Fig. 1

Generation of pure endothelial cultures from human brain tissue. Brain tissue was cultured to generate mixed glial and endothelial cultures. Cells were fixed, and purity of cultures was assessed at passage two using immunocytochemistry. a Processing of biopsy specimens, followed by b mechanical and c–e enzymatic digestion at 37 °C. Suspension was f triturated and g passed through cell strainers h which were washed. i Debris were collected, spun, and j plated onto a matrigel-coated flask. k Schematic of the method used to generate mixed glial (containing astrocytes/microglia/endothelia/pericytes), and isolated endothelial cultures. l Representative live images of passage 1 cultures throughout different growth stages. Arrowheads highlight blood vessels and endothelia while areas outlined in red are endothelial colonies, arrows indicate astrocytes and asterisks denote microglia. Scale bar = 200 μm. m Representative images and n quantification of staining for lineage markers (CD31, endothelial; PDGFRβ, pericyte; GFAP, astrocyte; PU.1, microglial) in passage two mixed glial and endothelial cultures. Scale bar = 500 μm, inset scale bar = 100 μm. d Cell lineage assessment of endothelial and mixed glial cultures (mean, n = 3 mixed glial, n = 5 endothelial)