Isolation of Human Brain Microvascular Endothelial Cells
[* All steps must be performed on ice.]
1. Collection of human brain tissues
Collection buffer : RPMI1640 media + 2% FBS + Penicillin/Streptomycin +
antibiotic/antimycotic [RPMI-S] in 50 mL centrifuge tube
Source : surgical resections of brain of patients with seizure disorders or postmortem
brains
Isolation should be performed less than 10 h after collection of brain tissues.
2. Isolation of gray matter from brain tissue
2-1. Wash the tissue with RPMI-S several times.
2-2. Remove the meninges, all visible large blood vessels, white matter, and other
impurities (e.g., laser burn, etc) using mass and sharp forceps on ice as complete as
you can.
2-3. Transfer the gray matter (pink or ivory color) to 15 mL centrifuge tube on ice.
3. Isolation of brain microvessels from gray matter
3-1. Homogenize the gray matter in 2 volume of RPMI-S using a sterile Dounce
homogenizer with stroke several times.
3-2. Pipetting homogenates several times.
3-3. Mix 1 vol. of homogenate and 1 vol. of 30% dextran dissolve in RPMI-S (filtersterilized) thoroughly [final dextran concentration : 15%].
3-4. Centrifuge the homogenate in 15% dextran at 10,000  g for 10 min at 4 C and
remove the cake (uppermost part : lipid, astrocytes, glial cells, etc.) and supernatant
using a 25 mL pipette.
3-5. Resuspend the pellet (crude microvessels) with 5 mL of RPMI-S (in 15 mL
centrifuge tube) and spin down (1,800 rpm, 5 min, 4 C) to remove dextran
completely.
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Dr. Lee’s Lab
4. Digest the pellet (crude microvessels) with 5 mL of 0.5 mg/mL collagenase/dispase
solution (dissolve in RPMI-S and filter-sterilized) for 1 h at 37 C with shaking and
centrifuge the mixture at 2,500 rpm for 5 min at 4 C.
5. Resuspend the pellet with complete RPMI1640 media (RPMI 1640-based medium
with 10% fetal bovine serum, 10% NuSerum, 30 g/mL of endothelial cell growth
supplement, 15 U/mL of heparin, 2 mM L-glutamine, 2 mM sodium pyruvate,
nonessential amino acids, vitamins, 100 U/mL of penicillin, and 100 g/mL of
streptomycin), plate the human brain microvessels on rat tail collagen-coated flasks,
and incubate them in CO2 incubator (HBMEC are extruded from microvessels and
attached to the bottom).
6. After incubation for 24 - 48 h, the cultures are washed with Hank’s and replenished
with complete RPMI1640 media. Cells are split in a ratio of 1:3. Experiments are
performed between passage 3 and 8.
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Dr. Lee’s Lab