CryoStor® CS10 is a serum- and animal component-free freezing medium, recommended for the cryopreservation of sensitive cell types, including human embryonic and induced pluripotent stem cells (ES and iPS cells).

For a more detailed version of this summary protocol, please refer to STEMCELL Technologies’ Technical Manual: Maintenance of Human Pluripotent Stem Cells in mTeSR™ Plus. For additional tips and tricks, refer to the Tech Tip: Cryopreservation and Thawing of ES/iPS Cells.

Materials

• CryoStor® CS10 (Catalog #07930)
• Gentle Cell Dissociation Reagent (Catalog #07174)
• mTeSR™ Plus (Catalog #100-0276)
• Cryovials (e.g. Corning® Cryogenic Vials with Orange Caps; Catalog #100-0091)
• 15 mL Conical tube (e.g. Falcon® Conical Tubes, 15 mL; Catalog #38009)
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• 2 mL Sterile pipette tips
• Isopropanol freezing container (e.g. Nalgene, Fisher Catalog #1535050)
• -150˚C Freezer or liquid nitrogen vapor tank
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Protocol

This procedure has been optimized for use with human pluripotent stem cell (hPSC) maintenance reagents and multiple ES and iPS cell lines passaged and frozen as aggregates. For upstream protocols and source materials, please see the mTeSR™ Plus Technical Manual and the Product Information Sheet for STEMCELL’s highly quality-controlled Healthy Control Human iPSC Line, Female, SCTi003-A. To freeze single cells, we recommend using FreSR™-S.

Before You Begin: The following procedure is based on human ES or iPS cell cultures grown in mTeSR™ Plus in 6-well plates. 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.
1. Mark regions of differentiation in the culture on the bottom of the plate.
2. Remove regions of differentiation by scraping with a pipette tip or by aspiration.
   
   
   Note: Avoid having the culture plate out of the incubator for more than 15 minutes at a time.
3. Aspirate remaining medium. Add 1 mL Gentle Cell Dissociation Reagent (GCDR) to each well and incubate at room temperature for 5 - 8 minutes.
   
   
   Note: The incubation time may vary when using different cell lines, other passaging reagents, or other culture media; dissociation should be monitored under the microscope until the optimal time is determined.
4. Aspirate the GCDR, and add 1 mL of mTeSR™ Plus. Gently detach the colonies by scraping with a serological glass pipette or a cell scraper, keeping cell aggregates large.
5. Transfer aggregates to a 15 mL conical tube using a 2 mL serological pipette.
6. Centrifuge at 300 x g for 5 minutes at room temperature. Gently aspirate the supernatant, keeping the pellet intact.
7. Add 1 mL cold CryoStor® CS10 per well harvested to the pellet.
   
   
   Note: Wipe down the outside of the bottle with 70% ethanol or isopropanol before opening.
8. Use a 2 mL serological pipette to dislodge the pellet, minimizing the break-up of cell aggregates.
9. Gently transfer the cell suspension to the cryopreservation vial using a 2 mL serological pipette.
10. Cryopreserve cell aggregates using either:
    1. A standard slow rate-controlled cooling protocol that reduces temperatures at approximately -1°C/min, followed by long-term storage at -135°C (liquid nitrogen) or colder. Long-term storage at -80°C is not recommended.
    2. A multi-step protocol where cells are kept at -20°C for 2 hours, followed by -80°C for 2 hours, followed by long-term storage at -135°C (liquid nitrogen) or colder.