This work was supported by the National Institutes of Health (NIH) grants MH128022, MH122235, MH072567, MH122235, HL126559, DA044579, DA039576, DA040537, DA050528, and DA047157.

NOTE: See the Table of Materials for details related to all cells, materials, equipment, and solutions used in this protocol.
1. Triple cell culture BBB model setting
<ol>
	<li>Seeding pericytes
	<ol>
		<li>Cultivate HBVP in T75 culture flasks with an activated surface for cell adhesion within a 5% CO2 incubator at 37 &#176;C until confluent. Once confluence is reached, aspirate the old pericyte medium and wash the cells with 5 mL of warm Dulbecco&#39;s phosphate-buffered saline (DPBS). Aspirate the DPBS and detach the cells from the flask using a combination of 4 mL of warm trypsin-EDTA solution and 1 mL of DPBS. 
		NOTE: Avoid using passages later than P7.</li>
		<li>Incubate the flask for 5 min at 37 &#176;C in a CO2 incubator. View under a microscope to confirm whether the cells are detached from the flask. Add 5 mL of warm pericyte medium (containing 2% fetal bovine serum [FBS]) to the flask and transfer the detached cells to a 50 mL centrifuge tube.</li>
		<li>Centrifuge the cell suspension for 3 min at 200 &#215; g, allowing the cells to form a pellet in the bottom of the tube. Aspirate the medium from the tube, ensuring the cell pellet remains intact.</li>
		<li>Resuspend the cell pellet in pericyte medium; calculate the amount of medium depending on the confluence of the cells and the number of well-inserts needed. Take 10 &#181;L of the resuspended cells, place them into a cell counting slide, and count the number of cells.</li>
		<li>Determine the cell density and seed 300,000 cells/insert in 1 mL of pericyte medium onto the abluminal side of the well-inserts (6-well format). 
		NOTE: When seeding HBVP on the underside of inserts, it is very important to first flip the well-insert plates upside down. While the plate is laid flat on a surface, remove the bottom section to expose the abluminal side of the well-inserts. After adding pericyte cell suspension onto the abluminal side, cover the well-inserts with the flipped plate to prevent evaporation. Keep all plates turned upside down in a 5% CO2 incubator at 37 &#176;C overnight.</li>
	</ol>
	</li>
	<li>Seeding astrocytes
	<ol>
		<li>Cultivate HA in T75 flasks within a 5% CO2 incubator at 37 &#176;C until confluence is reached. Follow the above steps 1.1.1-1.1.4 using astrocyte medium (also containing 2% FBS) instead of pericyte medium. 
		NOTE: Avoid using passages later than P9.</li>
		<li>Determine the cell density and seed 300,000 cells/well onto the bottom of the tissue culture 6-well plates. Cover the plate to prevent evaporation and keep all plates in a 5% CO2 incubator at 37 &#176;C overnight.</li>
	</ol>
	</li>
	<li>Seeding endothelial cells
	<ol>
		<li>Cultivate HBMEC in tissue culture dishes within a 5% CO2 incubator at 37 &#176;C until confluent. Follow the above steps 1.1.1-1.1.4 using complete classic medium (containing 10% FBS) instead of pericyte medium. 
		&#8203;NOTE: Avoid using passages later than P12.</li>
		<li>Take out the tissue culture 6-well plates containing astrocytes and the well-inserts (6-well format) containing pericytes from the 5% CO2 incubator at 37 &#176;C. Aspirate the astrocyte medium from the tissue culture 6-well plates. Add 1 mL of pericyte medium and 1 mL of astrocyte medium to each well.</li>
		<li>Aspirate the pericyte medium from the well-inserts and place them into the tissue culture 6-well plates containing the seeded astrocytes. Seed HBMEC at a density of 300,000 cells/well in 2 mL of complete classic medium onto the apical side of the same well-inserts. 
		NOTE: The endothelial cells should be seeded on the apical side of the well-inserts on the next day after seeding pericytes on the abluminal side of the well-inserts and astrocytes on tissue culture 6-well plates. Cells should be maintained in triple culture for 6 days to induce BBB-like properties. The cell culture medium should be changed in both well-insert compartments 24 h prior to experiments.</li>
	</ol>
	</li>
</ol>
2. Induction of the oxygen-glucose deprivation
<ol>
	<li>Wash the cells 3x with DPBS. For triple cell cultures subjected to OGD, add glucose-free medium (without L-glutamine and phenol red) to both the apical and basolateral compartments. Replace the culture medium with fresh medium in normoxic control cell cultures. Place control triple cultures in the 5% CO2 incubator at 37 &#176;C. 
	NOTE: Medium changes prior to the induction of OGD or immediately after OGD might create mechanical stress that would further impact the endothelial cell monolayers. Thus, the steps with medium replacement are included for normoxic control cell cultures.</li>
	<li>Place a Petri dish containing 20 mL of sterile water in the hypoxia incubator chamber to provide adequate humidification of the cultures. Open the chamber by releasing the ring clamp. Arrange the cell cultures on the shelves. Seal the chamber by securing the ring clamp.</li>
	<li>Open both the inlet and outlet ports of the chamber. Attach the tubing coming from the top of the flow meter to the chamber. Attach the tubing coming from the bottom of the flow meter to the gas tank containing the gas mixture of 95% N2/5% CO2&#160;via an air filter.</li>
	<li>Open the tank flow control valve by turning it counterclockwise to allow minimum gas flow. Slowly open the pressure regulator valve by turning clockwise.</li>
	<li>Flush the chamber with the gas mixture at a flow rate of 20 L/min for 5 min. Disconnect the chamber from the gas source and firmly close both white plastic clamps.</li>
	<li>Turn off the tank flow control valve by turning clockwise. Close the pressure regulator valve by turning counterclockwise.</li>
	<li>Place the hypoxia chamber in a conventional incubator at 37 &#176;C for 4 h. 
	&#8203;NOTE: For adding subsequent reoxygenation period, wash the cells 3x with DPBS, add fresh medium in all triple cultures, and keep for an additional 24 h in the 5% CO2 incubator at 37&#176;C. According to the manufacturer&#39;s instructions, the oxygen concentration remaining in the chamber is 0% after no less than 4 min of purging with anaerobic gas mixture at 20 L/min.</li>
</ol>
3. TEER measurements
<ol>
	<li>Place the sterilized TEER instrument into the biosafety cabinet and plug the electrodes into the epithelial voltohmmeter. Sterilize the electrodes in 30 mL of 70% isopropyl alcohol solution for a minimum of 30 min.</li>
	<li>Turn on the TEER instrument and set the function to ohms.</li>
	<li>Remove the electrodes from the 70% isopropyl alcohol solution and place them in 20 mL of DPBS for a minimum of 30 min until the digital readout on the TEER device reads 0 ohms.</li>
	<li>Insert the long prong of the electrode through one of the three openings in the well-insert hanger of the blank well-insert control, lowering it until it touches the bottom of the well. Ensure that the short prong is resting above the apical culture on the bottom of the well-insert. 
	NOTE: The blank well-insert control is comprised of 2 mL of complete classic medium in the apical compartment and a combination of 1 mL of pericyte medium and 1 mL of astrocyte medium in the basolateral compartment. Ascertain that the electrodes are held at a 90&#176; to the well-insert while taking the TEER measurement. Using the average of two or three readings obtained in the same well (per opening) can help to decrease the variability.</li>
	<li>Wait until the digital readout values on the TEER instrument level off before recording the value. Place the electrodes back into DPBS to wash them between measurements. Continue to collect all the TEER measurements for two more blank well-insert controls.</li>
	<li>Collect the TEER measurements of the sample plates using steps 3.4-3.5 taken for the control measurements. Once all measurements have been taken, place the electrode back into the 70% isopropyl alcohol solution for 30 min. Disconnect the electrodes from the TEER instrument and allow them to air-dry.</li>
	<li>Calculate the TEER values. Use equation (1) to subtract the mean ohm value of the blank well-insert control from the ohm value of the sample, and then multiply this resistance value by the area of the membrane insert (cm2) to give the reported TEER value in &#937;&#8729;cm2. 
	<img alt="Equation 1" src="/files/ftp_upload/64469/64469eq01.jpg" style="margin: auto;" /> (1)</li>
</ol>
4. Assessment of the BBB paracellular permeability
NOTE: Perform all steps involving FITC-dextran in a cell culture biosafety cabinet with the lights turned off. Cover the FITC-dextran solutions with aluminum foil to minimize photobleaching.
<ol>
	<li>Prepare solutions containing FITC-dextrans of 20 and 70 kDa (0.1 mg/mL) using phenol red-free endothelial cell growth medium and allow to stir on a rocking platform shaker for 1 h. Filter the solutions using a 0.22 &#181;m syringe filter.</li>
	<li>Aspirate the medium from the basolateral compartment and replace it with 2 mL of phenol red-free endothelial cell growth medium in the triple cell culture BBB model. Wash the cells in the apical compartment twice with Hanks&#39; balanced salt solution (HBSS).</li>
	<li>Add 1 mL of the FITC-dextran solution in the apical compartment and cover the plate with aluminum foil. Place the plate into a 5% CO2 incubator at 37 &#176;C for 1 h.</li>
	<li>Take 100 &#181;L of medium from the basolateral compartments and transfer it into a black 96-well plate. Measure the fluorescence using a microplate reader with the excitation and emission wavelengths set to 480 nm and 530 nm, respectively.</li>
</ol>
5. Detection of hypoxia in live cells
<ol>
	<li>Seed 200 &#181;L of HBMEC, HA, and HBVP in the center of&#160;poly-d-lysine-coated, 35 mm glass bottom dishes at a density of 150,000 cells/dish. Before seeding HBMEC, coat&#160;the bottom of the dishes with the attachment factor. Allow the cells to attach to the glass surface by leaving them overnight at 37 &#176;C in a CO2 incubator. 
	NOTE:&#160;It is important to place dishes with human primary cells at the same time with a triple well-insert BBB model&#160;in a hypoxia chamber for OGD.</li>
	<li>Once confluence is reached, discard the culture medium and add 2 mL of prewarmed glucose-free medium (for OGD) or normal medium (for controls) containing 2 &#181;L of 1 mM&#160;Hoechst 33342 (final concentration 0.2 &#181;M) and 0.5 &#181;L of 5 mM stock solution of the referenced Image-iT green hypoxia reagent&#160;(final concentration 1 &#181;M). After OGD treatment, replace the medium with imaging optimized medium in all dishes.</li>
	<li>Perform fluorescence live-cell imaging with the GFP filter and top-stage confocal microscope incubator as described previously14,15.</li>
</ol>

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All the authors disclosed that there are no conflicts of interest.

In this protocol, we describe a method to set up a reliable triple endothelial cell-pericyte-astrocyte culture BBB model for&#160;studying BBB dysfunction in the setting of ischemic stroke in vitro. Considering that pericytes are the nearest neighbors of endothelial cells in vivo, HBVP are plated on the underside of the well-inserts in this model16. Although this configuration lacks the direct cell-to-cell communication between astrocytes and endothelial cells, this arrangement allows for indirect cell-to-cell communication between cell types via secreted soluble factors. The presence of astrocytes and pericytes in this system greatly improves HBMEC integrity, limiting paracellular permeability of the monolayer to small size tracers. Additionally, astrocytes and pericytes have a protective effect on HBMEC in OGD conditions.
In the developed protocol, we optimized the cell ratio (1:1:1) and culture medium mixture for the basolateral compartment. The triple BBB model displayed almost&#160;2.5x higher TEER&#160;values (239 &#177; 9 &#937;&#183;cm2) than the control HBMEC monoculture (112 &#177; 11 &#937;&#183;cm2) after 6 days of co-culturing, the time necessary for the proper HBMEC monolayer formation17,18.&#160;The TEER values achieved in this triple model are comparable to those in the other previously described triple model, in which&#160;HA were in contact with HBMEC and HBVP were seeded on the bottom of the plate wells19. In the other human primary cell triple culture model with the same cell configuration and OGD exposure time, the baseline TEER values were substantially lower than those in this work&#160;and did not reach the standard levels (100 &#937;&#183;cm2)20. The use of 6-well well-insert plates with larger compartments allowed us to decrease the medium change frequency to avoid the disruption of substance exchange between the cell populations.
OGD experiments were performed using a sealed hypoxia incubator chamber containing an anaerobic gas mixture (95% nitrogen and 5% carbon dioxide). One of the main advantages of this protocol is that the severity of the ischemic injury can be easily adjusted to specific needs by varying the length of the OGD period. After evaluating the TEER and dextran permeability, 4 h of OGD did not compromise BBB integrity in the constructed triple model. Therefore, to study ischemic stroke in vitro, longer exposure to OGD needs to be applied to induce BBB breakdown. Live-cell imaging, provided in the protocol, allows real-time monitoring of different cell types and verification of OGD injury, which represents an advantage in many studies.
In this protocol, 0.4 &#181;m polyester well-inserts were chosen as a basis for this triple BBB model. A polyester membrane is considered the best insert type for cell visualization in fluorescence-imaging applications, which provides an excellent opportunity to visualize modifications of tight junction proteins and transporters if needed. While the selected small pore diameter creates a low permeability to small hydrophilic molecules across endothelial monolayers, it also limits the potential application of the present triple BBB model for transendothelial migration assays, in which 3 &#181;m or 8 &#181;m pore sizes are required.
Finally, another limitation of this model can be related to the lack of shear stress, which has been shown to promote the formation of tight junctions in dynamic triple BBB in vitro systems21. In conclusion, the established robust and physiologically relevant triple culture BBB model, composed of primary human cells, represents a valuable tool for drug screening and studying BBB dysfunction associated with ischemic stroke.

Stroke is one of the leading causes of death and long-term disability worldwide1. The incidence of stroke rapidly increases with age, doubling every 10 years after the age of 552. Ischemic stroke occurs as a result of cerebral blood flow disruption due to thrombotic and embolic events, which encompasses more than 80% of all stroke cases3. Even now, there are relatively few treatment options available to minimize tissue death following ischemic stroke. The treatments that do exist are time-sensitive and consequentially do not always lead to good clinical outcomes. Therefore, research on complex cellular mechanisms of ischemic stroke that affect poststroke recovery is urgently needed.
The BBB is a dynamic interface for the exchange of molecules between the blood and the brain parenchyma. Structurally, the BBB is comprised of brain microvascular endothelial cells interconnected by junctional complexes surrounded by a basement membrane, pericytes, and astrocytic endfeet4. Pericytes and astrocytes play an essential role in the maintenance of BBB integrity through the secretion of various factors necessary for the formation of strong, tight junctions5,6. The breakdown of the BBB is one of the hallmarks of ischemic stroke. Acute inflammatory response and oxidative stress associated with cerebral ischemia results in the disruption of tight junction protein complexes and dysregulated crosstalk between astrocytes, pericytes, and endothelial cells, which leads to increased paracellular solute permeability across the BBB7. BBB dysfunction further promotes the formation of brain edema and increases the risk of hemorrhagic transformation8. Considering all of the above, there is great interest in understanding the molecular and cellular changes that occur at the BBB level during and after ischemic stroke.
Although many in vitro BBB models have been developed over recent decades and used in a variety of studies, none of them can fully replicate in vivo conditions9. While some models are based on endothelial cell monolayers cultured on well-insert permeable supports alone or in combination with pericytes or astrocytes, only more recent studies have introduced triple cell culture model designs. Almost all existing triple culture BBB models incorporate primary brain endothelial cells along with astrocytes and pericytes isolated from animal species or cells derived from human pluripotent stem cells10,11,12,13.
Recognizing the need to better recapitulate the human BBB in vitro, we established a triple cell culture in vitro BBB model composed of human brain microvascular endothelial cells (HBMEC), primary human astrocytes (HA), and primary human brain vascular pericytes (HBVP). This triple culture BBB model is set up on 6-well plate, polyester membrane inserts with 0.4 &#181;m pore size. These well-inserts provide an optimal environment for cell attachment and enable easy access to both apical (blood) and basolateral (brain) compartments for medium sampling or compound application. The features of this proposed triple cell culture BBB model are assessed by measuring TEER and paracellular flux post OGD mimicking ischemic stroke in vitro, with a shortage of oxygen (&#60;1% O2) and nutrients (by using glucose-free medium) achieved by using a humidified, sealed chamber. Additionally, induced ischemic-like conditions in this model are accurately verified by direct visualization of hypoxic cells.

<table><tbody><tr><td>24 mm Transwell with 0.4 &amp;micro;m Pore Polyester Membrane Insert</td><td>Corning</td><td>3450</td><td></td></tr><tr><td>35 mm Glass Bottom Dishes</td><td>MatTek Life Sciences (FISHERSCI)</td><td>P35GC-1.5-14-C</td><td></td></tr><tr><td>Astrocyte Medium</td><td>Science Cell</td><td>1801</td><td></td></tr><tr><td>Attachment Factor</td><td>Cell Systems (Fisher Scientific)</td><td>4Z0-201</td><td></td></tr><tr><td>BD 60 mL Syringe</td><td>BD</td><td>309653</td><td></td></tr><tr><td>BrainPhys Imaging Optimized Medium</td><td>STEMCELL Technologies</td><td>5791</td><td></td></tr><tr><td>Complete Classic Medium With Serum and CultureBoost</td><td>4Z0-500</td><td>Cell Systems</td><td></td></tr><tr><td>Corning 50 mL PP Centrifuge Tubes (Conical Bottom with CentriStar Cap</td><td>VWR</td><td>430829</td><td></td></tr><tr><td>Corning 75cm&amp;sup2; U-Shaped Canted Neck Not Treated Cell Culture Flask&amp;nbsp;</td><td>Corning</td><td>431464U</td><td></td></tr><tr><td>Corning CellBIND 96-well Flat Clear Bottom Black Polystyrene Microplates</td><td>Corning</td><td>3340</td><td></td></tr><tr><td>Countes Cell Counting Chamber Slides</td><td>Thermo Fisher Scientific</td><td>C10228</td><td></td></tr><tr><td>Countess II FL Automated Cell Counter</td><td>Thermo Fisher Scientific</td><td>ZGEXSCCOUNTESS2FL</td><td></td></tr><tr><td>Decon CiDehol 70 Isopropyl Alcohol Solution&amp;nbsp;</td><td>Fisher Scientific&amp;nbsp;</td><td>04-355-71</td><td></td></tr><tr><td>Disposable Petri Dishes</td><td>VWR</td><td>25384-088</td><td></td></tr><tr><td>DMEM Medium (No glucose, No glutamine, No phenol red)</td><td>ThermoFisher</td><td>A14430-01</td><td>Glucose-free medium</td></tr><tr><td>DPBS (No Calcium, No Magnesium)</td><td>ThermoFisher</td><td>14190250</td><td></td></tr><tr><td>EBM Endothelial Cell Growth Basal Medium, Phenol Red Free, 500 mL</td><td>Lonza</td><td>CC-3129</td><td></td></tr><tr><td>EVOM2 Epithelial Volt/Ohm (TEER) Meter with STX2 electrodes</td><td>World Precison Instruments</td><td>NC9792051</td><td>Epithelial voltohmmeter&amp;nbsp;</td></tr><tr><td>Fluorescein isothiocyanate&amp;ndash;dextran (wt 20,000)</td><td>Millipore Sigma</td><td>FD20-250MG</td><td></td></tr><tr><td>Fluorescein isothiocyanate&amp;ndash;dextran (wt 70,000)</td><td>Millipore Sigma</td><td>FD70S-250MG</td><td></td></tr><tr><td>Fluorview FV3000 Confocal Microscope</td><td>Olympus</td><td>FV3000</td><td></td></tr><tr><td>Gas Tank (95% N&lt;sub&gt;2&lt;/sub&gt;, 5% CO&lt;sub&gt;2&lt;/sub&gt;)</td><td>Airgas</td><td>X02NI95C2003071</td><td></td></tr><tr><td>HBSS (No calcium, No magnesium, no phenol red)</td><td>Thermofisher</td><td>14025092</td><td></td></tr><tr><td>Hoechst 33342, Trihydrochloride, Trihydrate - 10 mg/mL Solution in Water</td><td>ThermoFisher</td><td>H3570</td><td></td></tr><tr><td>Human Astrocytes</td><td>Science Cell</td><td>1800</td><td></td></tr><tr><td>Human Brain Vascular Pericytes</td><td>Science Cell</td><td>1200</td><td></td></tr><tr><td>Hypoxia Incubator Chamber</td><td>STEMCELL Technologies</td><td>27310</td><td></td></tr><tr><td>Image-iT Green Hypoxia Reagent</td><td>ThermoFisher</td><td>I14834</td><td></td></tr><tr><td>Pericyte Medium</td><td>Science Cell</td><td>1201</td><td></td></tr><tr><td>Primary Human Brain Microvascular Endothelial Cells</td><td>ACBRI 376</td><td>Cell Systems</td><td></td></tr><tr><td>Rocking Platform Shaker, Double</td><td>VWR</td><td>10860-658</td><td></td></tr><tr><td>Single Flow Meter</td><td>STEMCELL Technologies</td><td>27311</td><td></td></tr><tr><td>SpectraMax iD3 Microplate Reader</td><td>Molecular Devices</td><td>75886-128</td><td></td></tr><tr><td>Syringe Filter, 25 mm, 0.22 &amp;mu;m, PVDF, Sterile</td><td>NEST Scientific</td><td>380121</td><td></td></tr><tr><td>TPP Mutli-well Plates (6 wells)</td><td>MidSci</td><td>TP92406</td><td></td></tr><tr><td>TPP Tissue Culture Flasks T-75 Flasks</td><td>MidSci</td><td>TP90075</td><td>Flasks with activated surface for cell adhesion</td></tr><tr><td>Trypsin-EDTA (0.25%), phenol red</td><td>ThermoFisher</td><td>25200056</td><td></td></tr><tr><td>UltraPure Distilled Water</td><td>Invitrogen (Life Technologies)</td><td>10977-015</td><td></td></tr><tr><td>Uno Stage Top Incubator-</td><td>Oko Lab</td><td>UNO-T-H-CO2-TTL</td><td></td></tr></tbody></table>

a triple primary cell culture model of the human blood-brain barrier for studying ischemic stroke in vitro

Ischemic stroke is a major cause of death and disability worldwide with limited therapeutic options. The neuropathology of ischemic stroke is characterized by an interruption in blood supply to the brain leading to cell death and cognitive dysfunction. During and after ischemic stroke, blood-brain barrier (BBB) dysfunction facilitates injury progression and contributes to poor patient recovery. Current BBB models primarily include endothelial monocultures and double co-cultures with either astrocytes or pericytes.
Such models lack the ability to fully imitate a dynamic brain microenvironment, which is essential for cell-to-cell communication. Additionally, commonly used BBB models often contain immortalized human endothelial cells or animal-derived (rodent, porcine, or bovine) cell cultures that pose translational limitations. This paper describes a novel well-insert-based BBB model containing only primary human cells (brain microvascular endothelial cells, astrocytes, and brain vascular pericytes) enabling the investigation of ischemic brain injury in vitro. 
The effects of oxygen-glucose deprivation (OGD) on barrier integrity were assessed by passive permeability, transendothelial electrical resistance (TEER) measurements,and direct visualization of hypoxic cells. The presented protocol offers a distinct advantage inmimicking the intercellular environment of the BBB in vivo, serving as a more realistic in vitro BBB model for developing new therapeutic strategies in the setting of ischemic brain injury.

To examine the effects of astrocytes and pericytes on the barrier function of HBMEC, we constructed the triple cell culture BBB model on cell culture inserts (Figure 1A) along with HBMEC monoculture and two double co-culture models as controls (Figure 1B). Double co-culture controls included a non-contact co-culture of HBMEC with HA and contact co-culture of HBMEC with HBVP. After 6 days in co-culture, all experimental setups were subjected to OGD for 4 h. The barrier integrity of the endothelial monolayer in the indicated BBB configurations was assessed by determining TEER before and after OGD, as well as after 24 h of reoxygenation (Figure 1C). For 4 h, the OGD caused a significant decrease in TEER values only in HBMEC monoculture and the co-culture model with HBMEC and HBVP. These decreased levels reached the baseline levels following 24 h of reoxygenation. No effect of OGD on the co-culture model with HBMEC and HA pointed tothe protective role of astrocytes against ischemic conditions in vitro.
Although there was no difference in baseline TEER between triple and HBMEC/HBVP double co-culture models, the TEER values in the triple co-culture model were significantly higher than those in the double co-culture controls or monoculture control immediately after OGD, suggesting that integration of HA and HBVP into the triple BBB model plays a critical role in maintaining the BBB functional integrity under pathological conditions (Figure 1C). We also investigated the permeability of small (20 kDa) and large (70 kDa) molecular mass FITC-dextrans across the endothelial monolayer in the developed triple culture model. The paracellular permeability of endothelial monolayers to 20 kDa molecular mass FITC-dextran&#160;was drastically increased in HBMEC monoculture and to a lesser extent in the co-culture model with HBMEC and HBVP as compared to normoxic controls (Figure 1D). Furthermore, 20 kDa&#160;FITC-dextran permeability levels were the lowest in the triple BBB model among all models under normoxic and OGD conditions. No changes were observed in 70 kDa FITC-dextran flux across endothelial monolayers in any of the models (Figure 1E). These data suggest that ischemic-hypoxic damage was not so severe to cause changes in paracellular permeability to large molecules such as plasma proteins.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64469/64469fig01.jpg" /> 
Figure 1: Effects of oxygen-glucose deprivation on transendothelial electrical resistance and endothelial permeability in human primary mono-, co-, and triple culture BBB models. (A)&#160;Schematic timeline for the setting of the triple primary cell culture BBB model. (B)&#160;Schematic representations of the configurations for in vitro static human primary cell-based BBB models.&#160;A monoculture model contains HBMEC seeded on the apical side of the well-insert porous membrane. A non-contact co-culture contains HBMEC seeded on the upper surface of the well-insert support and HA seeded at the bottom of the culture well. A contact co-culture model includes HBVP seeded on the lower surface of the well-insert support with HBMEC on the upper surface. For the triple culture model, HBMEC are seeded on the upper surface of the support with HBVP seeded on the lower surface and HA seeded on the bottom of the culture wells. (C)&#160;Changes in TEER monitored immediately before OGD, after 4 h of OGD, and after 24 h of reoxygenation. Data represented as mean &#177; SEM (n = 5-6). ****P &#60; 0.0001 compared with mono- and co-culture models (two-way ANOVA). (D) Paracellular permeability to 20 kDa FITC-dextran across endothelial monolayers was measured after 4 h of OGD. (E) Paracellular permeability to 70 kDa FITC-dextran across endothelial monolayers was measured after 4 h of OGD. Data represented as mean &#177; SD (n = 5-6). *P &#60; 0.05. ****P &#60; 0.0001 (two-way ANOVA). Abbreviations: OGD = oxygen-glucose deprivation; TEER = transendothelial electrical resistance; HBMEC = human brain microvascular endothelial cells; HA = human astrocytes; HBVP = human brain vascular pericytes; FITC-dextran = dextran conjugated to fluorescein isothiocyanate; arb. units = arbitrary units. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64469/64469fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
To verify OGD-induced hypoxic injury, HBMEC, HA, and HBVP were cultured in the wells of 35mm glass&#160;bottom dishes and loaded with the hypoxia reagent, whose fluorescent signal increased with reduced oxygen levels. All primary human types exposed to OGD exhibited strong green fluorescence, proving that the imaged live cells were hypoxic (Figure 2).&#160;After estimating the effects of having different perivascular cell types (astrocytes and pericytes), or their combination in the BBB model on the barrier properties following an OGD protocol, we concluded that the triple model was superior to the other BBB models tested.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64469/64469fig02.jpg" /> 
Figure 2: Live staining of hypoxic primary human brain microvascular endothelial cells, human astrocytes, and human brain vascular pericytes following 4 h of oxygen-glucose deprivation. OGD exposures were carried out on all primary cells (HBMEC, HA, and HBVP) using a hypoxia reagent and glucose-free medium for 4 h. Results from four independent experiments are shown. Cultures were imaged at 20x magnification. Scale bars = 100 &#956;m. Abbreviations: OGD = oxygen-glucose deprivation; HBMEC = human brain microvascular endothelial cells; HA = human astrocytes; HBVP = human brain vascular pericytes; DAPI = 4&#39;,6-diamidino-2-phenylindole. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64469/64469fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Triple Primary Cell Culture Model of the Human Blood-Brain Barrier for Studying Ischemic Stroke In Vitro at JoVE.com

A Triple Primary Cell Culture Model of the Human Blood-Brain Barrier for Studying Ischemic Stroke In Vitro

Here, we describe the method for establishing a triple cell culture model of the blood-brain barrier based on primary human brain microvascular endothelial cells, astrocytes, and pericytes. This multicellular model is suitable for studies of neurovascular unit dysfunction during ischemic stroke in vitro or for the screening of drug candidates.

A Triple Primary Cell Culture Model of the Human Blood-Brain Barrier for Studying Ischemic Stroke In Vitro

Ischemic stroke is a major cause of death and disability worldwide with limited therapeutic options. The neuropathology of ischemic stroke is ...

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Nanyang Technological University

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JoVE Journal

Biochemistry

3.8K Views.  University of Miami. Ons protocol erkent de noodzaak om de menselijke bloed-hersenbarrière in vitro beter samen te vatten voor de studie van complexe cellulaire mechanismen die het herstel na een beroerte beïnvloeden. Ons drievoudige model vertoont een robuuste integriteit die deels verband houdt met grote compartimentering van putinzetstukken, waardoor we de veranderingsfrequentie kunnen verlagen en verstoring van populaties kunnen voorkomen. Ons model is een geweldig hulpmiddel voor het bestuderen van de mole...

Video: A Triple Primary Cell Culture Model of the Human Blood-Brain Barrier for Studying Ischemic Stroke In Vitro

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

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

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm&#183;cm2 on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 &#177;&#160;0.11 x 10-3 cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.

Setting-up an In Vitro Model of Rat Blood-brain Barrier BBB: A Focus on BBB Impermeability and Receptor-mediated Transport

The aim of the present study was to validate the reproducibility of an in vitro BBB model involving a rat syngeneic co-culture of endothelial cells and astrocytes. The endothelial cell monolayer presented high TEER and low LY permeability. Expression of specific TJ proteins, functional responses to inflammation and functionality of transporters and receptors were assessed.

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and ...

Medicine

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

An In Vivo Assessment of Blood-Brain Barrier Disruption in a Rat Model of Ischemic Stroke

Ischemic stroke leads to vasogenic cerebral edema and subsequent primary brain injury, which is mediated through destruction of the blood-brain barrier (BBB). Rats with induced ischemic stroke were established and used as in vivo models to investigate the functional integrity of the BBB. Spectrophotometric detection of Evans blue (EB) in the brain samples with ischemic injury could provide reliable justification for the research and development of novel therapeutic modalities. This method generates reproducible results, and is applicable in any laboratory without a need for special equipment. Here, we present a visualized and technical guideline on the detection of the extravasation of EB following induction of ischemic stroke in rats.

An In Vivo Assessment of Blood-Brain Barrier Disruption in a Rat Model of Ischemic Stroke

The overall goal of this procedure is to provide a highly reproducible technique for in vivo assessment of the blood-brain barrier disruption in rat models of ischemic stroke.

Ischemic stroke leads to vasogenic cerebral edema and subsequent primary brain injury, which is mediated through destruction of the blood-brain barrier ...

Neuroscience

Isolation of Cerebral Capillaries from Fresh Human Brain Tissue

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic strategies that hold the promise to enhance brain drug delivery, improve brain protection, and treat brain disorders. However, studying the human blood-brain barrier function is challenging. Thus, there is a critical need for appropriate models. In this regard, brain capillaries isolated from human brain tissue represent a unique tool to study barrier function as close to the human in vivo situation as possible. Here, we describe an optimized protocol to isolate capillaries from human brain tissue at a high yield and with consistent quality and purity. Capillaries are isolated from fresh human brain tissue using mechanical homogenization, density-gradient centrifugation, and filtration. After the isolation, the human brain capillaries can be used for various applications including leakage assays, live cell imaging, and immune-based assays to study protein expression and function, enzyme activity, or intracellular signaling. Isolated human brain capillaries are a unique model to elucidate the regulation of the human blood-brain barrier function. This model can provide insights into central nervous system (CNS) pathogenesis, which will help the development of therapeutic strategies for treating CNS disorders.

Isolated brain capillaries from human brain tissue can be used as a preclinical model to study barrier function under physiological and pathophysiological conditions. Here, we present an optimized protocol to isolate brain capillaries from fresh human brain tissue.

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic ...

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) ...

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 ...

A Cell Culture Model for Studying the Role of Neuron-Glia Interactions in Ischemia

Ischemic stroke is a clinical condition characterized by hypoperfusion of brain tissue, leading to oxygen and glucose deprivation, and the consequent neuronal loss. Numerous evidence suggests that the interaction between glial and neuronal cells exert beneficial effects after an ischemic event. Therefore, to explore potential protective mechanisms, it is important to develop models that allow studying neuron-glia interactions in an ischemic environment. Herein we present a simple approach to isolate astrocytes and neurons from the rat embryonic cortex, and that by using specific culture media, allows the establishment of neuron- or astrocyte-enriched cultures or neuron-glia cultures with high yield and reproducibility.
To study the crosstalk between astrocytes and neurons, we propose an approach based on a co-culture system in which neurons cultured in coverslips are maintained in contact with a monolayer of astrocytes plated in multiwell plates. The two cultures are maintained apart by small paraffin spheres. This approach allows the independent manipulation and the application of specific treatments to each cell type, which represents an advantage in many studies.
To simulate what occurs during an ischemic stroke, the cultures are subjected to an oxygen and glucose deprivation protocol. This protocol represents a useful tool to study the role of neuron-glia interactions in ischemic stroke.

Here, we present a simple approach using specific culture media that allows the establishment of neuron- and astrocyte-enriched cultures, or neuron-glia cultures from the embryonic cortex, with high yield and reproducibility.

Ischemic stroke is a clinical condition characterized by hypoperfusion of brain tissue, leading to oxygen and glucose deprivation, and the consequent ...

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

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

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

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

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

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

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

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

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

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

A Triple Primary Cell Culture Model of the Human Blood-Brain Barrier for Studying Ischemic Stroke In Vitro

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