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).

Cryopreservation should be done approximately one day before cells are ready for passaging. Human ES and iPS cells demonstrate improved post-thaw survival if cryopreserved in large aggregates.

For a more detailed version of this summary protocol, please refer to STEMCELL Technologies’ Technical Manual: Maintenance of Human Pluripotent Stem Cells in mTeSR™1. 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)
• DMEM/F-12 with 15 mM HEPES (Catalog #36254) or mTeSR™1 (Catalog #85850)
• 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. 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.

Before You Begin: The following procedure is based on human ES or iPS cell cultures grown in mTeSR™1 in 6-well plates (60 - 70% confluence, < 20% differentiation).
1. Bring the required amount of CryoStor® CS10 to room temperature (15 - 25˚C).
2. Mark regions of differentiation in the culture on the bottom of the plate.
3. Scrape off the marked regions with a pipette tip (or aspirate).
4. Aspirate remaining medium. Rinse wells with 2 mL DMEM/F-12 with 15 mM HEPES, then aspirate.
5. Add 1 mL Gentle Cell Dissociation Reagent to each well and incubate at room temperature for 5 - 8 minutes.
   
   
   Note: The incubation time may vary when using different cell lines, other enzymatic passaging reagents, or other culture media; dissociation should be monitored under the microscope until the optimal time is determined.
6. Remove Gentle Cell Dissociation Reagent.
7. Add 1 mL DMEM/F-12 with 15 mM HEPES or mTeSR™1 to each well. Scrape colonies off, keeping cell aggregates large.
8. Transfer aggregates to a 15 mL conical tube. Centrifuge at 300 x g for 5 minutes at room temperature. Aspirate the supernatant, keeping the pellet intact.
9. Add 1 mL CryoStor® CS10 per well harvested to the pellet. Using a 1 mL pipette tip, pipette up and down once to dislodge the pellet. Gently transfer the cell suspension to the cryopreservation vial.
10. Place vial in an isopropanol freezing container at -80˚C to -150˚C overnight. Transfer to liquid nitrogen the next day.
    
    
    Note: Long-term storage at -80˚C is not recommended.