Query: high content screening

title: High throughput isolation of giant viruses using high content screening
abstract: The race to discover and isolate giant viruses began 15 years ago. Metagenomics is counterbalancing coculture, with the detection of giant virus genomes becoming faster as sequencing technologies develop. Since the discovery of giant viruses, many efforts have been made to improve methods for coculturing amebas and giant viruses, which remains the key engine of isolation of these microorganisms. However, these techniques still lack the proper tools for high speed detection. In this paper, we present advances in the isolation of giant viruses. A new strategy was developed using a high throughput microscope for real time monitoring of cocultures using optimized algorithms targeting infected amebas. After validating the strategy, we adapted a new tabletop scanning electron microscope for high speed identification of giant viruses directly from culture. The speed and isolation rate of this strategy has raised the coculture to almost the same level as sequencing techniques in terms of detection speed and sensitivity. Francis et al. develop a new strategy for the identification and isolation of giant viruses in real time by using optimized algorithms targeting infected amebas in co cultures. This new approach, which does not require sequencing techniques, is highly sensitive and 100 fold faster than other high throughput procedures.

title: High Content Screening in hPSC Neural Progenitors Identifies Drug Candidates that Inhibit Zika Virus Infection in Fetal like Organoids and Adult Brain.
abstract: Zika virus (ZIKV) infects fetal and adult human brain and is associated with serious neurological complications. To date, no therapeutic treatment is available to treat ZIKV infected patients. We performed a high content chemical screen using human pluripotent stem cell derived cortical neural progenitor cells (hNPCs) and found that hippeastrine hydrobromide (HH) and amodiaquine dihydrochloride dihydrate (AQ) can inhibit ZIKV infection in hNPCs. Further validation showed that HH also rescues ZIKV induced growth and differentiation defects in hNPCs and human fetal like forebrain organoids. Finally, HH and AQ inhibit ZIKV infection in adult mouse brain in vivo. Strikingly, HH suppresses viral propagation when administered to adult mice with active ZIKV infection, highlighting its therapeutic potential. Our approach highlights the power of stem cell based screens and validation in human forebrain organoids and mouse models in identifying drug candidates for treating ZIKV infection and related neurological complications in fetal and adult patients.

title: Scalable Production of iPSC Derived Human Neurons to Identify Tau Lowering Compounds by High Content Screening
abstract: Lowering total tau levels is an attractive therapeutic strategy for Alzheimer's disease and other tauopathies. High throughput screening in neurons derived from human induced pluripotent stem cells (iPSCs) is a powerful tool to identify tau targeted therapeutics. However, such screens have been hampered by heterogeneous neuronal production, high cost and low yield, and multi step differentiation procedures. We engineered an isogenic iPSC line that harbors an inducible neurogenin 2 transgene, a transcription factor that rapidly converts iPSCs to neurons, integrated at the AAVS1 locus. Using a simplified two step protocol, we differentiated these iPSCs into cortical glutamatergic neurons with minimal well to well variability. We developed a robust high content screening assay to identify tau lowering compounds in LOPAC and identified adrenergic receptors agonists as a class of compounds that reduce endogenous human tau. These techniques enable the use of human neurons for high throughput screening of drugs to treat neurodegenerative disease.

title: A novel multi parametric high content screening assay in ciPTEC OAT1 to predict drug induced nephrotoxicity during drug discovery
abstract: Drug induced nephrotoxicity is a major concern in the clinic and hampers the use of available treatments as well as the development of innovative medicines. It is typically discovered late during drug development, which reflects a lack of in vitro nephrotoxicity assays available that can be employed readily in early drug discovery, to identify and hence steer away from the risk. Here, we report the development of a high content screening assay in ciPTEC OAT1, a proximal tubular cell line that expresses several relevant renal transporters, using five fluorescent dyes to quantify cell health parameters. We used a validation set of 62 drugs, tested across a relevant concentration range compared to their exposure in humans, to develop a model that integrates multi parametric data and drug exposure information, which identified most proximal tubular toxic drugs tested (sensitivity 75% without any false positives (specificity 100% Due to the relatively high throughput (straight forward assay protocol, 96 well format, cost effective) the assay is compatible with the needs in the early drug discovery setting to enable identification, quantification and subsequent mitigation of the risk for nephrotoxicity.

title: Bioprinted pluripotent stem cell derived kidney organoids provide opportunities for high content screening
abstract: Recent advances in the directed differentiation of human pluripotent stem cells to kidney brings with it the prospect of drug screening and disease modelling using patient derived stem cell lines. Development of such an approach for high content screening will require substantial quality control and improvements in throughput. Here we demonstrate the use of the NovoGen MMX 3D bioprinter for the generation of highly reproducible kidney organoids from as few as 4,000 cells. Histological and immunohistochemical analyses confirmed the presence of renal epithelium, glomeruli, stroma and endothelium, while single cell RNAseq revealed equivalence to the cell clusters present within previously described organoids. The process is highly reproducible, rapid and transferable between cell lines, including genetically engineered reporter lines. We also demonstrate the capacity to bioprint organoids in a 96 well format and screen for response to doxorubicin toxicity as a proof of concept for high content compound screening.

title: Localization based super resolution imaging meets high content screening
abstract: Single molecule localization microscopy techniques have proven to be essential tools for quantitatively monitoring biological processes at unprecedented spatial resolution. However, these techniques are very low throughput and are not yet compatible with fully automated, multiparametric cellular assays. This shortcoming is primarily due to the huge amount of data generated during imaging and the lack of software for automation and dedicated data mining. We describe an automated quantitative single molecule based super resolution methodology that operates in standard multiwell plates and uses analysis based on high content screening and data mining software. The workflow is compatible with fixed and live cell imaging and allows extraction of quantitative data like fluorophore photophysics, protein clustering or dynamic behavior of biomolecules. We demonstrate that the method is compatible with high content screening using 3D dSTORM and DNA PAINT based super resolution microscopy as well as single particle tracking.

title: High Content Screening for Quantitative Cell Biology.
abstract: High content screening (HCS) which combines automated fluorescence microscopy with quantitative image analysis, allows the acquisition of unbiased multiparametric data at the single cell level. This approach has been used to address diverse biological questions and identify a plethora of quantitative phenotypes of varying complexity in numerous different model systems. Here, we describe some recent applications of HCS, ranging from the identification of genes required for specific biological processes to the characterization of genetic interactions. We review the steps involved in the design of useful biological assays and automated image analysis, and describe major challenges associated with each. Additionally, we highlight emerging technologies and future challenges, and discuss how the field of HCS might be enhanced in the future.

title: Live Cell High Content Screening in Drug Development.
abstract: In the past decade, automated microscopy has become an important tool for the drug discovery and development process. The establishment of imaging modalities as screening tools depended on technological breakthroughs in the domain of automated microscopy and automated image analysis. These types of assays are often referred to as high content screening or high content analysis (HCS/HCA) The driving force to adopt imaging for drug development is the quantity and quality of cellular information that can be collected and the enhanced physiological relevance of cellular screening compared to biochemical screening. Most imaging in drug development is performed on fixed cells as this allows uncoupling the preparation of the cells from the acquisition of the images. Live cell imaging is technically challenging, but is very useful for many aspects of the drug development pipeline such as kinetic studies of compound mode of action or to analyze the motion of cellular components. Most vendors of HCS microscopy systems offer the option of environmental chambers and onboard pipetting on their platforms. This reflects the wish and desire of many customers to have the ability to perform live cell assays on their HCS automated microscopes. This book chapter summarizes the challenges and advantages of live cell imaging in drug discovery. Examples of applications are presented and the motivation to perform these assays in kinetic mode is discussed.

title: Filling the drug discovery gap: is high content screening the missing link?
abstract: In recent years, questions about the sustainability of the current drug discovery process have triggered a revival of interest in phenotypic drug discovery approaches. This trend has clearly been amplified by the emergence of multiple cell based assay technologies enabling a higher degree of translatability between in vitro conditions and physio pathological situations, including induced pluripotent stem cells, three dimensional models, co culture and organ on a chip systems, complemented by advances in gene editing technologies. Progress in High Content Screening technology has also contributed to the recent excitement for phenotypic drug discovery approaches, bringing image capture and processing, and data analysis, to a level of content and throughput fully compatible with large scale drug discovery efforts. Nevertheless, implementation of HCS in discovery projects must be carefully considered, to ensure optimal performance and the generation of relevant data to enable the discovery of first in class medicines.

title: Microfluidic systems for high throughput and high content screening using the nematode Caenorhabditis elegans.
abstract: In a typical high throughput drug screening (HTS) process, up to millions of chemical compounds are applied to cells cultured in well plates, aiming to find molecules that exhibit a robust dose response, as evidenced for example by a fluorescence signal. In high content screening (HCS) one goes a step further by linking the tested compounds to phenotypic information, obtained, for instance, from microscopic cell images, thereby creating richer data sets that also require more advanced analysis methods. The nematode Caenorhabditis elegans came into the screening picture due to the wide availability of its mutants and human disease models, its relatively easy culture and short life cycle. Being a whole organism model, it allows drug testing under physiological conditions at multi tissue levels and provides additional observable phenotypes with respect to cell models, related, for instance, to development, aging, behavior or motility. Worm based HTS studies in liquid environments on microwell plates have been demonstrated, while microfluidic devices allowed surpassing the performance of plates by enabling more versatile and accurate assays, precise and dynamic dosing of compounds, and readouts down to single animal resolution. In this review, we discuss microfluidic devices for C. elegans analysis and related studies, published in the period from 2012 to 2017. After an introduction to the different screening approaches, we first focus on microfluidic systems with potential for screening applications. Various enabling technologies, e.g. electrophysiological on chip recordings or laser axotomy, have been implemented, as well as techniques for reversible worm immobilization and high resolution imaging, combined with algorithms for automated experimentation and analysis. Several devices for developmental or behavioral assays, and worm sorting based on different phenotypes, have been proposed too. In a subsequent section, we review the application of microfluidic based systems for medium and high throughput screens, including neurobiology and neurodegeneration studies, aging and developmental assays, toxicity and pathogenesis screens, as well as behavioral and motility assays. A thorough analysis of this work reveals a trend towards microfluidic systems more and more capable of offering high quality analyses of large worm populations, based on multi phenotypic and/or longitudinal readouts, with clear potential for their application in larger HTS/HCS contexts.