WO2025143240A1 - Method for producing cell structure - Google Patents

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

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

When evaluating the toxicity of compounds such as pharmaceuticals to blood vessels 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 toxicity to blood vessels 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 differences between species and social circumstances such as animal protection. To solve these problems, efforts are currently underway to develop artificial tissues with vascular structures using human-derived cells (for example, Patent Document 1).

International Publication No. 2021-100709

The inventors have developed a method for constructing a cell structure with a vascular structure by embedding cells in a gel (hydrogel) and culturing them (Patent Document 1). The inventors have newly discovered an issue with this conventional cell structure, in that even if the tissue is created using the same method and on the same day, there may be variation in the blood vessel formation ability (instability of blood vessel formation ability).

The present invention aims to provide a method for producing a cell structure that can suppress variation in blood vessel formation ability.

An embodiment of the present invention is as follows.
[1]
A method for producing a cell structure having a vascular structure, comprising the steps of:
a gelling step of gelling a cell-containing liquid containing cells including vascular endothelial cells to form a gel containing the cells;
A culturing step of culturing the cells in the gel,
The method, wherein the cell-containing gel is formed by contacting a plurality of gelling agents with a gelation promoter.
[2]
The method according to [1], wherein the cell-containing fluid further contains extracellular matrix components and polyelectrolytes.
[3]
The method according to [2], wherein the extracellular matrix component comprises collagen.
[4]
The method according to [2], wherein the polyelectrolyte comprises heparin.
[5]
The method according to any one of [1] to [4], wherein the multiple gelling agents include fibrinogen and sodium alginate.
[6]
The method according to any one of [1] to [5], wherein the gelation accelerator comprises thrombin and calcium chloride.
[7]
The method according to any one of [1] to [6], wherein the gelation step is a step of forming the gel by injecting the cell-containing liquid containing the cells and the gelation promoter into a droplet of a reaction liquid containing the multiple types of gelling agents.
[8]
The method according to [7], wherein the volume ratio of the droplet volume of the reaction liquid to the injection volume of the cell-containing liquid is 2 to 10.
[9]
The method according to any one of [2] to [4], comprising, prior to the gelation step, a step of mixing the cells, the extracellular matrix components, and the polymer electrolyte to obtain a mixed solution.
[10]
The method according to [9], further comprising a step of obtaining a precipitate containing the cells, the extracellular matrix components, and the polyelectrolytes by centrifuging the mixture prior to the gelation step.
[11]
The method according to any one of [2] to [4] and [9] to [10], wherein the concentration of the polyelectrolyte is more than 0 mg/mL and not more than 1.5 mg/mL based on the total amount of the cell-containing liquid.
[12]
The method according to any one of [2] to [4] and [9] to [11], wherein the concentration of the extracellular matrix component is greater than 0 mg/mL and less than or equal to 1.5 mg/mL based on the total amount of the cell-containing liquid.
[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:
A step of mixing cells including vascular endothelial cells, extracellular matrix components, and polymer electrolytes to obtain a mixed solution;
obtaining a precipitate containing the cells, the extracellular matrix components, and the polyelectrolytes by centrifuging the mixture;
Obtaining a cell-containing liquid containing the precipitate and a gelation promoter;
forming a gel by injecting the cell-containing liquid into a droplet of a reaction liquid containing multiple types of gelling agents;
A culturing step of culturing the cells in the gel,
The concentration of the polyelectrolyte is more than 0 mg/mL and not more than 1.5 mg/mL based on the total amount of the cell-containing liquid;
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 liquid,
the plurality of gelling agents includes fibrinogen and sodium alginate;
The fibrinogen content is 1 mg/ml to 10 mg/ml based on the total amount of the reaction solution;
The content of the sodium alginate is 0.01% by mass to 2% by mass based on the total amount of the reaction solution,
the gelation promoter comprises thrombin and calcium ions;
the content of the thrombin is 10 units/ml to 30 units/ml based on the total volume of the cell-containing liquid;
the calcium ion content is 1 mM to 10 mM based on the total amount of the cell-containing liquid;
The volume ratio of the droplet volume of the reaction liquid to the injection volume of the cell-containing liquid is 2 to 10.
[17]
A method for producing a cell structure having a vascular structure, comprising the steps of:
A step of mixing cells including vascular endothelial cells, extracellular matrix components, and polymer electrolytes to obtain a mixed solution;
obtaining a precipitate containing the cells, the extracellular matrix components, and the polyelectrolytes by centrifuging the mixture;
Obtaining a cell-containing liquid containing the precipitate and a gelation promoter;
forming a gel by injecting the cell-containing liquid into a droplet of a reaction liquid containing multiple types of gelling agents;
A culturing step of culturing the cells in the gel,
the polyelectrolyte comprises heparin;
The concentration of the polyelectrolyte is more than 0 mg/mL and not more than 1.5 mg/mL based on the total amount of the cell-containing liquid;
the extracellular matrix component comprises 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 liquid,
the plurality of gelling agents includes fibrinogen and sodium alginate;
The method, wherein the gelation promoter comprises thrombin and calcium ions.

The present invention provides a method for producing a cell structure that can suppress variation in blood vessel formation ability.

1 is an image showing the results of evaluating blood vessel formation in a cell structure prepared by the method of Test Example 1. 13 is an image showing the results of evaluating blood vessel formation in a cell structure prepared by the method of Test Example 2. 13 is an image showing the results of evaluating blood vessel formation in a cell structure prepared by the method of Test Example 3. 13 is an image showing the results of evaluating blood vessel formation in a cell structure prepared by the method of Test Example 4. 13 is an image showing the observation results of a cell structure prepared by the method of Test Example 5. 13 is an image showing the observation results of a cell structure prepared by the method of Test Example 6. 13 is an image showing the observation results of a cell structure prepared by the method of Test Example 7. 1 is a schematic diagram illustrating one embodiment of a method for producing a cell structure, in which (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.

Below, we will explain in detail the form for implementing the present invention. However, the present invention is not limited to the following embodiment.

[Method for producing cell structures]
The method according to the present 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, and the gel containing the cells is formed by contacting a plurality of gelling agents with a gelling promoter. The method according to the present embodiment may further include a preparation step of preparing a cell-containing liquid prior to the gelling step.

In this specification, the term "cell structure" refers to a cell aggregate (agglomerated cell mass) in which multiple cells are arranged three-dimensionally, and is an aggregate artificially created by cell culture. The cell structure may contain only one type of cell, or may contain two or more types of cells. Figure 8 is a schematic diagram for explaining one embodiment of a method for producing a cell structure including a gelling step and a culture step. In Figure 8, (a) shows a gel containing cells after the gelling step and before the culture step. In Figure 8, (b) shows a cell structure formed by culturing the gel containing the cells shown in (a) in a medium. As shown in Figure 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 Figure 8(b), in the cell structure, a part of the cell aggregate is in contact with the bottom surface of the culture vessel, and a gel is formed so as to cover the cell aggregate.

In this specification, 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 are observed to have multiple branching points and form a mesh structure.

The shape of the cell structure is not particularly limited, and examples 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, and the like. Here, biological tissue includes sweat glands, lymphatic vessels, sebaceous glands, and the like, and has a more complex structure than a cell structure. Therefore, cell structures and biological tissues can be easily distinguished.

In this specification, the term "gelling agent" refers to a material that forms a gel (hydrogel) by reacting with a gelling accelerator, and refers to a material that has not yet formed a gel. In this specification, the term "gelling accelerator" refers to a component that gels a liquid that contains a gelling agent by reacting with the gelling agent. There are no particular limitations on the gelling agent and gelling accelerator, so 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.

Examples of combinations of gelling agents and gelling 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.

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 having different solidification speeds and elastic moduli after gelation when used alone at a predetermined concentration. For example, when two types of gelling agents with different solidification speeds are used, it is possible to compensate for the solidification speed that is insufficient when a single gelling agent having an appropriate parameter for elastic modulus is used. Alternatively, when gelling agents with different elastic moduli are used, it is possible to compensate for the elastic modulus that is insufficient when a single gelling agent having an appropriate parameter for solidification speed is used. The gelling agents may be two or more types, for example, five or less, four or less, or three or less types, and two types are preferable. By including multiple types of gelling agents, the amount of droplets during gel formation can be increased. This suppresses the diffusion and aggregation of cells in the gel, and stabilizes the blood vessel formation ability in the method for producing a cell structure. When multiple types of gelling agents are included, the shape retention of the cell structure produced is improved compared to when it is produced under conditions containing one type of gelling agent.

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. The ratio of the mass of the second gelling agent to the mass of the first gelling agent may be, for example, 5 or more, or 10 or more, or 10 or less, or 5 or less.

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 ratio of the mass of the second gelation accelerator to the mass of the first gelling accelerator can be adjusted appropriately depending on the type of gelation accelerator. The ratio of the mass of the second gelation accelerator to the mass of the first gelling accelerator may be, for example, 1 or more, or 2 or more, and may be 10 or less, or 5 or less.

In terms of cytotoxicity and stability, the multiple gelling agents may include fibrinogen and sodium alginate. In this case, the gelling promoter may include thrombin and calcium ions.

Below, an example of a method for producing a cell structure having a vascular structure, including a preparation process, a gelation process, and a culture process, is described.

<Preparation process>
In the preparation step, a cell-containing liquid containing cells including vascular endothelial cells is prepared.

The cell-containing liquid may contain two or more gelation promoters, or two or more gelling agents. When the cell-containing liquid contains two or more gelation promoters, the cell-containing liquid may contain, for example, thrombin and calcium ions. When the cell-containing liquid contains two or more gelling agents, 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 agent (a second gelling agent 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.

The amount of gelling agent and/or gelling promoter used can be appropriately set depending on the type of gelling agent and gelling promoter, etc. The total content of gelling agent and/or gelling promoter 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, based on the total amount of the cell-containing liquid, and 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.

When the cell-containing fluid contains fibrinogen, the fibrinogen concentration, based on the total volume of the cell-containing fluid, 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. When the cell-containing fluid contains fibrinogen, the fibrinogen concentration may be, based on the total volume of the cell-containing fluid, 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. 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 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). When the fibrinogen concentration is within the above-mentioned range, the three-dimensional structure of the cell structure after tissue creation is more stable.

When the cell-containing liquid contains thrombin, the thrombin concentration, based on the total volume of the cell-containing liquid, may be 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, based on the total volume of the cell-containing liquid, 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. 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 the formation of a gel is more stable, and a stable cell structure can be created with less risk of damage to the gel even if culture medium is added thereafter.

When the cell-containing liquid contains sodium alginate, the content of sodium alginate may be 0.01 mass% or more, 0.02 mass% or more, 0.03 mass% or more, or 0.04 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 mass% or less, 1.5 mass% or less, 1.0 mass% or less, 0.50 mass% or less, 0.10 mass% or less, 0.08 mass% or less, or 0.06 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% to 2% by mass, 0.01% to 1.5% by mass, 0.02% to 1.0% by mass, 0.02% to 0.50% by mass, 0.03% to 0.10% by mass, or 0.03% 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 until the formation of a gel is more stable, and a stable cell structure can be created with less risk of damage to the gel even if a culture medium is added thereafter.

When the cell-containing liquid contains calcium ions, the calcium ion concentration 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, based on the total amount of the cell-containing liquid. When the cell-containing liquid contains calcium ions, the calcium ion concentration 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, based on the total amount of the cell-containing liquid. When the cell-containing liquid contains calcium ions, the calcium ion concentration 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, based on the total amount of the cell-containing liquid. A cell-containing liquid containing calcium ions can be prepared by adding a calcium salt, such as calcium chloride, to the cell-containing liquid. When the calcium ion concentration is within the above-mentioned range, the time from the start of the reaction with sodium alginate until the formation of a gel is more stable, and a stable cell structure can be produced with less risk of damage to the gel even if a culture medium is added thereafter.

(cell)
The cells include at least vascular endothelial cells. Vascular endothelial cells refer to flat cells that constitute the surface of the vascular lumen. The vascular endothelial cells in the culture step may be, for example, sinusoidal endothelial cells, human umbilical vein-derived vascular endothelial cells (HUVEC), etc. Sinusoidal endothelial cells are non-parenchymal hepatic cells (cells that constitute the liver other than hepatic cells), and have a characteristic morphology different from other vascular endothelial cells, such as a large number of small pores (sieve plate structure) in the cytoplasm and lacking a basement membrane. The vascular endothelial cells in the culture step may be primary cells (primary vascular endothelial cells) collected from the liver of an animal (e.g., human), cells cultured from primary cells, cultured cell lines established from primary cells, or cells artificially differentiated from stem cells. The cultured cell lines may be those established by gene transfer, specifically cultured cell lines into which immortalization-inducing genes such as hTERT and SV40LT have been introduced. Examples of primary vascular endothelial cells include primary sinusoidal endothelial cells, such as product 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 effect of forming and maintaining blood vessels by growth factors involved in blood vessel formation will be further improved. Examples of cultured cell lines include the cultured cell line, product number T0056 manufactured by Applied Biological Materials. Examples of stem cells to be differentiated include embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), etc. The vascular endothelial cells in the culture step may be non-cancerous cells.

The cells may further include epithelial cells. Epithelial cells are cells that form epithelial tissues present in the skin, digestive tract, blood vessels, etc. Examples of epithelial cells include liver cells, 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, thymic epithelial cells, etc.

The cells preferably include hepatocytes. Hepatocytes are also called hepatocytes, and are cells that have functions such as secreting bile and plasma proteins. The hepatocytes constituting the cell structure may be primary hepatocytes collected from the liver of an animal, cultured cells of primary hepatocytes, cultured cell lines of primary hepatocytes, or hepatoblasts artificially differentiated from stem cells. Examples of primary hepatocytes include frozen primary hepatocytes, and primary hepatocytes such as PXB Cell (registered trademark) and HepaSH (registered trademark) collected from human hepatocyte chimera animals 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 to be differentiated into hepatoblasts include embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), and mesenchymal stem cells. As the hepatocytes, non-cancerous cells such as primary hepatocytes and hepatoblasts are preferred, and PXB cells are more preferred due to their ease of handling. When functionally normal and mature hepatocytes are used, the cell structure is able to exhibit high liver function similar to that of the original living body, and thus is preferred. Specifically, primary hepatocytes, HepaRG (registered trademark), and hepatocytes highly differentiated from stem cells can be used. In general, when such functionally mature hepatocytes are co-cultured in a cell structure containing vascular endothelial cells, it tends to be difficult for the vascular endothelial cells to form and maintain a vascular network in the tissue, but according to the method of one aspect of the present invention, it is also possible to form and maintain a vascular network.

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 a protein involved in liver function. All hepatocytes contained in the cells may have the same genotype for a protein involved in liver function. Examples of proteins involved in liver function include drug metabolizing enzymes.

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), have functions such as storing vitamin A, and are present in the space of Disse, which is the area between hepatocytes and sinusoids in the liver. The hepatic stellate cells may be primary hepatic stellate cells extracted from the liver, or may be cells cultured from primary hepatic stellate cells. The hepatic stellate cells may be a cell line 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.

The cells may further include other 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, for example, 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 may be cells such as cancer cells in which any cell function is enhanced or suppressed. A "cancer cell" is a cell that is derived from a somatic cell and has acquired the ability to proliferate indefinitely.

The origin of the cells is not particularly limited, but may be, for example, cells derived from mammals such as humans, monkeys, dogs, cats, rabbits, pigs, cows, mice, and rats.

The total number of cells (X0) is not particularly limited and is appropriately determined 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) may be 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 cells or more. The total number of cells (X0) may be 1 x 10 ⁹ cells or less, 1 x 10 ⁷ cells or less, or 1 x 10 ⁵ cells.

The ratio (X1/X0 x 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 even 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, from the viewpoint of being even more suitable as a cell structure having blood vessels.

The ratio (X2/X0 x 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 hepatic cells, it is desirable that the ratio of hepatic cells is 50% or more, which is a condition close to that of a living body.

The ratio (X3/X0 x 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.

(Extracellular matrix components)
The cell-containing liquid may further contain an extracellular matrix component. When the cell-containing liquid contains an extracellular matrix component, the accumulation of cells is promoted. This promotes adhesion between vascular endothelial cells, and makes it easier to further suppress the variation in angiogenesis ability.

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 the formation of cell aggregates. 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.

The extracellular matrix may be a modified or variant of the above-mentioned extracellular matrix, or may be a polypeptide such as a chemically synthesized peptide, so long as it does not adversely affect cell growth and cell aggregate formation. The extracellular matrix may have a repeat 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. The multiple Gly-XYs may be the same or different. By having a repeat of the sequence represented by Gly-X-Y, there is less restriction on the arrangement of the molecular chain, so that, for example, the function as a scaffold material during cell culture is further improved. In the extracellular matrix having a repeat of the sequence represented by Gly-X-Y, the ratio 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 is 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 scaffolding material for cell culture, for example. Examples of extracellular matrices that contain the sequence represented by Gly-X-Y and the RGD sequence include collagen, fibronectin, vitronectin, laminin, cadherin, etc.

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.

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

The shape of the extracellular matrix component may be, for example, fibrous. Fibrous means a shape composed of thread-like extracellular matrix components, or a shape composed of thread-like extracellular matrix components cross-linked with each other. At least a part of the extracellular matrix component may be fibrous. The shape of the extracellular matrix component is the shape of a mass of extracellular matrix components (aggregates of extracellular matrix components) observed under a microscope, and the extracellular matrix components preferably have an average diameter and/or average length as described below. Fibrous extracellular matrix components include thin threads (fibers) formed by the aggregation of multiple thread-like extracellular matrix molecules, threads formed by further aggregation of filaments, and defibrillated versions of these threads. When the extracellular matrix component has a fibrous shape, the RGD sequence is preserved without being destroyed in the fibrous extracellular matrix component, and the fibrous extracellular matrix component can function more effectively as a scaffold for cell adhesion.

The extracellular matrix component may contain at least one selected from the group consisting of collagen, laminin, and fibronectin, and preferably contains collagen. The collagen is preferably fibrous collagen, and 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 pig skin manufactured by Nippon Ham Ltd.

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 a combination of components derived from multiple types of animals.

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

(polymer electrolyte)
The cell-containing liquid may further contain a polyelectrolyte. The polyelectrolyte is a polymer compound having electrolytic properties. When the cell-containing liquid contains a polyelectrolyte, the accumulation of cells is promoted. This promotes adhesion between vascular endothelial cells, and makes it easier to further suppress the variation in blood vessel formation ability.

Polyelectrolytes 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 polyelectrolyte may consist of one of the above-mentioned types, or may contain a combination of two or more types.

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.

The concentration of the polyelectrolyte in the cell-containing liquid is not particularly limited as long as it does not adversely affect cell growth and the formation of cell structures. The concentration of the polyelectrolyte may be, for example, more than 0 mg/mL and not more than 1.5 mg/mL based on the total amount of the cell-containing liquid. The concentration of the polyelectrolyte may be 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 based on the total amount of the cell-containing liquid, and may be 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 concentration of the polyelectrolyte 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 amount of the cell-containing liquid. When the concentration of the polyelectrolyte is within the above-mentioned range, it is easier to promote adhesion between cells while suppressing excessive aggregation between cells, stabilizing the three-dimensional structure and stabilizing the formation of a vascular network.

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 of the mass 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 blood vessel formation ability is more easily suppressed.

(aqueous medium)
The cell-containing liquid includes an aqueous medium. The term "aqueous medium" means 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 a cationic substance. In addition, a culture medium may be used as the aqueous medium. Examples of the culture medium include liquid culture media such as Dulbecco's Modified Eagle medium (DMEM) and hepatocyte-specific medium (HCM). The liquid medium may be a mixed medium in which two types of culture media are mixed.

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, so 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, or 40 to 70 mM, or 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.

(Method for producing cell-containing liquid)
The cell-containing liquid can be obtained by a method including 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 liquid may include centrifuging the mixture, if necessary. A specific example of the method for producing the cell-containing liquid includes a step of mixing cells, an extracellular matrix component, and a polymer electrolyte to obtain a mixture, a step of centrifuging the mixture to obtain a precipitate containing the cells, the extracellular matrix component, and the polymer electrolyte, and a step of mixing the precipitate with multiple types of gelling agents to obtain the cell-containing liquid.

<Gelling step>
In the gelation step, the cell-containing liquid containing the cells is gelled to form a gel containing the cells. The gelation may be performed by contacting the cell-containing liquid with a reaction liquid containing an aqueous medium and a gelling agent and/or a gelling promoter. The type of the gelling agent and/or the gelling promoter in the reaction liquid is determined according to the type of the gelling agent and/or the gelling promoter in the cell-containing liquid. The aqueous medium may be as described above.

The amount of gelling agent and/or gelling promoter used in gelling the cell-containing liquid can be appropriately set depending on the type of gelling agent and gelling promoter, etc. The total content of gelling agent and/or gelling promoter in 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, based on the total amount of the reaction liquid, and 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.

If the reaction solution contains fibrinogen, the fibrinogen concentration, based on the total volume of the reaction solution, 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. 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 amount 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 amount of the reaction solution. When the fibrinogen concentration is within the above-mentioned range, the three-dimensional structure of the cell structure after tissue creation is more stable.

When the reaction solution contains thrombin, the thrombin concentration, based on the total volume of the reaction solution, may be 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 until 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.

When the reaction liquid contains sodium alginate, the content of sodium alginate may be 0.01 mass% or more, 0.02 mass% or more, 0.03 mass% or more, or 0.04 mass% or more, based on the total amount of the reaction liquid. When the reaction liquid contains sodium alginate, the content of sodium alginate may be 2.0 mass% or less, 1.5 mass% or less, 1.0 mass% or less, 0.50 mass% or less, 0.10 mass% or less, 0.08 mass% or less, or 0.06 mass% or less, based on the total amount of the reaction liquid. When the reaction solution contains sodium alginate, the content of sodium alginate may be 0.01% to 2% by mass, 0.01% to 1.5% by mass, 0.02% to 1.0% by mass, 0.02% to 0.50% by mass, 0.03% to 0.10% by mass, or 0.03% 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 until the formation of a gel is more stable, and a stable cell structure can be created with less risk of damage to the gel even if a culture medium is added thereafter.

When the reaction solution contains calcium ions, the calcium ion concentration 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, based on the total volume of the reaction solution. When the reaction solution contains calcium ions, the calcium ion concentration 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, based on the total volume of the reaction solution. When the reaction solution contains calcium ions, the calcium ion concentration 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, based on the total volume of the reaction solution. 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 until the formation of a gel is more stable, and a more stable cell structure can be created with less risk of the gel being damaged even if a culture medium is added thereafter.

When gelation is performed by contacting the cell-containing liquid with the reaction liquid, the gelation step may be performed 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), and may be performed by injecting the cell-containing liquid into a droplet of the reaction liquid, since this makes it easier to form a gel containing cells. There are no particular limitations on the injection method, and a typical injection method may be used.

The droplet volume of one of the liquids (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 from the viewpoint of further improving the effect of suppressing variation in blood vessel formation ability. The volume ratio of the droplet volume of one of the liquids 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 blood vessel formation ability.

The gelation step may be performed by injecting one of the cell-containing liquid and the reaction liquid into a droplet of the other liquid formed on the substrate. The substrate may be a container that has a surface capable of forming a gel and is capable of accommodating a culture medium. There are no particular limitations on the material or shape of the substrate surface. 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. There are no particular limitations on the method of surface treatment or the coating agent that may be used for the surface treatment. From the viewpoint of ease of tissue preparation, it is preferable that the substrate is a culture vessel having a flat bottom.

Since a gel containing 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 reaction liquid. It is preferable that the volume ratio of the amount of the reaction liquid droplet to the amount of the cell-containing liquid injected is within the above-mentioned range.

The gelation process may include incubating (gelation culture) for a certain period of time after contacting multiple types of 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 manufactured cell structure. On the other hand, according to the method of this embodiment, variation in the angiogenic ability of the cell structure due to the incubation time is suppressed.

<Culture process>
In the culturing step, cells are cultured in the gel. The culturing step can be performed after adding a medium to the culture substrate in which the gel containing the cells is placed. As used herein, "culturing cells" means maintaining the cells in the medium under conditions that do not cause the cells to die or decline. Culturing cells may or may not involve cell proliferation.

The medium can be selected from a variety of media depending on the type of cells to be cultured. Examples of such media include Eagle's MEM medium, DMEM, Modified Eagle Medium (MEM), Minimum Essential Medium, RPMI, GlutaMax medium, and hepatocyte medium (e.g., HCM medium (Lonza)). The medium may be a medium containing serum or a serum-free medium. Furthermore, the medium may be a mixed medium containing two or more types of media. In addition, supplemental factors may be added to the medium as necessary by diluting them to a predetermined concentration. 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).

The culture conditions for the cells in the culture process can be set to suitable conditions depending on the type of cells. 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.

The cell structures produced by the method according to this embodiment have reduced variation in blood vessel formation ability. Furthermore, the method according to this embodiment reduces the evaporation of liquid components that occurs during gelation and the damage to the gel when culture medium is added during the culture process. In addition, the method according to this embodiment does not require the use of special equipment for tissue production, and it is possible to produce cell structures manually. Therefore, the method according to this embodiment can easily produce cell structures with reduced variation in blood vessel formation ability.

[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 the gelation promoter described above, and may include, for example, fibrin gel and alginate gel. The cell structure according to this embodiment may include an extracellular matrix component, and may further include a polyelectrolyte. The cell structure according to this embodiment may be obtained, for example, by the method described above.

When the cell structure of this embodiment contains extracellular matrix components, the content of the extracellular matrix components 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, and 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 0.01-90% by mass, 10-90% by mass, 10-80% by mass, 10-70% by mass, 10-60% by mass, 1-50% by mass, 10-50% by mass, 10-30% by mass, or 20-30% by mass, based on the dry weight of the cell structure.

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

The present invention will be explained in more detail below based on examples. However, the present invention is not limited to these.

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

<Test Example 1>
LX2 and GFP-HUVEC were awakened and pre-cultured according to the manufacturer's protocol. LX2 and GFP-HUVEC were detached using 0.05% Trypsin/EDTA. PXB cells were detached using 0.25% Trypsin/EDTA. After detachment, LX2 and GFP-HUVEC 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 Countess (registered trademark) II FL (Invitrogen). After the measurement, taking into consideration the number of wells to be prepared, the amount of cells required for seeding per well was divided into 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 a 1.0 mg/ml heparin solution and a 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 centrifuged at 400 G for 2 minutes. Thereafter, the supernatant was 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. In this way, a cell-containing solution of Test Example 1 containing cells and thrombin as a gelation promoter was prepared.

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. That is, the volume ratio of the cell-containing solution injected to the volume of the droplets containing fibrinogen (reaction solution) was 1:1. In order to promote gelation due to the reaction between thrombin and fibrinogen, the droplets of the reaction solution into which the cell-containing solution was injected were cultured in a CO ₂ incubator at 37° C. The gelation culture time at this time was set to 30 minutes, 60 minutes, and 90 minutes, respectively. After the incubation was completed, 500 μL of HCM medium was added to the culture substrate and cultured for 7 days.

Figure 1 shows the results of verifying the blood vessel formation ability at each gelation culture time by fluorescent observation of GFP-HUVEC. It can be seen from Figure 1 that the longer the incubation time, the lower the blood vessel formation ability.

<Test Example 2>
Except for changing the droplet volume of the fibrinogen solution in Test Example 1 to 10 μL (the volume ratio of the injected volume of the cell-containing liquid to the droplet volume of the fibrinogen-containing droplet (reaction liquid) was set to 5:1), a cell structure was prepared in the same manner as in Test Example 1. The results when the gelation culture time was set to 30 minutes are shown in Figure 2.

Figure 2 shows that when only one type of gelling agent is used and the amount of fibrinogen solution droplet is increased, the injected cells spread throughout the entire droplet, the three-dimensional structure collapses, and the vascular structure is not observed.

<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.

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 droplet volume of the solution containing sodium alginate was 10 μL. The cell-containing solution of Test Example 3 containing calcium ions was dropped so as to be injected into the droplets (reaction solution) containing sodium alginate. The injection volume of the cell-containing solution was 2 μL. That is, the volume ratio of the injection volume of the cell-containing solution to the droplet volume of the reaction solution was 5:1. In order to advance gelation, the droplets of the reaction solution into which the cell-containing solution was injected were cultured in a CO ₂ incubator under conditions of 37 ° C. The gelation culture time at this time was set to 30 minutes. The subsequent process was performed in the same manner as in Test Example 1 to prepare a cell structure. The results are shown in FIG. 3.

Figure 3 shows that under other conditions, when only one type of gelling agent was used, the cells aggregated excessively, again preventing blood vessels from forming.

<Test Example 4>
A cell-containing solution of Test Example 4 containing thrombin and calcium ions as gelation promoters 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 amount of the mixed solution (cell-containing solution). The thrombin concentration in the mixed solution was 20 U/ml based on the total amount of the mixed solution (cell-containing solution).

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 content of sodium alginate in the mixed solution was 0.05% by mass based on the total volume of the mixed solution (reaction solution). The concentration of fibrinogen 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 dropped so as to be injected into 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 droplet volume of the reaction solution was 5:1. To promote gelation, the droplets of the reaction solution into which the cell-containing solution was injected were cultured in a _CO2 incubator at 37°C. The gelation culture time was set to 30, 60, and 90 minutes. The subsequent steps were the same as in Test Example 1 to prepare a cell structure. The results are shown in Figure 4.

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 blood vessel formation ability even when the incubation time (gelling culture time) is longer. As shown in Figures 2 and 3, blood vessels were not formed when a single gelling agent was used to enlarge the gel, whereas blood vessels were formed when multiple types of gelling agents were used as shown in Figure 4. This shows that by using multiple types of gelling agents, it is possible to form blood vessels even when the droplets are enlarged. As shown in Figure 4, the blood vessel formation ability of the cell structure produced by the method of Test Example 4 was not affected by the incubation time.

<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 culture 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.

In Figure 5, when one type of gelling agent (only fibrinogen) is used and the amount of gel is small, it can be observed that 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.

<Test Example 6>
Tissue preparation was performed in the same manner as in Test Example 5, except that 10 μL of a gelling agent composed of fibrinogen and sodium alginate was used and that the cell-containing liquid contained 5 mM calcium ions, that is, in the same manner as in 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.

As can be seen from Figure 6, the use of multiple types of gelling agents prevents tissue damage and also shows that cells are properly aggregated.

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

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 content of sodium alginate in the mixed solution was 0.05% by mass based on the total volume of the mixed solution (reaction solution). The concentration of fibrinogen 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 20 μL. The cell-containing solution of Test Example 4 containing thrombin and calcium ions was dropped so as to be injected into the droplet (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 droplet volume of the reaction solution was 10:1. In order to promote gelation, the droplets of the reaction solution into which the cell-containing solution was injected were cultured in a _CO2 incubator at 37°C. The gelation culture time was set to 30 minutes. After the incubation, 500 μL of HCM medium was added to the culture substrate, and the culture was continued for 14 days.

The GFP-SEC images in Figure 7 (from three trials) are the results of verifying blood vessel formation ability on Day 14 by fluorescent observation of GFP-SE. From the GFP-SEC images in Figure 7, it can be seen that even on Day 14, the vascular network was sufficiently formed and maintained, the three-dimensional structure was formed without collapse, and there was no excessive aggregation.

After culturing for 14 days, 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 the expression of MRP2 and albumin was evaluated using a confocal microscope. MRP2 is a transporter expressed on the bile canaliculi that form between functionally mature hepatocytes, and albumin is a protein produced within hepatocytes that serves as a marker for hepatocytes.

In Figure 7, MRP2 and Albumin are images of cell structures stained with the respective antibodies. From the image in Figure 7, the hepatocytes are accumulated within the cell structure without excessive aggregation, and the formation of bile canaliculi between the hepatocytes, as indicated by the MRP2 staining image, is clearly and precisely confirmed. From the above results, it can be seen that even when a cell structure is formed using sinusoidal endothelial cells by the method according to one aspect 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 (17)

A method for producing a cell structure having a vascular structure, comprising the steps of:
a gelling step of gelling a cell-containing liquid containing cells including vascular endothelial cells to form a gel containing the cells;
A culturing step of culturing the cells in the gel,
The method, wherein the cell-containing gel is formed by contacting a plurality of gelling agents with a gelation promoter. The method of claim 1, wherein the cell-containing fluid further comprises extracellular matrix components and polyelectrolytes. The method of claim 2, wherein the extracellular matrix component comprises collagen. The method of claim 2, wherein the polyelectrolyte comprises heparin. The method of claim 1, wherein the multiple gelling agents include fibrinogen and sodium alginate. The method of claim 1, wherein the gelation promoter comprises thrombin and calcium chloride. 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 liquid containing the cells and the gelation promoter into a droplet of a reaction liquid containing the multiple types of gelling agents. The method according to claim 7, wherein the volume ratio of the droplet volume of the reaction liquid to the injected volume of the cell-containing liquid is 2 to 10. The method according to any one of claims 2 to 4, further comprising a step of mixing the cells, the extracellular matrix components, and the polyelectrolyte to obtain a mixed solution prior to the gelling step. The method according to claim 9, further comprising a step of obtaining a precipitate containing the cells, the extracellular matrix components, and the polyelectrolytes by centrifuging the mixture before the gelation step. The method according to any one of claims 2 to 4, wherein the concentration of the polyelectrolyte is greater than 0 mg/mL and less than or equal to 1.5 mg/mL based on the total volume of the cell-containing liquid. The method according to any one of claims 2 to 4, wherein the concentration of the extracellular matrix component is greater than 0 mg/mL and less than or equal to 1.5 mg/mL based on the total volume of the cell-containing liquid. The method according to any one of claims 1 to 6, wherein the cells further comprise hepatocytes. The method of any one of claims 1 to 6, wherein the cells further comprise hepatic stellate cells. 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. A method for producing a cell structure having a vascular structure, comprising the steps of:
A step of mixing cells including vascular endothelial cells, extracellular matrix components, and polymer electrolytes to obtain a mixed solution;
obtaining a precipitate containing the cells, the extracellular matrix components, and the polyelectrolytes by centrifuging the mixture;
Obtaining a cell-containing liquid containing the precipitate and a gelation promoter;
forming a gel by injecting the cell-containing liquid into a droplet of a reaction liquid containing multiple types of gelling agents;
A culturing step of culturing the cells in the gel,
The concentration of the polyelectrolyte is more than 0 mg/mL and not more than 1.5 mg/mL based on the total amount of the cell-containing liquid;
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 liquid,
the plurality of gelling agents includes fibrinogen and sodium alginate;
The fibrinogen content is 1 mg/ml to 10 mg/ml based on the total amount of the reaction solution;
The content of the sodium alginate is 0.01% by mass to 2% by mass based on the total amount of the reaction solution,
the gelation promoter comprises thrombin and calcium ions;
the content of the thrombin is 10 units/ml to 30 units/ml based on the total volume of the cell-containing liquid;
the calcium ion content is 1 mM to 10 mM based on the total amount of the cell-containing liquid;
The volume ratio of the droplet volume of the reaction liquid to the injection volume of the cell-containing liquid is 2 to 10. A method for producing a cell structure having a vascular structure, comprising the steps of:
A step of mixing cells including vascular endothelial cells, extracellular matrix components, and polymer electrolytes to obtain a mixed solution;
obtaining a precipitate containing the cells, the extracellular matrix components, and the polyelectrolytes by centrifuging the mixture;
Obtaining a cell-containing liquid containing the precipitate and a gelation promoter;
forming a gel by injecting the cell-containing liquid into a droplet of a reaction liquid containing multiple types of gelling agents;
A culturing step of culturing the cells in the gel,
the polyelectrolyte comprises heparin;
The concentration of the polyelectrolyte is more than 0 mg/mL and not more than 1.5 mg/mL based on the total amount of the cell-containing liquid;
the extracellular matrix component comprises 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 liquid,
the plurality of gelling agents includes fibrinogen and sodium alginate;
The method, wherein the gelation promoter comprises thrombin and calcium ions.

Images