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2.2 Reagents
RPMI-1640, DMEM containing 4000mg/L glucose and FBS were obtained from
Gbico; L-Glutamine (200mmol/L), Penicillin-Streptomycin Mixture (10KU/L),
25% Trypsin - EDTA mixture were obtained from Genom; Dextran (clinical grade,
average mol. wt. 70,000) were obtained from Seebio; DNase , Heparin(10KU/ml
PBS) were obtained from Sigma; Collagen Type І (100mg/28.65ml) were obtained
from Upstate; Sodium Pyruvate (100mmol/L), MEM non-essential amino acids,
MEM vitamins were obtained from Irvine; Nu-Serum were obtained from B&D;
Endothelial cell growth factor (ECGF), and Collagenase/ Dispase were obtained
from Roche.
2.3 Culture Solution
Isolation medium: DMEM containing 2%FBS and 5% Penicillin-Streptomycin
Mixture; 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;
Digestive juice: Isolation medium containing 1mg/ml Collagenase/Dispase and
0.1mg/ml DNase
, filtered through 0.22µm filter membrane; Collagen Type 1:
Diluted it to 5 ~ 10µg/cm 2 for the surface of culture flask with PBS. 30% Dextran:
Dextran 60g dissolved in 200ml ddH 2 O, autoclaved (0.15KPa121
and then added 20ml PBS in dextran solution.
2.4 Culture Flask
Covered the surface of culture flasks with collagen type I about 5~10µg/cm 2 , and kept
the flasks at 4 overnight. Then dry them in 37 incubator, then kept at 4 .2.5
Isolation method.
2.5 Method
Rats were killed using cervical dislocated method, then, immersed them in 75% etha-
nol for 5 min. Then each brain of these rats was eased out of the skull, and the cere-
bellum and brain stem were removed. Meninges, associated vessels on the surface and
white matters of the brain were removed using forceps, and gray matter (Figure 2.) of
the brain was rolled slowly and carefully on a sterile filter paper. The isolated cerebral
cortex was placed into 25ml isolation medium.
Then, gray matters were passed through a common metal net gently to pulverize
them into smaller pieces. Translated the organization levitation medium into homoge-
nizer, homogenized the medium till it looks like milk shake. The homogenized sam-
ples were transferred into a 50ml centrifuge tube. Then 25ml 30% dextran solution
was added into this centrifuge tube. The mixture was centrifuged at 4500 g , 4 for 20
min. Discarded supernatant and resuspended the pellets with PBS, and centrifuged at
150 g , 4 for 5 min to wash dextran away. Then the step repeated twice.
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