This study was supported by internal grants from Institut Pasteur including an Incitative grant (PTR 435) and by a grant &#8220;Contrat de Soutien &#224; la Recherche&#8221; provided by Sanofi Pasteur to Institut Pasteur. A. da Costa was supported by the Sanofi-Pasteur grant and Florian Bakoa is recipient of a PhD grant provided by ANRT (Association Nationale de la Recherche et de la Technologie). We are indebted to Pr Pierre-Olivier Couraud and Dr Florence Miller for helpful discussions.

The intellectual property of the system was the patents referenced in 32, 35 and 38.

In this article we demonstrated how to build an in cellulo blood/brain interface, the BBB-Minibrain, by combining a BBB model and a culture of mixed brain cerebral cells (Minibrain) into a single kit. This system is biologically relevant, easy to set up and handle for experimenters well trained in cell culture.
As for any other in vitro model of BBB, reliable results would be obtained if drastic control of tightness of the barrier is applied. Inserts should be carefully tested for permeability...

The brain is separated from the systemic circulation by a non-permeable structure that restricts exchanges between the brain parenchyma and the blood, called the blood-brain barrier (BBB). Mostly composed of cerebral endothelial cells, the BBB dynamically interacts with astrocytes, perivascular microglia and neurons of the neighboring brain parenchyma. The three major functions of the BBB are the creation and maintenance of ionic homeostasis for neuronal functions, supply of the brain with nutrients, and protection from toxic injuries or entry of pathogens1,2, which contribute to the maintenance of brain homeostasis and its functions3. This barrier is so efficient that only few drugs can cross the BBB4,5. At present, the available methods to predict whether a molecule will pass the BBB and diffuse into the brain consist of ex vivo studies on autopsy material, image tracking in the brain of human volunteers by MRI (magnetic resonance imaging) or PET (positon emission tomography) or pharmacodynamics and pharmacokinetic preclinical studies in animals6,7,8. These techniques and models have some limitations, such as the limited resolution of PET and the low sensitivity of MRI6,8, the difficulty to quantify molecules (i.e., antibody based molecules for example) that poorly penetrate the brain7, and for the preclinical studies their high cost and resort of animal testing.
The last point is important because, according to the 3R&#8217;s rules, (replacement, reduction and refinement of animal testing) the regulatory administrations have asked that the researchers urgently develop scientifically accurate alternative to animal experimentation9,10,11,12,13,14,15.
Over the last decades, several in vitro models of BBB have been proposed16,17,18 by cultivating on filter membrane inserts endothelial cells from different species such as mouse, rat, bovine and pig. As far as the human species is concerned, the scarce and difficult availability of primary cells prompted the researchers to develop human models based on immortalized brain endothelial cells or human-derived stem cells19,20,21. These barriers are proper in vitro surrogates of BBB provided that they express endothelial cell markers, tight junction markers, efflux transporters, solute carriers, receptors, and respond to the endothelial stimuli 20. A few BBB models using filter membrane inserts coated with endothelial cells and other cell types (i.e., astrocytes, neurons or pericytes22,23,24) were assayed. The goal of these co-cultures was to increase the BBB physical characteristics by taking advantage of the secretion of soluble factors by astrocytes/neurons or pericytes.
Nevertheless, none of these models includes brain parenchyma to study and predict the fate of a drug candidate once it has passed the barrier. Therefore, our goal was to build an in cellulo blood/brain interface, the BBB-Minibrain, by combining a BBB model and a culture of mixed brain cells into a single kit. The BBB-Minibrain uses a culture system consisting of a porous filter inserted in a well of a multiwell cell culture plate. The filter is coated with hCMEC/D3 cells, a human brain endothelial cell line that has been proved highly reliable for BBB drug testing25,26,27, to form the BBB. The Minibrain, which is a co-differentiated culture of human neurons and astrocytes derived from the NTera/Cl2.D1 cell line28,29 mixed together with the human microglial cell line CHME/Cl530 in ratio corresponding to the microglia vs. neuron-astrocytes ratios of the brain31, is cultivated in the bottom of the plate well.
Besides studying passage of drugs across the BBB and their fate in the parenchyma, the blood-brain interface in cellulo model could be a powerful tool to address the entry of pathogens into the brain (neuroinvasiveness), the dispersion into the brain (neurotropism) and the toxicity (neurovirulence) they can exert on brain parenchyma cells. Neurovirulence and neuroinvasiveness studies would benefit from the development of an efficient in cellulo model and be advantageous to replace animal models. Using the BBB-Minibrain kit32, we demonstrated the neuroinvasive phenotype of rare viral mutants that accumulated in the French Neurotropic virus strain of Yellow Fever Virus (i.e., FNV-YFV33,34) used to prepare a discontinued live YFV vaccine and the passage of a neuroregenerative and neuroprotective biomolecule called Neurovita (referred as NV henceforth in the manuscript)35. Because NV neither naturally crosses the cell membrane nor the BBB, NV was fused with the variable part (VHH) of a single chain antibody of Llama that crosses the biological membranes including the BBB and functions as a cell penetrating molecule (CPM)36. The CPM property of VHH seems to depend upon the isoelectric point and the length of the VHH37.
This in cellulo test should make it possible to sort the molecules that could potentially cross the BBB before carrying out pharmacokinetic and pharmacodynamics analysis in animals, and ideally in the same time to be able to predict their behavior in the nervous parenchyma. This system is biologically relevant and easy to set up and handle by professionals well trained in cell culture26,29,30,38. The interest of such in cellulo testing would be two-fold: reducing the costs of preclinical tests on the one hand and reducing the use of animal testing on the other hand.

<table><tbody><tr><td>12 well plates</td><td>Corning</td><td>3336</td><td></td></tr><tr><td>5-fluoro-2&amp;rsquo;deoxyuridine</td><td>Merck-Sigma Aldrich</td><td>F0503</td><td></td></tr><tr><td>85mm Petri Dish</td><td>Sarstedt</td><td>83-3902-500</td><td></td></tr><tr><td>Anti-Nf200</td><td>Merck-Sigma Aldrich</td><td>N4142</td><td></td></tr><tr><td>&amp;beta;-mercapto-ethanol</td><td>Merck-Sigma Aldrich</td><td>M3148</td><td></td></tr><tr><td>CHME/Cl5</td><td>Unit&amp;eacute; de Neuroimmunologie Virale</td><td>On request to Dr Lafon</td><td></td></tr><tr><td>CMC</td><td>Calbiochem</td><td>217274</td><td></td></tr><tr><td>Cytosine &amp;beta;-D-arabinofuranoside</td><td>Merck-Sigma Aldrich</td><td>C1768</td><td></td></tr><tr><td>Dark 96 well plates</td><td>Corning</td><td>3915</td><td></td></tr><tr><td>DMEM F12</td><td>Thermofisher Scientific</td><td>31330-038</td><td></td></tr><tr><td>DMSO</td><td>Merck-Sigma Aldrich</td><td>D2650</td><td></td></tr><tr><td>Endogro IV</td><td>Millipore</td><td>SCME004</td><td>endothelial cell medium</td></tr><tr><td>Ethanol</td><td>Carlo Erba</td><td>529121</td><td></td></tr><tr><td>FBS</td><td>Hyclone</td><td>SV30015-04</td><td></td></tr><tr><td>Formaldehyde</td><td>Merck-Sigma Aldrich</td><td>252549</td><td></td></tr><tr><td>GIEMSA</td><td>RAL Diagnostic</td><td>320310</td><td></td></tr><tr><td>Goat-Anti Mouse</td><td>Jackson Immuno Research</td><td>115-545-003</td><td></td></tr><tr><td>Goat-Anti Rabbit</td><td>Thermofisher Scientific</td><td>R37117</td><td></td></tr><tr><td>HBSS with Ca2+-Mg2+</td><td>Thermofisher Scientific</td><td>14025-100</td><td></td></tr><tr><td>hCMEC/D3</td><td>Cedarlane</td><td>CLU512</td><td></td></tr><tr><td>Hepes 1M</td><td>Thermofisher Scientific</td><td>15630-070</td><td></td></tr><tr><td>Hoescht 33342</td><td>Merck-Sigma Aldrich</td><td>33263</td><td></td></tr><tr><td>Laminine</td><td>Merck-Sigma Aldrich</td><td>L6274</td><td></td></tr><tr><td>L-glutamin</td><td>Thermofisher Scientific</td><td>25030-024</td><td></td></tr><tr><td>Lucifer Yellow</td><td>Merck-Sigma Aldrich</td><td>L0259</td><td></td></tr><tr><td>MEM 10X</td><td>Thermofisher Scientific</td><td>21430</td><td></td></tr><tr><td>MEM 1X</td><td>Thermofisher Scientific</td><td>42360</td><td></td></tr><tr><td>Ntera/Cl2D.1</td><td>ATCC</td><td>CRL-1973</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Electron Microscopy Sciences</td><td>15714</td><td></td></tr><tr><td>PBS without Ca2+-Mg2+</td><td>Thermofisher Scientific</td><td>14190</td><td></td></tr><tr><td>PBS-Ca2+-Mg2+</td><td>Thermofisher Scientific</td><td>14040-091</td><td></td></tr><tr><td>Pen/Strep</td><td>Eurobio</td><td>CXXPES00-07</td><td></td></tr><tr><td>Poly-d-Lysine</td><td>Merck-Sigma Aldrich</td><td>P1149</td><td></td></tr><tr><td>Prolong Gold</td><td>Thermofisher Scientific</td><td>P36930</td><td></td></tr><tr><td>Qiashredder</td><td>QIAGEN</td><td>79656</td><td></td></tr><tr><td>Rat Collagen I</td><td>Cultrex</td><td>3443-100-01</td><td></td></tr><tr><td>Retinoic Acid All-Trans</td><td>Merck-Sigma Aldrich</td><td>R2625</td><td></td></tr><tr><td>RNA purification kit</td><td>QIAGEN</td><td>74104</td><td></td></tr><tr><td>SDS</td><td>Merck-Sigma Aldrich</td><td>L4509</td><td></td></tr><tr><td>Sodium bicarbonate 5.6%</td><td>Eurobio</td><td>CXXBIC00-07</td><td></td></tr><tr><td>Sodium Pyruvate</td><td>Thermofisher Scientific</td><td>11360</td><td></td></tr><tr><td>T75 Cell+ Flask</td><td>Sarstedt</td><td>83-1813-302</td><td>Tissue culture polystyrene flask with specific surface treatment (Cell+) for sensitive adherent cells</td></tr><tr><td>Transwell</td><td>Corning</td><td>3460</td><td>polyester porous membrane culture inserts</td></tr><tr><td>Trypsin-EDTA</td><td>Merck-Sigma Aldrich</td><td>T3924</td><td></td></tr><tr><td>Ultra Pure Water</td><td>Thermofisher Scientific</td><td>10977-035</td><td></td></tr><tr><td>Uridine</td><td>Merck-Sigma Aldrich</td><td>U3750</td><td></td></tr><tr><td>Versene</td><td>Thermofisher Scientific</td><td>15040-033</td><td>EDTA</td></tr><tr><td>YFV-FNV</td><td>IP Dakar</td><td>Vaccine vial</td><td></td></tr></tbody></table>

a human blood-brain interface model to study barrier crossings by pathogens or medicines and their interactions with the brain

The early screening of nervous system medicines on a pertinent and reliable in cellulo BBB model for their penetration and their interaction with the barrier and the brain parenchyma is still an unmet need. To fill this gap, we designed a 2D in cellulo model, the BBB-Minibrain, by combining a polyester porous membrane culture insert human BBB model with a Minibrain formed by a tri-culture of human brain cells (neurons, astrocytes and microglial cells). The BBB-Minibrain allowed us to test the transport of a neuroprotective drug candidate (e.g., Neurovita), through the BBB, to determine the specific targeting of this molecule to neurons and to show that the neuroprotective property of the drug was preserved after the drug had crossed the BBB. We have also demonstrated that BBB-Minibrain constitutes an interesting model to detect the passage of virus particles across the endothelial cells barrier and to monitor the infection of the Minibrain by neuroinvasive virus particles. The BBB-Minibrain is a reliable system, easy to handle for researcher trained in cell culture technology and predictive of the brain cells phenotypes after treatment or insult. The interest of such in cellulo testing would be twofold: introducing derisking steps early in the drug development on the one hand and reducing the use of animal testing on the other hand.

The BBB-Minibrain is an in cellulo experimental model of blood-brain interface.
The BBB-Minibrain is set up on the polyester membrane culture insert system to mimic a blood compartment on the upper level and a brain compartment on the lower level of the blood-brain interface (Figure 2A,B). It consists of a luminal compartment with the hCMEC/D3 endothelial cells on t...

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A Human Blood-Brain Interface Model to Study Barrier Crossings by Pathogens or Medicines and Their Interactions with the Brain

Here we present a protocol describing the setting of an in cellulo BBB (Blood brain barrier)-Minibrain polyester porous membrane culture insert system in order to evaluate the transport of biomolecules or infectious agents across a human BBB and their physiological impact on the neighboring brain cells.

The early screening of nervous system medicines on a pertinent and reliable in cellulo BBB model for their penetration and their interaction with the ...

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Research

JoVE Journal

Neuroscience

8.6K Views.  Institut Pasteur, CNRS UMR 3569, Unité de Neuroimmunologie Virale. Dit menselijke bloed-hersenen interface model is nuttig voor het bestuderen van de overdracht van moleculen in het neurosysteem. Evenals voor het bestuderen van microben bereiken een geïnfecteerde hersenpijn tumor. De toevoeging van de mini-hersenen aan deze bloed-hersenbarrière of BBB-model maakt de studie van onze ziekteverwekkers en moleculen die ervoor zorgen dat de BBB zich gedragen in de hersenen.Het degraderen van de procedure zal Florian Bakao zijn, de promovendi voor mijn la...

Video: A Human Blood-Brain Interface Model to Study Barrier Crossings by Pathogens or Medicines and Their Interactions with the Brain

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.

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 ...

Bioengineering

Generation of a Human iPSC-Based Blood-Brain Barrier Chip

The blood brain barrier (BBB) is formed by neurovascular units (NVUs) that shield the central nervous system (CNS) from a range of factors found in the blood that can disrupt delicate brain function. As such, the BBB is a major obstacle to the delivery of therapeutics to the CNS. Accumulating evidence suggests that the BBB plays a key role in the onset and progression of neurological diseases. Thus, there is a tremendous need for a BBB model that can predict penetration of CNS-targeted drugs as well as elucidate the BBB&#39;s role in health and disease.
We have recently combined organ-on-chip and induced pluripotent stem cell (iPSC) technologies to generate a BBB chip fully personalized to humans. This novel platform displays cellular, molecular, and physiological properties that are suitable for the prediction of drug and molecule transport across the human BBB. Furthermore, using patient-specific BBB chips, we have generated models of neurological disease and demonstrated the potential for personalized predictive medicine applications. Provided here is a detailed protocol demonstrating how to generate iPSC-derived BBB chips, beginning with differentiation of iPSC-derived brain microvascular endothelial cells (iBMECs) and resulting in mixed neural cultures containing neural progenitors, differentiated neurons, and astrocytes. Also described is a procedure for seeding cells into the organ chip and culturing of the BBB chips under controlled laminar flow. Lastly, detailed descriptions of BBB chip analyses are provided, including paracellular permeability assays for assessing drug and molecule permeability as well as immunocytochemical methods for determining the composition of cell types within the chip.

The blood-brain barrier (BBB) is a multicellular neurovascular unit tightly regulating brain homeostasis. By combining human iPSCs and organ-on-chip technologies, we have generated a personalized BBB chip, suitable for disease modeling and CNS drug penetrability predictions. A detailed protocol is described for the generation and operation of the BBB chip.

The blood brain barrier (BBB) is formed by neurovascular units (NVUs) that shield the central nervous system (CNS) from a range of factors found in the ...

Developmental Biology

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). This process is characterized by the transmigration of activated T cells across brain endothelial cells (ECs), the main constituents of the BBB. However, the consequences on brain EC function upon interaction with such T cells are largely unknown. Here we describe an assay that allows for the evaluation of primary mouse brain microvascular EC (MBMEC) function and barrier integrity during the interaction with T cells over time. The assay makes use of impedance cell spectroscopy, a powerful tool for studying EC monolayer integrity and permeability, by measuring changes in transendothelial electrical resistance (TEER) and cell layer capacitance (Ccl). In direct contact with ECs, stimulated but not na&#239;ve T cells are capable of inducing EC monolayer dysfunction, as visualized by a decrease in TEER and an increase in Ccl. The assay records changes in EC monolayer integrity in a continuous and automated fashion. It is sensitive enough to distinguish between different strengths of stimuli and levels of T cell activation and it enables the investigation of the consequences of a targeted modulation of T cell-EC interaction using a wide range of substances such as antibodies, pharmacological reagents and cytokines. The technique can also be used as a quality control for EC integrity in in vitro T-cell transmigration assays. These applications make it a versatile tool for studying BBB properties under physiological and pathophysiological conditions.

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Here, we describe an in vitro murine model of the blood-brain barrier that makes use of impedance cell spectroscopy, with a focus on the consequences on endothelial cell integrity and permeability upon interaction with activated T cells.

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal ...

Immunology and Infection

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

High-resolution Confocal Imaging of the Blood-brain Barrier: Imaging, 3D Reconstruction, and Quantification of Transcytosis

The blood-brain barrier (BBB) is a dynamic multicellular interface that regulates the transport of molecules between the circulation and the brain. Transcytosis across the BBB regulates the delivery of hormones, metabolites, and therapeutic antibodies to the brain parenchyma. Here, we present a protocol that combines immunofluorescence of free-floating sections with laser scanning confocal microscopy and image analysis to visualize subcellular organelles within endothelial cells at the BBB. Combining this data-set with 3D image analysis software allows for the semi-automated segmentation and quantification of capillary volume and surface area, as well as the number and intensity of intracellular organelles at the BBB. The detection of mouse endogenous immunoglobulin (IgG) within intracellular vesicles and their quantification at the BBB is used to illustrate the method. This protocol can potentially be applied to the investigation of the mechanisms controlling BBB transcytosis of different molecules in vivo.

Here we present a microscopy-based protocol for high-resolution imaging and a three-dimensional reconstruction of the mouse neurovascular unit and blood-brain barrier using brain free-floating sections. This method allows for the visualization, analysis, and quantification of intracellular organelles at the BBB.

The blood-brain barrier (BBB) is a dynamic multicellular interface that regulates the transport of molecules between the circulation and the brain. ...

Analyzing the Permeability of the Blood-Brain Barrier by Microbial Traversal through Microvascular Endothelial Cells

The human blood-brain barrier (BBB) is characterized by a very low permeability for biomolecules in order to protect and regulate the metabolism of the brain. The BBB is mainly formed out of endothelial cells embedded in collagen IV and fibronectin-rich basement membranes. Several pathologies result from dysfunction of the BBB followed by microbial traversal, causing diseases such as meningitis. In order to test the effect of multiple parameters, including different drugs and anesthetics, on the permeability of the BBB we established a novel human cell culture model mimicking the BBB with human brain microvascular endothelial cells. The endothelial cells are grown on collagen IV and fibronectin-coated filter units until confluence and can then be treated with different compounds of interest. In order to demonstrate a microbial traversal, the upper chamber with the apical surface of the endothelial cells is inoculated with bacteria. After an incubation period, samples of the lower chamber are plated on agar plates and the obtained colonies are counted, whereby the number of colonies correlate with the permeability of the BBB. Endogenous cellular factors can be analyzed in this experimental set-up in order to elucidate basic cellular mechanisms of the endothelial cells contributing to the BBB. In addition, this platform allows performing a screen for compounds that might affect the permeability of the endothelial cells. Finally, bacterial traversal can be studied and linked to different pathologies, such as meningitis. It might be possible to extend the model and analyze the pathways of the bacteria through the BBB. In this article, we provide a detailed protocol of the described method to investigate the permeability of the BBB.

The human blood-brain barrier selectively prevents penetration of hydrophilic molecules and pathogens into the brain. Several pathologies, including meningitis and postoperative delirium, are associated with an increased permeability of the blood-brain barrier. Here, we describe an endothelial cell culture model to test the barrier permeability by microbial traversal.

The human blood-brain barrier (BBB) is characterized by a very low permeability for biomolecules in order to protect and regulate the metabolism of the ...

Analysis of Blood-Brain Barrier Models via Immunofluorescence Staining

Use of the MicroSiM (&#181;SiM) Barrier Tissue Platform for Modeling the Blood-Brain Barrier

The microSiM (&#181;SiM) is a membrane-based culture platform for modeling the blood-brain barrier (BBB). Unlike conventional membrane-based platforms, the&#160;&#181;SiM provides experimentalists with new capabilities, including live cell imaging, unhindered paracrine signaling between &#39;blood&#39; and &#39;brain&#39; chambers, and the ability to directly image immunofluorescence without the need for the extraction/remounting of membranes. Here we demonstrate the basic use of the platform to establish monoculture (endothelial cells) and co-culture (endothelial cells and pericytes) models of the BBB using ultrathin nanoporous silicon-nitride membranes. We demonstrate compatibility with both primary cell cultures and human induced pluripotent stem cell (hiPSC) cultures. We provide methods for qualitative analysis of BBB models via immunofluorescence staining and demonstrate the use of the &#181;SiM for the quantitative assessment of barrier function in a small molecule permeability assay. The methods provided should enable users to establish their barrier models on the platform, advancing the use of tissue chip technology for studying human tissues.

Author Spotlight: Advancing the Use of Tissue Chip Technology for Studying Human Tissues 

This report provides protocols for assembly, cell culture, and assays on the &#181;SiM platform for the construction of blood-brain barrier models.

The microSiM (&#181;SiM) is a membrane-based culture platform for modeling the blood-brain barrier (BBB). Unlike conventional membrane-based platforms, ...

Transendothelial Resistance Measurement to Assess Cell Barrier Integrity

Barrier Functional Integrity Recording on bEnd.3 Vascular Endothelial Cells via Transendothelial Electrical Resistance Detection

The blood-brain barrier (BBB) is a dynamic physiological structure composed of microvascular endothelial cells, astrocytes, and pericytes. By coordinating the interaction between restricted transit of harmful substances, nutrient absorption, and metabolite clearance in the brain, the BBB is essential in preserving central nervous system homeostasis. Building in vitro models of the BBB is a valuable tool for exploring the pathophysiology of neurological disorders and creating pharmacological treatments. This study describes a procedure for creating an in vitro monolayer BBB cell model by seeding bEnd.3 cells into the upper chamber of a 24-well plate. To assess the integrity of cell barrier function, the conventional epithelial cell voltmeter was used to record the transmembrane electrical resistance of normal cells and CoCl2-induced hypoxic cells in real-time. We anticipate that the above experiments will provide effective ideas for the creation of in vitro models of BBB and drugs to treat disorders of central nervous system diseases.

Author Spotlight: Applications of TEER Detection to Assess Cell Barrier Integrity 

This protocol describes a dependable and efficient in vitro model of the brain blood barrier. The method uses mouse cerebral vascular endothelial cells bEnd.3 and measures transmembrane electrical resistance.

The blood-brain barrier (BBB) is a dynamic physiological structure composed of microvascular endothelial cells, astrocytes, and pericytes. By coordinating ...

A 3D Blood-Brain Barrier Model Derived from Human Pluripotent Stem-Cells

Reconstruction of the Blood-Brain Barrier In Vitro to Model and Therapeutically Target Neurological Disease

The blood-brain barrier (BBB) is a key physiological component of the central nervous system (CNS), maintaining nutrients, clearing waste, and protecting the brain from pathogens. The inherent barrier properties of the BBB pose a challenge for therapeutic drug delivery into the CNS to treat neurological diseases. Impaired BBB function has been related to neurological disease. Cerebral amyloid angiopathy (CAA), the deposition of amyloid in the cerebral vasculature leading to a compromised BBB, is a co-morbidity in most cases of Alzheimer&#39;s disease (AD), suggesting that BBB dysfunction or breakdown may be involved in neurodegeneration. Due to limited access to human BBB tissue, the mechanisms that contribute to proper BBB function and BBB degeneration remain unknown. To address these limitations, we have developed a human pluripotent stem cell-derived BBB (iBBB) by incorporating endothelial cells, pericytes, and astrocytes in a 3D matrix. The iBBB self-assembles to recapitulate the anatomy and cellular interactions present in the BBB. Seeding iBBBs with amyloid captures key aspects of CAA. Additionally, the iBBB offers a flexible platform to modulate genetic and environmental factors implicated in cerebrovascular disease and neurodegeneration, to investigate how genetics and lifestyle affect disease risk. Finally, the iBBB can be used for drug screening and medicinal chemistry studies to optimize therapeutic&#160;delivery to the CNS. In this protocol, we describe the differentiation of the three types of cells (endothelial cells, pericytes, and astrocytes) arising from human pluripotent stem cells, how to assemble the differentiated cells into the iBBB, and how to model CAA in vitro using exogenous amyloid. This model overcomes the challenge of studying live human brain tissue with a system that has both biological fidelity and experimental flexibility, and enables the interrogation of the human BBB and its role in neurodegeneration.

Author Spotlight: A Personalized Approach Towards Investigating Alzheimer's Disease Using an In Vitro Blood-Brain Barrier Model 

The blood-brain barrier (BBB) has a crucial role in sustaining a stable and healthy brain environment. BBB dysfunction is associated with many neurological diseases. We have developed a 3D, stem-cell-derived model of the BBB to investigate cerebrovascular pathology, BBB integrity, and how the BBB is altered by genetics and disease.

The blood-brain barrier (BBB) is a key physiological component of the central nervous system (CNS), maintaining nutrients, clearing waste, and protecting ...

A Triple Culture Cell System Modeling the Human Blood-Brain Barrier

The delivery of drugs to the brain remains a challenge due to the blood-brain barrier's (BBB) highly specific and restrictive properties, which controls and restrict access to the brain parenchyma. However, with the development of nanotechnologies, large panels of new nanomaterials were developed to improve drug delivery, highlighting the need for reliable in vitro microsystems to predict brain penetration in the frame of preclinical assays. Here is a straightforward method to set up a microphysiological system to model the BBB using solely human cells. In its configuration, the model consists of a triple culture including brain-like endothelial cells (BLECs), pericytes, and astrocytes, the three main BBB cellular actors necessary to induce and regulate the BBB properties in a more physiological manner without the requirement of tightening compounds. The model developed in a 12-well plate format, ready after 6 days of triple culture, is characterized in physical properties, gene, and protein expressions and used for polymeric nanogel transport measurement. The model can be used for an extensive range of experiments in healthy and pathological conditions and represents a valuable tool for preclinical assessments of molecule and particle transport, as well as inter-and intracellular trafficking.

This protocol describes a method to establish a human blood-brain barrier (BBB) in vitro model. The endothelial cells and pericytes are seeded on each side of an insert filter (blood compartment), and astrocytes are seeded in the bottom well (brain compartment). The model characterized was used for nanoparticles transport experiments.

The delivery of drugs to the brain remains a challenge due to the blood-brain barrier's (BBB) highly specific and restrictive properties, which controls ...

A Human Blood-Brain Interface Model to Study Barrier Crossings by Pathogens or Medicines and Their Interactions with the Brain

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