High-Throughput Microfluidic Extraction of Platelet-free Plasma for MicroRNA and Extracellular Vesicle Analysis.

Cell-free RNAs and extracellular vesicles (EVs) are valuable biomarkers in liquid biopsies, but they are prone to preanalytical variabilities such as nonstandardized centrifugation or ex vivo blood degradation. Herein, we report a high-throughput and label-free inertial microfluidic device (ExoArc) for isolation of platelet-free plasma from blood for RNA and EV analysis. Unlike conventional inertial microfluidic devices widely used for cell sorting, a submicrometer size cutoff (500 nm) was achieved which completely removed all leukocytes, RBCs, platelets, and cellular debris based on differential lateral migration induced by Dean vortices. The single-step operation also reduced platelet-associated miRNAs (∼2-fold) compared to centrifugation. We clinically validated ExoArc for plasma miRNA profiling (39 samples) and identified a 7-miRNA panel that detects non-small cell lung cancer with ∼90% sensitivity. ExoArc was also coupled with size exclusion chromatography (SEC) to isolate EVs within 50 min with ∼10-fold higher yield than ultracentrifugation. As a proof-of-concept for EV-based transcriptomics analysis, we performed miRNA analysis in healthy and type 2 diabetes mellitus (T2DM) subjects (n = 3 per group) by coupling ExoArc and ExoArc+SEC with quantitative polymerase chain reaction (RT-qPCR) assay. Among 293 miRNAs detected, plasmas and EVs showed distinct differentially expressed miRNAs in T2DM subjects. We further demonstrated automated in-line EV sorting from low volume culture media for continuous EV monitoring. Overall, the developed ExoArc offers a convenient centrifugation-free workflow to automate plasma and EV isolation for point-of-care diagnostics and quality control in EV manufacturing.

Label-free microfluidic cell sorting and detection for rapid blood analysis.

Blood tests are considered as standard clinical procedures to screen for markers of diseases and health conditions. However, the complex cellular background (>99.9% RBCs) and biomolecular composition often pose significant technical challenges for accurate blood analysis. An emerging approach for point-of-care blood diagnostics is utilizing "label-free" microfluidic technologies that rely on intrinsic cell properties for blood fractionation and disease detection without any antibody binding. A growing body of clinical evidence has also reported that cellular dysfunction and their biophysical phenotypes are complementary to standard hematoanalyzer analysis (complete blood count) and can provide a more comprehensive health profiling. In this review, we will summarize recent advances in microfluidic label-free separation of different blood cell components including circulating tumor cells, leukocytes, platelets and nanoscale extracellular vesicles. Label-free single cell analysis of intrinsic cell morphology, spectrochemical properties, dielectric parameters and biophysical characteristics as novel blood-based biomarkers will also be presented. Next, we will highlight research efforts that combine label-free microfluidics with machine learning approaches to enhance detection sensitivity and specificity in clinical studies, as well as innovative microfluidic solutions which are capable of fully integrated and label-free blood cell sorting and analysis. Lastly, we will envisage the current challenges and future outlook of label-free microfluidics platforms for high throughput multi-dimensional blood cell analysis to identify non-traditional circulating biomarkers for clinical diagnostics.

Studying the efficacy of antiplatelet drugs on atherosclerosis by optofluidic imaging on a chip.

Vascular stenosis caused by atherosclerosis instigates activation and aggregation of platelets, eventually resulting in thrombus formation. Although antiplatelet drugs are commonly used to inhibit platelet activation and aggregation, they unfortunately cannot prevent recurrent thrombotic events in patients with atherosclerosis. This is partially due to the limited understanding of the efficacy of antiplatelet drugs in the complex hemodynamic environment of vascular stenosis. Conventional methods for evaluating the efficacy of antiplatelet drugs under stenosis either fail to simulate the hemodynamic environment of vascular stenosis characterized by high shear stress and recirculatory flow or lack spatial resolution in their analytical techniques to statistically identify and characterize platelet aggregates. Here we propose and experimentally demonstrate a method comprising an in vitro 3D stenosis microfluidic chip and an optical time-stretch quantitative phase imaging system for studying the efficacy of antiplatelet drugs under stenosis. Our method simulates the atherogenic flow environment of vascular stenosis while enabling high-resolution and statistical analysis of platelet aggregates. Using our method, we distinguished the efficacy of three antiplatelet drugs, acetylsalicylic acid (ASA), cangrelor, and eptifibatide, for inhibiting platelet aggregation induced by stenosis. Specifically, ASA failed to inhibit stenosis-induced platelet aggregation, while eptifibatide and cangrelor showed high and moderate efficacy, respectively. Furthermore, we demonstrated that the drugs tested also differed in their efficacy for inhibiting platelet aggregation synergistically induced by stenosis and agonists (e.g., adenosine diphosphate, and collagen). Taken together, our method is an effective tool for investigating the efficacy of antiplatelet drugs under vascular stenosis, which could assist the development of optimal pharmacologic strategies for patients with atherosclerosis.

Hyaluronidase-1-mediated glycocalyx impairment underlies endothelial abnormalities in polypoidal choroidal vasculopathy.

BACKGROUND: Polypoidal choroidal vasculopathy (PCV), a subtype of age-related macular degeneration (AMD), is a global leading cause of vision loss in older populations. Distinct from typical AMD, PCV is characterized by polyp-like dilatation of blood vessels and turbulent blood flow in the choroid of the eye. Gold standard anti-vascular endothelial growth factor (anti-VEGF) therapy often fails to regress polypoidal lesions in patients. Current animal models have also been hampered by their inability to recapitulate such vascular lesions. These underscore the need to identify VEGF-independent pathways in PCV pathogenesis. RESULTS: We cultivated blood outgrowth endothelial cells (BOECs) from PCV patients and normal controls to serve as our experimental disease models. When BOECs were exposed to heterogeneous flow, single-cell transcriptomic analysis revealed that PCV BOECs preferentially adopted migratory-angiogenic cell state, while normal BOECs undertook proinflammatory cell state. PCV BOECs also had a repressed protective response to flow stress by demonstrating lower mitochondrial functions. We uncovered that elevated hyaluronidase-1 in PCV BOECs led to increased degradation of hyaluronan, a major component of glycocalyx that interfaces between flow stress and vascular endothelium. Notably, knockdown of hyaluronidase-1 in PCV BOEC improved mechanosensitivity, as demonstrated by a significant 1.5-fold upregulation of Krüppel-like factor 2 (KLF2) expression, a flow-responsive transcription factor. Activation of KLF2 might in turn modulate PCV BOEC migration. Barrier permeability due to glycocalyx impairment in PCV BOECs was also reversed by hyaluronidase-1 knockdown. Correspondingly, hyaluronidase-1 was detected in PCV patient vitreous humor and plasma samples. CONCLUSIONS: Hyaluronidase-1 inhibition could be a potential therapeutic modality in preserving glycocalyx integrity and endothelial stability in ocular diseases with vascular origin.

Microfluidic Size Exclusion Chromatography (µSEC) for Extracellular Vesicles and Plasma Protein Separation.

Extracellular vesicles (EVs) are recognized as next generation diagnostic biomarkers due to their disease-specific biomolecular cargoes and importance in cell-cell communications. A major bottleneck in EV sample preparation is the inefficient and laborious isolation of nanoscale EVs (≈50-200 nm) from endogenous proteins in biological samples. Herein, a unique microfluidic platform is reported for EV-protein fractionation based on the principle of size exclusion chromatography (SEC). Using a novel rapid (≈20 min) replica molding technique, a fritless microfluidic SEC device (µSEC) is fabricated using thiol-ene polymer (UV glue NOA81, Young's modulus ≈1 GPa) for high pressure (up to 6 bar) sample processing. Controlled on-chip nanoliter sample plug injection (600 nL) using a modified T-junction injector is first demonstrated with rapid flow switching response time (<1.5 s). Device performance is validated using fluorescent nanoparticles (50 nm), albumin, and breast cancer cells (MCF-7)-derived EVs. As a proof-of-concept for clinical applications, EVs are directly isolated from undiluted human platelet-poor plasma using µSEC and show distinct elution profiles between EVs and proteins based on nanoparticle particle analysis (NTA), Western blot and flow cytometry analysis. Overall, the optically transparent µSEC can be readily automated and integrated with EV detection assays for EVs manufacturing and clinical diagnostics.

A Facile and Scalable Hydrogel Patterning Method for Microfluidic 3D Cell Culture and Spheroid-in-Gel Culture Array.

Incorporation of extracellular matrix (ECM) and hydrogel in microfluidic 3D cell culture platforms is important to create a physiological microenvironment for cell morphogenesis and to establish 3D co-culture models by hydrogel compartmentalization. Here, we describe a simple and scalable ECM patterning method for microfluidic cell cultures by achieving hydrogel confinement due to the geometrical expansion of channel heights (stepped height features) and capillary burst valve (CBV) effects. We first demonstrate a sequential "pillar-free" hydrogel patterning to form adjacent hydrogel lanes in enclosed microfluidic devices, which can be further multiplexed with one to two stepped height features. Next, we developed a novel "spheroid-in-gel" culture device that integrates (1) an on-chip hanging drop spheroid culture and (2) a single "press-on" hydrogel confinement step for rapid ECM patterning in an open-channel microarray format. The initial formation of breast cancer (MCF-7) spheroids was achieved by hanging a drop culture on a patterned polydimethylsiloxane (PDMS) substrate. Single spheroids were then directly encapsulated on-chip in individual hydrogel islands at the same positions, thus, eliminating any manual spheroid handling and transferring steps. As a proof-of-concept to perform a spheroid co-culture, endothelial cell layer (HUVEC) was formed surrounding the spheroid-containing ECM region for drug testing studies. Overall, this developed stepped height-based hydrogel patterning method is simple to use in either enclosed microchannels or open surfaces and can be readily adapted for in-gel cultures of larger 3D cellular spheroids or microtissues.

Leucine-Rich α-2-Glycoprotein 1 Suppresses Endothelial Cell Activation Through ADAM10-Mediated Shedding of TNF-α Receptor.

Elevated serum concentrations of leucine-rich α-2-glycoprotein (LRG1) have been reported in patients with inflammatory, autoimmune, and cardiovascular diseases. This study aims to investigate the role of LRG1 in endothelial activation. LRG1 in endothelial cells (ECs) of arteries and serum of patients with critical limb ischemia (CLI) was assessed by immunohistochemistry and ELISA, respectively. LRG1 expression in sheared and tumor necrosis factor-α (TNF-α)-treated ECs was analyzed. The mechanistic role of LRG1 in endothelial activation was studied in vitro. Plasma of 37-week-old Lrg1 -/- mice was used to investigate causality between LRG1 and tumor necrosis factor receptor 1 (TNFR1) shedding. LRG1 was highly expressed in ECs of stenotic but not normal arteries. LRG1 concentrations in serum of patients with CLI were elevated compared to healthy controls. LRG1 expression was shear dependent. It could be induced by TNF-α, and the induction of its expression was mediated by NF-κB activation. LRG1 inhibited TNF-α-induced activation of NF-κB signaling, expression of VCAM-1 and ICAM-1, and monocyte capture, firm adhesion, and transendothelial migration. Mechanistically, LRG1 exerted its function by causing the shedding of TNFR1 via the ALK5-SMAD2 pathway and the subsequent activation of ADAM10. Consistent with this mechanism, LRG1 and sTNFR1 concentrations were correlated in the serum of CLI patients. Causality between LRG1 and TNFR1 shedding was established by showing that Lrg1 -/- mice had lower plasma sTNFR1 concentrations than wild type mice. Our results demonstrate a novel role for LRG1 in endothelial activation and its potential therapeutic role in inflammatory diseases should be investigated further.

A Multifunctional Role of Leucine-Rich α-2-Glycoprotein 1 in Cutaneous Wound Healing Under Normal and Diabetic Conditions.

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 TGFß 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.

Recapitulating atherogenic flow disturbances and vascular inflammation in a perfusable 3D stenosis model.

Blood vessel narrowing and arterial occlusion are pathological hallmarks of atherosclerosis, which involves a complex interplay of perturbed hemodynamics, endothelial dysfunction and inflammatory cascade. Herein, we report a novel circular microfluidic stenosis model that recapitulates atherogenic flow-mediated endothelial dysfunction and blood-endothelial cell (EC) interactions in vitro. 2D and 3D stenosis microchannels with different constriction geometries were fabricated using 3D printing to study flow disturbances under varying severity of occlusion and wall shear stresses (100 to 2000 dynecm-2). Experimental and fluid simulation results confirmed the presence of pathological shear stresses in the stenosis region, and recirculation flow post stenosis. The resultant pathological flow profile induced pro-inflammatory and pro-thrombotic EC state as demonstrated by orthogonal EC alignment, enhanced platelet adhesion at the stenosis, and aberrant leukocyte-EC interactions post stenosis. Clinical utility of the vascular model was further investigated by testing anti-thrombotic and immunomodulatory efficacy of aspirin and metformin, respectively. Overall, the platform enables multi-factorial analysis of critical atherogenic events including endothelial dysfunction, platelets and leukocyte adhesion, and can be further developed into a liquid biopsy tool for cardiovascular risk stratification.

A tunable microfluidic 3D stenosis model to study leukocyte-endothelial interactions in atherosclerosis.

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.

A Novel Microdevice for Rapid Neutrophil Purification and Phenotyping in Type 2 Diabetes Mellitus.

Neutrophil dysfunction is strongly linked to type 2 diabetes mellitus (T2DM) pathophysiology, but the prognostic potential of neutrophil biomarkers remains largely unexplored due to arduous leukocyte isolation methods. Herein, a novel integrated microdevice is reported for single-step neutrophil sorting and phenotyping (chemotaxis and formation of neutrophil extracellular traps (NETosis)) using small blood volumes (fingerprick). Untouched neutrophils are purified on-chip from whole blood directly using biomimetic cell margination and affinity-based capture, and are exposed to preloaded chemoattractant or NETosis stimulant to initiate chemotaxis or NETosis, respectively. Device performance is first characterized using healthy and in vitro inflamed blood samples (tumor necrosis factor alpha, high glucose), followed by clinical risk stratification in a cohort of subjects with T2DM. Interestingly, "high-risk" T2DM patients characterized by severe chemotaxis impairment reveal significantly higher C-reactive protein levels and poor lipid metabolism characteristics as compared to "low-risk" subjects, and their neutrophil chemotaxis responses can be mitigated after in vitro metformin treatment. Overall, this unique and user-friendly microfluidics immune health profiling strategy can significantly aid the quantification of chemotaxis and NETosis in clinical settings, and be further translated into a tool for risk stratification and precision medicine methods in subjects with metabolic diseases such as T2DM.

Preservation of Anticancer and Immunosuppressive Properties of Rapamycin Achieved Through Controlled Releasing Particles.

Rapamycin is commonly used in chemotherapy and posttransplantation rejection suppression, where sustained release is preferred. Conventionally, rapamycin has to be administered in excess due to its poor solubility, and this often leads to cytotoxicity and undesirable side effects. In addition, rapamycin has been shown to be hydrolytically unstable, losing its bioactivity within a few hours. The use of drug delivery systems is hypothesized to preserve the bioactivity of rapamycin, while providing controlled release of this otherwise potent drug. This paper reports on the use of microparticles (MP) as a means to tune and sustain the delivery of bioactive rapamycin for up to 30 days. Rapamycin was encapsulated (100% efficiency) in poly(lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL), or a mixture of both via an emulsion method. The use of different polymer types and mixture was shown to achieve a variety of release kinetics and profile. Released rapamycin was subsequently evaluated against breast cancer cell (MCF-7) and human lymphocyte cell (Jurkat). Inhibition of cell proliferation was in good agreement with in vitro release profiles, which confirmed the intact bioactivity of rapamycin. For Jurkat cells, the suppression of cell growth was proven to be effective up to 20 days, a duration significantly longer than free rapamycin. Taken together, these results demonstrate the ability to tune, sustain, and preserve the bioactivity of rapamycin using MP formulations. The sustained delivery of rapamycin could lead to better therapeutic effects than bolus dosage, at the same time improving patient compliance due to its long-acting duration.

Interference-free Micro/nanoparticle Cell Engineering by Use of High-Throughput Microfluidic Separation.

Engineering cells with active-ingredient-loaded micro/nanoparticles is becoming increasingly popular for imaging and therapeutic applications. A critical yet inadequately addressed issue during its implementation concerns the significant number of particles that remain unbound following the engineering process, which inadvertently generate signals and impart transformative effects onto neighboring nontarget cells. Here we demonstrate that those unbound micro/nanoparticles remaining in solution can be efficiently separated from the particle-labeled cells by implementing a fast, continuous, and high-throughput Dean flow fractionation (DFF) microfluidic device. As proof-of-concept, we applied the DFF microfluidic device for buffer exchange to sort labeled suspension cells (THP-1) from unbound fluorescent dye and dye-loaded micro/nanoparticles. Compared to conventional centrifugation, the depletion efficiency of free dyes or particles was improved 20-fold and the mislabeling of nontarget bystander cells by free particles was minimized. The microfluidic device was adapted to further accommodate heterogeneous-sized mesenchymal stem cells (MSCs). Complete removal of unbound nanoparticles using DFF led to the usage of engineered MSCs without exerting off-target transformative effects on the functional properties of neighboring endothelial cells. Apart from its effectiveness in removing free particles, this strategy is also efficient and scalable. It could continuously process cell solutions with concentrations up to 10(7) cells·mL(-1) (cell densities commonly encountered during cell therapy) without observable loss of performance. Successful implementation of this technology is expected to pave the way for interference-free clinical application of micro/nanoparticle engineered cells.