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help with cryopreservation - (Feb/12/2002 )

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-20 overnight, -80 overnight followed by liquid nitrogen


Dear carbio,
as mentioned by sia i used to use the old medium (conditioned medium) while thawing cells, the only precaution you should take is you should not grow cells more than 24 hours at an appropriate density. the reason is when you grow cells longer in medium they started releasing some cytotoxic mediators (may be from dead cells).
i would use condition medium, but it should be prepared in carefull manner, other wise when you add conditioned medium to thawed cells from liquid nitrogen the cytotoxic mediators will give boosting effect to celll death.


QUOTE (Carbio @ Jul 3 2005, 05:42 PM)
QUOTE (sia @ Jun 27 2005, 07:27 PM)
I general freezing cells using styrfoam boxes work fine however, results gets better if you do not keep your cells at 4C rather start from room temperature, then cover the styrofoam with another and place a rubber band, place it now in -20 C for 1-2 hour and then over night in -80c and then too liquid nitrogen.  Higher serum content in freezing medium helps.  You can also add old medium in which cells were growing that way when you will thaw they will have the natural growth factors available.

Interesting suggestion, Sia. I've never heard of using old, used medium to improve after-thawing culture. Has anybody else tried it?


QUOTE (nevermore1 @ Feb 12 2002, 10:36 PM)
I have had very good success in freezing cells by simply putting the Nunc vials (or whatever you're using) into a
stryofoam box in a -80 C freezer for 24 hours then transferring directly to liquid nitrogen storage.  The box should already be in the freezer before you start.  I've done this a couple of hundred times and always had viabilities of at least 70%.

hi... wat if i leave the cells in -70 for around 60 hrs... and then transfer them to liq nitrogen... wat can i expect wen i revive my cells.... which is NFS60 suspension cells


QUOTE (Daniel5306 @ Dec 17 2004, 12:06 AM)
My method is more simple. I directly put the tube into -80oC. Then keep there for years. The viability of the cells usually are more than 80%. I used this method for at least five kinds of different mammalian cells, it works very well. I have to mentioned that the medium what I used to store the cell were 90% of FBS and 10% of DMSO. Good luck.



Daniel's method works if you use 90 or 95% serum, it might not work if you use 90 or 95% complete medium and rest of the DMSO.


Hi everyone. This is my first post here...

What we do at our lab is to use what we call "Llit de cèl·lules" (cell bed).. it's a small styrofoam box in which we layer: (bottom to top)

- Dry Ice (1 layer)
- Lab filter paper (2 layers)
- Wrinkled tissue paper (1 layer)
- Lab filter paper (1 layer)
- Dry ice (1 layer)
- Lab filter paper (1 layer)

The cells go in the dry ice sandwhiched between the two layers of filter paper.
The box is kept shut at all times to ensure the contents are cold enough when the cryovials are put inside.

Oh, and I use 9.5 ml FBS + 0.5 ml DMSO.

And finally we put the styrofoam box at -80ºC for a few days (up to 1 week) then liquid nitrogen.
It works perfectly fine for all kinds of cells we use (both adherent and suspension cells; HeLa, Jurkat, HEK, COS, MEF, tec etc)


Hi everyone,

This is my first time posting and i am also quite new to cell culture. I just realized that the medium of the cells that i froze last nite turned into yellow. Does that mean that all my cells in the vial are dead? It happened to other previous cells that i've frozen too, but the medium turned into bright pink instead. Can someone tell me what's with the colour changes and do you think my cells are still alive? And how can i avoid this in the future?

I should have try thawing them and see whether they are still alive but i just want to get myself ready to face the disaster cause these cells are my precious sad.gif

I hope someone can help me.

Thanks in advance!


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