Blood-brain barrier model

Abstract

The present invention addresses the problem of providing: a blood-brain barrier model that can be produced in a relatively simple manner and in which a physiological barrier function and selective permeability are reproduced; and a method for producing the blood-brain barrier model.　A blood-brain barrier model in which a physiological barrier function and selective permeability are reproduced can be produced in a relatively simple manner in a blood-brain barrier model having three cell layers of perisites, astrocytes, and vascular endothelial cells by culturing vascular endothelial cells so as to form a sheet structure on the surface of a porous membrane and so that perisites are present in an internal structure layer of the porous membrane.

Description

Blood-brain barrier model

[0001] This invention relates to a blood-brain barrier (BBB) ​​model and a method for manufacturing 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] On the other hand, polyimide porous membranes (sometimes referred to as "PPI membranes" in this specification) are porous membranes based on polyimide, a polymer containing imide bonds in its repeating units, and are used in applications mainly related to batteries, such as filters, low dielectric constant films, and electrolyte membranes for fuel cells. Recently, a cell culture method has been reported that includes culturing cells in a polyimide porous membrane (Patent Document 4).

[0005] Japanese Patent Publication No. 2007-166915, International Publication No. 2016 / 202343, Brochure International Publication No. 2017 / 179375, Brochure International Publication No. 2015 / 012415

[0006] The object of the present invention is to provide a blood-brain barrier model that can be manufactured relatively easily and reproduces physiological barrier function and selective permeability, as well as a method for manufacturing the same.

[0007] In order to solve the above problems, the inventors of the present invention have diligently conducted research and have discovered that in a blood-brain barrier model having three types of cell layers—pericytes, astrocytes, and vascular endothelial cells—by culturing vascular endothelial cells to form a sheet structure on the surface of a porous membrane, and culturing the membrane so that pericytes are present in the internal structural layer, it is possible to produce a blood-brain barrier model that reproduces physiological barrier function and selective permeability, thus completing the present invention.

[0008] In other words, the present invention is as follows: [1] A blood-brain barrier model comprising a porous membrane having three types of cell layers: pericytes, astrocytes, and vascular endothelial cells, and a surface layer A having a plurality of pores and a surface layer B having a plurality of pores, and an internal structural layer sandwiched between the surface layer A and the surface layer B, wherein the vascular endothelial cells form a sheet structure on the surface layer B, and the pericytes are present in the internal structural layer. [2] The blood-brain barrier model according to [1], wherein the astrocytes are present on the surface layer A. [3] The blood-brain barrier model according to [1] or [2], wherein the maximum opening diameter of the pores in the surface layer B is 43 μm or less. [4] The blood-brain barrier model according to any one of [1] to [3], wherein the pericytes, astrocytes, and vascular endothelial cells are human-derived cells. [5] The blood-brain barrier model according to any one of [1] to [4], wherein the porous membrane is a polyimide porous membrane. [6] A method for manufacturing a blood-brain barrier model comprising the following steps (a) to (d), wherein the blood-brain barrier model is composed of a porous membrane having three types of cell layers: pericytes, astrocytes, and vascular endothelial cells, and a surface layer A having a plurality of pores and a surface layer B having a plurality of pores, and an internal structural layer sandwiched between the surface layer A and the surface layer B, wherein the vascular endothelial cells form a sheet structure on the surface layer B, the pericytes are present in the internal structural layer, and the astrocytes are present on the surface layer A. (a) A step of seeding pericytes into the internal structural layer of the porous membrane; (b) A step of seeding astrocytes on the surface layer A of the porous membrane; (c) A step of seeding vascular endothelial cells on the surface layer B of the porous membrane; (d) A step of culturing the porous membrane 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 layer B; [7] The manufacturing method according to [6] above, wherein in step (a), pericytes are seeded into the internal structural layer from the surface layer A side of the porous membrane. [8] The manufacturing method according to [7] above, wherein steps (a), (b), and (c) are carried out in that order.[9] The manufacturing method according to [8] above, wherein the porous membrane after step (b) is inverted 180 degrees and step (c) is carried out.

[10] The manufacturing method according to any one of [6] to [9] above, wherein the maximum opening diameter of the pores in the surface layer B is 43 μm or less.

[11] The manufacturing method according to any one of [6] to

[10] above, wherein the pericytes, astrocytes, and vascular endothelial cells are human-derived cells.

[12] The manufacturing method according to any one of [6] to

[11] above, wherein the porous membrane is a polyimide porous membrane.

[0009] According to the present invention, a blood-brain barrier model that reproduces physiological barrier function and selective permeability can be manufactured relatively easily, thereby providing a high-quality blood-brain barrier model that contributes to analysis using the blood-brain barrier model (for example, selection of candidate substances with good brain permeability). Furthermore, this BBB model can maintain physiological barrier function and selective permeability even after long-term culture and storage, providing a blood-brain barrier model with a long shelf life, which is useful for transporting blood-brain barrier models to remote locations and for long-term analysis using the blood-brain barrier model.

[0010] These are microscopic images of the BBB model manufactured using polyimide porous membrane No. 4 and three types of cells (pericytes, astrocytes, and vascular endothelial cells). This figure shows the results of analyzing the presence of pericytes and astrocytes in the three layers of the polyimide porous membrane (surface layer A, internal structural layer, and surface layer B). These are microscopic images of the formation of a sheet structure of vascular endothelial cells on surface layer B of polyimide porous membrane No. 1 (Figure 3A), on surface layer B of polyimide porous membrane No. 2 (Figure 3B), on surface layer B of polyimide porous membrane No. 3 (Figure 3C), and on surface layer B of polyimide porous membrane No. 4 (Figure 3D). The areas circled in white in Figure 3A indicate areas where voids were observed overall, and the areas circled in white in Figure 3B indicate areas where voids were observed. Figure 4A shows No. Figure 4B shows the results of analyzing the cell proliferation rate when pericytes seeded on polyimide porous membrane No. 4 ("Reference Example Sample 1" in the figure) or pericytes seeded on Transwell® cell culture inserts ("Comparative Example Sample 1" in the figure) were cultured for 1 day or 3 days, respectively. Figure 4B shows the results of analyzing the cell proliferation rate when astrocytes seeded on polyimide porous membrane No. 4 ("Reference Example Sample 2" in the figure) or astrocytes seeded on Transwell® cell culture inserts ("Comparative Example Sample 2" in the figure) were cultured for 1 to 6 days, respectively. Figure 4C shows the results of analyzing the cell proliferation rate when vascular endothelial cells seeded on polyimide porous membrane No. 4 ("Reference Example Sample 3" in the figure) or vascular endothelial cells seeded on Transwell® cell culture inserts ("Comparative Example Sample 3" in the figure) were cultured for 1 day or 3 days, respectively. No. This figure shows the results of analyzing the barrier function of vascular endothelial cells (Reference Example Sample 4) that formed a sheet structure on surface layer B of polyimide porous membrane 4, and the barrier function of vascular endothelial cells (Comparative Example Sample 4) that formed a sheet structure on ThinCert® 24-well cell culture insert, using transepithelial electrical resistance (TEER) measurement.This figure shows the results of analyzing the barrier function of five types of samples (vascular endothelial cells [HUVEC] [Comparative Example Sample 5] formed in a sheet structure on ThinCert® 24-well cell culture insert, Comparative Example Sample 4, Reference Example Sample 4, BBB model manufactured based on ThinCert® 24-well cell culture insert [Comparative Example BBB Model], and the present BBB model manufactured based on No. 5 polyimide porous membrane [Example BBB Model]) by TEER measurement. This figure shows the results of analyzing the barrier function of the above five types of samples by permeability test.

[0011] <The BBB Model of the Present Invention> The blood-brain barrier model of the present invention is a blood-brain barrier model composed of 1) and 2) a porous membrane having a surface layer A having multiple pores and a surface layer B having multiple pores and an internal structural layer sandwiched between the surface layer A and the surface layer B, wherein the vascular endothelial cells form a sheet structure on the surface layer B and the pericytes are present in the internal structural layer, and the BBB model of the present invention is a blood-brain barrier model (hereinafter referred to as the "BBB model of the present invention") that comprises 1) and 2), wherein the vascular endothelial cells form a sheet structure on the surface layer B and the pericytes are present in the internal structural layer, and the BBB model of the present invention is a blood-brain barrier model (hereinafter referred to as the "BBB model of the present invention") that comprises blood-brain barrier function (for example, barrier function [more specifically, physiological barrier function], selective permeability [more specifically, receptor-mediated transcytosis [Receptor-Mediated This is an in vitro BBB model with Transcytosis (RMT) function, transporter function, etc. Note that for convenience, the "surface layer" also includes the "surface."

[0012] 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).

[0013] In the BBB model, the BBB constituent cell layers can be stacked (laid up) due to the structure of the porous membrane, 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 may be stacked in the order of pericyte cell layer, astrocyte cell layer, and vascular endothelial cell layer from surface layer A to surface layer B, or in the order of astrocyte cell layer, pericyte cell layer, and vascular endothelial cell layer, but it is preferable that they be stacked in the order of astrocyte cell layer, pericyte cell layer, and vascular endothelial cell layer.

[0014] In the BBB model, the astrocyte cell population may be located anywhere in the BBB model, as long as it is distinguishable from the pericyte cell population and the vascular endothelial cell population. Possible locations for astrocytes include, for example, the surface layer A or the internal structural layer of the BBB model, with the surface layer A being preferred. In the BBB model, if astrocytes are present on surface layer A, astrocytes may also be present on the internal structural layer and / or surface layer B. However, it is preferable that the majority of the total astrocyte 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.) are present on surface layer A, and a portion of them (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.) are present on the internal structural layer and / or surface layer B.

[0015] In the BBB model, the pericyte cell population may be located on surface layer A and / or surface layer B, as long as it is present in at least the internal structural layer. 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.) is located in the internal structural layer, 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 surface layer A and / or surface layer B.

[0016] In the BBB model, the vascular endothelial cell population may be present on the surface layer A and / or the internal structural layer, as long as it forms a sheet structure on the surface layer B. 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 present on the surface layer B, 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 present on the surface layer A and / or the internal structural layer.

[0017] 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).

[0018] 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.

[0019] The pericytes that constitute the BBB cell layer in this case refer to cells that surround the walls of microvessels in tissues or organs such as the brain and retina, and are enclosed in the basement membrane; they are also called pericytes. As the effect of pericytes has been demonstrated in the embodiment described later, brain pericytes (i.e., cerebral pericytes) can be suitably exemplified.

[0020] The astrocytes that make up the BBB cell layer in this case are a type of glial cell found in the central nervous system and refer to cells that express GFAP (Glial fibrillary acid protein).

[0021] The vascular endothelial cells that constitute the BBB constituent cell layer in this case can be any cells that make up the inner surface of blood vessels, for example, microvascular endothelial cells in tissues or organs such as the brain, lungs, skin, heart, and uterus; umbilical vein endothelial cells; aortic endothelial cells; coronary artery endothelial cells; pulmonary artery endothelial cells; and, as the effect has been demonstrated in the embodiment described later, microvascular endothelial cells in the brain (i.e., cerebral microvascular endothelial cells) can be preferably given as an example.

[0022] 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 treatment 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 the literature "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 those prepared in-house by differentiating stem cells such as embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells), or commercially available products may be used.

[0023] <Manufacturing Method> The manufacturing method of the blood-brain barrier model of the present invention includes the steps (a) to (d) of: (a) seeding pericytes into the internal structural layer of a porous membrane (i.e., the porous membrane) having a surface layer A having a plurality of pores and a surface layer B having a plurality of pores, and an internal structural layer sandwiched between the surface layers A and B; (b) seeding astrocytes on surface layer A of the porous membrane; (c) seeding vascular endothelial cells on surface layer B of the porous membrane; and (d) culturing the porous membrane 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 surface layer B; and thus producing three types of cell layers of pericytes, astrocytes, and vascular endothelial cells (i.e., the BBB constituent cell layers of the present invention), The method for manufacturing a blood-brain barrier model (i.e., the BBB model in which astrocytes are present on surface layer A) is not particularly limited, as it is composed of a porous membrane (i.e., the porous membrane) having a surface layer A having a plurality of pores and a surface layer B having a plurality of pores, and an internal structural layer sandwiched between surface layers A and B, wherein the vascular endothelial cells form a sheet structure on surface layer B, the pericytes are present in the internal structural layer, and the astrocytes are present on surface layer A. In step (a), the pericytes may be seeded into the internal structural layer from either the surface layer A side or the surface layer B side of the porous membrane, but it is preferable to seed them into the internal structural layer from the surface layer A side.

[0024] Specifically, possible orders for carrying out the above steps (a) to (c) include: step (a), step (b), and step (c); step (a), step (c), and step (b); step (b), step (a), and step (c); step (b), step (c), and step (a); step (c), step (a), and step (b); and step (c), step (b), and step (a). In step (a), when perisite is seeded from the surface layer A side into the internal structural layer of the porous membrane, the order of step (a), step (b), and step (c) can be preferably exemplified. Because pericytes have the property of burrowing into the internal structural layer, even if pericytes are seeded into the internal structural layer from the surface layer A side where astrocytes are seeded, or from the surface layer B side where vascular endothelial cells are seeded, or if pericytes are seeded into the internal structural layer from the surface layer A side and then astrocytes are seeded on surface layer A, or if pericytes are seeded into the internal structural layer from the surface layer B side and then vascular endothelial cells are seeded on surface layer B, the pericyte cell population and the astrocyte cell population will not become a single, integrated state, and a cell layer containing the pericyte cell population and a cell layer containing the astrocyte cell population can be formed. Similarly, the pericyte cell population and the vascular endothelial cell population will not become a single, integrated state, and a cell layer containing the pericyte cell population and a cell layer containing the vascular endothelial cell population can be formed.

[0025] When the present manufacturing method is carried out in the order of process (a), process (b), and process (c), from the viewpoint of automation, it is preferable to rotate the porous membrane after process (b) by 180 degrees and then carry out process (c).

[0026] In this manufacturing method, the method for seeding pericytes, astrocytes, and vascular endothelial cells is not particularly limited. For example, a cell suspension containing a 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, pipetter, or pipetman, and then dropped onto the target for seeding (e.g., surface layer A, surface layer B). The temperature at which seeding takes place is not particularly limited and may be room temperature (e.g., within the range of 10°C to 30°C) or a temperature suitable for culturing pericytes, astrocytes, and vascular endothelial cells (e.g., within the range of 33°C to 38°C). The cell suspension at which seeding takes place may be any liquid containing pericytes, astrocytes, or vascular endothelial cells, but considering subsequent cell culture, a culture medium is preferred.

[0027] In (d) above, as a method of culturing the porous membrane 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 layer B, any culturing method may 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 layer B. The culturing period is not particularly limited and is, for example, within the range of 3 to 30 days, preferably 4 to 20 days, more preferably 4 to 10 days. The culture medium used for culturing is not particularly limited. For example, animal cell culture media (DMEM, EMEM, IMDM, RPMI1640, αMEM, F-12, F-10, M-199, AIM-V, Astrocyte Medium [manufactured by ScienCell Research Laboratories], etc.) containing 0.1 to 30 (v / v)% serum (fetal bovine serum [FBS], calf bovine serum [CS], etc.), EGM-2 Endothelial Cell Growth Medium-2 BulletKit (manufactured by LONZA), etc. can be mentioned. The culturing temperature is usually within the range of 30 to 40°C. When using immortalized cells under the above-described temperature conditions, 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. The CO ２ concentration is usually within the range of about 1 to 10%, preferably about 5%. Also, the humidity during culturing is usually within the range of about 70 to 100%, preferably within the range of about 95 to 100%.

[0028] <The present porous membrane> As the form of the internal structure layer in the present porous membrane, any form capable of culturing pericyte may be used. For example, in addition to a uniform structure in which polymer particles are densely packed and slightly voids remain, a sponge-like form having one or more (two or more) macrovoids inside the membrane can be mentioned. Further, as the internal structure layer in the present porous membrane, those having a plurality of macrovoids communicating with the pores in the surface layer A and the pores in the surface layer B are preferable.

[0029] In the present specification, "macrovoid" means a pore having a spherical approximate average diameter (average opening diameter) of 8 μm or more. The average opening diameter of the above macrovoid in the membrane plane direction is not particularly limited. For example, it is 10 to 500 μm, preferably 10 to 100 μm, and more preferably 10 to 80 μm. Further, the thickness of the partition wall in the internal structure layer in the present porous membrane is not particularly limited. For example, it is 0.01 to 50 μm, preferably 0.01 to 20 μm. In one embodiment, at least one partition wall in the internal structure layer has one or more pores with an average opening diameter of 0.01 to 100 μm, preferably 0.01 to 50 μm, that communicate adjacent macrovoids. In another embodiment, the partition wall in the internal structure layer has no pores.

[0030] The plurality of pores in the surface layer A may communicate with each other in the internal structure layer, the plurality of pores in the surface layer B may communicate with each other in the internal structure layer, and the plurality of pores in the surface layer A and the plurality of pores in the surface layer B may communicate with each other in the internal structure layer.

[0031] The maximum opening diameter of the pores in the surface layer A is not particularly limited. Examples of its lower limit include 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, 3.3 μm or more, 3.6 μm or more, 4.0 μm or more, etc. Examples of its upper limit include 200 μm or less, 160 μm or less, 130 μm or less, 100 μm or less, 60 μm or less, 40 μm or less, 36 μm or less, 33 μm or less, 30 μm or less, 26 μm or less, etc. These lower and upper limits can be arbitrarily combined with each other.

[0032] The maximum opening diameter of the pores in the surface layer B is not particularly limited. Examples of its lower limit include 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, 3.3 μm or more, 3.6 μm or more, 4.0 μm or more, etc. Examples of its upper limit include 100 μm or less, 50 μm or less, 47 μm or less, 43 μm or less, 42 μm or less, 41 μm or less, 40 μm or less, 36 μm or less, 33 μm or less, 30 μm or less, 26 μm or less, 23 μm or less, 20 μm or less, 19 μm or less, etc. These lower and upper limits can be arbitrarily combined with each other. The maximum opening diameter of the pores in the surface layer B is preferably 43 μm or less, more preferably 40 μm or less.

[0033] The number average opening diameter of the pores in the surface layer A is not particularly limited. Examples of its lower limit include 0.3 μm or more, 0.4 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 its upper limit include 100 μm or less, 80 μm or less, 60 μm or less, 40 μm or less, 20 μm or less, 16 μm or less, 13 μm or less, 10 μm or less, 6.0 μm or less, 4.3 μm or less, 4.0 μm or less, etc. These lower and upper limits can be arbitrarily combined with each other.

[0034] The average aperture diameter of the pores in surface layer B is not particularly limited. Examples of lower limits include 0.3 μm or more, 0.4 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, 40 μm or less, 20 μm or less, 16 μm or less, 13 μm or less, 11 μm or less, 5.0 μm or less, 4.3 μm or less, 4.0 μm or less, 3.6 μm or less, 3.3 μm or less, etc. These lower and upper limits can be combined arbitrarily.

[0035] The area-average aperture diameter of the pores in surface layer A is not particularly limited. Examples of lower limits include 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, 3.3 μm or more, 3.6 μm or more, 4.0 μm or more, etc. Examples of upper limits include 300 μm or less, 250 μm or less, 200 μm or less, 150 μm or less, 100 μm or less, 80 μm or less, 60 μm or less, 40 μm or less, 20 μm or less, 14 μm or less, 12 μm or less, 10 μm or less, 8 μm or less, etc. These lower and upper limits can be combined arbitrarily.

[0036] The area-average aperture diameter of the pores in surface layer B is not particularly limited. Examples of lower limits include 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, 3.3 μm or more, 3.6 μm or more, 4.0 μm or more, etc. Examples of upper limits include 400 μm or less, 350 μm or less, 300 μm or less, 250 μm or less, 200 μm or less, 150 μm or less, 100 μm or less, 80 μm or less, 60 μm or less, 40 μm or less, 30 μm or less, 16 μm or less, 13 μm or less, 10 μm or less, etc. These lower and upper limits can be combined arbitrarily.

[0037] The average surface opening ratio of pores in surface layer A is not particularly limited. Examples of its lower limit include 1.0% or more, 1.3% or more, 1.6% or more, 2.0% or more, 2.3% or more, 2.6% or more, 3.0% or more, 3.3% or more, 3.6% or more, 4.0% or more, etc. Examples of its upper limit include 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 38% or less, 33% or less, 30% or less, etc. These lower and upper limits can be combined arbitrarily.

[0038] The average surface opening ratio of pores in surface layer B is not particularly limited. Examples of lower limits include 0.1% or more, 0.3% or more, 0.6% or more, 1.0% or more, 2.3% or more, 2.6% or more, 2.0% or more, etc. Examples of upper limits include 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 17% or less, 13% or less, 10% or less, 6% or less, 3% or less, etc. These lower and upper limits can be combined arbitrarily.

[0039] In this specification, the number-average pore diameter and area-average pore diameter in the porous membrane can be determined according to (1) and (2) below. The average pore diameter (number-average pore diameter or area-average pore diameter) in parts of the porous membrane other than surface layers A and B can also be determined in the same manner.

[0040] (1) From scanning electron microscope images of surface layer A and surface layer B of the porous membrane in question, 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.

[0041]

[0042] (2) All hole diameters obtained by formula I above are applied to formulas II and III below to determine the number-average aperture diameter Sn and the area-average aperture diameter Sa (where n represents the total number of holes).

[0043]

[0044]

[0045] The average surface aperture ratio can be calculated as the ratio of the total area of ​​holes within an observation field of view (e.g., 1.28 mm × 0.96 mm) to the area of ​​that observation field of view. The maximum aperture diameter can be calculated as the maximum value of the hole diameter d within an observation field of view (e.g., 1.28 mm × 0.96 mm).

[0046] The substrate for the porous membrane in this case is not particularly limited, and examples include polycarbonate (PC), polyester (PET), polystyrene (PS), TAC (triacetylcellulose), polyimide (PI), polyketone (PK), nylon (Ny), low-density polyethylene (LDPE), medium-density polyethylene (MDPE), vinyl chloride, vinylidene chloride, polyphenylene sulfide, polyethersulfone (PES), polyethylene naphthalate, polypropylene, acrylic, etc. Preferably, it is a porous membrane with polyimide as the substrate (i.e., a polyimide porous membrane).

[0047] The porous membrane in question is composed of a single porous membrane (a single unit) and is different from a single fibrous structure or a composite structure composed of multiple porous membranes and / or fibrous structures stacked together.

[0048] If the substrate of the porous membrane is unsuitable for culturing adherent cells, it is preferable that the surfaces of surface layer A, the internal structural layer, and surface layer B are coated with cell adhesion components. If the substrate of the porous membrane is suitable for culturing adherent cells, it is preferable that the surfaces of surface layer A, the internal structural layer, and surface layer B are not coated with cell adhesion components. 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.

[0049] <Polyimide Porous Membrane> A polyimide porous membrane, which is a suitable material for the porous membrane in this invention, will be described in detail. In this specification, "polyimide" means a general term for polymers that contain imide bonds in their repeating units. The polyimide porous membrane that can be used in the present invention is preferably a polyimide porous membrane that contains (as the main component) a polyimide obtained from tetracarboxylic dianhydride and diamine, and more preferably a polyimide porous membrane consisting of a polyimide obtained from tetracarboxylic dianhydride and diamine. "Contained as the main component" means that, as a constituent component of the polyimide porous membrane, other components other than the polyimide obtained from tetracarboxylic dianhydride and diamine are not essentially contained, or may be contained, but are additional components that do not affect the properties of the polyimide obtained from tetracarboxylic dianhydride and diamine.

[0050] A porous polyimide membrane can be produced, for example, by a method comprising the steps of: casting a polyamic acid solution composition containing a polyamic acid solution (A) consisting of 0.3 to 60% by mass of polyamic acid composed of tetracarboxylic acid units and diamine units and 40 to 99.7% by mass of an organic polar solvent, and 0.1 to 200 parts by mass of an organic compound having polar groups (B) or a polymer compound having polar groups in its side chains per 100 parts by mass of the polyamic acid in a film form, and immersing or contacting it with a solidification solvent in which water is an essential component to produce a porous polyamic acid membrane; and heat-treating the porous polyamic acid membrane obtained in the above step to imide it. Here, the organic compound (B) and the polymer compound (C) are organic compounds that promote the penetration of water into the film-like cast of the polyamic acid solution composition.

[0051] Polyamic acids can be obtained by polymerizing tetracarboxylic dianhydrides and diamines. Polyamic acids are polyimide precursors that can be cyclized to polyimides by thermal or chemical imidation.

[0052] The above-mentioned polyamic acid may be used even if a portion of the amic acid is imidized, as long as this does not affect the present invention. In other words, the polyamic acid may be partially thermally imidized or chemically imidized.

[0053] The above tetracarboxylic dianhydrides specifically include pyromellitic dianhydride, biphenyltetracarboxylic dianhydride such as 3,3',4,4'-biphenyltetracarboxylic dianhydride (s-BPDA), 2,3,3',4'-biphenyltetracarboxylic dianhydride (a-BPDA), oxydiphthalic dianhydride, diphenylsulfone-3,4,3',4'-tetracarboxylic dianhydride, bis(3,4-dicarboxyphenyl) sulfide dianhydride, 2,2-bis(3,4-dicarboxyphenyl)-1,1,1,3,3,3-hexafluoropropane dianhydride, 2,3,3',4'-benzophenonetetracarboxylic dianhydride, 3,3',4,4'-benzophenonetetracarboxylic dianhydride, bis(3,4-dicarboxyphenyl)methane dianhydride, and 2,2-bis(3,4-dicarboxyphenyl)propane dianhydride. Examples include pan dianhydride, p-phenylenebis(trimellitic acid monoester acid anhydride), p-biphenylenebis(trimellitic acid monoester acid anhydride), m-terphenyl-3,4,3',4'-tetracarboxylic acid dianhydride, p-terphenyl-3,4,3',4'-tetracarboxylic acid dianhydride, 1,3-bis(3,4-dicarboxyphenoxy)benzene dianhydride, 1,4-bis(3,4-dicarboxyphenoxy)benzene dianhydride, 1,4-bis(3,4-dicarboxyphenoxy)biphenyl dianhydride, 2,2-bis[(3,4-dicarboxyphenoxy)phenyl]propane dianhydride, 2,3,6,7-naphthalenetetracarboxylic acid dianhydride, 1,4,5,8-naphthalenetetracarboxylic acid dianhydride, and 4,4'-(2,2-hexafluoroisopropylidene)diphthalic acid dianhydride. Furthermore, it is also preferable to use aromatic tetracarboxylic acids such as 2,3,3',4'-diphenylsulfonetetracarboxylic acid. These can be used individually or in combination of two or more.

[0054] Among these, at least one aromatic tetracarboxylic dianhydride selected from the group consisting of biphenyltetracarboxylic dianhydrides and pyromellitic dianhydrides is particularly preferred. As the biphenyltetracarboxylic dianhydride, 3,3',4,4'-biphenyltetracarboxylic dianhydride can be suitably used.

[0055] Specifically, the following can be listed as examples of the above diamines: 1) Benzene diamines with one benzene ring, such as 1,4-diaminobenzene (paraphenylenediamine), 1,3-diaminobenzene, 2,4-diaminotoluene, and 2,6-diaminotoluene; 2) Diaminodiphenyl ethers such as 4,4'-diaminodiphenyl ether and 3,4'-diaminodiphenyl ether, 4,4'-diaminodiphenylmethane, 3,3'-dimethyl-4,4'-diaminobiphenyl, 2,2'-dimethyl-4,4'-diaminobiphenyl, 2,2'-bis(trifluoromethyl)-4,4'-diaminobiphenyl, 3,3'-dimethyl-4,4'-diaminodiphenylmethane, 3,3'-dicarboxy-4,4'-diaminodiphenylmethane, 3 ,3',5,5'-tetramethyl-4,4'-diaminodiphenylmethane, bis(4-aminophenyl) sulfide, 4,4'-diaminobenzanilide, 3,3'-dichlorobenzidine, 3,3'-dimethylbenzidine, 2,2'-dimethylbenzidine, 3,3'-dimethoxybenzidine, 2,2'-dimethoxybenzidine, 3,3'-diaminodiphenyl ether, 3,4'-diaminodiphenyl ether, 4,4'-diaminodiphenyl ether, 3,3'-diaminodiphenyl Sulfide, 3,4'-diaminodiphenyl sulfide, 4,4'-diaminodiphenyl sulfide, 3,3'-diaminodiphenyl sulfone, 3,4'-diaminodiphenyl sulfone, 4,4'-diaminodiphenyl sulfone, 3,3'-diaminobenzophenone, 3,3'-diamino-4,4'-dichlorobenzophenone, 3,3'-diamino-4,4'-dimethoxybenzophenone, 3,3'-diaminodiphenylmethane, 3,4'-diaminodiphenylmethane, 4,4'-di Diamines with two benzene rings, such as aminodiphenylmethane, 2,2-bis(3-aminophenyl)propane, 2,2-bis(4-aminophenyl)propane, 2,2-bis(3-aminophenyl)-1,1,1,3,3,3-hexafluoropropane, 2,2-bis(4-aminophenyl)-1,1,1,3,3,3-hexafluoropropane, 3,3'-diaminodiphenyl sulfoxide, 3,4'-diaminodiphenyl sulfoxide, and 4,4'-diaminodiphenyl sulfoxide;3) 1,3-bis(3-aminophenyl)benzene, 1,3-bis(4-aminophenyl)benzene, 1,4-bis(3-aminophenyl)benzene, 1,4-bis(4-aminophenyl)benzene, 1,3-bis(4-aminophenoxy)benzene, 1,4-bis(3-aminophenoxy)benzene, 1,4-bis(4-aminophenoxy)benzene, 1,3-bis(3-aminophenoxy)-4-trifluoromethylbenzene, 3,3'-diamino-4-(4-phenyl)phenoxybenzophenone, 3,3'-diamino-4,4'-di(4-phenylphenoxy)benzophenone, 1,3 Benzene-nuclear diamines such as bis(3-aminophenylsulfide)benzene, 1,3-bis(4-aminophenylsulfide)benzene, 1,4-bis(4-aminophenylsulfide)benzene, 1,3-bis(3-aminophenylsulfone)benzene, 1,3-bis(4-aminophenylsulfone)benzene, 1,4-bis(4-aminophenylsulfone)benzene, 1,3-bis[2-(4-aminophenyl)isopropyl]benzene, 1,4-bis[2-(3-aminophenyl)isopropyl]benzene, and 1,4-bis[2-(4-aminophenyl)isopropyl]benzene;4) 3,3'-bis(3-aminophenoxy)biphenyl, 3,3'-bis(4-aminophenoxy)biphenyl, 4,4'-bis(3-aminophenoxy)biphenyl, 4,4'-bis(4-aminophenoxy)biphenyl, bis[3-(3-aminophenoxy)phenyl] ether, bis[3-(4-aminophenoxy)phenyl] ether, bis[4-(3-aminophenoxy)phenyl] ether, bis[4-(4-aminophenoxy)phenyl] ether, bis[3-(3-aminophenoxy)phenyl] ketone, bis[3-(4-A Minophenoxy)phenyl]ketone, bis[4-(3-aminophenoxy)phenyl]ketone, bis[4-(4-aminophenoxy)phenyl]ketone, bis[3-(3-aminophenoxy)phenyl]sulfide, bis[3-(4-aminophenoxy)phenyl]sulfide, bis[4-(3-aminophenoxy)phenyl]sulfide, bis[4-(4-aminophenoxy)phenyl]sulfide, bis[3-(3-aminophenoxy)phenyl]sulfone, bis[4-(3-aminophenoxy)phenyl]sulfone, bis[4-(3-aminophenoxy)phenyl]ketone, bis[4-(3-aminophenoxy)phenyl]ketone, bis[4-(3-aminophenoxy)phenyl]ketone [nophenoxy)phenyl]sulfone, bis[4-(4-aminophenoxy)phenyl]sulfone, bis[3-(3-aminophenoxy)phenyl]methane, bis[3-(4-aminophenoxy)phenyl]methane, bis[4-(3-aminophenoxy)phenyl]methane, bis[4-(4-aminophenoxy)phenyl]methane, 2,2-bis[3-(3-aminophenoxy)phenyl]propane, 2,2-bis[3-(4-aminophenoxy)phenyl]propane, 2,2- Diamines with four benzene rings, such as bis[4-(4-aminophenoxy)phenyl]propane, 2,2-bis[3-(3-aminophenoxy)phenyl]-1,1,1,3,3,3-hexafluoropropane, 2,2-bis[3-(4-aminophenoxy)phenyl]-1,1,1,3,3,3-hexafluoropropane, 2,2-bis[4-(3-aminophenoxy)phenyl]-1,1,1,3,3,3-hexafluoropropane, and 2,2-bis[4-(4-aminophenoxy)phenyl]-1,1,1,3,3,3-hexafluoropropane;

[0056] These can be used individually or in mixtures of two or more. The diamines used can be appropriately selected according to the desired properties.

[0057] Among these, aromatic diamine compounds are preferred, and 3,3'-diaminodiphenyl ether, 3,4'-diaminodiphenyl ether, 4,4'-diaminodiphenyl ether and paraphenylenediamine, 1,3-bis(3-aminophenyl)benzene, 1,3-bis(4-aminophenyl)benzene, 1,4-bis(3-aminophenyl)benzene, 1,4-bis(4-aminophenyl)benzene, 1,3-bis(4-aminophenoxy)benzene, and 1,4-bis(3-aminophenoxy)benzene can be suitably used. In particular, at least one diamine selected from the group consisting of benzenediamine, diaminodiphenyl ether and bis(aminophenoxy)phenyl is preferred.

[0058] From the viewpoint of heat resistance and dimensional stability at high temperatures, the above-mentioned porous polyimide membrane is preferably formed from a polyimide obtained by combining a tetracarboxylic dianhydride and a diamine, which has a glass transition temperature of 240°C or higher, or a tetracarboxylic dianhydride with no clear transition point at 300°C or higher.

[0059] From the viewpoint of heat resistance and dimensional stability at high temperatures, the above-mentioned porous polyimide membrane is preferably a porous polyimide membrane made of the following aromatic polyimides: (i) an aromatic polyimide made of at least one tetracarboxylic acid unit selected from the group consisting of biphenyltetracarboxylic acid units and pyromellitic acid units and an aromatic diamine unit; (ii) an aromatic polyimide made of a tetracarboxylic acid unit and at least one aromatic diamine unit selected from the group consisting of benzenediamine units, diaminodiphenyl ether units and bis(aminophenoxy)phenyl units; and / or (iii) an aromatic polyimide made of at least one tetracarboxylic acid unit selected from the group consisting of biphenyltetracarboxylic acid units and pyromellitic acid units and at least one aromatic diamine unit selected from the group consisting of benzenediamine units, diaminodiphenyl ether units and bis(aminophenoxy)phenyl units.

[0060] 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 pericyte culture medium, Astrocyte Medium (manufactured by ScienceCell Research Laboratories) containing 10% FBS was used as the astrocyte culture medium, and EGM-2 Endothelial Cell Growth Medium-2 Bullet Kit (manufactured by LONZA) was used as the endothelial cell culture medium. Unless otherwise specified, each procedure was performed at room temperature (20°C to 25°C).

[0061] 1. Materials and Methods 1-1 Primary astrocyte strains isolated and cultured from human BBB were introduced using a retroviral vector to introduce the temperature-sensitive SV40 large T antigen (tsA58) gene, according to the method described in the literature "J. Neurological Science 331 (2013) 136-144," thereby creating human-derived temperature-immortalized astrocyte strains. For the BBB model prepared and observed 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.

[0062] 1-2 Primary pericyte strains isolated and cultured from human BBB were introduced with the tsA58 gene 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. When the BBB model was manufactured and observed with a confocal microscope, the human-derived temperature-immortalized pericyte strains were used after being pre-stained with cyto-ID red (Enzo Life Sciences). 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.

[0063] 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. When the BBB model was manufactured and observed using a confocal microscope, the human-derived temperature-conditioned immortalized vascular endothelial cell line was used after being previously stained with Cell Tracker Orange (Thermo Fisher).

[0064] 1-4 The Porous Membrane The porous membrane in question was a three-layer porous membrane having a surface layer A and a surface layer B having multiple pores, and a macrovoid layer sandwiched between surface layers A and B, manufactured according to the method described in Japanese Patent Application Publication No. 2020-147685. Four indicators of the pores in surface layers A and B of the five types of porous membranes (Nos. 1 to 5) manufactured were measured (see Table 1). Specifically, the pore area S of 200 or more openings was measured from scanning electron microscope images of the porous membrane surface. The pore diameter d was calculated assuming the shape of the pores was perfectly circular according to Equation I above. Then, based on each pore diameter d, the number average pore diameter Sn and the area average pore diameter Sa were calculated according to Equations II and III above, respectively. Furthermore, for holes within an observation field of view of 1.28 mm × 0.96 mm, the ratio of the total area of ​​the pores within the observation field to the area of ​​the observation field was defined as the average surface aperture ratio, and the maximum pore diameter d within the observation field was defined as the maximum aperture diameter.

[0065]

[0066] 1-5 Manufacturing and Observation of the BBB Model The manufacturing and observation of the BBB model were carried out according to the following procedures [1] to [5]. [1] After placing porous membranes No. 2 to 4 in a multi-well cell culture plate with surface layer A facing upwards, 4 × 10 ６50 μL of perisite-containing culture medium with a concentration of cells / mL was seeded from the surface layer A side using a piston pipette, and the medium was left to stand at room temperature for 1 hour until the perisites migrated into the internal structural layer through the pores in the surface layer A. [2] 4 × 10 ６ 50 μL of astrocyte-containing culture medium at a concentration of cells / mL was seeded onto surface layer A using a piston pipette. [3] The porous membrane was rotated so that surface layer A 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 surface layer B using a piston pipette. [4] The porous membrane seeded with three types of cells (pericytes, astrocytes, and vascular endothelial cells) was heated at 33°C and 5% CO2. ２ The cells were cultured for 5 days under the following conditions: [5] 37°C, 5% CO2 ２ After culturing the cells under the specified conditions for 5 days to induce differentiation into mature cells, the BBB model was observed using a confocal microscope (Leica SP5 laser scanning confocal microscope) (Leica Wetzlar).

[0067] 1-6 Analysis of Cell Distribution The distribution of two types of cells (pericytes and astrocytes) on the surface layer A, within the internal structure, and on the surface layer B of the porous membrane was analyzed according to the following procedures [1] to [3]. [1] After placing the porous membrane No. 4 in a multi-well cell culture plate with surface layer A facing upwards, 1) 4 × 10 ６ 50 μL of perisite-containing culture medium containing cells / mL is seeded from the surface layer A side using a piston pipette, and left to stand at room temperature for 1 hour until the perisites migrate through the pores in surface layer A into the internal structural layer, or 2) 4 × 10 ６[1] 50 μL of astrocyte-containing culture medium at a concentration of cells / mL was seeded onto surface layer A using a piston pipette. [2] The porous membrane seeded with pericytes or astrocytes 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] After staining the cell nuclei with DAPI (4',6-diamidino-2-phenyllindole), they were observed using a confocal microscope (Leica SP5 laser scanning confocal microscope) (Leica Wetzlar).

[0068] 1-7 Optimization of Surface Layer B of Porous Membrane In order to optimize the surface layer B of the porous membrane, analysis was performed according to the following procedures [1] to [3]. [1] 1 × 10 ５ 200 μL of vascular endothelial cell culture medium containing cells / mL was seeded onto surface layer B of porous membranes No. 1-4 using a piston pipette, and the culture was maintained at 33°C and 5% CO2. ２ [2] The cells were cultured for 48 hours under the specified conditions. After fixing the porous membrane on which vascular endothelial cells were seeded with a 4% paraformaldehyde solution, 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. [3] After staining the cell nuclei with DAPI, they were observed using a confocal microscope (Leica SP5 laser scanning confocal microscope) (Leica Wetzlar).

[0069] 1-8 Analysis of Cell Proliferation Rate The cell proliferation rates of the three types of cells (pericytes, astrocytes, and vascular endothelial cells) constituting the BBB model in the porous membrane or the Transwell® cell culture insert were analyzed according to the following procedures [1] to [3]. In this example, the Transwell® cell culture insert used was collagen-coated. [1] Culture medium containing three types of cells (4 × 10 ６50 μL of perisite-containing culture medium with cells / mL, 4 × 10 ６ 50 μL of astrocyte-containing culture medium with cells / mL, and 2 × 10 ７ 50 μL of culture medium containing vascular endothelial cells (cells / mL) was seeded using a piston pipette onto the surface layer B of the porous membrane No. 4 and onto the Transwell® cell culture insert (PET, 3 μm pores: Corning International), respectively, at 33°C and 5% CO2. ２ The cells were cultured for 1 to 6 days under the specified conditions. [2] The membrane surface containing the cells was fixed with a 4% paraformaldehyde solution, and then immunocytochemistry was performed according to standard procedures using an anti-phalloidin antibody conjugated with a fluorescent dye (Acti-stain 488 phalloidin, manufactured by Cytoskeleton) to stain the cytoskeleton. [3] After staining the cell nuclei with DAPI, they were observed using an inverted fluorescence microscope (all-in-one fluorescence microscope BZ-X810) (manufactured by Keyence Corporation), and 10 ４ μm ２ The number of cells per unit was measured.

[0070] 1-9 Analysis of the barrier function of the vascular endothelial cell layer in the porous membrane The barrier function of the vascular endothelial cell layer in the porous membrane was analyzed according to the following procedure [1] to [3]. [1] After removing the PET membrane insert portion from the ThinCert® 24-well cell culture insert (made of PET, 0.4 μm pores, No. 662641: manufactured by Greiner bio-one), porous membrane No. 4 was placed in a multi-well cell culture plate with surface layer B facing upwards, and then 2 × 10 ７ 50 μL of culture medium containing vascular endothelial cells at a concentration of 1 / mL was seeded onto surface layer B using a piston pipette. [2] The porous membrane on which the vascular endothelial cells were seeded was heated at 33°C and 5% CO2. ２ The cells were cultured for 3 days under the following conditions: [3] 37°C, 5% CO2 ２After culturing the cells under the specified conditions for 1 to 4 days (4 to 7 days including the culture period at 33°C) to induce differentiation into mature cells, TEER measurements were performed on vascular endothelial cells (hereinafter referred to as "Reference Example Sample 4") that formed a sheet structure on surface layer B of porous membrane No. 4, using an EVOM resistance meter (WPI Corporation) according to the protocol provided with the product. For comparison, TEER measurements were also performed on vascular endothelial cells (hereinafter referred to as "Comparative Example Sample 4") that formed a sheet structure on ThinCert® 24-well cell culture insert (No. 662641, Greiner bio-one Corporation).

[0071] 1-10 Barrier Function Analysis of the BBB Model (1) The barrier function of the BBB model was analyzed by TEER measurement according to the following procedure [1] to [5]. [1] After placing the porous membrane No. 5 in the container of the insert frame in a multiwell plate for cell culture with surface layer A facing upwards, 4 × 10 ６ 25 μL of perisite-containing culture medium with a concentration of cells / mL was seeded from the surface layer A side using a piston pipette, and the medium was left to stand at room temperature for 1 hour until the perisites migrated into the internal structural layer through the pores in the surface layer A. [2] 4 × 10 ６ 25 μL of astrocyte-containing culture medium at a concentration of cells / mL was seeded onto surface layer A using a piston pipette. [3] The porous membrane was rotated so that surface layer A was facing downwards and placed in a multi-well plate for cell culture, then 2 × 10 ７ 25 μL of culture medium containing vascular endothelial cells at a concentration of 1 / mL was seeded onto surface layer B using a piston pipette. [4] The porous membrane seeded with three types of cells (pericytes, astrocytes, and vascular endothelial cells) was 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 for one day under the specified conditions (six days including the culture period at 33°C) to induce differentiation into mature cells, TEER measurements were performed on the BBB model manufactured based on the No. 5 polyimide porous membrane (hereinafter referred to as the "Example BBB Model") using an EVOM resistance meter (WPI) according to the protocol provided with the product. For comparison, TEER measurements were also performed on four other samples (Comparative Example Sample 4, human umbilical vein endothelial cells [HUVEC] [PromoCell] [hereinafter referred to as "Comparative Example Sample 5"] formed as a sheet structure on a ThinCert® 24-well cell culture insert, Reference Example Sample 4, and a BBB model manufactured based on a ThinCert® 24-well cell culture insert [hereinafter referred to as the "Comparative Example BBB Model"]). The comparative example BBB model was manufactured using a ThinCert® 24-well cell culture insert (No. 662641, manufactured by Greiner bio-one) and three types of cells (pericytes, astrocytes, and vascular endothelial cells) according to the method described in Patent Document 3.

[0072] 1-11 Barrier Function Analysis of the BBB Model (2) The barrier function of the BBB model was analyzed by a permeability test. Specifically, five types of samples (Comparative Example Sample 5, Comparative Example Sample 4, Reference Example Sample 4, Comparative Example BBB Model, and Example BBB Model) were placed in the insert frame container of a multi-well plate for cell culture so that the upper side would be vascular endothelial cells. Then, FITC (Fluorescein isothiocyanate)-labeled dextran (average molecular weight: 10 kDa) (FITC-10k-Dextran) (manufactured by Sigma-Aldrich) was added to the culture medium on the upper side of each sample to a final concentration of 1 mg / mL. After standing for 20 minutes, the culture medium was collected from the wells on the lower side (astrocyte side), and a microplate reader (FlexStation) was used. 3. Using a device manufactured by Molecular Devices Japan, the amount of FITC-10k-Dextran that permeated through the five types of samples was measured by analyzing the fluorescence level of FITC, and the transmission coefficient (Papp) of 10k-Dextran was calculated.

[0073] 1-12 RMT Function Analysis of the BBB Model The RMT function of the BBB model was analyzed using melanotransferrin (MTF), a substrate of low-density lipoprotein receptor-related protein (LRP) in the BBB. Specifically, the Example BBB model was placed in the insert frame container of a multi-well cell culture plate so that the upper side would be vascular endothelial cells. Then, MTF (Novatein Bioscience) and FITC-10k-Dextran (Sigma-Aldrich) were added to the culture medium on the upper side of the Example BBB model to a final concentration of 1 mg / mL. After standing for 20 minutes, the culture medium was collected from the wells on the lower side (astrocyte side), and the amount of MTF that permeated through the Example BBB model was measured using "Human microtransferrinuria MTF ELISA Kit 96 Wells" (NB-E10315, Novatein). The amount of FITC-10k-Dextran that permeated through the BBB model of the example was measured using the ELISA method with Bioscience Inc., and the amount of FITC-10k-Dextran that permeated through the BBB model of the example was also measured using the ELISA method with the "Fluorescein Competitive ELISA Kit (AKR-5141, Cell Biolabs Inc.)" to calculate the Papp of MTF and 10k-Dextran. Based on the calculated Papp of MTF (Papp[MTF]) and Papp of 10k-Dextran (Papp[Dextran]), the Transcellular index (=Papp[MTF] / Papp[Dextran]), which is an indicator of RMT, was calculated.

[0074] 1-13 Analysis of Transporter Function in the BBB Model The transporter function of the BBB model was analyzed using rhodamine 123, a substrate of the efflux transporter (P-glycoprotein [P-gp]) in the BBB. Specifically, the Example BBB model was placed in the insert frame container of a multi-well cell culture plate with the upper side facing astrocytes. Then, rhodamine 123 (Sigma-Aldrich) and paracellular permeability evaluation substance (sodium fluorescein [Na Fluorescein], Sigma-Aldrich) were added to the culture medium on the upper side of the Example BBB model to a final concentration of 0.1 mg / mL. After standing for 20 minutes, the culture medium was collected from the wells on the lower side (vascular endothelial cell side), and the amount of rhodamine 123 and sodium fluorescein that permeated through the Example BBB model was measured by analyzing the fluorescence levels of rhodamine 123 and sodium fluorescein using a microplate reader (FlexStation 3, Molecular Devices Japan). The Papp of rhodamine 123 and sodium fluorescein was then calculated. Based on the calculated Papp values ​​for rhodamine 123 (Papp[rhodamine 123]) and fluorescein sodium (Papp[fluorescein sodium]), the Transporter index (=Papp[rhodamine 123] / Papp[fluorescein sodium]), an indicator of transporter function, was calculated.

[0075] 2. Results 2-1 Manufacturing and Observation of the BBB Model The BBB model, manufactured using porous membranes No. 2-4 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 layer A, within the internal structure, and on the surface layer B of the porous membrane, respectively (see Figure 1). Furthermore, when steps [1] and [2] of item "1-5" above were reversed (i.e., astrocytes were seeded on the surface layer A, and then pericytes were seeded from the surface layer A side), it was similarly confirmed that three types of cell layers—astrocytes, pericytes, and vascular endothelial cells—were formed on the surface layer A, within the internal structure, and on the surface layer B of the porous membrane, respectively.

[0076] Furthermore, analysis of the distribution of two types of cells (pericytes and astrocytes) on the surface layer A, within the internal structure, and on the surface layer B of the porous membrane revealed that approximately 90% of the astrocyte population, representing the majority of the total astrocyte population, resided on surface layer A, while approximately 9% resided within the internal structure and approximately 1% resided on surface layer B (see Figure 2).

[0077] 2-2 Optimization of the surface layer B of porous membranes Endothelial cells were seeded onto the surface layer B of porous membranes No. 1 to 4 using a piston pipette, and the sheet structure formed by the endothelial cells was analyzed. The results showed that when seeded on the surface layer B of porous membranes No. 3 and No. 4, a sheet structure of endothelial cells with little or no voids was formed. In contrast, when seeded on the surface layer B of porous membrane No. 1, a sheet structure of endothelial cells with voids throughout was formed, and when seeded on the surface layer B of porous membrane No. 2, a sheet structure of endothelial cells with multiple voids was formed (see Figure 3). These results indicate that, in order to suppress the formation of a sheet structure of vascular endothelial cells with voids throughout the entire surface layer B of a porous membrane, it is necessary to set the maximum opening diameter of the pores in the surface layer B to at least 43 μm or less. Conversely, in order to form a sheet structure of vascular endothelial cells with little to no voids, it is necessary to set the maximum opening diameter of the pores in the surface layer B to at least 40 μm or less.

[0078] 2-3 Analysis of Cell Proliferation Rate The three types of cells constituting the BBB model (pericytes, astrocytes, and vascular endothelial cells) were seeded on the surface layer B of the porous membrane or on the Transwell® cell culture insert, and the cell proliferation rate was analyzed. The results showed that for all cell types, the cell proliferation rate was faster and the adhesion to the membrane was higher when seeded on the surface layer B of the porous membrane than when seeded on the Transwell® cell culture insert (see Figure 4). This result indicates that the cell proliferation and adhesion of the three types of cells constituting the BBB model (pericytes, astrocytes, and vascular endothelial cells) are maintained at a high level.

[0079] 2-4 Analysis of the barrier function of the vascular endothelial cell layer in the porous membrane of this study The TEER of comparative example sample 4 tended to decrease slightly as the culture period lengthened from 4 days to 7 days, whereas the TEER of reference example sample 4 was maintained without decrease even after 7 days of culture (see Figure 5). This result indicates that, of the three cell layers constituting the BBB model of this study, only the vascular endothelial cell layer can be cultured and stored for a long period of time, and its barrier function can be maintained without decrease.

[0080] 2-5 Barrier Function Analysis of the BBB Model (1) The barrier function of five types of samples (Comparative Example Sample 5, Comparative Example Sample 4, Reference Example Sample 4, Comparative Example BBB Model, and Example BBB Model) was analyzed by TEER measurement. The TEER of Reference Example Sample 4 was found to be higher than that of Comparative Example Sample 4, similar to the results in Figure 5 (see Figure 6 and Table 2). In addition, the TEER of the Example BBB Model was equivalent to or slightly higher than that of Reference Example Sample 4 and Comparative Example BBB Model (see Figure 6 and Table 2). These results indicate that the BBB model in this case is a BBB model with high barrier function that can be manufactured more easily than the Comparative Example BBB Model (i.e., the BBB model manufactured by the method described in Patent Document 3).

[0081] The values ​​in the table are shown as relative values ​​for the TEER values ​​of the four types of samples (Comparative Example Sample 4, Reference Example Sample 4, Comparative Example BBB Model, and Example BBB Model), with the TEER value of Comparative Example Sample 5 set to 1.

[0082] 2-6 Barrier Function Analysis of the BBB Model (2) The barrier function of five types of samples (Comparative Example Sample 5, Comparative Example Sample 4, Reference Example Sample 4, Comparative Example BBB Model, and Comparative Example BBB Model) was analyzed by permeability testing. The results showed that the Papp of Reference Example Sample 4 was lower than that of Comparative Example Sample 4 (see Figure 7 and Table 3). This result indicates that the barrier function of Reference Example Sample 4 is higher than that of Comparative Example Sample 4, supporting the results in Figures 5 and 6.

[0083] Furthermore, the Papp of the Example BBB model was shown to be lower than that of the Comparative Example BBB model (see Figure 7 and Table 3). This result indicates that the barrier function of the Example BBB model is higher than that of the Comparative Example BBB model. Although the difference in barrier function between the two could not be clearly detected in the aforementioned TEER measurement, the difference in barrier function between the two was detectable in this permeability test.

[0084] The values ​​in the table are shown as relative values ​​for the Papp of the four types of samples (Comparative Example Sample 4, Reference Example Sample 4, Comparative Example BBB Model, and Example BBB Model), with the Papp of Comparative Example Sample 5 set to 1.

[0085] 2-7 In the RMT functional analysis of the BBB model in this case, if there is no active transport in the cells, generally the larger the molecular weight, the smaller the Papp. Therefore, the Papp of 10k-Dextran with a molecular weight of approximately 10,000 Da is larger than that of MTF with a molecular weight of approximately 80,000 Da, and as a result, the Transcellular index (= Papp [MTF] / Papp [Dextran]) becomes less than 1.

[0086] On the other hand, in the BBB model of the example, RMT function analysis was performed using MTF, a substrate of LRP in BBB, and 10k-Dextran. The results showed that the Transcellular index (= Papp[MTF] / Papp[Dextran]) was a high value of 14.5 (>2). This result indicates that MTF, which was present on the vascular endothelial cell side (vascular lumen side), was actively transported to the astrocyte side (brain parenchyma side) via LRP, and that the BBB model in this case possesses RMT function.

[0087] 2-8 Transporter Function Analysis of the BBB Model In this case, transporter function analysis was performed on the BBB model using rhodamine 123, a substrate of P-gp in the BBB, and fluorescein sodium, a paracellular permeability evaluation substance. The result showed that the Transcellular index (= Papp [rhodamine 123] / Papp [fluorescein sodium]) was 2.1 (>2). This result indicates that rhodamine 123, which was present on the astrocyte side (brain parenchyma side), was excreted to the vascular endothelial cell side (vascular lumen side), indicating that the BBB model in this case possesses transporter function.

[0088] This invention contributes to analyses using blood-brain barrier models, such as the selection of candidate substances with good brain permeability.

Claims

1. A blood-brain barrier model comprising three cell layers: pericytes, astrocytes, and vascular endothelial cells, and a porous membrane having a surface layer A having multiple pores and a surface layer B having multiple pores, and an internal structural layer sandwiched between the surface layers A and B, wherein the vascular endothelial cells form a sheet structure on the surface layer B, and the pericytes reside in the internal structural layer.

2. The blood-brain barrier model according to claim 1, wherein astrocytes are present on surface layer A.

3. The blood-brain barrier model according to claim 1, wherein the maximum opening diameter of the pores in surface layer B is 43 μm or less.

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

5. The blood-brain barrier model according to any one of claims 1 to 4, wherein the porous membrane is a polyimide porous membrane.

6. A method for manufacturing a blood-brain barrier model, comprising the following steps (a) to (d), wherein the blood-brain barrier model is composed of three cell layers of pericytes, astrocytes, and vascular endothelial cells, and a porous membrane having a surface layer A having a plurality of pores and a surface layer B having a plurality of pores, and an internal structural layer sandwiched between the surface layer A and the surface layer B, wherein the vascular endothelial cells form a sheet structure on the surface layer B, the pericytes are present in the internal structural layer, and the astrocytes are present on the surface layer A. (a) A step of seeding pericytes into the internal structural layer of the porous membrane; (b) A step of seeding astrocytes on the surface layer A of the porous membrane; (c) A step of seeding vascular endothelial cells on the surface layer B of the porous membrane; (d) A step of culturing the porous membrane 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 layer B; 7. The manufacturing method according to claim 6, wherein in step (a), perisite is seeded into the internal structural layer from the surface layer A side of the porous membrane.

8. The manufacturing method according to claim 7, wherein steps (a), (b), and (c) are carried out in that order.

9. The manufacturing method according to claim 8, wherein the porous membrane after step (b) is inverted 180 degrees and step (c) is carried out.

10. The manufacturing method according to claim 6, wherein the maximum opening diameter of the pores in surface layer B is 43 μm or less.

11. The manufacturing method according to claim 6, wherein the pericytes, astrocytes, and vascular endothelial cells are human-derived cells.

12. The manufacturing method according to any one of claims 6 to 11, wherein the porous membrane is a polyimide porous membrane.

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