A New Immortalized Human Alveolar Epithelial Cell Model to Study Lung Injury and Toxicity on a Breathing Lung-On-Chip System

Abstract

The evaluation of inhalation toxicity, drug safety and efficacy assessment, as well as the investigation of complex disease pathomechanisms, are increasingly relying on in vitro lung models. This is due to the progressive shift towards human-based systems for more predictive and translational research. While several cellular models are currently available for the upper airways, modelling the distal alveolar region poses several constraints that make the standardization of reliable alveolar in vitro models relatively difficult. In this work, we present a new and reproducible alveolar in vitro model, that combines a human derived immortalized alveolar epithelial cell line (AXiAEC) and organ-on-chip technology mimicking the lung alveolar biophysical environment (AXlung-on-chip). The latter mimics key features of the in vivo alveolar milieu: breathing-like 3D cyclic stretch (10% linear strain, 0.2 Hz frequency) and an ultrathin, porous and elastic membrane. AXiAECs cultured on-chip were characterized for their alveolar epithelial cell markers by gene and protein expression. Cell barrier properties were examined by TER (Transbarrier Electrical Resistance) measurement and tight junction formation. To establish a physiological model for the distal lung, AXiAECs were cultured for long-term at air-liquid interface (ALI) on-chip. To this end, different stages of alveolar damage including inflammation (via exposure to bacterial lipopolysaccharide) and the response to a profibrotic mediator (via exposure to Transforming growth factor β1) were analyzed. In addition, the expression of relevant host cell factors involved in SARS-CoV-2 infection was investigated to evaluate its potential application for COVID-19 studies. This study shows that AXiAECs cultured on the AXlung-on-chip exhibit an enhanced in vivo-like alveolar character which is reflected into: 1) Alveolar type 1 (AT1) and 2 (AT2) cell specific phenotypes, 2) tight barrier formation (with TER above 1,000 Ω cm2) and 3) reproducible long-term preservation of alveolar characteristics in nearly physiological conditions (co-culture, breathing, ALI). To the best of our knowledge, this is the first time that a primary derived alveolar epithelial cell line on-chip representing both AT1 and AT2 characteristics is reported. This distal lung model thereby represents a valuable in vitro tool to study inhalation toxicity, test safety and efficacy of drug compounds and characterization of xenobiotics.

A multiplex inhalation platform to model in situ like aerosol delivery in a breathing lung-on-chip

“…ALI along with CS culture conditions has a tremendous influence on the alveolar barrier and made it highly sensitive to the exposed NPs and PHMG. From our previous study (Sengupta et al, 2022) we had observed changes with the actin fibers on CS, which might hint towards a cytoskeletal rearrangement which facilitates better sensitivity to the exposed toxicants. Recent studies have shown convincing evidence that cell-ECM interactions play an important role for particle uptake kinetics and subsequent inflammation (Augustine et al, 2020;Engin et al, 2017;A;Lee et al, 2022).…”

“…As described previously in Stucki et al, 2018 and Sengupta et al, 2022 , the AX Lung-on-chip system (AlveoliX AG, Switzerland) comprises of the Lung-on-Chip (AX12) consumable along with two electro-pneumatic devices ( AX Exchanger and AX Breather), and the interface platform ( AX Dock) ( Figure 1B ). Cell seeding and medium exchanges were performed as recommended by the manufacturer (AlveoliX AG).…”

“…This cell line represents a valuable tool for in vitro toxicity studies, as it is derived from primary human cells and retains some of their characteristics. These cells were cultured according to manufacturer instruction as previously described and characterized ( Sengupta et al, 2022 ).…”

“…Although these advanced transwell ALI exposure models can be well used for upper airways, they are not suited to model the dynamic microenvironment of the distal alveolar space where cyclic stretch plays an imminent role in alveolar cell differentiation and lung disease progression ( Desai et al, 2008 ; Wu et al, 2018 ; Sehlmeyer et al, 2020 ). Exposing lung cells to ALI culture conditions along with physiological levels of cyclic stretch has been recently established to affect particle uptake ( Doryab et al, 2021 ), maintain alveolar characteristics ( Sengupta et al, 2022 ) and contribute to lung inflammation ( Wu et al, 2013 ). However, due to the lack of proper exposure systems, the relevance and importance for including cyclic stretch (CS) with ALI culture conditions has not been studied in depth for inhalation studies.…”

“…These studies demonstrate the relevance and potential of microfluidic technology in reproducing in vivo conditions and studying the mechanisms of chronic lung injuries in a controlled and precise manner. There are a few advanced lung-on-chip models for the lower airways that are currently available which can recapitulate crucial features of the native alveolar in vivo milieu like topography ( Huang et al, 2021 ) and breathing motion ( Huh et al, 2010 ; Stucki et al, 2018 ; Sengupta et al, 2022 ). However, adequate inhalation-based lung-on-chip models for the distal lung are largely missing ( Artzy-Schnirman et al, 2021 ).…”