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Adhering CHO cell culture


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#1 toxog

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Posted 25 May 2010 - 01:10 PM

Hi, I am totally new to CHO (chinese hamster ovary) cell culture, and to make things worse, I am in charge now of five different mutant CHO cell lines received by donation (4 day-travel and customs) :wacko: . So the thing is that to not make mistakes I am growing them in a rich Ham's F12 medium containing 10% FBS, pen-streptomycin, glutamine, and non essential amino-acids. They grow quite well according to their passage number, even the cell line with 86 passages grows although slower than the one on passage 5. However the problem I am having, is that many cells just don't attach to the flask and remain floating, even after changing medium every 2 days so I don't think it has to do with medium. Are these floating cells dying or just not attaching, because CHO cells can also grow as a suspension culture, though i don't know how to optimize the conditions in this aspect.
Please help me! I have to start a lot of infection experiments on these cell lines and i need to be sure they are worth the effort!!!

#2 labrat612

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Posted 26 May 2010 - 06:59 AM

Have you done a viability assay to ensure that the floating cells are, in fact, dead?

CHO cells are damned hardy and will adhere to almost anything. However, it is possible that the mutant lines are more picky and will require a specific type of coating in order to adhere. Is it a majority of cells that are floating or just some? And does this number increase despite changing the media?

#3 toxog

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Posted 27 May 2010 - 02:02 PM

Have you done a viability assay to ensure that the floating cells are, in fact, dead?

CHO cells are damned hardy and will adhere to almost anything. However, it is possible that the mutant lines are more picky and will require a specific type of coating in order to adhere. Is it a majority of cells that are floating or just some? And does this number increase despite changing the media?

Well, today I counted them in a hemocytometer after trypsin treatment with trypan blue, and viability is about 97-98% and I get about 4 million cells per "confluent" T25 flask, so I think they are not dead, it's just that some of them simply don't attach, they are not the majority but they are a lot and the number of floating cells remains about the same depending on the total amount of cells. By the way, it also happens to the parental CHO-K1 line, so that's even trickier. I have read that non essential amino acids can favor suspension CHO cell cultures but I'm not so sure of it.




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