A Human Vascular Injury-on-a-Chip Model of Hemostasis.

Hemostasis is an innate protective mechanism that plays a central role in maintaining the homeostasis of the vascular system during vascular injury. Studying this essential physiological process is often challenged by the difficulty of modeling and probing the complex dynamics of hemostatic responses in the native context of human blood vessels. To address this major challenge, this paper describes a microengineering approach for in vitro modeling of hemostasis. This microphysiological model replicates the living endothelium, multilayered microarchitecture, and procoagulant activity of human blood vessels, and is also equipped with a microneedle that is actuated with spatial precision to simulate penetrating vascular injuries. The system recapitulates key features of the hemostatic response to acute vascular injury as observed in vivo, including i) thrombin-driven accumulation of platelets and fibrin, ii) formation of a platelet- and fibrin-rich hemostatic plug that halts blood loss, and iii) matrix deformation driven by platelet contraction for wound closure. Moreover, the potential use of this model for drug testing applications is demonstrated by evaluating the effects of anticoagulants and antiplatelet agents that are in current clinical use. The vascular injury-on-a-chip may serve as an enabling platform for preclinical investigation of hematological disorders and emerging therapeutic approaches against them.

Generalized On-Demand Production of Nanoparticle Monolayers on Arbitrary Solid Surfaces via Capillarity-Mediated Inverse Transfer.

Century-old Langmuir monolayer deposition still represents the most convenient approach to the production of monolayers of colloidal nanoparticles on solid substrates for practical biological and chemical-sensing applications. However, this approach simply yields arbitrarily shaped large monolayers on a flat surface and is strongly limited by substrate topography and interfacial energy. Here, we describe a generalized and facile method of rapidly producing uniform monolayers of various colloidal nanoparticles on arbitrary solid substrates by using an ordinary capillary tube. Our method is based on an interesting finding of inversion phenomenon of a nanoparticle-laden air-water interface by flowing through a capillary tube in a manner that prevents the particles from adhesion to the capillary sidewall, thereby presenting the nanoparticles face-first at the tube's opposite end for direct and one-step deposition onto a substrate. We show that our method not only allows the placement of a nanoparticle monolayer at target locations of solid substrates regardless of their surface geometry and adhesion but also enables the production of monolayers containing nanoparticles with different size, shape, surface charge, and composition. To explore the potential of our approach, we demonstrate the facile integration of gold nanoparticle monolayers into microfluidic devices for the real-time monitoring of molecular Raman signals under dynamic flow conditions. Moreover, we successfully extend the use of our method to developing on-demand Raman sensors that can be built directly on the surface of consumer products for practical chemical sensing and fingerprinting. Specifically, we achieve both the pinpoint deposition of gold nanoparticle monolayers and sensitive molecular detection from the deposited region on clothing fabric for the detection of illegal drug substances, a single grain of rice and an orange for pesticide monitoring, and a $100 bill as a potential anti-counterfeit measure, respectively. We believe that our method will provide unique opportunities to expand the utility of colloidal nanoparticles and to greatly improve the accessibility of nanoparticle-based sensing technologies.

In Vitro Granuloma Models of Tuberculosis: Potential and Challenges.

Despite intensive research efforts, several fundamental disease processes for tuberculosis (TB) remain poorly understood. A central enigma is that host immunity is necessary to control disease yet promotes transmission by causing lung immunopathology. Our inability to distinguish these processes makes it challenging to design rational novel interventions. Elucidating basic immune mechanisms likely requires both in vivo and in vitro analyses, since Mycobacterium tuberculosis is a highly specialized human pathogen. The classic immune response is the TB granuloma organized in three dimensions within extracellular matrix. Several groups are developing cell culture granuloma models. In January 2018, NIAID convened a workshop, entitled "3-D Human in vitro TB Granuloma Model" to advance the field. Here, we summarize the arguments for developing advanced TB cell culture models and critically review those currently available. We discuss how integrating complementary approaches, specifically organoids and mathematical modeling, can maximize progress, and conclude by discussing future challenges and opportunities.

Use of three-dimensional organoids and lung-on-a-chip methods to study lung development, regeneration and disease.

Differences in lung anatomy between mice and humans, as well as frequently disappointing results when using animal models for drug discovery, emphasise the unmet need for in vitro models that can complement animal studies and improve our understanding of human lung physiology, regeneration and disease. Recent papers have highlighted the use of three-dimensional organoids and organs-on-a-chip to mimic tissue morphogenesis and function in vitro Here, we focus on the respiratory system and provide an overview of these in vitro models, which can be derived from primary lung cells and pluripotent stem cells, as well as healthy or diseased lungs. We emphasise their potential application in studies of respiratory development, regeneration and disease modelling.

Roles of fluid shear stress and retinoic acid in the differentiation of primary cultured human podocytes.

Due to the distinct features that distinguish immortalized podocyte cell lines from their in vivo counterparts, primary cultured human podocytes might be a superior cell model for glomerular disease studies. However, the podocyte de-differentiation that occurs in culture remains an unresolved problem. Here, we present a method to differentiate primary cultured podocytes using retinoic acid (RA) and fluid shear stress (FSS), which mimic the in vivo environment of the glomerulus. RA treatment induced changes in the cell shape of podocytes from a cobblestone-like morphology to an arborized configuration with enhanced mobility. Moreover, the expression of synaptopodin and zonula occludens (ZO)-1 in RA-treated podocytes increased along with Krüppel-like factor 15 (KLF15) expression. Confocal microscopy revealed that RA increased the expression of cytoplasmic synaptopodin, which adopted a filamentous arrangement, and junctional ZO-1 expression, which showed a zipper-like pattern. To elucidate the effect of FSS in addition to RA, the podocytes were cultured in microfluidic devices and assigned to the static, static+RA, FSS, and FSS+RA groups. The FSS+RA group showed increased synaptopodin and ZO-1 expression with prominent spikes on the cell-cell interface. Furthermore, interdigitating processes were only observed in the FSS+RA group. Consistent with these data, the mRNA expression levels of synaptopodin, podocin, WT-1 and ZO-1 were synergistically increased by FSS and RA treatment. Additionally, the heights of the cells were greater in the FSS and FSS+RA groups than in the static groups, suggesting a restoration of the 3D cellular shape. Meanwhile, the expression of KLF15 increased in the RA-treated cells regardless of fluidic condition. Taken together, FSS and RA may contribute through different but additive mechanisms to the differentiation of podocytes. These cells may serve as a useful tool for mechanistic studies and the application of regenerative medicine to the treatment of kidney diseases.

A mechanosensitive transcriptional mechanism that controls angiogenesis.

Angiogenesis is controlled by physical interactions between cells and extracellular matrix as well as soluble angiogenic factors, such as VEGF. However, the mechanism by which mechanical signals integrate with other microenvironmental cues to regulate neovascularization remains unknown. Here we show that the Rho inhibitor, p190RhoGAP (also known as GRLF1), controls capillary network formation in vitro in human microvascular endothelial cells and retinal angiogenesis in vivo by modulating the balance of activities between two antagonistic transcription factors, TFII-I (also known as GTF2I) and GATA2, that govern gene expression of the VEGF receptor VEGFR2 (also known as KDR). Moreover, this new angiogenesis signalling pathway is sensitive to extracellular matrix elasticity as well as soluble VEGF. This is, to our knowledge, the first known functional cross-antagonism between transcription factors that controls tissue morphogenesis, and that responds to both mechanical and chemical cues.

Red blood cells induce necroptosis of lung endothelial cells and increase susceptibility to lung inflammation.

RATIONALE: Red blood cell (RBC) transfusions are associated with increased risk of acute respiratory distress syndrome (ARDS) in the critically ill, yet the mechanisms for enhanced susceptibility to ARDS conferred by RBC transfusions remain unknown. OBJECTIVES: To determine the mechanisms of lung endothelial cell (EC) High Mobility Group Box 1 (HMGB1) release following exposure to RBCs and to determine whether RBC transfusion increases susceptibility to lung inflammation in vivo through release of the danger signal HMGB1. METHODS: In vitro studies examining human lung EC viability and HMGB1 release following exposure to allogenic RBCs were conducted under static conditions and using a microengineered model of RBC perfusion. The plasma from transfused and nontransfused patients with severe sepsis was examined for markers of cellular injury. A murine model of RBC transfusion followed by LPS administration was used to determine the effects of RBC transfusion and HMGB1 release on LPS-induced lung inflammation. MEASUREMENTS AND MAIN RESULTS: After incubation with RBCs, lung ECs underwent regulated necrotic cell death (necroptosis) and released the essential mediator of necroptosis, receptor-interacting serine/threonine-protein kinase 3 (RIP3), and HMGB1. RIP3 was detectable in the plasma of patients with severe sepsis, and was increased with blood transfusion and among nonsurvivors of sepsis. RBC transfusion sensitized mice to LPS-induced lung inflammation through release of the danger signal HMGB1. CONCLUSIONS: RBC transfusion enhances susceptibility to lung inflammation through release of HMGB1 and induces necroptosis of lung EC. Necroptosis and subsequent danger signal release is a novel mechanism of injury following transfusion that may account for the increased risk of ARDS in critically ill transfused patients.

Cyr61 delivery promotes angiogenesis during bone fracture repair.

Compromised vascular supply and insufficient neovascularization impede bone repair, increasing risk of non-union. Cyr61, Cysteine-rich angiogenic inducer of 61kD (also known as CCN1), is a matricellular growth factor that is regulated by mechanical cues during fracture repair. Here, we map the distribution of endogenous Cyr61 during bone repair and evaluate the effects of recombinant Cyr61 delivery on vascularized bone regeneration. In vitro, Cyr61 treatment did not alter chondrogenesis or osteogenic gene expression, but significantly enhanced angiogenesis. In a mouse femoral fracture model, Cyr61 delivery did not alter cartilage or bone formation, but accelerated neovascularization during fracture repair. Early initiation of ambulatory mechanical loading disrupted Cyr61-induced neovascularization. Together, these data indicate that Cyr61 delivery can enhance angiogenesis during bone repair, particularly for fractures with stable fixation, and may have therapeutic potential for fractures with limited blood vessel supply.

Effects of various extracellular matrix proteins on the growth of HL-1 cardiomyocytes.

We present the physical and biochemical effects of extracellular matrixes (ECMs) on HL-1 cardiomyocytes. ECMs play major roles in cell growth, adhesion and the maintenance of native cell functions. We investigated the effects of 6 different cell culture systems: 5 different ECM-treated surfaces (fibronectin, laminin, collagen I, gelatin and a gelatin/fibronectin mixture) and 1 nontreated surface. Surface morphology was scanned and analyzed using atomic force microscopy in order to investigate the physical effects of ECMs. The attachment, growth, viability, proliferation and phenotype of the cells were analyzed using phase-contrast microscopy and immunocytochemistry to elucidate the biochemical effects of ECMs. Our study provides basic information for understanding cell-ECM interactions and should be utilized in future cardiac cell research and tissue engineering.

High performance inkjet printed embedded electrochemical sensors for monitoring hypoxia in a gut bilayer microfluidic chip.

Sensing devices have shown tremendous potential for monitoring state-of-the-art organ chip devices. However, challenges like miniaturization while maintaining higher performance, longer operating times for continuous monitoring, and fabrication complexities limit their use. Herein simple, low-cost, and solution-processible inkjet dispenser printing of embedded electrochemical sensors for dissolved oxygen (DO) and reactive oxygen species (ROS) is proposed for monitoring developmental (initially normoxia) and induced hypoxia in a custom-developed gut bilayer microfluidic chip platform for 6 days. The DO sensors showed a high sensitivity of 31.1 nA L mg-1 with a limit of detection (LOD) of 0.67 mg L-1 within the 0-9 mg L-1 range, whereas the ROS sensor had a higher sensitivity of 1.44 nA µm-1 with a limit of detection of 1.7 µm within the 0-300 µm range. The dynamics of the barrier tight junctions are quantified with the help of an in-house developed trans-epithelial-endothelial electrical impedance (TEEI) sensor. Immunofluorescence staining was used to evaluate the expressions of HIF-1α and tight junction protein (TJP) ZO-1. This platform can also be used to enhance bioavailability assays, drug transport studies under an oxygen-controlled environment, and even other barrier organ models, as well as for various applications like toxicity testing, disease modeling and drug screening.

Extracellular Matrix Optimization for Enhanced Physiological Relevance in Hepatic Tissue-Chips.

The cellular microenvironment is influenced explicitly by the extracellular matrix (ECM), the main tissue support biomaterial, as a decisive factor for tissue growth patterns. The recent emergence of hepatic microphysiological systems (MPS) provide the basic physiological emulation of the human liver for drug screening. However, engineering microfluidic devices with standardized surface coatings of ECM may improve MPS-based organ-specific emulation for improved drug screening. The influence of surface coatings of different ECM types on tissue development needs to be optimized. Additionally, an intensity-based image processing tool and transepithelial electrical resistance (TEER) sensor may assist in the analysis of tissue formation capacity under the influence of different ECM types. The current study highlights the role of ECM coatings for improved tissue formation, implying the additional role of image processing and TEER sensors. We studied hepatic tissue formation under the influence of multiple concentrations of Matrigel, collagen, fibronectin, and poly-L-lysine. Based on experimental data, a mathematical model was developed, and ECM concentrations were validated for better tissue development. TEER sensor and image processing data were used to evaluate the development of a hepatic MPS for human liver physiology modeling. Image analysis data for tissue formation was further strengthened by metabolic quantification of albumin, urea, and cytochrome P450. Standardized ECM type for MPS may improve clinical relevance for modeling hepatic tissue microenvironment, and image processing possibly enhance the tissue analysis of the MPS.

Surface-directed engineering of tissue anisotropy in microphysiological models of musculoskeletal tissue.

Here, we present an approach to model and adapt the mechanical regulation of morphogenesis that uses contractile cells as sculptors of engineered tissue anisotropy in vitro. Our method uses heterobifunctional cross-linkers to create mechanical boundary constraints that guide surface-directed sculpting of cell-laden extracellular matrix hydrogel constructs. Using this approach, we engineered linearly aligned tissues with structural and mechanical anisotropy. A multiscale in silico model of the sculpting process was developed to reveal that cell contractility increases as a function of principal stress polarization in anisotropic tissues. We also show that the anisotropic biophysical microenvironment of linearly aligned tissues potentiates soluble factor-mediated tenogenic and myogenic differentiation of mesenchymal stem cells. The application of our method is demonstrated by (i) skeletal muscle arrays to screen therapeutic modulators of acute oxidative injury and (ii) a 3D microphysiological model of lung cancer cachexia to study inflammatory and oxidative muscle injury induced by tumor-derived signals.

Real-time monitoring of liver fibrosis through embedded sensors in a microphysiological system.

Hepatic fibrosis is a foreshadowing of future adverse events like liver cirrhosis, liver failure, and cancer. Hepatic stellate cell activation is the main event of liver fibrosis, which results in excessive extracellular matrix deposition and hepatic parenchyma's disintegration. Several biochemical and molecular assays have been introduced for in vitro study of the hepatic fibrosis progression. However, they do not forecast real-time events happening to the in vitro models. Trans-epithelial electrical resistance (TEER) is used in cell culture science to measure cell monolayer barrier integrity. Herein, we explored TEER measurement's utility for monitoring fibrosis development in a dynamic cell culture microphysiological system. Immortal HepG2 cells and fibroblasts were co-cultured, and transforming growth factor ß1 (TGF-ß1) was used as a fibrosis stimulus to create a liver fibrosis-on-chip model. A glass chip-based embedded TEER and reactive oxygen species (ROS) sensors were employed to gauge the effect of TGF-ß1 within the microphysiological system, which promotes a positive feedback response in fibrosis development. Furthermore, albumin, Urea, CYP450 measurements, and immunofluorescent microscopy were performed to correlate the following data with embedded sensors responses. We found that chip embedded electrochemical sensors could be used as a potential substitute for conventional end-point assays for studying fibrosis in microphysiological systems.

Instantaneous fabrication of arrays of normally closed, adjustable, and reversible nanochannels by tunnel cracking.

A direct fabrication method capable of producing fully-reversible, tunable nanochannel arrays, without the use of a molding step, is described. It is based on tunnel cracking of a readily-prepared brittle layer constrained between elastomeric substrates. The resulting nanochannels have adjustable cross-sections that can be reversibly opened, closed, widened and narrowed merely by applying and removing tensile strains to the substrate. This permits reversible trapping and release of nanoparticles, and easy priming or unclogging of the nanochannels for user-friendly and robust operations. The ease of fabrication and operation required to open and close the nanochannels is superior to previous approaches.

Dynamics of liquid plugs of buffer and surfactant solutions in a micro-engineered pulmonary airway model.

We describe a bioinspired microfluidic system that resembles pulmonary airways and enables on-chip generation of airway occluding liquid plugs from a stratified air-liquid two-phase flow. User-defined changes in the air stream pressure facilitated by mechanical components and tuning the wettability of the microchannels enable generation of well-defined liquid plugs. Significant differences are observed in liquid plug generation and propagation when surfactant is added to the buffer. The plug flow patterns suggest a protective role of surfactant for airway epithelial cells against pathological flow-induced mechanical stresses. We discuss the implications of the findings for clinical settings. This approach and the described platform will enable systematic investigation of the effect of different degrees of fluid mechanical stresses on lung injury at the cellular level and administration of exogenous therapeutic surfactants.

Acoustically detectable cellular-level lung injury induced by fluid mechanical stresses in microfluidic airway systems.

We describe a microfabricated airway system integrated with computerized air-liquid two-phase microfluidics that enables on-chip engineering of human airway epithelia and precise reproduction of physiologic or pathologic liquid plug flows found in the respiratory system. Using this device, we demonstrate cellular-level lung injury under flow conditions that cause symptoms characteristic of a wide range of pulmonary diseases. Specifically, propagation and rupture of liquid plugs that simulate surfactant-deficient reopening of closed airways lead to significant injury of small airway epithelial cells by generating deleterious fluid mechanical stresses. We also show that the explosive pressure waves produced by plug rupture enable detection of the mechanical cellular injury as crackling sounds.

A fluorescent lateral flow biosensor for the quantitative detection of Vaspin using upconverting nanoparticles.

Vaspin is a protein present in human serum that can cause type-2 diabetes, obesity, and other cardiovascular diseases. We report fluorescent upconverting nanoparticles (UCNPs)-based lateral flow biosensor for ultrasensitive detection of Vaspin. A pair (primary and secondary) of cognate aptamers was used that has duo binding with Vaspin. UCNPs with a diameter of around 100 nm were used as a tag to label a detection probe (secondary aptamer). A primary aptamer (capture probe) was immobilized on the test zone. Sandwich type hybridization reactions among the conjugate probe, target Vaspin, and primary aptamer were performed on the lateral flow biosensor. In the presence of target Vaspin, UCNPs were captured on the test zone of the biosensor and the fluorescent intensity of the captured UCNPs was measured through a colorimetric app under NIR. Fluorescence intensity indicates the quantity of Vaspin present in the sample. A range of Vaspin concentration across 0.1-55 ng ml-1 with a Limit of detection (LOD) 39 pg ml-1 was tested through this UCNPs based LFSA with high sensitivity, reproducibility and repeatability, whereas it's actual range in human blood is from 0.1 to 7 ng ml-1. Therefore, this research provides a well-suited lateral flow strip with an ultrasensitive and low-cost approach for the early diagnosis of type-2 diabetes and this could be applied to any targets with a duo of aptamers generated.

Organoids-on-a-chip.

Recent studies have demonstrated an array of stem cell-derived, self-organizing miniature organs, termed organoids, that replicate the key structural and functional characteristics of their in vivo counterparts. As organoid technology opens up new frontiers of research in biomedicine, there is an emerging need for innovative engineering approaches for the production, control, and analysis of organoids and their microenvironment. In this Review, we explore organ-on-a-chip technology as a platform to fulfill this need and examine how this technology may be leveraged to address major technical challenges in organoid research. We also discuss emerging opportunities and future obstacles for the development and application of organoid-on-a-chip technology.

Polydopamine-Based Interfacial Engineering of Extracellular Matrix Hydrogels for the Construction and Long-Term Maintenance of Living Three-Dimensional Tissues.

Diverse biological processes in the body rely on the ability of cells to exert contractile forces on their extracellular matrix (ECM). In three-dimensional (3D) cell culture, however, this intrinsic cellular property can cause unregulated contraction of ECM hydrogel scaffolds, leading to a loss of surface anchorage and the resultant structural failure of in vitro tissue constructs. Despite advances in the 3D culture technology, this issue remains a significant challenge in the development and long-term maintenance of physiological 3D in vitro models. Here, we present a simple yet highly effective and accessible solution to this problem. We leveraged a single-step surface functionalization technique based on polydopamine to drastically increase the strength of adhesion between hydrogel scaffolds and cell culture substrates. Our method is compatible with different types of ECM and polymeric surfaces and also permits prolonged shelf storage of functionalized culture substrates. The proof-of-principle of this technique was demonstrated by the stable long-term (1 month) 3D culture of human lung fibroblasts. Furthermore, we showed the robustness and advanced application of the method by constructing a dynamic cell stretching system and performing over 100 000 cycles of mechanical loading on 3D multicellular constructs for visualization and quantitative analysis of stretch-induced tissue alignment. Finally, we demonstrated the potential of our technique for the development of microphysiological in vitro models by establishing microfluidic 3D co-culture of vascular endothelial cells and fibroblasts to engineer self-assembled, perfusable 3D microvascular beds.

iPreP is a three-dimensional nanofibrillar cellulose hydrogel platform for long-term ex vivo preservation of human islets.

Islet transplantation is an effective therapy for achieving and maintaining normoglycemia in patients with type 1 diabetes mellitus. However, the supply of transplantable human islets is limited. Upon removal from the pancreas, islets rapidly disintegrate and lose function, resulting in a short interval for studies of islet biology and pretransplantation assessment. Here, we developed a biomimetic platform that can sustain human islet physiology for a prolonged period ex vivo. Our approach involved the creation of a multichannel perifusion system to monitor dynamic insulin secretion and intracellular calcium flux simultaneously, enabling the systematic evaluation of glucose-stimulated insulin secretion under multiple conditions. Using this tool, we developed a nanofibrillar cellulose hydrogel-based islet-preserving platform (iPreP) that can preserve islet viability, morphology, and function for nearly 12 weeks ex vivo, and with the ability to ameliorate glucose levels upon transplantation into diabetic hosts. Our platform has potential applications in the prolonged maintenance of human islets, providing an expanded time window for pretransplantation assessment and islet studies.