Composition for cell culture containing microalgae extract and use thereof

Abstract

The present invention relates to a cell culture composition containing an extract of microalgae belonging to the Thraustochytrid family. It has been confirmed that when muscle cells are cultured in a medium containing an extract extracted from microalgae biomass belonging to the Thraustochytrid family of the present invention, the cell proliferation ability of the muscle cells is improved. Therefore, the cell culture composition containing the microalgae extract can be used in place of fetal bovine serum among the components of cell culture medium.

Description

[Technical Field]

[0001] [CROSS-REFERENCE TO RELATED APPLICATIONS] This application claims the benefit of priority based on Korean Patent Application No. 10-2022-0175629 dated December 15, 2022, and all contents disclosed in the documents of this Korean patent application are incorporated herein by reference.

[0002] The present invention relates to a composition for cell culture containing a microalgae extract and uses thereof. [Background technology]

[0003] Fetal bovine serum (FBS) is the most commonly used component of basal medium for animal cell culture. However, ethical issues inevitably arise during the manufacturing process of FBS, and there are various issues such as contamination and supply restrictions, making the development of alternatives essential. Although various research efforts are underway to develop serum-free media that do not use FBS (KR10-2021-0090560), various hormones or growth factors still need to be added to replace FBS, and further development is required for user convenience.

[0004] In this invention, an extract is obtained from biomass produced through the fermentation of Thraustochytrid microalgae and utilized as a component of cell culture medium. Unlike the existing fetal bovine serum production process, the fermentation-based microalgae biomass production and extraction process has the advantage of being free of ethical issues and avoiding stability issues such as contamination issues. Summary of the Invention [Problem to be solved by the invention]

[0005] An object of the present invention is to provide a cell culture medium composition comprising an extract of microalgae of the Thraustochytrid family.

[0006] Another object is to provide a cell culture medium additive comprising an extract of microalgae from the Thraustochytrid family.

[0007] Another example object is to provide a cell culture medium comprising the cell culture medium additive.

[0008] Another object is to provide a method for culturing cells using the cell culture medium composition.

[0009] Another object of the present invention is to provide a use of an extract of microalgae belonging to the Thraustochytrid family as a culture medium additive that can replace animal serum and promote cell growth and survival. [Means for solving the problem]

[0010] In one aspect to achieve the above object, the present invention relates to a cell culture medium composition containing an extract of microalgae of the Thraustochytrid family.

[0011] In another aspect, the present invention relates to a cell culture medium additive comprising an extract of microalgae of the Thraustochytrid family.

[0012] In another aspect, the present invention relates to a cell culture medium containing the medium additive.

[0013] In another aspect, the present invention relates to a method for culturing cells, comprising culturing cells in the medium.

[0014] In another aspect, the present invention relates to the use of an extract of microalgae belonging to the Thraustochytrid family as a culture medium additive that can replace animal serum and promote cell growth and survival.

[0015] The present invention will now be described in more detail.

[0016] The present invention provides a cell culture medium composition comprising an extract of microalgae from the Thraustochytrid family.

[0017] As used herein, the term "cell culture" refers to the process of artificially growing living cells outside the body under controlled conditions. It can also refer to the process of aseptically removing a portion of an individual tissue, using enzymes to decompose the intercellular connective tissue, releasing the resulting suspension, and spreading the cells on the flat bottom of a culture dish such as a bottle or Petri dish to grow and proliferate the cells.

[0018] The cell culture may include culturing cells for producing cell-cultured meat. Thus, the composition may be a medium composition for producing cultured meat. The medium composition of the present invention may include a microalgae extract from the Thraustochytrid family instead of commonly used fetal bovine serum. The Thraustochytrid microalgae extract is a material rich in nutrients such as protein and amino acids, which can eliminate stability issues and is expected to have a lower production cost and superior productivity compared to fetal bovine serum.

[0019] In this specification, the term "cultured meat" refers to edible meat obtained by harvesting animal cells and then growing them using cell engineering technology, and can be considered a branch of cellular agriculture, which produces meat without the process of raising livestock. In Korean, it is called cultured meat, alternative meat, or artificial meat, and in English, it is called in vitro meat, meaning grown in a test tube; artificial meat, meaning synthesized by humans using stem cells rather than natural methods; clean meat, meaning produced in clean production facilities rather than traditional farming facilities; and lab-grown meat, meaning produced in a laboratory.

[0020] As used herein, the term "culture media" refers to a medium capable of supporting the growth, survival, and differentiation of cells or stem cells in vitro, and includes all common media suitable for the culture and differentiation of cells or stem cells used in the art. The type of medium and culture conditions can be selected according to the state of the art in the field depending on the type of cell. The medium used for culture is specifically cell culture minimum medium (CCMM), which generally contains a carbon source, a nitrogen source, and trace element components. The minimal cell culture medium may be, for example, one or more selected from the group consisting of Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basal Medium Eagle's Medium (BME), RPMI 1640, F-10, F-12, DMEM / F12, α-Minimal Essential Medium (α-MEM), Glasgow's Minimal Essential Medium (G-MEM), Iscove's Modified Dulbecco's Medium (IMDM), MacCoy's 5A medium, AmnioMax complete medium, AminoMax ± medium, Endothelial Basal Medium (EBM) medium, Chang's Medium, MesenCult-XF, Dulbecco's Modified Eagle's Medium high glucose (DMEM / HG) medium, and MCDB+DMEM / LG (MCDB+Dulbecco's Modified Eagle's Medium low glucose) medium. The medium may also contain antibiotics such as penicillin, streptomycin, gentamicin, or a mixture of two or more thereof. Therefore, the cell culture medium composition of the present invention may comprise the minimal cell culture medium supplemented with an extract of a microalga belonging to the Thraustochytridaceae family.

[0021] As used herein, the term "Thraustochytrid microalgae" refers to heterotrophic microorganisms belonging to the order Thraustochytriales, which are known as oleaginous microorganisms capable of producing bio-oil containing unsaturated fatty acids such as DHA (docosahexaenoic acid), and are said to be a good protein source because they are rich in protein and amino acids.

[0022] The Thraustochytrid microalgae may be any genera available to those skilled in the art, specifically, any one selected from the group consisting of the genera Schizochytrium, Aurantiochytrium, Thraustochytrium, and Ulkenia, but is not limited thereto.

[0023] As used herein, the term "extract" may refer to a liquid component obtained by immersing a target substance in various solvents and then extracting for a certain period of time at room temperature or at elevated temperatures, or a solid component obtained by removing the solvent from the liquid component. In addition to the above-mentioned product, the term "extract" may be interpreted as encompassing all forms of extracts that can be prepared using the extract, including dilutions of the above-mentioned product, concentrates thereof, crude products thereof, purified products thereof, and mixtures thereof. The extract may also include fractions obtained therefrom.

[0024] The extract can be extracted from natural, hybrid or variant Thraustochytrid microalgae, and can also be extracted from tissue cultures of Thraustochytrid microalgae.

[0025] The extraction solvent for the extract can be a protic polar solvent or an aprotic polar and non-polar solvent. The protic polar solvent can be water, methanol, ethanol, propanol, isopropanol, or butanol. The aprotic polar solvent can be dichloromethane, tetrahydrofuran, ethyl acetate, acetonitrile, dimethylformamide, dimethyl sulfoxide, acetone, 2-butanone, or hexamethylphosphoramide. The non-polar solvent can be pentane, hexane, chloroform, or diethyl ether. Benzene is excluded from the non-polar solvents. The solvent can be a C1-C6 alcohol, a C3-C10 ester (e.g., a C3-C10 acetate), a C3-C10 ketone, a C1-C6 unsubstituted or halogenated hydrocarbon, a C2-C10 cyclic ether, a mixture thereof, or a mixture of one or more of the solvents with water. The solvent can be ethanol, propanol, acetonitrile, ethyl acetate, acetone, 2-butanone, chloroform, dichloromethane, hexane, a mixture thereof, or a mixture of one or more of the solvents with water. The hydrocarbon can be an alkane, alkene, or alkyne.

[0026] The extract may be extracted with one or more solvents selected from the group consisting of water, C1-C4 lower alcohols such as methanol, ethanol, propanol, and butanol, polyhydric alcohols such as glycerin, butylene glycol, and propylene glycol, and hydrocarbon solvents such as methyl acetate, ethyl acetate, acetone, benzene, hexane, diethyl ether, and dichloromethane. Specifically, the extract may be extracted using water as the solvent.

[0027] The Thraustochytridaceae microalgae extract may be extracted from a culture solution of Thraustochytridaceae microalgae itself, or may be extracted from a culture solution obtained by dehydrating the culture solution using a freeze-drying or centrifuge, followed by other drying processes such as freeze-drying, drum drying, spray drying, or a granulator, followed by production of biomass. However, the extract is not limited thereto.

[0028] As used herein, the term "biomass" may refer to, but is not limited to, Thraustochytridaceae microalgae themselves, a culture thereof, a dried product thereof, a crushed product thereof, or a product produced by cultivating or fermenting the microalgae, or a concentrate or structure of the biomass.

[0029] The Thraustochytridaceae microalgae extract can be obtained by extracting Thraustochytridaceae microalgae or Thraustochytridaceae microalgae biomass using a conventional method, such as hot water extraction, cold maceration extraction, reduced pressure high-temperature extraction, hot water extraction, room temperature extraction, fractional extraction, reflux cooling extraction, solvent extraction, steam distillation, ultrasonic extraction, elution, or squeezing, and is preferably extracted using hot water extraction. Furthermore, if necessary, commercially available Thraustochytridaceae microalgae extracts can also be used as the Thraustochytridaceae microalgae extract.

[0030] The hot water extraction method can be performed at 80 to 140°C, 85 to 135°C, 90 to 130°C, 95 to 125°C, 100 to 120°C, or 105 to 115°C, and in a specific example, 110°C, but is not limited thereto. The hot water extraction method can be performed for 3 minutes or more, 5 minutes or more, 10 minutes or more, 1 to 30 minutes, 2 to 30 minutes, 3 to 30 minutes, 4 to 30 minutes, 5 to 30 minutes, 6 to 30 minutes, 7 to 30 minutes, 8 to 30 minutes, 9 to 30 minutes, 1 to 25 minutes, 2 to 25 minutes, 3 to 25 minutes, 4 to 25 minutes, 5 to 25 minutes, 6 to 25 minutes, 7 to 25 minutes, 8 to 25 minutes, 9 to 25 minutes, 1 to 2 ...25 minutes, 5 to 25 minutes, 6 to 25 minutes, 7 to 25 minutes, 8 to 25 minutes, 9 to 25 minutes, 1 to 20 minutes, 2 to 30 minutes, 3 to 30 minutes, The duration may be 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 70 minutes, 80 minutes, 90 minutes, 15 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 70 minutes, 80 minutes, 90 minutes, 15 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 70 minutes, 80 minutes, or 90 minutes. In a specific example, the duration may be 10 minutes, but is not limited to this.

[0031] As used herein, the term "fraction" refers to the product obtained by fractionating a mixture containing a variety of components to separate a specific component or a specific group of components.

[0032] The fractionation method for obtaining the fractions is not particularly limited and can be a method commonly used in the art. Non-limiting examples of the fractionation method include fractionation using various solvents, ultrafiltration fractionation using ultrafiltration membranes with a specific molecular weight cutoff, chromatographic fractionation using various chromatographies (designed for separation according to size, charge, hydrophobicity, or affinity), and combinations thereof.

[0033] The fractionation solvent used to obtain the fraction is not particularly limited, and any solvent known in the art can be used. Non-limiting examples of fractionation solvents include polar solvents such as water and C1-C4 alcohols; non-polar solvents such as hexane, ethyl acetate, chloroform, and dichloromethane; and mixtures thereof. These solvents can be used alone or in combination, but are not limited thereto.

[0034] The above-mentioned スラウストキトリッドfamily microalgae extract is, the aforementioned nutrient composition for cell culture has a concentration of 25g / L or less, and specifically, the concentration is 23.6g / L or less. Example: えば, the concentration mentioned above, 0.001~25g / L, 0.005~25g / L, 0.01~25g / L, 0.05~25g / L, 0 .1~25g / L, 0.2~25g / L, 0.4~25g / L, 0.8~25g / L, 1~25g / L, 2~25g / L, 3~2 5g / L, 0.001~23.6g / L, 0.005~23.6g / L, 0.01~23.6g / L, 0.05~23.6g / L , 0.1~23.6g / L, 0.2~23.6g / L, 0.4~23.6g / L, 0.8~23.6g / L, 1~23.6g / L , 2~23.6g / L, 3~23.6g / L, 0.001~22g / L, 0.005~22g / L, 0.01~22g / L, 0.05~22g / L, 0.1~22g / L, 0.2~22g / L, 0.4~22g / L, 0.8~22g / L, 1~22g / L, 2~22g / L, 3~22g / L, 0.001~20g / L, 0.005~20g / L, 0.01~20g / L, 0.05~20 g / L, 0.1~20g / L, 0.2~20g / L, 0.4~20g / L, 0.8~20g / L, 1~20g / L, 2~20g / L, 3~20g / L, 0.001~15g / L, 0.005~15g / L, 0.01~15g / L, 0.05~15g / L, 0.1~15g / L, 0.2~15g / L, 0.4~15g / L, 0.8~15g / L, 1~15g / L, 2~15g / L, 3~1 5g / L, 0.001~12g / L, 0.005~12g / L, 0.01~12g / L, 0.05~12g / L, 0.1~12g / L, 0.2~12g / L, 0.4~12g / L, 0.8~12g / L, 1~12g / L, 2~12g / L, 3~12g / L, 0 .001~11g / L, 0.005~11g / L, 0.01~11g / L, 0.05~11g / L, 0.1~11g / L, 0.2 ~11g / L, 0.4~11g / L, 0.8~11g / L, 1~11g / L, 2~11g / L, 3~11g / L, 0.001~1 0g / L, 0.005~10g / L, 0.01~10g / L, 0.05~10g / L, 0.1~10g / L, 0.2~10g / L , 0.4~10g / L, 0.8~10g / L, 1~10g / L, 2~10g / L, 3~10g / L, 0.001~8g / L, 0.005~8g / L, 0.01~8g / L, 0.05~8g / L, 0.1~8g / L, 0.2~8g / L, 0.4~8g / L, 0.8~8g / L, 1~8g / L, 2~8g / L, 3~8g / L, 0.001~5g / L, 0.005~5g / L, 0.01~5g / L, 0.05~5g / L, 0.1~5g / L, 0.2~5g / L, 0.4~5g / L, The concentration may be 0.8 to 5 g / L, 1 to 5 g / L, 2 to 5 g / L, 3 to 5 g / L, 0.001 to 4 g / L, 0.005 to 4 g / L, 0.01 to 4 g / L, 0.05 to 4 g / L, 0.1 to 4 g / L, 0.2 to 4 g / L, 0.4 to 4 g / L, 0.8 to 4 g / L, 1 to 4 g / L, 2 to 4 g / L, or 3 to 4 g / L, and specifically 0.8 to 4 g / L.

[0035] The cells may be somatic cells, embryonic cells, germ cells, stem cells, cancer cells, cell lines, cultured cells (in vitro), graft cells, primary cultured cells (in vitro and ex vivo), and in vivo cells; cells of eukaryotic organisms or mammalian cells, including humans. For example, the cells may be selected from the group consisting of cancer cells, stem cells, vascular endothelial cells, leukocytes, immune cells, epithelial cells, germ cells, fibroblasts, muscle cells, bone marrow cells, epidermal cells, osteoblasts, and nerve cells. The eukaryotic organisms may be insects, amphibians, birds (chickens, turkeys, ducks, etc.), or mammals (humans, primates such as monkeys, dogs, pigs, cows, sheep, goats, mice, rats, etc.).

[0036] The cells may be cells isolated from an individual or a eukaryotic organism.

[0037] The cells may be undifferentiated cells, differentiating cells, or fully differentiated cells, and in particular may be stem cells.

[0038] As used herein, the term "stem cell" refers to a cell that has the potential to differentiate and self-renew. Stem cells are classified as pluripotent, multipotent, or unipotent depending on their differentiation potential. The stem cells may be one or more selected from the group consisting of embryonic stem cells (ESCs) (cells inside a preimplantation embryo), adult stem cells (undifferentiated cells present in various tissues and organs), and induced pluripotent stem cells (iPSCs) (cells induced to reverse differentiation by inserting genes and / or proteins into somatic cells, or induced pluripotent stem cells).

[0039] The stem cells may be of any type or origin, as long as they have differentiation and self-renewal capabilities. The stem cells may be derived from, for example, mammals, humans, monkeys, pigs, horses, cows, goats, dogs, cats, mice, or rabbits. The stem cells may be derived from isolated umbilical cords, placenta, fat, bone marrow, muscle, umbilical cord blood, or amniotic fluid. The term "isolated" means that the stem cells are present in an environment different from that of naturally occurring cells or tissues. Isolation of umbilical cords, placenta, fat, bone marrow, muscle, umbilical cord blood, or amniotic fluid and obtaining stem cells therefrom can be performed by conventional anatomical and known methods.

[0040] The cells may be muscle cells or muscle stem cells, and may be derived from livestock such as chicken, bovine, and porcine, and more specifically from bovine or porcine.

[0041] As used herein, the term "myogenic stem cell" refers to cells that have all of the characteristics of muscle stem cells, including proliferation without transformation, limitless proliferation, self-renewal, and the ability to differentiate into muscle. It includes, without limitation, cells that exhibit self-renewal or limitless proliferation and muscle differentiation. The self-renewal and muscle differentiation abilities can be confirmed using markers. Muscle stem cells can be used in combination with muscle satellite cells (SCs).

[0042] According to one example, it was confirmed that the medium composition containing the Thraustochytridaceae microalgae extract exhibited significantly superior cell proliferation efficacy in muscle stem cells.

[0043] The composition may further comprise serum, which may be one or more selected from the group consisting of fetal bovine serum (FBS), bovine calf serum (BCS), human serum, and horse serum (HS), and may specifically be fetal bovine serum.

[0044] The composition may contain serum and an extract of Thraustochytridaceae microalgae, and the serum may be contained in a weight ratio of 1000:1 to 1:1 relative to the extract of Thraustochytridaceae microalgae (serum: Thraustochytridaceae microalgae extract). For example, the weight ratio of the serum to the chlorella extract may be 1000:1 to 1:1, 800:1 to 1:1, 600:1 to 1:1, 500:1 to 1:1, 400:1 to 1:1, 350:1 to 1:1, 300:1 to 1:1, 250:1 to 1:1, 200:1 to 1:1, 150:1 to 1:1, 100:1 to 1:1, 50:1 to 1:1, 40:1 to 1:1, 30:1 to 1:1, 25:1 to 1:1, 20:1 to 1:1, 15:1 to 1:1, 12.5:1 to 1:1, 10:1 to 1:1, 7.5:1 to 1:1, 5:1 to 1:1, or 2.5:1 to 1:1.

[0045] The culture composition allows for subculturing of cells.

[0046] As used herein, the term "passage" refers to replacing a culture vessel or culturing a separate cell population in a method of culturing cells, specifically stem cells, in a healthy, long-term, continuous manner. Replacing a culture vessel once or culturing a separate cell population is referred to as one passage. In the present invention, the term "passage" can be used interchangeably with "separate passage."

[0047] The culture may be growth or proliferation. As used herein, the terms "growth" and "proliferation" refer to an increase in cell number. The culture may be undifferentiated proliferation. Undifferentiated proliferation refers to the proliferation of stem cells into cells with the same properties as the original cell, i.e., cells with potency and self-renewal, without differentiating into a specific cell. The term "differentiation" refers to the phenomenon in which cells specialize in structure and function as they divide and grow, i.e., the morphology and function of cells and tissues of an organism change to perform their assigned roles. The degree of differentiation into a specific cell type can be measured or determined by methods well known in the art. The differentiation can also be confirmed by examining cell morphology using light or confocal microscopy, measuring cell surface markers (e.g., staining cells with tissue-specific or cell marker-specific antibodies) and changes in cell morphology (e.g., nuclear / cytoplasmic ratio) using techniques such as flow cytometry or immunocytochemistry, or by measuring changes in gene expression using techniques well known in the art, such as polymerase chain reaction (PCR) and gene expression profiling.

[0048] The present invention also provides a cell culture medium additive comprising an extract of microalgae of the Thraustochytrid family.

[0049] The same parts as explained above also apply to the composition.

[0050] The medium additive can replace all or part of the animal serum contained in the medium, and the cell culture medium containing the medium additive can have a lower serum content than a general culture medium (containing about 10% serum). Furthermore, the low-serum culture medium containing the medium additive can exhibit cell growth rate, cell viability, and subculture efficiency that are equivalent to or significantly better than those of a 10% serum-containing culture medium.

[0051] The present invention also provides a cell culture method comprising culturing isolated cells using the cell culture medium composition.

[0052] The same parts as those explained above also apply to the method.

[0053] The isolated cells may be cells isolated from livestock, specifically cells isolated from livestock such as cows, pigs or chickens.

[0054] The cell culture method may be a method for culturing cells to produce cultured meat, specifically, for proliferating and culturing muscle stem cells to produce cultured meat. Therefore, cultured meat can be produced using the cells, specifically muscle stem cells, cultured by the method.

[0055] The culture format may be a conventional two-dimensional or three-dimensional culture known in the art. However, three-dimensional culture is preferred to realize tissues similar to actual living tissues through cell-cell interactions. Specific examples of three-dimensional culture include 3D porous scaffolds, scaffold-free platforms using cells themselves or cell sheet technology, methods of placing cells in microchips, methods using hydrogels, and methods using bioreactors.

[0056] The present invention also provides a use of an extract of microalgae belonging to the Thraustochytrid family as a culture medium additive that can replace animal serum and promote cell growth and survival.

[0057] In the present application, the Thraustochytridaceae microalgae, extract, medium additive, etc. are as described above. [Effects of the Invention]

[0058] It has been confirmed that the cell proliferation ability of bovine muscle cells is improved when muscle cells are cultured in a medium containing an extract extracted from microalgae biomass belonging to the Thraustochytrid family of the present invention. Therefore, the cell culture composition containing the microalgae extract can be used as a substitute for fetal bovine serum among the cell culture medium components. Furthermore, since the microalgae extract contained in the cell culture composition of the present invention is produced through a fermentation-based process, it is free from various ethical and contamination issues, unlike the production process of existing fetal bovine serum, and has the advantage of being able to be mass-produced sustainably while maintaining more consistent quality. [Brief explanation of the drawings]

[0059] [Figure 1] 1 shows the cytotoxicity and cell proliferation effects of a microalgae extract from the Thraustochytrid family on bovine muscle stem cells, expressed as the mean (N=3) ± standard deviation. [Figure 2] This figure shows the cell proliferation effect of culture media containing various concentrations of FBS and a microalgal extract from the Thraustochytrid family on bovine muscle stem cells. The results are shown as the mean (N=3) ± standard deviation. Statistical comparisons were made between cells cultured in a medium containing a microalgal extract from the Thraustochytrid family and cells cultured without the extract from the Thraustochytrid family for the same culture period. (*p<0.05, **p<0.01, ***p<0.001, Student's t-test) DETAILED DESCRIPTION OF THE INVENTION

[0060] The present invention will be described in more detail below with reference to examples. However, these examples are intended to illustrate one or more specific examples, and the scope of the present invention is not limited to these examples.

[0061] Example 1. Obtaining microalgae extract A Schizochytrium sp. strain belonging to the Thraustochytrid family of microalgae was dark-cultured and then dried to obtain biomass.

[0062] Specifically, Schizochytrium sp. strains were fermented in a 5L fermenter containing sterilized MJW01 medium (glucose 30g / L, MgSO4·7H2O 3.0g / L, Na2SO4 10g / L, NaCl 1.0g / L, yeast extract 9.0g / L, MSG·1H2O 1.0g / L, NaNO3 1.0g / L, KH2PO4 0.1g / L, K2HPO4 0.5g / L, CaCl2 0.5g / L, and vitamin mixture solution 10ml / L). The culture medium was either freeze-dried as is or dehydrated by centrifugation and then freeze-dried to obtain biomass. The obtained microalgae biomass was dispersed in purified water at a concentration of 1.5% and then heated to 110°C for 10 minutes for hot water extraction. The extract was filtered to remove residual suspended matter and then freeze-dried to obtain a Thraustochytrid microalgae extract. The individual amino acid contents and total amino acid contents per dry weight of the obtained Thraustochytrid microalgae extract were measured and are shown in Table 1 below.

[0063] [Table 1]

[0064] As a result, as shown in Table 1, the total amino acid content of the obtained Thraustochytrid microalgae extract was confirmed to be 52.17% by dry weight, and the amino acid content was highest in the extract, followed by arginine (Arg) and aspartic acid (Asp).

[0065] Example 2. Confirmation of cytotoxicity of microalgae extract To confirm the effect of Thraustochytrid microalgae extract on bovine muscle stem cells (bMuSCs) extracted from beef shank and sirloin, a WST-8 test was conducted as follows.

[0066] Specifically, cells from passages 3 or 5 were seeded at 5,000 cells per well into a 96-well culture plate. Triplicate wells were seeded for each condition and cultured at 37°C and 5% CO2 for one day. The medium was then replaced with fresh medium containing 10% FBS and 0.001 g / L to 40 g / L of Thraustochytrid extract and cultured for three days. Then, 5 μL of WST-8 was added per well and cultured for an additional three hours. Cell metabolic activity was determined by measuring optical density (OD450) at 450 nm using a microplate spectrophotometer. Wells containing only culture medium without cells served as blank controls, and wells not treated with Thraustochytrid extract served as negative controls. Relative cell viability was assessed by comparing the OD450 values ​​of cells treated with Thraustochytrid extract. The concentration of Thraustochytrid extract showing the highest viability was designated E max The IC50 was calculated using the Quest Graph™ IC50 calculator to determine the CC at which cell viability was 50% of the negative control group. 50The values ​​were calculated. The results of cell viability measurements as a function of the concentration of Thraustochytrid extract measured in this experiment are shown in Figure 1.

[0067] As a result, as shown in Figure 1, cell proliferation of bMuSCs was at a similar level to the control group when Thraustochytrid extract was included in the concentration range of 0.001-2 g / L. When Thraustochytrid extract was included in the concentration of 4 g / L, cell viability increased by 115.8% compared to the control group, showing the highest cell viability within the concentration range used in the experiment. Therefore, the 4 g / L concentration was selected as the E max It was also confirmed that CC 50 The value was determined to be 23.6 g / L.

[0068] In conclusion, when Thraustochytrid extract is added to the culture medium at a concentration of 4 g / L or less, it does not exhibit toxicity to cells, and in particular, when Korean beef cattle muscle cells (bMuSC) are added to the culture medium at this concentration, cell proliferation is excellent.

[0069] Example 3. Confirmation of serum replacement effect of Thraustochytrid extract In order to confirm the serum replacement effect of Thraustochytrid extract, the following experiment was carried out.

[0070] Specifically, under culture conditions in which fetal bovine serum (FBS) in the culture medium was reduced, the E confirmed in Example 2 was maxA WST-8 test was performed to determine the effect of adding Thraustochytrid extract at various concentrations on bMuSC cells. Bovine muscle cells at passages 5, 6, or 15 were seeded into 96-well culture plates, with 5,000 cells per well, in triplicate. After culturing for one day at 37°C and 5% CO2, half of the culture medium was replaced with medium containing 0% to 10% FBS and 0 g / L to 4 g / L Thraustochytrid extract. The weight ratios of added FBS and Thraustochytrid extract are shown in Table 2 below.

[0071] [Table 2]

[0072] The cells were cultured for one day to allow them to adapt to the new medium, and then the medium was replaced with new one again, and the WST-8 assay was performed on days 3 and 6. Cell proliferation on days 3 and 6 was determined by comparing the measured OD450 values ​​with the OD450 value measured on day 0, and the results are shown in Figure 2 and Table 3 below.

[0073] [Table 3]

[0074] As a result, as shown in Figure 2 and Table 3, it was confirmed that cell proliferation increased when bMuSC cells were cultured in a medium containing Thraustochytrid extract. maxWhen Thraustochytrid extract was added at a concentration of 10% FBS, the cell proliferation rate increased by 26.9% compared to the control group not treated with Thraustochytrid extract, 74.0% in the 5% FBS condition, and 127.4% in the 2% FBS condition, based on the results of the 6th day of culture. The 5% FBS condition showed a level of cell proliferation equal to or greater than that of the 10% FBS control group. Therefore, it was confirmed that Thraustochytrid extract can substitute for FBS and induce the proliferation of bMuSC cells, and specifically, Thraustochytrid extract was effective in E max When it is contained at a concentration of 4 g / L, it shows a 50% to 80% FBS replacement rate.

Claims

1. A cell culture medium composition comprising an extract of microalgae from the Thraustochytrid family.

2. The cell culture medium composition according to claim 1 , wherein the Thraustochytridaceae microalgae extract is contained at a concentration of 23.6 g / L or less.

3. 2. The cell culture medium composition according to claim 1, wherein the Thraustochytridaceae microalgae extract is contained at a concentration of 0.4 g / L to 10 g / L.

4. 2. The cell culture medium composition according to claim 1, wherein the Thraustochytridaceae microalgae is any one selected from the group consisting of the genus Schizochytrium, the genus Aurantiochytrium, the genus Thraustochytrium, and the genus Ulkenia.

5. 2. The cell culture medium composition according to claim 1, wherein the Thraustochytridaceae microalgae extract has a total amino acid content of 40% or more based on the dry weight.

6. The cell culture medium composition of claim 1 , wherein the cells are muscle stem cells or muscle cells.

7. The cell culture medium composition according to claim 1 , wherein the cells are derived from bovine or porcine.

8. 10. The cell culture medium composition of claim 1, wherein the composition further comprises serum.

9. 9. The cell culture medium composition according to claim 8, wherein the serum is at least one selected from the group consisting of fetal bovine serum (FBS), bovine calf serum (BCS), human serum, and horse serum (HS).

10. 9. The cell culture medium composition according to claim 8, wherein the serum is contained in a weight ratio of 1000:1 to 1:1 relative to the Thraustochytridaceae microalgae extract (serum: Thraustochytridaceae microalgae extract).

11. A cell culture medium additive comprising an extract of microalgae from the Thraustochytrid family.

12. A cell culture method comprising a step of culturing isolated cells using the cell culture medium composition according to any one of claims 1 to 10.

Images