Culturing of Rodent Brain Microvascular Endothelial Cells for In Vitro Modeling of the Blood-Brain Barrier

  • Malgorzata Burek
  • Carola Y. Förster
Part of the Neuromethods book series (NM, volume 142)


The blood-brain barrier (BBB) is important in the maintenance of the microenvironment of the brain and proper neuronal function. Apart from the protective function, BBB regulates entry of nutrients into the central nervous system by selective transport and metabolism of blood- and brain-borne substances. Successful modeling of BBB in vitro is established since 1970s and has been used to study mechanisms of transport, cellular interaction, and gene regulation. Rodent in vitro BBB models are widely used and have been proven to retain sufficiently the in vivo properties during culturing. In this chapter we describe methodological aspects of culturing the microvascular endothelial cells. Immortalized endothelial cell lines as well as primary brain microvascular endothelial cells in monoculture, co-culture, and triple-culture are discussed.

Key words

Blood-brain barrier Brain microvascular endothelial cells In vitro model TEER 


  1. 1.
    Pardridge W (2001) Brain drug targeting: the future of brain drug development. Cambridge University Press, CambridgeCrossRefGoogle Scholar
  2. 2.
    Pardridge W (2005) The blood–brain barrier: bottleneck in brain drug development. NeuroRx 2:3–14PubMedPubMedCentralCrossRefGoogle Scholar
  3. 3.
    Liebner S, Engelhardt B (2005) Development of the blood-brain barrier. In: Prat A, de Vries E (eds) The blood-brain barrier and its microenvironment: basic physiology to neurological diseases, 1st edn. Taylor & Francis Group, Boca Raton, FL, pp 1–26Google Scholar
  4. 4.
    Neuwelt E, Abbott NJ, Abrey L, Banks WA, Blakley B, Davis T et al (2008) Strategies to advance translational research into brain barriers. Lancet Neurol 7(1):84–96. Epub 21 Dec 2007PubMedCrossRefGoogle Scholar
  5. 5.
    Ballabh P, Braun A, Nedergaard M (2004) The blood–brain barrier: an overview. Structure, regulation, and clinical implications. Neurobiol Dis 16:1–13PubMedCrossRefGoogle Scholar
  6. 6.
    Begley D, Brightman MW (2003) Structural and functional aspects of the blood–brain barrier. Progr Drug Res 61:39–78Google Scholar
  7. 7.
    Wolburg H, Noell S, Mack A, Wolburg-Buchholz K, Fallier-Becker P (2009) Brain endothelial cells and the glio–vascular complex. Cell Tissue Res 335:75–96PubMedCrossRefGoogle Scholar
  8. 8.
    Helms HC, Abbott NJ, Burek M, Cecchelli R, Couraud PO, Deli MA et al (2016) In vitro models of the blood-brain barrier: an overview of commonly used brain endothelial cell culture models and guidelines for their use. J Cereb Blood Flow Metab 36(5):862–890PubMedPubMedCentralCrossRefGoogle Scholar
  9. 9.
    Butt A, Jones HC, Abbott NJ (1990) Electrical resistance across the blood–brain barrier in anaesthetised rats: a developmental study. J Physiol 429:47–62PubMedPubMedCentralCrossRefGoogle Scholar
  10. 10.
    Ribeiro M, Domingues MM, Freire JM, Santos NC, Castanho MARB (2012) Translocating the blood–brain barrier using electrostatics. Front Cell Neurosci 6:1–7CrossRefGoogle Scholar
  11. 11.
    Wilhelm I, Krizbai IA (2014) In vitro models of the blood-brain barrier for the study of drug delivery to the brain. Mol Pharm 11(7):1949–1963PubMedCrossRefGoogle Scholar
  12. 12.
    Patel M, Goyal BR, Bhadada SV, Bhatt JS, Amin AF (2009) Getting into the brain: approaches to enhance brain drug delivery. CNS Drugs 23:35–58PubMedCrossRefGoogle Scholar
  13. 13.
    Löscher W, Potschka H (2005) Role of drug efflux transporters in the brain for drug disposition and treatment of brain diseases. Prog Neurobiol 76:22–76PubMedCrossRefGoogle Scholar
  14. 14.
    Xiuli G, Meiyu G, Guanhua D (2005) Glucose transporter 1, distribution in the brain and in neural disorders: its relationship with transport of neuroactive drugs through the blood-brain barrier. Biochem Genet 43:175–187CrossRefGoogle Scholar
  15. 15.
    Espinoza-Rojo M, Iturralde-Rodriguez KI, Chanez-Cardenas ME, Ruiz-Tachiquin ME, Aguilera P (2010) Glucose transporters regulation on ischemic brain: possible role as therapeutic target. Cent Nerv Syst Agents Med Chem 10:317–325PubMedCrossRefGoogle Scholar
  16. 16.
    Neuhaus W, Burek M, Djuzenova CS, Thal SC, Koepsell H, Roewer N, Förster CY (2012) Addition of NMDA-receptor antagonist MK801 during oxygen/glucose deprivation moderately attenuates the upregulation of glucose uptake after subsequent reoxygenation in brain endothelial cells. Neurosci Lett 506(1):44–49PubMedCrossRefGoogle Scholar
  17. 17.
    Abbott N, Patabendige AAK, Dolman DEM, Yusof SR, Begley DJ (2010) Structure and function of the blood–brain barrier. Neurobiol Dis 37:13–25CrossRefGoogle Scholar
  18. 18.
    Engelhardt S, Patkar S, Ogunshola OO (2014) Cell-specific blood-brain barrier regulation in health and disease: a focus on hypoxia. Br J Pharmacol 171(5):1210–1230PubMedPubMedCentralCrossRefGoogle Scholar
  19. 19.
    Rubin L, Barbu K, Bard F, Cannon C, Hall DE, Horner H, Janatpour M, Liaw C, Manning K, Morales J et al (1991) Differentiation of brain endothelial cells in cell culture. Ann N Y Acad Sci 633:420–425PubMedCrossRefGoogle Scholar
  20. 20.
    Alvarez J, Dodelet-Devillers A, Kebir H, Ifergan I, Fabre PJ, Terouz S, Sabbagh M, Wosik K, Bourbonniere L, Bernard M, van Horssen J, de Vries HE, Charron F, Prat A (2011) The Hedgehog pathway promotes blood-brain barrier integrity and CNS immune quiescence. Science 334:1727–1731PubMedCrossRefGoogle Scholar
  21. 21.
    Haseloff RFBI, Bauer HC, Bauer H (2005) In search of the astrocytic factor(s) modulating blood-brain barrier functions in brain capillary endothelial cells in vitro. Cell Mol Neurobiol 25:25–39PubMedCrossRefGoogle Scholar
  22. 22.
    Siddharthan V, Kim YV, Liu S, Kim KS (2007) Human astrocytes/astrocyte-conditioned medium and shear stress enhance the barrier properties of human brain microvascular endothelial cells. Brain Res 1147:39–50PubMedPubMedCentralCrossRefGoogle Scholar
  23. 23.
    Thanabalasundaram G, Schneidewind J, Pieper C, Galla HJ (2011) The impact of pericytes on the blood-brain barrier integrity depends critically on the pericyte differentiation stage. Int J Biochem Cell Biol 43:1284–1293PubMedCrossRefGoogle Scholar
  24. 24.
    Hellstrom MGH, Kalen M, Li X, Eriksson U, Wolburg H, Betsholtz C (2001) Lack of pericytes leads to endothelial hyperplasia and abnormal vascular morphogenesis. J Cell Biol 153:543–553PubMedPubMedCentralCrossRefGoogle Scholar
  25. 25.
    Armulik A, Genove G, Mae M, Nisancioglu MH, Wallgard E, Niaudet C, He L, Norlin J, Lindblom P, Strittmatter K, Johansson BR, Betsholtz C (2010) Pericytes regulate the blood-brain barrier. Nature 468:557–561PubMedCrossRefGoogle Scholar
  26. 26.
    Hori S, Ohtsuki S, Hosoya K, Nakashima E, Terasaki T (2004) A pericyte-derived angiopoietin-1 multimeric complex induces occludin gene expression in brain capillary endothelial cells through Tie-2 activation in vitro. J Neurochem 89(2):503–513PubMedCrossRefGoogle Scholar
  27. 27.
    Uliasz TF, Hamby ME, Jackman NA, Hewett JA, Hewett SJ (2012) Generation of primary astrocyte cultures devoid of contaminating microglia. Methods Mol Biol 814:61–79PubMedCrossRefGoogle Scholar
  28. 28.
    Tigges U, Welser-Alves JV, Boroujerdi A, Milner R (2012) A novel and simple method for culturing pericytes from mouse brain. Microvasc Res 84(1):74–80PubMedPubMedCentralCrossRefGoogle Scholar
  29. 29.
    Burek M, Arias-Loza PA, Roewer N, Forster CY (2010) Claudin-5 as a novel estrogen target in vascular endothelium. Arterioscler Thromb Vasc Biol 30(2):298–304PubMedCrossRefGoogle Scholar
  30. 30.
    Brown RC, Morris AP, O’Neil RG (2007) Tight junction protein expression and barrier properties of immortalized mouse brain microvessel endothelial cells. Brain Res 1130(1):17–30PubMedCrossRefGoogle Scholar
  31. 31.
    Czupalla CJ, Liebner S, Devraj K (2014) In vitro models of the blood-brain barrier. Methods Mol Biol 1135:415–437PubMedCrossRefGoogle Scholar
  32. 32.
    Wilhelm I, Fasakas C, Krizbai IA (2011) In vitro models of the blood-brain barrier. Acta Neurobiol Exp 71:113–128Google Scholar
  33. 33.
    Deli M, Abraham CS, Kataoka Y, Niwa M (2005) Permeability studies on in vitro blood–brain barrier models: physiology, pathology, and pharmacology. Cell Mol Neurobiol 25:59–127PubMedCrossRefGoogle Scholar
  34. 34.
    Reichel A, Begley DJ, Abbott NJ (2003) An overview of in vitro techniques for blood–brain barrier studies. Methods Mol Med 89:307–324PubMedGoogle Scholar
  35. 35.
    Toth A, Veszelka S, Nakagawa S, Niwa M, Deli MA (2011) Patented in vitro blood-brain barrier models in CNS drug discovery. Recent Pat Drug Discov 6:107–118CrossRefGoogle Scholar
  36. 36.
    Liu R, Zhang T, Wu C, Lan X, Du G (2011) Targeting the neurovascular unit: development of a new model and consideration for novel strategy for Alzheimer’s disease. Brain Res Bull 86:13–21PubMedCrossRefGoogle Scholar
  37. 37.
    Gaillard P, Voorwinden LH, Nielsen JL, Ivanov A, Atsumi R, Engman H, Ringbom C, Boer AG, Breimer DD (2001) Establishment and functional characterization of an in vitro model of the blood–brain barrier, comprising a co-culture of brain capillary endothelial cells and astrocytes. Eur J Pharm Sci 12:215–222PubMedCrossRefGoogle Scholar
  38. 38.
    Hellinger E, Veszelka S, Toth AE, Walter F, Kittel A, Bakk ML, Tihanyi K, Hada V, Nakagawa S, Duy TDH, Niwa M, Deli MA, Vastag M (2012) Comparison of brain capillary endothelial cell-based and epithelial (MDCK-MDR1, Caco-2, and VB-Caco-2) cell-based surrogate blood–brain penetration models. Eur J Pharm Biopharm 82:340–351PubMedCrossRefGoogle Scholar
  39. 39.
    Naik P, Cucullo L (2012) In vitro blood–brain barrier models: current and perspective technologies. J Pharm Sci 101(4):1337–1354PubMedCrossRefGoogle Scholar
  40. 40.
    Burek M, Salvador E, Forster CY (2012) Generation of an immortalized murine brain microvascular endothelial cell line as an in vitro blood brain barrier model. J Vis Exp 66:e4022. Epub 7 Sept 2012Google Scholar
  41. 41.
    Abbott N, Couraud PO, Roux E et al (1995) Studies on an immortalized brain endothelial cell line: characterization, permeability and transport. In: Greenwood J, Begley DJ, Segal MB (eds) New concepts of a blood-brain barrier. Plenum Press, New York, pp 239–249CrossRefGoogle Scholar
  42. 42.
    Greenwood J, Pryce G, Devine L, Male DK, dos Santos WL, Calder VL et al (1996) SV40 large T immortalised cell lines of the rat blood-brain and blood-retinal barriers retain their phenotypic and immunological characteristics. J Neuroimmunol 71(1–2):51–63. Epub 1 Dec 1996PubMedCrossRefGoogle Scholar
  43. 43.
    Muruganandam A, Herx LM, Monette R, Durkin JP, Stanimirovic DB (1997) Development of immortalized human cerebromicrovascular endothelial cell line as an in vitro model of the human blood-brain barrier. FASEB J 11(13):1187–1197. Epub 21 Nov 1997PubMedCrossRefGoogle Scholar
  44. 44.
    Teifel M, Friedl P (1996) Establishment of the permanent microvascular endothelial cell line PBMEC/C1-2 from porcine brains. Exp Cell Res 228(1):50–57. Epub 10 Oct 1996PubMedCrossRefGoogle Scholar
  45. 45.
    Lechardeur D, Scherman D, Schwartz B (1997) Development and characterization of cellular models of the blood-brain barrier. STP Pharma Sci 7:5–11Google Scholar
  46. 46.
    Forster C, Silwedel C, Golenhofen N, Burek M, Kietz S, Mankertz J et al (2005) Occludin as direct target for glucocorticoid-induced improvement of blood-brain barrier properties in a murine in vitro system. J Physiol 565(Pt 2):475–486. Epub 26 Mar 2005PubMedPubMedCentralCrossRefGoogle Scholar
  47. 47.
    Silwedel C, Forster C (2006) Differential susceptibility of cerebral and cerebellar murine brain microvascular endothelial cells to loss of barrier properties in response to inflammatory stimuli. J Neuroimmunol 179(1–2):37–45PubMedCrossRefGoogle Scholar
  48. 48.
    Yang T, Roder KE, Abbruscato TJ (2007) Evaluation of bEnd5 cell line as an in vitro model for the blood-brain barrier under normal and hypoxic/aglycemic conditions. J Pharm Sci 96(12):3196–3213. Epub 11 Sept 2007PubMedCrossRefGoogle Scholar
  49. 49.
    Kochi S, Takanaga H, Matsuo H, Naito M, Tsuruo T, Sawada Y (1999) Effect of cyclosporin A or tacrolimus on the function of blood-brain barrier cells. Eur J Pharmacol 372(3):287–295. Epub 8 Jul 1999PubMedCrossRefGoogle Scholar
  50. 50.
    Hosoya K, Tetsuka K, Nagase K, Tomi M, Saeki S, Ohtsuki S et al (2000) Conditionally immortalized brain capillary endothelial cell lines established from a transgenic mouse harboring temperature-sensitive simian virus 40 large T-antigen gene. AAPS PharmSci 2(3):E27. Epub 14 Dec 2001PubMedCrossRefGoogle Scholar
  51. 51.
    Harkness KA, Adamson P, Sussman JD, Davies-Jones GA, Greenwood J, Woodroofe MN (2000) Dexamethasone regulation of matrix metalloproteinase expression in CNS vascular endothelium. Brain 123(Pt 4):698–709. Epub 29 Mar 2000PubMedCrossRefGoogle Scholar
  52. 52.
    Regina A, Romero IA, Greenwood J, Adamson P, Bourre JM, Couraud PO et al (1999) Dexamethasone regulation of P-glycoprotein activity in an immortalized rat brain endothelial cell line, GPNT. J Neurochem 73(5):1954–1963. Epub 28 Oct 1999PubMedGoogle Scholar
  53. 53.
    Roux F, Durieu-Trautmann O, Chaverot N, Claire M, Mailly P, Bourre JM et al (1994) Regulation of gamma-glutamyl transpeptidase and alkaline phosphatase activities in immortalized rat brain microvessel endothelial cells. J Cell Physiol 159(1):101–113. Epub 1 Apr 1994PubMedCrossRefGoogle Scholar
  54. 54.
    Kido Y, Tamai I, Okamoto M, Suzuki F, Tsuji A (2000) Functional clarification of MCT1-mediated transport of monocarboxylic acids at the blood-brain barrier using in vitro cultured cells and in vivo BUI studies. Pharm Res 17(1):55–62. Epub 14 Mar 2000PubMedCrossRefGoogle Scholar
  55. 55.
    Blasig IE, Giese H, Schroeter ML, Sporbert A, Utepbergenov DI, Buchwalow IB et al (2001) *NO and oxyradical metabolism in new cell lines of rat brain capillary endothelial cells forming the blood-brain barrier. Microvasc Res 62(2):114–127. Epub 23 Aug 2001PubMedCrossRefGoogle Scholar
  56. 56.
    Terasaki T, Hosoya K (2001) Conditionally immortalized cell lines as a new in vitro model for the study of barrier functions. Biol Pharm Bull 24(2):111–118. Epub 24 Feb 2001PubMedCrossRefGoogle Scholar
  57. 57.
    Lyck R, Ruderisch N, Moll AG, Steiner O, Cohen CD, Engelhardt B et al (2009) Culture-induced changes in blood-brain barrier transcriptome: implications for amino-acid transporters in vivo. J Cereb Blood Flow Metab 29(9):1491–1502PubMedCrossRefGoogle Scholar
  58. 58.
    Calabria AR, Shusta EV (2008) A genomic comparison of in vivo and in vitro brain microvascular endothelial cells. J Cereb Blood Flow Metab 28(1):135–148PubMedCrossRefGoogle Scholar
  59. 59.
    Blecharz KG, Haghikia A, Stasiolek M, Kruse N, Drenckhahn D, Gold R et al (2010) Glucocorticoid effects on endothelial barrier function in the murine brain endothelial cell line cEND incubated with sera from patients with multiple sclerosis. Mult Scler 16(3):293–302PubMedCrossRefGoogle Scholar
  60. 60.
    Kleinschnitz C, Blecharz K, Kahles T, Schwarz T, Kraft P, Gobel K et al (2011) Glucocorticoid insensitivity at the hypoxic blood-brain barrier can be reversed by inhibition of the proteasome. Stroke 42(4):1081–1089PubMedCrossRefGoogle Scholar
  61. 61.
    Salvador E, Burek M, Forster CY (2015) Stretch and/or oxygen glucose deprivation (OGD) in an in vitro traumatic brain injury (TBI) model induces calcium alteration and inflammatory cascade. Front Cell Neurosci 9:323PubMedPubMedCentralGoogle Scholar
  62. 62.
    Neuhaus W, Gaiser F, Mahringer A, Franz J, Riethmuller C, Forster C (2014) The pivotal role of astrocytes in an in vitro stroke model of the blood-brain barrier. Front Cell Neurosci 8:352PubMedPubMedCentralCrossRefGoogle Scholar
  63. 63.
    Nakagawa S, Deli MA, Nakao S, Honda M, Hayashi K, Nakaoke R et al (2007) Pericytes from brain microvessels strengthen the barrier integrity in primary cultures of rat brain endothelial cells. Cell Mol Neurobiol 27(6):687–694PubMedCrossRefGoogle Scholar

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Authors and Affiliations

  1. 1.Department of Anaesthesia and Critical CareUniversity of WürzburgWürzburgGermany

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