Pneumacult ali maintenance medium

Human ALI airway tissue models were established as described previously (Xiong et al. 2018 (link)). Normal human bronchial epithelial (NHBE) cells (MatTek, Ashland, MA) were expanded in PneumaCult™-Ex Medium (STEMCELL Technologies, Vancouver, Canada). Cells were seeded at a density of 4.0 × 10⁴ cells onto 24-well Transwell^® culture inserts and grown in the Expansion Medium until they reached 100% confluence. Medium from the apical side was then removed and cultures were fed from the basolateral side with PneumaCult™-ALI Maintenance Medium (STEMCELL Technologies) for 4 weeks.

Low–passage primary human bronchial epithelial cells (NHBE, Lonza) from six genet-ically independent donors (n = 6) were grown in PneumaCult-Ex Plus expansion medium (Stemcell) on Corning transwell polyester membrane cell culture inserts (precoated with 1% collagen, Merck, Kenilworth, NJ, USA) according to the manufacturer’s instruction. Medium was applied to the basal and apical chamber until cells were grown 100% confluence. An airlift was performed by removing the apical medium and the basal medium was exchanged to Pneumacult-ALI maintenance medium (Stemcell). When the transepithelial electrical resistance (TEER), measured using EVOM2 instrument (World Precision Instruments), reached the threshold of 700 Ω, cells each donor were stimulated with IFNα (300 IU/mL, Roche, Basel, Switzerland), IFNβ (100 IU/mL, Peprotech, Rocky Hill, NJ, USA), IFNγ (200 IU/mL, Promocell, Heidelberg, Germany), IFNλ1 (100 ng/mL, Biotechne, Minneapolis, MN, USA), IFNλ3 (100 ng/mL, Biotechne) or medium as control for 24 h. Each negative control was genetically matched. TEER measurement was used to identify epithelial integrity prior stimulation and samples showing a TEER >700 Ω were classified as integer. Cells were harvested and RNA was extracted using AllPrep DNA/RNA Micro Kit (Qiagen, Hilden, Germany). Figure 1 and Supplementary Figure S1 was created with BioRender.com (accessed on 26 August 2022).