The authors acknowledge Dr. Maryam Hussain for previous work on this method, the group of PD Dr. Kerstin Danker (Charit&#233;-Universit&#228;tsmedizin, Berlin) for providing the THBMECs and Juliane Weber for critically reading the manuscript. This study was supported by the RTK 2155 (ProMoAge).

1. Preparation of Buffer and Reagents
<ol>
	<li>Prepare 10x phosphate buffer saline (10x PBS) by adding 80 g of sodium chloride (NaCl), 2 g of potassium chloride (KCl), 14.4 g of disodium-hydrogen-phosphate dihydrate (Na2HPO4 · 2H2O) and 2 g of potassium-dihydrogen phosphate (KH2PO4) in a 1 L glass flask in 1 L of double distilled H2O. Autoclave the 10x PBS solution and dilute 100 mL of this solution in 900 mL of double distilled water to get 1x PBS.
	<ol>
		<li>Use the autoclave to sterilize solutions. Put the glass flask into the basket, close the lid and sterilize it for 15 min at 121 °C and 98.9 kPa. 
		NOTE: This protocol is always used for autoclaving solutions in further steps.</li>
	</ol>
	</li>
	<li>Prepare a 10 µg/mL collagen IV and 10 µg/mL fibronectin solution by diluting 0.5 mg/mL fibronectin solution and the 0.3 mg/mL collagen IV solution each with 1x PBS to 100 mg/µL aliquots in 1.5 mL micro tubes. Thereafter, mix 100 µL of both aliquots with 1,800 µL of 1x PBS in 2 mL micro tubes and store them at -20 °C.</li>
	<li>Prepare DMEM/F-12 medium by adding 4% fetal bovine serum and 2 mM L-glutamine and 100 mg/L penicillin/streptomycin to the medium and store it at 4 °C.</li>
	<li>Prepare 1x trypsin-EDTA solution by diluting 5 mL of the 10x concentrated trypsin-EDTA solution with 45 mL of 1x PBS in a 50 mL tube and store it at 4 °C.</li>
	<li>Prepare 500 mL of LB medium by weighing 10 g of LB broth base in a 500 mL glass flask. Add 500 mL of sterilized water and autoclave it.</li>
	<li>Prepare LB agar by weighing 10 g of LB broth base and 7.5 g of agar-agar in a 500 mL glass flask. Add 500 mL of sterilized water before autoclaving and do not close the lid of the flask. Autoclave and leave the solution to cool down until it is warm to the touch.</li>
	<li>Prepare antibiotic-free medium by adding 4% fetal bovine serum and 2 mM L-glutamine, but no penicillin/streptomycin to the DMEM/F-12 medium and store it at 4 °C as in step 1.3.</li>
</ol>
2. Growth of the Blood-brain Barrier Mimicking Cells
<ol>
	<li>To assemble the 12 well plate, put the cell culture inserts into the wells (Figure 2A).
	<ol>
		<li>Unpack the plate and each insert in a biological safety cabinet and perform further steps there. Use sterilized forceps to grab the insert at its broad base to move it.</li>
	</ol>
	</li>
	<li>Coat the porous membrane of each insert with 90 µL of 10 µg/mL collagen IV and 10 µg/mL fibronectin mixture. Incubate the 12 well plate for 24 h at 37 °C in a cell culture incubator (Figure 2B).</li>
	<li>Wash the inserts twice by pipetting 1 mL of 1x PBS into each insert and aspirate the solution with a vacuum pump for cell cultures.</li>
	<li>Equilibrate the membranes by pipetting 0.5 mL of prewarmed DMEM/F-12 medium in the upper and 1.5 mL in the lower chamber. Incubate the plate for 30 min at 37 °C in a cell culture incubator with 5% CO2 atmosphere (Figure 2C).</li>
	<li>Seed 2 x 105 human microvascular endothelial cells into each upper chamber and incubate the 12 well plate at 37 °C in a cell culture incubator (Figure 2D).
	<ol>
		<li>Aspirate the medium from the cell culture flask using a vacuum pump for cell cultures and wash the monolayer by pipetting 10 mL of 1x PBS and aspirating the solution afterwards with a vacuum pump.</li>
		<li>Cover the cells completely with 1x concentrated trypsin-EDTA by pipetting 5 mL of the solution into the cell culture flask. Incubate the flask for 3-5 min at 37 °C in a cell culture incubator. 
		NOTE: If the cells are not dissociated, firmly tap the flask against the palm of the hand to dislodge the cells.</li>
		<li>Take 5 mL with a pipette as an aliquot from the cell suspension in a 15 mL tube and add 5 mL of the FCS-containing medium to stop the enzymatic reaction.</li>
		<li>Centrifuge the suspension for 3 min at 210 x g, remove the supernatant with a vacuum pump, and resuspend the pellet in 5 mL of medium with a pipette.</li>
		<li>Use the cell counter by mixing 10 µL of the cell suspension with 10 µL of 0.4% trypan blue stain in a 1.5 mL micro tube. Add 10 µL of the mixture into a counting chamber slide, put it into the cell counter, and start counting.
		<ol>
			<li>Focus the counter on the cells so that their edge is dark blue and the middle white. Thereafter, start the appropriate program for cell counting.</li>
		</ol>
		</li>
		<li>To calculate the volume for each insert, divide the obtained 2 x 105 cells per insert with the calculated concentration of the cell suspension.</li>
	</ol>
	</li>
</ol>
3. Cultivation of the Blood-brain Barrier Model
<ol>
	<li>Incubate the 12 well plate for 14 days at 37 °C.</li>
	<li>Change 0.5 mL of medium for the upper chamber and 1.5 mL of medium for the lower one every 2-3 days. Warm the medium before putting it in the cell flask. Aspirate with the vacuum pump (Figure 2E). 
	NOTE: Work carefully to avoid touching the membrane.</li>
	<li>Check the status of the cells by imaging them with a microscope and determine the confluency. Ensure that the confluency is 100% after 14 days.</li>
</ol>
4. Preparation of Bacteria
<ol>
	<li>A day before measurement, put a colony of the E. coli strain GM2163 into LB medium. Incubate the culture tube for 24 h at 37 °C with 180 rpm in an incubation shaker (Figure 2F).
	<ol>
		<li>Cultivate the E. coli strain on an LB agar plate at 4 °C. Take one colony with a sterilized pick and put the pick in the prepared culture tube with 3 mL of LB medium.</li>
		<li>Prepare an LB agar plate for each insert with warm LB agar solution and fill the Petri dishes to half their total volume. Let them become solid and store them at 4 °C.</li>
	</ol>
	</li>
</ol>
5. Treatment of Cells
<ol>
	<li>On day 14 after seeding, treat cells with compounds or measure the transendothelial electrical resistance (TEER), if planned (Figure 2G). 
	NOTE: Always have some untreated cells as a control.
	<ol>
		<li>To treat cells with the compound of interest, dilute the compound to the final concentration in DMEM/F-12 medium. Add 0.5 mL of this mixture into the upper chamber and 1.5 mL into the lower chamber. Incubate the plate in a cell culture incubator for the desired time.</li>
	</ol>
	</li>
	<li>Afterwards, exchange the complete medium with antibiotic-free medium by aspirating with the vacuum pump and pipetting (Figure 2H).</li>
</ol>
6. Measurement of Permeability
<ol>
	<li>To get constant concentrations of bacteria, measure the optical density with a photometer at a wavelength of 600 nm. Dilute the overnight solution of bacteria with LB medium in a 50 mL falcon tube to an OD600 of 0.5 ± 0.05. Work on ice.
	<ol>
		<li>Fill 1 mL of LB medium into a cuvette. Start the photometer and put the cuvette in, marked side forward. Press the bottom "Blank" for measuring the blank value afterwards.</li>
		<li>Measure the density of the bacterial solution by filling it into a cuvette, putting it in, and pressing the bottom sample. Repeat the measurement during the diluting until obtaining the final concentration.</li>
	</ol>
	</li>
	<li>Work in a biological safety cabinet where bacteria can be handled with the prepared 12 well plate and bacterial solution at OD600 = 0.5. Add 450 µL of bacterial solution only into each upper chamber containing 0.5 mL of medium (Figure 2I).</li>
	<li>Incubate the 12 well plate for 6 h at 37 °C in an incubator. 
	NOTE: The protocol dictates to pause here.</li>
	<li>Sample 50 µL of the medium with a pipette from each lower chamber by removing the insert with forceps (Figure 2J). Take care not to spill the medium from the upper chambers to the lower ones.</li>
	<li>Plate each sample on a separate agar-plate (Figure 2K). Drop the sample on the plate and streak out the solution with a cell spreader.</li>
	<li>Incubate the agar plates for 24 h at 37 °C in an incubator.</li>
	<li>Count the colonies in each plate (Figure 2L).</li>
</ol>
7. Analyzing Data
<ol>
	<li>Write the data in a table and calculate the average and standard deviation of the observed colonies of treated and untreated cells.</li>
	<li>Display the average as the absolute number of colonies.
	<ol>
		<li>To normalize the results, calculate the relative number of colonies by dividing all results with the control value.</li>
	</ol>
	</li>
</ol>
<img alt="Microbial culture process diagram; inoculation, incubation, extraction, and analysis steps shown." class="xfigimg" src="/files/ftp_upload/60692/60692fig2a.jpg" data-altupdatedat="1747929244000" data-alt="Solid state fermentation, diagram, inoculation and growth of microorganisms on substrate in bioprocess."> 
Figure 2: Detailed presentation of the individual steps in the protocol. (A) Put the inserts with sterilized forceps into the 12 well plate. (B) Coat each insert with 90 µL of fibronectin and collagen IV mixture and incubate for 24 h. (C) Equilibrate the membranes with prewarmed medium for 30 min. (D) Seed 2 x 105 human brain microvascular endothelial cells per insert. (E) Cultivate the plate for the appropriate amount of time. (F) One day before measuring, put an E. coli colony into a LB medium culture tube and incubate for 24 h. (G) Treat cells or measure TEER. (H) Exchange complete medium with antibiotic-free medium. (I) Add 450 µL of bacterial solution (OD600 = 0.5) into each upper chamber and incubate for 6 h. (J) Sample 50 µL of medium from each lower chamber removing insert with forceps. (K) Plate the sample on agar plates and incubate for 24 h. (L) Count the colonies and analyze the data. <a href="https://www.jove.com/files/ftp_upload/60692/60692fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

The authors have nothing to disclose.

Limited insight into the pathogenesis of microbial traversal limits further development of therapies for POD or meningitis. The mortality and morbidity of these diseases demand better patient treatment, require research of the underlying mechanisms, and need a robust platform for compound screening. The multifactorial events can be studied with human BMECs. Several successful reported isolation procedures of BMECs from a number of species have shown a loss of the cells&#39; characteristics molecular signature52,53. The described THBMECs in this procedure were transfected in very early passages, where they exhibited specific brain endothelial cell characteristics and preserved them43. This is important, because not all steps in the affected pathways have been discovered so far, and this model seems to mimic conventional BMECs. Our presented model shows direct influences on BMECs and the microbial traversal through the BBB.
The handling of THBMEC cells is straightforward, and the required technical equipment exists in most life science laboratories. Our model allows for an immediate start of investigative procedures after the THBMECs have built a tight monolayer. The field of applications can be extensive because of the possible combinations between new tests and conventional assays such as TEER measurement or labeling with tracers54. It is also possible to add astrocytes or pericytes to make a co- or triple-culture model. The influence of drugs on the microbial traversal could also be tested in our model by treating the THBMECs with compounds before inoculating the upper chamber with bacteria. In fact, it is possible to purchase inserts with filters for 96 well plates allowing the automation of the procedure. This can facilitate the implementation of high-throughput drug screening systems to accelerate the discovery of drugs against the mentioned diseases and to reduce side effects on the BBB during drug development.
A critical step in the presented method is the incubation time after adding the bacteria to the upper chamber. It is important to use hours as timelines in the protocol, because the generation time of E. coli is only 20 min55. Otherwise, use of different time points could lead to misleading results. There is also a possible risk of contamination between upper and lower chamber during the bacterial exposure if the plates are not handled with care. Any alterations to the 12 well plate at this point could contaminate the medium in the lower chamber.
E. coli is one well-known, very common cause of bacterial meningitis. Further investigations should test different bacteria that are also associated with meningitis, such as Neisseria meningitidis56 or Streptococcus pneumoniae57. These seem to use different mechanisms to cross the BBB and need to be better understood for the treatment of patients. In elderly patients, the incidence for POD increases26 as well as the number of occurring comorbidities. It is known that there are interactions between different diseases, especially systemic ones like diabetes. In our model, it is possible to simulate those conditions or treat the cells before adding the bacteria.
The model is limited by the direct contact of THBMECs and bacteria, and further research is necessary to investigate potential mechanisms of contact to detect the involved pathways and proteins. However, it is possible to remove inserts and harvest the cells for further analysis. The TEER of the model is lower in comparison to stem cell models38,39,40. We confirmed this by using a bacterial concentration that did not cross the BBB in untreated cells after 6 h.
In summary, this method represents a robust platform to analyze the traversal of bacteria through the BBB with the potential to expand it for high-throughput drug screenings.

The human BBB is a unique boundary of brain tissue, separating the brain from the blood. It strictly regulates the passage of larger and hydrophilic molecules, blocks paracellular diffusion, and maintains brain homeostasis. It also protects the brain from plasma fluctuations, toxins, microbes, and guides inflammatory cells as part of the central nervous system (CNS) immunity. Since its discovery a century ago1, many studies have been carried out to understand the structure and function of the BBB. The complex interactions of cells, proteins, and signals from brain and blood demand still further investigation and models.
The human BBB is composed of three cell types: brain microvascular endothelial cells (BMECs), pericytes, and astrocytes2,3. The BMECs differ from the majority of the endothelial cells in the body in that they possess a high number of tight junctions and adherens junctions4, low pinocytotic activity2,5, and a continuous basement membrane6,7 to block paracellular diffusion. Small lipophilic molecules can diffuse and pass the BBB following their concentration gradient; larger and hydrophilic molecules enter or leave the brain only through polarized expressed selective transport systems8. This regulation results in a high transendothelial electrical resistance (TEER) of 1,500-2,000 Ω·cm2 that is inversely correlated to permeability9,10. Although BMECs build a tight barrier, they can react to local and peripheral signals11,12. There is a close interaction between BMECs and astrocytes13; the astrocyte end-feet build a layer around the vessels and induce the formation of tight junctions13,14. They are involved in BBB maturation with different factors, including transforming growth factor-β (TGF-β)15,16. In addition, pericytes play a key role in the regulation of angiogenesis17 and preventing apoptosis of the endothelium in cellular differentiation18 (Figure 1). They are embedded in the basement membrane and provide structural stability of the vessel wall19.
<img alt="Blood-brain barrier diagram; shows endothelial cells, tight junctions, diffusion, and transport process." class="xfigimg" src="/files/ftp_upload/60692/60692fig1.jpg" data-altupdatedat="1747929244000" data-alt="Blood-brain barrier diagram showing cell layers, paracellular diffusion, and regulated transport."> 
Figure 1: Schematic structure of the blood-brain barrier. The unique structure of the human BBB is composed of three different cell types. The microvessel lumen is surrounded by endothelial cells, which are enriched in tight junctions, and are not fenestrated. They are embedded in the basement membrane, like the pericytes. These cells are important for structural stability of the vessel wall and play a role in the development of the BBB next to the astrocytes. Their end-feet build a close layer around the vessel and support the building of tight junctions. All components of the BBB are important for physiological functionality. <a href="https://www.jove.com/files/ftp_upload/60692/60692fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Many different pathologies are related to the collapse of the BBB (e.g., septic encephalopathy). The affected patients have increased protein levels in the cerebrospinal fluid20, and the brain parenchyma in affected rodents shows an increased uptake of marked colloidal iron oxide and amino acids21,22. These results point towards an increased permeability of the BBB that occurs alongside an increased pinocytosis in BMECs21 and endothelial activation23. Another associated pathology related to an altered BBB is meningitis, a medical emergency and a complex inflammation accompanied with cerebral edema that can lead to neuronal cell death. The primary entry site of circulating bacteria is supposed to be the microvessels24; however, the BBB prevents the entry of bacteria. The permeability of the BBB is not always linked to an increase in experimental hematogenous meningitis25 and the mechanisms can be multifactorial. Coincidence of sepsis with postoperative delirium (POD)26 and the association with preoperative infections27,28 indicates the need for a BBB model that enables the direct exposure to bacteria to get a better understanding into bacterial pathogenesis.
There are many gaps in understanding and quantifying the microbial traversal through the BBB. Therefore, we developed a model that allows a convenient testing of different factors and conditions with a direct correlation between bacterial traversal and influences on the permeability of the BBB. Previous work focused on the paracellular permeability and included TEER measurement and tracer flux. In addition, macromolecule transport was analyzed by conjugated molecules or antibodies, whereby different models using only endothelial cells or combinations with astrocytes and pericytes were developed. Due to the difficulty in obtaining human tissue on a regular basis, many animal-based models are used. Brain endothelial cells of bovine and porcine origin form tight monolayers with a high TEER that form well-shaped apical-basal polarity and are suited for investigations of small molecule transport through the BBB. The proteins differ in sequence from their human homologues29,30, making investigation of therapeutic antibodies difficult. For this reason, murine or human culture models may be preferable. Mouse or rats as sample sources have the advantage of being obtained from well characterized species but yield few cells for study purposes. This can be circumvented by the use of immortalized mouse brain endothelioma (END) cell lines bEND.3, bEND.5 or cEND31,32,33.
Primary cultured cells from human tissue are difficult to obtain and to handle on a regular basis. Therefore, most human cellular models used in research investigating the human BBB are immortalized endothelial cell lines. A published cell line is human cerebral microvascular endothelial cell line hCMEC/D3, which is well suited for studying drug uptake and is easy to handle. The cells build a monolayer and express the characteristic tight junction proteins of the BBB34, whereas the expression level of claudin-5 is reported to be lower than in intact microvessels35 and many specific transporters have been detected at transcript level36 as well as in proteomic studies34. A relatively low TEER in the range of 30-50 Ω·cm2 is still a challenge37. Another source for brain endothelial cells are human pluripotent stem cells (hPSCs)38 and human cord blood-derived stem cells of circulating endothelial progenitor and hematopoietic lineages39,40. Both protocols of differentiation result in tight cell monolayers and high TEER values (e.g., 1,450 Ω·cm2 in co-cultures)38. These stem cell models require extreme care for cultivation, yet offer the opportunity to study the influence of regulating hormones41 or diseases with genetic backgrounds42 on BBB development.
In this study, we established an immortalized transfected human brain microvascular endothelial cell line, THBMEC43, to mimic the BBB and to study bacterial traversal. Cells are seeded on a filter and grown to 100% confluency in this cell culture model. Bacteria are inoculated in the upper part of the cell culture chamber. We use Escherichia coli (E. coli) in our sample study because of the high incidence of E. coli meningitis44. It has been shown that the lowest permeability of cell monolayer occurs between day 13 and day 15 after seeding45. Therefore, treatment of the THBMEC monolayer is performed after this time and bacteria are inoculated afterwards in the medium on the apical surface of the monolayer. After an incubation time, bacteria that were able to cross the barrier are quantified via plating medium with the bacteria on agar plates and counting the colonies. An increased number of colonies correlates with higher bacterial traversal through the BBB. The TEER is about 70 Ω·cm2 46. However, it is not necessary to measure the TEER in the described method. Although it is a well-established value for the permeability of the BBB, it seems to have no impact on the traversal of bacteria through the BBB. Untreated cells serve as a control of tightness in our model. It has been shown in previous work that the cells are able to react to proinflammatory cytokines and express typical tight junction proteins47. This allows for compound screening and validation of a larger set of transporter substrates and receptors.

<table><tbody><tr><td>Agar - Agar</td><td>Carl Roth</td><td>6494.3</td><td>BioScience-Grade</td></tr><tr><td>Autoclave</td><td>Systec</td><td>VX-150</td><td></td></tr><tr><td>Bacteria &lt;em&gt;E.coli&lt;/em&gt; strain GM2163</td><td>Fermentas Life Sciences, Lithuania</td><td></td><td></td></tr><tr><td>Photometer</td><td>Eppendorf</td><td>6131</td><td></td></tr><tr><td>Cells THBMEC</td><td>Group of M. F. Stins</td><td></td><td></td></tr><tr><td>Cell culture flasks</td><td>Greiner Bio-One</td><td>658175</td><td></td></tr><tr><td>Centrifuge Universal 320</td><td>Hettrichlab</td><td>1401</td><td></td></tr><tr><td>Collagen IV</td><td>SIGMA Aldrich</td><td>C6745</td><td>from human cell culture</td></tr><tr><td>Countess Automated Cell Counter with cell counting chamber slides and Trypan Blue stain 0.4%</td><td>Invitrogen</td><td>C10311</td><td></td></tr><tr><td>Culture tubes</td><td>Greiner Bio-One</td><td>191180</td><td></td></tr><tr><td>Cuvettes</td><td>BRAND</td><td>759015</td><td></td></tr><tr><td>Di sodium hydrogen phosphate di hydrate</td><td>MERCK</td><td>1065800500</td><td></td></tr><tr><td>DMEM/F-12</td><td>GIBCO/ Thermo Sc.</td><td>11330032</td><td>HEPES</td></tr><tr><td>Falcon tubes 15 ml</td><td>Greiner Bio-One</td><td>188271</td><td></td></tr><tr><td>Falcon tubes 50 ml</td><td>Greiner Bio-One</td><td>227261</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>GIBCO/ Thermo Sc.</td><td>10270</td><td>value FBS -Brazil</td></tr><tr><td>Fibronectin</td><td>SIGMA Aldrich</td><td>F0556</td><td>solution human fibroblasts</td></tr><tr><td>Heracell 150 CO2 Incubator</td><td>Heraeus</td><td>50116047</td><td></td></tr><tr><td>Incubator shaker I 26 New Brunswick</td><td>Eppendorf</td><td>M1324-0000</td><td></td></tr><tr><td>Inoculation loop</td><td>Dr. Ilona Schubert - Laborfachhandel</td><td>641000</td><td></td></tr><tr><td>LB Broth Base</td><td>GIBCO/ Thermo Sc.</td><td>12780029</td><td></td></tr><tr><td>L-Glutamine</td><td>GIBCO/ Thermo Sc.</td><td>25030-081</td><td></td></tr><tr><td>Microbial incubator B 6200</td><td>Heraeus</td><td>51015192</td><td></td></tr><tr><td>Microbiological Safety Cabinet AURA 2000 M.A.C. Class II</td><td>BIOAIR</td><td>12469</td><td></td></tr><tr><td>Microscope inverse</td><td>Zeiss</td><td>TELAVAL 31</td><td></td></tr><tr><td>Micro tubes 2 ml</td><td>Sarstedt</td><td>72,695,400</td><td></td></tr><tr><td>Micro tubes 1,5 ml</td><td>Sarstedt</td><td>72,706,400</td><td></td></tr><tr><td>Penicillin / Streptomycin</td><td>GIBCO/ Thermo Sc.</td><td>15140122</td><td></td></tr><tr><td>Petri dish</td><td>Dr. Ilona Schubert - Laborfachhandel</td><td>464-800</td><td></td></tr><tr><td>Potassium chloride</td><td>Roth</td><td>HN02.3</td><td></td></tr><tr><td>Potassium-di-hydrogen phosphate</td><td>Roth</td><td>P018.2</td><td></td></tr><tr><td>Sodium chloride</td><td>Roth</td><td>9265.2</td><td></td></tr><tr><td>ThinCerts + Multiwell Plates</td><td>Greiner Bio-One</td><td>665631</td><td>12 well, pore size 3.0 &amp;micro;m</td></tr><tr><td>Trypsin - EDTA</td><td>GIBCO/ Thermo Sc.</td><td>15400054</td><td></td></tr><tr><td>Vacuumpump Laboport</td><td>KNF</td><td>N 86 KT.18</td><td></td></tr></tbody></table>

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.

Following the protocol, the cells were seeded, and the BBB model was built. At day 14 after seeding, the cells were treated with glyoxal as a reactive aldehyde. The aim of the experiment was to investigate the correlation between age and diabetes in POD27 and the high incidence of meningitis in elderly patients48. The increased levels of advanced glycation end products (AGEs) in both age and diabetes49 demand further examination of the effect of glycation in the pathogenesis of microbial traversal through the BBB. Glycation is a non-enzymatic reaction of free amino groups in proteins with carbonyl groups of reducing carbohydrates or other carbonyl compounds. Glucose is well known as donor of carbonyl groups; however, there are more reactive ones known. After building an unstable Schiff's base, they rearrange to more stable and reactive dicarbonyl compounds like glyoxal. AGEs, the final products, can cause crosslinks between proteins50. They can damage cellular structures and alter cellular function by interaction with the receptor of AGEs (RAGE)51.
Cells were treated with a 0.05 and 0.15 mM glyoxal (GO) solution for 1 h, and untreated cells served as a control. Glycation was detected via immunoblotting and detection with an anti-AGE antibody (Figure 3). The obtained bacterial colonies were counted and represented as the absolute number of colonies (Figure 4A) or the relative number of colonies normalized to the control (Figure 4B). The medium taken from wells with the untreated cells formed very few colonies. This result demonstrated that the untreated cells were able to build a barrier and could serve as a control. Samples treated with glyoxal displayed an increased number of colonies, leading to the conclusion that there is an effect of glyoxal on the THBMECs and the cellular barrier density, because the number of colonies demonstrated a significant difference between untreated and treated cells. The increased bacterial crossing of the barrier after the treatment with glyoxal could explain why diabetes is correlated to diseases with a BBB breakdown.
<img alt="SDS-PAGE gel electrophoresis showing protein bands; anti-CML antibody, Ponceau S staining, THBMEC." class="xfigimg" src="/files/ftp_upload/60692/60692fig3a.jpg" data-altupdatedat="1747929244000" data-alt="Western blot result; anti-CML antibody, protein detection. Comparative lanes with Ponceau S stain."> 
Figure 3: Detection of protein glycation via immunoblotting. THBMECs were treated with GO at different concentrations for 1 h. Total protein was isolated and separated using SDS-PAGE. Glycation of the proteins was detected via immunoblotting using anti-AGE-antibody (CML-26). <a href="https://www.jove.com/files/ftp_upload/60692/60692fig3alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
In a different setting, protein glycation of THBMECs was induced by glucose. Sterilized glucose was added to the DMEM/F-12 medium to increase glucose concentration from normal glucose medium (NG) with 17.5 mM to high glucose medium (HG) with 42.5 mM. THBMECs were cultivated in two different cell culture flasks: one in normal glucose (NG) medium and the other in high glucose (HG) medium. These two different media were also used for growth of the BBB on filters in 12 well plates. Cells cultivated in NG medium served as a control. The obtained colonies are represented as the absolute number of colonies (Figure 4C) or the relative number of colonies normalized to the control (Figure 4D). The results indicate no significant effect on the traversal of bacteria through the human BBB, leading to the conclusion that the effect of NG vs. HG was not severe enough to affect the integrity of the BBB. The different scenarios were designed to prove the model and the integrity of the cells mimicking the BBB.
<img alt="Colony growth comparison bar charts; glyoxal, glucose effects on absolute, relative numbers." class="xfigimg" src="/files/ftp_upload/60692/60692fig4d.jpg" data-altupdatedat="1747929244000" data-alt="Bar charts comparing colony numbers, glyoxal treatment, glucose conditions; statistical analysis shown."> 
Figure 4: Absolute and relative numbers of counted bacterial colonies in the BBB model with THBMECs. THBMECs were treated with 0.15 mM GO for 1 h, untreated cells served as a control. A total of 450 µL of E. coli suspension (OD600 = 0.5) was added to each upper chamber. Medium from the lower chambers was plated on agar plates after 6 h. (A) The graph shows the average mean +/- SEM of counted colonies. (B) The graph shows the counted colonies normalized to the untreated cells as control +/- SEM (n = 4). In (C) and (D), THBMECs were cultivated in NG and HG medium. A total of 450 µL of E. coli suspension (OD600 = 0.5) was added to each upper chamber. Medium from the lower chamber was plated on agar plates after 6 h. (C) The graph shows the average mean +/- SEM of counted colonies. (D) The graph shows the counted colonies normalized to the untreated cells as control +/- SEM (n = 3). <a href="https://www.jove.com/files/ftp_upload/60692/60692fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

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 ...

analyzing-permeability-blood-brain-barrier-microbial-traversal

Research

JoVE Journal

Immunology and Infection

15.3K Views.  Martin Luther University Halle-Wittenberg. Deze methode maakt het mogelijk om de menselijke bloed-hersenbarrière te analyseren door het meten van bacteriële traversal op verschillende behandelingen. Het kan helpen om de pathogenese van bacteriële meningitis en postoperatieve delirium te ontdekken. Het is een eenvoudige methode en de effecten van de behandeling worden direct vertegenwoordigd door het aantal bacteriën.Het kan worden gewijzigd om een hoge doorvoeranalyse van verbindingen op de endotheelcellen mogelijk te maken...

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

Isolation of Primary Murine Brain Microvascular Endothelial Cells

The blood-brain-barrier is ultrastructurally assembled by a monolayer of brain microvascular endothelial cells (BMEC) interconnected by a junctional complex of tight and adherens junctions. Together with other cell-types such as astrocytes or pericytes, they form the neurovascular unit (NVU), which specifically regulates the interchange of fluids, molecules and cells between the peripheral blood and the CNS. Through this complex and dynamic system BMECs are involved in various processes maintaining the homeostasis of the CNS. A dysfunction of the BBB is observed as an essential step in the pathogenesis of many severe CNS diseases. However, specific and targeted therapies are very limited, as the underlying mechanisms are still far from being understood. 
Animal and in vitro models have been extensively used to gain in-depth understanding of complex physiological and pathophysiological processes. By reduction and simplification it is possible to focus the investigation on the subject of interest and to exclude a variety of confounding factors. However, comparability and transferability are also reduced in model systems, which have to be taken into account for evaluation. The most common animal models are based on mice, among other reasons, mainly due to the constantly increasing possibilities of methodology. In vitro studies of isolated murine BMECs might enable an in-depth analysis of their properties and of the blood-brain-barrier under physiological and pathophysiological conditions. Further insights into the complex mechanisms at the BBB potentially provide the basis for new therapeutic strategies.
This protocol describes a method to isolate primary murine microvascular endothelial cells by a sequence of physical and chemical purification steps. Special considerations for purity and cultivation of MBMECs as well as quality control, potential applications and limitations are discussed.

Brain microvascular endothelial cells (BMEC) are interconnected by specific junctional proteins forming a highly regulated barrier separating blood and the central nervous system (CNS), the so-called blood-brain-barrier (BBB). The isolation of primary murine brain microvascular endothelial cells, as discussed in this protocol, enables detailed in vitro studies of the BBB.

The blood-brain-barrier is ultrastructurally assembled by a monolayer of brain microvascular endothelial cells (BMEC) interconnected by a junctional ...

Neuroscience

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 ...

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 ...

Assessment of Blood-brain Barrier Permeability by Intravenous Infusion of FITC-labeled Albumin in a Mouse Model of Neurodegenerative Disease

Disruption of blood-brain barrier (BBB) integrity is a common feature for different neurological and neurodegenerative diseases. Although the interplay between perturbed BBB homeostasis and the pathogenesis of brain disorders needs further investigation, the development and validation of a reliable procedure to accurately detect BBB alterations may be crucial and represent a useful tool for potentially predicting disease progression and developing targeted therapeutic strategies.
Here, we present an easy and efficient procedure for evaluating BBB leakage in a neurodegenerative condition like that occurring in a preclinical mouse model of Huntington disease, in which defects in the permeability of BBB are clearly detectable precociously in the disease. Specifically, the high molecular weight fluorescein isothiocyanate labelled (FITC)-albumin, which is able to cross the BBB only when the latter is impaired, is acutely infused into a mouse jugular vein and its distribution in the vascular or parenchymal districts is then determined by fluorescence microscopy.
Accumulation of green fluorescent-albumin in the brain parenchyma functions as an index of aberrant BBB permeability and, when quantitated by using Image J processing software, is reported as Green Fluorescence Intensity.

In this study, we present an easy and efficient procedure for evaluating the disruption of the blood-brain barrier under neurodegenerative conditions. To achieve our objective, we infused high molecular weight fluorescein isothiocyanate labelled (FITC)-albumin into mouse jugular vein and evaluated its leakage into the brain parenchyma by fluorescence microscopy.

Disruption of blood-brain barrier (BBB) integrity is a common feature for different neurological and neurodegenerative diseases. Although the interplay ...

An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

Blood-brain barrier (BBB) is a specialized barrier that protects the brain microenvironment from toxins and pathogens in the circulation and maintains brain homeostasis. The principal sites of the barrier are endothelial cells of the brain capillaries whose barrier function results from tight intercellular junctions and efflux transporters expressed on the plasma membrane. This function is regulated by pericytes and astrocytes that together form the neurovascular unit (NVU). Several neurological diseases such as stroke, Alzheimer&#39;s disease (AD), brain tumors are associated with an impaired BBB function. Assessment of the BBB permeability is therefore crucial in evaluating the severity of the neurological disease and the success of the treatment strategies employed.
We present here a simple yet robust permeability assay that have been successfully applied to several mouse models both, genetic and experimental. The method is highly quantitative and objective in comparison to the tracer fluorescence analysis by microscopy that is commonly applied. In this method, mice are injected intraperitoneally with a mix of aqueous inert fluorescent tracers followed by anesthetizing the mice. Cardiac perfusion of the animals is performed prior to harvesting brain, kidneys or other organs. Organs are homogenized and centrifuged followed by fluorescence measurement from the supernatant. Blood drawn from the cardiac puncture just before perfusion serves for normalization purpose to the vascular compartment. The tissue fluorescence is normalized to the wet weight and serum fluorescence to obtain a quantitative tracer permeability index. For additional confirmation, the contralateral hemi-brain preserved for immunohistochemistry can be utilized for tracer fluorescence visualization purposes.

An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

Here we present a mouse brain vascular permeability assay using intraperitoneal injection of fluorescent tracers followed by perfusion that is applicable to animal models of blood-brain barrier dysfunction. One hemi-brain is used for assessing permeability quantitatively and the other for tracer visualization/immunostaining. The procedure takes 5 - 6 h for 10 mice.

Blood-brain barrier (BBB) is a specialized barrier that protects the brain microenvironment from toxins and pathogens in the circulation and maintains ...

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) ...

Medicine

A Simple and Reproducible Method to Prepare Membrane Samples from Freshly Isolated Rat Brain Microvessels

The blood-brain barrier (BBB) is a dynamic barrier tissue that responds to various pathophysiological and pharmacological stimuli. Such changes resulting from these stimuli can greatly modulate drug delivery to the brain and, by extension, cause considerable challenges in the treatment of central nervous system (CNS) diseases. Many BBB changes that affect pharmacotherapy, involve proteins that are localized and expressed at the level of endothelial cells. Indeed, such knowledge on BBB physiology in health and disease has sparked considerable interest in the study of these membrane proteins. From a basic science research standpoint, this implies a requirement for a simple but robust and reproducible method for isolation of microvessels from brain tissue harvested from experimental animals. In order to prepare membrane samples from freshly isolated microvessels, it is essential that sample preparations be enriched in endothelial cells but limited in the presence of other cell types of the neurovascular unit (i.e., astrocytes, microglia, neurons, pericytes). An added benefit is the ability to prepare samples from individual animals in order to capture the true variability of protein expression in an experimental population. In this manuscript, details regarding a method that is utilized for isolation of rat brain microvessels and preparation of membrane samples are provided. Microvessel enrichment, from samples derived, is achieved by using four centrifugation steps where dextran is included in the sample buffer. This protocol can easily be adapted by other laboratories for their own specific applications. Samples generated from this protocol have been shown to yield robust experimental data from protein analysis experiments that can greatly aid the understanding of BBB responses to physiological, pathophysiological, and pharmacological stimuli.

Here, a method for isolation of rat brain microvessels and for the preparation of membrane samples is described. This protocol has the clear advantage of producing enriched microvessel samples with acceptable protein yield from individual animals. Samples can then be used for robust protein analyses at the brain microvascular endothelium.

The blood-brain barrier (BBB) is a dynamic barrier tissue that responds to various pathophysiological and pharmacological stimuli. Such changes resulting ...

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 ...

Derivation, Expansion, Cryopreservation and Characterization of Brain Microvascular Endothelial Cells from Human Induced Pluripotent Stem Cells

Brain microvascular endothelial cells (BMECs) can be differentiated from human induced pluripotent stem cells (iPSCs) to develop ex vivo cellular models for studying blood-brain barrier (BBB) function. This modified protocol provides detailed steps to derive, expand, and cryopreserve BMECs from human iPSCs using a different donor and reagents than those reported in previous protocols. iPSCs are treated with essential 6 medium for 4 days, followed by 2 days of human endothelial serum-free culture medium supplemented with basic fibroblast growth factor, retinoic acid, and B27 supplement. At day 6, cells are sub-cultured onto a collagen/fibronectin matrix for 2 days. Immunocytochemistry is performed at day 8 for BMEC marker analysis using CLDN5, OCLN, TJP1, PECAM1, and SLC2A1. Western blotting is performed to confirm BMEC marker expression, and absence of SOX17, an endodermal marker. Angiogenic potential is demonstrated with a sprouting assay. Trans-endothelial electrical resistance (TEER) is measured using chopstick electrodes and voltohmmeter starting at day 7. Efflux transporter activity for ATP binding cassette subfamily B member 1 and ATP binding cassette subfamily C member 1 is measured using a multi-plate reader at day 8. Successful derivation of BMECs is confirmed by the presence of relevant cell markers, low levels of SOX17, angiogenic potential, transporter activity, and TEER values ~2000 &#937; x cm2. BMECs are expanded until day 10 before passaging onto freshly coated collagen/fibronectin plates or cryopreserved. This protocol demonstrates that iPSC-derived BMECs can be expanded and passaged at least once. However, lower TEER values and poorer localization of BMEC markers was observed after cryopreservation. BMECs can be utilized in co-culture experiments with other cell types (neurons, glia, pericytes), in three-dimensional brain models (organ-chip and hydrogel), for vascularization of brain organoids, and for studying BBB dysfunction in neuropsychiatric disorders.

This protocol details an adapted method to derive, expand, and cryopreserve brain microvascular endothelial cells obtained by differentiating human induced pluripotent stem cells, and to study blood brain barrier properties in an ex vivo model.

Brain microvascular endothelial cells (BMECs) can be differentiated from human induced pluripotent stem cells (iPSCs) to develop ex vivo cellular models ...

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 ...

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

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