We are culturing BJ fibroblasts in MEM on TPP yellow culture plates (10 and 15 cm). Recently our cells started to proliferate at a much lower rate than usual, and we checked that the problem was not coming from the incubators, medium, serum... Even freshly thawed cells eventually stop proliferating after 2 passages. When routinely looking at the cells under the microscope, I noticed that the number of dividing cells in the plate was normal, the same as you would expect if the cells were proliferating at their normal rate. However, many mitotic cells were detached and floating in the medium (particularly in telophase). I suspect a problem with the coating of the plates (I mean the normal surface treatment performed by the manufacturer, we don't additionally coat the plates ourselves for culturing the cells), but culturing the cells on different TPP plate lots didn't solve the problem. However, the cells grow normally in 96- or 24-well plates... Have you dealt with similar issues with BJ cells or other cell lines? Or had a problem with culture plate adhesion? Any feedback or help would be greatly appreciated!
Dear Valentin
you can check if your cells are cobtaminated by micoplasma.
a simple pcr protocool is available on my blog: ProteoCool ( https://proteocool.blogspot.com/?m=1)
ProteoCool n°18 Simple PCR test to detect Mycoplasma contaminations at Page 5
good luck
Manuele
Dear Manuele,
Thank you for your answer and your link. I will check this.
Cheers,
Valentin
How long have you been propagating those cells? They might be on the onset of senescence.
Dear Valentin,
What about the cell density? It might be that the cells are very scarce in the big dishes.
"Cell density has a significant influence on the growth of cells in vitro. Cells secrete a variety of beneficial factors into the medium, and as such are able to condition the media in which they are growing. Increased cell-cell contact also directly stimulates a variety of membrane-bound receptors. When seeding cells, there is a key level at which the cell density is sufficient for this beneficial effect to function. All media and cells have a minimum cell density below which initial attachment and growth begins to decline. "
Dear Rubén,
Thanks a lot for your answer! However we finally identified the problem, which was related to a particular medium lot. My cells are now growing normally!
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