We received a stock of contaminated vials. After discovering contamination in the fifth thawed vial, we must assume the entire stock is affected (12 vials). This issue appears to be isolated to the vials themselves, as other iPSC cultures remain unaffected.
Unfortunately, discarding the entire batch is not an option, so we will attempt to save at least one vial.
We have acquired Normocure and plan to conduct tests over the next few days. At the point of thawing, the medium (mTeSR Plus) contains antibiotic pen/strep, which appears to be not enough.
After reading other inquiries and responses on this blog, I discovered that the decontamination process is highly dissuaded.
However, I would like to ask if anyone else has had a similar experience and what was the solution to the problem? What approaches to decontamination did you employ? Did you carry out any unique examinations to validate the bacteria that contaminated your cultures?
Thank you in advance.
This is the situation at d2 post-thawing, using mTeSR Plus and penicillin/streptomycin on matrigel. I am yet to start the Normocure antibiotic treatment from Invivogen. I have recorded a video where some small dots are clearly moving, but unfortunately, I could not upload it.
Additionally, I dispensed some medium drops on an LB agar plate and incubated it overnight at 37°C. However, no colony was obtained this morning. Does it not appear inconsistent?
UPDATE
Following extensive research, it appears that Black Swimmin Dots (BSDs) have contaminated my iPSC cultures.
The most recent study on this symbiotic contaminant dates back to November 2022 and can be found at:
https://microbiologyjournal.org/elucidating-the-effectiveness-of-antibiotic-treatments-in-eliminating-the-black-dots-contamination-from-vero-cell-line-culture/
I also found this second article interesting, which analyses the phenomenon in depth and also provides a lot of multimedia material that may be of interest.
https://www.biorxiv.org/content/10.1101/366906v1.full
Unfortunately, our lab cannot commit to investigating and decontaminating these BSDs, so we will be eliminating the stock of contaminated vials.
This discussion will remain open for future reference, and additional information on the matter is welcome.
We have been dealing with this in our lab for over a year now. We tossed out all cell cultures and started fresh and it will not go away. Please advise if you have found a solution.
Skylar King
Thank you for sharing your experiences.
Could you specify which cell line is affected by the black dots? Also, when you say "start fresh", do you mean ordering a new batch from e.g. ATCC or thawing another vial from your liquid nitrogen supply?
Hek293-T, 10T1/2, Smooth muscle cells, monocytes. Every single line that we work with is contaminated. We ordered new cells from ATTC or cultured more primary cells. We tried switching the antibiotics to piperacillin and cipro like the article that you posted suggested. We finally have had to shut down our cell culture and have been extensively cleaning the room, hood and incubators because it continues to come back and now all lab members are affected. I think what happened was someone was unfamiliar with what to look for regarding contamination and now it is in the hood, incubator, etc.
Hi Skylar King
I have some pictures from the last iPSC culture that I thawed, and I noticed that the usual dots present in the medium were moving inside the cell. Also, I have some videos - which illustrate the situation more clearly - but unfortunately I can't post them on Research Gate.
I will conduct further investigation in the coming days, with the hope of uncovering something advantageous for both of us.
Can you send me the video? Via message? We are also still troubleshooting. This is the most annoying thing ever; it seems as if the cells will look fine for a bit but then once they are split using Trypsin the BSD return. Your BSD seem larger than ours. Are your cells dying?
Skylar King
The cell picture have been taken three hours after thawing and seeding, so maybe there are actually a lot of dead cell and debris.
I am reporting the state of this new culture daily, we can discuss via message, I am going to write you.
I just found this and I think we will try it. This will rescue cell lines within 3 passages. Normocure. I also think we will use Normocin as a preventative for a bit to make sure we are rid of everything. Please see the attachment from Invitrogen. Hope this helps!
I have already tried Normocure, but I did not get lucky. I was thinking to try with a higher concentration, and together with Fungin at the same time. I hope this could work!
Good luck, and keep me posted!
We ordered the Normocure, and plan to also use the Normocin as part of a "routine addition" with the other things we add to the media. Fingers crossed for us both!
Eric Perouzel
Yes, I did. After one month of troubleshooting, I managed to solve the problem, even if I can't really say what the source was
You can't test quality into a material. You can't test every drop of the material and whatever treatment you apply to mitigate potential contamination has to be validated and offers a confounding variable to your experiment.
The batch is adulterated and using it is not appropriate. Are you willing to report in a potential publication the contamination risk you attempted to control?
This supplier is not giving you quality material and using their material - whatever your data - fails in scientific rigor.
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