Establishing An Epithelial-Endothelial Co-Culture Lung-on-a-chip For Investigation In Healthy and COPD Conditions.

Two dynamic lung-on-a-chip co-culture models were developed to recreate the airway-vascular interface by incorporating primary airway epithelial and pulmonary microvascular endothelial cells. One model uses commercially available cells from healthy donors, whereas the other employs cells derived from patients with chronic obstructive pulmonary disease (COPD), enabling direct comparison of healthy and diseased conditions under identical culture conditions. Both models support epithelial differentiation under continuous perfusion and air-liquid interface (ALI) conditions, resulting in a physiologically relevant epithelial barrier supported by an underlying endothelial layer. The workflow includes extracellular matrix coating, cell seeding, ALI establishment, barrier function assessment, cell phenotyping, and cell type-specific RNA extraction. Permeability assays demonstrated robust barrier integrity in both systems, while immunofluorescence confirmed continuous localization of Zonula Occludens-1 (ZO-1) and vascular endothelial cadherin (VE-cadherin) at cell junctions. Epithelial differentiation was confirmed by β-tubulin IV staining and visualization of ciliary beating. High-quality RNA was obtained separately from each compartment, supporting cell type-specific molecular analyses. The COPD model recapitulated key features of the disease, notably enhanced mucus production, without compromising barrier function or cell viability. The use of patient-derived epithelial and endothelial cells preserves individual pathological characteristics, enabling investigation of intercellular mechanisms in COPD. This approach provides a physiologically relevant alternative to static in vitro and animal models and supports preclinical studies of inhaled drugs, pathogen exposure, and epithelial-endothelial interactions. Importantly, the healthy and COPD lung-on-a-chip models can be used either in combination for direct comparative studies or independently for mechanistic investigations, therapeutic testing, and drug discovery. The system can also be adapted to other respiratory conditions involving altered epithelial or endothelial compartments, offering a versatile and translational platform for respiratory research.