I know that a "passage" in cell culture practice can be rather arbitrary, but I hope to get some experts opinion on the matter.
Example: Cells in "Passage 4" reach 80% confluency. Instead of splitting the cells they are frozen. In this example, I would label the cryo vials "Passage 5". When recovering the "Passage 5" cells, I would label the culture flask "Passage 5" as this would have been the case, had I continued culturing them instead of freezing. Is this the correct way?
Cheers,
Tina
Actually, only about 50-60% cells recover on rethawing - so sometimes people do add 1 more passage to the culture age..., but whatever you do, n´be consistent. The number of passages or population doublings a culture goes through is often variable from person to person and from time to time - this number is only for guidance, nothing magical about it.
Hi Tina,
Some labs/people keep tabs on the passage number since certain cells behave differently when continuously passed for some time like HepG2. I've seen some people reset the passage number to 1 once they thaw their cells. What's been bothering you about the number?
-ED
Thanks to both of you for taking time to answer my question.
@ED I'm helping out a colleague of mine who's on maternity leave at the moment and the lab tech helping her out is working part time which makes cell culture experiments difficult. According to the protocol, that I've recieved from my colleague, the cells should be harvested at passage 7 for analysis. Culturing my way, that means harvesting after splitting 3 times. If we culture according to the lab tech the cells are ready for harvest after 2 splits (She adds a passage number after recovery).
@Suresh your explanation about viability after thawing may explain the lab techs practice. We will continue doing it her way, if that is how it has always been done.
Have a nice day.
Tina
Hi Tina,
In my lab we add 1 passage each time we add trypsin to detach the cells, when they are adherent. When you frozen them, you would detach them, so we add +1 passage. If they are in suspension (for example monocytes), you would have to count each time you spin them to harvest again.
Hi Tina,
As you see everyone's practice is bit different. We add +1 every time we detached cells. When recovering cells from frozen we would state "from frozen" +1 as we not always have information how long they had been passaged before freezing. If it is important for analysis that your cells are at certain passage number, your best choice is to speak to the person who established the protocol at your lab.
As mentioned by others it is an arbitrary numbers but sometime it does make sense. If you split and dilute them at low level seeding then the numbers will messed up. Therefore it would be approximate passage number for the experiment. If the other lab member say 7th passage, but in your case 9th may work. Depending on the seeding density and the health of cells, passage number differs.
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