Open-source, three-dimensionally printed manifolds for exposure studies using human airway epithelial cells

Area of Science:

• Exposure science
• 3D printing
• Airway epithelial cell biology

Background:

• *In vitro* air-liquid interface (ALI) cultures are crucial for studying human airway epithelial cell (HAEC) responses.
• Commercial exposure systems are effective but costly.
• There is a need for accessible, customizable exposure systems in exposure science.

Purpose of the Study:

• To develop and validate 3D printed manifolds for uniform application of exposures to ALI cultures in standard well plates.
• To provide an open-source, cost-effective alternative to commercial exposure systems.
• To assess the system's performance with different agents like cannabis concentrate vapor and whole tobacco smoke.

Main Methods:

• 3D stereolithography printing was used to manufacture chamber-style exposure systems and custom manifolds.
• Exposure uniformity was assessed using simulations and fluorescein isothiocyanate (FITC)-labeled dextran deposition.
• Calu-3 cells and primary HAECs were exposed to cannabis concentrate vapor and whole tobacco smoke using the manifold system.

Main Results:

• The 3D printed manifolds demonstrated superior uniformity compared to the chamber system in simulations and physical deposition.
• The system delivered distinct doses of cannabis concentrate vapor with minimal well-to-well variation.
• Whole tobacco smoke exposure induced changes in Calu-3 cell barrier function, cytokine production (IL-6, IL-8), and membrane potential.
• Cannabis smoke exposure reduced primary HAEC barrier function in a dose- and strain-dependent manner.

Conclusions:

• The developed open-source 3D printed manifolds are feasible and valid for *in vitro* exposure studies.
• These designs offer an accessible alternative to commercial systems for investigating diverse exposures.
• The system enables reproducible research on cellular responses to environmental agents.