Development and validation of an open-source, disposable, 3D-printed in vitro environmental exposure system for Transwell culture inserts

A schematic depicting the in vitro exposure system (IVES) connected to air and smoke sources. A three-way valve connects the cannabis cigarette to IVES through a 50-mL syringe. Another three-way valve connects room air to IVES. Smoke is drawn through the smoke exposure syringe and expelled with predetermined rate and volume into IVES. Room air is introduced with the fresh air syringe in a similar fashion. A heating pad positioned below IVES maintains the experimental system at 37°C (figure generated with BioRender).

Quantitative simulation for in vitro exposure system (IVES) using COMSOL Multiphysics with air used as the gas of interest for simulation. a) A three-dimensional view of the IVES with air flow streamlines showing vortices in IVES and how gases distribute. b) Top view with gas flow streamlines. c and d) The side views with gas flow streamlines. e) The top view of the velocity profile (m·s⁻¹) for the modelled gas presenting a uniform flow distribution among all four exposure chambers with a gentle velocity decrease. f) The velocity profile (μm·s⁻¹) at the close approximation to the surface where the cells were cultured. g) The shear stress (Pa) profile at the location that the cells were cultured. It should be noted that both air and smoke inlets are merged into a larger duct which is only shown in this figure.

Three-dimensional quantitative modelling results of the gas (CO₂) concentration distribution in an in vitro exposure system chamber: a) real-time CO₂ concentration distribution over 5 s exposure of smoke (initial CO₂ concentration modelled at 1.0 mol·L⁻¹) showing a gentle gas distribution in the exposure chamber; b) real-time CO₂ concentration distribution over 5 s exposure of fresh air (the initial CO₂ concentration was the final CO₂ concentration in the exposure chamber from the previous smoke exposure and it was assumed that there was no CO₂ in the fresh air exposed to the chamber); c) volume average concentration of CO₂ in the chamber for one smoke–fresh air cycle would lead to a drop in CO₂ concentration in the chamber back to zero; and d) average outlet concentration of CO₂ after one smoke–fresh air cycle confirming that exposure kinetics were sufficient to reach a repeatable smoke–air exposure cycle (zero concentration at the outlet).

a) Change in transepithelial electrical resistance (TEER) from baseline after room air versus whole cannabis smoke exposure. Analysed with paired t-test, p=0.029, n=10. b) Lactate dehydrogenase (LDH) expression as a proportion of maximal LDH release, analysed via ANOVA and Tukey's post hoc test. Shows representative microscopy (×40) of c) Transwell inserts with human bronchial epithelial cells (HBECs) prior to room air exposure, d) Transwell with HBECs after room air exposure, e) Transwell with HBECs prior to whole cannabis smoke exposure, and f) Transwell with HBECs after whole cannabis smoke exposure (product weight of 0.7 g). Representative microscopy (×100) of g) Transwell inserts with HBECs prior to room air exposure, h) Transwell with HBECs after room air exposure, i) Transwell with HBECs prior to whole cannabis smoke exposure, and j) Transwell with HBECs after whole cannabis smoke exposure (product weight of 0.7 g). *: p<0.05; ***: p<0.001.

Interleukin (IL)-1 cytokine family member quantification in apical washing of primary human airway epithelial cells exposed to air or whole cannabis smoke. a) IL-1α, p=0.054; b) IL-1β, p=0.296; c) IL-18, p=0.064; and d) IL-1R antagonist (IL-1Ra), p<0.05 (analysed via paired t-tests). *: p<0.05.

Antiviral cytokine quantification in apical washing of primary human airway epithelial cells exposed to air or whole cannabis smoke. a) CXCL10, p=0.110; and b) CCL5, p=0.252 (n=5, analysed via paired t-tests).