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cell culture question - passage number - (Oct/20/2008 )

I have a relatively basic question about cell culture. I am using HEK293T cells and was wondering what is the highest recommended passage that I can use these cells before they get too "old?" From my experience, the HEK cells are pretty stable and still look good, but I was wondering if there is a general guideline on how many passages is good and how many is too many. I am at a kind of high passage now (60), but like I said they still look good. Is this too high? Can I continue to split these for some time or should I thaw out fresh cells? Thanks.

-elenacg-

Are you saying that you have had these cells up for 60 passages, or that they have been in the lab and frozen, then thawed etc. so that the total passage is 60?

If you have had your cells up for 60 passages continuously, I suspect they will have significantly diverged from the parent line unless you have been exceptional with your cell culture. Minor changes in such things as density after passage, confluency before passage and media replacement frequency will all act on some of the cells to create a selective pressure that slowly changes the cell population over time.

The general rule is not more than 5-10 passages, depending on passage density, before getting more out from a frozen stock. You should also freeze down more cells each time you get up a cell line, so as to not deplete the low passage stocks too much.

Having said that, 293 is a very common cell line and does not show too much drift over a wide range of passages, and I think the parent line at the ATCC is somewhere up in the over 300 passage range so you may not have too much trouble.

-bob1-

QUOTE (bob1 @ Oct 20 2008, 06:40 PM)
Are you saying that you have had these cells up for 60 passages, or that they have been in the lab and frozen, then thawed etc. so that the total passage is 60?

If you have had your cells up for 60 passages continuously, I suspect they will have significantly diverged from the parent line unless you have been exceptional with your cell culture. Minor changes in such things as density after passage, confluency before passage and media replacement frequency will all act on some of the cells to create a selective pressure that slowly changes the cell population over time.

The general rule is not more than 5-10 passages, depending on passage density, before getting more out from a frozen stock. You should also freeze down more cells each time you get up a cell line, so as to not deplete the low passage stocks too much.

Having said that, 293 is a very common cell line and does not show too much drift over a wide range of passages, and I think the parent line at the ATCC is somewhere up in the over 300 passage range so you may not have too much trouble.



Thanks for your reply. What I meant by the 60 is that they have been frozen, thawed and now I am at a total passage of 60. The frozen cells are actually at passage 18, which based on what you said seems like it's kind of high, but that's when they were given to me (they were given to me by another lab). Would you say that's even too high for a frozen stock?
I guess I will go ahead and thaw out some fresh cells (and also freeze some down). Thanks again!

-elenacg-

QUOTE (elenacg @ Oct 21 2008, 08:37 AM)
QUOTE (bob1 @ Oct 20 2008, 06:40 PM)
Are you saying that you have had these cells up for 60 passages, or that they have been in the lab and frozen, then thawed etc. so that the total passage is 60?

If you have had your cells up for 60 passages continuously, I suspect they will have significantly diverged from the parent line unless you have been exceptional with your cell culture. Minor changes in such things as density after passage, confluency before passage and media replacement frequency will all act on some of the cells to create a selective pressure that slowly changes the cell population over time.

The general rule is not more than 5-10 passages, depending on passage density, before getting more out from a frozen stock. You should also freeze down more cells each time you get up a cell line, so as to not deplete the low passage stocks too much.

Having said that, 293 is a very common cell line and does not show too much drift over a wide range of passages, and I think the parent line at the ATCC is somewhere up in the over 300 passage range so you may not have too much trouble.





Thanks for your reply. What I meant by the 60 is that they have been frozen, thawed and now I am at a total passage of 60. The frozen cells are actually at passage 18, which based on what you said seems like it's kind of high, but that's when they were given to me (they were given to me by another lab). Would you say that's even too high for a frozen stock?
I guess I will go ahead and thaw out some fresh cells (and also freeze some down). Thanks again!



Dear Elenacg,

I am afraid that bob1 is wrong when he says that the " general rule is 5-10 passages". We would all have to continously go back to the ATCC/ECACC for cells if that was the case. The most important thing to be aware of is that all cell lines are different and will lose characteristics at different rates. I use J774.A1, a murine cell line to induce iNOS enzyme. The cells at P5 will produce similar amounts of iNOS compared to cells that have been passaged over 200 times. I am only using the cells to produce this enzyme. I am not looking at receptor expression, metabolism, RNA/DNA synthesis etc etc. If I was and say the passage number affected a receptor expression that I was interested in, then of course I would get out fresh cell stocks. It is very important to remember that a cell/cell line is only an INVITRO MODEL of what MAY happen in vivo.
I do agree with bob1 that you should freeze down as many cells as possible at the LOWEST passage.
I also agree with him about selection pressure....you need to keep the cellular environment CONSTANT, to reduce this risk.....i.e. split ratio, density, serum batch, CO2 levels, humidification, PLASTICWARE, type of trypsin etc etc.

Rhombus

-Rhombus-

QUOTE (elenacg)
Thanks for your reply. What I meant by the 60 is that they have been frozen, thawed and now I am at a total passage of 60. The frozen cells are actually at passage 18, which based on what you said seems like it's kind of high, but that's when they were given to me (they were given to me by another lab). Would you say that's even too high for a frozen stock?


Your cells should be fine if you have been freezing them down regularly. P60 for frozen stocks is no problem.

What I was trying to say is that if you have had cells up for 5-10 passages continuously, you should get up some more, making sure that you freeze down more as often as possible. This keeps the passage number of the stocks low and makes sure that your cells aren't diverging from the parent line while you are working on them.

I work on proteins that are involved in cell cycle regulation and DNA/RNA replication and we can see differences in cells that have been continuously passaged for some time. However, if you have tested this and found that it doesn't affect your experiments, then it isn't a problem.

We bought in some cells like MCF7 14 years ago (I think), and they are used in the lab about 75% of the time by one or more people and still have them at very low passage (I'm currently using P10 stocks), but we have stocks going back as far as P5 - I think they started them as P1 when they were bought in. Anyway, no need to go back to the ATCC at all. The theory is that your cells will eventually drift from the parent line no matter what you do, you just have to aim to minimise that, and freezing down stocks at as low a passage as possible is the only way to do this.

-bob1-

It depends on the context of what you want to look at. If you are using 293T's for lentivirus production then yeah it sure as hell matters. The titre drops after 10 passages, and gets worse the higher passage number.
If you are looking at signalling pathways - yeah it matters aswell. mutations are preserved because the cells are immortalised - you often find very different responses to high passage cells then low.
A549 cells for example spontaneously derive inhibition of adenovirus about passage 50.
There are lots of cell-specific differences in passage numbers.
Me personally, I would never use anything above passage 50 if I had the choice.

-Aaron I-