5 ml tubes

For the nebulisation of previously produced liposomes, two nebulisers representing two different techniques of nebulisation were used and compared: PARI VELOX^® (PARI GmbH, Starnberg, Germany), a metal-based vibrating-mesh nebuliser with a resonance frequency of 160 kHz and PARI BOY^® SX utilising compressed air with an operating pressure of approximately 1.6 bar and a nozzle size of 0.48 mm. Briefly, 2 mL of each liposomal formulation were pipetted in the sample reservoir and collected again after nebulisation directly into 5 mL tubes (Sarstedt AG & Co. KG, Nümbrecht, Germany). The nebulisation time and volume of nebulised liposomes were measured for each formulation to calculate the aerosol output rate (L/min) and emitted volume (%). All formulations were nebulised at a total lipid concentration of 1 mg/mL.

The preparation of the tracheal organ cultures (TOC) has been described previously [43 (link)]. Briefly, tracheae were dissected from humanely sacrificed 20-day-old specific-pathogen-free (SPF) chicken embryos (Valo BioMedia GmbH, Osterholz-Scharmbeck, Lower Saxony, Germany). They were subsequently cut into equal rings of 0.8 mm and placed separately into 5mL-tubes (Sarstedt AG & Co KG, Nümbrecht, North Rhine-Westphalia, Germany) filled with 1 mL pre-warmed (~37 °C) 199 Hanks’ salts medium (Sigma-Aldrich, Taufkirchen, Bavaria, Germany), supplemented with 1% L-glutamine (200mM, Biochrome, Berlin, Germany) and 1% Penicillin G (10,000 U/mL, Biochrome, Berlin, Germany). The TOCs were placed in an overhead shaker and incubated at 37 °C for four days. After microscopical evaluation, tracheal rings with 100% ciliary activity were randomly assigned to different groups.

The phagocytic activity of macrophages was analyzed using 2 µm green fluorescence-labeled latex beads (Sigma-Aldrich, Munich (D)). The beads were opsonized in pooled human serum for 30 min prior to addition to the macrophage culture in a ratio of 10 beads per seeded cell and incubated in a humidified atmosphere for two h at 37 °C and 5% CO₂. Cells were washed three times with serum-free macrophage culture medium to remove non-phagocytosed beads.
Bead uptake was analyzed via flow cytometry. Therefore, macrophages were scraped off the well plate, transferred into suitable 5 ml tubes (Sarstedt, Nümbrecht (D)), and centrifuged (300 × g, 5 min, 4 °C). The supernatant containing free-floating beads was discarded, whereas the pellet was resuspended in 500 µl of FC-buffer and the number of positive cells was quantified. For the discrimination of cell types in co-culture studies with hMSCs, cells were stained against leukocyte-specific CD45 (Milteny Biotec; labeled with APC) according to the manufacturer’s instruction. Double-positive cells were determined as bead-containing macrophages.