Membrane-Free Alveolus-on-a-Chip via Biodegradable Scaffold Recapitulates Interstitial Mechanics, Immune Trafficking, and Aerosolized mRNA Delivery

Abstract

The pulmonary alveolus is a highly specialized microenvironment where epithelial, interstitial, and immune components interact to maintain gas exchange and tissue homeostasis. In vivo, the air-blood barrier consists of an epithelial layer and a capillary endothelium separated by an ultrathin interstitium composed of extracellular matrix (ECM) and lung fibroblasts. However, most existing lung-on-a-chip platforms rely on permanent synthetic membranes, which fail to recapitulate the dynamic biological and mechanical properties of the native interstitium. Here, we present a membrane-free human alveoli-on-a-chip enabled by a biodegradable poly(lactic-co-glycolic acid) (PLGA) scaffold that is progressively replaced by fibroblast-derived ECM. This process reconstructs a biologically formed interstitial layer while preserving an alveolus-like dome architecture. The resulting system supports multicellular organization under air-liquid interface conditions, enabling epithelial barrier formation and surfactant-related phenotypes. Additionally, direct epithelial-fibroblast interactions enhanced surfactant-related phenotypes, as evidenced by increased SPC and LAMP3 expression. Importantly, we demonstrate that conventional rigid substrates promote fibroblast-to-myofibroblast differentiation, leading to elevated reactive oxygen species (ROS) production, increased epithelial cell death, and compromised barrier integrity. In contrast, the membrane-free PLGA system mitigates stiffness-driven myofibroblast activation, preserving epithelial viability and maintaining barrier function. These findings highlight the critical role of interstitial mechanics in regulating alveolar homeostasis and reveal limitations of conventional membrane-based platforms. The platform further enables chemokine-driven monocyte migration across the alveolar barrier, recapitulating key immune trafficking processes observed in vivo. In addition, aerosolized metal-organic framework (MOF) nanoparticles efficiently mediated mRNA delivery to epithelial and interstitial cells with minimal cytotoxicity and modest inflammatory responses. Together, this membrane-free alveoli-on-a-chip reconstructs essential structural, mechanical, and functional features of the human alveolar microenvironment and provides a physiologically relevant platform for studying pulmonary biology, fibrosis-related mechanisms, immune cell trafficking, and inhaled nanomedicine delivery.