Development of a biomimetic Alveolus-on-chip to model the physiological alveolar barrier by supporting multicellular culture and air-liquid interface implementation.

The respiratory system is exposed to harmful substances reaching the lung during the breathing cycle. For this reason, studying the role of alveolar-capillary barrier in the pulmonary homeostasis plays a fundamental role to understand the pathophysiology of diseases associated with this barrier, such as the asthma, Idiopathic pulmonary fibrosis (IPF), Chronic obstructive pulmonary disease (COPD) and Coronavirus disease 2019 (COVID-19). To achieve this purpose, engineering microdevices platforms called Organ-on-chips (OoCs) are implemented to study the mechanism underlying the functions of a tissue, analyzing the cells environment and fluidic behavior. OoCs are high-throughput in vitro models that describe the architecture, the functionality and dynamic physiological environment into a miniaturized scale in order to analyze in vivo pathological conditions. The objective of this Master thesis work is the development of an alveolus-on-chip system to mimic the alveolar barrier. The main goal is focused to recreate in vitro the thickest portion of the alveolar barrier characterized by the presence of a collagen hydrogel and the implementation of the air-liquid interface (ALI) in the apical compartment of the device to mimic the natural environment inside the alveolus. The system comprises a microfluidic platform made of polydimethylsiloxane (PDMS), which incorporates a nanofibrous PCL/Gelatin membrane (80:20) obtained by electrospinning to mimic the basement membrane of the alveolar barrier and a type I collagen hydrogel loading fibroblasts to reproduce the stromal tissue. The starting point for the development of this work is the optimization of a functional geometry of the PDMS device. The PDMS layers constituting the alveolus-on-chip are obtained from poly(methyl methacrylate) (PMMA) molds, designed through Rhinoceros software and realized by laser ablation technique with a Poly-Jet 3D printer. In order to ensure the ALI and to reduce the tendence of liquid to flow back towards the inlet and/or to rise above the nanofibrous PCL/Gelatin membrane, the two microfluidic channels are connected to valvular conduits designed on a Nikola Tesla’s original patent that are tested with colored water via microscope comparing the final device with and without valvular conduits.. A co-culture was set within the here design OoC by pouring collagen based hydrogel loading MRC-5 fibroblasts above the electrospun membrane and then seeding A549 epithelial cells atop. The resulting model is characterized at different time pointing through fluorescence imaging (DAPI, to perform the staining of the nuclei, and Phalloidin, to evaluate the staining of the actin filaments). The images show that the adopted multi-culture protocol allows the epithelial cells as well as fibroblasts inside the hydrogel to reach confluence with close cell-cell contact and a spread morphology, respectively. In addition, immunostaining tests were conducted to assess the barrier function of the model. These tests demonstrate the confluence of epithelial cells, which is determined by detecting the expression of E-cadherin (epithelial cadherin), and the enhanced proliferation of fibroblasts inside the bioinspired collagen hydrogel through the detection of Vimentin expression. Moreover, live/dead staining is carried out to evaluate the cell viability using fluorescent microscopy and, in order to prove the 3-Dimensions structure of in vitro alveolar barrier, Z-stack images are analyzed.