generation-3d-collagen-embedded-a549mrc-5-alveolar-organoids-using

Generation of 3D Collagen-Embedded A549/MRC-5 Alveolar Organoids using a Mechanical Cell-Stretching Bioreactor

Although breathing dynamics and mechanical strain play critical roles in alveolar biology, their contributions to the pathogenesis of respiratory diseases ...

University of British Columbia - Okanagan

University of British Columbia

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.

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

Mechanical strain plays a significant role in lung pathophysiology. Current two-dimensional (2D)  models fail to capture the lung's dynamic mechanical environment. We developed mechanically strained 2D and more complex three-dimensional (3D) alveolar epithelial-fibroblast co-cultures and organoids using the Flexcell cell stretching bioreactor. To do this we used readily available human A549 epithelial cells and MRC-5 lung fibroblasts to establish 2D and 3D alveolar co-cultures and collagen-I-gel-embedded organoid models in the Flexcell and then strained them at 18% amplitude, 0.4 Hz for 24 h to mimic a pathological environment. The impact of mechanical strain on cell proliferation, morphology, cytoskeletal and tight junctional protein expression, interleukin-6 and-8 (IL-6, IL-8) inflammatory cytokine release, and cell death were assessed. Mechanical strain significantly increased total cell counts in 3D co-cultures but not in 2D co-cultures, potentially signifying increased proliferation. Morphological analysis revealed a marked transition of fibroblasts into broadened shape cells under strain in the 3D co-cultures. This was in line with increased F-actin in 3D co-cultures after strain. The tight junctional protein zonula occludens-1 expression decreased after strain in all 2D and 3D models. Furthermore, exposure to strain increased the release of IL-6 and IL-8. Strain-induced cell death was also elevated across all models, particularly in 3D cultures. This study presents exploratory findings suggesting that  mechanical multicellular alveolar models using the Flexcell system may replicate the dynamic environment of  lung tissue. These multicellular models offer a valuable platform for investigating strain-induced cellular responses relevant to inflammatory and fibrotic mechanisms in lung diseases, particularly in exploring epithelial-mesenchymal interactions that may underlie disease progression.

Article	Year
Establishing a 3D Vascularized Tri-Culture Model of the Human Airways via a Digital Light Processing Bioprinter. Biotechnology and bioengineering\| PubMed ID: 40298351	2025
Dynamic mechanical stimulation of alveolar epithelial-fibroblast models using the Flexcell tension system to study of lung disease mechanisms. Frontiers in medicine\| PubMed ID: 40901507	2025

Tony J. F. Guo

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