Dynamic Vortex Generation, Pulsed Injection, and Rapid Mixing of Blood Samples in Microfluidics Using the Tube Oscillation Mechanism.

Here, we describe the generation of dynamic vortices in micro-scale cavities at low flow rates. The system utilizes a computer-controlled audio speaker to axially oscillate the inlet tube of the microfluidic system at desired frequencies and amplitudes. The oscillation of the tube induces transiently high flow rates in the system, which facilitates the generation of dynamic vortices inside the cavity. The size of the vortices can be modulated by varying the tube oscillation frequency or amplitude. The vortices can be generated in single or serial cavities and in a wide range of cavity sizes. We demonstrate the suitability of the tube oscillation mechanism for the pulsed injection of water-based solutions or whole blood into the cavity. The injection rate can be controlled by the oscillation characteristics of the tube, enabling the injection of liquids at ultralow flow rates. The dynamic vortices facilitate the rapid mixing of the injected liquid with the main flow. The controllability and versatility of this technology allow for the development of programmable inertial microfluidic systems for performing multistep biological assays.

Transcatheter Aortic Valve Implantation Represents an Anti-Inflammatory Therapy Via Reduction of Shear Stress-Induced, Piezo-1-Mediated Monocyte Activation.

BACKGROUND: Aortic valve stenosis is an increasingly prevalent degenerative and inflammatory disease. Transcatheter aortic valve implantation (TAVI) has revolutionized its treatment, thereby avoiding its life-threatening/disabling consequences. Whether aortic valve stenosis is accelerated by inflammation and whether it is itself a cause of inflammation are unclear. We hypothesized that the large shear forces exerted on circulating cells, particularly on the largest circulating cells, monocytes, while passing through stenotic aortic valves result in proinflammatory effects that are resolved with TAVI. METHODS: TAVI provides a unique opportunity to compare the activation status of monocytes under high shear stress (before TAVI) and under low shear stress (after TAVI). The activation status of monocytes was determined with a single-chain antibody, MAN-1, which is specific for the activated ß2-integrin Mac-1. Monocyte function was further characterized by the adhesion of myocytes to stimulated endothelial cells, phagocytic activity, uptake of oxidized low-density lipoprotein, and cytokine expression. In addition, we designed a microfluidic system to recapitulate the shear rate conditions before and after TAVI. We used this tool in combination with functional assays, Ca2+ imaging, siRNA gene silencing, and pharmacological agonists and antagonists to identify the key mechanoreceptor mediating the shear stress sensitivity of monocytes. Last, we stained for monocytes in explanted stenotic aortic human valves. RESULTS: The resolution of high shear stress through TAVI reduces Mac-1 activation, cellular adhesion, phagocytosis, oxidized low-density lipoprotein uptake, and expression of inflammatory markers in monocytes and plasma. Using microfluidics and pharmacological and genetic studies, we could recapitulate high shear stress effects on isolated human monocytes under highly controlled conditions, showing that shear stress-dependent calcium influx and monocyte adhesion are mediated by the mechanosensitive ion channel Piezo-1. We also demonstrate that the expression of this receptor is shear stress dependent and downregulated in patients receiving TAVI. Last, we show monocyte accumulation at the aortic side of leaflets of explanted aortic valves. CONCLUSIONS: We demonstrate that high shear stress, as present in patients with aortic valve stenosis, activates multiple monocyte functions, and we identify Piezo-1 as the mainly responsible mechanoreceptor, representing a potentially druggable target. We demonstrate an anti-inflammatory effect and therefore a novel therapeutic benefit of TAVI.

Microfluidic Skin-on-a-Chip Models: Toward Biomimetic Artificial Skin.

The role of skin in the human body is indispensable, serving as a barrier, moderating homeostatic balance, and representing a pronounced endpoint for cosmetics and pharmaceuticals. Despite the extensive achievements of in vitro skin models, they do not recapitulate the complexity of human skin; thus, there remains a dependence on animal models during preclinical drug trials, resulting in expensive drug development with high failure rates. By imparting a fine control over the microenvironment and inducing relevant mechanical cues, skin-on-a-chip (SoC) models have circumvented the limitations of conventional cell studies. Enhanced barrier properties, vascularization, and improved phenotypic differentiation have been achieved by SoC models; however, the successful inclusion of appendages such as hair follicles and sweat glands and pigmentation relevance have yet to be realized. The present Review collates the progress of SoC platforms with a focus on their fabrication and the incorporation of mechanical cues, sensors, and blood vessels.

Tunable Harmonic Flow Patterns in Microfluidic Systems through Simple Tube Oscillation.

Generation of tunable harmonic flows at low cost in microfluidic systems is a persistent and significant obstacle to this field, substantially limiting its potential to address major scientific questions and applications. This work introduces a simple and elegant way to overcome this obstacle. Harmonic flow patterns can be generated in microfluidic structures by simply oscillating the inlet tubes. Complex rib and vortex patterns can be dynamically modulated by changing the frequency and magnitude of tube oscillation and the viscosity of liquid. Highly complex rib patterns and synchronous vortices can be generated in serially connected microfluidic chambers. Similar dynamic patterns can be generated using whole or diluted blood samples without damaging the sample. This method offers unique opportunities for studying complex fluids and soft materials, chemical synthesis of various compounds, and mimicking harmonic flows in biological systems using compact, tunable, and low-cost devices.

Oscillation and self-propulsion of Leidenfrost droplets enclosed in cylindrical cavities.

Leidenfrost droplets can be considered as soft engines capable of directly transforming heat into mechanical energy. Despite remarkable advancements in understanding the propulsion of Leidenfrost droplets on asymmetric structures, the complex dynamics of droplets in enclosed structures is not fully understood. To address this fundamental gap, we investigated the dynamics of Leidenfrost droplets restricted by metal disks. The disk alters the accumulation and release of the vapour generated by the droplet, and substantially changes its dynamic characteristics. Our experiments reveal the formation of oscillating multi-lobed structures when restricting the droplet within a disk. In comparison, patterning offset radial grooves on the surface of the disk rectifies the vapour flow and facilitates the self-propulsion of the droplet along the edge of the disk. Our work offers opportunities for developing soft and short-living actuators, which can operate at high temperatures.

Temperature-Controlled Microfluidic System Incorporating Polymer Tubes.

Here, we demonstrate a multilayered microfluidic system integrated with commercially available polymer tubes for controlling the temperature of the sample under various static and dynamic conditions. Highly controllable temperature profiles can be produced by modulating the flow rate or inlet temperature of the water passing through the tubes. Customised temperature gradients can be created across the length or width of a channel by mismatching the inlet temperature of the tubes. Temperature cycles can also be produced by repeatedly switching the tubes between hot and cold flasks. Proof-of-concept experiments demonstrate the utility of this system for studying the drug-induced calcium signaling of human monocytes under dynamic thermal conditions. The versatility and simplicity of our system provides opportunities for studying temperature-sensitive chemical, biochemical, and biological samples under various operating conditions.

Studying the Response of Aortic Endothelial Cells under Pulsatile Flow Using a Compact Microfluidic System.

We describe a piezoelectric pumping system for studying the mechanobiology of human aortic endothelial cells (HAECs) under pulsatile flow in microfluidic structures. The system takes advantage of commercially available components, including pumps, flow sensors, and microfluidic channels, which can be easily integrated, programmed, and operated by cellular biologists. Proof-of-concept experiments were performed to elucidate the complex mechanotransduction processes of endothelial cells to pulsatile flow. In particular, we investigated the effect of atheroprone and atheroprotective pulsatile shear stress on endothelial cytoskeleton remodeling and distribution of ß-catenin, as well as nuclear shape and size. The system is simple to operate, relatively inexpensive, portable, and controllable, providing opportunities for studying the mechanobiology of endothelial cells using microfluidic technologies.

Reconfigurable, Self-Sufficient Convective Heat Exchanger for Temperature Control of Microfluidic Systems.

Here, we demonstrate a modular, reconfigurable, and self-sufficient convective heat exchanger for regulation of temperature in microfluidic systems. The heat exchanger consists of polymer tubes wrapped around a plastic pole and fully embedded in an elastomer block, which can be easily mounted onto the microfluidic structure. It is compatible with various microfluidic geometries and materials. Miniaturized, battery-powered piezoelectric pumps are utilized to drive the heat carrying liquid through the heat exchanger at desired flow rates and temperatures. Customized temperature profiles can be generated by changing the configuration of the heat exchanger with respect to the microfluidic structure. Tailored dynamic temperature profiles can be generated by changing the temperature of the heat carrying liquid in successive cycles. This feature is used to study the calcium signaling of endothelial cells under successive temperature cycles of 24 to 37 °C. The versatility, simplicity, and self-sufficiency of the heat exchanger makes it suitable for various microfluidic based cellular assays.

Label-Free Optofluidic Nanobiosensor Enables Real-Time Analysis of Single-Cell Cytokine Secretion.

Single-cell analysis of cytokine secretion is essential to understand the heterogeneity of cellular functionalities and develop novel therapies for multiple diseases. Unraveling the dynamic secretion process at single-cell resolution reveals the real-time functional status of individual cells. Fluorescent and colorimetric-based methodologies require tedious molecular labeling that brings inevitable interferences with cell integrity and compromises the temporal resolution. An innovative label-free optofluidic nanoplasmonic biosensor is introduced for single-cell analysis in real time. The nanobiosensor incorporates a novel design of a multifunctional microfluidic system with small volume microchamber and regulation channels for reliable monitoring of cytokine secretion from individual cells for hours. Different interleukin-2 secretion profiles are detected and distinguished from single lymphoma cells. The sensor configuration combined with optical spectroscopic imaging further allows us to determine the spatial single-cell secretion fingerprints in real time. This new biosensor system is anticipated to be a powerful tool to characterize single-cell signaling for basic and clinical research.

A Gallium-Based Magnetocaloric Liquid Metal Ferrofluid.

We demonstrate a magnetocaloric ferrofluid based on a gadolinium saturated liquid metal matrix, using a gallium-based liquid metal alloy as the solvent and suspension medium. The material is liquid at room temperature, while exhibiting spontaneous magnetization and a large magnetocaloric effect. The magnetic properties were attributed to the formation of gadolinium nanoparticles suspended within the liquid gallium alloy, which acts as a reaction solvent during the nanoparticle synthesis. High nanoparticle weight fractions exceeding 2% could be suspended within the liquid metal matrix. The liquid metal ferrofluid shows promise for magnetocaloric cooling due to its high thermal conductivity and its liquid nature. Magnetic and thermoanalytic characterizations reveal that the developed material remains liquid within the temperature window required for domestic refrigeration purposes, which enables future fluidic magnetocaloric devices. Additionally, the observed formation of nanometer-sized metallic particles within the supersaturated liquid metal solution has general implications for chemical synthesis and provides a new synthetic pathway toward metallic nanoparticles based on highly reactive rare earth metals.

Liquid metal enabled pump.

Small-scale pumps will be the heartbeat of many future micro/nanoscale platforms. However, the integration of small-scale pumps is presently hampered by limited flow rate with respect to the input power, and their rather complicated fabrication processes. These issues arise as many conventional pumping effects require intricate moving elements. Here, we demonstrate a system that we call the liquid metal enabled pump, for driving a range of liquids without mechanical moving parts, upon the application of modest electric field. This pump incorporates a droplet of liquid metal, which induces liquid flow at high flow rates, yet with exceptionally low power consumption by electrowetting/deelectrowetting at the metal surface. We present theory explaining this pumping mechanism and show that the operation is fundamentally different from other existing pumps. The presented liquid metal enabled pump is both efficient and simple, and thus has the potential to fundamentally advance the field of microfluidics.

Dynamic drag force based on iterative density mapping: A new numerical tool for three-dimensional analysis of particle trajectories in a dielectrophoretic system.

Dielectrophoresis is a widely used means of manipulating suspended particles within microfluidic systems. In order to efficiently design such systems for a desired application, various numerical methods exist that enable particle trajectory plotting in two or three dimensions based on the interplay of hydrodynamic and dielectrophoretic forces. While various models are described in the literature, few are capable of modeling interactions between particles as well as their surrounding environment as these interactions are complex, multifaceted, and computationally expensive to the point of being prohibitive when considering a large number of particles. In this paper, we present a numerical model designed to enable spatial analysis of the physical effects exerted upon particles within microfluidic systems employing dielectrophoresis. The model presents a means of approximating the effects of the presence of large numbers of particles through dynamically adjusting hydrodynamic drag force based on particle density, thereby introducing a measure of emulated particle-particle and particle-liquid interactions. This model is referred to as "dynamic drag force based on iterative density mapping." The resultant numerical model is used to simulate and predict particle trajectory and velocity profiles within a microfluidic system incorporating curved dielectrophoretic microelectrodes. The simulated data are compared favorably with experimental data gathered using microparticle image velocimetry, and is contrasted against simulated data generated using traditional "effective moment Stokes-drag method," showing more accurate particle velocity profiles for areas of high particle density.

Controlled rotation and vibration of patterned cell clusters using dielectrophoresis.

The localized motion of cells within a cluster is an important feature of living organisms and has been found to play roles in cell signaling, communication, and migration, thus affecting processes such as proliferation, transcription, and organogenesis. Current approaches for inducing dynamic movement into cells, however, focus predominantly on mechanical stimulation of single cells, affect cell integrity, and, more importantly, need a complementary mechanism to pattern cells. In this article, we demonstrate a new strategy for the mechanical stimulation of large cell clusters, taking advantage of dielectrophoresis. This strategy is based on the cellular spin resonance mechanism, but it utilizes coating agents, such as bovine serum albumin, to create consistent rotation and vibration of individual cells. The treatment of cells with coating agents intensifies the torque induced on the cells while reducing the friction at the cell-cell and cell-substrate interfaces, resulting in the consistent motion of the cells. Such localized motion can be modulated by varying the frequency and voltage of the applied sinusoidal AC signal and can be achieved in the absence and presence of flow. This strategy enables the survival and functioning of moving cells within large-scale clusters to be investigated.

Using dielectrophoresis to study the dynamic response of single budding yeast cells to Lyticase.

Budding yeast cells are quick and easy to grow and represent a versatile model of eukaryotic cells for a variety of cellular studies, largely because their genome has been widely studied and links can be drawn with higher eukaryotes. Therefore, the efficient separation, immobilization, and conversion of budding yeasts into spheroplast or protoplast can provide valuable insight for many fundamentals investigations in cell biology at a single cell level. Dielectrophoresis, the induced motion of particles in non-uniform electric fields, possesses a great versatility for manipulation of cells in microfluidic platforms. Despite this, dielectrophoresis has been largely utilized for studying of non-budding yeast cells and has rarely been used for manipulation of budding cells. Here, we utilize dielectrophoresis for studying the dynamic response of budding cells to different concentrations of Lyticase. This involves separation of the budding yeasts from a background of non-budding cells and their subsequent immobilization onto the microelectrodes at desired densities down to single cell level. The immobilized yeasts are then stimulated with Lyticase to remove the cell wall and convert them into spheroplasts, in a highly dynamic process that depends on the concentration of Lyticase. We also introduce a novel method for immobilization of the cell organelles released from the lysed cells by patterning multi-walled carbon nanotubes (MWCNTs) between the microelectrodes.

Microfluidic platforms for the investigation of intercellular signalling mechanisms.

Intercellular signalling has been identified as a highly complex process, responsible for orchestrating many physiological functions. While conventional methods of investigation have been useful, their limitations are impeding further development. Microfluidics offers an opportunity to overcome some of these limitations. Most notably, microfluidic systems can emulate the in-vivo environments. Further, they enable exceptionally precise control of the microenvironment, allowing complex mechanisms to be selectively isolated and studied in detail. There has thus been a growing adoption of microfluidic platforms for investigation of cell signalling mechanisms. This review provides an overview of the different signalling mechanisms and discusses the methods used to study them, with a focus on the microfluidic devices developed for this purpose.

Microfluidics and Raman microscopy: current applications and future challenges.

Raman microscopy systems are becoming increasingly widespread and accessible for characterising chemical species. Microfluidic systems are also progressively finding their way into real world applications. Therefore, it is anticipated that the integration of Raman systems with microfluidics will become increasingly attractive and practical. This review aims to provide an overview of Raman microscopy-microfluidics integrated systems for researchers who are actively interested in utilising these tools. The fundamental principles and application strengths of Raman microscopy are discussed in the context of microfluidics. Various configurations of microfluidics that incorporate Raman microscopy methods are presented, with applications highlighted. Data analysis methods are discussed, with a focus on assisting the interpretation of Raman-microfluidics data from complex samples. Finally, possible future directions of Raman-microfluidic systems are presented.

Bioengineered models of cardiovascular diseases.

Age-associated cardiovascular diseases (CVDs), predominantly resulting from artery-related disorders such as atherosclerosis, stand as a leading cause of morbidity and mortality among the elderly population. Consequently, there is a growing interest in the development of clinically relevant bioengineered models of CVDs. Recent developments in bioengineering and material sciences have paved the way for the creation of intricate models that closely mimic the structure and surroundings of native cardiac tissues and blood vessels. These models can be utilized for basic research purposes and for identifying pharmaceutical interventions and facilitating drug discovery. The advancement of vessel-on-a-chip technologies and the development of bioengineered and humanized in vitro models of the cardiovascular system have the potential to revolutionize CVD disease modelling. These technologies offer pathophysiologically relevant models at a fraction of the cost and time required for traditional experimentation required in vivo. This progress signifies a significant advancement in the field, transitioning from conventional 2D cell culture models to advanced 3D organoid and vessel-on-a-chip models. These innovative models are specifically designed to explore the complexities of vascular aging and stiffening, crucial factors in the development of cardiovascular diseases. This review summarizes the recent progress of various bioengineered in vitro platforms developed for investigating the pathophysiology of human cardiovascular system with more focus on advanced 3D vascular platforms.

A microfluidic model to study the effects of arrhythmic flows on endothelial cells.

Atrial fibrillation (AF) is the most common type of cardiac arrhythmia and an important contributor to morbidity and mortality. Endothelial dysfunction has been postulated to be an important contributing factor in cardiovascular events in patients with AF. However, how vascular endothelial cells respond to arrhythmic flow is not fully understood, mainly due to the limitation of current in vitro systems to mimic arrhythmic flow conditions. To address this limitation, we developed a microfluidic system to study the effect of arrhythmic flow on the mechanobiology of human aortic endothelial cells (HAECs). The system utilises a computer-controlled piezoelectric pump for generating arrhythmic flow with a unique ability to control the variability in both the frequency and amplitude of pulse waves. The flow rate is modulated to reflect physiological or pathophysiological shear stress levels on endothelial cells. This enabled us to systematically dissect the importance of variability in the frequency and amplitude of pulses and shear stress level on endothelial cell mechanobiology. Our results indicated that arrhythmic flow at physiological shear stress level promotes endothelial cell spreading and reduces the plasma membrane-to-cytoplasmic distribution of ß-catenin. In contrast, arrhythmic flow at low and atherogenic shear stress levels does not promote endothelial cell spreading or redistribution of ß-catenin. Interestingly, under both shear stress levels, arrhythmic flow induces inflammation by promoting monocyte adhesion via an increase in ICAM-1 expression. Collectively, our microfluidic system provides opportunities to study the effect of arrhythmic flows on vascular endothelial mechanobiology in a systematic and reproducible manner.

Modifying dielectrophoretic response of nonviable yeast cells by ionic surfactant treatment.

Nonviable cells are essential biosystems, due to the functionalities they offer and their effects on viable cells. Therefore, the separation and immobilization of nonviable cells separately or in the vicinity of viable cells is of great importance for many fundamentals investigations in cell biology. However, most nonviable cells become less polarizable than the surrounding medium at conductivities above 0.01 S/m. This means that in such a medium, dielectrophoresis, despite its great versatilities for manipulation of cells, cannot be employed for immobilizing nonviable cells. Here, we present a novel approach to change the dielectrophoretic (DEP) response of nonviable yeast cells by treating them with low concentrations of ionic surfactants such as sodium dodecyl sulfate. After this treatment, they exhibit a strong positive DEP response, even at high medium conductivities. The capability of this treatment is demonstrated in two proof-of-concept experiments. First, we show the sorting and immobilization of viable and nonviable yeast cells, along consecutive microelectrode arrays. Second, we demonstrate the immobilization of viable and nonviable cells in the vicinity of each other along the same microelectrode array. The proposed technique allows DEP platforms to be utilized for the immobilization and subsequent postanalysis of both viable and nonviable cells with and without the presence of each other.

Reorientation of microfluidic channel enables versatile dielectrophoretic platforms for cell manipulations.

Dielectrophoresis is a versatile tool for the sorting, immobilization, and characterization of cells in microfluidic systems. The performance of dielectrophoretic systems strongly relies on the configuration of microelectrodes, which produce a nonuniform electric field. However, once fabricated, the microelectrodes cannot be reconfigured to change the characteristics of the system. Here, we show that the reorientation of the microfluidic channel with respect to the microelectrodes can be readily utilized to alter the characteristics of the system. This enables us to change the location and density of immobilized viable cells across the channel, release viable cells along customized numbers of streams within the channel, change the deflection pattern of nonviable cells along the channel, and improve the sorting of viable and nonviable cells in terms of flow throughput and efficiency of the system. We demonstrate that the reorientation of the microfluidic channel is an effective tool to create versatile dielectrophoretic platforms using the same microelectrode design.