JP7556194B2 - Cellular structures and uses thereof - Google Patents

Description

The present invention relates to a cell structure and its use. More specifically, the present invention relates to a cell structure, a method for inhibiting proliferation rate, and a method for evaluating anticancer effect.

In general, immortalized cells and established cell lines have mutations in their genomes, and these cells cultured ex vivo (outside the living body) may grow in a manner different from that of cells growing in the living body.

Meanwhile, as disclosed in Patent Document 1, the applicants have invented a simple method for producing a cell structure containing cells, extracellular matrix components, and a polymer electrolyte by mixing and assembling these materials. In addition, as disclosed in Patent Document 2, the applicants have invented a method for forming a cell structure using cancer cells by this method and evaluating the efficacy of an anticancer drug.

International Publication No. 2017/146124 International Publication No. 2019/039452

However, because the proliferation rate of immortalized cells and cell lines is significantly faster than that of cells in the body, there are cases in which the effects of drugs or toxic substances cannot be accurately evaluated using immortalized cells or cell lines cultured ex vivo.

The problem to be solved by the present invention is to provide a technology for culturing established cell lines or immortalized cells ex vivo under conditions more similar to those found in vivo than conventional methods.

The present invention includes the following aspects.
[1] A cell structure comprising a first cell population and an extracellular matrix component, wherein the first cell population contains the extracellular matrix component between cells and the first cell population contains at least interstitial cells, and when a second cell population is cultured in contact with the cell structure, the cell structure is used to culture the second cell population under conditions similar to those in a living body.
[2] The cell structure described in [1], wherein the first cell population further contains vascular endothelial cells, and the interstitial cells are fibroblasts.
[3] The cell structure described in [1] or [2], wherein the second cell population comprises an established cell line.
[4] The cell structure described in any one of [1] to [3], wherein the second cell population comprises cancer cells.
[5] The cell structure described in any one of [1] to [4], wherein the thickness of the cell structure is 40 μm or more.
[6] The cell structure described in any of [1] to [5], wherein the ratio of the number of cells of the first cell population to the number of cells of the second cell population (number of cells of the first cell population:number of cells of the second cell population) is 3:1 to 300:1.
[7] A method for inhibiting the proliferation rate of a second cell population, comprising the step of contacting a second cell population with a cell structure comprising a first cell population and an extracellular matrix component and culturing the second cell population, wherein the first cell population contains the extracellular matrix component between cells and the first cell population contains at least interstitial cells.
[8] The method for inhibiting the proliferation rate of the second cell population described in [7], wherein the first cell population further contains vascular endothelial cells and the interstitial cells are fibroblasts.
[9] The method for suppressing the proliferation rate of the second cell population described in [7] or [8], wherein the second cell population includes an established cell line.
[10] The method for suppressing the proliferation rate of the second cell population described in any one of [7] to [9], wherein the second cell population includes cancer cells.
[11] The method for suppressing the proliferation rate of the second cell population described in any of [7] to [10], wherein the cell structure is produced by a method comprising the steps of mixing the first cell population with a cationic substance, an extracellular matrix component and a polymer electrolyte to obtain a mixture, and culturing the cells in the obtained mixture to obtain the cell structure.
[12] A method for evaluating a test substance, comprising the steps of: culturing a cell population in the presence of the test substance by contacting the cell structure according to any one of [1] to [6] and analyzing the state of the cell population; and comparing the state in the presence of the test substance with the state in the absence of the test substance to evaluate an effect of the test substance.

The present invention provides a technology that allows established or immortalized cells to be cultured ex vivo under conditions more similar to those found in vivo than conventional methods.

[Cell Structure]
In one embodiment, the present invention provides a cell structure comprising a first cell population and an extracellular matrix component, wherein the first cell population contains the extracellular matrix component between cells and the first cell population comprises at least interstitial cells, and when a second cell population is cultured in contact with the cell structure, the cell structure is used to culture the second cell population under conditions similar to those in vivo.

In this embodiment and in this specification, a "cell structure" is a three-dimensional structure in which multiple cell layers are stacked. A "cell layer" is a layer that is composed of a group of cells and stroma that exist in a direction perpendicular to the thickness direction and in which the cell nuclei do not overlap in the thickness direction when observed at a magnification at which cell nuclei can be recognized in a slice image of a cross section of the cell structure in the thickness direction, that is, at a magnification at which the entire thickness of the stained slice is visible. In addition, "layered" means that two or more different cell layers are stacked in the thickness direction.

By culturing the second cell population in contact with the cell structure according to this embodiment, the second cell population can be cultured in a state similar to that in a living body. As a result, for example, the proliferation rate of the second cell population can be made to approach (inhibit) the proliferation rate of the second cell population in a living body. The lifespan of the second cell population may also be made to approach the lifespan of the second cell population in a living body. The degree of differentiation, maturation, etc. of the second cell population may also be made to approach the degree of differentiation, maturation, etc. of the second cell population in a living body. In other words, the type and amount of mRNA, protein, etc. expressed by the second cell population, as well as the morphology, function, etc. of the second cell population may be made to approach those in a living body. This makes it possible to make the response of the second cell population to drugs and poisons closer to those in a living body, and to perform more accurate efficacy evaluation and toxicity evaluation.

The cell structure according to this embodiment may be a multi-layer structure in which the cell types constituting each cell layer are different for each layer, or the cell types constituting each cell layer may be the same for all layers of the structure.

The size and shape of the cell structure according to this embodiment are not particularly limited. The thickness of the cell structure is preferably 5 μm or more, more preferably 10 μm or more, more preferably 20 μm or more, more preferably 30 μm or more, more preferably 40 μm or more, even more preferably 50 μm or more, and even more preferably 100 μm or more. The thickness of the cell structure is also preferably 500 μm or less, more preferably 400 μm or less, and even more preferably 300 μm or less. When the thickness of the cell structure is equal to or greater than the above lower limit, the second cell population can be cultured in a state more similar to that in vivo. As described later in the examples, when the thickness of the cell structure is equal to or greater than 40 μm, the proliferation of cancer cells can be significantly suppressed. When the thickness of the cell structure is equal to or less than the above upper limit, the cost of manufacturing the cell structure can be reduced. 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 even more preferably about 10 to 60 layers.

The number of cell layers constituting a cell structure may be measured by dividing the total number of cells constituting the three-dimensional structure by the number of cells per layer (the number of cells required to constitute one layer). The number of cells per layer can be determined by culturing cells confluently in advance in a planar manner in a cell culture vessel used to form the cell structure. Specifically, the number of cell layers of a cell structure formed in a certain cell culture vessel can be calculated by counting the total number of cells constituting the cell structure and dividing it by the number of cells per layer of the cell culture vessel.

In this embodiment, the ratio of the number of cells in the first cell population to the number of cells in the second cell population (number of cells in the first cell population: number of cells in the second cell population) is preferably 3:1 to 300:1, more preferably 5:1 to 200:1, and even more preferably 6:1 to 100:1. By having the ratio of the number of cells in the first cell population to the number of cells in the second cell population within the above range, the second cell population can be cultured under conditions more similar to those in vivo.

The cell structure according to this embodiment includes a first cell population and an extracellular matrix component. The first cell population also includes the extracellular matrix component between the cells. That is, the first cell population is covered with the extracellular matrix component, and the three-dimensional structure including the first cell population forms a cell structure.

First Cell Population
The first cell population includes at least interstitial cells, and may further include cells other than interstitial cells, such as immune cells, nerve cells, hepatic cells, pancreatic cells, cardiac muscle cells, smooth muscle cells, bone cells, alveolar epithelial cells, and spleen cells.

(Interstitial cells)
In this embodiment, the cell type contained in the first cell population is not particularly limited, and may be cells collected from an animal, cells obtained by culturing cells collected from an animal, cells obtained by subjecting cells collected from an animal to various treatments, or a cultured cell line. In the case of cells collected from an animal, the site of collection is not particularly limited, and may be somatic cells derived from bone, muscle, internal organs, nerves, brain, bone, skin, blood, etc., germ cells, or embryonic stem cells (ES cells). In addition, the species from which the cells constituting the cell structure according to this embodiment are derived is not particularly limited, and cells derived from animals such as humans, monkeys, dogs, cats, rabbits, pigs, cows, mice, and rats can be used. Cells cultured from cells collected from animals may be primary culture cells or subculture cells. In addition, examples of cells that have been subjected to various treatments include induced pluripotent stem cells (iPS cells) and cells after differentiation induction. In addition, the cell structure according to this embodiment may be composed of only cells derived from the same species of organism, or may be composed of cells derived from multiple species of organisms.

Examples of interstitial cells include immune cells, endothelial cells, fibroblasts, nerve cells, mast cells, epithelial cells, cardiomyocytes, hepatocytes, pancreatic islet cells, tissue stem cells, smooth muscle cells, etc. The interstitial cells contained in the cell structure according to this embodiment may be of one type or of two or more types. The type of interstitial cells contained in the cell structure according to this embodiment is not particularly limited, and can be appropriately selected taking into consideration the types of cells other than the interstitial cells contained in the first cell population.

The first cell population may further include vascular endothelial cells, and the interstitial cells may be fibroblasts. A vascular network structure or a lymphatic network structure may be important for the proliferation and activity of the second cell population. For this reason, the cell structure according to this embodiment may have a vascular network structure.

In other words, the cell structure according to this embodiment may be one in which a vascular network structure such as lymphatic vessels and/or blood vessels is constructed three-dimensionally inside a laminate of cells that does not form blood vessels, constructing tissue that is closer to that found in a living body. The vascular network structure may be formed only inside the cell structure, or may be formed so that at least a portion of the vascular network structure is exposed on the surface or bottom surface of the cell structure. In this embodiment and this specification, the term "vascular network structure" refers to a network structure having branches, such as a blood vessel network or lymphatic network in biological tissue.

The vascular network structure can be formed by further including in the first cell population endothelial cells that constitute blood vessels. Such endothelial cells may be vascular endothelial cells or lymphatic endothelial cells. Furthermore, the first cell population may include both vascular endothelial cells and lymphatic endothelial cells as endothelial cells.

When the cell structure according to this embodiment has a vascular network structure, since endothelial cells are likely to form a vascular network that retains its original function and shape, it is preferable that the cells other than endothelial cells in the cell structure are cells that constitute the tissue surrounding the blood vessels in the living body, and since this is more similar to the cancer microenvironment in the living body, it is more preferable that the cell structure contains at least fibroblasts as cells other than endothelial cells, and it is even more preferable that the cell structure contains cells containing vascular endothelial cells and fibroblasts, cells containing lymphatic endothelial cells and fibroblasts, or cells containing vascular endothelial cells, lymphatic endothelial cells and fibroblasts. Note that the cells other than endothelial cells contained in the cell structure may be cells derived from the same biological species as the endothelial cells, or may be cells derived from a different biological species.

The number of endothelial cells in the cell structure according to this embodiment is not particularly limited as long as the second cell population can be cultured under conditions similar to those in the body, and can be appropriately determined taking into consideration the size of the cell structure, the cell types of endothelial cells and cells other than endothelial cells, and the like. For example, a cell structure having a vascular network structure can be prepared by setting the abundance ratio (cell number ratio) of endothelial cells to the total cells constituting the cell structure according to this embodiment to 0.1% or more. Furthermore, when fibroblasts are used as cells other than endothelial cells, the number of endothelial cells in the cell structure according to this embodiment is preferably 0.1% or more of the number of fibroblasts, and more preferably 0.1 to 5.0%. When both vascular endothelial cells and lymphatic endothelial cells are included as endothelial cells, the total number of vascular endothelial cells and lymphatic endothelial cells is preferably 0.1% or more of the number of fibroblasts, and more preferably 0.1 to 5.0%.

(Extracellular matrix components)
Examples of the extracellular matrix components contained in the cell structure include collagen, laminin, fibronectin, vitronectin, elastin, tenascin, entactin, fibrillin, proteoglycan, or modified or variant thereof. Examples of proteoglycan include chondroitin sulfate proteoglycan, heparan sulfate proteoglycan, keratan sulfate proteoglycan, and dermatan sulfate proteoglycan. In addition, the cell structure may contain modified or variant extracellular matrix components as long as they do not adversely affect cell growth and cell structure formation. The cell structure may contain one type of extracellular matrix component, or may contain two or more types of cell matrices. The cell structure preferably contains collagen, laminin, and fibronectin, and more preferably uses collagen. The above-mentioned proteins may be extracted from cells or may be artificially synthesized by peptide synthesis or the like.

Extracellular matrix components include proteins having an arginine-glycine-aspartic acid (RGD) sequence, or modified or variants thereof. Proteins having an RGD sequence have cell adhesion ability. Examples of proteins having an RGD sequence include fibronectin, vitronectin, collagen, osteopontin, laminin, etc. Proteins having an RGD sequence are not particularly limited as long as they can form a cell structure, and may be composed of an artificially designed amino acid sequence.

Second Cell Population
The cells contained in the second cell population are not particularly limited, and may be cells collected from an animal, cells obtained by culturing cells collected from an animal, cells obtained by subjecting cells collected from an animal to various treatments, or cultured cell lines. In the case of cells collected from an animal, the site of collection is not particularly limited, and may be somatic cells derived from bone, muscle, internal organs, nerves, brain, bone, skin, blood, etc., germ cells, or embryonic stem cells (ES cells). In addition, the species from which the cells contained in the second cell population are derived is not particularly limited, and cells derived from animals such as humans, monkeys, dogs, cats, rabbits, pigs, cows, mice, and rats can be used. Cells cultured from cells collected from animals may be primary cultured cells, subcultured cells, or established cells. In addition, examples of cells that have been subjected to various treatments include induced pluripotent stem cells (iPS cells) and cells after differentiation induction. The second cell population may be composed of only one type of cell, or may be composed of multiple types of cells.

The second cell population may include, for example, cancer cells. The type of cancer cells included in the second cell population may be one type, or may be two or more types. Note that cancer cells are cells that are derived from somatic cells and have acquired infinite proliferation ability. The cancer cells included in the second cell population may be cultured cells that have been established, or may be cancer cells collected from a cancer patient. The cancer cells collected from a cancer patient may be cells that have been cultured and grown in advance. Specifically, primary cancer cells collected from a cancer patient, artificially cultured cancer cells, iPS cancer stem cells, cancer stem cells, and cancer cell lines that have been prepared in advance for use in research on cancer treatment and development of anticancer drugs, etc. may be included. In addition, the cancer cells may be derived from humans, or may be derived from animals other than humans. Note that when the second cell population includes cancer cells collected from a cancer patient, cells other than the cancer cells collected from the cancer patient may also be included together with the cancer cells. Examples of cells other than cancer cells include one or more types of cells contained in solid tissue removed after surgery.

When the second cell population contains cancer cells, examples of the cancers from which the cancer cells are derived include breast cancer (e.g., invasive ductal carcinoma, ductal carcinoma in situ, inflammatory breast cancer, etc.), prostate cancer (e.g., hormone-dependent prostate cancer, hormone-independent prostate cancer, etc.), pancreatic cancer (e.g., pancreatic ductal carcinoma, etc.), gastric cancer (e.g., papillary adenocarcinoma, mucinous adenocarcinoma, adenosquamous carcinoma, etc.), lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, malignant mesothelioma, etc.), colon cancer (e.g., gastrointestinal stromal tumor, etc.), rectal cancer (e.g., gastrointestinal stromal tumor, etc.), colon cancer (e.g., familial colorectal cancer, hereditary nonpolyposis colorectal cancer, gastrointestinal stromal tumor, etc.), small intestine cancer (e.g., non-Hodgkin's lymphoma, gastrointestinal stromal tumor, etc.), esophageal cancer, duodenal cancer, tongue cancer, pharyngeal cancer (e.g., nasopharyngeal cancer, oropharyngeal cancer, hypopharyngeal cancer, etc.), head and neck cancer, salivary gland cancer, brain tumors (e.g., pineal astrocytoma, pilocytic astrocytoma, diffuse astrocytoma, anaplastic astrocytoma, etc.), neurilemmoma, liver cancer (e.g., primary liver cancer, extrahepatic bile duct cancer, etc.), Kidney cancer (e.g., renal cell carcinoma, transitional cell carcinoma of the renal pelvis and ureter, etc.), gallbladder cancer, bile duct cancer, pancreatic cancer, liver cancer, endometrial cancer, cervical cancer, ovarian cancer (e.g., epithelial ovarian cancer, extragonadal germ cell tumor, ovarian germ cell tumor, ovarian low malignant potential tumor, etc.), bladder cancer, urethral cancer, skin cancer (e.g., intraocular (eye) melanoma, Merkel cell carcinoma, etc.), hemangioma, malignant lymphoma (e.g., reticulum cell sarcoma, lymphosarcoma, Hodgkin's disease, etc.), melanoma, thyroid cancer (e.g., medullary thyroid cancer, etc.), parathyroid These include, but are not limited to, cancer of the nasal cavity, cancer of the paranasal sinuses, bone tumors (e.g., osteosarcoma, Ewing's tumor, uterine sarcoma, soft tissue sarcoma, etc.), metastatic medulloblastoma, angiofibroma, dermatofibrosarcoma protuberans, retinal sarcoma, penile cancer, testicular tumor, pediatric solid tumors (e.g., Wilms' tumor, pediatric renal tumor, etc.), Kaposi's sarcoma, Kaposi's sarcoma caused by AIDS, maxillary sinus tumor, fibrous histiocytoma, leiomyosarcoma, rhabdomyosarcoma, chronic myeloproliferative disease, leukemia (e.g., acute myeloid leukemia, acute lymphoblastic leukemia, etc.), etc.

The second cell population may include immune cells. By including cancer cells and immune cells in the second cell population, the environment of the cancer cells cultured in contact with the cell structure becomes more similar to the environment of the cancer cells in a living body.

The second cell population may be located outside or inside the cell structure. When the second cell population is outside the cell structure, the position of the second cell population is not particularly limited as long as the second cell population is in contact with the cell structure. For example, the second cell population may be located on the top surface (upper surface) of the cell structure, or on the bottom surface of the cell structure, or the cell structure may be in contact with the top and bottom surfaces of the cell layer containing the second cell population. When the second cell population is inside the cell structure, the second cell population may be scattered throughout the cell structure.

(Polymer electrolyte)
The cell structure may contain a polyelectrolyte. Examples of the polyelectrolyte include, but are not limited to, glycosaminoglycans such as heparin, chondroitin sulfate (e.g., chondroitin 4-sulfate, chondroitin 6-sulfate), heparan sulfate, dermatan sulfate, keratan sulfate, and hyaluronic acid; dextran sulfate, rhamnan sulfate, fucoidan, carrageenan, polystyrene sulfonic acid, polyacrylamide-2-methylpropane sulfonic acid, and polyacrylic acid, or derivatives thereof. The cell structure may contain only one type of polyelectrolyte, or may contain two or more types of polyelectrolytes. The polyelectrolyte is preferably a glycosaminoglycan. Furthermore, it is more preferable to use at least one of heparin, dextran sulfate, chondroitin sulfate, and dermatan sulfate as the polyelectrolyte. It is even more preferable that the polyelectrolyte is heparin.

(Cationic Substances)
The cell structure may contain a cationic substance. Any positively charged substance may be used as the cationic substance as long as it does not adversely affect cell growth and the cell structure. Examples of the cationic substance include, but are not limited to, cationic buffers such as Tris-HCl buffer, Tris-maleic acid buffer, Bis-Tris buffer, and HEPES, as well as ethanolamine, diethanolamine, triethanolamine, polyvinylamine, polyallylamine, polylysine, polyhistidine, and polyarginine. In a preferred embodiment, the cationic substance used in the present invention is a cationic buffer. In a more preferred embodiment, the cationic substance used in the present invention is a Tris-HCl buffer.

The concentration of the cationic substance is not particularly limited as long as it does not adversely affect cell growth and cell structures. The concentration of the cationic substance is preferably 10 to 100 mM. For example, the concentration of the cationic substance used in this embodiment is preferably 20 to 90 mM, 30 to 80 mM, 40 to 70 mM, or 45 to 60 mM. It is more preferable that the concentration of the cationic substance used in this embodiment is 50 mM.

When a cationic buffer is used as the cationic substance, the pH of the cationic buffer is not particularly limited as long as it does not adversely affect cell growth and cell structures. The pH of the cationic buffer used in this embodiment is preferably 6.0 to 8.0. For example, the pH of the cationic buffer used in this embodiment is 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0. The pH of the cationic buffer used in this embodiment is more preferably 7.2 to 7.6. The pH of the cationic buffer used in this embodiment is even more preferably 7.4.

[method]
In one embodiment, the present invention provides a method for inhibiting the proliferation rate of a second cell population, comprising contacting and culturing a cell structure comprising a first cell population and an extracellular matrix component, wherein the first cell population contains the extracellular matrix component between cells, and the first cell population comprises at least interstitial cells.

As the cell structure used in this embodiment, the one described in [Cell structure] can be used. Furthermore, as the first cell population, the one described in [Cell structure] can be used. Furthermore, as the second cell population, the one described in [Cell structure] can be used.

By culturing the second cell population in contact with the cell structure, the second cell population can be cultured in a state similar to that in a living body. As a result, for example, the proliferation rate of the second cell population can be made to approach (inhibit) the proliferation rate of the second cell population in a living body. The lifespan of the second cell population can also be made to approach the lifespan of the second cell population in a living body. The degree of differentiation, maturation, etc. of the second cell population can also be made to approach the degree of differentiation, maturation, etc. of the second cell population in a living body. In other words, the type and amount of mRNA, protein, etc. expressed by the second cell population, as well as the morphology, function, etc. of the second cell population can be made to approach those in a living body. This makes it possible to make the response of the second cell population to drugs and poisons closer to those in a living body, and to perform more accurate efficacy evaluation and toxicity evaluation.

In this embodiment, the cell structure may contain interstitial cells and extracellular matrix components. The cell structure may be manufactured (constructed) by, for example, the following method. The method for manufacturing the cell structure may be, for example, a method of constructing one layer at a time and stacking them in order, a method of constructing two or more cell layers at once, or a method of constructing multiple cell layers by appropriately combining both construction methods. In addition, the cell structure according to this embodiment may be a multi-layer structure in which the cell types constituting each cell layer are different for each layer, or the cell types constituting each cell layer may be a common cell type for all layers of the structure. For example, the cell structure may be constructed by forming a layer for each cell type and stacking the cell layers for each cell type in order, or a method in which a cell mixture containing multiple types of cells is prepared in advance and a multi-layered cell structure is constructed at once from the cell mixture containing the previously prepared multiple types of cells.

As a method of constructing layers one by one and stacking them in sequence, for example, there is a method described in Japanese Patent No. 4919464, that is, a method of stacking cell layers continuously by alternately repeating a step of forming a cell layer and a step of contacting the formed cell layer with a solution containing ECM (extracellular matrix) components. For example, when carrying out this method, a cell mixture is prepared in advance by mixing all the cells constituting the cell structure and the second cell population, and each cell layer is formed with this cell mixture, thereby constructing a cell structure in which a vascular network structure is formed throughout the cell structure and the second cell population is scattered throughout the cell structure. In addition, by forming each cell layer for each cell type, a cell structure in which a vascular network structure is formed only in the layer formed from endothelial cells, the second cell population is present only in a specific cell layer, and the cell layer containing the second cell population is in contact with the cell layer can be constructed.

An example of a method for constructing two or more cell layers at once is the method described in Japanese Patent Publication No. 5850419. In this method, the entire surface of a cell is coated in advance with a polymer containing an arginine-glycine-aspartic acid (RGD) sequence to which integrins bind and a polymer that interacts with the polymer containing the RGD sequence, and the coated cells coated with this adhesive film are placed in a cell culture vessel, and then the coated cells are accumulated by centrifugation or the like to construct a cell structure formed from multiple cell layers. For example, when carrying out this method, a cell mixture is prepared in advance by mixing all the cells constituting the cell structure and a second cell population, and an adhesive component is added to this cell mixture to prepare the coated cells. This allows a cell structure in which the second cell population is scattered throughout the entire structure to be constructed by a single centrifugation process. Also, for example, coated cells that coat endothelial cells, coated cells that coat fibroblasts, and coated cells that coat cells of a second cell population are each prepared separately, and a multilayer composed of coated cells of fibroblasts is formed, and then a layer formed of coated cells of endothelial cells is layered on top of that, a multilayer formed of coated cells of fibroblasts is layered on top of that, and a layer formed of coated cells of the second cell population is layered on top of that. This allows the construction of a cell structure that has a vascular network structure sandwiched between thick fibroblast layers and has a cell layer containing the second cell population in contact with the top surface.

In this embodiment, the cell structure may be produced by a method including a step (A) of mixing the first cell population with a cationic substance, an extracellular matrix component, and a polyelectrolyte to obtain a mixture, and a step (B) of incubating the cells in the obtained mixture to obtain a cell structure.

In this embodiment, the cell structure may be produced by a method including the steps of: (a) mixing the first cell population with a cationic substance, an extracellular matrix component, and a polyelectrolyte to obtain a mixture; (b) collecting the first cell population from the mixture to form a cell aggregate; and (c) incubating the cell aggregate.

By carrying out steps (a) to (c), a cell structure with few large internal voids can be obtained. Furthermore, the obtained cell structure is relatively stable, and can be cultured for at least several days, and the tissue is unlikely to collapse even when the medium is replaced. Furthermore, in this embodiment, step (b) may include seeding the mixture in a cell culture vessel and allowing it to settle in the cell culture vessel. The settling of the cell mixture may be achieved by actively sedimenting the cells using centrifugation or the like, or by allowing them to settle naturally.

As the cationic substance, those described in [Cell Structure] can be used. As the extracellular matrix component, those described in [Cell Structure] can be used. As the polyelectrolyte, those described in [Cell Structure] can be used.

In this embodiment, the concentration of the polyelectrolyte can be greater than 0 mg/mL (higher than 0 mg/mL) and less than 1.0 mg/mL, preferably 0.025 to 0.5 mg/mL, and more preferably 0.05 to 0.3 mg/mL. In this embodiment, the above mixture can also be prepared without mixing the polyelectrolyte to produce a cell structure.

In this embodiment, the blend ratio of the polymer electrolyte to the extracellular matrix components is 1:5 to 5:1. In constructing the cell structure according to this embodiment, the blend ratio of the polymer electrolyte to the extracellular matrix components is more preferably 1:2 to 2:1, more preferably 1:1.5 to 1.5:1, and even more preferably 1:1.

By repeating steps (a) to (c), specifically, by placing the mixture prepared in step (a) on the upper surface of the cell structure obtained in step (c) as step (b), gathering the first cell population to form a cell aggregate, and then repeating step (c), a cell structure of sufficient thickness can be constructed. The cell composition of the mixture newly placed on the cell structure obtained in step (c) may be the same as or different from the cell composition constituting the already constructed cell structure.

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

After step (a), (a'-1) a step of removing the liquid portion from the obtained mixture to obtain a mixture, and (a'-2) a step of suspending the obtained mixture in a solvent may be performed, and then step (b) may be proceeded to. By performing the above-mentioned steps (a) to (c), the desired cell structure can be obtained, but a more homogeneous cell structure can be obtained by performing (a'-1) and (a'-2) after step (a) and then performing step (b).

In addition, after step (a), the following steps (b'-1) and (b'-2) may be performed instead of step (b). By performing steps (b'-1) and (b'-2), a more homogeneous cell structure can also be obtained. Like step (b), step (b'-2) may also include sedimenting the cell mixture seeded in the cell culture vessel in the cell culture vessel. The sedimentation of the cell mixture may be performed by actively sedimenting the cells by centrifugation or the like, or by allowing the cells to sediment naturally. In this embodiment and the present specification, the "cell viscous body" refers to a gel-like cell aggregate (see, for example, Nishiguchi et al., Macromol Bioscience, 2015, vol.15(3), p.312-317.).
(b'-1) seeding the mixture obtained in step (a) into a cell culture vessel, and then removing liquid components from the mixture to obtain a viscous cell mass;
(b'-2) A step of suspending the cell viscous body in a solvent in a cell culture vessel.

The solvent for preparing the cell suspension is not particularly limited as long as it is non-toxic to cells and does not impair their proliferation or function, and water, a buffer solution, a cell culture medium, etc. can be used. Examples of the buffer solution include phosphate-buffered saline (PBS), HEPES, Hanks buffer, etc. Examples of the culture medium include D-MEM, E-MEM, MEMα, RPMI-1640, Ham's F-12, etc. When a cell culture medium is used as the solvent for preparing the cell suspension, the cells can be cultured without removing the liquid components in step (c) described below.

Instead of the step (c), the following step (c') may be carried out.
(c'): Incubating the cell structure on the substrate.

The method for removing the liquid components in steps (c) and (c') is not particularly limited as long as it does not adversely affect cell growth and the construction of cell structures, and can be appropriately performed using a method known to those skilled in the art as a method for removing liquid components from a suspension of liquid and solid components. Examples of such methods include aspiration, centrifugation, magnetic separation, and filtration. For example, when a cell culture insert is used as a cell culture vessel, the cell culture insert seeded with the mixture can be centrifuged at 10°C and 400 x g for 1 minute, causing the cell mixture to settle, and the liquid components can be removed by aspiration.

The cell structure according to this embodiment may be constructed in a cell culture vessel. The cell culture vessel is not particularly limited as long as it is capable of constructing a cell structure and culturing the constructed cell structure. Specific examples of the cell culture vessel include dishes, cell culture inserts (e.g., Transwell (registered trademark) inserts, Netwell (registered trademark) inserts, Falcon (registered trademark) cell culture inserts, Millicell (registered trademark) cell culture inserts, etc.), tubes, flasks, bottles, plates, etc. In constructing the cell structure according to this embodiment, dishes or various cell culture inserts are preferred because they allow for more appropriate evaluation using the cell structure.

More specifically, the cell structure may be manufactured, for example, as follows. For example, first, in step (a), a mixture containing only fibroblasts as cells is prepared, and steps (b) and (c) are performed to obtain a cell structure formed of 10 fibroblast layers in a cell culture vessel. Next, in step (a), a mixture containing only vascular endothelial cells as cells is prepared, and steps (b) and (c) are performed to laminate one vascular endothelial cell layer on the fibroblast layer in the cell culture vessel. Furthermore, in step (a), a mixture containing only fibroblasts as cells is prepared, and steps (b) and (c) are performed to laminate 10 fibroblast layers on the vascular endothelial cell layer in the cell culture vessel. This allows the construction of a cell structure in which layers are laminated in order for each cell type, such as 10 fibroblast layers - 1 vascular endothelial cell layer - 10 fibroblast layers. By adjusting the number of cells contained in the mixture in step (b), the thickness of the cell layers laminated in step (c) can be adjusted. The more cells contained in the mixture in step (b), the more cell layers will be stacked in step (c).

For example, a cell layer containing the second cell population can be laminated on the fibroblast layer of the above-mentioned cell structure of 10 fibroblast layers - 1 vascular endothelial cell layer - 10 fibroblast layers, in the same manner as in steps (a) to (c). In addition, in step (a), a mixture is prepared in which fibroblasts for 20 fibroblast layers and vascular endothelial cells for 1 vascular endothelial cell layer are all mixed, and steps (b) and (c) are performed. By laminating a mixture containing the second cell population prepared in the same manner on the formed multi-layered cell structure, a cell structure having a thickness of 21 layers and in which a vascular network structure is scattered inside can be constructed. Furthermore, in step (a), a mixture is prepared by mixing fibroblasts from 20 fibroblast layers, vascular endothelial cells from one vascular endothelial cell layer, and a second cell population from one cell layer containing the second cell population, and steps (b) and (c) are carried out to construct a cell structure with a thickness of 22 layers, with the second cell population and a vascular network structure dispersed therein.

[Evaluation method]
In one embodiment, the present invention provides a method for evaluating a test substance, comprising the steps of culturing a cell population in the presence of the test substance by contacting it with the above-mentioned cell structure and analyzing the state of the cell population, and comparing the state in the presence of the test substance with the state in the absence of the test substance to evaluate the effect of the test substance.

In this embodiment, by culturing the cell population in contact with the cell structure, the cell population can be cultured in a state similar to that in a living body. As a result, for example, the proliferation rate of the cell population can be made to approach (inhibit) the proliferation rate of the cell population in a living body. The lifespan of the cell population can also be made to approach the lifespan of the cell population in a living body. The degree of differentiation, maturation, etc. of the cell population can also be made to approach the degree of differentiation, maturation, etc. of the cell population in a living body. In other words, the type and amount of mRNA, protein, etc. expressed by the cell population, as well as the morphology, function, etc. of the cell population can be made to approach those in a living body. This makes it possible to make the response of the cell population to the test substance closer to that in a living body, and to more accurately evaluate the effect of the test substance.

The cell population used in this embodiment includes cells similar to the second cell population described above in the section "Cell Structure."

The test substances used in this embodiment are not particularly limited, and examples include substances derived from natural compound libraries, synthetic compound libraries, existing drug libraries, metabolite libraries, etc.

According to the evaluation method of this embodiment, if the state of the cell population in the presence of the test substance differs from the state of the cell population in the absence of the test substance, it can be determined that the test substance is effective against the cell population.

In the process of analyzing the state of a cell population, for example, cell viability, cell proliferation rate, cell morphology, state of cell components, state of secretions from cells, cell function, etc. may be analyzed.

Methods for analyzing cell viability, cell proliferation rate, and cell morphology may be any method known to those skilled in the art.

Cell constituents include, but are not limited to, genetic material such as genomic DNA and mitochondrial DNA; gene products such as RNA and proteins; and low molecular weight metabolic products such as second messengers and ions.

The state of the components of a cell may be analyzed, for example, as follows. To analyze the state of genetic material, for example, the frequency of DNA methylation may be analyzed. To analyze the state of gene products, for example, the expression level of RNA, protein, etc., the state of modification, the strength of protein activity, etc. may be analyzed. To analyze the state of small molecular weight metabolites, for example, the abundance of small molecular weight metabolites may be analyzed.

Secretions from cells are not particularly limited, and examples thereof include hormones such as peptide hormones and fat-soluble hormones; signal transduction substances such as cytokines; neurotransmitters such as glutamic acid, GABA, acetylcholine, monoamines, and neuropeptides; proteins such as antibodies; and secretory vesicles such as exosomes. In order to analyze the state of secretions from cells, for example, the secretions in the culture medium in which the cell population is cultured may be detected and quantified.

To analyze the function of a cell, for example, the degree of cell differentiation, maturity, etc. may be analyzed. The degree of cell differentiation, maturity, etc. may be determined based on the results of an analysis of the state of the constituent components of the cell described above and the state of secretions from the cell.

In addition, to analyze the function of a cell, the cell may be stimulated and the cell's response to the stimulation may be analyzed. For example, if the cell is a muscle cell, the contraction of the muscle cell, calcium dynamics within the muscle cell, etc. may be analyzed, and if the cell is a nerve cell, the action potential of the nerve cell, etc. may be analyzed.

In this embodiment, when the cell population forms a structure, the state of the cell population may be the state of the structure formed by the cell population. In order to analyze the state of the structure formed by the cell population, the morphology of the structure, the function of the structure, etc. may be analyzed. For example, when the cell population includes nerve cells, the morphology of the neural network formed by the nerve cells may be analyzed in order to analyze the state of the cell population.

In this embodiment, the cell population may include cancer cells. The evaluation method of this embodiment may include a culture step of culturing cancer cells by contacting them with the cell structure in the presence of an anticancer drug, and an evaluation method for evaluating the anticancer effect of the anticancer drug using the number of live cancer cells after the culture step as an index. The anticancer drug may be contained in the medium used to culture the cell structure.

According to the method for evaluating anticancer effects of this embodiment, cancer cells can be cultured under conditions more similar to those in vivo than conventional methods. This allows the types and amounts of mRNA, proteins, etc. expressed by cancer cells cultured in contact with a cell structure, as well as the morphology, function, etc. of the cancer cells to be closer to those in vivo. As a result, the anticancer effect of cancer cells cultured in contact with a cell structure against an anticancer agent can be made similar to the effect of cancer cells in vivo against the anticancer agent.

In the culture step of this embodiment, the cancer cells that are brought into contact with the cell structure and cultured in the presence of an anticancer drug may be co-cultured with immune cells. By co-culturing the cancer cells with immune cells, the environment of the cancer cells becomes more similar to the environment of cancer cells in a living body.

(Anti-cancer drugs)
The anticancer drug used in the evaluation method according to the present embodiment may be any drug used in cancer treatment, including not only drugs that act directly on cancer cells, such as cytotoxic drugs, but also drugs that do not have cytotoxicity but suppress the proliferation of cancer cells. Examples of anticancer drugs that do not have cytotoxicity include drugs that do not directly attack cancer cells, but rather suppress the proliferation of cancer cells, slow down the activity of cancer cells, or kill cancer cells by acting cooperatively with other drugs, and drugs that suppress the proliferation of cancer cells by damaging cells or tissues other than cancer cells. The anticancer drug used in the present embodiment may be a drug known to have an anticancer effect, or may be a candidate compound for a novel anticancer drug. When the cancer cells of the present embodiment are co-cultured with immune cells, the anticancer drug may be a drug that acts cooperatively with immune cells in the living body.

Cytotoxic anticancer drugs include, but are not limited to, molecular targeted drugs, alkylating agents, metabolic antagonists such as 5-FU anticancer drugs, plant alkaloids, anticancer antibiotics, platinum derivatives, hormones, topoisomerase inhibitors, microtubule inhibitors, and compounds classified as biological response modifiers.

Examples of anticancer drugs that do not have cytotoxicity include, but are not limited to, angiogenesis inhibitors, prodrugs of anticancer drugs, drugs that regulate the activity of intracellular metabolic enzymes involved in the metabolism of anticancer drugs or their prodrugs (hereinafter referred to as "intracellular enzyme regulators" in the specification), immunotherapy drugs, etc. Other examples include drugs that ultimately contribute to anticancer effects by enhancing the function of anticancer drugs or improving immune function in the body.

The angiogenesis inhibitor may be any compound that is expected to have angiogenesis inhibitory activity, and may be a known angiogenesis inhibitor or a candidate compound for a novel angiogenesis inhibitor. Known angiogenesis inhibitors include Avastin, EYLEA, Suchibaga, CYRAMZA® (Eli Lilly, also known as ramucirumab), BMS-275291 (Bristol-Myers), celecoxib (Pharmacia/Pfizer), EMD121974 (Merck), Endostatin (EntreMed), Erbitaux (ImCloneSystems), Interferon-α (Roche), LY317615 (Eli Lilly), Neovastat (Aeterna), Laboratories), PTK787 (Abbott), SU6688 (Sugen), Thalidamide (Celgene), VEGF-Trap (Regeneron), Iressa (registered trademark) (Astrazeneca, also known as gefitinib), Caplerusa (registered trademark) (Astrazeneca, also known as pandetanib), Recentin (registered trademark) (Astrazeneca, also known as cediranib), VGX-100 (Circadian Technologies), VD1andcVE199, VGX-300 (Circadian Technologies), sVEGFR2, hF4-3C5, Nexavar (registered trademark) (manufactured by Bayer Yakuhin, also known as sorafenib), Vortrient (registered trademark) (manufactured by GlaxoSmithKline, also known as pazopanib), Sutent (registered trademark) (manufactured by Pfizer, also known as sunitinib), Inlyta (registered trademark) (manufactured by Pfizer, also known as axitinib), CEP-11981 (manufactured by Teva Pharmaceutical Industries), AMG-386 (Takeda Examples include anti-NRP2B (manufactured by Genentech), Ofev (registered trademark) (manufactured by Boehringer-Ingelheim, also known as nintatenib), and AMG706 (manufactured by Takeda Yakuhin, also known as motesanib).

Anticancer drug prodrugs are drugs that are converted into active forms with anticancer effects by intracellular enzymes in organs such as the liver and in cancer cells. The cytokine network increases the enzymatic activity of intracellular enzymes, increasing the amount of the active form and enhancing the antitumor effect, so they are considered to be drugs that are involved in anticancer effects.

An example of an intracellular enzyme regulator is gimeracil, which does not have a direct antitumor effect when used alone, but is involved in anticancer effects by inhibiting the enzyme that breaks down 5-FU anticancer drugs (dihydropyrimidine dehydrogenase: DPD).

Immunotherapeutic agents are drugs that obtain anti-cancer effects by improving immune function through activation of immune function or motility of immune cells. Examples of immunotherapeutic agents include drugs used in biological response modifier therapy (hereinafter abbreviated as "BRM preparations"), cytokine-based preparations formed from cytokines secreted from immune cells and involved in migration and infiltration, cancer immune checkpoint inhibitors that have attracted attention in recent years, cancer vaccines, and cancer viruses. Examples of BRM preparations include krestin, lentinan, OK-432, and the like. Examples of cytokine-based preparations include interleukins such as IL-8 and IL-2; interferons such as IFN-α, IFN-β, and IFN-γ; and chemokines such as CCL3, CCL4, CCL5, CXCL9, CXCL10, CXCL11, CXCL16/CXCR6, and CX3CL1/CX3CR1.

Cancer immune checkpoint inhibitors are substances that specifically inhibit the function of proteins that are present on the surface of cancer cells and immune cells and are involved in the reduction of immune function against cancer cells. Examples of such proteins include PD-1, PD-L1, PD-L2, CD4, CD8, CD19, CD28, CD80/86, B7, Galectin-9, HVEM, CTLA-4, TIM-3, BTLA, MHC-II, LAG-3, and TCR. As cancer immune checkpoint inhibitors, specific monoclonal antibody drugs against these are preferred. Specifically, cancer immune checkpoint inhibitors include nivolumab (Opdivo), pembrolizumab (Keytruda), atezolizumab (Tecentriq), ipilimumab (Yervoy), tremelimumab, durvalmab, and avelumab.

In the evaluation method according to this embodiment, in the aspect in which cancer cells and immune cells are co-cultured in the presence of an anticancer drug by contacting them with a cell structure, the anticancer drug used is preferably an immunotherapy drug, and among them, a cancer immune checkpoint inhibitor is more preferable since its efficacy is thought to be significantly affected by immune function.

In the evaluation method according to this embodiment, one type of anticancer drug may be used, or two or more types of anticancer drugs may be used in combination. In addition, an anticancer drug may be used in combination with a drug other than an anticancer drug. For example, even if an anticancer drug has an anticancer effect when administered alone, if the anticancer drug is administered in combination with another drug in an actual clinical setting, the evaluation method according to this embodiment may be performed using the anticancer drug in combination with the other drug.

The present invention will be described below with reference to examples, but the present invention is not limited to the following examples.

[Experimental Example 1]
(Cancer cell proliferation suppression effect of cell structures)
The cell construct, which is composed of fibroblasts and vascular endothelial cells and has a vascular network structure, was used to evaluate the effect of inhibiting the proliferation of cancer cells after culturing the cancer cells for 7 days.

<Cell structure>
The following cells were used in forming the cell structures:
NHDF: Human neonatal dermal fibroblasts (Normal Human Dermal Fibroblasts, Lonza, product number: CC-2509)
HUVEC: Human Umbilical Vein Endothelial Cell (Lonza, product number: CC-2517A)

<Cancer cells>
The following cancer cells were used.
HT29: human colorectal adenocarcinoma cell line (ATCC number: CCL-247)
HCT116: human colorectal adenocarcinoma cell line (ATCC number: CCL-247)
DLD-1: human colorectal adenocarcinoma cell line (ATCC number: CCL-221)
A549: Human lung cancer cell line (ATCC number: CCL-185)
NCI-H1975: human lung cancer cell line (ATCC number: CCL-5908)
MCF-7: human breast cancer cell line (ATCC number: HTB-22)

<Cell culture container, culture medium>
The following cell culture vessels and culture media were used.
Cell culture vessel: Transwell cell culture insert (Corning, product number: #3470)
Culture medium: D-MEM containing 10% by volume of bovine serum (Corning, product number: #35-010-CV) and 1% by volume of penicillin/streptomycin (Wako Pure Chemical Industries, Ltd., product number: 168-23191). (Wako Pure Chemical Industries, Ltd., product number: 043-30085)

<Formation of cell structures>
First, 2×10 ⁶ NHDFs and 3×10 ⁴ HUVECs were suspended in a Tris-HCl buffer solution containing heparin and collagen (0.1 mg/mL heparin, 0.1 mg/mL collagen, 50 mM Tris, pH 7.4) to prepare a cell suspension. This cell suspension was centrifuged at 1000×g for 1 minute at room temperature, the supernatant was removed, and the cells were resuspended in an appropriate amount of culture medium. Next, this cell suspension was seeded in a Transwell cell culture insert, and the Transwell cell culture insert was centrifuged at 400×g for 1 minute at room temperature. Then, an appropriate amount of culture medium was added to the Transwell cell culture insert, and the insert was cultured in a CO ₂ incubator (37° C., 5% CO ₂ ) for 24 hours.

<Cancer cell seeding>
^2x104 cancer cells suspended in an appropriate amount of culture medium were seeded into the Transwell cell culture insert with the structure formed, and then cultured for 7 days in a _CO2 incubator (37°C, 5% _CO2 ). After the culture was completed, a cell structure was obtained in which a cancer cell layer was laminated on a layer with a vascular network structure. The cancer cells used had been fluorescently labeled (SIGMA, product number: PKH26GL) in advance.

For comparison, instead of the cell structure, the same number of cancer cells were cultured in a monolayer in a general culture vessel (2D culture) and similarly cultured.

<Living cell count analysis>
After the culture, the cell structure and the cancer cells were immersed in a trypan blue solution and stained with trypan blue, and the cells that emitted fluorescence and were not stained with trypan blue were counted as live cancer cells. The cell count was performed using the fluorescence mode of a cell counter "Countess II" (manufactured by Life Technologies).

The cell counts for each culture are shown in Table 1. In the table, the "2D" column shows the results for 2D cultures, and the "3D" column shows results for cancer cells cultured in contact with the cell structure of the present invention (3D cultures).

The results showed that, compared to 2D cultures, 3D cultures significantly suppressed cell proliferation in all cancer cell types used after 7 days of culture.

[Experimental Example 2]
(Cancer cell proliferation suppression effect of cell structures)
The cell structure, which is composed of fibroblasts and vascular endothelial cells and has a vascular network structure, was used to evaluate the effect of inhibiting the proliferation of cancer cells. The evaluation was performed after culturing the cancer cells for 4 days and then 7 days.

<Cell structure>
The following cells were used in forming the cell structures:
NHDF: Human neonatal dermal fibroblasts (Normal Human Dermal Fibroblasts, Lonza, product number: CC-2509)
HUVEC: Human Umbilical Vein Endothelial Cell (Lonza, product number: CC-2517A)

<Cancer cells>
The following cancer cells were used.
HT29: human colorectal adenocarcinoma cell line (ATCC number: CCL-247)
NCI-H1975: human lung cancer cell line (ATCC number: CCL-5908)
MCF-7: human breast cancer cell line (ATCC number: HTB-22)

<Cell culture container, culture medium>
The following cell culture vessels and culture media were used.
Cell culture vessel: Transwell cell culture insert (Corning, product number: #3470)
Culture medium: D-MEM containing 10% by volume of bovine serum (Corning, product number: #35-010-CV) and 1% by volume of penicillin/streptomycin (Wako Pure Chemical Industries, Ltd., product number: 168-23191). (Wako Pure Chemical Industries, Ltd., product number: 043-30085)

<Formation of cell structures>
First, 2×10 ⁶ NHDFs and 3×10 ⁴ HUVECs were suspended in a Tris-HCl buffer solution containing heparin and collagen (0.1 mg/mL heparin, 0.1 mg/mL collagen, 50 mM Tris, pH 7.4) to prepare a cell suspension. This cell suspension was centrifuged at 1000×g for 1 minute at room temperature, the supernatant was removed, and the cells were resuspended in an appropriate amount of culture medium. Next, this cell suspension was seeded in a Transwell cell culture insert, and the Transwell cell culture insert was centrifuged at 400×g for 1 minute at room temperature. Then, an appropriate amount of culture medium was added to the Transwell cell culture insert, and the insert was cultured in a CO ₂ incubator (37° C., 5% CO ₂ ) for 24 hours.

<Cancer cell seeding>
^2x104 cancer cells suspended in an appropriate amount of culture medium were seeded into the Transwell cell culture insert with the structure formed, and then cultured for 4 or 7 days in a _CO2 incubator (37°C, 5% _CO2 ). After the culture was completed, a cell structure was obtained in which a cancer cell layer was laminated on a layer with a vascular network structure. The cancer cells used had been fluorescently labeled in advance (manufactured by SIGMA, product number: PKH26GL).

For comparison, instead of the cell structure, the same number of cancer cells were cultured in a monolayer in a general culture vessel (2D culture) and similarly cultured.

<Living cell count analysis>
After the culture, the cell structure and the cancer cells were immersed in a trypan blue solution and stained with trypan blue, and the cells that emitted fluorescence and were not stained with trypan blue were counted as live cancer cells. The cell count was performed using the fluorescence mode of a cell counter "Countess II" (manufactured by Life Technologies).

The cell counts for each culture are shown in Table 2. In the table, the "2D" column shows the results for 2D cultures, and the "3D" column shows results for cancer cells cultured in contact with the cell structure of the present invention (3D cultures).

The results showed that, compared to 2D cultures, 3D cultures significantly suppressed cell proliferation in all cancer cell types used after 4 and 7 days of culture.

[Experimental Example 3]
(Relationship between the number of cell layers in a cell structure and the effect of inhibiting cancer cell proliferation)
The effect of inhibiting the proliferation of cancer cells was evaluated using a cell structure that was composed of fibroblasts and vascular endothelial cells and had a vascular network structure. The cell structures were formed with cell layers equivalent to 1.25 layers, 2.5 layers, 5 layers, 10 layers, and 20 layers.

<Cell structure>
The following cells were used in forming the cell structures:
NHDF: Human neonatal dermal fibroblasts (Normal Human Dermal Fibroblasts, Lonza, product number: CC-2509)
HUVEC: Human Umbilical Vein Endothelial Cell (Lonza, product number: CC-2517A)

<Cancer cells>
The following cancer cells were used.
NCI-H1975: human lung cancer cell line (ATCC number: CCL-5908)

<Cell culture container, culture medium>
The following cell culture vessels and culture media were used.
Cell culture vessel: Transwell cell culture insert (Corning, product number: #3470)
Culture medium: D-MEM containing 10% by volume of bovine serum (Corning, product number: #35-010-CV) and 1% by volume of penicillin/streptomycin (Wako Pure Chemical Industries, Ltd., product number: 168-23191). (Wako Pure Chemical Industries, Ltd., product number: 043-30085)

<Formation of cell structures>
First, NHDF and HUVEC were suspended in a Tris-HCl buffer solution containing heparin and collagen (0.1 mg/mL heparin, 0.1 mg/mL collagen, 50 mM Tris, pH 7.4) to prepare a cell suspension. This cell suspension was centrifuged at room temperature at 1000×g for 1 minute, the supernatant was removed, and the cells were resuspended in an appropriate amount of culture medium. Next, this cell suspension was seeded in a Transwell cell culture insert, and the Transwell cell culture insert was centrifuged at room temperature at 400×g for 1 minute. Then, an appropriate amount of culture medium was added to the Transwell cell culture insert, and the insert was cultured in a CO ₂ incubator (37° C., 5% CO ₂ ) for 24 hours. The number of cells to be seeded was adjusted to form cell structures with cell layers equivalent to 1.25 layers, 2.5 layers, 5 layers, 10 layers, and 20 layers. The number of cell layers was calculated assuming that the number of cells corresponding to one cell layer of 1 ^cm2 is 3 x ^105. Furthermore, the cell structure produced by the method of this experimental example has a thickness of about 40 μm per 10 layers. That is, the thicknesses of the cell structures corresponding to 1.25 layers, 10 layers, and 20 layers are about 5 μm, about 40 μm, and about 80 μm, respectively.

<Cancer cell seeding>
^2x104 cancer cells suspended in an appropriate amount of culture medium were seeded into the Transwell cell culture insert with the structure formed, and then cultured for 7 days in a _CO2 incubator (37°C, 5% _CO2 ). After the culture was completed, a cell structure was obtained in which a cancer cell layer was laminated on a layer with a vascular network structure. The cancer cells used had been fluorescently labeled (SIGMA, product number: PKH26GL) in advance.

For comparison, instead of the cell structure, the same number of cancer cells were cultured in a monolayer in a general culture vessel (2D culture) and similarly cultured.

<Living cell count analysis>
After the culture, the cell structure and the cancer cells were treated with an anti-cytokeratin 7 antibody (Invitrogen: OV-TL 12/30) and a secondary antibody (Invitrogen: A-21235), and only the cancer cells were fluorescently stained and measured. The cell measurements were performed using the fluorescence mode of a confocal microscope system (PerkinElmer: Operetta CLS), and the area of the cancer cells was calculated by taking the fluorescent region on the container as the region where the cancer cells existed.
The 2D cultures were also stained and measured in the same manner. Measurements were repeated three times for each culture condition.

The cell counts for each culture are shown in Table 3. In the table, the "2D" column shows the results for 2D cultures, and the "3D" column shows the results for cancer cells cultured in contact with the cell structure of the present invention (3D cultures).

The results showed that, compared to 2D cultures, 3D cultures showed that cancer cell proliferation was suppressed when cell structures with 10 or more cell layers were used.

[Experimental Example 4]
(Cancer cell proliferation suppression effect of cell structures without vascular network structure)
The effect of inhibiting the proliferation of cancer cells was evaluated using a cell construct with a vascular network structure composed of fibroblasts and vascular endothelial cells, and a cell construct without a vascular network structure composed of fibroblasts. The evaluation was performed after culturing the cancer cells for 7 days.

<Cell structure>
The following cells were used in forming the cell structures:
NHDF: Human neonatal dermal fibroblasts (Normal Human Dermal Fibroblasts, Lonza, product number: CC-2509)
HUVEC: Human Umbilical Vein Endothelial Cell (Lonza, product number: CC-2517A)

<Cancer cells>
The following cancer cells were used.
HCT116: human colorectal adenocarcinoma cell line (ATCC number: CCL-247)

<Cell culture container, culture medium>
The following cell culture vessels and culture media were used.
Cell culture vessel: Transwell cell culture insert (Corning, product number: #3470)
Culture medium: D-MEM containing 10% by volume of bovine serum (Corning, product number: #35-010-CV) and 1% by volume of penicillin/streptomycin (Wako Pure Chemical Industries, Ltd., product number: 168-23191). (Wako Pure Chemical Industries, Ltd., product number: 043-30085)

<Formation of cell structures>
First, 2×10 ⁶ NHDFs and 3×10 ⁴ HUVECs, or NHDFs alone, were suspended in a Tris-HCl buffer solution containing heparin and collagen (0.1 mg/mL heparin, 0.1 mg/mL collagen, 50 mM Tris, pH 7.4) to prepare a cell suspension. This cell suspension was centrifuged at room temperature at 1000×g for 1 minute, the supernatant was removed, and the cells were resuspended in an appropriate amount of culture medium. Next, this cell suspension was seeded in a Transwell cell culture insert, and the Transwell cell culture insert was centrifuged at room temperature at 400×g for 1 minute. Then, an appropriate amount of culture medium was added to the Transwell cell culture insert, and the insert was cultured in a CO ₂ incubator (37° C., 5% CO ₂ ) for 24 hours.

<Cancer cell seeding>
^2x104 cancer cells suspended in an appropriate amount of culture medium were seeded into the Transwell cell culture insert with the structure formed, and then cultured for 7 days in a _CO2 incubator (37°C, 5% _CO2 ). After the culture was completed, a cell structure was obtained in which a cancer cell layer was laminated on a layer with a vascular network structure. The cancer cells used had been fluorescently labeled (SIGMA, product number: PKH26GL) in advance.

For comparison, instead of the cell structure, the same number of cancer cells were cultured in a monolayer in a general culture vessel (2D culture) and similarly cultured.

<Living cell count analysis>
After the culture, the cell structure and the cancer cells were immersed in a trypan blue solution and stained with trypan blue, and the cells that emitted fluorescence and were not stained with trypan blue were counted as live cancer cells. The cell count was performed using the fluorescence mode of a cell counter "Countess II" (manufactured by Life Technologies).
In addition, the 2D cultures were similarly stained with trypan blue, and unstained cells were counted as live cancer cells. Each culture condition was measured three times.

The cell counts for each culture are shown in Table 4. In the table, the "2D" column shows the results for 2D cultures, and the "3D" column shows cancer cells cultured in contact with the cell structure of the present invention (3D cultures). "With 3D vascular network" shows the results when a cell structure with a vascular network structure consisting of fibroblasts and vascular endothelial cells was used, and "without 3D vascular network" shows the results when a cell structure consisting of fibroblasts and no vascular network structure was used.

The results showed that, compared to 2D cultures, cancer cell proliferation was significantly suppressed in 3D cultures, regardless of whether the 3D cultures had a vascular network or not.

The present invention provides a technology that allows established or immortalized cells to be cultured ex vivo under conditions more similar to those found in vivo than conventional methods.

Claims (5)

A method for suppressing the proliferation rate of a second cell population compared to when cultured in a monolayer, comprising:
The method includes a step of contacting a second cell population with a cell structure comprising a first cell population and an extracellular matrix component and culturing the second cell population;
the first cell population contains the extracellular matrix component between the cells;
the first cell population comprises at least stromal cells;
The method for inhibiting the proliferation rate of the second cell population , wherein the cell structure has 10 to 60 cell layers . The method for suppressing the proliferation rate of the second cell population according to claim 1, wherein the first cell population further comprises vascular endothelial cells and the interstitial cells are fibroblasts. The method for suppressing the proliferation rate of the second cell population according to claim 1 or 2, wherein the second cell population includes an established cell line. The method for suppressing the proliferation rate of the second cell population according to any one of claims 1 to 3, wherein the second cell population includes cancer cells. The cell structure,
mixing the first cell population with a cationic substance, an extracellular matrix component, and a polyelectrolyte to obtain a mixture;
and a step of culturing the cells in the obtained mixture to obtain the cell structure.