Using an American Type Culture Collection (ATCC) reference strain for every experiment would be great, but not all that practical. So, most labs subculture their cells into a new vessel. This subculture is also known as a “passage.” A passage number is the number of times a cell culture has been subcultured, and knowing the passage number can make or break an experiment.
All cell cultures start somewhere; this “somewhere” is the reference strain, or reference culture. These are fresh cells that come from a reliable source, like the ATCC. While many labs may passage cells dozens, even hundreds of times, this many passages probably results in cells that have little in common with the original reference strain. These “working cultures,” if passaged enough times, can show evidence of genetic drift—changes in genotype from the original reference strain which may or may not result in observable changes in phenotype. Other genotype changes may not show any phenotypic variation immediately, but could result in changes after further subculturing. In addition, genetic changes caused by subculturing could create epigenetic changes that could affect how genes are regulated. More passages also increase the risk of contamination. Not good.
One study showed that high- and low-passage adenocarcinoma cells had different responses to androgens and retinoids, indicating alterations in gene expression. Researchers in Belgium compared how two strains of LNCaP prostate cancer cells responded to androgens and retinoids, depending on passage numbers. The cells with high passage numbers showed higher-amplitude response curves to 3H-thymidine (measuring cell proliferation), while cells with low passage numbers showed greater growth inhibition by the synthetic androgen R1881, greater PSA mRNA expression and PAP expression (prostatic acid phosphatase). For responses to retinoic acid (atRA), lower-passage cells showed a marked stimulation of 3H-thymidine incorporation in the cells, while lower passage cells only showed growth inhibition. Clearly, passage number affected cellular physiology, which in this case caused the researchers to caution about the use of prostate drugs containing these molecules!
Good cell practice calls for starting any experiment with low-passage cell culture, and limit the number of passages you’ll accept in your experiment. But what is a good passage number (besides “zero,” that is)? The numbers have differed over the years. Some standards recommend three stock subcultures and three “working culture” subcultures—those add up to seven passages, including the original passage from the reference. Meanwhile, some cell culture producers charge more for cultures of two passages or less. However, the ATCC warns researchers to assume that a cell culture from a commercial source may be already one or two passages away from the reference strain. Generally, the ATCC recommends that cell culture should be limited to five passages, at least for use in medical and biopharmaceutical applications.
You’ll always need to look for any changes in phenotype or other signs of gene expression changes as you subculture. But, we can’t always work with reference cultures. The ATCC recommends replicate subcultures—just make, say, five replicates of a single subculture. These replicates are just multiple cultures of the same reference strain stock, so cells in those cultures should be closer in characteristics to the reference strain. That gives you enough to work with later. In addition, thawing and rehydrating reference cultures is not considered a “passage” by the ATCC.
In a large, busy lab, it’s entirely possible to lose track of how many passages your cell culture has gone through. Keep a record, both on the vessel and in your computer or a notebook. The easier it is to check on passage number, the less likely you’ll be working with altered cells. And trust your gut; if your experiments start producing unusual and unexplained results, you may have a passage problem.
Esquenent M., et al. (1997). LNCaP prostatic adenocarcinoma cells derived from low and high passage numbers display divergent responses not only to androgens but also to retinoids. J. Steroid Biochem. Mol. Bio. 62(5-6): 391-399.
ATCC Reference Strains: How many passages are too many? Technical Bulletin no. 6. http://www.atcc.org/Portals/1/Pdf/tb06.pdf
ATCC Cell Line authentication test recommendations. Technical Bulletin no. 8. http://www.atcc.org/Portals/1/Pdf/tb08.pdf