What is Cell Culture Forensics?

Cell culture forensics emerged as a result of the widespread contamination that plagued the work of scientists around the world during the 1970s and 1980s.

Cell Culture

Cell Culture. Image Credit: Hakat/Shutterstock.com

Recent advances in cell identification technologies have created a powerful forensic approach aimed at ensuring that these contamination issues are not repeated.  

Widespread global contamination

In 1951, George Gey of Johns Hopkins Medical School obtained primary cervical cancer cells from a 31-year-old woman named Henrietta Lacks.

These cells otherwise referred to as HeLa cells, grew at an exponential rate, which was a remarkable discovery to scientists at the time who were unable to maintain cell cultures for extended periods.

As more laboratories around the world began utilizing these highly useful HeLa cells for their given research purposes, its rapid growth, unfortunately, led to widespread contamination of the other cells that were being grown in these labs at the time.

As a result, researchers were unable to discern which cell lines were untouched by HeLa, thereby discrediting much of their published research. It is estimated that between the 1970s and 1980s, one in every three cell lines was overtaken or masquerading as another.

While HeLa cells were considered to be the most notorious culprit of this widespread contamination, several other cell lines were also contributing to this issue.

Early attempts to confirm contamination


One of the earliest ways in which researchers attempted to confirm HeLa cell contamination was by measuring the expression of the allozyme genotype GGPD-A, which is only found in African Americans, like Henrietta Lacks.

Since all of the other cell lines that were tested during this early experiment were obtained from Caucasian patients, this assay validated the source of their contamination to be HeLa cells.

As a backup to this identification method, three highly rearranged chromosomes were also used to confirm HeLa cell contamination.  

DNA fingerprints

In 1990, Dennis Gilbert and his team of researchers developed a multilocus DNA fingerprinting method that was aimed towards assisting in cell contamination monitoring endeavors.

This method, which was the first major DNA individual identification method to be used in forensic legal cases, provided a quantitative estimate of the genetic difference that exists between individuals and their respective cells.

In their work, the DNA fingerprints of 46 human cell lines were assessed, and the average percent difference (APD) between each of the cell line pair combinations, which amount to 1,035 lines, was also assessed.

The methodology behind DNA fingerprinting of these cell lines first involved the digestion of genomic DNA, which was followed by electrophoretic separation and hybridization by radiolabeled hypervariable probes.

On average, about 17.4 HAEIII and 15.6 HinfI restriction enzyme fragments were obtained from each cell line, with a mean allele frequency of 0.047 and 0.063, respectively.

The allele frequency represents the probability that a randomly selected cell line would match a given DNA fingerprint.

When comparing unrelated cell lines, the researchers found an average APD of 76.9%, whereas HeLa derivative cell lines were found to almost completely exhibit an identical DNA fingerprint to the pure HeLa cell line.

This DNA fingerprinting technique, therefore, provides researchers with a verifiable and rigorous method capable of differentiating cell lines and confirming the presence of contamination.


Since its approval as an American National Standard by the American National Standards Institute, short tandem repeat (STR) profiling has been widely used for the authentication of human cell lines.

The STR profiling approach is a rapid, economical, and highly sensitive technology that has demonstrated its specificity advantages over other authentication technologies like karyotyping, isoenzyme analysis, immunotyping, and human leukocyte antigen typing.

The STR profiles of cell lines are continuously updated onto a public database, thereby allowing users to accurately compare their cell stocks to reduce the occurrence of misidentification of cell lines.

Importance of cell culture forensics

Despite the various efforts that have been made over the past several decades to prevent cell culture contamination from occurring by identifying potential contaminants early on, cell contamination remains a difficult challenge for many research laboratories.

In a German cell line repository, for example, 18% of their 252 cell culture lines were found to be contaminated by another cell line.

While this may be true, the highly regulated biopharmaceutical industry requires thorough phenotypic analysis and confirmation of animal species of origin tests to be conducted on all cell lines, thereby ensuring a significantly lower frequency of cell line misidentification.

Cell line integrity is crucial for every research laboratory that depends upon cell culture data, such as vaccinology studies, tumor research, and other cell biology areas of interest.

As the number of isolated cell lines continues to rise around the world through gene discovery and the development of specified disease cohorts, cell culture forensic techniques, such as those discussed here, will remain crucial in maintaining the genetic integrity of these cell lines.

Such techniques should not only be accurate and reliable but should also be inexpensive, standardized, and facilitate proper identity testing for a wide range of cell populations.


  • O’Brien, S. J. (2001). Cell culture forensics. PNAS 98(14); 7656-7658. DOI: 10.1073/pnas.141237598.
  • Gilbert, D. A., Reid, Y. A., Gail, M. H., Pee, D., White, C., Hay, R. J., & O’Brien, S. J. (1990). Application of DNA fingerprints for cell-line individualization. American Journal of Human Genetics 47(3); 499-514. PMCID: PMC1683851.
  • Barallon, R., Bauer, S. R., Butler, J., Capes-Davis, A., Dirks, W. G., Elmore, E., et al. (2010). Recommendation of short tandem repeat profiling for authenticating human cell lines, stem cells, and tissues. In Vitro Cellular & Developmental Biology – Animal 46; 727-732. DOI: 10.1007/s11626-010-9333-z.

Further Reading

Last Updated: Aug 17, 2022

Benedette Cuffari

Written by

Benedette Cuffari

After completing her Bachelor of Science in Toxicology with two minors in Spanish and Chemistry in 2016, Benedette continued her studies to complete her Master of Science in Toxicology in May of 2018. During graduate school, Benedette investigated the dermatotoxicity of mechlorethamine and bendamustine; two nitrogen mustard alkylating agents that are used in anticancer therapy.


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