I was looking for ingredients of serum free freezing medium but I cannot find anywhere or its discussion in this research gate.
So Can anyone share your knowledege on making serum free freezing medium for cryopreservation. Thank you.
Dear Jamail Lu!
Here is the complete protocol, together with freezing of suspension culture and adherent
culture! Good luck!
Cryopreservation of mammalian cells
Mammalian cells are cryopreserved to avoid loss by contamination, to minimize a genetic change in continuous cell lines, and to avoid ageing and transformation infinite cell lines. Before cryopreservation, cells should be characterized and checked for contamination. Several media are commonly 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 consist 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 you 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 BSA.
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 achieved using a programmable cooler 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 should be frozen slowly at 1°C/min. This can be achieved using a programmable cooler or by placing vials in an insulated box placed in a –70°C to –90°C freezer, then transferring to liquid nitrogen storage.
If there are some unclear things, please contact me again1
Sincerely Yours!
Dr. Bratko Filipič
Email: [email protected]
Dear Jamail Lu!
Here is the complete protocol, together with freezing of suspension culture and adherent
culture! Good luck!
Cryopreservation of mammalian cells
Mammalian cells are cryopreserved to avoid loss by contamination, to minimize a genetic change in continuous cell lines, and to avoid ageing and transformation infinite cell lines. Before cryopreservation, cells should be characterized and checked for contamination. Several media are commonly 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 consist 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 you 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 BSA.
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 achieved using a programmable cooler 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 should be frozen slowly at 1°C/min. This can be achieved using a programmable cooler or by placing vials in an insulated box placed in a –70°C to –90°C freezer, then transferring to liquid nitrogen storage.
If there are some unclear things, please contact me again1
Sincerely Yours!
Dr. Bratko Filipič
Email: [email protected]
We are using 90% KnockOut-Serum Replacement (Gibco)+ 10% DMSO for our stem cells (pluripotent and neural) and have a good recovery rate (frozen down at -1°C/min). Just to be sure, filter it before use and make fresh freezing medium everytime you need it. We have an internal "shelf-life" of 1 week for this medium (@4°C).
Dear Jamal, you have to telll what type of cell you are going to freeze. The colleagues gave a very strong idea of media for freezing cells, but they may not be the right ones for freezing sperm by example. In this case, you have to tell the species too, because media are very different for each ones. Cheers
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