Protocol for isolation of HCEC.

摘要: Microvascular HCEC are isolated from fresh biopsies of normal human brain following a modified version the isolation protocol by Bowman (1). The samples tissue gained on occasion surgical approach to deep seated lesions or after resection epileptogeneic foci. Informed consent patients has be obtained in each case and approved local ethical committee. Tissue is immediately transferred laboratory cold culture medium for isolation. pia mater arachnoidea removed cerebrum. Large blood vessels not used endothelial cell grey matter areas homogenized washed M199 containing 10% fetal calf serum (both Bio Whittaker, Belgium), amphotericin (2.5 μg/ml) penicillin/streptomycin (100 µg/ml, both GIBCO BRL life technology, Germany). enrichment cerebral microvessels performed centrifugation step 15% dextran (Sigma, U.S.A.) followed dissociation with collagenase/dispase (Boehringer Mannheim, Germany) 16 – 20 hours. Endothelial cells separated other types percoll gradient (Amersham Pharmacia, Sweden) several washing steps 5% FCS. Cells plated gelatine coated 24- 6-well plates (Costar, 20% FCS, heparin ECGS (endothelial growth supplement, 30 μg/ml, Sigma, Germany), (250 μg/ml), penicillin (50 U/ml), streptomycin gentamycin all BRL) cultured at 37ΰC/5% C0 2/l 00% humidity. Medium changed every two days. During further culturing FCS content reduced 10%. split subconfluent stages maximum 1:4 ratio. For experiments only passage 2 8 used. Cell purity over 90% confirmed microscopy inspection staining appropriate antibodies experiments.