Cryopreservation of Mammalian Cells

Mammalian cells are cryopreserved to avoid loss by contamination, to minimize genetic change in continuous cell lines, and to avoid aging and transformation in finite cell lines.  Before cryopreservation, cells should be characterized and checked for contamination.  There are several common media used to freeze cells.

For serum-containing medium, the constituents may be as follows:

• complete medium containing 10% glycerol
• complete medium containing 10% DMSO (dimethylsulfoxide)
• 50% cell-conditioned medium with 50% fresh medium with 10% glycerol or 10 % DMSO

Cryopreservation media generally consists of a base medium, cryopreservative, and a protein source.  The crypreservative and protein protect the cells from the stress of the freeze-thaw process.  A serum-free medium has generally low or no protein; but one can still use it as a base for a cryopreservative medium in the following formulations:

For serum-free medium, some of the common media constituents may be:

• 50% cell-conditioned serum free medium with 50% fresh serum-free medium containing 7.5% DMSO
• fresh serum-free medium containing 7.5% DMSO and 10% cell culture grade DMSO.

Protocol:  Suspension Cultures

1. Count the number of viable cells to be cryopreserved.  Cells should be in log phase.  Centrifuge the cells at ~200 to 400 x g for 5 min to pellet cells.  Using a pipette, remove the supernate down to the smallest volume without disturbing the cells.
2. Resuspend cells in freezing medium to a concentration of 1 x 10⁷ to 5 x 10⁷ cells/ml for serum containing medium, or 0.5 x 10⁷ to 1 x 10⁷ cells/ml for serum-free medium.
3. Aliquot into cryogenic storage vials.  Place vials on wet ice or in a 4°C refrigerator, and start the freezing procedure within 5 minutes.
4. Cells are frozen slowly at 1°C /min.  This can be done by programmable coolers or by placing vials in an insulated box placed in a -70°C to -90°C freezer, then transferring to liquid nitrogen storage.

Protocol:  Adherent Cultures

1. Detach cells from the substrate with dissociation agents.  Detach as gently as possible to minimize damage to the cells.
2. Resuspend the detached cells in a complete growth medium and establish the viable cell count.
3. Centrifuge at ~200 x g for 5 min to pellet cells.  Using a pipette, withdraw the supernate down to the smallest volume without disturbing the cells.
4. Resuspend cells in freezing medium to a concentration of 5 x 10⁶ to 1 x 10⁷ cells/ml.
5. Aliquot into cryogenic storage vials.  Place vials on wet ice or in a 4°C refrigerator, and start the freezing procedure within 5 min.
6. Cells are frozen slowly at 1°C /min.  This can be done by programmable coolers or by placing vials in an insulated box placed in a -70°C to -90°C freezer, then transferring to liquid nitrogen storage.

Reference:

1. Freshney, R. (1987) Culture of Animal Cells: A Manual of Basic Technique, p. 220, Alan R. Liss,Inc., New York.

To thaw cryopreserved cells, see this protocol