EP3665264A1

EP3665264A1 - Device that can serve as a hemato-encephalitic barrier model

Info

Publication number: EP3665264A1
Application number: EP18756178.2A
Authority: EP
Inventors: Guylène PAGE; Hanitriniaina RABEONY; Damien CHASSAING; Emilie DUGAST; Thierry Janet
Original assignee: Universite de Poitiers; Centre Hospitalier Universitaire de Poitiers
Current assignee: Universite de Poitiers; Centre Hospitalier Universitaire de Poitiers
Priority date: 2017-08-10
Filing date: 2018-08-10
Publication date: 2020-06-17
Also published as: FR3070045B1; JP2020529871A; FR3070045A1; US20220010258A1; WO2019030380A1

Abstract

The present invention concerns a device that can serve as a hemato-encephalitic barrier (HEB) model comprising two compartments in which certain cell types are arranged. The invention also concerns a method for preparing said device and its use as an HEB model.

Description

Device that can serve as a model for a blood-brain barrier

The present invention relates to a device that can serve as a hematoencephalic (BBB) barrier model comprising two compartments in which certain cell types are arranged.

The brain is isolated from the bloodstream by a particular structure, the blood-brain barrier (BBB). This barrier is mainly composed of endothelial cells that interact with neighboring cells, particularly pericytes and astrocytes. These interact with microglia and neurons. The BBB maintains an environment that allows neurons to function properly by performing various key functions: by finely controlling the passage of molecules and ions, instantly delivering nutrients and oxygen to the needs of neurons and protecting the brain from toxins and pathogens.

In order for medications to act on the brain to be effective, they must be able to easily pass the BBB. To study the passage of molecules through the BBB and optimize it, several in vitro models of this barrier have been developed to date. These models most often include the culture of several cell types in which the endothelial cells are separated from other cell types by a porous synthetic membrane.

However, there is still a need for barrier models that are even closer to the BBB in vivo and can be used to carry out various studies such as the study of the physiopathology of certain degenerative diseases, the study of the aging of the BBB and the study of the passage of molecules in particular for therapeutic or diagnostic purposes, etc.

The inventors have sought and succeeded in putting pericytes and endothelial cells in direct contact, which has favored the appearance of structures organized in vessels, and has thus made it possible to obtain a tight model that is very close to the BBB in vivo.

An object of the invention is therefore a device comprising two compartments separated by a porous synthetic membrane: a so-called luminal compartment comprising endothelial cells and pericytes, and a so-called abluminal compartment comprising astrocytes and microglia. On the luminal side, peripheral blood mononuclear cells (PBMCs) can be deposited. This type of device makes it possible to obtain models of BBB with a seal and a functionality very similar to what is observed in vivo. The inventors have even observed that the endothelial cells organize themselves into vessels in this device.

The porous synthetic membrane may be tubular or planar.

By "luminal compartment", it is understood the compartment formed by the light of a tubular device or the upper compartment in a planar device.

By "abluminal compartment" is meant the compartment outside the luminal compartment in a tubular device or the lower compartment in a planar device.

This device comprising the major cellular actors of the BBB reproduces the neurovascular microenvironment forming the BBB in vivo and makes it possible to take into account and reproduce the multiple cellular and molecular interactions that can occur in vivo.

According to one embodiment, the pericytes and the endothelial cells are arranged in superposed layers in the luminal compartment. In a preferred manner, the pericytes are placed on or in contact with the porous synthetic membrane and the endothelial cells are placed above the pericytes, the latter therefore being in very close contact with the endothelial cells. The pericyte / endothelial cell seeding ratio may vary, in particular it may be chosen so as to correspond to the ratio present in the BBB studied, for example the human BBB. Preferably, the pericyte / endothelial seeding ratio is between about 1/2 to 1/4 and is preferably about 1/3 (corresponding to the ratio of pericytes / endothelial cells of human BBB).

In a specific embodiment, the luminal compartment further comprises blood cells, and preferably peripheral blood mononuclear cells (PBMCs). These are then arranged above the endothelial cells.

By "porous synthetic membrane" is meant a permeable support, which may be traversed by small molecules or ions or in a particular embodiment that may be traversed by cell extensions or cells depending on the size of the selected pores. This support allows the cells of each compartment to interact remotely. Around the membrane, a cellular structure similar to a BBB is gradually being put in place, under the combined action of the development of the seeded cells, and this once mature structure further comprises two extracellular matrices: the vascular basement membrane and the parenchymal basement membrane.

This porous synthetic membrane can be made of polyester (clear support allowing good visibility of the cells under the microscope) or polycarbonate (translucent support allowing low visibility of the cells under the microscope). This membrane may be previously coated ("coating") with constituents of the extracellular matrix, and in particular with collagen, laminin, fibronectin or a mixture thereof according to the desired applications. The size of the pores must be chosen according to the desired applications. If it is desired to conduct permeability, molecule transport, cell polarity, endocytosis of protein and receptor and / or cell interaction studies, it is preferable to take supports with a pore diameter of approximately 0.4 μηι and about 3 μηι in diameter and preferably about 0.4 μηι in diameter. Thus, in one embodiment, the support has a pore diameter of between about 0.4 and about 3 μηι, preferably about 0.4 μηι. For studies aimed at studying the passage of cells through the support, such as PBMCs for example, it will be preferable to take supports with a pore diameter of between approximately 3 μηι and approximately 8 μηι, preferably between approximately 5 μηι and approximately 8 μηι. Thus, in one embodiment, the support has a pore diameter of between about 3 μηι and about 8 μηι, preferably about 5 μηι about 8 μηι.

In a specific embodiment, the abluminal compartment comprising astrocytes further comprises microglia. The microglia / astrocyte seeding ratio may vary, in particular it may be chosen so as to correspond to the ratio present in the BBB studied, for example the human BBB. In the embodiment in which astrocytes and microglia are used, preferably the microglia / astrocyte seeding ratio is between about 1% and about 10% and is preferably about 5%. Preferably, the abluminal compartment comprising astrocytes is free of pericytes.

In a preferred manner, all the cells of the device according to the invention come from the same animal species, in particular from mammals. According to a mode of the invention, the cells are rodent cells, preferably mouse cells. According to another embodiment of the invention, the cells are primate cells, preferably human cells. According to one embodiment, one or more cell types of the device according to the invention are derived from immortal cell cultures, in a particular embodiment all cell types are derived from immortal cells.

By "immortal cells" is meant immortal cells derived from tumors, spontaneously immortal cells and / or cells rendered immortal ("immortalized") by introduction of at least one viral or cellular oncogene. According to a specific embodiment, one or more cell types of the device according to the invention are derived from primary cultures of cells made immortal by introduction of at least one viral or cellular oncogene.

In a preferred embodiment, one or more cell types of the device according to the invention are derived from primary cultures, preferably all cell types are derived from primary cultures.

By "primary culture" is meant a culture of cells directly from the tissue and / or cells of an individual. Alternatively, one or more cell types of the device according to the invention are derived from primary cultures of tissues and / or cells taken from individuals of the same species and of the same age, preferably all cell types are derived from cultures. tissues and / or cells taken from individuals of the same species and age. According to one embodiment of the invention, one or more and preferably all cell types are derived from adult individuals. In this way the device according to the invention also makes it possible to study the variations due to age or the impact on the BBB of diseases developing during the life of the individual. Cells from primary cultures retain contact inhibition in contrast to immortalized cells, so the use of these cells limits cell proliferation in the device. In addition, the use of primary cultures makes it possible to approach still more in vivo conditions.

By "individual" is meant a subject of an animal species, in particular mammals. According to a mode of the invention, the individual or individuals are rodents, preferably mice. According to another embodiment of the invention, the individuals are primates, preferably humans.

In a variant of the device according to the invention the endothelial cells and the pericytes are derived from primary cultures of mouse cells and, preferably, from adult mice. Particularly preferably, the endothelial cells and the pericytes are derived from primary cultures of mouse cells aged at least 3 months, more particularly at least 6 months and in a preferred mode of at least 12 months.

Thus, according to a variant of the process according to the invention, all cell types are derived from primary cultures of mouse cells and, preferably, from adult mice. Particularly preferably, all cell types are derived from primary cultures of mouse cells at least 3 months old, more preferably at least 6 months old and in a preferred mode of at least 12 months.

When one or more cell types of the device according to the invention are derived from primary cultures, then, according to a particular embodiment, one or more of these cell types are model cell types of pathologies.

"Cellular model disease types" means cell types derived from animal models reproducing pathologies occurring spontaneously or induced by genetic engineering methods (such as, for example, transgenesis) or with pharmacological tools to reproduce cell characteristics. individuals suffering from these particular pathologies. By way of example, we can cite cell types derived from APPswePS1 dE9 mouse models for Alzheimer's disease, hemi-parkinsonian or parkinsonian models (MPTP, 6-OHDA toxic injections) or mutated transgenic mice for Γα-synuclein for Parkinson's disease, mutated mouse models for the huntingtin gene for Huntington's disease, mutation of the C90RF72 gene for amyoptrophic lateral sclerosis (ALS). Preferably, the pathologies according to the invention are pathologies that have or are suspected of having an influence on the BBB, such as: neurodegenerative diseases (Alzheimer's, Parkinson's, Huntington's, ALS, etc.), cerebrovascular accident and cancers brain.

According to one possible embodiment, the porous synthetic membrane is in the form of a tube, in which a fluid can be introduced, renewed, circulated.

According to a preferred embodiment, the porous synthetic membrane is a plane membrane disposed horizontally. In this embodiment the compartments are one above the other.

According to one embodiment, the device can be cryopreserved to facilitate its transport or to delay its use.

The invention also relates to a method for preparing the device according to the invention. According to one embodiment of the invention, this method comprises the following steps:

a) astrocyte seeding on a surface and / or on one side of the porous synthetic membrane (face which will become the abluminal face),

b) insertion or deposition of the porous synthetic membrane on said surface, such that if the porous synthetic membrane comprises astrocytes, then the face comprising the astrocytes is opposite the surface, c) seeding the face of the synthetic membrane porous luminal with pericytes and endothelial cells.

Step b) thus creates the abluminal compartment between the surface and the porous synthetic membrane.

In this embodiment of the invention, the surface is a support on which the device rests. When the porous synthetic membrane is flat, the surface may be for example the bottom of a culture box.

According to another embodiment of the invention, this method comprises the following steps:

a) astrocyte seeding on one side of the porous synthetic membrane,

b) seeding the other side of the porous synthetic membrane with endothelial cells and pericytes and optionally,

c) depositing the porous synthetic membrane on a surface so that the face comprising astrocytes is facing the surface.

According to one embodiment, the method of preparation of the device according to the invention comprises an additional step of cryopreservation of the device.

According to one embodiment, the pericytes and the endothelial cells are arranged in superposed layers in the luminal compartment. Preferably in the methods according to the invention the pericytes are seeded before the endothelial cells. In a preferred manner, the pericytes are placed on or in contact with the porous synthetic membrane and the endothelial cells are placed above the pericytes, the latter therefore being in close contact with the endothelial cells. The pericyte / endothelial cell seeding ratio may vary, in particular it may be chosen so as to correspond to the ratio present in the BBB studied, for example the human BBB. Preferably, the pericyte / endothelial seeding ratio is about 1/2 to 1/4 and is preferably about 1/3 (corresponding to the ratio in human BBB). Preferably the porous synthetic membrane is in contact with the cells of each compartment.

In a specific embodiment, the step of seeding the porous synthetic membrane with pericytes and endothelial cells further comprises adding blood cells, and preferably peripheral blood mononuclear cells (PBMCs). Preferably, the addition of blood cells takes place after seeding the pericytes and the endothelial cells.

In a specific embodiment, the one or more inoculation steps with astrocytes also include microglia seeding. Preferably astrocytes and microglia are cultured together before being seeded in the device. The microglia / astrocyte seeding ratio may vary, in particular it may be chosen so as to correspond to the ratio present in the BBB studied, for example the human BBB. Preferably, the microglia / astrocyte seeding ratio is about 5% (corresponding to the ratio in human BBB).

Preferably, the seeding step (s) with astrocytes do not include seeding pericytes.

In a preferred manner, all the cells seeded according to the process of the invention come from the same animal species, in particular from mammals. According to a mode of the invention, the cells are rodent cells, preferably mouse cells. According to another embodiment of the invention, the cells are primate cells, preferably human cells.

According to one embodiment, one or more cell types of the process according to the invention are derived from immortal cell cultures. In a particular embodiment, all cell types are derived from immortal cells.

In a preferred embodiment, one or more cell types of the method according to the invention are derived from primary cultures, preferably all the cell types are derived from primary cultures. In one variant, one or more cell types of the process according to the invention are derived from primary cultures of tissues taken from individuals of the same age, preferably all cell types are derived from primary cultures of tissues taken from individuals of the same age. According to one embodiment of the invention, one or more and preferably all cell types are derived from adult individuals.

Thus, according to one variant of the method according to the invention, endothelial cells and pericytes are derived from primary cultures of mouse cells and, preferably, from adult mice. Particularly preferably, endothelial cells and pericytes are derived from primary mouse cell cultures at least 3 months old, more preferably at least 6 months old and in a preferred mode of at least 12 months.

When one or more cell types of the method according to the invention are derived from primary cultures, then, according to a particular embodiment, one or more of these cell types are model cell types of pathologies.

The invention also relates to the use of the device according to the invention and in particular its use as a model of the BBB. The invention also relates to the use of the device according to the invention as a model of pathological BBB.

In particular the device can be used to test the permeability of the model. Indeed, this device can be used to study its permeability to:

molecules, especially therapeutic molecules (in particular small biological or synthetic molecules, antibodies, psychotropic substances, etc.),

- viruses (especially HIV),

spores (such as bacterial spores, fungal spores, plant spores, etc., for example for the study of fungal infections such as certain meningitis), or

exosomes (especially for studying the spread of cancers),

liposomes or nanoparticles,

naked or vectorized nucleic acids, cells (in particular PBMCs, cancer cells, bacteria, etc.);

hereinafter referred to as 'compounds'.

Thus, the invention relates to the use of the device according to the invention for testing the permeability of the model to a compound comprising the steps:

a) adding said compound to one of the compartments of the device,

b) incubation of the device,

c) detection and analysis of the presence of said compound and / or its metabolites in the compartment where the addition of said compound has not occurred, and optionally

d) deduce the permeability of the model vis-à-vis this compound.

Advantageously, during step a), a known quantity of the compound is added and step c) makes it possible to measure the amount of compound or its metabolites in the compartment where the addition has not taken place. In addition it is also possible to detect and analyze the presence of said compound or its metabolites in the compartment where the addition took place, in particular to determine the remaining amount of this compound in said compartment.

Techniques for detecting and analyzing the presence of the compound (or its metabolites) mentioned above are well known in the art. For example, the detection or ^analysis of the presence of the compound may be effected by various analytical chemistry techniques following the test compound whose HPLC coupled to one or even two mass spectrometry. Advantageously, the compound may be labeled in order to facilitate its detection. Indeed, if we have fluorescent or radiolabeled compounds (in particular tracers that would be used in diagnosis and radiolabeled, for example Fluor 18 or Carbon 1 1), readers of the fluorescence intensity or radioactivity counters respectively, can be used to quantify the labeled compound or its labeled metabolites in the luminal and abluminal compartments.

The invention furthermore relates to the use of the device according to the invention for studying the physiopathology of a disease, testing preventive, therapeutic or diagnostic molecules targeting the cellular and molecular actors of the BBB or testing physical conditions or testing protocols. By "studying the pathophysiology of a disease" is meant to study the impact of diseases on the characteristics of the BBB, such as permeability, selectivity, electrical resistance, cell morphology, etc. In this embodiment the device then comprises at least one model cell type of the pathology studied. In one embodiment the expression of proteins and / or the functionality of BBB transporters such as, for example, glycoprotein P (P-gp) or glucose transporter GLUT1 are compared in devices comprising at least one model cell type of the pathology studied and devices comprising no model cell type of this pathology. Protein expression can be assayed by Western Blot, ELISA, or gene expression (RTqPCR).

By "testing preventive, therapeutic or diagnostic molecules targeting the cellular and molecular actors of the BBB" we intend to study the impact of these molecules on the BBB and possibly their passage through the BBB. In this embodiment, at least one molecule to be tested is applied to the device according to the invention, and after a time of exposure or incubation, the device is analyzed in order to determine the changes that have been caused by said at least one a molecule tested. These changes may in particular concern the permeability, the selectivity, the electrical resistance, the morphology of the cells, etc. It is possible in particular to study the result of the addition of the molecule on the target by comparing the functionality and / or the expression of this target in devices in which the test molecule has been applied to devices in which it has not been applied. It is also possible to study the passage through the BBB of the molecule in a device according to the invention and in particular by additionally adding at least one carrier inhibitor of the BBB or a physical condition.

By "testing physical conditions", one intends to study the impact of these conditions on the BBB and possibly their influence on the passage of compounds through the BBB. In this embodiment, at least one physical condition is applied to the device according to the invention. After a time of exposure or incubation, the device is analyzed to determine the changes that have been caused by said at least one tested physical condition. These changes may in particular concern the permeability, the selectivity, the electrical resistance, the morphology of the cells, etc. One can notably study the result of the addition of the physical condition by comparing it with a device on which the physical condition has not been applied. By "Physical condition" refers in particular to the use of waves such as magnetic waves, electromagnetic waves or ultrasounds.

By "test protocols" we intend to study the impact of a treatment at the level of the BBB. In this embodiment, at least one treatment, i.e., a physical condition or a molecule, is applied to a compound as defined above, which compound is then contacted with the BBB. After a time of exposure or incubation, the device is analyzed to determine the changes that have been caused by said treatment. These changes may in particular relate to the permeability, the selectivity, the electrical resistance, the morphology of the cells, etc. The treatment result can in particular be studied by comparing it with a device put in contact with a compound which has not been treated. .

The invention will now be described in more detail using non-limiting examples.

Figures:

Figure 1: Diagram of preparation of a device comprising primary cell cultures according to the invention.

On D1, astrocytes and microglia are thawed and endothelial cells and pericytes are purified from mouse brains. PBMCs are extracted from the peripheral blood of mice.

On D3, the culture medium of the cells is renewed with cessation of the effect of puromycin in the endothelial cell medium.

On D5, the cell culture medium is renewed again.

On D8, astrocytes and microglia, represented by dots ". Are seeded in a culture dish. The culture media of the other cells are renewed. On D10, the microglia cells are observable.

On day 11, astrocytes and microglia are seeded on the porous synthetic membrane.

On D12, the porous synthetic membrane is deposited in the culture dish so that the two astrocyte cultures are in contact, thus forming the abluminal compartment. Then pericytes and endothelial cells, represented by squares and rounds ("□", "o") are seeded on the upper side of the membrane porous synthetic, thus forming the luminal compartment. The treatment of the hydrocortisone device begins.

On D15, treatment of the hydrocortisone device is complete, PBMCs, represented by "+" crosses, are seeded in the luminal compartment. The model is ready to use.

Figure 2: Paracellular permeability of dextran-FITC on a device according to the invention comprising primary cell cultures derived from model mice for Alzheimer (AD) or wild mice (Wild Type, WT).

The device permeability test is performed with dextran-FITC 4kD. The culture media are replaced by 1 mL of HBSS Ca ² 7Mg ²⁺ in the abluminal compartment and 500 μί of dextran-FITC in the luminal compartment (ie 2.10 ^"6 moles) 50 μί samples in the luminal compartments and abluminal are performed at 0 min, 10 min, 20 min, 30 min, 1 h and 1 h 30 and deposited in wells of a 96 well black plate, read on the player Varioskan (Thermo Scientific). (Aex) of dextran-FITC is 485 nm and the emission length (Aem) is 515 nm Figure 2 shows the results obtained for the abluminal compartment as a curve. ^* p <0.05 , ^** p <0.01 compared to the control device which corresponds to a device without cell but covered with the same "coating" matrix as the other devices studied (AD versus WJ).

The permeability of the device AD remains higher compared to the WT device (46%), indicating a poorer sealing of the pathological device compared to the healthy device. The statistical test used is the Kruskal-Wallis test followed by the Dunn test for multiple comparisons.

Figure 3: Paracellular permeability coefficient of dextran-FITC on a device according to the invention comprising primary cell cultures derived from wild-type (WT) mice.

The calculation of the coefficient of permeability Pe is done with this formula:

Pe = dQ / (dT * A * Co)

Pe: coefficient of permeability (cm / s)

dQ: quantity transported (mol)

dT: incubation time (second) A: surface of the porous synthetic membrane (here 1, 12 cm ² )

Co: initial concentration (4.10 ⁶ mol / cm ³ )

The determination of dQ is performed according to the following calculation:

(Abluminal fluorescence intensity at 1 h) * 2.10 ^{~ 6} / (luminescence fluorescence intensity at T0)

The fluorescence intensity is proportional to the amount of dextran-FITC present in each compartment (abluminal and luminal).

The coefficient of permeability is shown in Figure 3 and was calculated after one hour. The results represent the mean ± SEM (mean standard deviation) of the permeability coefficient of 3 to 4 devices in each group. ^* p <0.01 compared to the control device which corresponds to a device without cell but covered with the same "coating" matrix as the WT device. The statistical test used is the Mann Whitney test.

Figure 4: Functionality of the P glycoprotein in the device according to the invention.

This functionality test is carried out with rhodamine 123 because it is known that this molecule as a substrate of the glycoprotein P is expelled by the P-glycoprotein to the luminal compartment, which limits its passage through the device. The culture media are renewed: 1 ml in the abluminal compartment and 250 μl in the luminal compartment containing or not the Zosuquidar inhibitor of the 5 μ-glycoprotein P (Dantzig et al., 1996). The devices are incubated for 2 hours in the incubator. Then, 250 μl of medium containing rhodamine 123 at 2 μl with or without Zosuquidar at 5 μl are added to the luminal compartment and incubated for 1 hour in the incubator. Samples of 50 μί in the luminal and abluminal compartments are made just after the addition of the medium containing Rhodamine 123 and after 1 hour of incubation at 37 ° C. The samples are deposited in wells of a black plate of 96 wells. The fluorescence intensity is read on the same apparatus as for the permeability test (previously described). The Aex of rhodamine is 500nm and the Aem is 524nm.

In FIG. 4, the results represent the mean ± SEM of the fluorescence intensity after 1 h incubation of the cells with rhodamine 123 (n = 4 to 8 in each group). The passage of rhodamine 123 through the WT decreases by 72.6% compared to the device without cell ( ^** p <0.01). The presence of the inhibitor P-glycoprotein specificity inhibited efflux by 57.3%. The statistical test used is the Kruskal-Wallis test followed by the Dunn test for multiple comparisons.

Figure 5: Trans-endothelial electrical resistance (TEER) in murine devices according to the invention of 12 and 24 wells.

In Figure 5, the results represent the mean ± SEM. For the 12-well format (Figure 5A), n = 16 controls and n = 18 12-well devices. For the 24-well format (Figure 5B), n = 12 controls and n = 74 24-well devices. The statistical test used is the Mann Whitney test: ^**** p <0.0001.

Figure 6: Coefficient of permeability of Dextran-FITC on the murine devices according to the invention in 24 and 96 well format. The coefficient of permeability is shown in FIG. 6 and in the same manner as in FIG. 3 after one hour. In Figure 6, the results represent the mean ± SEM. For the 24-well format (Figure 6A), n = 12 controls and n = 50 devices. For the 96-well format (Figure 6B), n = 8 controls and n = 54 devices. The statistical test used is the Mann Whitney test: ^**** p <0.0001.

Figure 7: Functionality of the P-glycoprotein in the murine device according to the invention in 24 and 96 well format.

In Figure 7, the results represent the mean ± SEM. For the 24-well format (Figure 7A), n = 12 controls and n = 9 24-well devices without Zosuquidar and n = 10 24-well devices with Zosuquidar. For the 96-well format (Figure 7B), n = 7 controls and n = 6 96-well devices without Zosuquidar and 9 96-well devices with Zosuquidar. The statistical test used is the Kruskal-Wallis test followed by a Dunn test for multiple comparisons: ^* p <0.05, ^** p <0.01, ^*** p <0.001.

FIG. 8: Coefficient of permeability of 8 molecules on the murine devices according to the invention in 24 and 96 well format.

The molecules were added to the luminal compartment and incubated for 48 hours. The luminal and abluminal media were then taken for the determination of the molecules. In FIG. 8, the different calculated permeability coefficients are represented (the results represent the mean ± SEM). The results obtained for the 24-well format are represented in FIG. 8A, and the results obtained for the 96-well format in FIG. 8B. Figure 9: Trans-endothelial electrical resistance (TEER) and coefficient of permeability of DEXTRAN-FITC in the commercial device.

In Figure 9A is shown the trans-endothelial electrical resistance (TEER) (n = 14 controls and n = 36 24-well devices). FIG. 9B shows the coefficient of permeability of DEXTRAN-FITC (n = 8 controls and n = 23 24-well devices). The results represent the mean ± SEM. The statistical test used is the Mann Whitney test: ^**** p <0.0001.

Figure 10: Functionality of P-glycoprotein in the commercial device.

In Figure 10, the results represent the mean ± SEM of the fluorescence intensity after 1 h incubation of cells with rhodamine 123 (n = 4 to 8 in each group), n = 3 controls and n = 4 devices trade 24 wells without or with Zosuquidar. The statistical test used is the Kruskal-Wallis test followed by a Dunn test for multiple comparisons: ^* p <0.05.

Figure 11: Trans-endothelial electrical resistance (TEER) in 24-well formats after cryopreservation. In Figure 1 1 is shown the trans-endothelial electrical resistance (TEER)

(n = 3-4 controls and n = 2-15 devices in 24-well format). The 24-well format devices were cryopreserved for 7, 15 or 30 days and then used 4, 5 or 6 days post-thawing. The results represent the mean ± SEM. The statistical test used is the Mann Whitney test: ^* p <0.05, ^** p <0.01.

Figure 12: Permeability coefficient of DEXTRAN-FITC in 24-well formats after cryopreservation.

In FIG. 8 the different calculated permeability coefficients are represented (n = 8 controls and n = 2-5 24-well devices). The results represent the mean ± SEM). 24-well devices were cryopreserved 7, 15 or 30 days, then used 4, 5 or 6 days post-thawing. The statistical test used is the Mann Whitney test: ^* p <0.05, ^** p <0.01.

Figure 13: Trans-endothelial electrical resistance (TEER) in murine devices with immortalized cell lines according to the invention in the format 12 and 24 wells.

In Figure 13, the results represent the mean ± SEM. For the 12-well format (Figure 13A), n = 20 controls and n = 20 devices in 12-well format. For the 24-well format (Figure 13B), n = 10 controls and n = 97 devices in 24-well format. The statistical test used is the Mann Whitney test: ^**** p <0.0001.

Figure 14: Coefficient of permeability of Dextran-FITC on murine devices with immortalized cell lines according to the invention in 12, 24 and 96 well format.

In Figure 14, the results represent the mean ± SEM. For the 12-well format (Figure 14A), n = 10 controls and n = 6 devices in 12-well format. For the 24-well format (Figure 14B), n = 17 controls and n = 92 24-well devices. For the 96-well format (Figure 14C), n = 12 controls and n = 63 devices in 96-well format. The statistical test used is the Mann Whitney test: ^** p <0.01, ^**** p <0.0001.

Figure 15: Functionality of P-glycoprotein on murine devices with immortalized cell lines according to the invention in 12, 24 and 96 well format. In Figure 15, the results represent the mean ± SEM. For the 12-well format (Figure 15A), n = 4 controls and n = 2 device in 12-well format with or without Zosuquidar. For the 24-well format (Figure 15B), n = 7 controls and n = 9 and 12 24-well device without or with Zosuquidar. For the 96-well format (Figure 15C), n = 12 controls and n = 13 and 14 96-well format device with or without Zosuquidar.

The statistical test used is the Kruskal-Wallis test followed by a Dunn test for multiple comparisons: ^* p <0.05, ^** p <0.01, ^*** p <0.001.

Example 1: Preparation of a device according to the invention This device comprises primary cultures of endothelial cells, pericytes, and PBMCs harvested from mouse brains. The realization of this device has gone through the following technical stages:

Endothelial cells and pericytes were purified using magnetic beads to exclude myelin and a Percoll gradient to dissociate endothelial cells from pericytes.

The PBMCs were extracted from the peripheral blood of the same mice on a Ficoll gradient identical to that used in human medical hematology, and then recovered by centrifugation.

A primary co-culture stock of astrocytes and microglia was created as follows. Primary cultures of astrocytes and mouse microglia were obtained from brains of newborn mice between D1 and D3. Cell dissociation of brain tissue was mechanically performed. Astrocytes and microglia were selected by selective culture medium. One week after seeding a dissociated newborn brain and cultured in a 75 cm ² flask coated (coated) with Poly-L-Lysine, the astrocytes forming a confluent mat were detached with trypsin and cryopreserved. Thawing of a cell cone was performed in a 25 cm ² flask and 72 hr after the astrocytes can be used for mounting the device. These cells can be sub-cultured 3 times. The cells were cultured in selective media for each cell type until a cellular mat covering the entire surface of the chosen culture dish was obtained.

Astrocytes and microglia were seeded in a culture dish

(30000 cells per well for a 12-well box).

Then astrocytes and microglia were also seeded under a porous synthetic membrane incubated for 24 hours (see Figure 1).

The membrane was deposited in the culture dish comprising the astrocytes and microglia so that the two astrocyte cultures were in contact, thus forming the abluminal compartment. These cells model the glial parenchyma.

Endothelial cells and pericytes were seeded on the membrane

(endothelial cells = 10 ⁶ cells / membrane and pericytes = 350,000 cells / membrane in a 12-well well).

The device was incubated for 72 h in the presence of hydrocortisone to promote the expression of P-glycoprotein in the endothelial cells. Indeed, the glycoprotein P is an important efflux pump in the functionality of the BBB. After incubation, the model is ready. It can in particular receive PBMCs.

EXAMPLE 2 Immunolabel Observation of Devices Made According to Example 1 After the permeability and functionality tests, the porous synthetic membranes of the devices made according to example 1 are washed twice for 5 min with 500 μl of PBS. 500 μl of a paraformaldehyde solution (4% PFA) are then added to the abluminal and luminal compartments for 15 min at room temperature. Two new washes of 5 min are carried out with 500 μΙ of PBS. The cells are then blocked and permeabilized with 500 μl 0.5% PBS / Triton / 5% BSA in the abluminal and luminal compartments for 1 hour at room temperature. On paraffin plastic film (parafilm) stretched on a petri dish, 30 μί of primary antibodies (Acl) are deposited. The membranes are then cut and deposited so that the cells are in contact with the Acl. The antibodies used are anti-ZO-1 antibodies (1: 50 dilution, tight junction marker, used as endothelial cell marker), anti-aSMA (1/50 dilution, pericyte marker), anti-vWF (dilution 1 / 50, endothelial cell marker) and anti-GFAP (1/100 dilution, astrocyte marker). The incubation lasts one night at 4 ° C in a humid chamber. The next day, each membrane is gently deposited in a well of a 12-well box with the face of the cells studied above and washed twice for 5 min in PBS without stirring. 30 μl of Ac II are deposited on a new paraffin plastic film before being incubated for 1 hour at room temperature with the membranes. The Ac II solution contains fluorochrome RRX-coupled anti-mouse antibodies II (red fluorescence) and Alexa 488 fluorochrome-conjugated anti-rabbit II antibodies (green fluorescence) all diluted 1:50. After 1 h of incubation, the washes are carried out under the same conditions as above then the membranes are incubated with 30 μl of DAPI solution (4,6-Diamino-2-phenylindole) for 15 min at room temperature, under cover. of light, on paraffin plastic film in a humid chamber to mark the nuclei of the cells. The membranes are again recovered to undergo three washes for 5 min with H ₂ 0 UHQ to remove the salts. The membranes are then glued on a glass slide with DAPI glue so that the glued side matches that of the non-immunolabeled cells. A glass slide is then glued to the membrane. The slides are observed under an epifluorescence microscope (Olympus BX 51). Immunostarisms highlight the cell types that make up the BBB model. In addition, we observed that endothelial cells organize into vessels and form tight junctions between them (ZO-1 labeling), thus spontaneously reproducing important characteristics of the BBB in vivo. Pericytes are organized around endothelial cells by establishing points of contact.

Example 3 Use of a Device According to the Invention as a Model of the BBB in the Case of Cells Derived from Model Alzheimer's Mice (AD)

A device was prepared according to Example 1, in which endothelial cells and pericytes were prepared from Alzheimer's mice (APPswePS1 dE9, AD) aged 4 to 8 weeks and astrocytes and microglia from wild mice. (WT), here called Alzheimer device (AD device). This device has been compared to a similar device formed completely from wild-type mouse (WT) cells, here called wild-type device and to a similar device devoid of cells, here called control device.

The results are shown in Figure 2.

The presence of cells reduces the passage of dextran-FITC (Figure

2). Indeed, there is a significant decrease of 82% of the passage of dextran-FITC through the wild-type device at 1 hour, and then a 78% decrease at 1 hour 30 compared to the control device without cells.

This difference in permeability is also observed with the device AD where a reduction of 60% is obtained at 1 hour and 1 hour 30 in comparison with the control.

However, these results are not statistically significant.

Moreover, even if the observed difference in permeability between the AD and wild-type devices does not reach a value considered significant by the statistical test, a 47% decrease in the passage of dextran-FITC through the wild-type device is observed. compared to the AD device at 1 hour 30 minutes.

EXAMPLE 4 Test of permeability and functionality of a device according to the invention

To test the permeability of the device according to the invention, a device was prepared according to Example 1, and dextran-FITC was added in the luminal compartment of the device. The presence of dextran-FITC was detected and analyzed by fluorescence. In FIG. 3, it can be seen that the coefficient of permeability is higher with the control device. There is a significant decrease in the permeability coefficient of 98% respectively for the WT device. This difference is statistically significant. It demonstrates the relative permeability of the device according to the invention.

To test the functionality of the device according to the invention, a device was prepared according to Example 1, and rhodamine 123 was added to the luminal compartment of said device in the presence or absence of Zosuquidar. Indeed it is known that rhodamine 123 as a substrate of the glycoprotein P is expelled by the glycoprotein P to the luminal compartment, which limits its passage through the device. Zosuquidar is an inhibitor of P-glycoprotein, so its use should limit the efflux of rhodamine 123 to the luminal compartment.

In FIG. 4, it is observed that the passage of Rhodamine 123 towards the abluminal compartment decreases by 72.6% for the wild-type device relative to the control device (insert without cells). These results are statistically significant. The presence of the specific inhibitor of P-glycoprotein inhibited efflux by 57.3%. These results demonstrate the functionality of the device according to the invention. Example 5 Validation of a Device According to the Invention in 24 and 96 Well Plate Format

The method of preparation of the device followed on these two new formats of BBB is identical to that described in Example 1. Only the densities of each cell type had to be adapted.

The relevance of these formats was validated through four tests: trans-endothelial electrical resistance (TEER), paracellular permeability, G-glycoprotein (P-gp) functionality and selectivity to 8 molecules.

1 - Trans-endothelial electrical resistance (TEER) Trans-endothelial electrical resistance was only measured on 12- and 24-well BBBs because the electrode is not suitable for the 96-well format. It is expressed in ohm.cm ² taking into account the surface of the insert: 1 .12 and 0.33 cm ² for the inserts of 12 and 24 wells, respectively. It was measured using an ohmeter (Millicell Electrical Resistance System-2, Millipore - [Molsheim] France) using two electrodes STX01: the largest is placed in the abluminal compartment and the smallest in the luminal compartment. The system carrying the two electrodes is connected to the ohmeter to measure the electrical resistance between the two compartments. The value displayed on the device is expressed in ohm, then it is multiplied by the surface of the insert to obtain the results in ohm.cm ² .

Figure 5 shows that the TEER is significantly increased in the devices according to the invention compared to control (i.e., a device without a cell).

2- Paracellular permeability

As in Example 4, the passage of Dextran-FITC was studied and the coefficient of permeability (Pe) of this molecule as a control of the paracellular permeability was calculated (FIG. 6).

The results obtained on both the 24-well format and the 96-well format show a permeability coefficient of less than 4.10 ^-6 cm / s as for the 12-well format (see Figure 3) 3- Functionality of the P-glycoprotein (P -gp)

As in Example 4, the passage of rhodamine 123 in the presence or absence of Zosuquidar was studied. P-gp pumps are functional in both 24- and 96-well devices because Rhodamine123 is significantly effluent towards the luminal side and thus passes very little on the abluminal side. This functionality is well inhibited by Zosuquidar known as a specific inhibitor of P-gp. This shows that as for the 12-well format, the endothelial cells that express the P-gp pump are well polarized, the addition of Rhodamine123 in the abluminal compartment results in a passage to the luminal side comparable to cell-less BBBs. The results are shown in Figure 7.

4- Selectivity vis-à-vis 8 molecules On 24 and 96 well format devices, the passage of 8 molecules known to be able or not to cross the BBB has been studied.

Dopamine (DA), Levodopa (L-DOPA), Bromazepam (BROMO), caffeine (CAF), sucrose (SUC), cyclosporine A (CYCLA), Zosuquidar (ZOSU), and Mitotane (MITO) ) have been tested at a physiological and / or therapeutic concentration known in the literature in humans.

In Table 2 below are indicated the concentrations chosen for each molecule, as well as whether they are known for their ability to cross the BBB (+) or not to cross the BBB (-).

Table 2: Concentration of the molecules used for the test and ability to pass through the BBB

The molecules were added to the luminal compartment at the indicated concentration and incubated for 48 hours. The luminal and abluminal media were then taken for the determination of the molecules.

For each molecule, the coefficient of permeability was calculated. The results are presented in FIG. 8. The results validate the passage of these molecules through the devices of the invention which serve here as a model of murine BBB, namely the first 4 molecules pass through and the last 4 do not pass (hence a very low coefficient of permeability for these last four).

Example 6 Comparison of the Device According to the Invention to a Commercial Device The device according to Example 1 in 24-well format was compared with a commercial model (BBB Kit ™ (RBT-24H) of Pharmaco-Cell®) which corresponds to a model of Primary BBB prepared from rat brain cells. In this commercial device the endothelial cells are seeded on the insert, the pericytes under the insert and rat astrocytes at the bottom of the well.

Measurement of TEER in this model shows a good transendothelial electrical resistance on average of 247 ± 17.76 Q.cm ² (see Figure 9 A). However the permeability of FITC-Dextran conjugate is higher than that of the device according to the invention (see Figure 9 B) (25.850 ± 2.308 x10 ^"6 cm / s versus device of the invention 3.867 ± 0.333 ^{x10" 6} cm / s). The seal would therefore be better on the device of the invention by a factor 6.7 compared to that of the commercial model.

Here again the functionality of the commercial model was evaluated by studying the passage of Rhodamine 123 in the presence or absence of the specific inhibitor of the glycoprotein P-gp, Zosuquidar.

The results show that the pump is present on both the luminal and the intestinal side, so the cells are not properly polarized. Thus Rhodamine123 is as much effluent on the luminal as on the luminal side when it is deposited either in the luminal compartment or in the abluminal compartment (% efflux of 97% whatever the side of the deposit).

Thus, no effect is observed during inhibition by Zosuquidar of the pump P-gp in the commercial model as shown in Figure 10 unlike the device according to the invention (see Figure 7). Thus the device according to the invention makes it possible to obtain a model more similar to the BBB than the commercial model that has been tested here. Example 7 Cryopreservation of the Device According to the Invention in a 24-Well Format

Cryopreservation of the device would allow on-demand preparation and delivery of the frozen device to the customer. The 24-well device was cryopreserved with a solution

CRYOSTOR ^® marketed by Sigma® (REF: C2874-100ML). A measurement of the TEER was carried out before the cryopreservation at J15 of the mounting of the device. The 100 and 200 μl volumes of CRYOSTOR were chosen for cryopreservation of the cells in the luminal and abluminal compartment, respectively. Thawing was carried out on day 7, day 15 and day 30 post-cryopreservation according to the following protocol and the leakproofness of the thawed devices was studied 4, 5 and 6 days after thawing (TEER and Dextran-FITC permeability coefficient).

- On the day of thawing, preheat the complete EndogroTM culture medium with serum at 37 ° C,

- During the entire defrosting process, do not touch the membrane of the insert with pastel tips or pipettes. Do not move the inserts. Treat device by device,

- Once the culture medium is warmed, immediately add 150 and 300 μΙ of complete EndogroTM culture medium with serum, on the luminal and abluminal side, respectively,

- Incubate for 3 hours at 37 ° C under 5% C0 ₂ .

- After 3h, gently aspirate the culture media (MC) on the luminal and abluminal side and add 200 and 500 μΙ of MC luminal and abluminal, respectively,

- Incubate at 37 ° C under 5% C0 ₂ ,

- The day after thawing, renew the culture medium,

- At D4, D5 and D6 TEER measurement tests and the coefficient of paracellular permeability were performed. The results are shown in FIGS. 11 and 12 respectively.

Compared to the non-cryopreserved 24-well device, we observe that the TEER resistance after 7 days of freezing is of the same order of magnitude as that measured just before freezing. For 15 and 30 days of freezing, an average decrease of 26% is observed but remains insignificant.

As for the paracellular permeability, we find that the coefficients of permeability are also of the same order of magnitude after 7 days of freezing as those obtained on the non-cryreserved BBBs (3,278 ± 0.925 versus 3,867 ± 0.333 10 ^{~ 6} cm / s, respectively) . For 15 and 30 days of freezing, the permeability coefficient is between 6 and 20.10 ^"6 cm / s.

Example 8: Murine BBB Device with Immortalized Cell Lines

Murine endothelial cell line and murine pericyte after immortalization of primary cells using a method already published in the literature (Burek et al., 2012) were obtained. The immortality of these lines was verified by performing a karyotype showing a change in the number of chromosomes. Normally, in the mouse, 2n = 40 chromosomes. In immortal lines, the reading of 16 metaphase plates shows several chromosome number anomalies validating immortalization with 2n = 39 to 77 chromosomes depending on the plates read.

These lines have a replication time of 48 hours. These lines can be cryopreserved. The culture media used are the same as those used for primary cultures. Their immortal character leads to a very high cell adhesion.

Mounting the device with these cells follows the following kinetics:

- J1 seeding of astrocytes / microglia I under the insert and at the bottom of the well

- D2 seeding endothelial and pericyte cells on the insert

- D3-J4 incubation of the 48-hour model in the incubator

- At J4 the device is ready for any experimentation.

In Table 3 below, the cell densities used for each cell type and for each format of the device (12, 24 and 96 wells) are indicated.

On 12- and 24-well formats, the TEER was measured (see Figure 13) and on all formats the paracellular permeability (see Figure 14) and the functionality of the P-gp efflux pump (see Figure 15) were evaluated.

For this model of BBB, co-immunomarks were performed to demonstrate the expression of molecular markers specific for each cell type. The inventors have thus observed that the immortalized endothelial cells express in the device: the glycoprotein P,

tight junction proteins: ZO-1, Claudine-5,

the vonWillebrand factor (vWF),

the receiver LRP-1, and

the transferrin receptor.

They do not express markers of pericytes α-SMA, NG2, PDGF3R, indicating that the culture is pure. In contrast pericytes express these 3 markers well, and LRP-1 and do not express GFAP and vWF factor, indicating a pure culture of pericytes uncontaminated by endothelial cells and astrocytes.

Waterproofing of BBB

Figure 13 shows the TEER results for 12 and 24-well formats.

The TEER on the 12-well format is quite comparable to that obtained on the device with primary cultures (see Figure 5: 12 well TEER average 218 ± 7.17 n.cm ² and here for the model with immortal cell lines the average TEER is of 184.60 ± 6.65 Q.cm ² ). For the 24-well format, the average is 63.30 ± 1 .35 Q.cm ² whereas the model of BHE with primary cultures had an average ERER of 125.50 ± 2.34 Q.crn ² .

For paracellular permeability, the permeability coefficient values of Dextran-FITC are shown in Fig. 14 for each of the 3 formats (12, 24 and 96 wells, Fig. 14A, B and C respectively). Whatever the format used with the immortalized endothelial and pericyte cell lines in place of the primary cultures, the results show that the devices are watertight and the average permeability coefficient is 12.15 ± 0.92, 10.19 ± 0.44, 9.66 ± 0.50 .10 ^{~ 6} cm / s for 12, 24 and 96 well formats, respectively. Functionality of the BBB

The 12, 24 and 96 well formats are functional because they significantly limit the passage of Rhodamine ¹²³ on the abluminal side by more than 60% (see Figure 15). Unlike the BBBs with primary cultures, the specific inhibitor of Glycoprotein P shows no effect on all formats, this may be related to a different efflux pump type "Muiti drug resistance" very often encountered in cell lines.

Reference:

Burek M, Salvador E, Fôrster CY. Generation of an immortalized murine brain microvascular endothelial cell line in vitro blood brain barrier model. J Exp. 2012 Aug 29; (66): e4022. doi: 10.3791 / 4022

Danzig AH, Shepard RL, Cao J, Law KL, Ehlhardt WJ, Baughman TM, Bumol TF, Starling JJ. Reversal of P-glycoprotein-mediated resistance muitidrug by a potent cyclopropyldibenzosuberane modulator, LY335979. Cancer Res. 1996 Sep 15; 56 (18): 4171-9.

Claims

1. Device comprising two compartments separated by a porous synthetic membrane, a luminal compartment comprising endothelial cells and pericytes, and an abluminal compartment comprising astrocytes.

2. - Device according to claim 1, wherein the compartment comprising astrocytes further comprises microglia.

3.- Device according to one of claims 1 to 2, wherein all the cells come from the same animal species.

4. - Device according to one of claims 1 to 3, wherein one or more cell types are derived from primary cultures.

5. - Device according to one of claims 1 to 3, wherein one or more cell types are derived from immortal cell cultures.

6. - Device according to claim 4, wherein the endothelial cells and pericytes are derived from primary cultures of self of the same age.

7. - Device according to one of claims 4 and 6, wherein one or more of said type (s) cell (s) from primary cultures are model cell types of pathologies.

8. - Process for preparing a device according to any one of claims 1 to 7, comprising the following steps:

a) seeding astrocytes on a surface and / or on one side of the porous synthetic membrane,

b) insertion or deposition of the porous synthetic membrane on said surface, such that if the porous synthetic membrane comprises astrocytes then the face comprising the astrocytes is opposite the surface, c) seeding of the face of the porous synthetic membrane luminal with endothelial cells and pericytes.

9. A method for preparing a device according to claim 1, comprising the following steps:

a) astrocyte seeding on one side of the porous synthetic membrane,

b) seeding the other side of the porous synthetic membrane with endothelial cells and pericytes, and optionally

10.- Use of the device according to one of claims 1 to 7 as a model of the blood-brain barrier (BBB).

1 1 .- Use according to claim 10 for testing the permeability of the model.

Use according to claim 10 for testing the permeability of the model to a compound comprising the steps of:

a) adding said compound to one of the compartments of the device,

b) incubation of the device,

c) detecting and analyzing the presence of said compound and / or its metabolites in the compartment where the addition of said compound has not occurred.

13 - Use according to claim 10 for studying the pathophysiology of a disease or testing preventive, therapeutic or diagnostic molecules targeting the cellular and molecular players in the BBB or testing physical conditions or testing protocols.