The Fine Cell Line

Are cells grown in the lab capable of replicating to the extent that they do inside the human body? One would think that if they make up our tissues for a lifetime, it should be easy to maintain their growth for equally as long in a dish. And yet, this is not the case, making it difficult for scientists to have constant access to a large enough number of viable cells for their experiments. Luckily, cell lines with an extended or “immortal” lifetime exist to avoid having to extract them repeatedly from fresh tissues. While this has undoubtedly propelled forward the use of cell culture within biomedical science, it also comes with drawbacks that are often blissfully or willfully ignored. These drawbacks may have been tolerated so far – but as cellular assays get ever closer to impacting the patient, is it still acceptable to “turn a blind eye”?

Hayflick’s limit

In 1921, Alexis Carrel, one of the founders of cell culture, suggested cells in culture can be immortal if cultured under the right conditions. This was later supported by some of his work which showed that fibroblasts had been successfully kept in culture for 34 years. Yet, no one was ever able to reproduce such findings, and much later it was proposed that their long-term culture was most likely the result of the daily addition of new cells present in the chick embryo extract used to feed the cells.¹

In the 1960's, Leonard Hayflick proved once and for all that not all cells are able to replicate indefinitely in vitro. This was so hard to believe back then that their paper was first rejected [1]. Thereafter, Hayflick published a series of seminal articles, including his 1965 study written with Moorhead where they showed that fetal fibroblasts degenerated after roughly 50 doublings in one year of culture,² a threshold later called the “Hayflick limit”. Their findings suggested that cells were capable of “remembering” how many times they had already doubled, pointing to the existence of an internal cellular clock.³ It took 33 additional years for scientists to completely solve the mystery. Long story short, with each round of cell division, the “caps” at the ends of the DNA strand, called telomeres, are shortened. This process enables cells to keep track of the number of replications they have gone through, such that when their telomeres become too short it indicates that they must stop replicating. But it took decades for scientists to unravel these mechanisms. In the meantime, they could observe that cancer cells replicated for much longer, and thus circumvented this death. The question was therefore, how do cancer cells seem to by-pass this mechanism, and can we transfer this “superpower” to other cells to extend their lifetime?

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Cell lines and cancer

While the use of cell lines has advanced our knowledge of many diseases, their development is particularly intertwined with that of cancer research.⁴ This is because cancer cells are essentially immortalized cells that can in theory grow indefinitely, which translates into an abnormal and aggressive cell proliferation. This facilitates the survival, replication and study of cells in non-physiological laboratory settings, which can be harder for non-cancerous cells. As such, the first cancer cell line established in vitro was isolated from Henrietta Lacks in 1951.⁵ Of 30 biopsies received from cervical cancer patients, the cells of Henrietta Lacks were the only ones able to grow well using the roller-tube technique.

Cancer cells were not only the first “natural” cell lines to be used in the lab, they also played a big part in enabling the development of new cell lines.

Before HeLa cells, Earle et al. created the first cell line in the lab by transforming fibroblasts with a carcinogenic compound to extend their lifespan.^6,7,8 But instead of “inducing” immortality, could there be a way to genetically modify cells to precisely transfer the superpower of cancer cells to other cells? To answer that question, we needed to understand how cancer cells found a way to prevent telomeres from shortening.¹ In 1985, Carol Greider and Elizabeth Blackburn discovered the enzyme that synthesizes and elongates telomeres, called telomerase.⁹ This enzyme was later found in most human tumors,⁹ and upon its ectopic expression in normal cells, their lifespan was increased.¹⁰ It turns out telomere length is maintained by telomerase which is downregulated in most cells, except germ cells and other post-mitotic cells.¹ For example, in the case of stem cells, telomerase shortening has been associated with loss of self-renewal properties.¹¹ In addition to unraveling the molecular mechanisms underlying immortality, cancer cells were also used to create the first hybridoma cell line that enabled the production of monoclonal antibodies at large scale, which is now key to biomanufacturing. In 1975, Kohler and Milstein fused a B cell (responsible for producing antibodies in the body) with a myeloma cell (a cancerous lymphocyte). This provided the B cell with the ability to divide rapidly and produce large quantities of the same monoclonal antibody.¹²

Thus, cancer research and cell line development are intimately tied. It is safe to say that cancer and all of in vitro research have benefited from the use of cell lines. And yet, close to 70 years after the first cancer cell line was established, could it be time to grasp its shortcomings too?

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Immortal cell lines: endless source of cells, proteins… and errors?

Today, a myriad of established cancer cell lines exist and are commonly used by labs around the world. They have been powerful in many regards, including standardization of results, as explained by Roger D. Kamm, PhD, professor at Massachusetts Institute of Technology (MIT): “It is essential that different labs be able to compare their results, and standardization and common use of cell lines helps enable that”. And yet, he adds that “cancer is so diverse, it’s a mistake to think that one cell line can represent the behavior of all patients.” Cancer cell lines are either the result of a selection for rapid growth in culture or of the exogenous expression of viral or mammalian oncogenes.¹³ Often, they represent transformed and aggressive cancer cells from advanced stage cancers, warranting their use only for studying such late-stage cancer development. One of their main limitations is that they do not retain the genetic and phenotypic tumor heterogeneity present in patients, and as Ran Li, PhD, postdoctoral fellow at Massachusetts General Hospital points out: “Genetic heterogeneity is important for assessing the efficacy of drug treatments.” At any rate, it is now understood that cell lines alone cannot be expected to represent the patient’s response: we know that the microenvironment plays an important role in drug response and should be reproduced in 3D in vitro to recapitulate the drug response along with cancer cell lines.

Furthermore, cell lines do accumulate mutations during culture over time,¹⁴ and too often this genetic drift is not taken into account by scientists and can induce uncontrolled variability. More generally, the failure of cells lines to represent the drug response of patients in vitro in and outside of cancer research might come from the insufficient number of cell lines tested per study, or from having chosen the wrong cell line. For example, Domcke et al. demonstrate that the most commonly used ovarian cell lines in vitro are probably not good models of high-grade serous ovarian carcinoma, the most common human form of ovarian cancer.¹⁵ 

Li also points to two major practical issues that can often come from the handling of cell lines, rather than from inherent limitations: “Contamination (micro plasma) and miss-identifying/cross-contamination is still a big issue”. Horbach et al. report that more than 30,000 studies might have misidentified cell lines¹⁶ due to contamination with HeLa cells. This warrants the use of regular validation and screening procedures (for example using short tandem repeats¹⁷). Hopefully progress in lab automation could help facilitate these tasks to ensure proper cell culture quality control.

Simply recognizing the limits of cell lines is a step in the right direction. But how can we address these limitations? There is a push towards using patient-derived cells but, as Kamm points out: “These are hard to come by, expensive and often not available in the quantity or cancer cell type that one would like. In addition, there’s such variability between patients and even within a single patient, that it becomes difficult to compare results from one study to another.” According to Kamm, one solution could consist in “finding ways to make them more widely available, perhaps by setting up cell banks across the nation that researchers can draw upon”. Different types of patient-derived cells can be extracted but stem cells and induced pluripotent stem cells in particular have been gaining ground. This is because they offer a unique alternative to both cancerous and non-cancerous cell lines due to their differentiation and self-renewal properties. More specifically, they offer the advantage of being able to generate both healthy and unhealthy cells of different tissues from the same patient.  

But expanding patient-derived cells in the lab can exert selective pressure and favor a subpopulation of cells that happens to thrive under these non-physiological conditions. Thus, an increasingly popular alternative consists in using patient-derived xenografts (PDX) models, which as Li explains: “offer heterogeneity and genetic variation that cell line models cannot.” PDX models are based on the transfer of primary tumors directly from the patient into an immunodeficient mouse. They are maintained by passaging cells directly from mouse to mouse once the tumor burden becomes too high.¹⁸ Li explains that: “current PDX have to be grown on mice, which is expensive and problematic because mouse cells can invade into human PDX. If microfluidic systems can be used to culture and maintain PDX, it will be ground-breaking as it will potentially expand PDX use to many different applications and labs.”

In spite of these issues, one must acknowledge the crucial role that cell lines are playing in mass-producing protein therapeutics.¹⁹ While these can also be produced in non-mammalian organisms (e.g. bacteria), cell lines are often preferred for the manufacturing of large, complex proteins with post-translational modifications. When it comes to producing proteins, the use of cell lines is a clear asset that allows for a high and uniform yield. Current challenges consist in optimizing the use of human rather than animal cell lines and ensuring the absence of human-specific viral contamination.  

Conclusion

Cell lines have brought unprecedented insights for in vitro studies – especially within the cancer research field. And yet, a shift might be under way because cell lines are now known to not fully represent the cellular heterogeneity of patients, an issue that becomes especially important with the onset of personalized medicine. At any rate, cell lines are bound to be used in the near future until other protocols for extracting, growing and expanding patient-specific cells in vitro are optimized. According to Li, the speed of this shift might very well: “depend on the resources and connection of a given lab seeing that cell lines are cheap to buy and maintain, and easy to propagate. In contrast, patient-derived or primary cells are hard to culture, often require supplement of expensive growth factors, and grow a lot slower.”

There is a fine line between being able to obtain large amounts of cells continuously and simultaneously ensuring the native heterogeneity of patients is truly represented. With the onset of organs-on-chips and miniaturized assays, there might be a chance for scientists to focus efforts towards using smaller quantities of cells that are more representative, even though they are harder to come by. In contrast, for large-scale industrial applications such as protein or viral production,immortalized cell lines will remain key.

Is it time to draw a line in the sand for more personalized clinical applications and push for systematic validation with primary cells?

References

1. Shay, J.W. & Wright, W.E. Hayflick, its limit, and cellular ageing. Nat Rev Mol Cell Biol. 1:72-6 (2000)

2. Hayflick, L . & Moorhead, P. The serial cultivation of human diploid cell strains Exp Cell Res 25, 585-621 (1961)

3. Hayflick, L. The limited in vitro lifetime of human diploid cell strains. Exp Cell Res. 37, 616-636 (1965)

4. Boussommier-Calleja, A. 2019. In Vitro models of cancer, Ladame, S. & Chang, J.Y. (Eds).  Bioengineering Innovative Solutions for Cancer (Chapter 4.1). Elsevier (In press).

5. Gey C., Coffman, W., & Kubicek, M. “Tissue culture studies of the

proliferative capacity of cervical carcinoma and normal epithelium,” Cancer Res. 12: 264-65 (1952)

6. Earle, B.W.R., Stark, T.H., Straus, N.P, Brown, M.F., & Shelton, E. Production of Malignancy in Vitro . IV . The Mouse Fibroblast Cultures and Changes Seen in the Living Cells. no. 2.

7. Hayflick, L. Myth-busting about first mass-produced human cell line. Nature. 464:30 (2010)

8. Greider, C.W. & Blackburn F.H. Identification of a specific telomere terminal transferase enzyme with two kinds of primer specificity. Cell 51:405-13 (1985)

9. Kim, N.W., et al. Specific association of human telomerase activity with immortal cells and cancer. Science. 266:2011-15 (1994)

10. Bodnar, A.G., et al. Extension of life span by introduction of telomerase into normal human cells. Science. 279:349-52 (1998)

11. Batista, L.F., et al. Telomere shortening and loss of self-renewal in dyskeratosis congenita induced pluripotent stem cells. Nature. 474:399-402 (2011)

12. Köhler, G., & Milstein, C. Continuous cultures of fused cells secreting

antibody of predefined specificity. Nature, 256(5517):495–7 (1975)

13. Maqsood, M.I., Matin, M.M., Bahrami, A.R. & Ghasroldasht, M.M. Immortality of cell lines: Challenges and advantages of establishment. Cell Biol. Int. 37:1038-45 (2013)

14. Wilding, J.L. & Bodmer, W.F. Cancer cell lines for drug discovery and development. Cancer Res. 74:2377-84 (2014)

15. Domcke, S., Sinha, R., Levine, D. A., Sander, C. & Schultz, N. Evaluating cell lines as tumour models by comparison of genomic profiles. Nat. Commun. 4:2126 (2013)

16. Horbach, S.P.J.M. & Halffman, W. The ghosts of HeLa: How cell line misidentification contaminates the scientific literature. PLoS One 12:1-16 (2017)

17. Masters, J.R., et al. Short tandem repeat profiling provides an international reference standard for human cell lines. Proc. Natl. Acad. Sci. U.S.A. 98:8012-7 (2001)

18. Siolas, D. & Hannon G.J. Patient Derived Tumor Xenografts: transforming clinical samples into mouse models. Cancer Res. 73(17):5315-9 (2013)

19. Dumont, J., et al. Human cell lines for biopharmaceutical manufacturing: history, status, and future perspectives. Crit Rev Biotechnol. 36(6):1110-22 (2016)