Engineered in vitro models to mimic the human alveolar barrier and their role in assessing toxicity of chemicals/mineral fibers

The exposure to chemicals and pollutants of the lung tissue affects the properties and functions of the alveolar wall leading to serious and often fatal outcomes. The study of biological events related to toxic substances is currently limited due to the lack of relevant preclinical models for testing and validation. To overcome ethical, experimental and technological limitations of the traditional in vivo and in vitro models, we exploit tissue engineering approaches to develop innovative platforms that provide an appropriate mimicking of the extracellular environment, multicellular architecture and physiological stimuli of human alveoli. We propose the implementation of a biomimetic in vitro models to reproduce the alveolar wall where an alveolar interstitial space is interposed between the alveolar epithelial and pulmonary endothelial basement membranes. A realistic barrier model was obtained by integrating an electrospun PCL-Gel membrane in ad-hoc modified transwell insert to mimic the alveolar basement membrane. A tri-culture was set by combining PCL-Gel membrane and fibroblasts (MRC-5)-laden collagen hydrogel where alveolar epithelial cells (A549) were seeded atop the fibroblasts (MRC-5)-laden collagen, under physiologic air-liquid interface (ALI) conditions, and lung microvascular endothelial (HULEC-5a) cells were cultured on the basolateral side of the PCL-Gel membrane. In vitro cell tests demonstrated that this multilayered model allowed to recreate a physiological-like environment promoting the expression of alveolar type I and type II epithelial cell markers for A549 cells as well as improving cell viability and barrier function. In addition, fluorescence images of the barrier model showed that cells maintained their typical shape after 7 days at ALI. Moreover, immunofluorescence staining of tight and adherent junctions demonstrated the formation of a tight barrier. The effect of different mineral fibers and chemical insults on barrier properties, inflammatory hallmarks and cell behaviour was evaluated to monitor the biological events associated to potentially toxic substances.