How many passages cells lines can survive before senescence? - (Mar/04/2006 )

Hi all,
Is there any agreed number of passages suitable for cell lines before they start to scenece? I work with the NIH3T3 line and after about 4 months the transfection efficiency was reduced dramatically. I figured it might be because they are too old so I thawed a new batch and it was fine now. So, Is there a known number of passages, after which you need to throw away the cells?

Thanks

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hi
that point depends of cell type. Actually i know that for IMR90 cells (fibroblast), they don't support more than 40 passages.

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Hi

The number you are after is called the Hayflick limit after Hayflick who was the first to observe it in primary cell lines. The Hayflick limit varies with each cell line and cell type. Try going to the ATCC websiete, where they may be able to tell you more about your cells. As far as I know 3T3 cells are immortalised, but they will still slowly die/scenesce after a lot of passages.

BTW I have found it best to transfect with cells that have undergone as few passages as possible.

Cheers,
bob

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Thank tou guys

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Hi, me also, I would like to know, how many times can I passage my Vero cells?

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Alesia, as Bob and Fred have mentioned, perhaps doing research on your specific cell lines (contact either ATCC or the company where you bought them) and find out; it is different with each type of cells

with my little primary cells, we don't passage more than 5-6 times or they go into senesence...but it is measured by generation, not by passage; it is important to understand the difference

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Hi, could you please tell me the difference between "generation" and "passage"? Usally we just consider the passage times of the cells?

Thanks!

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If you pass your cells always 1:2, then one passage equals 1 cell doubling. If you pass your cells 1:4, then each passage equals 2 cell divisions. For senescens, the only thing that counts is the number of times a cell has divided and not the number of times it has been passed to a new dish.

Fibroblasts may have from 20 up to 80 divisions before they go into senescense. For epithelial cells, it is already difficult to obtain a few cell doublings.

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Thank you very much for you suggestions.

Just one stupid question: how exactly to calculate the split ratio:

I have adherent cells in T75, during the passage i throw away the 25 ml of medium, I have finally all my cells in 5 ml ( 1 ml of TV+ 4 ml of serum), and I take 1 ml of this into fresh 24 ml of medium. Is it calculated by the general dilution of my cells in the flask (1:4), or by their dilution with the medium (1:24)?

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