Bioprinted Ventilated 3D Alveoliform Epithelial Sacculoids.

Understanding the human distal lung requires ex-vivo models that capture both the structural hierarchy and mechanical environment of alveolar sacs, yet current ex-vivo systems fall short. Manually cultured organoids and two-dimensional cell cultures lack structural hierarchy, apical access, and physiologic actuation via ventilation, limiting their use in modeling infection and mechano-transduction. Here, we established three-dimensional (3D) alveolar epithelial sacculoids (AES) by bioprinting pluripotent stem cell-derived alveolar epithelial type II cells (ATIIs) into defined 3D geometries in high density. AES reproducibly self-organized into multi-unit, lumenized sacs with polarized epithelia, surfactant secretion, and functional heterogeneity including proliferative ATIIs, surfactant-producing ATIIs, and transitional pre-alveolar type I transitional cell state (PATS)-like cells. A custom air-driven platform enabled fluid-mediated 3D ventilation, producing volumetric oscillations across closed sacs. This actuation engaged canonical (Ser127) and non-canonical (Tyr357, integrin-FAK-dependent) Hippo signaling, driving ATII-to-ATI remodeling and junctional stabilization. Upon apical infection with influenza virus, AES recapitulated canonical antiviral responses and epithelial plasticity resembling injury-induced alveolar repair, including depletion of functional ATIIs and emergence of proliferative ATIIs and transitional states. AES represent a physiologically ventilated model of the human alveolar niche, enabling mechanistic studies of epithelial plasticity, viral pathogenesis, and biomechanical signaling under physiologically-relevant conditions. Overall, our model provides a foundation for future integration of stromal, vascular, and immune components toward full alveolar mimicry to facilitate ex-vivo translational respiratory research.