It seems some fungal contamination

try adding some antifungal agent to the media.

to confirm the presence of fungi you can culture the mycelium (the thread like structure) in suitable fungal medium like PDA

These look familiar. I have seen these in cell cultures that produce a lot of extracellular matrix and these substances often float off above the cellular monolayer. Considering the nature of your primary cells it could indeed be something along the lines of mucus? It definitively isn't bacteria and it doesn't look like fungi as they tend to grow in one large furry spot and look dense and fluffy under the microscope with hyphae.

Are these structures affecting how your cells grow - maybe changing the media everyday or other day would help if this is affecting your cell growth?

Dear Hedstrom,

More likely, it seems fungal contamination. Try to add some antifungal agent like Amphotericin-B along with your antibiotic. Decontaminate your culture room at specific interval along with your culture incubator. More importantly check all your culture media components individually by incubating each and every component individually on the same incubation temperature on which you are growing yours cells. I am sure you will get rid of this problem.

Don't know for sure, but I would agree with Dr. Burns. It does not look like the typical fungal contamination I have seen. Too organized.

mee too think that its not any organism. could be like extracellular matrix

If you separate the film from the cells, do they grow a new one? If yes you may have a model for lung surface in your hands. It then should contain surfactant proteins.

That seems to be a fungal structure as some collegues have stated in previous commens. A common práctica is to add some amphotericin B to your cultures. Just check that the concentration used is not too high as it may be toxic for your cells.

It doesn't look like a fungal contamination... But to confirm the doubt I would try to isolate it and make it grow back to confirm to have more and more... Do you have an higher magnification? Looking at those picture we can't see the usual fungus morphology... 

Il looks like mucus to me. Do all cells appear to have these filaments in contact with their edges?.Have you stained the monolayers?

Im a mycologist and it doesnt look like fungal mycelium to me, but to exclude this possibility you could put a sample of a plate of Sabourauds agar and see if anything grows 

What magnification do you see them? Have you considered it for mycoplasma?

Thank you everyone for your feedback and suggestions.

Amphotericin B has been present in my medium all along, but the threads still appear. I once tried increasing the amphotericin B concentration but then my cells started dying. I clean the laminar flow hood and decontaminate the incubator at regular intervals.

When I transfer all the medium from an almost fully confluent flask, containing a lot of threads, to a new flask without cells, the threads stop growing in number in the cell-free flask. This would indeed be expected if the threads are made up of extracellular matrix molecules. Thank you Alana for that suggestion.

Dr. Urbano, the threads/filaments are generally not connected to the edges of the cells, based on my observations. I have also considered mucus but I have not at all been sure whether it can have this appearance in a culture. Would you say that mucus can look like this?

Regarding the magnification, I'm pretty sure this is 20x.

By the way, the cell bodies you see in one of the pictures are dead epithelial cells entangled in the threads. I can't really see any cell bodies connected to the threads. Are fungal cell bodies always expected to also be visible when a fungal mycelium is present?

Thanks Dr. Nicklin for you suggestion about using Sabourauds agar. Would such a test be completely conclusive, i.e. would you expect all possible fungal organisms to grow in a Sabourauds agar plate?

Dr. Agrawal, I haven't considered mycoplasma as a candidate here. As far as I know, mycoplasma is generally not visible in a cell culture.

Glad you are managing to rule a few things out. If it is fungi it should grow without the cell layer beneath; transfer the media with thread like structures into a new flask and culture to see if anything grows out. If there is no change it would be conclusive that it is from the cell themselves and not another microorganism. You are correct in saying that you cannot see mycoplasma. We test our cells regularly for mycoplasma and it can affect cell growth and wellbeing which isn't the case here. 

Maybe you should consider a better prep for the epithelial cells as you may have cultured mostly goblet cells. 

Here is an EM picture of mucins; althougth this is very high magnification these strctures could like what you have in your flask at 20x. http://www.nature.com/mi/journal/v6/n2/fig_tab/mi201281f4.html

Futheremore here: http://www.nature.com/nmeth/journal/v10/n8/fig_tab/nmeth.2523_F1.html in figure 1C there is a picture of ECM (extracellular matrices in cluture).

Hope this helps,

Alana

Yes, I have already tried to transfer the media from a culture flask with a lot of threads to a new empty flask, and when I do that the threads do not increase in number in the cell-free flask.

I doubt that the majority of the prepared cells are goblet cells, but if the threads actually are mucins I wouldn't be too worried, since possible microbiological contamination was my primary concern.

Interesting pictures of deposited ECM in the Nature paper. It looks fairly similar to what I see.

Thanks again Alana,

Ulf

Hi Jules,

I still have not confirmed what it is but at least now I know what it's not. I sent samples to a clinical microbiology lab for testing and they told me it's definitely not fungus. They tested for bacteria and those tests were also negative.

Since then I have also seen these structures appearing in cultures of human bronchial epithelial cells set up by various colleagues of mine.

I think this is something the cells are producing, possibly extracellular matrix molecules like collagen. These cells have a natural production of ECM (e.g. synthesis of the basement membrane they are attached to in vivo) so it would make sense.

So now I actually see these threads as a sign of cellular health rather than a problem :)

It would be interesting to find out exactly what they are though. Good luck with you cells!

Thanks

Ulf

Hi Ulf,

thanks for the interesting question and sharing your data and views!

I am no expert on bronchial epithelial cells or lung, but since these structures that you see and describe are no fungi or bacteria (and I suspect also not eukaryotes), they may be protein filaments like in Reissners fiber (produced by the subcommissural organ close to the aequaeduct in brain`s ventricular system in all vertebrates except humans and higher primates). My other guess (conforming to yours) is filament forming proteins like surfactant proteins. Did you give a sample to a mass spec analysis?

Just curious,

best,

Erik

Hi Erik,

No, I haven't done that. It would be interesting but for the time being I have decided to prioritize my project since I feel pretty confident that these structures will not be an issue in my experiments.

Best,

Ulf

Hi Ulf,

its good to set priorities, but investigating the unexpected findings may be the better (or more interesting) long term perspective. Anyway I respect your decision and wish you all the best for your future as a scientist! Best, Erik

I am very curious about this. Do you coat your cell culture flasks with anything?

Hi Willy,

Sometimes I coat my flasks with collagen (PureCol from Advanced Biomatrix), but that is not what is causing these threads to appear, as they are also present when I use uncoated flasks.

Hi Ulf, just wondering if you ever found out what these threads are?  I have recently begun culturing primary endocervical epithelial and vaginal epithelial cells and have seen this same thing.  None of my tissue-culture-savvy colleagues have seen anything like them... do you still suspect that they are something produced by the epithelial cells?  Thanks!

Hi Mel,

No, I haven't yet tried to find out what the threads are made of or where they originate from, but based on my observations I still consider it likely that they are the result of something produced by the cells. Epithelial cells produce extracellular matrix molecules in vivo to support the structure of the basement membrane they are attached to and if they can retain this ability also in vitro it would make sense that you're also seeing them in your endocervical and vaginal epithelial cell cultures.

I have also observed these threads in cultures of the bronchial epithelial cell line BEAS-2B, but interestingly they were only present when the cells were cultured in BEGM medium (Lonza), but not when I used DMEM medium with 10 % FBS.

Hi Ulf, I work with lung epithelial cells from Cystic Fibrosis patients and have encountered this same problem with some of the cell lines Isolated but not all. I do see it more when I use antibiotics for resistant bacteria.  I am wondering if you have found out more about what it is? I use BEGM media and pre-collagen coated flask. 

I have used all suggestions you have received with the many anti-fungal treatments with no success.  I have figured out a technique to get rid of it, but would still like to now what it is and a better way to remove it.

My procedure is using liquid toenail fungal treatment with 25%v/v with active ingredient: Undecylenic Acid. Add 10ul/ml to PBS and wash flask twice with treatment solution, followed by two washes with PBS without added solution.  It breaks down the threads and your able to remove from flask over a couple of feedings with washes. Do not use more than 10ul, this amount does not harm the cells.     

Hi Jackie,

No, I haven't pursued this further, since I got confirmation that it's not a fungal or bacterial contamination and because the threads are not really a problem for me. They always show up in my cultures of bronchial epithelial cells, at least when they are grown in BEGM.

I have noticed that you can get rid of them temporarily by thorough washing with PBS, but they always come back within a day or two.

It's good that you found a procedure to get rid of them if they are an issue in your experiments, but I have decided to just ignore them.

We contacted the manufacturer about this. Apparently this is normal for this type of media and the debris should not be filtered out.

Any idea what this could be?

Hi Anjali,

I don't really know but it looks like it might be a fungal contamination.

Good luck,

Ulf

Hi all! That was very informative discussion. Thank you! Also, I want to make a contribution.

We are working with HEK293 cells. We faced a contamination and we don't know what it is. Could you make comment about it?

Ulf,

These images remind me of the 'ocular debris' I see in my vision. I have been told that they're pieces of broken down cells/cell 'waste'. When you mentioned the possibility of something your cells are making, and the fact that your cultures came back negative for fungal and bacterial contamination, I really started to think perhaps the structures are coming from your cells.

Thanks for sharing. Very interesting.

Bridget

Hi Ulf,

I, too, have these structures form in my human iPSC cultures. Sent them to a microbiologist colleague, but they were unable to culture it concluding it is not a microorganism. Did your cultures appear healthy despite the 'threads'? Did they appear to have any impact on your experiments?

Thanks for sharing

Chloe

Chloe Christensen Thanks for sharing your observations. My cultures certainly appeared healthy no matter how much I saw of these threads, and I did not see anything in the data suggesting they had an impact. Good luck with your research!

Theses are most probably fungal contamination. You will find nothing for 2..3 days after sub culture. Then suddenly you may observe one or two thread. But surprisingly your cell will grow normally. But after 6 to..7 days you will find your media turns yellow and the threads become mature fungal colonies.

Hi, I work at a biobank of human bronchial epithelial cells and we have a similar problem, can you tell me if what you saw is something similar to this? (sorry for the bad quality pics) I used multiple yeast medications but still they are there... My cells grew normal, However they are affected by so much antifungal medication...

Hi Jessica Elizabeth De La Torre

Morpologically I think this looks different from what I saw in my cultures. I never confirmed what it was, but I eventually started to assume that it was something produced by the cells rather than a contamination with a microorganism. I hope you will be able to solve your problem.