I am doing a cytotoxicity study for a group of drugs in different cell lines using an MTT assay. I am wondering about cell passage (Subculturing). One time I worked in an early passage (such as no 3) and another time I worked in a late passage (such as no.12). Does the number of passages show a type of resistant or not to the cytotoxic drugs? The shape and the identity of a cell still conserved. How many passages after it should I withdraw the cell?
In general, passage number is a greater concern for primary cells than established cell lines. So the answer likely depends on what cell types or cell line you are culturing.
Do you know whether the cytotoxic effect of your drug is depend on mitosis? If so, then you had better test the growth rate of the cells at different passages. It is not hard to imagine that growth rates of primary cells would vary between passage 3 and 12 in a way that could change their susceptibility to a cytotoxic drug that acts specifically on mitotic cells.
If you're working with an established cell line and/or the mechanism of action is independent of mitosis, then the likelihood of a passage dependent effect is probably low.
In my opinion, if you are working with primary cells, then passage number is important. Im working with primary cells and I would not use passage no. 12. I think its too much, because of many inner events you dont know what "kind of cell line" finally it is in such a high passage no. and also this cell line could react different on drugs as low passage no. After so many DNA-damages inside the cells in high passage level there could be many mutations and such cells are never the same like in low passage level. So if you are working with late passages, and you have some results about drug cytotoxicity, these results will be never reproductable on cells in low passage level. In our lab we are trying to work with cells just in low passage level, the lower the better. Cause passage number really matters.
Thanks Daniel and Petra, I am working in established cell line (non-small lung cancer cell NCI H1650 and Gastric cancer cell GSC7901), and I observed a little difference in susceptability toward anticancer agents when I use early or late passage.
So I don't know exactly what the reason even the cell keep it's shape and identity?
and I want to know at what passage I have to throw the cells and not working in it?
Yes, to an unpredictable extent sometimes. You can avoid that by working with in the variability range of 2-3 passages for continuous cell-lines..
Bashir- you stated, " I observed a little difference in susceptability toward anticancer agents". What is the magnitude of the effect? Have you tested for statistical significance?
It could be that the variability that you attribute to passage number is merely noise in your assay. Have you since repeated your experiment with both early and late passage cells and demonstrated that the effect is reproducible? It may be shear coincidence that you got slightly different cytotoxicity results on different days, and not be casually related to the difference in passage number.
For passage 3 vs 12, i dont think there will be much difference; if passage 3 vs 300, there might be. Culturing cells in the flask/plate is not an ideal condition for any cells to growth, even for stabilised cell line. Genetic expression may altered in longer passage number for the cells to adapt to the "in vitro" growing condition. For transfected cell line, usually it will be recommended not more than 20 passages for the same reason.
Ideally, you should perhaps keep many stock of cells with low passage numbers.
You could always discard the cells if you observe any abnormal morphological changes in cells prior any treatment.
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