2.3. Generation of 3D NVU

For co‐culture, medium contained neurospheres was centrifuged at 100 × g for 5 min and dissociated the obtained neurospheres to single cell using ACCUTASE. About 80%–90% confluent BMECs were trypsinized (with 0.02% EDTA) and centrifuged at 150 × g for 5 min and resuspension with serum‐free culture medium. NSCs and BMECs were mixed and resuspension with Matrigel (BD Biosciences, growth factor reduced) at a density ratio of 1:10. The final density of BMECs was 2.0 × 10⁶·ml⁻¹, and the final density of NSCs was 2.0 × 10⁵·ml⁻¹. The 100 μl cell/Matrigel mixture was then evenly spread on the bottom of the confocal dishes (NEST, 15 mm) or cell culture inserts (Greiner Bio‐One, 24‐well), and then, placed in an incubator to solidify. After 1 h, 50 μl mixture of NSCs/Matrigel was evenly spread on the surface of the solidified gels, which was further placed in the incubator for 2 h, and then, the culture medium was added. BMECs‐NSCs co‐culture system was grown in DMEM/F12: Neurobasal medium 1:1, 2% B27 (without vitamin A), 20 ng·ml⁻¹ rh EGF, 10 ng ml⁻¹ rh bFGF and the medium was supplemented with 1% FBS. After 3 days, B27 in the medium was replaced with B27 containing vitamin A. The BMEC‐NSC co‐cultures or control BMEC or NSC monocultures were analyzed following 3 or 7 days of culture, using live cell microscopy and immunofluorescence staining.