Cell structure and method for producing the same

Abstract

To provide a cell structure including a cancer cell in the state closer to an organism, and a method for producing the cell structure.SOLUTION: A cell structure is provided in which an endothelial cell layer consisting of vascular endothelial cells is formed on a surface, cells other than the vascular endothelial cell (cancer cell is excluded) are present inside, and cancer cells are present on an external surface of the endothelial cell layer, where the cancer cells are present as a cell cluster on the external surface of the endothelial cell layer, and the ellipticity of a cross-sectional shape of the cell cluster in a thickness direction of the cell structure is 3.0 or less. A method for producing a cell structure is provided in which after producing a three-dimensional structure in which an endothelial cell layer consisting of vascular endothelial cells is formed on a surface, and cells other than the vascular endothelial cell (cancer cell is excluded) are present inside, cancer cells are disseminated and cultured on the three-dimensional structure, by which a cell structure in which cancer cells are present on an external surface of the endothelial cell layer is produced.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This invention relates to a cellular structure used for analyzing the properties and functions of cancer cells. [Background technology]

[0002] In recent years, the advantages of using three-dimensional cell tissues, which are organized in three dimensions, over cells grown on flat plates have been demonstrated, not only in regenerative medicine but also in drug assay systems that require an environment close to that of a living organism (Non-Patent Literature 1). For this reason, various technologies have been developed to construct three-dimensional cell tissues outside the body. In particular, blood vessels and other vascular structures play an important role in supplying oxygen, nutrients, hormones, etc., which are necessary for the normal functioning of tissues in the body, and further elucidation of their function is desired. Furthermore, blood vessels and lymphatic vessels in particular are known to be deeply involved in the growth, invasion, metastasis, and other malignant processes and progression of cancer (tumors), and cell structures with vascular structures formed in addition to cancer cells are used in the evaluation of anticancer drugs (Patent Literature 1).

[0003] One method for producing a cell structure in which a vascular network is formed is to continuously stack cell layers by alternately repeating the steps of preparing a cell mixture of vascular endothelial cells and stromal cells, forming a cell layer from the cell mixture, and contacting the formed cell layer with a solution containing extracellular matrix components (Patent Document 2). Another method involves preparing coated cells by alternately coating vascular endothelial cells and stromal cells with two types of extracellular matrix components, stacking these to adhere to each other, and then culturing them to form a cell structure in which a vascular network is constructed (Patent Document 3). Furthermore, there is a method of forming a cell structure in which a vascular network is constructed by collecting a cell mixture in which vascular endothelial cells and stromal cells are suspended in a solution containing at least a cationic buffer, extracellular matrix components, and polyelectrolytes on a substrate and then culturing it (Patent Document 4). [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] International Publication No. 2017 / 183673 [Patent Document 2] Patent No. 4919464 [Patent Document 3] Patent No. 5850419 [Patent Document 4] Patent No. 6427836 [Non-patent literature]

[0005] [Non-Patent Document 1] Imamura et al, ONCOLOGY REPORTS, 2015, vol.33, p.1837-1843. [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] The problems to be solved by the present invention include providing a cell structure containing cancer cells in a state closer to that of a living organism, and a method for producing the same. [Means for solving the problem]

[0007] The inventors of this invention conducted extensive research to solve the above problems and discovered that by increasing the ratio of vascular endothelial cells to the total number of cells that serve as raw materials for constructing the cell structure to a much higher proportion than that used to form a conventional vascular network, a cell structure is formed in which a cell layer consisting of vascular endothelial cells is formed on the surface and cells other than vascular endothelial cells are present inside. Furthermore, they discovered that when cancer cells are brought into contact with this cell structure, a cell structure is obtained in which the vascular endothelial cell layer on the surface is in contact with cancer cells, thus completing the present invention.

[0008] The cell construct according to the first aspect of the present invention has an endothelial cell layer composed of vascular endothelial cells formed on its surface, cells other than the vascular endothelial cells (excluding cancer cells) present inside, and cancer cells present on the outer surface of the endothelial cell layer. The method for producing a cell construct according to the second aspect of the present invention is as follows: A three-dimensional structure having an endothelial cell layer composed of vascular endothelial cells formed on its surface and cells other than the vascular endothelial cells (excluding cancer cells) present inside is (A) suspended in a mixture in which cells are suspended in a solution containing at least a cationic substance, an extracellular matrix component, and a polyelectrolyte to obtain a mixture; (B) collecting the cells from the obtained mixture and forming a cell aggregate on a substrate; and (C) culturing the cells to obtain a cell construct. After performing steps (A) and (B) at least once and then performing step (C), a cell construct having cancer cells present on the outer surface of the endothelial cell layer is produced by seeding and culturing cancer cells on the three-dimensional structure. The cells in (A) include at least vascular endothelial cells but do not include cancer cells, and the ratio of the number of the vascular endothelial cells to the total number of cells other than the vascular endothelial cells is 6% or more.

Effects of the Invention

[0009] The cell construct according to the present invention has a unique structure in which an endothelial cell layer is exposed on the surface and cancer cells are present on the surface of a three-dimensional structure in which cells other than endothelial cells, such as stromal cells, are covered by the endothelial cell layer. Therefore, the cell construct according to the present invention is suitably used as a model of cancer cells present in blood vessels and lymphatic vessels for elucidating the behavior and functions of cancer cells in blood vessels and evaluating medicinal components such as anticancer agents. In addition, the cell construct according to the present invention can be efficiently produced by the method for producing the cell construct.

Brief Description of the Drawings

[0010] [Figure 1]The images show the CD31 staining and EpCAM staining of each cell structure produced from cells with an NE ratio (ratio of GFP-HUVEC to NHDF) of 10% (left column) and 1.5% (right column) in Example 1. [Modes for carrying out the invention]

[0011] In this embodiment and in this specification, "cellular structure" refers to a three-dimensional structure in which multiple cell layers are stacked. A "cell layer" is a layer 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 with the thickness direction, when observed in a section image of the cellular structure in the thickness direction at a magnification that allows for the recognition of cell nuclei, that is, at a magnification that allows the entire thickness of the stained section to be in the field of view. Furthermore, "layered" means that two or more different cell layers are stacked in the thickness direction.

[0012] <Cell structure> A cell structure according to one embodiment of the present invention is a cell structure having an endothelial cell layer made up of vascular endothelial cells formed on its surface, with cells other than vascular endothelial cells (excluding cancer cells) present inside, and cancer cells present on the outer surface of the endothelial cell layer.

[0013] In conventional cell structures containing a vascular network and cancer cells, blood vessels are formed in a network within the stromal cell layer, and cancer cells reside within the stromal cell layer. It is difficult to construct cell structures in which cancer cells reside within blood vessels, and therefore, it has been difficult to analyze the behavior and function of cancer cells within blood vessels. In contrast, the cell structure according to the present invention has a structure surface covered with vascular endothelial cells, and is an inverted version of a conventional cell structure containing a vascular network, with cancer cells adhering to the vascular endothelial cell layer on the surface of the structure. Thus, in the cell structure according to the present invention, cancer cells that normally reside in or near the endothelial cell layer inside blood vessels are present on or near the surface of the cell structure, making analysis easy. For this reason, the cell structure according to the present invention is suitable as a cell model for analyzing and evaluating the physiological phenomena of blood vessels and cancer cells mediated through them. Examples of such physiological phenomena include the invasion of cancer cells and the intravascular behavior of free cancer cells (CTCs) in the blood, as well as the physiological phenomena of cancer cells on the basement membrane of endothelial cells.

[0014] The endothelial cells included in the cell structure according to the present invention may be vascular endothelial cells or lymphatic endothelial cells. Furthermore, it may include both vascular endothelial cells and lymphatic endothelial cells.

[0015] The cell types of cells other than endothelial cells included in the cell structure according to the present invention are not particularly limited, as long as they do not hinder the endothelial cells from forming the surface layer of the cell structure in close proximity to each other. The cells other than endothelial cells in the cell structure according to the present invention are preferably cells that constitute the surrounding tissue of blood vessels in vivo, as this allows the endothelial cell layer to easily maintain its original function. Such cells are preferably stromal cells, more preferably one or more selected from the group consisting of fibroblasts, immune cells, and mast cells, and even more preferably one or more selected from the group consisting of fibroblasts, dendritic cells, macrophages, and fibroblasts. The cells other than endothelial cells included in the cell structure may be one type or two or more types. The cells other than endothelial cells included in the cell structure may be cells derived from the same species as the endothelial cells, or cells derived from a different species.

[0016] The cell structure according to the present invention preferably includes at least fibroblasts as cells other than endothelial cells, and more preferably includes vascular endothelial cells and fibroblasts, lymphatic endothelial cells and fibroblasts, or vascular endothelial cells, lymphatic endothelial cells and fibroblasts. In particular, the cell structure according to the present invention preferably consists of vascular endothelial cells and fibroblasts, lymphatic endothelial cells and fibroblasts, or vascular endothelial cells, lymphatic endothelial cells and fibroblasts.

[0017] The cell structure according to the present invention may include cells other than endothelial cells, such as stromal cells and cancer cells. Examples of such cells include epithelial cells, nerve cells, cardiomyocytes, hepatocytes, pancreatic islet cells, mesenchymal stem cells, smooth muscle cells, and osteocytes.

[0018] The cell structure according to the present invention has cancer cells present on the surface of a three-dimensional structure covered with an endothelial cell layer composed of vascular endothelial cells. The cancer cells may be a cell layer that covers at least a portion of the endothelial cell layer, but it is preferable that they exist as a cell aggregate. A cancer cell aggregate that forms a three-dimensional structure similar to that in vivo is a better model of cancer tissue in vivo than a cancer cell layer (Non-Patent Literature 1).

[0019] Whether or not cancer cells present on the outer surface of the endothelial cell layer of the cell structure according to the present invention form a cell aggregate can be determined by the cross-sectional shape in the thickness direction of the cell structure. The cross-sectional shape in the thickness direction of the cell structure of the aggregate of cancer cells in the cell structure can be determined from the cell image of a section in the thickness direction of the cell structure. The ellipticity of the cross-sectional shape in the thickness direction of the cell structure of the aggregate of cancer cells in the cell structure is larger for layered cells and smaller as the cells become more aggregated. For the shape of cancer cells present on the outer surface of the endothelial cell layer of the cell structure according to the present invention, it is preferable that the ellipticity of the cross-sectional shape in the thickness direction of the cell structure is 3.0 or less.

[0020] Furthermore, the ellipticity of the cross-sectional shape of cancer cells in a cell structure in the thickness direction can be determined by using image analysis software such as ImageJ on a stained image of cancer cells in a section of the cell structure in the thickness direction. In addition, in the present invention and this specification, the ellipticity of cancer cells in a cell structure refers to the ratio of the major axis to the minor axis (major axis / minor axis ratio) of the smallest ellipse that encloses the aggregate of cancer cells in the section image in the thickness direction of the cell structure.

[0021] Cancer cells are cells that have evolved from somatic cells and acquired unlimited proliferative capacity. Examples of cancers from which cancer cells originate include breast cancer (e.g., invasive ductal carcinoma, non-invasive ductal carcinoma, 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 cell 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.), and colorectal cancer. Cancer (e.g., familial colorectal cancer, hereditary nonpolyposis colorectal cancer, gastrointestinal stromal tumors, etc.), small intestine cancer (e.g., non-Hodgkin lymphoma, gastrointestinal stromal tumors, 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 tumor (e.g., pineal astrocytoma, pilocytic astrocytoma, diffuse astrocytoma, anaplastic astrocytoma, etc.), schwannoma, 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, low-grade ovarian tumor, etc.), bladder cancer, urethral cancer, skin cancer (e.g., intraocular melanoma, Merkel cell carcinoma, etc.), hemangioma, malignant lymphoma (e.g., reticulum sarcoma, lymphosarcoma, Hodgkin's disease, etc.), melanoma (malignant melanoma), thyroid cancer (e.g., medullary thyroid carcinoma, etc.), parathyroid cancer, nasal cavity This includes, but is not limited to, cancer, sinus cancer, bone tumors (e.g., osteosarcoma, Ewing's tumor, uterine sarcoma, soft tissue sarcoma, etc.), metastatic medulloblastoma, angiofibroma, dermatofibrosarcoma protuberance, retinal sarcoma, penile cancer, testicular tumors, pediatric solid tumors (e.g., Wilms' tumor, pediatric renal tumors, etc.), Kaposi's sarcoma, Kaposi's sarcoma caused by AIDS, maxillary sinus tumors, fibrous histiocytoma, leiomyosarcoma, rhabdomyosarcoma, chronic myeloproliferative disorders, leukemia (e.g., acute myeloid leukemia, acute lymphoblastic leukemia, etc.), etc.

[0022] The type of cells constituting the cell structure according to the present invention is not particularly limited, and may be cells collected from animals, cells obtained by culturing cells collected from animals, cells obtained by subjecting cells collected from animals to various treatments, or cultured cell lines. In the case of cells collected from animals, the collection site is not particularly limited, and may be somatic cells derived from bone, muscle, internal organs, nerves, brain, bone, skin, blood, etc., germ cells, embryonic stem cells (ES cells), or tissue stem cells. Further, the biological species from which the cells constituting the cell structure according to the present invention are derived is not particularly limited, and for example, cells derived from animals such as humans, monkeys, dogs, cats, rabbits, pigs, cows, mice, rats, etc. can be used. The cells obtained by culturing cells collected from animals may be primary cultured cells or subcultured cells. Further, examples of the cells subjected to various treatments include induced pluripotent stem cells (iPS cells) and cells after differentiation induction. Further, the cell structure according to the present invention may be composed only of cells derived from the same biological species, or may be composed of cells derived from a plurality of biological species.

[0023] The number of vascular endothelial cells in the cell structure according to the present invention is preferably a number such that the ratio of the number of vascular endothelial cells to the total number of cells other than vascular endothelial cells and cancer cells among all the cells constituting the cell structure is 6% or more. Specifically, when the total number of cells constituting the cell structure is N A , the number of vascular endothelial cells is N E , and the number of cancer cells is N C , then N E / (N A - N C - N E ) × 100 (%) (hereinafter sometimes referred to as the N E ratio) is preferably 6% or more. When the N E ratio is 6% or more, the abundance ratio of vascular endothelial cells and other cells becomes appropriate, and a three-dimensional structure in which a layer of vascular endothelial cells encloses other cell populations can be formed. When the N E ratio is about 1 to 3%, a cell structure in which a conventional vascular network is formed can be obtained, but N EWhen the ratio exceeds 5%, aggregation of vascular endothelial cells likely becomes more frequent, making vascular formation difficult.

[0024] The number of vascular endothelial cells in the cell structure according to the present invention is N E While there are no particular limitations as long as the cell count is 6% or more, vascular endothelial cells tend to form layers on the surface of structures, N E A cell count of 10-40% is preferred, N E A cell count that results in a ratio of 10-3.0% is more preferable. E A cell count that accounts for 10-25% is even more preferable, N E A cell count that accounts for 10-20% is even more preferable.

[0025] The total number of cells constituting the cell structure according to the present invention is not particularly limited, but it is preferable that the number of cells be such that the theoretical number of cell layers is 15 to 25, in terms of the ease with which a structure is formed in which layers of vascular endothelial cells contain other cell populations, and the ease with which the formed cell structure is observed. The theoretical number of cell layers is expressed by the following formula. In the formula, "N A " is the total number of cells that make up the cellular structure, "N S " represents the number of cells per layer when constructing a cell structure using the same type of cells on the same type of substrate, and "L" represents the theoretical number of cell layers.

[0026] [L] = [N] A ] / [N S ]

[0027] N S This can be determined experimentally. For example, using a 96-well plate as the substrate, 0.9 × 10 6 Individual NHDF and 0.0135 × 10 6 When a cell suspension is prepared using HUVEC, the constructed cell structure has approximately 20 nuclei stacked vertically. Therefore, in the case of a cell structure constructed using NHDF and HUVEC with a 96-well plate as the substrate, N A is 0.45 × 10 5 Let's consider them as one.

[0028] Generally, the cell structure according to the present invention is constructed in a cell culture vessel. The cell culture vessel is not particularly limited as long as it is capable of constructing the cell structure and culturing the constructed cell structure.

[0029] <Method for manufacturing cell structures> A method for producing a cell structure according to one embodiment of the present invention is a method for producing a cell structure in which a three-dimensional structure (i.e., a three-dimensional structure covered with an endothelial cell layer made of vascular endothelial cells) is produced, having an endothelial cell layer made of vascular endothelial cells formed on its surface and containing cells other than vascular endothelial cells (excluding cancer cells) inside, and then cancer cells are seeded onto the three-dimensional structure and cultured to produce a cell structure in which cancer cells are present on the outer surface of the endothelial cell layer. The three-dimensional structure having an endothelial cell layer made of vascular endothelial cells formed on its surface and containing cells other than vascular endothelial cells (excluding cancer cells) inside is produced by performing steps (A) and (B) at least once, and then performing step (C). (A) A step of obtaining a mixture in which cells are suspended in a solution containing at least a cationic substance, extracellular matrix components, and a polyelectrolyte, (B) A step of collecting the cells from the obtained mixture and forming a cell aggregate on a substrate, (C) A step of culturing the cells to obtain a cell structure.

[0030] In this specification and the present application, “cell aggregate” means a group of cells. Cell aggregates also include cell precipitates (aggregates of cells formed by precipitating cells) obtained by centrifugation or filtration. In some embodiments, the cell aggregate is a slurry-like viscous substance. In this specification, “slurry-like viscous substance” refers to a gel-like cell aggregate as described in Akihiro Nishiguchi et al., Macromol Biosci. 2015 Mar;15(3):312-7.

[0031] Step (A) is carried out by suspending cells to be used to construct a cell structure in a solution (hereinafter sometimes referred to as "cell suspension solution") containing at least a cationic substance, extracellular matrix components, and a polyelectrolyte. The cell population suspended in the cell suspension solution may be all the cells used to construct a three-dimensional structure covered with an endothelial cell layer consisting of vascular endothelial cells, or only a portion thereof. If the cell population suspended in the cell suspension solution is all the cells used to manufacture the three-dimensional structure, it may include vascular endothelial cells and cells other than vascular endothelial cells, and N E It is preferable to obtain a mixture by suspending a cell population in a cell suspension solution in a proportion of 6% or more, preferably 10-25%. The cells used in the method according to this embodiment can be the same cells as those that constitute the cell structure according to the present invention.

[0032] In this embodiment, any positively charged substance can be used as the cationic substance, as long as it does not adversely affect cell growth and cell aggregate formation. Examples of cationic substances include cationic buffers such as Tris-HCl buffer, Tris-maleate buffer, bis-Tris-buffer, and HEPES, as well as ethanolamine, diethanolamine, triethanolamine, polyvinylamine, polyallylamine, polylysine, polyhistidine, and polyarginine. In this embodiment, the cationic substance is preferably a cationic buffer.

[0033] The concentration of the cationic substance is not particularly limited, as long as it does not adversely affect cell growth and cell aggregate formation. The concentration of the cationic substance used in this embodiment is preferably 10 to 100 mM. For example, the concentrations of the cationic substance used in this embodiment are preferably 20 to 90 mM, 3.0 to 80 mM, 40 to 70 mM, and 45 to 60 mM. A concentration of 50 mM is more preferable.

[0034] 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 aggregate formation. 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.

[0035] In the present invention and this specification, "polymer electrolyte" means a polymer having dissociable functional groups in its polymer chain. Any polymer electrolyte can be used as the polymer electrolyte in this embodiment, as long as it does not adversely affect cell growth and the formation of cell structures. Examples of polymer electrolytes include, but are not limited to, heparin, glycosaminoglycans such as 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, and polyacrylamide-2-methylpropanesulfonic acid, polyacrylic acid, etc. The mixture prepared in step (A) may contain only one type of polymer electrolyte, or a combination of two or more types. In the production of cell structures according to the present invention, glycosaminoglycans are preferably used, and heparin and / or dextran sulfate are more preferably used. The amount of polyelectrolyte mixed into the cell suspension solution is not particularly limited, as long as it does not adversely affect cell growth and the production of cell structures. For example, the concentration of polyelectrolyte in the cell suspension solution may be greater than 0 mg / mL, preferably 0.010 mg / mL or more, more preferably 0.020 mg / mL or more, even more preferably 0.025 mg / mL or more, and even more preferably 0.05 mg / mL or more. Furthermore, the concentration of polyelectrolyte in the cell suspension solution may be less than 1.0 mg / mL, more preferably 0.75 mg / mL or less, even more preferably 0.5 mg / mL or less, even more preferably 0.25 mg / mL or less, and particularly preferably 0.1 mg / mL or less.

[0036] In this embodiment, any component constituting the extracellular matrix (ECM) can be used as the extracellular matrix component, as long as it does not adversely affect cell growth and cell aggregate formation. Examples include collagen, laminin, fibronectin, vitronectin, elastin, tenascin, entactin, fibrillin, proteoglycans, glycosaminoglycans, or their modifications or variants. Examples of proteoglycans include chondroitin sulfate proteoglycan, heparan sulfate proteoglycan, keratan sulfate proteoglycan, and dermatan sulfate proteoglycan. Examples of glycosaminoglycans include hyaluronic acid, chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate, keratan sulfate, heparan sulfate, and heparin. The mixture prepared in step (A) may contain only one type of extracellular matrix component, or a combination of two or more types. In the production of cell structures according to the present invention, it is preferable to use one or more selected from the group consisting of collagen, laminin, and fibronectin, with collagen being particularly preferred. The amount of extracellular matrix component mixed into the cell suspension solution is not particularly limited as long as it does not adversely affect cell growth and the production of cell structures. For example, the concentration of the extracellular matrix component in the cell suspension solution may be greater than 0 mg / mL, preferably 0.010 mg / mL or more, more preferably 0.020 mg / mL or more, even more preferably 0.025 mg / mL or more, and even more preferably 0.05 mg / mL or more. Furthermore, the concentration of the extracellular matrix component in the cell suspension solution may be less than 1.0 mg / mL, more preferably 0.75 mg / mL or less, even more preferably 0.5 mg / mL or less, even more preferably 0.25 mg / mL or less, and particularly preferably 0.1 mg / mL or less.

[0037] The mixing ratio of the polymer electrolyte and extracellular matrix component to be mixed in the cell suspension solution is 1:2 to 2:1. In the production of cell structures according to the present invention, the mixing ratio of the polymer electrolyte and extracellular matrix component is preferably 1:1.5 to 1.5:1, and more preferably 1:1.

[0038] In the cell structure according to the present invention, the production of a three-dimensional structure covered with an endothelial cell layer consisting of vascular endothelial cells involves performing steps (A) and (B) at least once, followed by step (C). By repeating steps (A) to (C), specifically by seeding the mixture prepared in step (A) as step (B) onto the cell structure obtained in step (C), and then repeating step (C), a three-dimensional structure of sufficient thickness can be constructed. The cell composition of the mixture newly seeded onto the three-dimensional structure obtained in step (C) may be the same as or different from the cell composition constituting the already constructed three-dimensional structure.

[0039] The method according to this embodiment includes, after step (A), (A'-1) a step of removing the liquid portion from the obtained mixture to obtain a cell aggregate, and (A'-2) a step of suspending the cell aggregate in a solution, and instead of step (B), a step of (B') of precipitating cells from the obtained suspension to form a cell precipitate on a substrate.

[0040] In the method of this embodiment, the mixing of cells and the cell suspension solution may be carried out in a suitable container such as a dish, tube, flask, bottle, or plate, or on the substrate used in step (B). The suspension in step (A'-2) may also be carried out in a suitable container such as a dish, tube, flask, bottle, or plate, or on the substrate used in step (B').

[0041] In the method of this embodiment, methods known to those skilled in the art can be used as means for removing the liquid portion in step (A'-1). For example, the liquid portion may be removed by centrifugation or filtration. The conditions for centrifugation are not particularly limited, as long as they do not adversely affect cell growth and cell aggregate formation. For example, the liquid portion may be removed by separating the liquid portion from the cell aggregate by centrifugation of a microtube containing the mixture at room temperature and 400 × g for 1 minute. Alternatively, the liquid portion may be removed after the cells have been collected by natural sedimentation.

[0042] The solution used in step (A'-2) of the above-described method of this embodiment is not particularly limited, as long as it does not adversely affect cell growth and cell aggregate formation. For example, a cell culture medium or buffer suitable for the cells being used may be used.

[0043] The substrate used in step (B) or (B') of the above method of this embodiment includes a culture vessel for cell culture. The culture vessel may be a container having a material and shape that is commonly used for culturing cells or microorganisms. Examples of materials for the culture vessel include, but are not limited to, glass, stainless steel, and plastic. Examples of culture vessels include, but are not limited to, dishes, tubes, flasks, bottles, and plates. The substrate is, for example, a material that does not allow cells in a liquid to pass through but allows liquid to pass through.

[0044] The substrate used in this embodiment is preferably a permeable membrane. Examples of containers having such a permeable membrane include, but are not limited to, cell culture inserts such as Transwell® inserts, Netwell® inserts, Falcon® cell culture inserts, and Millicell® cell culture inserts.

[0045] In the method of this embodiment, methods known to those skilled in the art can be used as means for collecting cells in step (B) or (B'). For example, cells may be collected by centrifugation, magnetic separation, or filtration. The conditions for centrifugation are not particularly limited as long as they do not adversely affect cell growth. For example, cells may be collected by seeding a mixture or suspension onto a cell culture insert and centrifugating at room temperature (25°C) at 400 × g for 1 minute. Alternatively, cells may be collected by natural sedimentation. In step (B'), a cell precipitate may be formed on the substrate by removing the liquid portion from the suspension, for example, by centrifugation or filtration. Alternatively, a cell precipitate may be formed on the substrate by natural sedimentation. The cell aggregate in step (B) or the cell precipitate in step (B') may be layered.

[0046] In the culture in step (C) of the method described above in this embodiment, one or more three-dimensional structures are formed on the substrate, each having an endothelial cell layer on its surface composed of vascular endothelial cells and containing cells other than vascular endothelial cells internally. The cell culture in step (C) can be carried out under culture conditions suitable for the cells to be cultured. Those skilled in the art can select an appropriate culture medium according to the type of cell and the desired function. Various conditions such as culture temperature and culture time can also be easily determined by those skilled in the art.

[0047] The culture medium used for culturing in step (C) is not particularly limited as long as it is a medium in which cells constituting the three-dimensional structure can grow. However, a medium with a low or no content of growth factors such as epidermal growth factor (EGF), vascular epithelial growth factor (VEGF), fibroblast growth factor (FGF), and insulin-like growth factor (IGF), or a medium with a low or no serum content is preferred. If the content of various growth factors is high, vascular formation may be promoted. In this embodiment, a medium without added growth factors is preferred, and a serum-free medium is more preferred.

[0048] By seeding cancer cells onto a three-dimensional structure covered with an endothelial cell layer made of vascular endothelial cells produced in step (C) and culturing them, a cell structure is obtained in which cancer cells adhere to the endothelial cell layer on the surface of the three-dimensional structure. Seeding of cancer cells is preferably performed after culturing for at least 24 hours in step (C). The number of cancer cells to be seeded is not particularly limited, but for example, 100 or more, preferably 500 or more, per container forming the three-dimensional structure is preferred. Furthermore, since cancer cells seeded on the three-dimensional structure tend to form cell aggregates, the number of cancer cells to be seeded is N S / 2(N S (The number of cells per layer when constructing a cell structure with the same type of cells on the same type of substrate is preferably less than or equal to N S / 4 or fewer is more preferable, N S / 10 or fewer is even more preferable. [Examples]

[0049] The present invention will be described in more detail and specifically below with reference to examples, but these examples are not intended to limit the scope of the present invention.

[0050] [Example 1] Human umbilical vein endothelial cells labeled with the fluorescent protein GFP (GFP-HUVEC) (model number: cAP-0001GFP, Funakoshi Co., Ltd.) were used as vascular endothelial cells, human neonatal-derived dermal fibroblasts (NHDF) (model number: CC-2509, Lonza Co., Ltd.) were used as stromal cells, and the colorectal cancer cell line HT29 (model number: HTB-38, ATCC Co., Ltd.) was used as cancer cells. Cell structures were prepared in which NHDF was coated with GFP-HUVEC. In manufacturing the cell structures, heparin (Heparin sodium salt from porcine intestinal mucosa Grade IA) (model number: H3393-100KU, Sigma Co., Ltd.) was used as the polyelectrolyte, and collagen (Collagen Type I, Bovine Skin, Acid Soluble) (model number: ASC-1-100-100, NIP Co., Ltd.) was used as the extracellular matrix component.

[0051] First, a cell suspension solution was prepared by mixing equal volumes of 0.1 mg / mL heparin / 50 mM Tris-HCl buffer (pH 7.4) and 0.1 mg / mL collagen / acetic acid solution (pH 3.7) (i.e., the final concentrations of collagen and heparin were each 0.05 mg / mL). Next, a cell population mixed with GFP-HUVEC at a ratio of 1.5, 3, 4.5, 6, 10, 15, or 20% relative to NHDF was suspended in this cell suspension solution to prepare a cell mixture. Then, the cell mixture was centrifuged at room temperature at 15000 rpm for 5 minutes, the supernatant was removed, and culture medium was added to prepare 20-layer cell suspensions and 30-layer cell suspensions. The 20-layer cell suspension contained 0.9 × 10⁶ NHDF per 135 μL. 6 The amount of cells included was adjusted so that the total number of cells equals 20 theoretical cell layers. The cell suspension for 30 layers contains 1.35 × 10¹⁶ NHDF per 135 μL. 6 The amount was adjusted so that it contained 30 cells and the total number of cells equaled 30 theoretical cell layers. The culture medium used was 10% FBS-containing high-glucose D-MEM (model number: 043-3.0085, Wako) supplemented with penicillin-streptomycin solution (×100) (model number: 168-23191, Wako).

[0052] Next, 135 μL of either a 20-layer or 30-layer cell suspension was dispensed into a Transwell insert-integrated 96-well (0.4 μm, model number: 7369, Corning) and the cells were seeded. After that, the culture medium outside the insert was changed, and the cells were cultured for 24 hours in a CO2 incubator (37°C, 5% CO2). Subsequently, HT29 cancer cells were added, with 1000 cells dispensed into each well and the cells were seeded. After that, the culture medium outside the insert was changed, and the cells were cultured in a CO2 incubator (37°C, 5% CO2). The start day of culture (the day the cell suspension was seeded into the wells) was defined as day 0 of culture. Cancer cells were seeded on day 1 of culture, and the culture medium was changed on days 2, 5, and 8 of culture. GFP fluorescence images were taken from the top of each well of the cellular structure after the culture medium change on day 8 of culture. Subsequently, after fixing each cell structure with formalin, sections were prepared perpendicular to the bottom surface of the insert. These sections were then stained with EpCAM (immunostaining using anti-EpCAM antibody) and CD31 (immunostaining using anti-CD31 antibody) to examine the localization of cancer cells and HUVEC cells.

[0053] From the GFP imaging results of each cell structure, in a cell structure constructed with a total number of NHDF and GFP-HUVEC cells equaling 20 theoretical cell layers, the ratio of GFP-HUVEC cells to NHDF cells (N E When the ratio was between 1.5% and 4.5%, GFP images confirmed that GFP-HUVECs were present in a tubular structure, i.e., forming a vascular network. CD31 staining of the sections also showed that the surface of tubular spaces within the cellular structure was stained, confirming that GFP-HUVECs were localized on the surface of these tubes. In contrast, N E In cell structures where the proportion was 6% or more, GFP-HUVEC was present throughout the entire cell structure, and no vascular network formation was observed. CD31 staining of the sections confirmed that almost all unstained cells (NHDF) were covered by stained cells (GFP-HUVEC). E The left column of Figure 1 shows stained images of CD31 staining and EpCAM staining at a ratio of 10%. EThe staining images for 1.5% CD31 and EpCAM staining are shown in the right column of Figure 1, respectively. Similar results were observed in cell structures constructed with a total number of NHDF and GFP-HUVEC cells equaling 30 theoretical cell layers.

[0054] The upper row of Table 1 shows whether or not a structure in which NHDF was coated with GFP-HUVEC was observed in each cell structure. In the table, "○" indicates that a structure in which NHDF was coated with GFP-HUVEC was observed, and "×" indicates that the formation of a vascular network was observed.

[0055] [Table 1]

[0056] From these results, N E It was found that by setting the ratio to 6% or more, it is possible to produce a cellular structure in which an endothelial cell layer consisting of vascular endothelial cells is formed on the surface, and stromal cells are present inside.

[0057] Furthermore, the localization of cancer cells was investigated from EpCAM-stained images of each cell structure. Additionally, the ellipticity of cancer cells in each EpCAM-stained image was determined using image analysis software "ImageJ". The results of measuring the ellipticity of cancer cells in each cell structure are shown in the lower section of Table 1.

[0058] As a result, in a cell structure constructed with a total number of NHDF and GFP-HUVEC cells equal to 20 theoretical cell layers, N E In cell structures with a ratio of 1.5-4.5%, cancer cells were present in layers (ellipticity of 5.6 or higher) on the top surface of a three-dimensional structure composed of NHDF and GFP-HUVEC, which had a vascular network formed inside. On the other hand, N E Cancer cell aggregates (with an ellipticity of 3.0 or less) were formed on the surface of three-dimensional structures coated with GFP-HUVECs containing NHDF at a ratio of 6% or more. Similar results were observed in cell structures constructed with a total number of NHDF and GFP-HUVEC cells equivalent to 30 theoretical cell layers.

Claims

1. An endothelial cell layer consisting of vascular endothelial cells is formed on the surface, and cells other than the vascular endothelial cells (excluding cancer cells) are present inside. Cancer cells are present as a cell cluster on the outer surface of the endothelial cell layer. A cell structure in which the ellipticity of the cross-sectional shape in the thickness direction of the cell structure of the cell aggregate is 3.0 or less.

2. The cell structure according to claim 1, wherein the interior contains at least stromal cells.

3. The cell structure according to claim 2, wherein the stromal cells are one or more selected from the group consisting of fibroblasts, dendritic cells, macrophages, and mast cells.

4. The cell structure according to any one of claims 1 to 3, wherein the inside contains one or more cells selected from the group consisting of epithelial cells, nerve cells, cardiomyocytes, hepatocytes, islet cells, smooth muscle cells, mesenchymal stem cells, and osteocytes.

5. The cell structure according to any one of claims 1 to 4, wherein the ratio of the number of vascular endothelial cells to the total number of cells other than vascular endothelial cells and cancer cells among all cells constituting the cell structure is 6% or more.

6. A cell structure according to any one of claims 1 to 5, which is a model of cancer cells present in a blood vessel.

7. A method for producing cell structures, A three-dimensional structure having an endothelial cell layer formed on its surface consisting of vascular endothelial cells, and containing cells other than the vascular endothelial cells (excluding cancer cells) inside, (A) A step of obtaining a mixture in which cells are suspended in a solution containing at least a cationic substance, extracellular matrix components, and a polyelectrolyte, (B) A step of collecting the cells from the obtained mixture and forming a cell aggregate on a substrate, (C) A step of culturing the cells to obtain a cell structure, The method includes, and after performing steps (A) and (B) at least once, then performing step (C), By seeding cancer cells onto the three-dimensional structure and culturing them, a cell structure is produced in which cancer cells exist on the outer surface of the endothelial cell layer as cell aggregates, the ellipticity of the cross-sectional shape in the thickness direction of the cell structure being 3.0 or less. The cells in (A) above include at least vascular endothelial cells but do not include cancer cells, and the ratio of the number of vascular endothelial cells to the total number of cells other than vascular endothelial cells is 6% or more. A method for producing cell structures.

8. The method for producing a cell structure according to claim 7, wherein the cells in (A) above include at least stromal cells.

9. The method for producing a cell structure according to claim 8, wherein the stromal cells are one or more selected from the group consisting of fibroblasts, dendritic cells, macrophages, and mast cells.

10. A method for producing a cell structure according to any one of claims 7 to 9, wherein the total number of cells in (A) is the number of cells such that the theoretical number of cell layers is 15 to 25.

11. A cell structure having an endothelial cell layer made of vascular endothelial cells formed on its surface, cells other than vascular endothelial cells (excluding cancer cells) present inside, and cancer cells present on the outer surface of the endothelial cell layer as a cell mass having an ellipticity of 3.0 or less in the cross-sectional shape in the thickness direction of the cell structure, manufactured by the method for manufacturing a cell structure according to any one of claims 7 to 10. A method for using the obtained cell structures to elucidate the behavior or function of cancer cells within blood vessels, or to evaluate the active ingredients of anticancer drugs.

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