ICLAC: Obtaining Cell Lines from Reliable Sources

  • Tab. 1: Cell Repositories Performing Comparative Authentication: The organizations listed are Biological Resource Centers (BRCs) that operate as cell line repositories with high standards of quality control. Human cell lines are tested using STR profiling, with comparison to consensus datasets and sharing of data to resolve cases where authenticity may be difficult to determine.Tab. 1: Cell Repositories Performing Comparative Authentication: The organizations listed are Biological Resource Centers (BRCs) that operate as cell line repositories with high standards of quality control. Human cell lines are tested using STR profiling, with comparison to consensus datasets and sharing of data to resolve cases where authenticity may be difficult to determine.
  • Tab. 1: Cell Repositories Performing Comparative Authentication: The organizations listed are Biological Resource Centers (BRCs) that operate as cell line repositories with high standards of quality control. Human cell lines are tested using STR profiling, with comparison to consensus datasets and sharing of data to resolve cases where authenticity may be difficult to determine.
  • Fig. 1: Configuration of STR Profiling Used by Major Cell Repositories. STR markers generally contain short sequences (2-4 bases) arranged in tandem, 3-40 repeats long. Alleles are labelled according to the number of repeats (e.g. STR locus D5S818 has alleles 3,6 present in the example). Consensus STR loci (D5S818, D13S317, D7S820, D16S539, vWA, THO1, TPOX and FGA) are located on the chromosomes shown. Fluorescent PCR primers are annealed to unique flanking sequences for each STR locus, producing fluorescent DNA fragments that are analysed using capillary electrophoresis. Amelogenin gender identification is also commonly performed in conjunction with STR typing, using PCR products generated from the amelogenin gene on both the X- and Y-chromosome (which differ in size by 6 bp).

ICLAC - the International Cell Line Authentication Committee -presents here its second article. In the first article (published G.I.T. Laboratory Journal Europe 1-2/2014, p. 12 and online here), we discussed why quality is important for cell lines used in research laboratories. But where can you obtain good quality cell lines? And how can you keep good quality lab stocks? In the present article, we discuss the advantages of obtaining cell lines from a cell repository, the authentication testing that repositories perform, and the reference datasets from those repositories that labs can now access.

Where Should I Get My Cell Line?
Cell lines are readily available from a variety of sources; however, some sources are more trustworthy than others. Laboratories often maintain their own personal bank of cell lines, and some are happy to share cell cultures with their colleagues. Though obtaining cells from a neighbouring lab, or from the lab that originally described the stock, may seem like an easy and cost-effective method of setting up an experiment, there are inherent risks to working with untested materials. An unacceptably high percentage of new cell lines obtained from originators may be cross-contaminated by other cell lines, as it is unlikely that thorough authentication testing will be performed prior to depositing at a cell bank. It is important to quarantine all cell cultures coming into a laboratory until tested for microbial contamination and misidentification.

Training and experience levels in the laboratory setting may run the gamut from newly-minted graduate student to highly-experienced veteran technician. The quality of their contributions to the laboratory's personal cell bank will likely match their level of experience. Additionally, since individual labs are not required to adhere to a standardized method of book-keeping, the quality of the records may change with laboratory personnel. Thus, there is no reliable way to ensure that the cells coming from a lab are free from contamination and misidentification without spending the time and money to test the material yourself.

Do not distribute cell lines from your own laboratory if they can be obtained from a Biological Resource Center (BRC) operating as a cell repository with high standards of quality control (Tab.

1). Qualified BRCs specialize in the authentication, production, preservation, and secure distribution of biological materials. Additionally, BRCs often hold ISO quality certifications, which demonstrate an institutional commitment to quality products, customer service, and continued improvement.

Investigators who obtain their stocks from a qualified BRC know that their cell line is tested for misidentification and microbial contamination, handled using procedures that promote high post-thaw recovery rates, and supported by the institution from which it was received.

If you receive cell lines from colleagues, collaborators, or other sources such as unauthorized "pirate" resellers of repository cell lines, there are always questions regarding the identity and quality of the cells. In particular, laboratories should be wary of purchasing from "unauthorized resellers" of cell lines because they may have been obtained without the permission or knowledge of the cell line originator or BRC and may be in breach of material transfer agreements.

Guidance for Young Scientists is Essential
Contamination and misidentification of cell cultures remain global problems. Mitigation of these problems requires a consistent laboratory quality control program and the acquisition of authenticated cell lines from recognized BRCs or trusted sources.

Though cell lines obtained from a cell repository may start out free from contamination, this can quickly change if the end-user does not practice good culture techniques. Microbial contamination spreads easily once it is introduced to the environment, so one poorly trained culturist can undo the good work of the entire laboratory. Good technique, however, is nearly impossible to develop on one's own; it must be learned through active mentorship.

The best method of passing on good technique is for an experienced cell culturist to sit with the novice as they work, so that any improper technique can be explained and corrected. This way, the experienced culturist not only passes on their valuable culture skills, they also prevent accidental contamination and protect the entire lab's culture supply.

Cell Repositories Perform Comparative Authentication Using Consensus Reference STR Profile Datasets
Cross-contaminated cell lines are used at an unacceptably high frequency. One reason for this is the failure of the global scientific community to tackle the problem from the beginning, using quality standards for identity. Early biochemical and cytogenetic approaches for tracking cell line identities were implemented at the first culture collection, but unfortunately, later established BRCs used mutually incompatible methods for data generation. Recent efforts to implement a global standard for the authentication of human cell lines using short tandem repeat (STR) profiling (Fig. 1) has been a key step to address this problem [1]. Today, all major BRCs worldwide use STR profiling for the identification and authentication of human cell lines [2, 3].

Ideally, authentication involves STR profiling of both donor and derived cell line samples in parallel. This requirement is met by only a few recently established cell lines. Most widely used classical tumor cell lines are decades old; their identification is largely retrospective and multidisciplinary, combining diverse immunological, cytogenetic, and molecular criteria. But how to unmask cross-contaminated cell lines established from the same tissues or genetic diseases? Comparison to other stocks of the same cell line offers a solution.

ATCC, DSMZ, JCRB, and RIKEN have merged their large databases of STR cell line profiles, obtained using compatible loci, as a tool to uncover discrepant STR profiles of shared human cell lines [4]. False negative matches were initially seen due to discrepant representation of single STR alleles by losses of heterozygosity (LOH) or microsatellite instability (MSI). Once addressed, the merged STR datasets allowed for the design of consensus STR reference databases. These offer a new tool for the cell biology community, fostering reproducibility and comparability of cell lines used in different laboratories.

ICLAC is now working towards a global interactive database for STR profiles of human cell lines [1, 2]. Inevitably, equivocal STR reference profiles remain subject to revision until all commonly held cell lines have been compared by STR profiling. We exhort labs to use these databases as resources to ensure that lab stocks are not misidentified.

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

References
[1] American Type Culture Collection Standards Development Organization Workgroup ASN-0002: Nat. Rev. Cancer 10, 441-8 (2010)
[2] ANSI/ATCC ASN-0002-2011 Authentication of Human Cell Lines: Standardization of STR Profiling. ANSI eStandards Store (2012)
[3] Masters J.R. et al.: Proc. Natl. Acad. Sci. 98, 8012-7 (2001)
[4] Dirks W.G. et al.: Int. J. Cancer 126, 302-4 (2010)

Authors
Wilhelm G. Dirks1, Liz Kerrigan2, Carolyn Peluso2, Cara Wilder2
1Leibniz-Institute, Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), Braunschweig, Germany
2American Type Culture Collection (ATCC), Manassas VA, USA

 

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