DOI: 10.5061/dryad.0rxwdbs56
title: Evaluation of in vitro rat and human airway epithelial models for acute inhalation toxicity testing

description: 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.
description: <strong>Transepithelial Electrical Resistance (TEER)
 Assessment</strong> 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 cm<sup>2</sup> by multiplying
 the observed resistance by the surface area of the culture insert
 (0.6 cm<sup>2</sup>). <strong>MTT
 Assay of Rat and Human EpiAirway™ Tissues</strong>
 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<sub>570-650</sub>) were corrected for background (wells
 containing MTT extractant solution only), then expressed as a percentage
 of the appropriate vehicle control.