Quality Matters

Cell Lines and their Use in Research

  • Fig. 1: Spectral karyotyping of human lymphoma cell line U-2932 (DSMZ ACC 633) shows complex yet stable Chromosome changes.Fig. 1: Spectral karyotyping of human lymphoma cell line U-2932 (DSMZ ACC 633) shows complex yet stable Chromosome changes.
  • Fig. 1: Spectral karyotyping of human lymphoma cell line U-2932 (DSMZ ACC 633) shows complex yet stable Chromosome changes.
  • Fig. 2: Cancer cell lines as in vitro therapeutic models and resources. Top: Spectral karyotyping of a leukemia cell line (HT-93) shows chromosomal translocation between chromosomes 15 and 17 (arrows), forming the leukemic PML/RARA fusion gene. Cells bearing this mutation are hypersensitive to retinoic acid therapy. Bottom: Arrows show leukemic fusion genes comprising BCR (red) and ABL (green) on chromosomes from a CML cell line (K-562). CCLs carrying this rearrangement were used to develop “intelligent” drugs against CML and related neoplasms.

Why should quality matter when we talk about cell lines and their use in research? Continuous cell lines (CCLs), cultures derived from human or animal donors that continue to proliferate indefinitely, are model systems where scientists can explore their ideas in a reproducible way. However, cell lines have several problems that many scientists are unaware of. Here, we will discuss what CCLs are used for and their common problems.

International Cell Line Authentication Committee
This article is the first of three from members and colleagues of the International Cell Line Authentication Committee (ICLAC) – a group focused on one of the most common problems affecting CCLs. Take a look at the second article called "Choose Wisely - Obtaining Cell Lines from Reliable Sources".

Cross-contamination is an invasion by foreign cells (from the same species or a different species) into a culture. If the foreign cells have a high proliferative potential, they may grow faster than the authentic cells originally present in that culture; in only a few passages the authentic cells will be replaced by the contaminating cells.

Authentication testing compares the culture at the DNA level to other reference samples. If testing is performed routinely, cross-contamination will be detected. But if testing is not done, the problem will persist unchecked in that culture.

The end result of cross-contamination is a false or misidentified cell line; one that the scientist may think represents a particular cell type or disease, but in reality fails to do so. More than 400 false CCLs are known to exist and continue to be used in publications worldwide.

ICLAC is a voluntary committee of scientists working together to address this problem. The committee curates a database of known crosscontaminated, or otherwise misidentified, cell lines [1].

So knowing these cell line problems, are they worth the effort? Why do we use them?

What are Cell Lines Used for?
CCLs are widely used in biomedical research and industry, mainly for cancer modeling and biopharmaceutical production.

The Chinese Hamster Ovary (CHO) cell line is a favorite protein factory.

Subject to intense selection and mutagenic treatment over 60 years, the cell line has given rise to several genomically distinct subclones, e.g., CHO-K1 and DG-44. It is used for vaccine production, which requires safe cell substrates that retain immunogenicity. In addition to CHO, Madin Darby Canine Kidney (MDCK), Vero (monkey), and MRC-5 (human) cells are often used as protein factories. The last cell line, while not immortal, proliferates extensively and is well suited for biopharmaceutical uses.

Cancer CCL panels (e.g., NCI-60) are used to discover new cancer drugs by high throughput testing in vitro. “Intelligent” drugs are now emerging that kill cancer cells bearing targetable mutations, while normal cells are spared. Ones with targetable mutations are of heightened interest as therapeutic models. Examples include the PML/RARA gene fusion specific to acute promyelocytic leukemia (Fig. 2 top panel); and the BCR/ABL gene fusion (Fig. 2 bottom panel) in chronic myeloid leukemia (CML), which is targeted by the tyrosine kinase inhibitor ST1571 [2].

Panels of cancer CCLs are now commonly being sequenced to uncover cancer-causing genetic mutations. An early example is BRAF, discovered in CCLs established from tumors including malignant melanoma, where its presence allows targeted therapy with vemurafenib, dabrafenib, and other agents [3].

What Types of Cell Lines are Available?
Human cancer CCLs comprise the largest group with approximately 10,0000 examples. Most major types are represented; but tumors come in many forms, which microarray profiling has further stratified, leaving many entities unrepresented. Among existing ones, high-grade poor prognosis cancer types are over-represented. For example, from over 40 CML cell lines available, none are from the early (chronic) phase [4] of this disease.

Unfortunately, the pace of establishment has slowed to a trickle. Most human CCLs date from the 1980s or earlier, carrying outdated pathologies and often ambiguous provenances. Most animal cancer cell lines also date from past decades, established from tumors induced in rodents by mutagenesis. There are very few spontaneous tumor CCLs derived from pets which share human lifestyles, or from non-human primates. These would help analyze cancer evolution and regulation in models more relevant than rodents.

There is a significant lack of normal human CCLs from major tissues to serve as controls for high-throughput array and mutation analyses. Hitherto, this obstacle has been circumvented by virus immortalization. However, viruses share oncogenic pathways with cancer cells, compromising their usefulness. It has recently become possible to extend cellular lifespan by transfection with telomerase (hTERT) [5]. Besides normal cells, cells from pre-cancerous conditions such as Barrett´s esophagus have been immortalized with hTERT.

Hence, a surge in CCL establishment is overdue. Is it unrealistic to hope that spurred on by novel methods, scientists will now be motivated to generate them to fill the holes where specific tissue types and cancers have been missed? Both journals and granting agencies must accord a higher priority to “difficult” cell lines establishment for these holes to be filled.

Main Problems: Cross-contamination and Infection by Virus and Mycoplasma
To assure reliable data are obtained from human and animal cell cultures, it is essential to monitor for cross-contamination and bacterial or viral infection.

PCR-based assays are now the methods of choice to detect or – importantly – to document the exclusion of such contaminations. Neglecting quality control can provoke serious consequences, as shown recently by a panel of 13 cell lines modeling tumorigenesis of the esophagus. Ten cell lines were shown to be esophageal, while three originated in reality from lung, stomach, and intestinal tissues [6]. Experiments using the three false cell lines led to eleven patents in the U.S. and more than 100 professional publications worldwide.

To prevent this risk, short tandem repeat (STR) genotyping is recommended to authenticate human cell lines, comparing the results to comprehensive online STR databases [7].

Microbiological contamination is also a significant problem. Here, mycoplasma, other bacteria, yeasts, and viruses represent common adventitious organisms. These organisms can be detected by applying specific PCR methods [8, 9]. Bacterial and yeast infections are usually curable through treatment with the appropriate antimicrobials, and implementation of good cell culture procedures will prevent new contaminations.

Most viral infections originate from the primary material and are species and cell type specific (e.g., Epstein-Barr virus, human papillomavirus). However, some retroviruses have contaminated different species and cell types (e.g., xenotropic murine leukemia viruses) through secondary sources such as xenotransplants or feeder cells [10]. These cell lines must therefore be assessed individually for possible viral contamination with consideration given to species, cell type, patient data, and CCL history.

Overall, detection of contamination is important for risk assessment, particularly at a time when cell cultures are increasingly used for the production of pharmaceuticals and medical applications.

CCLs, though irreplaceable tools in biomedicine, demand awareness regarding the ever present risks of cross-contamination and microbial contamination. Future articles will discuss options for minimising these risks.

Part 3 on Cell Banking can be found here: http://bit.ly/ICLAC-3

References
[1] Masters J.R. et al.: Nature 492, 186 (2012)
[2] Druker B.J. et al.: Nat. Med. 2, 561–6 (1996)
[3] Davies H. et al.: Nature 417, 949–54 (2002)
[4] MacLeod R.A. et al.: Int. J. Cancer 132, 1232–4 (2013)
[5] Ramirez R.D. et al.: Cancer Res. 64, 9027–34 (2004)
[6] Boonstra J.J. et al.: J. Natl. Cancer Inst. 102, 271–4 (2010)
[7] Dirks W.G. et al.: Int. J. Cancer 126, 302–4 (2010)
[8] Uphoff C.C. & Drexler H.G.: Methods Mol. Biol. 946, 1–13 (2013)
[9] Uphoff C.C. et al.: J. Biomed. Biotechnol. 904767 (2010)
[10] Zhang Y.A. et al.: Cancer Biol. Ther. 12, 617–62 (2011)

Contact
Amanda Capes-Davis
acapes-davis@cmri.org.au

Authors

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Children's Medical Research Inst.


Australia

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