Microfluidic Shear Research Articles

A Microfluidic Design for Quantitative Measurements of Shear Stress-Dependent Adhesion and Motion of Dictyostelium discoideum Cells.

Shear stress plays a crucial role in modulating cell adhesion and signaling. We present a microfluidic shear stress generator used to investigate the adhesion dynamics of Dictyostelium discoideum, an amoeba cell model organism with well-characterized adhesion properties. We applied shear stress and tracked cell adhesion, motility, and detachment using time-lapse videomicroscopy. In the precise shear conditions generated on-chip, our results show cell migration patterns are influenced by shear stress, with cells displaying an adaptive response to shear forces as they alter their adhesion and motility behavior. Additionally, we observed that DH1-10 wild-type D. discoideum cells exhibit stronger adhesion and resistance to shear-induced detachment compared to phg2 adhesion-defective mutant cells. We also highlight the influence of cell density on detachment kinetics.

Using microfluidic shear to assess transfusion requirements in trauma patients

BackgroundViscoelastic assays have widely been used for evaluating coagulopathies but lack the addition of shear stress important to in vivo clot formation. Stasys technology subjects whole blood to shear forces...

Single-cell mechanical assay unveils viscoelastic similarities in normal and neoplastic brain cells

Understanding cancer cell mechanics allows for the identification of novel disease mechanisms, diagnostic biomarkers, and targeted therapies. In this study, we utilized our previously established fluid shear stress assay to investigate and compare the viscoelastic properties of normal immortalized human astrocytes and invasive human glioblastoma (GBM) cells when subjected to physiological levels of shear stress that are present in the brain microenvironment. We used a parallel-flow microfluidic shear system and a camera-coupled optical microscope to expose single cells to fluid shear stress and monitor the resulting deformation in real time, respectively. From the video-rate imaging, we fed cell deformation information from digital image correlation into a three-parameter generalized Maxwell model to quantify the nuclear and cytoplasmic viscoelastic properties of single cells. We further quantified actin cytoskeleton density and alignment in immortalized human astrocytes and GBM cells via fluorescence microscopy and image analysis techniques. Results from our study show that contrary to the behavior of many extracranial cells, normal and cancerous brain cells do not exhibit significant differences in their viscoelastic properties. Moreover, we also found that the viscoelastic properties of the nucleus and cytoplasm as well as the actin cytoskeletal densities of both brain cell types are similar. Our work suggests that malignant GBM cells exhibit unique mechanical behaviors not seen in other cancer cell types. These results warrant future studies to elucidate the distinct biophysical characteristics of the brain and reveal novel mechanical attributes of GBM and other primary brain tumors.

Effects of Microfluidic Shear on the Plasmid DNA Structure: Implications for Polymeric Gene Delivery Vectors.

Microfluidic manufacturing of advanced gene delivery vectors necessitates consideration of the effects of microfluidic shear forces on the structural integrity of plasmid DNA (pDNA). In this paper, we expose pDNA to variable shear forces in a two-phase, gas-liquid microfluidic reactor and apply gel electrophoresis to analyze the products of on-chip shear-induced degradation. The effects of shear rate, solvent environment, pDNA size, and copolymer complexation on shear-induced degradation are investigated. We find that small naked pDNA (pUC18, 2.7 kb) exhibits shear rate-dependent shear degradation in the microfluidic channels in a mixed organic solvent (dioxane/water/acetic acid; 90/10/<0.1 w/w/w), with the extents of both supercoil isoform relaxation and complete fragmentation increasing as the maximum shear rates increase from 4 × 105 to 2 × 106 s-1. However, over the same range of shear rates, the same pDNA sample shows no evidence of microfluidic shear-induced degradation in a pure aqueous environment. Quiescent control experiments in the same mixed organic solvent prove that a combination of solvent and shear forces is involved in the observed shear-induced degradation. Furthermore, we show that shear degradation effects in mixed organic solvents can be significantly attenuated by complexation of pDNA with the block copolymer polycaprolactone-block-poly(2-vinylpyridine) prior to exposure to microfluidic shear. Finally, we demonstrate that medium (pDSK519, 8.1 kb) and large (pRK290, 20 kb) naked pDNA are more sensitive to shear-induced microfluidic degradation in the mixed organic solvent environment than small pDNA, with both plasmids showing complete fragmentation even at the lowest shear rate, although we found no evidence of shear-induced damage in water for the largest investigated naked pDNA even at the highest flow rate. The resulting understanding of the interplay of the solvent and shear effects during microfluidic processing should inform microfluidic manufacturing routes to new gene therapy formulations.

Nematic Fibrin Fibers Enabling Vascularized Thrombus Implants Facilitate Scarless Cutaneous Wound Healing (Adv. Mater. 25/2023)

Wound Healing As reported by Joo H. Kang and co-workers in article number 2211149, implantable vascularized engineered thrombi (IVET) using autologous blood can improve wound healing by creating robust microcapillary networks. By aligning fibrin fibers and activating platelets through microfluidic shear stress, the engineered thrombi create a moderate stiffness microenvironment that can promote endothelial cell maturation and vascularization. In full-thickness skin wound models, IVET implantation facilitates rapid and scar-free wound recovery.

Versatile Microfluidics Separation of Colloids by Combining External Flow with Light-induced Chemical Activity.

Separation of particles by size, morphology, or material identity is of paramount importance in fields such as filtration or bioanalytics. Up to now separation of particles distinguished solely by surface properties or bulk/surface morphology remains a very challenging process. Here we propose a combination of pressure driven microfluidic flow and local self-phoresis/osmosis via light-induced chemical activity of a photoactive azobenzene-surfactant solution. This process induces a vertical displacement of the sedimented particles and depends on the size and surface properties of the particle, thus, consequently, different particles experience different regions of the ambient microfluidic shear flow. Accordingly, simple, versatile method for separation of colloids can be achieved by elution times in a sense of particle chromatography. The concepts are illustrated via experimental studies, complemented by theoretical analysis, which include separation of bulk-porous from bulk-compact particles and separation of particles distinguished solely by slight differences in their surface physico-chemical properties. This article is protected by copyright. All rights reserved.

Nematic Fibrin Fibers Enabling Vascularized Thrombus Implants Facilitate Scarless Cutaneous Wound Healing.

Autologous implantable scaffolds that induce vasculogenesis have shown great potential in tissue regeneration; however, previous attempts mainly relied on cell-laden hydrogel patches using fat tissues or platelet-rich plasma, which are insufficient for generating a uniform vasculature in a scalable manner. Here, implantable vascularized engineered thrombi (IVETs) are presented using autologous whole blood, which potentiate effective skin wound healing by constructing robust microcapillary vessel networks at the wound site. Microfluidic shear stresses enable the alignment of bundled fibrin fibers along the direction of the blood flow streamlines and the activation of platelets, both of which offer moderate stiffness of the microenvironment optimal for facilitating endothelial cell maturation and vascularization. Rodent dorsal skin wounds patched with IVET present superior wound closure rates (96.08±1.58%), epidermis thickness, collagen deposition, hair follicle numbers, and neutrophil infiltration, which are permitted by enhanced microvascular circulation. Moreover, IVET treatment accelerates wound healing by recruiting M2phenotype macrophages.

A Fluid Multivalent Magnetic Interface for High-Performance Isolation and Proteomic Profiling of Tumor-Derived Extracellular Vesicles.

Isolation and analysis of tumor-derived extracellular vesicles (T-EVs) are important for clinical cancer management. Here, we develop a fluid multivalent magnetic interface (FluidmagFace) in a microfluidic chip for high-performance isolation, release, and protein profiling of T-EVs. The FluidmagFace increases affinity by 105-fold with fluidity-enhanced multivalent binding to improve isolation efficiency by 13.9% compared with a non-fluid interface. Its anti-adsorption property and microfluidic hydrodynamic shear minimize contamination, increasing detection sensitivity by two orders of magnitude. Moreover, its reversibility and expandability allow high-throughput recovery of T-EVs for mass spectrometric protein analysis. With the chip, T-EVs are detected in all tested cancer samples with identification of differentially expressed proteins compared with healthy controls. The FluidmagFace opens a new avenue to isolation and release of targets for cancer diagnosis and biomarker discovery.

A Fluid Multivalent Magnetic Interface for High‐Performance Isolation and Proteomic Profiling of Tumor‐Derived Extracellular Vesicles

AbstractIsolation and analysis of tumor‐derived extracellular vesicles (T‐EVs) are important for clinical cancer management. Here, we develop a fluid multivalent magnetic interface (FluidmagFace) in a microfluidic chip for high‐performance isolation, release, and protein profiling of T‐EVs. The FluidmagFace increases affinity by 105‐fold with fluidity‐enhanced multivalent binding to improve isolation efficiency by 13.9 % compared with a non‐fluid interface. Its anti‐adsorption property and microfluidic hydrodynamic shear minimize contamination, increasing detection sensitivity by two orders of magnitude. Moreover, its reversibility and expandability allow high‐throughput recovery of T‐EVs for mass spectrometric protein analysis. With the chip, T‐EVs were detected in all tested cancer samples with identification of differentially expressed proteins compared with healthy controls. The FluidmagFace opens a new avenue to isolation and release of targets for cancer diagnosis and biomarker discovery.

Microfluidic jet impact: Spreading, splashing, soft substrate deformation and injection

HypothesisNeedle-free injections using microfluidic jets could be optimized by reducing splashing and controlling injection depth. However, this is impeded by an incomplete understanding on how jet characteristics influence impact outcome. We hypothesise that exploring the relation between microfluidic jet characteristics and substrate shear modulus on impact behavior will assist in predicting and giving insights on the impact outcome on skin and injection endpoints. ExperimentsTo do so, a setup using microfluidic chips, at varying laser powers and stand-off distances, was used to create thermocavitation generated microfluidic jets with ranging characteristics (velocity: 7–77 m/s, diameter: 35–120 μm, Weber-number: 40–4000), which were impacted on substrates with different shear modulus. FindingsSeven impact regimes were found, depending on jet Weber-number and substrate shear modulus, and we identified three thresholds: i) spreading/splashing threshold, ii) dimple formation threshold, and iii) plastic/elastic deformation threshold. The regimes show similarity to skin impact, although the opacity of skin complicated determining the threshold values. Additionally, we found that jet velocity has a higher predictive value for injection depth compared to the Weber-number, and consequently, the jet-diameter. Our findings provide fundamental knowledge on the interaction between microfluidic jets and substrates, and are relevant for optimizing needle-free injections.

Microfluidic T Cell Selection by Cellular Avidity.

No T cell receptor (TCR) T cell therapies have obtained clinical approval. The lack of strategies capable of selecting and recovering potent T cell candidates may be a contributor to this. Existing protocols for selecting TCR T cell clones for cell therapies such as peptide multimer methods have provided effective measurements on TCR affinities. However, these methods lack the ability to measure the collective strength of intercellular interactions (i.e., cellular avidity) and markers of T cell activation such as immunological synapse formation. This study describes a novel microfluidic fluid shear stress-based approach to identify and recover highly potent T cell clones based on the cellular avidity between living T cells and tumor cells. This approach is capable of probing approximately up to 10000 T cell-tumor cell interactions per run and can recover potent T cells with up to 100% purity from mixed populations of T cells within 30 min. Markers of cytotoxicity, activation, and avidity persist when recovered high cellular avidity T cells are subsequently exposed to fresh tumor cells. These results demonstrate how microfluidic probing of cellular avidity may fast track the therapeutic T cell selection process and move the authors closer to precision cancer immunotherapy.

Microfluidic Study of Bacterial Attachment on and Detachment from Zinc Oxide Nanopillars.

Nanopillars can influence how bacterial cells attach to a surface. Herein, we investigated whether self-assembled zinc oxide (ZnO) nanopillars synthesized on glass substrates via the conventional hydrothermal route possess anti-biofouling properties either by reducing the amount of initially attached cells or promoting the detachment of cells from the surface or both. To avoid complications associated with manual intervention methods of assessing bacterial attachment on nanopillar surfaces, we implemented a microfluidic approach. In our study, we synthesized two nanopillar topographies: a low surface density of ZnO nanopillars and a high surface density of ZnO nanopillars. Next, we mounted microfluidic channels to each of these substrates. This microfluidic approach allowed us to gently flow Pseudomonas aeruginosa, Staphylococcus aureus, or Bacillus subtilis cells onto the nanopillars for initial attachment before systematically increasing the flowrate to attempt to detach remaining attached cells without introducing air-liquid interface artefacts during the assay. Generally, initial bacterial attachment was similar across all substrates. However, cells consistently detached more readily from high-surface-density nanopillars compared to low-surface-density nanopillars. Electron microscopy revealed that cells that attached to high-surface-density nanopillars rested atop the nanopillars, fully exposed to microfluidic shear, whereas many cells became trapped in the void space between neighboring low-surface-density nanopillars, shielding these cells from detachment. Our findings indicate that self-assembled ZnO nanopillars can provide anti-biofouling properties under submerged flow but only if synthesized at high surface density.

Zwitterionic surface chemistry enhances detachment of bacteria under shear.

The ubiquitous nature of microorganisms, especially of biofilm-forming bacteria, makes biofouling a prevalent challenge in many settings, including medical and industrial environments immersed in liquid and subjected to shear forces. Recent studies have shown that zwitterionic groups are effective in suppressing bacteria and protein adhesion as well as biofilm growth. However, the effect of zwitterionic groups on the removal of surface-bound bacteria has not been extensively studied. Here we present a microfluidic approach to evaluate the effectiveness in facilitating bacteria detachment by shear of an antifouling surface treatment using (3-(dimethyl;(3-trimethoxysilyl)propyl)ammonia propane-1-sulfonate), a sulfobetaine silane (SBS). Control studies show that SBS-functionalized surfaces greatly increase protein (bovine serum albumin) removal upon rinsing. On the same surfaces, enhanced bacteria (Pseudomonas aeruginosa) removal is observed under shear. To quantify this enhancement a microfluidic shear device is employed to investigate how SBS-functionalized surfaces promote bacteria detachment under shear. By using a microfluidic channel with five shear zones, we compare the removal of bacteria from zwitterionic and glass surfaces under different shear rates. At times of 15 min, 30 min, and 60 min, bacteria adhesion on SBS-functionalized surfaces is reduced relative to the control surface (glass) under quiescent conditions. However, surface-associated bacteria on the SBS-functionalized glass and control show similar percentages of live cells, suggesting minimal intrinsic biocidal effect from the SBS-functionalized surface. Notably, when exposed to shear rates ranging from 104 to 105 s-1, significantly fewer bacteria remain on the SBS-functionalized surfaces. These results demonstrate the potential of zwitterionic sulfobetaine as effective antifouling coatings that facilitate the removal of bacteria under shear.

Mechanically Strong Proteinaceous Fibers: Engineered Fabrication by Microfluidics

Lightweight and mechanically strong natural silk fibers have been extensively investigated over the past decades. Inspired by this research, many artificial spinning techniques (wet spinning, dry spinning, electrospinning, etc.) have been developed to fabricate robust protein fibers. As the traditional spinning methods provide poor control over the as-spun fibers, microfluidics has been integrated with these techniques to allow the fabrication of biological fibers in a well-designed manner, with simplicity and cost efficiency. The mechanical behavior of the developed fibers can be precisely modulated by controlling the type iop and size of microfluidic channel, flow rate, and shear force. This technique has been successfully used to manufacture a broad range of protein fibers, and can accelerate the production and application of protein fibers in various fields. This review outlines recent progress in the design and fabrication of protein-based fibers based on microfluidics. We first briefly discuss the natural spider silk-spinning process and the microfluidics spinning process. Next, the fabrication and mechanical properties of regenerated protein fibers via microfluidics are discussed, followed by a discussion of recombinant protein fibers. Other sourced protein fibers are also reviewed in detail. Finally, a brief outlook on the development of microfluidic technology for producing protein fibers is presented.

A high-resolution real-time quantification of astrocyte cytokine secretion under shear stress for investigating hydrocephalus shunt failure

It has been hypothesized that physiological shear forces acting on medical devices implanted in the brain significantly accelerate the rate to device failure in patients with chronically indwelling neuroprosthetics. In hydrocephalus shunt devices, shear forces arise from cerebrospinal fluid flow. The shunt’s unacceptably high failure rate is mostly due to obstruction with adherent inflammatory cells. Astrocytes are the dominant cell type bound directly to obstructing shunts, rapidly manipulating their activation via shear stress-dependent cytokine secretion. Here we developed a total internal reflection fluorescence microscopy combined with a microfluidic shear device chip (MSDC) for quantitative analysis and direct spatial-temporal mapping of secreted cytokines at the single-cell level under physiological shear stress to identify the root cause for shunt failure. Real-time secretion imaging at 1-min time intervals enabled successful detection of a significant increase of astrocyte IL-6 cytokine secretion under shear stress greater than 0.5 dyne/cm2, validating our hypothesis and highlighting the importance of reducing shear stress activation of cells.

Sensing morphogenesis of bone cells under microfluidic shear stress by holographic microscopy and automatic aberration compensation with deep learning.

We present sensing time-lapse morphogenesis of living bone cells under micro-fluidic shear stress (FSS) by digital holographic (DH) microscopy. To remove the effect of aberrations on quantitative measurements, we propose a numerical and automatic method to compensate for aberrations based on a convolutional neural network (CNN). For the first time, the aberration compensation issue is considered as a regression task where optimal coefficients for constructing the phase aberration map act as responses corresponding to the input aberrated phase image. We adopted tens of thousands of living cells' phase images reconstructed from digital holograms for training the CNN. The experiments demonstrate that, based on the trained network, phase aberrations can be totally removed in real-time without any hypothesis of object and aberration phase, knowledge of the setup's physical parameters, and the operation of selecting background regions; hence, the morphogenesis of the bone cells under FSS is accurately detected and quantitatively analyzed. The results show that the proposed method could provide a highly efficient and versatile way to investigate the effects of micro-FSS on living biological cells in microfluidic lab-on-chip platforms thanks to the combination of phase-contrast label-free microcopy with artificial intelligence.

Evaluation of bacterial adhesion strength on phospholipid copolymer films with antibacterial ability using microfluidic shear devices.

Biomimetic phospholipid copolymer films are known to possess antifouling properties against protein adsorption and biofilm formation. However, the interactions between bacterial cells and material surfaces are not fully understood. This work investigated the bacterial adhesion strength of phospholipid copolymer films using a shear stress-tunable microfluidic device. The copolymer, comprising 2-methacryloyloxyethyl phosphorylcholine (MPC), 3-methacryloxypropyl trimethoxysilane (MPTMSi), and 3-(methacryloyloxy) propyl-tris(trimethylsilyloxy) silane (MPTSSi), formed crosslinked films on glass substrates; the thickness of the coating film was controlled by the polymer concentration during dip-coating. Polymer films with two typical thicknesses, 20 and 40 nm (denoted as C-20 and C-40, respectively), were prepared on the bottom wall of the microfluidic device. After seeding S. aureus in the microfluidic device, several shear stresses were applied to evaluate the adhesion strength of the polymer films. S. aureus was found to have weaker adhesion strength on the C-40 surface than on the C-20 surface; numerous bacterial cells detached from the C-40 surface on application of identical shear stress. To mimic the presence of plasma protein, fibrinogen (Fg) was introduced into the device before performing the bacterial adhesion assay. The results showed that the adsorption of Fg promoted S. aureus adhesion and strong interactions under shear stress. However, the adhesion strength of S. aureus did not affect the Fg adsorption for both the C-20 and C-40 surfaces. Using the shear stress-tunable microfluidic device, we found that the adhesion of S. aureus on the thicker and softer phospholipid copolymer was weak, and the cells easily detached under high shear stress.

Adhesive stem cell coatings for enhanced retention in the heart tissue.

Injection into the heart tissue is a direct route for optimally placing mesenchymal stem cells (MSC) to regulate local inflammation following a heart attack. The retention of MSCs at the injection site is severely limited by the fluid flows that rapidly wash cells away and minimize their capacity to modulate cardiac inflammation. To prevent this loss of MSCs and their function, antibody coatings were designed for the surface of MSCs to enhance their adhesion to the inflamed tissue. MSCs were biotinylated, and biotinylated antibodies against intercellular cell adhesion molecules were conjugated to the cell surface through an intermediate layer of streptavidin. MSC surfaces were modified with ~7,000 biotin/μm2 and ~23 antibodies/μm2. The heart tissue injection of antibody-coated MSCs offered a 3-fold increase of cell retention in an infarcted heart over the injection of uncoated MSCs. We supported the mechanism of adhesion through analysis of MSC adhesion to inflamed endothelial cells and also surfaces of purified adhesion molecules on glass under microfluidic shear flow.

Toxoplasma gondii Dysregulates Barrier Function and Mechanotransduction Signaling in Human Endothelial Cells.

Toxoplasma gondii can infect and replicate in vascular endothelial cells prior to entering host tissues. However, little is known about the molecular interactions at the parasite-endothelial cell interface. We demonstrate that T. gondii infection of primary human umbilical vein endothelial cells (HUVEC) altered cell morphology and dysregulated barrier function, increasing permeability to low-molecular-weight polymers. T. gondii disrupted vascular endothelial cadherin (VE-cadherin) and β-catenin localization to the cell periphery and reduced VE-cadherin protein expression. Notably, T. gondii infection led to reorganization of the host cytoskeleton by reducing filamentous actin (F-actin) stress fiber abundance under static and microfluidic shear stress conditions and by reducing planar cell polarity. RNA sequencing (RNA-Seq) comparing genome-wide transcriptional profiles of infected to uninfected endothelial cells revealed changes in gene expression associated with cell-cell adhesion, extracellular matrix reorganization, and cytokine-mediated signaling. In particular, genes downstream of Hippo signaling and the biomechanical sensor and transcriptional coactivator Yes-associated protein (YAP) were downregulated in infected endothelial cells. Interestingly, T. gondii infection activated Hippo signaling by increasing phosphorylation of LATS1, leading to cytoplasmic retention of YAP, and reducing YAP target gene expression. These findings suggest that T. gondii infection triggers Hippo signaling and YAP nuclear export, leading to an altered transcriptional profile of infected endothelial cells.IMPORTANCE Toxoplasma gondii is a foodborne parasite that infects virtually all warm-blooded animals and can cause severe disease in individuals with compromised or weakened immune systems. During dissemination in its infected hosts, T. gondii breaches endothelial barriers to enter tissues and establish the chronic infections underlying the most severe manifestations of toxoplasmosis. The research presented here examines how T. gondii infection of primary human endothelial cells induces changes in cell morphology, barrier function, gene expression, and mechanotransduction signaling under static conditions and under the physiological conditions of shear stress found in the bloodstream. Understanding the molecular interactions occurring at the interface between endothelial cells and T. gondii may provide insights into processes linked to parasite dissemination and pathogenesis.

Endothelial cell polarization and orientation to flow in a novel microfluidic multimodal shear stress generator.

Endothelial cells (EC) respond to shear stress to maintain vascular homeostasis, and a disrupted response is associated with cardiovascular diseases. To understand how different shear stress modalities affect EC morphology and behavior, we developed a microfluidic device that concurrently generates three different levels of uniform wall shear stress (WSS) and six different WSS gradients (WSSG). In this device, human umbilical vein endothelial cells (HUVECs) exhibited a rapid and robust response to WSS, with the relative positioning of the Golgi and nucleus transitioning from a non-polarized to polarized state in a WSS magnitude- and gradient-dependent manner. By contrast, polarized HUVECs oriented their Golgi and nucleus polarity to the flow vector in a WSS magnitude-dependent manner, with positive WSSG inhibiting and negative WSSG promoting upstream orientation. Having validated this device, this chip can now be used to dissect the mechanisms underlying EC responses to different WSS modalities, including shear stress gradients, and to investigate the influence of flow on a diverse range of cells during development, homeostasis and disease.