Additive composition for culture medium, culture medium comprising additive composition, and method for culturing cells

Abstract

There is provided an additive composition for a culture medium, the additive composition comprising a first component comprising at least one sample extract prepared from at least one natural substance, wherein the at least one natural substance is selected from the group consisting of bird's nest (BN), ginseng, Ganoderma lucidum, Mushroom, Cordyceps sinensis (CS), South African Solanum Aquilaria (ASH), and combinations thereof. The additive composition may act as an alternative source of growth factors and hormones for cell culture media to improve consumer acceptance and / or minimize production costs.

Description

Cross - Reference to Related Applications

[0001] This application claims the priority and benefit of U.S. Provisional Application Serial No. 63 / 381,761, filed on November 1, 2022. The entire content of the foregoing application is hereby incorporated by reference in its entirety for all purposes. Technical Field

[0002] This application relates to compositions for culture media, culture media, kits, methods, and uses. In particular, this application relates to additive compositions for culture media, culture media, kits, methods, and uses. Background Art

[0003] Growth factors and hormones are essential for cell culture media (e.g., for culturing animal cells to produce animal tissues). For example, fetal bovine serum (FBS) is widely used in cell culture as a source of growth factors and hormones for the culture medium. However, FBS is highly unethical and expensive. Attempts have been made to produce recombinant growth factors and hormones to reduce costs. The over - use of recombinant growth factors may cause significant health problems in consumers and consumer rejection in food products of cell culture, such as cultured - meat. There is a great need to provide alternative media additive compositions that are all - natural or mostly natural as alternative sources of growth factors and hormones for cell culture media to increase consumer acceptance and / or minimize production costs. Summary of the Invention

[0004] In some embodiments, there is provided an additive composition for a culture medium, the additive composition comprising a first component, the first component comprising at least one sample extract prepared from at least one natural substance, wherein the at least one natural substance is selected from the group consisting of bird's nest (BN), ginsengs, Ganoderma lucidum (Ling Zhi), miscellaneous mushrooms, Cordyceps sinensis (CS), Withania somnifera (ASH), and combinations thereof.

[0005] In some embodiments, there is provided an additive composition for a culture medium for culturing myoblasts, the additive composition comprising: a first component, the first component comprising at least one sample extract prepared from at least one natural substance, wherein the at least one natural substance is selected from the group consisting of bird's nest (BN), American Ginseng (AGN), Korean Ginseng (KGN), and combinations thereof, and wherein the individual sample extract has a final concentration of about 1 - 500 μg / mL in the culture medium.

[0006] In some embodiments, there is provided an additive composition for a medium for culturing porcine skeletal muscle cells, the additive composition comprising: a first component, the first component comprising at least one sample extract prepared from at least one natural substance, wherein the at least one natural substance is selected from the group consisting of bird's nest (BN), American Ginseng (AGN), Korean Ginseng (KGN), and combinations thereof, wherein the individual sample extract has a final concentration of about 1 - 500 μg / mL in the medium.

[0007] In some embodiments, there is provided an additive composition for a medium for culturing bovine skeletal muscle cells, the additive composition comprising: a first component, the first component comprising at least one sample extract prepared from at least one natural substance, wherein the at least one natural substance is selected from the group consisting of bird's nest (BN), ginsengs, Ling Zhi (Ganoderma Lucidum), variegated mushroom, Cordyceps sinensis (CS), Withania somnifera (ASH), and combinations thereof, wherein the individual sample extract has a final concentration of about 1 - 500 μg / mL in the medium.

[0008] In some embodiments, there is provided a medium comprising a basal medium or a minimal medium and the medium additive composition as described in any one of the foregoing embodiments.

[0009] In some embodiments, there is provided a method for culturing cells, the method comprising the steps of: (i) providing a basal medium or a minimal medium and the medium additive composition as described in any one of the foregoing embodiments to form a medium; and (ii) treating and culturing the cells with the medium.

[0010] Advantages of the present disclosure

[0011] In certain embodiments, the provided additive composition contains one or more extracts from biological or natural (such as non - mammalian, plant, or fungal) sources (such as bird's nest and ginsengs, etc.), and the one or more extracts are used to regulate the production of growth factors in cells, stabilize those growth factors to increase and prolong their activity, or regulate the same pathways as those regulated by certain growth factors. In certain embodiments, the extracts can also increase cell proliferation, increase cell stemness, inhibit cell differentiation, and / or induce cell differentiation. In addition to this, these extracts can provide additional health benefits, and from the perspective of traditional medicine (such as traditional Chinese medicine (TCM) or Ayurveda), these extracts bring balance to both the cells in culture and the consumer's body when consumed.

[0012] In certain embodiments, the bird's nest is an edible bird's nest made by the swiftlet.

[0013] In certain embodiments, alternatives / novel compositions, culture media, kits, methods, and uses are provided to minimize or even replace the use of one or more of the following major and most expensive components in cell culture media for growing cells: growth factors such as fibroblast growth factor 2 (FGF-2) (such as basic fibroblast growth factor, bFGF) and TGF-β1. In certain embodiments, these alternatives can be derived from natural sources (such as non-mammalian / plant / fungal extracts), and they act in the following ways: as homologs (proteins with protein sequences similar to those of growth factors with the same function), regulating the production of growth factors in cells, regulating the same pathways as those regulated by certain growth factors, and / or stabilizing growth factors for greater effectiveness. In certain embodiments, they can also act as alternatives to hormones (e.g., insulin) or transferrin, which have antimicrobial properties (e.g., preventing contamination in large-scale production), anticancer properties, and / or promote the production of extracellular matrix such as elastin and collagen in the case of skin care serums. Using these alternatives, it may not be necessary to add exogenous growth factors, hormones, and antibiotics during cell culture, which in turn minimizes cost and safety issues. In certain embodiments, they can also act as alternatives to serum (such as fetal bovine serum) to produce additive compositions for serum-free, substantially serum-free, or serum-reduced media. Production costs are significantly reduced.

[0014] In certain embodiments, the use of these high-quality natural extracts can make them more attractive to consumers and can be very advantageous from a marketing perspective.

[0015] In certain embodiments, the novel compositions, culture media, kits, methods, and uses provided are more consumer-friendly and natural compared to the use of external or recombinant growth factors. In certain embodiments, they can also achieve the same goal of reducing the cost of cultured meat to reach (or be close to or even lower than) the same price level as ordinary meat.

[0016] In certain embodiments, the novel compositions, culture media, kits, methods, and uses provided also reduce concerns during regulatory approval because these components have a long history of consumption without any substantial health / safety issues.

[0017] In certain embodiments, the novel compositions, culture media, kits, methods, and uses provided can generate cells, cell cultures, cell tissues in various applications such as, but not limited to, gene therapy, microfluidics, pharmaceuticals, nutraceuticals, diagnostics, transplantation, biotechnology, etc.

[0018] In certain embodiments, the novel compositions, culture media, kits, methods, and uses provided may also be more nutritionally healthy because some of these components can prevent, treat diseases (such as cancer or diabetes), disorders, or symptoms.

[0019] In certain embodiments, the provided additive composition contains one or more extracts that increase the final cell yield by inhibiting differentiation and promoting cell proliferation and stemness. For example, in certain embodiments, stemness in skeletal muscle satellite cells can be increased, such as via regulation of the Pax7 gene.

[0020] In certain embodiments, the provided additive composition contains one or more extracts that promote cell differentiation (e.g., maturation of muscle fibers or adipocytes).

[0021] In certain embodiments, the provided additive composition contains extracts that are used to partially or fully replace growth factors such as vascular endothelial growth factor (VEGF) and / or fibroblast growth factor (FGF). In other words, the amount of exogenous growth factors in the additive composition is reduced.

[0022] In certain embodiments, the provided additive composition contains extracts that are used to partially or fully replace serum (such as FBS). In other words, the amount of exogenous serum in the additive composition is reduced.

[0023] In certain embodiments, there are provided alternative / novel additive compositions, culture media, kits, methods, and uses for regulating, mimicking, stabilizing, minimizing, or even replacing the use of one or more of growth factors, hormones, and / or serum. In certain embodiments, there are provided alternative / novel additive compositions, culture media, kits, methods, and uses for regulating one or more pathways that have the same or similar effects as growth factors, hormones, and / or serum.

[0024] In certain embodiments, the growth factors are fibroblast growth factor 2 (FGF-2) (such as basic fibroblast growth factor, bFGF), TGF-β1, VEGF, IGF, insulin, EGF, PDGF, and / or IL-6.

[0025] In certain embodiments, the novel compositions, culture media, kits, methods, and uses provided are for producing cultured meat. In certain embodiments, the cultured meat is edible and safe for human or animal consumption.

[0026] In certain embodiments, the novel compositions, culture media, kits, methods, and uses provided reduce the cost of generating cells during cell culture (such as in cultured meat).

[0027] In certain embodiments, the novel compositions, culture media, kits, methods, and uses provided show that sample extracts from Panax ginseng (KGN), Panax quinquefolius (AGN), Ashwagandha (ASH), Cordyceps sinensis (CS), edible bird's nest (BN), Coriolus versicolor (YZ), and Ganoderma lucidum (GL) have the potential to stimulate the proliferation of various types of cells, such as L6, primary porcine myoblasts, and bovine skeletal muscle fibroblasts, under various conditions, such as serum-reduced / growth factor-reduced media and serum-free / growth factor-free media.

[0028] In certain embodiments, the additive composition provided contains a specific combination and effective concentration / range of various sample extracts, and the sample extracts provide an unexpected synergistic effect to the culture medium and have one or more of the above advantages. Brief Description of the Drawings

[0029] Figure 1 is a graph showing the cell growth of porcine myoblasts stimulated by different exemplary additive compositions containing various sample extracts (provided at 300 μg / mL) at the 56 h time point according to an exemplary embodiment.

[0030] Figure 2 is a graph showing the cell growth of bovine fibroblasts stimulated by different exemplary additive compositions containing various sample extracts (provided at 160 μg / mL) at the 48 h time point according to an exemplary embodiment.

[0031] Figures 3A - 3B is a graph showing the cell growth of L6 myoblasts stimulated by exemplary additive compositions containing different concentrations of the sample extracts, bird's nest extract or ginseng extract, at the 48 h time point according to an exemplary embodiment. Detailed Description

[0032] Although the description refers to specific embodiments, the present disclosure should not be construed as limited to the embodiments set forth herein.

[0033] As used herein and in the claims, the term "comprising" (or any related form such as "comprise" and "comprises"), "including" (or any related form such as "include" or "includes"), "containing" (or any related form such as "contain" or "contains") means including the following elements but not excluding other elements. It should be understood that for each embodiment in which the term "comprising" (or any related form such as "comprise" and "comprises"), "including" (or any related form such as "include" or "includes"), or "containing" (or any related form such as "contain" or "contains") is used, this disclosure / application also includes alternative embodiments in which the term "comprising", "including", or "containing" is replaced by "consisting essentially of" or "consisting of". These alternative embodiments using "consisting of" or "consisting essentially of" are understood to be embodiments of a smaller scope of the "comprising", "including", or "containing" embodiments.

[0034] For clarity, "including", "comprising", "containing", and "having" and any related forms are open - ended terms that allow additional elements or features in addition to the specified essential elements, while "consisting of" is a closed - ended term that is limited to the elements recited in the claim and does not include any element, step, or ingredient not specified in the claim.

[0035] "Consisting essentially of" limits the scope of the claim to specific materials, components, or steps ("essential elements") that do not materially affect one or more of the basic characteristics of the claimed invention. In some embodiments, the basic characteristics are one or more of the basic and novel characteristics of the claimed invention.

[0036] As used herein, the singular forms "a / an" and "the" are intended to also include the plural forms unless the context clearly indicates otherwise. When a range is recited in the specification, the range is understood to include every discrete point within the recited range. For example, 1 to 7 means 1, 2, 3, 4, 5, 6, and 7.

[0037] As used herein, the term "about" is understood to be within normal tolerances in the art and not exceeding ±20% of the specified value. By way of example only, about 50 means from 40 to 60, including all values therebetween. As used herein, the phrase "about" a particular value also includes the particular value, e.g., about 50 includes 50.

[0038] As used herein and in the claims, the terms "substantially" or "substantially" or "essentially" or "essentially" mean that the recited characteristics, angles, shapes, states, structures or values need not be achieved exactly, but deviations or variations, including for example, tolerances, measurement errors, measurement precision limitations and other factors known to those skilled in the art, may occur in an amount that does not preclude the effects expected to be provided by the recited characteristics. For example, a composition "substantially" free of serum or "substantially does not contain" serum means that the composition is completely free of serum, almost completely free of serum (e.g., an amount below the detection limit), or less than 1% of the total composition.

[0039] As used herein, the terms "cultured meat", "cultivated meat", "in vitro meat", "cell-based meat", "clean meat", "synthetic meat" and "lab-grown meat" are used interchangeably and refer to meat products that contain tissues derived from cell culture. In some instances, cultured meat is produced by culturing animal cells or tissues in vitro without growing the whole animal (also referred to as "cellular agriculture"). For example, the method involves isolating meat-specific cells from an animal, an amplification step (where the cells are grown in a nutrient-rich medium to cause cell proliferation), and subsequently finally harvesting the cells to form the final meat product.

[0040] As used herein, the term "proliferation" or "amplification" refers to the process of increasing the number of cells and cell populations by culturing the desired cells.

[0041] As used herein, the term "culture medium" refers to a composition that supports the growth and / or other cellular activities of cells or cell tissues.

[0042] As used herein, the terms "basal medium" or "basic medium" refer to a growth medium that provides the essential nutrients for the survival and cultivation of the desired cells. The basal medium can serve as a starting point or basis on which specific additives can be added to meet the specific requirements of the desired cell culture. Examples thereof include, but are not limited to, Dulbecco's Modified Eagle Medium (DMEM), Ham's F12 Medium (F12), Dulbecco's Modified Eagle Medium F12 (DMEM:F12), Ham's F10 Medium (F10), Roswell Park Memorial Institute 1640 Medium (RPMI 1640), Minimal Essential Medium (MEM), Serum-Free Medium (SFM), or combinations thereof.

[0043] As used herein, the term "additive composition" refers to a formulation containing one or more additives or ingredients to meet one or more specific requirements of a cell culture. The additive composition can be added to a basal medium to form a culture medium.

[0044] As used herein, the term "serum" refers to the fluid component of blood, which can contain substances required for cell growth (such as proliferation and / or differentiation), such as growth factors and hormones. In some instances, serum (such as fetal bovine serum) is an exogenous additive to the culture medium.

[0045] As used herein, the terms "growth factor" and "hormone" refer to substances, such as proteins, that regulate signal transduction pathways and / or stimulate cell functions (such as cell growth, proliferation, and / or differentiation). Examples of growth factors or hormones include fibroblast growth factor (FGF) (such as FGF-2), transforming growth factor (TGF) (such as TGF-β1 and TGF-β3), vascular endothelial growth factor (VEGF) (such as VEGF-A), insulin-like growth factor (IGF) (such as IGF-1), insulin, epidermal growth factor (EGF), platelet-derived growth factor (PDGF) (such as PDGF-BB), hepatocyte growth factor (HGF), and interleukin (IL) (such as interleukin 6 (IL-6)).

[0046] As used herein, the term "natural substance" refers to a material derived from a naturally occurring source (such as animals, fungi, and plants) and not significantly altered or artificially / chemically synthesized. In some instances, natural substances are pharmaceutical substances, such as traditional medicine, traditional Chinese medicine (TCM), and / or Ayurvedic medicine. In some embodiments, natural substances are derived from plants and fungi containing a wide variety of proteins, polysaccharides, or other bioactive substances.

[0047] As used herein, the term "sample extract" refers to a partial or representative sample that has been extracted or prepared from a natural substance. In some instances, the sample extract is prepared by preparing a sample solution of the natural substance in powder form.

[0048] As used herein, the term "bird's nest" or "edible bird's nest" refers to a natural substance mainly made from the solidified saliva of the swiftlet and is generally considered TCM.

[0049] As used herein, the term "ginseng species" refers to perennial plants belonging to the genus Panax. In some instances, only the root portion is used. For example, Korean ginseng, Japanese ginseng or Chinese ginseng (Panax ginseng) and American ginseng (Panax quinquefolius) are two of the example ginseng species used as TCM. Other ginseng species may be used. In some instances, ginseng species from other regions such as the Korean Peninsula, Northeast China, the Russian Far East, Canada, Vietnam and the United States may be used. In some embodiments, Korean ginseng includes Korean Red Ginseng.

[0050] As used herein, the term "Ganoderma lucidum" or "Lingzhi" refers to a polypore mushroom belonging to the genus Ganoderma and is considered to be TCM. For example, Ganoderma lucidum and Ganoderma sichuanense are two of the example Ganoderma species used as TCM.

[0051] As used herein, the term "variegated mushroom" refers to a polypore mushroom belonging to the genus Trametes. For example, Coriolus versicolor (or Trametes versicolor) is one of the example variegated mushroom species used as TCM and may also be referred to as Yunzhi.

[0052] As used herein, the term "Cordyceps sinensis" or "Ophiocordyceps sinensis" refers to an entomogenous fungus in the family Ophiocordycipitaceae and is generally considered to be TCM.

[0053] As used herein, the term "Ashwagandha" or "Withania somnifera" refers to a herb belonging to the genus Withania and is generally considered to be Ayurvedic medicine.

[0054] As used herein, the term "exogenous" refers to some component or factor that originates from outside or external to the composition. For clarity, if a component (such as a growth factor from serum, a chemically synthesized or recombinant growth factor or hormone) is added to the additive composition, the component is considered to be exogenous. On the other hand, if a growth factor or hormone is naturally present in or derived from an extract of a sample of a natural substance, the growth factor or hormone is not considered to be exogenous.

[0055] As used herein, the term "final concentration" refers to the concentration of a substance or component (such as a first component or a second component) when it is present in a solution or mixture (such as a culture medium) after all necessary dilutions or reactions have occurred. In some examples, the total concentration of the culture medium is calculated as a weight / volume ratio (100% (w / v)). In some other examples, the total concentration of the culture medium is calculated as a volume / volume ratio (100% (v / v)).

[0056] As used herein, the term "animal" refers to eukaryotic species such as mammals (such as pigs, cows, sheep, horses, dogs, cats, rodents), birds or fowl, reptiles, fish, amphibians, crustaceans, mollusks, cephalopods, and the like. Examples of the present invention Example 1

[0057] In certain embodiments, the novel compositions, culture media, kits, methods, and uses provided comprise extracts of samples selected from the group consisting of bird's nest, ginsengs, Withania somnifera, Ganoderma lucidum, Psathyrella miscella, Cordyceps sinensis, and combinations thereof, which extracts have been verified to increase cell numbers and can be used as additives to a basal cell culture medium such as DMEM medium (or another in-house formulated basal medium having essential salts, buffers, minerals, amino acids, lipids, minerals, etc.) for the purpose of producing meat tissue (e.g., on an industrial scale).

[0058] In certain embodiments, the novel compositions, culture media, kits, methods, and uses provided comprise one or more of the following features: 1. Using natural extracts (in certain embodiments, edible bird's nest, Korean ginseng, and American ginseng), the novel compositions are more consumer-friendly, healthy, natural, and can potentially reduce costs. 2. Without being bound by any theory, these extracts may contain active ingredients useful for upregulating specific growth factors or hormones, and / or they may have protein structures homologous to essential growth factors or hormones. Thus, they fully or partially replace the need for non-natural sources of growth factors or hormones (e.g., recombinant sources), and can also reduce the cost of producing cell cultures (such as cultured meat). 3. Specific compounds from the extracts can be further isolated and / or purified for use in place of the intact extracts. 4. The extracts can be added to cell culture media to replace the functions of certain growth factors or hormones. 5. These extracts are used to maintain the stemness of meat cells in culture. Higher cell yields can be obtained, such as by delaying uncontrolled cell differentiation. 6. These extracts are used to increase the proliferation rate of cells in culture. Higher cell yields can be obtained in a shorter period of time.

[0059] In certain embodiments, there is provided a culture medium additive composition comprising at least one sample extract prepared from at least one natural substance. In certain embodiments, the at least one natural substance is a sample selected from the group consisting of bird's nest, ginseng species, Withania somnifera, Ganoderma lucidum, Coprinus variegatus, Cordyceps sinensis, and combinations thereof. In some embodiments, the ginseng species is American ginseng or Korean ginseng. Other embodiments are described below.

[0060] In certain embodiments, the composition substantially does not contain one or more exogenous growth factors or hormones, or combinations thereof.

[0061] In certain embodiments, the at least one sample extract is bird's nest. In certain embodiments, the at least one sample extract is American ginseng. In certain embodiments, the at least one sample extract is Korean ginseng. In certain embodiments, the at least one sample extract is Ganoderma lucidum. In certain embodiments, the at least one sample extract is Coprinus variegatus. In certain embodiments, the at least one sample extract is Coriolus versicolor. In certain embodiments, the at least one sample extract is Cordyceps sinensis. In certain embodiments, the at least one sample extract is Withania somnifera. In certain embodiments, Withania somnifera is derived from only the roots or the whole plant.

[0062] In certain embodiments, the sample extract is prepared from a solution of a filtrate or supernatant. In certain embodiments, the solution comprises water. In certain embodiments, the solution comprises an organic solvent.

[0063] In certain embodiments, the composition consists essentially of at least one sample extract prepared from at least one sample selected from the group consisting of bird's nest, ginseng species, Ganoderma lucidum, Coprinus variegatus, Cordyceps sinensis, Withania somnifera, and combinations thereof.

[0064] In certain embodiments, the at least one sample extract comprises a concentration of 0.001 ug / mL - 1000 mg / mL. In certain embodiments, the at least one sample extract comprises a concentration of 20 mg / mL - 100 mg / mL. In certain embodiments, the at least one sample extract comprises a concentration of 20 mg / mL. In certain embodiments, the at least one sample extract comprises a concentration of 60 mg / mL. In certain embodiments, the at least one sample extract comprises a concentration of 100 mg / mL. In certain embodiments, the sample extract is prepared from an aqueous solution of a filtrate or supernatant.

[0065] In certain embodiments, a culture medium is provided, which comprises a basal medium or a basic medium and a culture medium additive composition as disclosed in any one of the above embodiments.

[0066] In certain embodiments, the culture medium substantially does not contain one or more exogenous growth factors or hormones, or a combination thereof. In certain embodiments, the culture medium further contains one or more exogenous growth factors or hormones, or a combination thereof.

[0067] In certain embodiments, the basal medium is Dulbecco's Modified Eagle Medium (DMEM) and / or F12.

[0068] In certain embodiments, the composition further contains FBS at a concentration of 5%.

[0069] In certain embodiments, a method for culturing cells is provided, which comprises the following steps: (i) providing a culture medium, which comprises a basal medium and at least one sample extract prepared from at least one natural substance; and (ii) treating and culturing the cells with the at least one natural substance. In certain embodiments, the at least one natural substance is a sample composed of bird's nest, ginseng species, Withania somnifera, Ganoderma lucidum (e.g., Ganoderma lucidum), Coriolus versicolor (e.g., Trametes versicolor), Cordyceps sinensis, and combinations thereof. In certain embodiments, the method further comprises the following steps: (1) after step (ii), treating the cell line with a dissociating agent (e.g., trypsin) to form a cell suspension. In certain embodiments, the method further comprises the following steps: (2) before step (ii), neutralizing the cell suspension with a neutralizing agent (e.g., trypsin neutralization solution, TrypLE) to obtain a neutralized cell suspension. In certain embodiments, the method further comprises the following steps: (3) obtaining a cell pellet from the neutralized cell suspension and resuspending the cell pellet in the culture medium.

[0070] In certain embodiments, the sample extract is prepared by the following steps: (a) preparing at least one powder of the at least one sample; (b) dissolving the at least one powder to form at least one sample solution; (c) purifying the at least one sample solution to obtain the at least one sample extract. In certain embodiments, the sample extract is prepared by the following steps: (a) preparing at least one powder of the at least one sample; (b) dissolving the at least one powder in water to form at least one sample solution; (c) filtering and / or centrifuging the at least one sample solution to obtain the at least one sample extract.

[0071] In some embodiments, the culture medium comprises a final sample extract concentration of about 0.001 μg / mL - 1000 mg / mL. In some embodiments, the culture medium comprises a final sample extract concentration of about 0.1 mg / mL - 0.5 mg / mL. In some embodiments, the culture medium comprises a final sample extract concentration of about 0.1 mg / mL. In some embodiments, the culture medium comprises a final sample extract concentration of about 0.3 mg / mL. In some embodiments, the culture medium comprises a final sample extract concentration of about 0.5 mg / mL.

[0072] In some embodiments, cultured cells are used to produce animal tissue. In some embodiments, the animal tissue is human tissue. In some embodiments, cultured cells are used to produce cultured meat. In some embodiments, the at least one sample solution comprises a solvent. In some embodiments, the at least one sample solution comprises water. In some embodiments, the at least one sample solution comprises an organic solvent. Example 2

[0073] In some embodiments, an additive composition for a culture medium is provided, the additive composition comprising a first component, the first component comprising at least one sample extract prepared from at least one natural substance, wherein the at least one natural substance is selected from the group consisting of bird's nest (BN), ginseng species, Ganoderma lucidum, Trametes versicolor, Cordyceps sinensis (CS), Withania somnifera (ASH), and combinations thereof.

[0074] In some embodiments, the ginseng species are American ginseng, ginseng, and combinations thereof.

[0075] In some embodiments, the Trametes versicolor is Coriolus versicolor (YZ).

[0076] In some embodiments, the Ganoderma lucidum (Ling Zhi) is Ganoderma lucidum (GL).

[0077] In some embodiments, the Trametes versicolor is Coriolus hirsutus.

[0078] In some embodiments, the at least one natural substance is bird's nest.

[0079] In some embodiments, the at least one natural substance is American ginseng.

[0080] In some embodiments, the at least one natural substance is Korean ginseng.

[0081] In some embodiments, the at least one natural substance is Ganoderma lucidum.

[0082] In some embodiments, the at least one natural substance is Trametes versicolor.

[0083] In some embodiments, the at least one natural substance is Cordyceps sinensis.

[0084] In some embodiments, the at least one natural substance is Withania somnifera.

[0085] In some embodiments, Withania somnifera is derived from the root or the whole plant.

[0086] In some embodiments, the first component substantially does not contain one or more exogenous sera, growth factors or hormones, or a combination thereof.

[0087] In some embodiments, the culture medium is for culturing animal cells.

[0088] In some embodiments, the culture medium is for culturing cells of mammals, birds, crustaceans or fish.

[0089] In some embodiments, the culture medium is for culturing fibroblasts, vascular cells, pericytes, endothelial cells, myoblasts, skeletal muscle satellite cells, muscle cells, smooth muscle cells, fat cells, adipose-derived stem cells, skin cells, tendons, liver, brain, bone, heart, kidney, stem cells or a combination thereof.

[0090] In some embodiments, the culture medium is for culturing fibroblasts or myoblasts.

[0091] In some embodiments, a separate sample extract is prepared from a solution of the filtrate or supernatant of a separate natural substance.

[0092] In some embodiments, the first component consists essentially of at least one sample extract prepared from at least one natural substance, wherein the at least one natural substance is selected from the group consisting of bird's nest, ginseng, Ganoderma lucidum, Miscellaneous Colored Mushroom, Cordyceps sinensis, Withania somnifera and combinations thereof.

[0093] In some embodiments, the separate sample extract has a final concentration of about 0.01 ng / mL - 1000 mg / mL in the culture medium.

[0094] In some embodiments, the separate sample extract has a final concentration of about less than 1000 or 500 μg / mL in the culture medium.

[0095] In some embodiments, the additive composition further comprises a second component, the second component comprising at least one serum, growth factor or hormone, amino acids, vitamins, salts, minerals, glucose, buffers, or a combination thereof.

[0096] In some embodiments, the second component further comprises fetal bovine serum (FBS) at a final concentration in the culture medium of less than about 20%, 10%, 5% or 2%.

[0097] In some embodiments, the second component further comprises bovine serum albumin (BSA) at a final concentration in the culture medium of about 0.01 ng / mL - 10 mg / mL.

[0098] In some embodiments, the second component further comprises bovine serum albumin (BSA) at a final concentration in the culture medium of less than about 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 mg / mL.

[0099] In some embodiments, the at least one growth factor or hormone is FGF-2, TGF-b1, vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF), insulin, EGF, platelet-derived growth factor (PDGF), interleukin 6 (IL-6) or a combination thereof.

[0100] In some embodiments, the second component further comprises TGF-b1 at a final concentration in the culture medium of less than about 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0.5 ng / mL.

[0101] In some embodiments, the second component further comprises fetal bovine serum (FBS), fetal calf serum, bovine serum albumin (BSA) or a combination thereof.

[0102] In some embodiments, provided is an additive composition for a culture medium for culturing myoblasts, the additive composition comprising: a first component, the first component comprising at least one sample extract prepared from at least one natural substance, wherein the at least one natural substance is selected from the group consisting of bird's nest (BN), American Ginseng (AGN), Korean Ginseng (KGN) and combinations thereof, wherein the individual sample extract has a final concentration in the culture medium of about 1 - 500 μg / mL.

[0103] In some embodiments, the additive composition further comprises a second component, the second component comprising FBS at a final concentration in the culture medium of about 2% - 5%.

[0104] In some embodiments, an additive composition for a medium for culturing porcine skeletal muscle cells is provided, the additive composition comprising: a first component, the first component comprising at least one sample extract prepared from at least one natural substance, wherein the at least one natural substance is selected from the group consisting of bird's nest (BN), American Ginseng (AGN), Korean Ginseng (KGN), and combinations thereof, wherein the individual sample extract has a final concentration of about 1 - 500 μg / mL in the medium.

[0105] In some embodiments, the additive composition further comprises a second component, the second component comprising FBS having a final concentration of about 2% - 5% in the medium.

[0106] In some embodiments, the additive composition further comprises a second component, the second component comprising BSA having a final concentration of about 1 - 10 mg / ml in the medium and optionally TGF - b1 having a final concentration of about 0.5 - 5 ng / ml.

[0107] In some embodiments, an additive composition for a medium for culturing bovine skeletal muscle cells is provided, the additive composition comprising: a first component, the first component comprising at least one sample extract prepared from at least one natural substance, wherein the at least one natural substance is selected from the group consisting of bird's nest (BN), ginsengs, Ling Zhi (Ganoderma Lucidum), miscellaneous mushrooms, Cordyceps sinensis (CS), Withania somnifera (ASH), and combinations thereof, wherein the individual sample extract has a final concentration of about 1 - 500 μg / mL in the medium.

[0108] In some embodiments, the additive composition further comprises a second component, the second component comprising FBS having a final concentration of about 2% - 5% in the medium.

[0109] In some embodiments, a medium is provided, the medium comprising a basal medium or a minimal medium and the medium additive composition as described in any one of the foregoing embodiments.

[0110] In some embodiments, the medium substantially does not contain or contains a reduced amount of one or more exogenous sera, growth factors or hormones, or combinations thereof.

[0111] In some embodiments, the basal medium or the basic medium is Dulbecco's Modified Eagle Medium (DMEM), Ham's F12 medium (F12), Dulbecco's Modified Eagle Medium F12 (DMEM:F12), Ham's F10 medium (F10), Roswell Park Memorial Institute 1640 medium (RPMI 1640), Minimal Essential Medium (MEM), serum-free medium (SFM), or a combination thereof.

[0112] In some embodiments, a method for culturing cells is provided, the method comprising the steps of: (i) providing a basal medium or a basic medium and a culture medium additive composition as described in any one of the foregoing embodiments to form a culture medium; and (ii) treating and culturing the cells with the culture medium.

[0113] In some embodiments, the method further comprises the step of: (1) treating the cell line with a dissociating agent to form a cell suspension.

[0114] In some embodiments, the method further comprises the step of: (2) neutralizing the cell suspension with a neutralizing agent to obtain a neutralized cell suspension.

[0115] In some embodiments, the method further comprises the step of: (3) obtaining a cell pellet from the neutralized cell suspension and resuspending the cell pellet in the culture medium.

[0116] In some embodiments, a sample extract is prepared by one or more of the following steps: (a) preparing at least one powder of the at least one natural substance; (b) dissolving the at least one powder to form at least one sample solution; (c) purifying the at least one sample solution to obtain the at least one sample extract.

[0117] In some embodiments, purifying the at least one sample solution in step (c) is filtering and / or centrifuging the at least one sample solution.

[0118] In some embodiments, the cultured cells are used to produce animal tissue.

[0119] In some embodiments, the animal tissue is tissue of a mammal, bird, crustacean, or fish.

[0120] In some embodiments, the cultured cells are used to produce cultured meat.

[0121] In some embodiments, the method further comprises the step of incubating the cells in a CO2 incubator.

[0122] In some embodiments, the cell line is derived from a mammal, bird, crustacean, or fish.

[0123] In some embodiments, the cell line is derived from fibroblasts, vascular cells, pericytes, endothelial cells, myoblasts, satellite cells, skeletal muscle cells, muscle cells, smooth muscle cells, adipocytes, adipose-derived stem cells, skin cells, tendons, liver, brain, bone, heart, kidney, stem cells, and combinations thereof.

[0124] In some embodiments, the cell line is derived from a mammal, bird, crustacean, or fish. In some embodiments, the cell line is derived from a crustacean (such as a shrimp), an avian (such as a chicken), an actinopterygii (such as a loach, salmon), or a mammal (such as a cow or pig).

[0125] In some embodiments, the cell line is derived from muscle cells (such as skeletal muscle cells, smooth muscle cells), myoblasts, skeletal muscle satellite cells, adipocytes, adipose-derived stem cells, fibroblasts, vascular cells (such as endothelial cells, pericytes), skin cells, tendons, liver, brain, bone, heart, kidney, stem cells, and combinations thereof. In some embodiments, the cell line is derived from skeletal muscle cells, satellite cells, and myoblasts and / or cells produced therefrom (such as muscle cells, muscle fibers). In some embodiments, the cell line is derived from adipose cells (adipose cell), such as adipose-derived stem cells (also known as preadipocytes), mature adipocytes, and / or cells produced therefrom. In some embodiments, the cell line is derived from fibroblasts. In some embodiments, the cell line is derived from induced pluripotent stem cells (iPSCs).

[0126] In some embodiments, the additive composition and the basal medium together form a medium with a total concentration of 100%.

[0127] In some embodiments, the additive composition comprises a first component, the first component comprising one or more sample extracts prepared from one or more natural substances as described herein, wherein the individual or each sample extract has a final concentration of about 0-99% (or higher) in the culture medium. For example, suitable concentration ranges are any ranges between 0-99%, such as 0.00001%-1%, 0.0001%-0.1%, 0.1%-5%, 0.01%-10%, 0.001%-20%, 1%-30%, 5%-40%, 10%-50%, 20%-60%, 30%-70%, 40%-80% or higher. In some embodiments, the individual or each sample extract has the following concentrations: about 0.00005%, about 0.0001%, about 0.0002%, about 0.0003%, about 0.0004%, about 0.0005%, about 0.0006%, about 0.0007%, about 0.0008%, about 0.0009%, about 0.001%, about 0.002%, about 0.003%, about 0.004%, about 0.005%, about 0.006%, about 0.007%, about 0.008%, about 0.009%, about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%,About 96%, about 97%, about 98% or about 99%. In some additional embodiments, the individual or each sample extract has a final concentration of 0.01 ng / mL - 1000 mg / mL (or higher) in the culture medium. In some additional embodiments, the individual or each sample extract has a concentration of about 0.01 ng / mL - 100 ng / mL. In some additional embodiments, the individual or each sample extract has a concentration of about 0.01 μg / mL - 100 μg / mL. In some additional embodiments, the individual or each sample extract has a concentration of about 0.01 mg / mL - 100 mg / mL. In some additional embodiments, the individual or each sample extract has a concentration of about 0.2 mg / mL - 100 mg / mL. In some additional embodiments, the individual or each sample extract has a first component with a concentration of about 10 μg / mL - 1000 μg / mL. In some additional embodiments, the individual or each sample extract has the following concentrations: about 0.01 ng / mL, 0.02 ng / mL, 0.03 ng / mL, 0.04 ng / mL, 0.05 ng / mL, 0.06 ng / mL, 0.07 ng / mL, 0.08 ng / mL, 0.09 ng / mL, 0.1 ng / mL, 0.2 ng / mL, 0.3 ng / mL, 0.4 ng / mL, 0.5 ng / mL, 0.6 ng / mL, 0.7 ng / mL, 0.8 ng / mL, 0.9 ng / mL, 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL, 15 ng / mL, 16 ng / mL, 17 ng / mL, 18 ng / mL, 19 ng / mL, 20 ng / mL, 21 ng / mL, 22 ng / mL, 23 ng / mL, 24 ng / mL, 25 ng / mL, 26 ng / mL, 27 ng / mL, 28 ng / mL, 29 ng / mL, 30 ng / mL, 31 ng / mL, 32 ng / mL, 33 ng / mL, 34 ng / mL, 35 ng / mL, 36 ng / mL, 37 ng / mL, 38 ng / mL, 39 ng / mL, 40 ng / mL, 41 ng / mL, 42 ng / mL, 43 ng / mL, 44 ng / mL, 45 ng / mL, 46 ng / mL, 47 ng / mL, 48 ng / mL, 49 ng / mL, 50 ng / mL, 51 ng / mL, 52 ng / mL, 53 ng / mL, 54 ng / mL, 55 ng / mL, 56 ng / mL, 57 ng / mL, 58 ng / mL, 59 ng / mL, 60 ng / mL, 61 ng / mL,62 ng / mL, 63 ng / mL, 64 ng / mL, 65 ng / mL, 66 ng / mL, 67 ng / mL, 68 ng / mL, 69 ng / mL, 70 ng / mL, 71 ng / mL, 72 ng / mL, 73 ng / mL, 74 ng / mL, 75 ng / mL, 76 ng / mL, 77 ng / mL, 78 ng / mL, 79 ng / mL, 80 ng / mL, 81 ng / mL, 82 ng / mL, 83 ng / mL, 84 ng / mL, 85 ng / mL, 86 ng / mL, 87 ng / mL, 88 ng / mL, 89 ng / mL, 90 ng / mL, 91 ng / mL, 92 ng / mL, 93 ng / mL, 94 ng / mL, 95 ng / mL, 96 ng / mL, 97 ng / mL, 98 ng / mL, 99 ng / mL, 0.1 μg / mL, 0.2 μg / mL, 0.3 μg / mL, 0.4 μg / mL, 0.5 μg / mL, 0.6 μg / mL, 0.7 μg / mL, 0.8 μg / mL, 0.9 μg / mL, 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL, 5 μg / mL, 6 μg / mL, 7 μg / mL, 8 μg / mL, 9 μg / mL, 10 μg / mL, 11 μg / mL, 12 μg / mL, 13 μg / mL, 14 μg / mL, 15 μg / mL, 16 μg / mL, 17 μg / mL, 18 μg / mL, 19 μg / mL, 20 μg / mL, 21 μg / mL, 22 μg / mL, 23 μg / mL, 24 μg / mL, 25 μg / mL, 26 μg / mL, 27 μg / mL, 28 μg / mL, 29 μg / mL, 30 μg / mL, 31 μg / mL, 32 μg / mL, 33 μg / mL, 34 μg / mL, 35 μg / mL, 36 μg / mL, 37 μg / mL, 38 μg / mL, 39 μg / mL, 40 μg / mL, 41 μg / mL, 42 μg / mL, 43 μg / mL, 44 μg / mL, 45 μg / mL, 46 μg / mL, 47 μg / mL, 48 μg / mL, 49 μg / mL, 50 μg / mL, 51 μg / mL, 52 μg / mL, 53 μg / mL, 54 μg / mL, 55 μg / mL, 56 μg / mL, 57 μg / mL, 58 μg / mL, 59 μg / mL, 60 μg / mL, 61 μg / mL, 62 μg / mL, 63 μg / mL, 64 μg / mL, 65 μg / mL66 ug / mL, 67 ug / mL, 68 ug / mL, 69 ug / mL, 70 ug / mL, 71 ug / mL, 72 ug / mL, 73 ug / mL, 74 ug / mL, 75 ug / mL, 76 ug / mL, 77 ug / mL, 78 ug / mL, 79 ug / mL, 80 ug / mL, 81 ug / mL, 82 ug / mL, 83 ug / mL, 84 ug / mL, 85 ug / mL, 86 ug / mL, 87 ug / mL, 88 ug / mL, 89 ug / mL, 90 ug / mL, 91 ug / mL, 92 ug / mL, 93 ug / mL, 94 ug / mL, 95 ug / mL, 96 ug / mL, 97 ug / mL, 98 ug / mL, 99 ug / mL, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL, 20 mg / mL, 21 mg / mL, 22 mg / mL, 23 mg / mL, 24 mg / mL, 25 mg / mL, 26 mg / mL, 27 mg / mL, 28 mg / mL, 29 mg / mL, 30 mg / mL, 31 mg / mL, 32 mg / mL, 33 mg / mL, 34 mg / mL, 35 mg / mL, 36 mg / mL, 37 mg / mL, 38 mg / mL, 39 mg / mL, 40 mg / mL, 41 mg / mL, 42 mg / mL, 43 mg / mL, 44 mg / mL, 45 mg / mL, 46 mg / mL, 47 mg / mL, 48 mg / mL, 49 mg / mL, 50 mg / mL, 51 mg / mL, 52 mg / mL, 53 mg / mL, 54 mg / mL, 55 mg / mL, 56 mg / mL, 57 mg / mL, 58 mg / mL, 59 mg / mL, 60 mg / mL, 61 mg / mL, 62 mg / mL, 63 mg / mL, 64 mg / mL, 65 mg / mL, 66 mg / mL, 67 mg / mL, 68 mg / mL, 69 mg / mL, 70 mg / mL, 71 mg / mL72 mg / mL, 73 mg / mL, 74 mg / mL, 75 mg / mL, 76 mg / mL, 77 mg / mL, 78 mg / mL, 79 mg / mL, 80 mg / mL, 81 mg / mL, 82 mg / mL, 83 mg / mL, 84 mg / mL, 85 mg / mL, 86 mg / mL, 87 mg / mL, 88 mg / mL, 89 mg / mL, 90 mg / mL, 91 mg / mL, 92 mg / mL, 93 mg / mL, 94 mg / mL, 95 mg / mL, 96 mg / mL, 97 mg / mL, 98 mg / mL, 99 mg / mL, 100 mg / mL, 110 mg / mL, 120 mg / mL, 130 mg / mL, 140 mg / mL, 150 mg / mL, 160 mg / mL, 170 mg / mL, 180 mg / mL, 190 mg / mL, 200 mg / mL, 210 mg / mL, 220 mg / mL, 230 mg / mL, 240 mg / mL, 250 mg / mL, 260 mg / mL, 270 mg / mL, 280 mg / mL, 290 mg / mL, 300 mg / mL, 310 mg / mL, 320 mg / mL, 330 mg / mL, 340 mg / mL, 350 mg / mL, 360 mg / mL, 370 mg / mL, 380 mg / mL, 390 mg / mL, 400 mg / mL, 410 mg / mL, 420 mg / mL, 430 mg / mL, 440 mg / mL, 450 mg / mL, 460 mg / mL, 470 mg / mL, 480 mg / mL, 490 mg / mL, 500 mg / mL, 510 mg / mL, 520 mg / mL, 530 mg / mL, 540 mg / mL, 550 mg / mL, 560 mg / mL, 570 mg / mL, 580 mg / mL, 590 mg / mL, 600 mg / mL, 610 mg / mL, 620 mg / mL, 630 mg / mL, 640 mg / mL, 650 mg / mL, 660 mg / mL, 670 mg / mL, 680 mg / mL, 690 mg / mL, 700 mg / mL, 710 mg / mL, 720 mg / mL, 730 mg / mL, 740 mg / mL, 750 mg / mL, 760 mg / mL, 770 mg / mL, 780 mg / mL, 790 mg / mL, 800 mg / mL, 810 mg / mL, 820 mg / mL, 830 mg / mL, 840 mg / mL, 850 mg / mL, 860 mg / mL, 870 mg / mL, 880 mg / mL, 890 mg / mL, 900 mg / mL, 910 mg / mL, 920 mg / mL, 930 mg / mL, 940 mg / mL, 950 mg / mL,960 mg / mL, 970 mg / mL, 980 mg / mL, 990 mg / mL, 1000 mg / mL or higher. In some embodiments, the additive composition comprises a first component at a concentration of about 0.2 mg / mL, 0.4 mg / mL, 0.8 mg / mL, 1.6 mg / mL, 3.2 mg / mL, 6.4 mg / mL, 20 mg / mL, 60 mg / mL or 100 mg / mL. The one or more sample extracts are prepared from at least one natural substance selected from the group consisting of: bird's nest (BN), ginsengs (such as American ginseng (Panax quinquefolius), Korean ginseng or Chinese ginseng (Panax ginseng)), Ganoderma lucidum (such as Ganoderma lucidum, GL), variegated mushrooms (such as Coriolus versicolor (YZ)), Cordyceps sinensis (CS), Withania somnifera (ASH) and combinations thereof.

[0128] In a further embodiment, the additive composition comprises or consists essentially of bird's nest at a concentration of about 0.01 ng / mL - 1000 mg / mL or any of the above concentrations.

[0129] In a further embodiment, the additive composition comprises or consists essentially of ginsengs (such as American ginseng or Panax quinquefolius) at a concentration of about 0.01 ng / mL - 1000 mg / mL or any of the above concentrations.

[0130] In one embodiment, the culture medium additive composition comprises or consists essentially of ginsengs (such as Korean ginseng, Chinese ginseng or Panax ginseng) at a concentration of about 0.01 ng / mL - 1000 mg / mL or any of the above concentrations.

[0131] In one embodiment, the additive composition comprises or consists essentially of variegated mushrooms (such as Coriolus versicolor) at a concentration of about 0.01 ng / mL - 1000 mg / mL or any of the above concentrations.

[0132] In one embodiment, the additive composition comprises or consists essentially of Ganoderma lucidum (such as Ganoderma lucidum) at a concentration of about 0.01 ng / mL - 1000 mg / mL or any of the above concentrations.

[0133] In one embodiment, the additive composition comprises or consists essentially of Cordyceps sinensis at a concentration of about 0.01 ng / mL - 1000 mg / mL or any of the above concentrations.

[0134] In one embodiment, the culture medium additive composition comprises or consists essentially of Withania somnifera at a concentration of about 0.01 ng / mL - 1000 mg / mL or any of the above concentrations.

[0135] In some embodiments, the culture medium comprises a first component at a final concentration of about 0.01 ng / mL - 1000 mg / mL or any of the above concentrations. In some embodiments, the culture medium comprises a first component at a final concentration of about 20 μg / mL - 0.5 mg / mL. In some embodiments, the culture medium comprises a first component at a final concentration of about 1 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, 40 μg / mL, 80 μg / mL, 100 μg / mL, 160 μg / mL, 300 μg / mL or 500 μg / mL.

[0136] In a further embodiment, the culture medium comprises an additive composition and a basal medium (such as DMEM or DMEM:F12), and the additive composition comprises one or more sample extracts prepared from one or more natural substances as described herein at a final concentration of about 0.01 ng / mL - 1000 mg / mL or any of the above concentrations.

[0137] In additional embodiments, the culture medium optionally comprises a second component as described herein. In some additional embodiments, the second component comprises one or more exogenous components such as serum, growth factors, hormones, amino acids, vitamins, salts, glucose, buffers, or combinations thereof. The individual or each component in the second component has a reduced amount or concentration in the culture medium, such as a final concentration in the culture medium of about 0-99% (or higher). For example, any suitable concentration range in the culture medium is between 0-99%, such as between 0.0001%-50%, 1%-20%, or between 0.01 ng / mL-1000 mg / mL (or higher). In some additional embodiments, the individual or each component in the second component has a concentration of about 0.0000151%-1%, 0.0001%-0.1%, 0.1%-5%, 0.01%-10%, 0.001%-20%, 0.01%-10%, 0.1%-5%, 1%-30%, 5%-40%, 10%-50%, 20%-60%, 30%-70%, or 40%-80% or higher. In some embodiments, the individual or each sample extract has the following concentrations: about 0.00005%, about 0.0001%, about 0.0002%, about 0.0003%, about 0.0004%, about 0.0005%, about 0.0006%, about 0.0007%, about 0.0008%, about 0.0009%, about 0.001%, about 0.002%, about 0.003%, about 0.004%, about 0.005%, about 0.006%, about 0.007%, about 0.008%, about 0.009%, about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%About 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99%. In some additional embodiments, the second component, individually or each component, has a concentration of about 0.01 ng / mL - 100 ng / mL. In some additional embodiments, the second component, individually or each component, has a concentration of about 0.01 μg / mL - 100 μg / mL. In some additional embodiments, the second component, individually or each component, has a concentration of about 0.01 mg / mL - 100 mg / mL. In some additional embodiments, the second component, individually or each component, has a concentration of about 0.2 mg / mL - 100 mg / mL. In some additional embodiments, the second component, individually or each component, has a first component with a concentration of about 10 μg / mL - 1000 μg / mL. In some additional embodiments, the second component, individually or each component, has the following concentrations: about 0.01 ng / mL, 0.02 ng / mL, 0.03 ng / mL, 0.04 ng / mL, 0.05 ng / mL, 0.06 ng / mL, 0.07 ng / mL, 0.08 ng / mL, 0.09 ng / mL, 0.1 ng / mL, 0.2 ng / mL, 0.3 ng / mL, 0.4 ng / mL, 0.5 ng / mL, 0.6 ng / mL, 0.7 ng / mL, 0.8 ng / mL, 0.9 ng / mL, 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL, 15 ng / mL, 16 ng / mL, 17 ng / mL, 18 ng / mL, 19 ng / mL, 20 ng / mL, 21 ng / mL, 22 ng / mL, 23 ng / mL, 24 ng / mL, 25 ng / mL, 26 ng / mL, 27 ng / mL, 28 ng / mL, 29 ng / mL, 30 ng / mL, 31 ng / mL, 32 ng / mL, 33 ng / mL, 34 ng / mL, 35 ng / mL, 36 ng / mL, 37 ng / mL, 38 ng / mL, 39 ng / mL, 40 ng / mL, 41 ng / mL, 42 ng / mL, 43 ng / mL, 44 ng / mL, 45 ng / mL,46 ng / mL, 47 ng / mL, 48 ng / mL, 49 ng / mL, 50 ng / mL, 51 ng / mL, 52 ng / mL, 53 ng / mL, 54 ng / mL, 55 ng / mL, 56 ng / mL, 57 ng / mL, 58 ng / mL, 59 ng / mL, 60 ng / mL, 61 ng / mL, 62 ng / mL, 63 ng / mL, 64 ng / mL, 65 ng / mL, 66 ng / mL, 67 ng / mL, 68 ng / mL, 69 ng / mL, 70 ng / mL, 71 ng / mL, 72 ng / mL, 73 ng / mL, 74 ng / mL, 75 ng / mL, 76 ng / mL, 77 ng / mL, 78 ng / mL, 79 ng / mL, 80 ng / mL, 81 ng / mL, 82 ng / mL, 83 ng / mL, 84 ng / mL, 85 ng / mL, 86 ng / mL, 87 ng / mL, 88 ng / mL, 89 ng / mL, 90 ng / mL, 91 ng / mL, 92 ng / mL, 93 ng / mL, 94 ng / mL, 95 ng / mL, 96 ng / mL, 97 ng / mL, 98 ng / mL, 99 ng / mL, 0.1 μg / mL, 0.2 μg / mL, 0.3 μg / mL, 0.4 μg / mL, 0.5 μg / mL, 0.6 μg / mL, 0.7 μg / mL, 0.8 μg / mL, 0.9 μg / mL, 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL, 5 μg / mL, 6 μg / mL, 7 μg / mL, 8 μg / mL, 9 μg / mL, 10 μg / mL, 11 μg / mL, 12 μg / mL, 13 μg / mL, 14 μg / mL, 15 μg / mL, 16 μg / mL, 17 μg / mL, 18 μg / mL, 19 μg / mL, 20 μg / mL, 21 μg / mL, 22 μg / mL, 23 μg / mL, 24 μg / mL, 25 μg / mL, 26 μg / mL, 27 μg / mL, 28 μg / mL, 29 μg / mL, 30 μg / mL, 31 μg / mL, 32 μg / mL, 33 μg / mL, 34 μg / mL, 35 μg / mL, 36 μg / mL, 37 μg / mL, 38 μg / mL, 39 μg / mL, 40 μg / mL, 41 μg / mL, 42 μg / mL, 43 μg / mL, 44 μg / mL, 45 μg / mL, 46 μg / mL, 47 μg / mL, 48 μg / mL, 49 μg / mL50 ug / mL, 51 ug / mL, 52 ug / mL, 53 ug / mL, 54 ug / mL, 55 ug / mL, 56 ug / mL, 57 ug / mL, 58 ug / mL, 59 ug / mL, 60 ug / mL, 61 ug / mL, 62 ug / mL, 63 ug / mL, 64 ug / mL, 65 ug / mL, 66 ug / mL, 67 ug / mL, 68 ug / mL, 69 ug / mL, 70 ug / mL, 71 ug / mL, 72 ug / mL, 73 ug / mL, 74 ug / mL, 75 ug / mL, 76 ug / mL, 77 ug / mL, 78 ug / mL, 79 ug / mL, 80 ug / mL, 81 ug / mL, 82 ug / mL, 83 ug / mL, 84 ug / mL, 85 ug / mL, 86 ug / mL, 87 ug / mL, 88 ug / mL, 89 ug / mL, 90 ug / mL, 91 ug / mL, 92 ug / mL, 93 ug / mL, 94 ug / mL, 95 ug / mL, 96 ug / mL, 97 ug / mL, 98 ug / mL, 99 ug / mL, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL, 20 mg / mL, 21 mg / mL, 22 mg / mL, 23 mg / mL, 24 mg / mL, 25 mg / mL, 26 mg / mL, 27 mg / mL, 28 mg / mL, 29 mg / mL, 30 mg / mL, 31 mg / mL, 32 mg / mL, 33 mg / mL, 34 mg / mL, 35 mg / mL, 36 mg / mL, 37 mg / mL, 38 mg / mL, 39 mg / mL, 40 mg / mL, 41 mg / mL, 42 mg / mL, 43 mg / mL, 44 mg / mL, 45 mg / mL, 46 mg / mL, 47 mg / mL, 48 mg / mL, 49 mg / mL, 50 mg / mL, 51 mg / mL, 52 mg / mL, 53 mg / mL, 54 mg / mL, 55 mg / mL, 56 mg / mL, 57 mg / mL,58 mg / mL, 59 mg / mL, 60 mg / mL, 61 mg / mL, 62 mg / mL, 63 mg / mL, 64 mg / mL, 65 mg / mL, 66 mg / mL, 67 mg / mL, 68 mg / mL, 69 mg / mL, 70 mg / mL, 71 mg / mL, 72 mg / mL, 73 mg / mL, 74 mg / mL, 75 mg / mL, 76 mg / mL, 77 mg / mL, 78 mg / mL, 79 mg / mL, 80 mg / mL, 81 mg / mL, 82 mg / mL, 83 mg / mL, 84 mg / mL, 85 mg / mL, 86 mg / mL, 87 mg / mL, 88 mg / mL, 89 mg / mL, 90 mg / mL, 91 mg / mL, 92 mg / mL, 93 mg / mL, 94 mg / mL, 95 mg / mL, 96 mg / mL, 97 mg / mL, 98 mg / mL, 99 mg / mL, 100 mg / mL, 110 mg / mL, 120 mg / mL, 130 mg / mL, 140 mg / mL, 150 mg / mL, 160 mg / mL, 170 mg / mL, 180 mg / mL, 190 mg / mL, 200 mg / mL, 210 mg / mL, 220 mg / mL, 230 mg / mL, 240 mg / mL, 250 mg / mL, 260 mg / mL, 270 mg / mL, 280 mg / mL, 290 mg / mL, 300 mg / mL, 310 mg / mL, 320 mg / mL, 330 mg / mL, 340 mg / mL, 350 mg / mL, 360 mg / mL, 370 mg / mL, 380 mg / mL, 390 mg / mL, 400 mg / mL, 410 mg / mL, 420 mg / mL, 430 mg / mL, 440 mg / mL, 450 mg / mL, 460 mg / mL, 470 mg / mL, 480 mg / mL, 490 mg / mL, 500 mg / mL, 510 mg / mL, 520 mg / mL, 530 mg / mL, 540 mg / mL, 550 mg / mL, 560 mg / mL, 570 mg / mL, 580 mg / mL, 590 mg / mL, 600 mg / mL, 610 mg / mL, 620 mg / mL, 630 mg / mL, 640 mg / mL, 650 mg / mL, 660 mg / mL, 670 mg / mL, 680 mg / mL, 690 mg / mL, 700 mg / mL, 710 mg / mL, 720 mg / mL, 730 mg / mL, 740 mg / mL, 750 mg / mL, 760 mg / mL, 770 mg / mL, 780 mg / mL, 790 mg / mL, 800 mg / mL, 810 mg / mL, 820 mg / mL,830 mg / mL, 840 mg / mL, 850 mg / mL, 860 mg / mL, 870 mg / mL, 880 mg / mL, 890 mg / mL, 900 mg / mL, 910 mg / mL, 920 mg / mL, 930 mg / mL, 940 mg / mL, 950 mg / mL, 960 mg / mL, 970 mg / mL, 980 mg / mL, 990 mg / mL, 1000 mg / mL or higher. In some embodiments, the additive composition comprises a first component at a final concentration of about 1 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, 40 μg / mL, 80 μg / mL, 100 μg / mL, 160 μg / mL, 300 μg / mL or 500 μg / mL. In additional embodiments, the second component comprises FBS at a final concentration of about 0.01%-50% (such as 0.1%-20%, or 2%-5%) or higher in the culture medium. In other embodiments, the second component comprises BSA at a final concentration of about 1-100 mg / mL (such as 6 mg / mL) and / or TGF-β1 at a final concentration of about 0.1-100 ng / mL (such as 2 ng / mL). The second component comprises at least one serum (such as FBS), growth factor or hormone (such as FGF-2, TGF-β1, vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF), insulin, EGF, platelet-derived growth factor (PDGF), interleukin 6 (IL-6)), amino acid (such as BSA), vitamin, salt, glucose, buffer or a combination thereof. Example

[0138] Examples are provided herein that describe certain embodiments of the present disclosure in more detail. The examples provided herein are for illustrative purposes only and are not meant to limit the scope of the invention in any way. All references given below and elsewhere in this application are hereby incorporated by reference herein. Instance 1

[0139] Preparation of example sample extract Table 1. Samples used in this example embodiment Sample number Sample type Form Usage amount Identity 1 Red Ginseng (KG) Dried herbal root part 500 mg Ginseng 2 American Ginseng (AG) Dried herbal root part 500 mg Panax quinquefolius 3 Bird's Nest (BN) Dried edible bird's nest 500 mg Bird's nest made from swiftlets 1. Grind the samples into a powder form (e.g., using an electric food chopper and then a coffee grinder). 2. Place each sample in a 50 mL tube and add 5 mL of water separately, and dissolve them in an oscillating incubator at 100 rpm for 24 hours. (Final concentration = 500 mg / 5 mL = 100 mg / mL) 3. Filter the sample through a sterile 0.2 µm filter into a 15 ml tube. This step and the following steps are carried out in a biosafety cabinet to ensure sterility. In the case where any sample cannot be filtered, centrifuge the sample and then filter it to obtain the supernatant as the sample extract. Optionally, before filtration, first filter the sample through a 100 µm cell strainer. 4. Prepare three 1.5 mL centrifuge tubes (in triplicate) for each sample extract. 5. Aliquot each sample extract into centrifuge tubes and add water as needed to obtain the following final concentrations: a. Tube 1: 100 mg / ml b. Tube 2: 60 mg / ml c. Tube 3: 20 mg / ml

[0140] Test sample extract 1. In this example, the example cultured cells used for testing are L6 myoblasts (non-bovine) cultured in DMEM supplemented with 10% FBS. Ensure serum starvation is introduced by replacing the medium with DMEM only for 4 hours before proceeding with the subsequent steps. For the positive control, the medium used is DMEM supplemented with 10% FBS. 2. After 4 hours, rinse the cells twice with PBS. 3. Remove the PBS and digest the cells with trypsin. Treat the cells with trypsin (Gibco, 0.25% trypsin-EDTA, phenol red) for 3 - 5 min while gently tapping the flask as usual to facilitate cell dissociation. 4. Once the trypsin is neutralized with TNS (trypsin neutralization solution), perform cell counting. The ratio of the volume of TNS used for neutralization to trypsin is 1:1. 5. Centrifuge the cells (RCF = 150 g) to form a pellet. 6. Resuspend the cell pellet in DMEM such that there are 2×10 4 cells / mL. Add 500 µL of the cell suspension to each well for testing in a 24-well plate. 7. Well arrangement: a. Column 1: Sample extract 1: Korean ginseng b. Column 2: Sample extract 2: American ginseng c. Column 3: Sample extract 3: Bird's nest d. Row A (for columns 1 - 3): Add 5 µL of the extract from tube 1 to the well. Also add 495 µL of DMEM in 10% FBS. e. Row B (for columns 1 - 3): Add 5 μL of the extract from tube 2. Also add 495 μL of DMEM in 10% FBS. f. Row C (for columns 1 - 3): Add 5 μL of the extract from tube 3. Also add 495 μL of DMEM in 10% FBS. g. Negative control: Row D (for columns 1 - 3): Add 500 μL of DMEM only h. Positive control 1: Rows 1 - 3, column 4 (wells 4A + 4B + 4C): Add 480 μL of DMEM. Add 20 μL of FGF - 2 (from a 5 μg / mL FGF - 2 aliquot) so that the final concentration of FGF - 2 in the well is 0.1 μg / mL. i. Additional positive control 2: Rows 1 - 3, column 5 (wells 5A + 5B + 5C): Add 500 μL of DMEM + 20% FBS. (Final concentration: 10% FBS) 8. Repeat steps 1 - 7 on another plate a. Incubate the first plate for 24 hours. b. Incubate the second plate for 48 hours. 9. Use trypan blue as a stain and evaluate proliferation by cell counting using a hemocytometer.

[0141] Results

[0142] (1) 24 - hour time point

[0143] Before performing cell counting, incubate the samples with the cells in the first plate for 24 hours. Table 2. Cell count and cell viability of cells treated with sample extract for 24 hours (0.3 ml T / E + 0.3 ml TNS = 0.6 ml total volume; initial cell number = 10,000 cells / well).

[0144] (2) 48 - hour time point

[0145] Before performing cell counting, incubate the sample extract with the cells for 48 hours Table 3. Cell count and cell viability of cells treated with sample extract for 48 hours (0.38 ml T / E + 0.38 ml TNS = 0.76 ml total volume; initial cell number = 10,000 cells / well).

[0146] Results and Discussion:

[0147] The above results showed that during the first 24 hours, the cell number remained relatively the same as the negative control (DMEM only), indicating that the cells might not have been upregulated by the samples. Only after 24 - 48 hours, the cell number treated with the three sample extracts increased unexpectedly, indicating that the sample extracts stimulated cell proliferation. Among the three sample extracts, the sample extract of American ginseng was unexpectedly superior to the other two, followed by bird's nest and Korean ginseng. By the 48 - hour time point, all sample extracts even unexpectedly exceeded the performance of the positive control (including DMEM supplemented with 0.05 μg / mL FGF - 2 and DMEM supplemented with 10% FBS).

[0148] In summary, the growth factor upregulation capabilities of sample extracts of bird's nest, American ginseng (AG), and Korean ginseng (KG) were tested. In 48 - hour myocyte cultures, KG and AG at a concentration of 100 μg / mL resulted in 24.09 - fold and 70.00 - fold increases in cell number, respectively, while 0.1 μg / mL of FGF - 2 resulted in a 9.58 - fold increase. If similar cell proliferation numbers were maintained even without the addition of 5% FBS, this translated to a significant (e.g., 22,285 - fold and 18,665 - fold, respectively) cost reduction in using KG and AG as alternatives to the FGF - 2 component, since in some instances, FGF - 2 is one of the most expensive components in cell culture.

[0149] The percentage (%) increase in proliferation within 24 hours and 48 hours normalized to the negative control is summarized in Table 4. The % increase in proliferation normalized to the control was calculated as follows:

[0150] % increase in proliferation = {[(total cell count in sample) - (total cell count in negative control with DMEM only)] / (total cell count in negative control with DMEM only)} * 100% Table 4. Increase in cell proliferation within 24 hours and 48 hours (%)

[0151] The results in Table 4 show that the culture medium containing the basal medium DMEM, the first component containing approximately 100 μg / ml of KGN, AGN, or BN, and the second component with a final concentration of 5% FBS of the exemplary additive composition had excellent proliferation increase percentages at 48 hours (66.207%, 382.759%, and 106.207% respectively). The results indicate that the exemplary additive compositions containing these sample extracts (AGN, KGN, BN) have unexpected effects on the cell proliferation of cells such as L6 myoblasts. This example demonstrates that the sample extracts can significantly reduce the final concentration of serum (FBS) used in the culture medium from 10% to 5%. The sample extracts in the presence of 5% serum can unexpectedly even outperform the positive control group with 10% serum. Instance 2

[0152] In this example, the effect of using culture medium compositions containing various exemplary additive compositions on the proliferation of porcine skeletal muscle cells was demonstrated. In this example, the culture medium was tested with porcine skeletal muscle cells. The culture medium is also applicable to both myoblasts and skeletal muscle satellite cells.

[0153] The materials used in this example include:

[0154] Cell line: Porcine skeletal muscle cells (PIG-iCell-S005) (Cellverse Bioscience Technology Co., Ltd.)

[0155] Basal medium: Dulbecco's Modified Eagle Medium (DMEM) (Thermofisher, D6429)

[0156] Second component: Fetal bovine serum (ScienCell, 0500), bovine serum albumin (BSA) (Sigma, A7030-10g), TGF-β1 (Thermofisher, PHG9204), FGF-2 (Thermofisher, PHG0026) Other reagents: 0.25% Trypsin / EDTA (Gibco, 25200-056), CyQuant MTT (Invitrogen, V13154), and gelatin (Sigma, G1890)

[0157] Exemplary sample extracts: Edible bird's nest, Ginseng (also known as "Korean ginseng" or "KGN") (Jung Kwan Jang) extract, and The extract of American ginseng (also known as "Panax quinquefolius" or "AGN") (President’s Brand) was prepared in a similar manner as described in Example 1.

[0158] In this example, the following steps were performed: 1. Prepare stock solutions of sample extracts (AGN, KGN) at concentrations of approximately 1 mg / mL, 3 mg / mL, and 5 mg / mL. 2. Prepare a stock solution of bovine serum albumin (BSA) at a concentration of approximately 80 mg / mL in a smaller volume (<1 mL) of distilled water (H 2 O). The high-concentration BSA stock solution allows for better maintenance of cell osmotic balance. 3. Prepare the following solutions: a. M1 medium: DMEM + 2 ng / mL TGF-b1 + 1 mg / mL BSA + 40 ng / mL FGF-2 b. M3 medium: DMEM + 2 ng / mL TGF-b1 + 1 mg / mL BSA 4. Coat all wells to be used in a 24-well plate with 2% gelatin. 5. Culture porcine skeletal muscle cells in DMEM supplemented with approximately 10% FBS until a final cell concentration of at least 1×10 6 cells / mL is reached. 6. Harvest porcine skeletal muscle cells using an appropriate volume of approximately 0.25% trypsin / EDTA, then neutralize with a trypsin neutralizer solution (also known as "neutralizer"), and centrifuge at 300 g for 5 minutes. Discard the supernatant and resuspend the cells in M3 medium at a final cell density of approximately 1×10 6 cells / mL. 7. Add approximately 10 uL of the cell suspension (final cell density: approximately 1×10 4 cells / well) to each well to be used. 8. Add approximately 840 uL of M3 medium and place the cells for approximately 1 hour to allow serum starvation. For the control groups using M3 medium (negative control) or M1 medium (also known as "positive control" or "M1"), add the same amount of M3 or M1 medium, respectively. 9. Add approximately 50 uL of approximately 100 mg / mL BSA such that the final concentration in each well is approximately 5 - 6 mg / mL. The same operation was performed for the control. 10. Add approximately 100 uL of the stock sample extract into each well. For the control, add 100 uL of phosphate-buffered saline (PBS) instead of the sample extract. The component concentrations of each medium are shown in Table 5 (KGN = Korean ginseng, AGN = American ginseng). Table 5. Final concentrations of the example culture medium compositions used in Instance 2 11. Repeat Step 10 as needed to obtain replicates. In this example, two replicates for each well were obtained. 12. After approximately 56 hours, transfer the medium from the wells to separate centrifuge tubes. When removing the used medium, also remove the non-attached cells. Extract the adherent cells remaining on the well plate using the following steps: 13. Then add 2 mL of 0.25% trypsin / EDTA to each well and incubate for 15 - 30 min. Gently tap the sides and bottom of the wells. 14. Mix 20 uL of the dissociated cells in 0.25% trypsin / EDTA with 20 uL of trypan blue. 15. Add 15 uL of the dissociated cell and trypan blue mixture to each side of a cell counting chamber slide. Then evaluate the cell density and total cell number. 16. Repeat Steps 14 - 15 for the medium transferred in Step 12.

[0159] Results

[0160] In this example, the effects of media with approximately 300 ug / mL KGN or approximately 300 ug / mL AGN as the first component and BSA and TGF-b1 as the second component, as well as the positive control (M1), on the cell number were evaluated and summarized in the results. Each sample was run in duplicate, and the average total cell number was compared to the positive control. The cells adhered to the well plate were counted. The percentage increase in proliferation (%) was calculated using the equation {[(number of cells in the sample) - (number of cells in M1)] / (number of cells in M1)} * 100%. The results are shown in Figure 1 and summarized in Table 6. In this experiment, cell viability was not evaluated because the trypsin / EDTA was not neutralized with a trypsin neutralizer solution.

[0161] Now refer to Figure 1, which shows the growth of porcine skeletal muscle cells stimulated by different sample extracts provided at the 56 - hour time point at approximately 300 μg / mL (i.e., sample A2 and B2 according to Table 5, respectively). The percentage increase in proliferation normalized to the control (%) is listed in Table 6. The results show that A2 (KGN at approximately 300 μg / mL) and B2 (AGN at approximately 300 μg / mL) perform better than the positive control (which contains a much higher level of FGF - 2 (approximately 34 ng / mL) in M1 medium) in the presence of TGF - b1 at a reduced final concentration of approximately 1.7 ng / mL and BSA at approximately 5.85 mg / mL (as the second component). After approximately 56 hours, B2 (AGN at approximately 300 μg / mL) reaches a cell number of approximately 1.58×10 5 cells, while A2 (KGN at approximately 300 μg / mL) reaches approximately 1.46×10 5 cells. This is approximately 50% more than the final cell number (with approximately 9.38×10 4 cells) in the positive control (M1) with FGF - 2. Additionally, no serum was used in these test groups, which demonstrates that the exemplary additive compositions containing the first component (such as AGN and / or KGN) are effective substitutes for serum in the preparation of serum - free (or substantially serum - free or serum - reduced) media for culturing cells (such as porcine skeletal muscle cells). Similar experiments can be further conducted on other sample extracts (such as Ganoderma lucidum, Coriolus versicolor, edible bird's nest, Withania somnifera, and / or Cordyceps sinensis) to show the unexpected technical effects of these natural substances in the preparation of additive compositions for serum - free (or substantially serum - free or serum - reduced) media for culturing samples (e.g., for culturing myoblasts or satellite cells located in a skeletal muscle cell mixture). Table 6 summarizes the percentage increase in cell proliferation (%) of the media containing the sample extracts KGN and AGN within 56 hours. Table 6. Increase in cell proliferation within 56 hours (%)

[0162] The results in Table 6 show that the media of the exemplary additive compositions containing the basal medium DMEM, the first component containing approximately 300 μg / ml of KGN or AGN, and the second component with a final concentration of approximately 5.85 mg / mL of BSA and approximately 1.7 ng / mL of TGF - b1 have excellent percentage increases in proliferation (approximately 55.22% and 68.44% respectively) at approximately 56 hours. The results indicate that these sample extracts (AGN and KGN) achieve unexpected effects in the cell proliferation of skeletal muscle cells. This example demonstrates that sample extracts can stimulate cell growth in a manner even exceeding that of the medium supplemented with FGF - 2. Instance 3

[0163] In this example, the effect of using the exemplary additive composition and the culture medium composition on the cell proliferation of bovine skeletal muscle cells was demonstrated. In this example, the culture medium was tested with bovine skeletal muscle cells. The culture medium is also suitable for both fibroblasts and skeletal muscle satellite cells.

[0164] The materials used in this example include:

[0165] Cell line: Bovine skeletal muscle cells (COW-iCell-S005) (Saiwei Biotechnology Co., Ltd.)

[0166] Basal medium: Dulbecco's Modified Eagle Medium / Nutrient Mixture F-12 (DMEM:F12) (Thermo Fisher Scientific, 21041-025)

[0167] Second component: Fetal bovine serum (ScienCell, 0500)

[0168] Other reagents: 0.25% Trypsin / EDTA (Gibco, 25200-056), CyQuant MTT (Invitrogen, V13154), Trypsin neutralization solution (Gibco, R-002-100), Gelatin (Sigma, G1890)

[0169] Sample extracts: Bird's nest extract (Hunan New Mstar Biotechnology Co., Ltd, batch MS230404), Korean ginseng (Xi’an Demeter Biotech Co., Ltd, batch DMTB20230106), Ginseng extract (Xi’an Demeter Biotech Co., Ltd, batch DMTB230115), Ganoderma lucidum (Xi’an Demeter Biotech Co., Ltd, batch 2022-10-12), Withania somnifera root extract (Xi’an Demeter Biotech Co., Ltd, batch DMTB230320), Cordyceps sinensis Yarsagumba polysaccharide 10% (Xi’an Demeter Biotech Co., Ltd, batch DMTB230223), and Yunzhi essence capsules (Vita Green Pharmaceuticals (HK) Ltd., batch HKNY2301A).

[0170] In this example, the following steps were carried out: 1. Prepare a stock solution of the extract at a concentration of about 1.6 mg / mL dissolved in deionized water. 2. Sterile filter each extract through a 0.22 µm filter and then store it at approximately 4°C. 3. After receiving bovine skeletal muscle cells, dissociate the cells in 0.25% trypsin / EDTA and neutralize them in a trypsin neutralizer solution (also known as "neutralizer"). 4. Centrifuge the dissociated cells at 300 g for 5 minutes. 5. Aspirate the supernatant, and then resuspend the cell pellet in DMEM:F12 + 10% FBS. 6. Then preplate the cell mixture in an uncoated T-flask (ideally a T75 flask) for 40 - 60 minutes and then transfer it to a 2% gelatin-coated T-flask (ideally a T75 flask). 7. Then add fresh medium (DMEM:F12 + 10% FBS) to the uncoated flask containing purified bovine skeletal muscle fibroblasts. The coated flask contains purified bovine skeletal muscle cells. 8. Replace the used medium with fresh medium every 48 hours until the cells reach 80% confluence. 9. After reaching confluence, add 4 mL of 0.25% trypsin / EDTA to the flask for 5 minutes, gently tapping the flask occasionally. 10. Add an equal volume of trypsin neutralizer solution to the flask and centrifuge the cell mixture at 300 g for 5 minutes. 11. Remove the supernatant from the centrifuged cell mixture and resuspend the cell pellet in 1 mL of fresh medium. 12. Perform cell counting using trypan blue. 13. Add fresh medium to the cell suspension until a cell density of 1e5 cells / mL is reached. 14. Then add 100 µL of the cell suspension to each well of a 96-well tissue culture plate so that each well has 10,000 cells. 15. Then incubate the cells at approximately 37°C + 5% carbon dioxide (CO 2 ) overnight. 16. The next day, remove the medium from the wells and add approximately 20 µL of the stock extract to each corresponding well. Then, add approximately 180 µL of fresh DMEM / F12 + approximately 2% FBS to all wells. 17. Then incubate the cells at approximately 37°C + 5% CO 2 for approximately 48 hours. 18. Approximately 48 hours later, perform MTT to evaluate cell proliferation. Select the rapid protocol; to eliminate background signals from the wells / media, subtract the absorbance reading at 650 nm from the reading at 570 nm.

[0171] Table 7 shows the final concentrations of each medium having red ginseng (Extract 1.3), ginseng extract (Extract 2.1), ganoderma lucidum (Extract 6.1), withania somnifera (Extract 7.1), cordyceps sinensis (rich in polysaccharides) (Extract 8), bird's nest extract (Extract BN), and coriolus versicolor extract (Extract YZ). Table 7: Final concentrations of the culture medium compositions

[0172] Results

[0173] Bovine skeletal muscle fibroblasts were cultured using different extracts at a concentration of approximately 160 μg / mL to study the effectiveness of the extracts in the presence of reduced serum. The MTT absorbance proportional to the cell number was compared to a control and expressed as a percentage, as Figure 2 shown and summarized in Table 8. The percent increase in proliferation normalized to the negative control was calculated as follows:

[0174] Percent increase in proliferation = {[(MTT absorbance in sample) - (MTT absorbance in negative control with DMEM:12)] / (MTT absorbance in negative control with DMEM:12)} * 100%

[0175] Now referring to Figure 2 , the figure shows cell growth at the 48 - hour time point stimulated by the following example additive compositions containing extracts (provided at approximately 160 μg / mL). As shown below, the extracts can exhibit various proliferative activities on the tested fibroblasts. Additionally, the cells were seeded at approximately 1×10 4 cells / well (96 - well plate) and were near 100% confluent. These extracts may require a reduced cell seeding density to stimulate fibroblasts because high density causes contact inhibition. In any case, these extracts show beneficial effects on fibroblasts, which may be an interesting approach for cell manufacturing. Table 8: Increase in cell proliferation within 48 hours (%)

[0176] The results in Table 8 show the corresponding results with the culture medium containing the basal medium DMEM:F12 and the example additive compositions, where the example additive compositions contain a first component of about 160 μg / ml of Panax ginseng (Extract 1.3), ginseng extract (Extract 2.1), Ganoderma lucidum (Extract 6.1), Withania somnifera (Extract 7.1), Cordyceps sinensis (rich in polysaccharides) (Extract 8), bird's nest extract (Extract BN) or Coriolus versicolor extract (Extract YZ) respectively, and a second component of FBS with a final concentration of about 2%. Among them, the best-performing extracts are Panax ginseng, Withania somnifera, bird's nest and Cordyceps sinensis, which have excellent proliferation increase percentages of 31.23%, 30.02%, 23.62% and 23.08% respectively at 56 hours. The results indicate that these sample extracts (KGN, BN, LZ, YZ, ASH, CS) have unexpected effects on the cell proliferation of myocytes under the condition of significantly reduced serum concentration (2% FBS). Instance 4

[0177] In this example, the effect of using a culture medium composition without the second component on the proliferation of L6 myoblasts was demonstrated.

[0178] Unless otherwise specified, the materials used in this example are similar or identical to those described in Example 1. For the sake of clarity, the list of materials is not repeated here.

[0179] In this example, the following steps are carried out: 1. Prepare a stock solution of the extract with a concentration of 1.6 mg / mL dissolved in deionized water. 2. Sterile filter each extract through a 0.22 μm filter and then store it at 4°C. 3. Thaw L6 myoblasts from liquid nitrogen, dilute them with DMEM medium, centrifuge, and resuspend the pellet in DMEM + 10% FBS to a cell density of 3×10 4 cells / mL. 4. Add 500 μL of cells to each well of a 24-well plate (coated with 2% gelatin) so that there are 1.5×10 4 cells per well. 5. Then incubate the cells overnight at 37°C + 5% CO 2 . 6. The next day, remove the medium from the wells and wash the cells twice with PBS. 7. Add DMEM with edible bird's nest extract (Hunan Newheixin Biomedicine Co., Ltd., batch MS230404) (extract BN) or ginseng extract (Xi'an Demeter Biotechnology Co., Ltd., batch DMTB230115) (extract 2.1) to the well plate so that the final volume per well is 500 μL. The final concentrations of the compositions are shown in Table 9. Table 9: Final concentrations of the culture medium compositions without the second component 8. Then incubate the cells at about 37 °C + 5% CO 2 for about 48 hours. 9. After 48 h, remove the medium and store it in a tube, and wash the cells with about 0.1 mL of PBS. 10. Then add about 0.3 mL of trypsin / EDTA and incubate at about 37 °C with occasional gentle tapping. Once the cells are completely dissociated, add back the medium stored in step 9. 11. Perform cell counting with a hemocytometer for analysis.

[0180] Results

[0181] Evaluate the final cell number after 48 h of treatment, and the results are shown in Table 10 and Figures 3A - 3B in. Table 10. Increase in cell proliferation within 48 hours (%)

[0182] Now refer to Figures 3A - 3B , which shows the final cell numbers of L6 myoblasts after 48 hours of treatment in media with different concentrations of ginseng extract (extract 2.1) and edible bird's nest extract (extract BN) according to Table 9. For the media of the example additive compositions with the first component containing different final concentrations of ginseng extract (extract 2.1) (and without any second component), it was observed that all the media of the example additive compositions with the first component containing different final concentrations of ginseng extract from 1 - 80 μg / ml showed an overall increase in the proliferation %. It was also observed that as the dose increased from 1 μg / ml to 40 μg / ml, there was an initial increase in the cell number. According to Table 10, the final cell number reached a peak at about 40 μg / mL of extract 2.1, with a 44.12% increase in proliferation compared to the negative control. This was accompanied by a sharp decrease in the final cell number at 80 μg / mL of extract 2.1. This shows that in the absence of serum, growth factors, or hormones in the medium, extract 2.1 at a final concentration of 40 μg / mL has a surprising effect on cell proliferation.

[0183] For the culture medium of an exemplary additive composition with a first component containing bird's nest (extract BN) at different final concentrations (1 - 80 μg / ml) and without any second component, as the concentration of extract BN increased from 5 μg / ml to 80 μg / ml, higher final cell numbers were observed, indicating a positive proliferative effect of extract BN in myoblasts. According to Table 10, in the presence of approximately 80 μg / mL extract BN, a 52.94% increase in proliferation was achieved. This shows that in the absence of serum, growth factors, or hormones in the culture medium, extract BN has a surprising effect on cell proliferation.

[0184] It was noted that according to Table 9, the final cell numbers of DMEM with BSA and DMEM without BSA were approximately 1.35×10 4 and approximately 1.91×10 4 cells, respectively. This indicates that 5 mg / mL BSA may be a concentration harmful to cells, and BSA should be eliminated or reduced.

[0185] In summary, the results show that when cells are treated with a sample additive composition containing bird's nest (extract BN) and ginseng (extract 2.1), there are surprising beneficial effects, as most of the test groups have a higher cell proliferation rate compared to when cells are not treated with the sample extract.

[0186] This example demonstrates that even in a serum - free culture medium, L6 cells treated with BN and ginseng extract can still outperform untreated controls in stimulating cell proliferation.

[0187] The examples disclosed herein show that at various concentrations and time points, various sample extracts can have different cellular effects, such as cell proliferation and differentiation.

[0188] In some embodiments, different types of extracts with different concentrations, different culture time points, and different extract suppliers will affect the proliferation, differentiation, and gene expression (e.g., Pax7, MyoD, MHC) of different cell types (e.g., fibroblasts, skeletal muscle satellite cells, skeletal muscle myoblasts, adipocytes, etc.).

[0189] In some embodiments, an increased dose and treatment time correspond to changes in the cell number. In some embodiments, the combination of extracts in the additive composition shows antagonistic, additive, or synergistic effects.

[0190] In some embodiments, after evaluating the relationship between various extract factors (concentration, time point, etc.) and cell growth / differentiation, an experimental method using multivariate statistics can be utilized to create a model showing the synergistic activity between extracts. The optimal combination of extracts that produce various cell effects (cell growth, differentiation, long-term passage, stemness) can be selected by using this model.

[0191] The exemplary embodiments of the present invention have thus been fully described. Although the description refers to specific embodiments, it will be apparent to those skilled in the art that the present invention can be practiced with variations of these specific details. Therefore, the present invention should not be construed as limited to the embodiments set forth herein.

[0192] For example, other cell lines of animal tissues can be used (e.g., muscle, fat, fibroblasts, vascular cells, pericytes, endothelial cells, myoblasts, satellite cells, adipocytes, adipose-derived stem cells, smooth muscle cells, skin cells, tendon, liver, brain, bone, heart, kidney, stem cells, and combinations thereof).

[0193] For example, other selected culture media and additives can be used.

[0194] For example, water is used as a solvent to extract the sample to form an aqueous solution, but other solvents such as DMSO, methanol, ethanol, etc. can be used.

[0195] For example, the sample extract can be commercially available or prepared from natural substances. Numbered embodiments Numbered Example 1

[0196] Example 1. A culture medium additive composition, the culture medium additive composition comprising at least one sample extract prepared from at least one natural substance, wherein the at least one natural substance is a sample selected from the group consisting of bird's nest, ginsengs, Ganoderma lucidum, Coprinus variegatus, Cordyceps sinensis, Withania somnifera, and combinations thereof.

[0197] Example 2. The composition according to any one of the preceding examples, wherein the ginsengs are American ginseng or Korean ginseng.

[0198] Example 3. The composition according to any one of the preceding examples, wherein the composition substantially does not contain one or more exogenous growth factors or hormones, or combinations thereof.

[0199] Example 4. The composition according to any one of the preceding examples, wherein the at least one sample extract is bird's nest.

[0200] Example 5. The composition according to any one of the preceding examples, wherein the at least one sample extract is American ginseng.

[0201] Example 6. The composition according to any one of the foregoing examples, wherein the at least one sample extract is Korean ginseng.

[0202] Example 7. The composition according to any one of the foregoing examples, wherein the at least one sample extract is Ganoderma lucidum.

[0203] Example 8. The composition according to any one of the foregoing examples, wherein the at least one sample extract is Hypholoma sublateritium.

[0204] Example 9. The composition according to any one of the foregoing examples, wherein the at least one sample extract is Cordyceps sinensis.

[0205] Example 10. The composition according to any one of the foregoing examples, wherein the at least one sample extract is Withania somnifera.

[0206] Example 11. The composition according to any one of the foregoing examples, wherein the Withania somnifera is derived from roots or the whole plant.

[0207] Example 12. The composition according to any one of the foregoing examples, wherein the sample extract is prepared from a solution of filtrate or supernatant.

[0208] Example 13. The composition according to any one of the foregoing examples, wherein the composition consists essentially of at least one sample extract, and the at least one sample extract is prepared from at least one sample selected from the group consisting of edible bird's nest, ginseng species, Ganoderma lucidum, Hypholoma sublateritium, Cordyceps sinensis, Withania somnifera, and combinations thereof.

[0209] Example 14. The composition according to any one of the foregoing examples, wherein the at least one sample extract has a concentration of 0.001 μg / mL - 1000 mg / mL.

[0210] Example 15. The composition according to any one of the foregoing examples, wherein the at least one sample extract has a concentration of 20 mg / mL - 100 mg / mL.

[0211] Example 16. A culture medium, which comprises a basal medium or a minimal medium and the culture medium additive composition according to any one of the foregoing examples.

[0212] Example 17. The culture medium according to any one of the foregoing examples, wherein the culture medium substantially does not contain one or more exogenous growth factors or hormones, or combinations thereof.

[0213] Example 18. The culture medium according to any one of the foregoing examples, wherein the culture medium further comprises one or more exogenous growth factors or hormones, or a combination thereof.

[0214] Example 19. The culture medium according to any one of the foregoing examples, wherein the basal medium is DMEM and / or F12.

[0215] Example 20. The culture medium according to any one of the foregoing examples, wherein the culture medium further comprises FBS at a final concentration of 0.01%-20%.

[0216] Example 21. A method for culturing cells, the method comprising the steps of: (i) providing a culture medium comprising a basal medium and at least one sample extract, the at least one sample extract being prepared from at least one sample selected from the group consisting of bird's nest, ginseng, Ganoderma lucidum, Hypsizygus marmoreus, Cordyceps sinensis, Withania somnifera, and combinations thereof; and (ii) treating and culturing the cells with the at least one natural substance.

[0217] Example 22. The method according to any one of the foregoing examples, wherein the method further comprises the step of: (1) treating the cell line with a dissociating agent to form a cell suspension before step (ii).

[0218] Example 23. The method according to any one of the foregoing examples, wherein the method further comprises the step of: (2) neutralizing the cell suspension with a neutralizing agent to obtain a neutralized cell suspension before step (ii).

[0219] Example 24. The method according to any one of the foregoing examples, wherein the method further comprises the step of: (3) obtaining a cell pellet from the neutralized cell suspension and resuspending the cell pellet in the culture medium.

[0220] Example 25. The method according to any one of the foregoing examples, wherein the sample extract is prepared by the steps of: (a) preparing at least one powder of the at least one sample; (b) dissolving the at least one powder to form at least one sample solution; (c) purifying the at least one sample solution to obtain the at least one sample extract.

[0221] Example 26. The method according to any one of the foregoing examples, wherein the purification of the at least one sample solution in step (c) is filtering and / or centrifuging the at least one sample solution.

[0222] Example 27. The method according to any one of the foregoing examples, wherein the culture medium has a final sample extract concentration of 0.001 ug / mL - 1000 mg / mL.

[0223] Example 28. The method according to any one of the foregoing examples, wherein the culture medium has a final sample extract concentration of 0.1 mg / mL - 0.5 mg / mL.

[0224] Example 29. The method according to any one of the foregoing examples, wherein the cultured cells are used to produce animal tissue.

[0225] Example 30. The method according to any one of the foregoing examples, wherein the animal tissue is human tissue.

[0226] Example 31. The method according to any one of the foregoing examples, wherein the cultured cells are used to produce cultured meat. Numbered Example 2

[0227] Example 1. An additive composition for a culture medium, the additive composition comprising a first component, the first component comprising at least one sample extract prepared from at least one natural substance, wherein the at least one natural substance is selected from the group consisting of: bird's nest (BN), ginsengs, Ganoderma lucidum, Trametes versicolor, Cordyceps sinensis (CS), Withania somnifera (ASH), and combinations thereof.

[0228] Example 2. The composition according to Example 1, wherein the ginsengs are American ginseng, Panax ginseng, and combinations thereof.

[0229] Example 3. The composition according to any one of the foregoing examples, wherein the Trametes versicolor is Coriolus versicolor (YZ).

[0230] Example 4. The composition according to any one of the foregoing examples, wherein the Ganoderma lucidum (Ling Zhi) is Ganoderma lucidum (GL).

[0231] Example 5. The composition according to any one of the foregoing examples, wherein the Trametes versicolor is Coriolus versicolor.

[0232] Example 6. The composition according to any one of the foregoing examples, wherein the first component substantially does not contain one or more exogenous sera, growth factors or hormones, or combinations thereof.

[0233] Example 7. The composition according to any one of the foregoing examples, wherein the culture medium is used to culture animal cells.

[0234] Example 8. The composition according to any one of the foregoing examples, wherein the culture medium is used to culture cells of mammals, birds, crustaceans or fish.

[0235] Example 9. The composition according to any one of the foregoing examples, wherein the culture medium is used for culturing fibroblasts, vascular cells, pericytes, endothelial cells, myoblasts, skeletal muscle satellite cells, muscle cells, smooth muscle cells, adipocytes, adipose-derived stem cells, skin cells, tendons, liver, brain, bone, heart, kidney, stem cells or a combination thereof.

[0236] Example 10. The composition according to any one of the foregoing examples, wherein the culture medium is used for culturing fibroblasts or myoblasts.

[0237] Example 11. The composition according to any one of the foregoing examples, wherein the individual sample extract is prepared from a solution of the filtrate or supernatant of the individual natural substance.

[0238] Example 12. The composition according to any one of the foregoing examples, wherein the first component consists essentially of at least one sample extract prepared from at least one natural substance, wherein the at least one natural substance is selected from the group consisting of bird's nest, ginseng species, Ganoderma lucidum, miscellaneous mushrooms, Cordyceps sinensis, Withania somnifera and combinations thereof.

[0239] Example 13. The composition according to any one of the foregoing examples, wherein the individual sample extract has a final concentration of about 0.01 ng / mL - 1000 mg / mL in the culture medium.

[0240] Example 14. The composition according to any one of the foregoing examples, wherein the individual sample extract has a final concentration of less than about 1000 or 500 μg / mL in the culture medium.

[0241] Example 15. The composition according to any one of the foregoing examples, the composition further comprises a second component, the second component comprising at least one serum, growth factor or hormone, amino acid, vitamin, salt, mineral, glucose, buffer, or a combination thereof.

[0242] Example 16. The composition according to any one of the foregoing examples, wherein the second component further comprises fetal bovine serum (FBS) at a final concentration of less than about 20%, 10%, 5% or 2% in the culture medium.

[0243] Example 17. The composition according to any one of the foregoing examples, wherein the second component further comprises bovine serum albumin (BSA) at a final concentration of about 0.01 ng / mL - 10 mg / mL in the culture medium.

[0244] Example 18. The composition according to any one of the foregoing examples, wherein the second component further comprises bovine serum albumin (BSA) at a final concentration of less than about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mg / mL in the culture medium.

[0245] Example 19. The composition according to any one of the foregoing examples, wherein the at least one growth factor or hormone is FGF-2, TGF-β1, vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF), insulin, EGF, platelet-derived growth factor (PDGF), interleukin 6 (IL-6), or a combination thereof.

[0246] Example 20. The composition according to any one of the foregoing examples, wherein the second component further comprises TGF-β1 present at a final concentration of less than about 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0.5 ng / mL in the culture medium.

[0247] Example 21. The composition according to any one of the foregoing examples, wherein the second component further comprises fetal bovine serum (FBS), fetal calf serum, bovine serum albumin (BSA), or a combination thereof.

[0248] Example 22. An additive composition for a culture medium for culturing myoblasts, the additive composition comprising: a first component, the first component comprising at least one sample extract prepared from at least one natural substance, wherein the at least one natural substance is selected from the group consisting of bird's nest (BN), American ginseng (AGN), and Korean ginseng (KGN) and combinations thereof, wherein the individual sample extract has a final concentration of about 1 - 500 μg / mL in the culture medium.

[0249] Example 23. The composition according to any one of the foregoing examples, the composition further comprising a second component, the second component comprising FBS at a final concentration of about 2% - 5% in the culture medium.

[0250] Example 24. An additive composition for a culture medium for culturing porcine skeletal muscle cells, the additive composition comprising: a first component, the first component comprising at least one sample extract prepared from at least one natural substance, wherein the at least one natural substance is selected from the group consisting of bird's nest (BN), American ginseng (AGN), and Korean ginseng (KGN) and combinations thereof, wherein the individual sample extract has a final concentration of about 1 - 500 μg / mL in the culture medium.

[0251] Example 25. The composition according to any one of the foregoing examples, the composition further comprising a second component, the second component comprising FBS at a final concentration of about 2% - 5% in the culture medium.

[0252] Example 26. The composition according to any one of the foregoing examples, wherein the composition further comprises a second component, and the second component comprises BSA having a final concentration of about 1-10 mg / ml in the medium and optionally TGF-β1 having a final concentration of about 0.5-10 ng / ml.

[0253] Example 27. An additive composition for a medium for culturing bovine skeletal muscle cells, the additive composition comprising: a first component, the first component comprising at least one sample extract prepared from at least one natural substance, wherein the at least one natural substance is selected from the group consisting of bird's nest (BN), ginseng species, Ganoderma lucidum, Hypsizygus marmoreus, Cordyceps sinensis (CS), Withania somnifera (ASH), and combinations thereof, and wherein the individual sample extract has a final concentration of about 1-500 μg / mL in the medium.

[0254] Example 28. The composition according to any one of the foregoing examples, wherein the composition further comprises a second component, and the second component comprises FBS having a final concentration of about 2%-5% in the medium.

[0255] Example 29. A medium comprising a basal medium or a basal medium and a medium additive composition according to any one of the foregoing examples.

[0256] Example 30. The medium according to any one of the foregoing examples, wherein the medium comprises a reduced amount of or substantially no exogenous serum, growth factor or hormone, or a combination thereof.

[0257] Example 31. The medium according to any one of the foregoing examples, wherein the basal medium or the basal medium is Dulbecco's Modified Eagle Medium (DMEM), Ham's F12 Medium (F12), Dulbecco's Modified Eagle Medium F12 (DMEM:F12), Ham's F10 Medium (F10), Roswell Park Memorial Institute 1640 Medium (RPMI 1640), Minimal Essential Medium (MEM), Serum-Free Medium (SFM), or a combination thereof.

[0258] Example 32. A method for culturing cells, the method comprising the steps of: (i) providing a basal medium or a basal medium and a medium additive composition according to any one of the foregoing examples to form a medium; and (ii) treating and culturing cells with the medium.

[0259] Example 33. The method according to any one of the foregoing examples, wherein the method further comprises the step of: (1) treating a cell line with a dissociating agent to form a cell suspension.

[0260] Example 34. The method according to any one of the preceding examples, wherein the method further comprises the step of: (2) neutralizing the cell suspension with a neutralizing agent to obtain a neutralized cell suspension.

[0261] Example 35. The method according to any one of the preceding examples, wherein the method further comprises the step of: (3) obtaining a cell pellet from the neutralized cell suspension and resuspending the cell pellet in the culture medium.

[0262] Example 36. The method according to any one of the preceding examples, wherein the sample extract is prepared by one or more of the following steps: (a) preparing at least one powder of the at least one natural substance; (b) dissolving the at least one powder to form at least one sample solution; (c) purifying the at least one sample solution to obtain the at least one sample extract.

[0263] Example 37. The method according to any one of the preceding examples, wherein in step (c), purifying the at least one sample solution is filtering and / or centrifuging the at least one sample solution.

[0264] Example 38. The method according to any one of the preceding examples, wherein the cultured cells are used to produce animal tissue.

[0265] Example 39. The method according to any one of the preceding examples, wherein the animal tissue is tissue of a mammal, bird, crustacean or fish.

[0266] Example 40. The method according to any one of the preceding examples, wherein the cultured cells are used to produce cultured meat.

[0267] Example 41. The method according to any one of the preceding examples, the method further comprising the step of incubating the cells in a CO 2 incubator.

[0268] Example 42. The method according to any one of the preceding examples, wherein the cell line is derived from a mammal, bird, crustacean or fish.

[0269] Example 43. The method according to any one of the preceding examples, wherein the cell line is derived from fibroblasts, vascular cells, pericytes, endothelial cells, myoblasts, satellite cells, skeletal muscle cells, muscle cells, smooth muscle cells, adipocytes, adipose-derived stem cells, skin cells, tendons, liver, brain, bone, heart, kidney, stem cells and combinations thereof.

Claims

1. An additive composition for a culture medium, the additive composition comprising a first component, the first component comprising at least one sample extract prepared from at least one natural substance, wherein the at least one natural substance is selected from the group consisting of: bird's nest (BN), ginseng species, Ganoderma lucidum, Trametes versicolor, Cordyceps sinensis (CS), Withania somnifera (ASH), and combinations thereof.

2. The composition according to claim 1, wherein the ginseng species are American ginseng, Panax ginseng, and combinations thereof.

3. The composition according to any one of the preceding claims, wherein the Trametes versicolor is Coriolus versicolor (YZ).

4. The composition according to any one of the preceding claims, wherein the Ganoderma lucidum (Ling Zhi) is Ganoderma lucidum (GL).

5. The composition according to any one of the preceding claims, wherein the Trametes versicolor is Coriolus versicolor.

6. The composition according to any one of the preceding claims, wherein the first component substantially does not contain one or more exogenous sera, growth factors, or hormones, or combinations thereof.

7. The composition according to any one of the preceding claims, wherein the culture medium is for culturing animal cells.

8. The composition according to any one of the preceding claims, wherein the culture medium is for culturing cells of mammals, birds, crustaceans, or fish.

9. The composition according to any one of the preceding claims, wherein the culture medium is for culturing fibroblasts, vascular cells, pericytes, endothelial cells, myoblasts, skeletal muscle satellite cells, muscle cells, smooth muscle cells, adipocytes, adipose-derived stem cells, skin cells, tendons, liver, brain, bone, heart, kidney, stem cells, or combinations thereof.

10. The composition according to any one of the preceding claims, wherein the culture medium is for culturing fibroblasts or myoblasts.

11. The composition according to any one of the preceding claims, wherein the individual sample extract is prepared from a solution of the filtrate or supernatant of the individual natural substance.

12. The composition according to any one of the preceding claims, wherein the first component consists essentially of at least one sample extract prepared from at least one natural substance, wherein the at least one natural substance is selected from the group consisting of: bird's nest, ginseng species, Ganoderma lucidum, Trametes versicolor, Cordyceps sinensis, Withania somnifera, and combinations thereof.

13. The composition according to any one of the preceding claims, wherein the individual sample extract has a final concentration in the culture medium of about 0.01 ng / mL - 1000 mg / mL.

14. The composition according to any one of the preceding claims, wherein the individual sample extract has a final concentration in the culture medium of about less than 1000 or 500 μg / mL.

15. The composition according to any one of the preceding claims, the composition further comprising a second component, the second component comprising at least one serum, growth factor, or hormone, amino acids, vitamins, salts, minerals, glucose, buffers, or combinations thereof.

16. The composition according to claim 15, wherein the second component further comprises fetal bovine serum (FBS) at a final concentration of less than about 20%, 10%, 5% or 2% in the culture medium.

17. The composition according to any one of claims 15 - 16, wherein the second component further comprises bovine serum albumin (BSA) at a final concentration of about 0.01 ng / mL - 10 mg / mL in the culture medium.

18. The composition according to any one of claims 15 - 17, wherein the second component further comprises bovine serum albumin (BSA) at a final concentration of less than about 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 mg / mL in the culture medium.

19. The composition according to any one of claims 15 - 18, wherein the at least one growth factor or hormone is FGF - 2, TGF - b1, vascular endothelial growth factor (VEGF), insulin - like growth factor (IGF), insulin, EGF, platelet - derived growth factor (PDGF), interleukin 6 (IL - 6) or a combination thereof.

20. The composition according to any one of claims 15, 17, 18, wherein the second component further comprises TGF - b1 at a final concentration of less than about 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0.5 ng / mL in the culture medium.

21. The composition according to any one of claims 15 - 20, wherein the second component further comprises fetal bovine serum (FBS), fetal calf serum, bovine serum albumin (BSA) or a combination thereof.

22. An additive composition for a culture medium for culturing myoblasts, the additive composition comprising: A first component, the first component comprising at least one sample extract prepared from at least one natural substance, wherein the at least one natural substance is selected from the group consisting of bird's nest (BN), American ginseng (AGN) and Korean ginseng (KGN) and combinations thereof, and wherein the individual sample extract has a final concentration of about 1 - 500 μg / mL in the culture medium.

23. The composition according to claim 22, the composition further comprising a second component, the second component comprising FBS at a final concentration of about 2% - 5% in the culture medium.

24. An additive composition for a culture medium for culturing porcine skeletal muscle cells, the additive composition comprising: A first component, the first component comprising at least one sample extract prepared from at least one natural substance, wherein the at least one natural substance is selected from the group consisting of bird's nest (BN), American ginseng (AGN) and Korean ginseng (KGN) and combinations thereof, and wherein the individual sample extract has a final concentration of about 1 - 500 μg / mL in the culture medium.

25. The composition according to claim 24, the composition further comprising a second component, the second component comprising FBS at a final concentration of about 2% - 5% in the culture medium.

26. The composition according to any one of claims 24-25, wherein the composition further comprises a second component, and the second component comprises BSA having a final concentration of about 1-10 mg / ml in the medium and optionally TGF-β1 having a final concentration of about 0.5-10 ng / ml.

27. An additive composition for a medium for culturing bovine skeletal muscle cells, the additive composition comprising: a first component, the first component comprising at least one sample extract prepared from at least one natural substance, wherein the at least one natural substance is selected from the group consisting of bird's nest (BN), ginseng species, Ganoderma lucidum, Hypsizygus marmoreus, Cordyceps sinensis (CS), Withania somnifera (ASH), and combinations thereof, and the individual sample extract has a final concentration of about 1-500 μg / mL in the medium.

28. The composition according to claim 27, wherein the composition further comprises a second component, and the second component comprises FBS having a final concentration of about 2%-5% in the medium.

29. A medium, the medium comprising a basal medium or a basic medium and a medium additive composition according to any one of the preceding claims.

30. The medium according to claim 29, wherein the medium substantially does not contain or contains a reduced amount of one or more exogenous sera, growth factors or hormones, or combinations thereof.

31. The medium according to any one of claims 29-30, wherein the basal medium or the basic medium is Dulbecco's Modified Eagle Medium (DMEM), Ham's F12 Medium (F12), Dulbecco's Modified Eagle Medium F12 (DMEM:F12), Ham's F10 Medium (F10), Roswell Park Memorial Institute 1640 Medium (RPMI1640), Minimal Essential Medium (MEM), Serum-Free Medium (SFM), or combinations thereof.

32. A method for culturing cells, the method comprising the steps of: (i) providing a basal medium or a basic medium and a medium additive composition according to any one of the preceding claims to form a medium; and (ii) treating and culturing the cells with the medium.

33. The method according to claim 32, wherein the method further comprises the steps of: (1) treating a cell line with a dissociating agent to form a cell suspension.

34. The method according to any one of claims 32-33, wherein the method further comprises the steps of: (2) neutralizing the cell suspension with a neutralizing agent to obtain a neutralized cell suspension.

35. The method according to any one of claims 32-34, wherein the method further comprises the steps of: (3) obtaining a cell pellet from the neutralized cell suspension and resuspending the cell pellet in the medium.

36. The method according to any one of claims 32-35, wherein the sample extract is prepared by one or more of the following steps: (a) preparing at least one powder of the at least one natural substance; (b) Dissolve the at least one powder to form at least one sample solution; (c) Purify the at least one sample solution to obtain the at least one sample extract.

37. The method according to any one of claims 32-36, wherein purifying the at least one sample solution in step (c) is filtering and / or centrifuging the at least one sample solution.

38. The method according to any one of claims 32-37, wherein the cultured cells are used to produce animal tissue.

39. The method according to claim 38, wherein the animal tissue is tissue of a mammal, bird, crustacean or fish.

40. The method according to claim 38, wherein the cultured cells are used to produce cultured meat.

41. The method according to any one of claims 32-40, the method further comprising incubating the cells in a CO 2 incubator.

42. The method according to any one of claims 33-41, wherein the cell line is derived from a mammal, bird, crustacean or fish.

43. The method according to any one of claims 33-42, wherein the cell line is derived from fibroblasts, vascular cells, pericytes, endothelial cells, myoblasts, satellite cells, skeletal muscle cells, muscle cells, smooth muscle cells, adipocytes, adipose-derived stem cells, skin cells, tendons, liver, brain, bone, heart, kidney, stem cells and combinations thereof.

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