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Finding the Balance: Minimizing the Impact of Solvent and Volume on Toxicity Testing Endpoints During Liquid Application Dosing on Air-Liquid Interface Bronchial Epithelial Cultures
Citation:
Huber, E., O. Rice, L. Dailey, AND S. McCullough. Finding the Balance: Minimizing the Impact of Solvent and Volume on Toxicity Testing Endpoints During Liquid Application Dosing on Air-Liquid Interface Bronchial Epithelial Cultures. Society of Toxicology Annual Meeting, Nashville, TN, March 19 - 23, 2023.
Impact/Purpose:
The respiratory epithelium provides a physical barrier and immune defense against inhaled chemicals and pathogens. Damage to the epithelium results in a loss of barrier integrity and secretion of pro-inflammatory cytokines. The physiology and barrier functions of the respiratory epithelium can be recapitulated under air-liquid interface (ALI) conditions in differentiated primary human bronchial epithelial cell (pHBEC) culture. Inhalation is a primary route of exposure to chemicals in humans, however, there are challenges faced to bio-mimic the delivery of test agents to the respiratory epithelium in ALI cultures. A common exposure method used in chemical testing is liquid application which involves suspending a test agent in an aqueous solution and applying that suspension to the apical layer of cells, thus submerging cultures that once were exposed to air. The application of ALI medium to pHBECs caused a significant decrease in barrier integrity and significant increases in the secretion of pro-inflammatory cytokines and growth factors, as well as significant alternative regulation of more than 4,000 and 10,000 genes at 6 and 24 hours, respectively. These observations indicate that the effects of liquid application dosing alone may be a confounding factor in the use of this approach for inhaled chemical hazard identification. To refine dosing applications using liquid, we tested the impact of buffered salt solutions that are commonly used to resuspend test agents on ALI-differentiated pHBEC cultures. Additionally, we assessed the influence of volume (10, 30, and 50 µL) and time (1, 6, and 24 hours) in 6.5 mm Transwell inserts to minimize the effects of liquid application on physiologically relevant endpoints while maintaining uniform test agent distribution. We hypothesized that the application of buffered salt solutions would reduce epithelial barrier integrity and the release of pro-inflammatory cytokines. Unexpectedly, given that 0.9% saline is a commonly used aqueous solvent for liquid application dosing, cultures exposed to 0.9% saline during apical washing exhibited significantly decreased TEER values when matched to comparable exposures to ALI medium. TEER is a common physiological endpoint in in vitro pulmonary models for chemical testing and can be measured using either chamber or chopstick electrodes. While chamber and chopstick electrodes are used readily in the field, the position of the current electrodes differ leading to variations in successive measurements. When comparing chamber and chopstick methods in TEER following an apical wash with a respective buffered salt solution, the chopstick method over predicted TEER values. The factor of volume must balance the dispersion of a test agent and aim to maintain the physiology of cultures. The volume of 10 µL was unable to fully cover the apical surface of the cells, while 30 µL lead to a nonuniform distribution of test solutions. Following 24 hours of liquid exposure using 50 µL, pHBECs had decreased TEER values. As a result of the disruption of the tight junctions, future work will be analyzing the secretion of pro-inflammatory cytokines. Our findings indicate that methodology used within the inhalation field, such as volume and solvent selection during liquid application, may consequently be impacting the use of in vitro ALI systems for hazard identification. Does not reflect US EPA policy.
Description:
The United States of America produces an estimated 1500 new chemical substances per year with many of these chemicals released into the air. With a gap in data of these inhaled chemicals there is a need to develop scientifically reproducible and translational models for chemical testing to protect human health. The respiratory epithelium provides a physical barrier and immune defense against inhaled chemicals and pathogens. Damage to the epithelium results in a loss of barrier integrity and secretion of pro-inflammatory cytokines. The physiology and barrier functions of the respiratory epithelium can be recapitulated under air-liquid interface (ALI) conditions in differentiated primary human bronchial epithelial cell (pHBEC) culture. Inhalation is a primary route of exposure to chemicals in humans, however, there are challenges faced to bio-mimic the delivery of test agents to the respiratory epithelium in ALI cultures. A common exposure method used in chemical testing is liquid application which involves suspending a test agent in an aqueous solution and applying that suspension to the apical layer of cells, thus submerging cultures that once were exposed to air. The application of ALI medium to pHBECs caused a significant decrease in barrier integrity and significant increases in the secretion of pro-inflammatory cytokines and growth factors, as well as significant alternative regulation of more than 4,000 and 10,000 genes at 6 and 24 hours, respectively. These observations indicate that the effects of liquid application dosing alone may be a confounding factor in the use of this approach for inhaled chemical hazard identification. To refine dosing applications using liquid, we tested the impact of buffered salt solutions that are commonly used to resuspend test agents on ALI-differentiated pHBEC cultures. Additionally, we assessed the influence of volume (10, 30, and 50 µL) and time (1, 6, and 24 hours) in 6.5 mm Transwell inserts to minimize the effects of liquid application on physiologically relevant endpoints while maintaining uniform test agent distribution. We hypothesized that the application of buffered salt solutions would reduce epithelial barrier integrity and the release of pro-inflammatory cytokines. Unexpectedly, given that 0.9% saline is a commonly used aqueous solvent for liquid application dosing, cultures exposed to 0.9% saline during apical washing exhibited significantly decreased TEER values when matched to comparable exposures to ALI medium. TEER is a common physiological endpoint in in vitro pulmonary models for chemical testing and can be measured using either chamber or chopstick electrodes. While chamber and chopstick electrodes are used readily in the field, the position of the current electrodes differ leading to variations in successive measurements. When comparing chamber and chopstick methods in TEER following an apical wash with a respective buffered salt solution, the chopstick method over predicted TEER values. The factor of volume must balance the dispersion of a test agent and aim to maintain the physiology of cultures. The volume of 10 µL was unable to fully cover the apical surface of the cells, while 30 µL lead to a nonuniform distribution of test solutions. Following 24 hours of liquid exposure using 50 µL, pHBECs had decreased TEER values. As a result of the disruption of the tight junctions, future work will be analyzing the secretion of pro-inflammatory cytokines. Our findings indicate that methodology used within the inhalation field, such as volume and solvent selection during liquid application, may consequently be impacting the use of in vitro ALI systems for hazard identification. Does not reflect US EPA policy.