Complete human endothelial cell medium

Endothelial barrier function was continuously recorded using the 8W10E + electrode chamber arrays and ECIS Z-Theta system (both Applied Biophysics, United States) with associated software, as described in (Tiruppathi et al., 1992 (link)). Human brain endothelial cells (purchased from CellBiologics, United States) were cultured in Complete Human Endothelial Cell Medium (CellBiologics, United States), plated in fibronectin-coated (Merck, United Kingdom; 10 μg/ml) 8W10E + array, and grown to confluency to form an endothelial monolayer. The cells were pre-treated with N2-01 for 48 h and then stimulated with LPS from E. coli O111:B4 (Merks, United Kingdom; 1 mg/ml) for 24 h. The capacity of N2-01 to restore barrier function in response to LPS has been monitored and recorded for 24 h.

HULEC-5a cells were obtained from the American Type Culture Collection (ATCC) and maintained in MCDB131 Medium (Gibco, Thermo Fisher) supplemented with 10ng/ml epidermal growth factor (Thermo Fisher), 1µg/ml hydrocortisone (Sigma Aldrich), 10mM L-glutamine (Thermo Fisher), and 10% (v/v) FCS (GeminiBio). Primary human small airway epithelial cells (HSAEC) were purchased (Lifeline Cell Technology) and maintained in BronchiaLife Medium (cat. no. LKL-0023, Lifeline Cell Technology). Primary human alveolar epithelial cells (HAEC) and primary human kidney glomerular endothelial cells (HKGEC) were purchased (CellBiologics) and maintained in Complete Human Epithelial Cell Medium (cat. no. H6621, CellBiologics) and Complete Human Endothelial Cell Medium (cat. no. H1168, CellBiologics), respectively. Primary human small intestine microvascular endothelial cells were purchased (Neuromics) and maintained in ENDO-Growth Medium (cat. no. EGK001, Neuromics). All cells were kept at 37°C in a humidified incubator supplemented with 5% CO₂ and maintained between 50–80% confluence. Primary cells were grown in cell culture flasks coated with gelatin (cat. no. 6950, CellBiologics) and used between 3–7 passages.

Primary human breast tumor associated endothelial cell (HBTAEC, Cell Biologics) was cultured in the vascular compartment of the bMTM to realistically represent the tumor microenvironment. Both MDA-MB-231 and MCF-7 tumor cells obtained from ATCC were cultured in the Dulbecco’s Modified Eagle Medium (DMEM, Invitrogen) supplemented with 10% FBS, 1% Penicillin/Streptomysin. HBTAECs were cultured in Complete Human Endothelial Cell Medium (Cell Biologics). All cells were incubated at 37 °C, 95% humidity with 5% CO₂ supplement. Confluent cells were sub-cultured at a 1:4 ratio until experiments.