Hello guys, I'm trying to grow some hybridomes which were frozen 10 years ago, I have been trying to grow them in RPMI + 10 % of FBS, but after one week they start to die, do you have some recommendations or a nice protocol which has worked for you with difficult hybridomes to grow.
Edwin,
When I was at Cold Spring Harbor Labs we used to grow the hybridoma cell lines in 96-well plates. Here are some strategies:
http://www.cshlpress.com/pdf/sample/2013/Antibodies2/AB2Intro7Part2.pdf
Good Luck
If the hybridoma cells were reselected in HAT medium prior to storage (which is usually a good idea, assuming this is how they were generated in the first place) they may require HT medium after thawing in order to grow. They may even require HAT medium, with slow dilution of aminopterin for a couple of weeks and then slow weaning from HT for another couple of weeks. Also, make sure you are using the right FBS concentration. Some hybridoma clones (especially if they were adapted to serum-free medium) are sensitive to excessive FBS concentrations (more is not necessarily better!). If properly stored in liq N2, how many years they were stored should not matter much, but some fraction of the cells will not survive. The best information on how to revive the cells is usually from the source. Have you contacted them?
See:
https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Product_Information_Sheet/2/h0137pis.pdf
The protocol of cell thawing is important: 10 min at 37°C and then add fresh medium; centrifuge and use 96-well plates to grow the hybridoma cell lines in HT medium with 10% FCS . Change the medium every 3-4 days. After 2-3 weeks use medium without HT. The mortality can be reduced then.
good luck
I suggest trying to thaw them with a higher concentration of FBS (15% - 20%) in your complete media then gradually decreasing once the cells revive. I hope this helps!
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