how we count the passages in cell cultures? - (Jun/22/2010 )

Hi to all!

A small detail (but important one!) that after 3 years of doing cell cultures, comes up.
When we say passage 0? when we defrost a cell vial from -80 degrees? Or the passage number continues from where we left it before we froze the cells?
How we should count then??

Thanks in advance!

I have always continued with the number at which the cells were frozen down, adding 1 for each passage. I know some people do it the other way, starting again fresh each time they thaw cells, and some people start the passage from when the cells were brought into the lab as some indication of how long they have been active in the lab/lab drift from parent line.

I don't think starting again when thawing is a good idea, as it gives a false measure of the newness of the cell line, compared to the rest of the labs using them around the world. For instance, some cell lines are sold from the ATCC as passage 400 (or whatever), I think starting again would be stupid as passage 400 is as early as you are going to get them if you need new ones, so saying that yours are passage 3 is wrong.

I agree with bob1 that you should continue the numerical sequence as this gives an immediate indication of how long the cells have been cultured. However although passage will give you a rough guide of how old a cell line is, you have no idea of the number of cell generations i.e. population doublings(pd's), the cells have undergone as you dont know the split ratio(s) used i.e. 1:2, 1:3, 1:4 etc. Pd's are a much more accurate measure of a cells age.

I would advise seeding at a specific density/cm2, and then counting the cells at confluence, measuring the time (hours) it takes. This way you can estimate the pd's and it will give you an idea of the maximum number achievable per vessel (cm2) or volume for suspension cells. You can then use this info to determine how many pd's your cells will undergo by splitting back at various ratios and the time it will take to reach confluence again.

Also if you are routinely storing cryopreserved cells at -80c, you should consider storage in liquid or gas phase nitrogen i.e. -130c to -196c, as this is the only way you can guarantee long term survival of cells!

Thank you Bob and Retroman!

In this way though, which I don't think has been followed so far in my lab, somebody can keep on working with a cell line at passage 500 which can be tricky, or misleading. But I don't see another way...
Would you have a limit lets say of passages that you would work with?
I know that this depends strongly on the cell type. For example for HEK293?

Personally, i would stop using the cells once i see some changes to the cell morphology and growth pattern (how quickly they grow, in particular). No experience with HEK293 cells so can't comment...

Obviously it depends on the passage number...
It depends on wht kind of work u r doing with those cells and kind of cells...
For Cell based assays I use hek with very low passage numbers... higher passage number gives discrepancy in the data compared to previous data with lower passage numbers... Some cell lines like huvec ppl dont use more than 10th passage...
For other kinds of work... like protein and molecular studies.. its okay with higher passages... just search the papers for the kind of cell line u r using, kind of assay and max passage number they used.....

Rgds
Jo