This study was funded by grants awarded to R.L.M. from the National Health and Medical Research Council of Australia (grant # 606658).

1. Cell Culture (3-7 Days Prior to BBB Assembly)
1.1. Primary Human Brain Microvascular Endothelial Cells (BECs)
BECs were commercially obtained. The cells were produced by dispase dissociation of normal human brain cortex tissue and provided frozen at passage 3 (&#60;12 population doublings).
<ol>
	<li>Substratum: Coat tissue-culture vessels with &#34;Attachment Factor&#34; as per the manufacturer instructions.</li>
	<li>Cell maintenance: Maintain ...

<ol>
	<li>Hawkins, B. T., Davis, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+blood-brain+barrier/neurovascular+unit+in+health+and+disease.">The blood-brain barrier/neurovascular unit in health and disease.</a> Pharmacol. Rev. 57, 173-185 (2005).</li><li>Abbott, N. J., Patabendige, A. A., Dolman, D. E., Yusof, S. R., Begley, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+function+of+the+blood-brain+barrier.">Structure and function of the blood-brain barrier.</a> Neurobiol. Dis. 37, 13-25 (2010).</li><li>Zlokovic, B. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+blood-brain+barrier+in+health+and+chronic+neurodegenerative+disorders.">The blood-brain barrier in health and chronic neurodegenerative disorders.</a> Neuron. 57, 178-201 (2008).</li><li>Vivien, D., Gauberti, M., Montagne, A., Defer, G., Touze, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+tissue+plasminogen+activator+on+the+neurovascular+unit:+from+clinical+data+to+experimental+evidence.">Impact of tissue plasminogen activator on the neurovascular unit: from clinical data to experimental evidence.</a> J. Cereb. Blood Flow Metab. 31, 2119-2134 (2011).</li><li>Stewart, P. A., Wiley, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developing+nervous+tissue+induces+formation+of+blood-brain+barrier+characteristics+in+invading+endothelial+cells:+a+study+using+quail--chick+transplantation+chimeras.">Developing nervous tissue induces formation of blood-brain barrier characteristics in invading endothelial cells: a study using quail--chick transplantation chimeras.</a> Dev. Biol. 84, 183-192 (1981).</li><li>Janzer, R. C., Raff, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocytes+induce+blood-brain+barrier+properties+in+endothelial+cells.">Astrocytes induce blood-brain barrier properties in endothelial cells.</a> Nature. 325, 253-257 (1987).</li><li>Abbott, N. J., Ronnback, L., Hansson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocyte-endothelial+interactions+at+the+blood-brain+barrier.">Astrocyte-endothelial interactions at the blood-brain barrier.</a> Nat. Rev. Neurosci. 7, 41-53 (2006).</li><li>Dalkara, T., Gursoy-Ozdemir, Y., Yemisci, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+microvascular+pericytes+in+health+and+disease.">Brain microvascular pericytes in health and disease.</a> Acta Neuropathol. 122, 1-9 (2011).</li><li>Engelhardt, B., Sorokin, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+blood-brain+and+the+blood-cerebrospinal+fluid+barriers:+function+and+dysfunction.">The blood-brain and the blood-cerebrospinal fluid barriers: function and dysfunction.</a> Semin. Immunopathol. 31, 497-511 (2009).</li><li>Girouard, H., Iadecola, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurovascular+coupling+in+the+normal+brain+and+in+hypertension,+stroke,+and+Alzheimer+disease.">Neurovascular coupling in the normal brain and in hypertension, stroke, and Alzheimer disease.</a> J. Appl. Physiol. 100, 328-335 (2006).</li><li>Deli, M. A., Abraham, C. S., Kataoka, Y., Niwa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Permeability+studies+on+in+vitro+blood-brain+barrier+models:+physiology,+pathology,+and+pharmacology.">Permeability studies on in vitro blood-brain barrier models: physiology, pathology, and pharmacology.</a> Cell. Mol. Neurobiol. 25, 59-127 (2005).</li><li>Booth, R., Kim, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+microfluidic+in+vitro+model+of+the+blood-brain+barrier+(muBBB).">Characterization of a microfluidic in vitro model of the blood-brain barrier (muBBB).</a> Lab Chip. 12, 1784-1792 (2012).</li><li>Gaillard, P. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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.</a> Eur. Pharm. Sci. 12, 215-222 (2001).</li><li>Hurwitz, A. A., Berman, J. W., Rashbaum, W. K., Lyman, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+fetal+astrocytes+induce+the+expression+of+blood-brain+barrier+specific+proteins+by+autologous+endothelial+cells.">Human fetal astrocytes induce the expression of blood-brain barrier specific proteins by autologous endothelial cells.</a> Brain Res. 625, 238-243 (1993).</li><li>Demeuse, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compartmentalized+coculture+of+rat+brain+endothelial+cells+and+astrocytes:+a+syngenic+model+to+study+the+blood-brain+barrier.">Compartmentalized coculture of rat brain endothelial cells and astrocytes: a syngenic model to study the blood-brain barrier.</a> J. Neurosci. Methods. 121, 21-31 (2002).</li><li>Coisne, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+syngenic+in+vitro+blood-brain+barrier+model:+a+new+tool+to+examine+inflammatory+events+in+cerebral+endothelium.">Mouse syngenic in vitro blood-brain barrier model: a new tool to examine inflammatory events in cerebral endothelium.</a> Lab. Invest. 85, 734-746 (2005).</li><li>Cohen-Kashi Malina, K., Cooper, I., Teichberg, V. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Closing+the+gap+between+the+in+vivo+and+in+vitro+blood-brain+barrier+tightness.">Closing the gap between the in vivo and in vitro blood-brain barrier tightness.</a> Brain Res. 1284, 12-21 (2009).</li><li>Kroll, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+the+blood-brain+barrier+by+glucocorticoids+and+the+cells+of+the+neurovascular+unit.">Control of the blood-brain barrier by glucocorticoids and the cells of the neurovascular unit.</a> Ann. N. Y. Acad. Sci. 1165, 228-239 (2009).</li><li>Cucullo, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immortalized+human+brain+endothelial+cells+and+flow-based+vascular+modeling:+a+marriage+of+convenience+for+rational+neurovascular+studies.">Immortalized human brain endothelial cells and flow-based vascular modeling: a marriage of convenience for rational neurovascular studies.</a> J. Cereb. Blood Flow Metab. 28, 312-328 (2008).</li><li>Li, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Permeability+of+endothelial+and+astrocyte+cocultures:+in+vitro+blood-brain+barrier+models+for+drug+delivery+studies.">Permeability of endothelial and astrocyte cocultures: in vitro blood-brain barrier models for drug delivery studies.</a> Ann. Biomed. Eng. 38, 2499-2511 (2010).</li><li>Antonic, A., Sena, E., Donnan, G., Howells, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+In+Vitro+Models+of+Ischaemic+Stroke:+a+Test+Bed+for+Translation.">Human In Vitro Models of Ischaemic Stroke: a Test Bed for Translation.</a> Transl. Stroke Res. 3, 306-309 (2012).</li><li>Major, E. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+a+line+of+human+fetal+glial+cells+that+supports+JC+virus+multiplication.">Establishment of a line of human fetal glial cells that supports JC virus multiplication.</a> Proc. Natl. Acad. Sci. U.S.A. 82, 1257-1261 (1985).</li><li>Eugenin, E. A., Berman, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemokine-dependent+mechanisms+of+leukocyte+trafficking+across+a+model+of+the+blood-brain+barrier.">Chemokine-dependent mechanisms of leukocyte trafficking across a model of the blood-brain barrier.</a> Methods. 29, 351-361 (2003).</li><li>Dehouck, M. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+transfer+across+the+blood-brain+barrier:+correlation+between+in+vitro+and+in+vivo+models.">Drug transfer across the blood-brain barrier: correlation between in vitro and in vivo models.</a> J. Neurochem. 58, 1790-1797 (1992).</li><li>Perriere, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Puromycin-based+purification+of+rat+brain+capillary+endothelial+cell+cultures.+Effect+on+the+expression+of+blood-brain+barrier-specific+properties.">Puromycin-based purification of rat brain capillary endothelial cell cultures. Effect on the expression of blood-brain barrier-specific properties.</a> J. Neurochem. 93, 279-289 (2005).</li><li>Niego, B., Samson, A. L., Petersen, K. U., Medcalf, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thrombin-induced+activation+of+astrocytes+in+mixed+rat+hippocampal+cultures+is+inhibited+by+soluble+thrombomodulin.">Thrombin-induced activation of astrocytes in mixed rat hippocampal cultures is inhibited by soluble thrombomodulin.</a> Brain Res. 1381, 38-51 (2011).</li><li>Ma, S. H., Lepak, L. A., Hussain, R. J., Shain, W., Shuler, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+endothelial+and+astrocyte+co-culture+model+of+the+blood-brain+barrier+utilizing+an+ultra-thin,+nanofabricated+silicon+nitride+membrane.">An endothelial and astrocyte co-culture model of the blood-brain barrier utilizing an ultra-thin, nanofabricated silicon nitride membrane.</a> Lab Chip. 5, 74-85 (2005).</li><li>Niego, B., Freeman, R., Puschmann, T. B., Turnley, A. M., Medcalf, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=t-PA-specific+modulation+of+a+human+BBB+model+involves+plasmin-mediated+activation+of+the+Rho-kinase+pathway+in+astrocytes.">t-PA-specific modulation of a human BBB model involves plasmin-mediated activation of the Rho-kinase pathway in astrocytes.</a> Blood. , (2012).</li><li>Deli, M. A., Abraham, C. S., Niwa, M., Falus, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N,N-diethyl-2-[4-(phenylmethyl)phenoxy]ethanamine+increases+the+permeability+of+primary+mouse+cerebral+endothelial+cell+monolayers.">N,N-diethyl-2-[4-(phenylmethyl)phenoxy]ethanamine increases the permeability of primary mouse cerebral endothelial cell monolayers.</a> Inflamm. Res. 52, 39-40 (2003).</li><li>Su, E. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+PDGF-CC+by+tissue+plasminogen+activator+impairs+blood-brain+barrier+integrity+during+ischemic+stroke.">Activation of PDGF-CC by tissue plasminogen activator impairs blood-brain barrier integrity during ischemic stroke.</a> Nat. Med. 14, 731-737 (2008).</li><li>Yepes, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-type+plasminogen+activator+induces+opening+of+the+blood-brain+barrier+via+the+LDL+receptor-related+protein.">Tissue-type plasminogen activator induces opening of the blood-brain barrier via the LDL receptor-related protein.</a> J. Clin. Invest. 112, 1533-1540 (2003).</li><li>Abu Fanne, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood-brain+barrier+permeability+and+tPA-mediated+neurotoxicity.">Blood-brain barrier permeability and tPA-mediated neurotoxicity.</a> Neuropharmacology. 58, 972-980 (1016).</li><li>Wang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipoprotein+receptor-mediated+induction+of+matrix+metalloproteinase+by+tissue+plasminogen+activator.">Lipoprotein receptor-mediated induction of matrix metalloproteinase by tissue plasminogen activator.</a> Nat. Med. 9, 1313-1317 (2003).</li><li>Wang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+hemorrhagic+transformation+after+tissue+plasminogen+activator+reperfusion+therapy+for+ischemic+stroke.">Mechanisms of hemorrhagic transformation after tissue plasminogen activator reperfusion therapy for ischemic stroke.</a> Stroke. 35, 2726-2730 (2004).</li><li>Donnan, G. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+make+better+use+of+thrombolytic+therapy+in+acute+ischemic+stroke.">How to make better use of thrombolytic therapy in acute ischemic stroke.</a> Nat. Rev. Neurol. 7, 400-409 (2011).</li><li>Acheampong, P., Ford, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacokinetics+of+alteplase+in+the+treatment+of+ischaemic+stroke.">Pharmacokinetics of alteplase in the treatment of ischaemic stroke.</a> Expert Opin. Drug Metab. Toxicol. 8, 271-281 (2012).</li><li>Derex, L., Nighoghossian, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracerebral+haemorrhage+after+thrombolysis+for+acute+ischaemic+stroke:+an+update.">Intracerebral haemorrhage after thrombolysis for acute ischaemic stroke: an update.</a> J. Neurol. Neurosurg. Psychiatry. 79, 1093-1099 (2008).</li><li>Garber, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stroke+treatment--light+at+the+end+of+the+tunnel.">Stroke treatment--light at the end of the tunnel.</a> Nat. Biotechnol. 25, 838-840 (2007).</li><li>Liebeskind, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversing+Stroke+in+the+2010s.">Reversing Stroke in the 2010s.</a> Stroke. 40, 3156-3158 (2009).</li><li>Weksler, B. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood-brain+barrier-specific+properties+of+a+human+adult+brain+endothelial+cell+line.">Blood-brain barrier-specific properties of a human adult brain endothelial cell line.</a> FASEB J. 19, 1872-1874 (2005).</li><li>Song, L., Pachter, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+of+murine+brain+microvascular+endothelial+cells+that+maintain+expression+and+cytoskeletal+association+of+tight+junction-associated+proteins.">Culture of murine brain microvascular endothelial cells that maintain expression and cytoskeletal association of tight junction-associated proteins.</a> In Vitro Cell. Dev. Biol. Anim. 39 (2003), 313-320 (2003).</li><li>Steiner, O., Coisne, C., Engelhardt, B., Lyck, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+immortalized+bEnd5+and+primary+mouse+brain+microvascular+endothelial+cells+as+in+vitro+blood-brain+barrier+models+for+the+study+of+T+cell+extravasation.">Comparison of immortalized bEnd5 and primary mouse brain microvascular endothelial cells as in vitro blood-brain barrier models for the study of T cell extravasation.</a> J. Cereb. Blood Flow Metab. 31, 315-327 (2011).</li></ol>

Authors have nothing to disclose.

Medical research into brain pathologies suffers much translational difficulty. In the area of acute ischemic stroke, for example, many drugs which showed great promise in animal models failed at the clinic38,39. The reasons for these disappointing results are diverse and include infidelity of the preclinical test systems to the human stroke scenario and overstatement of results obtained from animal studies21. One strategy to improve the predictive value of preclinical findings to the clinical phase ...

The BBB is a specialized interface between the peripheral blood circulation and the central nervous system, crucially responsible for the maintenance of brain hemostasis. It comprises distinct brain microvascular endothelial cells (BECs) which are functionally influenced by few cellular and acellular components (below) to form a tight and dynamic gateway into the brain. Under physiological conditions the BBB restricts the passage of blood cells, plasma components and harmful substances, all potentially neurotoxic, into the brain. In parallel, the BBB selectively exchanges key ions and nutrients (glucose and amino-acids) and metabolic waste products between the brain and the circulation to precisely maintain the brain environment1,2. In recent years it is becoming evident that failure of the BBB occurs in a variety of chronic brain pathologies, such as neurodegenerative or inflammatory-related diseases (e.g. Alzheimer&#39;s disease and multiple sclerosis, respectively)3, as well as in acute conditions like ischemic stroke4.
The unique BBB properties of brain endothelial cells (BECs) are largely induced by their cerebral environment5, and in particular by astrocytes6,7. There is a growing understanding that other cell-types, such as pericytes8, neurons and microglia1,3, as well as the basement membrane9, support BECs and form together a functional unit termed the &#34;neurovascular unit&#34; (NVU) which simultaneously couples neuronal metabolic demands to their supplying capillaries10.
The involvement of the BBB in pathological situations underlies numerous attempts to develop in vitro BBB models to assist in BBB-related research11,12. These models aim to mimic as close as possible in vivo BBB characteristics according to the NVU principle. In vitro BBB models generally rely on a monolayer of tight-junction-forming BECs (mainly from bovine13, human14, rat15, mouse16, and porcine17,18 origins), cultured on a porous membrane together with supporting astrocytes (extensively reviewed by Deli et al. 200511).
Astrocytes can be grown in non-contact conditions on the bottom of a tissue culture well, separated from BECs (cultivated on the upper surface of the membrane) by the culture medium yet communicating with BECs via soluble factors16. In more advanced models which better resemble the anatomical structure of the BBB in vivo, astrocytes are maintained in contact conditions and cultured directly on the opposite side of the membrane in close proximity to BECs13,15,17 (Figure 1). This configuration enables physical contact between BECs and astrocytes, established when astrocytes project their processes through the porous membrane. Importantly, for a true contact to occur the pores should be &#8805;1&#181;m in diameter, since astrocytic end-feet cannot pass through smaller pore sizes (i.e. 0.4 &#181;m)14,15. Notably, contact BBB systems are demonstrated in some studies to be superior to their non-contact counterparts regarding their trans-endothelial electrical resistance (TEER) and endothelial permeability values of various tracers13,17,18. An additional dimension of media flow was recently added in a number of in vitro BBB models to apply shear forces to the endothelium for closer simulation of the brain vasculature12,19.
One technical obstacle to overcome when generating a contact BBB model is the seeding of astrocytes against gravity on the abluminal surface of the porous membrane. Previous protocols13,20, where astrocytes were simply seeded in a drop of media on top of an inverted insert, allowed only short seeding times (i.e. 10 min13 or 2 hr20) which were found in our hands to be insufficient for proper cell attachment. Using this basic method, a longer astrocyte attachment period requires constant monitoring of the inserts by frequent opening of the incubator (causing fluctuations in temperature, pH and humidity) and is also prone to uneven cell seeding due to leakage of media through the pores, especially if pores larger than 1 &#181;m are employed.
Here, we describe a general protocol for preparation of a contact BBB model. Our procedure includes an alternative method for cell-seeding on inverted inserts, which addresses the above mentioned limitations. The method permits undisturbed adherence of astrocytes onto the abluminal membrane surface, in an equilibrated incubator, for an extended period of time. As a result, a uniform seeding of astrocytes is achieved which increases barrier quality and minimizes basal permeability variations between inserts.
As the use of human cells is important for human-relevant research21, we additionally demonstrate in this article the specific utilization of a novel combination of primary human BECs and immortalized human astrocytes for establishment of a contact human BBB model with a high throughput screening capacity. Since viewing of cells on porous membranes can be difficult, we also detail staining techniques which can assist in determination of confluence and cell morphology on the porous membranes. Finally, we exemplify how our human BBB model can be utilized to examine the effect of tissue-type plasminogen activator (t-PA) - a clot busting enzyme which serves as a sole treatment option for acute ischemic stroke - on the BBB.

<table border="0" cellpadding="0" cellspacing="0" width="863">
	<colgroup>
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		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Attachment Factor</td>
			<td style="width:193px;">Cell-Systems Corporation</td>
			<td style="width:171px;">4Z0-210</td>
			<td style="width:243px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Brain microvascular endothelial cells (BECs), primary, human</td>
			<td style="width:193px;">Cell-Systems Corporation</td>
			<td style="width:171px;">ACBRI 376</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Complete medium</td>
			<td style="width:193px;">Cell-Systems Corporation</td>
			<td style="width:171px;">4Z0-500</td>
			<td style="width:243px;">Supplemented with CSC JetFuel</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Complete serum-free medium</td>
			<td style="width:193px;">Cell-Systems Corporation</td>
			<td style="width:171px;">SF-4Z0-500</td>
			<td style="width:243px;">Supplemented with CSC RocketFue</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">DMEM/F-12 with 15 mM HEPES</td>
			<td style="width:193px;">Life Technologies</td>
			<td style="width:171px;">11330-032</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Endothelial cells growth supplement (from bovine origin)</td>
			<td style="width:193px;">Sigma-Aldrich</td>
			<td style="width:171px;">E2759-15MG</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">External silicone tubing</td>
			<td style="width:193px;">Watson-Marlow</td>
			<td style="width:171px;">913.A080.016</td>
			<td style="width:243px;">Pumpsil brand, 8 mm internal diameter, 1.6 mm wall</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Foetal calf serum</td>
			<td style="width:193px;">Lonza</td>
			<td style="width:171px;">14-501F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Gentamycin sulfate</td>
			<td style="width:193px;">Life Technologies</td>
			<td style="width:171px;">15750-060</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Heparin sodium</td>
			<td style="width:193px;">Pfizer</td>
			<td style="width:171px;"></td>
			<td style="width:243px;">1,000 U/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Hexamethyldisilazane (HMDS)</td>
			<td style="width:193px;">Sigma-Aldrich</td>
			<td style="width:171px;">H4875</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Human brain microvascular endothelial cells</td>
			<td style="width:193px;">Cell-Systems Corporation</td>
			<td style="width:171px;">ACBRI 376</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Internal silicone tubing</td>
			<td style="width:193px;">Watson-Marlow</td>
			<td style="width:171px;">913.A032.016</td>
			<td style="width:243px;">Pumpsil brand, 3.2 mm internal diameter, 1.6 mm wall</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">L-Glutamine</td>
			<td style="width:193px;">Life Technologies</td>
			<td style="width:171px;">25030</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Mayer&#8217;s hematoxylin solution</td>
			<td style="width:193px;">Amber Scientific</td>
			<td style="width:171px;">MH</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Minimum essential medium with Earle&#8217;s balanced salt solution</td>
			<td style="width:193px;">HyClone Laboratories</td>
			<td style="width:171px;">SH30244.01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Penicillin/Streptomycin</td>
			<td style="width:193px;">Life Technologies</td>
			<td style="width:171px;">15140</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Rat collagen I</td>
			<td style="width:193px;">Trevigen</td>
			<td style="width:171px;">3440-100-01</td>
			<td style="width:243px;">Cultrex brand</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:257px;">Tissue culture inserts</td>
			<td style="width:193px;">Corning Life Sciences</td>
			<td style="width:171px;">3472</td>
			<td style="width:243px;">Transwell brand, 6.5 mm in diameter, with polyester porous membrane, 3 &#181;m pore-size</td>
		</tr>
	</tbody>
</table>

improved method for the preparation of a human cell-based, contact model of the blood-brain barrier

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has been implied in the etiology of many brain pathologies, creating a need for development of human in vitro BBB models to assist in clinically-relevant research. Among the numerous BBB models thus far described, a static (without flow), contact BBB model, where astrocytes and brain endothelial cells (BECs) are cocultured on the opposite sides of a porous membrane, emerged as a simplified yet authentic system to simulate the BBB with high throughput screening capacity. Nevertheless the generation of such model presents few technical challenges. Here, we describe a protocol for preparation of a contact human BBB model utilizing a novel combination of primary human BECs and immortalized human astrocytes. Specifically, we detail an innovative method for cell-seeding on inverted inserts as well as specify insert staining techniques and exemplify how we use our model for BBB-related research.

In order to establish a human, contact BBB model we had to cultivate SVGs and BECs on porous membranes with a 3 &#181;m pore-size, shown to permit passage of astrocyte end-feet for contact with endothelial cells14,15,27,28. A schematic representation of the complete contact model is illustrated in Figure 1 (left illustration). The main technical challenge presented by a contact system is the need to seed astrocytes against gravity on the abluminal surface of the membrane. We have succeeded in ...

Watch this Scientific Journal Video about Improved Method for the Preparation of a Human Cell-based, Contact Model of the Blood-Brain Barrier at JoVE.com

Improved Method for the Preparation of a Human Cell-based, Contact Model of the Blood-Brain Barrier

Establishment of human models of the blood-brain barrier (BBB) can benefit research into brain conditions associated with BBB failure. We describe here an improved technique for preparation of a contact BBB model, which permits coculturing of human astrocytes and brain endothelial cells on the opposite sides of a porous membrane.

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has ...

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Research

JoVE Journal

Bioengineering

16.1K visualizações.  Monash University.  Estabelecimento de modelos humanos da barreira sangue-cérebro (BBB) ​​pode beneficiar de pesquisa em condições cerebrais associadas com insuficiência certificação. Descrevemos aqui uma técnica melhorada para a preparação de um modelo de certificação de contacto, o que permite coculturing de astrócitos e células endoteliais cerebrais sobre os lados opostos de uma membrana porosa. 