how we count the passages in cell cultures?
Posted 22 June 2010 - 04:18 AM
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!
Posted 22 June 2010 - 04:47 PM
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.
Posted 23 June 2010 - 05:22 AM
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!
Posted 24 June 2010 - 12:44 AM
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?
Posted 24 June 2010 - 06:38 AM
Posted 29 June 2010 - 03:28 AM
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.....