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amaxa eNews #5
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Authenticated, Low-passage Cell Lines Optimize Cancer Research
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Established animal cell lines occupy a critical place in the cancer research toolbox, and ATCC, as the world’s largest biological resource center (BRC), is proud of the role it plays in supplying these important tools. The ability of cell lines to help investigators advance cancer research, however, comes with some forewarning. A growing body of literature demonstrates that when most cancer cell lines have been subcultured too often they can give rise to questionable experimental results. ATCC actively promotes a heightened awareness of the importance of using authenticated, low-passage cancer cell lines to improve experimental reliability and validity.
The ability of continuous cell lines to propagate almost indefinitely makes these cultures susceptible to genetic drift. Cancer cell lines are even more susceptible to changes with long-term subculturing. The literature is peppered with studies demonstrating that increased passage numbers can alter the properties of cancer cell lines over time. Compared to lower-passage cells, cell lines at higher passage numbers experience changes in cell morphology, response to stimuli, growth rates, gene expression and transfection capacities [1-7].
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Effects of Passage Number
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| Characteristic | High passage vs. low passage
| | | | | Morphology | Less heterogeneity | | | | | Transepithelial electrical resistance | Higher | | | | | Transcellular diffusion | Higher | | | | | Paracellular diffusion | Lower | | | | | Carrier mediated | Lower | | | | | Alkaline phosphatase | Lower |
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Figure 1: Effect of passaging on the activity of alkaline phosphatase in a Caco-2* cell line. Data represents mean ± SEM from three measurements from the same batch of culture. Asterisks indicate significant difference between populations (p < 0.05, t-test). (Data reproduced from Yu, H et al. Evidence for diminished functional expression of intestinal transporters in Caco-2 Cell monolayers at high passages. Pharmaceutical Research (1997) 14; 6:757-762.)
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The basic problem of cell line contamination can be divided into two categories: contamination of a cell line by microorganisms — primarily mycoplasma — and cellular-cross-contamination or misidentification of cells. The later problem was first described in the 1960s, when early pioneers Stanley Gartler and Walter Nelson-Rees used isoenzymology to determine that many of the earliest established human cell lines were identical to the HeLa cell line, rather than the cell types they were purported to be. From the late 1990s to the early 2000s, several studies have documented the persistence of cell line misidentification or cellular-cross-contamination. [8-13]
An example of a contemporary case of cell line misidentification in cancer biology can be found among the so-called “NCI-60” panel of tumor cell lines maintained by the National Cancer Institute Developmental Therapeutics Program for distribution to investigators for cancer drug screening. The panel includes leukemia and melanoma as well as other cancer cell lines derived from the lung, colon, brain, ovary, breast, prostate and kidney. DNA fingerprinting studies in 2000 on the breast cancer cell line MCF-7 revealed that its drug-resistant variant was unrelated. [14-15]
To help ensure reliable, reproducible experimental results and avoid passage-dependent effects and contamination, a good first step is to obtain cultures from established BRCs whenever possible. Unlike most cultures obtained from individual laboratories, ATCC cultures have undergone comprehensive quality control procedures that confirm viability and identity, as well as ensure that the cultures are free from contaminants. Furthermore, ATCC uses a systematic seed-stock method to produce virtually identical distribution lots, which ensures consistent materials at the lowest passage possible. [16-17]
If cell lines of interest cannot be obtained from a BRC, investigators should take necessary steps to avoid passage-dependent effects on their data. This includes preparing a token stock to be used as source material for all subsequent experiments. Such a step ensures that only low-passage cell relatives of the source material are used, resulting in consistent and reproducible results. Additional steps include confirmation of morphology, confirmation of species via STR (short tandem repeats) and COI (cytochrome oxidase I) analyses and verification that the cell cultures are free of microbial contaminants (bacteria, fungi and mycoplasma). For more information on how to avoid passage-related effects and contamination, view the technical literature at www.atcc.org (click on Technical Support).
Investing modest effort to find passage levels that are “safe” for a given cell type in a given application will pay dividends in the form of higher reliability in the research results obtained. By taking basic precautions with regard to cell line passage number and contamination, investigators can protect the integrity of their experimental data.
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References
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[1] Hughes P et al. The costs of using unauthenticated, over-passaged cell lines: how much more data do we need? Biotechniques. 43 (5): 575-586. 2007.
[2] Esquenet M et al. LNCaP prostatic adenocarcinoma cells derived from high and low passage numbers display divergent responses not only to androgens but also to retinoids. Journal of Steroid Biochemistry and Molecular Biology. 62: 391-399. 1997.
[3] Briske-Anderson MJ et al. Influence of culture time and passage number on morphological and physiological development of Caco-2 cells. Proceedings of the Society for Experimental Biology and Medicine. 214(3): 248-257. 1997.
[4] Chang-Liu CM et al. Effect of passage number on cellular response to DNA-damaging agents: cell survival and gene expression. Cancer Letters. 26(113):77-86. 1997.
[5] Yu H et al. Evidence for diminished functional expression of intestinal transporters in Caco-2 cell monolayers at high passages. Pharmaceutical Research. 14(6): 757-762. 1997.
[6] Sambuy Y et al. The Caco-2 cell line as a model of the intestinal barrier; influence of cell and culture-related factors on Caco-2 cell functional characteristics. Cell Biology and Toxicology. 21: 1-26. 2005.
[7] Wenger SL et al. Comparison of established cell lines at different passages by karyotype and comparative genomic hybridization. Bioscience Reports. 24(6): 631-639. 2004.
[8] Buehring GC et al. Cell line cross-contamination: how aware are mammalian cell culturists of the problem and how to monitor it? In Vitro Cellular and Developmental Biology: Animal. 40(7): 211-215. 2004.
[9] Drexler HG et al. False leukemia-lymphoma cell lines: An update on over 500 cell lines. Leukemia. 17(2): 416-426. 2003.
[10] Macleod RA et al. Widespread intraspecies cross-contamination of human tumor cell lines arising at source. International Journal of Cancer. 83(4): 555-563. 1999.
[11] Markovic O et al. Cell line cross-contamination in cell cultures: the silent and neglected danger. In Vitro Cellular and Developmental Biology: Animal. 34(1): 1-8. 1998.
[12] No authors listed. Contamination of cell lines—a conspiracy of silence. Lancet Oncology. 2(7): 393. 2001.
[13] Thompson EW et al. LCC15 cells are MDA-MB-435: A review of misidentified breast and prostate cell lines. Clinical & Experimental Metastasis. 21: 535-541. 2004.
[14] Pirnia F et al. Uncertain Identity of Doxorubicin-resistant MCF-7 cell lines expressing mutated p53. Journal of the National Cancer Institute. 92(18): 1535-1536. 2000.
[15] Scudiero DA et al. Cell line designation change: multidrug-resistant cell line in the NCI anticancer screen [letter]. Journal of the National Cancer Institute. 90:862. 1998.
[16] ATCC. Cell biology catalog 2007. Available from ATCC.
[17] ATCC. Maintaining high standards in cell culture. Brochure 2006. Available from ATCC.
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*Caco-2 was developed by the Memorial Slan Kettering Cancer Center and is accordingly subject to certain third party restrictions. Please contact Memorial Sloan Kettering Cancer Center or Navicyte Scientific for licensing information.
The Nucleofector Technology, comprising Nucleofection Process, Nucleofector Device, Nucleofector Solutions, Nucleofector 96-well Shuttle System and Nucleocuvette plates and modules is covered by patent and/or patent pending rights owned by amaxa AG.
amaxa, Nucleofector, nucleofection, maxGFP, 96-well Shuttle and Nucleocuvette are either registered trademarks or trademarks of amaxa AG in the U.S. and/or Germany and/or other countries. ATCC‚ and the ATCC Catalog Marks are trademarks of ATCC used under License. Other product and company names mentioned herein are the trademarks of their respective owners.
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