In vivo models (mostly rodents) are currently accepted by regulatory authorities for assessing acute inhalation toxicity. Considerable efforts have been made in recent years to evaluate in vitro human airway epithelial models (HAEM) as replacements for in vivo testing. In the current work, an organotypic in vitro rat airway epithelial model (RAEM), rat EpiAirway™, was developed and characterized to allow a direct comparison with the available HAEM, human EpiAirway™, in order to address potential interspecies variability in responses to harmful agents. The rat and human models were evaluated in two independent laboratories with 14 reference chemicals, selected to cover a broad range of chemical structures and reactive groups, as well as known acute animal and human toxicity responses, in three replicate rounds of experiments. Toxicity endpoints included changes in tissue viability (MTT assay), epithelial barrier integrity (TEER, transepithelial electrical resistance), and tissue morphology (histopathology). The newly developed rat EpiAirway™ model produced reproducible results across all replicate experiments in both testing laboratories. Furthermore, a high level of concordance was observed between the RAEM and HAEM toxicity responses (determined by IC₂₅) in both laboratories, with R² = 0.78 and 0.88 when analyzed by TEER; and R² = 0.92 for both when analyzed by MTT. These results indicate that rat and human airway epithelial tissues respond similarly to acute exposures to chemicals. The new in vitro RAEM will help extrapolate to in vivo rat toxicity responses and support screening as part of a 3Rs program.

Transepithelial Electrical Resistance (TEER) Assessment

Transepithelial electrical resistance (TEER) was measured immediately prior to dose application (untreated control tissues only) and following the 21 h recovery period (all samples) to assess barrier function. AEM tissues were transferred into TEER buffer (700 µL) in 24 well plates at room temperature. TEER buffer (250 µL) was also applied to the apical surface of the AEM tissue. For the rat tissues assessed at CRL, if the TEER readings were not stable, an additional rinse with TEER buffer (400 μL) was performed. TEER was then measured using a MilliCell ERS-2 meter (Millipore™, with chopstick electrodes, CRL) or an EVOM-X (World Precision Instruments with EndOhm-12 chamber, MatTek). Resistance (Ω) readings were corrected for background by subtracting the mean resistance of cell-free insert membranes from each reading. The TEER buffer was removed, and the AEM tissues were transferred to fresh, pre-warmed assay medium (1 mL in six-well plates) and returned to culture (standard conditions, prior to dose application) or were used to determine post-exposure viability using the MTT assay, as applicable. TEER was then converted to Ω x cm² by multiplying the observed resistance by the surface area of the culture insert (0.6 cm²).

MTT Assay of Rat and Human EpiAirway™ Tissues

The viability of EpiAirway™ tissues was measured using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. MTT reagent and extractant solution were supplied as a kit (MTT-100, MatTek Corporation) and prepared following the supplier’s recommendations. The AEM tissues were transferred into 24 well plates containing MTT reagent (300 µL per well) and incubated for 1.5 h ± 15 min at standard cell culture conditions. At the end of the MTT incubation, AEM tissues were tapped dry on absorbent paper and transferred to a new 24-well plate containing MTT extract solution (1 mL/well). Additional MTT extract solution (1 mL) was added to the apical chamber of each tissue insert. Samples were protected from light and evaporation using aluminum foil and Parafilm® and incubated at ambient temperature on a plate shaker for ca 2 h ± 15 min, or overnight without shaking. Following extraction, the AEM tissue inserts were removed from the wells and discarded. The upper and lower extracts were mixed and duplicate aliquots (200 µL) from each sample were transferred to 96 well plates and measured in a plate reader at 570 nm and a reference wavelength of 650 nm. Sample results (A_570-650) were corrected for background (wells containing MTT extractant solution only), then expressed as a percentage of the appropriate vehicle control.