JP7702137B2 - Hierarchical spheroid blood-brain barrier model using immortalized human cells and its manufacturing method - Google Patents

Description

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

The central nervous system (CNS) is physiologically isolated from the periphery by the blood-brain barrier (BBB). The BBB is a unique feature of the cerebrovascular system characterized by intercellular tight junctions (tight junctions and adherens junctions) and efflux transporters (P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP)) that limit the movement of endogenous and exogenous compounds, including drugs, from the blood to the brain (Non-Patent Document 1). Thus, the BBB essentially functions as a protective barrier against blood-borne substances that may be involved in CNS diseases (Non-Patent Document 1), but at the same time, it also represents an obstacle to the development of effective drugs for CNS diseases (Non-Patent Document 1).

However, on the other hand, the BBB has certain substance influx transport systems, such as glucose transporter 1 (GLUT1) and receptor-mediated transcytosis (RMT). A representative example of a receptor involved in RMT is the transferrin receptor (TfR), which is required for the transport of transferrin to the brain, but also transports macromolecules that bind to the receptor, such as anti-transferrin antibodies. Thus, RMT is considered a promising CNS delivery route for therapeutic macromolecules (Non-Patent Document 2).

The BBB also restricts the movement of immune cells into the CNS, and therefore plays an essential role in maintaining low-level immune surveillance in the brain (Non-Patent Document 3). However, the entry of immune cells into the CNS has been observed in various diseases in which the BBB is disrupted, such as multiple sclerosis and stroke, and this event plays an important role in the progression of the disease (Non-Patent Document 4).

For these reasons, the BBB is an important research target for understanding CNS diseases, identifying therapeutic approaches, and developing drugs for CNS diseases.

Facilitating such research will require extensive studies to explore the molecular mechanisms behind the development and maintenance of the BBB and to test various drug candidates and findings to advance the treatment of CNS disorders.

Because the in vivo BBB is formed by brain microvascular endothelial cells (BMEC) covered by pericytes and astrocytes (Non-Patent Document 1), in vitro BBB models (Non-Patent Document 5) are constructed using BMEC or a combination of these three cell types. Conventional in vitro BBB models have used a transwell culture system. In addition to being easy to prepare, this system has many experimental advantages, such as the ability to easily analyze intercellular junctions and drug permeability of the BBB.

However, because the transwell culture system lacks an in vivo microenvironment (i.e., cells live in a two-dimensional environment and there is no direct communication between each of the three cell types), its functions are not fully mature, resulting in a low level of BBB function and the risk of false negative/positive results in certain experiments.

In contrast, three-dimensional culture methods such as BBB on a chip, organoids and spheroids (Non-Patent Document 5) have attracted much attention as promising approaches to develop BBB models with better functions. These new systems aim to more faithfully mimic the in vivo BBB and are therefore expected to achieve higher levels of BBB function than ever before. However, in order for such models to be implemented in the field of BBB research and drug discovery and contribute to its promotion, they must be adaptable and convenient for various experiments.

Abbott NJ, Patabendige AA, Dolman DE, Yusof SR, Begley DJ. Structure and function of the blood-brain barrier. Neurobiol Dis. 2010;37(1):13-25. Preston JE, Joan Abbott N, Begley DJ. Transcytosis of macromolecules at the blood-brain barrier. Adv Pharmacol. 2014;71:147-63. Shechter R, London A, Schwartz M, Orchestrated leukocyte recruitment to immune-privileged sites: absolute barriers versus educational gates. Nat. Rev. Immunol. 13(3) (2013) 206-18. Lopes Pinheiro MA, Kooij G, Mizee MR, Kamermans A, Enzmann G, Lyck R, Schwaninger M, Engelhardt B, de Vries HE, Immune cell trafficking across the barriers of the central nervous system in multiple sclerosis and stroke. Biochim. Biophys. Acta. 1862(3) (2016) 461-71. Kaisar MA, Sajja RK, Prasad S, Abhyankar VV, Liles T, Cucullo L. New experimental models of the blood-brain barrier for CNS drug discovery. Expert Opin Drug Discov. 2017;12(1):89-103.

The present invention aims to provide a hierarchical spheroid blood-brain barrier model using human immortalized cells and a method for producing the same.

In light of the growing interest in three-dimensional (3D) BBB models, the present inventors have succeeded in establishing a hierarchical spheroid blood-brain barrier model using human conditionally immortalized cells. As previously reported by the present inventors (References 1-4), the immortalized cells used have high proliferation capacity and cryopreservability, and can be used in a transwell-type BBB model that can distinguish between high and low BBB-permeable drugs. The method for producing the model presented in this specification allows the construction of a three-dimensional BBB model with further improved BBB characteristics, which can serve as a useful research tool for various BBB studies.

That is, the present invention relates to:
[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 the two types of cells;
(iii) a second seeding step of seeding human conditionally immortalized brain microvascular endothelial cells into a medium containing a 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 with the human conditionally immortalized brain microvascular endothelial cells to obtain a multicellular three-dimensional blood-brain barrier model;
[2] The method according to [1], wherein the medium of the first seeding step (i) contains a thickening agent;
[3] The method according to [1] or [2], wherein the first co-culture step (ii) and the second co-culture step (iv) are carried out at a temperature within a range of 35 to 39°C;
[4] The method according to any one of [1] to [3], wherein in the first co-culture step (ii), a co-culture containing the two types of cells forms ellipsoidal, spheroidal, or spherical cell masses;
[5] The manufacturing method according to [4], wherein the longest diameter among three diameters of the ellipsoid, spheroid, or sphere is within a range of 180 to 350 μm;
[6] The method according to [4] or [5], wherein in the second co-culture step (iv), the human conditionally immortalized brain microvascular endothelial cells surround the cell mass 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 an ellipsoid, spheroid, or spherical shape,
The method according to any one of [1] to [6], wherein the longest diameter of the three diameters of the ellipsoid, spheroid, or sphere of the multicellular three-dimensional blood-brain barrier model is within the range of 180 to 350 μm;
[8] The method according to any one of [2] to [7], wherein the concentration of the thickener in the medium in the first seeding step (i) is 0.1 to 20 mg/mL;
[9] The method according to any one of [1] to [8], wherein the second seeding step (iii) is carried out 12 to 72 hours after the first seeding step (i);
[10] The method according to any one of [1] to [9], wherein in the second co-culture step (iv), the co-culture is carried out for 12 to 72 hours;
[11] The method according to any one of [1] to [10], wherein at least one of the steps (i) to (iv) is carried out in a vessel having a substantially V-shaped or V-shaped bottom;
[12] A multicellular three-dimensional blood-brain barrier model produced by the production 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 surrounding the cell mass;
[14] The multicellular three-dimensional blood-brain barrier model according to [13], wherein the cell mass (A) has human conditionally immortalized astrocytes positioned on the inside and human conditionally immortalized pericytes positioned on the 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 ellipsoidal, spheroidal, or spherical;
[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 within the range of 180 to 350 μm.

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

Figure 1A shows an overview of the preparation of a hierarchical spheroid blood-brain barrier model (hereinafter referred to as the "hiMCS-BBB90 model"). The hiMCS-BBB90 model was prepared using either the 33°C or 37°C protocol. The 33°C protocol was used only in Figure 1 for comparison. Figure 1B shows the diameter of the hiMCS-BBB90 model measured over 6 days. Each value is the mean (and standard deviation [SD]) of values obtained from 10 spheroids in one cell culture experiment. (**P < 0.01, one-way analysis of variance (ANOVA) followed by Student's t test.) Figure 1C shows hypoxic regions in the hiMCS-BBB90 model detected by pimonidazole staining (red), with 4',6-diamidino-2-phenylindole (DAPI) used as nuclear counterstain (blue). FIG. 1D shows the results of analysis of the three-dimensional structure of the hiMCS-BBB90 model using an OCT imaging system. Figure IE is a fluorescence microscopy image of a monolayer of human conditionally immortalized brain capillary endothelial cells clone 18 (hereafter referred to as "HBMEC/ci18") in the hiMCS-BBB90 model. HBMEC/ci18 cells were pre-labeled with a green fluorescent cell labeling reagent. FIG. 1F shows the results of a fluorescence microscopy analysis of the localization of each cell type (green: HBMEC/ci18 cells, blue: HBPC/ci37 cells, and red: HASTR/ci35 cells) in the hiMCS-BBB90 model created using the 37°C protocol. This experiment was repeated three times independently, and representative photographs are shown in the figure. HBPC/ci37 refers to human conditionally immortalized brain pericyte clone 37, and HASTR/ci35 refers to human conditionally immortalized brain astrocyte clone 35. FIG. 2A shows the HBMEC/ci18 sorting scheme from the hiMCS-BBB90 model. Figure 2B shows the results of qPCR testing of cell-cell junction gene mRNA expression levels in HBMEC/ci18 cells derived from the hiMCS-BBB90 model or the transwell model. Target mRNAs are claudin-5 [CLDN-5], zonula occludens 1 [ZO-1], vascular endothelial (VE)-cadherin, and β-catenin. Values were normalized using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels, and comparative data are presented as relative expression ratios (mean and standard deviation [SD] of triplicate mRNA quantifications) from a single cell culture experiment (hiMCS/transwell, where transwell = 1). Each mRNA quantification was performed using duplicate samples (**P<0.01, *P<0.05; one-way analysis of variance (ANOVA) followed by Student's t-test). FIG. 2C shows the results of qPCR testing of transporter gene mRNA expression levels in HBMEC/ci18 cells in the transwell model and hiMCS-BBB90 model. The target mRNAs are multidrug-resistance (MDR)-1, breast cancer resistance protein (BCRP), glucose transporter (GLUT1), LDL receptor-related protein (LRP)-1, major facilitator superfamily domain (MFSD) 2A, L-type amino acid transporter (LAT) 1, multidrug resistance protein (MRP)-4, and monocarboxylate transporter (MCT) 8. Values were normalized using GAPDH mRNA levels, and comparative data are presented as relative expression ratios (mean and standard deviation [SD] of triplicate mRNA quantifications) from a single cell culture experiment (hiMCS/transwell, where transwell=1). Each mRNA quantification was performed using duplicate samples (**P<0.01, *P<0.05; one-way analysis of variance (ANOVA) followed by Student's t-test). Figure 2D shows the results of immunocytochemical staining (ICC) performed to confirm the protein expression of cell junction genes. HBMEC/ci18 cells in the hiMCS-BBB90 model expressed tight junction proteins (CLDN-5 and ZO-1) and adherens junction proteins (VE-cadherin and β-catenin) at significantly higher levels than those in the transwell model. Figure 2E shows the results of ICC performed to confirm the protein expression of BBB transporter genes. HBMEC/ci18 cells in the hiMCS-BBB90 model expressed transporter proteins (P-gp, BCRP, and GLUT1) at significantly higher levels than those in the Transwell model. FIG. 3A shows the hiMCS-BBB90 model and spheroids without endothelial cells (ie, spheroids containing only HASTR/ci35 and HBPC/ci37 cells) (hereinafter referred to as "ΔBMEC-hiMCS-BBB90 model" or simply "ΔBMEC"). FIG. 3B shows the results of microscopic examination of the permeability of fluorescein isothiocyanate (FITC)-labeled dextran (70 kDa, 75 μg/mL) after 12 hours of incubation. FIG. 3C shows the permeability of FITC-dextran (5 kDa, 75 μg/mL) examined by microscopy after 12 hours of incubation. FIG. 3D shows the results of microscopic examination of the permeability of 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose (2-NBDG, 300 μM) after 30 minutes of incubation. Figure 3E shows the results of R123 permeability (1.0 μM) after 90 min of incubation in the presence or absence of elacridar (10 nM, a P-gp inhibitor) examined by fluorescence microscopy. All experiments (Figures 3B-3E) show representative images of intraspheroid fluorescence, and the intraspheroid fluorescence signal intensity was determined by Zeiss Efficient Navigation software. Relative signal intensity ratios (ΔBMEC/hiMCS, where ΔBMEC=1) are shown using the mean and SD obtained from 10 spheroids in one permeability experiment. (**P<0.01; one-way analysis of variance (ANOVA) followed by Student's t-test). Figure 4A shows the protein expression of LRP-1 and TfR in the hiMCS-BBB90 model examined by ICC. Nuclear counterstaining (blue) was performed with 4',6-diamidino-2-phenylindole (DAPI). For comparison, the transwell BBB model was used. FIG. 4B shows the results of a permeability assay using fluorescently labeled scrambled peptide and LRP1 ligand peptide Angiopep-2 (each 5.0 μM) at 37° C. or 4° C. (where RMT activity is thought to be largely suppressed). The permeability of Angiopep-2 was higher than that of the scrambled peptide. The permeability of Angiopep-2 also decreased to the same level as that of the scrambled peptide at 4° C. These results indicate that the hiMCS-BBB90 model has the function of LRP-1-mediated transcytosis. FIG. 4C shows the results of the permeability assay shown in FIG. 4B as numerical values of fluorescence intensity. FIG. 4D shows the results of a permeability assay using fluorescently labeled control (CTRL) IgG and anti-transferrin receptor antibodies (MEM-75 and MEM-189) (10 μg/mL) at 37° C. or 4° C. The permeability of MEM-189 and MEM-75 was higher than that of CTRL IgG, and that of MEM-189 was higher than that of MEM-75. The permeability of MEM-189 and MEM-75 decreased to the same level as that of CTRL IgG at 4° C. These results indicate that the hiMCS-BBB90 model has the function of LRP-1 and TfR-mediated transcytosis. Note that FIGS. 4B and 4D show representative images of intraspheroid fluorescence analyzed 3 hours after peptide or antibody was added to each well. Figure 4E shows the results of the permeability assay shown in Figure 4D as numerical values of fluorescence intensity. Note that in Figures 4C and 4E, two independent experiments were performed, and the signal intensity is shown as the mean (and SD) of the relative values obtained from 10 or 5 spheroids in one experiment (Angiopep-2/scrambled or MEM/CTRL IgG, where scrambled or CTRL IgG at 37°C = 1) (**P<0.01; one-way analysis of variance (ANOVA) followed by Student's t-test). Figure 5A shows the results of qPCR testing of mRNA expression levels of inflammatory cytokines/chemokines and endothelial adhesion molecules in isolated HBMEC/cil8 cells exposed to TNF-α (20 ng/mL) for 24 hours in the hiMCS-BBB90 model. Target mRNAs are CXCL8, IL-6, CXCL10, CCL2, E-selectin, ICAM-1 and VCAM-1. Values were normalized using GAPDH mRNA levels and comparative data are expressed as the mean and SD of triplicate mRNA quantification values from one cell culture experiment as fold difference (hiMCS/transwell, where transwell=1). The mRNA quantification experiments were performed using duplicate samples (**P<0.01, *P<0.05; one-way ANOVA followed by Student's t-test). (B) ICAM-1 and E-selectin protein expression was examined by ICC in the hiMCS-BBB90 model with or without TNF-α treatment. 4',6-diamidino-2-phenylindole (DAPI) was used for nuclear counterstaining (blue). The transwell BBB model was used for comparison. FIG. 5C shows the results of co-culturing THP-1 cells (a cell line often used as a monocyte model in immune studies) labeled with a red fluorescent cell labeling reagent with the hiMCS-BBB90 model for 48 hours in the absence (CTRL, sterile water) or presence of TNF-α (20 ng/mL). Serum-free medium was used in this assay. THP-1 cells recruited to the surface of the hiMCS-BBB90 were observed under a fluorescent microscope. The results showed that TNF-α stimulation promoted the adhesion of THP-1 cells to the surface of the hiMCS-BBB90 model. Note that in FIGS. 5B and 5C, the experiment was repeated two or three times, and representative images are shown. Figure 6A shows three types of hiMCS-BBB90 models. EPA, EP0 and E0A models represent hiMCS-BBB90, hiMCS-BBB90 without HASTR/ci35, and hiMCS-BBB90 without HBPC/ci37, respectively. The localization of HBMEC/ci18 cells is shown by green fluorescence. Figure 6B shows the results of testing the permeability of FITC-dextran (70 kDa) by the microscope shown in Figure 3. The figure shows a representative image from two independent assays. Figure 6C is a graph of the fluorescence signal intensity of Figure 6B. The signal intensity of intraspheroid fluorescence was measured by Zeiss Efficient Navigation software. Signal intensity ratios (EP0 or E0A/EPA, where EPA=1) were calculated and shown as the mean (and SD) of values obtained from one of two independent cell culture experiments with 10 spheroids. (**P<0.01; one-way analysis of variance (ANOVA) followed by Student's t-test). Figure 6D shows a table in which genes that showed characteristic changes in expression were classified into three categories by RNA sequencing using HBMEC/ci18 cells isolated from each hiMCS-BBB90 model: genes that were upregulated solely by astrocytes ("Up by astrocytes"), genes that were upregulated solely by pericytes ("Up by pericytes"), and genes that were upregulated only in the presence of both astrocytes and pericytes ("Up by tri-culture"). FIG. 6E is a heatmap showing 22 expression profiles classified as "Up by tri-culture" as confirmed by qPCR. Figure 6F shows the expression levels of genes classified as "Up by tri-culture" (COL11A1, ZNF287, BEND6, NTM, DCN, and BEX1) confirmed by qPCR. Values were normalized using GAPDH mRNA levels, and comparative data are expressed as fold differences (EP0 or E0A/EPA, where EPA=1) using the mean and SD of triplicate mRNA quantification experiments from one cell culture experiment. The mRNA quantification experiments were performed with duplicate samples (**P<0.01, *P<0.05; one-way analysis of variance (ANOVA) followed by Student's t-test). Figure 7A shows the shape of the hiMCS-BBB90 model constructed in a V-shaped well. The left side shows an example of a normal shape, and the right side shows an example of an abnormal shape. Only the normal shape was used in the experiment. FIG. 7B shows the results of constructing hiMCS-BBB90 models in all wells of five 96 V-shaped well plates, observing their shapes, and calculating the percentage of wells that showed normal morphology in each plate. FIG. 8 is a table showing measured values of viscosity of the medium.

The present invention is described in more detail below.

A 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.

That is, the method for producing a multicellular three-dimensional blood-brain barrier model according to the present invention comprises the steps of:
(i) a first seeding step of seeding human conditionally immortalized astrocytes and human conditionally immortalized pericytes in a culture 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 into a medium containing a 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 with the human conditionally immortalized brain microvascular endothelial cells to obtain a multicellular three-dimensional blood-brain barrier model. Each step is described in detail below.

<First seeding step>
In this step, human conditionally immortalized astrocytes and human conditionally immortalized pericytes are seeded in a medium. Human conditionally immortalized astrocytes and human conditionally immortalized pericytes are seeded together in a medium, and in a subsequent step, they are co-cultured.

The ratio of the number of human conditionally-immortalized astrocytes to the number of human conditionally-immortalized pericytes seeded in this process may be appropriately adjusted within a range that allows the human conditionally-immortalized astrocytes and the human conditionally-immortalized pericytes to form cell masses by self-aggregation in the subsequent co-culture process.

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 the subsequent co-culture.

The thickener used is one that imparts an appropriate viscosity to the medium and does not impair the function of the cells being cultured. Examples of thickeners 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, keratosulfuric acid, succinoglucan, caronic acid, chitin, chitosan, carboxymethyl chitin, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, alkyl-modified carboxyvinyl polymer, and bentonite. The thickener may be a combination of two or more of these. In one embodiment, the thickener is methylcellulose.

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

The concentration of the thickening agent in the medium in step (i) is, for example, 0.1 to 20 mg/mL. When the thickening agent 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, even 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 in step (i) is 0.48 mg/mL.

The medium used in this step 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, even 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. The viscosity of the medium is determined by measuring the value 1 minute after the start of stirring at a rotation speed of 60 rpm using a B-type viscometer (Toki Sangyo Co., Ltd. "TVB10" viscometer) equipped with an M2 rotor (Toki Sangyo Co., Ltd.) as the stirring bar. Specifically, the measured value is measured 10 times every 1 to 2 seconds from 1 minute after the start of stirring, and the average of the measured values is determined as the viscosity.

<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 the two types of cells.

The co-culture containing the two types of cells obtained in this process forms a cell mass. Specifically, human conditionally immortalized astrocytes and human conditionally immortalized pericytes form a cell mass by self-assembly. The cell mass has, for example, an ellipsoidal shape, preferably a spheroidal or spherical shape.

In this specification, an "ellipsoid" is a three-dimensional shape obtained by expanding an ellipse into three dimensions, and its surface is a quadratic curved surface. If the half lengths of the diameters of an ellipsoid in the x-axis, y-axis, and z-axis directions are a, b, and c, respectively, an ellipsoid in which any two of a, b, and c are equal is a body of revolution obtained by rotating an ellipse around its axis, and both an ellipsoid whose major axis is the axis of revolution (a prolate spheroid) and an ellipsoid whose minor axis is the axis of revolution (an oblate spheroid) are referred to as "ellipsoids of revolution". An ellipsoid in which a, b, and c are all equal is a "sphere". In this specification, the term "ellipsoid" includes both "ellipsoid of revolution" and "sphere".

The longest diameter of the three diameters of the ellipsoid, spheroid, or sphere is, for example, within 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 occurrence of hypoxic regions in the cell mass.

In the cell mass formed in this process, 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 (see Figure 1A).

In this step, the co-culture is carried out at, for example, 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 aggregates increases, and the core region where the cell aggregates form becomes hypoxic. On the other hand, by carrying out the co-culture in the above-mentioned temperature range, the action of the immortalization signal is weakened, cell proliferation is suppressed, differentiation is promoted, and cell aggregates of suitable size can be obtained, and cell aggregates with good sphericity can be obtained. Furthermore, the cells have temperature resistance within the above-mentioned temperature range. In one embodiment, the co-culture is carried out at 36 to 38°C. In another embodiment, the co-culture is carried out at 36.5 to 37.5°C.

As described later in the Examples, in a specific experiment, the present inventors succeeded in obtaining cell aggregates of suitable size by performing co-culture at 37°C, a temperature condition that suppresses proliferation while promoting differentiation. Until now, it has been reported that the immortalization signal of cells is completely released at 39°C, but it has been shown that differentiation can be promoted by weakening the action of the immortalization signal even at 37°C. In this respect, the production method of the present invention can be said to be a method with higher versatility in that differentiation can be promoted at a lower temperature. In addition, it was found that the sphericity was improved by performing co-culture at 37°C. An example of the co-culture condition in this step is 37°C, 5% _CO2 /95% air.

In this step, 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.

<Second seeding step>
In this step, human conditionally immortalized brain microvascular endothelial cells are seeded in a medium containing a co-culture containing the two types of cells obtained in the previous step. The seeding of human conditionally immortalized brain microvascular endothelial cells is performed, 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 after seeding human conditionally immortalized astrocytes and human conditionally immortalized pericytes in the first seeding step. In a specific embodiment, the seeding of human conditionally immortalized brain microvascular endothelial cells is performed 48 hours after seeding human conditionally immortalized astrocytes and human conditionally immortalized pericytes.

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

In this step, the co-culture of the cells is carried out at, for example, 34 to 40°C, preferably 35 to 39°C. By carrying out the co-culture in the above-mentioned temperature range, it is possible to weaken the action of the immortalization signal and suppress cell proliferation while promoting differentiation, thereby obtaining cell aggregates of suitable size and cell aggregates with good sphericity. Furthermore, the cells have temperature resistance within the above-mentioned temperature range. In one embodiment, the co-culture in 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.

As described later in the Examples, in specific experiments, the inventors found that by performing co-culture at 37°C, a temperature condition that suppresses proliferation while promoting differentiation, cell aggregates of an appropriate size can be obtained, the sphericity is improved, and the human conditionally immortalized brain microvascular endothelial cells can uniformly cover the periphery of the co-culture containing the two types of cells.

In this step, the human conditionally immortalized brain microvascular endothelial cells coat the periphery of the cell clusters of the co-culture containing the 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 coat the periphery of the cell clusters of the co-culture containing the two types of cells formed in the first co-culture step in a monolayer to form the model (FIG. 1A).

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 culture medium, and then human conditionally immortalized brain microvascular endothelial cells are seeded, thereby obtaining a hierarchical spheroid blood-brain barrier model in which the cell mass formed from human conditionally immortalized astrocytes and human conditionally immortalized pericytes is surrounded by human conditionally immortalized brain microvascular endothelial cells.

The multicellular three-dimensional blood-brain barrier model thus formed has, for example, an ellipsoidal shape, preferably a spheroid or a sphere. The longest diameter of the three diameters of the ellipsoid, spheroid or sphere of the multicellular three-dimensional blood-brain barrier model is, for example, within 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.

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 within a range in which the human conditionally-immortalized astrocytes and the human conditionally-immortalized pericytes can form cell clusters by self-assembly in the co-culture step, and the cell clusters containing the human conditionally-immortalized astrocytes and the human conditionally-immortalized pericytes can be sufficiently surrounded by the human conditionally-immortalized brain microvascular endothelial cells.

In one embodiment, at least one of the steps is performed in a vessel having a substantially V-shaped or V-shaped bottom (see FIG. 1A). Here, the shape of the bottom refers to a shape defined based on a cross-sectional view when the vessel is cut along a plane formed along the depth direction. If the steps are performed in a vessel having a bottom other than a substantially V-shaped or V-shaped shape, for example, a vessel having a U-shaped bottom, the effect of concentrating the cells to the center is weak, and the endothelial cells do not adhere uniformly to the spheroid surface. At least one of the steps is performed, for example, in a plate having a substantially V-shaped or V-shaped bottom. The plate has a desired number of wells. In a preferred embodiment, all of the steps are performed in a vessel having a substantially V-shaped or V-shaped bottom (see FIG. 1A).

<Other processes>
The production method of the present invention may further include a freezing step of freezing the prepared multicellular three-dimensional blood-brain barrier model, a step of maintaining the shape, performance, etc. of the prepared model, and a step of transporting the model to a location where it is needed.

A second aspect of the present invention relates to a multicellular three-dimensional blood-brain barrier model produced by the aforementioned manufacturing method.

A third aspect of the present invention is a method for producing a composition comprising the steps of:
(A) A cell mass comprising human conditionally immortalized astrocytes and human conditionally immortalized pericytes;
(B) A layer of human conditionally immortalized brain microvascular endothelial cells surrounding the cell mass.

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 (see Figure 1A). The multicellular three-dimensional blood-brain barrier model has the shape and size described above.

As mentioned above, in vitro blood-brain barrier (BBB) models are important research tools for developing brain-targeting drugs and understanding 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. This model retains basic human BBB characteristics and is expected to provide a useful and highly applicable experimental platform for accelerating various BBB research based on the unlimited proliferation capacity of immortalized cells.

The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to these examples in any way.

<1> Samples and test methods

[Preparation of immortalized cells]
Human BMEC/conditionally immortalized clone 18 (HBMEC/ci18), human astrocyte/conditionally immortalized clone 35 (HASTR/ci35), and human brain pericyte/conditionally immortalized clone 37 (HBPC/ci37) were generated according to the methods described in references 1 to 4. They have the temperature-sensitive simian virus 40 large T antigen (tsSV40T) and human telomerase catalytic subunit (hTERT) as immortalization genes. tsSV40T confers a conditional (reversible) immortalization property to cells that promotes cell cycle at a culture temperature of 33°C but is unstable at non-permissive temperatures (37-39°C) and thus is lost. Thus, the immortalized cells were cultured at 33°C for growth and at 37°C for differentiation. Specifically, the culture was carried out according to the methods described in References 2 to 4 and shown in Figure 1. The medium composition used is shown in Table 1 below.

Human monocytic leukemia cell line (THP-1) was obtained from the JCRB Cell Bank (Japanese Collection of Research Bioresources, Osaka, Japan) and cultured in RPMI-1640 (Wako, Osaka, Japan) containing 10% fetal bovine serum (FBS) and penicillin-streptomycin at 37°C in 5% _CO2 /95% air.

[Preparation of BBB model]
The hiMCS-BBB model was generated by sequentially combining three human immortalized BBB cell lines in a Primesurface 96V plate (Sumitomo Bakelite, Tokyo, Japan) (see FIG. 1A), but was generated using a procedure different from that used to generate known BBB spheroids (e.g., see references 5 and 6).

Specifically, as shown in FIG. 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 "spheroid medium."

Next, on Day 3, HBMEC/ci18 cells (1.0 x 10 ³ ) were seeded into the HASTR/ci35/HBPC/ci37 spheroid cores containing 100 μL of spheroid medium, and the cells were further cultured for 48 hours. During this time, the cells were left to stand at 37°C in 5% CO ₂ /95% air. Although hiMCS-BBB spheroids were basically constructed at 37°C, some models were constructed at 33°C for comparison in the following studies.

Phase-contrast images of the BBB spheroids were then taken using a BZ-X710 fluorescence microscope (Keyence, Osaka, Japan), and the diameters of the BBB spheroids were measured using a BZ-H3M analysis application measurement module software (Keyence). The diameters of the BBB spheroids were all between 200 and 280 μm. For comparison, a transwell-type BBB model (12 wells) was also prepared. The transwell-type model was prepared according to the method described in reference 4.

[Detection of hypoxic regions in the hiMCS-BBB model]
Hypoxic regions in hiMCS-BBB spheroids were examined using the Hypoxyprobe-1 kit (Hypoxyprobe, Burlington, MA). hiMCS-BBB spheroids were incubated in spheroid medium containing pimonidazole (200 μM) at 37°C for 3 hours. They were then embedded in O.C.T. compound (Sakura Finetek, Tokyo, Japan), frozen, and sliced into 14 μm sections. The sections were incubated 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 under a confocal microscope. Fluorescence was detected using a Zeiss LSM780 confocal microscope (Carl Zeiss, Oberkochen, Germany), and fluorescent images were taken at a depth of 30-50 μm in the BBB spheroids.

[3D imaging of the hiMCS-BBB model]
Label-free 3D images of hiMCS-BBB were captured by optical coherence tomography (OCT) using a Cell ³ iMager Etier (SCREEN, Kyoto, Japan). To eliminate noise from the culture medium, the original OCT images were processed using Cell Visualizer software (SCREEN).

[Cell localization analysis]
Cells were pre-labeled with CellTracker Green CMFDA Dye (Thermo Fisher Scientific, Waltham, MA) and red and blue fluorescent cytoplasmic membrane staining kits (Promocell, Heidelberg, Germany). The hiMCS-BBB model with pre-labeled cells was fixed with 4% paraformaldehyde (PFA) for 15 min at room temperature and then mounted on a glass slide 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 to a depth of up to 50 μm.

[Isolation of HBMEC/ci18 cells from BBB spheroids]
The prepared hiMCS-BBB model (approximately 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. Undigested cell aggregates were removed with a 40 μm cell strainer (BD falcon), and the cells were resuspended in PBS(-) containing 0.5% BSA and 2 mM ethylenediaminetetraacetic acid (isolation buffer).

HBMEC/ci18 cells were prelabeled with human CD31 MicroBead Kit (Miltenyi Biotec, North Rhine-Westphalia, Germany) and 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 enriched in the isolated cells.

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

[Immune cell staining (ICC)]
ICC was performed using pooled hiMCS-BBB models (approximately 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 concentrations with CanGetSignal immunostain solution A (TOYOBO, Osaka, Japan). 4',6-diamidino-2-phenylindole (DAPI) was used for nuclear counterstaining.

[Dextran Permeability Assay]
In addition to the BBB spheroids, spheroids without HBMEC/ci18 cells (i.e., spheroids containing only HASTR/ci35 and HBPC/ci37, hereafter referred to as "ΔBMEC-hiMCS") were generated for comparison. The spheroids were left 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. The 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).

[Transporter functional assay]
The functions of GLUT1 and P-gp in BBB spheroids were verified by permeability assays using 2-(N-[7-Nitrobenz-2-oxa-1,3-diazol-4-yl]amino)-2-deoxyglucose (2-NBDG; a GLUT1 substrate) and rhodamine123 (R123; a P-gp substrate), 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 min. In addition, 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) was used at a final concentration of 0.1%. The fluorescence intensity in the spheroids was quantified according to the method described above.

Functional Assay of Receptor-Mediated Transcytosis (RMT)
Low density lipoprotein receptor-related protein 1 (LRP1)-mediated transcytosis was examined using the ligand of the protein, Angiopep-2 (amino acid sequence: TFFYG GSRGK RNNFK TEEY) and a 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 BBB spheroids and incubated at 37°C for 2 hours.

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

Tumor necrosis factor alpha (TNF-α) treatment assay
At the time 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 three times with PBS(-), the spheroids were treated with TNF-α for 24 hours. HBMEC/ci18 cells were then isolated from the BBB spheroids by MACS as described above, and RNA was extracted therefrom.

TNF-α treatment assays were 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 BBB spheroids 1 day after seeding of HBMEC/ci18 cells. They were co-cultured in serum-free spheroid medium with or without TNF-α (20 ng/mL) for 48 h. Fluorescence was then detected using a Zeiss LSM780 confocal microscope, and confocal z-stack images were taken at 2.0 μm intervals to a depth of 50 μm.

RNA Sequencing and Data Analysis
Total RNA was extracted from HBMEC/ci18 cells isolated from BBB spheroids using RNeasy Plus Micro Kit (Qiagen, Hilden, Germany), and cDNA libraries were generated using 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 human genome (UCSC/mm10 or UCSC/hg19) mapping of the obtained data and matching with known gene information were performed using TopHat (version 2.0.13 with default parameters). Gene expression levels were then quantified using Cuffdiff (Cufflinks version 2.2.1, default parameters). Gene clustering, heat map creation, and biological function prediction were performed on all mRNA expression data using Subio platform64 software (version 1.22, Subio, Kagoshima, Japan).

[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 assays were first analyzed by one-way analysis of variance (ANOVA), and then the statistical significance of differences between two values was verified by Student's t-test.

[Measurement of viscosity of culture medium]
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 rotation speed of 60 rpm, and the viscosity of the medium was measured at room temperature 10 times every 1 to 2 seconds from 1 minute after the start of rotation. A B-type viscometer (Toki Sangyo Co., Ltd. "TVB10" type viscometer) was used for the viscosity measurement, and the samples used for the 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.

<2> Results [Creation of multicellular spheroids using three types of immortalized BBB cells]
First, as shown in FIG. 1A, the hiMCS-BBB model was created in a 96-well V-bottom plate at 33° C. When seeded, HBPC/ci37 and HASTR/ci35 cells self-assembled to form a spheroid core within 24 hours. HBMEC/ci18 cells were then added to the core to create multicellular spheroids (FIG. 1A). However, at 33° C., the spheroids were large in size and hypoxic conditions were observed in the core region (FIGS. 1B and 1C). Furthermore, the spheroids at this temperature had low sphericity and were not uniformly covered by HBMEC/ci18 cells (FIGS. 1C and 1E). Based on these results, it was determined that the creation method or conditions needed to be changed to improve spheroid formation.

[Effect of removal of immortalization signal in the creation of hiMCS-BBB model]
In response to the above, the following modifications were made by utilizing the conditionally immortalized properties of BBB cells. Conditionally immortalized BBB cells can have their immortalization signal removed by changing the culture temperature from 33°C to 37°C, which causes proliferation to cease and differentiation at the same time (References 1-3). Therefore, we attempted to culture spheroids at 37°C. As a result, a culture temperature of 37°C clearly inhibited spheroid growth (Figure 1B), and hypoxic regions were no longer detectable (Figure 1C). Furthermore, 3D imaging analysis showed that the sphericity of spheroids was higher at 37°C than at 33°C (Figure 1D). The spherical structure of spheroids created under these conditions was maintained for at least 3 days.

Since spheroids formed better at 37°C, we then labeled each cell with a fluorescent dye and examined their localization within the sphere. The results showed that HASTR/ci35 cells (red) were located in the center of the spheroids, while the majority of HBPC/ci37 cells (blue) were located outside the core, with HBMEC/ci18 cells (green) forming on the surface along the center (Figures 1E and 1F).

Taking these results together, it is believed that the 37°C culture condition is an important factor in forming complete multicellular spheroids. 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). In order to confirm the stability of the hiMCS-BBB90 model creation method, spheroid models (480 in total) were created on five separate plates and observed morphologically. It was determined that more than 90% of the spheroids were formed without any problems (Figures 7A and 7B). Therefore, it was demonstrated that the hiMCS-BBB90 model creation method is stable. The BBB characteristics of this model were analyzed below.

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

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

[Intercellular barrier function and transporter function of hiMCS-BBB90 model]
Based on the results of the gene expression analysis, the intercellular permeability barrier function and transporter function were then tested. First, a permeability assay was performed using 70 kDa and 5 kDa FITC-dextran. For comparison, the hiMCS-BBB90 model without HBMEC/ci18 cells (ΔBMEC-hiMCS model) was used (FIG. 3A). As a result, the permeability of the hiMCS-BBB90 model was shown to be significantly lower than that of 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) (FIGS. 3B and 3C). This result suggests that the hiMCS-BBB90 model has intercellular permeability barrier function.

Next, to examine the transporter function of the hiMCS-BBB90 model, we performed permeability assays for 2-NBDG (a substrate for GLUT1) and R123 (a substrate for P-gp). The results showed that the hiMCS-BBB90 model exhibited higher 2-NBDG permeability (2.00 ± 0.40, P < 0.01) than the ΔBMEC-hiMCS model (Fig. 3D), indicating that GLUT1 was functional 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) than the ΔBMEC-hiMCS model (Fig. 3E). Furthermore, treatment with elacridar (a P-gp inhibitor) increased R123 influx into the hiMCS-BBB90 model (1.48 ± 0.31, P < 0.05) (Figure 3E), suggesting that the hiMCS-BBB90 model has P-gp function.

[RMT activity of the hiMCS-BBB90 model]
RMT is a BBB function that has been attracting attention in recent years, and representative receptors involved in this include TfR and LRP-1 (Reference 7). To analyze the RMT activity of these receptors in the hiMCS-BBB model, we first examined their protein expression by immunocytochemical staining. As a result, the hiMCS-BBB90 model expressed higher levels of TfR and LRP-1 proteins than the transwell model (Figure 4A).

Next, a macromolecular permeability assay was performed. As shown in Figures 4B and 4C, at 37°C, angiopep-2 (LRP-1 binding peptide) permeated the hiMCS-BBB90 model more than the scrambled peptide (4.54 ± 1.43 times, P < 0.01). However, at 4°C, where RMT function is significantly suppressed, the permeability of angiopep-2 decreased to a level equivalent to that of the scrambled peptide. Similarly, the permeability of MEM-75 and MEM-189 (anti-TfR antibodies) in the hiMCS-BBB90 model was higher than that of the control IgG at 37°C (1.66 ± 0.24 times, P < 0.05 and 3.46 ± 0.48 times, P < 0.01, respectively). As previously reported (Reference 8), the permeability of MEM-189 was higher than that of MEM-75. Moreover, these permeabilities decreased to levels comparable to those of control IgG at 4° C. (FIGS. 4D and 4E), indicating that the hiMCS-BBB90 model is functional for LRP-1- and TfR-mediated transcytosis.

Response to TNF-α treatment in the hiMCS-BBB90 model
Because the BBB is involved in the formation of brain inflammation (eg, inflammatory cytokine secretion and lymphocyte recruitment), we examined how the hiMCS-BBB90 model responds to inflammatory stimuli.

HBMEC/ci18 cells were isolated from the hiMCS-BBB90 model with or without treatment with the proinflammatory 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. qPCR results showed that the mRNA levels of all analyzed genes 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 immunocytochemical staining (Figure 5C).

Next, to examine whether TNF-α stimulation induces lymphocyte adhesion to activated HBMEC/ci18 cells in the hiMCS-BBB90 model, a TNF-α treatment assay was performed in the presence or absence of THP-1 cells (monocyte model cells). The results showed that TNF-α stimulation promoted adhesion of THP-1 cells to the surface of the hiMCS-BBB90 model (Figure 5D). Thus, these results indicate that the hiMCS-BBB90 model responds to TNF-α, suggesting that the hiMCS-BBB90 model can serve as a model that reproduces BBB inflammation.

[Special role of HBPC/ci37 and HASTR/ci35 cells in the formation of the hiMCS-BBB90 model]
To gain new insight into how cerebral pericytes and astrocytes affect the formation of the hiMCS-BBB90 model, three types of hiMCS-BBB90 models were established:
EPA: Three-kind culture model (hiMCS-BBB90 model),
EP0: co-culture model without HASTR/ci35 cells;
E0A: A co-culture model without HBPC/ci37 cells was created ( FIG. 6A ).

First, to confirm 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. The results showed that the permeability in the EP0 and EA0 models was higher than 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 necessary for the expression of BBB function in the hiMCS-BBB90 model (see Figures 6B and 6C).

Next, HBMEC/ci18 cells were isolated from each model and gene expression profiles were analyzed by RNA sequencing. The results showed that the expression of 182 genes in EP0 and 104 genes in E0A were altered compared to the EPA model, indicating that the expression of genes in each group was regulated by HASTR/ci35 or HBPC/ci37 cells (Figure 6D).

Furthermore, 22 mRNAs were highly expressed only in the EPA model (Fig. 6D and 6E). In other words, these are considered to be genes whose expression is induced only in the presence of both HASTR/ci35 cells and HBPC/ci37 cells. qPCR analysis of COL11A1, ZNF287, BEND6, NTM, DCN, and BEX1 mRNAs included in these 22 genes revealed that their expression levels were higher in HBMEC/ci18 cells of the EPA model than in the EP0 and E0A models, consistent with the results of RNA sequencing (Fig. 6F). These results indicate that HASTR/ci35 cells and HBPC/ci37 cells each have unique roles as well as cooperative roles in the expression of BBB function in the hiMCS-BBB90 model.

[Viscosity of medium]
The measurement results of the viscosity of EBM-2 comp containing 0.48 mg/mL methylcellulose, EBM-2 comp containing no methylcellulose, and tap water are shown in FIG.

<3> Discussion The present inventors have newly established an MCS-BBB model (i.e., "hiMCS-BBB90 model") using human conditionally immortalized cells. The model has a three-layer structure, and from the outside to the inside, it has BMEC, pericytes, and astrocytes, in that order. Through this example, the present inventors have demonstrated that the model is valid as a BBB model and can be applied to various BBB research.

In this model, dextran, a cell-permeable marker, barely penetrates into the spheroids, suggesting that HBMEC/ci18 cells uniformly cover the spheroid core and form intercellular junctions to form a gap-free monolayer. This layered structure is believed to provide a platform for appropriate cell-cell communication required for BBB function expression. 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, in order for HBMEC/ci18 cells to exhibit high BBB performance, a special structure such as that exemplified by the hierarchical spheroids shown in this invention is believed to be necessary.

Furthermore, this example demonstrated that the hiMCS-BBB90 model has transporter function. In HBMEC/ci18 cells, P-gp (and BCRP) are localized on the apical membrane as in vivo, which is believed to effectively restrict the migration of its substrate (R123) into the spheroid (i.e., into the brain). On the other hand, such localization is not observed for GLUT1, as in vivo, which is believed to promote the transcellular transport of its substrate (2-NBDG). Thus, in the hiMCS-BBB90 model, BBB-related transporters are functionally expressed with properties similar to those in vivo, which is believed to promote or restrict the transport of compounds between the inside and outside of the spheroid.

In recent years, RMT has been attracting 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 recognition of RMT activity related to these in the hiMCS-BBB90 model is significant in that it indicates the possibility that this model may contribute not only to basic BBB function but also to the development of drugs for treating brain diseases.

The BBB model created by the inventors is the first spheroid-type BBB model using human conditionally immortalized cells. Since immortalized BBB cells have excellent proliferation capabilities, the hiMCS-BBB90 model can be constructed indefinitely. Furthermore, the hiMCS-BBB90 model can be easily constructed without the need for complex operations or specific culture equipment, reducing the burden on users in terms of time, effort, and finances. These features make it possible to conduct large-scale experiments in BBB research with this model.

Furthermore, the usefulness of the hiMCS-BBB90 model for various BBB research studies was demonstrated by constructing an inflammation model and revealing new insights into BBB biology.

Proinflammatory cytokines are known to play an important role in the development and progression of neuroinflammation through various actions, including the recruitment of immune cells to the BBB. It was revealed that HBMEC/ci18 cells in the hiMCS-BBB90 model respond to TNF-α by increasing the expression levels of adhesion molecules as well as cytokines/chemokines, which in turn recruit THP-1 cells to the BBB. Therefore, the hiMCSC-BBB model is believed to be able to reproduce the inflammatory response reaction at the BBB. This model is therefore believed to be a valuable tool for understanding the molecular mechanisms of BBB inflammation and for exploring new drug targets.

Furthermore, it is expected that the hiMCS-BBB90 model will be useful in elucidating the molecular basis of BBB formation. It has been reported that both astrocytes and pericytes are necessary for BBB formation, but their roles have not been fully elucidated. The inventors have demonstrated that these cells have unique roles in the expression of BBB function in this model, and also exert cooperative effects.

The hiMCS-BBB90 model of the present invention retains basic BBB characteristics while offering excellent scalability and ease of handling. This model is useful for evaluating new CNS drug modalities and for research aimed at elucidating the mechanisms of BBB formation and breakdown. Therefore, the BBB model according to the present invention is expected to become a new research platform for accelerating various human BBB research, including CNS drug development.

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 (15)

(i) a first seeding step of seeding human conditionally immortalized astrocytes and human conditionally immortalized pericytes in a culture 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 into a medium containing a 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 the multicellular three-dimensional blood-brain barrier model having a shape of an ellipsoid, a spheroid or a sphere . The method according to claim 1, wherein the medium in the first seeding step (i) contains a thickening agent. The method according to claim 1 or 2, wherein the first co-culture step (ii) and the second co-culture step (iv) are carried out at a temperature in the range of 35 to 39°C. The 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 ellipsoidal, spheroidal or spherical cell masses. The manufacturing method according to claim 4, wherein the longest diameter of the three diameters of the ellipsoid, spheroid, or sphere is within the range of 180 to 350 μm. The method according to claim 4 or 5, wherein in the second co-culture step (iv), the human conditionally immortalized brain microvascular endothelial cells surround the cell mass formed in the first co-culture step (ii) to form a multicellular three-dimensional blood-brain barrier model. The method according to any one of claims 1 to 6, wherein the longest diameter of the three diameters of the ellipsoid, spheroid or sphere of the multicellular three-dimensional blood-brain barrier model is within the range of 180 to 350 µm. The method according to any one of claims 2 to 7, wherein the concentration of the thickener in the medium in the first seeding step (i) is 0.1 to 20 mg/mL. The method according to any one of claims 1 to 8, wherein the second seeding step (iii) is carried out 12 to 72 hours after the first seeding step (i). The method according to any one of claims 1 to 9, wherein in the second co-culture step (iv), the co-culture is carried out for 12 to 72 hours. The manufacturing method according to claims 1 to 10, wherein at least one of steps (i) to (iv) is carried out in a vessel having a substantially V-shaped or V-shaped bottom. A multicellular three-dimensional blood-brain barrier model produced by the method according to any one of claims 1 to 11. (A) a cell mass containing human conditionally immortalized astrocytes and human conditionally immortalized pericytes;
(B) A multicellular three-dimensional blood-brain barrier model having a spheroidal, spheroidal or spherical shape, comprising a layer of human conditionally immortalized brain microvascular endothelial cells surrounding the cell mass. The multicellular three-dimensional blood-brain barrier model according to claim 13, wherein human conditionally immortalized astrocytes are located on the inside and human conditionally immortalized pericytes are located on the outside of the cell mass (A). The multicellular three-dimensional blood-brain barrier model according to claim 13 or 14 , wherein the longest diameter of the three diameters of the ellipsoid, spheroid or sphere of the multicellular three-dimensional blood-brain barrier model is within the range of 180 to 350 μm.