Puromycin-based purification of rat brain capillary endothelial cell cultures. Effect on the expression of blood-brain barrier-specific properties

J Neurochem. 2005 Apr;93(2):279-89. doi: 10.1111/j.1471-4159.2004.03020.x.


One of the main difficulties with primary rat brain endothelial cell (RBEC) cultures is obtaining pure cultures. The variation in purity limits the achievement of in vitro models of the rat blood-brain barrier. As P-glycoprotein expression is known to be much higher in RBECs than in any contaminating cells, we have tested the effect of five P-glycoprotein substrates (vincristine, vinblastine, colchicine, puromycin and doxorubicin) on RBEC cultures, assuming that RBECs would resist the treatment with these toxic compounds whereas contaminating cells would not. Treatment with either 4 microg/mL puromycin for the first 2 days of culture or 3 microg/mL puromycin for the first 3 days showed the best results without causing toxicity to the cells. Transendothelial electrical resistance was significantly increased in cell monolayers treated with puromycin compared with untreated cell monolayers. When cocultured with astrocytes in the presence of cAMP, the puromycin-treated RBEC monolayer showed a highly reduced permeability to sodium fluorescein (down to 0.75 x 10(-6) cm/s) and a high electrical resistance (up to 500 Omega x cm(2)). In conclusion, this method of RBEC purification will allow the production of in vitro models of the rat blood-brain barrier for cellular and molecular biology studies as well as pharmacological investigations.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Blood-Brain Barrier / cytology
  • Blood-Brain Barrier / drug effects*
  • Blood-Brain Barrier / metabolism
  • Capillary Permeability / drug effects
  • Capillary Permeability / physiology
  • Cell Culture Techniques / methods
  • Cell Separation / methods
  • Cells, Cultured
  • Cerebral Cortex / cytology
  • Cerebral Cortex / drug effects*
  • Cerebral Cortex / metabolism
  • Coculture Techniques / methods
  • Dose-Response Relationship, Drug
  • Endothelium, Vascular / cytology*
  • Endothelium, Vascular / drug effects*
  • Gene Expression Regulation / drug effects
  • Gene Expression Regulation / physiology
  • Organ Specificity / drug effects
  • Organ Specificity / physiology
  • Puromycin / metabolism
  • Puromycin / pharmacology*
  • Rats
  • Rats, Wistar


  • Puromycin