B77-07 Ferret Precision-Cut Lung Slices: An Advanced Ex Vivo Model for Translational Lung Disease Research

Abstract Rationale Despite the extensive use of mouse models, studies on human lung development and disease mechanisms remain limited by anatomical differences across species and access to human lung tissue. Ferret lungs, in contrast, more closely resemble human lungs, thus providing a valuable alternative, but with limited experimental throughput. To overcome this limitation, we established a high-throughput ex vivo Precision-Cut Lung Slices (PCLSs) system to investigate structural cell responses to injury and drug treatments relevant to human lung diseases. Methods We established standardized protocols to generate PCLSs from ferrets between 5.5 and 14.5 weeks of age. The accessory lobe of the ferret was inflated with 3mL of 4% low-melting agarose and immediately submerged in cold PBS. Afterwards, 300-450 μm PCLSs were generated using a Compresstome and cultured overnight. Downstream analysis included whole-mount immunofluorescence (IF) staining and RNA quality assessment. Whole-mount IF staining was optimized for ferret PCLSs, enabling 3D reconstruction of the respiratory bronchioles and submucosal glands. We further established live-cell labeling and time-lapse imaging for dynamic cell tracking. Finally, we assessed RNA quality from the PCLSs cultured for 24 hours to 7 days to benchmark suitability for transcriptomic analyses. Results We successfully generated approximately 40 high-quality slices from each accessory lung lobe of ferrets. Whole-mount IF staining visualized multiple cell types in respiratory bronchioles and submucosal glands, two key respiratory structures in lung disease studies, that are absent in mouse lungs. Furthermore, we demonstrated the feasibility of labeling and tracking the erratic behavior of airway epithelial cells through live-cell imaging. In addition, RNA quality analysis identified the optimal culture window of the PCLSs for drug screening and potential gene modification studies. Conclusions Ferret PCLSs provide a novel and essential platform for studying lung development and diseases involving respiratory bronchioles and submucosal glands, two structures central to human pathology but absent in mouse models. By enabling stem cell differentiation, live-cell tracking, drug responses, and transcriptomic profiling, this system bridges basic, translational, and clinical research. Ultimately, it provides a powerful tool to advance mechanistic studies and the development of targeted therapies for human lung diseases. This abstract is funded by: NIH