JP2022105712A - Method for evaluating angiogenesis inhibitory activity of test compound - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an evaluation method for evaluating the angiogenesis inhibitory activity of a test compound, in which more reliable evaluation can be performed without using animal models.

SOLUTION: Provided is a method for evaluating the angiogenesis inhibitory activity of a test compound, the method comprising: a culturing step for culturing, in the presence of the test compound, a cell structure having a vascular network structure, the cell structure produced by the steps (a) of mixing, in a cationic buffer, a cell, an extracellular matrix component and a strong electrolyte polymer to give a mixture, (b) inoculating the mixture resulting from the step (a) into a cell culture container, and (c) preparing a cell structure with multilayers of cells laminated in the cell culture vessel following the step (b); and an evaluation step for evaluating whether or not the test compound has angiogenesis inhibitory activity, by using, as an index, the state of vessels in the cell structure cultured in the culturing step.

SELECTED DRAWING: None

COPYRIGHT: (C)2022,JPO&INPIT

Description

The present invention relates to a method for evaluating the angiogenesis inhibitory activity of a test compound in an in vitro system without using an animal model.
This application claims priority based on Japanese Patent Application No. 2016-083947 filed in Japan on April 19, 2016, the contents of which are incorporated herein by reference.

In recent years, in cancer chemotherapy, not only chemotherapy targeting the cancer cells themselves but also treatment methods targeting the microenvironment around the cancer have been developed. One of them is a therapy in which an angiogenesis inhibitor is administered alone or in combination with a general anticancer drug having cytotoxicity. It is known that the new generation of blood vessels such as blood vessels and lymph vessels is deeply involved in the growth of cancer (tumor) and the malignant transformation and progression of infiltration and metastasis.

Angiogenesis inhibitors are agents that suppress or inhibit angiogenesis by inhibiting factors essential for angiogenesis and protein signal pathways essential for angiogenesis such as their receptors. Typical examples include Avastin (registered trademark) (Genentech, also known as bevacizumab) approved in the United States in 2004, and EYLEA (registered trademark) (Bayer Yakuhin, also known as aflibercept) thereafter. ), Suchibaga (registered trademark) (manufactured by Bayer Yakuhin, also known as regorafenib), and various other drugs have been developed and introduced into clinical practice.

Although it lags behind angiogenesis, lymphangiogenesis is also a target for cancer chemotherapy. In particular, lymphangiogenesis is regarded as important as a prognostic factor for patients with lymph node metastasis, and the development of lymphangiogenesis inhibitors is also progressing.

Currently, as an evaluation system for these angiogenesis inhibitors, it is common to basically use an in vivo model of an immunodeficient mouse or the like. As an in vitro evaluation system, a single cell layer having endothelial cells and fibroblasts is formed on a substrate such as glass or plastic, and a test compound is used in a system in which a blood vessel is formed in this cell layer. (Refer to Patent Document 1), a method for evaluating vascular neoplasia by adding blood vessels, and fine processing on a substrate such as glass or plastic to form a vascular skeleton, and endothelial cells are grown in the skeleton. A method of adding a test compound to a system for forming a blood vessel to evaluate the ability of blood vessel formation, or a system for growing endothelial cells in a gel of an in vivo sample such as extracellular matrix to form a blood vessel. There is a method of adding a test compound and evaluating the vasogenic ability (see Non-Patent Document 1).

Japanese Patent No. 3404572

Goodwin, NIH Public Access Microvasc Research, 2007, vol.74 (2-3), p.172-183. Nishiguchi et al., Macromol Bioscience, 2015, vol.15 (3), p.312-317.

When the efficacy of a drug is evaluated by an in vitro evaluation system, the reliability of the obtained evaluation becomes a problem. That is, it is important that the evaluation in the evaluation system reflects the drug efficacy obtained when the drug is actually administered to the living body, and the evaluation in the evaluation system and the effect obtained by administering the drug to the living body are important. An evaluation system with a high probability of matching with is a highly reliable evaluation system.

Since the in vitro evaluation system described in Patent Document 1 and Non-Patent Document 1 uses a blood vessel in a state where there is no tissue surrounding the blood vessel such as stromal cells, an evaluation that can correctly reproduce the actual in vivo body. It cannot be said to be a system. Therefore, even compounds evaluated to have an inhibitory activity on angiogenesis in these evaluation systems often do not exhibit an appropriate inhibitory activity on angiogenesis when actually administered to a living body.

An object of the present invention is to provide an evaluation method capable of performing a more reliable evaluation without using an animal model when evaluating the angiogenesis inhibitory activity of a test compound.

The method for evaluating the vessel formation inhibitory activity of the test compound according to the first aspect of the present invention is a culture step of culturing a cell structure having a vessel network structure in the presence of the test compound, and after the culture step. With the state of the vessel in the cell structure as an index, the test compound has an evaluation step of evaluating whether or not the test compound has a vessel formation inhibitory activity, and in the evaluation step, the test is performed. When the number of cells constituting the vessels in the cell structure is smaller than when cultured in the absence of the compound, or when the vessel network structure in the cell structure is damaged. , The test compound is evaluated to have an inhibitory activity on angiogenesis.
In the first aspect, the cell structure may contain one or more selected from the group consisting of vascular endothelial cells and lymphatic endothelial cells.
In the first aspect, the cell structure may further contain one or more selected from the group consisting of fibroblasts, nerve cells, dendritic cells, macrophages, and mast cells.
In the first aspect, the cell structure contains fibroblasts, and the total number of vascular endothelial cells and lymphatic endothelial cells in the cell structure is 0.1% or more of the number of fibroblasts. May be.
The kit for evaluating the vascular renewal inhibitory activity according to the second aspect of the present invention is a kit for evaluating the vasculonesis inhibitory activity of the test compound according to the first aspect, and has a vasculature network structure. A cell structure to be provided and a cell culture container for accommodating the cell structure are provided.
In the second aspect, the cell structure may have a vasculature structure sandwiched between fibroblast layers.

The method for evaluating the vasculogenesis inhibitory activity of the test compound according to the above aspect of the present invention is an in vitro evaluation system using a cell structure containing a vasculogenic structure closer to the state in the living body, and the cells thereof. This is a method for evaluating the vasculogenesis inhibitory activity of a test compound using the effect on vasculogenesis in the structure as an index. Therefore, by using this evaluation method, the angiogenesis inhibitory activity of candidate compounds for angiogenesis inhibitors and lymphangiogenesis inhibitors, which have been attracting attention as cancer chemotherapeutic agents in recent years, can be trusted without using an animal model. It is possible to obtain a highly evaluated evaluation.
In addition, the evaluation method can be carried out more easily by using the kit for evaluating the vasogenic inhibitory activity according to the present invention.

Hereinafter, a method for evaluating the angiogenesis inhibitory activity of the test compound according to the embodiment of the present invention will be described. The method for evaluating the vessel formation inhibitory activity of the test compound according to the present embodiment (hereinafter, may be referred to as “evaluation method according to the present embodiment”) is to test a cell structure having a vasculature network structure. Evaluation to evaluate whether or not the test compound has an inhibitory activity on angiogenesis, using the culture step of culturing in the presence of the compound and the state of the blood vessels in the cell structure after the culture step as an index. It has a process. The evaluation method according to the present embodiment uses a cell structure having a vascular network structure in the three-dimensional structure, and evaluates the vasculogenesis inhibitory activity of the test compound using the effect on the vasculogenesis in the cell structure as an index. do. As described above, since the influence on the blood vessels formed in a state close to the environment in the actual living body is used as an index, highly reliable evaluation can be obtained by the evaluation method according to the present embodiment. In the present invention and the present specification, the "vascular network structure" refers to a network-like structure such as a vascular network or a lymphatic network in a living tissue.

<Cell structure>
In the present invention and the present specification, the "cell structure" is a three-dimensional structure in which a plurality of cell layers are laminated. The cell structure used in the present embodiment has a vasculature network structure, and is composed of endothelial cells that constitute blood vessels and cells that do not constitute vasculature (cells other than endothelial cells). .. That is, the cell structure used in the present embodiment (hereinafter, may be referred to as “cell structure according to the present embodiment”) is a lymphatic vessel and a lymphatic vessel inside a stack of cells that do not form a vessel. / Or the vascular network structure such as blood vessels is constructed three-dimensionally, and a tissue closer to the living body is constructed. The vessel network structure may be formed only inside the cell structure, or at least a part thereof may be formed so as to be exposed on the surface or the bottom surface of the cell structure.

The endothelial cells contained in the cell structure according to the present embodiment may be vascular endothelial cells or lymphatic endothelial cells. It may also contain both vascular endothelial cells and lymphatic endothelial cells.

The cell type of cells other than the endothelial cells contained in the cell structure according to the present embodiment is not particularly limited as long as it does not inhibit the formation of the vasculature of the endothelial cells, and is not particularly limited. It can be appropriately selected in consideration of the type of the test compound, the environment in the living body in which the desired angiogenesis inhibitory activity is exhibited, and the like. As cells other than the endothelial cells in the cell structure according to the present embodiment, since the endothelial cells easily form a vascular network that retains the original function and shape, they constitute a tissue surrounding the blood vessels in the living body. It is preferably a cell. Examples of such cells include fibroblasts, nerve cells, dendritic cells, macrophages, obese cells, epithelial cells, myocardial cells, hepatocytes, pancreatic islet cells, tissue stem cells, smooth muscle cells and the like. The cells other than the endothelial cells contained in the cell structure may be one type or two or more types. As the cell structure according to the present embodiment, a cell structure containing at least fibroblasts is preferable as cells other than endothelial cells, a cell structure containing vascular endothelial cells and fibroblasts, lymphatic endothelial cells and fibroblasts. A cell structure containing cells or a cell structure containing vascular endothelial cells, lymphatic endothelial cells and fibroblasts is more preferable. The cells other than the endothelial cells contained in the cell structure may be cells derived from the same species as the endothelial cells or cells derived from different species.

The number of endothelial cells in the cell structure according to the present embodiment is not particularly limited as long as the number is sufficient to form a vasculature network structure, and the size of the cell structure, endothelial cells, and the like. It can be appropriately determined in consideration of cell types and the like of cells other than endothelial cells. For example, by setting the abundance ratio (cell number ratio) of endothelial cells to all cells constituting the cell structure according to the present embodiment to 0.1% or more, a cell structure in which a vessel network structure is formed can be obtained. Can be prepared. When fibroblasts are used as cells other than endothelial cells, the number of endothelial cells in the cell structure according to this embodiment is preferably 0.1% or more of the number of fibroblasts, and is 0.1 to 10.0. %, More preferably 0.1 to 5.0%. When both vascular endothelial cells and lymphatic endothelial cells are included as endothelial cells, the total number of vascular endothelial cells and lymphatic endothelial cells is preferably 0.1% or more of the number of fibroblasts. It is more preferably 1 to 10.0%, and even more preferably 0.1 to 5.0%.

The cell structure according to the present embodiment may contain cells other than the cells constituting the peripheral tissue of the blood vessel in the living body as cells other than the endothelial cells. Examples of the cells include cancer cells and the like.

Cancer cells are cells that are derived from somatic cells and have acquired infinite proliferative capacity. Cancers from which cancer cells are derived include, for example, breast cancer (for example, invasive duct cancer, non-invasive duct cancer, inflammatory breast cancer, etc.) and prostate cancer (eg, hormone-dependent prostate). , Hormone-independent prostate cancer, etc.), Pancreatic cancer (eg, pancreatic duct cancer, etc.), Gastric cancer (eg, papillary adenocarcinoma, mucinous adenocarcinoma, adenoflat epithelial cancer, etc.), Lung cancer (eg, pancreatic epithelial cancer, etc.) Non-small cell lung cancer, small cell lung cancer, malignant mesotheloma, etc.), colon cancer (eg, gastrointestinal stromal tumor, etc.), rectal cancer (eg, gastrointestinal stromal tumor, etc.), colon cancer (eg, gastrointestinal stromal tumor, etc.), colon cancer (eg, gastrointestinal stromal tumor, etc.) Familial colon cancer, hereditary non-polyposis colon cancer, gastrointestinal stromal tumor, etc.), small intestinal cancer (eg, non-Hodikin lymphoma, gastrointestinal stromal tumor, etc.), esophageal cancer, duodenal cancer, tongue Hmm, pharyngeal cancer (eg, nasopharyngeal cancer, mesopharyngeal cancer, hypopharyngeal cancer, etc.), head and neck cancer, salivary adenocarcinoma, brain tumor (eg, pineapple stellate cell tumor, hairy cell stellar) Cell tumor, diffuse stellate cell tumor, degenerative stellate cell tumor, etc.), nerve sheath tumor, liver cancer (eg, primary liver cancer, extrahepatic bile duct cancer, etc.), kidney cancer (eg, renal cells) Cancer, transitional epithelial cancer of the renal pelvis and urinary tract, etc.), bile sac cancer, bile duct cancer, pancreatic cancer, liver cancer, endometrial cancer, cervical cancer, ovarian cancer (eg, epithelial cancer) Ovarian cancer, extragonal embryonic cell tumor, ovarian embryonic cell tumor, low-grade ovarian tumor, etc.), bladder cancer, urinary tract cancer, skin cancer (for example, intraocular (eye) melanoma, Mercel cell carcinoma) Etc.), hemangiomas, malignant lymphomas (eg, reticular sarcoma, lymphosarcoma, Hodgkin's disease, etc.), melanoma (malignant melanoma), thyroid cancer (eg, thyroid medullary cancer, etc.), parathyroid cancer, nasal cavity Cancer, sinus cancer, bone tumor (eg, osteosarcoma, Ewing tumor, uterine sarcoma, soft tissue sarcoma, etc.), metastatic myeloma, vascular fibroma, elevated cutaneous fibrosarcoma, retinal sarcoma, penis cancer, Testis tumor, pediatric solid cancer (eg Wilms tumor, pediatric kidney tumor, etc.), Kaposi sarcoma, Kaposi sarcoma caused by AIDS, maxillary sinus tumor, fibrous histiocytoma, smooth muscle tumor, rhabdomyomyoma, chronic bone marrow Proliferative diseases, leukemia (eg, acute myeloid leukemia, acute lymphoblastic leukemia, etc.) and the like, but are not limited thereto.

The type of cell constituting the cell structure according to the present embodiment is not particularly limited, and may be a cell collected from an animal, a cell obtained by culturing a cell collected from an animal, or a cell from an animal. The collected cells may be subjected to various treatments, or may be cultured cell lines. In the case of cells collected from animals, the collection site is not particularly limited, and may be somatic cells derived from bone, muscle, internal organs, nerve, brain, skin, blood, etc., or may be germ cells. It may be an embryonic stem cell (ES cell). Further, the species from which the cells constituting the cell structure according to the present embodiment are derived is not particularly limited, and for example, animals such as humans, monkeys, dogs, cats, rabbits, pigs, cows, mice, and rats. Cell derived from can be used. The cell obtained by culturing the cell collected from the animal may be a primary cultured cell or a subcultured cell. Examples of the cells subjected to various treatments include induced pluripotent stem cell cells (iPS cells) and cells after induction of differentiation. Further, the cell structure according to the present embodiment may be composed only of cells derived from the same species, or may be composed of cells derived from a plurality of species.

The size and shape of the cell structure according to the present embodiment are not particularly limited as long as the vasculature structure can be formed. Since it is possible to form a vascular network structure that is closer to the blood vessels formed in the tissue in the living body, the thickness of the cell structure is preferably 5 μm or more, more preferably 10 μm or more, and 50 μm or more. More preferably, 100 μm or more is even more preferable. The thickness of the cell structure is preferably 500 μm or less, more preferably 400 μm or less, still more preferably 300 μm or less. The number of cell layers of the cell structure according to the present embodiment is preferably about 2 to 60 layers, more preferably about 5 to 60 layers, still more preferably about 10 to 60 layers.

The number of cell layers constituting the cell structure is measured by dividing the total number of cells constituting the three-dimensional structure by the number of cells per layer (the number of cells required to form one layer). To. The number of cells per layer can be examined by culturing cells in a cell container used for constructing a cell structure in a plane so as to become confluent in advance. Specifically, the number of cell layers of a cell structure formed in a cell container is obtained by measuring the total number of cells constituting the cell structure and dividing by the number of cells per layer of the cell container. Can be calculated.

Generally, the cell structure according to this embodiment is constructed in a cell culture vessel. The cell culture container is not particularly limited as long as it is a container capable of constructing a cell structure and capable of culturing the constructed cell structure. Specific examples of the cell culture vessel include a dish, a cell culture insert (for example, a Transwell® insert, a Netwell® insert, a Falcon® cell culture insert, and a Millicell® cell culture. Inserts, etc.), tubes, flasks, bottles, plates, etc. In the construction of the cell structure according to the present embodiment, a dish or various cell culture inserts are preferable because the test compound can be evaluated more appropriately using the cell structure.

The cell structure according to the present embodiment may be a structure composed of a multi-layered cell in which a vasculature structure is formed, and the method for constructing the cell structure is not particularly limited. For example, it may be a method of constructing one layer at a time and sequentially stacking them, or a method of constructing two or more cell layers at a time, or a method of appropriately combining both construction methods to form a multi-layered cell layer. It may be a method of construction. Further, the cell structure according to the present embodiment may be a multi-layered structure in which the cell types constituting each cell layer are different for each layer, and the cell types constituting each cell layer are all layers of the structure. It may be a common multi-layer structure. For example, it may be a method of forming a layer for each cell type and constructing by sequentially stacking the cell layers. A cell mixed solution in which a plurality of types of cells are mixed is prepared in advance, and a multi-layered solution is obtained from the cell mixed solution. It may be a method of constructing a cell structure of a structure at a time.

As a method of constructing one layer at a time and sequentially stacking them, for example, the method described in Japanese Patent No. 4919464, that is, a step of forming a cell layer and an ECM (cell) of the formed cell layer. A method of continuously laminating cell layers by alternately repeating the steps of contacting with a solution containing the components of the outer matrix) can be mentioned. For example, when performing the method, a cell mixture in which all the cells constituting the cell structure are mixed is prepared in advance, and each cell layer is formed by the cell mixture, whereby the vessel network is formed over the entire structure. A cell structure in which a structure is formed can be constructed. Further, by forming each cell layer for each cell type, it is possible to construct a cell structure in which a vascular network structure is formed only in a layer formed from endothelial cells.

Examples of the method for constructing two or more cell layers at one time include the method described in Japanese Patent No. 5850419. That is, the entire surface of the cell is previously coated with a polymer containing an arginine-glycine-aspartic acid (RGD) sequence to which integrin binds and a polymer that interacts with the polymer containing the RGD sequence. A method of constructing a cell structure formed from a multi-layered cell layer by accommodating the coated cells coated with the adhesive film in a cell culture vessel and then accumulating the coated cells by centrifugation or the like can be mentioned. For example, when performing the method, a cell mixture prepared by mixing all the cells constituting the cell structure is prepared in advance, and coated cells prepared by adding an adhesive component to the cell mixture are used. As a result, a cell structure in which a vascular network structure is formed in the entire structure can be constructed by a single centrifugation treatment. Further, for example, a coated cell coated with an endothelial cell and a coated cell coated with a fibroblast are prepared separately to form a multilayer composed of the coated cell of the fibroblast, and then the endothelium is formed on the multilayer. One layer formed from the coated cells of the cells is laminated, and then a multilayer formed from the coated cells of the fibroblasts is laminated on the layer. This makes it possible to construct a cell structure having a vasculature structure sandwiched between thick fibroblast layers.

The cell structure according to the present embodiment can also be constructed by the method having the following steps (a) to (c).
(A) A step of mixing cells and extracellular matrix components in a cationic buffer to obtain a mixture, and
(B) A step of seeding the mixture obtained in the above step (a) in a cell culture vessel, and a step of seeding.
(C) After the step (b), a step of removing a liquid component from a cell mixture in the cell culture vessel to obtain a cell structure in which cells are laminated in multiple layers in the cell culture vessel.

In the present invention, in step (a), cells are mixed with a buffer solution containing a cationic substance and an extracellular matrix component, and a cell aggregate is formed from this cell mixture, whereby a solid having few large voids inside is formed. Target cell tissue can be obtained. In addition, since the obtained three-dimensional cell tissue is relatively stable, it can be cultured for at least several days, and the tissue does not easily collapse even when the medium is exchanged.
Further, in the present invention, the step (b) may include precipitating the cell mixture seeded in the cell culture vessel in the cell culture vessel. As for the sedimentation of the cell mixture, the cells may be positively sedimented by centrifugation or the like, or may be spontaneously sedimented.

In step (a), it is preferable to further mix the cells with the strong electrolyte polymer. By mixing the cells with a cationic substance, a strong electrolyte polymer, and an extracellular matrix component, the cells were naturally settled without requiring a treatment such as centrifugation for actively assembling the cells in step (b). However, a thick three-dimensional cell tissue with few voids can be obtained.

Examples of the cationic buffer solution include Tris-hydrochloric acid buffer solution, Tris-maleic acid buffer solution, Bis-Tris-buffer solution, HEPES and the like. The concentration and pH of the cationic substance (eg, Tris in Tris-hydrochloric acid buffer) in the cationic buffer is not particularly limited as long as it does not adversely affect the growth of cells and the construction of cell structures. For example, the concentration of the cationic substance in the cationic buffer can be 10 to 100 mM, preferably 40 to 70 mM, and more preferably 50 mM. The pH of the cationic buffer solution can be 6.0 to 8.0, preferably 6.8 to 7.8, and more preferably 7.2 to 7.6. ..

Examples of the strong electrolyte polymer include glycosaminoglycans such as heparin, chondroitin sulfate (for example, chondroitin 4-sulfate, chondroitin 6-sulfate), heparan sulfate, dermatan sulfate, keratane sulfate, and hyaluronic acid; dextran sulfate and the like. , Ramnan sulfate, fucoidan, caraginan, polystyrene sulfonic acid, polyacrylamide-2-methylpropane sulfonic acid, polyacrylic acid and the like, or derivatives thereof and the like. In the mixture prepared in the step (a), only one type of the strong electrolyte polymer may be mixed, or two or more types may be mixed in combination. In the construction of the cell structure according to the present embodiment, the polyelectrolyte is preferably glycosaminoglycan. Further, it is preferable to use at least one of heparin dextran sulfate, chondroitin sulfate, and dermatan sulfate. It is more preferable that the polyelectrolyte used in this embodiment is heparin. The amount of the strong electrolyte polymer mixed with the cationic buffer solution is not particularly limited as long as it does not adversely affect the growth of cells and the construction of cell structures. For example, the concentration of the strong electrolyte polymer in the cationic buffer solution can be more than 0 mg / mL and less than 1.0 mg / mL, preferably 0.025 to 0.1 mg / mL, preferably 0.05. It is more preferably ~ 0.1 mg / mL. Further, as one aspect of the present embodiment, it is also possible to prepare the mixture without mixing the strong electrolyte to construct a cell structure.

Examples of the extracellular matrix component include collagen, laminin, fibronectin, vitronectin, elastin, tenascin, entactin, fibrillin, proteoglycan, and variants or variants thereof. Examples of proteoglycans include chondroitin sulfate proteoglycan, heparan sulfate proteoglycan, keratan sulfate proteoglycan, and dermatan sulfate proteoglycan. In the mixture prepared in the step (a), only one type of extracellular matrix component may be mixed, or two or more types may be mixed in combination. In the construction of the cell structure according to the present embodiment, collagen, laminin and fibronectin are preferably used, and collagen is more preferable. The amount of the extracellular matrix component mixed with the cationic buffer is not particularly limited as long as it does not adversely affect the growth of cells and the construction of cell structures. For example, the concentration of extracellular matrix components in the cationic buffer can be greater than 0 mg / mL and less than 1.0 mg / mL, preferably 0.025 to 0.1 mg / mL, preferably 0.05. More preferably, it is ~ 0.1 mg / mL.

The compounding ratio of the strong electrolyte polymer mixed with the cationic buffer solution and the extracellular matrix component is 1: 2 to 2: 1. In the construction of the cell structure according to the present embodiment, the compounding ratio of the strong electrolyte polymer and the extracellular matrix component is preferably 1: 1.5 to 1.5: 1, preferably 1: 1. Is more preferable.

Repeat steps (a) to (c), specifically, after seeding the mixture prepared in step (a) as step (b) on the cell structure obtained in step (c). By repeating the step (c), a cell structure having a sufficient thickness can be constructed. The cell composition of the mixture newly seeded on the cell structure obtained in step (c) may be the same as or different from the cell composition constituting the already constructed cell structure. ..

For example, first, in step (a), a mixture containing only fibroblasts is prepared, and then steps (b) and (c) are performed to form cells formed from 10 fibroblast layers in a cell culture vessel. Get the structure. Then, as step (a), a mixture containing only vascular endothelial cells as cells is prepared, and steps (b) and (c) are carried out to perform one layer of blood vessels on the ten layers of fibroblasts in the cell culture vessel. Laminate the endothelial cell layer. Further, as step (a), a mixture containing only fibroblasts as cells is prepared, and steps (b) and (c) are carried out to perform 10 layers of fibroblasts on the vascular endothelial cell layer in the cell culture vessel. Laminate the layers. This makes it possible to construct a cell structure in which 10 layers of fibroblast layer-10 layers of vascular endothelial cell layer-10 layers of fibroblast layer are sequentially laminated in layers for each cell type. By adjusting the number of cells seeded in the step (b), the thickness of the cell layer laminated in the step (c) can be adjusted. The larger the number of cells seeded in the step (b), the larger the number of cell layers laminated in the step (c). Further, in the step (a), a mixture in which all the fibroblasts for 20 layers of the fibroblast layer and the vascular endothelial cells for one layer of the vascular endothelial cell layer are mixed is prepared, and steps (b) and (c) are performed. This makes it possible to construct a cell structure having a thickness of 21 layers and having a vascular network structure scattered inside the structure.

When constructing a cell structure containing cancer cells, for example, a mixture containing only cancer cells as cells is prepared, and the fibroblast layer 10 layers-vascular endothelial cell layer 1 layer-fibers constructed as described above are prepared. A cancer cell layer can be further laminated on a cell structure formed from 10 layers of blast cells. Further, in step (a), a mixture containing all of fibroblasts, vascular endothelial cells and cancer cells is prepared, and by performing steps (b) and (c), cancer cells and vascular network structure are performed. It is possible to construct a cell structure in which both of them are scattered independently inside the structure.

Vascular neoplasia plays an important role in the growth of cancer cells. When the cell structure contains cancer cells, the angiogenesis is inhibited when the test compound has an angiogenesis inhibitory activity. As a result, the growth of cancer cells in the cell structure is suppressed, and the number of cancer cells is smaller than that in the case of culturing in the absence of the test compound.

When the steps (a) to (c) are repeated, the obtained cell structure may be cultured after the step (c) and before the step (b). The culture conditions such as the composition of the culture medium used for culturing, the culturing temperature, the culturing time, and the atmospheric composition at the time of culturing are performed under the conditions suitable for culturing the cells constituting the cell structure. Examples of the culture medium include D-MEM, E-MEM, MEMα, RPMI-1640, Ham's F-12 and the like.

After step (a), (a'-1) a step of removing a liquid portion from the obtained mixture to obtain a cell aggregate, and (a'-2) a step of suspending the cell aggregate in a solution are performed. , You may proceed to step (b).
Further, after the step (a), the following steps (b'-1) and (b'-2) may be performed instead of the step (b). In the present invention and the present specification, the "cell viscous body" refers to a gel-like cell aggregate as described in Non-Patent Document 2.
(B'-1) A step of seeding the mixture obtained in step (a) in a cell culture vessel and then removing a liquid component from the mixture to obtain a cell viscous body.
(B'-2) A step of suspending a cell viscous substance in a solvent in a cell culture vessel.
A desired structure can be obtained by carrying out the above-mentioned steps (a) to (c), but after the step (a), (a'-1) and (a'-2) are carried out, and the steps are carried out. By carrying out (b), a more homogeneous structure can be obtained.

The solvent for preparing the cell suspension is not particularly limited as long as it is not toxic to the cells and does not impair the proliferation and function, and water, a buffer solution, a cell culture medium and the like are used. be able to. Examples of the buffer solution include phosphate saline (PBS), HEPES, and Hanks buffer solution. Examples of the culture medium include D-MEM, E-MEM, MEMα, RPMI-1640, Ham's F-12 and the like.

Instead of the step (c), the following step (c') may be performed.
(C') A step of removing a liquid component from a seeded mixture to form a layer of cells on a substrate.

The method for removing the liquid component in the steps (c) and (c') is not particularly limited as long as it does not adversely affect the growth of cells and the construction of the cell structure, and the method is not particularly limited, and the suspension of the liquid component and the solid component is used. As a method for removing the liquid component, a method known to those skilled in the art can be appropriately used. Examples of the method include centrifugal separation treatment, magnetic separation treatment, filtration treatment and the like.
For example, when a cell culture insert is used as a cell culture container, the liquid component can be removed by subjecting the cell culture insert seeded with the mixture to a centrifugation treatment at 10 ° C. and 400 × g for 1 minute. can.

<Test compound>
In the evaluation method according to the present embodiment, the test compound to be evaluated may be any compound expected to have an inhibitory activity on angiogenesis, and may be a known inhibitor of angiogenesis, and is novel. It may be a candidate compound for an inhibitor of angiogenesis. Known angiogenic inhibitors include Avastin, EYLEA, Sunitinib, CYRAMZA® (registered trademark) (Eli Lilly, also known as ramsilmab), BMS-275291 (Bristol-Myers), Celecoxib (Pfizer). , EMD121974 (Merck), Endostatin (EndreMed), Erbitaux (ImCloneSystems), Interferon-α (Roche), LY317615 (Eli Lilylily), Neovasatat (A. (Manufactured by), SU6688 (manufactured by Sugen), Talidomide (manufactured by Celgene), VEGF-Trap (manufactured by Regeneron), Iressa (registered trademark) (manufactured by AstraZeneca, also known as gefitinib), Caplerusa (registered trademark). , Also known as Pandetanib), Recentin® (AstraZeneca, also known as Sedilanib) VGX-100 (Circadian Technologies), VD1andcVE199, VGX-300 (Circadian Technology) Registered with VGX-300 (Circadian Technology) ) (Bayer Yakuhin, also known as sorafenib), Vaultrient (registered trademark) (GlaxoSmithKline, also known as pazopanib), Stent (registered trademark) (Pfizer, also known as sunitinib), Inlyta (registered trademark) (Pfizer). Axitinib), CEP-11981 (Teva Pharmaceutical Industries), AMG-386 (Takeda Yakuhin, also known as Trevananib), anti-NRP2B (Genentech), Ofev (registered trademark) ), AMG706 (manufactured by Takeda Yakuhin, also known as motesanib) and the like.

<Culture process>
In the evaluation method according to the present embodiment, first, as a culturing step, a cell structure having a vasculature structure is cultured in the presence of a test compound. Specifically, the cell structure is cultured in a culture medium mixed with the test compound. The amount of the test compound to be mixed in the culture medium can be experimentally determined in consideration of the type and number of cells constituting the cell structure, the type of the culture medium, the culture temperature, the culture time, and other culture conditions. .. The culturing time is not particularly limited, and can be, for example, 24 to 96 hours, preferably 48 to 96 hours, and more preferably 48 to 72 hours. Further, as long as the culture environment is not significantly changed, hydrodynamic addition such as reflux may be added as necessary.

<Evaluation process>
Using the state of blood vessels in the cell structure after the culture step as an index, it is evaluated whether or not the test compound has an inhibitory activity on vasculogenesis. Specifically, when the number of cells constituting the vessels in the cell structure is smaller or when the cells are cultured in the presence of the test compound, the number of cells constituting the vessels is smaller than that in the case of culturing in the absence of the test compound. When the vessel network structure in the structure is damaged, the test compound is evaluated to have an inhibitory activity on angiogenesis. On the other hand, when culturing in the presence of the test compound, vascular neoplasia occurred as in the case of culturing in the absence of the test compound, that is, when a decrease in the number of cells constituting the blood vessel was not confirmed. In addition, if no damage to the vasculature structure is confirmed, it can be evaluated that the test compound does not have vasculogenesis inhibitory activity.

The presence or absence of vascular neoplasia and the presence or absence of damage to the vascular network structure can be examined, for example, by labeling the cells constituting the blood vessel so as to distinguish them from other cells, and using the signal from the label as an index. For example, by fluorescently labeling the cells constituting the blood vessel, the blood vessel in the cell structure can be directly observed. In addition, using image analysis technology, the fluorescent image of the cell structure cultured in the presence of the test compound is compared with the fluorescent image of the cell structure cultured in the absence of the test compound, and the pulse is determined by the test compound. It is possible to investigate whether the vascular network structure has been damaged and whether vascular neoplasia has occurred. The fluorescent label of the cells constituting the vessel is, for example, an antibody against a substance specifically expressed on the cell surface of the cells constituting the vessel as a primary antibody, and fluorescence that specifically binds to the primary antibody. It can be carried out by a known method such as an immunostaining method using a labeled secondary antibody.

Similarly, the number of cells constituting the blood vessel can be examined by labeling the cells constituting the blood vessel and using the signal from the label as an index. For example, when the cells constituting the vessel are fluorescently labeled, the total fluorescence intensity or the area of the fluorescence emission region in the fluorescence image of the cell structure depends on the number of cells constituting the vessel. When the number of cells constituting the blood vessel is small, the total fluorescence intensity is small and the fluorescence emission region area is small. Therefore, by comparing the fluorescence intensity or the fluorescence emission region area of the fluorescence image of the cell structure cultured in the presence of the test compound and the fluorescence image of the cell structure cultured in the absence of the test compound, the vessel tube. The number of cells constituting the cell can be compared.
In addition, after destroying the three-dimensional structure of the cell structure after labeling the cells constituting the vessel, only the cells labeled by FACS (fluorescence activated cell sorting) or the like can be directly counted.

The evaluation method according to this embodiment uses a cell structure having a vascular network structure similar to that of an actual vascular structure in a living body. Therefore, it is possible to perform a highly reliable evaluation of the action and effect of the test compound on vasculogenesis. According to the evaluation method according to the present embodiment, the test compound evaluated to have an inhibitory activity on angiogenesis can be expected to show an effective inhibitory activity on angiogenesis even when administered in vivo. Therefore, the evaluation method according to the present embodiment can be applied to the evaluation and screening of a novel angiogenesis inhibitor.

In addition, using endothelial cells, fibroblasts, and cell structures formed using cancer cells collected from cancer patients, various known angiogenesis inhibitors are evaluated by the evaluation method according to the present embodiment. can do. This makes it possible to select a vasculonesis inhibitor that can exhibit effective angiogenesis inhibitory activity when actually administered to the cancer patient.

The cell structure used in the evaluation method according to the present embodiment and the cell culture vessel containing the cell structure can form a kit. By using such a kit for evaluating angiogenesis inhibitory activity, the evaluation method according to the present embodiment can be carried out more easily. Instead of the cell structure, the kit may include endothelial cells constituting the cell structure and cells other than the endothelial cells.

The kit can also further include other substances used in the evaluation method. Examples of the other substance include a culture medium for a cell structure, a labeling substance for labeling vascular cells formed by endothelial cells, and a substance used for constructing the cell structure (for example, a cationic buffer). Liquids, strong electrolyte polymers, extracellular matrix components, etc.).

Hereinafter, the present invention will be specifically described with reference to Examples, but the present invention is not limited to the following Examples.

[Example 1] Preparation of a cell structure having a vasculature A cell structure formed from fibroblasts and vascular endothelial cells and having a vascular network structure was prepared, and the vascular network structure was observed.
Examples of the cell structure including the vascular network include human neonatal skin fibroblasts (Lonza, CC-2509, Normal Human Dermal Fibroblasts: NHDF), human umbilical vein endothelial cells (Lonza, CC-2517A, Human). A cell structure formed from two types of cells (Umbilical Vein Endothelial Cell: HUVEC) was used. As a cell culture container, Transwell Cell Culture Insert (manufactured by Corning, product number: # 3470) was used, and as a culture medium, 10 volume% fetal bovine serum (manufactured by System Biosciences, EXO-FBS-50A-1) was used. ) And 1 volume% penicillin / streptomycin (manufactured by Wako Junyaku Co., Ltd., product number: 168-23191) and D-MEM (manufactured by Wako Junyaku Co., Ltd., product number: 043-30085) were used. Bevacizumab (manufactured by R & D Systems, product number: MAB293) was used as the angiogenesis inhibitor to be evaluated.

<Construction of cell structure>
First, 2 × 10 ⁶ NHDF and HUVEC of 0.05, 0.1, 0.25, 0.5, 1.0, 1.5, 5.0% of the number of NHDF cells, heparin and collagen. A cell suspension was prepared by suspending in a Tris-hydrochloride buffer containing (0.1 mg / mL heparin, 0.1 mg / mL collagen, 50 mM Tris, pH 7.4) (ratio of HUVEC number to NHDF number: 5). %) (Step (a)). This cell suspension was centrifuged at room temperature at 400 × g for 1 minute to remove the supernatant, and then resuspended in an appropriate amount of culture medium (steps (a'-1) (a'-2)). ). The cell suspension was then inoculated into the Transwell cell culture insert (step (b)) and then the Transwell cell culture insert was centrifuged at room temperature, 400 xg for 1 minute. Then, an appropriate amount of culture medium was added to the Transwell cell culture insert, and the cells were cultured in a CO ₂ incubator (37 ° C., 5% CO ₂ ) for 96 hours (step (c)).

<Fluorescent labeling and evaluation of cells constituting blood vessels>
The cell structure after culturing was subjected to fluorescent immunostaining using an anti-CD31 antibody (manufactured by DAKO, product number: JC70A M082329) and a secondary antibody (manufactured by Invitrogen, product number: A-11001). The blood vessels in the structure were fluorescently labeled with green. The presence or absence of vascular network formation was confirmed by directly observing this fluorescently labeled cell structure.

From Table 1, the formation of the vascular network structure could be confirmed under all conditions except the case where HUVEC of 0.05% of the NHDF number was contained.

[Example 2]
The angiogenesis inhibitory activity of the angiogenesis inhibitor bevacizumab was evaluated using a cell structure formed of fibroblasts and vascular endothelial cells and having a vascular network structure.
As constituent cells of the cell structure including the vascular network structure, human neonatal-derived skin fibroblasts (NHDF) (manufactured by London, product number: CC-2509), human umbilical vein endothelial cells (Human Umbilical) Two types of Vein Endothelial Cell: HUVEC (manufactured by London, product number: CC-2517A) were used. As the cell culture container, a Transwell cell culture insert (manufactured by Corning, product number: # 3470) was used. As the culture medium, D-MEM containing 10% by volume bovine serum (Corning, product number: # 35-010-CV) and 1 volume% penicillin / streptomycin (Wako Pure Chemical Industries, Ltd., product number: 168-23191). (Manufactured by Wako Pure Chemical Industries, Ltd., product number: 043-30085) was used. As the angiogenesis inhibitor to be evaluated, bevacizumab (manufactured by R & D Systems, product number: MAB293) was used.

<Construction of cell structure>
First, 2 × 10 ⁶ NHDF and 3 × 10 ⁴ HUVEC are mixed with tris-hydrochloric acid buffer (0.1 mg / mL heparin, 0.1 mg / mL collagen, 50 mM Tris, pH 7) containing heparin and collagen. 4. Suspended in 4) to prepare a cell suspension (step (a)). This cell suspension was centrifuged at room temperature at 400 × g for 1 minute to remove the supernatant, and then resuspended in an appropriate amount of culture medium (steps (a'-1) (a'-2)). ). The cell suspension is then seeded in the Transwell cell culture insert (step (b)) and then the Transwell cell culture insert is centrifuged at room temperature at 400 xg for 1 minute to remove liquid components. did. Then, an appropriate amount of culture medium was added to the Transwell cell culture insert, and the cells were cultured in a CO ₂ incubator (37 ° C., 5% CO ₂ ) for 24 hours (step (c)). After completion of the culture, a cell structure having a vascular network structure (a mixed layer (21 layers) of NHDF (20 layers) and HUVEC (1 layer)) was obtained.

<Culturing in the presence of bevacizumab>
The obtained cell structure was cultured at 37 ° C. and 5% CO ₂ for 72 hours in a culture medium in which the amount of bevacizumab added per well of the Transwell cell culture insert was 0, 1, or 2 μg.

<Fluorescent labeling and evaluation of cells constituting blood vessels>
The cell structure after culturing was subjected to fluorescent immunostaining using an anti-CD31 antibody (manufactured by DAKO, product number: JC70A M082329) and a secondary antibody (manufactured by Invitrogen, product number: A-11001). The blood vessels in the structure were fluorescently labeled with green. The fluorescently labeled cell structure was directly observed, and image analysis was performed on the captured fluorescent image to measure the occupied area of the fluorescent light emitting region in the fluorescent image. The more cells that make up a blood vessel, the larger the area occupied by the fluorescent light emitting region. Each concentration was repeatedly measured 3 times. The measurement results are shown in Table 2.

As a result of direct observation, it was found that the final concentration of 1 μg / well and 2 μg / well significantly inhibited the vascular network formation with respect to the final concentration of bevacizumab of 0 μg / well (not shown). The occupied area of the fluorescence emission region was 158.0 × 105 μm ^{2 at a final concentration of 0 μg / well, whereas it was 106.6 × 105 μm 2 at} a ^final concentration of ¹ μg / well. At ^{2 μg / well, it was 47.5 × 105 μm 2 ,} and the occupied area of the fluorescent light emitting region was significantly reduced by the addition of bevasizumab. From these results, bevacizumab was evaluated to have an angiogenesis inhibitory effect. Since bevacizumab is a known angiogenesis inhibitor, it was confirmed that the evaluation method according to the present invention can evaluate the angiogenesis inhibitory effect of the test compound.

[Example 3]
The angiogenesis inhibitory activity of the angiogenesis inhibitor bevacizumab was evaluated using a cell structure formed from fibroblasts, vascular endothelial cells, and cancer cells and having a vascular network structure.
In addition to NHDF and HUVEC used in Example 2, there are three types of cells constituting the cell structure including the vascular network structure: HT29 (ATCC number: HTB-38TM), which is a human colon-rectal adenocarcinoma cell line. The cell culture vessel, culture medium, and bevasizumab used in Example 2 were the same as those used in Example 2.

<Construction of cell structure>
As a cell suspension, instead of a suspension containing only 2 × 10 ⁶ NHDF and 3 × 10 ⁴ HUVEC, 2 × 10 ⁶ NHDF and 3 × 10 ⁴ HUVEC and 2 × 10 A cell structure having a vascular network structure was obtained in the same manner as in Example 2 except that a suspension containing ⁴ cancer cells was used. As the HUVEC cells, those labeled with a fluorescent label (manufactured by SIGMA, product number PKH26GL) were used.

<Culturing in the presence of bevacizumab>
The obtained cell structure was cultured at 37 ° C. and 5% CO ₂ for 72 hours in a culture medium in which the amount of bevacizumab added per well of the Transwell cell culture insert was 0, 1, or 2 μg.

<Dispersion of cell structure>
Next, an appropriate amount of Tris buffer solution (50 mM, pH 7.4) was added to the Transwell cell culture insert, and then the liquid component was removed. This series of steps was repeated three times. Then, 300 μL of 0.25% trypsin-EDTA solution (manufactured by Invitrogen) was added to the Transwell cell culture insert and incubated in a CO ₂ incubator (37 ° C., 5% CO ₂ ) for 15 minutes. Then, the whole solution was recovered and transferred to a recovery 1.5 mL tube to which 300 μL of 0.25% trypsin-EDTA solution (manufactured by Invitrogen) was previously added. Then, 100 μL of 0.25% trypsin-EDTA solution (manufactured by Invitrogen) was added to the Transwell cell culture insert, and the CO ₂ incubator (37 ° C., 5% CO ₂ ) was used for 5 minutes together with a 1.5 mL tube for recovery. Incubated. Then, the entire solution is recovered, transferred to a 1.5 mL tube for recovery, and 300 μL of a 0.25% trypsin-EDTA solution (manufactured by Invitrogen) is added, and the mixture is placed in a CO ₂ incubator (37 ° C., 5% CO ₂ ). Incubation was carried out for 5 minutes to obtain a cell structure dispersion.

<Analysis and evaluation of living cell number>
After immersing the obtained dispersion of the cell structure in a trypan blue solution and staining it with trypan blue, cells that were fluorescent and not stained with trypan blue were counted as living HUVECs. Cell counting was performed using the fluorescence mode of the cell counter "Countess II" (manufactured by Life Technologies). Table 3 shows the counting results of the number of viable cells of HUVEC and the calculated survival rate (ratio of the number of viable cells when the number of viable cells of bevacizumab 0 μg is 100%) (%).

The number of viable cells of HUVEC is remarkable at the final concentration of 1 μg / well and 2 μg / well where the inhibition of angiogenesis is confirmed in Example 2 as opposed to the final concentration of bevacizumab of 0 μg / well in which angiogenesis is not inhibited. It was confirmed that the survival rate of HUVEC was reduced by the addition of bevacizumab. The higher the angiogenesis-inhibiting effect of bevacizumab, the lower the survival rate of HUVEC. Therefore, it can be said that the presence or absence of the angiogenesis-inhibiting effect of the test compound can be evaluated from the survival rate of HUVEC.

[Example 4] When a cell structure composed of lung-derived stromal cells is used A cell structure formed from fibroblasts and vascular endothelial cells and having a vascular network structure is used with the angiogenesis inhibitor bevasizumab. evaluated.
Examples of the cell structure including the vasculature include human lung fibroblasts (NHPF) (Promocell, product number: C-12360) and human pulmonary microvascular endothelial cells. : HMVEC-L) (manufactured by London, product number: CC-2527), a cell structure formed from two types of cells was used. As a cell culture container, Transwell Cell Culture Insert (manufactured by Corning, product number: # 3470) was used, and as a culture medium, 10 volume% fetal bovine serum (manufactured by System Biosciences, EXO-FBS-50A-1) was used. ) And 1 volume% penicillin / streptomycin (manufactured by Wako Junyaku Co., Ltd., product number: 168-23191) and D-MEM (manufactured by Wako Junyaku Co., Ltd., product number: 043-30085) were used. Bevacizumab (manufactured by R & D Systems, product number: MAB293) was used as the angiogenesis inhibitor to be evaluated.

<Construction of cell structure>
First, 2 × 10 ⁶ NHPF and 1 × 10 ⁵ HMVEC-L, Tris-hydrochloric acid buffer containing heparin and collagen (0.1 mg / mL heparin, 0.1 mg / mL collagen, 50 mM Tris, The cells were suspended at pH 7.4) to prepare a cell suspension (ratio of HMVEC-L number to NHPF number: 5%) (step (a)). This cell suspension was centrifuged at room temperature at 400 × g for 1 minute to remove the supernatant, and then resuspended in an appropriate amount of culture medium (steps (a'-1) (a'-2)). ). The cell suspension was then seeded in the Transwell cell culture insert (step (b)) and then the Transwell cell culture insert was centrifuged at room temperature, 400 xg for 1 minute. Then, an appropriate amount of culture medium was added to the Transwell cell culture insert, and the cells were cultured in a CO ₂ incubator (37 ° C., 5% CO ₂ ) for 24 hours (step (c)).

<Culturing in the presence of bevacizumab>
The obtained cell structure was cultured at 37 ° C. and 5% CO ₂ for 144 hours in a culture medium in which the amount of bevacizumab added per well of the Transwell cell culture insert was 0, 1, or 2 μg.

<Fluorescent labeling and evaluation of cells constituting blood vessels>
The cell structure after culturing was subjected to fluorescent immunostaining using an anti-CD31 antibody (manufactured by DAKO, product number: JC70A M082329) and a secondary antibody (manufactured by Invitrogen, product number: A-11001). The blood vessels in the structure were fluorescently labeled with green. The fluorescently labeled cell structure was directly observed, and image analysis was performed on the captured fluorescent image to measure the occupied area of the fluorescent light emitting region in the fluorescent image. The results are shown in Table 4. The more cells that make up a blood vessel, the larger the area occupied by the fluorescent light emitting region. Each concentration was repeatedly measured 3 times.

As a result, it was 252 × 10 ⁵ μm ² at the final concentration of 0 μg / well, 178 × 10 ⁵ μm ² at the final concentration of 1 μg / well, and 131 × 10 ⁵ μm ² at the final concentration of 2 μg / well. , The occupied area of the fluorescent light emitting region was significantly reduced by the addition of bevacizumab. From these results, it was evaluated that bevacizumab has an angiogenesis inhibitory effect even when using lung-derived fibroblasts and vascular endothelial cells.

Claims (4)

(A) A step of mixing cells, extracellular matrix components, and a polyelectrolyte in a cationic buffer solution to obtain a mixture.
(B) A step of seeding the mixture obtained in the above step (a) in a cell culture vessel, and a step of seeding.
(C) After the step (b), a step of obtaining a cell structure in which cells are laminated in multiple layers in the cell culture vessel, and a step of obtaining the cell structure.
After producing a cell structure with a vasculature structure
A culture step of culturing the cell structure in the presence of a test compound, and
Using the state of the blood vessels in the cell structure after the culture step as an index, an evaluation step of evaluating whether or not the test compound has a vessel formation inhibitory activity, and an evaluation step.
Have,
In the evaluation step, when the number of cells constituting the vessel in the cell structure is smaller than in the case of culturing in the absence of the test compound, or the vessel network structure in the cell structure. The test compound is evaluated to have an inhibitory activity on angiogenesis when the cell is damaged.
The cell structure comprises one or more selected from the group consisting of vascular endothelial cells and lymphatic endothelial cells and fibroblasts.
The total number of vascular endothelial cells and lymphatic endothelial cells in the cell structure is 0.1% or more of the number of fibroblasts.
The extracellular matrix component is selected from the group consisting of collagen, laminin, fibronectin, vitronectin, elastin, tenascin, entactin, fibrillin, proteoglycan, and combinations thereof.
The polyelectrolyte is selected from the group consisting of glycosaminoglycan, dextran sulfate, ramnan sulfate, fucoidan, caraginan, polystyrene sulfonic acid, polyacrylamide-2-methylpropane sulfonic acid, polyacrylic acid, and combinations thereof.
The concentration of the extracellular matrix component in the mixture is 0.025 mg / mL or more and less than 1.0 mg / mL.
The concentration of the polyelectrolyte in the mixture is 0.025 mg / mL or more and less than 1.0 mg / mL.
A method for evaluating the angiogenesis inhibitory activity of a test compound. The method for evaluating angiogenesis-inhibiting activity of a test compound according to claim 1, wherein in the step (a), the mixture contains all the cells constituting the cell structure. The vasculogenesis inhibitory activity of the test compound according to claim 1 or 2, wherein the cell structure further comprises one or more selected from the group consisting of nerve cells, dendritic cells, macrophages, and mast cells. Evaluation method. After the step (a) and before the step (b), (a'-1) a step of removing a liquid portion from the obtained mixture to obtain a cell aggregate, and (a'-2) a cell assembly. The method for evaluating angiogenesis-inhibiting activity of a test compound according to any one of claims 1 to 3, wherein the step of suspending the body in a solution is performed.