A Capillary-Endothelium-Mimetic Microfluic Chip for the Study of Chemotactic Response (original) (raw)
A capillary-endothelium-mimetic microfluidic chip for the study of immune responses
Sensors and Actuators B: Chemical, 2015
The transwell system is the most widely used tool for studying chemotaxis and understanding chemotactic responses. It has been suggested that chemotactic gradients attract neutrophils, leading to extravasation, but recent findings also implicate vascular hydrodynamic forces in chemotactic responses. With this motivation, we developed a Labchip that mimics the dynamic three-dimensional microenvironment of a blood vessel. This capillary-endothelium-mimetic (CEM) microfluidic chip serves as a dynamic transwell system for studying neutrophil migration at different flow velocities. Under lower flow rates, the chemotactic factor dominates over the flow rate, increasing the extravasation of neutrophil-like cells; at higher flow rates, the neutrophil-like cells aggregate near the side wall of the chamber due to a hydrodynamic force, limiting extravasation. In this report, we demonstrate the use of this Labchip for studying extravasation behavior over an extended period of time, under conditions of continuous flow and a stable concentration gradient. This Labchip-based approach is also applicable to the study of cancer metastasis, atherosclerosis and other angiopathies.
Bioinspired microfluidic assay for in vitro modeling of leukocyte-endothelium interactions
Current in vitro models of the leukocyte adhesion cascade cannot be used for real-time studies of the entire leukocyte adhesion cascade, including rolling, adhesion, and migration in a single assay. In this study, we have developed and validated a novel bioinspired microfluidic assay (bMFA) and used it to test the hypothesis that blocking of specific steps in the adhesion/migration cascade significantly affects other steps of the cascade. The bMFA consists of an endothelialized microvascular network in communication with a tissue compartment via a 3 μm porous barrier. Human neutrophils in bMFA preferentially adhered to activated human endothelial cells near bifurcations with rolling and adhesion patterns in close agreement with in vivo observations. Treating endothelial cells with monoclonal antibodies to E-selectin or ICAM-1 or treating neutrophils with wortmannin reduced rolling, adhesion, and migration of neutrophils to 60%, 20%, and 18% of their respective control values. Antibody blocking of specific steps in the adhesion/migration cascade (e.g., mAb to E-selectin) significantly downregulated other steps of the cascade (e.g., migration). This novel in vitro assay provides a realistic human cell based model for basic science studies, identification of new treatment targets, selection of pathways to target validation, and rapid screening of candidate agents.
Microfluidic kit-on-a-lid: a versatile platform for neutrophil chemotaxis assays
Blood, 2012
Improvements in neutrophil chemotaxis assays have advanced our understanding of the mechanisms of neutrophil recruitment; however, traditional methods limit biologic inquiry in important areas. We report a microfluidic technology that enables neutrophil purification and chemotaxis on-chip within minutes, using nanoliters of whole blood, and only requires a micropipette to operate. The low sample volume requirements and novel lid-based method for initiating the gradient of chemoattractant enabled the measurement of human neutrophil migration on a cell monolayer to probe the adherent and migratory states of neutrophils under inflammatory conditions; mouse neutrophil chemotaxis without sacrificing the animal; and both 2D and 3D neutrophil chemotaxis. First, the neutrophil chemotaxis on endothelial cells revealed 2 distinct neutrophil phenotypes, showing that endothelial cell-neutrophil interactions influence neutrophil chemotactic behavior. Second, we validated the mouse neutrophil che...
Traffic of leukocytes in microfluidic channels with rectangular and rounded cross-sections
2011
Traffic of leukocytes in microvascular networks (particularly through arteriolar bifurcations and venular convergences) affects the dynamics of capillary blood flow, initiation of leukocyte adhesion during inflammation, and localization and development of atherosclerotic plaques in vivo. Recently, a growing research effort has been focused on fabricating microvascular networks comprising artificial vessels with more realistic, rounded cross-sections. This paper investigated the impact of the crosssectional geometry of microchannels on the traffic of leukocytes flowing with human whole blood through a non-symmetrical bifurcation that consisted of a 50 mm mother channel bifurcating into 30 mm and 50 mm daughter branches. Two versions of the same bifurcation comprising microchannels with rectangular and rounded cross-sections were fabricated using conventional multi-layer photolithography to produce rectangular microchannles that were then rounded in situ using a recently developed method of liquid PDMS/air bubble injection. For microchannels with rounded crosssections, about two-thirds of marginated leukocytes traveling along a path in the top plane of the bifurcation entered the smallest 30 mm daughter branch. This distribution was reversed in microchannels with rectangular cross-sections -the majority of leukocytes traveling along a similar path continued to follow the 50 mm microchannels after the bifurcation. This dramatic difference in the distribution of leukocyte traffic among the branches of the bifurcation can be explained by preferential margination of leukocytes towards the corners of the 50 mm mother microchannels with rectangular cross-sections, and by the additional hindrance to leukocyte entry created by the sharp transition from the 50 mm mother microchannel to the 30 mm daughter branch at the intersection. The results of this study suggest that the trajectories of marginated leukocytes passing through non-symmetrical bifurcations are significantly affected by the cross-sectional geometry of microchannels and emphasize the importance of using microfludic systems with geometrical configurations closely matching physiological configurations when modeling the dynamics of whole blood flow in the microcirculation.
Microfluidic tools to investigate pathologies in the blood microcirculation
International Journal of Nanotechnology, 2012
We show how microfluidics technology can be used to fabricate simple and innovative biomimetic tools to shed new light on physiopathological events occurring in the blood microcirculation. Examples of applications are given in the context of the acute respiratory distress Syndrome (ARDS), an inflammatory disease of the lung triggered by a massive arrest of white blood cells in the lung microvasculature. The main challenge consists in building relevant micro-devices to reproduce key biological characteristics of blood capillaries. We present a series of tools that permit us to decouple the role of the multiple parameters involved in complex biological events. Straight narrow channels with non-adherent walls are used to characterise the passage of a cell in 4 µm wide constrictions in the absence of adhesion, whereas channels covered by endothelial cells allow a quantitative measurement of cell adhesion in the absence of mechanical constraints. We show that incubation of white blood cells in sera of ARDS patients increases their stiffness, confirming the role of stiffness on the abnormal sequestration of white blood cells, whereas we could not bring to light a significant adhesion increase. The multiple branches and constrictions of the blood microvasculature network are mimicked here by series of interconnected crenelled constrictions with different symmetries. In symmetric crenels, cells adopt a stable deformed shape after a few constrictions and travel fast through successive constrictions with a constant orientation. In asymmetric channels, cell orientation and trajectory are perturbed between two constrictions. Unfavourable orientations upon entry can yield temporary or even definitive arrests with the stiffest cells. Finally, we present a new artificial micro-vessel with porous walls to mimic the porosity of real blood vessels. This new tool is useful to observe directly under a microscope the late stages of inflammation in the microvasculature such as immune cells transmigration, or the infection of a micro-vessel by pathogenic bacteria.
Endothelial cell culture in microfluidic devices for investigating microvascular processes
Biomicrofluidics, 2018
Numerous conditions and disease states such as sickle cell disease, malaria, thrombotic microangiopathy, and stroke significantly impact the microvasculature function and its role in disease progression. Understanding the role of cellular interactions and microvascular hemodynamic forces in the context of disease is crucial to understanding disease pathophysiology. In vivo models of microvascular disease using animal models often coupled with intravital microscopy have long been utilized to investigate microvascular phenomena. However, these methods suffer from some major drawbacks, including the inability to tightly and quantitatively control experimental conditions, the difficulty of imaging multiple microvascular beds within a living organism, and the inability to isolate specific microvascular geometries such as bifurcations. Thus, there exists a need for in vitro microvascular models that can mitigate the drawbacks associated with in vivo systems. To that end, microfluidics has...
Neutrophil Migration in Opposing Chemoattractant Gradients Using Microfluidic Chemotaxis Devices
Annals of Biomedical Engineering, 2005
Neutrophils migrating in tissue respond to complex overlapping signals generated by a variety of chemotactic factors (CFs). Previous studies suggested a hierarchy between bacteriaderived CFs and host-derived CFs but could not differentiate neutrophil response to potentially equal host-derived CFs (IL-8 and LTB 4 ). This paper reports neutrophil migration in conflicting gradients of IL-8 and LTB 4 using a microfluidic chemotaxis device that can generate stable and well-defined gradients. We quantitatively characterized the movement of cells from time-lapse images. Neutrophils migrate more efficiently toward single IL-8 gradients than single LTB 4 gradients as measured by the effective chemotactic index (ECI). In opposing gradients of IL-8 and LTB 4 , neutrophils show obvious chemotaxis toward a distant gradient, consistent with previous reports. When an opposing gradient of LTB 4 is present, neutrophils show less effective chemotaxis toward IL-8 than when they are in a gradient of IL-8 alone. In contrast, the chemotactic response of neutrophils to LTB 4 is not reduced in opposing gradients as compared to that in a single LTB 4 gradient. These results indicate that the presence of one host-derived CF modifies the response of neutrophils to a second CF suggesting a subtle hierarchy between them.
Computational and Functional Evaluation of a Microfluidic Blood Flow Device
ASAIO Journal, 2007
The development of microfluidic devices supporting physiological blood flow has the potential to yield biomedical technologies emulating human organ function. However, advances in this area have been constrained by the fact that artificial microchannels constructed for such devices need to achieve maximum chemical diffusion as well as hemocompatibility. To address this issue, we designed an elastomeric microfluidic flow device composed of poly (dimethylsiloxane) to emulate the geometry and flow properties of the pulmonary microcirculation. Our chip design is characterized by high aspect ratio (width > height) channels in an orthogonally interconnected configuration. Finite element simulations of blood flow through the network design chip demonstrated that the apparent pressure drop varied in a linear manner with flow rate. For simulated flow rates <250 l min ؊1 , the simulated pressure drop was <2000 Pa, the flow was laminar, and hemolysis was minimal. Hemolysis rate, assayed in terms of [total plasma hemoglobin (TPH) (sample ؊ control)/(TPH control)] during 6 and 12 hour perfusions at 250 l/min, was <5.0% through the entire period of device perfusion. There was no evidence of microscopic thrombus at any channel segment or junction under these perfusion conditions. We conclude that a microfluidic blood flow device possessing asymmetric and interconnected microchannels exhibits uniform flow properties and preliminary hemocompatibility. Such technology should foster the development of miniature oxygenators and similar biomedical devices requiring both a microscale reaction volume and physiological blood flow. ASAIO Journal 2007; 53:447-455.
Lab on a Chip, 2006
Experimental systems that provide temporal and spatial control of chemical gradients are required for probing into the complex mechanisms of eukaryotic cell chemotaxis. However, no current technique can simultaneously generate stable chemical gradients and allow fast gradient changes. We developed a microfluidic system with microstructured membranes for exposing neutrophils to fast and precise changes between stable, linear gradients of the known chemoattractant Interleukin-8 (IL-8). We observed that rapidly lowering the average concentration of IL-8 within a gradient, while preserving the direction of the gradient, resulted in temporary neutrophil depolarization. Fast reversal of the gradient direction while increasing or decreasing the average concentration also resulted in temporary depolarization. Neutrophils adapted and maintained their directional motility, only when the average gradient concentration was increased and the direction of the gradient preserved. Based on these observations we propose a two-component temporal sensing mechanism that uses variations of chemokine concentration averaged over the entire cell surface and localized at the leading edge, respectively, and directs neutrophil responses to changes in their chemical microenvironment.