Hello everybody,
i hope somebody can help me with my cells.
Since 2 weeks they started to change morphologically, as you can see in the two pictures with the smaller magnification (they started to build a vessel like formation combined with some really dark round structures (this is no pigment!)). I really dont know what the problem is but i have had this now every time after RPE isolation. (I tried new media, we cleaned the incubator etc. but still it looks kind of contaminated). In the third picture (the one with the larger magnification) you can see that there are a lot of small black dots which even moved (bacteria ?).
So my overall question here is, if those cells look contaminated to you ? And if yes what can i do to prevent it in the future even though i already threw away all the old media, and openend new bottles for everything, cleaned the incubator etc. ???
Thanks a lot for your help,
Peter
https://pubmed.ncbi.nlm.nih.gov/12752057/
check this out.
Not an expert on such things, especially not on primary cell lines, but maybe you can add a mixture of antibiotics to your media? The stuff you see swimming around could also be cell contents from disintegrating cells. Did your media look turbid, any changes in color and does it smell when you open the incubator with your incubated cells?
Thanks for your answer ! I already have Pen-Strep. in my media, i tried antimykotika which stressed the cells and let to an even higher cell death !
The media looked totally fine and the incubator was already cleaned 2 weeks ago. I did not smell anything, neither before the cleaning or now.
I am kinda curious what it is. What i forgot to mention is that only some small dots that you can see in the picture with the large magnification are moving. The small dots which are on the cells are not moving.
Definitely looks like bacteria in the bottom right, especially if moving
Moving black dots is never a good sign...if something is moving in your plate you should throw it away ASAP! Make sure it is moving in a random pattern and not just something floating in the media.
Your top picture looks pretty normal but the black areas scare me. It sort of looks like mold or some fungus contamination.
Is your media cloudy? Even if it is not cloudy, you could still have contamination.
It is hard to tell, but the last one sort of looks like yeast to me.
I'm sorry this has happened to you, I feel that every cell culturist has at least one encounter with contamination, and then you learn from it, and it never happens again.
Check your waterbath, this is usually a very high source of contamination. Wipe all counters with ethanol/other detergent and even mop floors with a diluted detergent. Clean incubator using it's built in cycle for cleaning.
I also recommend using 50 mL tubes to aliquot media every time you feed cells. this has helped me a ton. Are you sterile filtering your media? If you use 1X PBS ae you autoclaving it?
I like using 1X anti-anti in my media. I order it from thermo. https://www.thermofisher.com/order/catalog/product/15240062#/15240062
I also work with epithelial cells and it works great for me. I've never had an issue with bacteria, but I have had an issue with yeast.
Sorry if this answer is all over the place, hopefully some of it is helpful to you.
- Badges
- Science method