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Basophils are the rarest circulating white blood cells (WBCs), but they play important roles in allergic disorders and other diseases. To enhance diagnostic capabilities, it would be desirable to isolate and analyze basophils efficiently from small blood samples. In 100 µL of whole blood, there are typically ~103 basophils, outnumbered by ~105 WBCs and ~108 red blood cells (RBCs). Basophils' low abundance has therefore presented a significant challenge in their isolation from whole blood. Conventional in-bulk basophil isolation methods require lengthy processing steps and cannot work with small volumes of blood. Here we report a parallelized integrated basophil isolation device (pi-BID) for the negative immunomagnetic selection of basophils directly from 4 samples of 100 µL of whole blood, in parallel, within 14 minutes including sample preparation time. The pi-BID interfaces directly with standard sample tubes, and uses a single pressure source to drive the flow in parallel microfluidic channels. Compared with conventional in-bulk basophil isolation, the pi-BID is >3× faster, and has higher purity (~93%) and similar recovery (~67%). Compared with other microfluidic devices for the immunomagnetic isolation of WBC sub-types, our pi-BID achieves 10× higher enrichment of target cells from whole blood, with no prior removal of RBCs necessary.
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The ability to regenerate after the loss of a part is a hallmark of living systems and occurs at both the tissue and organ scales, but also within individual cells. Regeneration entails many processes that are physical and mechanical in nature, including the closure of wounds, the repositioning of material from one place to another, and the restoration of symmetry following perturbations. However, we currently know far more about the genetics and molecular signaling pathways involved in regeneration, and there is a need to investigate the role of physical forces in the process. Here, we will provide an overview of how physical forces may play a role in wound healing and regeneration, in which we compare and contrast regenerative processes at the tissue and cell scales.
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Ciliates are powerful unicellular model organisms that have been used to elucidate fundamental biological processes. However, the high motility of ciliates presents a major challenge in studies using live-cell microscopy and microsurgery. While various immobilization methods have been developed, they are physiologically disruptive to the cell and incompatible with microscopy and/or microsurgery. Here, we describe a Simple Microfluidic Operating Room for the Examination and Surgery of Stentor coeruleus (SMORES). SMORES uses Quake valve-based microfluidics to trap, compress, and perform surgery on Stentor as our model ciliate. Compared with previous methods, immobilization by physical compression in SMORES is more effective and uniform. The mean velocity of compressed cells is 24 times less than that of uncompressed cells. The compression is minimally disruptive to the cell and is easily applied or removed using a 3D-printed pressure rig. We demonstrate cell immobilization for up to 2 h without sacrificing cell viability. SMORES is compatible with confocal microscopy and is capable of media exchange for pharmacokinetic studies. Finally, the modular design of SMORES allows laser ablation or mechanical dissection of a cell into many cell fragments at once. These capabilities are expected to enable biological studies previously impossible in ciliates and other motile species.
Sujet(s)
Ciliophora , Microfluidique , Blocs opératoires , Ciliophora/physiologieRÉSUMÉ
Ciliates are powerful unicellular model organisms that have been used to elucidate fundamental biological processes. However, the high motility of ciliates presents a major challenge in studies using live-cell microscopy and microsurgery. While various immobilization methods have been developed, they are physiologically disruptive to the cell and incompatible with microscopy and/or microsurgery. Here, we describe a Simple Microfluidic Operating Room for the Examination and Surgery of Stentor coeruleus (SMORES). SMORES uses Quake valve-based microfluidics to trap, compress, and perform surgery on Stentor as our model ciliate. Compared with previous methods, immobilization by physical compression in SMORES is more effective and uniform. The mean velocity of compressed cells is 24 times less than that of uncompressed cells. The compression is minimally disruptive to the cell and is easily applied or removed using a 3D-printed pressure rig. We demonstrate cell immobilization for up to 2 hours without sacrificing cell viability. SMORES is compatible with confocal microscopy and is capable of media exchange for pharmacokinetic studies. Finally, the modular design of SMORES allows laser ablation or mechanical dissection of a cell into many cell fragments at once. These capabilities are expected to enable biological studies previously impossible in ciliates and other motile species.
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BACKGROUND: Conventional basophil activation tests (BATs) measure basophil activation by the increased expression of CD63. Previously, fluorophore-labeled avidin, a positively-charged molecule, was found to bind to activated basophils, which tend to expose negatively charged granule constituents during degranulation. This study further compares avidin versus CD63 as basophil activation biomarkers in classifying peanut allergy. METHODS: Seventy subjects with either a peanut allergy (N = 47), a food allergy other than peanut (N = 6), or no food allergy (N = 17) were evaluated. We conducted BATs in response to seven peanut extract (PE) concentrations (0.01-10,000 ng/mL) and four control conditions (no stimulant, anti-IgE, fMLP (N-formylmethionine-leucyl-phenylalanine), and anti-FcεRI). We measured avidin binding and CD63 expression on basophils with flow cytometry. We evaluated logistic regression and XGBoost models for peanut allergy classification and feature identification. RESULTS: Avidin binding was correlated with CD63 expression. Both markers discriminated between subjects with and without a peanut allergy. Although small by percentage, an avidin+ /CD63- cell subset was found in all allergic subjects tested, indicating that the combination of avidin and CD63 could allow a more comprehensive identification of activated basophils. Indeed, we obtained the best classification accuracy (97.8% sensitivity, 96.7% specificity) by combining avidin and CD63 across seven PE doses. Similar accuracy was obtained by combining PE dose of 10,000 ng/mL for avidin and PE doses of 10 and 100 ng/mL for CD63. CONCLUSIONS: Avidin and CD63 are reliable BAT activation markers associated with degranulation. Their combination enhances the identification of activated basophils and improves the classification accuracy of peanut allergy.
Sujet(s)
Test de dégranulation des basophiles , Hypersensibilité aux arachides , Humains , Hypersensibilité aux arachides/diagnostic , Hypersensibilité aux arachides/métabolisme , Avidine/métabolisme , Immunoglobuline E/métabolisme , Granulocytes basophiles/métabolisme , Cytométrie en flux , Arachis , Antigène CD63/métabolismeRÉSUMÉ
The giant ciliate Stentor coeruleus is a classical model system for studying regeneration and morphogenesis in a single cell. The anterior of the cell is marked by an array of cilia, known as the oral apparatus, which can be induced to shed and regenerate in a series of reproducible morphological steps, previously shown to require transcription. If a cell is cut in half, each half regenerates an intact cell. We used RNA sequencing (RNAseq) to assay the dynamic changes in Stentor's transcriptome during regeneration, after both oral apparatus shedding and bisection, allowing us to identify distinct temporal waves of gene expression including kinases, RNA -binding proteins, centriole biogenesis factors, and orthologs of human ciliopathy genes. By comparing transcriptional profiles of different regeneration events, we identified distinct modules of gene expression corresponding to oral apparatus regeneration, posterior holdfast regeneration, and recovery after wounding. By measuring gene expression after blocking translation, we show that the sequential waves of gene expression involve a cascade mechanism in which later waves of expression are triggered by translation products of early-expressed genes. Among the early-expressed genes, we identified an E2F transcription factor and the RNA-binding protein Pumilio as potential regulators of regeneration based on the expression pattern of their predicted target genes. RNAi-mediated knockdown experiments indicate that Pumilio is required for regenerating oral structures of the correct size. E2F is involved in the completion of regeneration but is dispensable for earlier steps. This work allows us to classify regeneration genes into groups based on their potential role for regeneration in distinct cell regeneration paradigms, and provides insight into how a single cell can coordinate complex morphogenetic pathways to regenerate missing structures.
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Ciliophora , Séquence nucléotidique , Ciliophora/génétique , Humains , Interférence par ARN , Analyse de séquence d'ARN , TranscriptomeRÉSUMÉ
Stentor coeruleus, a single-cell ciliated protozoan, is a model organism for wound healing and regeneration studies. Despite Stentor's large size (up to 2 mm in extended state), microdissection of Stentor remains challenging. In this work, we describe a hydrodynamic cell splitter, consisting of a microfluidic cross junction, capable of splitting Stentor cells in a non-contact manner at a high throughput of â¼500 cells per minute under continuous operation. Introduction of asymmetry in the flow field at the cross junction leads to asymmetric splitting of the cells to generate cell fragments as small as â¼8.5 times the original cell size. Characterization of cell fragment viability shows reduced 5-day survival as fragment size decreases and as the extent of hydrodynamic stress imposed on the fragments increases. Our results suggest that cell fragment size and composition, as well as mechanical stress, play important roles in the long-term repair of Stentor cells and warrant further investigations. Nevertheless, the hydrodynamic splitter can be useful for studying phenomena immediately after cell splitting, such as the closure of wounds in the plasma membrane which occurs on the order of 100-1000 seconds in Stentor.
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Ciliophora , Microfluidique , Membrane cellulaire , HydrodynamiqueRÉSUMÉ
Despite their rarity in peripheral blood, basophils play important roles in allergic disorders and other diseases including sepsis and COVID-19. Existing basophil isolation methods require many manual steps and suffer from significant variability in purity and recovery. We report an integrated basophil isolation device (i-BID) in microfluidics for negative immunomagnetic selection of basophils directly from 100 µL of whole blood within 10 minutes. We use a simulation-driven pipeline to design a magnetic separation module to apply an exponentially increasing magnetic force to capture magnetically tagged non-basophils flowing through a microtubing sandwiched between magnetic flux concentrators sweeping across a Halbach array. The exponential profile captures non-basophils effectively while preventing their excessive initial buildup causing clogging. The i-BID isolates basophils with a mean purity of 93.9% ± 3.6% and recovery of 95.6% ± 3.4% without causing basophil degradation or unintentional activation. Our i-BID has the potential to enable basophil-based point-of-care diagnostics such as rapid allergy assessment.
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COVID-19 , Hypersensibilité , Granulocytes basophiles , Humains , Hypersensibilité/diagnostic , Numération des leucocytes , MicrofluidiqueRÉSUMÉ
Recent far-reaching advances in synthetic biology have yielded exciting tools for the creation of new materials. Conversely, advances in the fundamental understanding of soft-condensed matter, polymers and biomaterials offer new avenues to extend the reach of synthetic biology. The broad and exciting range of possible applications have substantial implications to address grand challenges in health, biotechnology and sustainability. Despite the potentially transformative impact that lies at the interface of synthetic biology and biomaterials, the two fields have, so far, progressed mostly separately. This Perspective provides a review of recent key advances in these two fields, and a roadmap for collaboration at the interface between the two communities. We highlight the near-term applications of this interface to the development of hierarchically structured biomaterials, from bioinspired building blocks to 'living' materials that sense and respond based on the reciprocal interactions between materials and embedded cells.
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Matériaux biocompatibles , Biologie synthétique , PolymèresRÉSUMÉ
Microscale surgery on single cells and small organisms has enabled major advances in fundamental biology and in engineering biological systems. Examples of applications range from wound healing and regeneration studies to the generation of hybridoma to produce monoclonal antibodies. Even today, these surgical operations are often performed manually, but they are labor intensive and lack reproducibility. Microfluidics has emerged as a powerful technology to control and manipulate cells and multicellular systems at the micro- and nanoscale with high precision. Here, we review the physical and chemical mechanisms of microscale surgery and the corresponding design principles, applications, and implementations in microfluidic systems. We consider four types of surgical operations: (1) sectioning, which splits a biological entity into multiple parts, (2) ablation, which destroys part of an entity, (3) biopsy, which extracts materials from within a living cell, and (4) fusion, which joins multiple entities into one. For each type of surgery, we summarize the motivating applications and the microfluidic devices developed. Throughout this review, we highlight existing challenges and opportunities. We hope that this review will inspire scientists and engineers to continue to explore and improve microfluidic surgical methods.
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Techniques d'analyse microfluidique , Microfluidique , Ingénierie , Laboratoires sur puces , Microfluidique/méthodes , Reproductibilité des résultatsRÉSUMÉ
Micro-blade design is an important factor in the cutting of single cells and other biological structures. This paper describes the fabrication process of three-dimensional (3D) micro-blades for the cutting of single cells in a microfluidic "guillotine" intended for fundamental wound repair and regeneration studies. Our microfluidic guillotine consists of a fixed 3D micro-blade centered in a microchannel to bisect cells flowing through. We show that the Nanoscribe two-photon polymerization direct laser writing system is capable of fabricating complex 3D micro-blade geometries. However, structures made of the Nanoscribe IP-S resin have low adhesion to silicon, and they tend to peel off from the substrate after at most two times of replica molding in poly(dimethylsiloxane) (PDMS). Our work demonstrates that the use of a secondary mold replicates Nanoscribe-printed features faithfully for at least 10 iterations. Finally, we show that complex micro-blade features can generate different degrees of cell wounding and cell survival rates compared with simple blades possessing a vertical cutting edge fabricated with conventional 2.5D photolithography. Our work lays the foundation for future applications in single cell analyses, wound repair and regeneration studies, as well as investigations of the physics of cutting and the interaction between the micro-blade and biological structures.
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When granular materials, colloidal suspensions, and even animals and crowds exit through a narrow outlet, clogs can form spontaneously when multiple particles or entities attempt to exit simultaneously, thereby obstructing the outlet and ultimately halting the flow. Counterintuitively, the presence of an obstacle upstream of the outlet has been found to suppress clog formation. For soft particles such as emulsion drops, clogging has not been observed in the fast flow limit due to their deformability and vanishing interparticle friction. Instead, they pinch off each other and undergo break up when multiple drops attempt to exit simultaneously. Similar to how an obstacle reduces clogging in a rigid particle system, we hypothesize and demonstrate that an obstacle could suppress break up in the two-dimensional hopper flow of a microfluidic crystal consisting of dense emulsion drops by preventing the simultaneous exit of multiple drops. A regime map plotting the fraction of drops that undergo break up in a channel with different obstacle sizes and locations delineates the geometrical constraints necessary for effective break up suppression. When optimally placed, the obstacle induced an unexpected ordering of the drops, causing them to alternate and exit the outlet one at a time. Droplet break up is suppressed drastically by almost three orders of magnitude compared to when the obstacle is absent. This result can provide a simple, passive strategy to prevent droplet break up and can find use in improving the robustness and integrity of droplet microfluidic biochemical assays as well as in extrusion-based three-dimensional printing of emulsion or foam-based materials.
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BACKGROUND: Wound healing is one of the defining features of life and is seen not only in tissues but also within individual cells. Understanding wound response at the single-cell level is critical for determining fundamental cellular functions needed for cell repair and survival. This understanding could also enable the engineering of single-cell wound repair strategies in emerging synthetic cell research. One approach is to examine and adapt self-repair mechanisms from a living system that already demonstrates robust capacity to heal from large wounds. Towards this end, Stentor coeruleus, a single-celled free-living ciliate protozoan, is a unique model because of its robust wound healing capacity. This capacity allows one to perturb the wounding conditions and measure their effect on the repair process without immediately causing cell death, thereby providing a robust platform for probing the self-repair mechanism. RESULTS: Here we used a microfluidic guillotine and a fluorescence-based assay to probe the timescales of wound repair and of mechanical modes of wound response in Stentor. We found that Stentor requires ~ 100-1000 s to close bisection wounds, depending on the severity of the wound. This corresponds to a healing rate of ~ 8-80 µm2/s, faster than most other single cells reported in the literature. Further, we characterized three distinct mechanical modes of wound response in Stentor: contraction, cytoplasm retrieval, and twisting/pulling. Using chemical perturbations, active cilia were found to be important for only the twisting/pulling mode. Contraction of myonemes, a major contractile fiber in Stentor, was surprisingly not important for the contraction mode and was of low importance for the others. CONCLUSIONS: While events local to the wound site have been the focus of many single-cell wound repair studies, our results suggest that large-scale mechanical behaviors may be of greater importance to single-cell wound repair than previously thought. The work here advances our understanding of the wound response in Stentor and will lay the foundation for further investigations into the underlying components and molecular mechanisms involved.
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Ciliophora/physiologie , Microfluidique , Régénération , Cicatrisation de plaieRÉSUMÉ
Inanimate objects or surfaces contaminated with infectious agents, referred to as fomites, play an important role in the spread of viruses, including SARS-CoV-2, the virus responsible for the COVID-19 pandemic. The long persistence of viruses (hours to days) on surfaces calls for an urgent need for effective surface disinfection strategies to intercept virus transmission and the spread of diseases. Elucidating the physicochemical processes and surface science underlying the adsorption and transfer of virus between surfaces, as well as their inactivation, is important for understanding how diseases are transmitted and for developing effective intervention strategies. This review summarizes the current knowledge and underlying physicochemical processes of virus transmission, in particular via fomites, and common disinfection approaches. Gaps in knowledge and the areas in need of further research are also identified. The review focuses on SARS-CoV-2, but discussion of related viruses is included to provide a more comprehensive review given that much remains unknown about SARS-CoV-2. Our aim is that this review will provide a broad survey of the issues involved in fomite transmission and intervention to a wide range of readers to better enable them to take on the open research challenges.
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Integrated bioassay systems that combine microfluidics and radiation detectors can deliver medical radiopharmaceuticals to live cells with precise timing, while minimizing radiation dose and sample volume. However, the spatial resolution of many radiation imaging systems is limited to bulk cell populations. Here, we demonstrate microfluidics-coupled radioluminescence microscopy (µF-RLM), a new integrated system that can image radiotracer uptake in live adherent cells growing inside microincubators with spatial resolution better than 30 µm. Our method enables on-chip radionuclide imaging by incorporating an inorganic scintillator plate (CdWO4) into a microfluidic chip. We apply this approach to investigate the factors that influence the dynamic uptake of [18F]fluorodeoxyglucose (FDG) by cancer cells. In the first experiment, we measured the effect of flow on FDG uptake of cells and found that a continuous flow of the radiotracer led to fourfold higher uptake than static incubation, suggesting that convective replenishment enhances molecular radiotracer transport into cells. In the second set of experiments, we applied pharmacokinetic modeling to show that lactic acidosis inhibits FDG uptake by cancer cells in vitro and that this decrease is primarily due to downregulation of FDG transport into the cells. The other two rate constants, which represent FDG export and FDG metabolism, were relatively unaffected by lactic acidosis. Lactic acidosis is common in solid tumors because of the dysregulated metabolism and inefficient vasculature. In conclusion, µF-RLM is a simple and practical approach for integrating high-resolution radionuclide imaging within standard microfluidics devices, thus potentially opening venues for investigating the efficacy of radiopharmaceuticals in in vitro cancer models.
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Microfluidique , Microscopie , Fluorodésoxyglucose F18 , Cinétique , Tomographie par émission de positons , RadiopharmaceutiquesRÉSUMÉ
Food allergy has reached epidemic proportions and has become a significant source of healthcare burden. Oral food challenge, the gold standard for food allergy assessment, often is not performed because it places the patient at risk of developing anaphylaxis. However, conventional alternative food allergy tests lack a sufficient predictive value. Therefore, there is a critical need for better diagnostic tests that are both accurate and safe. Microfluidic methods have the potential of helping one to address such needs and to personalize the diagnostics. This article first reviews conventional diagnostic approaches used in food allergy. Second, it reviews recent efforts to develop novel biomarkers and in vitro diagnostics. Third, it summarizes the microfluidic methods developed thus far for food allergy diagnosis. The article concludes with a discussion of future opportunities for using microfluidic methods for achieving precision diagnostics in food allergy, including multiplexing the detection of multiple biomarkers, sampling of tissue-resident cytokines and immune cells, and multi-organ-on-a-chip technology.
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Transcription polymerases can exhibit an unusual mode of regenerating certain RNA templates from RNA, yielding systems that can replicate and evolve with RNA as the information carrier. Two classes of pathogenic RNAs (hepatitis delta virus in animals and viroids in plants) are copied by host transcription polymerases. Using in vitro RNA replication by the transcription polymerase of T7 bacteriophage as an experimental model, we identify hundreds of new replicating RNAs, define three mechanistic hallmarks of replication (subterminal de novo initiation, RNA shape-shifting, and interrupted rolling-circle synthesis), and describe emergence from DNA seeds as a mechanism for the origin of novel RNA replicons. These results inform models for the origins and replication of naturally occurring RNA genetic elements and suggest a means by which diverse RNA populations could be propagated as hereditary material in cellular contexts.
Sujet(s)
DNA-directed RNA polymerases/métabolisme , ARN/biosynthèse , Réplicon , Transcription génétique , Protéines virales/métabolisme , BiocatalyseRÉSUMÉ
In soft matter consisting of many deformable objects, object shapes often carry important information about local forces and their interactions with the local environment, and can be tightly coupled to the bulk properties and functions. In a concentrated emulsion, for example, the shapes of individual droplets are directly related to the local stress arising from interactions with neighboring drops, which in turn determine their stability and the resulting rheological properties. Shape descriptors used in prior work on single drops and dilute emulsions, where droplet-droplet interactions are largely negligible and the drop shapes are simple, are insufficient to fully capture the broad range of droplet shapes in a concentrated system. This paper describes the application of a machine learning method, specifically a convolutional autoencoder model, that learns to: (1) discover a low-dimensional code (8-dimensional) to describe droplet shapes within a concentrated emulsion, and (2) predict whether the drop will become unstable and undergo break-up. The input consists of images (N = 500 002) of two-dimensional droplet boundaries extracted from movies of a concentrated emulsion flowing through a confined microfluidic channel as a monolayer. The model is able to faithfully reconstruct droplet shapes, as well as to achieve a classification accuracy of 91.7% in the prediction of droplet break-up, compared with â¼60% using conventional scalar descriptors based on droplet elongation. It is observed that 4 out of the 8 dimensions of the code are interpretable, corresponding to drop skewness, elongation, throat size, and surface curvature, respectively. Furthermore, the results show that drop elongation, throat size, and surface curvature are dominant factors in predicting droplet break-up for the flow conditions tested. The method presented is expected to facilitate follow-on work to identify the relationship between drop shapes and the interactions with other drops, and to identify potentially new modes of break-up mechanisms in a concentrated system. Finally, the method developed here should also apply to other soft materials such as foams, gels, and cells and tissues.
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This book chapter describes the use of droplet microfluidics to phenotype single cells. The basic process flow includes the encapsulation of single cells with a specific probe into aqueous micro-droplets suspended in a biocompatible oil. The probe is chosen to measure the phenotype of interest. After incubation, the encapsulated cell turns the probe fluorescent and renders the entire droplet fluorescent. Enumerating drops that are fluorescent quantifies the concentration of cells possessing the phenotype of interest. Examining the distribution of fluorescence further allows one to quantify the heterogeneity among the cell population.