Differentiation media

To further test the biological effects of these meshes, we induced the neuronal differentiation of hASCs in contact with FG meshes during 10 days of culture. For this purpose, hASCs were seeded on FG samples at a density of 2 × 10⁴ cells/cm² and after 24 h, they were treated with the corresponding differentiation media (PromoCell, Heidelberg, Germany) for 7/10 days. The medium was changed every 2 days. The expression levels of the neuronal maker, β-III tubulin, was evaluated by immunofluorescence and confocal microscopy. After fixation and permeabilization, the samples were incubated with β-III tubulin antibody (Cell Signaling Technology, Danvers, MA, USA) overnight at 4 °C and then incubated with anti-rabbit Alexa Flour 546 secondary antibody (Invitrogen) for 2 h at RT and in the dark. Finally, the samples were incubated with Hoechst 33342 (ThermoFisher Scientific, Foster City, CA, USA) for 5 min in order to show the cell nuclei. A laser-scanning confocal microscope (Nikon A1/A1R Confocal Laser Microscope System) was used for visualization and the images were analyzed using the corresponding software.

Primary human nasal epithelial cells (HNECs) were purchased from PromoCell (Heidelberg, Germany) and cultured at passage two in the proliferation media (PromoCell) until >90% confluent. For air–liquid interface (ALI) studies, HNECs were seeded at a density of 16.5 × 10⁴ cells/mL in 0.5 mL differentiation media (PromoCell) in 12 mm Transwell^® plates (7.4 × 10⁴ cells/cm²) with 0.4 µM pore polyester membrane inserts and differentiation media in the basal compartment. When confluent, apical media was removed to facilitate ALI (day 0), where cells differentiate and become ciliated and mucus secreting. Cells were maintained at ALI until day 14, at which point they were exposed to A. fumigatus (Af293) conidia in a similar manner to submerged 16HBE14o- cells.