High-throughput Screening Applications Research Articles

Fully automated OCT-based tissue screening system

This study introduces an innovative optical coherence tomography (OCT) imaging system dedicated to high-throughput screening applications using ex vivo tissue culture. Leveraging OCT’s non-invasive, high-resolution capabilities, the system is equipped with a custom-designed motorized platform and tissue detection ability for automated, successive imaging across samples. Transformer-based deep-learning segmentation algorithms further ensure robust, consistent, and efficient readouts meeting the standards for screening assays. Validated using retinal explant cultures from a mouse model of retinal degeneration, the system provides robust, rapid, reliable, unbiased, and comprehensive readouts of tissue response to treatments. This fully automated OCT-based system marks a significant advancement in tissue screening, promising to transform drug discovery, as well as other relevant research fields.

A multiplexed sensing system with multimodal readout electronics and thread-based electrochemical sensors for high throughput screening

High throughput screening (HTS) based on microwell (or bioreactor) arrays is a well-established approach for rapid screening and identification of lead molecules or target analytes for drug discovery and other applications in biotechnology. However, it has been challenging to scale up these platforms due to the lack of integrated sensors to monitor the microenvironment of each microwell in the array needed. In this work, we present a practical solution for a miniaturized multiplexed sensing system with integrated circuitry capable of performing continuous electrochemical potentiometric and amperometric measurements. The platform uses lightweight multiplexed electrochemical sensors implemented on thin textile threads, all of which share a single operational amplifier (op-amp) and a switch network for both voltage and current measurements. The resulting platform forgoes the need for large-area sensors or bulky multichannel potentiostat with a simple readout circuitry capable of multimodal measurements. The design and validation of the multiplexed sensing system have been reported in the context of measuring pH and oxygen (O2), which are crucial biomarkers for any HTS application. The thread-based pH and O2 sensors possess sensitivities of ∼65.87 mV/pH for the pH sensor (n = 3) and 0.6621 nA/mg L-1 for the O2 sensor (n = 3). This architecture is modular such that it can be easily extended to monitoring multiple biomarkers employing thread-based sensors and sharing the same readout circuitry, making it a practical and versatile tool for biotechnology research.

Data-science-guided calibration curve prediction of an MLCT-based ee determination assay for chiral amines

Circular dichroism (CD) based enantiomeric excess (ee) determination assays are optical alternatives to chromatographic ee determination in high-throughput screening (HTS) applications. However, the implementation of these assays requires calibration experiments using enantioenriched materials. We present a data-driven approach that circumvents the need for chiral resolution and calibration experiments for an octahedral Fe(II) complex (1) used for the ee determination of α-chiral primary amines. By computationally parameterizing the imine ligands formed in the assay conditions, a model of the circular dichroism (CD) response of the Fe(II) assembly was developed. Using this model, calibration curves were generated for four analytes and compared to experimentally generated curves. In a single-blind ee determination study, the ee values of unknown samples were determined within 9% mean absolute error, which rivals the error using experimentally generated calibration curves.

Rational Design of a Highly Sensitive Carboxylesterase Probe and Its Application in High-Throughput Screening for Uncovering Carboxylesterase Inhibitors.

Tracking carboxylesterases (CESs) through noninvasive and dynamic imaging is of great significance for diagnosing and treating CES-related metabolic diseases. Herein, three BODIPY-based fluorescent probes with a pyridine unit quaternarized via an acetoxybenzyl group were designed and synthesized to detect CESs based on the photoinduced electron transfer process. Notably, among these probes, BDPN2-CES exhibited a remarkable 182-fold fluorescence enhancement for CESs within 10 min. Moreover, BDPN2-CES successfully enabled real-time imaging of endogenous CES variations in living cells. Using BDPN2-CES, a visual high-throughput screening method for CES inhibitors was established, culminating in the discovery of an efficient inhibitor, WZU-13, sourced from a chemical library. These findings suggest that BDPN2-CES could provide a new avenue for diagnosing CES-related diseases, and WZU-13 emerges as a promising therapeutic candidate for CES-overexpression pathological processes.

Development of a novel high-throughput culture system for hypoxic 3D hydrogel cell culture.

Animal models lack physiologic relevance to the human system which results in low clinical translation of results derived from animal testing. Besides spheroids or organoids, hydrogel-based 3D in vitro models are used to mimic the in vivo situation increasing the relevance while reducing animal testing. However, to establish hydrogel-based 3D models in applications such as drug development or personalized medicine, high-throughput culture systems are required. Furthermore, the integration of oxygen-reduced (hypoxic) conditions has become increasingly important to establish more physiologic culture models. Therefore, we developed a platform technology for the high-throughput generation of miniaturized hydrogels for 3D cell culture. The Oli-Up system is based on the shape of a well-plate and allows for the parallel culture of 48 hydrogel samples, each with a volume of 15µl. As a proof-of-concept, we established a 3D culture of gelatin-methacryloyl (GelMA)-encapsulated mesenchymal stem/stromal cells (MSCs). We used a hypoxia reporter cell line to establish a defined oxygen-reduced environment to precisely trigger cellular responses characteristicof hypoxia in MSCs. In detail, the expression of hypoxia response element (HRE) increased dependent on the oxygen concentration and cell density. Furthermore, MSCs displayed an altered glucose metabolism and increased VEGF secretion upon oxygen-reduction. In conclusion, the Oli-Up system is a platform technology for the high-throughput culture of hydrogel-based 3D models in a defined oxygen environment. As it is amenable for automation, it holds the potential for high-throughput screening applications such as drug development and testing in more physiologic 3D in vitro tissue models.

Efficient approach to cyclic scheduling of high throughput screening systems for bioengineering

As a standard technology, high throughput screening (HTS) is widely applied for the analysis and detection of new biological substances and drugs in pharmaceutical industries and life science research. Efficient scheduling of HTS systems is a crucial issue for reducing the cost of their operations. As a typical application of high throughput screening, this work studies the scheduling problem of the enzyme-linked immunoassay (ELISA) processes in HTS systems. For this process, it requires a one-microplate cyclic schedule and a microplate visits some processing resources multiple times, making the process deadlock-prone such that its scheduling problem is very challenging. To tackle this problem, the process is modeled by a kind of Petri nets. Based on this model, by analyzing the dynamic properties of the process, it is able to directly deduce an activity sequence to form an optimal cyclic schedule. In this way, it not only efficiently solves the challenging scheduling problem without building a mathematical programming model, but also the obtained schedule is very easy to understand and implement.

Abstract 3093: Combining lab automation with data analysis automation to enable high-throughput screening of patient-derived organoids

Abstract Although patient-derived tumor organoids (PDOs) better-represent patient tumors compared to traditional in vitro cell cultures, limitations of PDO analysis remain a major hurdle for high-throughput screening (HTS) applications. In particular, current PDO analysis fall short in at least 1 of 2 major criteria: 1) accurate recapitulation of patient tumor or 2) scalability. To address this, we have developed an automation-compatible protocol to satisfy these 2 major criteria to facilitate the adoption of PDOs for HTS applications1. Our novel protocol leverages live-cell imaging techniques to capture the dynamic interactions and intricate complexities of PDOs, which allowed for more precise and comprehensive analysis. To further enhance the accuracy and efficiency of our approach, we developed a fully automated image and data analysis pipeline (Orbits). Using label-free detection of organoids from brightfield images, Orbits integrates growth-rate-based drug response metrics with drug synergy metrics to significantly improve the identification of synergistic drug interactions. Here, present the results of our HTS protocol on 10 distinct drug combination strategies, specifically focusing on the repurposed drug candidate Auranofin, a Thioredoxin reductase inhibitor. We screened on 10 PDOs sourced from healthy lung, non-small cell lung cancer, and pancreatic ductal adenocarcinoma. Not only were we able to distinguishing cytostatic from cytotoxic drug responses at a single-organoid level, we identified drug candidates that can synergistically enhanced the efficacy of Auranofin in a tumor selective manner. Our study presents a major step forward in the field of PDO-based drug discovery, offering a robust protocol for HTS and automated data analysis. The implementation of our method will accelerate the discovery of effective therapeutic strategies, ultimately translating to improved patient outcomes. 1 Le Compte, M., et al JoVE 190 (2022) Citation Format: Abraham Lin, Maxim Le Compte, Tyler Gilcrest, Edgar Cardenas De La Hoz, Geert Roeyen, Jeroen Hendriks, Filip Lardon, Christophe Deben. Combining lab automation with data analysis automation to enable high-throughput screening of patient-derived organoids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3093.

XPORT ENTRAP: A droplet microfluidic platform for enhanced DNA transfer between microbial species

A significant hurdle for the widespread implementation and use of synthetic biology is the challenge of highly efficient introduction of DNA into microorganisms. This is especially a barrier for the utilization of non-model organisms and/or novel chassis species for a variety of applications, ranging from molecular biology to biotechnology and biomanufacturing applications. Common approaches to episomal and chromosomal gene editing, which employ techniques such as chemical competence and electroporation, are typically only amenable to a small subset of microbial species while leaving the vast majority of microorganisms in nature genetically inaccessible. To address this challenge, we have employed the previously described B. subtilis broad-host conjugation strain, XPORT, which was modularly designed for loading DNA cargo and conjugating such DNA into recalcitrant microbes. In this current work, we have leveraged and adapted the XPORT strain for use in a droplet microfluidic platform to enable increased efficiency of conjugation-based DNA transfer. The system named DNA ENTRAP (DNA ENhanced TRAnsfer Platform) utilizes cell-encapsulated water-in-oil emulsion droplets as pico-liter-volume bioreactors that allows controlled contacts between the donor and receiver cells within the emulsion bioreactor. This allowed enhanced XPORT-mediated genetic transfer over the current benchtop XPORT process, demonstrated using two different Bacillus subtilis strains (donor and receiver), as well as increased throughput (e.g., number of successfully conjugated cells) due to the automated assay steps inherent to microfluidic lab-on-a-chip systems. DNA ENTRAP paves the way for a streamlined automation of culturing and XPORT-mediated genetic transfer processes as well as future high-throughput cell engineering and screening applications.

A Stable Cell Line Co-expressing hTRPV1 and GCaMP6s: A Novel Cell-based Assay For High-throughput Screening of hTRPV1 Agonists.

Transient receptor potential vanilloid-1 (TRPV1) is a non-selective cation channel capable of integrating various noxious chemical and physical stimuli. Recently, human TRPV1 (hTRPV1) has attracted wide attention from researchers because it is closely related to pain, inflammation, temperature perception, and tumors. Our study was aimed at generating a stable cell line co-expressing hTRPV1 receptor and GCaMP6s calcium indicator protein and, based on this, developing high-throughput screening methods for targeting hTRPV1 agonists. The CHO-hTRPV1-GCaMP6s cell line stably expressing hTRPV1 and GCaMP6s was generated by co-transfection of hTRPV1 and GCaMP6s into Chinese hamster ovary (CHO) cells. The high-throughput screening methods were developed based on detecting the concentration of intracellular calcium ions ([Ca2+]i) by using chemically synthesized dyes and genetically encoded calcium indicator (GECI). Meanwhile, the sensitivity and adaptability of these methods in the evaluation of capsaicinoids were also compared. A stable cell line co-expressing hTRPV1 and GCaMP6s was generated and used to establish a functional high-throughput screening assay based on the measurement of [Ca2+]i by fluorometric imaging plate reader (FLIPR). The GECI exhibited a higher sensitivity and applicability than that of chemically synthesized dyes in detecting the changes in [Ca2+]i induced by capsaicin. The CHO-hTRPV1-GCaMP6s cell line was further used to detect the dose-dependent relationships of various hTRPV1 agonists (comparison of EC50 values: capsaicin (39 ± 1.67 nM) < nonivamide (67 ± 3.05 nM) < piperine (9222 ± 1851 nM)), and this order is consistent with the pharmacological properties of hTRPV1 activation by these agonists. The successful establishment of the CHO-hTRPV1-GCaMP6s cell lines and their application in high-throughput screening of hTRPV1 agonists.

Benchmarking DFT-based excited-state methods for intermolecular charge-transfer excitations.

Intermolecular charge-transfer is a highly important process in biology and energy-conversion applications where generated charges need to be transported over several moieties. However, its theoretical description is challenging since the high accuracy required to describe these excited states must be accessible for calculations on large molecular systems. In this benchmark study, we identify reliable low-scaling computational methods for this task. Our reference results were obtained from highly accurate wavefunction calculations that restrict the size of the benchmark systems. However, the density-functional theory based methods that we identify as accurate can be applied to much larger systems. Since targeting charge-transfer states requires the unambiguous classification of an excited state, we first analyze several charge-transfer descriptors for their reliability concerning intermolecular charge-transfer and single out the charge-transfer distance calculated based on the variation of electron density upon excitation (DCT) as an optimal choice for our purposes. In general, best results are obtained for orbital-optimized methods and among those, the maximum overlap method proved to be the most numerically stable variant when using the initial MOs as reference orbitals. Favorable error cancellation with optimally-tuned range-separated hybrid functionals and a rather small basis set can provide an economical yet reasonable wavefunction when using time-dependent density functional theory, which provides relevant information about the excited-state character to be used in the orbital-optimized methods. The qualitative agreement makes these fast calculations attractive for high-throughput screening applications.

PhaseFIT: live-organoid phase-fluorescent image transformation via generative AI

Organoid models have provided a powerful platform for mechanistic investigations into fundamental biological processes involved in the development and function of organs. Despite the potential for image-based phenotypic quantification of organoids, their complex 3D structure, and the time-consuming and labor-intensive nature of immunofluorescent staining present significant challenges. In this work, we developed a virtual painting system, PhaseFIT (phase-fluorescent image transformation) utilizing customized and morphologically rich 2.5D intestinal organoids, which generate virtual fluorescent images for phenotypic quantification via accessible and low-cost organoid phase images. This system is driven by a novel segmentation-informed deep generative model that specializes in segmenting overlap and proximity between objects. The model enables an annotation-free digital transformation from phase-contrast to multi-channel fluorescent images. The virtual painting results of nuclei, secretory cell markers, and stem cells demonstrate that PhaseFIT outperforms the existing deep learning-based stain transformation models by generating fine-grained visual content. We further validated the efficiency and accuracy of PhaseFIT to quantify the impacts of three compounds on crypt formation, cell population, and cell stemness. PhaseFIT is the first deep learning-enabled virtual painting system focused on live organoids, enabling large-scale, informative, and efficient organoid phenotypic quantification. PhaseFIT would enable the use of organoids in high-throughput drug screening applications.

Rapid Prototyping of Thermoplastic Microfluidic 3D Cell Culture Devices by Creating Regional Hydrophilicity Discrepancy.

Microfluidic 3D cell culture devices that enable the recapitulation of key aspects of organ structures and functions in vivo represent a promising preclinical platform to improve translational success during drug discovery. Essential to these engineered devices is the spatial patterning of cells from different tissue types within a confined microenvironment. Traditional fabrication strategies lack the scalability, cost-effectiveness, and rapid prototyping capabilities required for industrial applications, especially for processes involving thermoplastic materials. Here, an approach to pattern fluid guides inside microchannels is introduced by establishing differential hydrophilicity using pressure-sensitive adhesives as masks and a subsequent selective coating with a biocompatible polymer. Optimal coating conditions are identified using polyvinylpyrrolidone, which resulted in rapid and consistent hydrogel flow in both the open-chip prototype and the fully bonded device containing additional features for medium perfusion. The suitability of the device for dynamic 3D cell culture is tested by growing human hepatocytes in the device under controlled fluid flow for a 14-day period. Additionally, the study demonstrated the potential of using the device for pharmaceutical high-throughput screening applications, such as predicting drug-induced liver injury. The approach offers a facile strategy of rapid prototyping thermoplastic microfluidic organ chips with varying geometries, microstructures, and substrate materials.

NanoFIRE: A NanoLuciferase and Fluorescent Integrated Reporter Element for Robust and Sensitive Investigation of HIF and Other Signalling Pathways.

The Hypoxia Inducible Factor (HIF) transcription factors are imperative for cell adaption to low oxygen conditions and development; however, they also contribute to ischaemic disease and cancer. To identify novel genetic regulators which target the HIF pathway or small molecules for therapeutic use, cell-based reporter systems are commonly used. Here, we present a new, highly sensitive and versatile reporter system, NanoFIRE: a NanoLuciferase and Fluorescent Integrated Reporter Element. Under the control of a Hypoxic Response Element (HRE-NanoFIRE), this system is a robust sensor of HIF activity within cells and potently responds to both hypoxia and chemical inducers of the HIF pathway in a highly reproducible and sensitive manner, consistently achieving 20 to 150-fold induction across different cell types and a Z' score > 0.5. We demonstrate that the NanoFIRE system is adaptable via substitution of the response element controlling NanoLuciferase and show that it can report on the activity of the transcriptional regulator Factor Inhibiting HIF, and an unrelated transcription factor, the Progesterone Receptor. Furthermore, the lentivirus-mediated stable integration of NanoFIRE highlights the versatility of this system across a wide range of cell types, including primary cells. Together, these findings demonstrate that NanoFIRE is a robust reporter system for the investigation of HIF and other transcription factor-mediated signalling pathways in cells, with applications in high throughput screening for the identification of novel small molecule and genetic regulators.

Enhancing cardiomyocytes contraction force measuring in drug testing: Integration of a highly sensitive single-crystal silicon strain sensor into SU-8 cantilevers

The development of efficient tools for predicting drug-induced cardiotoxicity in the preclinical phase would greatly benefit the drug development process. This study presents an SU-8 cantilever integrated with a single-crystal silicon strain sensor to enhance force sensitivity in toxicity screening methods based on changes in the contraction force of cardiomyocytes. The proposed cantilever device enables real-time measurements of cardiomyocytes contraction force with high sensitivity, thereby facilitating the assessment of drug cardiotoxicity. The experimental results obtained herein demonstrate the responsiveness of the proposed platform in detecting forces smaller than 0.02 μN with a force sensitivity that is nearly 17 times higher than those of conventional metal-based strain sensors. Moreover, the integration of strain sensors demonstrates the potential for manufacturing cantilever arrays that can be used in high-throughput screening applications. The developed methodology successfully facilitates in vitro culturing of cardiomyocytes and allows for continuous monitoring of their contraction force. The practical applicability of the proposed platform is further validated through cardiotoxicity analysis. The cultured cardiomyocytes are treated with two cardiovascular drugs, namely verapamil (an L-type calcium channel blocker) and isoproterenol (a sympathomimetic drug targeting β1 and β2 adrenergic receptors), to analyze the drug induced effects in the cardiomyocytes.

Towards modular engineering of cell signalling: Topographically-textured microparticles induce osteogenesis via activation of canonical hedgehog signalling

Polymer microparticles possess great potential as functional building blocks for advanced bottom-up engineering of complex tissues. Tailoring the three-dimensional architectural features of culture substrates has been shown to induce osteogenesis in mesenchymal stem cells in vitro, but the molecular mechanisms underpinning this remain unclear. This study proposes a mechanism linking the activation of Hedgehog signalling to the osteoinductive effect of surface-engineered, topographically-textured polymeric microparticles. In this study, mesenchymal progenitor C3H10T1/2 cells were cultured on smooth and dimpled poly(D,l-lactide) microparticles to assess differences in viability, cellular morphology, proliferation, and expression of a range of Hedgehog signalling components and osteogenesis-related genes. Dimpled microparticles induced osteogenesis and activated the Hedgehog signalling pathway relative to smooth microparticles and 2D-cultured controls without the addition of exogenous biochemical factors. We observed upregulation of the osteogenesis markers Runt-related transcription factor2 (Runx2) and bone gamma-carboxyglutamate protein 2 (Bglap2), as well as the Hedgehog signalling components, glioma associated oncogene homolog 1 (Gli1), Patched1 (Ptch1), and Smoothened (Smo). Treatment with the Smo antagonist KAAD-cyclopamine confirmed the involvement of Smo in Gli1 target gene activation, with a significant reduction in the expression of Gli1, Runx2 and Bglap2 (p ≤ 0.001) following KAAD-cyclopamine treatment. Overall, our study demonstrates the role of the topographical microenvironment in the modulation of Hedgehog signalling, highlighting the potential for tailoring substrate topographical design to offer cell-instructive 3D microenvironments. Topographically-textured microparticles allow the modulation of Hedgehog signalling in vitro without adding exogenous biochemical agonists, thereby eliminating potential confounding artefacts in high-throughput drug screening applications.

Rational design of supramolecular self-assembly sensor for living cell imaging of HDAC1 and its application in high-throughput screening

Supramolecular chemistry offers new insights in bioimaging, but specific tracking of enzyme in living cells via supramolecular host-guest reporter pair remains challenging, largely due to the interference caused by the complex cellular environment on the binding between analytes and hosts. Here, by exploiting the principle of supramolecular tandem assay (STA) and the classic host-guest reporter pair (p-sulfonatocalix[4]arene (SC4A) and lucigenin (LCG)) and rationally designing artificial peptide library to screen sequence with high affinity of the target enzyme, we developed a “turn-on” fluorescent sensing system for intracellular imaging of histone deacetylase 1 (HDAC1), which is a potential therapeutic target for various diseases, including cancer, neurological, and cardiovascular diseases. Based on computational simulations and experimental validations, we verified that the deacetylated peptide by HDAC1 competed LCG, freeing it from the SC4A causing fluorescence increase. Enzyme kinetics experiments were further conducted to prove that this assay could detect HDAC1 specifically with high sensitivity (the LOD value is 0.015 μg/mL, ten times lower than the published method). This system was further applied for high-throughput screening of HDAC1 inhibitors over a natural compound library containing 147 compounds, resulting in the identification of a novel HDAC1 down-regulator (Ginsenoside RK3). Our results demonstrated the sensitivity and robustness of the assay system towards HDAC1. It should serve as a valuable tool for biochemical studies and drug screening.

EnzyHTP Computational Directed Evolution with Adaptive Resource Allocation.

Directed evolution facilitates enzyme engineering via iterative rounds of mutagenesis. Despite the wide applications of high-throughput screening, building "smart libraries" to effectively identify beneficial variants remains a major challenge in the community. Here, we developed a new computational directed evolution protocol based on EnzyHTP, a software that we have previously reported to automate enzyme modeling. To enhance the throughput efficiency, we implemented an adaptive resource allocation strategy that dynamically allocates different types of computing resources (e.g., GPU/CPU) based on the specific need of an enzyme modeling subtask in the workflow. We implemented the strategy as a Python library and tested the library using fluoroacetate dehalogenase as a model enzyme. The results show that compared to fixed resource allocation where both CPU and GPU are on-call for use during the entire workflow, applying adaptive resource allocation can save 87% CPU hours and 14% GPU hours. Furthermore, we constructed a computational directed evolution protocol under the framework of adaptive resource allocation. The workflow was tested against two rounds of mutational screening in the directed evolution experiments of Kemp eliminase (KE07) with a total of 184 mutants. Using folding stability and electrostatic stabilization energy as computational readout, we identified all four experimentally observed target variants. Enabled by the workflow, the entire computation task (i.e., 18.4 μs MD and 18,400 QM single-point calculations) completes in 3 days of wall-clock time using ∼30 GPUs and ∼1000 CPUs.

A Comparative Study on the Lysosomal Cation Channel TMEM175 Using Automated Whole-Cell Patch-Clamp, Lysosomal Patch-Clamp, and Solid Supported Membrane-Based Electrophysiology: Functional Characterization and High-Throughput Screening Assay Development.

The lysosomal cation channel TMEM175 is a Parkinson's disease-related protein and a promising drug target. Unlike whole-cell automated patch-clamp (APC), lysosomal patch-clamp (LPC) facilitates physiological conditions, but is not yet suitable for high-throughput screening (HTS) applications. Here, we apply solid supported membrane-based electrophysiology (SSME), which enables both direct access to lysosomes and high-throughput electrophysiological recordings. In SSME, ion translocation mediated by TMEM175 is stimulated using a concentration gradient at a resting potential of 0 mV. The concentration-dependent K+ response exhibited an I/c curve with two distinct slopes, indicating the existence of two conducting states. We measured H+ fluxes with a permeability ratio of PH/PK = 48,500, which matches literature findings from patch-clamp studies, validating the SSME approach. Additionally, TMEM175 displayed a high pH dependence. Decreasing cytosolic pH inhibited both K+ and H+ conductivity of TMEM175. Conversely, lysosomal pH and pH gradients did not have major effects on TMEM175. Finally, we developed HTS assays for drug screening and evaluated tool compounds (4-AP, Zn as inhibitors; DCPIB, arachidonic acid, SC-79 as enhancers) using SSME and APC. Additionally, we recorded EC50 data for eight blinded TMEM175 enhancers and compared the results across all three assay technologies, including LPC, discussing their advantages and disadvantages.

Droplet dynamics in asymmetric microfluidic junctions

Droplet-based microfluidics has received increasing interest over the last two decades, due to its extensive applications in drug delivery, cell encapsulation, protein crystallization, biochemical synthesis and high-throughput screening. As one of the key units in the microfluidic channels, asymmetric microfluidic junctions provide more opportunities to control and organize the droplets with different sizes, however, few studies have been reported to explore the effect of the junction angle on droplet behaviour in the asymmetric junctions. This work presents a numerical investigation of the droplet behaviour in asymmetric microfluidic junctions. The effect of the junction angle on the droplet breakup is explored in detail. Droplet breakup and non-breakup are found in asymmetric junctions. In the regime of the asymmetric breakup, the volume of the daughter droplet in the uphill branching channel is always larger than that in the downhill branching channel. Interestingly, in the regime of non-breakup, droplets are sorted into the uphill or downhill branching channels, suggesting that this asymmetric junction could be used for passive droplet sorting. The physical mechanisms behind droplet breakup and sorting are explored. Both the junction angle and the initial tip-to-tip length of the droplet play essential roles in the droplet behaviour in the asymmetric junctions. A phase diagram of the droplet behaviour is presented in this work. A scaling model is proposed to predict the transition between the breakup and non-breakup of the droplets in the asymmetric junctions.

Integrating spheroid-on-a-chip with tubeless rocker platform: A high-throughput biological screening platform.

Spheroid-on-a-chip platforms are emerging as a promising in-vitro model that enables screening of the efficacy of biologically active ingredients. Generally, the supply of liquids to spheroids occurs in the steady flow mode with the use of syringe pumps; however, the utilization of tubing and connections, especially for multiplexing and high-throughput screening applications, makes spheroid-on-a-chip platforms labour- and cost-intensive. Gravity-induced flow using rocker platforms overcomes these challenges. Here, we develop a robust gravity-driven technique to culture arrays of cancer cell spheroids and dermal fibroblast spheroids in a high-throughput manner using a rocker platform. We benchmark the efficiency of the developed rocker-based platform to syringe pumps for generating multicellular spheroids and their use for screening biologically active ingredients. We study cell viability, internal spheroid structure, as well as the effect of vitamin C on the production of collagen type I and fibronectin by dermal fibroblast spheroids. We demonstrate that the rocker-based platform not only offers comparable or enhanced performance in terms of cell viability, spheroids formation, and protein production by dermal fibroblast spheroids but also, from a practical perspective it offers a smaller footprint, requires a lower cost, and offers an easier method for handling. These results support the application of rocker-based microfluidic spheroid-on-a-chip platforms for in-vitro screening in a high-throughput manner with industrial scaling-up opportunities. This article is protected by copyright. All rights reserved.