1. 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 Isolation and Primary Culture of Rat Brain Microvascular Endothelial Cell

2. 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. Introduction

3. White Matter and Gray Matter

4. 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. Introduction

5. 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.

6. 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.

7. 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.

8. Methods and results

9. 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. Preparation

10. Isolation Medium: DMEM containing 2% FBS and 5% Penicillin- Streptomycin 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 ddH 2 O, autoclaved (0.15KPa ， 121 ℃， 15min), and then added 20ml PBS in dextran solution.

11. 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.

12. 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.

13. The pellets was resuspended and dissociated with 2ml digestive juice, at 37 ℃ for 25-30min. Discarded supernatant and resuspended the pellets with PBS, and centrifuged at 150g, 4 ℃ for 5 min

14. 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 × ）

15. Results Culture within 24 hours of the BMEC: 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). After 5 to 6 hours of culture, some microvessels had adhered (Figure A). A B

16. 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 "road- metal" arrangement.

17. Summary

18. 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.

19. 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.

20. 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 collagen- coated surface.

21. 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.

22. 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.