JP7650480B2 - Blood-brain barrier model using human conditionally immortalized cells and its production method - Google Patents

Description

(1) ▲1▼Publication date: June 20, 2019 ▲2▼Publication: Published in the abstracts of the 13th International conference on Cerebral Vascular Biology (Location: Miami, Florida, USA, Mariott Miami Biscayne Bay Hotel, Date and time: June 25, 2019)

(2) ▲1▼Date: June 25, 2019 ▲2▼Meeting name and venue: 13th International conference Cerebral Vascular Biology, Miami, Florida, USA, held at the Mariott Miami Biscayne Bay Hotel

Applicable to Article 30, Paragraph 2 of the Patent Act (3) ▲1▼Publication date: October 15, 2019 ▲2▼Publication: Published in Molecular Pharmaceuticals, 2019 Nov 4; 16 (11): 4461-4471

(4) ▲1▼Publication date: November 26, 2019 ▲2▼Publication: Published in the abstracts of the 34th Annual Meeting of the Japanese Society for the Study of Xenobiotics (Location: Tsukuba International Congress Center, Tsukuba, Ibaraki Prefecture, Date and time: December 10, 2019)

Article 30, paragraph 2 of the Patent Act applies (5) ▲1▼Date: December 10, 2019 ▲2▼Meeting name and venue: The 34th Annual Meeting of the Japanese Society for the Study of Xenobiotics, Tsukuba International Congress Center, Tsukuba, Ibaraki Prefecture

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

The blood-brain barrier (BBB) is formed by brain microvascular endothelial cells (BMEC) and supporting cells such as astrocytes and pericytes that exist around them (Non-Patent Document 1, Non-Patent Document 2). BMEC is a major component 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).

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 BMEC (Non-Patent Document 6, Non-Patent Document 7, Non-Patent Document 8, Non-Patent Document 9).

Due to these special features, identifying drug candidates with excellent BBB permeability has been a major challenge in the development of CNS drugs. 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.

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 between the BBB functions of animals and humans, as indicated by the different expression levels of BBB transporters (Non-Patent Document 13, Non-Patent Document 14, Non-Patent Document 15). Therefore, a human-derived BBB model is necessary for the development of CNS drugs.

Petzold, G. C.; Murthy, V. N. Role of astrocytes in neurovascular coupling. Neuron 2011, 71 (5), 782-797. Sa-Pereira, I.; Brites, D.; Brito, M. A. Neurovascular unit: a focus on pericytes. Mol. Neurobiol. 2012, 45 (2), 327-347. Engelhardt, B.; Liebner, S. Novel insights into the development and maintenance of the blood-brain barrier. Cell Tissue Res. 2014, 355 (3), 687-699. Pardridge, W. M. The blood-brain barrier: bottleneck in brain drug development. NeuroRx 2005, 2 (1), 3-14. Pardridge, W. M. Blood-brain barrier delivery. Drug Discovery Today 2007, 12 (1-2), 54-61. Nitta, T.; Hata, M.; Gotoh, S.; Seo, Y.; Sasaki, H.; Hashimoto, N.; Furuse, M.; Tsukita, S. Size-selective loosening of the blood-brain barrier in claudin-5-deficient mice. J. Cell Biol. 2003, 161 (3), 653-660. Stamatovic, S. M.; Johnson, A. M.; Keep, R. F.; Andjelkovic, A. V. Junctional proteins of the blood-brain barrier: New insights into function and dysfunction. Tissue Barriers 2016, 4 (1), e1154641. Muzi, M.; Mankoff, D. A.; Link, J. M.; Shoner, S.; Collier, A. C.; Sasongko, L.; Unadkat, J. D. Imaging of cyclosporine inhibition of Pglycoprotein activity using 11C-verapamil in the brain: studies of healthy humans. J. Nucl. Med. 2009, 50 (8), 1267-1275. Kodaira, H.; Kusuhara, H.; Ushiki, J.; Fuse, E.; Sugiyama, Y. Kinetic analysis of the cooperation of P-glycoprotein (P-gp/Abcb1) and breast cancer resistance protein (Bcrp/Abcg2) in limiting the brain and testis penetration of erlotinib, flavopiridol, and mitoxantrone. J. Pharmacol. Exp. Ther. 2010, 333 (3), 788-796. Megard, I.; Garrigues, A.; Orlowski, S.; Jorajuria, S.; Clayette, P.; Ezan, E.; Mabondzo, A. A co-culture-based model of human blood-brain barrier: application to active transport of indinavir and in vivo-in vitro correlation. Brain Res. 2002, 927 (2), 153-167. Thomsen, L. B.; Burkhart, A.; Moos, T. A Triple Culture Model of the Blood-Brain Barrier Using Porcine Brain Endothelial cells, Astrocytes and Pericytes. PLoS One 2015, 10 (8), e0134765. Nakagawa, S.; Deli, M. A.; Kawaguchi, H.; Shimizudani, T.; Shimono, T.; Kittel, A.; Tanaka, K.; Niwa, M. A new blood-brain barrier model using primary rat brain endothelial cells, pericytes and astrocytes. Neurochem. Int. 2009, 54 (3-4), 253-263. Syvanen, S.; Lindhe, O.; Palner, M.; Kornum, B. R.; Rahman, O.; Langstrom, B.; Knudsen, G. M.; Hammarlund-Udenaes, M. Species differences in blood-brain barrier transport of three positron emission tomography radioligands with emphasis on P-glycoprotein transport. Drug Metab. Dispos. 2009, 37 (3), 635-643. Uchida, Y.; Ohtsuki, S.; Katsukura, Y.; Ikeda, C.; Suzuki, T.; Kamiie, J.; Terasaki, T. Quantitative targeted absolute proteomics of human blood-brain barrier transporters and receptors. J. Neurochem. 2011, 117 (2), 333-345. Warren, M. S.; Zerangue, N.; Woodford, K.; Roberts, L. M.; Tate, E. H.; Feng, B.; Li, C.; Feuerstein, T. J.; Gibbs, J.; Smith, B.; de Morais, S. M.; Dower, W. J.; Koller, K. J. in five species including human. Pharmacol. Res. 2009, 59 (6), 404-413.

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

In the context of the above, the present inventors established human immortalized cells derived from BMEC, 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, and 26).

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. This allows cells to redifferentiate as soon as the immortalization signal disappears (References 24, 25, 26, 30, 31). Based on the function of the above 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.

These human immortalized BBB cells are expected to contribute to the development of a human BBB model for 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 previously established, and characterized the BBB model.

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 culture 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;
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 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;
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 production method according to [1] or [2], wherein the culture in the pericyte culture step (i) is performed at a temperature range of 32 to 34°C, and the differentiation in the pericyte differentiation step (ii) is performed at a temperature range of 35 to 39°C, and/or the culture in the astrocyte culture step (iii) is performed at a temperature range of 32 to 34°C, and the differentiation in 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 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 in the brain microvascular endothelial cell culture step (v) 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 production 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 human conditionally immortalized pericytes and the culture containing human conditionally immortalized brain microvascular endothelial cells are formed on each surface of a porous substrate, so that the culture containing human conditionally immortalized pericytes and the culture containing human conditionally immortalized brain 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 production 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 production 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) 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 multicellular blood-brain barrier model comprising the human conditionally immortalized pericytes and a culture medium for the human conditionally immortalized astrocytes,
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 placed 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 described in [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:
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 [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 [19] to [22], wherein the medium (E) is a medium for nerve cells.

The present invention provides a blood-brain barrier model using human immortalized cells.

FIG. 1A shows the cell morphology of the conditionally immortalized human brain microvascular endothelial cell line HBMEC/ci18 observed by microscopy. 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 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 ID 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 count was performed in duplicate. 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-brain 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 glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels and results are the mean ± SD of values obtained from three independent experiments, qPCR in each experiment was performed in duplicate. *p<0.05, **p<0.01. 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 astrocyte (HASTR)/ci35 cells and human brain pericyte (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 the 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 onto the culture inserts (apical membrane side) using VascuLife complete medium (Vas-V/E-free medium) that does not contain VEGF or 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"). FIG. 2B shows six types of BBB models made 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 alphabet means 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 inside the insert and HBPC/ci137 cells or HASTR/ci35 cells outside the insert. The E0A model is co-cultures of HBMEC/ci18 cells inside the insert and HASTR/ci35 cells at the bottom of the well. The EPA model was 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 seeded with 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. FIG. 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 in FIG. 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" indicates "HBMEC/ci18 cells", "A" indicates "HASTR/ci35 cells", and "P" indicates "HBPC/ci37". **p<0.01, ***p<0.001 vs. E00, ##, p<0.01; ###, p<0.001 vs. E0A. 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 shows the expression profile of cell-cell junction-related genes in the human immortalized BBB model. 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 folds relative to the values in the E00 model. Data are the means (and SD) of values obtained from three independent experiments. Each qPCR was performed using duplicate samples (*P<0.05). 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. FIG. 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 minutes. The medium was collected from the opposite side and the R123 concentration was measured. Based on the measured concentrations, the values of the permeability coefficient PeAB from the apical membrane to the basolateral membrane and the permeability coefficient 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 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 difference relative to the values in 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 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, x-y plane images are shown in the center of each figure, and y-z and x-z 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 shows the results of permeability assay of Lucifer Yellow (BBB impermeability marker) in E00 model (12 wells) and EPA model (12 wells). Lucifer Yellow (10 μM) was added to the apical membrane side and then left for 30, 60 or 90 minutes. The medium was collected from the wells and the concentration of Lucifer Yellow was determined. Permeability coefficient (Pe) values were calculated based on this concentration. Data are shown as the mean (and SD) from three independent experiments. Each experiment was performed with duplicate samples (*P<0.05; **P<0.01 vs E00). FIG. 8 shows the results of the transepithelial electrical resistance (TEER) values measured on Day 1 after the start of co-culture for the EPA models prepared using various media on the basal side. The E00 model and the EP0 model were used for comparison. 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.

The present invention will be described in more detail below.

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.

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 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. Each step will be described in detail below.

<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 in the range of 32 to 34°C, and more preferably at 33°C. Cultivation at a lower or higher temperature may result in a decrease in the cell proliferation rate.

The culture in the pericyte culture step (i) is carried out, for example, for 12 to 72 hours after seeding, preferably 18 to 60 hours, more preferably 20 to 48 hours, and even more preferably 22 to 36 hours. 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 model creation takes time and is not efficient.

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

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

<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 by 1° C. or more, preferably at a temperature higher by 2° C. or more, more preferably at a temperature higher by 3° C. or more, and particularly preferably at a temperature higher by 4° C. or more. More specifically, the differentiation of the human conditionally immortalized pericytes is carried out at a temperature higher by, for example, 1 to 7° C., preferably at a temperature higher by 2 to 6° C., more preferably at a temperature higher by 3 to 5° C., even more preferably at a temperature higher by 3.5 to 4.5° C., and most preferably at a temperature higher by 4° C. than the culture temperature in the pericyte culture step.

In this process, 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.

As described later in the Examples, in specific experiments, the 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 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.

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 even more preferably 22 to 36 hours. In one embodiment, the differentiation in the pericyte differentiation step (iii) is carried out for 24 hours.

In this step, a medium known in the art and commonly used for differentiation of pericytes 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 Fetal Bovine Serum (FBS) (Thermo Fisher Scientific, Waltman, CA), growth factors include Pericyte Growth Factors (ScienCell), and antibiotics include, but are not limited to, penicillin/streptomycin (pen/st, Wako, Osaka) and blasticidin S (nacali tesque, Kyoto).

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 differ 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.

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.

When the term "layer" is used in this specification 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 areas 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.

<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 culture of human conditionally immortalized astrocytes in this step is 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.

The culture in the astrocyte culture step (iii) is carried out, for example, for 12 to 72 hours after seeding, preferably 18 to 60 hours, more preferably 20 to 48 hours, and even more preferably 22 to 36 hours. 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.

The human conditionally immortalized astrocytes are preferably seeded in a container into which a further container can be inserted, for example as shown in FIG. 2B. In one embodiment, the container has a flat bottom.

The third medium into which the astrocytes are seeded in this process may 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.). Antibiotics, serum, N2 supplement (Wako), etc. may be added to the third medium as appropriate.

<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 by 1° C. or more, preferably at a temperature higher by 2° C. or more, more preferably at a temperature higher by 3° C. or more, and particularly preferably at a temperature higher by 4° C. or more. More specifically, the differentiation of the human conditionally immortalized astrocytes is carried out at a temperature higher by, for example, 1 to 7° C., preferably at a temperature higher by 2 to 6° C., more preferably at a temperature higher by 3 to 5° C., even more preferably at a temperature higher by 3.5 to 4.5° C., and most preferably at a temperature higher by 4° C. than the culture temperature in the astrocyte culture step.

Specifically, the differentiation of human conditionally immortalized astrocytes in this process is 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.

As described later in the Examples section, in specific experiments, the 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 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.

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 even more 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.

The fourth medium used to differentiate astrocytes in this process can be a differentiation medium known in the art that is commonly used for astrocyte differentiation, 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.).

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.

Here, examples of serum include, but are not limited to, FBS. The third culture medium contains serum, which promotes cell proliferation of astrocytes, and the fourth culture medium does not contain serum, which prevents inhibition of astrocyte differentiation.

In another embodiment, the medium used in the astrocyte differentiation step (fourth medium) may contain an astrocyte differentiation promoter. Examples of astrocyte differentiation promoters include, but are not limited to, cAMP and its analogs (dBcAMP, etc.).

In one embodiment, the fourth medium may optionally contain an antibiotic. In another embodiment, if 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.

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. In addition, the culture containing human conditionally immortalized astrocytes may be a cell mass or a complex three-dimensional shape.

The culture in the pericyte culture step (i) and the culture in the astrocyte culture step (iii) are carried out independently within the above-mentioned time range, for example. In a preferred embodiment, the culture in the pericyte culture step (i) and the culture in the astrocyte culture step (iii) are carried out simultaneously within the above-mentioned time range. By carrying out these cultures simultaneously, the time required for model preparation can be shortened.

In addition, the differentiation in the pericyte differentiation step (ii) and the differentiation in the astrocyte differentiation step (iv) are carried out independently, for example, within the above-mentioned time range. 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 differentiations simultaneously, it is possible to shorten the time required for model production.

<Brain microvascular endothelial cell culture step (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.

In this process, 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 mimicked.

In this process, 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.

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 differentiation in the astrocyte differentiation step (step (iv)). They are carried out at a temperature 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, they are carried out at a temperature lower by, for example, 1 to 7°C, 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.

In one embodiment, the culture of the human conditionally immortalized brain microvascular endothelial cells in this process, as well as the further culture of the human conditionally immortalized pericytes and human conditionally immortalized astrocytes, is carried out, for example, at a temperature in the range of 31 to 35°C, preferably in the range of 32 to 34°C, and more preferably at 33°C.

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 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 human conditionally immortalized astrocytes are carried out for 24 hours after the seeding of the human conditionally immortalized brain microvascular endothelial cells.

Here, the culture containing human conditionally immortalized brain microvascular endothelial cells mimics the apical membrane side of the blood-brain barrier (BBB) structure in vivo, and 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.

In the culture of human conditionally immortalized brain microvascular endothelial cells in this process, 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 to this. 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.

In the further culture of the human conditionally immortalized pericytes and human conditionally immortalized astrocytes in this process, 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. Examples of the sixth medium include, but are not limited to, neurobasal medium (Thermo Fisher Scientific). The medium may be appropriately supplemented with antibiotics, N2 supplements, etc. In one embodiment, the sixth medium can be Neurobasal medium (Thermo Fisher Scientific), to which pen/st, N2 supplement containing apotransferrin, and L-glutamine may be added (the medium prepared in this manner is also called brain parenchymal medium (BPM)).

In the multicellular blood-brain barrier model produced 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.

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.

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 in communication with 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 of the material include, but are not limited to, porous membranes commonly used in the field, such as polyethylene terephthalate.

The size of the pores in the porous substrate may be appropriately selected so long as it 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 with the formation of the cell culture (e.g., layer or mass). An example of a commercially available porous substrate is translucent polyethylene terephthalate, 0.4 μm high-density pores (BD Falcon, Franklin Lakes, NJ).

The porous substrate is preferably coated with molecules constituting the 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.

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 appropriately adjusted.

<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.

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

A third aspect of the present invention is a method for producing a composition comprising the steps of:
(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 multicellular blood-brain barrier model comprising said human conditionally immortalized pericytes and a culture medium for said human conditionally immortalized astrocytes.

In this model, the culture (A) containing human conditionally immortalized brain microvascular endothelial cells and the culture (B) containing human conditionally immortalized pericytes are arranged so as to be capable of interacting 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 case 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.

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

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

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

In one aspect, 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 carries 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.

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, thereby reproducing a BBB model that mimics the structure in vivo (e.g., Figures 2B, 3A, and 8). The first container and the second container preferably have a flat bottom.

Here, the arrangement of the cultures (A), (B), and (C) and the arrangement of the first container and the second container are not limited to those described above, and may be changed as appropriate to the extent that the structure or function of the BBB in the body can be mimicked. In addition, the shapes of the first container and the second container may also be changed as appropriate to the extent that the structure or function of the BBB in the body can be mimicked.

As mentioned above, in vitro blood-brain barrier (BBB) models are important research tools for developing brain-targeted 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. 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.

<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 compositions of the media used are shown in Table 1.

Vas-comp is a mixture of VascuLife basal medium (KURABO, Osaka, Japan) containing 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, and 10 mM L-glutamine (Wako, Osaka, Japan). 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).

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

Pericyte medium (PM) was made by adding 2% (v/v) FBS, 1% (w/v) pericyte growth factor, and 1% pen/st (all purchased from ScienCell, San Diego, CA) to pericyte basal medium.

All immortalized cells were cultured in type I collagen-coated dishes under conditions of 5% CO ₂ /95% air at 33° C. All cells were subcultured every 3 days using 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA).

[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.

[Cell proliferation analysis]
HASTR/ci18 were seeded at a concentration of 5.0×10 ⁴ 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.

[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 sections of this specification. The amplification efficiency of each qPCR was confirmed to be close to 1. The level of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA in each cell type was determined as a standardization control. The delta-delta CT method was used to determine the relative expression levels of these values.

[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). For nuclear counterstaining, 4',6-diamidino-2-phenylindole (DAPI) (Dojindo, Kumamoto, Japan) was used. The cells were encapsulated 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).

[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 a 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 hour (extracellular matrix coating). The insert was air-dried and washed twice with phosphate-buffered saline (PBS).

For the co-culture model, HASTR/ci35 (2.5× ¹⁰⁴ cells/ ^cm2 ) and HBPC/ci37 (3.0× ¹⁰⁴ cells/ ^cm2 ) were seeded on the outside of the membrane of the insert (0.33 ^cm2 for 24-well, 0.9 ^cm2 for 12-well) or on the bottom of the plate (BD Falcon) (2.0 ^cm2 for 24-well or 4.0 ^cm2 for 12-well).

HASTR/ci35 were cultured in AGM and HBPC/ci37 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 the AGM was replaced with astrocyte differentiation medium (ADM) to differentiate HASTR/ci35 and HBPC/ci37 cells.

Here, the ADM is
It has the same composition as AGM except that (i) it does not contain FBS and blasticidin S, and (ii) it contains 1 mM dibutyryl cyclic adenosine monophosphate (dBcAMP, Santa Cruz Biotech, Santa Cruz, Calif.) (see FIG. 2A, Step 2).

On Day 0, to start the co-culture (see FIG. 2A, Step 3), HBMEC/ci18 cells (1.3× ¹⁰⁵ cells/ ^cm2 ) were seeded inside the insert in which the HBPC/ci37 (or HASTR/ci35) cells were cultured, and immediately thereafter the insert containing the two cell types was moved to the well in which the HASTR/ci35 (or HBPC/ci37) cells were cultured. For the sole culture of HBMEC/ci18 cells, the cells were seeded inside the insert in the absence of other cells (see FIG. 2B).

In all types of BBB models (see FIG. 2B),
On the apical membrane side (insert), VascuLife complete medium (Vas-V/E-free medium) not containing VEGF or hEGF was used.
Brain parenchymal medium (BPM) was used on the basal side (well).

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).

BPM consists of Neurobasal medium (Thermo Fisher Scientific) supplemented with 100 units/mL penicillin-100 μg/mL streptomycin, 1% (v/v) N2 supplement containing 1% (v/v) apotransferrin, and 2 mM L-glutamine (see Table 1-4).

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

[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 TEER values (Ω×cm ² ).

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

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

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

The above formula used the apical (C _apical ) and basal (C _basal ) compound concentrations, and the apical (V _apical =750 μL) and basal (V _basal =2250 μL) volumes. Cleared volume was plotted against time to obtain the slope of the clearance line for the BBB model (PS _total ) and cell-free inserts (PS _insert ). For the endothelial monolayer (PS _e ), the permeability×area value was determined using the following formula:

Finally, the Pe value was obtained by dividing the PS _e value by the surface area A (for 12-well inserts A: 0.9 cm ² ) as shown below.

[In vitro BBB permeability test]
Lucifer Yellow permeability assay was performed using HBMEC/ci18 cells cultured alone (E00), HASTR/ci35 cells cocultured with 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 pre-incubated at 33°C for 10 minutes. Lucifer Yellow (LY, 10 μM, Wako) was then added to the apical membrane.

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

[Statistical analysis]
Statistical analysis was performed using Excel with ystat2006 (Igakutosho Publishing 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.

[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, but without serum)
・BPM (Table 1-4)

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

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).

To examine whether HBMEC/ci18 cells exhibited gene expression characteristics specific to brain vascular endothelial cells, we then 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 BMEC (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).

In summary, HBMEC/ci18 cells were considered to have basic BMEC characteristics. Therefore, we subsequently developed an in vitro BBB culture model using HBMEC/ci18.

[Development of an in vitro BBB model based on human immortalized cells]
Astrocytes and pericytes are known to play important roles 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).

To achieve this, the inventors have worked on developing a new culture method. For example, to promote the maturation of HASTR/ci35 and HBPC/ci3 cells, serum-free medium and a culture temperature of 37°C were used (Step 2). In addition, to enhance the function of vascular endothelial cells, an endothelial medium without VEGF and hEGF (References 35, 36), which act suppressively on BBB formation, was used.

Under these conditions, six BBB model types were generated, 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 co-cultured with HASTR/ci35 (HASTR/ci35 is on the outside of the insert)
(4) E0A: HBMEC/ci18 cells co-cultured with HASTR/ci35 (HASTR/ci35 is 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, HBPC/ci37 on the bottom of the well)

The results of TEER analysis shown in Figure 3A indicated that the TEER value of the co-culture model was higher than that of the monoculture E00 model (60.3 ± 4.1 Ω × cm ² ). Among the co-culture models, the EPA model had the highest TEER value (93.0 ± 3.4 Ω × cm ² ). Thus, as shown in Figure 3B, these TEER levels were maintained at least until Day 5. Therefore, the EPA model is the most preferred configuration for the development of a BBB model.

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

[Functional expression of cell junctions in a human immortalized cell BBB model]
To clarify whether intercellular junctions were formed and functioning 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).

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

[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).

The expression levels of P-gp in the EPA and E00 models were then compared by qPCR and immunocytochemistry. The results showed 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).

Furthermore, analysis of the intracellular localization of P-gp and BCRP in the EPA model from images of vertical sections (Z-axis plane) revealed that P-gp and BCRP were mainly localized to the apical membrane. These localization patterns were in good agreement with the results of the bidirectional transport analysis described above. On the other hand, β-catenin, an adherens junction protein, was localized to the cell-cell boundary, unlike P-gp and BCRP (Figure 6B).

In summary, the EPA model had better efflux transporter function compared to 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.

[Evaluation of compound permeability using a human immortalized cell BBB model]
Whether the BBB model using human immortalized cells can be used for compound permeability tests was examined 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 the value was lower in the EPA model than in the E00 model. Therefore, it was revealed that the BBB model using human immortalized cells can be used for compound permeability tests, and furthermore, the EPA model has better functions than the E00 model.

[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 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.

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

Regarding cell junction (barrier) function, the TEER value of 90 (Ω × cm ² ) in the hiBBB90 model indicates 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 a rat primary cell-based BBB model (~350) (Reference 14), the TEER value of the hiBBB90 model is close to that of a human primary cell BBB model (140-260) (References 12, 37). Although the minimum TEER level required for a BBB model has not yet been determined, it has been reported that the Pe value of fluorescent compounds reaches a certain level in BBB models showing TEER values exceeding 150-400 (Ω × cm ² ) (References 38, 39), and these values can be considered as the benchmark TEER in BBB models at present. Furthermore, this model showed low permeability to LY, a BBB non-permeable marker, suggesting that a functional barrier was formed.

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

In addition, by optimizing the culture medium, we were able to further improve the functionality of this model. Its most distinctive feature is the use of a neuronal culture medium to reproduce the environment of the brain. This allowed us to reproduce the brain environment more closely compared to conventional models, which is thought to be an important factor in establishing this model.

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

From the above, the hiBBB90 model has both BBB functionality and practicality, and is expected to be applicable to various central nervous system drug development research, including BBB permeability screening of drugs.

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. The inventors have previously 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 increases the co-culture effect on HBMEC/ci18 cells and improves their function as a BBB model. The molecular mechanism of the co-culture effect is unknown, but referring to References 2, 3, 5, 14, etc., it is possible that, for example, glial-derived neurotrophic factor, sonic hedgehog, and Wnt protein secreted from astrocytes, or angiopep-1 secreted from pericytes, etc. Therefore, it is possible that the expression levels of these trophic factors in HASTR/ci35 and HBPC/ci37 cells increase during differentiation.

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, a method for predicting the in vivo steady-state K _p,uu,brain from in vitro BBB model data has been recently proposed, albeit in rats (Reference 40). Therefore, by using the hiBBB90 model, it may be possible to predict the human in vivo brain concentration-time profile of a drug from human in vitro data (Reference 41).

The hiBBB90 model invented in this study retains basic BBB characteristics while offering excellent scalability and ease of handling. This model may be widely applicable to evaluating the brain delivery of new CNS drugs and for research into the molecular mechanisms of BBB biology. Therefore, the hiBBB90 model of the present invention is expected to become a new tool for accelerating various human BBB research projects, including the development of CNS pharmaceuticals.

References

Claims (21)

(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,
The culture comprising the human conditionally immortalized pericytes and the culture comprising the human conditionally immortalized brain microvascular endothelial cells are arranged so as to be capable of interacting with each other,
A method for producing a multicellular blood-brain barrier model, 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 and is a culture medium for neural cells . 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 performed 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). The method according to claim 1 or 2, wherein the culture in the pericyte culture step (i) is performed at a temperature range of 32 to 34°C, the differentiation in the pericyte differentiation step (ii) is performed at a temperature range of 35 to 39°C, and/or the culture in the astrocyte culture step (iii) is performed at a temperature range of 32 to 34°C, and the differentiation in the astrocyte differentiation step (iv) is performed at a temperature range of 35 to 39°C. The method according to any one of claims 1 to 3, wherein the culture of the seeded human conditionally immortalized brain microvascular endothelial cells, the further culture of the human conditionally immortalized pericytes, and the further culture of the human conditionally immortalized astrocytes in the brain microvascular endothelial cell culture step (v) is carried out at a temperature range of 32 to 34°C. The method according to any one of claims 1 to 4, wherein the culture in the pericyte culture step (i) and the culture in the astrocyte culture step (iii) are each performed independently for 12 to 72 hours after seeding. The method according to any one of claims 1 to 5, wherein the differentiation in the pericyte differentiation step (ii) and the differentiation in the astrocyte differentiation step (iv) are each performed independently for 12 to 72 hours. The method according to any one of claims 1 to 6, wherein the brain microvascular endothelial cell culture step (v) is carried out for 12 to 240 hours after seeding. The method according to any one of claims 1 to 7, wherein the pericyte culture step (i) and the astrocyte culture step (iii) are performed simultaneously. The method according to any one of claims 1 to 8, wherein differentiation in the pericyte differentiation step (ii) and differentiation in the astrocyte differentiation step (iv) are carried out simultaneously. 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 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. 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 monolayer. 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 11, 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. The method according to any one of claims 1 to 12, wherein 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, and are arranged so that the culture containing human conditionally immortalized pericytes and the culture containing human conditionally immortalized brain microvascular endothelial cells can interact with each other. The method according to claim 13, wherein the porous substrate is coated with molecules that constitute the extracellular matrix. The manufacturing method according to any one of claims 1 to 14, wherein the third medium and the fourth medium differ in that the serum contained in the third medium is not contained in the fourth medium. The method according to any one of claims 1 to 15, wherein the fifth culture medium is a culture medium that does not contain growth factors. A multicellular blood-brain barrier model produced by the method according to any one of claims 1 to 16. (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 multicellular blood-brain barrier model comprising the human conditionally immortalized pericytes and a culture medium for the human conditionally immortalized astrocytes,
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);
The medium (D) is a medium containing no growth factors,
The multicellular blood-brain barrier model, wherein the medium (E) is a medium for neural cells. The culture (A) containing human conditionally immortalized brain microvascular endothelial cells is a layer, and/or
19. The multicellular blood-brain barrier model of claim 18, wherein the culture (B) containing human conditionally immortalized pericytes is a layer. The multicellular blood-brain barrier model according to claim 18 or 19, wherein the culture (A) containing human conditionally immortalized brain microvascular endothelial cells is a monolayer. 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 18 to 20, wherein the culture (B) containing the human conditionally immortalized pericytes covers the other side of the porous substrate.

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