______________________________________________________________________Development of new therapeutics for chronic respiratory diseases, such as asthma and chronic obstructive pulmonary disease (COPD), which pose a huge public health burden 1 , have been hindered by the inability to study organ-level complexities of lung inflammation in vitro. While hospitalization and mortality due to these diseases are often the consequences of exacerbations triggered by pathogens 2, 3 , there is currently no way to study these processes in human lung outside of the clinical setting. Animal models of asthma and COPD exist; however, their clinical relevance is questionable because the anatomy, immune system and inflammatory responses exhibited by animal lungs differ greatly from those in humans [4][5][6] . For example, mucin-producing cells, which are central to the development of asthma, are less frequent in the respiratory tree of mice and rats compared with humans 6 . Neutrophils that increase dramatically in the lungs of patients with COPD and severe asthma 7-9 also comprise only 10-25% of circulating leukocytes in mice, whereas they represent 50-70% in humans 5 . Because many animal models fail to predict drug activities in humans, the pharmaceutical and biotechnology industries strive to reduce or replace animal models for drug testing whenever possible 10 .Airway inflammatory diseases have been modeled in vitro using cultures of primary or immortalized human epithelial cells, sometimes positioned at an air-liquid interface to induce epithelial differentiation 11 or using co-cultures of airway epithelium and tissue-resident immune cells (e.g., macrophages or dendritic cells) 12 . However, lung inflammation is mediated by organ-level responses that involve complex tissuetissue interactions between the lung airway epithelium and underlying microvascular endothelium that modulate immune reactions to respiratory pathogens and allergens [13][14][15] and alter the vascular cell adhesion molecular machinery that recruits circulating immune cells, such as neutrophils. This is important because neutrophil accumulation in the lung is associated with enhanced severity of airflow limitation in COPD patients 7 and it plays a critical role in severe asthma as well 8 . Unfortunately, it is not possible to study complex interactions among airway epithelium, endothelium and circulating neutrophils using existing in vitro lung models because most fail to recapitulate normal functional coupling between the epithelium and endothelium, and none enable analysis of recruitment of circulating immune cells under active fluid flow. This latter point is crucial because neutrophil adhesion to inflamed endothelium involves initial rolling along the luminal surface of endothelium mediated by E-selectin, which is then followed by firm adhesion to and this dynamic shear stress-dependent response cannot be studied in a physiologically relevant way using static cell cultures.Advances in microsystems engineering have recently made it possible to create bio...

The ultimate goal of most biomedical research is to gain greater insight into mechanisms of human disease or to develop new and improved therapies or diagnostics. Although great advances have been made in terms of developing disease models in animals, such as transgenic mice, many of these models fail to faithfully recapitulate the human condition. In addition, it is difficult to identify critical cellular and molecular contributors to disease or to vary them independently in whole-animal models. This challenge has attracted the interest of engineers, who have begun to collaborate with biologists to leverage recent advances in tissue engineering and microfabrication to develop novel in vitro models of disease. As these models are synthetic systems, specific molecular factors and individual cell types, including parenchymal cells, vascular cells, and immune cells, can be varied independently while simultaneously measuring system-level responses in real time. In this article, we provide some examples of these efforts, including engineered models of diseases of the heart, lung, intestine, liver, kidney, cartilage, skin and vascular, endocrine, musculoskeletal, and nervous systems, as well as models of infectious diseases and cancer. We also describe how engineered in vitro models can be combined with human inducible pluripotent stem cells to enable new insights into a broad variety of disease mechanisms, as well as provide a test bed for screening new therapies.

Trans-Epithelial Electrical Resistance (TEER) is broadly used as an experimental readout and a quality control assay for measuring the integrity of epithelial monolayers cultured under static conditions in vitro, however, there is no standard methodology for its application to microfluidic Organ-on-a-Chip (Organ Chip) cultures. Here, we describe a new microfluidic Organ Chip design that contains embedded electrodes, and we demonstrate its utility for assessing formation and disruption of barrier function both within a human Lung Airway Chip lined by a fully differentiated mucociliary human airway epithelium and in a human Gut Chip lined by intestinal epithelial cells. These chips with integrated electrodes enable real-time, non-invasive monitoring of TEER and can be applied to measure barrier function in virtually any type of cultured cell.

Respiratory syncytial virus (RSV) is the major viral cause of severe pulmonary disease in young infants worldwide. However, the mechanisms by which RSV causes disease in humans remain poorly understood. To help bridge this gap, we developed an ex vivo/in vitro model of RSV infection based on well-differentiated primary pediatric bronchial epithelial cells (WD-PBECs), the primary targets of RSV infection in vivo. Our RSV/WD-PBEC model demonstrated remarkable similarities to hallmarks of RSV infection in infant lungs. These hallmarks included restriction of infection to noncontiguous or small clumps of apical ciliated and occasional nonciliated epithelial cells, apoptosis and sloughing of apical epithelial cells, occasional syncytium formation, goblet cell hyperplasia/metaplasia, and mucus hypersecretion. RSV was shed exclusively from the apical surface at titers consistent with those in airway aspirates from hospitalized infants. Furthermore, secretion of proinflammatory chemokines such as CXCL10, CCL5, IL-6, and CXCL8 reflected those chemokines present in airway aspirates. Interestingly, a recent RSV clinical isolate induced more cytopathogenesis than the prototypic A2 strain. Our findings indicate that this RSV/WD-PBEC model provides an authentic surrogate for RSV infection of airway epithelium in vivo. As such, this model may provide insights into RSV pathogenesis in humans that ultimately lead to successful RSV vaccines or therapeutics.

Analysis of enterovirus infection is difficult in animals because they express different virus receptors than humans, and static cell culture systems do not reproduce the physical complexity of the human intestinal epithelium. Here, using coxsackievirus B1 (CVB1) as a prototype enterovirus strain, we demonstrate that human enterovirus infection, replication and infectious virus production can be analyzed in vitro in a human Gut-on-a-Chip microfluidic device that supports culture of highly differentiated human villus intestinal epithelium under conditions of fluid flow and peristalsis-like motions. When CVB1 was introduced into the epithelium-lined intestinal lumen of the device, virions entered the epithelium, replicated inside the cells producing detectable cytopathic effects (CPEs), and both infectious virions and inflammatory cytokines were released in a polarized manner from the cell apex, as they could be detected in the effluent from the epithelial microchannel. When the virus was introduced via a basal route of infection (by inoculating virus into fluid flowing through a parallel lower ‘vascular’ channel separated from the epithelial channel by a porous membrane), significantly lower viral titers, decreased CPEs, and delayed caspase-3 activation were observed; however, cytokines continued to be secreted apically. The presence of continuous fluid flow through the epithelial lumen also resulted in production of a gradient of CPEs consistent with the flow direction. Thus, the human Gut-on-a-Chip may provide a suitable in vitro model for enteric virus infection and for investigating mechanisms of enterovirus pathogenesis.