An optically transparent membrane supports shear stress studies in a three-dimensional microfluidic neurovascular unit model

We report a microfluidic blood-brain barrier model that enables both physiological shear stress and optical transparency throughout the device. Brain endothelial cells grown in an optically transparent membrane-integrated microfluidic device were able to withstand physiological fluid shear stress using a hydrophilized polytetrafluoroethylene nanoporous membrane instead of the more commonly used polyester membrane. A functional three-dimensional microfluidic co-culture model of the neurovascular unit is presented that incorporates astrocytes in a 3D hydrogel and enables physiological shear stress on the membrane-supported endothelial cell layer.     

Microfluidic endothelial cell culture model to replicate disturbed flow conditions seen in atherosclerosis susceptible regions

Atherosclerotic lesions occur non-randomly at vascular niches in bends and bifurcations where fluid flow can be characterized as "disturbed" (low shear stress with both forward and retrograde flow). Endothelial cells (ECs) at these locations experience significantly lower average shear stress without change in the levels of pressure or strain, which affects the local balance in mechanical stresses. Common in vitro models of atherosclerosis focus primarily on shear stress without accounting for pressure and strain loading. To overcome this limitation, we used our microfluidic endothelial cell culture model (ECCM) to achieve accurate replication of pressure, strain, and shear stress waveforms associated with both normal flow seen in straight sections of arteries and disturbed flow seen in the abdominal aorta in the infrarenal segment at the wall distal to the inferior mesenteric artery (IMA), which is associated with high incidence of atherosclerotic lesion formation. Human aortic endothelial cells (HAECs) were cultured within the ECCM under both normal and disturbed flow and evaluated for cell shape, cytoskeletal alignment, endothelial barrier function, and inflammation using immunofluorescence microscopy and flow cytometry. Results clearly demonstrate quantifiable differences between cells cultured under disturbed flow conditions, which are cuboidal with short and randomly oriented actin microfilaments and show intermittent expression of β-Catenin and cells cultured under normal flow. However, in the absence of pro-inflammatory stimulation, the levels of expression of activation markers: intra cellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), platelet endothelial cell adhesion molecule-1 (PECAM-1), and vascular endothelial cell growth factor - receptor 2 (VEGF-R2) known to be involved in the initiation of plaque formation were only slightly higher in HAECs cultured under disturbed flow in comparison to cells cultured under normal flow.     

Examination of the role of transient receptor potential vanilloid type 4 in endothelial responses to shear forces

Shear stress is the major mechanical force applied on vascular endothelial cells by blood flow, and is a crucial factor in normal vascular physiology and in the development of some vascular pathologies. The exact mechanisms of cellular mechano-transduction in mammalian cells and tissues have not yet been elucidated, but it is known that mechanically sensitive receptors and ion channels play a crucial role. This paper describes the use of a novel and efficient microfluidic device to study mechanically-sensitive receptors and ion channels in vitro, which has three independent channels from which recordings can be made and has a small surface area such that fewer cells are required than for conventional flow chambers. The contoured channels of the device enabled examination of a range of shear stresses in one field of view, which is not possible with parallel plate flow chambers and other previously used devices, where one level of flow-induced shear stress is produced per fixed flow-rate. We exposed bovine aortic endothelial cells to different levels of shear stress, and measured the resulting change in intracellular calcium levels ([Ca²⁺]_i) using the fluorescent calcium sensitive dye Fluo-4AM. Shear stress caused an elevation of [Ca²⁺]_i that was proportional to the level of shear experienced. The response was temperature dependant such that at lower temperatures more shear stress was required to elicit a given level of calcium signal and the magnitude of influx was reduced. We demonstrated that shear stress-induced elevations in [Ca²⁺]_i are largely due to calcium influx through the transient receptor potential vanilloid type 4 ion channel.     

A microfluidic membrane device to mimic critical components of the vascular microenvironment

Vascular function, homeostasis, and pathological development are regulated by the endothelial cells that line blood vessels. Endothelial function is influenced by the integrated effects of multiple factors, including hemodynamic conditions, soluble and insoluble biochemical signals, and interactions with other cell types. Here, we present a membrane microfluidic device that recapitulates key components of the vascular microenvironment, including hemodynamic shear stress, circulating cytokines, extracellular matrix proteins, and multiple interacting cells. The utility of the device was demonstrated by measuring monocyte adhesion to and transmigration through a porcine aortic endothelial cell monolayer. Endothelial cells grown in the membrane microchannels and subjected to 20 dynes∕cm(2) shear stress remained viable, attached, and confluent for several days. Consistent with the data from macroscale systems, 25 ng∕ml tumor necrosis factor (TNF)-α significantly increased RAW264.7 monocyte adhesion. Preconditioning endothelial cells for 24 h under static or 20 dynes∕cm(2) shear stress conditions did not influence TNF-α-induced monocyte attachment. In contrast, simultaneous application of TNF-α and 20 dynes∕cm(2) shear stress caused increased monocyte adhesion compared with endothelial cells treated with TNF-α under static conditions. THP-1 monocytic cells migrated across an activated endothelium, with increased diapedesis in response to monocyte chemoattractant protein (MCP)-1 in the lower channel of the device. This microfluidic platform can be used to study complex cell-matrix and cell-cell interactions in environments that mimic those in native and tissue engineered blood vessels, and offers the potential for parallelization and increased throughput over conventional macroscale systems.     

A microfluidic shear device that accommodates parallel high and low stress zones within the same culturing chamber

Fluid shear stress (FSS) plays a critical role in regulating endothelium function and maintaining vascular homeostasis. Current microfluidic devices for studying FSS effects on cells either separate high shear stress zone and low shear stress zone into different culturing chambers, or arranging the zones serially along the flow direction, which complicates subsequent data interpretation. In this paper, we report a diamond shaped microfluidic shear device where the high shear stress zone and the low shear stress zone are arranged in parallel within one culturing chamber. Since the zones with different shear stress magnitudes are aligned normal to the flow direction, the cells in one stress group are not substantially affected by the flow-induced cytokine/chemokine releases by cells in the other group. Cell loading experiments using human umbilical vein endothelial cells show that the device is able to reveal stress magnitude-dependent and loading duration-dependent cell responses. The co-existence of shear stress zones with varied magnitudes within the same culturing chamber not only ensures that all the cells are subject to the identical culturing conditions, but also allows the resemblance of the differential shear stress pattern in natural arterial conditions. The device is expected to provide a new solution for studying the effects of heterogeneous hemodynamic patterns in the onset and progression of various vascular diseases.     

A multichannel acoustically driven microfluidic chip to study particle-cell interactions

Microfluidic devices have emerged as important tools for experimental physiology. They allow to study the effects of hydrodynamic flow on physiological and pathophysiological processes, e.g., in the circulatory system of the body. Such dynamic in vitro test systems are essential in order to address fundamental problems in drug delivery and targeted imaging, such as the binding of particles to cells under flow. In the present work an acoustically driven microfluidic platform is presented in which four miniature flow channels can be operated in parallel at distinct flow velocities with only slight inter-experimental variations. The device can accommodate various channel architectures and is fully compatible with cell culture as well as microscopy. Moreover, the flow channels can be readily separated from the surface acoustic wave pumps and subsequently channel-associated luminescence, absorbance, and/or fluorescence can be determined with a standard microplate reader. In order to create artificial blood vessels, different coatings were evaluated for the cultivation of endothelial cells in the microchannels. It was found that 0.01% fibronectin is the most suitable coating for growth of endothelial monolayers. Finally, the microfluidic system was used to study the binding of 1 μm polystyrene microspheres to three different types of endothelial cell monolayers (HUVEC, HUVECtert, HMEC-1) at different average shear rates. It demonstrated that average shear rates between 0.5 s⁻¹ and 2.25 s⁻¹ exert no significant effect on cytoadhesion of particles to all three types of endothelial monolayers. In conclusion, the multichannel microfluidic platform is a promising device to study the impact of hydrodynamic forces on cell physiology and binding of drug carriers to endothelium.     

Effects of shear stresses and antioxidant concentrations on the production of reactive oxygen species in lung cancer cells

Reactive oxygen species (ROS) are known to be a key factor in the development of cancer, and many exogenous sources are supposed to be related to the formation of ROS. In this paper, a microfluidic chip was developed for studying the production of ROS in lung cancer cells under different chemical and physical stimuli. This chip has two unique features: (1) five relative concentrations of 0, 1/8, 1/2, 7/8, and 1 are achieved in the culture regions; (2) a shear stress gradient is produced inside each of the five culture areas. Lung cancer cells were seeded inside this biocompatible chip for investigating their response to different concentrations of H₂O₂, a chemical stimulus known to increase the production of ROS. Then the effect of shear stress, a physical stimulus, on lung cancer cells was examined, showing that the production of ROS was increased in response to a larger shear stress. Finally, two antioxidants, α-tocopherol and ferulic acid, were used to study their effects on reducing ROS. It was found that high-dose α-tocopherol was not able to effectively eliminate the ROS produced inside cells. This counter effect was not observed in cells cultured in a traditional chamber slide, where no shear stress was present. This result suggests that the current microfluidic chip provides an in vitro platform best mimicking the physiological condition where cells are under circulating conditions.     

Study of flow behaviors on single-cell manipulation and shear stress reduction in microfluidic chips using computational fluid dynamics simulations

Various single-cell retention structures (SCRSs) were reported for analysis of single cells within microfluidic devices. Undesirable flow behaviors within micro-environments not only influence single-cell manipulation and retention significantly but also lead to cell damage, biochemical heterogeneity among different individual cells (e.g., different cell signaling pathways induced by shear stress). However, the fundamentals in flow behaviors for single-cell manipulation and shear stress reduction, especially comparison of these behaviors in different microstructures, were not fully investigated in previous reports. Herein, flow distribution and induced shear stress in two different single-cell retention structures (SCRS I and SCRS II) were investigated in detail to study their effects on single-cell trapping using computational fluid dynamics (CFD) methods. The results were successfully verified by experimental results. Comparison between these two SCRS shows that the wasp-waisted configuration of SCRS II has a better performance in trapping and manipulating long cylinder-shaped cardiac myocytes and provides a safer “harbor” for fragile cells to prevent cell damage due to the shear stress induced from strong flows. The simulation results have not only explained flow phenomena observed in experiments but also predict new flow phenomena, providing guidelines for new chip design and optimization, and a better understanding of the cell micro-environment and fundamentals of microfluidic flows in single-cell manipulation and analysis.     

A microfluidic model to study fluid dynamics of mucus plug rupture in small lung airways

Fluid dynamics of mucus plug rupture is important to understand mucus clearance in lung airways and potential effects of mucus plug rupture on epithelial cells at lung airway walls. We established a microfluidic model to study mucus plug rupture in a collapsed airway of the 12th generation. Mucus plugs were simulated using Carbopol 940 (C940) gels at concentrations of 0.15%, 0.2%, 0.25%, and 0.3%, which have non-Newtonian properties close to healthy and diseased lung mucus. The airway was modeled with a polydimethylsiloxane microfluidic channel. Plug motion was driven by pressurized air. Global strain rates and shear stress were defined to quantitatively describe plug deformation and rupture. Results show that a plug needs to overcome yield stress before deformation and rupture. The plug takes relatively long time to yield at the high Bingham number. Plug length shortening is the more significant deformation than shearing at gel concentration higher than 0.15%. Although strain rates increase dramatically at rupture, the transient shear stress drops due to the shear-thinning effect of the C940 gels. Dimensionless time-averaged shear stress, T_xy, linearly increases from 3.7 to 5.6 times the Bingham number as the Bingham number varies from 0.018 to 0.1. The dimensionless time-averaged shear rate simply equals to T_xy/2. In dimension, shear stress magnitude is about one order lower than the pressure drop, and one order higher than yield stress. Mucus with high yield stress leads to high shear stress, and therefore would be more likely to cause epithelial cell damage. Crackling sounds produced with plug rupture might be more detectable for gels with higher concentration.     

A microfluidic platform for real-time and in situ monitoring of virus infection process

Microfluidic chip is a promising platform for studying virus behaviors at the cell level. However, only a few chip-based studies on virus infection have been reported. Here, a three-layer microfluidic chip with low shear stress was designed to monitor the infection process of a recombinant Pseudorabies virus (GFP-PrV) in real time and in situ, which could express green fluorescent protein during the genome replication. The infection and proliferation characteristics of GFP-PrV were measured by monitoring the fluorescence intensity of GFP and determining the one-step growth curve. It was found that the infection behaviors of GFP-PrV in the host cells could hardly be influenced by the microenvironment in the microfluidic chip. Furthermore, the results of drug inhibition assays on the microfluidic chip with a tree-like concentration gradient generator showed that one of the infection pathways of GFP-PrV in the host cells was microtubule-dependent. This work established a promising microfluidic platform for the research on virus infection.     

Individually addressable multi-chamber electroporation platform with dielectrophoresis and alternating-current-electro-osmosis assisted cell positioning

A multi-functional microfluidic platform was fabricated to demonstrate the feasibility of on-chip electroporation integrated with dielectrophoresis (DEP) and alternating-current-electro-osmosis (ACEO) assisted cell/particle manipulation. A spatial gradient of electroporation parameters was generated within a microchamber array and validated using normal human dermal fibroblast (NHDF) cells and red fluorescent protein-expressing human umbilical vein endothelial cells (RFP-HUVECs) with various fluorescent indicators. The edge of the bottom electrode, coinciding with the microchamber entrance, may act as an on-demand gate, functioning under either positive or negative DEP. In addition, at sufficiently low activation frequencies, ACEO vortices can complement the DEP to contribute to a rapid trapping/alignment of particles. As such, results clearly indicate that the microfluidic platform has the potential to achieve high-throughput screening for electroporation with spatial control and uniformity, assisted by DEP and ACEO manipulation/trapping of particles/cells into individual microchambers.     

The role of acoustofluidics in targeted drug delivery

With the fast development of acoustic systems in clinical and therapeutic applications, acoustically driven microbubbles have gained a prominent role as powerful tools to carry, transfer, direct, and target drug molecules in cells, tissues, and tumors in the expanding fields of targeted drug delivery and gene therapy. The aim of the present study is to establish a biocompatible acoustic microfluidic system and to demonstrate the generation of an acoustic field and its effects on microbubbles and biological cells in the microfluidic system. The acoustic field creates non-linear oscillations of the microbubble-clusters, which results in generation of shear stress on cells in such microsystems. This effectively helps in delivering extracellular probes in living cells by sonoporation. The sonoporation is investigated under the combined effects of acoustic stress and hydrodynamic stress during targeted drug and gene delivery.     

A microfluidic model for organ-specific extravasation of circulating tumor cells

Circulating tumor cells (CTCs) are the principal vehicle for the spread of non-hematologic cancer disease from a primary tumor, involving extravasation of CTCs across blood vessel walls, to form secondary tumors in remote organs. Herein, a polydimethylsiloxane-based microfluidic system is developed and characterized for in vitro systematic studies of organ-specific extravasation of CTCs. The system recapitulates the two major aspects of the in vivo extravasation microenvironment: local signaling chemokine gradients in a vessel with an endothelial monolayer. The parameters controlling the locally stable chemokine gradients, flow rate, and initial chemokine concentration are investigated experimentally and numerically. The microchannel surface treatment effect on the confluency and adhesion of the endothelial monolayer under applied shear flow has also been characterized experimentally. Further, the conditions for driving a suspension of CTCs through the microfluidic system are discussed while simultaneously maintaining both the local chemokine gradients and the confluent endothelial monolayer. Finally, the microfluidic system is utilized to demonstrate extravasation of MDA-MB-231 cancer cells in the presence of CXCL12 chemokine gradients. Consistent with the hypothesis of organ-specific extravasation, control experiments are presented to substantiate the observation that the MDA-MB-231 cell migration is attributed to chemotaxis rather than a random process.     

One step antibody-mediated isolation and patterning of multiple cell types in microfluidic devices

Cell-cell interactions play a key role in regeneration, differentiation, and basic tissue function taking place under physiological shear forces. However, current solutions to mimic such interactions by micro-patterning cells within microfluidic devices have low resolution, high fabrication complexity, and are limited to one or two cell types. Here, we present a microfluidic platform capable of laminar patterning of any biotin-labeled peptide using streptavidin-based surface chemistry. The design permits the generation of arbitrary cell patterns from heterogeneous mixtures in microfluidic devices. We demonstrate the robust co-patterning of α-CD24, α-ASGPR-1, and α-Tie2 antibodies for rapid isolation and co-patterning of mixtures of hepatocytes and endothelial cells. In addition to one-step isolation and patterning, our design permits step-wise patterning of multiple cell types and empty spaces to create complex cellular geometries in vitro. In conclusion, we developed a microfluidic device that permits the generation of perfusable tissue-like patterns in microfluidic devices by directly injecting complex cell mixtures such as differentiated stem cells or tissue digests with minimal sample preparation.     

A method to integrate patterned electrospun fibers with microfluidic systems to generate complex microenvironments for cell culture applications

The properties of a cell’s microenvironment are one of the main driving forces in cellular fate processes and phenotype expression invivo. The ability to create controlled cell microenvironments invitro becomes increasingly important for studying or controlling phenotype expression in tissue engineering and drug discovery applications. This includes the capability to modify material surface properties within well-defined liquid environments in cell culture systems. One successful approach to mimic extra cellular matrix is with porous electrospun polymer fiber scaffolds, while microfluidic networks have been shown to efficiently generate spatially and temporally defined liquid microenvironments. Here, a method to integrate electrospun fibers with microfluidic networks was developed in order to form complex cell microenvironments with the capability to vary relevant parameters. Spatially defined regions of electrospun fibers of both aligned and random orientation were patterned on glass substrates that were irreversibly bonded to microfluidic networks produced in poly-dimethyl-siloxane. Concentration gradients obtained in the fiber containing channels were characterized experimentally and compared with values obtained by computational fluid dynamic simulations. Velocity and shear stress profiles, as well as vortex formation, were calculated to evaluate the influence of fiber pads on fluidic properties. The suitability of the system to support cell attachment and growth was demonstrated with a fibroblast cell line. The potential of the platform was further verified by a functional investigation of neural stem cell alignment in response to orientation of electrospun fibers versus a microfluidic generated chemoattractant gradient of stromal cell-derived factor 1 alpha. The described method is a competitive strategy to create complex microenvironments invitro that allow detailed studies on the interplay of topography, substrate surface properties, and soluble microenvironment on cellular fate processes.     

Continuously perfused microbubble array for 3D tumor spheroid model

Multi-cellular tumor spheroids (MCTSs) have been established as a 3D physiologically relevant tumor model for drug testing in cancer research. However, it is difficult to control the MCTS testing parameters and the entire process is time-consuming and expensive. To overcome these limitations, we developed a simple microfluidic system using polydimethylsiloxane (PDMS) microbubbles to culture tumor spheroids under physiological flow. The flow characteristics such as streamline directions, shear stress profile, and velocity profile inside the microfluidic system were first examined computationally using a COMSOL simulation. Colo205 tumor spheroids were created by a modified hanging drop method and maintained inside PDMS microbubble cavities in perfusion culture. Cell viability inside the microbubbles was examined by live cell staining and confocal imaging. E-selectin mediated cell sorting of Colo205 and MDA-MB-231 cell lines on functionalized microbubble and PDMS surfaces was achieved. Finally, to validate this microfluidic system for drug screening purposes, the toxicity of the anti-cancer drug, doxorubicin, on Colo205 cells in spheroids was tested and compared to cells in 2D culture. Colo205 spheroids cultured in flow showed a threefold increase in resistance to doxorubicin compared to Colo205 monolayer cells cultured under static conditions, consistent with the resistance observed previously in other MCTS models. The advantages presented by our microfluidic system, such as the ability to control the size uniformity of the spheroids and to perform real-time imaging on cells in the growth platform, show potential for high throughput drug screening development.     

Life under flow: A novel microfluidic device for the assessment of anti-biofilm technologies

In the current study, we have developed and fabricated a novel lab-on-a-chip device for the investigation of biofilm responses, such as attachment kinetics and initial biofilm formation, to different hydrodynamic conditions. The microfluidic flow channels are designed using computational fluid dynamic simulations so as to have a pre-defined, homogeneous wall shear stress in the channels, ranging from 0.03 to 4.30 Pa, which are relevant to in-service conditions on a ship hull, as well as other man-made marine platforms. Temporal variations of biofilm formation in the microfluidic device were assessed using time-lapse microscopy, nucleic acid staining, and confocal laser scanning microscopy (CLSM). Differences in attachment kinetics were observed with increasing shear stress, i.e., with increasing shear stress there appeared to be a delay in bacterial attachment, i.e., at 55, 120, 150, and 155 min for 0.03, 0.60, 2.15, and 4.30 Pa, respectively. CLSM confirmed marked variations in colony architecture, i.e.,: (i) lower shear stresses resulted in biofilms with distinctive morphologies mainly characterised by mushroom-like structures, interstitial channels, and internal voids, and (ii) for the higher shear stresses compact clusters with large interspaces between them were formed. The key advantage of the developed microfluidic device is the combination of three architectural features in one device, i.e., an open-system design, channel replication, and multiple fully developed shear stresses.     

Transport and shear in a microfluidic membrane bilayer device for cell culture

Microfluidic devices have been established as useful platforms for cell culture for a broad range of applications, but challenges associated with controlling gradients of oxygen and other soluble factors and hemodynamic shear forces in small, confined channels have emerged. For instance, simple microfluidic constructs comprising a single cell culture compartment in a dynamic flow condition must handle tradeoffs between sustaining oxygen delivery and limiting hemodynamic shear forces imparted to the cells. These tradeoffs present significant difficulties in the culture of mesenchymal stem cells (MSCs), where shear is known to regulate signaling, proliferation, and expression. Several approaches designed to shield cells in microfluidic devices from excessive shear while maintaining sufficient oxygen concentrations and transport have been reported. Here we present the relationship between oxygen transport and shear in a "membrane bilayer" microfluidic device, in which soluble factors are delivered to a cell population by means of flow through a proximate channel separated from the culture channel by a membrane. We present an analytical model that describes the characteristics of this device and its ability to independently modulate oxygen delivery and hemodynamic shear imparted to the cultured cells. This bilayer configuration provides a more uniform oxygen concentration profile that is possible in a single-channel system, and it enables independent tuning of oxygen transport and shear parameters to meet requirements for MSCs and other cells known to be sensitive to hemodynamic shear stresses.     

A standalone perfusion platform for drug testing and target validation in micro-vessel networks

Studying the effects of pharmacological agents on human endothelium includes the routine use of cell monolayers cultivated in multi-well plates. This configuration fails to recapitulate the complex architecture of vascular networks in vivo and does not capture the relationship between shear stress (i.e. flow) experienced by the cells and dose of the applied pharmacological agents. Microfluidic platforms have been applied extensively to create vascular systems in vitro; however, they rely on bulky external hardware to operate, which hinders the wide application of microfluidic chips by non-microfluidic experts. Here, we have developed a standalone perfusion platform where multiple devices were perfused at a time with a single miniaturized peristaltic pump. Using the platform, multiple micro-vessel networks, that contained three levels of branching structures, were created by culturing endothelial cells within circular micro-channel networks mimicking the geometrical configuration of natural blood vessels. To demonstrate the feasibility of our platform for drug testing and validation assays, a drug induced nitric oxide assay was performed on the engineered micro-vessel network using a panel of vaso-active drugs (acetylcholine, phenylephrine, atorvastatin, and sildenafil), showing both flow and drug dose dependent responses. The interactive effects between flow and drug dose for sildenafil could not be captured by a simple straight rectangular channel coated with endothelial cells, but it was captured in a more physiological branching circular network. A monocyte adhesion assay was also demonstrated with and without stimulation by an inflammatory cytokine, tumor necrosis factor-α.