Medium composition for culturing animal cells comprising d-chiro inositol or derivative thereof

Abstract

The present specification relates to: a medium composition for culturing animal cells, comprising D-chiro inositol (DCI) or a derivative thereof; and a method for culturing animal cells using same. Cells cultured in the medium composition can achieve excellent improvement in cell proliferation ability and reduction in lactic acid, which is a by-product in a culture medium.

Description

A culture medium composition for animal cells comprising D-chiroinositol or a derivative thereof

[0001] This specification provides a culture medium composition for animal cells comprising D-chiro-inositol (D-chiro-inositol / DCI) or a derivative thereof, and a method for culturing animal cells using the same. The present invention relates to a specific research task, and the information regarding the research task is as follows:

[0002] Assignment No.: 20026699

[0003] Ministry Name: Ministry of Trade, Industry and Energy

[0004] Name of Project Management Agency: Korea Institute of Industrial Technology Planning and Evaluation

[0005] Research Project Name: Material and Component Technology Development - Material Component Package Type

[0006] Research Project Title: Development of Customized Animal-Derived Component Substitute Culture Media Technology for Clinical Mesenchymal Stem Cell Therapeutics

[0007] Contribution rate: 100%

[0008] Project Implementing Organization Name: Amikogen

[0009] Research Period: 2023.09.01~2026.12.31

[0010] Animal cell culture is one of the core technologies of modern life science research and industry, including the manufacturing of pharmaceuticals and the development of cell therapies. In particular, large-scale animal cell culture is essential for the production of protein-based drugs, vaccines, and cell therapies; currently, BME media, or media based on it such as DMEM and MEM, are widely used for animal cell culture.

[0011] The culture medium composition required for animal cell culture includes various nutrients, growth factors, and auxiliary substances to enhance cell growth, proliferation, and productivity. It is important to develop an appropriate composition that does not exhibit cytotoxicity and does not reduce culture efficiency, in order to achieve high cell growth and proliferation rates and growth rates.

[0012]

[0013] Against the background described above, the inventors completed the present invention by incorporating D-chiro-inositol (DCI) or a derivative thereof into feed media, confirming the effect of reducing lactate, a byproduct in the culture medium, and improving animal cell productivity.

[0014] One example of the present specification provides a culture medium composition comprising D-chiro inositol (D-chiro inositol / DCI) or a derivative thereof.

[0015] The above-mentioned culture medium composition may be used for the culture of animal cells.

[0016] Another example provides a method for culturing cells, comprising the step of culturing cells in the above-mentioned medium composition.

[0017] The above cell may be an animal cell.

[0018] One example of the present specification provides a culture medium composition for animal cells comprising D-chiro inositol (D-chiro inositol / DCI) or a derivative thereof.

[0019] One example of the present specification provides a cell culture medium composition for cultured meat comprising D-chiro-inositol (D-chiro inositol / DCI) or a derivative thereof.

[0020] One example of the present specification provides a medium composition for stem cell culture comprising D-chiro-inositol (D-chiro inositol / DCI) or a derivative thereof.

[0021] One example of the present specification provides a culture medium composition for immune cells comprising D-chiro-inositol (D-chiro inositol / DCI) or a derivative thereof.

[0022] Another example provides a method for culturing animal cells, comprising the step of culturing animal cells in the above-mentioned culture medium composition.

[0023] Another example provides the use of D-chiro-inositol (D-chiro inositol / DCI) or its derivatives in animal cell culture.

[0024] Another example provides the use of D-chiro inositol (D-chiro inositol / DCI) for preparing compositions for animal cell culture.

[0025]

[0026] The present invention will be described in more detail below.

[0027]

[0028] Composition for animal cell culture

[0029] One example of the present specification provides a culture medium composition for animal cells comprising D-chiro inositol (D-chiro inositol / DCI) or a derivative thereof.

[0030] In this specification, "culture" refers to the process of maintaining or proliferating a specific organism (e.g., cells (animal cells), microorganisms, tissues, etc.) in an appropriate environment to induce survival, growth, proliferation, or the production of specific substances. The culture process may be carried out by controlling various conditions, such as temperature, pH, oxygen concentration, and nutrient concentration, to optimize the metabolic activity of the organism. The culture may include fed-batch culture, batch culture, continuous culture, repeated fed-batch culture, etc. "Fed-batch culture" refers to a culture method that maximizes cell growth and productivity by supplying a fixed amount or gradually of nutrients necessary for cell growth during culture without removing the culture medium, thereby enabling efficient cell culture by reducing the accumulation of metabolic products and nutrient depletion within the culture medium.

[0031] In this specification, "D-chiro inositol (D-chiro inositol / DCI)" refers to a cyclohexanehexol compound having a D-chiro stereoconfiguration as one of the stereoisomers of inositol. In this specification, unless specifically defined otherwise, D-chiro inositol includes anhydrous or hydrated forms, crystalline or amorphous forms, and solvates; and may also be used to mean acceptable salts of D-chiro inositol, various forms considering the in vivo / in vitro forms of D-chiro inositol (e.g., phosphorylated forms), isomers of D-chiro inositol, or mixtures thereof. In this specification, "derivatives of D-chiro-inositol" means compounds in which a part of the chemical structure of D-chiro-inositol is substituted, added, deleted, or chemically modified, but which maintain or improve the intrinsic activity of D-chiro-inositol (e.g., enhancement of cell culture efficiency) to a substantially equivalent level. The derivatives of D-chiro-inositol may be in a methylated, phosphorylated, and / or acylated form of D-chiro-inositol, and may be salts, esters, ethers, and / or pharmaceutically acceptable forms thereof, but are not limited thereto.

[0032] In this specification, "animal cell culture" refers to the process of culturing cells derived from animals in an artificial environment for the purpose of survival, growth, proliferation, or the production of specific substances, and "animal cells" include cells derived from mammals, birds, fish, or other vertebrates and invertebrates, and specifically may include adhesion-dependent cells (e.g., fibroblasts, muscle cells, etc.), suspension cells (e.g., blood cells, etc.), cultured meat cells (e.g., muscle cells, fat cells, or related cells, etc.), stem cells (e.g., embryonic stem cells, adult stem cells, induced pluripotent stem cells (iPSCs), etc.), and immune cells (e.g., T cells, NK cells, dendritic cells, etc.). Specifically, the animal cell culture may include cultured meat (cultured meat cells), stem cell culture, immune cell culture, etc. The above "cells for cultured meat" refers to animal-derived cells that are cultured, proliferated, and / or differentiated in vitro to produce cultured meat.

[0033] In this specification, animal cells refer to cells derived from vertebrates or invertebrates, and may mean unit cells or cell populations capable of being cultured in an external environment, or cells derived from multicellular animals, such as mammals.

[0034] The above animal cells are hamster-derived cells (e.g., CHO (Chinese Hamster Ovary) cells, CHO-K1, CHO-DXB11, BHK cells (Baby Hamster Kidney Cell), GS gene-defective CHO cells, DHFR gene-defective CHO cells, etc.), mouse-derived cells (e.g., COP cells (Copulatory Organ Progenitor Cell), L cells (L Fibroblast Cell), C127 cells (C127 Mouse Mammary Gland Cell), Sp2 / 0 cells (Sp2 / 0-Ag14 Myeloma Cell), NS-0 cells (Non-Secreting 0 Myeloma Cell), NS-1 cells (Non-Secreting 1 Myeloma Cell), AtT-20 cells (AtT-20 Mouse Pituitary Tumor Cell), NIH3T3 cells (National Institutes of Health 3-Day Transfer, Inoculum 3 Fibroblast), etc.), rat-derived cells (e.g., PC12 cells) (Pheochromocytoma Cell), PC12h cells (PC12 Highly Differentiated Cell), GH3 cells (Growth Hormone Cell Line 3), MtT (Mouse Thyrotropic Tumor) cells, etc.), monkey-derived cells (e.g., COS1 cells (CV-1 Origin, SV40-Transformed Cell Line 1), COS3 cells (CV-1 Origin, SV40-Transformed Cell Line 3), COS7 cells (CV-1 Origin, SV40-Transformed Cell Line 7), CV1 cells (Cercopithecus aethiops Vero Kidney Cell Line), Vero cells), and human-derived cells (e.g., HeLa cells (Henrietta Lacks Cell Line),It may be one or more selected from the group consisting of HEK-293 cells (Human Embryonic Kidney 293 Cell), retinal-derived PER-C6, cells derived from diploid fibroblasts, myeloma cells, HepG2 cells, etc., but is not limited thereto.

[0035] In this specification, the expressions "comprising (a specified component)" or "consisting of (a specified component)" mean that the described component is essentially included, and may not exclude the inclusion of additional and / or auxiliary components as long as they produce an effect equivalent to the intended effect.

[0036] In this specification, "medium" may refer to a liquid or gel-like (solid or semi-solid) mixture containing components necessary for the growth and proliferation of cells or microorganisms in vitro. The medium may further include cells (animal cells) to be cultured.

[0037] In this specification, the term “about” is an expression intended to include all ranges of figures equivalent to or similar to the following figures, and may be interpreted as including, but not limited to, ranges of ±20%, ±15%, ±10%, ±5%, ±4%, ±3%, ±2%, or ±1% based on the following figures.

[0038] The above composition comprises DCI or a derivative thereof in an amount of 0.01 to 10,000 mg / L, 0.01 to 5,000 mg / L, 0.01 to 2,000 mg / L, 0.01 to 1,500 mg / L, 0.01 to 1,000 mg / L, 0.01 to 500 mg / L, 0.01 to 200 mg / L, 0.01 to 150 mg / L, 0.01 to 100 mg / L, 0.01 to 50 mg / L, 0.01 to 10 mg / L, 0.01 to 5 mg / L, 0.01 to 2 mg / L, 0.01 to 1 mg / L, 0.01 to 0.5 mg / L, 0.01 to 0.2 mg / L, 0.01 based on the total volume of the composition. to 0.1 mg / L, 0.05 to 10,000 mg / L, 0.05 to 5,000 mg / L, 0.05 to 2,000 mg / L, 0.05 to 1,500 mg / L, 0.05 to 1,000 mg / L, 0.05 to 500 mg / L, 0.05 to 200 mg / L, 0.05 to 150 mg / L, 0.05 to 100 mg / L, 0.05 to 50 mg / L, 0.05 to 10 mg / L, 0.05 to 5 mg / L, 0.05 to 2 mg / L, 0.05 to 1 mg / L, 0.05 to 0.5 mg / L, 0.05 to 0.2 mg / L, 0.05 to 0.1 mg / L, 0.08 to 10,000 mg / L, 0.08 to 5,000 mg / L, 0.08 to 2,000 mg / L, 0.08 to 1,500 mg / L, 0.08 to 1,000 mg / L, 0.08 to 500 mg / L, 0.08 to 200 mg / L, 0.08 to 150 mg / L, 0.08 to 100 mg / L, 0.08 to 50 mg / L, 0.08 to 10 mg / L, 0.08 to 5 mg / L, 0.08 to 2 mg / L, 0.08 to 1 mg / L, 0.08 to 0.5 mg / L, 0.08 to 0.2 mg / L, 0.08 to 0.1 mg / L, 0.1 to 10,000 mg / L, 0.1 to 5,000 mg / L, 0.1 to 2,000 mg / L, 0.1 to 1,500 mg / L, 0.1 to 1,000 mg / L, 0.1 to 500 mg / L, 0.1 to 200 mg / L, 0.1 to 150 mg / L, 0.1 to 100 mg / L, 0.1 to 50 mg / L, 0.1 to 10 mg / L, 0.1 to 5 mg / L, 0.1 to 2 mg / L, 0.1 to 1 mg / L, 0.1 to 0.5 mg / L, 0.1 to 0.2 mg / L, 0.5 to 10,000 mg / L, 0.5 to 5,000 mg / L, 0.5 to 2,000 mg / L, 0.5 to 1,500 mg / L, 0.5 to 1,000 mg / L, 0.5 to 500 mg / L, 0.5 to 200 mg / L, 0.5 to 150 mg / L, 0.5 to 100 mg / L, 0.5 to 50 mg / L, 0.5 to 10 mg / L, 0.5 to 5 mg / L, 0.5 to 2 mg / L, 0.5 to 1 mg / L, 0.8 to 10,000 mg / L, 0.8 to 5,000 mg / L, 0.8 to 2,000 mg / L, 0.8 to 1,500 mg / L, 0.8 to 1,000 mg / L, 0.8 to 500 mg / L, 0.8 to 200 mg / L, 0.8 to 150 mg / L, 0.8 to 100 mg / L, 0.8 to 50 mg / L, 0.8 to 10 mg / L, 0.8 to 5 mg / L, 0.8 to 2 mg / L, 0.8 to 1 mg / L, 1 to 10,000 mg / L, 1 to 5,000 mg / L, 1 to 2,000 mg / L, 1 to 1,500 mg / L, 1 to 1,000 mg / L, 1 to 500 mg / L, 1 to 200 mg / L, 1 to 150 mg / L, 1 to 100 mg / L, 1 to 50 mg / L, 1 to 10 mg / L, 1 to 5 mg / L, 1 to 2 mg / L, 10 to 10,000 mg / L, 10 to 5,000 mg / L, 10 to 2,000 mg / L, 10 to 1,500 mg / L,10 to 1,000 mg / L, 10 to 500 mg / L, 10 to 200 mg / L, 10 to 150 mg / L, 10 to 100 mg / L, 10 to 50 mg / L, 50 to 10,000 mg / L, 50 to 5,000 mg / L, 50 to 2,000 mg / L, 50 to 1,500 mg / L, 50 to 1,000 mg / L, 50 to 500 mg / L, 50 to 200 mg / L, 50 to 150 mg / L, 50 to 100 mg / L, 80 to 10,000 mg / L, 80 to 5,000 mg / L, 80 to 2,000 mg / L, 80 to 1,500 mg / L, 80 to 1,000 mg / L, 80 to 500 mg / L, 80 to 200 mg / L, 80 to 150 mg / L, 80 to 100 mg / L, 100 to 10,000 mg / L, 100 to 5,000 mg / L, 100 to 2,000 mg / L, 100 to 1,500 mg / L, 100 to 1,000 mg / L, 100 to 500 mg / L, 100 to 200 mg / L, 100 to 150 mg / L, 500 to 10,000 mg / L, 500 to 5,000 mg / L, 500 to 2,000 mg / L, 500 to 1,500 mg / L, 500 It may be included at concentrations of up to 1,000 mg / L, 800 to 10,000 mg / L, 800 to 5,000 mg / L, 800 to 2,000 mg / L, 800 to 1,500 mg / L, 800 to 1,000 mg / L, 1,000 to 10,000 mg / L, 1,000 to 5,000 mg / L, 1,000 to 2,000 mg / L, or 1,000 to 1,500 mg / L, for example, 0.1, 1, 100, or 1,000 mg / L, but is not limited thereto.

[0039] The above culture medium composition may have one or more characteristics selected from the group consisting of (1) to (3) below, but is not limited thereto.

[0040] (1) Inhibition of byproduct generation in animal cells;

[0041] (2) Enhancement of antibody productivity in animal cells; and

[0042] (3) Enhancement of the linear growth rate of animal cells.

[0043] In this specification, "by-product" refers to a compound that is generated secondarily during the process of cell growth, metabolism, or protein production, rather than being a major reaction product; it has a significant effect on the culture environment, particularly the pH control of the culture medium, and may have a positive or negative effect on cell growth and productivity. The by-product may be lactic acid, but is not limited thereto.

[0044] In this specification, "antibody productivity" refers to an indicator representing the amount of antibodies produced per unit time, unit volume, or number of units of cells. The antibody productivity of the animal cells can be confirmed by inoculating the animal cells into the medium composition and culturing them, and then measuring the antibody titer of the culture medium.

[0045] In this specification, "linear growth rate" is an indicator describing a cell growth pattern in which the number of cells or biomass increases at a constant rate over a constant time interval. Specifically, it can be calculated by the following Equation 1 by measuring the Viable cell density (VCD) in the culture medium on days 2 and 5 of cell culture.

[0046] [Mathematical Formula 1]

[0047] Linear growth rate = (T: cell culture day)

[0048] The above-mentioned culture medium composition may be characterized by not including extracts derived from animals, plants, and / or microorganisms (e.g., proteins, protein hydrolysates, etc.), thereby not causing side effects such as inducing unwanted immune responses.

[0049] The above animal cells may be for the production of cultured meat. In this specification, "cultured meat" refers to a meat-like product produced by artificially culturing cells extracted from animals (animal cells, e.g., muscle cells, fat cells, connective tissue cells, etc.).

[0050] Specifically, when lactic acid accumulates at a certain concentration (e.g., 20 to 30 mM) or higher in a culture medium (e.g., a culture medium for animal cell culture), cell viability decreases rapidly, cell growth is inhibited, and eventually, apoptosis may be induced. When animal cells are cultured in a medium composition containing the DCI or a derivative thereof, the effect of reducing lactic acid, a byproduct in the culture medium, is obtained, thereby improving the productivity of cultured meat.

[0051] The above-mentioned culture medium composition may further include, but is not limited to, water, a carbon source (e.g., glucose, fructose, sucrose, pyruvate and / or salts thereof), a nitrogen source (e.g., amino acids, peptone, yeast extract, ammonia compounds, etc.), inorganic salts (e.g., sodium chloride, magnesium sulfate, potassium phosphate, iron sulfate, copper sulfate, zinc salts, etc.), vitamins (e.g., B vitamins (thiamine, riboflavin, pyridoxine, etc.), vitamin C, etc.), cofactors (e.g., NAD+, CoA, or other essential metabolic cofactors, etc.), pH regulators (e.g., hydrochloric acid, sodium hydroxide, Tris-hydrochloric acid buffer, etc.) and / or additives (e.g., antibiotics, antioxidants).

[0052]

[0053] Animal cell culture method

[0054] Another example of the present specification provides a method for culturing animal cells, comprising the step of culturing animal cells in the medium composition.

[0055] The above culture method may be performed by subculture and may include, but is not limited to, culture methods such as fed-batch culture, batch culture, continuous culture, and repeated fed-batch culture.

[0056] The above subculture may be performed for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days, and may be repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times, but is not limited thereto.

[0057] The above medium composition may be one or more selected from basic medium, main culture medium (main medium), initial medium, maintenance medium, growth medium, production medium, differentiation medium, feed medium, replacement medium and perfusion culture medium, and temporary expression medium, but is not limited thereto.

[0058] The above animal cell culture may be a culture in which animal cells are included in or added to a culture medium composition in the form of a basic medium.

[0059] The above animal cell culture may be performed by adding (administering, inoculating) the medium composition to the animal cells in the form of a feed medium.

[0060] The above animal cell culture method may further include the step of adding (administering, inoculating) animal cells to the medium composition and / or the step of adding the medium composition to animal cells.

[0061] The step of adding a culture medium composition to the animal cells may involve adding the culture medium composition to the culture medium in which the animal cell culture is carried out. Adding the culture medium composition to the culture medium may involve adding the culture medium composition at a concentration of 1 to 5 (v / v)%, 1 to 4 (v / v)%, 1 to 3 (v / v)%, 2 to 5 (v / v)%, 2 to 4 (v / v)%, or 2 to 3 (v / v)%, for example, 2.5 (v / v)%, based on the volume of the culture medium, but is not limited thereto. Adding the culture medium composition to the culture medium may involve adding it repeatedly at intervals of 1, 2, 3, 4, or 5 days, but is not limited thereto.

[0062] The above animal cell culture is 1 to 20% CO2, 1 to 15% CO2, 1 to 12% CO2, 1 to 10% CO2, 1 to 9% CO2, 3 to 20% CO2, 3 to 15% CO2, 3 to 12% CO2, 3 to 10% CO2, 3 to 9% CO2, 5 to 20% CO2, 5 to 15% CO2, 5 to 12% CO2, 5 to 10% CO2, 5 to 9% CO2, 7 to 20% CO2, 7 to 15% CO2, 7 to 12% CO2, 7 to 10% CO2, or 7 to 9% CO2 2, For example, it may be performed under conditions of a carbon dioxide concentration of 8% CO2, but is not limited thereto.

[0063] The above animal cell culture may be performed under humidity conditions of 60 to 99%, 60 to 90%, 60 to 85%, 60 to 82%, 70 to 99%, 70 to 90%, 70 to 85%, 70 to 82%, 75 to 99%, 75 to 90%, 75 to 85%, 75 to 82%, 78 to 99%, 78 to 90%, 78 to 85%, or 78 to 82%, for example, 80%, but is not limited thereto.

[0064] The above animal cell culture is 20 to 50°C, 20 to 45°C, 20 to 40°C, 20 to 38°C, 20 to 36°C, 20 to 34°C, 25 to 50°C, 25 to 45°C, 25 to 40°C, 25 to 38°C, 25 to 36°C, 25 to 34°C, 30 to 50°C, 30 to 45°C, 30 to 40°C, 30 to 38°C, 30 to 36°C, 30 to 34°C, 32 to 50°C, 32 to 45°C, 32 to 40°C, 32 to 38°C, 32 to 36°C, 32 to 34°C, 34 to 50°C, 34 to 45°C, 34 to It may be performed under temperature conditions of 40°C, 34 to 38°C, 34 to 36°C, 36 to 50°C, 36 to 45°C, 36 to 40°C, or 36 to 38°C, for example, 33°C, or 37°C, but is not limited thereto.

[0065] The above animal cell culture may change temperature conditions during culture, and may be changed on day 1, day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, or day 10 of culture, but is not limited thereto. For example, on day 6 of culture, the culture may be carried out by changing the temperature condition from 37°C to 33°C.

[0066] The present invention relates to a culture medium composition comprising D-chiro-inositol (DCI) or a derivative thereof, wherein culture in the culture medium composition rapidly reduces lactate, a culture byproduct, and the cultured cells can achieve excellent cell proliferation ability.

[0067] Figure 1 shows the vector map of the pOzBio vector.

[0068] Figure 2 is a graph showing the measured viable cell density (VCD) among the results of the fed batch culture test of CHO-K1 cells with the addition of DCI to the feed medium.

[0069] Figure 3 is a graph showing the viability of CHO-K1 cells in the fed batch culture test results following the addition of DCI to the feed medium.

[0070] Figure 4 is a graph showing the lactate concentration in the culture medium during the fed batch culture test of CHO-K1 cells with the addition of DCI to the feed medium.

[0071] Figure 5 is a graph showing the pH level of the culture medium in the fed batch culture test results of CHO-K1 cells with the addition of DCI to the feed medium.

[0072] Figure 6 is a graph showing the antibody concentration in the culture medium during the fed batch culture test of CHO-K1 cells following the addition of DCI to the feed medium.

[0073] Figure 7 is a graph showing the results of a batch culture test of CHO-K1 cells with the addition of DCI to Basal medium, measuring viable cell density (VCD), viability, and linear growth rate.

[0074] Figure 8 is a graph showing the results of a batch culture test of CHO-DXB11 cells with the addition of DCI to Basal medium, measuring viable cell density (VCD), viability, and linear growth rate.

[0075] The present invention will be explained in more detail below through examples. However, the following examples are intended to illustrate the present invention and do not limit the scope of the invention.

[0076]

[0077] Example 1. Lactic acid reduction effect and cell viability improvement of D-Chiro Inositol (DCI)

[0078] For the experiment, CHO-K1-derived cells expressing Nivolumab (DrugBank Accession Number DB09035) were constructed. Specifically, the floating cell line CHO-K1 (ECACC Cell Line Bank, Cat. No. 85051005) and the expression vector pOzBio were provided by Artiabio, Inc., USA. The LC (Light Chain) and HC (Heavy Chain) genes of Nivolumab were cloned into the pOzBio vector, and the cell line was constructed by transforming the genes into CHO cells using electroporation. The vector map of the pOzBio vector is shown in Figure 1.

[0079] For the cell line prepared above, Forma under conditions of 37℃, 8% CO2, and 80% humidity TM Steri-Cult TM Culture was performed in a CO2 incubator (Thermo Fisher Scientific, Cat No. 3311).

[0080] Specifically, a working volume of 30 mL of basal medium (UltiMax Pro, Amikogen Co., Ltd.) was added to a 125 mL Erlenmeyer flask in the above-mentioned incubator, 100 X HT (Gibco, Cat No. 11067030) was titrated to 1 X and added, and the above-mentioned CHO-K1 cell line was added in 2 X 10 5 cell / mL or 3 x 10⁶ 5 Inoculated at a concentration of cell / mL, and subcultured using a suspension culture method at 130 rpm.

[0081] Specifically, the above CHO-K1 cell line was subcultured for a total of 17 days according to the following cell concentrations and culture periods:

[0082] (1) 2 X 10 5 Add cell / mL and culture for 3 days;

[0083] (2) Recover the cultured cells from (1) above, 2 x 10 5 Culture for 3 days in the same manner at a cell / mL concentration;

[0084] (3) Recover the cultured cells from (2) above, 3 x 10 5 Culture for 4 days in the same manner at a cell / mL concentration;

[0085] (4) Recover the cultured cells from (3) above, 3 x 10 5 Culture for 4 days in the same manner at a cell / mL concentration;

[0086] (5) Recover the cultured cells from (4) above, 2 x 10 5 Culture for 3 days in the same manner at a cell / mL concentration.

[0087] Collect the CHO-K1 cells currently being subcultured and place them in a 125 mL Erlenmeyer flask containing 40 mL of basal medium (4 x 10⁶). 5Cells were inoculated at a concentration of cell / mL. 30 to 40 minutes after inoculation, 1 mL was sampled, and pH, glucose, and lactate concentrations were measured using a metabolite analyzer (NOVA Biomedical, BioFlex2). Viable cell density (VCD) and viability were analyzed using an automatic cell counter (Invitrogen, Countess 3 FL). The above process was repeated daily for sampling and analysis. Fed-batch culture was performed by adding glucose daily based on the measured glucose concentration to maintain a glucose concentration of 4.5 g / L in the culture medium of all experimental groups.

[0088] Ultimax feed and Ultimax CY solution (Amikogen Co., Ltd.) were prepared as feed media, and D-chiro-inositol (DCI, Amikogen Co., Ltd.) was added to the Ultimax feed media to final concentrations of 100 mg / L (Experimental Group 1-1), 1 mg / L (Experimental Group 1-2), and 0.1 mg / L (Experimental Group 1-3), respectively. As a control, a medium without added D-chiro-inositol was used (Control Group 1).

[0089] From the 3rd day of fed-batch culture of the harvested cells, 2.5 (v / v)% of the Ultimax feed medium of experimental groups 1-1 to 1-3 and control group 1, based on the volume of the culture medium, and Ultimax CY solution (UltiMax TM CY solution (Amikogen Co., Ltd.) was fed every other day at a concentration of 0.25 (v / v)% based on the volume of the culture medium.

[0090] On the 6th day from the initial culture, the temperature conditions were changed from 37°C to 33°C, and culture was terminated on the 14th day or when the viability of each cell line was 70% or less. After termination, antibody productivity was measured by measuring antibody titer using samples collected during culture, and the measured results are shown in Figures 2 to 6.

[0091] As a result of measuring VCD, CHO-K1 cultured with feed medium containing DCI was able to be cultured for a longer period and grew more than the control group without DCI and growth factor.

[0092] As a result of measuring the concentration of lactate, a culture byproduct that has a negative effect on protein quality and lowers the pH of the culture medium, the control group without growth factor and DCI added showed a continuous increase in lactate until the end of culture, and consequently, the pH of the culture medium dropped to about 6.2, whereas the experimental groups treated with DCI in the feed showed a decrease in lactate during culture and normalized the pH starting from the 7th day of culture.

[0093] As a result of measuring antibody titer, the experimental group fed with DCI had a higher antibody titer compared to the control group without added DCI.

[0094] Based on the above results, it was confirmed that DCI was highly effective in reducing lactate, a culture byproduct, during cultivation. By preventing the pH of the culture medium from dropping to acidity due to lactate, it minimized cell damage caused by acidic pH in the medium, thereby confirming that DCI has a positive effect on cell viability and productivity.

[0095]

[0096] Example 2. Enhancement of cell growth rate by D-Chiro Inositol (DCI)

[0097] To observe the animal cell proliferation effect of DCI, host cell lines derived from CHO-K1 and CHO-DXB11 were used. Suspended CHO-K1 cells (ECACC Cell Line Bank, Cat. No. 85051005) and CHO-DXB11 (ECACC Cell Line Bank, Cat. No. 94060607) were provided by Artiabio, USA.

[0098] The CHO-K1 cell line prepared above was subcultured in substantially the same manner as in Test Example 1 above, and the CHO-DXB11 cell line was subcultured for a total of 17 days in a suspension culture manner under the following conditions by adding 5 mM L-glutamine instead of 1 X HT.

[0099] (1) 5 X 10 5 Add cell / mL and culture for 3 days;

[0100] (2) Recover the cultured cells from (1) above, 5 x 10 5 Culture for 3 days in the same manner at a cell / mL concentration;

[0101] (3) Recover the cultured cells of (2) above, 10 x 10 5 Culture for 4 days in the same manner at a cell / mL concentration;

[0102] (4) Recover the cultured cells from (3) above, 10 x 10 5 Culture for 4 days in the same manner at a cell / mL concentration;

[0103] (5) Recover the cultured cells of (4) above, 5 X 10 5 Culture for 3 days in the same manner at a cell / mL concentration

[0104] Cell lines 3 days after subculture were subjected to Forma under conditions of 37℃, 8% CO2, and 80% humidity. TM Steri-CultTM Cultures were performed in a CO2 incubator. Specifically, a working volume of 40 mL of UltiMax Pro (Amicogen, Inc.) was added to a 125 mL flask in the incubator, and the subcultured CHO-K1 cell line was suspended in 1 X HT, while the CHO-DXB11 line was suspended in 5 mM L-glutamine, at 130 rpm. For each cell line, CHO-K1 was suspended in 3 X 10 5 cell / mL, CHO-DXB11 is 5 x 10 5 Cells were inoculated at a concentration of cell / mL, and batch culture tests were conducted for 6 days. Ultimax Pro (Amikogen Co., Ltd.) was used as the basal medium for the above culture, and D-chiro-inositol (DCI, Amikogen Co., Ltd.) was added to Ultimax Pro for each cell line culture according to the concentrations in Table 1 below. A medium without D-chiro-inositol was used as a control.

[0105] Cell Line Type Final Concentration of Added DCI (mg / L) CHOK1 Experimental Group 2-1-K 1,000 Experimental Group 2-2-K 100 Control Group 2-CK-DX B11 Experimental Group 2-1-D 1,000 Experimental Group 2-2-D 100 Control Group 2-CD-

[0106] During the batch culture test, viable cell density (VCD) and viability were measured daily using an automatic cell counter (Invitrogen, Countess 3 FL). Based on the VCD measurements on days 2 and 5 after culture, the linear growth rate was calculated using the VCD values ​​on day 2 (when the lag phase ends) and day 5 (when the exponential phase ends) using the following Equation 1 to measure the growth rate during the exponential phase. The measurement results for CHO-K1 and CHO-DXB11 cell lines are shown in Figures 7 and 8, respectively.

[0107] [Mathematical Formula 1]

[0108] Linear growth rate = (T: cell culture day)

[0109] As a result of measuring VCD and viability, it was confirmed that cell growth of both CHO-K1 and CHO-DXB11 increased more rapidly in the medium supplemented with DCI than in the medium without DCI, and the results of linear growth rate calculations confirmed that the cell growth rate was higher in the medium supplemented with DCI.

Claims

1. A culture medium composition for animal cells comprising D-chiro-inositol (D-chiro inositol / DCI) or a derivative thereof.

2. A culture medium composition according to claim 1, wherein the animal cells are cultured meat cells.

3. A culture medium composition according to claim 1, wherein the animal cells are stem cells.

4. A culture medium composition according to claim 1, wherein the animal cells are immune cells.

5. A culture medium composition according to any one of claims 1 to 4, wherein the DCI or a derivative thereof is contained at a concentration of 0.01 to 10,000 mg / L based on the total volume of the culture medium composition.

6. A culture medium composition according to any one of claims 1 to 4, wherein the animal cells are one or more selected from the group consisting of mouse-derived cells, rat-derived cells, hamster-derived cells, monkey-derived cells, and human-derived cells.

7. A culture medium composition according to any one of claims 1 to 4, wherein the composition has one or more characteristics selected from the group consisting of (1) to (3) below: (1) Inhibition of byproduct generation in animal cells; (2) Enhancement of antibody productivity in animal cells; and (3) Enhancement of the linear growth rate of animal cells.

8. A culture medium composition according to claim 7, wherein the byproduct is lactic acid.

9. A method for culturing animal cells, comprising the step of culturing animal cells in a culture medium composition according to any one of claims 1 to 4.

10. In claim 9, the animal cell culture is (i) the above medium composition is in the form of a basic medium in which animal cells are included in the medium composition and cultured; (ii) A culture method in which the above-mentioned culture medium composition is added to animal cells in the form of a feed medium and cultured.

11. A culture method according to claim 9, wherein the animal cells are one or more selected from the group consisting of mouse-derived cells, rat-derived cells, hamster-derived cells, monkey-derived cells, and human-derived cells.

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