Hi! The literature (even here in RG) does not provide a clear answer about the best composition for cryopreservative medium to freeze mammal cell lines, specially regarding to the serum component. In your experience, is FBS recommended or not? if yes, which percentage? Does it change between different cell lines or are there standar compositions? Thank you
The best method is to use your growth media with 10-20% FBS plus 10% DMSO.
You can use glycerol instead of DMSO but in my opinion does not work as well.
Cells must be frozen slowly (use an insulated container) at -80oC before transfer to Liquid N2.
Hi Md,
your freezing media is good and I have used it as well, however, it is very expensive if you are freezing a lot of cells. 10-20% FBS in normal medial really does work as well.
Thank you both for your answers. This is exactly the discrepancy I meant: 90-95 vs 10-20% FBS. Do you think that the reason of putting 10 instead of 90% is just to save money? Because, for example, I tried to freeze 293T cells with 95% FBS + 5% DMSO and they all died; but they were well cryopreserved when I did with 20% FBS and 10% DMSO in DMEM
I would propose a simple 20:1 solution of FBS and DMSO (sterile) for cell cryopreservation.
Lucas, your cells died for different reasons.
All the freezing media suggested should work for 293 cells.
Lucas Taoro Mammalian cell lines varies considerably in their growth and storage requirements. For this simple reason, the composition of freezing media needs to be modified to a certain extent according to the mammalian cell line to applied to. Regarding the specific percentage of FBS, it is usually 10-20% in most of the cases. One the reason why FBS is used in freezing media is partly that it possesses antioxidative effecrs which is used to counter the oxidative stress generated from cryopreservation during the freeze-thawing process. Next, to answer your question on the necessity of FBS, usually it will be better if alternatives are used. This is primarily due to few concerns: 1) Batch to batch variations that exist in FBS. 2) Possibility of viral contamination has been raised regarding the use of FBS. 3) There have been considerable amount of concerns that bovine spongiform encephalopathy may be transmitted through the use of FBS. FBS is currently a undefined media (meaning its composition has not been completely identified). FBS is now still widely used. As such i would say the use of FBS will still continue although the supplementation of alternatives is a increasing trend. Hope all your questions have been answered fully.
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