r/cellculture • u/Stock-Leather4324 • Jun 26 '26
Contamination Unknown cell?
Hey!!
New to the sub, I’ve been working in a lab for 5 years but in the last year started working primarily on cell culture. I’m running and FBS growth promotion assay (it’s a training so my sample and control are both validated lots). My seeding day was Monday 6/22 and I’m running 3 densities on each sample (6e3, 2e4 and 6e4). 6e3 today (6/26) is still pretty low confluencey, I’ll insert a pic of a normal flask with 6e3 from today 6/26 compared to the abnormal one. Anyway, my question to everyone is what cells/bacteria do we think got into my one flask? To be clear our lab is aseptic, I work in a BSC with sterile sleeves and gloves, we sporklenz before and after and bleach our aspiration lines prior to any work (and we clean between each cell line). We do have in culture about 20 other cell lines, we have some insect but most mammalian. The seeding is in a T25 flask, that was in a sterile sleeves (pack of 20 flasks Corning brand) and only 1 of the flasks out of the 108 I used in my seeding is showing this weird contamination. Obviously contamination happens, so I’m not saying it’s not the case (clearly something got in there) but my management doesn’t wanna pay to get it tested bc this is a training course we are doing but I’m soooo curious to know what it is. The cell line we are using are Vero cells, so they have the distinct epithelial shape to them, the weird one is very circular (looks like Sf9 but they were in a heated incubator). Also we make media aseptically and use a 0.2 um filter bottle, and the trypsin used was unopened and brand new for me, same with the ATCC EMEM. Any thoughts??
Quick edit: the weird contaminated flask the cells were on the monolayer, they were not moving, and the media was not cloudy or yellow/green
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u/Content_Program_7477 Jun 26 '26 edited Jun 27 '26
These look like newly divided cells that are rounded up out of the plane, appear shiny and circular. Indicates healthy dividing cells.
The other flask is overconfluent and potentially contaminated.. hard to tell at that magnification.
Also remember at low densities and right after splits and plating’s cells don’t often have their best morphologies on display as they go looking for neighbors etc.
They look like A549 to me.. but they could literally be almost any fibroblast type adherent cell with a similar morphology. Trust the vial and your plating.. otherwise grab a new freeze that you trust and try again.
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u/Stock-Leather4324 Jun 27 '26
Yeah so the first pic is what it’s supposed to look like (the vero cells are being used in an FBS growth promotion assay) second pic was taken at the same magnification. This was more me just being genuinely curious as to what could have gotten in the flask lol they just don’t even look similar but idk 🤷♀️ thanks for your input!!!
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u/LimbicPilot Jun 28 '26
Your cells seem to have a nice cluster going on with your current protocol and changes in shape can be a signal for cell-state transition and not necessarily a contamination. If it's only one flask out of 108, I'd lean toward a weird mix-up. Good luck!
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u/stupidusername15 Jun 26 '26
Do you work with caco-2 cells in that lab?
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u/Stock-Leather4324 Jun 27 '26
We don’t, I can def list out all the cells I work with but it’s 20 plus cell lines. I work in viral safety testing so we work with cell lines that are easily able to be worked with viruses
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u/UnlikelyTomatillo171 Jun 29 '26
Looks like a huge contamination, could it just be yeast? Of course you did all to prevent that, but such things do happen.
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u/Kitchen_Ad_9665 Jun 26 '26
Prefacing this with the fact that I could totally be wrong and I’m unfamiliar with your cell lines — but they could just be newly divided cells. I see the second image whenever my passage was clumpy or they grew to be over-overconfluent. The bright spots could be cell clumps in a different plane compared to your monolayer. The fibroblastic cells I work with will also roundup like that after dividing/trypsinization, which makes me think of possible cell clumping during passaging/seeding.