EP3665264A1 - Dispositif pouvant servir de modèle de barrière hémato-encéphalique - Google Patents
Dispositif pouvant servir de modèle de barrière hémato-encéphaliqueInfo
- Publication number
- EP3665264A1 EP3665264A1 EP18756178.2A EP18756178A EP3665264A1 EP 3665264 A1 EP3665264 A1 EP 3665264A1 EP 18756178 A EP18756178 A EP 18756178A EP 3665264 A1 EP3665264 A1 EP 3665264A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- cells
- astrocytes
- bbb
- porous synthetic
- synthetic membrane
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
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Classifications
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- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
- C12M35/08—Chemical, biochemical or biological means, e.g. plasma jet, co-culture
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/34—Internal compartments or partitions
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
- C12M3/04—Tissue, human, animal or plant cell, or virus culture apparatus with means providing thin layers
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0618—Cells of the nervous system
- C12N5/0622—Glial cells, e.g. astrocytes, oligodendrocytes; Schwann cells
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- C12N5/06—Animal cells or tissues; Human cells or tissues
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- C12N5/069—Vascular Endothelial cells
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- C—CHEMISTRY; METALLURGY
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0697—Artificial constructs associating cells of different lineages, e.g. tissue equivalents
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- C12N2502/08—Coculture with; Conditioned medium produced by cells of the nervous system
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- C12N2502/28—Vascular endothelial cells
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- G01N2500/00—Screening for compounds of potential therapeutic value
- G01N2500/10—Screening for compounds of potential therapeutic value involving cells
Definitions
- 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.
- BBB hematoencephalic
- the brain is isolated from the bloodstream by a particular structure, the blood-brain barrier (BBB).
- BBB blood-brain barrier
- 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.
- 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.
- a so-called luminal compartment comprising endothelial cells and pericytes
- a so-called abluminal compartment comprising astrocytes and microglia.
- PBMCs peripheral blood mononuclear cells
- the porous synthetic membrane may be tubular or planar.
- luminal compartment it is understood the compartment formed by the light of a tubular device or the upper compartment in a planar device.
- luminal 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.
- the pericytes and the endothelial cells are arranged in superposed layers in the luminal compartment.
- 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.
- 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).
- the luminal compartment further comprises blood cells, and preferably peripheral blood mononuclear cells (PBMCs). These are then arranged above the endothelial cells.
- PBMCs peripheral blood mononuclear cells
- 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.
- 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 ⁇ and about 3 ⁇ in diameter and preferably about 0.4 ⁇ in diameter.
- the support has a pore diameter of between about 0.4 and about 3 ⁇ , preferably about 0.4 ⁇ .
- the support has a pore diameter of between about 3 ⁇ and about 8 ⁇ , preferably about 5 ⁇ about 8 ⁇ .
- 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.
- the microglia / astrocyte seeding ratio is between about 1% and about 10% and is preferably about 5%.
- the abluminal compartment comprising astrocytes is free of pericytes.
- all the cells of the device according to the invention come from the same animal species, in particular from mammals.
- the cells are rodent cells, preferably mouse cells.
- the cells are primate cells, preferably human cells.
- 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.
- immortal cells immortal cells derived from tumors, spontaneously immortal cells and / or cells rendered immortal (“immortalized”) by introduction of at least one viral or cellular oncogene.
- 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.
- 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.
- primary culture is meant a culture of cells directly from the tissue and / or cells of an individual.
- 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.
- one or more and preferably all cell types are derived from adult individuals.
- 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.
- the use of primary cultures makes it possible to approach still more in vivo conditions.
- ⁇ is meant a subject of an animal species, in particular mammals.
- the individual or individuals are rodents, preferably mice.
- the individuals are primates, preferably humans.
- the endothelial cells and the pericytes are derived from primary cultures of mouse cells and, preferably, from adult mice.
- 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.
- all cell types are derived from primary cultures of mouse cells and, preferably, from adult mice.
- 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.
- 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.
- 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.
- neurodegenerative diseases Alzheimer's, Parkinson's, Huntington's, ALS, etc.
- cerebrovascular accident cerebrovascular accident and cancers brain.
- the porous synthetic membrane is in the form of a tube, in which a fluid can be introduced, renewed, circulated.
- the porous synthetic membrane is a plane membrane disposed horizontally.
- the compartments are one above the other.
- 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:
- 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 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.
- the surface is a support on which the device rests.
- the surface may be for example the bottom of a culture box.
- the method of preparation of the device according to the invention comprises an additional step of cryopreservation of the device.
- the pericytes and the endothelial cells are arranged in superposed layers in the luminal compartment.
- the pericytes are seeded before the endothelial cells.
- 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.
- 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).
- the porous synthetic membrane is in contact with the cells of each compartment.
- 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).
- blood cells preferably peripheral blood mononuclear cells (PBMCs).
- PBMCs peripheral blood mononuclear cells
- the addition of blood cells takes place after seeding the pericytes and the endothelial cells.
- the one or more inoculation steps with astrocytes also include microglia seeding.
- 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.
- the microglia / astrocyte seeding ratio is about 5% (corresponding to the ratio in human BBB).
- the seeding step (s) with astrocytes do not include seeding pericytes.
- all the cells seeded according to the process of the invention come from the same animal species, in particular from mammals.
- the cells are rodent cells, preferably mouse cells.
- the cells are primate cells, preferably human cells.
- one or more cell types of the process according to the invention are derived from immortal cell cultures.
- all cell types are derived from immortal cells.
- 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.
- 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.
- one or more and preferably all cell types are derived from adult individuals.
- endothelial cells and pericytes are derived from primary cultures of mouse cells and, preferably, from adult mice.
- 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.
- all cell types are derived from primary cultures of mouse cells and, preferably, from adult mice.
- 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.
- 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.
- the device can be used to test the permeability of the model.
- this device can be used to study its permeability to:
- therapeutic molecules especially therapeutic molecules (in particular small biological or synthetic molecules, antibodies, psychotropic substances, etc.),
- spores such as bacterial spores, fungal spores, plant spores, etc., for example for the study of fungal infections such as certain meningitis), or
- naked or vectorized nucleic acids, cells in particular PBMCs, cancer cells, bacteria, etc.
- 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:
- 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.
- 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.
- 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 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).
- 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.
- 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.
- test physical conditions 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.
- 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.
- Physical condition refers in particular to the use of waves such as magnetic waves, electromagnetic waves or ultrasounds.
- test protocols we intend to study the impact of a treatment at the level of the BBB.
- at least one treatment i.e., a physical condition or a molecule
- a compound as defined above which compound is then contacted with the BBB.
- 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. .
- Figure 1 Diagram of preparation of a device comprising primary cell cultures according to the invention.
- astrocytes and microglia are thawed and endothelial cells and pericytes are purified from mouse brains.
- PBMCs are extracted from the peripheral blood of mice.
- 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.
- astrocytes and microglia are seeded on the porous synthetic membrane.
- 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.
- 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 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.
- dT incubation time (second)
- A surface of the porous synthetic membrane (here 1, 12 cm 2 )
- 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 ⁇ l in the luminal compartment containing or not the Zosuquidar inhibitor of the 5 ⁇ -glycoprotein P (Dantzig et al., 1996).
- the devices are incubated for 2 hours in the incubator.
- 250 ⁇ l of medium containing rhodamine 123 at 2 ⁇ l with or without Zosuquidar at 5 ⁇ l are added to the luminal compartment and incubated for 1 hour in the incubator.
- Samples of 50 ⁇ 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.
- the passage of rhodamine 123 through the WT decreases by 72.6% compared to the device without cell ( ** p ⁇ 0.01).
- the statistical test used is the Kruskal-Wallis test followed by the Dunn test for multiple comparisons.
- Figure 7 Functionality of the P-glycoprotein in the murine device according to the invention in 24 and 96 well format.
- FIG. 8 Coefficient of permeability of 8 molecules on the murine devices according to the invention in 24 and 96 well format.
- Figure 10 Functionality of P-glycoprotein in the commercial device.
- the statistical test used is the Kruskal-Wallis test followed by a Dunn test for multiple comparisons: * p ⁇ 0.05.
- FIG 11 Trans-endothelial electrical resistance (TEER) in 24-well formats after cryopreservation.
- Figure 1 1 is shown the trans-endothelial electrical resistance (TEER)
- 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.
- FIG. 13 Trans-endothelial electrical resistance (TEER) in murine devices with immortalized cell lines according to the invention in the format 12 and 24 wells.
- TEER Trans-endothelial electrical resistance
- 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.
- Figure 15 Functionality of P-glycoprotein on murine devices with immortalized cell lines according to the invention in 12, 24 and 96 well format.
- the results represent the mean ⁇ SEM.
- 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.
- 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 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.
- the device was incubated for 72 h in the presence of hydrocortisone to promote the expression of P-glycoprotein in the endothelial cells.
- the glycoprotein P is an important efflux pump in the functionality of the BBB.
- the model is ready. It can in particular receive PBMCs.
- EXAMPLE 2 Immunolabel Observation of Devices Made According to Example 1
- the porous synthetic membranes of the devices made according to example 1 are washed twice for 5 min with 500 ⁇ l of PBS.
- 500 ⁇ l 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 ⁇ of PBS.
- the cells are then blocked and permeabilized with 500 ⁇ l 0.5% PBS / Triton / 5% BSA in the abluminal and luminal compartments for 1 hour at room temperature.
- 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.
- 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 ⁇ l 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.
- the washes are carried out under the same conditions as above then the membranes are incubated with 30 ⁇ l 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.
- 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).
- WT Alzheimer 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.
- 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.
- 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.
- 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.
- 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.
- TEER trans-endothelial electrical resistance
- P-gp G-glycoprotein
- 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 .
- TEER Trans-endothelial electrical resistance
- 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).
- DA Dopamine
- L-DOPA Bromazepam
- BROMO Bromazepam
- CAF caffeine
- SUC sucrose
- SUC sucrose
- CYCLA cyclosporine A
- ZOSU Zosuquidar
- MISO Mitotane
- 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.
- 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 TM (RBT-24H) of Pharmaco-Cell®) which corresponds to a model of Primary BBB prepared from rat brain cells.
- BBB Kit TM RBT-24H
- Pharmaco-Cell® a commercial model of Primary BBB prepared from rat brain cells.
- the endothelial cells are seeded on the insert, the pericytes under the insert and rat astrocytes at the bottom of the well.
- 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).
- 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
- 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) .
- the permeability coefficient is between 6 and 20.10 "6 cm / s.
- 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.
- 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.
- VWF vonWillebrand factor
- pericytes do not express markers of pericytes ⁇ -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.
- 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 ).
- 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 .
- 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.
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