The Establishment of in vitro Model of Primary Rat Brain Microvascular Endothelial Cells - Advances in Computational Science and Engineering - page 112

microvessels by once centrifugation and have less inhibition to cell growth activity.
Besides, many labs use BSA which is considered better than dextran in this process.
But according to our attempt and test, the result of culture using BSA is not much
better than that of dextran. As to the ingredient of culture fluid, in the absence of Nu-
serum or ECGF circumstances, we can use only one of them for our culture. But using
both of them can promote the cell growing better. The planting density should be in-
creased if only one of them to be used, the proper density is three culture flasks for
BMEC from eight rats.
The growing ability was restrained obviously by miscellaneous cells, especially fi-
broblast after passage, thus BMEC need to be purified after passage. Several methods
of purification such as digestion lag separation, adherence lag separation, miscellane-
ous cells curettage method, monoclonal culture and suppression medium culture
methods were tried to remove fibroblasts. Digestion lag separation method and mono-
clonal culture method do not apply to remove fibroblasts. Curettage method could kill
fibroblasts effectively, easily, but only partly. Because of adherence between BMECs
and fibroblast, curettage fibroblasts often embroiled the endothelial cells around.
Because medium D-Viline MEM can lead to fibroblast's growth inhibiting, which
inhibit endothelial cells' growth as well, so cultivating the primary cell with D-Viline
MEM when dominant growth of endothelial cells appeared can inhibit the growth of
fibroblasts effectively and won't restrain the growth activity of endothelial cells
which own the overall advantages.
The cells we cultured were fusiform or polygonal, and presented monolayer spiral,
formed a cell aggregate. These cell aggregate presented "road-metal" arrangement
when they reached confluence (Figure5). These characters are the same as reported
before.
It has begun to attempt to cultivate endothelial cells abroad since 1960s, it mainly
drew materials from the cerebral cortex tissues of mice, rats, cattle, pigs, goats, dogs
and human. The source of rat brain is plentiful, it is relatively easy to perform sterile
operation and draw materials, but the gray matter is comparatively little, it needs a
large number of animals at one time. The gray matter of cattle brain is plentiful, it is
easy to obtain comparatively pure gray matter tissue, but the source of brain tissue is
relatively difficult, the cost is comparatively high, it is comparatively difficult to draw
materials and it is easily to be polluted, the domestic and abroad documents of
isolation and culture methods are quite different, the cultivated cell shapes are also
different, so it seldom uses cattle as sampling animals in China. After the isolation
technique of brain microvascular endothelial cells of animals becomes more and more
mature, human's will also be isolated and cultivated, all the founded indexes of in
vitro blood-brain barrier is closer to the blood-brain barrier inside human's body. But
compared to the experimental animals, the source of human's healthy brain tissue is
less, it is more difficult to draw materials and perform cells isolation and culture. In
contrast, the source of rats is plentiful, which contain multiple kinds of mature patho-
logical models, so rats are always usual sampling objects.
In fact, pigs and cows have been used to isolate brain microvascular endothelial
cells in our laboratory, too. In our opinion, these experiments appeared well result of
culture, but these animals are costly, and the operation of taking out brain is more
difficult, and the brains of animals are much easier to be polluted by microbe. And we
find that infancy animals' brain microvascular endothelial cells showed more excellent