Implementation and Use of State-of-the-Art, Cell-Based In Vitro Assays

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...

Bioengineered 3D Glial Cell Culture Systems and Applications for Neurodegeneration and Neuroinflammation

“…Several groups have now performed HTS and high content imaging (HCI) screens in various 3D culture formats, largely in non-CNS contexts. [176][177][178][179][180][181][182][183] It is now possible to clearly envision taking similar approaches to identify new compounds and targets for neurodegeneration and neuroinflammation. We are thus now in the exciting early stages of being able to screen disease-relevant 3D CNS systems.…”

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“…Assay formats that have enhanced the capabilities relative to phenotypic assays include label-free impedance-based [39, 40] dynamic mass redistribution [41, 42] and multiplex assays [43, 44], and these have been successfully applied in screening against small-molecule libraries. More recent state-of-the-art screening compatible assays use three-dimensional spheroids that offer the potential to represent the microenvironment of cells in the body [45].

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Discovery and Characterization of BAY 1214784, an Orally Available Spiroindoline Derivative Acting as a Potent and Selective Antagonist of the Human Gonadotropin-Releasing Hormone Receptor as Proven in a First-In-Human Study in Postmenopausal Women

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“…N- 13 Frozen Cell Assays. 59 All cell lines used were routinely monitored for the presence of mycoplasma and shown to be free of any contamination. Two assay procedures were used for compound testing which differed in the time required for functional recovery of the frozen cells at 37 °C (i.e., [a] a one-day protocol based on a short, 1 h functional recovery period in the case of hGnRH-R and cGnRH-R cells or [b] a two-day protocol employing overnight incubation of the rGnRH-R cells to achieve functional recovery; see the individual assay protocols below).…”