Name: an in vitro model for studying pathogen crossing of the blood-brain barrier and brain cell infection
Uploaded: 2025-03-31T15:00:44.000+00:00
Duration: 1 min 30 s
Description: Source: da Costa, A., et al. A Human Blood-Brain Interface Model to Study Barrier Crossings by Pathogens or Medicines and Their Interactions with the ...

an in vitro model for studying pathogen crossing of the blood-brain barrier and brain cell infection

An In Vitro Model for Studying Pathogen Crossing of the Blood-Brain Barrier and Brain Cell Infection

After setting up a BBB mini brain device, as demonstrated, add 3,500 plaque forming units of the French neurotropic virus strain of yellow fever virus, diluted in 50 microliters of 2% FBS endothelial cell medium very carefully on top of the luminal compartment. Inoculate the control BBB mini brain with 50 microliters of 2% FBS endothelial cell medium without virus. Then, place the virus exposed BBB mini brain device in the incubator for 24 hours.
The next day, remove the inserts to determine the endothelial cell permeability in a companion well, as just demonstrated, and save 1 milliliter of sample from each well to determine the virus titer in the abluminal compartment.
Under the insert gently to avoid leakage of the luminal compartment into the abluminal compartment.

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1. Construction and quality control of the BBB-Minibrain (Blood-brain barrier - Minibrain) (Figure 1)
<ol>
	<li>Setting up a BBB-Minibrain Polyester membrane culture insert device (Figure 1B)
	<ol>
		<li>Grow the human brain endothelial cell line (hCMEC/D3 cells) on 12 well Polyester membrane culture insert filters for 6 days on endothelial cell medium before using them for the experiment.</li>
		<li>Coat a 12 well plate with poly-D-lysine (1 mL/well, 10 &#181;g/mL, 4 h at RT) and then laminin (1 mL/well, 1 &#181;g/mL, overnight at RT).</li>
		<li>Remove laminin and add 1 mL of endothelial cell medium supplemented with 5% fetal bovine serum (FBS) (5% endothelial cell medium).</li>
		<li>Incubate for 1 h at 37 &#176;C, 5% CO2 and 95% humidity.</li>
		<li>Gently trypsinize the post-mitotic human neurons (hNeurons) and human astrocytes (hAstrocytes) (NT2-N/A) and human microglial cell line (CHME/Cl5) and dissociate the cell pellets with complete endothelial cell medium.</li>
		<li>Count the cells, mix 3.6 x 105 NT2-N/A and 0.4 x 105 CHME/Cl5 cells/well and seed the 12 well plate.</li>
		<li>At T=24 h (24 h after seeding): change the used medium with fresh complete endothelial cell medium (Minibrain cells and endothelial cells) and transfer the hCMEC/D3 Polyester membrane culture insert filter on the top of the Minibrain cells. Incubate the BBB-Minibrain at 37 &#176;C, 5% CO2 and 95% humidity. 
		NOTE: The BBB-Minibrain will then consist of a layer of human endothelial cells hCMEC/D3 on filter isolating the luminal compartment (or &#34;blood&#34; compartment) and of a mixed culture of human cells hNT2-N/A and hCHME/Cl5 (Minibrain) at the bottom of the well defining the abluminal compartment (or &#34;brain&#34; compartment) (Figure 1B).</li>
	</ol>
	</li>
	<li>Validation of the endothelial permeability of the BBB-Minibrain (Quality Control) (Figure 1C)
	<ol>
		<li>Prepare the transport buffer (TB), which is HBSS (Hanks&#39; Balanced Salt Solution) buffer with Ca2+ and Mg2+ supplemented with 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and 1 mM sodium pyruvate.</li>
		<li>Prepare 12 well plates with 1.5 mL of transport buffer per well.</li>
		<li>At T= 0, make fresh transport buffer supplemented with 50 &#181;M Lucifer Yellow (LY-TB).</li>
		<li>Reverse each filter upside down to remove carefully the medium without affecting the endothelial cell barrier.</li>
		<li>Place the filter on the filled 12 well plate and add 0.5 mL of LY-TB.</li>
		<li>Incubate the plates in an incubator at 37&#176;C, 5% CO2 and 95% humidity.</li>
		<li>At T=10 min transfer the filters on new TB filled 12 well plates and keep the abluminal compartment of the first plate for OD reading.</li>
		<li>At T= 25 min, repeat the step 1.2.7.</li>
		<li>At T= 45 min, stop the transport by removing the filters from the plates. Keep abluminal and luminal compartments for OD measures.</li>
		<li>Transfer in a dark 96 well plates the samples: 10 &#181;L with 190 &#181;L TB for the LY-TB and the luminal compartments, 200 &#181;L of sample for the abluminal compartments.</li>
		<li>Measure the fluorescence of the LY-TB present in the different samples at &#955;428 nm &#955;535 nm (excitation and emission length waves respectively).</li>
		<li>Calculate the endothelial permeability (Pe) toward LY-TB (PeLY) by using the formula: 
		1/PSe= (1/PSt) &#8211; (1/PSf) and PeLY= PSe/S. 
		NOTE: PSf is the permeability of the filter without cells, PSt is the permeability of the filter with cells, PSe is the permeability of the endothelial monolayer time the surface of the monolayer, S is the surface of the monolayer (for a 12 well filter=1.12 cm2), PeLY is expressed in cm/min. For hCMEC/D3 the PeLY should be between 0.7 and 1.2 x 10-3 cm/min depending mainly of the FBS used in the experiments. Important: a PeLY higher than 1.2 means that the BBB is not tight enough and some leakage can be observed. Discard these barriers.</li>
	</ol>
	</li>
</ol>
2. Use of BBB-Minibrain to highlight the presence of neuro-invasive viral particles in a Yellow Fever Virus vaccine sample, the French Neurotropic virus, YFV-FNV 34 (Figure 2)
<ol>
	<li>BBB crossing and multiplication of Yellow fever viruses in the Minibrain
	<ol>
		<li>Use the BBB-Minibrain prepared as described in step 1.1.7 24 h before the addition of the virus; replace the medium by 2% FBS endothelial cell medium. 
		NOTE: It is extremely important to avoid changing the medium just before adding the virus. Changing the medium can activate the human endothelial cells and transiently open the barrier which will allow the passage of the virus. Here the medium is changed 24 h before starting the experiment.</li>
		<li>At T= 0, add 3500 Plaque Forming Units (PFU) of YFV-FNV diluted in 50 &#181;L of 2% FBS endothelial cell medium very carefully on the top of the luminal compartment. The control BBB-Minibrain is inoculated with 50 &#181;L of 2% FBS endothelial cell medium without virus. Determine PeLY on companion well.</li>
		<li>Incubate the BBB-Minibrain in an incubator at 37 &#176;C, 5% CO2 and 95% humidity.</li>
		<li>After 24 h, remove the polyester membrane culture inserts filter device and determine PeLY, sample 1 mL from the abluminal compartment and titrate the virus. Replace the medium with fresh 2% FBS endothelial cell medium.</li>
		<li>Incubate the BBB-Minibrain at 37 &#176;C, 5% CO2 and 95% humidity.</li>
		<li>After 72 h, sample the medium from the abluminal compartment and titrate the virus, and/or extract the RNA from the Minibrain cells for gene expression analysis.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>12 well plates</td>
			<td>Corning</td>
			<td>3336</td>
			<td></td>
		</tr>
		<tr>
			<td>85mm Petri Dish</td>
			<td>Sarstedt</td>
			<td>83-3902-500</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>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></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>endothelial cell medium</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>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>RNA purification kit</td>
			<td>QIAGEN</td>
			<td>74104</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>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>

Source: da Costa, A., et al. <a href="https://app.jove.com/v/59220">A Human Blood-Brain Interface Model to Study Barrier Crossings by Pathogens or Medicines and Their Interactions with the Brain.</a> J. Vis. Exp. (2019).
This video demonstrates an in vitro model to study pathogens crossing the blood-brain barrier (BBB). Microvascular endothelial cells, which express tight junctions, form an endothelial layer in the upper chamber, while neurons, astrocytes, and microglia represent the brain parenchyma in the lower chamber, separated by a porous membrane. Virus infection in the BBB model mimics the pathogen crossing from blood to the brain. Upon introducing a neurotropic virus, it crosses the BBB via transcytosis, infects neural cells, replicates, and releases viral progeny, demonstrating successful neuroinvasion.

<img alt="Figure 1" src="/files/ftp_upload/23203/23203_Figure1.jpg" /> 
Figure 1: the BBB-Minibrain model. A. Time schedule of the hCMEC/D3 cultures and photograph of the BBB-Minibrain device: a polyester membrane culture inserts-filter with hCMEC/D3 endothelial barrier is held with a forcep before to be inserted in to one well of a 12 wells cell culture plate. B. Cartoon describing the device with the luminal (Blood) compartment containing the endot...

Source: da Costa, A., et al. A Human Blood-Brain Interface Model to Study Barrier Crossings by Pathogens or Medicines and Their Interactions with the ...

Take an in vitro blood-brain barrier, or BBB, model comprising two compartments separated by a porous membrane.
The upper or luminal compartment contains microvascular endothelial cells. These cells express tight junctions, mimicking the blood-brain barrier endothelium that limits the passage of cells and molecules.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;
The lower or abluminal compartment contains neurons, astrocytes, and microglia, representing the brain parenchyma.
Introduce a neurotropic virus into the luminal compartment to simulate a blood-borne infection and incubate.
The virus is endocytosed by the endothelial cells, undergoes intracellular trafficking within vesicles, and is released on the abluminal side.
Post-incubation, remove the upper chamber. Sample the medium from the abluminal side to assess the viral quantity, confirming the successful crossing of the endothelial layer.
Add fresh medium and incubate again.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;
The virus enters the neural cells via endocytosis, replicates, and releases viral progeny.
Quantify the increased extracellular viral load to confirm viral neuroinvasion and replication.

14 vues. An In Vitro Model for Studying Pathogen Crossing of the Blood-Brain Barrier and Brain Cell Infection

Vidéo: An In Vitro Model for Studying Pathogen Crossing of the Blood-Brain Barrier and Brain Cell Infection - Expérience

A Benchtop Approach to the Location Specific Blood Brain Barrier Opening using Focused Ultrasound in a Rat Model

Stereotaxic surgery is the gold standard for localized drug and gene delivery to the rodent brain. This technique has many advantages over systemic delivery including precise localization to a target brain region and reduction of off target side effects. However, stereotaxic surgery is highly invasive which limits its translational efficacy, requires long recovery times, and provides challenges when targeting multiple brain regions. Focused ultrasound (FUS) can be used in combination with circulating microbubbles to transiently open the blood brain barrier (BBB) in millimeter sized regions. This allows intracranial localization of systemically delivered agents that cannot normally cross the BBB. This technique provides a noninvasive alternative to stereotaxic surgery. However, to date this technique has yet to be widely adopted in neuroscience laboratories due to the limited access to equipment and standardized methods. The overall goal of this protocol is to provide a benchtop approach to FUS BBB opening (BBBO) that is affordable and reproducible and can therefore be easily adopted by any laboratory.

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An In Vitro Model for Studying Pathogen Crossing of the Blood-Brain Barrier and Brain Cell Infection

Transcription

An In Vitro Model for Studying Pathogen Crossing of the Blood-Brain Barrier and Brain Cell Infection