Method for producing cell structure

Abstract

The present invention pertains to a method for producing a cell structure, said method comprising a step for culturing a plurality of types of cells including at least epithelial cells and vascular endothelial cells in a culture medium containing one or more types of growth factors involved in angiogenesis.

Description

Technical Field

[0001] The present invention relates to a method for producing a cell structure. Background Art

[0002] Various studies have been conducted so far on methods for manufacturing cell structures having blood vessels (for example, Patent Document 1). Patent Document 1 discloses a method for manufacturing a three-dimensional cell culture having a microvascular-like structure, which includes specified steps 1 to 3. Step 1 in the manufacturing method is a step of inoculating fibroblasts that have been subjected to oscillation treatment into a culture container to form a first fibroblast layer, step 2 is a step of adding collagen IV to the first fibroblast layer, then inoculating vascular endothelial cells for culture, and forming a vascular-like structure layer on the first fibroblast layer, and step 3 is a step of inoculating fibroblasts that have been subjected to oscillation treatment together with VEGF and FGF into the vascular-like structure layer to form a second fibroblast layer with a vascular-like structure extending upward from the vascular-like structure layer.

[0003] Furthermore, for example, Patent Document 2 discloses a biologically active placenta-derived liquid matrix (hpS) containing extracellular matrix (ECM) proteins, cytokines, and growth factors. It also describes that in an experiment in which HUVECs were seeded on the hpS, HUVECs were detected to aggregate into interconnected cell networks (angiogenesis).

[0004] Prior art literature

[0005] Patent Literature

[0006] Patent Document 1: Japanese Patent Application Laid-Open No. 2017-176025

[0007] Patent Document 2: Japanese Patent Application No. 2022-527648 Summary of the Invention

[0008] Problems to be solved by the invention

[0009] The present inventors have discovered the following new problems in conventional liver models: when a part of non-parenchymal cells (hepatic stellate cells) is present to further improve liver function through co-culture, the liver function of the liver model cannot be maintained, and when hepatic stellate cells are absent, blood vessels are not formed (see Test Example 3 and Test Example 1). In addition, even if blood vessels are formed in a liver model without hepatic stellate cells, the blood vessels may spread to the outside of the cell structure during culture (see Reference Examples, Figure 7 Thus, it cannot be said that sufficient research has been conducted so far on methods for producing cell structures containing epithelial cells and having blood vessels that do not diffuse to the outside.

[0010] Therefore, an object of the present invention is to provide a method for producing a cell structure comprising epithelial cells and having formed blood vessels that do not proliferate to the outside.

[0011] Means for solving problems

[0012] The present inventors have discovered that by culturing hepatocytes and vascular endothelial cells in a culture medium containing VEGF-A, a cell structure with blood vessels that do not diffuse to the outside can be produced. Based on this finding, it is believed that the invention can also be applied to the production of cell structures containing epithelial cells and with blood vessels that do not diffuse to the outside. The present invention is based on this new finding.

[0013] That is, the present invention includes the following inventions. [1]

[0015] A method for producing a cell structure comprises the step of culturing a plurality of cells including at least epithelial cells and vascular endothelial cells in a culture medium containing one or more growth factors involved in angiogenesis. [2]

[0017] The method according to [1], wherein

[0018] The above culture medium contains two or more growth factors involved in angiogenesis,

[0019] The two or more growth factors involved in angiogenesis include angiogenic factors and angiogenic maintenance factors. [3]

[0021] The method according to [1] or [2], wherein

[0022] The content of the growth factor involved in angiogenesis in the culture medium is 10 ng / mL or more. [4]

[0024] The method according to any one of [1] to [3], wherein

[0025] The above-mentioned culturing step is performed under conditions in which the cell structure can take up the medium components from all directions. [5]

[0027] The method according to any one of [1] to [4], wherein

[0028] The above-mentioned epithelial cells are hepatocytes, and the above-mentioned cells do not include hepatic stellate cells. [6]

[0030] The method according to any one of [1] to [5], wherein

[0031] Prior to the above-mentioned culturing step, a step of obtaining a mixture containing the above-mentioned plurality of cells and extracellular matrix components is provided. [7]

[0033] The method according to [6], wherein

[0034] The above-mentioned extracellular matrix components include collagen components. [8]

[0036] The method according to [6] or [7], wherein

[0037] The above mixture further contains a polymer electrolyte. [9]

[0039] The method according to [8], wherein

[0040] The above-mentioned polymer electrolyte contains heparin.

[0041] Effects of the Invention

[0042] According to the present invention, a method for producing a cell structure containing epithelial cells and having formed blood vessels that do not diffuse to the outside can be provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 These are images showing the results of evaluating angiogenesis in cell structures.

[0044] Figure 2 These are images showing the results of evaluating angiogenesis in cell structures.

[0045] Figure 3 These are images showing the results of evaluating angiogenesis in cell structures.

[0046] Figure 4 This is a graph showing the results of analyzing the length of blood vessels in cell structures.

[0047] Figure 5 This is a graph showing the results of analyzing the expression levels of the CYP3A4 gene and the CYP2C9 gene in cell structures.

[0048] Figure 6 These are images showing the results of evaluating angiogenesis in cell structures.

[0049] Figure 7 These are images showing the results of evaluating angiogenesis in cell structures.

[0050] Figure 8 These are images showing the results of observing the vascular structure in a cell structure.

[0051] Figure 9 These are images showing the results of observing the vascular structure in a cell structure.

[0052] Figure 10 This is a graph showing the results of analyzing the length of blood vessels in cell structures.

[0053] Figure 11 This is a graph showing the results of analyzing the survival rate of cell structures.

[0054] Figure 12 This is a graph showing the results of analyzing CYP3A4 activity in cell structures. DETAILED DESCRIPTION

[0055] Hereinafter, the embodiment for implementing the present invention will be described in detail. However, the present invention is not limited to the following embodiment.

[0056] [Method for producing cell structure]

[0057] In this specification, a "cell structure" refers to a cell aggregate (a cell mass) in which multiple cells are arranged three-dimensionally, and is an aggregate artificially produced by cell culture. The cell structure may contain only one type of cell or two or more types of cells.

[0058] The shape of the cell structure is not particularly limited, and examples thereof include spherical, approximately spherical, ellipsoidal, approximately ellipsoidal, hemispherical, approximately hemispherical, semicircular, approximately semicircular, rectangular, and approximately rectangular. Here, biological tissues include sweat glands, lymphatic vessels, and fat glands, and are more complex than cell structures. Therefore, it is easy to distinguish between cell structures and biological tissues. In addition, the cell structure can be assembled into a block in a state attached to a support, or it can be assembled into a block in a state not attached to a support.

[0059] The method for producing a cell structure according to this embodiment comprises culturing a plurality of cells comprising at least epithelial cells and vascular endothelial cells in a culture medium containing one or more growth factors involved in angiogenesis (culturing step). As used herein, "culturing cells" means maintaining the cells in the culture medium under conditions that prevent their death or decline. Culturing cells may or may not be accompanied by cell proliferation.

[0060] The method for producing a cell structure according to the present embodiment can produce a cell structure comprising epithelial cells and having blood vessels that do not diffuse to the outside by having the above-mentioned configuration. A "cell structure having blood vessels" refers to a cell structure having a tubular structure formed by vascular endothelial cells. Blood vessels can be confirmed by, for example, immunohistochemical staining, microscopic observation using vascular endothelial cells expressing fluorescent proteins, etc. The formation of blood vessels can be, for example, the formation of blood vessels to the extent that multiple branch points and a mesh structure are observed when the cell structure is observed from the upper surface under a microscope.

[0061] A "cell structure having blood vessels that do not proliferate to the outside" may refer to a cell structure having blood vessels that do not separate from the cell structure even after 14 days of culture. Alternatively, as in the reference example described below, in which blood vessels separated from the cell structure were observed outside the cell structure, a cell structure may be formed in which, when the cell structure is observed from above under a microscope after 14 days of cell culture, no blood vessels separated from the cell structure are observed outside the cell structure.

[0062] In the culturing step, a plurality of cells including at least epithelial cells and vascular endothelial cells are cultured in a medium containing one or more growth factors involved in angiogenesis to obtain a cell structure including epithelial cells and having formed blood vessels that do not spread to the outside.

[0063] Epithelial cells are cells that form epithelial tissues present in the skin, digestive organs, blood vessels, etc. Examples of epithelial cells used in the culture step include hepatocytes, epidermal keratinocytes, renal epithelial cells, vascular epithelial cells, alveolar epithelial cells, intestinal epithelial cells, retinal epithelial cells, neural epithelial cells, gingival epithelial cells, bile duct epithelial cells, and thymic epithelial cells, with hepatocytes being preferred.

[0064] Hepatocytes are also referred to as hepatocytes, and are cells having functions such as bile secretion and plasma protein secretion. As the hepatocytes constituting the cell structure, it can be primary hepatocytes collected from the liver of an animal, it can be cells obtained by culturing primary hepatocytes, it can be a cultured cell line obtained by transforming primary hepatocytes into a line, or it can be hepatoblasts obtained by artificial differentiation of stem cells. As primary hepatocytes, primary human hepatocytes such as PXB cells can be mentioned. As cultured cell lines, cell lines derived from inactivated hepatoma cells such as HepG2 can be mentioned. As stem cells differentiated into hepatoblasts, embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), mesenchymal stem cells, etc. can be mentioned. As hepatocytes, it is preferably non-cancerous cells such as primary hepatocytes and hepatoblasts, and from the simplicity of processing, PXB cells are more preferred.

[0065] The epithelial cells used in the culture step may be one type or two or more types. For example, if the epithelial cells used in the culture step are hepatocytes, the hepatocytes may include a plurality of hepatocytes having different genotypes for proteins involved in liver function. Conversely, all cultured hepatocytes may have the same genotype for proteins involved in liver function. Examples of proteins involved in liver function include drug-metabolizing enzymes.

[0066] The ratio (X1 / X0×100) of the number of epithelial cells (X1) to the total number of the plurality of cells cultured in the culture step (X0) can be 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, or 65% or more, and can be 95% or less, 90% or less, 80% or less, or 75% or less. From the perspective of being more suitable as a cell structure containing epithelial cells, the ratio (X1 / X0×100) of the number of epithelial cells (X1) to the total number of multiple cells cultured in the culture step (X0) can be 60% or more and 80% or less, or 60% or more and 70% or less, or 60% or more and 95% or less, 65% or more and 95% or less, 65% or more and 90% or less, 75% or more and 90% or less, or 80% or more and 88% or less. When the epithelial cells are hepatocytes, the ratio of hepatocytes is preferably 50% or more, which is close to the conditions in a living body.

[0067] Vascular endothelial cells refer to flat cells that constitute the surface of the vascular lumen. The vascular endothelial cells in the culture process can be, for example, sinusoidal endothelial cells, human umbilical vein-derived vascular endothelial cells (HUVEC), etc. Sinusoidal endothelial cells are liver non-parenchymal cells (cells other than hepatocytes in the cells that constitute the liver), and are cells with characteristic morphologies different from other vascular endothelial cells such as a plurality of small pores (cribriform plate structure) in the cytoplasm and basement membrane defects. As the vascular endothelial cells in the culture process, it can be primary cells (primary vascular endothelial cells) collected from the liver of an animal (such as a human), or it can be cells obtained by culturing primary cells, or it can be a cultured cell line obtained by transforming primary cells into a cell line, or it can be a cell obtained by artificially differentiating from stem cells. The cultured cell line can be a cell line established by gene introduction, specifically, it can be a cultured cell line introduced with immortalization-inducing genes such as hTERT or SV40LT. As primary vascular endothelial cells, for example, primary sinusoidal endothelial cells such as product model 5000 made by Sciencell can be mentioned. When using a cultured cell line immortalized by gene transfer into primary sinusoidal endothelial cells, it is expected that the angiogenesis and maintenance effects brought about by growth factors involved in angiogenesis will be further improved. As a cultured cell line, for example, the cultured cell line of product model T0056 manufactured by Applied Biological Materials can be cited. As differentiated stem cells, embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), etc. can be cited. The vascular endothelial cells in the culture process can be non-cancerous cells.

[0068] The ratio (X2 / X0×100) of the number of vascular endothelial cells (X2) to the total number of the plurality of cells cultured in the culture step (X0) may be 5% or more, 10% or more, 12% or more, 14% or more, 15% or more, 20% or more, or 25% or more, and may be 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, or 18% or less. From the perspective of being more suitable as a cell structure having blood vessels, the ratio (X2 / X0×100) of the number of vascular endothelial cells (X2) to the total number of the plurality of cells cultured in the culture step (X0) may be 5% or more and 40% or less, 5% or more and 35% or less, 10% or more and 35% or less, 10% or more and 25% or less, or 12% or more and 20% or less, and may be 20% or more and 40% or less, or 30% or more and 40% or less.

[0069] The various cells in the culture step can include other cells other than epithelial cells and vascular endothelial cells within the scope of not damaging the effects of the present invention. Other cells, for example, can be mature somatic cells or undifferentiated cells such as stem cells. As specific examples of somatic cells, for example, nerve cells, dendritic cells, immune cells, lymphatic endothelial cells, fibroblasts, cardiomyocytes, pancreatic islet cells, smooth muscle cells, osteocytes, spleen cells, etc. can be enumerated. As stem cells, ES cells, iPS cells, mesenchymal stem cells, etc. can be enumerated. Other cells can be normal cells or any cells such as cancer cells with hyperfunction or suppressed cell function. Cancer cells refer to cells derived from somatic cells and having acquired unlimited proliferation ability.

[0070] Hepatic stellate cells are liver non-parenchymal cells (cells other than hepatocytes in the cells constituting the liver), have the function of storing vitamin A, and are present in the hepatocytes in the liver and the region between the sinusoids, i.e., the perisinusoidal space. All along, hepatic stellate cells have been considered to further improve liver function by co-culturing with hepatocytes in previous culture systems. However, it has been gradually clarified that when the hepatic stellate cells isolated from an organism, the cell strain set up from the hepatic stellate cells become activated state under an in vitro culture environment, and are cultured together with hepatocytes, the expression level of metabolic enzymes peculiar to hepatocytes as culture is carried out can significantly reduce, etc., and hepatic stellate cells have a negative effect, and when hepatic stellate cells are not included, blood vessels are not formed. Therefore, when the epithelial cells in the culture process are hepatocytes, as cells other than epithelial cells and vascular endothelial cells, it is preferably not included that hepatic stellate cells display an activated state. As cells displaying such an activated state, for example, hepatic stellate cell strains such as LX2 can be enumerated.

[0071] The origin of epithelial cells, vascular endothelial cells, or other cells contained in the cell structure is not particularly limited, and may be, for example, cells derived from mammals such as humans, monkeys, dogs, cats, rabbits, pigs, cows, mice, and rats.

[0072] The total number of the various cells (X0) in the culture step is not particularly limited and can be appropriately determined in consideration of the thickness and shape of the constructed cell structure, the size of the cell culture container used for construction, etc. The total number of the various cells cultured in the culture step (X0) can be 1×10 3 More than 1×10 cells 4 More than 1×10 cells 5 More than 1×10 cells, or 1×10 6 In addition, the total number of cells cultured in the culture step (X0) can be 1×10 9 Less than 1×10 8 Less than 1×10 7In addition, for example, the cell density in the culture medium during the culture step can be 10 3 ~10 7 cells / mL, or 10 4 ~10 6 cells / mL.

[0073] The culture conditions for the various cells in the culture step can be any conditions that prevent cell death or decline, and appropriate culture conditions can be set depending on the cell type. For example, the culture temperature can be 20°C to 40°C, or 30°C to 37°C. The pH of the culture medium can be 6 to 8, or 7.2 to 7.4. The culture time can be 1 day to 2 weeks, or 1 to 2 weeks.

[0074] The culture medium in the culture process contains one or more growth factors involved in angiogenesis. "Growth factors involved in angiogenesis" refer to growth factors that act at any stage in the process of forming new blood vessels from existing blood vessels or where there are no existing blood vessels, and maturing and maintaining them. "Growth factors" are a general term for endogenous proteins that promote the proliferation or differentiation of specific cells. Growth factors involved in angiogenesis can contain at least angiogenic factors or angiogenic maintenance factors. "Angiogenic factors" refer to growth factors involved in angiogenesis that act in the initial stage of angiogenesis, that is, growth factors that have the function of promoting the vascular network formation of vascular endothelial cells. "Vascular maintenance factors" will be described later.

[0075] The growth factors involved in angiogenesis contained in the culture medium may be 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, or 13 or more.

[0076] The culture medium in the culturing step preferably contains two or more growth factors involved in angiogenesis. In this case, the growth factors involved in angiogenesis more preferably contain angiogenic factors and vascular maintenance factors. "Vascular maintenance factors" refer to growth factors involved in angiogenesis that act in the later stages of angiogenesis, that is, in the stage of maintaining formed blood vessels. These are growth factors that have the effect of stabilizing and maintaining the vascular structure. When the culture medium in the culturing step contains angiogenic factors and vascular maintenance factors as growth factors involved in angiogenesis, in addition to forming blood vessels that do not spread to the outside of the cell structure, the decline of the formed blood vessels is also suppressed.

[0077] Examples of angiogenic factors include vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF). Examples of VEGF include VEGF-A to VEGF-E. Examples of FGF include FGF1 to FGF10.

[0078] Examples of vascular maintenance factors include angiopoietin (e.g., angiopoietin-1 to -4), insulin, hemopexin, epidermal growth factor (EGF), melanotransferrin, heparan sulfate proteoglycan (HSPG2), insulin-like growth factor-1 (IGF-1), and leucine-rich 2α glycoprotein 1 (LRG1). The vascular maintenance factor may be one or more selected from the above-mentioned vascular maintenance factors.

[0079] From the perspective of further promoting angiogenesis, the concentration of growth factors involved in angiogenesis in the culture medium may be, for example, 10 ng / mL or higher, 20 ng / mL or higher, 30 ng / mL or higher, 40 ng / mL or higher, 50 ng / mL or higher, 60 ng / mL or higher, 70 ng / mL or higher, 80 ng / mL or higher, 90 ng / mL or higher, or 100 ng / mL or higher. Furthermore, the concentration of growth factors involved in angiogenesis in the culture medium may be, for example, 1000 ng / mL or lower, 900 ng / mL or lower, 800 ng / mL or lower, 700 ng / mL or lower, 600 ng / mL or lower, 500 ng / mL or lower, 400 ng / mL or lower, 300 ng / mL or lower, or 200 ng / mL or lower.

[0080] When the culture medium contains angiogenic factors and vascular maintenance factors, the ratio of the concentration of the vascular maintenance factors in the culture medium (ng / mL) to the concentration of the angiogenic factors in the culture medium (ng / mL) can be, for example, 0.5 or more, 0.6 or more, 0.7 or more, 0.8 or more, 0.9 or more, 1 or more, 2 or more, 3 or more, or 4 or more, and can be 20 or less, 15 or less, 10 or less, 9 or less, 8 or less, 7 or less, or 5 or less.

[0081] There are no particular limitations on the culture medium as long as it contains the growth factors involved in angiogenesis. An appropriate culture medium can be selected based on the type of cells being cultured. Examples of the culture medium include Eagle's MEM, DMEM, Modified Eagle Medium (MEM), Minimum Essential Medium, RPMI, GlutaMax Medium, and hepatocyte culture medium (e.g., HCM medium (manufactured by Lonza)). The culture medium may be serum-supplemented or serum-free. Alternatively, the culture medium may be a mixed medium containing two or more culture media.

[0082] The culture container (support) is not particularly limited and may include, for example, a culture dish, a well insert, a low-adhesion plate, or a plate having a U-shaped or V-shaped bottom surface. For example, a culture container may include a container having a substrate (permeable membrane) that does not allow cells in a liquid to pass through but allows the liquid to pass through. Examples of containers having a permeable membrane include, but are not limited to, cell culture inserts such as Transwell (registered trademark) inserts, Netwell (registered trademark) inserts, Falcon (registered trademark) cell culture inserts, and Millicell (registered trademark) cell culture inserts.

[0083] The above-mentioned various cells may be cultured in a state of being adhered to the support, in a state of being non-adherent to the support, or separated from the support during the culture process. When the above-mentioned various cells are cultured in a state of being non-adherent to the support, or when the cells are cultured after being separated from the support during the culture process, it is preferable to use a plate having a bottom surface shape such as a U-shaped or V-shaped plate or a low-adsorption plate that inhibits cell adhesion to the support.

[0084] The culturing step in the method for manufacturing the cell structure of the present embodiment is preferably carried out under conditions in which the cell structure can take in culture medium components from all directions. By culturing a variety of cells or cell structures under such conditions, the variety of cells or cell structures can take in more culture medium components (mainly growth factors involved in angiogenesis), thereby further promoting angiogenesis in the cell structure. In addition, the decline of the formed blood vessels can also be suppressed in the cell structure. Here, a variety of cells are cultured to form a cell structure. Furthermore, in this specification, "cell structure" includes not only a cell structure that is formed with blood vessels that do not diffuse to the outside, but also a cell structure in the stage before the formation of blood vessels that do not diffuse to the outside, and a cell structure in the middle of the formation of blood vessels that do not diffuse to the outside.

[0085] Regarding methods for culturing under conditions where the cell structure can take in culture medium components from all directions, for example, there can be cited methods of culturing cells or cell structures using a container (such as the above-mentioned container with a plug) having a substrate (permeable membrane) that allows liquid to pass through; methods of culturing cells or cell structures by embedding the cells in a hydrogel and immersing the gel in a culture medium. Embedding cells in a hydrogel means that a hydrogel is present on the outside of the cells or at least part or all of the outside and intercellular spaces. The method of embedding cells in a hydrogel will be described later. On the contrary, for example, when cells are cultured in a state adhered to a culture dish, a plate, etc., cells adhered to the bottom surface of the culture vessel, etc. cannot take in culture medium components from the lower surface.

[0086] The manufacture method of the cell structure of the present embodiment can possess the process (mixing process) of obtaining the mixture comprising the above-mentioned multiple cells and extracellular matrix components before the above-mentioned culture process. In the mixing process, the mixture is obtained by mixing the multiple cells with the extracellular matrix components. By possessing the mixing process, the multiple cells are configured via the extracellular matrix, and therefore the cell structure of the multiple cells is easily obtained to be three-dimensionally configured.

[0087] The mixing process can be carried out in an aqueous medium. "Aqueous medium" refers to a liquid with water as an essential component. As an aqueous medium, for example, it can be an aqueous medium containing a cationic substance. The aqueous medium containing a cationic substance can be, for example, a cationic buffer such as Tris-hydrochloric acid buffer, Tris-maleic acid buffer, Bis-Tris-buffer or HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid). As a cationic substance, it can be a medium containing cationic compounds such as ethanolamine, diethanolamine, triethanolamine, polyethyleneamine, polyallylamine, polylysine, polyhistidine, polyarginine and water. In addition, as the above-mentioned aqueous medium, a culture medium can also be used. As a culture medium, for example, liquid culture media such as Dulbecco's Modified Eagle medium (DMEM) and hepatocyte-specific culture medium (HCM) can be cited. The liquid culture medium can be a mixed culture medium mixed with two culture media.

[0088] The concentration and pH of the cationic substance (e.g., Tris in a Tris-HCl buffer) in an aqueous medium containing a cationic substance are not particularly limited as long as they do not adversely affect cell growth and the construction of cell structures. For example, the concentration of the cationic substance can be 10 to 100 mM, 40 to 70 mM, or even 50 mM, based on the total amount of the aqueous medium containing the cationic substance. The pH of the aqueous medium (e.g., a cationic buffer) can be 6.0 to 8.0, 6.8 to 7.8, or 7.2 to 7.6.

[0089] In this manual, " extracellular matrix composition " refers to the aggregate of extracellular matrix molecules that are formed by multiple extracellular matrix molecules. Extracellular matrix molecules refer to the extracellular material that is present in biology. As extracellular matrix, as long as the formation of cell growth and cell aggregate does not have a negative effect, just can use arbitrary material. As the object lesson of extracellular matrix molecules, can enumerate collagen, elastin, proteoglycan, fibronectin, hyaluronic acid, laminin, vitronectin, cell adhesion element, nidogen, fibrillin and cadherin etc., but are not limited to these. Extracellular matrix composition can use a kind among them separately, also can be used in combination.

[0090] As long as it does not adversely affect cell growth and cell aggregate formation, the extracellular matrix can be a modified or mutant form of the above-mentioned extracellular matrix, or a polypeptide such as a chemically synthesized peptide. The extracellular matrix can have repetitions of the sequence represented by Gly-XY, a characteristic of collagen. Here, Gly represents a glycine residue, and X and Y each independently represent an arbitrary amino acid residue. Multiple Gly-XY residues can be the same or different. The repetition of the sequence represented by Gly-XY reduces constraints on the configuration of the molecular chain, thereby improving its function as a scaffold material during cell culture, for example. In an extracellular matrix having repetitions of the sequence represented by Gly-XY, the proportion of the sequence represented by Gly-XY in the total amino acid sequence can be 80% or more, preferably 95% or more. Alternatively, the extracellular matrix can be a polypeptide having the RGD sequence. The RGD sequence refers to the sequence represented by Arg-Gly-Asp (arginine residue-glycine residue-aspartic acid residue). By having the RGD sequence, cell adhesion is further promoted, and thus, for example, it is more suitable as a scaffold material for cell culture. Examples of extracellular matrices containing a sequence represented by Gly-XY and an RGD sequence include collagen, fibronectin, ponectin, laminin, and cadherin.

[0091] As collagen, for example, fibrous collagen and non-fibrous collagen can be mentioned. Fibrous collagen refers to collagen that becomes the main component of collagen fibers, specifically type I collagen, type II collagen, type III collagen, etc. As non-fibrous collagen, for example, type IV collagen can be mentioned.

[0092] Examples of proteoglycans include chondroitin sulfate proteoglycans, heparin sulfate proteoglycans, keratan sulfate proteoglycans, and dermatan sulfate proteoglycans, but are not limited thereto.

[0093] As the shape of extracellular matrix components, for example, fibrous can be enumerated. Fibrous refers to the shape consisting of filamentous extracellular matrix components, or the shape consisting of filamentous extracellular matrix components in intermolecular crosslinking. At least a portion of extracellular matrix components can be fibrous. The shape of extracellular matrix components is the shape of a piece of extracellular matrix components (aggregate of extracellular matrix components) observed during microscopic observation, and extracellular matrix components preferably have the size of average diameter and / or average length described later. The fibrous extracellular matrix components include filaments (thin fibers) formed by the collection of multiple filamentous extracellular matrix molecules, filaments further collected and formed, materials obtained by defibrating these filaments, etc. When comprising a fibrous extracellular matrix component, in the fibrous extracellular matrix component, the RGD sequence is not destroyed and is preserved, and as a scaffold material for cell adhesion, can further effectively perform functions.

[0094] The extracellular matrix component can include at least one selected from collagen, laminin and fibronectin, preferably collagen. Collagen is preferably fibrous collagen, more preferably type I collagen. As fibrous collagen, commercially available collagen can be used, and as its specific example, type I collagen derived from pigskin manufactured by Ham Co., Ltd. of Japan can be enumerated.

[0095] The extracellular matrix component can be an extracellular matrix component of animal origin. As the animal species that becomes the source of the extracellular matrix component, for example, humans, pigs, cattle, etc. can be enumerated, but are not limited to these. The extracellular matrix component can use a component of one animal origin, or can use the component of multiple animal origins in combination.

[0096] The extracellular matrix component can include a fragmented extracellular matrix component. "Fragmentation" refers to a process in which the aggregate of the extracellular matrix component is reduced to a smaller size. The fragmented extracellular matrix component can include a fibrillated extracellular matrix component. The fibrillated extracellular matrix component is a component obtained by applying a physical force to fibrillate the above-mentioned extracellular matrix component. For example, fibrillation is carried out without cutting the bonds within the extracellular matrix molecules.

[0097] The fragmented extracellular matrix component can be produced, for example, by a method including a step of fragmenting the extracellular matrix component (fragmentation step).

[0098] The method for fragmenting extracellular matrix components is not particularly limited, and can be fragmented by applying physical force. The extracellular matrix fragmented by applying physical force is different from enzyme treatment, and usually the molecular structure is not changed (molecular structure is maintained) compared with before fragmentation. The method for fragmenting extracellular matrix components can be, for example, a method for finely pulverizing bulk extracellular matrix components. Extracellular matrix components can be fragmented with solid phase, or fragmented in aqueous medium. For example, physical force can be applied by ultrasonic homogenizer, stirring homogenizer and high pressure homogenizer etc. to fragment extracellular matrix components. When using stirring homogenizer, extracellular matrix components can be directly homogenized, or homogenized in aqueous mediums such as physiological saline. In addition, by adjusting the time, number of times etc. of homogenization, it is also possible to obtain millimeter-sized, nanometer-sized fragmented extracellular matrix components. When the extracellular matrix components are fragmented in an aqueous medium, the fragmented extracellular matrix components can be produced, for example, by a method comprising a step of fragmenting the extracellular matrix components in an aqueous medium and a step of removing the aqueous medium from a liquid containing the fragmented extracellular matrix components and the aqueous medium (removal step). The removal step can be carried out, for example, by freeze-drying. "Removal of the aqueous medium" does not mean that the fragmented extracellular matrix components are completely free of water, but rather means that water is not attached to a degree that is generally achieved by the conventional drying method described above.

[0099] The diameter and length of the fragmented extracellular matrix components can be determined by analyzing each fragmented extracellular matrix component using an electron microscope.

[0100] The average length of the fragmented extracellular matrix component can be more than 100 nm and less than 400 μm, or more than 100 nm and less than 200 μm. In one embodiment, from the viewpoint of easily forming a thick cell structure, the average length of the fragmented extracellular matrix component can be more than 5 μm and less than 400 μm, can be more than 10 μm and less than 400 μm, can be more than 100 μm and less than 400 μm. In another embodiment, the average length of the fragmented extracellular matrix component can be less than 100 μm, can be less than 50 μm, can be less than 30 μm, can be less than 15 μm, can be less than 10 μm, can be less than 1 μm, and can be more than 100 nm. In the entire fragmented extracellular matrix component, the average length of most of the fragmented extracellular matrix components is preferably within the above numerical range. Specifically, in the entire fragmented extracellular matrix component, the average length of the fragmented extracellular matrix components of more than 50% is preferably within the above numerical range, and the average length of the fragmented extracellular matrix components of more than 95% is more preferably within the above numerical range. The fragmented extracellular matrix component is preferably a fragmented collagen component having an average length within the above range.

[0101] The average diameter of the fragmented extracellular matrix component may be 50 nm to 30 μm, 4 μm to 30 μm, or 5 μm to 30 μm. The fragmented extracellular matrix component is preferably a fragmented collagen component having an average diameter within the above range.

[0102] The average length and average diameter of the fragmented extracellular matrix components can be determined by measuring each fragmented extracellular matrix component using an optical microscope and performing image analysis. In this specification, "average length" refers to the average value of the length of the sample in the longitudinal direction, and "average diameter" refers to the average value of the length of the sample in a direction perpendicular to the longitudinal direction.

[0103] Fragmented collagen components are also referred to as "fragmented collagen components." "Fragmented collagen components" refer to components obtained by fragmenting collagen components such as fibrous collagen components, which maintain a triple helical structure. The average length of the fragmented collagen components is preferably 100 nm to 200 μm, more preferably 22 μm to 200 μm, and even more preferably 100 μm to 200 μm. The average diameter of the fragmented collagen components is preferably 50 nm to 30 μm, more preferably 4 μm to 30 μm, and even more preferably 20 μm to 30 μm.

[0104] At least a portion of the extracellular matrix components can be cross-linked intermolecularly or intramolecularly. The extracellular matrix components can be cross-linked intermolecularly or intramolecularly of the extracellular matrix molecules that constitute the extracellular matrix components. In the case where the extracellular matrix components include fragmented extracellular matrix components, at least a portion of the fragmented extracellular matrix components can be cross-linked intermolecularly or intramolecularly.

[0105] The extracellular matrix component of at least a part of intermolecular or intramolecular crosslinking can be manufactured by the method for comprising the process (crosslinking process) of crosslinking the extracellular matrix component. The extracellular matrix component can for example comprise the extracellular matrix component through fragmentation and crosslinking. The extracellular matrix component through fragmentation and crosslinking can for example be manufactured by the method for the process of successively possessing the process of fragmenting the extracellular matrix component and the process of crosslinking the fragmented extracellular matrix component or successively possessing the process of crosslinking the extracellular matrix component and the process of crosslinking the extracellular matrix component fragmentation.

[0106] As the method for cross-linking, for example, physical cross-linking by applying heat, ultraviolet rays, radiation, etc., chemical cross-linking by cross-linking agents, enzyme reactions, etc. can be enumerated, and the method is not particularly limited. From the viewpoint of not hindering cell growth, physical cross-linking is preferred. Cross-linking (physical cross-linking and chemical cross-linking) can be cross-linking via covalent bonds.

[0107] In the case where the extracellular matrix component comprises collagen composition, crosslinking can be formed between collagen molecules (triple helical structure), can also be formed between the collagen fibrils formed by collagen molecules.Crosslinking can be the crosslinking (thermal crosslinking) performed by heat.Thermal crosslinking can be implemented by using a vacuum pump to carry out heat treatment under reduced pressure.In the case of carrying out the thermal crosslinking of collagen composition, the extracellular matrix component can be crosslinked by forming a peptide bond (NH-CO) with the amino group of collagen molecules and the same or the carboxyl group of other collagen molecules.

[0108] The extracellular matrix component can also be cross-linked by using a cross-linking agent. Cross-linking agent can be, for example, a cross-linking agent that can cross-link a carboxyl group and an amino group or a cross-linking agent that can cross-link an amino group to each other. As a cross-linking agent, for example, from the viewpoint of economy, safety and operability, preferably aldehydes, carbodiimides, epoxides and imidazoles cross-linking agents, specifically, water-soluble carbodiimides such as glutaraldehyde, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, 1-cyclohexyl-3-(2-morpholinyl-4-ethyl) carbodiimide sulfonate can be enumerated.

[0109] The quantitative degree of cross-linking can be appropriately selected based on the type of extracellular matrix components, the means of cross-linking, etc. The degree of cross-linking can be 1% or more, 2% or more, 4% or more, 8% or more, or 12% or more, and can be 30% or less, 20% or less, or 15% or less. By keeping the degree of cross-linking within the above range, the extracellular matrix molecules can be appropriately dispersed, and the redispersibility after drying and storage is good.

[0110] When amino groups in the extracellular matrix components are used for crosslinking, the degree of crosslinking can be quantified based on the TNBS (2,4,6-trinitrobenzenesulfonic acid) method described in Acta Biomaterialia, 2015, vol. 25, pp. 131-142, etc. The degree of crosslinking obtained by the TNBS method can be within the above range. The degree of crosslinking obtained by the TNBS method is the ratio of amino groups in the extracellular matrix that are used for crosslinking. When the extracellular matrix components include collagen components, the degree of crosslinking determined by the TNBS method is preferably within the above range.

[0111] The degree of cross-linking can be calculated by quantifying carboxyl groups. For example, in the case of water-insoluble extracellular matrix components, quantification can be performed using the TBO (Toluidine Blue O) method. The degree of cross-linking obtained by the TBO method can also be within the above range.

[0112] In the process of cross-linking, the temperature (heating temperature) and time (heating time) when heating the extracellular matrix component can be appropriately determined. The heating temperature can be, for example, more than 100 ° C, and can be below 200 ° C, or can be below 220 ° C. Specifically, the heating temperature can be, for example, 100 ° C, 110 ° C, 120 ° C, 130 ° C, 140 ° C, 150 ° C, 160 ° C, 170 ° C, 180 ° C, 190 ° C, 200 ° C or 220 ° C. The heating time (the time kept at the above-mentioned heating temperature) can be appropriately set according to the heating temperature. The heating time can be, for example, more than 6 hours and less than 72 hours, more preferably more than 24 hours and less than 48 hours, when heating at 100 ° C ~ 200 ° C. In the process of cross-linking, heating can be performed under the condition of not having a solvent, or it can be performed under reduced pressure.

[0113] The content of the extracellular matrix component in the mixture in the mixing step can be appropriately determined according to the shape, thickness, etc. of the target cell structure. The content of the extracellular matrix component in the mixture can be based on the total amount of the mixture, for example, 0.005 mg / mL or more, 0.01 mg / mL or more, 0.025 mg / mL or more, 0.05 mg / mL or more, 0.10 mg / mL or more, 0.15 mg / mL or more, 0.20 mg / mL, 0.25 mg / mL, 0.30 mg / mL or more, 0.35 mg / mL or more, 0.40 mg / mL or more, or 0.45 mg / mL or more, and can be 1.5 mg / mL or less, 1.25 mg / mL or less, 1.0 mg / mL or less, 0.8 mg / mL or less, or 0.6 mg / mL or less.

[0114] The mixture in the mixing step may further include a polymer electrolyte. A polymer electrolyte is a polymer compound having the properties of an electrolyte. As a polymer electrolyte, glycosaminoglycans such as heparin, chondroitin sulfate (such as 4-chondroitin sulfate or 6-chondroitin sulfate), heparin sulfate, dermatan sulfate, keratan sulfate, hyaluronic acid, etc. can be cited; dextran sulfate, rhamnose sulfate, fucoidan, carrageenan, polystyrene sulfonic acid, polyacrylamide-2-methylpropanesulfonic acid, and polyacrylic acid, or their derivatives, etc., but are not limited to these. The polymer electrolyte can be composed of one of the above, or it can be combined to include two or more.

[0115] The polymer electrolyte is preferably a glycosaminoglycan, more preferably comprises at least one selected from heparin, dextran sulfate, chondroitin sulfate, and dermatan sulfate, and further preferably comprises heparin. When the mixture in the mixing step comprises a polymer electrolyte, excessive aggregation of extracellular matrix components can be more effectively suppressed, and as a result, a cell structure with excellent responsiveness to a prescribed substance can be more easily obtained. When the cell structure comprises heparin as the polymer electrolyte, the effect is even more significant.

[0116] The concentration of the polymer electrolyte in the mixture in the mixing process is not particularly limited as long as it does not adversely affect the growth of cells and the formation of cell structures. The concentration of the polymer electrolyte in the mixture can be, for example, more than 0 mg / mL and less than 1.5 mg / mL based on the total amount of the mixture. The concentration of the polymer electrolyte in the mixture can be more than 0.005 mg / mL, more than 0.01 mg / mL, more than 0.02 mg / mL, more than 0.03 mg / mL, or more than 0.04 mg / mL, and can be less than 1.5 mg / mL, less than 1.0 mg / mL, less than 0.1 mg / mL, less than 0.08 mg / mL, or less than 0.06 mg / mL. The concentration of the polymer electrolyte in the mixture can be, for example, 0.025 mg / mL, 0.05 mg / mL, 0.075 mg / mL, or 0.1 mg / mL.

[0117] The ratio of the mass C2 of the polymer electrolyte to the mass C1 of the extracellular matrix component (C2 / C1) may be 1 / 100 to 100 / 1, 1 / 10 to 10 / 1, 1 / 5 to 5 / 1, or 1 / 2 to 2 / 1, or 1 / 1.5 to 1.5 / 1.

[0118] The mixture in the mixing step may further contain a hydrogel precursor. In this case, the method for producing a cell structure of this embodiment may include a step of gelling the hydrogel precursor in the mixture (gelling step) after the mixing step and before the culturing step.

[0119] The gelation of the mixture comprising the hydrogel precursor may comprise an incubation step for a certain period of time.

[0120] A hydrogel precursor refers to a substance that forms a hydrogel under certain external stimuli such as chemical stimulation or physical stimulation, and is a substance in a state where a hydrogel is not formed. Examples of hydrogels include fibrin gel, collagen gel, gelatin gel, hyaluronic acid gel, alginate gel, and pectin gel. Examples of hydrogel precursors include fibrinogen and thrombin, collagen, gelatin, hyaluronic acid, alginate, and pectin. As a hydrogel precursor, it is preferred to include fibrinogen and thrombin. Fibrin is a component generated by thrombin acting on fibrinogen and releasing A chain and B chain from the N-terminal ends of Aα chain and Bβ chain. Fibrin is formed by contacting fibrinogen with thrombin.

[0121] The concentration of the hydrogel precursor in the mixture during the mixing step may be 0.1 mg / mL or more, 0.2 mg / mL or more, 0.3 mg / mL or more, 0.4 mg / mL or more, 0.5 mg / mL or more, 0.6 mg / mL or more, 0.7 mg / mL or more, 0.8 mg / mL or more, 0.9 mg / mL or more, or 1.0 mg / mL or more, based on the total amount of the mixture. The concentration of the hydrogel precursor in the mixture during the mixing step may be 10.0 mg / mL or less, 8.0 mg / mL or less, 6.0 mg / mL or less, 5.0 mg / mL or less, 3.0 mg / mL or less, 1.0 mg / mL or less, 0.8 mg / mL or less, or 0.6 mg / mL or less, based on the total amount of the mixture.

[0122] For example, when the hydrogel precursor contains two or more substances, such as a combination of fibrinogen and thrombin, a mixture containing cells and a hydrogel precursor can be obtained by mixing a first composition containing cells and a first hydrogel precursor (e.g., thrombin) with a second composition containing a second hydrogel precursor (e.g., fibrinogen). It should be noted that the second composition may contain cells, and both the first composition and the second composition may contain cells.

[0123] [Method for preventing cell structures from spreading to the outside by forming blood vessels]

[0124] As described above, the production method of this embodiment can produce a cell structure comprising epithelial cells and having blood vessels that are not externally proliferating. Therefore, the present invention can also be understood as a method for forming a cell structure comprising epithelial cells into blood vessels that are not externally proliferating, comprising culturing a plurality of cells comprising at least epithelial cells and vascular endothelial cells in a culture medium containing one or more growth factors involved in angiogenesis. As specific aspects of the method, the aforementioned aspects can be applied without particular limitation.

[0125] 〔Cell structure〕

[0126] The cell structure of this embodiment comprises a plurality of cells including epithelial cells and vascular endothelial cells and forms blood vessels that do not diffuse to the outside. The cell structure of this embodiment may comprise an extracellular matrix component and may further comprise a polymer electrolyte. The cell structure of this embodiment may be obtained, for example, by the above-mentioned method.

[0127] When the cell structure of this embodiment contains an extracellular matrix component, the content of the extracellular matrix component in the cell structure can be 0.01 mass% or more, 0.05 mass% or more, 0.1 mass% or more, 0.5 mass% or more, 1 mass% or more, 2 mass% or more, 3 mass% or more, 4 mass% or more, 5 mass% or more, 6 mass% or more, 7 mass% or more, 8 mass% or more, 9 mass% or more, 10 mass% or more, 15 mass% or more, 20 mass% or more, 25 mass% or more, or 30 mass% or more, and can be 90 mass% or less, 80 mass% or less, 70 mass% or less, 60 mass% or less, 50 mass% or less, 30 mass% or less, 20 mass% or less, or 15 mass% or less, based on the dry weight of the cell structure. The content of the extracellular matrix components in the cell structure can be 0.01 to 90 mass%, 10 to 90 mass%, 10 to 80 mass%, 10 to 70 mass%, 10 to 60 mass%, 1 to 50 mass%, 10 to 50 mass%, 10 to 30 mass%, or 20 to 30 mass%, based on the dry weight of the cell structure.

[0128] The blood vessels formed in the cell structure of this embodiment may be blood vessels whose vascular decline is further suppressed under the action of the above-mentioned growth factors involved in angiogenesis.

[0129] The cell structure of this embodiment may have an average length of blood vessels formed in the cell structure of, for example, 500 μm or more, 600 μm or more, 700 μm or more, or 800 μm or more. Alternatively, the average length of blood vessels formed in the cell structure may be 3000 μm or less, 2500 μm or less, 2000 μm or less, or 1500 μm or less.

[0130] The cell structure of this embodiment, when the epithelial cells are hepatocytes, can express metabolic enzyme genes belonging to the cytochrome P450 superfamily, which is unique to hepatocytes, or fully maintain the activity of the metabolic enzymes. Examples of metabolic enzymes belonging to the cytochrome P450 superfamily include CYP3A4, CYP2C9, CYP1A1, CYP1A2, CYP2E1, and CYP27A1. CYP3A4 is an enzyme involved in the metabolism of various compounds, and therefore maintaining its expression and activity during the culture period is particularly important. The cell structure of this embodiment, when the epithelial cells are hepatocytes, can particularly fully maintain the activity of the CYP3A4 enzyme.

[0131] In the cell structure of this embodiment, when the epithelial cells are hepatocytes, the expression level of the gene can be more than 0.6 times, more than 0.65 times, more than 0.7 times, more than 0.75 times, or more than 0.8 times, based on the activity of the metabolic enzyme gene after the cell structure is manufactured (for example, 4 days after the start of cell culture), and can be less than 2 times, less than 1.9 times, less than 1.8 times, less than 1.7 times, less than 1.6 times, less than 1.5 times, less than 1.4 times, or less than 1.3 times.

[0132] The thickness of the cell structure of this embodiment can be 10 μm or more, 30 μm or more, 50 μm or more, 100 μm or more, 300 μm or more, or 1000 μm or more. Such a cell structure is a structure closer to biological tissue and is suitable as a substitute for experimental animals and a transplant material. The upper limit of the thickness of the three-dimensional hepatocyte tissue is not particularly limited, for example, it can be 10 mm or less, 3 mm or less, 2 mm or less, 1.5 mm or less, 1 mm or less, 300 μm or less, 200 μm or less, 150 μm or less, or 100 μm or less.

[0133] Here, the "thickness of the cell structure" refers to the distance between both ends in a direction perpendicular to the main surface when the cell structure is a rectangular parallelepiped. When the main surface has irregularities, the thickness refers to the distance at the thinnest portion of the main surface.

[0134] When the cell structure is spherical or roughly spherical, the thickness of the cell structure refers to the diameter of the cell structure. When the cell structure is ellipsoidal or roughly ellipsoidal, the thickness of the cell structure refers to the minor diameter of the cell structure. When the cell structure is roughly spherical or roughly ellipsoidal and has uneven surfaces, the thickness of the cell structure refers to the shortest distance between the two points where a straight line passing through the center of gravity of the cell structure intersects the surface.

[0135] Example

[0136] Hereinafter, the present invention will be described in more detail based on examples, but the present invention is not limited to these.

[0137] [Test Example 1: Confirmation of the Effect of Growth Factors Involved in Angiogenesis on Angiogenesis]

[0138] 5.85×10 5 Human hepatocytes (PXB cells (registered trademark), manufactured by PhoenixBio) and 2.25×10 5A cell mixture of 100 vascular endothelial cells (GFP-HUVEC, manufactured by ANGIO-PROTEOMIE) was suspended in a mixture of 50 μL of an equal volume of 1.0 mg / mL heparin / 200 mM Tris-HCl buffer (pH 7.4) and 50 μL of 0.6 mg / mL collagen / 5 mM acetic acid solution (pH 3.7) (heparin-collagen solution). The resulting mixture was centrifuged at 400 × g for 2 minutes at room temperature, the supernatant was removed, and the cell density was adjusted to 2.7 × 10 4 The cells were resuspended in HCM culture medium (model "CC-3198", manufactured by Lonza) containing 10 U / mL thrombin (model "R4648", manufactured by Sigma-Aldrich) and 1 v / v% endothelial cell growth supplement (ECGS) (model "1052", manufactured by Sciencell) at a cell density of 1.95 × 10 cells / 2 μL to obtain a cell suspension. 4 The cell density of endothelial cells was 7.5×10 3 / 2μL.

[0139] 2 μL of HCM culture medium containing 5 mg / mL fibrinogen and 1 v / v% ECGS was added to a 48-well microplate (model "3830-048", manufactured by AGC TECHNO GLASS) to form droplets. The above-mentioned cell suspension was then added to the inside of the droplets, and the 48-well microplate was then left to stand in an incubator for 40 minutes to form a fibrin gel that embedded human hepatocytes and vascular endothelial cells. 50 ng / mL VEGF-A as an angiogenic factor, or 50 ng / mL VEGF-A as an angiogenic factor and 200 ng / mL angiopoietin-1 as a vascular maintenance factor, and 0.5 mL of HCM culture medium containing 1 v / v% ECGS were added to each well of the fibrin gel, and cultured at 37°C and 5% CO2 to obtain a cell structure. During the culture, the culture medium was replaced every 2 days after the start of the culture. As a control, a cell structure was obtained in the same manner as above except that a fibrin gel was formed to embed human hepatocytes and vascular endothelial cells and the cells were cultured in HCM medium containing no ECGS or growth factors involved in angiogenesis.

[0140] The obtained cell structure was observed by confocal microscopy to observe vascular endothelial cells (GFP-HUVEC) and evaluate the angiogenesis in the cell structure. Figure 1 .

[0141] like Figure 1As shown in FIG, the cell structure cultured in a medium containing VEGF-A formed blood vessels, but no blood vessel proliferation to the outside was observed ( Figure 1 (B)). In addition, the cell structures cultured in the medium containing VEGF-A and angiopoietin-1 further promoted angiogenesis. Moreover, even on the 14th day of culture, the cell structures cultured in the medium containing VEGF-A and angiopoietin-1 showed less vascular decline than the cell structures cultured in the medium containing VEGF-A, and no outward spread of blood vessels was observed ( Figure 1 (C) No angiogenesis was observed in the cell structure cultured in a conventional medium that does not contain growth factors involved in angiogenesis ( Figure 1 (A)).

[0142] [Test Example 2: Confirmation of the Effect of Culture Method on Angiogenesis]

[0143] A fibrin gel encapsulating human hepatocytes and vascular endothelial cells was formed in the same manner as in Experimental Example 1, except that a 48-well microplate or a 24-well Transwell (manufactured by Corning) was used as a culture container. A composition containing insulin, hemopexin, EGF, melanotransferrin, IGF-1, LRG1, and HSPG2 as vascular maintenance factors (hereinafter referred to as the "vascular maintenance factor mixture") was prepared. Cells were then cultured in the same manner as in Experimental Example 1, except that a culture medium containing 50 ng / mL VEGF-A and 1 v / v% of the vascular maintenance factor mixture as angiogenic factors was used, or a culture medium containing 50 ng / mL VEGF-A and 50 ng / mL VEGF-C and 1 v / v% of the vascular maintenance factor mixture as angiogenic factors was used, to obtain a cell structure.

[0144] First, the obtained cell structure was observed under a microscope for vascular endothelial cells (GFP-HUVEC) in the same manner as in Test Example 1 to evaluate the formation of blood vessels. Figure 2 and Figure 3 .

[0145] like Figure 2 and Figure 3 As shown, compared with cell structures produced by culturing using 48-well microplates, cell structures produced by culturing using containers with inserts such as 24-well Transwells, where cells can take up culture medium components from all directions, exhibited further suppression of vascular decline ( Figure 2 Middle (A) and Figure 3 (A), or Figure 2 (B) with Figure 3 (B) comparison).

[0146] Next, the length of blood vessels in the cell structures was measured using Image J in the microscope images taken on the 14th day of cell structure culture (number of cell structure samples: 6). The results are shown in Figure 4 .like Figure 4 As shown, the length of blood vessels in cell structures cultured using 24-well Transwell plates was statistically significantly longer than that in cell structures cultured using 48-well microplates. This result suggests that culturing cells under conditions that allow them to take up culture medium components from all directions, such as by using a container equipped with a permeable membrane, further suppresses vascular decline.

[0147] [Test Example 3: Confirmation of the Effect of Growth Factors Involved in Angiogenesis on the Expression of Metabolism-Related Genes]

[0148] Cells were cultured in the same manner as in Experimental Example 1, except that 50 ng / mL VEGF-A and 200 ng / mL angiopoietin-1 (ANG), 50 ng / mL VEGF-A and a 1 v / v% mixture of vascular maintenance factors, 50 ng / mL VEGF-A, or 50 ng / mL VEGF-C and 200 ng / mL angiopoietin-1, or a culture medium containing no growth factors involved in angiogenesis, to obtain a cell structure.

[0149] Separately, cells were cultured in the same manner as in Experimental Example 2, except that a 24-well Transwell was used as a culture vessel and a culture medium containing 50 ng / mL VEGF-A and 200 ng / mL angiopoietin-1 (ANG), 50 ng / mL VEGF-A and a 1 v / v% mixture of vascular maintenance factors, or 50 ng / mL VEGF-A, 50 ng / mL VEGF-C and a 1 v / v% mixture of vascular maintenance factors was used to obtain a cell structure.

[0150] In addition, using 5.85×10 5 Human hepatocytes, 2.25×10 5 Human vascular endothelial cells (HUVEC) and 9.0×10 4 A mixture of hepatic stellate cells (LX2, manufactured by Sigma-Aldrich) was prepared and the cell density was adjusted to 3×10 4 A cell structure containing hepatic stellate cells was obtained in the same manner as in Test Example 1, except that the cells were resuspended at 100 μL / 2 μL to obtain a cell suspension.

[0151] RNA was extracted from the cell structures on day 1, day 4, and day 8 of culture, and the expression levels of the CYP3A4 and CYP2C9 genes in each of the cell structures were analyzed. RNA was extracted using the Direct-zol RNA Kit (model "R2061", manufactured by Zymo Research). The extracted RNA was reverse transcribed to synthesize complementary DNA (cDNA). cDNA was synthesized using the SuperScript (registered trademark) VILO (registered trademark) cDNA Synthesis Kit (model "11754050", manufactured by Thermo Fisher Scientific). The synthesized cDNA was subjected to quantitative PCR analysis to analyze the expression levels of CYP3A4, CYP2C9, and GAPDH. Quantitative PCR analysis was performed using Taqman (registered trademark) Fast Advanced Master Mix (model "4444556", manufactured by Thermo Fisher Scientific) and Taqman (registered trademark) Gene Expression assay (model "4331182", manufactured by Thermo Fisher Scientific). In addition, as primers for quantitative PCR, oligonucleotides with Assay ID: Hs00604506_m1 for CYP3A4, Assay ID: Hs00426397_m1 for CYP2C9, and Assay ID: Hs99999905_m1 for GAPDH were used. The analysis was performed using the StepOnePlus Real-Time PCR System (Thermo Fisher Scientific) according to the manufacturer's recommended thermal profile.

[0152] Figure 5 To express the CYP3A4 gene ( Figure 5 (A)) and CYP2C9 gene ( Figure 5 (B) is a graph showing the results of expression level analysis. Figure 5 The vertical axis represents the relative value (ΔCt) of the Ct value of CYP3A4 or CYP2C9 to the amplification cycle number (Ct value) of GAPDH.

[0153] like Figure 5 As shown, no difference in the expression of metabolism-related genes was observed between culture medium conditions. Thus, no direct adverse effects of growth factors involved in angiogenesis on liver function were observed. Furthermore, it was shown that when cell structures containing hepatic stellate cells were cultured, the expression of metabolism-related genes gradually decreased, making it difficult to maintain liver function ( Figure 5 On the other hand, it can be seen that in the cell structure without hepatic stellate cells, the expression of metabolism-related genes does not decrease, and liver function is maintained.

[0154] [Test Example 4: Confirmation of the Effect of Vascular Endothelial Cell Type on Angiogenesis]

[0155] A 24-well Transwell was used as a culture vessel, and sinusoidal endothelial cells (SEC, manufactured by Sciencell, product model "5000") immortalized by the introduction of hTERT and SV40LT were used instead of HUVECs. A cell structure was obtained in the same manner as in Experimental Example 2 except for these conditions.

[0156] Since the SEC used in Test Example 4 expressed GFP, the cell structure produced using SEC as vascular endothelial cells was also observed in the same manner as in Test Example 2 to evaluate the formation of blood vessels. The results are shown in Figure 6 .

[0157] Referring to the results of Experimental Example 2, compared with the cell structure produced using HUVEC as the vascular endothelial cell, the cell structure produced using SEC further promoted angiogenesis (see Figure 3 and Figure 6 In addition, in the cell structure produced by SEC, no diffusion of blood vessels to the outside was confirmed ( Figure 6 ).

[0158] Next, similarly to Experimental Example 2, the length of the blood vessels in the cell structures produced using SEC was measured in microscopic images on the 14th day of culture (number of cell structure samples: 6). The results are shown in Table 1. When the results of Experimental Example 2 are also referred to, the length of the blood vessels in the cell structures produced using SEC was statistically significantly longer than that in the cell structures produced using HUVEC (see Figure 4 and Table 1 ).

[0159] [Table 1]

[0160]

[0161] [Reference Example: Confirmation of the Effect of Hepatic Stellate Cell Secretions on Angiogenesis]

[0162] By culturing human hepatocytes and HUVECs for a predetermined period of time using the same method as in the control of Experimental Example 1, a cell structure 1 containing human hepatocytes and HUVECs was obtained. Cell structure 1 is a cell structure at a stage before the formation of blood vessels that do not proliferate to the outside. In addition, instead of human hepatocytes and HUVECs, hepatic stellate cells (LX2) were cultured at a cell density of 3×10 4A cell structure 2 consisting solely of hepatic stellate cells was obtained by the same method as the control in Test Example 1, except that a cell suspension was prepared at 100 cells / 2 μL. The obtained cell structures 1 and 2 were then cultured in the same well for a predetermined period.

[0163] Cell structure 1 cultured in the same well as cell structure 2 was observed under a microscope in the same manner as in Test Example 1 to evaluate the formation of blood vessels. The results are shown in FIG. Figure 7 .

[0164] like Figure 7 As shown, blood vessels were formed by culturing the cell structure 1 in the same well as the cell structure 2, that is, culturing the cell structure 1 in a culture medium containing secretions of hepatic stellate cells. Figure 7 The blood vessels observed (inside the middle dotted line) spread to the outside of the cell structure during culture, but no cell structure with blood vessels that did not spread to the outside was obtained.

[0165] [Test Example 5: Confirmation of the Effect of Cell Type on Vascular Structure]

[0166] 24-well Transwell was used as a culture container, and 1.95×10 cells were cultured in HCM medium containing 50 ng / mL VEGF-A and 50 ng / mL VEGF-C as angiogenic factors and a 1 v / v% vascular maintenance factor mixture. 4 Individual hepatocytes (PXB cells (registered trademark), manufactured by PhoenixBio) and 3.0×10 3 A cell mixture of immortalized sinusoidal endothelial cells (SEC, manufactured by Sciencell, product model "5000") was prepared, and hepatocytes (PXB cells (registered trademark), manufactured by Phoenix Bio) were cultured in HCM medium containing 1 v / v% of a mixture of vascular maintenance factors. 7.5 × 10 3 immortalized sinusoidal endothelial cells (SEC, manufactured by Sciencell, product model “5000”), and 3.0×10 3 A mixture of hepatic stellate cells (LX2, Sigma-Aldrich) or 2.1×10 3 immortalized sinusoidal endothelial cells (SEC, manufactured by Sciencell, product model “5000”), and 9.0×10 3A cell mixture of hepatic stellate cells (LX2, manufactured by Sigma-Aldrich) was prepared. Otherwise, the cell mixture was cultured in the same manner as in Experimental Example 4 to obtain a cell structure. On the 7th day of culturing the cell structure, 0 μM, 7.3 μM, 22 μM, 66 μM, 200 μM or 600 μM of monocrotaline was added to the culture medium, and the cell structure was further cultured in the culture medium for 7 days. Then, the cell structure was fixed with 4% paraformaldehyde (PFA), immunostaining of CD31 was performed according to conventional methods, and the vascular structure was observed using a confocal microscope. The results are shown in Figures 8-9 .

[0167] In addition, from Figures 8-9 The vascular structure was extracted from the microscope image and the vascular length of the cell structure was measured. The results are shown in Figure 10 . Figure 10 The vascular length of each cell structure is represented by the value obtained when the vascular length in the culture medium without monocrotaline is taken as 100%. Hereinafter, a cell structure prepared using a mixture of hepatocytes and immortalized sinusoidal endothelial cells will be referred to as a "vascular liver model," a cell structure prepared using a mixture of hepatocytes, immortalized sinusoidal endothelial cells, and hepatic stellate cells will be referred to as a "vascular liver model containing hepatic stellate cells," and a cell structure prepared using a mixture of immortalized sinusoidal endothelial cells and hepatic stellate cells will be referred to as a "vascular model."

[0168] It should be noted that monocrotaline is metabolized by CYP enzymes that are strongly expressed in hepatocytes and converted into a compound with vascular toxicity. Even when monocrotaline is administered to a model with reduced CYP enzyme activity or a model without hepatocytes, vascular toxicity is not exhibited.

[0169] In addition, for the cell structures made in the same manner, quantification of cell viability based on ATP analysis was also performed. ATP analysis was performed using the CellTiter-Glo (registered trademark) 3D Cell Viability Assay kit according to the following steps. The culture medium containing the compound was removed from each well of the plate used in the culture of each cell structure, 100 μL of DMEM at room temperature was added, and then 100 μL of the ATP analysis reagent attached to the kit was added at room temperature. The plate to which the ATP analysis reagent was added was shaken in a constant temperature oscillator for 5 minutes (1000 rpm, room temperature). The plate was then further allowed to stand at room temperature for 25 minutes. The total amount of the mixed solution of DMEM and ATP analysis reagent was transferred from each well to a 96-well plate for luminescence measurement, and the luminescence intensity was measured with a microplate reader. The results are shown in Figure 11 .

[0170] like Figure 10As shown in Figure 2, the vascular liver model also showed sensitivity to low concentrations of monocrotaline compared to other models, especially the shortening of blood vessel length. Figure 11 As shown, the survival rate in the vascularized liver model was reduced in a monocrotaline-dependent manner compared to the other models. These results suggest that the vascularized liver model is a liver model that further maintains the ability to metabolize monocrotaline and is susceptible to vascular toxicity.

[0171] [Test Example 6: Quantification of CYP3A4 Enzyme Activity in Cell Structures]

[0172] 1.95 × 10 4 Human hepatocytes (PXB cells (registered trademark), manufactured by PhoenixBio) were cultured in HCM medium containing 50 ng / mL VEGF-A and 50 ng / mL VEGF-C as angiogenic factors and a 1 v / v% mixture of vascular maintenance factors at a density of 1.95 × 10 4 Human hepatocytes (PXB cells (registered trademark), manufactured by PhoenixBio) and 3.0×10 3 A mixture of immortalized sinusoidal endothelial cells (derived from SEC, manufactured by Sciencell, product model "5000") or 1.95×10 4 Human hepatocytes (PXB cells (registered trademark), manufactured by PhoenixBio), 7.5×10 3 immortalized sinusoidal endothelial cells (SEC, manufactured by Sciencell, product model “5000”), and 3.0×10 3 A cell mixture of human hepatocytes (LX2, manufactured by Sigma-Aldrich) and hepatic stellate cells was prepared. The cell mixture was cultured in the same manner as in Experimental Example 5, except that the mixture was prepared using human hepatocytes to obtain a cell structure. Hereinafter, the cell structure prepared using human hepatocytes will also be referred to as a "liver model," the cell structure prepared using human hepatocytes and immortalized sinusoidal endothelial cells will be referred to as a "vascular liver model," and the cell structure prepared using human hepatocytes, immortalized sinusoidal endothelial cells, and hepatic stellate cells will be referred to as a "vascular liver model containing hepatic stellate cells."

[0173] On the 4th, 7th and 14th day of culturing the cell structure, the CYP3A4 enzyme activity in the cell structure was quantified using the P450-Glo (trademark) CYP3A4 Assay and Screening System. 72 hours before the CYP3A4 activity assay (day 1, day 4, day 11), the culture medium was replaced with a medium containing rifampicin, a compound that induces CYP3A4 expression. After the cell structure was cultured in the culture medium, the CYP3A4 activity in each cell structure was measured using the above-mentioned kit. In this method, the fluorescein precursor (a substrate specific for CYP3A4) taken into the cell is hydrolyzed by CYP3A4 and converted into fluorescein. By mixing the fluorescein detection reagent, the fluorescein is converted into a luminescent substance, and the CYP3A4 activity of the cell can be quantified based on its luminescence intensity. The results are shown in Figure 12 . Figure 12 In the table, samples in which activity was not detected (luminescence intensity was below the detection limit) were indicated as “ND”.

[0174] like Figure 12 As shown, CYP3A4 activity decreased in both the liver model and the vascular liver model containing hepatic stellate cells on culture day 14. On the other hand, CYP3A4 activity was maintained in the vascular liver model even on culture day 14, indicating that CYP3A4 gene expression is maintained in the vascular liver model.

Claims

1. A method for producing a cell structure, comprising the step of culturing a plurality of cells including at least epithelial cells and vascular endothelial cells in a culture medium containing one or more growth factors involved in angiogenesis.

2. The method according to claim 1, wherein The culture medium contains two or more growth factors involved in angiogenesis, The two or more growth factors involved in angiogenesis include angiogenic factors and vascular maintenance factors.

3. The method according to claim 1 or 2, wherein: The content of the growth factor involved in angiogenesis in the culture medium is 10 ng / mL or more.

4. The method according to claim 1 or 2, wherein: The culturing step is performed under conditions in which the cell structure can take up medium components from all directions.

5. The method according to claim 1 or 2, wherein: The epithelial cells are hepatocytes, and the cells do not include hepatic stellate cells.

6. The method according to claim 1 or 2, wherein: Prior to the culturing step, there is provided a step of obtaining a mixture containing the plurality of cells and an extracellular matrix component.

7. The method according to claim 6, wherein: The extracellular matrix component contains collagen.

8. The method according to claim 6, wherein: The mixture further comprises a polymer electrolyte.

9. The method according to claim 8, wherein The polymer electrolyte contains heparin.

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