The hydrophilic concentrate of BMGLS used within this study was purchased from Conforma NV (Belgium) that used the method of Dynafyt for BMGLS production, and complies with European GMP and Chinese Pharmacopeia standards [29]. The ingredients are listed in Table 1 (www.theplantlist.org). The correct botanical identity of the components was tested using microscopy, thin-layer chromatography (TLC) and high-performance layer chromatography (HPLC) as reported in the Certificate of Analysis. Additional tests were performed to preclude contamination with harmful substances (e.g., aflatoxins, aristolochid acid, bacteria, heavy metals, pesticides, pyrrolizidine alkaloids and sulfur dioxide). The carrier solution of the hydrophilic concentrate (HCS; 30% glycerol, 0.1% propylene glycol, 0.1% methyl-4-hydroxybenzoate, 0.05% propyl-4-hydroxybenzoate), as indicated by Conforma NV, was used as control in all experiments. In this study, we used concentrations of 0.07%, 0.15%, 0.3%, 0.6%, 1.25%, 2.5% and 5% (v/v) of the hydrophilic concentrate BMGLS in PneumaCult™ ExPlus or PneumaCult™ ALI medium (Stemcell Technologies, Vancouver, Canada) supplemented with MycoZAP (Lonza, Basel, Switzerland), and Anti-Anti (Gibco, Dublin, Ireland) as instructed by the supplier as well as 40 µg/ml ticarcillin (Merck, Darmstadt, Germany).

Table 1 Herbal formulation of BMGLS. Herbal ingredients and their proportion in BMGLS including traditional usage and effects reported in literature. Major active compounds are givenCalu-3 cells and culture

The Calu-3 human bronchial epithelial cell line was provided from the Center for Medical Research (Medical University of Graz, Austria, MUG) and used between passages 20 and 40. Cells were cultured in Minimum Essential Medium (Gibco; MEM) completed with 10% fetal bovine serum (HyClone, Lot no RC35965; FBS), 100 µM penicillin-streptomycin (Sigma-Aldrich) and 2 mM L-Glutamine (Gibco), and maintained in a humidified, 5% CO2-95% atmospheric air incubator at 37 °C. Medium was exchanged twice per week and cells were passaged weekly at a 1:3 split ratio by using a trypsin-EDTA solution.

Specimen collection

Tracheal tissue was obtained from organ donors from the Division of Transplantation Surgery or during routinely performed autopsies from the Institute of Pathology (MUG) in accordance with the Austrian law BGBl. 1 Nr. 108/2012 following approval of the ethics committee of the Medical University of Graz (EK30-377ex17/18). We confirm that informed consent was obtained by participants. Donors diagnosed with bronchial carcinoma, acute or chronic pulmonary disease or current nicotine abuse were excluded. The mean age was 67.17 ± 18.17 years for female (19 samples) and 63.52 ± 15.30 for male (22 samples) donors.

Isolation of NHBE cells

NHBE cells were isolated as previously described [28]. Briefly, the tracheal tissue was washed in sterile 0.9% NaCl / 3% penicillin-streptomycin before mechanically removing soft tissue or lung parenchyma and incubation in MEM (Gibco) supplemented with 1x MycoZAP (Lonza), 1x Antibiotic-Antimycotic (Anti-Anti, Gibco), 40 µg/mL ticarcillin (Merck), 500 µg/mL dithiothreitol and 10 µg/mL DNase (in PBS) for 4 h at 4 °C followed by incubation in the aforementioned medium supplemented with collagenase solution (185 U/ml collagenase type II, 2 mg/ml BSA, 0.5 mM calcium chloride, 1 U amino acids 100x, 5% FCS in DMEM) and 10 µg/ml DNase (Gibco) over night at room temperature (RT). The epithelial cells were gently scraped off from the luminal surface of the trachea using fresh MEM and suspended in MEM supplemented with 5x Anti-Anti solution for incubation for 2 h at 37 °C to remove any residual contaminants. The cells were then suspended in PneumaCult™ ExPlus medium (Stemcell) supplemented with 1x Anti-Anti, 1x MycoZAP and 40 µg/mL ticarcillin and seeded into gelatin-coated cell culture flasks.

ALI cultures of Calu-3 and NHBE cells

Following cell harvesting (Calu-3) or isolation (NHBE), the cells were seeded into fibronectin-coated (2 µg/cm2; air dry for 1 h; Sigma-Aldrich) transwell cell culture supports (VWR, Dublin, Ireland) at a density of 3.5 × 105 cells (Calu-3) or 3 × 105 cells (NHBE) per 12-well insert/cell culture support in 0.5 mL medium with 1 mL medium added to the basolateral chamber and incubated at 37 °C and 5% CO2. As suggested by the manufacturer, the apical compartment was cleared from medium after three days and PneumaCult™ ALI medium, including BMGLS, was henceforth supplied only in the basal compartment with medium changes every other day.

Epithelial height measurement

The epithelial height was measured using ImageJ and OlyVIA (Olympus) software on three different locations per membrane and sample and converted to µm according to the specifications of the microscope. The epithelial height of the ALI cultures was tracked throughout the 3 weeks’ cultivation period.

Transepithelial electric resistance

The trans-epithelial electric resistance (TEER) was tracked using the EVOM voltohmmeter equipped with STX-2 chopstick electrodes (World Precision Instruments, Stevenage, UK) during wet (days − 3, -2 and − 1) and ALI culture (days 0, 3, 7, 10, 14 and 21). The apical and basolateral side contained 0.5 and 1 mL of ALI medium, respectively, during the measurement. TEER values were corrected for the blank, as posed by the fibronectin-coated non-cellularized insert, and the surface area.

Cytotoxicity assessment of BMGLS

The hydrophilic concentrate BMGLS was tested for cytotoxicity using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay (EZ4U Assay, Biomedica, Vienna, Austria) according to manufacturer’s instructions. The Calu-3 cells or primary NHBE cells were seeded in a 96-well culture plate (Nunc, ThermoFisher Scientific, San Francisco, USA) at a density of 5 × 104 cells/well, let adhere for 48 h and incubated for 24 h with BMGLS at concentrations of 0.07%, 0.15%, 0.3%, 0.6%, 1.25%, 2.5%, and 5% (v/v) with HCS at 5% (v/v) as solvent control. The absorbance of dissolved formazan was measured at 584 nm (Flustar Optima, BMG Labtechnology, Offenburg, Germany) and is given as percentage of the untreated control.

BMGLS treatment of ALI cultures

BMGLS was supplied basolateral at concentrations of 0.15%, 0.3% and 0.6% (v/v) in PneumaCult™ ALI culture medium, supplemented as described above, with HCS at 0.6% (v/v) and medium only as the controls over three weeks starting with the air-lift of the ALI culture (post 3 days of wet culture). All cultures were kept at 37 °C and 5% CO2.

Sample processing for histology and automated image-based analysis

ALI cultures were fixed using Carnoy’s reagent (ethanol, chloroform, glacial acetic acid; 6:3:1, v/v) for 20 min, flushed in PBS, paraffin-embedded using an Excelsior™ AS Tissue Processor (ThermoFisher Scientific), sectioned to 4 μm and mounted on SuperFrost Plus™ slides before dewaxing using Histolab Clear (Histolab®, Askim, Sweden) and rehydration in a descending series of ethanol (100%, 96%, 70% and 50%). Antigen retrieval was done using 0.1 M sodium citrate buffer solution (pH 6) for 15 min at 93° C by using a KOS Microwave Multifunctional Tissue Processor (Milestone, Sorisole, Italy).

Alcian blue (AlB) staining was conducted by incubation in 3% acetic acid for 3 min before incubation in 1% alcian blue (in 3% acetic acid) for 10 min and flushed in 3% acetic acid and dH2O. Nuclear fast red (NFR) was used for counterstaining. No NFR was used for combinational staining of AlB and IF. Here, the slides were directly subjected to background blocking using the Ultra Vision Protein Block for 5 min (ThermoFisher Scientific) and exposed to primary and secondary antibodies for 45 min and 30 min at RT, respectively. 4′,6-diamidino-2-phenylindole (DAPI) was used for nuclei counterstaining (1:2000). Samples were mounted using ProLong Gold Antifade Reagent (both ThermoFisher Scientific).

Immunostaining was performed using the UltraVision Detection System HRP Polymer Kit (ThermoFisher Scientific) following the manufacturer’s instructions. Mouse and/or rabbit IgG (1:1000, 1 µg/mL, diluted in antibody diluent, Agilent Technologies) served as negative control. Antibody specifications are given in Table 2.

Table 2 Antibody specificities for IHC and IF.Scanning (SEM) and transmission (TEM) electron microscopy

The ALI cell culture was fixed in 2.5% (w/v) glutardialdehyde and 2% (w/v) paraformaldehyde in 0.1 M cacodylate-buffer for 30 min covering both sides of the membrane, followed by post-fixation in 2% (w/v) osmium tetroxide in 0.1 M cacodylate-buffer for 30 min and dehydration in graded series of ethanol. SEM samples were critical point dried (CPD 030, Bal-Tec, Balzers, Liechtenstein), sputter coated with gold/ palladium (SCD 500, Bal-Tec, Balzers, Liechtenstein) and analyzed using a Zeiss Sigma 500 field emission scanning electron microscope (Zeiss, Oberkochen, Germany).TEM samples were embedded in TAAB epoxy resin (Agar Scientific Ltd., Essex, England) and ultrathin sections (70 nm) were cut with a UC7 Ultramicrotome (Leica Microsystems, Vienna, Austria) stained with platin blue and lead citrate. The ultrathin sections were investigated using a Zeiss EM 902 transmission electron microscope (Zeiss).

Image acquisition and automated mucus analysis

Histological and immunostained images were captured by bright field and fluorescence microscopy using a Leica CRT6000 (Leica Microsystems, Wetzlar, Germany) or an Olympus BX63 microscope with a DP73 camera and the DP2-TWAIN software (version 2.1, all Olympus, Tokyo, Japan, if not stated otherwise). The open source programs GNU Image Manipulation Program (GIMP, version 2.8.22, http://www.gimp.org) and Cell Profiler (version 3.1.5., [35]) were used for image and graphical data analysis. Cell Profiler uses a number of consecutive modules to generate an algorithm known as pipeline to detect and analyze target areas. The pipelines for analysis of airway mucus and mucosal protein content were previously described in Groiss et al., 2021 [28]. The Cell-Profiler pipeline used within this study including template data sets are available at http://cellprofiler.org. In short, the mucosal area as indicated by AlB staining in bright field images was identified and used to calculate the fluorescence signal intensity of the mucosal proteins within that AlB area. Downstream calculations to assess the area and optical density of both AlB and the mucosal proteins, respectively, were conducted using the same pipeline.

RNA isolation and quantitative reverse transcription PCR

Total RNA was isolated using the peqGOLD Total RNA Kit (Peqlab, Erlangen, Germany), tested on concentration and integrity using an ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, USA) and reverse transcribed with the ReverseAid First Strand cDNA Synthesis Kit (ThermoFisher Scientific). mRNA expression levels of target genes were analyzed by quantitative reverse transcription PCR (RT-qPCR) using the iTaq™ Universal SYBR® Green Supermix on a CFX384™ Real-Time System (both BioRad, Hercules, CA, USA) including melting curve analysis for quality assessment. Primers were purchased from Microsynth (Vienna, Austria; MUC5AC forward primer: 5’-GGGACTTCTCCTACCAAT-3’, reverse primer: 5’-TATATGGTGGATCCTGCAGGGTAG-3’; MUC5B forward primer: 5’-CACATCCACCCTTCCAAC-3’, reverse primer: 5’-GGCTCATTGTCGTCTCTG-3’; ALOX15 forward primer: 5’-GGGCAAGGAGACAGAACTCAA-3’, reverse primer: 5’-CAGCGGTAACAAGGGAACCT-3’). Gene expression was normalized against β-actin and quantified by standard \(^\) method.

Statistical analysis

Prism 8 (GraphPad, San Diego, United States) was used for data presentation and statistical analysis. Data are given as means ± SEM if not stated otherwise. Data were tested for normal distribution using a Shapiro-Wilk test followed by an unpaired two-tailed Student’s t-test (normal distribution) or a Welch’s t-test (data not normally distributed). Differences between more than two groups were assessed by two-way ANOVA with Tukey’s multiple comparison test or by non-parametric Kruskal-Wallis test. Statistical significance was considered at p < 0.05 (* p = 0.05, ** p = 0.01, *** p = 0.001, and **** p = 0.0001). Experiments were performed in 3–7 independent biological replicates in 2 (qPCR) or 3 (TEER, height) technical replicates (N = 3–7, n = 2–3). For automated analysis of airway mucus, the entire specimens were scanned and data is given as mean ± SEM (N = 12–50 images) per sample.