High-Throughput Screening of Encapsulated Islets Using Wide-Field Lens-Free On-Chip Imaging

On-chip imaging of intact three-dimensional tissues within microfluidic devices is fundamentally hindered by intratissue optical scattering, which impedes their use as tissue models for high-throughput screening assays. Here, we engineered a microfluidic system that preserves and converts tissues into optically transparent structures in less than 1 d, which is 20× faster than current passive clearing approaches. Accelerated clearing was achieved because the microfluidic system enhanced the exchange of interstitial fluids by 567-fold, which increased the rate of removal of optically scattering lipid molecules from the cross-linked tissue. Our enhanced clearing process allowed us to fluorescently image and map the segregation and compartmentalization of different cells during the formation of tumor spheroids, and to track the degradation of vasculature over time within extracted murine pancreatic islets in static culture, which may have implications on the efficacy of beta-cell transplantation treatments for type 1 diabetes. We further developed an image analysis algorithm that automates the analysis of the vasculature connectivity, volume, and cellular spatial distribution of the intact tissue. Our technique allows whole tissue analysis in microfluidic systems, and has implications in the development of organ-on-a-chip systems, high-throughput drug screening devices, and in regenerative medicine.

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High-throughput screening of biomolecules using cell-free gene expression systems

Synthetic Biology ◽

10.1093/synbio/ysy012 ◽

2018 ◽

Vol 3 (1) ◽

Cited By ~ 15

Author(s):

Luis E Contreras-Llano ◽

Cheemeng Tan

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

Screening Method ◽

Screening Methods ◽

Free Gene ◽

On Chip ◽

Translation Systems ◽

Selection Of

Abstract The incorporation of cell-free transcription and translation systems into high-throughput screening applications enables the in situ and on-demand expression of peptides and proteins. Coupled with modern microfluidic technology, the cell-free methods allow the screening, directed evolution and selection of desired biomolecules in minimal volumes within a short timescale. Cell-free high-throughput screening applications are classified broadly into in vitro display and on-chip technologies. In this review, we outline the development of cell-free high-throughput screening methods. We further discuss operating principles and representative applications of each screening method. The cell-free high-throughput screening methods may be advanced by the future development of new cell-free systems, miniaturization approaches, and automation technologies.

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High-throughput screening of blood samples based on structured illumination on-chip imaging

CLEO: 2013 ◽

10.1364/cleo_si.2013.cth3i.1 ◽

2013 ◽

Author(s):

Serap Altay Arpali ◽

Caglar Arpali ◽

Ahmet F. Coskun ◽

Hsin-Hao Chiang ◽

Aydogan Ozcan

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

Structured Illumination ◽

Blood Samples ◽

On Chip

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Development of a Fluorescence-Based, Ultra High-Throughput Screening Platform for Nanoliter-Scale Cytochrome P450 Microarrays

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057109336592 ◽

2009 ◽

Vol 14 (6) ◽

pp. 668-678 ◽

Cited By ~ 24

Author(s):

Sumitra M. Sukumaran ◽

Benjamin Potsaid ◽

Moo-Yeal Lee ◽

Douglas S. Clark ◽

Jonathan S. Dordick

Keyword(s):

Enzyme Activity ◽

Cytochrome P450 ◽

High Throughput ◽

High Throughput Screening ◽

Imaging System ◽

Early Stage ◽

Wide Field ◽

Cytochrome P450 Enzyme ◽

Assay Sensitivity ◽

Screening Platform

Cytochrome P450 enzyme (CYP450s) assays are critical enzymes in early-stage lead discovery and optimization in drug development. Currently available fluorescence-based reaction assays provide a rapid and reliable method for monitoring CYP450 enzyme activity but are confined to medium-throughput well-plate systems. The authors present a high-throughput, integrated screening platform for CYP450 assays combining enzyme encapsulation techniques, microarraying methods, and wide-field imaging. Alginate-containing microarrays consisting of up to 1134 CYP450 reaction elements were fabricated on functionalized glass slides (reaction volumes 20 to 80 nL, total enzyme content in pg) and imaged to yield endpoint activity, stability, and kinetic data. A charge-coupled device imager acquired quantitative, high-resolution images of a 20 × 20 mm area/snapshot using custom-built wide-field optics with telecentric lenses and easily interchangeable filter sets. The imaging system offered a broad dynamic intensity range (linear over 3 orders of magnitude) and sensitivity down to fluorochrome quantities of <5 fmols, with read accuracy similar to a laser scanner or a fluorescence plate reader but with higher throughput. Rapid image acquisition enabled analysis of CYP450 kinetics. Fluorogenic assays with CYP3A4, CYP2C9, and CYP2D6 on the alginate microarrays exhibited Z′ factors ranging from 0.75 to 0.85, sensitive detection of inhibitory compounds, and reactivity comparable to that in solution, thereby demonstrating the reliability and accuracy of the microarray platform. This system enables for the first time a significant miniaturization of CYP enzyme assays with significant conservation of assay reagents, greatly increased throughput, and no apparent loss of enzyme activity or assay sensitivity. ( Journal of Biomolecular Screening 2009:668-678)

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Microfluidic Technologies for High Throughput Screening Through Sorting and On-Chip Culture of C. elegans

Molecules ◽

10.3390/molecules24234292 ◽

2019 ◽

Vol 24 (23) ◽

pp. 4292 ◽

Cited By ~ 5

Author(s):

Daniel Midkiff ◽

Adriana San-Miguel

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

Model Organism ◽

Large Data ◽

Large Data Sets ◽

Data Sets ◽

C Elegans ◽

Drug Candidates ◽

On Chip ◽

And Behavior

The nematode Caenorhabditis elegans is a powerful model organism that has been widely used to study molecular biology, cell development, neurobiology, and aging. Despite their use for the past several decades, the conventional techniques for growth, imaging, and behavioral analysis of C. elegans can be cumbersome, and acquiring large data sets in a high-throughput manner can be challenging. Developments in microfluidic “lab-on-a-chip” technologies have improved studies of C. elegans by increasing experimental control and throughput. Microfluidic features such as on-chip control layers, immobilization channels, and chamber arrays have been incorporated to develop increasingly complex platforms that make experimental techniques more powerful. Genetic and chemical screens are performed on C. elegans to determine gene function and phenotypic outcomes of perturbations, to test the effect that chemicals have on health and behavior, and to find drug candidates. In this review, we will discuss microfluidic technologies that have been used to increase the throughput of genetic and chemical screens in C. elegans. We will discuss screens for neurobiology, aging, development, behavior, and many other biological processes. We will also discuss robotic technologies that assist in microfluidic screens, as well as alternate platforms that perform functions similar to microfluidics.

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Solid State High Throughput Screening Microscopy

10.1101/602011 ◽

2019 ◽

Author(s):

M. Ashraf ◽

S. Mohanan ◽

B Sim ◽

A. Tam ◽

D. Brousseau ◽

...

Keyword(s):

Solid State ◽

High Throughput ◽

High Throughput Screening ◽

Imaging System ◽

Focal Point ◽

Collection Efficiency ◽

Frame Rate ◽

Single Sample ◽

Wide Field ◽

Led Array

We introduce a solid state high throughput screening (ssHTS) imaging modality that uses a novel Newtonian telescope design to image multiple spatially separated samples without moving parts or robotics. Conventional high-throughput imaging modalities either require movement of the sample to the focal plane of the imaging system1–3 or movement of the imaging system itself4,5, or use a wide-field approach to capture several samples in one frame. Schemes which move the sample or the imaging system can be mechanically complex and are inherently slow, while wide-field imaging systems have poor light collection efficiency and resolution compared to systems that image a single sample at a given time point. Our proposed ssHTS system uses a large parabolic reflector and an imaging lenses positioned at their focal distances above each sample. A fast LED array sequentially illuminate samples to generate images that are captured with a single camera placed at the focal point of the reflector. This optical configuration allows each sample to completely fill a sensors field of view. Since each LED illuminates a single sample and LED switch times are very fast, images from spatially separated samples can be captured at rates limited only by the camera’s frame rate. The system is demonstrated by imaging cardiac monolayer and Caenorhabditis elegans (C. elegans) preparations.

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Vectorchip: Microfluidic platform for highly parallel bite by bite profiling of mosquito-borne pathogen transmission

10.1101/2020.10.19.345603 ◽

2020 ◽

Author(s):

Shailabh Kumar ◽

Felix J. H. Hol ◽

Sujit Pujhari ◽

Clayton Ellington ◽

Haripriya Vaidehi Narayanan ◽

...

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

Mosquito Species ◽

Simultaneous Detection ◽

Pathogen Transmission ◽

Membrane Thickness ◽

Human Pathogens ◽

Microfluidic Platform ◽

Mechanical Filter ◽

On Chip

AbstractMosquito bites transmit a number of human pathogens resulting in potentially fatal diseases including malaria, dengue, chikungunya, West Nile encephalitis, and Zika. Although female mosquitoes transmit pathogens via salivary droplets deposited during blood feeding on a host, very little is known about the genomic content of these nanoliter scale droplets, including the transmission dynamics of live pathogens. Here we introduce Vectorchip, a low-cost, scalable microfluidic platform for molecular interrogation of individual mosquito bites in a high-throughput fashion. An ultra-thin PDMS membrane coupled to a microfluidic chip acts as a biting interface, through which freely-behaving mosquitoes deposit saliva droplets by biting into isolated arrayed micro-wells enabling molecular interrogation of individual bites. By modulating membrane thickness, the device enables on-chip comparison of biting capacity and provides a mechanical filter allowing selection of a specific mosquito species. Utilizing Vectorchip, we show on-chip simultaneous detection of mosquito DNA as well as viral RNA from Zika infected Aedes aegypti mosquitoes – demonstrating multiplexed high-throughput screening of vectors and pathogens. Focus-forming assays performed on-chip quantify number of infectious viral particles transmitted during mosquito bites, enabling assessment of active virus transmission. The platform presents a promising approach for single-bite-resolution laboratory and field characterization of vector pathogen communities, to reveal the intricate dynamics of pathogen transmission, and could serve as powerful early warning artificial “sentinel” for mosquito-borne diseases.

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A Multi-Omics Human Liver Organoid Screening Platform for DILI Risk Prediction

10.1101/2021.08.26.457824 ◽

2021 ◽

Author(s):

Charles J. Zhang ◽

Matthew J. O’Meara ◽

Sophia R. Meyer ◽

Sha Huang ◽

Meghan M. Capeling ◽

...

Keyword(s):

Drug Development ◽

Risk Prediction ◽

High Throughput ◽

High Throughput Screening ◽

Human Liver ◽

Drug Induced ◽

Predictive Capacity ◽

On Chip

AbstractBackground and AimsDrug-induced liver injury (DILI) is a prominent failure mode in drug development resulting in clinical trial failures and post-approval withdrawal. Improved in vitro models for DILI risk prediction that can model diverse genetics are needed to improve safety and reduce high attrition rates in drug development. In this study, we evaluated the utility of human liver organoids (HLOs) for high-throughput DILI risk prediction and in an organ-on-chip system. The recent clinical failure of inarigivir soproxil due to DILI underscores the need for improved models.MethodsHLOs were adapted for high-throughput drug screening in dispersed-cell 384-well format and a collection of DILI-associated drugs were screened. HLOs were also adapted to a liver-chip system to investigate enhanced in vivo-like function. Both platforms were benchmarked for their ability to predict DILI using combined biochemical assays, microscopy-based morphological profiling, and transcriptomics.ResultsDispersed HLOs retained DILI predictive capacity of intact HLOs and are amenable to high-throughput screening allowing for measurable IC50 values for cytotoxicity. Distinct morphological differences were observed in cells treated with drugs exerting differing mechanisms of action. HLOs on chips were shown to increase albumin production, CYP450 expression and also release ALT/AST when treated with known DILI drugs. Importantly, HLO liver chips were able to predict hepatotoxicity of tenofovir-inarigivir and showed steatosis and mitochondrial perturbation via phenotypic and transcriptomic analysis.ConclusionsThe high throughput and liver-on-chip system exhibit enhanced in vivo-like function and demonstrate the utility of the platforms in early and late-stage drug development. Tenofovir-inarigivr associated hepatotoxicity was observed and highly correlates with the clinical manifestation of DILI.

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