A bioengineered lung-on-a-chip platform for in vitro modeling and mechanistic investigation of chlorine inhalation toxicology in human lungs

Introduction:

Introduction: Despite remarkable advances in our knowledge of human health and disease, it remains a significant challenge to study and predict how the human body reacts to extreme environments encountered in extraordinary circumstances. Among the best-known examples of such conditions is environmental, occupational, or accidental exposure to gaseous compounds with known or suspected toxicities, which represents a major public health concern. Although studies of human victims have demonstrated the capacity of inhaled toxic gases to induce lung injury and dysfunction with long-term health consequences, the scarcity and unpredictability of such events make it very challenging to acquire human data with sufficient breadth and depth needed to probe, interrogate, and mitigate adverse biological effects of these materials on human lungs. Here, we demonstrate the feasibility of addressing this critical challenge by developing a bioengineered platform that enables in vitro modeling and multidimensional in-depth analysis of how the distal region of the human lung responds to toxic gases using chlorine gas as a model toxicant.

Materials and

Methods: Materials and

Methods: Our microengineered lung models were designed to emulate small airways and alveoli in the distal regions of human lungs. These systems were created in a three-dimensional (3D) microdevice that consists of two layers of microfabricated chambers separated by a thin, semi-permeable membrane with 1μm pores. To construct a small airway model, we established long-term, co-culture of primary human small epithelial cells, primary human pulmonary microvascular endothelial cells, and lung fibroblasts in a physiological microenvironment to engineer a fully differentiated airway epithelium with airspace and the underlying stroma interspersed with fibroblasts and a perfusable network of blood vessels (Figs. 1a, 1b). The alveolar model was generated in the same device and used primary human type II pneumocytes isolated from iPSC-derived human alveolar organoids. These models were treated with computationally predicted physiologically relevant concentrations of chlorine gas using a custom-designed exposure system that permitted precise measurement and controlled delivery of chlorine gas into the epithelial compartment. Responses of our models were examined by various analytical techniques, including immunofluorescence, video microscopy, ELISA, and single-cell RNA sequencing (scRNA-seq).

Results, Conclusions, and Discussions: Results and Discussions: The design principles and tissue engineering techniques described above allowed us to generate vascularized, perfusable human airway and alveolar tissues with high viability (Fig. 1). The bioengineered small airway epithelium exhibited morphological and functional properties of differentiated tissue, including columnar architecture, ciliogenesis and secretion of proteinaceous airway surface liquid, mucociliary clearance, and barrier function (Fig. 1g-l). Similar results indicating tissue-specific differentiation were observed in the alveolar model in which some of type II alveolar epithelial cells cultured in the device transdifferentiated into type I alveolar epithelial cells with physiological phenotype (Fig. 1m-o). In our preliminary study, exposure of these models to chlorine gas elicited various types of adverse biological responses. In the small airway model, for example, our data showed a dose-dependent loss of ciliated cells in the chlorine-treated epithelium (Fig. 2b). The capacity of chlorine gas to induce airway inflammation was demonstrated by the dose-dependent production of various pro-inflammatory cytokines in the effluent of the epithelial and stromal compartments (Fig. 2c). Importantly, scRNA-seq of this model revealed previously unknown differential susceptibility to chlorine-induced tissue injury exhibited by distinct subtypes of ciliated small airway epithelial cells (Fig. 2d). Similar to the small airway system, the alveolar model displayed adverse biological responses indicating acute tissue injury, inflammation, and functional deterioration.

Conclusions: Our preliminary results demonstrate that our bioengineered lung models provide potentially powerful tools for basic and translational research in inhalation toxicology of chlorine and other toxic gases. Further development of this work is underway with the goal of i) developing a more comprehensive, in-depth understanding of chlorine toxicities, ii) identifying specific biomarkers and molecular signatures of chlorine-induced lung injury, and iii) developing and testing effective countermeasures.

Acknowledgements (Optional): Acknowledgements (Optional): This project has been supported in whole or in part with federal funds from the Department of Health and Human Services; Administration for Strategic Preparedness and Response; Biomedical Advanced Research and Development Authority, under contract number 75A50120C00134. The contract and federal funding are not an endorsement of the study results, product, or company.