I am trying to grow an immortalized cell line that has done well before, but now are no longer attaching. The cells attach fine post thaw, but over the course of 48-72 hours, all the cells detach. I used the following:
- Pricoat coated flasks
- DMEM/F12 medium
- 10% FBS
- 1% Penicillin/Streptomycin solution
- 2% ITX
I also tried coating the plates for better attachment with different coatings (Fibronectin, Poly-L-Lysine, Collagen, Gelatin) but just cannot seem to expand the cells. Any suggestions?
You commented that the freeze thawed cells have done well before. If you were using culture dishes instead of flasks at that time, try using the dishes again. Then you might try to obtain flasks that are comparable to the dishes that work. One key may be to get the dishes and flasks from the same source. If you did not coat the dishes then add no coating to the flasks. One suggestion might be to use the largest size of dishes that work. Another suggestion would be to start over with a fresh stock of the immortalized cell line, if possible.
Since they worked well before it might have to do with the composition of your FBS. Was it the same batch? Maybe try different batches. Also is the dmso removed during thawing by centrifugation? If not you need to change the medium 24h after thawing.
Make sure you wash the cells after thawing. Long term exposure to DMSO can kill them!
In what conditions were the cells grown before (when they were attaching)? It seems to me like some type of toxic effect is going on, so the suggestions to wash cells to get rid of DMSO and review your media composition are valid. However, some cell lines just have a tendency to detach, like the 293s. Here are some suggestions from ATCC that might be helpful to manage this issue:
https://www.atcc.org/support/faqs/f0c93/Detachment+of+293+HEK-293+cells-313.aspx
Hope it works!
Recombinant Human AFP purified from the goat milk is in clinical trials:
http://alpha-cancer.com/
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