Ec growth supplement

The in vitro model was set up using stem cells as described previously [10 (link)]. Endothelial cells were derived from human umbilical cord blood CD34⁺-cells. In accordance with French legislation, the infant’s parents signed informed consent. The collection protocol was approved by the French Ministry of Higher Education and Research (CODECOH number DC2011-1321).
Briefly, CD34⁺-hematopoietic stem cells were isolated and differentiated into endothelial cells as described by Pedroso et al., [23 (link)]. Then these endothelial cells were seeded onto matrigel (BD Biosciences, Franklin Lakes, NJ, USA, 354230) coated Transwell inserts (Corning, NY, USA, 3401). Endothelial cells (8 × 10⁴ cells/cm²) were seeded onto the insert and cultured in the presence of 5 × 10⁴ cells/cm² bovine brain pericytes seeded at bottom of a well (12-well format). The medium of this co-culture [ECM-5 composed of ECM basal medium (Sciencell, Carlsbad, CA, USA) supplemented with 5% (v/v) fetal calf serum, 1% (v/v) EC growth supplement (Sciencell) and 50 µg/mL gentamycin (Biochrom AG, Berlin, Germany)] was renewed every 2 days. After 6 days of co-culture with pericytes, the endothelial cells differentiated into BBB endothelial cells and were ready for experiments. This model named BLECs reproduces several features of the in vivo BBB [24 (link),25 (link),26 (link)].

Human umbilical vein ECs (HUVECs) were purchased from Procell Life Science&Technology Co., Ltd (Wuhan, China). HUVECs were cultured in EC medium (ScienCell, USA) supplemented with 10% exosome-depleted foetal bovine serum (SBI, USA), 1% EC growth supplement (ScienCell) and 1% penicillin/streptomycin solution (ScienCell), and cells were cultured at 37 ℃ in humidified air containing 5% CO₂. HUVECs were performed for the experiments in passages 4–6.
Rat SCs (RSC96 cells) were purchased from Procell Life Science&Technology Co., Ltd. SCs were cultured in high-glucose Dulbecco’s modified Eagle’s medium (Gibco, USA). Moreover, the culture mediums were supplemented with 10% exosome-depleted FBS (SBI). RSC96 cells were performed for the experiments in passages 4–6.

The technique for isolating mouse BMECs was adapted from published protocols (16 (link)). Mice were euthanized and perfused with saline. And brains were finely minced with 1 ml of medium and homogenized by passing through a 23-gauge needle. The homogenate was mixed with an equal volume of 30% dextran (MW 70,000, BBI) in PBS and centrifuged at 10,000 g for 15 min at 4°C. The pellet was resuspended in PBS and passed through a 40 μm cell strainer that retained the microvessels. After washing, the cell strainer was back-flushed with 2 ml PBS over a 6-well plate to collect the microvessels, which were rocked at room temperature with 2% FBS, 1 mg/ml collagenase II (02100502, MP Biomedicals) and 20 μg/ml DNase I (10104159001, Sigma-Aldrich) for 90 min. Vessel fragments were collected and resuspended in EC medium (0.1 mg/ml EC growth supplement from ScienCell, catalog #1001) with 4 μg/ml puromycin and seeded into a collagen-coated 6-well plate. The medium was replaced (without puromycin) 3 days later and every 3–4 days thereafter. The purity of BMECs was identified with CD31 by flow cytometry. For cytokine activation of BMECs, 20 ng/ml IFN-γ was added to the cell medium 24 h prior to subsequent analysis.