Hierarchical spheroid blood-brain barrier model using immortalized human cells and method for producing the same

Abstract

Provided is a method for producing a multicellular, three-dimensional blood brain barrier model, comprising: (i) a first seeding step for seeding a human conditionally immortalized astrocyte and a human conditionally immortalized pericyte on a medium; (ii) a first coculturing step for coculturing the seeded human conditionally immortalized astrocyte and human conditionally immortalized pericyte to obtain a coculture containing two types of cells; (iii) a second seeding step for seeding a human conditionally immortalized brain microvascular endothelial cell on a medium that contains the coculture containing the two types of cells; and (iv) a second coculturing step for coculturing the coculture containing the two types of cells and the human conditionally immortalized brain microvascular endothelial cell to obtain a multicellular, three-dimensional blood brain barrier model.

Description

Technical Field

[0001] The present invention relates to a hierarchical spheroid blood-brain barrier model using human immortalized cells and a method for producing the same.

Background Art

[0002] The central nervous system (CNS) is physiologically isolated from the periphery by the blood-brain barrier (BBB). The BBB is a unique function of the cerebral blood vessels characterized by intercellular tight junctions (tight junctions and adherens junctions) and efflux transporters (P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP)) that restrict the movement of endogenous and exogenous compounds, including drugs, from the blood to the brain (Non-Patent Document 1). Therefore, the BBB inherently functions as a protective barrier against blood-derived substances that may be involved in CNS diseases (Non-Patent Document 1), but at the same time, it also poses an obstacle in developing drugs effective for CNS diseases (Non-Patent Document 1).

[0003] However, on the other hand, the BBB has specific substance influx transport systems such as glucose transporter 1 (GLUT1) and receptor-mediated transcytosis (RMT). A typical example of a receptor involved in RMT is the transferrin receptor (TfR), which is required for the transport of transferrin into the brain, and at the same time, transports macromolecules that bind to the receptor, such as anti-transferrin antibodies. Thus, RMT is regarded as a promising CNS delivery pathway for therapeutic macromolecules (Non-Patent Document 2).

[0004] The BBB also plays an essential role in maintaining a low-level immune surveillance mechanism in the brain by restricting the movement of immune cells into the CNS (Non-Patent Document 3). However, the invasion of immune cells into the CNS has been observed in various diseases such as multiple sclerosis and stroke where the BBB breaks down, and this event plays an important role in the progression of the diseases (Non-Patent Document 4).

[0005] For these reasons, BBB is an important target for research in terms of understanding CNS diseases, identifying treatment approaches, and developing drugs for CNS diseases.

[0006] To promote such research, extensive studies are required to explore the molecular mechanisms underlying the development and maintenance of the BBB and to test various drug candidates and findings for advancing the treatment of CNS diseases.

[0007] Since the in vivo BBB is formed by brain microvascular endothelial cells (BMECs) covered with pericytes and astrocytes (Non-Patent Document 1), in vitro BBB models (Non-Patent Document 5) are constructed using BMECs or a combination of these three cell types. Conventional in vitro BBB models have used the transwell culture system. This system has many experimental advantages such as being easy to prepare and allowing for easy analysis of the intercellular junctions and drug permeability of the BBB.

[0008] However, since the transwell culture system lacks the in vivo microenvironment (i.e., cells survive two-dimensionally and there is no direct communication between each of the three cell types), its function is not fully mature, resulting in a low BBB function level and leaving the possibility of false-negative / false-positive results in specific experiments.

[0009] In contrast, three-dimensional culture methods such as BBB-on-a-chip, organoids, and spheroids (Non-Patent Document 5) have attracted great attention as promising approaches for developing BBB models with better functions. Since these new systems aim to more faithfully mimic the in vivo BBB, they are expected to achieve a higher BBB function level than ever before. However, for such models to be implemented in the field of BBB research and drug discovery and contribute to its advancement, compatibility and convenience for various experiments are required.

Prior Art Documents

Non-Patent Literature

[0010]

Non-Patent Literature 1

Non-Patent Literature 2

Non-Patent Literature 3

Non-Patent Literature 4

Non-Patent Literature 5

Summary of the Invention

Problems to be Solved by the Invention

[0011] An object of the present invention is to provide a hierarchical spheroid blood-brain barrier model using human immortalized cells and a method for producing the same.

Means for Solving the Problems

[0012] Under the increasing interest in three-dimensional (3D) BBB models as described above, the present inventors have successfully established a hierarchical spheroid blood-brain barrier model using human conditionally immortalized cells. As the present inventors have reported so far (References 1 to 4), the immortalized cells used have high proliferative ability and cryopreservation properties, and can also be used in a transwell-type BBB model that can distinguish highly BBB-permeable drugs and low BBB-permeable drugs. According to the method for producing the model presented in this specification, a three-dimensional BBB model can be constructed, which further enhances the characteristics of the BBB and serves as a research tool useful for various BBB studies.

[0013] That is, the present invention provides: [1] (i) A first seeding step of seeding human conditionally immortalized astrocytes and human conditionally immortalized pericytes in a medium, (ii) A first co-culture step of co-culturing the seeded human conditionally immortalized astrocytes and the human conditionally immortalized pericytes to obtain a co-culture containing two types of cells, (iii) A second seeding step of seeding human conditionally immortalized brain microvascular endothelial cells in a medium containing the co-culture containing the two types of cells, (iv) A second co-culture step of co-culturing a co-culture containing the two types of cells with the human conditionally immortalized brain microvascular endothelial cells to obtain a multicellular three-dimensional blood-brain barrier model; A method for producing a multicellular three-dimensional blood-brain barrier model, comprising; [2] The production method according to [1], wherein the medium in the first seeding step (i) contains a thickening agent; [3] The production method according to [1] or [2], wherein the first co-culture step (ii) and the second co-culture step (iv) are performed within the range of 35 to 39 °C; [4] The production method according to any one of [1] to [3], wherein in the first co-culture step (ii), the co-culture containing the two types of cells forms cell aggregates in an ellipsoidal shape, a prolate ellipsoidal shape or a spherical shape; [5] The production method according to [4], wherein the longest diameter among the three diameters of the ellipsoid, prolate ellipsoid or sphere is within the range of 180 to 350 μm; [6] The production method according to [4] or [5], wherein in the second co-culture step (iv), the human conditionally immortalized brain microvascular endothelial cells cover the periphery of the cell aggregate formed in the first co-culture step (ii) to form a multicellular three-dimensional blood-brain barrier model; [7] The multicellular three-dimensional blood-brain barrier model has a shape of an ellipsoid, a prolate ellipsoid or a sphere, The production method according to any one of [1] to [6], wherein the longest diameter among the three diameters of the ellipsoid, prolate ellipsoid or sphere of the multicellular three-dimensional blood-brain barrier model is within the range of 180 to 350 μm; [8] The production method according to any one of [2] to [7], wherein the concentration of the thickening agent in the medium of the first seeding step (i) is 0.1 to 20 mg / mL; [9] The production method according to any one of [1] to [8], wherein the seeding of the second seeding step (iii) is performed 12 to 72 hours after the first seeding step (i);

[10] The production method according to any one of [1] to [9], wherein in the second co-culture step (iv), the co-culture is performed for 12 to 72 hours;

[11] The manufacturing method according to [1] to

[10] , wherein at least one of the steps (i) to (iv) is performed in a container having a bottom portion in a substantially V-shaped or V-shaped form;

[12] A multicellular three-dimensional blood-brain barrier model produced by the manufacturing method according to any one of [1] to

[11] ;

[13] (A) A cell mass containing human conditionally immortalized astrocytes and human conditionally immortalized pericytes, (B) A layer of human conditionally immortalized brain microvascular endothelial cells covering the periphery of the cell mass and a multicellular three-dimensional blood-brain barrier model;

[14] The multicellular three-dimensional blood-brain barrier model according to

[13] , wherein in the cell mass (A), human conditionally immortalized astrocytes are located inside and human conditionally immortalized pericytes are located outside;

[15] The multicellular three-dimensional blood-brain barrier model according to

[13] or

[14] , wherein the multicellular three-dimensional blood-brain barrier model is in an ellipsoidal shape, a spheroid shape or a spherical shape;

[16] The multicellular three-dimensional blood-brain barrier model according to

[15] , wherein the longest diameter among the three diameters of the ellipsoid, spheroid or sphere of the multicellular three-dimensional blood-brain barrier model is in the range of 180 to 350 μm relating to.

Advantages of the Invention

[0014] According to the present invention, a hierarchical spheroid blood-brain barrier model using human immortalized cells can be provided.

Brief Description of the Drawings

[0015]

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Mode for Carrying Out the Invention

[0016] Hereinafter, the present invention will be described in more detail.

[0017] The first aspect of the present invention provides a method for producing a multicellular three-dimensional blood-brain barrier model using human conditionally immortalized cells. Specifically, the multicellular three-dimensional blood-brain barrier model provided by this production method is a human in vitro blood-brain barrier model. This production method includes the steps described below.

[0018] That is, the method for producing a multicellular three-dimensional blood-brain barrier model according to the present invention is as follows: (i) A first seeding step of seeding human conditionally immortalized astrocytes and human conditionally immortalized pericytes in a medium, (ii) A first co-culture step of co-culturing the seeded human conditionally immortalized astrocytes and the human conditionally immortalized pericytes to obtain a co-culture containing the two types of cells; (iii) A second seeding step of seeding human conditionally immortalized brain microvascular endothelial cells in a medium containing the co-culture containing the two types of cells; (iv) A second co-culture step of co-culturing the co-culture containing the two types of cells and the human conditionally immortalized brain microvascular endothelial cells to obtain a multicellular three-dimensional blood-brain barrier model and the like. Hereinafter, each step will be described in detail.

[0019] <First seeding step> In this step, human conditionally immortalized astrocytes and human conditionally immortalized pericytes are seeded in a medium. The human conditionally immortalized astrocytes and the human conditionally immortalized pericytes are seeded together in a medium and co-cultured in subsequent steps.

[0020] The ratio of the number of human conditionally immortalized astrocytes seeded in this step to the number of human conditionally immortalized pericytes can be appropriately adjusted within a range in which the human conditionally immortalized astrocytes and the human conditionally immortalized pericytes can form cell aggregates by self-assembly in the subsequent co-culture step.

[0021] In a preferred embodiment, the medium contains a thickening agent. By including a thickening agent in the medium, the formation of cell aggregates is promoted in subsequent co-cultures.

[0022] A thickener that imparts an appropriate viscosity to the medium and does not impair the function of the cells to be cultured is used. Examples of the thickener include, but are not limited to, methylcellulose, hydroxymethylcellulose, ethylcellulose, hydroxyethylcellulose, agarose, carboxymethylcellulose, methylhydroxypropylcellulose, dextran, polyethylene glycol, xanthan gum, carrageenan, guar gum, tamarind seed gum, locust bean gum, gellan gum, gum arabic, quince seed gum, galactan, pectin, mannan, starch, curdlan, glycogen, hydroxyethyl guar gum, carboxymethyl guar gum, keratan sulfate, succinoglucan, carroninic acid, chitin, chitosan, carboxymethyl chitin, polyvinyl alcohol, polyvinyl pyrrolidone, carboxyvinyl polymer, alkyl-modified carboxyvinyl polymer, bentonite. The thickener may be used in combination of a plurality of these. In one embodiment, the thickener is methylcellulose.

[0023] Methylcellulose has, for example, one or more properties selected from the following: an average degree of polymerization of 300 to 500, an average molecular weight of 60,000 to 100,000, and a methoxy group content of 20 to 40%. Preferred methylcellulose has one or more properties selected from the following: an average degree of polymerization of 400 to 500, an average molecular weight of 70,000 to 90,000, and a methoxy group content of 25 to 35%. Most preferred methylcellulose has one or more properties selected from the following: an average degree of polymerization of 440, an average molecular weight of 84,000, and a methoxy group content of 26 to 33%.

[0024] The concentration of the thickener in the medium of the step (i) is, for example, 0.1 to 20 mg / mL. When the thickener is methylcellulose, the concentration of methylcellulose is, for example, 0.1 to 10 mg / mL, preferably 0.2 to 5.0 mg / mL, more preferably 0.3 to 3.0 mg / mL, still more preferably 0.4 to 2.0 mg / mL, and most preferably 0.4 to 1.0 mg / mL. In one embodiment, the concentration of methylcellulose in the medium of the step (i) is 0.48 mg / mL.

[0025] The medium used in this project has a viscosity at room temperature of, for example, 6.3 mPa·s or more, preferably 6.4 mPa·s or more, more preferably 6.5 mPa·s or more, even more preferably 6.7 mPa·s or more, still more preferably 6.8 mPa·s or more, and most preferably 7.0 mPa·s or more. If the viscosity of the medium is too low, the formation of cell aggregates may not be promoted. Using a B-type viscometer (manufactured by Toki Sangyo Co., Ltd., "TVB10" type viscometer) equipped with an M2 rotor (manufactured by Toki Sangyo Co., Ltd.), the value measured 1 minute after the start of stirring at a rotational speed of 60 rpm is taken as the viscosity of the medium. Specifically, starting from 1 minute after the start of stirring, the measured values were measured 10 times every 1 to 2 seconds, and the average of the measured values was taken as the viscosity.

[0026] <First co-culture step> In this step, the human conditionally immortalized astrocytes and human conditionally immortalized pericytes seeded in the first seeding step are co-cultured to obtain a co-culture containing two types of cells.

[0027] The co-culture containing two types of cells obtained in this step forms cell masses. Specifically, the human conditionally immortalized astrocytes and human conditionally immortalized pericytes form cell masses by self-assembly. The cell masses have, for example, an ellipsoidal shape, preferably a prolate ellipsoidal or spherical shape.

[0028] In this specification, an "ellipsoid" is a three-dimensional extended solid shape of an ellipse, and its surface is a quadratic surface. When the half-lengths of the diameters in the x-axis, y-axis, and z-axis directions of the ellipsoid are respectively a, b, and c, an ellipsoid in which any two of a, b, and c are equal is a solid of revolution obtained by rotating the ellipse around the axis of the ellipse, and the one with the major axis as the axis of rotation (prolate spheroid) and the one with the minor axis as the axis of rotation (oblate spheroid) are collectively referred to as a "prolate ellipsoid". An ellipsoid in which all of a, b, and c are equal is a "sphere". In this specification, the term "ellipsoid" shall include "prolate ellipsoid" and "sphere".

[0029] The longest of the three diameters of the ellipsoid, ellipsoid of revolution or sphere is, for example, in the range of 180 to 350 μm, preferably 200 to 320 μm, more preferably 220 to 300 μm, and most preferably 240 to 280 μm. When the longest diameter is within this range, it is possible to prevent the formation of a hypoxic region in the cell mass.

[0030] In the cell mass formed in this step, human conditionally immortalized astrocytes are located inside and human conditionally immortalized pericytes are located outside. Specifically, the cell mass has a structure in which a mass with human conditionally immortalized astrocytes as the core is formed inside, and human conditionally immortalized pericytes are located outside thereof (see Fig. 1A).

[0031] In this step, the co-culture is carried out, for example, at 34 to 40 °C, preferably 35 to 39 °C. For example, when these cells are co-cultured at a temperature condition of 33 °C, the size of the cell mass increases, resulting in a hypoxic state in the core region formed by the cell mass. On the other hand, by performing co-culture within the above temperature range, the function of the immortalization signal can be weakened to suppress cell growth while promoting differentiation, and a cell mass with a suitable size can be obtained, and a cell mass with good sphericity can be obtained. Furthermore, the cells have temperature tolerance within the above temperature range. In one aspect, the co-culture is carried out at 36 to 38 °C. In another aspect, the co-culture is carried out at 36.5 to 37.5 °C.

[0032] As will be described later in the examples, the inventors of the present invention succeeded in obtaining a cell mass with a suitable size by performing co-culture under a temperature condition of 37 °C, which suppresses growth while promoting differentiation, in a specific experiment. Heretofore, it has been reported that the immortalization signal of cells is completely released at 39 °C, but it was shown that the function of the immortalization signal can be weakened and differentiation can be promoted even at 37 °C. In this regard, it can be said that the production method according to the present invention is a method with higher versatility in that differentiation can be promoted at a lower temperature. In addition, it was found that the sphericity is improved by performing co-culture at 37 °C. An example of the co-culture conditions in this step is 37 °C, 5% CO2 / 95% air.

[0033] In this project, the co-culture is carried out for, for example, 12 to 72 hours, preferably 24 to 60 hours, more preferably 30 to 60 hours, particularly preferably 36 to 54 hours, and most preferably 40 to 50 hours. In a specific embodiment, the co-culture is carried out for 48 hours.

[0034] <Second seeding step> In this step, human conditionally immortalized brain microvascular endothelial cells are seeded into the medium containing the co-culture containing the two types of cells obtained in the previous step. The seeding of human conditionally immortalized brain microvascular endothelial cells is carried out, for example, 12 to 72 hours after seeding human conditionally immortalized astrocytes and human conditionally immortalized pericytes, preferably 24 to 60 hours after, more preferably 30 to 60 hours after, particularly preferably 36 to 54 hours after, and most preferably 40 to 50 hours after. In a specific embodiment, the seeding of human conditionally immortalized brain microvascular endothelial cells is carried out 48 hours after seeding human conditionally immortalized astrocytes and human conditionally immortalized pericytes.

[0035] <Second co-culture step> In this step, a co-culture containing the two types of cells obtained in the first co-culture step and human conditionally immortalized brain microvascular endothelial cells are co-cultured to obtain a multicellular three-dimensional blood-brain barrier model.

[0036] In this step, the co-culture of the above cells is carried out at, for example, 34 to 40 °C, preferably 35 to 39 °C. By co-culturing within the above temperature range, the effect of the immortalization signal can be weakened to suppress cell growth while promoting differentiation, and cell aggregates with a suitable size can be obtained, and cell aggregates with good sphericity can be obtained. Furthermore, if it is within the above temperature range, the cells have temperature tolerance. In one embodiment, the co-culture of this step is carried out at 36 to 38 °C. In another embodiment, the co-culture is carried out at 36.5 to 37.5 °C.

[0037] As will be described later in the examples, in specific experiments, the inventors of the present invention carried out co-culture under temperature conditions of 37°C, which promotes differentiation while suppressing growth, and were able to obtain cell aggregates of a suitable size, with an improved sphericity, and found that human conditionally immortalized brain microvascular endothelial cells can uniformly cover the periphery of a co-culture containing two types of cells.

[0038] In this step, human conditionally immortalized brain microvascular endothelial cells cover the periphery of the cell aggregates of the co-culture containing two types of cells formed in the first co-culture step to form a multicellular three-dimensional blood-brain barrier model. Preferably, the human conditionally immortalized brain microvascular endothelial cells cover the periphery of the cell aggregates of the co-culture containing two types of cells formed in the first co-culture step in a single layer to form the model (Figure 1A).

[0039] As described above, in the method for producing a multicellular three-dimensional blood-brain barrier model according to the present invention, first, human conditionally immortalized astrocytes and human conditionally immortalized pericytes are seeded in a medium, and then human conditionally immortalized brain microvascular endothelial cells are seeded, so that a hierarchical spheroid blood-brain barrier model in which the periphery of the cell aggregates formed from human conditionally immortalized astrocytes and human conditionally immortalized pericytes is covered with human conditionally immortalized brain microvascular endothelial cells can be obtained.

[0040] The formed multicellular three-dimensional blood-brain barrier model has a shape such as an ellipsoid, preferably a prolate ellipsoid or a sphere. The longest diameter among the three diameters of the ellipsoid, prolate ellipsoid or sphere of the multicellular three-dimensional blood-brain barrier model is, for example, in the range of 180 to 350 μm, preferably 200 to 320 μm, more preferably 220 to 300 μm, and most preferably 240 to 280 μm.

[0041] The ratio of the number of human conditionally immortalized astrocytes seeded in the first seeding step, the number of human conditionally immortalized pericytes seeded in the first seeding step, and the number of human conditionally immortalized brain microvascular endothelial cells seeded in the second seeding step may be appropriately adjusted in the co-culture step so that the human conditionally immortalized astrocytes and the human conditionally immortalized pericytes can form cell aggregates by self-assembly, and the human conditionally immortalized brain microvascular endothelial cells can sufficiently cover the periphery of the cell aggregates containing the human conditionally immortalized astrocytes and the human conditionally immortalized pericytes.

[0042] In one aspect, at least one of the above steps is performed in a container having a bottom in a substantially V-shaped or V-shaped configuration (see FIG. 1A). Here, the shape of the bottom refers to the shape defined based on the cross-sectional view when cut along the depth direction of the container. When performed in a container having a bottom in a shape other than a substantially V-shaped or V-shaped configuration, for example, a container having a U-shaped bottom, the action of gathering around the cells is weak, and endothelial cells do not adhere uniformly to the spheroid surface. At least one of the above steps is performed, for example, in a plate having a bottom in a substantially V-shaped or V-shaped configuration. The plate has a desired number of wells. In a preferred aspect, all of the above steps are performed in a container having a bottom in a substantially V-shaped or V-shaped configuration (see FIG. 1A).

[0043] <Other steps> The manufacturing method of the present invention may further include a freezing step of freezing the produced multicellular three-dimensional blood-brain barrier model, a step of maintaining the shape, performance, etc. of the produced model, and a step of transporting the model to a required location.

[0044] The second aspect of the present invention relates to a multicellular three-dimensional blood-brain barrier model produced by the above-described manufacturing method.

[0045] The third aspect of the present invention is as follows: (A) A cell aggregate containing human conditionally immortalized astrocytes and human conditionally immortalized pericytes, (B) A layer of human conditionally immortalized brain microvascular endothelial cells covering the periphery of the cell mass and relates to a multicellular three-dimensional blood-brain barrier model containing the same.

[0046] In the cell mass (A), human conditionally immortalized astrocytes are located on the inside and human conditionally immortalized pericytes are located on the outside. Specifically, the cell mass has a structure in which human conditionally immortalized astrocytes form a core mass on the inside and human conditionally immortalized pericytes are located on the outside thereof (see Fig. 1A). The multicellular three-dimensional blood-brain barrier model has the above-described shape and size.

[0047] As described above, the in vitro blood-brain barrier (BBB) model is an important research tool for the development of brain-targeted drugs and the understanding of the physiological and pathophysiological functions of the BBB. According to the present invention, a multicellular three-dimensional blood-brain barrier model using human conditionally immortalized cells can be provided. The model is expected to maintain basic human BBB characteristics and provide a useful and highly applicable experimental platform for accelerating various BBB studies based on the infinite proliferation ability of immortalized cells.

Examples

[0048] Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples in any way.

[0049] <1>Sample and Test Method

[0050] [Preparation of Immortalized Cells] Human brain microvascular endothelial cells / conditionally immortalized clone 18 (HBMEC / ci18), human astrocytes / conditionally immortalized clone 35 (HASTR / ci35), and human brain pericytes / conditionally immortalized 37 (HBPC / ci37) were prepared according to the methods described in References 1 to 4. These have temperature-sensitive simian virus 40 large T antigen (tsSV40T) and human telomerase catalytic subunit (hTERT) as immortalizing genes. tsSV40T confers conditional (reversible) immortalization characteristics on cells, which promote the cell cycle at a culture temperature of 33°C but disappear because they are unstable at non-permissive temperatures (37 to 39°C). Therefore, immortalized cells were cultured at 33°C for growth and at 37°C for differentiation. Specifically, the culture was performed according to the methods described in References 2 to 4 and the method shown in FIG. 1. The medium composition used is shown in Table 1 below.

[0051] [Table 1] TIFF0007702137000002.tif204167

[0052] The human monocytic leukemia cell line (THP-1) was obtained from the JCRB Cell Bank (Japanese Collection of Research Bioresources (Osaka, Japan)) and cultured at 37°C, 5% CO2 / 95% air in RPMI-1640 (Wako, Osaka, Japan) containing 10% fetal bovine serum (FBS) and penicillin-streptomycin.

[0053] [Preparation of BBB model] The hiMCS-BBB model was created by sequentially combining three immortalized human BBB cell lines in Primesurface96V plates (Sumitomo Bakelite, Tokyo, Japan) (see Figure 1A), but was created by a procedure different from the known procedures for creating BBB spheroids (see, for example, References 5 and 6).

[0054] Specifically, as shown in Figure 1A, on Day 1, HASTR / ci35 cells (1.0 × 10 3 ) and HBPC / ci37 cells (1.0 × 10 3 ) were seeded together into each well of the plate containing endothelial cell basal medium-2 (Lonza, Basel, Switzerland). The medium contained endothelial cell growth medium-2 Single Quots (Lonza), penicillin-streptomycin, and 0.48 mg / mL methylcellulose-400 (Wako). The medium used here is hereinafter referred to as the "spheroid medium".

[0055] Next, on Day 3, HBMEC / ci18 cells (1.0 × 10 3 ) were seeded onto the HASTR / ci35 / HBPC / ci37 spheroid core containing 100 μL of the spheroid medium, and the cells were further cultured for 48 hours. During this period, the cells were left standing at 37°C, 5% CO2 / 95% air. The hiMCS-BBB spheroid was basically constructed at 37°C, but in subsequent studies, the model was constructed at 33°C for comparison purposes in part.

[0056] Thereafter, phase-contrast images of the BBB spheroids were taken using a BZ-X710 fluorescence microscope (Keyence, Osaka, Japan), and the diameters of the BBB spheroids were measured using BZ-H3M analysis application measurement module software (Keyence). Each of the diameters of the BBB spheroids was between 200 and 280 μm. For comparison, a transwell-type BBB model (12 wells) was also created. The transwell-type model was created according to the method described in Reference 4.

[0057] [Detection of Hypoxic Regions in the hiMCS-BBB Model] The Hypoxyprobe-1 kit (Hypoxyprobe, Burlington, MA) was used to examine hypoxic regions in hiMCS-BBB spheroids. The hiMCS-BBB spheroids were incubated in spheroid medium containing pimonidazole (200 μM) at 37 °C for 3 hours. Thereafter, they were embedded in O.C.T. compound (Sakura Finetek, Tokyo, Japan), frozen, and sliced into 14-μm sections. The sections were left standing overnight at 4 °C in phosphate-buffered saline (PBS)(-) containing anti-pimonidazole monoclonal antibody (1:50 dilution), bovine serum albumin (BSA) (Sigma, St. Louis, MO), and 0.1% tween-20. The sections were then reacted with Alexa555-labeled anti-mouse IgG antibody (see Table 2) for 2 hours at room temperature (RT) and observed with a confocal microscope. Fluorescence was detected using a Zeiss LSM780 confocal microscope (Carl Zeiss, Oberkochen, Germany), and fluorescence images were taken at a depth of 30 - 50 μm in the BBB spheroids.

[0058] [Table 2]

[0059] [3D Imaging of the hiMCS-BBB Model] Label-free 3D images of hiMCS-BBB were taken by optical coherence tomography (OCT) using a Cell 3 iMager Estier (SCREEN, Kyoto, Japan). The original OCT images were processed using Cell Visualizer software (SCREEN) to remove medium-derived noise.

[0060] [Cell Localization Analysis] Cells were pre-labeled with CellTracker Green CMFDA Dye (Thermo Fisher Scientific, Waltham, MA), as well as a red and blue fluorescent cell membrane staining kit (Promocell, Heidelberg, Germany). The hiMCS-BBB model with pre-labeled cells was fixed with 4% paraformaldehyde (PFA) at room temperature for 15 minutes and then placed on a slide glass together with Fluoro-KEEPER Antifade Reagent (Nacalai tesque, Kyoto, Japan). Fluorescence was detected using a Zeiss LSM780 confocal microscope, and confocal z-stack images were taken at 2.0 μm intervals up to a depth of 50 μm at most.

[0061] [Isolation of HBMEC / ci18 cells from BBB spheroids] The prepared hiMCS-BBB model (about 500 spheroids) was washed with PBS(-), and then HBMEC / ci18 cells were detached with Accumax (Nacalai tesque) while shaking at 37°C for 20 minutes. After removing undigested cell clumps with a 40-μm cell strainer (BD falcon), the cells were resuspended in PBS(-) containing 0.5% BSA and 2 mM ethylenediaminetetraacetic acid (isolation buffer).

[0062] Pre-labeled with the Human CD31 MicroBead Kit (Miltenyi Biotec, North Rhine-Westphalia, Germany), HBMEC / ci18 cells were sorted using a Midi magnetic-activated cell sorting (MACS) separator (Miltenyi Biotec) and an LS column set. Flow cytometry analysis confirmed that more than 90% of HBMEC / ci18 cells were concentrated in the isolated cells.

[0063] [Total RNA extraction, cDNA synthesis and qPCR] Total RNA extraction and cDNA synthesis were performed according to the methods described in References 2-4. qPCR was performed using the primers shown in Table 3 and cDNA. The amplification efficiency of each qPCR was confirmed to be close to 1. The target mRNA is shown in the following results. The data obtained were acquired using the delta-delta-CT method. In this method, the mRNA level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a normalization control.

[0064]

Table 3

[0065] [Immunocytochemistry (ICC)] ICC was performed using pooled hiMCS-BBB models (about 30 spheroids) according to the methods described in References 2-4. The primary and secondary antibodies used are shown in Table 2. All antibodies were diluted to the specified concentration with CanGetSignal immunostain solution A (TOYOBO, Osaka, Japan). 4’,6-Diamidino-2-phenylindole (DAPI) was used for nuclear counterstaining.

[0066] [Dextran permeability assay] In addition to the BBB spheroids, spheroids without HBMEC / ci18 cells (i.e., spheroids containing only HASTR / ci35 and HBPC / ci37, hereinafter referred to as "ΔBMEC-hiMCS") were prepared for comparison. The spheroids were incubated in spheroid medium containing fluorescein isothiocyanate (FITC)-labeled dextran (5 kDa or 70 kDa, 75 μg / mL each) (Sigma) for 12 hours. After washing three times with PBS(-), the spheroids were fixed with 4% PFA and mounted for microscopic observation. Fluorescence of FITC-dextran that had penetrated into the cells was detected using a Zeiss LSM780 confocal microscope. Fluorescence intensity was quantified using ZEISS Efficient Navigation software (Carl Zeiss).

[0067] [Assay of Transporter Function] The functions of GLUT1 and P-gp in the BBB spheroids were verified by permeability assays using 2-(N-[7-Nitrobenz-2-oxa-1,3-diazol-4-yl]amino)-2-deoxyglucose (2-NBDG; GLUT1 substrate) and rhodamine123 (R123; substrate of P-gp), respectively. The hiMCS-BBB model and ΔBMEC-hiMCS were incubated in spheroid medium containing 2-NBDG (100 μM, Wako) or R123 (1.0 μM, Wako) for 90 minutes. Furthermore, the permeability assay of R123 was also performed in the presence of elacridar (10 nM, P-gp inhibitor) (Wako). As a control, dimethyl sulfoxide (DMSO) with a final concentration of 0.1% was used. Fluorescence intensity within the spheroids was quantified according to the method described above.

[0068] [Assay of Receptor-Mediated Transcytosis (RMT) Function] Low-density lipoprotein receptor-related protein 1 (LRP1)-mediated transcytosis was verified using the ligands of this protein, Angiopep-2 (amino acid sequence: TFFYG GSRGK RNNFK TEEY) and scrambled peptide (amino acid sequence: GNYTS RFERE YGKFN KFGT). These peptides were synthesized by Scrum (Tokyo, Japan) and labeled with 5 / 6-carboxyfluorescein. Either Angiopep-2 (5.0 μM) or scrambled peptide (5.0 μM) was added to the medium of the BBB spheroids and allowed to stand at 37°C for 2 hours.

[0069] Transferrin receptor (TfR)-mediated transcytosis was verified using anti-TfR antibodies (MEM-75 and MEM-189) and their isotype control IgG (IgG1). These were purchased from Thermo Fisher Scientific, labeled with the Alexa Fluoro-488 Monoclonal Antibody Labeling Kit (Thermo Fisher Scientific), and the labeling efficiency was examined with a spectrophotometer. Either control IgG, MEM-75 antibody, or MEM-189 was added to the medium of the BBB spheroids at 10 μg / mL and allowed to stand at 37°C for 3 hours. These assays were also performed at 4°C (suppressive conditions for RMT). The fluorescence intensity within the spheroids was quantified according to the method described above.

[0070] [Tumor necrosis factor α (TNF-α) treatment assay] At the timing of BBB spheroid formation (2 days after seeding of HBMEC / ci18 cells), the medium was changed to serum-free spheroid medium containing TNF-α (20 ng / mL, Peprotech, Rocky hills, NJ). After washing 3 times with PBS(-), the spheroids were treated with TNF-α for 24 hours. Then, HBMEC / ci18 cells were isolated from the BBB spheroids by MACS described above, and RNA was extracted therefrom.

[0071] The TNF-α treatment assay was also performed in the presence and absence of THP-1 cells. Specifically, THP-1 cells (1.0 × 10 3 ) were labeled with a red fluorescent cell membrane staining kit and added to the BBB spheroids 1 day after seeding of HBMEC / ci18 cells. These were co-cultured for 48 hours in serum-free spheroid medium with or without TNF-α (20 ng / mL). Subsequently, fluorescence was detected using a Zeiss LSM780 confocal microscope, and confocal z-stack images were taken at 2.0 μm intervals up to a depth of 50 μm.

[0072] [RNA Sequencing and Data Analysis] Total RNA was extracted from HBMEC / ci18 cells isolated from BBB spheroids using the RNeasy Plus Micro Kit (Qiagen, Hilden, Germany), and a cDNA library was prepared using the NEBNext Ultra RNA Library Prep Kit (New England BioLabs, Beverly, MA). RNA sequencing was performed with a single read length of 60 bp using HiSeq1500 (Illumina, San Diego, CA). The alignment of the obtained data with the human genome (UCSC / mm10 or UCSC / hg19) mapping and known gene information was performed using TopHat (version 2.0.13 with default parameters). Subsequently, gene expression levels were quantified using Cuffdiff (Cufflinks version 2.2.1, default parameters). Gene clustering, heatmap creation, and biological function prediction were performed for all mRNA expression data using Subio platform64 software (version 1.22, Subio, Kagoshima, Japan).

[0073] [Statistical Analysis] Statistical analysis was performed using a statistical software package (Statcel, OMS, Saitama, Japan). Values obtained from spheroid volume, qPCR, permeability analysis, and transcytosis assay were first analyzed by one-way analysis of variance (ANOVA), and then the statistical significance of the difference between two values was verified by Student's t-test.

[0074] [Measurement of medium viscosity] A stirrer M2 (manufactured by Toki Sangyo Co., Ltd.) and 30 mL of the measurement sample were placed in a centrifuge tube. The stirrer M2 was rotated at a rotational speed of 60 rpm, and the viscosity of the medium was measured at room temperature 10 times every 1 - 2 seconds starting 1 minute after the start of rotation. A B-type viscometer (TVB10 type viscometer manufactured by Toki Sangyo Co., Ltd.) was used for viscosity measurement, and the samples used for measurement were EBM-2 comp with a methylcellulose concentration of 0.48 mg / mL (0.048% (w / v)), EBM-2 comp without methylcellulose, and tap water.

[0075] <2>Results [Preparation of multicellular spheroids using three types of immortalized BBB cells] First, as shown in Figure 1A, the hiMCS-BBB model was created in a 96-well V-bottom plate under the condition of 33°C. At the time of seeding, HBPC / ci37 cells and HASTR / ci35 cells formed the core of the spheroid by self-aggregation within 24 hours. Next, HBMEC / ci18 cells were added to the core to create multicellular spheroids (Figure 1A). However, under the condition of 33°C, the size was large, and a hypoxic state was observed in the core region (Figures 1B and 1C). Furthermore, the spheroids under this temperature condition had a low sphericity, and the coating of the spheroids by HBMEC / ci18 cells was also not uniform (Figures 1C and 1E). From these results, it was determined that it was necessary to change the preparation method or conditions to improve spheroid formation.

[0076] [Effect of removal of immortalization signal in the creation of the hiMCS-BBB model] In response to the above, the following modifications were made by utilizing the conditional immortalization characteristics of BBB cells. Conditional immortalized BBB cells can remove the immortalization signal by changing the culture temperature from 33 °C to 37 °C. By removing the immortalization signal, proliferation stops and differentiation occurs simultaneously (References 1-3). Therefore, an attempt was made to culture at 37 °C for spheroid construction. As a result, the culture temperature of 37 °C clearly suppressed spheroid growth (Figure 1B), and the hypoxic region was no longer detected (Figure 1C). Furthermore, the results of 3D imaging analysis showed that the sphericity of the spheroids was higher at 37 °C than at 33 °C (Figure 1D). Under these conditions the spherical structure of the spheroids created was maintained for at least 3 days.

[0077] Since a better morphology of the spheroids was formed at 37 °C, each cell was then labeled with a fluorescent dye and tested for its localization within the spheres. The results showed that HASTR / ci35 cells (red) were located in the center of the spheroids, most of the HBPC / ci37 cells (blue) were located outside the core, and along this, HBMEC / ci18 cells (green) were formed on the surface (Figures 1E and 1F).

[0078] Taking the above results together, the culture condition of 37 °C is considered to be an important factor for forming a complete multicellular spheroid. This complete multicellular spheroid is hereinafter referred to as the "hiMCS-BBB90 model". Here, "90" means the total number of cell clones (18 + 35 + 37). To confirm the stability of the hiMCS-BBB90 model creation method, spheroid models (a total of 480) were created in 5 separate plates and morphologically observed. As a result, it was determined that they were formed without problems in more than 90% of the cases (Figures 7A and 7B). Therefore, the hiMCS-BBB90 model creation method was shown to be stable. Subsequently, the BBB characteristics of this model were analyzed.

[0079] [Gene Expression Profile of the hiMCS-BBB90 Model] HBMEC / ci18 cells were isolated from the hiMCS-BBB90 model by MACS using an anti-CD31 antibody (endothelial cell marker) (Figure 2A), and their gene expression profiles were examined by qPCR. A transwell model was used for comparison. From the results, various BBB-related mRNAs, including intercellular gene mRNAs and drug efflux / uptake transporter mRNAs, were expressed in the HBMEC / ci18 cells of the hiMCS-BBB90 model, and their expression levels were significantly higher than those in the transwell BBB model (Figures 2B and 2C).

[0080] To confirm the protein expression of these BBB-related genes, ICC was performed. HBMEC / ci18 cells in the hiMCS-BBB90 model expressed tight junction proteins (CLDN-5 and zonula occludens 1 [ZO-1]), adherens junction proteins (vascular endothelial (VE) cadherin and β-catenin) (Figure 2D), and transporter proteins (P-gp, BCRP, and GLUT1) (Figure 2E) at significantly higher levels than those in the transwell model. Furthermore, the junction proteins were present at the cell-cell boundary, and P-gp and BCRP were localized to the apical membrane side (medium side).

[0081] [Intercellular Space Permeability Barrier Ability and Transporter Function of the hiMCS-BBB90 Model] Based on the results of the above gene expression analysis, next, the intercellular space permeation barrier ability and transporter function were tested. First, a permeability assay was performed using 70 kDa and 5 kDa FITC-dextran. For comparison, a hiMCS-BBB90 model without HBMEC / ci18 cells (ΔBMEC-hiMCS model) was used (Figure 3A). As a result, it was shown that the permeability of the hiMCS-BBB90 model was significantly lower compared to the ΔBMEC-hiMCS model (0.34 ± 0.04-fold for 70 kDa FITC-dextran, P < 0.01; 0.31 ± 0.07-fold for 5 kDa FITC-dextran, P < 0.01) (Figures 3B and 3C). From these results, it is suggested that the hiMCS-BBB90 model has an intercellular space permeation barrier ability.

[0082] Next, to examine the transporter function of the hiMCS-BBB90 model, permeability assays of 2-NBDG (a substrate of GLUT1) and R123 (a substrate of P-gp) were performed. As a result, the hiMCS-BBB90 model showed higher 2-NBDG permeability (2.00 ± 0.40, P < 0.01) compared to the ΔBMEC-hiMCS model (Figure 3D), which means that GLUT1 is functioning in the hiMCS-BBB90 model. On the other hand, the hiMCS-BBB90 model had significantly lower R123 permeability (0.53 ± 0.11, P < 0.01) compared to the ΔBMEC-hiMCS model (Figure 3E). Furthermore, treatment with elacridar (a P-gp inhibitor) increased the influx of R123 into the hiMCS-BBB90 model (1.48 ± 0.31, P < 0.05) (Figure 3E). From these results, it is suggested that the hiMCS-BBB90 model has P-gp function.

[0083] [RMT Activity of the hiMCS-BBB90 Model] RMT is a BBB function that has attracted attention in recent years. Representative receptors involved in this are TfR and LRP-1 (Reference 7). To analyze the RMT activity of these receptors in the hiMCS-BBB model, their protein expression was first examined by immunocytochemistry. As a result, the hiMCS-BBB90 model expressed TfR and LRP-1 proteins at a higher level compared to the transwell model (Figure 4A).

[0084] Next, a permeability assay for macromolecules was performed. As shown in Figures 4B and 4C, under the condition of 37 °C, angiopep-2 (an LRP-1 binding peptide) permeated the hiMCS-BBB90 model higher than the scrambled peptide (4.54 ± 1.43 times, P < 0.01). However, under the condition of 4 °C where the RMT function was greatly suppressed, the permeability of angiopep-2 decreased to a level equivalent to that of the scrambled peptide. Similarly, the permeation rates of MEM-75 and MEM-189 (anti-TfR antibodies) in the hiMCS-BBB90 model were higher at 37 °C compared to the control IgG (1.66 ± 0.24 times, P < 0.05 and 3.46 ± 0.48 times, P < 0.01, respectively). In addition, as reported previously (Reference 8), the permeation rate of MEM-189 was higher than that of MEM-75. Also, these permeation rates decreased to a level equivalent to that of the control IgG at 4 °C (Figures 4D and 4E). Therefore, these results indicate that the hiMCS-BBB90 model has the functions of LRP-1- and TfR-mediated transcytosis.

[0085] [Response to TNF-α treatment in the hiMCS-BBB90 model] Since the BBB is involved in the formation of brain inflammation (e.g., inflammatory cytokine secretion and lymphocyte mobilization), a test was conducted on how the hiMCS-BBB90 model responds to inflammatory stimuli.

[0086] HBMEC / ci18 cells were isolated from the hiMCS-BBB90 model with or without treatment with the inflammatory cytokine TNF-α, and the mRNA expression profiles of inflammatory mediators (C-X-C motif ligand 8 [CXCL8], interleukin-6 [IL-6], CXCL10, and CC chemokine ligand 2) and endothelial adhesion molecules (E-selectin, ICAM-1, and VCAM-1) were compared. From the results of qPCR, for all the genes analyzed, their mRNA levels were significantly higher in TNF-α-treated HBMEC / ci18 cells than in untreated cells (Figures 5A and 5B). Furthermore, TNF-α-induced ICAM-1 and E-selectin protein expression was confirmed by immunocytostaining (Figure 5C).

[0087] Next, to examine whether lymphocytes adhere to activated HBMEC / ci18 cells upon TNF-α stimulation in the hiMCS-BBB90 model, TNF-α treatment assays were performed in the presence or absence of THP-1 cells (monocyte model cells). As a result, it was shown that TNF-α stimulation promoted the adhesion of THP-1 cells to the surface of the hiMCS-BBB90 model (Figure 5D). Therefore, from these results, it was shown that the hiMCS-BBB90 model responds to TNF-α, suggesting that the hiMCS-BBB90 model can be a model that reproduces BBB inflammation.

[0088] [Special Roles of HBPC / ci37 Cells and HASTR / ci35 Cells in the Formation of the hiMCS-BBB90 Model] To obtain new insights into how brain pericytes and astrocytes affect the formation of the hiMCS-BBB90 model, three types of hiMCS-BBB90 models were created, namely, · EPA: Three-cell co-culture model (hiMCS-BBB90 model), · EP0: Co-culture model without HASTR / ci35 cells, · E0A: Co-culture model without HBPC / ci37 cells (Figure 6A).

[0089] First, to determine whether HBPC / ci37 cells and HASTR / ci35 cells contribute to the expression of BBB function in the hiMCS-BBB model, a 70 kDa FITC-dextran permeability assay was performed. As a result, the permeability in the EP0 and EA0 models was higher compared to that in the EPA model (1.35 ± 0.22-fold, P < 0.05 and 1.88 ± 0.21-fold, P < 0.01, respectively), indicating that both HBPC / ci37 cells and HASTR / ci35 cells are required for the expression of BBB function in the hiMCS-BBB90 model (see Figures 6B and 6C).

[0090] Next, HBMEC / ci18 cells were isolated from each model and their gene expression profiles were analyzed by RNA sequencing. As a result, it was revealed that the expression of 182 genes in EP0 or 104 genes in E0A fluctuated compared to the EPA model, meaning that the genes belonging to each group are regulated by HASTR / ci35 cells or HBPC / ci37 cells (Figure 6D).

[0091] Furthermore, it was shown that 22 mRNAs were highly expressed only in the EPA model (Figures 6D and 6E). That is, these are considered to be genes whose expression is induced only when both HASTR / ci35 cells and HBPC / ci37 cells are present. qPCR analysis of COL11A1, ZNF287, BEND6, NTM, DCN, and BEX1 mRNAs included in these 22 genes showed that, as in the RNA sequencing results, their expression levels were highest in the HBMEC / ci18 cells of the EPA model compared to the EP0 and E0A models (Figure 6F). These results indicate that in the expression of BBB function in the hiMCS-BBB90 model, HASTR / ci35 cells and HBPC / ci37 cells have both specific and cooperative roles.

[0092] [Viscosity of the medium] The measurement results of the viscosity of EBM-2 comp with a methylcellulose concentration of 0.48 mg / mL, EBM-2 comp without methylcellulose, and tap water are shown in Fig. 8.

[0093] <3>Discussion The inventors newly established an MCS-BBB model (i.e., "hiMCS-BBB90 model") using human conditionally immortalized cells. The model has a three-layer structure and has BMEC, pericytes, and astrocytes in this order from the outside to the inside. Through this example, the inventors demonstrated that the model has validity as a BBB model and is further applicable to various BBB studies.

[0094] Since dextran, a cell permeability marker, hardly enters the spheroid in this model, it is considered that HBMEC / ci18 cells uniformly cover the spheroid core and form cell junctions to form a monolayer without gaps. This layer structure is considered to be a platform that enables appropriate cell-cell communication necessary for the expression of BBB functions. This is consistent with the fact that the mRNA and protein expression levels of BBB-related genes in this model were significantly higher than those found in the transwell model. Therefore, it is considered that a special structure such as the hierarchical spheroid exemplified in the present invention is required for HBMEC / ci18 cells to exhibit high performance as a BBB.

[0095] Furthermore, in this example, it was demonstrated that the hiMCS-BBB90 model has transporter functions. In HBMEC / ci18 cells, P-gp (and BCRP) is localized on the apical membrane as observed in vivo, and thus the influx of its substrate (R123) into spheroids (i.e., into the brain) is considered to be effectively restricted. On the other hand, such localization was not observed for GLUT1 as in vivo, and thus the transcellular transport of its substrate (2-NBDG) is considered to be promoted. Therefore, in the hiMCS-BBB90 model, BBB-related transporters are functionally expressed with the same characteristics as in vivo, and thus it is considered that the transport of compounds between the inside and outside of spheroids is promoted or restricted by this.

[0096] In recent years, RMT has attracted attention as a route for drug delivery to the brain, and Angiopep-2 and TfR receptor-binding antibodies are also promising drug carriers for drug delivery to the brain (Reference 7). The fact that RMT activities related to these were observed in the hiMCS-BBB90 model is of great significance in that it indicates that this model can contribute not only to the basic BBB function but also to the development of therapeutic drugs for brain diseases.

[0097] The BBB model created by the present inventors is the first spheroid-type BBB model using human conditionally immortalized cells. Since immortalized BBB cells have excellent proliferative ability, the hiMCS-BBB90 model can be constructed without limit. Furthermore, since the hiMCS-BBB90 model does not require complicated operations or specific culture devices and can be easily prepared, it can reduce the burden on the user in terms of time, labor, and economy. Due to these features, large-scale experiments can be conducted using this model in BBB research.

[0098] Furthermore, it was demonstrated that the hiMCS-BBB90 model is useful for various BBB studies by constructing an inflammation model and revealing new findings in BBB biology.

[0099] Inflammatory cytokines are known to play important roles in the occurrence and progression of neuroinflammation through various actions such as mobilizing immune cells to the BBB. The HBMEC / ci18 cells of the hiMCS-BBB90 model respond to TNF-α, increase the expression levels of cytokines / chemokines together with adhesion molecules, and furthermore, it has been clarified that THP-1 cells are mobilized there through this. Therefore, it is considered that the hiMCSC-BBB model can reproduce the inflammatory response reaction in the BBB. From this, it is considered that this model will be a valuable tool for understanding the molecular mechanism of BBB inflammation and exploring new drug targets.

[0100] Furthermore, the hiMCS-BBB90 model is expected to become a model useful for elucidating the molecular basis of BBB formation. It has been reported so far that both astrocytes and pericytes are required for the formation of the BBB, but their roles have not been fully elucidated. The inventors clarified that these cells have unique roles and also exert cooperative effects on the expression of BBB functions in this model.

Industrial Applicability

[0101] The hiMCS-BBB90 model of the present invention retains basic BBB characteristics and at the same time has excellent scalability and ease of handling. This model is useful for evaluating the modalities of new CNS drugs and for research aiming to elucidate the formation and breakdown mechanisms of the BBB. Therefore, the BBB model according to the present invention is expected to become a new research platform for accelerating various human BBB studies including CNS drug development.

[0102] References 1. Kamiichi A, Furihata T, Kishida S, Ohta Y, Saito K, Kawamatsu S, Chiba K. Establishment of a new conditionally immortalized cell line from human brain microvascular endothelial cells: a promising tool for human blood - brain barrier studies. Brain Res. 2012;1488:113 - 22. 2. Furihata T, Ito R, Kamiichi A, Saito K, Chiba K. Establishment and characterization of a new conditionally immortalized human astrocyte cell line. J Neurochem. 2016 Jan;136(1):92 - 105. 3. Umehara K, Sun Y, Hiura S, Hamada K, Itoh M, Kitamura K, Oshima M, Iwama A, Saito K, Anzai N, Chiba K, Akita H, Furihata T. A New Conditionally Immortalized Human Fetal Brain Pericyte Cell Line: Establishment and Functional Characterization as a Promising Tool for Human Brain Pericyte Studies. Mol Neurobiol. 2018;55(7):5993 - 6006. 4. Ito R, Umehara K, Suzuki S, Kitamura K, Nunoya KI, Yamaura Y, Imawaka H, Izumi S, Wakayama N, Komori T, Anzai N, Akita H, Furihata T. A Human Immortalized Cell-Based Blood-Brain Barrier Triculture Model: Development and Characterization as a Promising Tool for Drug-Brain Permeability Studies. Mol Pharm. 2019;16(11):4461-4471. 5. Urich E, Patsch C, Aigner S, Graf M, Iacone R, Freskgard PO. Multicellular self-assembled spheroidal model of the blood brain barrier. Sci Rep. 2013;3:1500. 6. Cho CF, Wolfe JM, Fadzen CM, Calligaris D, Hornburg K, Chiocca EA, Agar NYR, Pentelute BL, Lawler SE. Blood-brain-barrier spheroids as an in vitro screening platform for brain-penetrating agents. Nat Commun. 2017 Jun 6;8:15623. 7. Preston JE, Joan Abbott N, Begley DJ. Transcytosis of macromolecules at the blood-brain barrier. Adv Pharmacol. 2014;71:147-63. 8.Sade H, Baumgartner C, Hugenmatter A, Moessner E, Freskgard PO, Niewoehner J, A human blood-brain barrier transcytosis assay reveals antibody transcytosis i nfluenced by pH-dependent receptor binding. PLoS ONE 9(4) (2014) 96340.

Claims

1. (i)A first seeding step of seeding a human conditionally immortalized astrocyte and a human conditionally immortalized pericytes in a culture medium; (ii)A first co-culture step of co-culturing the seeded human conditionally immortalized astrocyte and the human conditionally immortalized pericytes to obtain a co-culture containing two types of cells; (iii)A second seeding step of seeding human conditionally immortalized brain microvascular endothelial cells in a culture medium containing the co-culture containing the two types of cells; (iv)A second co-culture step of co-culturing the co-culture containing the two types of cells and the human conditionally immortalized brain microvascular endothelial cells to obtain a multicellular three-dimensional blood-brain barrier model A method for manufacturing a multicellular three-dimensional blood-brain barrier model having a shape of an ellipsoid, a spheroid or a sphere, comprising the above steps.

2. The manufacturing method according to claim 1, wherein the culture medium in the first seeding step (i) contains a thickening agent.

3. The manufacturing method according to claim 1 or 2, wherein the first co-culture step (ii) and the second co-culture step (iv) are performed within a range of 35 to 39 °C.

4. The manufacturing method according to any one of claims 1 to 3, wherein in the first co-culture step (ii), the co-culture containing the two types of cells forms a cell mass in an ellipsoidal shape, a spheroidal shape or a spherical shape.

5. The manufacturing method according to claim 4, wherein the longest diameter among the three diameters of the ellipsoid, the spheroid or the sphere is within a range of 180 to 350 μm.

6. The manufacturing method according to claim 4 or 5, wherein in the second co-culture step (iv), the human conditionally immortalized brain microvascular endothelial cells cover the periphery of the cell mass formed in the first co-culture step (ii) to form a multicellular three-dimensional blood-brain barrier model.

7. The manufacturing method according to any one of claims 1 to 6, wherein the longest diameter among the three diameters of the ellipsoid, the spheroid or the sphere of the multicellular three-dimensional blood-brain barrier model is within a range of 180 to 350 μm.

8. The manufacturing method according to any one of claims 2 to 7, wherein the concentration of the thickening agent in the culture medium in the first seeding step (i) is 0.1 to 20 mg / mL.

9. The manufacturing method according to any one of claims 1 to 8, wherein the seeding in the second seeding step (iii) is performed 12 to 72 hours after the first seeding step (i).

10. The manufacturing method according to any one of claims 1 to 9, wherein in the second co-culture step (iv), the co-culture is performed for 12 to 72 hours.

11. The manufacturing method according to any one of claims 1 to 10, wherein at least one of the steps (i) to (iv) is carried out in a container having a bottom with a substantially V-shaped or V-shaped configuration.

12. A multicellular three-dimensional blood-brain barrier model manufactured by the manufacturing method according to any one of claims 1 to 11.

13. (A) A cell mass containing human conditionally immortalized astrocytes and human conditionally immortalized pericytes, (B) A layer of human conditionally immortalized brain microvascular endothelial cells covering the periphery of the cell mass A multicellular three-dimensional blood-brain barrier model that is ellipsoidal, spheroidal or spherical and includes the above.

14. The multicellular three-dimensional blood-brain barrier model according to claim 13, wherein in the cell mass (A), human conditionally immortalized astrocytes are located inside and human conditionally immortalized pericytes are located outside.

15. The multicellular three-dimensional blood-brain barrier model according to claim 13 or 14, wherein the longest diameter among the three diameters of the ellipsoid, spheroid or sphere of the multicellular three-dimensional blood-brain barrier model is in the range of 180 to 350 μm.

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