Endothelial Cell Culture from Rat Brain Microvessels: A Procedure to Isolate and Culture Endothelial Cells from Microvessels of Rat Brain

研究方案

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Isolation of Rat Brain Microvessels

1. For each preparation and for one experimenter, use 3 five-week-old Wistar rats.
2. Monday of the first week, prepare 2 T75 flasks by coating with collagen type IV and fibronectin, both at 1 µg/cm² in sterile culture water (10 ml per T75). Allow to adhere at 37 °C until microvessel seeding.
3. Wednesday morning of the first week, euthanize the rats under an increasing flux of CO₂ to induce drowsiness, loss of posture, and breathing interruption, then followed by cervical dislocation. Spray the heads with 70% ethanol. Cut the heads with a pair of scissors and transfer them into a dry Petri dish under a laminar flow hood.
4. Remove the brains from the skull without the cerebellum and the optic nerves, then transfer them into a Petri dish cooled on ice and filled with cold dissection buffer (HBSS supplemented with 1% BSA, penicillin 100 units/ml, and streptomycin 100 µg/ml).
5. Cut the brains in half to separate the 2 cerebral hemispheres and separate midbrain from forebrain. Transfer the forebrains into a clean Petri dish on ice with dissection buffer.
6. Take a couple of hemispheres in a new Petri dish with cold dissection buffer to carefully remove the meninges from the forebrains under a stereomicroscope with N° 5 curved forceps. Then, clean the interior of the brain to remove the duvet of myelin and obtain a shell of cortex. These steps should not take more than 2 hr for tissue preservation and cell survival.
7. Dissociate the cortex from 3 brains into 6 mL of cold dissection buffer in a 7 mL dounce homogenizer by 10 up and down strokes with each of 2 pestles of different clearances, 71 µm followed by 20 µm.
8. Divide the suspension to obtain the equivalent of 1 cortex in 1 mL of a 50 mL Falcon tube and centrifuge at 1,000 x g for 5 min at RT. Discard the supernatant.
9. Digest the suspension from 1 cortex with 1 mL of an enzymatic solution containing a mix of collagenases/dispase (60 µg/mL–0.3 U/mL), DNase type I (35 µg/mL–20 K units/mL), and gentamicin (50 µg/mL) in a shaker for 30 min at 37 °C.
10. Mix the 1 mL digest from 1 cortex with 10 mL of 25 % BSA/HBSS 1X and separate by density-dependent centrifugation at 3,600 x g for 15 min at RT. Carefully transfer the upper disk (myelin and brain parenchyma) and the supernatant into a clean 50 mL Falcon tube and repeat the centrifugation. Keep the resulting pellet containing the brain microvessels at 4 °C.
11. Carefully discard the upper disk and the supernatant. Resuspend both resulting pellets containing the brain microvessels with 1 mL of cold HBSS 1X and transfer into a clean 50 mL Falcon tube.
12. Wash the microvessels by addition of 20 mL of cold HBSS 1X and centrifuge at 1,000 x g for 5 min. Discard the supernatant.
13. Further, digest the microvessels from 1 cortex with 1 mL of the same enzymatic solution as described in step 1.9 during 1 hr at 37 °C in a shaker.
14. Then, mix the digested microvessels from each of the 3 cortices into one tube and further separate into two 50 mL Falcon tubes to obtain microvessels extracted from the equivalent of one and a half (1.5) cortex per tube. Add 30 mL of cold dissection buffer and centrifuge at 1,000 x g for 5 min at RT.
15. Resuspend the microvessel pellet in 10 mL of DMEM/F12 supplemented with 20% bovine platelet-poor plasma-derived serum, basic fibroblast growth factor (bFGF, 2 ng/mL), heparin (100 µg/mL), gentamicin (50 µg/mL) and HEPES (2.5 mM), named endothelial cell media (ECM) supplemented with puromycin at 4 µg/mL.
16. Take the 2 coated T75 flasks from the incubator, aspirate the excess of coating, and plate the microvessels from each tube in one T75 flask (microvessels extracted from 1.5 cortex per T75 flask).

材料

Name	Company	Catalog Number	Comments
Fetal bovine serum	Invitrogen	10270-098	-20 °C / 1 year
BSA fraction V low endotoxin	PAA	K45-011-500g	4 °C
Penicillin-streptomycin	Invitrogen	15140-122	-20 °C
T75 flasks	Becton Dickinson Falcon	353135
HEPES buffer (1 M)	Invitrogen	15630-056	4 °C / 1 year
Collagen type IV mouse (10 x 1 mg)	Becton Dickinson	356233	-20 °C / 1 month
Fibronectin humain plasma (5 mg)	Becton Dickinson	356008	-20 °C / 1 month
Vannas spring scissors, straight (9 cm)	Fine Science Tools	91500-09
Scissors, straight (9 cm)	Fine Science Tools	14568-09
Forceps Dumont #5/45 (11 cm)	Fine Science Tools	11251-35
Graefe Forceps, tip width 0.8 mm	Fine Science Tools	11050-10
Graefe Forceps, curve tip width 0.8 mm	Fine Science Tools	11051-10
Collagenases / dispase mix (2 x 5 mg)	Roche Diagnostics	05 401 054 001	-20 °C / 3 months
DNase I (100 mg / 569 KU / mg)	Sigma Aldrich	DN25-100 mg	-20 °C / 1 year
Gentamicin (10 mg/ml)	Invitrogen	15710-049	4 °C / 1 year
DMEM/F12	Invitrogen	31331-028	4 °C
Bovine serum from platelet poor plasma (500 ml)	Clinisciences	BT-214-100	-20 °C / 1 year
bFGF (10 µg)	Invitrogen	13256-029	-20 °C / 6 months
Heparine Na salt from porcine mucosa Grade I	Sigma Aldrich	H3149-100KU	4 °C / 1 year
Puromycin	Sigma Aldrich	P8833-10 mg	-20 °C / 6 months