could freezing/thawing make mammalian cells competent - could it change phenotype back? (Jul/08/2011 )
hello everyone, regarding the cryopreservation of cell lines, I got several questions.
usually when I got a new cell line or newly thawed one from a frozen vial, the first thing I do is to wait the cells proliferate and then freeze the stock. Normally it will be done in 1 week. Besides making stocks, I think that would also maintain the competency (I dont know if the word is correct) of the cells because they would provide you the same transfection efficiency when you thawed them again. So basically, I will thaw a new batch when the old cells are giving low transfection efficiency. But since every freezing/thawing cycle will make cells about 1 week older (it takes time for cells to recover and reach log-phase), eventually you will get a quite different cell line if you do a lot of freezing/thawing cycles. But in fact, that is not the case. They are very constant. that is where I am lost.
the reason I asked this question is because I have a cell line which has been cultured for a long time. recently I found the phenotype has changed as some cells became adherent, which should all be in suspension. unfortunately I dont have any stock, so I am wondering
thank you.
It depends on the cell line a bit, but in general the longer the cell line is in culture, the lower the transfection efficiency and the less able they are to proliferate. This has a bit to do with laboratory evolution, but also to do with just general damage from the the culture - remember in the body there are all sorts of processes to prevent cellular damage, that are absent in culture. As a general rule you should only use cells for about 10-15 passages before you get some more up. Some immortalized and transformed cells can escape this, such as HeLa and 293.
Freezing the cells does not usually restore this property. Many primary cells will not function at all after about 60 population doublings (generally about 10 passages) and other non-transformed cell lines that are immortalized will proliferate continuously, but will still loose characteristics with continuous culture.
bob1 on Sat Jul 9 06:31:54 2011 said:
It depends on the cell line a bit, but in general the longer the cell line is in culture, the lower the transfection efficiency and the less able they are to proliferate. This has a bit to do with laboratory evolution, but also to do with just general damage from the the culture - remember in the body there are all sorts of processes to prevent cellular damage, that are absent in culture. As a general rule you should only use cells for about 10-15 passages before you get some more up. Some immortalized and transformed cells can escape this, such as HeLa and 293.
Freezing the cells does not usually restore this property. Many primary cells will not function at all after about 60 population doublings (generally about 10 passages) and other non-transformed cell lines that are immortalized will proliferate continuously, but will still loose characteristics with continuous culture.
Thank you very much. Do you mean only some cell lines could maintain the characteristics even after long culture, for example 293 and HeLa as you have mentioned? I am using T cell lines now...
Correct. Have a look for information about Hayflick limits and continuous culture.
Thank you very much, bob1. But still one thing not very clear. I have cultured many cell lines and they are all immortal. Do they all have lengthened telomeres or other mechanisms exist?
IIRC there are more than one mechanisms for telomere lengthening, but essentially it seems that most immortal cells have some maintenance of telomere length, usually through telomerase activity.
bob1 on Sun Jul 24 04:54:21 2011 said:
IIRC there are more than one mechanisms for telomere lengthening, but essentially it seems that most immortal cells have some maintenance of telomere length, usually through telomerase activity.
thank you.