Recovery™ Cell Culture Freezing Medium

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:
1) Complete medium containing 10% DMSO (dimethylsulfoxide)
2) 50% cell-conditioned medium with 50% fresh medium with 10% DMSO

Cryopreservation media generally consists of a base medium, cryopreservative, and a protein source. The cryopreservative 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:

1) 50% cell-conditioned serum free medium and 50% fresh serum-free medium containing 7.5% DMSO
2) Fresh serum-free medium containing 7.5% DMSO and 10% cell culture grade BSA

Protocol for Suspension Cultures:
1. Count the number of viable cells to be cryopreserved. Cells should be in log phase.
2. Centrifuge the cells at ~200 to 400 x g for 5 min to pellet cells.
3. Using a pipette, remove the supernatant down to the smallest volume without disturbing the cells.
4. Resuspend cells in freezing medium to a concentration of 1 x 10E7 to 5 x 10E7cells/ml for serum-containing medium, or 0.5 x 10E7to 1 x 10E7 cells/ml for serum-free medium.
5. Aliquot into cryogenic storage vials. Place vials on wet ice or in a 4°C refrigerator, and start the freezing procedure within 5 minutes.
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.

Protocol for 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.
4. Using a pipette, withdraw the supernatant down to the smallest volume without disturbing the cells.
5. Resuspend cells in freezing medium to a concentration of 5 x 10E6 to 1 x 10E7 cells/ml. Aliquot into cryogenic storage vials.
6. Place vials on wet ice or in a 4°C refrigerator, and start the freezing procedure within 5 min.
7. 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: Freshney, R. (1987) Culture of Animal Cells: A Manual of Basic Technique, p. 220, Alan R. Liss,Inc., New York.

Thawing of Cryopreserved Cells:
Cryopreserved cells are fragile and require gentle handling. Thaw cells quickly and plate directly into complete growth medium. If cells are particularly sensitive to cryopreservation (DMSO or glycerol), they are centrifuged to remove cryopreservative and then plated into growth medium. The following are suggested procedures for thawing cryopreserved cells:

- Centrifugation Method: Remove cells from storage and thaw quickly in a 37°C water bath. Place 1 or 2 ml of frozen cells in ~25 ml of complete growth medium. Mix very gently. Centrifuge cells at ~80 x g for 2 to 3 min. Discard supernatant. Gently Resuspend cells in complete growth medium and perform a viable cell count. Plate the cells. Cell innoculum should be at least 3 x 10E5 cells/ml.
- Direct Plating Method: Remove cells from storage and thaw quickly in a 37°C water bath. Plate cells directly with complete growth media.

Our Cell Culture Freezing Media is composed of DMEM, FBS, calf serum, and 10% DMSO. This is useful for many mammalian cells for freezing and is the same percentage of DMSO used in most methods. This product will work fine for most adherent cell lines grown with serum. A good general protocol for freezing cells can be found on our website by searching "Freezing cells protocol" from the home page.

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Recovery Cell Culture Freezing Medium is meant for general cell culture applications where a wide range of growth media will be used. We recommend that you aspirate Recovery Cell Culture Freezing Medium before seeding, although other protocols can be substituted (this will need to be determined by the end user).

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Recovery Cell Culture Freezing Medium should be stable at 2 to 8 degrees C for 1 week, although we have no data to support this.

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There have not been any bench studies at this time. It is best to aliquot this product.

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