Get documents about "In vitro Blood-Brain Barrier model
Document Sample
In vitro Blood-Brain Barrier model
Pharmaceutical R&D
A primary rat in vitro blood-brain barrier model
to predict drug candidate entrance to the brain
nicolas.perriere@vigicell.fr
Blood-Brain Barrier: Complex and active interface between blood and brain
The Blood-Brain Barrier: Paracellular Passive Efflux Influx Trancytosis
Foot astrocyte route diffusion transporters transporters specific routes
Pericyte
• Maintains brain homeostasis
BLOOD
• Controls all flux between blood and brain
Tight
Brain
• Protects from xenobiotics junction
us
endothelial cell
le
uc
N
Particular properties of BBB’s endothelial cells are
Lumen conferred by the cerebral microenvironment: BRAIN
Tight Hydrophilic Lipophilic Specific
junction • Expression of tight junctions compounds compounds substrates
Basal
lamina • Only specific passage Properties of BBB are a combination of
Endothelial cell passive diffusion, active and facilitated transport (efflux and influx).
• Expression of polarized transporters
VigiCell offers a physiological in vitro model that maintains these specific properties
1 Similar to in vivo 2 Quality & Reproducibility
In vivo characteristics preserved using our in vitro standardized model Standard animal
• Primary culture • Strain: Crl:OFA(SD) rat, male, 2 weeks old
Brain endothelial cell • Fresh primary cells
• Rat syngenic coculture BLOOD
(endothelial cells [RBECs] + glial cells) Astrocytes Protocol strategy
BRAIN
• Freshly isolated cells Preservation of brain endothelium properties
• Soft enzymatic digestions
Tight junction expression, localization and functionality • Short amplification
Claudin-3 Claudin-5 Occludin ZO-1 • Reduced culture time to avoid dedifferentiation
Standardized protocol
in vivo
Paracellular permeability Minimization of cell culture intra- and inter-variability
(Pe : 10-3 cm/min)
fluorescein
• Positive selection of brain endothelial cells with BBB phenotype
sucrose inulin FD4 FD40 FD70
0.10 0.05 0.12 0.05 0.015 0.01 • Seeding on inserts at D4 with a defined cellular density
in vitro
• Permeability study realized at D10
Low permeability values
similar to in vivo
Internal control
Tight junction staining in freshly isolated brain capillaries and RBECs.
Junctional localization in both cases • A fluorescent dye is coadministrated with the drug candidate in each
BBB insert
Transporter expression, polarization and functionality
Pe
Perm(Fluorescein)AtoB = 0.12 ± 0.02 10-3 cm/min (n=40)
P-gp Bcrp Oatp-2 400
A to B w/o P-gp inhibitor
Efflux ratio = 3.24
(10-3 cm/min)
B to A w/o P-gp inhibitor
0.813 ± 0.056
A to B w/ P-gp inhibitor
B to A w/ P-gp inhibitor
in vivo
300
3 Services : screening & customized studies
Quantity (pmol)
0.533 ± 0.060
200 0.409 ± 0.022
0.251 ± 0.014
CELL CULTURE
100 • Production of 2 cultures per week (up to 96 inserts)
in vitro
0 ADAPTABILITY
0 20 40 60 80 100 120
Time (minutes) • Shipment and protocol adapted to your needs
Transporter staining in freshly isolated brain capillaries and Polarized transport of Vinblastin (concentrations, controls, reproducibility, mechanistic studies…)
RBECs. Membrane expression of transporters similar to in vivo • Access to mass spectrometry facility
* VALIDATION
150 *
[Daunorubicin] (% of control)
[Daunorubicin] (% of control)
100
A to B 125
A to B vs B to A B to A • Control of membrane integrity for each culture insert
Daunorubicin transport in the *
Daunorubicin 100 presence or not of specific 75
Daunorubicin • Control of P-gp functionality for each culture
transport efflux transporter inhibitors transport
75
50
50
Polarized transport OUR COMMITMENT
25
25 of Daunorubicin • Permeability experiments within 8 days after reception of the drug
0
- P-gp Mrps Bcrp
0
- P-gp Mrps Bcrp candidate
Transporter inhibited Transporter inhibited • Your analysis within 24h after assay results are received
Predictive pharmacokinetics
in vivo In vivo Kin
(µL/s/g) Diffusion
R = 0.94 4 Publications
100 Imipramine
Glut-1influx • Perrière N et al. Puromycin-based purification of rat brain capillary endothelial cell cultures. Effect on the
P-gp efflux Diazepam expression of blood-brain barrier-specific properties. J Neurochem. 2005 Apr;93(2):279-89.
10 Paracellular route • Perrière N et al. A functional in vitro model of rat blood-brain barrier for molecular analysis of efflux
In situ Brain Perfusion * in vitro brain
transporters. Brain Res. 2007 May 30;1150:1-13.
Combined passages D-Glucose endothelial
Prazosine cells
In vivo technique which permits
to strictly measure passage of a
1 • Adenot M et al. Applications of a blood-brain barrier technology platform to predict CNS penetration of
chosen molecule through the
Vinblastine astrocytes
BBB by radioactive detection Colchicine Syngenic co-culture of various chemotherapeutic agents. 1. Anti-infective drugs. Chemotherapy. 2007;53(1):70-2.
without peripheral influences freshly isolated rat cells
0.1 Vincristine
*mice data from JM Scherrmann
M6G
(astrocytes and
capillary brain
• Pifferi F et al. n-3 Fatty acids modulate brain glucose transport in endothelial cells of the blood-brain
Inulin Sucrose
endothelial cells) barrier.Prostaglandins Leukot Essent Fatty Acids. 2007 Nov-Dec;77(5-6):279-86.
0.01 In vitro Pe
-3
0.01 0.1 1 10 (10 cm/min)
nicolas.perriere@vigicell.fr
Strong correlation between in vitro and in vivo permeabilities, independent of the passage mechanisms Tel. : +33 1 49 58 34 79
7 rue Guy Moquet 94800 Villejuif FRANCE
Copyright VigiCell
www.vigicell.fr & www.vigicell.eu
Related docs
