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 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

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Related Resources

Protocol

How to Cryopreserve Human Pluripotent Stem Cells in CryoStor® CS10

Brochure

Cryopreservation Media for Stem Cell Research

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Video

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

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Webinar

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

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