Complete culture medium

Human GBM cells U87MG, U251, and HS683 and endothelial cell HUV-EC were obtained from the National Infrastructure of Cell Line Resource in Beijing, China. Cell lines were authenticated at the National Infrastructure of Cell Line Resource, Beijing, China. GBM cells were cultured in a DMEM medium containing 10% fetal bovine serum and 1% penicillin/streptomycin at 5% CO₂ and 37 °C. HUV-EC cells were cultured in complete culture medium (Procell Life Science & Technology Co., Ltd., Wuhan, China). Cells were tested mycoplasma free using a Mycoplasma Test Kit (Cellorlab, China). All materials for cell culture were purchased from Gibco Invitrogen (Grand Island, NY, USA) unless otherwise noted.

The male mice at the age of 6–8 weeks were purchased from the Central Animal Facility of Southern Medical University and used for cell isolation. All the procedures conducted on the animals were approved by the Animal Care and Use Committee of Southern Medical University. The mice were anesthetized with pentobarbital at 0.07 mg/kg, and then their kidneys were collected. This was followed by renal capsule removement in PBS containing 1% penicillin/streptomycin (Sigma–Aldrich, St Louis, MO, U.S.A.). The medullas were removed and the kidneys were minced into small pieces of 1–3 mm³ sized pieces, and then gently ground. The suspension was filtered through a succession of cell strainers of 80, 120, and 200 mesh. The filtrate was centrifuged at 50 g for 2 min. The pellet was resuspended with 0.1% collagenase I in RPMI 1640 and then incubated at 37°C in a shaker for 20 min. The cell suspension was then centrifuged at 100 g for 5 min. The cells were resuspended in complete culture medium (Procell, Wuhan, China). Thereafter, the cells were cultured in the complete medium on dishes precoated with polylysine at 37°C, 5% CO₂, and the medium was refreshed every 3 days.