Gel composition, method for manufacturing the same, and three-dimensional structure, method for manufacturing the same

Abstract

To provide a gel composition that is transparent and has a high modulus of elasticity, containing extracellular matrix components and / or fragmented extracellular matrix components. [Solution] A gel composition containing at least one selected from the group consisting of extracellular matrix components and fragmented extracellular matrix components, and ions of a metal element.

Description

[Technical Field]

[0001] The present invention relates to a gel composition and a method for producing the same. The present invention also relates to a three-dimensional structure using the gel composition according to the present invention and a method for producing the same. [Background technology]

[0002] Gels of extracellular matrix components such as collagen are commonly used as scaffold materials in cell culture. Collagen gels are usually prepared by dissolving collagen under acidic conditions, followed by neutralization and heating to around 37°C to gel the collagen. Another known method involves cross-linking collagen using cross-linking agents such as formaldehyde and glutaraldehyde to induce gelation.

[0003] On the other hand, for example, Patent Document 1 discloses an extracellular matrix-containing composition comprising fragmented extracellular matrix components and an aqueous medium. The extracellular matrix-containing composition disclosed in Patent Document 1 exhibits a thermally reversible sol-gel transition, making it possible to gel and solize by temperature control. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] International Publication No. 2019 / 208831 [Overview of the project] [Problems that the invention aims to solve]

[0005] Conventional methods for producing collagen gels have problems such as cytotoxicity due to acidic conditions or crosslinking agents, difficulty in controlling the elastic modulus, and difficulty in obtaining gels with high elastic modulus. On the other hand, the method using fragmented extracellular matrix components disclosed in Patent Document 1 does not have the problem of cytotoxicity, allows for a certain degree of control over the elastic modulus, and can produce gels with high elastic modulus. However, the gel obtained with the method disclosed in Patent Document 1 is opaque, making it difficult to observe its internal structure.

[0006] The present invention aims to provide a gel composition that is transparent and has a high elastic modulus, and that contains extracellular matrix components and / or fragmented extracellular matrix components. [Means for solving the problem]

[0007] The present invention relates to a gel composition containing at least one selected from the group consisting of extracellular matrix components and fragmented extracellular matrix components, and ions of a metal element.

[0008] The gel composition according to the present invention contains extracellular matrix components and / or fragmented extracellular matrix components, and further contains ions of a metal element, and is therefore transparent and has a high elastic modulus. The gel composition according to the present invention is based on the finding that a transparent and highly elastic (hard) gel can be obtained by contacting (for example, dropping) a solution containing ions of a metal element with a solution containing extracellular matrix components and / or fragmented extracellular matrix components.

[0009] In the above gel composition, the metal element is preferably at least one metal element selected from the group consisting of transition metal elements and base metal elements, more preferably at least one metal element selected from the group consisting of transition metals of Group 10, transition metals of Group 11, and metals of Group 12 of the periodic table, and even more preferably at least one selected from the group consisting of copper, zinc, palladium, platinum, and gold. This allows the above-mentioned effects to be exhibited more significantly.

[0010] In the above gel composition, it is preferable that the extracellular matrix component includes collagen. This allows the effects described above to be exhibited more significantly.

[0011] In the above gel composition, it is preferable that the fragmented extracellular matrix component includes fragmented collagen. This allows the effects described above to be exhibited more significantly.

[0012] The above gel composition may be one in which at least one selected from the group consisting of the above extracellular matrix component and fragmented extracellular matrix component is crosslinked.

[0013] The present invention also relates to a method for producing a gel composition, comprising the step of contacting a solution containing at least one selected from the group consisting of extracellular matrix components and fragmented extracellular matrix components with a solution containing ions of a metal element.

[0014] According to the method for producing a gel composition of the present invention, a transparent gel composition with a high elastic modulus (hardness) can be obtained.

[0015] In the method for producing the above-mentioned gel composition, the metal element is preferably at least one metal element selected from the group consisting of transition metal elements and base metal elements, more preferably at least one metal element selected from the group consisting of transition metals of Group 10, transition metals of Group 11, and metals of Group 12 of the periodic table, and even more preferably at least one selected from the group consisting of copper, zinc, palladium, platinum, and gold. This allows the above-mentioned effects to be exhibited more significantly.

[0016] In the method for producing the above-mentioned gel composition, it is preferable that the extracellular matrix component includes collagen. This allows the effects described above to be exhibited more significantly.

[0017] In the method for producing the above-described gel composition, it is preferable that the fragmented extracellular matrix component includes fragmented collagen. This allows the effects described above to be exhibited more significantly.

[0018] In the method for producing the gel composition described above, the contact step may be carried out at a temperature of -3°C to 10°C. This avoids excessive reactions and allows for efficient production of the gel composition.

[0019] The present invention further relates to a method for producing a three-dimensional tissue, comprising the steps of: obtaining a cell-containing gel composition by contacting at least one selected from the group consisting of extracellular matrix components and fragmented extracellular matrix components, cells, and ions of a metal element in an aqueous medium; and culturing the cell-containing gel composition.

[0020] The above method for manufacturing a three-dimensional tissue allows for the production of a three-dimensional tissue having a transparent appearance. Therefore, cells and other elements inside the three-dimensional tissue can be easily observed. Furthermore, the above method for manufacturing a three-dimensional tissue allows for the production of a three-dimensional tissue with a high elastic modulus.

[0021] In the method for producing the three-dimensional tissue described above, the step of obtaining the cell-containing gel composition may include contacting a suspension containing at least one selected from the group consisting of the extracellular matrix component and the fragmented extracellular matrix component, the cells, and a first aqueous medium with a solution containing ions of the metal element and a second aqueous medium.

[0022] In the above-described method for producing a three-dimensional tissue, the culturing step may be carried out under conditions in which at least a portion of the cells remain viable.

[0023] The above method for producing the three-dimensional tissue may further include a step of incubating the cell-containing gel composition at 20°C to 30°C after the step of obtaining the cell-containing gel composition and before the step of culturing the cell-containing gel composition.

[0024] In the method for manufacturing the three-dimensional structure described above, the metal element may be at least one metal element selected from the group consisting of transition metal elements and base metal elements.

[0025] The present invention further relates to a three-dimensional tissue that contains at least one selected from the group consisting of extracellular matrix components and fragmented extracellular matrix components, cells, and ions of a metal element, and is transparent at 37°C.

[0026] The above three-dimensional structure may have a total light transmittance of 80% or more at 37°C.

[0027] In the above three-dimensional structure, the metal element may be at least one metal element selected from the group consisting of transition metal elements and base metal elements. [Effects of the Invention]

[0028] According to the present invention, it is possible to provide a gel composition that is transparent and has a high elastic modulus, and that contains extracellular matrix components and / or fragmented extracellular matrix components. [Brief explanation of the drawing]

[0029] [Figure 1] This is a photograph showing the results of Test Example 1. [Figure 2] This is a photograph showing the results of Test Example 2. [Figure 3] This is a photograph showing the results of Test Example 3. [Figure 4] This is a photograph showing the results of Test Example 4. [Figure 5] These are photographs showing the external appearance (Figure 5(A)) and internal structure (Figure 5(B)) of the three-dimensional tissue produced in Test Example 6. [Figure 6] This graph shows the elastic modulus of gel compositions prepared using solutions of various metal ions. [Figure 7](A) A graph showing the transmittance at a wavelength of 500 nm for gel compositions prepared with fragmented collagen solution and Pt2+ solution at various concentrations. (B) Images of the gels prepared with fragmented collagen solution and Pt2+ solution at various concentrations, observed from above. [Figure 8] This is a photograph showing the results of Test Example 9. [Modes for carrying out the invention]

[0030] The following describes in detail embodiments for carrying out the present invention. However, the present invention is not limited to the following embodiments.

[0031] [Gel composition] The gel composition according to this embodiment contains at least one selected from the group consisting of extracellular matrix components and fragmented extracellular matrix components, and ions of a metal element.

[0032] Extracellular matrix components are aggregates of extracellular matrix molecules, formed by multiple extracellular matrix molecules. Extracellular matrix molecules may be substances that exist outside the cell in multicellular organisms. Any substance can be used as an extracellular matrix molecule, as long as it does not adversely affect cell growth or the formation of cell aggregates. Examples of extracellular matrix molecules include, but are not limited to, collagen, laminin, fibronectin, vitronectin, elastin, tenascin, enteractin, fibrillin, and proteoglycans. These extracellular matrix molecules may be used individually or in combination of two or more.

[0033] The extracellular matrix component may include, for example, collagen, or may consist of collagen. When the extracellular matrix component includes collagen, the effects of the present invention, such as the gel composition being transparent and having a high elastic modulus (hardness), can be more pronounced. Furthermore, when the extracellular matrix component includes collagen, and the gel composition according to this embodiment is used as a scaffold material during cell culture, the collagen functions as a scaffold for cell adhesion, further promoting the formation of three-dimensional cell structures.

[0034] The extracellular matrix molecule may be a modified or variant of the extracellular matrix molecule described above, or it may be a polypeptide such as a chemically synthesized peptide. The extracellular matrix molecule may have a repeating sequence represented by Gly-XY, which is characteristic of collagen. Here, Gly represents a glycine residue, and X and Y each independently represent any amino acid residue. Multiple Gly-XY sequences may be identical or different. Having a repeating sequence represented by Gly-XY reduces constraints on the arrangement of the molecular chain, resulting in improved functionality as a scaffold material during cell culture, for example. In an extracellular matrix molecule having a repeating sequence represented by Gly-XY, the proportion of the sequence represented by Gly-XY may be 80% or more of the total amino acid sequence, preferably 95% or more. The extracellular matrix molecule may also be a polypeptide having an RGD sequence. An RGD sequence is a sequence represented by Arg-Gly-Asp (arginine residue-glycine residue-aspartic acid residue). The presence of the RGD sequence further promotes cell adhesion, making it even more suitable as a scaffold material for cell culture, for example. Examples of extracellular matrix molecules containing both the Gly-XY sequence and the RGD sequence include collagen, fibronectin, vitronectin, laminin, and cadherin.

[0035] Examples of collagen include fibrous collagen and non-fibrous collagen. Fibrous collagen refers to collagen that is the main component of collagen fibers, and specifically includes type I collagen, type II collagen, type III collagen, etc. An example of non-fibrous collagen is type IV collagen.

[0036] Examples of proteoglycans include, but are not limited to, chondroitin sulfate proteoglycans, heparan sulfate proteoglycans, keratan sulfate proteoglycans, and dermatan sulfate proteoglycans.

[0037] The extracellular matrix components may include at least one selected from the group consisting of collagen, laminin, and fibronectin, as this enhances the effects of the present invention, and it is preferable that they include collagen. The collagen is preferably fibrous collagen, and more preferably type I collagen. Commercially available collagen may be used as the fibrous collagen, and a specific example of this is porcine skin-derived type I collagen manufactured by Nippon Ham Co., Ltd.

[0038] The extracellular matrix components may be extracellular matrix components derived from animals. Examples of animal species from which the extracellular matrix components may be derived include, but are not limited to, humans, pigs, and cattle. The extracellular matrix components may be components derived from one type of animal, or components derived from multiple types of animals may be used in combination.

[0039] Fragmented extracellular matrix components can be obtained by fragmenting the extracellular matrix components described above. "Fragmentation" means reducing the size of aggregates of extracellular matrix molecules. Fragmentation may be carried out under conditions that cleave the bonds within the extracellular matrix molecules, or under conditions that do not cleave the bonds within the extracellular matrix molecules. Fragmented extracellular matrix components may include defibrated extracellular matrix components (defibrated extracellular matrix components), which are components obtained by defibrating the extracellular matrix components described above by applying physical force. Defibration is one form of fragmentation, and is carried out, for example, under conditions that do not cleave the bonds within the extracellular matrix molecules.

[0040] There are no particular limitations on the method for fragmenting extracellular matrix components. For example, extracellular matrix components may be defibrated by applying physical force, such as using an ultrasonic homogenizer, agitator homogenizer, or high-pressure homogenizer. When using an agitator homogenizer, the extracellular matrix components may be homogenized directly or in an aqueous medium such as physiological saline. Furthermore, by adjusting the homogenization time and number of repetitions, it is possible to obtain defibrated extracellular matrix components of millimeter or nanometer size. Defibrated extracellular matrix components can also be obtained by repeated freeze-thaw cycles.

[0041] The fragmented extracellular matrix component may contain at least a portion of the defibrated extracellular matrix component. Alternatively, the fragmented extracellular matrix component may consist solely of the defibrated extracellular matrix component. In other words, the fragmented extracellular matrix component may be the defibrated extracellular matrix component. The defibrated extracellular matrix component preferably contains the defibrated collagen component (defibrated collagen component). The defibrated collagen component preferably maintains the triple helix structure derived from collagen. The defibrated collagen component may be a component that partially maintains the triple helix structure derived from collagen.

[0042] Examples of the shape of fragmented extracellular matrix components include fibrous structures. Fibrous refers to a shape composed of thread-like collagen components, or a shape composed of thread-like extracellular matrix components cross-linked between molecules. At least a portion of the fragmented extracellular matrix components may be fibrous. Fibrous extracellular matrix components include thin threads (fibrillaries) formed by the aggregation of multiple thread-like extracellular matrix molecules, threads formed by further aggregation of fibrillaries, and defibrillated versions of these threads. In fibrous extracellular matrix components, the RGD sequence is preserved without disruption.

[0043] The average length of the fragmented extracellular matrix components may be between 100 nm and 400 μm, or between 100 nm and 200 μm. In one embodiment, the average length of the fragmented extracellular matrix components may be between 5 μm and 400 μm, between 10 μm and 400 μm, between 22 μm and 400 μm, or between 100 μm and 400 μm. In another embodiment, from the viewpoint of achieving even better redispersibility, the average length of the fragmented extracellular matrix components may be 100 μm or less, 50 μm or less, 30 μm or less, 15 μm or less, 10 μm or less, 1 μm or less, or 100 nm or more. It is preferable that the average length of most of the fragmented extracellular matrix components is within the above numerical range. Specifically, it is preferable that the average length of 95% of the fragmented extracellular matrix components is within the above numerical range. The fragmented extracellular matrix component is preferably a fragmented collagen component having an average length within the above range, and more preferably a defibrated collagen component having an average length within the above range.

[0044] The average diameter of the fragmented extracellular matrix components may be 10 nm to 30 μm, 30 nm to 30 μm, 50 nm to 30 μm, 100 nm to 30 μm, 1 μm to 30 μm, 2 μm to 30 μm, 3 μm to 30 μm, 4 μm to 30 μm, or 5 μm to 30 μm. The fragmented extracellular matrix components are preferably fragmented collagen components with an average diameter within the above range, and more preferably defibrated collagen components with an average diameter within the above range.

[0045] The average length and average diameter of fragmented extracellular matrix components can be determined by measuring individual fragmented extracellular matrix components using an optical microscope and performing image analysis. In this specification, "average length" refers to the average value of the length in the longitudinal direction of the measured sample, and "average diameter" refers to the average value of the length in the direction perpendicular to the longitudinal direction of the measured sample.

[0046] While fragmented extracellular matrix components typically form an opaque gel upon gelation, the gel composition according to the present invention forms a transparent gel even when containing fragmented extracellular matrix components.

[0047] In the gel composition according to this embodiment, at least a portion of the extracellular matrix components and / or fragmented extracellular matrix components (hereinafter collectively referred to as "extracellular matrix components, etc.") may be crosslinked intermolecularly or intramolecularly. The extracellular matrix components, etc. may be crosslinked within the molecules constituting the extracellular matrix components, etc., or they may be crosslinked between the molecules constituting the extracellular matrix components, etc.

[0048] The mode of crosslinking of extracellular matrix components or the like may be at least partly due to the formation of hydrogen bonds between carboxyl groups of extracellular matrix molecules and ions of metal elements. The crosslinking of extracellular matrix components or the like may also include crosslinking by physical crosslinking such as the application of heat, ultraviolet rays, radiation, etc., chemical crosslinking by crosslinking agents, enzymatic reactions, etc.

[0049] The type of ions of metal elements contained in the gel composition according to the present embodiment is not particularly limited, and ions of any metal element can be used.

[0050] From the viewpoint of more significantly exhibiting the effect of the present invention that the gel composition is transparent and has a high (hard) elastic modulus, the metal element is preferably at least one metal element selected from the group consisting of transition metal elements and base metal elements. Specific examples of at least one metal element selected from the group consisting of transition metal elements and base metal elements include, for example, titanium (Ti), copper (Cu), zinc (Zn), palladium (Pd), platinum (Pt), gold (Au), etc. Specific examples of ions of at least one metal element selected from the group consisting of transition metal elements and base metal elements include, for example, Ti + 、Ti 2+ 、Ti 3+ 、Ti 4+ 、Cu + 、Cu 2+ 、Zn + 、Zn 2+ 、Pd 2+ 、Pd 4+ 、Pt 2+ 、Pt 4+ 、Au + 、Au 2+ 、Au 4+ etc.

[0051] From the viewpoint of being able to exhibit the effects of the present invention described above even more clearly, it is more preferable that the metal element is at least one metal element selected from the group consisting of transition metals of Group 10, transition metals of Group 11, and metals of Group 12 of the periodic table. Specific examples of at least one metal element selected from the group consisting of transition metals of Group 10, transition metals of Group 11, and metals of Group 12 of the periodic table include, for example, copper (Cu), zinc (Zn), palladium (Pd), platinum (Pt), and gold (Au). Specific examples of ions of at least one metal element selected from the group consisting of transition metals of Group 10, transition metals of Group 11, and metals of Group 12 of the periodic table include, for example, Cu 2+ Zn 2+ , Pd 2+ , Pd 4+ Pt 2+ Pt 4+ Au + Au 2+ Au 4+ These are some examples.

[0052] The metal element ions used in the gel composition according to this embodiment are preferably selected from titanium (Ti), copper (Cu), zinc (Zn), platinum (Pt), and gold (Au), from the viewpoint of being non-cytotoxic, or having low cytotoxicity, and being safe for living organisms. A gel composition that is safe for living organisms can be suitably used, for example, as a scaffold material during cell culture when forming artificial tissues.

[0053] The gel composition according to this embodiment is transparent, making it easy to observe its internal structure. Therefore, it can be suitably used, for example, as a scaffold material during cell culture when forming artificial tissues. The total light transmittance of the gel composition according to this embodiment may be, for example, 80% or more, 85% or more, 90% or more, or 95% or more. Total light transmittance is defined as the ratio of transmitted light flux (including the diffusive component) to parallel incident light flux of a test piece made of the gel composition with a thickness of 10 mm.

[0054] The gel composition according to this embodiment has a high elastic modulus and can therefore be suitably used, for example, as a scaffold material during cell culture when artificially forming cancer tissue. This makes it possible to form artificial cancer tissue with a high elastic modulus (hardness). The gel composition according to this embodiment may have an elastic modulus of 1.5 kPa or more, 2.0 kPa or more, 5.0 kPa or more, 10 kPa or more, or 50 kPa or more. There is no particular upper limit to the elastic modulus, but it is usually 250 kPa or less, preferably 200 kPa or less, and more preferably 150 kPa or less. The elastic modulus is a value measured by the method described in the examples below.

[0055] In the gel composition according to this embodiment, the content of at least one selected from the group consisting of extracellular matrix components and fragmented extracellular matrix components may be, for example, 0.01% by weight or more, 0.05% by weight or more, 0.1% by weight or more, 0.15% by weight or more, 0.2% by weight or more, 0.5% by weight or more, 1.0% by weight or more, 2.0% by mass or more, or 3.0% by mass or more, based on the total amount of the gel composition. The upper limit of the content of at least one selected from the group consisting of extracellular matrix components and fragmented extracellular matrix components is not particularly limited as long as it is possible to prepare, but may be, for example, 10.0% by weight or less, or 5.0% by weight or less, based on the total amount of the gel composition.

[0056] In the gel composition according to this embodiment, the content of metal element ions may be, for example, 5 mg or more, 10 mg or more, 20 mg or more, 30 mg or more, or 40 mg or more per 1 g of the total content of at least one selected from the group consisting of extracellular matrix components and fragmented extracellular matrix components. There are no particular restrictions on the content of metal element ions, but for example, it may be 100 mg or less, 80 mg or less, or 60 mg or less per 1 g of the total content of at least one selected from the group consisting of extracellular matrix components and fragmented extracellular matrix components.

[0057] The gel composition according to this embodiment may further contain at least one selected from the group consisting of extracellular matrix components and fragmented extracellular matrix components, and other components other than ions of metal elements, depending on the application of the gel composition. Examples of other components include, when used as a scaffold material during cell culture, nutrients for the cells to be cultured, pH adjusters, etc.

[0058] [Method for producing gel composition] The gel composition according to this embodiment can be obtained, for example, by a manufacturing method comprising a step (contact step) of contacting a solution containing at least one selected from the group consisting of extracellular matrix components and fragmented extracellular matrix components with a solution containing ions of a metal element.

[0059] A solution containing at least one selected from the group consisting of extracellular matrix components and fragmented extracellular matrix components can be obtained by dissolving or dispersing the above-mentioned extracellular matrix components and / or fragmented extracellular matrix components in a solvent. The solvent is not particularly limited as long as it can dissolve or disperse the extracellular matrix components and / or fragmented extracellular matrix components, but specific examples include water, saline solutions such as phosphate-buffered saline (PBS), and liquid media such as Dulbecco's Modified Eagle medium (DMEM).

[0060] A solution containing ions of a metal element is not particularly limited as long as it contains the ions of the metal element as described above. The ion source for the metal element ions is also not particularly limited and may be, for example, an inorganic salt or an organic salt. A solution containing ions of a metal element can be obtained, for example, by dissolving the ion source for the metal element ions in a solvent. The solvent is not particularly limited as long as it can dissolve the ion source for the metal element ions and may be, for example, an organic solvent such as water, ethanol, or dimethyl sulfoxide (DMSO).

[0061] The contact step can employ any method that involves contacting a solution containing at least one selected from the group consisting of extracellular matrix components and fragmented extracellular matrix components with a solution containing ions of a metal element. Specifically, for example, one method is to bring the two solutions into contact by mixing a solution containing at least one selected from the group consisting of extracellular matrix components and fragmented extracellular matrix components with a solution containing ions of a metal element. When mixing, it is preferable to mix both solutions so that they become uniform. Furthermore, from the viewpoint of increasing the efficiency of gel formation, it is preferable to bring the two solutions into contact by stirring the solution containing at least one selected from the group consisting of extracellular matrix components and fragmented extracellular matrix components while dropping the solution containing ions of a metal element into it. The dropping rate can be exemplified by, for example, 1 drop / second.

[0062] While there are no particular restrictions on the temperature during the contact process, from the viewpoint of avoiding excessive reactions, it is preferable to carry it out at, for example, 10°C or lower, and more preferably at 5°C or lower. There are no particular restrictions on the lower limit of the temperature, but it is usually -3°C or higher, and preferably 0°C or higher.

[0063] The contact time between the two solutions during the contact process is appropriately set to allow gel formation, depending on the type of extracellular matrix component and / or fragmented extracellular matrix component used, and the type of metal element ions. The contact time is typically between 0 seconds (gelation immediately after contact) and 30 minutes. For example, fragmented collagen solution and Pd 2+When in contact with a solution, gelation occurs instantly upon contact, so a contact time of 0 to 10 seconds is sufficient. Also, for example, collagen solution and Pt 4+ When in contact with a solution, a gel can be formed by ensuring a contact time of 10 to 20 minutes.

[0064] [How to use the gel composition] The gel composition according to this embodiment is transparent and has a high elastic modulus (hardness), and is therefore suitable for use as a scaffold material for forming cell structures (three-dimensional tissues), for example.

[0065] [Three-dimensional organization] A three-dimensional tissue is an aggregate of cells in which cells are arranged three-dimensionally via extracellular matrix components, and is an aggregate artificially created by cell culture. There are no particular restrictions on the shape of the three-dimensional tissue; for example, it can be sheet-like, spherical, ellipsoidal, or rectangular.

[0066] The three-dimensional tissue body according to this embodiment contains at least one selected from the group consisting of extracellular matrix components and fragmented extracellular matrix components, cells, and ions of a metal element. At least a portion of the cells may be in contact with the extracellular matrix components and / or fragmented extracellular matrix components. One form of contact is adhesion.

[0067] The three-dimensional structure according to this embodiment may be transparent at 37°C, and more specifically, its total light transmittance at 37°C may be 80% or more, 85% or more, 90% or more, or 95% or more. The total light transmittance is defined in accordance with the total light transmittance of the gel composition.

[0068] The cells included in the three-dimensional tissue according to this embodiment are not particularly limited, but may be derived from animals such as humans, monkeys, dogs, cats, rabbits, pigs, cows, mice, and rats. The site of origin of the cells is also not particularly limited, and may be somatic cells derived from bone, muscle, internal organs, nerves, brain, skin, blood, etc., or germ cells. Furthermore, the cells may be induced pluripotent stem cells (iPS cells), embryonic stem cells (ES cells), or cultured cells such as primary cultured cells, subcultured cells, and cell line cells. Specifically, examples of cells include, but are not limited to, nerve cells, dendritic cells, immune cells, vascular endothelial cells (e.g., human umbilical vein-derived vascular endothelial cells (HUVEC)), lymphatic endothelial cells, fibroblasts, cancer cells such as colorectal cancer cells (e.g., human colorectal cancer cells (HT29)), hepatocytes, epithelial cells (e.g., human gingival epithelial cells), keratinocytes, cardiomyocytes (e.g., human iPS cell-derived cardiomyocytes (iPS-CM)), hepatocytes, pancreatic islet cells, tissue stem cells, and smooth muscle cells (e.g., aortic smooth muscle cells (Aorta-SMC)). Cells may be used individually or in combination of multiple types.

[0069] The specific embodiments of the extracellular matrix components and fragmented extracellular matrix components, as well as the ions of metal elements, contained in the three-dimensional tissue according to this embodiment are the same as those described in the gel composition according to the present invention.

[0070] The thickness of the three-dimensional tissue according to this embodiment is preferably 10 μm or more, more preferably 100 μm or more, and even more preferably 1000 μm or more. Such a three-dimensional tissue has a structure closer to living tissue and is suitable as a substitute for experimental animals and as a transplant material. The upper limit of the thickness of the three-dimensional tissue according to this embodiment is not particularly limited, but for example it may be 10 mm or less, 3 mm or less, 2 mm or less, 1.5 mm or less, or 1 mm or less. Here, the thickness of the three-dimensional tissue means the distance between both ends in a direction perpendicular to the main surface when the three-dimensional tissue is in the form of a sheet or a rectangular parallelepiped. If the main surface has irregularities, the thickness means the distance at the thinnest part of the main surface. If the three-dimensional tissue is spherical, it means its diameter. Furthermore, if the three-dimensional tissue is ellipsoidal, it means its minor axis. If the three-dimensional tissue is substantially spherical or ellipsoidal and has irregularities on its surface, the thickness means the shortest distance between two points where a line passing through the centroid of the three-dimensional tissue intersects with the surface.

[0071] [Method for manufacturing three-dimensional tissues] The three-dimensional tissue according to this embodiment can be obtained by a manufacturing method comprising, for example, a step of obtaining a cell-containing gel composition by contacting at least one selected from the group consisting of extracellular matrix components and fragmented extracellular matrix components (hereinafter also referred to as "extracellular matrix components, etc."), cells, and ions of a metal element in an aqueous medium (contact step), and a step of culturing the cell-containing gel composition obtained in the contact step (cultivation step). The manufacturing method according to this embodiment may further include a step of incubating the cell-containing gel composition at 20°C to 30°C after the contact step and before the culturing step (incubation step).

[0072] The contact step is a process of obtaining a cell-containing gel composition by bringing extracellular matrix components, cells, and ions of metal elements into contact in an aqueous medium. The aqueous medium refers to a liquid in which water is an essential component. Examples of aqueous media include physiological salines such as phosphate-buffered saline (PBS) and liquid media such as Dulbecco's Modified Eagle medium (DMEM). The liquid medium may also be a mixed medium obtained by mixing two types of media. From the viewpoint of reducing the burden on cells, the aqueous medium is preferably a liquid medium.

[0073] There are no particular restrictions on the liquid culture medium, and 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 medium (MEM), Minimum Essential medium, RPMI medium, and GlutaMax medium. The medium may be a serum-added medium or a serum-free medium. Furthermore, the liquid medium may be a mixed medium obtained by mixing two or more types of media.

[0074] The specific embodiments of the extracellular matrix components and metal element ions used in the contact process are the same as those described in the gel composition according to the present invention.

[0075] The contact step may include, for example, contacting a suspension containing extracellular matrix components, cells, and a first aqueous medium with a solution containing ions of a metal element and a second aqueous medium. In this case, the first aqueous medium and the second aqueous medium may be the same or different. The suspension can be obtained, for example, by mixing a solution in which extracellular matrix components are dissolved in the first aqueous medium with cells.

[0076] The concentration of extracellular matrix components, etc., in the contact process can be appropriately determined according to the shape, thickness, size of the culture vessel, etc., of the target three-dimensional tissue. For example, the concentration of extracellular matrix components, etc., in the aqueous medium in the contact process may be 0.1 to 30% by mass or 0.1 to 10% by mass, based on the total amount of the aqueous medium (for example, the sum of the first aqueous medium and the second aqueous medium). Also, for example, the amount of extracellular matrix components, etc., may be 1 × 10⁻⁶ 6 The amount of the substance relative to the cells may be 0.01 to 1000 μg, 0.1 to 100 μg, or 0.1 to 5 μg.

[0077] The culture step involves culturing the cell-containing gel composition obtained in the contact step. Preferably, the culture step is carried out under conditions that maintain the viability of at least some of the cells contained in the cell-containing gel composition. The conditions for maintaining viability can be appropriately set depending on the type of cell. Specifically, the culture conditions exemplified below are examples.

[0078] The culture conditions in the culture process can be set according to 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 culture medium may be 6 to 8 or 7.2 to 7.4. The culture time may be 1 to 14 days, 7 to 14 days, 14 to 30 days, 30 to 60 days, or 60 to 90 days.

[0079] The cell density in the cell-containing gel composition can be appropriately determined according to the shape, thickness, etc., of the target three-dimensional tissue. For example, the cell density in the cell-containing gel composition can be 1 to 10 8 It may be cells / ml or 10 3 ~10 7 Cells / ml is also acceptable.

[0080] The incubation step is a process in which the cell-containing gel composition is incubated at 20°C to 30°C after the contact step and before the culture step. The incubation step is performed to ensure more reliable gel formation by the extracellular matrix components and metal element ions. The incubation step may be performed as needed. The incubation time can be appropriately set depending on the type of extracellular matrix components and metal element ions used, but for example, 30 minutes to 24 hours can be exemplified. [Examples]

[0081] The present invention will be described in more detail below based on test examples. However, the present invention is not limited to the following test examples.

[0082] [Test Example 1: Manufacturing of Gel Composition] (Preparation of collagen solution) 1 g of mixed type I and type III collagen derived from porcine skin, manufactured by Nippon Ham Co., Ltd., was added to 500 mL of ultrapure water and incubated at 4°C for 12 hours to prepare a 0.2 wt% collagen solution. Subsequently, 0.45 M NaCl and 5 mM Tris-HCl were added to the collagen solution and incubated at 4°C for 12 hours, after which 1.2 M NaCl was added and incubated for another 12 hours at 4°C. After incubation, the solution was centrifuged at 10,000 rpm for 15 minutes, and the supernatant was collected to obtain a purified type I collagen solution. The obtained type I collagen solution was dialyzed with ultrapure water for 7 days (molecular weight cutoff (MWCO): 15 kDa), and then freeze-dried for 3 days to obtain purified type I collagen.

[0083] The purified type I collagen obtained was added to PBS (pH 7) and incubated at 4°C for 12 hours to prepare a 0.2% by weight type I collagen solution.

[0084] (Au 3+ (Preparation of solutions) To an aqueous solution of tetrachloride-augnolic acid (HAuCl4), add an aqueous solution of sodium hydroxide (NaOH) or water, and then add 12.5 mM Au. 3+Solution (pH 6.8) or 12.5 mM Au 3+ Solutions (pH 2.2) were prepared separately. Hydrochloric acid (HCl aqueous solution, pH 2.2) was prepared as a control.

[0085] (Preparation of gel composition) <Example 1> 6 mL of 0.2 wt% type I collagen solution was placed in a container with a stirrer bar and stirred while cooling in an ice bath. Then, while stirring the collagen solution in the ice bath, 240 μL of Au was added to it. 3+ The solution (pH 6.8) was added dropwise (Au 3+ :3 μmol).

[0086] <Example 2> Au 3+ Instead of the solution (pH 6.8), use 240 μL of Au 3+ The solution (pH 2.2) was added dropwise (Au 3+ The procedure was the same as in Example 1, except that (3 μmol) was used.

[0087] <Comparative Example 1> Au 3+ The procedure was the same as in Example 1, except that 240 μL of hydrochloric acid (pH 2.2) was added dropwise instead of the solution (pH 6.8).

[0088] (Measurement of elastic modulus) The elastic modulus of the gel was measured using a small benchtop testing machine, EZ-test (manufactured by Shimadzu Corporation). Specifically, a rod-shaped jig (tip area: 11 mm²) set in the load cell of the testing machine was used to measure the elastic modulus of the gel. 2 Compression was performed at a temperature of 25°C and a compression rate of 1 mm / min to obtain a stress-strain curve. Young's modulus (kPa) was calculated from the slope of the elastic deformation region at the initial stage of stress rise in the obtained stress-strain curve and was defined as the modulus of elasticity.

[0089] (result) Figure 1 shows Au 3+ These are photographs showing the appearance of each composition 10 minutes after the addition of the solution or hydrochloric acid. In Figure 1, container 1 is Au 3+The image shows the composition with the solution (pH 6.8) added dropwise, and container 2 is Au 3+ The first container shows the composition with a solution (pH 2.2) added dropwise, and container 3 shows the composition with hydrochloric acid (pH 2.2) added dropwise.

[0090] As shown in Figure 1, Au is added to type I collagen. 3+ The composition in container 1, to which the solution (pH 6.8) was dropped, became a transparent gel (elastic modulus 2.2 kPa). Similarly, Au was added to type I collagen. 3+ The composition in container 2, to which the solution (pH 2.2) was added dropwise, became a transparent gel (elastic modulus 2.2 kPa). On the other hand, the composition in container 3, to which hydrochloric acid (pH 2.2) was added dropwise to type I collagen, did not gel. From these results, it can be concluded that the gelation of collagen is not dependent on pH, but on metal ions (Au). 3+ It becomes clear that the presence of ) is necessary.

[0091] [Test Example 2: Manufacturing of Gel Composition] (Preparation of collagen solution) A 0.2% by weight type I collagen solution was prepared using the same procedure as in Test Example 1. In addition, 1 g of mixed type I and type III collagen derived from porcine skin, manufactured by Nippon Ham Co., Ltd., was added to 500 mL of ultrapure water and incubated at 4°C for 12 hours to prepare a 0.2% by weight mixed type I and type III collagen solution.

[0092] (Au 3+ (Preparation of solutions) Using the same procedure as in Test Example 1, 12.5 mM Au 3+ A solution (pH 6.8) was prepared.

[0093] (Preparation of gel composition) <Example 3> 6 mL of 0.2 wt% type I collagen solution was placed in a container with a stirrer bar and stirred while cooling in an ice bath. Then, while stirring the collagen solution in the ice bath, 240 μL of Au was added to it. 3+ The solution (pH 6.8) was added dropwise (Au 3+ :3 μmol).

[0094] <Example 4> 6 mL of a 0.2 wt% mixed type I and type III collagen solution was placed in a container with a stirrer bar and stirred while cooling in an ice bath. Then, while stirring the collagen solution in the ice bath, 240 μL of Au was added to it. 3+ The solution (pH 6.8) was added dropwise (Au 3+ :3 μmol).

[0095] (result) Figure 2 shows Au 3+ This is a photograph showing the appearance of each composition 10 minutes after the solution was added. In Figure 2, container 1 contains Au in type I collagen solution. 3+ The composition is shown with a solution (pH 6.8) added dropwise. Container 6 contains a mixed collagen solution of type I and type III with Au 3+ The composition is shown with the solution (pH 6.8) added dropwise.

[0096] As shown in Figure 2, Au in type I collagen solution 3+ The composition in container 1, to which the solution (pH 6.8) was dropped, had become a transparent gel (elastic modulus 2.2 kPa). Au in a mixed collagen solution of type I and type III. 3+ The composition in container 6, to which the solution (pH 6.8) was added, also became a transparent gel, but it was a softer gel than the gel composition in container 1.

[0097] [Test Example 3: Manufacturing of Gel Composition] (Preparation of collagen solution) A 0.2% by weight type I collagen solution was prepared using the same procedure as in Test Example 1.

[0098] (Preparation of fragmented collagen solution) 50 mg of purified type I collagen, obtained using the same procedure as in Test Example 1, was suspended in 5 mL of 1 × PBS (pH=7), homogenized at room temperature for 6 minutes using a stirring homogenizer, and then incubated at 4°C for 3 days to obtain a 1% by weight fragmented collagen solution. The average diameter of the fragmented collagen in the obtained fragmented collagen solution was 84.4 ± 43.0 nm (N=25).

[0099] (Pd 2+(Preparation of solutions) Dissolve palladium chloride (H2PdCl4) in water and add 12.5 mM palladium. 2+ A solution (pH 1.1) was prepared.

[0100] (Preparation of gel composition) <Example 5> 6 mL of 0.2 wt% type I collagen solution was placed in a container with a stirrer bar and stirred while cooling in an ice bath. Then, while stirring the collagen solution in the ice bath, 240 μL of Pd was added to it. 2+ The solution was added dropwise (Pd 2+ :3 μmol).

[0101] <Example 6> 6 mL of 1% by weight fragmented collagen solution was placed in a container with a stirrer bar and stirred while cooling in an ice bath. Then, 240 μL of Pd was added to the fragmented collagen solution while stirring in the ice bath. 2+ The solution was added dropwise (Pd 2+ :3 μmol).

[0102] (result) Figure 3 shows Pd 2+ This is a photograph showing the appearance of each composition 10 minutes after the solution was added. In Figure 3, container 7 contains Pd in ​​type I collagen solution. 2+ The composition with the solution dropped into it is shown, and container 8 contains Pd in ​​the fragmented collagen solution. 2+ This shows the composition to which the solution has been added dropwise.

[0103] As shown in Figure 3, Pd 2+ The composition of container 7 into which the solution was dropped is Pd 2+ Although some discoloration originating from Pd was observed, the gel was transparent (elastic modulus 2.4 kPa). 2+ The composition in container 8, into which the solution was dropped, also contains Pd 2+ Although some discoloration originating from Pd was observed, the gel was transparent (elastic modulus 116 kPa). In particular, the composition of container 8 was Pd 2+ The area where the solution was dropped instantly gelled, forming a harder gel.

[0104] [Test Example 4: Manufacturing of Gel Composition] (Preparation of collagen solution) A 0.2% by weight type I collagen solution was prepared using the same procedure as in Test Example 1.

[0105] (Au + (Preparation of solutions) Chloro[(tetrahydrothiophen-1-ium)-1-yl]gold(III) was dissolved in dimethyl sulfoxide (DMSO), and 12.5 mM Au was added. + A solution was prepared.

[0106] (Preparation of gel composition) <Example 7> 6 mL of 0.2 wt% type I collagen solution was placed in a container with a stirrer bar and stirred while cooling in an ice bath. Then, while stirring the collagen solution in the ice bath, 240 μL of Au was added to it. + The solution was added dropwise (Au + :3 μmol).

[0107] <Comparative Example 2> 6 mL of 0.2 wt% type I collagen solution was placed in a container containing a stirrer bar and stirred while cooling in an ice bath. Then, DMSO was added dropwise to the collagen solution while stirring it in the ice bath.

[0108] <Comparative Example 3> Six mL of a 0.2 wt% type I collagen solution was placed in a container containing a stirrer bar and stirred while cooling in an ice bath. Next, while stirring the collagen solution in the ice bath, chloro[(tetrahydrothiophen-1-ium)-1-yl]gold(III) powder was added. Although stirring continued after the addition, the powder did not dissolve in the solution.

[0109] (result) Figure 4 shows Au +These are photographs showing the appearance of each composition 10 minutes after the addition or dropwise addition of the solution, DMSO, or chloro[(tetrahydrothiophen-1-ium)-1-yl]gold(III) powder. In Figure 4, container 9 shows the composition obtained by adding DMSO dropwise to the type I collagen solution, and container 10 shows the composition obtained by adding Au to the type I collagen solution. + The diagram shows the composition with the solution added dropwise, and container 11 shows the composition obtained by adding chloro[(tetrahydrothiophen-1-ium)-1-yl]gold(III) powder to a type I collagen solution.

[0110] As shown in Figure 4, in the composition of container 9, in which DMSO was dropped into the type I collagen solution, localized gelation was observed at the dropped portion, but gelation of the entire solution was not confirmed. Au + The composition in container 10, to which the solution was dropped, formed a transparent gel. On the other hand, in the composition in container 11, to which chloro[(tetrahydrothiophen-1-ium)-1-yl]gold(III) powder was added to the type I collagen solution, the powder did not dissolve in the solution.

[0111] [Test Example 5: Manufacturing of Gel Composition] (Preparation of collagen solution) A 1% by weight fragmented collagen solution was prepared using the same procedure as in Test Example 3.

[0112] (Pt 4+ (Preparation of solutions) Hexachloroplatinic acid (H2PtCl6) is dissolved in water, and 12.5M Pt 4+ A solution was prepared.

[0113] (Preparation of gel composition) <Example 8> 6 mL of 1% by weight fragmented collagen solution was placed in a container with a stirrer bar and stirred while cooling in an ice bath. Next, the fragmented collagen solution was left to stand in the ice bath, and 240 μL of Pt was added to it. 4+ The solution was added dropwise (Pt 4+ :3 μmol).

[0114] (result) Type I collagen solution with Pt 4+ The composition to which the solution was added did not show gelation immediately after addition, but after standing in an ice bath for 15 minutes, it became a transparent gel (elastic modulus 89 kPa).

[0115] [Test Example 6: Manufacturing and Evaluation of Three-Dimensional Tissues] (Preparation of collagen solution) A 0.2% by weight type I collagen solution was prepared using the same procedure as in Test Example 1.

[0116] (Preparation of fragmented collagen solution) 50 mg of purified type I collagen, obtained by the same procedure as in Test Example 1, was suspended in 5 mL of 1 × PBS (pH=7), homogenized at room temperature for 6 minutes using a stirring homogenizer, and then incubated at 4°C for 3 days to obtain a 1.0 wt% fragmented collagen solution. Furthermore, the fragmented collagen solution was diluted with RPMI-1640 medium to obtain a 0.5 wt% fragmented collagen solution.

[0117] (Pt 2+ (Preparation of solutions) Dissolve potassium tetrachloride platinum (K2PtCl4) in PBS and add 12.5 mM Pt 2+ A solution was prepared.

[0118] (Preparation of cell suspension) 1.0 × 10 6 Human mammary gland cancer cells (MDA-MB-231) from cells were suspended in 64 μL of RPMI-1640 medium containing 10% FBS, and the entire amount was seeded into the inserts of a 24-well insert (CORNING 3470).

[0119] (Preparation and evaluation of three-dimensional tissue models) <Example 9> In the insert of a 24-well insert seeded with cells, 224 μL of 0.5 wt% fragmented collagen solution and 12.5 mM Pt were added. 2+An additional 12 μL of Solution was added and suspended, and the cell-containing gel composition was obtained by incubating at room temperature for 30 minutes. Thereafter, 2 mL of RPMI-1640 medium was added outside the insert, the medium was changed every other day, and the cells were cultured for 4 days to prepare a three-dimensional tissue construct. At the time point of culturing for 4 days (Day 4), the appearance and internal structure of the three-dimensional tissue construct were observed using a phase-contrast microscope (CKX53 manufactured by Olympus).

[0120] (Results) Figure 5(A) is a photograph showing the appearance of the three-dimensional tissue construct. Figure 5(B) is a photograph showing the inside of the three-dimensional tissue construct. As shown in Figure 5(A) and Figure 5(B), the three-dimensional tissue construct obtained in Example 9 had a transparent appearance although there was coloring derived from the medium components and the like, and the internal cells could be observed. In addition, when the cell suspension and only the Pt 2+ solution were mixed to culture the cells and the cell viability was confirmed, the viability was 81% - 87% under the condition of the final concentration of Pt 2+ of 0.05 mM to 0.5 mM (no concentration dependence was observed), and no significant cytotoxicity was observed.

[0121] [Test Example 7: Production of Gel Composition] (Preparation of Fragmented Collagen Solution) A 1 wt% fragmented collagen solution was prepared by the same operation as in Test Example 3.

[0122] (Preparation of Pt 2+ Solution) Potassium tetrachloroplatinate (K2PtCl4) was dissolved in PBS to prepare Pt 2+ solutions of 25 mM, 12.5 mM, 2.5 mM, 1.25 mM, 0.25 mM, and 0.​​​​​​​​​​​

[0124] (Preparation of Gel Composition) <Example 10> 288 μL of 1 wt% fragmented collagen solution and Pt solution or Au solution at each concentration were added to the insert of a 24-well insert, and the mixture was suspended and incubated at room temperature for 30 minutes to obtain a gel composition. Further, after incubation at 37 °C for 24 hours, the elastic modulus was evaluated. The measurement of the elastic modulus was carried out in the same procedure as in Test Example 1.

[0125] <000058-0> (Results) Figure 6 is a graph showing the elastic modulus of the gel composition prepared with the solution of each metal ion. The horizontal axis indicates the final concentration of each metal ion. As shown in Figure 6, the elastic modulus of the gel improved as the concentration of the metal ion increased. Also, compared with the case of using Au solution, a gel with a higher elastic modulus could be prepared when using Pt solution. In both cases of using either metal ion solution, the elastic modulus of the gel improved depending on the concentration of the metal ion until the final concentration reached 0.1 mM, and showed an approximately constant elastic modulus in the concentration range of 0.1 mM or higher.<000058-2> <000058-3> <000058-4> [Test Example 8: Production of Gel Composition] (Preparation of Fragmented Collagen Solution) A 1 wt% fragmented collagen solution was prepared in the same operation as in Test Example 3. Then, 0.2 wt% and 0.5 wt% fragmented collagen solutions were prepared by diluting the prepared fragmented collagen solution with PBS.

[0127] (Preparation of Pt Solution) 2+ (Preparation of Pt Solution) Potassium tetrachloroplatinate (K2PtCl4) was dissolved in PBS to prepare Pt solutions of 12.5 mM, 2.5 mM, 1.25 mM, 0.25 mM, and 0.125 mM, respectively.

[0128] (Preparation of Gel Composition) <Example 11> Each well of a 96-well microplate contains 288 μL of fragmented collagen solution at various concentrations, and each well contains Pt at various concentrations. 2+ The solution was further suspended in 12 μL and incubated at room temperature for 30 minutes to obtain the gel composition. After incubation at 37°C for 24 hours, the absorbance at a wavelength of 500 nm was measured and the transmittance was calculated. The elastic modulus was then measured using the same procedure as in Example 1.

[0129] (result) Figure 7(A) shows fragmented collagen solution and Pt at various concentrations. 2+ This graph shows the transmittance at a wavelength of 500 nm for gel compositions prepared using a solution. The horizontal axis represents the transmittance of metal ions (Pt). 2+ The final concentrations of the fragmented collagen solution and Pt are shown. As shown in Figure 7(A), the permeability of the gel improved as the concentration of metal ions increased. In particular, when the final concentration of metal ions was 0.05 mM or higher, a significant improvement in permeability was observed compared to cases below that level. Figure 7(B) shows the final concentrations of the fragmented collagen solution and Pt at each concentration. 2+ This image shows the top surface of a gel prepared using a solution. In Figure 7(B), the numerical values ​​at the center of each well indicate the elastic modulus of the gel composition in that well. An interesting result was observed: despite the improvement in elastic modulus, the transmittance also improved.

[0130] [Test Example 9: Manufacturing of Gel Composition] (Preparation of fragmented collagen solution) A 0.5% by weight fragmented collagen solution was prepared using the same procedure as in Test Example 6.

[0131] (Pd 2+ (Preparation of solutions) Dissolve palladium(II) chloride (PdCl2) in water and add 12.5 mM Pd 2+ A solution was prepared.

[0132] (Cu 2+ (Preparation of solutions) Dissolve copper(II) chloride (CuCl2) in water and add 12.5 mM Cu 2+ A solution was prepared.

[0133] (Zn 2+ (Preparation of solutions) Dissolve zinc(II) chloride (ZnCl2) in water to form a 12.5 mM Zn solution. 2+ A solution was prepared.

[0134] (Preparation of gel composition) <Example 12> A gel composition was obtained by suspending 480 μL of 0.5 wt% fragmented collagen solution and 20 μL of each metal ion solution in a glass container and incubating at room temperature for 30 minutes. After further incubation at 4°C for 24 hours, the appearance was evaluated.

[0135] (result) Figure 8 shows photographs of the appearance of each composition after one day following the addition of each metal solution. All compositions prepared using any of the metal ions were transparent gels.

Claims

1. It contains at least one selected from the group consisting of extracellular matrix components and fragmented extracellular matrix components, and an ion of a metal element. The extracellular matrix component includes collagen, The aforementioned fragmented extracellular matrix component includes defibrated collagen, The aforementioned metallic element is at least one metallic element selected from the group consisting of transition metals of Group 10, transition metals of Group 11, and metals of Group 12 of the periodic table. A gel composition having an elastic modulus of 1.5 kPa or higher.

2. The gel composition according to claim 1, wherein the elastic modulus is 1686 kPa or less.

3. The gel composition according to claim 1 or 2, wherein the metal element is at least one selected from the group consisting of copper, zinc, palladium, platinum, and gold.

4. The gel composition according to any one of claims 1 to 3, wherein at least one selected from the group consisting of the extracellular matrix component and the fragmented extracellular matrix component is crosslinked.

5. A method for producing a gel composition, The method comprises a step of contacting a solution containing at least one selected from the group consisting of extracellular matrix components and fragmented extracellular matrix components with a solution containing ions of a metal element. The extracellular matrix component includes collagen, The aforementioned fragmented extracellular matrix component includes defibrated collagen, The aforementioned metal elements are transition metals of Group 10, Group 11, and Group 12 of the periodic table. At least one metallic element selected from the group consisting of the following metals, A method for producing the gel composition wherein the elastic modulus of the gel composition is 1.5 kPa or more.

6. The manufacturing method according to claim 5, wherein the content of at least one selected from the group consisting of the extracellular matrix component and the fragmented extracellular matrix component in the gel composition is 0.01% by weight or more and 1% by weight or less based on the total amount of the gel composition.

7. The manufacturing method according to claim 5 or 6, wherein the content of ions of the metal element in the gel composition is 5 mg or more and 60 mg or less per 1 g of the total content of at least one selected from the group consisting of the extracellular matrix component and the fragmented extracellular matrix component.

8. A step of obtaining a cell-containing gel composition by contacting at least one selected from the group consisting of extracellular matrix components and fragmented extracellular matrix components, cells, and ions of a metal element in an aqueous medium. The process comprises a step of culturing the cell-containing gel composition, The extracellular matrix component includes collagen, The aforementioned fragmented extracellular matrix component includes defibrated collagen, The aforementioned metallic element is at least one metallic element selected from the group consisting of transition metals of Group 10, transition metals of Group 11, and metals of Group 12 of the periodic table. A method for producing a three-dimensional structure, wherein the elastic modulus of the gel composition is 1.5 kPa or more.

9. The method for producing a three-dimensional tissue according to claim 8, wherein the step of obtaining the cell-containing gel composition comprises contacting a suspension containing at least one selected from the group consisting of the extracellular matrix component and the fragmented extracellular matrix component, the cells, and a first aqueous medium with a solution containing ions of the metal element and a second aqueous medium.

10. The method for producing a three-dimensional tissue according to claim 8 or 9, wherein the culturing step is carried out under conditions in which at least a portion of the cells remain viable.

11. A method for producing a three-dimensional tissue according to any one of claims 8 to 10, further comprising the step of incubating the cell-containing gel composition at 20°C to 30°C after the step of obtaining the cell-containing gel composition and before the step of culturing.

12. It contains at least one selected from the group consisting of extracellular matrix components and fragmented extracellular matrix components, cells, and ions of a metal element. The extracellular matrix component includes collagen, The aforementioned fragmented extracellular matrix component includes defibrated collagen, The aforementioned metal element is at least one metal element selected from the group consisting of transition metals of Group 10, transition metals of Group 11, and metals of Group 12 of the periodic table, and has a total light transmittance of 80% or more at 37°C. A three-dimensional tissue with a thickness of 10 μm or more.

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