Hi everyone !
I am working with Thermo Expi293 cells for a while for high-yield protein production in lab scale. Sometimes, viability of the cells decreases day by day although nothing was changed in culture conditions. Is there a way I can increase the viability again or can I somehow remove dead cells from the healthy ones? Also, I freeze the cells as Thermo recommends (10%DMSO, 45% fresh media and 45% conditioned media) but after thawing, viability is generally low (70-80%).
If you have any idea or recommendation please share with me.
Best wishes
Miray
Hello, Miray!
I think since the dead cells are weakly adherent or float in the medium, the medium can be carefully drained and replaced with a new medium. Together with the environment, the cells will be removed. To increase the viability of cells after thawing, they can simply be cultured for several passages in a medium with an increased serum content (20-30%).
Dear Miray Turk
For the removal of the dead cells wash thrice with 1x PBS buffer (1% of Penicillin G sodium salt, Ampicillin, Amphotericin B). At the same time, retrieval time you have to use 106 medium with 10 to 20 % of Low serum growth supplement. It will give a good ratio of cell retrieval.
Hi Miray! I agree with Alexandr and Niranjan. You should be able to rinse 2-3 times with 1X PBS (if you need a recipe let me know) and then prepare your cells for a passage (with trypsin or whatever you use).
For freezing, I can help a little more. I've had great success using freezing media from Sigma. It is very easy to use. https://www.sigmaaldrich.com/catalog/product/mm/s002m?lang=en®ion=US
We use cryovials and place them in a special container in the -80 before placing in liquid Nitrogen. The protocol should be listed with the product on Sigma's website.
Let me know if I can help more.
Hello guys !
Thank you for your recommendations. These cells grow as suspension culture. Does washing with PBS also suitable for suspension cells? Does it help for these cells?
Thank you?
Hi! I have not worked with suspended cell culture before, but I found this on another thread on researchgate:
https://www.researchgate.net/post/How-can-I-change-the-medium-of-suspension-cell
There is one comment about washing with 1XPBS when centrifuging.
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