Development of an advanced in vitro lung model for mimicking healthy and pathological states

The development of physiologically relevant in vitro models is essential for investigating tissue-specific functions and disease mechanisms while reducing reliance on animal testing. These models serve as fundamental tools in modern biomedical research, offering reproducibility, ethical compliance, and the potential for high-throughput screening, thanks to their ability to replicate the topographical, biomechanical and biochemical behavior of the physiological tissue. In this thesis, work focuses on the development of two biomimetic in vitro models to reproduce the bronchial epithelial and alveolar-capillary barriers using tissue engineering approaches. A Poly(ε-caprolactone)/gelatin (PCL-Gel) electrospun membrane was fabricated to more closely mimic the basement membrane of lung barriers. This membrane was accommodated in a Transwell® insert to obtain a platform that provides an appropriate mimicking of the extracellular environment, physiological stimuli and multicellular architecture of human lungs. First, the bronchial epithelial barrier was engineered by combining human bronchial epithelial cells (HBECs) with an MRC-5 fibroblasts-laden collagen hydrogel to reproduce the bronchial microenvironment by studying the interactions of adjacent cell types. The co-culture model was implemented on engineered transwell (coded as PCL-Gel TW) for 3-4 weeks under physiological air-liquid-interface condition (ALI). Immunofluorescence staining demonstrated the presence of both cell types and the formation of a tight barrier. Then to simulate healthy alveolar tissue, co-culture of human alveolar epithelial cells (A549), endothelial cells (HUVECs), and tri-culture of HUVECs, A549 and MRC-5 were developed. These were integrated into static (PCL-Gel TW) systems to simulate physiological conditions, supporting ALI conditions for 7 days. The model was optimized by considering variables such as coating procedures, HUVEC seeding densities, and timing strategies, all of which were determined to significantly influence barrier integrity, cell viability, and morphological organization. Additionally, a pathological model was developed by exposing the constructs to silica nanoparticles (SiNPs) and bleomycin (BLM) used as a positive control. These are commonly used in vitro to simulate lung inflammation and fibrosis. Finally, the biomimetic electrospun membrane was integrated into a commercial dynamic system (LiveBox2, IVTech S.R.L.) and the co- and tri-culture alveolar-capillary barrier models were reproduced to further enhance physiological relevance through the introduction of regulated fluid movement. These models were characterized through immunofluorescence staining for junctional proteins, confirming the formation of tight, polarized cellular barriers with appropriate marker expression under both static and dynamic conditions. This validation underscores the models’ ability to support tissue-like behavior in vitro. In the pathological model, altered expression patterns were observed, including the disruption or reduced localization of proteins such as ZO-1, indicating compromised barrier integrity. Additionally, a marked increase in the expression of inflammatory and fibrotic markers, such as alpha-smooth muscle actin (α-SMA), was detected, reflecting the inflammation and epithelial-to-mesenchymal transition (EMT) processes.