Airway Organ Culture: A Model to Study Pulmonary Biology

Introduction:: Discoveries in pulmonary biology and development of new therapies for airway diseases rely heavily on airway models. However, conventional models with animals or cell cultures have inherent limitations. Cell cultures lack three-dimensional (3D) anatomic structures and intact airway functions. On the other hand, animal models suffer from low experimental throughput, limited interventional approaches, and ethical concerns. In this work, we developed an airway organ culture model that preserves 3D anatomical structures, key airway defense functions, and offers enhanced experimental throughput. After two weeks of culture, we evaluated this model using histological and immunochemical staining, gene expression, and tissue function studies.


Materials and Methods:: Airway Organ Culture. Tracheal segments were harvested from newborn pigs 1-3 days after birth (Figure 1A). The trachea was opened ventrally, and 4-8 tissue specimen were collected with a 6 mm biopsy punch (Figure 1B). These biopsies were embedded basolaterally with Matrigel (25% v/v), exposed apically to 100% humidified air with 5% CO₂, and placed in 12 mm diameter Transwell inserts (Figure 1C). Tissues were cultured with basolaterally applied USG media. To model gene therapy, adenovirus carrying green fluorescent protein (GFP, 5x10^8 pfu/mL) and lysophosphatidylcholine (LPC, 0.1% v/v) were applied apically. After culturing up to 2-weeks, tissues were harvested.

Morphological, Molecular, and Functional Studies. Hematoxylin and eosin (H&E) stains were used to evaluate the morphology of airway organ culture. After culture, tissues were placed in 10% neutral buffered formalin, paraffin-embedded, sectioned (5 μm thickness), and stained with H&E. Mucins MUC5AC and MUC5B were stained to evaluate airway secretion function. After culture, tissues were placed in 4% paraformaldehyde, dehydrated in 30% sucrose solution, OCT-embedded, sectioned (5 μm thickness), and stained with MUC5AC and MUC5B antibodies. The delivery and expression of GFP was evaluated with confocal microscopy, using EpCAM and DAPI as counterstains. To study mucociliary clearance function, cultured tissues were removed from the Transwell inserts, pinned to surgical wax, and submerged in either Krebs or HEPES buffer with bumetanide (10 μM). Mucus secretion was stimulated with methacholine (2.5 μg/mL). Mucus was stained with green-fluorescent nanoparticles (20 nm diameter) and imaged with a confocal microscope over the course of 1 hr.


Results, Conclusions, and Discussions:: Airway organ explants survived the two-week culture and integrated with the Matrigel matrix. After 2-weeks of culture, tissue maintained physiological anatomical structures, including ciliated epithelia and submucosal glands, as seen in H&E-stained sections (Figure 2A). MUC5AC and MUC5B are key secreted mucins. MUC5AC localized to goblet cells on the airway surface, and MUC5B localized to submucosal glands, as shown by antibody staining (Figure 2B). Imaging of GFP, with EpCAM and DAPI counterstains revealed that the GFP was successfully transferred to and expressed in the epithelium (Figure 2C). After culture, tissues submerged in Krebs buffer and stimulated with methacholine displayed normal ciliary activity that swept mucus stands off the airways. In striking contrast, disrupting submucosal gland HCO₃^- and Cl^- secretion as a model of cystic fibrosis (with HCO₃^--free buffer and bumetanide) displayed impaired mucus strand detachment from submucosal gland ducts, leading to mucus accumulation on the airway surface (Figure 2D).

Our results suggest that this airway organ culture technique can maintain key organ-level structures and mucin secretion over the course of the two-week culture period. Key host defense functions (e.g., mucociliary clearance) are also conserved during culture. The disruption of mucus strands clearance in HCO₃^--free solution and bumetanide suggests our model can allow the study of cystic fibrosis pathogenesis. The selective transduction of GFP onto airway epithelia suggests that this method can be used to understand the role of epithelia in disease pathogenesis or be used as a rescue mechanism to investigate possible therapies for airway disease.

In summary, the airway organ culture technology has demonstrated its advantages in preserving airway structure and functions, allowing various interventions, and enabling enhanced throughput. With these advantages, we expect that this technology can be a powerful tool to study airway physiology and disease therapeutics.


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