I am enriching breast cancer patient blood samples for circulating tumour cells (CTCs) that i would like to store and work on later. I have read a paper where glycerol is used but no concentration has been specified (i have tried contacting the researcher but no luck), as these are very rare and precious samples, i would like to be sure of the concentration s as not to induce cytotoxicity.
Thank you!
Dear Allison,
Please read the following text:
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:
https://www.thermofisher.com/ps/en/home/references/gibco-cell-culture-basics/cell-culture-protocols/cryopreservation-of-mammalian-cells.html
Another reference indicates the following:
Glycerol is generally used at a final concentration of between 5 and 20% (v/v).
Sterilize by autoclaving for 15 minutes in small volumes (5mL) and refrigerate in
the dark. Although less toxic to cells than DMSO, glycerol frequently causes osmotic
problems, especially after thawing. Always add it at room temperature or above and
remove slowly by dilution.
https://www.corning.com/media/worldwide/cls/documents/t_cryoanimalcc.pdf
Hoping this will be helpful,
Rafik
It might be best to try with some similar cells, or a cancer cell line first, to refine the amounts and the freezing/thawing times. When cryopreserving PBLs we trialled several freezing programs, mediums and thawing times to ensure maximum viability from our cells.
We found the best viability at 80%, worked with antibody negative human serum (bovine might be ok for CTCs), 10% DMSO, no glycerol, and an incredibly gradual freezing program, (0.5 degrees per minute for the first half 45 mins from memory). The thawing process is also critical, best done gently and quickly.
Are you using a graduated cooling system, or in an insulated container in the minus 80 freezer?
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