Hello, I have A549 cells in culture. Normally they attached really nice on normal flask, T25 or T75 without coating the flask. Now I freeze some of these cells and after thawing they don't attached. They are not dead, they grow in the media.
Any idea?
Hi Sven,
are your flasks, media etc. from the same vendor as before? Have you performed a trypan blue staining to check their viability?
This is certainly a point of concern when adherent cell line like A549 becomes non adherent. It is an obvious sign of some sort of contamination like mycoplsma or mollicutes as they are commonly known. Many labs involved in cell culture face this problem and as it is usual for contamination to spread from lab to lab as cell lines, media and media components are shared among researchers. You can test your culture for contamination if you have in house facility for testing but most contaminants me remain undetectible at earlier stages. Such contaminations are difficult to elimnate, as they could be antibiotic resisitant. I would suggest you to dicard your culture and media as it may contaminate your other sources and start afresh using clean culture after following decontamination procedures. As prevention is the best strategy and your attempts at trying to eliminate contamination may waste your time and resources. But testing wil help you in decontamination and adopting elimnation strategy. Good luck!!!
You may go to the fololwing link for detailed information:
http://www.the-iom.org/assets/files/IRPCM_Team_Mycoplasma_contamination.pdf
@Tamas: Trypan blue staining shows that the cells are ok.
@Syed: Thanks for your answer. I froze the cells for about two weeks and when I froze them they look ok, maybe you are right but normally I don't know where the contamination can be come from. I just put again new cells in culture with new media and new vendor of flasks.
Hi Seven, please control your media again. i had the same problem with HEK293T. I was using low Glu media insted High Glu mistakenly becouse my media had changed without I khow.GOOD LUCK
Hi Sven. do you normally centrifuge the cells after thawing , in order to eliminate DMSO, before put the cells in the flask?
@Maryam Bikhof: The media I use was complete media (F-12K) so this can't be the reason.
@Sarah: Yes, I allways centrifuge.
I think something go from in the freezing step. Because now I thaw an older stock and everything works fine. In the older freezing steps I don't use a freezing aid, these cells works. The last time I use a freezing aid and this cells don't attach. Maybe the freezing with the freezing aid is not fast enough?
Thank at all for your comments!!!
i always use a cryoprotective medium in the freezing steps (also for A549 cells) without problem. I had the same problem in the past and i resolved it skipping the centrifugation step. The next time you can try putting all the cells directly in the flask with the medium and the day after change the medium. good luck!
I work with NHBE cells (Bronchial primary cells) and I culture them directly in the flask. But, If you have cultured A549 cells already and everything have gone fine, so the problem probably is not the centrifugation. Something is wrong with your media, maybe contamination.
Hi! Sven. Could you solve this problem you had lived ? If you solved, You could say what did you do in order to overcome the problem. I have already lived this problem on my A549 cells. They are not attach to the flasks. I tried everthing. Probably, somethings escape our attention. I hope you detected the causes of the problem. Could you help me about it?
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