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WO2016202343A1 - Modèle de coculture triple de la barrière hémato-encéphalique utilisant des cellules endothéliales cérébrales porcines primaires, péricytes porcins et astrocytes porcins - Google Patents

Modèle de coculture triple de la barrière hémato-encéphalique utilisant des cellules endothéliales cérébrales porcines primaires, péricytes porcins et astrocytes porcins Download PDF

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Publication number
WO2016202343A1
WO2016202343A1 PCT/DK2016/050190 DK2016050190W WO2016202343A1 WO 2016202343 A1 WO2016202343 A1 WO 2016202343A1 DK 2016050190 W DK2016050190 W DK 2016050190W WO 2016202343 A1 WO2016202343 A1 WO 2016202343A1
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culture
porcine
pericytes
astrocytes
triple
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Louiza Bohn THOMSEN
Annette Burkhart LARSEN
Torben MOOS
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Aalborg Universitet AAU
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Aalborg Universitet AAU
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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
    • 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
    • C12N5/069—Vascular Endothelial cells
    • C12N5/0691—Vascular smooth muscle cells; 3D culture thereof, e.g. models of blood vessels
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5058—Neurological cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5064—Endothelial cells
    • 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
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/08—Coculture with; Conditioned medium produced by cells of the nervous system
    • C12N2502/081—Coculture with; Conditioned medium produced by cells of the nervous system neurons
    • 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
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/08—Coculture with; Conditioned medium produced by cells of the nervous system
    • C12N2502/086—Coculture with; Conditioned medium produced by cells of the nervous system glial cells

Definitions

  • the present invention relates to a blood brain barrier (BBB) model.
  • BBB blood brain barrier
  • the present invention relates to a BBB model comprising three different porcine cell types. Background of the invention
  • BECs Brain endothelial cells denote the blood-brain barrier (BBB) and form a major physical restraint on the transport into the brain of several molecules present in blood plasma for transport.
  • BBB blood-brain barrier
  • the BECs are non-fenestrated, rich in mitochondria, high in concentrations of drug- and nutrient metabolizing enzymes, but low in vesicles involved in endocytotic and transcytotic activity.
  • the BECs are also closely connected with intermingling tight junctions and adherence junctions. Pericytes and end-feet of astrocytes form close contact with the BECs and participate in the formation, regulation and maintenance of the integrity of the BBB.
  • astrocytes are known to secrete a number of substances that participates in the induction of the BBB, e.g. basic fibroblast growth factor (bFGF) and angiopoietin 1 (ANG1).
  • BFGF basic fibroblast growth factor
  • ANG1 angiopoietin 1
  • Primary BECs are often isolated from small mammals.
  • PBEC's have mainly been cultured in either monoculture or in co- culture with primary rat astrocytes isolated from neonatal rats or rat astrocytes cell lines e.g. C6 glioma.
  • Such co-cultures were therefore, constructed from two different species, which could influence the barrier function and gene expression of the PBEC's.
  • rat astrocytes are most often derived from rat pups due to their ability to grow faster than astrocytes obtained from older animals. Deriving cells from laboratory animals is very expensive and requires large amounts of animal sacrifices only for this single purpose.
  • an improved BBB model would be advantageous, and in particular, a more efficient and/or reliable BBB model derived entirely from porcine cells would be advantageous.
  • PBEC's porcine brain endothelial cells
  • PBEC's have several advantages, when compared to those of in vitro rodent BBB models: i) higher cellular yield per animal, ii) PBEC's retain many of the important BBB features, iii) human and porcine genome, anatomy, physiology, and disease progression are more comparable, iv) porcine brains are by-products from the abattoir and, therefore, inexpensive and their usage for research more ethically acceptable.
  • PBEC's, astrocytes and pericytes were isolated from 6 months old domestic pig brains donated and considered a waste product by an abattoir.
  • the aim of the present study was to establish a triple co-culture based entirely on porcine cells i.e. PBEC's, astrocytes and pericytes and to determine if this preferable cellular combination for BBB formation would compare to PBEC's co- cultured with rat astrocytes and pericytes isolated from new-born rat pups.
  • porcine cells i.e. PBEC's, astrocytes and pericytes
  • Porcine or rat astrocytes and pericytes were cultured in both contact and non- contact co-culture with PBEC's to examine their effects on the PBEC's for barrier formation as revealed by formation of trans-endothelial electric resistance (TEER), loss of passive permeability, and expression patterns of BEC specific proteins.
  • TEER trans-endothelial electric resistance
  • the results show that primary porcine astrocytes and pericytes are useable for triple co-culture with PBEC's instead of primary rat astrocytes and pericytes, as equally high TEER values, low passive permeability and expression of hallmarks of BECs were observed. Because astrocytes may need culturing for two-three weeks before being co-cultured in most in vitro BBB models, they are often isolated from a different animal than the PBEC's and pericytes, which may impair the overall function of the model.
  • an object of the present invention relates to the provision of a blood-brain barrier model constituted entirely of porcine cells.
  • one aspect of the invention relates to an isolated triple co-culture
  • the isolated triple co-culture comprises (or is provided in)
  • a culture plate (2) comprising the culture of porcine astrocytes or porcine pericytes
  • porous culture membrane (3) comprising the culture of BECs (5) on one side and a culture of the porcine pericytes or the porcine astrocytes on the opposite side; with the proviso that the triple co-culture comprises porcine astrocytes and porcine pericytes.
  • Another aspect of the present invention relates to the use of the isolated triple co- culture according to the invention as an in vitro model of a mammalian blood- brain barrier.
  • Yet another aspect of the present invention is to provide a method of
  • Still another aspect of the present invention is to provide a method for producing an isolated triple co-culture according to the present invention, the method comprising
  • a further aspect of the present invention is to provide a method for
  • a yet further aspect of the present invention is to provide an isolated triple co- culture comprising porcine primary brain endothelial cells (BEC's), porcine astrocytes and porcine pericytes obtained/obtainable by a process according to the method for producing an isolated triple co-culture comprising porcine primary brain endothelial cells (BEC's), porcine astrocytes and porcine pericytes.
  • the present invention serves to provide technical progress in several important areas compared to prior art.
  • a porcine triple co-culture blood brain barrier model is a more anatomical correct model than competing technologies based on cultures of lower mammals. Consequently, it is to be expected that the present invention will yield results that are more reliable than competing models.
  • the present invention is constructed from pig brain material obtained from pigs sacrificed at an abattoir.
  • pig brain material is usually perceived as a waste product and the present invention therefore represents a new ethical benchmark compared to technologies in which pigs are bred with the sole purpose of harvesting the brain.
  • the ability to use pig brain material from local abattoirs was only possible because the inventors surprisingly found that the pig brain material could be cultured subsequent to cooled transport from the abattoir.
  • the inventors surprisingly discovered that the pig brain material did not have to originate from piglets, as commonly presumed, but also animals as old as 6 months yielded viable brain cell cultures.
  • the technical progress provided by the present invention offer not only a more anatomically correct in vitro model of the human blood brain barrier compared to competing technologies, but also supplies it in a more ethical acceptable manner.
  • Figure 1A shows an in-vitro blood-brain barrier model in different cell
  • Figure IB also shows a schematic representation of the in-vitro blood-brain barrier model with a preferred combination and placement of the different porcine cell types, corresponding to number 10 from 1A.
  • Figure 2 The overall BBB model, (2) astrocytes, (3) porous culture membrane, (4) pericytes, (5) BEC's, and (6) culture plate.
  • Figure 2 shows characterization of primary cell cultures by immunocytochemistry.
  • PBEC's were found to express the tight junction proteins Claudin-5 (light grey) and Zonula occludens 1 (light grey) at the cell borders.
  • Porcine mixed glial cells (C) were found to mainly consist of astrocytes, which express GFAP (light grey).
  • Rat astrocytes (D) were found to express GFAP (light grey).
  • Porcine pericytes (E) and rat pericytes (F) were found to express alpha-smooth muscle actin (light grey).
  • Porcine pericytes cultured in monoculture (G) and porcine pericytes cultured in a triple culture with porcine pericytes and PBECs in a triple co-culture (H) was stained for PDGFR-beta (light grey, without filament
  • FIG 3 shows trans-endothelial electric resistance (TEER) measurement made across PBEC's in thirteen co-culture combinations.
  • Figure 4 shows mannitol permeability measurements on PBEC's in thirteen co- culture combinations as a function of their TEER.
  • TEER was measured just before the permeability experiment was conducted.
  • Each point represents one hanging culture insert with PBEC's.
  • Figure 5 shows gene expression of A) claudin-5, B), Occludin, C), transferrin, D) BCRP, and E) P-gp, in PBEC's.
  • RT-qPCR was performed on the PBEC's from all thirteen different culture combinations.
  • the relative gene expression of Claudin-5, Occludin, P-gp, BCRP and transferrin receptor- 1 is shown for each culture combination.
  • Figure 6 shows gene expression of A) claudin-5, B), Occludin, C), transferrin, D) BCRP, and E) P-gp
  • the blood brain barrier is a permeability barrier formed from brain endothelial cells (BEC's) connected by tight junctions that limits the transport of material from the blood into the brain extracellular fluid.
  • BEC brain endothelial cells
  • the BBB is highly selective and thereby protects the brain from entry of foreign substances such a bacterial material.
  • both astrocytes and pericytes play crucial supporting roles in the establishment of the BBB. Consequently, in the present context, a BBB model comprises three types of cells; BEC's, astrocytes and pericytes.
  • a triple co-culture is a conglomerate of three distinctly different cell types cultured together in a single medium.
  • these three cell types are porcine brain endothelial cells, porcine astrocytes and porcine pericytes. It is noted that the different cells are preferably grown at physically distinct locations in the medium.
  • a confluent monolayer is a single layer of cells that covers an entire surface, such as a culture plate.
  • Confluency is the fraction of a surface covered by cells and is measured in percent; such that 50% confluency means that half the area of a surface is covered with cells and 100% confluency means that the entire surface is covered with cells. It is preferred that the cells are 100% confluent, especially the BEC cells should be confluent.
  • a porous culture membrane is a membrane comprising pores of a defined size and is suitable for culturing cells on.
  • the size of the pores decides what size of molecules will be allowed to pass the membrane, and accordingly serves as an effective molecular cut-off.
  • membrane can be part of an insert used together with a culture plate, which enables the seeding of triple co-cultures.
  • a culture plate When used together with a culture plate, all transport of molecules from one side of the culture plate medium to the other side is restricted to be conveyed through the porous culture membrane and the cells attached to it.
  • the present inventors have constructed a BBB model comprising a porcine triple co-culture that fulfil the benchmarks of electrical resistance, permeability and expression of proteins. Furthermore, the inventors surprisingly found that they could produce this model from pig brain material considered a waste product from the abattoir. Together, the present invention solves both a technical need of an anatomically correct BBB model and the ethical dilemma of sacrificing piglets with the sole purpose of harvesting their brain material.
  • the cell culture used for production of the in vitro blood brain barrier is a central element of the present invention.
  • the ultimate object is to produce a BBB model that closely imitates the real life human (or pig) BBB. To achieve that goal it is necessary to build a culture comprising not only brain endothelial cells but also astrocytes and pericytes.
  • a first aspect of the invention relates to an isolated triple co-culture comprising porcine primary brain endothelial cells (BEC's), porcine astrocytes and porcine pericytes.
  • BEC's porcine primary brain endothelial cells
  • porcine astrocytes porcine pericytes.
  • one reason for preferring a porcine triple co-culture BBB model to a competing rat triple co-culture BBB model has been to obtain a model that anatomically and physiologically mimic the human BBB as closely as possible.
  • the native BBB is shielding the brain from foreign substances in the blood by a single layer of brain endothelial cells connected by tight junctions. For formation of a selective and tight BBB model, it is therefore necessary to regulate the growth of cells in the triple co-culture to one single layer of cells.
  • At least the BEC's of the isolated triple co-culture are present as a monolayer, such as a confluent monolayer.
  • each type of porcine cells of the isolated triple co-culture is present as individual monolayers, such as confluent
  • the BEC's, the astrocytes and the pericytes of the isolated triple co-culture are viable.
  • Co-cultures can be grown as either contact or non-contact co- cultures, whereas triple co-cultures only grow as contact co-cultures.
  • triple co- cultures one type of cells are seeded at the culture plate and the two remaining types of cells are seeded on the either side of the porous culture membrane.
  • the different cell types of the isolated triple co-culture are grown at different locations in the co-culture.
  • the isolated triple co-culture comprises
  • a culture plate (2) comprising the culture of porcine astrocytes or porcine pericytes
  • porous culture membrane (3) comprising the culture of BEC's (5) on one side and a culture of the porcine pericytes or the porcine astrocytes on the opposite side; with the proviso that the triple co-culture comprises porcine astrocytes and porcine pericytes.
  • the isolated triple co-culture comprises
  • a culture plate (2) comprising the culture of porcine astrocytes or porcine pericytes
  • FIG. 1 shows a schematic overview of such co-cultures comprising both a culture plate and a culture membrane.
  • the triple co-culture is an in vitro blood-brain-barrier (BBB) model (1) or is suitable for use as an in vitro blood- brain-barrier (BBB) model (1).
  • BBB blood-brain-barrier
  • Figure IB shows a schematic overview of a preferred embodiment according to the invention, where the different cell types are indicated in their preferred location in the model.
  • the isolated triple co-culture are arranged such that substances passing from one side of the porous culture membrane to the other side pass through the porous culture membrane.
  • the porous culture membrane is a hanging cell culture insert.
  • the brain endothelial cells are lined by pericytes, with astrocyte endfeets placed adjacent to pericytes and brain endothelial cells. It is therefore desirable to imitate this configuration of the cells in the BBB model.
  • the culture plate (2) comprises a culture of porcine astrocytes (6)
  • said porous culture membrane (3) comprises a culture of porcine pericytes (4) on the side facing towards the culture plate.
  • a hanging culture can be inserted in a regular culture plate.
  • brain endothelial cells and either astrocytes or pericytes can be seeded to form the actual cellular permeability barrier.
  • Conditions can be adjusted to facilitate simultaneous growth of all three cell types and ensure that each type of cells form a satisfactory monolayer of cells. Therefore, in one embodiment of the invention, the porous culture membrane (3) is a hanging culture that are not in direct physical contact with the culture plate (2).
  • the porcine triple co-culture is immersed in a liquid medium, such as a growth medium allowing for growth of all three cell types.
  • the BEC's, the astrocytes and the pericytes are monolayers of 50-100% confluency, such as 70-100% confluency, such as 90-100% confluency, preferably 100% confluency.
  • the porous culture membrane (3) is of a material selected from the group consisting of polycarbonate,
  • the porous culture membrane (3) has pores of a diameter in the range 0.2-2 ⁇ , such as 0.5-2 ⁇ , such as 0.5-1.5 ⁇ or such as 0.7-1.2.
  • the cells are exposed to a flow of liquid, such as a continuous flow.
  • the liquid of the continuous flow is selected from the group consisting of blood, plasma, serum, cell growth media or cerebrospinal fluid.
  • animals are bred with the sole purpose of harvesting the brain cells of the animals. These animals, such as piglets and rat pups, are then sacrificed at a very early age to supply cells that have not taken damage over time. In addition, it usually takes more than 10 rat pups to obtain enough brain material to construct a single BBB model. Pigs gives a higher cellular yield per animal and thereby alleviates some of the ethical concerns of sacrificing animals with the sole purpose of harvesting brain material. However, it would be preferable if viable brain cells could be retrieved in a more acceptable manner.
  • the porcine astrocytes (4), the porcine BEC's (5), and the porcine pericytes (6) are obtained from one or more pigs, which is at least two months old, such as at least, 4 months old or such as at least six months old, e.g. 4-12 months old, such as 4-10 months old or such as 5- 7 months old.
  • the porcine astrocytes (4), the porcine BEC's (5), and the porcine pericytes (6) are obtained from the same pig.
  • the porcine astrocytes (4), the porcine BEC's (5), and the porcine pericytes (6) are obtained from the same pig, which is at least 4 months old, preferably at least 6 months old.
  • the cells of the triple co-culture are obtained from a pig sacrificed at an abattoir/slaughterhouse, a laboratory, or an animal facility, preferably from an abattoir/slaughterhouse.
  • Pig brains are considered to be a waste product of many abattoirs, which essentially means that all the ethical concerns of the prior art are circumvented when utilizing such waste material for the construction of in vitro BBB models.
  • the cooled pig brains have been cooled to a temperature below 10°C, such as below 5°C, such as below 1°C, such as in the range 0.1-10°C, such as 2-10°C, or such as 5-10°C.
  • Such cooling could e.g. take place by placing a pig head on ice, and storing it in an insulated container, e.g. under transport to a location for further processing.
  • the cooled pig brains are instantly cooled after sacrificing the pig, such as within 4 hours from death of the animal, such as within 2 hours from the death of the animal, preferably within 30 minutes from the death of the animal.
  • the cells of the isolated triple co-culture are harvested from the cooled pig brains for further processing within 24 hours from the death of the animal, such as within 12 hours from the death of the animal, such as 4 hours from the death of the animal, preferably within 3 hours from the death of the animal.
  • the cooled pig brains are instantly cooled, within 30 minutes from the death of the animal, to a temperature in the range of 0.1-10°C and the cells of the isolated triple co-culture are harvested from the cooled pig brains for further processing within 3 hours from the death of the animal.
  • TEER trans-epithelial electrical resistance
  • passive permeability may be measured for a wide range of different substances.
  • the sugar alcohol mannitol is a substance that has been used to facilitate the transport of therapeutic molecules into the brain, by shrinking the brain endothelial cells, thereby inducing a stretch of the tight junctions. Consequently, BBB passive permeability to mannitol is a valid benchmark for the integrity of the BBB model.
  • the isolated triple co-culture has a TEER value of at least 700 ⁇ x cm 2 , such as in the range of 700-2000 ⁇ x cm 2 , or such as 700-1200 ⁇ x cm 2 across the porous culture membrane.
  • the TEER value is measured by using Millicell epithelial-volt-ohm meter and chopstick electrodes (Millipore), the TEER value is calculated as the measured values minus measurements of coated but cell free culture inserts and the difference is multiplied with the area of the culture insert.
  • the isolated triple co-culture has a passive permeability of mannitol across the porous culture membrane below 4.0xl0 6 cm*s _1 , such as 0-4.0xl0 ⁇ 6 cm*s _1 , preferably below 2.5xl0 ⁇ 6 cm*s _1 , such as 0-2.5xl0 6 cm*s _1 .
  • a passive permeability of mannitol across the porous culture membrane below 4.0xl0 6 cm*s _1 , such as 0-4.0xl0 ⁇ 6 cm*s _1 , preferably below 2.5xl0 ⁇ 6 cm*s _1 , such as 0-2.5xl0 6 cm*s _1 .
  • the passive permeability of [3H] mannitol is measured by addition of radioactive labelled mannitol, [3H] mannitol, to the luminal compartment of the BBB and measuring radioactivity in the lower, abluminal compartment. The measurements are performed on a scintillation counter. In example 1, it is described how passive permeability of mannitol is measured. Further data is provided in example 5.
  • the isolated triple co-culture has a TEER value of at least 700 ⁇ x cm 2 and a passive permeability of mannitol across the porous culture membrane below 2.5xl0 ⁇ 6 cm*s _1 .
  • the isolated triple co-culture also has a low permeability towards FLU.
  • the isolated triple co-culture of the invention has a passive permeability of FLU across the porous culture membrane below 7.0*10 ⁇ 6 Papp (Cm-s or such as below 6.0*10 ⁇ 6 Papp (Cm-s ⁇
  • the isolated triple co-culture can be stored before use.
  • Many pharmaceutical products are stored in frozen forms or as freeze- dried products.
  • a routine for storing the triple co- culture within the culture plate and porous culture membrane will be
  • the isolated triple co-culture is in a frozen state, such as below -20°C, such as below -50°C or such as below -80°C.
  • a frozen state such as below -20°C, such as below -50°C or such as below -80°C.
  • proteins may play crucial roles in anchoring the brain endothelial cells to each other via tight junctions and other proteins may be part of signaling pathways through transport across the BBB.
  • the expression of these proteins can be dependent on the composition of cells in the BBB model, as astrocytes and pericytes within a triple co-culture can influence the protein expression pattern of brain endothelial cells.
  • the expression of a set of proteins may be used as an additional benchmark of the quality of the BBB model.
  • the BEC's of the isolated triple co- culture express all of the proteins selected from the group consisting of claudin 5, occluding, P-glycoprotein, breast cancer related protein, and transferrin receptor.
  • expression pattern in different cultures are determined.
  • the isolated triple co-culture of the present invention is of porcine origin, which has a great anatomical and physiological resemblance to higher mammals. This similarity translates directly into its usefulness as an in vitro model of the blood brain barrier of higher mammals.
  • a second aspect of the invention relates to the use of the isolated triple co-culture as an in vitro model of a mammalian blood-brain barrier.
  • the mammal is selected from human and pig. Test method
  • the present invention comprises an isolated porcine triple co-culture that can be used as an in vitro BBB model, which both meets the criteria of high TEER values and low passive permeability, but also express proteins important to the native functioning of the BBB, such as transporters and receptors. Therefore, it is possible to utilize the present invention to screen substances for their ability to cross or bind the BBB model.
  • a third aspect of the invention relates to a method of measuring/evaluating blood-brain barrier permeability of a substance, the method comprising
  • the substance (such as a compound or drug) can be added at either side of the porous culture membrane, but preferably, it is added at the side facing towards the BEC's.
  • Another embodiment of the invention relates to the method of measuring BBB permeability of a substance, wherein said substance is intended to be able to cross the mammalian blood-brain barrier.
  • Yet another embodiment of the invention relates to the method of measuring BBB permeability of a substance, wherein said substance is not intended to be able to cross the mammalian blood-brain barrier.
  • a further embodiment of the invention relates to the method of measuring BBB permeability of a substance, wherein said substance is a molecule intended to bind one or more constituents of the blood-brain barrier.
  • one embodiment of the invention relates to the method of measuring BBB permeability of a substance, wherein said substance is a medicament or a compound intended to be a medicament.
  • Another embodiment of the invention relates to the method of measuring BBB permeability of a substance, wherein said substance is for or intended for the treatment or amelioration of diseases in the central nervous system.
  • Yet another embodiment of the invention relates to the method of measuring BBB permeability of a substance, wherein said diseases in the central nervous system are selected from the group consisting of ADHD, autism, Alzheimer's disease, Parkinson's disease, multiple sclerosis, Krabbe's disease, Huntington's disease, stroke and brain cancer.
  • Critical parameters for pharmaceutical substances include their release profiles, bio-distribution and half-lives, all of which are time dependent characteristics. It is therefore important to keep in mind over what time spans permeability and binding of substances are measured.
  • one embodiment of the invention relates to the method of measuring BBB permeability of a substance, wherein the amount of permeating substances are determined after a predetermined period of time, such as within 24 hours.
  • Another embodiment of the invention relates to the method of measuring BBB permeability of a substance, wherein the amount of said substance permeating across the culture insert is measured by a technique selected from the group consisting of radio-imaging, fluorescence, HPLC, FPLC, NMR, MS, ELISA, PCR and/or Western blot.
  • Yet another embodiment of the invention relates to the method of measuring BBB permeability of a substance, wherein the amount of said substance binding to the BEC's is measured by a technique selected from the group consisting of radio- imaging and fluorescence.
  • a fundamental objective for all development of pharmaceutical lead structures is to minimize adverse side effects that could abolish the usability of the substance as a medicament.
  • One key indicator of side effects is the toxicity of a substance to the affected cells.
  • a fourth aspect of the invention relates to a method for
  • the method comprising incubating said substance with the triple cell co-culture according to the invention and subsequently assessing the viability of one or more of the cell types in the triple cell co-culture.
  • An embodiment of the invention relates to a method for determining/evaluating the toxicity of a substance toward the blood brain barrier, wherein the viability of one or more of the cell types in the triple cell co-culture is measured by an assay selected from the group consisting of life/dead assays, Elisa, fluorescence spectrometry, TEER, permeability measurements,.
  • the present invention also provides a method for producing the triple co-culture necessary for establishment of an efficient BBB model.
  • the method revolves around a triple co-culture in which brain endothelial cells and pericytes are in direct contact through the pores of a porous culture membrane, while astrocytes are located in the bottom of the culture plate.
  • the astrocytes and pericytes stimulate the expression of proteins integral to the formation of the BBB.
  • a fifth aspect of the invention relates to a method for producing an isolated triple co-culture comprising porcine primary brain endothelial cells (BEC's), porcine astrocytes and porcine pericytes, the method comprising
  • porcine brain cells a) providing porcine brain cells; b) proliferating from said porcine brain cells viable BEC's, viable porcine astrocytes and viable porcine pericytes; and
  • An embodiment of the invention relates to the method for producing an isolated triple co-culture, further comprising seeding primary cultured brain endothelial cells onto one surface side of a porous culture membrane; seeding primary cultured pericytes or primary cultured astrocytes onto the other surface side of the porous culture membrane; seeding primary cultured astrocytes or primary cultured pericytes onto the inside surface of a culture plate; and co-culturing these cells in a culture medium.
  • a preferred embodiment of the invention relates to the method for producing an isolated triple co-culture, wherein primary cultured pericytes or primary cultured astrocytes are seeded on culture plate and primary cultured pericytes or primary cultured astrocytes are seeded on the side of the porous culture membrane facing towards the culture plate.
  • primary cultured pericytes or primary cultured astrocytes are seeded on culture plate and primary cultured pericytes or primary cultured astrocytes are seeded on the side of the porous culture membrane facing towards the culture plate.
  • one embodiment of the invention relates to the method for producing an isolated triple co-culture, wherein the porcine astrocytes (4), the porcine BEC's (5), and the porcine pericytes (6) are obtained from one or more pigs, which is at least two months old, such as at least, 4 months old or such as at least six months old.
  • conditions also involve handling the pig material at adequate temperatures and periods of time.
  • Prior art teaches that cell material for use in the production of BBB models should be cultured as close as possible to the time of harvesting the cells from the pig brains.
  • the present invention provides several conditions at which cell material may be stored or transported over longer time spans and still yield cellular material suitable for culturing.
  • one embodiment of the invention relates to a method for producing an isolated triple co-culture, wherein the cells are obtained from cooled pig brains.
  • Another embodiment of the invention relates to a method for producing an isolated triple co-culture, wherein said cooled pig brains have been cooled to a temperature below 10°C, such as below 5°C, such as below 1°C, such as in the range 0.1-10°C, such as 2-10°C, or such as 5-10°C.
  • Yet another embodiment of the invention relates to a method for producing an isolated triple co-culture, wherein said cooled pig brains are cooled after sacrificing the pig, such as within 4 hours from death of the animal, such as within 2 hours from the death of the animal, preferably within 30 minutes from the death of the animal.
  • a further embodiment of the invention relates to a for producing an isolated triple co-culture, wherein cells are harvested from the cooled pig brains for further processing within 24 hours from the death of the animal, such as within 12 hours from the death of the animal, such as 4 hours from the death of the animal, preferably within 3 hours from the death of the animal.
  • one embodiment of the invention relates to a method for producing an isolated triple co-culture, wherein the astrocytes are seeded before the BEC's and the pericytes, such as 1-10 weeks before, such 1-4 weeks before, such as 1-3 weeks before, such as 14-21 days before.
  • cryoprotecting may for example be achieved by slowly cooling the cell material or quickly by immersion of the cell material into liquid nitrogen. Cell material is typically frozen together with a cryoprotectant, such as DMSO, to preserve the sample.
  • a cryoprotectant such as DMSO
  • one embodiment of the invention relates to a method for producing an isolated triple co-culture, wherein one or more of the BEC's, astrocytes and pericytes are cryoprotected as a monoculture after step b) and optionally before step c).
  • Another embodiment of the invention relates to a method for producing an isolated triple co-culture, wherein one or more of the BEC's, astrocytes and pericytes are provided from a cryoprotected frozen monoculture before step c).
  • a further embodiment of the invention relates to a method for producing an isolated triple co-culture, wherein the BEC's or pericytes are provided from a cryoprotected frozen monoculture.
  • a sixth aspect of the invention is an isolated triple co-culture comprising porcine primary brain endothelial cells (BEC's), porcine astrocytes and porcine pericytes obtained/obtainable by a process according to the method for producing an isolated triple co-culture comprising porcine primary brain endothelial cells
  • PBEC's derived from 6 months old domestic pig brains were obtained from the local abattoir (Danish Crown, DK), which are obligated to follow the Danish regulations within animal welfare and are under constant supervision by the Danish and European Food Standard Agency.
  • the brains were collected and transported on ice to the Laboratory of Neurobiology, Aalborg University,
  • Microvessels were collected using a 33% Percoll gradient (Sigma-Aldrich, Brondby, Denmark, DK). The isolated microvessel fragments were finally plated on to 60 mm 2 plastic dishes coated with collagen IV (Sigma-Aldrich) and fibronectin (Sigma-Aldrich).
  • PBEC's were maintained in DMEM/F12 supplemented with 10% plasma-derived serum (First Link, Wolverhampton, United Kingdom, UK), basic fibroblast growth factor (Roche), heparin (Sigma-Aldrich), insulin, transferrin, sodium selenite (Roche) and gentamicin sulphate (10 pg/ml) and cultured in an incubator with humidified 5 % CO 2 / 95 % air at 37 °C. Puromycin (Sigma-Aldrich) was added to the media for the first 3 days to obtain a pure culture of PBEC's.
  • Cerebral porcine pericytes were obtained by culturing a cell fraction obtained from the PBEC's isolation protocol. When the microvessels were collected from the Percoll gradient, the underlying cell fraction in the gradient was collected as pericytes. Pericyte survival and proliferation were favored over PBEC's by i) using uncoated dishes, ii) addition of puromycin, and iii) DMEM supplemented with 10% fetal calf serum and gentamicin sulphate. Only passage 1 or 2 of primary porcine pericytes were used in this study. It is noted that (like astrocytes) pericytes can also be cryoprotected and frozen before use.
  • Porcine brain endothelial cells can also be cryoprotected and frozen before use.
  • the cerebral endothelial cells, astrocytes and pericytes can all be cryoprotected and frozen before they are used in a triple culture. Therefore, cells from the same animal can be stored and later thawed for new/different purposes without being limited by a short timeframe.
  • Rat pericytes were derived from 2-3 weeks old Sprague Dawley rats as previously described by Nakagawa et al. A new blood-brain barrier model using primary rat brain endothelial cells, pericytes and astrocytes. Neurochem Int. 2009;54: 253- 263.
  • the rats were deeply anesthetized by a subcutaneous injection of 0.5 ml / 10 g body weight of Hypnorm/Dormicum (Fentanyl/Fluanisone mixed with Midazolam and sterile water in a ratio of 1 : 1 :2).
  • the rat heads were rinsed with 70 % ethanol and 10 % poly(vinylpurrolidone)-iodine complex.
  • porcine glia cells were additionally obtained from the brain of the 6 months old domestic pigs. Approximately 1.5-2 g of cortical pieces were collected and mechanically dissociated in DMEM supplemented with 10% fetal bovine serum and gentamicin sulphate. Dissociated cells were seeded into culture flasks until they reached confluence, frozen in media supplemented with DMSO and FCS in a -80 °C freezer for 24 hours, and then moved to a -140°C freezer until use. It was evidenced by immunocytochemistry that the mixed glial cell cultures consisted mainly of astrocytes and only a few microglial cells, and therefore the mixed glial cell population is referred to below as porcine astrocytes.
  • the porcine astrocytes were thawed two-three weeks before establishment of co- culture models and seeded in 12 well dishes to obtain a confluent layer for co- culture.
  • the antibiotic chloramphenicol Sigma-Aldrich
  • MRSA methicillin resistance staphylococcus aureus
  • rat glial cells were also isolated from neonatal Sprague Dawley rats as previously described by Nakagawa et al. The rats were obtained from the Animal Facility at Aalborg University Hospital. Their mothers were fed and housed under a 12/12 h dark/light cycle and had free access to food and water until they were euthanized. All animals were put down following the directions given by the Danish Experimental Animal Inspectorate.
  • the rats were rapidly decapitated by scissor, and their brains removed from the scull. From here on the procedure for isolation of astrocytes and pericytes described above was followed.
  • the thirteen different models were subdivided into four different types of in vitro BBB models.
  • the first and simplest in vitro BBB model was a monoculture of PBEC's, in which the PBEC's were cultured on the upper side of the hanging culture inserts.
  • the second type of in vitro BBB model was a non-contact co-culture model in which the culture insert containing PBEC's were cultured together with porcine astrocytes, rat astrocytes, porcine pericytes or rat pericytes, which were located on the bottom of the 12 well culture dish.
  • the third type was a contact co-culture models in which porcine astrocytes, rat astrocytes, porcine pericytes or rat pericytes was cultured on the bottom of the culture insert, together with PBEC's, which were cultured on the upper side of the culture insert.
  • the fourth and final type of in vitro BBB model was a triple co-culture model. In this model, the PBEC's were cultured on the upper side of the culture inserts, while porcine or rat pericytes were cultured on the bottom side of the culture inserts and porcine or rat astrocytes were seeded on the bottom of the culture dish.
  • the PBEC's in all thirteen in vitro BBB models were supplied once with 550nM hydrocortisone (Sigma-Aldrich), 250 ⁇ cAMP (Sigma-Aldrich) and 17.5 ⁇ RO-201724 (Sigma-Aldrich) to further induce BBB characteristics, when the PBEC's had reached confluence approximately 24 hours after seeding.
  • the astrocytes or pericytes were seeded in a density of 80.000 cells per insert.
  • the hanging cell culture insert was turned upside down in a large petri dish and coated with poly-l-lysine for seeding astrocytes. The appropriate amount of cells was resuspended in 100 ⁇ media per insert and seeded on the insert.
  • the closed petri dish with the hanging cell culture inserts were then placed in an incubator for 3-4 hours until attached.
  • the inserts were then placed hanging into a 12 well culture dish supplied with media in both insert and well and incubated for three days, until PBEC's were seeded in the inserts as described previously.
  • the barrier integrity of the different in vitro BBB models was evaluated by measurement of TEER and permeability to radiolabeled mannitol (Pelkin Elmer, Skovlunde, Denmark, DK).
  • Passive permeability was analyzed by the addition of 1 ⁇ 3H-D-Mannitol (Specific activity 14.2 Ci/mol) to the upper chamber of a culture insert.
  • the culture plate was placed on a rocking table at 37 °C for 120 min. Samples of 100 ⁇ were collected from the upper chamber at 0 and 120 min, and from the lower chamber at 0, 15, 30, 60 and 120 min. The samples were replaced with 100 ⁇ fresh culture medium.
  • Pericytes were stained with monoclonal mouse anti-a-smooth muscle actin (a- SMA) (Sigma-Aldrich, cat. no. A5228, lot 091M4832), polyclonal rabbit anti-ZO-1 and rabbit anti-platelet-derived growth factor receptor-beta (PDGFR- ⁇ ) (Santa Cruz, cat.no.Sc-432, lot K1113).
  • a- SMA monoclonal mouse anti-a-smooth muscle actin
  • PDGFR- ⁇ rabbit anti-platelet-derived growth factor receptor-beta
  • the cells were subsequently stained with goat anti-rabbit Alexa 488 or goat anti-mouse Alexa 585 (Invitrogen) as the secondary antibodies. All cells were counterstained with DAPI.
  • the Millicell membranes were cut out of the inserts and mounted on glass slides in fluorescent mounting media (Dako, Denmark) and cover slips were placed upon the membranes.
  • the qPCR reactions were 95 °C for 10 min, 40 cycles of 95 °C for 30 sec, 60 °C for 30 sec and 72 °C for 30 sec, which were performed using the Stratagene Mx3000PTM QPCR system (Agilent Technologies, Horsholm, Denmark, DK).
  • the relative expression of mRNA was calculated and analysed in the GraphPad Prism 5.0 software using a 1-way ANOVA with Tukey's multiple comparisons post hoc test.
  • Table 1 displays the reference sequence numbers and primer sequences of the six primers used in this study.
  • PBEC's, astrocytes and pericytes were isolated from 6 months old domestic pigs.
  • the present in vitro BBB model gives the advantage of having all three cell types forming an in vitro BBB derived from the same species.
  • astrocytes need culturing for two-three weeks before being co-cultured in most in vitro BBB models, they are often isolated from a different animal than the PBEC's and pericytes. This disadvantage can be avoided in the present model by freezing PBEC's and pericytes, and thereby the present in vitro BBB model contained all three cell-types from the same animal.
  • the average yield of PBEC's per isolation from 12-15 grams of brain tissues was 8-10 x 10 6 cells and for porcine pericytes the yield was on average 5.0 x 10 6 cells.
  • Concerning porcine astrocytes, 5.0 x 10 7 cells were isolated from 2 grams of brain.
  • Such high cellular yield allows for many experiments on the in vitro BBB model using cells from the same isolation.
  • the porcine astrocytes grew slowly in the first week of culture compared to rat astrocytes, but after two weeks of continued culture no difference in growth was detected between the porcine astrocytes and rat astrocytes.
  • PBEC's acquired the characteristic morphology of brain endothelial cells, seen as a tightly connected polarized monolayer.
  • BBB model comprising a triple co-culture of PBEC's, porcine astrocytes and porcine pericytes wherein all three cell-types may be derived from the same animal.
  • Example 3 Immunocytochemical stain of PBEC's, porcine astrocytes and pericytes
  • the porcine mixed glial cells mainly consisted of GFAP positive astrocytes (Fig. 2c).
  • the rat mixed glial cells mainly consisted of GFAP positive cells (Fig. 2d) and a few microglia (data not shown).
  • porcine pericytes stained positive for a-SMA and PDGFR- ⁇ , when cultured in monoculture (Fig. 2g). Porcine pericytes co-cultured in a triple co-culture with PBEC's and astrocytes stained positive for PDGFR- ⁇ but only a minority of the pericytes were a-SMA positive (Fig. 2h). These observations are in good accordance with studies on differential stages of pericytes, which have shown that pericytes always express PDGFR- ⁇ , irrespective of differential stage but turn into a-SMA negative pericytes when subjected to bFGF. Furthermore, a-SMA negative pericytes have been shown to induce higher TEER than a-SMA positive pericytes.
  • Example 4 trans-endothelial electrical resistance of PBECs in thirteen culture conditions
  • TEER measurements denote a valid real-time monitor of the BBB integrity.
  • PBECs form TEER values between 70-1800 ⁇ x cm 2 depending on their culture conditions.
  • PBECs were previously mainly cultured as pure monocultures or as co-cultures with primary rat astrocytes or astrocytic cell lines like C6 glioma.
  • primary rat astrocytes or astrocytic cell lines like C6 glioma.
  • porcine astrocytes and pericytes from 6 months old domestic pigs, and would, therefore, like to
  • the TEER values of the present study was measured by chopstick electrodes, and the PBECs grown on hanging cell culture inserts with a pore size of 1 ⁇ , which differs from the before mentioned studies, which all used a pore size of 0.4 ⁇ and two of the studies measured TEER with an Endohm electrode chamber and the rest with chopstick electrodes. As the pore size and TEER measuring devices affect TEER values, this should be accounted for when comparing studies. Porcine brain endothelial cells in monoculture
  • TEER of PBEC's varied from 831 ⁇ 29 ⁇ x cm 2 for the co-contact porcine pericytes to 1192 ⁇ 113 ⁇ x cm 2 for the contact rat astrocytes. This indicates that rat astrocytes are better at inducing high TEER in PBEC's if they are cultured in contact co-culture, which has also been found by Malina et al with a mean TEER at approximately 1100 ⁇ x cm 2 [35].
  • the PBEC's cultured in non-contact co-cultures varied from 778 ⁇ 33 ⁇ x cm 2 for the non-contact porcine pericytes to 1093 ⁇ 60 ⁇ x cm 2 for the non-contact porcine astrocytes. This means that porcine astrocytes should be preferred if PBEC's are cultured in non-contact co-cultures.
  • Studies made on PBEC's cultured in non- contact co-culture with primary rat astrocytes has reported mean TEER values from ⁇ 400-800 ⁇ x cm 2 , which is lower than the mean TEER value of 881 ⁇ 33 ⁇ x cm 2 obtained in the present study.
  • TEER values for PBEC's cultured in triple culture varied from 1052 ⁇ 55 ⁇ x cm 2 for the triple porcine culture to 1171 ⁇ 55 ⁇ x cm 2 for the triple culture with rat astrocytes and rat pericytes (Fig. 3).
  • the small non-significant differences found in TEER between the PBEC's cultured in the four different triple co-cultures (Fig. 1) indicates that rat astrocytes and pericytes cannot be preferred over porcine astrocytes and pericytes. Comparison of the influence of porcine versus rat pericytes and astrocytes on TEER values
  • TEER values obtained on PBEC's co-cultured with either rat or porcine astrocytes or pericytes were also compared. There was no significant difference between the TEER obtained on PBEC's cultured in non-contact co-cultures when comparing the inductive properties of rat and porcine astrocytes or pericytes. Only TEER values of PBEC's that had been cultured in contact co-cultures with porcine pericytes were significantly lower (P ⁇ 0.05) than TEER values obtained from PBEC's cultured in contact with rat pericytes (Fig. 3). Rat pericytes are therefore better at inducing high TEER in PBEC's when cultured in contact co-culture in comparison with porcine pericytes.
  • Optimal properties of an in vitro BBB model are reflected in high expression of tight junction proteins that do not just lead to a high TEER, but also lead to a low permeability of e.g. sodium fluorescein or mannitol from the luminal to the abluminal side of the in vitro BBB model.
  • the apparent mannitol permeability was measured on PBEC's in thirteen different culture conditions and plotted against TEER values measured on the same PBEC's just before the permeability
  • the permeability showed an inverse relation to TEER.
  • the lowest permeability and hence highest integrity was found in PBEC's cultured in co-contact with rat astrocytes, which had an average steady state mannitol permeability of 0.87 ⁇ 0.04 x 10 "6 cm x s "1 .
  • this culture setup had the highest TEER before the permeability experiment of an average of 1994 ⁇ 79 ⁇ x cm 2 . All the co- and triple co-culture setups were in the range of 4.10 - 0.87 x 10 ⁇ 6 cm x s 1 and no significant difference was found within these culture conditions.
  • Zhang et al have reported a comparable apparent mannitol permeability of 9.4 x 10 ⁇ 5 cm x s 1 for PBEC's in non-contact co-cultures with mean TEER of ⁇ 500 ⁇ x cm 2 .
  • Franke et al reported a permeability coefficient to mannitol in PBEC's in monoculture as low as 1.8 x 10 ⁇ 6 cm x s _1 , but with corresponding high peak TEER values of ⁇ 1500 ⁇ x cm 2 . This corresponds well with our permeability values of PBEC's with a TEER of ⁇ 1500 ⁇ x cm 2 (Fig. 4).
  • Naklband and Ohmidi reported a mannitol permeability coefficient for PBEC's in non-contact co-culture with rat C6 glioma cells at
  • Gaillard and de Boer found that there was an inverse relation between Papp and TEER, and found that at a certain level the Papp would not decrease any further despite of increasing TEER.
  • Franke et al found that PBEC's with a TEER of 600 ⁇ x cm 2 or above had reached a low permeability that did not decrease with further increasing TEER. Patabendige et al found that the apparent
  • TEER should therefore preferably be above 700 ⁇ x cm 2 to ensure a low permeability with the PBEC's in the present study.
  • Example 6 Differences in relative mRNA expression in PBEC's in the thirteen different culture conditions
  • claudin-5, occludin, transferrin receptor, P-gp and BCRP was confirmed by RT-qPCR (Fig. 5).
  • claudin-5 was significantly increased in PBEC's when they were cultured in contact co-culture with either porcine astrocytes (P ⁇ 0.01), porcine pericytes (P ⁇ 0.0001) and triple culture with rat astrocytes and porcine pericytes (P ⁇ 0.05).
  • the claudin-5 expression was significantly higher when PBEC's were cultured in a contact co-culture with porcine pericytes (P ⁇ 0.0001) compared to rat pericytes. This indicates that claudin-5 expression in PBEC's depends on induction from porcine pericytes, which cannot be substituted by rat pericytes.
  • porcine astrocytes were included in the triple porcine culture, the inductive properties of the porcine pericyte were reversed, which was not seen when rat astrocytes were used instead for a triple culture with PBEC's and porcine pericytes.
  • porcine astrocytes should be in contact with the PBEC's for inducing the expression of claudin-5 due to a significant increase in expression (P ⁇ 0.05) seen when comparing with claudin-5 expression in PBEC's in non-contact co-culture with porcine astrocytes.
  • occludin was only significantly increased by non-contact co-culture with rat pericytes (P ⁇ 0.0001) and in triple co-culture with rat astrocytes and rat pericytes (P ⁇ 0.05) when compared to monoculture. Furthermore, in non-contact co-culture rat pericytes significantly increased the occludin expression in PBEC's compared to porcine pericytes in non-contact co-culture (P ⁇ 0.0001). It should though be noted that there were no significant difference between the occludin expression in PBEC's cultured in contact co-culture with either porcine or rat pericytes.
  • occludin expression in PBEC's is highly upregulated by co-culture with rat pericytes in a non-contact co-culture.
  • Malina et al showed that the protein expression of occludin was increased although not significant when PBEC's where co-cultured in contact with the astrocytes compared with PBEC's in non-contact co-cultures with rat astrocytes.
  • PBEC's co-cultured with rat astrocytes in non- contact co-culture have a higher relative mRNA expression of occludin than if they were co-cultured together in a contact co-culture. This difference could be due to differences in isolation and culture methods and this indicates that it is very important to have knowledge of exactly how the PBEC's react in different culture setups to determine which conditions are the most optimal for different research purposes.
  • P-gp (ABCBl) expression in PBEC's was not significantly increased by co-culture or triple co-culture with either porcine or rat astrocytes or pericytes.
  • P-gp expression was increased significantly in PBEC's by non-contact co-culture with rat pericytes when compared to non-contact co-culture with porcine pericytes
  • BCRP (ABCG2) expression was significantly increased in PBEC's in non-contact co- culture with rat astrocytes (P ⁇ 0.01) and rat pericytes (P ⁇ 0.05). Furthermore, BCRP expression was significantly increased in all four types of triple co-culture setups (P ⁇ 0.05). There was no significant difference between co-culturing PBEC's with rat or porcine astrocytes or pericytes. Although not significant, the BCRP expression is upregulated in PBEC's when they are cultured in co-culture and the expression is even higher when they are cultured in triple co-cultures. This up regulation seems to be independent of astrocyte and pericyte origin.
  • Transferrin receptor expression was only significantly decreased in PBEC's by non- contact co-culture with rat astrocytes (P ⁇ 0.01) and in triple co-culture with rat astrocytes and porcine pericytes (P ⁇ 0.05).
  • Example 7 Pericytes impact on TEER, gene expression and permeability in co-culture and triple culture.
  • rat pericytes significantly increased TEER and decreased permeability of primary rat BECs in contact co-culture and triple culture with rat pericytes in contact with the endothelial cells and astrocytes in the bottom of the well when compared to rat BECs in either monoculture or in co- culture with only rat astrocytes.
  • porcine pericytes significantly increase the claudin-5 expression in contact culture with PBEC's when compared to PBEC's in non-contact co-culture with porcine astrocytes (P ⁇ 0.01) and in triple culture with porcine astrocytes and porcine pericytes (contact) (P ⁇ 0.01) (Fig. 5). This indicates that the porcine pericytes are favorable to use in a co-culture with PBEC's if claudin-5 should be highly expressed. Furthermore, rat pericytes are also important for obtaining a high expression of occludin and P-gp in PBEC's when cultured in a non-contact co- culture (Fig. 5).
  • Example 8 Impact of astrocytes vs pericytes on PBEC's in co-culture.
  • rat and porcine astrocytes do not have higher inductive skills on TEER in PBEC's than rat and porcine pericytes do (Fig. 3).
  • astrocytes and pericytes of either porcine or rat origin are equally good at inducing high TEER, low permeability and gene expression of the five investigated genes in the present study.
  • Example 9 Is monoculture, co-culture or triple co-culture the most optimal culture condition for PBEC's for establishing an in vitro BBB model?
  • Example 10 Investigations of the functionality of the P-gp efflux transporter using rhodamine-123 (R123) as a Pgp-substrate, and C4 and Verapamil as P-gp inhibitors.
  • the P-gp efflux transporter is very important for a functional BBB. If the P-gp transporter is inactive, drugs that would normally be effluxed by the P-gp transporter would instead be able to pass through the BBB and get access to the brain.
  • the in vitro BBB model will therefore not be a useful model for predicting drug permeability if the P-gp efflux transporter is not active.
  • Functionality of the Pgp-efflux transporter was analyzed by the addition of 20 ⁇ of the Pgp-substrate R123 in the upper chamber with or without a Pgp-inhibitor (10 ⁇ C4 or 25 ⁇ verapamil) in order to block the efflux transport.
  • the insert was the added 1ml Ringer-Hepes buffer with the calculated amount of R123 and either C4 or verapamil and incubated on a rocking table at 37 °C for two hours. Following the incubation, samples were collected. The samples were diluted 1 : 100 in Ringer-Hepes buffer and loaded in black 96well plate ( ⁇ pr. well) before analyzed using
  • R123 was measured in the wells with or without addition of C4 and verapamil.
  • the accumulation of R123 was increased to 212 ⁇ 50.80% by inhibiting the P-gp efflux transporter with C4.
  • Verapamil inhibited the P-gp efflux transporter slightly by increasing the accumulation of R123 to 125 ⁇ 40.14%
  • porcine in vitro BBB model has a functional P-gp efflux transporter.
  • Example 11 Passive permeability using sodium fluorescein (FLU).
  • the tightness of the in vitro BBB model is determined by measuring the permeability of molecules with different sizes.
  • the BBB should only be permeable to very small molecules ⁇ 70 Da, therefore larger molecules like Mannitol (182 Da) and sodium fluorescein (FLU) (376 Da) should not be able to easily pass through the BBB.
  • Mannitol permeability has previously been described (see e.g. example 5).
  • Passive permeability of the PBECs cultured in either a non-contact co-culture with porcine astrocytes or in a porcine triple-culture was analyzed by the addition of 1 Mg/ml FLU in the upper chamber of a culture insert, in order to investigate the amount of detectable fluorescence in the bottom chamber following a one hour period of incubation.
  • the insert was then added 1ml Ringer-Hepes buffer with the calculated amount of each transport molecule and incubated on a rocking table at 37 °C for 15 min. After the first 15 min of incubation, each insert was transferred to a new well containing 1.5ml Ringer-Hepes buffer and again incubated for 15 min until one hour had passed. After 60 min, the insert was transferred once more to a well with buffer constituting the final step. Afterwards samples were diluted 1 : 100 in Ringer-Hepes buffer and loaded in black 96well plates ( ⁇ pr. well) before analyzed using fluorescence spectrometry (Enspire, Perkin Elmer).
  • the porcine in vitro BBB has a low permeability to FLU, which has a weight of 376 Da.
  • the PBECs cultured in triple porcine culture are less permeable compared to the non-contact co-culture.

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Abstract

La présente invention concerne un modèle in vitro de barrière hémato-encéphalique (BBB) comprenant une coculture porcine triple comprenant des astrocytes porcins, des cellules endothéliales cérébrales (BEC) primaires porcines et des péricytes porcins. L'invention porte également sur des procédés de production de telles cocultures triples porcines et leurs utilisations.
PCT/DK2016/050190 2015-06-19 2016-06-16 Modèle de coculture triple de la barrière hémato-encéphalique utilisant des cellules endothéliales cérébrales porcines primaires, péricytes porcins et astrocytes porcins Ceased WO2016202343A1 (fr)

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US10143187B2 (en) 2017-02-17 2018-12-04 Denali Therapeutics Inc. Transferrin receptor transgenic models
US10457717B2 (en) 2017-02-17 2019-10-29 Denali Therapeutics Inc. Engineered polypeptides
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CN114507642A (zh) * 2022-03-17 2022-05-17 上海纽仁生物医药科技有限公司 一种动物神经系统周细胞单细胞的分离方法
CN114703120A (zh) * 2022-03-17 2022-07-05 上海纽仁生物医药科技有限公司 一种动物神经系统血管平滑肌细胞单细胞的分离方法
CN114507642B (zh) * 2022-03-17 2024-02-02 上海纽仁生物医药科技有限公司 一种动物神经系统周细胞单细胞的分离方法
CN114703120B (zh) * 2022-03-17 2024-02-02 上海纽仁生物医药科技有限公司 一种动物神经系统血管平滑肌细胞单细胞的分离方法

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