Bioreactor-derived EVs from placental MSCs show context- and donor-specific immunomodulatory trends in human 3D lung inflammatory models

BACKGROUND: Extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) have garnered attention as cell-free therapeutics due to their regenerative and immunomodulatory potential. Human placenta-derived stromal cells (hPSCs) are a particularly promising source owing to their accessibility, scalability, and superior proliferative capacity-yet the functional behavior of their EVs, especially in inflammatory disease contexts, remains poorly defined. This study introduces a novel integrated approach, combining a 3D microcarrier bioreactor for scalable EV production with advanced 3D human airway disease models, to resolve how donor variability and inflammatory context shape the immunomodulatory activity of hPSC-EVs. METHODS: hPSCs were expanded under Good Manufacturing Practice (GMP)-compliant conditions in both conventional 2D cultures and 3D stirred-tank bioreactors using microcarrier technology. EVs were isolated from conditioned media via tangential flow filtration and characterized by fluorescent nanoparticle tracking analysis (fNTA), flow cytometry and protein content. Functional effects of hPSCs and EVs were assessed in PBMC co-cultures and two human 3D airway models-cystic fibrosis (CF) and acute respiratory distress syndrome (ARDS)-based on the Epithelix SmallAir™ platform, integrating primary airway epithelium and macrophages at the air-liquid interface. Inflammation was induced with TNF-α (CF) or LPS (ARDS), and EVs or hPSCs were administered to both apical and basal compartments. RESULTS: 3D culture significantly increased EV yield without compromising quality. A key finding was a substantial donor-dependent variability in both hPSC and EV activity, which translated into distinct, model-specific immunomodulatory profiles. Notably, EV- and hPSC-mediated responses diverged across immune and epithelial compartments, indicating that EVs do not simply recapitulate parental cell function. In the 3D models, despite substantial heterogeneity, induction of IL-10 and Arginase-1 (up to 25-fold) in the macrophage compartment emerged as a consistent trend across experimental conditions. In contrast, parental hPSCs showed broader but less predictable cytokine modulation, including variable TNF-α suppression and context-specific effects across donors. CONCLUSIONS: Our findings demonstrate that hPSC- and EV-mediated immunomodulation is highly context-dependent and cannot be predicted solely from donor identity or culture format. Rather than identifying a single optimal condition, this study highlights the need for larger donor cohorts and functional profiling in advanced human models and supports the use of EVs as distinct, cell-free immunomodulatory entities with compartment-specific activity. Together, this work provides a translational framework linking GMP-compliant EV manufacturing with functionally relevant human disease modeling.