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Development of a microfluidic alveolus-on-chip supporting epithelial- endothelial cells co-culture and air-liquid interface implementation for the modelling of the physiological alveolar-capillary barrier

Martina Cicolini

Development of a microfluidic alveolus-on-chip supporting epithelial- endothelial cells co-culture and air-liquid interface implementation for the modelling of the physiological alveolar-capillary barrier.

Rel. Matteo Cocuzza, Gianluca Ciardelli. Politecnico di Torino, Corso di laurea magistrale in Ingegneria Biomedica, 2022

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The respiratory system, being the largest interface with the external environment, is constantly exposed to harmful substances reaching the lungs by inhalation, other than via the blood stream. The alveolar-capillary barrier, that constitutes the functional interface between air and blood and mediates the gas exchange in the lung, plays a fundamental role in the defence against pathogens and xenobiotics. Diseases associated with alveolar-capillary barrier dysfunction include asthma, idiopathic pulmonary fibrosis (IPF) and Coronavirus disease 2019 (COVID-19), caused by the RNA virus SARS-CoV-2: studying the role of alveolar-capillary barrier in pulmonary homeostasis may help to understand the pathophysiology of diseases and to discover new therapeutic targets. In this respect, in vitro models are useful alternatives to animal models, that present limiting species-specific differences in organogenesis, tissue organization and susceptibility to diseases, to study the mechanisms underlying the functions of a tissue and to develop personalized treatments for different pathological conditions. Organs-on-Chip are high-throughput in vitro models that strive to mimic the architecture, functionality, and mechanical and chemical cues of the physiological environment at a miniaturized scale, processing microscale fluids in channels that range in size from tens to hundreds of microns. The aim of the present work was the realization of an alveolus-on-chip that would face the challenge of developing a device able to support both the presence of an air-liquid interface (ALI) in the apical compartment of the device and the implementation of a mechanical stimulation of the barrier model, that would recapitulate the effects of the breathing motion on the physiological tissue. The device consists of a polydimethylsiloxane (PDMS) microfluidic platform able to embed a nanofibrous scaffold, hosting an alveolar-capillary barrier model. The model is a co-culture of epithelial (A549 cell line) and human lung microvascular endothelial cells (HULEC-5a) on the two sides of an electrospun bioartificial membrane, mimicking the human alveolar wall. Each of the three PDMS layers of the device was designed using the software Rhinoceros and fabricated using the replica molding technique, by casting PDMS in 3D printed molds obtained with a Poly-Jet 3D printer. The nanofibrous scaffold was fabricated electrospinning a blend solution of polycaprolactone (PCL) and gelatin, and its morphology was analysed through scanning electron microscopy. The cellular adhesion on the two sides of the electrospun membrane was evaluated performing the staining of the nuclei, with DAPI, and the actin filaments, with phalloidin, of A549 and HULEC-5a at different time points, before and after the implementation of the ALI condition. Immunostaining and permeability tests were performed to evaluate the barrier function of the model. At seven days after air-liquid interface implementation, the epithelial layer appears confluent and homogeneous over the nanofibrous membrane. The endothelial cells reach confluence as well and show a spread cytoskeleton. Results suggest that the design of the PDMS bottom layer impacts on the ability of the endothelial cells to reach confluence and long-term viability. The pattern of each component was therefore optimized to minimize the impact on cell migration and not to obstruct medium passage, crucial for cells survival.

Relators: Matteo Cocuzza, Gianluca Ciardelli
Academic year: 2021/22
Publication type: Electronic
Number of Pages: 93
Corso di laurea: Corso di laurea magistrale in Ingegneria Biomedica
Classe di laurea: New organization > Master science > LM-21 - BIOMEDICAL ENGINEERING
Aziende collaboratrici: UNSPECIFIED
URI: http://webthesis.biblio.polito.it/id/eprint/23802
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