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Thawing Hek 233t cells- but cells dying!!


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#1 Lenti_NN

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Posted 24 April 2012 - 07:54 AM

Hi Guys,

I froze a batch of Hek 293t cells few months ago and have been using them since then and they were fine till recently when I wanted to thaw a new vial from the same batch the cells would not adhere or grow and all the cells almost died within 12 hours and media was completely pink. I am not sure what is going on with these cells, I froze them in DMSO in -80.
I tried thawing 3 vials and all of them had the same outcome, I tried using a new bottle of media still the same.
I am completely puzzled any suggestions/advice would be really helpful.
Thanks so much

#2 bob1

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Posted 25 April 2012 - 04:42 PM

Cells do not survive more than a few months at -80. They should be stored in the vapour phase of a LN2 container to ensure continued viability.

If you wish to store at -80, you will need to freeze cells down at frequent intervals and not store for more than a couple of months to ensure that they have not changed from the parent line!

#3 Lenti_NN

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Posted 26 April 2012 - 06:53 AM

Its been liitle over 5 months that they have been sitting in -80. Do you think 5 months period in -80 is harmful to the viability of the cells?

#4 bob1

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Posted 26 April 2012 - 01:30 PM

yes!




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