I am recently beginning to understand more or less how to culture suspension cells, but I get confused in regards with passage number in these cells.
My question regards to:
Thawing: Whenever you thaw a cell and culture them to expand them, is it consider a passage?
Subculturing: Subculturing seems to be very easy to record, taking an aliquot of desired cells and transferring them to a new flask with fresh media counts as a new passage.
Freezing: Once cells have reached confluency, around 1,000,000 cells/mL, it would be time to freeze them. If I started at passage number 6 and it is time to freeze them, are they frozen at passage number 6?
My main confusion is with freezing and thawing in recording passage number.
A freeze is just a passage with a cold spell in between. If you have a culture that currently is passage 6, then whether you decide to subculture it or freeze it, the cells will be identified as P7. Cryovials and plates or flasks would be labeled as P7.
When you thaw one of the P7 cryovials into a plate or flask, it remains P7 because you did not passage or subculture it again just because you thawed it..
A freeze is just a passage with a cold spell in between. If you have a culture that currently is passage 6, then whether you decide to subculture it or freeze it, the cells will be identified as P7. Cryovials and plates or flasks would be labeled as P7.
When you thaw one of the P7 cryovials into a plate or flask, it remains P7 because you did not passage or subculture it again just because you thawed it..
Hi, the passage numbers are as Ron mentioned, but if you want to exactly record age of your cells and pergorm experiments with cells with the same age you should use cumulative population doubling (how many times did the first cell divide) instead of passage number.
The reason is simple - researchers passage cells differently
e.g.
researcher 1 passage cells after reaching 800 000 cells/mL and always use 100 000 cells/mL for next passage
researcher 2 passage cells after reaching 800 000 cells/mL and always use 400 000 cells/mL for next passage
both researchers use the same cells passage 10 but they find significant differences in activity of some genes and protein expression - simply because researcher 1 has much older cells
researcher 1 use cells passage 10 cPD 27 (27x divided before experiment)
rersearcher 2 use cells passage 10 cPD 9 (9x divided before experiment)
So if you use cells which are known to change in passages such as endothelial cells I would recommend to use cPD
And if you want to be 100% sure you should also test viability of your cells after thawing to know how many live cells did you get.
I use cPD for endothelial cells
passage number for tumor cells (but all the cells are passaged exactly the same way with the same person)
hope this helps a little
I agree with Matej Vicen, passage number is a crude measure of cell culture age. It is better to use cumulative doublings, since non-immortal cells (i.e., not expressing telomerase), are subject to a Hayflick limit (about 50 doublings) and one wants to use young cultures for one's experiments to avoid complication from aged cells near their Hayflick limit. Cumulative doublings number is a much better indicator of this "age". For immortalized cells, this is no longer a consideration. But one should keep a frozen stock and go back every 3-6 months to replace existing cultured stocks to prevent genetic drift and its consequent changes in your cells.
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