This study was supported by internal grants from Institut Pasteur including an Incitative grant (PTR 435) and by a grant &#8220;Contrat de Soutien &#224; la Recherche&#8221; provided by Sanofi Pasteur to Institut Pasteur. A. da Costa was supported by the Sanofi-Pasteur grant and Florian Bakoa is recipient of a PhD grant provided by ANRT (Association Nationale de la Recherche et de la Technologie). We are indebted to Pr Pierre-Olivier Couraud and Dr Florence Miller for helpful discussions.

1. Cell culture work of Ntera/CL2.D1 to prepare a co-culture of post-mitotic hNeurons and hAstrocytes (NT2-N/A)
NOTE: This is the component of the Minibrain (Figure 1).
<ol>
	<li>Culturing the Ntera/Cl2.D1
	<ol>
		<li>Remove a vial of frozen cells from liquid nitrogen tank. Keep on ice.</li>
		<li>Thaw the cells rapidly in a 37 &#176;C water bath.</li>
		<li>Transfer the cells in a 15 mL tube containing 10 mL of complete DMEM F12 medium (Dulbecco&#39;s Modified ...

The intellectual property of the system was the patents referenced in 32, 35 and 38.

In this article we demonstrated how to build an in cellulo blood/brain interface, the BBB-Minibrain, by combining a BBB model and a culture of mixed brain cerebral cells (Minibrain) into a single kit. This system is biologically relevant, easy to set up and handle for experimenters well trained in cell culture.
As for any other in vitro model of BBB, reliable results would be obtained if drastic control of tightness of the barrier is applied. Inserts should be carefully tested for permeability...

The brain is separated from the systemic circulation by a non-permeable structure that restricts exchanges between the brain parenchyma and the blood, called the blood-brain barrier (BBB). Mostly composed of cerebral endothelial cells, the BBB dynamically interacts with astrocytes, perivascular microglia and neurons of the neighboring brain parenchyma. The three major functions of the BBB are the creation and maintenance of ionic homeostasis for neuronal functions, supply of the brain with nutrients, and protection from toxic injuries or entry of pathogens1,2, which contribute to the maintenance of brain homeostasis and its functions3. This barrier is so efficient that only few drugs can cross the BBB4,5. At present, the available methods to predict whether a molecule will pass the BBB and diffuse into the brain consist of ex vivo studies on autopsy material, image tracking in the brain of human volunteers by MRI (magnetic resonance imaging) or PET (positon emission tomography) or pharmacodynamics and pharmacokinetic preclinical studies in animals6,7,8. These techniques and models have some limitations, such as the limited resolution of PET and the low sensitivity of MRI6,8, the difficulty to quantify molecules (i.e., antibody based molecules for example) that poorly penetrate the brain7, and for the preclinical studies their high cost and resort of animal testing.
The last point is important because, according to the 3R&#8217;s rules, (replacement, reduction and refinement of animal testing) the regulatory administrations have asked that the researchers urgently develop scientifically accurate alternative to animal experimentation9,10,11,12,13,14,15.
Over the last decades, several in vitro models of BBB have been proposed16,17,18 by cultivating on filter membrane inserts endothelial cells from different species such as mouse, rat, bovine and pig. As far as the human species is concerned, the scarce and difficult availability of primary cells prompted the researchers to develop human models based on immortalized brain endothelial cells or human-derived stem cells19,20,21. These barriers are proper in vitro surrogates of BBB provided that they express endothelial cell markers, tight junction markers, efflux transporters, solute carriers, receptors, and respond to the endothelial stimuli 20. A few BBB models using filter membrane inserts coated with endothelial cells and other cell types (i.e., astrocytes, neurons or pericytes22,23,24) were assayed. The goal of these co-cultures was to increase the BBB physical characteristics by taking advantage of the secretion of soluble factors by astrocytes/neurons or pericytes.
Nevertheless, none of these models includes brain parenchyma to study and predict the fate of a drug candidate once it has passed the barrier. Therefore, our goal was to build an in cellulo blood/brain interface, the BBB-Minibrain, by combining a BBB model and a culture of mixed brain cells into a single kit. The BBB-Minibrain uses a culture system consisting of a porous filter inserted in a well of a multiwell cell culture plate. The filter is coated with hCMEC/D3 cells, a human brain endothelial cell line that has been proved highly reliable for BBB drug testing25,26,27, to form the BBB. The Minibrain, which is a co-differentiated culture of human neurons and astrocytes derived from the NTera/Cl2.D1 cell line28,29 mixed together with the human microglial cell line CHME/Cl530 in ratio corresponding to the microglia vs. neuron-astrocytes ratios of the brain31, is cultivated in the bottom of the plate well.
Besides studying passage of drugs across the BBB and their fate in the parenchyma, the blood-brain interface in cellulo model could be a powerful tool to address the entry of pathogens into the brain (neuroinvasiveness), the dispersion into the brain (neurotropism) and the toxicity (neurovirulence) they can exert on brain parenchyma cells. Neurovirulence and neuroinvasiveness studies would benefit from the development of an efficient in cellulo model and be advantageous to replace animal models. Using the BBB-Minibrain kit32, we demonstrated the neuroinvasive phenotype of rare viral mutants that accumulated in the French Neurotropic virus strain of Yellow Fever Virus (i.e., FNV-YFV33,34) used to prepare a discontinued live YFV vaccine and the passage of a neuroregenerative and neuroprotective biomolecule called Neurovita (referred as NV henceforth in the manuscript)35. Because NV neither naturally crosses the cell membrane nor the BBB, NV was fused with the variable part (VHH) of a single chain antibody of Llama that crosses the biological membranes including the BBB and functions as a cell penetrating molecule (CPM)36. The CPM property of VHH seems to depend upon the isoelectric point and the length of the VHH37.
This in cellulo test should make it possible to sort the molecules that could potentially cross the BBB before carrying out pharmacokinetic and pharmacodynamics analysis in animals, and ideally in the same time to be able to predict their behavior in the nervous parenchyma. This system is biologically relevant and easy to set up and handle by professionals well trained in cell culture26,29,30,38. The interest of such in cellulo testing would be two-fold: reducing the costs of preclinical tests on the one hand and reducing the use of animal testing on the other hand.

<table>
	<tbody>
		<tr>
			<td>12 well plates</td>
			<td>Corning</td>
			<td>3336</td>
			<td></td>
		</tr>
		<tr>
			<td>5-fluoro-2&#8217;deoxyuridine</td>
			<td>Merck-Sigma Aldrich</td>
			<td>F0503</td>
			<td></td>
		</tr>
		<tr>
			<td>85mm Petri Dish</td>
			<td>Sarstedt</td>
			<td>83-3902-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Nf200</td>
			<td>Merck-Sigma Aldrich</td>
			<td>N4142</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-mercapto-ethanol</td>
			<td>Merck-Sigma Aldrich</td>
			<td>M3148</td>
			<td></td>
		</tr>
		<tr>
			<td>CHME/Cl5</td>
			<td>Unit&#233; de Neuroimmunologie Virale</td>
			<td>On request to Dr Lafon</td>
			<td></td>
		</tr>
		<tr>
			<td>CMC</td>
			<td>Calbiochem</td>
			<td>217274</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytosine &#946;-D-arabinofuranoside</td>
			<td>Merck-Sigma Aldrich</td>
			<td>C1768</td>
			<td></td>
		</tr>
		<tr>
			<td>Dark 96 well plates</td>
			<td>Corning</td>
			<td>3915</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM F12</td>
			<td>Thermofisher Scientific</td>
			<td>31330-038</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Merck-Sigma Aldrich</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>Endogro IV</td>
			<td>Millipore</td>
			<td>SCME004</td>
			<td>endothelial cell medium</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Carlo Erba</td>
			<td>529121</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Hyclone</td>
			<td>SV30015-04</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>Merck-Sigma Aldrich</td>
			<td>252549</td>
			<td></td>
		</tr>
		<tr>
			<td>GIEMSA</td>
			<td>RAL Diagnostic</td>
			<td>320310</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat-Anti Mouse</td>
			<td>Jackson Immuno Research</td>
			<td>115-545-003</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat-Anti Rabbit</td>
			<td>Thermofisher Scientific</td>
			<td>R37117</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS with Ca2+-Mg2+</td>
			<td>Thermofisher Scientific</td>
			<td>14025-100</td>
			<td></td>
		</tr>
		<tr>
			<td>hCMEC/D3</td>
			<td>Cedarlane</td>
			<td>CLU512</td>
			<td></td>
		</tr>
		<tr>
			<td>Hepes 1M</td>
			<td>Thermofisher Scientific</td>
			<td>15630-070</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoescht 33342</td>
			<td>Merck-Sigma Aldrich</td>
			<td>33263</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminine</td>
			<td>Merck-Sigma Aldrich</td>
			<td>L6274</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamin</td>
			<td>Thermofisher Scientific</td>
			<td>25030-024</td>
			<td></td>
		</tr>
		<tr>
			<td>Lucifer Yellow</td>
			<td>Merck-Sigma Aldrich</td>
			<td>L0259</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM 10X</td>
			<td>Thermofisher Scientific</td>
			<td>21430</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM 1X</td>
			<td>Thermofisher Scientific</td>
			<td>42360</td>
			<td></td>
		</tr>
		<tr>
			<td>Ntera/Cl2D.1</td>
			<td>ATCC</td>
			<td>CRL-1973</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>15714</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS without Ca2+-Mg2+</td>
			<td>Thermofisher Scientific</td>
			<td>14190</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS-Ca2+-Mg2+</td>
			<td>Thermofisher Scientific</td>
			<td>14040-091</td>
			<td></td>
		</tr>
		<tr>
			<td>Pen/Strep</td>
			<td>Eurobio</td>
			<td>CXXPES00-07</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-d-Lysine</td>
			<td>Merck-Sigma Aldrich</td>
			<td>P1149</td>
			<td></td>
		</tr>
		<tr>
			<td>Prolong Gold</td>
			<td>Thermofisher Scientific</td>
			<td>P36930</td>
			<td></td>
		</tr>
		<tr>
			<td>Qiashredder</td>
			<td>QIAGEN</td>
			<td>79656</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat Collagen I</td>
			<td>Cultrex</td>
			<td>3443-100-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Retinoic Acid All-Trans</td>
			<td>Merck-Sigma Aldrich</td>
			<td>R2625</td>
			<td></td>
		</tr>
		<tr>
			<td>RNA purification kit</td>
			<td>QIAGEN</td>
			<td>74104</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Merck-Sigma Aldrich</td>
			<td>L4509</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate 5.6%</td>
			<td>Eurobio</td>
			<td>CXXBIC00-07</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Pyruvate</td>
			<td>Thermofisher Scientific</td>
			<td>11360</td>
			<td></td>
		</tr>
		<tr>
			<td>T75 Cell+ Flask</td>
			<td>Sarstedt</td>
			<td>83-1813-302</td>
			<td>Tissue culture polystyrene flask with specific surface treatment (Cell+) for sensitive adherent cells</td>
		</tr>
		<tr>
			<td>Transwell</td>
			<td>Corning</td>
			<td>3460</td>
			<td>polyester porous membrane culture inserts</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Merck-Sigma Aldrich</td>
			<td>T3924</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra Pure Water</td>
			<td>Thermofisher Scientific</td>
			<td>10977-035</td>
			<td></td>
		</tr>
		<tr>
			<td>Uridine</td>
			<td>Merck-Sigma Aldrich</td>
			<td>U3750</td>
			<td></td>
		</tr>
		<tr>
			<td>Versene</td>
			<td>Thermofisher Scientific</td>
			<td>15040-033</td>
			<td>EDTA</td>
		</tr>
		<tr>
			<td>YFV-FNV</td>
			<td>IP Dakar</td>
			<td>Vaccine vial</td>
			<td></td>
		</tr>
	</tbody>
</table>

a human blood-brain interface model to study barrier crossings by pathogens or medicines and their interactions with the brain

The early screening of nervous system medicines on a pertinent and reliable in cellulo BBB model for their penetration and their interaction with the barrier and the brain parenchyma is still an unmet need. To fill this gap, we designed a 2D in cellulo model, the BBB-Minibrain, by combining a polyester porous membrane culture insert human BBB model with a Minibrain formed by a tri-culture of human brain cells (neurons, astrocytes and microglial cells). The BBB-Minibrain allowed us to test the transport of a neuroprotective drug candidate (e.g., Neurovita), through the BBB, to determine the specific targeting of this molecule to neurons and to show that the neuroprotective property of the drug was preserved after the drug had crossed the BBB. We have also demonstrated that BBB-Minibrain constitutes an interesting model to detect the passage of virus particles across the endothelial cells barrier and to monitor the infection of the Minibrain by neuroinvasive virus particles. The BBB-Minibrain is a reliable system, easy to handle for researcher trained in cell culture technology and predictive of the brain cells phenotypes after treatment or insult. The interest of such in cellulo testing would be twofold: introducing derisking steps early in the drug development on the one hand and reducing the use of animal testing on the other hand.

The BBB-Minibrain is an in cellulo experimental model of blood-brain interface.
The BBB-Minibrain is set up on the polyester membrane culture insert system to mimic a blood compartment on the upper level and a brain compartment on the lower level of the blood-brain interface (Figure 2A,B). It consists of a luminal compartment with the hCMEC/D3 endothelial cells on t...

Watch this Scientific Journal Video about A Human Blood-Brain Interface Model to Study Barrier Crossings by Pathogens or Medicines and Their Interactions with the Brain at JoVE.com

A Human Blood-Brain Interface Model to Study Barrier Crossings by Pathogens or Medicines and Their Interactions with the Brain

Here we present a protocol describing the setting of an in cellulo BBB (Blood brain barrier)-Minibrain polyester porous membrane culture insert system in order to evaluate the transport of biomolecules or infectious agents across a human BBB and their physiological impact on the neighboring brain cells.

The early screening of nervous system medicines on a pertinent and reliable in cellulo BBB model for their penetration and their interaction with the ...

This human blood-brain interface model is useful for studying the transfer of molecules into the neuro system. As well as for studying of microbes reach an infected brain pain tumor. The addition of the mini brain to this blood brain barrier or BBB model allows the study of our pathogens and molecules that cause the BBB behave in the brain.Demostrating the procedure will be Florian Bakao, the PhD students for my laboratory. To set up a BBB mini brain polyester membrane culture insert device, dilute the cells to a five times 10 to the fourth cells per insert concentration incomplete endothelial cell medium and add the appropriate volume of cells to each polyester membrane culture insert in a 12 well plate. Then, place the inserts and the cell culture incubator until the cells reach 100%confluency.Hold a 12 well plate with one milliliter of poly-D-lysine her well for four hours at room temperature. Followed by one milliliter of laminin per well overnight. Replace the laminin with one milliliter of endothelial cell medium supplemented with 5%fetal bovine serum, or FBS.Place the place in an incubator for one hour. To prepare the mini grain gently trypsinize the human neurons astrocytes in CHME clone five cells. Associate the cell pellets with complete endothelial cell medium.After counting at 3.6 times 10 to the fifth neurons and astrocytes to 0.4 times 10 to the fifth CHME clone five cells per well have a 12 well plate. And seed the 12 well plate. To construct the BBB mini brain 24 hours after seeding replace the used medium with fresh complete endothelial cell medium and transfer the cerebral micro vascular endothelial cell coated polyester membrane culture inserts over the mini brain cells.Then, place the BBB many brain device into the incubator. To validate the BBB mini brain endothelial permeability add 1.5 milliliters of transport buffer per well to a new 12 well plate. And flip each filter upside down to carefully remove the medium without affecting the endothelial cell barrier.Place each filter in one well of the transport buffer filled plate, and add 0.5 milliliters of Lucifer yellow into each well. Then place the plate into the incubator for 10 minutes before transferring the filters into a new transport buffer filled 12 well plate. After setting up a BBB many brain device as demonstrated, add 3500 platforming units of the French neurotrophic virus strain of yellow fever virus diluted and 50 microliters of 2%FPS endothelial cell medium, very carefully, on top of luminal compartment.Inoculate the control BBB mini brain with 50 microliters of 2%FBS endothelial cell medium without virus. Then place the virus expose BBB many brain device in the incubator for 24 hours. The next day, remove the inserts to determine the endothelial cell permeability and a companion well as just as demonstrated.Save one milliliter of sample from each well to determine the virus titer in the middle compartment. Under the insert gently to avoid leakage of the luminal compartment into the abluminal compartments. To study the transport of a neuron targeting biomolecular across the blood brain barrier, add the bio molecule of interest to the inserts and return the plate to the cell culture incubator.After 24 hours remotely inserts and determine endothelial permeability. Then stain the many brain cells with the appropriate antibody for detecting the presence of the bio molecule of interest within the mini brain. Human cerebral micro vascular endothelial cells express all the subsets of rececptors, Efflux transporters or transporters that are key relevant proteins for their biological functions.Some French neuro tropic virus strain of yellow fever virus can cross the blood brain barrier at 24 hours. The viruses then amplify for the next two days by the multiplication of the virus inside the brain cells. Further, the expression of specific viral biomarkers is stimulated when this neuro invasive viral population is serially passaged on the mini brain cells and strictly correlate to the viral load.After being added to the limit of compartment cell penetrating molecule Neuro-Tag Neurovita crosses the blood brain barrier and is able to target human neurons. Cell penetrating molecule neuro tag Delta neurovita crosses the endothelial cell barrier, but targets less efficiently the human neurons. After being added to the little middle compartment, cell penetrating molecule Neuro-Tag Neurovita crosses the blood brain barrier and is able to regenerate the axons of human neurons, after wounding.In contrast to cell penetrating molecule Neuro-Tag Neurovita Delta, the inactive form of Neurovita. When applied after the axon scratching cell penetrating molecule Neuro-Tag Neurovita triggers the neuro protection of the wounded neurons and the x zonal regeneration. Strictly for the good sake of practices.do not over use I-cell passages. Try to maintain some semblance of medium and regions and check for the absence of mycoplasma. Interactions between the mini brain and the drugs are microbes of interest can be further studied by transcript that chemicalize this or classical biology called techniques.

a-human-blood-brain-interface-model-to-study-barrier-crossings

Research

JoVE Journal

Neuroscience

8.5K Views.  Institut Pasteur, CNRS UMR 3569, Unité de Neuroimmunologie Virale. This human blood-brain interface model is useful for studying the transfer of molecules into the neuro system. As well as for studying of microbes reach an infected brain pain tumor. The addition of the mini brain to this blood brain barrier or BBB model allows the study of our pathogens and molecules that cause the BBB behave in the brain.Demostrating the procedure will be Florian Bakao, the PhD students for my laboratory. To set up a BBB mini brain polyester membrane culture insert d...