Cell cryopreservation and resuscitation

Huaqiang Square crystal original spot stock

Cryopreservation and resuscitation of cells

Cells should be suspended at a density of 5x10⁵ to 1x10⁶ cells/mL in a freezing medium containing DMSO or glycerol. Alternatively, for lower concentrations (0.5x10⁵ to 1x10⁵ cells/mL), use serum-free medium for resuspension.

Transfer the cell suspension into cryovials and place them on wet ice or in a 4°C refrigerator. Begin the freezing process within 5 minutes to ensure optimal preservation.

The cells are then frozen at a controlled rate of 1°C per minute. They can be stored in a programmable freezer or directly in a -70°C to -90°C freezer before being transferred to liquid nitrogen for long-term storage.

Adherent Cell Cryopreservation

1. Gently detach the cells from the culture surface using a suitable dissociation reagent to minimize cellular damage.

2. Resuspend the cells in complete growth medium and determine the viable cell count.

3. Centrifuge the cells at approximately 200g for 5 minutes. Carefully remove the supernatant, leaving only a minimal volume to avoid disturbing the cell pellet.

4. Resuspend the cells in cryopreservation medium at a concentration of 5x10⁵ to 1x10⁶ cells/mL.

5. Transfer the cell suspension into cryotubes and place them on wet ice or in a 4°C refrigerator. Start the freezing process within 5 minutes.

6. Freeze the cells at a controlled rate of 1°C per minute and store them in a -70°C to -90°C freezer, or transfer to liquid nitrogen for long-term storage.

Resuscitation of Cryopreserved Cells

Cryopreserved cells are more fragile and should be handled with care. Thaw the vials rapidly in a 37°C water bath. Immediately add the cells to pre-warmed complete growth medium.

If the cells are sensitive to cryopreservation agents like DMSO or glycerol, centrifuge the cells to remove the cryopreservation medium before resuspending in fresh growth medium.

Direct Plating Method

1. Remove the cryovial from storage and thaw it quickly in a 37°C water bath.

2. Add the thawed cells directly to 10–20 mL of pre-warmed complete growth medium. Ensure the cell density is at least 3x10⁵ live cells/mL.

3. Incubate the cells for 12–24 hours. Replace the medium with fresh complete growth medium to remove any residual cryopreservation agents.

Centrifugation Method

1. Thaw the cryovial in a 37°C water bath.

2. Add 1–2 mL of the cryopreserved cell suspension to approximately 25 mL of pre-warmed complete growth medium. Mix gently.

3. Centrifuge at around 80g for 2–3 minutes.

4. Carefully discard the supernatant without disturbing the cell pellet.

5. Resuspend the cells in fresh complete growth medium and perform a viability count.

6. Plate the cells at a minimum of 3x10⁵ live cells/mL for optimal recovery and growth.