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

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 pathogens^1,2, which contribute to the maintenance of brain homeo....

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 .......

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.......

Materials

Name	Company	Catalog Number	Comments
12 well plates	Corning	3336
5-fluoro-2’deoxyuridine	Merck-Sigma Aldrich	F0503
85mm Petri Dish	Sarstedt	83-3902-500
Anti-Nf200	Merck-Sigma Aldrich	N4142
β-mercapto-ethanol	Merck-Sigma Aldrich	M3148
CHME/Cl5	Unité de Neuroimmunologie Virale	On request to Dr Lafon
CMC	Calbiochem	217274
Cytosine β-D-arabinofuranoside	Merck-Sigma Aldrich	C1768
Dark 96 well plates	Corning	3915
DMEM F12	Thermofisher Scientific	31330-038
DMSO	Merck-Sigma Aldrich	D2650
Endogro IV	Millipore	SCME004	endothelial cell medium
Ethanol	Carlo Erba	529121
FBS	Hyclone	SV30015-04
Formaldehyde	Merck-Sigma Aldrich	252549
GIEMSA	RAL Diagnostic	320310
Goat-Anti Mouse	Jackson Immuno Research	115-545-003
Goat-Anti Rabbit	Thermofisher Scientific	R37117
HBSS with Ca2+-Mg2+	Thermofisher Scientific	14025-100
hCMEC/D3	Cedarlane	CLU512
Hepes 1M	Thermofisher Scientific	15630-070
Hoescht 33342	Merck-Sigma Aldrich	33263
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
Qiashredder	QIAGEN	79656
Rat Collagen I	Cultrex	3443-100-01
Retinoic Acid All-Trans	Merck-Sigma Aldrich	R2625
RNA purification kit	QIAGEN	74104
SDS	Merck-Sigma Aldrich	L4509
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
Uridine	Merck-Sigma Aldrich	U3750
Versene	Thermofisher Scientific	15040-033	EDTA
YFV-FNV	IP Dakar	Vaccine vial