*2.1. Cell Culture*

MucilAirTM primary human bronchial epithelial cells were purchased from Epithelix (Epithelix, Sárl, Geneve, Switzerland) and cultured using air−liquid interface conditions. Cultures were established from three different healthy non-smoking donors (two males and one female, aged 15, 41, and 71 years, respectively) and three different asthmatic non-smoking donors (two males and one female, aged 36, 50, and 55 years, respectively). MucilAirTM cell culture medium (Epithelix) was exchanged and the cells were washed carefully with a medium from the apical side to remove residual mucus on a regular basis, according to the manufacturer's instructions. The cell culture medium was continuously collected over one month from the basolateral and apical cell sides. While basal samples were directly retrieved from the bottom chamber of the culture system, for collection of the apical samples, the cells were incubated with 200 μL apically applied cell culture medium for 30 min at 37 ◦C, and afterwards they were washed by carefully pipetting up and down. All samples were stored at −80 ◦C until further use. The general experimental downstream workflow is shown in Figure 1.

**Figure 1.** Schematic overview of EV preparation steps.
