Abstract

Rationale In vitro studies using air-liquid interface (ALI) cultures enable controlled investigation of human airway epithelial cell (HAEC) responses to clinically relevant exposures. Commercial in vitro exposure systems provide precise and reproducible dosage but require significant investment. Exposure science may benefit from a more accessible customizable open-source exposure system. We present 3D printed manifolds for applying a range of exposures uniformly across standard, commercially available 6- and 24-well plates with ALI culture inserts. Methods A chamber-style exposure system and designed manifolds were evaluated for exposure uniformity via simulations and deposition of nebulized FITC-labelled dextran. Chamber and manifolds were manufactured using 3D stereolithography printing. Cannabis concentrate vapour was generated from 3 different vaporizers and applied to well plates using the manifold system. Calu-3 cells and primary HAECs were cultured on Transwell™ inserts for exposure studies. Results The manifolds produced less variation in simulations and physical deposition of FITC-dextran aerosol across well plates compared to the chamber system. Distinct doses of cannabis concentrate vapour were delivered to well plates with minimal variation among wells. Whole tobacco smoke exposure using the manifold system induced functional changes in Calu-3 barrier function, cytokine production (IL-6 and IL-8), and cell membrane potential. Cannabis smoke led to reduced primary HAEC barrier function in a dose- and strain-dependent manner. Conclusions Our data demonstrate the feasibility and the validity of our open-source 3D printed manifolds for use in studying multiple exposures and position our designs as an accessible option in parallel with commercially available systems. All article content is licensed under a Creative Commons Attribution (CC BY-NC 4.0) license ( https://creativecommons.org/licenses/by-nc/4.0/ ).