Purpose
To establish a method for isolating highly purified brain capillary endothelial cells (BCECs) from rat brain by using magnetic cell sorting, and clarify the expression levels of multidrug resistance-associated protein (Mrp) subtypes in these highly purified BCECs.
Methods
The cells were prepared from the capillary enriched-fraction by enzyme digestion, and reacted with anti-PECAM-1 antibody. The cell sorting was performed by autoMACS. The mRNA levels were measured by quantitative real-time PCR analysis.
Results
From five rats, 2.3 × 106 cells were isolated in the PECAM-1(+) fraction and the percentage of labeled cells in this was 85.9%. PECAM-1, claudin-5 and Tie-2 mRNA were concentrated in the PECAM-1(+) fraction compared with rat brain. The contamination by neurons and astrocytes was markedly less than in the brain capillary fraction prepared by the glass bead column method. Mrp1 and 4 were predominantly expressed in the PECAM-1(+) fraction at similar levels to Mdr1a. The mRNA levels of Mrp5 and 3 were 10.6 and 7.60% of that of Mrp1, respectively.
Conclusions
This new purification method provides BCECs with less contamination by neural cells. In the isolated BCECs, Mrp1 and 4 are predominantly expressed, suggesting that they play an important role at the rat blood-brain barrier.
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Abbreviations
- 6-mp:
-
6-mercaptopurine
- ABC:
-
ATP binding cassette
- AZT:
-
azidothymidine
- BBB:
-
blood-brain barrier
- BCECs:
-
brain capillary endothelial cells
- DHEAS:
-
dehydroepiandrosterone sulfate
- E217βG:
-
17β-estradiol-D-17β-glucuronide
- FL2:
-
fluorescence channel 2
- Mrp:
-
multidrug resistance-associated protein
- PBS:
-
phosphate buffered saline
- PE:
-
Phycoerythrin
- PECAM-1:
-
platelet endothelial cellular adhesion molecule-1
- SSC:
-
side scattered light
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Acknowledgement
This study was supported, in part, by a Grant-in-Aid for Scientific Research on Priority Areas 17081002 from The Ministry of Education, Culture, Sports, Science and Technology of Japan and a 21st Century Center of Excellence (COE) Program grant from the Japan Society for the Promotion of Science.
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Ohtsuki, S., Yamaguchi, H., Asashima, T. et al. Establishing a Method to Isolate Rat Brain Capillary Endothelial Cells by Magnetic Cell Sorting and Dominant mRNA Expression of Multidrug Resistance-associated Protein 1 and 4 in Highly Purified Rat Brain Capillary Endothelial Cells. Pharm Res 24, 688–694 (2007). https://doi.org/10.1007/s11095-006-9188-x
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DOI: https://doi.org/10.1007/s11095-006-9188-x