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The addition of reagents into preformed droplets is a crucial yet intricate task in droplet-based applications where sequential reactions is required. Pico-injection offers high throughput and robustness in accomplishing this task, but the existing pico-injection techniques work in an indiscriminate manner, making it difficult to target particular groups of droplets. Here we report image-activated pico-injection (imgPico) for label-free, on-demand reagent supplementation into droplets. The imgPico detects the droplets of interest by real-time image analysis and makes decisions for the downstream pico-injection operation. We studied the performance of different algorithms for the image analysis and optimized the experimental settings of the imgPico. In the validation experiment, the imgPico successfully injected fluorescent dyes into droplets encapsulating one, two, and three cells, respectively, as expected. We further demonstrated the utility of imgPico by targeting droplets encapsulating single cells in droplet-based single-cell RNA sequencing (scRNA-seq) using exceedingly high cell density, and the results showed that the imgPico effectively reduced the presence of doublets in the scRNA-seq data. With the merits of being label-free and versatile, the imgPico represents a technical advance with potential applications in single-cell analysis.
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Algoritmos , Análisis de la Célula Individual , Análisis de la Célula Individual/métodos , Recuento de CélulasRESUMEN
The utilization of microfluidic technology for miniaturized and efficient particle sorting holds significant importance in fields such as biology, chemistry, and healthcare. Passive separation methods, achieved by modifying the geometric shapes of microchannels, enable gentle and straightforward enrichment and separation of particles. Building upon previous discussions regarding the effects of column arrays on fluid flow and particle separation within microchips, we introduced a column array structure into an H-shaped microfluidic chip. It was observed that this structure enhanced mass transfer between two fluids while simultaneously intercepting particles within one fluid, satisfying the requirements for particle interception. This enhancement was primarily achieved by transforming the originally single-mode diffusion-based mass transfer into dual-mode diffusion-convection mass transfer. By further optimizing the column array, it was possible to meet the basic requirements of mass transfer and particle interception with fewer microcolumns, thereby reducing device pressure drop and facilitating the realization of parallel and high-throughput microfluidic devices. These findings have enhanced the potential application of microfluidic systems in clinical and chemical engineering domains.
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Multiplex, digital nucleic acid detections have important biomedical applications, but the multiplexity of existing methods is predominantly achieved using fluorescent dyes or probes, making the detection complicated and costly. Here, we present the StratoLAMP for label-free, multiplex digital loop-mediated isothermal amplification based on visual stratification of the precipitate byproduct. The StratoLAMP designates two sets of primers with different concentrations to achieve different precipitate yields when amplifying different nucleic acid targets. In the detection, deep learning image analysis is used to stratify the precipitate within each droplet and determine the encapsulated targets for nucleic acid quantification. We investigated the effect of the amplification reagents and process on the precipitate generation and optimized the assay conditions. We then implemented a deep-learning image analysis pipeline for droplet detection, achieving an overall accuracy of 94.3%. In the application, the StratoLAMP successfully achieved the simultaneous quantification of two nucleic acid targets with high accuracy. By eliminating the need for fluorescence, StratoLAMP represents a unique concept toward label-free, multiplex nucleic acid assays and an analytical tool with great cost-effectiveness.
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Técnicas de Amplificación de Ácido Nucleico , Ácidos Nucleicos , Técnicas de Amplificación de Ácido Nucleico/métodos , Técnicas de Diagnóstico Molecular/métodos , Cartilla de ADN , Sensibilidad y EspecificidadRESUMEN
Antibody arrays have great implications in many biomedical settings. However, commonly used patterning methods have difficulties in generating antibody arrays with both high resolution and multiplexity, limiting their applications. Here, a convenient and versatile technique for the patterning of multiple antibodies with resolution down to 20 µm is reported using micropillar-focused droplet printing and microcontact printing. Droplets of antibody solutions are first printed and stably confined on the micropillars of a stamp, and then the antibodies absorbed on the micropillars are contact-printed to the target substrate, generating antibody patterns faithfully replicating the micropillar array. The effect of different parameters on the patterning results is investigated, including hydrophobicity of the stamps, override time of the droplet printing, incubation time, and the diameters of the capillary tips and micropillars. To demonstrate the utility of the method, multiplex arrays of anti-EpCAM and anti-CD68 antibodies is generated to capture breast cancer cells and macrophages, respectively, on the same substrate, and successful capturing of individual cell types and enrichment among the cells are achieved. It is envision that this method would serve as a versatile and useful protein patterning tool for biomedical applications.
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Anticuerpos , Interacciones Hidrofóbicas e HidrofílicasRESUMEN
Multiplex, digital nucleic acid tests have important biomedical applications, but existing methods mostly use fluorescent probes that are target-specific and difficult to optimize, limiting their widespread applications. Here, we report color-encoded, intelligent digital loop-mediated isothermal amplification (CoID-LAMP) for the coidentification of multiple nucleic acid targets. CoID-LAMP supplements different primer solutions with different dyes, generates primer droplets and sample droplets, and collectively pairs these two types of droplets in a microwell array device to perform LAMP. After imaging, the droplet colors were analyzed to decode the primer information, and the precipitate byproducts within droplets were detected to determine the target occupancy and calculate the concentrations. We first established an image analysis pipeline based on a deep learning algorithm for reliable droplet detection and validated the analytical performance in nucleic acid quantification. We then implemented CoID-LAMP using fluorescent dyes as the coding materials and established an 8-plex digital nucleic acid assay, confirming the reliable coding performance and the capability of multiplex nucleic acid quantification. We further implemented CoID-LAMP using brightfield dyes for a 4-plex assay, suggesting that the assay could be realized solely by brightfield imaging with minimal demand on the optics. Leveraging the advantages of droplet microfluidics in multiplexing and deep learning in intelligent image analysis, CoID-LAMP offers a useful tool for multiplex nucleic acid quantification.
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Técnicas de Amplificación de Ácido Nucleico , Ácidos Nucleicos , Técnicas de Amplificación de Ácido Nucleico/métodos , Microfluídica/métodos , Colorantes FluorescentesRESUMEN
Combinatorial drug therapy reduces drug resistance and disease relapse, but informed drug combinations are lacking due to the high scale of possible combinations and the relatively simple phenotyping strategies. Here we report combinatorial perturbation sequencing (CP-seq) on single cells using microwell-base droplet random pairing. CP-seq uses oligonucleotides to barcode drugs, encapsulates drugs and cells in separate droplets, and pairs cell droplets with two drug droplets randomly on a microwell array chip to complete combinatorial drug treatment and barcode-tagging on cells. The subsequent single-cell RNA sequencing simultaneously detects the single-cell transcriptomes and drug barcodes to demultiplex the corresponding drug treatment. The microfluidic droplet operations had robust performance, with the overall utilization rate of the microwells being up to 83%. We then progressively validated the CP-seq by performing single-drug treatments and then combinatorial-drug treatments, confirming the CP-seq's capability in the collection and analysis of drug-perturbed transcriptomes. Leveraging the advantage of droplet microfluidics in massive multiplexing, the CP-seq represents a great technology for combinatorial perturbation screening with high throughput and comprehensive profiling.
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Técnicas Biosensibles , Neoplasias Cutáneas , Humanos , Microfluídica , Oligonucleótidos , ARNRESUMEN
Bone diseases, such as osteoporosis and bone defects, often lead to structural and functional deformities of the patient's body. Understanding the complicated pathophysiology and finding new drugs for bone diseases are in dire need but challenging with the conventional cell and animal models. Bone-on-a-chip (BoC) models recapitulate key features of bone at an unprecedented level and can potentially shift the paradigm of future bone research and therapeutic development. Nevertheless, current BoC models predominantly rely on off-chip analysis which provides only endpoint measurements. To this end, integrating biosensors within the BoC can provide non-invasive, continuous monitoring of the experiment progression, significantly facilitating bone research. This review aims to summarize research progress in BoC and biosensor integrations and share perspectives on this exciting but rudimentary research area. We first introduce the research progress of BoC models in the study of bone remodeling and bone diseases, respectively. We then summarize the need for BoC characterization and reported works on biosensor integration in organ chips. Finally, we discuss the limitations and future directions of BoC models and biosensor integrations as next-generation technologies for bone research.
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The rapid promotion of single-cell omics in various fields has begun to help solve many problems encountered in research, including precision medicine, prenatal diagnosis, and embryo development. Meanwhile, single-cell techniques are also constantly updated with increasing demand. For some specific target cells, the workflow from droplet screening to single-cell sequencing is a preferred option and should reduce the impact of operation steps, such as demulsification and cell recovery. We developed an all-in-droplet method integrating cell encapsulation, target sorting, droplet picoinjection, and single-cell transcriptome profiling on chips to achieve labor-saving monitoring of TCR-T cells. As a proof of concept, in this research, TCR-T cells were encapsulated, sorted, and performed single-cell transcriptome sequencing (scRNA-seq) by injecting reagents into droplets. It avoided the tedious operation of droplet breakage and re-encapsulation between droplet sorting and scRNA-seq. Moreover, convenient device operation will accelerate the progress of chip marketization. The strategy achieved an excellent recovery performance of single-cell transcriptome with a median gene number over 4000 and a cross-contamination rate of 8.2 ± 2%. Furthermore, this strategy allows us to develop a device with high integrability to monitor infused TCR-T cells, which will promote the development of adoptive T cell immunotherapy and their clinical application.
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Nanobodies, also known as VHHs, originate from the serum of Camelidae. Nanobodies have considerable advantages over conventional antibodies, including smaller size, more modifiable, and deeper tissue penetration, making them promising tools for immunotherapy and antibody-drug development. A high-throughput nanobody screening platform is critical to the rapid development of nanobodies. To date, droplet-based microfluidic systems have exhibited improved performance compared to the traditional phage display technology in terms of time and throughput. In realistic situations, however, it is difficult to directly apply the technology to the screening of nanobodies. Requirements of plasma cell enrichment and high cell viability, as well as a lack of related commercial reagents, are leading causes for impeding the development of novel methods. We overcame these obstacles by constructing a eukaryotic display system that secretes nanobodies utilizing homologous recombination and eukaryotic transformation technologies, and the significant advantages are that it is independent of primary cell viability and it does not require plasma cell enrichment in advance. Next, a signal capture system of "SA-beads + Biotin-antigen + nanobody-6 × His + fluorescence-labeled anti-6 × His (secondary antibody)" was designed for precise localization of the eukaryotic-expressed nanobodies in a droplet. Based on this innovation, we screened 293T cells expressing anti-PD-L1 nanobodies with a high positive rate of targeted cells (up to 99.8%). Then, single-cell transcriptomic profiling uncovered the intercellular heterogeneity and BCR sequence of target cells at a single-cell level. The complete complementarity determining region (CDR3) structure was obtained, which was totally consistent with the BCR reference. This study expanded the linkage between microfluidic technology and nanobody applications and also showed potential to accelerate the rapid transformation of nanobodies in the large-scale market.
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Anticuerpos de Dominio Único , Animales , Anticuerpos , Camelidae , Biblioteca de Genes , Inmunoterapia , MicrofluídicaRESUMEN
Monitoring of the coagulation function has applications in many clinical settings. Routine coagulation assays in the clinic are sample-consuming and slow in turnaround. Microfluidics provides the opportunity to develop coagulation assays that are applicable in point-of-care settings, but reported works required bulky sample pumping units or costly data acquisition instruments. In this work, we developed a microfluidic coagulation assay with a simple setup and easy operation. The device continuously generated droplets of blood sample and buffer mixture and reported the temporal development of blood viscosity during coagulation based on the color appearance of the resultant droplets. We characterized the relationship between blood viscosity and color appearance of the droplets and performed experiments to validate the assay results. In addition, we developed a prototype analyzer equipped with simple fluid pumping and economical imaging module and obtained similar assay measurements. This assay showed great potential to be developed into a point-of-care coagulation test with practical impact.
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Microfluídica , Sistemas de Atención de Punto , Coagulación Sanguínea , Pruebas de Coagulación Sanguínea , Viscosidad Sanguínea , Microfluídica/métodosRESUMEN
The monitoring of coagulation function has great implications in many clinical settings. However, existing coagulation assays are simplex, sample-consuming, and slow in turnaround, making them less suitable for point-of-care testing. In this work, we developed a novel blood coagulation assay that simultaneously assesses both the tendency of clotting and the stiffness of the resultant clot using printed circuit board (PCB)-based digital microfluidics. A drop of blood was actuated to move back and forth on the PCB electrode array, until the motion winded down as the blood coagulated and became thicker. The velocity tracing and the deformation of the clot were calculated via image analysis to reflect the coagulation progression and the clot stiffness, respectively. We investigated the effect of different hardware and biochemical settings on the assay results. To validate the assay, we performed assays on blood samples with hypo- and hyper-coagulability, and the results confirmed the assay's capability in distinguishing different blood samples. We then examined the correlation between the measured metrics in our assays and standard coagulation assays, namely prothrombin time and fibrinogen level, and the high correlation supported the clinical relevance of our assay. We envision that this method would serve as a powerful point-of-care coagulation testing method.
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Sistemas de Atención de Punto , Trombosis , Coagulación Sanguínea , Pruebas de Coagulación Sanguínea/métodos , Humanos , MicrofluídicaRESUMEN
Digital nucleic acid amplification tests enable absolute quantification of nucleic acids, but the generation of uniform compartments and reading of the fluorescence requires specialized instruments that are costly, limiting their widespread applications. Here, the authors report deep learning-enabled polydisperse emulsion-based digital loop-mediated isothermal amplification (deep-dLAMP) for label-free, low-cost nucleic acid quantification. deep-dLAMP performs LAMP reaction in polydisperse emulsions and uses a deep learning algorithm to segment and determine the occupancy status of each emulsion in images based on precipitated byproducts. The volume and occupancy data of the emulsions are then used to infer the nucleic acid concentration based on the Poisson distribution. deep-dLAMP can accurately predict the sizes and occupancy status of each emulsion and provide accurate measurements of nucleic acid concentrations with a limit of detection of 5.6 copies µl-1 and a dynamic range of 37.2 to 11000 copies µl-1 . In addition, deep-dLAMP shows robust performance under various parameters, such as the vortexing time and image qualities. Leveraging the state-of-the-art deep learning models, deep-dLAMP represents a significant advancement in digital nucleic acid tests by significantly reducing the instrument cost. We envision deep-dLAMP would be readily adopted by biomedical laboratories and be developed into a point-of-care digital nucleic acid test system.
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The lack of an efficient method for the identification of tumor antigen-specific T cell receptors (TCRs) impedes the development of T cell-based cancer immunotherapies. Here, we introduce a droplet-based microfluidic platform for function-based screening and sorting of tumor antigen-specific T cells with high throughput. We built a reporter cell line by co-transducing the TCR library and reporter genes at the downstream of TCR signaling, and reporter cells fluoresced upon functionally binding with antigens. We co-encapsulated reporter cells and antigen-presenting cells in droplets to allow for stimulation on a single-cell level. Functioning reporter cells specific against the antigen were identified in the microfluidic channel based on the fluorescent signals of the droplets, which were immediately sorted out using dielectrophoresis. We validated the reporter system and sorting results using flow cytometry. We then performed single-cell RNA sequencing on the sorted cells to further validate this platform and demonstrate the compatibility with genetic characterizations. Our platform provides a means for precise and efficient T cell immunotherapy, and the droplet-based high-throughput TCR screening method could potentially facilitate immunotherapeutic screening and promote T cell-based anti-tumor therapies.
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Microfluídica , Linfocitos T , Antígenos de Neoplasias/metabolismo , Citometría de Flujo , Ensayos Analíticos de Alto Rendimiento , Microfluídica/métodos , Linfocitos T/metabolismoRESUMEN
Embryonic development is a fundamental physiological process that can provide tremendous insights into stem cell biology and regenerative medicine. In this process, cell fate decision is highly heterogeneous and dynamic, and investigations at the single-cell level can greatly facilitate the understanding of the molecular roadmap of embryonic development. Rapid advances in the technology of single-cell sequencing offer a perfectly useful tool to fulfill this purpose. Despite its great promise, single-cell sequencing is highly interdisciplinary, and successful applications in specific biological contexts require a general understanding of its diversity as well as the advantage versus limitations for each of its variants. Here, the technological principles of single-cell sequencing are consolidated and its applications in the study of embryonic development are summarized. First, the technology basics are presented and the available tools for each step including cell isolation, library construction, sequencing, and data analysis are discussed. Then, the works that employed single-cell sequencing are reviewed to investigate the specific processes of embryonic development, including preimplantation, peri-implantation, gastrulation, and organogenesis. Further, insights are provided on existing challenges and future research directions.
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Desarrollo Embrionario/fisiología , Análisis de la Célula Individual , Diferenciación Celular , Implantación del Embrión , Desarrollo Embrionario/genética , Femenino , Humanos , Organogénesis , EmbarazoRESUMEN
Diagnostic tools play significant roles in the fight against COVID-19 and other pandemics. Existing tests, such as RT-qPCR, have limitations including long assay time, low throughput, inadequate sensitivity, and suboptimal portability. Emerging biosensing technologies hold the promise to develop tests that are rapid, highly sensitive, and suitable for point-of-care testing, which could significantly facilitate the testing of COVID-19. Despite that, practical applications of such biosensors in pandemics have yet to be achieved. In this review, we consolidate the newly developed diagnostic tools for COVID-19 using emerging biosensing technologies and discuss their application promise. In particular, we present nucleic acid tests and antibody tests of COVID-19 based on both conventional and emerging biosensing methods. We then provide perspectives on the existing challenges and potential solutions.
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Técnicas Biosensibles/métodos , COVID-19/diagnóstico , ARN Viral/genética , SARS-CoV-2/genética , COVID-19/epidemiología , COVID-19/virología , Prueba de COVID-19/métodos , Humanos , Pandemias , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Reproducibilidad de los Resultados , SARS-CoV-2/fisiología , Sensibilidad y EspecificidadRESUMEN
During blood clotting, clot retraction alters its mechanical properties and critically affects hemostasis. Despite that, existing clot retraction assays hold limitations such as large footprint and low throughput. In this work, we report the design of flexural post rings for a miniaturized assay of clot retraction force (CRF) with high throughput. Leveraging surface tensions, the post rings hold blood samples in a highly reproducible fashion while simultaneously serving as cantilever beams to measure the CRF. We investigated the effect on the device performance of major parameters, namely, surface hydrophobicity, post number, and post stiffness. We then tested the devices using 14 patient samples and revealed the correlation between CRF and fibrinogen levels. We further implemented an automated liquid handler and developed a high-throughput platform for clot retraction assay. The device's small sample consumption, simple operation, and good compatibility with existing automation facilities make it a promising high-throughput clot retraction assay.
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Coagulación Sanguínea , Pruebas de Coagulación Sanguínea , Retracción del Coagulo , HumanosRESUMEN
Exposure to estrogen is strongly associated with increased breast cancer risk. While all women are exposed to estrogen, only 12% are expected to develop breast cancer during their lifetime. These women may be more sensitive to estrogen, as rodent models have demonstrated variability in estrogen sensitivity. Our objective was to determine individual variation in expression of estrogen receptor (ER) and estrogen-induced responses in the normal human breast. Human breast tissue from female donors undergoing reduction mammoplasty surgery were collected for microarray analysis of ER expression. To examine estrogen-induced responses, breast tissue from 23 female donors were cultured ex- vivo in basal or 10 nM 17ß-estradiol (E2) media for 4 days. Expression of ER genes (ESR1 and ESR2) increased significantly with age. E2 induced consistent increases in global gene transcription, but expression of target genes AREG, PGR, and TGFß2 increased significantly only in explants from nulliparous women. E2-treatment did not induce consistent changes in proliferation or radiation induced apoptosis. Responses to estrogen are highly variable among women and not associated with levels of ER expression, suggesting differences in intracellular signaling among individuals. The differences in sensitivity to E2-stimulated responses may contribute to variation in risk of breast cancer.
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Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/patología , Estrógenos/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Receptores de Estrógenos/metabolismo , Adolescente , Adulto , Anciano , Apoptosis , Biomarcadores de Tumor/genética , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Proliferación Celular , Femenino , Humanos , Persona de Mediana Edad , Pronóstico , Receptores de Estrógenos/genética , Células Tumorales Cultivadas , Adulto JovenRESUMEN
Early human embryonic development involves extensive lineage diversification, cell-fate specification and tissue patterning1. Despite its basic and clinical importance, early human embryonic development remains relatively unexplained owing to interspecies divergence2,3 and limited accessibility to human embryo samples. Here we report that human pluripotent stem cells (hPSCs) in a microfluidic device recapitulate, in a highly controllable and scalable fashion, landmarks of the development of the epiblast and amniotic ectoderm parts of the conceptus, including lumenogenesis of the epiblast and the resultant pro-amniotic cavity, formation of a bipolar embryonic sac, and specification of primordial germ cells and primitive streak cells. We further show that amniotic ectoderm-like cells function as a signalling centre to trigger the onset of gastrulation-like events in hPSCs. Given its controllability and scalability, the microfluidic model provides a powerful experimental system to advance knowledge of human embryology and reproduction. This model could assist in the rational design of differentiation protocols of hPSCs for disease modelling and cell therapy, and in high-throughput drug and toxicity screens to prevent pregnancy failure and birth defects.
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Amnios/embriología , Estratos Germinativos/embriología , Modelos Biológicos , Células Madre Pluripotentes/citología , Amnios/citología , Diferenciación Celular , Embrión de Mamíferos/citología , Embrión de Mamíferos/embriología , Femenino , Estratos Germinativos/citología , Humanos , Embarazo , Línea Primitiva/citologíaRESUMEN
During early post-implantation human embryogenesis, the epiblast (EPI) within the blastocyst polarizes to generate a cyst with a central lumen. Cells at the uterine pole of the EPI cyst then undergo differentiation to form the amniotic ectoderm (AM), a tissue essential for further embryonic development. While the causes of early pregnancy failure are complex, improper lumenogenesis or amniogenesis of the EPI represent possible contributing factors. Here we report a novel AM microtissue array platform that allows quantitative phenotyping of lumenogenesis and amniogenesis of the EPI and demonstrate its potential application for embryonic toxicity profiling. Specifically, a human pluripotent stem cell (hPSC)-based amniogenic differentiation protocol was developed using a two-step micropatterning technique to generate a regular AM microtissue array with defined tissue sizes. A computer-assisted analysis pipeline was developed to automatically process imaging data and quantify morphological and biological features of AM microtissues. Analysis of the effects of cell density, cyst size and culture conditions revealed a clear connection between cyst size and amniogenesis of hPSC. Using this platform, we demonstrated that pharmacological inhibition of ROCK signaling, an essential mechanotransductive pathway, suppressed lumenogenesis but did not perturb amniogenic differentiation of hPSC, suggesting uncoupled regulatory mechanisms for AM morphogenesis vs. cytodifferentiation. The AM microtissue array was further applied to screen a panel of clinically relevant drugs, which successfully detected their differential teratogenecity. This work provides a technological platform for toxicological screening of clinically relevant drugs for their effects on lumenogenesis and amniogenesis during early human peri-implantation development, processes that have been previously inaccessible to study.
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Amnios/citología , Evaluación Preclínica de Medicamentos , Ectodermo/citología , Células Madre Pluripotentes/citología , Análisis de Matrices Tisulares , Amnios/efectos de los fármacos , Amnios/metabolismo , Línea Celular , Evaluación Preclínica de Medicamentos/métodos , Ectodermo/efectos de los fármacos , Ectodermo/metabolismo , Humanos , Células Madre Pluripotentes/efectos de los fármacos , Células Madre Pluripotentes/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Análisis de Matrices Tisulares/métodos , Ingeniería de Tejidos/métodos , Quinasas Asociadas a rho/antagonistas & inhibidoresRESUMEN
Droplet microfluidics-based platform (Drop-seq) has been shown to be a powerful tool for single cell expression profiling. Nevertheless, this platform required the simultaneous encapsulation of single cell and single barcoded bead, the incidence of which was very low, limiting its efficiency. Spiral channels were reported to focus the barcoded beads and thus increased the efficiency, but focusing of cells was not demonstrated, which could potentially further enhance the performance. Here, we designed spiral and serpentine channels to focus both bead and cell solutions and implemented this microfluidic design on Drop-seq. We characterized the effect of cell/bead concentration on encapsulation results and tested the performance by coencapsulating barcoded beads and human-mouse cell mixtures followed by sequencing. The results showed â¼300% and â¼40% increase in cell utilization rate compared to the traditional Drop-seq device and the device focusing beads alone, respectively. This chip design showed great potential for high efficiency single cell expression profiling.