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Abstract

Rationale – The increased incidence of chronic respiratory diseases globally without the accompanying drug discovery has resulted in an increased demand for in vitro models closely mimicking the in vivo lung environment. However, the focus so far has mainly been on developing non-perfusable co-culture models. Interestingly, pulmonary vasculature, the main player in respiratory diseases is not accounted for. To bridge this knowledge gap, the present study utilized digital light processing bioprinting (DLP) to establish a three-dimensional (3D) vascularized tri-culture model to closely mimic the complex in vivo environment of the human airways. Methods - To create a 3D vascularized tri-culture model of the human airways, lung fibroblasts were first mixed with photopolymerizable bioink and printed using LUMENX+ bioprinter into the model designed via CAD modelling software. Endothelial and epithelial cells were then seeded in lumens and on top of models. Perfusion experiments were set up to flow cell culture media through models and develop endothelialization. Models were structurally characterized using immunofluorescence staining and cell viability analysis. Results - To establish tri-culture airway models, a unique combination of 80% Polyethylene glycol diacrylate (PEGDA) and 20% gelatin methacrylate (GelMa) photopolymerizable bioinks was prepared and mixed with lung fibroblasts and bioprinted into a monolithic structure with patterned lumens after which endothelial cells were seeded in the lumen coated with basement membrane proteins and flipped every 15 minutes for 4 hours. After this airway epithelial cells were seeded on top of the model while a specialized peristaltic pump was used to establish flow to endothelialize the lumens. Immunocytochemistry analysis demonstrated an intact apical epithelial and luminal endothelial layer demonstrated by junctional protein (e-cadherin) staining. Lung fibroblasts remained spindle-shaped with dendritic extensions as demonstrated by F-actin staining. Viability analysis with propidium iodide staining demonstrated 80-90% cell viability. Conclusion - The present study successfully engineered and characterized a working 3D vascularized tri-culture model closely mimicking human airways using DLP bioprinting. The model can also be easily adapted by the addition of other cell-types such as primary cells, and alterations to designs or the protocols for future studies.