Medium composition, medium for muscle hypertrophy, and method for muscle hypertrophy

Abstract

To provide a medium composition, a medium for muscle hypertrophy, and a muscle hypertrophy method, which enable enlargement of three-dimensional muscle tissue and production of artificial muscle tissue resembling biologically derived muscle in morphology.SOLUTION: The present invention provides a medium composition for use in a medium for culturing a muscle fiber-like tissue and hypertrophying the muscle fiber-like tissue, the composition containing a hormone and an antioxidant, and a medium for muscle hypertrophy containing the composition, and a muscle hypertrophy method. In some embodiments, the medium composition consists only of the hormone and the antioxidant; or the medium composition does not contain linoleic acid and linolenic acid.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a composition for a medium, a medium for muscle hypertrophy, and a method for muscle hypertrophy for producing a muscle fiber-like tissue used as a raw material for artificial muscle tissue by culturing.

Background Art

[0002] Artificial muscle tissue has many applications such as edible cultured meat, materials for bioactuators, etc., and artificial muscle tissue for treatment, etc., and its production is useful. For the production of artificial muscle tissue, it is required to culture a muscle fiber-like tissue to make it in a state close to the muscle tissue in a living body. So far, a technique for constructing a three-dimensional muscle tissue from myoblasts and a scaffold has been established. As a differentiation induction medium for myoblasts, a differentiation medium supplemented with horse serum has been used.

[0003] However, even when culturing skeletal muscle cells in vitro to form muscle fiber-like structures, compared with the morphology of in vivo muscle fibers in a living body, the fibers are thinner and often have inferior muscle strength and contraction amount. Also, when used as edible cultured meat, the texture is different from that of muscle tissue in a living body (meat collected from organisms such as livestock). Therefore, means for thickening muscle fibers (muscle hypertrophy) of in vitro cultured muscle fibers similar to those of muscle tissue in a living body are required.

[0004] To thicken the muscle fibers of cultured skeletal muscle, for example, techniques for applying physical operations such as mechanical load or electrical stimulation to the muscle fibers are considered. For example, as shown in Non-Patent Document 1, a technique for applying tension to muscle fibers by an anchor is considered. Also, as shown in Non-Patent Document 2, a technique for applying electrical stimulation to muscle fibers is considered. Furthermore, as shown by the present inventors in Non-Patent Document 3, a technique for applying a load similar to muscle training in organisms to muscle fibers has also been developed.

[0005] As a culture method for culturing muscle fibers, the B27 medium described in Non-Patent Document 4 and the like is known. In this technique, an action of differentiating cells derived from iPS cells into muscle fiber shapes is known.

Prior Art Documents

Non-Patent Documents

[0006]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Summary of the Invention

Problems to be Solved by the Invention

[0007] However, in techniques that physically manipulate muscle fibers such as Non-Patent Documents 1-3, sufficient muscle hypertrophy could not be obtained for the cultured muscle fiber-like tissue, and it was difficult to construct a mature muscle tissue having thick muscle fibers like those of biological muscle and a morphology close to that of a hypertrophied and developed muscle. For example, when mechanical stimulation or electrical stimulation was applied in the conventional technique, the muscle tissue sometimes became thicker depending on the presence or absence of the stimulation, but morphologically, the formation of endomysium and the migration of nuclei to the periphery of muscle fibers were not observed, and it was not possible to obtain cultured meat close to the morphology of biological muscle.

[0008] Therefore, as another aspect separate from physical operations, the inventors examined culture conditions, particularly the composition of the culture medium, to investigate whether the action of muscle hypertrophy could be obtained. If the action of muscle hypertrophy can be obtained by culturing, it was considered possible to obtain a cultured muscle fiber-like tissue having a structure similar to that of muscle collected from a living organism by culturing or by combining culturing with the above-described physical operation. In addition, in artificial edible meat, the use of a serum-free medium is required from the perspective of animal welfare, and it was considered useful if an artificial medium could be used for culturing cultured meat instead of the conventional equine serum medium. Then, attention was paid to the possibility that the B27 medium involved in the differentiation into muscle tissue in Non-Patent Document 4 has the action of muscle hypertrophy.

[0009] However, according to the research of the inventors, although the culture of muscle fiber-like tissue using the B27 medium showed an effect of thickening the muscle tissue, the action of muscle hypertrophy that was significantly recognized in three-dimensional muscle hypertrophy was not obtained. Furthermore, the B27 medium is a commercially available off-the-shelf product with an unclear composition and is also expensive, and there was a cost problem for mass production such as culturing artificial edible meat.

[0010] Therefore, the inventors further studied with the goal of obtaining a more effective action of muscle hypertrophy by specifying the composition of the medium having the action of muscle hypertrophy and optimizing the composition.

[0011] The present invention has been made in view of the above circumstances, and an object thereof is to provide a composition for a medium, a medium for muscle hypertrophy, and a method for muscle hypertrophy that can hypertrophy a three-dimensional muscle tissue and obtain an artificial muscle tissue having a form similar to that of a living body-derived muscle.

Means for Solving the Problems

[0012] To solve the above problems, the present invention has the following aspects. Aspect 1 of the present invention is A composition for use in a medium for culturing a muscle fiber-like tissue to hypertrophy the muscle fiber-like tissue, It is a composition for a medium containing a hormone and an antioxidant.

[0013] Aspect 2 of the present invention is The composition for a medium according to Aspect 1, which consists only of the hormone and the antioxidant.

[0014] Aspect 3 of the present invention is The composition for a medium according to Aspect 1 or 2, wherein the hormone contains one or more selected from the group consisting of insulin, transferrin, corticosteron, progesterone, and T3.

[0015] Aspect 4 of the present invention is The composition for a medium according to any one of Aspects 1 to 3, wherein the antioxidant contains one or more selected from the group consisting of SOD (superoxide dismutase) enzyme, catalase, glutathione, vitamin C, and vitamin E.

[0016] Aspect 5 of the present invention is The composition for a medium according to any one of Aspects 1 to 4, which does not contain linoleic acid and linolenic acid.

[0017] Aspect 6 of the present invention is Including insulin, transferrin, corticosteron, progesterone, and T3 as the hormone, The composition for a medium according to any one of Aspects 1 to 5, which contains vitamin A acetate, α-tocopherol acetate, α-tocopherol (vitamin E), ascorbic acid (vitamin C), SOD (superoxide dismutase) enzyme, catalase, and reduced glutathione as the antioxidant.

[0018] Aspect 7 of the present invention is A medium for muscle hypertrophy, containing the composition for a medium according to any one of Aspects 1 to 6.

[0019] Aspect 8 of the present invention is The medium for muscle hypertrophy according to Aspect 7, which is used for improving muscle contraction force by culturing the muscle fiber-like tissue.

[0020] Aspect 9 of the present invention is the muscle hypertrophy medium according to Aspect 7, which is used for increasing the muscle fiber area by culturing the muscle fiber-like tissue.

[0021] Aspect 10 of the present invention is the muscle hypertrophy medium according to Aspect 7, which is used for improving the traction force by culturing the muscle fiber-like tissue and applying a physical load to the muscle fiber-like tissue.

[0022] Aspect 11 of the present invention is the muscle hypertrophy medium according to Aspect 7, which is used for inducing the expression of muscle development-related factors by culturing the muscle fiber-like tissue.

[0023] Aspect 12 of the present invention is a muscle hypertrophy method for culturing a muscle fiber-like tissue to hypertrophy the muscle fiber-like tissue, the muscle hypertrophy method, wherein the muscle fiber-like tissue is cultured in a muscle hypertrophy medium containing a medium composition containing a hormone and an antioxidant.

[0024] Aspect 13 of the present invention is the muscle hypertrophy method according to Aspect 12, wherein the medium composition consists of only the hormone and the antioxidant.

[0025] Aspect 14 of the present invention is the muscle hypertrophy method according to Aspect 12 or 13, wherein the medium composition does not contain linoleic acid and linolenic acid.

[0026] Aspect 15 of the present invention is the medium composition contains insulin, transferrin, corticosterone, progesterone and T3 as the hormone, The method for muscle hypertrophy according to the above-mentioned aspect 12 or 13, wherein the antioxidant substance includes vitamin A acetate, α-tocopherol acetate, α-tocopherol (vitamin E), ascorbic acid (vitamin C), SOD (superoxide dismutase) enzyme, catalase, and reduced glutathione.

[0027] Aspect 16 of the present invention is The method for muscle hypertrophy according to the above-mentioned aspect 12 or 13, wherein the muscle fibrous tissue is cultured to improve muscle contraction force.

[0028] Aspect 17 of the present invention is The method for muscle hypertrophy according to the above-mentioned aspect 12 or 13, wherein the muscle fibrous tissue is cultured to increase muscle fiber area.

[0029] Aspect 18 of the present invention is The method for muscle hypertrophy according to the above-mentioned aspect 12 or 13, wherein the muscle fibrous tissue is cultured and a physical load is applied to the muscle fibrous tissue to improve traction force.

[0030] Aspect 19 of the present invention is The method for muscle hypertrophy according to the above-mentioned aspect 12 or 13, wherein the muscle fibrous tissue is cultured to induce the expression of muscle development-related factors.

Advantages of the Invention

[0031] According to the present invention, a culture medium composition, a culture medium for muscle hypertrophy, and a method for muscle hypertrophy are obtained, which can hypertrophy three-dimensional muscle tissue and obtain an artificial muscle tissue similar to living body-derived muscle.

Brief Description of the Drawings

[0032]

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Embodiments for Carrying Out the Invention

[0033] Hereinafter, embodiments of the composition for culture medium, the culture medium for muscle hypertrophy, and the method for muscle hypertrophy according to the present invention will be shown and described. However, the present invention is not limited to the following embodiments.

[0034] (Composition for culture medium) The composition for culture medium of the present embodiment is a composition for use in a culture medium for culturing muscle fiber-like tissue to hypertrophy the muscle fiber-like tissue, and contains a hormone and an antioxidant.

[0035] The composition for culture medium is a composition that may be contained in the culture medium. The composition for culture medium of the present embodiment is used in a culture medium for culturing muscle fiber-like tissue to hypertrophy the muscle fiber-like tissue. This culture medium is preferably the culture medium for muscle hypertrophy described later.

[0036] The muscle fiber-like tissue is a tissue composed of muscle cells and the like. As the cells, various cells constituting the muscle fiber-like tissue can be used to prepare it. Specific examples of the cells constituting the muscle fiber-like tissue include skeletal muscle cells, myoblasts, fibroblasts, adipocytes, or adipose stem cells. As the origin of the cells, livestock, experimental animals, etc. can be used. For example, cows are preferably used as livestock, and mice are preferably used as experimental animals.

[0037] Hypertrophy of muscle fiber-like tissue means that the fibers of the muscle fiber-like tissue become thicker. That the fibers become thicker may mean that it can be confirmed that the fibers become thicker in observations with a microscope or the like, or it may also be confirmed by an increase in the value measured for the thickness of the fibers. The thickness may be determined from an increase in the diameter or cross-sectional area of the fibers.

[0038] In particular, it is preferable that hypertrophy of three-dimensional muscle tissue can be confirmed. The three-dimensional muscle tissue refers to a tissue that constitutes a three-dimensional tissue, different from the two-dimensional tissue cultured on a plate or the like.

[0039] In addition, when muscle hypertrophy occurs, since the contraction distance (the difference before and after muscle fiber contraction) increases, it may be determined that muscle hypertrophy has occurred when the contraction distance increases. The contraction distance may be measured, for example, as the distance between devices that fix both ends of the muscle fiber-like tissue.

[0040] The composition for the medium contains a hormone and an antioxidant. The hormone can be appropriately selected from those conventionally known. In particular, the hormone preferably contains at least one selected from the group consisting of insulin, transferrin, corticosterone, progesterone, and T3. Among these, it is more preferable to contain insulin or transferrin, and even more preferable to contain insulin and transferrin. It is also preferable to contain all of insulin, transferrin, corticosterone, progesterone, and T3. The antioxidant can be appropriately selected from those conventionally known. In particular, the antioxidant preferably contains at least one selected from the group consisting of SOD (superoxide dismutase) enzyme, catalase, glutathione (including reduced glutathione), vitamin C, and vitamin E (including tocopherol and reduced tocopherol). It is also preferable to contain all of vitamin A acetate, α-tocopherol acetate, α-tocopherol (vitamin E), ascorbic acid (vitamin C), SOD (superoxide dismutase) enzyme, catalase, and reduced glutathione as the antioxidant. Furthermore, it is also preferable to contain insulin, transferrin, corticosterone, progesterone, and T3 as the hormone, and to contain vitamin A acetate, α-tocopherol acetate, α-tocopherol (vitamin E), ascorbic acid (vitamin C), SOD (superoxide dismutase) enzyme, catalase, and reduced glutathione as the antioxidant. The medium containing these may be generically referred to as NODA medium in this specification.

[0041] The inventors have found that by including the hormone and antioxidant in the medium, it has a remarkable effect on muscle hypertrophy.

[0042] The composition for the medium preferably consists only of a hormone and an antioxidant. Consisting only of a hormone and an antioxidant means that, in addition to the hormone and antioxidant that have a remarkable effect on the above-mentioned muscle hypertrophy, it contains substantially no other components. The medium composition may not contain progesterone among the above-mentioned hormones. Not containing progesterone means that it contains substantially no or hardly any detectable components. By the medium composition not containing progesterone, it is highly effective for two-dimensional muscle differentiation and can be expected to be applied to muscle hypertrophy in three dimensions.

[0043] The composition for the medium preferably does not contain linoleic acid and linolenic acid in particular. Fatty acids such as linoleic acid and linolenic acid have conventionally been included as nutrients for the medium, but the inventors have found that these components are not necessary for the above-mentioned muscle hypertrophy.

[0044] (Medium for muscle hypertrophy) The medium for muscle hypertrophy of the present embodiment contains the above-mentioned composition for the medium. In addition to the above-mentioned composition for the medium, the medium for muscle hypertrophy may appropriately contain medium components included for culturing within a range that does not deviate from the physiological conditions of muscle hypertrophy and cell culture.

[0045] The medium for muscle hypertrophy of the present embodiment may be a serum-free medium that does not contain animal serum. In cultured meat, it is considered preferable not to use serum in the medium from the perspective of animal welfare, so a medium that does not use serum is useful for culturing for cultured meat. When the muscle fiber tissue to be cultured is not for food, etc., the medium for muscle hypertrophy of the present embodiment may contain animal serum in addition to the above-mentioned composition for the medium. As the animal serum, horse serum etc. can be used.

[0046] The muscle hypertrophy medium of this embodiment is preferably a muscle hypertrophy medium for improving muscle contraction force by culturing the muscle fiber-like tissue. Here, muscle contraction force is also referred to as the traction force due to muscle contraction. Specifically, muscle contraction force is the force applied to both ends of the muscle fiber-like tissue when an electrical stimulus is applied to the muscle fiber-like tissue. The improvement of muscle contraction force means that the force applied to the muscle fiber-like tissue cultured with the muscle hypertrophy medium is greater than that of the control tissue cultured with the control medium. Specifically, when an electrical stimulus is applied at 100 Hz and 10 V / cm, the muscle contraction force of the muscle fiber-like tissue may be 5 times or more, preferably 10 times or more, more preferably 19 times or more greater than that of the control tissue.

[0047] The muscle hypertrophy medium of this embodiment is preferably a muscle hypertrophy medium for increasing the muscle fiber area by culturing the muscle fiber-like tissue. Here, the increase in muscle fiber area means that when measuring the cross-sectional area of the muscle fiber, the muscle fiber-like tissue cultured with the muscle hypertrophy medium has increased compared to the control tissue cultured with the control medium (for example, general horse serum, etc.), preferably 1.5 times or more, more preferably 2.5 times or more. The measurement of the cross-sectional area of the muscle fiber may be performed using an electron microscope or by using immunostaining, etc.

[0048] The muscle hypertrophy medium of this embodiment may be a muscle hypertrophy medium for improving muscle contraction force by culturing the muscle fiber-like tissue and applying a physical load to the muscle fiber-like tissue. The increase in muscle contraction force is the same as described above. Applying a physical load to the muscle fiber-like tissue preferably involves performing an operation of applying a physical force in the stretching direction to the muscle fiber-like tissue. Specifically, by fixing both ends of the muscle fiber-like tissue and moving at least one end away from the other end, a physical force in the stretching direction is applied to the muscle fiber tissue. The physical load may be applied intermittently or continuously. In particular, it is preferable to continuously apply the physical load for 1 day or more, preferably 3 days or more, more preferably 10 days or more. By culturing the muscle fiber-like tissue and applying a physical load to the muscle fiber-like tissue, a muscle contraction force of 1.2 times or more, preferably 1.5 times or more, more preferably 2 times or more can be obtained compared to when only culturing is performed. Specifically, the muscle contraction force when applying electrical stimulation at 100 Hz and 10 V / cm may be 10 times or more, preferably 20 times or more, more preferably 38 times or more compared to the control tissue in the muscle fiber-like tissue.

[0049] The culture medium for muscle hypertrophy of the present embodiment may be a culture medium for muscle hypertrophy used for inducing the expression of muscle development-related factors by culturing the muscle fiber-like tissue. Induction of the expression of muscle development-related factors means, for example, that the muscle fiber-like tissue cultured in the culture medium for muscle hypertrophy has the expression of muscle development-related factors induced in the analysis result of RNA-seq compared to the control muscle fiber-like tissue (control tissue) cultured in a control medium (for example, normal horse serum). The induction of the expression of muscle development-related factors may be, for example, an increase in the expression levels of Acta2, Myl4, Grem1, Spp1, Myl2, Mustn1, Pgm5, Thbs1, Fxyd6, etc., which are muscle fiber-related genes. When performing expression analysis of mouse cells, the expression levels of MYH1, 3, 7, 8 that make up myofibrils, Lama2, Nid2, Dcn which are basement membrane factors expressed at maturity, Sgca, Sgcb, Sgcg, Utrn, DMD, NOS, Cav3, Dysf, Des, Itg7, 6, 1, etc. related to the cell membrane structure of muscular dystrophy may also increase. Here, the expression level may be determined, for example, in DEG detection, that genes with an FDR value of less than 0.05 have a significant increase or decrease in expression level. The muscle development-related factors may be these genes. The induction of the expression of muscle development-related factors may also be that in gene ontology (GO) analysis, a significant increase in the expression of genes related to muscle cells and contraction (either or both in the number of genes Genes and the fold enrichment of expression level Fold Enrichment) can be confirmed. The induction of the expression of muscle development-related factors may also be that in Gene Set Enrichment Analysis (GSEA) analysis, when performing an Enrichment Plot in GOBP_MUSCLE_HYPERTROPHY, a significant induction of the increase in the expression of muscle hypertrophy-related genes (such as igf1, etc.) can be confirmed.

[0050] (Method for muscle hypertrophy) The method for muscle hypertrophy of the present embodiment is a method for muscle hypertrophy in which a fibrous tissue is cultured to hypertrophy the fibrous tissue, and the fibrous tissue is cultured in a culture medium for muscle hypertrophy containing a composition for a culture medium containing a hormone and an antioxidant.

[0051] The composition for a culture medium and the culture medium for muscle hypertrophy may be appropriately used as described above. For example, the composition for a culture medium may consist only of the hormone and the antioxidant. Also, the composition for a culture medium may not contain linoleic acid and linolenic acid. The medium is preferably used for three-dimensional culture. For three-dimensional culture, techniques for culturing three-dimensional muscle tissues such as conventional cultured meat and artificial muscle tissues can be used. For example, a cell suspension obtained by performing two-dimensional culture or the like of cells and a cell suspension containing a hydrogel solution may be prepared and added to a culture device equipped with an anchor or the like, which is a member for holding muscle tissue, and cultured. As the hydrogel used in the hydrogel solution, an alginate gel, fibrin, Matrigel, a collagen gel, or the like can be used.

[0052] As described above, the composition for the medium contains insulin, transferrin, corticosterone, progesterone, and T3 as the hormones, and vitamin A acetate, α-tocopherol acetate, α-tocopherol (vitamin E), ascorbic acid (vitamin C), SOD (superoxide dismutase) enzyme, catalase, and reduced glutathione as the antioxidants. The NODA medium may be used.

[0053] The muscle hypertrophy method of the present embodiment may be the muscle hypertrophy method of culturing the muscle fiber-like tissue to improve muscle contraction force.

[0054] The muscle hypertrophy method of the present embodiment may be the muscle hypertrophy method of culturing the muscle fiber-like tissue to increase the muscle fiber area.

[0055] The muscle hypertrophy method of the present embodiment may be the muscle hypertrophy method of culturing the muscle fiber-like tissue and applying a physical load to the muscle fiber-like tissue to improve muscle contraction force.

[0056] The muscle hypertrophy method of the present embodiment may be the muscle hypertrophy method of culturing the muscle fiber-like tissue to induce the expression of muscle development-related factors.

[0057] (Effect of the present embodiment) According to this embodiment, a culture medium composition, a muscle hypertrophy medium, and a muscle hypertrophy method are obtained that can hypertrophy three-dimensional muscle tissue and obtain an artificial muscle tissue having a morphology similar to that of living body-derived muscle.

[0058] This embodiment provides a method for preparing a medium that brings about muscle hypertrophy of three-dimensional muscle tissue and a method for constructing a three-dimensional skeletal muscle tissue having a thick muscle fiber diameter like that of a living body. Currently, in the context of the aging population, research on mechanisms for maintaining motor function, particularly research on sarcopenia and muscular dystrophy, has become important. In skeletal muscle research, experimental animals and three-dimensional skeletal muscle tissue are used as research models. However, the muscle fiber diameter of the three-dimensional skeletal muscle tissue produced so far is thinner than that of a living body, and it is an immature tissue. The present invention is useful in the fields of drug discovery for muscle diseases, bioactuators, and cultured meat for food use, etc., which are application fields of skeletal muscle research, in order to achieve muscle hypertrophy and construction of mature tissue of three-dimensional skeletal muscle tissue.

[0059] In addition, the muscle hypertrophy medium and muscle hypertrophy method of this embodiment can obtain the effects of increasing muscle contractile force (traction force) by culturing with the muscle hypertrophy medium, increasing the cross-sectional area of muscle fiber-like tissue, further increasing muscle contractile force (traction force) by using physical load in combination, and inducing the expression of muscle development-related factors.

Examples

[0060] Examples are shown below. Note that the present invention is not limited to the examples.

[0061] In this example, first, the preparation of the novel differentiation medium of this embodiment and the culture of C2C12 cells were performed. Subsequently, the construction and culture of three-dimensional skeletal muscle tissue were performed. Finally, the cultured three-dimensional skeletal muscle tissue was evaluated.

[0062] [Formation of two-dimensional skeletal muscle fibers] (Preparation of differentiation medium) The differentiation medium of this example used DMEM high glucose as the basal medium and added a serum-free cocktail containing hormones, vitamins, and antioxidants.

[0063] As a commercially available serum-free cocktail for reference in the composition study of the control and this example, B27 supplement (B27, Sigma-Aldrich) was used. Although some of the contained compositions of B27 are publicly available as described later, all compositions and concentrations are unknown (not disclosed by the manufacturer). B27 was aliquoted and stored at -80 °C until use. After thawing promptly in a thermostatic bath, it was added to DMEM to achieve 0.1%, 2%, and 5%.

[0064] Subsequently, regarding the preparation of the differentiation medium of this example, five hormones that are publicly known to be contained in B27, an antioxidant shown to be useful in muscle tissue culture, and albumin serving as a carrier for fat-soluble components were combined to prepare a serum-free cocktail. As the five hormones, Insulin, Transfferin, Thyronine (T3), Progesterone, and Corticosterone were added, and as antioxidants, Vitamin A Acetate, α-Tocopherol acetate, α-Tocopherol, Ascorbic Acid, Superoxide Dismutase, Catarase, and Reduced Glutathione were added. This medium was tentatively named NODA medium.

[0065] As shown in Table 1, a serum-free cocktail (NODA cocktail) used in the NODA medium was prepared and stored at -20 °C until immediately before use. At the time of preparing the differentiation medium, the NODA cocktail was promptly thawed in a thermostatic bath and then added to DMEM to achieve 0.1%, 2%, and 5% to obtain the NODA medium, which was used for culture.

[0066]

Table 1

[0067] (Culture of myoblasts) The C2C12 cells (CRL1772, ATCC), a mouse sarcoma-derived myoblast cell line, and bovine myoblasts collected from bovine skeletal muscle were used. As the growth medium for myoblasts, Dulbecco’s Modified Eagle Medium (DMEM, High Glucose, Fujifilm Wako Pure Chemical Industries), Fetal bovine serum (10%, FBS, Gibco), and the antibiotics penicillin / streptomycin (1%, P / S, Sigma-Aldrich) were added and used.

[0068] The myoblasts were seeded onto a 24-well plate (BioCoat(TM), BD) coated with highly cell-adhesive Collagen-type 1 at 5.0×10 4 cells / cm 2 and cultured in the growth medium. After reaching confluence, they were switched to the aforementioned differentiation medium to induce differentiation, thereby forming two-dimensional skeletal muscle fibers. The cells were cultured in an incubator at 37 °C and 5% CO 2 and the medium was changed every two days.

[0069] [Construction of three-dimensional skeletal muscle tissue] A device for constructing three-dimensional skeletal muscle tissue was fabricated, and then three-dimensional skeletal muscle tissue was constructed.

[0070] (Method for fabricating the device) The device used for the construction and culture of skeletal muscle tissue was composed of three parts: a mold for shaping the hydrogel in which the cells were suspended, an anchor for the tissue fixation part, and an anchor holder for fixing the anchor. As an overview of these, the mold is a frame for shaping the hydrogel and has a structure that can be removed when adding the growth medium after the construction of three-dimensional skeletal muscle tissue. Also, the anchor has a structure for fixing both ends of the tissue to make the skeletal muscle tissue tensile and muscle-like. Note that all 3D models of the devices used in this example were designed with Fusion360, a 3D CAD software.

[0071] (Mold and Anchor Holder) The mold is a frame for shaping hydrogel and was used by being placed on the anchor holder. The anchor holder is a substrate for fixing an anchor that holds tissues in place and was designed to be circular so as to fit inside a 6-well plate. 3D models of both devices were fabricated using ClearV4 (Fromlabs) resin with a 3D printer (Form 3+, FormLabs).

[0072] After fabricating both devices, they were washed with a washing machine (Form Wash, Formlabs) to roughly remove the support material. The fabricated devices were immersed in the cleaning agent isopropyl alcohol (IPA) and washed with an ultrasonic cleaner for 15 minutes. They were blown off using an air blower so that there would be no residue of the support material on the surface. They were washed again with the ultrasonic cleaner for 5 minutes, the IPA on the surface was blown off using an air blower, and then dried at room temperature for 2 hours to completely remove the penetrated IPA. After drying, in order to sufficiently complete the curing reaction of the unreacted part of the resin, post-treatment was performed using a post-treatment device (Form Cure, Formlabs) with UV irradiation at 60 °C for 30 minutes.

[0073] Thereafter, both devices were coated with parylene C to a thickness of 4 μm using a vacuum deposition apparatus (LABCOTER PDS2010, Nippon Parylene). The parylene C polymer has resistance to chemical substances and moisture and is a highly biocompatible and stable substance. Also, by coating with a uniform thin film of parylene C polymer by vacuum deposition, leakage of the 3D printed resin can be prevented.

[0074] Subsequently, a non-adhesive treatment coating was applied to prevent cells from adhering to the mold and the anchor holder. A 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer (LIPIDURE(R), NOF Corporation) was dissolved in 99.5% ethanol at 0.5% w / v to prepare an MPC polymer coating solution. After completely immersing the device in the coating solution, excess MPC polymer coating solution was blotted up by arranging it on a Kimwipe. Then, it was heated and dried at 65 °C for 45 min on a hot plate.

[0075] (Anchor) The anchor is a device for fixing both ends of a tissue in order to suppress shrinkage due to cell aggregation and maintain tensile force. It is installed in the holding part of the anchor holder and used to tie up the tissue by utilizing cell adhesion. The designed 3D model was fabricated using Aqua Grey 8K (Phrozen) resin with a 3D printer (Phrozen Sonic Mini 8K, Phrozen). After roughly removing the support material after fabrication, it was immersed in IPA and washed with an ultrasonic cleaner for 15 min. It was blown off using an air blower so that there would be no residue of the support material on the surface.

[0076] Next, the fabricated anchor member was washed again with an ultrasonic cleaner for 5 min, the IPA was blown off using an air blower, and then it was dried at room temperature for 2 hours to completely remove the penetrated IPA. After drying, in order to sufficiently complete the curing reaction of the unreacted part of the resin, UV irradiation treatment was performed at 60 °C for 30 min using a post-treatment device (Form Cure, Formlabs). Also, parylene coating was performed to a thickness of 4 μm. Finally, a coating treatment was performed to promote cell adhesion to the anchor. The anchor was sterilized with an ozone / UV sterilizer for 30 min and immersed in a 50 μg / mL fibronectin (FUJIFILM Wako Pure Chemical Industries, Ltd.) / PBS solution and reacted at 37 °C for 60 min.

[0077] [Method for constructing three-dimensional tissue] Using C2C12 cells and hydrogel, three-dimensional skeletal muscle tissue was fabricated. The cells were detached from the culture dish, suspended in the hydrogel, and then molded using a mold.

[0078] (Cell recovery) The C2C12 cells cultured in the culture of the myoblasts were aspirated as much as possible with an aspirator from the growth medium in the culture dish, and the remaining medium was washed with 10 mL of PBS. 2 mL of Trypsin-EDTA solution (FUJIFILM Wako Pure Chemical Corporation) was added and reacted at 37 °C for 5 min. After confirming that all the cells were detached under a microscope, 8 mL of the growth medium was added to stop the enzymatic reaction of Trypsin. Then, by repeating pipetting with a 10 mL pipette, the detachment of the cells adhering to the bottom surface of the dish and the dispersion of the cell mass were performed. The cell suspension was collected, the cell concentration was calculated using a hemocytometer, the required amount of the suspension was divided into a 50 mL tube, and centrifuged at 200 g for 5 min.

[0079] (Construction of three-dimensional skeletal muscle tissue) The device was fabricated according to the procedure shown in the method for fabricating the device, and immediately before tissue construction, it was sterilized again using an ozone / UV sterilizer for 30 min, and then an anchor holder (1 piece), anchors (6 pieces), and a mold (1 piece) were assembled. Subsequently, a hydrogel solution containing 5 mg / ml of fibrin and 20% of Matrigel was prepared, and a cell suspension containing this hydrogel solution and C2C12 cells at a concentration of 2.5×10 7 cells / mL was prepared and dropped onto the device. Since the hydrogel solution gels depending on the temperature, the preparation of the cell suspension was carried out on ice.

[0080] As shown in Table 2, a cell pellet of 2.5×10 7 cells was suspended in 590 μL of the growth medium, 200 μL of the Matrigel solution and 10 μL of the Thorombin solution were added, and gently pipetted so that no bubbles were formed. Finally, 200 μL of the Fibrinogen solution was added and mixed, and then immediately dropped onto the device. Then, at 37 °C, 5% CO 2It was allowed to stand in the incubator for 30 minutes to gel. After confirming that the light transmittance of the tissue had changed due to gelation, 20 mL of the growth medium was added, and the mold was removed with sterilized forceps.

[0081]

Table 2

[0082] (Culture of three-dimensional skeletal muscle tissue) Using the day when the three-dimensional skeletal muscle tissue was constructed in the above step as day 0, it was cultured in the growth medium until day 3. On day 3, after washing once with PBS so that no growth medium remained, the above-mentioned differentiation medium was added and the culture was continued. In addition, the medium was changed every two days.

[0083] (Electrical stimulation culture of three-dimensional skeletal muscle tissue) The method of electrical stimulation during the culture of three-dimensional skeletal muscle tissue is shown. An electrical stimulation culture system (C-PaceEM, IonOptix) was used to culture while applying electrical stimulation. The electrode plate (C-Dish, CLD6WFC, IonOptix) for 6-well plates of the electrical stimulation culture system is an instrument that is repeatedly used and may cause contamination, so it was washed and sterilized before use.

[0084] First, the electrode plate was washed with 70% ethanol and dried, and then sterilized for 30 minutes using an ozone / UV sterilizer. The sterilized electrode plate was placed in a 6-well plate (Falcon) in which three-dimensional skeletal muscle tissue was being cultured, and cultured in the incubator while applying electrical stimulation. Electrical stimulation was applied for 10 days from the 4th day of culture, with a voltage of 12 V, a frequency of 1 Hz, and a pulse width of 1 ms, for 2 hours every day.

[0085] [Evaluation method] (Formation of two-dimensional skeletal muscle fibers) The myotube fusion rate in two-dimensional culture was used as an index of muscle differentiation, and the effect of promoting myotube formation by the differentiation medium was evaluated.

[0086] (Immunostaining of two-dimensional skeletal muscle fiber) As described above, after culturing myoblasts in a 24-well plate, immunostaining was performed. The differentiation medium was removed with an aspirator and washed with 1 mL of PBS. Then, 250 μL of 4% PFA / PBS (FUJIFILM Wako Pure Chemical Corporation) was added, and the cells were left standing at room temperature for 10 min for fixation. After fixation, the cells were washed twice with PBS for 3 min each, then 250 μL of 0.1% TritonX-100 (Sigma-Aldrich) / PBS was added, and the cells were left standing at room temperature for 10 min for membrane permeabilization treatment. Membrane permeabilization treatment is a process of increasing the permeability of the cell membrane using a surfactant so that the antibody can penetrate into the cell interior.

[0087] Next, after washing twice with PBS for 3 min each, 250 μL of 3% BSA (Bovine Serum Albumin, FUJIFILM Wako Pure Chemical Corporation) / PBS was added, and blocking treatment was performed at room temperature for 30 min. Blocking treatment is a process performed to prevent non-specific binding of the antibody.

[0088] Subsequently, 250 μL of each primary antibody diluted with 3% BSA / PBS described below was added, and the reaction was allowed to proceed overnight at 4 °C while infiltrating. After washing three times with PBS for 3 min again, 250 μL of the secondary antibody was added, and the cells were left standing at room temperature for 120 min while shielding from light to prevent fading of the fluorescent substance. Finally, after washing twice with PBS for 3 min, 1 mL of PBS was added, and the samples were stored at 4 °C until observation.

[0089] [Myogenic marker expression rate] [Preparation of frozen sections] [Freezing of samples] The cultured samples were immersed in 4% paraformaldehyde / PBS and fixed with shaking at 4 °C for 180 min. To prevent excessive reaction of 4% paraformaldehyde / PBS, the samples were replaced with PBS and washed three times for 5 min each. Then, by replacing the PBS infiltrated into the tissue with a Sucrose solution, the samples were immersed in 20% Sucrose / PBS and left standing at 4 °C for 2 days while shaking to prevent cracking during freezing.

[0090] The sample was placed on a Kimwipe to remove the excess Sucrose solution. Then, Tissue-Tek OCT compound (Sakura Finetek) was dropped onto the sample and allowed to penetrate for 5 min. The sample cut into 2-mm segments in the long axis direction was placed on the bottom surface of Cryomold No. 1 (Sakura Finetek), and OCT compound was dropped from above to fill the Cryomold.

[0091] Subsequently, in order to observe the cross-section of the skeletal muscle tissue, the sample was moved using forceps so that it was vertical and at the center of the Cryomold. Since the presence of air bubbles in the OCT compound would cause cracking during freezing, they were removed with forceps. Finally, the bottom of the Cryomold was immersed in liquid nitrogen and frozen over 1 min. The frozen sample was placed in a resin-sealed bag (Ziplock (registered trademark)) until sectioning and stored at -80 °C.

[0092] (Preparation of sections) The temperature inside the cryostat was set to -25 °C, and it was waited for 30 min until the temperature dropped. A little OCT compound was applied to the sample stage, and the frozen sample was attached and fixed. Using a microtome knife (s35, FEATHER), trimming was performed until skeletal muscle tissue appeared, and thin sections were cut at a thickness of 8 μm. The sections were attached to Crest-coated slides (Matsunami Glass Industry Co., Ltd.), dried at room temperature for 120 min, and then used for staining. Also, if staining was not performed within 2 days of sectioning, they were stored at -80 °C.

[0093] (Immunostaining of sections) The periphery of the section on the glass slide was surrounded with a liquid blocker (Daito Sangyo Co., Ltd.) and dried at room temperature for 10 min. The glass slide was placed in a staining bottle (KENIS) and washed 3 times with PBS for 5 min each while shaking. 600 μL of 0.1% TritonX-100 / PBS was dropped each time, and it was left standing in a moisture chamber for 15 min. After washing again with PBS for 5 min twice while shaking, 600 μL of 3% BSA / PBS was dropped each time, and it was left standing in the moisture chamber for 120 min.

[0094] After removing 3% BSA / PBS, 400 μL of the primary antibody solution was dropped one by one, and it was left standing at 4 °C overnight in a moisture chamber. The next day, after removing the primary antibody solution, it was washed 3 times with PBS for 5 min while shaking. 400 μL of the fluorescently labeled secondary antibody solution was dropped one by one, and it was left standing for 120 min in a light-shielded moisture chamber. Then, the secondary antibody solution was removed, and it was washed 3 times with PBS for 5 min while shaking.

[0095] Finally, Entellan New (mounting medium, Merck-Millipore) was dropped onto a cover glass (No. 1, Matsunami Glass Industry Co., Ltd.), and it was overlaid and mounted on a slide glass so that no bubbles would enter. After sucking up the excess mounting medium with filter paper, the edge of the cover glass was fixed with nail polish (AC MAKEUP).

[0096] [Test Results] (Comparison of Two-Dimensional Media) Figure 1 is a photographic diagram showing a comparison of the formation of two-dimensional skeletal muscle fibers using a conventional medium and the medium of this embodiment. In the figure, DMEM is used as the basal medium, and (a) is horse serum, (b) is horse serum and an ITS-X cocktail containing insulin, transferrin, and serine, (c) is the aforementioned NODA cocktail, and (d) is a medium supplemented with B27. For each of (a) to (d), the upper part of the figure shows an enlarged view with a scale bar of 200 μm, and the lower part shows an enlarged view with a scale bar of 50 μm.

[0097] Each culture was cultured in the proliferation medium from Day 0 to Day 1 and in the differentiation medium from Day 1 to Day 4, and then immunostaining was performed. The primary antibody for immunostaining was the MF20 antibody, a myosin heavy chain antibody, diluted 2-fold, the secondary antibody Alexa488 was diluted 1000-fold, and Hoechst was diluted 1000-fold for use.

[0098] In the figure, the dark color (Blue) indicates the signal of Hoechst, and the light color (Green) indicates the signal of MF20. That is, the dotted dark color indicates the cell nucleus, and the linear light color indicates the cytoplasm (muscle fiber). In the figure, for those cultured with the addition of horse serum, the muscle fibers are thicker and denser in the order of ITS-X cocktail and B27, and it can be seen that those with the addition of NODA cocktail have the densest and hypertrophied muscle fibers. Therefore, it was shown that the NODA medium containing the specific component NODA cocktail has a higher effect on muscle hypertrophy than culturing with B27 or serum.

[0099] (Comparison of three-dimensional skeletal muscle tissues) Using the device, three-dimensional tissues were constructed using the NODA medium of this example and a conventional medium, and immunological and morphological comparisons were performed.

[0100] Figure 2 is a photographic diagram showing a comparison of three-dimensional skeletal muscle tissues using a conventional medium (DMEM + 2% horse serum medium) and the medium of this embodiment. In the figure, (a) shows α-actinin, (b) shows laminin, (c) shows Hoechst, and (d) shows the merged signal of these. In the figure, the upper row is the medium with the addition of conventional horse serum to the basal medium, and the lower row is the NODA medium with the addition of the NODA cocktail of this example. The scale bar is 100 μm each. For each antibody in the immunostaining, the primary antibody was α-actinin (manufactured by abcam) diluted 500-fold, and the secondary antibody Alexa488 was diluted 1000-fold and used. Or, the primary antibody was laminin (manufactured by abcam) diluted 500-fold, and the secondary antibody Alexa546 was diluted 1000-fold and used. Hoechst was diluted 1000-fold and used.

[0101] α-Actinin is an actin-binding protein that is strongly expressed on the surface of skeletal muscle. In contrast, laminin and Hoechst are expressed throughout the tissue as signals in the cytoplasm and cell nucleus. When observing the signal of α - actinin and Merge, α - actinin is particularly strongly developed near the surface of the three - dimensional skeletal muscle tissue, and it is even more strongly developed in the NODA medium than in the horse serum medium. This result indicates that the NODA medium is developing muscle tissue in the three - dimensional skeletal muscle tissue.

[0102] Figure 3 is a partially enlarged view under the same conditions as Figure 2. (a) shows the conventional medium (horse serum - added medium), and (b) shows the NODA medium of this example. The scale bar indicates 50 μm.

[0103] In the figure, the light color is the signal of α - actinin. In the NODA medium, the signal of actinin is more prominent than in the horse serum medium, indicating that the NODA medium is developing muscle tissue in the three - dimensional skeletal muscle tissue.

[0104] Figure 4 is another photographic view showing a comparison of three - dimensional skeletal muscle tissues in the conventional medium for reference. It is a stereoscopic image observed by Whole IF confocal. In the figure, for the basal medium, (a) is the medium with horse serum added, and (b) is the medium with B27 added. The scale bar indicates 50 μm. In the figure, the dark color (Blue) indicates Hoechst, and the light color (Green) indicates the signal of MF20. In the B27 medium, slightly more muscle hypertrophy is observed compared to horse serum.

[0105] (Comparison of muscle fiber cross - sectional area) Regarding the muscle fibers, the light - colored signal in the tissue observation figure was analyzed by image analysis using a demonstration machine of Keyence, the cross - sectional area of each muscle fiber was measured, and the muscle fibers cultured in the conventional medium and the NODA medium of this example were compared respectively. Regarding the signal of immunostained α - actinin, the positive part was binarized automatically, the boundary was added manually, and the cross - sectional areas of 10 fibers each were measured and used as representative values.

[0106] Figure 5 is a graph showing the comparison of muscle fiber cross - sectional areas. HS represents the conventional medium (horse serum added to the basal medium), and Nоda represents the tissue cultured in the NODA medium of this example. The cross - sectional areas of the top 10 average values are shown for the muscle fibers. As shown in the figure, in the NODA medium, the average cross-sectional area of muscle fibers was significantly increased compared to the conventional medium, indicating that there is an effect of muscle hypertrophy.

[0107] (Measurement of contraction function) Using the device, the contraction function, which is a property of muscle, was examined for muscle tissues cultured using the conventional medium (with horse serum added) and the NODA medium of this example.

[0108] Figure 6 is a photographic diagram showing an outline of a method for measuring the contraction function of a three-dimensional skeletal muscle tissue. (a) shows the device used. The container is filled with a medium, a three-dimensional skeletal muscle tissue is housed, and electrodes are connected to the tissue. The electrodes are connected to an electrical stimulation device. Electrical stimulation was applied at 100 Hz and 10 V / cm. (b) is an enlarged view of the container in (a) and is a view observed from the side. In the state where no electrical stimulation is applied, the three-dimensional skeletal muscle tissue is not contracted or curved and is in a rod shape. (c) is a further enlarged view of the three-dimensional skeletal muscle tissue in the container in (b) and shows the state where electrical stimulation is applied.

[0109] As shown in Figure 6(c), the three-dimensional skeletal muscle tissue cultured in the HS (conventional horse serum) medium shows little contraction even when electrical stimulation is applied, while the three-dimensional skeletal muscle tissue cultured in the NODA medium contracts significantly by electrical stimulation. The distance between the dashed lines in the figure indicates the length by which the tissue contracts by applying electrical stimulation, that is, the contraction distance.

[0110] Figure 7 is a graph showing the contraction distances of muscle tissues cultured using the NODA medium and the conventional medium. It was shown that the muscle tissue using the NODA medium has a larger contraction distance and higher contraction performance compared to that using the conventional medium.

[0111] Figure 8 is a graph showing the contraction distances of muscle tissues. (a), (b), and (c) show the cases where the tissue width at the time of tissue construction is 1, 2, and 3 mm, respectively. The medium used is the NODA medium. It was shown that the smaller the tissue width at the time of tissue construction, the larger the contraction distance.

[0112] (Examination of culture medium composition) In this embodiment, it has been shown that using a NODA medium containing each hormone and each antioxidant can obtain the effect of muscle hypertrophy. Among these hormones and antioxidants, the components that contribute to muscle hypertrophy were investigated. Two-dimensional cultures were performed and compared for HS (conventional medium supplemented with horse serum), medium supplemented with B27, NODA4 (NODA medium), and medium with various components not added to the NODA medium.

[0113] Figure 9 is a photographic diagram comparing the formation of two-dimensional muscle fibers cultured using media with each component. "-(component name)" in the figure indicates the medium with each component reduced (not added) from the NODA medium. The number of cells is 3.0×10 4 cells / cm 2 . The scale bar is 400 μm. The density of linear muscle fibers was visually compared.

[0114] According to the figure, the medium without insulin added shows a significant decrease in the development of muscle fibers compared to the NODA medium. Also, the medium without transferrin added shows a slight decrease in the development of muscle fibers compared to the NODA medium. It is considered preferable that insulin and transferrin are included in the development of muscle fibers in the NODA medium.

[0115] (Improvement of contractile force by muscle hypertrophy medium) Regarding the muscle fibers cultured with the NODA medium, it was investigated whether the improvement of muscle contractile force (traction force) occurred. For the muscle fiber tissue cultured in the same way as the examination of the above culture medium composition, the contractile force was measured using a force measurement system. The force measurement system fixed the muscle fibers, applied electrical stimulation to this muscle fiber tissue every 3000 seconds, and measured the force (N) applied between both ends, that is, the muscle contractile force (traction force by the muscle) due to the stimulation. As the measurement operation, one end of the muscle tissue was connected to a capacitance-type micro force sensor, and the other end was fixed. Electrical stimulation of 100 HZ and 30 V / 3 cm was applied to the muscle tissue, and force measurement was performed from the voltage signal of the micro force sensor.

[0116] Figure 10 is a graph showing the muscle contraction force of muscle fibers cultured in NODA medium. (a) shows the muscle contraction force over time. (b) is a graph showing the statistical results of the contraction force for every 5 samples of muscle fibers. The contraction force of the control muscle group measured in the normal HS medium was about 0.1 mN, while that of the muscle fiber group cultured in NODA medium was about 1.7 mN. The contraction function was improved 19.8 times by culturing in NODA medium. That is, by culturing in NODA medium, the contraction force of muscle fibers was significantly improved.

[0117] (Increase in muscle fiber cross-sectional area by the muscle hypertrophy medium) Regarding the muscle fibers cultured in NODA medium, the morphology of the muscle tissue, particularly the change in the muscle fiber cross-sectional area, was examined by immunostaining and its image analysis. Figure 11 is a photographic view showing the immunostaining of muscle fibers cultured in NODA medium. Culturing was performed in the same manner as the examination of the medium composition, and the skeletal muscle tissue was formed and the cross-sectional image was observed. Control is the culture with HS (normal horse serum). In the figure, the immunostaining of white (Myosin heavy chain, muscle fiber), dark (Laminin, basement membrane component), and gray (DNA, cell nucleus) is shown. As shown in Control in the figure, the muscle fibers of a normal living body are wrapped by a basement membrane represented by dark color. On the other hand, in the tissue cultured with NODA medium this time, the formation of a laminin membrane, which is a basement membrane component, was observed around some muscle fibers.

[0118] Figure 12 is a graph showing the analysis results of the muscle fiber cross-sectional area. (a) shows the distribution of the muscle fiber cross-sectional area (n≥60). (b) shows the average value of the top 10. (c) shows the ratio of muscle fibers having a basement membrane-like structure. As shown in (a), the average value of the muscle fiber cross-sectional area was 149 μm in the control 2 , and in the muscle fiber tissue cultured with NODA medium of the present embodiment, it was 240 μm 2Moreover, the average value of the cross-sectional area increased, and the proportion of tissues with a large cross-sectional area also increased in terms of distribution. As shown in (b), in the cross-sectional area of the upper muscle fibers, the muscle fibers of this embodiment were approximately 2.6 times that of the control. As shown in (c), when measuring the ratio of all muscle fibers surrounded by the laminin membrane to all muscle fibers, it was significantly increased in the muscle fiber tissue cultured with the NODA medium of this embodiment.

[0119] (Improvement of contraction force by muscle hypertrophy medium and physical load) It was examined whether the muscle contraction force (traction force) was improved for muscle fibers cultured with the NODA medium and subjected to physical load. For the muscle fiber tissue cultured in the same manner as in the examination of the medium composition, a periodic stretching stimulus was applied as a physical load for 10 days during the culture. As a specific operation of the physical load, one end of the muscle tissue was pulled at 0.1 mm / s to 0.3 mm / s by a linear stage. A physical load was applied by stretching from the length at the time of culture. An electrical stimulus of 100 HZ and 30 V / 3 cm was also applied to the muscle tissue during stretching. The contraction force of this muscle fiber tissue was measured using the force measurement system.

[0120] Figure 13 is a graph showing the muscle contraction force of muscle fibers cultured with the NODA medium and subjected to physical load. (a) shows the muscle contraction force over time. (b) is a graph showing the contraction force statistically analyzed for every 5 samples of muscle fibers. The contraction force showed approximately twice the contraction force for the muscle fiber group cultured with the NODA medium compared to the muscle fiber group combined with the same culture and physical load. That is, by culturing with the NODA medium and further combining with physical load, the contraction force of the muscle fibers was further significantly improved.

[0121] (Upregulation of muscle development-related genes) The expression of muscle development-related genes was verified from the results of RNA-seq analysis comparing muscle fiber-like tissues differentiated with the NODA medium and muscle fiber-like tissues differentiated with normal horse serum. Figure 14 is a graph showing the analysis of RNA expression in muscle fiber-like tissue by principal component analysis (PCA). In HS (control muscle fibers, cultured with horse serum) and ND (muscle fibers cultured in NODA medium), it is shown that signals at different locations, i.e., gene expressions, vary in PCA. That is, it was speculated that the variation in gene expression was induced by the NODA medium. Figure 15 is a graph showing the analysis of RNA expression in muscle fiber-like tissue by extracting differentially expressed genes (DEGs). In muscle fibers cultured in NODA medium, the expression levels of Acta2, Myl4, Grem1, Spp1, Myl2, Mustn1, Pgm5, Thbs1, or Fxyd6, etc. were increased. In DEG detection, genes with an FDR value less than 0.05 were regarded as genes with significant increases or decreases. Although there may be other genes expressed in muscle tissues constructed from various other muscle cells, in the experiment of the mouse C2C12 cell line this time, the following genes varied. An increase in the expression of MYH1, 3, 7, 8, which constitute myofibrils, was confirmed. An increase in the expression of Lama2, Nid2, Dcn, which are basement membrane factors expressed at maturity, was confirmed. An increase in the expression of Sgca, Sgcb, Sgcg, Utrn, DMD, NOS, Cav3, Dysf, Des, Itg7, 6, 1, which are related to the cell membrane structure of muscular dystrophy, was confirmed.

[0122] Figure 16 is a graph showing the gene ontology (GO) analysis of RNA expression in muscle fiber-like tissue. An enrichment analysis was performed on the results of the analysis of expression variation using RNA-seq, and in muscle fibers cultured in NODA medium, it is shown that the tissues named in (a) and (b) increase in terms of the number of genes (Genes) and the fold enrichment of the expression level. As shown in the figure, it was shown that many gene names related to the formation, growth, and hypertrophy of muscle tissue were enhanced.

[0123] Figure 17 is a graph showing the Gene Set Enrichment Analysis (GSEA) of RNA expression in muscle fiber-like tissue. The changes in expression levels between two groups of HS (control muscle fibers) and muscle fibers cultured in NODA medium were analyzed by GSEA. The horizontal axis of the figure represents the gene rank, with greater changes in expression levels towards the left. The Enrichment score (ES) on the vertical axis of the figure examines all the genes included in the group in order from left to right, and adds or subtracts scores depending on whether the target gene set (here, genes related to muscle hypertrophy, GOBP MUSCLE HYPERTROPHY) is included or not. That is, if the large part of the value on the vertical axis is biased towards the left, it means that many genes with large changes in expression variation are included in the comparison between the two groups. As shown in the figure, a large part of the value on the vertical axis was seen to be towards the left. The graph of the GSEA results shows that the genes on the left are genes that promote muscle hypertrophy, and in the group with NODA medium, the expression of many genes that promote muscle hypertrophy was shown to be increased. The TMP (expression level score) of igf1, which is a gene related to muscle hypertrophy, increased by 19.13 times. From this result, it was shown that when comparing HS and NODA medium, the expression of genes related to muscle hypertrophy increased significantly, that is, culturing with NODA medium can induce an increase in the expression of genes related to muscle hypertrophy.

[0124] From the results of the above test examples, it was shown that culturing in NODA medium increases the expression of genes related to muscle cells and contraction.

Industrial Applicability

[0125] According to the present invention, a culture medium composition, a medium for muscle hypertrophy, and a method for muscle hypertrophy can be obtained, which can hypertrophy three-dimensional muscle tissue and obtain an artificial muscle tissue in a form similar to that of biological muscle.

Claims

1. A composition for use in a medium for culturing muscle fiber-like tissue to hypertrophy the muscle fiber-like tissue, The composition for the medium, comprising a hormone and an antioxidant.

2. The composition for the medium according to claim 1, consisting only of the hormone and the antioxidant.

3. The composition for the medium according to claim 1 or 2, wherein the hormone comprises one or more selected from the group consisting of insulin, transferrin, corticosteron, progesterone, and T3.

4. The composition for the medium according to claim 1 or 2, wherein the antioxidant comprises one or more selected from the group consisting of SOD (superoxide dismutase) enzyme, catalase, glutathione, vitamin C, and vitamin E.

5. The composition for the medium according to claim 1 or 2, which does not contain linoleic acid and linolenic acid.

6. Including insulin, transferrin, corticosteron, progesterone, and T3 as the hormone, The composition for the medium according to claim 1 or 2, including vitamin A acetate, α-tocopherol acetate, α-tocopherol (vitamin E), ascorbic acid (vitamin C), SOD (superoxide dismutase) enzyme, catalase, and reduced glutathione as the antioxidant.

7. A medium for muscle hypertrophy, comprising the composition for the medium according to claim 1 or 2.

8. The medium for muscle hypertrophy according to claim 7, for use in improving muscle contraction force by culturing the muscle fiber-like tissue.

9. The medium for muscle hypertrophy according to claim 7, for use in increasing muscle fiber area by culturing the muscle fiber-like tissue.

10. The medium for muscle hypertrophy according to claim 7, for use in improving muscle contraction force by culturing the muscle fiber-like tissue and applying a physical load to the muscle fiber-like tissue.

11. The medium for muscle hypertrophy according to claim 7, for use in inducing the expression of muscle development-related factors by culturing the muscle fiber-like tissue.

12. A muscle hypertrophy method for culturing muscle fiber-like tissue to hypertrophy the muscle fiber-like tissue, The muscle hypertrophy method, wherein the muscle fiber-like tissue is cultured in a medium for muscle hypertrophy comprising a composition for the medium comprising a hormone and an antioxidant.

13. The muscle hypertrophy method according to claim 12, wherein the composition for the medium consists only of the hormone and the antioxidant.

14. The method for muscle hypertrophy according to claim 12 or 13, wherein the composition for the medium does not contain linoleic acid and linolenic acid.

15. The composition for the medium contains insulin, transferrin, corticosterone, progesterone and T3 as the hormone, The method for muscle hypertrophy according to claim 12 or 13, wherein the antioxidant contains vitamin A acetate, α-tocopherol acetate, α-tocopherol (vitamin E), ascorbic acid (vitamin C), SOD (superoxide dismutase) enzyme, catalase and reduced glutathione.

16. The method for muscle hypertrophy according to claim 12 or 13, wherein the muscle fibrous tissue is cultured to improve muscle contraction force.

17. The method for muscle hypertrophy according to claim 12 or 13, wherein the muscle fibrous tissue is cultured to increase muscle fiber area.

18. The method for muscle hypertrophy according to claim 12 or 13, wherein the muscle fibrous tissue is cultured and a physical load is applied to the muscle fibrous tissue to improve muscle contraction force.

19. The method for muscle hypertrophy according to claim 12 or 13, wherein the muscle fibrous tissue is cultured to induce the expression of muscle development-related factors.

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