3 replies to this topic

[CMIRC]

Dear All

I am confused as to how cell passaging works. My question is:

If I thaw a vial of PC3 cells (prostate cancer immortal cell line) and culture them. The passage number would be 0 when I thaw them first. However the passage number would increase to 1 when I sub culture them. Say after subsequent cell culturing I decide to freeze the cells at passage 4. The next time I take out a the PC3 vial from liquid nitrogen and culture would the passage start from 4 or from 0?

I know that for primary cell lines the passage number does not reset and continous even after freezing and thawing. however does the same apply for immortal cancer cells. And if it does then upto what passage can one do experiments without having any genetic alterations due to the passaging. ie upto what passage can one use an immortal cell line before a buying a new one?

Also for non adherent cell lines when would a passage be considered. If I take flask of 50% confluent THP1 cells and centrifuge them and put them back in the same flask with new media without splitting would that be a passage, secondly if I have 2 flasks of THP1 cells 25% confluence and I put them into one flask without changing the medium or centrifuging, then would that be a passage?

Hope to hear from somebody soon.

CMIRC

[bob1]

CMIRC, on 30 July 2012 - 06:22 AM, said:

If I thaw a vial of PC3 cells (prostate cancer immortal cell line) and culture them. The passage number would be 0 when I thaw them first. However the passage number would increase to 1 when I sub culture them. Say after subsequent cell culturing I decide to freeze the cells at passage 4. The next time I take out a the PC3 vial from liquid nitrogen and culture would the passage start from 4 or from 0?

The passage number always increases for all cell lines - restart your culture from 4.

CMIRC, on 30 July 2012 - 06:22 AM, said:

I know that for primary cell lines the passage number does not reset and continous even after freezing and thawing. however does the same apply for immortal cancer cells. And if it does then upto what passage can one do experiments without having any genetic alterations due to the passaging. ie upto what passage can one use an immortal cell line before a buying a new one?

Immortal cell lines still increase the passage number, however, they tend to be a bit better off after freezing than a primary line, so you can keep them up for about 5-10 passages before you should thaw a fresh tube.  In theory (and practise) you should be able to freeze several fresh tubes down at the passage after you thaw the tube, which should mean that you have a more or less continuous supply of cells that are good for your experiments.  I would suggest you read R.I. Freshney's "Culture of animal cells: a laboratory manual" for a scheme of how to do this.

CMIRC, on 30 July 2012 - 06:22 AM, said:

Also for non adherent cell lines when would a passage be considered. If I take flask of 50% confluent THP1 cells and centrifuge them and put them back in the same flask with new media without splitting would that be a passage, secondly if I have 2 flasks of THP1 cells 25% confluence and I put them into one flask without changing the medium or centrifuging, then would that be a passage?

Technically, the first scenario is called "feeding" the cells, analagous to putting fresh medium on an adherent line without lifting the cells.  I don't think the other would be considered a passage, as technically you havn't reduced the number to make more growing space - quite the opposite, so you couldn't call it a passage.

[CMIRC]

Dear Bob1

I would like to say thank you for your brilliant informative reply. I will keep your words of wisdom in mind when culturing.

Just one more question,

When you buy a cell line from a company and when you thaw to culture it for the first time at what passage do you freeze them for future use and how many vials do you make of them.

[bob1]

I freeze them down at the next passage if possible (p+1).  From a t-75 flask I would be able to freeze 4-8 tubes with enough cells to easily seed out t-25 flasks when thawed.