Liquid Application Dosing Alters the Physiology of Air-Liquid Interface (ALI) Primary Human Bronchial Epithelial Cell/Lung Fibroblast Co-Cultures and In Vitro Testing Relevant Endpoints

Differentiated primary human bronchial epithelial cell (dpHBEC) cultures grown under air-liquid interface (ALI) conditions exhibit key features of the human respiratory tract and are thus critical for respiratory research as well as efficacy and toxicity testing of inhaled substances (e.g., consumer products, industrial chemicals, and pharmaceuticals). Many inhalable substances (e.g., particles, aerosols, hydrophobic substances, reactive substances) have physiochemical properties that challenge their evaluation under ALI conditions in vitro. Evaluation of the effects of these methodologically challenging chemicals (MCCs) in vitro is typically conducted by “liquid application,” involving the direct application of a solution containing the test substance to the apical, airexposed surface of dpHBEC-ALI cultures. We report that the application of liquid to the apical surface of a dpHBEC-ALI co-culture model results in significant reprogramming of the dpHBEC transcriptome and biological pathway activity, alternative regulation of cellular signaling pathways, increased secretion of proinflammatory cytokines and growth factors, and decreased epithelial barrier integrity. Given the prevalence of liquid application in the delivery of test substances to ALI systems, understanding its effects provides critical infrastructure for the use of in vitro systems in respiratory research as well as in the safety and efficacy testing of inhalable substances.

Impact/Purpose

Primary bronchial epithelial cell (pHBEC) cultures differentiated under air-liquid interface (ALI) conditions are the most physiologically relevant in vitro model available for regular use in inhalation toxicology research and testing.  The maintenance of ALI conditions is critical to the differentiation of these cultures into in vitro bronchial epithelial tissues and their ability to represent the corresponding tissue in the human respiratory tract.  Exposures to methodologically challenging test agents (e.g., aerosols and particles) are often conducted by the application of aqueous liquid (e.g., saline or cell culture medium) containing the test agent to the differentiated culture.  Despite the relatively common use of liquid application dosing of differentiated ALI cultures, the effect of liquid application alone on commonly used toxicity endpoints is poorly understood.  Using an organotypic differentiated pHBEC co-culture ALI tissue model, the study described in this manuscript examined the effects of liquid application alone (i.e., in the absence of any test agent) on common in vitro toxicology endpoints.  The application of liquid resulted in significant effects on global gene expression/transcriptional reprogramming, decreased epithelial barrier integrity, and increased pro-inflammatory cytokine and growth factor secretion.  The magnitude of the effect of liquid application on these common endpoints increased over time and was greater than that often observed with many known toxicants thus indicating a likelihood that this dosing method will confound the accuracy, sensitivity, and translation of data resulting from the use of liquid dosing of dpHBEC-ALI systems for inhaled chemical testing.  Additional information is available in the attached fact sheet. Inhalation is one of the three primary routes of chemical exposure, and many inhaled toxicants elicit adverse portal of entry effects in the lung.  Primary human airway epithelial cell cultures grown under air-liquid interface (ALI) conditions exhibit key features of the human respiratory tract in vivo and are thus a valuable resource for basic science research and inhaled chemical toxicity testing.  Many types of inhalable agents (e.g., particles, aerosols, hydrophobic substances, reactive agents, et cetera) have physical and/or chemical properties that pose significant challenges to their evaluation under ALI conditions using existing and accessible in vitro exposure apparatus.  In vitro testing to evaluate the potential toxicity of these methodologically challenging chemicals (MCCs) within the respiratory tract is typically conducted by “liquid application,” which involves the direct application of an aqueous solution containing the test agent to the apical surface of ALI differentiated primary human airway epithelial cell cultures.  Here, we report that the application liquid to the apical surface of a dpHBEC-ALI co-culture model results in significant reprogramming of the dpHBEC transcriptome and biological pathway activity, alternative regulation of cellular signaling pathways, increased secretion of pro-inflammatory cytokines and growth factors, and decreased epithelial barrier integrity.  Given the large number of data poor MCCs and the prevalence of liquid application in the delivery of test agents and biological stimuli, understanding its effects provides critical infrastructure for the use of in vitro systems in respiratory biology research and human health risk assessment.

Citation

Mallek, N., E. Martin, L. Dailey, AND S. McCullough. Liquid Application Dosing Alters the Physiology of Air-Liquid Interface (ALI) Primary Human Bronchial Epithelial Cell/Lung Fibroblast Co-Cultures and In Vitro Testing Relevant Endpoints. Frontiers, Lausanne, SWITZERLAND, 5:1264331, (2023). [DOI: 10.3389/ftox.2023.1264331]

DOI: Liquid Application Dosing Alters the Physiology of Air-Liquid Interface (ALI) Primary Human Bronchial Epithelial Cell/Lung Fibroblast Co-Cultures and In Vitro Testing Relevant Endpoints