Add to single-cell cryopreserved cultures for 24 hours post-thaw

Aggregates

  • 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

STEMCELL Products

Product
Catalog #
CryoStor® CS10
mFreSR™
FreSR™-S
mTeSR™1
mTeSR™ Plus
TeSR™-E8™
DMEM/F-12 with 15 mM HEPES
Y-27632
Gentle Cell Dissociation Reagent (GCDR)
ReLeSR™
ACCUTASE™
Falcon® Serological Pipettes, 2 mL
  1. Draper JS et al. (2004) Recurrent gain of chromosomes 17q and 12 in cultured human embryonic stem cells. Nat Biotechnol 22(1): 53–4.
  2. Buzzard JJ et al. (2004) Karyotype of human ES cells during extended culture. Nat Biotechnol 22(4): 381–2.