An in vitro blood-brain barrier model for high throughput (HTS) toxicological screening

Culot, Maxime; Lundquist, Stefan; Vanuxeem, Dorothée; Nion, Stéphane; Landry, Christophe; Delplace, Y; Dehouck, Marie‐Pierre; Bérézowski, Vincent; Fénart, Laurence; Cecchelli, Roméo

doi:10.1016/j.tiv.2007.12.016

Cited by 118 publications

(83 citation statements)

References 29 publications

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“…This correlated with a decreased mannitol permeability, which confirmed a functional tightening of the junctions towards small molecules. Permeability values of small compounds like sodium fluorescein or sucrose in the ranges of 1-6×10 −6 cm/s have been reported (13)(14)(15)(16)(17). In the present study, we obtained values ranging from 0.48 (TEER average of 1,640 Ωcm 2 ) to 3.2× 10 −6 cm/s (TEER average of 630 Ω cm 2 ), and, to our knowledge, only two studies have reported equally low permeability values (18,19) These values were obtained from porcine endothelial cultures, which have generally Fig.…”

Section: Discussionmentioning

confidence: 99%

“…Functional integrity of the monolayers was investigated by addition of 14 C mannitol (specific activity 56.5 mCi/mmol) to the culture medium to a final concentration of 1 μCi/ml. pH measurements of the media during the culture period and transcellular transport experiments were performed using a PHM 240 PH/ION meter equipped with a PHC2401-8 combined electrode (Meterlab-Radiometer Analytical, Lyon, France).…”

Section: Characterization Of Blood-brain Barrier Propertiesmentioning

confidence: 99%

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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

Helms

Waagepetersen

Nielsen

et al. 2010

AAPS J

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Abstract. 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 cocultured using astrocyte-conditioned media with cAMP increasing agonists and dexamethasone. The coculture had average TEER values from 261±26 Ωcm 2 to 760±46 Ωcm 2 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 Ωcm 2 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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Section: Discussionmentioning

confidence: 99%

Section: Characterization Of Blood-brain Barrier Propertiesmentioning

confidence: 99%

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

Helms

Waagepetersen

Nielsen

et al. 2010

AAPS J

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“…Brain endothelial cells have been extracted from various species to implement in vitro BBB models based on low passage primary cultures from bovine 6,7 , porcine 8,9 . In vitro BBB models are usually used to compare the transport of drugs with: i) known penetration into the CNS or with low permeability across the BBB and no central effects, and ii) the apparent Pe of the same drugs measured in animal models of the same species.…”

Section: Introductionmentioning

confidence: 99%

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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“…According to the Lucifer yellow Papp, paracellular leakage was minimal during the experiments, thereby indicating the absence of fenestration in the cell barrier and the lack of cytotoxicity of the peptides at the concentrations tested. [12] Therefore, the transport measured during the permeability assay was attributable mainly to a transcellular mechanism.…”

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confidence: 99%

Applying the Retro‐Enantio Approach To Obtain a Peptide Capable of Overcoming the Blood–Brain Barrier

et al. 2015

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The blood-brain barrier (BBB) is a formidable physical and enzymatic barrier that tightly controls the passage of molecules from the blood to the brain. In fact, less than 2 % of all potential neurotherapeutics are able to cross it. Here, by applying the retro-enantio approach to a peptide that targets the transferrin receptor, a full protease-resistant peptide with the capacity to act as a BBB shuttle was obtained and thus enabled the transport of a variety of cargos into the central nervous system.

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An in vitro blood-brain barrier model for high throughput (HTS) toxicological screening

Cited by 118 publications

References 29 publications

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

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

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

Applying the Retro‐Enantio Approach To Obtain a Peptide Capable of Overcoming the Blood–Brain Barrier

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