Help with potential Fungal contamination of non-adherent line - (Jun/27/2011 )

We are currently experiencing what we believe to be a contamination problem in our cell culture. About 6 months ago we noticed our cells (U87 glioblastoma line grown as neurospheres) were not giving reproducible results in assays standard in the lab. We began noticing what appeared to be fiber like strings throughout the media which appear to become longer with time and there are black dots throughout the culture. The color of the media never changes and the cultures never become turbid. We have done PCR test for mycoplasma and they consistently come back negative. Growth of supernatant on tryptic soy agar with 5% sheep blood gave rise to no growth. It does not look bacteria and adding pen/strep to our culture did nothing. We have extensively cleaned out our incubators and hoods, as well as ordered new cells from ATTC 3 times. Recently I added fungizone to my cultures but as I was changing the media on a daily basis I could not see any fibers, however, when I went back to changing media when we split the cells (once a week as required by our protocol) I began to see the fibers again. We even tried to establish the line using a completely different hood and incubator without ever bringing it in contact with anything in our cell culture room. We just started seeing long fibers again in this media. The U87 cells are shipped as an adherent line which we grown in DMEM, 10%FBS, Na pyruvate and convert into spheres by culturing in DMEM:F12, B27 supplement, HEPES, EGF, FGF. We have tried to change media we use to convert into spheres to NeuroCult media sold from stem cell technologies and that didnt not help. I have attached a powerpoint presentation with numerous pictures depicting the fiber-like floaties we see as well as pictures of our cells showing how unhealthy they look.

Your powerpoint doesn't appear to be attached. You can just attach the images as JPG or PNG if you prefer. I have seen fibres that are from precipitates in the FCS before; these don't usually look like fungal hyphae though.

Fungal hyphae are usually quite obvious in culture, they typically form a fuzzy ball with extending hyphae which will grow over time. It is quite common not to see changes in the medium colour etc..

Not trying to be patronising, as it seems that you probably know reasonably well what you are doing...

As these are commercial cell lines, it is best to throw them out and get some new ones in if you can. Treatment with drugs only damages the cells and often doesn't really work, especially for heavy contamnation like you seem to have. You should also throw out all your stocks of medium and any supplements you add. Get fresh boxes of gloves.

It maybe a good idea to de-contaminate the fridge/cold room where you keep your medium stocks too, as these are often good sources of fungi with the moisture present in them. Your common source for the different hood etc. experiments you described above is likely to be a fridge/cold room.

Who froze down the stocks of cells after they were brought in? Do they have the same contamination problems?

How recently were your hoods serviced - were they de-contaminated and filters changed during the service?
Do you wipe your flasks/gloves/everything down with 70% ethanol or IMS before putting them in the hood?

Thanks so much for your input and sorry for the powerpoint file, I have attached the images below. We have been having a really difficult time getting our cells to grow. When this started back in January we began seeing that our cells were not behaving as they had hundreds of time in a standard assay. When we began to suspect we had a contamination we threw away all our cultures and carried out our first large clean-up of the tissue culture room. Incubators, hoods, and floors were all cleaned, all the reagents thrown away. At this time we ordered a new vial from ATTC and brand new reagents to propagate the line. THis went well for about 5 weeks before we started seeing floaties again and the cells didnt quite look 100% healthy (there was no mass dying off of the culture or decrease in proliferation, however, the spheres looked like they were covered with little black dots but we were unsure). At this time we ordered 2 new vias from ATCC and again new reagents but began propagating the lins in a totally different cell culture room located in a different part of the floor. THis room has its own hoods, incubators, fridge, and microscope so there was no need to ever bring anything in from the old room into this new room. These cells grew well for about 4 weeks until this past weekend I saw again that the cultures looked unhealthy as there were more single cells and less neurospheres forming, with the presence of long fiber looking floaties again. When we first got the cells we did freeze down cells that did not have any floaties or visiblel sign of contamination. We obatain the cells as an adherent line which we grownin DMEM+Na pyruvate+10%FBS and they are fine. Once we convert, about a week or two, into expanding the culture we switch the media to DMEM:F12+HEPES+B27 supplement+EGF+FGF so we can grow them as neurospheres. It is after a few weeks here that we see the floaties. I have gone back and evaluated my adherent line and they still have no floaties but they do have black dots that are sparce throughout the culture (not sure if these are spores?). We have tried 3 different sources of media and components of our reagnts and the floaties are in all of them so we dont believe its coming from one of the reagents. We have tried growing supernants in plates for bacteria and fungus and we get nothing. We are extremely careful as we normally dont even have pen/strep in our media and have never gotten contamination before. Both a post-doc in my lab and myself are having the same problems, however, we are both using the cells from the same vial when we obtain them from ATTC. The only reason we have added fungizone is to see if we can see a decrease in the floaties and maybe determine if this is fungus or not. I will look into what we can do for the cold room as we have not tackled that. Our hoods are serviced on a yearly basis but we are looking into bombing the whole room along with incubators and hoods. I just wanted to know if others have seen what I am seeing or could this potentially be a properties of neurospheres? I find it hard to believe as I have never seen it before but I have heard stranger things.

bob1 on Tue Jun 28 01:18:53 2011 said:

Those pictures look very like precipitates that you sometimes see in FBS, especially if it has been freeze/thawed more than twice. They do not look alive at all. The last images appears to be something to do with the flask.

Google image search for fungal hyphae and compare...

bob1 on Wed Jun 29 01:33:39 2011 said:

Thanks so much for your reply however they can not be a result of FBS precipitation as our neurospheres ar grown in media lacking FBS.

rhombus on Thu Jun 30 12:30:17 2011 said:

PPT from some other additive then (inicdentally what additives are you using?), definitely not fungus all the same.

Then try to filter your complete Medium with a 0,2µm membran. This should eliminate all precipitates but keep the necessary stuff in.
Also have a look at the pipets you use. In some cases fibres from the filter at the top come into the culture. They are sterile and look almost like some of your pictures, but you do not want them to be there, do you?

Good Luck.

Hi,
we had the same issue. After repeated screening on all of the reagents, we found out that it was coming from the B27. After reporting and investigation on brand new vials, the Company did not confirm the contamination issue and did not give a clear description of the nature of these "flocculent debris".
That is.
Regards