This work is granted by European Union&#39;s Horizon 2020 research and innovation program under grant agreement No 764958, as part of the NANOSTEM project, a Marie Sk&#322;odowska-Curie Innovative Training Network (ITN) (Fellowship Eleonora Rizzi). This study is granted by the &#39;Conseil r&#233;gional du Nord-Pas-de-Calais&#39; (Fellowship to Cl&#233;mence Deligne), the &#34;Soci&#233;t&#233; Fran&#231;aise de lutte contre les Cancers et les leuc&#233;mies de l&#39;Enfant et de l&#39;adolescent&#34;(SFCE), the Association &#34;l&#39;&#233;toile de Martin&#34; and the Association&#34;Cassandra contre la leuc&#233;mie&#34;.

The protocol was approved by the French Ministry of Higher Education and Research (reference: CODECOH DC2011-1321) and by the local investigational review board (B&#233;thune Maternity Hospital, Beuvry, France). For obtaining the endothelial cells (ECs), written and informed consent from the donor&#39;s parents was obtained to collect umbilical cord blood, in compliance with the French Legislation. The pericytes are provided by Professor Takashi Kanda (Department of Neurology and Clinical Neuroscience, Yamaguchi University Graduate School of Medicine, Ube, Japan) that were isolated as per Reference19. Primary human brain cortex astrocytes are purchased from a commercial provider (see Table of Materials).
1. Cell culture
<ol>
	<li>Culturing of endothelial cells 
	NOTE: Endothelial cells (ECs) are derived from CD34+ hematopoietic stem cells isolated from human umbilical cord blood, according to the method described by Pedroso et al.20. The endothelial phenotype of the ECs is described in Pedroso et al.20.
	<ol>
		<li>Cultivate human ECs using endothelial cell&#160;medium&#160;supplemented with 5% of fetal calf serum (FCS), 0.5% of gentamicin, and 1% of endothelial cell growth supplement (ECM) (see Table of Materials).</li>
		<li>For sub-culturing ECs, two days before the setting of the model, coat one dish with 10 mL of 2% gelatin for 15 min at 37 &#176;C and then replace it with 20 mL of warm ECM. Thaw one vial of ECs containing 1 million cells (cells were manually counted as described in step 2.3.3 before cell freezing) in the pre-coated cell culture dish.</li>
		<li>After 3 h at 37 &#176;C, renew the medium and maintain the cells until the setting of the triculture in a humidified atmosphere inside an incubator at 37 &#176;C under 5% CO2 and 21% O2.</li>
	</ol>
	</li>
	<li>Culturing of&#160;pericytes 
	NOTE: The pericytes are isolated from the human brain according to the protocol published by Shimizu et al.19, whose isolation procedure follows the method published by Kanda et al.21 with modifications.
	<ol>
		<li>Cultivate the pericytes using Dulbecco&#39;s modified Eagle medium supplemented with 4.5 g/L of glucose (DMEM HG), 10% of FCS, 1% de penicillin/streptomycin, and 1% of L-glutamine (see Table of Materials).</li>
		<li>For pericyte sub-culturing, five days before the setting of the model, coat two dishes with 8 mL/dish of 100 &#181;g/mL of collagen type I solution in 0.02 N acetic acid for 1 h at room temperature (RT) and then wash twice with RT DMEM HG. Thaw one vial of pericytes containing 1 million cells in a conical tube containing 10 mL of warm medium and centrifuge the suspension for 5 min at 190 x g at 20 &#176;C.</li>
		<li>Resuspend the pellet in 10 mL of warm medium and seed in the pre-coated cell culture dishes prefilled with 15 mL of warm medium/dish. The medium is renewed after 3 days, and the cells are maintained until the setting of the triculture in a humidified incubator at 37 &#176;C under 5% CO2 and 21% O2.</li>
	</ol>
	</li>
	<li>Culturing of&#160;astrocytes
	<ol>
		<li>Cultivate the astrocytes using a&#160;astrocyte medium supplemented&#160;with 20% of FCS, 1% of astrocyte growth supplement, and 1% of penicillin/streptomycin solution (AM) (see Table of Materials).</li>
		<li>For sub-culturing the astrocytes, one week before the setting of the model, coat one T75 cell culture flask with 10 mL of 2 &#181;g/cm2 poly-L-lysine (PLL) for 1 h at 37 &#176;C and wash twice with RT sterile water. Thaw one vial of astrocytes containing 1 million cells in 20 mL of warm medium and seed in the pre-coated T75 cell culture flask. 
		NOTE: The commercially obtained cell vials confirm the presence of ~1 million cells, so the counting of cells was not performed here.</li>
		<li>Maintain the cells in a humidified incubator at 37 &#176;C under 5% CO2 and 21% O2. The medium is renewed after 24 h and then every 2 days until the setting of the triculture.</li>
	</ol>
	</li>
</ol>
2. Triple culture model setting
NOTE: The assembly of the three cell types is performed on the same day. The day before the setting of the triculture, perform the collagen type I coating on the reverted insert filters (see Table of Materials) and seed the astrocytes in the PLL-precoated wells of a 12-well plate.
<ol>
	<li>Seeding of astrocytes in the wells
	<ol>
		<li>Coat the wells with 500 &#181;L of 2 &#181;g/cm2 PLL solution as described in step 1.3.2.</li>
		<li>Wash the cells once with 10 mL of warm phosphate buffer saline - calcium and magnesium-free 1X&#160;(1X&#160;PBS-CMF) (Table 1) before incubating for 3 min at 37 &#176;C with 10 mL of warm 20% trypsin/EDTA (T/E) solution and mechanically detach the cells from the flask. Transfer the suspension to a conical tube containing 5 mL of warm non-diluted FCS. 
		NOTE: According to the provider&#39;s protocol22, the collection of astrocytes can be optimized by placing the flask in the incubator for 1 min and tapping the flask to help complete the detachment. The remaining cells should be collected with 5 mL of T/E neutralization solution and placed in the FCS-containing conical tube.</li>
		<li>Centrifuge the suspension for 5 min at 20 &#176;C at 190 x g.</li>
		<li>Resuspend the cell pellet in 5 mL of warm AM. Count the cells by diluting 20 &#181;L of the cell suspension in 80 &#181;L of 1X&#160;PBS-CMF using a manual counting chamber under a microscope (see Table of Materials). Plate around 40,000 cells/cm2 in each PLL-precoated well in a volume of 1.5 mL of warm AM.</li>
	</ol>
	</li>
	<li>Seeding of pericytes on the reverted insert filters
	<ol>
		<li>Add 250 &#181;L of collagen type I solution (100 &#181;g/mL) on the reverted insert filters, placed at the periphery of a covered 25 mm high dish (see Table of Materials) using sterile tweezers. Leave the coating for 1 h at RT under sterile conditions. 
		NOTE: The used dish needs to be high enough to ensure the maintenance of sterility when outside the hood and avoid contact between the solutions on the reverted filter and the cover of the dish.</li>
		<li>Remove carefully the collagen type I solution with a glass pipette connected to an aspirating system. Wash twice with 250 &#181;L of RT DMEM HG and then carefully remove all the solution from the insert filters. Leave the coated insert filters at RT under sterile conditions until the seeding of the cells. 
		NOTE: During the coating procedure, be careful not to touch the filter to avoid membrane damage. Once coated with collagen type I, the insert filters can be stored overnight at RT.</li>
		<li>On the day of the triculture setting, wash the pericytes twice with 10 mL of warm 1X&#160;PBS-CMF and incubate the cells with 2 mL of warm trypsin. Monitor the action of the trypsin by observing the cells under the microscope. Once the cells start to detach, remove the trypsin and add 5 mL of warm ECM before mechanical dissociation.</li>
		<li>Count the cells by diluting 20 &#181;L of the cell suspension in 80 &#181;L of 1X&#160;PBS-CMF using a manual counting chamber under a microscope, and seed 44,500 cells/cm2 on the pre-coated reverted insert filters in a volume of 250 &#181;L. Keep the insert filters in a humidified incubator at 37 &#176;C for 3 h under 5% CO2 and 21% O2.</li>
		<li>Carefully revert the insert filters using sterile tweezers in a 12-well plate containing 1.5 mL of warm ECM/well. The insert filters are now ready to be coated on the other side.</li>
	</ol>
	</li>
	<li>Seeding of endothelial cells on the insert filters
	<ol>
		<li>Coat the upper side of the insert filters with 500 &#181;L of extracellular matrix-based hydrogel (1/48 v/v) (see Table of Materials). After 1 h, in a humidified incubator at 37 &#176;C under 5% CO2 and 21% O2, wash once with 500 &#181;L of RT DMEM HG.</li>
		<li>Wash once with 10 mL of warm 1X&#160;PBS-CMF and incubate the cells with 2 mL of warm trypsin. Once the cells start to detach, remove the trypsin and add 5 mL of warm ECM before mechanical dissociation.</li>
		<li>Count the cells by diluting 20 &#181;L of the cell suspension in 80 &#181;L of 1X&#160;PBS-CMF using a manual counting chamber under microscope and seed the ECs at a density of 71,500 cells/cm2 on the pre-coated insert filters in a volume of 500 &#181;L of warm ECM.</li>
		<li>Replace AM with 1.5 mL of warm ECM/well and then transfer the seeded insert filters (ECs + pericytes) upon the wells containing the astrocytes.</li>
		<li>Place the triculture cell systems in a humidified incubator at 37 &#176;C under 5% CO2 and 21% O2.</li>
	</ol>
	</li>
	<li>Maintenance of the triple cell culture for the induction of the BBB properties 
	NOTE: For the induction of the BBB properties in the ECs, 6 days of triple culture are necessary.
	<ol>
		<li>Renew the medium every other day until day 6, taking off carefully the medium from the upper and bottom compartment using a glass pipette connected with an aspiration system.</li>
		<li>Quickly replace with warm ECM in a volume of 500 &#181;L in the upper compartment and 1.5 mL in the bottom compartment, and put back the cells in a humidified incubator at 37 &#176;C under 5% CO2 and 21% O2.</li>
	</ol>
	</li>
</ol>
3. BBB phenotype validation
NOTE: After 6 days of triple culture, the time necessary to induce the BBB phenotype in the ECs, the human BBB model is ready for experiments. The physical integrity of the brain-like endothelial cells (BLECs) is visualized by immunofluorescence staining of TJ proteins evaluated using permeability assay to BBB integrity markers. The BBB phenotype validation also includes genes/proteins expression analysis and efflux pumps functionality according to the procedure described in Deligne et al.,23. Pericytes and astrocytes are visualized by respective staining markers according to the procedure described in Deligne et al. 202023.
<ol>
	<li>Immunofluorescence staining
	<ol>
		<li>Fix the insert filters and astrocytes in ice-cold methanol/acetone (50/50 v/v) for 1 min and wash twice with RT 1X&#160;PBS-CMF.</li>
		<li>Carefully separate the filter from the insert by cutting the membrane using a scalpel. Perform immunocytochemistry on the membrane and bottom wells according to Deligne et al.23. 
		NOTE: For the blocking step, use 250 &#181;L of SEA BLOCK Blocking buffer (see Table of Materials) for 30 min at RT.</li>
	</ol>
	</li>
	<li>BBB integrity assay
	<ol>
		<li>Assess the physical integrity of the BLECs by a permeability assay using BBB integrity markers with different molecular weights, like Sodium fluorescein (NaF) and 20 kDa Dextran (FD20) (see Table of Materials). 
		NOTE: The experiment can be performed according to the procedure described in Deligne et al.23.</li>
	</ol>
	</li>
	<li>Efflux pump functionality
	<ol>
		<li>Assess the functionality of P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) by measuring the intracellular accumulation of Rhodamine 123 (R123) with and without Elacridar, a P-gp and BCRP inhibitor (see Table of Materials). 
		NOTE: The experiment can be performed according to the procedure described in Deligne et al.23.</li>
	</ol>
	</li>
	<li>Gene and protein expressions
	<ol>
		<li>Perform gene and protein sample collection on ice after a quick wash with cold Ringer HEPES (RH) (Table 1) of the cells. Before EC sample collection, scrape off the pericytes from the inverted insert filters20.</li>
	</ol>
	</li>
</ol>
4. Nanogel transport
NOTE: To estimate the passage of polymeric nanogels (NGs) from the luminal to the abluminal compartment of the triculture BLEC model, 0.1 mg/mL of NG solution was added at day 6 on the luminal compartment for 24 h. Studied NGs were fluorescently tagged N-Isopropylacrylamide (NIPAM)-based hydrogels with an average size of 8-10 nm (see Table of Materials).
<ol>
	<li>Weigh the nanogel powder and solubilize it in ECM at a concentration of 1 mg/mL. Sonicate the solution for 10 min and filter using a 0.2 &#181;m PTFE filter. 
	NOTE: Prepare a fresh NG solution the day of the experiment.</li>
	<li>Change the medium in the luminal compartment and add 50 &#181;L of NGs solution in the upper compartment for a final concentration of 0.1 mg/mL. 
	NOTE: Perform a dilution of 1:10 from the original solution.</li>
	<li>After 24 h of incubation, collect aliquots from luminal (20 &#181;L) and abluminal (200 &#181;L) compartments and place them in a black 96-well plate.</li>
	<li>Quantify the fluorescence using a fluorescent multiplate reader (see Table of Materials) with a black 96-well plate using the setting of excitation/emission wavelengths at 477/540 nm. Calculate the percentage of crossing referred to the initial working solution added at time = 0 h (t0)6,15. 
	NOTE: To prepare the 96-well plate for the fluorescence measurement, add 200 &#181;L of solution from the abluminal compartment and 20 &#181;L of solution from the luminal compartment and t0 preparation (add 180 &#181;L of ECM to reach a final volume of 200 &#181;L). Include also a calibration curve and a blank to the reading plate. Instrumental parameters: Detection method - Fluorescence, Optical Position - Top, Read Type - Endpoint, Excitation wavelength - 477 nm, Emission wavelength - 540 nm, Sensitivity - 100, Shake - Double Orbital for 5 s.</li>
</ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrocortisone+reinforces+the+blood-brain+properties+in+a+serum+free+cell+culture+system.">Hydrocortisone reinforces the blood-brain properties in a serum free cell culture system.</a> Biochemical and Biophysical Research Communications. 247 (2), 312-315 (1998).</li><li>Drolez, A., 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=Selection+of+a+relevant+in+vitro+blood-brain+barrier+model+to+investigate+pro-metastatic+features+of+human+breast+cancer+cell+lines.">Selection of a relevant in vitro blood-brain barrier model to investigate pro-metastatic features of human breast cancer cell lines.</a> PloS One. 11 (3), 0151155 (2016).</li><li>Neuhaus, W., Gaiser, F., Mahringer, A., Franz, J., Riethm&#252;ller, C., F&#246;rster, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pivotal+role+of+astrocytes+in+an+in+vitro+stroke+model+of+the+blood-brain+barrier.">The pivotal role of astrocytes in an in vitro stroke model of the blood-brain barrier.</a> Frontiers in Cellular Neuroscience. 8, 352 (2014).</li><li>Mysiorek, C., 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=Peroxisome+proliferator-activated+receptor-&#945;+activation+protects+brain+capillary+endothelial+cells+from+oxygen-glucose+deprivation-induced+hyperpermeability+in+the+blood-brain+barrier.">Peroxisome proliferator-activated receptor-&#945; activation protects brain capillary endothelial cells from oxygen-glucose deprivation-induced hyperpermeability in the blood-brain barrier.</a> Current Neurovascular Research. 6 (3), 181-193 (2009).</li><li>Culot, M., 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=Cerebrovascular+protection+as+a+possible+mechanism+for+the+protective+effects+of+NXY-059+in+preclinical+models:+An+in+vitro+study.">Cerebrovascular protection as a possible mechanism for the protective effects of NXY-059 in preclinical models: An in vitro study.</a> Brain Research. 1294, 144-152 (2009).</li><li>Vandenhaute, E., Drolez, A., Sevin, E., Gosselet, F., Mysiorek, C., Dehouck, M. -. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adapting+co-culture+in+vitro+models+of+the+blood-brain+barrier+for+use+in+cancer+research:+Maintaining+an+appropriate+endothelial+monolayer+for+the+assessment+of+transendothelial+migration.">Adapting co-culture in vitro models of the blood-brain barrier for use in cancer research: Maintaining an appropriate endothelial monolayer for the assessment of transendothelial migration.</a> Laboratory Investigation. 96 (5), 588-598 (2016).</li><li>Cucullo, L., Hossain, M., Puvenna, V., Marchi, N., Janigro, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+shear+stress+in+Blood-Brain+Barrier+endothelial+physiology.">The role of shear stress in Blood-Brain Barrier endothelial physiology.</a> BMC Neuroscience. 12, 40 (2011).</li></ol>

The authors declare that they have no conflict of interest.

Treatment of brain diseases remains a challenge considering the difficulty of the drugs to hurdle over the BBB to reach their cellular and molecular targets in the brain parenchyma.
Drug development for brain diseases currently exhibits a low success rate since most drugs displaying promising results in preclinical models failed to show any benefit when used in the clinic. Following the &#34;3R rule,&#34; which aims at reducing the number of animals used for experimentation, in vitro models of the BBB are developed to study brain pathologies and to predict brain penetration of drugs29. In vitro models of BBB have mainly been developed using animal cells and have become more sophisticated to improve the relevance of the results obtained16. One of the significant advances in the use of human cells, which brings undeniable new insight and more specificity, at the cellular and molecular levels, to study human disease mechanisms16. However, the development of relevant models requires considering the improvement of the BBB in vitro model settings and the knowledge arising, thanks to animal models. Hence, it needs to consider the complexity of the BBB architecture and the importance of the cell-cell communications to study the BBB under physiological and pathological conditions30.
The protocol presented here describes a method to set up a full human BBB in vitro model comprising the three main cell types of the BBB, without limitation of access to brain tissue. As a multiple cell system, the induction and the maintenance of BBB properties, without the artificial use of tightening compounds, but instead induced by cell-cell communications is more physiologically relevant and in line with the in vivo induction of the BBB properties31. Hence, the respect of the chronology of the protocol is prime of importance for the success of the protocol. Moreover, the incubation times during the setting of the triple culture and once the three cell types are assembled represent the main critical steps of the protocol.
The BBB properties in ECs are induced by the co-culture with pericytes, as described for the co-culture model24. Hence, the culture of pericytes at the reverse side of the insert filter is the most critical point and requires strictly following the protocol at the risk of not having enough pericytes for the induction of the BBB properties. First of all, during the coating procedure and also cell seeding, attention has to be put not to have the cover of the Petri dish in contact with the coating and also the medium once the cells are seeded to ensure a good coating of the filter and not to lose cells (steps 2.2.1 and 2.2.4). Moreover, once the pericytes are seeded, it is essential to wait the indicated time for the attachment of the pericytes (step 2.2.4) before reverting the insert filter for the coating and seeding of ECs on the other side (steps 2.2.5 and 2.3). Once seeded, six days are required to induce the BBB properties through cell-cell communications (step 2.4).
The model is validated in terms of restricted permeability (associated with the setting of the tight junctions) since the ECs of the triple culture model display permeability values to BBB integrity markers similar to the validated co-culture model and also measured in validated animal or human models16,27,32. Moreover, the validation of an in vitro BBB model requires, in addition to the restricted permeability, the responsiveness to other cell types of the NVU and the expression of functional receptors and transporters16. In addition, the model is reproducible and produces multiple insert filters and wells to perform numerous analyses (gene and protein expression, fluorescent staining, toxicity tests) on each cell type separately without requiring a cell sorting method.
The model was developed using a 0.4 &#181;m pore size filter to have one cell type on each side of the insert filter. The insert filter system allowed the study of cell-cell communications in physiological conditions by transferring it upon well-containing astrocytes. The presence of astrocytes in the system represents a plus value compared to the initial co-culture in vitro model24. Indeed, considering the importance of astrocytes in the physiology of the BBB, this third cell type allows further understanding of the cell-cell communications within the BBB. Moreover, the triple cell culture system can also be studied in pathological conditions such as stroke, in which the astrocytes play an essential role33,34,35. In addition, the design of BLECs/ pericytes on both sides of the insert filter can easily be placed upon other cell types to mimic pathological conditions such as brain cancer23.
The pore size of the insert filter can bring limitations with some experiments, such as cell transmigration across the BBB. However, the development of the model with a larger pore size requires the adaptation of the protocol to ensure the formation of a physiological monolayer of ECs and not multiple layers, which is not physiologically relevant to mimic the BBB36.
The model&#39;s applicability has been demonstrated using NGs transport experiment exhibiting the possibility to do transport experiment using a multicellular system. Nevertheless, one should be aware of the difficulties in having a control compound or molecule for the transport experiment, sharing comparable properties with NGs since each nanostructure exhibits a unique set of properties (molecular weight, charge, shape, physical properties, protein corona formation).
One limitation of the model is the absence of shear stress, which was demonstrated to influence the differentiation of ECs and the expression of TJ proteins37. However, developing a fluidic system mimicking the brain capillary is challenging considering the complexity of adding a fluidic part, requiring a specific device, in a multiple cell system. Moreover, the particular device is usually not commercially available and does not allow many replicates, thus making fluidic systems less adapted for high-throughput use.
In summary, this triple culture system consisting of human cells reproduces in vitro the architecture of the BBB. It allows the generation of many inserts that can be used for extensive screening of compounds.

The BBB, localized at the level of brain capillary endothelial cells (ECs), controls and regulates access to the brain parenchyma, which is crucial for maintaining brain homeostasis and the function of neural cells1,2. However, in the case of brain pathology, the lack of access to the brain parenchyma represents a real obstacle to developing therapeutic strategies.
The BBB ECs possess a complex set of properties, including tight junction (TJ) proteins, which seal the intercellular space, associated with a system of efflux pumps, specific transporters, and receptors, which control the transcellular pathway1,2,3. Moreover, all these properties are induced and maintained, thanks to communications with the pericytes embedded in the BBB EC basement membrane and the astrocytes, whose end-feet surround the brain capillaries1,2,3. Hence, studying the BBB in vitro is a challenge considering the complexity of its architecture and the communications among the different cell types constituting the neurovascular unit (NVU)2. Moreover, the different cell types are crucial for the induction and maintenance of BBB properties and consequently impact the prediction of the crossing through the BBB. Different strategies for drug delivery to the brain were already tested using a large panel of tactics to bypass the BBB restricted properties4. More recently, with the progress of nanotechnologies, new materials are being developed for applications as drug carriers5,6. In addition to their higher load, reduced toxicity, and increased drugs&#39; bioavailability, these new nanomaterials can be functionalized for a Trojan horse strategy to cross the BBB and specifically target cells in the parenchyma5,6. Among the different types of nanomaterials being evaluated, nanogels have attracted considerable attention, mainly due to their colloidal properties and ability to tailor the chemical structure to introduce stimuli-responsive properties7,8,9,10,11,12,13,14,15.
In vitro models are now developed for preclinical studies using human cells to predict brain penetration of drugs16. Different settings of these models are available, from monolayers of brain ECs to multiple cell systems16. Considering the importance of the NVU cells in the BBB induction and maintenance and the coordinated response to the pathological environment, BBB in vitro models need to consider all these protagonists to improve the relevance of the prediction2,17.
The current method describes setting up a triple culture in vitro model of the human BBB, which is fully developed with human cells to study specific cellular and human molecular mechanisms. To be physiologically relevant, the model consists of the main three cellular actors of the BBB (ECs, pericytes, and astrocytes) necessary to induce and maintain the BBB properties, without the use of tightening compounds and displaying a set of properties required to be considered as an in vitro BBB model16,18. The model is set up in a configuration delimiting the blood and brain compartment, suitable for preclinical studies of drug and particle transport to predict brain penetration. The usefulness of the model is illustrated by measuring the transport of polymeric nanogels.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Cell Culture</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Astocyte Medium (AM)</td>
			<td>ScienCell&#160;</td>
			<td>1801</td>
			<td></td>
		</tr>
		<tr>
			<td>Astrocyte Growth Supplement</td>
			<td>ScienCell&#160;</td>
			<td>1852</td>
			<td>Astrocyte Growth Supplement is provided in the AM set.</td>
		</tr>
		<tr>
			<td>Cell culture dish 100 mm</td>
			<td>Corning&#160;</td>
			<td>430293</td>
			<td>100 mm x 20 mm; dish used for the thawing of ECs and PCs before the triculture setting</td>
		</tr>
		<tr>
			<td>Cell culture dish 150 mm</td>
			<td>Corning&#160;</td>
			<td>430599</td>
			<td>The height of these dishes (25 mm) allows the seeding of PCs in the reverted insert for the setting of the triculture model.</td>
		</tr>
		<tr>
			<td>Collagen I&#160;</td>
			<td>Corning</td>
			<td>354236</td>
			<td>Rat tail&#160;</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium</td>
			<td>Gibco</td>
			<td>31600-083</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>Endothelial Cell Growth Supplement</td>
			<td>ScienCell&#160;</td>
			<td>1051</td>
			<td>Endothelial Cell Growth Supplement is provided in the ECM set.</td>
		</tr>
		<tr>
			<td>Endothelial Cell Medium (ECM)</td>
			<td>ScienCell&#160;</td>
			<td>1001</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Calf Serum&#160;</td>
			<td>Sigma</td>
			<td>F7524</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>Sigma</td>
			<td>G2500</td>
			<td>2% gelatin from porcine skin in PBS-CMF</td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>BiochromA6</td>
			<td>A-2712</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G6152</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>Glutamine</td>
			<td>Merck</td>
			<td>1002891000</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Brain Cortex Astrocytes</td>
			<td>ScienCell&#160;</td>
			<td>1800-SC</td>
			<td></td>
		</tr>
		<tr>
			<td>Malassez cell counting chamber</td>
			<td>vWR</td>
			<td>HECH40453702</td>
			<td>The count was performed manually.</td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>354230</td>
			<td>Extracellular matrix-based hydrogel</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>ScienCell&#160;</td>
			<td>0503</td>
			<td>Penicillin/Streptomycin solution is provided in the ECM and AM sets.</td>
		</tr>
		<tr>
			<td>Poly-L-lysine</td>
			<td>ScienCell&#160;</td>
			<td>0413</td>
			<td></td>
		</tr>
		<tr>
			<td>Steritop</td>
			<td>Millipore System</td>
			<td>SCGPT0SRE</td>
			<td>0.22 &#181;m pore size</td>
		</tr>
		<tr>
			<td>Transwell insert&#160;</td>
			<td>Corning</td>
			<td>3401</td>
			<td>0.4 &#181;m pore polycarbonate filter&#160;</td>
		</tr>
		<tr>
			<td>Trypsin/EDTA neutralization solution</td>
			<td>ScienCell&#160;</td>
			<td>0113</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin/EDTA solution</td>
			<td>ScienCell&#160;</td>
			<td>0103</td>
			<td></td>
		</tr>
		<tr>
			<td>Immunocytochemistry</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SEA BLOCK blocking buffer</td>
			<td>ThermoScientific</td>
			<td>37527</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 568 anti-Mouse secondary antibody</td>
			<td>Thermofisher</td>
			<td>A11031</td>
			<td>Dilution 1:500</td>
		</tr>
		<tr>
			<td>Alexa Fluor 568 anti-Rabbit secondary antibody</td>
			<td>Thermofisher</td>
			<td>A11036</td>
			<td>Dilution 1:500</td>
		</tr>
		<tr>
			<td>Anti-Claudin-5 primary antibody</td>
			<td>InVitrogen</td>
			<td>34-1600</td>
			<td>Dilution 1:100</td>
		</tr>
		<tr>
			<td>Anti-Desmin primary antibody</td>
			<td>Abcam</td>
			<td>ab6322</td>
			<td>Dilution 1:200</td>
		</tr>
		<tr>
			<td>Anti-Glial Fibrillary Acidic Protein primary antibody</td>
			<td>Dako</td>
			<td>Z0334</td>
			<td>Dilution 1:500</td>
		</tr>
		<tr>
			<td>Anti-Platelet-Derived Growth Factor-&#946; primary antibody</td>
			<td>Abcam</td>
			<td>ab51090</td>
			<td>Dilution 1:200</td>
		</tr>
		<tr>
			<td>Anti-VE-cadherin primary antibody</td>
			<td>Abcam</td>
			<td>ab207732</td>
			<td>Dilution 1:200</td>
		</tr>
		<tr>
			<td>Anti-Zona Occludens-1 primary antibody</td>
			<td>InVitrogen</td>
			<td>61-7300</td>
			<td>Dilution 1:200</td>
		</tr>
		<tr>
			<td>Normal Goat Serum</td>
			<td>Sigma</td>
			<td>G6767</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Diamond Antifade Mountant with DAPI</td>
			<td>Invitrogen&#160;</td>
			<td>P36962</td>
			<td></td>
		</tr>
		<tr>
			<td>Gene expression</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NucleoSpin Rna/Protein Macherey Nagel Kit</td>
			<td>Macherey-Nagel</td>
			<td>740,933,250</td>
			<td></td>
		</tr>
		<tr>
			<td>96 multiplate well</td>
			<td>Biorad</td>
			<td>HSP9601</td>
			<td></td>
		</tr>
		<tr>
			<td>iSCRIPT</td>
			<td>Biorad</td>
			<td>1708841</td>
			<td></td>
		</tr>
		<tr>
			<td>Sealer sheet&#160;</td>
			<td>Biorad</td>
			<td>MSB1001</td>
			<td></td>
		</tr>
		<tr>
			<td>SsoFast EvaGreen Supermix</td>
			<td>Biorad</td>
			<td>172-5201</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein expression</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2x Laemli Sample Buffer</td>
			<td>Biorad</td>
			<td>161-0737</td>
			<td>Add 50 &#181;L of bMercaptoetanol to 950 &#181;L of Laemmli Buffer and store at -20&#176;C. Dilute 1:1 with the protein sample for the assay.</td>
		</tr>
		<tr>
			<td>Anti-Breast Cancer Resistance Protein primary antibody</td>
			<td>Abcam</td>
			<td>ab207732</td>
			<td>Pre-treatment 15 minutes at RT under agitation; dilution 1:1000, O.N. at 4&#176;C</td>
		</tr>
		<tr>
			<td>Anti-Claudin-5 primary antibody</td>
			<td>Abcam</td>
			<td>ab15106</td>
			<td>Pre-treatment 5 minutes at 95&#176;C; dilution 1:1000, O.N. at 4&#176;C</td>
		</tr>
		<tr>
			<td>Anti-Glucose Transporter 1 primary antibody</td>
			<td>Millipore</td>
			<td>07-1401</td>
			<td>Pre-treatment 5 minutes at 95&#176;C; dilution 1:1000, O.N. at 4&#176;C</td>
		</tr>
		<tr>
			<td>Anti-Mouse secondary antibody</td>
			<td>Dako</td>
			<td>P0447</td>
			<td>Dilution 1:5000 in TBS-Tween</td>
		</tr>
		<tr>
			<td>Anti-P-glycoprotein primary antibody</td>
			<td>Genetex</td>
			<td>GTX23364</td>
			<td>Pre-treatment 15 minutes at RT under agitation; dilution 1:400, 3 hours at RT</td>
		</tr>
		<tr>
			<td>Anti-Rabbit secondary antibody</td>
			<td>Dako</td>
			<td>P0448</td>
			<td>Dilution 1:8000 in TBS-Tween</td>
		</tr>
		<tr>
			<td>Anti-Transferrin Receptor primary antibody</td>
			<td>Abcam</td>
			<td>ab84036</td>
			<td>Pre-treatment 5 minutes at 95&#176;C; dilution 1:1000, O.N. at 4&#176;C</td>
		</tr>
		<tr>
			<td>Anti-Zona occludens-1 primary antibody</td>
			<td>Abcam</td>
			<td>ab216880</td>
			<td>Pre-treatment 5 minutes at 95&#176;C; dilution 1:1000, O.N. at 4&#176;C</td>
		</tr>
		<tr>
			<td>Criterion TGX Gel&#160;</td>
			<td>Biorad</td>
			<td>5678083</td>
			<td></td>
		</tr>
		<tr>
			<td>ECL Prime Solution</td>
			<td>Amersham&#160;</td>
			<td>RPN2236</td>
			<td>Revelation solution to keep in the dark</td>
		</tr>
		<tr>
			<td>Phospatase inhibitor cocktail 2</td>
			<td>Sigma</td>
			<td>P5726</td>
			<td></td>
		</tr>
		<tr>
			<td>Phospatase inhibitor cocktail 3</td>
			<td>Sigma</td>
			<td>P0044</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease Inhibitor</td>
			<td>Sigma</td>
			<td>P8340</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein Standards</td>
			<td>Biorad</td>
			<td>161-0373</td>
			<td>Molecular weight markers</td>
		</tr>
		<tr>
			<td>RIPA 10x</td>
			<td>Millipore</td>
			<td>20-188</td>
			<td></td>
		</tr>
		<tr>
			<td>TBS 10x</td>
			<td>Biorad</td>
			<td>1706435</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIS-Glycine</td>
			<td>Biorad</td>
			<td>1610771</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween</td>
			<td>Biorad</td>
			<td>1706531</td>
			<td></td>
		</tr>
		<tr>
			<td>BBB integrity assay</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Fluorescein</td>
			<td>Ampresco</td>
			<td>0681</td>
			<td>&#955;ex= 490 nm; &#955;em= 525 nm&#160;&#160;</td>
		</tr>
		<tr>
			<td>Elacridar</td>
			<td>Sigma</td>
			<td>SML0486</td>
			<td>GF120918</td>
		</tr>
		<tr>
			<td>FITC-Dextran 20 kDa</td>
			<td>Sigma</td>
			<td>FD-20S</td>
			<td>&#955;ex= 490 nm; &#955;em=&#160; 525 nm&#160;&#160;</td>
		</tr>
		<tr>
			<td>Rhodamine 123</td>
			<td>Sigma</td>
			<td>R8004</td>
			<td>&#955;ex= 501 nm; &#955;em= 538 nm&#160;&#160;</td>
		</tr>
		<tr>
			<td>SynergyTM H1&#160;</td>
			<td>BioTek Instruments</td>
			<td></td>
			<td>Fluorescent multiplate reader</td>
		</tr>
		<tr>
			<td>Nanogel Transport</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>Terumo</td>
			<td>SS+01T1</td>
			<td>1 mL syringe&#160;</td>
		</tr>
		<tr>
			<td>Filter</td>
			<td>FisherScientific</td>
			<td>15161499</td>
			<td>0.2 &#181;m PTFE membrane filter, 15 mm diameter</td>
		</tr>
		<tr>
			<td>N-Isopropylacrylamide (NIPAM)-based hydrogels&#160;&#160;</td>
			<td></td>
			<td></td>
			<td>The nanogels (NGs) used in the study are provided by our collaborator in Queen Mary University London, Department of Chemistry. The NGs are covalently tagged with a fluorescent molecule (&#955;ex= 477 nm; &#955;em= 540 nm). NGs are freeze dried and shipped as powder, in this state they are stable at room temperature for long period of time.&#160;</td>
		</tr>
	</tbody>
</table>

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.

Setting of the human triple culture BBB model 
The protocol required for the setting of the human BBB in vitro model is described in Figure 1 and includes successive steps whose order must be strictly respected. First, the three cell types are cultivated individually in cell culture dishes (Figure 1A) before being assembled in an insert filter system. The triple culture setting begins with seeding the first cell type, astrocytes, in the pre-coated bottom well. The following day, pericytes and ECs are seeded on the insert filter&#39;s pre-coated abluminal and luminal surfaces, respectively. The insert filter is then transferred over the astrocytes. The model is maintained in culture for 6 days, the time necessary to induce the BBB properties in ECs, with a renewal of medium every other day according to the patented co-culture model24. The ECs are then renamed as BLECs (Figure 1B).
Characterization of the human BBB model 
The triple cell culture model has been characterized for the presence of a set of BBB-specific properties. First of all, immunocytochemistry data confirmed the expression of conventional markers such as platelet-derived growth factor receptor &#946; (PDGFR-&#946;)25,26 and desmin for pericytes and glial fibrillary acidic protein (GFAP)26 for astrocytes (Figure 2A). Hence, after the 6 days of culture with the pericytes and astrocytes, the monolayer of BLECs, visualized with the adherent junction staining of VE-Cadherin, displays a continuous localization of TJ proteins, Claudin-5 and ZO-1, at the cell borders (Figure 2A). The setting-up of the TJs is correlated with low paracellular permeability coefficients measured using BBB integrity markers of low molecular weight, i.e., NaF (376 Da)16,27 and high molecular weight, i.e., FD20 (20 kDa)27, as shown in Figure 2B. The values measured are comparable with validated BBB in vitro models using the exact source of ECs23,24,28. Altogether, these results highlight the low paracellular permeability of the triple culture BLEC monolayer, which is characteristic of the in vivo BBB. Additionally, R123 intracellular accumulation in BLECs exhibited a significant increase in the presence of the efflux pump inhibitor Elacridar23,24 compared to the control condition with its absence (Figure 2C). This indicates the presence of active efflux pump molecules, namely P-gp and BCRP, in the BLECs.
To further characterize the BLECs, gene expression and protein level of key BBB features were studied (Figure 3). The data obtained with the triple culture model were compared with the validated and patented co-culture model consisting of ECs and pericytes24 used as a control model. The astrocytes represent the third cell type added in the initial co-culture model in the triple culture model. Hence, the gene expression analysis (Figure 3A) of triple culture BLECs, compared with co-culture BLECs, showed the maintenance of expression of key BBB features such as TJ proteins (claudin-5 and zonula occludens-1) and efflux pumps (P-gp and BCRP), and the upregulation of most studied BBB transporters (glucose transporter 1) and receptors (transferrin receptor). Protein quantification data (Figure 3B) were found to be in line with the transcriptional results. Overall, these data support the positive induction of BBB properties in the triple culture BLEC layer similar to the validated co-culture model. Altogether, the triple culture model displays the required physical and metabolic properties for an in vitro microphysiological system to model the BBB.
Applicability to drug delivery strategies - measurement of nanogel transport 
To assess the possibility of using the triple culture model to study new brain delivery strategies, the transport of fluorescently-tagged NIPAM-based neutral NGs was evaluated6,15. At time 0, NGs were placed in the luminal compartment at a concentration of 0.1 mg/mL (Figure 4A). After 24 h of incubation, 5.82% of the NGs were found in the abluminal compartment (Figure 4B), proving their ability to cross the BLECs.
The results demonstrate the suitability of the model to measure the permeability of small and larger compounds, as described with the integrity markers, and evaluate the transport of nanomaterials such as polymeric NGs.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63134/63134fig01.jpg" /> 
Figure 1: Representation of critical steps for the setting of the triple culture in vitro model of the human BBB. (A) Phase-contrast images of the three cell components of the BBB model: endothelial cells (EC), pericytes (PC), and astrocytes (AC).&#160;Scale bar = 250 &#181;m. (B)&#160;Schematic and illustrative timeline for the setting of the triple culture human BBB in vitro model. The highlighted box represents the coating procedure for the inverted insert filter. <a href="https://www.jove.com/files/ftp_upload/63134/63134fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63134/63134fig02.jpg" /> 
Figure 2: Assessment of the properties of the triple culture BBB model. (A) Representative immunostaining images of the distinctive markers for BLECs (Claudin-5: CLD5, Zona Occludens-1: ZO1 and VE-Cadherin: Ve-Cadh), pericytes (Platelet-Derived Growth Factor Receptor-&#946;: PDGFR-&#946; and desmin), and astrocytes (Glial Fibrillary Acidic Protein: GFAP). Scale bar = 10 &#181;m. (B) Paracellular permeability of BLECs to fluorescent BBB integrity markers, Sodium Fluorescein (NaF, 376 Da, Pe: 0.61 &#177; 0.062) and FITC-Dextran (FD20, 20 kDa, Pe: 0.04 &#177; 0.005). N = 3; n = 9. Mean &#177; SEM. (C) P-gp and BCRP functionality in ECs was assessed by quantifying intracellular R123 with (124.2% &#177; 3.39%) and without (100% &#177; 8.79%) Elacridar. N = 4; n = 12. Mean &#177; SEM. p = 0.017 using an unpaired t-test. <a href="https://www.jove.com/files/ftp_upload/63134/63134fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63134/63134fig03.jpg" /> 
Figure 3: Evaluation of BLEC gene expression and protein level of distinctive markers in the triple culture model compared with the co-culture BBB model. (A) Gene expression of tight junction proteins (Claudin-5:&#160;CLD5, and Zona Occludens-1:&#160;ZO1), transporters (Glucose Transporter-1:&#160;GLUT1, P-glycoprotein:&#160;PGP, and Breast Cancer Resistance Protein:&#160;BCRP), and large molecule-receptors (Transferrin Receptor:&#160;TRFR), normalized by the expression of RPLP0. N = 3; n = 9. (B) Protein level of tight junction proteins (CLD5 and ZO1), transporters (GLUT1, PGP, and BCRP), and large molecule-receptors (TRFR), normalized by the expression of &#946;-actin. N = 3; n = 9. Mean &#177; SEM.&#160;For (A)&#160;and (B), values&#62;1 correspond to higher gene expression or protein levels in the triple culture model. The red line corresponds to a value of 1 where the expression level (genes or proteins) of the two models is equivalent. <a href="https://www.jove.com/files/ftp_upload/63134/63134fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63134/63134fig04.jpg" /> 
Figure 4: Measuring nanogel transport in the triple culture model. (A) Schematic representation of the nanogel transport assay. (B)&#160;Percentage of nanogel transport after 24 h of incubation in the triple culture model (5.82% &#177; 0.09%). N = 2; n = 6. Mean &#177; SEM. <a href="https://www.jove.com/files/ftp_upload/63134/63134fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="2" rowspan="2">Name of the Buffer</td>
			<td colspan="3">Composition</td>
			<td colspan="2" rowspan="2">Note</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>Molecular Weight</td>
		</tr>
		<tr>
			<td rowspan="5">Phosphate Buffer Saline, Calcium Magnesium Free</td>
			<td rowspan="5">PBS-CMF</td>
			<td>NaCl</td>
			<td>8 g/L</td>
			<td>58.4</td>
			<td colspan="2" rowspan="5">Add all the compound to sterile water and wait for complete solubilization. The pH of the obtained solution has to be in the range of 7.3-7.4. Filtrate the solution using a 0.22 &#181;m membrane and store the sterile solution at 4 &#176;C.</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>0.2 g/L</td>
			<td>74.55</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>0.2 g/L</td>
			<td>136.09</td>
		</tr>
		<tr>
			<td>NaHPO4-12 H2O</td>
			<td>2.87 g/L</td>
			<td>358.14</td>
		</tr>
		<tr>
			<td>water</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="8">Ringer HEPES</td>
			<td rowspan="8">RH</td>
			<td>NaCl</td>
			<td>8.8 g/L</td>
			<td>58.4</td>
			<td colspan="2" rowspan="8">Add all the compound to sterile water and wait for complete solublization. Adjust the pH to 7.4 (starting solution pH around 6.8). Filtrate the solution using a 0.22 &#181;m membrane and store the sterile solution at 4 &#176;C.</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>0.387 g/L</td>
			<td>74.55</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>0.244 g/L</td>
			<td>110.99</td>
		</tr>
		<tr>
			<td>MgCl2 6H2O</td>
			<td>0.0406 g/L</td>
			<td>203.3</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>0.504 g/L</td>
			<td>84.1</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>1.19 g/L</td>
			<td>238.3</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>0.504 g/L</td>
			<td>180.16</td>
		</tr>
		<tr>
			<td>water</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1: Composition of the different buffers used in the protocol.

Watch this Scientific Journal Video about A Triple Culture Cell System Modeling the Human Blood-Brain Barrier at JoVE.com

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

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 ...

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3.3K Views.  Univ. Artois, UR 2465, Laboratoire de la Barrière Hémato-Encéphalique (LBHE). 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.

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

Human ES cells: Starting Culture from Frozen Cells

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.  First, a one to two day old ten cm plate of  approximately one (to two) million irradiated mouse embryonic fibroblast feeder cells is rinsed with HuES media to remove residual serum and cell debris, and then HuES media added and left to equilibrate in the cell culture incubator.  A frozen vial of cells from long term liquid nitrogen storage or a -80C freezer is sourced and quickly submerged in a 37C water bath for quick thawing.  Cells in freezing media are then removed from the vial and placed in a large volume of HuES media.  The large volume of HuES media facilitates removal of excess serum and DMSO, which can cause HuES human embryonic stem cells to differentiate.  Cells are gently spun out of suspension, and then re-suspended in a small volume of fresh HuES media that is then used to seed the MEF plate.  It is considered important to seed the MEF plate by gently adding the HuES cells in a drop wise fashion to evenly disperse them throughout the plate.  The newly established HuES culture plate is returned to the incubator for 48 hrs before media is replaced, then is fed every 24 hours thereafter.

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.  First, a one to two day old ten cm plate of  ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present methods for the development of assays to query potentially clinically significant nonsynonymous changes using in vivo complementation in zebrafish. Zebrafish (Danio rerio) are a useful animal system due to their experimental tractability; embryos are transparent to enable facile viewing, undergo rapid development ex vivo, and can be genetically manipulated.1 These aspects have allowed for significant advances in the analysis of embryogenesis, molecular processes, and morphogenetic signaling. Taken together, the advantages of this vertebrate model make zebrafish highly amenable to modeling the developmental defects in pediatric disease, and in some cases, adult-onset disorders. Because the zebrafish genome is highly conserved with that of humans (~70% orthologous), it is possible to recapitulate human disease states in zebrafish. This is accomplished either through the injection of mutant human mRNA to induce dominant negative or gain of function alleles, or utilization of morpholino (MO) antisense oligonucleotides to suppress genes to mimic loss of function variants. Through complementation of MO-induced phenotypes with capped human mRNA, our approach enables the interpretation of the deleterious effect of mutations on human protein sequence based on the ability of mutant mRNA to rescue a measurable, physiologically relevant phenotype. Modeling of the human disease alleles occurs through microinjection of zebrafish embryos with MO and/or human mRNA at the 1-4 cell stage, and phenotyping up to seven days post fertilization (dpf). This general strategy can be extended to a wide range of disease phenotypes, as demonstrated in the following protocol. We present our established models for morphogenetic signaling, craniofacial, cardiac, vascular integrity, renal function, and skeletal muscle disorder phenotypes, as well as others.

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present a systematic approach for developing physiologically relevant, sensitive and specific in vivo assays for interpreting variation in human pathology. Transient genetic manipulation via microinjection of WT and mutant human mRNA and morpholino (MO) antisense oligonucleotides harness the tractability of the developing zebrafish embryo to rapidly assay pathogenic mutations, especially, but not exclusively, in the context of human developmental disorders.

Here,  we present methods for the development of assays to query potentially clinically  significant nonsynonymous changes using in  vivo complementation ...

Mouse Embryonic Development in a Serum-free Whole Embryo Culture System

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in an oxygenated rolling bottle culture system. Mouse embryos at E10.5 were carefully isolated from the uterus with intact yolk sac and in a process involving precise surgical maneuver the embryos were gently exteriorized from the yolk sac while maintaining the vascular continuity of the embryo with the yolk sac. Compared to embryos prepared with intact yolk sac or with the yolk sac removed, these embryos exhibited superior survival rate and developmental progression when cultured under similar conditions. We show that these mouse embryos, when cultured in a defined medium in an atmosphere of 95% O2 / 5% CO2 in a rolling bottle culture apparatus at 37 &#176;&#8203;C for 16-40 hr, exhibit morphological growth and development comparable to the embryos developing in utero. We believe this method will be useful for investigators needing to utilize whole embryo culture to study signaling interactions important in embryonic organogenesis.

Serum utilized in embryo cultures contains unknown components that can affect the outcome of experiments especially in studies involving signaling interactions. Here we utilized a serum-free oxygenated culture system and show that mid-gestation mouse embryos cultured for 16-40 hr&#160;exhibit morphological development comparable to embryos developing in utero.

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in ...

The Slice Culture Method for Following Development of Tooth Germs In Explant Culture

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental biologist. For many organs it is difficult to access developing tissue to allow monitoring during ex vivo culture. The slice culture method allows access to tissue so that morphogenetic movements can be followed and specific cell populations can be targeted for manipulation or lineage tracing.
In this paper we describe a method of slice culture that has been very successful for culture of tooth germs in a range of species. The method provides excellent access to the tooth germs, which develop at a similar rate to that observed in vivo, surrounded by the other jaw tissues. This allows tissue interactions between the tooth and surrounding tissue to be monitored. Although this paper concentrates on tooth germs, the same protocol can be applied to follow development of a number of other organs, such as salivary glands, Meckel&#39;s cartilage, nasal glands, tongue, and ear.

Here we detail a method to culture tooth germs in mandible slices using a tissue chopper. This method allows unique access to the tooth during development, providing excellent opportunity for manipulation and lineage tracing, not available using more traditional culture methods.

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental ...

An Ex vivo Culture System to Study Thyroid Development

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are produced by differentiated thyrocytes, organized in closed spheres called follicles, while calcitonin is synthesized by C-cells, interspersed in between the follicles and a dense network of blood capillaries. Although adult thyroid architecture and functions have been extensively described and studied, the formation of the &#8220;angio-follicular&#8221; units, the distribution of C-cells in the parenchyma and the paracrine communications between epithelial and endothelial cells is far from being understood.
This method describes the sequential steps of mouse embryonic thyroid anlagen dissection and its culture on semiporous filters or on microscopy plastic slides. Within a period of four days, this culture system faithfully recapitulates in vivo thyroid development. Indeed, (i) bilobation of the organ occurs (for e12.5 explants), (ii) thyrocytes precursors organize into follicles and polarize, (iii) thyrocytes and C-cells differentiate, and (iv) endothelial cells present in the microdissected tissue proliferate, migrate into the thyroid lobes, and closely associate with the epithelial cells, as they do in vivo.
Thyroid tissues can be obtained from wild type, knockout or fluorescent transgenic embryos. Moreover, explants culture can be manipulated by addition of inhibitors, blocking antibodies, growth factors, or even cells or conditioned medium. Ex vivo development can be analyzed in real-time, or at any time of the culture by immunostaining and RT-qPCR.
In conclusion, thyroid explant culture combined with downstream whole-mount or on sections imaging and gene expression profiling provides a powerful system for manipulating and studying morphogenetic and differentiation events of thyroid organogenesis.

An Ex vivo Culture System to Study Thyroid Development

This protocol describes dissection of mouse embryonic thyroid anlagen and the culture of explants on semiporous filters or on microscopy plastic slides. This system is ideal to study morphogenetic or differentiation events occurring during thyroid development of wild type or knockout embryos, and is amenable to gain- and loss-of-function experiments.

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are ...

Establishment of an Extracellular Acidic pH Culture System

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the role of acidic extracellular pH in tumor aggressiveness and its underlying mechanism has not been extensively studied compared to hypoxic or nutrient starvation conditions. In addition, a well-defined culture method to mimic the acidic extracellular tumor microenvironment has not been fully reported.
Here we present a simple in vitro culture method to maintain acidic extracellular pH using reduced bicarbonate and increased lactate or HCl concentrations in the culture medium. The medium pH was sustained for at least 24 h and gradually decreased by 72 h following culture of PANC-1 and AsPC-1 pancreatic cancer cells. Three distinct acidic media conditions in this study highly upregulated pH-responsive genes such as MSMO1, INSIG1, and IDI1 compared to hypoxia or nutrient starvation. The upregulation of these genes can be used as a marker of acidic pH. These simple techniques are beneficial to elucidate underlying mechanisms of tumor malignancy under acidic tumor microenvironment. Therefore, our extracellular acidic pH culture system enables discovery of cellular acidic pH responses not only in cancer cells but also in primary cells, such as renal tubular cells, in relation to the other acidic disorders including, diabetic ketoacidosis, lactic acidosis, renal tubular acidosis, and respiratory acidosis.

The acidic tumor microenvironment plays critical roles in tumor progression. To assess the effects of acidic extracellular pH on cancer cells in vitro, we established simple acidic culture systems.

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the ...

A High Output Method to Isolate Cerebral Pericytes from Mouse

In recent years, cerebral pericytes have become the focus of extensive research in vascular biology and pathology. The importance of pericytes in blood brain barrier formation and physiology is now demonstrated but its molecular basis remains largely unknown. As the pathophysiological role of cerebral pericytes in neurological disorders is intriguing and of great importance, the in vitro models are not only sufficiently appropriate but also able to incorporate different techniques for these studies. Several methods have been proposed as in vitro models for the extraction of cerebral pericytes, although an antibiotic-free protocol with high output is desirable. Most importantly, a method that has increased output per extraction reduces the usage of more animals.
Here, we propose a simple and efficient method for extracting cerebral pericytes with sufficiently high output. The mouse brain tissue homogenate is mixed with a BSA-dextran solution for the separation of the tissue debris and microvascular pellet. We propose a three-step separation followed by filtration to obtain a microvessel rich filtrate. With this method, the quantity of microvascular fragments obtained from 10 mice is sufficient to seed 9 wells (9.6 cm2 each) of a 6-well plate. Most interestingly with this protocol, the user can obtain 27 pericyte rich wells (9.6 cm2 each) in passage 2. The purity of the pericyte cultures are confirmed with the expression of classical pericyte markers: NG2, PDGFR-&#946; and CD146. This method demonstrates an efficient and feasible in vitro tool for physiological and pathophysiological studies on pericytes.

We present a protocol for the extraction of murine cerebral pericytes. Based on an antibiotic-free enrichment oriented pericyte extraction, this protocol is a valuable tool for in vitro studies providing high purity and high yield, thus decreasing the number of experimental animals used.

In recent years, cerebral pericytes have become the focus of extensive research in vascular biology and pathology. The importance of pericytes in blood ...

Isolation of Human Primary Valve Cells for In vitro Disease Modeling

Calcific aortic valve disease (CAVD) is present in nearly a third of the elderly population. Thickening, stiffening, and calcification of the aortic valve causes aortic stenosis and contributes to heart failure and stroke. Disease pathogenesis is multifactorial, and stresses such as inflammation, extracellular matrix remodeling, turbulent flow, and mechanical stress and strain contribute to the osteogenic differentiation of valve endothelial and valve interstitial cells. However, the precise initiating factors that drive the osteogenic transition of a healthy cell into a calcifying cell are not fully defined. Further, the only current therapy for CAVD-induced aortic stenosis is aortic valve replacement, whereby the native valve is removed (surgical aortic valve replacement, SAVR) or a fully collapsible replacement valve is inserted via a catheter (transcatheter aortic valve replacement, TAVR). These surgical procedures come at a high cost and with serious risks; thus, identifying novel therapeutic targets for drug discovery is imperative. To that end, the present study develops a workflow where surgically removed tissues from patients and donor cadaver tissues are used to create patient-specific primary lines of valvular cells for in vitro disease modeling. This protocol introduces the utilization of a cold storage solution, commonly utilized in organ transplant, to reduce the damage caused by the often-lengthy procurement time between tissue excision and laboratory processing with the benefit of greatly stabilizing cells of the excised tissue. The results of the present study demonstrate that isolated valve cells retain their proliferative capacity and endothelial and interstitial phenotypes in culture upwards of several days after valve removal from the donor. Using these materials allows for the collection of control and CAVD cells, from which both control and disease cell lines are established.

This protocol describes the collection of human aortic valves extracted during surgical aortic valve replacement procedures or from cadaveric tissue, and the subsequent isolation, expansion, and characterization of patient specific primary valve endothelial and interstitial cells. Included are important details regarding the processes needed to ensure cell viability and phenotype specificity.

Calcific aortic valve disease (CAVD) is present in nearly a third of the elderly population. Thickening, stiffening, and calcification of the aortic valve ...

Tissue Collection and RNA Extraction from the Human Osteoarthritic Knee Joint

Osteoarthritis (OA) is a chronic and degenerative joint disease most often affecting the knee. As there is currently no cure, total knee arthroplasty (TKA) is a common surgical intervention. Experiments using primary human OA tissues obtained from TKA provide the capability to investigate disease mechanisms ex vivo. While OA was previously thought to impact mainly the cartilage, it is now known to impact multiple tissues in the joint. This protocol describes patient selection, sample processing, tissue homogenization, RNA extraction, and quality control (based on RNA purity, integrity, and yield) from each of seven unique tissues to support disease mechanism investigation in the knee joint. With informed consent, samples were obtained from patients undergoing TKA for OA. Tissues were dissected, washed, and stored within 4 h of surgery by flash freezing for RNA or formalin fixation for histology. Collected tissues included articular cartilage, subchondral bone, meniscus, infrapatellar fat pad, anterior cruciate ligament, synovium, and vastus medialis oblique muscle. RNA extraction protocols were tested for each tissue type. The most significant modification involved the method of disintegration used for low-cell, high-matrix, hard tissues (considered as cartilage, bone, and meniscus) versus relatively high-cell, low-matrix, soft tissues (considered as fat pad, ligament, synovium, and muscle). It was found that pulverization was appropriate for hard tissues, and homogenization was appropriate for soft tissues. A proclivity for some subjects to yield higher RNA integrity number (RIN) values than other subjects consistently across multiple tissues was observed, suggesting that underlying factors such as disease severity may impact RNA quality. The ability to isolate high-quality RNA from primary human OA tissues provides a physiologically relevant model for sophisticated gene expression experiments, including sequencing, that can lead to clinical insights that are more readily translated to patients.

Primary tissues obtained from patients following total knee arthroplasty provide an experimental model for osteoarthritis research with maximal clinical translatability. This protocol describes how to identify, process, and isolate RNA from seven unique knee tissues to support mechanistic investigation in human osteoarthritis.

Osteoarthritis (OA) is a chronic and degenerative joint disease most often affecting the knee. As there is currently no cure, total knee arthroplasty ...