Development of a Rat Trachea Explant Model for Cystic Fibrosis Research
Bishop-Gylys, Matthew
Bishop-Gylys, Matthew
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Abstract
Cystic fibrosis (CF) is characterized by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that impair chloride transport. This causes the production of thick, sticky mucus which impairs the function of multiple organs, such as the lungs. While CFTR modulators have greatly improved patient outcomes, about 10% of patients with rare mutations are ineligible because they synthesize no CFTR protein. Developing effective therapeutics for this population requires more physiologically accurate pre-clinical airway models. Thus, this project aims to develop a rat trachea explant model that maintains in vivo-like tissue architecture over the course of a week. Tracheas were cultured at an air liquid interface on Gelfoam�. We evaluated media formulations (DMEM vs. DMEM/F12), handling methods (pinning whole trachea vs. pre-sectioning with no pins), and fixation protocols (storage in 4% PFA vs. 48-hour fixation in 4% PFA followed by storage in 70% ethanol). Viability and contamination were assessed over 7 days via H&E staining to verify the preservation of ciliated, pseudostratified epithelium. Initial parameters utilizing standard DMEM, pinned whole tracheas, and prolonged PFA storage resulted in complete epithelium loss in day 0 controls and within 24 hours of culture. By pre-sectioning tracheas and removing the need for pins to reduce mechanical stress, along with culturing in DMEM/F12 (with Amphotericin B removed after 24 hours to reduce toxicity), and utilizing ethanol storage, we drastically improved outcomes. This approach successfully maintained an intact, ciliated, pseudostratified epithelium without contamination for 7 days. This protocol establishes a baseline for extending explant cultures for longer term. Ultimately, this platform will be used to evaluate engineered epithelial cell therapies as a CF treatment and provides a translatable model for broader pulmonary research.
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Date
1/1/2026
Student Status
Senior (Graduating in 2026)
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Poster
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Biomedical Engineering
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College of Engineering and Mathematical Sciences
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Engineering