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  • NLM标题: Biomicrofluidics
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1461条结果
  • 机译 带电纳米粒子抑制高剪切动脉血栓形成
    摘要:Platelet accumulation under high shear rates at the site of atherosclerotic plaque rupture leads to myocardial infarction and stroke. Current antiplatelet therapies remain ineffective within a large percentage of the population, while presenting significant risks for bleeding. We explore a novel way to inhibit arterial thrombus formation by biophysical means without the use of platelet inactivating drugs. Our computational multi-scale dynamics model has predicted that charged particles of a specific size may entangle von Willebrand Factor (vWF) polymers and reduce the amount of elongation at high shear rates. We tested this hypothesis experimentally for negatively charged nanoparticles (CNP) to inhibit arterial thrombus formation. CNP of a particular size and charge inhibited thrombus formation, with a 10-fold peak inhibition over control conditions of thrombotic occlusion. Particles of differing material composition, size, and charge had little effect as predicted by computational studies. Surprisingly, the dose response curve was not sigmoidal, but exhibited a peak at 1.5 CNP:vWF proteins, which was not predicted by the model. This study describes a new antithrombotic agent that may have a different mechanism of action than current pharmaceutical therapies.
  • 机译 液体栓塞诱发的机械性气道损伤的生物启发性微流模型
    摘要:Occlusion of distal airways due to mucus plugs is a key pathological feature common to a wide variety of obstructive pulmonary diseases. Breathing-induced movement of airway mucus plugs along the respiratory tract has been shown to generate abnormally large mechanical stresses, acting as an insult that can incite acute injury to the airway epithelium. Here, we describe a unique microengineering strategy to model this pathophysiological process using a bioinspired microfluidic device. Our system combines an air-liquid interface culture of primary human small airway epithelial cells with a microengineered biomimetic platform to replicate the process of mucus exudation induced by airway constriction that leads to the formation of mucus plugs across the airway lumen. Specifically, we constructed a compartmentalized three-dimensional (3D) microfluidic device in which extracellular matrix hydrogel scaffolds reminiscent of airway stroma were compressed to discharge fluid into the airway compartment and form liquid plugs. We demonstrated that this plug formation process and subsequent movement of liquid plugs through the airway channel can be regulated in a precisely controlled manner. Furthermore, we examined the detrimental effect of plug propagation on the airway epithelium to simulate acute epithelial injury during airway closure. Our system allows for a novel biomimetic approach to modeling a complex and dynamic biophysical microenvironment of diseased human airways and may serve as an enabling platform for mechanistic investigation of key disease processes that drive the progression and exacerbation of obstructive pulmonary diseases.
  • 机译 组织切片中的快速微荧光原位杂交
    摘要:This paper describes a micro fluorescence in situ hybridization (μFISH)-based rapid detection of cytogenetic biomarkers on formalin-fixed paraffin embedded (FFPE) tissue sections. We demonstrated this method in the context of detecting human epidermal growth factor 2 (HER2) in breast tissue sections. This method uses a non-contact microfluidic scanning probe (MFP), which localizes FISH probes at the micrometer length-scale to selected cells of the tissue section. The scanning ability of the MFP allows for a versatile implementation of FISH on tissue sections. We demonstrated the use of oligonucleotide FISH probes in ethylene carbonate-based buffer enabling rapid hybridization within <1 min for chromosome enumeration and 10–15 min for assessment of the HER2 status in FFPE sections. We further demonstrated recycling of FISH probes for multiple sequential tests using a defined volume of probes by forming hierarchical hydrodynamic flow confinements. This microscale method is compatible with the standard FISH protocols and with the Instant Quality FISH assay and reduces the FISH probe consumption ∼100-fold and the hybridization time 4-fold, resulting in an assay turnaround time of <3 h. We believe that rapid μFISH has the potential of being used in pathology workflows as a standalone method or in combination with other molecular methods for diagnostic and prognostic analysis of FFPE sections.
  • 机译 跨血脑屏障的分子和纳米颗粒运输的体外模型
    摘要:The blood-brain barrier (BBB) is the tightest endothelial barrier in humans. Characterized by the presence of tight endothelial junctions and adherens junctions, the primary function of the BBB is to maintain brain homeostasis through the control of solute transit across the barrier. The specific features of this barrier make for unique modes of transport of solutes, nanoparticles, and cells across the BBB. Understanding the different routes of traffic adopted by each of these is therefore critical in the development of targeted therapies. In an attempt to move towards controlled experimental assays, multiple groups are now opting for the use of microfluidic systems. A comprehensive understanding of bio-transport processes across the BBB in microfluidic devices is therefore necessary to develop targeted and efficient therapies for a host of diseases ranging from neurological disorders to the spread of metastases in the brain.
  • 机译 实时可变形细胞计数的统计数据:聚类,降维和重要性测试
    摘要:Real-time deformability (RT-DC) is a method for high-throughput mechanical and morphological phenotyping of cells in suspension. While analysis rates exceeding 1000 cells per second allow for a label-free characterization of complex biological samples, e.g., whole blood, data evaluation has so far been limited to a few geometrical and material parameters such as cell size, deformation, and elastic Young's modulus. But as a microscopy-based technology, RT-DC actually generates and yields multidimensional datasets that require automated and unbiased tools to obtain morphological and rheological cell information. Here, we present a statistical framework to shed light on this complex parameter space and to extract quantitative results under various experimental conditions. As model systems, we apply cell lines as well as primary cells and highlight more than 11 parameters that can be obtained from RT-DC data. These parameters are used to identify sub-populations in heterogeneous samples using Gaussian mixture models, to perform a dimensionality reduction using principal component analysis, and to quantify the statistical significance applying linear mixed models to datasets of multiple replicates.
  • 机译 使用微流控平台分析癌细胞在发炎内皮细胞上的粘附动力学
    摘要:Metastasis is the ultimate cause of death among the vast majority of cancer patients. This process is comprised of multiple steps, including the migration of circulating cancer cells across microvasculature. This trans-endothelial migration involves the adhesion and eventual penetration of cancer cells to the vasculature of the target organ. Many of these mechanisms remain poorly understood due to poor control of pathophysiological conditions in tumor models. In this work, a microfluidic device was developed to support the culture and observation of engineered microvasculature with systematic control of the environmental characteristics. This device was then used to study the adhesion of circulating cancer cells to an endothelium under varying conditions to delineate the effects of hemodynamics and inflammations. The resulting understanding will help to establish a quantitative and biophysical mechanism of interactions between cancer cells and endothelium.
  • 机译 通过微流体控制双向细胞信号传导,在纤维蛋白水凝胶构建物中构建三维血管前网络
    摘要:Reengineering functional vascular networks in vitro remains an integral part in tissue engineering, since the incorporation of non-perfused tissues results in restricted nutrient supply and limited waste removal. Microfluidic devices are routinely used to mimic both physiological and pathological vascular microenvironments. Current procedures either involve the investigation of growth factor gradients and interstitial flow on endothelial cell sprouting alone or on the heterotypic cell-cell interactions between endothelial and mural cells. However, limited research has been conducted on the influence of flow on co-cultures of these cells. Here, we exploited the ability of microfluidics to create and monitor spatiotemporal gradients to investigate the influence of growth factor supply and elution on vascularization using static as well as indirect and direct flow setups. Co-cultures of human adipose-derived stem/stromal cells and human umbilical vein endothelial cells embedded in fibrin hydrogels were found to be severely affected by diffusion limited growth factor gradients as well as by elution of reciprocal signaling molecules during both static and flow conditions. Static cultures formed pre-vascular networks up to a depth of 4 mm into the construct with subsequent decline due to diffusion limitation. In contrast, indirect flow conditions enhanced endothelial cell sprouting but failed to form vascular networks. Additionally, complete inhibition of pre-vascular network formation was observable for direct application of flow through the hydrogel with decline of endothelial cell viability after seven days. Using finite volume CFD simulations of different sized molecules vital for pre-vascular network formation into and out of the hydrogel constructs, we found that interstitial flow enhances growth factor supply to the cells in the bulk of the chamber but elutes cellular secretome, resulting in truncated, premature vascularization.
  • 机译 刚性红色存在下血管靶向颗粒的结合功效血细胞:对患病血液表现的影响
    摘要:The field of drug delivery has taken an interest in combating numerous blood and heart diseases via the use of injectable vascular-targeted carriers (VTCs). However, VTC technology has encountered limited efficacy due to a variety of challenges associated with the immense complexity of the in vivo blood flow environment, including the hemodynamic interactions of blood cells, which impact their margination and adhesion to the vascular wall. Red blood cell (RBC) physiology, i.e., size, shape, and deformability, drive cellular distribution in blood flow and has been shown to impact VTC margination to the vessel wall significantly. The RBC shape and deformability are known to be altered in certain human diseases, yet little experimental work has been conducted towards understanding the effect of these alterations, specifically RBC rigidity, on VTC dynamics in physiological blood flow. In this work, we investigate the impact of RBCs of varying stiffnesses on the adhesion efficacy of particles of various sizes, moduli, and shapes onto an inflamed endothelial layer in a human vasculature-inspired, in vitro blood flow model. The blood rigid RBC compositions and degrees of RBC stiffness evaluated are analogous to conditions in diseases such as sickle cell disease. We find that particles of different sizes, moduli, and shapes yield drastically different adhesion patterns in blood flow inthe presence of rigid RBCs when compared to 100% healthy RBCs. Specifically, up to 50%reduction in the localization and adhesion of non-deformable 2 μm particles to the vessel wall was observed in the presence of rigid RBCs.Interestingly, deformable 2 μm particles showed enhancedvessel wall localization and adhesion, by up to 85%, depending on the rigidity of RBCsevaluated. Ultimately, this work experimentally clarifies the importance of consideringRBC rigidity in the intelligent design of particle therapeutics and highlights possibleimplications for a wide range of diseases relating to RBC deformability.
  • 机译 片上屏障:片上器官中组织屏障功能的测量
    摘要:Disruption of tissue barriers formed by cells is an integral part of the pathophysiology of many diseases. Therefore, a thorough understanding of tissue barrier function is essential when studying the causes and mechanisms of disease as well as when developing novel treatments. In vitro methods play an integral role in understanding tissue barrier function, and several techniques have been developed in order to evaluate barrier integrity of cultured cell layers, from microscopy imaging of cell-cell adhesion proteins to measuring ionic currents, to flux of water or transport of molecules across cellular barriers. Unfortunately, many of the current in vitro methods suffer from not fully recapitulating the microenvironment of tissues and organs. Recently, organ-on-chip devices have emerged to overcome this challenge. Organs-on-chips are microfluidic cell culture devices with continuously perfused microchannels inhabited by living cells. Freedom of changing the design of device architecture offers the opportunity of recapitulating the in vivo physiological environment while measuring barrier function. Assessment of barriers in organs-on-chips can be challenging as they may require dedicated setups and have smaller volumes that are more sensitive to environmental conditions. But they do provide the option of continuous, non-invasive sensing of barrier quality, which enables better investigation of important aspects of pathophysiology, biological processes, and development of therapies that target barrier tissues. Here, we discuss several techniques to assess barrier function of tissues in organs-on-chips, highlighting advantages and technical challenges.
  • 机译 与高电导率解决方案中产生的长程直流偏置交流电动向心涡流集成的阻抗式生物亲和感测芯片
    摘要:Immunoreaction of specific antibodies to antigens is widely used in numerous immunoanalysis applications. However, diffusion-dominated transport in stationary solutions limits the rate and binding density of immunoreaction. This research describes the construction of chip-type concentric multi-double ring electrodes and single central disk electrode. A +1 V-biased 6 Vpp voltage was applied to the multi-double ring electrodes to induce a long-range DC-biased AC electrokinetic flow (ACEKF). The immunoreaction was quantified by electrochemical impedance spectrum (EIS). Fluorescence-labeled secondary antibody (FLSA) and protein A were exemplified as an immunoreacting model to demonstrate the effect of ACEKF on immunoreaction efficiency. The results showed that FLSA binding can reach a plateau in 8 min with the DC-biased ACEKF vortex, and the increment of electron transfer resistance is 2.26 times larger than that obtained in the unstirred solution. The sensitivity of the calibration curves obtained by EIS detection with the aid of DC-biased ACEKF vortex is 1.51 times larger than that obtained in an unstirred solution. The label-free EIS-based sensing chip integrated with the long-range DC-biased ACEKF vortex promises to facilitate immunoreaction efficiency, which is beneficial for the development of a miniature and fast-detection in vitro diagnostic device.
  • 机译 使用等势交流电场分析细胞的介电泳特性
    摘要:Various microfluidic devices utilizing the principle of dielectrophoresis (DEP) have been developed to separate, concentrate, and characterize biological cells; however, their performance is still limited by a lack of quantitative characterization. We addressed this limitation by employing a method capable of accurately quantifying a cell's response to an imposed field gradient. In this study, a simple method using a newly designed Creek-gap electrode was proposed, and the electrokinetic behavior of cells was characterized by DEP velocimetry under the exposure of an approximately constant gradient of electric field square established along the gap of the electrodes. Together with the numerical prediction of the electric field based on three-dimensional electric field analysis, the magnitude of DEP forces and the real part of the Clausius-Mossotti factor of cells were deduced from their movement. Results demonstrated that the proposed method was applicable to the determination of the dielectrophoretic properties of cells.
  • 机译 Dean结构的样品核与线性惯性聚焦的串行集成,可增强颗粒和细胞的分选
    摘要:In this contribution, a channel aspect ratio of >2 was used to access high velocity regimes to provide confined sample cores by Dean focussing in advance of linear inertial focussing. This produces a singular separation origin with a mirrored transport path for efficient particle and blood cell sorting, while also increasing the spatial resolution for multiscale sorting.
  • 机译 基于细胞附着和毒性测定的改性聚二甲基硅氧烷表面的生物学特性
    摘要:Poly(dimethylsiloxane) (PDMS) is a material applicable for tissue and biomedical engineering, especially based on microfluidic devices. PDMS is a material used in studies aimed at understanding cell behavior and analyzing the cell adhesion mechanism. In this work, biological characterization of the modified PDMS surfaces based on cell attachment and toxicity assays was performed. We studied Balb 3T3/c, HMEC-1, and HT-29 cell adhesion on poly(dimethylsiloxane) surfaces modified by different proteins, with and without pre-activation with plasma oxygen and UV irradiation. Additionally, we studied how changing of a base and a curing agent ratios influence cell proliferation. We observed that cell type has a high impact on cell adhesion, proliferation, as well as viability after drug exposure. It was tested that the carcinoma cells do not require a highly specific microenvironment for their proliferation. Cytotoxicity assays with celecoxib and oxaliplatin on the modified PDMS surfaces showed that normal cells, cultured on the modified PDMS, are more sensitive to drugs than cancer cells. Cell adhesion was also tested in the microfluidic systems made of the modified PDMS layers. Thanks to that, we studied how the surface area to volume ratio influences cell behavior. The results presented in this manuscript could be helpful for creation of proper culture conditions during in vitro tests as well as to understand cell response in different states of disease depending on drug exposure.
  • 机译 基于微量移液器的液滴微流控技术
    摘要:Droplet-based microfluidics, using water-in-oil emulsion droplets as micro-reactors, is becoming a widespread method for performing assays and especially in the cell biology field. Making a simple and highly portable system for creating emulsion droplets would help to continue the popularization of such a technique. Also, the ability to emulsify all the samples would strengthen this compartimenlization technique to handle samples with limited volume. Here, we propose a strategy of droplet formation that combines a classical flow-focusing microfluidic chip, which could be commercially available, with a standard laboratory adjustable micropipette. The micropipette is used as a negative pressure generator for controlling liquid flows. In that way, emulsification does neither require any electrical power supply nor a cumbersome device and functions with small liquid volumes. Droplet formation can be easily and safely performed in places with limited space, opening a wide range of applications especially in biological laboratory environments with higher level of safety regulations, i.e., BSL-3/4. Fortunately, the present methodology that involves small fluid volumes, and thus possible time dependent flow conditions, allows to minimize dead volume while keeping drops' size homogeneous. A physical characterization of droplet production and a model that describes the emulsion features, in terms of drop size and size distribution, are proposed for rationalizing the performances of the micropipette-powered emulsification process.
  • 机译 流体剪切应力对输尿管芽细胞体外培养的影响
    摘要:Most kidney cells are continuously exposed to fluid shear stress (FSS) from either blood flow or urine flow. Recent studies suggest that changes in FSS could contribute to the function and injury of these kidney cells. However, it is unclear whether FSS influences kidney development when urinary flow starts in the embryonic kidneys. In this study, we evaluated the influence of FSS on in vitro cultured ureteric bud (UB) cells by using a pumpless microfluidic device, which offers the convenience of conducting parallel cell culture experiments while also eliminating the need for cumbersome electronic driven equipment and intricate techniques. We first validated the function of the device by both mathematical model and experimental measurements. UB cells dissected from E15.5 mouse embryonic kidneys were cultured in the pumpless microfluidic device and subjected to FSS in the range of 0.4–0.6 dyn mm−2 for 48 h (dynamic). Control UB cells were similarly cultured in the device and maintained under a no-flow condition (static). We found from our present study that the exposure to FSS for up to 48 h led to an increase in mRNA expression levels of UB tip cell marker genes (Wnt11, Ret, Etv4) with a decrease in stalk cell marker genes (Wnt7b, Tacstd2). In further support of the enrichment of UB tip cell population in response to FSS, we also found that exposure to FSS led to a remarkable reduction in the binding of lectin Dolichos Biflorus Agglutinin. In conclusion, results of our present study show that exposure to FSS led to an enrichment in UB tip cell populations, which could contribute to the development and function of the embryonic kidney when urine flow starts at around embryonic age E15.5 in mouse. Since UB tip cells are known to be the proliferative progenitor cells that contribute to the branching morphogenesis of the collecting system in the kidney, our finding could imply an important link between the FSS from the initiation of urine flow and the development and function of the kidney.
  • 机译 气室增强了片上密度混合器
    摘要:This paper proposes an on-chip density mixer that can achieve even density in a target chamber with a swirling flow enhanced by an air chamber. The system is composed of a main channel, a target chamber where two liquids with different densities are included, an isolated air chamber, and an external vibration pump driven by a piezo actuator at the entrance of the main channel. The air chamber is expected to amplify the vibration owing to structure softening. The amplification would be more pronounced at the resonance frequencies of the structure. We developed the system and conducted experiments. We showed that the swirling motion in the target chamber with an air chamber is stronger than that without an air chamber. We also confirmed that the time resulting in even density is shorter when the pump is driven at a resonance frequency. An air-based virtual valve is introduced for maintaining a constant density in the target chamber.
  • 机译 使用基于液滴接触的球体转移进行高通量培养和球体阵列嵌入
    摘要:Spheroids are one of the most representative models of 3D cell culture, which can be easily formed using conventional hanging drop method. However, medium change and spheroid transferring process are the bottlenecks that reduce the throughput of the entire process in the hanging drop culture. In addition, the embedment of spheroid into hydrogel still depends on the individual pipetting process. To overcome these issues, we present poly(dimethylsiloxane) (PDMS)-based simple devices which can exploit droplet contact-based spheroid transfer using a drop array chip (DAC) having an array of well structures and peripheral rims. When the upper spheroid-containing drops were in contact with the lower liquid drops, the air–liquid interface disappeared at the merged surface and the spheroids settled down due to gravitational force. This method was applied to repetitive medium change and live/dead staining of spheroids cultured with the hanging drop method. To simultaneously embed the spheroids into the corresponding collagen hydrogel drops, a PDMS-based pillar array chip (PAC) was contacted in advance with the spheroid-containing DAC. The contacted PAC then contained the spheroids trapped in small drops of liquid reduced in volume to around 0.5 μl. Consequently, the spheroids were embedded into the collagen drops at once by contacting the spheroid-containing PAC with the collagen-loaded DAC. The embedded spheroids using the DAC–PAC contacting method showed a reliable invasion behavior compared to the embedded spheroids using conventional manual pipetting.
  • 机译 笼养浮游生物在微流体环境中进行亚致死水生毒性试验
    摘要:Quantification of neuro-behavioural responses of intact small model organisms has been proposed as a sensitive, sub-lethal alternative to conventional toxicity testing. Such bioassays are characterized by a high physiological and ecological relevance, short response times, increased sensitivity, and non-invasive nature. Despite a significant potential for predictive aquatic toxicology analysis of behavioural traits of micro-invertebrates in microfluidic environment has received little attention. In this work, we demonstrate a new Lab-on-a-Chip technology capable of effectively caging freshwater rotifers Brachionus calyciflorus for real-time video-microscopy analysis. We demonstrate that behavioural bioassays performed under microfluidic perfusion can significantly enhance the sensitivity of conventional ecotoxicology test protocols.
  • 机译 功能性细胞计数微珠的微流控实施,可改善多重细胞因子的定量
    摘要:Functional microbeads have been widely applied in molecular identification and other biochemical applications in the past decade, owing to the compatibility with flow cytometry and the commercially available microbeads for a wide range of molecular identification. Nevertheless, there is still a technical hurdle caused by the significant sample volume required (∼50 μl), limited molecular detection limit (∼20 pg/ml), complicated liquid/microbead handling procedures, and the long reaction time (>2 h). In this work, we optimize the operation of an automated microbead-based microfluidic device for the reagent mixing and the dynamic cytokine detection. In particular, we adopt fluorescence microscopy for quantification of multiple microbeads in each microchamber instead of flow cytometry for a lower detection limit. The operation parameters are then configured for improved measurement performance. As demonstrated, we consider the cytokine secretion of human macrophage-differentiating lymphocytes stimulated by lipopolysaccharides. We examine requirements on the mixing duration, minimal sample volume, and the image analysis scheme for the smaller biosample volume (<5 μl), the lower cytokine detection limit (∼5 pg/ml), and shorter process time (∼30 min). Importantly, this microfluidic strategy can be further extended in the molecular profiling using other functional microbeads for a broad range of biomedical applications.
  • 机译 沉浸式交流电喷雾(iACE)用于单分散水液滴的产生
    摘要:We report a new immersed alternating current (AC) electrospray droplet generation method that can generate monodispersed water-in-oil droplets, with diameters ranging from 5 μm to 150 μm, in a stationary oil phase. This method offers high through-put, easy size tuning, and droplets with a viscous aqueous phase at high ionic strengths (raw physiological samples). Yet, it does not require coordinated flows of the dispersed/continuous phases or even a microfluidic chip. The design relies on a small constant back pressure (less than 0.1 atm) to drive the water phase through a nozzle (glass micropipette) and a non-isotropic AC electric Maxwell pressure to eject it into the oil phase. Undesirable field-induced discharge and nanojet formation at the tip are suppressed with a biocompatible polymer, polyethylene oxide. Its viscoelastic property favors the monodispersed dripping mechanism, with a distinct neck forming at the capillary tip before pinch-off, such that the tip dimension is the only controlling length scale. Consecutive droplets are connected by a whipping filament that disperses the drops away from the high-field nozzle to prevent electro-coalescence. A scaling theory is developed to correlate the droplet size with the applied pressure, the most important tuning parameter, and to determine the optimum frequency. The potential applications of this technology to biological systems are demonstrated with a digital loop-mediated isothermal amplification experiment, with little damage to the nucleic acids and other biomolecules, but with easy adaptive tuning for the optimum droplet number for accurate quantification.

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