Dynamic mechanical stimulation of alveolar epithelial-fibroblast models using the Flexcell tension system to study of lung disease mechanisms

Mechanical strain plays a significant role in lung pathophysiology. Current two-dimensional (2D) <i>in vitro</i> 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 <i>in vitro</i> mechanical multicellular alveolar models using the Flexcell system may replicate the dynamic environment of <i>in vivo</i> 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.