cell culture - passage number (Sep/17/2007 )
how important is passage number? why? thanks you in advance
Well, the higher the passage number the "older" are your cells.
When you passage your cells, there's always the chanche of not selecting a representative sample of the initial population.
Moreover, some cell lines tend to lose their initial characteristics such as membrane proteins/receptors etc...
I had cells changing their shape!
the higher passage number, more likely to lose the characteristics of your cell, I am currently working on CHO cells , and I can clearly feel that my cells do NOT attach like the 1st day and they need 2 days to attach, besides, transfection efficiency was low.
basically I make stocks of my cells when they are at low passage number and culture them til max. passage 20. Then I thaw new cells.
I do a lot of immunofluorescence with CHO, HeLa and U2OS cells. As these cells get higher and higher in passage number (above 20) there are major changes. First, as previously mentioned, they just don't attach quite as well and transfection efficiency drops like a stone. Second, all three of these cell lines will start to become multinucleated or there will be clusters of cells with nuclear bridges. I've seen up to 10 cells all connected by their nuclei.. as visualized with DAPI. Cool to look at, bad for cell cycle analysis. Most likely, such a profound change in cellular morphology is going to be accompanied by major changes in signaling, protein expression/interaction, etc. It's just in your best interest, as a researcher, to use low passage cells.
ok..now i have a better idea about it. thanks a lot guys. erm, does passage 20 is consider old for a cell? what is the maximum? yes some of the cell, such as hct116 have change in their morphology when it above 20.
It is totally dependent upon the cell type AND the marker you are looking for. Use these 2 as examples:
J774.A1 are murine macrophages and are an immortalised cell line. I use these cells to produce an inducible enzyme used for drug screening. They can be passaged 200-300 times and still give the same amount of enzyme as P1 cells.
PAEC are primary cells isloated from pig aorta. I have used them to look at Nitric Oxide release from endothelail cells. I have to use them at P1 as the ability to release NO is not seen after this. Also if you look at another marker such as Prostacylin this is also NOT seen after P1.
Normally researchers are looking at Receptor Expression, enzymes and other secondary messenger sysyems. Cells are used as a tool for research and as a STARTING MODEL. Normally you would go from an invitro system like cell culture to an in vivo model and then to human models i.e. clinical trials (Phase 1-4).
The J774 cells being murine gave us a starting model for NO release. We then went from this to Human primary cells and then to go onto the human tissue and clinical trials. However you have to be careful in choosing models because of the inherent changes that can take place.
Hope this is useful
Thank you, Rhombus
Very clear explanation.