Bovine collagen 1

The invasive properties of WI-38 and MRC-5 fibroblasts were investigated using a spheroid invasion assay performed and quantified as previously described [48 (link)]. Briefly, 2500 cells per well were seeded in 100 µl of DMEM containing 10% FBS into a cell-repellent 96-well microplate (650790, Greiner Bio-one, Frickenhausen, Germany). After overnight incubation at 37 °C, the cells formed spheroids, 70 μL of medium was removed from each well and the remaining medium with spheroid was overlaid with 2.5% bovine collagen I (5005, Advanced BioMatrix, Carlsbad, CA, USA). Following the polymerization of collagen, fresh serum-free DMEM supplemented with PBS, 20 µg/mL 2D EVs or 20 ng/mL of PDGF-B (100-14B, Peprotech, London, UK) was added. The cells were allowed to invade the collagen matrix for 48 h, after which they were stained with Hoechst (62249, Thermo Fischer Scientific, Waltham, MA, USA). Images were acquired on an Axio Observer 2 fluorescence microscope (Zeiss) using a 5× objective. Cell invasion is determined as the average of the distance invaded by the cells from the center of the spheroid as determined by the cell dissemination counter software aSDIcs. Three to five spheroids per condition were analyzed and three independent experiments were performed.

NIH 3T3 fibroblasts were from The American Type Culture Collection (ATCC). Transwells were purchased from Corning Incorporated, Coring, NY. Bovine collagen I was obtained from Advanced BioMatrix (Carlsbad, CA). Submerged cell culture media consisted of Bronchial Epithelial Cell Growth Medium (BEGM) with antibiotics from Lonza (Walkersville, MD). Air–liquid interface (ALI) media consisted of the PneumaCult kit purchased from Stem Cell Technologies (Vancouver Canada). Recombinant human IL-13 from R&D systems, Minneapolis, MN was reconstituted in 0.1% bovine serum albumin (BSA) aliquots and stored at − 80 °C. Human rhinovirus 16 (HRV16) was propagated in H1-Hela cells (ATCC) and purified as previously described [8 (link)]. RNA lysis buffer (RLT) was from Qiagen (Hilden, Germany). IL-8 and IP-10 ELISA kits were purchased from R&D systems (Minneapolis, MN).

MCF10A cells were maintained in MCF10A growth medium^{97 (link)} (DMEM/F12 supplemented with 5% horse serum, 20 ng/mL EGF (Peprotech Cat# AF100-15), 0.5 μg/mL Hydrocortisone (Sigma Cat# H-0888), 100 ng/mL Cholera toxin (Sigma Cat# C-8052), 10 μg/mL insulin (Sigma Cat# I-1882) and 1× penicillin/streptomycin). Stable ectopic expression of wild-type BAD (WT) or phosphomutant (3SA) was done in MCF10A BAD^−/− (BAD knockout) cells we previously described^{13 (link)}. For 3D tubulogenesis assay gel preparation, bovine collagen-I (Advanced BioMatrix Cat# 5005) was neutralized and incubated for 1 h as detailed above for branching organoid assay. MCF10A single cells were then embedded (50 cell per μl) in pre-thawed 1 parts volume growth factor reduced Matrigel (Corning Cat# 354230) and 9 parts volume collagen-I solution (1 mg/mL final concentration). The cell-gel suspension was gently plated in wells of a 12-well plate (50–100 μl per well) placed on top of a heating block (37 °C) to aid with gelation for a few minutes before placing the plate in a cell incubator (5% CO₂, 37 °C) for 45 min. Pre-warmed MCF10A growth medium was then added and replenished every 3 days.

Recombinant human IL-13 protein from the R&D Systems, Minneapolis, MN was reconstituted in 0.1% bovine serum albumin (BSA) and stored at -80°C. Bronchial epithelial cell growth medium (BEGM) with antibiotics was purchased from Lonza, Walkersville, MD, USA. Air-liquid interface culture media (F6 media) consisted of 1:1 ratio of bronchial epithelial basal medium (BEBM) and Dulbecco's modified eagle medium (DMEM) plus insulin, transferrin, epinephrine, bovine pituitary extract (BPE), gentamicin and amphotericin, bovine albumin (0.5 μg/ml, ethanolamine (80 μM), MgCl₂ (0.3 mM), MgSO₄ (0.4 mM), CaCl2 (1 mM), retinoic acid (30 ng/ml), hEGF (10 ng/ml). Bovine collagen I (3 mg/ml) was obtained from Advanced BioMatrix (San Diego, CA). RNA lysis buffer (RLT) was from Qiagen (Hilden, Germany). RIPA Western lysis buffer was purchased from Thermo-Fisher Scientific (Waltham, MA).

Bovine collagen I (6 mg/mL; 5010, Advanced BioMatrix) was mixed on ice with UltraPure Distilled Water (Invitrogen), PBS 10X and 1M NaOH according to the manufacturer protocol to obtain a 5 mg/mL collagen solution. Pelleted cells were resuspended and vortexed with the collagen solution to obtain a concentration of 4 × 10⁶ cells/mL. Hydrogels were molded as described above and incubated for 30 min at 37°C until complete gelation.