Establishing a 3D Vascularized Tri‐Culture Model of the Human Airways via a Digital Light Processing Bioprinter

ABSTRACT The rise in chronic lung diseases globally and the corresponding lag in drug discovery in this field highlights the need for In Vitro models closely mimicking In Vivo lung tissue. Efforts to date have largely focused on In Vitro coculture models, often neglecting the pulmonary vasculature's role in lung physiology and lacking perfusability. To address this gap, we utilized digital light processing bioprinting to establish a complex three‐dimensional (3D) vascularized tri‐culture airway model. Models were generated using a photopolymerizable bioink consisting of 80% polyethylene glycol diacrylate (PEGDA) and 20% gelatin methacrylate (GelMa) and printed using the LUMENX+ bioprinter. Stiffness, diffusivity, and gel expansion were characterized. Models were printed with MRC‐5 lung fibroblasts embedded in hydrogels, while EA.hy926 endothelial cells and 1HAEo‐ epithelial cells were seeded on the luminal surface and on the apical domain, respectively. Endothelialization was achieved by coating lumens with matrix proteins, followed by perfusion‐based endothelial cell seeding and uniform distribution via rotating the model. Structural characterization, including immunofluorescence imaging, lactate dehydrogenase (LDH) viability, interleukin‐6 and interleukin‐8 quantification was performed following cigarette smoke extract (CSE) exposure. PEGDA/GelMa 80:20 hydrogels had a Young's modulus of 10.7 kPa, expanded by 101.5% in volume and 107% in weight after 24 h in phosphate‐buffered saline, and turned completely blue following 12 h of exposure to 0.1% methylene blue. Immunofluorescence staining revealed an intact apical epithelial and luminal endothelial layer demonstrated by E‐cadherin expression. Lung fibroblasts retained their spindle shape with dendritic extensions as shown by F‐actin staining. Propidium iodide staining demonstrated 80‐90% cell viability. Cigarette smoke exposure significantly increased IL‐6 and IL‐8 release, but not LDH release. A multiplex assay revealed distinct immune mediator profiles and clustering between co‐cultures and tri‐cultures at baseline, underscoring differences in intercellular communication. This study successfully engineered and characterized a 3D bioprinted vascularized tri‐culture model that mimics human airways. The model is adaptable to future studies by incorporating additional cell types, primary cells, or modified designs and protocols.