Passage number and behaviour of cell line - (Nov/06/2005 )

Hi,
I've been using A549 cells and am having trouble reproducing some results I got a couple of months back. Ive thawed out a new aliquot of the cells and will try my experiment with this. Meanwhile I was wondering if any one could shed more light on this. Do the cells behave differently if they have been split too many times. If so what is the threshold.
Do let me know.
thanks and regards.

Hi,

I haven't worked with this specific cell line, but yes, high passage number can alter behaviour of cell lines. The threshold is dependent on your cell line itself. Difference in behaviour after passaging too many times is the major reason why you should always after 2 or 3 passages preserve several vials in cryo.

Other factors that can influence the behaviour of your cell line is a possible mycoplasma contamination, the quality of your serum (there can be a difference between different batches of serum), and several others probably.
Try a fresh batch of cells and see what happens.

thanks, am in the process of setting up the expt with the new cells . will get to know in a few days.

Hi

You say you cannot reproduce the results you had a couple of months ago.
Did you then do the experiement how many times. Were the results consistent then?

You are assuming there is something with the cells, which could be true but try to think well, and check whether you are really reproducing the experiment, didn't you change something?
Sometimes, we don't take notes of everything we do in detail because we think we will not forget.
But check up everything again. Like cell density, medium (company suplier), serum, incubation time.

I don't know whether you are transfecting cells but if you are, is the plasmid DNA the same....?

Good luck.

Have you checked for Mycoplasma contamination???

how many passages are we talking? I would guess senescence if it is not something like mycoplasma contamination. did your cells change with multiple passages? any difference in morphology, growth rate, anything like that?

when our keratinocytes approach senescence, they grow slowly and after a couple passages they no longer divide much at all, and their morphology moves from the characteristic cobblestone appearance to a somewhat stringy-looking, longer cell shape. for primary HNEK, this happens at roughly 6 to 8 passages out of the initial tube, but sometimes this value will vary depending on seeding density and things like that.

Oh really 8 passages only!?

Do you know any web page or catalogue that says something about number of passages for common used cells.
Thank you

I used to thaw new A549 cells after ~30 passages. That's been my general rule for cancerous/transformed cell lines like A549s.

I use HepG2 cells up to 20 passages, but every cell line is different.

our cell line is primary and I know that limits it, but they are all different. I do not know of a link to any common information like passage number you can expect...I noticed our cells were flaky after around 7 or 8 passages, so I did all the appropriate mycoplasma testing and such because we were assuming some sort of contamination. I got nowhere with that, and finally emailed the vendor's tech support out of frustration. A very helpful lady told me about the senesence thing; she said the cells we use are only guaranteed through about 12-15 generations, or about 3 passages using 10cm dishes. so yeah, I bet it's different for every cell and media system.