Literature to support use of passage numbers - (Apr/10/2007 )
Hi there everyone,
I've seen that a number of people have posted questions about the 'appropriate' number of times to passage cells during experimentation.
The general concensus is clear - it depends on which cells your using, but you probably don't want to use over 20 - 30 passages during your experimentation period.
Here's my question - where does this concensus arise from? Apart from written on here, plus a protocol I've found on Google written by some anonymous person, I can't find any literature to support this. I've found a few papers that describe various metabolic/morphologic differences that occur during yeh number of passages - but none of these are comparable, as they use different, often not immortal, cell lines. (I'm using HT29).
Surely there must be some supportive statements out there that say "don't passage cells more than --- times"?
You may supect the reason for this query - yep, I'm trying to reference this in my thesis! I don't think it's permissible to reference (somebody nice on discussion forum 2007) - albeit a very friendly thing to do!!!! 
TTFN,
I've seen that a number of people have posted questions about the 'appropriate' number of times to passage cells during experimentation.
The general concensus is clear - it depends on which cells your using, but you probably don't want to use over 20 - 30 passages during your experimentation period.
Here's my question - where does this concensus arise from? Apart from written on here, plus a protocol I've found on Google written by some anonymous person, I can't find any literature to support this. I've found a few papers that describe various metabolic/morphologic differences that occur during yeh number of passages - but none of these are comparable, as they use different, often not immortal, cell lines. (I'm using HT29).
Surely there must be some supportive statements out there that say "don't passage cells more than --- times"?
You may supect the reason for this query - yep, I'm trying to reference this in my thesis! I don't think it's permissible to reference (somebody nice on discussion forum 2007) - albeit a very friendly thing to do!!!!
TTFN,
One can carry cell lines till they beave fine, but once you notice difference in the cell line behavior, you get new cells out.
i usually passage my cells till around 20, but sometimes they r fine till passage 22 or even 18. So there is no definite number as the confluency the cells reach each time before splitting them can influence the behavior.
If you notice any deifference in the cells, that when you get new cells out.
I've seen that a number of people have posted questions about the 'appropriate' number of times to passage cells during experimentation.
The general concensus is clear - it depends on which cells your using, but you probably don't want to use over 20 - 30 passages during your experimentation period.
Here's my question - where does this concensus arise from? Apart from written on here, plus a protocol I've found on Google written by some anonymous person, I can't find any literature to support this. I've found a few papers that describe various metabolic/morphologic differences that occur during yeh number of passages - but none of these are comparable, as they use different, often not immortal, cell lines. (I'm using HT29).
Surely there must be some supportive statements out there that say "don't passage cells more than --- times"?
You may supect the reason for this query - yep, I'm trying to reference this in my thesis! I don't think it's permissible to reference (somebody nice on discussion forum 2007) - albeit a very friendly thing to do!!!!
TTFN,
One can carry cell lines till they beave fine, but once you notice difference in the cell line behavior, you get new cells out.
i usually passage my cells till around 20, but sometimes they r fine till passage 22 or even 18. So there is no definite number as the confluency the cells reach each time before splitting them can influence the behavior.
If you notice any deifference in the cells, that when you get new cells out.
Thanks for that. Still curious to know though, if this altered behaviour is a phenomenon that anyone has discussed in any published literature. I spoke to my supervisor about this today too - and she's of the same opinion - you keep going 'til your cells behave differently, but she's also not sure where I might find this specified in the lit. I did notice that a few of the cell-culture protocol attachments that other people have left (on other topics) make some reference to passaging; I'll spend a bit more time looking through those too.
Thanks again for your reply,
Sue
Altered behaviour can be many things. Some cells grow much faster and some cells grow slower. Some of them dont produce as much virus as before or some of them show more cell death upon transfection. And each cell line differs. I am not sure if some one would actually categorize them.
Look at literature and if you find some reference, please do let us know.
I've seen that a number of people have posted questions about the 'appropriate' number of times to passage cells during experimentation.
The general concensus is clear - it depends on which cells your using, but you probably don't want to use over 20 - 30 passages during your experimentation period.
Here's my question - where does this concensus arise from? Apart from written on here, plus a protocol I've found on Google written by some anonymous person, I can't find any literature to support this. I've found a few papers that describe various metabolic/morphologic differences that occur during yeh number of passages - but none of these are comparable, as they use different, often not immortal, cell lines. (I'm using HT29).
Surely there must be some supportive statements out there that say "don't passage cells more than --- times"?
You may supect the reason for this query - yep, I'm trying to reference this in my thesis! I don't think it's permissible to reference (somebody nice on discussion forum 2007) - albeit a very friendly thing to do!!!!
TTFN,
Dear Suebee,
Having contributed to many published papers over the past 25 years, here is my take on your questions.
Firstly, on the whole only positive results are published. This means that many experiments are done to optimise conditions such as passage number and not put into the paper.
Secondly, in my opinion these are left out because these are the "tricks of the trade". It is very difficult sometimes to reproduce results from the literature, mainly because the optimisation steps are not published.
Thirdly, researchers look for conditions that will ultimately "fit" with a hypothesis they hold and the type of experiments they want to do.
I can give you some examples of what I mean by some optimisations that we did in the 1980's for our experiments concerning Nitric Oxide/EDRF release from cells (Moncada et al Nature 1987).
Firstly we looked at primary Bovine and Porcine Endothelial cells. NO/EDRF release was different in both types of cells i.e. Bovine cells when stimulated with Bradykinin (BK) would release very small amounts but over a 5-8 minute time frame. Porcine cells when stimulated with BK would release a relatively massive amount of NO/EDRF but over 5-8 seconds. We were wanting to do Bioassay using strips of Rabbit aorta, and IT FITTED OUR PURPOSE that porcine cells were a more convenient experimental tool. This was never publsihed but was an extremely important detail.
Secondly and important to your question about passage number. NO/EDRF in porcine cells was only present in the first passage. This made life difficult as we had to obtain fresh tissue daily to obtain enough cells to do our research.
Thirdly, we looked at passage number and Prostanoid release from these cells. In vivo and in the first passage, the predominant prostaglandin released is Prostacyclin. This is an important vasodilator and anti aggregator in the body. However over 7-10 days in culture this completely dissappears, and Prostaglandin E2 becomes the predominant prostanoid. Again if you were interested in PGI2, you would not choose these primary porcine cells.
You must always be careful in what cells to choose, how many populations doublings to allow, and make sure your specific marker is always present. Cell Culture is just a tool and cells will always change in culture, so you have to be very careful about conclusions to draw.
Kindest regards
Rhombus
Hi,
Maybe this could help you a little: www.atcc.org/common/documents/pdf/tb07.pdf (or search in google for atcc number of passage). I believe that they do have some references there.
I remember that I had read about the recommended number of passage for cell culturing, also from atcc. In that they describe how to define a 'passage' (essentially each time cells are place into a viable environtment and allow to grow is considered one passage, so that when you thaw cells into TC flask or disc, it is 1 passage, but when you freeze down cells, it is not counted as 1). Then they start about how many passage is recommended. In that, they recommend much less passage, I think only 10-20, nearer to 10. I am trying to look for that but cannot find it, so don't think it will be much help for you.
Anyway, practically, it depends on what you are doing, what cells you are using, the purpose of your experiments...
Maybe this could help you a little: www.atcc.org/common/documents/pdf/tb07.pdf (or search in google for atcc number of passage). I believe that they do have some references there.
I remember that I had read about the recommended number of passage for cell culturing, also from atcc. In that they describe how to define a 'passage' (essentially each time cells are place into a viable environtment and allow to grow is considered one passage, so that when you thaw cells into TC flask or disc, it is 1 passage, but when you freeze down cells, it is not counted as 1). Then they start about how many passage is recommended. In that, they recommend much less passage, I think only 10-20, nearer to 10. I am trying to look for that but cannot find it, so don't think it will be much help for you.
Anyway, practically, it depends on what you are doing, what cells you are using, the purpose of your experiments...
The problem with counting passage number is that you can never be sure of it especially if you obtain cells from different labs. Researchers are notouriously bad at writing down passage numbers everytime they subculture.