Transcript Isolation of primary BMECs

Isolation and Primary Culture
of Rat Brain Microvascular
Endothelial Cell
The 2nd International Conference on BSBT 2008
December 14, 2008
Li Jun, Cao Hong
Department of Microbiology, School of public health and tropical
medicine, Southern medical university,
Guangzhou, 510515 China
Introduction
• The blood-brain barrier (BBB) is a metabolic
and cellular structure in the central nervous
system (CNS) that restricts the passage of
various chemical substances and microscopic
objects (e.g. bacteria) between the bloodstream
and the neural tissue itself, while still allowing
the passage of substances essential to
metabolic function.
White Matter and Gray Matter
Introduction
• The brain microvascular endothelial cells
(BMEC) are the main and the most important
component of blood-brain barrier (BBB). They
are joined by tight junctions of high electrical
resistance providing an effective barrier against
molecules.
• The BMEC monolayer in vitro model system are
morphologically similar to endothelial cells of the
BBB in vivo: characterized by tight intercellular
junction, few pinocytic vesicles, and lacking
fenestra.
The BBB: Brain microvascular endothelial cell is the main
component of the BBB. Source: Sheng-He Huang and Ambrose
Y. Jong. Cellular mechanisms of microbial proteins contributing
to invasion of the blood–brain barrier.
Introduction
• Markers for cells of endothelial origin, for
example, angiotensin-converting enzyme, and
factor VIII antigen, have been demonstrated in
this model.
• The transfer function studies on the model in vitro
showed that whether passive or active transport
dispersion process is found similar with that in
vivo.
• So, isolated BMEC provide an in vitro model for
studying the pathogenesis of microbial meningitis
and physiopathology, Pharmacology of the BBB.
Introduction
• There are many methods to isolate BMEC, but
spending long time, big workload, demanding
instruments, survival difficulty of BMEC and
growth of mixed fibroblasts are still common
problems.
• We tried and found out a method of separation
and culture of BMEC with high purity, growth
activity and characteristics of the BBB.
Methods and results
Preparation
• Culture Flasks:
Covered the surface of culture flasks with collagen
type I about 5~10μg/cm2, and kept the flasks at 4℃
overnight. Then dry them in 37℃ incubator, then kept
at 4℃.
• Growth Medium:
RMPI 1640 containing 10% FBS, 10% NuSerum,
ECGS 30mg/L, 1% Penicillin-Streptomycin Mixture,
Heparin 5 U/ml, 1% L-Glutamine, 1% Sodium
Pyruvate, 1% MEM non-essential amino acids, 1%
MEM vitamins.
• Isolation Medium:
DMEM containing 2% FBS and 5% PenicillinStreptomycin Mixture.
• Digestive Juice:
Isolation medium containing 1mg/ml
Collagenase/Dispase and 0.1mg/ml DNase Ⅰ, filtered
through 0.22µm filter membrane.
• 30% Dextran:
Dextran 60g dissolved in 200ml ddH2O, autoclaved
(0.15KPa，121℃，15min), and then added 20ml PBS
in dextran solution.
Methods ：
Meninges associated vessels on
the surface and white matters
of the brain were removed .
Gray matters were passed through
a common metal net gently.
Translated the organization levitation
Medium into homogenizer,
homogenized the medium
till it looks like milk shake.
Same volume of 30% dextran solution
was added into this centrifuge tube.
The mixture was centrifuged at 4500g,
4℃ for 20 min.
Discarded supernatant and
resuspended the pellets with PBS,
and centrifuged at 150g,
4℃ for 5 min
The pellets was resuspended
and dissociated with
2ml digestive juice,
at 37℃ for 25-30min.
Till most microvessels beaded
and shorter then before.
The microvessels were centrifuged
at 150g, room temperature for
5min, three times. Then the pellet
was resuspended with culture
medium, plated in collagen-coated
flask.
The isolated microvessels
after digesting: digested
using Collagenase/dispase
and DNase Ⅰ, till most
microvessels beaded and
shorter then before.（200
×）
Results
Culture within 24 hours of the BMEC:
After 5 to 6 hours of culture,
some microvessels had
adhered (Figure A).
A
About 12 hours later, the most
microvessels had attached.
Twenty hours after the vessels
planted, cells had climbed out and
proliferated, gradually formed cell
aggregates (Figure B).
B
BMEC after 1 week of culture
Cultivated in the growth medium longer than 24 hours, the
cells climbed out from the microvessels increased obviously,
and presented monolayer spiral arrangement, formed a cell
aggregate. These cell aggregates proliferated gradually,
would reach confluence at the 7th day, and presented "roadmetal" arrangement.
Summary
• The BMEC monolayer in vitro model system
is the one of most important models of the
BBB in vitro. The means we reported included
homogenate, centrifuge and digestion.
characterized by tight intercellular junction,
few pinocytic vesicles, and lacking fenestra,
angiotensin-converting enzyme, and factor
VIII antigen.
• To establish the BBB model system in vitro,
the brain of 2- to 3-week-old SD rats were
choosed for this study.
• Meninges, vessels on the surface and white
matters of the brain were removed, then gray
matters were pulverized into smaller pieces.
• The organization levitation medium of gray
matters were homogenized, and centrifuged
with dextran to separate microvessels and
other components of cerebral cortex.
• The microvessels were washed with PBS, then
digested by digestion juice.
• When the microvessels were much shorter then
before and looked like beads, the process of
digestion could be stopped, and resuspented in
culture medium and planted on the collagencoated surface.
• The digested microvessels could adhere
culture-surface after cultivated five hours. The
microvessels in flask after incubated 20 hours,
cells had climbed out, and then gradually
formed cell aggregate.
• The BMEC after 7 days’ culture, the cells
aggregated proliferate gradually, the cells
reached confluence after 7 days, and presented
“road-metal” arrangement. It is the same with
the reported before.
• Comparing other separation methods
reported interiorly and abroad, the
method of isolation and culture BMECs
effectively but relatively low
requirements for the instrument was
explored. BMEC could be separated
without high-speed centrifuge, and this
method consumes less time, too.