Abstract
The impressive advances in the generation and interpretation of functional omics data have greatly contributed to a better understanding of the (patho-)physiology of many biological systems and led to a massive increase in the number of specific targets and phenotypes to investigate in both basic and applied research. The obvious complexity revealed by these studies represents a major challenge to the research community and asks for improved target characterisation strategies with the help of reliable, high-quality assays. Thus, the use of living cells has become an integral part of many research activities because the cellular context more closely represents target-specific interrelations and activity patterns. Although still predominant, the use of traditional two-dimensional (2D) monolayer cell culture models has been gradually complemented by studies based on three-dimensional (3D) spheroid (Sutherland 1988) and other 3D tissue culture systems (Santos et al. 2012; Matsusaki et al. 2014) in an attempt to employ model systems more closely representing the microenvironment of cells in the body. Hence, quite a variety of state-of-the-art cell culture models are available for the generation of novel chemical probes or the identification of starting points for drug development in translational research and pharma drug discovery. In order to cope with these information-rich formats and their increasing technical complexity, cell-based assay development has become a scientific research topic in its own right and is used to ensure the provision of significant, reliable and high-quality data outlasting any discussions related to the current “irreproducibility epidemic” (Dolgin 2014; Prinz et al. 2011; Schatz 2014). At the same time the use of cells in microplate assay formats has become state of the art and greatly facilitates rigorous cell-based assay development by providing the researcher with the opportunity to address the multitude of factors affecting the actual assay results in a systematic fashion and a timely manner. This microplate-based assay development strategy should result in the setting up of more robust and reliable test systems that ensure and increase the confidence in the statistical significance of the actual data generated. And, although assay miniaturisation is essential in order to achieve this, most, if not all, cell-based assays can be easily reformatted and adapted to be used in this format in a straightforward manner. This synopsis aims at summarising valuable, general observations made when implementing a diverse set of functional cellular in vitro assays at Bayer Pharma AG without claiming to deeply review all of the literature available in each and every detail. In addition, phenotypic assays (Moffat et al. 2014) or label-free detection methods (Minor 2008) are not discussed. Although this essay tries to cover the most relevant technological developments in the field, it nevertheless may express personal preferences and peculiarities of the author’s approach to state-of-the-art cell-based assay development. For additional reviews covering the actual field, see Wunder et al. (2008) and Michelini et al. (2010).
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That is, the 96-well plate described by Gyula Takátsy in 1951 already
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Aside from the growing public debate over the disproportionate use of experimental animals in certain research areas
- 3.
See: Foundation of the Society for Biomolecular Sciences (SBS) in 1994 (http://www.slas.org/about/who-we-are/society-for-biomolecular-sciences/) now an integral part of the Society for Laboratory Automation and Screening (SLAS) established in 2010 (https://www.slas.org/).
- 4.
As exemplified by the foundation of (a) the NIH Chemical Genomic Center (NCGC) in 2004, now a part of the National Center For Advancing Translational Science (NCATS; established 2012; http://www.ncats.nih.gov/research/reengineering/ncgc/ncgc.html) or (b) the Innovative Medicines Initiative (IMI; http://www.imi.europa.eu/content/home) established in 2008 as well as (c) the establishment of the National Cancer Institute Chemical Genomics Consortium in 2009 (http://dctd.cancer.gov/CurrentResearch/cbc/20090810_meeting.htm)
- 5.
See Freshney (2000b).
- 6.
The term frozen cell describes the use of freshly resuscitated cells shortly after their recovery from liquid nitrogen storage and should not to be mistaken for the use of division-arrested cells, as exemplified/described by Digan et al. (2005).
- 7.
Demanding for the supply of 1–5 × 108 cells per screening day
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From routine pharmacological profiling on a weekly basis to approaches based on frozen cells transiently transfected with mutant receptors to test hypotheses from computational chemistry
- 9.
That is, a medium version based on charcoal-treated serum, medium only or even just PBS
- 10.
For chloramphenicol acetyltransferase (CAT), see Devinoy et al. (1991).
- 11.
For secreted alkaline phosphatase (SEAP), see: Cullen and Malim (1992).
- 12.
For secreted urokinase, see Langer et al. (1995).
- 13.
For a discussion of FRET artefacts and signal proper, see (Vogel et al. 2006).
- 14.
For a thorough examination of potential BRET-related artefacts, see Marullo and Bouvier (2007).
- 15.
That is, from milliseconds to seconds and from a few minutes to a few hours
- 16.
A process called complementation that has been reported in 1965 already, see Ullmann et al. (1965).
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- 19.
For dissociation-enhanced lanthanide fluorescent immunoassays (Delfia) marketed by PerkinElmer, see http://www.perkinelmer.com/catalog/category/id/delfia%20trf%20assays%20and%20reagents.
- 20.
The use of lanthanide cryptates is called HTRF (homogeneous, time-resolved FRET) and marketed by the French company Cisbio http://www.cisbio.com/other.
- 21.
For more details, see http://www.cisbio.com/other/drug-discovery as well as http://www.perkinelmer.com.
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- 30.
That is, average signal intensity, which can be the product of multiple sub-parameters of course
- 31.
In a sense permitting population studies otherwise known from fluorescence-activated cell sorter (FACS) analysis
- 32.
Molecular Probes® Handbook – A Guide to Fluorescent Probes and Labeling Technologies; the printed version is available free of charge via http://www.lifetechnologies.com/de/de/home/references/molecular-probes-the-html.
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Acknowledgements
The author thanks B. Bader, U. Nguyen, K. Parczyk, P. Steigemann and S. Prechtl for critical reading and helpful comments while preparing the manuscript.
Competing Financial Interests The author is an employee of Bayer Pharma AG.
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Langer, G. (2015). Implementation and Use of State-of-the-Art, Cell-Based In Vitro Assays. In: Nielsch, U., Fuhrmann, U., Jaroch, S. (eds) New Approaches to Drug Discovery. Handbook of Experimental Pharmacology, vol 232. Springer, Cham. https://doi.org/10.1007/164_2015_18
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