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

Helms, Hans Christian Cederberg; Waagepetersen, Helle S.; Nielsen, Carsten Uhd; Brodin, Birger

doi:10.1208/s12248-010-9237-6

Cited by 58 publications

(54 citation statements)

References 40 publications

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“…Important cues for cell proliferation are provided by the extracellular matrix such as integrin and growth factor (bFGF) activation 49 . Buffer concentration and pH of the culture media have been described to impact positively on paracellular tightness 50 and we observed a better reproducibility between cultures with the addition of HEPES buffer at 5 mM.…”

Section: Other In Vitro Bbb Models Based On Endothelial Cells From Rasupporting

confidence: 62%

Setting-up an <em>In Vitro</em> Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport

Molino¹,

Jabès²,

Lacassagne

et al. 2014

JoVE

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Citation: Molino, Y., Jabès, F., Lacassagne, E., Gaudin, N., Khrestchatisky, M. Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport. J. Vis. Exp. (88), e51278, doi:10.3791/51278 (2014). AbstractThe blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm·cm 2 on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 ± 0.11 x 10 -3 cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.

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Section: Other In Vitro Bbb Models Based On Endothelial Cells From Rasupporting

confidence: 62%

Setting-up an <em>In Vitro</em> Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport

Molino¹,

Jabès²,

Lacassagne

et al. 2014

JoVE

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“…STX100C) and hence better reproducibility. The "Endohm" chamber system with large plate electrodes to fi t in the fi lter cup (above) and the well (below) the cells on the fi lter, samples a larger area of membrane including the more uniform central area, and can give more reproducible readings (Cohen-Kashi Malina et al 2009 ;Helms et al 2010Helms et al , 2012Patabendige et al 2013a , b ); however, the "plunger" action of inserting the upper electrode can disturb the cells, particularly the brain endothelial cells, which are much thinner and more fragile than the CNS barrier epithelial cells.…”

Section: Methods To Measure Barrier Permeability and Teermentioning

confidence: 99%

In Vitro Models of CNS Barriers

Abbott

Dolman

Yusof

et al. 2013

AAPS Advances in the Pharmaceutical Sciences Series

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This chapter reviews the history and modern applications of isolated preparations of the three main CNS barrier layers and cell culture preparations derived from them. In vitro models give valuable mechanistic information but also provide useful assay systems for drug discovery and delivery programmes. However, it is important to take into account practical issues including species differences and the degree to which the differentiated state of the in vivo barrier is retained. The range of models available is reviewed, with a critical evaluation of their strengths and weaknesses, and guidance in selecting and optimizing a suitable model for particular applications. New understanding of the unstirred water layers and paracellular leak pathway in in vitro preparations gives greater insights into the "intrinsic permeability" of the membrane, and a variety of techniques permit characterization of the transport systems and enzymes contributing to barrier function. Increasingly, aspects of CNS pathology are being modelled in cell culture, aiding the optimization of drug delivery regimes in pathological conditions. Chapter 6In Vitro Models of CNS Barriers IntroductionFrom the earliest demonstration of restricted exchange between the blood and the brain (Ehrlich 1885 ) leading to the modern understanding of the blood-CNS barriers, animal experiments and clinical observations have provided valuable information about the physiology and pathology of the barrier layers. However, obtaining mechanistic information from such studies at the cellular and molecular level is complex and time-consuming, and it is often diffi cult to obtain suffi cient spatial and temporal resolution. The situation was dramatically improved by the introduction of in vitro methods (reviewed in Joó 1992 ). Background and Early HistoryThe fi rst successful isolation of cerebral microvessels (Siakotos and Rouser 1969 ;Joó and Karnushina 1973 ) prepared the way for development of in vitro models of the blood-brain barrier (BBB), which have contributed to current understanding of its physiology, pharmacology and pathophysiology (reviewed in Joó 1992 ). Methods have also been developed for in vitro models of the choroid plexus and of the arachnoid epithelium (blood-CSF barrier, BCSFB). However, this proliferation of in vitro models and techniques causes problems for attempts at comparison between models and transferability of results obtained with different models, and makes it hard for scientists entering the fi eld to select an optimal model for their particular interests. This chapter gives an overview of the current status of the most widely used in vitro CNS barrier models, with an update on an earlier review (Reichel et al. 2003 ), and offers guidance in model selection for specifi c applications, including permeability assay for drugs and "new chemical entities" (NCEs). Isolated brain microvessels were the fi rst model system for studying the BBB in vitro, offering new opportunities to investigate physiological and pathological processes at...

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“…The general TEER levels in these models have ranged from approximately 100-800 Ω·cm 2 , which is reasonably tight compared to other models. However, we previously demonstrated that TEER across a bovine endothelial/rat astrocyte co-culture BBB model was raised approximately two-fold by culture in highly buffered differentiation medium reaching values above 1,600 Ω·cm 2 [32]. Low TEER in cell culture models may cause lack of polarized transport because of a large paracellular flux contributing to the overall transport of the substrate [28].…”

Section: Polarized Transport Of Efflux Transporter Substrates May Bementioning

confidence: 99%

“…Recently, our group published a new culture protocol based on the model published by Gaillard et al [31] to improve the tightness of a bovine endothelium/rat astrocyte BBB model [32,33]. The co-cultured endothelial cells displayed high TEER values, low mannitol fluxes, and polarized transport of the small molecules, L-glutamate and L-aspartate favouring the abluminal-to-luminal direction [34].…”

Section: Introductionmentioning

confidence: 99%

An Electrically Tight In Vitro Blood–Brain Barrier Model Displays Net Brain-to-Blood Efflux of Substrates for the ABC Transporters, P-gp, Bcrp and Mrp-1

Helms

Hersom

Kuhlmann

et al. 2014

AAPS J

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Abstract. Efflux transporters of the ATP-binding cassette superfamily including breast cancer resistance protein (Bcrp/Abcg2), P-glycoprotein (P-gp/Abcb1) and multidrug resistance-associated proteins (Mrp's/ Abcc's) are expressed in the blood-brain barrier (BBB). The aim of this study was to investigate if a bovine endothelial/rat astrocyte in vitro BBB co-culture model displayed polarized transport of known efflux transporter substrates. The co-culture model displayed low mannitol permeabilities of 0.95±0.1·10 H-etoposide revealed polarized transport favouring the brain-to-blood direction for all substrates. Steady state efflux ratios of 2.5±0.2 for digoxin, 4.4±0.5 for estrone-3-sulphate and 2.4±0.1 for etoposide were observed. These were reduced to 1.1±0.08, 1.4±0.2 and 1.5±0.1, by addition of verapamil (digoxin), Ko143 (estrone-3-sulphate) or zosuquidar+reversan (etoposide), respectively. Brain-toblood permeability of all substrates was investigated in the presence of the efflux transporter inhibitors verapamil, Ko143, zosuquidar, reversan and MK 571 alone or in combinations. Digoxin was mainly transported via P-gp, estrone-3-sulphate via Bcrp and Mrp's and etoposide via P-gp and Mrp's. The expression of P-gp, Bcrp and Mrp-1 was confirmed using immunocytochemistry. The findings indicate that P-gp, Bcrp and at least one isoform of Mrp are functionally expressed in our bovine/rat co-culture model and that the model is suitable for investigations of small molecule transport.KEY WORDS: blood-brain barrier; breast cancer resistance protein; multidrug resistance-associated protein; p-glycoprotein; polarized small molecule transport.

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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

Cited by 58 publications

References 40 publications

Setting-up an <em>In Vitro</em> Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport

Setting-up an <em>In Vitro</em> Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport

In Vitro Models of CNS Barriers

An Electrically Tight In Vitro Blood–Brain Barrier Model Displays Net Brain-to-Blood Efflux of Substrates for the ABC Transporters, P-gp, Bcrp and Mrp-1

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