WO2017183676A1 - Method for evaluating angiogenesis inhibitory activity of test compound - Google Patents

Abstract

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; 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, wherein, in the evaluation step, when the number of cells constituting the vessels in the cell structure is small or the vascular network structure in the cell structure is damaged compared to the case in which the cell structure is cultured in the absence of the test compound, the test compound is evaluated to have angiogenesis inhibitory activity.

Description

Method for evaluating angiogenesis inhibitory activity of test compound

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 for which it applied to Japan on April 19, 2016, and uses the content here.

In recent cancer chemotherapy, not only chemotherapy targeting cancer cells themselves, but also treatment methods targeting the microenvironment around cancer have been developed. One of them is a therapy in which an angiogenesis inhibitor is administered as a single agent or in combination with an anticancer agent having general cytotoxicity. It is known that neovascularization such as blood vessels and lymphatic vessels is deeply related to cancer (tumor) growth, malignant transformation and progression such as invasion and metastasis.

An angiogenesis inhibitor is a drug that suppresses or inhibits angiogenesis by inhibiting a protein signal pathway essential for angiogenesis such as a factor essential for angiogenesis and its receptor. Typical examples include Avastin (registered trademark) (Genentech, also known as bevacizumab) approved in the United States in 2004, and thereafter EYLEA (registered trademark) (Bayer Yakuhin, also known as Afribercept). ), And various drugs such as Schibaga (registered trademark) (manufactured by Bayer Yakuhin, also known as regorafenib) have been developed and introduced into clinical settings.

Angiogenesis is delayed, but lymphangiogenesis is also a target for cancer chemotherapy. In particular, lymphangiogenesis is regarded as important as a prognostic factor for patients such as lymph node metastasis, and development of lymphangiogenesis inhibitors is also progressing.

Currently, as an evaluation system for these angiogenesis inhibitors, in vivo models such as immunodeficient mice are generally used. As an in vitro evaluation system, a test compound is formed by forming a monolayer cell layer having endothelial cells and fibroblasts on a substrate such as glass or plastic and forming a vascular vessel in this cell layer. A method for evaluating vascularization ability (see Patent Document 1), or by applying fine processing on a substrate such as glass or plastic to create a vascular skeleton, and growing endothelial cells in the skeleton In this method, a test compound is added to a system that forms a blood vessel to evaluate the angiogenic ability, or a system in which endothelial cells are grown in a gel of an in vivo sample such as an extracellular matrix to form a blood vessel. In addition, there is a method of adding a test compound and evaluating angiogenic ability (see Non-Patent Document 1).

Japanese Patent No. 3404572

場合 When the efficacy of a drug is evaluated in 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 effect obtained when the drug is actually administered to the living body, and the effect obtained by the evaluation in the evaluation system and the administration to the living body. An evaluation system with a high probability of matching with is a highly reliable evaluation system.

The in vitro evaluation system described in Patent Document 1 and Non-Patent Document 1 uses a blood vessel in the absence of tissue surrounding the blood vessel such as stromal cells, so that the evaluation can be accurately reproduced in the actual living body. Not a system. For this reason, even if it is a compound evaluated as having angiogenesis inhibitory activity in these evaluation systems, when it is actually administered to a living body, an appropriate angiogenesis inhibitory activity is often not achieved.

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 angiogenesis inhibitory activity of a test compound according to the first aspect of the present invention includes a culturing step of culturing a cell structure having a vascular network structure in the presence of the test compound, and after the culturing step. An evaluation step of evaluating whether or not the test compound has an angiogenesis inhibitory activity using the state of the vasculature in the cell structure as an index, and in the evaluation step, the test When the number of cells constituting the vasculature in the cell structure is small or when the vascular network structure in the cell structure is damaged as compared with the case where it is cultured in the absence of a compound The test compound is evaluated to have angiogenesis inhibitory activity.
In the first aspect, the cell structure may include 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 include 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 includes 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. It may be.
The kit for evaluating angiogenesis inhibitory activity according to the second aspect of the present invention is a kit for performing the method for evaluating the angiogenesis inhibitory activity of the test compound according to the first aspect, wherein the vasculature has a vascular network structure. A cell structure provided with the cell structure, and a cell culture container containing the cell structure.
In the second aspect, the cell structure may have a vascular network structure sandwiched between fibroblast layers.

The method for evaluating angiogenesis inhibitory activity of a test compound according to the above aspect of the present invention is an in vitro evaluation system using a cell structure containing a vascular network structure closer to a state in a living body, and the cell This is a method for evaluating the angiogenesis inhibitory activity of a test compound using an influence on angiogenesis in a structure as an index. Therefore, by using this evaluation method, the angiogenesis inhibitory activity of candidate compounds for angiogenesis inhibitors and lymphangiogenesis inhibitors that have been attracting attention as cancer chemotherapeutic drugs in recent years can be trusted without using animal models. High evaluation can be obtained.
Moreover, the said evaluation method can be implemented more simply by using the angiogenesis inhibitory activity evaluation kit according to the present invention.

Hereinafter, a method for evaluating the angiogenesis inhibitory activity of a test compound according to an embodiment of the present invention will be described. The evaluation method of the angiogenesis inhibitory activity of the test compound according to the present embodiment (hereinafter sometimes referred to as “evaluation method according to the present embodiment”) is performed on a cell structure having a vascular network structure. Cultivation step of culturing in the presence of the compound, and evaluation for evaluating whether the test compound has angiogenesis inhibitory activity using as an index the state of the vasculature in the cell structure after the culturing step And a process. The evaluation method according to the present embodiment uses a cell structure having a vascular network structure in a three-dimensional structure, and evaluates the angiogenesis inhibitory activity of a test compound using the influence on angiogenesis in the cell structure as an index. To do. Thus, since the influence on the vascular formed in a state approximate to the actual in vivo environment 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 structure such as a vascular network or a lymphatic network in a living tissue.

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

The cell types of the cells other than the endothelial cells contained in the cell structure according to the present embodiment are not particularly limited as long as the endothelial cells do not inhibit the formation of the vascular network. In consideration of the type of test compound and the in vivo environment in which the target angiogenesis inhibitory activity is exhibited, it can be selected as appropriate. 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 the tissue surrounding the vascular in vivo. A cell is preferred. Examples of such cells include fibroblasts, nerve cells, dendritic cells, macrophages, mast cells, epithelial cells, cardiomyocytes, hepatocytes, pancreatic islet cells, tissue stem cells, smooth muscle cells and the like. The number of 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 as cells other than endothelial cells is preferable, 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 preferred. 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 it is a sufficient number to form a vascular network structure. It can be appropriately determined in consideration of cell types of cells other than endothelial cells. For example, a cell structure in which a vascular network structure is formed by setting an 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. 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, 0.1 to 10.0. % Is more preferable, and 0.1 to 5.0% is more preferable. When both vascular endothelial cells and lymphatic endothelial cells are included as the 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 further preferably 0.1 to 5.0%.

The cell structure according to the present embodiment may include cells other than cells constituting the peripheral tissue of the vascular in vivo as cells other than endothelial cells. Examples of the cells include cancer cells.

Cancer cells are cells that are derived from somatic cells and have acquired unlimited proliferative capacity. Examples of cancers from which cancer cells are derived include breast cancer (eg, invasive breast cancer, non-invasive breast cancer, inflammatory breast cancer, etc.), prostate cancer (eg, hormone-dependent prostate). Cancer, hormone-independent prostate cancer, etc.), pancreatic cancer (eg, pancreatic duct cancer, etc.), stomach cancer (eg, papillary adenocarcinoma, mucinous adenocarcinoma, adenosquamous carcinoma, etc.), lung cancer (eg, Non-small cell lung cancer, small cell lung cancer, malignant mesothelioma, etc.), colon cancer (eg, gastrointestinal stromal tumor), rectal cancer (eg, gastrointestinal stromal tumor), colorectal cancer (eg, Familial colorectal cancer, hereditary nonpolyposis colorectal cancer, gastrointestinal stromal tumor, etc.), small intestine cancer (eg, non-Hodgkin lymphoma, gastrointestinal stromal tumor, etc.), esophageal cancer, duodenal cancer, tongue Cancer, pharyngeal cancer (eg, nasopharyngeal cancer, oropharyngeal cancer, hypopharyngeal cancer), head and neck cancer, saliva Cancer, brain tumor (eg, pineal astrocytoma, ciliary astrocytoma, diffuse astrocytoma, anaplastic astrocytoma), schwannoma, liver cancer (eg, primary liver) Cancer, extrahepatic bile duct cancer, etc.), kidney cancer (eg, renal cell cancer, transitional cell carcinoma of the renal pelvis and ureter), gallbladder cancer, bile duct cancer, pancreatic cancer, liver cancer, Endometrial cancer, cervical cancer, ovarian cancer (eg, epithelial ovarian cancer, extragonadal germ cell tumor, ovarian germ cell tumor, ovarian low grade tumor, etc.), bladder cancer, urethral cancer , Skin cancer (eg, intraocular (eye) melanoma, Merkel cell carcinoma, etc.), hemangioma, malignant lymphoma (eg, reticulosarcoma, lymphosarcoma, Hodgkin's disease, etc.), melanoma (malignant melanoma), thyroid Cancer (eg, medullary thyroid cancer), parathyroid cancer, nasal cavity cancer, sinus cancer, bone tumor (eg, osteosarcoma, Ewing tumor, uterus) Tumors, soft tissue sarcomas, etc.), metastatic medulloblastoma, hemangiofibroma, elevated dermal fibrosarcoma, retinal sarcoma, penile cancer, testicular tumor, childhood solid cancer (eg Wilms tumor, childhood kidney tumor, etc.), Kaposi Sarcoma, Kaposi sarcoma caused by AIDS, maxillary sinus tumor, fibrous histiocytoma, leiomyosarcoma, rhabdomyosarcoma, chronic myeloproliferative disease, leukemia (eg, acute myeloid leukemia, acute lymphoblastic leukemia, etc.) ) And the like, but is 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 an animal. The collected cells may be cells that have been 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, viscera, nerves, brain, skin, blood, etc., or germ cells, Embryonic stem cells (ES cells) may also be used. In addition, the biological species from which the cells constituting the cell structure according to the present embodiment are derived is not particularly limited. For example, animals such as humans, monkeys, dogs, cats, rabbits, pigs, cows, mice, rats, etc. Cells derived from can be used. A cell obtained by culturing a cell collected from an 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 differentiation induction. Moreover, 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 vascular network structure can be formed. Since it is possible to form a vascular network structure that is closer to the vasculature 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 more preferably 50 μm or more. More preferably, it is more preferably 100 μm or more. The thickness of the cell structure is preferably 500 μm or less, more preferably 400 μm or less, and even more preferably 300 μm or less. The number of cell layers of the cell structure according to this embodiment is preferably about 2 to 60 layers, more preferably about 5 to 60 layers, and further preferably about 10 to 60 layers.

Note that 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 necessary for constituting one layer). The The number of cells per layer can be examined by previously culturing the cells in a planar manner so as to be confluent in a cell container used for constructing the cell structure. 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. It can be calculated.

Generally, the cell structure according to this embodiment is constructed in a cell culture container. The cell culture vessel is not particularly limited as long as the cell structure can be constructed and the constructed cell structure can be cultured. Specific examples of the cell culture container include dishes, cell culture inserts (eg, Transwell (registered trademark) insert, Netwell (registered trademark) insert, Falcon (registered trademark) cell culture insert, Millicell (registered trademark) cell culture). Inserts, etc.), tubes, flasks, bottles, plates and the like. 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 using the cell structure can be more appropriately evaluated.

The cell structure according to the present embodiment is not particularly limited as long as it is a structure composed of multi-layered cells in which a vascular network structure is formed. For example, it may be a method of constructing one layer at a time and sequentially laminating it, or a method of constructing two or more cell layers at a time, and combining the two construction methods as appropriate to form a multilayer cell layer. It may be a method of construction. In addition, the cell structure according to the present embodiment may be a multilayer 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. A common multilayer structure may be used. For example, a method may be used in which a layer is formed for each cell type and the cell layers are sequentially stacked. A cell mixture solution in which a plurality of types of cells are mixed is prepared in advance, and a multilayer is prepared from the cell mixture solution. A method of constructing a structural cell structure at a time may be used.

As a method of constructing one layer at a time and sequentially laminating, for example, a method described in Japanese Patent No. 4919464, that is, a step of forming a cell layer, and the formed cell layer is converted into an ECM (cell The method of laminating | stacking a cell layer continuously by repeating the process made to contact the solution containing the component of an outer matrix) alternately is mentioned. For example, when performing the method, a cell mixture in which all cells constituting the cell structure are mixed is prepared in advance, and each cell layer is formed by this cell mixture, whereby the vascular network is formed throughout the structure. A cell structure in which a structure is formed can be constructed. Further, by forming each cell layer for each cell type, a cell structure in which a vascular network structure is formed only in a layer formed from endothelial cells can be constructed.

Examples of a method for constructing two or more cell layers at a time include a method described in Japanese Patent No. 5850419. That is, the whole cell surface is previously coated with a polymer containing an arginine-glycine-aspartic acid (RGD) sequence to which integrin binds, and a polymer interacting with the polymer containing the RGD sequence, A method of constructing a cell structure formed from multiple cell layers by accumulating the coated cells coated with the adhesive film in a cell culture container and then accumulating the coated cells by centrifugation or the like is mentioned. For example, when performing the method, a cell mixture prepared by mixing all cells constituting the cell structure is prepared in advance, and coated cells prepared by adding an adhesive component to the cell mixture are used. Thereby, a cell structure in which a vascular network structure is formed in the entire structure can be constructed by a single centrifugation process. In addition, for example, a coated cell coated with endothelial cells and a coated cell coated with fibroblasts are separately prepared to form a multilayer composed of coated cells of fibroblasts, and then endothelial cells are formed thereon. A single layer formed from cell-coated cells is laminated, and a multilayer formed from fibroblast-coated cells is further laminated thereon. Thereby, a cell structure provided with a vascular network structure sandwiched between thick fibroblast layers can be constructed.

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

In the present invention, in the step (a), the cells are mixed with a buffer solution containing a cationic substance and an extracellular matrix component, and a cell aggregate is formed from the cell mixture. 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 is difficult to collapse even when the medium is changed.
In the present invention, in the step (b), the cell mixture seeded in the cell culture container may be precipitated in the cell culture container. In sedimentation of the cell mixture, the cells may be actively sedimented by centrifugation or the like, or may be spontaneously sedimented.

In the step (a), it is preferable to further mix the cells with a strong electrolyte polymer. This is a case where the cells are naturally precipitated by mixing cells with a cationic substance, a strong electrolyte polymer and an extracellular matrix component without requiring a process such as centrifugation to actively collect the cells in the step (b). Even so, a thick three-dimensional cellular tissue can be obtained.

Examples of the cationic buffer include tris-hydrochloric acid buffer, tris-maleic acid buffer, bis-tris-buffer, and HEPES. The concentration and pH of the cationic substance (for example, Tris in Tris-HCl buffer) in the cationic buffer are not particularly limited as long as they do not adversely affect cell growth and cell structure construction. 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. In addition, the pH of the cationic buffer 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 heparin, chondroitin sulfate (eg, chondroitin 4-sulfate, chondroitin 6-sulfate), heparan sulfate, dermatan sulfate, keratan sulfate, hyaluronic acid and other glycosaminoglycans; dextran sulfate, , Rhamnan sulfate, fucoidan, carrageenan, polystyrene sulfonic acid, polyacrylamide-2-methylpropane sulfonic acid, polyacrylic acid and the like, or derivatives thereof. In the mixture prepared in the step (a), only one type of strong electrolyte polymer may be mixed, or two or more types may be combined and mixed. In the construction of the cell structure according to this embodiment, the polyelectrolyte is preferably a glycosaminoglycan. In addition, it is preferable to use at least one of heparin dextran sulfate, chondroitin sulfate, and dermatan sulfate. More preferably, the polymer electrolyte used in this embodiment is heparin. The amount of the strong electrolyte polymer mixed with the cationic buffer is not particularly limited as long as it does not adversely affect the cell growth and the construction of the cell structure. For example, the concentration of the strong electrolyte polymer in the cationic buffer can be more than 0 mg / mL and less than 1.0 mg / mL, preferably 0.025 to 0.1 mg / mL, 0.05 More preferably, it is ˜0.1 mg / mL. Further, as one aspect of the present embodiment, the cell structure can be constructed by adjusting the mixture without mixing the strong electrolyte.

Examples of the extracellular matrix component include collagen, laminin, fibronectin, vitronectin, elastin, tenascin, entactin, fibrillin, proteoglycan, or a modified or variant thereof. Examples of proteoglycans include chondroitin sulfate proteoglycan, heparan sulfate proteoglycan, keratan sulfate proteoglycan, dermatan sulfate proteoglycan and the like. 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 and mixed. In the construction of the cell structure according to this embodiment, it is preferable to use collagen, laminin, or fibronectin, and more preferably to use collagen. The amount of the extracellular matrix component mixed with the cationic buffer is not particularly limited as long as it does not adversely affect cell growth and cell structure construction. For example, the concentration of the extracellular matrix component 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, 0.05 More preferably, it is ˜0.1 mg / mL.

The compounding ratio of the strong electrolyte polymer and the extracellular matrix component mixed in the cationic buffer 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, and is 1: 1. It is more preferable.

Steps (a) to (c) are repeated. 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 the 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 as cells, and cells formed from 10 fibroblast layers in a cell culture container by performing steps (b) and (c) Get a structure. Next, as a step (a), a mixture containing only vascular endothelial cells as a cell is prepared, and steps (b) and (c) are performed to form a single blood vessel on the 10 fibroblast layers in the cell culture container. The endothelial cell layer is laminated. Furthermore, as a step (a), a mixture containing only fibroblasts as a cell is prepared, and steps (b) and (c) are performed to form 10 layers of fibroblasts on the vascular endothelial cell layer in the cell culture container. Laminate the layers. As a result, a cell structure in which 10 layers of fibroblasts, 1 layer of vascular endothelial cell layer, 10 layers of fibroblasts, and layers in order for each cell type can be constructed. 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 greater the number of cells seeded in step (b), the greater the number of cell layers stacked in step (c). In step (a), a mixture is prepared by mixing all fibroblasts for 20 layers of fibroblasts and vascular endothelial cells for 1 layer of vascular endothelial cells, and steps (b) and (c) are performed. Thus, a cell structure having a thickness of 21 layers and having a vascular network structure scattered inside the structure can be constructed.

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-fiber constructed as described above A cancer cell layer can be further laminated on a cell structure formed from 10 blast cell layers. In step (a), a mixture of all fibroblasts, vascular endothelial cells, and cancer cells is prepared, and steps (b) and (c) are performed, whereby cancer cells and vascular network structures are prepared. A cell structure can be constructed in which both and are scattered independently inside the structure.

Angiogenesis plays an important role in the growth of cancer cells. When the cell structure contains cancer cells, angiogenesis is inhibited when the test compound has 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 when cultured 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 the culture, the culture temperature, the culture time, and the atmospheric composition during the culture are those 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) removing the liquid portion from the resulting mixture to obtain cell aggregates, and (a′-2) suspending the cell aggregates in the solution are performed. The process 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, “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 container and then removing a liquid component from the mixture to obtain a cell viscous body;
(B′-2) A step of suspending a cell viscous material in a cell culture vessel in a solvent.
A desired tissue body can be obtained by carrying out the above steps (a) to (c). However, after step (a), (a′-1) and (a′-2) are carried out, By carrying out (b), a more homogeneous tissue can be obtained.

The solvent for preparing the cell suspension is not particularly limited as long as it is not toxic to cells and does not impair the growth ability or function, and water, buffer solution, cell culture medium, etc. are used. be able to. Examples of the buffer include phosphate physiological saline (PBS), HEPES, Hanks buffer, and the like. 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 ′) removing liquid components from the seeded mixture to form a cell layer on the 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 the cell and the construction of the cell structure. From the suspension of the liquid component and the solid component. As a method of removing the liquid component, it can be appropriately performed by a method known to those skilled in the art. Examples of the technique include centrifugation, magnetic separation, or filtration.
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. it can.

<Test compound>
In the evaluation method according to the present embodiment, the test compound to be evaluated may be a compound that is expected to have angiogenesis inhibitory activity, and may be a known angiogenesis inhibitor. It may be a candidate compound for an angiogenesis inhibitor. Known angiogenesis inhibitors include Avastin, EYLEA, Suchibaga, CYRAMZA (registered trademark) (manufactured by Eli Lilly, also known as Ramsilmab), BMS-275291 (manufactured by Bristol-Myers), and Celecoxib (Pharmacia / FarmicaP) , EMD121974 (manufactured by Merck), Endostatin (manufactured by EntreMed), Erbitaux (manufactured by ImClone Systems), Interferon-α (manufactured by Roche), LY317615 (manufactured by Eli Lilly, 78) ), SU6688 (manufactured by Sugen), Thalidomide (manufactured by Celgene) VEGF-Trap (manufactured by Regeneron), Iressa (registered trademark) (manufactured by Astrazenca, also known as gefitinib), Caplerusa (registered trademark) (manufactured by Astrazeneca, also known as pandetanib), Recentin (registered trademark) (also known as Astrazenec) VGX-100 (manufactured by Circadian Technologies), VD1andcVE199, VGX-300 (manufactured by Circadian Technologies), sVEGFR2, hF4-3C5, Nexavar (registered trademark) (Bayer Yak, registered trademark) (Bayer Yak Manufactured by the company, also known as Pazopanib), Sutent (registered trademark) (Pfiz) er, also known as sunitinib), Inlyta (registered trademark) (manufactured by Pfizer, also known as axitinib), CEP-11981 (manufactured by Teva Pharmaceutical Industries), AMG-386 (manufactured by Takeda Yakuhin, aka tibanani RP, B) Genevent), Ofev (registered trademark) (boehringer-ingelheim, also known as nintatinib), 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 vascular network structure is cultured in the presence of a test compound. Specifically, the cell structure is cultured in a culture medium mixed with a test compound. The amount of the test compound to be mixed in the culture medium can be experimentally determined in consideration of the culture conditions such as the type and number of cells constituting the cell structure, the type of culture medium, the culture temperature, and the culture time. . The culture 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. In addition, hydrodynamic additions such as reflux may be added as necessary as long as the culture environment is not significantly changed.

<Evaluation process>
Whether or not the test compound has angiogenesis inhibitory activity is evaluated using the vascular state in the cell structure after the culturing step as an index. Specifically, when the number of cells constituting the vessel in the cell structure is small when cultured in the presence of the test compound, compared to when cultured in the absence of the test compound, or the cell When the vascular network structure in the structure is damaged, the test compound is evaluated as having angiogenesis inhibitory activity. On the other hand, when culturing in the presence of a test compound, if angiogenesis has occurred as in the case of culturing in the absence of the test compound, that is, if a decrease in the number of cells constituting the vasculature is not confirmed When no damage is confirmed in the vascular network structure, it can be evaluated that the test compound does not have angiogenesis inhibitory activity.

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

Similarly, the number of cells constituting the vasculature can be examined by labeling the cells constituting the vasculature and using the signal from the label as an index. For example, when cells constituting the vascular are fluorescently labeled, the total fluorescence intensity or the fluorescence emission region area in the fluorescence image of the cell structure depends on the number of cells constituting the vascular. When the number of cells constituting the vessel is small, the total fluorescence intensity is small and the area of the fluorescence emission region is small. Therefore, by comparing 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, by comparing the fluorescence intensity or the fluorescence emission area area, Can be compared.
In addition, it is also possible to directly count only the cells labeled by FACS (fluorescence activated cell sorting) after destroying the three-dimensional structure of the cell structure after labeling the cells constituting the vessel.

The evaluation method according to the present embodiment uses a cell structure having a vascular network structure close to the vascular structure in an actual living body. Therefore, highly reliable evaluation can be performed about the effect of the test compound on the angiogenesis. The test compound evaluated as having angiogenesis inhibitory activity by the evaluation method according to the present embodiment can be expected to show effective angiogenesis inhibitory activity even when administered in vivo. For this reason, the evaluation method according to the present embodiment can be applied to the evaluation and screening of novel angiogenesis inhibitors.

In addition, using endothelial cells and fibroblasts collected from cancer patients, cell structures formed using cancer cells, various known angiogenesis inhibitors are evaluated by the evaluation method according to this embodiment. can do. Thereby, the angiogenesis inhibitor which can show an effective angiogenesis inhibitory activity when actually administered to the said cancer patient can be selected.

The cell structure used in the evaluation method according to the present embodiment and the cell culture container containing the cell structure can constitute a kit. By using such an angiogenesis inhibitory activity evaluation kit, the evaluation method according to this embodiment can be carried out more easily. In place of the cell structure, the kit may include endothelial cells constituting the cell structure and cells other than the endothelial cells.

The kit may further include other substances used in the evaluation method. Examples of the other substance include a culture medium for cell structures, a labeling substance for labeling vascular cells formed by endothelial cells, and a substance used when constructing a cell structure (for example, a cationic buffer). Liquid, strong electrolyte polymer, extracellular matrix component, etc.).

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

Example 1 Production of Cell Structure Having Vascular Structure 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.
As a cell structure including a vascular network structure, 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. In addition, Transwell cell culture insert (Corning, product number: # 3470) was used as the cell culture container, and 10% bovine serum (EXO-FBS-50A-1, manufactured by System Biosciences) was used as the culture medium. ) And 1% by volume penicillin / streptomycin (Wako Pure Chemical Industries, product number: 168-23191) -containing D-MEM (Wako Pure Chemical Industries, product number: 043-30085). 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 0.05, 0.1, 0.25, 0.5, 1.0, 1.5, 5.0% HUVEC of NHDF cells, heparin and collagen The cell suspension was prepared by suspending in the contained Tris-HCl buffer (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 400 × g for 1 minute at room temperature, the supernatant was removed, and then resuspended in an appropriate amount of culture medium (steps (a′-1) and (a′-2)). ). Subsequently, this cell suspension was seeded in a Transwell cell culture insert (step (b)), and then the Transwell cell culture insert was centrifuged at 400 × g for 1 minute at room temperature. Thereafter, an appropriate amount of culture medium was added to the Transwell cell culture insert, followed by culturing 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 the culture 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 vessel in the structure was labeled with green fluorescence. This fluorescently labeled cell structure was directly observed to confirm the presence or absence of vascular network formation.

From Table 1, the formation of the vascular network structure was confirmed under all conditions except for the case where HUVEC containing 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 from fibroblasts and vascular endothelial cells and having a vascular network structure.
As constituent cells of a cell structure including a vascular network structure, human neonatal dermal fibroblasts (NHDF) (manufactured by Lonza, product number: CC-2509), human umbilical vein endothelial cells (Human Umbilical) Two types of Vein Endothelial Cell (HUVEC) (manufactured by Lonza, product number: CC-2517A) were used. As a cell culture vessel, Transwell cell culture insert (Corning, product number: # 3470) was used. As culture media, D-MEM containing 10% by volume bovine serum (Corning, product number: # 35-010-CV) and 1% by volume penicillin / streptomycin (product number: 168-23191). (Product number: 043-30085 manufactured by Wako Pure Chemical Industries, Ltd.) 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 NHDFs and 3 × 10 ⁴ HUVECs were added to Tris-HCl buffer (0.1 mg / mL heparin, 0.1 mg / mL collagen, 50 mM Tris, pH 7) containing heparin and collagen. 4) to prepare a cell suspension (step (a)). This cell suspension was centrifuged at 400 × g for 1 minute at room temperature, the supernatant was removed, and then resuspended in an appropriate amount of culture medium (steps (a′-1) and (a′-2)). ). The cell suspension is then seeded in a transwell cell culture insert (step (b)), and then the transwell cell culture insert is centrifuged at 400 × g for 1 minute at room temperature to remove liquid components. did. Thereafter, an appropriate amount of culture medium was added to the Transwell cell culture insert, followed by culturing for 24 hours in a CO ₂ incubator (37 ° C., 5% CO ₂ ) (step (c)). After completion of the culture, a cell structure having a vascular network structure (mixed layer (21 layers) of NHDF (20 layers) and HUVEC (1 layer)) was obtained.

<Culture in the presence of bevacizumab>
The obtained cell structure was cultured at 37 ° C., 5% CO _{2 for} 72 hours in a culture medium in which the addition amount of bevacizumab 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 the culture 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 vessel in the structure was labeled with green fluorescence. This fluorescently labeled cell structure was directly observed, and further, image analysis was performed on the captured fluorescent image, and the area occupied by the fluorescent emission region in the fluorescent image was measured. The more cells that make up the blood vessel, the larger the area occupied by the fluorescence emission region. Each concentration was measured three times repeatedly. The measurement results are shown in Table 2.

As a result of direct observation, it was found that vascular network formation was significantly inhibited in the final concentration of 1 μg / well and 2 μg / well compared to the final concentration of bevacizumab of 0 μg / well (not shown). Also, the area occupied by the fluorescence emission region was 158.0 × 10 ⁵ μm ² at the final concentration of 0 μg / well, whereas it was 106.6 × 10 ⁵ μm ² at the final concentration of 1 μg / well. At 2 μg / well, it was 47.5 × 10 ⁵ μm ² , and the area occupied by the fluorescence emission region was significantly reduced by adding bevacizumab. From these results, bevacizumab was evaluated to have an angiogenesis inhibitory effect. Since bevacizumab is a known angiogenesis inhibitor, it was confirmed that the angiogenesis inhibitory action of the test compound can be evaluated by the evaluation method according to the present invention.

[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, HT29 (ATCC number: HTB-38TM), which is a human colorectal adenocarcinoma cell line, is used as a constituent cell of a cell structure including a vascular network structure. The cell culture container, culture medium, and bevacizumab 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, 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 HUVEC cells, those previously fluorescently labeled (manufactured by SIGMA, product number PKH26GL) were used.

<Culture in the presence of bevacizumab>
The obtained cell structure was cultured at 37 ° C., 5% CO _{2 for} 72 hours in a culture medium in which the addition amount of bevacizumab 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. Next, 300 μL of a 0.25% trypsin-EDTA solution (manufactured by Invitrogen) was added to the Transwell cell culture insert, and incubated for 15 minutes in a CO ₂ incubator (37 ° C., 5% CO ₂ ). Thereafter, the entire amount of the solution was recovered and transferred to a 1.5 mL tube for recovery to which 300 μL of a 0.25% trypsin-EDTA solution (manufactured by Invitrogen) was added in advance. Next, 100 μL of 0.25% trypsin-EDTA solution (manufactured by Invitrogen) is added to the Transwell cell culture insert, and together with a 1.5 mL tube for collection for 5 minutes in a CO ₂ incubator (37 ° C., 5% CO ₂ ). Incubated. Thereafter, the entire amount of the solution was recovered, transferred to a 1.5 mL tube for recovery, and 300 μL of a 0.25% trypsin-EDTA solution (manufactured by Invitrogen) was added, and in a CO ₂ incubator (37 ° C., 5% CO ₂ ). Incubation for 5 minutes gave a cell structure dispersion.

<Live cell count analysis and evaluation>
The obtained dispersion of cell structures was immersed in trypan blue solution and stained with trypan blue, and then the cells that emitted fluorescence and were not stained with trypan blue were counted as living HUVECs. The cells were counted using the fluorescence mode of a cell counter “Countess II” (manufactured by Life Technologies). Table 3 shows the results of counting the number of viable cells of HUVEC and the calculated survival rate (ratio of viable cell counts when the viable cell count of 0 μg of bevacizumab is 100%) (%).

In contrast to the final concentration of 0 μg / well of bevacizumab in which angiogenesis is not inhibited, the number of viable cells of HUVEC is more pronounced in the final concentrations of 1 μg / well and 2 μg / well in which inhibition of angiogenesis was confirmed in Example 2. It was confirmed that the addition of bevacizumab decreases the survival rate of HUVEC. Since wells with higher anti-angiogenic effects of bevacizumab have a lower HUVEC survival rate, it can be said that the presence or absence of the angiogenesis inhibitory action of the test compound can also be evaluated from the HUVEC survival rate.

[Example 4] In the case of using a cell structure composed of lung-derived stromal cells A cell structure formed from fibroblasts and vascular endothelial cells and having a vascular network structure was used with bevacizumab, an angiogenesis inhibitor. evaluated.
Examples of cell structures including a vascular network structure include normal human fibroblasts (NHPF) (manufactured by Promocell, product number: C-12360), and human lung microvascular endothelial cells (Human Dermal Microvascular Endothelial Cell). : HMVEC-L) (manufactured by Lonza, product number: CC-2527), a cell structure formed from two types of cells was used. In addition, Transwell cell culture insert (Corning, product number: # 3470) was used as the cell culture container, and 10% bovine serum (EXO-FBS-50A-1, manufactured by System Biosciences) was used as the culture medium. ) And 1% by volume penicillin / streptomycin (Wako Pure Chemical Industries, product number: 168-23191) -containing D-MEM (Wako Pure Chemical Industries, product number: 043-30085). 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 NHPF and 1 × 10 ⁵ HMVEC-L were added to Tris-HCl buffer (0.1 mg / mL heparin, 0.1 mg / mL collagen, 50 mM Tris, containing heparin and collagen, The suspension was 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 400 × g for 1 minute at room temperature, the supernatant was removed, and then resuspended in an appropriate amount of culture medium (steps (a′-1) and (a′-2)). ). Subsequently, this cell suspension was seeded in a Transwell cell culture insert (step (b)), and then the Transwell cell culture insert was centrifuged at 400 × g for 1 minute at room temperature. Thereafter, an appropriate amount of culture medium was added to the Transwell cell culture insert, followed by culturing for 24 hours in a CO ₂ incubator (37 ° C., 5% CO ₂ ) (step (c)).

<Culture in the presence of bevacizumab>
The obtained cell structure was cultured for 144 hours at 37 ° C. and 5% CO ₂ in a culture medium in which the addition amount of bevacizumab 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 the culture 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 vessel in the structure was labeled with green fluorescence. This fluorescently labeled cell structure was directly observed, and further, image analysis was performed on the captured fluorescent image, and the area occupied by the fluorescent emission region in the fluorescent image was measured. The results are shown in Table 4. The more cells that make up the blood vessel, the larger the area occupied by the fluorescence emission region. Each concentration was measured three times repeatedly.

As a result, it was 252 × 10 ⁵ μm ² at the final concentration of 0 μg / well, whereas it was 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 area occupied by the fluorescence emission region was significantly reduced by the addition of bevacizumab. From these results, it was evaluated that bevacizumab has an angiogenesis inhibitory effect even when lung-derived fibroblasts and vascular endothelial cells were used.

Claims (6)

1. A culture step of culturing a cell structure having a vascular network structure in the presence of a test compound;
   An evaluation step for evaluating whether the test compound has an angiogenesis inhibitory activity, using as an index the state of the vasculature in the cell structure after the culturing step;
   Have
   In the evaluation step, when the number of cells constituting the vasculature in the cell structure is small as compared to when cultured in the absence of the test compound, or the vascular network structure in the cell structure A method for evaluating an anti-angiogenic activity of a test compound, wherein the test compound is evaluated to have an anti-angiogenic activity when the test compound is damaged.
2. The method for evaluating angiogenesis inhibitory activity of a test compound according to claim 1, wherein the cell structure comprises one or more selected from the group consisting of vascular endothelial cells and lymphatic endothelial cells.
3. The angiogenesis of the test compound according to claim 2, wherein the cell structure further comprises one or more selected from the group consisting of fibroblasts, nerve cells, dendritic cells, macrophages, and mast cells. Evaluation method of inhibitory activity.
4. The cellular structure comprises fibroblasts;
   The angiogenesis inhibitory activity of the test compound according to claim 2, wherein 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. Evaluation method.
5. A kit for performing the method for evaluating angiogenesis inhibitory activity of a test compound according to any one of claims 1 to 4,
   A kit for evaluating angiogenesis inhibitory activity, comprising a cell structure having a vascular network structure and a cell culture container containing the cell structure.
6. The kit according to claim 5, wherein the cell structure comprises a vascular network structure sandwiched between fibroblast layers.