Freeze human cells at -80°C

[Mega [Andreas Stuermer]]

Hi everyone,

Today at University I heard that human cells from the -80°C freezer often don't work after thawing. Especially when being frozen for a year or more.
I always thought -80°C would work well and -130°C would be better but optional.

Is this due to our freezer, or do you have similar experience?

[SC]

Hi Mega,

I would think it would have something more to do with the preparation process than with the freezer.  -80 is commonly used for vertebrate cells.  It may also have to do with the condition of the cells when they are frozen.  Was it a cell culture taken at exponential growth phase?  Or just some cells from a person?
Stacy

[Mega [Andreas Stuermer]]

Thanks! The cells mentioned were stocks of HeLa and thelike...

How about primary human cells? Just harvest, add DMSO and freeze? Does the temperature gradient play a big role?

[Ravasz]

Hi,

This is definitely true for all the cell line storages I have ever dealt with. The key is long-term storage. Human cells are okay at -80 for a month or two, but after a year of storage at -80 it is strongly advised to discard them. I was told that viability decreases, and even if the cells do thaw, they will contain epigenetic and other changes that will ultimately change the cell line and this is not what we want. I never tested this though, but I accepted it as this is the norm everywhere.

Liquid nitrogen (-200) is the recommended long term storage for human cells.

Mate

[Claudio Tiecher]

Hi,

in my lab mammalian cell lines are stored in liquid nitrogen.

Cheers,
Claudio

[Michael Tellier]

It seems also to depends on the cell line. HeLa, HEK293, or U2OS survive longer at -80 than immortalized B cells from what I have observed. Even afte ra year and half at -80C, some of my U2OS cell lines were still alive but with a good 80/90% death rate on thawing so it is likely they had mutations/epigenetic changes (but depending of the experiment you want to perform it can be not important).  

Freezing medium also play a role in thawing survival. Some of my cell lines survived better in 50% fresh medium, 40% FBS, 10% DMSO than 90% FBS and 10% DMSO for example. DMSO is also toxic for the cells at 37 degrees (should also be at room temperature) which is why the cells need to be put in a medium as soon as possible after thawing (warm medium also improve cell survival). For the freezing medium, it is better to follow what is written on the ATCC website since it is tailored for each cell line.

But yeah as a rule, it is better to keep cell lines in liquid nitrogen. 

[Mega [Andreas Stuermer]]

Thanks to all for sharing your knowledge and expertise!!