Method for producing collagen-containing structure

Abstract

The present invention relates to a method for producing collagen-containing anisotropic fibers, the method comprising: a step for bringing an anionic polymer into contact with an aqueous collagen solution in which collagen has been dissolved to form an interface, so that the collagen is formed into fibrils; and a step for pulling up the fibrillated collagen from the aqueous collagen solution with which the anionic polymer has been brought into contact so as to form anisotropic fibers. The present invention also relates to a method for producing a collagen-containing structure having in the interior thereof a hollow structure filled with an aqueous medium, the method comprising: a step for forming, in an aqueous solution containing an anionic polymer, droplets of an aqueous solution in which at least collagen is dissolved; and after the step for forming the droplets, a step for allowing the droplets to stand for a certain period of time to aggregate collagen molecules at the interface between the droplets and the aqueous solution containing the anionic polymer.

Description

Method for producing collagen-containing structures

[0001] The present invention relates to a method for producing a collagen-containing structure.

[0002] Collagen has the ability to form fibers under specific conditions, but it is mostly used in the form of randomly oriented gels. Methods for forming anisotropic fibers using collagen include, for example, electrospinning using organic solvents (Non-Patent Document 1) and a method of extruding a gel into an organic solvent and drying it (Non-Patent Document 2).

[0003] Furthermore, in recent years, technologies have been developed to construct three-dimensional tissues of cells in vitro. For example, Patent Document 1 discloses a method for producing a three-dimensional tissue, which includes (1) a step of bringing fragmented collagen derived from exogenous collagen into contact with cells in an aqueous medium, and (2) a step of culturing the cells that have come into contact with the fragmented collagen.

[0004] International Publication No. 2018 / 143286

[0005] Y. Wakuda, S. Nishimoto, S.Suye and S. Fujita, Sci Rep, 2018, 8, 6248.S. Yunoki, M. Kishimoto, Y.Mandai, Y. Hiraoka and E. Kondo, Biomedical Materials, 2024, 19, 045010.

[0006] This embodiment aims to provide a novel method for producing a collagen-containing structure having a desired shape. Specifically, for example, the present invention aims to provide the following method.

[0007] This embodiment aims to provide a novel method for producing anisotropic fibers containing collagen.

[0008] Furthermore, because electrospinning requires organic solvents, it is not possible to mix cells simultaneously with spinning, and a challenge remains that the cells remain on the surface of the scaffold material.

[0009] Therefore, this embodiment also aims to provide a method for producing anisotropic fibers (fibrous tissue) containing cells, and anisotropic fibers (fibrous tissue) obtained by this method.

[0010] Furthermore, three-dimensional tissues manufactured by mixing cells and collagen are prone to necrosis because the interaction between cells and collagen makes the interior densely packed with cells, leading to oxygen deprivation. In addition, porous collagen scaffolds depend on the structure of the scaffolding material, which hinders the free movement of cells.

[0011] Therefore, this embodiment was made in view of the above circumstances and aims to provide a new method for manufacturing a collagen-containing structure having a hollow structure inside.

[0012] This disclosure encompasses the following embodiments: [1] A method for producing anisotropic fibers containing collagen, comprising the steps of: contacting an anionic polymer with an aqueous collagen solution in which collagen is dissolved to form an interface and fibrillate the collagen; and pulling up the fibrillated collagen from the aqueous collagen solution in which the anionic polymer has been contacted to form the anisotropic fibers. [2] The method according to [1], wherein the anionic polymer comprises at least one selected from the group consisting of poly(meth)acrylic acid, heparin, chondroitin sulfate, hyaluronic acid, dextran sulfate, poly(p-styrene sulfonic acid), carrageenan, alginic acid, and salts thereof. [3] The method according to [1] or [2], wherein the anionic polymer is at least one selected from the group consisting of poly(meth)acrylic acid, heparin, and salts thereof. [4] The method according to any one of [1] to [3], wherein the anionic polymer is at least one selected from the group consisting of poly(meth)acrylic acid and salts thereof. [5] The method according to any one of [1] to [4], wherein the collagen is atelocollagen. [6] The method according to any one of [1] to [5], wherein the step of fibrillating the collagen is carried out by contacting the collagen aqueous solution with a polymer aqueous solution containing the anionic polymer. [7] The method according to any one of [1] to [6], wherein the pH of the collagen aqueous solution is 3.0 or more and 6.0 or less, and the collagen content in the collagen aqueous solution is 0.31 to 10 μM based on the total amount of the collagen aqueous solution. [8] The method according to [6] or [7], wherein the pH of the polymer aqueous solution is 3.0 or more and less than 7.0, and the anionic polymer content in the polymer aqueous solution is 0.31 mM or more based on the total amount of the polymer aqueous solution. [9] The method according to any one of [1] to [8], wherein in the step of pulling up the fibrillated collagen from the collagen aqueous solution that has been contacted with the anionic polymer to form the anisotropic fibers, the pulling speed of the fibrillated collagen is 11 mm / second or less.

[10] The method according to any one of [1] to [9], wherein the collagen aqueous solution contains cells and is a cell-containing collagen aqueous solution in which collagen is dissolved.

[11] The method according to

[10] , wherein the anisotropic fibers encapsulate the cells.

[12] The method according to

[10] or

[11] , wherein the cells include at least one selected from the group consisting of stem cells, fibroblasts, vascular endothelial cells and chondrocytes.

[13] The method according to any one of

[10] to

[12] , wherein the cells are satellite cells.

[14] The method according to any one of

[10] to

[13] , wherein the cell-containing collagen aqueous solution includes at least one selected from the group consisting of lactic acid and citric acid.

[15] The method according to any one of

[10] to

[14] , wherein the cells include vascular endothelial cells and the collagen includes type III collagen.

[16] The method according to any one of

[10] to

[15] , wherein the cells include chondrocytes and the collagen includes type II collagen.

[17] The method according to any one of

[10] to

[16] , wherein the cells include satellite cells, the collagen includes type I collagen and type IV collagen, and the cell-containing collagen aqueous solution further includes laminin.

[18] The permeability of a 5 mM HEPES solution containing FITC-dextran, which is an anisotropic fiber containing collagen, has a fiber length of 10 mm or more, has a storage modulus (G') of 10 Pa or more and 1000 Pa or less at a strain of 0.1%, and has a molecular weight of 4000 at 0.5 mg / mL, is 1.0 × 10. －７Anisotropic fiber having a storage modulus (G') of cm / s or less.

[19] Anisotropic fiber according to

[18] , wherein the storage modulus (G') is 50 or more and 300 Pa or less.

[20] Anisotropic fiber (fibrous tissue) according to

[18] or

[19] containing cells.

[21] Anisotropic fiber (fibrous tissue) according to any one of

[18] to

[20] , wherein the proportion of collagen having an orientation angle with respect to the reference direction within ±5° is 50% or more.

[22] Anisotropic fiber (fibrous tissue) according to

[20] or

[21] , wherein the cells include stem cells.

[23] Anisotropic fiber (fibrous tissue) according to any one of

[20] to

[22] , wherein the cells include satellite cells.

[24] Anisotropic fiber (fibrous tissue) according to any one of

[20] to

[23] , wherein the cells include fibroblasts.

[25] A method for producing a collagen-containing structure having a hollow structure filled with an aqueous medium, comprising: a step of forming droplets of an aqueous solution containing at least collagen in an aqueous solution containing an anionic polymer; and a step of allowing the solution to stand for a certain period of time after the step of forming the droplets to allow the collagen molecules to assemble at the interface between the droplets and the aqueous solution containing the anionic polymer.

[26] The method according to

[25] , wherein the anionic polymer comprises at least one selected from the group consisting of poly(meth)acrylic acid and its salts.

[27] The method according to

[25] or

[26] , wherein the aqueous solution containing at least collagen is an aqueous solution in which at least collagen is dissolved in a solution containing at least one acid selected from the group consisting of lactic acid, citric acid, succinic acid, tartaric acid, gluconic acid, malic acid, fumaric acid, and phosphoric acid.

[28] The method according to any one of

[25] to

[27] , wherein the pH of the aqueous solution in which at least collagen is dissolved is 3.0 or more and 6.0 or less, the collagen content in the aqueous solution in which at least collagen is dissolved is 0.31 to 10 μM based on the total amount of the collagen aqueous solution, and the pH of the aqueous solution containing the anionic polymer is 3.0 or more and less than 7.0, and the anionic polymer content in the aqueous solution containing the anionic polymer is 0.31 mM or more based on the total amount of the aqueous solution containing the anionic polymer.

[29] The method according to any one of

[25] to

[28] , wherein the aqueous solution in which at least collagen is dissolved contains cells.

[30] A collagen-containing structure having a hollow structure inside which is filled with an aqueous medium.

[31] The structure according to

[30] , wherein cells are adhered to the outside of the structure, or cells are contained within the hollow structure of the structure.

[32] A composition comprising the structure according to

[30] or

[31] and an aqueous medium, wherein the structure is suspended in the aqueous medium.

[0013] The present invention provides a novel method for producing a collagen-containing structure having a desired shape. Specifically, the present invention provides a novel method for producing anisotropic fibers containing collagen. This embodiment also provides a method for producing a fibrous tissue containing cells and a fibrous tissue obtained by this method. This embodiment further provides a novel method for producing a collagen-containing structure having a hollow structure inside.

[0014] This is a photograph showing the appearance of fibers spun using polyacrylic acid and collagen. This is a microscopic image of the fibers from the example. This is a microscopic image of fibers formed using polymer and collagen under neutral conditions. This is a fluorescence microscopic image of fibers formed using fluorescent collagen or fluorescent PAA. This is a graph showing the results of confirming the effect of the pulling speed of fibrous collagen on the length of the formed fibers. This is a graph showing the results of confirming the effect of the collagen concentration in the collagen aqueous solution on the length of the formed fibers. This is a graph showing the results of confirming the dependence of interface formation (fiber formation) time on collagen concentration by measuring absorbance. This is a graph showing the absorbance (turbidity) at 180 minutes in Figure 7. This is a graph showing the results of viscoelasticity measurements performed on fibers spun using polyacrylic acid and collagen. This is a graph showing the storage modulus (A) and modulus crossover point (B) at a strain of 0.1% during viscoelasticity measurement. This is a graph showing the results of a permeability test. This is an image showing the results of microscopic observation of a fibrous tissue prepared by the method of Experiment 2-1-1 in Test Example 2-1. This graph shows the results of measuring the cell viability of fibrous tissue prepared using the method of Test Example 2-2. This graph shows the results of measuring the cell viability of fibrous tissue prepared using the method of Test Example 2-3. This is an observation image showing the appearance of fibrous tissue prepared using the method of Test Example 2-4, where (A) shows the results when heparin is used as the anionic polymer, and (B) shows the results when polymethacrylic acid is used as the anionic polymer (fibrous tissue within the circle). This is a phase-contrast observation image of fibrous tissue prepared using bovine satellite cells (bSC). This is a fluorescence observation image showing the results of staining Actin, COL1, and the nucleus (Nuclei) in fibrous tissue prepared using human fibroblasts (NHDF). This is a fluorescence observation image showing the results of Live Dead staining of fibrous tissue prepared using bSC, NHDF, or human umbilical vein endothelial cells (HUVEC). These are fluorescence images showing the results of staining Actin, COL1, or Nuclei in fibrous tissue prepared using bSC, NHDF, or HUVEC.This graph shows the evaluation results of fibrous tissue prepared using bSC, NHDF, or HUVEC, where (A) shows the evaluation results for fiber length, (B) shows cell viability, (C) shows the cell number, and (D) shows the proliferation rate. This is a fluorescence observation image showing the results of live dead staining of fibrous tissue prepared using bSC. This is a fluorescence observation image showing the results of staining for Actin, MYH4, or Nuclei in fibrous tissue prepared using bSC. This graph shows the evaluation results of fibrous tissue prepared using bSC, where (A) shows the evaluation results for fiber length, (B) shows cell viability on day 7 of culture, (C) shows the cell number, and (D) shows the proliferation rate. These are fluorescence observation images showing the results of Livedead Assay in fibrous tissue prepared using an aqueous collagen solution containing acetic acid (AA), lactic acid (LA), or citrate (CA). These are evaluation results of fibrous tissue prepared using an aqueous collagen solution containing acetic acid (AA), lactic acid (LA), or citrate (CA), where (A) shows the evaluation results of fiber length and (B) shows the quantitative results of the number of living cells by Livedead Assay. These are observation images of the interface between an aqueous collagen solution and a colored aqueous polymer solution, where (A) shows the observation results of the interface between a 10 μM aqueous collagen solution and a 0.00–10.00 mM aqueous polymer solution, and (B) shows the observation results of the interface between a 10 mM aqueous polymer solution and a 0.00–10.0 μM aqueous collagen solution. These are fluorescence images showing the results of staining Actin, CD31, or Nuclei in fibrous tissue prepared using HUVEC and NHDF, and using type I or type III collagen. These are observation images of fibrous tissue prepared using bSC and type I collagen, or fibrous tissue prepared using bSC, and type I and IV collagen and laminin. These are fluorescence images showing the results of staining Actin, MYH4, or Nuclei in fibrous tissue prepared using ATDC5 cells and type I or type II collagen. This is a graph showing the results of orientation measurements of cell fibers.This is a fluorescence observation image showing the results of preparing bovine adipose-derived stem cell fibers, specifically the staining of Lipid, Actin, and Nuclei in the bovine adipose-derived stem cell fibers. This is a schematic diagram showing a manufacturing method according to one embodiment. This image shows the results of immunostaining of a collagen-containing structure using anti-collagen 1 antibody (anti-COL-1 antibody), and shows an XZ planar view of the collagen-containing structure. This image shows the results of immunostaining of a collagen-containing structure using anti-collagen 1 antibody (anti-COL-1 antibody), and shows a ZY planar view of the collagen-containing structure, with (B) showing an XY planar view of the collagen-containing structure. This image shows the results of immunostaining of a collagen-containing structure using anti-collagen 1 antibody (anti-COL-1 antibody), and shows an XY planar view of the collagen-containing structure. This is a merged image showing the results of immunostaining using anti-collagen 1 antibody, DAPI (4',6-diamidino-2-phenylindole) staining, and immunostaining using anti-actin antibody on a collagen-containing structure containing cells. This image shows the results of DAPI staining on a structure containing collagen and cells. This image shows the results of immunostaining using an anti-actin antibody on a structure containing collagen and cells. This image shows the results of immunostaining using an anti-collagen 1 antibody on a structure containing collagen and cells. This image shows (A) a merged image of the results of immunostaining using an anti-COL-1 antibody, DAPI staining, and immunostaining using an anti-actin antibody on a structure containing collagen and cells, and (B) an image showing the result of DAPI staining, with an XZ planar view of the structure containing collagen and cells. This image shows (A) the results of immunostaining using an anti-actin antibody and (B) the results of immunostaining using an anti-COL-1 antibody on a structure containing collagen and cells, with an XZ planar view of the structure containing collagen and cells.The images show the ZY planar view of a structure containing collagen containing cells, with (A) a merged image of the results of immunostaining using anti-COL-1 antibody, DAPI staining, and immunostaining using anti-actin antibody, and (B) an image showing the result of DAPI staining. The images show the ZY planar view of a structure containing collagen containing cells, with (A) a merged image of the results of immunostaining using anti-actin antibody, and (B) an image showing the result of immunostaining using anti-COL-1 antibody, and the ZY planar view of a structure containing collagen containing cells. The images show the XY planar view of a structure containing collagen containing cells, with (A) a merged image of the results of immunostaining using anti-COL-1 antibody, DAPI staining, and immunostaining using anti-actin antibody, and (B) an image showing the result of DAPI staining. The images show the XY planar view of a structure containing collagen containing cells, with (A) a merged image of the results of immunostaining using anti-actin antibody, and (B) an image showing the result of immunostaining using anti-COL-1 antibody. This is a schematic diagram of the centrifuge tube used to produce the collagen-containing structure in Test Example 3-3. Figure 46 shows the results of producing the collagen-containing structure using the centrifuge tube shown. This graph shows the results of measuring the outer diameter of the produced collagen structure.

[0015] The following describes this embodiment in detail.

[0016] In this disclosure, numerical ranges indicated using "~" represent a range that includes the numbers before and after "~" as the minimum and maximum values, respectively. Unless otherwise explicitly stated, the units of the numbers before and after "~" are the same. In numerical ranges described in stages within this disclosure, the upper or lower limit of one stage of the range may be replaced by the upper or lower limit of another stage. Furthermore, in numerical ranges described within this disclosure, the upper or lower limit of that range may be replaced by the values ​​shown in the examples. Also, individually described upper and lower limits can be combined in any way. Additionally, "A or B" may include either A or B, or both.

[0017] A method according to the first embodiment of the present invention is a method for producing anisotropic fibers containing collagen, comprising: a step of contacting an anionic polymer with an aqueous collagen solution in which collagen is dissolved to form an interface and fibrillate the collagen (contact step); and a step of pulling up the fibrillated collagen from the aqueous collagen solution in which the anionic polymer has been contacted to form anisotropic fibers (fiber formation step).

[0018] The collagen aqueous solution may be a cell-containing collagen aqueous solution containing cells and dissolved collagen. In that case, according to the method of the first embodiment, anisotropic fibers (fibrous tissue) containing cells and collagen can be produced. That is, if the collagen aqueous solution is a cell-containing collagen aqueous solution containing cells and dissolved collagen, the method of the first embodiment may be a method for producing fibrous tissue. In this specification, "fibrous tissue" means a fibrous tissue containing cells and oriented collagen fibers (anisotropic collagen fibers).

[0019] <Contact Process> In the contact process, an anionic polymer is brought into contact with an aqueous collagen solution in which collagen is dissolved. In the contact process, two-phase separation (liquid-liquid phase separation) occurs due to the above contact, and at the interface in the liquid (hereinafter also referred to as "raw material liquid") in which collagen and anionic polymer are dissolved in each phase, collagen and anionic polymer interact, and an aggregate of fibrous collagen is formed at the interface of the two-phase separated liquid. It is thought that the collagen becomes fibrous by the anionic polymer taking water (water of hydration) adsorbed on the collagen molecules, but the mechanism is not limited to this.

[0020] The fiber formation mechanism according to the present invention is characterized by the formation of an interface driven by a polyionic complex (PIC) formed by collagen molecules and an anionic polymer. The electrostatic interaction between the anionic groups of the anionic polymer (e.g., carboxyl groups or sulfo groups) and the amino groups of collagen controls the bonding state between polymers according to the pH condition, leading to fiber formation. The ability to form PICs increases with higher polymer concentrations and lower molecular weights, promoting polymer supply to the interface and enabling the generation of long fibers. Fiber formation is not observed with polymers other than anionic polymers (neutral or cationic polymers), suggesting that PIC formation is one of the necessary conditions for fiber generation. Furthermore, the properties of the interface, particularly hardness and formation rate, change depending on the type of anionic polymer, which can lead to differences in fiber length and structure. Therefore, the anisotropic fibers (including fibrous tissues) containing collagen according to the present invention are primarily formed by the PIC interface formed between oriented collagen molecules and anionic polymers, and the fiber formation efficiency can be adjusted by pH, polymer concentration, molecular weight, and polymer type.

[0021] The raw material liquid preferably has a phase containing an anionic polymer on the lower vertical side and a phase containing collagen on the upper vertical side, as this facilitates the formation of anisotropic fibers.

[0022] At the interface of the two-phase separated liquid, collagen and anionic polymer interact, causing the collagen to fibrillate. The contact process creates aggregates of fibrillated collagen at the interface of the two-phase separated liquid. While it is thought that the anionic polymer removes water (water of hydration) adsorbed onto the collagen molecules, the mechanism is not limited to this.

[0023] Similarly, even if the collagen aqueous solution contains cells and is a cell-containing collagen aqueous solution in which collagen is dissolved, the contact step involves two-phase separation (also called liquid-liquid phase separation) due to the contact, and at the interface in the liquid (raw material liquid) in which collagen and anionic polymer are dissolved in each phase, the collagen and anionic polymer interact, causing the collagen to fibrillate and a mixture of collagen fibers and cells to be formed. If the collagen aqueous solution contains cells and is a cell-containing collagen aqueous solution in which collagen is dissolved, the contact step may also be a step of contacting the cell-containing collagen aqueous solution containing cells and dissolved collagen with a polymer aqueous solution in which anionic polymer is dissolved to fibrillate the collagen and obtain a mixture of collagen fibers and cells.

[0024] A collagen aqueous solution is a liquid containing collagen and an aqueous medium in which collagen is dissolved. In this specification, "aqueous medium" means a liquid in which water is an essential component. Examples of aqueous media include physiological salines such as phosphate-buffered saline (PBS), sterile water, and pH buffers such as Good's buffer. The collagen aqueous solution does not need to contain organic solvents. A culture medium can also be used as the aqueous medium. Examples of culture media include liquid media such as Dulbecco's Modified Eagle medium (DMEM) and hepatocyte culture medium (HCM). The liquid medium may be a serum-added medium or a serum-free medium. The liquid medium may be a mixed medium obtained by mixing two types of media.

[0025] Collagen may be fibrous collagen or non-fibrous collagen. Fibrous collagen refers to collagen that is the main component of collagen fibers. Specific examples of fibrous collagen include type I collagen, type II collagen, and type III collagen. The influence of the type of fibrous collagen (e.g., type I, type II, or type III) on fiber formation is relatively small, and various types of collagen can be used. Examples of non-fibrous collagen include type IV collagen.

[0026] As described below, when producing fibrous tissue, collagen can be appropriately selected depending on the type of cell. By selecting collagen that matches the environment in which the cells exist in vivo, it is possible to produce fibrous tissue that is closer to that of living organisms. For example, when using chondrocytes (e.g., ATDC5), the collagen may be type II collagen, which is abundant in cartilage. When using bovine satellite cells (bSCs), the collagen may be type IV collagen and / or type I collagen, which are components of the basement membrane. When using human umbilical vein endothelial cells (HUVECs), the collagen may be type III collagen, which constitutes the reticular fibers of blood vessels.

[0027] Collagen may be atelocollagen or tropocollagen. Atelocollagen is collagen from which telopeptides have been removed. Atelocollagen can be obtained, for example, by pepsin treatment of tropocollagen. Collagen may be atelocollagen because it is more likely to form longer anisotropic fibers.

[0028] Collagen may be derived from skin, cartilage, or tendon. Because collagen readily forms longer, anisotropic fibers, it may be derived from tendon.

[0029] Examples of animal species from which collagen can be derived include mammals, birds, and fish. Examples of mammals include humans, pigs, and cattle. Collagen may be derived from a single animal species, or from multiple animal species in combination. The animal species from which collagen is derived may be mammals or cattle, as they are more likely to form longer anisotropic fibers.

[0030] Specific examples of collagen include bovine skin-derived type I collagen, bovine cartilage-derived type I collagen, fish-derived type I collagen, bovine skin-derived tropocollagen, bovine tendon-derived type I collagen, bovine dermis-derived type III collagen, and human placenta-derived type IV collagen. Commercially available collagen can be used.

[0031] Collagen does not need to be cross-linked. In other words, collagen does not need to contain cross-linked collagen. Examples of cross-linked collagen include collagen cross-linked by chemical cross-linking agents or physical cross-linking. Examples of chemical cross-linking agents include glutaraldehyde and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). Examples of physical cross-linking methods include thermal dehydration, ultraviolet light, or the use of radiation (e.g., gamma rays).

[0032] Cell-containing collagen aqueous solution is a cell suspension containing cells, collagen, and an aqueous medium in which the collagen is dissolved. The collagen and the aqueous medium in which the collagen is dissolved are as described above.

[0033] 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.

[0034] 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 (e.g., human-derived fibroblasts (NHDF)), chondrocytes, chondrocytes (e.g., ATDC5), 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, and colorectal cancer cells (e.g., human colorectal cancer cells (HCT116, HT29)). Examples include cancer cells such as liver cancer cells. Cells may be used individually or in combination of multiple types. Preferably, the cells include satellite cells (for example, bovine satellite cells (bSCs)). When the cultured cells include satellite cells, fibrous tissue suitable for cultured meat applications can be produced. In addition, the cells in the fibrous tissue may include bovine adipose-derived stem cells (bADSCs), human dermal fibroblasts (NHDFs), human umbilical vein endothelial cells (HUVECs), or chondrocytes. By selecting these cells, fibrous tissues can be produced according to the target tissue, such as fat, skin, blood vessels, or cartilage, and effects suitable for a wide range of applications such as biomedical engineering and regenerative medicine can be obtained.

[0035] The number of cells in the cell-containing collagen aqueous solution can be appropriately selected depending on the type of cell, the intended use of the fibrous tissue, and other factors.

[0036] The cell concentration in the cell-containing collagen aqueous solution is set to 0.5 × 10⁻⁶ from the viewpoint of facilitating the creation of anisotropic fibers and improving cell viability. ６ cells / mL or more 8.0×10 ６ cells / mL or less, 0.5×10 ６ cells / mL or more 3.5×10 ６ cells / mL or less, 1.0×10 ６ cells / mL or more 6.0×10 ６ cells / mL or less, 1.0×10６ cells / mL or more, 3.5×10 ６ cells / mL or less, 1.0×10 ６ cells / mL or more, 3.0×10 ６ cells / mL or less, 2.0×10 ６ cells / mL or more, 5.0×10 ６ cells / mL or less, or 2.5×10 ６ cells / mL or more, 3.5×10 ６ cells / mL or less may be sufficient.

[0037] When the cell concentration in the aqueous collagen solution containing cells is around 6.0×10 ６ cells / mL (for example, 1.0×10 ６ cells / mL or more and 1.0×10 ７ cells / mL or less, 2.0×10 ６ cells / mL or more and 9.0×10 ６ cells / mL or less, or 4.0×10 ６ cells / mL or more and 8.0×10 ６ cells / mL or less, etc.), the number of cells in the collagen fibers at the time of producing the fibrous tissue body increases, and since there is little space for growth, the cells do not grow and differentiation is promoted. Further, if the number of cells is too small, it takes a long culture period to reach the cell density required for differentiation, and in order to avoid a decrease in the cell survival rate due to the solvent of collagen, an appropriate number of cells is 3.0×10 ６ / mL or around (for example, 1.0×10 ６ / mL or more and 1.0×10 ７ cells / mL or less, 2.0×10 ６ / mL or more and 8.0×10 ６ cells / mL or less, 2.0×10 ６ / mL or more and 5.0×10 ６ cells / mL or less, etc.), which is considered appropriate.

[0038] The collagen aqueous solution may also be an aqueous solution containing other biocompatible materials. The biocompatible material is one that does not adversely affect cell growth and does not hinder the formation of anisotropic fibers (including fibrous tissues) according to the first embodiment and collagen structures according to the second embodiment. Specifically, for example, it may be an extracellular matrix component other than collagen.

[0039] The extracellular matrix component is an aggregate of extracellular matrix molecules, formed by multiple extracellular matrix molecules. Extracellular matrix molecules may be substances present outside the cell in multicellular organisms. Any substance can be used as the extracellular matrix molecule, as long as it does not adversely affect cell growth and the formation of the collagen structure according to the first embodiment. Examples of extracellular matrix molecules include, but are not limited to, laminin, fibronectin, vitronectin, elastin, tenascin, enteractin, fibrillin, and proteoglycans. These extracellular matrix molecules may be used individually or in combination of two or more.

[0040] 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-X-Y, which is characteristic of collagen. Here, Gly represents a glycine residue, and X and Y each independently represent any amino acid residue. Multiple Gly-X-Y sequences may be identical or different. Having a repeating sequence represented by Gly-X-Y reduces constraints on the arrangement of the molecular chain. In an extracellular matrix molecule having a repeating sequence represented by Gly-X-Y, the proportion of the sequence represented by Gly-X-Y 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 refers to a sequence represented by Arg-Gly-Asp (arginine residue - glycine residue - aspartic acid residue). Extracellular matrix molecules containing a Gly-X-Y sequence and an RGD sequence include collagen, fibronectin, vitronectin, laminin, and cadherin.

[0041] Examples of proteoglycans include, but are not limited to, chondroitin sulfate proteoglycans, heparan sulfate proteoglycans, keratan sulfate proteoglycans, and dermatan sulfate proteoglycans.

[0042] 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 derived from one type of animal, or components derived from multiple types of animals may be used in combination.

[0043] The polymer aqueous solution is an aqueous solution containing an anionic polymer and an aqueous medium in which the anionic polymer is dissolved. The aqueous medium may be any of the examples given above, or it may be a physiological saline solution such as phosphate-buffered saline (PBS). The polymer aqueous solution does not need to contain an organic solvent.

[0044] Anionic polymers are polymers that contain monomer units with anionic functional groups (anionic groups). Examples of anionic groups include carboxyl groups (-COOH) and sulfo groups (-SO). ３ H) is an example. The anionic polymer may have one type of anionic group alone, or it may have two or more types of anionic groups.

[0045] Examples of monomers containing anionic groups include (meth)acrylic acid, sulfonic acid monomers (e.g., p-styrenesulfonic acid), monosaccharides having anionic groups, or monosaccharides to which anionic groups have been added. In this specification, "(meth)acrylic acid" and similar terms mean either acrylic acid and methacrylic acid, or both.

[0046] Examples of anionic polymers include poly(meth)acrylic acid, poly(p-styrenesulfonic acid), polysaccharides containing anionic groups, and salts thereof. The anionic polymer may be included alone or in combination of two or more types. Examples of salts in the anionic polymer include alkali metal salts such as sodium salts and potassium salts.

[0047] Poly(meth)acrylic acid is a polymer of (meth)acrylic acid. Poly(meth)acrylic acid is preferably polyacrylic acid. Poly(p-styrenesulfonic acid) is a polymer of p-styrenesulfonic acid.

[0048] Examples of polysaccharides containing anionic groups include heparin, chondroitin sulfate (e.g., chondroitin sulfate A, chondroitin sulfate C, chondroitin sulfate E, etc.), hyaluronic acid, dextran sulfate, carrageenan, alginic acid, and fucoidan.

[0049] Heparin is a mucopolysaccharide in which uronic acid (D-glucuronic acid and L-iduronic acid) and glucosamine (D-N-acetylglucosamine and D-N-sulfate glucosamine) are alternately linked.

[0050] Chondroitin sulfate is a glycosaminoglycan having a structure in which a sulfate group is added to a sugar chain in which D-glucuronic acid and N-acetyl-D-galactosamine (GalNAc) are repeatedly linked. Examples of chondroitin sulfate include chondroitin sulfate A, which contains glucuronic acid and acetylgalactosamine 4-sulfate as structural units; chondroitin sulfate C, which contains glucuronic acid and acetylgalactosamine 6-sulfate as structural units; and chondroitin sulfate E, which contains glucuronic acid and acetylgalactosamine 4,6-disulfate as structural units.

[0051] Hyaluronic acid is a linear polysaccharide in which N-acetylglucosamine and D-glucuronic acid are alternately linked by β-1,3 and β-1,4 bonds. Dextran sulfate is a polysaccharide having a structure in which dextran, a polymer of glucose, is sulfated.

[0052] Carrageenan is a polysaccharide in which D-galactose and 3,6-anhydro-D-galactose are alternately linked and sulfate groups are added. Examples of carrageenan include kappa-carrageenan, iota-carrageenan, and lambda-carrageenan. Alginic acid is a polymer of uronic acid in which L-guluronic acid and D-mannuronic acid are alternately or continuously linked. Fucoidan is a polysaccharide mainly composed of L-fucose and sulfate groups.

[0053] The anionic polymer is preferably at least one selected from the group consisting of poly(meth)acrylic acid, heparin, chondroitin sulfate, hyaluronic acid, dextran sulfate, poly(p-styrene sulfonic acid), carrageenan, alginic acid, and salts thereof, as it facilitates the formation of anisotropic fibers. It is more preferably at least one selected from the group consisting of poly(meth)acrylic acid, heparin, and salts thereof, even more preferably poly(meth)acrylic acid, and particularly preferably polyacrylic acid.

[0054] The molecular weight (average molecular weight) of the anionic polymer may be 2,000 or more, 4,000 or more, 4,500 or more, 5,000 or more, 7,000 or more, 9,000 or more, 10,000 or more, 12,000 or more, 14,000 or more, 16,000 or more, 18,000 or more, 20,000 or more, 22,000 or more, or 24,000 or more. The molecular weight of the anionic polymer may be 5,000,000 or less, 4,000,000 or less, 3,000,000 or less, 2,000,000 or less, 1,000,000 or less, 800,000 or less, 600,000 or less, 400,000 or less, 300,000 or less, 200,000 or less, 100,000 or less, 80,000 or less, 60,000 or less, 40,000 or less, 30,000 or less, 26,000 or less, 25,000 or less, 20,000 or less, 10,000 or less, 8,000 or less, or 6,000 or less, as this facilitates the formation of longer anisotropic fibers. The molecular weight of the anionic polymer may be 2,000 to 30,000, 2,000 to 25,000, 2,000 to 10,000, 2,000 to 8,000, 4,000 to 30,000, 4,000 to 25,000, or 4,000 to 6,000. If the anionic polymer is a commercially available product, the molecular weight (or average molecular weight) of the anionic polymer may be the value listed in the catalog of the commercially available product.

[0055] Anionic polymers are preferable to have a pKa of 5 or less, as this facilitates the formation of anisotropic fibers.

[0056] Methods for contacting an anionic polymer with a collagen aqueous solution include, for example, contacting the anionic polymer itself with the collagen aqueous solution, and contacting a polymer aqueous solution in which the anionic polymer is dissolved with the collagen aqueous solution. The polymer aqueous solution is an aqueous solution containing an anionic polymer and an aqueous medium in which the anionic polymer is dissolved. The aqueous medium may be any of the examples given above, or it may be a physiological saline solution such as phosphate-buffered saline (PBS). The polymer aqueous solution does not need to contain an organic solvent.

[0057] In the method according to the first embodiment, it is preferable to contact the polymer aqueous solution with the collagen aqueous solution because it facilitates the formation of anisotropic fibers. The preferred method for contacting the collagen aqueous solution with the anionic polymer is to add the polymer aqueous solution to a container, and then add or drop the collagen aqueous solution to the polymer aqueous solution. The method for adding the collagen aqueous solution to the polymer aqueous solution may be, for example, by layering the collagen aqueous solution on top of the polymer aqueous solution. By adding the polymer aqueous solution and the collagen aqueous solution to a container in this order, a raw material solution is formed having a phase containing the anionic polymer on the lower vertical side and a phase containing collagen on the upper vertical side. When such a raw material solution is used, there is a tendency for anisotropic fibers to be formed more easily.

[0058] The preferred method for contacting the cell-containing collagen aqueous solution with the polymer aqueous solution is to add the polymer aqueous solution to a container, and then add or drop the cell-containing collagen aqueous solution to the polymer aqueous solution. The method of adding the cell-containing collagen aqueous solution to the polymer aqueous solution may be, for example, by layering the cell-containing collagen aqueous solution on top of the polymer aqueous solution. By adding the polymer aqueous solution and the cell-containing collagen aqueous solution to a container in this order, a raw material solution is formed having a phase containing an anionic polymer on the lower vertical side and a phase containing collagen on the upper vertical side. When such a raw material solution is used, fibrous tissue tends to form more easily.

[0059] The collagen content in the collagen aqueous solution may be 1 mg / mL or more, 2 mg / mL or more, or 3 mg / mL or more, and 10 mg / mL or less, 8 mg / mL or less, 6 mg / mL or less, or 4 mg / mL or less, based on the total volume of the collagen aqueous solution. When the collagen content in the collagen aqueous solution is within the above range, anisotropic fibers are more easily formed. The collagen content in the collagen aqueous solution may be 1 to 10 mg / mL, 1 to 8 mg / mL, 1 to 5 mg / mL, 2 to 6 mg / mL, or 3 to 4 mg / mL, based on the total volume of the collagen aqueous solution. When the collagen content in the collagen aqueous solution is within the above range, anisotropic fibers are more easily formed.

[0060] The collagen content in the collagen aqueous solution may be, for example, 0.1 to 10 μM, 0.16 to 10 μM, 0.31 to 10 μM, 0.6 to 10 μM, 1.2 to 10 μM, 2.5 to 10 μM, 5 to 10 μM, 0.10 to 0.50 μM, 0.20 to 0.45 μM, or 0.25 to 0.40 μM, based on the total amount of the collagen aqueous solution. When the collagen content in the collagen aqueous solution is within the above range, anisotropic fibers are more easily formed.

[0061] The collagen aqueous solution may further contain extracellular matrix other than collagen. Examples of extracellular matrix other than collagen include laminin, fibronectin, vitronectin, elastin, tenascin, enteractin, fibrillin, and proteoglycans. Examples of proteoglycans include chondroitin sulfate proteoglycan, heparan sulfate proteoglycan, keratan sulfate proteoglycan, and dermatan sulfate proteoglycan.

[0062] The ratio of the mass of extracellular matrix other than collagen to the mass of collagen may be 1 / 3 to 3 / 1, 1 / 2 to 2 / 1, or 3 / 4 to 5 / 4.

[0063] The collagen aqueous solution may further contain an acid. The acid may be an organic acid or an inorganic acid. The organic acid may be, for example, a carboxylic acid. The carboxylic acid may be a carboxylic acid having a hydroxyl group. Examples of acids include lactic acid, citric acid, succinic acid, tartaric acid, gluconic acid, malic acid, fumaric acid, and phosphoric acid.

[0064] The acid may be at least one selected from the group consisting of lactic acid and citrate, and may be lactic acid, from the viewpoint of further reducing cytotoxicity and promoting the formation of anisotropic fibers with longer fiber lengths. When the acid is lactic acid or citrate, under the same proton conditions, cell viability is further improved compared to when the acid is acetic acid. The factors contributing to this effect are thought to be a combination of factors, such as the fact that the acid of that type is in an ionic state under in vivo conditions, resulting in low cell membrane permeability, and that under the same proton conditions, the molar concentrations of lactic acid and citrate are lower than the molar concentration of acetic acid, but the factors contributing to this effect are not limited to these.

[0065] The acid content may be 1 to 10 mg / mL, 2 to 8 mg / mL, 3 to 7 mg / mL, or 4 to 6 mg / mL, based on the total amount of the cell-containing collagen aqueous solution.

[0066] The acid content may be 0.1 to 10 mM, 0.5 to 6.0 mM, 0.5 to 4.0 mM, or 0.5 to 2.0 mM, based on the total amount of the collagen aqueous solution.

[0067] Cell-containing collagen aqueous solution can be obtained by a method that includes mixing cells with a collagen aqueous solution. The collagen aqueous solution is a liquid containing the collagen described above and an aqueous medium in which the collagen is dissolved. The collagen aqueous solution may contain, for example, an acid as a component in the aqueous medium. Specific examples of acids are as described above. As the collagen aqueous solution, for example, a commercially available collagen aqueous solution or a liquid obtained by dissolving collagen powder in an aqueous medium can be used. If the commercially available collagen aqueous solution contains an acid (e.g., acetic acid), a collagen aqueous solution obtained by substituting the acid (e.g., acetic acid) in the commercially available collagen aqueous solution with another acid (e.g., lactic acid, citric acid) can also be used. Methods for substituting the acid in the collagen aqueous solution with another acid include, for example, removing the acid in the collagen aqueous solution by dialysis, then obtaining collagen powder by freeze-drying, and dissolving the collagen powder in the other acid.

[0068] The collagen content in the collagen aqueous solution may be 1 to 10 mg / mL or more, 1 to 8 mg / mL or more, 1 to 5 mg / mL, 2 to 6 mg / mL, or 2 to 4 mg / mL, based on the total volume of the collagen aqueous solution.

[0069] The cells are 0.5 × 10⁶ per 1 mL of collagen aqueous solution. ６ cells~8.0×10 ６ cells, 1.0×10 ６ cells~6.0×10 ６ cells, 2.0×10 ６ cells~5.0×10 ６ cells, or 2.5 × 10 ６ cells~3.5×10 ６The cells may be mixed to the concentration described above. Mixing the cells to the aforementioned concentration makes it easier to create fibrous tissue and improves cell viability. Mixing the cells to the aforementioned concentration increases the number of cells within the collagen fibers at the time of fibrous tissue creation, and because there is less space for proliferation, the cells do not proliferate and differentiation is promoted. Furthermore, if the number of cells is too low, it will take longer to culture to reach the cell density necessary for differentiation. Also, to avoid a decrease in cell viability due to the collagen solvent, the cell number is appropriate, making it easier to create fibrous tissue and increasing cell viability.

[0070] The pH of the collagen aqueous solution may be 2.0 or higher, 2.5 or higher, 3.0 or higher, 3.5 or higher, 4.0 or higher, or 5.5 or higher, and may be 7.5 or lower, 7.0 or lower, less than 7.0, 6.5 or lower, 6.0 or lower, 5.5 or lower, 5.0 or lower, 4.5 or lower, 4.0 or lower, or 3.5 or lower. The pH of the collagen aqueous solution may be 3.5 or higher and 7.5 or lower, 4.0 or higher and 6.5 or lower, or 3.0 or higher and 6.0 or lower, 3.0 or higher and 5.0 or lower, or 3.5 or higher and 4.5 or lower. When the pH of the collagen aqueous solution is within the above range, anisotropic fibers are more easily formed.

[0071] The pH of the cell-containing collagen aqueous solution may be 3.5 or higher, 4.0 or higher, or 5.5 or higher, and may be 7.5 or lower, 7.0 or lower, less than 7.0, 6.5 or lower, 6.0 or lower, 5.5 or lower, 5.0 or lower, or 4.5 or lower. The pH of the cell-containing collagen aqueous solution may be 3.5 to 7.5, 4.0 to 6.5, or 3.0 to 6.0, 3.0 to 5.0, or 3.5 to 4.5. When the pH of the collagen aqueous solution is within the above range, fibrous tissue is more easily formed.

[0072] The content of anionic polymer in the polymer aqueous solution may be 1 mg / mL or more, 2 mg / mL or more, 3 mg / mL or more, 5 mg / mL or more, 8 mg / mL or more, 10 mg / mL or more, 15 mg / mL or more, 20 mg / mL or more, 25 mg / mL or more, or 28 mg / mL or more, based on the total amount of the polymer aqueous solution, and may be 40 mg / mL or less, 38 mg / mL or less, 36 mg / mL or less, 34 mg / mL or less, 32 mg / mL or less, 30 mg / mL or less, 25 mg / mL or less, 20 mg / mL or less, 15 mg / mL or less, 10 mg / mL or less, 8 mg / mL or less, 6 mg / mL or less, or 4 mg / mL or less, based on the total amount of the polymer aqueous solution. When the content of anionic polymers in the aqueous polymer solution is within the range described above, anisotropic fibers are more easily formed.

[0073] The content of anionic polymer in the polymer aqueous solution may be 0.30 mM or more, 0.31 mM or more and below the saturation concentration, 30 mM or less, 20 mM or less, 15 mM or less, or 12 mM or less, based on the total amount of the polymer aqueous solution; 1.0 mM or more and below the saturation concentration, 30 mM or less, 20 mM or less, 15 mM or less, or 12 mM or less; 5.0 mM or more and below the saturation concentration, 30 mM or less, 20 mM or less, 15 mM or less, or 12 mM or less; or 8.0 mM or more and below the saturation concentration, 30 mM or less, 20 mM or less, 15 mM or less, or 12 mM or less. The content of the anionic polymer in the polymer aqueous solution may be 0.30 mM or more, 0.31 mM or more, 1.0 mM or more, 5.0 mM or more, or 8.0 mM or more, based on the total amount of the polymer aqueous solution, and may be below the saturation concentration, 30 mM or less, 20 mM or less, 15 mM or less, or 12 mM or less.

[0074] The pH of the polymer aqueous solution may be 2.0 or higher, 2.5 or higher, 3.0 or higher, 3.5 or higher, 4.0 or higher, 4.5 or higher, 5.0 or higher, or 5.5 or higher, and may be less than 7.0, 6.5 or lower, 6.0 or lower, 5.5 or lower, 5.0 or lower, 4.5 or lower, 4.0 or lower, or 3.5 or lower. For better suitability in fiber formation, the pH of the polymer aqueous solution may be 2.0 to 6.0, 3.0 to 6.0, or 4.0 to 5.0, and may be 2.0 or higher but less than 7.0, or 3.0 or higher but less than 7.0.

[0075] When producing fibrous tissues, the pH of the polymer aqueous solution may be 3.5 or higher, 4.0 or higher, 4.5 or higher, 5.0 or higher, or 5.5 or higher, and may be less than 7.0, 6.5 or lower, 6.0 or lower, 5.5 or lower, 5.0 or lower, or 4.5 or lower. The pH of the polymer aqueous solution may be 3.5 to 6.0, 3.5 to 5.5, or 4.0 to 5.0, as this is more suitable for tissue formation.

[0076] The ratio of the volume of the polymer aqueous solution to the volume of the collagen aqueous solution may be 0.1 or more, 0.2 or more, 0.4 or more, 0.6 or more, 0.8 or more, or 0.9 or more, and may be 2.0 or less, 1.8 or less, 1.6 or less, 1.4 or less, 1.2 or less, or 1.1 or less.

[0077] The ratio of the volume of the polymer aqueous solution to the volume of the cell-containing collagen aqueous solution may be 0.01 or more, 0.05 or more, 0.08 or more, 0.10 or more, 0.15 or more, or 0.18 or more, and may be 2.0 or less, 1.5 or less, 1.0 or less, 0.80 or less, 0.60 or less, 0.40 or less, or 0.25 or less.

[0078] <Settling Step> The method according to the first embodiment may further include a setting step after the contact step and before the fiber formation step. When the method according to the first embodiment includes a setting step, fiber formation is facilitated.

[0079] The standing process may involve a standing time of, for example, 10 seconds or more, 15 seconds or more, 20 seconds or more, 25 seconds or more, 30 seconds or more, 35 seconds or more, 40 seconds or more, 45 seconds or more, 50 seconds or more, or 55 seconds or more. Alternatively, the standing time in the standing process may be, for example, 5 minutes or less, 4 minutes or less, 3 minutes or less, 2 minutes or less, or 1 minute or less. From the viewpoint of facilitating fiber formation, it is preferable that the standing time be 10 seconds to 5 minutes, 10 seconds to 4 minutes, 10 seconds to 3 minutes, 10 seconds to 2 minutes, or 10 seconds to 1 minute. The standing time in the standing process may be 24 hours (overnight) or more. Since interface formation quickly reaches a plateau in a certain amount of time, the standing period may be extended for as long as necessary after the plateau is reached.

[0080] <Fiber Formation Process> In the fiber formation process, fibrous collagen is pulled from a collagen aqueous solution that has been contacted with an anionic polymer to form anisotropic fibers containing collagen (collagen-containing anisotropic fibers). When a cell-containing collagen aqueous solution is used, this process can form collagen-containing anisotropic fibers and simultaneously encapsulate cells within the collagen-containing anisotropic fibers.

[0081] Anisotropic fibers (fibrous tissue) formed by a fiber formation process using a cell-containing collagen aqueous solution contain cells and anisotropic collagen fibers (oriented collagen fibers). The cells in the fibrous tissue are supported by the oriented collagen fibers inside the fibrous tissue. Immediately after spinning, the fibrous tissue has a hollow, sheath-like membrane structure. The cells in the fibrous tissue are adhered to the inside of the membrane and are arranged in a single row along the axial direction of the fibrous tissue. Subsequently, the hollow portion gradually closes through processes such as culture, and finally, the hollow portion is eliminated by the collagen fibers and cells, forming a dense fibrous tissue. In the fiber formation process, cells can be encapsulated within the fibrous tissue in a state where they are arranged in a single axial direction.

[0082] Collagen-containing anisotropic fibers are fibers composed of fibrous collagen (oriented collagen fibers) whose longitudinal direction is aligned in one direction. Through a fiber formation process, the collagen fibers can be aligned so that their longitudinal direction (length direction) aligns with the direction of the upward pulling force. In other words, the fiber formation process makes it possible to align the collagen fibers in the direction in which the upward pulling force is applied.

[0083] The anisotropy (orientation) of collagen fibers and the quantification of anisotropy (orientation) can be measured by the following method: Take a microscopic image of anisotropic fibers (fibrous tissue) containing collagen fibers. The long axis direction of the outermost layer of collagen fibers in the microscopic image is set as the reference direction (0°). Measure the angle difference (degree of inclination) of other collagen fibers present within the anisotropic fibers (fibrous tissue) containing collagen fibers from their reference direction. By evaluating the percentage of collagen fibers whose measured angle difference falls within -15° to +15°, the anisotropy (orientation) can be quantified.

[0084] Specifically, "orientation" as used herein is defined and measured by the following sample preparation, image acquisition, and image analysis methods. The same procedure was followed in the examples described later.

[0085] 1. Sample Preparation (Fixation and Staining) After the differentiation (or culture) period is complete, fix the fiber (or fiber containing cells) with a 4% paraformaldehyde (PFA) solution (e.g., at room temperature for 30 minutes). Then, perform permeabilization with, for example, a 0.1% Triton X-100 / PBS solution, and then stain the cytoskeleton (e.g., actin filaments) (e.g., stain with Phalloidin-Alexa Fluor 488) and counterstain the nucleus (e.g., counterstain with DAPI). 2. Image Acquisition Observe the sample using a light-sheet microscope (e.g., Zeiss Z.1, LaVision BioTec UltraMicroscope II, or equivalent instrument) or a confocal microscope. A 3D fluorescence image is obtained by scanning the sample surface from a depth of 100 μm to 4000 mm with a Z-step of 1 μm to 10 μm. For 2D images, for example, 1024 × 1024 pixel 2D fluorescence images are obtained for any five regions of the sample. 3. Image analysis (quantification of orientation) (a) The acquired image (for example, a 2D image processed with Max Intensity Projection in the Z-axis direction) is opened in the image analysis software "ImageJ" (for example, the Fiji distribution). (b) For example, the orientation angle of the stained cytoskeleton in the image is analyzed using the Skeletonize plugin, which is a plugin for ImageJ. The analysis is performed, for example, based on local gradients, and the orientation angle (-90° to +90°) and intensity of each pixel (or region) are calculated. (c) The long axis direction of the outermost layer of collagen fibers in the microscope image was defined as the reference direction (0°). (d) A histogram (frequency distribution) of angles relative to the reference 0° was created from all obtained data points (pixels). In this histogram, the percentage of data points distributed within the specified angle range was calculated by dividing the total number of data points included in the specified angle range by the total number of data points and multiplying by 100.

[0086] Collagen-containing anisotropic fibers have high mechanical strength because they contain oriented collagen fibers. By using oriented collagen fibers, it becomes possible to form collagen-containing anisotropic fibers that are poorly soluble in water and culture medium without using denatured collagen such as chemically crosslinked collagen, which is in a different state from the undenatured collagen in the body. By forming fibers by lifting the interface, it is possible to reduce equipment costs by not using equipment such as electrospinning or two-photon microscopes. Furthermore, when using a cell-containing collagen aqueous solution, cells can be simultaneously encapsulated within the fibers during spinning, rather than seeding cells into pre-spun collagen fibers, thus enabling the formation of fibrous tissue containing cells internally.

[0087] One method for lifting the fibrous collagen is to lift the fibrous collagen-containing membrane that forms at the interface between the anionic polymer-containing phase and the collagen-containing phase using tweezers or the like. The collagen-containing anisotropic fibers obtained after lifting the fibrous collagen may be wound up as needed.

[0088] The pulling speed of the fibrous collagen may be 25 mm / sec or less, 20 mm / sec or less, 18 mm / sec or less, 16 mm / sec or less, 14 mm / sec or less, 12 mm / sec or less, 11 mm / sec or less, 10 mm / sec or less, 8 mm / sec or less, 6 mm / sec or less, or 5 mm / sec or less, and may be 1 mm / sec or more, 2 mm / sec or more, 3 mm / sec or more, or 4 mm / sec or more. The pulling speed is calculated by the formula: length of collagen-containing anisotropic fiber (unit: mm) ÷ spinning time (seconds).

[0089] The maximum longitudinal length (fiber length) of the collagen-containing anisotropic fiber can be appropriately selected depending on the application. The fiber length of the collagen-containing anisotropic fiber may be, for example, 1 mm or more, 3 mm or more, 5 mm or more, 10 mm or more, 20 mm or more, 40 mm or more, 60 mm or more, 80 mm or more, 100 mm or more, 120 mm or more, 140 mm or more, 160 mm or more, 180 mm or more, 200 mm or more, or 210 mm or more, and may be 300 mm or less, 250 mm or less, 200 mm or less, 180 mm or less, 160 mm or less, 140 mm or less, 120 mm or less, 100 mm or less, 80 mm or less, 60 mm or less, 40 mm or less, 20 mm or less, 15 mm or less, 10 mm or less, 8 mm or less, 6 mm or less, 4 mm or less, or 2 mm or less.

[0090] <Other steps> The method according to the first embodiment may further include a step of culturing cells in anisotropic fibers (fibrous tissue).

[0091] The culture method can be one that is suitable for the type of cells to be cultured. If the cells include stem cells, differentiation induction culture may be performed during the cell culture process. Differentiation induction culture can be performed under conditions suitable for the type of stem cells.

[0092] The culture medium is selected appropriately depending on the cell type, etc. 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, GlutaMax medium, EGM2, and Ham's F-12. The culture medium may be serum-added or serum-free. The culture medium may be a mixed medium (for example, DMEM / F12 medium) obtained by mixing two types of media. The culture medium may contain additives as needed.

[0093] 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.

[0094] 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 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.

[0095] According to the method of the first embodiment, collagen-containing anisotropic fibers can be produced without using organic solvents. Therefore, the collagen-containing anisotropic fibers obtained by the method of the first embodiment are suitable for use in cultured meat and the like. Furthermore, the collagen-containing anisotropic fibers obtained by the method of the first embodiment can be used for culture applications.

[0096] The method according to the first embodiment allows for the production of collagen-containing anisotropic fibers through the contact step and fiber formation step described above, and does not require complicated steps such as dialysis to remove high concentrations of salt, thus making it possible to produce collagen-based fibers more easily.

[0097] The present invention may include, instead of a fiber formation step, a step (removal step) to remove the solution present around the fibrous collagen-containing membrane formed at the interface between a phase containing an anionic polymer and a phase containing collagen. In that case, the present invention can also be considered as a method for producing a collagen-containing membrane tissue comprising a contact step and the removal step. The method for removing the solution present around the fibrous collagen-containing membrane formed at the interface between a phase containing an anionic polymer and a phase containing collagen may include an embodiment in which a collagen aqueous solution and a polymer aqueous solution are layered to form an interface, and a membrane-like structure (hereinafter referred to as a membrane-like structure) is obtained by allowing the interface to stand while in contact. Specifically, when left to stand overnight (for example, 8 to 24 hours) at room temperature or 37°C under conditions of pH 3 to 4, a precipitated film based on a polyion complex (PIC) is formed at the interface. The formed membrane-like structure can be transferred to a support substrate (Petri dish, well bottom, glass, polystyrene, etc.) or formed in situ, and after removing unreacted polymers by washing with PBS, etc., it can be used for cell culture.

[0098] [Anisotropic Fibers (Fibrous Tissue)] The anisotropic fibers according to the first embodiment may be anisotropic fibers containing collagen (collagen-containing anisotropic fibers), or they may be anisotropic fibers containing collagen that do not contain cells (collagen-containing anisotropic fibers), or they may be anisotropic fibers containing collagen that do contain cells (collagen-containing anisotropic fibers). Anisotropic fibers containing collagen that do contain cells (collagen-containing anisotropic fibers) are also called "fibrous tissue."

[0099] In this embodiment, the collagen-containing anisotropic fibers may contain 50% or more of collagen whose orientation angle with respect to the reference direction is within ±5°. The proportion of collagen distributed within ±5° of the orientation angle with respect to the reference direction may be 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, or 90% or more, and may be 100% or less. The proportion of collagen distributed within ±5° of the orientation angle with respect to the reference direction is measured by the orientation measurement method described above.

[0100] In the collagen-containing anisotropic fiber according to the first embodiment, the proportion of collagen distributed within ±10° of the reference direction may be 60% or more. The proportion of collagen distributed within ±10° of the reference direction may be 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, or 90% or more, and may be 100% or less. The proportion of collagen distributed within ±10° of the reference direction is measured by the orientation measurement method described above.

[0101] In the collagen-containing anisotropic fiber according to the first embodiment, the proportion of collagen distributed within ±15° of the reference direction may be 70% or more. The proportion of collagen distributed within ±15° of the reference direction may be 75% or more, 80% or more, 85% or more, or 90% or more, and may be 100% or less. The proportion of collagen distributed within ±15° of the reference direction is measured by the orientation measurement method described above.

[0102] The collagen-containing anisotropic fiber according to the first embodiment may, for example, have a storage modulus (G') at a strain of 0.1% of 2000 Pa or less, 1800 Pa or less, 1600 Pa or less, 1400 Pa or less, 1200 Pa or less, 1000 Pa or less, 800 Pa or less, 600 Pa or less, 400 Pa or less, 300 Pa or less, 200 Pa or less, or 160 Pa or less, and may also have a storage modulus (G') of 10 Pa or more, 15 Pa or more, 20 Pa or more, 25 Pa or more, 30 Pa or more, 35 Pa or more, 40 Pa or more, 45 Pa or more, or 50 Pa or more. The collagen-containing anisotropic fiber according to the first embodiment may have a storage modulus (G') at a strain of 0.1%, for example, 10 Pa to 2000 Pa, 10 Pa to 1000 Pa, 15 Pa to 1000 Pa, 20 Pa to 400 Pa, 30 Pa to 160 Pa, or 50 Pa to 300 Pa.

[0103] The collagen-containing anisotropic fiber according to the first embodiment may, for example, have elastic modulus cross points located at strains of 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 100% or more, 105% or more, 110% or more, 115% or more, 120% or more, 125% or more, 130% or more, 135% or more, 140% or more, 145% or more, or 150% or more, or it may have elastic modulus cross points located at strains of 300% or less, 290% or less, 280% or less, 270% or less, 260% or less, 250% or less, 240% or less, 230% or less, 220% or less, 210% or less, or 200% or less. The collagen-containing anisotropic fiber according to the first embodiment may have elastic modulus cross points with strains of, for example, 30% to 300%, 50% to 280%, 100% to 250%, or 150% to 210%.

[0104] In this specification, "storage modulus (G')", "loss modulus (G'')", and "modulus cross-point" are defined and measured by the following dynamic viscoelastic measurement (oscillation measurement). The same procedure was followed in the examples described later.

[0105] 1. Measurements are performed using a rheometer, such as the MCR series from Anton Paar, the DHR series from TA Instruments, or a rheometer with equivalent functionality. 2. After preparing the sample and spinning (or extruding) the fibers (or fiber bundles) obtained, wash them with ultrapure water or PBS, for example, before using them for measurement. The fibers are placed in the center of a parallel plate geometry with a diameter of 25 mm, for example. The gap between the plates (measurement gap) is set to 1.0 mm, for example, and a suitable amount of PBS is dropped to maintain a moist state so that the sample fills the gap. For measurements involving stretching the fibers, the sample is attached to a dedicated fiber stretching jig, for example, and an initial load of 0.01 N is applied. 3. Strain-dependent measurements are performed at a temperature of 25°C, for example. Under the condition that the frequency is fixed at 1 Hz, the applied strain is logarithmically increased, for example, in the range of 0.01% to 1000%, and the storage modulus (G') and loss modulus (G'') at each strain are recorded. 4. Definition of characteristic values ​​(a) Storage modulus at 0.1% strain: From the data obtained from the strain dependence measurement in 3. above, the value of the storage modulus (G') at 0.1% strain (unit: Pa) is read. (b) Modulus crossover point: In the measurement in 3. above, regression analysis is performed from the values ​​in the linear region, and the strain value at which G' - G'' = 0 is defined as the "modulus crossover point". The value of strain (%) at this point is adopted as the yield point (yield strain) of the structure.

[0106] The collagen-containing anisotropic fiber according to the first embodiment has a permeability of 1.0 × 10⁻¹⁶ to a 5 mM HEPES solution containing 0.5 mg / mL FITC-dextran (molecular weight 4k). －７ cm / s or less, preferably 0.6 × 10 －７ The pressure may be less than or equal to cm / s. The collagen-containing anisotropic fiber according to the first embodiment has a permeability of 2.0 × 10⁻¹⁰ to a 5 mM HEPES solution containing 0.5 mg / mL FITC-dextran (molecular weight 2000 k). －７ cm / s or less, preferably 1.2 × 10 －７The pressure may be less than or equal to cm / s. The collagen-containing anisotropic fiber according to the first embodiment has a permeability of 10 × 10 to a 5 mM PBS solution containing 0.5 mg / mL of FITC-dextran (molecular weight 4k). －７ cm / s or less, preferably 9.0 × 10 －７ The pressure may be less than or equal to cm / s. The collagen-containing anisotropic fiber according to the first embodiment has a permeability of 2.0 × 10⁻¹⁰ to a 5 mM PBS solution containing 0.5 mg / mL of FITC-dextran (molecular weight 2000 k). －７ It may be less than cm / s.

[0107] The collagen-containing anisotropic fiber according to the first embodiment has a permeability of 1.0 × 10⁻¹⁶ to a 5 mM HEPES solution containing 0.5 mg / mL of FITC-dextran (molecular weight 4k). －７ (1) The permeability of (1) above is 0.6 × 10 －７ (1) The permeability of a 5 mM HEPES solution containing 0.5 mg / mL FITC-dextran (molecular weight 2000 k) is 2.0 × 10 －７ (2) The permeability of (2) above is 1.2 × 10 －７ (3) The permeability of a 5 mM PBS solution containing 0.5 mg / mL of FITC-dextran (molecular weight 4k) is 10 × 10 －７ (3') The permeability of (3) above is 9.0 × 10 －７ (4) The permeability of a 5 mM PBS solution containing 0.5 mg / mL of FITC-dextran (molecular weight 2000 k) is 2.0 × 10 －７The collagen-containing anisotropic fiber of the present invention may satisfy at least one property selected from the group consisting of (1), (1'), (2), (2'), (3), (3'), and (4). The present invention may satisfy any combination of two or more properties selected from the group consisting of (1), (1'), (2), (2'), (3), (3'), and (4). This description clarifies that any combination of these properties (e.g., a combination of (1') and (2), a combination of (1) and (3'), a combination of (1'), (2'), and (3), etc.) is intended as individually disclosed embodiments.

[0108] The collagen-containing anisotropic fiber according to the first embodiment may have a ratio of the permeability of a 5 mM HEPES solution containing 0.5 mg / mL rhodamine-dextran (molecular weight 2000 k) to the permeability of a 5 mM HEPES solution containing 0.5 mg / mL FITC-dextran (molecular weight 4 k) to a ratio of 6.5 or more, 7.0 or more, or 7.5 or more.

[0109] The collagen-containing anisotropic fiber according to the first embodiment may have a ratio of the permeability of a PBS solution containing 0.5 mg / mL rhodamine-dextran (molecular weight 2000 k) to the permeability of a PBS solution containing 0.5 mg / mL FITC-dextran (molecular weight 4), which may be, for example, 8.0 or more, 8.5 or more, 9.0 or more, 9.5 or more, 10.0 or more, 10.5 or more, 11.0 or more, 11.5 or more, 12.0 or more, or 12.5 or more.

[0110] In this specification, permeability is defined and measured by the following permeability tests. The same procedure was used in the examples described later.

[0111] <Permeability Test> Dissolve FITC-dextran (molecular weight 4k) and Rhodamine-dextran (molecular weight 2000k) in PBS and 5 mM HEPES, respectively, to a concentration of 0.5 mg / mL. Wash the membrane and gel on the culture insert twice with ultrapure water, aspirate, and then add PBS or HEPES to the reservoir. Add the fluorescent dextran solution in the same solvent as the reservoir to the membrane and gel, and sample 50 μL from the reservoir at 1, 5, 15, 30, 45, 60, 75, 90, 105, 120, and 180 minutes. Add 50 μL of the respective solvent to the reservoir each time you sample.

[0112] The apparent transmittance is calculated from the calibration curve using the following formula (1).

[0113] In this experiment, the same amount of buffer solution was replenished as the sampled amount. Therefore, the true cumulative permeation amount (mg) at each sampling point is corrected using the following formula (2).

[0114] In the above formulas (1) and (2), each letter represents the following: A: Membrane area (cm²) ２ , Example: 0.33cm ２ ) C ０ : Initial concentration on the donor side (mg / mL, e.g., 0.5 mg / mL) dQ / dt: Slope (permeation rate, mg / s) in the steady state (linear portion) when the cumulative permeation amount Q (mg) on ​​the reservoir side is plotted against time t (seconds) Q ｎ : Corrected cumulative permeation amount (mg) at time n C ｎ : Measured concentration (mg / mL) on the reservoir side at time point n V Ｒ : Total liquid volume on the reservoir side (mL) (e.g., 0.6 mL) V Ｓ : Sampling and replenishment volume (mL) (e.g., 0.05 mL) Σ C ｉ : The sum of measured concentrations (mg / mL) from time point 0 to the most recent sampling time point (n-1).

[0115] The fibrous tissue according to the first embodiment is an anisotropic fiber according to the first embodiment, and includes oriented collagen fibers (also called anisotropic collagen fibers) and cells. The anisotropic fiber (also called fibrous tissue) can be produced, for example, by the method described above. The anisotropic fiber (also called fibrous tissue) can also be described as a fibrous tissue that includes an aggregate of oriented collagen fibers (also called anisotropic collagen fibers) and cells contained within the aggregate. The specific forms of the oriented collagen fibers (also called anisotropic collagen fibers) and cells can be the forms described as the forms of the method for producing the anisotropic fiber (also called fibrous tissue) or the forms of the anisotropic fiber described above. The cells may be fibroblasts, stem cells or cells differentiated from stem cells, satellite cells or cells differentiated from satellite cells.

[0116] Anisotropic fibers (also called fibrous tissues) can be used as, for example, muscle tissue, vascular tissue, and cartilage-like tissue. Fibrous tissues can be used in applications such as cultured meat and transplant materials.

[0117] [Method for producing a collagen-containing structure] A second embodiment of the present invention will now be described. The production method according to the second embodiment is a method for producing a collagen-containing structure having a hollow structure filled with an aqueous medium inside, comprising: a step of forming droplets of an aqueous solution containing at least collagen in an aqueous solution containing an anionic polymer (also called a droplet formation step); and a step of allowing the structure to stand for a certain period of time after the droplet formation step to allow the collagen molecules to assemble at the interface between the droplets and the aqueous solution containing the anionic polymer (a standing step).

[0118] The manufacturing method according to the second embodiment allows for the simple production of a collagen-containing structure having a hollow structure filled with an aqueous medium inside, by comprising the above-described steps.

[0119] Figure 32 is a schematic diagram showing a manufacturing method according to one embodiment. In the manufacturing method shown in Figure 32, first, a PBS aqueous solution containing polyacrylic acid (PAA) in an amount sufficient to completely cover droplets of an aqueous collagen solution (hereinafter also referred to as "collagen aqueous solution") is added to a dish to prepare a PAA bath. Droplets are then intermittently formed in the PAA bath by repeatedly dispensing and stopping the aqueous collagen solution with a pipette (droplet formation step). Next, by allowing it to stand at 25°C to 40°C for a certain period of time (for example, 60 seconds) (standing step), collagen molecules are aggregated at the interface between the collagen aqueous solution droplets and the PAA bath, forming a droplet-shaped collagen structure.

[0120] A collagen-containing structure (hereinafter also referred to as "collagen structure") means a solid object having any shape formed with collagen as the main component. The collagen content in the collagen structure according to the second embodiment may be, for example, 50% by mass or more, 55% by mass or more, 60% by mass or more, 65% by mass or more, 70% by mass or more, 75% by mass or more, 80% by mass or more, 85% by mass or more, 90% by mass or more, or 95% by mass or more, or 100% by mass, based on the total amount of the collagen structure according to the second embodiment.

[0121] <Droplet Formation Process> In the droplet formation process, droplets of an aqueous solution containing at least collagen are formed in an aqueous solution containing an anionic polymer. In this specification, "droplet" means a mass (particle, drip) of liquid held together by surface tension. The method for forming the above droplets may be, for example, intermittently or continuously dispensing droplets of an aqueous solution containing at least collagen into an aqueous solution containing an anionic polymer, or intermittently or continuously dropping droplets of an aqueous solution containing at least collagen. From the viewpoint of being able to produce multiple collagen structures, it is preferable to drop droplets of an aqueous solution containing at least collagen rather than dispensing droplets of an aqueous solution containing at least collagen. The method for dispensing or dropping droplets of an aqueous solution containing at least collagen is not particularly limited and can be, for example, by using a pipette, dropper, or syringe to dispensing or dropping the above aqueous solution droplets. When using a syringe, for example, an adapter, syringe, and needle may be attached to a centrifuge tube. A syringe can be attached to an adapter, and the syringe with the adapter attached can be attached to a centrifuge tube containing a polymer aqueous solution. By attaching a needle to the tip of each syringe and centrifuging using the centrifuge tube, the collagen solution filled in the syringe can be dispensed by centrifugal force.

[0122] The size of the droplets formed in the droplet formation step is not particularly limited, but for example, the diameter may be 10 μm or more, 50 μm or more, 100 μm or more, 200 μm or more, 300 μm or more, 400 μm or more, 500 μm or more, 600 μm or more, 700 μm or more, 800 μm or more, 900 μm or more, or 1000 μm or more, and the diameter may be 3 cm or less, 2.5 cm or less, 2 cm or less, 1.5 cm or less, 1 cm or less, 5 mm or less, or 1 mm or less. When the size of the droplets formed in the droplet formation step is within the above range, the collagen structure according to the second embodiment is more easily formed.

[0123] (Aqueous solution containing anionic polymer) An aqueous solution containing anionic polymer (hereinafter also referred to as "polymer aqueous solution") is an aqueous solution containing an aqueous medium in which the anionic polymer is dissolved. Examples of aqueous media include physiological saline such as phosphate-buffered saline (PBS), sterile water, and pH buffers such as Good's buffer. Culture media can also be used as the aqueous medium. Examples of culture media include liquid media such as Eagle's MEM medium, Dulbecco's Modified Eagle medium (DMEM), Modified Eagle medium (MEM), Minimum Essential medium, RPMI medium, and GlutaMax medium. The liquid medium may be a serum-added medium or a serum-free medium. The liquid medium may be a mixed medium obtained by mixing two types of media. The polymer aqueous solution does not need to contain organic solvents.

[0124] Anionic polymers are polymers that contain monomer units with anionic functional groups (anionic groups). Examples of anionic groups include carboxyl groups (-COOH) and sulfo groups (-SO). ３ Examples include H). The anionic polymer may have only one type of anionic group, or it may have two or more types of anionic groups.

[0125] Examples of monomers containing anionic groups include (meth)acrylic acid, sulfonic acid monomers (e.g., p-styrenesulfonic acid), monosaccharides having anionic groups, or monosaccharides to which anionic groups have been added. In this specification, "(meth)acrylic acid" and similar terms mean either acrylic acid and methacrylic acid, or both.

[0126] Examples of anionic polymers include poly(meth)acrylic acid, poly(p-styrenesulfonic acid), polysaccharides containing anionic groups, and salts thereof. The anionic polymer may be included alone or in combination of two or more types. Examples of salts in the anionic polymer include alkali metal salts such as sodium salts and potassium salts.

[0127] Poly(meth)acrylic acid is a polymer of (meth)acrylic acid. Poly(meth)acrylic acid is preferably polyacrylic acid. Poly(p-styrenesulfonic acid) is a polymer of p-styrenesulfonic acid.

[0128] Examples of polysaccharides containing anionic groups include heparin, chondroitin sulfate (e.g., chondroitin sulfate A, chondroitin sulfate C, chondroitin sulfate E, etc.), hyaluronic acid, dextran sulfate, carrageenan, alginic acid, and fucoidan. Heparin, chondroitin sulfate, hyaluronic acid, dextran sulfate, carrageenan, alginic acid, and fucoidan are as described above.

[0129] The anionic polymer is preferably at least one selected from the group consisting of poly(meth)acrylic acid, heparin, chondroitin sulfate, hyaluronic acid, dextran sulfate, poly(p-styrene sulfonic acid), carrageenan, and salts thereof, and more preferably at least one selected from the group consisting of poly(meth)acrylic acid and its salts, since this facilitates the formation of the collagen structure according to the second embodiment. The anionic polymer may not contain alginic acid.

[0130] The molecular weight (average molecular weight) of the anionic polymer may be 2,000 or more, 4,000 or more, 4,500 or more, 5,000 or more, 7,000 or more, 9,000 or more, 10,000 or more, 12,000 or more, 14,000 or more, 16,000 or more, 18,000 or more, 20,000 or more, 22,000 or more, or 24,000 or more. The molecular weight of the anionic polymer may be 5,000,000 or less, 4,000,000 or less, 3,000,000 or less, 2,000,000 or less, 1,000,000 or less, 800,000 or less, 600,000 or less, 400,000 or less, 300,000 or less, 200,000 or less, 100,000 or less, 80,000 or less, 60,000 or less, 40,000 or less, 30,000 or less, 26,000 or less, 25,000 or less, 20,000 or less, 10,000 or less, 8,000 or less, or 6,000 or less, in order to more easily form the collagen structure of the second embodiment. The molecular weight of the anionic polymer may be 2,000 to 30,000, 2,000 to 25,000, 2,000 to 10,000, 2,000 to 8,000, 4,000 to 30,000, 4,000 to 25,000, or 4,000 to 6,000.

[0131] Anionic polymers are preferable to have a pKa of 5 or less, as this facilitates the formation of collagen structures.

[0132] The content of the anionic polymer in the polymer aqueous solution may be 1 mg / mL or more, 2 mg / mL or more, 3 mg / mL or more, 5 mg / mL or more, 8 mg / mL or more, 10 mg / mL or more, 15 mg / mL or more, 20 mg / mL or more, 25 mg / mL or more, or 28 mg / mL or more, based on the total amount of the polymer aqueous solution. It may also be 40 mg / mL or less, 38 mg / mL or less, 36 mg / mL or less, 34 mg / mL or less, 32 mg / mL or less, 30 mg / mL or less, 25 mg / mL or less, 20 mg / mL or less, 15 mg / mL or less, 10 mg / mL or less, 8 mg / mL or less, 6 mg / mL or less, or 4 mg / mL or less, as this further improves cell viability. When the content of the anionic polymer in the polymer aqueous solution is within the above range, the collagen structure according to the second embodiment is more easily formed.

[0133] The content of anionic polymer in the polymer aqueous solution may be 0.30 mM or more, 0.31 mM or more and below the saturation concentration, 30 mM or less, 20 mM or less, 15 mM or less, or 12 mM or less, based on the total amount of the polymer aqueous solution; 1.0 mM or more and below the saturation concentration, 30 mM or less, 20 mM or less, 15 mM or less, or 12 mM or less; 5.0 mM or more and below the saturation concentration, 30 mM or less, 20 mM or less, 15 mM or less, or 12 mM or less; or 8.0 mM or more and below the saturation concentration, 30 mM or less, 20 mM or less, 15 mM or less, or 12 mM or less. The content of the anionic polymer in the polymer aqueous solution may be 0.30 mM or more, 0.31 mM or more, 1.0 mM or more, 5.0 mM or more, or 8.0 mM or more, based on the total amount of the polymer aqueous solution, and may be below the saturation concentration, 30 mM or less, 20 mM or less, 15 mM or less, or 12 mM or less.

[0134] The pH of the polymer aqueous solution may be 2.0 or higher, 3.0 or higher, 3.5 or higher, 4.0 or higher, 4.5 or higher, 5.0 or higher, or 5.5 or higher, and may be less than 7.0, 6.5 or lower, 6.0 or lower, 5.5 or lower, 5.0 or lower, or 4.5 or lower. The pH of the polymer aqueous solution may be, for example, 2.0 to 6.0, 3.0 to 6.0, 0, or 4.0 to 5.0, and may be 2.0 or higher but less than 7.0, or 3.0 or higher but less than 7.0, as it is more suitable for the production of the structure.

[0135] The volume of the polymer aqueous solution can be appropriately selected depending on the volume of the collagen aqueous solution droplets, as long as the droplets of collagen aqueous solution are coated by the polymer aqueous solution. The volume of the polymer aqueous solution may be, for example, 200 μL or more, 300 μL or more, 400 μL or more, 500 μL or more, 1 mL or more, 5 mL or more, or 10 mL or more, and may also be 1000 mL or less, 900 mL or less, 800 mL or less, 700 mL or less, 600 mL or less, 500 mL or less, 400 mL or less, 300 mL or less, 200 mL or less, or 100 mL or less. From the viewpoint of making it easier to form the collagen structure according to the second embodiment, the volume of the polymer aqueous solution is preferably 500 μL or more.

[0136] (An aqueous solution containing at least collagen) An aqueous solution containing at least collagen (also called a collagen aqueous solution) is an aqueous solution containing an aqueous medium containing at least collagen. The aqueous medium in the collagen aqueous solution may be any of the aqueous mediums exemplified above. The collagen aqueous solution does not need to contain any organic solvents.

[0137] Collagen does not dissolve under neutral conditions, but it dissolves under acidic conditions (pH 2 to 4). The collagen aqueous solution is not particularly limited as long as it is an aqueous solution in which collagen is dissolved, and may be an aqueous solution in which collagen is dissolved in an acid-containing solution (pH 2 to 4). From the viewpoint of high biocompatibility compared to other acids (specifically, for example, not interfering with cell survival), the collagen aqueous solution is preferably an aqueous solution in which at least collagen is dissolved in a solution containing at least one acid selected from the group consisting of lactic acid, citric acid, succinic acid, tartaric acid, gluconic acid, malic acid, fumaric acid, and phosphoric acid, more preferably an aqueous solution in which at least collagen is dissolved in a solution containing lactic acid, and even more preferably an aqueous solution in which at least collagen is dissolved in a solution containing lactic acid.

[0138] Collagen may be fibrous collagen or non-fibrous collagen. Fibrous collagen refers to collagen that is the main component of collagen fibers. Specific examples of fibrous collagen include type I collagen, type II collagen, and type III collagen. The influence of the type of fibrous collagen (e.g., type I, type II, or type III) on the structure formation is relatively small, and various types of collagen can be used. Examples of non-fibrous collagen include type IV collagen.

[0139] If the collagen aqueous solution further contains cells, the collagen can be appropriately selected depending on the type of cell. By selecting collagen that matches the environment in which cells exist in vivo, it is possible to produce fibrous tissue that is closer to that of living organisms. For example, when using chondrocytes (e.g., ATDC5), the collagen may be type II collagen, which is abundant in cartilage. When using bovine satellite cells (bSCs), the collagen may be type IV collagen and / or type I collagen, which are components of the basement membrane. When using human umbilical vein endothelial cells (HUVECs), the collagen may be type III collagen, which constitutes the reticular fibers of blood vessels.

[0140] Collagen may be atelocollagen or tropocollagen.

[0141] Collagen may be derived from skin, cartilage, or tendons.

[0142] Examples of animal species from which collagen can be derived include mammals, birds, and fish. Examples of mammals include humans, pigs, and cattle. Collagen may be derived from a single animal species, or from multiple animal species in combination. The animal species from which collagen can be derived may be mammals or cattle, as they readily form collagen structures.

[0143] Specific examples of collagen include bovine skin-derived type I collagen, bovine cartilage-derived type I collagen, fish-derived type I collagen, bovine skin-derived tropocollagen, and bovine tendon-derived type I collagen. Commercially available collagen can be used.

[0144] Collagen does not need to be cross-linked. In other words, collagen does not need to contain cross-linked collagen. Examples of cross-linked collagen include collagen cross-linked by chemical cross-linking agents or physical cross-linking. Examples of chemical cross-linking agents include glutaraldehyde and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). Examples of physical cross-linking methods include thermal dehydration, ultraviolet light, or the use of radiation (e.g., gamma rays).

[0145] The collagen content in the collagen aqueous solution may be 90 μg or more, 96 μg / mL or more, 100 μg / mL or more, 500 μg / mL or more, 0.75 mg / mL or more, 1 mg / mL or more, 1.5 mg / mL or more, 2 mg / mL or more, 2.5 mg / mL or more, or 3 mg / mL or more, based on the total amount of the collagen aqueous solution, and may be 10 mg / mL or less, 9 mg / mL or less, 8 mg / mL or less, 7 mg / mL or less, 6 mg / mL or less, 5 mg / mL or less, 4 mg / mL or less, or 3 mg / mL or less. When the collagen content in the collagen aqueous solution is within the above range, the collagen structure according to the second embodiment is more easily formed. Furthermore, from the viewpoint of making it easier to form the collagen structure according to the second embodiment, it is preferable that the collagen content in the collagen aqueous solution is 0.75 mg / mL or more and 10 mg / mL or less, based on the total amount of the collagen aqueous solution.

[0146] The collagen content in the collagen aqueous solution may be, for example, 0.1 to 10 μM, 0.16 to 10 μM, 0.31 to 10 μM, 0.6 to 10 μM, 1.2 to 10 μM, 2.5 to 10 μM, 5 to 10 μM, 0.10 to 0.50 μM, 0.20 to 0.45 μM, or 0.25 to 0.40 μM, based on the total amount of the collagen aqueous solution. When the collagen content in the collagen aqueous solution is within the above range, the collagen structure according to the second embodiment is more easily formed.

[0147] The pH of the collagen aqueous solution may be 3.0 or higher, 3.5 or higher, 4.0 or higher, or 5.5 or higher, and may be 7.5 or lower, 7.0 or lower, less than 7.0, 6.5 or lower, 6.0 or lower, 5.5 or lower, 5.0 or lower, or 4.5 or lower. When the pH of the collagen aqueous solution is within the above range, the collagen structure according to the second embodiment is more easily formed. Furthermore, from the viewpoint of making it easier to form the collagen structure according to the second embodiment, it is preferable that the pH of the collagen aqueous solution is 3.0 or higher and 6.0 or lower.

[0148] The volume of the collagen aqueous solution droplet (for example, the amount of collagen aqueous solution dispensed in one go) can be appropriately selected depending on the volume of the polymer aqueous solution, as long as the collagen aqueous solution droplet is coated with the polymer aqueous solution. The volume of the collagen aqueous solution droplet may be, for example, 1 μL or more, 5 μL or more, 10 μL or more, or 50 μL or more, and may also be 1000 μL or less, 900 μL or less, 800 μL or less, 700 μL or less, 600 μL or less, or 500 μL or less. From the viewpoint of making it easier to form the collagen structure according to the second embodiment, the volume of the collagen aqueous solution droplet may be 1 μL or more and 100 μL or less.

[0149] The ratio of the volume of the polymer aqueous solution to the volume of the collagen aqueous solution droplet (volume of polymer aqueous solution / volume of collagen aqueous solution droplet) is such that the collagen aqueous solution droplet is coated with the polymer aqueous solution (i.e., the collagen aqueous solution droplet can be formed in the polymer aqueous solution). For example, it may be 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 45 or more, or 50 or more, and may be 1000 or less, 900 or less, 800 or less, 700 or less, 600 or less, 500 or less, 400 or less, 300 or less, 200 or less, or 100 or less. Furthermore, from the viewpoint of making it easier to form the collagen structure according to the second embodiment, the ratio of the volume of the polymer aqueous solution to the volume of the collagen aqueous solution droplet is preferably 5 or more.

[0150] The collagen aqueous solution may also be an aqueous solution containing a biocompatible material other than collagen. The biocompatible material is one that does not adversely affect cell growth and does not hinder the formation of the collagen structure according to the second embodiment. Specifically, for example, it may be an extracellular matrix component other than collagen. The specific form of the extracellular matrix component is as described above.

[0151] The collagen aqueous solution may further contain cells. That is, the collagen aqueous solution may be a cell suspension containing at least an aqueous medium in which collagen is dissolved and cells.

[0152] 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.

[0153] 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)), and cancer cells such as hepatocytes. The cells may be used individually or in combination of multiple types.

[0154] The number of cells in the collagen aqueous solution can be appropriately selected depending on the type of cell, the intended use of the collagen structure according to the second embodiment, etc. The cell density in the collagen aqueous solution can be, for example, 1 to 10 ８ cells / mL may be 10 ３ ~10 ７ cells / mL may be 10 ４ ~10 ６ The cell count may be cells / mL. When the number of cells in the collagen aqueous solution is within the above range, it is easier to form collagen structures that have a hollow structure inside, and furthermore, compared to collagen structures that do not have a hollow structure inside, it is easier to form collagen structures that are less prone to oxygen deficiency when cells are cultured inside the collagen and that allow cells to move freely.

[0155] The collagen aqueous solution may contain oil used in cell culture. Examples of oils used in cell culture include edible oils (e.g., vegetable oils such as salad oil, soybean oil, rapeseed oil (canola oil), sesame oil, olive oil, sunflower oil, corn oil, rice oil, and palm oil; animal oils such as lard, beef tallow, and fish oil), and fatty acids (e.g., oleic acid, palmitic acid, stearic acid, linoleic acid, alpha-linolenic acid, arachidonic acid, docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA)). When the collagen aqueous solution contains oil used in cell culture, the collagen structure formed according to the second embodiment may contain the oil (oil droplets) within its hollow structure. Cells may be encapsulated within the oil (oil droplets) contained within the hollow structure of the collagen structure according to the second embodiment.

[0156] The oil content in the collagen aqueous solution may be, for example, 0.1% by mass or more, 0.5% by mass or more, 1% by mass or more, 5% by mass or more, or 10% by mass or more, based on the total amount of the collagen aqueous solution, or it may be 30% by mass or less, 25% by mass or less, 20% by mass or less, 15% by mass or less, 10% by mass or less, 5% by mass or less, or 1% by mass or less.

[0157] <Settling Process> In the setting process, after the droplet formation process, the mixture is allowed to stand for a certain period of time to allow collagen molecules to assemble at the interface between the droplets of the collagen aqueous solution and the polymer aqueous solution. This forms a collagen structure having a hollow structure filled with an aqueous medium inside. The shape of the collagen structure obtained by the manufacturing method according to the second embodiment is droplet-shaped, and can also be described as spherical, approximately spherical, teardrop-shaped, ellipsoidal, approximately ellipsoidal, hemispherical, or approximately hemispherical.

[0158] During the standing process, at the interface in the liquid where the collagen aqueous solution and the polymer aqueous solution have separated into liquid-liquid phases, the collagen and the anionic polymer interact, and the anions of the anionic polymer (e.g., -COO) － (etc.) and collagen cation (-NH ３ ＋A complex (polyion complex) is rapidly formed through electrostatic interaction with the above interface, and a film is formed at the interface. In this film, the anionic polymer removes water (water of hydration) adsorbed on the collagen molecules, thereby strengthening the hydrophobic interactions of the collagen molecules themselves (for example, it is thought that the hydrophobic interactions of the collagen molecules themselves are strengthened by standing at a temperature of 20°C to 40°C). It is thought that the collagen molecules in the collagen aqueous solution gather towards the interface with the polymer aqueous solution, and a hollow structure is formed inside, but the mechanism is not limited to this.

[0159] The standing time in the standing process may be, for example, 1 second or more, 5 seconds or more, 10 seconds or more, 15 seconds or more, 20 seconds or more, 25 seconds or more, 30 seconds or more, 35 seconds or more, 40 seconds or more, 45 seconds or more, 50 seconds or more, 55 seconds or more, or 60 seconds or more. Alternatively, the standing time in the standing process may be, for example, 24 hours or less, 12 hours or less, 6 hours or less, 1 hour or less, 50 minutes or less, 40 minutes or less, 30 minutes or less, 20 minutes or less, 10 minutes or less, 5 minutes or less, or 1 minute or less. If the collagen aqueous solution contains cells, it is preferable that the standing time be between 1 second and 15 minutes from the viewpoint of improving cell viability. The hardness of the structure or the film thickness described later can be changed to a desired range depending on the standing time in the standing process. The standing time in the standing process may be 24 hours (overnight) or more. Since the hardness of the structure or the film thickness described later reaches a plateau in a certain amount of time, it may be left to stand for any amount of time after the plateau is reached.

[0160] The temperature during the standing process may be, for example, 20°C to 40°C, 25°C to 37°C, or 30°C to 37°C.

[0161] The pH conditions during the standing process may be neutral, and specifically, the pH may be between 6 and 8.

[0162] <Other steps> The manufacturing method according to the second embodiment may further include a step of mixing cells (cell mixing step) after the standing step. The cell mixing step makes it possible to obtain a collagen structure according to the second embodiment in which cells are attached to the outside. The cells are as described above.

[0163] If the collagen aqueous solution in the manufacturing method according to the second embodiment contains cells, or if the manufacturing method according to the second embodiment includes the cell mixing step, the manufacturing method according to the second embodiment may further include a step of culturing cells (culture step). The culture temperature in the culture step may be, for example, 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 6 hours or more, 6 hours to 3 weeks, 1 day to 2 weeks, or 1 week to 2 weeks. The culture medium is as described above.

[0164] The manufacturing method according to the second embodiment may further include a step of replacing the aqueous medium that fills the hollow structure in the collagen structure according to the second embodiment formed in the standing step (replacement step). The replacement step may include standing the collagen structure according to the second embodiment in a desired aqueous medium. The aqueous medium is as described above.

[0165] [Collagen-containing structure] The collagen-containing structure (collagen structure) according to the second embodiment has a hollow structure filled with an aqueous medium inside. This hollow structure is a closed hollow structure located inside the collagen structure according to the second embodiment. Furthermore, this hollow structure is a cavity where the collagen network structure or fibrous structure is substantially absent.

[0166] The collagen structure according to the second embodiment has a hollow structure inside, and compared to collagen structures without a hollow structure, it is less prone to oxygen deficiency, thus maintaining cell viability, and cells can move freely. Furthermore, it is possible to observe cell proliferation while keeping the cells alive.

[0167] The collagen structure according to the second embodiment may have cells attached to its exterior or contain cells within its hollow structure. If the collagen structure according to the second embodiment contains cells within its hollow structure, the cells may be attached to the collagen structure, not attached, or both.

[0168] The collagen structure according to the second embodiment may also contain biocompatible materials other than collagen. The biocompatible materials are as described above.

[0169] The film thickness of the collagen structure according to the second embodiment may be 1 μm or more, 5 μm or more, 10 μm or more, 20 μm or more, 30 μm or more, 40 μm or more, 45 μm or more, 50 μm or more, 60 μm or more, 70 μm or more, 80 μm or more, 90 μm or more, 100 μm or more, 200 μm or more, 300 μm or more, 400 μm or more, or 500 μm or more, and may also be 5000 μm or less, 4000 μm or less, 3000 μm or less, 2000 μm or less, 1000 μm or less, 900 μm or less, 800 μm or less, 700 μm or less, 600 μm or less, or 500 μm or less. "Film thickness" refers to the distance from the outer surface (the surface on the interface side between the polymer aqueous solution and the collagen aqueous solution) to the inner surface (the surface on the hollow structure side) in the collagen structure according to the second embodiment. In this specification, the film thickness of the collagen structure is measured by fixing the collagen structure in formalin, obtaining a cross section, and measuring and observing the said cross section.

[0170] The diameter of the hollow structure of the collagen structure according to the second embodiment may be, for example, 10 μm or more, 30 μm or more, 50 μm or more, 100 μm or more, 200 μm or more, 300 μm or more, 400 μm or more, 500 μm or more, 600 μm or more, 700 μm or more, 800 μm or more, 900 μm or more, 1000 μm or more, 1500 μm or more, 2000 μm or more, 2500 μm or more, or 3000 μm or more, and may be 5 mm or less, 4 mm or less, 3 mm or less, 2 mm or less, 1.5 mm or less, or 1 mm or less.

[0171] In the collagen structure according to the second embodiment, the volume of the hollow structure is, for example, 0.1 mm ３ or more, 0.2 mm ３ or more, 0.3 mm ３ or more, 0.4 mm ３ or more, 0.5 mm ３ or more, 0.6 mm ３ or more, 0.7 mm ３ or more, 0.8 mm ３ or more, 0.9 mm ３ or more, or 1 mm ３ or more, and may be 10 mm ３ or less, 9 mm ３ or less, 8 mm ３ or less, 7 mm ３ or less, 6 mm ３ or less, or 5 mm ３ or less.

[0172] The thickness of the collagen structure according to the second embodiment may be, for example, 10 μm or more, 15 μm or more, 30 μm or more, 50 μm or more, 100 μm or more, 300 μm or more, or 1000 μm or more, and may be 10 mm or less, 5 mm or less, 3 mm or less, 2 mm or less, 1.5 mm or less, or 1 mm or less.

[0173] The permeability of the collagen structure according to the second embodiment to a 5 mM HEPES solution containing 0.5 mg / mL of FITC-dextran (molecular weight 4k) is 1.0×10 －７ cm / s or less, preferably 0.6×10 －７ cm / s or less. The permeability of the collagen structure according to the second embodiment to a 5 mM HEPES solution containing 0.5 mg / mL of FITC-dextran (molecular weight 2000k) is 2.0×10 －７ cm / s or less, preferably 1.2×10 －７ cm / s or less. The permeability of the collagen structure according to the second embodiment to a 5 mM PBS solution containing 0.5 mg / mL of FITC-dextran (molecular weight 4k) is 10×10 －７ cm / s or less, preferably 9.0×10 －７It may be 0 cm / s or less. The collagen structure according to the second embodiment has a permeability of 2.0×10 －７ cm / s or less for a 5 mM PBS solution containing 0.5 mg / mL of FITC-dextran (molecular weight 2000k).

[0174] The collagen structure according to the second embodiment has (1) a permeability of 1.0×10 －７ cm / s or less for a 5 mM HEPES solution containing 0.5 mg / mL of FITC-dextran (molecular weight 4k); (1') the permeability of (1) above is 0.6×10 －７ cm / s or less; (2) a permeability of 2.0×10 －７ cm / s or less for a 5 mM HEPES solution containing 0.5 mg / mL of FITC-dextran (molecular weight 2000k); (2') the permeability of (2) above is 1.2×10 －７ cm / s or less; (3) a permeability of 10×10 －７ cm / s or less for a 5 mM PBS solution containing 0.5 mg / mL of FITC-dextran (molecular weight 4k); (3') the permeability of (3) above is 9.0×10 －７ cm / s or less; (4) a permeability of 2.0×10 －７ cm / s or less for a 5 mM PBS solution containing 0.5 mg / mL of FITC-dextran (molecular weight 2000k); and may satisfy at least one characteristic selected from the group consisting of these. Here, (1') is a preferred embodiment of (1), (2') is a preferred embodiment of (2), and (3') is a preferred embodiment of (3). The collagen-containing anisotropic fibers of the present invention may satisfy any combination of two or more characteristics selected from the group consisting of (1), (1'), (2), (2'), (3), (3'), and (4) above. This description clarifies that any combination of these characteristics (for example, the combination of (1') and (2), the combination of (1) and (3'), the combination of (1'), (2'), and (3), etc.) is also intended as an individually disclosed embodiment.

[0175] In the collagen structure according to the second embodiment, the ratio of the permeability of a 5 mM HEPES solution containing 0.5 mg / mL rhodamine-dextran (molecular weight 2000 k) to the permeability of a 5 mM HEPES solution containing 0.5 mg / mL FITC-dextran (molecular weight 4 k) may be, for example, 6.5 or more, 7.0 or more, or 7.5 or more.

[0176] In the collagen structure according to the second embodiment, the ratio of the permeability of a PBS solution containing 0.5 mg / mL rhodamine-dextran (molecular weight 2000 k) to the permeability of a PBS solution containing 0.5 mg / mL FITC-dextran (molecular weight 4 may be, for example, 8.0 or more, 8.5 or more, 9.0 or more, 9.5 or more, 10.0 or more, 10.5 or more, 11.0 or more, 11.5 or more, 12.0 or more, or 12.5 or more.

[0177] Here, "thickness of the collagen structure" means the diameter of the collagen structure if it is spherical or nearly spherical. If the collagen structure is teardrop-shaped, the thickness of the collagen structure means the major axis of the collagen structure. If the collagen structure is ellipsoidal or nearly ellipsoidal, the thickness of the collagen structure means the minor axis of the collagen structure. If the collagen structure is hemispherical or nearly hemispherical, the thickness of the collagen structure means the height of the collagen structure (the distance perpendicular to the base (ground surface)). If the collagen structure is nearly spherical, nearly ellipsoidal, or nearly hemispherical and has irregularities on its surface, the thickness of the collagen structure means the shortest distance between two points where a line passing through the centroid of the collagen structure intersects with the surface.

[0178] [Composition] The composition according to the second embodiment comprises a collagen structure according to the second embodiment and an aqueous medium. Here, the collagen structure according to the second embodiment is suspended in the aqueous medium. The collagen structure and aqueous medium according to the second embodiment are as described above.

[0179] The number of collagen structures of the second embodiment in the composition of the second embodiment may be, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 45 or more, or 50 or more, and may also be 500 or less, 400 or less, 300 or less, 200 or less, or 100 or less.

[0180] The density of the collagen structure according to the second embodiment in the composition according to the second embodiment may be, for example, 1 or more units / 100 μL, 1 or more units / 1 mL, or 1 or more units / 10 mL, or 10 or fewer units / 100 μL, 5 or fewer units / 1 mL, or 20 or fewer units / 10 mL.

[0181] The present invention will be described more specifically below based on examples. However, the present invention is not limited to the following examples.

[0182] [Test Example 1: Fabrication of Anisotropic Fibers] Table 1 shows the polymers used in Test Examples 1-1 to 1-16.

[0183] [Test Example 1-1] 100 μL of a polymer aqueous solution, in which the concentration of polyacrylic acid was adjusted to 3 mg / mL using PBS, was added to a microtube, and 500 μL of a collagen aqueous solution, in which the concentration of bovine skin-derived collagen was adjusted to 3 mg / mL, was gently dropped onto it. The interface between the polymer aqueous solution and the collagen aqueous solution was scooped up with tweezers and gently lifted. Images of the spun fibers were taken with a phase-contrast microscope, and their length was measured with a ruler. The results are shown in Figures 1(A) and (B). The polymer aqueous solution was colored with food coloring.

[0184] Figure 1(A) is a photograph showing the phase separation of the collagen aqueous solution and the polymer aqueous solution. Figure 1(B) is a photograph showing the results of observation of the spun fibers. As shown in Figures 1(A) and (B), it was confirmed that fibers that can be grasped with tweezers can be formed at the interface between the collagen aqueous solution and the polyacrylic acid solution. The length of the spun collagen fibers was approximately 14 centimeters.

[0185] Figure 2 is a photograph showing the results of a phase-contrast microscope observation of spun fibers. As shown in Figure 2, it became clear that the spun collagen fibers were aligned in one direction.

[0186] [Test Example 1-2] Polymer aqueous solutions (pH 3) were obtained by adjusting the polymers shown in Table 2 to 3 mg / mL with 5 mM acetic acid. 100 μL of each polymer aqueous solution was added to a microtube, and 500 μL of collagen aqueous solution (pH 3), adjusted to a concentration of 3 mg / mL, was gently added dropwise. The interface between the polymer-containing phase and the collagen-containing phase was scooped up with tweezers, gently lifted, and evaluated whether fibers could be spun into the gas phase. The results of spinning feasibility and the length of the spun fibers are shown in Tables 2 and 3. In Tables 2 and 3, "-" is written if fibers could not be spun into the gas phase, and the length of the fibers is written if fibers could be spun into the gas phase.

[0187]

[0188]

[0189] When using neutral polymers and cationic polymers, it was not possible to spin fibers into the gas phase. However, when using specific anionic polymers (Hep, CSA, HA, DexS, PSSA, Cag, Alg, and PAA), it was possible to spin fibers into the gas phase.

[0190] [Test Example 1-3] 500 μL of a polymer aqueous solution (pH 7) with a polymer concentration of 3 mg / mL adjusted using PBS was added to a microtube and mixed with a collagen aqueous solution (pH 3) with a collagen concentration of 3 mg / mL. Fiber formation was evaluated under a microscope. The evaluation results are shown in Figure 3.

[0191] We defined "-" as indicating no observable fiber formation, "+" as indicating fiber formation but with a length of less than 1 mm based on image evaluation, and "++" as indicating fiber formation and a length of 1 mm or more. The results are shown in Table 4.

[0192]

[0193] It was confirmed that collagen fibrillation is possible even when the pH of the polymer aqueous solution is changed to 7 by using specific anionic polymers (Hep, CSA, CSC, CSE, DexS, PSSA, Cag, Alg, and PAA).

[0194] [Test Example 1-4: Analysis of Fibers Using Fluorescent Collagen and Fluorescent PAA] Using a collagen aqueous solution containing 1 mg / mL of FITC-collagen (K21 / Collagen Technology Training), a polymer aqueous solution (PBS aqueous solution) containing 3 mg / mL of FITC-PAA (PAA-FC-1 / NANOCS), a collagen aqueous solution containing collagen at a concentration of 3 mg / mL, and a polymer aqueous solution (PBS solution) containing PAA at a concentration of 3 mg / mL, fibers were spun by pulling up the interface in the same manner as in Test Example 1. After washing with ultrapure water, the fibers were observed using a fluorescence microscope. Figure 4 shows fluorescence microscope images of fibers formed with fluorescent collagen or fluorescent PAA.

[0195] When FITC-PAA was used, no fluorescence signal was observed within the formed fibers. On the other hand, when FITC-collagen was used, a strong fluorescence signal was detected from the formed fibers. Therefore, the fibers produced by spinning were derived solely from collagen, and no residual PAA was observed.

[0196] [Test Example 1-5: Molecular Weight of Polymer] PAA with molecular weights of 5 k, 25 k, or 400 k was adjusted with PBS to a concentration of 3 mg / mL to prepare an aqueous polymer solution. Collagen fibers were spun using this aqueous polymer solution and an aqueous collagen solution prepared by adjusting bovine skin-derived collagen type I to a concentration of 3 mg / mL. The length of the obtained collagen fibers was measured with a ruler. The results are shown in Table 5.

[0197]

[0198] Lowering the molecular weight of the polymer resulted in longer fibers. It was shown that lower polymer molecular weight is associated with higher spinnability.

[0199] [Test Example 1-6: Concentration of Polymer Aqueous Solution] A polymer aqueous solution (pH 7) was prepared by adjusting the concentration of PAA with a molecular weight of 25 k to 30 mg / mL using PBS. Fiber spinning was performed using this polymer aqueous solution and a collagen aqueous solution prepared by adjusting the concentration of bovine skin-derived collagen type I to 3 mg / mL. The length of the obtained fibers was measured with a ruler. The results of measuring the length of the fibers (n=3) showed that the length of the fibers produced under the condition of a PAA concentration of 30 mg / mL was 100 mm. It was confirmed that fiber spinning is possible even when the concentration of PAA is changed.

[0200] [Test Example 1-7: Types of Collagen] Fibers were spun using bovine skin-derived collagen type I, type III, bovine cartilage-derived collagen type II, fish-derived collagen type I, bovine tendon-derived collagen type I, and bovine skin-derived tropocollagen (collagen that retains the telopeptide of the collagen molecule due to differences in extraction method) in a PAA aqueous solution (PBS aqueous solution) with a molecular weight of 25 k and a PAA concentration of 3 mg / mL. The length of the obtained fibers was measured with a ruler. The results are shown in Table 6. The unit of length in Table 6 is "mm".

[0201]

[0202] We confirmed that collagen fibers can be produced even when the type of collagen and the animal from which it originates are changed. It was shown that when the collagen is atelocollagen, the spinnability is even higher compared to tropocollagen. It was also shown that when the collagen is tendon-derived, the spinnability is even higher.

[0203] [Test Example 1-8: pH of Polymer Aqueous Solution] A polymer aqueous solution was prepared by adjusting PAA with a molecular weight of 25 k to a concentration of 3 mg / mL using PBS. The pH of this aqueous solution was adjusted using 5 N sodium hydroxide or 1 N hydrochloric acid, and collagen fibers were spun using collagen containing 3 mg / mL of bovine skin-derived collagen type I. The length of the obtained fibers was measured with a ruler. The results are shown in Table 7. The unit of length in Table 7 is "mm".

[0204]

[0205] It was confirmed that anisotropic fibers containing collagen can be produced when the pH of the polymer aqueous solution is varied within the range of 2 to less than 7. Longer fibers were formed when the pH of the polymer aqueous solution was between 3 and 4.

[0206] [Test Example 1-9: Production of anisotropic fibers using a polymer aqueous solution containing Mw 25k or 5k PAA at pH 3 or 7 and a collagen aqueous solution at pH 3] The length of fibers spun using a collagen aqueous solution at pH 3 with a polymer aqueous solution containing Mw 25k or 5k PAA (concentration 3 mg / mL or 30 mg / mL) was measured. The pH of the polyacrylic acid was adjusted with 0.05N NaOH. The results are shown in Table 8.

[0207] It was confirmed that anisotropic fibers containing collagen can also be produced by combining a polymer aqueous solution containing Mw25k or 5k PAA with a collagen aqueous solution at pH 3.

[0208] [Test Example 1-10: Production of anisotropic fibers using a polymer aqueous solution containing Mw 25k or 5k PAA at pH 3 and a collagen aqueous solution at pH 6] The length of fibers spun using a collagen aqueous solution at pH 6 was measured with a polymer aqueous solution (concentration 30 mg / mL) containing Mw 25k or 5k PAA. The pH of the polyacrylic acid was adjusted with 0.05N NaOH, and the pH of the collagen was adjusted to pH 6 by dissolving freeze-dried collagen (pH 3) in PBS. The results are shown in Table 9.

[0209]

[0210] It was confirmed that anisotropic fibers containing collagen can also be produced by combining a polymer aqueous solution containing Mw25k or 5k PAA with a collagen aqueous solution at pH 6.

[0211] [Test Example 1-11: Production of anisotropic fibers using a polymer aqueous solution containing Mw5k PAA with a pH of 4-7 and a collagen aqueous solution with a pH of 4 or 5] The length of fibers spun with collagen (pH 4 or 5) was measured for polymer aqueous solutions containing Mw5k PAA (30 mg / mL, pH 4, 5, 7). The pH of the polyacrylic acid was adjusted with 0.05N NaOH, and for the pH of the collagen side, for pH 4 and 5, pH 3 collagen was adjusted with 0.05N NaOH. The results are shown in Table 10. In the table, "◎" indicates that the spun fibers were 10 cm or longer, and "〇" indicates that fibers were spun, but the length of the fibers was less than 10 cm.

[0212]

[0213] It was confirmed that anisotropic fibers containing collagen can be produced by combining a polymer aqueous solution containing Mw5k PAA with a pH of 4-7 and a collagen aqueous solution with a pH of 4 or 5.

[0214] [Test Example 1-12: Optimization of Pulling Speed ​​and Length] Fiber spinning was performed using a polymer aqueous solution (PBS aqueous solution) containing PAA at Mw 25k and a concentration of 3 mg / mL, and a collagen aqueous solution containing 3 mg / mL of bovine tendon-derived collagen type I. The speed at which the yarn was lifted by hand was varied. The spinning time was measured from the time the tweezers were placed in the solution until the interface was lifted and the spinning collagen broke. The length of the spun fiber was measured with a ruler. The pulling speed was calculated as the quotient of the length of the spun fiber and the spinning time, and the relationship with the length was plotted. The results are shown in Figure 5.

[0215] The overall trend revealed that the faster the spinning speed, the shorter the spun fiber. The figure confirms that a spinning speed of 11 mm / sec or less is required to spin fibers longer than 10 cm.

[0216] [Test Example 1-13: Optimization of Collagen Concentration and Length] A 3 mg / mL collagen aqueous solution (pH 4) was diluted to prepare 0.5 or 1 mg / mL collagen aqueous solutions. High-concentration collagen aqueous solutions were obtained by dissolving freeze-dried collagen after dialysis in 5 mM acetic acid at predetermined concentrations (5, 10, or 20 mg / mL). Using a polymer aqueous solution (PBS aqueous solution; pH 4) containing 30 mg / mL PAA (MW 5000), fibers were spun by pulling up the interface between the polymer aqueous solution and the collagen aqueous solution using the same method as in Test Example 1-1, and their lengths were measured. The results are shown in Figure 6.

[0217] As shown in Figure 6, fibers could be spun under all collagen concentration conditions, and particularly long fibers could be spun under collagen concentration conditions of 1 to 10 mg / mL.

[0218] [Test Example 1-14: Investigation of the Dependence of Interface Formation Time on Collagen Concentration] 100 μL of collagen aqueous solutions at concentrations of 0, 0.02, 0.09, 0.19, 0.38, 0.75, 1.5, 3, 5, or 10 mg / mL, prepared using a 3 mg / mL collagen aqueous solution (pH 4), were added to a 96-well plate, and a PBS solution (pH 4) containing 30 mg / mL PAA (Mw 5000) was gently overlaid. Immediately thereafter, the absorbance at 600 nm was measured over time every minute using a plate reader. The absorbance value reflects the amount of collagen fibers (turbidity) formed at the interface. The results are shown in Figures 7 and 8.

[0219] As shown in Figure 7, the rate of increase in absorbance and the final absorbance were strongly dependent on the collagen concentration. In particular, the higher the collagen concentration, the higher the absorbance reached. Furthermore, at collagen concentrations of 0.38 mg / mL or higher, the absorbance increased significantly from immediately after the start of measurement (0 minutes). This suggests that in these collagen concentration ranges, rapid fibrillation begins simultaneously with interface formation. Thus, it is suggested that efficient fibrillation progresses when the collagen concentration exceeds a certain threshold (e.g., 0.3 mg / mL or higher).

[0220] As shown in Figure 8, a clear positive correlation was observed between collagen concentration and absorbance at 180 minutes. In other words, it was shown that the higher the collagen concentration, the greater the amount (thickness) of collagen fibers that were ultimately formed.

[0221] [Test Example 1-15: Viscoelasticity Measurement] Using a 3 mg / mL collagen solution (pH 4) and a PBS solution (pH 4) containing 30 mg / mL PAA (MW5000), fibers were spun in the same manner as in Test Example 1-1, and strain-dependence measurements were performed at a frequency of f = 1 Hz. As a comparative example, according to the reference (Kim J et al., “Fabrication of fully aligned self-assembled cell-laden collagen filaments for tissue engineering via a hybrid bioprinting process.”, Bioact Mater. 2024 Feb 21;36:14-29.), a PBS solution containing 20 mg / mL collagen was dispensed into a 50 wt% PEG (MW6000) aqueous solution using a syringe with a 25 G (φ: 260 μm) blunt needle to produce fibers. After incubation at 37°C for 2 hours, the fibers were removed from the PEG solution, washed with ultrapure water, and subjected to the same viscoelasticity test. The results are shown in Figure 9. In Figure 9, the results for fibers manufactured using the PAA method are indicated as "PAA," and the results for fibers manufactured using the comparative example method are indicated as "Comparative Example." Furthermore, the loss modulus is indicated as "G''" and the storage modulus as "G'." The specific test methods were carried out according to the procedure described above.

[0222] Furthermore, Figure 10 shows the storage modulus and modulus crossover point at a strain of 0.1% for fibers produced using the PAA method and the comparative method, as bar graphs.

[0223] As shown in Figures 9-10, the storage modulus (G') was significantly higher in the comparative example, but the modulus cross-point was significantly higher in the present method (the method using PAA). The fibers produced by the comparative example method exhibited very high storage modulus of approximately 2250 Pa at low strain, i.e., when the strain was 0.1%, showing a hard characteristic. As the strain increased from 0.1% to 10%, the storage modulus decreased rapidly. At approximately 23% strain, the storage modulus (G') and loss modulus (G'') were almost identical, indicating a crossover. On the other hand, in the case of fibers manufactured using the PAA method, the storage modulus was approximately 150 Pa at 0.1% strain, indicating that it was softer than the comparative example. Furthermore, even when the strain was increased from 0.1% to 10%, the storage modulus of the fibers manufactured using PAA hardly decreased, maintaining approximately 150 Pa. The crossover point was at approximately 200% strain. These results suggest that this method produces a gel-like material that is softer but more resilient than the comparative example.

[0224] [Test Example 1-16: Permeability Test] <Sample Preparation> PAA (Example): 100 μL of 3 mg / mL collagen aqueous solution (pH 4) was added to a culture insert (Corning / #3470), and 1 mL of PBS solution (pH 4) containing 30 mg / mL PAA was added to a 24-well plate to serve as a reservoir, bringing the collagen and PAA solution into contact via a membrane. The mixture was incubated overnight at 4°C.

[0225] Gel (Comparative Example): 100 μL of 3 mg / mL collagen aqueous solution was added to the culture insert, and 25 μL of a neutralization solution prepared by mixing 25 mM HEPES and 0.05 M NaOH in a 1:1 ratio was added and mixed thoroughly. 1 mL of PBS was added to a 24-well plate to serve as the reservoir, and the mixture was incubated overnight at 37°C.

[0226] Control: 300 μL and 2 mL of 5 mM HEPES or PBS were added to the culture insert and reservoir, respectively, and the membranes were incubated overnight at 4°C to equilibrate.

[0227] <Permeability Test> FITC-dextran (Sigma-aldrich / 46944, molecular weight 4k) and Rhodamine-dextran (Creative PEG works / DE-616, molecular weight 2000k) were dissolved in PBS and 5 mM HEPES, respectively, to a concentration of 0.5 mg / mL. The membranes and gels on the culture insert were washed twice with ultrapure water, aspirated, and then PBS or HEPES was added to the reservoir. Fluorescent dextran solution in the same solvent as the reservoir was added to the membranes and gels, and 50 μL was sampled from the reservoir at 1, 5, 15, 30, 45, 60, 75, 90, 105, 120, and 180 minutes. 50 μL of each solvent was added to the reservoir each time a sample was taken.

[0228] The apparent transmittance was calculated from the calibration curve using the following formula (1).

[0229] In this experiment, the same amount of buffer solution was replenished as the sampled amount. Therefore, the true cumulative permeation amount (mg) at each sampling time point was corrected using the following formula (2).

[0230] In the above equations (1) and (2), each letter represents the following: A: Membrane area (0.33 cm²) ２ ) C ０ : Initial concentration on the donor side (0.5 mg / mL) dQ / dt: Slope (permeation rate, mg / s) in the steady state (linear portion) when the cumulative permeation amount Q (mg) on ​​the reservoir side is plotted against time t (seconds) Q ｎ : Corrected cumulative permeation amount (mg) at time n C ｎ : Measured concentration (mg / mL) on the reservoir side at time point n V Ｒ Total liquid volume on the reservoir side (1.0 mL) V Ｓ : Sampling and replenishment volume (0.05 mL) Σ C ｉ : The sum of measured concentrations (mg / mL) from time point 0 to the most recent sampling time point (n-1).

[0231] The results are shown in Figure 11.

[0232] Regardless of the molecular weight of dextran, collagen membranes prepared using this method showed lower substance permeability compared to conventional collagen gels. However, this permeability increased in the presence of salt (PBS), resulting in leakage.

[0233] Specifically, the apparent permeability coefficient in HEPES for a marker with a molecular weight of 4 kDa in a collagen composition prepared with PAA is 0.6 × 10⁻⁶. －７ While the control is approximately 40 x 10⁻⁶ cm / s, the control is approximately 40 x 10⁻⁶. －７ cm / s, collagen gel is approximately 28 x 10 －７ The value is cm / s, which exceeds this specified value. In the case of PBS, the apparent permeability coefficient for a marker with a molecular weight of 4 kDa in a collagen composition prepared with PAA is approximately 9.0 × 10⁻⁶. －７ While the control is below cm / s, the control is approximately 52 x 10 －７ cm / s, collagen gel is approximately 25 x 10 －７ The value was cm / s. The apparent permeability coefficient in HEPES for a marker with a molecular weight of 2000 kDa in the collagen composition prepared with PAA was approximately 1.2 × 10⁻⁶. －７ While the control is approximately 9.0 x 10⁻⁶ cm / s, the control is approximately 9.0 x 10⁻⁶. －７ At cm / s, the collagen gel is approximately 5.0 x 10 －７ The value is cm / s, which exceeds this specified value. In the case of PBS, the apparent permeability coefficient for a marker with a molecular weight of 2000 kDa in a collagen composition prepared with PAA is approximately 2.0 × 10⁻⁶. －７ While the rate is cm / s, the control is approximately 7 x 10 －７ cm / s, collagen gel is approximately 5 x 10 －７The pressure was cm / s. Furthermore, when examining the ratio of permeability that the collagen composition prepared with PAA can distinguish between small molecules (4k) and large molecules (2000k), the ratio of permeability of a molecular weight 4kDa marker to that of a molecular weight 2000kDa marker (Papp(4k) / Papp(2000k)) in PBS solution was 12.5. Since the ratios for collagen gel and control were 7.8 and 3.9, respectively, it is considered that the collagen composition prepared with PAA has higher selectivity. In addition, the ratio of permeability of the same marker in HEPES solution (Papp(4k) / Papp(2000k)) was 7.5 for the collagen composition prepared with PAA, compared to 4.55 for collagen gel and 5.67 for control. This result indicates that, regardless of the solution system used, the collagen composition prepared with PAA has a superior ability to distinguish differences in molecular size compared to the control and conventional collagen gel.

[0234] [Test Example 2: Preparation of Anisotropic Fibers (Fibrous Tissues) Containing Cells] Materials Used The main materials used in Test Examples 2-1 to 2-11 are shown in Tables 11 and 12 below.

[0235]

[0236] Evaluation Method <Live-dead staining> LIVE / DEAD ＴＭ 2.5 μL of Calcein, 10 μL of Ethidium Homodimmer (EthD-1), and 5 μL of Hoechst from the Viability / Cytotoxicity Kit, for mammalian cells were added to 5 mL of phenol red-free medium containing 10% FBS. The resulting medium was incubated at 37°C for 3 hours. After incubation, washing with PBS was performed. The specimens were spread onto glass slides, PBS was dropped on to prevent drying, and the specimens were observed while lightly pressing with a coverslip. The viability rate was calculated using the following formula: Viability rate (%) = (1 - (Number of EthD-1 positive cells / Number of Hoechst positive cells)) × 100

[0237] <ATP Assay> Fibrous tissue (fibers) were cut into 10 mm lengths using scissors. Each 10 mm length was used as a sample, and 100 μL of culture medium was added to 96 wells. CellTiter-Glo® 3D Cell Viability Assay Working Solution was dissolved at room temperature and added to each well containing the sample in 100 μL. The sample was pipetted to break down the general structure of the tissue. Then, it was homogenized using a plate shaker at 1000 rpm for 10 minutes. After homogenization, the sample was incubated at room temperature for 30 minutes. After incubation, the luminescence was measured using a microplate reader.

[0238] <Immunostaining> The culture medium of the fibrous tissue was aspirated, and the fibrous tissue was washed twice with PBS. With the fibrous tissue extended as much as possible, 4% paraformaldehyde was added and fixed for 3 hours. The fixed samples were washed twice with PBS and blocked for 1 hour with a 10-fold diluted blocking reagent containing 10% Normal Goat Serum (0.2% TritonX). Primary antibodies (CD31: 1000-fold, MYH4: 400-fold, COL1: 1000-fold), diluted to the desired concentrations using the blocking reagent, were reacted with the samples overnight at 4°C. After washing twice with PBS, secondary antibodies diluted 200-fold, 400-fold diluted Phalloidin, and 1000-fold diluted DAPI were added to the samples using the blocking reagent and reacted with the samples overnight at 4°C. After washing three times with PBS, 1 mL of RapiClear 1.47 was added, and the mixture was inverted and mixed overnight at room temperature in the dark. RapiClear CS Mounting gel was dissolved at 75°C and poured into the mold together with the sample to embed the sample.

[0239] Test Example 2-1. Examination of the possibility of fibrous tissue formation <Experiment 2-1-1> 5.0 × 10 ５Bovine satellite cells (bSCs) were mixed with 500 μL of a collagen aqueous solution containing bovine tendon-derived collagen at a concentration of 3 mg / mL to obtain a cell-containing collagen aqueous solution. 100 μL of a polymer aqueous solution, in which polyacrylic acid (MW: 25k) was adjusted to a concentration of 3 mg / mL using PBS, was added to a microtube, and 500 μL of the cell-containing collagen aqueous solution was gently dropped onto it. The interface between the polyacrylic acid-containing phase and the collagen-containing phase was quickly scooped up with tweezers and gently lifted to obtain a fibrous tissue.

[0240] 3 mL of 20% serum-containing DMEM (p38i, bFGF) at 37°C was added to a 6-well plate, and cell-containing fibers were immersed and cultured. After 7 days of culture, the fibrous tissue was cultured for 3 days in differentiation medium (2% serum-containing DMEM), and then the fibrous tissue was fixed.

[0241] Figure 12 shows images of microscopic observation results of fibrous tissue. It was observed that bSCs extended within collagen fibers from Day 1 and increased in number as the culture progressed. The post-differentiation photograph shows that they are arranged uniaxially, forming muscular tissue.

[0242] <Experiment 2-1-2> 1.0 × 10 ６ Bovine satellite cells (bSCs) were mixed with 1000 μL of a collagen aqueous solution containing bovine tendon-derived collagen at a concentration of 3 mg / mL.

[0243] A polymer aqueous solution containing polyacrylic acid (MW: 5k) at a concentration of 3 mg / mL was prepared using PBS.

[0244] 100 μL of polymer aqueous solution was added to a microtube, and 500 μL of cell-containing collagen aqueous solution was gently dropped onto it. The interface between the polymer aqueous solution and the cell-containing collagen aqueous solution was quickly scooped up with tweezers and gently lifted. This created a fibrous tissue containing cells and oriented collagen fibers.

[0245] Three mL of 20% serum-containing DMEM (p38i, bFGF) at 37°C was added to a 6-well plate (BM#2 medium), and fibrous tissue samples were immersed in the DMEM and cultured. After 7 days of culture, the tissues were cultured for 3 days in differentiation medium (2% serum-containing DMEM) to fix them.

[0246] Figure 16 shows a phase-contrast microscope image of the fibrous tissue. The results shown in Figure 16 reveal that cells are encapsulated within collagen fibers by raising the interface between the cell-containing collagen aqueous solution (obtained by suspending bSCs in a collagen aqueous solution) and the polymer aqueous solution. When cells were contained in the polymer aqueous solution, fewer cells were encapsulated within the collagen fibers. Cells in the fibrous tissue were observed to proliferate as the culture period progressed. This suggests the possibility of cell culture within collagen fibers. Furthermore, after 5 days of culture, the cells were switched to a differentiation medium and cultured again. This resulted in observations of uniaxial fusion and differentiation of the cells. This suggests that cells spontaneously align uniaxially within the anisotropic collagen fibers, resulting in the creation of oriented muscle fibers. The fabricated fibrous tissue had a total length of approximately 5 cm. While the length of three-dimensional tissues that could be fabricated using printing methods was previously limited to approximately 2 cm, this method makes it possible to fabricate longer fibrous tissues containing more cells.

[0247] <Experiment 2-1-3> The same experiment was conducted using NHDF, which is a fibroblast, instead of bSC, and the cell morphology was evaluated using immunostaining with collagen. Figure 17 shows the observed image of the prepared fibrous tissue stained with fluorescence. When the inside of the fibrous tissue was observed using actin, a cytoskeletal marker, COL1, which stains collagen itself, or DAPI, which indicates the position of the cell nucleus, it was observed that the cytoskeleton extended along the direction of the collagen fibers, and that cells were distributed even inside the fibers.

[0248] Until now, there have been few reports on the production of anisotropic collagen fibers other than by electrospinning using organic solvents. However, this method does not allow for simultaneous spinning with cells, so anisotropic cell fibers are produced by crosslinking after spinning and then attaching cells (Non-Patent Literature 1). As a result, a problem with these anisotropic cell fibers is that cells cannot penetrate into the interior and only adhere to the surface. The present invention demonstrates that cells can be distributed into the interior of uncrosslinked collagen fibers, and that cell proliferation is possible within them.

[0249] Test Example 2-2: Investigation of Molecular Weight and Content of Anionic Polymers Each polymer aqueous solution containing polyacrylic acid, prepared under conditions of molecular weight (25k or 3k) and concentration of 3mg / mL or 30mg / mL, contained 4 × 10¹⁶ of each polymer. ６ 500 μL of a cell-containing collagen aqueous solution, in which bovine satellite cells were suspended, was added to a concentration of 500 μL / mL. The interface between the collagen-containing phase and the polyacrylic acid-containing phase was lifted with tweezers to obtain a fibrous tissue.

[0250] The obtained fibrous tissue was cultured overnight in a 6-well plate. The following day, the fibrous tissue was collected in a 24-well plate, washed with PBS, and Accumax (17087-54 Nacalai Tesque) was added. After resuspending by pipetting, it was incubated at 37°C for 5 minutes. The incubated fibrous tissue was transferred to a 1.5 mL tube and centrifuged at 10,000 rpm for 1 minute, and the supernatant was removed. After removing the supernatant, the fibrous tissue was again transferred to a 24-well plate, resuspended in 2 mg / mL collagenase (037-17603 / Fujifilm Wako Pure Chemical Industries) / PBS solution, and incubated at 37°C for 5 minutes with shaking at 400 rpm. The obtained fibrous tissue was filtered through a 40 μm cell strainer, the number of cells was counted, and the viability was measured.

[0251] Figure 13 shows the effect of polymer aqueous solution conditions on cell viability. It was confirmed that changing the molecular weight of PAA did not significantly affect cell viability. It was confirmed that lowering the polymer concentration resulted in higher cell viability.

[0252] Test Example 2-3: Examination of pH conditions 1 × 10⁶ of each polymer solution containing polyacrylic acid, prepared under pH 4 or 5 conditions, were used. ６ 1000 μL of an aqueous collagen solution (pH 4) containing human-derived fibroblasts (NHDF) suspended in a solution was added to a concentration of 1 / mL. The interface between the polyacrylic acid-containing phase and the collagen-containing phase was lifted with tweezers to obtain a fibrous tissue.

[0253] The obtained fibrous tissue was cultured overnight in a 6-well plate. After one day, it was washed with PBS and then the fibrous tissue was cut into 5 mm pieces using a scalpel. For each tissue piece obtained from the cutting, the number of viable cells was calculated from the ATP level using CellTiter-Glo® 3D Cell Viability Assay in a 96-well plate.

[0254] Figure 14 shows the effect of the pH of the polymer aqueous solution on the number of living cells. It was confirmed that fibrous tissue formation is possible even when the pH of the polymer aqueous solution is changed.

[0255] Test Example 2-4: Examination of Anionic Polymer Species We evaluated whether similar fibrous tissue formation was possible using an aqueous polymer solution containing heparin or polymethacrylic acid (polymer concentration: 30 mg / mL, pH: 4) and NHDF and collagen at the same concentrations as in Experiment 3. The evaluation results are shown in Figure 15. In Figure 15, (A) shows the results when heparin was used, and (B) shows the results when polymethacrylic acid was used (fibrous tissue within the circle).

[0256] As shown in Figure 15, fibrous tissue formation was possible even when the type of polymer was changed. When polymethacrylic acid was used, thin fibers (fibrous tissue) with a fiber length of 1 to 2 cm could be spun, and when heparin was used, it was possible to produce fibers almost equivalent to those produced when polyacrylic acid was used.

[0257] Test Example 2-5: Cell Type Examination 1.0 × 10 ６bSC, human skin-derived fibroblasts (NHDF), or human umbilical vein-derived endothelial cells (HUVEC) were mixed in 1000 μL of a collagen aqueous solution containing bovine tendon-derived collagen at a concentration of 3 mg / mL. Fibrous tissue was prepared and cultured in the same manner as in Experiment 1-2 of Test Example 1. Note that bSC was cultured in BM#2 medium, NHDF in DMEM + 10% FBS medium, and HUVEC in EGM medium. ＴＭ -2MV BulletKit ＴＭ Culture was performed using [a specific method / tool].

[0258] Cell behavior within fibrous tissues prepared using NHDF, bSC, or HUVEC was evaluated. Evaluation was performed using Livedead staining, ATP Assay, and immunohistochemistry.

[0259] Figure 18 shows phase-contrast microscope images (Ph), calcein staining results, EthD-1 staining results, and cell nucleus staining results of fibrous tissue. Figure 19 shows images of the results of immunohistochemical evaluation of fibrous tissue. In immunohistochemical evaluation, NHDF shows staining results for Actin, COL1, or the nucleus; bSC shows staining results for Actin, MYH4, or the nucleus; and HUVEC shows staining results for Actin, CD31, or the nucleus. Figure 20 shows the evaluation results for fiber length, cell viability, cell number, and proliferation rate of the fibrous tissue (A, B, C, D).

[0260] At day 7 of culture, cell extension within collagen fibers was observed in all cell types. Calcein signaling revealed the presence of numerous positive cells. While some cells were positive for EthD signaling, the proportion of EthD-positive cells (percentage of dead cells) relative to the total number of nuclei remained at around 10-20%, indicating that approximately 80% of cells survived under all conditions. ATP assays showed that NHDF and bSC showed a high rate of increase, approximately 6-7 times, at day 7 (D7) of culture. HUVEC also showed proliferation, though not as pronounced as NHDF and bSC, approximately 3 times.

[0261] In fibrous tissue prepared using bSCs, co-staining with actin in the cytoskeleton revealed that most of the extending cells expressed MYH4, indicating normal differentiation.

[0262] In fibrous tissue fabricated using HUVEC, there were more CD31-positive cells than actin-expressing cells, and it was revealed that these cells proliferated without elongation.

[0263] The results above suggest that cell fibers using polyacrylic acid and collagen can be applied not only to bSCs but also to other fibroblasts and endothelial cells, along with cytoskeletal signaling and the number of viable cells.

[0264] Test Example 2-6: Cell Density Study 1.0 × 10 ６ , 3.0 x 10 ６ , or 6.0 x 10 ６ Bovine satellite cells were mixed with 1000 μL of a collagen aqueous solution containing bovine tendon-derived collagen at a concentration of 3 mg / mL to prepare a cell-containing collagen aqueous solution. Except for using this cell-containing collagen aqueous solution, the preparation and culture of fibrous tissue was carried out in the same manner as in Experiment 1-2 of Test Example 1.

[0265] Figure 21 shows phase-contrast microscopy images (Ph), calcein staining results, EthD-1 staining results, and cell nucleus staining results of fibrous tissue. Figure 22 shows images of immunohistochemical staining results for Actin, MYH4, or the nucleus of fibrous tissue. Figure 23 shows the evaluation results for fiber length, cell viability, cell number, and proliferation rate of fibrous tissue (A, B, C, D, and D).

[0266] The fiber length measurement results in Figure 23(A) show that as the initial cell concentration decreases, it becomes easier to form fibrous tissues with longer fiber lengths.

[0267] As shown in the results of the proliferation rate in Figure 23(D), 1.0 × 10 ６ , and 3.0 × 10 ６Under these conditions, the number of cells increased significantly after 7 days of culture compared to the start of culture. From the proliferation rate results in Figure 23(D), 6.0 × 10⁻⁶ ６ Under these conditions, the number of cells remained unchanged even after 7 days of culture, suggesting that there was little cell proliferation within the cells. 6.0 × 10 ６ Under these conditions, the fluorescent staining of MYH4 revealed a positive area that was more elongated in the longitudinal direction. From this result, 6.0 × 10 ６ Under these conditions, differentiation efficiency is considered to be better than under other conditions. 6.0 × 10 ６ Under these conditions, it is thought that the number of cells within the collagen fibers was already high at the time of fibrous tissue formation, leaving no space for further proliferation, thus promoting differentiation rather than cell proliferation. Furthermore, if the number of cells is too low, it takes longer to reach the cell density necessary for differentiation in muscle cells, and the cells are affected by acetic acid, the solvent for collagen, leading to decreased cell viability. Therefore, an appropriate cell count of 3.0 × 10⁻⁶ is desirable, as this facilitates the formation of fibrous tissue and maintains high cell viability. ６ / mL is considered appropriate.

[0268] Test Example 2-7: Verification of the effect of collagen solvent substitution on spinnability. A type I collagen solution with a collagen concentration of 3 mg / ml was placed in a dialysis tube and immersed in 5 L of ultrapure water, and dialysis was performed for 3 hours x 2 times. The sample after dialysis was collected in the tube and freeze-dried for approximately 18 hours. The dried sample was weighed and dispersed with lactic acid (LA): 0.63 mM or citrate (CA): 1.93 mM to a concentration of 3 mg / ml. The concentrations of each acid were specified so that the proton amount was equivalent to that of the 5 mM acetic acid used as a comparison. After dispersion, spinning was examined using the method shown in Test Example 1 Experiment 1-2, and if spinning was possible, the orientation was quantified. Also, NHDF 1.0 × 10 ６ Fibrous tissue samples were prepared using [a specific method], and the survival rate was quantified the following day using Livedead Assay.

[0269] Figure 24 shows the staining results of fibrous tissue with DAPI, Calcein (Calc), or EthD. Figure 25(A) shows the results of fiber length measurement of fibrous tissue. Lactic acid showed a significantly higher tendency to form fibers. Citric acid also tended to produce longer fibers than acetic acid.

[0270] Cell fibers were prepared using acetic acid, lactic acid, and citrate, and their viability was evaluated on the first day of culture. The results are shown in Figure 25(B). As shown in Figure 25(B), lactic acid and citrate showed significantly higher cell viability compared to acetic acid.

[0271] Test Example 2-8: Verification of Collagen and PAA Concentrations A polymer aqueous solution was prepared, colored with blue food coloring and containing PAA at a concentration of 30 mg / mL. 500 μL of the obtained polymer aqueous solution and aqueous solutions obtained by serially diluting the polymer aqueous solution with PBS were added to microtubes, and 500 μL of collagen acetate solution with a collagen concentration of 3 mg / mL was added on top, and the formation of the interface was observed.

[0272] A polymer aqueous solution colored with blue food coloring and containing PAA at a concentration of 30 mg / mL was added to a microtube in a volume of 500 μL. Then, a collagen acetate solution with a collagen concentration of 3 mg / mL was added, which was serially diluted with an aqueous acetic acid solution with an acetic acid concentration of 5 mM, and the formation of an interface was observed.

[0273] Figure 26(A) is a photograph showing the interface observation results between a collagen aqueous solution containing collagen at a concentration of 3 mg / mL (upper phase) and various polymer aqueous solutions with different PAA content (lower phase). Figure 26(B) is a photograph showing the interface observation results between various collagen aqueous solutions with different collagen content (upper phase) and polymer aqueous solutions containing PAA at a concentration of 30 mg / mL (lower phase).

[0274] Assuming a molecular weight of 300,000 for collagen and 5,000 for PAA, when using a polymer aqueous solution containing 30 mg / mL (10 mM) of PAA aqueous solution, interface formation was confirmed even when the collagen solution concentration was 0.31 μM. When calculating the molecular weights of collagen and PAA in the same manner as above, when using a collagen aqueous solution containing 3 mg / mL (10 μM) of collagen, interface formation was confirmed even when the PAA concentration was 0.31 mM. When the collagen solution concentration was less than 0.31 μM and the PAA concentration was less than 0.31 mM, no interface was formed, and it was confirmed that the collagen and PAA were mixed.

[0275] Test Example 2-9: Preparation of collagen fibers containing collagen other than type I. Fibrous tissue was prepared in the same manner as in Test Example 1 Experiment 1-2, except that type II collagen was used as the collagen and chondrocytes (ATDC5) were used as the cells.

[0276] Fibrous tissue was prepared using the same method as in Experiment 1-2 of Test Example 1, except that type III collagen was used as the collagen and HUVEC and NHDF were used as the cells.

[0277] Fibrous tissue was prepared in the same manner as in Experiment 1, Experiment 1-2, except that instead of type I collagen, a mixture of type IV collagen and type I collagen in a 1:1 (mass ratio) with 2% laminin was used, and bSCs were used as the cells.

[0278] ATDC5, HUVEC, and bSC are each 3.0 × 10⁻⁶ ６ Each cell was suspended separately. The culture conditions for each cell type are shown in the table below.

[0279] Immunostaining was performed and the samples were compared with fibrous tissue prepared using Type I. For chondrocytes, they were stained overnight with Alcian blue (pH 2.5), washed three times with 3% acetic acid, and then observed.

[0280] Depending on the cell type, there are several tissues in which the niche within the body is collagen other than type I. In this study, we investigated whether fibrous tissue could be created using collagen other than type I, depending on the tissue niche, and evaluated the function of the tissues using immunohistochemical staining images, along with fibrous tissue created using type I collagen. The results are shown in Figures 27, 28, and 29.

[0281] Fibrous tissues prepared using type III collagen, which is the niche for vascular endothelial cells, tended to have a slightly higher number of positive sites compared to those prepared using type I collagen (Figure 27).

[0282] Fibrous tissue spun using a combination of type IV collagen and laminin, components of the muscle basement membrane, showed significantly higher expression of MYH4, a muscle cell marker, compared to tissue using type I collagen, indicating a high degree of differentiation (Figure 28). In particular, type IV collagen is a rare collagen that dissolves in culture medium, making it difficult to add to tissues at high concentrations until now. These results demonstrate that it is possible to spin long fibers using type IV collagen when combined with type I collagen, and that more highly differentiated tissues can be produced by combining it with ECM such as laminin.

[0283] Figures 29(A) and (B) show microscopic observations of fibrous tissue prepared using type I collagen and type II collagen, respectively. In fibrous tissue prepared using ATDC5, a chondroprogenitor cell, culturing within type II collagen, which is abundant in cartilage, significantly increased the number of Alcian blue-positive sites (mucopolysaccharide-producing staining reagent), a differentiation marker, compared to culturing within type I collagen.

[0284] Test Example 2-10: Measurement of cell fiber orientation in bovine muscle satellite cells (bSC; 1.0 × 10⁻¹⁰) ６The cells were suspended on ice in 1,000 μL of a 3 mg / mL aqueous solution of bovine skin-derived collagen type I containing 2% laminin. Separately, polyacrylic acid (Mw 5 kDa) was dissolved in PBS at a concentration of 3 mg / mL and dispensed into sterile 1.5 mL microcentrifuge tubes. Subsequently, to avoid mixing, the collagen-cell suspension was gently overlaid on top of the polyacrylic acid solution. Immediately after overlaying, the interface between the two phases was grasped with sterile, fine-tipped tweezers and slowly lifted to generate a continuous fiber embedded with collagen, polyacrylic acid, and bSCs. For NHDF, only the aqueous solution of bovine skin-derived collagen type I was used to prepare the fiber. The total cell concentration was kept the same as that used for the bSC fiber.

[0285] Each fiber was transferred to a well in a 6-well plate containing 3 mL of pre-warmed growth medium. Constructs were kept at 37°C and 5% CO2. ２ The cells were incubated under the specified conditions for 7 days, after which the culture medium was replaced with differentiation medium (DMEM containing 2% serum). At the end of the differentiation period, the fibers were fixed with 4% paraformaldehyde, the cytoskeleton was stained, and observed using a light-sheet microscope. The degree of orientation was quantified by measuring the angle of the fiber relative to the long axis using ImageJ from the obtained microscopic images. The results are shown in Figure 30. The specific method for quantifying the degree of orientation was performed according to the procedure described above.

[0286] As shown in Figure 30, when using bSC, the proportion of orientation angles distributed within ±5° relative to the reference direction was 65% or more, the proportion within ±10° was 85% or more, and the proportion within ±15° was 90% or more, demonstrating high orientationability. Similarly, when using NHDF, the proportion of orientation angles distributed within ±5° relative to the reference direction was 65% or more, the proportion within ±10° was 85% or more, and the proportion within ±15° was 95% or more, demonstrating high orientationability.

[0287] Test Example 2-11: Preparation of bovine adipose-derived stem cell fibers. Bovine adipose-derived stem cells (bSC; 1.0 × 10) were prepared in the same manner as in Test Example 2-5. ６Adipose fibers (fibrous tissue) were prepared using [number] cells. After proliferation culture for 3 days, they were cultured for 14 days in differentiation medium (DMEM containing 500 μM oleic acid + 10% FBS). The fibers were fixed with 4% paraformaldehyde, and the lipid droplets, cytoskeleton, and nucleus were stained and observed using a light-sheet microscope. The results are shown in Figure 31.

[0288] As shown in Figure 31, accumulation of lipid droplets was observed in the orientation direction, confirming differentiation into lipid fibers.

[0289] [Test Example 3: Fabrication of Collagen-Containing Structures] In Test Examples 3-1 to 3-3, the materials shown in Table 14 below were used.

[0290]

[0291] [Test Example 3-1: Production of Collagen-Containing Structures (1)] A polyacrylic acid bath was prepared by adding 10 mL of a PBS aqueous solution containing 30 mg / mL of polyacrylic acid as a polymer aqueous solution to a 100 mL dish. Into the polyacrylic acid bath, a bovine skin-derived collagen solution (collagen / lactic acid solution) prepared to a concentration of 1.5 mg / mL using lactic acid was intermittently dispensed and stopped using a pipette to form droplets. Next, it was left to stand at room temperature for 60 seconds. As a result, the collagen / lactic acid solution droplets rapidly formed spherical structures in the polyacrylic acid bath. Subsequently, the formed collagen-containing structures (hereinafter also referred to as "collagen structures") were transferred to DMEM + 10% FBS medium heated to 37°C using a spatula and incubated overnight.

[0292] The collagen structures cultured overnight were subjected to immunostaining using an anti-COL-1 antibody according to the following procedure. The culture medium in which the collagen structures were cultured was aspirated and washed twice with PBS. Then, fixation was performed for 3 hours by adding 4% paraformaldehyde. After washing twice with PBS, blocking was performed for 1 hour with a 10-fold diluted blocking reagent containing 10% normal goat serum (0.2% TritonX). The anti-COL-1 antibody, diluted 1000-fold using the blocking reagent, was reacted with the collagen structures overnight at 4°C. After washing twice with PBS, a secondary antibody, diluted 200-fold using the blocking reagent, was added to the collagen structures and reacted with the collagen structures overnight at 4°C.

[0293] Visualization images of three-dimensional fluorescence imaging data obtained using a confocal laser microscope were acquired for immunostained collagen structures. The results are shown in Figures 33 to 35. Figure 33 shows the XZ plane image of the three-dimensional image. Figure 34 shows the ZY plane image of the three-dimensional image, and Figure 35 shows the XY plane image of the three-dimensional image.

[0294] As shown in Figures 33-35, in the collagen structure, only the side where the interface between the collagen / lactic acid solution droplet and the polyacrylic acid bath was formed was stained by immunostaining using an anti-COL-1 antibody, while the interior of the collagen structure was not stained. Therefore, it was found that the obtained collagen structure had a hollow structure inside. Furthermore, the film thickness of the obtained collagen structure was 45 μm, and the diameter of the hollow structure was 3000-4000 μm.

[0295] [Test Example 3-2: Production of Cell-Containing Collagen Structure] Instead of the collagen / lactic acid solution used in Test Example 1, bovine adipose-derived stem cells were used in 5 × 10⁶ solutions. ５ Collagen structures (cell-containing collagen structures) were formed in the same manner as in Test Example 1, except that a bovine skin-derived collagen solution (collagen / lactic acid solution) containing cells, prepared to a concentration of 3 mg / mL, was used. The formed cell-containing collagen structures were transferred to DMEM + 10% FBS medium heated to 37°C using a spatula and cultured at rest for 10 days.

[0296] The cultured cell-containing collagen structures were immunostained using an anti-COL-1 antibody following the same procedure as in Test Example 1. Furthermore, the cultured cell-containing collagen structures were stained with DAPI according to the following procedure. The cultured cell-containing collagen structures were fixed according to the same procedure as in Test Example 1, and DAPI diluted 1000-fold using a 10-fold diluted blocking reagent containing 10% normal goat serum (0.2% TritonX) was added to the cell-containing collagen structures and reacted with them overnight at 4°C. In addition, immunostaining using an anti-actin antibody was performed in the same manner as the immunostaining using the anti-COL-1 antibody.

[0297] Visualization images of 3D fluorescence imaging data acquired using a confocal laser microscope were obtained for cell-containing collagen structures that were immunostained with anti-COL-1 antibody and DAPI staining. The results are shown in Figures 36 to 45. Figure 36 shows the entire cell-containing collagen structure and is a merged image of the results of immunostaining with anti-COL-1 antibody, DAPI (4',6-diamidino-2-phenyllindole) staining, and immunostaining with anti-actin antibody. Figure 37 shows the entire cell-containing collagen structure and the result of DAPI staining. Figure 38 shows the entire cell-containing collagen structure and the result of immunostaining with anti-actin antibody. Figure 39 shows the entire cell-containing collagen structure and the result of immunostaining with anti-COL-1 antibody. Figures 40 to 41 show the XZ planar images of the obtained 3D images. Figure 40(A) shows a merged image of the results of immunostaining using anti-COL-1 antibody, DAPI staining, and immunostaining using anti-actin antibody, and Figure 40(B) shows the results of DAPI staining on a collagen-containing structure containing cells. Figure 41(A) shows the results of immunostaining on a collagen-containing structure containing cells using anti-actin antibody, and Figure 41(B) shows the results of immunostaining using anti-COL-1 antibody. Figures 42-43 show the ZY planar images of the obtained three-dimensional images. Figure 42(A) shows a merged image of the results of immunostaining using anti-COL-1 antibody, DAPI staining, and anti-actin antibody on a collagen-containing structure containing cells, and Figure 42(B) shows the results of DAPI staining on a collagen-containing structure containing cells. Figure 43(A) shows the results of immunostaining of a collagen-containing structure containing cells using an anti-actin antibody, and Figure 43(B) shows the results of immunostaining of a collagen-containing structure containing cells using an anti-COL-1 antibody. Figures 44-45 show the XY plane images of the obtained three-dimensional images.Figure 44(A) shows a merged image of the results of immunostaining using an anti-COL-1 antibody, DAPI staining, and anti-actin antibody on a collagen-containing structure containing cells. Figure 44(B) shows the results of DAPI staining on a collagen-containing structure containing cells. Figure 45(A) shows the results of immunostaining using an anti-actin antibody on a collagen-containing structure containing cells. Figure 45(B) shows the results of immunostaining using an anti-COL-1 antibody on a collagen-containing structure containing cells.

[0298] As shown in Figures 36-45, by incorporating bovine adipose-derived stem cells into a collagen / lactic acid solution, forming droplets in a polyacrylic acid bath, and allowing them to stand and culture for a certain period of time, a collagen structure containing cells within a hollow structure was obtained. The obtained collagen structure had a film thickness of 40 μm, and the diameter of the hollow structure was 3000-4000 μm.

[0299] [Test Example 3-3: Production of Collagen-Containing Structures (2)] As shown in Figure 46, an adapter, syringe, and needle were attached to a centrifuge tube. First, a 1 mL syringe filled with 500 μL of 3 mg / mL collagen aqueous solution was attached to the designed adapter. Then, the syringe with the adapter attached was attached to a 15 mL centrifuge tube containing 2 mL of polymer aqueous solution (PBS aqueous solution) containing 30 mg / mL PAA (MW 5000). Then, a 22, 25, 27, or 30 gauge (G) needle was attached to the tip of the syringe, respectively. The centrifuge tube was centrifuged at 2000 rpm (750 G) for 1 minute, causing the collagen solution filled in the syringe to be ejected by centrifugal force, thereby forming a collagen structure. Subsequently, the fiber diameter (tube diameter) was measured from the phase-contrast microscope image of the obtained collagen structure. The results are shown in Figures 47 and 48.

[0300] As shown in the upper part of Figure 47, fibrous collagen structures were formed regardless of the needle gauge used. Furthermore, from the phase-contrast microscope images in the lower part of Figure 47, it was confirmed that these collagen structures were tubular collagen structures with a hollow internal structure. In addition, as shown in Figure 48, the outer diameter of the collagen structures tended to decrease as the needle gauge number increased (the inner diameter of the needle decreased). Also, as shown in Figure 48, even when needles of different gauge numbers were used, there was no significant difference in the outer diameter of the collagen structures produced using needles of the same gauge number, indicating that collagen structures with a constant outer diameter can be stably produced. Moreover, by performing centrifugation at at least 700G to 800G, collagen structures with a constant outer diameter could be stably produced.

Claims

A method for producing anisotropic fibers containing collagen, A step of forming an interface by contacting an anionic polymer with an aqueous collagen solution containing dissolved collagen, thereby causing the collagen to fibrillate. A method comprising the step of pulling up the fibrous collagen from the aqueous collagen solution in which the anionic polymer has been contacted to form the anisotropic fibers. 　 The method according to claim 1, wherein the anionic polymer comprises at least one selected from the group consisting of poly(meth)acrylic acid, heparin, chondroitin sulfate, hyaluronic acid, dextran sulfate, poly(p-styrene sulfonic acid), carrageenan, alginic acid, and salts thereof. 　 The method according to claim 1 or 2, wherein the anionic polymer is at least one selected from the group consisting of poly(meth)acrylic acid, heparin, and salts thereof. 　 The method according to claim 1 or 2, wherein the anionic polymer is at least one selected from the group consisting of poly(meth)acrylic acid and salts thereof. 　 The method according to claim 1 or 2, wherein the collagen is atelocollagen. 　 The method according to claim 1 or 2, wherein the step of fibrillating the collagen is carried out by contacting the collagen aqueous solution with a polymer aqueous solution containing the anionic polymer. 　 The method according to claim 1 or 2, wherein the pH of the collagen aqueous solution is 3.0 or higher and 6.0 or lower, and the collagen content in the collagen aqueous solution is 0.31 to 10 μM based on the total amount of the collagen aqueous solution. 　 The method according to claim 6, wherein the pH of the polymer aqueous solution is 3.0 or more and less than 7.0, and the content of the anionic polymer in the polymer aqueous solution is 0.31 mM or more, based on the total amount of the polymer aqueous solution. 　 The method according to claim 1 or 2, wherein, in the step of forming the anisotropic fibers by pulling up the fibrous collagen from the collagen aqueous solution in which the anionic polymer has been contacted, the pulling speed of the fibrous collagen is 11 mm / second or less. 　 The method according to claim 1 or 2, wherein the collagen aqueous solution contains cells and is a cell-containing collagen aqueous solution in which collagen is dissolved. 　 The method according to claim 10, wherein the anisotropic fiber encapsulates the cell. 　 The method according to claim 10, wherein the cells include at least one selected from the group consisting of stem cells, fibroblasts, vascular endothelial cells, and chondrocytes. 　 The method according to claim 10, wherein the cells are satellite cells. 　 The method according to claim 10, wherein the aqueous collagen solution containing cells comprises at least one selected from the group consisting of lactic acid and citric acid. 　 The method according to claim 10, wherein the cells include vascular endothelial cells and the collagen includes type III collagen. 　 The method according to claim 10, wherein the cells include chondrocytes and the collagen includes type II collagen. 　 The method according to claim 10, wherein the cells include satellite cells, the collagen includes type I collagen and type IV collagen, and the cell-containing collagen aqueous solution further includes laminin. 　 Anisotropic fibers containing collagen, The fiber length is 10 mm or more. The storage modulus (G') at a strain of 0.1% is between 10 Pa and 1000 Pa. The permeability of a 5 mM HEPES solution containing FITC-dextran with a molecular weight of 4000 at a concentration of 0.5 mg / mL is 1.0 × 10⁻⁶. －７ Anisotropic fibers with a density of less than cm / s. 　 The anisotropic fiber according to claim 18, wherein the storage modulus (G') is 50 or more and 300 Pa or less. 　 An anisotropic fiber according to claim 18 or 19, comprising cells. 　 The anisotropic fiber according to claim 18 or 19, wherein the proportion of collagen whose orientation angle with respect to the reference direction is within ±5° is 50% or more. 　 The anisotropic fiber according to claim 20, wherein the cells include stem cells. 　 The anisotropic fiber according to claim 20, wherein the cells include satellite cells. 　 The anisotropic fiber according to claim 20, wherein the cells include fibroblasts. 　 A method for producing a collagen-containing structure having a hollow structure filled with an aqueous medium, A step of forming droplets of an aqueous solution containing at least collagen in an aqueous solution containing an anionic polymer, A method comprising the step of forming droplets, followed by the step of allowing the droplets to stand for a certain period of time to allow the collagen molecules to assemble at the interface between the droplets and the aqueous solution containing the anionic polymer. 　 The method according to claim 25, wherein the anionic polymer comprises at least one selected from the group consisting of poly(meth)acrylic acid and salts thereof. 　 The method according to claim 25 or 26, wherein the aqueous solution in which at least collagen is dissolved is an aqueous solution in which at least collagen is dissolved in a solution containing at least one acid selected from the group consisting of lactic acid, citric acid, succinic acid, tartaric acid, gluconic acid, malic acid, fumaric acid, and phosphoric acid. 　 The pH of the aqueous solution containing at least collagen is 3.0 or higher and 6.0 or lower, and the collagen content in the aqueous solution containing at least collagen is 0.31 to 10 μM based on the total amount of the collagen aqueous solution. The method according to claim 25 or 26, wherein the pH of the aqueous solution containing the anionic polymer is 3.0 or more and less than 7.0, and the content of the anionic polymer in the aqueous solution containing the anionic polymer is 0.31 mM or more, based on the total amount of the aqueous solution containing the anionic polymer. 　 The method according to claim 25 or 26, wherein the aqueous solution in which at least collagen is dissolved contains cells. A collagen-containing structure having a hollow structure inside that is filled with an aqueous medium. 　 The structure according to claim 30, wherein cells are adhered to the outside of the structure, or cells are contained within the hollow structure of the structure. 　 A composition comprising the structure described in claim 30 or 31 and an aqueous medium, wherein the structure is suspended in the aqueous medium.

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