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 output: Contribution to journalArticlepeer-review

30 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)688-694
Number of pages7
JournalPharmaceutical research
Volume24
Issue number4
DOIs
Publication statusPublished - 2007 Apr

Keywords

  • Brain capillary endothelial cells
  • Magnetic cell sorting
  • Multidrug resistance-associated protein
  • PECAM-1
  • Purification

ASJC Scopus subject areas

  • Biotechnology
  • Molecular Medicine
  • Pharmacology
  • Pharmaceutical Science
  • Organic Chemistry
  • Pharmacology (medical)

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