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

プロトコル

1. Construction and quality control of the BBB-Minibrain (Blood-brain barrier - Minibrain) (Figure 1)

1. Setting up a BBB-Minibrain Polyester membrane culture insert device (Figure 1B)
   1. 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.
   2. Coat a 12 well plate with poly-D-lysine (1 mL/well, 10 µg/mL, 4 h at RT) and then laminin (1 mL/well, 1 µg/mL, overnight at RT).
   3. Remove laminin and add 1 mL of endothelial cell medium supplemented with 5% fetal bovine serum (FBS) (5% endothelial cell medium).
   4. Incubate for 1 h at 37 °C, 5% CO₂ and 95% humidity.
   5. 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.
   6. 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.
   7. 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 °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 "blood" 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 "brain" compartment) (Figure 1B).
2. Validation of the endothelial permeability of the BBB-Minibrain (Quality Control) (Figure 1C)
   1. Prepare the transport buffer (TB), which is HBSS (Hanks' Balanced Salt Solution) buffer with Ca²⁺ and Mg²⁺ supplemented with 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and 1 mM sodium pyruvate.
   2. Prepare 12 well plates with 1.5 mL of transport buffer per well.
   3. At T= 0, make fresh transport buffer supplemented with 50 µM Lucifer Yellow (LY-TB).
   4. Reverse each filter upside down to remove carefully the medium without affecting the endothelial cell barrier.
   5. Place the filter on the filled 12 well plate and add 0.5 mL of LY-TB.
   6. Incubate the plates in an incubator at 37°C, 5% CO₂ and 95% humidity.
   7. 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.
   8. At T= 25 min, repeat the step 1.2.7.
   9. At T= 45 min, stop the transport by removing the filters from the plates. Keep abluminal and luminal compartments for OD measures.
   10. Transfer in a dark 96 well plates the samples: 10 µL with 190 µL TB for the LY-TB and the luminal compartments, 200 µL of sample for the abluminal compartments.
   11. Measure the fluorescence of the LY-TB present in the different samples at λ428 nm λ535 nm (excitation and emission length waves respectively).
   12. Calculate the endothelial permeability (Pe) toward LY-TB (Pe_LY) by using the formula:
       1/PSe= (1/PSt) – (1/PSf) and Pe_LY= 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), Pe_LY is expressed in cm/min. For hCMEC/D3 the Pe_LY should be between 0.7 and 1.2 x 10^-3 cm/min depending mainly of the FBS used in the experiments. Important: a Pe_LY higher than 1.2 means that the BBB is not tight enough and some leakage can be observed. Discard these barriers.

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)

1. BBB crossing and multiplication of Yellow fever viruses in the Minibrain
   1. 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.
   2. At T= 0, add 3500 Plaque Forming Units (PFU) of YFV-FNV diluted in 50 µL of 2% FBS endothelial cell medium very carefully on the top of the luminal compartment. The control BBB-Minibrain is inoculated with 50 µL of 2% FBS endothelial cell medium without virus. Determine Pe_LY on companion well.
   3. Incubate the BBB-Minibrain in an incubator at 37 °C, 5% CO₂ and 95% humidity.
   4. After 24 h, remove the polyester membrane culture inserts filter device and determine Pe_LY, sample 1 mL from the abluminal compartment and titrate the virus. Replace the medium with fresh 2% FBS endothelial cell medium.
   5. Incubate the BBB-Minibrain at 37 °C, 5% CO₂ and 95% humidity.
   6. 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.

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結果

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 endothelial cell barrier and the abluminal (Brain) compartment containing the Minibrain cells (human neurons, astrocytes and microglial cells). C. Representative measures of the BBB-Minibrain permeabililty by TEER (i.e., TransEndothelial Electrical Resistance) on three devices or permeability to LY (i.e., PeLY) on five filters. D. expression analysis by quantitative reverse transcription polymerase chain reaction (qRT-PCR) of the Receptors, efflux Transporters and Transporters on the endothelial cells hCMEC/D3.

Figure 2: The BBB-Minibrain model allows identification of neuroinvasive variants among live YFV vaccine preparation. A. Schedule of the experiment. B. Quantification of the viruses which have crossed the BBB by plaque forming unit (PFU) titration of live virus (each point represents one polyester membrane culture inserts experiment). C. Plot of Interferon Stimulated Gene 15 (ISG15) and Interferon Regulatory Factor 7 (IRF7) gene expression measured by qRT-PCR as a function of the number of viral particles in the viral load.

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資料

Name	Company	Catalog Number	Comments
12 well plates	Corning	3336
85mm Petri Dish	Sarstedt	83-3902-500
CHME/Cl5	Unité de Neuroimmunologie Virale	On request to Dr Lafon
CMC	Calbiochem	217274
Dark 96 well plates	Corning	3915
DMEM F12	Thermofisher Scientific	31330-038
DMSO	Merck-Sigma Aldrich	D2650
Endogro IV	Millipore	SCME004
Ethanol	Carlo Erba	529121
FBS	Hyclone	SV30015-04	endothelial cell medium
HBSS with Ca2+-Mg2+	Thermofisher Scientific	14025-100
hCMEC/D3	Cedarlane	CLU512
Hepes 1M	Thermofisher Scientific	15630-070
Laminine	Merck-Sigma Aldrich	L6274
L-glutamin	Thermofisher Scientific	25030-024
Lucifer Yellow	Merck-Sigma Aldrich	L0259
MEM 10X	Thermofisher Scientific	21430
MEM 1X	Thermofisher Scientific	42360
Ntera/Cl2D.1	ATCC	CRL-1973
Paraformaldehyde	Electron Microscopy Sciences	15714
PBS without Ca2+-Mg2+	Thermofisher Scientific	14190
PBS-Ca2+-Mg2+	Thermofisher Scientific	14040-091
Pen/Strep	Eurobio	CXXPES00-07
Poly-d-Lysine	Merck-Sigma Aldrich	P1149
Prolong Gold	Thermofisher Scientific	P36930
RNA purification kit	QIAGEN	74104
Sodium bicarbonate 5.6%	Eurobio	CXXBIC00-07
Sodium Pyruvate	Thermofisher Scientific	11360
T75 Cell+ Flask	Sarstedt	83-1813-302	Tissue culture polystyrene flask with specific surface treatment (Cell+) for sensitive adherent cells
Transwell	Corning	3460	polyester porous membrane culture inserts
Trypsin-EDTA	Merck-Sigma Aldrich	T3924
Ultra Pure Water	Thermofisher Scientific	10977-035
Versene	Thermofisher Scientific	15040-033	EDTA
YFV-FNV	IP Dakar	Vaccine vial