A Microengineered Airway Lung Chip Models Key Features of Viral-induced Exacerbation of Asthma.

Viral-induced exacerbation of asthma remains a major cause of hospitalization and mortality. New human-relevant models of the airways are urgently needed to understand how respiratory infections may trigger asthma attacks and to advance treatment development. Here, we describe a new human-relevant model of rhinovirus-induced asthma exacerbation that recapitulates viral infection of asthmatic airway epithelium and neutrophil transepithelial migration, and enables evaluation of immunomodulatory therapy. Specifically, a microengineered model of fully differentiated human mucociliary airway epithelium was stimulated with IL-13 to induce a T-helper cell type 2 asthmatic phenotype and infected with live human rhinovirus 16 (HRV16) to reproduce key features of viral-induced asthma exacerbation. We observed that the infection with HRV16 replicated key hallmarks of the cytopathology and inflammatory responses observed in human airways. Generation of a T-helper cell type 2 microenvironment through exogenous IL-13 stimulation induced features of asthmatic airways, including goblet cell hyperplasia, reduction of cilia beating frequency, and endothelial activation, but did not alter rhinovirus infectivity or replication. High-resolution kinetic analysis of secreted inflammatory markers revealed that IL-13 treatment altered IL-6, IFN-λ1, and CXCL10 secretion in response to HRV16. Neutrophil transepithelial migration was greatest when viral infection was combined with IL-13 treatment, whereas treatment with MK-7123, a CXCR2 antagonist, reduced neutrophil diapedesis in all conditions. In conclusion, our microengineered Airway Lung-Chip provides a novel human-relevant platform for exploring the complex mechanisms underlying viral-induced asthma exacerbation. Our data suggest that IL-13 may impair the hosts' ability to mount an appropriate and coordinated immune response to rhinovirus infection. We also show that the Airway Lung-Chip can be used to assess the efficacy of modulators of the immune response.

Motile cilia create fluid-mechanical microhabitats for the active recruitment of the host microbiome.

We show that mucociliary membranes of animal epithelia can create fluid-mechanical microenvironments for the active recruitment of the specific microbiome of the host. In terrestrial vertebrates, these tissues are typically colonized by complex consortia and are inaccessible to observation. Such tissues can be directly examined in aquatic animals, providing valuable opportunities for the analysis of mucociliary activity in relation to bacteria recruitment. Using the squid-vibrio model system, we provide a characterization of the initial engagement of microbial symbionts along ciliated tissues. Specifically, we developed an empirical and theoretical framework to conduct a census of ciliated cell types, create structural maps, and resolve the spatiotemporal flow dynamics. Our multiscale analyses revealed two distinct, highly organized populations of cilia on the host tissues. An array of long cilia ([Formula: see text]25 [Formula: see text]m) with metachronal beat creates a flow that focuses bacteria-sized particles, at the exclusion of larger particles, into sheltered zones; there, a field of randomly beating short cilia ([Formula: see text]10 [Formula: see text]m) mixes the local fluid environment, which contains host biochemical signals known to prime symbionts for colonization. This cilia-mediated process represents a previously unrecognized mechanism for symbiont recruitment. Each mucociliary surface that recruits a microbiome such as the case described here is likely to have system-specific features. However, all mucociliary surfaces are subject to the same physical and biological constraints that are imposed by the fluid environment and the evolutionary conserved structure of cilia. As such, our study promises to provide insight into universal mechanisms that drive the recruitment of symbiotic partners.

Flow Physics Explains Morphological Diversity of Ciliated Organs.

Organs that pump fluids by the coordinated beat of motile cilia through the lumen are integral to animal physiology. Such organs include the human airways, brain ventricles, and reproductive tracts. Although cilia organization and duct morphology vary drastically in the animal kingdom, ducts are typically classified as either carpet or flame designs. The reason behind this dichotomy and how duct design relates to fluid pumping remain unclear. Here, we demonstrate that two structural parameters -- lumen diameter and cilia-to-lumen ratio -- organize the observed duct diversity into a continuous spectrum that connects carpets to flames across all animal phyla. Using a unified fluid model, we show that carpet and flame designs maximize flow rate and pressure generation, respectively. We propose that convergence of ciliated organ designs follows functional constraints rather than phylogenetic distance, along with universal design rules for ciliary pumps.

STRUCTURE-FUNCTION RELATIONSHIPS OF MUCOCILIARY CLEARANCE IN HUMAN AIRWAYS.

Mucociliary clearance is a key mechanical defense mechanism of human airways, and clearance failure is linked to major respiratory diseases, such as chronic obstructive pulmonary disease (COPD) and asthma. While single-cell transcriptomics have unveiled the cellular complexity of the human airway epithelium, our understanding of the mechanics that link epithelial structure to clearance function mainly stem from animal models. This reliance on animal data limits crucial insights into human airway barrier function and hampers the human-relevant in vitro modeling of airway diseases. Our study fills this crucial knowledge gap and for the first time (1) maps the distribution of ciliated and secretory cell types on the mucosal surface along the proximo-distal axis of the rat and human airway tree, (2) identifies species-specific differences in ciliary beat and clearance function, and (3) elucidates structural parameters of airway epithelia that predict clearance function in both native and in vitro tissues alike. Our broad range of experimental approaches and physics-based modeling translate into generalizable parameters to quantitatively benchmark the human-relevancy of mucociliary clearance in experimental models, and to characterize distinct disease states.

STRUCTURE-FUNCTION RELATIONSHIPS OF MUCOCILIARY CLEARANCE IN HUMAN AIRWAYS.

Our study focuses on the intricate connection between tissue-level organization and ciliated organ function in humans, particularly in understanding the morphological organization of airways and their role in mucociliary clearance. Mucociliary clearance is a key mechanical defense mechanism of human airways, and clearance failure is associated with many respiratory diseases, including chronic obstructive pulmonary disease (COPD) and asthma. While single-cell transcriptomics have unveiled the cellular complexity of the human airway epithelium, our understanding of the mechanics that link epithelial structure to clearance function mainly stem from animal models. This reliance on animal data limits crucial insights into human airway barrier function and hampers the human-relevant in vitro modeling of airway diseases. This study, for the first time, maps the distribution of ciliated and secretory cell types along the airway tree in both rats and humans, noting species-specific differences in ciliary function and elucidates structural parameters of airway epithelia that predict clearance function in both native and in vitro tissues alike. By uncovering how tissue organization influences ciliary function, we can better understand disruptions in mucociliary clearance, which could have implications for various ciliated organs beyond the airways.

Spontaneous body wall contractions stabilize the fluid microenvironment that shapes host-microbe associations.

The freshwater polyp Hydra is a popular biological model system; however, we still do not understand one of its most salient behaviors, the generation of spontaneous body wall contractions. Here, by applying experimental fluid dynamics analysis and mathematical modeling, we provide functional evidence that spontaneous contractions of body walls enhance the transport of chemical compounds from and to the tissue surface where symbiotic bacteria reside. Experimentally, a reduction in the frequency of spontaneous body wall contractions is associated with a changed composition of the colonizing microbiota. Together, our findings suggest that spontaneous body wall contractions create an important fluid transport mechanism that (1) may shape and stabilize specific host-microbe associations and (2) create fluid microhabitats that may modulate the spatial distribution of the colonizing microbes. This mechanism may be more broadly applicable to animal-microbe interactions since research has shown that rhythmic spontaneous contractions in the gastrointestinal tracts are essential for maintaining normal microbiota.

Sorafenib inhibits invasion of multicellular organoids that mimic Lymphangioleiomyomatosis nodules.

Lymphangioleiomyomatosis (LAM) is a debilitating, progressive lung disease with few therapeutic options, largely due to a paucity of mechanistic knowledge of disease pathogenesis. Lymphatic endothelial cells (LECs) are known to envelope and invade clusters of LAM-cells, comprising of smooth muscle α-actin and/or HMB-45 positive "smooth muscle-like cells" however the role of LECs in LAM pathogenesis is still unknown. To address this critical knowledge gap, we investigated wether LECs interact with LAM-cells to augment their metastatic behaviour of LAM-cells. We performed in situ spatialomics and identified a core of transcriptomically related cells within the LAM nodules. Pathway analysis highlights wound and pulmonary healing, VEGF signaling, extracellular matrix/actin cytoskeletal regulating and the HOTAIR regulatory pathway enriched in the LAM Core cells. We developed an organoid co-culture model combining primary LAM-cells with LECs and applied this to evaluate invasion, migration, and the impact of Sorafenib, a multi-kinase inhibitor. LAM-LEC organoids had significantly higher extracellular matrix invasion, decreased solidity and a greater perimeter, reflecting increased invasion compared to non-LAM control smooth muscle cells. Sorafenib significantly inhibited this invasion in both LAM spheroids and LAM-LEC organoids compared to their respective controls. We identified TGFß1ι1, a molecular adapter coordinating protein-protein interactions at the focal adhesion complex and known to regulate VEGF, TGFß and Wnt signalling, as a Sorafenib-regulated kinase in LAM-cells. In conclusion we have developed a novel 3D co-culture LAM model and have demonstrated the effectiveness of Sorafenib to inhibit LAM-cell invasion, identifying new avenues for therapeutic intervention.

Breathing on chip: Dynamic flow and stretch accelerate mucociliary maturation of airway epithelium in vitro.

Human lung function is intricately linked to blood flow and breathing cycles, but it remains unknown how these dynamic cues shape human airway epithelial biology. Here we report a state-of-the-art protocol for studying the effects of dynamic medium and airflow as well as stretch on human primary airway epithelial cell differentiation and maturation, including mucociliary clearance, using an organ-on-chip device. Perfused epithelial cell cultures displayed accelerated maturation and polarization of mucociliary clearance, and changes in specific cell-types when compared to traditional (static) culture methods. Additional application of airflow and stretch to the airway chip resulted in an increase in polarization of mucociliary clearance towards the applied flow, reduced baseline secretion of interleukin-8 and other inflammatory proteins, and reduced gene expression of matrix metalloproteinase (MMP) 9, fibronectin, and other extracellular matrix factors. These results indicate that breathing-like mechanical stimuli are important modulators of airway epithelial cell differentiation and maturation and that their fine-tuned application could generate models of specific epithelial pathologies, including mucociliary (dys)function.

Modeling alcohol-associated liver disease in a human Liver-Chip.

Alcohol-associated liver disease (ALD) is a global health issue and leads to progressive liver injury, comorbidities, and increased mortality. Human-relevant preclinical models of ALD are urgently needed. Here, we leverage a triculture human Liver-Chip with biomimetic hepatic sinusoids and bile canaliculi to model ALD employing human-relevant blood alcohol concentrations (BACs) and multimodal profiling of clinically relevant endpoints. Our Liver-Chip recapitulates established ALD markers in response to 48 h of exposure to ethanol, including lipid accumulation and oxidative stress, in a concentration-dependent manner and supports the study of secondary insults, such as high blood endotoxin levels. We show that remodeling of the bile canalicular network can provide an in vitro quantitative readout of alcoholic liver toxicity. In summary, we report the development of a human ALD Liver-Chip as a powerful platform for modeling alcohol-induced liver injury with the potential for direct translation to clinical research and evaluation of patient-specific responses.

Multiscale mechanics of mucociliary clearance in the lung.

Mucociliary clearance (MCC) is one of the most important defence mechanisms of the human respiratory system. Its failure is implicated in many chronic and debilitating airway diseases. However, due to the complexity of lung organization, we currently lack full understanding on the relationship between these regional differences in anatomy and biology and MCC functioning. For example, it is unknown whether the regional variability of airway geometry, cell biology and ciliary mechanics play a functional role in MCC. It therefore remains unclear whether the regional preference seen in some airway diseases could originate from local MCC dysfunction. Though great insights have been gained into the genetic basis of cilia ultrastructural defects in airway ciliopathies, the scaling to regional MCC function and subsequent clinical phenotype remains unpredictable. Understanding the multiscale mechanics of MCC would help elucidate genotype-phenotype relationships and enable better diagnostic tools and treatment options. Here, we review the hierarchical and variable organization of ciliated airway epithelium in human lungs and discuss how this organization relates to MCC function. We then discuss the relevancy of these structure-function relationships to current topics in lung disease research. Finally, we examine how state-of-the-art computational approaches can help address existing open questions. This article is part of the Theo Murphy meeting issue 'Unity and diversity of cilia in locomotion and transport'.

Stem cell-based Lung-on-Chips: The best of both worlds?

Pathologies of the respiratory system such as lung infections, chronic inflammatory lung diseases, and lung cancer are among the leading causes of morbidity and mortality, killing one in six people worldwide. Development of more effective treatments is hindered by the lack of preclinical models of the human lung that can capture the disease complexity, highly heterogeneous disease phenotypes, and pharmacokinetics and pharmacodynamics observed in patients. The merger of two novel technologies, Organs-on-Chips and human stem cell engineering, has the potential to deliver such urgently needed models. Organs-on-Chips, which are microengineered bioinspired tissue systems, recapitulate the mechanochemical environment and physiological functions of human organs while concurrent advances in generating and differentiating human stem cells promise a renewable supply of patient-specific cells for personalized and precision medicine. Here, we discuss the challenges of modeling human lung pathophysiology in vitro, evaluate past and current models including Organs-on-Chips, review the current status of lung tissue modeling using human pluripotent stem cells, explore in depth how stem-cell based Lung-on-Chips may advance disease modeling and drug testing, and summarize practical consideration for the design of Lung-on-Chips for academic and industry applications.

An energy budget for the olfactory glomerulus.

Energy demands are becoming recognized as an important constraint on neural signaling. The olfactory glomerulus provides a well defined system for analyzing this question. Odor stimulation elicits high-energy demands in olfactory glomeruli where olfactory axons converge onto dendrites of olfactory bulb neurons. We performed a quantitative analysis of the energy demands of each type of neuronal element within the glomerulus. This included the volumes of each element, their surface areas, and ion loads associated with membrane potentials and synaptic activation as constrained by experimental observations. In the resting state, there was a high-energy demand compared with other brain regions because of the high density of neural elements. The activated state was dominated by the energy demands of action potential propagation in afferent olfactory sensory neurons and their synaptic input to dendritic tufts, whereas subsequent dendritic potentials and dendrodendritic transmission contributed only a minor share of costs. It is proposed therefore that afferent input and axodendritic transmission account for the strong signals registered by 2-deoxyglucose and functional magnetic resonance imaging, although postsynaptic dendrites comprise at least one-half of the volume of the glomerulus. The results further suggest that presynaptic inhibition of the axon terminals by periglomerular cells plays an important role in limiting the range of excitation of the postsynaptic cells. These results provide a new quantitative basis for interpreting olfactory bulb activation patterns elicited by odor stimulation.

Automated fabrication of photopatterned gelatin hydrogels for organ-on-chips applications.

Organ-on-chip platforms aim to improve preclinical models for organ-level responses to novel drug compounds. Heart-on-a-chip assays in particular require tissue engineering techniques that rely on labor-intensive photolithographic fabrication or resolution-limited 3D printing of micropatterned substrates, which limits turnover and flexibility of prototyping. We present a rapid and automated method for large scale on-demand micropatterning of gelatin hydrogels for organ-on-chip applications using a novel biocompatible laser-etching approach. Fast and automated micropatterning is achieved via photosensitization of gelatin using riboflavin-5'phosphate followed by UV laser-mediated photoablation of the gel surface in user-defined patterns only limited by the resolution of the 15 µm wide laser focal point. Using this photopatterning approach, we generated microscale surface groove and pillar structures with feature dimensions on the order of 10-30 µm. The standard deviation of feature height was 0.3 µm, demonstrating robustness and reproducibility. Importantly, the UV-patterning process is non-destructive and does not alter gelatin micromechanical properties. Furthermore, as a quality control step, UV-patterned heart chip substrates were seeded with rat or human cardiac myocytes, and we verified that the resulting cardiac tissues achieved structural organization, contractile function, and long-term viability comparable to manually patterned gelatin substrates. Start-to-finish, UV-patterning shortened the time required to design and manufacture micropatterned gelatin substrates for heart-on-chip applications by up to 60% compared to traditional lithography-based approaches, providing an important technological advance enroute to automated and continuous manufacturing of organ-on-chips.

A linked organ-on-chip model of the human neurovascular unit reveals the metabolic coupling of endothelial and neuronal cells.

The neurovascular unit (NVU) regulates metabolic homeostasis as well as drug pharmacokinetics and pharmacodynamics in the central nervous system. Metabolic fluxes and conversions over the NVU rely on interactions between brain microvascular endothelium, perivascular pericytes, astrocytes and neurons, making it difficult to identify the contributions of each cell type. Here we model the human NVU using microfluidic organ chips, allowing analysis of the roles of individual cell types in NVU functions. Three coupled chips model influx across the blood-brain barrier (BBB), the brain parenchymal compartment and efflux across the BBB. We used this linked system to mimic the effect of intravascular administration of the psychoactive drug methamphetamine and to identify previously unknown metabolic coupling between the BBB and neurons. Thus, the NVU system offers an in vitro approach for probing transport, efficacy, mechanism of action and toxicity of neuroactive drugs.

Laminar ventricular myocardium on a microelectrode array-based chip.

Pharmaceutical screening based on human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and multi electrode arrays (MEAs) have been proposed as a complementary method for electrophysiological safety and efficacy assessment in drug discovery and development. Contrary to animal models, these cells offer a human genetic background but, at present, fail to recapitulate the mechanical and structural properties of the native human myocardium. Here, we report that topographical cues on soft micromolded gelatin can coax hiPSC-CMs to form laminar cardiac tissues that resemble the native architecture of the heart. Importantly, using this method we were able to record tissue-level electrophysiological responses with a commercially available MEA setup. To validate this platform, we recorded cardiac field potentials at baseline and after pharmacological interventions with a ß-adrenergic agonist (isoproterenol). Further, we tested the ability of our system to predict the response of laminar human cardiac tissues to a cardiotoxic pro-drug (terfenadine) and its non-cardiotoxic metabolite (fexofenadine). Finally, we integrated our platform with microfluidic components to build a heart-on-a-chip system that can be fluidically linked with other organs-on-chips in the future.

Design standards for engineered tissues.

Traditional technologies are required to meet specific, quantitative standards of safety and performance. In tissue engineering, similar standards will have to be developed to enable routine clinical use and customized tissue fabrication. In this essay, we discuss a framework of concepts leading towards general design standards for tissue-engineering, focusing in particular on systematic design strategies, control of cell behavior, physiological scaling, fabrication modes and functional evaluation.

Modeling of cardiac muscle thin films: pre-stretch, passive and active behavior.

Recent progress in tissue engineering has made it possible to build contractile bio-hybrid materials that undergo conformational changes by growing a layer of cardiac muscle on elastic polymeric membranes. Further development of such muscular thin films for building actuators and powering devices requires exploring several design parameters, which include the alignment of the cardiac myocytes and the thickness/Young's modulus of elastomeric film. To more efficiently explore these design parameters, we propose a 3-D phenomenological constitutive model, which accounts for both the passive deformation including pre-stretch and the active behavior of the cardiomyocytes. The proposed 3-D constitutive model is implemented within a finite element framework, and can be used to improve the current design of bio-hybrid thin films and help developing bio-hybrid constructs capable of complex conformational changes.

A tissue-engineered jellyfish with biomimetic propulsion.

Reverse engineering of biological form and function requires hierarchical design over several orders of space and time. Recent advances in the mechanistic understanding of biosynthetic compound materials, computer-aided design approaches in molecular synthetic biology 4,5 and traditional soft robotics, and increasing aptitude in generating structural and chemical micro environments that promote cellular self-organization have enhanced the ability to recapitulate such hierarchical architecture in engineered biological systems. Here we combined these capabilities in a systematic design strategy to reverse engineer a muscular pump. We report the construction of a freely swimming jellyfish from chemically dissociated rat tissue and silicone polymer as a proof of concept. The constructs, termed 'medusoids', were designed with computer simulations and experiments to match key determinants of jellyfish propulsion and feeding performance by quantitatively mimicking structural design, stroke kinematics and animal-fluid interactions. The combination of the engineering design algorithm with quantitative benchmarks of physiological performance suggests that our strategy is broadly applicable to reverse engineering of muscular organs or simple life forms that pump to survive.

High-resolution three-dimensional extracellular recording of neuronal activity with microfabricated electrode arrays.

Microelectrode array recordings of neuronal activity present significant opportunities for studying the brain with single-cell and spike-time precision. However, challenges in device manufacturing constrain dense multisite recordings to two spatial dimensions, whereas access to the three-dimensional (3D) structure of many brain regions appears to remain a challenge. To overcome this limitation, we present two novel recording modalities of silicon-based devices aimed at establishing 3D functionality. First, we fabricated a dual-side electrode array by patterning recording sites on both the front and back of an implantable microstructure. We found that the majority of single-unit spikes could not be simultaneously detected from both sides, suggesting that in addition to providing higher spatial resolution measurements than that of single-side devices, dual-side arrays also lead to increased recording yield. Second, we obtained recordings along three principal directions with a multilayer array and demonstrated 3D spike source localization within the enclosed measurement space. The large-scale integration of such dual-side and multilayer arrays is expected to provide massively parallel recording capabilities in the brain.