JP2021159015A - Blood brain barrier model using human conditionally immortalized cell, and method for manufacturing the same - Google Patents

Abstract

To provide a blood brain barrier model using human immortalized cells, and a method for manufacturing the model.SOLUTION: A method for manufacturing a multicellular blood brain barrier model includes the steps of: (i) disseminating and culturing human conditionally immortalized pericytes on a first culture medium; (ii) differentiating the cultured pericytes on a second culture medium; (iii) disseminating and culturing the human conditionally immortalized astrocytes on a third culture medium; (iv) differentiating the cultured astrocytes on a fourth culture medium; and (v) disseminating the human conditionally immortalized brain microvascular endothelial cells on a fifth culture medium and culturing the disseminated brain microvascular endothelial cells.SELECTED DRAWING: Figure 2A

Description

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

The blood-brain barrier (BBB) is formed by brain microvascular endothelial cells (Brain Microvasual Endothelial Cells; BMEC) and supporting cells such as astrocytes and pericytes existing around the blood-brain barrier (BBB) (Non-Patent Document 1). , Non-Patent Document 2). Although BMEC is a major component of BBB, both astrocytes and pericytes are required to acquire BBB-specific properties, both of which play important roles in the control and maintenance of BBB. Fulfill (Non-Patent Document 3).

The BBB provides an important interface between the peripheral and central nervous system (CNS), maintains independent physiological homeostasis, and contains potentially harmful chemicals such as drugs. Prevents entry into the brain (Non-Patent Document 4, Non-Patent Document 5). The two major molecular entities that make up the BBB are (1) intercellular junctions (tight junctions and tight junctions) that strictly limit the paracellular influx of the drug into the brain; and (2) on the apical membrane side of the BMEC. A plurality of efflux transporters (including P-glycoprotein (P-gp) and breast cancer resistant protein (BCRP)) that actively carry out various drugs (Non-Patent Document 6, Non-Patent Document 7, Non-Patent Document 8, Non-Patent Document 9).

Due to these special functions, identification of drug candidates with excellent BBB permeability has been a major challenge in the development of CNS drugs. Under these circumstances, the in vitro BBB model is expected to provide a valuable tool for facilitating the specific process of BBB-permeable CNS drugs.

Since the single culture system of BMEC does not accurately mimic in vivo BBB, co-culture with astrocytes and / or pericytes is considered to be a useful method for developing BBB models with advanced and special functions. (Non-Patent Document 10, Non-Patent Document 11, Non-Patent Document 12). To achieve this, BBB cells from a variety of animal species have been widely used, but how many animal and human BBB functions are indicated by different levels of expression of the BBB transporter. It has been clarified that there is such a difference (Non-Patent Document 13, Non-Patent Document 14, Non-Patent Document 15). Therefore, a human-derived BBB model is required 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, DA; Link, JM; Shoner, S .; Collier, AC; Sasongko, L .; Unadkat, JD 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, MA; 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, BR; Rahman, O .; Langstrom, B .; Knudsen, GM; 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, MS; Zerangue, N .; Woodford, K .; Roberts, LM; Tate, EH; Feng, B .; Li, C .; Feuerstein, TJ; Gibbs, J .; Smith, B .; de Morais, SM Dower, WJ; Koller, KJ Comparative gene expression profiles of ABC transporters in brain microvessel endothelial cells and brain in five species including human. Pharmacol. Res. 2009, 59 (6), 404-413.

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

Against this background, we have two immortalizing genes: (1) human telomerase that extends telomere length and counters genomic damage caused by telomere shortening associated with growth arrest and cell division. Reverse transcriptase (hTERT) gene; (2) Human immortality derived from BMEC, astrosites and brain pericite by introducing the temperature-sensitive Simianvirus 40 macrotumor antigen (tsSV40T) gene into human primary BBB cells. Telomerase cells were established (Reference 24, Reference 25, Reference 26).

The tsSV40T imparts conditional immortalization properties to cells. Specifically, it is stable at a culture temperature of 33 ° C. and promotes the cell cycle, but is rapidly decomposed at a temperature of 37 ° C. or higher. As a result, the cells redifferentiate at the same time as the immortalization signal disappears (Reference 24, Reference 25, Reference 26, Reference 30, Reference 31). Based on the functions of the above two genes, human immortalized BBB cells retain important cellular properties that exhibit a continuous proliferative profile. This is a clear advantage over conventional cell types in drug development in view of various applicability.

These human immortalized BBB cells are expected to contribute to the development of human BBB models in drug permeability studies. The present inventors have established the human immortalized astrocyte cell line (HASTR / ci35), the human immortalized brain pericite cell line (HBPC / ci37), and the human immortalized brain microvascular endothelium. Using a cell line (HBMEC / ci18), a multi-cell BBB model based on human immortalized cells was developed, and the characteristics of the BBB model were determined.

That is, the present invention is:
[1] (i) A pericyte culture step of seeding and culturing human conditionally immortalized pericyte in a first medium, and
(Ii) A pericyte differentiation step of differentiating the cultured human conditionally immortalized pericyte in a second medium,
(Iii) An astrocyte culturing step in which human conditionally immortalized astrocytes are seeded and cultured in a third medium, and
(Iv) An astrocyte differentiation step of differentiating the cultured human conditional immortalized astrocytes in a fourth medium.
(V) Including a step of culturing brain microvascular endothelial cells in which human conditional immortalized cerebral microvascular endothelial cells are seeded in a fifth medium and the seeded human conditional immortalized cerebral microvascular endothelial cells are cultured.
In the pericyte culture step (i) and / or the pericyte differentiation step (ii), a culture containing human conditionally immortalized pericyte was formed.
In the astrocyte culture step (iii) and / or the astrocyte differentiation step (iv), a culture containing human conditionally immortalized astrocytes was formed.
The differentiation in the pericyte differentiation step (ii) is performed at a temperature different from the culture temperature in the pericyte culture step (i).
Differentiation in the astrocyte differentiation step (iv) is performed 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 performed, and the step (iv) is performed.
In the cerebral microvascular endothelial cell culture step (v), a culture containing human conditional immortalized cerebral microvascular endothelial cells was formed.
In the cerebral microvascular endothelial cell culturing step (v), the culturing of the seeded human conditional immortalized cerebral microvascular endothelial cells further cultivates the human conditioned immortalized pericyte in a sixth medium and Performed with further culture of the human conditionally immortalized astrocytes,
A multicellular blood-brain barrier model in which the culture containing the human conditionally immortalized pericyte and the culture containing the human conditionally immortalized brain microvascular endothelial cells are arranged so as to be able to interact with each other. Manufacturing method;
[2] Culture of the seeded human conditional immortalized brain microvascular endothelial cells in the brain microvascular endothelial cell culture step (v), further culture of the human conditional immortalized pericyte, and the human. The production according to [1], wherein further culturing of the conditional immortalized astrocytes is carried out at a temperature different from the differentiation in the pericyte differentiation step (ii) and / or the differentiation in the astrocyte differentiation step (iv). Method;
[3] Culturing in the pericyte culturing step (i) is carried out in a temperature range of 32 to 34 ° C., differentiation in the pericyte differentiation step (ii) is carried out in a temperature range of 35 to 39 ° C., and / or. Culturing in the astrocyte culturing step (iii) is carried out in a temperature range of 32 to 34 ° C., and differentiation in the astrocyte differentiation step (iv) is carried out in a temperature range of 35 to 39 ° C. [1] or [2]. ] The manufacturing method described in
[4] Culture of the seeded human conditionally immortalized brain microvascular endothelial cells in the brain microvascular endothelial cell culture step (v), further culture of the human conditionally immortalized pericyte, and the human. The production method according to any one of [1] to [3], wherein further culture of conditional immortalized astrocytes is carried out in a temperature range of 32 to 34 ° C.;
[5] Any of [1] to [4], wherein the culture in the pericyte culture step (i) and the culture in the astrocyte culture step (iii) are independently performed for 12 to 72 hours after sowing. The manufacturing method according to item 1;
[6] 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 independently performed for 12 to 72 hours, respectively. The manufacturing method described in
[7] The production method according to any one of [1] to [6], wherein the cerebral 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 performed at the same time;
[9] The production 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 performed at the same time;
[10] The culture containing the human conditionally immortalized pericyte formed in the pericyte culture step (i) and / or the pericyte differentiation step (ii) is a layer and / or.
The production according to any one of [1] to [9], wherein the culture containing the human conditionally immortalized brain microvascular endothelial cells formed in the brain microvascular endothelial cell culture step (v) is a layer. Method;
[11] 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 single layer. The manufacturing method described in
[12] On one side of the culture containing the human conditionally immortalized pericyte, the culture containing the human conditionally immortalized astrocyte is located.
In any one of [1] to [11], the culture containing the human conditionally immortalized brain microvascular endothelial cells is located on the other side of the culture containing the human conditionally immortalized pericyte. The manufacturing method described;
[13] By forming the culture containing the human conditionally immortalized pericyte and the culture containing the human conditionally immortalized brain microvascular endothelial cells on the respective surfaces of the porous substrate. , The culture containing the human conditionally immortalized pericyte and the culture containing the human conditionally immortalized brain microvascular endothelial cells are arranged so as to be able to interact with each other, [1] to The production method according to any one of [12];
[14] The production method according to [13], wherein the porous substrate is coated with molecules constituting the extracellular matrix;
[15] The third medium and the fourth medium differ in that the serum contained in the third medium is not contained in the fourth medium, according to [1] to [14]. The manufacturing method according to any one of the above;
[16] The production method according to any one of [1] to [15], wherein the fifth medium is a medium containing no growth factor;
[17] 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. The manufacturing method described in the section;
[18] A multicellular blood-brain barrier model produced by the production method according to any one of [1] to [17];
[19] (A) A culture containing human conditionally immortalized brain microvascular endothelial cells and
(B) Cultures containing human conditioned immortalized pericytes and
(C) Cultures containing human conditionally immortalized astrocytes,
(D) The medium for the human conditionally immortalized brain microvascular endothelial cells and
(E) A multicellular blood-brain barrier model comprising the human conditioned immortalized pericyte and a medium for the human conditioned immortalized astrocyte.
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 able to interact with each other.
On one side of the culture (B) containing the human conditionally immortalized pericyte, the culture (A) containing the human conditionally immortalized brain microvascular endothelial cells is located.
A multicellular blood-brain barrier model in which the culture containing the human conditionally immortalized astrocytes (C) is located on the other side of the culture containing the human conditionally immortalized pericytes (B);
[20] The culture (A) containing the 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 pericyte 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 single layer.
[22] Further containing a porous substrate,
The culture (A) containing the human conditionally immortalized brain microvascular endothelial cells covered one surface of the 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 pericyte covers the other surface of the porous substrate. ;
[23] The medium (D) is a medium containing no growth factor.
The multicellular blood-brain barrier model according to any one of [19] to [22], wherein the medium (E) is a medium for nerve cells.

According to the present invention, it is possible to provide 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 as observed under a microscope. FIG. 1B shows protein expression of endothelial cell markers (vWF and CD31) in HBMEC / ci18 cells detected by immunocytochemistry. 4', 6-Diamidino-2-phenylindole (DAPI) was used for the counterstain of the nucleus (blue). The test was repeated 3 times to show representative images. FIG. 1C shows protein expression of immortalizing genes (tsSV40T and hTERT) in HBMEC / ci18 cells detected by immunocytochemistry. The test was repeated 3 times to show representative images. FIG. 1D shows the proliferative capacity of HBMEC / ci18 cells. The proliferative capacity of HBMEC / ci18 cells cultured at 33 ° C. or 37 ° C. was examined by direct counting of cell numbers 3, 5 and 9 days after seeding. Each value was the mean ± SD of the values obtained from three independent tests, and each measurement was performed twice. FIG. 1E shows the results of comparative analysis of various mRNA expression levels evaluated by qPCR. The mRNA expression levels of genes characteristic of BBB in HBMEC / ci18 cells and human hepatic sinusoidal endothelial cell lines (HLSEC / ciJ, used as an example of non-cerebral vascular endothelial cells) are shown. Targets are low density lipoprotein receptor-related protein 1 (LRP1), insulin receptor (INSR), multidrug resistance-related protein 4 (MRP4), glucose transporter 1 (GLUT1), major facilityator superior domain (MFSD) 2A, P-Glycoprotein (P-gp), monocarboxylic acid transporter 8 (MCT8) and transferrin receptor (TfR). Values were standardized for mRNA levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the results were mean ± SD of values obtained from three independent studies, with qPCR in each study being 2 I went there times. * p <0.05, ** p <0.01. FIG. 2A outlines the procedure for creating a 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 added to astrocyte growth medium (AGM) and pericyte medium (PM), respectively. Was seeded on type IV collagen / fibronectin coated medium inserts or plates. Then, on Day-1, the culture temperature was changed from 33 ° C. to 37 ° C., and the astrocyte growth medium (AGM) was changed to the astrocyte differentiation medium (ADM) (“Step 2: 35/37 differentiation” in the figure). ). On Day 0, HBMEC / ci18 cells were seeded into 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 well (basal side). The cells were maintained at 33 ° C. from the start of co-culture to each test (“Step 3: 18/35/37 co-culture” in the figure). FIG. 2B shows six BBB models made with different cell arrangement patterns. In the figure, "E", "A", "P" and "0" mean "HBMEC / ci18 cells", "HASTR / ci35 cells", "HBPC / ci37 cells" and "no cells", respectively. The alphabetical order means the culture position of each cell type, in the order of the inside of the insert, the outside of the insert and the bottom of the well. The E00 model is a single culture model of HBMEC / ci18 cells. The EP0 and EA0 models are co-cultured of HBMEC / ci18 inside the insert and HBPC / ci137 cells or HASTR / ci35 cells outside the insert. The E0A model is a co-culture of HBMEC / ci18 cells inside the insert and HASTR / ci35 cells at the bottom of the well. In the EPA model, HBMEC / ci18 cells were seeded inside the insert, HBPC / ci37 cells were seeded outside the insert, and HASTR / ci35 cells were seeded at the bottom of the well. In the EAP model, HBMEC / ci18 cells were seeded inside the insert, HSTR / ci35 cells were seeded outside the insert, and HBPC / ci37 cells were seeded at the bottom of the well at the bottom of the plate. FIG. 3A shows the value of transendothelial electrical resistance (TEER) measured on Day 1 after the start of co-culture for 6 BBB models prepared in a 24-well transwell culture system according to the method described with respect to FIG. .. The result is shown as a magnification difference with respect to the value obtained from the E00 model. The values were expressed as the average (SD) of the three independent tests, and the resistance values were measured 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. FIG. 3B shows the TEER values of the E00 and EPA models (12 wells) measured 1 day after co-culture. The result was an average of ± SD of the values obtained from three independent trials, with each trial performed twice. * p <0.001. FIG. 4A shows the expression profile of intercellular junction-related genes in a BBB model by human immortalized cells. Relative mRNA expression levels of intercellular junction-related genes (zonula occludens 1 [ZO-1], vascular endothelium [VE] -cadherin and β-catenin) in E00 and EPA models (12 wells) were tested by qPCR. Values are standardized using glyceraldehyde 3-phosphate dehydogenesis (GAPDH) mRNA levels and expressed in multiples relative to the values in the E00 model. The data are the average (and SD) of the values obtained from three independent experiments. Each qPCR was performed using double duplicate samples (* P <0.05). FIG. 4B shows the results of testing the protein expression and localization of ZO-1, E-cadherin and β-catenin in the E00 model (12 wells) and EPA model (12 wells) by immune cell staining (ICC). 4', 6-diamidino-2-phenylindole (DAPI) was used for nuclear counterstain (blue). All experiments were repeated 3 times independently, and the figure shows a representative photograph. FIG. 5 shows the results of testing the function of the efflux transporter in the BBB model with human immortalized cells. Specifically, Rhodamine 123 (R123; P) in the E00 model (12 wells) and the EPA model (12 wells). It is the result of a bidirectional transport assay of glycoprotein (P-gp) substrate). R123 (5 μM) was added to the bottom of the insert chamber or well and allowed to stand for 20, 40 or 60 minutes. 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 side to the basal side and the permeability coefficient PeBA from the basal side to the apical membrane side were calculated. The emission rate (ER) was calculated according to ER = PeBA / PeAB. The data are the average (and SD) of the values obtained from three independent experiments. Each experiment was performed with duplicate duplicates (* P <0.05; ** P <0.01). FIG. 6A shows the results of qPCR testing of P-gp and BCRP mRNA expression levels in the E00 model (12 wells) and EPA model (12 wells). Values are standardized using GAPDH mRNA levels and expressed as magnification differences relative to the values in the E00 model. Data are shown as means (and SD) obtained from 3 independent experiments. Each qPCR was performed using double duplicate samples (* P <0.05; ** P <0.01). FIG. 6B shows the results of ICC testing of protein expression and intracellular localization of P-gp and BCRP in the E00 model (12 wells) and EPA model (12 wells). For comparison, β-catenin was also tested. For the EPA model, the xy plane image is in the center of each figure, and the yz plane image and the xx plane image (the x and y positions are indicated by horizontal and vertical white lines, respectively) are on the top and right. It was shown to. 4', 6-diamidino-2-phenylindole (DAPI) was used for nuclear counterstain (blue). All experiments were repeated 3 times independently, and the figure shows a representative photograph. FIG. 7 shows the results of a permeability assay for Lucifer Yellow (BBB opaque marker) in the E00 model (12 wells) and the EPA model (12 wells). After adding Lucifer Yellow (10 μM) to the apical membrane side, it was allowed to stand for 30 minutes, 60 minutes or 90 minutes. Medium was harvested from the wells to determine the concentration of Lucifer Yellow. Based on this concentration, the transmission coefficient (Pe) value was calculated. Data are shown as means (and SD) obtained from 3 independent experiments. Each experiment was performed with duplicate duplicates (* P <0.05; ** P <0.01 vs E00). FIG. 8 shows the results of transepithelial electrical resistance (TEER) values measured on Day 1 after the start of co-culture for 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 value, the co-culture effect with astrocytes and pericytes was confirmed by using BPM (sixth medium) as the basal medium. The test was performed twice.

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

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

That is, the method for producing the multicellular blood-brain barrier model according to the present invention is as follows:
(I) A pericyte culture step of seeding and culturing human conditionally immortalized pericyte in a first medium, and
(Ii) A pericyte differentiation step of differentiating the cultured human conditionally immortalized pericyte in a second medium,
(Iii) An astrocyte culturing step in which human conditionally immortalized astrocytes are seeded and cultured in a third medium, and
(Iv) An astrocyte differentiation step of differentiating the cultured human conditional immortalized astrocytes in a fourth medium.
(V) Includes a brain microvascular endothelial cell culture step in which human conditionally immortalized brain microvascular endothelial cells are seeded in a fifth medium and the seeded human conditionally immortalized brain microvascular endothelial cells are cultured. Hereinafter, each step will be described in detail.

<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. Culturing of human conditionally immortalized pericytes in this step is carried out, for example, in the range of 31 to 35 ° C, preferably in the range of 32 to 34 ° C, and more preferably in the range of 33 ° C. Culturing at lower or higher temperatures can reduce the rate of cell growth.

The culture in the pericyte culture step (i) is carried out, for example, 12 to 72 hours after sowing, preferably 18 to 60 hours, more preferably 20 to 48 hours, still more preferably 22 to 36 hours. In one embodiment, the culture in the pericyte culture step (i) is carried out for 24 hours after sowing. If the cells are cultured for a shorter period of time, the cells may not adhere, develop, or proliferate sufficiently, and if the cells are cultured for a longer period of time, overgrowth may occur, and model preparation takes time, which is not efficient.

Human conditionally immortalized pericytes are preferably, for example, seeded on a substrate having a flat surface, as shown in FIG. 2B, but are not limited to this as long as the culture is formed. When seeded on a substrate having a flat surface, a culture containing human conditional immortalized brain microvascular endothelial cells in the brain microvascular endothelial cell culture step on the opposite side of the flat surface, as described later. Objects are formed in layers.

In this step, a medium known in the art that is usually used for culturing pericytes can be used. For example, a commercially available product includes a pericyte medium (manufactured by Scien Cell).

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

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

As will be described later in the examples, in a specific experiment, the present inventors obtained a suitable blood-brain barrier model by performing differentiation under a temperature condition of 37 ° C., which suppresses proliferation but promotes differentiation. I succeeded in getting it. So far, it has been reported that the immortalization signal of cells is completely released at 39 ° C., but the present inventors have shown that the function of the immortalization signal can be weakened and differentiation can be promoted even at 37 ° C. The production method according to the present invention is a more versatile method in that differentiation can be promoted at a lower temperature.

Differentiation in the pericyte differentiation step (ii) is carried out, for example, for 12 to 72 hours, preferably 18 to 60 hours, more preferably 20 to 48 hours, still more preferably 22 to 36 hours. In one aspect, differentiation in the pericyte differentiation step (iii) takes place for 24 hours.

In this step, a medium known in the art that is usually used for pericyte differentiation can be used, and examples thereof include, but are not limited to, a medium for pericytes (ScienCell, San Diego, CA). Serum, growth factors, antibiotics and the like may be further added to the medium. Fetal bovine serum (FBS) (Thermo Fisher Scientific, Waltman, CA) as a serum, Pericyte Growth Factors (ScienCell) as a growth factor, penicillin / streptomycin (penicillin / streptomycin) as an antibiotic. , Blast scidin S (nacali tesque, Kyoto) and the like, but are not limited thereto.

In a preferred embodiment, the medium used in the perisite differentiation step (second medium) is the same as the medium used in the perisite culture step (first medium), or is the first medium and the second medium. The medium is different from the above medium in that the serum contained in the first medium is not contained in the second medium. When the second medium does not contain serum, pericyte differentiation is promoted as compared with the case where it contains serum. In one aspect, the medium used in the pericyte differentiation step (second medium) is the same as the medium used in the pericyte culturing step (first medium).

In the pericyte culture step (i) and / or the pericyte differentiation step (ii), a culture containing human conditionally immortalized pericyte is formed. The shape of the culture containing human conditionally immortalized pericytes may be a layer or mass. In one aspect, the culture containing human conditionally immortalized pericytes is a layer containing human conditionally immortalized pericytes. The layer containing the human conditionally immortalized pericyte may be a single layer or a plurality of layers, and is preferably a single layer. If the layer containing human conditionally immortalized pericytes is a single layer, overestimation of the barrier function formed in the endothelium can be avoided.

When the term "layer" is used herein with respect to human conditioned immortalized pericytes, human conditioned immortalized astrocytes and human conditioned immortalized brain microvascular endothelial cells, the term "layer" is used. , A layer having a portion where cells partially overlap each other and a layer having a gap portion where cells do not exist are also included. That is, in the present specification, the term "layer" includes a layer-like structure and a sheet-like structure in addition to a layer 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. Culturing of human conditionally immortalized astrocytes in this step is carried out, for example, in the range of 31 to 35 ° C, preferably in the range of 32 to 34 ° C, and more preferably in the range of 33 ° C.

Culturing in the astrocyte culturing step (iii) is carried out, for example, 12 to 72 hours after sowing, preferably 18 to 60 hours, more preferably 20 to 48 hours, still more preferably 22 to 36 hours. Prolonged culture can reduce cell function. In one aspect, culturing in the astrocyte culturing step (iii) is carried out 24 hours after sowing.

Human conditional immortalizing astrocytes are preferably seeded, for example, in a container into which an additional container can be inserted, as shown in FIG. 2B. In one aspect, the container has a flat bottom surface.

As the third medium for seeding astrocytes in this step, a medium known in the art that is usually used for culturing astrocytes can be used. For example, as a commercial product, Dulbecco's modified Eagle's medium (DMEM) can be used. (LONZA, Sigma-Aldrich, Thermo Fisher Scientific, etc.), but are not limited thereto. Antibacterial agents, serum, N2 supplement (Wako) and the like may be added to the third medium as appropriate.

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

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

As will be described later in the section of Examples, in a specific experiment, the present inventors performed a suitable blood-brain barrier by performing differentiation under a temperature condition of 37 ° C., which suppresses proliferation but promotes differentiation. I succeeded in getting a model. So far, it has been reported that the immortalization signal of cells is completely released at 39 ° C., but the present inventors have shown that the function of the immortalization signal can be weakened and differentiation can be promoted even at 37 ° C. The production method according to the present invention is a more versatile method in that differentiation can be promoted at a lower temperature.

Differentiation in the astrocyte differentiation step (iv) is carried out, for example, for 12 to 72 hours, preferably 18 to 60 hours, more preferably 20 to 48 hours, still more preferably 22 to 36 hours. Differentiation for a longer period of time can reduce cell function. In one aspect, differentiation in the astrocyte differentiation step (iv) is carried out for 24 hours.

As the fourth medium for differentiating astrocytes in this step, a differentiation medium known in the art, which is usually used for astrocyte differentiation, can be used. For example, as a commercial product, Dulbecco's modified Eagle's medium ( DMEM) (LONZA, Sigma-Aldrich, Thermo Fisher Scientific, etc.), but is not limited thereto.

In a preferred embodiment, the medium used in the astrocyte differentiation step (fourth medium) is different from the medium used in the astrocyte culture step (third medium) in that it does not contain serum contained in the third medium. Is different.

Here, the serum includes, for example, FBS and the like, but is not limited thereto. Serum-containing third medium promotes cell proliferation of astrocytes, and serum-free fourth medium prevents astrocyte differentiation from being suppressed.

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

In one aspect, the fourth medium may optionally contain an antibiotic. In another aspect, if the third medium contains an antibiotic, the fourth medium may not contain the antibiotic contained in the third medium. Examples of the antibiotic include, but are not limited to, blastidin.

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 mass. In one embodiment, the culture containing human conditionally immortalized astrocytes is a layer containing human conditionally immortalized astrocytes. The layer containing the human conditional immortalizing astrocyte may be a single layer or a plurality of layers. In addition, the culture containing human conditionally immortalized astrocytes may have 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 performed independently, for example, within the above time range. 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 time range. By performing these cultures at the same time, the time required for model preparation can be shortened.

Further, the differentiation in the pericyte differentiation step (ii) and the differentiation in the astrocyte differentiation step (iv) are performed independently within the above 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 time range. By performing these differentiations at the same time, it is possible to shorten the time required for model preparation.

<Cerebral microvascular endothelial cell culture step (step (v))>
This step is performed 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 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 monolayer. If the layer containing human conditionally immortalized brain microvascular endothelial cells is a single layer, the structure of the blood-brain barrier in vivo can be more faithfully mimicked.

In this step, the culture of seeded human conditionally immortalized brain microvascular endothelial cells is combined with a further culture of human conditionally immortalized pericytes and a further culture of human conditionally immortalized astrocytes in a sixth medium. Will be done. By co-culturing in this way, a state closer to the brain environment in the living body can be reproduced, and the blood-brain barrier function in human conditionally immortalized brain microvascular endothelial cells can be improved.

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

In one embodiment, the culture of human conditionally immortalized brain microvascular endothelial cells in this step and the further culture of human conditionally immortalized pericytes and human conditionally immortalized astrocytes are, for example, in the range of 31-35 ° C. Of these, the temperature is preferably in the range of 32 to 34 ° C, more preferably 33 ° C.

The culture of human conditionally immortalized brain microvascular endothelial cells in this step, and the further culture of human conditionally immortalized pericytes and human conditionally immortalized astrocytes, are performed on human conditionally immortalized brain microvascular endothelial cells. After sowing, for example, 12 hours or more, preferably 12 to 240 hours, more preferably 12 to 150 hours, still more preferably 18 to 120 hours, particularly preferably 24 to 120 hours. If the culture time is longer than the above range, the function of the brain microvascular endothelial cells may not be maintained, and the model is too time-consuming and inefficient. On the other hand, if the culture time is shorter than the above range, the brain microvascular endothelial cells may not proliferate sufficiently or form a layer. In one embodiment, culturing human conditionally immortalized brain microvascular endothelial cells and further culturing human conditionally immortalized pericytes and human conditionally immortalized astrocytes disseminate human conditionally immortalized brain microvascular endothelial cells. It will be done for another 24 hours.

Here, the culture containing the human conditioned immortalized cerebral microvascular endothelial cells formed here mimics the astrocyte side of the blood-brain barrier (BBB) structure in vivo and was formed in the previous step. The cultures containing human conditionally immortalized pericytes and the cultures containing human conditionally immortalized astrocytes mimic the basal side of the BBB structure.

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, as a commercially available product, VascuLife basic medium (KURABO, Osaka, etc.) Japan), but is not limited to this. Preferably, the fifth medium in culturing human conditionally immortalized brain microvascular endothelial cells is a growth factor-free medium. In one aspect, as the fifth medium, VascuLife complete medium (Vas-V / E-free medium) containing no VEGF and hEGF or Blasticidin S can be used. The fifth medium may optionally contain an antibiotic. Here, examples of the growth factor include, but are not limited to, VEGF, hEGF, bFGF, IGF, and the like. Further, examples of the antibiotic include, but are not limited to, blastsidedin, penicillin, streptomycin and the like.

In the further culture of 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. As the sixth medium, for example, a commercially available product may be used. The sixth medium includes, but is not limited to, a neurobasal medium (Thermo Fisher Scientific). Antibacterial agents, N2 supplements and the like may be added to the medium as appropriate. In one embodiment, as the sixth medium, a neurobasal medium (Thermo Fisher Scientific) can be used, and pen / st, N2 supplement containing apotransferrin, and L-glutamine are added to the medium. (The medium thus prepared is also referred to as brain parenchymal medium (BPM)).

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

Further, in one embodiment, the culture containing human conditionally immortalized pericyte and the culture containing human conditionally immortalized brain microvascular endothelial cells are communicated so as to be able to 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 at least an interaction mediated by a humoral factor, but pericytes. The site and brain microvascular endothelial cells may be in direct contact 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 interact. Communicate so that it can be done.

In one embodiment, the culture containing human conditionally immortalized pericytes and the culture containing human conditionally immortalized cerebral microvascular endothelial cells are formed on the respective surfaces of a porous substrate, whereby humans. The culture containing the conditionally immortalized pericytes and the culture containing the human conditionally immortalized brain microvascular endothelial cells are communicated so as to be able to interact with each other. The porous substrate is, for example, a porous membrane. The material of the porous substrate is appropriately selected as long as it does not interfere with the direct interaction between the culture containing human conditionally immortalized pericytes and the culture containing human conditionally immortalized brain microvascular endothelial cells. For example, general porous membranes used in the art such as polyethylene terephthalate can be mentioned, but the present invention is not limited thereto.

The size of the pores of the porous substrate 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, and the cell culture. It may be appropriately selected as long as it does not interfere with the formation of (for example, a layer or a mass). Examples of commercially available products of the porous substrate include transfluent polyethylene terephthalate, 0.4 μm high-density poses (BD Falcon, Franklin Lakes, NJ).

The porous substrate is preferably coated with molecules that make up the extracellular matrix in order to promote cell adhesion. The porous substrate is coated with, for example, at least one selected from the group consisting of collagen, fibronectin and laminin. Coating with such a substance improves cell adhesion and differentiation traits. In one embodiment, the porous substrate is coated with collagen and / or fibronectin.

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

<Other processes>
The manufacturing method further includes 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 transporting step of transporting the model to a required place. May include.

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

The third aspect of the present invention is as follows:
(A) Cultures containing human conditioned immortalized brain microvascular endothelial cells and
(B) Cultures containing human conditioned immortalized pericytes and
(C) Cultures containing human conditionally immortalized astrocytes,
(D) The medium for the human conditionally immortalized brain microvascular endothelial cells and
(E) The present invention relates to a multicellular blood-brain barrier model containing the human conditional immortalized pericyte and a medium for the human conditional immortalized astrocyte.

In this model, the culture containing human conditionally immortalized brain microvascular endothelial cells (A) and the culture containing human conditionally immortalized pericytes (B) are arranged so as to be able to interact with each other. There is. In one embodiment, even if a porous substrate is present between the culture (A) containing human conditionally immortalized brain microvascular endothelial cells and the culture (B) containing human conditionally immortalized pericytes. Often, in this case, the culture (A) containing human conditionally immortalized brain microvascular endothelial cells covers one surface of the porous substrate, and the culture (B) containing human conditionally immortalized pericytes It covers the other surface of the porous substrate.

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

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

The medium (D) corresponds to the above-mentioned fifth medium and does not contain a growth factor. Medium (D) may also be free of antibiotics. The medium (E) corresponds to the above-mentioned sixth medium and is a medium for nerve cells.

In one aspect, the multicellular blood-brain barrier model according to the present invention comprises a first container that internally holds a culture (C) containing human conditionally immortalized astrocytes and a human conditionally immortalized brain microvascular endothelium. Includes a second vessel carrying a culture containing cells (A) and a culture containing human conditional immortalized pericytes (B). For example, the first container may have a structure in which a second container can be installed, and may have, for example, a well structure having a certain depth. In this case, the second container may be an insert placed inside the first container.

The second container contains the culture (A) containing human conditionally immortalized brain microvascular endothelial cells on one side (eg, inside the second container) and the other side (eg, inside the second container). A culture (B) containing human conditioned immortalized pericytes is carried on the outside). 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 (B) containing human conditioned immortalized pericytes. ) Is located on one side of the culture layer (A) containing human conditionally immortalized brain microvascular endothelial cells, and on the other side of the culture layer (B) containing human conditionally immortalized pericytes. Has a structure in which a culture layer containing human conditional immortalized astrocytes (C) is located, and a BBB model that mimics the structure in vivo can be reproduced (for example, FIGS. 2B, 3A and 8). The first container and the second container preferably have a flat bottom surface.

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 the structure or function of the BBB in vivo. It may be changed as appropriate as long as it can imitate. Further, the shapes of the first container and the second container may be appropriately changed as long as the structure or function of the BBB in the living body can be imitated.

As mentioned earlier, the in vitro blood-brain barrier (BBB) model is an important research tool for the development of brain-targeted drugs and the understanding of the physiological and pathophysiological functions of BBB. According to the present invention, it is possible to provide a multicellular blood-brain barrier model using human conditioned immortalized cells. The model is expected to retain basic human BBB properties and provide a useful and highly applicable experimental platform for accelerating various BBB studies based on the infinite proliferative capacity of immortalized cells. Will be done.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

<1> Sample and test method [Cell maintenance]
HBMEC / ci18 (described below) and human liver sinusoid endothelial cell line (HLSEC / ciJ (Reference 32) with VascuLife complete medium (Vas-comp), HASTR / ci35 with astrocyte growth medium (AGM)) , HBPC / ci37 cells were cultured using pericyte medium (PM). The composition of the medium used is shown in Table 1.

Vas-comp is added to VascuLife basic medium (KURABO, Osaka, Japan), 5 ng / mL human fibroblast growth factor-basic (hFGF-b), 50 μg / mL ascorbic acid, 1 μg / mL ascorbic acid, 1 μg / mL Insulin-like growth factor-1 (hIGF-1), 5 ng / mL human epidermal growth factor (hEGF), 5 ng / mL vascular endothelial cell growth factor (VEGF), 0.75 unit / mL heparin, 10 mM L-glutamine (Wako, Osaka) , Japan), 2% (v / v) bovine fetal serum (FBS) (all but glutamine purchased from KURABO), 100 unit / mL penicillin / streptomycin (pen / st) (Thermo Fisher Scientific, Waltherm, MA) and 4 μg / mL Blast Saidin S (InvivoGen, San Diego, CA) was added.

AGM is Dulbecco's Modified Eagle's Medium (DMEM) High Glucose GlutaMAX Pilbate (Thermo Fisher Scientific) containing transferrin in 1% (v / v) N2 suplement (Wako), 10% (v / v) FBS (Therciliter). , 100 units / mL to 100 μg / mL pen / st and 4 μg / mL Blastsaidin S added.

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

All immortalized cells were cultured in a Type I collagen coated dish at 33 ° C. and 5% CO ₂ /95% air. All cells were subcultured every 3 days with 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA).

[Isolation of Conditionally Immortalized Human BMEC Clone 18 (HBMEC / ci18)]
The HBMEC / ci clone 18 (HBMEC / ci18, FIG. 1) was isolated from the HBMEC / ci parent cells by the serial dilution method as described in Reference 24 except that Vas-comp was used.

[Cell proliferation analysis]
HASTR / ci18 was ^{seeded at a concentration of 5.0 × 10 4} cells / mL (Day 0) and cultured at either 33 ° C or 37 ° C. The number of cells was counted using a cytometer on Day 3, Day 5 and Day 9.

[Total RNA isolation, cDNA synthesis, and real-time Quantative PCR (qPCR)]
Total RNA extraction, genomic DNA contamination checks, and cDNA synthesis were performed according to the methods described in References 24, 26, 33. qPCR was performed using the HBMEC / ci18 cDNA as a template using the primers shown in Table 2. Target mRNAs are described in the description of the figure and in the corresponding sections herein. It was confirmed that the amplification efficiency of each qPCR was close to 1. The mRNA level of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in each cell type was determined as a standardized control. Relative expression levels were determined for these values using delta-delta CT.

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

[Development of in vitro BBB model]
As shown in FIG. 2, human immortalized BBB cells were combined in a transwell culture system (12 or 24 wells) to develop a BBB model. Cell culture insert membrane (translucent polythylene thermoslate, 0.4 μm high-density pores, BD Falcon, Franklin Lakes, NJ), 100 μg / mL type-ive-IV collagen (Nitta) Incubated with Thermo Fisher Scientific solution at 37 ° C. for 1 hour (extracellular matrix coating). The inserts were air dried and washed twice with phosphate buffered saline (PBS).

For co-culture models, HASTR / ci35 (2.5 × 10 ⁴ cells / cm ² ) and HBPC / ci37 (3.0 × 10 ⁴ cells / cm ² ) were placed outside the insert membrane (0. For 24 wells). for 33cm ^2, 12 well 0.9 cm ²⁾ or plates (for bottom (24 wells BD Falcon) for 2.0 cm ² or 12 wells were 4.0 cm ²⁾ seeding.

For 2 days (Fig. 2, Step 1) before seeding HBMEC / ci18 cells (Day2), HASTR / ci35 was cultured with AGM and HBPC / ci37 was cultured with PM. After sowing, the cells were cultured at 33 ° C. for 1 day, and then (Day 1), the culture temperature was shifted from 33 ° C. to 37 ° C., and the AGM was replaced with an astrocyte differentiation medium (ADM) to HASTR / ci35 and HBPC /. Cell differentiation of ci37 was performed.

Here, ADM is
(I) Free of FBS and Blasticidin S (ii) Same composition as AGM except that it contains 1 mM dibutyryl cyclic adenosine monophosphate (dBcAMP, Santa Cruz Biotech, Santa Cruz, CA) (FIG. 2A, Fig. 2A, See Step 2).

On Day 0, in order to start co-culture (see FIG. 2A, Step ³ ), HBMEC / ci18 cells (1.3x10 5 cells / cm ² ) are inserted into the above-mentioned HBPC / ci37 (or HASTR / ci35) cells. Immediately after seeding inside the cells, inserts holding the two cell types were transferred to wells culturing HASTR / ci35 (or HBPC / ci37) cells. For single culture of HBMEC / ci18 cells, cells were seeded inside inserts without other cells (see FIG. 2B).

For all types of BBB models (see Figure 2B)
-VascuLife complete medium (Vas-V / E-free medium) containing no VEGF and hEGF was placed on the apical membrane side (insert).
-For the basal side (well), brain parenchymal medium (BPM) was used.

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

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

In Step 3, cell culture was performed 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, Day 2 and Day 5.

[Measurement of transendothelial electrical resistance (TEER)]
TER was measured using a Millicell ERS-2 (Millipore, Darmstadt, Germany) equipped with an STX01 electrode (Millipore). The TEER value (Ω x cm ² ) was calculated using the area of the culture insert.

[Functional assay of P-gp]
The efflux transporter function (P-gp) was determined by bidirectional transport experiments with rhodamine 123 (R123, P-gp substrate).

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

In order to determine the Pe value, the cleared volume was calculated using the following formula.

In the above formula, _{the compound concentration on the apical} side and the basal (C _basolaternal ) side, and the volume on the _apical membrane side (V apical = 750 μL) and the volume on the basal side (V _basolaternal = 2250 μL) were used. Cleared volume was plotted against the time axis to obtain clearance straight slopes for _{the BBB model (PS total} ) and cell-free inserts (PS _insert). For the endothelial monolayer (PS _e ), the value of permeability × area was determined using the following formula.

Finally, as shown below, the PS _e value was divided by the surface area A to obtain the Pe value (A: 0.9 cm ² for a 12-well insert).

[In vitro BBB permeability test]
A lucifer yellow permeability assay was performed using a single culture of HBMEC / ci18 cells (E00), a co-culture with HASTR / ci35 cells and HBPC / ci37 cells (EPA). After washing the inserts once with PBS (+), the medium was replaced with a Hanks buffered saline solution (Thermo Fisher Scientific) containing calcium and magnesium, followed by preincubation at 33 ° C. for 10 minutes. Then, Lucifer Yellow (LY, 10 μM, Wako) was added to the apical membrane side.

After incubation at 37 ° C. for 30, 60 or 90 minutes, medium was withdrawn from the basal well. For quantification of LY, the fluorescence intensity of each medium was determined using Infinite 200 PRO (Tecan, Mannedorf, Switzerland) (wavelength 428/536 (nm / nm)).

[Statistical analysis]
Statistical analysis was performed by ystat2006 (Igakutosho Shuppan Ltd., Tokyo, Japan) using Excel. A comparison between the plurality of values was performed by one-way analysis of variance (ANOVA), followed by Bonferroni correction. The two values were compared by an independent Student's t-test.

[Media optimization]
The following media were examined as the basal medium (that is, the medium for culturing pericytes and astrocytes) in the co-culture of the three types of cells. 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 (Table 1-3, but serum-free here)
・ BPM (Table 1-4)

<2> Results [Isolation and characterization of novel clone HBMEC / ci18 of conditional immortalized human brain microvascular endothelial cell line]
We have isolated human brain microvascular endothelial cells / conditional immortalized clone β, HBMEC / ciβ (Reference 24) so far, but in order to prepare clones with better BBB characteristics. , Cloning was performed again from the parent cells. The present inventors selected clone 18 from the clones obtained from this clone and used it as HBMEC / ci18 (FIG. 1A).

HBMEC / ci18 cells exhibited a typical elongated weight-shaped cell morphology (FIG. 1A) and expressed the representative endothelial marker proteins von Willebrand factor (vWF) and CD31 (FIG. 1B). In addition, HBMEC / ci18 cells expressed the immortalizing proteins tsSV40T and hTERT (FIG. 1C). In addition, this cell showed excellent proliferative ability (Fig. 1D).

Next, in order to investigate whether HBMEC / ci18 cells exhibit the characteristics of cerebrovascular endothelial cell-specific gene expression, the mRNA expression level characteristic of BBB was tested, and conditional immortalized human hepatic sinusoidal endothelial cells. The expression level of the strain (HLSEC / ciJ, non-cerebral blood vessel-derived cell line) was compared. As a result, HBMEC / ci18 cells were found to have BMEC-characteristic mRNAs (low-density lipoprotein receptor-related protein 1, insulin receptor, multidrug resistance-related protein 4, glucose transporter 1 (GLUT1), major facilityator superfamily domain (low density lipoprotein receptor-related protein 1, insulin receptor, multidrug resistance-related protein 4, glucose transporter 1 (GLUT1)) MFSD) 2A, P-gp, monocarboxylic acid transporter 8 and transferrin receptor) were expressed at higher levels than HLSEC / ciJ (FIG. 1E).

In summary, HBMEC / ci18 cells were considered to have basic BMEC properties. Therefore, thereafter, an in vitro BBB culture model was developed using HBMEC / ci18.

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

To achieve this, the present inventors have worked on the development of a new culture method. For example, serum-free medium and a culture temperature of 37 ° C. were used to promote the maturation of HASTR / ci35 and HBPC / ci3 cells (Step 2). In addition, in order to enhance the function of vascular endothelial cells, an endothelial medium to which VEGF and hEGF (References 35 and 36), which act suppressively on BBB formation, were not added was used.

Under these conditions, the six BBB model types shown in FIG. 2B were made. Details of the six model types are as follows.
(1) E00: HBMEC / ci18 cells only cultured (2) EP0: HBMEC / ci18 cells co-cultured with HBPC / ci37 (3) EA0: HBMEC / ci18 cells co-cultured with HASTR / ci35 (1) 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: Co-cultured all three types of cells (HBPC / ci37 is on the outside of the insert, HSTR / ci35 is on the bottom of the well)
(6) EAP: Co-cultured all three types of cells (HASTR / ci35 is on the outside of the insert, HBPC / ci37 is on the bottom of the well)

From the results of the TEER analysis shown in FIG. 3A, it was shown that the TEER value of the co-culture model was higher than ^{the TEER value of the E00 model of the single culture (60.3 ± 4.1 Ω × cm 2).} rice field. In addition, among the co-culture models, the EPA model had the highest TER value (93.0 ± 3.4 Ω × cm ² ). Therefore, as shown in FIG. 3B, these TEER levels were maintained up to at least Day 5. Therefore, the EPA model is the most preferred configuration for the development of the 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 the human immortalized cell BBB model]
To clarify whether cell-cell junctions are formed and function in HBMEC / ci18 cells of the EPA model, we present adhesion-related genes (VE-cadherin and β-catenin) and tight-junction-related genes (ZO-1). , Claudin-5 and occludin) were examined by qPCR and immunohistochemistry. As shown in FIG. 4A, all these mRNA levels were higher in the EPA model than in the E00 model. Consistent with this result, expression levels of VE-cadherin and β-catenin proteins were also higher in the EPA model than in the E00 model (Fig. 4B).

From these results, it is considered that HASTR / ci35 and HBPC / ci37 cells promote the formation of intercellular junctions (mainly AJ) in HBMEC / ci18 cells and are involved in the formation of a stronger intercellular barrier.

[Functional analysis of excretion transporter in human immortalized cell BBB model]
P-gp activity in the EPA and E00 models was examined by bidirectional transport analysis of R123 (the substrate for P-gp). The emission rate (ER) of R123 was shown to be higher in the EPA model than in the E00 model (R123: 1.72 vs 1.31) (FIG. 5).

Subsequently, the expression levels of P-gp in the EPA and E00 models were compared by qPCR and immunocytochemistry. As a result, the mRNA and protein expression levels of P-gp and BCRP in the EPA model were significantly higher than those in the E00 model (FIGS. 6A and 6B).

Furthermore, when the intracellular localization of P-gp and BCRP in the EPA model was analyzed from the image of the vertical cross section (Z-axis plane), it was revealed that P-gp and BCRP were mainly localized in the apical membrane. It became. These localizations were in good agreement with the results of the bidirectional transport analysis above. On the other hand, the β-catenin protein, which is an adhesion-binding protein, was localized at the cell-cell boundary, unlike P-gp and BCRP (Fig. 6B).

In summary, the EPA model has better excretion transporter function compared to the E00 model, which is due to co-culture with HASTR / ci35 cells and HBPC / ci37 cells, which is the HBMEC of the EPA model. / It suggests that the expression and function of P-gp and BCRP in ci18 cells were induced.

[Evaluation of compound permeability using human immortalized cell BBB model]
Whether the BBB model using human immortalized cells could be used for the compound permeability test was examined using Lucifer Yellow, which is a typical permeability marker. The results of the permeability test 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 using human immortalized cells can be used for the compound permeability test, and that the EPA model has better functions than the E00 model.

[Evaluation of medium]
From the results shown in FIG. 8, it was shown that the TEER value was the 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 / and pericytes. Was done. The effect of the three-kind co-culture had a peak on Day 1 in all the models.

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

With respect to cell junction (barrier) function, the hiBBB90 model having a TEER value of 90 (Ω × cm ² ) indicates that HBMEC / ci18 cells have a functional barrier function. Although it does not reach the value observed in vivo (more than 1000) (references 21 and 22) and the value observed in the BBB model based on rat primary cells (about 350) (reference 14), hiBBB90 The TEER value of the model is close to that of the human primary cell BBB model (140-260) (references 12 and 37). The minimum TEER level required for the BBB model has not yet been determined, but so far, the Pe value of the fluorescent compound may reach a certain level in the BBB model showing a TEER value exceeding 150 to 400 ^{(Ω × cm 2).} It has been reported (references 38, 39) and these values can be considered as benchmark TEERs in the current BBB model. In addition, this model also shows low permeability to LY, which is a BBB impermeable marker, and from this point as well, it can be seen that a functional barrier is formed.

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

In addition, by optimizing the medium, the function of this model could be further enhanced. The most characteristic point is the use of a neural medium to reproduce the environment on the brain side. As a result, it is considered that the ability to reproduce the brain environment more than the conventional model is an important factor in the establishment of this model.

In addition to the above BBB features, its excellent scalability is also a unique advantage of the hiBBB90 model. The three immortalized cell lines were capable of long-term culture or repeated freezing / thawing, and no phenotypic abnormalities associated with them were observed. Furthermore, in this research, we developed a simple spheroid construction method, which is also a great merit in carrying out the research.

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

The main points of the development of the hiBBB90 model in this study are changes in the medium composition and co-culture conditions. In particular, the point of differentiating HASTR / ci35 cells and HBPC / ci37 cells is unique to this model. So far, the inventors have reported that the culture temperature of 37 ° C. significantly promotes the differentiation traits of HASTR / ci35 cells and HBPC / ci37 cells (References 26 and 33). It is presumed that the improvement of this differentiation trait increased the co-culture effect on HBMEC / ci18 cells and improved the function as a BBB model. Although the molecular mechanism of co-culture effect is unknown, referring to References 2, 3, 5, 14 and the like, for example, glial-derived neurotrophic factor, sonic hedgehog, and Wnt protein secreted from astrocytes, or Angiopep-1 secreted from pericytes and the like can be considered. Therefore, the expression levels of these trophic factors in HASTR / ci35 and HBPC / ci37 cells may increase with differentiation.

Finally, we briefly mention the further utilization of the hiBBB90 model in CNS drug development. In general, the pharmacological action of a drug in the brain cannot be clearly associated with its plasma concentration, but rather with the drug concentration in the brain. Therefore, although it is a rat, _{a method for predicting the in vivo steady state Kp, uu, and brain} from in vivo BBB model data has been recently proposed (Reference 40). Therefore, by using the hiBBB90 model, it may be possible to predict the brain concentration-time profile of a drug in human in vivo from human in vitro data (Reference 41).

The hiBBB90 model invented in this study retains basic BBB characteristics, and at the same time has excellent scalability and ease of handling. This model has the potential to be widely used in the evaluation of brain migration of new CNS drugs and in research to elucidate the molecular mechanism of BBB biology. Therefore, the hiBBB90 model according to the present invention is expected to be a new tool for accelerating various human BBB studies including CNS drug development.

References

Claims (23)

(I) A pericyte culture step of seeding and culturing human conditionally immortalized pericyte in a first medium, and
(Ii) A pericyte differentiation step of differentiating the cultured human conditionally immortalized pericyte in a second medium,
(Iii) An astrocyte culturing step in which human conditionally immortalized astrocytes are seeded and cultured in a third medium, and
(Iv) An astrocyte differentiation step of differentiating the cultured human conditional immortalized astrocytes in a fourth medium.
(V) Including a step of culturing brain microvascular endothelial cells in which human conditional immortalized cerebral microvascular endothelial cells are seeded in a fifth medium and the seeded human conditional immortalized cerebral microvascular endothelial cells are cultured.
In the pericyte culture step (i) and / or the pericyte differentiation step (ii), a culture containing human conditionally immortalized pericyte was formed.
In the astrocyte culture step (iii) and / or the astrocyte differentiation step (iv), a culture containing human conditionally immortalized astrocytes was formed.
The differentiation in the pericyte differentiation step (ii) is performed at a temperature different from the culture temperature in the pericyte culture step (i).
Differentiation in the astrocyte differentiation step (iv) is performed 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 performed, and the step (iv) is performed.
In the cerebral microvascular endothelial cell culture step (v), a culture containing human conditional immortalized cerebral microvascular endothelial cells was formed.
In the cerebral microvascular endothelial cell culturing step (v), the culturing of the seeded human conditional immortalized cerebral microvascular endothelial cells further cultivates the human conditioned immortalized pericyte in a sixth medium and Performed with further culture of the human conditionally immortalized astrocytes,
A multicellular blood-brain barrier model in which the culture containing the human conditionally immortalized pericyte and the culture containing the human conditionally immortalized brain microvascular endothelial cells are arranged so as to be able to interact with each other. Manufacturing method. In the brain microvascular endothelial cell culture step (v), the seeded human conditionally immortalized brain microvascular endothelial cells are cultured, and the human conditionally immortalized pericyte is further cultured and the human conditionally immortalized. The production method according to claim 1, wherein further culturing of astrocytes is carried out at a temperature different from the differentiation in the pericyte differentiation step (ii) and / or the differentiation in the astrocyte differentiation step (iv). Culturing in the pericyte culturing step (i) is carried out in a temperature range of 32 to 34 ° C., differentiation in the pericyte differentiation step (ii) is carried out in a temperature range of 35 to 39 ° C., and / or the astrocyte. The production according to claim 1 or 2, wherein the culture in the culturing step (iii) is carried out in a temperature range of 32 to 34 ° C., and the differentiation in the astrocyte differentiation step (iv) is carried out in a temperature range of 35 to 39 ° C. Method. In the brain microvascular endothelial cell culture step (v), the seeded human conditionally immortalized brain microvascular endothelial cells are cultured, and the human conditionally immortalized pericyte is further cultured and the human conditionally immortalized. The production method according to any one of claims 1 to 3, wherein further culture of astrocytes is carried out in a temperature range of 32 to 34 ° C. The invention 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 independently performed for 12 to 72 hours after sowing. Production method. The production 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 independently performed for 12 to 72 hours. .. The production method according to any one of claims 1 to 6, wherein the cerebral microvascular endothelial cell culture step (v) is carried out for 12 to 240 hours after seeding. The production method according to any one of claims 1 to 7, wherein the culture in the pericyte culture step (i) and the culture in the astrocyte culture step (iii) are performed at the same time. The production method according to any one of claims 1 to 8, wherein the differentiation in the pericyte differentiation step (ii) and the differentiation in the astrocyte differentiation step (iv) are performed at the same time. The culture containing the human conditionally immortalized pericytes formed in the pericyte culture step (i) and / or the pericyte differentiation step (ii) is a layer and / or.
The production method according to any one of claims 1 to 9, wherein the culture containing the human conditionally immortalized brain microvascular endothelial cells formed in the brain microvascular endothelial cell culture step (v) is a layer. The production method according to any one of claims 1 to 10, wherein the culture containing human conditional immortalized brain microvascular endothelial cells formed in the brain microvascular endothelial cell culture step (v) is a single layer. .. On one side of the culture containing the human conditionally immortalized pericyte, the culture containing the human conditionally immortalized astrocyte is located.
The one according to any one of claims 1 to 11, wherein the culture containing the human conditionally immortalized brain microvascular endothelial cells is located on the other side of the culture containing the human conditionally immortalized pericyte. Production method. The human conditionally immortalized pericyte-containing culture and the human conditionally immortalized brain microvascular endothelial cell-containing culture are formed on the respective surfaces of the porous substrate, whereby the human being. Any of claims 1 to 12, wherein the culture containing the conditionally immortalized pericyte and the culture containing the human conditionally immortalized brain microvascular endothelial cells are arranged so as to be able to interact with each other. The manufacturing method according to item 1. The production method according to claim 13, wherein the porous substrate is coated with molecules constituting the extracellular matrix. The third medium and the fourth medium are different from each other in that the serum contained in the third medium is not contained in the fourth medium, according to any one of claims 1 to 14. The manufacturing method described. The production method according to any one of claims 1 to 15, wherein the fifth medium is a medium containing no growth factor. The production according to any one of claims 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. Method. A multicellular blood-brain barrier model produced by the production method according to any one of claims 1 to 17. (A) Cultures containing human conditioned immortalized brain microvascular endothelial cells and
(B) Cultures containing human conditioned immortalized pericytes and
(C) Cultures containing human conditionally immortalized astrocytes,
(D) The medium for the human conditionally immortalized brain microvascular endothelial cells and
(E) A multicellular blood-brain barrier model comprising the human conditioned immortalized pericyte and a medium for the human conditioned immortalized astrocyte.
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 able to interact with each other.
On one side of the culture (B) containing the human conditionally immortalized pericyte, the culture (A) containing the human conditionally immortalized brain microvascular endothelial cells is located.
A multicellular blood-brain barrier model in which the culture containing the human conditionally immortalized astrocytes (C) is located on the other side of the culture containing the human conditionally immortalized pericytes (B). The culture (A) containing the human conditionally immortalized brain microvascular endothelial cells is a layer and / or
The multicellular blood-brain barrier model according to claim 19, wherein the culture (B) containing the human conditionally immortalized pericyte is a layer. The multicellular blood-brain barrier model according to claim 19 or 20, wherein the culture (A) containing the human conditionally immortalized brain microvascular endothelial cells is monolayer. Further containing a porous substrate,
The culture (A) containing the human conditionally immortalized brain microvascular endothelial cells covered one surface of the 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 pericyte covers the other surface of the porous substrate. The medium (D) is a medium containing no growth factor, and the medium (D) is a medium containing no growth factor.
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.

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