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Paracellular Tightness and Claudin-5 Expression is Increased in the BCEC/Astrocyte Blood–Brain Barrier Model by Increasing Media Buffer Capacity During Growth


Most attempts to develop in vitro models of the blood–brain barrier (BBB) have resulted in models with low transendothelial electrical resistances (TEER), as compared to the native endothelium. The aim of the present study was to investigate the impact of culture pH and buffer concentration on paracellular tightness of an established in vitro model of the BBB consisting of bovine brain capillary endothelial cells (BCEC) co-cultured with rat astrocytes. BCEC and rat astrocytes were isolated and co-cultured using astrocyte-conditioned media with cAMP increasing agonists and dexamethasone. The co-culture had average TEER values from 261 ± 26 Ω cm2 to 760 ± 46 Ω cm2 dependent on BCEC isolation batches. Furthermore, mRNA of occludin, claudin-1, claudin-5, JAM-1, and ZO-1 were detected. Increased buffer concentration by addition of HEPES, MOPS, or TES to the media during differentiation increased the TEER up to 1,638 ± 256 Ω cm2 independent of the type of buffer. This correlated with increased expression of claudin-5, while expression of the other tight junction proteins remained unchanged. Thus, we show for the first time that increased buffer capacity of the medium during differentiation significantly increases tightness of the BCEC/astrocyte in vitro BBB model. This regulation may be mediated by increased claudin-5 expression. The observations have practical implications for generating tighter BBB cell culture models, and may also have physiological implications, if similar sensitivity to pH-changes can be demonstrated in vivo.

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  1. 1.

    Reese TS, Karnovsky MJ. Fine structural localization of a blood–brain barrier to exogenous peroxidase. J Cell Biol. 1967;34(1):207–17.

    CAS  Article  PubMed  Google Scholar 

  2. 2.

    Crone C, Olesen SP. Electrical resistance of brain microvascular endothelium. Brain Res. 1982;241(1):49–55.

    CAS  Article  PubMed  Google Scholar 

  3. 3.

    Brightman MW, Reese TS. Junctions between intimately apposed cell membranes in the vertebrate brain. J Cell Biol. 1969;40(3):648–77.

    CAS  Article  PubMed  Google Scholar 

  4. 4.

    de Boer AG, Gaillard PJ, Breimer DD. The transference of results between blood–brain barrier cell culture systems. Eur J Pharm Sci. 1999;8(1):1–4.

    Article  PubMed  Google Scholar 

  5. 5.

    el-Bacha RS, Minn A. Drug metabolizing enzymes in cerebrovascular endothelial cells afford a metabolic protection to the brain. Cell Mol Biol (Noisy-le-grand). 1999;45(1):15–23.

    CAS  Google Scholar 

  6. 6.

    Cecchelli R, Berezowski V, Lundquist S, Culot M, Renftel M, Dehouck MP, et al. Modelling of the blood–brain barrier in drug discovery and development. Nat Rev Drug Discov. 2007;6(8):650–61.

    CAS  Article  PubMed  Google Scholar 

  7. 7.

    Deli MA, Abraham CS, Kataoka Y, Niwa M. Permeability studies on in vitro blood–brain barrier models: physiology, pathology, and pharmacology. Cell Mol Neurobiol. 2005;25(1):59–127.

    Article  PubMed  Google Scholar 

  8. 8.

    Taylor AC. Responses of cells to pH changes in the medium. J Cell Biol. 1962;15:201–9.

    CAS  Article  PubMed  Google Scholar 

  9. 9.

    Mackenzie CG, Mackenzie JB, Beck P. The effect of pH on growth, protein synthesis, and lipid-rich particles of cultured mammalian cells. J Biophys Biochem Cytol. 1961;9:141–56.

    CAS  Article  PubMed  Google Scholar 

  10. 10.

    Lo CM, Keese CR, Giaever I. pH changes in pulsed CO2 incubators cause periodic changes in cell morphology. Exp Cell Res. 1994;213(2):391–7.

    CAS  Article  PubMed  Google Scholar 

  11. 11.

    Dickinson PA, Evans JP, Farr SJ, Kellaway IW, Appelqvist TP, Hann AC, et al. Putrescine uptake by alveolar epithelial cell monolayers exhibiting differing transepithelial electrical resistances. J Pharm Sci. 1996;85(10):1112–6.

    CAS  Article  PubMed  Google Scholar 

  12. 12.

    Gaillard PJ, de Boer AG. 2B-Trans technology: targeted drug delivery across the blood–brain barrier. Methods Mol Biol. 2008;437:161–75.

    CAS  Article  PubMed  Google Scholar 

  13. 13.

    Boveri M, Berezowski V, Price A, Slupek S, Lenfant AM, Benaud C, et al. Induction of blood–brain barrier properties in cultured brain capillary endothelial cells: comparison between primary glial cells and C6 cell line. Glia. 2005;51(3):187–98.

    Article  PubMed  Google Scholar 

  14. 14.

    Culot M, Lundquist S, Vanuxeem D, Nion S, Landry C, Delplace Y, et al. An in vitro blood–brain barrier model for high throughput (HTS) toxicological screening. Toxicol In Vitro. 2008;22(3):799–811.

    CAS  Article  PubMed  Google Scholar 

  15. 15.

    Franke H, Galla HJ, Beuckmann CT. An improved low-permeability in vitro-model of the blood–brain barrier: transport studies on retinoids, sucrose, haloperidol, caffeine and mannitol. Brain Res. 1999;818(1):65–71.

    CAS  Article  PubMed  Google Scholar 

  16. 16.

    Gaillard PJ, Voorwinden LH, Nielsen JL, Ivanov A, Atsumi R, Engman H, et al. 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. 2001;12(3):215–22.

    CAS  Article  PubMed  Google Scholar 

  17. 17.

    Nakagawa S, Deli MA, Nakao S, Honda M, Hayashi K, Nakaoke R, et al. Pericytes from brain microvessels strengthen the barrier integrity in primary cultures of rat brain endothelial cells. Cell Mol Neurobiol. 2007;27(6):687–94.

    CAS  Article  PubMed  Google Scholar 

  18. 18.

    Hoheisel D, Nitz T, Franke H, Wegener J, Hakvoort A, Tilling T, et al. Hydrocortisone reinforces the blood–brain properties in a serum free cell culture system. Biochem Biophys Res Commun. 1998;247(2):312–5.

    CAS  Article  PubMed  Google Scholar 

  19. 19.

    Malina KCK, Cooper I, Teichberg VI. Closing the gap between the in-vivo and in-vitro blood–brain barrier tightness. Brain Res. 2009;1284:12–21.

    Article  Google Scholar 

  20. 20.

    Bratlid D, Cashore WJ, Oh W. Effect of acidosis on bilirubin deposition in rat brain. Pediatrics. 1984;73(4):431–4.

    CAS  PubMed  Google Scholar 

  21. 21.

    Nagy Z, Szabo M, Huttner I. Blood–brain barrier impairment by low pH buffer perfusion via the internal carotid artery in rat. Acta Neuropathol. 1985;68(2):160–3.

    CAS  Article  PubMed  Google Scholar 

  22. 22.

    Asai M, Takeuchi K, Saotome M, Urushida T, Katoh H, Satoh H, et al. Extracellular acidosis suppresses endothelial function by inhibiting store-operated Ca2+ entry via non-selective cation channels. Cardiovasc Res. 2009;83(1):97–105.

    CAS  Article  PubMed  Google Scholar 

  23. 23.

    Gueffroy DE, editor. Buffers a guide for preparation and use of buffers in biological systems. San Diego: Calbiochem-Novabiochem Corporation; 1993.

  24. 24.

    Altura BM, Carella A, Altura BT. Adverse effects of Tris. HEPES and MOPS buffers on contractile responses of arterial and venous smooth muscle induced by prostaglandins. Prostaglandins Med. 1980;5(2):123–30.

    CAS  Article  PubMed  Google Scholar 

  25. 25.

    Lelong IH, Rebel G. pH drift of “physiological buffers” and culture media used for cell incubation during in vitro studies. J Pharmacol Toxicol Methods. 1998;39(4):203–10.

    CAS  Article  PubMed  Google Scholar 

  26. 26.

    Luo S, Pal D, Shah SJ, Kwatra D, Paturi KD, Mitra AK. Effect of HEPES buffer on the uptake and transport of P-glycoprotein substrates and large neutral amino acids. Mol Pharm. Apr 5;7(2):412–20

  27. 27.

    Poole CA, Reilly HC, Flint MH. The adverse effects of HEPES, TES, and BES zwitterion buffers on the ultrastructure of cultured chick embryo epiphyseal chondrocytes. In Vitro. 1982;18(9):755–65.

    CAS  Article  PubMed  Google Scholar 

  28. 28.

    Morita K, Sasaki H, Furuse M, Tsukita S. Endothelial claudin: Claudin-5/TMVCF constitutes tight junction strands in endothelial cells. J Cell Biol. 1999;147(1):185–94.

    CAS  Article  PubMed  Google Scholar 

  29. 29.

    Nitta T, Hata M, Gotoh S, Seo Y, Sasaki H, Hashimoto N, et al. Size-selective loosening of the blood–brain barrier in claudin-5-deficient mice. J Cell Biol. 2003;161(3):653–60.

    CAS  Article  PubMed  Google Scholar 

  30. 30.

    Chen Z, Zandonatti M, Jakubowski D, Fox HS. Brain capillary endothelial cells express MBEC1, a protein that is related to the Clostridium perfringens enterotoxin receptors. Lab Investig. 1998;78(3):353–63.

    CAS  PubMed  Google Scholar 

  31. 31.

    Ishizaki T, Chiba H, Kojima T, Fujibe M, Soma T, Miyajima H, et al. Cyclic AMP induces phosphorylation of claudin-5 immunoprecipitates and expression of claudin-5 gene in blood–brain-barrier endothelial cells via protein kinase A-dependent and -independent pathways. Exp Cell Res. 2003;290(2):275–88.

    CAS  Article  PubMed  Google Scholar 

  32. 32.

    Koto T, Takubo K, Ishida S, Shinoda H, Inoue M, Tsubota K, et al. Hypoxia disrupts the barrier function of neural blood vessels through changes in the expression of claudin-5 in endothelial cells. Am J Pathol. 2007;170(4):1389–97.

    CAS  Article  PubMed  Google Scholar 

  33. 33.

    Soma T, Chiba H, Kato-Mori Y, Wada T, Yamashita T, Kojima T, et al. Thr(207) of claudin-5 is involved in size-selective loosening of the endothelial barrier by cyclic AMP. Exp Cell Res. 2004;300(1):202–12.

    CAS  Article  PubMed  Google Scholar 

  34. 34.

    Liebner S, Fischmann A, Rascher G, Duffner F, Grote EH, Kalbacher H, et al. Claudin-1 and claudin-5 expression and tight junction morphology are altered in blood vessels of human glioblastoma multiforme. Acta Neuropathol. 2000;100(3):323–31.

    CAS  Article  PubMed  Google Scholar 

  35. 35.

    Amasheh S, Schmidt T, Mahn M, Florian P, Mankertz J, Tavalali S, et al. Contribution of claudin-5 to barrier properties in tight junctions of epithelial cells. Cell Tissue Res. 2005;321(1):89–96.

    CAS  Article  PubMed  Google Scholar 

  36. 36.

    Wen H, Watry DD, Marcondes MC, Fox HS. Selective decrease in paracellular conductance of tight junctions: role of the first extracellular domain of claudin-5. Mol Cell Biol. 2004;24(19):8408–17.

    CAS  Article  PubMed  Google Scholar 

  37. 37.

    Krause G, Winkler L, Mueller SL, Haseloff RF, Piontek J, Blasig IE. Structure and function of claudins. Biochim Biophys Acta. 2008;1778(3):631–45.

    CAS  PubMed  Google Scholar 

  38. 38.

    Favre CJ, Mancuso M, Maas K, McLean JW, Baluk P, McDonald DM. Expression of genes involved in vascular development and angiogenesis in endothelial cells of adult lung. Am J Physiol Heart Circ Physiol. 2003;285(5):H1917–38.

    CAS  PubMed  Google Scholar 

  39. 39.

    Wang F, Daugherty B, Keise LL, Wei Z, Foley JP, Savani RC, et al. Heterogeneity of claudin expression by alveolar epithelial cells. Am J Respir Cell Mol Biol. 2003;29(1):62–70.

    CAS  Article  PubMed  Google Scholar 

  40. 40.

    Gumbleton M, Audus KL. 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. 2001;90(11):1681–98.

    CAS  Article  PubMed  Google Scholar 

  41. 41.

    Garberg P, Ball M, Borg N, Cecchelli R, Fenart L, Hurst RD, et al. In vitro models for the blood–brain barrier. Toxicol In Vitro. 2005;19(3):299–334.

    CAS  Article  PubMed  Google Scholar 

  42. 42.

    Calabria AR, Shusta EV. A genomic comparison of in vivo and in vitro brain microvascular endothelial cells. J Cereb Blood Flow Metab. 2008;28(1):135–48.

    CAS  Article  PubMed  Google Scholar 

  43. 43.

    Lyck R, Ruderisch N, Moll AG, Steiner O, Cohen CD, Engelhardt B, et al. Culture-induced changes in blood–brain barrier transcriptome: implications for amino-acid transporters in vivo. J Cereb Blood Flow Metab. 2009;29(9):1491–502.

    CAS  Article  PubMed  Google Scholar 

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The authors would like to thank Carlsberg foundation, the Predicting Drug Absorption Consortium and the Novo Scholarship Programme for financial support. Furthermore, we would like to thank Assoc. Professor—Ph.D. A.G. de Boer for generous help and collaboration during the start up of the co-culture system as well as senior laboratory technicians Heidi Nielsen, Maria D. Læssøe Pedersen and Bettina Dinitzen for their expert technical assistance.

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Correspondence to Birger Brodin.

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Helms, H.C., Waagepetersen, H.S., Nielsen, C.U. et al. Paracellular Tightness and Claudin-5 Expression is Increased in the BCEC/Astrocyte Blood–Brain Barrier Model by Increasing Media Buffer Capacity During Growth. AAPS J 12, 759–770 (2010).

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Key words

  • blood–brain barrier
  • buffer capacity
  • drug delivery
  • in vitro model
  • tight junction regulation