Pharmaceutical Research

, Volume 24, Issue 4, pp 688–694

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

  • Sumio Ohtsuki
  • Hirofumi Yamaguchi
  • Tomoko Asashima
  • Tetsuya Terasaki
Research Paper

DOI: 10.1007/s11095-006-9188-x

Cite this article as:
Ohtsuki, S., Yamaguchi, H., Asashima, T. et al. Pharm Res (2007) 24: 688. doi:10.1007/s11095-006-9188-x
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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.

Key words

brain capillary endothelial cellsmagnetic cell sortingmultidrug resistance-associated proteinPECAM-1purification

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

Copyright information

© Springer Science+Business Media, LLC 2007

Authors and Affiliations

  • Sumio Ohtsuki
    • 1
    • 2
    • 3
  • Hirofumi Yamaguchi
    • 1
  • Tomoko Asashima
    • 1
    • 2
    • 3
  • Tetsuya Terasaki
    • 1
    • 2
    • 3
  1. 1.Department of Molecular Biopharmacy and Genetics, Graduate School of Pharmaceutical SciencesTohoku UniversitySendaiJapan
  2. 2.New Industry Creation Hatchery CenterTohoku UniversitySendaiJapan
  3. 3.CREST and SORST of the Japan Science and Technology Agency (JST)SaitamaJapan