Culture of brain microvascular endothelial cells in normal rabbits

Culture of brain microvascular endothelial cells in normal rabbits

Experimental Materials:

1. Normal rabbit cerebral cortical tissue;

2. 1×PBS containing no Ca 2+ and Mg 2+ , adding 200,000 IU/L penicillin, 200 mg/L streptomycin, pH 7.2;

3. M199 medium (containing 20% ​​calf serum, heparin 25U/ml, HEPES 10mmol/L, 100IU/ml penicillin and 100μg/ml streptomycin); D-Hanks solution; 0.05% trypsin solution; 0.05% collagen Enzyme solution; 15% dextran (prepared with Hanks, pH 7.4);

4. Homogenizer, scalpel, anatomical scissors, anatomical sputum, ophthalmic scissors, ophthalmic sputum, hemostat, sterile sterile surgical instruments;

5. Centrifuge tube (15ml, 50ml)

6. Mesh screen: 110μm nylon mesh screen

experimental method:

1. The rabbit is sacrificed by carotid bloodletting and the scalp is removed. The skull was removed and immersed in 75% ethanol for about 1 min. The rabbit cerebral cortex was quickly removed under aseptic conditions. Care must be taken to remove large blood vessels and pia mater;

2. Place the cortex in D-Hanks solution, rinse it several times, and then cut it. Then transferred into a vial containing 0.05% trypsin solution, and digested in a 37 ° C water bath for 10-20 min;

3. Stop the digestion with serum culture medium. It was ground in a glass grinder to prepare a coarse suspension. Filtration through a nylon mesh having a pore size of 110 μm, and centrifuging the filtrate at 4000 r/min for 10 min;

4. Discard the supernatant after centrifugation. A 15% dextran solution was added to the pellet and the pellet was resuspended. Then, it was centrifuged at 4000 r/min for 20 min. Collecting microvascular fragments;

5. Digest with 5% collagenase solution for 2-4 hours. Washed with Hanks solution and centrifuged, and added M199 medium to the microvascular fragments;

6. Inoculate into a culture flask and incubate in a 37 ° C, 5% CO 2 incubator (humidity 100%).

7. After 24h, change the fluid and move the unattached microvascular segments into other culture flasks or dishes to continue adherent growth. After that, the liquid is changed every 3 days, and when the cells are grown to the fusion state, subculture can be carried out;