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
• For best cell recovery, add CloneR™2 to your plating medium after thaw

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 hPSCs as aggregates, since serial single-cell passaging can increase the risk of karyotype abnormalities^1,2. If single-cell passaging is preferred or required for downstream applications, use eTeSR™ to enhance the genetic stability of your cultures.

Thawing

• Quickly thaw cells in a 37°C water bath or the ThawSTAR® CFT2 Automated Thawing System for ensured sample sterility and consistent thawing performance. 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

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

Protocol

How to Cryopreserve Human Pluripotent Stem Cells in CryoStor® CS10

Brochure

Cryopreservation Media for Stem Cell Research

7:57

Video

How to Generate Cell Aggregates and Passage Human Pluripotent Stem Cells (hPSCs) in mTeSR™ Plus

21:53

Webinar

Improving Reproducibility of Your hPSC Research by Generating a High-Quality Cell Bank

View All Resources

View More Resources for Your PSC Research >