I am trying to revive my cells and I don't know what they're being affected by. I have thoroughly cleaned every corner of the culture room and incubator; done all possible cleaning practices to avoid all possible sources of contamination. Now my cells are not adhering to the surfaces of the plates. They become rounded and are floating in the media. What additional techniques can I try to establish my culture? I don't know whether it is some biological contamination or some physical factor that is playing a major role.
Are you sure that you use the correct cultivation medium? Every cell line likes different cultivation media. It can happen that for the cell line A, there are say three suitable media X,Y,Z. If the cell line was grown in X in the past and you are trying to revive it in Y or Z, it will not work.
Thank you all for your kind suggestion....I am using cos7 and A549 cell lines. But the problem is same for both cell lines. I am using correct and well prescribed media for both. And I am working on them since 8 months with same media. But I don't know why they are not adhering now. I have tried different tissue culture plates from different suppliers also, checked pH of media, CO2 level in the incubator. Everything is perfectly fine. Then what the other factor could be, I m not able to understand. Please answer I you have any such experience. Your suggestions will help me a lot.
Dear Tomas
I haven't checkrd it yet for mycoplasma infection. But as far as I know, in case of mycoplasma, I don't think such severe adherence problem arises. What do you think?? Pls explain.....
Well
1-you use correct media
2-you checked growing conditions
everything OK.....
what you did not check are infections (virus, bacteria, fungi). The most common infection (except ordinary bacteria and fungi-you would see it in microscope) is mycoplasma.
There is also other possibility. Do you mix your media yourself o do you buy commercial products? Something might change. For example you bought new chemicals and start using them for mixing media.
We should also consider the freezing media composition and storage condition.
There is also the possibility of a change in the FBS?
The ECM proteins required for cells to stick down come from the FBS (Fibronectin etc).
If you made up media without FBS the cells won't stick down regardless of whether you are using TC plates or not. Also, as Megha has implied, if they were not frozen down correctly they may just all be dead upon revival. You mentioned they are not dead - have you checked this with i.e. Trypan blue?
To reiterate what has already been said:
1) Add trypsin neutralizing solution or FBS after trypsinizing your cells
2) Check your antifreeze concentrations prior to freeze backs
3) Check the ATCC website for more details on your cell line specifications
Were you ever able to resolve this problem? I am seeing the exact same phenomenon for over 2 months now and after going through all of the same things you did, I am at a loss as to how to resolve this. Any insights would be greatly appreciated!
Dear Arun,
I am now dealing with a similar issue with my own cells, BJ fibroblasts. They suddenly stopped proliferating, and would detach from the plate when going through mitosis (especially in telophase). Freshly thawed cells behave the same, so this must be due to an external stress factor. I checked the incubators, media, serum... Had you finally solved your problem?
Valentin
I have the same problem. I consider liquid nitrogen to run out and I expect all cells to die. What do you think about it?
I am not an expert in cell culture. Looking at your problem, I assume to try to perform single-cell isolation and start culturing them. We should end up with some single cell starts to adhere back.
The first things to ask yourself when this happens:
1. Are you using appropriate media?
2. Are you using the right type of FBS?
3. Does your cell line require collagen coated flasks?
I've had this happen to me twice to me for two different reasons. The first time it happened to me, it was with an HEK cell line. And the cells had decent viability, but were not adhering to the flask. This was due to me filtering my FBS for the media, which filtered out key components the cells required for attachment. This also happened to a coworker using the same cell line when they used complete media that was older than 6 months.
I also experienced it this year, when I had use a substitute base media due to COVID supply shortages. The cell line had a decent viability, but took several days to attach and grew slowly. It turned out that my substitute base media had a 10 times more glutamine than correct base media, which inhibited the cell growth. The way I was able to work around this was to replace the substitute media every day, until my correct base media arrived.
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