I do not have any experience culturing hybridoma cells and I need to expand a clone to purify the respective mAb. I am wondering if anyone has had success recovering hybridoma cells from cryopreservation directly in serum free medium (i.e. Gibco Hybridoma-SFM)? Some protocol advice to recover the cells, first in 10% FBS RPMI and then try direct adaptation in serum free media, but I do not have FBS right now and I wonder if I recover in Hybridoma-SFM medium, would it work?
Would it work? Probably not. I have attempted to do this several times -- even with clones that had previously been accommodated to serum-free conditions prior to freezing, and it hasn't ever worked. So now I always raise them in 10% (or even 20%) FBS, and once the cells are well established (i.e. numerous large, glassy, round cells, growing in grape-like aggregates) I halve the serum, eventually eliminating it.
So, I recommend you just ask for a few ml of FBS from a colleague -- you don't have to supplement the whole bottle of media, just what you use in the culture vessel.
In general the hybridoma protocols mean what they say. You should do whatever the instructions told you. Good luck!
I have the same experience. We have never recovered the frozen hybdridoma cells in serum-free medium. Usually, the conctration of 15-20% of FBS is necessary.
Alright, thanks everybody, this is a good forum. I asked for some FBS and I used Hybridoma SFM-10 % FBS-P/S. The frozen vial had a good viability 83% and 2x106 viable cells/mL. If they adapt well to current medium I will try direct and sequential adaptation in serum free media.
thank you
I have tried to do so with Sigma serum free medium but it did not work. The best thing to do is to retrieve the hybridoma in medium containing 10% FCS and then gradually decreasing the concentration of FCS and finally cultivating the cells in serum free medium.
Good luck
To Jan Brauner
Can you give the amounts of the supplements you use in the Hybridoma SFM--IL-6 and Cholesterol? I am really interested in supplements added to the mediim.
Thanks,
Tommye Jordan
Many thanks, The hybridoma cells are producing low levels of IgG and one producing low level of IgM. I am hoping the change from the Opti-mem-Glutamax with 1% low level IgG FCS to the Hybridoma SFM will change this low level to a really see an increase in antibody. I will also try some cells with you supplements.
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