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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

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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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Correspondence to Tetsuya Terasaki.

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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

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