Assessing Blood–Brain Barrier Function Using In Vitro Assays

  • Joseph Bressler
  • Katherine Clark
  • Cliona O’Driscoll
Part of the Methods in Molecular Biology book series (MIMB, volume 1066)


The impermeability of the blood–brain barrier (BBB) is due to a number of properties including tight junctions on adjoining endothelial cells, absence of pinocytic vesicles, and expression of multidrug transporters. Although the permeability of many chemicals can be predicted by their polarity, or oil/water partition coefficient, many lipophilic chemicals are not permeable because of multidrug transporters at the luminal and abluminal membranes. In contrast, many nutrients, which are usually polar, cross the BBB more readily than predicted by their oil/water partition coefficients due to the expression of specific nutrient transporters. In vitro models are being developed because rodent models are of low input and relatively expensive. Isolated brain microvessels and cell culture models each offers certain advantages and disadvantages. Isolated brain microvessels are useful in measuring multidrug drug transporters and tight junction integrity, whereas cell culture models allow the investigator to measure directional transport and can be genetically manipulated. In this chapter, we describe how to isolate large batches of brain microvessels from freshly slaughtered cows. The different steps in the isolation procedure include density gradient centrifugations and filtering. Purity is determined microscopically and by marker enzymes. Permeability is assessed by measuring the uptake of fluorescein-labeled dextran in an assay that has been optimized to have a large dynamic range and low inter-day variability. We also describe how to evaluate transendothelial cell electrical resistance and paracellular transport in cell culture models.

Key words

Blood–brain barrier Microvessels Tight junctions Transporters Electrical resistance 


  1. 1.
    Roux F, Couraud PO (2005) Rat brain endothelial cell lines for the study of blood–brain barrier permeability and transport functions. Cell Mol Neurobiol 25:41–58PubMedCrossRefGoogle Scholar
  2. 2.
    Gumbleton M, Audus KL (2001) Progress and limitations in the use of in vitro cell cultures to serve as a permeability screen for the blood–brain barrier. J Pharm Sci 90: 1681–1698PubMedCrossRefGoogle Scholar
  3. 3.
    Aschner M, Fitsanakis VA, dos Santos AP et al (2006) Blood–brain barrier and cell-cell interactions: methods for establishing in vitro models of the blood–brain barrier and transport measurements. Methods Mol Biol (Clifton, NJ) 341:1–15Google Scholar
  4. 4.
    Patabendige A, Skinner RA, Abbott NJ (2013) Establishment of a simplified in vitro porcine blood-brain barrier model with high transendothelial electrical resistance. Brain Res 1521:1–15Google Scholar
  5. 5.
    Milton SG, Knutson VP (1990) Comparison of the function of the tight junctions of endothelial cells and epithelial cells in regulating the movement of electrolytes and macromolecules across the cell monolayer. J Cell Physiol 144:498–504PubMedCrossRefGoogle Scholar
  6. 6.
    Handler JS (1983) Use of cultured epithelia to study transport and its regulation. J Exp Biol 106:55–69PubMedGoogle Scholar
  7. 7.
    Crone C, Olesen SP (1982) Electrical resistance of brain microvascular endothelium. Brain Res 241:49–55PubMedCrossRefGoogle Scholar
  8. 8.
    Lippmann ES, Azarin SM, Kay JE et al (2012) Derivation of blood–brain barrier endothelial cells from human pluripotent stem cells. Nat Biotechnol 30:783–791PubMedCrossRefGoogle Scholar
  9. 9.
    Cucullo L, Couraud PO, Weksler B et al (2008) Immortalized human brain endothelial cells and flow-based vascular modeling: a marriage of convenience for rational neurovascular studies. J Cereb Blood Flow Metab 28:312–328PubMedCrossRefGoogle Scholar
  10. 10.
    Felix RA, Barrand MA (2002) P-glycoprotein expression in rat brain endothelial cells: evidence for regulation by transient oxidative stress. J Neurochem 80:64–72PubMedCrossRefGoogle Scholar
  11. 11.
    Seetharaman S, Barrand MA, Maskell L et al (1998) Multidrug resistance-related transport proteins in isolated human brain microvessels and in cells cultured from these isolates. J Neurochem 70:1151–1159PubMedCrossRefGoogle Scholar
  12. 12.
    Perriere N, Yousif S, Cazaubon S et al (2007) A functional in vitro model of rat blood–brain barrier for molecular analysis of efflux transporters. Brain Res 1150:1–13PubMedCrossRefGoogle Scholar
  13. 13.
    Bachmeier CJ, Trickler WJ, Miller DW (2006) Comparison of drug efflux transport kinetics in various blood–brain barrier models. Drug Metab Dispos 34:998–1003PubMedGoogle Scholar
  14. 14.
    Shapiro AB, Ling V (1998) The mechanism of ATP-dependent multidrug transport by P-glycoprotein. Acta Physiol Scand 643: 227–234Google Scholar
  15. 15.
    Polgar O, Robey RW, Bates SE (2008) ABCG2: structure, function and role in drug response. Expert Opin Drug Metab Toxicol 4:1–15PubMedCrossRefGoogle Scholar
  16. 16.
    Wang Q, Strab R, Kardos P et al (2008) Application and limitation of inhibitors in drug-transporter interactions studies. Int J Pharm 356:12–18PubMedCrossRefGoogle Scholar
  17. 17.
    Yang Z, Horn M, Wang J et al (2004) Development and characterization of a recombinant madin-darby canine kidney cell line that expresses rat multidrug resistance-associated protein 1 (rMRP1). AAPS J 6:77–85PubMedGoogle Scholar
  18. 18.
    Wolff JE, Belloni-Olivi L, Bressler JP et al (1992) Gamma-glutamyl transpeptidase activity in brain microvessels exhibits regional heterogeneity. J Neurochem 58:909–915PubMedCrossRefGoogle Scholar
  19. 19.
    Vernon H, Clark K, Bressler JP (2011) In vitro models to study the blood–brain barrier. Methods Mol Biol (Clifton, NJ) 758:153–168CrossRefGoogle Scholar
  20. 20.
    Shivers RR, Betz AL, Goldstein GW (1984) Isolated rat brain capillaries possess intact, structurally complex, interendothelial tight junctions; freeze-fracture verification of tight junction integrity. Brain Res 324:313–322PubMedCrossRefGoogle Scholar
  21. 21.
    White FP, Dutton GR, Norenberg MD (1981) Microvessels isolated from rat brain: localization of astrocyte processes by immunohistochemical techniques. J Neurochem 36: 328–332PubMedCrossRefGoogle Scholar
  22. 22.
    Erdlenbruch B, Alipour M, Fricker G et al (2003) Alkylglycerol opening of the blood–brain barrier to small and large fluorescence markers in normal and C6 glioma-bearing rats and isolated rat brain capillaries. Br J Pharmacol 140:1201–1210PubMedCrossRefGoogle Scholar
  23. 23.
    Hartz AM, Bauer B, Fricker G et al (2004) Rapid regulation of P-glycoprotein at the blood–brain barrier by endothelin-1. Mol Pharmacol 66:387–394PubMedCrossRefGoogle Scholar
  24. 24.
    Bauer B, Hartz AM, Lucking JR et al (2008) Coordinated nuclear receptor regulation of the efflux transporter, Mrp2, and the phase-II metabolizing enzyme, GSTpi, at the blood–brain barrier. J Cereb Blood Flow Metab 28:1222–1234PubMedCrossRefGoogle Scholar
  25. 25.
    Hartz AM, Mahringer A, Miller DS et al (2010) 17-beta-Estradiol: a powerful modulator of blood–brain barrier BCRP activity. J Cereb Blood Flow Metab 30:1742–1755PubMedCrossRefGoogle Scholar
  26. 26.
    Zhang Y, Schuetz JD, Elmquist WF et al (2004) Plasma membrane localization of multidrug resistance-associated protein homologs in brain capillary endothelial cells. J Pharmacol Exp Ther 311:449–455PubMedCrossRefGoogle Scholar
  27. 27.
    Miller DS, Nobmann SN, Gutmann H et al (2000) Xenobiotic transport across isolated brain microvessels studied by confocal microscopy. Mol Pharmacol 58:1357–1367PubMedGoogle Scholar
  28. 28.
    Dallaire L, Tremblay L, Beliveau R (1991) Purification and characterization of metabolically active capillaries of the blood–brain barrier. Biochem J 276(Pt 3):745–752PubMedGoogle Scholar
  29. 29.
    Goldstein GW, Wolinsky JS, Csejtey J et al (1975) Isolation of metabolically active capillaries from rat brain. J Neurochem 25: 715–717PubMedCrossRefGoogle Scholar
  30. 30.
    Hargreaves KM, Pardridge WM (1988) Neutral amino acid transport at the human blood–brain barrier. J Biol Chem 263: 19392–19397PubMedGoogle Scholar
  31. 31.
    Forster C (2008) Tight junctions and the modulation of barrier function in disease. Histochem Cell Biol 130:55–70PubMedCrossRefGoogle Scholar
  32. 32.
    Anderson JM, Van Itallie CM (1995) Tight junctions and the molecular basis for regulation of paracellular permeability. Am J Physiol 269:G467–G475PubMedGoogle Scholar
  33. 33.
    Haorah J, Heilman D, Knipe B et al (2005) Ethanol-induced activation of myosin light chain kinase leads to dysfunction of tight junctions and blood–brain barrier compromise. Alcohol Clin Exp Res 29:999–1009PubMedCrossRefGoogle Scholar
  34. 34.
    Yeh TH, Hsu LW, Tseng MT et al (2011) Mechanism and consequence of chitosan-mediated reversible epithelial tight junction opening. Biomaterials 32:6164–6173PubMedGoogle Scholar
  35. 35.
    McCall IC, Betanzos A, Weber DA et al (2009) Effects of phenol on barrier function of a human intestinal epithelial cell line correlate with altered tight junction protein localization. Toxicol Appl Pharmacol 241:61–70PubMedCrossRefGoogle Scholar
  36. 36.
    Rapoport SI (2000) Osmotic opening of the blood–brain barrier: principles, mechanism, and therapeutic applications. Cell Mol Neurobiol 20:217–230PubMedCrossRefGoogle Scholar
  37. 37.
    Iversen PW, Eastwood BJ, Sittampalam GS et al (2006) A comparison of assay performance measures in screening assays: signal window, Z′ factor, and assay variability ratio. J Biomol Screen 11:247–252PubMedCrossRefGoogle Scholar
  38. 38.
    Parran DK, Magnin G, Li W et al (2005) Chlorpyrifos alters functional integrity and structure of an in vitro BBB model: co-cultures of bovine endothelial cells and neonatal rat astrocytes. Neurotoxicology 26:77–88PubMedCrossRefGoogle Scholar
  39. 39.
    Abbott NJ, Dolman DE, Drndarski S et al (2012) An improved in vitro blood–brain barrier model: rat brain endothelial cells co-cultured with astrocytes. Methods Mol Biol (Clifton, NJ) 814:415–430CrossRefGoogle Scholar
  40. 40.
    Weksler BB, Subileau EA, Perriere N et al (2005) Blood–brain barrier-specific properties of a human adult brain endothelial cell line. FASEB J 19:1872–1874PubMedGoogle Scholar
  41. 41.
    Griepp EB, Dolan WJ, Robbins ES et al (1983) Participation of plasma membrane proteins in the formation of tight junctions by cultured epithelial cells. J Cell Biol 96: 693–702PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, New York 2013

Authors and Affiliations

  • Joseph Bressler
    • 1
    • 2
  • Katherine Clark
    • 2
  • Cliona O’Driscoll
    • 3
    • 2
  1. 1.Hugo Moser Laboratory at the Kennedy KriegerKennedy Krieger InstituteBaltimoreUSA
  2. 2.Department of Environmental Health Sciences, Center In Alternatives In Animal Testing, Bloomberg School of Public HealthJohns Hopkins UniversityBaltimoreUSA
  3. 3.Hugo Moser LaboratoryKennedy Krieger InstituteBaltimoreUSA

Personalised recommendations