Wnt activation of immortalized brain endothelial cells as a tool for generating a standardized model of the blood brain barrier in vitro

PLoS One. 2013 Aug 5;8(8):e70233. doi: 10.1371/journal.pone.0070233. Print 2013.


Reproducing the characteristics and the functional responses of the blood-brain barrier (BBB) in vitro represents an important task for the research community, and would be a critical biotechnological breakthrough. Pharmaceutical and biotechnology industries provide strong demand for inexpensive and easy-to-handle in vitro BBB models to screen novel drug candidates. Recently, it was shown that canonical Wnt signaling is responsible for the induction of the BBB properties in the neonatal brain microvasculature in vivo. In the present study, following on from earlier observations, we have developed a novel model of the BBB in vitro that may be suitable for large scale screening assays. This model is based on immortalized endothelial cell lines derived from murine and human brain, with no need for co-culture with astrocytes. To maintain the BBB endothelial cell properties, the cell lines are cultured in the presence of Wnt3a or drugs that stabilize β-catenin, or they are infected with a transcriptionally active form of β-catenin. Upon these treatments, the cell lines maintain expression of BBB-specific markers, which results in elevated transendothelial electrical resistance and reduced cell permeability. Importantly, these properties are retained for several passages in culture, and they can be reproduced and maintained in different laboratories over time. We conclude that the brain-derived endothelial cell lines that we have investigated gain their specialized characteristics upon activation of the canonical Wnt pathway. This model may be thus suitable to test the BBB permeability to chemicals or large molecular weight proteins, transmigration of inflammatory cells, treatments with cytokines, and genetic manipulation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Blood-Brain Barrier / metabolism*
  • Brain / cytology*
  • Brain / metabolism*
  • Endothelial Cells / metabolism*
  • Mice
  • Microscopy, Fluorescence
  • Reverse Transcriptase Polymerase Chain Reaction
  • Wnt Proteins / metabolism*
  • beta Catenin / metabolism


  • Wnt Proteins
  • beta Catenin

Grant support

This study was supported by grants from: Fondation Leducq Transatlantic Network of Excellence, Associazione Italiana per la Ricerca sul Cancro (AIRC), and “Special Program Molecular Clinical Oncology 5×1000” to AGIMM (AIRC-Gruppo Italiano Malattie Mieloproliferative), the European Community (EUSTROKE-contract-202213, OPTISTEM-contract-223098, ANGIOSCAFF-NMP3-LA-2008-214402 Networks; ENDOSTEM-HEALTH-2009-241440; JUSTBRAIN-HEALTH-2009-241861; ITN Vessels), the European Research Council and CARIPLO Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.