A novel integrated inertial-impedance cytometer for rapid and label-free electrical profiling of neutrophil extracellular trap formation (NETosis).
Advanced management of dysmetabolic syndromes such as diabetes will benefit from a timely mechanistic insight enabling personalized medicine approaches. Herein, we present a rapid microfluidic neutrophil sorting and functional phenotyping strategy for type 2 diabetes mellitus (T2DM) patients using small blood volumes (fingerprick ~100 μL). The developed inertial microfluidics technology enables single-step neutrophil isolation (>90% purity) without immuno-labeling and sorted neutrophils are used to characterize their rolling behavior on E-selectin, a critical step in leukocyte recruitment during inflammation. The integrated microfluidics testing methodology facilitates high throughput single-cell quantification of neutrophil rolling to detect subtle differences in speed distribution. Higher rolling speed was observed in T2DM patients (P < 0.01) which strongly correlated with neutrophil activation, rolling ligand P-selectin glycoprotein ligand 1 (PSGL-1) expression, as well as established cardiovascular risk factors (cholesterol, high-sensitive C-reactive protein (CRP) and HbA1c). Rolling phenotype can be modulated by common disease risk modifiers (metformin and pravastatin). Receiver operating characteristics (ROC) and principal component analysis (PCA) revealed neutrophil rolling as an important functional phenotype in T2DM diagnostics. These results suggest a new point-of-care testing methodology, and neutrophil rolling speed as a functional biomarker for rapid profiling of dysmetabolic subjects in clinical and patient-oriented settings.
Atherosclerosis, a chronic inflammatory disorder characterized by endothelial dysfunction and blood vessel narrowing, is the leading cause of cardiovascular diseases including heart attack and stroke. Herein, we present a novel tunable microfluidic atherosclerosis model to study vascular inflammation and leukocyte-endothelial interactions in 3D vessel stenosis. Flow and shear stress profiles were characterized in pneumatic-controlled stenosis conditions (0%, 50% and 80% constriction) using fluid simulation and experimental beads perfusion. Due to non-uniform fluid flow at the 3D stenosis, distinct monocyte (THP-1) adhesion patterns on inflamed [tumor necrosis factor-α (TNF-α) treated] endothelium were observed, and there was a differential endothelial expression of intercellular adhesion molecule-1 (ICAM-1) at the constriction region. Whole blood perfusion studies also showed increased leukocyte interactions (cell rolling and adherence) at the stenosis of healthy and inflamed endothelium, clearly highlighting the importance of vascular inflammation, flow disturbance, and vessel geometry in recapitulating atherogenic microenvironment. To demonstrate inflammatory risk assessment using leukocytes as functional biomarkers, we perfused whole blood samples into the developed microdevices (80% constriction) and observed significant dose-dependent effects of leukocyte adhesion in healthy and inflamed (TNF-α treated) blood samples. Taken together, the 3D stenosis chip facilitates quantitative study of hemodynamics and leukocyte-endothelial interactions, and can be further developed into a point-of-care blood profiling device for atherosclerosis and other vascular diseases.
Delayed wound healing is commonly associated with diabetes. It may lead to amputation and death if not treated in a timely fashion. Limited treatments are available partially due to the poor understanding of the complex disease pathophysiology. Here, we investigated the role of leucine-rich α-2-glycoprotein 1 (LRG1) in normal and diabetic wound healing. First, our data showed that LRG1 was significantly increased at the inflammation stage of murine wound healing, and bone marrow–derived cells served as a major source of LRG1. LRG1 deletion causes impaired immune cell infiltration, reepithelialization, and angiogenesis. As a consequence, there is a significant delay in wound closure. On the other hand, LRG1 was markedly induced in diabetic wounds in both humans and mice. LRG1-deficient mice were resistant to diabetes-induced delay in wound repair. We further demonstrated that this could be explained by the mitigation of increased neutrophil extracellular traps (NETs) in diabetic wounds. Mechanistically, LRG1 mediates NETosis in an Akt-dependent manner through TGFβ type I receptor kinase ALK5. Taken together, our studies demonstrated that LRG1 derived from bone marrow cells is required for normal wound healing, revealing a physiological role for this glycoprotein, but that excess LRG1 expression in diabetes is pathogenic and contributes to chronic wound formation.
Efficient separation of sub-micrometer synthetic or biological components is imperative in particle-based drug delivery systems and purification of extracellular vesicles for point-of-care diagnostics. Herein, we report a novel phenomenon in spiral inertial microfluidics, in which the particle transient innermost distance (D inner ) varies with size during Dean vortices-induced migration and can be utilized for small microparticle (MP) separation; aptly termed as high-resolution Dean flow fractionation (HiDFF). The developed technology was optimized using binary bead mixtures (1-3 μm) to achieve~100-to 1000-fold enrichment of smaller particles. We demonstrated tunable size fractionation of polydispersed drug-loaded poly(lactic-co-glycolic acid) particles for enhanced drug release and anti-tumor effects. As a proof-of-concept for microvesicles studies, circulating extracellular vesicles/ MPs were isolated directly from whole blood using HiDFF. Purified MPs exhibited well-preserved surface morphology with efficient isolation within minutes as compared with multi-step centrifugation. In a cohort of type 2 diabetes mellitus subjects, we observed strong associations of immune cell-derived MPs with cardiovascular risk factors including body mass index, carotid intima-media thickness and triglyceride levels (Po0.05). Overall, HiDFF represents a key technological progress toward highthroughput, single-step purification of engineered or cell-derived MPs with the potential for quantitative MP-based health profiling. NPG Asia Materials (2017) 9, e434; doi:10.1038/am.2017.175; published online 29 September 2017 INTRODUCTIONEnabling technologies for continuous, size-based separation of submicrometer engineered or biological components are highly desirable in clinical applications, such as particle-based drug delivery systems 1 and the purification of extracellular vesicles in clinical diagnostics. 2 In microparticle fabrication, conventional 'bottom-up' self-assembly emulsification techniques yield a broad particle size distribution, which can affect the drug release kinetics and biotransport in blood. 3,4 Although well-controlled and monodisperse particles can be produced by 'top-down' approaches using specific lithographic techniques 5,6 and microfluidics, 7,8 microfabricated particles are prone to damage during mechanical harvesting, a problem further aggravated at the smaller/nanoscale level. Similarly, microfluidic synthesis of drug-loaded polymeric particles requires compatible drug/surfactant chemistry with additional steps to remove solvent prior use. Developing novel tools to achieve tunable size fractionation of polydispersed synthetic particles would enable optimal biodistribution and controlled drug release. Such technologies also facilitate physical isolation of smaller biological targets (o2 μm) including platelets, microbes and extracellular vesicles in a label-free manner for unbiased downstream analysis.
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