The Establishment of in vitro Model of Primary Rat Brain Microvascular Endothelial Cells - Advances in Computational Science and Engineering - page 111

4 Discussions
When Panula and other people first proved that BMEC can be cultivated by tissue
culture method, including that primary and genetic individual BMEC had been ap-
plied in the following future experimental external BBB model system. Compared
with the individual microvessel which is the earliest in vitro model system of the
BBB, its main advantage is that it can be applied to research the transport process of
the transversal cells under the proper experimental conditions. At the mean time, the
external experiments of central nervous system infection related to invading BMEC is
also mainly dominated by such model.
A method of isolation and culture BMECs effectively but relatively low require-
ments for the instrument was explored. BMEC could be separated without high-speed
centrifuge, and this method consumes less time, too.
Comparing other separation methods interiorly and abroad, the step of passing the
gray matter through the metal gauze instead of the fine pruning, and tissue suspension
obtained after this grinding process was more uniform, the pieces were smaller, the
operation is more finely and timesaving. The use of tissue homogenizer which oper-
ated gently and slowly can crush tissue with less damage of cells, and the suspension
can be stratified by centrifugation directly. Compare with twice digestions method,
this process result in shorter operation time, and less complicated steps. But the me-
chanical damage to microvessels would be more severe than digestion's if operated
roughly, which even affect the activity of cells. So this process must operate gently
and in low temperature. In addition, the use of screen mesh suggested by many labs
was attempted and tested. It is considered that screen mesh can reduce the immixing
with blood cell effectively, but the function of filtering off gliacyte and astrocyte is
not obvious, oppositely, the loss of blood vessels in the process increased. If men-
ingeal fragments were remained apparently because of rough operation when strip-
ping meninge, we can filter it once using the screen mesh with 150 micron aperture
after homogenate to remove meningeal fragments.
Dextran zonal centrifugation can get good isolation effect. According to previous
reports, we attempted the step of centrifuge at different centrifugal speed and time [1,
3~5], including 7000g, 10min; 3500g, 20min; and 4500g, 10min, repeated three times.
Of the three plans, the first one have higher centrifugal force which can isolated cells
effectively, and also have short contact time between microvascular fragments and
dextran, but it demand high requirement to equipment, and the high centrifugal force
would decline the growth activity of vascular fragments. According to the culture ex-
perience, many microvascular fragments can't adhere to culture surface and grow
slowly which usually show growing ability after have been cultivated between 24 to 48
hours. The second plan asks lower centrifugal force and keeps high microvascular
growth activity, but microvascular can still found in upper layer of liquid and there are
too many impurities in vascular layer because of the incomplete isolation. This plan
can be taken if there were equipments for percoll operation for further isolation. The
third plan use the method of repeated centrifugation to remove impurities as possible,
but repeated centrifugation and long contact with dextran are the inhibitory factors of
growth activity of cells, those steps also increased the loss of vascular fragment. Thus,
according to these protocols we take a compromise centrifugal rate and centrifugal
time which can isolate microvascular fragments well and show well pick-up rate of