Roy J. Carver Department of Biomedical Engineering, University of Iowa, United States
Introduction:: Cystic Fibrosis (CF) lung disease is caused by mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR). A hallmark of CF is impaired mucociliary clearance (MCC) in the airways, which results in chronic infections and inflammation [1], causing the majority of mortality and morbidity. However, how MCC removes inhaled particles from the airway remains largely unknown. In this work, we discuss a “trachea-on-a-chip” approach, by integrating a porcine trachea explant into a microfluidic device, to analyze MCC. This approach has the following advantages in study of MCC. First, the movement of inhaled-like particles are studied on wildtype (WT) and CF pig trachea ex-vivo. The device allows for a large-scale analysis of hundreds of particles respective clearance off the airway surface. Second, the trachea-on-a-chip approach is advantageous in precisely controlling the biochemical and biophysical environment of the trachea, allowing real-time pharmacologic interventions on the ex-vivo tissue with strict temporal and spatial control. Third, porcine trachea explants are utilized as they share similar respiratory anatomy, biochemistry, physiology, and genetics with humans[2].
Materials and Methods:: Both WT and CF trachea explants were obtained from newborn pigs (< 3 days) via necroscopy. Trachea explants were opened ventrally (Figure 1A) and mounted onto an aluminum device, fabricated by CNC machining (Figure 1B). The device is composed of a basal component that allows for perfusion of Krebs buffer via integrated channels, as well as a supporting mesh platform to accept a trachea explant. In a subset of experiments, methacholine was applied through channels in the basolateral component at discrete timepoints. Air perfusion was applied through integrated channels in the apical component. The center of the apical component contains a window to image the trachea surface. Fluorescent particles of varied sizes (Green 6 µm and Red 102 µm, Fluoro-max, Thermo Scientific) were applied to both WT and CF trachea surfaces (Figure 1C). The device was placed in an environmental chamber to maintain temperature and imaged using a Nikon A1R confocal microscope. The resulting images obtained through confocal microscopy were stitched together and analyzed with Imaris software for particle tracking.
Results, Conclusions, and Discussions:: For all particle sizes on both WT and CF trachea explants, motions of particles were characterized by their minimum, median, and maximum velocities. The particle clearance was calculated by percentage of particles remaining (Figure 2). All measurements of CF MCC velocities were found to be statistically slower in comparison to WT. The percent clearance of particles on the explant surface were also demonstrated to be impaired in comparison to WT.
Prior to our work, clearance of inhaled particles on the airway surface has proven difficult to analyze due to experimental shortcomings. To overcome these shortcomings, our trachea-on-a-chip method utilizes tissues with diseased phenotypes, in parallel to WT, allowing for characterization of unique diseased conditions. To rescue CF MCC impairment, pharmacologic interventions were applied in real-time while tracking various sized particles. Future studies pertaining to MCC clearance will utilize particles with differing solubility profiles to probe the interaction of isolated mucus layers found in the airway surface liquid (ASL). The role of ASL fluid regulation and its implication on particle MCC can further be probed by modifying the microfluidic device. Pharmacologic interventions can be further applied to understand both pathophysiologic mechanisms of MCC impairment as well as serve to investigate therapeutic strategies.
In summary, the trachea-on-a-chip device allows for direct investigation of MCC phenomena. This method demonstrates that CF tissue has an intrinsically impaired capability of clearing particles regardless of size. This impairment is shown across minimum, median, and maximum values of tracked particles. CF MCC is incapable of reaching clearance speeds seen in wild type tissue and is unable to remove inhaled particles from a majority of its surface.
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References (Optional): : 1. Donaldson, S.H., et al., Mucociliary Clearance as an Outcome Measure for Cystic Fibrosis Clinical Research. Proceedings of the American Thoracic Society, 2007. 4(4): p. 399-405.
2. Rogers, C.S., et al., The porcine lung as a potential model for cystic fibrosis. American Journal of Physiology-Lung Cellular and Molecular Physiology, 2008. 295(2): p. L240-L263.
