Hi all!
I have been thawing a few cell line stocks we had kept in liquid nitrogen and faced a couple of problems not being able to get the cells adhere to the T25 flask easily. I am not sure if it is because the freezing procedure might have been done differently by the previous members (I don't have access to them now). So when I thawed HEK293T that was frozen in Jul 2020 but kept in -80 deg celsius, the cells adhered properly and reached a good level of confluence in about 3 days. But for the following cell lines that were stored in liquid nitrogen (which I expect to see better viability), they were rounded even after 3 days. If I try to change the media, the cells would just be washed away:
HCT 116 (frozen down Mar 2017)
HEK 293T (frozen down Jul 2019)
Beta TC-6 (frozen down Mar 2017)
As for the following two cell lines, there was some growth only after 3 days. Prior to that, they were rounded and unattached.
MCF7 (frozen down Apr 2020 and kept in -80)
HT29 (frozen down Apr 2020 and kept in -80)
So I am not sure if it is because of the liquid nitrogen storage that this is happening. I could wait a couple more days to see how the lines from LN2 turn out, but I am also worried about changing the media as it might very well wash the cells away.
As for thawing, I do it quickly (2 mins at 37 deg cel) for any cell line that has been frozen down (both LN2 and -80).
I would really appreciate it if someone could share their insights into this issue!
Best Regards,
Mathangi
Dear Mathangi,
Some cell lines take more than three days to grow. But it should attach within 24 hours.
There are some reasons for cells, not attachment
Overly trypsinized cells (use less trypsin or trypsinize for short time)
Mycoplasma contamination
No attachment factor (serum formulation) in the medium
And please double-check the storage media which they used to store the cells previously.
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