I was freezing spontaneously immortalized mouse embryonic fibroblasts and forgot them in -20°C overnight (stupid, I know, but it happened). I moved them to -80°C in the morning, but I am not sure whether it is worth it to keep these cells or it is better to get rid of them as they might not be able to set a culture anymore. What do you think? What is your experience?
Many years ago before the Mr Frosty was invented we put the vials containing the cells and freezing media on ice for 30 mins, then -20 overnight, then -80 for 1 day then moved to liquid nitrogen. Freeze slow...thaw fast... was what I was always told! Good luck.
Thank you Miao. These cells are in freezing medium to store aliquots if I wanted to start cell culture in the future. Normally, I freeze like this: about 20 minutes in fridge, then 20 minutes in -20, then I move the cells to -80 at least overnight, then to liquid nitrogen. This time I accidentaly forgot them in -20 overnight...
It might be a problem, if you have sensitive cells. You could try to thaw one aliquot of cells to see if the cells are alive.
To freeze cells, you could use a "Mr frostie" box or you could put your tubes with cells in a box filled with cotton. In both case, you have a slow decrease of the temperature when the box is put at -80°C.
Sometimes, when we don't have available "Mr frostie" box, we just do exactly the same - keep the cells (cell lines!) at -20°C overnight before transferring them to -80°C and everything goes fine. Of course, might be a problem for primary and more sensitive cells, but I would definitely give it a try anyway after more deep freezing.
Thank you for your answers. I will thaw one vial and check the viability. These are already cell lines (immortalized cells) so it might work.
once you add freezing medium to the cells, we should immediately transfer the cells to Frostie, or any box with cotton (for support) and Iso-propyl alcohol . the concept is It reduces temperature gradually (1degree C per hour). Anyways, the aim is slow cooling of the sample to avoid temperature shock to cells. so, 12hrs in frostie wil reduce 12 degree C no matter whether its in -80 or -20. So clearly it doesn make difference . You can keep it in -20 for one day and later to -80 for at least two days and then to liquid nitrogen.You can revive and check the viability of your samples, for your confirmation.
Anyways, give your cells a try storing it and once revived , do post your observation :)
Thanks :) I checked the viability in one of the vials (after 12 hours in -20, about 8 hours in -80) and it is about 62 % - no big success, but also not a complete mess. Thanks for all your answers once more :)
For fast and safe freezing, you can use Cryo freezing container (Nalgene Mr.Frosty cryo 1℃ Freezing container, Thermo scientific, Cat No. 5100-0001). In this container, you can directly place the cryogenic vials contains cells with freezing medium and keep the container in -80 C for 4 hours and then transfer the frozen vial to liquid nitrogen storage.
https://extranet.fisher.co.uk/webfiles/fr/Pjointes/Mdemploi/NAL001_EN%20MINICOOLER%20MR%20FROSTY.pdf
Many years ago before the Mr Frosty was invented we put the vials containing the cells and freezing media on ice for 30 mins, then -20 overnight, then -80 for 1 day then moved to liquid nitrogen. Freeze slow...thaw fast... was what I was always told! Good luck.
What if you left the cells in -4C overnight (~12h) by mistake and then used Mr. Frosty next day. I would appreciate it if you could let me know whether the viability is still worth keeping. These are precious cells.
Thanks.
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