For any scientist’s work to have meaning and value, it is imperative that the materials they use, including cell lines, are reliable and are what the are supposed to be. Without this, results may be worthless or even worse damaging if they mislead and misinform future work. It has been estimated that over 30,000 scientific papers, which have each been referenced an average of 15 times each present incorrect results as a consequence of the use of misidentified cell lines. Conservative figures suggest that irreproducibility in biology, to which misidentified cell lines contributes greatly, equates to $28 billion per year of wasted research funding – staggering statistics.
The provision of an accessible resource that can offer strains and cell lines that have been checked and confirmed to be what it says on the tube is therefore invaluable, saving untold time and money and reinforcing the integrity and validity of research output.
We spoke to Mindy Goldsborough, Ph.D., Chief Science and Technology Officer, VP and General Manager, ATCC Cell Systems about the importance and challenges of maintaining authenticated cell lines for scientific research.
Karen Steward (KS): Can you tell us a bit about the history of ATCC and how their cell biology collection came to be one of the largest bioresources in the world?
Mindy Goldsborough (MG): Founded in 1925, ATCC is a non-profit organization with headquarters in Manassas, VA, and an R&D and Services center in Gaithersburg, MD. During the first few decades following its inception, ATCC focused exclusively on microorganisms as cell culture was yet to be a widely practiced technique. That changed in the late 1940s when Julius Younger—a member of Jonas Salk’s lab where the first effective polio vaccine was developed, and later a member of the ATCC Board of Directors—used cell culture methods to expand polio viral production. The first formulations for cell culture media were published during this same time frame, and over the next decade cell culture became a more routine technology.
As it had done for the microbial collection, ATCC set out to develop standardized methods for the authentication, culture, cryopreservation, quality control, and distribution of cell lines deposited to ATCC by the scientific community. ATCC continues its mission to this day by adding to the microbial and cell line portfolios on a constant basis. We have become the largest and most diverse collection by being the most trusted biocollection: the standard for the industry. We earned that trust every day of our 94-year history by using the most current technologies to provide the highest-quality biomaterials to the research community: the credible standards upon which our customers build their incredible work.
KS: How are cell lines validated and can anyone submit their cell lines if they are not already held by ATCC for validation?
MG: Cells are authenticated using a number of techniques, including short tandem repeat profiling (STR). Once established, a cell line’s STR profile becomes the unique “fingerprint” of that cell line. Using STR technology, ATCC can identify if a cell line is misidentified (i.e., a cell line being misrepresented as another one). Cell lines are also checked for microbial contamination, including mycoplasma, and for cross-contamination (i.e., where you’d have more than a single cell line in a culture). Researchers can have cell lines that are not part of the ATCC collection authenticated using one of ATCC’s testing services; we currently have services for human and mouse STR analysis as well as mycoplasma testing. It is also a general best practice for researchers to routinely authenticate the cell lines within their laboratory.
KS: What are the biggest challenges for authenticating cell lines? Are there any cell or species types that are particularly problematic?
MG: STR analysis for authenticating human cell lines is a very well-established technology as it has been used in forensic work for decades. For cell line authentication, there are a few instances where additional tests beyond STR analysis may be required. Some cancer cell lines are known to undergo chromosomal changes, such as cell lines with microsatellite instability, and those changes can impact the STR profile. If more than one cell line is generated from a single donor, all the resulting cell lines would have the same STR profile. In this case, if one cell line was from a “normal” sample and the other a tumor sample, additional tests (e.g., a tumor specific biomarker) could be used to authenticate each sample independently. The same is generally true for isogenic cell lines where a desired genetic mutation has been introduced. Depending on the genomic site of the change, this may or may not cause a change in STR alleles. In an isogenic cell line, the genetic change is known and therefore a PCR-based test can be developed for distinguishing the isogenic from the parental cell line.
Currently, reliable STR analysis is available only for human and mouse cell lines, but this covers a large percentage of the cell lines routinely used in scientific research. Developing the STR test for mouse cell lines had unique challenges, as so many of the mouse strains from which cell lines are established are inbred strains. For common cell line species where STR is currently not available, ATCC uses the species-specific cytochrome oxidase 1 (CO1) gene to identify cell lines down to the species level. As needed, additional authentication tests such as PCR or immunocytochemistry can be performed.
KS: Why it is so important to have validated cell lines?
MG: The old saying “garbage in, garbage out” sums it up perfectly. How can one do incredible research if you are not sure what you are starting with? In every cell culture lab throughout the world, there are liquid nitrogen freezers full of cryovials from past experiments and staff. There is a label on that vial saying what it is, or rather what the person who froze it down thought it was. If you are about to start an experiment that will take a few days, a week, a month, or a year, are you willing to lose all that time if you find out the cell line you pulled out of that freezer wasn’t what is was supposed to be, or that it was contaminated with mycoplasma? In addition, using questionable material puts one of the basic tenants of scientific research, which is to have others build upon one’s work, at risk. ATCC provides the standards—the cell lines that everyone can start with—so that results can be compared, and work can be extended and expanded upon. Without a standard, it is not possible to do this reproducibly.
KS: Can you tell us about some of the problems that are occurring due to the use of unvalidated cell lines?
MG: Part of the reproducibility issue with translating basic research to clinical outcomes stems from the use of unvalidated cell lines. The reproducibility issue has been estimated to cost tens of billions of dollars a year; solving even just a small part of the issue can have significant financial and scientific benefits. However, those are just the societal problems and benefits. The use of unvalidated cell lines can be very personal, from the scientist who can’t get a paper published (or worse, must retract a publication) to those that can’t move a drug target along because a cell line they thought was something turned out not to be.
In our authentication services business, for example, we hear from a fair number of shocked researches; they have sent in the cell line for testing not because they think it’s a good practice, but because the journal suggests or requires it before publication, and then they find out it’s not the right cell line and their work is potentially wasted.
The cost of starting experiments with authenticated materials is inconsequential when compared with the cost of not doing so. Further, it is important to remember that authentication shouldn’t just be performed at the start of an experiment; researchers need to continuously monitor cells under culture, especially cell lines that are in culture for extended periods of time. But the cause and effect are often underappreciated and thus, the practice and waste continue.
KS: How do you see the issue of cell line authenticity changing in the future, short and long-term?
MG: I believe there will always be a need for access to highly authenticated cells, and I know that ATCC intends to be there for the next 94 years, and beyond, to provide that access. But it’s not just authenticated cells, it is also the bioinformation that accompanies those biomaterials that is important, whether it’s characterization data, genomic data, or some other information—it all needs to be of the highest quality. Modeling and other in silico methods mean that the starting material for research may not always be a biomaterial, so the quality of bioinformation is also something that ATCC takes quite seriously. Other changes in authentication will follow technology development. As new methods evolve that enable more precise authentication, those will be tested and incorporated, as appropriate. When ATCC started accessioning cell lines about 70 years ago, cell line morphology was one of the most common ways to authenticate cell lines; today we use methods such as STR analysis, PCR, sequencing, and immunocytochemistry. In another 70 years, these methods may seem equally as simplistic.
Dr Mindy Goldsborough was speaking to Dr Karen Steward, Science Writer for Technology Networks.