Colored fragmented extracellular matrix components, methods for producing the same, and structures

Abstract

To provide a colored, fragmented extracellular matrix component capable of forming a structure with high visibility and suppressed color transfer. [Solution] A colored fragmented extracellular matrix component comprising a fragmented extracellular matrix component and a coloring agent adsorbed to the fragmented extracellular matrix component, wherein the coloring agent comprises at least one selected from the group consisting of water-insoluble and ethanol-soluble dyes and lipid-soluble dyes.

Description

Technical Field

[0001] The present invention relates to a colored fragmented extracellular matrix component, a method for producing the same, and a structure.

Background Art

[0002] Techniques for producing a structure that mimics a living tissue in vitro are known. As a method for obtaining such a structure, a method using a fragmented extracellular matrix component obtained by fragmenting an extracellular matrix component such as collagen is known (Patent Document 1).

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0004] Conventional fragmented extracellular matrix components are white, and it has been difficult to visually confirm the boundary between structures containing the fragmented extracellular matrix components. When the fragmented extracellular matrix component is colored with a coloring agent and used for producing a structure, migration into an aqueous solution or migration into another structure in contact with the structure may occur.

[0005] An object of the present invention is to provide a colored fragmented extracellular matrix component and a method for producing the same that can form a structure with high visibility and suppressed migration. Another object of the present invention is to provide a structure containing the colored fragmented extracellular matrix component.

Means for Solving the Problems

[0006] This disclosure includes, for example, the following inventions. [1] A colored fragmented extracellular matrix component comprising a fragmented extracellular matrix component and a coloring agent adsorbed to the fragmented extracellular matrix component, wherein the coloring agent comprises at least one selected from the group consisting of water-insoluble and ethanol-soluble dyes and lipid-soluble dyes. [2] The colored fragmented extracellular matrix component according to [1], wherein the coloring agent is a curcuminoid. [3] The colored fragmented extracellular matrix component according to [1], wherein the coloring agent is capsicum pigment. [4] The colored fragmented extracellular matrix component according to any one of [1] to [3], wherein the fragmented extracellular matrix component contains a fragmented collagen component. [5] The colored fragmented extracellular matrix component according to any one of [1] to [4], wherein the fragmented extracellular matrix component is a defibration extracellular matrix component. A structure comprising a colored fragmented extracellular matrix component as described in any of [6], [1], to [5]. [7] The structure described in [6], which is cultured meat. [8] A method for producing a colored fragmented extracellular matrix component, comprising: a fragmentation step of obtaining a fragmented extracellular matrix component by fragmenting an extracellular matrix component in a first solution containing a first polar organic solvent; and a coloring step of obtaining a colored fragmented extracellular matrix component by homogenizing the fragmented extracellular matrix component in a second solution containing a second polar organic solvent and a coloring agent to adsorb the coloring agent onto the fragmented extracellular matrix component, wherein the coloring agent comprises at least one selected from the group consisting of water-insoluble and ethanol-soluble dyes and lipid-soluble dyes. [9] The manufacturing method according to [8], wherein the coloring agent is a curcuminoid.

[10] The method of production according to [8], wherein the coloring agent is chili pepper pigment.

[11] The manufacturing method according to any one of [8] to

[10] , wherein the extracellular matrix component is a collagen component.

[12] The manufacturing method according to any one of [8] to

[11] , wherein the fragmentation includes defibrillating the extracellular matrix components.

[13] The manufacturing method according to any one of [8] to

[12] , wherein the first polar organic solvent and the second polar organic solvent contain ethanol.

[14] A manufacturing method according to any one of [8] to

[13] , further comprising, after the coloring step, a substitution step of replacing the liquid component of a liquid containing the colored fragmented extracellular matrix component and the second polar organic solvent with water, and after the substitution step, a drying step of removing the liquid component from the liquid containing the colored fragmented extracellular matrix component and water. [Effects of the Invention]

[0007] According to the present invention, it is possible to provide a colored fragmented extracellular matrix component capable of forming a structure with high visibility and suppressed color transfer, and a method for producing the same. According to the present invention, it is possible to provide a structure containing the above-mentioned colored fragmented extracellular matrix component. [Brief explanation of the drawing]

[0008] [Figure 1] This graph shows the evaluation results of the amount of dye eluted from colored fragmented extracellular matrix components. [Figure 2] This photograph shows the results of the evaluation of the transfer of colored fragmented extracellular matrix components. [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] [Colored fragmented extracellular matrix components] The colored fragmented extracellular matrix component according to this embodiment comprises a fragmented extracellular matrix component and a coloring agent adsorbed to the fragmented extracellular matrix component. In the colored fragmented extracellular matrix component, the coloring agent comprises at least one selected from the group consisting of water-insoluble and ethanol-soluble dyes and lipid-soluble dyes.

[0011] "Fragmented extracellular matrix components" can be obtained by fragmenting extracellular matrix components. 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 and 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 as extracellular matrix components, or in combination of two or more types.

[0012] 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. 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). Extracellular matrix molecules containing both a Gly-XY sequence and an RGD sequence include collagen, fibronectin, vitronectin, laminin, and cadherin.

[0013] 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. Collagen is preferably fibrous collagen.

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

[0015] The extracellular matrix component may contain at least one selected from the group consisting of collagen, laminin, and fibronectin, and preferably contains collagen from the viewpoint of excellent cell adhesion. Collagen is preferably fibrous collagen, more preferably type I collagen. Fibrous collagen may be commercially available collagen, and specific examples thereof include type I collagen derived from porcine skin manufactured by Nippon Ham Foods Co., Ltd.

[0016] The extracellular matrix component may be an extracellular matrix component derived from an animal. Examples of the animal species from which the extracellular matrix component is derived include, but are not limited to, humans, pigs, cows, etc. The extracellular matrix component may be a component derived from one type of animal, or components derived from multiple types of animals may be used in combination.

[0017] In this specification, "fragmentation" means making the aggregate of extracellular matrix molecules into a smaller size. Fragmentation may be performed under conditions that cut the bonds within the extracellular matrix molecule, or may be performed under conditions that do not cut the bonds within the extracellular matrix molecule. Unlike enzymatic treatment, the extracellular matrix fragmented by the application of physical force usually does not change in molecular structure before and after fragmentation (the molecular structure is maintained). The fragmented extracellular matrix component may include the defibrated extracellular matrix component (defibrated extracellular matrix component), which is a component obtained by defibrating the above-mentioned extracellular matrix component by applying physical force. Defibration is one aspect of fragmentation and is, for example, performed under conditions that do not cut the bonds within the extracellular matrix molecule.

[0018] There are no particular restrictions on the method of fragmenting extracellular matrix components; fragmentation may be performed by applying physical force. For example, extracellular matrix components may be fragmented by applying physical force using a homogenizer such as an ultrasonic homogenizer, agitator homogenizer, or high-pressure homogenizer. By adjusting the homogenization time, number of repetitions, etc., it is possible to obtain fragmented extracellular matrix components of millimeter size or nanometer size.

[0019] 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 under which equivalent physical force can be applied.

[0020] The fragmented extracellular matrix component may contain at least a portion of the defibrated extracellular matrix component. 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. The defibrated collagen component preferably maintains the triple helix structure derived from collagen. The defibrated collagen component may be a component that completely or partially maintains the triple helix structure derived from collagen.

[0021] Examples of the shape of fragmented extracellular matrix components include fibrous structures. Fibrous refers to a shape composed of thread-like fragmented extracellular matrix components, or a shape composed of thread-like fragmented 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 thread-like structures (fibrillaries) formed by the aggregation of multiple thread-like extracellular matrix molecules, thread-like structures formed by further aggregation of fibrillaries, and defibrillated versions of these thread-like structures. In fibrous extracellular matrix components, the RGD sequence is preserved without disruption.

[0022] 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, 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. The average length of most of the fragmented extracellular matrix components may fall within the above numerical range. Specifically, the average length of 95% of the fragmented extracellular matrix components may fall within the above numerical range. The fragmented extracellular matrix component may be a fragmented collagen component having an average length within the above range, or a defibrillated collagen component having an average length within the above range.

[0023] 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.

[0024] 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.

[0025] The fragmented extracellular matrix component may include, for example, a fragmented collagen component, or may consist of a fragmented collagen component. "Fragmented collagen component" means a collagen component, such as a fibrous collagen component, that has been fragmented and maintains 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.

[0026] The coloring agent comprises at least one selected from the group consisting of water-insoluble and ethanol-soluble dyes and lipid-soluble dyes. The coloring agent is adsorbed to the fragmented extracellular matrix components. The adsorption of the coloring agent may be chemiadsorption or physiadsorption.

[0027] In this specification, "water-insoluble" means that the amount soluble in 100g of water is 0.1g or less at 25°C. The amount soluble in 100g of water for a water-insoluble and ethanol-soluble dye may be 0.05g or less, or 0.01g or less, at 25°C.

[0028] In this specification, "ethanol-soluble" means that the amount that can be dissolved in 100g of ethanol is 0.1g or more at 25°C. The amount that can be dissolved in 100g of ethanol for a water-insoluble and ethanol-soluble dye may be 0.5g or more at 25°C.

[0029] Examples of water-insoluble and ethanol-soluble pigments include curcuminoids (turmeric pigment), marigold pigment, annatto (bixin, norbixin), and paprika pigment.

[0030] Curcuminoids include curcumin and its analogs (demethoxycurcumin and bisdemethoxycurcumin). The water-insoluble and ethanol-soluble dye may be turmeric pigment (also called curcumin dye), which has curcumin as its main component.

[0031] Examples of lipid-soluble pigments include chili pepper pigment, β-carotene (carotene pigment), lycopene (tomato pigment), canthaxanthin (canthaxanthin pigment), paprika pigment, astaxanthin (astaxanthin pigment), and lutein (marigold pigment).

[0032] Capsicum pigment is a pigment whose main component is capsanthin. It is also called capsicum pigment or paprika pigment. A commercially available example of capsicum pigment is High Orange LH (manufactured by Daiwa Chemical Co., Ltd.).

[0033] The coloring agent may be a carotenoid that is water-insoluble and ethanol-soluble, and / or a lipid-soluble dye.

[0034] The coloring agent preferably contains at least one selected from the group consisting of curcuminoids and capsicum pigments.

[0035] The amount of coloring agent can be set appropriately depending on the type of coloring agent, etc.

[0036] The colored fragmented extracellular matrix component according to this embodiment can be suitably used as a component of a structure, particularly a three-dimensional tissue.

[0037] When the colored fragmented extracellular matrix component according to this embodiment is used to form a three-dimensional tissue, the colored fragmented extracellular matrix component functions as a scaffolding material and also functions as a component that improves the visibility of the three-dimensional tissue.

[0038] In this embodiment, a structure colored with a colored fragmented extracellular matrix component has a visible boundary with other materials in contact with it (a structure that does not contain the colored fragmented extracellular matrix component, or a structure formed by a colored fragmented extracellular matrix component containing a different coloring agent). Therefore, by using a colored fragmented extracellular matrix component, it becomes easier to distinguish between regions when they are divided by cell type or the like.

[0039] The colored fragmented extracellular matrix component according to this embodiment makes it possible to form structures in aqueous solutions or with suppressed transfer of color to other materials. Other materials include structures that do not contain the colored fragmented extracellular matrix component, or structures formed by a colored fragmented extracellular matrix component containing a different coloring agent.

[0040] The colored fragmented extracellular matrix component according to this embodiment, because it contains fragmented extracellular matrix components, possesses not only high cell affinity but also the physical property of being able to be mixed with culture medium and extruded by a nozzle-type 3D printer. Therefore, the colored fragmented extracellular matrix component according to this embodiment can be suitably used as a bio-ink material for use in nozzle-type 3D printers.

[0041] [Method for producing colored fragmented extracellular matrix components] Colored fragmented extracellular matrix components can be produced by a method that includes adsorbing a coloring agent onto the fragmented extracellular matrix components.

[0042] A method for producing a colored fragmented extracellular matrix component includes, for example, a fragmentation step of obtaining a fragmented extracellular matrix component by fragmenting an extracellular matrix component in a first solution containing a first polar organic solvent, and a coloring step of obtaining a colored fragmented extracellular matrix component by homogenizing the fragmented extracellular matrix component in a second solution containing a second polar organic solvent and a coloring agent to adsorb the above-mentioned coloring agent onto the fragmented extracellular matrix component.

[0043] A method for producing colored fragmented extracellular matrix components may further include, as a step prior to the fragmentation step, a neutralization step in which the solution in which the extracellular matrix components are dissolved is neutralized, a gelation step in which the neutralized solution is gelled, and a solvent removal step in which the solvent in the solution is removed by freeze-drying to obtain a mass containing the extracellular matrix components.

[0044] A method for producing a colored fragmented extracellular matrix component may further include, as a step after the coloring step, a substitution step in which the liquid component of a liquid containing the colored fragmented extracellular matrix component and a second polar organic solvent is replaced with water, and a drying step after the substitution step in which the liquid component is removed from the liquid containing the colored fragmented extracellular matrix component and water.

[0045] The following describes a method for producing a colored, fragmented extracellular matrix component, in which the neutralization step, gelation step, freeze-drying step, fragmentation step, coloring step, substitution step, and drying step are performed in this order.

[0046] (neutralization process) In the neutralization step, the solution containing the dissolved extracellular matrix components is neutralized. The solvent for the solution containing the dissolved extracellular matrix components is not particularly limited as long as it can dissolve the extracellular matrix components. Specific examples of solvents include water and buffer solutions (e.g., phosphate-buffered saline (PBS), Tris-HCl buffer).

[0047] The concentration of extracellular matrix components in the solution in which the extracellular matrix components are dissolved is not particularly limited, but for example, it may be between 1 mg / mL and 50 mg / mL.

[0048] Since the solution containing the extracellular matrix components 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. 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 suitably used. More specifically, examples of alkaline solutions include aqueous solutions of potassium hydroxide (KOH) and sodium hydroxide (NaOH).

[0049] If the solution containing the dissolved extracellular matrix components is alkaline, the neutralization step can be carried out, for example, by adding an acidic solution to the solution containing the dissolved extracellular matrix components. There are no particular restrictions on the acidic solution, but examples include hydrochloric acid, sulfuric acid, acetic acid, and carbonic acid.

[0050] Neutralization should be carried out in a manner that causes structural changes in the extracellular matrix, and it is not necessary to ensure that the pH of the solution after neutralization is 7. Specifically, the pH of the solution after neutralization may be in the range of 6 to 8, preferably in the range of 6.5 to 7.5, more preferably in the range of 6.9 to 7.1, and even more preferably in the range of 6.95 to 7.05.

[0051] (Gelation process) In the gelation step, the neutralized solution is gelled after the neutralization step and before the solvent removal step. The gelation step may be performed as needed.

[0052] The gelation process can be carried out, for example, by heating the neutralized solution to induce gelation. The temperature and heating time can be appropriately set according to the type and concentration of the extracellular matrix components, but examples include a temperature of 25-45°C and a heating time of 1-3 hours.

[0053] By performing a gelation step, it is possible to visually determine whether the formed gel is uniform, thereby confirming whether the solution was uniformly neutralized in the neutralization step. Furthermore, by performing a gelation step, the aggregate containing extracellular matrix components obtained in the solvent removal step becomes a porous material, allowing for more efficient fragmentation in the fragmentation step.

[0054] (Solvent removal process) The solvent removal step involves removing the solvent by freeze-drying after the neutralization step (or after the gelation step, if one is included) to obtain a mass containing extracellular matrix components.

[0055] The freeze-drying treatment of the neutralized solution or the gel after gelation can be carried out according to conventional methods. The solvent removal step removes the solvent, and a mass (solid) containing extracellular matrix components is obtained. Note that the removal of the solvent does not mean that no solvent is attached at all to the mass (solid) containing extracellular matrix components, but rather that the amount of solvent attached is reduced to a level that can be reasonably achieved through general freeze-drying.

[0056] (Fragmentation process) In the fragmentation step, extracellular matrix components are fragmented in a first solution containing a first polar organic solvent to obtain fragmented extracellular matrix components. The method and conditions for fragmenting the extracellular matrix components may be those described above.

[0057] The extracellular matrix component used in the fragmentation step may be the aggregate obtained in the solvent removal step. The fragmentation step may consist of dispersing the aggregate obtained in the solvent removal step in a first solution containing a first polar organic solvent, fragmenting the extracellular matrix component in the solution, and obtaining a liquid containing the fragmented extracellular matrix component. The liquid containing the fragmented extracellular matrix component includes the fragmented extracellular matrix component as well as a solution containing the first polar organic solvent, etc.

[0058] The first solution may consist only of the first polar organic solvent, or it may contain the first polar solvent and water. In this specification, "water" refers to tap water, distilled water, ultrapure water, etc.

[0059] The first 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.

[0060] The concentration of the first polar organic solvent in the first solution 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, 75 v / v% or more, 80 v / v% or more, or 85 v / v% or more. The concentration of the first polar organic solvent in the first solution may be, for example, 100 v / v% or less, 99 v / v% or less, 95 v / v% or less, 90 v / v% or less, 85 v / v% or less, 80 v / v% or less, or 75 v / v% or less.

[0061] During the fragmentation process, the concentration of the first polar organic solvent in the first solution may be changed as needed. For example, the fragmentation process may include performing a first fragmentation treatment under the condition that the concentration of the first polar organic solvent in the first solution is 80 v / v% or higher, and then performing a second fragmentation treatment under the condition that the concentration of the first polar organic solvent in the first solution is less than 80 v / v%. Between the first and second fragmentation treatments, the liquid obtained after the first fragmentation treatment may be centrifuged to settle the fragmented extracellular matrix components in the liquid, and then the liquid components may be removed.

[0062] The fragmentation step may consist of a step (defibration step) in which the aggregate obtained in the solvent removal step is dispersed in a solution containing a polar organic solvent, the extracellular matrix components are defibrated in the solution, and a liquid containing the defibrated extracellular matrix components is obtained.

[0063] (Coloring process) In the coloring step, the fragmented extracellular matrix component is homogenized in a second solution containing a second polar organic solvent and a coloring agent to adsorb the coloring agent onto the fragmented extracellular matrix component, thereby obtaining a colored fragmented extracellular matrix component. The coloring step may be performed after removing at least a portion of the liquid component from the liquid containing the fragmented extracellular matrix component.

[0064] The second solution may consist only of the second polar organic solvent, or it may contain the second polar organic solvent and water.

[0065] The second 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.

[0066] The second polar organic solvent may be the same type of polar organic solvent as the first polar organic solvent, or it may be a different type of polar organic solvent. Preferably, the second polar organic solvent is the same type of polar organic solvent as the first polar organic solvent. Preferably, both the first and second polar organic solvents are ethanol.

[0067] The concentration of the second polar organic solvent in the second solution may be, for example, 70 v / v% or higher, 75 v / v% or higher, 80 v / v% or higher, or 85 v / v% or higher. The concentration of the first polar organic solvent in the first solution may be, for example, 100 v / v% or lower, 99 v / v% or lower, 95 v / v% or lower, 90 v / v% or lower, or 85 v / v% or lower.

[0068] The concentration of the coloring agent in the second solution can be appropriately set depending on the type of coloring agent, etc. If the coloring agent is a curcuminoid, the concentration of the coloring agent in the second solution may be, for example, 10-50 mg / 25 mL or 20-40 mg / 25 mL. If the coloring agent is capsicum pigment, the concentration of the coloring agent in the second solution may be, for example, 10-50 μL / 25 mL or 20-40 μg / 25 mL.

[0069] The amount of the second solution used may be 10 mL or 30 mL per 5 mg of fragmented extracellular matrix component.

[0070] Methods for homogenization in the coloring process include, for example, using homogenizers such as ultrasonic homogenizers, agitated homogenizers, and high-pressure homogenizers.

[0071] A liquid containing colored fragmented extracellular matrix components can be obtained through a coloring process. This liquid contains, in addition to the colored fragmented extracellular matrix components, a solution containing a second polar organic solvent, etc.

[0072] (Replacement process) The substitution step is a process that takes place after the coloring step and before the drying step, in which the liquid components in the liquid obtained in the coloring step are replaced with water.

[0073] There are no particular restrictions on the method for replacing the liquid components in the liquid obtained in the coloring step with water; the usual methods used in solvent replacement can be used. For example, one method of replacement with water is to centrifuge the liquid obtained in the coloring step to settle the fragmented extracellular matrix components in the liquid, remove the liquid components, and then add water to the fragmented extracellular matrix components. It should be noted that replacing liquid components with water does not mean that all liquid components present in the liquid are replaced with water, but rather that the main solvent in the liquid is replaced with water, and trace amounts of liquid components other than water may remain after replacement.

[0074] The liquid obtained through the substitution process contains water in addition to colored, fragmented extracellular matrix components.

[0075] (drying process) The drying process involves removing the liquid component from the liquid containing the colored fragmented extracellular matrix component obtained in the coloring process.

[0076] The removal of liquid components can be carried out, for example, by air drying, freeze-drying, vacuum drying, or vacuum freeze-drying. The removal of liquid components may be carried out by air drying or freeze-drying.

[0077] The drying process removes the liquid components, yielding a solid containing dried colored fragmented extracellular matrix components. It should be noted that the removal of liquid components does not mean that no liquid components are present at all on the solid containing the dried fragmented extracellular matrix components; rather, it means that the amount of liquid components present is limited to what can be reasonably achieved through the general drying methods described above.

[0078] (Washing process) The washing step involves dispersing the solid obtained in the drying step into an aqueous medium. Since the colored fragmented extracellular matrix component in this embodiment exhibits suppressed transfer of color into aqueous solutions, the washing step can be performed. The washing step removes unwanted components (e.g., salts) from the solid containing the colored fragmented extracellular matrix component, thereby obtaining a colored fragmented extracellular matrix component of higher purity.

[0079] The aqueous medium used in the washing process is preferably one that can disperse fragmented extracellular matrix components and dissolve the aforementioned unwanted components. Specific examples of aqueous mediums include, for example, water, phosphate-buffered saline (PBS), and Tris buffer (Tris).

[0080] [Composition] The composition according to this embodiment contains the above-described colored fragmented extracellular matrix component. The composition can be suitably used, for example, as a bio-ink material for 3D bioprinting. The composition according to this embodiment may further contain components that are typically found in bio-ink materials.

[0081] According to the composition containing the colored fragmented extracellular matrix component of this embodiment, structures described later can be formed by 3D bioprinting.

[0082] [Structure] The structure according to this embodiment includes the above-described colored fragmented extracellular matrix component.

[0083] The structure may further contain cells. At least a portion of the cells may be in contact with the colored, fragmented extracellular matrix components. One form of contact is adhesion.

[0084] The cells 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; they 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 lines. 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.

[0085] The structure may be a three-dimensional tissue. A three-dimensional tissue is an aggregate of cells in which cells are arranged three-dimensionally via an extracellular matrix component containing colored fragmented extracellular matrix components, and is an aggregate artificially created by cell culture.

[0086] A three-dimensional tissue can be produced, for example, by a method comprising a culture step of culturing cells in a culture medium containing a colored fragmented extracellular matrix component and cells. The culture medium can be obtained by mixing the colored fragmented extracellular matrix component and cells in an aqueous medium.

[0087] An 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) 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, it is preferable that the aqueous medium be a liquid medium. There are no particular restrictions on the liquid 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.

[0088] The amount of colored fragmented extracellular matrix component used to prepare a three-dimensional tissue can be appropriately determined according to the shape, thickness, and size of the culture vessel of the desired three-dimensional tissue. For example, the concentration of colored fragmented extracellular matrix component in the culture medium may be 0.1% by mass or more, 30% by mass or less, or 10% by mass or less, based on the total amount of aqueous medium. The amount of colored fragmented extracellular matrix component used is 1 × 10⁻⁶ 6 The amount relative to the cells may be 0.01 μg or more, and may be 1000 μg or less, 100 μg or less, or 5 μg or less.

[0089] The culture process is preferably carried out under conditions that maintain the viability of at least a portion of the cells. The conditions for maintaining viability can be appropriately set depending on the type of cell. Specifically, the culture conditions exemplified below are examples.

[0090] 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.

[0091] The cell density in the culture medium can be appropriately determined according to the shape, thickness, and size of the culture vessel of the target three-dimensional tissue. For example, the cell density in the culture medium can be 1 to 10 8 It may be cells / mL, 10 3 ~10 7 It may be cells / mL.

[0092] There are no particular restrictions on the shape of the structure and the three-dimensional organization; for example, they can be sheet-like, spherical, ellipsoidal, rectangular, etc.

[0093] The thickness of the structure may be 10 μm or more, 100 μm or more, or 1000 μm or more, and may be 10 mm or less, 3 mm or less, 2 mm or less, 1.5 mm or less, or 1 mm or less. If the structure is in the form of a sheet or a rectangular parallelepiped, the thickness refers to the distance between the two ends in a direction perpendicular to the main surface. If the main surface has irregularities, the thickness refers to the distance at the thinnest part of the main surface. If the structure is spherical, it refers to its diameter. Furthermore, if the structure is ellipsoidal, it refers to its minor axis. If the structure is roughly spherical or ellipsoidal and has irregularities on its surface, the thickness refers to the shortest distance between two points where a line passing through the centroid of the structure intersects the surface.

[0094] In the structure according to this embodiment, the boundary between the structure and the material in contact with it can be visually confirmed. In the structure according to this embodiment, color transfer into aqueous solution or to other materials in contact with the structure (for example, a structure that does not contain colored fragmented extracellular matrix components, and a cell structure formed by colored fragmented extracellular matrix components containing different colorants) is suppressed.

[0095] The structure can be suitably used for applications such as cultured meat, substitutes for laboratory animals, and transplant materials. The cultured meat structure (three-dimensional tissue) may further contain components that are normally present in cultured meat, in addition to fragmented extracellular matrix components. [Examples]

[0096] The present invention will be described more specifically below based on examples. However, the present invention is not limited to the following examples.

[0097] 1. Experiment 1-1. Pigments used [Table 1]

[0098] Each dye was dissolved in ethanol (EtOH) or an aqueous ethanol solution in the following amounts. Food coloring red 30mg / 25mL (85v / v% EtOH aqueous solution) Food color yellow 30mg / 25mL (85v / v% EtOH aqueous solution) Curcumin 30mg / 25mL (100% EtOH) Capsicum pigment (95% pigment weight) 31.5 μL / 25 mL (100% EtOH)

[0099] 1-2. Preparation of colored defibrillated collagen component (colored CMF) The defibrated collagen component (CMF), which had been freeze-dried after gelling, was homogenized with 85% ethanol. After centrifugation at 10,000 rpm for 3 minutes, the supernatant was removed and homogenized with 70% EtOH. After centrifugation at 10,000 rpm for 3 minutes, the supernatant was removed and 10 mL of dye dissolved in ethanol at the above concentration was added to each tube and homogenized. After centrifugation at 10,000 rpm for 3 minutes, the supernatant was removed and homogenized with 10 mL of ultrapure water. The samples were then frozen at -20°C and freeze-dried.

[0100] 1-3. Evaluation of colored CMF transfer into water 5 mg of lyophilized colored CMF was weighed into 1.5 mL tubes, and 300 μL of ultrapure water was added to completely disperse the mixture by sonication. The mixture was centrifuged at 10,000 rpm for 1 minute, and the supernatant was transferred to a 96-well plate. The absorbance of each colorant (yellow food coloring: 430 nm, curcumin: 430 nm, red food coloring: 520 nm, capsicum pigment: 450 nm) was measured. From the obtained absorbance values, the amount of eluted colorant was calculated using a calibration curve created by diluting each colorant. Statistical comparisons were performed using one-way ANOVA, followed by Tukey-Kramer multiple comparison tests.

[0101] 1-4. Evaluation of stain transfer of structures fabricated using colored CMF 5 mg each of colored CMF and uncolored CMF was weighed out and dispersed in 500 μL of ultrapure water. The mixture was dropped onto a 24-well culture insert and centrifuged at 1,100 × g for 5 minutes to form structures containing CMF. The supernatant was removed and incubated at 37°C overnight. 1 mL of 4% paraformaldehyde was added to each well to fix the structures. After washing twice with PBS, the structures were removed with a scalpel. Each colored CMF was layered onto the uncolored CMF using tweezers and kept at 4°C for 3 days. After removing the colored CMF, each uncolored CMF was visually observed.

[0102] 2.Results 2-1. Evaluation of color transfer from colored CMF to water We attempted to stain CMF by dissolving four pigments—yellow food coloring, red food coloring, curcumin (a water-insoluble and ethanol-soluble pigment), and capsicum pigment (a lipid-soluble pigment)—in ethanol and adding them to the fragmentation step in the CMF manufacturing method. After freeze-drying, a portion of the dried colored CMF was separated, and the fluorescence intensity of the supernatant suspended in ultrapure water was measured. The amount of pigment eluted was calculated from a calibration curve. The results are shown in Figure 1. It was found that 10 to 30 times more pigment was eluted when using yellow food coloring and red food coloring compared to when using curcumin and capsicum pigment. With curcumin and capsicum pigment, the elution from the colored CMF was minimal because these pigments are almost insoluble in water. With yellow food coloring and red food coloring, since these pigments are soluble in both ethanol and water, it was found that a large amount of the pigment adsorbed on the CMF eluted after washing with water.

[0103] 2-2. Evaluation of color transfer from colored CMF to uncolored CMF In cultured meat, when muscle fibers stained with cochineal dye (water-soluble food coloring) and unstained fat fibers are stored in layers, color transfer can occur between fibers even after washing. To evaluate color transfer between structures containing colored CMF and structures containing uncolored CMF, structures containing colored CMF and structures containing uncolored CMF were fixed on a culture insert, contacted for 3 days, and then returned to their original state, and the degree of color transfer was visually confirmed. The results are shown in Figure 2.

[0104] When yellow food coloring was used, the pigment almost completely transferred to the water during the structure formation process, and virtually no coloring of the CMF was observed. Uncolored CMF that came into contact with red food coloring showed red color transfer. Colored CMF using curcumin and capsicum pigment showed almost no color transfer to uncolored CMF, suggesting that it can maintain a vivid color.

[0105] 3. Summary Taking advantage of the characteristics of the fragmented extracellular matrix component preparation process, which involves "fragmenting extracellular matrix components in a solution containing a polar organic solvent," we prepared CMF colored with water-insoluble and ethanol-soluble dyes, as well as CMF colored with lipid-soluble dyes. Unlike CMF colored with water-soluble dyes, these colored CMFs showed less dye elution into the washing water, suggesting that inter-tissue color transfer is less likely to occur even after tissue formation. Since curcumin and capsicum pigment are approved as food additives, they have high cell affinity and are expected to be applicable to cultured meat.

Claims

1. The system comprises a fragmented extracellular matrix component and a colorant adsorbed onto the fragmented extracellular matrix component. A colored fragmented extracellular matrix component comprising at least one colorant selected from the group consisting of water-insoluble and ethanol-soluble dyes and lipid-soluble dyes.

2. The colored fragmented extracellular matrix component according to claim 1, wherein the coloring agent is a curcuminoid.

3. The colored fragmented extracellular matrix component according to claim 1, wherein the coloring agent is capsicum extract.

4. The colored fragmented extracellular matrix component according to any one of claims 1 to 3, wherein the fragmented extracellular matrix component contains a fragmented collagen component.

5. The colored fragmented extracellular matrix component according to any one of claims 1 to 3, wherein the fragmented extracellular matrix component is a defibrated extracellular matrix component.

6. A structure comprising a colored fragmented extracellular matrix component according to any one of claims 1 to 3.

7. The structure according to claim 6, wherein it is cultured meat.

8. A method for producing colored fragmented extracellular matrix components, A fragmentation step to obtain fragmented extracellular matrix components by fragmenting extracellular matrix components in a first solution containing a first polar organic solvent, The system includes a coloring step of homogenizing the fragmented extracellular matrix component in a second solution containing a second polar organic solvent and a coloring agent to adsorb the coloring agent onto the fragmented extracellular matrix component, thereby obtaining a colored fragmented extracellular matrix component. A method for producing a colored fragmented extracellular matrix component, wherein the coloring agent comprises at least one selected from the group consisting of water-insoluble and ethanol-soluble dyes and lipid-soluble dyes.

9. The manufacturing method according to claim 8, wherein the coloring agent is a curcuminoid.

10. The manufacturing method according to claim 8, wherein the coloring agent is chili pepper pigment.

11. The manufacturing method according to any one of claims 8 to 10, wherein the extracellular matrix component is a collagen component.

12. The manufacturing method according to any one of claims 8 to 10, wherein the fragmentation includes defibrillating the extracellular matrix components.

13. The method for producing a product according to any one of claims 8 to 10, wherein the first polar organic solvent and the second polar organic solvent include ethanol.

14. After the coloring step, a substitution step is performed in which the liquid component of the liquid containing the colored fragmented extracellular matrix component and the second polar organic solvent is replaced with water. The manufacturing method according to any one of claims 8 to 10, further comprising a drying step of removing the liquid component from the liquid containing the colored fragmented extracellular matrix component and water after the substitution step.

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