Penicillin streptomycin

PDX-derived cell lines were supplied by Charles River from Charles River’s Cancer Model Database (https://compendium.criver.com/ (accessed on 18 November 2021)) and were grown at 37 °C in a humidified atmosphere with 5% CO₂ in RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum and 100 U/mL Penicillin-Streptomycin. PDX cells were used within 10 passages and are listed in Table 1 [16 (link)].
Human dermal fibroblasts (HDF, ScienCell Research Laboratories, Carlsbad, CA, USA) were grown at 37 °C in a humidified atmosphere with 5% CO₂ in Fibroblast growth medium 3 supplemented with 10% fetal bovine serum, 100 U/mL Penicillin-Streptomycin, 1 ng/mL basic fibroblast growth factor and 5 μg/mL insulin (PromoCell, Heidelberg, Germany).

EA.hy926 cells (“EAHy”, ATCC^® CRL-292) were cultured in Dulbecco modified Eagle medium (DMEM; Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS), 2 mmol/L L-glutamine, 1× HAT (5 mmol/L sodium hypoxanthine, 20 µM aminopterin, and 0.8 mmol/L thymidine) and 100 U/mL penicillin-streptomycin (Thermo Fisher Scientific). Cells were used between passage 7–25. Human Umbilical Vein Endothelial Cells (HUVEC) (Promocell GmbH) were grown in endothelial cell growth medium (Promocell, Heidelberg, Germany) supplemented with 100 U/mL penicillin-streptomycin. Cells were used between passage 4 and 7. MonoMac-6 cells (DSMZ, ACC124) were cultured in RPMI supplemented with 10% FBS, 2 mmol/L L-glutamine, non-essential amino acids, 1 mmol/L sodium pyruvate, and 10 μg/mL human insulin. Cells were used between passage 7 and 25. Bimonthly samples were measured for the absence of mycoplasma using the MycoAlert Mycoplasma Detection Kit as per manufacturer’s protocol (Lonza, Walkersville, MD, USA). All cells were maintained in a humidified incubator at 37 °C and 5% CO₂.

Human umbilical vein endothelial cells (HUVECs) from a single donor (C-12200, PromoCell GmbH, Heidelberg, Germany) were expanded at 37 °C in a 5% CO₂ incubator with a humidified atmosphere. Cells were seeded at passage 4 at a density of 30,000 cells/cm² on DBPs and functionalized samples (n = 3) and cultivated in static conditions with endothelial growth medium supplemented with 1% (v/v) penicillin-streptomycin (PromoCell GmbH, Heidelberg, Germany). Samples were collected on days 1, 7, and 14. Untreated DBPs were considered as the control.

Human umbilical vein endothelial cells (HUVEC, Pelobiotech, Planegg, Germany) were cultured in complete endothelial cell growth medium (Promocell GmbH, Heidelberg, Germany) supplemented with 10% fetal calf serum (FBS, Gibco, Life Technologies GmbH, Darmstadt, Germany) and 1% penicillin-streptomycin (Promocell). Hematopoietic progenitor Hoxb8 neutrophils^{19 (link)} were a generous gift from Prof. Häcker (Institute of Medical Microbiology and Hygiene, UMC, Freiburg, Germany). Cells were cultured in Opti-modified Eagle medium (Gibco) supplemented with 10% heat-inactivated FBS (Gibco), 30 µM 2-mercaptoethanol (Thermo Fischer, Paisley, UK), 1 µM estradiol (Sigma) and 1% stem cell factor. Stem cell factor was harvested from chinese hamster ovary cells (a generous gift from Prof. Häcker) as described earlier^{19 (link)}. Prior to the experiments, progenitor Hoxb8 neutrophils were cultured for four days in differentiating medium (estradiol free medium)^{19 (link)}. All cells were grown and experiments were performed at standard growing conditions (37 °C, 5% CO₂, sufficient humidity). All neutrophil assays were performed in the presence of FBS.

For cell-based studies to assess effects of patients microparticles on endothelial cell signalling and function we used human aortic endothelial cell (HAEC) (ATCC^®, Middlesex, UK; PCS-100-011). HAEC were cultured in Endothelial Cell Growth Medium (Promocell^®) supplemented with Penicillin/Streptomycin (50 µg/mL) and Endothelial Cell Growth Medium Supplement (10 mL; Promocell^®). For functional studies, confluent cells were made quiescent for 2 h in low-serum medium containing 0.5% foetal bovine serum (FBS) and stimulated with total microparticles (10⁶ MPs/mL) isolated from patients pre-VEGFi and post-VEGFi. Quiescent cells were used because this provides a system where molecular events can be studied in controlled conditions, where cells are not actively dividing. In addition, serum-free conditions avoid the potential for cell activation because serum contains growth factors and other proteins that trigger signalling and cell growth. This is especially relevant in our study, where we are interfering with growth factor (VEGF) signalling through VEGFi.
All studies comparing effects of pre-treatment and post-treatment MPs were performed using the same set of patients. In some experiments, cells were pre-treated with BQ123 (ETA receptor antagonist; 1 µmol/L) or BQ788 (ET_B receptor antagonist; 1 µmol/L).

Human aortic endothelial cells (HAEC) were isolated from aneurysmal aorta of BAV and TAV patients, as described previously 18, and cultured in Endothelial Cell Basal media with growth supplements, including 10% foetal calf serum and penicillin/streptomycin (PromoCell, Heidelberg, Germany) with 5% CO² at 37 °C. Primary aortic cells were used in passage 3–4. Human EA.hy926 cells 19 were cultured in Dulbecco's modified Eagle's high glucose medium (DMEM) supplemented with 10% foetal calf serum and penicillin/streptomycin at 37 °C with 5% CO². EA.hy926 cells were transfected with commercially available LNA™ Oligonucleotides (Exiqon, Vedbaek, Denmark, #450012) for hsa‐miR‐200 family or negative control (Exiqon, #199006‐001) using Lipofectamine RNAiMAX Transfection Reagent (Thermofisher, #13778075). Expression levels of ZEB1 and SNAI1 were measured to establish the effect of microRNA‐200 family on the target transcription factors.