Cryopreservation of Lymphocytes

[pipette_man]

Does anyone have any ideas of how to do cryopreservation?

thank you all,
pipette_man

[danfive]

Hope this helps, it came from http://www.invitrogen.com/contentFra...p?p_faqid=1890

Cryopreservation of Mammalian Cells----From Invitrogen FAQ Database

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% DMSO (dimethylsulfoxide)
---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: For serum-free medium, some of the common media constituents may be:
---50% cell-conditioned serum free medium and 50% fresh serum-free medium containing 7.5% DMSO
---fresh serum-free medium containing 7.5% DMSO and 10% cell culture grade BSA


Protocol for Suspension Cultures:
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. Resuspend cells in freezing medium to a concentration of 1 x 10^7 to 5 x 10^7 cells/ml for serum containing medium, or 0.5 x 10^7 to 1 x 10^7 cells/ml for serum-free medium. Aliquot into cryogenic storage vials. Place vials on wet ice or in a 4°C refrigerator, and start the freezing procedure within 5 minutes. 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:
Detach cells from the substrate with dissociation agents. Detach as gently as possible to minimize damage to the cells. Resuspend the detached cells in a complete growth medium and establish the viable cell count. 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. Resuspend cells in freezing medium to a concentration of 5 x 10^6 to 1 x 10^7 cells/ml. Aliquot into cryogenic storage vials. Place vials on wet ice or in a 4°C refrigerator, and start the freezing procedure within 5 min. 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 supernate. Gently Resuspend cells in complete growth medium and perform a viable cell count. Plate the cells. Cell inoculum should be at least 3 x 10^5 cells/ml.


Direct Plating Method:
Remove cells from storage and thaw quickly in a 37°C water bath. 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 10^5 cells/ml. Culture cells for 12 to 24 h. Replace medium with fresh complete growth medium to remove cryopreservative.