Abstract
The blood–brain barrier (BBB) proper is composed of endothelial cells (ECs) of the cerebral microvasculature, which are interconnected by tight junctions (TJs) that in turn form a physical barrier restricting paracellular flux. Tight control of vascular permeability is essential for the homeostasis and functionality of the central nervous system (CNS). In vitro BBB models have been in use for decades and have been of great benefit in the process of investigating and understanding the cellular and molecular mechanisms underlying BBB establishment. BBB integrity changes can be addressed in vitro by determining cell monolayer permeability (Pe) to different solutes and measuring trans-endothelial electrical resistance (TEER).
This chapter describes procedures that can be utilized for both freshly isolated mouse brain microvascular ECs (MBMECs) and murine or human brain EC lines (bEnd5 or hCMEC/D3), cultivated either as a single monolayer or in cocultivation with primary mouse astrocytes (ACs). It starts with detailed information on how to perform transwell cell culture, including coating of inserts and seeding of the ECs and ACs. Moreover, it encompasses instructions for electrical assessment of the in vitro BBB using the more recent cellZscope® device, which was traditionally performed with chopstick electrodes of voltohmmeter type (EVOM). From continuous impedance measurements, the cellZscope® device provides TEER (paracellular resistance) and cell membrane capacitance (Ccl—transcellular resistance), two independent measures of monolayer integrity. Additionally, this chapter provides guidance through subsequent experiments such as permeability analysis (Pe, flux), expression analysis (qRT-PCR and Western blotting), and localization analysis of BBB junction proteins (immunocytochemistry) using the same inserts subjected earlier to impedance analysis.
As numerous diseases are associated with BBB breakdown, researchers aim to continuously improve and refine in vitro BBB models to mimic in vivo conditions as closely as possible. This chapter summarizes protocols with the intention to provide a collection of BBB in vitro assays that generate reproducible results not only with primary brain ECs but also with EC lines to open up the field for a broader spectrum of researchers who intend to investigate the BBB in vitro particularly aiming at therapeutic aspects.
Cathrin J. Czupalla and Kavi Devraj contributed equally.
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References
Abbott NJ, Patabendige AA, Dolman DE, Yusof SR, Begley DJ (2010) Structure and function of the blood-brain barrier. Neurobiol Dis 37:13–25
Pardridge WM (2003) Molecular biology of the blood-brain barrier. Methods Mol Med 89:385–399
Pardridge WM (2005) The blood-brain barrier: bottleneck in brain drug development. NeuroRx 2:3–14
Neuwelt E, Abbott NJ, Abrey L, Banks WA, Blakley B, Davis T, Engelhardt B, Grammas P, Nedergaard M, Nutt J, Pardridge W, Rosenberg GA, Smith Q, Drewes LR (2008) Strategies to advance translational research into brain barriers. Lancet Neurol 7:84–96
Deli MA, Abraham CS, Kataoka Y, Niwa M (2005) Permeability studies on in vitro blood-brain barrier models: physiology, pathology, and pharmacology. Cell Mol Neurobiol 25:59–127
Lippmann ES, Azarin SM, Kay JE, Nessler RA, Wilson HK, Al-Ahmad A, Palecek SP, Shusta EV (2012) Derivation of blood-brain barrier endothelial cells from human pluripotent stem cells. Nat Biotechnol 30:783–791
Lyck R, Ruderisch N, Moll AG, Steiner O, Cohen CD, Engelhardt B, Makrides V, Verrey F (2009) Culture-induced changes in blood-brain barrier transcriptome: implications for amino-acid transporters in vivo. J Cereb Blood Flow Metab 29:1491–1502
Calabria AR, Shusta EV (2008) A genomic comparison of in vivo and in vitro brain microvascular endothelial cells. J Cereb Blood Flow Metab 28:135–148
Tilling T, Korte D, Hoheisel D, Galla HJ (1998) Basement membrane proteins influence brain capillary endothelial barrier function in vitro. J Neurochem 71:1151–1157
Benson K, Cramer S, Galla HJ (2013) Impedance-based cell monitoring: barrier properties and beyond. Fluids Barriers CNS 10:5
Gunzel D, Zakrzewski SS, Schmid T, Pangalos M, Wiedenhoeft J, Blasse C, Ozboda C, Krug SM (2012) From TER to trans- and paracellular resistance: lessons from impedance spectroscopy. Ann N Y Acad Sci 1257:142–151
Nitta T, Hata M, Gotoh S, Seo Y, Sasaki H, Hashimoto N, Furuse M, Tsukita S (2003) Size-selective loosening of the blood-brain barrier in claudin-5-deficient mice. J Cell Biol 161:653–660
Van Itallie C, Rahner C, Anderson JM (2001) Regulated expression of claudin-4 decreases paracellular conductance through a selective decrease in sodium permeability. J Clin Invest 107:1319–1327
Van Itallie CM, Anderson JM (2011) Measuring size-dependent permeability of the tight junction using PEG profiling. Methods Mol Biol 762:1–11
Liebner S, Corada M, Bangsow T, Babbage J, Taddei A, Czupalla CJ, Reis M, Felici A, Wolburg H, Fruttiger M, Taketo MM, von Melchner H, Plate KH, Gerhardt H, Dejana E (2008) Wnt/beta-catenin signaling controls development of the blood-brain barrier. J Cell Biol 183:409–417
Rohnelt RK, Hoch G, Reiss Y, Engelhardt B (1997) Immunosurveillance modelled in vitro: naive and memory T cells spontaneously migrate across unstimulated microvascular endothelium. Int Immunol 9:435–450
Weksler BB, Subileau EA, Perriere N, Charneau P, Holloway K, Leveque M, Tricoire-Leignel H, Nicotra A, Bourdoulous S, Turowski P, Male DK, Roux F, Greenwood J, Romero IA, Couraud PO (2005) Blood-brain barrier-specific properties of a human adult brain endothelial cell line. FASEB J 19:1872–1874
Uliasz TF, Hamby ME, Jackman NA, Hewett JA, Hewett SJ (2012) Generation of primary astrocyte cultures devoid of contaminating microglia. Methods Mol Biol 814:61–79
Liebner S, Czupalla CJ, Wolburg H (2011) Current concepts of blood-brain barrier development. Int J Dev Biol 55:467–476
Crone C, Olesen SP (1982) Electrical resistance of brain microvascular endothelium. Brain Res 241:49–55
Li G, Simon MJ, Cancel LM, Shi ZD, Ji X, Tarbell JM, Morrison B 3rd, Fu BM (2010) Permeability of endothelial and astrocyte cocultures: in vitro blood-brain barrier models for drug delivery studies. Ann Biomed Eng 38:2499–2511
Antonetti DA, Wolpert EB (2003) Isolation and characterization of retinal endothelial cells. Methods Mol Med 89:365–374
Kroll S, El-Gindi J, Thanabalasundaram G, Panpumthong P, Schrot S, Hartmann C, Galla HJ (2009) Control of the blood-brain barrier by glucocorticoids and the cells of the neurovascular unit. Ann N Y Acad Sci 1165:228–239
Zhang Y, Li CS, Ye Y, Johnson K, Poe J, Johnson S, Bobrowski W, Garrido R, Madhu C (2006) Porcine brain microvessel endothelial cells as an in vitro model to predict in vivo blood-brain barrier permeability. Drug Metab Dispos 34:1935–1943
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Czupalla, C.J., Liebner, S., Devraj, K. (2014). In Vitro Models of the Blood–Brain Barrier. In: Milner, R. (eds) Cerebral Angiogenesis. Methods in Molecular Biology, vol 1135. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0320-7_34
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DOI: https://doi.org/10.1007/978-1-4939-0320-7_34
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