Type 1 collagen coated plates

Mouse lung and tumor endothelial cells (EC) were isolated from C57/BL6 mice as previously described [24 (link),25 (link)]. Alternatively, B16 or LLC1 tumors were isolated from Tie2-Cre^ERT2; Wt1^Lox/Lox mice treated with Tamoxifen or vehicle. Briefly, lung and tumor tissues were cut into small fragments and digested with 1 mg/mL collagenase A and 100 IU/mL type I DNase (Roche Diagnostics, Meylan, France) for 45 min at 37 °C. ECs were then purified from the cell suspension using a rat anti-CD31 antibody (clone MEC 13.3; BD Biosciences, San Jose, CA, USA) conjugated to Dynabeads (Life Technologies, Courtaboeuf, France) using a magnetic particle concentrator and cultured on 0.2% type I collagen-coated plates (Sigma Aldrich, St. Louis, MO, USA) in DMEM medium supplemented with 20% FCS, 100 IU/mL penicillin, and 100 µg/mL streptomycin. Endothelial cell purity was confirmed by FACS analysis using Alexa Fluor 647 anti-mouse VE-cadherin antibody (Clone: BV13; BioLegend, San Diego, CA, USA) and anti-mouse Alexa Fluor 488 Fab′2 recognizing the VE-cadherin antibody.

Separated liver cells were processed as follows. HEPs were pelleted by low-speed centrifugation (50 g, 3 min, 4 °C), the viable cells were isolated with a 40% Percoll solution (Sigma, USA, 150 g, 7 min, 4 °C). The cells were cultured in Type I collagen-coated plates (15 µg/cm², Sigma, USA) and were seeded in either 24-well plates for EV uptake studies (1.5 × 10⁵ cells/well) or in 6-well plates for EV production studies (10 × 10⁵ cells/well) in a seeding medium (DMEM (high glucose, 4.5 g/L), 10% fetal bovine serum (FBS, Gibco), 1000 U/L penicillin, 1000 µg/L streptomycin, 2 mmol/L l-glutamine, 7.5 mg/L Hydrocortisone, 0.02 mg/L epidermal growth factor, 0.014 mg/L glucagon, 1 mM sodium pyruvate, 0.5 ml/L insulin (all from Sigma, USA)). The cells were allowed to attach to the plate surface for 2–3 h at 37 °C and 5% CO₂. After the incubation, the medium was changed to a FBS-free seeding medium.