Blood-brain barrier model using human conditional immortalized cell, and method for producing the same

Abstract

To provide a blood-brain barrier model using a human immortalized cell, and a method for producing the same.SOLUTION: A method for producing a multicellular blood-brain barrier model includes: (i) a culture step of disseminating human conditional immortalized pericytes onto a first culture medium to culture the pericytes; (ii) a pericyte differentiation step of differentiating the cultured human conditional immortalized pericyte on a second culture medium; (iii) an astrocyte culture step of disseminating human conditional immortalized astrocytes onto a third culture medium to culture the astrocytes; (iv) an astrocyte differentiation step of differentiating the cultured human conditional immortalized astrocytes on a fourth culture medium; and (v) a brain microvascular endothelial cell culture step of disseminating human conditional immortalized brain microvascular endothelial cells onto a fifth culture medium to culture the disseminated human conditional immortalized brain microvascular endothelial cells.SELECTED DRAWING: Figure 2A

Description

[Technical field]

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

[0002] The blood brain barrier (BBB) ​​is formed by brain microvascular endothelial cells (BMECs) and supporting cells such as astrocytes and pericytes that are present around them (Non-Patent Document 1, Non-Patent Document 2). BMECs are the main components of the BBB, but both astrocytes and pericytes are required to acquire BBB-specific properties, and both play important roles in regulating and maintaining the BBB (Non-Patent Document 3).

[0003] The BBB provides an important interface between the periphery and the central nervous system (CNS), maintaining independent physiological homeostasis and preventing potentially harmful chemicals such as drugs from entering the brain (Non-Patent Document 4, Non-Patent Document 5). The two main molecular entities that compose the BBB are (1) intercellular junctions (tight junctions and adherens junctions) that strictly restrict the paracellular influx of drugs into the brain; and (2) multiple efflux transporters (including P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP)) that actively export various drugs at the apical membrane side of BMECs (Non-Patent Document 6, Non-Patent Document 7, Non-Patent Document 8, Non-Patent Document 9).

[0004] Due to these special features, identifying drug candidates with excellent BBB permeability has been a major challenge in CNS drug development. Under these circumstances, in vitro BBB models are expected to provide a valuable tool to facilitate the process of identifying CNS drugs with BBB permeability.

[0005] Because the monoculture system of BMECs does not accurately mimic the in vivo BBB, coculture with astrocytes and / or pericytes is considered to be a useful method for developing BBB models with advanced and specialized functions (Non-Patent Document 10, Non-Patent Document 11, Non-Patent Document 12). To achieve this, BBB cells obtained from various animal species have been widely used, but it has become clear that there are some differences in the BBB functions of animals and humans, as indicated by different expression levels of BBB transporters (Non-Patent Document 13, Non-Patent Document 14, Non-Patent Document 15). Therefore, human-derived BBB models are necessary for the development of CNS drugs. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Petzold, GC; Murthy, VN Role of astrocytes in neurovascular coupling. Neuron 2011, 71 (5), 782-797. [Non-Patent Document 2] Sa-Pereira, I.; Brites, D.; Brito, MA Neurovascular unit: a focus on pericytes. Mol. Neurobiol. 2012, 45 (2), 327-347. [Non-Patent Document 3] Engelhardt, B.; Liebner, S. Novel insights into the development and maintenance of the blood-brain barrier. Cell Tissue Res. 2014, 355 (3), 687-699. [Non-Patent Document 4] Pardridge, WM The blood-brain barrier: bottleneck in brain drug development. NeuroRx 2005, 2(1), 3–14.

Direct Environment 5

Outdoor Configuration6

Direct Environment 7

Outdoor Track 8

Outdoor Tools9

Outdoor Tools 10

Outdoor Content11

Outdoor Tools 12

Outdoor Content13

Outdoor Tools 14

Outdoor Tools 15

[0007] An object of the present invention is to provide a blood-brain barrier model using human conditionally immortalized cells and a method for producing the same. [Means for solving the problem]

[0008] Under the above-mentioned background, the inventors established human immortalized cells derived from BMECs, astrocytes and brain pericytes by introducing two immortalization genes, namely, (1) the human telomerase reverse transcriptase (hTERT) gene, which extends telomere length and counteracts genomic damage caused by telomere shortening associated with growth arrest and cell division; and (2) the temperature-sensitive simian virus 40 major tumor antigen (tsSV40T) gene, into primary human BBB cells (References 24, 25, 26).

[0009] tsSV40T confers conditional immortalization properties to cells. Specifically, it is stable at a culture temperature of 33°C and promotes cell cycle, but is rapidly degraded at temperatures above 37°C, allowing cells to redifferentiate as soon as the immortalization signal disappears (References 24, 25, 26, 30, 31). Based on the function of these two genes, human immortalized BBB cells retain important cellular characteristics that show a continuous proliferation profile. This is a clear advantage over conventional cell types in drug development, given the diverse potential applications.

[0010] These human immortalized BBB cells are expected to contribute to the development of a human BBB model in drug permeability research. The inventors have developed a multicellular BBB model based on human immortalized cells using the human immortalized astrocyte cell line (HASTR / ci35), human immortalized brain pericyte cell line (HBPC / ci37), and human immortalized brain microvascular endothelial cell line (HBMEC / ci18) that they have established, and characterized the BBB model.

[0011] That is, the present invention provides: [1] (i) a pericyte culture step of seeding and culturing human conditionally immortalized pericytes in a first medium; (ii) a pericyte differentiation step of differentiating the cultured human conditionally-immortalized pericytes in a second medium; (iii) an astrocyte culturing step of seeding and culturing human conditionally immortalized astrocytes in a third medium; (iv) an astrocyte differentiation step of differentiating the cultured human conditionally-immortalized astrocytes in a fourth medium; (v) a brain microvascular endothelial cell culture step of seeding human conditionally immortalized brain microvascular endothelial cells in a fifth medium and culturing the seeded human conditionally immortalized brain microvascular endothelial cells; Including, In the pericyte culture step (i) and / or the pericyte differentiation step (ii), a culture comprising human conditionally immortalized pericytes is formed, In the astrocyte culture step (iii) and / or the astrocyte differentiation step (iv), a culture comprising human conditionally immortalized astrocytes is formed, The differentiation in the pericyte differentiation step (ii) is carried out at a temperature different from the culture temperature in the pericyte culture step (i), the differentiation in the astrocyte differentiation step (iv) is carried out at a temperature different from the culture temperature in the astrocyte culture step; After the step (ii) and the step (iv), the brain microvascular endothelial cell culture step (v) is carried out; In the brain microvascular endothelial cell culture step (v), a culture containing human conditionally immortalized brain microvascular endothelial cells is formed, In the brain microvascular endothelial cell culture step (v), the seeded human conditionally immortalized brain microvascular endothelial cells are cultured together with a further culture of the human conditionally immortalized pericytes and a further culture of the human conditionally immortalized astrocytes in a sixth culture medium; a method for producing a multicellular blood-brain barrier model, the method comprising: arranging a culture comprising said human conditionally immortalized pericytes and a culture comprising said human conditionally immortalized brain microvascular endothelial cells so as to be capable of interacting with each other; [2] The method according to [1], wherein the culture of the seeded human conditionally immortalized brain microvascular endothelial cells in the brain microvascular endothelial cell culture step (v), as well as the further culture of the human conditionally immortalized pericytes and the further culture of the human conditionally immortalized astrocytes, are carried out at a temperature different from that of the differentiation in the pericyte differentiation step (ii) and / or the differentiation in the astrocyte differentiation step (iv); [3] The culture in the pericyte culture step (i) is carried out at a temperature range of 32 to 34°C, and the differentiation in the pericyte differentiation step (ii) is carried out at a temperature range of 35 to 39°C, and / or The method according to [1] or [2], wherein the astrocyte culture step (iii) is performed at a temperature range of 32 to 34°C, and the astrocyte differentiation step (iv) is performed at a temperature range of 35 to 39°C; [4] The method according to any one of [1] to [3], wherein in the brain microvascular endothelial cell culture step (v), the culture of the seeded human conditionally immortalized brain microvascular endothelial cells, as well as the further culture of the human conditionally immortalized pericytes and the further culture of the human conditionally immortalized astrocytes are carried out at a temperature range of 32 to 34°C; [5] The method according to any one of [1] to [4], wherein the culture in the pericyte culture step (i) and the culture in the astrocyte culture step (iii) are each independently performed for 12 to 72 hours after seeding; [6] The method according to any one of [1] to [5], wherein the differentiation in the pericyte differentiation step (ii) and the differentiation in the astrocyte differentiation step (iv) are each independently carried out for 12 to 72 hours; [7] The method according to any one of [1] to [6], wherein the brain microvascular endothelial cell culture step (v) is carried out for 12 to 240 hours after seeding; [8] The method according to any one of [1] to [7], wherein the culture in the pericyte culture step (i) and the culture in the astrocyte culture step (iii) are carried out simultaneously; [9] The method according to any one of [1] to [8], wherein the differentiation in the pericyte differentiation step (ii) and the differentiation in the astrocyte differentiation step (iv) are carried out simultaneously;

[10] The culture comprising human conditionally immortalized pericytes formed in the pericyte culture step (i) and / or the pericyte differentiation step (ii) is a layer, and / or The method according to any one of [1] to [9], wherein the culture containing human conditionally immortalized brain microvascular endothelial cells formed in the brain microvascular endothelial cell culture step (v) is a layer;

[11] The method according to any one of [1] to

[10] , wherein the culture containing human conditionally immortalized brain microvascular endothelial cells formed in the brain microvascular endothelial cell culture step (v) is a monolayer;

[12] A culture containing human conditionally immortalized astrocytes is located on one side of a culture containing human conditionally immortalized pericytes; The method according to any one of [1] to

[11] , wherein a culture containing human conditionally immortalized brain microvascular endothelial cells is located on the other side of the culture containing human conditionally immortalized pericytes;

[13] The method according to any one of [1] to

[12] , wherein the culture containing the human conditionally immortalized pericytes and the culture containing the human conditionally immortalized cerebral microvascular endothelial cells are formed on each surface of a porous substrate, and are arranged so that the culture containing the human conditionally immortalized pericytes and the culture containing the human conditionally immortalized cerebral microvascular endothelial cells can interact with each other;

[14] The method according to

[13] , wherein the porous substrate is coated with molecules constituting an extracellular matrix;

[15] The method according to any one of [1] to

[14] , wherein the third medium and the fourth medium are different in that the serum contained in the third medium is not contained in the fourth medium;

[16] The method according to any one of [1] to

[15] , wherein the fifth culture medium is a culture medium that does not contain a growth factor;

[17] The method according to any one of [1] to

[16] , wherein the sixth medium is different from any of the first medium, the second medium, the third medium, the fourth medium, and the fifth medium;

[18] A multicellular blood-brain barrier model produced by the production method according to any one of [1] to

[17] ;

[19] (A) A culture comprising human conditionally immortalized brain microvascular endothelial cells; (B) Cultures containing human conditionally immortalized pericytes; (C) Cultures containing human conditionally immortalized astrocytes. (D) a culture medium for the human conditionally immortalized brain microvascular endothelial cells; (E) A culture medium for the human conditionally immortalized pericytes and the human conditionally immortalized astrocytes. A multicellular blood-brain barrier model comprising: The culture (A) containing the human conditionally immortalized brain microvascular endothelial cells and the culture (B) containing the human conditionally immortalized pericytes are arranged so as to be capable of interacting with each other; A culture (A) containing human conditionally immortalized brain microvascular endothelial cells is located on one side of a culture (B) containing human conditionally immortalized pericytes; a multicellular blood-brain barrier model, in which a culture comprising human conditionally immortalized pericytes (B) is on the other side of a culture comprising human conditionally immortalized astrocytes (C);

[20] The culture (A) containing human conditionally immortalized brain microvascular endothelial cells is a layer, and / or The multicellular blood-brain barrier model according to

[19] , wherein the culture (B) containing the human conditionally immortalized pericytes is a layer;

[21] The multicellular blood-brain barrier model according to

[19] or

[20] , wherein the culture (A) containing the human conditionally immortalized brain microvascular endothelial cells is a monolayer.

[22] The method according to claim 1, further comprising the steps of: A culture (A) containing the human conditionally immortalized brain microvascular endothelial cells covers one side of a porous substrate; The multicellular blood-brain barrier model according to any one of

[19] to

[21] , wherein the culture (B) containing the human conditionally immortalized pericytes covers the other surface of the porous substrate;

[23] The medium (D) is a medium containing no growth factors; The multicellular blood-brain barrier model according to any one of

[19] to

[22] , wherein the medium (E) is a medium for nerve cells. Effect of the Invention

[0012] According to the present invention, a blood-brain barrier model using human immortalized cells can be provided. [Brief description of the drawings]

[0013] [Figure 1A] FIG. 1A shows the cell morphology of the conditionally immortalized human brain microvascular endothelial cell line HBMEC / ci18 observed by microscopy. [Figure 1B]Figure 1B shows the protein expression of endothelial cell markers (vWF and CD31) in HBMEC / ci18 cells detected by immunocytochemistry. Nuclear counterstaining was performed with 4',6-Diamidino-2-phenylindole (DAPI) (blue). Experiments were repeated three times, and representative images are shown. [Figure 1C] Figure 1C shows the protein expression of immortalization genes (tsSV40T and hTERT) in HBMEC / ci18 cells detected by immunocytochemistry. The experiment was repeated three times, and representative images are shown. [Figure 1D] Figure 1D shows the proliferation ability of HBMEC / ci18 cells. The proliferation ability of HBMEC / ci18 cells cultured at 33°C or 37°C was examined by direct cell counting 3, 5, and 9 days after seeding. Each value is the mean ± SD of values ​​obtained from three independent experiments, and each measurement was performed in duplicate. [Figure 1E] Figure 1E shows the comparative analysis of various mRNA expression levels assessed by qPCR. The mRNA expression levels of genes characteristic of the BBB in HBMEC / ci18 cells and a human liver sinusoidal endothelial cell line (HLSEC / ciJ, used as an example of non-cerebral endothelial cells) are shown. The targets are low-density lipoprotein receptor-related protein 1 (LRP1), insulin receptor (INSR), multidrug resistance-associated protein 4 (MRP4), glucose transporter 1 (GLUT1), major facilitator superfamily domain (MFSD) 2A, P-glycoprotein (P-gp), monocarboxylate transporter 8 (MCT8) and transferrin receptor (TfR). Values ​​were normalized to the mRNA levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and results are the mean ± SD of values ​​obtained from three independent experiments, and qPCR in each experiment was performed in duplicate. *p<0.05, **p<0.01. [Figure 2A]Figure 2A shows an outline of the procedure for constructing the BBB model based on human immortalized cells. Two days before the start of co-culture (Day-2), human astrocytes (HASTR) / ci35 cells and human brain pericytes (HBPC) / ci37 were seeded on type IV collagen / fibronectin-coated culture inserts or plates using astrocyte growth medium (AGM) and pericyte medium (PM), respectively. Then, on Day-1, the culture temperature was changed from 33°C to 37°C, and astrocyte growth medium (AGM) was replaced with astrocyte differentiation medium (ADM) (Step 2: 35 / 37 differentiation in the figure). On Day 0, HBMEC / ci18 cells were seeded on the culture inserts (apical membrane side) using VascuLife complete medium (Vas-V / E-free medium) without VEGF and hEGF, while brain parenchymal medium (BPM) was used for the culture wells (basal side). The cells were maintained at 33°C from the start of coculture until each test was performed (in the figure, "Step 3: 18 / 35 / 37 coculture"). [Figure 2B]Figure 2B shows six types of BBB models created with different cell arrangement patterns. In the figure, "E", "A", "P" and "0" respectively mean "HBMEC / ci18 cells", "HASTR / ci35 cells", "HBPC / ci37 cells" and "no cells". The order of the alphabets refers to the culture position of each cell type, which is the inside of the insert, the outside of the insert and the bottom of the well. The E00 model is a monoculture model of HBMEC / ci18 cells. The EP0 and EA0 models are co-cultures of HBMEC / ci18 cells on the inside of the insert and HBPC / ci137 cells or HASTR / ci35 cells on the outside of the insert. The E0A model is co-cultures of HBMEC / ci18 cells on the inside of the insert and HASTR / ci35 cells on the bottom of the well. The EPA model is seeded with HBMEC / ci18 cells on the inside of the insert, HBPC / ci37 cells on the outside of the insert and HASTR / ci35 cells on the bottom of the well. The EAP model was prepared by seeding HBMEC / ci18 cells on the inside of the insert, HASTR / ci35 cells on the outside of the insert, and HBPC / ci37 cells on the bottom of the well. [Figure 3A] Figure 3A shows the values ​​of transendothelial electrical resistance (TEER) measured on day 1 after the start of co-culture for six BBB models prepared in a 24-well transwell culture system according to the method described for Figure 2. Results are shown as fold difference relative to the values ​​obtained from the E00 model. Values ​​are expressed as the mean (SD) of three independent experiments, and resistance measurements were performed twice each time. In the figure, "E" stands for "HBMEC / ci18 cells," "A" for "HASTR / ci35 cells," and "P" for "HBPC / ci37." **p<0.01, ***p<0.001 vs E00, ##, p < 0.01; ###, p < 0.001 vs E0A. [Figure 3B] Figure 3B shows the TEER values ​​of E00 and EPA models (12 wells) measured 1 day after co-culture. Results are the mean ± SD of values ​​obtained from three independent experiments, each performed in duplicate. *p<0.001. [Figure 4A]Figure 4A shows the expression profile of cell-cell junction-related genes in the BBB model with human immortalized cells. The relative mRNA expression levels of cell-cell junction-related genes (zonula occludens 1 [ZO-1], vascular endothelial [VE]-cadherin, and β-catenin) in the E00 and EPA models (12 wells) were examined by qPCR. Values ​​were normalized using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA levels and expressed as a factor relative to the value in the E00 model. Data are the mean (and SD) of values ​​obtained from three independent experiments. Each qPCR was performed using duplicate samples (*P<0.05). [Figure 4B] Figure 4B shows the protein expression and localization of ZO-1, E-cadherin, and β-catenin in the E00 model (12 wells) and the EPA model (12 wells) examined by immunocytochemistry (ICC). 4',6-diamidino-2-phenylindole (DAPI) was used for nuclear counterstaining (blue). All experiments were repeated three times independently, and representative photographs are shown in the figures. [Diagram 5] Figure 5 shows the results of testing the function of efflux transporters in the BBB model using human immortalized cells, specifically, the results of bidirectional transport assay of rhodamine 123 (R123; a substrate of P-glycoprotein (P-gp)) in the E00 model (12 wells) and the EPA model (12 wells). R123 (5 μM) was added to the insert chamber or the bottom of the well and left to stand for 20, 40 or 60 min. The medium was collected from the opposite side and the R123 concentration was measured. Based on the measured concentrations, the values ​​of the permeability coefficients PeAB from the apical membrane to the basolateral membrane and PeBA from the basolateral membrane to the apical membrane were calculated. The efflux rate (ER) was calculated according to ER = PeBA / PeAB. Data are the mean (and SD) of values ​​obtained from three independent experiments. Each experiment was performed with duplicate samples (*P<0.05; **P<0.01). [Figure 6A]Figure 6A shows the results of testing the mRNA expression levels of P-gp and BCRP in the E00 model (12 wells) and the EPA model (12 wells) by qPCR. Values ​​were normalized with GAPDH mRNA levels and expressed as fold differences relative to the values ​​of the E00 model. Data are shown as the mean (and SD) obtained from three independent experiments. Each qPCR was performed with duplicate samples (*P<0.05; **P<0.01). [Figure 6B] Figure 6B shows the results of testing the protein expression and subcellular localization of P-gp and BCRP in the E00 model (12 wells) and the EPA model (12 wells) by ICC. For comparison, β-catenin was also tested. For the EPA model, xy plane images are shown in the center of each figure, and yz and xz plane images (x and y positions are indicated by horizontal and vertical white lines, respectively) are shown at the top and right. 4',6-diamidino-2-phenylindole (DAPI) was used for nuclear counterstaining (blue). All experiments were repeated three times independently, and representative photographs are shown in the figures. [Figure 7] Figure 7 shows the results of the permeability assay of Lucifer Yellow (BBB impermeability marker) in the E00 model (12 wells) and the EPA model (12 wells). Lucifer Yellow (10 μM) was added to the apical membrane side and then left for 30, 60 or 90 min. The medium was collected from the wells and the concentration of Lucifer Yellow was determined. Based on this concentration, the permeability coefficient (Pe) value was calculated. Data are shown as the mean (and SD) obtained from three independent experiments. Each experiment was performed with duplicate samples (*P<0.05; **P<0.01 vs E00). [Figure 8]Figure 8 shows the results of the transepithelial electrical resistance (TEER) values ​​measured on Day 1 after the start of co-culture for EPA models created using various media on the basal side. For comparison, the E00 model and the EP0 model were used. From the obtained TEER values, the effect of co-culture with astrocytes and pericytes was confirmed by using BPM (the sixth medium) as the medium on the basal side. The test was performed twice. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] The present invention will now be described in more detail.

[0015] The first aspect of the present invention provides a method for producing a multicellular blood-brain barrier model using human conditionally immortalized cells. Specifically, the multicellular 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.

[0016] That is, the method for producing a multicellular blood-brain barrier model according to the present invention comprises the steps of: (i) a pericyte culture step of seeding and culturing human conditionally immortalized pericytes in a first medium; (ii) a pericyte differentiation step of differentiating the cultured human conditionally-immortalized pericytes in a second medium; (iii) an astrocyte culturing step of seeding and culturing human conditionally immortalized astrocytes in a third medium; (iv) an astrocyte differentiation step of differentiating the cultured human conditionally-immortalized astrocytes in a fourth medium; (v) a brain microvascular endothelial cell culture step of seeding human conditionally immortalized brain microvascular endothelial cells in a fifth medium and culturing the seeded human conditionally immortalized brain microvascular endothelial cells; Each step will be described in detail below.

[0017] <Pericyte culture step (step (i))> In this step, human conditionally immortalized pericytes are seeded in a medium and cultured. The human conditionally immortalized pericytes cultured in this step are subjected to differentiation in the subsequent pericyte differentiation step. The human conditionally immortalized pericytes in this step are cultured at, for example, a temperature in the range of 31 to 35°C, preferably 32 to 34°C, and more preferably 33°C. If cultured at a lower or higher temperature, the cell proliferation rate may decrease.

[0018] The culture in the pericyte culture step (i) is carried out for, for example, 12 to 72 hours, preferably 18 to 60 hours, more preferably 20 to 48 hours, and further preferably 22 to 36 hours after seeding. In one embodiment, the culture in the pericyte culture step (i) is carried out for 24 hours after seeding. If the culture is carried out for a shorter time, the cells may not adhere, develop, or proliferate sufficiently, whereas if the culture is carried out for a longer time, there is a concern of overgrowth, and furthermore, model creation takes time and is not efficient.

[0019] For example, the human conditionally immortalized pericytes are preferably seeded on a substrate having a flat surface as shown in Figure 2B, but are not limited thereto as long as a culture is formed. When seeded on a substrate having a flat surface, a culture containing human conditionally immortalized cerebral microvascular endothelial cells is formed in a layer on the opposite side of the flat surface in the cerebral microvascular endothelial cell culture step, as described below.

[0020] In this step, a medium that is commonly used for culturing pericytes and known in the art can be used, and an example of a commercially available product is Pericyte Medium (manufactured by Scien Cell).

[0021] <Pericyte differentiation step (step (ii))> In this step, the human conditionally immortalized pericytes cultured in the pericyte culture step are differentiated. The differentiation of the human conditionally immortalized pericytes in this step is carried out at a temperature different from the culture temperature in the pericyte culture step. The differentiation of the human conditionally immortalized pericytes is carried out at a temperature higher than the culture temperature in the pericyte culture step, for example, at a temperature higher than the culture temperature in the pericyte culture step, for example, at a temperature higher than 1°C, preferably at a temperature higher than 2°C, more preferably at a temperature higher than 3°C, and particularly preferably at a temperature higher than 4°C. More specifically, the differentiation of the human conditionally immortalized pericytes is carried out at a temperature higher than the culture temperature in the pericyte culture step, for example, at a temperature higher than 1 to 7°C, preferably at a temperature higher than 2 to 6°C, more preferably at a temperature higher than 3 to 5°C, even more preferably at a temperature higher than 3.5 to 4.5°C, and most preferably at a temperature higher than 4°C.

[0022] In this step, the differentiation of human conditionally-immortalized pericytes is specifically carried out within a temperature range of, for example, 34 to 40° C., preferably 35 to 39° C., and more preferably 36 to 38° C. In one embodiment, the differentiation is carried out within a temperature range of 36.5 to 37.5° C. In another embodiment, the differentiation is carried out at 37° C.

[0023] As described later in the Examples, in specific experiments, the present inventors succeeded in obtaining a suitable blood-brain barrier model by performing differentiation at 37°C, a temperature condition that suppresses proliferation while promoting differentiation. It has been reported that the immortalization signal of cells is completely released at 39°C, but the present inventors have demonstrated that differentiation can be promoted by weakening the action of the immortalization signal even at 37°C. The production method of the present invention is a more versatile method in that it can promote differentiation at a lower temperature.

[0024] The differentiation in the pericyte differentiation step (ii) is carried out for, for example, 12 to 72 hours, preferably 18 to 60 hours, more preferably 20 to 48 hours, and further preferably 22 to 36 hours. In one embodiment, the differentiation in the pericyte differentiation step (iii) is carried out for 24 hours.

[0025] In this step, a medium commonly used for differentiation of pericytes and known in the art can be used, for example, but not limited to, a medium for pericytes (ScienCell, San Diego, CA). The medium may further contain serum, growth factors, antibiotics, etc. Examples of serum include, but are not limited to, Fetal Bovine Serum (FBS) (Thermo Fisher Scientific, Waltman, CA), growth factors include, for example, Pericyte Growth Factors (ScienCell), and antibiotics include, but are not limited to, penicillin / streptomycin (pen / st, Wako, Osaka) and blasticidin S (nacali tesque, Kyoto).

[0026] In a preferred embodiment, the medium (second medium) used in the pericyte differentiation step is the same as the medium (first medium) used in the pericyte culture step, or the first medium and the second medium are different in that the serum contained in the first medium is not contained in the second medium. When the second medium does not contain serum, the differentiation of pericytes is promoted compared to when it contains serum. In one embodiment, the medium (second medium) used in the pericyte differentiation step is the same as the medium (first medium) used in the pericyte culture step.

[0027] In the pericyte culture step (i) and / or the pericyte differentiation step (ii), a culture containing human conditionally immortalized pericytes is formed. The shape of the culture containing human conditionally immortalized pericytes may be a layer or a mass. In one embodiment, the culture containing human conditionally immortalized pericytes is a layer containing human conditionally immortalized pericytes. The layer containing human conditionally immortalized pericytes may be a single layer or multiple layers, and is preferably a single layer. If the layer containing human conditionally immortalized pericytes is a single layer, it is possible to avoid overestimation of the barrier function formed in the endothelium.

[0028] In this specification, when the term "layer" is used with respect to human conditionally immortalized pericytes, human conditionally immortalized astrocytes, and human conditionally immortalized brain microvascular endothelial cells, it is intended to include layers having overlapping portions between cells and layers having gaps where no cells are present. In other words, in this specification, the term "layer" includes layer-like structures and sheet-like structures in addition to layers having a complete and uniform structure.

[0029] <Astrocyte culture step (step (iii))> In this step, human conditionally immortalized astrocytes are seeded in a medium and cultured. The human conditionally immortalized astrocytes cultured in this step are subjected to differentiation in the subsequent astrocyte differentiation step. The human conditionally immortalized astrocytes in this step are cultured at a temperature within the range of, for example, 31 to 35°C, preferably within the range of 32 to 34°C, and more preferably at 33°C.

[0030] The culture in the astrocyte culture step (iii) is carried out for, for example, 12 to 72 hours, preferably 18 to 60 hours, more preferably 20 to 48 hours, and further preferably 22 to 36 hours after seeding. If the culture is carried out for a longer period, the function of the cells may be reduced. In one embodiment, the culture in the astrocyte culture step (iii) is carried out for 24 hours after seeding.

[0031] The human conditionally-immortalized astrocytes are preferably seeded in a container inside which a further container can be inserted, for example as shown in Figure 2B. In one embodiment, the container has a flat bottom.

[0032] The third medium in which the astrocytes are seeded in this step can be a medium commonly used in the art for culturing astrocytes, and examples of commercially available products include, but are not limited to, Dulbecco's Modified Eagle Medium (DMEM) (LONZA, Sigma-Aldrich, Thermo Fisher Scientific, etc.). The third medium may be supplemented with antibiotics, serum, N2 supplement (Wako), etc., as appropriate.

[0033] <Astrocyte differentiation step (step (iv))> In this step, the human conditionally immortalized astrocytes cultured in the astrocyte culture step are differentiated. The differentiation of the human conditionally immortalized astrocytes in this step is carried out at a temperature different from the culture temperature in the astrocyte culture step. The differentiation of the human conditionally immortalized astrocytes is carried out at a temperature higher than the culture temperature in the astrocyte culture step, for example, at a temperature higher than 1°C, preferably at a temperature higher than 2°C, more preferably at a temperature higher than 3°C, and particularly preferably at a temperature higher than 4°C. More specifically, the differentiation of the human conditionally immortalized astrocytes is carried out at a temperature higher than the culture temperature in the astrocyte culture step, for example, at a temperature higher than 1 to 7°C, preferably at a temperature higher than 2 to 6°C, more preferably at a temperature higher than 3 to 5°C, even more preferably at a temperature higher than 3.5 to 4.5°C, and most preferably at a temperature higher than 4°C.

[0034] In this step, the differentiation of human conditionally-immortalized astrocytes is specifically carried out within a temperature range of, for example, 34 to 40° C., preferably 35 to 39° C., and more preferably 36 to 38° C. In one embodiment, the differentiation is carried out within a temperature range of 36.5 to 37.5° C. In another embodiment, the differentiation is carried out at 37° C.

[0035] As described later in the Examples section, in specific experiments, the present inventors succeeded in obtaining a suitable blood-brain barrier model by performing differentiation at 37°C, a temperature condition that suppresses proliferation while promoting differentiation. It has been reported that the immortalization signal of cells is completely released at 39°C, but the present inventors have demonstrated that differentiation can be promoted by weakening the action of the immortalization signal even at 37°C. The production method of the present invention is a more versatile method in that it can promote differentiation at a lower temperature.

[0036] The differentiation in the astrocyte differentiation step (iv) is carried out for, for example, 12 to 72 hours, preferably 18 to 60 hours, more preferably 20 to 48 hours, and further preferably 22 to 36 hours. Differentiation for a longer period of time may result in a decrease in the function of the cells. In one embodiment, the differentiation in the astrocyte differentiation step (iv) is carried out for 24 hours.

[0037] The fourth medium used to differentiate astrocytes in this step can be a differentiation medium commonly used for astrocyte differentiation and known in the art, for example, commercially available products include, but are not limited to, Dulbecco's Modified Eagle Medium (DMEM) (LONZA, Sigma-Aldrich, Thermo Fisher Scientific, etc.).

[0038] In a preferred embodiment, the medium used in the astrocyte differentiation step (fourth medium) differs from the medium used in the astrocyte culture step (third medium) in that it does not contain the serum contained in the third medium.

[0039] Here, examples of serum include, but are not limited to, FBS, etc. When the third medium contains serum, the cell proliferation of astrocytes is promoted, and when the fourth medium does not contain serum, the inhibition of differentiation of astrocytes is prevented.

[0040] In another embodiment, the medium (fourth medium) used in the astrocyte differentiation step may contain an astrocyte differentiation promoter, such as, but not limited to, cAMP and its analogs (dBcAMP, etc.).

[0041] In one embodiment, the fourth medium may contain an antibiotic, if necessary. In another embodiment, when the third medium contains an antibiotic, the fourth medium may not contain the antibiotic contained in the third medium. Examples of antibiotics include, but are not limited to, blasticidin.

[0042] In the astrocyte culture step (iii) and / or the astrocyte differentiation step (iv), a culture containing human conditionally immortalized astrocytes is formed. The shape of the culture containing human conditionally immortalized astrocytes may be a layer or a mass. In one embodiment, the culture containing human conditionally immortalized astrocytes is a layer containing human conditionally immortalized astrocytes. The layer containing human conditionally immortalized astrocytes may be a single layer or multiple layers. The culture containing human conditionally immortalized astrocytes may also be a cell mass or a complex three-dimensional shape.

[0043] Pericyte culture step (i) and astrocyte culture step (iii) The culturing is carried out, for example, independently within the above-mentioned time range. In the pericyte culture step (i) and the astrocyte culture step (iii), The cultures in the above are carried out simultaneously within the above time range. This can reduce the time required to create a model.

[0044] In addition, the differentiation in the pericyte differentiation step (ii) and the differentiation in the astrocyte differentiation step (iv) are carried out independently within the above-mentioned time range, for example. In a preferred embodiment, the differentiation in the pericyte differentiation step (ii) and the differentiation in the astrocyte differentiation step (iv) are carried out simultaneously within the above-mentioned time range. By carrying out these differentiation steps simultaneously, it is possible to shorten the time required for model preparation.

[0045] <Brain microvascular endothelial cell culture process (step (v))> This step is carried out after the pericyte differentiation step (step (ii)) and the astrocyte differentiation step (step (iv)). In this step, human conditionally immortalized brain microvascular endothelial cells are seeded in a medium, and the seeded human conditionally immortalized brain microvascular endothelial cells are cultured.

[0046] In this step, a culture containing human conditionally immortalized brain microvascular endothelial cells is formed. Here, preferably, the culture containing human conditionally immortalized brain microvascular endothelial cells is a layer. In one embodiment, the culture containing brain microvascular endothelial cells is a monolayer. If the layer containing human conditionally immortalized brain microvascular endothelial cells is a monolayer, the structure of the blood-brain barrier in vivo can be more faithfully imitated.

[0047] In this step, the seeded human conditionally immortalized brain microvascular endothelial cells are cultured together with the further culture of human conditionally immortalized pericytes and the further culture of human conditionally immortalized astrocytes in the sixth medium. By co-culturing in this manner, a state closer to the in vivo brain environment can be reproduced, and the blood-brain barrier function in the human conditionally immortalized brain microvascular endothelial cells can be improved.

[0048] In the brain microvascular endothelial cell culture step, the culture of the seeded human conditionally immortalized brain microvascular endothelial cells, as well as the further culture of the human conditionally immortalized pericytes and the further culture of the human conditionally immortalized astrocytes are preferably carried out at a temperature different from that of the differentiation in the pericyte differentiation step (step (ii)) and / or the astrocyte differentiation step (step (iv)). The temperature is lower than the differentiation temperature in the differentiation step (ii) and / or the differentiation step (iv), for example, at a temperature lower by 1°C or more, preferably at a temperature lower by 2°C or more, more preferably at a temperature lower by 3°C or more, and particularly preferably at a temperature lower by 4°C or more. More specifically, the temperature is, for example, 1 to 7°C lower, preferably at a temperature lower by 2 to 6°C, more preferably at a temperature lower by 3 to 5°C, even more preferably at a temperature lower by 3.5 to 4.5°C, and most preferably at a temperature lower by 4°C.

[0049] In one embodiment, the culture of the human conditionally-immortalized brain microvascular endothelial cells in this step, and the further culture of the human conditionally-immortalized pericytes and human conditionally-immortalized astrocytes are carried out at a temperature within the range of, for example, 31 to 35°C, preferably within the range of 32 to 34°C, and more preferably at 33°C.

[0050] In this step, the culture of the human conditionally immortalized brain microvascular endothelial cells and the further culture of the human conditionally immortalized pericytes and the human conditionally immortalized astrocytes are carried out for, for example, 12 hours or more, preferably 12 to 240 hours, more preferably 12 to 150 hours, even more preferably 18 to 120 hours, and particularly preferably 24 to 120 hours, after the seeding of the human conditionally immortalized brain microvascular endothelial cells. If the culture time is longer than the above range, the function of the brain microvascular endothelial cells may not be maintained, and in addition, the preparation of the model takes too much time and is not efficient. On the other hand, if the culture time is shorter than the above range, the brain microvascular endothelial cells may not grow sufficiently or may not form a layer. In one embodiment, the culture of the human conditionally immortalized brain microvascular endothelial cells and the further culture of the human conditionally immortalized pericytes and the human conditionally immortalized astrocytes are carried out for 24 hours after the seeding of the human conditionally immortalized brain microvascular endothelial cells.

[0051] Here, the culture containing human conditionally immortalized brain microvascular endothelial cells mimics the apical membrane side of the blood-brain barrier (BBB) ​​structure in the body, while the culture containing human conditionally immortalized pericytes and the culture containing human conditionally immortalized astrocytes formed in the previous step mimic the basal side of the BBB structure.

[0052] In the culture of human conditionally immortalized brain microvascular endothelial cells in this step, a medium known in the art can be used as the fifth medium, for example, VascuLife basal medium (KURABO, Osaka, Japan) is a commercially available product, but is not limited thereto. Preferably, the fifth medium in the culture of human conditionally immortalized brain microvascular endothelial cells is a medium that does not contain growth factors. In one embodiment, VascuLife complete medium (Vas-V / E-free medium) that does not contain VEGF, hEGF, or blasticidin S can be used as the fifth medium. The fifth medium may contain an antibiotic as appropriate. Examples of growth factors include, but are not limited to, VEGF, hEGF, bFGF, IGF, etc. Examples of antibiotics include, but are not limited to, blasticidin, penicillin, streptomycin, etc.

[0053] In the further culture of the human conditionally immortalized pericytes and human conditionally immortalized astrocytes in this step, a medium known in the art can be used as the sixth medium. Preferably, the sixth medium is different from any of the first medium, the second medium, the third medium, the fourth medium, and the fifth medium. For example, a commercially available product may be used as the sixth medium. The sixth medium may be, but is not limited to, Neurobasal medium (Thermo Fisher Scientific). The medium may be appropriately supplemented with antibiotics, N2 supplements, and the like. In one embodiment, the sixth medium may be Neurobasal medium (Thermo Fisher Scientific), which may be supplemented with pen / st, N2 supplement containing apotransferrin, and L-glutamine (the medium thus prepared is also called brain parenchymal medium (BPM)).

[0054] In the multicellular blood-brain barrier model prepared as described above, a layer containing human conditionally immortalized astrocytes is located on one side of the culture containing human conditionally immortalized pericytes, and a culture containing human conditionally immortalized brain microvascular endothelial cells is located on the other side of the culture containing human conditionally immortalized pericytes.

[0055] In one embodiment, the culture containing human conditionally immortalized pericytes and the culture containing human conditionally immortalized brain microvascular endothelial cells are in communication with each other so that they can interact with each other. Here, the interaction between the culture containing human conditionally immortalized pericytes and the culture containing human conditionally immortalized brain microvascular endothelial cells is an interaction mediated by at least a humoral factor, but the pericytes and the brain microvascular endothelial cells may be in direct contact with each other through the pores of the porous membrane. In another embodiment, when the culture is a layer, the layer of the culture containing human conditionally immortalized pericytes and the layer of the culture containing human conditionally immortalized brain microvascular endothelial cells are in communication with each other so that they can interact with each other.

[0056] In one embodiment, the culture containing human conditionally immortalized pericytes and the culture containing human conditionally immortalized brain microvascular endothelial cells are formed on the respective surfaces of a porous substrate, so that the culture containing human conditionally immortalized pericytes and the culture containing human conditionally immortalized brain microvascular endothelial cells are connected to each other so that they can interact with each other. The porous substrate is, for example, a porous membrane. The material of the porous substrate is appropriately selected within a range that does not prevent direct interaction between the culture containing human conditionally immortalized pericytes and the culture containing human conditionally immortalized brain microvascular endothelial cells, and examples thereof include, but are not limited to, those commonly used in the field of porous membranes, such as polyethylene terephthalate.

[0057] The size of the pores of the porous substrate may be appropriately selected within a range that does not interfere with the interaction between the culture containing human conditionally immortalized pericytes and the culture containing human conditionally immortalized brain microvascular endothelial cells, or does not interfere with the formation of the cell culture (e.g., layer or mass). Commercially available examples of the porous substrate include translucent polyethylene terephthalate, 0.4 μm high-density pores (BD Falcon, Franklin Lakes, NJ).

[0058] The porous substrate is preferably coated with a molecule constituting an extracellular matrix to promote cell adhesion. For example, the porous substrate is coated with at least one selected from the group consisting of collagen, fibronectin, and laminin. By coating with such a substance, cell adhesion and differentiation characteristics are improved. In one embodiment, the porous substrate is coated with collagen and / or fibronectin.

[0059] In the production method of the present invention, the number of human conditionally immortalized pericytes, the number of human conditionally immortalized astrocytes, the number of human conditionally immortalized brain microvascular endothelial cells to be seeded, and the ratio of these numbers may be adjusted as appropriate.

[0060] <Other processes> The production method may further include a freezing step of freezing the prepared multicellular blood-brain barrier model, a step of maintaining the shape, performance, etc. of the prepared model, and a transport step of transporting the model to a location where it is needed.

[0061] A second aspect of the present invention relates to a multicellular blood-brain barrier model produced by the aforementioned production method.

[0062] A third aspect of the present invention provides a method for producing a method for the production of a medicament for use in a pharmaceutical composition comprising the (A) A culture comprising human conditionally immortalized brain microvascular endothelial cells; (B) Cultures containing human conditionally immortalized pericytes; (C) Cultures containing human conditionally immortalized astrocytes. (D) a culture medium for the human conditionally immortalized brain microvascular endothelial cells; (E) A culture medium for the human conditionally immortalized pericytes and the human conditionally immortalized astrocytes. The present invention relates to a multicellular blood-brain barrier model comprising:

[0063] In this model, a culture (A) containing human conditionally immortalized brain microvascular endothelial cells and a culture (B) containing human conditionally immortalized pericytes are arranged so that they can interact with each other. In one embodiment, a porous substrate may be present between the culture (A) containing human conditionally immortalized brain microvascular endothelial cells and the culture (B) containing human conditionally immortalized pericytes, in which the culture (A) containing human conditionally immortalized brain microvascular endothelial cells covers one side of the porous substrate, and the culture (B) containing human conditionally immortalized pericytes covers the other side of the porous substrate.

[0064] On one side of the culture containing human conditionally immortalized pericytes (B) is a culture containing human conditionally immortalized brain microvascular endothelial cells (A), and on the other side of the culture containing human conditionally immortalized pericytes (B) is a culture containing human conditionally immortalized astrocytes (C).

[0065] In a preferred embodiment, the culture (A) comprising human conditionally immortalized brain microvascular endothelial cells is a layer and / or the culture (B) comprising human conditionally immortalized pericytes is a layer. In another preferred embodiment, the culture (A) comprising human conditionally immortalized brain microvascular endothelial cells is a monolayer and / or the culture (B) comprising human conditionally immortalized pericytes is a monolayer.

[0066] The medium (D) corresponds to the fifth medium described above and is a medium that does not contain growth factors. The medium (D) may further be free of antibiotics. The medium (E) corresponds to the sixth medium described above and is a medium for neural cells.

[0067] In one embodiment, the multicellular blood-brain barrier model of the present invention includes a first container that holds a culture (C) containing human conditionally immortalized astrocytes therein, and a second container that holds a culture (A) containing human conditionally immortalized brain microvascular endothelial cells and a culture (B) containing human conditionally immortalized pericytes. For example, the first container may have a structure in which the second container can be placed, for example, a well structure having a certain depth. In this case, the second container may be an insert that is placed inside the first container.

[0068] The second container carries a culture (A) containing human conditionally immortalized brain microvascular endothelial cells on one side (e.g., inside the second container) and a culture (B) containing human conditionally immortalized pericytes on the other side (e.g., outside the second container). By carrying the culture (A) and the culture (B) in this way, when the first container and the second container are combined, the culture layer (A) containing human conditionally immortalized brain microvascular endothelial cells is located on one side of the culture layer (B) containing human conditionally immortalized pericytes, and the culture layer containing human conditionally immortalized astrocytes (C) is located on the other side of the culture layer (B) containing human conditionally immortalized pericytes, and a BBB model that mimics the structure in vivo can be reproduced (e.g., Figures 2B, 3A, and 8). The first container and the second container preferably have a flat bottom surface.

[0069] Here, the arrangement of the cultures (A), (B) and (C) and the arrangement of the first and second containers are not limited to those described above, and may be appropriately changed as long as the structure or function of the BBB in vivo can be imitated. The shapes of the first and second containers may also be appropriately changed as long as the structure or function of the BBB in vivo can be imitated.

[0070] 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 blood-brain barrier model using human conditionally immortalized cells can be provided. The 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 ability of immortalized cells. EXAMPLES

[0071] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples in any way.

[0072] <1> Samples and Test Methods [Cell maintenance] HBMEC / ci18 (described below) and the human hepatic sinusoidal endothelial cell line (HLSEC / ciJ (Reference 32)) were cultured in VascuLife complete medium (Vas-comp), HASTR / ci35 in astrocyte growth medium (AGM), and HBPC / ci37 cells in pericyte medium (PM). The composition of the media used is shown in Table 1.

[0073] Vas-comp was prepared by adding 5 ng / mL human fibroblast growth factor-basic (hFGF-b), 50 μg / mL ascorbic acid, 1 μg / mL hydrocortisone (HC), 15 ng / mL human insulin-like growth factor-1 (hIGF-1), 5 ng / mL human epidermal growth factor (hEGF), 5 ng / mL vascular endothelial growth factor (VEGF), 0.75 unit / mL heparin, 10 mM L-glutamine (Wako, Osaka, Japan), 2% (v / v) fetal bovine serum (FBS) (all except glutamine were purchased from KURABO), 100 unit / mL penicillin / streptomycin (pen / st) (Thermo Fisher Scientific, Waltham, MA), and 4 μg / mL blasticidin S (InvivoGen, San Diego, CA) to VascuLife basal medium (KURABO, Osaka, Japan).

[0074] AGM was prepared by adding 1% (v / v) N2 supplement (Wako) containing transferrin, 10% (v / v) FBS (Thermo Fisher Scientific), 100 units / mL to 100 μg / mL pen / st, and 4 μg / mL blasticidin S to Dulbecco's modified Eagle's medium (DMEM) high glucose GlutaMAX pyruvate (Thermo Fisher Scientific).

[0075] Pericyte medium (PM) consisted of pericyte basal medium supplemented with 2% (v / v) FBS, 1% (w / v) pericyte growth factor, and 1% pen / st (all purchased from ScienCell, San Diego, CA).

[0076] [Table 1] TIFF2025017371000003.tif210144

[0077] All immortalized cells were cultured in type I collagen-coated dishes at 33°C in 5% CO2 / 95% air. All cells were subcultured every 3 days using 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA).

[0078] [Isolation of conditionally immortalized human BMEC clone 18 (HBMEC / ci18)] HBMEC / ci clone 18 (HBMEC / ci18, Fig. 1 ) was isolated from parental HBMEC / ci cells by serial dilution as described in reference 24 , except that Vas-comp was used.

[0079] [Cell proliferation analysis] HASTR / ci18, 5.0×10 4 The cells were seeded at a concentration of 1000 cells / mL (Day 0) and cultured at either 33° C. or 37° C. On Days 3, 5, and 9, the cell numbers were counted using a cytometer.

[0080] [Total RNA isolation, cDNA synthesis, and Real-Time Quantitative PCR (qPCR)] Total RNA extraction, genomic DNA contamination check, and cDNA synthesis were performed according to the methods described in references 24, 26, and 33. qPCR was performed using the primers shown in Table 2 and HBMEC / ci18 cDNA as a template. Target mRNAs are described in the figure legends and corresponding parts of this specification. The amplification efficiency of each qPCR was confirmed to be close to 1. The mRNA level of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in each cell type was determined as a normalization control. The delta-delta CT method was used to determine the relative expression level of these values.

[0081] [Table 2]

[0082] [Immunocytochemistry (ICC)] ICC was performed according to the methods described in references 25, 26, 33, 34. The primary and secondary antibodies used are shown in Table 3. All antibodies were diluted to appropriate concentrations with Can Get Signal immunostain solution A (TOYOBO, Osaka, Japan). 4',6-diamidino-2-phenylindole (DAPI) (Dojindo, Kumamoto, Japan) was used for nuclear counterstaining. Cells were mounted with antifade (0.1% "w / v" p-phenylenediamine and 68.8% "v / v" glycerol, pH 8.0), and fluorescence was detected using a Zeiss LSM780 confocal microscope (Carl Zeiss, Oberkochen, Germany).

[0083] [Table 3]

[0084] [Development of an in vitro BBB model] As shown in Figure 2, human immortalized BBB cells were combined in a transwell culture system (12 or 24 wells) to develop the BBB model. The membrane of the cell culture insert (translucent polyethylene terephthalate, 0.4 μm high-density pores, BD Falcon, Franklin Lakes, NJ) was incubated with 100 μg / mL type-IV collagen (Nitta Gelatin, Osaka, Japan) and 100 μg / mL fibronectin (Thermo Fisher Scientific) solution at 37°C for 1 h (extracellular matrix coating). The insert was air-dried and washed twice with phosphate-buffered saline (PBS).

[0085] For the co-culture model, HASTR / ci35 (2.5 × 10 4 cells / cm 2 ) and HBPC / ci37 (3.0 × 10 4cells / cm 2 ) on the outside of the membrane of the insert (0.33 cm for 24 wells) 2 , 0.9 cm for 12 wells 2 ) or on the bottom of the plate (BD Falcon) (2.0 cm for 24 wells) 2 or 4.0 cm for 12 wells 2 ) seeds were sown.

[0086] HASTR / ci35 cells were cultured in AGM and HBPC / ci37 cells in PM for two days (Figure 2, Step 1) before seeding HBMEC / ci18 cells (Day 2). After seeding, the cells were cultured at 33°C for one day, and then (Day 1), the culture temperature was shifted from 33°C to 37°C and AGM was replaced with astrocyte differentiation medium (ADM) to differentiate HASTR / ci35 and HBPC / ci37 cells.

[0087] Here, ADM is (i) Contains no FBS or blasticidin S (ii) containing 1 mM dibutyryl cyclic adenosine monophosphate (dBcAMP, Santa Cruz Biotech, Santa Cruz, CA); Other than that, the composition is the same as that of AGM (see Figure 2A, Step 2).

[0088] On day 0, to start the co-culture (see Figure 2A, Step 3), HBMEC / ci18 cells (1.3x10 5 cells / cm 2 ) were seeded inside the inserts containing HBPC / ci37 (or HASTR / ci35) cells, and immediately afterwards the inserts containing the two cell types were moved to wells containing HASTR / ci35 (or HBPC / ci37) cells. For cultures of HBMEC / ci18 cells alone, the cells were seeded inside the inserts without any other cells (see Figure 2B).

[0089] In all types of BBB models (see Figure 2B), The apical membrane side (insert) was filled with VascuLife complete medium (Vas-V / E-free medium) that does not contain VEGF or hEGF. The basal side (well) is filled with brain parenchymal medium (BPM). Used.

[0090] The Vas-V / E-free medium has the same composition as Vascomp except that it does not contain VEGF, hEGF, or blasticidin S (see Table 1-1).

[0091] BPM is composed of Neurobasal medium (Thermo Fisher Scientific) supplemented with 100 units / mL penicillin-100 μg / mL streptomycin, 1% (v / v) apotransferrin, 1% (v / v) N2 supplement, and 2 mM L-glutamine (see Tables 1-4).

[0092] In Step 3, cells were cultured at 33° C. On Day 1, RNA extraction, ICC, and permeability assay were performed, and on Days 1, 2, and 5, transendothelial electrical resistance (TEER) analysis was performed.

[0093] [Measurement of transendothelial electrical resistance (TEER)] TEER measurements were performed using a Millicell ERS-2 (Millipore, Darmstadt, Germany) equipped with an STX01 electrode (Millipore). The area of ​​the culture insert was used to calculate the TEER value (Ω x cm 2 ) was calculated.

[0094] [Functional assay of P-gp] Efflux transporter function (P-gp) was determined by bidirectional transport experiments using rhodamine 123 (R123, a substrate for P-gp).

[0095] Specifically, R123 (5 μM, Wako) was added to the apical side (A). After 20, 40, or 60 min of incubation, the medium was collected from the apical side (A) and basolateral side (B), respectively, and the Pe values ​​(PeAB for the apical-basolateral direction and PeBA for the basolateral-apical direction) were calculated to determine the efflux rate (ER) (PeBA / PeAB).

[0096] To determine the Pe value, the cleared volume was calculated using the following formula:

[0097]

number

[0098] In the above formula, the apical membrane (C apical ) lateral and basal (C basolateral ) side, and the volume of the apical membrane (V apical = 750 μL) and the basal volume (V basolateral The cleared volume was plotted against the time axis, and the BBB model (PS total ) and cell-free inserts (PS insert The slope of the clearance line was obtained for the endothelial monolayer (PS e ) the permeability x area value was determined using the following formula:

[0099]

number

[0100] Finally, as shown below, PS e The Pe value was obtained by dividing the value by the surface area A (for a 12-well insert, A: 0.9 cm 2 ).

[0101]

number

[0102] [In vitro BBB permeability test] Lucifer Yellow permeability assay was performed using HBMEC / ci18 cells cultured alone (E00) and cocultured with HASTR / ci35 and HBPC / ci37 cells (EPA). After washing the inserts once with PBS(+), the medium was replaced with calcium and magnesium-containing Hanks' buffered saline solution (Thermo Fisher Scientific) and preincubated at 33°C for 10 min. Lucifer Yellow (LY, 10 μM, Wako) was then added to the apical membrane.

[0103] After incubation at 37°C for 30, 60 or 90 min, the medium was collected from the basal wells. For LY quantification, the fluorescence intensity of each medium was determined using an Infinite 200 PRO (Tecan, Mannedorf, Switzerland) at wavelengths of 428 / 536 (nm / nm).

[0104] [Statistical analysis] Statistical analysis was performed using Excel with ystat2006 (Igakutosho Shuppan Ltd., Tokyo, Japan). Comparisons between multiple values ​​were performed by one-way analysis of variance (ANOVA) followed by Bonferroni correction. Two values ​​were compared by unpaired Student's t-test.

[0105] [Medium optimization] The following media were examined as the basal medium for the co-culture of the three types of cells (i.e., the medium for culturing pericytes and astrocytes). The composition of each medium is as follows: ·Vas-comp (Table 1-1) ·Vas-V / E-free medium (Table 1-1) ·ADM (Table 1-2) PM (Tables 1-3, except that serum is not included here) ·BPM (Table 1-4)

[0106] <2> result [Isolation and characterization of a novel clone, HBMEC / ci18, of a conditionally immortalized human brain microvascular endothelial cell line] The present inventors previously isolated human brain microvascular endothelial cell / conditionally immortalized clone β, HBMEC / ciβ (Reference 24), but to generate clones with better BBB properties, they re-cloned the parent cells. From the clones obtained, the present inventors selected clone 18, which they named HBMEC / ci18 (Figure 1A).

[0107] HBMEC / ci18 cells showed a typical elongated spindle-shaped cell morphology (Figure 1A) and expressed the representative endothelial marker proteins von Willebrand factor (vWF) and CD31 (Figure 1B). Furthermore, HBMEC / ci18 cells expressed the immortalization proteins tsSV40T and hTERT (Figure 1C). These cells also showed excellent proliferation ability (Figure 1D).

[0108] To examine whether HBMEC / ci18 cells show gene expression characteristics specific to brain vascular endothelial cells, we examined the expression levels of mRNAs characteristic of the BBB and compared them with those of a conditionally immortalized human liver sinusoidal endothelial cell line (HLSEC / ciJ, a non-brain vascular cell line). As a result, HBMEC / ci18 cells expressed higher levels of mRNAs characteristic of BMECs (low-density lipoprotein receptor-related protein 1, insulin receptor, multidrug resistance-associated protein 4, glucose transporter 1 (GLUT1), major facilitator superfamily domain (MFSD) 2A, P-gp, monocarboxylate transporter 8, and transferrin receptor) than HLSEC / ciJ (Figure 1E).

[0109] In summary, HBMEC / ci18 cells were considered to have basic characteristics of BMECs. Therefore, we developed an in vitro BBB culture model using HBMEC / ci18.

[0110] [Development of an in vitro BBB model based on human immortalized cells] Astrocytes and pericytes are known to play an important role in inducing BBB properties in BMECs (References 4 and 5). Several in vitro studies have reported that the BBB function of BMECs cocultured with astrocytes and / or pericytes is higher than that of BMECs cultured alone (References 12-14). Therefore, we decided to develop a coculture BBB model by combining HBMEC / ci18 cells with HASTR / ci35 cells and HBPC / ci37 cells (Figure 2).

[0111] To achieve this, the inventors have developed a new culture method. For example, to promote the maturation of HASTR / ci35 and HBPC / ci3 cells, we used serum-free medium and a culture temperature of 37°C (Step 2). In addition, to enhance the function of vascular endothelial cells, we used VEGF and hEGF, which act suppressively on BBB formation (References 35, 36). Unsupplemented endothelial medium was used.

[0112] Under these conditions, six BBB model types were created, as shown in Figure 2B. The details of the six model types are as follows: (1) E00: HBMEC / ci18 cells cultured alone (2) EP0: HBMEC / ci18 cells co-cultured with HBPC / ci37 (3) EA0: HBMEC / ci18 cells cocultured with HASTR / ci35 cells (HASTR / ci35 cells are on the outside of the insert). (4) E0A: HBMEC / ci18 cells co-cultured with HASTR / ci35 cells (HASTR / ci35 cells are on the bottom side of the well) (5) EPA: All three types of cells were co-cultured (HBPC / ci37 on the outside of the insert, HASTR / ci35 on the bottom of the well). (6) EAP: All three types of cells were co-cultured (HASTR / ci35 on the outside of the insert, and HBPC / ci37 on the bottom of the well).

[0113] From the results of the TEER analysis shown in Figure 3A, the TEER value of the co-culture model was 60.3 ± 4.1 Ω × cm 2 ), and the EPA model had the highest TEER value (93.0 ± 3.4 Ω × cm) among the co-culture models. 2 ), and these TEER levels were maintained at least until Day 5, as shown in FIG. 3B. Therefore, the EPA model is the most preferable configuration for the development of a BBB model.

[0114] Based on these data, we used the EPA model in further characterization of the BBB model and the E00 model for comparison.

[0115] [Functional expression of cell junctions in a human immortalized BBB model] To clarify whether intercellular junctions were formed and functioned in HBMEC / ci18 cells in the EPA model, we examined the expression levels and localization of adherens junction-related genes (VE-cadherin and β-catenin) and tight junction-related genes (ZO-1, claudin-5, and occludin) by qPCR and immunohistochemistry. As shown in Figure 4A, the mRNA levels of all of these genes were higher in the EPA model than in the E00 model. Consistent with this result, the expression levels of VE-cadherin and β-catenin proteins were also higher in the EPA model than in the E00 model (Figure 4B).

[0116] These results suggest that HASTR / ci35 and HBPC / ci37 cells promote the formation of cell-cell junctions (mainly AJs) in HBMEC / ci18 cells and are involved in the formation of a stronger intercellular barrier.

[0117] [Functional analysis of efflux transporters in a human immortalized BBB model] P-gp activity in the EPA and E00 models was examined by bidirectional transport analysis of R123 (a substrate of P-gp).The efflux rate (ER) of R123 was shown to be higher in the EPA model than in the E00 model (R123: 1.72 vs 1.31) (Figure 5).

[0118] We then compared the expression levels of P-gp in the EPA and E00 models by qPCR and immunocytochemistry, and found that the mRNA and protein expression levels of P-gp and BCRP in the EPA model were significantly higher than those in the E00 model (Figures 6A and 6B).

[0119] Furthermore, the subcellular localization of P-gp and BCRP in the EPA model was analyzed from images of vertical sections (Z-axis plane), and it was found that P-gp and BCRP were mainly localized at the apical membrane. These localization patterns were consistent with the results of the bidirectional transport analysis described above. On the other hand, the adherens junction protein β-catenin was localized at the cell-cell boundary, unlike P-gp and BCRP (Fig. 6B).

[0120] Taken together, the EPA model had better efflux transporter function compared with the E00 model, suggesting that co-culture with HASTR / ci35 and HBPC / ci37 cells induced the expression and function of P-gp and BCRP in HBMEC / ci18 cells in the EPA model.

[0121] [Evaluation of compound permeability using a human immortalized BBB model] We investigated whether the BBB model based on human immortalized cells can be used for compound permeability tests using Lucifer Yellow, a representative permeability marker. The results of the permeability tests using the E00 model and the EPA model showed that the permeability of Lucifer Yellow was low in both models, and that the value was lower in the EPA model than in the E00 model. Therefore, it was revealed that the BBB model based on human immortalized cells can be used for compound permeability tests, and that the EPA model has better functions than the E00 model.

[0122] [Evaluation of the medium] The results shown in Figure 8 indicate that the TEER value was highest when BPM was used as the basal medium in the EPA model, and it was confirmed that BPM enhances the co-culture effect of astrocytes and / or pericytes. The effect of the triple co-culture peaked on Day 1 in both models.

[0123] <3> Consideration As described above, an in vitro human BBB model was established by co-culturing three conditionally immortalized human BBB cell lines. This human immortalized cell-based BBB model is hereinafter referred to as "hiBBB90 model". The hiBBB90 model has unique advantages for BBB permeability screening of CNS drug candidates.

[0124] Regarding cell junction (barrier) function, the hiBBB90 model had a TEER value of 90 (Ω × cm 2 ), indicating that HBMEC / ci18 cells have a functional barrier function. Although it does not reach the values ​​observed in vivo (>1000) (References 21, 22) or in primary cell-based rat BBB models (~350) (Reference 14), the TEER value of the hiBBB90 model is close to that of primary human cell BBB models (140-260) (References 12, 37). The minimum TEER level required for a BBB model has not yet been determined, but to date, it has been estimated that the TEER value is between 150 and 400 (Ω × cm).2 It has been reported that in BBB models with TEER values ​​exceeding 100%, the Pe value of fluorescent compounds reaches a constant value (References 38, 39), and these values ​​can be considered as the benchmark TEER in the current BBB model. In addition, this model also shows low permeability to LY, a BBB impermeability marker, which also suggests that a functional barrier is formed.

[0125] Furthermore, the hiBBB90 model has efflux transporter function, which is supported by the results of immunocytochemical detection of P-gp / BCRP and the ER of R123 (1.72, respectively).

[0126] In addition, by optimizing the culture medium, we were able to further improve the function of this model. The most distinctive feature is the use of a culture medium for nerve tissue to reproduce the environment of the brain. This allows us to reproduce the brain environment more closely than conventional models, which is thought to be an important factor in establishing this model.

[0127] In addition to the BBB functions mentioned above, the excellent scalability is another unique advantage of the hiBBB90 model. The three immortalized cell lines can be cultured for a long period of time or repeatedly frozen / thawed without any phenotypic abnormalities. Furthermore, in this study, we developed a simple method for constructing spheroids, which is also a major advantage in carrying out research.

[0128] From the above, it is expected that the hiBBB90 model possesses both BBB functionality and practicality, and can be applied to various central nervous system drug development research, including BBB permeability screening of drugs.

[0129] The key to the development of the hiBBB90 model in this study is the change in the medium composition and co-culture conditions. In particular, the differentiation of HASTR / ci35 cells and HBPC / ci37 cells is unique to this model. Previously, the inventors have reported that the differentiation characteristics of HASTR / ci35 cells and HBPC / ci37 cells are significantly promoted by a culture temperature of 37°C (References 26, 33). It is presumed that this improvement in differentiation characteristics leads to a greater co-culture effect on HBMEC / ci18 cells and improved function as a BBB model. The molecular mechanism of the co-culture effect is unknown, but referring to References 2, 3, 5, 14, etc., for example, glial-derived neurotrophic factor, sonic hedgehog, and Wnt proteins secreted from astrocytes, or angiopep-1 secreted from pericytes, etc., are considered. Therefore, it is possible that the expression levels of these trophic factors in HASTR / ci35 and HBPC / ci37 cells increase with differentiation.

[0130] Finally, we briefly mention further utilization of the hiBBB90 model in CNS drug development. In general, the pharmacological action of a drug in the brain cannot be clearly related to its plasma concentration, but rather to the drug concentration in the brain. Therefore, we can obtain the in vivo steady-state K values ​​from the in vitro BBB model data, albeit in rats. p,uu,brain A method to predict the brain concentration-time profile of a drug in vivo has recently been proposed (Reference 40). Therefore, by using the hiBBB90 model, it may be possible to predict the brain concentration-time profile of a drug in vivo from human in vitro data (Reference 41). [Industrial Applicability]

[0131] The hiBBB90 model invented in this study retains basic BBB characteristics while also having excellent scalability and ease of handling. This model may be widely used in studies to evaluate the brain penetration of new CNS drugs and to elucidate the molecular mechanisms of BBB biology. Therefore, the hiBBB90 model according to the present invention is expected to become a new tool for accelerating various human BBB research, including CNS drug development.

[0132] References TIFF2025017371000009.tif227170TIFF2025017371000010.tif229170TIFF2025017371000011.tif226169TIFF2025017371000012.tif110168

Claims

1. (i) a pericyte culture step of seeding and culturing human conditionally immortalized pericytes in a first medium; (ii) a pericyte differentiation step of differentiating the cultured human conditionally-immortalized pericytes in a second medium; (iii) an astrocyte culturing step of seeding and culturing human conditionally-immortalized astrocytes in a third medium; (iv) an astrocyte differentiation step of differentiating the cultured human conditionally-immortalized astrocytes in a fourth culture medium; (v) a brain microvascular endothelial cell culture step of seeding the human conditionally immortalized brain microvascular endothelial cells in a fifth medium and culturing the seeded human conditionally immortalized brain microvascular endothelial cells; (vi) a further culturing step of further culturing the human conditionally immortalized pericytes and the human conditionally immortalized astrocytes in a sixth culture medium; Including, In the pericyte culture step (i) and / or the pericyte differentiation step (ii), a culture comprising human conditionally immortalized pericytes is formed, In the astrocyte culture step (iii) and / or the astrocyte differentiation step (iv), a culture comprising human conditionally immortalized astrocytes is formed, The differentiation in the pericyte differentiation step (ii) is carried out at a temperature different from the culture temperature in the pericyte culture step (i); the differentiation in the astrocyte differentiation step (iv) is carried out at a temperature different from the culture temperature in the astrocyte culture step (iii); After the steps (ii) and (iv), the brain microvascular endothelial cell culture step (v) is carried out; In the brain microvascular endothelial cell culture step (v), a culture containing human conditionally immortalized brain microvascular endothelial cells is formed; The brain microvascular endothelial cell culture step (v) and the further culture step (vi) are carried out together, A method for producing a multicellular blood-brain barrier model, in which a culture comprising the human conditionally immortalized pericytes and a culture comprising the human conditionally immortalized brain microvascular endothelial cells are arranged so as to be capable of interacting with each other.

2. The manufacturing method described in claim 1, wherein the sixth culture medium is different from any of the first culture medium, the second culture medium, the third culture medium, the fourth culture medium and the fifth culture medium.

3. 3. The method according to claim 1, wherein the culture of the seeded human conditionally-immortalized brain microvascular endothelial cells in the brain microvascular endothelial cell culture step (v), as well as the further culture of the human conditionally-immortalized pericytes and the further culture of the human conditionally-immortalized astrocytes, are carried out at a temperature different from that of the differentiation in the pericyte differentiation step (ii) and / or the differentiation in the astrocyte differentiation step (iv).

4. The culture in the pericyte culture step (i) is carried out at a temperature range of 32 to 34° C., and the differentiation in the pericyte differentiation step (ii) is carried out at a temperature range of 35 to 39° C., and / or The method according to any one of claims 1 to 3, wherein the culture in the astrocyte culture step (iii) is carried out at a temperature range of 32 to 34°C, and the differentiation in the astrocyte differentiation step (iv) is carried out at a temperature range of 35 to 39°C.

5. The method according to any one of claims 1 to 4, wherein in the brain microvascular endothelial cell culture step (v), the culture of the seeded human conditionally immortalized brain microvascular endothelial cells, as well as the further culture of the human conditionally immortalized pericytes and the further culture of the human conditionally immortalized astrocytes, are carried out at a temperature range of 32 to 34°C.

6. The method according to any one of claims 1 to 5, wherein the culture in the pericyte culture step (i) and the culture in the astrocyte culture step (iii) are each independently performed for 12 to 72 hours after seeding.

7. The method according to any one of claims 1 to 6, wherein the differentiation in the pericyte differentiation step (ii) and the differentiation in the astrocyte differentiation step (iv) are each independently performed for 12 to 72 hours.

8. The method according to any one of claims 1 to 7, wherein the brain microvascular endothelial cell culture step (v) is carried out for 12 to 240 hours after seeding.

9. The method according to any one of claims 1 to 8, wherein the culture in the pericyte culture step (i) and the culture in the astrocyte culture step (iii) are carried out simultaneously.

10. The method according to any one of claims 1 to 9, wherein the differentiation in the pericyte differentiation step (ii) and the differentiation in the astrocyte differentiation step (iv) are carried out simultaneously.

11. the culture comprising human conditionally immortalized pericytes formed in the pericyte culture step (i) and / or in the pericyte differentiation step (ii) is a layer; and / or The method according to any one of claims 1 to 10, wherein the culture containing human conditionally immortalized brain microvascular endothelial cells formed in the brain microvascular endothelial cell culture step (v) is a layer.

12. The method according to any one of claims 1 to 11, wherein the culture comprising human conditionally immortalized brain microvascular endothelial cells formed in the brain microvascular endothelial cell culture step (v) is a monolayer.

13. A culture comprising said human conditionally immortalized astrocytes is positioned on one side of the culture comprising said human conditionally immortalized pericytes; The method according to any one of claims 1 to 12, wherein a culture containing human conditionally immortalized pericytes is located on the other side of the culture containing human conditionally immortalized brain microvascular endothelial cells.

14. The method according to any one of claims 1 to 13, wherein the culture containing the human conditionally immortalized pericytes and the culture containing the human conditionally immortalized brain microvascular endothelial cells are formed on each surface of a porous substrate, and the culture containing the human conditionally immortalized pericytes and the culture containing the human conditionally immortalized brain microvascular endothelial cells are arranged so as to be capable of interacting with each other.

15. The method according to claim 14 , wherein the porous substrate is coated with molecules that constitute an extracellular matrix.

16. The method according to any one of claims 1 to 15, wherein the third medium and the fourth medium differ from each other in that the serum contained in the third medium is not contained in the fourth medium.

17. The method according to any one of claims 1 to 16, wherein the fifth culture medium is a culture medium that does not contain growth factors.

18. A multicellular blood-brain barrier model produced by the method according to any one of claims 1 to 17.

19. (A) a culture comprising human conditionally immortalized brain microvascular endothelial cells; (B) A culture comprising human conditionally immortalized pericytes; (C) A culture comprising human conditionally immortalized astrocytes; (D) a culture medium for the human conditionally immortalized brain microvascular endothelial cells; (E) a medium for the human conditionally immortalized pericytes and the human conditionally immortalized astrocytes; A multicellular blood-brain barrier model comprising: The culture (A) containing the human conditionally immortalized brain microvascular endothelial cells and the culture (B) containing the human conditionally immortalized pericytes are arranged so as to be capable of interacting with each other; A culture (A) containing human conditionally immortalized brain microvascular endothelial cells is located on one side of a culture (B) containing human conditionally immortalized pericytes; A culture containing human conditionally immortalized pericytes (B) is placed on the other side of the culture containing human conditionally immortalized astrocytes (C); A multicellular blood-brain barrier model, in which the medium (D) is different from the medium (E).

20. The culture (A) containing human conditionally immortalized brain microvascular endothelial cells is a layer, and / or 20. The multicellular blood-brain barrier model of claim 19, wherein the culture (B) containing human conditionally immortalized pericytes is a layer.

21. 21. The multicellular blood-brain barrier model of claim 19 or 20, wherein the culture (A) containing human conditionally immortalized brain microvascular endothelial cells is a monolayer.

22. Further comprising a porous substrate; The culture (A) containing human conditionally immortalized brain microvascular endothelial cells covers one side of a porous substrate; The multicellular blood-brain barrier model according to any one of claims 19 to 21, wherein the culture (B) containing the human conditionally immortalized pericytes covers the other side of the porous substrate.

23. The medium (D) is a medium containing no growth factors, The multicellular blood-brain barrier model according to any one of claims 19 to 22, wherein the medium (E) is a medium for nerve cells.

Images