Cell culture compositions comprising microalgae extracts and uses thereof

Abstract

The present invention relates to a cell culture composition comprising an extract of thraustochytriaceae microalgae. It has been found that the proliferative capacity of muscle cells is enhanced when the muscle cells are cultured in a culture medium according to the present disclosure comprising an extract derived from a microalgae biomass belonging to the Thraustochytridae family. Thus, the cell culture composition comprising the microalgae extract can be used as a substitute for fetal calf serum in the cell culture medium component.

Description

Technical Field

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority based on Korean Patent Application No. 10-2022-0175629 filed on December 15, 2022, and all contents disclosed in the corresponding Korean patent application document are incorporated herein by reference as a part of this specification.

[0003] The present invention relates to a composition for cell culture comprising a microalgae extract and uses thereof. Background Art

[0004] So far, fetal bovine serum (FBS) is the most commonly used component in the basic culture medium components for animal cell culture. However, ethical issues inevitably occur during the preparation process of fetal bovine serum, and various problems such as contamination problems and supply limitations occur from time to time, so it is inevitable to develop its substitute. Various studies have been conducted to develop serum-free culture media (KR 10-2021-0090560) that do not use fetal bovine serum, but there is still a need to add various hormones or growth factors to replace fetal bovine serum, so for the convenience of users, additional development is required.

[0005] In the present invention, an extract is obtained from biomass produced by fermenting Thraustochytrid-based microalgae and used as a medium component for cell culture. The fermentation-based microalgae biomass production and extraction process has the advantages of being different from the conventional fetal bovine serum production process without ethical issues and avoiding stability issues such as contamination issues. Summary of the invention

[0006] Technical issues

[0007] The present invention provides a culture medium composition for cell culture, comprising a Thraustochytridaceae microalgae extract.

[0008] Another embodiment provides a medium supplement for cell culture, comprising a Thraustochytridaceae microalgae extract.

[0009] Other embodiments provide a culture medium for cell culture, comprising a culture medium supplement for cell culture.

[0010] Other embodiments provide a method for culturing cells using a medium composition for cell culture.

[0011] Other embodiments provide the use of a Thraustochytridaceae microalgae extract as a culture medium supplement capable of promoting cell proliferation and survival while replacing animal serum.

[0012] Technical Solution

[0013] As one aspect to achieve the above object, the present invention relates to a medium composition for cell culture, comprising a Thraustochytridaceae microalgae extract.

[0014] As another aspect, the present invention relates to a culture medium supplement for cell culture, comprising a Thraustochytridaceae microalgae extract.

[0015] As another aspect, the present invention relates to a culture medium for cell culture, comprising the culture medium supplement.

[0016] As a further aspect, the present invention relates to a method for culturing cells, comprising culturing cells in the culture medium.

[0017] As another aspect, the present invention relates to use of a Thraustochytridaceae microalgae extract as a culture medium additive capable of promoting cell proliferation and survival while replacing animal serum.

[0018] Hereinafter, the present invention will be described in more detail.

[0019] The present invention provides a culture medium composition for cell culture, comprising a Thraustochytridaceae microalgae extract.

[0020] In the description herein, the term "cell culture" refers to a process of artificially culturing living cells in vitro under regulated conditions. In addition, it is possible to grow and proliferate these cells by aseptically removing a portion of individual tissues and applying the suspension released by degrading the connecting substances between cells with enzymes on the flat bottom of a culture dish such as a bottle or a petri dish.

[0021] Cell culture may include culturing cells to prepare cell cultured meat. Therefore, the composition may be a medium composition for preparing cultured meat. The medium composition of the present invention may contain a thraustochytridaceae microalgae extract instead of generally used fetal bovine serum. The thraustochytridaceae microalgae extract is a substance rich in nutrients such as protein and amino acids, and stability problems can be eliminated, and excellent productivity can be expected because of its low production cost compared to fetal bovine serum.

[0022] In the description herein, the term "cultured meat" refers to edible meat to be obtained by collecting animal cells and then proliferating them through cell engineering technology, and it can be described as a field of cell agriculture in which meat is obtained without the process of raising livestock. In Korean, it is called cultured meat, meat analogs, or artificial meat, and in English, it is also called in vitro meat (meaning grown in vitro), artificial meat (meaning synthesized by humans using stem cells), non-natural clean meat (meaning produced in a clean production facility rather than a traditional breeding facility), and lab-grown meat (because it is produced in a laboratory).

[0023] In the description herein, the term "culture medium" refers to a matrix that enables the growth, survival and differentiation of supporting cells or stem cells in vitro, and includes all common culture media used in the art that are suitable for culturing and differentiating cells or stem cells. The type of culture medium and the culture conditions can be selected based on the type of cells at the technical level in the corresponding technical field. The culture medium used for cultivation is particularly a cell culture minimum medium (CCMM), and it generally contains a carbon source, a nitrogen source and trace element components. The cell culture minimum medium can be, for example, at least one selected from the group consisting of DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimum Essential Medium), BME (Basal Medium Eagle), RPMI 1640, F-10, F-12, DMEM / F12, α-MEM (α-Minimum Essential Medium), G-MEM (Glasgow's Minimum Essential Medium), IMDM (Iscove's Modified Dulbecco's Medium), MacCoy's 5A Medium, AmnioMax Complete Medium, AminoMax± Medium, EBM (Endothelial Basal Medium) Medium, Chang's Medium, MesenCult-XF, DMEM / HG (Dulbecco's Modified Eagle's Medium High Glucose) Medium, and MCDB+DMEM / LG (MCDB+Dulbecco's Modified Eagle's Medium Low Glucose) Medium. In addition, the medium may contain antibiotics such as penicillin, streptomycin, gentamicin, or a mixture of two or more thereof, or the like. Therefore, the medium composition for cell culture of the present invention may include a Thraustochytridaceae microalgae extract added to a minimum medium for cell culture.

[0024] In the description herein, the term "Thraustochytridaceae microalgae" refers to a heterotrophic microorganism belonging to the order Thraustochytriales, and is known as an oleaginous microorganism capable of producing bio-oil containing unsaturated fatty acids such as DHA (docosahexaenoic acid), and is considered a good protein source because it is rich in protein and amino acids.

[0025] The Thraustochytriaceae microalgae may be any genus available to those skilled in the art. In particular, it may be any one selected from the group consisting of genus Schizochytrium, genus Aurantiochytrium, genus Thraustochytrium, and genus UIkenia, but is not limited thereto.

[0026] In the description herein, the term "extract" may refer to the resulting product, such as a liquid fraction obtained by immersing the target substance in various solvents and then extracting it at room temperature or an elevated temperature state for a certain period of time, and a solid obtained by removing the solvent from the liquid component, etc. In addition, in addition to the resulting product, it may be comprehensively understood to include the extracted solution itself, such as a diluted solution of the resulting product, a concentrate thereof, a crude purified substance, a purified substance or a mixture thereof, etc., and an extract in the form of all preparations that can be formed using the extracted solution. In addition, the extract may include fractions obtained therefrom.

[0027] The extract may be extracted from native, hybrid or variant Thraustochytridaceae microalgae, and it may be extracted from tissue culture of Thraustochytridaceae microalgae.

[0028] The extraction solvent of 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. The non-polar solvent excludes benzene. The solvent can be a C1-C6 alcohol, a C3-C10 ester, such as 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 at least one of these solvents and water. The solvent can be ethanol, propanol, acetonitrile, ethyl acetate, acetone, 2-butanone, chloroform, dichloromethane, hexane, a mixture thereof or a mixture of at least one of these solvents and water. The hydrocarbon can be an alkane, an alkene or an alkyne.

[0029] The extract may be extracted with at least one solvent selected from the group consisting of: water; C1 to C4 lower alcohols such as methanol, ethanol, propanol, butanol, etc.; polyols such as glycerol, butanediol, propylene glycol, etc.; and hydrocarbon solvents such as methyl acetate, ethyl acetate, acetone, benzene, hexane, diethyl ether, dichloromethane, etc. In particular, the extract may be extracted with water as a solvent.

[0030] The Thraustochytridaceae microalgae extract may be extracted from the Thraustochytridaceae microalgae culture solution itself, and may be extracted after being prepared into biomass by freeze-drying or dehydrating the culture solution with a centrifuge and then introducing other drying processes using freeze drying, drum drying, spray drying, a granulator, etc. (but not limited thereto).

[0031] In the description herein, the term "biomass" may be the Thraustochytridaceae microalgae itself, a culture thereof, a dry matter thereof, a lysate thereof, or a product produced by culturing or fermenting the microalgae, or it may be a concentrate or dry matter of the biomass, but is not limited thereto.

[0032] The thraustochytriaceae microalgae extract may be used in the form of thraustochytriaceae microalgae or thraustochytriaceae microalgae biomass, which is extracted by a common method and, for example, an extraction method such as hot water extraction, cold extraction, reduced pressure high temperature extraction, boiling water extraction, room temperature extraction, fraction extraction, reflux cooling extraction, solvent extraction, steam distillation, ultrasonic extraction, blasting, compression, etc., and preferably, it may be extracted using hot water extraction. In addition, if necessary, a commercially available thraustochytriaceae microalgae extract may be used as the thraustochytriaceae microalgae extract.

[0033] The hot water extraction may 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 embodiment, it may be performed at 110° C., but is not limited thereto. In addition, the hot water extraction may 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 In some embodiments, the present invention relates to a method for preparing the granulated powder or the like. The granulated powder or the like may be carried out for 1 to 20 minutes, 2 to 20 minutes, 3 to 20 minutes, 4 to 20 minutes, 5 to 20 minutes, 6 to 20 minutes, 7 to 20 minutes, 8 to 20 minutes, 9 to 20 minutes, 1 to 15 minutes, 2 to 15 minutes, 3 to 15 minutes, 4 to 15 minutes, 5 to 15 minutes, 6 to 15 minutes, 7 to 15 minutes, 8 to 15 minutes or 9 to 15 minutes, and in a specific embodiment, it can be carried out for 10 minutes, but is not limited thereto.

[0034] In the description herein, the term "fraction" refers to a resultant product obtained by performing fractionation to separate a specific component or a group of specific components from a mixture containing various components.

[0035] The fractionation method for obtaining these fractions is not particularly limited and can be performed according to methods generally used in the art. Non-limiting examples of the fractionation method include a fractionation method by treating various solvents, an ultrafiltration fractionation method by passing through an ultrafiltration membrane having a certain molecular weight cutoff value, a chromatographic fractionation method by performing various chromatography (generated for separation based on size, charge, hydrophobicity or affinity), and combinations thereof, etc.

[0036] The type of fractionation solvent used to obtain these fractions is not particularly limited, and any solvent known in the art can be used. Non-limiting examples of fractionation solvents may include polar solvents such as water, alcohols having 1 to 4 carbon atoms, etc.; non-polar solvents such as hexane, ethyl acetate, chloroform, dichloromethane, etc.; or mixed solvents thereof. They may be used alone or in combination of one or more, but are not limited thereto.

[0037] The Thraustochytridaceae microalgae extract may be included in a medium composition for cell culture at a concentration of 25 g / L or less, specifically, at a concentration of 23.6 g / L or less. For example, the concentration can be 0.001 to 25 g / L, 0.005 to 25 g / L, 0.01 to 25 g / L, 0.05 to 25 g / L, 0.1 to 25 g / L, 0.2 to 25 g / L, 0.4 to 25 g / L, 0.8 to 25 g / L, 1 to 25 g / L, 2 to 25 g / L, 3 to 25 g / L, 0.001 to 23.6 g / L, 0.005 to 23.6 g / L, 0.01 to 23.6 g / L, 0.05 to 23.6 g / L, 0.1 to 23.6 g / L, 0.2 to 23.6 g / L, 0.4 to 23.6 g / L, 0.8 to 23.6 g / L, 1 to 23.6 g / L, 2 to 23.6g / L, 3 to 23.6g / L, 0.001 to 22g / L, 0.005 to 22g / L, 0.01 to 22g / L, 0.05 to 22g / L, 0.1 to 22g / L, 0.2 to 22g / L, 0.4 to 22g / L, 0.8 to 22g / L, 1 to 22g / L, 2 to 22g / L, 3 to 22g / L, 0.001 to 20g / L, 0.005 to 20g / L, 0.01 to 20g / L, 0.05 to 20g / L, 0.1 to 20g / L, 0.2 to 20g / L, 0.4 to 20g / L, 0.8 to 20g / L, 1 to 20g / L, 2 to 20g / L, 3 to 20 g / L, 0.001 to 15g / L, 0.005 to 15g / L, 0.01 to 15g / L, 0.05 to 15g / L, 0.1 to 15g / L, 0.2 to 15g / L, 0.4 to 15g / L, 0.8 to 15g / L, 1 to 15g / L, 2 to 15g / L, 3 to 15g / L, 0.001 to 12g / L, 0.005 to 12g / L, 0.01 to 12g / L, 0.05 to 12g / L, 0.1 to 12g / L, 0.2 to 12g / L, 0.4 to 12g / L, 0.8 to 12g / L, 1 to 12g / L, 2 to 12g / L, 3 to 12g / L, 0.001 to 11g / L, 0.005 to 11 g / L, 0.01 to 11 g / L, 0.05 to 11 g / L, 0.1 to 11 g / L, 0.2 to 11 g / L, 0.4 to 11 g / L, 0.8 to 11 g / L, 1 to 11 g / L, 2 to 11 g / L, 3 to 11 g / L, 0.001 to 10 g / L, 0.005 to 10 g / L, 0.01 to 10 g / L, 0.05 to 10 g / L, 0.1 to 10 g / L, 0.2 to 10 g / L, 0.4 to 10 g / L, 0.8 to 10 g / L, 1 to 10 g / L, 2 to 10 g / L, 3 to 10 g / L, 0.001 to 8 g / L, 0.005 to 8 g / L, 0.01 to 8g / L, 0.05 to 8g / L, 0.1 to 8g / L, 0.2 to 8g / L, 0.4 to 8g / L, 0.8 to 8g / L, 1 to 8g / L, 2 to 8g / L, 3 to 8g / L, 0.001 to 5g / L, 0.005 to 5g / L, 0.01 to 5g / L, 0.05 to 5g / L, 0.1 to 5g / L, 0.2 to 5g / L, 0.4 to 5g / L, 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 in particular, it may be 0.8 to 4 g / L. .

[0038] The cell can be a somatic cell, a germ cell, a stem cell, a cancer cell, a cell line, a cultured cell (in vitro), a transplanted cell, and a primary cultured cell (in vitro and in vitro), as well as an in vivo cell, a cell of a eukaryotic organism, or a mammalian cell (including a human being), and for example, the cell can be selected from the group consisting of a cancer cell, a stem cell, a vascular endothelial cell, a leukocyte, an immune cell, an epithelial cell, a germ cell, a fibroblast, a muscle cell, a bone marrow cell, an epidermal cell, an osteoblast, and a neural cell. In addition, the eukaryotic organism can be an insect, an amphibian, a bird (chicken, turkey, duck, etc.), or a mammal (primates such as humans, monkeys, etc., dogs, pigs, cattle, sheep, goats, mice, rats, etc.).

[0039] The cell may be a cell isolated from an individual or a eukaryotic organism.

[0040] The cell may be an undifferentiated cell, a differentiating cell, or a fully differentiated cell, and in particular, it may be a stem cell.

[0041] In the description herein, the term "stem cell" refers to a cell with potential and self-renewal ability. Stem cells are divided into pluripotent, multipotent or unipotent according to their differentiation ability. Stem cells can be at least one selected from the group consisting of embryonic stem cells (ESC) (preimplantation embryonic cells), adult stem cells (undifferentiated cells present in various tissues and organs) and induced pluripotent stem cells (iPSC) (wherein dedifferentiated cells are induced by inserting genes and / or proteins into somatic cells, or induced pluripotent stem cells).

[0042] The type and source of stem cells are not limited, as long as they have potential and self-renewal ability. Stem cells can be derived from, for example, mammals, humans, monkeys, pigs, horses, cattle, sheep, dogs, cats, mice or rabbits. Stem cells can be derived from separated umbilical cord, placenta, fat, bone marrow, muscle, umbilical cord blood or amniotic fluid. The term "separated" refers to being present in an environment different from the environment of the cell or tissue. Separation of umbilical cord, placenta, fat, bone marrow, muscle, umbilical cord blood or amniotic fluid and obtaining stem cells can be carried out by conventional dissection methods and known methods.

[0043] The cell may be a muscle cell or a myogenic stem cell, and may particularly be derived from livestock such as chicken, cattle and pigs, and more particularly may be a muscle cell or a myogenic stem cell derived from cattle or pigs.

[0044] In the description herein, the term "myogenic stem cell" refers to a cell having the characteristics of myogenic stem cells, including proliferation without transformation, unlimited proliferation, self-reproduction ability and the ability to differentiate into muscle, and may include any cell showing self-reproduction ability or unlimited proliferation ability and muscle differentiation ability without limitation. The self-reproduction ability and muscle differentiation ability can be confirmed with markers. Myogenic stem cells can be used interchangeably with satellite cells (SC).

[0045] According to one example, it has been confirmed that a culture medium composition comprising an extract of Thraustochytridaceae microalgae shows significantly excellent cell proliferation efficiency in myogenic stem cells.

[0046] The composition may further comprise serum. The serum may be at least one selected from the group consisting of fetal bovine serum (FBS), calf calf serum (BCS), human serum and horse serum (HS), and in particular, it may be fetal bovine serum.

[0047] The composition may include serum and a Thraustochytriaceae microalgae extract, and the serum may be included at a weight ratio of 1000:1 to 1:1 based on the Thraustochytriaceae microalgae extract (serum:Thraustochytriaceae microalgae extract). For example, the weight ratio of serum to chlorella extract can 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.

[0048] The culture composition allows subculture of cells.

[0049] In the description herein, the term "passage" refers to replacing a culture container or culturing by separating a cell group, which is a method of culturing by continuous cell generation, in order to continuously culture cells in a healthy state for a long time, especially stem cells. Replacing a culture container once or culturing by separating a cell group is called one passage. In the present invention, passage can be used interchangeably with generation.

[0050] Cultivation can be growth and proliferation. In the description herein, the term "growth and proliferation" refers to the increase in the number of cells. Cultivation can be undifferentiated proliferation. Undifferentiated proliferation refers to the proliferation of stem cells into cells with the same characteristics (i.e., potential and self-renewal) as the original cells, without differentiation into specific cells. The term "differentiation" refers to a phenomenon in which cells become specialized in structure or function, while they divide and proliferate and grow, i.e., change the form or function of cells, tissues, etc. of living organisms to carry out their respective tasks. The degree of differentiation measured or determined to a specific cell type can be carried out by methods well known in the art. In addition, differentiation can be confirmed by the following: using an optical microscope or confocal microscope to study cell form, while using methods such as flow cytometry or immunocytochemistry to measure cell surface markers (e.g., staining cells with tissue-specific or cell tag-specific antibodies) and changes in cell form (e.g., nuclear ratio / cytoplasm ratio) or using methods well known in the art such as polymerase chain reaction (PCR) and gene expression profiles to measure changes in gene expression.

[0051] In addition, the present invention provides a culture medium additive for cell culture, comprising a Thraustochytridaceae microalgae extract.

[0052] The same parts as those described above are actually applicable to this method.

[0053] The medium supplement can replace all or part of the animal serum contained in the medium, so the medium for cell culture containing the medium supplement can have a reduced serum content compared to a general medium (containing about 10% serum). In addition, the low-serum medium containing the medium supplement can show cell growth rate, cell viability and subculture efficiency that are equal to or significantly superior to that of a medium containing 10% serum.

[0054] Furthermore, the present invention provides a method for cell culture, which comprises culturing the separated cells using the medium composition for cell culture.

[0055] The same parts as those described above are actually applicable to this method.

[0056] These isolated cells may be cells isolated from livestock, and in particular may be cells isolated from livestock such as cows, pigs or chickens.

[0057] The method for cell culture can be a method for culturing cells to prepare cultured meat, and in particular can be a method for proliferating and culturing myogenic stem cells to prepare cultured meat. Therefore, cultured meat can be prepared using cells cultured by this method, in particular myogenic stem cells.

[0058] The culture form can be a general two-dimensional or three-dimensional culture known in the art. However, three-dimensional culture may be preferred to achieve an organization similar to actual biological tissue through cell-to-cell interaction. Specific examples of three-dimensional culture include methods for arranging cells in a 3D porous scaffold, a scaffold-free platform or a microchip by cells themselves or cell sheet technology, methods using hydrogels, and methods using bioreactors.

[0059] In addition, the present invention provides a use of a Thraustochytridaceae microalgae extract as a culture medium additive capable of promoting cell proliferation and survival while replacing animal serum.

[0060] In the use of the present application, the Thraustochytridaceae microalgae, culture medium additives, etc. are as described above.

[0061] Beneficial effects

[0062] It has been confirmed that when bovine muscle cells are cultured in a medium containing the extract extracted from the biomass of microalgae belonging to the Thraustochytridaceae family of the present invention, the cell proliferation ability of the muscle cells is improved, and therefore the composition for cell culture containing the microalgae extract can be used by replacing fetal bovine serum in the medium components for cell culture. In addition, the microalgae extract contained in the composition for cell culture of the present invention undergoes a production process based on fermentation, so it is free from various ethical issues and pollution issues unlike the conventional production process of fetal bovine serum, and has the advantage that continuous quality production is possible while maintaining more consistent quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 This is a graph showing the cytotoxicity and cell proliferation effects of the Thraustochytridaceae microalgae extract on bovine myogenic stem cells. They are expressed as mean values ​​(N=3)±standard deviations.

[0064] Figure 2This is a graph showing the effect of a culture solution containing FBS and various concentrations of a thraustochytriaceae microalgae extract on the cell proliferation of bovine myogenic stem cells. It is presented as the mean (N=3) ± standard deviation, and statistical comparisons are made between cells cultured in a medium containing a thraustochytriaceae microalgae extract and cells cultured for the same culture period without a thraustochytriaceae microalgae extract. (*p<0.05, **p<0.01, ***p<0.001, Student's T test) DETAILED DESCRIPTION

[0065] Hereinafter, the present invention will be described in more detail by way of examples. However, these examples are intended to illustratively describe at least one specific embodiment, and the scope of the present invention is not limited by these examples.

[0066] Example 1. Obtaining microalgae extract

[0067] A Schizochytrium sp. strain belonging to the Thraustochytriaceae family of microalgae was dark-cultured and then dried to obtain a biomass.

[0068] Specifically, the Schizochytrium species strain was fermented in a 5L fermenter containing a sterilized MJW01 medium (glucose 30 g / L, MgSO4·7H2O 3.0 g / L, Na2SO4 10 g / L, NaCl 1.0 g / L, yeast extract 9.0 g / L, MSG·1H2O 1.0 g / L, NaNO3 1.0 g / L, KH2PO4 0.1 g / L, K2HPO4 0.5 g / L, CaCl2 0.5 g / L, vitamin mixed solution 10 ml / L). The microbial cell culture solution was freeze-dried directly or freeze-dried after dehydration by a centrifuge to obtain biomass. The obtained microalgae biomass was dispersed in pure water at a concentration of 1.5%, and then heated at 110°C for 10 minutes for hot water extraction. The extracted solution was filtered to remove the remaining floating matter, and then freeze-dried to obtain a thraustochytrid microalgae extract. The individual amino acid contents and the total amino acid content of the obtained thraustochytrid microalgae extract were measured on a dry weight basis and are shown in Table 1 below.

[0069] Table 1

[0070] Types of Amino Acids Amino acid content based on dry weight (%) Asp 3.19 Thr 1.17 Ser 1.10 Glu 24.58 Gly 1.59 Ala 1.75 Cys 0.31 Val 1.92 Met 0.57 Ile 1.13 Leu 1.96 Tyr 0.91 Phe 1.25 Lys 1.91 His 0.64 Arg 7.45 Pro 0.74 Total amino acids 52.17

[0071] As a result, as shown in Table 1, it was confirmed that the total amino acid content based on the dry weight of the obtained thraustochytrid microalgae extract was 52.17%, and it was confirmed that the content of glutamic acid was the highest, and the amino acid content was high in the order of arginine (Arg) and aspartic acid (Asp).

[0072] Example 2. Confirmation of cytotoxicity of microalgae extracts

[0073] To confirm the effect of the Thraustochytrid microalgae extract on bovine myogenic stem cells (bMuSCs) extracted from shank and loin, a WST-8 test was performed as follows.

[0074] Specifically, cells with a subculture number of 3 or 5 were seeded in a 96-well culture dish at 5,000 cells per well. Three wells were inoculated for each condition and cultured for one day at 37°C and 5% CO2. Afterwards, a new culture solution containing 10% FBS and 0.001 g / L to 40 g / L of a thraustochytrid extract was replaced and cultured for 3 days, and then each well was treated with 5 μL of WST-8 and cultured for an additional 3 hours. The metabolic activity of the cells was determined by measuring the absorbance (OD450) at a wavelength of 450 nm using a microplate spectrophotometer. Wells containing culture solution but no cells were used as blank controls, and wells not treated with thraustochytrid extracts were used as negative controls, and relative cell viability was compared by comparing the OD450 values ​​of cells treated with thraustochytrid extracts. The concentration of thraustochytrid extract that showed the highest viability was named E. max , and CC when its cell viability was 50% of that of the negative control group 50 The value is using the Quest Graph TM The results of measuring cell viability based on the concentration of the thraustochytrid extract measured by this experiment are shown in Figure 1 Displayed in.

[0075] As a result, Figure 1 The results showed that when the thraustochytrid extract was included in the concentration range of 0.001 to 2 g / L, the cell proliferation of bMuSC was similar to that of the control group. When the thraustochytrid extract was included in the concentration of 4 g / L, the highest cell viability was shown to be increased by 115.8% compared to the control group in the concentration range used in the experiment, and based on this, it was confirmed that the concentration of 4 g / L was the best. max In addition, confirm that CC 50 The value is 23.6g / L.

[0076] In summary, when the thraustochytrid extract was included in the culture solution at a concentration of 4 g / L or less, no toxicity to cells was shown, and in particular, it was seen that when the thraustochytrid extract was included in the culture solution at the above concentration, excellent cell proliferation was observed in Korean native livestock muscle cells (bMuSC).

[0077] Example 3. Confirmation of serum replacement effect of Thraustochytrid extract

[0078] In order to confirm the serum replacement effect of the Thraustochytrid extract, the following experiment was performed.

[0079] Specifically, the WST-8 test was performed to confirm that the addition of E max What is the effect of the concentration of thraustochytrid extract in the culture solution on bMuSC cells under the culture condition of reduced fetal bovine serum (FBS). Bovine muscle cells with a subculture number of 5, 6 or 15 were seeded in 3 wells of a 96-well culture plate at a condition of 5,000 cells per well. After culturing for one day at 37°C and 5% CO2, half of the culture solution was replaced with a culture solution containing FBS at a concentration of 0% to 10% and thraustochytrid extract at a concentration of 0g / L to 4g / L, and the weight ratio of FBS to thraustochytrid extract added was as shown in Table 2 below.

[0080] Table 2

[0081]

[0082] Cultivation was performed for one day to allow cells to adapt to the new culture solution, and then the new culture solution was replaced once again, and the WST-8 test was performed on days 3 and 6 after the replacement time. Cell proliferation on days 3 and 6 was determined by comparing the measured OD450 value with the OD450 value measured on day 0, and the measured results were compared in Table 1. Figure 2 and are shown in Table 3 below.

[0083] Table 3

[0084]

[0085] As a result, Figure 2 As shown in Table 3, it was confirmed that when bMuSC cells were cultured in a medium containing a thraustochytrid extract, cell proliferation was increased. max When the thraustochytrid extract was included at a concentration of 10 g / L, the cell proliferation rate increased by 26.9% under the 10% FBS condition compared to the control group not treated with the thraustochytrid extract, and it increased by 74.0% under the 5% FBS condition, and increased by 127.4% under the 2% FBS condition, based on the culture results on the 6th day. Under the 5% FBS condition, the cell proliferation was shown to be at an equal or higher level than that of the 10% FBS control group. Therefore, it can be confirmed that the thraustochytrid extract can induce the proliferation of bMuSC cells by replacing FBS, and in particular, it can be seen that when the thraustochytrid extract is used at 4 g / L E max Concentrations including thraustochytrid extract showed 50% to 80% FBS replacement.

Claims

1. A culture medium composition for cell culture, comprising a Thraustochytridaceae microalgae extract. 2 . The medium composition for cell culture according to claim 1 , comprising the Thraustochytridaceae microalgae extract at a concentration of 23.6 g / L or less. 3 . The medium composition for cell culture according to claim 1 , wherein the Thraustochytridaceae microalgae extract is contained at a concentration of 0.4 g / L to 10 g / L or less.

4. The medium composition for cell culture according to claim 1, wherein the Thraustochytridaceae microalgae is any one selected from the group consisting of Schizochytrium species, Aurochrysum species, Thraustochytrium species, and Ukenichthys species. 5 . The medium composition for cell culture according to claim 1 , wherein the Thraustochytridaceae microalgae extract has a total amino acid content of 40% or more based on dry weight. The medium composition for cell culture according to claim 1 , wherein the cells are myogenic stem cells or muscle cells. The medium composition for cell culture according to claim 1 , wherein the cells are cells derived from bovine or porcine. The medium composition for cell culture according to claim 1 , wherein the composition further comprises serum. 9 . The medium composition for cell culture according to claim 8 , wherein the serum is any one or more selected from the group consisting of fetal bovine serum (FBS), calf serum (BCS), human serum and horse serum (HS). 10 . The medium composition for cell culture according to claim 8 , wherein the serum is included in a weight ratio of 1000:1 to 1:1 (serum:Thraustochytriaceae microalgae extract) based on the Thraustochytriaceae microalgae extract.

11. A medium supplement for cell culture, comprising an extract of Thraustochytridaceae microalgae. 12 . A method for cell culture, comprising culturing isolated cells using the medium composition for cell culture according to claim 1 .

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