Skip to main content
SpringerLink
Log in

Assessing Blood–Brain Barrier Function Using In Vitro Assays

  • Protocol
  • First Online:
Cell-Cell Interactions

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1066))

Abstract

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.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  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 25:41–58

    Article  PubMed  Google Scholar 

  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: 1681–1698

    Article  PubMed  CAS  Google Scholar 

  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) 341:1–15

    CAS  Google Scholar 

  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 1521:1–15

    Google Scholar 

  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 144:498–504

    Article  PubMed  CAS  Google Scholar 

  6. Handler JS (1983) Use of cultured epithelia to study transport and its regulation. J Exp Biol 106:55–69

    PubMed  CAS  Google Scholar 

  7. Crone C, Olesen SP (1982) Electrical resistance of brain microvascular endothelium. Brain Res 241:49–55

    Article  PubMed  CAS  Google Scholar 

  8. Lippmann ES, Azarin SM, Kay JE et al (2012) Derivation of blood–brain barrier endothelial cells from human pluripotent stem cells. Nat Biotechnol 30:783–791

    Article  PubMed  CAS  Google Scholar 

  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 28:312–328

    Article  PubMed  CAS  Google Scholar 

  10. Felix RA, Barrand MA (2002) P-glycoprotein expression in rat brain endothelial cells: evidence for regulation by transient oxidative stress. J Neurochem 80:64–72

    Article  PubMed  CAS  Google Scholar 

  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 70:1151–1159

    Article  PubMed  CAS  Google Scholar 

  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 1150:1–13

    Article  PubMed  CAS  Google Scholar 

  13. Bachmeier CJ, Trickler WJ, Miller DW (2006) Comparison of drug efflux transport kinetics in various blood–brain barrier models. Drug Metab Dispos 34:998–1003

    PubMed  CAS  Google Scholar 

  14. Shapiro AB, Ling V (1998) The mechanism of ATP-dependent multidrug transport by P-glycoprotein. Acta Physiol Scand 643: 227–234

    CAS  Google Scholar 

  15. Polgar O, Robey RW, Bates SE (2008) ABCG2: structure, function and role in drug response. Expert Opin Drug Metab Toxicol 4:1–15

    Article  PubMed  CAS  Google Scholar 

  16. Wang Q, Strab R, Kardos P et al (2008) Application and limitation of inhibitors in drug-transporter interactions studies. Int J Pharm 356:12–18

    Article  PubMed  CAS  Google Scholar 

  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 6:77–85

    PubMed  Google Scholar 

  18. Wolff JE, Belloni-Olivi L, Bressler JP et al (1992) Gamma-glutamyl transpeptidase activity in brain microvessels exhibits regional heterogeneity. J Neurochem 58:909–915

    Article  PubMed  CAS  Google Scholar 

  19. Vernon H, Clark K, Bressler JP (2011) In vitro models to study the blood–brain barrier. Methods Mol Biol (Clifton, NJ) 758:153–168

    Article  CAS  Google Scholar 

  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 324:313–322

    Article  PubMed  CAS  Google Scholar 

  21. White FP, Dutton GR, Norenberg MD (1981) Microvessels isolated from rat brain: localization of astrocyte processes by immunohistochemical techniques. J Neurochem 36: 328–332

    Article  PubMed  CAS  Google Scholar 

  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 140:1201–1210

    Article  PubMed  CAS  Google Scholar 

  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 66:387–394

    Article  PubMed  CAS  Google Scholar 

  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 28:1222–1234

    Article  PubMed  CAS  Google Scholar 

  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 30:1742–1755

    Article  PubMed  CAS  Google Scholar 

  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 311:449–455

    Article  PubMed  CAS  Google Scholar 

  27. Miller DS, Nobmann SN, Gutmann H et al (2000) Xenobiotic transport across isolated brain microvessels studied by confocal microscopy. Mol Pharmacol 58:1357–1367

    PubMed  CAS  Google Scholar 

  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):745–752

    PubMed  Google Scholar 

  29. Goldstein GW, Wolinsky JS, Csejtey J et al (1975) Isolation of metabolically active capillaries from rat brain. J Neurochem 25: 715–717

    Article  PubMed  CAS  Google Scholar 

  30. Hargreaves KM, Pardridge WM (1988) Neutral amino acid transport at the human blood–brain barrier. J Biol Chem 263: 19392–19397

    PubMed  CAS  Google Scholar 

  31. Forster C (2008) Tight junctions and the modulation of barrier function in disease. Histochem Cell Biol 130:55–70

    Article  PubMed  Google Scholar 

  32. Anderson JM, Van Itallie CM (1995) Tight junctions and the molecular basis for regulation of paracellular permeability. Am J Physiol 269:G467–G475

    PubMed  CAS  Google Scholar 

  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 29:999–1009

    Article  PubMed  CAS  Google Scholar 

  34. Yeh TH, Hsu LW, Tseng MT et al (2011) Mechanism and consequence of chitosan-mediated reversible epithelial tight junction opening. Biomaterials 32:6164–6173

    PubMed  CAS  Google Scholar 

  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 241:61–70

    Article  PubMed  CAS  Google Scholar 

  36. Rapoport SI (2000) Osmotic opening of the blood–brain barrier: principles, mechanism, and therapeutic applications. Cell Mol Neurobiol 20:217–230

    Article  PubMed  CAS  Google Scholar 

  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 11:247–252

    Article  PubMed  CAS  Google Scholar 

  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 26:77–88

    Article  PubMed  CAS  Google Scholar 

  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) 814:415–430

    Article  CAS  Google Scholar 

  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 19:1872–1874

    PubMed  CAS  Google Scholar 

  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: 693–702

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Hugo Moser Laboratory at the Kennedy Krieger, Kennedy Krieger Institute, Baltimore, MD, USA

    Joseph Bressler

  2. Department of Environmental Health Sciences, Center In Alternatives In Animal Testing, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD, USA

    Joseph Bressler, Katherine Clark & Cliona O’Driscoll

  3. Hugo Moser Laboratory, Kennedy Krieger Institute, Baltimore, MD, USA

    Cliona O’Driscoll

Authors
  1. Joseph Bressler
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Katherine Clark
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Cliona O’Driscoll
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. Texas A&M Health Science Center, Temple, Texas, USA

    Troy A. Baudino

Rights and permissions

Reprints and permissions

Copyright information

© 2013 Springer Science+Business Media, New York

About this protocol

Cite this protocol

Bressler, J., Clark, K., O’Driscoll, C. (2013). Assessing Blood–Brain Barrier Function Using In Vitro Assays. In: Baudino, T. (eds) Cell-Cell Interactions. Methods in Molecular Biology, vol 1066. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-604-7_6

Download citation

  • DOI: https://doi.org/10.1007/978-1-62703-604-7_6

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-603-0

  • Online ISBN: 978-1-62703-604-7

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation