Contamination. Help needed!

[Cindy GRANDJENETTE]

Dear all, 

We have problems of contamination in several of our cell lines. Cell viability is pretty good, however, cell morphology is affected whatever the cell line. In addition and importantly, we observe a lot of small particles in the medium. Their number increase over time.

Mycoplasma tests were negative. We also sent our samples to 2 microbiological labs. Nevertheless, they were not able to identify the source of our contamination. We are wondering whether these contaminations are due to (myco)bacteria, viruses, or even yeast.

You may find attached movies showing how these particles "appear". The morphology of this cell line is normally homogeneous with round cells.

https://www.dropbox.com/s/n1p711dwfxnzxgy/150724%20tf1%202h30%20mes%205%20min%202%28converti%29.mov?dl=0

https://www.dropbox.com/s/ggbt1u8i3piau0q/150724%20tf1%202h30%20mes%205%20min.avi?dl=0

https://www.dropbox.com/s/sp2ncal7szl2kkr/150726%20tf1%202%20min%20mes%20100ms.mov?dl=0

https://www.dropbox.com/s/c0okt4k3twgzg4z/150726%20tf1%204h%205%20min.avi?dl=0

https://www.dropbox.com/s/6ngjhzcy9fejzjn/TF-1%20big%20cell.avi?dl=0

Does somebody have an idea concerning this contamination ? 

Thanks for your help,

Cindy

[Ravasz]

Hi,

No idea what this is, but I can tell you what its not, maybe that helps:

- It's definitely not yeast, that looks very different. Yeast would consist of small groups (4-10) of oval particles, smaller than a mammalian cell, with a well defined, smooth outline. They would also outgrow your cells and kill the culture in a matter of days. This is not like that.

- It's not mycoplasma or a virus as both are too small to resolve in a light microscope. They would simply not be visible even in a rampant infection.

- It is not usual cell debris as it is moving.

My best bet is that it is some form of bacteria. Were you using penicillin-streptomycin in your medium? If not, try adding that. You can also add some Fungizone too if you are desperate, as it might be some fungus.

However, such "resurrected" cultures are never really perfect as the cells were put under stress and they might be permanently altered, even if appearing completely cleaned at some point. I would just discard the whole culture along with any media and buffers that are in use, autoclave any equipment that came into contact with it and thoroughly wash incubators and culture hoods with Virkon, or your disinfectant of choice.

If this is showing up in multiple cultures, then the source of contamination is not within the culture itself.

Maybe this helps.

Mate

[Cindy GRANDJENETTE]

Thanks for your answer.

We have this problem for 4 months.  At the beginning we thought that the particles we observed were mycobacteria. But the gram tests from the microbiological labs appeared negative. Of note, our cells look grainy.
We are not able to determine whether these particles are the source of contamination or just a consequence. 
We use penicillin-streptomycin and fungizone (Amphotericin B)  in our culture media. 
We already cleaned the lab (laminar flows, incubators, etc), autoclaved everything, and threw our cells away. But the problem is still here. And we do not know how to solve it.

Cindy

[Dakota Hamill]

Check some controls.  Look at just the growth media fresh, then after a few days.  Aerosols from pipettes could be building up but I imagine you use filter tips. 

Also, just spin everything down, extract DNA, and run 16s or ITS bacteria/fungal barcoding to look for a band.  Sequence it if you have one and you could get the exact species

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[Mike Horwath]

A few questions to help with diagnosis:

What cell lines are they? (mammalian, insect, etc)
Are these particles actually motile/swimming, or just drifting?  It is hard to tell from the video.
If you remove the  cell line (straining, hypososmotic lysis, etc) is the contamination able to replicate by itself in fresh media?
If you streak the culture on agar, do you get colonies?

What did you mean by this: "At the beginning we thought that the particles we observed were mycobacteria. But the gram tests from the microbiological labs appeared negative."  Mycoplasma will NOT show up on a gram stain.  Was there another test the lab did for mycoplasma?

Dakota's suggestions to check for contamination in your media and check for 16S RNA are good ones.

One of the hardest kinds of contamination to detect and get rid of is contamination from another cell line. It's possible you could have a 2nd cell line mixed in, that has a different morphology and might be producing more apoptotic blebs...

Mike

[Cindy GRANDJENETTE]

Thanks for your answer.

We already looked if something grow in the fresh media, but we did not observe anything after few days.

For the second point, we thought to do it but unfortunately we do not have the material to perform it in the lab.

[CodonAUG]

I was under the impression that the only real strong test for mycoplasma was PCR (and even it can miss if you dont use diverse enough primers as there are many species of mycoplasma).

[Cindy GRANDJENETTE]

We are working with mammalian cells and we observe the contamination above all in haematological cell lines (K-562, TF-1 and also in CD34+ cells isolated from cord blood).

Normally, these cells are round but, after contamination, we observe really big cells, cells that look like fibroblasts, cells with protrusions (pictures attached).

Particles do not seem to move a lot but it is difficult to determine whether this movement is "autonomous" or not. They also seem to adhere to the support.

We try to observe if something grow in fresh media but we did not observe anything. It is a good idea to remove the cell line but we do not know how to distinguish these particles from the debris.

We do not have the material to study bacteria but we sent some samples to several labs:

- Nothing grew on their media

- 2 labs dit not observe anything with gram staining but one found gram + bacteria (but the staining was not good). These Gram + bacteria are found inside and outside the cells (pictures attached)

- We hypothesised that coud be mycobacteria. Then, we sent samples to a another lab to perform Ziehl staining. However, this test appeared negative.

- We do not know exactly whether microbiological tests were done on our media our on cells.

- We did mycoplasma test in our lab and the results were negative.

- We do not have the material to perform the universal PCR.

- Concerning cross-contamination, we don't think so because we are also working on primary cells, which are also contaminated.

[Cindy GRANDJENETTE]

This is an additional movie we performed on another cell line.

https://www.dropbox.com/s/99g92mlslfguhdu/150727%20k%20562%2024h%2015%20min.avi?dl=0

[Mike Horwath]

Hi Cindy,

My best guess is that you are getting apoptosis without an infection.

• Your contamination test results have been negative.
• Your media does not grow anything by itself
• You are seeing apoptosis/weird cells even with chord blood cells, not just cell lines
• Your TF1 stained cells look like they are in early apoptosis, with apoptotic blebs on the surface
• On the videos, especially the most recent one, you can see cells breaking apart into blebs.
• I don't see any evidence of the debris multiplying, it's just getting produced from dying cells.
  

Figuring out exactly why cells are becoming apoptotic can be challenging...I have had to deal with this question in the past when working with cell lines.  Your media might not be quite right to keep them happy.  Pay attention to any changes you might have made around the time you started seeing this issue.   Also pay attention to necessity for pyruvate, glutamine, etc for a particular line, as well as expiration dates on these reagents.

Also, depending on your microscopy setup it's possible the cells may be fine until you start trying to take live images.  Change in temperature, PH, even the bright light itself can be damaging over time.

Another possibility is the antibiotics you are adding to reduce contamination, especially amphotericin.  "amphoterrible" is somewhat toxic to mammalian cells too.  Try a culture with no antibiotics.

The really big cells in your TF1 giemsa stain look like multinucleated giant cells.  These can occur in some cancer cell lines, although I have no idea if this is common in TF1 or K562.  Primary myeloid cells will also join together to make giant cells under certain cytokine and stress stimuli.  I see this in mouse macrophages and dendritic cells, especially if stimulated with IL4.

Good luck!

Mike