For the first time I'm gonna start culturing some eukaryotic cell lines and the passage thing is confusing me a little bit... in the case of well-established secondary cell lines (Hela, MCF7 or 293T, CHO, Cos-7), when you purchase them from a manufacturer, and as we don't know its passage, what's the point of labeling our cultures with the passage number? I mean, a cell line provided 10 years ago by a given manufacturer is no longer gonna be the same cell line 10 years later... Many passages will have been produced that would have led to some genetic shift, right?
Quick answer YES.
Long answer:
The Manufacturer or provider only passages very seldom. just to keep up a certain stock. Furthermore they recharacterize the celllines they provide regularly (sequencing, expression, growth behaviour, customer feedback, etc.) So the cells mainly stay the same.
You should lable your cell culture, so you know, that you have for example passaged this and that cell-line already 30 times and should "possibly" think about unfreezing some new ones for experiments. Most groups do it for example after a maximum of 20 passages regularly or if they feel the cells do not behave as they should anymore possibly earlier.
And even thou it mit still seem a lot to do 20 passages, this often only means 20 weeks or even less if you have really fast growing cells. So if you passage 2x a week, you have to trow the cells away after 10 weeks already.
I often came across the case, that cells changed behaviour over time quite a bit, because even thou, thay are labled as cell-lines they are often actually containing multiple phenotypse, if one grows slower but sticks more to your cellculture dish you will most likely slowly enrich that one over time. Some cell lines have to be changed ater 10-15 passages, which is annoying but often necessary.
Thank you for the long explanation Thomas. However, if cell senescence is related to culture aging and to how many times the cell have divided, the cell culture dilution (in each of the passages) should be as important as the passage number, right? A cell line with a passage number of 20 that has always been 1:10 diluted would bring more divisions than the same line with a passage number of 20 that has always been 1:2 diluted...
And when you mention phenotype, does it mean different genotypes as well?
It is not cell senescence, we are talking about, the cell lines are either artificially or naturally immortalized, they however are mostly fast deviding cancer cells with multiple chages in their genome, so the tend to change over time or as mentioned sometimes consist of slightly different subspecies.
The more you devide the cells, the more selective you get. So with a higher dilution, you also reduce the chance of keeping cells which have changed and also you select for the fastest growing cells, which is most of the time the main population which you want to have since this was most of the time also the original process, by which exactly these cells were selected. This is however not neccessaryly the case, so you are partially right but as explained in most instances this effect is farily well buffered by the higher dilution. Additionally YES it is important, to note how much you diluted your culture! It is common, to put not that on the cell culture dish and absolutly obligatory, to do so in your Lab journal as well, since in some instances it can give you valuable information. (if you cells die for example you may have diluted them too much, some cells are senitive). Additionally you could note, about how loong your passaging takes (same reason) ans other thins you may do differently.
I've not herd of such a case so far. It is possible, depending on how the cell lines are characterized but today genome sequencing is fairly common for these charaterizations so i would expect no. (Unless you mean during your cultivation? In this case of course both is possible)
Thank you again. So if I have understood correctly these kind of cell lines are not going to stop dividing, and die (as the primary cell lines do), after 20-30 passages but they are just going to acquire some mutations (that will not affect its survival as they are immortal cell lines)!
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