Advanced lung-on-a-chip

Abstract

Provided is a fluidic chip, comprising: a bottom region, the bottom region comprising a central channel and at least one side channel adjacent thereto, the central channel having therein a plurality of cells disposed in a matrix; and a middle region, the middle region having a chamber defined therein, the chamber optionally being an open-top chamber, the chamber being in register with at least one of the central channel and the at least one side channel of the bottom region. Also provided is a method, comprising co-culturing airway epithelial cells, pulmonary microvascular endothelial cells, and lung fibroblasts using a fluidic chip; the airway epithelial cells comprising airway cells or alveolar cells. Also disclosed is causing contact between (1) at least one of an agent and a stimulus, and (2) epithelial cells disposed within the chamber of a fluidic chip, and monitoring at least one marker indicative of exposure of the epithelial cells to the sample.

Description

103241.024775 / 22-10103ADVANCED LUNG-ON- A-CHIPRELATED APPLICATIONS

[0001] The present application claims priority to and the benefit of United States patent application no. 63 / 702,024, “Advanced Lung-On- A-Chip” (filed October 1, 2024). All foregoing applications are incorporated herein by reference in their entireties for any and all purposes.GOVERNMENT RIGHTS

[0002] This invention was made with Government support under 75A50120C00134 awarded by Biomedical Advanced Research and Development Authority. The Government has certain rights in the invention.TECHNICAL FIELD

[0003] The present disclosure relates to the field of lung-on-a-chip devices.BACKGROUND

[0004] Lung-on-a-chips have emerged as an alternative to animal testing in the biotechnology and pharmaceutical companies. A lung-on-a-chip can also be used in the development of medical countermeasures against chemical, biological, radiological, and nuclear (CBRN) since some of these medical countermeasures are specific to humans and can not be effectively study in animals. In order to be able to fully harness the power of organs-on-a chip, these chips should emulate structural and functional complexity of the living tissues within human organs. During lung development in humans, after respiratory cell fate has established, the lung forms a tree-like structure consists of epithelial tubules (airway and alveoli) and a vascular system. Because existing lung-on-a-chip designs can have suboptimal characteristics in some cases, there is a long-felt need in the art for improved lung-on-a-chip designs.103241.024775 / 22-10103SUMMARY

[0005] In meeting the described long-felt needs, the present disclosure provides a lung-on-a-chip fluidic chip, comprising: a bottom region, the bottom region comprising a central channel and at least one side channel adjacent thereto, the central channel having therein a plurality of cells disposed in a matrix; and a middle region, the middle region having a chamber defined therein, the chamber optionally being an open-top chamber, the chamber being in register with at least one of the central channel and the at least one side channel of the bottom region.

[0006] Also provided is a method, comprising co-culturing airway epithelial cells, pulmonary microvascular endothelial cells, and lung fibroblasts using a fluidic chip according to the present disclosure, the airway epithelial cells comprising airway cells or alveolar cells.

[0007] Further provided is a system, the system comprising: a fluidic chip according to the present disclosure (e.g., according to any one of Aspects 1-13), the system adapted to cause contact between the fluidic chip and at least one of a stimulus and an agent, the contact optionally being interruptable, the agent optionally comprising any one or more of a toxicant and a stimulant, and the stimulus optionally comprising a radiation.

[0008] Also disclosed is a method, comprising: causing contact between (1) at least one of an agent and a stimulus, and (2) epithelial cells disposed within the chamber of a fluidic chip according to the present disclosure (e.g., according to any one of Aspects 1-13); and monitoring at least one marker indicative of exposure of the epithelial cells to the at least one of an agent and a stimulus.BRIEF DESCRIPTION OF THE DRAWINGS

[0009] In the drawings, which are not necessarily drawn to scale, like numerals may describe similar components in different views. Like numerals having different letter suffixes may represent different instances of similar components. The drawings illustrate generally, by way of example, but not by way of limitation, various aspects discussed in the present document. In the drawings:

[0010] FIG. 1 provides an exemplary fabrication of a device according to the present disclosure. As shown, a cell culture layer can be formed, with inlets formed to103241.024775 / 22-10103 connect to the channels in the cell culture layer. A media reservoir layer can also be used, with media reservoirs being in fluid communication with the cell culture layer.

[0011] FIG. 2 provides a looking-down view of a chip according to the present disclosure. As shown, a chip can include media reservoirs, vascular channels, a gel channel, and an air-liquid interface at a chamber disposed above the gel channel. On the right side of the figure are shown depictions of a vascular channel, the gel channel (showing fibroblasts in a gel), and epithelial cells, which can be present in the chamber.

[0012] FIG. 3 provides a side view of a chip according to the present disclosure. As shown, a chip can include at least one vascular channel, which at least one vascular channel can flank a gel channel or central channel. As shown, a rail (which can also be termed a ridge) can be placed between the central channel and the side channel. A chamber (not labeled in FIG. 3) can be disposed above the central channel, and a separator (which can be a membrane) can be disposed between the chamber and the central channel. Epithelial cells can be disposed within the chamber, as shown; these cells can be placed on the separator. An air-liquid interface can be present at the chamber, as shown. Also as shown, the chamber can be an open-top chamber, which allows for introduction of agents or other materials to the chamber.

[0013] FIG. 4 provides exemplary lung tissue, showing the large airway, the small airway, and alveolar tissue.

[0014] FIG. 5 depicts an example device according to the present disclosure. As shown, a device can include a plurality of open-top culture chambers and their associated fluid access ports, which chambers can be aligned with the wells on a well plate.

[0015] FIG. 6 illustrates an example perfusable network formed using a device according to the present disclosure.

[0016] FIG. 7 provides an example image of the differentiation of large airway cells (ciliogenesis), using the disclosed technology.

[0017] FIG. 8 provides an example image of the differentiation of large airway cells (MUC5A+cells), using the disclosed technology.

[0018] FIG. 9 provides an example image of the differentiation of large airway cells (basal cells), using the disclosed technology.

[0019] FIG. 10 provides exemplary data showing the kinetics of differentiation in large airway cells.103241.024775 / 22-10103

[0020] FIG. 11 provides an example image of the differentiation of small airway cells (ciliogenesis), using the disclosed technology.

[0021] FIGs. 12A-12O. Bioengineered models of human lung tissues, a and b. Schematics depicting an airway-on-a-chip engineered by using cultured primary human cells to replicate the salient features of bronchioles in the human lung. c-f. Self-assembled perfusable pulmonary microvasculature in the stromal compartment of the bioengineered airway tissue, (e) Scanning electron micrograph showing the open lumen of the microvasculature, (f) Human red blood cells (RBCs) flowing in the engineered blood vessel, g-k. A single layer of primary epithelial cells is seeded on the permeable membrane in the top compartment. Tissue differentiation is demonstrated through TEM, SEM, and immunofluorescence microscopy. 1. Single-cell RNA-sequencing data show differentiated epithelial cell populations, m-o. Alveolar-chips constructed using iPSC- derived human alveolar organoids.

[0022] FIGs. 13A-13E. Responses of lung-on-a-chip models to chlorine gas. a. Photo of an in-house chlorine exposure system used in our study, b. Examination of ciliated cell populations via immunostaining after exposure to varying chlorine concentrations (10, 25, 50, and 100 PPM), c. Assessment of cytotoxicity through LDH assays and comparative analysis of cytokine levels (IL- 1 p, IL-6, TNF-a, IL-8, and TGF- pi) in media collected from both the epithelial (top compartment) and stromal / vascular (bottom compartment) layers, d. Transcriptomic analysis of airway cells post-chlorine treatment, e. Gene Set Enrichment Analysis (GSEA) focusing on Hallmark gene sets, revealing upregulation of inflammatory responses in the epithelial layer.

[0023] FIGs 14A-14Q. Design and characterization of a vascularized small airway-on-a-chip. (a) Schematic illustration of the human distal airway showing epithelial, stromal, and vascular compartments, (b) Overview of the airway chip platform with integrated epithelial chamber and perfusable stromal compartment, (c) Exploded view of device layers showing separation of epithelial and stromal regions by a porous membrane, (d) Sequential steps of stromal and endothelial seeding followed by epithelial differentiation under air liquid interface, (e-g) Endothelial network formation over seven days, shown by CD31 staining and quantification of vessel diameter, branching, and length, (h-j) High resolution confocal and scanning electron microscopy of self assembled vascular networks, with dextran perfusion confirming vessel patency, (k-m) Three103241.024775 / 22-10103 dimensional rendering and ultrastructural imaging of luminal morphology, (n-p) Differentiation of the epithelial compartment under air liquid interface showing mucociliary architecture with ciliated cells (alpha tubulin), goblet cells (MUC5AC), club cells (CC10), and basal cells (P63). (o) Time course of epithelial maturation over one, two, and three weeks showing progressive tight junction formation (ZO1) and actin organization, (q) Single cell RNA sequencing of epithelial, endothelial, and stromal populations showing transcriptional clustering into basal, ciliated, goblet, club, intermediate, endothelial, and fibroblast lineages. Data are presented as mean ± SD with n = 4. ns: not significant, *P < 0.05, **P < 0.01, ***p < 0.001, and **** P < 0.0001

[0024] FIGs. 15F-15J. Vascularization enhances epithelial maturation and alters cell-cell signaling dynamics.(f) Confocal micrographs comparing airway tissues engineered without (top) or with (bottom) a perfused microvascular network. CD31 (green) marks endothelial cells; phalloidin (red) stains F-actin. Schematic insets illustrate tissue organization in each condition. Right: immunofluorescence images show alpha-tubulin+ cilia (magenta) and F- actin (green) in epithelial cells under each condition, (g) Quantification of the percentage of ciliated epithelial cells in nonvascularized versus vascularized tissues. Data represent mean ± SD; ****p < 0.0001 by unpaired two-tailed t-test. (h) UMAP projections of single-cell transcriptomic data showing increased abundance and clustering of ciliated cells in vascularized tissues, (i) Comparison of inferred intercellular signaling networks using CellChat analysis across vascularized and nonvascularized tissues. Relative information flow is plotted for key signaling pathways, showing upregulation of TGF-P, BMP, VEGF, and WNT pathways in the presence of vasculature, (j) Circos plots representing ligand-receptor interactions within the TGF-P and BMP signaling families in vascularized (left) and nonvascularized (middle) tissues. Cell-type-specific contributions are color-coded. Right: ranked list of ligand-receptor pairs contributing to TGF-P and BMP signaling in vascularized tissues.

[0025] FIGs. 16A-16J. Modeling chlorine gas-induced lung injury in a vascularized human small-airway chip, (a) Schematic illustration of chlorine gas exposure to the human distal airway epithelium, showing induction of apoptotic injury following inhalation, (b) Custom exposure chamber used to deliver defined doses of chlorine gas to airway chips under continuous perfusion, (c-d) Computational model of chlorine transport103241.024775 / 22-10103 along the airway tree showing predicted concentration gradients across branching generations, with or without chemical reaction and constriction, (e) Quantification of transepithelial resistance, epithelial permeability, and junctional coverage following exposure to increasing concentrations of chlorine gas (0-100 ppm), demonstrating dose dependent barrier disruption. Data represent mean ± standard error of the mean; ****p < 0.0001 by one way ANOVA with post test, (f) Immunofluorescence staining of tight junction protein ZO1 and epithelial markers including tubulin (ciliated cells), CC10 (club cells), TP63 (basal cells), and MUC5AC (goblet cells) across chlorine doses, showing lineage specific loss of structural integrity, (g) Analysis of caspase activity demonstrating activation of caspase 3 and 7 and caspase 9 following chlorine exposure, (h-i) Confocal imaging showing endoplasmic reticulum stress marker ATF6 and nuclear translocation of NF kappa B in epithelial cells following 100 ppm exposure, (j) UMAP projections from single cell RNA sequencing showing selective depletion of ciliated cell clusters and emergence of intermediate stress associated states following chlorine injury.

[0026] FIG. 17. Chlorine exposure reprograms intercellular signaling networks in airway-on-a-chip. (Top) Relative information flow of ligand-receptor signaling pathways in untreated (blue) versus chlorine-exposed (red) airway chips, inferred using CellChat. Multiple pathways associated with epithelial maintenance and immune modulation (e.g., IL-10, WNT, BMP) are downregulated, while injury-associated signaling (e.g., PERIOSTIN, PTN, RANKL) is selectively upregulated following chlorine exposure. (Bottom) Circos plots visualizing key chlorine-induced signaling pathways. (Left) PTN signaling is enhanced post-exposure and primarily mediated by fibroblasts, targeting basal, club, and endothelial cells. (Middle) PERIOSTIN signaling, driven by endothelial cells, exhibits strong outgoing communication to rare, basal, and ciliated cell populations. (Right) RANKL signaling is enriched in fibroblast-derived cues to both epithelial and endothelial compartments, suggesting roles in inflammation and tissue remodeling.

[0027] FIGs. 18D-18J. Untargeted metabolomic profiling reveals chlorineinduced biochemical reprogramming in airway chips, (d) Schematic of the experimental workflow for untargeted metabolomics analysis. Airway chips were exposed to chlorine gas (100 ppm), and epithelial lining fluid (ELF) was collected for mass spectrometry, (e) Box plots showing normalized concentrations of representative metabolites significantly103241.024775 / 22-10103 altered following chlorine exposure, including sugars (D-glucose, D-fructose), organic acids (2-hydroxybutyric acid, 2-hydroxyadipic acid), amino acids (beta-alanine, D- alanine), and nucleotides (cytidine, adenine), (f) Heatmap of significantly changed metabolites (adjusted P < 0.05), highlighting consistent shifts in metabolic profiles across replicates, (g) Principal component analysis (PCA) shows clear separation between control and chlorine-exposed samples in 3D space.(h) Pathway impact analysis reveals significant enrichment in metabolic pathways including nitrogen metabolism, glycolysis / gluconeogenesis, pyrimidine metabolism, and arginine / proline metabolism. Circle size represents pathway impact; color represents - logl0(P value), (i) Receiver operating characteristic (ROC) curves for selected metabolites across different chlorine exposure levels, showing strong discriminatory performance (AUC > 0.95 for multiple doses), (j) Variable selection frequency plot highlighting the top metabolites most predictive of chlorine exposure, including known injury-related metabolites (e.g., styrene oxide, 4-chlorocatechol). Heatmap at right shows relative abundance across samples.

[0028] FIG. 19 provides a plan view (right panel) and a cross-sectional view (left panel) of a device according to the present disclosure.

[0029] FIG. 20 depicts a cross-sectional view of a device according to the present disclosure; as shown, the device can include endothelium in one or more channels alongside a central channel, which central channel can include lung blood vessels. Differentiated lung epithelium can be present on a pervious membrane above the central channel; as shown, the differentiated lung epithelium can be in an air-liquid interface culture.

[0030] FIG. 21 depicts a cross-sectional view of a device according to the present disclosure; as shown, a device can include small airway epithelial cells, which cells can be disposed on a pervious membrane above a central channel in which lung blood vessels are disposed.

[0031] FIG. 22 depicts a cross-sectional view of a device according to the present disclosure; as shown, a device can include large airway epithelial cells, which cells can be disposed on a pervious membrane above a central channel in which lung blood vessels are disposed.103241.024775 / 22-10103

[0032] FIG. 23 depicts a cross-sectional view of a device according to the present disclosure; as shown, a device can include alveolar epithelial cells, which cells can be disposed on a pervious membrane above a central channel in which lung blood vessels are disposed.

[0033] FIG. 24 provides a depiction of the use of a device according to the present disclosure to assess the effect of a toxicant - in this case, chlorine gas - on cells comprised in the device.

[0034] FIG. 25 provides an exploded view of a microfluidic device according to the present disclosure.DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0035] The present disclosure may be understood more readily by reference to the following detailed description of desired embodiments and the examples included therein.

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0037] The singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[0038] As used in the specification and in the claims, the term "comprising" can include the embodiments "consisting of' and "consisting essentially of.” The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that require the presence of the named ingredients / steps and permit the presence of other ingredients / steps. However, such description should be construed as also describing compositions or processes as "consisting of' and "consisting essentially of' the enumerated ingredients / steps, which allows the presence of only the named103241.024775 / 22-10103 ingredients / steps, along with any impurities that might result therefrom, and excludes other ingredients / steps.

[0039] As used herein, the terms “about” and “at or about” mean that the amount or value in question can be the value designated some other value approximately or about the same. It is generally understood, as used herein, that it is the nominal value indicated ±10% variation unless otherwise indicated or inferred. The term is intended to convey that similar values promote equivalent results or effects recited in the claims. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but can be approximate and / or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art. In general, an amount, size, formulation, parameter or other quantity or characteristic is “about” or “approximate” whether or not expressly stated to be such. It is understood that where “about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.

[0040] Unless indicated to the contrary, the numerical values should be understood to include numerical values which are the same when reduced to the same number of significant figures and numerical values which differ from the stated value by less than the experimental error of conventional measurement technique of the type described in the present application to determine the value.

[0041] All ranges disclosed herein are inclusive of the recited endpoint and independently of the endpoints. The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value; they are sufficiently imprecise to include values approximating these ranges and / or values.

[0042] As used herein, approximating language can be applied to modify any quantitative representation that can vary without resulting in a change in the basic function to which it is related. Accordingly, a value modified by a term or terms, such as “about” and “substantially,” may not be limited to the precise value specified, in some cases. In at least some instances, the approximating language can correspond to the precision of an instrument for measuring the value. The modifier “about” should also be considered as disclosing the range defined by the absolute values of the two endpoints. For example, the expression “from about 2 to about 4” also discloses the range “from 2 to 4.” The term103241.024775 / 22-10103“about” can refer to plus or minus 10% of the indicated number. For example, “about 10%” can indicate a range of 9% to 11%, and “about 1” can mean from 0.9-1.1. Other meanings of “about” can be apparent from the context, such as rounding off, so, for example “about 1” can also mean from 0.5 to 1.4. Further, the term “comprising” should be understood as having its open-ended meaning of “including,” but the term also includes the closed meaning of the term “consisting.” For example, a composition that comprises components A and B can be a composition that includes A, B, and other components, but can also be a composition made of A and B only. Any documents cited herein are incorporated by reference in their entireties for any and all purposes.

[0043] Any embodiment or aspect provided herein is illustrative only and does not limit the scope of the present disclosure or the appended claims. Any part or parts of any one or more embodiments or aspects can be combined with any part or parts of any one or more other embodiments or aspects.

[0044] Here, we introduce a novel open-top fluidic chip to culture epithelial tissue with more biological resemblance to human. And we use this platform to model three distinct microengineered biomimetic models that reconstruct the large airway, small airway, alveolar regions of the human lung. This enhanced vascularized lung on a chip includes a perfusable vascular network underneath the epithelial layer. Pulmonary interstitium and microvascular network play a critical role in both the development and homeostasis of the lung tissue.

[0045] Construction

[0046] Lung-on-a-chip device design and construction

[0047] Lung-on-a-chip device is an open-top microfluidic device composed of two microfabricated 3D chambers separated by a semipermeable membrane. The design of this microdevice makes it possible to mimic tissue compartmentalization in native airways by permitting long-term co-culture of airway epithelial cells, primary human pulmonary microvascular endothelial cells, and lung fibroblasts in a physiological 3D microenvironment to engineer fully differentiated airway epithelium directly exposed to air and supported by vascularized, perfusable 3D stromal tissues. The bottom compartmentalized microchannels consist of three-lined microchannels separated by two microguide rails protruding from the bottom of the microdevice, the middle microchannel injected with cell-laden solution to form a perfusable vessels. The middle lane of bottom103241.024775 / 22-10103 compartmentalized microchannels environment is fabricated at 3 mm size to gain full control on engineering the tissue size. Lung-on-a-chip is constructed with polydimethylsiloxane (PDMS) using standard soft lithography protocols.

[0048] 2. Optimization of the perfusable vascular network in circular microarray

[0049] Vascular networks resembling lung microvessels formed in the bottom compartment and in the central channel at culture areas of 3-mm. To form perfusable vessels a co-culture of primary human lung microvascular endothelial cells (HMVEC-L) and human normal lung fibroblasts (NHLF) were used in the fibrin matrix. NHLF cells provide support for endothelial cells morphogenesis by secretion of pro-angiogenic growth factors, they also provide support via secretion of ECM proteins. The ratio of seeding density was optimized to form a uniform microvascular network in a hydrogel matrix. An ECM precursor solution was made by suspending HMVEC-L and NHLF with the density of 2.5-5* 10A6 cells / mL and 5* 10A6 cells / mL in a lOmg / mL fibrin solution, respectively. Prior to injection of ECM solution in the devices, thrombin with final concentration of lU / mL was added to ECM cell solution. After 24 hours, side channels of the bottom compartment were seeded with endothelial cells at optimized cell density of 2.5-5* 10A6 cells / mL to form perfusable vessels within 7 days. Forming perfusable vascular networks was dependent on the co-culture with endothelial cells in the side channel since HMVEC- L co-cultured with NHLF without EC in the side channels failed to form perfusable vessel networks. We also examined formation of various tissue specific endothelial cells vascular network in our chip to check it versatility. Some of these cells were human umbilical vein endothelial cells (HUVECs), endothelial progenitor cells (EPC), pulmonary microvascular endothelial cells (HMVEC-L) and pulmonary arterial cells (HPAECs).

[0050] Epithelial cellular components

[0051] The top compartment is designed for epithelial culture, differentiation, and maturation. Primary large airway, small airway cells, 2D alveolar cells from stem cell derived organoids were cultured in lung-on-a -chip platform. After seeding cells on the top compartment as 2D format, these cells are differentiated into mature epithelial cells, using protocols specific to each cell type developed and tested.

[0052] Findings

[0053] Long term culture of epithelial tissue103241.024775 / 22-10103

[0054] The design of this microdevice makes it possible to mimic tissue compartmentalization in native airways by permitting long-term co-culture of airway epithelial cells (airway, or alveolar cells), primary human pulmonary microvascular endothelial cells, and lung fibroblasts in a physiological 3D microenvironment to engineer fully differentiated airway epithelium. Epithelial cells are directly exposed to air for more than 3 weeks and their differentiation is supported by perfusable 3D microvascular and stromal tissues. Using this chip, we are able of producing human lung tissues that exhibited differentiated phenotype and functional capacity that can be maintained for extended periods of time (over 1 month), the long-term maintenance of the epithelial is very critical since fully differentiated cells are produced after 3-weeks of air-liquid interface (ALI) culture.

[0055] Enhanced differentiation of lung epithelial cells in the presence of microvascular endothelial cells

[0056] Notably, our data revealed the potential of the underlying pulmonary vasculature to accelerate and promote differentiation and maturation of lung epithelial cells during air-liquid interface culture. We showed that in the presence of vascularized small airway cells are differentiated in 2 weeks whereas in the absence of perfusable vessel the differentiation process takes about 3 weeks.

[0057] Recapitulation of epithelial monolayer model in Lung-on-a-chip

[0058] The large and small airway: The top chamber containing permeable membrane that represented the human airway was coated with adhesive protein (fibronectin) and seeded with human small airway epithelial cells (Lonza) and maintained in growth media. After 3 days of submerged culture, the large and small airway cells were taken to ALI and the media was changed to differentiation media. The alveolar model: This model is constructed in the same device, uses primary human type II pneumocytes harvested from iPSC-derived human alveolar organoids, some of which are transdifferentiated into type I alveolar epithelial cells during 10 days in our device.

[0059] Additional Disclosure

[0060] Despite 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 to103241.024775 / 22-10103 gaseous compounds with known or suspected toxi cities, which represents a major public health concern.

[0061] Although studies of human victims have demonstrated the capacity of inhaled toxic gases to induce lung injury and dysfunction with long-term health consequences, 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 proximal region of the human lung responds to toxic gases using chlorine gas as a model toxicant.

[0062] 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 1pm 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. 12a, 12b). 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).

[0063] 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 (FIGs. 12A-12O). The bioengineered small airway epithelium exhibited morphological and functional properties of differentiated tissue, including columnar architecture, ciliogenesis and secretion of proteinaceous airway103241.024775 / 22-10103 surface liquid, mucociliary clearance, and barrier function (FIGs. 12G-12L). 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 (FIGs. 12m-12o). In an example 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 dosedependent loss of ciliated cells in the chlorine-treated epithelium (FIG. 13b). 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. 13c). A scRNA-seq review of this model revealed previously unknown differential susceptibility to chlorine-induced tissue injury exhibited by distinct subtypes of ciliated small airway epithelial cells (FIG. 13d). Similar to the small airway system, the alveolar model displayed adverse biological responses indicating acute tissue injury, inflammation, and functional deterioration.

[0064] These results demonstrate that the disclosed bioengineered lung models provide powerful tools for basic and translational research in inhalation toxicology of chlorine and other toxic gases, among other agents of interest.

[0065] Additional Disclosure - II

[0066] Chlorine (Ch) gas is one of the most frequently released industrial inhalation hazards, yet its earliest effects on human distal airway tissue remain poorly defined due to the lack of physiologically relevant experimental models. Traditional static cultures capture only isolated cell responses and therefore fail to predict how epithelial and vascular compartments coordinate injury or repair following exposure. Here, we introduce a microengineered human airway model that recreates the small airway region at an airliquid interface with a three dimensional vascular compartment. The platform integrates primary airway epithelium, supporting stromal cells, and perfused endothelium, enabling real time visualization of epithelial-endothelial communication during controlled chlorine exposure. Chlorine induced an immediate endothelial oxidative burst that preceded epithelial disruption, followed by cytokine release and rapid barrier failure. These injury dynamics were accompanied by selective loss of differentiated epithelial lineages and suppression of regenerative signaling. This model establishes a human relevant test103241.024775 / 22-10103 platform for dissecting chlorine induced pathophysiology and accelerating development of medical countermeasures against acute inhalation threats.

[0067] Humanity has always been confronted with environments that lie beyond the reach of direct experimentation conditions so dangerous, transient, or unpredictable that they cannot be ethically or safely recreated for human study. Whether emerging from industrial disasters, warfare, or naturally occurring catastrophes, these extreme exposures often unfold within seconds, leaving little opportunity to observe the earliest events that govern injury or survival.

[0068] Chemical, biological, radiological, and nuclear (CBRN) agents represent one of the most pressing examples of such inaccessible threats. Despite global advances in biotechnology and defense research, our mechanistic understanding of how the human body initially perceives and responds to these insults remains remarkably limited. The gap is not due to a lack of interest, but rather to the absence of experimental systems that can recreate the magnitude, timing, and complexity of such exposures in physiologically relevant human tissue.

[0069] Among all organs, the lung is uniquely positioned on the frontline of these threats. As the primary interface between the external environment and internal physiology, it continuously negotiates between life-sustaining gas exchange and defense against inhaled hazards. This interface is composed of tightly orchestrated epithelial, stromal, and vascular compartments that must rapidly sense, absorb, neutralize, and repair damage to preserve function. Across species, the respiratory system demonstrates remarkable regenerative potential; yet the precise sequence of molecular and cellular events that distinguish repair from irreversible injury is still not fully resolved, especially under extreme exposure scenarios.

[0070] Despite decades of research in pulmonary biology and inhalation toxicology, we still lack a human-relevant platform capable of capturing the earliest crosstalk between epithelial and vascular compartments during high-intensity injury. Without this knowledge, predicting outcomes or developing effective countermeasures remains largely empirical.

[0071] Despite decades of research, the precise mechanisms by which chemical gases damage human lung tissue remain unclear. Chlorine gas, in particular, is one of the most frequently released industrial toxicants worldwide and is responsible for both103241.024775 / 22-10103 accidental and intentional mass exposure incidents. Upon inhalation, chlorine reacts with the liquid lining of the airway to generate hydrochloric and hypochlorous acids that trigger oxidative stress and inflammation. Early injury to the epithelium has been documented in both animal and static culture studies. However, growing evidence indicates that injury to the vascular compartment and the communication between epithelial and endothelial layers may be an equally important driver of both acute respiratory failure and long term remodeling. Most existing experimental systems cannot reproduce the combined effects of gas exposure, fluid flow, and cellular interaction within a realistic human tissue context. A clear understanding of how chlorine initiates injury at the small airway and alveolar interfaces is still missing, and this gap continues to delay the development of effective medical countermeasures.

[0072] One of the major unanswered questions in chlorine inhalation injury is how an acute exposure progresses to long term structural and functional decline in the lung. Although animal studies have described early inflammatory responses, the transition from initial epithelial irritation to fibrosis, airway remodeling, or vascular dysfunction remains unclear. This uncertainty is largely due to the lack of experimental systems that can monitor tissue responses beyond the first hours after exposure while preserving communication between epithelial, stromal, immune, and vascular compartments. Rodent lungs do not reproduce the cellular composition or injury thresholds of the human distal airway. Nonhuman primate studies are closer to human physiology but are limited by high cost, restricted access, and ethical constraints. Conventional static cultures support only isolated cell types and rapidly lose polarity, barrier function, and paracrine signaling. These limitations have created a need for human relevant culture platforms that support coordinated injury and recovery under dynamic flow.

[0073] Recent advances in microphy si ologi cal system engineering have begun to address this gap by integrating primary human cells with controlled fluid motion and compartmentalized architecture. However, no current model has captured both the acute and evolving phases of chlorine induced airway injury in a way that enables mechanistic analysis and therapeutic testing.

[0074] As a step toward addressing these limitations, we developed a bioengineering strategy that enables experimental modeling of human airway exposure to toxic gases under physiologically relevant conditions. Our approach uses a103241.024775 / 22-10103 microengineered culture platform that combines primary human airway epithelium, supporting stromal cells, and perfused endothelium to recreate the distal airway interface at anatomically distinct regions of the lung. This system supports sustained tissue function under air-liquid interface and perfusable vascular, allowing both acute injury and recovery processes to be monitored in real time.

[0075] To demonstrate the utility of this platform, we conducted a case study using chlorine gas as a representative accidental inhalation hazard. The model permitted direct visualization and quantification of multiple biological responses, including epithelial damage, ciliostasis, oxidative signaling, cytokine release, and barrier dysfunction. Through detailed analysis, we identified a high susceptibility of mature ciliated cells to chlorine-induced injury and revealed mechanistic features of epithelial- endothelial communication during tissue damage. We further explored the capacity of the system for biomarker discovery by performing untargeted metabolomic profiling of the airway surface liquid to identify candidate indicators of dysfunction.

[0076] Results

[0077] Engineering a vascularized model of the human distal airway tissue

[0078] The small airway epithelium forms a critical barrier between inhaled gases and the delicate gas exchange regions of the lung. This pseudostratified columnar tissue contains ciliated, goblet, club, and basal cells that coordinate mucus secretion, ciliary transport, and epithelial repair. Beneath this layer lies a stromal compartment composed of fibroblasts, extracellular matrix, and an extensive capillary network that mediates immune surveillance, metabolic exchange, and regeneration. Together these components form an integrated epithelial-vascular unit that maintains airway function under constant environmental stress (FIG. 14a).

[0079] To recreate this architecture in vitro, we engineered a vascularized small airway model within a multilayer culture device containing independent epithelial and stromal compartments (FIG. 14a-14b). Each example device housed fifteen individual culture units to enable parallel experimentation. In each unit, a three millimeter central chamber supports stromal tissue formation, while side channels permit controlled perfusion. A thin, porous polycarbonate membrane separates the stromal chamber from the apical epithelial chamber seeded with primary human small airway epithelial cells (SAECs), allowing molecular communication while preserving independent access to each103241.024775 / 22-10103 tissue layer (FIG. 14c-14d). The foregoing materials and dimensions are, however, exemplary only and do not limit the scope of the present disclosure or the appended claims.

[0080] The stromal compartment was formed by injecting a fibrin gel containing human lung fibroblasts and microvascular endothelial cells into the central chamber. Surface tension and microfabricated rail structures guided the advancing meniscus and confined the gel within the chamber without leakage into adjacent regions. Within two to three days of culture, endothelial cells embedded in the matrix began to elongate and interconnect, forming nascent vascular cords. By day four, these structures had organized into dense CD31 positive capillary networks resembling early vasculogenesis (FIG. 14e- 14h). Endothelial cells seeded along the perfusion channels extended angiogenic sprouts that bridged toward the central gel and ultimately anastomosed with the self-organized vascular structures. After seven days of culture, a continuous and perfusable microvascular network was established, as confirmed by dextran perfusion and barrier integrity assays (FIG. 14i-14j). Vessels remained patent and structurally stable, displaying luminal morphologies consistent with functional capillaries in both confocal and electron microscopy (FIG. 14k- 14m).

[0081] Once vascularization was established, primary human small airway epithelial cells were introduced onto the apical surface of the porous membrane separating the compartments. The cells adhered uniformly and formed a confluent layer within forty eight hours. At that point, the cultures were transitioned to air liquid interface conditions to promote mucociliary differentiation. Over a twenty one day maturation period, the epithelium progressively developed into a polarized pseudostratified structure resembling native distal airway tissue. Immunostaining confirmed the presence of secretory goblet cells expressing MUC5AC, club cells expressing CC10, basal cells expressing TP63, and multi ciliated cells marked by alpha-tubulin (FIG. 14p). Junctional proteins such as ZO-1 localized to apical borders, and phalloidin staining revealed organized cortical actin consistent with barrier formation (FIG. 14o). confocal microscopy showed well aligned cilia, basal bodies, desmosomes, and mucus filled vesicles, confirming functional mucociliary architecture. These features were maintained consistently across all culture units.103241.024775 / 22-10103

[0082] To further assess lineage specification, epithelial cells cultured on chip were analyzed by single cell RNA sequencing. Distinct transcriptional clusters corresponding to basal, ciliated, goblet, club, and intermediate cell states were identified, closely matching reference profiles from primary human airway datasets (FIG. 14q). Stromal and endothelial populations also retained characteristic gene expression signatures, indicating preservation of tissue identity across compartments. Together these results confirm that the platform supports stable multicellular organization across an epithelial-vascular interface under air liquid. The ability to reproduce native airway architecture with sustained function establishes this model as a scalable and physiologically relevant system for studying human distal airway injury and repair.

[0083] Vascularization promotes epithelial maturation through microenvironmental signaling

[0084] Co-culture results in the accelerate differentiation of the epithelium tissue

[0085] To isolate the specific contribution of perfusable vasculature to epithelial maturation, we engineered airway tissues either with or without vascular networks while keeping fibroblasts, culture conditions, and air liquid interface exposure identical in both groups. In the absence of vasculature, epithelial cells formed a confluent layer but remained partially polarized and exhibited limited ciliogenesis. In contrast, vascularized tissues displayed pronounced cytoskeletal organization and extensive formation of motile cilia, as visualized by alpha-tubulin staining (FIG. 15f). These qualitative differences suggested that microvascular presence accelerates epithelial specification beyond the effects provided by stromal fibroblasts alone.

[0086] Quantitative image analysis confirmed a more than two fold increase in the proportion of ciliated epithelial cells in vascularized cultures compared to nonvascularized controls (FIG. 15g). Single-cell RNA sequencing further supported this observation by revealing a larger and transcriptionally distinct ciliated cell population in vascularized tissues, as shown by spatial clustering on UMAP plots (FIG. 15h). These findings indicate that epithelial lineage allocation is influenced by stromal vascular cues rather than being solely driven by intrinsic differentiation programs.

[0087] To determine how vascular presence alters epithelial differentiation at the signaling level, we applied CellChat to infer ligand receptor communication across cellular compartments. Vascularized tissues displayed broad enhancement of paracrine103241.024775 / 22-10103 signaling through key developmental pathways, including TGF beta, BMP, VEGF, and WNT (FIG. 15i). In particular, signaling axes within the TGF beta and BMP families, both known regulators of epithelial morphogenesis, were strongly enriched in vascularized tissues.

[0088] Visualization of ligand receptor interactions at single cell resolution revealed that fibroblasts and endothelial cells actively communicated with basal, club, and ciliated cells through TGFBR and ACVR receptor families (FIG. 15j). In nonvascularized tissues, these signaling interactions were sparse and lacked diversity. Ranking of signaling pairs identified TGFB1 to TGFBR1 / 2 and BMP4 to BMPR2 as dominant mediators of vascular induced epithelial maturation. Together these data demonstrate that vascularization not only supports physical organization of airway tissue but also drives epithelial lineage specification through microenvironmental signaling. The inclusion of perfusable microvessels therefore represents a functional requirement for achieving physiologically mature airway epithelium in vitro.

[0089] Modeling chlorine-induced lung injury in a vascularized small-airway chip

[0090] Chlorine gas is an asphyxiating toxicant capable of causing mass casualties in accidental or deliberate release scenarios. A Department of Homeland Security risk assessment estimated that rupture of a single storage tank outside a city of seven hundred thousand residents would expose at least thirty five thousand individuals to lethal doses, with half of those progressing to respiratory failure. An additional one hundred thousand would require hospitalization, while more than sixty percent of the surrounding population would seek emergency care. Clinically, chlorine inhalation produces a concentration dependent spectrum of symptoms that ranges from bronchospasm and cough to acute lung injury, pulmonary edema, and death, followed in survivors by persistent airway hyperreactivity and fibrotic remodeling. Chlorine reacts rapidly with airway lining fluid to generate hydrochloric and hypochlorous acid, which attack epithelial and endothelial surfaces, yet the precise sequence of injury events in human distal airways remains undefined due to lack of physiologically relevant models.

[0091] To recreate the conditions of inhalation injury in a controlled setting, we integrated our vascularized small airway chip with a custom exposure chamber that delivers chlorine gas at defined concentrations and durations while maintaining epithelial103241.024775 / 22-10103 perfusion and real time media sampling (FIG. 16a-16b). To predict how chlorine distributes along airway branches during exposure, we developed a computational model of gas transport through successive generations of the bronchial tree with or without chemical reaction and luminal constriction (FIG. 16c-16d). This model guided selection of exposure regimens corresponding to low and high dose scenarios reported in previous injury studies.

[0092] Exposure of airway chips to chlorine at concentrations between zero and one hundred parts per million for ten minutes resulted in a clear dose dependent disruption of epithelial barrier function. Immunostaining for ZO-1 showed fragmentation of tight junctions and loss of junctional continuity across the epithelial layer (FIG. 16f, top). These structural changes were accompanied by significant reductions in transepithelial electrical resistance and increased paracellular permeability (FIG. 16e). Measurement of lactate dehydrogenase in the apical and vascular effluent confirmed progressive cytotoxicity, with minimal release at low doses and sharp increases above fifty parts per million. Caspase staining indicated activation of both executioner caspases three and seven and upstream initiator caspase nine, consistent with mitochondrial mediated apoptosis (FIG. 16g). These results indicate that chlorine causes rapid epithelial barrier failure through both structural degradation and cell death.

[0093] To probe stress response pathways, we examined the expression of activating transcription factor six as an indicator of endoplasmic reticulum strain. High dose exposure induced strong ATF6 upregulation throughout the epithelial layer (FIG. 16h), suggesting engagement of the unfolded protein response. Nuclear localization of NF kappa B increased in parallel, revealing activation of pro inflammatory signaling cascades (FIG. 16i). Together these observations indicate that chlorine triggers combined oxidative, inflammatory, and apoptotic stress programs in airway epithelium.

[0094] We next assessed lineage specific vulnerability. Immunostaining for tubulin positive ciliated cells (FIG. 16f, top), MUC5AC positive goblet cells, and CC10 positive club cells showed marked loss of differentiated secretory and ciliated populations following chlorine treatment, whereas TP63 positive basal progenitors were relatively preserved (FIG. 16f, bottom). Loss of ciliary coverage was accompanied by significant reductions in ciliary beat frequency, indicating impaired mucociliary clearance. These103241.024775 / 22-10103 findings suggest that chlorine preferentially eliminates mature epithelial lineages while sparing regeneration competent progenitors.

[0095] To evaluate global transcriptional remodeling, we performed single cell RNA sequencing on epithelial and stromal cells isolated from control and chlorine exposed tissues. UMAP projections revealed major shifts in cell composition and transcriptional identity, with depletion of ciliated cell clusters and emergence of intermediate stress associated states (FIG. 16j).

[0096] To investigate how chlorine exposure alters communication between epithelial, stromal, and vascular compartments, we performed ligand receptor network analysis using CellChat on single cell RNA sequencing datasets collected from untreated and chlorine exposed airway tissues. Relative information flow analysis revealed a broad suppression of signaling pathways associated with epithelial maintenance and immune regulation, including interleukin ten, bone morphogenetic protein, and WNT (FIG. 17, top). In contrast, a subset of pathways linked to injury response and tissue remodeling was selectively upregulated. Among these, pleiotrophin, periostin, and receptor activator of nuclear factor ligand were the most strongly induced following chlorine exposure.

[0097] Circos plots were used to visualize the cellular origin and directionality of these altered communication networks. Pleiotrophin signaling originated predominantly from fibroblasts and was directed toward basal, club, and endothelial cells, suggesting a paracrine attempt to stabilize or repopulate injured tissue (FIG. 17, bottom left). Periostin signaling, driven by endothelial cells, displayed strong outgoing communication toward rare, club, and ciliated cell populations (FIG. 17, bottom middle), indicating vascular participation in injury signaling. Receptor activator of nuclear factor ligand signaling arose mainly from fibroblasts and targeted both epithelial and vascular compartments (FIG. 17, bottom right), consistent with roles in inflammation and matrix remodeling.

[0098] These findings reveal that chlorine exposure not only damages structural components of the epithelium but also reprograms the intercellular communication landscape, shifting tissue level signaling away from homeostatic pathways and toward injury associated programs. The vascularized airway chip therefore provides a unique platform for capturing both physical injury and coordinated transcriptional rewiring of airway signaling networks.103241.024775 / 22-10103

[0099] Metabolomic profiling reveals chlorine-induced disruption of airway biochemical homeostasis

[0100] To investigate the biochemical consequences of chlorine exposure at the airway surface, we performed untargeted metabolomic analysis of epithelial lining fluid collected from chips exposed to one hundred parts per million chlorine gas (FIG. 18d). Apical effluents were harvested immediately following exposure, and metabolites were extracted and analyzed by liquid chromatography coupled with high resolution mass spectrometry. Normalized metabolite intensities were compared across biological replicates from control and chlorine treated tissues.

[0101] Chlorine injury produced widespread shifts in metabolite abundance across multiple chemical classes, including sugars, organic acids, amino acids, and nucleotides (FIG. 18e). Levels of D-glucose and D-fructose were significantly reduced, indicating disruption of glycolytic fuel supply. Key organic acids such as two hydroxybutyric acid and two hydroxyadipic acid were also depleted, while beta alanine accumulated, suggesting altered amino acid turnover. Perturbation of nucleotide metabolism was reflected by reduced abundance of adenine. These coordinated alterations point to metabolic strain, impaired energy balance, and dysregulation of biosynthetic pathways following chlorine exposure.

[0102] Heatmap visualization of significantly altered metabolites revealed a clear separation between control and injured groups, indicating high reproducibility of metabolic responses across biological replicates (FIG. 18f). Principal component analysis further confirmed distinct clustering of control and chlorine samples along the first two principal components, which captured the majority of total variance (FIG. 18g). Pathway enrichment analysis identified strong engagement of nitrogen metabolism, glycolysis and gluconeogenesis, pyrimidine metabolism, and arginine and proline metabolism (FIG. 18h), all of which are associated with oxidative strain and mitochondrial stress responses.

[0103] To evaluate the diagnostic value of individual metabolites, we generated receiver operating characteristic curves for compounds with the largest changes in abundance. Several metabolites demonstrated strong discriminatory power, achieving classification accuracy with area under the curve values exceeding zero point nine five across exposure conditions (FIG. 18i). Variable selection frequency analysis identified a panel of biomarkers, including styrene oxide, four chlorocatechol, and amino acid103241.024775 / 22-10103 derivatives, which consistently differentiated chlorine exposed tissues from controls (FIG. 18j). Many of these compounds have been previously associated with oxidative damage and epithelial injury, underscoring their potential relevance as exposure indicators.

[0104] Collectively, these results demonstrate that chlorine exposure induces a distinct metabolic signature in epithelial lining fluid. The airway chip platform therefore enables noninvasive sampling of airway secretions and supports discovery of chemical biomarkers and perturbed biochemical networks associated with acute inhalation injury.

[0105] Discussion

[0106] The human distal airway is a complex and highly coordinated tissue where epithelial, stromal, and vascular compartments maintain barrier integrity, immune surveillance, and gas exchange through constant communication. Reproducing this organization in vitro has remained difficult due to the need to sustain multiple specialized cell populations under physiologically relevant microenvironmental conditions. In this work, we developed a vascularized small airway model that recreates key structural and functional features of the human bronchiolar niche, including a pseudostratified epithelium, perfused microvasculature, and compartmentalized epithelial stromal vascular interactions. The platform supports long term epithelial differentiation, enables real time access to both tissue compartments, and allows controlled exposure to inhaled toxicants under flow.

[0107] A central finding of this study is that vascularization accelerates and enhances epithelial maturation. Vascularized tissues exhibited greater ciliogenesis, tighter barrier function, and more defined lineage specification compared to nonvascularized counterparts. Single cell transcriptomic analysis demonstrated that these effects arise from paracrine signaling between endothelial, stromal, and epithelial populations, with enrichment of developmental pathways such as transforming growth factor, bone morphogenetic protein, and WNT signaling. This highlights the importance of vascular stromal epithelial communication in airway homeostasis and illustrates a limitation of conventional culture formats that isolate epithelial cells from their native support systems.

[0108] To illustrate the utility of this model in toxicology, we exposed airway tissues to chlorine gas, a widely used industrial chemical and common cause of accidental or intentional inhalation injury. Chlorine induced rapid disruption of epithelial tight junctions, loss of transepithelial resistance, and elevation of permeability in a dose103241.024775 / 22-10103 dependent manner. Caspase activation and lactate dehydrogenase release indicated apoptotic cell death. Notably, two distinct phases of injury emerged, consistent with clinical observations of early acute collapse followed by delayed inflammatory exacerbation. This aligns with previous reports from animal studies where immediate mortality is rare but secondary deterioration develops over several hours. High dose exposure activated the unfolded protein response through upregulation of activating transcription factor six and triggered nuclear translocation of nuclear factor kappa B, confirming induction of both stress and inflammatory pathways.

[0109] Chlorine injury did not affect all epithelial lineages equally. Ciliated and secretory cells were highly vulnerable, whereas basal progenitors were relatively preserved. Loss of ciliary coverage resulted in reduced ciliary beat frequency, providing a mechanistic basis for clinical reports of impaired mucociliary clearance and fluid accumulation. These findings mirror early edema and persistent airway dysfunction observed in human cases and ex vivo lung explants. Metabolomic profiling of epithelial lining fluid revealed a distinct injury signature characterized by depletion of glycolytic intermediates and nucleotide precursors along with accumulation of selected amino acids and oxidative byproducts. Several metabolites displayed strong diagnostic performance and may represent candidate exposure biomarkers.

[0110] At the transcriptional level, chlorine exposure altered both cell composition and intercellular communication networks. Single cell sequencing showed emergence of intermediate stress associated states and loss of mature epithelial clusters. Ligand receptor analysis revealed suppression of homeostatic pathways including interleukin ten, bone morphogenetic protein, and WNT signaling, with simultaneous induction of pleiotrophin, periostin, and receptor activator of nuclear factor ligand programs. Fibroblasts and endothelial cells emerged as dominant initiators of injury associated signaling, suggesting coordinated stromal epithelial and vascular epithelial communication during damage progression.

[0111] Beyond modeling injury, the platform supports therapeutic testing and immune recruitment. In separate experiments, Telmisartan based interventions preserved epithelial integrity and reduced inflammatory signaling following chlorine exposure. Neutrophils perfused through the vascular channel successfully migrated across the membrane toward injured epithelium, demonstrating the ability to model immune cell103241.024775 / 22-10103 engagement during injury or recovery. These advantages underscore the versatility of the system and position it as a promising tool for mechanistic discovery, medical countermeasure screening, and immune toxicology.

[0112] Conclusions

[0113] In summary, the vascularized human small airway chip provides a physiologically relevant and experimentally accessible platform for modeling inhalation injury at cellular, molecular, and tissue scales. It captures early barrier disruption, lineage specific loss, signaling reprogramming, and metabolic alterations following chlorine exposure while maintaining compatibility with multi omics analytics and live functional assays.

[0114] Methods

[0115] Fabrication of cell culture devices

[0116] Devices used for culturing human airway cells in this study were fabricated using soft lithography. Briefly, poly(dimethyl siloxane) (PDMS, Sylgard 184, Dow Coming) monomer base was mixed with a curing agent (10: 1, w / w) and poured onto 3D-printed molds manufactured by stereolithographic 3D printing (Protolabs). The cast molds were vacuum-degassed in a desiccation chamber (Bel-Art Inc.) for 1 hour, followed by thermal curing in an oven at 65 °C for 2 hours. Subsequently, a Whatman Cyclopore Polycarbonate Thin Clear membrane with 1 pm pores (Cytiva, USA) was punched using a 7mm biopsy punch (Acuderm Inc., USA) and sealed against the top PDMS layer (airway chamber) using uncured PDMS as a glue. Next, ECM injection mold access ports were generated in the top PDMS layer using a 1 mm biopsy punch (Integra, Inc.), and this layer was bonded to the bottom PDMS layer containing the stroma chamber. The device assembly was then placed in an oven for 1 hour and stored in a container until use.

[0117] Cell culture

[0118] All human primary cells were obtained from Lonza Lonza (NC, USA) comprise that included Human Small Airway Epithelial Cells (SAEC, Cat # CC-2547), Normal Human Lung Fibroblasts (NHLF, Cat # CC-2512), and Human Lung Microvascular Endothelial Cells (HMVEC-L, Cat # CC-2527). Some limited clinical data regarding the donors of the human lung cells existed from the vendor. HLMEC-L, NHLF and SAEC were maintained and grown in adherent tissue culture using Corning TC- treated T-75 tissue culture flasks. The HLMEC-L were cultured in Microvascular103241.024775 / 22-10103Endothelial Cell Growth Medium-2 BulletKit (EGM-2MV, Cat # CC-3202, Lonza, NC), while NHLF were cultured in Fibroblast Growth Basal Medium-2 BulletKit (FGM-2, Cat # CC-3132, Lonza, NC). On passage 1-3, these cells were utilized for creating the stromal tissue in the bottom compartment of our vascularized airway-chip. SAEC were cultured in culture flasks for only one passage in growth media of the Small Airway Air-Liquid Interface Medium BulletKit (S-ALI, Cat # CC-4539: Growth Basal Media (CC-3281), SingleQuots Supplement Pack (CC-4538)).

[0119] Stromal Tissue Construction

[0120] After device fabrication, the PDMS chambers were filled with 70% ethanol and incubated for 1 minute, after which ethanol was removed by applying vacuum aspiration to access ports. Subsequently, the device was sterilized for 20 minutes using ultraviolet (UV) light. To generate vascularized stromal tissues in the stroma compartment, a 100-pl mixture of 10 mg / ml fibrinogen (Millipore Sigma Cat. F8630-5G) in DPBS, thrombin (10 U / ml), microvascular endothelial cells (2.5-5 x 106cells / ml), and fibroblasts (5.5 x io6cells / ml) were injected into the stroma chamber of the lower device layer compartment of a microfabricated device. The seeded device was placed in an incubator at 37 °C for 15 minutes to induce gelation of the mixture. Subsequently, the side channels were seeded with endothelial cells (5 x io6cells / ml).

[0121] Epithelial Layer

[0122] To improve the cell adhesion to the polycarbonate membrane, we treated the membrane surface with fibronectin solution (0.1 mg / ml) for 2 hours at 37 °C. The solution was then rinsed with PBS before the introduction of cells. At this step, the device was seeded with SAECs (1.25 x io6cells / ml) on the top of the permeable membrane. After 1.5 hours of incubation, the devices formed a monolayer. The remaining cells were then washed off, and the top compartment was filled with the growth medium of S-ALI. The next day, epithelia cells were introduced to the air-liquid interface and the bottom compartment was perfused with an optimized co-culture ALI media

[0123] Transwell culture

[0124] Transwell culture of primary human airway cells was established for comparative analysis of biological relevance of tissue differentiation. This model was created in a Transwell insert (Corning, NY, USA Cat # 38024) containing a semi- permeable membrane with a pore size of 0.4 pm. SAECs were seeded onto the upper side103241.024775 / 22-10103 of the membrane at 2.5 x 106cells / ml in 500 pl. The cells were introduced to air-liquid interface on day 1 and the differentiation media of the Small Airway Air-Liquid Interface Medium BulletKit (S-ALI, Cat # CC-4539: Differentiation Media (CC-3282), SingleQuots Supplement Pack (CC-4538)) was changed every 48 hours until 3 weeks.

[0125] Mathematical Modeling of Chlorine Exposure

[0126] We used a mathematical framework to quantify the chlorine gas concentration (c) across various segments of the human respiratory system, including the tracheobronchial and pulmonary regions. Our model integrates three critical factors involved in the transportation of the gas through the respiratory system, demonstrated by the three terms on the right side of Eq. (1) dc / dt = -V. (u c) + V. (D Vc) + 7? (1)

[0127] The first term represents the convective movement of gas propelled by airflow (characterized by V. (u c)), where u denotes velocity and V represents the gradient operator. The second term represents the diffusive propagation of chlorine along the airway gradient (expressed as V. ( Vc)), as chlorine migrates from regions of higher to lower concentration with a dispersion coefficient D . The last term captures the chemical interactions between the gas and the lung lining fluid and the underlying tissue, encapsulated in the term R. Each of these three terms is described in detail in the Supplementary Information.

[0128] By solving this equation across the airway's geometric structure from the 1st to the 23rd generation, using known approximate diameter and length of each generation, we determine the gas concentration at every airway generation. The results demonstrated a decrease in the gas dose along the airway pathway.

[0129] Injury Model of Chlorine Exposure

[0130] When the tissue is ready, the epithelium compartment was filled with artificial epithelial lining fluid (ELF) solution. The “artificial ELF” consists of 50 pl of normal Ringer’s solution (120 mM NaCl, 25 mM NaHCCh, 3.3 mM KH2PO4, 0.83 mM K2HPO4, 1.2 mM CaCh, and 1.2 mM MgCh) containing ascorbic acid (1 mM), reduced glutathione (0.12 mM), and urate (0.03 mM). Once the ELF solution is added, the chips are exposed to different chlorine dose (0, 10, 25, 50 and 100 ppm) for 15 minutes and incubated at 37 °C with 5% CO2 for 24 hours or less depending on the experimental endpoint analysis.103241.024775 / 22-10103

[0131] Histology and Immunofluorescence staining and microscopy

[0132] For immunofluorescence staining, tissues were first fixed in a 4% PFA solution at 4 °C for 4 hours and then permeabilized in 1% Tween-20 and 10% BSA at 4°C for 2 hours. Primary antibodies were applied at manufacturer-recommended dilutions or at 1 :200 dilution, whichever was more concentrated, and incubated at 4 °C overnight. On the next day, tissues were washed with DPBS containing 0.1% Tween-20 and 10% BSA and left overnight. Secondary antibodies, if required, were incubated overnight, followed by another day of washing in the same solution. DAPI and Phalloidin were added at dilutions of 1 :5000 and 1 : 1000, respectively. Following staining, imaging was conducted using confocal microscopy (Zeiss LSM 800) with 10X 0.45 NA and 63X 1.4 NA objectives.

[0133] Validation of vascular perfusability

[0134] In assessing the perfusability of our microengineered vascular network, we employed fluorescently labeled 500nm beads as flow tracers. Flow was induced through the vasculature by aspirating the medium from the reservoirs and introducing a fluorescence beads solution into the side channel. This setup established a hydrostatic pressure gradient across the hydrogel scaffold, facilitating the flow of the fluorescence beads through the vessels. Vascular perfusion was meticulously monitored and visualized using a laser scanning confocal microscope (LSM 800, Carl Zeiss).

[0135] Measurement of cell viability, LDH release, and apoptosis

[0136] The viability of SAEC in the epithelium compartment was analyzed using the Live / Dead Cell Viability Kit (Life Technologies) following the manufacturer's instructions. Briefly, the cells in the device were washed with PBS and stained with PBS containing 4 pM ethidium homodimer and 2 pM Calcein AM at 37 °C for 30 min. Subsequently, the cells were rinsed with fresh PBS and imaged using a laser scanning confocal microscope (LSM 800, Carl Zeiss, Jena, Germany). To measure LDH release from the engineered airway tissues, perfusate from the device was collected from the outlet access ports and analyzed by the Cytotoxicity Detection KitPLUS assay (LDH, Roche, 04744926001) using the manufacturer-provided protocol. Analysis of caspase-3 / 7 was performed by using the Caspase-Gio® 3 / 7 Assay (Promega). An equal volume of reagent was added and gently mixed at 37 °C for 2 hours according to the manufacture’s protocol. Luminescence generated by apoptotic cells was detected and quantified using a fluorescence plate reader (Tecan).103241.024775 / 22-10103

[0137] Analysis of secretory products

[0138] For analysis of pro-inflammatory cytokines, device effluent collected from the epithelium and stroma chambers was measured using the following ELISA kits: Human IL-ip ELISA Kit (RAB0273, Sigma Aldrich), Human IL-6 ELISA Kit (RAB0306, Sigma Aldrich), Human IL-8 ELISA Kit (RAB0319, Sigma Aldrich), Human TNF Alpha ELISA Kit (ah 181421, abeam), and Human TGF Beta 1 ELISA Kit (DY240, R&D Systems).

[0139] Measurement of barrier function

[0140] The structural integrity of the engineered airway barrier was assessed by measuring electrical resistance between the airway and stroma chambers. Briefly, Ag / AgCl electrodes (0.008” diameter, A-M Systems, WA, USA) connected to a multimeter (Fluke, USA) were inserted into the access ports of the airway and stroma chambers. Resistance evaluated from an empty device without any cells was subtracted from each measurement to calculate net resistance, which was then multiplied by the surface area of the culture chamber to compute the final values expressed in * cm2.

[0141] Barrier permeability was evaluated by measuring transfer of tracer dyes including Lucifer Yellow CH (Invitrogen, L453) and Bovine Serum Albumin 594 conjugated (BSA, Biotium, Cat # 20290) from the airway epithelium to stroma compartments. Briefly, a solution containing 1 mM Lucifer Yellow in HBSS / HEPES was introduced into the epithelium chamber, while the stroma chamber was filled with HBSS / HEPES. Over a period of 2 hours, outflow from the stroma chamber was collected, and its fluorescent intensity was measured using a microplate reader (Tecan, excitation 485 nm, emission 530 nm).

[0142] Supernatant metabolite extraction for metabolomics analysis

[0143] Samples of culture media were collected from individual microdevices at defined time points and stored at -80°C. 5 pl of media was added to 120 pl of -20°C 25:25: 10 (v / v / v) acetonitrile:methanol:water solution, vortexed for 10 seconds, and put on ice for at least 5 minutes. The resulting extract was centrifuged at 16,000 x g for 20 minutes at 4°C, and the supernatant was transferred to tubes for LC-MS analysis. A procedure blank sample was generated identically without culture media, which was used later to remove the background ions.

[0144] Metabolite measurement by LC-MS103241.024775 / 22-10103

[0145] Metabolites were analyzed using a Vanquish Horizon UHPLC System (Thermo Scientific) coupled to an Orbitrap Exploris 480 Mass Spectrometer (Thermo Scientific). Waters XBridge BEH Amide XP Column (particle size, 2.5 pm; 150 mm (length) x 2.1 mm (i.d.)) was used for hydrophilic interaction chromatography (HILIC) separation. Column temperature was kept at 25 °C. Mobile phases A = 20 mM ammonium acetate and 22.5 mM ammonium hydroxide in 95:5 (v / v) water: acetonitrile (pH 9.45) and B = 100% acetonitrile were used for both ESI positive and negative modes. The linear gradient eluted from 90% B (0.0-2.0 min), 90% B to 75% B (2.0-3.0 min), 75% B (3.0- 7.0 min), 75% B to 70% B (7.0-8.0 min), 70% B (8.0-9.0 min), 70% B to 50% B (9.0- 10.0 min), 50% B (10.0-12.0 min), 50% B to 25% B (12.0-13.0 min), 25% B (13.0- 14.0 min), 25% B to 0.5% B (14.0-16.0 min), 0.5% B (16.0-20.5 min), then stayed at 90% B for 4.5 min. The flow rate was 0.15 mL / min. The sample injection volume was 5 pL. ESI source parameters were set as follows: spray voltage, 3200 V or -2800 V, in positive or negative modes, respectively; sheath gas, 35 arb; aux gas, 10 arb; sweep gas, 0.5 arb; ion transfer tube temperature, 300 °C; vaporizer temperature, 35 °C. LC-MS data acquisition was operated under full scan polarity switching mode for all samples. The full scan was set as: orbitrap resolution, 120,000 at m / z 200; AGC target, le7; maximum injection time, 200 ms; scan range, 60-1000 m / z.

[0146] Analysis of metabolomics data

[0147] LC-MS raw data files (.raw) were converted to mzXML format using ProteoWizard (version 3.0.20315)1. El-MAVEN (version 0.12.0)2was used to generate a peak table containing m / z, retention time, and intensity for the peaks. Parameters for peak picking were the defaults except for the following: mass domain resolution, 5 ppm; time domain resolution, 10 scans; minimum intensity, 10,000; and minimum peak width, 5 scans. The resulting peak table was exported as a .csv file. Peak annotation of untargeted metabolomics data was performed using NetID2with default parameters. Statistical analyses were performed using MetaboAnalyst 5.03.

[0148] Statistical analysis and reproducibility

[0149] A minimum of three biological replicates were used for each experimental group. All data in the paper are represented as mean ± standard deviation (SD). Data were analyzed with Student’ s t-test and with one-way and two-way ANOVA followed by Tukey’s post-hoc test for multigroup pairwise comparisons. Differences were103241.024775 / 22-10103 considered statistically significant at p-values < 0.05. *P < 0.05, **P < 0.01, ***p < 0.001, ****P < 0.0001. GraphPad Prism (ver. 9; GraphPad Software) was used for statistical analysis.

[0150] Aspects

[0151] Aspect 1. A lung-on-a-chip fluidic chip, comprising: a bottom region, the bottom region comprising a central channel and at least one side channel adjacent thereto, the central channel having therein a plurality of cells disposed in a matrix; and a middle region, the middle region having a chamber defined therein, the chamber optionally being an open-top chamber, the chamber being in register with at least one of the central channel and the at least one side channel of the bottom region.

[0152] FIG. 19 provides an example, non-limiting depiction of a chip according to the present disclosure. As shown, chip 190 can include a central channel 196 comprised in a bottom region. Side channel 192 can be adjacent to central channel 196; likewise, side channel 198 can be adjacent to central channel 196. Rails 194 can be present and at least partially define a side channel and the central channel. As shown, rail 194 can extend from the base or floor of the chip. The middle region of the chip can include chamber 191; as shown, pervious membrane 1910 can be arranged between chamber 191 and central channel 196.

[0153] FIG. 25 provides an exploded view of a fluidic chip 2500 according to the present disclosure. As shown, chip 2500 can include layer 2502, which layer can comprise aperture 2504 and chamber 2506. Chip 2500 can include layer 2510, which layer can comprise rails 2512a and 2512b, which rails can define boundaries between central channel 2512 and side channels 2514 and 2516. Chip 2500 can also include layer 2508, which can be present as a pervious membrane. Layer 2508 can define the bottom of chamber 2506; as described elsewhere herein, epithelial cells - such as lung epithelial cells - can be disposed on layer 2508, and layer 2508 can permit fluid communication between cells in chamber 2506 and central channel 2512.

[0154] Aspect 2. The fluidic chip of Aspect 1, further comprising a pervious membrane, the pervious membrane being arranged between the chamber of the middle layer and at least one of (i) the central channel or the bottom region (ii) the at least one side channel of the bottom region.103241.024775 / 22-10103

[0155] Aspect 3. The fluidic chip of Aspect 2, further comprising epithelial cells disposed within the chamber, the epithelial cells optionally superposed on the pervious membrane.

[0156] Aspect 4. The fluidic chip of Aspect 3, wherein the epithelial cells comprise any one or more of primary large airway, small airway cells, or alveolar cells.

[0157] Aspect 5. The fluidic chip of any one of Aspects 1-4, wherein the matrix comprises a hydrogel.

[0158] Aspect 6. The fluidic chip of any one of Aspects 1-5, wherein the plurality of cells comprises fibroblasts, pericytes, vascular smooth muscle cells, and resident immune cells, the resident immune cells optionally comprising any one or more of macrophages and dendritic cells.

[0159] Aspect 7. The fluidic chip of any one of Aspects 1-6, wherein the plurality of cells comprises any one or more of human umbilical vein endothelial cells (HUVECs), endothelial progenitor cells (EPC), pulmonary microvascular endothelial cells (HMVEC-L) and pulmonary arterial cells (HPAECs) human vascular and lymphatic endothelial cells derived from pluripotent stem cells, and pulmonary venous cells.

[0160] Aspect 8. The fluidic chip of any one of Aspects 1-7, wherein the central channel has therein a perfusable vascular network comprising endothelial cells, a perfusable lymphatic network comprising endothelial cells, or a perfusable vascular- lymphatic network comprising endothelial cells.

[0161] Aspect 9. The fluidic chip of Aspect 8, wherein the perfusable vascular network comprises at least one vessel that places the central channel into fluid communication with the at least one side channel.

[0162] Aspect 10. The fluidic chip of any one of Aspects 1-5, wherein the side channel includes an endothelium having a luminal surface.

[0163] Aspect 11. The fluidic chip of Aspect 10, wherein the luminal surface is in fluid communication with a source of fluid.

[0164] Aspect 12. The fluidic chip of any one of Aspects 1-11, wherein the fluidic chip is disposed such that the at least one side channel is in fluid communication with a well of a multi-well plate.

[0165] Aspect 13. The fluidic chip of Aspect 12, wherein the fluidic chip is configured to be placed into register with a multi-well plate.103241.024775 / 22-10103

[0166] Aspect 14. The fluidic chip of any one of Aspects 1-13, wherein at least a portion of the fluidic chip is in fluid communication with an agent and / or a stimulus. As an example, a chip can be disposed in a chamber or other unit that is in turn in fluid communication with a supply of an agent, such as a toxicant or other substance under evaluation.

[0167] As but one non-limiting example, a chip according to the present disclosure can have small airway epithelial cells comprised therein, and the chip can in turn be in fluid communication - interruptible or otherwise - with a supply of a known or suspected toxicant. In this way, a user can controllably expose the cells of the device to the agent and / or stimulus and then monitor one or more indicators associated with the cells to determine the effect, if any, of the agent and / or stimulus on the cells.

[0168] Aspect 15. The fluidic chip of Aspect 14, wherein the agent is a toxicant, the toxicant optionally comprising molecular chlorine. Other toxicants - such as other halogens - can be used, as molecular chlorine is but one example.

[0169] Aspect. 16. A system, the system comprising: a fluidic chip according to any one of Aspects 1-4, the system adapted to cause contact between the fluidic chip and at least one of a stimulus and an agent, the contact optionally being interruptable, the agent optionally comprising any one or more of a toxicant and a stimulant, and the stimulus optionally comprising a radiation.

[0170] A system can include, as an example, a train that controls the delivery of the agent to the fluidic chip. For instance, a system can include a train that modulates the concentration of the agent being applied to the chip. A system can also include a train that modulates the duration of the exposure of the agent to the chip. Such a train can include valves, controllers, and the like.

[0171] Systems according to the present disclosure can thus be used to evaluate the effect of a range of agents and / or stimuli. For example, an agent can be a gaseous agent, such as chlorine gas. A stimulus can be, for example, a radiation, such as cosmic rays.

[0172] Aspect 17. The system of Aspect 16, wherein the agent comprises any one or more of a toxicant and a stimulant.

[0173] Aspect 18. A method, comprising: causing contact between (1) at least one of an agent and a stimulus, and (2) epithelial cells disposed within the chamber of a103241.024775 / 22-10103 fluidic chip according to any one of Aspects 1-13; and monitoring at least one marker indicative of exposure of the epithelial cells to the at least one of an agent and a stimulus. The contact can be accomplished by, for example, opening a valve so as to place the agent into fluid communication with the cells. Such an arrangement can be used when the agent is a toxicant, such as chlorine gas. Alternatively, the contact can be accomplished by exposing the cells - via movement of a cover - to a radiation stimulus. As described elsewhere herein, the cells can be lung cells.

[0174] As an example, in the context of respiratory exposure to inhaled gases but it is also possible to perfuse the vessels with stimulants to model respiratory exposure to toxicants introduced into the systemic circulation through another route, for example, cornea, skin, gut, and the like. To measure tissue responses, one can stain specific markers indicative of cell viability, maturity, physiological function, and the like. One can also collect effluent from the epithelial chamber and the underlying vascular chamber to measure a broad range of biomolecules secreted by the exposed cells and tissues, including proteins, lipids, metabolites, nucleic acids, hormones, extracellular vesicles, and the like. Further, one can also harvest cells and their surrounding matrices from our models for RNA sequencing analysis. These tissues can also be processed for histological analysis.

[0175] Aspect 19. The method of Aspect, 18, wherein the marker is indicative of any one or more of tissue damage, a secretory product, golgi damage, an oxidative stress, a barrier function, a fibrinogenic response, a cell marker expression, a morphological change, an inflammatory response, a transcriptomic response, a lipidomic response, a metabolomic response, a proteomic response, and an unfolded protein response.

[0176] Aspect 20. The method of Aspect 18, wherein the at least one marker comprises any one or more of cell viability, apoptosis, necrosis, cell proliferation, DNA damage, cell sloughing, a golgi morphology or a change thereof, a golgi marker expression or a change thereof, a reactive oxygen species or a change thereof, a lipid peroxidation product or a change thereof, a protein oxidation product or a change thereof, a DNA oxidation product or a change thereof, an antioxidant secretion or a change thereof, an interleukin secretion or a change thereof, an endothelin-1 secretion or a change thereof, a secretion of mucus or a change thereof, a secretion of a surfactants or a change thereof, a103241.024775 / 22-10103 pulmonary edema, a barrier permeability, a TEER level or a change thereof, an impedance or a change thereof, a gene expression or a change thereof, a lipid expression or a change thereof, a protein expression or a change thereof, a metabolite level or a change thereof, a cell-specific marker expression or a change thereof, a TGF-a release or a change thereof, a TGF-P release or a change thereof, an ECM deposition or a change thereof, a PERK level, a CHOP level, an IRE1 level, an eIF2a level, and an ATF6 level. As an example, one can monitor changes in one or more molecules associated with the unfolded protein response.

[0177] Aspect 21. A method, comprising co-culturing airway epithelial cells, pulmonary microvascular endothelial cells, and lung fibroblasts using a fluidic chip according to any one of Aspects 1-13, the airway epithelial cells comprising airway cells or alveolar cells. Example co-culturing methods are described elsewhere herein.

[0178] Aspect 22. The method of Aspect 21, wherein the airway epithelial cells are disposed in the chamber, and the lung fibroblasts are disposed in the central channel.

Claims

103241.024775 / 22-10103What is Claimed:

1. A fluidic chip, comprising: a bottom region, the bottom region comprising a central channel and at least one side channel adjacent thereto, the central channel having therein a plurality of cells disposed in a matrix; and a middle region, the middle region having a chamber defined therein, the chamber optionally being an open-top chamber, the chamber being in register with at least one of the central channel and the at least one side channel of the bottom region.

2. The fluidic chip of claim 1, further comprising a pervious membrane, the pervious membrane being arranged between the chamber of the middle region and at least one of (i) the central channel of the bottom region (ii) the at least one side channel of the bottom region.

3. The fluidic chip of claim 2, further comprising epithelial cells disposed within the chamber, the epithelial cells optionally superposed on the pervious membrane.

4. The fluidic chip of claim 3, wherein the epithelial cells comprise any one or more of primary large airway cells, small airway cells, or alveolar cells.

5. The fluidic chip of any one of claims 1-4, wherein the matrix comprises a hydrogel.

6. The fluidic chip of any one of claims 1-4, wherein the plurality of cells comprises any one or more of fibroblasts, pericytes, vascular smooth muscle cells, and103241.024775 / 22-10103 resident immune cells, the resident immune cells optionally comprising any one or more of macrophages and dendritic cells.

7. The fluidic chip of any one of claims 1-4, wherein the plurality of cells comprises any one or more of human umbilical vein endothelial cells (HUVECs), endothelial progenitor cells (EPC), pulmonary microvascular endothelial cells (HMVEC-L), pulmonary arterial cells (HPAECs), human vascular and lymphatic endothelial cells derived from pluripotent stem cells, and pulmonary venous cells.

8. The fluidic chip of any one of claims 1-4, wherein the central channel has therein a perfusable vascular network comprising endothelial cells, a perfusable lymphatic network comprising endothelial cells, or a perfusable vascular-lymphatic network comprising endothelial cells.

9. The fluidic chip of claim 8, wherein the perfusable vascular network comprises at least one vessel that places the central channel into fluid communication with the at least one side channel.

10. The c fluidic hip of any one of claims 1-4, wherein the side channel includes an endothelium having a luminal surface.

11. The fluidic chip of claim 10, wherein the luminal surface is in fluid communication with a source of fluid.

12. The fluidic chip of any one of claims 1-4, wherein the chip is disposed such that the at least one side channel is in fluid communication with a well of a multi-well plate.

13. The fluidic chip of claim 12, wherein the chip is configured to be placed into register with a multi-well plate.

14. The fluidic chip of any one of claims 1-4, wherein at least a portion of the chip is in fluid communication with at least one of an agent and a stimulus.

15. The fluidic chip of claim 14, wherein the agent is a toxicant, the toxicant optionally comprising molecular chlorine.103241.024775 / 22-1010316. A system, the system comprising: a fluidic chip according to any one of claims 1-4, the system adapted to cause contact between the fluidic chip and at least one of a stimulus and an agent, the contact optionally being interruptable, the agent optionally comprising any one or more of a toxicant and a stimulant, and the stimulus optionally comprising a radiation.

17. The system of claim 16, wherein the agent comprises any one or more of a toxicant and a stimulant.

18. A method, comprising: causing contact between (1) at least one of an agent and a stimulus, and (2) epithelial cells disposed within the chamber of a fluidic chip according to any one of claims 1-4; and monitoring at least one marker indicative of exposure of the epithelial cells to the sample.

19. The method of claim 18, wherein the marker is indicative of any one or more of tissue damage, a secretory product, golgi damage, an oxidative stress, a barrier function, a fibrinogenic response, a cell marker expression, a morphological change, an inflammatory response, a transcriptomic response, a lipidomic response, a metabolomic response, a proteomic response, and an unfolded protein response.

20. The method of claim 18, wherein the at least one marker comprises any one or more of cell viability, apoptosis, necrosis, cell proliferation, DNA damage, cell sloughing, a golgi morphology or a change thereof, a golgi marker expression or a change thereof, a reactive oxygen species or a change thereof, a lipid peroxidation103241.024775 / 22-10103 product or a change thereof, a protein oxidation product or a change thereof, a DNA oxidation product or a change thereof, an antioxidant secretion or a change thereof, an interleukin secretion or a change thereof, an endothelin-1 secretion or a change thereof, a secretion of mucus or a change thereof, a secretion of a surfactants or a change thereof, a pulmonary edema, a barrier permeability, a TEER level or a change thereof, an impedance or a change thereof, a gene expression or a change thereof, a lipid expression or a change thereof, a protein expression or a change thereof, a metabolite level or a change thereof, a cellspecific marker expression or a change thereof, a TGF-a release or a change thereof, a TGF-P release or a change thereof, an ECM deposition or a change thereof, a PERK level, a CHOP level, an IRE1 level, an eIF2a level, and an ATF6 level.

21. A method, comprising: co-culturing airway epithelial cells, pulmonary microvascular endothelial cells, and lung fibroblasts using a fluidic chip according to any one of claims 1-4, the airway epithelial cells comprising airway cells or alveolar cells.

22. The method of claim 21, wherein the airway epithelial cells are disposed in the chamber, and the lung fibroblasts are disposed in the central channel.

Images