Three-dimensional muscular tissue and method for producing same

Abstract

Provided is a method for producing a three-dimensional muscle tissue, comprising: a step for producing a substantially rectangular first cell module that contains skeletal muscle myoblasts in a hydrogel and has a plurality of substantially rectangular holes parallel to each other; a second cell module containing skeletal muscle myoblasts in a hydrogel and having a plurality of substantially rectangular pores parallel to each other at positions different from those of the first cell module in the vertical direction; a step for obtaining a laminate by alternately laminating the produced first cell modules and second cell modules; a step in which the skeletal muscle myoblasts included in the obtained laminate are subjected to proliferation culture; and a step for inducing differentiation of the proliferated skeletal muscle myoblasts into myotubes.

Description

Related Applications This application is a divisional application of Chinese Patent Application No. 202080018038.5 (the filing date is deemed to be January 22, 2020) with the invention name of “Three-dimensional muscle tissue and its manufacturing method”, and claims all priorities and rights of the original application and cites the entire contents of the original application specification, etc. in its entirety. Technical Field

[0001] The invention mainly relates to a method for manufacturing three-dimensional muscle tissue. Background Art

[0002] The demand for meat is increasing as the population grows and incomes rise in emerging countries. On the other hand, it is difficult to increase the supply of meat due to the rising prices of grains used as livestock feed and the problem of securing breeding sites, so there is a desire to develop meat substitutes.

[0003] Cultured meat (3D muscle tissue) is made by growing skeletal muscle cells through culture. Since it can be produced in a laboratory, it can be produced without being affected by climate change. In addition, compared with traditional livestock farming, it emits less greenhouse gases and has less impact on the environment. Prior art literature Patent Literature

[0004] Patent Document 1: Japanese Patent Application Publication No. 2018-000194 Patent Document 2: Japanese Patent No. 6427836 Summary of the invention Problem that the invention aims to solve

[0005] In order to produce edible meat with a good taste such as steak from cells, it is necessary to construct mature three-dimensional muscle tissue. In the field of regenerative medicine, it is reported that skeletal muscle cells of humans or mice are used to construct three-dimensional muscle tissue (Patent Documents 1 and 2). However, research on the conditions of cultured meat specifically for consumption is still insufficient.

[0006] The object of the present invention is to provide a method for producing three-dimensional muscle tissue that is particularly suitable for consumption. Methods used to solve problems

[0007] In view of the above problems, the inventors have repeatedly conducted intensive research and found that the above problems can be solved by the following manufacturing method. The manufacturing method includes the following steps: the process of making a roughly rectangular first cell module and a roughly rectangular second cell module, the first cell module contains skeletal muscle myoblasts in a hydrogel and has a plurality of roughly rectangular holes parallel to each other, and the second cell module contains skeletal muscle myoblasts in a hydrogel and has a plurality of roughly rectangular holes parallel to each other at positions different from the first cell module in the vertical direction; the process of alternately stacking the first cell module and the second cell module to obtain a stacked body; the process of proliferating and culturing the skeletal muscle myoblasts contained in the obtained stacked body; and the process of inducing the differentiation of the proliferated skeletal muscle myoblasts into myotubes. The present invention is completed by further repeated research on the above findings.

[0008] The present invention includes the following technical solutions.

[0009] Item 1. A method for producing a three-dimensional muscle tissue, comprising the following steps: a step of preparing a first cell module having a substantially rectangular shape and a second cell module having a substantially rectangular shape, wherein the first cell module contains skeletal muscle myoblasts in a hydrogel and has a plurality of substantially rectangular holes parallel to each other, and the second cell module contains skeletal muscle myoblasts in a hydrogel and has a plurality of substantially rectangular holes parallel to each other, wherein at least a portion of the holes of the second cell module are located at a different position in the vertical direction from the holes of the first cell module; a step of alternately stacking the first cell modules and the second cell modules thus prepared to obtain a stack; and a step of inducing the skeletal muscle myoblasts to differentiate into myotubes.

[0010] Item 2. In the production method according to Item 1, the hydrogel contains 0.3 mg / mL or more of collagen.

[0011] Item 3. In the production method according to Item 1 or 2, the hydrogel contains 10 to 1000 μM of ascorbic acid, a salt thereof, or a derivative thereof.

[0012] Item 4. In the production method according to any one of Items 1 to 3, the width of the holes in the short-side direction of the first cell module and the second cell module is 200 to 2000 μm.

[0013] Item 5. In the production method according to any one of Items 1 to 4, the interval between adjacent holes of the first cell module and the second cell module in the short-side direction is 200 to 2000 μm.

[0014] Item 6. In the manufacturing method according to any one of Items 1 to 5, the sizes of the first cell module and the second cell module are 3 mm or more in the short-side direction and 9 mm or more in the long-side direction.

[0015] Item 7. In the production method according to any one of Items 1 to 6, a total of six or more first cell modules and second cell modules are stacked.

[0016] Item 8. In the production method according to any one of Items 1 to 7, both ends of the first cell module and the second cell module in the stacked body in the longitudinal direction are fixed.

[0017] Item 9. In any one of Items 1 to 8, the production method is a method for producing three-dimensional muscle tissue for edible meat.

[0018] Item 10. In the production method according to any one of Items 1 to 9, the skeletal muscle myoblasts are bovine skeletal muscle myoblasts.

[0019] Item 11. A three-dimensional muscle tissue obtained by the production method according to any one of Items 1 to 10. Effects of the Invention

[0020] Based on the present invention, a method for producing a three-dimensional muscle tissue that is particularly suitable for consumption can be provided. The three-dimensional muscle tissue produced by the production method of the present invention has a myometrial structure similar to that of a muscle derived from a living body. In addition, a three-dimensional muscle tissue having a sufficient size suitable for consumption can be produced. Therefore, the three-dimensional muscle tissue produced by the production method of the present invention can be expected to have a taste close to that of livestock meat when consumed. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is an example of the shape of the first cell module 1 and the second cell module 2 . Figure 2 This is an example of how a cell module is made. Figure 3 This is an example of a layered approach to cell modules. Figure 4 It is the calculation result of the fusion index; the VC on the horizontal axis is ascorbic acid phosphate. Figure 5 It is a method of forming three-dimensional muscle tissue. Figure 6 These are the results of the hydrogel composition study; (A) MATRIGEL 100%, (B) MATRIGEL:collagen=1:1, (C) collagen 100%, (D) immunostaining (blue: cell nucleus; green: SAA). Figure 7 is the result of the cell concentration study; (A) 2.5×10 7 cells / mL, (B) 5.0×10 7cells / mL, (C) 10.0×10 7 cells / mL, (D) immunostaining (blue: cell nucleus; green: SAA), (E) 5.0×10 7 cells / mL (magnification), (F) 10.0×10 7 cells / mL (magnification). Figure 8 It is a method for forming three-dimensional large muscle tissue. Fig. 9 The results of stacking 3 layers of bovine myoblasts; (A) Day 0 (stacking), (B) Day 1 (DM transition), (C) Day 6, (D) Day 12, (E) Immunostaining (green: SAA; blue: nucleus), (F) Myotube formation, (G) Myomere-like structure. Fig.10 The results of stacking 25 layers of mouse myoblasts; (A) Day 0, (B) Day 9, (C) Day 10, (D) Culture container and images during culture. After 12 days of culture, a single piece of tissue (about 1 cm × 1 cm × 6 mm thick) was formed. DETAILED DESCRIPTION

[0022] The method for producing a three-dimensional muscle tissue of the present invention includes the following steps: a step of preparing a first cell module having a substantially rectangular shape and a second cell module having a substantially rectangular shape, wherein the first cell module contains skeletal muscle myoblasts in a hydrogel and has a plurality of substantially rectangular holes parallel to each other, and the second cell module contains skeletal muscle myoblasts in a hydrogel and has a plurality of substantially rectangular holes parallel to each other, wherein at least a portion of the holes of the second cell module are located at a different position from the holes of the first cell module in the vertical direction; a step of alternately stacking the first cell module and the second cell module to obtain a stack; and a step of inducing the skeletal muscle myoblasts to differentiate into myotubes.

[0023] In the present invention, the three-dimensional muscle tissue mainly refers to muscle that is not derived from a living body but is artificially produced. The three-dimensional muscle tissue of the present invention is composed of skeletal muscle cells (striated muscle cells). Skeletal muscle cells are myotubes (myotube cells) or muscle fibers in the form of multinucleated myoblasts as their precursors.

[0024] Usually, muscle fibers are composed of myofibrils, which are composed of fibers (actin filaments) of actin, a protein constituting muscle, and fibers (myosin filaments) of myosin, a protein constituting muscle. In addition, myofibrils have a structure in which a plurality of sarcomere structures are connected along the long axis direction. It is known that the contraction and relaxation of muscles are generated based on the interaction (sliding) of actin and myosin in the sarcomere.

[0025] The three-dimensional muscle tissue of the present invention has a sarcomere structure. However, it does not matter whether or not the sliding of the sarcomere structure occurs.

[0026] Whether the three-dimensional muscle tissue has a sarcomere structure can be evaluated by a well-known method. For example, the presence of sarcomere α-actinin (SAA), which is a protein constituting the Z membrane of the sarcomere structure, can be evaluated by immunostaining of SAA. If the SAA immunostaining is positive and the SAA distribution is regular stripes, it can be judged that the sarcomere structure is present. In addition, in the three-dimensional muscle tissue of the present invention, the muscle fibers are aligned and oriented in the same direction. The orientation of the muscle fibers can be evaluated by immunostaining of SAA, for example.

[0027] The three-dimensional muscle tissue of the present invention is preferably an edible three-dimensional muscle tissue. Edible three-dimensional muscle tissue can also be referred to as "cultured meat", "artificial edible meat (artificial meat)", etc. In the case where the three-dimensional muscle tissue of the present invention is an edible three-dimensional muscle tissue, the ingredients (preferably all ingredients) used in the production method of the present invention are preferably ingredients that meet the prescribed criteria and ensure safety for food production and / or food use, but are not limited thereto.

[0028] The first step of the production method of the present invention is a step of preparing a first cell module and a second cell module. Hereinafter, the first cell module and the second cell module may be collectively referred to as a "cell module".

[0029] The cell module of the present invention contains skeletal muscle myoblasts in the hydrogel. Skeletal muscle myoblasts can be prepared by well-known methods. For example, primary myoblasts obtained by treating muscle tissue derived from an organism with a degrading enzyme (e.g., collagenase) can be used. In order to remove impurities such as connective tissue, it is preferred to filter the primary myoblasts.

[0030] In addition, skeletal muscle myoblasts may be cells induced to differentiate from pluripotent stem cells such as ES cells and iPS cells, or adult stem cells having the ability to differentiate into skeletal muscle myoblasts.

[0031] Skeletal muscle myoblasts are derived from vertebrates such as mammals, birds, reptiles, amphibians, and fish. As mammals, non-human mammals such as monkeys, cows, horses, pigs, sheep, goats, dogs, cats, guinea pigs, rats, and mice can be mentioned. As birds, ostriches, chickens, ducks, sparrows, etc. can be mentioned. As reptiles, snakes, crocodiles, lizards, turtles, etc. can be mentioned. As amphibians, frogs, newts, salamanders, etc. can be mentioned. As fish, salmon, tuna, sharks, sea bream, carp, etc. can be mentioned. In the case of edible three-dimensional muscle tissue, skeletal muscle myoblasts are preferably derived from mammals raised for livestock production such as cows, pigs, sheep, goats, and horses, and are more preferably derived from cows.

[0032] Skeletal muscle myoblasts can be skeletal muscle myoblasts that have been genetically modified by a genome editing method such as homologous recombination, CRISPR / Cas9, or skeletal muscle myoblasts that have not been genetically modified. In one embodiment of edible three-dimensional muscle tissue, skeletal muscle myoblasts are preferably skeletal muscle myoblasts that have not been genetically modified from the perspective of safety and consumer preference.

[0033] As the hydrogel constituting the cell module of the present invention, a gel of components constituting the extracellular basement membrane matrix such as fibrin, fibronectin, laminin, collagen (e.g., type I, type II, type III, type V, type XI, etc.), agar, agarose, glycosaminoglycan, hyaluronic acid, proteoglycan, etc. can be used. As the hydrogel, a commercially available product can also be used, for example, a component based on a mouse EHS tumor extract (including type IV collagen, laminin, heparan sulfate proteoglycan, etc.) sold under the trade name of "MATRIGEL" can be used.

[0034] In the present specification, "collagen" includes undenatured collagen and denatured collagen. Examples of denatured collagen include gelatin.

[0035] In particular, when the skeletal muscle myoblasts are derived from bovine, the hydrogel preferably contains collagen, more preferably undenatured type I collagen. When type I collagen is contained, its content is preferably 0.3 mg / mL or more, more preferably 1.0 to 3.0 mg / mL, and even more preferably 1.0 to 1.5 mg / mL.

[0036] For skeletal muscle myoblasts in hydrogels, it is ideal that the cell density is, for example, about 1.0 × 10 6 / mL or more, preferably about 1.0×10 7 / mL~about 1.0×10 8 / mL, more preferably 5.0×10 7 / mL~about 1.0×108 Pieces / mL.

[0037] The cell module of the present invention may contain components (additives) other than the above-mentioned skeletal muscle myoblasts and hydrogel. Examples of such additives include culture medium components (e.g., various amino acids, inorganic salts, vitamins, etc.), serum components (e.g., growth factors such as IGF-1, bFGF, insulin, testosterone, etc.), and antibiotics.

[0038] As a preferred example of such additives, ascorbic acid (vitamin C) or its salt or derivatives thereof can be cited. As ascorbic acid or its salt, specifically, ascorbic acid, sodium ascorbate, potassium ascorbate, calcium ascorbate, etc. can be cited. As ascorbic acid derivatives, isoascorbic acid, ascorbic acid phosphate and its salts, ascorbic acid sulfate and its salts, ascorbic acid palmitate phosphate and its salts, L-ascorbic acid-2-glucoside and other L-ascorbic acid glycosides can be cited. As salts, sodium salts, potassium salts, calcium salts, magnesium salts, etc. can be cited. The content of ascorbic acid (vitamin C) or its salt or derivatives thereof can be 10 to 1000 μM, more preferably 50 to 250 μM, and particularly preferably 50 to 100 μM. By including ascorbic acid (vitamin C) or its salt or derivatives thereof in the above content, especially when skeletal muscle myoblasts are derived from cattle, differentiation induction from skeletal muscle myoblasts to myotubes can be effectively performed.

[0039] The first cell module is roughly rectangular in shape and has a plurality of roughly rectangular holes parallel to each other. Similar to the first cell module, the second cell module is roughly rectangular in shape and has a plurality of roughly rectangular holes parallel to each other. At least a portion of the plurality of holes in the second cell module is preferably all located at a different position from the first cell module in the vertical direction. Between the holes parallel to each other, a roughly rectangular and narrow band portion is formed. Figure 1 An example of the shape of the first cell module 1 and the second cell module 2 is shown in FIG.

[0040] The shape of the cell module is roughly rectangular. As the size of each module, the short side direction is preferably 3 mm or more, more preferably 5 mm or more, and further preferably 10 mm or more. The long side direction is preferably 9 mm or more, more preferably 13 mm or more, and further preferably 16 mm or more.

[0041] The cell modules are in a substantially rectangular sheet shape. The thickness of each cell module is preferably about 300 μm to 2000 μm, and more preferably about 500 to 1000 μm.

[0042] The number of the plurality of wells in the cell module is preferably about 1 to 30, more preferably about 2 to 25, and even more preferably about 4 to 10.

[0043] The shape of the holes of the cell module is roughly rectangular. As the size of each hole, the short side direction is preferably 200 to 2000 μm wide, more preferably 200 to 1000 μm wide, and further preferably 300 to 700 μm wide. In addition, as the size of the roughly rectangular and narrow width band portion formed between adjacent holes, the short side direction is preferably 200 to 2000 μm wide, more preferably 200 to 1000 μm wide, and further preferably 500 to 1000 μm wide. The multiple holes of the cell module are parallel to each other, preferably parallel to each other and roughly equally spaced. The holes of the cell module are preferably holes that penetrate the cell module.

[0044] There is no particular limitation on the method for making the cell module of the above-mentioned shape. For example, a cell module of a desired shape can be made by sandwiching a hydrogel solution containing skeletal muscle myoblasts between a model stamp obtained by patterning the shape of the module and a substrate. There is no particular limitation on the material of the model stamp and the substrate, and for example, thermosetting resins such as silicone rubber (dimethylpolysiloxane, PDMS), thermoplastic resins such as various plastics, glass, etc. can be used. Figure 2 An example of a method for producing a cell module is shown in FIG.

[0045] The second step of the production method of the present invention is a step of alternately stacking the first cell modules and the second cell modules obtained in the first step to obtain a stacked body.

[0046] Here, "alternately" means stacking the first cell module, the second cell module, the first cell module, the second cell module, etc. in sequence. By stacking alternately, in the stacked body, except when there is no cell module directly above or below any first cell module, the cell modules directly above and below any first cell module are the second cell modules.

[0047] In a preferred embodiment of stacking the first cell module and the second cell module, the holes of the first cell module and the holes of the second cell module directly above and directly below are stacked in a manner that at least a portion (or all) of them do not overlap with each other. In other words, in a preferred embodiment of stacking, the narrow-width strip-shaped portion of the first cell module and the narrow-width strip-shaped portion of the second cell module directly above and directly below are stacked in a manner that at least a portion (or all) of them do not overlap with each other. By stacking in this way, a bundle of myotubes / myofibers having an orientation and a large cross-sectional area can be produced. Figure 3 An example of a cell module stacking method is schematically shown in FIG.

[0048] There is no particular limitation on the number of the stacked first cell modules and the second cell modules. From the viewpoint of manufacturing a three-dimensional muscle tissue with sufficient thickness, it is preferred to stack a total of 6 or more of the first cell modules and the second cell modules, and more preferably stack a total of 10 or more. From the viewpoint of ease of operation, the upper limit of the number of stacks may preferably be 50 in total, more preferably 30, and particularly preferably 20, but is not limited thereto.

[0049] For example, the stacking can be performed on an appropriate substrate. That is, the first cell module (either the first cell module or the second cell module) is placed on the substrate, and a cell module (a cell module different from the first module) is stacked thereon. The material of the substrate is preferably a material that is non-adhesive to cells, such as dimethyl polysiloxane (PDMS), or a material that is rendered non-adhesive by surface treatment.

[0050] The stacking of the present invention is preferably performed in a manner that the two ends of the long side direction of the cell module are fixed. A person skilled in the art can appropriately select a method for fixing. For example, a component having adhesive force (e.g., a hydrogel such as fibrin) can be used to fix the two end portions of the cell module. In another embodiment, the cell module can be fixed by allowing a fixing member (pile, anchor) provided on the substrate to penetrate a hole pre-set near the end of the cell module.

[0051] In the production method of the present invention, each cell module after production and before stacking or the stacked body obtained by stacking can be subjected to growth culture to proliferate the skeletal muscle myoblasts contained therein.

[0052] For example, a sufficient amount (e.g., 1.0×10 8 In the case of skeletal muscle myoblasts of 100 μg / mL or more, the following differentiation induction step may be performed without proliferation culture. For example, in the case of skeletal muscle myoblasts that need to be proliferated, the following differentiation induction step may be performed after proliferation culture.

[0053] From the viewpoint of further improving the stability of the cell module, it is also preferred to subject each cell module after preparation and before stacking to growth culture. From the viewpoint of further promoting the adhesion of cells between the cell modules, it is also preferred to subject the stacked body obtained by stacking to growth culture.

[0054] For example, the above-mentioned culture can be carried out in the above-mentioned culture medium for proliferation culture by a method well known to those skilled in the art. As a method for carrying out appropriate culture, a method of carrying out culture under the conditions of about 37°C and a carbon dioxide concentration of about 5 to 10% (V / V) can be exemplified, but it is not limited thereto. For example, the culture under the above-mentioned conditions can also be carried out using a well-known CO2 incubator.

[0055] As a culture medium for proliferation culture, a culture medium obtained by adding serum components (such as horse serum, fetal bovine serum (FBS) and human serum), growth factors and other components, and antibiotics such as penicillin and streptomycin to a common liquid culture medium such as DMEM (Dulbecco's Modified Eagle's Medium), EMEM (Eagle's minimal essential medium), αMEM (alpha Modified Minimum Essential Medium), etc., can be used.

[0056] When a serum component is added to the medium for proliferation culture, fetal bovine serum can be used as the serum component. The concentration of the serum component can be about 10% (V / V).

[0057] The culture period may be, for example, about 1 day to 2 weeks.

[0058] The culture medium can be exchanged as needed, and the culture conditions can be in accordance with conventional methods.

[0059] The third step of the production method of the present invention is a step of inducing skeletal muscle myoblasts to differentiate into myotubes. In this step, skeletal muscle myoblasts and surrounding cells become multinucleated through cell fusion to form myotubes. Myotubes further mature to form muscle fibers.

[0060] For example, the above-mentioned culture can be carried out in a culture medium for differentiation induction (multinucleation) by a method well known to those skilled in the art. As a method for suitable culture, a method of culture under the conditions of about 37°C and a carbon dioxide concentration of about 5 to 10% (V / V) can be exemplified, but it is not limited thereto. For example, a well-known CO2 incubator can be used for culture under the above-mentioned conditions.

[0061] It is known that when nutrients become low, myoblasts will roll up to surrounding cells and begin to multinucleate. Therefore, it is preferred to use a medium with fewer nutrients than the aforementioned proliferation culture to induce differentiation into myotubes. Since it is known that horse serum has fewer nutrients than fetal bovine serum, horse serum can be used. The concentration of serum components can be about 2% (V / V).

[0062] In this way, three-dimensional muscle tissue is produced. Example

[0063] The present invention will be further specifically described below by way of examples. However, the following examples do not limit the scope of the present invention.

[0064] 1. Vitamin C-based Differentiation Induction of Bovine Myoblasts The effect of vitamin C on differentiation culture of bovine myoblasts was studied. Vitamin C derivatives were added at concentrations varying from 0 to 1000 μM during differentiation culture, and the myotube formation rate was measured.

[0065] [method] Myoblasts were collected from bovine masseter muscle and cultured at 1.0 × 10 5 Cells / well were seeded into a 12-well collagen-coat dish.

[0066] After culturing for 3 days in a growth medium (10% FBS F12 / DMEM, hereinafter referred to as GM), the medium was replaced with a differential medium (2% HSF12 / DMEM, hereinafter referred to as DM) supplemented with a vitamin C derivative (ascorbic acid phosphate), and further cultured for 10 days. The concentration of ascorbic acid phosphate added was 0 μM, 10 μM, 100 μM, and 1000 μM, and the medium was exchanged every other day.

[0067] After 10 days of culture in DM, the cells were fixed with 4% PFA, immunostained for α-actinin (SAA) of the sarcomere, and stained for the nucleus with Hoechst staining. SAA is a protein of the Z membrane that constitutes the sarcomere structure (the smallest unit of muscle), and is expressed with the formation of myotubes. Therefore, cells stained by SAA staining were judged as myotubes, and the fusion index (number of nuclei in myotubes / total number of nuclei) was calculated based on the staining results. In addition, for each ascorbic acid phosphate (VC) concentration, the fusion index was calculated from 10 stained images with a total number of nuclei of 100 or more, and the average was obtained.

[0068] Figure 4 The results are shown in .

[0069] [Study] Myotube formation was observed on the fourth day after transition to DM in both the case of ascorbyl phosphate addition and the case of no ascorbyl phosphate addition. The number of myotubes formed increased in a manner dependent on the ascorbyl phosphate concentration, and the length and thickness of the formed myotubes also increased, suggesting that ascorbyl phosphate is also involved in the maturation of myotubes.

[0070] 2. Formation of 3D Muscle Tissue Using Bovine Myoblasts Bovine myoblasts were embedded in hydrogels, fixed at both ends and cultured to produce muscle tissues with a length of 7 mm. The hydrogel composition and cell density were varied to study the conditions for the formation and maturation of muscle tissues.

[0071] [method] Anchors 9 were fixed on PDMS, and cell-containing hydrogels were seeded in a manner that connected the anchors, thereby forming elongated muscle tissues.

[0072] PDMS was poured into a model made using a stereolithography device and solidified to make an anchor fixing member 10 and a width adjustment stamp 11. The length was fixed to 7 mm by the anchor fixing member 10, and the width of the tissue at the start of culture was fixed to 1 mm by the width adjustment stamp 11. In addition, anchor 9 was made using a stereolithography device. Anchor 9 had 18 cylindrical fixing parts with a diameter of 100 μm and a height of 1.0 mm. Cell adhesion was promoted by covering the fixing parts with fibronectin, and both ends of the tissue were fixed.

[0073] (1) Study on hydrogel composition Different hydrogels were prepared by mixing type 1 collagen and matrigel at ratios of 1:0, 1:1, and 0:1.

[0074] Myoblasts 2.0×10 6 The cells were mixed with 40 μL of each of the three hydrogels (5.0×10 7 Cells / mL), and the width was adjusted to 1 mm by adjusting the width of the stamp 11. In order to solidify the hydrogel, after culturing at 37°C for 10 minutes in a CO2 incubator, 10% FBS F12 / DMEM was added and cultured at 37°C and 5% CO2. On the day after tissue formation, the culture medium was replaced with 2% HS F12 / DMEM and cultured for a further 12 days. The culture medium was replaced every other day. After 12 days, immunostaining was performed to confirm maturity and orientation.

[0075] (2) Cell concentration study Myoblasts were embedded in type 1 collagen and cultured. At this time, the number of embedded cells was 1.0×10 6 cells, 2.0×106 cells, 4.0×10 6 cells (2.5×10 7 cells / mL, 5.0×10 7 cells / mL, 10.0×10 7 The culture method was the same as in (1), and the cells were cultured in 2% HS F12 / DMEM supplemented with 100 μM ascorbic acid phosphate from the second day of culture.

[0076] Figure 5 The method is schematically shown in FIG. Figure 6 The results of the study on the composition of the hydrogel are shown in Figure 7 The results of the cell concentration studies are shown in .

[0077] [Investigation] (1) Hydrogel composition Cell orientation was confirmed in the composition containing collagen. On the other hand, Matrigel tended to have uneven cell distribution and was not good at maintaining tissue shape. No significant difference was observed in the maturity of tissue among the three conditions.

[0078] (2) Cell concentration study In 5.0×10 7 Sarcomere-like structures were confirmed under conditions of 5.0×10 cells / mL collagen gel or more. 7 The maximum value was 1 cell / mL collagen gel.

[0079] 3. Formation of Three-Dimensional Large Muscle Tissues Using Myoblast Modules In order to make a larger tissue, myoblasts were embedded in a three-dimensional module and stacked. If the tissue width of the muscle tissue increases, the movement direction of the muscle tissue cells becomes random, so the directionality cannot be maintained. Therefore, in order to maintain the directionality of the tissue, a PDMS mold was used to produce: a roughly rectangular first cell module with parallel roughly rectangular holes, and a second cell module with holes in different positions from the first cell module.

[0080] By alternately stacking two types of cell modules with different hole positions, tissues designed to be narrow in width fill the gaps between each other and build a piece of muscle tissue.

[0081] At a concentration of 5.0×10 7Cells / mL collagen (collagen) were mixed with myoblasts and type 1 collagen gel to obtain a collagen gel containing myoblasts. The collagen gel containing myoblasts was seeded on a rubber module, and a model stamp made of PDMS was placed to make a muscle cell module 15. In order to solidify the collagen gel (about 170 μL of collagen gel exists in each module), it was cultured at 37°C in a CO2 incubator for 10 minutes at 37°C, and then 10% FBS F12 / DMEM was added, and cultured for 24 hours at 37°C and 5% CO2. After 24 hours, the stamp was removed, and the muscle cell modules were stacked in such a way that the anchor fixing part of the muscle cell module overlapped with the anchor of the culture container. The culture container used was a culture container made by a 3D printer and treated with cell adhesion in advance by O2 plasma treatment to improve the adhesion of the cells. Starting from the day after the myoblast module was stacked, the culture medium was replaced with 2% HS F12 / DMEM, and the culture was carried out for 12 days while the culture medium was replaced every other day.

[0082] Figure 8 The method is schematically shown in FIG.

[0083] Fig. 9 The results of stacking three layers of bovine myoblasts are shown in FIG. Fig.10 The results of stacking 25 layers of mouse myoblasts are shown in FIG.

[0084] [Investigation] It was found that a single piece of oriented tissue can be produced by stacking myoblast modules. SAA staining confirmed at least the orientation of the outer side and the formation of myotubes in part. Description of Reference Numerals

[0085] 1: First cell module; 2: Second cell module; 3: Model impression; 4: Base material; 5: Hydrogel solution containing skeletal muscle myoblasts; 6: Clamping; 7: Overview diagram; 8: A-A' section view; 9: Anchor; 10: Anchor fixing parts; 11: Width adjustment die; 12: Muscle tissue; 13: Collagen gel containing cells (1 mm width); 14: gap (0.5 mm width); 15: Myocyte module; 16: Alternately stack the modules; 17: Design drawing; 18: Top view; 19: Section view.

[0086] The parent case of this divisional application also involves the following technical solutions. Item 1. A method for producing three-dimensional muscle tissue, comprising the following steps: Prepare a first cell module having a substantially rectangular shape and a second cell module having a substantially rectangular shape, wherein the first cell module contains skeletal muscle myoblasts in a hydrogel and has a plurality of substantially rectangular holes parallel to each other, and the second cell module contains skeletal muscle myoblasts in a hydrogel and has a plurality of substantially rectangular holes parallel to each other, wherein at least a portion of the holes of the second cell module are located at a different position in the vertical direction from the holes of the first cell module; Alternately stacking the manufactured first cell modules and the second cell modules to obtain a stacked body; and Induce skeletal muscle myoblasts to differentiate into myotubes. Item 2. The manufacturing method according to Item 1, wherein: The hydrogel contains more than 0.3 mg / mL of collagen. Item 3. The production method according to Item 1 or 2, wherein: The hydrogel contains 10 μM to 1000 μM ascorbic acid or a salt thereof or a derivative thereof. Item 4. The production method according to any one of Items 1 to 3, wherein The width of the holes of the first cell module and the second cell module in the short-side direction is 200 μm to 2000 μm. Item 5. The production method according to any one of Items 1 to 4, wherein The interval between adjacent holes of the first cell module and the second cell module is 200 μm to 2000 μm in the short-side direction. Item 6. The production method according to any one of Items 1 to 5, wherein The sizes of the first cell module and the second cell module are 3 mm or more in the short-side direction and 9 mm or more in the long-side direction. Item 7. The production method according to any one of Items 1 to 6, wherein A total of six or more of the first cell modules and the second cell modules are stacked. Item 8. The production method according to any one of Items 1 to 7, wherein The first cell module and the second cell module in the stacked body are fixed at both ends in the long-side direction. Item 9. The production method according to any one of Items 1 to 8, wherein The manufacturing method is a method for manufacturing edible three-dimensional muscle tissue. Item 10. The production method according to any one of Items 1 to 9, wherein The skeletal muscle myoblasts are bovine skeletal muscle myoblasts. Item 11. A three-dimensional muscle tissue obtained by the production method according to any one of Items 1 to 10.

Claims

1. A method for producing three-dimensional muscle tissue, comprising the following steps: Prepare a first cell module having a substantially rectangular shape and a second cell module having a substantially rectangular shape, wherein the first cell module contains skeletal muscle myoblasts in a hydrogel and has a plurality of substantially rectangular holes parallel to each other, and the second cell module contains skeletal muscle myoblasts in a hydrogel and has a plurality of substantially rectangular holes parallel to each other, wherein at least a portion of the holes of the second cell module are located at a different position in the vertical direction from the holes of the first cell module; The first cell module and the second cell module are alternately stacked to obtain a stacked body, In the above method, a substantially rectangular narrow band-shaped portion is formed between mutually parallel holes, and the holes of the cell module are holes that penetrate the cell module.

2. The manufacturing method according to claim 1, wherein: The hydrogel contains more than 0.3 mg / mL of collagen.

3. The manufacturing method according to claim 1, further comprising: In the first cell module, skeletal muscle myoblasts are induced to differentiate into myotubes.

4. The manufacturing method according to claim 3, wherein: The cell concentration of the cell module is 5.0×10 7 The hydrogel contains 10 μM to 1000 μM ascorbic acid or a salt thereof or a derivative thereof.

5. The manufacturing method according to claim 1 or 2, wherein: The width of the holes of the first cell module and the second cell module in the short-side direction is 200 μm to 2000 μm.

6. The manufacturing method according to claim 1 or 2, wherein: The interval between adjacent holes of the first cell module and the second cell module is 200 μm to 2000 μm in the short-side direction.

7. The manufacturing method according to claim 1 or 2, wherein: The sizes of the first cell module and the second cell module are 3 mm or more in the short-side direction and 9 mm or more in the long-side direction.

8. The manufacturing method according to claim 1 or 2, wherein: A total of six or more of the first cell modules and the second cell modules are stacked.

9. The manufacturing method according to claim 1 or 2, wherein: The first cell module and the second cell module in the stacked body are fixed at both ends in the long-side direction.

10. The manufacturing method according to claim 1 or 2, wherein: The manufacturing method is a method for manufacturing edible three-dimensional muscle tissue.

11. The manufacturing method according to claim 1 or 2, wherein: The skeletal muscle myoblasts are bovine skeletal muscle myoblasts.

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