Before cryopreservation cells should be checked for contamination. Cryopreservation reagents of the CryoMaxx product line can be used with any standard freezing protocol.

1. Count the number of viable cells to be cryopreserved. Cells should be in mid-log phase of growth. Centrifuge the cells for 5 min to pellet cells (200 to 400 x g). Remove the supernatant down to the smallest volume without disturbing the cells.
2. Resuspend cells in pre-cooled (4°C to 8°C) CryoMaxx to a concentration of 5 x 10⁶  to 1 x 10⁷ cells/ml.
3. Aliquot into cryogenic storage vials. Place vials at 4°C and start the freezing procedure within 5 min.
4. Cells are frozen slowly at 1°C/min (by programmable coolers or by placing vials in an insulated box in a ?70°C to ?90°C freezer).
5. Then transfer storage vials to liquid nitrogen storage.

1. Detach cells from the substrate with a gentle dissociating agent. Especially with sensitive cells use Accutase (Cat. No. L11-007) or Alfazyme (Cat. No. L11-012) to avoid cell damage. Inactivate dissociating agent if necessary.
2. Resuspend the detached cells in complete growth medium and establish the viable cell count.
3. Centrifuge for 5 min to pellet cells (200 to 400 x g). Remove the supernatant down to the smallest volume without disturbing the cells.
4. Resuspend cells in pre-cooled (4°C to 8°C) CryoMaxx to a concentration of 5 x 10⁶ to 1 x 10⁷ cells/ml.
5. Cells are frozen slowly at 1°C/min (by programmable coolers or by placing vials in an insulated box in a ?70°C to ?90°C freezer).
6. Then transfer storage vials to liquid nitrogen storage.

Cryopreserved cells can be thawed by the following procedures:

• Remove cells from storage and thaw quickly in a 37°C water bath. PAA recommends eye protection by using approved safety goggles. We also suggest the use of safety gloves to protect uncovered skin.
• Place 1 to 2 ml of thawed cells in ~25 ml of complete growth medium. Mix cell suspension gently.
• Centrifuge the cells at ~80 x g for 2 to 3 min.
• Check clarity of the supernatant and visibility of a consolidated cell pellet. Discard supernatant without disturbing the cells.
• Gently resuspend the cells in complete growth medium and perform a viable cell count.
• Plate the cells. Cell inoculum should be at least 3 x 105 viable cells/ml.

• Remove cells from storage and thaw quickly in a 37°C water bath.
• PAA recommends eye protection by using approved safety goggles. We also suggest the use of safety gloves to protect uncovered skin.
• Plate cells directly with complete growth medium. Use 10 to 20 ml of complete medium per 1 ml of frozen cells. Cell inoculum should be at least 3 x 10⁵ cells/ml.
• Culture cells for 12 to 24 h. Replace medium with fresh complete growth medium to remove cryopreservative.

We recommend thawing procedure 1, especially when handling sensitive cells.

For in vitro laboratory use or further manufacturing only. Not for human use.