In Vitro primary human airway epithelial whole exhaust exposure

Abstract

The method outlined in this article is a customization of the whole exhaust exposure method generated by Mullins et al. (2016) using reprogrammed primary human airway epithelial cells as described by Martinovich et al. (2017). It has been used successfully to generate recently published data (Landwehr et al. 2021). The goal was to generate an exhaust exposure model where exhaust is collected from a modern engine, real-world exhaust concentrations are used and relevant tissues exposed to assess the effects of multiple biodiesel exposures. Exhaust was generated, gently vacuumed into a dilution chamber where it was diluted 1/15 with air and then vacuumed into an incubator containing the primary cell cultures for exposure. Exhaust physico-chemical properties including combustion gas concentrations and particle spectra were then analyzed using a combustion gas analyzer and a Universal Scanning Mobility Particle Sizer. 24 h after exposure, cellular viability and mediator release were measured using Annexin-V/PI staining and meditator multiplexing kits respectively. This method was generated to test biodiesel exhaust exposures but can be easily adapted for any type of engine exhaust exposure or even potentially other respirable environmental exposures such as woodsmoke. The main customization points for this method are:•Exhaust generated by a diesel engine equipped with EURO VI exhaust after treatment devices including diesel particulate filter and diesel oxidation catalyst.•The generated exhaust was diluted 1/15 with air to replicate real world exposure concentrations.•Used primary human airway epithelial cells obtained from bronchoscope brushings from multiple volunteers and reprogrammed to allow multiple, comparative exposures from the same individual.

Keywords: Airway epithelial cells; Biodiesel; Environmental exposure; Exhaust exposure; Exposure protocol.

Graphical abstract

Fig. 1

Example chromatograms for (a) Soy biodiesel, (b) Tallow biodiesel and (c) ultra-low sulfur diesel for comparison.

Fig. 2

A diagram of our exposure set-up, adapted from . This diagram was generated using Biorender.com.

Fig. 3

A diagram of our customized baffle plate. Cell culture dishes (35 mm wide) fit inside the bigger holes and exhaust is suctioned through the smaller holes.

Fig. 4

The gating strategy used to analyze the flow cytometry data. This figure was taken from supplementary. First cells were selected using forward and side scatter area (S2a). Next single cells populations were selected using forward scatter area and forward scatter height (S2b). Finally, cells were separated into Viable, Early Apoptotic, Late Apoptotic and Necrotic populations using the FIT-C channel to measure Alexa Fluor 488 Annexin V staining and the TEXAS Red channel to measure (S2c).

Fig. 5

Exhaust gas concentrations for the 1/10 diluted exhaust exposures. Results for a) nitrogen oxides (NO_x) and b) carbon dioxide (CO₂) are shown for the three tested biodiesel and commercial mineral diesel (ULSD). Significant results were obtained using gas concentrations and GAM methodologies (*= p < 0.05, **= p < 0.01, ***= p < 0.001, ****= p < 0.0001).

Fig. 6

Exhaust particle spectra for the concentrations for the 1/10 diluted exhaust exposures. The spectra for the three tested biodiesel and commercial mineral diesel (ULSD) are shown. Significant differences were obtained with total particle number using GLM methodologies (*= p < 0.05, **= p < 0.01).

Fig. 7

Raw viability data for 4 samples exposed to the 1/10 diluted exhaust of the three tested biodiesel and commercial mineral diesel (ULSD). No significant differences were obtained in comparison to Air controls, likely due to small sample size.

Fig. 8

Inflammatory mediator release for 4 samples exposed to the 1/10 diluted exhaust of the three tested biodiesel and commercial mineral diesel (ULSD). The results for a) IL-6, b) IL-8 and c) TNF-α are shown. No significant differences were obtained in comparison to Air controls, likely due to small sample size.