Genetic Engineering & Biotechnology News

JUL 2017

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8 | JULY 2017 | GENengnews.com | Genetic Engineering & Biotechnology News Lubna Hussain Cell culture methods are showing great prom- ise in advancing biomedical research, particu- larly in the fields of drug discovery, cancer biology, and regenerative medicine. These methods typically involve culturing two dis- tinct types of cells in vitro: cell lines and pri- mary cells. Cell lines are populations of cells that have been continually passaged over ex- tended periods of time to evade normal cel- lular senescence and to acquire homogenous genotypic and phenotypic characteristics (ex- amples include A549, HeLa, and HEK 293 cells). In contrast, primary cells are isolated directly from donor tissue and are non-trans- formed and non-immortalized. Traditional two-dimensional (2D) cell culture methods are increasingly being en- hanced by new three-dimensional (3D) tech- nologies that mimic the natural cellular in vivo environment, which is one of the main reasons it is taking off as an experimental ap- proach in biomedical research. 1 Primary cells are also considered to be more biologically representative than cell lines due to their typically identical (or at least similar) charac- teristics to the original donor tissue. 2 Therefore, it is becoming increasingly clear that using primary cells in 3D cell culture can produce more biologically representative models of in vivo multicellular environments compared with using cell lines. 3 Another ad- vantage is that primary cells are more suitable than cell lines for research into personalized therapeutics. Because they remain in a state practically unchanged from that of the origi- nal donor tissue, primary cells possess in vitro characteristics that inform targeted therapies more reliably than do cell lines. 4 Primary cells are, therefore, key to the advancement of 3D cell culture in drug discovery, biomedical re- search, and precision medicine. Cell Lines Are Losing Credibility for Cell Culture Cell lines have traditionally been used for cell culture mostly for convenience, be- cause they can be easily handled, are well established, and are relatively inexpensive. In particular, they have been frequently used for high-throughput anticancer drug screen- ing because of their wide availability and the ease with which they can be propagated. 5 However, various concerns are starting to be raised about using cell lines for cell culture. First, several biological pathways cannot be represented by cell lines, and we do not have cell lines available to model every type of cancer, which limits the application of cell lines for cancer research. 2 Second, cell lines often mutate so that their genotypic and phenotypic characteris- tics no longer represent those of the original donor cells. For example, Shaw et al. found that in the cell line HEK 293, the properties of the cells had been changed by adenovi- rus transformation so they more closely re- sembled immature neurons than embryonic kidney cells. 6 Finally, many cell lines are often misiden- tified and can also be contaminated with other cells. For example, Drexler et al. (2003) found that in over 500 reported human leu- kemia-lymphoma cell lines, 15% were mis- identified, 7 and Hughes et al. (2007) found that 18–36% of cell lines may be contaminat- ed or misidentified. 8 Cross-contamination of cell lines has persisted as a result of mishan- dling and a lack of attention to best practices in tissue culture. Since cell lines can be contaminated and often have different genotypic and phenotypic characteristics compared to the original donor cells, their use in biomedical research is likely to produce unreliable and inconsistent results that are irreproducible or induce additional studies of questionable value. 9,10 An open let- ter prepared by leading cell culture scientists and addressed to Michael O. Leavitt, Secre- tary of the U.S. Department of Health and Human Services, suggested that as many as 20% of scientific publications using cultured cells might be "blemished" as a result of cross- contamination. 11 In turn, this could cause significant downstream problems. For exam- ple, drug screening using cell lines could give false-positive results, leading to increased costs through needless animal testing and clinical trials, and potentially risking patients' lives. These concerns have resulted in funding agencies and publishers requiring authentica- tion of cell lines for their use in research and in grant applications. For example, in 2015 the U.S. National Institutes of Health revised its guidelines to applications for funding 12 and provided guidelines for reporting and en- dorsement by major journals. 13 For example, since 2013, the Nature Publishing Group has required authors to report the authentication status of all cell lines used. The Advantages of Using Primary Cells for 3D Cell Culture Unlike cell lines, primary cells have a lim- ited lifespan, so they maintain identical (or at least, very similar) characteristics to the origi- nal donor tissue. Thus, cultures that use pri- mary cells, such as fibroblasts and epithelial cells, can produce more biologically repre- sentative models of cells and tissues than cell lines. For this reason, primary cells have ap- plications in cancer biology and for screening anticancer drug candidates (Figure 1). Indeed, many cancer research initiatives, such as the Cancer Genome Atlas, prefer to use primary cells rather than cell lines to se- quence cancer genomes because they are more biologically relevant. 2 Cultured primary cells that mimic the target tissue are also essential in drug screening, because they can more reliably identify potential drug candidates, and us- ing primary cells reduces the costs associated with downstream animal testing and human clinical trials. 14 Indeed, cytotoxic responses to EC 50 doses of the anticancer drug camptoth- ecin have been found to be much different in primary cells compared to cell lines (Figure 2), and primary cell cultures often better mimic the in-vivo tumor response to drugs. 15 Culturing some types of primary cells in traditional two-dimensional systems can be difficult, however, particularly if the media composition is not optimal (primary cells, unlike cell lines, typically require additional growth factors in their culture medium). Pri- mary hepatocytes cultured as a monolayer on plastic become undifferentiated and die within just four days. In contrast, they survive for three weeks and maintain differentiation longer when entrapped in a three-dimensional collagen gel matrix. 16 Moreover, primary cells cultured using certain 3D cell culture technol- ogies are showing better success in engineering physiologically relevant cell and tissue mod- els. 17 For example, biomimetic 3D prostate organoids can be generated by culturing hu- man prostate luminal and basal cells and cir- culating tumor cells in a protein gel matrix to enable the study prostate cancer and facilitate 3D Culture Models Mimic the Natural In Vivo Environment Advancing 3D Cell Culture for Biomedical Research Using Primary Cells Drug Discovery Tutorial Figure 2. Primary cells and cell lines show variability in responses to anticancer drug camptothecin, so data acquired through cell lines cannot easily be replicated in an in vivo model. Figure 1. Primary cells have several advantages over cell lines in cell culture for biomedical research and drug discovery.

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