Human Cell Lines as Tools of Our Trade: “Laying It on the (Cell) Line”

Human cell lines are commonly used for research investigation. For decades, cell lines have been the workhorse of programs to identify and interrogate mechanisms of action, discover and/or test drug/compounds/factors, and show relevance of findings to human disease. In this editorial, I pause for thought to reflect on how we should revere the cell lines we use and consider how best and better to incorporate such tools into our research programs.

Most laboratories routinely use human cell lines. Many were established decades ago and distributed within the research community. Herein lies the first of our problems. Human cell lines cultures have become so routine that there is a common assumption that cultured lines in one laboratory equal those in another. This is not so. Variations in passage number or culture conditions can result in a drift in the cellular and molecular phenotype; on occasions, cell lines are even misidentified or cross-contaminated. To provide consistency, Endocrine Society editorial policy now requires that all cell lines used and described in all newly submitted and revised manuscripts are authenticated. Effective from January 1, 2015, our policy is similar to guidelines for Nature and American Association for Cancer Research journal publications and concurs with the American Type Culture Collection Standards Development Organization. Providing data on where and when the cells were obtained, if and how they were tested and authenticated, and when they were last tested enables comparison and reproducibility between investigators.

“The time has come for us to culture our cells better and move from 2D to 3D models and attempt to more closely replicate the environment in which cells normally reside and then change the environment to mimic disease.”

Another assumption is that a cell line comprises cells that are equal and similar. In fact, most cells lines are heterogeneous and may include stem/progenitor cells. Although the stem/progenitor cells may be present at low frequency, these cell subpopulations are often the therapeutic target. A global reduction in cell proliferation/transcription/translation may be 90%–95% and be statistically significant, but the change that is measured may occur only in the differentiated cells that make up the bulk of the cell lines in culture. What about and how do we show alteration to the subpopulations, including stem/progenitors that may constitute a minority of cells? This is a moot point for drug targeting studies, in which a highly significant effect on the bulk of the cells is frequently favored compared with any decline in smaller cell subpopulations.

Heterogeneity of another type is a further significant issue. Human cell lines are usually obtained from a patient and therefore represent the diseased organ or tissue from that one patient. In 2014 patient variability is a complexity that is acknowledged as being significant. There is a focus on targeted therapies and personalized medicine, but cell lines in culture fail to represent the diversity of patient cohorts. Furthermore, diseases may be chronic, and even in cancer, for example, solid tumors such as breast and prostate cancer develop over years/decades. There is a need to use cell lines that most accurately represent the stage of disease under investigation. Thus, cell lines from metastatic sites may be insightful for end-stage disease but provide little relevance to the mechanisms of transition from the organ confined, clinically insignificant to clinically significant disease. What is observed in one cell line cannot legitimately be extrapolated as evidence of efficacy for treatment for cancer/disease X. Common or different responses by different human cell lines may provide insight, but the use of primary lines from clinically defined patients might be even better. Nevertheless, generating primary lines is challenging and ultimately brings me to my next point: the common use of two-dimensional (2D) culture protocols.

It is common practice to culture cells on plastic culture dishes devoid of any other cell type with which they normally coexist. Although the use of single-cell line cultures may identify molecular mechanisms that could be insightful, this approach is limited by the failure to mimic the condition in which cells coexist with other cell types in a tissue or organ. Therefore, our reductionist approach is only one of our tools of the trade, and there is a need to temper sweeping statements such as “these findings suggest drug X or Y could treat cancer/diabetes” when based on single-cell line studies.

The time has come for us to culture our cells better and move from 2D to three-dimensional (3D) models and attempt to more closely replicate the environment in which cells normally reside and then change the environment to mimic disease. I proffer an analogy: thoughtful neighbors make us happy and normal but noisy neighbors often make us grumpy and encourage us to behave badly, doing things that are out of character. I suggest putting our cells into a plastic dish that puts them without any neighbors and/or in a bad location that encourages abnormal features. There is a need to upgrade our common 2D culture approach, and it is very likely that cell behavior will change as we use 3D and include and promote cell-to-cell interaction and as we change the environment to mimic disease/pathology. This topic was important enough for the? European Association For Cancer Research to convene an interdisciplinary meeting entitled Goodbye Flat Biology in November 2014. The meeting was a forum “to review recent advances and to discuss the challenge of creating reductionist in vitro models of complex biology.” It showcased contemporary approaches to improving cell culture and 3D models involving the biofabrication of new structures for biologists to incorporate into cell cultures. The contributions of mechanobiology, the lymphatic system, matrix remodeling, and angiogenesis collectively enforce the central role of the tumor microenvironment in cell culture. Similarly but differently, a recent issue of Nature Reviews Molecular Cell Biology is recommended reading to remind us of the importance of the extracellular matrix in regulating cell and tissue health through the microenvironment (1).

To return to the challenge to establish human patient primary cell cultures, how can we do this better? The realization that 3D architecture is a critical component of cell viability led to efforts to develop spheroid/organoid assays using human patient tissues. Coupled with the ability to tailor and supplement culture conditions to support the growth and phenotype of the specific organ or disease being studied, organoids provide a key advance in culturing cells to more realistically mimic biological process. A further advantage of organoid cultures is the derivation of lines that could previously survive only as xenografts, thus increasing the number and diversity of cell lines (2). Another approach is to include coculture of isolated human cell types on biofabricated scaffolds or support structures such as those used in regenerative medicine to grow stem or progenitors with cells of the niche or surrounding microenvironment (3).

Whatever we do, I urge you in 2015 to give more thought to how we culture cells so that our human cell lines more accurately reflect the full spectrum of disease progression and patient diversity. In doing so, we will gain greater confidence that panels of human cell lines can meaningfully evaluate therapeutic responses.

Gail P. Risbridger, PhD

Funding Statement

The Author is a Research Fellow funded by the National Health and Medical Research Council of Australia.