In situ-Like Aerosol Inhalation Exposure for Cytotoxicity Assessment Using Airway-on-Chips Platforms

Abstract

Lung exposure to inhaled particulate matter (PM) is known to injure the airway epithelium via inflammation, a phenomenon linked to increased levels of global morbidity and mortality. To evaluate physiological outcomes following PM exposure and concurrently circumvent the use of animal experiments, in vitro approaches have typically relied on traditional assays with plates or well inserts. Yet, these manifest drawbacks including the inability to capture physiological inhalation conditions and aerosol deposition characteristics relative to in vivo human conditions. Here, we present a novel airway-onchip exposure platform that emulates the epithelium of human bronchial airways with critical cellular barrier functions at an air-liquid interface (ALI). As a proof-of-concept for in vitro lung cytotoxicity testing, we recapitulate a well-characterized cell apoptosis pathway, induced through exposure to 2 µm airborne particles coated with αVR1 antibody that leads to significant loss in cell viability across the recapitulated airway epithelium. Notably, our in vitro inhalation assays enable simultaneous aerosol exposure across multiple airway chips integrated within a larger bronchial airway tree model, under physiological respiratory airflow conditions. Our findings underscore in situ-like aerosol deposition outcomes where patterns depend on respiratory flows across the airway tree geometry and gravitational orientation, as corroborated by concurrent numerical simulations. Our airway-on-chips not only highlight the prospect of realistic in vitro exposure assays in recapitulating characteristic local in vivo deposition outcomes, such platforms open opportunities toward advanced in vitro exposure assays for preclinical cytotoxicity and drug screening applications.

PerfuPul—A Versatile Perfusable Platform to Assess Permeability and Barrier Function of Air Exposed Pulmonary Epithelia

“…Especially for substances that are rapidly absorbed after aerosol deposition, such delays could aggravate subsequent analysis. Sophisticated aerosol deposition on cells grown at an ALI was described by Artzy-Schnirman et al (2019a) for a morphologically inspired acinus-on-chip and also recently for a bronchial bifurcation mimic by Elias-Kirma et al (2020). Unfortunately, these devices were not characterized in terms of TEER measurements nor in the application of perfusion.…”

“…An anatomically inspired true-scale acini-on-chip allowed the investigation of the immune response of alveolar epithelial cells in co-culture with differentiated THP-1 macrophage-like cells to nebulized lipopolysaccharide (LPS) as a surrogate for bacterial infections (Artzy-Schnirman et al, 2019a). Recently, the same group used a branching airway-on-chip platform to realistically mimic the transport and deposition of aerosolized particulate matter and study its cytotoxic effect on normal human bronchial epithelial (NHBE) cells (Elias-Kirma et al, 2020). Other devices worth mentioning enable the displacement of a flexible membrane in a diaphragm-like motion (Stucki et al, 2015) in combination with perfusion (Cei et al, 2020) as well as the membrane-free culture of airway smooth muscle cells in coculture with epithelial cells embedded within a hydrogel (Humayun et al, 2018) or the culture of human alveolar epithelial cells in a gelatin methacryloyl hydrogel resembling alveoli-like hemispheres in a breathing lung-on-chip (Huang et al, 2021).…”

Ventilation-induced epithelial injury drives biological onset of lung traumain vitroand is mitigated with anti-inflammatory therapeutics

“…We chose Calu-3, a well-established lung cell line for preclinical pulmonary research that grows confluent layers in immersed conditions and forms barrier junctions. Previous lung-on-chip models have recapitulated other essential aspects of epithelial cell function such as ciliation 61 and mucus 41 secretion by culturing primary cells at an air-liquid-interface, but have excluded physiologically-relevant flow conditions critical to our study. Mucus is an aqueous secretion that offers a critical protective lining to epithelial cells covering the in vivo airways and serves to clear contaminants upwards via ciliary motion.…”

Emerging Technologies for In Vitro Inhalation Toxicology

“…The airway-on-chip platforms in this direction contributed to reliably mimic the exposure on in vitro models of respiratory inhalation. [9]…”