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.

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.

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.

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.

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.

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.

Primary Ciliary Dyskinesia Patient-Specific hiPSC-Derived Airway Epithelium in Air-Liquid Interface Culture Recapitulates Disease Specific Phenotypes In Vitro.

Primary ciliary dyskinesia (PCD) is a rare heterogenic genetic disorder associated with perturbed biogenesis or function of motile cilia. Motile cilia dysfunction results in diminished mucociliary clearance (MCC) of pathogens in the respiratory tract and chronic airway inflammation and infections successively causing progressive lung damage. Current approaches to treat PCD are symptomatic, only, indicating an urgent need for curative therapeutic options. Here, we developed an in vitro model for PCD based on human induced pluripotent stem cell (hiPSC)-derived airway epithelium in Air-Liquid-Interface cultures. Applying transmission electron microscopy, immunofluorescence staining, ciliary beat frequency, and mucociliary transport measurements, we could demonstrate that ciliated respiratory epithelia cells derived from two PCD patient-specific hiPSC lines carrying mutations in DNAH5 and NME5, respectively, recapitulate the respective diseased phenotype on a molecular, structural and functional level.

Ciliated epithelia are key elements in the recruitment of bacterial partners in the squid-vibrio symbiosis.

The Hawaiian bobtail squid, Euprymna scolopes, harvests its luminous symbiont, Vibrio fischeri, from the surrounding seawater within hours of hatching. During embryogenesis, the host animal develops a nascent light organ with ciliated fields on each lateral surface. We hypothesized that these fields function to increase the efficiency of symbiont colonization of host tissues. Within minutes of hatching from the egg, the host's ciliated fields shed copious amounts of mucus in a non-specific response to bacterial surface molecules, specifically peptidoglycan (PGN), from the bacterioplankton in the surrounding seawater. Experimental manipulation of the system provided evidence that nitric oxide in the mucus drives an increase in ciliary beat frequency (CBF), and exposure to even small numbers of V. fischeri cells for short periods resulted in an additional increase in CBF. These results indicate that the light-organ ciliated fields respond specifically, sensitively, and rapidly, to the presence of nonspecific PGN as well as symbiont cells in the ambient seawater. Notably, the study provides the first evidence that this induction of an increase in CBF occurs as part of a thus far undiscovered initial phase in colonization of the squid host by its symbiont, i.e., host recognition of V. fischeri cues in the environment within minutes. Using a biophysics-based mathematical analysis, we showed that this rapid induction of increased CBF, while accelerating bacterial advection, is unlikely to be signaled by V. fischeri cells interacting directly with the organ surface. These overall changes in CBF were shown to significantly impact the efficiency of V. fischeri colonization of the host organ. Further, once V. fischeri has fully colonized the host tissues, i.e., about 12-24 h after initial host-symbiont interactions, the symbionts drove an attenuation of mucus shedding from the ciliated fields, concomitant with an attenuation of the CBF. Taken together, these findings offer a window into the very first interactions of ciliated surfaces with their coevolved microbial partners.

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.

Stem cells and lung regeneration.

The ability to replace defective cells in an airway with cells that can engraft, integrate, and restore a functional epithelium could potentially cure a number of lung diseases. Progress toward the development of strategies to regenerate the adult lung by either in vivo or ex vivo targeting of endogenous stem cells or pluripotent stem cell derivatives is limited by our fundamental lack of understanding of the mechanisms controlling human lung development, the precise identity and function of human lung stem and progenitor cell types, and the genetic and epigenetic control of human lung fate. In this review, we intend to discuss the known stem/progenitor cell populations, their relative differences between rodents and humans, their roles in chronic lung disease, and their therapeutic prospects. Additionally, we highlight the recent breakthroughs that have increased our understanding of these cell types. These advancements include novel lineage-traced animal models and single-cell RNA sequencing of human airway cells, which have provided critical information on the stem cell subtypes, transition states, identifying cell markers, and intricate pathways that commit a stem cell to differentiate or to maintain plasticity. As our capacity to model the human lung evolves, so will our understanding of lung regeneration and our ability to target endogenous stem cells as a therapeutic approach for lung disease.

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.

Robotic fluidic coupling and interrogation of multiple vascularized organ chips.

Organ chips can recapitulate organ-level (patho)physiology, yet pharmacokinetic and pharmacodynamic analyses require multi-organ systems linked by vascular perfusion. Here, we describe an 'interrogator' that employs liquid-handling robotics, custom software and an integrated mobile microscope for the automated culture, perfusion, medium addition, fluidic linking, sample collection and in situ microscopy imaging of up to ten organ chips inside a standard tissue-culture incubator. The robotic interrogator maintained the viability and organ-specific functions of eight vascularized, two-channel organ chips (intestine, liver, kidney, heart, lung, skin, blood-brain barrier and brain) for 3 weeks in culture when intermittently fluidically coupled via a common blood substitute through their reservoirs of medium and endothelium-lined vascular channels. We used the robotic interrogator and a physiological multicompartmental reduced-order model of the experimental system to quantitatively predict the distribution of an inulin tracer perfused through the multi-organ human-body-on-chips. The automated culture system enables the imaging of cells in the organ chips and the repeated sampling of both the vascular and interstitial compartments without compromising fluidic coupling.

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'.

Reproducing human and cross-species drug toxicities using a Liver-Chip.

Nonclinical rodent and nonrodent toxicity models used to support clinical trials of candidate drugs may produce discordant results or fail to predict complications in humans, contributing to drug failures in the clinic. Here, we applied microengineered Organs-on-Chips technology to design a rat, dog, and human Liver-Chip containing species-specific primary hepatocytes interfaced with liver sinusoidal endothelial cells, with or without Kupffer cells and hepatic stellate cells, cultured under physiological fluid flow. The Liver-Chip detected diverse phenotypes of liver toxicity, including hepatocellular injury, steatosis, cholestasis, and fibrosis, and species-specific toxicities when treated with tool compounds. A multispecies Liver-Chip may provide a useful platform for prediction of liver toxicity and inform human relevance of liver toxicities detected in animal studies to better determine safety and human risk.

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.

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.

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.

Organ-on-a-Chip Systems for Women's Health Applications.

Biomedical research, for a long time, has paid little attention to the influence of sex in many areas of study, ranging from molecular and cellular biology to animal models and clinical studies on human subjects. Many studies solely rely on male cells/tissues/animals/humans, although there are profound differences in male and female physiology, which can significantly impact disease mechanisms, toxicity of compounds, and efficacy of pharmaceuticals. In vitro systems have been traditionally very limited in their capacity to recapitulate female-specific physiology and anatomy such as dynamic sex-hormone levels and the complex interdependencies of female reproductive tract organs. However, the advent of microphysiological organ-on-a-chip systems, which attempt to recreate the 3D structure and function of human organs, now gives researchers the opportunity to integrate cells and tissues from a variety of individuals. Moreover, adding a dynamic flow environment allows mimicking endocrine signaling during the menstrual cycle and pregnancy, as well as providing a controlled microfluidic environment for pharmacokinetic modeling. This review gives an introduction into preclinical and clinical research on women's health and discusses where organ-on-a-chip systems are already utilized or have the potential to deliver new insights and enable entirely new types of studies.

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.

Matched-Comparative Modeling of Normal and Diseased Human Airway Responses Using a Microengineered Breathing Lung Chip.

Smoking represents a major risk factor for chronic obstructive pulmonary disease (COPD), but it is difficult to characterize smoke-induced injury responses under physiological breathing conditions in humans due to patient-to-patient variability. Here, we show that a small airway-on-a-chip device lined by living human bronchiolar epithelium from normal or COPD patients can be connected to an instrument that "breathes" whole cigarette smoke in and out of the chips to study smoke-induced pathophysiology in vitro. This technology enables true matched comparisons of biological responses by culturing cells from the same individual with or without smoke exposure. These studies led to identification of ciliary micropathologies, COPD-specific molecular signatures, and epithelial responses to smoke generated by electronic cigarettes. The smoking airway-on-a-chip represents a tool to study normal and disease-specific responses of the human lung to inhaled smoke across molecular, cellular and tissue-level responses in an organ-relevant context.