An Open-Source, 3D-Printable On-Plate Aerosol Delivery Array (OPADA) for In Vitro Aerosol Research

Inhaled therapies or delivery of pharmacological aerosols to the respiratory tract are central to medicine and more specifically, respiratory diseases. As such, in vitro aerosol exposure systems have become indispensable for initial drug screening and toxicity evaluation. While differentiated airway epithelial cell cultures at air-liquid interface (ALI) effectively model the respiratory tract, conventional exposure methods, such as adding dissolved compounds to culture media or bulk application to the apical surface, fail to replicate physiological aerosol deposition. These approaches either ignore polarized cellular responses or disrupt the ALI, causing hypoxic conditions that may alter toxicity and efficacy outcomes. While several commercial in vitro aerosol exposure systems are available, they can be cost-prohibitive, contain proprietary components, have limited throughput, and are not adaptable to different conditions. Therefore, open-source, standardized methods for in vitro aerosol delivery are urgently needed to accelerate innovation in respiratory toxicology and drug development. To address these limitations, we developed OPADA (On-Plate Aerosol Delivery Array), a novel open-source system for in vitro evaluation of inhaled drugs and toxicants. OPADA advances previous open-source designs by increasing deposition efficiency and enabling simultaneous multi-well, multi-compound exposures, enhancing throughput and experimental flexibility. We validated OPADA through proof-of-principle experiments demonstrating uniform and reproducible deposition. Furthermore, we confirmed OPADA's biological relevance using inhaled delivery of a nutraceutical (calcitriol) in airway epithelial cells at ALI, and pharmacokinetic evaluation of aerosolized pirfenidone in a complex donor-matched co-culture system. These findings establish OPADA as an effective, reproducible platform for in vitro evaluation of inhaled compounds.