Hi,

in general long-term storage of cell lines should be done at ultra-low temperatures less than -135 °C. You can maintain this temperatures in an electric freezer or by using liquid nitrogen. You should store the cells as cold as possible, at least -70 °C, to prevent a deterioration of the cells. This would result in a low viability when thawing the cells. So a storage in the gas phase of liquid nitrogen would be the best option.

For cryopreservation you should add a cryoprotective agent to your media, like dimethyl sulphoxide (DMSO) or glycerol, usually at a final concentration of 10 %. This reduces the formation of ice crystals, which can destroy the cells, and will result in a higher viability when thawing the cells. You should also ensure a constant and slow cooling rate for your cells, usually 1 °C per minute.

You can find generall informations and protocols for cryopreservation of cell lines in the links below

http://www.sigmaaldrich.com/technical-documents/protocols/biology/cryopreservation-and.html

https://www.thermofisher.com/de/de/home/references/gibco-cell-culture-basics/cell-culture-protocols/freezing-cells.html

It was a very detailed explanation Simon but I really want to know can we skip -70*C step and replace it with -20*C????

Hello Zain,

To be specific. No, you can't replace -70 with -20 as even an overnight storage in -20 will result in the compromise in cell viability. So it's best that you use -70/-80 for cell storage as nicely explained by Simon but in many cell lines snap freeze (-1 per minute) is not required. I have also published a paper on the same. You can put cells directly into -70/-80 but thawing step is really important.

Hope it helps!

Hi Ajay..!!

So nice of you to share your thoughts. Thanks :)