Genetic Engineering & Biotechnology News

AUG 2017

Genetic Engineering & Biotechnology News (GEN) is the world's most widely read biotech publication. It provides the R&D community with critical information on the tools, technologies, and trends that drive the biotech industry.

Issue link:

Contents of this Issue


Page 25 of 37

24 | AUGUST 2017 | | Genetic Engineering & Biotechnology News Patrick Schneider, Ph.D. The contamination of cell lines by other cell lines or by microbes is a plague on the biomedical research community. 1 Cell-line misidentification was first acknowledged nearly half a century ago upon the discovery of HeLa contamination of 18 cell lines. 2 The problem persists to this day, as an estimated 15–20% of all cell lines used in biomedical research continue to be misidentified. 3 A PubMed query of five cell lines identified in the 1960s as HeLa-contaminated (KB, HEp- 2, Chang liver, Int-407, and WISH) found nearly 400 citations between March 2009 and February 2014. Despite peer review, many of the published articles still described these cell lines as "normal" human cells, not cervical cancer cells. The reproducibility of research findings is greatly impacted by contaminated cells, but the influence of contamination extends far beyond the laboratory bench. For example, results generated from the use of misidenti- fied esophageal cell lines led to at least three NIH grants, 100+ scientific publications, 11 U.S. patents, and patient recruitment for clinical trials. 4 Moreover, these events could also damage patients' and the public's trust in scientific research. Microbial contamination of cell lines pos- es a serious challenge to obtaining reliable research data. The most dangerous of micro- bial contaminants are mycoplasma. These microbes cause no discernible change in turbidity or pH even at high concentrations. Mycoplasma can induce abnormal behavior in cultured cells, including altered growth rates, morphological changes, chromosomal aberrations, and altered cell metabolism. Contamination is difficult to control because mycoplasma lack cell walls and thus cannot be treated with most antibiotics. 5 If left un- checked, mycoplasma can contaminate an entire operation or facility. Cell-Line Integrity Asking a few simple questions before purchasing cell lines from a vendor can help ensure that the cells are authentic and free of contamination (Figure 1). A reputable ven- dor will be able to adequately address each of the following questions: 1. Is the cell line authenticated? 2. Is the cell line found in the misidentified cell-line database (that is, in the Database of Cross-Contaminated or Misidentified Cell Lines, which is compiled by the International Cell Line Authentication Committee [ICLAC])? 3. Is the short tandem repeat (STR) profile known? Does the vendor supply a Certificate of Analysis (CofA) showing the STR profile of a cell? 4. Has every batch been tested for mycoplasma? 5. Does the vendor supply references and journal citations for the cell lines? 6. Is technical support available? Will spe- cialists answer specific questions about cell-line properties and identification? The vendor should also demonstrate that upfront measures have been performed to validate cell-line quality and health. Estab- lished cell repositories such as the European Collection of Authenticated Cell Cultures (ECACC) and the American Type Culture Collection (ATCC) have already performed extensive tests on the cell lines in their librar- ies; thus, purchasing cells from these organi- zations is secure. Validation Techniques The consistent application of validation methods and proper laboratory techniques are necessary for maintaining reliable cell cultures upon receipt from a vendor. Estab- lishing a laboratory-wide plan of action for when contamination is detected will mini- mize research disruptions. Mycoplasma testing should be performed weekly to monthly, and cell-line authentica- tion should be performed before the first use, every six months to one year, and when a mix-up is suspected. If cell lines are obtained from a colleague (this is not recommended), they must be tested for authenticity and the presence of mycoplasma before use. The more reliable route is to purchase cell lines from a reputable repository, such as ECACC or ATCC. Mycoplasma Detection Low levels of mycoplasma contamination are difficult to detect and may require two or more methods for detection. Mycoplasma Culture: Mycoplasma cul- ture is a standard method of detection. The FDA/European Pharmacopeia-approved protocol, the most sensitive method, relies on a selective and highly enriched growth medi- um on standard agar plates. Resulting colo- nies have a distinctive "fried egg" appear- ance, and a positive result using this method is conclusive proof of mycoplasma contami- nation. However, because this method does not detect all species, such as Mycoplasma hyorhinis, DNA staining and/or polymerase chain reaction (PCR) analysis are required to assure the absence of contamination. DNA Staining: DNA staining relies on the Hoechst 33258 stain, which causes DNA-rich nuclei and any mycoplasma in the cytoplasm to fluoresce. Mycoplasma and mitochondria are easily differentiated because mycoplasma has 10 times the DNA content, and thus their fluorescence is much brighter. Commercial kits for DNA staining are available. Using this method, false positives can be caused by cell detritus or cells undergoing apoptosis. False negatives are also possible, as this is the least sensitive method. Unlike the culture method, however, DNA staining will detect all types of mycoplasma. PCR: PCR-based mycoplasma detection can be very sensitive, capable of detecting as few as 20 copies of a mycoplasma genome within a 2-μL sample. Mycoplasma detection is achieved by a primer/probe system that am- plifies the highly conserved 16S rRNA operon coding region of the mycoplasma genome. PCR detection is effective for 19 species of mycoplasma, including M. hyorhinis, which is not detectable by the culture method. If a lab routinely performs PCR, a commercial kit will work well. If not, several services are available to perform PCR-based mycoplasma testing, for a fee. Cell-Line Authentication In 2012, ECACC cofounded ICLAC. ICLAC members include representatives from the ECACC and other international cell culture organizations, as well as respected scientific researchers committed to champi- oning the importance of cell-line verification. The ICLAC maintains a searchable database of cross-contaminated/misidentified cell lines. The comprehensive ICLAC database is up- dated periodically and serves as a valuable re- source to the research community, but retrac- tions do occur. After searching the database, it is also worth querying Google for the cell line name and the keyword "retraction." Short Tandem Repeat Profiling (DNA Fingerprinting) A short tandem repeats (STRs) is a repeat- ing unit of two to six nucleotides in a DNA sequence. The number of STRs at any locus is highly variable within the human popula- tion, and these variations are heritable. The ECACC, the ATCC, and other cell repositories use the ASN-0002-2011 Standard Method based on the Combined DNA Index System Best Practices for Validing, Storing, and Handling Cell Culture Uphold Research Integrity Addressing Cell-Line Contamination to Improve Data Reproducibility Bioprocessing Tutorial Figure 1. The steps that should be taken to ensure cell-line authentication vary depending on how cell lines are sourced. Ideally, cell lines should be obtained from established repositories, such as those maintained by The European Collection of Authenticated Cell Cultures (ECACC). ICLAC = International Cell Line Authentication Committee; STR = short tandem repeat.

Articles in this issue

Links on this page

Archives of this issue

view archives of Genetic Engineering & Biotechnology News - AUG 2017