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...

<ol>
	<li>Mozaffarian, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+disease+and+stroke+statistics-2016+update:+a+report+from+the+American+Heart+Association.">Heart disease and stroke statistics-2016 update: a report from the American Heart Association.</a> Circulation. 133 (94), 38 (2016).</li><li>Yousufuddin, M., Young, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aging+and+ischemic+stroke.">Aging and ischemic stroke.</a> Aging. 11 (9), 2542-2544 (2019).</li><li>Donkor, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stroke+in+the+21st+century:+a+snapshot+of+the+burden,+epidemiology,+and+quality+of+life.">Stroke in the 21st century: a snapshot of the burden, epidemiology, and quality of life.</a> Stroke Research and Treatment. , 3238165 (2018).</li><li>Kadry, H., Noorani, B., Cucullo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+blood-brain+barrier+overview+on+structure,+function,+impairment,+and+biomarkers+of+integrity.">A blood-brain barrier overview on structure, function, impairment, and biomarkers of integrity.</a> Fluids and Barriers of the CNS. 17 (1), 69 (2020).</li><li>Brown, L. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pericytes+and+neurovascular+function+in+the+healthy+and+diseased+brain.">Pericytes and neurovascular function in the healthy and diseased brain.</a> Frontiers in Cellular Neuroscience. 13, 282 (2019).</li><li>Cabezas, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocytic+modulation+of+blood+brain+barrier:+perspectives+on+Parkinson's+disease.">Astrocytic modulation of blood brain barrier: perspectives on Parkinson's disease.</a> Frontiers in Cellular Neuroscience. 8, 211 (2014).</li><li>Abdullahi, W., Tripathi, D., Ronaldson, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood-brain+barrier+dysfunction+in+ischemic+stroke:+targeting+tight+junctions+and+transporters+for+vascular+protection.">Blood-brain barrier dysfunction in ischemic stroke: targeting tight junctions and transporters for vascular protection.</a> American Journal of Physiology-Cell Physiology. 315 (3), 343-356 (2018).</li><li>Candelario-Jalil, E., Dijkhuizen, R. M., Magnus, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroinflammation,+stroke,+blood-brain+barrier+dysfunction,+and+imaging+modalities.">Neuroinflammation, stroke, blood-brain barrier dysfunction, and imaging modalities.</a> Stroke. 53 (5), 1473-1486 (2022).</li><li>He, Y., Yao, Y., Tsirka, S. E., Cao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-culture+models+of+the+blood-brain+barrier.">Cell-culture models of the blood-brain barrier.</a> Stroke. 45 (8), 2514-2526 (2014).</li><li>Thomsen, L. B., Burkhart, A., Moos, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+triple+culture+model+of+the+blood-brain+barrier+using+porcine+brain+endothelial+cells,+astrocytes+and+pericytes.">A triple culture model of the blood-brain barrier using porcine brain endothelial cells, astrocytes and pericytes.</a> PLoS One. 10 (8), 0134765 (2015).</li><li>Song, Y., Cai, X., Du, D., Dutta, P., Lin, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+blood-brain+barrier+models+for+in+vitro+biological+analysis:+one+cell+type+vs+three+cell+types.">Comparison of blood-brain barrier models for in vitro biological analysis: one cell type vs three cell types.</a> ACS Applied Bio Materials. 2 (3), 1050-1055 (2019).</li><li>Xu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silver+nanoparticles+induce+tight+junction+disruption+and+astrocyte+neurotoxicity+in+a+rat+blood-brain+barrier+primary+triple+coculture+model.">Silver nanoparticles induce tight junction disruption and astrocyte neurotoxicity in a rat blood-brain barrier primary triple coculture model.</a> International Journal of Nanomedicine. 10, 6105-6118 (2015).</li><li>Appelt-Menzel, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+a+human+blood-brain+barrier+co-culture+model+mimicking+the+neurovascular+unit+using+induced+pluri-+and+multipotent+stem+cells.">Establishment of a human blood-brain barrier co-culture model mimicking the neurovascular unit using induced pluri- and multipotent stem cells.</a> Stem Cell Reports. 8 (4), 894-906 (2017).</li><li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rational+construction+of+a+reversible+arylazo-based+NIR+probe+for+cycling+hypoxia+imaging+in+vivo.">Rational construction of a reversible arylazo-based NIR probe for cycling hypoxia imaging in vivo.</a> Nature Communications. 12 (1), 2772 (2021).</li><li>Palacio-Casta&#241;eda, V., Kooijman, L., Venzac, B., Verdurmen, W. P. R., Le Gac, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+switching+of+tumor+cells+under+hypoxic+conditions+in+a+tumor-on-a-chip+model.">Metabolic switching of tumor cells under hypoxic conditions in a tumor-on-a-chip model.</a> Micromachines. 11 (4), 382 (2020).</li><li>Ramsauer, M., Krause, D., Dermietzel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenesis+of+the+blood-brain+barrier+in+vitro+and+the+function+of+cerebral+pericytes.">Angiogenesis of the blood-brain barrier in vitro and the function of cerebral pericytes.</a> FASEB Journal. 16 (10), 1274-1276 (2002).</li><li>Lyck, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ALCAM+(CD166)+is+involved+in+extravasation+of+monocytes+rather+than+T+cells+across+the+blood-brain+barrier.">ALCAM (CD166) is involved in extravasation of monocytes rather than T cells across the blood-brain barrier.</a> Journal of Cerebral Blood Flow &amp; Metabolism. 37 (8), 2894-2909 (2017).</li><li>Rizzi, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+triple+culture+cell+system+modeling+the+human+blood-brain+barrier.">A triple culture cell system modeling the human blood-brain barrier.</a> Journal of Visualized Experiments. (177), (2021).</li><li>Kumar, S., Shaw, L., Lawrence, C., Lea, R., Alder, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=P50:+Developing+a+physiologically+relevant+blood+brain+barrier+model+for+the+study+of+drug+disposition+in+glioma.">P50: Developing a physiologically relevant blood brain barrier model for the study of drug disposition in glioma.</a> Neuro-Oncology. 16 (6), (2014).</li><li>Stone, N. L., England, T. J., O'Sullivan, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+transwell+blood+brain+barrier+model+using+primary+human+cells.">A novel transwell blood brain barrier model using primary human cells.</a> Frontiers in Cellular Neuroscience. 13, 230 (2019).</li><li>Al Ahmad, A., Taboada, C. B., Gassmann, M., Ogunshola, O. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocytes+and+pericytes+differentially+modulate+blood-brain+barrier+characteristics+during+development+and+hypoxic+insult.">Astrocytes and pericytes differentially modulate blood-brain barrier characteristics during development and hypoxic insult.</a> Journal of Cerebral Blood Flow &amp; Metabolism. 31 (2), 693-705 (2011).</li></ol>

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 a...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>24 mm Transwell with 0.4 &#181;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&#178; U-Shaped Canted Neck Not Treated Cell Culture Flask&#160;</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&#160;</td>
			<td>Fisher Scientific&#160;</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&#160;</td>
		</tr>
		<tr>
			<td>Fluorescein isothiocyanate&#8211;dextran (wt 20,000)</td>
			<td>Millipore Sigma</td>
			<td>FD20-250MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescein isothiocyanate&#8211;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% N2, 5% CO2)</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 &#956;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 b...

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 ...

Our protocol recognizes the need to better recapitulate the human blood-brain barrier in vitro for the study of complex cellular mechanisms that affect post-stroke recovery. Our triple model exhibits robust integrity which is partly related to large compartmenting of well inserts, allowing us to decrease the change frequency and avoid disruption of populations. Our model is a great tool for studying the molecular and cellular changes that occur at the blood-brain barrier during and after ischemic stroke.Our protocol allows for the discovery of novel targets and therapeutics to improve post-stroke recovery. Current treatments for ischemic stroke are time sensitive and consequentially are not always effective. Begin by cultivating HBVP in T75 culture flasks with an activated surface for cell adhesion within a 5%carbon dioxide incubator at 37 degrees Celsius until confluent.Once confluence is reached, aspirate the old pericyte medium and wash the cells with five milliliters of warm Dulbecco's PBS. Aspirate the Dulbecco's PBS and detach the cells from the flask using a combination of four milliliters of warm trypsin EDTA solution and one milliliter of Dulbecco's PBS. Incubate the flask for five minutes at 37 degree Celsius in a carbon dioxide incubator.View under a microscope to confirm whether the cells are detached from the flask. Add five milliliters of warm pericyte medium containing 2%FBS to the flask and transfer the detached cells to a 50 milliliter centrifuge tube. Centrifuge the cell suspension for three minutes at 200 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. Re-suspend 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 microliters of the re-suspended cells, place them into a cell counting slide, and count the number of cells. Determine the cell density and seed 300, 000 cells per insert in one milliliter of pericyte medium onto the abluminal side of the well inserts. Cultivate primary human astrocytes using the astrocyte medium containing 2%FBS as described in the text manuscript.Determine the cell density and seed 300, 000 cells per well onto the bottom of the tissue culture six-well plates. Cover the plate to prevent evaporation and incubate overnight. Similarly, cultivate HBMEC in tissue culture dishes using complete classic medium containing 10%FBS as described in the text manuscript.Take out the tissue culture six-well plates containing astrocytes and the well inserts containing pericytes from the incubator. Aspirate the astrocyte medium from the tissue culture six-well plates and add one milliliter of pericyte medium and one milliliter of astrocyte medium to each well. Aspirate the pericyte medium from the well inserts and place them into the tissue culture six-well plates containing the seeded astrocytes.Seed HBMEC at a density of 300, 000 cells per well in two milliliters of complete classic medium onto the apical side of the same well inserts. Wash the cells thrice with Dulbecco's PBS. For triple cell cultures subjected to oxygen-glucose deprivation, add glucose-free medium to both the apical and basolateral compartments.Replace the culture medium with fresh medium and normoxic control cell cultures. Place control triple cultures in the 5%carbon dioxide incubator at 37 degrees Celsius. Place a Petri dish containing 20 milliliters of sterile water in the hypoxia incubator chamber to provide adequate humidification of the cultures.Open the chamber by releasing the ring clamp, then arrange the cell cultures on the shelves. Seal the chamber by securing the ring clamp. Open the inlet and outlet ports of the chamber and attach the tubing from the top of the chamber's flow meter.Attach the tubing from the bottom of the flow meter to the gas tank via an air filter. Open the tank flow control valve by turning it counterclockwise to allow minimum gas flow. Slowly open the pressure regulator valve by turning clockwise.Flush the chamber with the gas mixture for five minutes at a flow rate of 20 liters per minute. Disconnect the chamber from the gas source and close both white plastic clamps firmly. Turn off the tank flow control valve by turning clockwise.Close the pressure regulator valve by turning counterclockwise. Place the hypoxia chamber in a conventional incubator set at 37 degrees Celsius for four hours. Place the sterilized TEER instrument into the biosafety cabinet and plug the electrodes into the epithelial volt ohm meter.Sterilize the electrodes in 30 milliliters of 70%isopropyl alcohol solution for a minimum of 30 minutes. Turn on the TEER instrument and set the function to ohms. Remove the electrodes from the 70%isopropyl alcohol solution and place them in 20 milliliters of Dulbecco's PBS for a minimum of 30 minutes until the digital readout on the TEER device reads zero ohms.Insert the long prong of the electrode through one of the three openings in the well insert hangar 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. Wait until the digital readout values on the TEER instrument level off before recording the value.Place the electrodes back into Dulbecco's PBS to wash them between measurements. Continue to collect all the TEER measurements for two more blank well insert controls. Collect the TEER measurements of the sample plates as demonstrated earlier.Once all the measurements have been taken place the electrode back into the 70%isopropyl alcohol solution for 30 minutes. Then disconnect the electrodes from the TEER instrument and allow them to air dry. Calculate the TEER values.Aspirate the medium from the basolateral compartment and replace it with two milliliters of phenol red free endothelial cell growth medium in the triple cell culture blood-brain barrier model. Wash the cells in the apical compartment twice with HBSS. Add one milliliter of the FITC dextran solution in the apical compartment and cover the plate with aluminum foil, then place the plate in the incubator set at 37 degrees Celsius with 5%carbon dioxide for one hour.Take 100 microliters 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 the emission wavelengths set to 480 and 530 nanometers, respectively. Seed 200 microliters of HBMEC, primary astrocytes, and HBVP in the center of poly-D-lysine coated 35 millimeter glass bottom dishes at a density of 150, 000 cells per dish.Before seeding HBMEC, coat the bottom of the dishes with the attachment factor. Allow the cells to attach to the glass surface by leaving them overnight at 37 degree Celsius in a carbon dioxide incubator. Once confluence is reached, discard the culture medium and add two milliliters of pre-warmed glucose-free medium for oxygen-glucose deprivation or normal medium for controls.After oxygen-glucose deprivation treatment, replace the medium with the imaging optimized medium in all dishes, then perform fluorescence live-cell imaging with the GFP filter and top stage confocal microscope incubator as described in the text manuscript. The barrier integrity of the endothelial monolayer in the indicated blood-brain barrier model configurations was assessed by determining TEER before and after oxygen-glucose deprivations, as well as after 24 hours of re-oxygenation. For four hours the oxygen-glucose deprivation 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 hours of re-oxygenation. 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 oxygen-glucose deprivation. The paracellular permeability of endothelial monolayers to 20 kilodalton molecular mass, FITC dextran, was drastically increased in the HBMEC monoculture and to a lesser extent in the co-culture model with HBMEC and HBVP as compared to normoxic controls.Furthermore, 20 kilodalton FITC dextran permeability levels were the lowest in the triple brain-blood barrier model among all models under normoxic and oxygen-glucose deprivation conditions. No changes were observed in 70 kilodalton, FITC dextran flux across endothelial monolayers in any of the models. All primary human types exposed to oxygen-glucose deprivation exhibited strong green fluorescence, proving that the imaged live cells were hypoxic.After seeding human brain vascular pericytes on the abluminal side of well inserts, it is very important to cover them with the flip plate to prevent evaporation. In our model we use a relatively small, 0.4 micrometer pore diameter of polyester well inserts making it suitable for the screening of drug candidates to any disease that requires crossing the blood-brain barrier for treatment. This technique provides an excellent opportunity to visualize modification of proteins and transporters if needed.

a-triple-primary-cell-culture-model-human-blood-brain-barrier-for

Research

JoVE Journal

Biochemistry

3.5K Views.  University of Miami. Il nostro protocollo riconosce la necessità di ricapitolare meglio la barriera emato-encefalica umana in vitro per lo studio dei complessi meccanismi cellulari che influenzano il recupero post-ictus. Il nostro modello triplo mostra una robusta integrità che è in parte correlata all'ampia compartimentazione degli inserti dei pozzi, permettendoci di ridurre la frequenza di cambio ed evitare interruzioni delle popolazioni. Il nostro modello è un ottimo strumento per studiare i cambiamenti mo...