CryoDefend-Cell Lines (5 x 10 mL)

Refer to the product datasheet for complete product details.

Reagents Provided

Reagents supplied in the CryoDefend^® Cell Lines Media (Catalog # CCM019):

• 5 x 10 mL vials of CryoDefend^® Cell Lines Media

Upon receipt, the CryoDefend^® Cell Lines Media should be stored at = -20 ^°C in a manual defrost freezer. The media can be thawed at 2 ^°C to 8 ^°C or at room temperature. Thawed media can be aliquoted and stored at = -20 ^°C in a manual defrost freezer for up to 3 months. Thaw a fresh aliquot for each use. Avoid repeated freeze-thaw cycles.

Other Supplies Required

Reagents

• Cell culture media

Materials

• Cultured cells
• Cryovials
• 15 mL centrifuge tubes
• Serological pipettes
• Pipette and pipette tips

Equipment

• 37 °C and 5% CO₂ incubator
• Centrifuge

Procedure Overview

R&D Systems Protocol for the Cryopreservation and Thawing of Stem Cells in CryoDefend^® Cell Lines Media (Catalog # CCM019)

Cryopreservation of Cultured Cells

1. Thaw CryoDefend^® Cell Lines Media.
2. Detach the cells from the cell culture dish.
3. Transfer the cells to a 15 mL conical tube.
4. Centrifuge at 200 x g for 5 minutes.
5. Remove and discard the supernatant.
6. Resuspendthe cells in CryoDefend^® Cell Lines Media at 0.5-1.0 x 10⁶ cells/mL.
7. Transfer the cells to a cryovial.
8. Freeze the cryovial at -80 ^°C overnight.
9. Transfer the frozen cryovial to liquid nitrogen for storage.

Thawing of Cryopreserved Cells

1. Warm appropriate cell culture media to 37 ^°C.
2. Add pre-warmed culture media to the cryovial containing cryopreserved cells.
3. Pipette up and down and as cells thaw, transfer the thawed cells to a 15 mL conical tube containing pre-warmed expansion media.
4. Centrifuge at 200 x g for 5 minutes.
5. Resuspend the cells in pre-warmed expansion media.
6. Plate the stem cells at the desired density.