I've some queries regarding assigning passage no in cell culture
During reviving : The passage no of vial = passage no of culture flask that was used for reviving.
During Freezing : The passage no of Cryopreserved vial is +1 to the passage no of flask that was used for freezing ?
4. For suspension culture :
During reviving : The passage no of vial = passage no of culture flask that was used for reviving.
During Freezing : The passage no of flask that was used for freezing = The passage no of vial ?
Hello Vikram Kumar , John's answer above is really complete so I will only add this for question 1. and 4. :
1. The splitting ratio that you use will depend on the growth rate of your cells. Some cells grow fast and can be diluted using a high ratio (1:15, 1:20 even), so you don't need to passage them daily, only every 3-4 days. But some other cells grow slowly, and if they are too diluted they will almost stop growing : I have seen it with patient fibroblasts (=primary culture) diluted at a ratio of 1:3 for example.
What doesn't change is that, once you detach your cells from the flask, you add +1 to your passage number, be it a 1:2 ratio or 1:15 ratio.
4. Personnally, where I work we don't count passage numbers for suspension cultures, because we consider that there is no chemical or mechanical action used to detach the cells. But for some particular cell lines (that I don't work with) I have seen that it is important to count the passages, because they might differentiate from the original population when diluted more than ~5-10 times after thawing.
Hope this helps !
1. Once cells are frozen, the passage # should come back to 0. Then when cells are thawed and seeded into dishes, it is passage #1. Then each time you detach cells via either trypsin or cell scraper and reseed/split cells into a new dish, it is p#+1.
2. For some cells, the consistent functions/features are limited to just a couple of passages (HUVEC in passages 2-4), others are not (RAW cells in passages 10 or 20). That depends, you have to check the literature. For GMP, it is another story.
Thank you John Hardy Lockhart Bénédicte Giroudot Feng Wang for your answers, I got a clarity.
Feng Wang
Your Response to Q1 makes absolutely no sense.
Please look at John Hardy Lockhart 's answer and question your mentioned protocols.
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