Adherent cells detach themselve from petri dish (problems in cell culture) - (Sep/01/2020 )
Dear all,
Since 4 weeks now I have the problem in my cell culture that my cells (human smooth muscle cells) detach from the petri dish and die (see pictures).
This happens after changing the medium, splitting cells or after thawing cells (I get new cells from my colleagues every time). The procedure is that after splitting/thawing the cells settle as usual after 24 hours, but after 1-2 days they partly separate again and become cell debris.
Since my colleagues use the same cells, media and incubators etc., many factors can be excluded (temperature, CO2, water bath, Petri dishes etc.).
There are no visible contaminations and a mycoplasma test was negative.
There is phenol red in the medium, which has not changed color. The pH-value of dishes with "dead" cells is the same as the pH-value of dishes from my colleagues.
I have been working with these cells for 3/4 of a year and never had such problems before (and I have not changed anything about the method/material).
Does anyone else have any idea what could be the cause?
-Tovusdavaz-
Have you checked the quality of the water you use?
-mdfenko-
Unfortunately I do not know exactly which water you mean: The one in the water bath or the one in the incubator. But since my colleagues have also used the same water bath and the same incubator as I did, and the problem does not occur in their cells, I think that this is not the reason.
Nevertheless, thank you very much for your comment
-Tovusdavaz-
I meant the water you use when preparing solutions
-mdfenko-
We buy our cell media, so there is no need for preparing solutions.
Since my colleagues also use the same medium, this cannot be the reason
cell media
-Tovusdavaz-
How about:
water used for PBS preparation? or do you purchase that as well?
are you and your colleagues using the same culture dish, trypsin concentration, etc?
-SF_HK-
You could try to use only ice-cold PBS w/o Ca/Mg for detaching your cells. Wash your cells with warm PBS, add prechilled PBS and keep the petri-plates on ice for 5-10 minutes. Then gently scrap the cells with a scrapper. In this way we used to detach macrophages for doing flow cytometry. But this process is little bit tricky. During scrapping there is always a probability of damaging the cells.
However, if you want cell culture plates at a reasonable price, you can visit our website i.e. Aaka Scientific
-AakaScientific-