Hope someone can help me out. We have a problem in my cell cultures. I had 3 separate cultures where the cells suddenly died (this never happened before). I was looking at a closer view (320x) to see if I have unwanted organisms. I see little balls, different sizes, mostly round or oval. Most of the times alone, sometimes two together. It moves a bit. But some balls are attached to the cell layer and not moving.
My thought was yeast, but some colleagues find it not typical yeast looking. Together with the fact that it is not overgrowing after a few days or even a week. We tested some supernatant on a SDA agar plate (kept at 37degrees), but nothing grew on that. This is indeed strange, but I am still not convinced it is not yeast. Now I am focused on these little balls, I see them in all my cultures more or less (still viable cell cultures). What are your thoughts? Yeast yes or no? What else can it be? Someone experienced something similar?
Thanks in advance!
Details: HepaRG cells in WME medium, cultured at 37C Celsius + 5% CO2
I attached 4 pictures. First three are from cultures where the cells died and not attached anymore. You can see dead cells and debris. And also the little round balls. The last one is from a viable cell culture.
yeast don't like to grow at 37°C they prefer 26-30.... try on agar plate. However what will you do if it is yeast? or bacteria? or fungus? it is almost impossible to get rid of eavy contaminations (bacteria or yeast or fugus) in cell culture and treatment to get rid of them could damage your cells (you don't know if the cells are really the same before and after treatment) : throw every thing away (after autoclaving) then check your mediums (throw the ones that are not too expenssive) and start a new culture ...
Hi Marielle Vdkaaij
I don’t see any yeast there at all. If you still suspect microbial contamination, a simple test would be to spread some media on Sheep Blood Agar and incubate the plates for 7 days at both 37°C and 30°C.
If I notice something similar in my cells, I typically test my media batches for formulation issues and run PCR for mycoplasma. In the past, we’ve also encountered viral contamination, which ended up killing our working cells but we also work with BSL3+ agents.
I would recommend discarding the current media and cells, fumigating the incubators (we use formaldehyde), and restarting the cultures from safe frozen stocks. A thorough deep clean and disinfection of pipettes, as well as changing lab coats, is essential.
Hope this helps!
Best, Povilas
Thanks for both the replies! I will place some on agar plates at different temperatures. Thanks for the tip.
Didier Poncet do you agree with Povilas that you don't see (possible) yeast in the pictures?
Povilas Kavaliauskas What would you think that the little balls are? They are too 'clean' to be cell debris, right? that is more rough
very difficult to see from a picture; with yeast sometimes you see bourgeonning figures ...
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