Method for producing cell structure

Abstract

The present invention relates to a method for producing a cell structure having a blood vessel structure, the method comprising: a gelation step in which a cell-containing liquid containing cells including vascular endothelial cells is gelled to form a gel containing the cells; and a culture step in which the cells are cultured in the gel, wherein the gel containing the cells is formed by bringing multiple types of gelling agents into contact with a gelling promoter.

Description

Method for producing a cell structure

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

[0002] When evaluating the vascular toxicity of compounds such as pharmaceuticals during the pharmaceutical manufacturing process, plate-cultured cells or animals such as mice are often used. However, because the mechanisms of side effects such as vascular toxicity are complex and diverse, it is difficult to accurately evaluate the toxicity of compounds using toxicity evaluation methods using plate-cultured cells. Animal tests using mice can also be difficult to use for risk assessment due to species differences and social issues such as animal protection. To address these issues, efforts are currently underway to develop artificial tissues with vascular structures using human-derived cells (e.g., Patent Document 1).

[0003] International Publication No. 2021-100709

[0004] The present inventors have developed a method for constructing a cell structure with a vascular structure by embedding cells in a gel (hydrogel) and then culturing them (Patent Document 1).The present inventors have newly discovered a problem with this conventional cell structure: even if the tissue is prepared by the same method and on the same day, variations in angiogenic activity may occur (instability of angiogenic activity).

[0005] An object of the present invention is to provide a method for producing a cell structure that can suppress variations in angiogenic ability.

[0006] One embodiment of the present invention is as follows: [1] A method for producing a cell structure having a vascular structure, comprising: a gelling step of gelling a cell-containing solution containing cells, including vascular endothelial cells, to form a gel containing the cells; and a culturing step of culturing the cells in the gel, wherein the gel containing the cells is formed by contacting multiple types of gelling agents with a gelation accelerator. [2] The method according to [1], wherein the cell-containing solution further contains an extracellular matrix component and a polyelectrolyte. [3] The method according to [2], wherein the extracellular matrix component contains collagen. [4] The method according to [2], wherein the polyelectrolyte contains heparin. [5] The method according to any one of [1] to [4], wherein the multiple types of gelling agents contain fibrinogen and sodium alginate. [6] The method according to any one of [1] to [5], wherein the gelation accelerator contains thrombin and calcium chloride. [7] The method according to any one of [1] to [6], wherein the gelling step is a step of forming the gel by injecting the cell-containing solution containing the cells and the gelation accelerator into a droplet of a reaction solution containing the multiple types of gelling agents. [8] The method according to [7], wherein the volume ratio of the volume of the reaction solution droplets to the volume of the cell-containing solution injected is 2 to 10. [9] The method according to any one of [2] to [4], comprising, prior to the gelling step, a step of mixing the cells, the extracellular matrix components, and the polyelectrolyte to obtain a mixed solution.

[10] The method according to [9], comprising, prior to the gelling step, a step of centrifuging the mixed solution to obtain a precipitate containing the cells, the extracellular matrix components, and the polyelectrolyte.

[11] The method according to any one of [2] to [4] and [9] to

[10] , wherein the concentration of the polyelectrolyte is greater than 0 mg / mL and not greater than 1.5 mg / mL, based on the total volume of the cell-containing solution.

[12] The method according to any one of [2] to [4] and [9] to

[11] , wherein the concentration of the extracellular matrix components is greater than 0 mg / mL and not greater than 1.5 mg / mL, based on the total volume of the cell-containing solution.

[13] The method according to any one of [1] to

[12] , wherein the cells further comprise hepatocytes.

[14] The method according to any one of [1] to

[13] , wherein the cells further comprise hepatic stellate cells.

[15] The method according to any one of [1] to

[14] , wherein the ratio of the number of vascular endothelial cells to the total number of cells is 5% or more and 50% or less.

[16] A method for producing a cell structure having a vascular structure, comprising the steps of: mixing cells, including vascular endothelial cells, an extracellular matrix component, and a polyelectrolyte to obtain a mixed solution; centrifuging the mixed solution to obtain a precipitate containing the cells, the extracellular matrix component, and the polyelectrolyte; obtaining a cell-containing solution containing the precipitate and a gelation promoter; injecting the cell-containing solution into a droplet of a reaction solution containing multiple types of gelling agents to form a gel; and culturing the cells in the gel, wherein the concentration of the polyelectrolyte is greater than 0 mg / mL and not greater than 1.5 mg / mL based on the total volume of the cell-containing solution; the concentration of the extracellular matrix component is greater than 0 mg / mL and not greater than 1.5 mg / mL based on the total volume of the cell-containing solution; the multiple types of gelling agents include fibrinogen and sodium alginate; and the content of the fibrinogen is 1 mg / mL to 10 mg / mL based on the total volume of the reaction solution. a content of the sodium alginate of 0.01% by mass to 2% by mass based on the total volume of the reaction solution; the gelation accelerator contains thrombin and calcium ions; the content of the thrombin of 10 units / ml to 30 units / ml based on the total volume of the cell-containing solution; the content of the calcium ions of 1 mM to 10 mM based on the total volume of the cell-containing solution; and a volume ratio of the volume of droplets of the reaction solution to the volume of injected cell-containing solution of 2 to 10.

[17] A method for producing a cell structure having a vascular structure, the method comprising the steps of: mixing cells, including vascular endothelial cells, an extracellular matrix component, and a polyelectrolyte to obtain a mixed solution; centrifuging the mixed solution to obtain a precipitate containing the cells, the extracellular matrix component, and the polyelectrolyte; obtaining a cell-containing solution containing the precipitate and a gelation promoter; injecting the cell-containing solution into a droplet of a reaction solution containing multiple types of gelling agents to form a gel; and culturing the cells in the gel, wherein the polyelectrolyte comprises heparin, and the concentration of the polyelectrolyte is greater than 0 mg / mL and not greater than 1.5 mg / mL, based on the total volume of the cell-containing solution; the extracellular matrix component comprises collagen, and the concentration of the extracellular matrix component is greater than 0 mg / mL and not greater than 1.5 mg / mL, based on the total volume of the cell-containing solution; the multiple types of gelling agents comprise fibrinogen and sodium alginate; and the gelation promoter comprises thrombin and calcium ions.

[0007] According to the present invention, a method for producing a cell structure that can suppress variations in angiogenic ability can be provided.

[0008] 1 is an image showing the results of evaluating angiogenesis in a cell structure produced by the method of Test Example 1. FIG. 2 is an image showing the results of evaluating angiogenesis in a cell structure produced by the method of Test Example 2. FIG. 3 is an image showing the results of evaluating angiogenesis in a cell structure produced by the method of Test Example 3. FIG. 4 is an image showing the results of evaluating angiogenesis in a cell structure produced by the method of Test Example 4. FIG. 5 is an image showing the results of observing a cell structure produced by the method of Test Example 5. FIG. 6 is an image showing the results of observing a cell structure produced by the method of Test Example 6. FIG. 7 is an image showing the results of observing a cell structure produced by the method of Test Example 7. FIG. 1 is a schematic diagram for explaining one embodiment of a method for producing a cell structure, where (a) shows a gel containing cells that are precursors of the cell structure, and (b) shows a cell structure obtained by culturing the gel containing the cells in a culture medium.

[0009] Hereinafter, embodiments of the present invention will be described in detail, but the present invention is not limited to the following embodiments.

[0010] [Method for producing a cell structure] The method according to this embodiment is a method for producing a cell structure having a vascular structure, and includes a gelling step of gelling a cell-containing liquid containing cells, including vascular endothelial cells, to form a gel containing the cells, and a culturing step of culturing the cells in the gel, where the gel containing the cells is formed by contacting multiple types of gelling agents with a gelation accelerator. The method according to this embodiment may further include a preparation step of preparing a cell-containing liquid prior to the gelling step.

[0011] As used herein, the term "cell structure" refers to a cell aggregate (agglomerated cell population) in which multiple cells are arranged three-dimensionally, and refers to an aggregate artificially created by cell culture. The cell structure may contain only one type of cell, or two or more types of cells. FIG. 8 is a schematic diagram illustrating one embodiment of a method for producing a cell structure, including a gelation step and a culture step. In FIG. 8, (a) shows a gel containing cells after the gelation step and before the culture step. In FIG. 8, (b) shows a cell structure formed by culturing the gel containing the cells shown in (a) in a medium. As shown in FIG. 8(b), the cell structure contains a cell aggregate composed of multiple cells, and the cell aggregate is embedded in a gel. As shown in FIG. 8(b), the cell structure has a portion of the cell aggregate in contact with the bottom surface of the culture vessel, and a gel is formed to cover the cell aggregate.

[0012] As used herein, a "cell structure having a vascular structure" refers to a cell structure having a tubular structure formed by vascular endothelial cells. Blood vessels can be confirmed, for example, by immunohistochemical staining, microscopic observation using vascular endothelial cells expressing fluorescent proteins, etc. Having a vascular structure may mean, for example, that when the cell structure is observed from above with a microscope, blood vessels are formed to the extent that they have multiple branching points and form a network structure.

[0013] The shape of the cell structure is not particularly limited, and examples thereof include a sphere, an approximately sphere, an ellipsoid, an approximately ellipsoid, a hemisphere, an approximately hemisphere, a semicircle, an approximately semicircle, a rectangular parallelepiped, an approximately rectangular parallelepiped, etc. Here, biological tissue includes sweat glands, lymphatic vessels, sebaceous glands, etc., and has a more complex structure than a cell structure. Therefore, a cell structure can be easily distinguished from biological tissue.

[0014] As used herein, the term "gelling agent" refers to a material that forms a gel (hydrogel) upon reaction with a gelling accelerator, and refers to a material in an ungelled state. As used herein, the term "gelling accelerator" refers to a component that reacts with a gelling agent to gel a liquid containing the gelling agent. There are no particular limitations on the gelling agent and gelling accelerator, as long as they are materials that can form a gel that contains cells inside. The gelling agent and gelling accelerator may each be a chemical substance or an enzyme.

[0015] Examples of combinations of gelling agents and gelation promoters include a combination of fibrinogen and thrombin, a combination of sodium alginate and calcium ions, a combination of collagen and sodium chloride, and a combination of pectin and calcium sulfate.

[0016] The multiple gelling agents include at least a first gelling agent and a second gelling agent different from the first gelling agent. The first gelling agent and the second gelling agent may be a combination of gelling agents that have different solidification rates or different elastic moduli after gelation when used alone at a predetermined concentration. For example, using two gelling agents with different solidification rates may compensate for the solidification rate that would be insufficient if a single gelling agent with an appropriate elastic modulus parameter were used. Alternatively, using gelling agents with different elastic moduli may compensate for the solidification rate that would be insufficient if a single gelling agent with an appropriate elastic modulus parameter were used. The gelling agents may be two or more types, for example, five or less, four or less, or three or less types, with two types being preferred. The inclusion of multiple gelling agents can increase the droplet volume during gel formation. This suppresses cell diffusion and aggregation in the gel, stabilizing angiogenicity in the method for producing a cell structure. When multiple types of gelling agents are contained, the shape retention of the cell structure produced is improved compared to when the cell structure is produced under conditions in which only one type of gelling agent is contained.

[0017] The ratio of the mass of the second gelling agent to the mass of the first gelling agent can be adjusted appropriately depending on the type of gelling agent, and may be, for example, 5 or more, or 10 or more, or 10 or less, or 5 or less.

[0018] The gelation accelerator may include at least a first gelation accelerator that reacts with the first gelling agent to accelerate the gelation reaction, and a second gelation accelerator that reacts with the second gelling agent to accelerate the gelation reaction. The mass ratio of the second gelation accelerator to the first gelation accelerator can be adjusted appropriately depending on the type of gelation accelerator. The mass ratio of the second gelation accelerator to the first gelation accelerator may be, for example, 1 or more, or 2 or more, and may be 10 or less, or 5 or less.

[0019] From the viewpoints of cytotoxicity and stability, the multiple gelling agents may include fibrinogen and sodium alginate. In this case, the gelation accelerator may include thrombin and calcium ions.

[0020] An example of a method for producing a cell structure having a vascular structure, including a preparation step, a gelation step, and a culture step, will be described below.

[0021] <Preparation Step> In the preparation step, a cell-containing solution containing cells including vascular endothelial cells is prepared.

[0022] The cell-containing liquid may contain two or more gelation accelerators or two or more gelling agents. When two or more gelation accelerators are contained, the cell-containing liquid may contain, for example, thrombin and calcium ions. When two or more gelling agents are contained, the cell-containing liquid may contain, for example, fibrinogen and sodium alginate. The cell-containing liquid may contain a first gelling agent and a second gelling accelerator (a second gelling accelerator that does not react with the first gelling agent to form a gel). In this case, the cell-containing liquid may contain thrombin and sodium alginate, or fibrinogen and calcium ions.

[0023] The amount of gelling agent and / or gelation accelerator used can be appropriately set depending on the type of gelling agent and gelation accelerator, etc. The total content of gelling agent and / or gelation accelerator in the cell-containing liquid may be 0.1 mg / mL or more, 0.5 mg / mL or more, 1 mg / mL or more, 2 mg / mL or more, 3 mg / mL or more, 4 mg / mL or more, or 5 mg / mL or more, or may be 100 mg / mL or less, 90 mg / mL or less, 80 mg / mL or less, 70 mg / mL or less, 60 mg / mL or less, or 50 mg / mL or less, based on the total amount of the cell-containing liquid.

[0024] When the cell-containing fluid contains fibrinogen, the fibrinogen concentration may be, based on the total volume of the cell-containing fluid, 0.1 mg / ml or more, 0.5 mg / ml or more, 1.0 mg / ml or more, 1.5 mg / ml or more, 2.0 mg / ml or more, 2.5 mg / ml or more, 3.0 mg / ml or more, 3.5 mg / ml or more, 4.0 mg / ml or more, or 4.5 mg / ml or more. When the cell-containing fluid contains fibrinogen, the fibrinogen concentration may be 100 mg / ml or less, 90 mg / ml or less, 80 mg / ml or less, 70 mg / ml or less, 60 mg / ml or less, 50 mg / ml or less, 40 mg / ml or less, 30 mg / ml or less, 20 mg / ml or less, 10 mg / ml or less, 9.0 mg / ml or less, 8.0 mg / ml or less, 7.0 mg / ml or less, 6.0 mg / ml or less, or 5.5 mg / ml or less, based on the total volume of the cell-containing fluid. When the cell-containing fluid contains fibrinogen, the fibrinogen concentration may be 0.1 mg / ml to 100 mg / ml, 0.5 mg / ml to 50 mg / ml, 1 mg / ml to 20 mg / ml, 2 mg / ml to 10 mg / ml, 3 mg / ml to 8 mg / ml, or 4 to 6 mg / ml (e.g., 5 mg / ml), based on the total volume of the cell-containing fluid. When the fibrinogen concentration is within the above range, the three-dimensional structure of the cell structure after tissue preparation is more stable.

[0025] When the cell-containing liquid contains thrombin, the thrombin concentration may be, based on the total volume of the cell-containing liquid, 1 unit / ml or more, 2 units / ml or more, 4 units / ml or more, 6 units / ml or more, 8 units / ml or more, 10 units / ml or more, 12 units / ml or more, 14 units / ml or more, 16 units / ml or more, 18 units / ml or more, 19 units / ml or more, or 19.5 units / ml or more. When the cell-containing liquid contains thrombin, the thrombin concentration may be 1000 units / ml or less, 800 units / ml or less, 600 units / ml or less, 400 units / ml or less, 200 units / ml or less, 100 units / ml or less, 80 units / ml or less, 60 units / ml or less, 40 units / ml or less, 30 units / ml or less, or 25 units / ml or less, based on the total volume of the cell-containing liquid. When the cell-containing liquid contains thrombin, the thrombin concentration may be 1 unit / ml to 1000 unit / ml, 2 unit / ml to 500 unit / ml, 5 unit / ml to 100 unit / ml, 10 unit / ml to 50 unit / ml, or 15 unit / ml to 30 unit / ml (e.g., 20 unit / ml) based on the total volume of the cell-containing liquid. When the thrombin concentration is within the above-mentioned range, the time from the start of the reaction with fibrinogen to gel formation is more stable, and a stable cell structure can be created with less risk of gel damage even if a medium is added thereafter.

[0026] When the cell-containing liquid contains sodium alginate, the content of sodium alginate may be 0.01% by mass or more, 0.02% by mass or more, 0.03% by mass or more, or 0.04% by mass or more based on the total amount of the cell-containing liquid. When the cell-containing liquid contains sodium alginate, the content of sodium alginate may be 2.0% by mass or less, 1.5% by mass or less, 1.0% by mass or less, 0.50% by mass or less, 0.10% by mass or less, 0.08% by mass or less, or 0.06% by mass or less based on the total amount of the cell-containing liquid. When the cell-containing liquid contains sodium alginate, the content of sodium alginate may be 0.01% by mass to 2% by mass, 0.01% by mass to 1.5% by mass, 0.02% by mass to 1.0% by mass, 0.02% by mass to 0.50% by mass, 0.03% by mass to 0.10% by mass, or 0.03% by mass to 0.08% by mass (e.g., 0.05% by mass) based on the total amount of the cell-containing liquid. When the content of sodium alginate is within the above-mentioned range, the time from the start of the reaction with calcium ions to gel formation is more stable, and a stable cell structure can be produced with less risk of gel damage even when a culture medium is added thereafter.

[0027] When the cell-containing fluid contains calcium ions, the calcium ion concentration, based on the total volume of the cell-containing fluid, may be 1.0 mM or more, 2.0 mM or more, 3.0 mM or more, 4.0 mM or more, 4.5 mM or more, or 5.0 mM or more. When the cell-containing fluid contains calcium ions, the calcium ion concentration, based on the total volume of the cell-containing fluid, may be 200 mM or less, 150 mM or less, 100 mM or less, 80 mM or less, 60 mM or less, 40 mM or less, 20 mM or less, 10 mM or less, 8 mM or less, or 6 mM or less. When the cell-containing fluid contains calcium ions, the calcium ion concentration, based on the total volume of the cell-containing fluid, may be 1 mM to 200 mM, 1 mM to 150 mM, 2 mM to 100 mM, 2 mM to 50 mM, 5 mM to 20 mM, or 5 mM to 10 mM. A cell-containing solution containing calcium ions can be prepared by adding a calcium salt, such as calcium chloride, to the cell-containing solution. When the calcium ion concentration is within the above-mentioned range, the time from the start of the reaction with sodium alginate to gel formation is more stable, and a stable cell structure can be produced with less risk of gel damage even if a culture medium is added thereafter.

[0028] (Cells) The cells include at least vascular endothelial cells. Vascular endothelial cells refer to flat cells that form the surface of the vascular lumen. The vascular endothelial cells used in the culture process may be, for example, sinusoidal endothelial cells, human umbilical vein-derived endothelial cells (HUVECs), etc. Sinusoidal endothelial cells are non-parenchymal hepatic cells (cells that make up the liver other than hepatocytes) that have a characteristic morphology different from other vascular endothelial cells, such as a collection of numerous small pores (cribriform structure) in the cytoplasm and a lack of a basement membrane. The vascular endothelial cells used in the culture process may be primary cells (primary vascular endothelial cells) collected from the liver of an animal (e.g., human), cells cultured from primary cells, a cultured cell line established from primary cells, or cells artificially differentiated from stem cells. The cultured cell line may be established by gene transfer, specifically, a cultured cell line into which an immortalization-inducing gene such as hTERT or SV40LT has been introduced. Examples of primary vascular endothelial cells include primary sinusoidal endothelial cells, such as product model number 5000 manufactured by Sciencell. When a cultured cell line immortalized by gene transfer into primary sinusoidal endothelial cells is used, it is expected that the effects of angiogenesis and maintenance by growth factors involved in angiogenesis will be further improved. Examples of cultured cell lines include the cultured cell line, product model number T0056 manufactured by Applied Biological Materials. Stem cells to be differentiated include embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), etc. The vascular endothelial cells used in the culture process may be non-cancerous cells.

[0029] The cells may further include epithelial cells. Epithelial cells are cells that form epithelial tissues present in the skin, digestive organs, blood vessels, etc. Examples of epithelial cells include hepatocytes, epidermal keratinocytes, renal epithelial cells, vascular epithelial cells, alveolar epithelial cells, intestinal epithelial cells, retinal epithelial cells, neuroepithelial cells, gingival epithelial cells, bile duct epithelial cells, and thymic epithelial cells.

[0030] The cells preferably include hepatocytes. Hepatocytes, also known as hepatocytes, are cells that have functions such as secreting bile and plasma proteins. The hepatocytes constituting the cell structure may be primary hepatocytes collected from an animal's liver, cultured primary hepatocytes, a cultured cell line established from primary hepatocytes, or hepatoblasts artificially differentiated from stem cells. Examples of primary hepatocytes include frozen primary hepatocytes and primary hepatocytes collected from human hepatocyte chimeric animals, such as PXB Cell (registered trademark) and HepaSH (registered trademark), in which primary human hepatocytes are transplanted into the liver of an animal such as a mouse. Examples of cultured cell lines include cell lines derived from inactivated hepatoma cells, such as HepG2. Examples of stem cells that can be differentiated into hepatoblasts include embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), and mesenchymal stem cells. Hepatocytes are preferably non-cancerous cells such as primary hepatocytes and hepatoblasts, with PXB cells being more preferred due to their ease of handling. Functionally normal and mature hepatocytes are preferred because they enable the cell structure to exhibit high liver function similar to that of the native organism. Specifically, primary hepatocytes, HepaRG (registered trademark), and hepatocytes highly differentiated from stem cells can be used. Generally, when such functionally mature hepatocytes are co-cultured within a cell structure containing vascular endothelial cells, it tends to be difficult for the vascular endothelial cells to form and maintain a vascular network within the tissue. However, according to a method of one embodiment of the present invention, the formation and maintenance of a vascular network is also possible.

[0031] The cells may include one or more types of epithelial cells. The cells may include, for example, a plurality of hepatocytes having different genotypes for proteins involved in liver function. All hepatocytes contained in the cells may have the same genotype for proteins involved in liver function. Examples of proteins involved in liver function include drug-metabolizing enzymes.

[0032] The cells may further include hepatic stellate cells. Hepatic stellate cells are non-parenchymal hepatic cells (cells other than hepatocytes among the cells that make up the liver), and have functions such as storing vitamin A. They are present in the space of Disse, which is the region between hepatocytes and sinusoids in the liver. The hepatic stellate cells may be primary hepatic stellate cells collected from the liver, or may be cells cultured from primary hepatic stellate cells. The hepatic stellate cells may be cell lines established from primary cells, or hepatic stellate cells artificially differentiated from stem cells. Examples of hepatic stellate cells include hepatic stellate cell lines such as LX2.

[0033] The cells may further include cells other than epithelial cells, vascular endothelial cells, and hepatic stellate cells. The other cells may be, for example, mature somatic cells, or undifferentiated cells such as stem cells. Specific examples of somatic cells include nerve cells, dendritic cells, immune cells, lymphatic endothelial cells, fibroblasts, cardiac myocytes, pancreatic islet cells, smooth muscle cells, bone cells, and spleen cells. Examples of stem cells include ES cells, iPS cells, and mesenchymal stem cells. The other cells may be normal cells, or cells in which any cell function is enhanced or suppressed, such as cancer cells. A "cancer cell" is a cell that is derived from a somatic cell and has the ability to proliferate indefinitely.

[0034] The origin of the cells 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.

[0035] The total number of cells (X0) is not particularly limited and is determined appropriately taking into consideration the thickness and shape of the cell structure to be constructed, the size of the cell culture vessel to be used for construction, etc. The total number of cells (X0) is 1 x 10 ３ cells or more, 1 x 10 ４ cells or more, 1 x 10 ５ cells or more, or 1 x 10 ６ The total number of cells (X0) may be 1 x 10 ９ Below cells, 1x10 ７ cells or less, or 1 x 10 ５ It may also be cells.

[0036] The ratio (X1 / X0×100) of the number of vascular endothelial cells (X1) to the total number of cells (X0) may be 5% or more, 10% or more, 15% or more, or 20% or more, and may be 50% or less, 45% or less, 40% or less, 35% or less, or 30% or less, from the viewpoint of being more suitable as a cell structure having blood vessels. From the viewpoint of being more suitable as a cell structure having blood vessels, the ratio of the number of vascular endothelial cells to the total number of cells is preferably 20% or more and 30% or less.

[0037] The ratio (X2 / X0×100) of the number of epithelial cells (X2) to the total number of cells (X0) may 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 may be 95% or less, 90% or less, 80% or less, 75% or less, or 70% or less, from the viewpoint of being more suitable as a cell structure containing epithelial cells. The ratio of the number of epithelial cells to the total number of cells is preferably 60% or more and 80% or less, from the viewpoint of being more suitable as a cell structure containing epithelial cells. When the epithelial cells are hepatocytes, the ratio of hepatocytes is desirably 50% or more, which is a condition close to that in vivo.

[0038] The ratio (X3 / X0×100) of the number of hepatic stellate cells (X3) to the total number of cells (X0) may be 1% or more, 3% or more, 5% or more, 8% or more, or 10% or more, and may be 30% or less, 25% or less, 20% or less, 15% or less, or 10% or less, from the viewpoint of being even more suitable as a cell structure containing hepatic stellate cells. From the viewpoint of being even more suitable as a cell structure containing hepatic stellate cells, the ratio of the number of hepatic stellate cells to the total number of cells is preferably 5% or more and 15% or less.

[0039] (Extracellular matrix components) The cell-containing solution may further contain extracellular matrix components. When the cell-containing solution contains extracellular matrix components, cell accumulation is promoted. This promotes adhesion between vascular endothelial cells, making it easier to further suppress variation in angiogenic ability.

[0040] As used herein, the term "extracellular matrix component" refers to an assembly of extracellular matrix molecules formed by multiple extracellular matrix molecules. An extracellular matrix molecule refers to a substance that exists outside cells in an organism. Any appropriate substance can be used as the extracellular matrix as long as it does not adversely affect cell growth and cell aggregate formation. Specific examples of extracellular matrix molecules include, but are not limited to, collagen, elastin, proteoglycan, fibronectin, hyaluronic acid, laminin, vitronectin, tenascin, entactin, fibrillin, and cadherin. The extracellular matrix component may be used alone or in combination.

[0041] The extracellular matrix may be a modified or variant of the above-mentioned extracellular matrix, or a polypeptide such as a chemically synthesized peptide, as long as it does not adversely affect cell growth and cell aggregate formation. The extracellular matrix may have repeats of a sequence represented by Gly-X-Y, which is characteristic of collagen. Here, Gly represents a glycine residue, and X and Y each independently represent an appropriate amino acid residue. Multiple Gly-X-Y residues may be the same or different. Having repeats of a sequence represented by Gly-X-Y reduces constraints on the molecular chain arrangement, thereby improving, for example, the function as a scaffold material during cell culture. In an extracellular matrix having repeats of a sequence represented by Gly-X-Y, the proportion of the sequence represented by Gly-X-Y may be 80% or more, preferably 95% or more, of the total amino acid sequence. The extracellular matrix may also be a polypeptide having an RGD sequence. The RGD sequence refers to a sequence represented by Arg-Gly-Asp (arginine residue-glycine residue-aspartic acid residue). Having the RGD sequence further promotes cell adhesion, making it even more suitable as a scaffold material for cell culture, for example. Examples of extracellular matrices containing a sequence represented by Gly-X-Y and an RGD sequence include collagen, fibronectin, vitronectin, laminin, and cadherin.

[0042] Examples of collagen include fibrous collagen and non-fibrous collagen. Fibrous collagen refers to collagen that is the main component of collagen fibers, and specific examples include type I collagen, type II collagen, and type III collagen. Examples of non-fibrous collagen include type IV collagen.

[0043] Proteoglycans include, but are not limited to, chondroitin sulfate proteoglycans, heparan sulfate proteoglycans, keratan sulfate proteoglycans, and dermatan sulfate proteoglycans.

[0044] Examples of the shape of the extracellular matrix components include fibrous shapes. Fibrous shapes refer to shapes composed of thread-like extracellular matrix components or shapes composed of thread-like extracellular matrix components cross-linked intermolecularly. At least a portion of the extracellular matrix components may be fibrous. The shape of the extracellular matrix component refers to the shape of a single extracellular matrix component (aggregate of extracellular matrix components) observed under a microscope, and the extracellular matrix component preferably has an average diameter and / or average length as described below. Fibrous extracellular matrix components include thin thread-like materials (fibers) formed by the aggregation of multiple thread-like extracellular matrix molecules, filaments formed by the further aggregation of filaments, and defibrillated versions of these filaments. When fibrous extracellular matrix components are included, the RGD sequences in the fibrous extracellular matrix components are preserved without being destroyed, allowing them to function more effectively as a scaffold for cell adhesion.

[0045] The extracellular matrix component may include at least one selected from the group consisting of collagen, laminin, and fibronectin, and preferably includes collagen. The collagen is preferably fibrous collagen, more preferably type I collagen. As the fibrous collagen, commercially available collagen may be used, and a specific example thereof is type I collagen derived from porcine skin manufactured by Nippon Meat Packers, Ltd.

[0046] The extracellular matrix components may be derived from animals. Examples of animal species from which the extracellular matrix components are derived include, but are not limited to, humans, pigs, and cows. The extracellular matrix components may be derived from a single type of animal, or may be derived from multiple types of animals in combination.

[0047] The content of the extracellular matrix component can be appropriately determined depending on the shape, thickness, etc. of the desired cell structure. The content of the extracellular matrix component may be, 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, or 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, based on the total volume of the cell-containing liquid. When the content of the extracellular matrix component is within the above-mentioned range, adhesion between cells is more easily promoted, the three-dimensional structure is more stabilized, and vascular network formation is also more stabilized.

[0048] (Polyelectrolyte) The cell-containing solution may further contain a polyelectrolyte. A polyelectrolyte is a polymer compound having electrolytic properties. When the cell-containing solution contains a polyelectrolyte, cell accumulation is promoted. This promotes adhesion between vascular endothelial cells, making it easier to further suppress variations in angiogenesis ability.

[0049] Examples of polymer electrolytes include, but are not limited to, glycosaminoglycans such as heparin, chondroitin sulfate (e.g., chondroitin 4-sulfate, chondroitin 6-sulfate), heparan sulfate, dermatan sulfate, keratan sulfate, and hyaluronic acid; dextran sulfate, rhamnan sulfate, fucoidan, carrageenan, polystyrene sulfonic acid, polyacrylamide-2-methylpropane sulfonic acid, and polyacrylic acid, or derivatives thereof. The polymer electrolyte may consist of one of the above-mentioned materials, or may contain two or more of them in combination.

[0050] The polymer electrolyte is preferably a glycosaminoglycan, more preferably contains at least one selected from the group consisting of heparin, dextran sulfate, chondroitin sulfate, and dermatan sulfate, and even more preferably contains heparin.

[0051] The concentration of the polyelectrolyte in the cell-containing solution is not particularly limited as long as it does not adversely affect cell growth and the formation of cell structures. The polyelectrolyte concentration may be, for example, greater than 0 mg / mL and less than 1.5 mg / mL, based on the total volume of the cell-containing solution. The polyelectrolyte concentration may be, based on the total volume of the cell-containing solution, 0.005 mg / mL or more, 0.01 mg / mL or more, 0.02 mg / mL or more, 0.03 mg / mL or more, or 0.04 mg / mL or more, or 1.5 mg / mL or less, 1.0 mg / mL or less, 0.1 mg / mL or less, 0.08 mg / mL or less, or 0.06 mg / mL or less. The polyelectrolyte concentration may be, for example, 0.025 mg / mL, 0.05 mg / mL, 0.075 mg / mL, or 0.1 mg / mL, based on the total volume of the cell-containing solution. When the concentration of the polyelectrolyte is within the above range, adhesion between cells is more easily promoted while excessive aggregation between cells is suppressed, and the three-dimensional structure and vascular network formation are stabilized.

[0052] The ratio of the mass C2 of the polymer electrolyte to the mass C1 of the extracellular matrix components (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 may be 1 / 1.5 to 1.5 / 1. When the ratio C2 of the polymer electrolyte to the mass C1 of the extracellular matrix components (C2 / C1) is within the above-mentioned range, adhesion between vascular endothelial cells is more easily promoted, and variation in angiogenic ability is more easily suppressed.

[0053] (Aqueous Medium) The cell-containing liquid includes an aqueous medium. "Aqueous medium" refers to a liquid containing water as an essential component. The aqueous medium may be, for example, an aqueous medium containing a cationic substance. The aqueous medium containing a cationic substance may be, for example, a cationic buffer solution such as Tris-hydrochloric acid buffer, Tris-maleic acid buffer, Bis-Tris buffer, or HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), or may be a medium containing water and a cationic compound such as ethanolamine, diethanolamine, triethanolamine, polyvinylamine, polyallylamine, polylysine, polyhistidine, or polyarginine as the cationic substance. Furthermore, a culture medium can also be used as the aqueous medium. Examples of the culture medium include liquid media such as Dulbecco's Modified Eagle Medium (DMEM) and Hepatocyte-specific Medium (HCM). The liquid medium may also be a mixed medium obtained by mixing two types of culture media.

[0054] The concentration and pH of the cationic substance (e.g., Tris in Tris-HCl buffer) in the aqueous medium containing the 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 may be 10 to 100 mM, 20 to 80 mM, 40 to 70 mM, 40 to 60 mM, or 50 mM, based on the total amount of the aqueous medium containing the cationic substance. The pH of the aqueous medium (e.g., cationic buffer) may be 6.0 to 8.0, 6.8 to 7.8, or 7.2 to 7.6.

[0055] (Method for producing cell-containing fluid) The cell-containing fluid can be obtained by a method comprising mixing cells, multiple types of gelling agents, an aqueous medium, and, if necessary, an extracellular matrix component and a polymer electrolyte. The order of mixing is not particularly limited. The method for producing the cell-containing fluid may also comprise centrifuging the mixture, if necessary. Specific examples of methods for producing cell-containing fluid include a method comprising the steps of: mixing cells, an extracellular matrix component, and a polymer electrolyte to obtain a mixture; centrifuging the mixture to obtain a precipitate containing the cells, the extracellular matrix component, and the polymer electrolyte; and mixing the precipitate with multiple types of gelling agents to obtain the cell-containing fluid.

[0056] <Gelling step> In the gelling step, a cell-containing liquid containing cells is gelled to form a cell-containing gel. Gelation may be carried out by contacting the cell-containing liquid with an aqueous medium and a reaction liquid containing a gelling agent and / or a gelation accelerator. The type of gelling agent and / or gelation accelerator in the reaction liquid is determined depending on the type of gelling agent and / or gelation accelerator in the cell-containing liquid. The aqueous medium may be as described above.

[0057] The amount of gelling agent and / or gelation accelerator used in gelling the cell-containing liquid can be appropriately set depending on the type of gelling agent and gelation accelerator, etc. The total content of gelling agent and / or gelation accelerator in the reaction liquid, based on the total volume of the reaction liquid, may be 0.1 mg / mL or more, 0.5 mg / mL or more, 1 mg / mL or more, 2 mg / mL or more, 3 mg / mL or more, 4 mg / mL or more, or 5 mg / mL or more, or may be 100 mg / mL or less, 90 mg / mL or less, 80 mg / mL or less, 70 mg / mL or less, 60 mg / mL or less, or 50 mg / mL or less.

[0058] When the reaction solution contains fibrinogen, the fibrinogen concentration may be 0.1 mg / ml or more, 0.5 mg / ml or more, 1.0 mg / ml or more, 1.5 mg / ml or more, 2.0 mg / ml or more, 2.5 mg / ml or more, 3.0 mg / ml or more, 3.5 mg / ml or more, 4.0 mg / ml or more, or 4.5 mg / ml or more, based on the total volume of the reaction solution. When the reaction solution contains fibrinogen, the fibrinogen concentration may be 100 mg / ml or less, 90 mg / ml or less, 80 mg / ml or less, 70 mg / ml or less, 60 mg / ml or less, 50 mg / ml or less, 40 mg / ml or less, 30 mg / ml or less, 20 mg / ml or less, 10 mg / ml or less, 9.0 mg / ml or less, 8.0 mg / ml or less, 7.0 mg / ml or less, 6.0 mg / ml or less, or 5.5 mg / ml or less, based on the total volume of the reaction solution. When the reaction solution contains fibrinogen, the fibrinogen concentration may be 0.1 mg / ml to 100 mg / ml, 0.5 mg / ml to 50 mg / ml, 1 mg / ml to 20 mg / ml, 2 mg / ml to 10 mg / ml, 3 mg / ml to 8 mg / ml, or 4 to 6 mg / ml (e.g., 5 mg / ml), based on the total volume of the reaction solution. When the fibrinogen concentration is within the above range, the three-dimensional structure of the cell structure after tissue preparation is more stable.

[0059] When the reaction solution contains thrombin, the thrombin concentration may be, based on the total volume of the reaction solution, 1 unit / ml or more, 2 units / ml or more, 4 units / ml or more, 6 units / ml or more, 8 units / ml or more, 10 units / ml or more, 12 units / ml or more, 14 units / ml or more, 16 units / ml or more, 18 units / ml or more, 19 units / ml or more, or 19.5 units / ml or more. When the reaction solution contains thrombin, the thrombin concentration may be 1000 units / ml or less, 800 units / ml or less, 600 units / ml or less, 400 units / ml or less, 200 units / ml or less, 100 units / ml or less, 80 units / ml or less, 60 units / ml or less, 40 units / ml or less, 30 units / ml or less, or 25 units / ml or less, based on the total volume of the reaction solution. When the reaction solution contains thrombin, the thrombin concentration may be 1 unit / ml to 1000 units / ml, 2 units / ml to 500 units / ml, 5 units / ml to 100 units / ml, 10 units / ml to 50 units / ml, or 15 units / ml to 30 units / ml (e.g., 20 units / ml), based on the total volume of the reaction solution. When the thrombin concentration is within the above-mentioned range, the time from the start of the reaction with fibrinogen to the formation of a gel is more stable, and a stable cell structure can be created with less risk of the gel being damaged even if culture medium is added thereafter.

[0060] When the reaction solution contains sodium alginate, the content of sodium alginate may be 0.01% by mass or more, 0.02% by mass or more, 0.03% by mass or more, or 0.04% by mass or more, based on the total amount of the reaction solution. When the reaction solution contains sodium alginate, the content of sodium alginate may be 2.0% by mass or less, 1.5% by mass or less, 1.0% by mass or less, 0.50% by mass or less, 0.10% by mass or less, 0.08% by mass or less, or 0.06% by mass or less, based on the total amount of the reaction solution. When the reaction solution contains sodium alginate, the content of sodium alginate may be 0.01% by mass to 2% by mass, 0.01% by mass to 1.5% by mass, 0.02% by mass to 1.0% by mass, 0.02% by mass to 0.50% by mass, 0.03% by mass to 0.10% by mass, or 0.03% by mass to 0.08% by mass (e.g., 0.05% by mass) based on the total amount of the reaction solution. When the content of sodium alginate is within the above-mentioned range, the time from the start of the reaction with sodium alginate to gel formation is more stable, and a stable cell structure can be produced with less risk of gel damage even when a culture medium is added thereafter.

[0061] When the reaction solution contains calcium ions, the calcium ion concentration, based on the total volume of the reaction solution, may be 1.0 mM or more, 2.0 mM or more, 3.0 mM or more, 4.0 mM or more, 4.5 mM or more, or 5.0 mM or more. When the reaction solution contains calcium ions, the calcium ion concentration, based on the total volume of the reaction solution, may be 200 mM or less, 150 mM or less, 100 mM or less, 80 mM or less, 60 mM or less, 40 mM or less, 20 mM or less, 10 mM or less, 8 mM or less, or 6 mM or less. When the reaction solution contains calcium ions, the calcium ion concentration, based on the total volume of the reaction solution, may be 1 mM to 200 mM, 1 mM to 150 mM, 2 mM to 100 mM, 2 mM to 50 mM, 5 mM to 20 mM, or 5 mM to 10 mM. A reaction solution containing calcium ions can be prepared by adding a calcium salt such as calcium chloride to the reaction solution. When the calcium ion concentration is within the above-mentioned range, the time from the start of the reaction with sodium alginate to gel formation is more stable, and a more stable cell structure can be produced with less risk of gel damage even when a culture medium is added thereafter.

[0062] When gelation is carried out by contacting the cell-containing liquid with the reaction liquid, the gelation step may be carried out by injecting the cell-containing liquid or the reaction liquid (injection liquid) into a droplet of the other liquid (cell-containing liquid or reaction liquid), or by injecting the cell-containing liquid into a droplet of the reaction liquid, since this more easily forms a gel containing cells. The injection method is not particularly limited, and a conventional injection method can be used.

[0063] The droplet volume of the one liquid (cell-containing liquid or reaction liquid) may be 2 μL or more, 5 μL or more, 8 μL or more, or 10 μL or more, and may be 30 μL or less, 25 μL or less, 20 μL or less, 15 μL or less, or 10 μL or less, from the viewpoint of ease of handling and further improving the effect of suppressing variation in angiogenic ability. The volume ratio of the droplet volume of the one liquid to the injection volume of the other liquid (injection liquid) (droplet volume (μL) / injection volume (μL)) may be 2 or more, 3 or more, or 4 or more, and may be 10 or less, 9 or less, 8 or less, 7 or less, or 6 or less, from the viewpoint of further improving the effect of suppressing variation in angiogenic ability.

[0064] The gelation step may be carried out by injecting one of the cell-containing liquid and the reaction liquid into a droplet of the other liquid formed on the substrate. A substrate commonly used as a culture vessel can be used. The substrate may be a vessel having a surface capable of forming a gel and capable of accommodating a culture medium. The material and shape of the substrate surface are not particularly limited. The substrate may be, for example, a dish, a well insert, or a plate having a bottom shape such as a U-shape or V-shape. The substrate may be surface-treated. The surface treatment method and the coating agent that can be used for the surface treatment are not particularly limited. From the viewpoint of ease of tissue preparation, the substrate is preferably a culture vessel having a flat bottom.

[0065] Since a gel encapsulating cells or a cell population can be more easily formed, the gelation step is preferably a step of forming a gel by injecting a cell-containing liquid into a droplet of a reaction liquid, and the volume ratio of the volume of the droplet of the reaction liquid to the volume of the injected cell-containing liquid is preferably within the above-mentioned range.

[0066] The gelation step may include incubating (gelation culture) for a certain period of time after contacting multiple gelling agents with a gelation accelerator. Incubation refers to a process of leaving a sample stationary to promote a reaction. The incubation temperature (gelation culture temperature) may be, for example, 20°C to 40°C, or 30°C to 37°C. The incubation time (gelation culture time) may be 10 minutes or more, 20 minutes or more, or 30 minutes or more, and may be 60 minutes or less, 50 minutes or less, or 40 minutes or less. In conventional methods, the incubation time after gelation may cause variation in the angiogenic ability of the produced cell structure. On the other hand, according to the method of the present embodiment, variation in the angiogenic ability of the cell structure due to the incubation time is suppressed.

[0067] <Culturing step> In the culturing step, cells are cultured in the gel. The culturing step can be performed after adding a medium to the culture substrate on which the gel containing the cells is placed. As used herein, "culturing cells" means maintaining the cells in the medium under conditions that prevent their death or decline. Culturing cells may or may not be accompanied by cell proliferation.

[0068] A suitable medium can be selected depending on the type of cells to be cultured. Examples of such media include Eagle's MEM medium, DMEM, Modified Eagle's Medium (MEM), Minimum Essential Medium, RPMI, GlutaMax medium, and hepatocyte medium (e.g., HCM medium (manufactured by Lonza)). The medium may be a serum-supplemented medium or a serum-free medium. Furthermore, the medium may be a mixed medium comprising a mixture of two or more types of media. In addition, supplemental factors may be diluted to a predetermined concentration and added to the medium as needed. The supplemental factors may be, for example, individual growth factors such as VEGF-A or VEGF-A, a cocktail of such supplemental factors, or an extract collected from a biological sample, such as Matrigel (registered trademark).

[0069] The cell culture conditions in the culture step can be set appropriately depending on the type of cell. For example, the culture temperature may be 20°C to 40°C, or 30°C to 37°C. The pH of the medium may be 6 to 8, or 7.2 to 7.4. The culture time may be 1 day to 2 weeks, or 1 week to 2 weeks.

[0070] The cell structures produced by the method of this embodiment have reduced variation in angiogenic activity. Furthermore, the method of this embodiment suppresses evaporation of liquid components that occurs during gelation and damage to the gel when culture medium is added during the culture process. In addition, the method of this embodiment does not require the use of special equipment for tissue production, and cell structures can be produced manually. Therefore, the method of this embodiment can easily produce cell structures with reduced variation in angiogenic activity.

[0071] [Cell Structure] The cell structure according to this embodiment includes cells, including vascular endothelial cells, and multiple types of gels encapsulating the cells. The gels may be gels formed by the reaction of the gelling agent and gelation promoter described above, and may include, for example, fibrin gel and alginate gel. The cell structure according to this embodiment may include extracellular matrix components and may further include a polyelectrolyte. The cell structure according to this embodiment can be obtained, for example, by the method described above.

[0072] When the cell structure of this embodiment contains an extracellular matrix component, the content of the extracellular matrix component in the cell structure may be, based on the dry weight of the cell structure, 0.01% by mass or more, 0.05% by mass or more, 0.1% by mass or more, 0.5% by mass or more, 1% by mass or more, 2% by mass or more, 3% by mass or more, 4% by mass or more, 5% by mass or more, 6% by mass or more, 7% by mass or more, 8% by mass or more, 9% by mass or more, 10% by mass or more, 15% by mass or more, 20% by mass or more, 25% by mass or more, or 30% by mass or more, or may be 90% by mass or less, 80% by mass or less, 70% by mass or less, 60% by mass or less, 50% by mass or less, 30% by mass or less, 20% by mass or less, or 15% by mass or less. The content of extracellular matrix components in the cell structure may be 0.01 to 90% by mass, 10 to 90% by mass, 10 to 80% by mass, 10 to 70% by mass, 10 to 60% by mass, 1 to 50% by mass, 10 to 50% by mass, 10 to 30% by mass, or 20 to 30% by mass, based on the dry weight of the cell structure.

[0073] The maximum length in a microscope image when the cell structure according to this embodiment is observed from above with a microscope may be 1000 μm or more, or 2000 μm or more, or may be 4000 μm or less, or 3000 μm or less. From the viewpoint of maintaining the three-dimensional shape of the cell structure, the maximum length is preferably 2000 μm to 3000 μm.

[0074] The present invention will be described in more detail below with reference to examples, although the present invention is not limited to these examples.

[0075] The following cells were used: Hepatocytes (PXB Cells (registered trademark), manufactured by Phoenix Bio) Hepatic stellate cells (LX2, manufactured by Sigma-Aldrich) Vascular endothelial cells (GFP-HUVEC, manufactured by ANGIO-PROTEOMIE)

[0076] Test Example 1 LX2 and GFP-HUVECs were awakened and pre-cultured according to the manufacturer's protocol. LX2 and GFP-HUVECs were detached using 0.05% Trypsin / EDTA. PXB cells were detached using 0.25% Trypsin / EDTA. After detachment, LX2 and GFP-HUVECs were suspended in 10% FBS-containing DMEM medium, and PXB cells were suspended in HCGM medium. A portion of the suspension was mixed with trypan blue, and the cell concentration was measured using a Countess® II FL (Invitrogen). After the measurement, taking into account the number of wells to be prepared, the necessary cell amounts were aliquoted so that the cell seeding amounts per well were 19,500 PXB cells, 7,500 GFP-HUVEC cells, and 3,000 LX2 cells, and the three types of cells were mixed. The resulting mixture was centrifuged at 1200 rpm for 5 minutes, the supernatant was carefully removed, and the cells were suspended in a heparin-collagen solution. The heparin-collagen solution was prepared by mixing equal amounts of 1.0 mg / ml heparin solution and 0.6 mg / ml collagen solution dissolved in 100 mM Tris buffer. After suspension, the cell suspension was left at room temperature for 3 minutes and then centrifuged at 400 G for 2 minutes. The supernatant was then carefully removed, and the cells were suspended in 20 U / ml thrombin solution dissolved in HCM medium supplemented with ECGS (hereinafter simply referred to as "HCM medium") to a concentration of 30,000 cells / 2 μL. This prepared a cell-containing solution of Test Example 1 containing cells and thrombin as a gelation promoter.

[0077] A 5 mg / ml fibrinogen solution was dropped onto a 48-well plate to form droplets containing fibrinogen as a gelling agent. The volume of the fibrinogen solution droplets was 2 μL. The cell-containing solution of Test Example 1 containing thrombin was dropped so as to be injected into the droplets containing fibrinogen (reaction solution). The volume of the cell-containing solution injected was 2 μL. In other words, the volume ratio of the injected volume of the cell-containing solution to the volume of the droplets containing fibrinogen (reaction solution) was 1:1. To promote gelation due to the reaction between thrombin and fibrinogen, the droplets of the reaction solution into which the cell-containing solution had been injected were heated at 37°C in CO 2 . ２ The cells were cultured in an incubator. The gelation incubation time was set to 30 minutes, 60 minutes, and 90 minutes. After incubation, 500 μL of HCM medium was added to the culture substrate, and the cells were cultured for 7 days.

[0078] Figure 1 shows the results of examining the angiogenic potential of GFP-HUVECs at each gelation incubation time by fluorescent observation. Figure 1 shows that the longer the incubation time, the lower the angiogenic potential.

[0079] Test Example 2 A cell structure was prepared in the same manner as in Test Example 1, except that the droplet volume of the fibrinogen solution in Test Example 1 was changed to 10 μL (the volume ratio of the injected volume of the cell-containing solution to the droplet volume of the fibrinogen-containing droplet (reaction solution) was set to 5:1). The results when the gelation incubation time was set to 30 minutes are shown in Figure 2.

[0080] As can be seen from Figure 2, when only one type of gelling agent is used and the amount of fibrinogen solution droplet is increased, the injected cells spread throughout the droplet, the three-dimensional structure is destroyed, and the vascular structure is not observed.

[0081] <Test Example 3> A cell-containing solution of Test Example 3 containing cells and calcium ions as a gelation promoter was prepared in the same manner as Test Example 1, except that an aqueous calcium solution prepared by dissolving calcium chloride in HCM medium to a concentration of 5 mM was used instead of the 20 U / ml thrombin solution dissolved in HCM medium.

[0082] A 0.05% by mass aqueous solution of sodium alginate, prepared by dissolving sodium alginate in HCM medium to a concentration of 0.05% by mass, was dropped onto a 48-well plate to form droplets containing sodium alginate as a gelling agent. The volume of the sodium alginate-containing solution droplets was 10 μL. The cell-containing solution of Test Example 3 containing calcium ions was dropped so as to be injected into the sodium alginate-containing droplets (reaction solution). The injection volume of the cell-containing solution was 2 μL. In other words, the volume ratio of the injection volume of the cell-containing solution to the volume of the reaction solution droplets was 5:1. To promote gelation, the reaction solution droplets into which the cell-containing solution had been injected were heated at 37°C in CO 2 . ２ The cells were cultured in an incubator. The gelation culture time was set to 30 minutes. The subsequent steps were the same as in Test Example 1, and a cell structure was produced. The results are shown in Figure 3.

[0083] From FIG. 3, it can be seen that under other conditions in which only one type of gelling agent was used, the cells aggregated excessively, which also prevented blood vessels from being formed.

[0084] Test Example 4 A cell-containing solution containing thrombin and calcium ions as gelation accelerators was prepared in the same manner as Test Example 1, except that a mixed solution containing thrombin and calcium ions was used instead of the 20 U / ml thrombin solution dissolved in HCM medium. The calcium ion concentration in the mixed solution was 5 mM based on the total volume of the mixed solution (cell-containing solution). The thrombin concentration in the mixed solution was 20 U / ml based on the total volume of the mixed solution (cell-containing solution).

[0085] A mixed solution of sodium alginate and fibrinogen prepared by dissolving sodium alginate and fibrinogen in HCM medium was dropped onto a 48-well plate to form droplets containing sodium alginate and fibrinogen as gelling agents. The sodium alginate content in the mixed solution was 0.05% by mass based on the total volume of the mixed solution (reaction solution). The fibrinogen concentration in the mixed solution was 5 mg / mL based on the total volume of the mixed solution. The droplet volume of the solution (reaction solution) containing sodium alginate and fibrinogen was 10 μL. The cell-containing solution of Test Example 4, containing thrombin and calcium ions, was added dropwise to the droplets (reaction solution) containing sodium alginate and fibrinogen. The injection volume of the cell-containing solution was 2 μL. The volume ratio of the injection volume of the cell-containing solution to the volume of the reaction solution droplet was 5:1. To promote gelation, the droplets of the reaction solution into which the cell-containing solution had been injected were incubated in CO at 37°C. ２ The cells were cultured in an incubator. The gelation culture time was set to 30, 60, and 90 minutes. The subsequent steps were the same as in Test Example 1, and a cell structure was produced. The results are shown in Figure 4.

[0086] Comparing the results shown in Figure 4 with those shown in Figure 1, it can be seen that when multiple types of gelling agents are used, there is no significant change in angiogenic ability even when the incubation time (gelling culture time) is extended. While blood vessels were not formed when a single gelling agent was used to enlarge the gel as shown in Figures 2 and 3, blood vessels were formed when multiple types of gelling agents were used as shown in Figure 4. This indicates that by using multiple types of gelling agents, blood vessels can be formed even when the droplets are enlarged. As shown in Figure 4, the angiogenic ability of the cell structure produced by the method of Test Example 4 was not affected by the incubation time.

[0087] Test Example 5 A cell structure was prepared in the same manner as in Test Example 1, except that only hepatocytes were used. The gelation incubation time was set to 30 minutes. The results are shown in Figure 5. The scale bar in the micrograph shown in Figure 5 indicates 1 mm.

[0088] Figure 5 shows that when only one gelling agent (fibrinogen alone) is used and the gel volume is small, the gel is damaged by the impact of adding the medium, causing cells to leak out. The arrows in Figure 5 indicate the areas where the gel is damaged and cells are leaking out.

[0089] Test Example 6 Tissue preparation was carried out in the same manner as Test Example 5, except that 10 μL of a gelling agent composed of fibrinogen and sodium alginate was used and the cell-containing solution contained 5 mM calcium ions, i.e., in the same manner as Test Example 4, except that only hepatocytes were used. The results are shown in Figure 6. The scale bar in the micrograph shown in Figure 6 indicates 1 mm.

[0090] From Figure 6, it can be observed that the use of multiple types of gelling agents prevents tissue damage and allows cells to aggregate appropriately.

[0091] Test Example 7 A cell-containing solution containing 20 U / ml thrombin and 5 mM calcium ions as a gelation accelerator was prepared in the same manner as in Test Example 4, except that GFP-SEC, which was obtained by introducing GFP into hepatocytes (PXB Cells) and sinusoidal endothelial cells (SEC, manufactured by Sciencell) by a general genetic recombination method using lentivirus, was used, and LX2 was not used.

[0092] A mixed solution of sodium alginate and fibrinogen prepared by dissolving sodium alginate and fibrinogen in HCM medium was dropped onto a 24-well Transwell insert to form droplets containing sodium alginate and fibrinogen as gelling agents. The sodium alginate content in the mixed solution was 0.05% by mass based on the total volume of the mixed solution (reaction solution). The fibrinogen concentration in the mixed solution was 5 mg / mL based on the total volume of the mixed solution. The droplet volume of the solution containing sodium alginate and fibrinogen (reaction solution) was 20 μL. The cell-containing solution of Test Example 4, containing thrombin and calcium ions, was added dropwise to the droplets containing sodium alginate and fibrinogen (reaction solution). The injection volume of the cell-containing solution was 2 μL. The volume ratio of the injection volume of the cell-containing solution to the volume of the reaction solution droplet was 10:1. To promote gelation, the droplets of the reaction solution into which the cell-containing solution had been injected were heated at 37°C in CO 2 . ２ The cells were cultured in an incubator. The gelation incubation time was set to 30 minutes. After incubation, 500 μL of HCM medium was added to the culture substrate, and the cells were cultured for 14 days.

[0093] The GFP-SEC images (from three trials) in Figure 7 are the results of verifying the angiogenic ability by fluorescent observation of GFP-SE on Day 14. The GFP-SEC images in Figure 7 show that the vascular network was sufficiently formed and maintained even on Day 14, the three-dimensional structure was formed without collapse, and there was no excessive aggregation.

[0094] After 14 days of culture, the cell structures were fixed with 10% neutral buffered formalin. After treatment with mouse anti-MRP2 antibody and rabbit anti-albumin antibody, immunostaining was performed with Alexa647-labeled anti-mouse IgG secondary antibody and Alexa546-labeled anti-rabbit IgG secondary antibody, and MRP2 and albumin expression was evaluated using a confocal microscope. MRP2 is a transporter expressed on bile canaliculi that form between mature, functional hepatocytes, and albumin is a protein produced within hepatocytes that serves as a hepatocyte marker.

[0095] Figure 7 shows images of cell structures stained with antibodies against MRP2 and albumin. From the image in Figure 7, it was confirmed that hepatocytes were accumulated within the cell structure without excessive aggregation, and that bile canaliculi were clearly and densely formed between the hepatocytes, as indicated by the MRP2 staining image. These results demonstrate that even when a cell structure is formed using sinusoidal endothelial cells by a method according to one embodiment of the present invention, the three-dimensional structure is appropriately maintained and the vascular network is maintained without adversely affecting the characteristics of the hepatocytes.

Claims

1. A method for manufacturing a cell structure having a vascular structure, comprising: a gelation step of gelling a cell-containing solution containing cells including vascular endothelial cells to form a gel containing the cells; and a culturing step of culturing the cells in the gel, wherein the gel containing the cells is formed by bringing a plurality of types of gelling agents into contact with a gelation promoter.

2. The method according to claim 1, wherein the cell-containing solution further contains an extracellular matrix component and a polyelectrolyte.

3. The method according to claim 2, wherein the extracellular matrix component contains collagen.

4. The method according to claim 2, wherein the polyelectrolyte contains heparin.

5. The method according to claim 1, wherein the plurality of types of gelling agents contain fibrinogen and sodium alginate.

6. The method according to claim 1, wherein the gelation promoter contains thrombin and calcium chloride.

7. The method according to any one of claims 1 to 6, wherein the gelation step is a step of forming the gel by injecting the cell-containing solution containing the cells and the gelation promoter into droplets of a reaction solution containing the plurality of types of gelling agents.

8. The method according to claim 7, wherein the volume ratio of the droplet amount of the reaction solution to the injection amount of the cell-containing solution is 2 to 10.

9. The method according to any one of claims 2 to 4, further comprising, before the gelation step, a step of mixing the cells, the extracellular matrix component, and the polyelectrolyte to obtain a mixed solution.

10. The method according to claim 9, further comprising, before the gelation step, a step of centrifuging the mixed solution to obtain a precipitate containing the cells, the extracellular matrix component, and the polyelectrolyte.

11. The method according to any one of claims 2 to 4, wherein the concentration of the polyelectrolyte is more than 0 mg / mL and 1.5 mg / mL or less based on the total amount of the cell-containing solution.

12. The method according to any one of claims 2 to 4, wherein the concentration of the extracellular matrix component is more than 0 mg / mL and 1.5 mg / mL or less based on the total amount of the cell-containing solution.

13. The method according to any one of claims 1 to 6, wherein the cells further include hepatocytes.

14. The method according to any one of claims 1 to 6, wherein the cells further include hepatic stellate cells.

15. The method according to any one of claims 1 to 6, wherein the ratio of the number of vascular endothelial cells to the total number of cells is 5% or more and 50% or less.

16. A method for manufacturing a cell structure having a vascular structure, comprising: a step of obtaining a mixed solution by mixing cells containing vascular endothelial cells, an extracellular matrix component, and a polyelectrolyte; a step of obtaining a precipitate containing the cells, the extracellular matrix component, and the polyelectrolyte by centrifuging the mixed solution; a step of obtaining a cell-containing solution containing the precipitate and a gelation accelerator; a step of forming a gel by injecting the cell-containing solution into droplets of a reaction solution containing a plurality of types of gelling agents; and a culturing step of culturing the cells in the gel, wherein the concentration of the polyelectrolyte is more than 0 mg / mL and 1.5 mg / mL or less based on the total amount of the cell-containing solution, the concentration of the extracellular matrix component is more than 0 mg / mL and 1.5 mg / mL or less based on the total amount of the cell-containing solution, the plurality of types of gelling agents include fibrinogen and sodium alginate, the content of fibrinogen is 1 mg / ml to 10 mg / ml based on the total amount of the reaction solution, the content of sodium alginate is 0.01% by mass to 2% by mass based on the total amount of the reaction solution, the gelation accelerator includes thrombin and calcium ions, the content of thrombin is 10 unit / ml to 30 unit / ml based on the total amount of the cell-containing solution, the content of calcium ions is 1 mM to 10 mM based on the total amount of the cell-containing solution, and the volume ratio of the droplet amount of the reaction solution to the injection amount of the cell-containing solution is 2 to 10.

17. A method for producing a cell structure having a vascular structure, comprising: a step of mixing cells containing vascular endothelial cells, an extracellular matrix component, and a polyelectrolyte to obtain a mixed solution; a step of centrifuging the mixed solution to obtain a precipitate containing the cells, the extracellular matrix component, and the polyelectrolyte; a step of obtaining a cell-containing solution containing the precipitate and a gelation accelerator; a step of forming a gel by injecting the cell-containing solution into droplets of a reaction solution containing a plurality of types of gelling agents; and a culturing step of culturing the cells in the gel, wherein the polyelectrolyte contains heparin, the concentration of the polyelectrolyte is more than 0 mg / mL and 1.5 mg / mL or less based on the total amount of the cell-containing solution, the extracellular matrix component contains collagen, the concentration of the extracellular matrix component is more than 0 mg / mL and 1.5 mg / mL or less based on the total amount of the cell-containing solution, the plurality of types of gelling agents contain fibrinogen and sodium alginate, and the gelation accelerator contains thrombin and calcium ions.

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