We used adult wild-type (WT) ferret tracheas for scratch injury assays ex vivo. Shortly after the animal was euthanized, the trachea was dissected and cut open longitudinally on the membranous side while the cartilaginous side was brushed with a stiff nylon brush (Justman Brush Company, cat#415140, d = 2 mm). Specifically, injury was performed either by making 3 overlapping streaks along the proximal-distal axis (limited injury) or by extensively brushing in all directions (total injury). The explants were then cultured in F-medium [3:1 (v/v) DMEM (Invitrogen): F-12 Nutrient Mixture (Gibco), 7.5% FBS, 1% Penicillin/Streptomycin, 125 ng/ml epidermal growth factor (Invitrogen), 25 μg/mL hydrocortisone (Sigma-Aldrich), 5 μg/mL insulin (Sigma-Aldrich), 0.01 mg/mL Gentamicin, 0.1% amphotericin B, 11.7 μM cholera toxin (Sigma-Aldrich) and 10 μmol/L Y-27632 (Tocris)] and pulsed with a nucleotide analog – 10 μM EdU (5-ethynyl-2′-deoxyuridine, Thermo Fisher Scientific C10340) on specified days after injury.

At various experimental timepoints the explants were fixed in 4% paraformaldehyde or 10% neutral buffered formalin for 2 h at room temperature on a rocker, then washed three times in PBS for 20 min each. For staining of proteins that require antigen retrieval, samples were additionally incubated in citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH = 6.0) at 55°C overnight. The samples were then washed in PBS three times and incubated in blocking buffer (20% donkey serum, 0.3% Triton X-100, and 1 mM CaCl₂ dissolved in PBS, pH 7.6), either overnight at 37°C or for 24–72 h at 4°C. Tissues were rinsed in PBS, after which EdU was detected using the Click-iT™ EdU Cell Proliferation Kit for Imaging (Thermo Fisher Scientific), in accordance with the supplied protocol. The tissues were then washed three times for 20 min each in PBS and incubated with primary antibodies (dissolved in diluent buffer: 1% donkey serum, 0.3% Triton X-100, and 1 mM CaCl₂ in PBS, pH 7.6) for 18–24 h at 37°C with agitation (see Table 1). The tracheas were then washed three times for 20 min in PBS and incubated with secondary antibodies (dissolved in diluent buffer) for 24 h at 37°C, with agitation. Next, the tissues were washed three times for 20 min in PBS and submerged in Ce3D tissue clearing solution (Biolegend cat# 427704) for 2 h at RT or overnight at 4°C. Samples were mounted under 0.33 mm coverslips or under 1 mm glass slides and the edges were sealed with Gorilla Glue™ and clamped with binder clips for about 20 min to ensure glue fixation. Tile scan and Z-projection modules of a Zeiss LSM 880 or 980 line-scanning confocal microscope (Carl Zeiss, Germany) were used for imaging of the entire explant.

RNA was isolated from brushed off cells using Trizol regent (Ambion) following the manufacturer’s protocol. A High-Capacity cDNA Synthesis Kit (Applied Biosystems) was used to synthesize cDNA following the protocol provided by the manufacturer. qPCR reactions were set up using 10 nM primers, the POWER SYBR master mix (Applied Biosystems), cDNA, and H₂O. Reactions were run on a CFX Connect Real-Time PCR Detection System (Bio-Rad). The Delta–Delta-CT method was used to normalize the expression data. Primers are listed in Table 2.

Merged tile scans were quantified using ImageJ. Epithelial coverage of the explants was scored in ImageJ by dividing the area stained with epithelial markers (KRT5, KRT14, KRT15 or Tomato) over the total area of the explant. The % fraction of actively proliferating SMGs was calculated by dividing the selected areas with discretely high EdU+ cell density by the total area of all the SMGs as defined morphologically by staining with Krt14/5 and/or αSMA and Sox9.

Unless stated otherwise in a figure legend, results are reported as mean +/− SEM, individual dots on the graphs represent biological replicates, and statistical analysis was conducted using Prism version 9 (GraphPad Software) where N equaled the number of independent animals. The statistical tests used are stated in each figure legend. Data were considered significant at p < 0.05.

Whole-mount tissue staining provides opportunities to capture aspects of the cellular architecture and organization that are often lost in 2D tissue sections. Furthermore, because a greater surface area is covered in such preparations than in tissue sections, this approach makes it possible to capture cellular events at the whole organ level. This approach is also well suited for capturing rare cell types and structures in the organ of choice, for example, the ionocytes in the airway epithelium. We developed an accessible and cost-effective method for whole-mount staining, clearing, and embedding of the ferret trachea (Figures 1A–C) that enables clear visualization of SMGs (Figures 1D–F). Specifically, we found that between each cartilage ring, ferrets have two rows of SMG acini, each perfused with blood vessels (20–25 μm in diameter) that branch laterally to the acini bundles (Figure 1D). Additionally, SMGs appear to be the most innervated structure in the trachea, with a dense network of thick Ac-Tubulin+ nerve bundles surrounding the acini (Figures 1E,F). This whole-mount staining approach can be used to evaluate anatomical structures over a large tissue area. For example, we imaged tracheal fragments 7–7.5 cm in length, from four animals, covering 100% of the width and about 90% of the length of an adult ferret trachea, to evaluate the SMG distribution. Our examination of ROSA-TG transgenic ferrets revealed that SMGs are located only on the ventral side of the trachea, between the cartilage rings, and that their abundance declines slightly along the distal axis (Figures 1G,H). In summary, our whole-mount staining and confocal microscopy experiments reveal the complexities of the structure, innervation, and vascularization of the trachea.

Next, we determined the culture conditions conducive to robust SAE repair and to proliferation of SMG progenitors after injury. We initially tested two media: DMEM containing antibiotics and antifungal agents; and F-medium, which contains DMEM, F12, 7.5% FBS, and other additives (see the methods for details). DMEM was chosen for its simplicity because we hypothesized that tracheal explants can produce everything that they require for prolonged survival and repair after injury. F-medium was chosen because it is permissive for the survival of both epithelial and mesenchymal cells when used in colony formation efficiency (CFE) assays of airway basal cells that are grown on 3 T3-J2 fibroblasts (15, 16).

The tracheal lumen of ferrets was subjected to mechanical injury using a stiff 2-mm-wide nylon brush. This left a gap in the SAE that was partially repaired by basal progenitor cells over the course of 15 days (Figure 2A). We observed that in basal cells KRT14 was upregulated at the leading edges of the scratch (orange dashed lines in Figures 2B–H). This is consistent with the observations that KRT14 is upregulated in different animal models of airway injury, for example with SO₂, Cl₂, naphthalene, and polidocanol as well as in human airway diseases, such as idiopathic pulmonary fibrosis (IPF), restrictive allograft syndrome (RAS), and bronchiolitis obliterans syndrome (BOS) (17–21). KRT14 is not the only airway injury response marker, but it has been one of the more robust and consistent injury-induced gene markers that our laboratory has focused on in the past (16–18). Cellular changes such as upregulation of KRT14 and cell proliferation, as well as altered cell morphology at the wound edges, were more pronounced in F-medium than in DMEM on days 2, 5 and 15 (Figures 2B–G). These findings suggest a more robust injury response in F-medium. Additionally, in the explants cultured in F-medium but not in those cultured in DMEM, a wide band of cells along the scratch boundary incorporated EdU, indicating that they were actively proliferating. Explants cultured in F-medium covered most of the denuded scratch by day 15, whereas those cultured in DMEM showed no signs of scratch re-epithelization by day 21 (Figures 2F,G). After 15 days of recovery while submerged in F-medium, the regenerated epithelium had not differentiated into ciliated cells; however, ciliated cells were retained at the uninjured periphery of the scratch (Figure 2H). We conclude that in the ferret ex vivo tracheal injury model F-medium is better suited than DMEM for regeneration studies.

The Rho Kinase inhibitor Y-27632 is a standard component of F-medium and is frequently used in culturing airway stem cells. We evaluated the effects of Y-27632 on SAE wound closure and SMG proliferation in our ex vivo tracheal injury model. When examining the SMGs following complete removal of the SAE by brushing, we noticed three distinct glandular phenotypes: “passive”, “active”, and “compromised” (Figure 3A). When explants were pulsed with EdU for 24 h on day-3 post injury (DPI3) and fixed on DPI5, “passive” glands contained few EdU+ cells whereas “active” glands contained many and this often coincided with increased KRT5 staining (Figures 3C,D). On DPI5 after total injury (extensive luminal brushing), “active” glands were typically found below KRT5+/EdU+ epithelium on the tracheal surface, suggesting that SAE regeneration involves migration of progenitors from these glands to the airway surface (Figure 3B). Glands of the “compromised” category were relatively rare, comprising <5% of the total gland volume; they incorporated no EdU and were negative for SOX9 (SMG marker, Figure 3E). This gland type most likely represents the SMGs that are no longer viable but retain keratin remnants, and thus also the distinct glandular morphology. We observed that the injured explants cultured in F-medium with Y-27632 contained higher percentages of ‘active’ glands (24.6% +/− 3.3% vs. 9.2% +/− 1.1%) and had more extensive SAE re-epithelialization (15.7% +/− 1.1% vs. 3.5% +/− 1.9% on DPI5 and 54.1% +/−3.0% vs. 22.5% +/− 7.3% on DPI9) than those cultured in F-medium without Y-27632 (Figures 3F–I). We conclude that Y-27632 stimulates SAE regeneration by glandular progenitors, and therefore selected F-medium with Y-27632 as the main culture method for subsequent experiments. Interestingly, most of the SMGs did not actively proliferate even when the explants were continuously cultured in the presence of EdU for 9 days after a total surface brushing. Only 37.3% +/−3.7% of the glands were actively proliferating (Figures 3I,K,L). If this phenomenon holds true in vivo, this has interesting implications as a natural mechanism to prevent replicative exhaustion of the glandular SC niche.

An important area of interest for cell therapy applications is the efficiency of transgenic cell engraftment (22, 23). With this in mind, we tested the ability of Tomato+ primary basal cells from ferrets to engraft onto intact, partially scratched, and fully scratched WT ferret tracheal explants. We hypothesized that reduced competition with endogenous airway epithelium would facilitate the engraftment of transgenic cells and therefore the efficiency will be higher in the context of total and partial injury versus uninjured explants. We tested this by seeding tracheal explants with 2*10⁶ Tomato+ passage-6 (p6) ferret surface airway basal cells immediately after partial or complete brush injury or onto uninjured control explants (Figure 4A). After the lumen was seeded with Tomato+ cells, the tissues were cultured for 2 or 15 days in F-medium containing Y-27632. We observed that infrequent patches of Tomato+ cells, that engrafted onto uninjured explants on DPI2, stained for KRT14 more intensely than the surrounding Tomato-native SAE (Figure 4B). This outcome was expected because KRT14 is rapidly induced in cultured SAE basal cells (16). In the explants with partially injured SAE, Tomato+ cells were incorporated primarily into the injured regions on day 2 (Figure 4C). In the explants with fully brushed surface after a total injury, SAE Tomato+ cells were incorporated throughout the surface as well as into the SMG ducts on day 2 (Figure 4D); Tomato+ cells were also incorporation into the SMG acini and their differentiation into αSMA+ MECs was observed, but this was very rare (Figure 4E). On day 15, Tomato+ SAE coverage was similar to that on day 2, and it was the highest in the explants subjected to a total injury, where engrafted cells covered 34.0% +/− 4.3% of the explant lumen (Figure 5A). In explants that received a limited scratch injury before cell engraftment, Tomato+ cells were incorporated mostly into the brushed region covering 29.6% +/− 5.1% of the scratch and 13.8% +/− 0.9% of the total explant. Uninjured explants incorporated the fewest Tomato+ cells in the SAE, covering 1.7% +/− 0.6% of the lumen (Figures 5A–C). These findings demonstrate that an injury that creates space for stem cells to attach prior to engraftment can improve their attachment efficiency. Staining of the explants with KRT5 and EdU click chemistry revealed that at least some of the engrafted cells were EdU+/KRT5+ (Figures 5D–G), suggesting that they were able to divide after engraftment which was evident by their incorporation of EdU that was pulsed for 24 h on DPI5.

To better understand the factors that influence the efficiency of cell engraftment, we assessed the differences in the transcription of cell adhesion related genes between Tomato+ cells seeded on a denuded tracheal graft and an ECM-coated cell culture dish. To this end, we removed the SAE from freshly excised tracheas using a series of SDS-containing detergent washes as described previously (24). Then, we engrafted 10⁷ p6 Tomato+ primary surface airway basal cells into a 3-cm ferret tracheal segment using a rotating bioreactor method developed by our group previously for ferret tracheal explant cultures (24). After 18 h, the explants were rigorously washed with PBS to remove any cells that did not fully engraft, and then the luminal surface of the tracheas was brushed to collect Tomato+ cells. RNA from these cells was compared to that of Tomato+ primary basal cells that had been seeded on 804G-coated culture dishes for 18 h (Figure 6A). Subsequent qPCR analysis revealed that expression of ITGB1, ITGA6 and ITGA3 was lower in the cells seeded on tracheal grafts than in those seeded on culture plates (Figure 6B). Expression of CD44, a surface receptor that binds the ECM and promotes cell adhesion was unchanged as was the expression of Tomato, which served as a control to ensure that we had primarily collected the newly engrafted Tomato+ cells and not the native cells of the Tomato-explant (Figure 6B). These data suggest that downregulated integrin expression could account for the decreased efficiency of engraftment of cells on the basal lamina of the tracheal explants versus ECM-coated culture dishes.

Efficient cell engraftment in injured airways is dependent not only on the donor cells but also the injury response of native epithelium that remains present. Therefore, we evaluated how different degrees of explant injury impact the native SAE during regeneration. To this end, we evaluated the cells on the explant surface after 14 days of regeneration following either partial or complete removal of the original SAE by luminal brushings. In the case of partial brush injury, regeneration of the SAE involved both the remaining surface basal cells and SMG progenitors, whereas in the case of total injury, regeneration was attributed primarily to the glandular progenitors. On DPI14 we collected the SAE cells from regenerating explants by brushing the lumen and isolated RNA from them for evaluation by qPCR (Figure 6C). Given that this was a proof-of-principle experiment, we focused on gene targets that we expected to be upregulated in regenerating airway epithelia. Although this method can be used for unbiased screens of transcriptional changes associated with regeneration using single-cell or bulk RNA-sequencing, in this experiment we focused on the expression of three genes. Two of them were MMP9 and LAMA3 that are both associated with ECM deposition and remodeling and are upregulated in epidermal wound healing (25, 26). We also assessed the expression of SOX9, a marker of airway SMG progenitors that have been previously shown to participate in SAE regeneration (15, 27, 28). As expected, all three of these targets were upregulated in both severely injured and moderately injured epithelia, with the level of upregulation corresponding to the level of injury (Figure 6D). We additionally verified that the proportion of basal cells (KRT5+) and non-basal cells (KRT8+) was similar between the treatment groups at the time of RNA collection by immunofluorescent staining (Figures 6E–G). Consistent with the published literature (29) and our prior observations (15, 16), we noted that epithelial cells on the airway surface were larger and incorporated EdU more frequently in explants that were injured versus uninjured (Figures 6E–G).

One of the greatest limitations of mammalian in vivo systems is the difficulty of performing high-resolution live imaging (30). An ex vivo culture system has brought us a step closer to circumventing this limitation. Existing methods for live imaging of the tracheal explants involve pinning the tissue to the support stage and imaging it under a dissecting microscope. This method works well for terminal assays such as mucociliary clearance, but is unsuitable for long-term culture of the explants. Additionally, the resolution and magnification of a dissecting microscope is limited even though individual cells can be resolved. For these reasons we sought to develop an explant culture chamber in which tissue can be positioned for imaging with a confocal laser scanning microscope while sterility is maintained. We designed and 3D-printed a 3-piece device that can keep epithelial tissue explants such as tracheas at an ALI and position them for confocal imaging (Figures 7A–I). Conceptually, an assembled device loaded with an explant resembles a transwell™ (Corning). Therefore, we will refer to it as a “tissue-transwell” for simplicity. The key difference between our device and traditional transwells that are available for in vitro culture is that instead of the permeable support for cells at the bottom, the tracheal explant itself fills the opening at the bottom of tissue-transwell acting as a barrier between the two compartments (which can be filled with media or be exposed to air). During assembly of the tissue transwell, the tissue is placed between the top and bottom parts of the cassette and this “sandwich” is screwed into the bracket (Figure 7B). The loaded tissue-transwell can be cultured in a 6-well dish or in a special 3D-printed housing chamber. Different sizes of this device were designed for mouse/rat, ferret, and human explants and printed using biocompatible plastic Med 610 HQ (31). After autoclaving the devices, we loaded them with fully brushed WT ferret tracheas and seeded the lumen with 2*10⁶ Tomato+ ferret airway basal cells. Seven days later we performed live imaging of the explants, confirming that this system is compatible with live imaging and that the newly engrafted cells were viable and able to migrate on day 7 after seeding (Figure 7J).

The main purpose of designing the tissue-transwell was to open the tracheal tube and position the airway lumen near the microscope objective for imaging. A secondary purpose was to compartmentalize the luminal and adventitial sides of the explant so that they can be treated with different media. We made two customizable versions of the top tissue cassette piece: one with a 1 cm² opening and another one with a thin cross-piece subdividing this opening into 4 smaller ones. In the version with the larger opening, the curvature caused by the tracheal cartilage is retained and an air bubble is trapped underneath, creating an ALI. In the version with a cross-piece, the tissue is held flat against the bottom of the dish for imaging. Tissue culture in the appropriate media at an ALI can stimulate differentiation of airway epithelia after basal cell engraftment or after an injury that removes differentiated luminal cells. As a proof of concept, we completely removed the native SAE by brushing and seeded it with Tomato+ SAE cells prior to transitioning the explant to an ALI. We used a version of the tissue-transwell with a 1 cm² opening without the cross piece in the middle (Figure 8A). Having a trapped air bubble ensures that the ALI forms even when the explant is loaded imperfectly or when the tissue is accidentally punctured. After 21 days of differentiation at an ALI, the engrafted Tomato+ ferret SAE cells differentiated into AC-TUB+ multi-ciliated cells and CCSP+ secretory cells, even without reaching 100% confluency (Figures 8B–D). On the other hand, loading the explants into the version of the tissue-transwell with a cross piece and flattening them for imaging made it possible to image cilia motility live after adding Sir Tubulin (a reagent for viable labeling of the cilia; Figures 8E,F). Finally, we evaluated how well the tracheal explants tolerate long-term culture at an ALI within the tissue-transwell. In these experiments, the explants were exposed to Pneumacult ALI medium on the adventitial side for 42 days (with media exchanges every 2–3 days). In this experiment, most of the SAE cells in the transwell opening were ciliated and secretory (Figures 8G,H), suggesting that the device can be used for ex vivo modeling of acute and chronic lung and airway diseases.