After reviving the cells from -80C and seeding the cells ~1 million in T25 flask when the cells reach 80% confluency then if I passage/subculture the cells for eg. spleen (finite) or CHO or Hybridoma cells 24 times (1:3 splits in T75 flask) twice a week. What would be the generation number of the cells? after 24th passage. Can I say the cells have passed 24 generations? And if I cryopreservate it as a cell bank after 24th passage? and again revive after several months and do 24 more passages?
Can I determine the generation number based on passage rather than doubling time?
And normally we split the cells when they reach 80% confluency so out of 1 million thawed cells which has a doubling time of 10hrs. If I take 300,000 cells (1:3 split) and wait until they reach confluecy. After 20 hrs my cells have passed two doubling times to reach 1.2 million and have passed two generations?
Hi Xin, usually you count the passage (or splitting) of the cells. If you split them 24 times then they should be labeled p24.
This is usually not important for immortalized cells, but for primary cells it is very important since they can change their phenotype depending on how often they are split and kept in culture.
For HUVECs, I don't keep them past p6.
Thank you for your response
Yes, I read this in a document today population doubling number is important for finite cells/primary cells as they reach senescence.
For continuous cell lines can I cryopreserve the cells after 24th passage? and revive and keep passage as per requirement.
Although more important for primary cells, the passage is also important for immortalized cells, since they accumulate changes during culture/proliferation. More passages mean the cell is more different than the original one.
I would never cryopreserve cells after so long in culture. I would rather freeze backup vials at the earliest passage possible. Then you keep your cells in culture, and when you need another vial (or when it stayed too long in the culture) you can thaw a vial with low passage to continue your experiments.
I agree with Mariana. Cells can change if kept a long time in culture.
I find it interesting that none of the answers so far have made any attempt at answering the question.
The generation number will be much higher than the passage number. The only way to estimate the generation number would be to perform cell counts at every passaging. In the example you gave using 300,000 cells that ended up at 1.2 million, yes it took two generations during one passage.
Of course generation number can be much higher than the passage number. Just look at HeLa cells. They were first isolated over 50 years ago.
That does not change the fact that many cells, especially primary cells, can lose their characteristics the longer they are kept in continuous culture.
Every time you split them there can be a subset of cells that grow faster than others. The more you split them, the more prominent these cells can become.
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