Endothelial cell growth media

This assay was performed using the Matrigel sandwich method. To polymerize the lower gel layer, 400 μL of Matrigel (BD biosciences) was placed into each well of the 24-well plates and incubated at 37 °C for 3 h. The thoracic aortas of 3-month-old male C57BL6/N mice (n = 3) were dissected out. After removing the surrounding fat tissues and rinsing with cold sterile PBS in petri dishes, the aortas were cut into 1 mm ring segments and placed in a lower layer of Matrigel. For the upper gel layer, 100 μL of Matrigel was added on top of each ring using pre-cooled pipette tips and incubated at 37 °C for 1 h. After solidification, the aortic rings were supplemented with endothelial cell growth media (Lonza) with/without 100 ng/mL mouse IL-17C (eBioscience). The aortic rings were grown on Matrigel for 14 days (d), and culture medium was replaced every other day. Aortic vessel outgrowth was monitored daily and photographed using the Nikon ECLIPSE TE 2000-U microscope (Nikon). The average vessel length was calculated using ImageJ v.1.47 software.

All cell lines used in this study were either purchased from ATCC (MSTO‐211H, HEK‐293, Met‐5A), provided by collaboration partners (MM05, Ren, SPC212, BEC, LEC), or established at the Medical University of Vienna (VMC23, VMC40, Meso62, and Meso84) as recently described [11 (link)], and authenticated by array comparative genomic hybridization and STR profiling within the past three years. Met‐5A is an immortalized, nonmalignant mesothelial cell line. Ren and VMC23 were established from epithelioid, Meso62, Meso84 from sarcomatoid and the other PM cell lines from biphasic PM. All PM and mesothelial cells were cultivated in RPMI medium supplemented with 10% heat‐inactivated fetal bovine serum (FBS) in a humidified atmosphere (37 °C, 5% CO₂) and regularly checked for Mycoplasma contamination. Immortalized endothelial cells (BEC, LEC) were grown in Endothelial Cell Growth Media (Lonza Group AG, Basel, Switzerland).

CPC clones were isolated from cardiac tissue of five neonatal (1 day–1 month) and five adult (57–72 years) patients undergoing cardiovascular surgery as previously described by our laboratory [23 (link)]. Briefly, discarded cardiac tissue was cut into small clumps (~1.0 mm³) then enzymatically digested using collagenase at a working concentration of 1.0 mg/mL for 2 h at 37 degrees Celsius. The resulting solution was then passed through a 40-um cell strainer. Cells were cloned in a 96 well plate by limiting dilution to a final concentration of 0.8 cells per well and were screened to identify Isl-1+ clones. These cells were expanded in media which contained 10% fetal bovine serum (Thermo Scientific, Waltham, MA, USA), 100 µg/mL Penicillin-Streptomycin (Life Technologies, Carlsbad, CA, USA), 1.0% minimum essential medium non-essential amino acids solution (Cat no. 11120052, Life Technologies, Carlsbad, CA, USA), and 22% endothelial cell growth media (Lonza, Basel, Switzerland) in Medium 199 (Life Technologies, Carlsbad, CA, USA) [23 (link)]. Twenty clones were expanded for use in the RT-PCR experiments described here and an additional six clones were analyzed by RNA sequencing