Muscle tissue produced through bioprinting

Abstract

The present invention aims to provide a method for manufacturing artificial three-dimensional muscle tissue that can stably, efficiently and easily manufacture muscle tissue, and to provide artificial three-dimensional muscle tissue manufactured by the method. The solution is a method for manufacturing artificial three-dimensional muscle tissue and artificial three-dimensional muscle tissue obtained by the method, the method comprising the following steps: (i) forming a three-dimensional muscle tissue precursor composed of a first muscle tissue support, a second muscle tissue support and muscle cells by arranging muscle cells linearly on a first muscle tissue support so that the muscle cells are positioned adjacent to the first muscle tissue support at one end of the line and adjacent to the second muscle tissue support at the other end of the line, and (ii) culturing the three-dimensional muscle tissue precursor to obtain artificial three-dimensional muscle tissue.

Description

Technical Field

[0001] The present invention relates to a method for manufacturing artificial three-dimensional muscle tissue and the artificial three-dimensional muscle tissue manufactured by the method. Background Art

[0002] As a method for producing artificial three-dimensional muscle tissue such as skeletal muscle, various methods are known in the past, including a multilayer three-dimensional cell culture scaffold system (Patent Document 1). Artificial three-dimensional muscle tissue is used, for example, as a medical muscle implant, etc. (Patent Document 2). Artificial three-dimensional muscle tissue is also used, for example, in food such as processed meat (Patent Document 3).

[0003] Reference List

[0004] Patent Literature

[0005] Patent document 1: Japanese Patent Laid-Open No. 2019-41755

[0006] Patent Document 2: Japanese Patent Laid-Open No. 2003-508130

[0007] Patent Document 3: Japanese translation of PCT International Application Publication No. 2014-521336. Summary of the invention

[0008] Technical issues

[0009] However, conventional methods for producing artificial three-dimensional muscle tissues are difficult to produce muscle tissues stably and efficiently. Therefore, there is a need for a method for producing artificial three-dimensional muscle tissues that can easily and stably produce muscle tissues, and artificial three-dimensional muscle tissues produced using the method.

[0010] Means of solving problems

[0011] In order to solve the above-mentioned problems, the present inventors conducted intensive research and discovered a method for manufacturing artificial three-dimensional muscle tissue, which includes the following steps: (i) arranging a first muscle tissue support, a second muscle tissue support and muscle cells to constitute a three-dimensional muscle tissue precursor, wherein the muscle cells are arranged in the form of a line so that one end of the line is adjacent to the first muscle tissue support and the other end of the line is adjacent to the second muscle tissue support, and (ii) culturing the three-dimensional muscle tissue precursor to obtain artificial three-dimensional muscle tissue. The present inventors have thus completed the present invention.

[0012] Specifically, the present invention provides the following contents.

[0013] [1] A method for producing artificial three-dimensional muscle tissue, comprising the following steps:

[0014] (i) arranging a first muscle tissue support, a second muscle tissue support, and muscle cells to constitute a three-dimensional muscle tissue precursor, wherein the muscle cells are arranged in the form of a line such that one end of the line is adjacent to the first muscle tissue support and the other end of the line is adjacent to the second muscle tissue support, and

[0015] (ii) Cultivating three-dimensional muscle tissue precursors to obtain artificial three-dimensional muscle tissue.

[0016] [2] A method for manufacturing artificial three-dimensional muscle tissue according to [1], wherein step (i) comprises arranging a first muscle tissue support and a second muscle tissue support, and then arranging muscle cells in the form of a line so that one end of the line is adjacent to the first muscle tissue support and the other end of the line is adjacent to the second muscle tissue support, so as to form a three-dimensional muscle tissue precursor.

[0017] [3] The method for manufacturing artificial three-dimensional muscle tissue according to [1], wherein step (i) comprises arranging muscle cells in the form of a line above a first muscle tissue support body so that one end of the line is adjacent to the first muscle tissue support body, and then arranging a second muscle tissue support body so that the other end of the line is adjacent to the second muscle tissue support body to form a three-dimensional muscle tissue precursor.

[0018] [4] The method according to any one of [1] to [3], wherein the muscle cells are arranged in a thixotropic gel.

[0019] [5] The method according to any one of [1] to [4], wherein the muscle cells are arranged in a line using a dispenser.

[0020] [6] The method according to [5], wherein the arranged lines of muscle cells mimic animal muscle fibers.

[0021] [7] The method according to any one of [1]-[6], wherein the first muscle tissue support and / or the second muscle tissue support comprises collagen.

[0022] [8] The method according to any one of [1] to [7], wherein the artificial three-dimensional muscle tissue is cultured by applying tensile tension to the artificial three-dimensional muscle tissue between a first muscle tissue support and a second muscle tissue support.

[0023] [9] The method according to any one of [1] to [8], wherein the artificial three-dimensional muscle tissue is used for medical care, drug evaluation or food.

[0024]

[10] An artificial three-dimensional muscle tissue, comprising: a first muscle tissue support, a second muscle tissue support, and muscle tissue, wherein the muscle tissue is linear, one end of the muscle tissue is connected to the first muscle tissue support, and the other end of the muscle tissue is connected to the second muscle tissue support.

[0025]

[11] The artificial three-dimensional muscle tissue according to

[10] , wherein the artificial three-dimensional muscle tissue has a sarcomere structure.

[0026]

[12] The artificial three-dimensional muscle tissue according to

[10] or

[11] , wherein the artificial three-dimensional muscle tissue is used for medical care, drug evaluation or food.

[0027] Advantageous Effects of the Invention

[0028] According to the present invention, artificial three-dimensional muscle tissue can be stably and efficiently manufactured. Artificial three-dimensional muscle tissue can be prepared with high quality and / or low cost. The present invention eliminates the need to obtain muscle tissue from living animals.

[0029] The artificial three-dimensional muscle tissue produced by the present invention can be used for medical care. The artificial three-dimensional muscle tissue can be used as, for example, a muscle implant, a myocardial sheet or a part or all of an organ. According to the present invention, the artificial three-dimensional muscle tissue can be used for medical care without being obtained from a living animal.

[0030] The artificial three-dimensional muscle tissue produced by the present invention can be used for drug (medicine) evaluation. For example, the target drug can be brought into contact with the artificial three-dimensional muscle tissue to evaluate the efficacy, toxicity, etc. of the drug.

[0031] The artificial three-dimensional muscle tissue produced by the present invention can be used for food. For example, the artificial three-dimensional muscle tissue can be used as steak meat or processed meat. According to the present invention, edible meat can be obtained without killing animals such as livestock. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] [ Figure 1 ] Figure 1 (a) Schematic diagram of muscle tissue fabrication via bioprinting. Figure 1 (b) shows the printed container. Figure 1 (c) is the printed photo. Figure 1 (d) shows a photograph of the prepared muscle tissue after culturing for 1 day (upper left), a photograph of the connection between the collagen layer and the muscle tissue confirmed by phase contrast microscopy (lower), and a three-dimensional fluorescent image after differentiation (upper right, green: myosin heavy chain, red: actin filaments, and blue: cell nuclei). Figure 1(e) shows the sarcomere structure observed in the fluorescent image of immunostained muscle tissue two weeks after differentiation (green: myosin heavy chain, red: actin filaments, and blue: cell nuclei).

[0034] [ Figure 2 ] Figure 2 (a) Another schematic diagram of muscle tissue fabricated by bioprinting. Figure 2 (b) is the printed photo.

[0035] [ Figure 3 ] Figure 3 Shown are images of muscle tissue fabricated using a support bath containing gelatin and measurements of the muscle tissue diameter.

[0036] [ Figure 4 ] Figure 4 Shown are images of muscle tissue produced using a support bath containing gellan gum and measurement results of the muscle tissue diameter.

[0037] [ Figure 5 ] Figure 5 A photograph showing cultured meat prepared by assembling fat cells, vascular cells, and manufactured muscle tissue. DETAILED DESCRIPTION

[0038] In one embodiment, the present invention provides a method for manufacturing artificial three-dimensional muscle tissue, which comprises the following steps: (i) arranging a first muscle tissue support, a second muscle tissue support, and muscle cells to constitute a three-dimensional muscle tissue precursor, wherein the muscle cells are arranged in the form of a line so that one end of the line is adjacent to the first muscle tissue support and the other end of the line is adjacent to the second muscle tissue support, and (ii) culturing the three-dimensional muscle tissue precursor to obtain artificial three-dimensional muscle tissue.

[0039] In one embodiment, the present invention provides a method for manufacturing artificial three-dimensional muscle tissue, which includes the following steps: (i) arranging a first muscle tissue support and a second muscle tissue support; (ii) arranging muscle cells in the form of a line so that one end of the line is adjacent to the first muscle tissue support and the other end of the line is adjacent to the second muscle tissue support to form a three-dimensional muscle tissue precursor; and (iii) culturing the three-dimensional muscle tissue precursor to obtain artificial three-dimensional muscle tissue.

[0040] In one embodiment, the present invention provides a method for manufacturing artificial three-dimensional muscle tissue, which includes the following steps: (i) arranging muscle cells in the form of a line above a first muscle tissue support so that one end of the line is adjacent to the first muscle tissue support; (ii) arranging a second muscle tissue support so that the other end of the line is adjacent to the second muscle tissue support to form a three-dimensional muscle tissue precursor; and (iii) culturing the three-dimensional muscle tissue precursor to obtain artificial three-dimensional muscle tissue.

[0041] Muscle cells can be from any animal. Muscle cells can be muscle cells from humans, or they can also be muscle cells from non-human animals. Animals are, for example, insects, fish, amphibians, reptiles, birds or mammals. Animals are, for example, frogs, chickens, humans, monkeys, pigs, horses, cattle, sheep, dogs, cats, mice or rabbits. Animals can be meat, and examples include cattle, pigs, sheep, chickens, etc. Muscle cells can be cells from muscle, or they can also be muscle cells from stem cells such as mesenchymal stem cells, embryonic stem cells, and induced pluripotent stem cells. Muscle cells can contain other types of cells such as adipocytes, vascular endothelial cells, fibroblasts, stem cells, adipocytes, and vascular endothelial cells. Muscle cells can be cultured cells. Examples of cultured cells include primary cultured cells, secondary cultured cells, and cell line cells. Muscle cells can be myoblasts. Muscle cells can be from muscle tissue or any cell of muscle tissue, such as cells from skeletal muscle, visceral muscle, striated muscle, voluntary muscle, involuntary muscle, smooth muscle, or cardiac muscle.

[0042] Artificial three-dimensional muscle tissue comprises an aggregate of cultured muscle cells, and comprises an extracellular matrix as required. Artificial three-dimensional muscle tissue may be similar to skeletal muscle tissue, or may also be similar to the muscle tissue of an organ such as a heart. Artificial three-dimensional muscle tissue preferably forms muscle fibers. Artificial three-dimensional muscle tissue may have a sarcomere structure, which is the smallest constituent unit of a muscle fiber. Artificial three-dimensional muscle tissue may have a vascular system. Artificial three-dimensional muscle tissue may have a three-dimensional structure arranged through an extracellular matrix. Artificial three-dimensional muscle tissue may be used as, for example, a muscle implant, a muscle sheet such as a myocardial sheet, a part or all of an organ, etc. Artificial three-dimensional muscle tissue may be used for drug (medical) evaluation, i.e., by contacting the target drug with the artificial three-dimensional muscle tissue to evaluate the efficacy, toxicity, etc. of the target drug. Artificial three-dimensional muscle tissue may be used for food such as steak meat or processed meat.

[0043] The size and shape of the artificial three-dimensional muscle tissue can be appropriately adjusted according to the purpose, the site of use, the type of muscle cells, the physical properties required of the muscle tissue, etc. If the artificial three-dimensional muscle tissue is cylindrical, its diameter can be, for example, several microns, tens of microns, hundreds of microns, several millimeters, several centimeters, or more. Its height can be, for example, several microns, tens of microns, hundreds of microns, several millimeters, several centimeters, or more.

[0044] The muscle tissue precursor is an aggregate of muscle cells before forming mature muscle tissue. In a specific embodiment, the muscle tissue precursor is a cell aggregate, wherein the first muscle tissue support and the second muscle tissue support are arranged adjacent to both ends of immature muscle cells arranged in a linear manner.

[0045] Muscle cells may be accompanied by an extracellular matrix. Extracellular matrix is ​​well known and may be natural or artificial. Extracellular matrix may include, for example, fibronectin, gelatin, collagen, laminin, polylysine, etc. The extracellular matrix may be one, or may be two or more. The extracellular matrix preferably promotes or forms intercellular adhesion between adjacent cells. By using an extracellular matrix, a sheath structure may be formed in the artificial three-dimensional muscle tissue of the present invention.

[0046] The muscle tissue support is a basic material for constituting a three-dimensional muscle tissue precursor or for manufacturing artificial three-dimensional muscle tissue. The three-dimensional muscle tissue precursor, the first muscle tissue support, and the second muscle tissue support are integrated to manufacture artificial three-dimensional muscle tissue. For example, a three-dimensional muscle tissue precursor that simulates muscle fibers and a first muscle tissue support and a second muscle tissue support that simulates tendons are integrated to manufacture artificial three-dimensional muscle tissue. Although in the present invention, at least the first muscle tissue support and the second muscle tissue support are used as muscle tissue supports, another support may be used.

[0047] Those skilled in the art may consider the use of artificial three-dimensional muscle tissue manufactured by integrating a three-dimensional muscle tissue precursor, a first muscle tissue support, a second muscle tissue support, etc. to appropriately determine the materials and sizes of the first muscle tissue support and the second muscle tissue support.

[0048] As long as the material of the muscle tissue support body can be connected to the three-dimensional muscle tissue precursor, the material of the muscle tissue support body can be any material, and can be, for example, collagen (e.g., selected from one or more of type I, type II, type III, type IV, type V, and type XI). The material of the muscle tissue support body can be appropriately selected according to the type or amount of muscle cells, the size, shape or physical properties of the muscle tissue, etc. The materials of the first muscle tissue support body and the second muscle tissue support body can be the same or different.

[0049] The size, shape and physical properties of the muscle tissue support can also be appropriately selected according to the type or amount of muscle cells, the size, shape or physical properties of the muscle tissue, etc. When the artificial three-dimensional muscle tissue is disc-shaped, its diameter is, for example, several microns, tens of microns, hundreds of microns, several millimeters, several centimeters or more, and its thickness is, for example, several microns, tens of microns, hundreds of microns, several millimeters, several centimeters or more. The size and shape of the first muscle tissue support and the second muscle tissue support can be the same or different.

[0050] The muscle cells can be arranged using known means and known methods. The muscle cells are typically arranged in a gel.

[0051] The gel preferably has thixotropy. When a thixotropic gel is used, for example, when a nozzle is used to arrange muscle cells, the gel is liquefied by the nozzle action and the cell discharge pressure from the nozzle, and the freedom of the size and shape of the three-dimensional muscle tissue precursor is increased. After the muscle cells are arranged, the thixotropic gel solidifies, and this solidification is beneficial to maintaining the shape of the three-dimensional muscle tissue precursor and protecting the muscle cells. Alternatively, for example, the muscle cells can be arranged in a liquid state in the gel before gelation, and then the gel is gelated. If the muscle cells are arranged in a liquid state in the gel, the nozzle is easy to move. The gel that is gelated after the muscle cells are arranged is beneficial to maintaining the shape of the three-dimensional muscle tissue precursor and protecting the muscle cells.

[0052] Thixotropy refers to a fluid property characterized in that a gel changes into a sol by a physical stimulus such as pressure, and when the sol is left to stand, it returns to a gel again (sol-gel transition). Specifically, thixotropy has a phenomenon of viscosity reduction during shearing of a liquid, a hysteresis phenomenon observed in a shear stress-shear rate curve of a liquid, and a phenomenon of having a yield value.

[0053] The gel used in the present invention may be a gel obtained by dispersing a polymer substance (for example, a natural polymer such as a protein or a polysaccharide or a synthetic polymer) in an aqueous medium. Examples of polymers constituting the gel include, but are not limited to, natural polymers such as agar, gelatin, agarose, xanthan gum, gellan gum, sclerotium gum, gum arabic, tragacanth gum, acacia gum, cellulose gum, tamarind gum, guar gum, locust bean gum, glucomannan, chitosan, carrageenan, quince seed, galactan, mannan, starch, dextrin, thermogelatin, casein, pectin, collagen, fibrin, peptide, matrigel, chondroitin sulfate such as sodium chondroitin sulfate, hyaluronic acid (mucopolysaccharide) and hyaluronates such as sodium hyaluronate, alginic acid, alginates such as sodium alginate and calcium alginate, and derivatives thereof; cellulose derivatives such as methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, carboxymethylcellulose and salts thereof; poly(meth)acrylic acid such as polyacrylic acid, polymethacrylic acid, acrylic acid-methacrylate copolymers and their salts. salts; synthetic polymers such as polyvinyl alcohol, polymers of polyethylene glycol di(meth)acrylate (PPEGDA and PPEGDM), polyhydroxyethyl methacrylate, polyacrylamide, poly(N,N-dimethylacrylamide), poly-2-acrylamide-2-methylpropane sulfonic acid, poly(N-isopropylacrylamide), polyvinyl pyrrolidone, polystyrene sulfonate, polyethylene glycol, carboxyvinyl polymers, alkyl-modified carboxyvinyl polymers, maleic anhydride copolymers, polyoxyalkylene resins, crosslinkers of poly(methyl vinyl ether-alt-maleic anhydride) and polyethylene glycol, crosslinkers of polyvinyl alcohol, crosslinkers of N-vinylacetamide, crosslinkers of acrylamide, and crosslinked materials of starch-acrylate graft copolymers; silicones; interpenetrating network structure hydrogels and semi-interpenetrating network structure hydrogels (DN hydrogels); and mixtures of two or more thereof.

[0054] By adjusting the amount (concentration) of the polymer in the medium, the viscosity of the gel can be adjusted, or thixotropy can be imparted. The amount (concentration) of the polymer in the medium can be adjusted according to the type of polymer, the size, shape or physical properties of the three-dimensional muscle tissue precursor or artificial three-dimensional muscle tissue, the size or movement speed of the nozzle used, etc. It is preferred to adjust the amount (concentration) of the polymer in the medium to obtain a viscosity that can keep the three-dimensional muscle tissue precursor or artificial three-dimensional muscle tissue in the gel. The selection of the type and amount (concentration) of the polymer is within the skill of those skilled in the art.

[0055] Three-dimensional bioprinting can be used to arrange muscle cells. Three-dimensional bioprinting is known. Three-dimensional bioprinting can be an automatic or semi-automatic process supported by a computer, or it can be a manual process. Using a dispenser with a nozzle, such as a multi-nozzle dispenser, a large number of cells can be arranged at one time. The nozzle caliber can be appropriately selected according to the size and shape of the desired artificial three-dimensional muscle tissue and the size and position of the cells.

[0056] Muscle cells can be arranged by imitating the muscle fibers of animals. Muscle cells can be arranged in the form of one or more lines, for example, 2 or more, 3 or more, 4 or more, 5 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 100 or more, 500 or more, 1000 or more, 5000 or more, or 10000 or more lines. Muscle cells can be arranged so as to form muscle fiber bundles. The thickness (diameter) of the line formed by muscle cells can be 100 μm or more, 200 μm or more, 500 μm or more, 5mm or more, 10 mm or more, or 100 mm or more. The thickness (diameter) of the line formed by muscle cells can be 100 mm or less, 10 mm or less, 5 mm or less, 500 μm or less, 200 μm or less, or 100 μm or less. The length of the line formed by the muscle cells can be 50 mm or more, 100 mm or more, or 200 mm or more. The muscle cells can be arranged in a straight line. The muscle cells can be arranged in a curved line. The line formed by the muscle cells can be one or more. Two or more (multiple) lines formed by the muscle cells can be cultivated to strengthen the line formed by the muscle cells.

[0057] The term "adjacent" as used herein refers to a state where a small gap exists between a muscle cell and a muscle tissue support, or a state where a muscle cell and a muscle tissue support are in contact. The size of the small gap is such that when the muscle cells proliferate, they will be in contact with the muscle tissue support. Although the size of the small gap varies depending on the size or shape of the muscle tissue, the cell type, or the culture conditions, the size can be, for example, several millimeters, hundreds of micrometers, tens of micrometers, several micrometers, or less. The muscle cell and the muscle tissue support are preferably in contact.

[0058] The term "in the form of a line" or "line" used herein means that muscle cells are arranged linearly or in an almost linear state. For the almost linear state, real muscle tissue in an animal may be referred to. Alternatively, the almost linear state may also refer to a state in which a tensile tension is generated between the first muscle tissue support and the second muscle tissue support of the artificial three-dimensional muscle tissue and the muscle tissue. "In the form of a line" or "line" may be in the form of a straight line or a straight line. "In the form of a line" or "line" may be in the form of a curved line or a curved line.

[0059] When the muscle cells are arranged in a line above the first muscle tissue support, this means that the angle between the first muscle tissue support and the arranged muscle tissue is, for example, 1 degree-90 degrees, 45 degrees-90 degrees, 60 degrees-90 degrees, 70 degrees-90 degrees, 80 degrees-90 degrees, 85-90 degrees, 86-90 degrees, 87-90 degrees, 88-90 degrees, 89-90 degrees, or 90 degrees (right angle). The second muscle tissue support and the arranged muscle tissue are also preferably at the above angles. Depending on the shape of the desired artificial three-dimensional muscle tissue, the angle between the first muscle tissue support and / or the second muscle tissue support and the muscle tissue is not limited to the above angles.

[0060] A specific example of muscle cell arrangement is as follows: the first muscle tissue support is placed at one end of the cylindrical gel, the second muscle tissue support is placed at the other end, and the muscle cells are arranged in the gel in the form of a line using a multi-nozzle dispenser. At this time, one end of the line of muscle cells is adjacent to the first muscle tissue support, and the other end of the line is adjacent to the second muscle tissue support. Another specific example of muscle cell arrangement is as follows: the first muscle tissue support is placed at one end of the cylindrical gel, and the muscle cells are arranged in the gel in the form of a line using a multi-nozzle dispenser. At this time, one end of the line of muscle cells is adjacent to the first muscle tissue support. Then the second muscle tissue support is arranged so that it is adjacent to the other end of the line of muscle cells. Another specific example of muscle cell arrangement is as follows: the muscle cells are arranged in the form of a line in the cylindrical gel using a multi-nozzle dispenser. At this time, the muscle cells are arranged so that the two ends of the line of muscle cells reach the two circular bases of the gel cylinder, or are close to the circular base. Then the first muscle tissue support and the second muscle tissue support are arranged on the two circular bases of the gel cylinder. Another specific example of muscle cell arrangement is as follows: a first muscle tissue support is printed at one end of a gel using a 3D printer, a second muscle tissue support is printed at the other end, and a multi-nozzle dispenser is used to arrange muscle cells in a line. At this time, one end of the line of muscle cells is adjacent to the first muscle tissue support, and the other end of the line is adjacent to the second muscle tissue support. A series of these steps are performed in the same gel. Another specific example of muscle cell arrangement is as follows: a first muscle tissue support is printed in a gel using a 3D printer, and muscle cells are arranged thereon in a line using a multi-nozzle dispenser. At this time, one end of the line of muscle cells is adjacent to the first muscle tissue support. Then a second muscle tissue support is printed using a 3D printer so that it is adjacent to the other end of the line of muscle cells. A series of these steps are performed in the same gel. Another specific example of muscle cell arrangement is as follows: muscle cells are arranged in a gel in a line using a multi-nozzle dispenser. At this time, muscle cells are arranged so that the two ends of the line of muscle cells reach the two circular bases of the cylindrical gel, or approach the circular bases, respectively. Using a 3D printer, the first muscle tissue support and the second muscle tissue support were printed on two circular bases of the gel, respectively. A series of these steps were performed in the same gel.

[0061] The constructed three-dimensional muscle tissue precursor is then cultured to obtain artificial three-dimensional muscle tissue. The culture conditions can be appropriately selected and changed according to the type, number, size, shape or physical properties (such as elasticity or texture) of muscle cells, etc. The selection and change of these culture conditions are within the skill range of those skilled in the art.

[0062] The three-dimensional muscle tissue precursor can be cultured in a liquid culture medium. If the three-dimensional muscle tissue precursor is formed in a gel containing culture medium components, the three-dimensional muscle tissue precursor can be removed from the gel and cultured in a liquid culture medium. Alternatively, the three-dimensional muscle tissue precursor formed in the gel can be cultured in the gel. The gel can contain culture medium components and / or components for promoting muscle cell differentiation induction. The gel can be a gel that can be dissolved or decomposed by physical and / or chemical treatment to be removed.

[0063] In a specific example of a three-dimensional muscle tissue precursor, the three-dimensional muscle tissue precursor is formed in a gel having thixotropy, and then the three-dimensional muscle tissue precursor is taken out of the gel and cultured in a liquid culture medium. In a specific specific example of the culture of a three-dimensional muscle tissue precursor, the three-dimensional muscle tissue precursor is formed in a gel having thixotropy and containing culture medium components and / or components for promoting differentiation induction of muscle cells, and then the gel is dissolved by physical and / or chemical treatment, and the three-dimensional muscle tissue precursor is cultured in the dissolved gel. Although examples of physical or chemical treatments include the addition of a gel decomposing agent, pH changes, temperature changes, and oscillations, they are not limited thereto. For example, gellan gum gel can be dissolved by adding a Tris buffer solution.

[0064] Preferably, tensile tension is generated between the first muscle tissue support and / or the second muscle tissue support and the muscle tissue in the artificial three-dimensional muscle tissue of the present invention. Although tensile tension can be generated in the step of culturing a three-dimensional muscle tissue precursor, tensile tension can also be generated before and / or after the culturing step. The generation of tensile tension promotes the formation of muscle fibers, muscle tissue or sarcomere structure, and the muscle tissue obtained can be similar to animal muscle. Tensile tension can be generated by the action of muscle cells, or can be applied from the outside. The size of tensile tension can be appropriately changed according to the type of muscle cells in the artificial three-dimensional muscle tissue, the number of muscle cells, the size, shape or physical properties (e.g., elasticity or texture) of the muscle tissue, etc.

[0065] In the step of culturing the three-dimensional muscle tissue precursor, or before and / or after the culturing step, the differentiation of muscle cells can be induced and / or the muscle tissue can be matured. Methods for inducing muscle cell differentiation and methods for maturing muscle tissue are known. For example, components for promoting the induction of muscle cell differentiation can be added to the culture medium. Muscle tissue can be matured using, for example, electrical stimulation. Cell culture medium components and components for inducing muscle cell differentiation are well known and can be appropriately selected.

[0066] The artificial three-dimensional muscle tissue obtained by the manufacturing method of the present invention can be used for food. "For food" as used herein means that the artificial three-dimensional muscle tissue is suitable for consumption by humans or non-human animals. The artificial three-dimensional muscle tissue of the present invention can be used for food such as steaks or processed meat. Protein, fat, carbohydrates, vitamins, minerals, fiber, fatty acids, amino acids, flavor raw materials, coloring raw materials, etc. can be added to the artificial three-dimensional muscle tissue, so that the artificial three-dimensional muscle tissue is suitable for food, for example, from the perspective of adjusting texture, nutritional value, taste, color, etc. The shape, weight, size, etc. of the artificial three-dimensional muscle tissue can be appropriately adjusted so that the artificial three-dimensional muscle tissue is suitable for food.

[0067] In another embodiment, the present invention provides artificial three-dimensional muscle tissue, which includes: a first muscle tissue support, a second muscle tissue support and muscle tissue, wherein the muscle tissue is linear, one end of the muscle tissue is connected to the first muscle tissue support, and the other end of the muscle tissue is connected to the second muscle tissue support.

[0068] The artificial three-dimensional muscle tissue of the present invention may have a sarcomere structure. The artificial three-dimensional muscle tissue of the present invention may have a vascular system. The artificial three-dimensional muscle tissue of the present invention may have a three-dimensional structure arranged through an extracellular matrix.

[0069] The artificial three-dimensional muscle tissue of the present invention can be used in medical care, drug evaluation or food.

[0070] Unless otherwise specified, the terms used herein are those commonly used in the art.

[0071] Hereinafter, the present invention will be described more specifically and in detail by way of examples, but the scope of the present invention is not limited to these examples. Example

[0072] Example 1

[0073] 1. Preparation of Collagen Nanofiber Solution

[0074] First, 1 wt% collagen sponge (manufactured by NH Foods Ltd.) was mixed in PBS 1x solution using a pulverizer (VH-10 homogenizer manufactured by AS ONE CORPORATION). The prepared collagen microfiber solution was then kept in a refrigerator at 4°C for several hours to prepare a collagen nanofiber (CFN) solution.

[0075] 2. Fabrication of the Support Bath

[0076] First, 4.5 wt% of gelatin was mixed in DMEM containing 10% FBS and 1% antibiotics (antibiotics / NACALAITESQUE, INC. / 02892-54), and gelatin was dissolved at 37°C. After dissolution, the product was gelled in a 4°C refrigerator. The gelatin gel was mixed with a DMEM (10% FBS and 1% antibiotic) solution having the same volume as the gelatin gel, and pulverized with a pulverizer. The DMEM (10% FBS and 1% antibiotic) having the same volume as the pulverized gelatin dispersion was added to the gelatin dispersion, and the mixture was centrifuged (4200 rpm / 3 minutes). After removing the supernatant, thrombin was added to make the thrombin concentration be 10U / mL.

[0077] 3. Provision of printing containers

[0078] The printed container is made of PDMS material, such as Figure 1 (b). Regarding the internal dimensions, the area of ​​the base is 1 cm 2 (1 cm × 1 cm) and 2 cm in height. The collagen layers are each 0.4 cm high and the support bath area is 0.8 cm high.

[0079] 4. Cell Culture

[0080] 4-1 Proliferation of bovine muscle satellite cells.

[0081] The muscle satellite cells isolated from bovine tissue were cultured in a cell culture flask (3-layer cell culture flask / Falcon / 353143). The satellite cell proliferation and subculture method was performed in the same manner as the conventional adherent cell method. 20% FBS (FBS / Corning / 35-010-CV), 1% antibiotics, 4 ng / mL of bFGF and 10 um / mL of p38 inhibitor (p38 inhibitor / selleck / S1076) were added to high glucose DMEM (DMEM / Thermo Fisher Scientific KK / 10569010) to prepare a culture medium.

[0082] 4-2. Differentiation of bovine muscle satellite cells

[0083] After the culture medium was replaced, differentiation of muscle satellite cells was induced. The culture medium was replaced with DMEM obtained by adding 2% horse serum and 1% antibiotics to a conventional proliferation medium. The culture medium was replaced every 2-3 days.

[0084] 5. Cell printing

[0085] 5-1. Preparation of printing bath

[0086] First, about 0.4 ml of the 1 wt% collagen nanofiber solution described in Example 1.1 was injected into the bottom area of ​​the printing container described in Example 1.3 and gelled at 37°C for about 2 hours. 4.5 wt% gelatin particles mixed with 10 U / mL thrombin were filled on the gelled collagen gel in the bottom area. Then, 0.4 mL of collagen nanofiber solution was further injected into the area above the support bath area.

[0087] 5-2 Preparation of bio-ink

[0088] Myosatellite cells were isolated on culture plates and then prepared into 5 × 10 7 The concentration of cells / ml was 20 mg / mL. Then, 20 mg / mL fibrinogen was mixed with the cells (60% of the total amount), and the remaining 40% of the total amount of matrix gel was further mixed to prepare the bio-ink. The mixing process was performed on ice at 4°C to minimize the gelation of the matrix gel.

[0089] 5-3 Printing

[0090] The printing container in which the support bath material and the collagen layer were formed as described in Example 5.1 was placed on a substrate of a printing device (manufactured by Musashi Engineering, Inc, NANO MASTER) maintained at 4°C and allowed to stand for about 10 minutes so that the collagen layer in the bottom area was liquid. The prepared bio-ink was inserted into a syringe and the syringe was installed in the printing device. Using a 16G nozzle (inner diameter 1.19 mm, outer diameter 1.65 mm), cells were printed within a range of 1.5 cm at a moving speed of 1 mm / s in the height direction of the printing container.

[0091] 5-4 Subsequent Operations

[0092] After cell printing, additional collagen solution was poured into the area where the nozzle passed through the collagen layer in the upper area, and the temperature was then kept at room temperature for 30 minutes. Subsequently, additional collagen solution was also poured into the collagen layer in the bottom area, and the temperature was then kept at room temperature for 30 minutes. The container was then kept in an incubator at 37°C for 2 hours to allow the collagen to gel. At the same time, the support bath was liquefied. After collagen gelation, additional culture medium (high glucose DMEM, 20% FBS, 1% antibiotics, and 4ng / mL bFGF) was added to the printing container in PDMS for culture.

[0093] 6. Confirmation of printed muscle tissue

[0094] 6-1. Induction of differentiation

[0095] The existing culture medium (high glucose DMEM, 20% FBS, 1% antibiotics, and 4 ng / ml bFGF) was replaced with the culture medium for inducing differentiation (high glucose DMEM, 2% horse serum, and 1% antibiotics) to induce differentiation into muscle fibers.

[0096] 6-2. Immunostaining and Fluorescence Imaging

[0097] The cultured muscle tissue was fixed in 4% paraformaldehyde for 20 minutes at room temperature and then washed with PBS 1x. Subsequently, the tissue was treated with 0.2 wt% Triton X-100 for 15 minutes and then washed once with PBS 1x. After treatment with 1 wt% BSA for 30 minutes at room temperature, the muscle tissue was initially stained with MF20 (myosin 4 monoclonal antibody) prepared at 1 μg / ml in 1 wt% BSA for 2 hours at room temperature. After washing with PBS 1x, fluorescence conjugated phalloidin and the secondary antibody corresponding to the first staining were mixed in 1% BSA and treated for 1 hour at room temperature. After washing with PBS 1x, the cell nuclei were stained with DAPI (4',6-diamidino-2-phenylindole dihydrochloride) and three-dimensional fluorescence photography was performed using a confocal laser microscope. The captured fluorescence images can be found in Figure 1 Confirmed in (d) and (e).

[0098] 6-3. Measurement of muscle fiber diameter

[0099] Measurements of the diameter of the cultured muscle tissue were also obtained ( Figure 3 ).

[0100] Example 2

[0101] 1. Preparation of Collagen Nanofiber Solution

[0102] First, 1 wt% of collagen sponge (manufactured by NH Foods Ltd.) was mixed in a PBS 1x solution, and collagen microfibers were prepared using a fiberizer (homogenizer). Then, the prepared collagen microfibers were kept in a refrigerator at 4°C for several hours to prepare a collagen nanofiber (CFN) solution.

[0103] 2. Fabrication of the Support Bath

[0104] First, 0.1 g of gellan gum was dissolved in 10 mL of PBS at 100°C, and the product was then gelled by incubation in a refrigerator at 4°C. The gelled gellan gum was pulverized into particles using a grinder. After centrifugation at 4200 rpm for 3 minutes, the supernatant was removed. Thrombin was added to the granulated gellan gum gel so that the concentration of thrombin was 10 U / mL.

[0105] 3. Preparation of Bio-ink

[0106] Then, 20 mg of fibrinogen was dissolved in 1 mL of DMEM at 37° C. to prepare a 20 mg / mL fibrinogen solution. During cell printing, cells and extracellular matrix materials were added.

[0107] 4. Print

[0108] The collagen nanofiber solution described in Example 2.1 and the bio-ink described in Example 2.3 were inserted into syringes, respectively. The syringe with the collagen nanofibers inserted was first loaded on the printing device, and 500 μL was discharged into a 3 mL vial. The collagen nanofibers were gelled at 37°C for 2 hours. After gelation, 2.5 mL of the support bath prepared in Example 2.2 was added to the collagen gel. The syringe was replaced with a syringe for bioprinting, and then printing was performed under the following conditions: nozzle: 19G (inner diameter 0.68 mm, outer diameter 1.00 mm) and moving speed: 1 mm / s. After printing, the fibrinogen gelation was accelerated at 37°C for 15 minutes. Figure 2 (b) shows the results.

[0109] 5. Measurement of muscle fiber diameter

[0110] Measurements of the diameter of the cultured muscle tissue were also obtained ( Figure 4 ).

[0111] Example 3

[0112] 1. Preparation of Cultured Meat

[0113] Based on the ratio among adipose tissue, vascular tissue and muscle tissue of commercially available beef, adipocytes, vascular cells and the muscle tissue obtained in Example 1 were assembled. Figure 5 The assembled cultured meat is shown. The vascular cells and muscle tissue are stained with carmine dye (red), while the fat cells are not stained.

[0114] Industrial Applicability

[0115] The present invention is useful in the fields of therapeutic materials for regenerative medicine, wound healing, recovery, maintenance and improvement of motor function, etc.; the field of medicine; the field of muscle-related research; the field of food and feed, etc.

Claims

1. A method for producing artificial three-dimensional muscle tissue, comprising the following steps: (i) arranging a first muscle tissue support, a second muscle tissue support, and muscle cells to constitute a three-dimensional muscle tissue precursor, wherein the muscle cells are arranged in the gel in the form of a line such that one end of the line is adjacent to the first muscle tissue support and the other end of the line is adjacent to the second muscle tissue support, (ii) dissolving or decomposing the gel, and (iii) Cultivating muscle cells of three-dimensional muscle tissue precursors in a culture medium to obtain artificial three-dimensional muscle tissue.

2. The manufacturing method according to claim 1, wherein step (ii) further comprises removing the gel.

3. The manufacturing method according to claim 2, comprising the steps of: (i) arranging a first muscle tissue support, a second muscle tissue support, and muscle cells to form a three-dimensional muscle tissue precursor, wherein the muscle cells are arranged in the gel in the form of a line, such that one end of the line is adjacent to the first muscle tissue support and the other end of the line is adjacent to the second muscle tissue support, (ii) dissolving or decomposing and removing the gel, and (iii) culturing muscle cells of three-dimensional muscle tissue precursors in a culture medium to obtain artificial three-dimensional muscle tissue, in, Step (i), step (ii) and step (iii) are performed sequentially.

4. The production method according to any one of claims 1 to 3, wherein step (iii) comprises replacing the culture medium.

5. The manufacturing method according to claim 1, characterized in that Artificial three-dimensional muscle tissue having muscle fiber bundles is manufactured, wherein step (i) comprises arranging muscle cells in the form of a plurality of threads in a gel.

6. The manufacturing method according to any one of claims 1 to 3, wherein the muscle cells are arranged in a gel having thixotropy.

7. The manufacturing method according to any one of claims 1 to 3, wherein the muscle cells are arranged in the form of a plurality of lines by being discharged using a multi-nozzle dispenser having a plurality of nozzles.

8. The manufacturing method according to any one of claims 1 to 3, further comprising gelling the first muscle tissue support and / or the second muscle tissue support.

9. The manufacturing method according to claim 8, wherein the first muscle tissue support and / or the second muscle tissue support is gelled after step (i).

10. The manufacturing method according to any one of claims 1 to 3, wherein the first muscle tissue support and / or the second muscle tissue support comprises collagen.

11. The manufacturing method according to any one of claims 1 to 3, further comprising adding and assembling fat cells and vascular cells based on the ratio among the fat tissue, vascular tissue and muscle tissue of commercially available beef.

12. The method according to any one of claims 1 to 3, further comprising adding at least one of protein, fat, carbohydrate, vitamin, mineral, fiber, fatty acid, amino acid, flavor raw material and coloring raw material to adjust at least one of texture, nutritional value, taste and color.

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