Journal articles on the topic 'Microfluidic blood vessels'
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1
Akhmetov, A. T., A. A. Valiev, A. A. Rakhimov, S. P. Sametov, and R. R. Habibullina. "Microfluidics of blood in blood vessels stenosis." Proceedings of the Mavlyutov Institute of Mechanics 11, no. 2 (2016): 210–17. http://dx.doi.org/10.21662/uim2016.2.031.
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It is mentioned in the paper that hydrodynamic conditions of a flow in blood vessels with the stenosis are abnormal in relation to the total hemodynamic conditions of blood flow in a vascular system of a human body. A microfluidic device developed with a stepped narrowing for studying of the blood flow at abnormal conditions allowed to reveal blood structure in microchannels simulating the stenosis. Microstructure change is observed during the flow of both native and diluted blood through the narrowing. The study of hemorheological properties allowed us to determine an increasing contribution of the hydraulic resistance of the healthy part of the vessel during the stenosis formation.
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Nam, Ungsig, Seunggyu Kim, Joonha Park, and Jessie S. Jeon. "Lipopolysaccharide-Induced Vascular Inflammation Model on Microfluidic Chip." Micromachines 11, no. 8 (July 31, 2020): 747. http://dx.doi.org/10.3390/mi11080747.
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Inflammation is the initiation of defense of our body against harmful stimuli. Lipopolysaccharide (LPS), originating from outer membrane of Gram-negative bacteria, causes inflammation in the animal’s body and can develop several diseases. In order to study the inflammatory response to LPS of blood vessels in vitro, 2D models have been mainly used previously. In this study, a microfluidic device was used to investigate independent inflammatory response of endothelial cells by LPS and interaction of inflamed blood vessel with monocytic THP-1 cells. Firstly, the diffusion of LPS across the collagen gel into blood vessel was simulated using COMSOL. Then, inflammatory response to LPS in engineered blood vessel was confirmed by the expression of Intercellular Adhesion Molecule 1 (ICAM-1) and VE-cadherin of blood vessel, and THP-1 cell adhesion and migration assay. Upregulation of ICAM-1 and downregulation of VE-cadherin in an LPS-treated condition was observed compared to normal condition. In the THP-1 cell adhesion and migration assay, the number of adhered and trans-endothelial migrated THP-1 cells were not different between conditions. However, migration distance of THP-1 was longer in the LPS treatment condition. In conclusion, we recapitulated the inflammatory response of blood vessels and the interaction of THP-1 cells with blood vessels due to the diffusion of LPS.
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Ren, Jifeng, Yi Liu, Wei Huang, and Raymond H. W. Lam. "A Narrow Straight Microchannel Array for Analysis of Transiting Speed of Floating Cancer Cells." Micromachines 13, no. 2 (January 26, 2022): 183. http://dx.doi.org/10.3390/mi13020183.
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Investigating floating cells along a narrow microchannel (e.g., a blood vessel) for their transiting speeds and the corresponding roles of cell physical properties can deepen our understanding of circulating tumor cells (CTCs) metastasis via blood vessels. Many existing studies focus on the cell transiting process in blood vessel-like microchannels; further analytical studies are desired to summarize behaviors of the floating cell movement under different conditions. In this work, we perform a theoretical analysis to establish a relation between the transiting speed and key cell physical properties. We also conduct computational fluid dynamics simulation and microfluidic experiments to verify the theoretical model. This work reveals key cell physical properties and the channel configurations determining the transiting speed. The reported model can be applied to other works with various dimensions of microchannels as a more general way to evaluate the cancer cell metastasis ability with microfluidics.
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Ahn, Jungho, Hyeok Lee, Habin Kang, Hyeri Choi, Kyungmin Son, James Yu, Jungseub Lee, et al. "Pneumatically Actuated Microfluidic Platform for Reconstituting 3D Vascular Tissue Compression." Applied Sciences 10, no. 6 (March 17, 2020): 2027. http://dx.doi.org/10.3390/app10062027.
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In vivo, blood vessels constitutively experience mechanical stresses exerted by adjacent tissues and other structural elements. Vascular collapse, a structural failure of vascular tissues, may stem from any number of possible compressive forces ranging from injury to tumor growth and can promote inflammation. In particular, endothelial cells are continuously exposed to varying mechanical stimuli, internally and externally, resulting in blood vessel deformation and injury. This study proposed a method to model biomechanical-stimuli-induced blood vessel compression in vitro within a polydimethylsiloxane (PDMS) microfluidic 3D microvascular tissue culture platform with an integrated pneumatically actuated compression mechanism. 3D microvascular tissues were cultured within the device. Histological reactions to compressive forces were quantified and shown to be the following: live/dead assays indicated the presence of a microvascular dead zone within high-stress regions and reactive oxygen species (ROS) quantification exhibited a stress-dependent increase. Fluorescein isothiocyanate (FITC)-dextran flow assays showed that compressed vessels developed structural failures and increased leakiness; finite element analysis (FEA) corroborated the experimental data, indicating that the suggested model of vascular tissue deformation and stress distribution was conceptually sound. As such, this study provides a powerful and accessible in vitro method of modeling microphysiological reactions of microvascular tissues to compressive stress, paving the way for further studies into vascular failure as a result of external stress.
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Virumbrales-Muñoz, María, Jiong Chen, Jose Ayuso, Moonhee Lee, E. Jason Abel, and David J. Beebe. "Organotypic primary blood vessel models of clear cell renal cell carcinoma for single-patient clinical trials." Lab on a Chip 20, no. 23 (2020): 4420–32. http://dx.doi.org/10.1039/d0lc00252f.
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Terrassoux, Lisa, Hugo Claux, Salimata Bacari, Samuel Meignan, and Alessandro Furlan. "A Bloody Conspiracy. Blood Vessels and Immune Cells in the Tumor Microenvironment." Cancers 14, no. 19 (September 21, 2022): 4581. http://dx.doi.org/10.3390/cancers14194581.
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Cancer progression occurs in concomitance with a profound remodeling of the cellular microenvironment. Far from being a mere passive event, the re-orchestration of interactions between the various cell types surrounding tumors highly contributes to the progression of the latter. Tumors notably recruit and stimulate the sprouting of new blood vessels through a process called neo-angiogenesis. Beyond helping the tumor cope with an increased metabolic demand associated with rapid growth, this also controls the metastatic dissemination of cancer cells and the infiltration of immune cells in the tumor microenvironment. To decipher this critical interplay for the clinical progression of tumors, the research community has developed several valuable models in the last decades. This review offers an overview of the various instrumental solutions currently available, including microfluidic chips, co-culture models, and the recent rise of organoids. We highlight the advantages of each technique and the specific questions they can address to better understand the tumor immuno-angiogenic ecosystem. Finally, we discuss this development field’s fundamental and applied perspectives.
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Ohta, Makoto, Naoya Sakamoto, Kenichi Funamoto, Zi Wang, Yukiko Kojima, and Hitomi Anzai. "A Review of Functional Analysis of Endothelial Cells in Flow Chambers." Journal of Functional Biomaterials 13, no. 3 (July 12, 2022): 92. http://dx.doi.org/10.3390/jfb13030092.
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The vascular endothelial cells constitute the innermost layer. The cells are exposed to mechanical stress by the flow, causing them to express their functions. To elucidate the functions, methods involving seeding endothelial cells as a layer in a chamber were studied. The chambers are known as parallel plate, T-chamber, step, cone plate, and stretch. The stimulated functions or signals from endothelial cells by flows are extensively connected to other outer layers of arteries or organs. The coculture layer was developed in a chamber to investigate the interaction between smooth muscle cells in the middle layer of the blood vessel wall in vascular physiology and pathology. Additionally, the microfabrication technology used to create a chamber for a microfluidic device involves both mechanical and chemical stimulation of cells to show their dynamics in in vivo microenvironments. The purpose of this study is to summarize the blood flow (flow inducing) for the functions connecting to endothelial cells and blood vessels, and to find directions for future chamber and device developments for further understanding and application of vascular functions. The relationship between chamber design flow, cell layers, and microfluidics was studied.
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Watanabe, Uran, Shinji Sugiura, Masayuki Kakehata, Fumiki Yanagawa, Toshiyuki Takagi, Kimio Sumaru, Taku Satoh, et al. "Fabrication of Hollow Structures in Photodegradable Hydrogels Using a Multi-Photon Excitation Process for Blood Vessel Tissue Engineering." Micromachines 11, no. 7 (July 13, 2020): 679. http://dx.doi.org/10.3390/mi11070679.
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Engineered blood vessels generally recapitulate vascular function in vitro and can be utilized in drug discovery as a novel microphysiological system. Recently, various methods to fabricate vascular models in hydrogels have been reported to study the blood vessel functions in vitro; however, in general, it is difficult to fabricate hollow structures with a designed size and structure with a tens of micrometers scale for blood vessel tissue engineering. This study reports a method to fabricate the hollow structures in photodegradable hydrogels prepared in a microfluidic device. An infrared femtosecond pulsed laser, employed to induce photodegradation via multi-photon excitation, was scanned in the hydrogel in a program-controlled manner for fabricating the designed hollow structures. The photodegradable hydrogel was prepared by a crosslinking reaction between an azide-modified gelatin solution and a dibenzocyclooctyl-terminated photocleavable tetra-arm polyethylene glycol crosslinker solution. After assessing the composition of the photodegradable hydrogel in terms of swelling and cell adhesion, the hydrogel prepared in the microfluidic device was processed by laser scanning to fabricate linear and branched hollow structures present in it. We introduced a microsphere suspension into the fabricated structure in photodegradable hydrogels, and confirmed the fabrication of perfusable hollow structures of designed patterns via the multi-photon excitation process.
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Akbari, Ehsan, Griffin B. Spychalski, Kaushik K. Rangharajan, Shaurya Prakash, and Jonathan W. Song. "Competing Fluid Forces Control Endothelial Sprouting in a 3-D Microfluidic Vessel Bifurcation Model." Micromachines 10, no. 7 (July 4, 2019): 451. http://dx.doi.org/10.3390/mi10070451.
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Sprouting angiogenesis—the infiltration and extension of endothelial cells from pre-existing blood vessels—helps orchestrate vascular growth and remodeling. It is now agreed that fluid forces, such as laminar shear stress due to unidirectional flow in straight vessel segments, are important regulators of angiogenesis. However, regulation of angiogenesis by the different flow dynamics that arise due to vessel branching, such as impinging flow stagnation at the base of a bifurcating vessel, are not well understood. Here we used a recently developed 3-D microfluidic model to investigate the role of the flow conditions that occur due to vessel bifurcations on endothelial sprouting. We observed that bifurcating fluid flow located at the vessel bifurcation point suppresses the formation of angiogenic sprouts. Similarly, laminar shear stress at a magnitude of ~3 dyn/cm2 applied in the branched vessels downstream of the bifurcation point, inhibited the formation of angiogenic sprouts. In contrast, co-application of ~1 µm/s average transvascular flow across the endothelial monolayer with laminar shear stress induced the formation of angiogenic sprouts. These results suggest that transvascular flow imparts a competing effect against bifurcating fluid flow and laminar shear stress in regulating endothelial sprouting. To our knowledge, these findings are the first report on the stabilizing role of bifurcating fluid flow on endothelial sprouting. These results also demonstrate the importance of local flow dynamics due to branched vessel geometry in determining the location of sprouting angiogenesis.
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Li, Zhongnan, Guiling Li, Yongjian Li, Yuexin Chen, Jiang Li, and Haosheng Chen. "Flow field around bubbles on formation of air embolism in small vessels." Proceedings of the National Academy of Sciences 118, no. 26 (June 21, 2021): e2025406118. http://dx.doi.org/10.1073/pnas.2025406118.
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An air embolism is induced by intravascular bubbles that block the blood flow in vessels, which causes a high risk of pulmonary hypertension and myocardial and cerebral infarction. However, it is still unclear how a moving bubble is stopped in the blood flow to form an air embolism in small vessels. In this work, microfluidic experiments, in vivo and in vitro, are performed in small vessels, where bubbles are seen to deform and stop gradually in the flow. A clot is always found to originate at the tail of a moving bubble, which is attributed to the special flow field around the bubble. As the clot grows, it breaks the lubrication film between the bubble and the channel wall; thus, the friction force is increased to stop the bubble. This study illustrates the stopping process of elongated bubbles in small vessels and brings insight into the formation of air embolism.
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Andrique, L., G. Recher, K. Alessandri, N. Pujol, M. Feyeux, P. Bon, L. Cognet, P. Nassoy, and A. Bikfalvi. "A model of guided cell self-organization for rapid and spontaneous formation of functional vessels." Science Advances 5, no. 6 (June 2019): eaau6562. http://dx.doi.org/10.1126/sciadv.aau6562.
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Most achievements to engineer blood vessels are based on multiple-step manipulations such as manual sheet rolling or sequential cell seeding followed by scaffold degradation. Here, we propose a one-step strategy using a microfluidic coextrusion device to produce mature functional blood vessels. A hollow alginate hydrogel tube is internally coated with extracellular matrix to direct the self-assembly of a mixture of endothelial cells (ECs) and smooth muscle cells (SMCs). The resulting vascular structure has the correct configuration of lumen, an inner lining of ECs, and outer sheath of SMCs. These “vesseloids” reach homeostasis within a day and exhibit the following properties expected for functional vessels (i) quiescence, (ii) perfusability, and (iii) contractility in response to vasoconstrictor agents. Together, these findings provide an original and simple strategy to generate functional artificial vessels and pave the way for further developments in vascular graft and tissue engineering and for deciphering the angiogenesis process.
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Brzoska, Tomasz, Tomasz W. Kaminski, Ravi Vats, Egemen Tutuncuoglu, Mark T. Gladwin, and Prithu Sundd. "Integrin αIIbβ3 Regulates Platelet-Procoagulant Activity in the Lung." Blood 136, Supplement 1 (November 5, 2020): 32. http://dx.doi.org/10.1182/blood-2020-142503.
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Pulmonary thrombosis is a major complication associated with high morbidity. Despite advances in diagnosis and treatment, the pathophysiology of pulmonary thrombosis remains incompletely understood. New clinical evidence suggests that in situ platelet activation resulting in enhanced procoagulant activity may promote pulmonary thrombosis. Improved understanding of the etiological mechanism would enable the development of new therapies for pulmonary thrombosis. Collagen and thromboplastin (TF) were administered intravascularly (IV) to C57BL/6 (WT) mice and the pulmonary microcirculation was visualized using quantitative fluorescence intravital fluorescence lung microscopy (qFILM). Fluorochrome-conjugated anti-mouse CD49b Ab and dextran was administered IV for in vivo staining of circulating platelets and visualization of blood vessels, respectively. Pulmonary thrombosis was defined as occlusion of blood vessels with platelet aggregates leading to pulmonary ischemia. Additionally, quantitative microfluidic fluorescence microscopy (qMFM) was used to study the effect of platelet αIIbβ3 inhibition on platelet procoagulant activity in human blood under vascular mimetic flow conditions. Collagen and TF triggered dose-dependent pulmonary thrombosis in mice in vivo, which involved development of platelet-rich thrombi in the pulmonary arteriolar bottlenecks (junction of pulmonary arteriole and capillaries), resulting in a transient ischemia in the arteriole and the down-stream capillary tree. The pulmonary arteriole thrombosis triggered by IV collagen or TF was protracted, lethal and completely abrogated following IV administration of αIIbβ3 receptor inhibitor (eptifibatide). Inhibition of platelet αIIbβ3 also significantly reduced platelet procoagulant activity, fibrin formation and thrombus formation in human blood flowing through microfluidic channels ex vivo. Our current findings suggest that αIIbβ3-dependent platelet procoagulant activity promotes pulmonary thrombosis. Both our models have potential application in investigating the molecular determinants of pulmonary thrombosis in diverse pulmonary disorders as well as evaluating efficacy of new antithrombotic drugs. Disclosures No relevant conflicts of interest to declare.
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Wang, Hongcheng, Chengxin Tang, Miaomiao Zhao, Liqun Wu, and Baohua Yu. "Numerical simulation and experiment of droplet formation in circular cross-section micro-channels." International Journal of Modern Physics B 33, no. 18 (July 20, 2019): 1950200. http://dx.doi.org/10.1142/s021797921950200x.
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In recent decades, microfluidics in biological applications have experienced significant growth due to their advantages of small volume, low cost, short reaction time and high throughput. Almost all cross-section shapes of micro-channels in microfluidic chips are rectangular or triangular by the existing chip fabricating technologies, including hot embossing, lithography, etching and injection molding, etc. However, compared with the above micro-channel shapes, the circular one has the advantages in aspects of fluid flow, droplet generating, heat transfer and its replication for blood vessels. This paper presents a T-junction droplet microfluidic chip with circular cross-section micro-channels. The effect of micro-channel wettability, interfacial tension, velocity and flow rate of continuous phase on droplet size are simulated and mechanism of droplets generating process is explored. Comparing with continuous phase viscosity and interfacial tension, flow rate plays a decisive role in determining the droplet size which is in the range of 100–350 [Formula: see text]m according to the simulation result. The Capillary number is affected by the above three parameters and an estimating numerical method for generated droplet size was proposed according to the above simulation results and calculated by Capillary number. The droplets, the sizes of which were in the range of 20–400 [Formula: see text]m, were produced by varying the parameters of water and oil flow rates in the designed T-junction droplet microfluidic chip with circular cross-section micro-channels.
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Sasaki, Satoko, Tomoko Suzuki, Kyojiro Morikawa, Michiya Matsusaki, and Kae Sato. "Fabrication of a Gelatin-Based Microdevice for Vascular Cell Culture." Micromachines 14, no. 1 (December 30, 2022): 107. http://dx.doi.org/10.3390/mi14010107.
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This study presents a novel technique for fabricating microfluidic devices with microbial transglutaminase-gelatin gels instead of polydimethylsiloxane (PDMS), in which flow culture simulates blood flow and a capillary network is incorporated for assays of vascular permeability or angiogenesis. We developed a gelatin-based device with a coverslip as the bottom, which allows the use of high-magnification lenses with short working distances, and we observed the differences in cell dynamics on gelatin, glass, and PDMS surfaces. The tubes of the gelatin microfluidic channel are designed to be difficult to pull out of the inlet hole, making sample introduction easy, and the gelatin channel can be manipulated from the cell introduction to the flow culture steps in a manner comparable to that of a typical PDMS channel. Human umbilical vein endothelial cells (HUVECs) and normal human dermal fibroblasts (NHDFs) were successfully co-cultured, resulting in structures that mimicked blood vessels with inner diameters ranging from 10 µm to 500 µm. Immunostaining and scanning electron microscopy results showed that the affinity of fibronectin for gelatin was stronger than that for glass or PDMS, making gelatin a suitable substrate for cell adhesion. The ability for microscopic observation at high magnification and the ease of sample introduction make this device easier to use than conventional gelatin microfluidics, and the above-mentioned small modifications in the device structure are important points that improve its convenience as a cell assay device.
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Kastrup, Christian J., Matthew K. Runyon, Feng Shen, and Rustem F. Ismagilov. "Microfluidic Tools To Probe the Spatial Dynamics of Coagulation." Blood 110, no. 11 (November 16, 2007): 3934. http://dx.doi.org/10.1182/blood.v110.11.3934.3934.
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Abstract To investigate the biophysical mechanisms that regulate the spatial dynamics of blood coagulation, we have developed a set of microfluidic tools that allow analysis and perturbation of blood coagulation on the micrometer scale with precise control of fluid flow, geometry, and surface chemistry. Physiological coagulation occurs in a localized manner; specifically, coagulation is believed to occur exclusively at regions of substantial vascular damage and does not spread throughout the entire vascular system. In vitro analysis and characterization of these spatial dynamics requires the ability to reproduce and perturb this system, an ability that is not provided by the mixed reactor systems commonly used for in vitro studies of blood coagulation. We developed microfluidic devices with micrometer-scale channels and methods to coat these channels with various phospholipids, including components of the blood coagulation network such as thrombomodulin and tissue factor, to reproduce in vitro the geometry and surface chemistry of blood vessels in vitro. In a microfluidic device with channels coated with phospholipids and thrombomodulin, we demonstrated that clots propagate in a wave-like fashion with a constant velocity in the absence of flow. We also showed that propagation of coagulation from an occluded channel to a channel with flowing blood plasma can be regulated by the geometry of the junction and the shear rate in the channel with flowing plasma. We also developed microfluidic tools to probe the spatial dynamics of initiation of clotting by patterning surfaces with tissue factor reconstituted into phospholipids bilayers. When human plasma or whole blood was exposed to these surfaces in a microfluidic device, clotting occurred only on patches of tissue factor larger than a threshold size. This threshold patch size is controlled by the rate of activation of clotting factors at the patch and the rate of transport of activated factors off the patch. These results suggest a mechanism for how tissue factor can circulate in blood without causing clotting, and how small regions of vascular damage can exist without causing clotting. These results also suggest new biophysical mechanisms that may control interactions between the coagulation cascade and bacterial surfaces.
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Low, Wan Shi, Nahrizul Adib Kadri, and Wan Abu Bakar bin Wan Abas. "Computational Fluid Dynamics Modelling of Microfluidic Channel for Dielectrophoretic BioMEMS Application." Scientific World Journal 2014 (2014): 1–11. http://dx.doi.org/10.1155/2014/961301.
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We propose a strategy for optimizing distribution of flow in a typical benchtop microfluidic chamber for dielectrophoretic application. It is aimed at encouraging uniform flow velocity along the whole analysis chamber in order to ensure DEP force is evenly applied to biological particle. Via the study, we have come up with a constructive strategy in improving the design of microfluidic channel which will greatly facilitate the use of DEP system in laboratory and primarily focus on the relationship between architecture and cell distribution, by resorting to the tubular structure of blood vessels. The design was validated by hydrodynamic flow simulation using COMSOL Multiphysics v4.2a software. Simulations show that the presence of 2-level bifurcation has developed portioning of volumetric flow which produced uniform flow across the channel. However, further bifurcation will reduce the volumetric flow rate, thus causing undesirable deposition of cell suspension around the chamber. Finally, an improvement of microfluidic design with rounded corner is proposed to encourage a uniform cell adhesion within the channel.
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Oseev, Aleksandr, Benoît Le Roy de Boiseaumarié, Fabien Remy-Martin, Jean-François Manceau, Alain Rouleau, Franck Chollet, Wilfrid Boireau, and Thérèse Leblois. "Integration of Microresonant Sensor into a Microfluidic Platform for the Real Time Analysis of Platelets-Collagen Interaction in Flow Condition." Proceedings 2, no. 13 (December 10, 2018): 940. http://dx.doi.org/10.3390/proceedings2130940.
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The contribution focuses on the development of microresonant sensor solution integrated in microfluidic platform for the haemostasis assessment at realistic rheological flow conditions similar to the one in blood vessels. A multi-parameter sensor performs real time analysis of interactions between immobilized collagen and platelets. The detection and characterization of such interactions at controlled flow rates provide information to evaluate the dynamic of each step of primary haemostasis. The microresonant sensor concept was developed and is described in the contribution.
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Sapuppo, F., M. Bucolo, M. Intaglietta, L. Fortuna, and P. Arena. "A cellular nonlinear network: real-time technology for the analysis of microfluidic phenomena in blood vessels." Nanotechnology 17, no. 4 (January 25, 2006): S54—S63. http://dx.doi.org/10.1088/0957-4484/17/4/009.
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Au, Sam H., Brian D. Storey, John C. Moore, Qin Tang, Yeng-Long Chen, Sarah Javaid, A. Fatih Sarioglu, et al. "Clusters of circulating tumor cells traverse capillary-sized vessels." Proceedings of the National Academy of Sciences 113, no. 18 (April 18, 2016): 4947–52. http://dx.doi.org/10.1073/pnas.1524448113.
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Multicellular aggregates of circulating tumor cells (CTC clusters) are potent initiators of distant organ metastasis. However, it is currently assumed that CTC clusters are too large to pass through narrow vessels to reach these organs. Here, we present evidence that challenges this assumption through the use of microfluidic devices designed to mimic human capillary constrictions and CTC clusters obtained from patient and cancer cell origins. Over 90% of clusters containing up to 20 cells successfully traversed 5- to 10-μm constrictions even in whole blood. Clusters rapidly and reversibly reorganized into single-file chain-like geometries that substantially reduced their hydrodynamic resistances. Xenotransplantation of human CTC clusters into zebrafish showed similar reorganization and transit through capillary-sized vessels in vivo. Preliminary experiments demonstrated that clusters could be disrupted during transit using drugs that affected cellular interaction energies. These findings suggest that CTC clusters may contribute a greater role to tumor dissemination than previously believed and may point to strategies for combating CTC cluster-initiated metastasis.
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de Graaf, Mees N. S., Aisen Vivas, Andries D. van der Meer, Christine L. Mummery, and Valeria V. Orlova. "Pressure-Driven Perfusion System to Control, Multiplex and Recirculate Cell Culture Medium for Organs-on-Chips." Micromachines 13, no. 8 (August 20, 2022): 1359. http://dx.doi.org/10.3390/mi13081359.
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Organ-on-chip (OoC) devices are increasingly used to mimic the tissue microenvironment of cells in intact organs. This includes microchannels to mimic, for example, fluidic flow through blood vessels. Present methods for controlling microfluidic flow in these systems rely on gravity, rocker systems or external pressure pumps. For many purposes, pressure pumps give the most consistent flow profiles, but they are not well-suited for high throughput as might be required for testing drug responses. Here, we describe a method which allows for multiplexing of microfluidic channels in OoC devices plus the accompanying custom software necessary to run the system. Moreover, we show the approach is also suitable for recirculation of culture medium, an essential cost consideration when expensive culture reagents are used and are not “spent” through uptake by the cells during transient unidirectional flow.
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Yue, Tao, Na Liu, Yuanyuan Liu, Yan Peng, Shaorong Xie, Jun Luo, Qiang Huang, Masaru Takeuchi, and Toshio Fukuda. "On-Chip Construction of Multilayered Hydrogel Microtubes for Engineered Vascular-Like Microstructures." Micromachines 10, no. 12 (December 1, 2019): 840. http://dx.doi.org/10.3390/mi10120840.
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Multilayered and multicellular structures are indispensable for constructing functional artificial tissues. Engineered vascular-like microstructures with multiple layers are promising structures to be functionalized as artificial blood vessels. In this paper, we present an efficient method to construct multilayer microtubes embedding different microstructures based on direct fabrication and assembly inside a microfluidic device. This four-layer microfluidic device has two separate inlets for fabricating various microstructures. We have achieved alternating-layered microtubes by controlling the fabrication, flow, and assembly time of each microstructure, and as well, double-layered microtubes have been built by a two-step assembly method. Modifications of both the inner and outer layers was successfully demonstrated, and the flow conditions during the on-chip assembly were evaluated and optimized. Each microtube was successfully constructed within several minutes, showing the potential applications of the presented method for building engineered vascular-like microstructures with high efficiency.
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van der Meer, Andries D., Kim Vermeul, André A. Poot, Jan Feijen, and István Vermes. "A microfluidic wound-healing assay for quantifying endothelial cell migration." American Journal of Physiology-Heart and Circulatory Physiology 298, no. 2 (February 2010): H719—H725. http://dx.doi.org/10.1152/ajpheart.00933.2009.
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Endothelial migration is an important process in the formation of blood vessels and the repair of damaged tissue. To study this process in the laboratory, versatile and reliable migration assays are essential. The purpose of this study was to investigate whether the microfluidic version of the conventional wound-healing assay is a useful research tool for vascular science. Endothelial cells were seeded in a 500-μm-wide microfluidic channel. After overnight incubation, cells had formed a viable and confluent monolayer. Then, a wound was generated in this monolayer by flushing the channel with three parallel fluid streams, of which the middle one contained the protease trypsin. By analyzing the closing of the wound over time, endothelial cell migration could be measured. Although the migration rate was two times lower in the microfluidic assay than in the conventional assay, an identical 1.5-times increase in migration rate was found in both assays when vascular endothelial growth factor (VEGF165) was added. In the microfluidic wound-healing assay, a stable gradient of VEGF165 could be generated at the wound edge. This led to a two-times increase in migration rate compared with the untreated control. Finally, when a shear stress of 1.3 Pa was applied to the wound, the migration rate increased 1.8 times. In conclusion, the microfluidic assay is a solid alternative for the conventional wound-healing assay when endothelial cell migration is measured. Moreover, it offers unique advantages, such as gradient generation and application of shear stress.
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Praljak, Niksa, Brandon Shipley, Ayesha Gonzales, Utku Goreke, Shamreen Iram, Gundeep Singh, Ailis Hill, Umut A. Gurkan, and Michael Hinczewski. "A Deep Learning Framework for Sickle Cell Disease Microfluidic Biomarker Assays." Blood 136, Supplement 1 (November 5, 2020): 15–16. http://dx.doi.org/10.1182/blood-2020-141693.
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Introduction: Vaso-occlusive crises (VOCs) are a leading cause of morbidity and early mortality in individuals with sickle cell disease (SCD). These crises are triggered by sickle red blood cell (sRBC) aggregation in blood vessels and are influenced by factors such as enhanced sRBC and white blood cell (WBC) adhesion to inflamed endothelium. Advances in microfluidic biomarker assays (i.e., SCD Biochip systems) have led to clinical studies of blood cell adhesion onto endothelial proteins, including, fibronectin, laminin, P-selectin, ICAM-1, functionalized in microchannels. These microfluidic assays allow mimicking the physiological aspects of human microvasculature and help characterize biomechanical properties of adhered sRBCs under flow. However, analysis of the microfluidic biomarker assay data has so far relied on manual cell counting and exhaustive visual morphological characterization of cells by trained personnel. Integrating deep learning algorithms with microscopic imaging of adhesion protein functionalized microfluidic channels can accelerate and standardize accurate classification of blood cells in microfluidic biomarker assays. Here we present a deep learning approach into a general-purpose analytical tool covering a wide range of conditions: channels functionalized with different proteins (laminin or P-selectin), with varying degrees of adhesion by both sRBCs and WBCs, and in both normoxic and hypoxic environments. Methods: Our neural networks were trained on a repository of manually labeled SCD Biochip microfluidic biomarker assay whole channel images. Each channel contained adhered cells pertaining to clinical whole blood under constant shear stress of 0.1 Pa, mimicking physiological levels in post-capillary venules. The machine learning (ML) framework consists of two phases: Phase I segments pixels belonging to blood cells adhered to the microfluidic channel surface, while Phase II associates pixel clusters with specific cell types (sRBCs or WBCs). Phase I is implemented through an ensemble of seven generative fully convolutional neural networks, and Phase II is an ensemble of five neural networks based on a Resnet50 backbone. Each pixel cluster is given a probability of belonging to one of three classes: adhered sRBC, adhered WBC, or non-adhered / other. Results and Discussion: We applied our trained ML framework to 107 novel whole channel images not used during training and compared the results against counts from human experts. As seen in Fig. 1A, there was excellent agreement in counts across all protein and cell types investigated: sRBCs adhered to laminin, sRBCs adhered to P-selectin, and WBCs adhered to P-selectin. Not only was the approach able to handle surfaces functionalized with different proteins, but it also performed well for high cell density images (up to 5000 cells per image) in both normoxic and hypoxic conditions (Fig. 1B). The average uncertainty for the ML counts, obtained from accuracy metrics on the test dataset, was 3%. This uncertainty is a significant improvement on the 20% average uncertainty of the human counts, estimated from the variance in repeated manual analyses of the images. Moreover, manual classification of each image may take up to 2 hours, versus about 6 minutes per image for the ML analysis. Thus, ML provides greater consistency in the classification at a fraction of the processing time. To assess which features the network used to distinguish adhered cells, we generated class activation maps (Fig. 1C-E). These heat maps indicate the regions of focus for the algorithm in making each classification decision. Intriguingly, the highlighted features were similar to those used by human experts: the dimple in partially sickled RBCs, the sharp endpoints for highly sickled RBCs, and the uniform curvature of the WBCs. Overall the robust performance of the ML approach in our study sets the stage for generalizing it to other endothelial proteins and experimental conditions, a first step toward a universal microfluidic ML framework targeting blood disorders. Such a framework would not only be able to integrate advanced biophysical characterization into fast, point-of-care diagnostic devices, but also provide a standardized and reliable way of monitoring patients undergoing targeted therapies and curative interventions, including, stem cell and gene-based therapies for SCD. Disclosures Gurkan: Dx Now Inc.: Patents & Royalties; Xatek Inc.: Patents & Royalties; BioChip Labs: Patents & Royalties; Hemex Health, Inc.: Consultancy, Current Employment, Patents & Royalties, Research Funding.
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Beyer, Sebastian, Anna Blocki, Matthew Chung Yin Cheung, Zoe Ho Ying Wan, Babak Mehrjou, and Roger Dale Kamm. "Lectin Staining of Microvascular Glycocalyx in Microfluidic Cancer Cell Extravasation Assays." Life 11, no. 3 (February 25, 2021): 179. http://dx.doi.org/10.3390/life11030179.
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The endothelial glycocalyx forms the inner-most lining of human microvasculature. It ensures the physiological function of blood vessels and plays a crucial role in the occurrence and progression of microvascular diseases. The present communication aims to highlight the usefulness of high-resolution imaging of lectin (Bandeiraea Simplicifolia) stained endothelial glycocalyx in 3-dimensional microfluidic cell cultures. The microfluidic system allowed visualizing cancer cell extravasation, which is a key event in metastasis formation in cancer pathologies. In brief, microvascular networks were created through spontaneous vasculogenesis. This occurred from 3 dimensional (3D) suspensions of human umbilical vein endothelial cells (HUVECs) in hydrogels confined within microfluidic devices. Extravasation of MDA-MB-231 breast cancer cells from perfusable endothelial lumens was observed with confocal imaging of lectin-stained microvascular networks. The present work provides guidance towards optimizing the methodology used to elucidate the role of the endothelial glycocalyx during cancer cell extravasation. In particular, a high-resolution view of the endothelial glycocalyx at the site of extravasation is presented. The occurrence of glycocalyx defects is well aligned with the contemporary notion in the field that glycocalyx shedding precedes cancer cell extravasation.
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Naserian, Sina, Mohamed Essameldin Abdelgawad, Julie Lachaux, Nassim Arouche, Fanny Loisel, Mazdak Afshar, Gilgueng Hwang, Mercier Olaf, Anne-Marie Haghiri-Gosnet, and Georges Uzan. "Development of Bio-Artificial Micro-Vessels with Immunosuppressive Capacities: A Hope for Future Transplantations and Organoids." Blood 134, Supplement_1 (November 13, 2019): 3610. http://dx.doi.org/10.1182/blood-2019-121395.
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Background: From creating artificial organs to transplantation and cancer, newly formed vessels are playing a critical role. Endothelial progenitor cells (EPCs) are non-differentiated endothelial cells which are present in circulation and are applied in neovascularization and correction of damaged endothelial sites. In culture, EPCs generate endothelial colony forming cells (ECFCs) that have endothelial features but still retain properties of stem/progenitor cells. However, these cells which are recognized as CD31+CD144+KDR+ are very rare in blood stream. Therefore, it is necessary to be ex-vivo expanded for further applications. Since EPCs from patients are proved to be impaired and inefficient, allogenic sources either from adult or cord blood are considered as good alternatives. Due to the reaction of immune response to allogenic cells which usually leads to increased immune response and inflammation and finally elimination of injected cells, we have focused on the exact role of EPCs on immune cells, particularly, T cells which are the most important cells applied in immune rejection. Aims: First we sought to design and produce a biomimetic micro-vessel device and to endothelialize it in static and under flow conditions. Second, we wanted to investigate the interaction between EPCs and T cells to further understand their potential immunogenicity. Results: For the first part of the study we have been able to produce a biomimetic micro-vessel device that is porous, biocompatible, soft and transparent for high-resolution microscopy techniques. We have developed a multi-scale microfluidic chips with a conventional design of successive branching for fluid injection into the capillaries following Murray's laws (Figure 1A). Furthermore, we have successfully endothelialized this artificial micro-vessel using cord blood derived EPCs both in static and under flow condition and kept them in an acceptable situation up to 2 weeks after the first seeding (Figure 1B and C). For the second part of our study, we have shown for the first time that in contrary to already differentiated endothelial cells, EPCs while co-cultured with T cells, not only do not increase T cell proliferation but also are extremely immunosuppressant (Figure 1D). Moreover, we have shown that EPCs could also reduce the activation markers expressed by both CD4+ and CD8+ conventional T cells. Conclusion: We have demonstrated for the first time the possibility of producing an endothelialized vessel-like micro chamber with remarkable immunosuppressive and immunomodulatory properties. This proves the importance of using EPCs for future bio-artificial organs since these cells not only do not increase allo-response but also can regulate it. In case of transplantation, our findings could open a door for ameliorating the transplant acceptance through faster endothelialization and suppression of alloreactive T cells aiming to reject the transplant. Disclosures No relevant conflicts of interest to declare.
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Williams, Vivienne, Dmitry Kashanin, Frank O’Dowd, and Alexander John Robinson. "Microfluidic platform simulation of leucocyte adhesion to the endothelium (97.8)." Journal of Immunology 178, no. 1_Supplement (April 1, 2007): S190. http://dx.doi.org/10.4049/jimmunol.178.supp.97.8.
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Abstract Leukocyte adhesion to endothelial cell bound proteins, such as ICAM-1 and VCAM-1, is an initial step of the inflammatory response. We have developed an in vitro microfluidic system which mimics conditions found in blood vessels in vivo during an immune response. Using this system, we can record leukocyte adhesion levels under physiologically relevant flow conditions (e.g. 0–5 dynes/cm2). The adhesion profiles of resting and PMA-stimulated peripheral blood lymphocytes (PBLs) were recorded, with respect to VCAM-1, ICAM-1, and BSA. Images at each shear stress level were captured using a digital camera, and analysed using our in-house Ducocell software package. Distinct morphological changes in PMA-stimulated PBLs, compared to non-stimulated cells, can be observed. These include a less rounded appearance of the PMA-stimulated PBLs, and evidence of “uropod” formation, which anchor the T cell to the endothelium as part of the migration process. Levels of adhesion to VCAM-1 are high (80–90%, compared to control), but there appears to be little difference between the adhesion profiles of non-stimulated and PMA-stimulated PBLs. However, there is a distinct difference between the adhesion levels of non-stimulated and PMA-stimulated PBLs to ICAM-1, with PMA-stimulated cells showing a higher affinity for ICAM-1 than non-stimulated cells (approx. 70% and 40%, respectively). PBL adhesion to BSA is negligible.
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Khalida Fakhruddin, Belal Yahya Hussein Al-Tam, Abdallah Nasser Sayed, Zarin Mesbah, Angelique Maryann Pereira Anthony Jerald Pereira, Al Ameerah Elza Toto Syaputri, and Mohamad Ikhwan Jamaludin. "3D Bioprinting: Introduction and Recent Advancement." Journal of Medical Device Technology 1, no. 1 (October 8, 2022): 25–29. http://dx.doi.org/10.11113/jmeditec.v1n1.13.
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In the additive manufacturing method known as 3D bioprinting, living cells and nutrients are joined with organic and biological components to produce synthetic structures that resemble natural human tissues. To put it another way, bioprinting is a type of 3D printing that can create anything from bone tissue and blood vessels to living tissues for a range of medical purposes, including tissue engineering and drug testing and discovery. During the bioprinting process, a solution of a biomaterial or a mixture of several biomaterials in the hydrogel form, usually encapsulating the desired cell types, which are termed as bioink, is used for creating tissue constructs. This bioink can be cross-linked or stabilised during or immediately after bioprinting to generate the designed construct's final shape, structure, and architecture. This report thus offers a comprehensive review of the 3D bioprinting technology along with associated 3D bioprinting methods including ink-jet printing, extrusion printing, stereolithography, laser-assisted bioprinting and microfluidic techniques. We also focus on the types of materials, cell source, maturing, the implant of various representative tissue and organs, including blood vessels, bone and cartilage as well as recent advancements related to 3D bioprinting technology.
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Bergevin, Michele d., Anna E. Boczula, Laura-lee Caruso, Henrik Persson, Craig A. Simmons, and Tara J. Moriarty. "A Live Cell Imaging Microfluidic Model for Studying Extravasation of Bloodborne Bacterial Pathogens." Cellular Microbiology 2022 (September 14, 2022): 1–12. http://dx.doi.org/10.1155/2022/3130361.
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Bacteria that migrate (extravasate) out of the bloodstream during vascular dissemination can cause secondary infections in many tissues and organs, including the brain, heart, liver, joints, and bone with clinically serious and sometimes fatal outcomes. The mechanisms by which bacteria extravasate through endothelial barriers in the face of blood flow-induced shear stress are poorly understood, in part because individual bacteria are rarely observed traversing endothelia in vivo, and in vitro model systems inadequately mimic the vascular environment. To enable the study of bacterial extravasation mechanisms, we developed a transmembrane microfluidics device mimicking human blood vessels. Fast, quantitative, three-dimensional live cell imaging in this system permitted single-cell resolution measurement of the Lyme disease bacterium Borrelia burgdorferi transmigrating through monolayers of primary human endothelial cells under physiological shear stress. This cost-effective, flexible method was 10,000 times more sensitive than conventional plate reader-based methods for measuring transendothelial migration. Validation studies confirmed that B. burgdorferi transmigrate actively and strikingly do so at similar rates under static and physiological flow conditions. This method has significant potential for future studies of B. burgdorferi extravasation mechanisms, as well as the transendothelial migration mechanisms of other disseminating bloodborne pathogens.
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Schuerle, S., A. P. Soleimany, T. Yeh, G. M. Anand, M. Häberli, H. E. Fleming, N. Mirkhani, et al. "Synthetic and living micropropellers for convection-enhanced nanoparticle transport." Science Advances 5, no. 4 (April 2019): eaav4803. http://dx.doi.org/10.1126/sciadv.aav4803.
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Nanoparticles (NPs) have emerged as an advantageous drug delivery platform for the treatment of various ailments including cancer and cardiovascular and inflammatory diseases. However, their efficacy in shuttling materials to diseased tissue is hampered by a number of physiological barriers. One hurdle is transport out of the blood vessels, compounded by difficulties in subsequent penetration into the target tissue. Here, we report the use of two distinct micropropellers powered by rotating magnetic fields to increase diffusion-limited NP transport by enhancing local fluid convection. In the first approach, we used a single synthetic magnetic microrobot called an artificial bacterial flagellum (ABF), and in the second approach, we used swarms of magnetotactic bacteria (MTB) to create a directable “living ferrofluid” by exploiting ferrohydrodynamics. Both approaches enhance NP transport in a microfluidic model of blood extravasation and tissue penetration that consists of microchannels bordered by a collagen matrix.
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Henderson, Aria R., Hyoann Choi, and Esak Lee. "Blood and Lymphatic Vasculatures On-Chip Platforms and Their Applications for Organ-Specific In Vitro Modeling." Micromachines 11, no. 2 (January 29, 2020): 147. http://dx.doi.org/10.3390/mi11020147.
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The human circulatory system is divided into two complementary and different systems, the cardiovascular and the lymphatic system. The cardiovascular system is mainly concerned with providing nutrients to the body via blood and transporting wastes away from the tissues to be released from the body. The lymphatic system focuses on the transport of fluid, cells, and lipid from interstitial tissue spaces to lymph nodes and, ultimately, to the cardiovascular system, as well as helps coordinate interstitial fluid and lipid homeostasis and immune responses. In addition to having distinct structures from each other, each system also has organ-specific variations throughout the body and both systems play important roles in maintaining homeostasis. Dysfunction of either system leads to devastating and potentially fatal diseases, warranting accurate models of both blood and lymphatic vessels for better studies. As these models also require physiological flow (luminal and interstitial), extracellular matrix conditions, dimensionality, chemotactic biochemical gradient, and stiffness, to better reflect in vivo, three dimensional (3D) microfluidic (on-a-chip) devices are promising platforms to model human physiology and pathology. In this review, we discuss the heterogeneity of both blood and lymphatic vessels, as well as current in vitro models. We, then, explore the organ-specific features of each system with examples in the gut and the brain and the implications of dysfunction of either vasculature in these organs. We close the review with discussions on current in vitro models for specific diseases with an emphasis on on-chip techniques.
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Ahn, Byungwook, Yumiko Sakurai, David R. Myers, Yongzhi Qiu, Elaissa Hardy, Reginald Tran, Robert Mannino, Alisa S. Wolberg, and Wilbur Lam. "An “Endothelialized” Microfluidic System That Distinguishes Procoagulant Mechanisms in Arterial and Venous Thrombosis." Blood 120, no. 21 (November 16, 2012): 1071. http://dx.doi.org/10.1182/blood.v120.21.1071.1071.
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Abstract Abstract 1071 Introduction: Hemostatic and thrombotic processes are dependent on platelets, coagulation factors, endothelial cells and hemodynamic flow. Current in vitro assays, however, only encompass one or two of these variables, rendering their results difficult to extrapolate to the in vivo setting. To that end, we have further refined our previously published endothelialized microfluidic system for studying thrombotic processes (Tsai et al, JCI, 2012) to specifically incorporate simultaneous differential flow rates spanning venous (10 s−1), capillary (100 s−1), and arterial/arteriolar (1000 s−1) flow conditions. Overall, key advantages of our system include: 1) successful integration of whole blood, an intact endothelium, and hemodynamic flow in a single microfluidic device, 2) simultaneous differential flow rates in a single experiment spanning 3 orders of magnitude, 3) use of corn trypsin inhibitor (CTI) as the sole anticoagulant, enabling calcium dependent processes to occur, and 4) minimal sample volume (1–2 mL) even for high shear conditions. We have applied our microsystem to elucidate some of the underlying mechanistic differences between venous and arterial thrombosis. Methods: We used photolithographic and microfabrication techniques. We previously employed to develop the silicone-based microfluidic device and applied our optimized protocol to culture human endothelial cells (HUVECs) to confluency throughout the entire inner surfaces of the system (Tsai et al, JCI, 2012 and Myers et al, JoVE, 2012) (Figure 1A, 1B). Once HUVECs are successfully cultured, whole blood with 5 % v/v fluorescently-labeled fibrinogen and cell membrane dyes is flowed into the microfluidic system. Our device can then be “activated” to induce 3 different simultaneous shear conditions (10 s−1, 100 s−1 and 1000 s−1) in 3 separate endothelialized microchannels by differentially varying the hydrodynamic resistance in each microchannel. Figure 1C shows a fibrin network that is formed under flow conditions (with whole blood anticoagulated with CTI) in one of the endothelialized microchannels. Results: Elevated levels of prothrombin are known to increase risk of venous, but not arterial thrombosis. We applied our novel system to investigate whether differences between arterial and venous thrombosis risk depend, at least in part, on differences in blood flow/shear in those vessels. By adding 1.38 μM of prothrombin to whole blood with CTI and flowing it into our system, we observed platelet-rich and fibrin rich thrombi form and occlude the 10 s−1 and 100 s−1 microchannels, but not the 1000 s−1 microchannel. In contrast, whole blood with CTI without prothrombin yielded minimal fibrin formation on the endothelial cells with no platelet adhesion/aggregation detected in all 3 shear conditions. Conclusions: Our results suggest that arterial and venous thrombosis risk is due, at least in part, to the differences in shear flow and highlights the utility of our novel endothelialized microfluidic system with simultaneous differential flow rates. Coupled with complementary in vivo experiments, our system is a powerful tool to investigate the underlying mechanisms of hemostatic and thrombotic processes involving flow. Disclosures: No relevant conflicts of interest to declare.
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Cai, Shengze, He Li, Fuyin Zheng, Fang Kong, Ming Dao, George Em Karniadakis, and Subra Suresh. "Artificial intelligence velocimetry and microaneurysm-on-a-chip for three-dimensional analysis of blood flow in physiology and disease." Proceedings of the National Academy of Sciences 118, no. 13 (March 24, 2021): e2100697118. http://dx.doi.org/10.1073/pnas.2100697118.
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Understanding the mechanics of blood flow is necessary for developing insights into mechanisms of physiology and vascular diseases in microcirculation. Given the limitations of technologies available for assessing in vivo flow fields, in vitro methods based on traditional microfluidic platforms have been developed to mimic physiological conditions. However, existing methods lack the capability to provide accurate assessment of these flow fields, particularly in vessels with complex geometries. Conventional approaches to quantify flow fields rely either on analyzing only visual images or on enforcing underlying physics without considering visualization data, which could compromise accuracy of predictions. Here, we present artificial-intelligence velocimetry (AIV) to quantify velocity and stress fields of blood flow by integrating the imaging data with underlying physics using physics-informed neural networks. We demonstrate the capability of AIV by quantifying hemodynamics in microchannels designed to mimic saccular-shaped microaneurysms (microaneurysm-on-a-chip, or MAOAC), which signify common manifestations of diabetic retinopathy, a leading cause of vision loss from blood-vessel damage in the retina in diabetic patients. We show that AIV can, without any a priori knowledge of the inlet and outlet boundary conditions, infer the two-dimensional (2D) flow fields from a sequence of 2D images of blood flow in MAOAC, but also can infer three-dimensional (3D) flow fields using only 2D images, thanks to the encoded physics laws. AIV provides a unique paradigm that seamlessly integrates images, experimental data, and underlying physics using neural networks to automatically analyze experimental data and infer key hemodynamic indicators that assess vascular injury.
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Cugno, Andrea, Alex Marki, and Klaus Ley. "Biomechanics of Neutrophil Tethers." Life 11, no. 6 (May 31, 2021): 515. http://dx.doi.org/10.3390/life11060515.
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Leukocytes, including neutrophils, propelled by blood flow, can roll on inflamed endothelium using transient bonds between selectins and their ligands, and integrins and their ligands. When such receptor–ligand bonds last long enough, the leukocyte microvilli become extended and eventually form thin, 20 µm long tethers. Tether formation can be observed in blood vessels in vivo and in microfluidic flow chambers. Tethers can also be extracted using micropipette aspiration, biomembrane force probe, optical trap, or atomic force microscopy approaches. Here, we review the biomechanical properties of leukocyte tethers as gleaned from such measurements and discuss the advantages and disadvantages of each approach. We also review and discuss viscoelastic models that describe the dependence of tether formation on time, force, rate of loading, and cell activation. We close by emphasizing the need to combine experimental observations with quantitative models and computer simulations to understand how tether formation is affected by membrane tension, membrane reservoir, and interactions of the membrane with the cytoskeleton.
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Wu, Jing, Qiushui Chen, Wu Liu, Yandong Zhang, and Jin-Ming Lin. "Cytotoxicity of quantum dots assay on a microfluidic 3D-culture device based on modeling diffusion process between blood vessels and tissues." Lab on a Chip 12, no. 18 (2012): 3474. http://dx.doi.org/10.1039/c2lc40502d.
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Hwang, Kyu-Seok, Yuji Son, Seong Soon Kim, Dae-Seop Shin, So Hee Lim, Jung Yoon Yang, Ha Neul Jeong, Byung Hoi Lee, and Myung Ae Bae. "Size-Dependent Effects of Polystyrene Nanoparticles (PS-NPs) on Behaviors and Endogenous Neurochemicals in Zebrafish Larvae." International Journal of Molecular Sciences 23, no. 18 (September 14, 2022): 10682. http://dx.doi.org/10.3390/ijms231810682.
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Microplastics, small pieces of plastic derived from polystyrene, have recently become an ecological hazard due to their toxicity and widespread occurrence in aquatic ecosystems. In this study, we exposed zebrafish larvae to two types of fluorescent polystyrene nanoparticles (PS-NPs) to identify their size-dependent effects. PS-NPs of 50 nm, unlike 100 nm PS-NPs, were found to circulate in the blood vessels and accumulate in the brains of zebrafish larvae. Behavioral and electroencephalogram (EEG) analysis showed that 50 nm PS-NPs induce abnormal behavioral patterns and changes in EEG power spectral densities in zebrafish larvae. In addition, the quantification of endogenous neurochemicals in zebrafish larvae showed that 50 nm PS-NPs disturb dopaminergic metabolites, whereas 100 nm PS-NPs do not. Finally, we assessed the effect of PS-NPs on the permeability of the blood–brain barrier (BBB) using a microfluidic system. The results revealed that 50 nm PS-NPs have high BBB penetration compared with 100 nm PS-NPs. Taken together, we concluded that small nanoparticles disturb the nervous system, especially dopaminergic metabolites.
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Mei, Xueting, Kevin Middleton, Dongsub Shim, Qianqian Wan, Liangcheng Xu, Yu-Heng Vivian Ma, Deepika Devadas, et al. "Microfluidic platform for studying osteocyte mechanoregulation of breast cancer bone metastasis." Integrative Biology 11, no. 4 (April 1, 2019): 119–29. http://dx.doi.org/10.1093/intbio/zyz008.
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AbstractBone metastasis is a common, yet serious, complication of breast cancer. Breast cancer cells that extravasate from blood vessels to the bone devastate bone quality by interacting with bone cells and disrupting the bone remodeling balance. Although exercise is often suggested as a cancer intervention strategy and mechanical loading during exercise is known to regulate bone remodeling, its role in preventing bone metastasis remains unknown. We developed a novel in vitro microfluidic tissue model to investigate the role of osteocytes in the mechanical regulation of breast cancer bone metastasis. Metastatic MDA-MB-231 breast cancer cells were cultured inside a 3D microfluidic lumen lined with human umbilical vein endothelial cells (HUVECs), which is adjacent to a channel seeded with osteocyte-like MLO-Y4 cells. Physiologically relevant oscillatory fluid flow (OFF) (1 Pa, 1 Hz) was applied to mechanically stimulate the osteocytes. Hydrogel-filled side channels in-between the two channels allowed real-time, bi-directional cellular signaling and cancer cell extravasation over 3 days. The applied OFF was capable of inducing intracellular calcium responses in osteocytes (82.3% cells responding with a 3.71 fold increase average magnitude). Both extravasation distance and percentage of extravasated side-channels were significantly reduced with mechanically stimulated osteocytes (32.4% and 53.5% of control, respectively) compared to static osteocytes (102.1% and 107.3% of control, respectively). This is the first microfluidic device that has successfully integrated stimulatory bone fluid flow, and demonstrated that mechanically stimulated osteocytes reduced breast cancer extravasation. Future work with this platform will determine the specific mechanisms involved in osteocyte mechanoregulation of breast cancer bone metastasis, as well as other types of cancer metastasis and diseases.
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Fay, Meredith E., David R. Myers, Amit Kumar, Cory T. Turbyfield, Rebecca Byler, Kaci Crawford, Robert G. Mannino, et al. "Cellular softening mediates leukocyte demargination and trafficking, thereby increasing clinical blood counts." Proceedings of the National Academy of Sciences 113, no. 8 (February 8, 2016): 1987–92. http://dx.doi.org/10.1073/pnas.1508920113.
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Leukocytes normally marginate toward the vascular wall in large vessels and within the microvasculature. Reversal of this process, leukocyte demargination, leads to substantial increases in the clinical white blood cell and granulocyte count and is a well-documented effect of glucocorticoid and catecholamine hormones, although the underlying mechanisms remain unclear. Here we show that alterations in granulocyte mechanical properties are the driving force behind glucocorticoid- and catecholamine-induced demargination. First, we found that the proportions of granulocytes from healthy human subjects that traversed and demarginated from microfluidic models of capillary beds and veins, respectively, increased after the subjects ingested glucocorticoids. Also, we show that glucocorticoid and catecholamine exposure reorganizes cellular cortical actin, significantly reducing granulocyte stiffness, as measured with atomic force microscopy. Furthermore, using simple kinetic theory computational modeling, we found that this reduction in stiffness alone is sufficient to cause granulocyte demargination. Taken together, our findings reveal a biomechanical answer to an old hematologic question regarding how glucocorticoids and catecholamines cause leukocyte demargination. In addition, in a broader sense, we have discovered a temporally and energetically efficient mechanism in which the innate immune system can simply alter leukocyte stiffness to fine tune margination/demargination and therefore leukocyte trafficking in general. These observations have broad clinically relevant implications for the inflammatory process overall as well as hematopoietic stem cell mobilization and homing.
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Barbot, Antoine, Dominique Decanini, and Gilgueng Hwang. "Local flow sensing on helical microrobots for semi-automatic motion adaptation." International Journal of Robotics Research 39, no. 4 (December 23, 2019): 476–89. http://dx.doi.org/10.1177/0278364919894374.
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Helical microrobots with dimensions below 100 µm could serve many applications for manipulation and sensing in small, closed environments such as blood vessels or inside microfluidic chips. However, environmental conditions such as surface stiction from the channel wall or local flow can quickly result in the loss of control of the microrobot, especially for untrained users. Therefore, to automatically adapt to changing conditions, we propose an algorithm that switches between a surface-based motion of the microrobot and a 3D swimming motion depending on the local flow value. Indeed swimming is better for avoiding obstacles and difficult surface stiction areas but it is more sensitive to the flow than surface motion such as rolling or spintop motion. First, we prove the flow sensing ability of helical microrobots based on the difference between the tracked and theoretical speed. For this, a 50 µm long and 5 µm diameter helical microrobot measures the flow profile shape in two different microchannels. These measurements are then compared with simulation results. Then, we demonstrate both swimming and surface-based motion using closed-loop control. Finally, we test our algorithm by following a 2D path using closed-loop control, and adapting the type of motion depending on the flow speed measured by the microrobot. Such results could enable simple high-level control that could expand the development of microrobots toward applications in complex microfluidic environments.
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Lee, Jungeun, Seungwoo Hong, and Byung-Kwon Kim. "Analysis of Single Blood Entities Using an Ultramicroelectrode through Single-Entity Electrochemistry." ECS Meeting Abstracts MA2022-01, no. 53 (July 7, 2022): 2217. http://dx.doi.org/10.1149/ma2022-01532217mtgabs.
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The research was conducted with the aim of analyzing individual human blood cells in the presence of redox species based on single-entity electrochemistry. Various blood cells (e.g., red blood cell, white blood cell, and platelets) perform a role directly related to survival, such as oxygen transport, hemostasis, and immune response, as they move through the whole body’s blood vessels. However, with the current lab-on-a-chip typed biosensor technology, the factors that can be measured using a small amount of blood are limited to uric acid, cholesterol, glucose, or hemoglobin in the blood. In clinical laboratories, common methods for measuring individual blood counts are automated hematology analyzers based on the automated Coulter cell counting method or flow cytometry, which produce a huge amount of blood count results, but are limited. For this reason, it is proposed a method for detecting individual blood cells that can be applied to a biosensor simply and quickly. References Ran, An. et al. (2021) ‘Emerging point-of-care technologies for anemia detection’, Lab on a Chip, 21(10), pp. 1843-1865. J-H, Lin. et al. (2020) ‘A Multifunctional Microfluidic Device for Blood Typing and Primary Screening of Blood Diseases’ ACS Sensors, 5(10), pp. 3082-3090. J, Lee. et al. (2020) ‘Determination of Serotonin Concentration in Single Human Platelets through Single-Entity Electrochemistry’, ACS Sensors, 5(7), pp. 1943-1948. T.L.T, Ho. et al. (2018) ‘Determining mean corpuscular volume and red blood cell count using electrochemical collision events’, Biosensors and Bioelectronics. 110, pp. 155-159. A-C, Villani. et al. (2017) ‘Single-cell RNA-seq reveals new types of human blood dendritic cells, monocytes, and progenitors’, Science. 356, No.6335.
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Marrella, Alessandra, Arianna Fedi, Gabriele Varani, Ivan Vaccari, Marco Fato, Giuseppe Firpo, Patrizia Guida, Nicola Aceto, and Silvia Scaglione. "High blood flow shear stress values are associated with circulating tumor cells cluster disaggregation in a multi-channel microfluidic device." PLOS ONE 16, no. 1 (January 14, 2021): e0245536. http://dx.doi.org/10.1371/journal.pone.0245536.
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Metastasis represents a dynamic succession of events involving tumor cells which disseminate through the organism via the bloodstream. Circulating tumor cells (CTCs) can flow the bloodstream as single cells or as multicellular aggregates (clusters), which present a different potential to metastasize. The effects of the bloodstream-related physical constraints, such as hemodynamic wall shear stress (WSS), on CTC clusters are still unclear. Therefore, we developed, upon theoretical and CFD modeling, a new multichannel microfluidic device able to simultaneously reproduce different WSS characterizing the human circulatory system, where to analyze the correlation between SS and CTC clusters behavior. Three physiological WSS levels (i.e. 2, 5, 20 dyn/cm2) were generated, reproducing values typical of capillaries, veins and arteries. As first validation, triple-negative breast cancer cells (MDA-MB-231) were injected as single CTCs showing that higher values of WSS are correlated with a decreased viability. Next, the SS-mediated disaggregation of CTC clusters was computationally investigated in a vessels-mimicking domain. Finally, CTC clusters were injected within the three different circuits and subjected to the three different WSS, revealing that increasing WSS levels are associated with a raising clusters disaggregation after 6 hours of circulation. These results suggest that our device may represent a valid in vitro tool to carry out systematic studies on the biological significance of blood flow mechanical forces and eventually to promote new strategies for anticancer therapy.
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Московцев, А. А., А. Н. Мыльникова, Д. В. Колесов, А. А. Микрюкова, Д. М. Зайченко, А. А. Соколовская, and А. А. Кубатиев. "Estimation of nitric oxide generation by endothelial cells EA.hy926 under flow-induced mechanical stress in a microfluidic system." Nauchno-prakticheskii zhurnal «Patogenez», no. 4 (December 25, 2020): 71–77. http://dx.doi.org/10.25557/2310-0435.2020.04.71-77.
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Эндотелиальные клетки, выстилающие стенки сосудов, преобразовывают деформацию собственных структур, вызванную током крови, в химические сигналы, одним из которых является важный регулятор просвета сосуда - оксид азота (NO). К настоящему моменту накоплен большой объём данных о клеточных механизмах активации продукции NO, однако сведений о динамике генерации оксида азота эндотелиальными клетками в зависимости от гидродинамических условий недостаточно. В этой связи разработка микрофлюидных систем in vitro, имитирующих кровеносное русло, и изучение в них эндотелия в сложных гидродинамических условиях является актуальной задачей. В данной работе для создания контролируемых гидродинамических условий для монослоя эндотелиоцитоподобных клеток EA.hy926 была спроектирована и разработана микрофлюидная система, имитирующая линейные участки микрососудистого русла. Методом непрямого определения содержания оксида азота (II) NO с использованием флуоресцентного зонда 4,5-диаминофлуоресцеина DAF-2 впервые получены данные об увеличении продукции NO клетками EA.hy926 при механическом стрессе, создаваемом потоком ростовой среды. Представлены расчетные гидродинамические характеристики микрофлюидной системы, а также методика измерения продукции NO. Возможность исследования функциональной активности эндотелия позволяет использовать разработанную микрофлюидную модельную систему как для изучения клеточно-автономных регуляторных свойств эндотелия при действии ряда вазоактивных фармакологических препаратов и других методов воздействия на эндотелий, так и при моделируемой дисфункции эндотелия. Endothelial cells lining vascular walls transform the flow-induced deformation of their own structures into chemical signals, one of which, nitric oxide (NO), is an important regulator of the vascular lumen diameter. By present, a large amount of data on cellular mechanisms for activation of NO production has been accumulated. However, there is insufficient information on changes in endothelial NO generation under different hydrodynamic conditions. Therefore, development of microfluidic systems that model blood vessels in vitro and using them to study the endothelium under complex hydrodynamic conditions are relevant tasks. In this study, a microfluidic system was developed to create controlled hydrodynamic conditions for a monolayer of endotheliocyte-like cells EAhy.926. This system simulates linear sections of the microvasculature. By indirect measurement of NO (II) content with a fluorescent 4,5-diaminofluorescein (DAF-2) probe, we showed an increase in the NO production by EAhy.926 cells under mechanical stress generated by the medium flow. The article presents the method for measuring NO production and the calculated hydrodynamic characteristics of the microfluidic system. The results showed that the developed microfluidic model system is promising for studying cell-autonomous regulatory properties of the endothelium both under the action of vasoactive agents and in simulated endothelial dysfunction.
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Hajal, Cynthia, Joelle Straehla, Giovanni Offeddu, Hannah Safford, Paula Hammond, and Roger Kamm. "TMOD-11. A PREDICTIVE MICROFLUIDIC MODEL OF VASCULARIZED GLIOMA TUMORS TO ASSESS TRAFFICKING OF THERAPEUTICS ACROSS THE BLOOD-BRAIN BARRIER." Neuro-Oncology 23, Supplement_6 (November 2, 2021): vi217—vi218. http://dx.doi.org/10.1093/neuonc/noab196.872.
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Abstract Although important distinctions exist between subtypes of HGGs, all patients fare poorly with a 5-year survival of < 5%. The infiltrative growth of HGGs imposes significant challenges to treatment, with the presence of residual microscopic tumors post-surgery and hampered chemotherapeutic penetration due to the blood-brain barrier (BBB), a highly selective endothelium regulating transport between blood and brain. Moreover, current preclinical models are costly, low throughput, and often fail to predict drug performance in the clinic. There is thus an increasing need for in vitro models of HGG tumors, surrounded by a realistic BBB vasculature, to recapitulate therapeutic trafficking. To this end, we engineered a high-throughput microfluidic model composed entirely of human cells where HGG spheroids are embedded in a BBB microvascular network of endothelial cells, pericytes, and astrocytes. Tumors co-opt the vasculature as observed in patients and BBB permeability measurements with perfused dextran demonstrate unaltered paracellular transport near and far from the tumors. Lipoprotein receptor protein 1 (LRP1) expression, however, is increased near HGG spheroids. These results were leveraged to synthesize layer-by-layer nanoparticles with angiopep-2 ligands exhibiting high affinity to LRP1 for targeted delivery. Angiopep-2 nanoparticles displayed the largest permeability across the BBB near tumors compared to bare nanoparticles. Knock-down of LRP1 in the BBB-HGG model decreased angiopep-2 nanoparticle permeability confirming LRP1-mediated transport. We next assessed the therapeutic implications of our findings by encapsulating cisplatin in the particles. Administration of cisplatin-loaded angiopep-2 nanoparticles resulted in significant tumor shrinkage with targeted apoptosis in the tumor area compared to bare cisplatin nanoparticles or free cisplatin which damaged the surrounding healthy BBB vessels. Validations in orthotopic tumor-bearing mice are ongoing. In summary, we propose a novel preclinical model with a functional human BBB vasculature surrounding HGG tumors for high-throughput investigations and validate that this platform predicts the transport of targeted drug-loaded carriers.
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Ghasemali, Samaneh, Safar Farajnia, Abolfazl Barzegar, Mohammad Rahmati-Yamchi, Roghayyeh Baghban, Leila Rahbarnia, and Hamid R. Y. Nodeh. "New Developments in Anti-Angiogenic Therapy of Cancer, Review and Update." Anti-Cancer Agents in Medicinal Chemistry 21, no. 1 (December 29, 2020): 3–19. http://dx.doi.org/10.2174/1871520620666200817103219.
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Background: Angiogenesis is one of the critical physiological processes, by which the new blood vessels are generated from the pre-existing vessels in the early stage of vasculogenesis. While normal angiogenesis is critical for development and tissue growth, pathologic angiogenesis is important for the growth and spread of cancers by supplying nutrients and oxygen as well as providing a conduit for distant metastasis. In the last two decades, angiogenesis has been the area of extensive researches, indicating antiangiogenic target therapy as an effective strategy for cancer therapy. At present, this field has become a major avenue for research and development of novel therapeutics. Objective: This review is dedicated to an updated review of the most prominent antiangiogenic agents, emphasizing the novel advancements and their applications, in particular, in the fields of antibodies, peptides, vaccines, endogenous inhibitors, Nanoparticles (NPs), antiangiogenic oligonucleotides and small molecules. Also, the potential role of 3D microfluidic models as an affordable and time-saving tool for angiogenesis investigations are discussed. Methods: Firstly, we collected and summarized new developments that have occurred in all review and research articles in databases. Then, we used important keywords related to antiangiogenic target therapy and their applications for retrieval of most relevant data. Results: This review is based on recent research and review articles and intended to cover all newly discovered agents inhibiting tumor angiogenesis and in particular, VEGF. Conclusion: New research studies have shown that anti-angiogenesis agents especially, in the form of combination therapy are effective in various cancers treatment.
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Vivas, Aisen, Julia Mikhal, Gabriela M. Ong, Anna Eigenbrodt, Andries D. van der Meer, Rene Aquarius, Bernard J. Geurts, and Hieronymus D. Boogaarts. "Aneurysm-on-a-Chip: Setting Flow Parameters for Microfluidic Endothelial Cultures Based on Computational Fluid Dynamics Modeling of Intracranial Aneurysms." Brain Sciences 12, no. 5 (May 5, 2022): 603. http://dx.doi.org/10.3390/brainsci12050603.
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Intracranial aneurysms are pouch-like extrusions from the vessels at the base of the brain which can rupture and cause a subarachnoid hemorrhage. The pathophysiological mechanism of aneurysm formation is thought to be a consequence of blood flow (hemodynamic) induced changes on the endothelium. In this study, the results of a personalized aneurysm-on-a-chip model using patient-specific flow parameters and patient-specific cells are presented. CT imaging was used to calculate CFD parameters using an immersed boundary method. A microfluidic device either cultured with human umbilical vein endothelial cells (HUVECs) or human induced pluripotent stem cell-derived endothelial cells (hiPSC-EC) was used. Both types of endothelial cells were exposed for 24 h to either 0.03 Pa or 1.5 Pa shear stress, corresponding to regions of low shear and high shear in the computational aneurysm model, respectively. As a control, both cell types were also cultured under static conditions for 24 h as a control. Both HUVEC and hiPSC-EC cultures presented as confluent monolayers with no particular cell alignment in static or low shear conditions. Under high shear conditions HUVEC elongated and aligned in the direction of the flow. HiPSC-EC exhibited reduced cell numbers, monolayer gap formation and cells with aberrant, spread-out morphology. Future research should focus on hiPSC-EC stabilization to allow personalized intracranial aneurysm models.
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Sandor, Barbara, Mickael Marin, Claudine Lapoumeroulie, Miklos Rabai, Sophie Lefevre, Nathalie Lemonne, Wassim El Nemer, et al. "Effects of Poloxamer 188 on Red Blood Cells Membrane Properties in Sickle Cell Disease." Blood 126, no. 23 (December 3, 2015): 2174. http://dx.doi.org/10.1182/blood.v126.23.2174.2174.
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Abstract Introduction: Sickle cell anemia (SCA) is a severe monogenic hereditary disorder characterized by chronic hemolytic anemia and the occurrence of frequent painful vaso-occlusive crises (VOC). SCA classical physiological scheme involves hemoglobin S polymerization under hypoxic conditions, which triggers red blood cells (RBCs) sickling. Recent studies demonstrated that the degree of hemorheological alterations, such as blood hyper-viscosity, determines the risk for VOC. Moreover, sickle RBCs abnormally adhere to the vascular endothelium, triggering microvascular occlusions. Despite extensive research very few drugs are available to efficiently treat VOCs or VOC-like events. A first clinical trial was performed to test the efficacy of poloxamer 188 (P188) in a large sickle cell cohort of adults and children (Orringer et al., 2001). This study demonstrated a significant reduction of pain duration in the children treated with P188. Recently, a new phase III multi-center trial has been started to test the efficacy of this drug during acute VOC in children (Humphries et al., 2015). We conducted in vitro experiments using the commercial formulation named Kolliphor P188 (Sigma-Aldrich) to test the effects of this drug on RBCs biophysical properties of SCA patients. Methods: To measure deformability and mechanical properties of RBCs, we used ektacytometry and microfluidic device mimicking the diameters of the micro vessels. RBCs adhesion assays were performed on HMEC-1 (Human Microvascular Endothelial Cell line) using dynamic flow adhesion platform. RBCs from healthy (AA) and SCA individuals were used for the different experiments. Results: While P188 did not significantly affect blood viscosity in AA, P188 treatment decreased blood viscosity at the lowest shear rates in SCA (Fig 1A). When measured in plasma, RBC aggregation decreased with P188 in SCA patients but not in AA (Fig 1B). RBC deformability assessed by both ektacytometry (not shown) and microfluidic device (Fig 2) was not affected by P188. This is in agreement with the mode of action of P188 suggesting that it binds to hydrophobic surfaces and lowers surface tension without any changes in the organization of the cytoskeleton. We examined the effect of P188 treatment on SCA-RBCs adhesion to the endothelial HMEC-1 cell line. We observed a mean adhesion of 40 cells/ mm2 for the untreated SCA-RBCs versus 20 cells/mm2 in the case of P188 treated RBCs, i.e. a 50% decrease upon P188 treatment (Fig 3). As for RBC aggregation, our findings suggest that the binding of P188 to SCA-RBCs membranes prevent the interaction with endothelial cells. This is of particular importance in the context of SCA since increased RBC adhesiveness has been demonstrated to trigger VOC. Conclusion: In parallel of the phase III clinical trial studying the profit of P188 for sickle cell patients during VOCs, our results bring clarifications regarding its mode of action on RBCs. We show that P188 directly reduces blood viscosity, RBC aggregation and adhesiveness to endothelial cells, making this drug as a potential beneficial therapy in SCA. References: Orringer, E.P., et al; (2001). JAMA, 286, 2099-2106. Humphries, J.D., et al; (2015). Trends Cell Biol, 25, 388-397. Figure 1. P188 treatment decreases blood viscosity (A) and RBC aggregation (B) in SS patients but not in AA controls. Figure 1. P188 treatment decreases blood viscosity (A) and RBC aggregation (B) in SS patients but not in AA controls. Figure 2. P188 treatment does not change SCA-RBC deformability. (A) Design of the microfluidic chip containing eight filtering units organized in parallel. Each filtering unit has a height of 5 µm and pillars are organized to allow an escape route of 20 µm around the unit to avoid occlusion. The surrounding pillars line has 5 µm slits. (B) Retention percentage of untreated and P188-treated SCA-RBC in 5 µm slits. Histograms represent mean of 5 µm slits from the 8 filtering units in one chip expressed in percent of total trapped RBCs for three patients. Figure 2. P188 treatment does not change SCA-RBC deformability. (A) Design of the microfluidic chip containing eight filtering units organized in parallel. Each filtering unit has a height of 5 µm and pillars are organized to allow an escape route of 20 µm around the unit to avoid occlusion. The surrounding pillars line has 5 µm slits. (B) Retention percentage of untreated and P188-treated SCA-RBC in 5 µm slits. Histograms represent mean of 5 µm slits from the 8 filtering units in one chip expressed in percent of total trapped RBCs for three patients. Figure 3. Graph representing adherent cells per mm2 at a flow rate of 1 dyne/mm2. The mean of the 5 patients is expressed as the average number of adherent cells/mm2 ± SD. Paired t test, P < 0.05. Figure 3. Graph representing adherent cells per mm2 at a flow rate of 1 dyne/mm2. The mean of the 5 patients is expressed as the average number of adherent cells/mm2 ± SD. Paired t test, P < 0.05. Disclosures No relevant conflicts of interest to declare.
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Villa, Carlos Hipolito, Daniel Pan, Ian Johnston, Colin F. Greineder, Marion E. Reid, Douglas B. Cines, Mortimer Poncz, Don L. Siegel, and Vladimir R. Muzykantov. "Coupling Therapeutics to Human Erythrocytes Demonstrates Target-Dependent Effects on Red Cell Physiology While Preserving Efficacy." Blood 128, no. 22 (December 2, 2016): 701. http://dx.doi.org/10.1182/blood.v128.22.701.701.
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Abstract Delivery of bio-therapeutics by red blood cells (RBCs) can greatly enhance pharmacokinetics and pharmacodynamics of the appended or loaded agents, and may even potentiate induction of immunologic tolerance. Our group and others have successfully used fusion proteins, antibodies, and peptides to couple therapeutics to murine, but not human, RBCs. It is known that extracellular ligands have the potential to induce marked, epitope-dependent changes in red cell physiology, including changes in deformability, phosphatidyl-serine (PS) exposure, and reactive oxygen species (ROS) production, particularly for agents targeted to glycophorin A and Band 3, two highly-expressed membrane proteins. To produce clinically translatable strategies for human RBCs, it is critical to identify optimal red cell target epitopes, understand their effects on red cell physiology, and create humanized or human-like ligands to minimize immunogenicity. We constructed single chain antibodies (scFv) against antigenic determinants on Band 3 protein (Wrb) and RHCE protein (Rh17/Hr0) on human erythrocytes using phage display libraries prepared from immunized cynamolgous macaques (Macacafascicularis). Both these antigens are present on essentially 100% of the human population. Unfused scFvs were produced in E.coli while fusions of scFv with the extracellular domain of human thrombomodulin (TM-scFv) were produced in Drosophila S2 cells. Binding of recombinant proteins to human RBCs was measured by radioimmunoassay and flow cytometry. Generation of activated protein (APC) by RBCs loaded with TM-scFv fusions was measured by colorimetric assay. RBCs pre-incubated with varying concentrations of anti-Band3 and anti-RHCE fusions were assessed for osmotic resistance and mechanical integrity by exposure to hypo-osmolar medium and rotation in the presence of glass beads, respectively. PS exposure was measured by annexin V binding, and ROS generation was measured by dihydrorhodamine-associated fluorescence. Effects on RBC rheology were measured by flowing through microfluidic channels under controlled shear rates. Efficacy of TM-scFv fusions in diseased micro-vessels was assessed using a TNF-alpha stimulated, endothelialized microfluidic model. Single-chain antibody fragments and TM fusion proteins targeted to conserved epitopes on Band 3 protein and RHCE protein bound to human, but not murine or porcine, RBCs with high specificity and affinity (~50 nM), and in numbers consistent with the expected level of target expression (105 and 106 copies/RBC for RHCE and Band3, respectively). Coating RBCs with proteins targeted to Band 3 lessened RBC hypo-osmolar hemolysis (20% reduction) but increased hemolysis (2-fold) under mechanical stress, changes compatible with decreased red cell deformability. Proteins targeted to RHCE did not induce significant changes in hemolysis of RBCs under either osmotic or mechanical stress. Targeting neither Band 3 nor RHCE induced significant exposure of PS or production of ROS. Target-dependent effects on RBC rheology were observed under varying shear stresses in a microfluidic system. Fusion proteins of TM targeted to both epitopes demonstrated dose- and surface-copy-number-dependent generation of APC in the presence of PC and thrombin. Both TM-scFv fusion proteins were efficacious in a microfluidic model of disseminated intravascular coagulation using whole human blood by demonstrating near complete abrogation of fibrin generation in response to endothelial activation with TNF-alpha. In summary, we designed human RBC-specific non-human primate single chain antibody fragments capable of fusion to therapeutic cargoes. The TM-scFv fusions maintained therapeutic activity when bound to human RBCs and showed effective thromboprophylaxis in a whole-blood model of vasculitic injury. These antibodies and fusion proteins bound to erythroid-specific epitopes, and demonstrated target-dependent effects on several aspects of red cell physiology. The non-human primate origin of the antibodies should minimize their potential immunogenicity and the findings provide a platform to translate red cell targeted drug delivery into the clinical realm. Disclosures No relevant conflicts of interest to declare.
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Gallab, Omata, Harada, Mitsuishi, Sugimoto, Ueta, Totsuka, et al. "Development of a Spherical Model with a 3D Microchannel: An Application to Glaucoma Surgery." Micromachines 10, no. 5 (April 30, 2019): 297. http://dx.doi.org/10.3390/mi10050297.
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Three-dimensional (3D) microfluidic channels, which simulate human tissues such as blood vessels, are useful in surgical simulator models for evaluating surgical devices and training novice surgeons. However, animal models and current artificial models do not sufficiently mimic the anatomical and mechanical properties of human tissues. Therefore, we established a novel fabrication method to fabricate an eye model for use as a surgical simulator. For the glaucoma surgery task, the eye model consists of a sclera with a clear cornea; a 3D microchannel with a width of 200–500 µm, representing the Schlemm’s canal (SC); and a thin membrane with a thickness of 40–132 µm, representing the trabecular meshwork (TM). The sclera model with a clear cornea and SC was fabricated by 3D molding. Blow molding was used to fabricate the TM to cover the inner surface of the sclera part. Soft materials with controllable mechanical behaviors were used to fabricate the sclera and TM parts to mimic the mechanical properties of human tissues. Additionally, to simulate the surgery with constraints similar to those in a real operation, the eye model was installed on a skull platform. Therefore, in this paper, we propose an integration method for fabricating an eye model that has a 3D microchannel representing the SC and a membrane representing the TM, to develop a glaucoma model for training novice surgeons.
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Kalagara, Thejaswi, Tracy Moutsis, Yi Yang, Karin I. Pappelbaum, Anne Farken, Lucia Cladder-Micus, Sabine Vidal-y-Sy, et al. "The endothelial glycocalyx anchors von Willebrand factor fibers to the vascular endothelium." Blood Advances 2, no. 18 (September 20, 2018): 2347–57. http://dx.doi.org/10.1182/bloodadvances.2017013995.
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Abstract The dynamic change from a globular conformation to an elongated fiber determines the ability of von Willebrand factor (VWF) to trap platelets. Fiber formation is favored by the anchorage of VWF to the endothelial cell surface, and VWF-platelet aggregates on the endothelium contribute to inflammation, infection, and tumor progression. Although P-selectin and ανβ3-integrins may bind VWF, their precise role is unclear, and additional binding partners have been proposed. In the present study, we evaluated whether the endothelial glycocalyx anchors VWF fibers to the endothelium. Using microfluidic experiments, we showed that stabilization of the endothelial glycocalyx by chitosan oligosaccharides or overexpression of syndecan-1 (SDC-1) significantly supports the binding of VWF fibers to endothelial cells. Heparinase-mediated degradation or impaired synthesis of heparan sulfate (HS), a major component of the endothelial glycocalyx, reduces VWF fiber–dependent platelet recruitment. Molecular interaction studies using flow cytometry and live-cell fluorescence microscopy provided further evidence that VWF binds to HS linked to SDC-1. In a murine melanoma model, we found that protection of the endothelial glycocalyx through the silencing of heparanase increases the number of VWF fibers attached to the wall of tumor blood vessels. In conclusion, we identified HS chains as a relevant binding factor for VWF fibers at the endothelial cell surface in vitro and in vivo.
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Zhang, Ke, Zhichang Du, Tianying Yuan, Jiajun Huang, Xiaoyu Zhao, and Shengli Mi. "Long-term cultured microvascular networks on chip for tumor vascularization research and drug testing." Biomicrofluidics 16, no. 4 (July 2022): 044101. http://dx.doi.org/10.1063/5.0090027.
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The vascular structure of the tumor microenvironment (TME) plays an essential role in the process of metastasis. In vitro microvascular structures that can be maintained for a long time will greatly promote metastasis research. In this study, we constructed a mimicking breast cancer invasion model based on a microfluidic chip platform, and the maintenance time of the self-assembled microvascular networks significantly improved by culturing with fibroblasts (up to 13 days). Using this model, we quantified the invasion ability of breast cancer cells and angiogenesis sprouts caused by cancer cells, and the intravasation behavior of cancer cells was also observed in sprouts. We found that cancer cells could significantly cause angiogenesis by promoting sprouting behaviors of the self-assembled human umbilical vein endothelial cells, which, in turn, promoted the invasion behavior of cancer cells. The drug test results showed that the drug resistance of the widely used anti-cancer drugs 5-Fluorouracil (5-FU) and Doxorubicin (DOX) in the 3D model was higher than that in the 2D model. Meanwhile, we also proved that 5-FU and DOX had the effect of destroying tumor blood vessels. The anti-angiogenic drug Apatinib (VEGFR inhibitor) enhanced the drug effect of DOX on MDA-MB-231 cells, further proving the promoting effect of angiogenesis on the invasion ability of cancer cells. These results indicate that our model is of great value in reconstructing TME and drug testing in vitro.
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Ko, Jihoon, Sujin Hyung, Minae An, Noo Li Jeon, and Jeeyun Lee. "Abstract 3206: Three-dimensional tumor angiogenesis mapping in metastatic gastric cancer patients." Cancer Research 82, no. 12_Supplement (June 15, 2022): 3206. http://dx.doi.org/10.1158/1538-7445.am2022-3206.
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Abstract Background: Patients with gastric cancer (GC) develop malignant ascites as the disease progresses due to peritoneal metastasis. The presence of malignant ascites is a critical prognostic sign of tumor progression. With an understanding of these patient subsets, better optimized treatment strategies are needed. Method: We analyzed whole exome and transcriptome sequences of ascites or primary tumor samples obtained from 46 patients with advanced gastric cancer. In addition, we engineered a microfluidic-based gastric cancer patient-on-a-chip (GRASP) to develop a tumor-induced patient-specific angiogenesis model and evaluate the ramucirumab responses. Results: Through single-cell sequencing, 46 patients were classified into two groups according to the level of KDR gene (VEGFR2) expression. (high KDR, N=25; low KDR, N=21) In the group with high VEGFR2 expression, 68% (17 of 25) samples were from ascites, and all samples with low KDR were from primary tumors. In the GRASP system, the quantitative results of random co-culture of PDCs from 46 patients with blood vessels demonstrated high concordances to the sequence results. In addition, the angiogenesis inhibitory effect of Ramucirumab was also high in the KDR high group. Conclusion: Understanding angiogenesis inhibition as part of a therapeutic strategy considering tumor microenvironment has important clinical implications. It would be a significant outcome for our analysis system to bring therapeutic benefits to GC patients. Citation Format: Jihoon Ko, Sujin Hyung, Minae An, Noo Li Jeon, Jeeyun Lee. Three-dimensional tumor angiogenesis mapping in metastatic gastric cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3206.