Cryopreservation and Thawing of Pluripotent Stem Cells: Technical Tips and Media Recommendations

The following technical tip is for the freezing and thawing of human embryonic stem (ES) cells or induced pluripotent stem (iPS) cells cultured in mTeSR™1, mTeSR™ Plus, or TeSR™-E8™ in 6-well plates using either mFreSR™ (for cells cultured in 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.

Not using TeSR™ pluripotent stem cell culture media yet? Learn more and see which TeSR™ medium is right for you.

Pluripotent stem cell (PSC) 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 media volumes accordingly.


Should I Freeze Aggregates or Single Cells?

 
Aggregates
Single Cells
 
Advantages
Aggregates
  • Faster recovery as no need to transition from single cells to clumps
  • Ease of use (no need for ROCK inhibitor)
Single Cells
  • Consistency between vials
  • More accurate cell count
 
Disadvantages
Aggregates
  • Different number of cells (aggregates) per vial
  • Time to first passage less predictable
Single Cells

Recommendations for Freezing and Thawing PSCs in Aggregates

  • CryoStor® CS10 or mFreSR™ (for PSCs cultured in mTeSR™1 /mTeSR™ Plus only) are the recommended pluripotent stem cell cryopreservation media
  • You may optimize your cryopreservation protocol to generate larger clumps (> 150 µm) by:
    • Using 2 mL serological pipettes
    • Reducing incubation time to ~1 - 2 minutes and minimizing scraping if using Gentle Cell Dissociation Reagent (GCDR)
    • Reducing incubation time to ~1 - 2 minutes if using ReLeSR™
  • 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

Recommendations for Freezing and Thawing PSCs as single cells

  • FreSR™-S is the recommended pluripotent cryopreservation medium
  • Use ACCUTASE™ or GCDR to generate single cells
  • Freeze 1 x 106 cells/cryovial, using a controlled-rate freezing protocol
  • Passage human ES/iPS cells as aggregates, since serial single-cell passaging can increase the risk of karyotype abnormalities1,2

General Tips for Freezing and Thawing PSCs

Cryopreservation

  • Chill cryopreservation medium before starting dissociation
  • Cryopreserve the contents of one well of a 6-well plate per cryovial at the time of passage (can vary depending on the density of the cultures)
  • Freeze 1 x 106 cells/cryovial
  • Passage single cells as aggregates, since serial single-cell passaging can increase the risk of karyotype abnormalities1,2

Thawing

  • Quickly warm cells to thaw, and wipe down the outside of the bottles/vials with 70% ethanol or isopropanol before opening
  • When only a small ice pellet remains, transfer the cells to an empty conical tube using a 2 mL serological pipette. Add the PSC maintenance media dropwise to cells to avoid osmotic shock and improve recovery
  • Thaw cells into the same culture system from which they were cryopreserved for one passage prior to transition into mTeSR™1/mTeSR™ Plus or TeSR™-E8™
  • Seed the equivalent of one cryovial of cells into 1 - 2 wells of a coated 6-well plate (depending on the number of aggregates or single cells cryopreserved)
  • The 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.

When to Use ROCK Inhibitor (Y-27632)

For Single Cells

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

For Aggregates

  • May be added to PSC cultures frozen as aggregates post-thaw for 24 hours. We do not recommend this method as this may increase the seeding density, forcing passage sooner than optimal, or may result in overgrowth or other issues
  • Will help attachment of human ES/iPS aggregates, but it is not necessary

STEMCELL Products for PSC Culture, Cryopreservation, and Thawing

Product
Catalog #
Product
CryoStor® CS10 Cell Freezing Medium
Catalog #
07930
Product
mFreSR™ PSC Cryopreservation Medium
Catalog #
05855
Product
FreSR™-S Single Cell Freezing Medium for PSCs
Catalog #
05859
Product
mTeSR™1 PSC Maintenance Medium
Catalog #
85850
Product
mTeSR™ Plus PSC Maintenance Medium
Catalog #
05825
Product
TeSR™-E8™ PSC Culture Medium
Catalog #
05990
Product
DMEM/F-12 with 15 mM HEPES
Catalog #
36254
Product
Y-27632 (ROCK Inhibitor)
Catalog #
72302
Product
Gentle Cell Dissociation Reagent (GCDR)
Catalog #
07174
Product
ReLeSR™ PSC Selection & Passaging Reagent
Catalog #
05872
Product
ACCUTASE™ Cell Detachment Solution
Catalog #
07920
Product
Falcon® Serological Pipettes, 2 mL
Catalog #
38002

Need help choosing the right media and reagents for your experiments? We can help. Contact us by phone or email, or use the LiveChat function on this page to discuss your specific needs and applications.


Related Resources

View More Resources for Your PSC Research >

  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.

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