Cell-Based Assay Design for High-Content Screening of Drug Candidates

Abstract

To reduce attrition in drug development, it is crucial to consider the development and implementation of translational phenotypic assays as well as decipher diverse molecular mechanisms of action for new molecular entities. High-throughput fluorescence and confocal microscopes with advanced analysis software have simplified the simultaneous identification and quantification of various cellular processes through what is now referred to as high-content screening (HCS). HCS permits automated identification of modifiers of accessible and biologically relevant targets and can thus be used to detect gene interactions or identify toxic pathways of drug candidates to improve drug discovery and development processes. In this review, we summarize several HCS-compatible, biochemical, and molecular biology-driven assays, including immunohistochemistry, RNAi, reporter gene assay, CRISPR-Cas9 system, and protein-protein interactions to assess a variety of cellular processes, including proliferation, morphological changes, protein expression, localization, post-translational modifications, and protein-protein interactions. These cell-based assay methods can be applied to not only 2D cell culture but also 3D cell culture systems in a high-throughput manner.

Advancing in vitro–in vivo toxicity correlations via high‐throughput three‐dimensional primary hepatocyte culture

“…Whereas 2D hepatic cultures quickly lose the ability to express DMEs at biologically relevant levels, 3D hepatic cultures are able to maintain stable DME levels for a matter of weeks . To that end, a 3D microscale cell culture system has been designed for high‐throughput testing of drugs and drug candidates at volumes of <100 nL . This system takes advantage of the expected high biological relevance of 3D culture systems due to cell–cell interactions and controlled matrix effects .…”

Botanicals and Their Bioactive Phytochemicals for Women’s Health

“…The compounds that have been isolated or identified through BGF can also be tested in several other assays including functional cell based assays that generally show less interference with fatty acids or chlorophyll leading to false-positive results. Also, using human cell culture models has the advantage that they might better predict human-compound interactions before preclinical and clinical trials ( Nierode et al, 2016 ). However, often BGF of a botanical extract does not lead to one strongly active compound but to several weakly active compounds due to possible interactive/synergistic processes ( Piersen et al, 2004 ).…”

High content analysis methods enable high throughput nematode discovery screening for viability and movement behavior in a multiplex sample in response to natural product treatment

“…Because the images are a permanent record of what is in each well at the time of image acquisition, we were able to compare manually determined worm phenotypes from the images to machine-determined phenotypes and found a very high level of agreement (Table 3). Based upon the robust statistically significant Z-factor 14, 15 and reproducible EC 50 results obtained for control treatments during our method development, we have concluded that the automated image analysis protocol for nematode high throughput screening described here is robust, reproducible and generates dependable data without variation inherent in manual worm observation (Figure 2 and 3, Tables 1 and 2). Inherent with the automated focus, plate movement and stage movement of HCA instrumentation is the ability to screen large sample libraries in a short time.…”