Composition, three-dimensional structure forming agent, and method for producing a three-dimensional structure

Abstract

To provide a composition suitable for a three-dimensional tissue-forming agent that can form a three-dimensional tissue with suppressed contraction while reducing the amount of extracellular matrix components used. [Solution] A composition comprising an extracellular matrix component and gelatin, wherein the extracellular matrix component comprises a fragmented extracellular matrix component, and the gelatin content is greater than 0 parts by mass and less than or equal to 50 parts by mass per 100 parts by mass of the total of the extracellular matrix component and the gelatin.

Description

Technical Field

[0001] The present invention relates to a composition, a three-dimensional tissue former, and a method for producing a three-dimensional tissue.

Background Art

[0002] Three-dimensional tissues are expected to be used as alternatives to experimental animals, transplantation materials, etc. Various studies have been made on methods for producing three-dimensional tissues. For example, Patent Document 1 discloses a method for producing a three-dimensional tissue, which includes a step of contacting fragmented collagen derived from exogenous collagen with cells in an aqueous medium, and a step of culturing the cells contacted with the fragmented collagen.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0004] When forming a three-dimensional tissue for cultured meat using fragmented extracellular matrix components such as fragmented collagen, it is desirable to reduce the amount of extracellular matrix components used from the viewpoint of cost and the like. However, when the amount of extracellular matrix components used is reduced, the formed tissue may shrink.

[0005] An object of the present invention is to provide a three-dimensional tissue former capable of forming a three-dimensional tissue with suppressed shrinkage while suppressing the amount of extracellular matrix components used, and a composition suitable for the three-dimensional tissue former. Another object of the present invention is to provide a method for producing a three-dimensional tissue with suppressed shrinkage while suppressing the amount of extracellular matrix components used.

Means for Solving the Problems

[0006] This invention encompasses the following inventions. [1] A composition comprising an extracellular matrix component and gelatin, wherein the extracellular matrix component comprises a fragmented extracellular matrix component, and the gelatin content is greater than 0 parts by mass and less than or equal to 50 parts by mass per 100 parts by mass of the total of the extracellular matrix component and the gelatin. [2] The composition according to claim 1 or 2, wherein the gelatin content is 5 parts by mass or more and 40 parts by mass or less with respect to 100 parts by mass of the total of the extracellular matrix components and the gelatin. [3] The composition according to [1] or [2], wherein the gelatin is derived from a non-human mammal. [4] The composition according to any one of [1] to [3], wherein the gelatin is derived from bovine. [5] The composition according to any one of [1] to [4], wherein the fragmented extracellular matrix component comprises a fragmented collagen component. [6] A three-dimensional tissue-forming agent comprising an extracellular matrix component and gelatin, wherein the extracellular matrix component includes a fragmented extracellular matrix component, and the gelatin content is greater than 0 parts by mass and less than or equal to 50 parts by mass per 100 parts by mass of the total of the extracellular matrix component and the gelatin. [7] The three-dimensional tissue-forming agent according to [6], wherein the gelatin content is 5 parts by mass or more and 40 parts by mass or less with respect to 100 parts by mass of the total of the extracellular matrix component and the gelatin. [8] The three-dimensional tissue-forming agent according to [6] or [7], wherein the gelatin is derived from a non-human mammal. [9] The three-dimensional tissue-forming agent according to any one of [6] to [8], wherein the gelatin is derived from bovine.

[10] The three-dimensional tissue-forming agent according to any one of [6] to [9], wherein the fragmented extracellular matrix component comprises a fragmented collagen component.

[11] A method for producing a three-dimensional tissue, comprising the step of culturing the cells in a culture medium containing cells, an extracellular matrix component, and gelatin, wherein the extracellular matrix component includes a fragmented extracellular matrix component, and the amount of gelatin used is greater than 0 parts by mass and less than or equal to 50 parts by mass relative to 100 parts by mass of the total of the extracellular matrix component and the gelatin.

[12] The method according to

[11] , wherein the amount of gelatin used is 5 parts by mass or more and 40 parts by mass or less, based on 100 parts by mass of the total of the extracellular matrix components and the gelatin.

[13] The method according to

[11] or

[12] , wherein the gelatin is derived from a non-human mammal.

[14] The method according to any one of

[11] to

[13] , wherein the gelatin is derived from bovine.

[15] The method according to any one of

[11] to

[14] , wherein the fragmented extracellular matrix component comprises a fragmented collagen component. [Effects of the Invention]

[0007] According to the present invention, it is possible to provide a three-dimensional tissue-forming agent that can form a three-dimensional tissue with suppressed contraction while reducing the amount of extracellular matrix components used, and a composition suitable for the three-dimensional tissue-forming agent. According to the present invention, it is possible to provide a method for producing a three-dimensional tissue with suppressed contraction while reducing the amount of extracellular matrix components used. [Brief explanation of the drawing]

[0008] [Figure 1] Figure 1 is a graph showing the evaluation results of the shrinkage rate of the three-dimensional tissue in the example. [Modes for carrying out the invention]

[0009] The embodiments for carrying out the present invention will be described in detail below. However, the present invention is not limited to the following embodiments.

[0010] [Composition] The composition according to this embodiment contains an extracellular matrix component and gelatin. In this composition, the extracellular matrix component contains a fragmented extracellular matrix component, and the content of gelatin is more than 0 part by mass and 50 parts by mass or less with respect to a total of 100 parts by mass of the extracellular matrix component and gelatin.

[0011] [Extracellular matrix component] As used herein, the "extracellular matrix component" is an aggregate of extracellular matrix molecules formed by a plurality of extracellular matrix molecules. The extracellular matrix molecule means a substance existing outside cells in an organism. As the extracellular matrix, any substance can be used 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.

[0012] The extracellular matrix may be a modified form or variant of the above-described extracellular matrix, or a polypeptide such as a chemically synthesized peptide, as long as it does not adversely affect cell growth and the formation of cell aggregates. The extracellular matrix may have a repeat of a sequence represented by Gly-X-Y characteristic of collagen. Here, Gly represents a glycine residue, and X and Y each independently represent an arbitrary amino acid residue. The plurality of Gly-XY may be the same or different from each other. By having a repeat of the sequence represented by Gly-X-Y, the restriction on the arrangement of the molecular chain is reduced, and for example, the function as a scaffold material during cell culture becomes even more excellent. 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 entire amino acid sequence. The extracellular matrix may be a polypeptide having an RGD sequence. The RGD sequence refers to a sequence represented by Arg-Gly-Asp (arginine residue-glycine residue-aspartic acid residue). By having the RGD sequence, cell adhesion is further promoted, and for example, it becomes more suitable as a scaffold material during cell culture. Examples of the extracellular matrix containing the sequence represented by Gly-X-Y and the RGD sequence include collagen, fibronectin, vitronectin, laminin, cadherin, and the like. [[ID=!

[0013] Examples of collagen include fibrous collagen and non-fibrous collagen. Fibrous collagen means collagen that is the main component of collagen fibers. Examples of fibrous collagen include type I collagen, type II collagen, type III collagen, and the like. Examples of non-fibrous collagen include type IV collagen.

[0014] Examples of proteoglycans include, but are not limited to, chondroitin sulfate proteoglycan, heparan sulfate proteoglycan, keratan sulfate proteoglycan, and dermatan sulfate proteoglycan.

[0015] Examples of extracellular matrix component shapes include fibrous structures. Fibrous refers to a shape composed of thread-like extracellular matrix components, or a shape composed of thread-like extracellular matrix components cross-linked between molecules. At least a portion of the extracellular matrix components may be fibrous. The shape of the extracellular matrix components is the shape of a single mass of extracellular matrix components (an aggregate of extracellular matrix components) observed under a microscope, and the extracellular matrix components preferably have an average diameter and / or average length, as described later. Fibrous extracellular matrix components include thin thread-like structures (fibers) formed by the aggregation of multiple thread-like extracellular matrix molecules, thread-like structures formed by further aggregation of fine fibers, and defibrations of these thread-like structures. When extracellular matrix components with a fibrous shape are included, the RGD sequence is preserved without being destroyed in the fibrous extracellular matrix components, and they can function even more effectively as a scaffold for cell adhesion.

[0016] The extracellular matrix components may include at least one selected from the group consisting of collagen, laminin, and fibronectin, and it is preferable that they include collagen. The collagen is preferably fibrous collagen, and more preferably type I collagen. As for the fibrous collagen, commercially available collagen may be used, and a specific example of this is porcine skin-derived type I collagen manufactured by Nippon Ham Co., Ltd.

[0017] 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.

[0018] Extracellular matrix components include fragmented extracellular matrix components. "Fragmentation" means reducing the size of aggregates of extracellular matrix components. Fragmented extracellular matrix components may also include defibrated extracellular matrix components. Defibrated extracellular matrix components are components obtained by defibrating the above-mentioned extracellular matrix components by applying physical force. For example, defibration is performed under conditions that do not break the bonds within the extracellular matrix molecules.

[0019] Fragmented extracellular matrix components can be produced, for example, by a method that includes a step of fragmenting extracellular matrix components (fragmentation step).

[0020] There are no particular restrictions on the method for fragmenting extracellular matrix components; fragmentation may be carried out by applying physical force. Unlike enzymatic treatment, the molecular structure of extracellular matrix components fragmented by physical force usually does not change from before fragmentation (the molecular structure is maintained). The method for fragmenting extracellular matrix components may be, for example, a method of finely crushing clumps of extracellular matrix components. Extracellular matrix components may be fragmented in a solid phase or in an aqueous medium. For example, extracellular matrix components may be fragmented by applying physical force 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. By adjusting the homogenization time, number of repetitions, etc., it is possible to obtain millimeter-sized or nanometer-sized fragmented extracellular matrix components. When fragmenting extracellular matrix components in an aqueous medium, the fragmented extracellular matrix components can be produced, for example, by a method comprising the steps of fragmenting the extracellular matrix components in an aqueous medium and removing the aqueous medium from a solution containing the fragmented extracellular matrix components and the aqueous medium (removal step). The removal step may be carried out, for example, by freeze-drying.

[0021] "Aqueous medium" refers to a liquid in which water is an essential component. Examples of aqueous mediums include physiological salines such as phosphate-buffered saline (PBS), sterile water, and pH buffers such as Good's buffer. "Removing the aqueous medium" does not mean that no moisture is attached to the fragmented extracellular matrix components, but rather that the amount of moisture attached is reduced to a level that can be reasonably achieved by the general drying methods described above.

[0022] The diameter and length of fragmented extracellular matrix components can be determined by analyzing individual fragmented extracellular matrix components using an electron microscope.

[0023] 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, or between 100 μm and 400 μm, from the viewpoint of facilitating the formation of thick three-dimensional tissues. In another embodiment, 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 within the total fragmented extracellular matrix components falls within the above numerical range. Specifically, it is preferable that the average length of 50% or more of the fragmented extracellular matrix components is within the above numerical range, and it is more preferable that the average length of 95% of the fragmented extracellular matrix components is within the above numerical range. The fragmented extracellular matrix components are preferably fragmented collagen components whose average length is within the above range.

[0024] The average diameter of the fragmented extracellular matrix components may be 50 nm or more, 100 nm or more, 200 nm or more, 300 nm or more, 400 nm or more, 500 nm or more, or 600 nm or more, and may be 30 μm or less, 20 μm or less, 10 μm or less, 8 μm or less, 6 μm or less, 4 μm or less, 2 μm or less, 1 μm or less, or 800 nm or less. The average diameter of the fragmented extracellular matrix components may be 50 nm to 30 μm, 600 nm or more 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.

[0025] 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 or the like, and then 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.

[0026] "Fragmented collagen component" refers to a collagen component, such as fibrous collagen component, that has been fragmented while maintaining a triple helix structure. The average length of the fragmented collagen component is preferably 100 nm to 200 μm, more preferably 22 μm to 200 μm, and even more preferably 100 μm to 200 μm. The average diameter of the fragmented collagen component is preferably 50 nm to 30 μm, more preferably 4 μm to 30 μm, and even more preferably 20 μm to 30 μm.

[0027] The content of extracellular matrix components may be 50 parts by mass or more, 60 parts by mass or more, 70 parts by mass or more, 80 parts by mass or more, 90 parts by mass or more, or 95 parts by mass or more, relative to 100 parts by mass of the total of extracellular matrix components and gelatin, in order to make it easier to obtain a three-dimensional tissue with suppressed shrinkage. The content of extracellular matrix components may be 99 parts by mass or less, 95 parts by mass or less, 90 parts by mass or less, 80 parts by mass or less, 75 parts by mass or less, 70 parts by mass or less, 65 parts by mass or less, 60 parts by mass or less, or 55 parts by mass or less, relative to 100 parts by mass of the total of extracellular matrix components and gelatin, in order to further facilitate mass production of three-dimensional tissues by reducing raw material costs, etc.

[0028] <Gelatin> Gelatin is a denatured form of collagen, a component in which the triple helix structure derived from collagen is destroyed through denaturation. Gelatin can be obtained by processes such as hot water treatment of collagen. Gelatin may or may not be fragmented.

[0029] Gelatin can be derived from, for example, mammals (human or non-human mammals), birds, and fish. Examples of non-human mammals include cattle and pigs. Gelatin can be derived from sources other than fish, preferably from non-human mammals, and more preferably from cattle, as it is easier to suppress denaturation under mammalian cell culture conditions (37°C) and is therefore more suitable for cultured meat applications.

[0030] The gelatin content is greater than 0 parts by mass and less than or equal to 50 parts by mass per 100 parts by mass of the total extracellular matrix components and gelatin. The lower limit of the gelatin content may be 1 part by mass or more, 3 parts by mass or more, 5 parts by mass or more, 10 parts by mass or more, 15 parts by mass or more, 20 parts by mass or more, 25 parts by mass or more, 30 parts by mass or more, 35 parts by mass or more, 40 parts by mass or more, or 45 parts by mass or more per 100 parts by mass of the total extracellular matrix components and gelatin, as this will further facilitate the mass production of three-dimensional tissues by reducing raw material costs, etc. The upper limit of the gelatin content may be 45 parts by mass or less, 40 parts by mass or less, 35 parts by mass or less, 30 parts by mass or less, 25 parts by mass or less, 20 parts by mass or less, 15 parts by mass or less, 10 parts by mass or less, 5 parts by mass or less, 3 parts by mass or less, or 1 part by mass or less, relative to 100 parts by mass of the total of extracellular matrix components and gelatin, in order to make it easier to obtain a three-dimensional tissue with suppressed shrinkage.

[0031] The extracellular matrix components and gelatin may be cross-linked, but it is preferable that they are not. When the extracellular matrix components and gelatin are not cross-linked, there is no concern about residual cross-linking agents. In addition, when the extracellular matrix components and gelatin are not cross-linked, their swelling in water is higher compared to when they are cross-linked, making it easier to increase the bulk and reduce the amount used. As a result, raw material costs can be reduced, and the mass production of three-dimensional tissues becomes easier.

[0032] The composition may be, for example, a powder, liquid, gel, lump, fibrous, or porous material.

[0033] If the composition is a liquid composition, the liquid composition may further contain the aqueous medium described above. The composition according to this embodiment can be suitably used as a bio-ink for 3D bioprinting by being a liquid medium.

[0034] [Method for manufacturing the composition] The composition can be produced, for example, by a method comprising the step of mixing a fragmented extracellular matrix component with gelatin, or by a method comprising the step of fragmenting a mixture comprising an extracellular matrix component and gelatin. The composition can be produced, for example, by a method comprising the following steps, but the production method is not limited to the following. (1) A step of neutralizing the solution containing the extracellular matrix components and gelatin. (2) Step to gel the neutralized solution (3) A step to remove the solvent by freeze-drying to obtain a mass containing extracellular matrix components and gelatin. (4) Disperse the aggregate in a solution containing a polar organic solvent, and in the solution, at least the extracellular matrix components are fragmented to obtain the first solution. (5) A step of obtaining a second liquid by replacing the liquid component in the first liquid with water. (6) The second liquid is subjected to ultrasonic fracturing. (7) Step of removing liquid components from the second liquid

[0035] <Process (1)> Step (1) is a step of neutralizing the solution containing the extracellular matrix components and gelatin. Examples of solvents for the solution containing the extracellular matrix components and gelatin include acetic acid, water, and buffer solutions (e.g., phosphate-buffered saline (PBS), Tris-HCl buffer). The solvent may be, for example, an aqueous solution of acetic acid. The concentration of the aqueous solution of acetic acid may be 1 to 10 mM or 3 to 8 mM.

[0036] The concentration of the extracellular matrix component in the solution in step (1) is not particularly limited, but may be, for example, 1 mg / mL or more and 50 mg / mL or less.

[0037] Since the solution containing the extracellular matrix components and gelatin is usually acidic, the neutralization step can be carried out, for example, by adding an alkaline solution to the solution containing the extracellular matrix components and gelatin. There are no particular restrictions on the alkaline solution, but solutions obtained by dissolving alkali metal hydroxides, alkaline earth metal hydroxides, etc. in water are preferably used. More specifically, examples of alkaline solutions include aqueous solutions of potassium hydroxide (KOH) and aqueous solutions of sodium hydroxide (NaOH).

[0038] If the solution containing the extracellular matrix components and gelatin is alkaline, the neutralization step can be carried out, for example, by adding an acidic solution to the solution containing the extracellular matrix components and gelatin. There are no particular restrictions on the acidic solution, but examples include hydrochloric acid solution, sulfuric acid solution, acetic acid solution, and carbonate solution.

[0039] The pH of the neutralized solution may be, for example, between 6 and 8, between 6.5 and 7.5, between 6.9 and 7.1, or between 6.95 and 7.05. It is not necessarily required to perform neutralization so that the pH of the neutralized solution becomes 7.

[0040] <Process (2)> Step (2) is a step of gelling the neutralized solution. Step (2) may be performed as needed. Gelation can be performed, for example, by heating the neutralized solution. The temperature and heating time can be set appropriately depending on the type and concentration of the extracellular matrix components, but examples include a heating temperature of 25-45°C and a heating time of 1-3 hours. By performing gelation, it is possible to confirm whether the solution was uniformly neutralized in step (2) by visually determining whether the formed gel is uniform.

[0041] <Process (3)> Step (3) is a step in which the solvent is removed by freeze-drying to obtain a mass containing extracellular matrix components and gelatin. Freeze-drying can be carried out according to conventional methods. Step (3) removes the solvent and yields a mass (solid) containing extracellular matrix components. The removal of the solvent does not mean that no solvent is attached to the mass (solid) containing extracellular matrix components and gelatin, but rather that the amount of solvent attached is reduced to a level that can be reasonably achieved by a typical freeze-drying process.

[0042] <Process (4)> The fragmentation step involves dispersing a mass containing extracellular matrix components and gelatin in a solution containing a polar organic solvent, thereby fragmenting the extracellular matrix components in the solution to obtain a first solution. The polar organic solvent is not particularly limited as long as it is a polar organic solvent, and examples include alcohols such as methanol, ethanol, n-propanol, and isopropanol, as well as acetone, acetonitrile, and diethyl ether.

[0043] The concentration of the polar organic solvent in a solution containing a polar organic solvent may be, for example, 20 v / v% or more, 30 v / v% or more, 40 v / v% or more, 50 v / v% or more, 60 v / v% or more, 70 v / v% or more, or 80 v / v% or more, and may be 100 v / v% or less, 95 v / v% or less, or 90 v / v% or less.

[0044] The fragmentation method can be the one described above. More specific conditions for fragmenting extracellular matrix components include, for example, using a homogenizer (AS ONE, VH-10) and processing at 20,000 rpm to 30,000 rpm for 3 to 10 minutes, or conditions in which an equivalent physical force can be applied.

[0045] <Process (5)> Step (5) is a step of obtaining a second solution by replacing the liquid component in the first solution with water. The second solution contains fragmented extracellular matrix components, gelatin, and water. Examples of water include tap water, distilled water, and ultrapure water. There are no particular restrictions on the method of replacing the liquid component in the first solution with water, and the usual method used in solvent replacement can be used. For example, one method of replacement with water is to centrifuge the first solution to settle the fragmented extracellular matrix components in the solution, then remove the liquid components, and then add water to the settled components. Replacing liquid components with water does not mean that all liquid components present in the solution are replaced with water, but rather that the main solvent in the solution is replaced with water, and trace amounts of liquid components other than water may remain after replacement.

[0046] <Process (6)> Step (6) is a step of performing ultrasonic fracturing on the second liquid. Ultrasonic fracturing may be performed as needed. Ultrasonic fracturing can be performed using an ultrasonic fracturing machine. As an ultrasonic fracturing machine, for example, a fully automatic ultrasonic fracturing machine such as the BIORUPTOR II manufactured by BM Kiki Co., Ltd. may be used. The ultrasonic fracturing treatment may be performed by repeatedly irradiating the second liquid with ultrasound and cooling the liquid. For example, the ultrasonic fracturing treatment may be performed by repeating ultrasonic irradiation for 10 to 30 seconds and cooling for 20 to 40 seconds 50 to 150 times under the condition of an ultrasonic frequency of 20 kHz.

[0047] <Process (7)> Step (7) is a step to remove liquid components from the second liquid. Removal of liquid components can be carried out by, for example, air drying, freeze-drying, vacuum drying, or vacuum freeze-drying. Removal of liquid components may be carried out by air drying or freeze-drying. Step (7) removes the liquid components and yields a solid containing fragmented extracellular matrix components and gelatin. Removal of liquid components does not mean that no liquid components are attached to the resulting solid, but rather that the amount of liquid components attached can be reasonably achieved by the general drying methods described above. The solid obtained in step (7) may be dispersed in an aqueous medium as needed to form a dispersion. Specific examples of aqueous mediums include, for example, water, phosphate-buffered saline (PBS), and Tris buffer (Tris).

[0048] [Three-dimensional tissue-forming agent] The compositions described above can be suitably used as three-dimensional tissue-forming agents. One embodiment of the present invention provides a three-dimensional tissue-forming agent comprising an extracellular matrix component and gelatin, wherein the extracellular matrix component comprises a fragmented extracellular matrix component, and the gelatin content is greater than 0 parts by mass and 50 parts by mass or less per 100 parts by mass of the total of the extracellular matrix component and gelatin. Specific embodiments of the three-dimensional tissue-forming agent can be those described as specific embodiments of the composition.

[0049] [Method for manufacturing three-dimensional tissue] A method for producing a three-dimensional tissue according to this embodiment comprises a step of culturing cells in a culture medium containing cells, extracellular matrix components, and gelatin (culture step). The extracellular matrix components in the culture step include fragmented extracellular matrix components.

[0050] In this specification, "three-dimensional tissue" means an aggregate of cells (a cluster of cells) in which cells are arranged three-dimensionally via extracellular matrix components, and is an aggregate artificially created by cell culture. The shape of the three-dimensional tissue is not particularly limited, and examples include sheet-like, spherical, nearly spherical, ellipsoidal, nearly ellipsoidal, hemispherical, nearly hemispherical, semicircular, nearly semicircular, rectangular parallelepiped, and nearly rectangular parallelepiped. The three-dimensional tissue may be aggregated in a cluster while attached to a support, or it may be aggregated in a cluster without being attached to a support.

[0051] In the culture process, the amount of gelatin used is greater than 0 parts by mass and less than or equal to 50 parts by mass per 100 parts by mass of the total of extracellular matrix components and gelatin. The amounts of gelatin and extracellular matrix components used in the culture process may be within the range specified as their content in the composition.

[0052] The cells are not particularly limited, but may be derived from mammals such as humans, monkeys, dogs, cats, rabbits, pigs, cows, mice, and rats. The site of origin of the cells is also not particularly limited; they may be somatic cells derived from bone, muscle, internal organs, nerves, brain, skin, blood, etc., or germ cells. Furthermore, the cells may be stem cells, or cultured cells such as primary cultured cells, subcultured cells, and cell line cells.

[0053] Specifically, the cells include, for example, skeletal muscle cells, smooth muscle cells (e.g., aortic smooth muscle cells (Aorta-SMC)), cardiomyocytes (e.g., human iPS cell-derived cardiomyocytes (iPS-CM)), adipocytes (e.g., mature adipocytes), vascular endothelial cells (e.g., human umbilical vein-derived vascular endothelial cells (HUVEC)), pericytes, lymphatic endothelial cells, nerve cells, dendritic cells, immune cells, fibroblasts, chondrocytes, osteoblasts, epithelial cells (e.g., human gingival epithelial cells), keratinocytes, hepatocytes, pancreatic islet cells, tissue stem cells (e.g., satellite cells, mesenchymal stem cells), astrocytes, colorectal cancer cells (e.g., human colorectal cancer cells (HCT116, HT29)), cancer cells such as hepatocytes, and so on. The cells may be used individually or in combination of multiple types. It is preferable that the cells include satellite cells. When the cultured cells include satellite cells, a three-dimensional tissue suitable for cultured meat applications can be produced.

[0054] Culture media can be prepared by mixing cells, extracellular matrix components, and gelatin. Mixing methods include, but are not limited to, mixing an aqueous medium containing fragmented extracellular matrix components and gelatin with an aqueous medium containing cells; adding cells to an aqueous medium containing fragmented extracellular matrix components and gelatin and mixing; adding an aqueous medium containing fragmented extracellular matrix components and gelatin to a culture medium containing cells and mixing; and adding fragmented extracellular matrix components, gelatin, and cells to a pre-prepared aqueous medium and mixing them separately.

[0055] The culture medium includes a culture medium. The culture medium can be appropriately selected depending on the type of cells to be cultured. Examples of culture media include liquid media such as IntegriCultureModified Eagle's Medium (IMEM), Eagle's MEM medium, DMEM, Modified Eagle medium (MEM), Minimum Essential medium, RPMI, and GlutaMax medium and EGM2. The culture medium may be a serum-added medium or a serum-free medium. The culture medium may also be a mixed medium, which is a mixture of two types of culture media.

[0056] The culture method can be one that is suitable for the type of cells being cultured. The culture temperature may be 20°C to 40°C or 30°C to 37°C. The pH of the culture medium may be 6.0 to 8.0 or 7.2 to 7.4. The culture time may be 1 day to 2 weeks or 1 week to 2 weeks.

[0057] The culture vessel (support) is not particularly limited and may be, for example, a well insert, a low-adhesion plate, or a plate with a bottom shape such as U-shaped or V-shaped. Cells may be cultured while attached to the support, or without being attached to the support, or they may be separated from the support during culture. When culturing cells without being attached to the support, or when separating them from the support during culture, it is preferable to use a plate with a bottom shape such as U-shaped or V-shaped that inhibits cell adhesion to the support, or a low-adhesion plate.

[0058] The cell density in the culture process is, for example, 1 to 10 8 cells / mL, or 10 3 ~10 7 Cells / mL may also be acceptable.

[0059] The three-dimensional tissue according to this embodiment comprises cells, an extracellular matrix component including fragmented extracellular matrix components, and gelatin. The three-dimensional tissue can be manufactured by the method described above.

[0060] Despite having a low content of extracellular matrix components, the three-dimensional tissue according to this embodiment exhibits suppressed tissue shrinkage over time.

[0061] The three-dimensional tissue can be suitably used for applications such as cultured meat and drug efficacy evaluation. [Examples]

[0062] The present invention will be described more specifically below based on examples. However, the present invention is not limited to the following examples.

[0063] 1.Material Table 1 shows the materials used, and Table 2 shows the culture media used. [Table 1] [Table 2]

[0064] 2. Experimental conditions 2-1. Preparation of the composition [Table 3]

[0065] Solutions containing gelatin and collagen were prepared in 15 mL centrifuge tubes in the mass ratios shown in Table 3. For the gelatin:collagen solution with a ratio of 100:0, 5 mM acetic acid, a solvent for collagen, was used as the solvent. 2.5 mL of a buffer prepared by mixing 0.05 N NaOH and ×10 PBS in a 1:1 ratio was added to each solution. The mixed solutions were heated at 37°C for 1 hour to gel. The resulting gels were frozen in liquid nitrogen and freeze-dried. 5 mL of 85 v / v% EtOH was added to the freeze-dried product and homogenized.

[0066] Under the condition where the gelatin:collagen ratio was 100:0 (Comparative Example 1), denaturation occurred during the homogenization process after adding 5 mL of 85% EtOH, and fiber formation was not possible.

[0067] The following procedure was performed on the samples (Examples 1-2 and Comparative Examples 2-3), excluding Comparative Example 1, which could not be fiberized. First, the samples were centrifuged at 10,000 rpm for 3 minutes, and then the supernatant was removed. 5 mL of 85% 0 v / v% EtOH was added to the sample after supernatant removal and homogenized. The homogenized sample was centrifuged at 10,000 rpm for 3 minutes and the supernatant was removed. 5 mL of 70 v / v% EtOH was added and homogenized. The homogenized sample was centrifuged at 10,000 rpm for 3 minutes and the supernatant was removed. Next, 5 mL of ultrapure water was added to the sample and homogenization was performed. The resulting sample was centrifuged at 10,000 rpm for 3 minutes and the supernatant was removed. Then, the sample was resuspended in 5 mL of ultrapure water and ultrasonic disruption (20 kHz, 10-second oscillations x 10 times) was performed. The ultrasonically disrupted sample was freeze-dried. Through the above operations, we obtained the freeze-dried compositions of Comparative Example 2 and Examples 1-2, which contain fragmented collagen components and gelatin, as well as the composition of Comparative Example 3, which contains fragmented collagen components but does not contain gelatin.

[0068] The average diameter of the fragmented collagen components, as measured by SEM image evaluation, was 560 nm ± 50 nm. The average length of the fragmented collagen components was 100–200 μm.

[0069] 2-2. Preparation of three-dimensional tissue models Each of the compositions from Examples 1-2 and Comparative Examples 2-3 was weighed to 1 mg and suspended in 1 mL of ultrapure water. The resulting suspension was then mixed with 3 × 10⁶ bovine-derived satellite cells isolated from a bovine. 6 The individual cells and the other were mixed. The resulting mixture was seeded into 24-well culture inserts (Corning / #3480) and cultured in growth medium at 37°C and 25% CO2. From day 4 of culture, the medium was switched to differentiation medium and differentiation culture was performed.

[0070] 2-3. Measurement of contraction rate The tissue preparation day was designated as Day 0, and the entire culture insert was photographed daily from the bottom using a phase-contrast microscope. The bottom area of ​​the tissue was calculated from the microscopic images, and the shrinkage rate was evaluated as a ratio to the bottom area of ​​the culture insert. Figure 1 shows the results of the shrinkage rate evaluation.

[0071] 3.Results When the gelatin content was 50 parts by mass or less per 100 parts by mass of the total extracellular matrix components and gelatin (Examples 1-2), the shrinkage of the three-dimensional tissue was suppressed compared to when it was 75 parts by mass (Comparative Example 2). It was shown that when the gelatin content is between 0 parts by mass and 50 parts by mass per 100 parts by mass of the total extracellular matrix components and gelatin, it is possible to form a three-dimensional tissue with suppressed shrinkage while reducing the amount of extracellular matrix components used.

Claims

1. It contains extracellular matrix components and gelatin. The extracellular matrix component includes fragmented extracellular matrix components, A composition in which the gelatin content is greater than 0 parts by mass and less than or equal to 50 parts by mass relative to 100 parts by mass of the total of the extracellular matrix component and the gelatin.

2. The composition according to claim 1, wherein the gelatin content is 5 parts by mass or more and 40 parts by mass or less with respect to 100 parts by mass of the total of the extracellular matrix component and the gelatin.

3. The composition according to claim 1 or 2, wherein the gelatin is derived from a non-human mammal.

4. The composition according to claim 1 or 2, wherein the gelatin is derived from bovine.

5. The composition according to claim 1 or 2, wherein the fragmented extracellular matrix component comprises a fragmented collagen component.

6. It contains extracellular matrix components and gelatin. The extracellular matrix component includes fragmented extracellular matrix components, A three-dimensional tissue-forming agent wherein the gelatin content is greater than 0 parts by mass and less than or equal to 50 parts by mass relative to 100 parts by mass of the total of the extracellular matrix component and the gelatin.

7. The three-dimensional tissue-forming agent according to claim 6, wherein the gelatin content is 5 parts by mass or more and 40 parts by mass or less with respect to 100 parts by mass of the total of the extracellular matrix component and the gelatin.

8. The three-dimensional tissue-forming agent according to claim 6 or 7, wherein the gelatin is derived from a non-human mammal.

9. The three-dimensional tissue-forming agent according to claim 6 or 7, wherein the gelatin is derived from bovine.

10. The three-dimensional tissue-forming agent according to claim 6 or 7, wherein the fragmented extracellular matrix component includes a fragmented collagen component.

11. The process includes culturing the cells in a culture medium containing cells, extracellular matrix components, and gelatin. The extracellular matrix component includes fragmented extracellular matrix components, A method for producing a three-dimensional tissue, wherein the amount of gelatin used is greater than 0 parts by mass and less than or equal to 50 parts by mass relative to 100 parts by mass of the total of the extracellular matrix components and the gelatin.

12. The method according to claim 11, wherein the amount of gelatin used is 5 parts by mass or more and 40 parts by mass or less, based on 100 parts by mass of the total of the extracellular matrix components and the gelatin.

13. The method according to claim 11 or 12, wherein the gelatin is derived from a non-human mammal.

14. The method according to claim 11 or 12, wherein the gelatin is derived from bovine.

15. The method according to claim 11 or 12, wherein the fragmented extracellular matrix component includes a fragmented collagen component.

Images