Development and Characterization of a Primary Ciliated Porcine Airway Model for the Evaluation of In Vitro Mucociliary Clearance and Mucosal Drug Delivery

Abstract

Background/Objectives: In vitro models play a crucial role in preclinical respiratory research, enabling the testing and screening of mucosal formulations, dosage forms, and inhaled drugs. Mucociliary clearance (MCC) is an essential defense mechanism in mucosal drug delivery but is often impaired in respiratory diseases. Despite its importance, standardized in vitro MCC assays are rarely reported. Furthermore, many published methods primarily measure cilia beat frequency (CBF), which requires high-speed cameras that are not accessible to all laboratories. Therefore, this study aimed to develop a physiologically relevant, differentiated in vitro model of the respiratory epithelium that incorporates both beating cilia and functional MCC. We chose porcine airway mucosa as an alternative to human tissue due to ethical considerations and limited availability. The established model is designed to provide a reproducible and accessible method for a broad range of research laboratories. Methods: The previously published tracheal mucosal primary cell (TMPC DS) model, derived from porcine tissue, lacked the presence of beating cilia, which are crucial for effective MCC analysis. For accurate MCC assessment, beating cilia are essential as they play a key role in mucus clearance. To address this limitation, the here-described ciliated tracheal mucosal primary cell (cTMPC) model was developed. cTMPCs were isolated from porcine tissue and cultured under air-liquid interface (ALI) conditions for 21 days to promote differentiation. This model was evaluated for cell morphology, tight junction formation, ciliated and mucus-producing cells, barrier function, gene expression, and tracer/IgG transport. MCC and the model's suitability for standardized MCC assays were assessed using an inverted microscope. In contrast to the TMPC DS model, which lacked beating cilia and thus could not support MCC analysis, the cTMPC model allows for comprehensive MCC studies. Results: The developed differentiated in vitro model demonstrated key structural and functional features of the respiratory epithelium, including well-differentiated cell morphology, tight junction integrity, ciliated and mucus-producing cells, and effective barrier function. Functional MCC was observed, confirming the model's potential for standardized clearance assays. Conclusions: This differentiated in vitro model closely replicates the structural and functional characteristics of in vivo airways. It provides a valuable platform for studying mucociliary clearance, toxicology, drug uptake, and evaluating mucosal formulations and dosage forms in respiratory research.

Keywords: biopharmaceutics; cilia; epithelial airway model; inhaled drug delivery; monoclonal antibodies; primary cell culture; therapeutic antibodies.

Figure 7

Using primary models for the preclinical evaluation of drugs, formulation, and dosage forms. Representative tracks of the chitosan-coated lumogen-capsulated PGLA particles (PBS on cTMPC) (a). Each yellow line represents a track of a single particle (violet dots). Particles were tracked using ImageJ plugin version 1.54 [47,128] and the median particle speed was determined per sample (b). The individual median is shown as a line in a box plot with min. to max. whiskers; the mean is displayed as ‘+’. While in the mucosa, median and mean differ largely, both more and less overlay in cTMPC (tracheal mucosa: PBS: n = 14, N = 3; 100 mg/mL IgG: n = 17, N = 3. cTMPC: PBS: n = 10, N = 3; 1% HPMC: n = 7, N = 3; 100 mg/mL IgG: n = 11, N = 3. Statistical analysis was performed using a nonparametric unpaired Mann–Whitney test for tracheal mucosa and a nonparametric unpaired Kruskal–Wallis test for cTMPC. * p ≤ 0.05; if not stated otherwise, there was no significant difference). Due to large variances, no dependence of particle speed with viscosity was observed for MCC in mucosa; mean ± SD (c). By contrast, in MCC on cTMPCs, a significant impact of viscosity on particle speed (* p ≤ 0.05; two-way ANOVA) was observed (d). Characterization of the barrier integrity of ciliated trachea mucosal primary cells (cTMPCs) and TMPC DS. (a) P_app was determined based on the permeability with 500 µg/mL FITC-dextran or 100 mg/mL IgG (e). Only cell culture inserts with a TEER ≥ 500 Ω × cm² were used. (cTMPC FITC-dextran n = 60–66, N = 3; cTMPC IgG n = 60–62, N = 3. TMPC DS FITC-dextran n = 15–25, N = 3; cTMPC IgG n = 3–5, N = 1; **** p ≤ 0.0001).

Figure 1

Workflow of cell cultivation procedures of ciliated trachea mucosal primary cells (cTMPC). For the extraction of cTMPC, all excess connective tissue was carefully removed and digested in Pronase. cTMPCs were then directly seeded into cell culture inserts and cultivated for 5 days under submerged conditions. After 5 days, the apical medium was removed, and cells were then cultivated for 21 days under air–liquid interface (ALI) conditions. See Supplementary Data S1 for details and Supplementary Table S1 for media composition. The sketch was created under license with BioRender.com.

Figure 2

Graphical representation of the tissue holder used for mucociliary clearance analysis in tracheal tissue. Circular tracheal tissue sections were extracted from the whole trachea using a tissue puncher. The excised tissue was then positioned in the tissue holder with the apical surface oriented upwards. After securely closing the holder, a particle dilution was applied to the apical surface. Finally, the tissue holder was inverted and placed into the Keyence fluorescence microscope for analysis. The sketch was created under license with BioRender.com.

Figure 3

Histological evaluation of native porcine tracheal tissue and in vitro ciliated trachea mucosal primary cells (cTMPCs), quantification of cilia length and epithelial thickness. Alcian blue staining of histological cross sections (thickness: 5 μm) was assessed for respiratory mucosa from the trachea (a) and cTMPCs (b) cultivated for 21 days at ALI. (a) Native trachea tissue showed a typical ciliated pseudostratified columnar epithelium, with a basal cell layer underneath. Goblet cells are marked with an asterisk (*) and cilia with an arrow. Fibroblasts were observed in the lamina propria. In the cTMPC model (b) a ciliated pseudostratified cuboidal morphology of the apical cell layer was observed. Many but not all epithelial cells are ciliated in cTMPCs and no goblet cells were detected. Similarly to respiratory mucosa, the cTMPC models also display a thin basal cell layer underneath the epithelial layer. Representative images are shown. Scale bar: 20 μm. Cilia length (c) and thickness of the epithelial cell layer (d) were quantified at three different areas of independent samples (n = 7), while the cTMPCs originated from three different animals (N = 3). The cilia length of the in vitro cTMPCs is comparable to that of the native tracheal mucosal cilia (c). The epithelial thickness is significantly reduced in the cTMPC model. Values are presented as mean ± SD. ns: not significant, **** p < 0.0001 by ordinary one-way ANOVA.

Figure 4

Evaluation of barrier integrity for trachea mucosal primary cells (TMPCs) cultured 21 days at air–liquid interface (ALI). Immunoreactivity against tight junction (ZO-1) in TMPC DS (a) and cTMPC (b) is shown in white. Nuclei were stained with DAPI and are shown in blue. Scale bar: 50 μm. Note the significantly smaller sizes of nuclei and cells in cTMPCs. Ultrastructural details of the junctional complex (c). The junctional complex in cTMPCs cultivated for 21 days at ALI cultivation was observed by TEM. Tight junctions (TJ), adherence junctions (AJ), and desmosomes (DS) were identified along the apicolateral borders of epithelial cells. Various other cell structures are also labeled: mitochondria (M), microvilli (MV), basal body (BB), and cilia. Additionally, the typical “9 + 2” axoneme arrangement in cilia from airway mucosa can be observed (C). Gene expression analysis of occludin (OCLN) and cadherin-1 (CDH1) (d). Both genes encode for proteins that are involved in barrier development. GAPDH was used as a housekeeping gene. X-fold change is shown as mean ± SD and was calculated in comparison to trachea tissue. The red dotted line indicates gene expression in porcine trachea tissue. cTMPCs: n = 2, N = 2. C: TMPC DS: n = 3, N = 3; * p ≤ 0.05, significant. If not mentioned otherwise in the graph, the p-value was >0.05, thus, no significant differences.

Figure 5

Characterization of basal cells in ciliated trachea mucosal primary cells (cTMPCs) cultured 21 days at air–liquid interface (ALI). Z-stack imaging series of the in vitro cultured cTMPCs to visualize tight junctions and basal cells (a–h). Z-stack images were captured at 2–3 μm intervals using confocal microscopy. Green represents zonula occludens-1 (ZO-1) immunoreactive tight junctions, red corresponds to TP63 immunoreactive basal cells, and blue corresponds to nuclear DNA DAPI stain. Tight junctions are present at the apicolateral side between cells while basal cells are located at the basolateral side. Scale bar: 20 μm. Overview of z-stack images and orientation of the apical and basolateral side (i). Immunoreactivity of ZO-1 and TP63 in respiratory mucosa from pig; scale bar 50 µm (j). Gene expression analysis of KRT5 and PDPN (k). Both genes encode for proteins that are mainly expressed in basal cells. GAPDH was used as a housekeeping gene. X-fold change is shown as mean ± SD and was calculated in comparison to tracheal tissue. The red dotted line indicates gene expression in porcine trachea tissue. *** p ≤ 0.001, significant; if not mentioned otherwise in the graph p-value was > 0.05 and therefore there was no significant difference; cTMPC: n = 2, N = 2. C: TMPC DS: n = 3, N = 3.

Figure 6

Characterization of MUC5AC (yellow) production in porcine trachea (a), (TMPC DS (b) and cTMPC (c); both 21 d ALI). Immunoreactivity against acetylated tubulin (green) as marker for cilia in porcine trachea and (d), (TMPC DS (e) and cTMPC (f); both 21 d ALI). Nuclei were counterstained with DAPI in blue. Scale bars: 50 µm (a–f). While both models show a reduced mucin production compared to respiratory mucosa, cTMPCs have a significantly increased number of ciliated cells. Ultrastructural analysis of the cilia by scanning electron microscopy (SEM) in porcine airway mucosa (g) and cTMPC after 21 ALI (h) demonstrate the ciliated cells in the cell model, though significantly lower than in mucosa. Transverse sections analyzed by transmission electron microscopy (TEM) show the typical “9 + 2” axoneme arrangement in cilia from airway mucosa (i) and cTMPC (j). Longitudinal sections analyzed by TEM show basal bodies anchoring the cilia in the cell highlighted by orange arrowheads in mucosa (k) and cTMPC (l).