3D Rat Airway Tissue Model for Translational Toxicology Studies

A scalable and reproducible in vitro 3-dimensional organotypic model of rat airway that will replicate biological responses is needed to provide an alternative to traditional in vivo rat toxicity tests. The aim of this study was to test the utility of a primary cell-based 3D reconstructed rat epithelial airway tissue (REAT) model to screen toxicants, inhaled drugs, and formulations for irritation potential. To achieve this goal, primary cells were expanded in monolayer culture and seeded onto microporous membrane inserts to reconstruct 3D organotypic REAT tissues. Then, tissues were characterized for polarity of epithelial cells (histology), epithelial cell markers (confocal microscopy), barrier integrity (transepithelial electrical resistance, TEER), and drug transporters and drug metabolizing enzymes (RT- PCR). Functionality for toxicological studies was also assessed using 4 chemicals at four concentrations (5, 25, 125, and 250mg/mL) of different classification categories. Analysis of the 3D REAT revealed well polarized epithelium with a physiological TEER value of >300 Ώ*cm2 mimicking the airway microenvironment. RT-PCR results showed expression of drug transporters (influx and efflux) and drug metabolizing enzymes: aldehyde dehydrogenases (2, 3a1, 3b1, 5a1, 6a1, 7a1, and 9a1), CYP450 enzymes (1b1, 26b1, 2c80, 2f4, 2s1, 2t1, 3a9, and 4f6), and esterases. Acute exposure to the chemicals showed varying levels of tissue viability (toxicity) by MTT assay. Based on the minimum concentration required to reduce tissue viability by 40%, the test chemicals were rank-ordered from maximal to minimal toxicity: Propamocarb (25 mg/mL), Diazinon (25 mg/mL)> Methomyl (125 mg/mL)> Permethrin (>250mg/mL), vehicle control. Thus, the REAT tissue model will be a useful tool to predict rat airway toxicity and bridge rodent to human translation of in vitro inhalation toxicology data. Such models can also reduce the use of animals for experimentation.