Multi Species 3D Airway Tissue Models for Translational Inhalation Toxicity Studies

Scalable, reproducible in vitro 3-dimensional organotypic models from different species are needed for translational studies to develop reliable alternatives to traditional in vivo animal inhalation toxicity tests. The aim of this study is to compare toxicity responses of 3D airway tissue models constructed using primary tracheobronchial epithelial cells harvested from rat, primate, and human tissues. Primary cells from the different species were expanded in monolayer culture and seeded onto microporous membrane inserts to reconstruct 3D organotypic tissue models. Tissues were characterized for polarity of epithelial cells (histology), epithelial cell markers (IHC), barrier integrity (transepithelial electrical resistance, TEER measurements), and functionality in inhalation toxicological studies by testing 3 well-characterized chemical toxicants (CT). Polyethylene glycol was used as a non-irritant control and water, or corn oil was used as the vehicle control. CTs (100 µL) were applied to the apical surface, and tissue inserts were sealed with insert caps (MILICEL-MTK-CAP, MatTek Life Sciences) for 4 hr to mimic in vivo rat exposure experiments. After 4 hr, dosed tissues were washed and allowed to recover for 20 hr at 37oC. Analysis of the 3D tissues from the different species showed a well polarized epithelium with a physiological TEER values of >300 Ώ*cm2, cilia formation on the apical surface, and mucin production mimicking the airway microenvironment. Acute exposure to CTs for 4 hr showed varying levels of tissue viability and membrane integrity by MTT and TEER assays, respectively. The effective dose concentration that reduced tissue viability by 50% (ED-50) for vinyl acetate and chloroacetaldehyde were comparable (<2 mg/tissue) for all species, but the ED-50 value for toluene showed differences: human >20 mg, primate 16.2±1.7 mg, and rat 13.8±0.1mg. Based on the MTT viability and TEER values the test chemicals were rank ordered from high to minimal toxicity: chloroacetaldehyde > vinyl acetate > toluene > propylene glycol and the vehicle controls. While the human and primate airway models showed comparable MTT values, the rat airway tissue was more sensitive to the higher concentration of toluene. Although more chemicals need to be tested, the multispecies 3D airway tissue models will be vital translational tools to predict airway inhalation toxicity and to bridge the in vitro in vivo knowledge gap to reliably predict human responses, while providing an alternative approach to animal experimentation.​

EpiAirway (AIR-100-DAY20), EpiAirway-Rat (AIR-100-DAY20-R), vapor cap (MILICEL-MTK-CAP), rhesus monkeys, primates, vinyl acetate, toluene, chloroacetaldehyde, propylene glycol, corn oil, b-tubulin, cadherin, cytokeratin 5 (CK5), Mucin 5B (MUC 5B), TEER, MTT, histology interspecies testing, translational toxicology

vinyl acetate, toluene, chloroacetaldehyde, propylene glycol, corn oil