Hello everyone. I need help. I am new to cell culture (2 months +) so I am not really experienced to distinguish these cells if they're healthy or not.
Is this HEK293 cells contaminated?
Context : I actually seed this cell at 4x10^3 density. However, after 24 hours when I want to start my treatment, I noticed that there's this traces of fungi (greenish colored, some looks like fiber). Therefore I washed the plate and changed my media. This is 24 hours after changing my media, I noticed that my cells has reduced confluency and I am not sure what is the structure in the image. My cells does not grow around the 'entity' as in the image.
My assumption is that these are an apoptotic cells because there's high chances that my cell is too stressed (Since this is a 96-well culture plate and I have maintained there in this plate for more than 24 hours). However, I am not sure if this assumption is correct.
If it is contaminated, what could be the source and what should I do?
Anyone has any idea on how can I fix this issue?
Hello! It looks like a cell spheroid, you could try washing the cells several times with PBS to flush out the spheroids that are not attached. Then reseed the cells and suspend more intensively. But you should watch the dynamics of change.
Hi dear friend
The main reason for the formation of such cell spheres is two important factors: one is the failure to properly trypsinize the cells and the cells are not separated properly, and the other is the repeated use of a cell culture flask to culture a specific cell.
Good luck
Hello Georgy Leonov and Kamran Hosseini . Thank you for the insight! I think this might be the cases because I noticed after I washed them, some of the cells started to detach from the plate surface. And after 24 hours, my cells loses some confluency (probably because the detached cells clumped together). Thank you!
I have a question, does this affect my result if I were to treat them with my samples to check for cytotoxicity?
In my opinion, before you want to use these cells for cytotoxicity assays or anything else, first trypsinize them well and pipet several times until the cells are detached and homogenized, then put it back into the flask and let it so that they grow well and get the correct morphology... if the cells were ok, then there is no problem and you can use the cells
Please look at this figure and compare the difference with your own figure
With conventional cytotoxic agents such as H202, this does not usually occur. Cellular spheroids can be stained with trypan blue for viability assessment.
HEK cells produce spheroids (organoid-like structures with no differentiation and single cell type). It happens when cells are not properly dissociated, and the clump fails to attach and proliferates in suspension. You can easily produce spheroids of various sizes by seeding HEK cells into low attachment plates. It doesn't indicate your cells are contaminated. If you have frequent spheroids formation and you don't want them, you can use a 50-um cell strainer after trypsinization before seeding your experiment.
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