Cc 4147

HUVECs were isolated from human umbilical cords as previously described (Schossleitner et al., 2019 (link)) and in accordance with the protocol approved by the ethics committee of the Medical University Vienna (approval number EK1621/2020). Informed consent was obtained from all donors and protocols were followed according to the Declaration of Helsinki principles. HPMECs were purchased from PromoCell GmbH (Heidelberg, Germany). Human bronchial epithelial cells (HBEs) and 293T cells were provided by Harald Renz (Klinikum der Philipps Universität Marburg, Germany) and Martin Bilban (Medical University of Vienna, Austria), respectively. Endothelial cells were cultured in endothelial growth medium (EGM-2; CC-3156, Lonza, Basel, Switzerland) containing 15% fetal bovine serum (FCS; 10500-064, Gibco, Karlsruhe, Germany) and supplements for microvascular cells (CC-4147, Lonza). Epithelial cells were cultured in RPMI 1640 (21875-034) supplemented with 10% FCS (10500-064), 2 mM L-glutamine (25030-024), and 50 U/ml streptomycin-penicillin (15070-063), which were all from Gibco. All cells were maintained in a humidified atmosphere containing 5% CO₂ at 37°C and passaged at 90% confluence. Prior to experiments, cells were authenticated and confirmed to be free of contamination by mycoplasma. Endothelial cells were used at passages 2 to 8.

HUVECs were purchased from Gibco. HUAECs were from Provitro. HUVECs and HUAECs were cultured in EGM-2 medium (CC-3156, Lonza) containing supplements (CC-4176 or CC-4147), respectively, and were used for a maximal number of 6 passages. All cells were cultured at 37 °C and 5% CO₂ and were tested negative for mycoplasma contamination before experiments.

Immediately after bonding, 10 μL of poly (D-lysine) solution (PDL, M.W. 70,000–150,000, 1.0 mg.mL⁻¹, Sigma-Aldrich, St. Louis, MO) was injected into the each compartment and incubated at room temperature to promote cell adhesion. After 2 hours, we rinsed with autoclaved and 0.2 μm filtered water (AM9920, Life Technologies, Grand Island, NY). The PDL-coated platforms were filled with pH 7.4 collagen Type 1 (354249, Becton Dickinson, Franklin Lakes, NJ) at 2 mg. mL⁻¹ and incubated at 37 °C for 30 minutes. The cured collagen in the endothelia compartments was replaced by a cell culturing medium (EGM-2 MV BulletKit: CC-3156 & CC-4147, Lonza Walkersville) supplemented with 1% P/S for overnight to promote the cellular growth. The flow of media was such that the gel in smaller migration channels remains inside the channels, to prevent spontaneous entrance of endothelial cells during plating.

Endothelial colony forming cells (ECFCs) were isolated from white adipose tissue, as described [28 (link)]. Briefly, ECFCs were cultured in 75 cm² flasks, treated with 1% gelatin (G1890, Sigma, Steinheim, Germany) and incubated in 20% FBS/ EBM-2 media (CC-3156, Lonza, Basel, Switzerland) containing 1% penicillin/streptomycin (P/S) and the necessary growth factors (VEGF, hFGF-B, hEGF and R3-IGF-1), ascorbic acid and heparin, without hydrocortisone (CC-4147, Lonza). Cells were grown until confluence (90%) was reached. Experiments were performed on passages 6–7.
Cell identity was confirmed by testing cloning-forming ability, as described [2 (link)], and also by flow cytometry, analyzing several specific antibodies against CD31, CD14, CD90, CD34, CD45, CD73, CD133, CD309 and CD146 (Supplementary Figure S3). An isotype IgG1 antibody was used for negative control. The full list of antibodies is shown in Supplementary Table S1. Fluorescence was measured using CytoFLEX cytometer (Beckam Coulter, West Sacramento, CA, USA) and CytExpert software. Finally, data were analyzed with FlowJo v10.4 software.