Authenticity Matters Cell-Line Development Begins at the Source


As director of biological content at the ATCC, Fang Tian oversees the preparation, authentication, characterization, quality control, and cryopreservation of more than 3,400 accessioned animal cell lines and hybridomas in the nonprofit organization’s cell-biology general collection. She has been with ATCC for 12 years, during which she served as a scientist and head of cell-biology research and development. Previously she was a research fellow at Harvard Medical School’s Massachusetts General Hospital, where she studied signaling pathways, working with high-throughput, real-time polymerase chain reaction (PCR) screening to study cell death and stress in oncology. Tian did her postdoctoral work at the University of Pittsburgh Cancer Institute after earning her PhD in pharmacology from the Chinese Academy of Sciences.

In April 2024, BPI associate editor Josh Abbott spoke with Tian about the importance of authentication to developing cell lines for biopharmaceutical discovery, production, and testing.

The Importance of Authentication

Cell-line authentication is critical to cell-line development (CLD), Tian said. The process must start with the right material. CLD requires multiple steps, manipulations, and processes. Things can go wrong along the way, especially as multiple processes happen simultaneously. So it’s wise to double-check throughout to ensure that the cell line you’re working on is what you expected it to be and has not been contaminated, switched, or mixed with other materials. “Once you have the model you want,” she said, “things don’t end there. Companies create a bank that will ensure continuity of critical materials.”

Cell banks are propagated and used to produce biologicals or provide models for drug screening, and so on. The goal is to produce a reliable cell stock or cell bank. That makes it critical to repeat some authentication work, double-checking that the bank remains as expected. “From beginning to the end, it’s one of the most important things to ensure that you’re working with the correct material.”

Problems can arise after the first authentication. Cell-line developers often culture many derivatives and subclones to select the final best performers. “Without proper documentation and training on best practices, it’s very easy to mix lots during the many steps involved,” Tian cautioned. “By the end, the frozen banked material might not reflect what was intended at the beginning.”

For the biopharmaceutical industry, it’s even more important to reauthenticate both during and at the end of a process. That’s true both for quality assurance and regulatory compliance. “You must document work done at the beginning, during development, and at the end to show what testing has been carried out and verified and that results meet expectations.”

Tian said that ATCC provides materials for many uses — basic research, translational research, and bioprocessing — and CLD is part of them all. The most relevant applications in the biopharmaceutical industry are disease modeling, therapeutic production, and drug testing.

Research Cell Lines: Researchers can create unique cell lines that are genetically modified to mimic certain diseases or resist certain drugs. So-called translational CLD provides cells for disease research and discovery of new candidate therapeutics. Human cells — e.g., human embryonic kidney (HEK293) cells — are used widely in such translational research. Other commonly used cell lines include Chinese hamster ovary (CHO), Madin–Darby canine kidney (MDCK) and Vero cells from African green monkey kidneys.

Such cells can be used later in drug development, as well. Cell-based assays are increasingly important for potency testing and other preclinical product-development studies.

Production Cell Lines: Other cell-line developers focus on cell culture to produce therapeutic antibodies or viral vectors. Often their work is improving an established host cell line — e.g., to create “superpowered CHO cells,” as Tian said. “We call this platform building or enhancement.”

It begins with an established parent cell line modified to improve production yield, for example, or to optimize for large-scale high-density culture and rapid growth (e.g., in producing antibiotics). Some companies use transient transfection of gene sequences for specific proteins or viruses. That strategy also requires CLD to select the cells with the best expression and productivity profiles. The resulting clones might be used to establish a stable cell line during later production stages.

Methods of Authentication

Authentication verifies the identity of a cell line: at the species and individual levels. For example, consider the well-known HeLa cell line. Not only can we say that HeLa cells are human cells, and human cancer cells in particular, but also that they are human cervical cancer cells from an individual woman, Henrietta Lacks. “We can distinguish cell lines to an individual level among billions of people on Earth,” Tian said. “That level of sensitivity is amazing.”

Cell authentication confirms that a cell comes from the identified source material and is free from contamination. Analysis begins with understanding the species of your working material, for which ATCC uses DNA barcoding. The standard method is based on cytochrome c oxidase (CO1), a mitochondrial enzyme expressed in all animal species because of its integral role in energy production. Because inheritance of mitochondrial genes is maternal, animals typically have only one variant of each CO1 gene. In different species, the size of the enzyme is significantly different, so scientists can use PCR to amplify host DNA and compare the size of the CO1 gene to identify its species of origin.

“We can use gel electrophoresis to test for the presence of cells from another species,” Tian said. “You might be working with multiple cell lines, not necessarily at the same time but maybe in the same biosafety cabinet or incubator. If a mix-up happens, you will catch that cross-contamination with this methodology.”

Using a PCR-amplified sample, scientists next apply a sequencing methodology to compare a specific sequence with others in a database. Such sequencing comparison will identify the species of origin exactly and with great confidence.

To separate cells further, Tian said, short-term repeat (STR) profiling is the gold standard for human cell-line authentication. STRs are highly polymorphic microsatellite sequences, and the numbers of repeats are different across cell types and individuals. “It’s almost like a combination locker,” Tian explained. “Imagine a lock with 18 numbers.” That broad array of possibilities makes the method supersensitive and highly individualized. The STR assay is also reliable, robust, and reproducible.

The method is beginning to be used for cells of nonhuman origin as well. ATCC has worked to apply STR profiling to mouse cell lines. “We established the first international standard and worked with the National Institute of Standards and Technology (NIST) in a consortium to establish a mouse STR authentication methodology. We developed a CO1 assay and are currently pushing CO1 standard publication in this area.”

Risk Management

Some high-impact journals that publish cell biology studies have claimed that over 70% of such work cannot be reproduced (1). The waste of such results is “astronomical,” Tian said, “billions of dollars.” One way to address the problem is to start with reliable biological materials. “Other aspects contribute to nonreproducible results, but publications have homed in on the message that use of contaminated or nonauthenticated cells is widespread.”

The economic burden of nonauthenticated cell lines has been “astonishing,” she said, to both the research community and the biotechnology industry. The negative impacts span from individual projects, wasting time and money, to larger economic scales. “The translational research community has paid so much for nonreproducible results.”

Now many cell-biology journals request that researchers provide authentication information before they can submit a paper. However, Tian said, people often do that work right before submitting just to get it published. “That’s really not the best practice.”

The sooner that significant problems can be caught, the better. “It can save a project, save labor and time. So each and every batch of the cells that ATCC produces always goes through cell authentication. People can start with those materials knowing that the work has been done. But many researchers create their own cell banks without any checking, and that’s a risk. When they’re culturing cells for several months, many things can go wrong. Without the authentication work, you risk finding something wrong at end.”

Scientists and companies take that risk ostensibly for financial reasons. But Tian said, “We need education about the problem. Genetic testing is new to many researchers. It requires changing habits, establishing new requirements, and using caution. Scientists need to be vigilant in their projects and careful with their materials throughout — then double-check at the end to ensure that everything’s correct.” Although authentication involves some cost, that is not prohibitively high. “People need to be aware of the negative impacts and the available tools and understand the benefits. Authentication can prevent future risks of wasted resources and ultimately data that aren’t useful. That certainly outweighs the [financial] cost.”

STR testing typically is outsourced. A number of contract laboratories offer that service in the United States and the United Kingdom, and other countries are establishing capabilities as well. Using a proven STR service is better than trying to do it in house, Tian said. “You want to be sure that it’s done properly, with the right algorithms to analyze the results and make the database matches. Well-established STR services have the expertise that individual development laboratories don’t have.”

ATCC reauthenticates cell lines every time cells are cultured from them. “We not only have a database for each type of cell, but also databases for each line and every lot. That enables us to investigate genetic drift and instability.” The nonprofit organization also provides STR services using those comprehensive databases to get accurate readouts.

“When I was at Massachusetts General Hospital,” Tian recalled, “we pursued novel, ‘hardcore’ science, so we never really thought about [something as basic as] cell culture. It didn’t feel like something that could have issues. But the reality is that they can arise. I have seen companies go through product development and then at the end find out that their materials have the wrong STR profile. In our work over the past 10 years, when global research laboratories send us high-value material to deposit with ATCC, we find that 30% of lines have been misidentified. That’s really striking.”

She said that sometimes even well-known laboratories will have that problem. “They think they’re working with cell line X, but on testing it turns out to be cell line Y. Often, they find unique features in those cells and now can explain those discrepancies because of the misidentification. My work at ATCC has made me aware of this widespread issue of misidentified and contaminated lines.”

So it’s important to ensure that such errors don’t happen in the first place. “I think that many researchers just want to get that certificate so that they can submit a paper,” Tian said. “But long before that, they should make starting materials from validated, reputable vendors. You don’t just borrow from somebody else across the hallway or grab cells from a tank — even though people often like to work with material that they have banked.”

When scientists cryopreserve some banked cell lines, said Tian, often they do not perform a thorough check. “They feel like it’s been done. They bank cells and then grow a group of them, freeze them down, and record that they’ve frozen a batch of cells. But they haven’t done proper testing to verify. When other people go to grab a vial of cells and start to do complex work with them, things can happen. Problems can amplify. That can cause a lot of unnecessary burden in cost, resources, and ultimately project delays.”


1 Hepkema WM, et al. Misidentified Biomedical Resources: Journal Guidelines Are Not a Quick Fix. Int. J. Cancer 150(8) 2022: 1233–1243;

Further Reading

Fantuzzo J, et al. Advances in Cell-Line Development, Banking, and Authentication. BioProcess Int. 20(7–8) 2022: insert;

Gazaille B, Castillo F, Nims RW. Cell Banking in the Spotlight: Advising Biologics Developers About Cell Bank Preparation and Characterization. BioProcess Int. 19(7–8) 2021: insert;

Josh Abbott is associate editor, and corresponding author Cheryl Scott is cofounder and editor in chief of BioProcess International (part of Informa Connect Life Sciences); 1-212-600-3429; [email protected].

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