WO2017183676A1 - Method for evaluating angiogenesis inhibitory activity of test compound - Google Patents
Method for evaluating angiogenesis inhibitory activity of test compound Download PDFInfo
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Definitions
- 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.
- cancer chemotherapy not only chemotherapy targeting cancer cells themselves, but also treatment methods targeting the microenvironment around cancer have been developed.
- 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.
- lymphangiogenesis is delayed, but lymphangiogenesis is also a target for cancer chemotherapy.
- lymphangiogenesis is regarded as important as a prognostic factor for patients such as lymph node metastasis, and development of lymphangiogenesis inhibitors is also progressing.
- 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
- 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.
- there is a method of adding a test compound and evaluating angiogenic ability (see Non-Patent Document 1).
- 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.
- 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.
- 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.
- 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.
- the said evaluation method can be implemented more simply by using the angiogenesis inhibitory activity evaluation kit according to the present invention.
- 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.
- 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.
- the “vascular network structure” refers to a network structure such as a vascular network or a lymphatic network in a living tissue.
- 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).
- the cell structure used in 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.
- 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.
- 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.
- 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).
- 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, hypophary
- 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.
- 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.
- the biological species from which the cells constituting the cell structure according to the present embodiment are derived is not particularly limited.
- 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.
- 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 ⁇ m or more, more preferably 10 ⁇ m or more, and more preferably 50 ⁇ m or more. More preferably, it is more preferably 100 ⁇ m or more. The thickness of the cell structure is preferably 500 ⁇ m or less, more preferably 400 ⁇ m or less, and even more preferably 300 ⁇ m 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.
- 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 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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
- ECM electro-chemical compound
- 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.
- 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.
- RGD arginine-glycine-aspartic acid
- a cell structure in which a vascular network structure is formed in the entire structure can be constructed by a single centrifugation process.
- 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.
- 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.
- 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.
- 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.
- 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.
- step (a) it is preferable to further mix the cells with a strong electrolyte polymer.
- 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.
- the cationic buffer examples include tris-hydrochloric acid buffer, tris-maleic acid buffer, bis-tris-buffer, and HEPES.
- 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.
- 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 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.
- chondroitin sulfate eg, chondroitin 4-sulfate, chondroitin 6-sulfate
- heparan sulfate eg, dermatan sulfate, keratan sulfate, hyaluronic acid and other glycosaminogly
- the polyelectrolyte is preferably a glycosaminoglycan.
- 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.
- 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.
- the cell structure can be constructed by adjusting the mixture without mixing the strong electrolyte.
- extracellular matrix component examples include collagen, laminin, fibronectin, vitronectin, elastin, tenascin, entactin, fibrillin, proteoglycan, or a modified or variant thereof.
- 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.
- 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.
- 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.
- 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. .
- 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.
- 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.
- steps (b) and (c) are performed to form 10 layers of fibroblasts on the vascular endothelial cell layer in the cell culture container.
- step (b) Laminate the layers.
- step (c) the thickness of the cell layer laminated in the step (c) can be adjusted.
- 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.
- steps (b) and (c) are performed.
- 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.
- 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.
- angiogenesis is inhibited when the test compound has angiogenesis inhibitory activity.
- 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.
- 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, MEM ⁇ , RPMI-1640, Ham's F-12, and the like.
- step (a) 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).
- “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.
- the buffer include phosphate physiological saline (PBS), HEPES, Hanks buffer, and the like.
- the culture medium include D-MEM, E-MEM, MEM ⁇ , RPMI-1640, Ham's F-12, and the like.
- step (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.
- 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.
- 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.
- 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.
- 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- ⁇ (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)
- a cell structure having a vascular network structure is cultured in the presence of a test compound.
- 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.
- 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.
- test compound 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.
- the cells in the cell structure can be directly observed by fluorescently labeling the cells constituting the vessel.
- 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.
- 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.
- 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.
- 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.
- the total fluorescence intensity is small and the area of the fluorescence emission region is small.
- 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.
- 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.
- 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.
- 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.).
- 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.
- 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.
- 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.
- bovine serum EXO-FBS-50A-1, manufactured by System Biosciences
- penicillin / streptomycin Wako Pure Chemical Industries, product number: 168-23191
- D-MEM Dako Pure Chemical Industries, product number: 043-30085
- 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 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)). ).
- 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)).
- 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.
- a cell structure formed from fibroblasts and vascular endothelial cells and having a vascular network structure.
- 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.
- NHDF neonatal dermal fibroblasts
- Human Umbilical Human Umbilical
- VEC Vein Endothelial Cell
- Transwell cell culture insert (Corning, product number: # 3470) was used.
- D-MEM 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.
- bevacizumab 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)). ).
- step (b) 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.
- a vascular network structure mixed layer (21 layers) of NHDF (20 layers) and HUVEC (1 layer
- 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 ⁇ g.
- 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.
- HT29 ATCC number: HTB-38TM
- HTB-38TM human colorectal adenocarcinoma cell line
- 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.
- HUVEC cells those previously fluorescently labeled (manufactured by SIGMA, product number PKH26GL) were used.
- 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 ⁇ g.
- 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.
- 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.
- 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.
- bovine serum EXO-FBS-50A-1, manufactured by System Biosciences
- penicillin / streptomycin Wako Pure Chemical Industries, product number: 168-23191
- D-MEM Dako Pure Chemical Industries, product number: 043-30085
- bevacizumab manufactured by R & D Systems, product number: MAB293 was used as the angiogenesis inhibitor to be evaluated.
- ⁇ Construction of cell structure> 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)). ).
- 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)).
- 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 ⁇ g.
- bevacizumab has an angiogenesis inhibitory effect even when lung-derived fibroblasts and vascular endothelial cells were used.
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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
本発明は、動物モデルを用いることなく、in vitroの系で被検化合物の脈管新生阻害活性を評価する方法に関する。
本願は、2016年4月19日に日本に出願された特願2016-083947号に基づき優先権を主張し、その内容をここに援用する。 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.
本願は、2016年4月19日に日本に出願された特願2016-083947号に基づき優先権を主張し、その内容をここに援用する。 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.
近年のがん化学療法においては、がん細胞自体を標的とする化学療法だけではなく、がんの周辺の微小環境を標的とした治療法も開発されている。そのひとつが、血管新生阻害剤を単剤で投与する、又は一般的な細胞障害性を有する抗がん剤と併用投与する療法である。血管やリンパ管等の脈管の新生は、がん(腫瘍)の成長や、浸潤、転移等の悪性化及び進行に深く係わっていることが知られている。
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.
血管新生阻害剤は、血管形成に必須となる因子やその受容体等血管形成に必須となるタンパク質シグナルパスウェイを阻害することによって血管新生を抑制、阻害する薬剤である。代表的なものとしては、2004年に米国で承認されたAvastin(登録商標)(Genentech社製、別名ベバシズマブ)等があり、それ以降、EYLEA(登録商標)(Bayer Yakuhin社製、別名アフリベルセプト)、Suchibaga(登録商標)(Bayer Yakuhin社製、別名レゴラフェニブ)等の様々な薬剤が開発され、臨床現場に導入されている。
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.
血管新生には遅れをとっているが、リンパ管新生についても同様に、がん化学療法の標的とされている。特にリンパ節転移などの患者の予後因子としてリンパ管新生は重要視されており、リンパ管新生阻害剤の開発も進んでいる。
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.
現在これら脈管新生阻害剤の評価系としては、基本的には免疫不全マウス等のin vivoモデルを用いるのが一般的である。in vitroの評価系としては、ガラス、プラスチック等の基材上に内皮細胞と繊維芽細胞とを有する単層の細胞層を形成し、この細胞層に脈管を形成させる系に、被検化合物を添加し、脈管新生能を評価する方法(特許文献1参照)や、ガラス、プラスチック等の基材上に微細加工を施して脈管の骨格を作り、当該骨格中で内皮細胞を成長させて脈管を形成させる系に、被検化合物を添加し、脈管新生能を評価する方法や、細胞外マトリックスなどの生体内試料のゲル内で内皮細胞を成長させて脈管を形成させる系に、被検化合物を添加し、脈管新生能を評価する方法(非特許文献1参照。)等がある。
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).
薬剤の薬効評価をin vitroの評価系で行う場合、得られる評価の信頼性が問題となる。すなわち、当該評価系での評価が、実際に当該薬剤を生体に投与した場合に得られる薬効を反映していることが重要であり、当該評価系での評価と生体に投与して得られる効果とが一致する確率が高い評価系が、信頼性の高い評価系である。
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.
特許文献1や非特許文献1に記載のin vitroの評価系では、間質細胞等の脈管の周辺組織がない状態の脈管を利用しているため、実際の生体内を正しく再現できる評価系とはいえない。このため、これらの評価系で脈管新生阻害活性を有すると評価された化合物であっても、実際に生体に投与された場合には適切な脈管新生阻害活性が奏されない場合も多い。
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.
本発明は、被検化合物の脈管新生阻害活性を評価するに際して、動物モデルを用いることなく、より信頼性の高い評価を行うことができる評価方法を提供することを目的とする。
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.
本発明の第一態様に係る被検化合物の脈管新生阻害活性の評価方法は、脈管網構造を備える細胞構造体を、被検化合物の存在下で培養する培養工程と、前記培養工程後の前記細胞構造体中の脈管の状態を指標として、前記被検化合物が、脈管新生阻害活性を有するか否かを評価する評価工程と、を有し、前記評価工程において、前記被検化合物の非存在下で培養した場合と比較して、前記細胞構造体中の脈管を構成する細胞数が少ない場合、又は前記細胞構造体中の脈管網構造が損傷を受けている場合に、前記被検化合物が脈管新生阻害活性を有すると評価する。
上記第一態様において、前記細胞構造体が、血管内皮細胞及びリンパ管内皮細胞からなる群より選択される1種以上を含んでいてもよい。
上記第一態様において、前記細胞構造体が、さらに、繊維芽細胞、神経細胞、樹状細胞、マクロファージ、及び肥満細胞からなる群より選択される1種以上を含んでいてもよい。
上記第一態様において、前記細胞構造体が、繊維芽細胞を含み、前記細胞構造体中の血管内皮細胞及びリンパ管内皮細胞の総細胞数が、繊維芽細胞の細胞数の0.1%以上であってもよい。
本発明の第二態様に係る脈管新生阻害活性評価用キットは、上記第一態様に係る被検化合物の脈管新生阻害活性の評価方法を行うためのキットであって、脈管網構造を備える細胞構造体と、前記細胞構造体を収容する細胞培養容器とを備える。
上記第二態様において、前記細胞構造体が、繊維芽細胞層に挟まれた脈管網構造を備えていていもよい。 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.
上記第一態様において、前記細胞構造体が、血管内皮細胞及びリンパ管内皮細胞からなる群より選択される1種以上を含んでいてもよい。
上記第一態様において、前記細胞構造体が、さらに、繊維芽細胞、神経細胞、樹状細胞、マクロファージ、及び肥満細胞からなる群より選択される1種以上を含んでいてもよい。
上記第一態様において、前記細胞構造体が、繊維芽細胞を含み、前記細胞構造体中の血管内皮細胞及びリンパ管内皮細胞の総細胞数が、繊維芽細胞の細胞数の0.1%以上であってもよい。
本発明の第二態様に係る脈管新生阻害活性評価用キットは、上記第一態様に係る被検化合物の脈管新生阻害活性の評価方法を行うためのキットであって、脈管網構造を備える細胞構造体と、前記細胞構造体を収容する細胞培養容器とを備える。
上記第二態様において、前記細胞構造体が、繊維芽細胞層に挟まれた脈管網構造を備えていていもよい。 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.
本発明の上記態様に係る被検化合物の脈管新生阻害活性の評価方法は、in vitroの評価系であって、生体内の状態により近い脈管網構造を含む細胞構造体を用い、当該細胞構造体内の脈管形成に対する影響を指標として被検化合物の脈管新生阻害活性を評価する方法である。このため、当該評価方法を用いることにより、近年がん化学治療薬として注目されている血管新生阻害剤及びリンパ管新生阻害剤の候補化合物の脈管新生阻害活性について、動物モデルを用いることなく信頼性の高い評価を得ることができる。
また、本発明に係る脈管新生阻害活性評価用キットを用いることにより、前記評価方法をより簡便に実施することができる。 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.
また、本発明に係る脈管新生阻害活性評価用キットを用いることにより、前記評価方法をより簡便に実施することができる。 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.
以下、本発明の一実施形態に係る被検化合物の脈管新生阻害活性の評価方法を説明する。本実施形態に係る被検化合物の脈管新生阻害活性の評価方法(以下、「本実施形態に係る評価方法」ということがある。)は、脈管網構造を備える細胞構造体を、被検化合物の存在下で培養する培養工程と、前記培養工程後の前記細胞構造体中の脈管の状態を指標として、前記被検化合物が、脈管新生阻害活性を有するか否かを評価する評価工程と、を有する。本実施形態に係る評価方法は、三次元構造内に脈管網構造を備える細胞構造体を用い、この細胞構造体内の脈管形成に対する影響を指標として被検化合物の脈管新生阻害活性を評価する。このように、実際の生体内の環境に近似した状態で形成された脈管に対する影響を指標とするため、本実施形態に係る評価方法により信頼性の高い評価が得られる。なお、本発明及び本願明細書において、「脈管網構造」とは、生体組織における血管網やリンパ管網のような、網状の構造を指す。
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.
<細胞構造体>
本発明及び本願明細書において、「細胞構造体」とは、複数の細胞層が積層された3次元構造体である。本実施形態において用いられる細胞構造体は、脈管網構造を備えており、脈管を構成する内皮細胞と、脈管を構成していない細胞(内皮細胞以外の細胞)と、により構成される。すなわち、本実施形態において用いられる細胞構造体(以下、「本実施形態に係る細胞構造体」ということがある。)は、脈管を形成していない細胞の積層体の内部に、リンパ管及び/又は血管等の脈管網構造が三次元的に構築され、より生体内に近い組織を構築している。脈管網構造は、細胞構造体の内部にのみ形成されていてもよく、少なくともその一部が細胞構造体の表面又は底面に露出されるように形成されていてもよい。 <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.
本発明及び本願明細書において、「細胞構造体」とは、複数の細胞層が積層された3次元構造体である。本実施形態において用いられる細胞構造体は、脈管網構造を備えており、脈管を構成する内皮細胞と、脈管を構成していない細胞(内皮細胞以外の細胞)と、により構成される。すなわち、本実施形態において用いられる細胞構造体(以下、「本実施形態に係る細胞構造体」ということがある。)は、脈管を形成していない細胞の積層体の内部に、リンパ管及び/又は血管等の脈管網構造が三次元的に構築され、より生体内に近い組織を構築している。脈管網構造は、細胞構造体の内部にのみ形成されていてもよく、少なくともその一部が細胞構造体の表面又は底面に露出されるように形成されていてもよい。 <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.
本実施形態に係る細胞構造体に含まれる内皮細胞としては、血管内皮細胞であってもよく、リンパ管内皮細胞であってもよい。また、血管内皮細胞とリンパ管内皮細胞の両方を含んでいてもよい。
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.
本実施形態に係る細胞構造体に含まれる内皮細胞以外の細胞の細胞種としては、内皮細胞が脈管網を形成することを阻害しないものであれば特に限定されるものではなく、評価対象となる被検化合物の種類や、目的の脈管新生阻害活性が奏される生体内の環境等を考慮して、適宜選択することができる。本実施形態に係る細胞構造体中の内皮細胞以外の細胞としては、内皮細胞が本来の機能及び形状を保持する脈管網を形成しやすいことから、生体内において脈管の周辺組織を構成する細胞であることが好ましい。このような細胞としては、例えば、繊維芽細胞、神経細胞、樹状細胞、マクロファージ、肥満細胞、上皮細胞、心筋細胞、肝細胞、膵島細胞、組織幹細胞、平滑筋細胞等が挙げられる。細胞構造体に含まれる内皮細胞以外の細胞は、1種類であってもよく、2種類以上であってもよい。本実施形態に係る細胞構造体としては、内皮細胞以外の細胞として少なくとも繊維芽細胞を含む細胞構造体が好ましく、血管内皮細胞と繊維芽細胞とを含む細胞構造体、リンパ管内皮細胞と繊維芽細胞とを含む細胞構造体、又は血管内皮細胞とリンパ管内皮細胞と繊維芽細胞とを含む細胞構造体がより好ましい。なお、細胞構造体に含まれる内皮細胞以外の細胞としては、内皮細胞と同種の生物種由来の細胞であってもよく、異種の生物種由来の細胞であってもよい。
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.
本実施形態に係る細胞構造体中の内皮細胞の数は、脈管網構造が形成されるのに充分な数であれば特に限定されるものではなく、細胞構造体の大きさ、内皮細胞や内皮細胞以外の細胞の細胞種等を考慮して適宜決定することができる。例えば、本実施形態に係る細胞構造体を構成する全細胞に対する内皮細胞の存在比(細胞数比)を0.1%以上に設定することによって、脈管網構造が形成された細胞構造体を調製できる。内皮細胞以外の細胞として繊維芽細胞を用いる場合、本実施形態に係る細胞構造体における内皮細胞数は、繊維芽細胞数の0.1%以上であることが好ましく、0.1~10.0%であることがより好ましく、0.1~5.0%であることがさらに好ましい。内皮細胞として血管内皮細胞とリンパ管内皮細胞との両方を含む場合、血管内皮細胞及びリンパ管内皮細胞の総細胞数が、繊維芽細胞数の0.1%以上であることが好ましく、0.1~10.0%であることがより好ましく、0.1~5.0%であることがさらに好ましい。
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%.
本実施形態に係る細胞構造体は、内皮細胞以外の細胞として、生体内において脈管の周辺組織を構成する細胞以外の細胞を含んでいてもよい。当該細胞としては、例えば、がん細胞等が挙げられる。
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.
がん細胞とは、体細胞から派生して無限の増殖能を獲得した細胞である。がん細胞の由来となるがんとしては、例えば、乳がん(例えば、浸潤性乳管がん、非浸潤性乳管がん、炎症性乳がん等)、前立腺がん(例えば、ホルモン依存性前立腺がん、ホルモン非依存性前立腺がん等)、膵がん(例えば、膵管がん等)、胃がん(例えば、乳頭腺がん、粘液性腺がん、腺扁平上皮がん等)、肺がん(例えば、非小細胞肺がん、小細胞肺がん、悪性中皮腫等)、結腸がん(例えば、消化管間質腫瘍等)、直腸がん(例えば、消化管間質腫瘍等)、大腸がん(例えば、家族性大腸がん、遺伝性非ポリポーシス大腸がん、消化管間質腫瘍等)、小腸がん(例えば、非ホジキンリンパ腫、消化管間質腫瘍等)、食道がん、十二指腸がん、舌がん、咽頭がん(例えば、上咽頭がん、中咽頭がん、下咽頭がん等)、頭頚部がん、唾液腺がん、脳腫瘍(例えば、松果体星細胞腫瘍、毛様細胞性星細胞腫、びまん性星細胞腫、退形成性星細胞腫等)、神経鞘腫、肝臓がん(例えば、原発性肝がん、肝外胆管がん等)、腎臓がん(例えば、腎細胞がん、腎盂と尿管の移行上皮がん等)、胆嚢がん、胆管がん、膵臓がん、肝がん、子宮内膜がん、子宮頸がん、卵巣がん(例、上皮性卵巣がん、性腺外胚細胞腫瘍、卵巣性胚細胞腫瘍、卵巣低悪性度腫瘍等)、膀胱がん、尿道がん、皮膚がん(例えば、眼内(眼)黒色腫、メルケル細胞がん等)、血管腫、悪性リンパ腫(例えば、細網肉腫、リンパ肉腫、ホジキン病等)、メラノーマ(悪性黒色腫)、甲状腺がん(例えば、甲状腺髄様がん等)、副甲状腺がん、鼻腔がん、副鼻腔がん、骨腫瘍(例えば、骨肉腫、ユーイング腫瘍、子宮肉腫、軟部組織肉腫等)、転移性髄芽腫、血管線維腫、隆起性皮膚線維肉腫、網膜肉腫、陰茎癌、精巣腫瘍、小児固形がん(例えば、ウィルムス腫瘍、小児腎腫瘍等)、カポジ肉腫、AIDSに起因するカポジ肉腫、上顎洞腫瘍、線維性組織球腫、平滑筋肉腫、横紋筋肉腫、慢性骨髄増殖性疾患、白血病(例えば、急性骨髄性白血病、急性リンパ芽球性白血病等)等が挙げられ、これらに限定されない。
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.
本実施形態に係る細胞構造体を構成する細胞の種類は特に限定されず、動物から採取された細胞であってもよく、動物から採取された細胞を培養した細胞であってもよく、動物から採取された細胞に各種処理を施した細胞であってもよく、培養細胞株であってもよい。動物から採取された細胞の場合、採取部位は特に限定されず、骨、筋肉、内臓、神経、脳、皮膚、血液などに由来する体細胞であってもよく、生殖細胞であってもよく、胚性幹細胞(ES細胞)であってもよい。また、本実施形態に係る細胞構造体を構成する細胞が由来する生物種は特に限定されるものではなく、例えば、ヒト、サル、イヌ、ネコ、ウサギ、ブタ、ウシ、マウス、ラット等の動物に由来する細胞を用いることができる。動物から採取された細胞を培養した細胞としては、初代培養細胞であってもよく、継代培養細胞であってもよい。また、各種処理を施した細胞としては、誘導多能性幹細胞細胞(iPS細胞)や、分化誘導後の細胞が挙げられる。また、本実施形態に係る細胞構造体は、同種の生物種由来の細胞のみから構成されていてもよく、複数種類の生物種由来の細胞により構成されていてもよい。
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.
本実施形態に係る細胞構造体の大きさや形状は、脈管網構造が形成可能な構成であれば特に限定されない。より生体内の組織に形成された脈管と近い状態の脈管網構造が形成可能であることから、当該細胞構造体の厚さは、5μm以上が好ましく、10μm以上がより好ましく、50μm以上がさらに好ましく、100μm以上がよりさらに好ましい。当該細胞構造体の厚さとしては、また、500μm以下が好ましく、400μm以下がより好ましく、300μm以下がさらに好ましい。本実施形態に係る細胞構造体の細胞層の数としては、2~60層程度が好ましく、5~60層程度がより好ましく、10~60層程度がさらに好ましい。
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.
なお、細胞構造体を構成する細胞層数は、三次元構造を構成する細胞の総数を、1層当たりの細胞数(1層を構成するために必要な細胞数)で除することにより測定される。1層当たりの細胞数は、細胞構造体を構成させる際に使用する細胞容器に、予め細胞をコンフルエントになるように平面的に培養して調べることができる。具体的には、ある細胞容器に形成された細胞構造体の細胞層数は、当該細胞構造体を構成する全細胞数を計測し、当該細胞容器の1層当たりの細胞数で除することにより算出できる。
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.
一般的に、本実施形態に係る細胞構造体は、細胞培養容器中に構築される。当該細胞培養容器としては、細胞構造体の構築が可能であり、かつ構築された細胞構造体の培養が可能な容器であれば特に限定されない。当該細胞培養容器としては、具体的には、ディッシュ、セルカルチャーインサート(例えば、Transwell(登録商標)インサート、Netwell(登録商標)インサート、Falcon(登録商標)セルカルチャーインサート、Millicell(登録商標)セルカルチャーインサート等)、チューブ、フラスコ、ボトル、プレート等が挙げられる。本実施形態に係る細胞構造体の構築においては、当該細胞構造体を用いた被検化合物の評価をより適正に行うことができるため、ディッシュ又は各種セルカルチャーインサートが好ましい。
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.
本実施形態に係る細胞構造体は、脈管網構造が形成された多層の細胞から構成される構造体であればよく、その構築方法は特に限定されない。例えば、一層ずつ構築して順次積層させて構築する方法であってもよく、2層以上の細胞層を一度に構築する方法であってもよく、両構築方法を適宜組み合わせて多層の細胞層を構築する方法であってもよい。また、本実施形態に係る細胞構造体は、各細胞層を構成する細胞種が層ごとに異なる多層構造体であってもよく、各細胞層を構成する細胞種が、構造体の全層で共通する多層構造体であってもよい。例えば、細胞種毎に層を形成し、この細胞層を順次積層させることによって構築する方法であってもよく、複数種類の細胞を混合した細胞混合液を予め調製し、この細胞混合液から多層構造の細胞構造体を一度に構築する方法であってもよい。
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.
一層ずつ構築して順次積層させて構築する方法としては、例えば、日本国特許第4919464号公報に記載されている方法、すなわち、細胞層を形成する工程と、形成された細胞層をECM(細胞外マトリックス)の成分を含有する溶液に接触させる工程と、を交互に繰り返すことにより、連続的に細胞層を積層する方法が挙げられる。例えば、当該方法を行うに際し、予め、細胞構造体を構成する全ての細胞を混合した細胞混合物を調製しておき、この細胞混合物によって各細胞層を形成することによって、構造体全体に脈管網構造が形成されている細胞構造体が構築できる。また、各細胞層を、細胞種ごとに形成することによって、内皮細胞から形成される層にのみ脈管網構造が形成されている細胞構造体が構築できる。
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.
2層以上の細胞層を一度に構築する方法としては、例えば、特許第5850419号公報に記載されている方法が挙げられる。すなわち、予め細胞の表面全体をインテグリンが結合するアルギニン-グリシン-アスパラギン酸(RGD)配列を含む高分子と、前記RGD配列を含む高分子に対する相互作用をする高分子と、によって被覆しておき、この接着膜で被覆された被覆細胞を細胞培養容器に収容した後、遠心処理等によって被覆細胞同士を集積させることにより、多層の細胞層から形成される細胞構造体を構築する方法が挙げられる。例えば、当該方法を行うに際し、予め、細胞構造体を構成する全ての細胞を混合した細胞混合物を調製しておき、この細胞混合物に接着性成分を添加することによって調製された被覆細胞を用いる。これにより、1度の遠心処理によって、構造体全体に脈管網構造が形成されている細胞構造体が構築できる。また、例えば、内皮細胞を被覆した被覆細胞と、繊維芽細胞を被覆した被覆細胞とを、それぞれ別個に調製し、繊維芽細胞の被覆細胞から構成される多層を形成した後、その上に内皮細胞の被覆細胞から形成された1層を積層させ、さらにその上に繊維芽細胞の被覆細胞から形成された多層を積層させる。これにより、厚みのある繊維芽細胞層に挟まれた脈管網構造を備える細胞構造体が構築できる。
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.
本実施形態に係る細胞構造体は、下記(a)~(c)の工程を有する方法により構築することもできる。
(a)カチオン性緩衝液中で、細胞と細胞外マトリックス成分とを混合して混合物を得る工程と、
(b)前記工程(a)により得られた混合物を、細胞培養容器中に播種する工程と、
(c)前記工程(b)の後、前記細胞培養容器中の細胞混合物から液体成分を除去し、当該細胞培養容器中に細胞が多層に積層された細胞構造体を得る工程。 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.
(a)カチオン性緩衝液中で、細胞と細胞外マトリックス成分とを混合して混合物を得る工程と、
(b)前記工程(a)により得られた混合物を、細胞培養容器中に播種する工程と、
(c)前記工程(b)の後、前記細胞培養容器中の細胞混合物から液体成分を除去し、当該細胞培養容器中に細胞が多層に積層された細胞構造体を得る工程。 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.
本発明においては、工程(a)において、細胞を、カチオン性物質を含む緩衝液および細胞外マトリックス成分と混合し、この細胞混合物から細胞集合体を形成することにより、内部に大きな空隙が少ない立体的細胞組織を得ることができる。また、得られた立体的細胞組織は、比較的安定であるため、少なくとも数日間の培養が可能であり、かつ培地交換時にも組織が崩壊し難い。
また、本発明においては、工程(b)において、細胞培養容器内に播種した細胞混合物を当該細胞培養容器内に沈降させることを含み得る。細胞混合物の沈降は、遠心分離等によって積極的に細胞を沈降させてもよいし、自然沈降させてもよい。 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.
また、本発明においては、工程(b)において、細胞培養容器内に播種した細胞混合物を当該細胞培養容器内に沈降させることを含み得る。細胞混合物の沈降は、遠心分離等によって積極的に細胞を沈降させてもよいし、自然沈降させてもよい。 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.
工程(a)において、細胞をさらに強電解質高分子と混合することが好ましい。細胞をカチオン性物質、強電解質高分子および細胞外マトリックス成分と混合することにより、工程(b)において遠心分離等の細胞を積極的に集合させる処理を要することなく、自然沈降させた場合であっても、空隙が少なく厚みのある立体的細胞組織が得られる。
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.
前記カチオン性緩衝液としては、例えば、トリス-塩酸緩衝液、トリス-マレイン酸緩衝液、ビス-トリス-緩衝液、又はHEPES等が挙げられる。当該カチオン性緩衝液中のカチオン性物質(例えば、トリス-塩酸緩衝液におけるトリス)の濃度及びpHは、細胞の生育及び細胞構造体の構築に悪影響を及ぼさない限り、特に限定されない。例えば、カチオン性緩衝液中のカチオン性物質の濃度は、10~100mMとすることができ、40~70mMであることが好ましく、50mMであることがより好ましい。また、当該カチオン性緩衝液のpHは、6.0~8.0とすることができ、6.8~7.8であることが好ましく、7.2~7.6であることがより好ましい。
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. .
前記強電解質高分子としては、例えば、ヘパリンや、コンドロイチン硫酸(例えば、コンドロイチン4-硫酸、コンドロイチン6-硫酸)、ヘパラン硫酸、デルマタン硫酸、ケラタン硫酸、ヒアルロン酸等のグリコサミノグリカン;デキストラン硫酸や、ラムナン硫酸、フコイダン、カラギナン、ポリスチレンスルホン酸、ポリアクリルアミド-2-メチルプロパンスルホン酸、及びポリアクリル酸等、又はこれらの誘導体等が挙げられる。工程(a)において調製される混合物には、強電解質高分子を1種類のみ混合させてもよく、2種類以上を組み合わせて混合させてもよい。本実施形態に係る細胞構造体の構築においては、高分子電解質はグリコサミノグリカンであることが好ましい。また、ヘパリンデキストラン硫酸、コンドロイチン硫酸、及びデルマタン硫酸の少なくともいずれか1つを用いることが好ましい。本実施形態で用いられる高分子電解質はヘパリンであることがさらに好ましい。前記カチオン性緩衝液に混合する強電解質高分子の量は、細胞の生育及び細胞構造体の構築に悪影響を及ぼさない限り、特に限定されない。例えば、カチオン性緩衝液中の強電解質高分子の濃度は、0mg/mL超1.0mg/mL未満とすることができ、0.025~0.1mg/mLであることが好ましく、0.05~0.1mg/mLであることがより好ましい。また、本実施形態の一態様としては、前記強電解質を混合せずに前記混合物を調整し、細胞構造体の構築の行うこともできる。
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.
前記細胞外マトリックス成分としては、例えば、コラーゲンや、ラミニン、フィブロネクチン、ビトロネクチン、エラスチン、テネイシン、エンタクチン、フィブリリン、プロテオグリカン、又はこれらの改変体若しくはバリアント等が挙げられる。プロテオグリカンには、コンドロイチン硫酸プロテオグリカン、ヘパラン硫酸プロテオグリカン、ケラタン硫酸プロテオグリカン、デルマタン硫酸プロテオグリカン等が挙げられる。工程(a)において調製される混合物には、細胞外マトリックス成分を1種類のみ混合させてもよく、2種類以上を組み合わせて混合させてもよい。本実施形態に係る細胞構造体の構築においては、コラーゲン、ラミニン、フィブロネクチンを用いることが好ましく、コラーゲンを用いることがより好ましい。前記カチオン性緩衝液に混合する細胞外マトリックス成分の量は、細胞の生育及び細胞構造体の構築に悪影響を及ぼさない限り、特に限定されない。例えば、カチオン性緩衝液中の細胞外マトリックス成分の濃度は、0mg/mL超1.0mg/mL未満とすることができ、0.025~0.1mg/mLであることが好ましく、0.05~0.1mg/mLであることがより好ましい。
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.
前記カチオン性緩衝液に混合する強電解質高分子と細胞外マトリックス成分と
の配合比は、1:2~2:1である。本実施形態に係る細胞構造体の構築においては、強電解質高分子と細胞外マトリックス成分との配合比が、1:1.5~1.5:1であることが好ましく、1:1であることがより好ましい。 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.
の配合比は、1:2~2:1である。本実施形態に係る細胞構造体の構築においては、強電解質高分子と細胞外マトリックス成分との配合比が、1:1.5~1.5:1であることが好ましく、1:1であることがより好ましい。 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.
工程(a)~(c)を繰り返す、具体的には、工程(c)で得られた細胞構造体の上に、工程(b)として、工程(a)で調製した混合物を播種した後、工程(c)を行うことを繰り返すことにより、充分な厚みの細胞構造体を構築することができる。工程(c)で得られた細胞構造体の上に新たに播種する混合物の細胞組成は、既に構築されている細胞構造体を構成する細胞組成と同じであってもよく、異なっていてもよい。
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. .
例えば、まず、工程(a)において細胞としては繊維芽細胞のみを含む混合物を調製し、工程(b)及び(c)を行って細胞培養容器に10層の繊維芽細胞層から形成される細胞構造体を得る。次いで、工程(a)として細胞として血管内皮細胞のみを含む混合物を調製し、工程(b)及び(c)を行って細胞培養容器内の10層の繊維芽細胞層の上に1層の血管内皮細胞層を積層させる。さらに、工程(a)として細胞として繊維芽細胞のみを含む混合物を調製し、工程(b)及び(c)を行って細胞培養容器内の血管内皮細胞層の上に、10層の繊維芽細胞層を積層させる。これにより、繊維芽細胞層10層―血管内皮細胞層1層―繊維芽細胞層10層と細胞種毎に順番に層状に積層された細胞構造体が構築できる。工程(b)において播種される細胞数を調節することにより、工程(c)において積層される細胞層の厚みを調整できる。工程(b)において播種される細胞数が多いほど、工程(c)において積層される細胞層の数が多くなる。また、工程(a)において、繊維芽細胞層20層分の繊維芽細胞と血管内皮細胞層1層分の血管内皮細胞を全て混合した混合物を調製し、工程(b)及び(c)を行うことにより、21層分の厚みを有し、血管網構造が構造体内部に散在している細胞構造体が構築できる。
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.
がん細胞を含む細胞構造体を構築する場合、例えば、細胞としてがん細胞のみを含む混合物を調製し、前述の通りに構築された繊維芽細胞層10層-血管内皮細胞層1層-繊維芽細胞層10層から形成される細胞構造体にさらにがん細胞層を積層させることができる。また、工程(a)において、繊維芽細胞と血管内皮細胞とがん細胞との全てを混合した混合物を調製し、工程(b)及び(c)を行うことにより、がん細胞と血管網構造との両方が構造体内部にそれぞれ独立して散在している細胞構造体が構築できる。
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.
がん細胞の増殖に脈管新生は重要な役割を果たしている。細胞構造体ががん細胞を含む場合、被検化合物が脈管新生阻害活性を有する場合には、脈管新生が阻害される。その結果、当該細胞構造体中のがん細胞の増殖が抑制され、被検化合物非存在下で培養した場合よりもがん細胞数が少なくなる。
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.
工程(a)~(c)を繰り返す場合に、工程(c)の後、工程(b)を行う前に、得られた細胞構造体を培養してもよい。培養に用いる培養培地の組成、培養温度、培養時間、培養時の大気組成等の培養条件は、当該細胞構造体を構成する細胞の培養に適した条件で行う。培養培地としては、例えば、D-MEM、E-MEM、MEMα、RPMI-1640、Ham’s F-12等が挙げられる。
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.
工程(a)の後に、(a’-1)得られた混合物から液体部分を除去し、細胞集合体を得る工程、および(a’-2)細胞集合体を溶液に懸濁する工程を行い、工程(b)へ進んでも良い。
また、工程(a)の後に、前記工程(b)に代えて、下記工程(b’-1)及び(b’-2)を行ってもよい。本発明及び本願明細書において、「細胞粘稠体」とは、非特許文献2に記載されるようなゲル様の細胞集合体を指す。
(b’-1)工程(a)で得られた混合物を細胞培養容器内に播種した後、混合物から液体成分を除去し細胞粘稠体を得る工程と、
(b’-2)細胞培養容器内に細胞粘稠体を溶媒に懸濁する工程。
上述の工程(a)~(c)を実施することで所望の組織体を得ることができるが、工程(a)の後に(a’-1)および(a’-2)を実施し、工程(b)を実施することで、より均質な組織体を得ることができる。 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.
また、工程(a)の後に、前記工程(b)に代えて、下記工程(b’-1)及び(b’-2)を行ってもよい。本発明及び本願明細書において、「細胞粘稠体」とは、非特許文献2に記載されるようなゲル様の細胞集合体を指す。
(b’-1)工程(a)で得られた混合物を細胞培養容器内に播種した後、混合物から液体成分を除去し細胞粘稠体を得る工程と、
(b’-2)細胞培養容器内に細胞粘稠体を溶媒に懸濁する工程。
上述の工程(a)~(c)を実施することで所望の組織体を得ることができるが、工程(a)の後に(a’-1)および(a’-2)を実施し、工程(b)を実施することで、より均質な組織体を得ることができる。 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.
細胞懸濁液を調製するための溶媒としては、細胞に対する毒性がなく、増殖性や機能を損なわないものであれば特に限定されるものではなく、水、緩衝液、細胞の培養培地等を用いることができる。当該緩衝液としては、例えば、リン酸生理食塩水(PBS)、HEPES、Hanks緩衝液等が挙げられる。培養培地としては、D-MEM、E-MEM、MEMα、RPMI-1640、Ham’s F-12等が挙げられる。
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.
前記工程(c)に代えて、下記工程(c’)を行ってもよい。
(c’)播種した混合物から液体成分を除去し、基材上に細胞の層を形成する工程。 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.
(c’)播種した混合物から液体成分を除去し、基材上に細胞の層を形成する工程。 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.
工程(c)及び(c’)における液体成分の除去処理の方法は、細胞の生育及び細胞構造体の構築に悪影響を及ぼさない限り、特に限定されず、液体成分と固体成分の懸濁物から液体成分を除去する方法として当業者に公知の手法により適宜行うことができる。当該手法としては、例えば、遠心分離処理、磁性分離処理、またはろ過処理等が挙げられる。
例えば、細胞培養容器としてセルカルチャーインサートを用いた場合には、混合物を播種したセルカルチャーインサートを、10℃、400×gで1分間の遠心分離処理に供することによって、液体成分を除去することができる。 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.
例えば、細胞培養容器としてセルカルチャーインサートを用いた場合には、混合物を播種したセルカルチャーインサートを、10℃、400×gで1分間の遠心分離処理に供することによって、液体成分を除去することができる。 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.
<被検化合物>
本実施形態に係る評価方法において、評価対象となる被検化合物は、脈管新生阻害活性を有することが期待される化合物であればよく、既知の脈管新生阻害剤であってもよく、新規な脈管新生阻害剤の候補化合物であってもよい。既知の脈管新生阻害剤としては、Avastin、EYLEA、Suchibaga、CYRAMZA(登録商標)(Eli Lilly社製、別名ラムシルマブ)、BMS-275291(Bristol-Myers社製)、Celecoxib(Pharmacia/Pfizer社製)、EMD121974(Merck社製)、Endostatin(EntreMed社製)、Erbitaux(ImCloneSystems社製)、Interferon-α(Roche社製)、LY317615(Eli Lilly社製)、Neovastat(Aeterna Laboratories社製)、PTK787(Abbott社製)、SU6688(Sugen社製)、Thalidomide(Celgene社製)、VEGF-Trap(Regeneron社製)、Iressa(登録商標)(Astrazeneca社製、別名ゲフィチニブ)、Caplerusa(登録商標)(Astrazeneca社製、別名パンデタニブ)、Recentin(登録商標)(Astrazeneca社製、別名セディラニブ)VGX―100(Circadian Technologies社製)、VD1andcVE199、VGX-300(Circadian Technologies社製)、sVEGFR2、hF4-3C5、Nexavar(登録商標)(Bayer Yakuhin社製、別名ソラフェニブ)、Vortrient(登録商標)(GlaxoSmithKline社製、別名パゾパニブ)、Sutent(登録商標)(Pfizer社製、別名スニチニブ)、Inlyta(登録商標)(Pfizer社製、別名アキシチニブ)、CEP-11981(Teva Pharmaceutical Industries社製)、AMG-386(Takeda Yakuhin社製、別名トレバナニブ)、anti-NRP2B(Genentech社製)、Ofev(登録商標)(boehringer-ingelheim社製、別名ニンタテニブ)、AMG706(Takeda Yakuhin社製、別名モテサニブ)等が挙げられる。 <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.
本実施形態に係る評価方法において、評価対象となる被検化合物は、脈管新生阻害活性を有することが期待される化合物であればよく、既知の脈管新生阻害剤であってもよく、新規な脈管新生阻害剤の候補化合物であってもよい。既知の脈管新生阻害剤としては、Avastin、EYLEA、Suchibaga、CYRAMZA(登録商標)(Eli Lilly社製、別名ラムシルマブ)、BMS-275291(Bristol-Myers社製)、Celecoxib(Pharmacia/Pfizer社製)、EMD121974(Merck社製)、Endostatin(EntreMed社製)、Erbitaux(ImCloneSystems社製)、Interferon-α(Roche社製)、LY317615(Eli Lilly社製)、Neovastat(Aeterna Laboratories社製)、PTK787(Abbott社製)、SU6688(Sugen社製)、Thalidomide(Celgene社製)、VEGF-Trap(Regeneron社製)、Iressa(登録商標)(Astrazeneca社製、別名ゲフィチニブ)、Caplerusa(登録商標)(Astrazeneca社製、別名パンデタニブ)、Recentin(登録商標)(Astrazeneca社製、別名セディラニブ)VGX―100(Circadian Technologies社製)、VD1andcVE199、VGX-300(Circadian Technologies社製)、sVEGFR2、hF4-3C5、Nexavar(登録商標)(Bayer Yakuhin社製、別名ソラフェニブ)、Vortrient(登録商標)(GlaxoSmithKline社製、別名パゾパニブ)、Sutent(登録商標)(Pfizer社製、別名スニチニブ)、Inlyta(登録商標)(Pfizer社製、別名アキシチニブ)、CEP-11981(Teva Pharmaceutical Industries社製)、AMG-386(Takeda Yakuhin社製、別名トレバナニブ)、anti-NRP2B(Genentech社製)、Ofev(登録商標)(boehringer-ingelheim社製、別名ニンタテニブ)、AMG706(Takeda Yakuhin社製、別名モテサニブ)等が挙げられる。 <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.
<培養工程>
本実施形態に係る評価方法では、まず、培養工程として、脈管網構造を備える細胞構造体を、被検化合物の存在下で培養する。具体的には、被検化合物を混合した培養培地中で、細胞構造体を培養する。培養培地に混合する被検化合物の量は、細胞構造体を構成する細胞の種類や数、培養培地の種類、培養温度、培養時間等の培養条件を考慮して実験的に決定することができる。培養時間は、特に限定されるものではなく、例えば、24~96時間とすることができ、48~96時間であることが好ましく、48~72時間であることがより好ましい。また、培養環境を著しく変化させない限度において、必要に応じて還流等の流体力学的な付加を加えてもよい。 <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.
本実施形態に係る評価方法では、まず、培養工程として、脈管網構造を備える細胞構造体を、被検化合物の存在下で培養する。具体的には、被検化合物を混合した培養培地中で、細胞構造体を培養する。培養培地に混合する被検化合物の量は、細胞構造体を構成する細胞の種類や数、培養培地の種類、培養温度、培養時間等の培養条件を考慮して実験的に決定することができる。培養時間は、特に限定されるものではなく、例えば、24~96時間とすることができ、48~96時間であることが好ましく、48~72時間であることがより好ましい。また、培養環境を著しく変化させない限度において、必要に応じて還流等の流体力学的な付加を加えてもよい。 <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.
<評価工程>
前記培養工程後の細胞構造体中の脈管の状態を指標として、前記被検化合物が、脈管新生阻害活性を有するか否かを評価する。具体的には、被検化合物の非存在下で培養した場合と比較して、被検化合物存在下で培養した場合に、細胞構造体中の脈管を構成する細胞数が少ない場合、又は細胞構造体中の脈管網構造が損傷を受けている場合に、当該被検化合物が脈管新生阻害活性を有すると評価する。一方で、被検化合物存在下で培養した場合に、被検化合物非存在下での培養と同様に脈管新生が生じていた場合、すなわち、脈管を構成する細胞数の減少が確認されない場合や、脈管網構造に損傷が確認されない場合には、当該被検化合物は脈管新生阻害活性を有さないと評価できる。 <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.
前記培養工程後の細胞構造体中の脈管の状態を指標として、前記被検化合物が、脈管新生阻害活性を有するか否かを評価する。具体的には、被検化合物の非存在下で培養した場合と比較して、被検化合物存在下で培養した場合に、細胞構造体中の脈管を構成する細胞数が少ない場合、又は細胞構造体中の脈管網構造が損傷を受けている場合に、当該被検化合物が脈管新生阻害活性を有すると評価する。一方で、被検化合物存在下で培養した場合に、被検化合物非存在下での培養と同様に脈管新生が生じていた場合、すなわち、脈管を構成する細胞数の減少が確認されない場合や、脈管網構造に損傷が確認されない場合には、当該被検化合物は脈管新生阻害活性を有さないと評価できる。 <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.
脈管新生の有無や脈管網構造の損傷の有無は、例えば、脈管を構成する細胞をその他の細胞と区別するように標識し、当該標識からのシグナルを指標として調べることができる。例えば、脈管を構成する細胞を蛍光標識することにより、細胞構造体中の脈管を直接観察することができる。また、画像解析技術を用いて、被検化合物存在下で培養した細胞構造体の蛍光画像と、被検化合物非存在下で培養した細胞構造体の蛍光画像とを比較し、被検化合物により脈管網構造が損傷を受けたかどうかや、脈管新生が起こったかどうかを調べることができる。なお、脈管を構成する細胞の蛍光標識は、例えば、脈管を構成する細胞の細胞表面に特異的に発現している物質に対する抗体を一次抗体とし、当該一次抗体と特異的に結合する蛍光標識二次抗体を用いる免疫染色法等の公知の手法で行うことができる。
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.
脈管を構成する細胞数も同様に、脈管を構成する細胞を標識し、当該標識からのシグナルを指標として調べることができる。例えば、脈管を構成する細胞を蛍光標識した場合には、細胞構造体の蛍光画像における総蛍光強度又は蛍光発光領域面積は、脈管を構成する細胞数に依存する。脈管を構成する細胞数が少ない場合には、総蛍光強度は小さくなり、蛍光発光領域面積は少なくなる。そこで、被検化合物存在下で培養した細胞構造体の蛍光画像と被検化合物非存在下で培養した細胞構造体の蛍光画像とについて、蛍光強度又は蛍光発光領域面積を比較することにより、脈管を構成する細胞数を比較することができる。
その他、脈管を構成する細胞を標識した後の細胞構造体の立体構造を破壊した後、FACS(fluorescence activated cell sorting)等により標識された細胞のみを直接計数することもできる。 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.
その他、脈管を構成する細胞を標識した後の細胞構造体の立体構造を破壊した後、FACS(fluorescence activated cell sorting)等により標識された細胞のみを直接計数することもできる。 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.
本実施形態に係る評価方法は、実際の生体内における脈管構造に近い脈管網構造を備える細胞構造体を用いる。そのため、被検化合物の脈管形成に対する作用効果について、信頼性の高い評価を行うことができる。本実施形態に係る評価方法により、脈管新生阻害活性があると評価された被検化合物は、生体内に投与した場合でも有効な脈管新生阻害活性を示すことが期待できる。このため、本実施形態に係る評価方法は、新規な脈管新生阻害剤の評価やスクリーニングに応用することができる。
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.
また、がん患者から採取された内皮細胞や繊維芽細胞、がん細胞を用いて形成された細胞構造体を用い、既知の様々な脈管新生阻害剤について本実施形態に係る評価方法により評価することができる。これにより、当該がん患者に実際に投与された場合に有効な脈管新生阻害活性を示すことができる脈管新生阻害剤を選択することができる。
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.
本実施形態に係る評価方法に用いられる細胞構造体と当該細胞構造体を収容する細胞培養容器とは、キットを構成することができる。このような脈管新生阻害活性評価用キットを用いることにより、本実施形態に係る評価方法をより簡便に実施することができる。当該キットには、細胞構造体に代えて、細胞構造体を構成する内皮細胞と内皮細胞以外の細胞とを備えることもできる。
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.
当該キットは、さらに、当該評価方法において用いられるその他の物質を備えることもできる。当該その他の物質としては、例えば、細胞構造体の培養培地、内皮細胞により形成される脈管細胞を標識するための標識物質、細胞構造体を構築する際に使用する物質(例えば、カチオン性緩衝液、強電解質高分子、細胞外マトリックス成分等)などが挙げられる。
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.).
以下、実施例を示し、本発明を具体的に説明するが、本発明は下記実施例に限定されるものではない。
Hereinafter, the present invention will be specifically described with reference to examples. However, the present invention is not limited to the following examples.
[実施例1]脈管構造を有する細胞構造体の作製
繊維芽細胞と血管内皮細胞から形成され、血管網構造を備える細胞構造体を作製し、血管網構造を観察した。
血管網構造を含む細胞構造体としては、ヒト新生児由来皮膚線維芽細胞(Lonza社製、CC-2509、Normal Human Dermal Fibroblasts:NHDF)、ヒト臍帯静脈内皮細胞(Lonza社製、CC-2517A、Human Umbilical Vein Endothelial Cell:HUVEC)の2種の2種類の細胞から形成された細胞構造体を用いた。また、細胞培養容器としては、トランズウェルセルカルチャーインサート(Corning社製、製品番号:#3470)を用い、培養培地としては、10容量%ウシ血清(System Biosciences社製、EXO-FBS-50A-1)及び1容量%ペニシリン/ストレプトマイシン(和光純薬社製、製品番号:168-23191)含有D-MEM(和光純薬社製、製品番号:043-30085)を用いた。評価対象となる血管新生阻害剤はベバシズマブ(R&D Systems社製、製品番号:MAB293)を用いた。 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.
繊維芽細胞と血管内皮細胞から形成され、血管網構造を備える細胞構造体を作製し、血管網構造を観察した。
血管網構造を含む細胞構造体としては、ヒト新生児由来皮膚線維芽細胞(Lonza社製、CC-2509、Normal Human Dermal Fibroblasts:NHDF)、ヒト臍帯静脈内皮細胞(Lonza社製、CC-2517A、Human Umbilical Vein Endothelial Cell:HUVEC)の2種の2種類の細胞から形成された細胞構造体を用いた。また、細胞培養容器としては、トランズウェルセルカルチャーインサート(Corning社製、製品番号:#3470)を用い、培養培地としては、10容量%ウシ血清(System Biosciences社製、EXO-FBS-50A-1)及び1容量%ペニシリン/ストレプトマイシン(和光純薬社製、製品番号:168-23191)含有D-MEM(和光純薬社製、製品番号:043-30085)を用いた。評価対象となる血管新生阻害剤はベバシズマブ(R&D Systems社製、製品番号:MAB293)を用いた。 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.
<細胞構造体の構築>
まず、2×106個のNHDFとNHDF細胞数の0.05、0.1、0.25、0.5、1.0、1.5、5.0%のHUVECを、ヘパリンとコラーゲンを含有するトリス-塩酸緩衝液(0.1mg/mL ヘパリン、0.1mg/mL コラーゲン、50mM トリス、pH7.4)に懸濁し、細胞懸濁液を調製した(NHDF数に対するHUVEC 数の割合:5%)(工程(a))。この細胞懸濁液を、室温、400×gで1分間、遠心処理し、上清を取り除いた後、適量の培養培地で再懸濁した(工程(a’-1)(a’-2))。次いで、この細胞懸濁液を、トランズウェルセルカルチャーインサート内に播種した後(工程(b))、当該トランズウェルセルカルチャーインサートを室温、400×gで1分間、遠心処理した。その後、当該トランズウェルセルカルチャーインサートに、適量の培養培地を追加した後、CO2インキュベーター(37℃、5%CO2)にて96時間培養した(工程(c))。 <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)).
まず、2×106個のNHDFとNHDF細胞数の0.05、0.1、0.25、0.5、1.0、1.5、5.0%のHUVECを、ヘパリンとコラーゲンを含有するトリス-塩酸緩衝液(0.1mg/mL ヘパリン、0.1mg/mL コラーゲン、50mM トリス、pH7.4)に懸濁し、細胞懸濁液を調製した(NHDF数に対するHUVEC 数の割合:5%)(工程(a))。この細胞懸濁液を、室温、400×gで1分間、遠心処理し、上清を取り除いた後、適量の培養培地で再懸濁した(工程(a’-1)(a’-2))。次いで、この細胞懸濁液を、トランズウェルセルカルチャーインサート内に播種した後(工程(b))、当該トランズウェルセルカルチャーインサートを室温、400×gで1分間、遠心処理した。その後、当該トランズウェルセルカルチャーインサートに、適量の培養培地を追加した後、CO2インキュベーター(37℃、5%CO2)にて96時間培養した(工程(c))。 <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)).
<血管を構成する細胞の蛍光標識及び評価>
培養後の細胞構造体に対して、anti-CD31抗体(DAKO社製、製品番号:JC70A M082329)と二次抗体(Invitrogen社製、製品番号:A-11001)を用いた蛍光免疫染色を行い、当該構造体中の血管を緑色蛍光標識した。この蛍光標識された細胞構造体を直接観察して血管網形成有無を確認した。 <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.
培養後の細胞構造体に対して、anti-CD31抗体(DAKO社製、製品番号:JC70A M082329)と二次抗体(Invitrogen社製、製品番号:A-11001)を用いた蛍光免疫染色を行い、当該構造体中の血管を緑色蛍光標識した。この蛍光標識された細胞構造体を直接観察して血管網形成有無を確認した。 <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.
表1よりNHDF数の0.05%のHUVECを含有する場合を除いた全ての条件で血管網構造の形成を確認できた。
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.
[実施例2]
繊維芽細胞と血管内皮細胞とから形成され、血管網構造を備える細胞構造体を用い、血管新生阻害剤ベバシズマブの血管新生阻害活性を評価した。
血管網構造を含む細胞構造体の構成細胞としては、ヒト新生児由来皮膚線維芽細胞(Normal Human Dermal Fibroblasts:NHDF)(Lonza社製、製品番号:CC-2509)、ヒト臍帯静脈内皮細胞(Human Umbilical Vein Endothelial Cell:HUVEC)(Lonza社製、製品番号:CC-2517A)の2種類を用いた。細胞培養容器としては、トランズウェルセルカルチャーインサート(Corning社製、製品番号:#3470)を用いた。培養培地としては、10容量%ウシ血清(Corning社製、製品番号:#35-010-CV)及び1容量%ペニシリン/ストレプトマイシン(和光純薬社製、製品番号:168-23191)含有D-MEM(和光純薬社製、製品番号:043-30085)を用いた。評価対象となる血管新生阻害剤は、ベバシズマブ(R&D Systems社製、製品番号:MAB293)を用いた。 [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.
繊維芽細胞と血管内皮細胞とから形成され、血管網構造を備える細胞構造体を用い、血管新生阻害剤ベバシズマブの血管新生阻害活性を評価した。
血管網構造を含む細胞構造体の構成細胞としては、ヒト新生児由来皮膚線維芽細胞(Normal Human Dermal Fibroblasts:NHDF)(Lonza社製、製品番号:CC-2509)、ヒト臍帯静脈内皮細胞(Human Umbilical Vein Endothelial Cell:HUVEC)(Lonza社製、製品番号:CC-2517A)の2種類を用いた。細胞培養容器としては、トランズウェルセルカルチャーインサート(Corning社製、製品番号:#3470)を用いた。培養培地としては、10容量%ウシ血清(Corning社製、製品番号:#35-010-CV)及び1容量%ペニシリン/ストレプトマイシン(和光純薬社製、製品番号:168-23191)含有D-MEM(和光純薬社製、製品番号:043-30085)を用いた。評価対象となる血管新生阻害剤は、ベバシズマブ(R&D Systems社製、製品番号:MAB293)を用いた。 [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.
<細胞構造体の構築>
まず、2×106個のNHDFと3×104個のHUVECとを、ヘパリン及びコラーゲンを含有するトリス-塩酸緩衝液(0.1mg/mL ヘパリン、0.1mg/mL コラーゲン、50mM トリス、pH7.4)に懸濁し、細胞懸濁液を調製した(工程(a))。この細胞懸濁液を、室温、400×gで1分間、遠心処理し、上清を取り除いた後、適量の培養培地で再懸濁した(工程(a’-1)(a’-2))。次いで、この細胞懸濁液を、トランズウェルセルカルチャーインサート内に播種した後(工程(b))、当該トランズウェルセルカルチャーインサートを室温、400×gで1分間、遠心処理し、液体成分を除去した。その後、当該トランズウェルセルカルチャーインサートに、適量の培養培地を追加した後、CO2インキュベーター(37℃、5%CO2)にて24時間培養した(工程(c))。培養終了後、血管網構造を備えた細胞構造体(NHDF(20層分)とHUVEC(1層分)との混合層(21層))が得られた。 <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.
まず、2×106個のNHDFと3×104個のHUVECとを、ヘパリン及びコラーゲンを含有するトリス-塩酸緩衝液(0.1mg/mL ヘパリン、0.1mg/mL コラーゲン、50mM トリス、pH7.4)に懸濁し、細胞懸濁液を調製した(工程(a))。この細胞懸濁液を、室温、400×gで1分間、遠心処理し、上清を取り除いた後、適量の培養培地で再懸濁した(工程(a’-1)(a’-2))。次いで、この細胞懸濁液を、トランズウェルセルカルチャーインサート内に播種した後(工程(b))、当該トランズウェルセルカルチャーインサートを室温、400×gで1分間、遠心処理し、液体成分を除去した。その後、当該トランズウェルセルカルチャーインサートに、適量の培養培地を追加した後、CO2インキュベーター(37℃、5%CO2)にて24時間培養した(工程(c))。培養終了後、血管網構造を備えた細胞構造体(NHDF(20層分)とHUVEC(1層分)との混合層(21層))が得られた。 <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.
<ベバシズマブ存在下での培養>
得られた細胞構造体を、トランズウェルセルカルチャーインサートの1ウェル当たりのベバシズマブ添加量が0、1、又は2μgである培養培地中で、37℃、5%CO2にて72時間培養した。 <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.
得られた細胞構造体を、トランズウェルセルカルチャーインサートの1ウェル当たりのベバシズマブ添加量が0、1、又は2μgである培養培地中で、37℃、5%CO2にて72時間培養した。 <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.
<血管を構成する細胞の蛍光標識及び評価>
培養後の細胞構造体に対して、anti-CD31抗体(DAKO社製、製品番号:JC70A M082329)と二次抗体(Invitrogen社製、製品番号:A-11001)を用いた蛍光免疫染色を行い、当該構造体中の血管を緑色蛍光標識した。この蛍光標識された細胞構造体を直接観察し、さらに撮像した蛍光画像に対して画像解析を行い、当該蛍光画像における蛍光発光領域の占有面積を計測した。血管を構成する細胞が多いほど、蛍光発光領域の占有面積は大きくなる。各濃度について、繰り返し3回ずつ測定した。測定結果を表2に示す。 <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.
培養後の細胞構造体に対して、anti-CD31抗体(DAKO社製、製品番号:JC70A M082329)と二次抗体(Invitrogen社製、製品番号:A-11001)を用いた蛍光免疫染色を行い、当該構造体中の血管を緑色蛍光標識した。この蛍光標識された細胞構造体を直接観察し、さらに撮像した蛍光画像に対して画像解析を行い、当該蛍光画像における蛍光発光領域の占有面積を計測した。血管を構成する細胞が多いほど、蛍光発光領域の占有面積は大きくなる。各濃度について、繰り返し3回ずつ測定した。測定結果を表2に示す。 <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.
直接観察の結果、ベバシズマブ終濃度0μg/ウェルに対して、終濃度1μg/ウェル、2μg/ウェルの方が、血管網形成が有意に阻害されていることがわかった(図示せず。)。また、蛍光発光領域の占有面積についても、終濃度0μg/ウェルでは158.0×105μm2であったのに対して、終濃度1μg/ウェルでは106.6×105μm2、終濃度2μg/ウェルでは47.5×105μm2であり、ベバシズマブ添加により蛍光発光領域の占有面積は顕著に減少していた。これらの結果から、ベバシズマブは血管新生阻害作用を有すると評価された。ベバシズマブは既知の血管新生阻害剤であることから、本発明に係る評価方法により、被検化合物の血管新生阻害作用を評価できることが確認できた。
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.
[実施例3]
繊維芽細胞、血管内皮細胞、及びがん細胞から形成され、血管網構造を備える細胞構造体を用い、血管新生阻害剤ベバシズマブの血管新生阻害活性を評価した。
血管網構造を含む細胞構造体の構成細胞としては、実施例2で用いたNHDFとHUVECとに加えて、ヒト結腸直腸腺がん細胞株であるHT29(ATCC番号:HTB-38TM)の3種類を用い、細胞培養容器、培養培地、及びベバシズマブは実施例2で使用した物と同じものを用いた。 [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.
繊維芽細胞、血管内皮細胞、及びがん細胞から形成され、血管網構造を備える細胞構造体を用い、血管新生阻害剤ベバシズマブの血管新生阻害活性を評価した。
血管網構造を含む細胞構造体の構成細胞としては、実施例2で用いたNHDFとHUVECとに加えて、ヒト結腸直腸腺がん細胞株であるHT29(ATCC番号:HTB-38TM)の3種類を用い、細胞培養容器、培養培地、及びベバシズマブは実施例2で使用した物と同じものを用いた。 [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.
<細胞構造体の構築>
細胞懸濁液として、2×106個のNHDF及び3×104個のHUVECのみを含む懸濁液に代えて、2×106個のNHDFと3×104個のHUVECと2×104個のがん細胞とを含む懸濁液を用いたこと以外は、実施例2と同様にして血管網構造を備えた細胞構造体を得た。HUVEC細胞は、予め、蛍光標識(SIGMA社製、製品番号PKH26GL)しておいたものを用いた。 <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.
細胞懸濁液として、2×106個のNHDF及び3×104個のHUVECのみを含む懸濁液に代えて、2×106個のNHDFと3×104個のHUVECと2×104個のがん細胞とを含む懸濁液を用いたこと以外は、実施例2と同様にして血管網構造を備えた細胞構造体を得た。HUVEC細胞は、予め、蛍光標識(SIGMA社製、製品番号PKH26GL)しておいたものを用いた。 <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.
<ベバシズマブ存在下での培養>
得られた細胞構造体を、トランズウェルセルカルチャーインサートの1ウェル当たりのベバシズマブ添加量が0、1、又は2μgである培養培地中で、37℃、5%CO2にて72時間培養した。 <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.
得られた細胞構造体を、トランズウェルセルカルチャーインサートの1ウェル当たりのベバシズマブ添加量が0、1、又は2μgである培養培地中で、37℃、5%CO2にて72時間培養した。 <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.
<細胞構造体の分散>
次に、当該トランズウェルセルカルチャーインサートにトリス緩衝溶液(50mM,pH7.4)を適量添加し、その後、液体成分を除去した。この一連の工程を繰り返し3回実施した。次いで、当該トランズウェルセルカルチャーインサートに0.25%トリプシン‐EDTA溶液(Invitrogen社製、)を300μL添加し、CO2インキュベータ(37℃,5%CO2)で15分間インキュベートした。その後、溶液全量を回収し、あらかじめ0.25%トリプシン‐EDTA溶液(Invitrogen社製、)が300μL添加された回収用1.5mLチューブに移した。次いで、当該トランズウェルセルカルチャーインサートに0.25%トリプシン‐EDTA溶液(Invitrogen社製、)を100μL添加し、回収用1.5mLチューブと共にCO2インキュベータ(37℃,5%CO2)で5分間インキュベートした。その後、溶液全量を回収し、回収用1.5mLチューブに移し、更に0.25%トリプシン‐EDTA溶液(Invitrogen社製、)を300μL添加し、CO2インキュベータ(37℃,5%CO2)で5分間インキュベートし、細胞構造体分散液を得た。 <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.
次に、当該トランズウェルセルカルチャーインサートにトリス緩衝溶液(50mM,pH7.4)を適量添加し、その後、液体成分を除去した。この一連の工程を繰り返し3回実施した。次いで、当該トランズウェルセルカルチャーインサートに0.25%トリプシン‐EDTA溶液(Invitrogen社製、)を300μL添加し、CO2インキュベータ(37℃,5%CO2)で15分間インキュベートした。その後、溶液全量を回収し、あらかじめ0.25%トリプシン‐EDTA溶液(Invitrogen社製、)が300μL添加された回収用1.5mLチューブに移した。次いで、当該トランズウェルセルカルチャーインサートに0.25%トリプシン‐EDTA溶液(Invitrogen社製、)を100μL添加し、回収用1.5mLチューブと共にCO2インキュベータ(37℃,5%CO2)で5分間インキュベートした。その後、溶液全量を回収し、回収用1.5mLチューブに移し、更に0.25%トリプシン‐EDTA溶液(Invitrogen社製、)を300μL添加し、CO2インキュベータ(37℃,5%CO2)で5分間インキュベートし、細胞構造体分散液を得た。 <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.
<生細胞数解析及び評価>
得られた細胞構造体の分散液をトリパンブルー溶液に浸漬させてトリパンブルー染色した後、蛍光を発しておりかつトリパンブルー染色されていない細胞を、生きているHUVECとして計数した。細胞の計数は、セルカウンター「CountessII」(ライフテクノロジーズ社製)の蛍光モードを使用して行った。HUVECの生細胞数の計数結果と、算出した生存率(ベバシズマブ0μgの生細胞数を100%とした場合の生細胞数の割合)(%)を表3に示す。 <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%) (%).
得られた細胞構造体の分散液をトリパンブルー溶液に浸漬させてトリパンブルー染色した後、蛍光を発しておりかつトリパンブルー染色されていない細胞を、生きているHUVECとして計数した。細胞の計数は、セルカウンター「CountessII」(ライフテクノロジーズ社製)の蛍光モードを使用して行った。HUVECの生細胞数の計数結果と、算出した生存率(ベバシズマブ0μgの生細胞数を100%とした場合の生細胞数の割合)(%)を表3に示す。 <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%) (%).
血管新生が阻害されていないベバシズマブ終濃度0μg/ウェルに対して、実施例2において血管新生の阻害が確認されている終濃度1μg/ウェル、2μg/ウェルの方が、HUVECの生細胞数は顕著に少なくなっており、ベバシズマブ添加により、HUVECの生存率が低下すること、が確認された。ベバシズマブによる血管新生阻害効果が高いウェルほど、HUVECの生存率が低いことから、HUVECの生存率からも、被検化合物の血管新生阻害作用の有無を評価できるといえる。
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.
[実施例4]肺由来の間質細胞で構成された細胞構造体を用いた場合
繊維芽細胞と血管内皮細胞から形成され、血管網構造を備える細胞構造体を用い、血管新生阻害剤ベバシズマブで評価した。
血管網構造を含む細胞構造体としては、ヒト肺線維芽細胞(Normal Human Pulomonary Fibroblasts:NHPF)(Promocell社製、製品番号:C-12360)、及びヒト肺微小血管内皮細胞(Human Dermal Microvascular Endothelial Cell:HMVEC-L)(Lonza社製、製品番号:CC-2527)の2種類の細胞から形成された細胞構造体を用いた。また、細胞培養容器としては、トランズウェルセルカルチャーインサート(Corning社製、製品番号:#3470)を用い、培養培地としては、10容量%ウシ血清(System Biosciences社製、EXO-FBS-50A-1)及び1容量%ペニシリン/ストレプトマイシン(和光純薬社製、製品番号:168-23191)含有D-MEM(和光純薬社製、製品番号:043-30085)を用いた。評価対象となる血管新生阻害剤はベバシズマブ(R&D Systems社製、製品番号:MAB293)を用いた。 [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.
繊維芽細胞と血管内皮細胞から形成され、血管網構造を備える細胞構造体を用い、血管新生阻害剤ベバシズマブで評価した。
血管網構造を含む細胞構造体としては、ヒト肺線維芽細胞(Normal Human Pulomonary Fibroblasts:NHPF)(Promocell社製、製品番号:C-12360)、及びヒト肺微小血管内皮細胞(Human Dermal Microvascular Endothelial Cell:HMVEC-L)(Lonza社製、製品番号:CC-2527)の2種類の細胞から形成された細胞構造体を用いた。また、細胞培養容器としては、トランズウェルセルカルチャーインサート(Corning社製、製品番号:#3470)を用い、培養培地としては、10容量%ウシ血清(System Biosciences社製、EXO-FBS-50A-1)及び1容量%ペニシリン/ストレプトマイシン(和光純薬社製、製品番号:168-23191)含有D-MEM(和光純薬社製、製品番号:043-30085)を用いた。評価対象となる血管新生阻害剤はベバシズマブ(R&D Systems社製、製品番号:MAB293)を用いた。 [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.
<細胞構造体の構築>
まず、2×106個のNHPF及び1×105個のHMVEC―Lを、ヘパリン及びコラーゲンを含有するトリス-塩酸緩衝液(0.1mg/mL ヘパリン、0.1mg/mL コラーゲン、50mM トリス、pH7.4)に懸濁し、細胞懸濁液を調製した(NHPF数に対するHMVEC―L数の割合:5%)(工程(a))。この細胞懸濁液を、室温、400×gで1分間、遠心処理し、上清を取り除いた後、適量の培養培地で再懸濁した(工程(a’-1)(a’-2))。次いで、この細胞懸濁液を、トランズウェルセルカルチャーインサート内に播種した後(工程(b))、当該トランズウェルセルカルチャーインサートを室温、400×gで1分間、遠心処理した。その後、当該トランズウェルセルカルチャーインサートに、適量の培養培地を追加した後、CO2インキュベーター(37℃、5%CO2)にて24時間培養した(工程(c))。 <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)).
まず、2×106個のNHPF及び1×105個のHMVEC―Lを、ヘパリン及びコラーゲンを含有するトリス-塩酸緩衝液(0.1mg/mL ヘパリン、0.1mg/mL コラーゲン、50mM トリス、pH7.4)に懸濁し、細胞懸濁液を調製した(NHPF数に対するHMVEC―L数の割合:5%)(工程(a))。この細胞懸濁液を、室温、400×gで1分間、遠心処理し、上清を取り除いた後、適量の培養培地で再懸濁した(工程(a’-1)(a’-2))。次いで、この細胞懸濁液を、トランズウェルセルカルチャーインサート内に播種した後(工程(b))、当該トランズウェルセルカルチャーインサートを室温、400×gで1分間、遠心処理した。その後、当該トランズウェルセルカルチャーインサートに、適量の培養培地を追加した後、CO2インキュベーター(37℃、5%CO2)にて24時間培養した(工程(c))。 <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)).
<ベバシズマブ存在下での培養>
得られた細胞構造体を、トランズウェルセルカルチャーインサートの1ウェル当たりのベバシズマブ添加量が0、1、又は2μgである培養培地中で、37℃、5%CO2にて144時間培養した。 <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.
得られた細胞構造体を、トランズウェルセルカルチャーインサートの1ウェル当たりのベバシズマブ添加量が0、1、又は2μgである培養培地中で、37℃、5%CO2にて144時間培養した。 <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.
<血管を構成する細胞の蛍光標識及び評価>
培養後の細胞構造体に対して、anti-CD31抗体(DAKO社製、製品番号:JC70A M082329)及び二次抗体(Invitrogen社製、製品番号:A-11001)を用いた蛍光免疫染色を行い、当該構造体中の血管を緑色蛍光標識した。この蛍光標識された細胞構造体を直接観察し、さらに撮像した蛍光画像に対して画像解析を行い、当該蛍光画像における蛍光発光領域の占有面積を計測した。結果を表4に示す。血管を構成する細胞が多いほど、蛍光発光領域の占有面積は大きくなる。各濃度について、繰り返し3回ずつ測定した。 <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.
培養後の細胞構造体に対して、anti-CD31抗体(DAKO社製、製品番号:JC70A M082329)及び二次抗体(Invitrogen社製、製品番号:A-11001)を用いた蛍光免疫染色を行い、当該構造体中の血管を緑色蛍光標識した。この蛍光標識された細胞構造体を直接観察し、さらに撮像した蛍光画像に対して画像解析を行い、当該蛍光画像における蛍光発光領域の占有面積を計測した。結果を表4に示す。血管を構成する細胞が多いほど、蛍光発光領域の占有面積は大きくなる。各濃度について、繰り返し3回ずつ測定した。 <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.
この結果、終濃度0μg/ウェルでは252×105μm2であったのに対して、終濃度1μg/ウェルでは178×105μm2、終濃度2μg/ウェルでは131×105μm2であり、ベバシズマブ添加により蛍光発光領域の占有面積は顕著に減少していた。これらの結果から、肺由来の線維芽細胞、血管内皮細胞を用いた場合でもベバシズマブは血管新生阻害作用を有すると評価された。
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)
- 脈管網構造を備える細胞構造体を、被検化合物の存在下で培養する培養工程と、
前記培養工程後の前記細胞構造体中の脈管の状態を指標として、前記被検化合物が、脈管新生阻害活性を有するか否かを評価する評価工程と、
を有し、
前記評価工程において、前記被検化合物の非存在下で培養した場合と比較して、前記細胞構造体中の脈管を構成する細胞数が少ない場合、又は前記細胞構造体中の脈管網構造が損傷を受けている場合に、前記被検化合物が脈管新生阻害活性を有すると評価する被検化合物の脈管新生阻害活性の評価方法。 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. - 前記細胞構造体が、血管内皮細胞及びリンパ管内皮細胞からなる群より選択される1種以上を含む、請求項1に記載の被検化合物の脈管新生阻害活性の評価方法。 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.
- 前記細胞構造体が、さらに、繊維芽細胞、神経細胞、樹状細胞、マクロファージ、及び肥満細胞からなる群より選択される1種以上を含む、請求項2に記載の被検化合物の脈管新生阻害活性の評価方法。 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.
- 前記細胞構造体が、繊維芽細胞を含み、
前記細胞構造体中の血管内皮細胞及びリンパ管内皮細胞の総細胞数が、繊維芽細胞の細胞数の0.1%以上である、請求項2に記載の被検化合物の脈管新生阻害活性の評価方法。 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. - 請求項1~4のいずれか一項に記載の被検化合物の脈管新生阻害活性の評価方法を行うためのキットであって、
脈管網構造を備える細胞構造体と、前記細胞構造体を収容する細胞培養容器とを備える、脈管新生阻害活性評価用キット。 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. - 前記細胞構造体が、繊維芽細胞層に挟まれた脈管網構造を備える、請求項5に記載のキット。 The kit according to claim 5, wherein the cell structure comprises a vascular network structure sandwiched between fibroblast layers.
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EP3674394A4 (en) * | 2017-08-21 | 2021-05-26 | Toppan Printing Co., Ltd. | Primary culture method |
US11781115B2 (en) | 2017-08-21 | 2023-10-10 | Toppan Printing Co., Ltd. | Primary culture method |
JP2019170469A (en) * | 2018-03-27 | 2019-10-10 | テルモ株式会社 | Lymphatic vessel function measuring apparatus and lymphatic vessel function measuring method |
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