If you could help reduce the waste of tens of billions of dollars per year in research spending, you’d do it, right?
This is the second in a series of two guest posts about the havoc misidentified cell lines can wreak on research, from Leonard P. Freedman, president of the Global Biological Standards Institute. Freedman who published a paper last summer detailing the financial costs of non-reproducible research — namely, tens of billions of dollars per year. Some of that non-reproducible research is due to the use of contaminated or misidentified cell lines. He writes about one key step to tackling the problem: Ask every scientist to use a relatively inexpensive technique to validate the identity of their cell lines.
Meanwhile, we have to deal with the issue of all the previously published papers that relied on problematic cell lines, now contaminating the scientific literature. Scroll down to the bottom of the post to take a poll on what you think should be done about those papers.
As new frontiers of science emerge, from Pluto to proteins, the very cornerstone of the scientific process—reproducibility—has also reared its head as a huge problem. Estimates of irreproducibility rates of published peer-reviewed papers range from 51% to 89%. An analysis that two colleagues and I recently published in PLOS Biology suggests the U.S. spends $28 billion per year on non-reproducible preclinical research; global spending could be up to $60 billion per year. This lack of reproducibility typically results from cumulative errors or flaws in one or more of the following areas: biological reagents and reference materials, study design, laboratory protocols, and data analysis and reporting. Given the size, scale, and especially the complexity of reproducing preclinical research, there is no single magic bullet fix. This is a difficult issue for scientists to own up to, and for the public to grasp.
However, an approach that has demonstrably addressed similar challenges in other complex and evolving industries, such as those involved in the founding of the Internet, is the expanded use of community-based voluntary standards and best practices. And here’s where we start: We should begin applying these standards to the use of cell lines in biomedical research.
The problem and cost of misidentified and contaminated cell lines – and their impact on reproducibility – cannot be overestimated. Immortalized cancer cell lines have been workhorses in the lab for decades. Accurate determination of species, sex, and tissue of origin is critical to study interpretation, validity, and translation of results. Yet cell lines are cultured, passaged, and processed in and among laboratories with widely varying quality control (QC) procedures, while casual sharing of cell lines between labs is endemic. For these reasons, contamination and misidentification errors occur frequently and persist for years (or decades). Additionally, genotypic and phenotypic drift as a result of over-passaging cells or through poor QC is also widespread.
Just how big is this problem? One survey reported combined rates of contaminated and misidentified cell lines as high as 36%; a more recent estimate in Science places the cross-contamination rate at 20%. Cross-contamination rates for cell lines established in China were recently reported to range from 25% to 85%. An NIH RePORTER search identified 9,000 active projects using cell lines, totaling $3.7B. Required use of authentication techniques would affect over $900M in research dollars annually.
In contrast to many other contributors to irreproducibility that pose complex solutions, the identity of a cell line can be readily determined by comparing its genetic signature or DNA fingerprint with those in established databases at major cell banks like ATCC or JCRB. There is also a technique, recommended by the American National Standards Institute, which authenticates human cell lines using short-tandem repeat (STR) DNA profiling techniques.
Yet despite the wide availability of this authentication gold standard, and its relatively low cost (approximately $150), a survey conducted in 2015 by my organization, the Global Biological Standards Institute (GBSI), and recently published in BioTechniques, found that only 25% of researchers use STR. Additionally, only half conduct species-related QC tests at all, and—to no one’s surprise—few receive specific training in the importance of authentication. And things aren’t improving: A 2004 survey reported that just one-third of laboratories authenticate their cell lines; 10 years later, a Sigma-Aldrich survey found that only 37% of respondents “validate the purity and identity before first use” of cell lines. Understanding the existing barriers that prevent implementation of universal cell authentication is central to changing this sad state of affairs. Part cultural, part financial, part “I have too many other things to deal with”, changing the mind-set of bench scientists as to the importance of cell authentication will go a long way toward solving an eminently solvable problem.
Here are three ways change could have a real impact.
Journals: While about 30 journals include various authentication-related requirements, they almost universally do not require documentation of authentication as a condition for publication. As it turns out, merely expecting that cell lines are authenticated—a staple of author/reporting guidelines—is clearly not doing the trick. Recently, some journals began requiring that authors check their cells against a database of commonly misidentified cells like that provided by the International Cell Line Authentication Committee (ICLAC). A good start, but journals must take the next step and require documented cell authentication.
Funders: As Retraction Watch readers recently learned, funders can play a key role in improving reproducibility. Authentication recommendations now appear in the U.S. National Institutes of Health’s Principles and Guidelines for Reporting Preclinical Research that are endorsed by many journals and societies. Notably, the NIH recently announced clarifications to their expectations for the rigor of proposed research, as well as additions to the review criteria used to evaluate proposals. Unfortunately, these authentication guidelines are not a scored part of the grant review process. Without some teeth, meaningful change is not likely to occur. The Prostate Cancer Foundation found a simple solution—withhold the next year’s payment until a certification of authentication has been received. Other funders need to follow PCF’s lead.
Training: A new approach to training the next generation of researchers (and the current scientific establishment) is also a key to addressing irreproducibility. In 2014 the NIH announced the “Training Modules to Enhance Data Reproducibility (R25) grant.” A recipient, GBSI is developing an active learning training module to educate researchers on why authentication is important – and, more importantly, how to do it. GBSI is building partnerships to disseminate this module widely to all researchers who use cells, particularly those at the start of their careers. But this module is also for PIs who need to both acknowledge the importance of cell authentication and make sure it is practiced in their labs. Most importantly, the training module will be freely available.
Taking these steps will help the scientific community understand why (and how) to authenticate cell lines, improve the efficiency of hundreds of millions of dollars in annual research expenditures, and accelerate the translation of research breakthroughs. We are in an era of unprecedented scientific discovery, but we are simultaneously being undone by our inability to reproduce preclinical research. Now is the time to curb the problem, and asking biomedical researchers to universally adopt and demonstrate the routine use of techniques to authenticate their cell culture lines—while requiring a modest amount of time and money—is the right thing to do.
Disclosure: GBSI receives core funding from ATCC’s BioNexus Foundation. ATCC provides cell authentication on a fee-for-service basis. ATCC also contributes to GBSI’s #authenticate campaign, as do the following cell authentication service providers: IDEXX BioResearch, Promega, and DDC Medical.
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