Culture medium composition for transdifferentiation of muscle stem cells into adipocytes, and method for producing cultured meat by using same

Abstract

The present invention relates to a culture medium composition for transdifferentiation of muscle stem cells into adipocytes, and a method for producing cultured meat by using same, and, more specifically, to: a culture medium composition for transdifferentiation of muscle stem cells into adipocytes, comprising a combination of two or more selected from the group consisting of oleic acid, thiazolidinedione, and lecithin; a method for transdifferentiation of muscle stem cells into adipocytes by using the medium composition; a method for producing cultured meat by using the transdifferentiation method; cultured meat produced by the cultured meat production method; and a food composition comprising same.

Description

Culture medium composition for cross-differentiation of muscle stem cells into adipocytes and method for producing cultured meat using the same

[0001] This application claims priority to Republic of Korea Patent Application No. 10-2023-0184632, filed December 18, 2023, the entire disclosure of which is incorporated herein by reference.

[0002] The present invention relates to a culture medium composition for cross-differentiating muscle stem cells into adipocytes and a method for producing cultured meat using the same, and more particularly, to a culture medium composition for cross-differentiating muscle stem cells into adipocytes, comprising a combination of two or more selected from the group consisting of oleic acid, thiazolidinedione and lecithin, a method for cross-differentiating muscle stem cells into adipocytes using the medium composition, a method for producing cultured meat using the cross-differentiating method, cultured meat produced using the cultured meat production method, and a food composition comprising the same.

[0003] According to a recent report published by the Food and Agriculture Organization of the United Nations (FAO), the world's population is expected to increase by 0.6% annually from 7.64 billion in July 2018 to 9.2 billion in 2050. In line with this population growth, human meat consumption is also estimated to reach 465 million tons.

[0004] Accordingly, the Food and Agriculture Organization of the United Nations (FAO) has announced that an additional 200 million tons of meat production is needed annually to meet growing demand. Furthermore, recent outbreaks of livestock diseases, such as mad cow disease and foot-and-mouth disease, as well as cases of pathogens contained in livestock intestines not being killed during processing, leading to various illnesses, are causing significant social problems.

[0005] Recently, 'cultured meat' has been attracting attention as a solution to problems such as population growth and the occurrence of diseases in slaughtered livestock.

[0006] Cultured meat refers to edible meat obtained by extracting cells from living animals and multiplying them using cell engineering technology. It can be considered a branch of cellular agriculture that obtains meat without going through the process of raising livestock. Cultured meat is also called alternative meat or artificial meat, and is called in vitro meat because it is grown in a test tube, clean meat because it is produced in a clean production facility rather than a traditional livestock facility, and lab-grown meat because some prototypes are made in a laboratory.

[0007] Cultured meat utilizes tissue engineering, a cell engineering technique. Cells are obtained from living animals, but the process involves painlessly harvesting and culturing stem cells. Therefore, cultured meat represents an ideal food production technology that preserves animal health while also reducing environmental pollution.

[0008] The biggest challenge in the production of cultured meat is the inability to accurately replicate the texture of real meat, which contains both intramuscular fat and fat. Currently, this requires culturing muscle and fat cells separately, then mixing the resulting muscle and fat. This means that the texture of meat with intramuscular fat cannot be replicated, and processing into forms like ground meat is the only option.

[0009] Accordingly, there have been attempts to produce cultured meat that mimics meat with intramuscular fat by co-culturing muscle and fat, but most of them have studied the interactions between different cells or within cells by culturing muscle cell lines such as C2C12 together with preadipocytes and factors necessary for culturing each fat and muscle cell (Non-patent Document 1).

[0010] Against this backdrop, the inventors of the present invention have made extensive efforts to develop a medium composition capable of cross-differentiating muscle stem cells into adipocytes in order to produce cultured meat in which intramuscular fat exists simultaneously. As a result, they have confirmed that when a combination of two or more of oleic acid, thiazolidinedione, and lecithin is treated in the medium composition, muscle stem cells are effectively cross-differentiated into adipocytes, thereby completing the present invention.

[0011] [Prior Art Literature]

[0012] [Non-patent literature]

[0013] (Non-patent literature 1) Anim Sci J. 2019 Mar;90(3):423-434.

[0014] Accordingly, an object of the present invention is to provide a culture medium composition for cross-differentiating muscle stem cells into adipocytes, the composition comprising two or more of oleic acid, thiazolidinedione, and lecithin.

[0015] Another object of the present invention is to provide a method for cross-differentiating muscle stem cells into adipocytes using the aforementioned medium composition.

[0016] Another object of the present invention is to provide a method for producing cultured meat using the method of cross-differentiating the aforementioned muscle stem cells into fat cells, and cultured meat produced therefrom.

[0017] Another object of the present invention is to provide a food composition comprising the cultured meat described above.

[0018] In order to solve the above-described problem, the present invention provides a culture medium composition for cross-differentiating muscle stem cells into adipocytes, comprising two or more selected from the group consisting of oleic acid, thiazolidinedione, and lecithin.

[0019] In the present invention, the thiazolidinedione may be at least one selected from the group consisting of pioglitazone, rosiglitazone, lobeglitazone, troglitazone, ciglitazone, darglitazone, englitazone, netoglitazone, and rivoglitazone.

[0020] In the present invention, the oleic acid may be included at a concentration of 100 to 250 μM.

[0021] In the present invention, the thiazolidinedione may be included at a concentration of 5 to 20 μM.

[0022] In the present invention, the lecithin may be included at a concentration of 5 to 35 μM.

[0023] In the present invention, when the medium composition includes oleic acid and thiazolidinedione, the oleic acid and thiazolidinedione may be included in a concentration ratio of 40:0.5 to 6.

[0024] In the present invention, when the medium composition includes oleic acid and lecithin, the oleic acid and lecithin may be included in a concentration ratio of 40:1 to 7.

[0025] In the present invention, when the medium composition includes oleic acid, thiazolidinedione and lecithin, the oleic acid, thiazolidinedione and lecithin may be included in a concentration ratio of 40:0.5 to 6:1 to 7.

[0026] In the present invention, the medium composition may additionally include at least one selected from the group consisting of insulin, 3-isobutyl-1-methylxanthine (IBMX), and dexamethasone.

[0027] In the present invention, the medium composition may not contain serum, or may additionally contain serum.

[0028] In the present invention, when the medium composition does not contain serum, it may additionally contain at least one selected from the group consisting of insulin, transferrin, and selenite.

[0029] In the present invention, the muscle stem cells may be cultured through passage 1 to 25 times.

[0030] The present invention also provides a method for cross-differentiating muscle stem cells into adipocytes using the aforementioned medium composition.

[0031] Additionally, the present invention provides a method for producing cultured meat using the method of cross-differentiating the aforementioned muscle stem cells into fat cells, and cultured meat produced therefrom.

[0032] In the present invention, the cultured meat may contain muscle cells and intramuscular fat at the same time.

[0033] Furthermore, the present invention provides a food composition comprising the cultured meat described above.

[0034] The culture medium composition for cross-differentiation of muscle stem cells into adipocytes according to the present invention can effectively induce cross-differentiation from muscle stem cells into adipocytes by a simple combination of treating two or more kinds of oleic acid, thiazolidinedione, and lecithin, thereby inducing cells in which intramuscular fat exists simultaneously, thereby producing cultured meat with improved flavor.

[0035] Figure 1 shows the rate of adipocyte cross-differentiation according to the type and concentration of additives included in the medium (left graph: compound combination added, right graph: compound combination not added).

[0036] Figure 2a shows the results of confirming the cross-differentiation pattern of fat cells by staining with Oil Red O after culturing muscle stem cells in the standard medium of DMEM (Dulbecco's Modified Eagle's Medium High glucose) containing FBS (Fetal Bovine Serum) treated with insulin, IBMX, and dexamethasone (control group), the control medium additionally treated with oleic acid at various concentrations (20, 100, 50, and 200 μM, respectively), and the control medium additionally treated with rosiglitazone at various concentrations (0.5, 1, 5, and 10 μM, respectively).

[0037] Figure 2b shows the results of culturing muscle stem cells in the control medium with lecithin at various concentrations (1.2, 2.4, 6, and 12 μg / ml, respectively) and in the control medium with combinations of oleic acid + rosiglitazone (OR), oleic acid + lecithin (OL), rosiglitazone + lecithin (RL), and oleic acid + rosiglitazone + lecithin (PRL), and confirming the cross-differentiation pattern into adipocytes by staining with Oil Red O.

[0038] Figure 3a shows the results of confirming the cross-differentiation pattern of fat cells by staining with Oil Red O after culturing muscle stem cells in DMEM standard medium containing FBS (control), the standard medium treated with oleic acid at various concentrations (20, 100, 50, and 200 μM, respectively), and the standard medium treated with rosiglitazone at various concentrations (0.5, 1, 5, and 10 μM, respectively).

[0039] Figure 3b shows the results of confirming the cross-differentiation pattern of fat cells by staining with Oil Red O after culturing muscle stem cells in the standard medium treated with lecithin at different concentrations (1.2, 2.4, 6, and 12 μg / ml, respectively), and in the standard medium treated with combinations of oleic acid + rosiglitazone (OR), oleic acid + lecithin (OL), rosiglitazone + lecithin (RL), and oleic acid + rosiglitazone + lecithin (ORL).

[0040] Figures 4a to 4f show the results of confirming the cross-differentiation pattern of muscle stem cells passaged 7 times into adipocytes by staining with Oil Red O. Figure 4a shows the result in the state (control) of treating insulin, IBMX, and dexamethasone (Chemical mix) in the DMEM standard medium containing FBS, Figure 4b shows the result in the state of treating combination 1 (oleic acid + rosiglitazone (OR): 200 μM + 5 / 10 / 15 / 20 μM) in the presence or absence of insulin, IBMX, and dexamethasone in the DMEM standard medium containing FBS, and Figure 4c shows the result in the state of treating combination 2 (oleic acid + lecithin (OL): 200 μM + 7.9 / 15.8 / 23.7 / 31.6) in the presence or absence of insulin, IBMX, and dexamethasone in the DMEM standard medium containing FBS. μM) in the presence or absence of insulin, IBMX and dexamethasone in DMEM standard medium containing FBS, and Fig. 4d shows the results in the presence or absence of combination 3 (rosiglitazone + lecithin (RL) 5 / 10 / 15 / 20 μM + 7.9 / 15.8 / 23.7 / 31.6 μM) in the presence or absence of insulin, IBMX and dexamethasone in DMEM standard medium containing FBS, and Fig. 4e shows the results in the presence or absence of combination 4 (oleic acid + rosiglitazone + lecithin (ORL): 200 μM + 5 / 10 / 15 / 20 μM + 7.9 / 15.8 / 23.7 / 31.6 μM) in the presence or absence of insulin, IBMX and dexamethasone in DMEM standard medium containing FBS, and Fig. 4f is a graph quantitatively showing the results of oil red O staining of each group.

[0041] Figures 5a to 5f show the results of confirming the cross-differentiation pattern of muscle stem cells passaged 12 times into adipocytes by staining with Oil Red O. Figure 5a shows the result in the state (control) of treating insulin, IBMX, and dexamethasone (Chemical mix) in the DMEM standard medium containing FBS, Figure 5b shows the result in the state of treating combination 1 (oleic acid + rosiglitazone (OR): 200 μM + 5 / 10 / 15 / 20 μM) in the presence or absence of insulin, IBMX, and dexamethasone in the DMEM standard medium containing FBS, and Figure 5c shows the result in the state of treating combination 2 (oleic acid + lecithin (OL): 200 μM + 7.9 / 15.8 / 23.7 / 31.6) in the presence or absence of insulin, IBMX, and dexamethasone in the DMEM standard medium containing FBS. μM) in the presence or absence of insulin, IBMX, and dexamethasone in DMEM standard medium containing FBS, and FIG. 5d shows the results in the presence or absence of insulin, IBMX, and dexamethasone in combination 3 (rosiglitazone + lecithin (RL): 5 / 10 / 15 / 20 μM + 7.9 / 15.8 / 23.7 / 31.6 μM) in the presence or absence of insulin, IBMX, and dexamethasone in DMEM standard medium containing FBS, and FIG. 5e shows the results in the presence or absence of insulin, IBMX, and dexamethasone in DMEM standard medium containing FBS in combination 4 (oleic acid + rosiglitazone + lecithin (ORL): 200 μM + 5 / 10 / 15 / 20 μM + 7.9 / 15.8 / 23.7 / 31.6 μM) in the presence or absence of insulin, IBMX, and dexamethasone, and FIG. 5f is a graph quantitatively showing the results of oil red O staining of each group.

[0042] Figure 6 is a graph comparing the results of Figures 4f and 5f according to the number of subcultures (left graph: P7, right graph: P12).

[0043] Figure 7 is a graph comparing the results of Figures 4f and 5f according to the presence or absence of a compound combination (insulin, IBMX, and dexamethasone) (left graph: without compound combination, right graph: with compound combination).

[0044] Figure 8 shows the results of staining with Oil Red O to determine the cross-differentiation rate and differentiation pattern of muscle stem cells into adipocytes depending on the presence or absence of serum in the medium.

[0045] Hereinafter, the present invention will be described in more detail.

[0046] All technical terms used in this invention, unless otherwise defined, have the same meaning as commonly understood by those skilled in the art. While preferred methods and samples are described herein, similar or equivalent methods are also included within the scope of this invention.

[0047] The present invention relates to a culture medium composition for cross-differentiating muscle stem cells into adipocytes, comprising two or more selected from the group consisting of oleic acid, thiazolidinedione, and lecithin.

[0048] In addition, the present invention relates to a culture medium composition comprising two or more selected from the group consisting of oleic acid, thiazolidinedione, and lecithin for use in cross-differentiating muscle stem cells into adipocytes.

[0049] Additionally, the present invention relates to the use of a composition comprising two or more selected from the group consisting of oleic acid, thiazolidinedione and lecithin in the preparation of a culture medium for cross-differentiating muscle stem cells into adipocytes.

[0050] It should be understood that although certain aspects are described herein with the term "comprising," other similar aspects described in terms of "consisting of" and / or "consisting essentially of" are also provided.

[0051] In the present invention, the terms “medium,” “culture medium,” “culture medium,” “medium composition,” “culture composition,” and “culture medium composition” refer to a culture solution containing nutrients capable of supporting the growth and survival of stem cells under in vitro culture conditions, and are not distinguished in this specification and may be used interchangeably.

[0052] The term "muscle stem cell" in the present invention includes precursor cells such as quiescent satellite cells and activated satellite cells called myoblasts. Muscle stem cells can differentiate into muscle cells, and livestock-derived muscle stem cells, in particular, can be used in actual cultured meat production, making them highly useful in agrobiological applications.

[0053] In the present invention, the term “cultivation of muscle stem cells” means that muscle stem cells proliferate while maintaining their differentiation potential, which is a stem cell potential.

[0054] In the present invention, the thiazolidinedione may be selected from the group consisting of pioglitazone, rosiglitazone, lobeglitazone, troglitazone, ciglitazone, darglitazone, englitazone, netoglitazone, and rivoglitazone.

[0055] In a specific embodiment of the present invention, Oil Red O staining was performed to confirm the adipocyte cross-differentiation rate when oleic acid, rosiglitazone, and lecithin were treated at different concentrations in the presence or absence of a compound combination (insulin, IBMX, and dexamethasone), and when two or more of these were treated in combination. As a result, as shown in Figs. 1, 2a 2b, 3a, and 3b, the adipocyte cross-differentiation efficiency increased as the concentrations of oleic acid, rosiglitazone, and lecithin increased regardless of the presence or absence of IBMX, insulin, and dexamethasone, and in particular, when IBMX, insulin, and dexamethasone were included, the adipocyte cross-differentiation efficiency increased significantly. It was confirmed that the combination showing the best adipocyte cross-differentiation rate was the case containing all of IBMX, insulin, dexamethasone, oleic acid, rosiglitazone, and lecithin.

[0056] Accordingly, the badge composition according to the present invention may include two or more selected from the group consisting of oleic acid, rosiglitazone and lecithin, in the presence or absence of IBMX, insulin and dexamethasone.

[0057] In another specific embodiment of the present invention, in order to confirm the efficiency of fat differentiation according to the concentration ratio of the combination of substances added to the medium in the presence or absence of a combination of compounds (insulin, IBMX, and dexamethasone), muscle stem cells passaged 7 times were cultured in a medium treated with a combination of substances at various concentration ratios, and then Oil red O staining was performed to evaluate the rate of fat cell cross-differentiation. As a result, it was confirmed that the adipocyte cross-differentiation rate was significantly increased when combination 1 (oleic acid + rosiglitazone (OR): 200 μM + 5 / 10 / 15 / 20 μM), combination 2 (oleic acid + lecithin (OL): 200 μM + 7.9 / 15.8 / 23.7 / 31.6 μM), or combination 4 (oleic acid + rosiglitazone + lecithin (ORL): 200 μM + 5 / 10 / 15 / 20 μM + 7.9 / 15.8 / 23.7 / 31.6 μM) was treated in the standard medium containing IBMX and dexamethasone, as shown in FIGS. 4a to 4f.

[0058] Accordingly, the medium composition according to the present invention may contain oleic acid at a concentration of 100 to 250 μM, preferably 150 to 250 μM, and most preferably 180 to 230 μM, but is not limited thereto. If oleic acid is contained in a concentration of less than 100 μM, the adipocyte cross-differentiation rate may be significantly reduced, and if oleic acid is contained in a concentration of more than 250 μM, the effect of increasing the adipocyte cross-differentiation rate according to the increase in concentration is minimal, and cytotoxicity may occur, resulting in the problem of muscle stem cells dying.

[0059] The medium composition according to the present invention may contain thiazolidinedione at a concentration of 5 to 20 μM, and the optimal concentration may be adjusted within the above range depending on the presence of other additional components.

[0060] The medium composition according to the present invention may contain lecithin at a concentration of 5 to 35 μM, and the optimal concentration may be adjusted within the above range depending on the presence of other additional components.

[0061] In the present invention, when the medium composition includes oleic acid and thiazolidinedione, the oleic acid and thiazolidinedione may be included in a concentration ratio of 40:0.5 to 6, preferably 40:1 to 4, and most preferably 40:2.5 to 4. When the concentration ratio is out of the above range, the synergistic effect of the adipocyte cross-differentiation rate according to the combination may decrease.

[0062] In the present invention, when the medium composition includes oleic acid and lecithin, the oleic acid and lecithin may be included in a concentration ratio of 40:1 to 7, preferably 40:1.4 to 6.6, and most preferably 40:3 to 6.4. When the concentration ratio is outside the above, the synergistic effect of the adipocyte cross-differentiation rate according to the combination may decrease.

[0063] In the present invention, when the medium composition comprises oleic acid, thiazolidinedione and lecithin, the oleic acid, thiazolidinedione and lecithin may be included in a concentration ratio of 40:0.5 to 6:1 to 7, preferably 40:2 to 6:3 to 6.6, more preferably 40:2 to 4:3 to 6.6, and most preferably 40:2 to 4:3 to 6.4. When the concentration ratio is out of the above range, the synergistic effect of the adipocyte cross-differentiation rate according to the combination may be significantly reduced.

[0064] In the present invention, the medium composition may additionally include at least one selected from the group consisting of insulin, 3-isobutyl-1-methylxanthine (IBMX), and dexamethasone.

[0065] In the present invention, the medium composition may not contain serum, or may additionally contain serum. In this case, the serum may be, for example, serum from various animals, such as chicken serum, horse serum, fetal calf serum (FCS), or fetal bovine serum (FBS), but is not limited thereto. The serum content may be 5% to 20%, preferably 5% to 15%, where % means volume (v / v)%.

[0066] In the present invention, when the medium composition does not contain serum, it may additionally contain at least one selected from the group consisting of insulin, transferrin, and selenite, and most preferably, it may contain all three of insulin, transferrin, and selenite.

[0067] The muscle stem cells used in the medium composition according to the present invention may be muscle stem cells that have been passaged 1 to 25 times, but are not limited thereto.

[0068] The term "subculture" used in the present invention refers to a method of continuously culturing cells in a healthy state for a long period of time by periodically transferring a portion of the cells to a new culture vessel and then changing the culture medium to continue culturing the cells for a long period of time. The term "passage" refers to the growth of cells from the initial seed culture in the culture vessel to the point where the cells grow vigorously (confluence) in the same culture vessel. As the number of cells increases in a culture vessel with a limited space, nutrients for growth are consumed or contaminants accumulate over a certain period of time, causing the cells to naturally die. Therefore, subculture is used as a method to increase the number of healthy cells. Typically, replacing the medium (culture vessel) once or dividing a cell group and culturing it is referred to as 1 passage. Methods known in the art may be used without limitation for subculture, but are preferably performed by mechanical separation or enzymatic separation.

[0069] In another specific embodiment of the present invention, in order to confirm the efficiency of fat differentiation according to the number of passages of muscle stem cells, muscle stem cells passaged 12 times were cultured in a medium treated with a combination of substances at various concentration ratios, and then Oil Red O staining was performed to evaluate the fat cell cross-differentiation rate. As a result, it was confirmed that the adipocyte cross-differentiation rate was significantly increased when combination 1 (oleic acid + rosiglitazone (OR): 200 μM + 5 / 10 / 15 / 20 μM), combination 2 (oleic acid + lecithin (OL): 200 μM + 7.9 / 15.8 / 23.7 / 31.6 μM), or combination 4 (oleic acid + rosiglitazone + lecithin (ORL): 200 μM + 5 / 10 / 15 / 20 μM + 7.9 / 15.8 / 23.7 / 31.6 μM) was treated in the standard medium containing IBMX and dexamethasone, as shown in FIGS. 5a to 5f.

[0070] Figure 6 shows a comparison of the adipocyte cross-differentiation rate according to the number of passages of muscle stem cells under the same conditions. It was confirmed that when combination 1 (oleic acid + rosiglitazone (OR): 200 μM + 5 / 10 / 15 / 20 μM), combination 2 (oleic acid + lecithin (OL) 200 μM + 7.9 / 15.8 / 23.7 / 31.6 μM) or combination 4 (oleic acid + rosiglitazone + lecithin (ORL): 200 μM + 5 / 10 / 15 / 20 μM + 7.9 / 15.8 / 23.7 / 31.6 μM) was treated in a standard medium containing insulin, IBMX and dexamethasone, an excellent adipocyte cross-differentiation rate was shown regardless of the number of passages.

[0071] Figure 7 shows the comparison of the adipocyte cross-differentiation rate of muscle stem cells passaged 7 and 12 times, respectively, according to the concentration ratio of the combination of substances added to the medium in the presence or absence of a combination of compounds (insulin, IBMX, and dexamethasone). When combination 1 (oleic acid + rosiglitazone (OR): 200 μM + 5 / 10 / 15 / 20 μM), combination 2 (oleic acid + lecithin (OL): 200 μM + 7.9 / 15.8 / 23.7 / 31.6 μM), or combination 4 (oleic acid + rosiglitazone + lecithin (ORL): 200 μM + 5 / 10 / 15 / 20 μM + 7.9 / 15.8 / 23.7 / 31.6 μM) was treated to the medium containing insulin, IBMX, and dexamethasone, overall excellent adipocyte It was confirmed that it shows the cross-differentiation rate.

[0072] It is common for the cross-differentiation rate to decrease as the number of cell passages increases, but when the medium composition of the present invention is used, the cross-differentiation rate into adipocytes can be maintained at a high level even when the number of muscle stem cells passages increases.

[0073] The cell culture medium that can be used in the present invention can be arbitrarily selected from among conventional media used in the relevant field suitable for stem cell culture. Furthermore, the culture conditions can also be arbitrarily selected from among appropriate conditions used in the relevant field. That is, the medium and culture conditions can be selected depending on the type of cell to be cultured. The medium used for culture is a cell culture minimum medium (CCMM), which can generally contain carbon sources, nitrogen sources, and trace elements.

[0074] The cell culture minimal media that can be used in the present invention include, but are not necessarily limited to, DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal essential Medium), BME (Basal Medium Eagle), RPMI1640, F-10, F-12, α-MEM (alpha-modified Minimum Essential Media), GMEM (Glasgow's Minimal essential Medium), IMDM (Iscove's Modified Dulbecco's Medium), DMEM / F12, etc. In the present invention, DMEM / F-12 media used in cell culture in the art can be used as the basic media.

[0075] In addition, the medium composition of the present invention preferably includes antibiotics, antifungals, and / or substances commonly used in the industry to prevent the growth of mycoplasma to prevent infection by bacteria, fungi, etc. As antibiotics, all antibiotics commonly used in cell culture, such as penicillin-streptomycin, can be used, and as antifungals, arforelicin B can be used, and as mycoplasma inhibitors, commonly used substances, such as gentamicin, ciprofloxacin, and azithromycin, can be used, but are not limited thereto. In addition, a commercially available antibiotic-antimycotic (AA) (Gibco) can be used.

[0076] The present invention also provides a method for cross-differentiating muscle stem cells into adipocytes using the aforementioned medium composition.

[0077] According to a specific embodiment of the present invention, the method for cross-differentiating muscle stem cells into adipocytes may include: (1) a culture medium comprising two or more selected from the group consisting of oleic acid, thiazolidinediones, and lecithin, (2) a culture medium further comprising one or more additives selected from the group consisting of insulin, IBX, and dexamethasone in (1), (3) a culture medium further comprising serum in (1) or (2), or (4) a medium further comprising one or more additives selected from the group consisting of insulin, transferrin, and selenite in (1).

[0078] The components included in the above culture medium and their effects are the same as those of the above-described medium composition, so their description is omitted.

[0079] The muscle stem cells used in the method for cross-differentiating muscle stem cells into fat cells according to the present invention may be undifferentiated muscle stem cells isolated from a non-human animal, and the method for isolating undifferentiated muscle stem cells may be performed using various methods known in the art, such as using cell growth rate and a sorter.

[0080] The method for cross-differentiating muscle stem cells of the present invention into adipocytes may include a step of proliferating the obtained muscle stem cells before culturing the muscle stem cells in the aforementioned culture medium. For example, the method for cross-differentiating muscle stem cells of the present invention into adipocytes may include a step of subculturing the muscle stem cells. As described above, the number of subculturing cycles may be 1 to 25, but is not limited thereto.

[0081] According to this specific embodiment, the method for cross-differentiating muscle stem cells into adipocytes may include: (a) a step of proliferating muscle stem cells; and (b) a step of culturing the proliferated stem cells in a culture medium defined in (1), (2), (3), or (4) to cross-differentiate the muscle stem cells into adipocytes.

[0082] According to another specific embodiment of the present invention, the method for cross-differentiating muscle stem cells into adipocytes may include: (a) a step of subculturing muscle stem cells; and (b) a step of culturing the subcultured stem cells in a culture medium defined in (1), (2), (3), or (4) to cross-differentiate the muscle stem cells into adipocytes.

[0083] The method for cross-differentiating muscle stem cells of the present invention into adipocytes may be, but is not limited to, culturing muscle stem cells in the culture medium described above for 2 to 21 days, preferably 7 to 14 days.

[0084] Additionally, the present invention provides a method for producing cultured meat using the method of cross-differentiating the aforementioned muscle stem cells into fat cells, and cultured meat produced therefrom.

[0085] Specifically, the method for producing cultured meat according to the present invention may include: a step of producing cultured meat by cross-differentiating muscle stem cells into adipocytes by culturing them in a culture medium defined in (1), (2), (3) or (4) above.

[0086] According to a specific embodiment of the present invention, the method for producing cultured meat may include: (a) a step of proliferating muscle stem cells; and (b) a step of culturing the proliferated stem cells in a culture medium defined in (1), (2), (3), or (4) to cross-differentiate the muscle stem cells into adipocytes to produce cultured meat.

[0087] According to another specific embodiment of the present invention, the method for cross-differentiating muscle stem cells into adipocytes may include: (a) a step of subculturing muscle stem cells; and (b) a step of culturing the sub-cultured stem cells in a culture medium defined in (1), (2), (3), or (4) to cross-differentiate muscle stem cells into adipocytes and produce cultured meat.

[0088] Here, the number of subcultures in step (a) and the culture period in step (b) are the same as described above, so their description is omitted.

[0089] In the present invention, the muscle stem cells used in the cultured meat production method are non-human animal muscle stem cells, and may be isolated from chicken, turkey, duck, quail, goose, pigeon, pheasant, ostrich, cow, deer, goat, sheep, horse, llama, camel, pig, rabbit, kangaroo, crocodile, turtle, lobster, salmon, tuna or whale, but are not limited thereto.

[0090] In the present invention, the cultured meat may contain muscle cells and intramuscular fat at the same time.

[0091] Furthermore, the present invention relates to a food composition comprising cultured meat produced by the cultured meat production method described above.

[0092] In the present invention, the food composition may be manufactured into snacks, dumplings, fried foods, stir-fried foods, soy sauces, seasonings, powder mixes, breads, beverages, processed canned foods, or processed noodles, but is not limited thereto. The form in which the cultured meat is added to a food may be ground into various particle sizes depending on the purpose of use in the food.

[0093] In the present invention, the cultured meat included in the food composition may be additionally treated with a coloring agent. Coloring agents refer to compounds that give color to food, and in order to reproduce the red meat color of beef or pork, artificial coloring agents, natural coloring agents, natural extracts [e.g., beet root extract, pomegranate fruit extract, cherry extract, carrot extract, red cabbage extract, red seaweed extract], modified natural extracts, natural juices (e.g., beet root juice, pomegranate juice, cherry juice, carrot juice, red cabbage juice, red seaweed juice), modified natural juices, FD&C (Food Drug Cosmetics) Red No. 3 (erythrosin), FD&C Green No. 3 (fast green FCF), FD&C Red No. 40 (allura red AC), FD&C Yellow No. 5 (tartazine), FD&C Yellow No. 6 (sunset yellow FCF), FD&C Blue No. 1 (brilliant blue FCF), FD&C Blue No. 2 (indigotine), titanium dioxide, Annatto, anthocyanins, betanins, beta-APE 8 carotenal, beta-carotene, black currant, burnt sugar, canthaxanthin, caramel, carmine / carminic acid, cochineal extract, curcumin, lutein, carotenoids, monascin, paprika, riboflavin, saffron, turmeric, and combinations thereof can be used, but are not particularly limited thereto. Additionally, a coloring agent such as nitrite and ascorbic acid, erythorbic acid, or a salt thereof that promotes the coloring of the nitrite can be further added as a coloring aid.

[0094] In the present invention, the cultured meat included in the food composition may further contain antioxidants, emulsifiers, salts, etc. to stabilize proteins in order to prevent fat oxidation, color change, or fat separation. The antioxidants, emulsifiers, salts, etc. may be used without limitation as long as they are widely used in the art.

[0095] The food composition comprising the cultured meat of the present invention can also be manufactured into a synthetic food product. In addition to the cultured meat, the synthetic food product may further include minerals, vitamins, supplementary vitamins, essential fatty acids, essential amino acids, enzymes, antioxidants, or a combination of two or more thereof.

[0096] Additionally, synthetic foods containing cultured meat may further include flavoring agents, flavor enhancers, sweeteners, coloring agents, colorants, bleaching agents, preservatives, sterilizers, antioxidants, leavening agents, coagulants, or thickeners.

[0097] The form of the above synthetic food may be a paste, puree, soup, pie, powder, granule, chip, tablet, capsule, or spread.

[0098] The cultured meat produced by the cultured meat production method of the present invention can be specifically processed or manufactured in the form of a meat product. The meat product may include, but is not limited to, sterilized meat products, ham, pressed ham, mixed pressed ham, sausage, mixed sausage, dried sausage (dried mixed sausage), semi-dried sausage (semi-dried mixed sausage), heated frozen sausage, bacon, dried stored meat, seasoned meat, ground processed meat, packaged meat, and other meat products.

[0099] [Example 1]

[0100] Confirmation of fat differentiation efficiency according to additive type and concentration

[0101] 1-1. Isolation and subculture of chicken muscle stem cells

[0102] Chicken muscle stem cells were obtained from cells extracted from the breasts and legs of chickens hatched on the 17th, 18th, and 19th days using fertilized eggs cultured in a sterile laboratory.

[0103] Specifically, muscle tissues isolated from chicken breasts and legs were minced into small pieces and treated with collagenase (final concentration 0.1%) in HBSS at 37°C for 45 min, while continuously stirring. The treated muscle tissues were forcibly dissociated using a 10-ml pipette, collected by centrifugation (300 g for 5 min), and washed once with PBS. The fragmented tissues were re-treated with 5 ml of trypsin (GIBCO, final concentration 0.1%) at 37°C for 45 min, while continuously stirring. After treatment, the suspension was diluted with 5 ml of standard medium (DMEM F / 12 90%, fetal bovine serum 10%, penicillin (100 IU / ml) and streptomycin (100 μg / ml)) and centrifuged at approximately 300 g for 10 min to collect the detached cells and tissue fragments. The trypsin suspension was diluted before centrifugation to further recover cells trapped in the viscous trypsin supernatant. The final pellet was resuspended in 5-10 ml of standard medium, and the tissue fragments were mechanically dissociated by passing them five times through a 10 ml pipette, then a 5 ml pipette, and finally a Pasteur pipette. Cells were collected from the resulting suspension by centrifugation (300 g, 10 min), resuspended in 2.5 ml of standard medium, and passaged five times through an 18-gauge needle to dissociate cell aggregates.

[0104] The suspension was then separated into 20% Percoll in a 15 ml tube. The 20% Percoll was added to the tube containing 60% Percoll to create a layer, which was treated with fetal bovine serum for approximately 2 hours to minimize cell adhesion to the wall. The cell suspension was layered on top of the 20% Percoll and centrifuged at 8°C and 15,000 g for 5 minutes. After centrifugation, cells at the 20 / 60% Percoll interface were collected, diluted with standard medium, and recovered by centrifugation (300 g for 10 minutes). The cell pellet was resuspended in 2 ml of standard medium by repeated passage through a Pasteur pipette and further passage through an 18-gauge needle. The cells were then counted and used for culture.

[0105] Cells were placed on collagen-coated tissue culture dishes and pre-incubated with 10% fetal bovine serum in DMEM F / 12 for 3 h to promote cell attachment. Cultures were maintained at 39°C in an atmosphere containing 5% CO2. For mass culture, cells were seeded at 2Y10 per 60 mm dish. 5 Cells were plated on a 60 mm dish. For sequential passage of cells, the culture medium was removed, the cells were washed with PBS, and then treated with 0.05% trypsin for 5 minutes at 37°C. The cells were then collected, centrifuged, and the cell pellet was resuspended in standard medium. The cells were seeded at 2Y10 per 60 mm dish. 5 The cells were replated at a concentration of 100 μg / mL. Briefly, they were cultured in 60 mm dishes for 4 to 5 days and then subcultured for the next passage.

[0106] 1-2. Manufacturing of badges

[0107] The culture medium for cross-differentiation contained 900 g / L DMEM (Dulbecco's Modified Eagle's Medium High glucose), a serum-free medium, 10% by volume of FBS, 11.2 g / L (500 μM) IBMX (3-Isobutyl-1-0methylxanthine), a phosphodiesterase inhibitor, 0.1 g / L (1.72 μM) insulin, and 4.0 g / L (0.1 μM) dexamethasone, a synthetic glucocorticoid.

[0108] Additionally, to determine the adipocyte differentiation rate under conditions without IBMX, insulin, and dexamethasone, a basal medium containing 900 g / L of DMEM (Dulbecco's Modified Eagle's Medium High glucose) and 10% by volume of FBS was also prepared.

[0109] To determine the differentiation efficiency of muscle stem cells into adipocytes according to the type and concentration of additives, the medium was treated with various combinations and concentrations of additives as shown in Table 1 below.

[0110] GroupBasic mediumFBSAdditives (with or without insulin, IBMX, dexamethasone)Control groupDMEMHigh glucose 10% FBSInsulin IBMX DexamethasoneOleic acidDMEMHigh glucose 10% FBS20 / 50 / 100 / 200 μM oleic acidInsulin, IBMX, dexamethasone + 20 / 50 / 100 / 200 μM oleic acidRosiglitazoneDMEMHigh glucose 10% FBS0.5 / 1 / 5 / 10 μM RosiglitazoneInsulin, IBMX, dexamethasone + 0.5 / 1 / 5 / 10 μM RosiglitazoneLecithinDMEMHigh glucose 10% FBS1.2 / 2.4 / 6 / 12 μM / ml lecithinInsulin, IBMX, Dexamethasone + 1.2 / 2.4 / 6 / 12 μM / ml lecithin Oleic acid + Rosiglitazone (OR) DMEM High glucose 10% FBS 200 μM oleic acid 10 μM Rosiglitazone Insulin, IBMX, dexamethasone + 200 μM oleic acid + 10 μM Rosiglitazone Oleic acid + Lecithin (OL) DMEM High glucose 10% FBS 200 μM oleic acid 12 μg / ml lecithin Insulin, IBMX, dexamethasone + 200 μM oleic acid + 12 μg / ml lecithin Rosiglitazone + Lecithin (RL) DMEM High glucose 10% FBS 10 μM Rosiglitazone 12 μg / ml lecithin Insulin, IBMX, dexamethasone + 10 μM Rosiglitazone +12μg / ml lecithin Oleic acid + rosiglitazone + lecithin (ORL) DMEM high glucose 10% FBS 200 μM oleic acid 10 μM rosiglitazone 12μg / ml lecithin Insulin, IBMX, dexamethasone + 200 μM oleic acid + 10 μM rosiglitazone + 12μg / ml lecithin

[0111] 1-3. Culturing muscle stem cells

[0112] Chicken muscle stem cells prepared in Example 1-1 were cultured in a medium containing 10% FBS in DMEM high glucose at 39°C for 3 days, and then seeded in a 12-well plate at 2.1×10 4 Cells were divided into wells. The following day, the cells were cultured in culture medium according to the conditions of each group in Table 1 at 39°C for 14 days.

[0113] 1-4. Oil Red O dyeing

[0114] After removing the medium from adipocytes cross-differentiated from chicken muscle stem cells, DPBS was added to wash the cells, mixed well, and then removed. Next, a 4% formalin solution was added at room temperature and left to stand for at least 1 hour. Next, after removing the formalin, the cells were washed three times with distilled water, 60% by volume isopropanol was added, left to stand for 5 minutes, then the isopropanol was removed and completely dried. Oil Red O working solution was added and left to stand at room temperature for 30 minutes. The Oil Red O solution was removed, and triple-distilled water was immediately added. After observing under a microscope, staining of the cells was confirmed, and photographs were taken. After discarding the water, the flask was dried, 100% isopropanol was added, and the flask was gently shaken for 10 minutes to ensure complete dissolution of the Oil Red O. Absorbance was measured at 490 nm using a microplate reader, and absorbance was measured again at 630 nm to correct the absorbance at 490 nm.

[0115] As a result, as confirmed in Figs. 1, 2a, and 2b, when IBMX, insulin, and dexamethasone were included, the adipocyte cross-differentiation efficiency increased as the concentration of oleic acid, rosiglitazone, and / or lecithin increased, and the best adipocyte cross-differentiation rate was shown when IBMX, insulin, dexamethasone, oleic acid, rosiglitazone, and lecithin were all included. In addition, as confirmed in Figs. 1, 3a, and 3b, when IBMX, insulin, and dexamethasone were not included, the adipocyte cross-differentiation efficiency decreased compared to when IBMX, insulin, and dexamethasone were included, but when two or more of oleic acid, rosiglitazone, and lecithin were added, the adipocyte cross-differentiation efficiency tended to increase.

[0116] [Example 2]

[0117] Confirmation of fat differentiation efficiency according to additive combination concentration

[0118] 2-1. Isolation and subculture of chicken muscle stem cells

[0119] Chicken muscle stem cells were prepared by passage 7 times using the same method as in Example 1-1.

[0120] 2-2. Preparation of media and cultivation of muscle stem cells

[0121] In order to confirm the efficiency of cross-differentiation of muscle stem cells into adipocytes according to the combined concentrations of additives, the media were prepared in the same manner as in Example 1-2, and oleic acid + rosiglitazone (combination 1), oleic acid + lecithin (combination 2), rosiglitazone + lecithin (combination 3), and oleic acid + rosiglitazone + lecithin (combination 4) were treated in various concentration combinations as shown in Table 2 below in DMEM media containing IBMX, insulin, and dexamethasone (insulin 1.72 μM, IBMX 500 μM, and dexamethasone 0.1 μM) and DMEM media not containing IBMX, insulin, and dexamethasone. As a control, DMEM media containing FBS was treated with insulin, IBMX, and dexamethasone.

[0122] GroupBasic mediumFBS supplement (with or without insulin, IBMX, dexamethasone)Control groupDMEM high glucose 10%FBSInsulin, IBMX, dexamethasoneCombination 1 (OR): Oleic acid + Rosiglitazone (200 μM + 5 / 10 / 15 / 20 μM)DMEM high glucose 10%FBSCombination 1Insulin, IBMX, dexamethasone + Combination 1Combination 2 (OL): Oleic acid + Lecithin (200 μM + 7.9 / 15.8 / 23.7 / 31.6 μM)DMEM high glucose 10%FBSCombination 2Insulin, IBMX, dexamethasone + Combination 2Combination 3 (RL): Rosiglitazone + Lecithin (5 / 10 / 15 / 20 μM + 7.9 / 15.8 / 23.7 / 31.6 μM)DMEM High Glucose 10% FBS Combination 3 Insulin, IBMX, Dexamethasone + Combination 3 Combination 4 (ORL): Oleic acid + Rosiglitazone + Lecithin (200 μM + 5 / 10 / 15 / 20 μM + 7.9 / 15.8 / 23.7 / 31.6 μM)DMEM High Glucose 10% FBS Combination 4 Insulin, IBMX, Dexamethasone + Combination 4

[0123] Chicken muscle stem cells, passaged 7 times, were cultured in DMEM high glucose medium containing 10% FBS at 39°C for 3 days, and then seeded in 12-well plates at 2.1X10 4 Cells were divided into wells. The next day after division, the cells were cultured at 39°C for 14 days in the culture medium according to the conditions of each group in Table 2, and the adipocyte differentiation rate was confirmed by Oil Red O staining as in Example 1-4.

[0124] Figure 4a shows the results of muscle stem cells cultured in a medium treated with insulin, IBMX, and dexamethasone, confirming that cross-differentiation into adipocytes did not occur.

[0125] Figure 4b shows the results for combination 1, which confirmed that the adipocyte cross-differentiation rate was significantly increased regardless of whether insulin, IBMX, and dexamethasone were included, and in particular, when combination 1 was included in DMEM high glucose medium containing insulin, IBMX, and dexamethasone, excellent adipocyte cross-differentiation rates were shown in all concentration combinations.

[0126] Figure 4c shows the results for combination 2, which confirmed that the adipocyte cross-differentiation rate was significantly increased regardless of whether insulin, IBMX, and dexamethasone were included, and in particular, when combination 2 was included in DMEM high glucose medium containing insulin, IBMX, and dexamethasone, excellent adipocyte cross-differentiation rates were shown in all concentration combinations.

[0127] Figure 4d shows the results for combination 3, which showed a tendency for the adipocyte cross-differentiation rate to increase when combination 3 was included in DMEM high glucose medium containing insulin, IBMX, and dexamethasone, but the effect was minimal compared to the combinations of other groups.

[0128] As a result for Combination 4 of Figure 4e, it was confirmed that the adipocyte cross-differentiation rate was significantly increased regardless of the inclusion of insulin, IBMX, and dexamethasone, and in particular, when Combination 4 was included in DMEM high glucose medium containing insulin, IBMX, and dexamethasone, an excellent adipocyte cross-differentiation rate was shown in all concentration combinations.

[0129] Figure 4f quantitatively shows the results of oil red O staining of each group above, and it can be confirmed that the adipocyte cross-differentiation rate significantly increases when combination 1, combination 2, or combination 4 is included in the DMEM high glucose medium containing insulin, IBMX, and dexamethasone.

[0130] [Example 3]

[0131] Determination of adipogenic differentiation efficiency according to the number of cell passages

[0132] Chicken muscle stem cells were prepared by passage 12 times using the same method as in Example 1-1, and the adipocyte cross-differentiation rate was confirmed using the same method as in Example 2.

[0133] Figure 5a shows the results of muscle stem cells cultured in a medium treated with insulin, IBMX, and dexamethasone, confirming that cross-differentiation into adipocytes did not occur.

[0134] Figure 5b shows the results for combination 1, which confirmed that the adipocyte cross-differentiation rate was significantly increased regardless of whether insulin, IBMX, and dexamethasone were included, and in particular, when combination 1 was included in DMEM high glucose medium containing insulin, IBMX, and dexamethasone, excellent adipocyte cross-differentiation rates were shown in all concentration combinations.

[0135] Figure 5c shows the results for combination 2, which confirmed that the adipocyte cross-differentiation rate was significantly increased regardless of whether insulin, IBMX, and dexamethasone were included, and in particular, when combination 2 was included in DMEM high glucose medium containing insulin, IBMX, and dexamethasone, excellent adipocyte cross-differentiation rates were shown in all concentration combinations.

[0136] Figure 5d shows the results for combination 3, which showed a tendency for the adipocyte cross-differentiation rate to increase when combination 3 was included in DMEM high glucose medium containing insulin, IBMX, and dexamethasone, but the effect was minimal compared to the combinations of other groups.

[0137] As a result for Combination 4 in Figure 5e, it was confirmed that the adipocyte cross-differentiation rate was significantly increased regardless of the inclusion of insulin, IBMX, and dexamethasone, and in particular, when Combination 4 was included in DMEM high glucose medium containing insulin, IBMX, and dexamethasone, an excellent adipocyte cross-differentiation rate was shown in all concentration combinations.

[0138] Figure 5f quantitatively shows the results of oil red O staining of each group above, and it can be confirmed that the adipocyte cross-differentiation rate significantly increases when combination 1, combination 2, or combination 4 is included in the DMEM high glucose medium containing insulin, IBMX, and dexamethasone.

[0139] Figure 6 shows the results of Examples 2 and 3 compared according to the number of subculture cycles, and it was confirmed that when Combination 1, Combination 2, or Combination 4 was included in DMEM high glucose medium containing insulin, IBMX, and dexamethasone, an excellent adipocyte cross-differentiation rate was shown regardless of the number of subculture cycles.

[0140] Figure 7 shows the results of Examples 2 and 3 compared with and without the compound combination (insulin, IBMX, and dexamethasone). Similarly, it was confirmed that when combination 1, combination 2, or combination 4 was included in the DMEM high glucose medium containing insulin, IBMX, and dexamethasone, an overall superior adipocyte cross-differentiation rate was exhibited.

[0141] [Example 4]

[0142] Confirmation of the effect of adipocyte cross-differentiation according to the presence or absence of serum in the medium

[0143] Instead of 10% by volume of FBS, DMEM / F12 medium containing 1% insulin-transferrin-selenite (ITS) was supplemented with 200 μM oleic acid, 10 μM rosiglitazone, 12 μg / ml lecithin, 0.5 mM IBMX, 0.1 μM dexamethasone, and 10 μg / ml insulin, and then muscle stem cells were cultured in the same manner as in Examples 1-3, and the efficiency of adipocyte cross-differentiation was confirmed by Oil Red O staining.

[0144] As a result, as confirmed in Fig. 8, even under conditions without serum, the cross-differentiation rate of fat cells was 96% compared to the condition with serum, indicating that the combination of insulin, IBMX, dexamethasone, oleic acid, rosiglitazone, and lecithin effectively induces cross-differentiation into fat cells.

[0145] While specific aspects of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A culture medium composition for cross-differentiating muscle stem cells into adipocytes, comprising two or more selected from the group consisting of oleic acid, thiazolidinedione, and lecithin.

2. A culture medium composition for cross-differentiating muscle stem cells into adipocytes, wherein the thiazolidinedione in paragraph 1 is selected from the group consisting of pioglitazone, rosiglitazone, lobeglitazone, troglitazone, ciglitazone, darglitazone, englitazone, netoglitazone, and rivoglitazone.

3. A culture medium composition for cross-differentiating muscle stem cells into adipocytes, wherein the oleic acid is contained in a concentration of 100 to 250 μM in the first paragraph.

4. A culture medium composition for cross-differentiating muscle stem cells into adipocytes, wherein the thiazolidinedione is included at a concentration of 5 to 20 μM in the first paragraph.

5. A culture medium composition for cross-differentiating muscle stem cells into adipocytes, wherein the lecithin is included in a concentration of 5 to 35 μM in the first paragraph.

6. A culture medium composition for cross-differentiating muscle stem cells into adipocytes, wherein in the first paragraph, when the medium composition comprises oleic acid and thiazolidinedione, the oleic acid and thiazolidinedione are contained in a concentration ratio of 40:0.5 to 6.

7. A culture medium composition for cross-differentiating muscle stem cells into adipocytes, wherein in the first paragraph, when the medium composition comprises oleic acid and lecithin, the oleic acid and lecithin are contained in a concentration ratio of 40:1 to 7.

8. A culture medium composition for cross-differentiating muscle stem cells into adipocytes, wherein in the first paragraph, when the medium composition comprises oleic acid, thiazolidinedione and lecithin, the oleic acid, thiazolidinedione and lecithin are contained in a concentration ratio of 40:0.5 to 6:1 to 7.

9. A culture medium composition for cross-differentiating muscle stem cells into adipocytes, further comprising at least one selected from the group consisting of insulin, 3-isobutyl-1-methylxanthine (IBMX), and dexamethasone in paragraph 1.

10. A culture medium composition for cross-differentiating muscle stem cells into adipocytes, the composition not containing serum or additionally containing serum in the first paragraph.

11. A culture medium composition for cross-differentiating muscle stem cells into adipocytes, wherein in clause 10, when the medium composition does not contain serum, the culture medium composition further contains at least one selected from the group consisting of insulin, transferrin, and selenite.

12. A culture medium composition for cross-differentiating muscle stem cells into adipocytes, wherein the muscle stem cells in the first paragraph are cultured by passage 1 to 25 times.

13. A method for cross-differentiating muscle stem cells into adipocytes using the medium composition of any one of claims 1 to 12.

14. Method for producing cultured meat using the method of Article 13.

15. Cultured meat produced by the cultured meat production method of Article 14.

16. In paragraph 15, the cultured meat is cultured meat in which muscle cells and intramuscular fat exist simultaneously.

17. A food composition comprising the cultured meat of claim 15 or 16.

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