Evaluation of in vitro rat and human airway epithelial models for acute inhalation toxicity testing

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 morpholo..., 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-we...,