Endopan 3

Monocytic THP-1 cells were cultured in RPMI (Sigma-Aldrich, St. Louis, USA) with 10% FBS (PAN-Biotech, Aidenbach, Germany) with a cell density of 0.2 to 2.0 × 10⁶ cells per ml.
Human pulmonary microvascular endothelial cells (HPMECs) were from PromoCell (Heidelberg, Germany) and cultured in Endothelial Cell Growth Medium MV2 (PromoCell) as recommended by the supplier. Human umbilical vein endothelial cells (HUVECs) and human umbilical artery endothelial cells (HUAECs) were isolated from the umbilical cord of caesarean sections in our laboratory as described [19 (link)] and cultured in Endopan-3 from PAN-Biotech (Aidenbach, Germany).
For flow experiments, cells were used in passages 4 to 6 and seeded with a density of 100,000 cells/cm² in ibidi μ-Slides of different types (0.1, 0.2, 0.4, and 0.8 mm μ-Slides, Martinsried, Germany) or 24 well plates for the static control. After 24 h, supernatants and cells were harvested or stimulated with 10 ng/ml TNFα for another 24 h. The flow in the μ-Slides was created with the ibidi pump system. Depending on the geometry of the μ-Slide type, the flow rate was adjusted as specified by the manufacturer to yield the desired laminar shear stress.

Human gingival fibroblasts were purchased from Provitro AG (HFIB-G, Berlin, Germany). Fibroblasts were maintained in DMEM/Ham’s F12 (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% FCS (fetal calf serum; Bovine Calf Serum Iron Supplemented, VWR International, Radnor, PA, USA) and antibiotics (100 U/mL penicillin and 100 mg/mL streptomycin) at 37 °C in 5% CO₂. Human umbilical vein endothelial cells (HUVECs) were isolated from human umbilical cord veins, as described earlier [31 (link)]. In short, HUVECs were retrieved by Collagenase H digestion (Roche Diagnostics, Mannheim, Germany) of perfused human umbilical veins and seeded into cell culture plates with a growth area of 25 cm² coated with 1% gelatin. HUVECs were cultured in specialized medium for endothelial cells (Endopan 3, PAN-Biotech GmbH, Aidenbach, Germany) and antibiotics. Cells were not used for experiments for longer than passage 10. Their typical morphology was closely monitored to exclude contamination and undesired effects due to cellular senescence. Tests to exclude mycoplasma contamination (Venor^® GeM Classic, Minerva Biolabs GmbH, Berlin, Germany) were carried out regularly.

Human pulmonary microvascular endothelial cells (HPMECs) were from Promocell (Heidelberg, Germany). Human adipose microvascular endothelial cells (HAMECs) were from ScienceCell (Carlsbad, USA). Human umbilical vein endothelial cells (HUVECs) and human umbilical artery endothelial cells (HUAECs) were isolated from the umbilical cord of cesarean sections in our laboratory as described (10 (link)) and cultured in Endopan-3 from PAN-Biotech (Aidenbach, Germany). This work was approved by the local ethic committee of the medical faculty RWTH Aachen with the ethical vote EK241/18. For a better comparison all endothelial cells were cultured in endothelial cell growth medium MV2 (Promocell). Each experiment with either HUVECs or HUAECs was performed with cells that were prepared from a different donor and each experiment with either HPMECs or HAMECs was performed with cells from two different donors.