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Abstract

Rationale: Mechanical strain plays a significant role in lung physiology and pathophysiology, influencing cellular behavior, tissue homeostasis, and mechanisms of disease such as inflammation and fibrosis. Current in-vitro models, particularly 2D monolayers and animal models, fail to capture the complexity of the lung's mechanodynamic environment. This study addressed this gap by developing advanced mechanodynamic 2D and 3D alveolar epithelial-fibroblast co-cultures and organoids to mimic the lung, using the unique Flexcell bioreactor that enables the application of strain to mimic the mechanical lung environment. Methods: Human alveolar epithelial cells (A549) and human lung fibroblasts (MRC-5) were used to establish 2D co-cultures in BioFlex plates as well as 3D alveolar co-cultures and organoids embedded in collagen-I-gels in Tissue Train plates. The models were then subjected to equibiaxial strain of 18% amplitude at 0.4Hz using the Flexcell tension system to mimic a pathological strain environment. The impact of mechanical strain on cell proliferation, morphology, cytoskeletal & tight junctional protein expression, IL-6 & IL-8 inflammatory cytokine release, and cell-viability were assessed via immunocytochemistry & confocal imaging, cell counts & identification, ELISAs, and lactate dehydrogenase (LDH) assays. Results: Mechanical strain of 18%, 0.4Hz, significantly caused predominantly increased cell proliferation rates in 3D co-culture models but not in 2D epithelial cell and fibroblast monolayers and co-cultures. Morphological analysis revealed a marked transition of fibroblasts into the broadened-shaped cells under strain, indicating myofibroblast differentiation in the 3D co-cultures. This was in line with increased F-actin in 3D co-cultures compared to decreased F-actin expression in epithelial cells and fibroblasts in 2D models after pathological strain. The expression of the tight junctional protein ZO-1 was decreased after strain in all 2D and 3D models. Further, there was increased release of pro-inflammatory cytokines, IL-6 and IL-8, in response to strain, and increased strain-induced cell death in all models, which was further heightened in 3D cultures. Conclusion: This study demonstrated that in-vitro mechanical multicellular alveolar models mimic the pathophysiological mechanodynamic lung environment. Specifically, 3D alveolar models in the Flexcell bioreactor provide a 3D configuration and multicellular environment necessary to replicate the in vivo mechanodynamic lung tissue, highlighting their importance for studying strain-induced cellular responses in line with inflammatory and fibrotic mechanisms in lung diseases. The developed models provide a valuable platform for exploring the mechanisms underlying lung disease progression and for testing therapeutic interventions aimed at restoring normal cellular responses to fatal injury from aberrant mechanical forces.