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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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’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.

Protocol

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).

  1. Culturing the Ntera/Cl2.D1
    1. Remove a vial of frozen cells from liquid nitrogen tank. Keep on ice.
    2. Thaw the cells rapidly in a 37 °C water bath.
    3. Transfer the cells in a 15 mL tube containing 10 mL of complete DMEM F12 medium (Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 medium) supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, 100 IU penicillin and 100 µg streptomycin (i.e., complete DMEM F12 medium).
    4. Centrifuge at 200 x g for 5 min at room temperature (RT). Dissociate the pellet in 2 mL of complete DMEM F12 medium.
    5. Transfer in a T75 tissue culture flask in polystyrene with specific treatment for sensitive adherent cells (T75Cell+) containing 13 mL of complete DMEM F12 medium.
    6. Culture cells in an incubator maintained at 37 °C, 5% CO2 and 95% humidity until 90% confluence (i.e., around 5 days). Change medium every 2-3 days.
  2. Subculturing the Ntera/Cl2.D1 and amplification
    1. Remove the medium from the flask.
    2. Rinse the cell monolayer with 10 mL of phosphate buffer saline (PBS) supplemented with Ca2+ and Mg2+.
    3. Rinse the cell monolayer with 10 mL of EDTA solution (kept at RT).
    4. Add 3 mL of trypsin-EDTA solution (0.05% trypsin, 0.02% EDTA) and incubate 2 min at RT.
    5. Shake the flask and make sure the cells are detached from the plastic surface.
    6. Add 10 mL of complete DMEM F12 medium to inactivate the trypsin, transfer in a 15 mL tube and centrifuge for 5 min at 200 x g at RT.
    7. Dissociate the pellet with 10 mL of complete medium and use 1 mL to inoculate each new flask containing 14 mL of complete DMEM F12 medium.
      NOTE: Thirty T75 Cell+ flasks are required for each differentiation.
    8. Incubate the flasks at 37 °C, 5% CO2 and 95% humidity until 90% confluence.
  3. Differentiation of NTera/Cl2.D1 in human neuron-astrocyte co-culture
    NOTE: This is the main component of the Minibrain.
    1. At Day 0, dissociate the cells with trypsin-EDTA as described in step 1.2 and dissociate the cell pellet of each flask with 2 mL of complete DMEM F12 medium.
    2. Add 1 mL of the cell suspension (corresponding to 5 x 106 cells) in 60 plastic Petri dishes of 85 mm diameter containing 11 mL of complete DMEM F12 medium.
      NOTE: The cells will not attach to the plastic and will aggregate to form pseudo neurospheres (see photograph in the lower panel of Figure 1). Incubate the 60 Petri dishes at 37 °C, 5% CO2 and 95% humidity for one day.
    3. At Day 1, add 1 mL of complete DMEM F12 medium supplemented with 130 µM of all-Trans Retinoic Acid (ATRA) per Petri dish (final concentration ATRA 10 µM) and return the dishes at 37 °C, 5% CO2 and 95% humidity for 24 h.
    4. At Day 2, transfer very carefully the medium containing cell spheres of each Petri Dish in a 50 mL tube and centrifuge for 10 min at 50 x g and RT. Dissociate carefully the loose pellet with 13 mL of complete DMEM F12 medium supplemented with 10 µM ATRA and add the 13 mL in a new 85 mm diameter Petri dish.
      NOTE: Over the different passages, it is extremely important to maintain the spheres density as high as the density obtained at Day 2 (i.e., around a density of 70%). Therefore, if the sphere density is lowering, reduce the total number of Petri dishes according to the number of cells.
    5. Repeat the procedure above (step 1.3.4) at days 4, 6 and 8.
    6. At Day 10, repeat the procedure above but add the spheres to the T75 Cell+ flasks instead of Petri dishes. Add the cells from one Petri dish to one T75 Cell+ flask.
    7. At Days 11, 13, 15 and 17, change the used medium with 14 mL of complete DMEM F12 supplemented with 10 µM ATRA.
    8. At Day 19, change medium with 14 mL of complete DMEM F12 medium without ATRA per flask.
    9. At Day 20, dissociate gently the cells with trypsin/EDTA as follows.
      1. For each T75 Cell+ flask, rinse the cells with 10 mL of PBS supplemented with Ca2+ and Mg2+; incubate the cells with 4 mL of EDTA for 5 min at RT.
      2. Remove carefully the EDTA and add 2 mL of trypsin-EDTA and incubate 7 min at RT. Shake very gently the flask and add carefully 8 mL of complete DMEM F12 medium.
      3. Centrifuge 10 for min at 160 x g and RT. Dissociate the cell pellet in 13 mL of complete DMEM F12 medium and transfer in a T75 Cell+ flask.
        NOTE: The very gentle dissociation with trypsin-EDTA will allow recovering the ATRA differentiated cells from the original teratocarcinoma cells, which are strongly attached to the plastic ware.
    10. At Day 21, remove the medium and the dead cells. Add 14 mL of complete DMEM F12 medium supplemented with 5% FBS, 2 mM glutamine, 100 IU penicillin, 100 µg streptomycin and 0.5 µM of AraC (Cytosine β-D-arabinofuranoside) (complete 5% FBS DMEM F12-AraC).
    11. At Days 23, 25 and 28, replace used medium with 14 mL of 5% FBS DMEM F12-AraC.
    12. At Days 29 up to 49 (every two days), replace used medium with 14 mL of complete DMEM F12 medium supplemented with 5% fetal bovine serum, 2 mM glutamine, 100 IU penicillin, 100 µg streptomycin, 5 µM of FudR (5-fluoro-2’deoxyuridine) and 10 µM Urd (Uridine) (complete 5% FBS DMEM F12-FudR-Urd).
    13. At Day 50, replace used medium with 14 mL of complete DMEM F12 medium supplemented with 5% FBS, 2 mM glutamine, 100 IU penicillin, 100 µg streptomycin and 10 µM Urd (complete 5% FBS DMEM F12-Urd).
    14. At Days 51 up to 95 (twice a week), replace used medium with 14 mL of complete 5% FBS DMEM F12-Urd.
      NOTE: Cells can be used for experiments from day 51 but not later than day 95. The use of serial treatments with mitosis inhibitors allows recovering pure population of co-culture of post-mitotic hNeuron and hAstrocytes (NT2-N/A).
    15. To seed the cells, proceed with gentle trypsinization as described for Day 20.

2. Cell culture work of human microglial cells CHME/Cl5

NOTE: This is the microglial component of the Minibrain (Figure 1).

  1. Culturing the human microglial cells CHME/Cl5
    1. Remove a vial of frozen cells from the liquid nitrogen tank. Keep on ice.
    2. Thaw rapidly in a 37 °C water bath.
    3. Transfer the cells in a 15 mL tube containing 10 mL of complete DMEM F12 medium supplemented with 5% fetal bovine serum, 2 mM glutamine, 100 IU penicillin and 100 µg streptomycin (i.e., complete 5% FBS DMEM F12 medium).
    4. Centrifuge at 200 x g for 5 min at RT. Dissociate the pellet with 2 mL of complete 5% FBS DMEM F12 medium.
    5. Transfer in a T75 Cell+ flask containing 13 mL of complete 5% FBS DMEM F12 medium.
    6. Culture cells at 37 °C, 5% CO2 and 95% humidity until 90% confluence. Change medium every 2-3 days.
  2. Subculturing the human microglial cells CHME/Cl5
    1. Remove medium from the flask.
    2. Rinse the cell monolayer with 10 mL of PBS supplemented with Ca2+ and Mg2+.
    3. Rinse the cell monolayer with 10 mL of EDTA solution (kept at RT).
    4. Add 3 mL of trypsin-EDTA solution and incubate 2 min at RT.
    5. Shake the flask and make sure the cells are detached from the plastic surface.
    6. Add 10 mL of complete 5% FBS DMEM F12 medium to inactivate the trypsin, transfer in a 15 mL tube and centrifuge for 5 min at 200 x g and RT.
    7. Dissociate the pellet with 10 mL of complete medium and use 1 mL to inoculate each new flask containing 14 mL of complete 5% FBS DMEM F12 medium.
    8. Incubate the flasks at 37 °C, 5% CO2 and 95% humidity until 90% confluence.

3. Culture work with the hCMEC/D3 to coat porous inserts and prepare BBB

  1. Culturing the human endothelial cells hCMEC/D3 (Figure 2)
    1. Dilute the type I rat Collagen to 1:30 with pure sterile water (cell culture grade).
    2. Transfer 10 mL into a T75 Cell+ flask and incubate 2 h in a 37 °C, 5% CO2 and 95% humidity incubator.
    3. Remove the collagen solution and replace it with 15 mL of endothelial cell medium supplemented with 10 mM of HEPES (referred as complete endothelial cell medium).
    4. Remove a cryo vial of cells from the liquid nitrogen tank. Keep on ice.
      NOTE: The cells were grown, and a seed lot was made as described by the manufacturer. The cell line is covered by a biological material transfer agreement. The cells are obtained at passage number 25 and should not be used further than passage 35.
    5. Thaw rapidly in a 37 °C water bath.
    6. Transfer the cells in the T75 Cell+ flask and return the flask at 37 °C, 5% CO2 and 95% humidity for 2 to 4 h.
    7. Remove the medium carefully without losing or removing cells. Rinse the cells once with 10 mL of complete endothelial cell medium.
    8. Add 15 mL of complete endothelial cell medium.
    9. Culture cells at 37 °C, 5% CO2 and 95% humidity until 100% confluence (not less than 4 days) without changing the medium.
  2. Subculturing the hCMEC/D3 to prepare the insert of the BBB
    1. Coat a new T75 Cell+ flask or inserts with the type I Rat Collagen as described above (step 3.1).
    2. Remove the medium from the flask. Rinse the cell monolayer with 10 mL of PBS supplemented with Ca2+ and Mg2+.
    3. Add 2 mL of trypsin-EDTA solution and incubate 5 min at 37 °C.
    4. Shake the flask and make sure that the cells are fully detached from the plastic surface.
    5. Add 4 mL of complete endothelial cell medium to inactivate the trypsin.
    6. With a 5 mL plastic pipette, mechanically dissociate the cells by aspirating and flushing the cell suspension while maintaining the 5 mL pipette to the bottom of the flask at least 5 times.
    7. Count the cells and use 5 x 104 cells/insert for a 12 well polyester membrane culture inserts insert and 2 x 106 cells for a T75 Cell+ flask. Prepare also three filters without cells for the PELy (i.e., endothelial cells permeability to Lucifer yellow see below paragraph 4.2) experiments with the Polyester membrane culture inserts.
    8. Incubate the flasks or inserts at 37 °C, 5% CO2 and 95% humidity until 100% confluence.
    9. Do not change the medium for T75 Cell+ flask until a new passage. On the contrary for BBB on polyester membrane culture inserts: change medium at days 2 and 4, use the BBB for experiments at day 6.

4. Construction and quality control of the BBB-Minibrain (Figure 2)

  1. Setting up a BBB-Minibrain Polyester membrane culture insert device (Figure 2B)
    1. Grow the 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% FBS (5% endothelial cell medium).
    4. Incubate for 1 h at 37 °C, 5% CO2 and 95% humidity.
    5. Gently trypsinize the NT2-N/A (as described in step 1.3.9) and CHME/Cl5 (as described in step 2.2) 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 2B).
  2. Validation of the endothelial permeability of the BBB-Minibrain (Quality Control) (Figure 2C)
    1. Prepare the transport buffer (TB), which is HBSS (Hanks' Balanced Salt Solution) buffer with Ca2+ and Mg2+ supplemented with 10 mM 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 4.2.7.
    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 (PeLY) according to Siflinger-Birnboim, A et al. (1987)39 and Da Costa, A et al. (2018)34 by using the formula:
      1/PSe= (1/PSt) – (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.

5. 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 3)

  1. BBB crossing and multiplication of Yellow fever viruses in the Minibrain
    1. Use the BBB-Minibrain prepared as described in step 4.1.7 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 PeLY 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 PeLY, sample 1 mL from the abluminal compartment and titrate the virus as described by A. da Costa et al. (2018)34. 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 as described by A. da Costa et al. (2018)34.
  2. Amplification of neurotropic variants of YFV-FNV on Minibrain cells by serial passages
    1. Use Minibrain cells coated 12 well plates (i.e., steps 4.1.6 and 4.1.7).
    2. At time point 0 h, add 3,500 Plaque Forming Units of YFV-FNV diluted in 50 µL of 2% FBS endothelial cell medium very carefully on the top of the cells (luminal compartment).
    3. After 1 h, remove the medium with the virus inoculum and replace with fresh 2% FBS endothelial cell medium.
    4. After 48 h, sample 500 µL of the culture medium and infect fresh Minibrain cells
    5. After 120 h, sample 500 µL of the culture medium and infect fresh Minibrain cells.
    6. After 192 h, save culture medium (=virus stock enriched) and extract the RNA from the Minibrain cells for gene expression analysis as described by A. da Costa et al. (2018)34.

6. Use of BBB-Minibrain to study BBB crossing and brain cell targeting of a biomolecule

  1. Transport across the BBB of a neuron targeting biomolecule
    1. Use the Minibrain-BBB prepared as described step 4.1.7.
    2. At time point 0 h, add the biomolecule (In the example provided in the result section, 58.75 ng/BBB of the cell permeant NeuroTag-NV molecules were added per BBB-Minibrain insert.
    3. Incubate the BBB-Minibrain at 37 °C, 5% CO2 and 95% humidity.
    4. After 24 h, remove the polyester membrane culture inserts filter device and determine PeLY, then stain the Minibrain cells for hNeurons neurofilament Nf200 and detect the presence of the biomolecule in the Minibrain (In the example provided in the result section, NeuroTag-NV was detected using an antibody directed against the Strep-Tag it contains35,40).
  2. Transport across the BBB of a neuroregenerative biomolecule and subsequent neuroprotective assay in the Minibrain after the biomolecule has crossed the BBB
    1. Use the Minibrain-BBB prepared as described in step 4.1.7.
      NOTE: For molecules targeting neurons only, Minibrain cells can be replaced by the Neurons cells (NT2-N) only38.
    2. At time point 0 h, add 58.75 ng/BBB-Minibrain well of the cell permeant NV molecules (active form CPM-NeuroTag-NV, non-active form CPM-NeuroTag-NVΔ) described by C. Prehaud et al. (2014)35.
    3. Incubate the BBB-Minibrain in an incubator at 37 °C, 5% CO2 and 95% humidity.
    4. After 4 h, make individual wounds with an injection needle (26GX1/2”, 12-4.5, Terumo, Belgium) on the Minibrain cells. Make at least 10 scratches on each individual well. Determine PeLY on the companion well.
    5. Incubate the BBB-Minibrain in an incubator at 37 °C, 5% CO2 and 95% humidity.
    6. After 8 h, replace the medium in the abluminal compartment by fresh complete endothelial cell medium.
      NOTE: It is important to replace the medium at this step since the wounding of the Minibrain cells can lead to cell death and then the release of cytotoxic compounds.
    7. Incubate the BBB-Minibrain in an incubator at 37 °C, 5% CO2 and 95% humidity.
    8. After 48 h, stained the cells for axon regeneration of hNeurons as described by C. Prehaud et al. (2013)40.

Results

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...

Discussion

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...

Disclosures

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

Acknowledgements

This study was supported by internal grants from Institut Pasteur including an Incitative grant (PTR 435) and by a grant “Contrat de Soutien à la Recherche” 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.

Materials

NameCompanyCatalog NumberComments
12 well platesCorning3336
5-fluoro-2’deoxyuridineMerck-Sigma AldrichF0503
85mm Petri DishSarstedt83-3902-500
Anti-Nf200Merck-Sigma AldrichN4142
β-mercapto-ethanolMerck-Sigma AldrichM3148
CHME/Cl5Unité de Neuroimmunologie ViraleOn request to Dr Lafon
CMCCalbiochem217274
Cytosine β-D-arabinofuranosideMerck-Sigma AldrichC1768
Dark 96 well platesCorning3915
DMEM F12Thermofisher Scientific31330-038
DMSOMerck-Sigma AldrichD2650
Endogro IVMilliporeSCME004endothelial cell medium
EthanolCarlo Erba529121
FBSHycloneSV30015-04
FormaldehydeMerck-Sigma Aldrich252549
GIEMSARAL Diagnostic320310
Goat-Anti MouseJackson Immuno Research115-545-003
Goat-Anti RabbitThermofisher ScientificR37117
HBSS with Ca2+-Mg2+Thermofisher Scientific14025-100
hCMEC/D3CedarlaneCLU512
Hepes 1MThermofisher Scientific15630-070
Hoescht 33342Merck-Sigma Aldrich33263
LaminineMerck-Sigma AldrichL6274
L-glutaminThermofisher Scientific25030-024
Lucifer YellowMerck-Sigma AldrichL0259
MEM 10XThermofisher Scientific21430
MEM 1XThermofisher Scientific42360
Ntera/Cl2D.1ATCCCRL-1973
ParaformaldehydeElectron Microscopy Sciences15714
PBS without Ca2+-Mg2+Thermofisher Scientific14190
PBS-Ca2+-Mg2+Thermofisher Scientific14040-091
Pen/StrepEurobioCXXPES00-07
Poly-d-LysineMerck-Sigma AldrichP1149
Prolong GoldThermofisher ScientificP36930
QiashredderQIAGEN79656
Rat Collagen ICultrex3443-100-01
Retinoic Acid All-TransMerck-Sigma AldrichR2625
RNA purification kitQIAGEN74104
SDSMerck-Sigma AldrichL4509
Sodium bicarbonate 5.6%EurobioCXXBIC00-07
Sodium PyruvateThermofisher Scientific11360
T75 Cell+ FlaskSarstedt83-1813-302Tissue culture polystyrene flask with specific surface treatment (Cell+) for sensitive adherent cells
TranswellCorning3460polyester porous membrane culture inserts
Trypsin-EDTAMerck-Sigma AldrichT3924
Ultra Pure WaterThermofisher Scientific10977-035
UridineMerck-Sigma AldrichU3750
VerseneThermofisher Scientific15040-033EDTA
YFV-FNVIP DakarVaccine vial

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