The authors would like to thank Prof. Hartwig Wolburg for performing the electron microscopy.

1. Prepare Cell Culture Filter Inserts for Seeding HIBCPP Cells in an Inverted Model System
<ol>
	<li>Pre-warm DMEM/ F12 (Ham) supplemented with 5 &#181;g/ml insulin, 100 U/ml penicillin, 100 &#181;g/ml streptomycin and 10% fetal calf serum (FCS).</li>
	<li>Use sterile forceps to place 0.33 cm&#178; growth area cell culture filter inserts with a pore size of 3 &#181;m upside down into a 12-well plate (Figure 1E).</li>
	<li>Fill medium into the lower compartment of the cell culture filter insert (about 3 ml) and 100 &#181;l on top of the filter insert. Flood the plate as well as the lower compartment with medium. Aspirate the excessive medium in such a way that the lower compartment of the filter insert stays filled (Figure 1E). 
	Note: It is recommended to use a serological pipette for this step.</li>
	<li>Cover 12-well plate with the lid and transfer the prepared cell culture filter inserts to the incubator, 37 &#176;C, 5% CO2 until addition of cells.</li>
</ol>
2. Cultivation and Passaging of HIBCPP Cells
<ol>
	<li>Prepare DMEM/ F12 (Ham) supplemented with 5 &#181;g/ml, 100 U/ml penicillin, 100 &#181;g/ml streptomycin and 10% FCS.</li>
	<li>Pre-warm medium and PBS in a 37 &#176;C water bath. Aspirate medium from flask. Add 10 ml PBS to the flask and swirl. Repeat this step once.</li>
	<li>Add 3 ml 0.25% trypsin-EDTA to the flask and swirl. Place into the incubator, 37 &#176;C, 5% CO2.</li>
	<li>After approximately 20 min remove the flask from the incubator. Ensure that the cells are detached from the bottom of the flask and display a round shape when surveyed with the microscope. 
	Note: The cells do not detach completely from each other and are often found in agglomerates. It is recommended to use the cells up to passage 38.</li>
	<li>To stop trypsinization add 17 ml medium. Resuspend the cells by pipetting up and down and transfer the suspension into a 50 ml tube. Centrifuge at 50 x g for 10 min at room temperature.</li>
	<li>Resuspend the cells in an appropriate volume of medium and count the cells by using a hemocytometer. 
	Note: The concentration of resuspended HIBCPP cells should be 1 x 106 cells/ml. For maintenance of HIBCPP cells it is suggested to transfer an amount of 1-6 x 106 cells in 10 ml medium to a T75-flask. Change medium every second day.</li>
</ol>
3. Seeding Inverted Cell Culture Filter Inserts with HIBCPP Cells
<ol>
	<li>On top of each inverted filter insert (i.e. the bottom side of the filter) add 80 &#181;l of cell suspension (i.e. 8 x 104 cells) (Figure 1E). 
	Note: Make sure that the cells are evenly distributed in the suspension by inverting the tube before seeding.</li>
	<li>Cover the seeded-cell culture filter inserts with the lid of the 12-well plate and transfer to the incubator, 37 &#176;C, 5% CO2.</li>
	<li>On the first day, fill 1 ml medium into the wells of a 24-well plate. Lift the cell culture filter inserts by using forceps out of the 12-well plate, discard the medium inside, turn the filter inserts and place them in the standard orientation into the prepared 24-well plate (Figure 1E).</li>
	<li>Place the cells into fresh medium every second day. Prepare 24-wells with fresh medium and transfer the filter inserts to it. Fill inserts with 0.5 ml fresh medium. Check TEER every day as described in section 4.</li>
	<li>When TEER values of HIBCPP seeded-cells on cell culture inserts exceeds 70 &#937; x cm&#178; (approximately 4 days after seeding), then continue cell culture in medium containing 1% FCS and 5 &#181;g/ml insulin. Prepare 24-well plates with 1 ml medium for each well. Transfer filter inserts to the prepared wells and exchange medium in the upper compartment. 
	Note: This serum withdrawal after confluency leads to the formation of a higher membrane potential.</li>
	<li>Aspirate medium from filter compartment, transfer to the prepared well and fill with 500 &#181;l medium. Repeat this step once. Put the cells overnight into the incubator, 37 &#176;C, 5% CO2.</li>
</ol>
4. Measurement of Transepithelial Electrical Resistance (TEER)
<ol>
	<li>Immerse the electrode tips of epithelial tissue voltohmmeter for 15 min in 80% ethanol. Transfer voltohmmeter under the hood and let electrode dry for a moment. Place electrode into respective culture medium used for the cells for another 15 min to equilibrate.</li>
	<li>Perform measurements by positioning the longer arm of the electrode so that it touches the bottom of the lower compartment each time, place the shorter arm into the filter insert compartment. 
	Note: Resistance values of cell culture filter inserts without cells in medium should be used as blank values ((measured value (&#8486;) &#8211; blank value (&#8486;)) x filter surface (i.e. 0.33 cm&#178;)).</li>
	<li>After measuring place the electrode back into 80% ethanol for 15 min. Store in a dry tube.</li>
</ol>
5. Determination of Paracellular Permeability
<ol>
	<li>Dissolve 1 g FITC-Inulin in 200 ml culture medium supplemented with 5 &#181;g/ml insulin 1% FCS. Apply 50 &#181;g/ml of the solution into the upper filter compartment before infection of the cells.</li>
	<li>After infection collect medium samples from the lower well to determine how much Inulin passed from the filter compartment through the cell layer.</li>
	<li>Prepare a FITC-Inulin standard solution and perform 1:2 dilutions, 10 times (100%, 50%, 25%, 12.5%, 6.25%, 3.13%, 1.56%, 0.78%, 0.39%, 0.2% and 0%). Determine fluorescence by measurement of all samples in microplate reader.</li>
</ol>
6. Preparation of Bacteria for Infection of HIBCPP Cells on Cell Culture Filter Inserts
<ol>
	<li>One day prior to the experiment, scrape the frozen N. meningitidis strains off a glycerol-stock and streak out on Chocolate Agar with Vitox. Grow overnight in the incubator, at 37 &#176;C, with 5% CO2.</li>
	<li>Extract 20-30 colonies from the overnight culture and transfer into a tube containing 8 ml Proteose Peptone Medium supplemented with 0.042% NaHCO3, 0.01 M MgCl2 and 1% Polyvitex.</li>
	<li>Shake the bacteria for 1.25 hr at 220 rpm, 37 &#176;C. Pellet the bacteria by centrifugation at 2,684 g for 10 min at room temperature. Discard supernatant and resuspend the bacterial pellet with 8 ml serum-free medium. Vortex to ensure an even suspension.</li>
	<li>To determine culture density, measure a dilution of 1:10 at an optical density (OD) of 600 nm. Adjust the bacterial suspension to an OD600 of 0.1. 
	Note: This suspension contains approx. 1 x 108 CFU/ml.</li>
	<li>To confirm the bacterial cell concentration, dilute the suspension stepwise (1:10) up to a dilution of 10-5 and plate onto Chocolate Agar plates. 
	Note: Similar serial dilutions need to be performed to calculate bacterial growth curves.</li>
</ol>
7. Infection of HIBCPP Cells on Cell Culture Filter Inserts and Determination of Bacterial Invasion by Double Immunofluorescence
<ol>
	<li>After continuing cell culture in medium containing 1% FCS and 5 &#181;g/ml insulin, measure cells every day using an epithelial tissue voltohmmeter. If cells have reached a TEER of around 500 &#937; x cm&#178; , carry out the infection.</li>
	<li>Infect the cells with the prepared bacterial suspension at a multiplicity of infection (MOI) of 10. Store the infected cells in the incubator, at 37 &#176;C, with 5% CO2 for the indicated period of time. 
	Note: The MOI can be calculated by taking into account the number of cells per cell culture filter insert at confluence (1.21 x 106 cells/cm2).</li>
	<li>Stop the infection by washing three times with 500 &#181;l serum-free medium (SFM) containing 1% bovine serum albumin (BSA) applied to the filter compartment, 1 ml to the lower compartment.</li>
	<li>Block with 500 &#181;l SFM containing 1% BSA buffer in the filter compartment and 1 ml in the lower compartment for 20 min at room temperature to prevent adherence of antibodies to unspecific binding sites.</li>
	<li>Incubate with 100 &#181;l primary antibody anti N. meningtidis &#945;-OMP (1:200) in the filter compartment and 500 &#181;l in the lower compartment for 20 min at room temperature. 
	Note: If required, the antibody concentration needs to be titrated.</li>
	<li>Wash the cells by aspirating the medium from the filter compartment, transfer the filter insert to a prepared well with 1 ml SFM containing 1% BSA and fill the emptied filter compartment with 500 &#181;l SFM containing 1% BSA. Repeat this step twice.</li>
	<li>Fix cells with 500 &#181;l 4% formaldehyde in the filter compartment, 1 ml in the lower compartment for 10 min at room temperature.</li>
	<li>Wash the cells by aspirating the medium from the filter compartment, transfer the filter insert to a prepared well with 1 ml PBS and fill the emptied filter compartment with 500 &#181;l PBS. Repeat this step once. 
	Note: Samples can be stored overnight in PBS at 4 &#176;C.</li>
	<li>Cut fixed cell culture filters out of the inserts and wash with 250 &#181;l PBS containing 1% BSA. Incubate cells for 15 min at room temperature with 250 &#181;l fluorescent labeled (excitation wavelength 594 nm) secondary antibody chicken anti rabbit (1:500) to stain extracellular bacteria. 
	Note: If required, the antibody concentration needs to be titrated.</li>
	<li>Permeabilize cells with 250 &#181;l PBS containing 1% BSA and 0.5% Triton X-100 for 1 hr at room temperature. Wash cells three times with 250 &#181;l PBS containing 1% BSA.</li>
	<li>Incubate with 250 &#181;l primary antibody anti N. meningitidis &#945;-OMP (1:200) for 30 min to stain extra- and intracellular bacteria. Wash cells three times with 250 &#181;l PBS containing 1% BSA.</li>
	<li>Apply 250 &#181;l of fluorescent labeled (excitation wavelength 488 nm) secondary antibody (1:500), fluorescent labeled (excitation wavelength 660 nm) Phalloidin (1:250) and 4&#180;,6-diamidino-2-phenylindole dihydrochloride (DAPI) (1:50,000) for 1 hr at room temperature to stain extra- and intracellular bacteria, actin cytoskeleton and nuclei. 
	Note: If required, the antibody concentration needs to be titrated.</li>
	<li>Wash cells three times with 250 &#181;l PBS containing 1% BSA. Embed the cells in mounting medium and store at 4 &#176;C until examination via microscope.</li>
	<li>Determine number of invaded bacteria per predefined field. Do this by counting 20 fields per filter membrane. Calculate the percentage of invaded bacteria. 
	Note: Multiply the mean bacterial count of the 20 microscopic fields with an area coefficient. The result expresses the amount of total bacteria present in a 0.33 cm&#178; cell culture filter insert. Divide this value by the amount of bacteria grown in media during the duration of infection.</li>
</ol>

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USA. 94, 3094-3099 (1997).</li><li>Vandenhaute, E., Stump-Guthier, C., Lasierra Losada, M., Tenenbaum, T., Rudolph, H., Ishikawa, H., 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=The+choroid+plexus+may+be+an+underestimated+site+of+tumor+invasion+to+the+brain:+an+in+vitro+study+using+neuroblastoma+cell+lines.">The choroid plexus may be an underestimated site of tumor invasion to the brain: an in vitro study using neuroblastoma cell lines.</a> Cancer Cell Int. , 15-102 (2015).</li></ol>

The authors have nothing to disclose.

The epithelial cells of the CP form the BCSFB that separates the CSF from the blood2,3. We recently established the HIBCPP cell line as a functional human model of the BCSFB. The cells display important barrier functions of the BCSFB in vitro, including the development of a high membrane potential, a low permeability for macromolecules, as well as the presence of continuous strands of TJs5. The TJ proteins contribute to an apical/basolateral polarity of the cells. The polarity is of high im...

The blood-cerebrospinal fluid barrier (BCSFB) is one of the three barrier sites between the blood and the brain1. Its morphological correlate are the epithelial cells of the choroid plexus (CP)2,3, an endothelial-epithelial convolute, which is strongly vascularized and located in the ventricles of the brain. The CP serves to produce the cerebrospinal fluid (CSF) as well as to separate the latter from the blood. In order to achieve barrier function, the CP epithelial cells show a low pinocytotic activity, express specific transporters, and are densely connected by a continuous network of tight junctions (TJs)2,3.
Human choroid plexus papilloma (HIBCPP) cells, derived from a malignant choroid plexus papilloma of a Japanese woman4, were used to construct a functional in vitro model of the BCSFB. HIBCPP cells show a couple of characteristics of a functional BCSFB as the formation of TJ strands, the development of a high transepithelial membrane potential that can be determined as transepithelial electrical resistance (TEER), and minor permeabilities for macromolecules. Moreover, HIBCPP cells express characteristic transporters, which may serve to regulate the ionic microenvironment, and show apical/basolateral polarity5,6,7.
The BCSFB has been shown to function as an entry site for pathogens (bacteria, viruses, and fungi) into the central nervous system (CNS)8. The invasion of pathogens, including Neisseria meningitidis (N. meningitidis), a Gram-negative bacterium, can cause severe diseases like meningitis. Evidence that it overcomes the protective epithelial barrier of the CP is supported by histopathological observations in patients with meningococcal disease exhibiting increased amounts of meningococci in the vessels and CP epithelial cells9,10. To gain entry into host cells bacteria often hijack endocytotic mechanisms, which are mediated or triggered by specific surface receptors located on the host cells. Since interactions of pathogens with these receptors can be species specific11, animal models can only be consulted to a restricted extent. The HIBCPP cell line provides the opportunity to study the invasion process as well as the underlying molecular mechanisms in a human model system. Employing cell culture inserts enables us to analyze interactions of pathogens with host cells from two distinct cell sides. Many bacteria, including N. meningitidis, are strongly subject to the impact of gravity during infection assays. For optimal interaction of pathogens with the HIBCPP cells during the assays, the bacteria are initially added into the upper compartment of the cell culture filter insert system. To enable infection from the apical or the basolateral cell side, respectively, two variations of the in vitro system have been established: In the standard system HIBCPP cells are seeded into the upper compartment of the filter insert, mimicking the situation when microorganisms are located on the CSF-side and get into contact with the apical side of the cells (Figure 1A, C). In contrast, using the HIBCPP cells in an inverted cell culture filter insert system reflects the conditions when bacteria have entered the blood stream. Microorganisms disseminate in the blood and encounter CP epithelial cells from the basolateral side (Figure 1B, D). Noteworthy, in this model system it has been shown that bacteria invade HIBCPP cells in a polar fashion specifically from the basolateral cell side5,7.
Subsequently to infection of the CP, the invaded pathogens can be recognized by the innate immune system through ligation to pattern-recognition receptors (PRRs). Well-described members of the PRRs belong to the Toll-like receptor (TLR) family. TLRs can bind to characteristic structures of infectious microorganisms, which are termed pathogen-associated molecular patterns (PAMPs). Ligation of the receptors leads to activation of host cell signaling cascades that trigger expression of cytokines and chemokines12, which in turn stimulate transmigration of immune cells across the BCSFB13,14. It has been shown that HIBCPP cells express several TLRs at mRNA level and that infection with N. meningitidis results in secretion of several cytokines and chemokines, including CXCL1-3, IL6, IL8 and TNF&#945;15,16.
Here, we describe cultivation and infection of the human cell line HIBCPP in an inverted cell culture insert system that mimics the BCSFB. This model system enables to study interactions of pathogens with the in vivo relevant basolateral cell side as well as the subsequent cellular response.

<table><tbody><tr><td>0.25% Trypsin-EDTA</td><td>Gibco</td><td>25200-056</td><td></td></tr><tr><td>4&amp;acute;,6 diamidino-2-phenylindole (DAPI)</td><td>Life Technologies</td><td>D1306</td><td></td></tr><tr><td>12-well plates</td><td>Starlab</td><td>CC7682-7512</td><td></td></tr><tr><td>24-well plates</td><td>Starlab</td><td>CC7682-7524</td><td></td></tr><tr><td>Anti Neisseria meningitidis &amp;alpha;-OMP</td><td></td><td></td><td>This antibody was a gift from Drs. H. Claus and U. Vogel (University of W&amp;uuml;rzburg, Germany)</td></tr><tr><td>Alexa Fluor 488 (chicken anti rabbit)</td><td>Invitrogen</td><td>A21441</td><td></td></tr><tr><td>Alexa Fluor 594 (chicken anti rabbit)</td><td>Invitrogen</td><td>A21442</td><td></td></tr><tr><td>Alexa Fluor 660 Phalloidin</td><td>Invitrogen</td><td>A22285</td><td></td></tr><tr><td>Bovine serum albumine (BSA)</td><td>Calbiochem</td><td>12659</td><td></td></tr><tr><td>Chocolate agar plates</td><td>Biomerieux</td><td>43109</td><td></td></tr><tr><td>Cytochalasin D</td><td>Sigma</td><td>C8273</td><td></td></tr><tr><td>DMEM/F12 + L-Glut + 15 mM HEPES</td><td>Gibco</td><td>31330-095</td><td></td></tr><tr><td>DMEM/F12 + L-Glut + 15 mM HEPES w/o Phenolred</td><td>Gibco</td><td>11039-047</td><td></td></tr><tr><td>Dimethyl sulfoxide</td><td>Sigma</td><td>D2650</td><td></td></tr><tr><td>Fetal calf serum (FCS)</td><td>Life Technologies</td><td>10270106</td><td></td></tr><tr><td>FITC-Inulin</td><td>Sigma</td><td>F3272</td><td></td></tr><tr><td>Insulin</td><td>Sigma</td><td>19278</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>2393</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma</td><td>55761</td><td></td></tr><tr><td>PBS + Mg + Ca</td><td>Gibco</td><td>14040-174</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>MP Biomedicals</td><td>1670049</td><td></td></tr><tr><td>Polyvitex</td><td>Biomerieux</td><td>55651</td><td></td></tr><tr><td>Proteose peptone</td><td>BD</td><td>211684</td><td></td></tr><tr><td>Serum-free medium</td><td>Gibco</td><td>10902-096</td><td></td></tr><tr><td>Thincert cell culture inserts for 24-well plates, pore size 3 &amp;micro;m</td><td>Greiner</td><td>662630</td><td></td></tr><tr><td>Tissue culture flask 75 cm&amp;sup2; red cap sterile</td><td>Greiner</td><td>658175</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T8787</td><td></td></tr><tr><td>Volt-Ohm Meter Millicell-ERS2 with MERSSTX01 electrode</td><td>Millipore</td><td>MERSSTX00</td><td></td></tr></tbody></table>

a choroid plexus epithelial cell-based model of the human blood-cerebrospinal fluid barrier to study bacterial infection from the basolateral side

The epithelial cells of the choroid plexus (CP), located in the ventricular system of the brain, form the blood-cerebrospinal fluid barrier (BCSFB). The BCSFB functions in separating the cerebrospinal fluid (CSF) from the blood and restricting the molecular exchange to a minimum extent. An in vitro model of the BCSFB is based on cells derived from a human choroid plexus papilloma (HIBCPP). HIBCPP cells display typical barrier functions including formation of tight junctions (TJs), development of a transepithelial electrical resistance (TEER), as well as minor permeabilities for macromolecules. There are several pathogens that can enter the central nervous system (CNS) via the BCSFB and subsequently cause severe disease like meningitis. One of these pathogens is Neisseria meningitidis (N. meningitidis), a human-specific bacterium. Employing the HIBCPP cells in an inverted cell culture filter insert system enables to study interactions of pathogens with cells of the BCSFB from the basolateral cell side, which is relevant in vivo. In this article, we describe seeding and culturing of HIBCPP cells on cell culture inserts. Further, infection of the cells with N. meningitidis along with analysis of invaded and adhered bacteria via double immunofluorescence is demonstrated. As the cells of the CP are also involved in other diseases, including neurodegenerative disorders like Alzheimer`s disease and Multiple Sclerosis, as well as during the brain metastasis of tumor cells, the model system can also be applied in other fields of research. It provides the potential to decipher molecular mechanisms and to identify novel therapeutic targets.

Here we describe culturing and infection of HIBCPP cells in an inverted cell culture insert system. This model allows us to study invasion mechanisms and the underlying molecular signaling pathways from the basolateral cell side, reproducing a physiological situation of bacteria disseminating and entering epithelial cells via the blood stream (Figure 1).
The HIBCPP cells display certain barrier functions, which ...

Watch this Scientific Journal Video about A Choroid Plexus Epithelial Cell-based Model of the Human Blood-Cerebrospinal Fluid Barrier to Study Bacterial Infection from the Basolateral Side at JoVE.com

A Choroid Plexus Epithelial Cell-based Model of the Human Blood-Cerebrospinal Fluid Barrier to Study Bacterial Infection from the Basolateral Side

The epithelial cells of the choroid plexus (CP) form the blood-cerebrospinal fluid barrier (BCSFB). An in vitro model of the BCSFB employs human choroid plexus papilloma (HIBCPP) cells. This article describes culturing and basolateral infection of HIBCPP cells using a cell culture filter insert system.

The epithelial cells of the choroid plexus (CP), located in the ventricular system of the brain, form the blood-cerebrospinal fluid barrier (BCSFB). The ...

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Research

JoVE Journal

Medicine

14.2K Views.  Heidelberg University. The epithelial cells of the choroid plexus (CP) form the blood-cerebrospinal fluid barrier (BCSFB). An in vitro model of the BCSFB employs human choroid plexus papilloma (HIBCPP) cells. This article describes culturing and basolateral infection of HIBCPP cells using a cell culture filter insert system.

Video: A Choroid Plexus Epithelial Cell-based Model of the Human Blood-Cerebrospinal Fluid Barrier to Study Bacterial Infection from the Basolateral Side

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

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 ...

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) models based on pBECs in mono-culture (MC), MC with astrocyte-conditioned medium (ACM), and non-contact co-culture (NCC) with astrocytes of porcine or rat origin. pBECs were isolated and cultured from fragments of capillaries from the brain cortices of domestic pigs 5-6 months old. These fragments were purified by careful removal of meninges, isolation and homogenization of grey matter, filtration, enzymatic digestion, and centrifugation. To further eliminate contaminating cells, the capillary fragments were cultured with puromycin-containing medium. When 60-95% confluent, pBECs growing from the capillary fragments were passaged to permeable membrane filter inserts and established in the models. To increase barrier tightness and BBB characteristic phenotype of pBECs, the cells were treated with the following differentiation factors: membrane permeant 8-CPT-cAMP (here abbreviated cAMP), hydrocortisone, and a phosphodiesterase inhibitor, RO-20-1724 (RO). The procedure was carried out over a period of 9-11 days, and when establishing the NCC model, the astrocytes were cultured 2-8 weeks in advance. Adherence to the described procedures in the protocol has allowed the establishment of endothelial layers with highly restricted paracellular permeability, with the NCC model showing an average transendothelial electrical resistance (TEER) of 1249 &#177; 80 &#937; cm2, and paracellular permeability (Papp) for Lucifer Yellow of 0.90 10-6 &#177; 0.13 10-6 cm sec-1 (mean &#177; SEM, n=55). Further evaluation of this pBEC phenotype showed good expression of the tight junctional proteins claudin 5, ZO-1, occludin and adherens junction protein p120 catenin. The model presented can be used for a range of studies of the BBB in health and disease and, with the highly restrictive paracellular permeability, this model is suitable for studies of transport and intracellular trafficking.

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of the protocol is to present an optimized procedure for the establishment of an in vitro blood-brain barrier (BBB) model based on primary porcine brain endothelial cells (pBECs). The model shows high reproducibility, high tightness, and is suitable for studies of transport and intracellular trafficking in drug discovery.

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) ...

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 ...

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.

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 ...

Neuroscience

Microdissection and Whole Mount Scanning Electron Microscopy Visualization of Mouse Choroid Plexus

The choroid plexus (CP), a highly vascularized structure protruding into the ventricles of the brain, is one of the most understudied tissues in neuroscience. As it is becoming increasingly clear that this tiny structure plays a crucial role in health and disease of the central nervous system (CNS), it is of utmost importance to properly dissect the CP out of the brain ventricles in a way that allows downstream processing, ranging from functional to structural analysis. Here, isolation of the lateral and fourth brain ventricle mouse CP without the need for specialized tools or equipment is described. This isolation technique preserves the viability, function, and structure of cells within the CP. On account of its high vascularization, the CP can be visualized floating inside the ventricular cavities of the brain using a binocular microscope. However, transcardial perfusion required for downstream analysis can complicate the identification of the CP tissue. Depending on the further processing steps (e.g., RNA and protein analysis), this can be solved by visualizing the CP via transcardial perfusion with bromophenol blue. After isolation, the CP can be processed using several techniques, including RNA, protein, or single cell analysis, to gain further understanding on the function of this special brain structure. Here, scanning electron microscopy (SEM) on whole mount CP is used to get an overall view of the structure.

The choroid plexus (CP), an understudied tissue in neuroscience, plays a key role in health and disease of the central nervous system. This protocol describes a microdissection technique for isolating the CP and the use of scanning electron microscopy to obtain an overall view of its cellular structure.

The choroid plexus (CP), a highly vascularized structure protruding into the ventricles of the brain, is one of the most understudied tissues in ...

A Simple Approach to Perform TEER Measurements Using a Self-Made Volt-Amperemeter with Programmable Output Frequency

Transepithelial/endothelial electrical resistance (TEER) has been used since the 1980s to determine confluency and permeability of in vitro barrier model systems. In most cases, chopstick electrodes are used to determine the electric impedance between the upper and lower compartment of a cell culture filter insert system containing cellular monolayers. The filter membrane allows the cells to adhere, polarize, and interact by building tight junctions. This technique has been described with a variety of different cell lines (e.g., cells of the blood-brain barrier, blood-cerebrospinal fluid barrier, or gastrointestinal and pulmonary tract). TEER measurement devices can be readily obtained from different laboratory equipment suppliers. However, there are more cost-effective and customizable solutions imaginable if an appropriate voltammeter is self-assembled. The overall aim of this publication is to set up a reliable device with programmable output frequency that can be used with commercially available chopstick electrodes for TEER measurement.

Here, we demonstrate how to set up an inexpensive volt-amperemeter with programmable output frequency that can be used with commercially available chopstick electrodes for transepithelial/endothelial electrical resistance measurements.

Transepithelial/endothelial electrical resistance (TEER) has been used since the 1980s to determine confluency and permeability of in vitro barrier model ...

Biology

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 ...

Developing a Triple Cell Culture Model of the Human Blood-Brain Barrier

Source: Fattakhov, N., et al. <a href="https://app.jove.com/v/64469">A triple primary cell culture model of the human blood-brain barrier for studying ischemic stroke in vitro.</a> J. Vis. Exp.&#160;(2022)
This video demonstrates the co-culturing of human brain pericyte, astrocyte, and endothelial cells. Each cell layer is formed separately and then cultured together, creating a three-layered system that mimics the cellular arrangement of the blood-brain barrier

1. Triple cell culture BBB model setting

	Seeding pericytes
	
		Cultivate HBVP (Human brain vascular pericytes) in T75 culture flasks with an activated ...

A Choroid Plexus Epithelial Cell-based Model of the Human Blood-Cerebrospinal Fluid Barrier to Study Bacterial Infection from the Basolateral Side

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