Hi, I am currently doing HT29 culture. Cells during initial passages appear clear, but after few lines of passages, something look like agglomerate appeared between cells. I was wondering if that thing is cell debris or contamination. I've tried rinse with media and PBS but that thing still there attached to the flask. The cells seem to be normal and are attached.
How many passeges they have? What kind of medium are you using? What is the percentage of living cells?
I don't think there are contaminations.
i think this is fungus. Throw it away and clean your laminar chamber and incubator. Do you use antibotics in your cell culture?
It's probably just debris. Can you see it moving under the microscope? Otherwise just do a simple test: Put your dish in the incubator and if the medium isn't cloudy after beeing on the dish for 48 or 72 hours, you probably do not have any kind of contamination.
I too think these are debris. As suggested by Sophie, if you do not find turbidity in the media; these are not contamination. Also, try to see whether these are increasing in numbers after incubating for 24 h.
It looks like debris to me too. And the media test is a great idea.
To me yours cells do not look very healthy tough. How long are they taling to reach the appropiate confluece ? Have you try to seed different amount of them?
many thanks
1. the cells are currently at passage 15. I use RPMI 1640 with 5% FBS and 1% penstrep. percentage of living usually >90%
2. I use only 1% penstrep in culture media
3. the debris are not moving freely. It seems floating but a part of it attach to the flask. as I can see the debris, the media is still clear and didn't change color, however I can see white precipitate in the flask or well plate
4. Yes, I've tried continue incubation and they increase in number
5. I've tried seeding at different amount. Usually took 3-5 days to reach confluency with living percentage >90%.
I have seen this multiple times when I have used primary cell cultures. Usually I notice this after high passage numbers. I believe it is response to stress, because I usually notice it is more prevalent after detaching with trypsin. Also, I do not usually see it when the cells are at a early passage and appear more "healthy". One more test you can do is take a sample of your media and place it into new media with no antibiotics and incubate it for 48hrs. If the media remains clear then it is not contamination.
Best of luck
It could be debris, due to stress. You may try giving a wash with PBS and give 10% FBS containing media and see whether they are getting better.
Maybe your cells naturally form aggregates (kind of spheroids) after some time in culture or under stress conditions. If by chance you can collect and disrupt this aggregates, you might be able to determine if it is cells (by counting them, doing viability test, or seeding).
Your experiments always deserve the best and healthiest cells. It is not worth your time to try to "cure" a contaminated culture. When in doubt, start a new flask from a vial of the back up cultures you stored in liquid nitrogen. Or buy a new vial from ATCC.
Having said that:
I have experienced an increase of clumps when I have "over-trypsinized" the cells (incubated them too long in trypsin). The trypsin digests the cadherins & integrins from the surface of the cells, which is why they release from the plastic surface. Be attentive to the time and temperature. Avoid trying to sub-culture too many flasks at once.
thanks everyone. Dr. Cynthia Ricard, I'll take note the trypsinized time. Good reminder for me. Thanks again
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