Spontaneously immortalized muscle stem cell line, and method for inducing spontaneous immortalization of muscle stem cells

Abstract

The present invention relates to a spontaneously immortalized muscle stem cell line, and a method for inducing spontaneous immortalization of muscle stem cells, and, more specifically, provides a muscle stem cell line in which spontaneous immortalization is induced without genetic manipulation, and a method for inducing spontaneous immortalization of muscle stem cells by controlling culture medium conditions.

Description

Naturally immortalized muscle stem cell line and method for inducing natural immortalization of muscle stem cells

[0001] This application claims priority to Republic of Korea Patent Application No. 10-2024-0104079, filed August 5, 2024, the entire disclosure of which is incorporated herein by reference.

[0002] The present invention relates to an immortalized muscle stem cell line and a method for inducing natural immortalization of muscle stem cells, and more particularly, to a muscle stem cell line in which natural immortalization is induced without genetic manipulation and a method for inducing natural immortalization of muscle stem cells by controlling the composition of a culture medium.

[0003] In the body, muscle stem cells exist in a quiescent state in muscle tissue. When muscle tissue is damaged, their proliferation function is activated, and they become myoblasts, which enter the myogenesis process, differentiate and fuse into myocytes to form muscle fibers, thereby repairing the damaged muscle. When cultured in vitro, myofiber differentiation can be artificially induced by treating with a low concentration of fetal bovine serum (FBS), or spontaneous myofiber differentiation can occur when muscle stem cells that have entered the myogenesis process proliferate and increase in density in the cell culture dish. Since muscle stem cells differentiated into myofibers lose their proliferative ability, repeating proliferation in an undifferentiated state is important for increasing cell culture efficiency. However, because it is difficult to control spontaneous muscle differentiation during in vitro culture, muscle stem cell culture efficiency is low.

[0004] Because of these characteristics, there are also culture methods that maintain the differentiation and proliferation abilities of muscle stem cells by treating FBS at a high concentration or adding growth factors known to have a direct effect on the proliferation of muscle stem cells, such as fibroblast growth factor 2 (FGF2), insulin or insulin-like growth factors (IGFs), such as IGF I or II, to high-concentration FBS. However, even when muscle stem cells are cultured using these methods, they typically lose their proliferation and differentiation abilities after 10 passages.

[0005] Animal cells are essential in the field of cultured meat. Animal cells suitable for cultured meat production must be able to maintain proliferative capacity for long periods, resulting in high cell proliferation efficiency. Simultaneously, they must also maintain differentiation potential, enabling differentiation into muscle fibers after mass proliferation. Various muscle stem cell culture methods have been reported. However, due to the low efficiency of in vitro culture of muscle stem cells, a method for culturing muscle stem cells for cultured meat has not yet been established. Furthermore, maintaining differentiation and proliferation potential in in vitro culture is difficult without high-concentration FBS treatment.

[0006] In this regard, non-patent document 1 discloses a method for preserving the stemness, proliferation, and differentiation of small muscle precursors by treating them with a p38 inhibitor, but it has not been confirmed whether the proliferation and differentiation abilities are maintained even when cultured for far more than 10 passages.

[0007] Against this backdrop, the present inventors obtained an immortalized muscle stem cell line capable of maintaining long-term differentiation and proliferation capabilities by controlling the composition of the culture medium in which muscle stem cells are cultured without genetic manipulation, thereby inducing natural immortalization of muscle stem cells, and completed the present invention by establishing a method for culturing muscle stem cells capable of inducing natural immortalization.

[0008] [Prior Art Literature]

[0009] [Non-patent literature]

[0010] (Non-patent Document 1) Ding, S., Swennen, GNM, Messmer, T. et al. Maintaining bovine satellite cells stemness through p38 pathway. Sci Rep 8, 10808 (2018).

[0011] Accordingly, the purpose of the present invention is to provide a naturally immortalized muscle stem cell line that maintains proliferative capacity and stem cell capacity for a long period of time.

[0012] Another object of the present invention is to provide a method for inducing spontaneous immortalization of muscle stem cells by controlling the composition of a culture medium and an immortalized muscle stem cell line produced by the method.

[0013] Another object of the present invention is to provide cultured meat obtained by culturing the naturally immortalized muscle stem cell line and a food composition containing the same.

[0014] Another object of the present invention is to provide a method for producing cultured meat or food using the naturally immortalized muscle stem cell line.

[0015] Another object of the present invention is to provide a use of the naturally immortalized muscle stem cell line for the production of cultured meat or food.

[0016] To solve the above-described problems, the present invention provides a naturally immortalized muscle stem cell line (accession number KCLRF-BP-00548) that maintains growth, proliferation, and differentiation for more than 10 passages.

[0017] In the present invention, the immortalized muscle stem cell line can be subcultured for at least 50 passages.

[0018] In the present invention, the immortalized muscle stem cell line can maintain stem cell capacity for at least 30 passages.

[0019] In the present invention, the naturally immortalized muscle stem cell line can be obtained by culturing muscle stem cells in a culture medium to which a composition including insulin, transferrin, fibroblast growth factor 2, transforming growth factor β1, albumin, selenite, and omega-6 fatty acids is added.

[0020] In addition, the present invention provides a method for inducing spontaneous immortalization of muscle stem cells, comprising a step of culturing muscle stem cells in a culture medium to which a composition comprising insulin, transferrin, fibroblast growth factor 2, transforming growth factor β1, albumin, selenite, and omega-6 fatty acids is added, and an immortalized muscle stem cell line produced by the method.

[0021] In the present invention, the composition may contain 0 to 20% (w / v) of serum.

[0022] In the present invention, the serum may be at least one selected from the group consisting of fetal bovine serum (FBS), human platelet lysate (hPL), human serum (HS), platelet-rich plasma (PRP), platelet poor plasma (PPP), calf serum, horse serum, porcine serum, and sheep serum.

[0023] In the present invention, the insulin may be bovine insulin and / or human insulin.

[0024] In the present invention, the albumin may be bovine serum albumin.

[0025] In the present invention, the omega-6 fatty acid may be selected from the group consisting of linoleic acid, α-linolenic acid, γ-linolenic acid, calendic acid, and arachidonic acid.

[0026] In the present invention, the composition may comprise 0 to 20% (w / v) of serum, 2 to 30 μg / ml of insulin, 3 to 8 μg / ml of transferrin, 0.5 to 10 ng / ml of fibroblast growth factor 2, 0.5 to 2 ng / ml of transforming growth factor β1, 0.5 to 5 mg / ml of albumin, 2 to 8 ng / ml of selenite, and 2 to 7 μg / ml of omega-6 fatty acids.

[0027] In the present invention, the method for inducing natural immortalization of the muscle stem cells may be a method for inducing a natural immortalized muscle stem cell line that can be cultured for more than 50 passages and maintains growth, proliferation, and differentiation even for more than 10 passages.

[0028] In the present invention, the naturally immortalized muscle stem cell line produced by the above method can be cultured for at least 50 passages, and the stem cell capacity can be maintained for at least 30 passages.

[0029] In the present invention, the naturally immortalized muscle stem cell line produced by the above method can exhibit a growth and proliferation level increased by 10 to 60% compared to a muscle stem cell line cultured in a culture medium under the same serum concentration condition without adding a composition comprising insulin, transferrin, fibroblast growth factor 2, transforming growth factor β1, albumin, selenite, and omega-6 fatty acid.

[0030] Additionally, the present invention provides cultured meat obtained by culturing the immortalized muscle stem cell line and a food composition comprising the cultured meat.

[0031] Furthermore, the present invention provides a method for producing cultured meat or food using the naturally immortalized muscle stem cell line.

[0032] The present invention also provides the use of the naturally immortalized muscle stem cell line for use in the production of cultured meat or food.

[0033] The immortalized muscle stem cell line according to the present invention is a muscle stem cell line in which natural immortalization is induced without genetic manipulation, and maintains proliferation ability and stem cell function even during long-term subculture, so that it can be utilized in high value-added fields such as cultured meat. In addition, through the method for inducing natural immortalization of muscle stem cells according to the present invention, by culturing muscle stem cells in a culture medium in which the types and concentrations of components are optimally combined without genetic manipulation, a naturally immortalized muscle stem cell line that can maintain proliferation ability and stem cell function for a long period of time can be easily produced.

[0034] Figure 1 is a schematic diagram showing the entire process of extracting muscle stem cells of the present invention.

[0035] Figure 2 shows changes in the cell growth rate of muscle stem cells depending on the addition of the NISF-1 composition including the components of Table 1 of the present invention and the difference in the concentration of fetal bovine serum (0, 2, 5, and 10% (w / v) respectively).

[0036] Figure 3 shows the results of comparing the cell growth rates of muscle stem cells cultured in media containing various growth factor mixtures 1 to 9 of Table 2 of the present invention and NISF-1 compositions.

[0037] Figure 4 shows the results of confirming the long-term proliferation ability of muscle stem cells cultured in a culture medium containing the NISF-1 composition including the components of Table 1.

[0038] Figure 5 shows the results of confirming the long-term differentiation potential of muscle stem cells cultured in a culture medium containing the NISF-1 composition including the components of Table 1.

[0039] Figure 6 is a photograph comparing the myofiber differentiation ability of muscle stem cells cultured in a culture medium containing a composition of NISF-1 including the components and a third-party muscle cell culture medium (Promocell Skeletal muscle cell growth media, C-23060).

[0040] Figure 7 shows the results of comparing the expression levels of PAX7, a marker for maintaining muscle stem cell differentiation ability, in muscle stem cells cultured in a culture medium containing a NISF-1 composition including components and a third-party muscle cell culture medium (Promocell Skeletal muscle cell growth media, C-23060).

[0041] Figure 8 shows the results of comparing the expression levels of MyoD, a marker for maintaining muscle stem cell differentiation ability, in muscle stem cells cultured in a culture medium containing a NISF-1 composition including components and a third-party muscle cell culture medium (Promocell Skeletal muscle cell growth media, C-23060).

[0042] Figure 9 shows the results of comparing the expression levels of myosin heavy chain (MyHC), a muscle differentiation marker, in muscle stem cells cultured in a culture medium containing a NISF-1 composition including components and a third-party muscle cell culture medium (Promocell Skeletal muscle cell growth media, C-23060).

[0043] Figure 10 shows the results of comparing the expression level of the muscle differentiation marker Desmin of muscle stem cells cultured in a culture medium containing a composition of NISF-1 including the components and a third-party muscle cell culture medium (Promocell Skeletal muscle cell growth media, C-23060).

[0044] Hereinafter, the present invention will be described in more detail.

[0045] 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.

[0046] All numbers expressing the sizes, quantities, and physical properties of features used in this specification and claims are to be understood as being modified by the term "about." Therefore, unless otherwise indicated, the numerical parameters disclosed in this specification and claims are approximations that may vary depending on the desired properties sought to be achieved by those skilled in the art utilizing the teachings disclosed herein.

[0047] As described above, there remains a need for the development of a naturally immortalized muscle stem cell line that maintains proliferation and differentiation capacity for more than 10 passages, and a method for producing the same. Therefore, the present inventors sought a solution to this problem by optimizing the composition of additives added to the culture medium without genetic manipulation, thereby establishing a naturally immortalized muscle stem cell line that exhibits excellent muscle differentiation rates and stem cell potential even at high passages.

[0048] Accordingly, the first aspect of the present invention relates to a naturally immortalized muscle stem cell line (accession number KCLRF-BP-00548) that maintains growth, proliferation and differentiation for more than 10 passages.

[0049] As used herein, the term "muscle stem cell line" refers to a cell line having the characteristics of muscle stem cells, including proliferation without transformation, infinite proliferation, self-renewal ability, and muscle differentiation ability, and may include, without limitation, cells that exhibit self-renewal ability or infinite proliferation ability and muscle differentiation ability. The self-renewal ability and muscle differentiation ability can be confirmed using a marker. In addition, the muscle stem cell line may be, for example, a cell line that exhibits self-renewal ability and expression of Pax7, MyoD, MyHC, and Desmin. The type and origin of the muscle stem cell line is not limited as long as it has differentiation ability and self-renewal ability. The muscle stem cell line may be derived from, for example, a mammal, such as a human, a monkey, a pig, a horse, a cow, a chicken, a duck, a sheep, a dog, a cat, a mouse, or a rabbit.

[0050] In the present invention, the muscle stem cell line may include precursor cells such as quiescent satellite cells in a growth-quiescent state and activated satellite cells called myoblasts.

[0051] The term "spontaneously immortalized muscle stem cell line" as used herein refers to a muscle stem cell line that can continuously proliferate without artificial genetic manipulation, and specifically may refer to a muscle stem cell line that proliferates for more than 50 passages, which is the HayFlick limit, in culture. In this case, the term "immortalized" is a concept that includes cells with "delayed senescence," i.e. cells with delayed senescence.

[0052] The naturally immortalized muscle stem cell line according to the present invention may be a muscle stem cell line that can be cultured for at least 50 passages.

[0053] As used herein, the term "subculture" refers to a method of continuously culturing cells in a healthy state for a long period of time by periodically transferring a portion of cells to a new culture vessel and replacing the culture medium. The term "passage" refers to the growth of muscle stem 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 proliferation are consumed or contaminants accumulate over a certain period of time, causing the cells to naturally die. Therefore, it 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. Subculture can be performed without limitation by a method known in the art, but is preferably performed by mechanical separation or enzymatic separation.

[0054] The terms "growth and proliferation" herein refer to an increase in cell number. The culture may be undifferentiated proliferation. The term "undifferentiated proliferation" refers to the proliferation of stem cells without differentiating into specific cells, i.e., cells with the same properties as the original cells, i.e., differentiation capacity and self-renewal capacity, and may also be expressed as "stemness."

[0055] The term "differentiation" as used herein refers to a phenomenon in which cells become specialized in structure or function during their growth through division and proliferation, i.e., a change in form or function in order for cells, tissues, etc. of an organism to perform their respective tasks. Measuring or determining the degree of differentiation into a specific cell type can be performed by a method well known in the art. In addition, the differentiation can be confirmed by examining cell morphology using a light microscope or confocal microscope while measuring changes in cell surface markers (e.g., staining cells with tissue-specific or cell-marker-specific antibodies) and cell morphology (e.g., nuclear / cytoplasmic ratio) using techniques such as flow cytometry or immunocytochemistry, or by measuring changes in gene expression using techniques well known in the art such as polymerase chain reaction (PCR) and gene expression profiling.

[0056] In the present invention, the naturally immortalized muscle stem cell line can maintain an undifferentiated state, i.e., stem cell capacity, for at least 30 passages, preferably 30 to 50 passages, and more preferably 30 to 40 passages.

[0057] In a specific embodiment of the present invention, the growth degree of muscle stem cells was comparatively evaluated according to the addition of the 'NISF-1 composition' described in Table 1 of Example 1-2 and the concentration of fetal bovine serum (0, 2, 5, and 10%, respectively). As a result, as confirmed in Fig. 2, in a culture environment with the same concentration of FBS, cell proliferation increased by 18.0 to 50.3% depending on the addition of NISF-1, and it was found that even in a low serum condition with 2% or less FBS, cell proliferation increased significantly with the addition of NISF-1, showing a cell proliferation effect similar to that of a culture environment with 5% FBS. In addition, even in a commonly added FBS concentration of 10% FBS, cell proliferation increased by 44.1% with the addition of NISF-1, showing that cell proliferation increased significantly when NISF-1 was added at the same FBS concentration.

[0058] In another specific embodiment of the present invention, the growth rate of muscle stem cells was compared and evaluated in a culture medium to which growth factor mixtures 1 to 9 described in Table 2 of Examples 1-4 were individually added, and in a culture medium to which a NISF-1 composition was added. As a result, as confirmed in Fig. 3, it was found that the growth rate of muscle stem cells was improved by 13 to 75% in a culture medium to which a NISF-1 composition was added, compared to growth factor mixtures 1 to 9.

[0059] In another specific embodiment of the present invention, the effect of adding NISF-1 composition on maintaining the proliferation capacity of muscle stem cells was evaluated. As a result, as confirmed in Fig. 4, when NISF-1 composition was added during muscle stem cell culture, it was found that passage for more than 50 passages was possible, significantly increasing the number of passages. This means that muscle stem cells proliferated beyond the Hayflick limit, indicating that naturally immortalized muscle stem cells were induced by the addition of NISF-1 composition.

[0060] In another specific embodiment of the present invention, muscle stem cells cultured in a culture medium supplemented with a NISF-1 composition were transferred to a differentiation-inducing medium, and differentiation patterns and muscle stem cell marker expression were observed. As a result, as confirmed in Fig. 5, muscle stem cells cultured in a culture medium supplemented with a NISF-1 composition were able to differentiate even after 40 passages, indicating that differentiation potential was still maintained even at high passages. In addition, as confirmed in FIGS. 6 to 10, muscle stem cells cultured in a culture medium with the NISF-1 composition added have an increased expression level of muscle stem cell differentiation ability maintenance markers and differentiation markers compared to muscle stem cells cultured in a commercially available muscle cell culture medium (Promocell Skeletal muscle cell growth media, C-23060) or a culture medium without the NISF-1 composition (DMEM / F12 containing 10% FBS and 10 ng / ml FGF2), so the composition according to the present invention can be added to a culture medium for the purpose of improving differentiation ability into muscle fibers.

[0061] Accordingly, the muscle stem cell line obtained by culturing for 120 to 200 days in a serum-containing medium supplemented with NISF-1 composition was named 'HS-Bo-A-Muscle 001' and deposited with the Korea Cell Line Research Foundation (KCLRF) on July 10, 2024, and was assigned the accession number KCLRF-BP-00548.

[0062] Therefore, the naturally immortalized muscle stem cell line (accession number KCLRF-BP-00548) according to the present invention can be obtained by culturing muscle stem cells in a culture medium to which a composition including insulin, transferrin, fibroblast growth factor 2, transforming growth factor β1, albumin, selenite, and omega-6 fatty acids is added.

[0063] As described above, in the present invention, a naturally immortalized muscle stem cell line is induced without genetic manipulation by using a specific composition added to a culture medium, and the composition added to the culture medium can be used to induce naturally immortalized muscle stem cells.

[0064] Accordingly, the second aspect of the present invention relates to a method for inducing spontaneous immortalization of muscle stem cells, comprising a step of culturing muscle stem cells in a culture medium to which a composition comprising insulin, transferrin, fibroblast growth factor 2, transforming growth factor β1, albumin, selenite, and omega-6 fatty acids is added, and to a spontaneous immortalized muscle stem cell line produced thereby.

[0065] In the present invention, culturing the muscle stem cells means that the muscle stem cells proliferate while maintaining their differentiation ability, which is a stem cell ability.

[0066] In the present invention, the composition may be added to a serum-free medium or serum-containing medium generally used for culturing muscle stem cells in vitro. Specifically, the serum-free or serum-containing medium used for culturing muscle stem cells may contain 0 to 20% (w / v) serum.

[0067] Referring to FIGS. 2 and 3, when culturing muscle stem cells in a culture medium to which the composition has been added, it can be seen that not only can the growth and proliferation of muscle stem cells be increased compared to culturing in a serum-free culture medium to which the composition has not been added or a culture medium containing serum at the same concentration, but also that cell proliferation can be significantly increased by the addition of the composition even under low serum conditions of 10% (w / v) or less. Therefore, the composition according to the present invention can be added to a culture medium for the purpose of increasing the growth and proliferation of muscle stem cells regardless of the presence or absence of serum, and can also be added to a medium containing no serum or a low concentration of serum (containing a serum concentration of 0 to 10% (w / v)) for the purpose of partially replacing serum.

[0068] Referring to FIG. 4, when culturing a muscle stem cell line using a culture medium to which the composition is added, passage culture for more than 50 passages is possible, and thus the composition according to the present invention can be added to a culture medium for the purpose of inducing naturally immortalized muscle stem cells.

[0069] In addition, referring to FIG. 5, when a muscle stem cell line cultured in a culture medium to which the composition is added is transferred to a differentiation induction medium and cultured, it exhibits excellent muscle differentiation rate and stem cell capacity at high passages. Therefore, the composition according to the present invention can be added to a culture medium for the purpose of maintaining differentiation capacity even at high passages of 10 passages or more.

[0070] Lastly, referring to FIGS. 6 to 10, when a muscle stem cell line cultured in a culture medium to which the composition has been added is transferred to a differentiation induction medium and cultured, the expression levels of muscle stem cell differentiation ability maintenance markers and differentiation markers increase, so the composition according to the present invention can be added to a culture medium for the purpose of improving differentiation ability into muscle fibers.

[0071] In the present invention, the serum may be derived from an animal, preferably a mammal such as a pig, a horse, a cow, a goat, a sheep, or a dog. For example, the serum may be at least one selected from the group consisting of fetal bovine serum (FBS), human platelet lysate (hPL), human serum (HS), platelet-rich plasma (PRP), platelet poor plasma (PPP), calf serum, horse serum, porcine serum, and sheep serum, but is not limited thereto.

[0072] In a specific embodiment of the present invention, FBS was used as the serum.

[0073] In the present invention, the insulin included in the composition may be bovine insulin and / or human insulin, and it is preferable that both bovine insulin and human insulin are included in the composition.

[0074] In the present invention, the albumin may be bovine serum albumin.

[0075] In the present invention, the omega-6 fatty acid may be at least one selected from the group consisting of linoleic acid, α-linolenic acid, γ-linolenic acid, calendic acid, and arachidonic acid, but is not limited thereto.

[0076] The above insulin is a hormone that promotes the uptake of sugar and amino acids into cells and plays a role in cell proliferation, and can promote cell proliferation.

[0077] The above transferrin is a protein that plays a role in moving iron into cells and can detoxify oxygen radicals and peroxides in the medium.

[0078] The above selenite is an inorganic salt form of selenium, which may include sodium selenite, calcium selenite, or potassium selenite. Selenium activates selenium-dependent enzymes to reduce lipid peroxidation and plays a role in protecting cell membranes.

[0079] In the present invention, the composition does not contain interleukin. Interleukin is a major group of hematopoietic cytokines known to play an important role in the proliferation of muscle stem cells. However, the composition according to the present invention can effectively proliferate muscle stem cells even without containing various types of interleukin.

[0080] The composition according to a preferred embodiment of the present invention may include, but is not limited to, 0 to 20% (w / v) serum, 2 to 30 μg / ml insulin, 3 to 8 μg / ml transferrin, 0.5 to 10 ng / ml fibroblast growth factor 2, 0.5 to 2 ng / ml transforming growth factor β1, 0.5 to 5 mg / ml albumin, 2 to 8 ng / ml selenite, and 2 to 7 μg / ml omega-6 fatty acids.

[0081] The method for inducing natural immortalization of muscle stem cells according to the present invention may include inducing naturally immortalized muscle stem cells that can be cultured for more than 50 passages and that maintain growth, proliferation, and differentiation even for more than 10 passages.

[0082] The above composition can be used as included in “medium”, “culture medium”, “culture medium”, “medium composition”, “culture composition”, and “culture medium composition”.

[0083] 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 that can support the growth and survival of stem cells under in vitro culture conditions, and are not distinguished in the present specification and may be used interchangeably. The basic medium that can be used in the present invention may be selected from DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal essential Medium), BME (Basal Medium Eagle), RPMI1640, F-10, F-12, α-MEM (α-modified Minimum Essential Media), GMEM (Glasgow's Minimal essential Medium), IMDM (Iscove's Modified Dulbecco's Medium), etc., but is not limited thereto.

[0084] In addition to the above medium, any medium used in the industry is sufficient, and the medium may contain amino acids, vitamins, minerals, and other ingredients. For example, non-essential amino acids and L-glutamine, vitamins such as vitamin B12, and minerals such as trace elements (CuSO 4· 5H2O, Fe(NO3) 3· 9H2O, ZnSO4), Phosphoenol pyruvate, Monoethanolamine, Sodium selenite and Sodium bicarbonate, Other ingredients may include D-glucose, Linoleic acid, Lipoic acid, Sodium pyruvate, Hypoxantine Na, Putrescine HCl and Polyamine solution.

[0085] In addition, the above composition preferably includes antibiotics, antifungals, and / or substances commonly used in the industry to prevent the growth of mycoplasma to prevent infections such as bacteria and fungi. Antibiotics commonly used in cell culture, such as penicillin-streptomycin, can be used, antifungals include alporelycin B, and mycoplasma inhibitors include, but are not limited to, commonly used substances such as gentamicin, ciprofloxacin, and azithromycin. In addition, a commercially available antibiotic-antimycotic (AA) (Gibco) can be used.

[0086] The naturally immortalized muscle stem cell line produced by the method according to the present invention exhibits the same characteristics as the naturally immortalized muscle stem cell line described in the first aspect, and thus, a description thereof is omitted.

[0087] Natural immortalized muscle stem cells produced by the above method can be utilized as a food material for producing cultured meat.

[0088] Accordingly, the third aspect of the present invention relates to cultured meat obtained by culturing the aforementioned naturally immortalized muscle stem cells and a food composition containing the same.

[0089] In relation to the third aspect, the present invention also provides a method for producing cultured meat, comprising a step of producing cultured meat by culturing the aforementioned natural immortalized muscle stem cells.

[0090] In addition, the present invention provides a method for producing a food product, comprising: a step of producing cultured meat by culturing the aforementioned naturally immortalized muscle stem cells; and a step of producing a food product by processing the produced cultured meat.

[0091] The present invention also provides the use of the naturally immortalized muscle stem cell line for use in the production of cultured meat or food.

[0092] In the method for producing cultured meat or food of the present invention, the step of producing the cultured meat can be performed under known conditions used in general muscle stem cell culture when producing cultured meat, and can be modified in various ways within a range that does not impair the viability and functionality of the cells, including, for example, appropriate media, growth factors, supports, etc.

[0093] According to one embodiment of the present invention, the "step of producing cultured meat by culturing the naturally immortalized muscle stem cells" may include a process of forming a muscle tissue analogue by proliferating and differentiating muscle stem cells having naturally immortalized properties under appropriate conditions and producing the same as cultured meat.

[0094] More specifically, the method for producing cultured meat of the present invention may include the following steps:

[0095] (a) Cell proliferation stage: By culturing naturally immortalized muscle stem cells under appropriate culture conditions, a sufficient number of cells can be secured. At this time, the medium used may be of the type described in the second aspect of the present invention, but is not limited thereto.

[0096] (b) Differentiation induction step: After reaching a sufficient cell density, myotube formation of naturally immortalized muscle stem cells can be induced by replacing the medium with differentiation induction medium.

[0097] (c) Organization or structuring step: The formed muscle cell aggregates can be cultured on a scaffold (e.g., collagen, gelatin, plant-derived hydrogel, etc.) or structured using 3D bioprinting technology, thereby producing a form similar to the texture of actual meat. If necessary, biophysical stimuli such as electrical stimulation or mechanical tension can be applied to improve muscle fiber arrangement and maturation. This organization or structuring step is optional and can be omitted.

[0098] (d) Harvesting and processing stage: After harvesting the formed cultured meat tissue, it can be manufactured into a final food or food material through processing processes such as shaping, cooking, drying, freezing, and mixing.

[0099] The naturally immortalized muscle stem cells of the present invention can proliferate and differentiate stably for a long period of time without genetic modification, and thus have higher production efficiency and consistency than existing primary cell-based cultured meat technology, and are also superior in terms of consumer acceptance and regulatory compliance.

[0100] According to a specific embodiment of the present invention, the conditions for culturing muscle stem cells described in the second aspect of the present invention can be applied to the cultured meat production step.

[0101] The step of manufacturing the above food may include a process of processing the manufactured cultured meat into various forms of food, and may include, for example, steps such as shaping, seasoning, heating, sterilizing, drying or freeze-drying the cultured meat, as well as mixing or adding it with other food raw materials.

[0102] The final food product may be a single cultured meat product or a composite food product containing cultured meat, and may be manufactured in the form of ready-to-eat foods, home meal replacements (HMR), snacks, health functional foods, etc.

[0103] The method for producing cultured meat using naturally immortalized muscle stem cells according to the present invention enables long-term continuous culture without genetic modification, thereby increasing the productivity and stability of cultured meat, and is also advantageous in terms of consumer acceptance and regulatory compliance.

[0104] In addition, the cultured meat can be used as a substitute for existing animal meat, and is suitable for mass production systems, so it has high potential for industrial application.

[0105] Cultured meat manufactured according to the manufacturing method of the present invention may further include a packaging or storage step, and methods such as refrigeration, freezing, and vacuum packaging may be used to maintain the freshness of the product and ensure distribution stability.

[0106] Cultured meat can be consumed on its own, or it can be combined with sauces, vegetables, grains, etc. to provide a complex meal replacement.

[0107] In the present invention, the muscle stem cells 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.

[0108] 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.

[0109] 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.

[0110] 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.

[0111] 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.

[0112] 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.

[0113] The form of the above synthetic food may be a paste, puree, soup, pie, powder, granule, chip, tablet, capsule, or spread.

[0114] 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 and frozen sausage, bacon, dried stored meat, seasoned meat, ground processed meat, packaged meat, and other meat products.

[0115] Hereinafter, the present invention will be described in more detail through examples. However, the present invention can be modified in various ways and can take various forms. Therefore, the specific examples and descriptions described below are only intended to aid in understanding the present invention and are not intended to limit the present invention to a specific disclosed form. It should be understood that the scope of the present invention includes all modifications, equivalents, and substitutes included in the spirit and technical scope of the present invention.

[0116] [Example 1]

[0117] Confirmation of cell growth effects of muscle stem cells according to medium composition

[0118] 1-1. Isolation of muscle stem cells

[0119] After disinfecting the beef rump tissue with 70% ethanol, any tissue suspected of external contamination was removed on a sterile bench. The tissue was transferred to a culture dish without internal contamination, washed three times with PBS, and then, using sterilized scissors and forceps, unnecessary tissues other than muscle, such as membranes and fat, were removed as much as possible. The tissue was transferred to a culture dish without internal contamination, washed three times with PBS, and then, using sterilized scissors and forceps, unnecessary tissues other than muscle, such as membranes and fat, were removed as much as possible. 2 g of the tissue was placed in 10 ml of an enzyme (0.25% trypsin) solution, cut into small pieces in the enzyme solution, and the solution containing the cut pieces was ground evenly with a grinder. 10 ml of an enzyme combination solution was added to the ground tissue, and shaking incubation was performed at 37.5°C and 180 rpm for 60 minutes to homogenize it. After treatment with 0.1% DNase I for 1 minute, the cells were filtered through 100 μm and 70 μm strainers to destroy any remaining red blood cells. The cells were washed with medium supplemented with 10% FBS, diluted with culture medium, plated, and then cultured.

[0120] 1-2. Preparation of badge composition

[0121] A medium composition for inducing natural immortalization of muscle stem cells was prepared with the composition shown in Table 1 below, and named 'NISF-1 composition'.

[0122] Ingredients Manufacturer Concentration Insulin Hanwha Solutions 20 μg / ml Transferrin Hanwha Solutions 5.5 μg / ml Fibroblast Growth Factor Hanwha Solutions 10 ng / ml Transforming Growth Factor Hanwha Solutions 2 ng / ml Bovine Serum Derived Protein Sigma-aldrich 1.5 mg / ml Selenite Sigma-aldrich 5 ng / ml α-Linoleic Acid Sigma-aldrich 4.7 μg / ml

[0123] 1-3. FBS concentration

[0124] The NISF-1 composition prepared in Example 1-2 was added to various concentrations of FBS (0, 2, 5, and 10%, respectively), and the cell growth of muscle stem cells was compared according to the presence or absence of the NISF-1 composition and the FBS concentration. The specific experimental process is as follows.

[0125] Muscle stem cells were seeded into each group in a 96-well cell culture plate coated with 0.01% collagen type 1. The amount of cell culture medium per well was 100 μl, and the cells were cultured in a CO2 incubator for 2 days with the culture medium composition for each group as shown in Table 2 below.

[0126] FBS concentration (%) Group with or without NISF1 addition 110% - Group 210%+ Group 35% - Group 45%+ Group 52% ​​- Group 62%+ Group 70% - Group 80%+

[0127] Following the manufacturer's instructions, 10 μl of CCK-8 reagent was dispensed per well and incubated in a CO2 incubator, shielded from light, for 1 to 4 hours. After sufficient reaction, cell growth was quantified as absorbance using a microplate reader set to a measurement wavelength of 450 nm and a reference wavelength of 620 nm. The differences in cell growth across the experimental groups can be seen in Figure 2.

[0128] As shown in Fig. 2, cell proliferation was significantly increased with the addition of NISF-1 in a culture environment with the same concentration of FBS, and it was confirmed that cell proliferation was significantly increased with the addition of NISF-1 even in a low serum condition with FBS of 2% or less, showing a cell proliferation effect similar to that of a culture environment with 5% FBS.

[0129] 1-4. Confirmation of cell growth effects according to combination of medium components

[0130] As shown in Table 3 below, growth factor mixtures 1 to 9 were prepared by variously combining the components of the culture medium, and the muscle stem cell growth effects in the culture medium to which each growth factor mixture was added and in the culture medium to which NISF-1 was added were compared in the same manner as in Examples 1 to 3. The culture medium to which 10% FBS was added was used as a negative control, and the culture medium to which 10% FBS and 10 ng / ml FGF2 were added was used as a positive control.

[0131] FBS (w / v%), Insulin (μg / ml), Transferrin (μg / ml), FGF2 (ng / ml), TGFβ1 (ng / ml), Bovine serum albumin (mg / ml), Selenite (ng / ml), α-Linoleic acid (μg / ml), Negative control group 10, Positive control group 10--10----, Growth factor mixture 12, 205.5-21.554.7, Growth factor mixture 2, 205.50.6, 2521.554.7, Growth factor mixture 3, 205.51.25, 21.554.7, Growth factor mixture 4, 205.52.5, 21.554.7, Growth factor mixture 5, 205.5521.554.7, Growth factor mixture 6205.5100.1251.554.7 Growth factor mixture 7205.5100.251.554.7 Growth factor mixture 8205.5100.51.554.7 Growth factor mixture 9205.51011.554.7 NISF-1 composition 205.51021.554.7

[0132] As a result, as shown in Fig. 3, it was confirmed that the growth rate of muscle stem cells was significantly improved in the culture medium containing NISF-1 compared to other growth factor mixture combinations.

[0133] [Example 2]

[0134] Confirmation of the effect of adding NISF-1 composition to maintain the proliferation ability of muscle stem cells.

[0135] The primary muscle stem cells isolated in Example 1 were cultured in a cell culture flask at 37.5°C in a 5% CO2 incubator for 1 to 2 weeks to a confluency of 80% or more. The cells attached to the cell culture flask were treated with trypsin / EDTA to detach all cells from the cell culture flask, and then centrifuged at 1500 rpm for 5 minutes to obtain a cell pellet. The cell pellet was diluted with 5 to 10 ml of a negative control culture medium (DMEM / F12 containing 10% FBS), and AO (Acridine Orange stain) / DAPI (4',6-diamidino-2-phenylindole) was mixed with the cell pellet dilution medium at a ratio of 19:1. 10 μl of the mixture was placed in a cell counting slide chamber, and the total number of cells and the number of viable cells were confirmed using an analysis device. After analysis, the cells were divided into a control group (DMEM / F12 containing 10% FBS) and an NISF-1 group (NISF-1 composition added to DMEM / F12 containing 10% FBS), and seeded in cell culture flasks and cultured. When the above process was repeated once, it was called 1 passage, and the increase in the total number of acquired cells compared to the initial seeding number for each passage experiment was calculated to calculate the cumulative population doubling level (CPDL). The CPDL derivation formula is as follows: Mathematical Formula 1.

[0136] [Mathematical Formula 1]

[0137] (LOG10(Nh)-LOG10(Ni)) / LOG10(2)

[0138] In mathematical expression 1, Nh is the total number of acquired cells, and Ni is the number of initially seeded cells.

[0139] As confirmed in Fig. 4, in the case of the control group (DMEM / F12 containing 10% FBS), the passage did not exceed 10, but when the NISF-1 composition was added during the muscle stem cell culture, passages exceeding 50 were possible, which significantly increased the number of passages. This means that the muscle stem cells proliferated beyond the Hayflick limit of 50 passages, and thus, naturally immortalized muscle stem cells were induced by adding the NISF-1 composition during the muscle stem cell culture.

[0140] Accordingly, the muscle stem cell line obtained by culturing for 120 to 200 days in a serum-containing medium supplemented with NISF-1 composition was named 'HS-Bo-A-Muscle 001' and deposited with the Korea Cell Line Research Foundation (KCLRF) on July 10, 2024, and was assigned the accession number KCLRF-BP-00548 on July 23, 2024.

[0141] [Example 3]

[0142] Confirmation of the effect of adding NISF-1 composition to maintain the differentiation ability of muscle stem cells.

[0143] 3-1. Confirmation of the effect of maintaining the differentiation ability of muscle stem cells by adding NISF-1 composition.

[0144] Muscle stem cells were cultured in cell culture flasks containing the control (DMEM / F12 containing 10% FBS) and NISF-1 medium (NISF-1 composition added to DMEM / F12 containing 10% FBS) until 80% confluent. After removing all culture supernatants, differentiation-inducing medium (DMEM / F12 containing 2% horse serum and 10 μg / ml bovine insulin) was applied, and muscle differentiation patterns were observed for 5 to 7 days.

[0145] As shown in Fig. 5, in the control group (DMEM / F12 containing 10% FBS), differentiation ability was lost and proliferation slowed down at passage 10, whereas when the NISF-1 composition was added, differentiation was confirmed to be possible even at passage 40.

[0146] 3-2. Confirmation of muscle stem cell marker expression following addition of NISF-1 composition

[0147] Cells were fixed in 4% paraformaldehyde (PFA) for 10 to 20 minutes at room temperature, and then the remaining fixative was removed. After washing twice with PBS to completely remove residue, the cells were incubated in a blocking solution (e.g., 3% bovine serum albumin solution or 10% normal goat serum solution) for 40 to 60 minutes at room temperature or overnight at 4°C to block nonspecific binding sites. The primary antibody, desmin antibody, was diluted in blocking solution according to the manufacturer's instructions. The diluted primary antibody was added to the cell culture plate from which the blocking solution had been removed, and the cells were incubated overnight at 4°C, protected from light. The cells were washed three times with washing buffer (e.g., PBS) for 5 minutes each to remove unbound primary antibody. Fluorescent-conjugated secondary antibodies were diluted in solution according to the manufacturer's instructions, and the cells were incubated with the diluted secondary antibodies at room temperature, protected from light, for 1 hour. Cells were washed three times with washing buffer (e.g., PBS) for 5 minutes each to remove unbound secondary antibodies. Nuclear staining was performed with a mounting solution containing DAPI, and coverslips were mounted on cell culture flasks. After the mounting solution attached to the coverslips was completely dried, immunofluorescently stained cells were visualized using a fluorescence microscope or confocal microscope equipped with an appropriate filter set.

[0148] As shown in Fig. 6, compared to a muscle stem cell culture medium from another company (Promocell Skeletal muscle cell growth media, C-23060) or a control medium (DMEM / F12 containing 10% FBS and 10 ng / ml FGF2), the addition of NISF-1 resulted in superior expression of Demsin, a muscle differentiation marker, and it was confirmed that long and uniformly shaped muscle fibers were formed overall.

[0149] In the same manner as the immunofluorescence staining described above, cells were fixed and a blocking solution was applied. To quantify the expression levels of muscle stem cell differentiation capacity maintenance markers and differentiation markers, the following method was performed. Primary antibodies, PAX7, MyoD, MyHC, and desmin antibodies, were diluted in blocking solution according to the manufacturer's instructions. The diluted primary antibodies were added to cell culture plates from which the blocking solution had been removed, and incubated overnight at 4°C, protected from light. Unbound primary antibodies were removed by washing the cells three times with washing buffer (e.g., PBS) for 5 minutes each. Fluorescent-conjugated secondary antibodies were diluted in solution according to the manufacturer's instructions, and the cells were incubated with the diluted secondary antibodies for 1 hour at room temperature, protected from light. Unbound secondary antibodies were removed by washing the cells three times with washing buffer (e.g., PBS) for 5 minutes each. The expression levels of the markers were quantified by setting the wavelength corresponding to the fluorescence of each secondary antibody in a microplate.

[0150] As shown in Figures 7 to 10, when the expression level of each muscle stem cell differentiation marker of muscle stem cells cultured in the control group (DMEM / F12 containing 10% FBS and 10 ng / ml FGF2) was taken as 100%, the expression levels of PAX7, MyoD, and MyHC of muscle stem cells cultured in a third-party muscle cell culture medium (Promocell Skeletal muscle cell growth media, C-23060) did not reach the muscle stem cell differentiation marker levels of the control group, and only the expression level of desmin was 12% higher than that of the control group.

[0151] In contrast, the expression levels of PAX7, MyoD, MyHC, and desmin in muscle stem cells cultured in culture medium supplemented with NISF-1 (NISF-1 supplemented to DMEM / F12 containing 10% FBS) all increased compared to muscle stem cells cultured in the control group, and in particular, the expression levels of MyHC and desmin were 2- and 4.5-fold higher, respectively, confirming that the differentiation ability into muscle fibers was significantly improved.

[0152] 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.

[0153] [Accession number]

[0154] Name of depositor: Korea Cell Line Research Foundation (KCLRF)

[0155] Accession number: KCLRFBP00548

[0156] Date of acceptance: 20240710

[0157]

Claims

A naturally immortalized muscle stem cell line (accession number KCLRF-BP-00548) that maintains growth, proliferation, and differentiation for more than 1.10 passages.

2. In the first paragraph, the naturally immortalized muscle stem cell line is a naturally immortalized muscle stem cell line capable of being cultured for at least 50 passages.

3. In the first paragraph, the naturally immortalized muscle stem cell line is a naturally immortalized muscle stem cell line in which stem cell potential is maintained for at least 30 passages.

4. In the first paragraph, the naturally immortalized muscle stem cell line is a naturally immortalized muscle stem cell line obtained by culturing muscle stem cells in a culture medium to which a composition containing insulin, transferrin, fibroblast growth factor 2, transforming growth factor β1, albumin, selenite, and omega-6 fatty acids is added.

5. A method for inducing natural immortalization of muscle stem cells, comprising the step of culturing muscle stem cells in a culture medium to which a composition comprising insulin, transferrin, fibroblast growth factor 2, transforming growth factor β1, albumin, selenite, and omega-6 fatty acids is added.

6. A method for inducing natural immortalization of muscle stem cells in the fifth paragraph, wherein the culture medium contains 0 to 20% (w / v) of serum.

7. A method for inducing natural immortalization of muscle stem cells in the 6th paragraph, wherein the serum is at least one selected from the group consisting of fetal bovine serum (FBS), human platelet lysate (hPL), human serum (HS), platelet-rich plasma (PRP), platelet poor plasma (PPP), calf serum, horse serum, porcine serum, and sheep serum.

8. A method for inducing natural immortalization of muscle stem cells in the fifth paragraph, wherein the insulin is bovine insulin and / or human insulin.

9. A method for inducing natural immortalization of muscle stem cells in paragraph 5, wherein the albumin is bovine serum albumin.

10. A method for inducing natural immortalization of muscle stem cells in the fifth paragraph, wherein the omega-6 fatty acid is selected from the group consisting of linoleic acid, α-linolenic acid, γ-linolenic acid, calendic acid, and arachidonic acid.

11. A method for inducing natural immortalization of muscle stem cells in claim 5, wherein the composition comprises 0 to 20% (w / v) of serum, 2 to 30 μg / ml of insulin, 3 to 8 μg / ml of transferrin, 0.5 to 10 ng / ml of fibroblast growth factor 2, 0.5 to 2 ng / ml of transforming growth factor β1, 0.5 to 5 mg / ml of albumin, 2 to 8 ng / ml of selenite, and 2 to 7 μg / ml of omega-6 fatty acids.

12. In the fifth paragraph, the method for inducing natural immortalization of muscle stem cells is a method for inducing natural immortalization of muscle stem cells, which is capable of being cultured for more than 50 passages and which maintains growth, proliferation, and differentiation even for more than 10 passages.

13. A naturally immortalized muscle stem cell line produced by a method for inducing naturally immortalized muscle stem cells according to any one of claims 6 to 12.

14. In the 13th paragraph, the immortalized muscle stem cell line is a naturally immortalized muscle stem cell line that can be cultured for at least 50 passages and maintains stem cell capacity for at least 30 passages.

15. A naturally immortalized muscle stem cell line according to claim 13, wherein the naturally immortalized muscle stem cell line exhibits a growth and proliferation level increased by 10 to 60% compared to muscle stem cells cultured in a culture medium under the same serum concentration condition without the addition of a composition comprising insulin, transferrin, fibroblast growth factor 2, transforming growth factor β1, albumin, selenite, and α-linoleic acid.

16. Cultured meat obtained by culturing a naturally immortalized muscle stem cell line according to any one of claims 1 to 3.

17. Cultured meat obtained by culturing the naturally immortalized muscle stem cell line of Article 13.

18. A food composition comprising the cultured meat of Article 16.

19. A food composition comprising the cultured meat of Article 18.

20. A method for producing cultured meat, comprising a step of producing cultured meat by culturing a naturally immortalized muscle stem cell line according to any one of claims 1 to 3.

21. A method for producing cultured meat, comprising a step of producing cultured meat by culturing the naturally immortalized muscle stem cell line of Article 13.

22. A step of producing cultured meat by culturing a naturally immortalized muscle stem cell line according to any one of clauses 1 to 3; and A step of manufacturing food by processing the cultured meat manufactured above; A food manufacturing method comprising:

23. A step of producing cultured meat by culturing the naturally immortalized muscle stem cell line of Article 13; and A step of manufacturing food by processing the cultured meat manufactured above; A food manufacturing method comprising:

24. Use of a naturally immortalized muscle stem cell line according to any one of claims 1 to 3 for use in the production of cultured meat or food.

25. Use of the naturally immortalized muscle stem cell line of paragraph 13 for use in the production of cultured meat or food.

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