Blood-brain barrier model

Abstract

The present invention addresses the problem of providing a blood-brain barrier model that is comparatively simple to produce and has high blood-brain barrier functionality and a production method for the blood-brain barrier model.　A blood-brain barrier model that has a pericyte layer, an astrocyte layer, and a vascular endothelial cell layer and has high blood-brain barrier functionality can be produced by culturing astrocytes to be present at the surface of a fiber structure of a layered body of a fiber structure and a porous membrane, culturing pericytes to be present inside the fiber structure of the layered body, and culturing vascular endothelial cells to form a sheet structure at the surface of the porous membrane of the layered body.

Description

Blood-brain barrier model

[0001] This invention relates to a blood-brain barrier (BBB) ​​model and a method for producing the same.

[0002] Blood vessels in nerve tissues such as the brain, spinal cord, and retina possess a highly sophisticated barrier function that controls the movement of substances between the blood and nerve tissue, preventing substances in the blood from easily entering the nerve tissue. This mechanism that controls the movement of substances between the blood and nerve tissue is called the blood-brain barrier (BBB). The vascular endothelial cells that make up the BBB are tightly bound together by a group of tight junction-forming proteins, which is thought to be responsible for this sophisticated barrier function. Furthermore, the vascular endothelial cells that make up the BBB express various transporters and are thought to be responsible for the exchange of substances via selective intracellular pathways, such as taking in nutrients from the blood and supplying them to the brain, as well as expelling foreign substances from the brain into the bloodstream. Although the BBB is essentially composed of vascular endothelial cells, the pericytes and astrocytes surrounding them are also thought to be involved in the maintenance and modification of BBB function.

[0003] When developing therapeutic drugs for central nervous system diseases such as Alzheimer's disease, cerebral infarction, and multiple sclerosis, it is necessary to select candidate substances with good brain penetration, and for this selection, an in vitro BBB model that more faithfully reproduces the BBB in vivo is required. For example, a BBB model has been reported in which vascular endothelial cells and pericytes are cultured on the upper and lower surfaces of a porous membrane, respectively, and astrocytes are cultured on the upper surface of the culture well (Patent Documents 1 and 2). However, in such a BBB model, since the astrocytes are located on a different culture well from the porous membrane in which the vascular endothelial cells and pericytes are cultured, there was doubt as to whether it adequately reflects the in vivo BBB model. Furthermore, the present inventors have reported a BBB model in which pericytes and astrocytes are cultured on the upper and lower surfaces of a porous membrane, respectively, and a cell sheet of vascular endothelial cells prepared separately is laminated on the layer of cultured pericytes (Patent Document 3).

[0004] Japanese Patent Publication No. 2007-166915, International Publication No. 2016 / 202343, International Publication No. 2017 / 179375

[0005] The object of the present invention is to provide a blood-brain barrier model that can be manufactured relatively easily and has high blood-brain barrier function, as well as a method for manufacturing the same.

[0006] In order to solve the above problems, the present inventors have diligently conducted research and have discovered that by culturing astrocytes to exist on the surface of the fibrous structure in a laminate of a fibrous structure and a porous membrane, culturing pericytes to exist inside the fibrous structure in the laminate, and culturing vascular endothelial cells to form a sheet structure on the surface of the porous membrane in the laminate, it is possible to produce a blood-brain barrier model with high blood-brain barrier function having three types of cell layers: pericytes, astrocytes, and vascular endothelial cells, thus completing the present invention.

[0007] In other words, the present invention is as follows: [1] A blood-brain barrier model comprising a laminate of three cell layers, pericytes, astrocytes, and vascular endothelial cells, and a fibrous structure and a porous membrane, wherein the astrocytes are located on the surface of the fibrous structure in the laminate, the pericytes are located inside the fibrous structure in the laminate, and the vascular endothelial cells form a sheet structure on the surface of the porous membrane in the laminate. [2] The blood-brain barrier model according to [1], wherein the average pore diameter of the pores on the surface of the porous membrane is 20 μm or less. [3] The blood-brain barrier model according to [1] or [2], wherein the pericytes, astrocytes, and vascular endothelial cells are human-derived cells. [4] The blood-brain barrier model according to any one of [1] to [3], wherein the fibrous structure is a nonwoven fabric. [5] A method for producing a blood-brain barrier model, comprising the following steps (a) to (e), wherein the blood-brain barrier model is composed of three cell layers: pericytes, astrocytes, and vascular endothelial cells, and a laminate of a fibrous structure and a porous membrane, wherein the astrocytes are located on the surface of the fibrous structure in the laminate, the pericytes are located inside the fibrous structure in the laminate, and the vascular endothelial cells form a sheet structure on the surface of the porous membrane in the laminate. (a) A step of preparing a laminate of a fibrous structure and a porous membrane; (b) A step of seeding pericytes into the interior of the fibrous structure from the surface side of the fibrous structure in the laminate; (c) A step of seeding astrocytes on the surface of the fibrous structure in the laminate; (d) A step of seeding vascular endothelial cells on the surface of the porous membrane in the laminate; (e) A step of culturing the laminate seeded with pericytes, astrocytes, and vascular endothelial cells until three types of cell layers of pericytes, astrocytes, and vascular endothelial cells are formed and the vascular endothelial cells form a sheet structure on the surface of the porous membrane; [6] The manufacturing method according to [5] above, carried out in the order of steps (b), (c), and (d). [7] The manufacturing method according to [6] above, wherein the laminate after step (c) is inverted 180 degrees and step (d) is carried out.[8] The manufacturing method according to any one of [5] to [7] above, wherein the average pore diameter of the pores on the surface of the porous membrane is 20 μm or less. [9] The manufacturing method according to any one of [5] to [8] above, wherein the pericytes, astrocytes, and vascular endothelial cells are human-derived cells.

[10] The manufacturing method according to any one of [5] to [9] above, wherein the fibrous structure is a nonwoven fabric.

[0008] Another embodiment of the present invention is a blood-brain barrier model comprising three cell layers, pericytes, astrocytes, and vascular endothelial cells, and a fibrous structure having a surface A and a surface B, and an interior sandwiched between the surfaces A and B, wherein the astrocytes are located on surface A, the pericytes are located in the interior, and the vascular endothelial cells form a sheet structure on surface B, the blood-brain barrier model (which may be referred to as "the BBB Model 2" in this specification);

[0009] Another embodiment of the present invention is a blood-brain barrier model comprising a laminate of three cell layers, pericytes, astrocytes, and vascular endothelial cells, and a fibrous structure A and a fibrous structure B, wherein the astrocytes are located on the surface of the fibrous structure A in the laminate, the pericytes are located inside the fibrous structure A in the laminate, and the vascular endothelial cells form a sheet structure on the surface of the fibrous structure B in the laminate (this blood-brain barrier model may be referred to as "the BBB Model 3" in this specification).

[0010] Another embodiment of the present invention is a method for producing a blood-brain barrier model, comprising the following steps (p) to (s): (p) seeding pericytes into the interior of the fibrous structure; (q) seeding astrocytes onto surface A of the fibrous structure; (r) seeding vascular endothelial cells onto surface B of the fibrous structure; (s) culturing the fibrous structure from which the pericytes, astrocytes, and vascular endothelial cells have been seeded until three cell layers of pericytes, astrocytes, and vascular endothelial cells have been formed and the vascular endothelial cells have been formed on surface B of the fibrous structure;

[0011] Another embodiment of the present invention is a method for manufacturing a blood-brain barrier model, comprising the following steps (t) to (y), wherein the blood-brain barrier model is composed of three cell layers: pericytes, astrocytes, and vascular endothelial cells, and a laminate of fibrous structure A and fibrous structure B, the astrocytes are located on the surface of fibrous structure A in the laminate, the pericytes are located inside fibrous structure A in the laminate, and the vascular endothelial cells form a sheet structure on the surface of fibrous structure B in the laminate. (t) A step of preparing a laminate of fiber structure A and fiber structure B; (u) A step of seeding pericytes into the interior of fiber structure A from the surface side of fiber structure A in the laminate; (v) A step of seeding astrocytes on the surface of fiber structure A in the laminate; (x) A step of seeding vascular endothelial cells on the surface of fiber structure B in the laminate; (y) A step of culturing the laminate seeded with pericytes, astrocytes, and vascular endothelial cells until three types of cell layers of pericytes, astrocytes, and vascular endothelial cells are formed and the vascular endothelial cells form a sheet structure on the surface of fiber structure B;

[0012] According to the present invention, a blood-brain barrier model with high blood-brain barrier function can be manufactured relatively easily. Therefore, a high-quality blood-brain barrier model can be provided without requiring skilled techniques or complex work processes, which contributes to analysis using the blood-brain barrier model (for example, selection of candidate substances with good brain permeability).

[0013] These are microscopic images of the BBB Model 1, which was manufactured using a laminate of nonwoven fabric and porous membrane, and three types of cells (pericytes, astrocytes, and vascular endothelial cells). The first image shows the results of an analysis of the presence of pericytes and astrocytes on the surface of the nonwoven fabric in the laminate of nonwoven fabric and porous membrane (Figures 2A and 2C, respectively), and the presence of pericytes and astrocytes inside the nonwoven fabric (Figures 2B and 2D, respectively). The second image shows the results of an analysis of the presence of endothelial cells (Figure 3A), pericytes (Figure 3B), and astrocytes (Figure 3C) on the surface of the porous membrane, on the surface of the nonwoven fabric, and inside the nonwoven fabric in the laminate of nonwoven fabric and porous membrane. The third image shows a microscopic image of a sheet structure of vascular endothelial cells formed on the surface of a porous membrane having pores with an average pore size of 20 μm (Figure 4A), 25 μm (Figure 4B), or 30 μm (Figure 4C). The white circles in the image indicate an example of voids. This figure shows the results of analyzing the barrier function of BBB Model 1 (n=7) and the comparative BBB Model (n=7). The asterisk (*) in the figure indicates a statistically significant difference (p < 0.05).

[0014] <The BBB Model of the Present> The blood-brain barrier model of the present invention is a blood-brain barrier model (hereinafter referred to as "the BBB constituent cell layers") composed of three types of cell layers: pericytes, astrocytes, and vascular endothelial cells, and a laminate of fibrous structures and porous membranes (hereinafter referred to as "the laminate 1"), wherein the astrocytes are located on the surface of the fibrous structures in the laminate 1 (i.e., the surface opposite to the surface of the fibrous structures stacked with the porous membrane), the pericytes are located inside the fibrous structures in the laminate 1, and the vascular endothelial cells form a sheet structure on the surface of the porous membrane in the laminate 1 (i.e., the surface opposite to the surface of the porous membrane stacked with the fibrous structures), and the blood-brain barrier model (hereinafter referred to as "the BBB Model 1"), 2) A blood-brain barrier model comprising a BBB constituent cell layer and a fibrous structure (which may be referred to as "the fibrous structure 2" in this specification) having a surface A and a surface B, and an interior sandwiched between the surfaces A and B, wherein the astrocytes are located on surface A of the fibrous structure 2, the pericytes are located in the interior of the fibrous structure 2, and the vascular endothelial cells form a sheet structure on surface B of the fibrous structure 2, and the blood-brain barrier model (i.e., the BBB model 2), 3) A blood-brain barrier model comprising the BBB constituent cell layer and a laminate of fibrous structure A and fibrous structure B (hereinafter referred to as "Laminate 3"), wherein the astrocytes are located on the surface of fibrous structure A in Laminate 3 (i.e., the surface opposite to the surface of fibrous structure A stacked with fibrous structure B), the pericytes are located inside fibrous structure A in Laminate 3, and the vascular endothelial cells form a sheet structure on the surface of fibrous structure B in Laminate 3 (i.e., the surface opposite to the surface of fibrous structure B stacked with fibrous structure A), and the blood-brain barrier model (i.e., BBB Model 3) is sufficient, and BBB Models 1 to 3 (hereinafter referred to simply as "BBB Models") are in vitro models.

[0015] In this specification, "three types of cell layers: pericytes, astrocytes, and vascular endothelial cells" means three types of cell layers formed in which the cell populations of pericytes, astrocytes, and vascular endothelial cells are not mixed together but can be distinguished from each other (i.e., a cell layer containing the cell population of pericytes, a cell layer containing the cell population of astrocytes, and a cell layer containing the cell population of vascular endothelial cells).

[0016] In the BBB Model 1, the BBB constituent cell layers can be stacked (laminated) due to the structure of the laminate 1, and a portion or all of each cell layer (for example, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more of the total of each cell layer, etc.) may be in direct contact with other cell layers or in a non-contact state. The BBB constituent cell layers are stacked in the order of astrocyte cell layer, pericyte cell layer, and vascular endothelial cell layer, from the surface of the fibrous structure toward the surface of the porous membrane.

[0017] In the BBB Model 2, the BBB constituent cell layers can be stacked (laid up) due to the structure of the fibrous structure 2, and a portion or all of each cell layer (for example, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more of the total of each cell layer, etc.) may be in direct contact with other cell layers or in a state of non-contact. The BBB constituent cell layers are stacked in the order of astrocyte cell layer, pericyte cell layer, and vascular endothelial cell layer in the direction from surface A to surface B of the fibrous structure 2.

[0018] In the BBB Model 3, the BBB constituent cell layers can be stacked (laid) due to the structure of the laminate 3, and a portion or all of each cell layer (for example, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more of the total of each cell layer, etc.) may be in direct contact with other cell layers or in a non-contact state. The BBB constituent cell layers are stacked in the order of astrocyte cell layer, pericyte cell layer, and vascular endothelial cell layer in the laminate 3, from the fibrous structure A to the surface of fibrous structure B.

[0019] In the BBB Model 1 of this case, the astrocyte cell population may be present inside the fiber structure and / or on the surface of the porous membrane in the laminate 1, as long as it is present on the surface of the fiber structure in the laminate 1. However, it is preferable that the majority of the total astrocyte cell population (e.g., more than 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, etc.) is present on the surface of the fiber structure, and a portion thereof (e.g., less than 50%, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, etc.) is present inside the fiber structure and / or on the surface of the porous membrane.

[0020] In the BBB Model 2 of this case, the astrocyte cell population may exist inside and / or on surface B of the fiber structure 2, as long as it exists on surface A of the fiber structure 2. However, it is preferable that the majority of the total astrocyte cell population (e.g., more than 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, etc.) exists on surface A of the fiber structure 2, and a portion of it (e.g., less than 50%, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, etc.) exists inside and / or on surface B of the fiber structure 2.

[0021] In the BBB Model 3 of this case, the astrocyte cell population may exist inside the fiber structure A and / or on the surface of the fiber structure B in the laminate 3, as long as it exists on the surface of the fiber structure A in the laminate 3. However, it is preferable that the majority of the total astrocyte cell population (e.g., more than 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, etc.) exists on the surface of the fiber structure A in the laminate 3, and a portion of it (e.g., less than 50%, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, etc.) exists inside the fiber structure A and / or on the surface of the fiber structure B in the laminate 3.

[0022] In the BBB Model 1, the perisite cell population may be located on the surface of the fiber structure in the laminate 1 and / or on the surface of the porous membrane, as long as it is located inside the fiber structure in the laminate 1. However, it is preferable that the majority of the total perisite cell population (e.g., more than 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, etc.) is located inside the fiber structure, and a portion of it (e.g., less than 50%, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, etc.) is located on the surface of the fiber structure and / or on the surface of the porous membrane.

[0023] In the BBB Model 2, the pericyte cell population may exist on surface A and / or surface B of the fiber structure 2, as long as it exists at least inside the fiber structure 2. However, it is preferable that the majority of the total pericyte cell population (e.g., more than 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, etc.) exists inside the fiber structure 2, and a portion of it (e.g., less than 50%, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, etc.) exists on surface A and / or surface B of the fiber structure 2.

[0024] In the BBB Model 3, the perisite cell population may exist on the surface of fiber structure A and / or fiber structure B in the laminate 3, as long as it exists at least inside fiber structure A in the laminate 3. However, it is preferable that the majority of the total perisite cell population (e.g., more than 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, etc.) exists inside fiber structure A in the laminate 3, and a portion of it (e.g., less than 50%, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, etc.) exists on the surface of fiber structure A and / or fiber structure B in the laminate 3.

[0025] In the BBB Model 1, the vascular endothelial cell population may be located on the surface and / or inside the fibrous structure of the laminate 1, as long as it forms a sheet structure on the surface of the porous membrane in the laminate 1. However, it is preferable that the majority of the total vascular endothelial cell population (e.g., at least 80%, at least 85%, at least 90%, at least 93%, at least 96%, at least 99%, etc.) is located on the surface of the porous membrane, and a portion of it (e.g., 20% or less, 15% or less, 10% or less, 7% or less, 4% or less, 1% or less, etc.) is located on the surface and / or inside the fibrous structure.

[0026] In the BBB Model 2, the vascular endothelial cell population may be located on and / or inside the surface A of the fibrous structure 2, as long as it forms a sheet structure on surface B of the fibrous structure 2. However, it is preferable that the majority of the total vascular endothelial cell population (for example, at least 80%, at least 85%, at least 90%, at least 93%, at least 96%, at least 99%, etc.) is located on surface B of the fibrous structure 2, and a portion of it (for example, 20% or less, 15% or less, 10% or less, 7% or less, 4% or less, 1% or less, etc.) is located on and / or inside the surface A of the fibrous structure 2.

[0027] In the BBB Model 3, the vascular endothelial cell population may be located on the surface and / or inside the fiber structure A in the laminate 3, as long as it forms a sheet structure on the surface of the fiber structure B in the laminate 3. However, it is preferable that the majority of the total vascular endothelial cell population (for example, at least 80%, at least 85%, at least 90%, at least 93%, at least 96%, at least 99%, etc.) is located on the surface of the fiber structure B in the laminate 3, and a portion of it (for example, 20% or less, 15% or less, 10% or less, 7% or less, 4% or less, 1% or less, etc.) is located on the surface and / or inside the fiber structure A in the laminate 3.

[0028] In this specification, "vascular endothelial cells...form a sheet structure" means that the cell density of vascular endothelial cells is high (for example, 0.5 × 10⁻⁶). ６ cells / cm ２ ~4.0 x 10 ６ cells / cm ２ Within the range [preferably 1.0 × 10 ６ cells / cm ２ ~2.0 x 10 ６ cells / cm ２ This refers to a state in which cells are tightly bound together in a sheet-like manner, with little to no intercellular space (for example, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, etc., of the total surface area of ​​the cell sheet composed of vascular endothelial cells).

[0029] Pericytes, astrocytes, and vascular endothelial cells may be derived from humans or from non-human mammals (for example, rodents such as mice, rats, hamsters, and guinea pigs; lagomorphs such as rabbits; ungulates such as pigs, cats, goats, horses, and sheep; carnivores such as dogs and cats; and primates such as monkeys, rhesus macaques, crab-eating macaques, marmosets, orangutans, and chimpanzees). Furthermore, each of the pericytes, astrocytes, and vascular endothelial cells may be derived from the same species or from different species, but it is preferable that all of them be derived from humans.

[0030] The pericytes that make up the BBB component cell layer refer to cells that exist around the microvascular walls present in tissues or organs such as the brain and retina, are surrounded by the basement membrane, and are also called perivascular cells. Since the effects of pericytes are demonstrated in the following examples, pericytes in the brain (i.e., brain pericytes) can be preferably exemplified.

[0031] The astrocytes that make up the BBB component cell layer are a type of glial cell present in the central nervous system and refer to cells that express GFAP (Glial fibrillary acidic protein).

[0032] The vascular endothelial cells that make up the BBB component cell layer may be any cells that constitute the inner surface of blood vessels. For example, microvascular endothelial cells in tissues or organs such as the brain, lung, skin, heart, uterus, etc.; umbilical vein endothelial cells; aortic endothelial cells; coronary artery endothelial cells; pulmonary artery endothelial cells; etc. can be listed. Since the effects are demonstrated in the following examples, microvascular endothelial cells in the brain (i.e., brain microvascular endothelial cells) can be preferably exemplified.

[0033] As pericytes, astrocytes, and vascular endothelial cells, cells that induce cell proliferation when cultured under certain conditions (e.g., specific temperature; presence of nutrients, growth factors, etc.) and suppress cell proliferation and promote differentiation into mature cells when cultured under other conditions (i.e., conditionally immortalized cells) are preferred. As this effect has been demonstrated in the embodiment described later, temperature-conditionally immortalized cells (i.e., cells that induce cell proliferation [become immortalized] at a specific temperature, and suppress cell proliferation and promote differentiation into mature cells under other temperature conditions) can be suitably exemplified. Temperature-induced immortalized cells can be produced by mutation or introduction of exogenous genes. For example, temperature-induced immortalized cells at approximately 33°C can be produced by introducing the gene for the temperature-sensitive SV40 large T antigen (specifically, a protein that binds to the p53 and Rb proteins, which are potent tumor suppressor genes, and inhibits their function under conditions of approximately 33°C) into primary cultured pericytes, astrocytes, and vascular endothelial cells, following the methods described in "J Neurological Science 331 136-144 2013", "J Cell Physiol 226 255-266 2010", or "J Cell Physiol 225 519-528 2010". Pericytes, astrocytes, and vascular endothelial cells may be self-produced or commercially available.

[0034] <Manufacturing Method> The manufacturing method of the blood-brain barrier model of the present invention includes the following steps: (a) a step of preparing a laminate of a fibrous structure and a porous membrane (i.e., the laminate 1); (b) a step of seeding pericytes into the interior of the fibrous structure in the laminate 1 from the surface side of the fibrous structure; (c) a step of seeding astrocytes on the surface of the fibrous structure in the laminate 1; (d) a step of seeding vascular endothelial cells on the surface of the porous membrane in the laminate 1; and (e) a step of culturing the laminate 1 seeded with pericytes, astrocytes, and vascular endothelial cells until three types of cell layers of pericytes, astrocytes, and vascular endothelial cells are formed and the vascular endothelial cells form a sheet structure on the surface of the porous membrane; and thus the three types of cell layers of pericytes, astrocytes, and vascular endothelial cells (i.e., the BBB constituent cell layers), and the laminate of a fibrous structure and a porous membrane (i.e., the laminate 1). A method for manufacturing a blood-brain barrier model (i.e., the BBB model 1) (hereinafter referred to as "the manufacturing method 1"), comprising: 1) a fibrous structure having surfaces A and B and an interior sandwiched between surfaces A and B (i.e., the fibrous structure 2); 2) a step of seeding pericytes into the interior of a fibrous structure having surfaces A and B and an interior sandwiched between surfaces A and B (i.e., the fibrous structure 2) (p); a step of seeding astrocytes onto surface A of the fibrous structure 2 (q); a step of seeding vascular endothelial cells onto surface B of the fibrous structure 2 (r); Step (s) of culturing the fibrous structure 2, which has been seeded with pericytes, astrocytes, and vascular endothelial cells, until three types of cell layers of pericytes, astrocytes, and vascular endothelial cells are formed, and the vascular endothelial cells form a sheet structure on the surface B of the fibrous structure 2;A method for manufacturing a blood-brain barrier model (i.e., the BBB model 2) (which may be referred to as "the manufacturing method 2" in this specification), comprising steps (p) to (s), wherein the BBB model 2 is composed of the BBB constituent cell layer and the fibrous structure 2, wherein the astrocytes are located on surface A, the pericytes are located inside the fibrous structure 2, and the vascular endothelial cells form a sheet structure on surface B of the fibrous structure 2; 3) Step (t) for preparing a laminate of fibrous structure A and fibrous structure B (i.e., the laminate 3); Step (u) for seeding pericytes from the surface side of fibrous structure A in the laminate 3 into the interior of fibrous structure A; Step (v) for seeding astrocytes on the surface of fibrous structure A in the laminate 3; Step (x) for seeding vascular endothelial cells on the surface of fibrous structure B in the laminate 3; A method for manufacturing a blood-brain barrier model (hereinafter referred to as "the manufacturing method 3") comprising steps (t) to (y), wherein the laminate 3, on which pericytes, astrocytes, and vascular endothelial cells have been seeded, is cultured until three cell layers of pericytes, astrocytes, and vascular endothelial cells are formed and the vascular endothelial cells form a sheet structure on the surface of the fibrous structure B, wherein the astrocytes are located on the surface of the fibrous structure A in the laminate 3, the pericytes are located inside the fibrous structure A in the laminate 3, and the vascular endothelial cells form a sheet structure on the surface of the fibrous structure B in the laminate 3. The manufacturing methods 1 to 3 are collectively referred to simply as "the manufacturing method" in this specification.

[0035] In the manufacturing method 1 of the present case, as the order of performing the steps (b) to (d) between the implementation of step (a) and the implementation of step (e), specifically, the order of step (b), step (c), and step (d); the order of step (b), step (d), and step (c); the order of step (c), step (b), and step (d); the order of step (c), step (d), and step (b); the order of step (d), step (b), and step (c); and the order of step (d), step (c), and step (b) can be cited. The order of step (b), step (c), and step (d) can be preferably exemplified. Since perisite has the property of潜入 into the interior of the fibrous structure, even if perisite is seeded into the fibrous structure from the surface side of the fibrous structure seeded with astrocyte, or after perisite is seeded into the fibrous structure from the surface side of the fibrous structure and then astrocyte is seeded on the surface of the fibrous structure, the cell population of perisite and the cell population of astrocyte do not become a浑然一体 state, and a cell layer containing the cell population of perisite and a cell layer containing the cell population of astrocyte can be formed.

[0036] In the above step (a), as a method for preparing the laminate 1 of the present case, any method can be used as long as it can prepare a laminate (stacked) of a fibrous structure and a porous membrane. For example, a method of applying a solution of a resin, which is a raw material of the porous membrane, on the surface of the fibrous structure or the porous membrane, and laminating the two by adhering the applied surface and the surface of the other (porous membrane or fibrous structure); a method of applying an adhesive on the surface of the fibrous structure or the porous membrane, and laminating the two by adhering the applied surface and the surface of the other (porous membrane or fibrous structure); a method of thermally fusing the fibrous structure and the porous membrane to laminate the two; a method of laminating the fibrous structure and the porous membrane by physically fixing them using a fixing member such as Cell Crown 24 NX insert (manufactured by scaffdex) and the like can be cited.

[0037] When the manufacturing method 1 of the present case is performed in the order of step (b), step (c), and step (d), from the viewpoint of automation, it is preferable to invert the laminate 1 of the present case 180 degrees after step (c) and perform step (d).

[0038] It should be noted that the Chinese expression "潜入" in the original text seems to be an incorrect or incomplete word. It might be a misspelling or an unclear term. The translation is made based on the best understanding of the context, but this part may need further clarification in the original text for a more accurate translation.In the manufacturing method of the present case, the method of seeding pericytes, astrocytes, and vascular endothelial cells is not particularly limited. For example, a cell suspension containing a cell population of pericytes, astrocytes, or vascular endothelial cells can be aspirated into a container such as a pipette tip or pipette using a piston pipette, automated electric pipette, pipettor, Pipetman, etc., and dropped onto the object to be seeded (for example, the fibrous structure or porous surface in the present laminate 1). The temperature at the time of seeding is not particularly limited and can be room temperature (for example, within the range of 10°C to 30°C) or a temperature suitable for culturing pericytes, astrocytes, and vascular endothelial cells (for example, within the range of 33°C to 38°C). The cell suspension at the time of seeding may be a liquid containing pericytes, astrocytes, or vascular endothelial cells, but considering subsequent cell culture, such a liquid is preferably a culture medium.

[0039] As the culture method in the above steps (e), step (s), and step (y), any culture method can be used as long as the seeded pericytes, astrocytes, and vascular endothelial cells proliferate, the BBB constituent cell layer of the present case is formed, and a sheet structure of vascular endothelial cells is formed on the surface of the porous membrane, on the surface B of the fibrous structure, or on the surface of the fibrous structure B. The culture period is not particularly limited and is, for example, within the range of 3 to 30 days, preferably 4 to 20 days, and more preferably 4 to 10 days. The culture medium used for culture is not particularly limited and examples include culture media for animal cell culture (DMEM, EMEM, IMDM, RPMI1640, αMEM, F-12, F-10, M-199, AIM-V, etc.) containing 0.1 to 30 (v / v)% serum (fetal bovine serum [Fetal bovine serum; FBS], calf bovine serum [Calf bovine serum; CS], etc.). The culture temperature is usually within the range of 30 to 40°C. When using immortalized cells under the temperature conditions described above, a combination of a temperature at which cell proliferation is induced (for example, about 33°C) and a temperature at which differentiation into mature cells is promoted (for example, about 37°C) is preferable. CO during culture ２The concentration is typically in the range of about 1-10%, preferably about 5%. The humidity during cultivation is typically in the range of about 70-100%, preferably about 95-100%.

[0040] <Fiber Structures> In this specification, "fiber structure" means a structure composed of fibers and having three-dimensionally connected voids on its surface. Examples of fiber structures include woven fabrics, knitted fabrics, nonwoven fabrics, braids, 3D printing, etc., with nonwoven fabrics being a preferred example. Such nonwoven fabrics may be long-fiber nonwoven fabrics or short-fiber nonwoven fabrics. Examples of long-fiber nonwoven fabrics include spunbond nonwoven fabrics, meltblown nonwoven fabrics, electrospun nonwoven fabrics, etc. Examples of short-fiber nonwoven fabrics include needle-punched nonwoven fabrics, chemical-bonded nonwoven fabrics, thermal-bonded nonwoven fabrics, spunlace nonwoven fabrics, etc. Examples of the shape of the fiber structure include hollow fiber shape, thread shape, flat membrane shape, etc.

[0041] The base material for the above-mentioned fibrous structure is not particularly limited, and examples include polymer compounds such as natural polymer compounds and synthetic polymer compounds. Examples of such natural polymer compounds include proteins and polysaccharides. Examples of proteins include gelatin, collagen (e.g., atelocollagen), fibronectin, fibrinogen, laminin, and fibrin. Examples of polysaccharides include natural polymers such as chitosan, alginic acid, calcium alginate, heparan sulfate, chondroitin sulfate, sialic acid, hyaluronic acid, heparin, starch, gellan gum, agarose, guar gum, xanthan gum, carrageenan, pectin, locust bean gum, tamarind gum, and dieutan gum, as well as derivatives of natural polymer compounds such as carboxymethylcellulose. Examples of the above-mentioned synthetic polymer compounds include non-absorbent synthetic polymer compounds such as polyester, polyethylene glycol, polypropylene glycol, polyethylene terephthalate, polyvinyl alcohol, thermoplastic elastomer, polypropylene, polyethylene, polystyrene, polymethyl methacrylate, polycarbonate, polydimethylsiloxane, cycloolefin polymer, and amorphous fluororesin, as well as bioabsorbable polymers such as polylactic acid, polyglycolic acid, polycaprolactone, and polydioxanone. These polymer compounds may be used individually or in combination of two or more.

[0042] As for the above fiber structure, from the viewpoint of versatility, a synthetic polymer nonwoven fabric mainly composed of the aforementioned synthetic polymer compounds (e.g., polyester, polyethylene, etc.) is preferred, and from the viewpoint of affinity with pericytes and astrocytes, a natural polymer nonwoven fabric mainly composed of the aforementioned natural polymer compounds (e.g., collagen [e.g., atelocollagen], gelatin, etc.) is preferred.

[0043] The average pore diameter of the voids on and / or inside the above-mentioned fiber structure is not particularly limited. Examples of lower limits include 0.3 μm or more, 0.4 μm or more, 0.5 μm or more, 0.6 μm or more, 1.0 μm or more, 1.5 μm or more, 2.0 μm or more, 2.5 μm or more, 3.0 μm or more, 3.5 μm or more, 4.0 μm or more, 4.5 μm or more, 5.0 μm or more, 5.5 μm or more, 6.0 μm or more, 6.5 μm or more, 7.0 μm or more, etc. Examples of upper limits include 300 μm or less, 200 μm or less, 100 μm or less, 60 μm or less, 50 μm or less, 40 μm or less, 30 μm or less, 20 μm or less, 10 μm or less, etc. These lower and upper limits can be combined arbitrarily. The average pore size of the voids on the surface of the above-mentioned fibrous structure can preferably be 5.0 to 10 μm.

[0044] <Porous membrane> In this specification, "porous membrane" means a membrane having a plurality of pores on the surface of the porous membrane in the laminate 1, and it is preferable that the porous membrane on the surface of the laminate 1 does not have voids in order to form a sheet structure of vascular endothelial cells.

[0045] The average pore diameter on the surface of the porous membrane is not particularly limited. Examples of lower limits include 0.3 μm or more, 0.4 μm or more, 0.5 μm or more, 0.6 μm or more, 1.0 μm or more, 1.3 μm or more, 1.6 μm or more, 2.0 μm or more, 2.3 μm or more, 2.6 μm or more, 3.0 μm or more, etc. Examples of upper limits include 100 μm or less, 80 μm or less, 60 μm or less, 30 μm or less, 25 μm or less, 20 μm or less, etc. These lower and upper limits can be combined arbitrarily. Preferably, the average pore diameter on the surface of the porous membrane is 20 μm or less, and more preferably between 0.5 μm and 20 μm.

[0046] In this specification, the average pore diameter (number average opening diameter) of voids in a fibrous structure and the average pore diameter (number average opening diameter) of pores in a porous membrane can be determined, for example, according to (1) and (2) below.

[0047] (1) From scanning electron microscope images of the fibrous structure and porous membrane, for example, the pore area S is measured for 200 or more open areas, and assuming that the pore area is a perfect circle, the pore diameter d is determined from the following formula I.

[0048]

[0049] (2) Apply all the hole diameters obtained by formula I above to formula II below to find the number-average aperture diameter Sn (where n represents the total number of holes).

[0050]

[0051] The substrate for the porous membrane described above is not particularly limited, and examples include polycarbonate (PC), polyester (PET), polystyrene (PS), TAC (triacetylcellulose), polyketone (PK), nylon (Ny), low-density polyethylene (LDPE), medium-density polyethylene (MDPE), vinyl chloride, vinylidene chloride, polyphenylene sulfide, polyether sulfone (PES), polyethylene naphthalate, polypropylene, acrylic, etc. Polyester and polystyrene can be preferably exemplified as their effects have been demonstrated in the embodiment described later. In the case of polyester, the porous membrane is a porous membrane based on polyester (i.e., a polyester porous membrane), and in the case of polystyrene, the porous membrane is a porous membrane based on polystyrene (i.e., a polystyrene porous membrane).

[0052] Laminate 1 is a composite composed of a fibrous structure and a porous membrane, and is different from a single porous membrane (single entity). Furthermore, fibrous structure 2 is composed of a fibrous structure, and is different from a porous membrane. Furthermore, laminate 3 is a composite composed of fibrous structure A and fibrous structure B, and is different from a single porous membrane (single entity).

[0053] For the laminate 1, it is preferable that the voids on the surface of the fibrous structure and the pores on the surface of the porous membrane are in communication, in order to allow the candidate substance having good brain transfer properties to pass from the surface of the fibrous structure to the surface of the porous membrane. Furthermore, for the fibrous structure 2, it is preferable that the voids on surface A and the voids on surface B are in communication, in order to allow the candidate substance having good brain transfer properties to pass from surface A to surface B of the fibrous structure. Furthermore, it is preferable that surface B of the fibrous structure 2 has voids that do not hinder the formation of a sheet structure of vascular endothelial cells. Furthermore, for the laminate 3, it is preferable that the voids on the surface of the fibrous structure A and the voids on the surface of the fibrous structure B are in communication, in order to allow the candidate substance having good brain transfer properties to pass from the surface of the fibrous structure A to the surface of the fibrous structure B.

[0054] If the substrates for the fiber structure and porous membrane in laminate 1, the substrates for the fiber structure in fiber structure 2, and the substrates for fiber structure A and fiber structure B in laminate 3 are unsuitable for culturing adherent cells, it is preferable that the surface of the fiber structure, the interior of the fiber structure, and the surface of the porous membrane in laminate 1, the surface A, interior, and surface B of the fiber structure in fiber structure 2, and the surface of fiber structure A, the interior, and the surface of fiber structure B in laminate 3 are coated with a cell adhesion component. If the substrates for the fiber structure and porous membrane in laminate 1, the substrates for the fiber structure in fiber structure 2, and the substrates for fiber structure A and fiber structure B in laminate 3 are suitable for culturing adherent cells, it is preferable that the surface of the fiber structure, the interior, and the surface of the porous membrane in laminate 1, the surface A, interior, and surface B of the fiber structure in fiber structure 2, and the surface of fiber structure A, the interior, and the surface of fiber structure B in laminate 3 are not coated with a cell adhesion component. Examples of such cell adhesion components include collagen, fibronectin, laminin, heparan sulfate proteoglycan, cadherin, gelatin, fibrinogen, fibrin, poly-L-lysine, poly-D-lysine, hyaluronic acid, platelet-rich plasma, and polyvinyl alcohol.

[0055] The present invention will be described more specifically below with reference to examples, but the technical scope of the present invention is not limited to these examples. In the following examples, DMEM (Dulbecco's Modified Eagle Medium) containing 10% FBS (Fetal Bovine Serum) was used as the perisite culture medium, Astrocyte Medium (ScienCell Research Laboratories) containing 10% FBS was used as the astrocyte culture medium, and EGM-2 Endothelial Cell Growth Medium-2 Bullet Kit (LONZA) was used as the endothelial cell culture medium. Unless otherwise specified, each procedure was performed at room temperature (20°C to 25°C).

[0056] 1. Materials and Methods 1-1 Primary astrocyte strains isolated and cultured from human BBB were introduced with the temperature-sensitive SV40 large T antigen (tsA58) gene using a retroviral vector, according to the method described in the literature "J. Neurological Science 331 (2013) 136-144", to create human-derived temperature-immortalized astrocyte strains. For the production of BBB Model 1 and observation using a confocal microscope, human-derived temperature-sensitive astrocytes were used that had been previously stained with Cell Tracker Green (Thermo Fisher). Under conditions of approximately 33°C, tsA58 can bind to the potent tumor suppressor genes p53 and Rb proteins, inhibiting their function and thus inducing cell proliferation in human-derived temperature-immortalized astrocyte strains. However, under conditions of 37°C, its metabolic function is lost, preventing cell proliferation in human-derived temperature-immortalized astrocyte strains and instead inducing differentiation into mature cells.

[0057] 1-2 The tsA58 gene was introduced into primary pericyte strains isolated and cultured from human BBB using a retroviral vector, according to the method described in the literature "Journal of Cell Physiology 226:255-266 (2011)," to create human-derived temperature-immortalized pericyte strains. For the BBB Model 1 prepared in this study and observed with a confocal microscope, cells that had been previously stained with cyto-ID red (Enzo Life Sciences) for living cells were used. Similar to human-derived temperature-immortalized astrocyte strains, the human-derived temperature-immortalized pericyte strains could induce cell proliferation under conditions of approximately 33°C, while they could be differentiated into mature cells under conditions of 37°C.

[0058] 1-3 Brain microvascular endothelial cells (BMECs) isolated and cultured from human BBB were introduced with the tsA58 gene using a retroviral vector according to the method described in the literature "J. Cell Physiol 225:519-528 (2010)" to create a human-derived temperature-conditioned immortalized vascular endothelial cell line. For the BBB Model 1 prepared in this study, the human-derived temperature-conditioned immortalized vascular endothelial cell line was prepared by first staining it with Cell Tracker Orange (Thermo Fisher) for living tissue staining beforehand when observing it with a confocal microscope.

[0059] 1-4 Laminate 1 In laminate 1, a polyester nonwoven fabric (Technowipe, manufactured by Nippon Paper Crecia Co., Ltd.) with an average pore size of 5 to 10 μm was used as the fibrous structure, and a polypropylene porous membrane (Polypropylene Membrane Filter, manufactured by Millipore Inc.) with an average pore size of 20 μm was used as the porous membrane in laminate 1. The nonwoven fabric and porous membrane were cut to the size of a Cell Crown 24 NX insert (manufactured by Scaffdex Inc.), and the nonwoven fabric and porous membrane were assembled into an ethylene oxide gas (EOG) sterilized Cell Crown 24 NX insert with the nonwoven fabric surface facing upwards and sandwiching both materials, thereby creating a laminate of nonwoven fabric and porous membrane (hereinafter referred to as the "nonwoven fabric / porous membrane laminate"). The average pore size of the pores in the polyester nonwoven fabric and polyester porous membrane was measured using an optical microscope.

[0060] 1-5 Manufacturing and Observation of BBB Model 1 The manufacturing and observation of BBB Model 1 were carried out according to the following procedures [1] to [5]. [1] 4 × 10 ６ 50 μL of perisite-containing culture medium at a concentration of cells / mL was seeded using a piston pipette from the surface side of the nonwoven fabric in a laminate of nonwoven fabric and porous membrane, and the mixture was left to stand at room temperature for 1 hour until the perisites migrated into the nonwoven fabric through the pores on the surface. [2] 4 × 10 ６ 50 μL of astrocyte-containing culture medium with a concentration of cells / mL was seeded onto the surface of the nonwoven fabric using a piston pipette. [3] The laminate of nonwoven fabric and porous membrane was rotated so that the surface of the nonwoven fabric was facing downwards and placed in a multi-well plate for cell culture, then 2 × 10 ７ 50 μL of culture medium containing vascular endothelial cells at a concentration of 1 / mL was seeded onto the surface of a porous membrane using a piston pipette. [4] Laminates of nonwoven fabric porous membrane seeded with three types of cells (pericytes, astrocytes, and vascular endothelial cells) were heated at 33°C and 5% CO2. ２ The cells were cultured for 5 days under the following conditions: [5] 37°C, 5% CO2 ２After culturing the cells under the specified conditions for five days to induce differentiation into mature cells, the BBB Model 1 was observed using a confocal microscope (Leica SP5 laser scanning confocal microscope) (Leica Wetzlar).

[0061] 1-6 Analysis of Cell Distribution The distribution of three types of cells (pericytes, astrocytes, and vascular endothelial cells) on the surface of the nonwoven fabric, inside the nonwoven fabric, and on the surface of the porous membrane in a laminate of nonwoven fabric and porous membrane was analyzed according to the following procedures [1] and [2]. [1] 1) 1 × 10 ５ 200 μL of perisite-containing culture medium at a concentration of cells / mL is seeded using a piston pipette from the surface side of the nonwoven fabric in a laminate of nonwoven fabric and porous membrane, and left to stand at room temperature for 1 hour until the perisite moves into the interior of the nonwoven fabric through the pores on the surface of the nonwoven fabric, or 2) 4 × 10 ６ 50 μL of astrocyte-containing culture medium containing cells / mL is seeded onto the surface of the nonwoven fabric in a laminate of nonwoven fabric and porous membrane using a piston pipette, or 3) the laminate of nonwoven fabric and porous membrane is rotated so that the surface of the nonwoven fabric is facing downwards, placed in a multi-well plate for cell culture, and then 2 × 10 ７ [2] 50 μL of culture medium containing vascular endothelial cells at a concentration of 1 / mL was seeded onto the surface of a porous membrane using a piston pipette. [2] Laminates of nonwoven fabric / porous membrane seeded with pericytes, astrocytes, or vascular endothelial cells were fixed with a 4% paraformaldehyde solution, and then the cytoskeleton was stained by immunocytochemistry using an anti-phalloidin antibody conjugated with a fluorescent dye (Acti-stain 488 phalloidin, Cytoskeleton) according to standard procedures.

[0062] 1-7 Optimization of the porous membrane surface in laminates of nonwoven fabrics and porous membranes To optimize the porous membrane surface of laminates of nonwoven fabrics and porous membranes, the following analysis was performed according to the following procedures [1] to [3]. [1] 2 × 10 ７ 50 μL of culture medium containing vascular endothelial cells / mL is seeded onto the surface of a porous membrane having an average pore size of 20 μm, 25 μm, or 30 μm using a piston pipette, and cultured at 33°C and 5% CO2. ２[2] The cells were cultured for 48 hours under the specified conditions. The surface of the porous membrane containing vascular endothelial cells was fixed with a 4% paraformaldehyde solution, and then the cytoskeleton was stained using immunocytochemistry with an anti-phalloidin antibody conjugated with a fluorescent dye (Acti-stain 488 phalloidin, Cytoskeleton) according to standard procedures. [3] The cell nuclei were stained with DAPI (4',6-diamidino-2-phenylindole) and then observed using a confocal microscope (Leica SP5 laser scanning confocal microscope) (Leica Wetzlar).

[0063] 1-8 Analysis of the Barrier Function of BBB Model 1 The barrier function of BBB Model 1 was analyzed according to the following procedure [1] to [5]. [1] 4 × 10 ６ 50 μL of perisite-containing culture medium at a concentration of cells / mL was seeded using a piston pipette from the surface side of the nonwoven fabric in a laminate of nonwoven fabric and porous membrane, and the mixture was left to stand at room temperature for 1 hour until the perisites migrated into the nonwoven fabric through the pores on the surface. [2] 4 × 10 ６ 50 μL of astrocyte-containing culture medium with a concentration of cells / mL was seeded onto the surface of the nonwoven fabric using a piston pipette. [3] The laminate of nonwoven fabric and porous membrane was rotated so that the surface of the nonwoven fabric was facing downwards and placed in a multi-well plate for cell culture, then 2 × 10 ７ 50 μL of culture medium containing vascular endothelial cells at a concentration of 1 / mL was seeded onto the surface of a porous membrane using a piston pipette. [4] Laminates of nonwoven fabric porous membrane seeded with three types of cells (pericytes, astrocytes, and vascular endothelial cells) were heated at 33°C and 5% CO2. ２ The cells were cultured for 5 days under the following conditions: [5] 37°C, 5% CO2 ２The cells were cultured under the specified conditions for one day (six days including the culture period at 33°C) to induce differentiation into mature cells. After this, trans-epithelial electrical resistance (TEER) was measured using an EVOM resistance meter (WPI) according to the protocol provided with the product. For comparison, TEER measurements were also performed on a BBB model (sometimes referred to as the "comparative BBB model" in this specification) prepared using the Transwell cell culture insert and three types of cells (pericytes, astrocytes, and vascular endothelial cells) according to the method described in Patent Document 3. The relative value (see vertical axis in Figure 5) was calculated with the TEER value of the comparative BBB model set to 1.

[0064] 2. Results 2-1 Manufacturing and Observation of BBB Model 1 The BBB Model 1 manufactured using a nonwoven fabric / porous membrane laminate and three types of cells (pericytes, astrocytes, and vascular endothelial cells) was observed using a confocal microscope. The results showed that three types of cell layers—astrocytes, pericytes, and vascular endothelial cells—were formed on the surface of the nonwoven fabric, inside the nonwoven fabric, and on the surface of the porous membrane, respectively, in the nonwoven fabric / porous membrane laminate (see Figure 1). Furthermore, when steps [1] and [2] of item "1-5" above were reversed (i.e., astrocytes were seeded on the surface of the nonwoven fabric, and then pericytes were seeded from the surface side of the nonwoven fabric), it was similarly confirmed that three types of cell layers—astrocytes, pericytes, and vascular endothelial cells—were formed on the surface of the nonwoven fabric, inside the nonwoven fabric, and on the surface of the porous membrane, respectively, in the nonwoven fabric / porous membrane laminate.

[0065] Furthermore, approximately 95% of the pericytes, representing the majority of the total pericyte population, were located inside the nonwoven fabric, while approximately 5% were located on the surface of the nonwoven fabric (see Figures 2B, 2A, and 3B). Similarly, approximately 90% of the astrocytes, representing the majority of the total astrocyte population, were located on the surface of the nonwoven fabric, while approximately 10% were located inside the nonwoven fabric (see Figures 2C, 2D, and 3C). Additionally, a sheet structure of vascular endothelial cells was observed on the surface of the porous membrane in the laminate of nonwoven fabric and porous membrane (see Figure 3A).

[0066] 2-2 Optimization of the surface of the porous membrane in laminates of nonwoven fabrics and porous membranes When vascular endothelial cells were seeded on the surface of a porous membrane having pores with an average opening diameter of 20 μm, 25 μm, or 30 μm, and the sheet structure formed by the vascular endothelial cells was analyzed, it was found that when seeded on the surface of a porous membrane with an average opening diameter of 20 μm, a sheet structure of vascular endothelial cells with almost no or no voids was formed, whereas when seeded on the surface of a porous membrane with an average opening diameter of 25 μm or 30 μm, a sheet structure of vascular endothelial cells with multiple voids was formed (see Figure 4). This result indicates that in order to form a sheet structure of vascular endothelial cells with almost no or no voids on the surface of the porous membrane in a laminate of nonwoven fabrics and porous membranes, it is necessary to make the average opening diameter of the pores on the surface 20 μm or less.

[0067] 2-3 Analysis of the Barrier Function of BBB Model 1 The TERE level of BBB Model 1 was significantly higher than that of the control BBB model (see Figure 5). This result indicates that BBB Model 1 has a higher barrier function than conventional BBB models.

[0068] This invention contributes to long-term analyses using blood-brain barrier models, such as the transport of blood-brain barrier models to remote locations and the selection of candidate substances with good brain permeability.

Claims

1. A blood-brain barrier model comprising a laminate of three cell layers: pericytes, astrocytes, and vascular endothelial cells, and a fibrous structure and a porous membrane, wherein the astrocytes are located on the surface of the fibrous structure in the laminate, the pericytes are located inside the fibrous structure in the laminate, and the vascular endothelial cells form a sheet structure on the surface of the porous membrane in the laminate.

2. The blood-brain barrier model according to claim 1, wherein the average pore diameter of the pores on the surface of the porous membrane is 20 μm or less.

3. The blood-brain barrier model according to claim 1, wherein the pericytes, astrocytes, and vascular endothelial cells are human-derived cells.

4. The blood-brain barrier model according to any one of claims 1 to 3, wherein the fibrous structure is a nonwoven fabric.

5. A method for producing a blood-brain barrier model, comprising the following steps (a) to (e), wherein the blood-brain barrier model is composed of three cell layers: pericytes, astrocytes, and vascular endothelial cells, and a laminate of fibrous structures and a porous membrane, wherein the astrocytes are located on the surface of the fibrous structures in the laminate, the pericytes are located inside the fibrous structures in the laminate, and the vascular endothelial cells form a sheet structure on the surface of the porous membrane in the laminate. (a) A step of preparing a laminate of a fibrous structure and a porous membrane; (b) A step of seeding pericytes into the interior of the fibrous structure from the surface side of the laminate; (c) A step of seeding astrocytes on the surface of the fibrous structure in the laminate; (d) A step of seeding vascular endothelial cells on the surface of the porous membrane in the laminate; (e) A step of culturing the laminate seeded with pericytes, astrocytes, and vascular endothelial cells until three cell layers of pericytes, astrocytes, and vascular endothelial cells are formed, and the vascular endothelial cells form a sheet structure on the surface of the porous membrane; 6. The manufacturing method according to claim 5, wherein steps (b), (c), and (d) are carried out in that order.

7. The manufacturing method according to claim 6, wherein the laminate after step (c) is inverted 180 degrees and step (d) is carried out.

8. The manufacturing method according to claim 5, wherein the average pore diameter of the pores on the surface of the porous membrane is 20 μm or less.

9. The manufacturing method according to claim 5, wherein the pericytes, astrocytes, and vascular endothelial cells are human-derived cells.

10. The manufacturing method according to any one of claims 5 to 9, wherein the fiber structure is a nonwoven fabric.

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