Scientists working with breast cancer cells purchased at the same time from the same cell bank say they discovered that the cells responded differently to chemicals, even though the researchers had not detected any difference when they tested them for authenticity at the time of purchase. Had the cells been the same, the exposure to chemicals would have produced similar results in the cells.
Instead, identical experiments in two different laboratories, one at the Johns Hopkins Bloomberg School of Public Health and another at Brown University, produced different results, even when the researchers swapped out the cells and replicated their experiment to rule out issues at the respective laboratories. Upon further testing, however, the scientists discovered that the cells had been genetically different from the time they acquired them.
Researchers have long worked under the assumption that cells purchased from the same lot of a cell bank are clones. The reliability of cells is the foundation of much scientific research.
The findings (“Genetic Variability in a Frozen Batch of MCF-7 Cells Invisible in Routine Authentication Affecting Cell Function”), published online in Scientific Reports, were made by a research team led by scientists at the Johns Hopkins Bloomberg School of Public Health. Even if this was one bad batch, the discovery raises uncertainty about the reproducibility of experiments using cells that researchers have assumed were the same and contained the same DNA. For the researchers, the flawed cells cost them several years of work and close to $1 million in NIH funding.
“Every researcher believes that if they use cells from the same line of cells, particularly cells from the same lot of a cell bank, then they have clones that should look and act the same,” says study leader Thomas Hartung, M.D., Ph.D., a professor in the Bloomberg School's departments of Environmental Health Sciences and Molecular Microbiology and Immunology. “We learned in this study that that is not always the case.”
Cell irregularities are not unprecedented. Last year, Italian researchers reported instances of genetic instability in another tumor cell line from different laboratories. However, they did not detect instability in cells from the same lot from a single cell bank, as the Bloomberg School researchers did. Separately, 3 years ago, an international study using the same cell line as the Bloomberg School researchers, also failed because the cells were inconsistent. This was not traced back to the cell source but blamed on the university laboratories.
The discovery came about when Dr. Hartung and colleagues were performing experiments for the Human Toxome Project, an effort led by Dr. Hartung and taking place at six study centers across the country to understand the toxicity of various chemicals better by testing them on cell cultures. For this experiment, the researchers worked with cells from the MCF-7 (Michigan Cancer Foundation-7) line, a well-established breast cancer line isolated in 1970. It has been used in original research published in more than 23,000 scientific articles. Many cancer cell lines, including this one, are immortalized.
To perform identical experiments at the two different schools, researchers from the Bloomberg School and Brown purchased frozen vials of cells of the same lot from the same cell bank. As a standard precaution to make sure that the cells they'd purchased weren't contaminated with others of a different type, the investigators at each school performed a test that sampled short segments of the genome, called short tandem repeats. The test suggested that the cells were genetically identical.
However, when the researchers began comparing notes from their experiments, they found that cells at the two different schools looked and behaved in vastly different ways, says Dr. Hartung, who also directs the Center for Alternatives to Animal Testing at the Bloomberg School. The MCF-7 cells at the Bloomberg School grew flat, with a cobblestone-like appearance, while those at Brown grew in large, heap-like aggregations. When the cells were exposed to estrogen, those at Brown proliferated wildly but those at the Bloomberg School remained static. Further tests at the two schools showed that the different batches of cells produced different amounts of metabolic by-products and had different patterns of gene activation.
As noted above, to rule out whether dissimilarities between the two labs' techniques were responsible for these variations, the schools swapped cells and tried the experiments again–with identical results to the original experiments at the opposite school.
When the scientists performed a comparative genomic hybridization (CGH) test to check for genomic differences, they found that the two batches of cells were riddled with variations, presumably the cause for their host of differences. Fortunately, both laboratories had an additional vial of the same lot as obtained from the cell bank in their freezers that they'd never touched. The big surprise: They already showed the same genetic differences, before they'd been exposed to anything.
The findings shed light on a potentially huge problem for the reproducibility of scientific experiments that use tumor cell cultures, according to Dr. Hartung. “This might explain, why 3 years ago a big international study using this very cell line failed because of lacking reproducibility,” he points out, adding that the results are a call to action for cell banks to rethink quality assurance procedures. By using detailed genetic testing, such as CGH, cell banks and their scientist customers can be reassured that cells from the same lot are indeed genetically identical, helping to reassure that they'll behave the same in identical experiments.
Dr. Hartung and colleagues have previously called for tighter quality controls. In 2005, he led a team that published recommendations for Good Cell Culture Practice aimed at quality standards for cell banks and researchers. These eventually led to the founding of the International Good Cell Culture Practice Collaboration, which has representatives form organizations and agencies from around the world and is formally launching at EuroScience Open Forum 2016.
“It is our goal to develop internationally agreed-upon standards for quality assurance of cell cultures and how we report on our experiments,” says Dr. Hartung.