Advanced human lung organoids-on-a-chip to model epithelial-immune responses to viral infection

Respiratory viral infections remain a leading cause of global mortality, yet effective treatments for many pathogens are still lacking. Furthermore, animal models present ethical and experimental constraints and frequently result in poor clinical translation due to interspecies differences. Consequently, there is a critical need for physiologically relevant human in vitro models of the respiratory system to advance our understanding of viral pathogenesis and facilitate targeted therapeutic strategies. Most existing in vitro models rely on epithelial cells cultured on synthetic polymer membranes that fail to replicate the mechanical and biochemical properties of the native extracellular matrix and create an artificial barrier impeding co-culture with relevant immune cells required for physiological infection response. Moreover, in vitro infection studies have largely focused on innate immune cell recruitment of neutrophils, while the contribution of adaptive components remains less explored. Notably, non-circulating tissue-resident memory T cells (TRMs) have been shown to play a crucial role in the frontline defense against invading viral pathogens in vivo but are currently neglected in most other models. To address these limitations, I developed a hydrogel-based lung organoids-on-a-chip platform by culturing human airway organoid-derived epithelial cells on the surface of a hydrogel compartment offering a membrane-free and in vivo-like microenvironment. This "ALIchip" supported the epithelial differentiation at air-liquid interface (ALI) and recapitulated key structural and functional aspects of the airway epithelium, including barrier integrity, mucus secretion and ciliary beating. Next, I augmented the biological complexity of the ALIchip by integrating a functional immune compartment. Leveraging the membrane-free design of the hydrogel system, I established two co-culture approaches using peripheral T cells and lung TRMs. Under homeostatic conditions, peripheral T cells introduced in the basal reservoir infiltrated the hydrogel and migrated toward the epithelium. Conversely, hydrogel-embedded TRMs integrated into the epithelial layer, recapitulating the intraepithelial localization observed in vivo. Upon rhinovirus (RV) infection, peripheral T cells exhibited a robust, chemokine-induced recruitment into the interstitium-mimicking hydrogel, a dynamic not reproducible in conventional in vitro models. In contrast, RV-challenged TRM co-cultures responded with rapid interferon gamma release, enhanced patrolling behavior and donor-specific attenuation of viral induced ciliary loss, demonstrating the platform's capacity to model complex human-specific immune responses. Finally, I applied these hydrogel-based platforms to model the distal human alveolar epithelium. By combining bioengineering approaches including laser-ablation and micropatterning of hydrogels with alveolar organoid-derived cells, I recapitulated a three dimensional, in vivo-like alve