Additive composition for culture medium, culture medium containing the additive composition, and method for culturing cells

Abstract

An additive composition for culture media is provided, the additive composition comprising a first component including at least one sample extract produced 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, versicolor mushroom, cordyceps sinensis (CS), ashwagandha (ASH), and combinations thereof. The additive composition can serve as an alternative source of growth factors and hormones for cell culture media, improving consumer acceptance and / or minimizing production costs.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 381,761, filed November 1, 2022, the entire contents of which are incorporated herein by reference in their entirety for all purposes. [Technical Field]

[0002] The present application relates to a composition for a culture medium, a culture medium, a kit, a method and an application thereof. In particular, the present application relates to an additive composition for a culture medium, a culture medium, a kit, a method and an application thereof. [Background technology]

[0003] Growth factors and hormones are essential for cell culture media (e.g., used to culture animal cells and produce animal tissues). For example, fetal bovine serum (FBS) is widely used in cell culture as a source of growth factors and hormones in the media. However, FBS is highly unethical and expensive. Attempts have been made to produce recombinant growth factors and hormones to reduce costs. Excessive use of recombinant growth factors can pose significant health problems to consumers and can lead to consumer rejection of cell-cultured food products (e.g., cultured meat). To improve consumer acceptance and / or minimize production costs, it is highly necessary to provide alternative, all-natural or mostly-natural media additive compositions as alternative sources of growth factors and hormones in cell culture media. Summary of the Invention

[0004] In some embodiments, an additive composition for a culture medium is provided, the additive composition comprising a first component including 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, lingzhi (Ganoderma lucidum), versicolor mushroom, cordyceps sinensis (CS), ashwagandha (ASH), and combinations thereof.

[0005] In some embodiments, an additive composition for a culture medium for culturing myoblasts is provided, the additive composition comprising a first component including 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 single sample extract has a final concentration of about 1 to 500 μg / mL in the culture medium.

[0006] In some embodiments, an additive composition for a culture medium for culturing porcine bone marrow muscle cells is provided, the additive composition comprising a first component including 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 single sample extract has a final concentration of about 1 to 500 μg / mL in the culture medium.

[0007] In some embodiments, an additive composition for a culture medium for culturing bovine bone marrow muscle cells is provided, the additive composition comprising a first component including 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, lingzhi (Ganoderma Lucidum), versicolor mushroom, cordyceps sinensis (CS), ashwagandha (ASH), and combinations thereof, wherein the single sample extract has a final concentration of about 1 to 500 μg / mL in the culture medium.

[0008] In some embodiments, a culture medium is provided, the culture medium comprising a basal or base medium and the medium additive composition of any one of the preceding embodiments.

[0009] In some embodiments, a method of culturing cells is provided, the method comprising: (i) providing a basal medium or base medium and the medium additive composition of any one of the preceding embodiments to form a medium; and (ii) treating and culturing cells in the medium.

[0010] Advantages of the present disclosure

[0011] In some embodiments, provided additive compositions contain one or more extracts derived from biological or natural (e.g., non-mammalian, plant, or fungal) sources (e.g., bird's nest and ginseng) that are used to modulate the production of growth factors in cells, stabilize those growth factors to increase or prolong their activity, or modulate pathways that are the same as those modulated by certain growth factors. In some embodiments, the extracts can also increase cell proliferation, increase cellular stemness, inhibit cell differentiation, and / or induce cell differentiation. In addition, these extracts can provide other health benefits, and from the perspective of traditional medicine (e.g., Traditional Chinese Medicine (TCM) or Ayurvedic medicine), upon consumption, these extracts bring balance to both the cells in culture and the consumer's body.

[0012] In some embodiments, the bird's nest is an edible bird's nest produced by swiftlets.

[0013] In some embodiments, alternative / novel compositions, media, kits, methods, and uses are provided to minimize or even replace the use of one or more of the major and most expensive components of cell culture media for growing cells: growth factors, such as fibroblast growth factor 2 (FGF-2) (e.g., basic fibroblast growth factor, bGF) and TGF-b1. In some embodiments, these alternatives may be derived from natural sources (e.g., non-mammalian / plant / fungal extracts), and they act as homologs (having protein sequences similar to those of functionally identical growth factors) by modulating the production of growth factors in cells, regulating pathways that are the same as those regulated by some growth factors, and / or stabilizing growth factors to make them more effective. In some embodiments, they may also function as replacements for hormones (e.g., insulin) or transferrin, which have antimicrobial properties (e.g., preventing contamination during scale-up), anticancer properties, and / or, in the case of skin care serums, promote the production of extracellular matrix, such as elastin and collagen. The use of these alternatives may eliminate the need to add exogenous growth factors, hormones, and antibiotics during cell culture, which further minimizes cost and safety issues. In some embodiments, they can also function as a substitute for serum (e.g., fetal bovine serum) to produce additive compositions for serum-free, substantially serum-free, or serum-reduced media. Production costs are significantly reduced.

[0014] In some embodiments, the use of these high quality natural extracts is more appealing to consumers and is highly advantageous from a marketing perspective.

[0015] In some embodiments, the novel compositions, media, kits, methods, and uses provided are more consumer-friendly and more natural than using exogenous or recombinant growth factors, and in some embodiments, they can also achieve the same goal: reducing the cost of cultured meat to reach (or approach or even be lower than) the same price level as regular meat.

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

[0017] In some embodiments, the novel compositions, media, kits, methods and uses provided can produce cells, cell cultures, and tissues for a variety of applications, including but not limited to gene therapy, microfluidics, drugs, nutraceuticals, diagnostics, transplantation, and biotechnology.

[0018] In some embodiments, the novel compositions, media, kits, methods and uses provided may be nutritionally healthier as some of these components may prevent or treat a disease (e.g., cancer or diabetes), disorder or condition.

[0019] In some embodiments, provided additive compositions include one or more extracts that inhibit differentiation and promote cell proliferation and stemness, thereby increasing final cell yield. For example, in some embodiments, stemness in skeletal muscle satellite cells can be increased, for example, by modulating the Pax7 gene.

[0020] In some embodiments, provided additive compositions contain one or more extracts that promote cell differentiation (e.g., maturation of muscle fibers or adipocytes).

[0021] In some embodiments, the additive compositions provided include extracts for partial or complete replacement of growth factors, such as vascular endothelial growth factor (VEGF) and / or fibroblast growth factor (FGF), i.e., the amount of exogenous growth factors in the additive composition is reduced.

[0022] In some embodiments, the additive compositions provided include extracts to partially or completely replace serum (e.g., FBS), i.e., the amount of exogenous serum in the additive composition is reduced.

[0023] In some embodiments, alternative / novel additive compositions, media, kits, methods and uses are provided to modulate, mimic, stabilize, minimize or even replace the use of one or more of growth factors, hormones and / or serum. In some embodiments, alternative / novel additive compositions, media, kits, methods and uses are provided that have the same or similar effect as growth factors, hormones and / or serum to modulate one or more pathways.

[0024] In some embodiments, the growth factor is fibroblast growth factor 2 (FGF-2) (e.g., basic fibroblast growth factor, bGF), TGF-b1, VEGF, IGF, insulin, EGF, PDGF, and / or IL-6.

[0025] In some embodiments, the novel compositions, media, kits, methods, and uses provided are used to produce cultivated meat, which in some embodiments is edible and safe for human or animal consumption.

[0026] In several embodiments, the novel compositions, media, kits, methods and uses provided reduce the cost of producing cells in cell culture (e.g., in cultivated meat).

[0027] In several examples, novel compositions, media, kits, methods, and uses are provided that demonstrate that sample extracts derived from ginseng (Panax ginseng, KGN), ginseng (Panax quinquefolius, AGN), ashwagandha (Ashwagandha, ASH), cordyceps sinensis (Cordyceps sinensis, CS), bird's nest (BN), Coriolus versicolor (Coriolus versicolor, YZ), and reishi mushroom (Ganoderma lucidum, GL) have the potential to stimulate proliferation of various cell types (e.g., L6, primary porcine myoblasts, bovine skeletal myofibroblasts) under various conditions (e.g., serum-reduced / growth factor-reduced medium and serum-free / growth factor-free medium).

[0028] In some embodiments, the additive compositions provided contain multiple sample extracts in specific combinations and effective concentrations / ranges, which provide unexpected synergistic effects for the culture medium, providing one or more of the benefits described above. [Brief explanation of the drawings]

[0029] [Figure 1] FIG. 1 shows cell growth of porcine myoblasts under stimulation of different exemplary additive compositions containing various sample extracts (provided at 300 μg / mL) at 56 h according to exemplary examples. [Figure 2] FIG. 1 shows cell growth of bovine fibroblasts under stimulation of different exemplary additive compositions containing various sample extracts (provided at 160 μg / mL) at 48 h according to exemplary examples. [Figure 3A] FIG. 1 shows cell growth of L6 myoblasts under stimulation of exemplary additive compositions containing different concentrations of sample extracts bird's nest extract or carrot extract at 48 hours according to exemplary embodiments. [Figure 3B] FIG. 1 shows cell growth of L6 myoblasts under stimulation of exemplary additive compositions containing different concentrations of sample extracts bird's nest extract or carrot extract at 48 hours according to exemplary embodiments. DETAILED DESCRIPTION OF THE INVENTION

[0030] Although the description refers to particular embodiments, the present disclosure should not be construed as being limited to the embodiments set forth herein.

[0031] As used in this specification and claims, the terms "comprise" (or any related form, e.g., "comprises" and "including"), "containing" (or any related form, e.g., "contains" or "containing"), and "comprises" (or any related form, e.g., "comprises" or "comprising") mean the inclusion of the following elements, but not the exclusion of other elements. It should be understood that for each example using the terms "comprises" (or any related form, e.g., "comprises" and "comprising"), "containing" (or any related form, e.g., "contains" or "containing"), and "comprises" (or any related form, e.g., "comprises" or "comprising"), this disclosure / application further includes alternative examples in which the terms "including," "comprises," or "comprising" are replaced with "consisting essentially of" or "consisting of." These alternative examples using "consisting of" or "consisting essentially of" are understood to be examples of a smaller scope than the examples using "comprising," "comprising," or "comprising."

[0032] For clarity, the terms "comprise," "comprising," "containing," and "having," and any related forms, are open terms, allowing for additional elements or features other than the required elements specified; and "consisting of" is closed terms, limited to the elements recited in the claim and not including any elements, steps, or ingredients not specified in the claim.

[0033] "Consisting essentially of" limits the claim to certain materials, components, or steps ("essential elements") that do not substantially affect one or more essential characteristics of the invention for which protection is sought. In some embodiments, the essential characteristics are one or more basic and novel characteristics of the invention for which protection is sought.

[0034] As used herein, the singular forms "a," "an," and "said" are intended to include the plural forms unless the context clearly dictates otherwise. When ranges are referred to in the specification, they are understood to include each discrete point within the range. For example, 1 to 7 means 1, 2, 3, 4, 5, 6, and 7.

[0035] As used herein, the term "about" is understood to mean within the normal tolerance range of the art and not more than ±20% of the stated value. For example, about 50 means 40 to 60, including all values ​​therebetween. As used herein, the term "about" a particular value also includes the particular value, for example, about 50 includes 50.

[0036] As used in this specification and claims, the terms "approximately" or "generally" or "substantially" or "substantially" mean that the recited feature, angle, shape, state, structure, or value need not be exactly achieved, but that deviations or variations, including, for example, tolerances, measurement errors, limitations in measurement accuracy, and other factors known to those skilled in the art, may occur in an amount that does not eliminate the intended effect of the feature. For example, a "substantially" serum-free composition or a composition that is "substantially free" of serum means that the composition contains no serum, very little serum (e.g., an amount below the detection limit), or less than 1% of the total composition.

[0037] As used herein, the terms "cultured meat," "cultured meat," "test tube meat," "cell-cultured meat," "clean meat," "synthetic meat," and "lab-grown meat" can be used interchangeably and refer to meat-like products that contain tissue derived from cell culture. In some instances, cultured meat is produced by culturing animal cells or tissues in vitro without growing a whole animal (also known as "cellular agriculture"). For example, the methods involve isolating and expanding meat-specific cells from an animal (where the cells are grown in a nutrient-rich medium to expand the cells), followed by eventual harvesting of the cells to form the final meat-like product.

[0038] As used herein, the terms "expansion" or "amplification" refer to the process of increasing cell numbers and cell populations by culturing the desired cells.

[0039] As used herein, the term "media" refers to a composition that supports the growth and / or other cellular activity of cells or cellular tissues.

[0040] As used herein, the term "basal medium" or "basal medium" refers to a growth medium that provides essential nutrients for the survival and growth of desired cells. Basal media can serve as a starting point or base to which specific additives can be added to meet the specific requirements of the desired cell culture. Examples include, but are not limited to, Dulbecco's Modified Eagle's Medium (DMEM), Ham's F12 Medium (F12), Dulbecco's Modified Eagle's Medium F12 (DMEM:F12), Ham's F10 Medium (F10), Roswell Park Memorial Institute 1640 Medium (RPMI 1640), Minimum Essential Medium (MEM), Serum-Free Medium (SFM), or combinations thereof.

[0041] As used herein, the term "additive composition" refers to a formulation containing one or more additives or ingredients to meet one or more specific needs of a cell culture. The additive composition can be added to a basal medium to form the medium.

[0042] As used herein, the term "serum" refers to the fluid component of blood, which may contain substances necessary for cell growth (e.g., proliferation and / or differentiation), such as growth factors and hormones. In some instances, serum (e.g., fetal bovine serum) is an exogenous additive to the culture medium.

[0043] As used herein, the terms "growth factor" and "hormone" refer to substances, e.g., proteins, that regulate signal transduction pathways and / or stimulate cellular functions (e.g., cell growth, proliferation, and / or differentiation). Examples of growth factors or hormones include fibroblast growth factors (FGFs) (e.g., FGF-2), transforming growth factors (TGFs) (e.g., TGF-b1 and TGF-3), vascular endothelial growth factors (VEGFs) (e.g., VEGF-A), insulin-like growth factors (IGFs) (e.g., IGF-1), insulin, epidermal growth factor (EGF), platelet-derived growth factors (PDGFs) (e.g., PDGF-BB), hepatocyte growth factor (HGF), and interleukins (ILs) (e.g., interleukin 6 (IL-6)).

[0044] As used herein, the term "natural substance" refers to a material that is derived from a naturally occurring source (e.g., an animal, fungus, or plant) and has not been significantly modified or artificially / chemically synthesized. In some instances, the natural substance is a pharmaceutical substance, such as a traditional medicine, traditional Chinese medicine (TCM), and / or Ayurvedic medicine. In some embodiments, the natural substance is derived from plants and fungi, which contain a wide variety of proteins, polysaccharides, or other biologically active substances.

[0045] As used herein, the term "sample extract" refers to a partial or representative sample that has been taken 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.

[0046] As used herein, the term "bird's nest" or "edible bird's nest" refers to a natural substance made primarily from the solidified saliva of swiftlets, and is generally recognized as a TCM.

[0047] As used herein, the term "ginseng" refers to perennial plants belonging to the genus Panax. In some instances, only the root is used. For example, Korean ginseng, Japanese ginseng or Chinese ginseng (Dongjiang) and American ginseng (Wangjiang) are two exemplary ginseng species used in TCM. Other ginseng species may also be used. In some instances, ginseng from other regions, such as the Korean Peninsula, Northeast China, Far East Russia, Canada, Vietnam, and the United States, may also be used. In some embodiments, ginseng includes Korean Red Ginseng.

[0048] As used herein, the term "Ling Zhi" or "Lingzhi" refers to a polypore fungus belonging to the genus Ganoderma and is recognized as a TCM. For example, Ganoderma sichuanense and Ganoderma sichuanense are two exemplary Ganoderma species used as TCM.

[0049] As used herein, the term "Trametes versicolor" refers to a polypore fungus belonging to the genus Trametes. For example, Trametes versicolor (or Trametes versicolor) is one of the exemplary versicolor mushroom species used in TCM and may also be called Yunzhi.

[0050] As used herein, the term "Cordyceps sinensis" or "Ophiocordyceps sinensis" refers to an entomopathogenic fungus in the family Ophiocordycipitaceae, and is generally recognized as a TCM.

[0051] As used herein, the term "ashwagandha" or "Withania somnifera" refers to an herb belonging to the Withania genus and generally recognized as an Ayurvedic medicine.

[0052] As used herein, the term "exogenous" refers to some component or element that originates outside or external to a composition. For clarity, a component (e.g., a serum-derived growth factor, a chemically synthesized or recombinant growth factor or hormone) is considered exogenous when added to an additive composition. In another aspect, a growth factor or hormone is not considered exogenous when it is naturally present in or derived from a sample extract of a natural substance.

[0053] As used herein, the term "final concentration" refers to the concentration of a substance or component (e.g., a first component or a second component) present in a solution or mixture (e.g., a medium) after all necessary dilutions or reactions have occurred. In some instances, the total concentration of the medium is calculated on a weight-to-volume basis (100% (w / v)). In some other instances, the total concentration of the medium is calculated on a volume-to-volume basis (100% (v / v)).

[0054] As used herein, the term "animal" refers to eukaryotic species such as mammals (e.g., porcine, bovine, ovine, equine, canine, feline, rodent), birds or avian species, reptiles, fish, amphibians, crustaceans, mollusks, cephalopods, etc. Examples of the present invention Example 1

[0055] In some embodiments, novel compositions, media, kits, methods, and uses are provided, which comprise an extract of a sample selected from the group consisting of bird's nest, ginseng, ashwagandha, reishi mushroom, versicolor mushroom, cordyceps, and combinations thereof, which extract has been validated to increase cell number and can be used as an additive in basal cell culture media, such as DMEM media (or another in-house formulated basal media having essential salts, buffers, minerals, amino acids, lipids, minerals, etc.), for the purpose of (e.g., scale-up) production of meat tissue.

[0056] In some embodiments, the novel compositions, media, kits, methods and uses provided include one or more of the following features: 1. By using natural extracts (in some embodiments, edible bird's nest, Korean ginseng, and American ginseng), the novel compositions are more consumer-friendly, healthier, natural, and potentially less costly. 2. Without being bound by any theory, these extracts may contain active ingredients to upregulate specific growth factors or hormones and / or they may have protein structures that are homologous to essential growth factors or hormones. As such, they may completely or partially replace the need for non-natural growth factor or hormone sources (e.g., recombinant sources) and may also reduce the cost of producing cell cultures (e.g., cultivated meat). 3. Certain compounds from the extract can be further isolated and / or purified and used in place of the whole extract. 4. The extract can be added to cell culture media to replace the function of some growth factors or hormones. 5. These extracts can be used to preserve stemness of meat cells in culture, for example by slowing uncontrolled cell differentiation, resulting in higher cell yields. 6. These extracts are used to increase the growth rate of cells in culture, resulting in higher cell yields in a shorter period of time.

[0057] In some embodiments, a culture medium additive composition is provided, the culture medium additive composition comprising at least one sample extract prepared from at least one natural substance. In some embodiments, the at least one natural substance is a sample selected from the group consisting of bird's nest, ginseng, ashwagandha, reishi mushroom, versicolor mushroom, cordyceps, and combinations thereof. In some embodiments, the ginseng is American ginseng or Korean ginseng. Other embodiments are described below.

[0058] In some embodiments, the composition is substantially free of one or more exogenous growth factors or hormones, or combinations thereof.

[0059] In some embodiments, the at least one sample extract is bird's nest. In some embodiments, the at least one sample extract is American ginseng. In some embodiments, the at least one sample extract is Korean ginseng. In some embodiments, the at least one sample extract is Reishi mushroom. In some embodiments, the at least one sample extract is Versicolor mushroom. In some embodiments, the at least one sample extract is Coriolus versicolor. In some embodiments, the at least one sample extract is Cordyceps sinensis. In some embodiments, the at least one sample extract is Ashwagandha. In some embodiments, Ashwagandha is derived from the root alone or the whole plant.

[0060] In some embodiments, a sample extract is prepared from a solution of the filtrate or supernatant. In some embodiments, the solution comprises water. In some embodiments, the solution comprises an organic solvent.

[0061] In some embodiments, the composition consists essentially of at least one sample extract, wherein the at least one sample extract is prepared from at least one sample selected from the group consisting of bird's nest, ginseng, reishi mushroom, versicolor mushroom, cordyceps, ashwagandha, and combinations thereof.

[0062] In some embodiments, the at least one sample extract comprises a concentration of 0.001 μg / mL to 1000 mg / mL. In some embodiments, the at least one sample extract comprises a concentration of 20 mg / mL to 100 mg / mL. In some embodiments, the at least one sample extract comprises a concentration of 20 mg / mL. In some embodiments, the at least one sample extract comprises a concentration of 60 mg / mL. In some embodiments, the at least one sample extract comprises a concentration of 100 mg / mL. In some embodiments, the sample extract is prepared from an aqueous solution of the filtrate or supernatant.

[0063] In some embodiments, a culture medium is provided, the culture medium comprising a basal or base medium and a medium additive composition disclosed in any one of the above embodiments.

[0064] In some embodiments, the medium is substantially free of one or more exogenous growth factors or hormones, or a combination thereof. In some embodiments, the medium further comprises one or more exogenous growth factors or hormones, or a combination thereof.

[0065] In some embodiments, the basal medium is Dulbecco's Modified Eagle's Medium (DMEM) and / or F12.

[0066] In some embodiments, the composition further comprises FBS at a concentration of 5%.

[0067] In some embodiments, a method for culturing cells is provided, the method comprising: (i) providing a medium containing a basal medium and at least one sample extract prepared from at least one natural substance; and (ii) treating and culturing cells with the at least one natural substance. In some embodiments, the at least one natural substance is a sample of bird's nest, ginseng, ashwagandha, Ling Zhi (e.g., Ganoderma lucidum), Versicolor mushroom (e.g., Yun Zhi), Cordyceps sinensis, and combinations thereof. In some embodiments, the method further comprises: (1) treating the cell line with a dissociation agent (e.g., trypsin) to form a cell suspension after step (ii). In some embodiments, the method further comprises: (2) neutralizing the cell suspension with a neutralizing agent (e.g., TrypLE, trypsin neutralizing solution) to obtain a neutralized cell suspension before step (ii). 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 a medium.

[0068] In some embodiments, the sample extract is produced by the steps of: (a) producing at least one powder of the at least one sample; (b) dissolving the at least one powder to form at least one sample solution; and (c) purifying the at least one sample solution to obtain the at least one sample extract. In some embodiments, the sample extract is produced by the steps of: (a) producing 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; and (c) filtering and / or centrifuging the at least one sample solution to obtain the at least one sample extract.

[0069] In some embodiments, the medium comprises a final sample extract concentration of about 0.001 μg / mL to 1000 mg / mL. In some embodiments, the medium comprises a final sample extract concentration of about 0.1 mg / mL to 0.5 mg / mL. In some embodiments, the medium comprises a final sample extract concentration of about 0.1 mg / mL. In some embodiments, the medium comprises a final sample extract concentration of about 0.3 mg / mL. In some embodiments, the medium comprises a final sample extract concentration of about 0.5 mg / mL.

[0070] In some embodiments, culturing the cells is used to produce animal tissue. In some embodiments, the animal tissue is human tissue. In some embodiments, culturing the cells is used to produce cultivated meat. In some embodiments, the at least one sample solution comprises a solvent, hi some embodiments, the at least one sample solution comprises water, hi some embodiments, the at least one sample solution comprises an organic solvent. Example 2

[0071] In some embodiments, an additive composition for a culture medium is provided, 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, versicolor mushroom, cordyceps sinensis (CS), ashwagandha (ASH), and combinations thereof.

[0072] In some embodiments, the carrots are ginseng, carrot, and combinations thereof.

[0073] In some embodiments, the versicolor mushroom is Yunzhi (YZ).

[0074] In some embodiments, the Ling Zhi is Ganoderma lucidum (GL).

[0075] In some embodiments, the versicolor mushroom is Coriolus versicolor.

[0076] In some embodiments, the at least one natural material is bird's nest.

[0077] In some embodiments, the at least one natural substance is American ginseng (Panax ginseng).

[0078] In some embodiments, the at least one natural substance is ginseng (ginseng).

[0079] In some embodiments, the natural substance is Ganoderma lucidum.

[0080] In some embodiments, the at least one natural substance is a versicolor mushroom.

[0081] In some embodiments, the at least one natural substance is Cordyceps sinensis.

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

[0083] In some embodiments, the ashwagandha is derived from the root or the whole plant.

[0084] In some embodiments, the first component is substantially free of one or more exogenous serum, growth factors or hormones, or combinations thereof.

[0085] In some embodiments, the medium is used to culture animal cells.

[0086] In some embodiments, the medium is used to culture mammalian, avian, crustacean, or fish cells.

[0087] In some embodiments, the medium is used to culture fibroblasts, vascular cells, pericytes, endothelial cells, myoblasts, skeletal muscle satellite cells, muscle cells, smooth muscle cells, fat cells, adipose-derived stem cells, skin cells, tendon, liver, brain, bone, heart, kidney, stem cells, or combinations thereof.

[0088] In some embodiments, the medium is used to culture fibroblasts or myoblasts.

[0089] In some embodiments, a single sample extract is prepared from a solution of a filtrate or supernatant of a single natural substance.

[0090] In some embodiments, the first component consists essentially of at least one sample extract made from at least one natural substance, wherein the at least one natural substance is selected from the group consisting of bird's nest, ginseng, reishi mushroom, versicolor mushroom, cordyceps, ashwagandha, and combinations thereof.

[0091] In some embodiments, the single sample extract has a final concentration in the medium of about 0.01 ng / mL to 1000 mg / mL.

[0092] In some embodiments, the single sample extract has a final concentration in the medium of less than about 1000 or 500 ug / mL.

[0093] In some embodiments, the additive composition further comprises a second component comprising at least one serum, growth factor or hormone, amino acid, vitamin, salt, mineral, glucose, buffer, or combination thereof.

[0094] In some embodiments, the second component further comprises fetal bovine serum (FBS) at a final concentration in the medium of less than about 20%, 10%, 5%, or 2%.

[0095] In some embodiments, the second component further comprises bovine serum albumin (BSA) at a final concentration in the medium of about 0.01 ng / mL to 10 mg / mL.

[0096] In some embodiments, the second component further comprises bovine serum albumin (BSA) at a final concentration in the medium of less than about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mg / mL.

[0097] 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.

[0098] In some embodiments, the second component further comprises TGF-b1 present in the medium at a final concentration of less than about 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0.5 ng / mL.

[0099] In some embodiments, the second component further comprises fetal bovine serum (FBS), fetal calf serum, bovine serum albumin (BSA), or a combination thereof.

[0100] In some embodiments, an additive composition for a culture medium for culturing myoblasts is provided, the additive composition comprising a first component including 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 single sample extract has a final concentration of about 1 to 500 μg / mL in the culture medium.

[0101] In some embodiments, the additive composition further comprises a second component comprising FBS having a final concentration of about 2% to 5% in the medium.

[0102] In some embodiments, an additive composition for a culture medium for culturing porcine bone marrow muscle cells is provided, the additive composition comprising a first component including 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 single sample extract has a final concentration of about 1 to 500 μg / mL in the culture medium.

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

[0104] In some embodiments, the additive composition further comprises a 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.

[0105] In some embodiments, an additive composition for a culture medium for culturing bovine bone marrow muscle cells is provided, the additive composition comprising a first component including 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, lingzhi (Ganoderma Lucidum), versicolor mushroom, cordyceps sinensis (CS), ashwagandha (ASH), and combinations thereof, wherein the single sample extract has a final concentration of about 1 to 500 μg / mL in the culture medium.

[0106] In some embodiments, the additive composition further comprises a second component comprising FBS having a final concentration of about 2% to 5% in the medium.

[0107] In some embodiments, a culture medium is provided, the culture medium comprising a basal or base medium and the medium additive composition of any one of the preceding embodiments.

[0108] In some embodiments, the medium is substantially free of or contains reduced amounts of one or more exogenous serum, growth factors or hormones, or combinations thereof.

[0109] In some embodiments, the basal medium or base medium is Dulbecco's Modified Eagle's Medium (DMEM), Ham's F12 Medium (F12), Dulbecco's Modified Eagle's Medium F12 (DMEM:F12), Ham's F10 Medium (F10), Roswell Park Memorial Institute 1640 Medium (RPMI 1640), Minimum Essential Medium (MEM), Serum-Free Medium (SFM), or a combination thereof.

[0110] In some embodiments, a method of culturing cells is provided, the method comprising: (i) providing a basal medium or base medium and the medium additive composition of any one of the preceding embodiments to form a medium; and (ii) treating and culturing cells in the medium.

[0111] In some embodiments, the method further comprises the step of: (1) treating the cell line with a dissociation agent to form a cell suspension.

[0112] 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.

[0113] 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 medium.

[0114] In some embodiments, the sample extract is produced by one or more of the following steps: (a) producing 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; and (c) purifying the at least one sample solution to obtain the at least one sample extract.

[0115] In some embodiments, in step (c), purifying the at least one sample solution comprises filtering and / or centrifuging the at least one sample solution.

[0116] In some embodiments, culturing cells is used to produce animal tissue.

[0117] In some embodiments, the animal tissue is mammalian, avian, crustacean, or fish tissue.

[0118] In some embodiments, culturing the cells is used to produce cultivated meat.

[0119] In some embodiments, the method further comprises incubating the cells in a CO 2 incubator.

[0120] In some embodiments, the cell line is derived from a mammal, bird, crustacean, or fish.

[0121] In some embodiments, the cell lines are derived from fibroblasts, vascular cells, pericytes, endothelial cells, myoblasts, satellite cells, bone muscle cells, muscle cells, smooth muscle cells, adipocytes, adipose-derived stem cells, skin cells, tendon, liver, brain, bone, heart, kidney, stem cells, and combinations thereof.

[0122] 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 (e.g., shrimp), bird (e.g., chicken), ray-finned fish (e.g., loach, salmon), or mammal (e.g., cow or pig).

[0123] In some embodiments, the cell line is derived from muscle cells (e.g., bone muscle cells, smooth muscle cells), myoblasts, skeletal muscle satellite cells, adipocytes, adipose-derived stem cells, fibroblasts, vascular cells (e.g., 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 bone muscle cells, satellite cells, and myoblasts and / or cells produced therefrom (e.g., muscle cells, muscle fibers). In some embodiments, the cell line is derived from adipose cells, such as adipose-derived stem cells (also called 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).

[0124] In some embodiments, the additive composition forms a medium with a basal medium at a total concentration of 100%.

[0125] In some embodiments, the additive composition includes a first component comprising one or more sample extracts prepared from one or more natural substances described herein, wherein the or each sample extract has a final concentration in the medium of about 0-99% (or higher). For example, suitable concentration ranges include any range 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 or each sample extract may contain 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%, approximately 0.01%, approximately 0.02%, approximately 0.03%, approximately 0.04%, approximately 0.05%, approximately 0.06%, approximately 0.07%, approximately 0.08%, approximately 0.09%, approximately 0.1%, approximately 0.2%, approximately 0.3%, approximately 0.4%, approximately 0.5%, approximately 0.6%, approximately 0.7%, approximately 0.8%, approximately 0.9%, approximately 1.0%, approximately 2%, approximately 3%, approximately 4%, approximately 5%, approximately 6%, approximately 7%, approximately 8%, approximately 9%, approximately 10%, approximately 11%, approximately 12%, approximately 13%, approximately 14%, approximately 15%, approximately 16%, approximately 17%, approximately 18%, approximately 19%, approximately 20%, approximately 21%, approximately 22%, approximately 23%, approximately 24%, approximately 25%, approximately 26%, approximately 27%, approximately 28%, approximately 29%, approximately 30%, approximately 31%, approximately 32%, approximately 33%, approximately 34%, approximately 35%, approximately 36%, approximately 37%, approximately 38%, approximately 39%, approximately 40%, approximately 41%, approximately 42%, approximately 43%, approximately 44%, approximately 45%, approximately 46%, approximately 47%, approximately 48%, approximately 49%, approximately 50%, approximately 51%, approximately 52%, approximately 53%, approximately 54%, approximately 55%, approximately 56%, approximately 57%, approximately 58%, approximately 59%, approximately 60%, approximately 61%, approximately 62%, approximately 63%, approximately 64%, approximately 65%, approximately 66%, approximately 67%, approximately 68%, approximately 69%, approximately 70%, approximately 71%, approximately 72%, approximately 73%, approximately 74%, approximately 75%, approximately 76%, approximately 77%, approximately 78%, approximately 79 4%, 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%, approximately 52%, approximately 53%, approximately 54%, approximately 55%, approximately 56%, approximately 57%, approximately 58%, approximately 59%, approximately 60%, approximately 61%, approximately 62%, approximately 63%, approximately 64%, approximately 65%, approximately 66%, approximately 67%, approximately 68%, approximately 69%, approximately 70%, approximately 71%, approximately 72%, approximately 73%, approximately 74%, approximately 75%, approximately 76%, approximately 77%, approximately 78%, approximately 79%, approximately 80%, approximately 81%, approximately 82%, approximately 83%, approximately 84%, approximately 85%, approximately 86%, approximately 87%,In some other embodiments, the single or each sample extract has a final concentration of 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 other embodiments, the single or each sample extract has a final concentration of 0.01 ng / mL to 1000 mg / mL (or higher) in the medium. In some other embodiments, the single or each sample extract has a concentration of about 0.01 ng / mL to 100 ng / mL. In some other embodiments, the single or each sample extract has a concentration of about 0.01 μg / mL to 100 μg / mL. In some other embodiments, the single or each sample extract has a concentration of about 0.01 mg / mL to 100 mg / mL. In some other embodiments, the single or each sample extract has a concentration of about 0.2 mg / mL to 100 mg / mL. In some other embodiments, the or each sample extract has a first component concentration of about 10 ug / mL to 1000 ug / mL, 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, 4 g / mL, 13ng / mL, 14ng / mL, 15ng / mL, 16ng / mL, 17ng / mL, 18ng / mL, 19ng / mL, 20ng / mL, 21ng / mL, 22ng / mL, 23ng / mL, 24ng / mL, 25ng / mL, 26ng / mL, 27ng / mL, 28ng / mL, 29ng / mL, 3 0ng / mL, 31ng / mL, 32ng / mL, 33ng / mL, 34ng / mL, 35ng / mL, 36ng / mL, 37ng / mL, 38ng / mL, 39ng / mL, 40ng / mL, 41ng / mL, 42ng / mL, 43ng / mL, 44ng / mL, 45ng / mL, 46ng / mL, 47ng / mL,48ng / mL、49ng / mL、50ng / mL、51ng / mL、52ng / mL、53ng / mL、54ng / mL、55ng / mL、56ng / mL、57ng / mL、58ng / mL、59ng / mL、60ng / mL、61ng / mL、62ng / mL、63ng / mL、64ng / mL、65ng / mL、66ng / mL、67ng / mL、68ng / mL、69ng / mL、70ng / mL、71ng / mL、72ng / mL、73ng / mL、74ng / mL、75ng / mL、76ng / mL、77ng / mL、78ng / mL、79ng / mL、80ng / mL、81ng / mL、82ng / mL、83ng / mL、84ng / mL、85ng / mL、86ng / mL、87ng / mL、88ng / mL、89ng / mL、90ng / mL、91ng / mL、92ng / mL、93ng / mL、94ng / mL、95ng / mL、96ng / mL、97ng / mL、98ng / mL、99ng / mL、0.1ug / mL、0.2ug / mL、0.3ug / mL、0.4ug / mL、0.5ug / mL、0.6ug / mL、0.7ug / mL、0.8ug / mL、0.9ug / mL、1ug / mL、2ug / mL、3ug / mL、4ug / mL、5ug / mL、6ug / mL、7ug / mL、8ug / mL、9ug / mL、10ug / mL、11ug / mL、12ug / mL、13ug / mL、14ug / mL、15ug / mL、16ug / mL、17ug / mL、18ug / mL、19ug / mL、20ug / mL、21ug / mL、22ug / mL、23ug / mL、24ug / mL、25ug / mL、26ug / mL、27ug / mL、28ug / mL、29ug / mL、30ug / mL、31ug / mL、32ug / mL、33ug / mL、34ug / mL、35ug / mL、36ug / mL、37ug / mL、38ug / mL、39ug / mL、40ug / mL、41ug / mL、42ug / mL、43ug / mL、44ug / mL、45ug / mL、46ug / mL、47ug / mL、48ug / mL、49ug / mL、50ug / mL、51ug / mL、52ug / mL、53ug / mL、54ug / mL、55ug / mL、56ug / mL、57ug / mL、58ug / mL、59ug / mL、60ug / mL、61ug / mL、62ug / mL、63ug / mL、64ug / mL、65ug / mL、66ug / mL、67ug / mL、68ug / mL、69ug / mL、70ug / mL、71ug / mL、72ug / mL、73ug / mL、74ug / mL、75ug / mL、76ug / mL、77ug / mL、78ug / mL、79ug / mL、80ug / mL、81ug / mL、82ug / mL、83ug / mL、84ug / mL、85ug / mL、86ug / mL、87ug / mL、88ug / mL、89ug / mL、90ug / mL、91ug / mL、92ug / mL、93ug / mL、94ug / mL、95ug / mL、96ug / mL、97ug / mL、98ug / mL、99ug / mL、0.1mg / mL、0.2mg / mL、0.3mg / mL、0.4mg / mL、0.5mg / mL、0.6mg / mL、0.7mg / mL、0.8mg / mL、0.9mg / mL、1mg / mL、2mg / mL、3mg / mL、4mg / mL、5mg / mL、6mg / mL、7mg / mL、8mg / mL、9mg / mL、10mg / mL、11mg / mL、12mg / mL、13mg / mL、14mg / mL、15mg / mL、16mg / mL、17mg / mL、18mg / mL、19mg / mL、20mg / mL、21mg / mL、22mg / mL、23mg / mL、24mg / mL、25mg / mL、26mg / mL、27mg / mL、28mg / mL、29mg / mL、30mg / mL、31mg / mL、32mg / mL、33mg / mL、34mg / mL、35mg / mL、36mg / mL、37mg / mL、38mg / mL、39mg / mL、40mg / mL、41mg / mL、42mg / mL、43mg / mL、44mg / mL、45mg / mL、46mg / mL、47mg / mL、48mg / mL、49mg / mL、50mg / mL、51mg / mL、52mg / mL、53mg / mL、54mg / mL、55mg / mL、56mg / mL、57mg / mL、58mg / mL、59mg / mL、60mg / mL、61mg / mL、62mg / mL、63mg / mL、64mg / mL、65mg / mL、66mg / mL、67mg / mL、68mg / mL、69mg / mL、70mg / mL、71mg / mL、72mg / mL、73mg / mL、74mg / mL、75mg / mL、76mg / mL、77mg / mL、78mg / mL、79mg / mL、80mg / mL、81mg / mL、82mg / mL、83mg / mL、84mg / mL、85mg / mL、86mg / mL、87mg / mL、88mg / mL、89mg / mL、90mg / mL、91mg / mL、92mg / mL、93mg / mL、94mg / mL、95mg / mL、96mg / mL、97mg / mL、98mg / mL、99mg / mL、100mg / mL、110mg / mL、120mg / mL、130mg / mL、140mg / mL、150mg / mL、160mg / mL、170mg / mL、180mg / mL、190mg / mL、200mg / mL、210mg / mL mL、220mg / mL、230mg / mL、240mg / mL、250mg / mL、260mg / mL、270mg / mL、280mg / mL、290mg / mL、300mg / mL、310mg / mL、320mg / mL、330mg / mL、340mg / mL、350mg / mL g / mL、360mg / mL、370mg / mL、380mg / mL、390mg / mL、400mg / mL、410mg / mL、420mg / mL、430mg / mL、440mg / mL、450mg / mL、460mg / mL、470mg / mL、480mg / mL、490mg / mL mg / mL、500mg / mL、510mg / mL、520mg / mL、530mg / mL、540mg / mL、550mg / mL、560mg / mL、570mg / mL、580mg / mL、590mg / mL、600mg / mL、610mg / mL、620mg / mL 30mg / mL、640mg / mL、650mg / mL、660mg / mL、670mg / mL、680mg / mL、690mg / mL、700mg / mL、710mg / mL、720mg / mL、730mg / mL、740mg / mL、750mg / mL、760mg / mL 770mg / mL、780mg / mL、790mg / mL、800mg / mL、810mg / mL、820mg / mL、830mg / mL、840mg / mL、850mg / mL、860mg / mL、870mg / mL、880mg / mL、890mg / mL、900mg / mL 、910mg / mL、920mg / mL、930mg / mL、940mg / mL、950mg / mL、960mg / mL、970mg / mL、980mg / mL、990mg / mL、1000mg / mL or with higher concentration。 In some examples, the additive composition isThe one or more sample extracts comprise a first component having 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 at least one natural substance is selected from the group consisting of bird's nest (BN), ginseng (e.g., American ginseng (Panax ginseng), Korean ginseng, or Chinese ginseng (Panax ginseng)), lingzhi (e.g., Ganoderma lucidum, GL), versicolor mushroom (e.g., Yunzhi mushroom (YZ)), cordyceps sinensis (CS), ashwagandha (ASH), and combinations thereof.

[0126] In one alternative embodiment, the additive composition comprises or consists essentially of bird's nest at a concentration of about 0.01 ng / mL to 1000 mg / mL, or any concentration therein.

[0127] In one alternative embodiment, the additive composition comprises or consists essentially of carrots (eg, American ginseng or European ginseng) at a concentration of about 0.01 ng / mL to 1000 mg / mL, or any concentration therein.

[0128] In one embodiment, the media additive composition comprises or consists essentially of ginseng (e.g., Korean ginseng, Chinese ginseng, or Japanese ginseng) at a concentration of about 0.01 ng / mL to 1000 mg / mL, or any concentration therein.

[0129] In one embodiment, the additive composition comprises or consists essentially of mushroom versicolor (eg, Unshiu mushroom) at a concentration of about 0.01 ng / mL to 1000 mg / mL, or any concentration therein.

[0130] In one embodiment, the additive composition consists essentially of, or consists of, Ling Zhi (eg, Ganoderma lucidum) at a concentration of about 0.01 ng / mL to 1000 mg / mL, or any concentration therein.

[0131] In one embodiment, the additive composition comprises or consists essentially of Cordyceps sinensis at a concentration of about 0.01 ng / mL to 1000 mg / mL, or any concentration therein.

[0132] In one embodiment, the media additive composition comprises or consists essentially of Ashwagandha at a concentration of about 0.01 ng / mL to 1000 mg / mL, or any concentration therein.

[0133] In some embodiments, the medium comprises the first component at a final concentration of about 0.01 ng / mL to 1000 mg / mL, or any concentration above. In some embodiments, the medium comprises the first component at a final concentration of about 20 μg / mL to 0.5 mg / mL. In some embodiments, the medium comprises the 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.

[0134] In another embodiment, the medium comprises an additive composition and a basal medium (e.g., DMEM or DMEM:F12), wherein the additive composition comprises one or more sample extracts prepared from one or more natural substances described herein at a final concentration of about 0.01 ng / mL to 1000 mg / mL, or any concentration therein.

[0135] In other embodiments, the medium optionally includes a second component described herein. In some other embodiments, the second component includes 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 medium, e.g., the final concentration in the medium is about 0-99% (or higher). For example, any suitable concentration range in the medium is between 0-99%, e.g., 0.0001%-50%, 1%-20%, or 0.01 ng / mL-1000 mg / mL (or higher). In some alternative embodiments, the or each component in the second component has a concentration of about 0.0000151% to 1%, 0.0001% to 0.1%, 0.1% to 5%, 0.01% to 10%, 0.001% to 20%, 0.01% to 10%, 0.1% to 5%, 1% to 30%, 5% to 40%, 10% to 50%, 20% to 60%, 30% to 70%, or 40% to 80% or higher. In some embodiments, the or each sample extract may contain 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 0.0008%, 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. ... 0.003%, approx. 0.004%, approx. 0.005%, approx. 0.006%, approx. 0.007%, approx. 0.008%, approx. 0.009%, approx. 0.01%, approx. 0.02%, approx. 0.03%, approx. 0.04%, approx. 0.05%, approx. 0.06%, approx. 0.07%, approx. 0.08%, approx. 0.09%, approx. 0.1%, approx. 0.2%, approx. 0.3%, approx. 0. 4%, approximately 0.5%, approximately 0.6%, approximately 0.7%, approximately 0.8%, approximately 0.9%, approximately 1.0%, approximately 2%, approximately 3%, approximately 4%, approximately 5%, approximately 6%, approximately 7%, approximately 8%, approximately 9%, approximately 10%, approximately 11%, approximately 12%, approximately 13%, approximately 14%, approximately 15%, approximately 16%, approximately 17%, approximately 18%, approximately 19%, approximately 20%, approximately 21%, approximately 22%, approximately 23%, approximately 24%, approximately 25%, approximately 26%, approximately 27%, approximately 28%, approximately 29%, approximately 30%, approximately 31%, approximately 32%, approximately 33%, approximately 34%, approximately 35%, approximately 36%, approximately 37%, approximately 38%, approximately 39%, approximately 40%, approximately 41%, approximately 42%, approximately 43%, approximately 44%, approximately 45%, approximately 46%, approximately 47%, approximately 48%, approximately 49%, approximately 50%, approximately 51%, approximately 52%, approximately 53%, approximately 54%, approximately 55%, approximately 56%, approximately 57%, approximately 58%, approximately 59%, approximately 60%, approximately 61%, approximately 62%, approximately 63%, approximately 64%, approximately 65%, approximately 66%, approximately 67%, approximately 68%, approximately 69%, approximately 70%, approximately 71%, approximately 72%, approximately 73%, approximately 74%, approximately 75%, approximately 76%, approximately 77%, approximately 78%, approximately 79%, approximately 80%, approximately 81%, approximately 82%, approximately 83%, approximately 84%, approximately 85%, approximately 86%, approximately 87%, approximately 88%, approximately 89%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, 3%, approximately 24%, approximately 25%, approximately 26%, approximately 27%, approximately 28%, approximately 29%, approximately 30%, approximately 31%, approximately 32%, approximately 33%, approximately 34%, approximately 35%, approximately 36%, approximately 37%, approximately 38%, approximately 39%, approximately 40%, approximately 41%, approximately 42%, approximately 43%, approximately 44%, approximately 45%, approximately 46%, approximately 47%, approximately 48%, approximately 49%, approximately 50%,Approximately 51%, approximately 52%, approximately 53%, approximately 54%, approximately 55%, approximately 56%, approximately 57%, approximately 58%, approximately 59%, approximately 60%, approximately 61%, approximately 62%, approximately 63%, approximately 64%, approximately 65%, approximately 66%, approximately 67%, approximately 68%, approximately 69%, approximately 70%, approximately 71%, approximately 72%, approximately 73%, approximately 74%, approximately 75%, approximately In some embodiments, the second component has a concentration of 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 other embodiments, the second component has a concentration of about 0.01 ng / mL to 100 ng / mL. In some other embodiments, the second component has a concentration of about 0.01 μg / mL to 100 μg / mL. In some other embodiments, the second component has a concentration of about 0.01 mg / mL to 100 mg / mL. In some other embodiments, the or each component in the second component has a concentration of about 0.2 mg / mL to 100 mg / mL. In some other embodiments, the or each component in the second component has a concentration of the first component of about 10 μg / mL to 1000 μg / mL. In some other embodiments, the or each component in the second component has a concentration of 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, or 2 ng / mL. , 3ng / mL, 4ng / mL, 5ng / mL, 6ng / mL, 7ng / mL, 8ng / mL, 9ng / mL, 10ng / mL, 11ng / mL, 12ng / mL, 13ng / mL, 14ng / mL, 15ng / mL, 16ng / mL, 17n g / mL, 18ng / mL, 19ng / mL, 20ng / mL, 21ng / mL, 22ng / mL, 23ng / mL, 24ng / mL, 25ng / mL, 26ng / mL, 27ng / mL, 28ng / mL, 29ng / mL, 30ng / mL,31ng / mL、32ng / mL、33ng / mL、34ng / mL、35ng / mL、36ng / mL、37ng / mL、38ng / mL、39ng / mL、40ng / mL、41ng / mL、42ng / mL、43ng / mL、44ng / mL、45ng / mL、46ng / mL、47ng / mL、48ng / mL、49ng / mL、50ng / mL、51ng / mL、52ng / mL、53ng / mL、54ng / mL、55ng / mL、56ng / mL、57ng / mL、58ng / mL、59ng / mL、60ng / mL、61ng / mL、62ng / mL、63ng / mL、64ng / mL、65ng / mL、66ng / mL、67ng / mL、68ng / mL、69ng / mL、70ng / mL、71ng / mL、72ng / mL、73ng / mL、74ng / mL、75ng / mL、76ng / mL、77ng / mL、78ng / mL、79ng / mL、80ng / mL、81ng / mL、82ng / mL、83ng / mL、84ng / mL、85ng / mL、86ng / mL、87ng / mL、88ng / mL、89ng / mL、90ng / mL、91ng / mL、92ng / mL、93ng / mL、94ng / mL、95ng / mL、96ng / mL、97ng / mL、98ng / mL、99ng / mL、0.1ug / mL、0.2ug / mL、0.3ug / mL、0.4ug / mL、0.5ug / mL、0.6ug / mL、0.7ug / mL、0.8ug / mL、0.9ug / mL、1ug / mL、2ug / mL、3ug / mL、4ug / mL、5ug / mL、6ug / mL、7ug / mL、8ug / mL、9ug / mL、10ug / mL、11ug / mL、12ug / mL、13ug / mL、14ug / mL、15ug / mL、16ug / mL、17ug / mL、18ug / mL、19ug / mL、20ug / mL、21ug / mL、22ug / mL、23ug / mL、24ug / mL、25ug / mL、26ug / mL、27ug / mL、28ug / mL、29ug / mL、30ug / mL、31ug / mL、32ug / mL、33ug / mL、34ug / mL、35ug / mL、36ug / mL、37ug / mL、38ug / mL、39ug / mL、40ug / mL、41ug / mL、42ug / mL、43ug / mL、44ug / mL、45ug / mL、46ug / mL、47ug / mL、48ug / mL、49ug / mL、50ug / mL、51ug / mL、52ug / mL、53ug / mL、54ug / mL、55ug / mL、56ug / mL、57ug / mL、58ug / mL、59ug / mL、60ug / mL、61ug / mL、62ug / mL、63ug / mL、64ug / mL、65ug / mL、66ug / mL、67ug / mL、68ug / mL、69ug / mL、70ug / mL、71ug / mL、72ug / mL、73ug / mL、74ug / mL、75ug / mL、76ug / mL、77ug / mL、78ug / mL、79ug / mL、80ug / mL、81ug / mL、82ug / mL、83ug / mL、84ug / mL、85ug / mL、86ug / mL、87ug / mL、88ug / mL、89ug / mL、90ug / mL、91ug / mL、92ug / mL、93ug / mL、94ug / mL、95ug / mL、96ug / mL、97ug / mL、98ug / mL、99ug / mL、0.1mg / mL、0.2mg / mL、0.3mg / mL、0.4mg / mL、0.5mg / mL、0.6mg / mL、0.7mg / mL、0.8mg / mL、0.9mg / mL、1mg / mL、2mg / mL、3mg / mL、4mg / mL、5mg / mL、6mg / mL、7mg / mL、8mg / mL、9mg / mL、10mg / mL、11mg / mL、12mg / mL、13mg / mL、14mg / mL、15mg / mL、16mg / mL、17mg / mL、18mg / mL、19mg / mL、20mg / mL、21mg / mL、22mg / mL、23mg / mL、24mg / mL、25mg / mL、26mg / mL、27mg / mL、28mg / mL、29mg / mL、30mg / mL、31mg / mL、32mg / mL、33mg / mL、34mg / mL、35mg / mL、36mg / mL、37mg / mL、38mg / mL、39mg / mL、40mg / mL、41mg / mL、42mg / mL、43mg / mL、44mg / mL、45mg / mL、46mg / mL、47mg / mL、48mg / mL、49mg / mL、50mg / mL、51mg / mL、52mg / mL、53mg / mL、54mg / mL、55mg / mL、56mg / mL、57mg / mL、58mg / mL、59mg / mL、60mg / mL、61mg / mL、62mg / mL、63mg / mL、64mg / mL、65mg / mL、66mg / mL、67mg / mL、68mg / mL、69mg / mL、70mg / mL、71mg / mL、72mg / mL、73mg / mL、74mg / mL、75mg / mL、76mg / mL、77mg / mL、78mg / mL、79mg / mL、80mg / mL、81mg / mL、82mg / mL、83mg / mL、84mg / mL、85mg / mL、86mg / mL、87mg / mL、88mg / mL、89mg / mL、90mg / mL、91mg / mL、92mg / mL、93mg / mL、94mg / mL、95mg / mL、96mg / mL、97mg / mL、98mg / mL、99mg / mL、100mg / mL、110mg / mL、120mg / mL、130mg / mL、140mg / mL、150mg / mL、160mg / mL、170mg / mL、180mg / mL、190mg / mL、200mg / mL、210mg / mL、220mg / mL、230mg / mL、240mg / mL、250mg / mL、260mg / mL、270mg / mL、280mg / mL、290mg / mL、300mg / mL、310mg / mL、320mg / mL、330mg / mL、340mg / mL、350mg / mL、360mg / mL、370mg / mL、380mg / mL、390mg / mL、400mg / mL、410mg / mL、420mg / mL、430mg / mL、440mg / mL、450mg / mL、460mg / mL、470mg / mL、480mg / mL、490mg / mL、500mg / mL、510mg / mL、520mg / mL、530mg / mL、540mg / mL、550mg / mL、560mg / mL、570mg / mL、580mg / mL、590mg / mL、600mg / mL、610mg / mL、620mg / mL、630mg / mL、640mg / mL、650mg / mL、660mg / mL、670mg / mL、680mg / mL、690mg / mL、700mg / mL、710mg / mL、720mg / mL、730mg / mL、740mg / mL、750mg / mL、760mg / mL、770mg / mL、780mg / mL、790mg / mL、800mg / mL、810mg / mL、820mg / mL、830mg / mL、840mg / mL、850mg / mL、860mg / mL、870mg / mL、880mg / mL、890mg / mL、In some embodiments, the concentration is 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 one example, the additive composition comprises a first component having 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 another example, the second component comprises FBS having a final concentration of about 0.01% to 50% (e.g., 0.1% to 20%, or 2% to 5%) or higher in the medium. In another example, the second component comprises BSA having a final concentration of about 1 to 100 mg / mL (e.g., 6 mg / mL) and / or TGF-b1 having a final concentration of about 0.1 to 100 ng / mL (e.g., 2 ng / mL). The second component comprises at least one serum (e.g., FBS), growth factor or hormone (e.g., 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)), amino acid (e.g., BSA), vitamin, salt, glucose, buffering agent, or combinations thereof. example

[0136] This specification provides examples that more fully describe some embodiments of the present disclosure. The examples provided herein are for purposes of clarity only and are not intended to limit the scope of the present invention in any way. All references given below and elsewhere in this application are incorporated herein by reference. Example 1

[0137] Exemplary Sample Extract Preparation [Table 1] 1. The sample was ground into powder form (e.g., using an electric food cutter and then a coffee grinder). 2. Each sample was placed in a 50 mL tube, 5 mL of water was added, and the samples were dissolved in a shaking incubator at 100 rpm for 24 hours (final concentration = 500 mg / 5 mL = 100 mg / mL). 3. The sample was filtered into a 15 ml tube using a sterile 0.2 μm filter. This and the following steps were performed in a biosafety cabinet to ensure sterility. If any sample was unfilterable, the sample was centrifuged and filtered, and the supernatant was obtained as the sample extract. Optionally, before filtration, the sample was first filtered through a 100 μm cell filter. 4. Three 1.5 mL centrifuge tubes were prepared (in triplicate) for each sample extract. 5. Each sample extract was aliquoted into centrifuge tubes and water was added as needed to obtain the following final concentrations: a. Tube 1: 100mg / ml b. Tube 2: 60mg / ml c. Tube 3: 20mg / ml

[0138] Testing of sample extracts 1. In this example, the exemplary cultured cells for testing are L6 myoblasts (non-bovine) cultured in DMEM supplemented with 10% FBS. The medium was replaced with DMEM-only medium for 4 hours to ensure serum starvation, before the subsequent steps were performed. For the positive control, the medium used was DMEM supplemented with 10% FBS. 2. After 4 hours, the cells were further washed twice with PBS. 3. The PBS was removed, and the cells were digested with trypsin (Gibco, 0.25% trypsin-EDTA, phenol red) for 3 to 5 minutes, while the flask was tapped routinely to promote cell dissociation. 4. After neutralizing trypsin with TNS (trypsin neutralizing solution), cells were counted. The volume ratio of TNS to trypsin for neutralization was 1:1. 5. The cells were centrifuged (RCF=150g) to form a pellet. 6. Resuspend the cell pellet in DMEM and collect at 2 x 10 4 Cells / mL were present. 500 uL of cell suspension was added to each well and used to perform the test in a 24-well plate. 7. Well array: a. Column 1: Sample Extract 1: 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): 5 uL of extract from tube 1 was added to the well. 495 uL of DMEM in 10% FBS was also added. e. Row B (for columns 1-3): 5 uL of extract from tube 2 was added. 495 uL of DMEM in 10% FBS was also added. f. Row C (for columns 1-3): 5 uL of extract from tube 3 was added. 495 uL of DMEM in 10% FBS was also added. g. Negative control: Row D (for columns 1-3): 500 uL of DMEM alone was added. h. Positive Control 1: Column 4, rows 1-3 (Wells 4A+4B+4C): 480 μL of DMEM was added. 20 μL of FGF-2 (from a 5 μg / mL FGF-2 aliquot) was added to bring the final concentration of FGF-2 in the well to 0.1 μg / mL. i. Separate positive control 2: Column 5, rows 1-3 (wells 5A, 5B, and 5C): Add 500 μL of DMEM + 20% FBS (final concentration: 10% FBS). 8. Repeat steps 1-7 on another plate. a. The first plate was incubated for 24 hours. b. The second plate was incubated for 48 hours. 9. Proliferation was assessed by cell counting using a hemocytometer using trypan blue as a stain.

[0139] result

[0140] (1) 24-hour period

[0141] The samples were incubated with the cells in the first plate for 24 hours before cell counting was performed.

[0142] [Table 2]

[0143] (2) 48 hours later

[0144] Sample extracts and cells were incubated for 48 hours before cell counting. [Table 3]

[0145] Results and Discussion

[0146] These results showed that during the first 24 hours, cell numbers remained relatively the same as the negative control (DMEM only), indicating that cells may not be upregulated by the samples. Only after 24 to 48 hours did cell numbers unexpectedly increase after treatment with the three sample extracts, indicating that the sample extracts stimulated cell proliferation. Of the three sample extracts, the American ginseng sample extract unexpectedly outperformed the other two, followed by bird's nest and Korean ginseng. At 48 hours, all sample extracts unexpectedly outperformed the positive controls (including DMEM supplemented with 0.05 μg / mL FGF-2 and DMEM supplemented with 10% FBS).

[0147] In brief, the growth factor upregulation abilities of bird's nest, American ginseng (AG), and Korean ginseng (KG) sample extracts were tested. In 48-hour muscle cell cultures, KG and AG at concentrations of 100 μg / mL resulted in a 24.09-fold and 70.00-fold increase in cell number, respectively, while 0.1 μg / mL FGF-2 resulted in a 9.58-fold increase. While similar cell proliferation numbers were maintained without the addition of 5% FBS, this means that, in some examples, the cost of FGF-2, one of the most expensive components in cell culture, was significantly reduced (e.g., 22,285-fold and 18,665-fold, respectively) when KG and AG were used as substitutes.

[0148] The percentage (%) increase in proliferation within 24 and 48 hours normalized to the negative control is summarized in Table 4. The % increase in proliferation after normalization to the control was calculated as follows:

[0149] % 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]

[0150] The results in Table 4 show that media containing the basal medium DMEM and exemplary additive compositions containing approximately 100 μg / ml of KGN, AGN, or BN as a first component and 5% FBS as a second component had significant percent growth increases (66.207%, 382.759%, and 106.207%, respectively) at 48 hours. The results also demonstrated that the exemplary additive compositions containing these sample extracts (AGN, KGN, and BN) had unexpected effects on cell proliferation in cells (e.g., L6 myoblasts). This example demonstrated that the sample extracts could significantly reduce the final serum (FBS) concentration used in the medium from 10% to 5%. The sample extracts in the presence of 5% serum unexpectedly outperformed the positive control group with 10% serum. Example 2

[0151] In this example, the effect of using a medium composition containing various exemplary additive compositions on porcine skeletal muscle cell proliferation was demonstrated. In this example, the medium was tested on porcine skeletal muscle cells. The medium is also applicable to both myoblasts and skeletal muscle satellite cells.

[0152] The materials used in this example include:

[0153] Cell line: Porcine bone muscle cells (PIG-iCell-S005) (Cellverse Bioscience Technology Co., Ltd.)

[0154] Basal medium: Dulbecco's modified Eagle's medium (DMEM) (Thermofisher, D6429)

[0155] Second component: fetal bovine serum (ScienCell, 0500), bovine serum albumin (BSA) (Sigma A7030-10g), TGF-b1 (ThermoFisher, PHG9204), FGF-2 (ThermoFisher, PHG0026) Other reagents: 0.25% trypsin / EDTA (Gibco, 25200-056), CyQuant MTT (Invitrogen, V13154), and gelatin (Sigma G1890).

[0156] Exemplary sample extracts: edible swallow's nest, Ginseng (also known as "Korean ginseng" or "KGN") (Jung Kwan Jang) extract and A ginseng (also known as "American ginseng" or "AGN") extract (President's Brand) was prepared in a similar manner as described in Example 1.

[0157] In this example, the following steps were performed: 1. Stock solutions of sample extracts (AGN, KGN) were prepared at concentrations of approximately 1 mg / mL, 3 mg / mL, and 5 mg / mL. 2. A stock solution of bovine serum albumin (BSA) was prepared in distilled water (HO) at a concentration of approximately 80 mg / mL in a small volume (<1 mL). A higher concentration of BSA stock solution allows for better maintenance of the osmotic balance of the cells. 3. The following solutions were prepared: a. M1 medium: DMEM+2ng / mL TGF-b1+1mg / mL BSA+40ng / mL FGF-2 b. M3 medium: DMEM+2ng / mL TGF-b1+1mg / mL BSA 4. All wells used in a 24-well plate were coated with 2% gelatin. 5. Culture porcine bone marrow cells in DMEM supplemented with approximately 10% FBS to a density of at least 1 × 10 6 This was continued until a final cell concentration of 100 cells / mL was reached. 6. Harvest porcine bone marrow myocytes using an appropriate volume of approximately 0.25% trypsin / EDTA and neutralize them with trypsin neutralizer solution (also called "neutralizer"), followed by centrifugation at 300 g for 5 minutes. Discard the supernatant and collect approximately 1 x 10 cells. 6The cells were resuspended in M3 medium at a final cell density of 100 cells / mL. 7. Add approximately 10 uL of cell suspension to each well to be used (final cell density: approximately 1 x 10 4 cells / well) were added. 8. Approximately 840 uL of M3 medium was added and the cells were allowed to serum starve for approximately 1 hour. To the control groups employing M3 medium (negative control) or M1 medium (also referred to as "positive control" or "M1"), the same amount of M3 or M1 medium was added, respectively. 9. Approximately 50 uL of approximately 100 mg / mL BSA was added to give a final concentration of approximately 5-6 mg / mL in each well. The same procedure was performed for the control. 10. Approximately 100 μL of stock sample extract was added to each well. For the control, 100 μL of phosphate-buffered saline (PBS) was added instead of the sample extract. The concentrations of the components in each medium are shown in Table 5 (KGN = Korean ginseng, carrot; AGN = American ginseng, ginseng). [Table 5] 11. Repeat step 10 as necessary to obtain replicates. In this example, two replicates of each well were obtained. 12. After approximately 56 hours, the medium from the wells was transferred to a separate centrifuge tube. When the used medium was removed, non-adherent cells were also removed. The adherent cells retained on the well plate were extracted in the following steps. 13. Then, 2 mL of 0.25% trypsin / EDTA was added to each well and incubated for 15 to 30 minutes. The sides and bottom of the wells were gently tapped. 14. 20 uL of dissociated cells in 0.25% trypsin / EDTA was mixed with 20 uL of trypan blue. 15. 15uL of the dissociated cells and trypan blue mixture was added to each side of the cell counting chamber slide, after which the cell density and total cell number were assessed. 16. Steps 14-15 were repeated for the medium transferred from step 12.

[0158] result

[0159] In this example, the effects of a medium containing approximately 300 μg / mL KGN or approximately 300 μg / mL AGN as the first component and BSA and TGF-b1 as the second component, as well as a positive control (M1), on cell number were evaluated and summarized in the results. Each sample was run in duplicate, and the average total cell number was compared with the positive control. Adherent cells were counted in the well plates. The percentage increase in proliferation (%) was calculated using the equation {[(cell number in sample) - (cell number in M1)] / (cell number in M1)} * 100%. The results are shown in Figure 1 and summarized in Table 6. In this experiment, cell viability was not assessed because trypsin / EDTA was not neutralized with trypsin neutralizer solution.

[0160] Referring to Figure 1, Figure 1 shows the growth of porcine bone marrow myocytes under stimulation with different sample extracts (i.e., samples A2 and B2, respectively, according to Table 5) provided at about 300 μg / mL at 56 hours. The percentage (%) increase in proliferation normalized to the control is listed in Table 6. The results showed that A2 (KGN at about 300 μg / mL) and B2 (AGN at about 300 μg / mL) were superior to the positive control (containing a significantly higher level of FGF-2 (about 34 ng / mL) in M1 medium) in the presence of reduced final concentrations of TGF-b1 at about 1.7 ng / mL and BSA (as a second component) at about 5.85 mg / mL. After about 56 hours, B2 (AGN at about 300 μg / mL) increased the proliferation rate by about 1.58 x 10 5 The cell number reached approximately 1.46 × 10 cells, while A2 (KGN approximately 300 μg / mL) reached approximately 1.46 × 10 cells. 5 This was significantly higher than the final cell number in the positive control (M1) with FGF-2 (approximately 9.38 × 10 4The cell proliferation rate was approximately 50% higher than that of the control (having 100 cells per 1000 cells). Furthermore, serum was not used in these test groups, demonstrating that the exemplary additive composition containing the first component (e.g., AGN and / or KGN) is an effective substitute for serum in serum-free (or substantially serum-free or serum-reduced) media for culturing cells (e.g., porcine bone marrow myocytes). Similar experiments can be performed on other sample extracts (e.g., Reishi mushroom, Coriolus versicolor, edible bird's nest, Ashwagandha, and / or Cordyceps sinensis) to demonstrate the unexpected technical effects of these natural substances in sample extracts (e.g., additive compositions for serum-free (or substantially serum-free or serum-reduced) media for culturing cells (e.g., myoblasts or satellite cells located in a bone marrow myocyte mixture). Table 6 summarizes the cell proliferation increase (%) within 56 hours for media containing the sample extracts KGN and AGN. [Table 6]

[0161] The results in Table 6 show that media containing the exemplary additive composition, which includes the basal medium DMEM and a first component of about 300 μg / ml of KGN or AGN and a second component of about 5.85 mg / mL of BSA and about 1.7 ng / mL of TGF-b1 at final concentrations, exhibited a significant increase in proliferation (about 55.22% and 68.44%, respectively) at about 56 hours. The results showed that these sample extracts (AGN and KGN) achieved unexpected effects on the cell proliferation of bone marrow myocytes. This example demonstrated that the sample extracts can stimulate cell growth more effectively than the effect of a medium supplemented with FGF-2. Example 3

[0162] In this example, the effect of using exemplary additive and media compositions on cell proliferation of bovine skeletal muscle cells was demonstrated. In this example, the media was tested on bovine skeletal muscle cells. The media is also applicable to both fibroblasts and skeletal muscle satellite cells.

[0163] The materials used in this example include:

[0164] Cell line: Bovine bone muscle cells (COW-iCell-S005) (Saiwei Biotechnology Co., Ltd.)

[0165] Basal medium: Dulbecco's Modified Eagle Medium / Nutrient Mixture F-12 (DMEM:F12) (Thermo Fisher, 21041-025)

[0166] Second component: fetal bovine serum (ScienCell, 0500)

[0167] Other reagents: 0.25% trypsin / EDTA (Gibco, 25200-056), CyQuant MTT (Invitrogen, V13154), trypsin neutralizer solution (Gibco, R-002-100), gelatin (Sigma, G1890).

[0168] Sample extracts: swallow's nest extract (Hunan New Mstar Biotechnology Co., Ltd., lot MS230404), ginseng (Xi'an Demeter Biotech Co., Ltd., lot MS230404), Ltd), lot DMTB20230106), carrot extract (Xian Demei Biotechnology Co., Ltd., lot DMTB230115), Ganoderma (Xi'an Demei Biotechnology Co., Ltd., lot 2022-10-12), ashwagandha root extract (Xi'an Demei Biotechnology Co., Ltd., lot DMTB230320), Cordyceps sinensis Yarsagumba) polysaccharide 10% (Xi'an Demei Biotechnology Co., Ltd., lot DMTB230223) and Yunji extract capsules (Vita Green Pharmaceuticals (HK) Ltd.), Lot HKNY2301A).

[0169] In this example, the following steps were performed: 1. A stock solution of the extract was prepared at a concentration of approximately 1.6 mg / mL dissolved in deionized water. 2. Each extract was sterile filtered through a 0.22 um filter and stored at approximately 4°C. 3. After receiving bovine bone marrow muscle cells, the cells were dissociated in 0.25% trypsin / EDTA and neutralized in trypsin neutralizer solution (also referred to as "neutralizer"). 4. Dissociated cells were centrifuged at 300 g for 5 minutes. 5. The supernatant was aspirated off, and the cell pellet was then resuspended in DMEM:F12 + 10% FBS. 6. The cell mixture was then pre-plated in an uncoated T-flask (preferably a T75 flask) for 40-60 minutes and then transferred to a 2% gelatin-coated T-flask (preferably a T75 flask). 7. Fresh medium (DMEM:F12 + 10% FBS) was then added to the uncoated flask containing purified bovine skeletal muscle fibroblasts. The coated flask contained purified bovine skeletal muscle cells. 8. Every 48 hours, the spent medium was replaced with fresh medium until the cells reached 80% confluence. 9. After confluence was reached, 4 mL of 0.25% trypsin / EDTA was added to the flask for 5 minutes, occasionally tapping the flask. 10. An equal volume of trypsin neutralizer solution was added to the flask and the cell mixture was centrifuged at 300 g for 5 minutes. 11. The supernatant was removed from the centrifuged cell mixture and the cell pellet was resuspended in 1 mL of fresh medium. 12. Cell counts were performed using trypan blue. 13. Fresh medium was added to the cell suspension and continued until a cell density of 1e5 cells / mL was reached. 14. 100 uL of the cell suspension was then added to each well of a 96-well tissue culture plate, with 10,000 cells per well. 15. The cells were then incubated overnight at approximately 37°C + 5% carbon dioxide (CO2). 16. The next day, the medium was removed from the wells and approximately 20 uL of stock extract was added to each appropriate well, followed by the addition of approximately 180 uL of fresh DMEM / F12 + approximately 2% FBS to all wells. 17. The cells were then incubated at about 37°C + 5% CO2 for about 48 hours. 18. After approximately 48 hours, MTT was performed to assess cell proliferation. Quick Plan was selected and the absorbance reading at 650 nm was subtracted from the absorbance reading at 570 nm to remove background signal from the well / medium.

[0170] Table 7 shows the final concentrations of each medium with ginseng (extract 1.3), carrot extract (extract 2.1), reishi (extract 6.1), ashwagandha (extract 7.1), cordyceps (rich in polysaccharides) (extract 8), bird's nest extract (extract BN) and Coriolus versicolor extract (extract YZ). [Table 7]

[0171] result

[0172] The effectiveness of different extracts in the presence of reduced serum was studied by culturing bovine skeletal muscle fibroblasts with a concentration of approximately 160 μg / mL. The MTT absorbance, which is proportional to the cell number, was compared to the control and expressed as a percentage, e.g., as shown in Figure 2 and summarized in Table 8. The calculation of the % increase in proliferation normalized to the negative control is as follows:

[0173] % Increase in Proliferation = {[(MTT absorbance in sample) - (MTT absorbance in negative control with DMEM:12)] / (MTT absorbance in negative control with DMEM:12)} * 100%

[0174] Referring to Figure 2, Figure 2 shows cell growth under stimulation of the following exemplary additive compositions containing the extract (provided at approximately 160 μg / mL) at 48 hours. As shown in the figure below, the extract can exhibit various proliferation activities on the tested fibroblasts. Also, approximately 1 x 10 cells were stimulated with the following additive compositions: 4 Cells were seeded at 100% cell / well (96-well plate) and reached nearly 100% confluence. Because high density induces contact inhibition, these extracts may require reduced cell seeding density to stimulate fibroblasts. In any case, these extracts showed beneficial effects on fibroblasts, which may be an interesting route for cell manufacturing. [Table 8]

[0175] The results in Table 8 show results corresponding to a medium containing basal medium DMEM:F12 and exemplary additive compositions, each containing about 160 μg / ml of a first component of ginseng (Extract 1.3), ginseng extract (Extract 2.1), ganoderma lucidum (Extract 6.1), ashwagandha (Extract 7.1), cordyceps (rich in polysaccharides) (Extract 8), bird's nest extract (Extract BN), or Coriolus versicolor extract (Extract YZ), and a second component of FBS at a final concentration of about 2%, where the most effective extracts were ginseng, ashwagandha, bird's nest, and cordyceps, which had a significant percent increase in proliferation of 31.23%, 30.02%, 23.62%, and 23.08%, respectively, at 56 hours. The results showed that these sample extracts (KGN, BN, LZ, YZ, ASH, and CS) had unexpected effects on muscle cell proliferation under conditions of significantly reduced serum concentration (2% FBS). Example 4

[0176] In this example, the effect on L6 myoblast proliferation of the use of a media composition without a second component was demonstrated.

[0177] Unless otherwise stated, the materials used in this example were similar or the same as those described in Example 1. For clarity, this specification will not repeat the materials list.

[0178] In this example, the following steps were performed: 1. A stock solution of the extract was prepared at a concentration of 1.6 mg / mL dissolved in deionized water. 2. Each extract was sterile filtered through a 0.22 um filter and stored at 4°C. 3. Thaw L6 myoblasts from liquid nitrogen, dilute with DMEM medium, and centrifuge to precipitate 3 x 10 cells. 4 The cells were resuspended in DMEM + 10% FBS to a cell density of cells / mL. 4. Add 500uL of cells to each well of a 24-well plate (coated with 2% gelatin) and add 1.5 x 10 cells to each well. 4 The cells were cultured in a 2000-well plate. 5. The cells were then incubated overnight at 37°C + 5% CO2. 6. The next day, the medium was removed from the wells and the cells were washed twice with PBS. 7. DMEM containing bird's nest extract (Hunan New Hexin Biomedical Co., Ltd., Lot MS230404) (Extract BN) or ginseng extract (Xi'an Demi Biotechnology Co., Ltd., Lot DMTB230115) (Extract 2.1) was added to the well plate to a final volume of 500 μL per well. The final concentrations of the compositions are shown in Table 9. [Table 9] 8. The cells were then incubated at about 37°C + 5% CO2 for about 48 hours. 9. After 48 hours, the medium was removed and stored in a tube, and the cells were washed with approximately 0.1 mL of PBS. 10. Then about 0.3 mL of trypsin / EDTA was added and incubated with occasional tapping at about 37° C. Once the cells were completely dissociated, the medium saved in step 9 was added again. 11. Cell counts were performed using a hemocytometer and used for analysis.

[0179] result

[0180] The final cell number after 48 h treatment was assessed and the results are shown in Table 10 and Figures 3A-3B. [Table 10]

[0181] Referring to Figures 3A-3B, the final cell counts of L6 myoblasts after 48 hours of treatment in media containing different concentrations of carrot extract (Extract 2.1) and bird's nest extract (Extract BN) according to Table 9 are shown. It was observed that for media containing the exemplary additive composition containing the first component of carrot extract (Extract 2.1) at different final concentrations (and without any second component), all media containing the exemplary additive composition containing the first component of carrot extract at different final concentrations from 1 to 80 μg / ml showed an overall increase in proliferation percentage. It was also observed that as the dose increased from 1 μg / ml to 40 μg / ml, the cell count initially increased. According to Table 10, the final cell count peaked at approximately 40 μg / mL Extract 2.1, representing a 44.12% increase in proliferation compared to the negative control. This was accompanied by a sharp decrease in the final cell count at 80 μg / mL Extract 2.1. This indicated that extract 2.1 at a final concentration of 40 ug / mL had an unexpected effect on cell proliferation in the absence of serum, growth factors or hormones in the medium.

[0182] For media with an exemplary additive composition containing different final concentrations (1-80 μg / ml) of the first component of bird's nest (extract BN) (and no second component), higher final cell numbers were observed as the concentration of extract BN increased from 5 μg / ml to 80 μg / ml, indicating the positive proliferation effect of extract BN on myoblasts. According to Table 10, a 52.94% increase in proliferation was achieved in the presence of approximately 80 μg / mL extract BN. This indicated that extract BN had an unexpected effect on cell proliferation in the absence of serum, growth factors, or hormones in the medium.

[0183] According to Table 9, the final cell numbers in DMEM with and without BSA were approximately 1.35 x 10 4 and approximately 1.91 x 10 4 This indicated that 5 mg / mL BSA may be a toxic concentration for cells and that BSA should be removed or reduced.

[0184] In summary, the results showed that treating cells with the sample additive composition containing bird's nest (extract BN) and carrots (extract 2.1) had an unexpected beneficial effect, as the majority of test groups had higher cell proliferation rates than when the cells were not treated with the sample extracts.

[0185] This example demonstrated that even in serum-free medium, L6 cells treated with BN and carrot extract were still superior to untreated controls in stimulating cell proliferation.

[0186] The examples disclosed herein demonstrated that at different concentrations and time points, different sample extracts can have different cellular effects, such as cell proliferation and differentiation.

[0187] In some examples, different types of extracts at different concentrations, at different culture times, and from different extract suppliers affect proliferation, differentiation, and gene expression (e.g., Pax7, MyoD, MHC) of different cell types (e.g., fibroblasts, skeletal satellite cells, skeletal myoblasts, adipocytes, etc.).

[0188] In some embodiments, increasing doses and treatment times correspond to changes in cell counts. In some embodiments, the combination of extracts in the additive composition exhibits antagonistic, additive, or synergistic effects.

[0189] In some examples, after evaluating the relationship between various extract factors (concentration, time point, etc.) and cell growth / differentiation, multivariate statistical experimental methods can be used to generate models that describe the synergistic activity between extracts, which can be used to select optimal extract combinations that produce various cellular effects (cell growth, differentiation, long-term passaging, stemness).

[0190] Illustrative embodiments of the present invention have now been fully described. Although described with reference to specific embodiments, it will be apparent to those skilled in the art that the present invention may be practiced with variations of these specific details. Therefore, the present invention should not be construed as being limited to the embodiments set forth herein.

[0191] For example, other cell lines from animal tissues (e.g., muscle, adipose, 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) may be used.

[0192] For example, other selected media and additives may be used.

[0193] For example, water is used as a solvent to extract the sample to form an aqueous solution, although other solvents such as DMSO, methanol, ethanol, etc. may also be used.

[0194] For example, sample extracts may be commercially available or prepared from natural materials. Numbered Examples Numbered Example 1

[0195] Example 1. A culture medium additive composition comprising at least one sample extract made 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, ginseng, ganoderma lucidum, versicolor mushroom, cordyceps sinensis, ashwagandha, and combinations thereof.

[0196] Example 2. The composition of any one of the preceding examples, wherein the carrot is American ginseng or Korean ginseng.

[0197] Example 3. The composition of any one of the preceding examples, wherein the composition is substantially free of one or more exogenous growth factors or hormones, or combinations thereof.

[0198] Example 4. The composition of any one of the preceding examples, wherein the at least one sample extract is bird's nest.

[0199] Example 5. The composition of any one of the preceding examples, wherein the at least one sample extract is American ginseng.

[0200] Example 6. The composition of any one of the preceding examples, wherein the at least one sample extract is ginseng.

[0201] Example 7. The composition of any one of the preceding examples, wherein the at least one sample extract is Ganoderma lucidum.

[0202] Example 8. The composition of any one of the preceding examples, wherein the at least one sample extract is Mushroom versicolor.

[0203] Example 9. The composition of any one of the preceding examples, wherein the at least one sample extract is Cordyceps sinensis.

[0204] Example 10. The composition of any one of the preceding examples, wherein the at least one sample extract is Ashwagandha.

[0205] Example 11. The composition of any one of the preceding examples, wherein the Ashwagandha is derived from the root or whole plant.

[0206] Example 12. The composition of any one of the preceding examples, wherein the sample extract is prepared from a solution of a filtrate or a supernatant.

[0207] Example 13. The composition of any one of the preceding examples, wherein the composition consists essentially of at least one sample extract, and the at least one sample extract is made from at least one sample selected from the group consisting of bird's nest, ginseng, reishi mushroom, versicolor mushroom, cordyceps, ashwagandha, and combinations thereof.

[0208] Example 14. The composition of any one of the preceding examples, wherein the at least one sample extract has a concentration of 0.001 ug / mL to 1000 mg / mL.

[0209] Example 15. The composition of any one of the preceding examples, wherein the at least one sample extract has a concentration of 20 mg / mL to 100 mg / mL.

[0210] Example 16. A culture medium comprising a basal or base medium and the medium additive composition of any one of the preceding examples.

[0211] Example 17. The medium of any one of the preceding examples, wherein said medium is substantially free of one or more exogenous growth factors or hormones, or combinations thereof.

[0212] Example 18. The medium of any one of the preceding examples, wherein the medium further comprises one or more exogenous growth factors or hormones, or a combination thereof.

[0213] Example 19. The medium of any one of the preceding examples, wherein the basal medium is DMEM and / or F12.

[0214] Example 20. The medium of any one of the preceding examples, wherein the medium further comprises FBS at a final concentration of 0.01% to 20%.

[0215] Example 21. A method for culturing cells, the method comprising: (i) providing a culture medium comprising a basal medium and at least one sample extract, wherein the at least one sample extract is prepared from at least one sample consisting of bird's nest, ginseng, ganoderma lucidum, versicolor mushroom, cordyceps, ashwagandha, and combinations thereof; and (ii) treating and culturing cells with the at least one natural substance.

[0216] Example 22. The method of any one of the preceding examples, wherein the method further comprises the step of: (1) treating the cell line with a dissociation agent to form a cell suspension prior to step (ii).

[0217] Example 23. The method of any one of the preceding examples, wherein the method further comprises (2) prior to step (ii), neutralizing the cell suspension with a neutralizing agent to obtain a neutralized cell suspension.

[0218] Example 24. The method of 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 medium.

[0219] Example 25. The method of any one of the preceding examples, wherein the sample extract is produced by the steps of: (a) producing at least one powder of the at least one sample; (b) dissolving the at least one powder to form at least one sample solution; and (c) purifying the at least one sample solution to obtain the at least one sample extract.

[0220] Example 26. The method of any one of the preceding examples, wherein in step (c), purifying the at least one sample solution comprises filtering and / or centrifuging the at least one sample solution.

[0221] Example 27 The method of any one of the preceding examples, wherein the medium has a final sample extract concentration of 0.001 ug / mL to 1000 mg / mL.

[0222] Example 28 The method of any one of the preceding examples, wherein the medium has a final sample extract concentration of 0.1 mg / mL to 0.5 mg / mL.

[0223] Example 29. The method of any one of the preceding Examples, wherein culturing the cells is used to produce animal tissue.

[0224] Example 30 The method of any one of the preceding examples, wherein the animal tissue is human tissue.

[0225] Example 31. The method of any one of the preceding examples, wherein culturing the cells is used to produce cultivated meat. Numbered Example 2

[0226] Example 1. An additive composition for a culture medium, said additive composition comprising a first component comprising at least one sample extract made from at least one natural substance, wherein said at least one natural substance is selected from the group consisting of bird's nest (BN), ginseng, ganoderma lucidum, versicolor mushroom, cordyceps sinensis (CS), ashwagandha (ASH), and combinations thereof.

[0227] Example 2. The composition of Example 1, wherein the carrots are ginseng, carrot, and combinations thereof.

[0228] Example 3. The composition of any one of the preceding examples, wherein the versicolor mushroom is Yunzhi (YZ).

[0229] Example 4. The composition of any one of the preceding examples, wherein the Ling Zhi is Ganoderma lucidum (GL).

[0230] Example 5. The composition of any one of the preceding examples, wherein the versicolor mushroom is Coriolus versicolor.

[0231] Example 6. The composition of any one of the preceding examples, wherein the first component is substantially free of one or more exogenous serum, growth factors or hormones, or combinations thereof.

[0232] Example 7. The composition of any one of the preceding examples, wherein the medium is used to culture animal cells.

[0233] Example 8. The composition of any one of the preceding examples, wherein the medium is used to culture mammalian, avian, crustacean, or fish cells.

[0234] Example 9. The composition of any one of the preceding examples, wherein the medium is used to culture fibroblasts, vascular cells, pericytes, endothelial cells, myoblasts, skeletal muscle satellite cells, muscle cells, smooth muscle cells, adipocytes, adipose-derived stem cells, skin cells, tendon, liver, brain, bone, heart, kidney, stem cells, or a combination thereof.

[0235] Example 10. The composition of any one of the preceding examples, wherein the medium is used to culture fibroblasts or myoblasts.

[0236] Example 11. A composition according to any one of the preceding examples, wherein the sample extract alone is prepared from a solution of a filtrate or supernatant of the natural substance alone.

[0237] Example 12. The composition of any one of the preceding examples, wherein the first component consists essentially of at least one sample extract made from at least one natural substance, wherein the at least one natural substance is selected from the group consisting of bird's nest, ginseng, reishi mushroom, versicolor mushroom, cordyceps, ashwagandha, and combinations thereof.

[0238] Example 13. The composition of any one of the preceding examples, wherein the single sample extract has a final concentration in the medium of about 0.01 ng / mL to 1000 mg / mL.

[0239] Example 14. The composition of any one of the preceding examples, wherein the single sample extract has a final concentration in the medium of less than about 1000 or 500 ug / mL.

[0240] Example 15. The composition of any one of the preceding examples, wherein the composition further comprises a second component comprising at least one serum, growth factor or hormone, amino acid, vitamin, salt, mineral, glucose, buffer, or combination thereof.

[0241] Example 16. The composition of any one of the preceding examples, wherein the second component further comprises fetal bovine serum (FBS) at a final concentration in the medium of less than about 20%, 10%, 5%, or 2%.

[0242] Example 17. The composition of any one of the preceding examples, wherein the second component further comprises bovine serum albumin (BSA) at a final concentration in the medium of about 0.01 ng / mL to 10 mg / mL.

[0243] Example 18. The composition of any one of the preceding examples, wherein the second component further comprises bovine serum albumin (BSA) at a final concentration in the medium of less than about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mg / mL.

[0244] Example 19. The composition of any one of the preceding examples, 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.

[0245] Example 20. The composition of any one of the preceding examples, wherein the second component further comprises TGF-b1 present in the medium at a final concentration of less than about 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0.5 ng / mL.

[0246] Example 21. The composition of any one of the preceding examples, wherein the second component further comprises fetal bovine serum (FBS), fetal calf serum, bovine serum albumin (BSA), or a combination thereof.

[0247] Example 22. An additive composition for a medium for culturing myoblasts, comprising: a first component containing at least one sample extract made 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 single sample extract has a final concentration of about 1 to 500 μg / mL in the medium.

[0248] Example 23. The composition of any one of the preceding examples, wherein the composition further comprises a second component comprising FBS having a final concentration of about 2% to 5% in the medium.

[0249] Example 24. An additive composition for a medium for culturing porcine bone marrow muscle cells, comprising a first component containing at least one sample extract made 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 a single sample extract has a final concentration of about 1 to 500 μg / mL in the medium.

[0250] Example 25. The composition of any one of the preceding examples, wherein the composition further comprises a second component comprising FBS having a final concentration of about 2% to 5% in the medium.

[0251] Example 26. The composition of any one of the preceding examples, wherein the composition further comprises a 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 0.5-10 ng / ml.

[0252] Example 27. An additive composition for a medium for culturing bovine bone marrow muscle cells, comprising a first component containing at least one sample extract made 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, versicolor mushroom, cordyceps sinensis (CS), ashwagandha (ASH), and combinations thereof, wherein a single sample extract has a final concentration of about 1 to 500 μg / mL in the medium.

[0253] Example 28. The composition of any one of the preceding examples, wherein the composition further comprises a second component comprising FBS having a final concentration of about 2% to 5% in the medium.

[0254] Example 29. A culture medium comprising a basal or base medium and the medium additive composition of any one of the preceding examples.

[0255] Example 30. The medium of any one of the preceding examples, wherein the medium contains reduced amounts of or is substantially free of one or more exogenous serum, growth factors or hormones, or combinations thereof.

[0256] Example 31. The medium of any one of the preceding examples, wherein the basal or base medium is Dulbecco's Modified Eagle's Medium (DMEM), Ham's F12 Medium (F12), Dulbecco's Modified Eagle's Medium F12 (DMEM:F12), Ham's F10 Medium (F10), Roswell Park Memorial Institute 1640 Medium (RPMI 1640), Minimum Essential Medium (MEM), Serum-Free Medium (SFM), or a combination thereof.

[0257] Example 32. A method of culturing cells, comprising the steps of: (i) providing a basal medium or base medium and a medium additive composition according to any one of the preceding Examples to form a medium; and (ii) treating and culturing cells in the medium.

[0258] Example 33. The method of any one of the preceding examples, wherein the method further comprises the step of: (1) treating the cell line with a dissociation agent to form a cell suspension.

[0259] Example 34. The method of 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.

[0260] Example 35. The method of 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 medium.

[0261] Example 36. The method of any one of the preceding examples, wherein the sample extract is produced by one or more of the following steps: (a) producing 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; and (c) purifying the at least one sample solution to obtain the at least one sample extract.

[0262] Example 37. The method of any one of the preceding examples, wherein in step (c), purifying the at least one sample solution comprises filtering and / or centrifuging the at least one sample solution.

[0263] Example 38 The method of any one of the preceding examples, wherein culturing the cells is used to produce animal tissue.

[0264] Example 39. The method of any one of the preceding examples, wherein the animal tissue is mammalian, avian, crustacean, or fish tissue.

[0265] Example 40. The method of any one of the preceding examples, wherein the culturing of the cells is used to produce cultivated meat.

[0266] Example 41 The method of any one of the preceding examples, wherein the method further comprises incubating the cells in a CO2 incubator.

[0267] Example 42. The method of any one of the preceding examples, wherein the cell line is derived from a mammal, bird, crustacean, or fish.

[0268] Example 43. The method of any one of the preceding examples, wherein the cell line is derived from fibroblasts, vascular cells, pericytes, endothelial cells, myoblasts, satellite cells, bone muscle cells, muscle cells, smooth muscle cells, adipocytes, adipose-derived stem cells, skin cells, tendon, liver, brain, bone, heart, kidney, stem cells, and combinations thereof.

Claims

1. 1. An additive composition for a culture medium, the additive composition comprising a first component comprising at least one sample extract produced 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, versicolor mushroom, cordyceps sinensis (CS), ashwagandha (ASH), and combinations thereof.

2. 2. The composition of claim 1, wherein the carrots are ginseng, carrot, and combinations thereof.

3. 10. The composition of claim 9, wherein the versicolor mushroom is Yunzhi (YZ).

4. 10. The composition of claim 1, wherein the Ling Zhi is Ganoderma lucidum (GL).

5. 10. A composition according to any one of the preceding claims, wherein the versicolor mushroom is Coriolus versicolor.

6. 10. The composition of any one of the preceding claims, wherein the first component is substantially free of one or more exogenous serum, growth factors or hormones, or combinations thereof.

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

8. 10. The composition according to any one of the preceding claims, wherein the medium is used for culturing mammalian, avian, crustacean or fish cells.

9. 10. The composition of any one of the preceding claims, wherein the medium is used to culture fibroblasts, vascular cells, pericytes, endothelial cells, myoblasts, skeletal satellite cells, muscle cells, smooth muscle cells, adipocytes, adipose-derived stem cells, skin cells, tendon, liver, brain, bone, heart, kidney, stem cells, or a combination thereof.

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

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

12. 10. The composition of claim 1, wherein the first component consists essentially of at least one sample extract made from at least one natural substance, wherein the at least one natural substance is selected from the group consisting of bird's nest, ginseng, reishi mushroom, versicolor mushroom, cordyceps, ashwagandha, and combinations thereof.

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

14. 10. The composition of claim 1, wherein the single sample extract has a final concentration in the medium of less than about 1000 or 500 ug / mL.

15. 10. The composition of any one of the preceding claims, further comprising a second component comprising at least one serum, growth factor or hormone, amino acid, vitamin, salt, mineral, glucose, buffer, or combination thereof.

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

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

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

19. 19. The composition of any one of claims 15 to 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. 19. The composition of any one of claims 15, 17, and 18, wherein the second component further comprises TGF-b1 present in the medium at a final concentration of less than about 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0.5 ng / mL.

21. 21. The composition of any one of claims 15 to 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 medium for culturing myoblasts, comprising: An additive composition for a culture medium for culturing myoblasts, comprising a first component containing at least one sample extract made 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 a single sample extract has a final concentration of about 1 to 500 ug / mL in the culture medium.

23. 23. The composition of claim 22, wherein the composition further comprises a second component comprising FBS having a final concentration of about 2% to 5% in the medium.

24. An additive composition for a medium for culturing porcine bone marrow muscle cells, comprising: An additive composition for a culture medium for culturing porcine bone marrow muscle cells, comprising a first component containing at least one sample extract made 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 a single sample extract has a final concentration of about 1 to 500 μg / mL in the culture medium.

25. 25. The composition of claim 24, wherein the composition further comprises a second component comprising FBS having a final concentration of about 2% to 5% in the medium.

26. 26. The composition of claim 24 or 25, wherein the composition further comprises a second component comprising BSA having a final concentration of about 1 to 10 mg / ml in the medium and optionally TGF-b1 having a final concentration of about 0.5 to 10 ng / ml.

27. An additive composition for a medium for culturing bovine bone marrow muscle cells, comprising:

1. An additive composition for a culture medium for culturing bovine bone marrow muscle cells, comprising: a first component containing at least one sample extract made 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, versicolor mushroom, cordyceps sinensis (CS), ashwagandha (ASH), and combinations thereof, wherein a single sample extract has a final concentration of about 1 to 500 μg / mL in the culture medium.

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

29. A culture medium comprising a basal or base medium and the medium additive composition of any one of the preceding claims.

30. 30. The medium of claim 29, wherein the medium is substantially free of or contains reduced amounts of one or more exogenous serum, growth factors or hormones, or combinations thereof.

31. 31. The medium of claim 29 or 30, wherein the basal medium or base medium is Dulbecco's Modified Eagle's Medium (DMEM), Ham's F12 Medium (F12), Dulbecco's Modified Eagle's Medium F12 (DMEM:F12), Ham's F10 Medium (F10), Roswell Park Memorial Institute 1640 Medium (RPMI 1640), Minimum Essential Medium (MEM), Serum Free Medium (SFM), or a combination thereof.

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

33. The method comprises:

33. The method of claim 32, further comprising the step of: (1) treating the cell line with a dissociation agent to form a cell suspension.

34. The method comprises: (2) The method according to claim 32 or 33, further comprising the step of neutralizing the cell suspension with a neutralizing agent to obtain a neutralized cell suspension.

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

36. (a) producing at least one powder of said at least one natural substance; (b) dissolving the at least one powder to form at least one sample solution; (c) purifying said at least one sample solution to obtain said at least one sample extract.

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

38. The method of any one of claims 32 to 37, wherein the culturing of cells is used to produce animal tissue.

39. 39. The method of claim 38, wherein the animal tissue is mammalian, avian, crustacean, or fish tissue.

40. 40. The method of claim 38, wherein said culturing cells is used to produce cultivated meat.

41. The method comprises: 2 41. The method of any one of claims 32 to 40, further comprising the step of incubating the cells in an incubator.

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

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

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