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

Abstract

Source: Molino, Y. et al. Setting-up an In Vitro Model of Rat Blood-Brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport. J. Vis. Exp. (2014)

This video presents the isolation of microvessels from a rat brain using a combination of density gradient centrifugation and enzymatic digestion mehods. The isolated microvessels can be cultured in a culture flask pre-coated with collagen and fibronectin matrix to obtain endothelial cell colonies.

Protocol

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
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Materials

List of materials used in this article

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