Cell cryopreservation and resuscitation

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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 another cryoprotectant. For optimal results, ensure the cell suspension is well mixed before proceeding.

Transfer the cell suspension into cryogenic vials and place them on wet ice or in a 4°C refrigerator. Begin the freezing process within 5 minutes to maintain cell viability.

The freezing process should be carried out at a controlled rate of 1°C per minute. This can be achieved using a programmable freezer or by placing the vials in a -70°C to -90°C freezer. Once frozen, transfer the vials 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 cell damage.

2. Resuspend the cells in complete growth medium and determine the viable cell count using a hemocytometer or automated cell counter.

3. Centrifuge the cells at approximately 200g for 5 minutes. Carefully remove the supernatant, leaving as little as possible without disturbing the cell pellet.

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

5. Transfer the cell suspension into cryovials 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 rate of 1°C per minute. Store the vials in a -70°C to -90°C freezer or a programmable cryo-freezer, then transfer to liquid nitrogen for long-term storage.

Resuscitation of Cryopreserved Cells

Cryopreserved cells are more fragile and require careful handling. Thaw the vials rapidly in a 37°C water bath, ensuring minimal exposure time. Immediately add the cells to pre-warmed complete growth medium.

If the cells are sensitive to cryopreservation agents like DMSO or glycerol, it is recommended to centrifuge the cells and 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 cell suspension directly to 10–20 mL of pre-warmed complete growth medium. Ensure gentle mixing to avoid cell damage.

3. Count the viable cells and seed them at a minimum of 3x10⁵ live cells/mL.

4. Incubate the cells for 12–24 hours, then replace the medium with fresh complete growth medium to remove any residual cryopreservation agent.

Centrifugation Method

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

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

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

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

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

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