I have tried multiple times to cryopreservation of SH-SY5Y cells, but afther thawing, no cells had survived. I bring the cells to cold (4 degrees Celsius) DMEM/F-12 medium with 5% DMSO. After that, I placed the cells directly in -20 degrees celsius for 10-20 minutes and after that 24h in -80 C freezer. After that, cells were stored in liquid nitrogen.
We have multiple times used this method, but the cells did not survive. What can I do the next time or does anyone have some tips to think about?
When you say you place directly at -20, you just put the tubes directly at -20?
You need to make a controlled rate freezing. If you don´t have any slow passive freezing device such as Mr Frosty or Coolcell, you can use a Styrofoam box filled with cotton surrounding the cryovials. With Mr frosty or coolcell you can put directly to -80, with a Styrofoam box with cotton I would use 30 minutes at -20 and then overnight to -80.
Additionally it was not clear from your description if you used serum in the freezing medium, I would advice you to do that. Using 10% serum, 5-10% DMSO and medium is a basic freezing media. Adding more % of serum sometimes helps too, even using 5-10% DMSO in serum works better for some cell lines!
Hope it helps!
Thank Diego for your answer. Yes, after adding the cells to the vials, I direct put the tubes in -20.
Okay, I will look at the possibilities in our department for the Mr Frosty, Coolcell and Styrofoam box. I use DMEM/F12 medium with 10% of serum and 5% DMSO.
I am also agree with Dr. Raffo suggestions. One more thing, you can use the FBS:DMSO at the ration of 9:1.
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