Endothelial cell growth medium mv2

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.

HUVECs were purchased from Lonza (from pooled donors) and cultured in Endothelial Cell Growth Medium 2 (EGM2, PromoCell). Cells were used between P3 and P6 for experiments. Human pericytes (from placenta) were purchased from PromoCell and cultured in Pericytes Growth Medium 2 (PGM2, PromoCell). Cells were used between P3 and P6 for experiments. Human cardiac endothelial cells and fibroblasts were purchased from PromoCell and cultured in Endothelial Cell Growth Medium MV 2 and Fibroblast Growth Medium 3 respectively (PromoCell). Endothelial cells were cultured on 0.1% gelatin treated T75 flasks. We note that although placental pericytes and HUVECs are not normally found in the cardiac microvasculature, they constitute a reliable source of relevant cells allowing the reproducible assembly of complex in vitro models.

Cells were grown on 1% gelatin‐coated tissue culture (TC)‐treated dishes (BD Falcon) at 37°C and 5% CO₂ in Endothelial Cell Growth Medium 2 (PromoCell) (for HUVEC and HBMEC) or Endothelial Cell Growth Medium MV2 (PromoCell) (for MS1 and HCMEC) supplemented with the respective SupplementMix (containing growth factors and 2 or 5% FBS, respectively) and 10 Units/ml penicillin/streptomycin.