I am working with MCF7 and 293T cells. Do I have to know the # of passages that they have passed? If yes, what is the maximum passage's number, in general to my cells?
Yes it is important to know N of passages for primary cells and for many other cell types. The question was very specific, about 2 specific cell lines, not random primary fibroblast line. For example, if the question was about primary mouse tail fibroblast culture, I would say 5-7 passages, no more.
However, when you are talking about Hela, MCF7 or 293T, CHO, Cos-7 available for decades, do you believe these cells only had 30-50 passages?
293T are immortalized so it does not matter. They will live "forever"
http://en.wikipedia.org/wiki/HEK_293_cells
I do not work with MCF7, but can guess that they are transformed as well based on the number of data produced with this cell line since 1970s. If the cell line is transformed (or immortalized), it does not matter how many passages made:
http://en.wikipedia.org/wiki/MCF-7
Yes, it is prudent to know. Most cancer cell lines can be passaged around ~30 times before they start picking up more genomic abnormalities. Some can even lose contact inhibition.
Even some cell lines divides very slowly after a certain no of passages. So it is always better to know the no of passages.
Yes, it is essential to know the passage number for the cell lines when they are cultured and must be mentioned properly while freezing. It is better complete doing experiments using cells with passage number ~30-35. So it is important to freeze cells (with lower passage number) in multiple aliquots in liquid nitrogen.
It's also important simply because it's good practice. Good practice with "regular" cell-lines leads to good practice when one begins to work with manipulated lines. Once one introduces expression and selection vectors, and creates one's own variants (shRNA knockouts, for example), it becomes much, much more important to monitor the pass number of cells and pay attention to how this affects the activity/behaviour/phenotype you've put into them. Good practice leads to good work leads to consistent, reproducible results.
Good luck!
G
ps - and speaking of good practice, you don't use antibiotics, do you?? ;)
It is Good Cell Culture Practice to note cell passages. For example it seems that transfection efficiency is reduce in MEF after 50 passages. It is good to have frozen aliquots of cells at early pasages to use in experiments.
I hope this will be helpful.
Best,
E
it is always good to record and note the passage. Each cell line has max in terms of passage.
Yes it is important to know N of passages for primary cells and for many other cell types. The question was very specific, about 2 specific cell lines, not random primary fibroblast line. For example, if the question was about primary mouse tail fibroblast culture, I would say 5-7 passages, no more.
However, when you are talking about Hela, MCF7 or 293T, CHO, Cos-7 available for decades, do you believe these cells only had 30-50 passages?
In addition to the number of the passages, it is good to determine the PDL (population doubling level). To calculate the PDL you can use the formula:
PDL = [log (number of the cells you have) - log (number of the cells you cultured)]/0.301
Best,
Juan.
For primary cell culture passage number really matters for experimental reproducibiluities for the data and I usually prefer lower passages 3-5 with those cells because at higher passages the cells usually loose their identity and express abnormal proteins. eg epithelial to messenchymal transformation (EMT).
I agree with Valentyn, for trasformed cells like Hela, MCF7 or 293T, CHO, Cos-7 we don't really know the exact number of passages we are working with since they have originated decades ago. The point we receive a vial for culturing cells we can mark it as passage 0 or 1, but that may not be the actual true passage number for those cells.
Surprisingly the transformed cells available from different sources always do not retain similar physiological characteristics. I myself tried with a melanoma cell (Mel 290) obtained from a lab in Germany and another lab from Emory University and both cultures differed a lot as because the way the cells have been maintained and fed with media were marginally different.
The bottom line is for transformed cells passage number do not make any sense to me, what seems important is the standard culturing and maintenance protocol consistent with the protocols suggested by the provider or the other laboratories following GLP.
Of course it is good to notify the P number, however, for immortalized cell lines should not be a big problem. Having enough cells ans using them in early passage would be best option .
Dear Ahmed
Yes, it is essential to know the passage number for the primary cell lines and for many other cell lines. When they are cultured and must be labeled properly before freezing. The best working passage numbers would be as low as possible (anything from passage 2 – 9) as these are the most similar to the original line. However, always make sure you have some low passage number tubes in storage, so that you can take a new one out and use them for several passages before the phenotypes change. It is important to freeze cells (with lower passage number) in multiple aliquots in liquid nitrogen.
Well.. when one is speaking of well-established cell-lines (and there is a current thread on RAW cells somewhere..), again, it is Good Practice to GET YOUR OWN FROM ATCC, ECACC or wherever. While this is not perfect, it gives you the reassurance that the cells represent some sort of defined start point, aren't likely to be contaminated with mycoplasma and so on.
Good Luck
G
There is another reason, why you should not use cell lines, you don't know the exact history and origin.
Nelson-Rees, W.A., et al. (1981) Cross-contamination of cells in culture. Science 212, 446–452
Yes, it is essential to know the number of passage for the cell lines when they are cultured and must be properly labelled. if you are using more than 7 passage the cells start behave abnormally and result will not come out properly.
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