Recently I’ve started seeing these clumps in my primary MSC flask (T175). I first noticed these compact clumps in a flask near 80% confluence but I’m also seeing it in less confluent flasks. The clumps vary in size. The medium is neither cloudy nor changing color. I’m using a basic growth media (DMEM, 10% FBS, and 1% antibiotic-antimycotic). When passaging I dissociate the cells using 0.25% trypsi-EDTA for 5 minutes. I’ve been washing the flasks with PBS + 3% antibiotic-antimycotic at media changes, but have not seen any change. Does this look like fungal contamination or could it be cellular contamination (for example, T cells or pluripotent cells clumping) or cellular debris? Whatever it is, it appears to sit a a level above the live, attached cells (with other dead cells).
Thank you!
The clumps are NOT fungal contamination. Most fungal contamination is a fuzzy looking ball with long hyphae coming out of it. Blatantly obvious.
The clumps are most likely some sort of cell debris. Once you harvest the bone marrow and attempt to break up the clumps (aspirating with a pipet?), do you pass it through a cell strainer (40, 70, 100um?) to remove large debris and obtain a single cell suspension before plating?
Tom Masi Thank you for your feedblack! we did suspend the marrow in media using a pipette before plating the marrow initially. We did not pass the suspension through any kind of cell filter. These images are not from the initial plating though, they are from passages 7-8. These clumps only recently started appearing in the culture, which was another concern of mine.
Thanks for the information. Since the cells are from passage 7-8 then I would say the clumps are definitely cell debris. I typically do not use MSCs after passage 6. Past passage 6 the cell morphology looks suspect, their growth slows down and you start to see a lot of floating cell debris compared to earlier passages. I just toss the flasks and break out new vials from LN2.
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