Cell Culture Collections | Biowire Spring 2011



Biowire Spring 2011 — Cell Lines — Models of Disease

Subscribe to Biowire and eBiowire
Download Biowire Spring 2011 (7.0 Mb PDF)
Biowire Spring 2011 Home



Cell line misidentification and Mycoplasma contamination are well known as the greatest risks to any practitioner of cell culture1,2.

Biological resource centers such as the European Collection of Cell Cultures (ECACC®) exist to ensure that life science researchers in both academia and industry have access to a collection of diverse cellular material of verifiable origin, ensuring the validity and integrity of any studies performed. ECACC’s thousands of human and animal cell lines represent a huge pool of potential biodiversity for research users.

Jim Cooper, General Collection Project Manager
European Collection of Cell Cultures (ECACC) HPA CEPR, Porton Down, Salisbury, Wiltshire UK SP4 0JG

ECACC is a registered trademark of the Health Protection agency in the U.K.

Upon deposition of a cell line at ECACC, a baseline for its identity is set. In the case of human cell lines, this will be through the generation of a forensic single-tandem repeat profile, which is cross-referenced with other cell lines held at ECACC and with those in other collections to ensure genetic uniqueness. During growth and cell banking, the cell line is examined and photo-documented for future reference. Any cells provided to end users are thoroughly tested for viability, ability to be sub-cultivated, and absence of microbial contaminants such as Mycoplasma. These rigorous tests will be repeated on any future cryopreserved banks of the cell line.

It is uncommon for any cell culture collection to confirm specific reported attributes for a particular cell line, due to the variety of specialized characterization techniques required and the fact that a given attribute may rarely, or never, be required by researchers. This presents a problem, as frequency of use is linked to the frequency of citation (Figure 1). Cell line characterization tends to be carried out in the public domain through peer-reviewed research and publication, leading to slow uptake and use of novel cell lines. Therefore, those cell lines that have low rates of publication are languishing, neglected in long-term storage. Furthermore, the most commonly used cell lines form only a small representation of the potential biodiversity available.

ECACC - over 20 human neuronal cell lines 

Figure 1. ECACC has over 20 human neuronal cell lines, but most purchases are represented by the neuroblastoma SH-SY5Y, a cell line that has over 1,700 citations in PubMed (a far greater number than other similar cell lines).

For example, 50% of ECACC’s cell line purchases are represented by only 3% of the total collection. This “iceberg” phenomenon is a concern, particularly in an era where pharmaceutical research is eager for novel, well-characterized cellular reagents. A single generic characterization test could offer a solution; in reality, however, this is an unachievable goal.

Recently, ECACC has made advances in characterizing its catalog by employing automated live cell microscopy to phenotypically characterize cell lines by capturing static and dynamic time-lapse images, growth profile metrics and plating efficiency data. This data is now being released to ECACC’s website and will be of great use to researchers with regard to the cultivation of cells and planning studies. Nevertheless, it does not shed light on the functional characteristics of the cell lines.

Where a cell line has a few key applications, such as Caco-2 in ADMET drug transport studies, specific characterization is still warranted. Thus, ECACC now offers batch-characterized Caco-2 cells (ECACC #09042001) that have been analyzed for the development of microvilli and tight junctions after 21 days seeding in transwells as demonstrated in trans-epithelial electrical resistance (TEER) testing and by electron microscopy (Figure 2).

ECACC: Batch-characterized Caco-2 

Figure 2. New from ECACC: Batch-characterized Caco-2, ECACC product number 09042001.

Microvilli formation Caco-2 culture, batch-characterized Caco-2 cells 

Figure 2A. Scanning Electron Micrograph (SEM) of microvilli formation in Caco-2 (ECACC #09042001) culture. Figure 2B. Transmission Electron Micrograph (TEM) of batch-characterized Caco-2 (ECACC#09042001) cells. (MV — microvilli; TJ — tight junction; LM —lateral membrane).

For the last 20 years, pharmaceutical research relied on the use of high-throughput screens (HTS ) and recombinant cell lines (such as CHO and HEK-293) overexpressing markers such as G-protein-coupled receptors (GPCRs) for much of its drug discovery pipeline. However, HTS has not delivered to expectation, and with the development of novel label-free screening instrumentation there is increasing interest in the endogenous receptor expression in native cell lines. Data on endogenous receptor expression in the public domain is limited3. With this specific need in mind, ECACC has embarked on a project to characterize cell lines for their endogenous receptors using a receptor panning technique and combining cellular dielectric spectroscopy (Molecular Devices CellKey® System) with Sigma® Life Science’s LOPAC™ 1280 library of 1280 pharmacologically active compounds. This novel label-free approach enables the identification of receptor signaling for real-time cell-based functional analysis of endogenous and recombinant receptors, allowing measurement of a wide range of targets in a single assay. This includes all families of GPCRs, as well as tyrosine kinase receptors, adhesion molecules, and indirect measurement of ion channel activation, overcoming many limitations of traditional assay formats. Initial experiments — subsequently supported by intracellular calcium flux (FLIPR® Calcium 5 Assay Kit) and cyclic AMP (CatchPoint cAMP Assay Kit) assays, carried out on a human astrocytoma cell line (132N1 ECACC#86030402) — confirm the presence of endogenous histamine, cholinergic M3, and a hitherto poorly reported beta-2-adrenergic receptor. There is also data to imply the presence of an adenosine receptor (Figure 3). In 2011, ECACC will be exploring more cell lines using this approach and employing the technique to QC-release custom assay ready-cryopreserved banks of cells for HTS .

Cellular Dielectric Spectroscopy Hit Analysis of the Human Astrocytoma Cell Line 132N1 ECACC#86030402 

Figure 3. Cellular Dielectric Spectroscopy Hit Analysis of the Human Astrocytoma Cell Line 132N1 ECACC#86030402.
By combining the Sigma Life Science LOPAC library and cellular dielectric spectroscopy (CellKey® System), we have been able to analyze the functional endogenous receptor expression of this cell line. Data here is from a duplicate set of exposures of the cell line to the LOPAC library. The Y axis labeled “% Hits” is a measure of the percentage of compounds specific for a particular receptor recognized as a hit by the CellKey System in both exposures. With positive hits from cholinergic, histamine, and adrenergic agonists, we confirmed the presence and function of their receptors. The kinetic traces of changes in impedance obtained indicate the coupling mechanism of the receptor — Gq (calcium coupled) in the case of the cholinergic and histamine receptors and Gs (cAMP) for the adrenergic receptor (this inference was subsequently verified by intracellular calcium flux [FLIPR® Calcium 5 Assay Kit] and cyclic AMP [CatchPoint cAMP Assay Kit] assays). Hit data obtained from cytoskeleton and extracellular matrix (ECM) inhibitors are included to demonstrate that, although positive hits can be seen from such compounds, the profile of impedance change indicates that these are not likely to be signals coupled through receptors, but artifacts of cellular trauma induced by these often-toxic compounds. The hit data from the adenosine agonists indicate that there may be a receptor present; however, this has yet to be verified and requires more investigation.

The functional analysis described above will be of interest to pharmaceutical small-molecule drug discovery, which represents a significant sector of the market for cell lines; however, there are many more areas of research in which cell culture is essential. At ECACC, we feel that transcriptomics is the approach that holds most promise to deliver characterization data relevant to all of these diverse areas of study. ECACC is in the process of delivering a project to generate panels of cDNA derived from tightly controlled cultures of cell lines. RNA will be extracted from the cell lines and reverse-transcribed to cDNA (Figure 4). Quantified, normalized, and quality-controlled cDNA from many cell lines will be presented as panels in multi-well format representing, for example, cell type, species, tissue of origin, etc. This novel reagent will allow researchers to interrogate many cell lines for expression products of interest, such as endogenously expressed receptors or intracellular proteins using techniques such as RT PCR. Prior to embarking on detailed cell culture experiments, end users will be able to rapidly verify reported characteristics of cell lines in their specific field of interest, as well as to determine if other similar cell lines that have been hitherto less well investigated share those characteristics, or show potential to serve as more appropriate models or better naturally occurring knockout models.

cDNA Panels of ECACC Cell Lines 

Figure 4. cDNA Panels of ECACC Cell Lines. Many cell lines will be available, grouped into panels, in the form of cDNAs, in multi-well format.

back to top Back to Top

Acknowledgements

Diane Fellows and Jamie Taylor (ECACC) for their work in the receptor characterization of 1321N1.

Dr. Peter Thraves (ECACC) for his work in characterizing Caco-2 cells.

Simon Lydford and the team at Molecular Devices for the use of the CellKey® System and support with pharmacology and data interpretation.

back to top Back to Top

About the Author

With a background in animal physiology and clinical immunology, Jim Cooper has over 20 years’ experience working with human and animal cells on a variety of scales and for many applications. He uses his wealth of experience in the management of internal and external customer-driven projects in the HPA and plays a key role in business development within ECACC.

back to top Back to Top

References

  1. MacLeod RAF, et al. Widespread intraspecies cross-contamination of human tumor cell lines arising at source. Int J Cancer. 1999;83(4):555–63.
  2. Capes-Davies A, et al. Check your cultures! A list of cross-contaminated or misidentified cell lines. Int J Cancer. 2010;127(1):1–8.
  3. Schonbrunn A, Steffen D. Endogenous GPCR List. Biomedical Computing, Inc. 2 Mar 2008. 28 Jan 2011 www.biomedcomp.com/GPCR.html.

back to Biowire main page

back to top Back to Top

Related Links