July 1, 2017 (Vol. 37, No. 13)

3D Culture Models Mimic the Natural In Vivo Environment

Cell culture methods are showing great promise in advancing biomedical research, particularly in the fields of drug discovery, cancer biology, and regenerative medicine. These methods typically involve culturing two distinct types of cells in vitro: cell lines and primary cells. Cell lines are populations of cells that have been continually passaged over extended periods of time to evade normal cellular senescence and to acquire homogenous genotypic and phenotypic characteristics (examples include A549, HeLa, and HEK 293 cells). In contrast, primary cells are isolated directly from donor tissue and are non-transformed and non-immortalized.

Traditional two-dimensional (2D) cell culture methods are increasingly being enhanced by new three-dimensional (3D) technologies that mimic the natural cellular in vivo environment, which is one of the main reasons it is taking off as an experimental approach 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) characteristics 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 advantage 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 original 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 research, and precision medicine.

Cell Lines Are Losing Credibility for Cell Culture

Cell lines have traditionally been used for cell culture mostly for convenience, because they can be easily handled, are well established, and are relatively inexpensive. In particular, they have been frequently used for high-throughput anticancer drug screening 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 characteristics 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 adenovirus transformation so they more closely resembled immature neurons than embryonic kidney cells.6

Finally, many cell lines are often misidentified and can also be contaminated with other cells. For example, Drexler et al. (2003) found that in over 500 reported human leukemia-lymphoma cell lines, 15% were misidentified,7 and Hughes et al. (2007) found that 18–36% of cell lines may be contaminated or misidentified.8 Cross-contamination of cell lines has persisted as a result of mishandling 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 letter prepared by leading cell culture scientists and addressed to Michael O. Leavitt, Secretary 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 example, 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 authentication 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 funding12 and provided guidelines for reporting and endorsement 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 limited lifespan, so they maintain identical (or at least, very similar) characteristics to the original donor tissue. Thus, cultures that use primary cells, such as fibroblasts and epithelial cells, can produce more biologically representative models of cells and tissues than cell lines. For this reason, primary cells have applications 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 sequence 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 using primary cells reduces the costs associated with downstream animal testing and human clinical trials.14 Indeed, cytotoxic responses to EC50 doses of the anticancer drug camptothecin 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

Figure 1. Primary cells have several advantages over cell lines in cell culture for biomedical research and drug discovery.

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). Primary 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 technologies are showing better success in engineering physiologically relevant cell and tissue models.17 For example, biomimetic 3D prostate organoids can be generated by culturing human prostate luminal and basal cells and circulating tumor cells in a protein gel matrix to enable the study prostate cancer and facilitate anticancer drug screening.18 Consequently, primary cells are increasingly becoming the focal point of 3D cell culture in cancer biology.19

In cancer research, many studies on 3D cultures have used established cancer cell lines. However, more recently, using patient-derived primary tumor cells instead of cell lines has generated advanced, more biologically representative 3D models of cancer to aid drug discovery and research.3 For example, one study based on a 3D model of primary human adult lung cancer-associated fibroblasts (LuCAFs) and human bronchial epithelial cells (HBECs) found that LuCAFs alter HBECs by modifying biochemical signals conveyed through the extracellular matrix.20 A preliminary study by Lonza has also demonstrated that human mammary fibroblasts and human mammary epithelial cells could be successfully co-cultured (using the RAFT™ System) to produce a reliable multicellular model of breast cancer that could be used to conduct anticancer drug screening studies.

Although using primary cells in 3D cell culture technology has not yet been optimized, it is recognized that its major advantage is the ability to use the same tumor model in vitro and in vivo for cancer research and drug screening. For example, Kondo et al. developed a new method for culturing primary colorectal cancer cells. Using this method, the investigators maintained cell-cell contact throughout the culture process, and they showed that these cultured cells could be used for the evaluation of chemosensitivity and signal pathway activation in cancer cells from individual patients.21 A 3D-tumor model system using primary patient-derived cells could, therefore, promulgate discoveries in cancer research and in early-stage drug discovery for personalized drug programs. 

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.

Primary Cell Culture: Tips and Tricks

There are clearly many compelling reasons why primary cells should be used instead of cell lines for research, but their adoption has been gradual because of the widespread belief that they can be difficult to culture. Yet, primary cells are just as easy to culture as cell lines, and simply require adherence to the specific protocols and commercially available growth media kits that are often provided by suppliers. Here are Lonza’s top tips and tricks to ensure the successful culture of primary cells.

  1. Before cells are cultured, they should be prepared correctly. The number of cells that will be needed (and thus the number of flasks) should be calculated beforehand, and the cells should be kept in liquid nitrogen for as long as possible before they are thawed
  2. Cells should be thawed quickly (i.e., in no more than 2 minutes) because they can be harmed if thawing takes longer. Similarly, once the cells are in the medium, they can be temperature sensitive, so repetitive warming and cooling should be avoided.
  3. After culture seeding and cell growth are established, cell proliferation should be stopped once the cells reach 70–80% confluency. Be careful not to reach 100% confluency, as this will make the cells enter senescence.
  4. To dissociate the cells from the culture, cells should be washed with trypsin at room temperature. Because this process can be harsh, the cells should be monitored carefully through a microscope (Versene-assisted detachment is a milder alternative to trypsinization, if required). Afterwards, a trypsin neutralizing solution (again at room temperature) should be used to fully inactivate the trypsin.
  5. If contamination is suspected for any reason, the culture should be checked regularly.


New three-dimensional cell culture systems are driving forward research in several key biomedical fields, including cancer biology and drug discovery. The cell types available for use in cell culture are either cell lines or primary cells. Increasingly, however, biologically relevant primary cells are becoming the preferred choice because of the various advantages they have over cell lines.

Specifically, although cell lines are the familiar choice, they require authentication prior to use, are likely to produce unreliable and inconsistent results, and could potentially cause increased costs from failed animal tests and clinical trials in drug development. In contrast, primary cells are showing great promise for biomedical research. For example, they are producing biologically representative 3D models of cancer to aid anticancer drug discovery. As such, using primary cells together with 3D cell culture systems could lead the way in advancing research and drug discovery, potentially helping to combat cancer and other life-threatening diseases.

Lubna Hussain is senior global product manager at Lonza.



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12. NIH. Enhancing Reproducibility through Rigor and Transparency (released June 9, 2015; effective Jan. 25, 2016). Notice Number: NOT-OD-15-103. Available at: https://grants.nih.gov/grants/guide/notice-files/NOT-OD-15-103.html (accessed: May 15, 2017).

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