How many cell passages to be safe ? - (Nov/18/2004 )

Hi !!

I don't have any experience in cell culture at all and I've just started to culture and transfect some cells for a few weeks. People from my lab gave me some SK-N-SH and C6 glioma cells.
The thing is nobody told me how many passages these cells have already been through... and this doesn't seem to worry anyone in the lab.
The cells look OK but I'm just wondering if they might lose some of their properties bit by bit with many passages...
What do you think ? Does anyone have experience with these cell lines especially ?
Thanks for your answers.

Anne

I've nerver worked with your cell lines, but what I personnaly do is working and 'pass' my cells for at least 40 passages, without a problem. I work with my cells as long as they "work well", and then I thaw some...
Hope this helped...

Simon

some of the cell lines out there, such as HeLa, CHO, Cos-7 etc etc have been through countless number of passages. I would bring up fresh cells as soon as I see morphological changes, or if by mistake they've gone too confluent.

A couple of years ago I got an undergraduate student working on endothelial cell receptor expression to look at the changes in expression of 3 receptors over 25 passages of EAHY926 endothelial cells. The expression of ERbeta dropped by 300 fold, IcamI by 250 fold and GR by 8 fold (qRT-PCR). This seemed to explain why a previous student couldn't detect any ERbeta in her EAHY cells which had been high passage.
After this we always went back to the early passage stocks every 15 passages.
I think different cells behave in different ways. always keep an eye out for changes in the phenotype you are studying.

I was wondering whether any phenotype changes have to be caused by genetic changes in the first place in high passage cells.

generally, some properties will change with the passages prolonged,

it is hard to say whether it is safe or not , because different cells have different situation cultured in vitro

We always "bank" our cells. Meaning thaw a vial out, grow it up in culture, passage at least 3-4 times all the while expanding it to be as large as possible then freeze down the cell line as one batch. This is your stock that you grab from once you have used cells to a high level of passage. Every time you passage a cell line a selection is made and genetic drift from the original stock cell line will undoubtedly occur.

Well, how about a stable cell line? In my lab we are still using some stable lines that someone made like 3-4 years ago. At some point you will run out of the original stock...

i
not directly related, but sf9 insect cells are supporting (as i was told) 35-40 passages...at most.
The relative way to estimate safety of cells for experiments is the number of cells in suspension. In fact, after a change of medium or a passage, when passage number increases, cells tends to get in suspension quicker... (and so dies early) kind of a increase of turbidity directl related to passage number.

for IMR 90 that are primary fibroblasts, it seems that things are close that for sf9 cells.