CN102021139A - Chinese hamster ovary culture medium as well as preparation method and application thereof - Google Patents

Abstract

The invention discloses a Chinese hamster ovary cell culture medium and its preparation method and application. The culture medium contains ascorbic acid, pyridoxine hydrochloride, vitamin B ₁₂ , nicotinamide, riboflavin, thiamine hydrochloride, and chole chloride Alkali, folic acid, calcium pantothenate, sodium pyruvate, reduced glutathione, inositol, biotin, hypoxanthine, putrescine, lipoic acid, linoleic acid, thymine, glucose, HEPES buffer, dextran sulfate Sugar, Pluronic-F68, various amino acids and their salts, and various inorganic salts. The cost of the medium is low, and the composition has no animal-derived components, no protein, and a definite chemical composition. Through the coordination of various components, it can not only meet the basic requirements of cell growth, but also effectively regulate, transmit and control the cultivation of cells. Good high-density cell culture is achieved, and its culture effect is at least equal to or even better than that of the existing culture medium.

Description

A kind of Chinese hamster ovary cell culture medium and its preparation method and application

technical field

The invention relates to a Chinese hamster ovary cell culture medium and its preparation method and application.

Background technique

Chinese hamster ovary cell (CHO cell) is currently the most widely used mammalian cell line and has been used to produce various genetically engineered protein products, for example, tissue plasminogen activator (t-PA) , human erythropoietin (rHuEPO), hepatitis B surface antigen (HBsAg), human thrombopoietin (rhTPO), various recombinant antibodies, etc.

CHO cells are usually cultured in a synthetic medium supplemented with fetal calf serum or calf serum. However, the added serum is a substance with unclear composition, which contains many proteins and other macromolecular substances with uncertain composition, which affects the separation and purification of the product and reduces the yield and purity of the product. At the same time, there is a potential risk of virus infection in the serum derived from animals. In addition, the addition of serum will greatly increase the cost of production. Therefore, many researchers have devoted themselves to developing low-serum or serum-free media.

Casser (In Vitro Cell Devel Biol 1985, 88:588) in the United States developed a serum-free medium suitable for the growth of CHO cells, which was based on DMEM/F12 and added transferrin, albumin, insulin and Sodium selenite and other components have become the basic formula of the widely used serum-free medium. However, the serum-free medium contains animal-derived proteins such as transferrin, albumin, and insulin. These proteins are not only potential sources of pollution, but also greatly increase the cost of the medium and increase the difficulty of separation and purification. Therefore, scientific researchers have begun to find inorganic salts that can replace these proteins, and have developed protein-free media.

Leopold Grillberger of the United States (US Patent 2006/0094104 A1) has developed a protein-free medium suitable for animal cell culture, which does not contain any animal-derived protein, and is replaced by protein hydrolyzate derived from animal or plant sources. Although the cost of this type of protein-free medium is already very low, and the potential risk of contamination is greatly reduced, it still contains components with uncertain chemical compositions, which still limits the research on cell growth, proliferation, differentiation regulation, etc. to a certain extent. Therefore, it is urgent to develop an animal-derived component-free, protein-free, chemically defined medium that is suitable for high-density cultivation of CHO cells and whose cultivation effect is equivalent to or better than that of serum-containing medium, serum-free medium, and protein-free medium. .

Contents of the invention

The technical problem to be solved by the present invention is to overcome the uncertain chemical composition of the existing Chinese hamster ovary cell culture medium, the use of animal source components or protein, so that the cultured cells have potential pollution threats, and the cost is high, increasing the separation and purification Difficulty, to a certain extent limit the development of cell research and other defects, provide a Chinese hamster ovary cell culture medium and its preparation method and its preparation method and application.

The invention relates to a Chinese hamster ovary cell culture medium, which contains: 9.02-141.06 mg/L L-tryptophan (L-Tryptophan), 31.29-148.74 mg/L L-cystine (L-Cystine), 17.56 ～159.87mg/L L-cysteine (L-Cystein), 38.4～377.28mg/L L-tyrosine disodium salt dihydrate (L-Tyrosine disodium salt dihydrate), 54.47～654mg/L L-iso Leucine (L-Isoleucine), 59.05～874.32mg/L L-Leucine (L-Leucine), 50～420mg/L L-Valine (L-Valine), 80～720mg/L L-Pine L-Arginine, 31~480mg/L L-Histidine, 80~852.3mg/L L-Lysine, 10~184.7mg/L L-Methionine (L-Methionine), 20～438.3mg/L L-Phenylalanine (L-Phenylalanine), 1～235.8mg/L L-Glutamic acid (L-Glutamic acid), 70～540mg/L L-Day Paragine (L-Asparagine), 1~40mg/L Glycine (Glycine), 10~620mg/L L-Proline (L-Proline), 0.1~63mg/L L-Alanine (L-Alanine), 10～223.6mg/L L-Apartic acid (L-Apartic acid), 20～661.2mg/L L-Serine (L-Serine), 40～893.4mg/L L-Threonine (L-Threonine) , 365～1168mg/L L-glutamine (L-Glutamine), 10～160mg/L anhydrous calcium chloride (Calcium Chloride (anhydrous), CaCl ₂ ), 0.001～0.003mg/L copper sulfate pentahydrate (Cupric Sulfate, CuSO ₄ 5H ₂ O), 0.05～1mg/L Ferric Nitrate (Fe(NO ₃ ) ₃ 9H ₂ O), 0.1～1mg/L Ferrous Sulfate (FeSO ₄ 7H ₂ O), 100～400mg/L Potassium Chloride (KCl), 20～60mg/L Magnesium Chloride (Magnesium Chloride, MgCl ₂ ), 5-200mg/L Sodium Phosphate Monobasic (Sodium Phosphate Monobasic, NaH ₂ PO ₄ ·H ₂ O), 50-300mg/L Sodium Phosphate Dibasic (anhydrous), Na ₂ HPO ₄ ), 0.1～2mg/L zinc sulfate heptahydrate (Zinc Sulfate, ZnSO ₄ 7H ₂ O), 8～50mg/L ferric citrate (Ferric Citrate, FeC ₆ H ₅ O ₇ ), 0.001～0.05mg/L L sodium selenite (Sodium Selenite, Na ₂ SeO ₃ ), 3000mg/L sodium bicarbonate (Sodium Bicarbonate), 2-20mg/L ascorbic acid (Ascorbic Acid), 0.1-4mg/L pyridoxine hydrochloride (Pyridoxine HCl) , 0.2～2.72mg/L vitamin B ₁₂ (Vitamin B ₁₂ ), 0.5～6.5mg/L niacinamide (Niacinamide), 0.05～1.5mg/L riboflavin (Riboflavin), 0.1～6.81mg/L Thiamine hydrochloride Thiamine HCl, 2.57~26.94mg/L Choline Chloride, 0.2~3mg/L Folic Acid, 0.1~2.5mg/L D-Ca Pantothenate, 2~150mg /L sodium pyruvate (Sodium Pyruvate), 0.3～2.25mg/L glutathione (reduced) (Glutathione (reduced)), 5～25mg/L inositol (i-Inositol), 0.0001～0.5mg/L Biotin, 0.008～8mg/L Na Hypoxanthine, 0.001～9mg/L Putrescine-2HCl, 0.003～0.8mg/L Lipoic Acid, 0.003～1.2mg /L Linoleic Acid (Linoleic Acid), 0.02～6mg/L Thymidine (Thymidine), 2150～7000mg/L Glucose (D-glucose (Dextrose)), 1500～4000mg/L HEPES Buffer, 5～70mg/L Dextran Sulfate Sodium Salt and 500～2000mg/LPluronic-F68 (Pluronic F6 8), and the rest is water (that is, water is added to 1L).

The Chinese hamster ovary cell culture medium in a preferred example of the present invention contains: 72mg/L L-tryptophan, 50mg/L L-cystine, 87.6mg/L L-cysteine, 130.2mg/L L L-tyrosine sodium salt dihydrate, 324.6mg/L L-isoleucine, 376.8mg/L L-leucine, 190.6mg/L L-valine, 321.2mg/L L-arginine amino acid, 206.1mg/L L-histidine, 425.3mg/L L-lysine, 84.7mg/L L-methionine, 196.7mg/L L-phenylalanine, 182.6mg/L L-glutamine Acid, 213mg/L-Asparagine, 13mg/L Glycine, 210mg/L L-Proline, 7mg/L L-Alanine, 113.4mg/L L-Aspartic Acid, 326mg/L L-Serine , 412mg/L L-threonine, 584mg/L L-glutamine, 116mg/L anhydrous calcium chloride, 0.0015mg/L copper sulfate pentahydrate, 0.09mg/L iron nitrate nonahydrate, 0.417mg/L Ferrous sulfate heptahydrate, 311.8mg/L potassium chloride, 40mg/L magnesium chloride, 62.5mg/L sodium dihydrogen phosphate monohydrate, 71mg/L disodium hydrogen phosphate anhydrous, 1mg/L zinc sulfate heptahydrate, 29.8mg /L Ferric Citrate, 0.01mg/L Sodium Selenite, 3000mg/L Sodium Bicarbonate, 12.5mg/L Ascorbic Acid, 2.6mg/L Pyridoxine Hydrochloride, 1.2mg/L Vitamin B ₁₂ , 4mg/L Niacinamide , 0.68mg/L riboflavin, 3.65mg/L thiamine hydrochloride, 19.8mg/L choline chloride, 1.78mg/L folic acid, 1.6mg/L calcium pantothenate, 98mg/L sodium pyruvate, 1.25mg/L L glutathione (reduced form), 12.6mg/L inositol, 0.01mg/L biotin, 4mg/L hypoxanthine, 1.8mg/L putrescine, 0.5mg/L lipoic acid, 0.08mg/L sub Oleic acid, 3.65mg/L thymine, 3150mg/L glucose, 2000mg/L HEPES buffer, 35mg/L dextran sulfate and 1200mg/L Pluronic-F68, the rest is water (that is, the water is made up to 1L).

In the present invention, the water is conventional water in this field, generally ultrapure water.

The Chinese hamster ovary cell culture medium of the present invention preferably further contains: 0.5-2.5 mg/L stannous chloride dihydrate (SnCl ₂ 2H ₂ O), 0.02-0.18 mg/L ammonium vanitate (NH ₄ VO ₃ ), 0.005～0.03mg/L barium acetate (Ba(C ₂ H ₃ O ₂ O) ₂ ), 0.001～0.04mg/L silver nitrate (AgNO ₃ ), 0.005～0.03mg/L sodium silicate (Na ₂ SiO ₃ ), 0.1～1.0mg/L aluminum chloride hexahydrate (AlCl ₃ 6H ₂ O), 0.001～0.01mg/L cadmium sulfate octahydrate (CdSO ₄ 8H ₂ O), 0.01～0.3mg/L hexahydrate Cobalt chloride (CoCl·6H ₂ O), 0.07～0.5mg/L manganese sulfate monohydrate (MnSO ₄ ·H ₂ O), 0.01～0.1mg/L ammonium molybdate tetrahydrate ((NH ₄ ) ₆ Mo ₇ O ₂₄ 4H ₂ O), 0.1～0.9mg/L nickel chloride hexahydrate (NiCl ₂ 6H ₂ O), 0.00001～0.0003mg/L potassium bromide (KBr), 0.001～0.01mg/L sodium fluoride ( NaF), 0.00005～0.00048mg/L potassium iodide (KI), 0.001～0.008mg/L arachidonic acid, 0.11～0.88mg/L cholesterol, 0.035～0.28mg/L DL-acetate α-tocopherol, 0.003～0.05 One or more of mg/L myristic acid, 0.002~0.04mg/L oleic acid, 0.005~0.04mg/L palmitene, 0.005~0.04mg/L palmitic acid and 0.001~0.09mg/L stearic acid .

The Chinese hamster ovary cell culture medium in another preferred embodiment of the present invention also contains: 1.5mg/L stannous chloride dihydrate, 0.08mg/L ammonium vulcanate, 0.02mg/L barium acetate, 0.01mg/L Silver nitrate, 0.01mg/L sodium silicate, 0.7mg/L aluminum chloride hexahydrate, 0.003mg/L cadmium sulfate octahydrate, 0.1mg/L cobalt chloride hexahydrate, 0.2mg/L manganese sulfate monohydrate, 0.033 mg/L ammonium molybdate tetrahydrate, 0.45mg/L nickel chloride hexahydrate, 0.00002mg/L potassium bromide, 0.0058mg/L sodium fluoride, 0.00023mg/L potassium iodide, 0.0035mg/L arachidonic acid, 0.52mg/L Cholesterol, 0.11mg/L DL-Acetate α-Tocopherol, 0.018mg/L Myristic Acid, 0.025mg/L Oleic Acid, 0.019mg/L Palmitic Acid, 0.024mg/L Palmitic Acid and 0.046mg/L One or more of L stearic acid.

The preparation method of the Chinese hamster ovary cell culture medium of the present invention can adopt conventional methods in the field, and preferably includes the following steps: dissolving the above-mentioned ingredients in water and simply uniformly mixing them. Wherein, after being uniformly mixed, it is generally sterilized according to a conventional method in the art. Among them, when ferric citrate or ammonium vitriol is involved in the culture medium, ferric citrate or ammonium vitriol need to be heated and dissolved in a boiling water bath before being uniformly mixed with other components; the culture medium involves lipoic acid or stannous chloride When using lipoic acid or stannous chloride, it is necessary to dissolve it in absolute ethanol, and then mix it evenly with other components.

The present invention also relates to the application of the Chinese hamster ovary cell culture medium of the present invention in culturing Chinese hamster ovary cells.

In the process of culturing CHO cells, the Chinese hamster ovary cell culture medium in the present invention can generally be used according to conventional methods in the art. Preferably, CHO cells are inoculated into the Chinese hamster ovary cell culture medium of the present invention. cultured under the culture conditions of the cells.

The reagents and raw materials used in the present invention are all commercially available.

The positive progress effect of the present invention is:

The invention provides a Chinese hamster ovary cell culture medium and application thereof. The culture medium of the present invention has low cost, no animal-derived components, no protein and definite chemical components in the composition. Through the coordination of various components, it can effectively regulate, transmit and control the growth of cells in addition to meeting the basic requirements of cell growth. Cultivate to achieve good high-density cell culture, and its culture effect is comparable to or even better than that of the existing serum medium, protein medium or protein hydrolyzate medium. The invention also relates to the application of the Chinese hamster ovary cell culture medium in cultivating Chinese hamster ovary cells, which can support the high-density growth of CHO cells and can completely replace the serum culture medium for the cultivation of CHO cells.

Description of drawings

Fig. 1 is the Chinese hamster ovary cell culture medium of embodiment 1 and comparative example 1 respectively culture CHO cell, its cell density changes with time graph; Wherein, ◆ is GIBCO CD CHO AGT ^TM medium; ■ is the culture of embodiment 1 base.

Detailed ways

The present invention is further illustrated below by means of examples, but the present invention is not limited to the scope of the examples.

Example 1

Prepare the animal-derived component-free, protein-free, and chemically defined culture medium of the present invention according to the following component concentrations, and dissolve each component in water, wherein ferric citrate needs to be heated and dissolved in a boiling water bath before being uniformly mixed with other substances; Caprylic acid is dissolved in absolute ethanol and then mixed uniformly; after uniform mixing, a 0.22 micron nitrocellulose membrane is used for filter sterilization.

The Chinese hamster ovary cell culture medium of the present invention contains: 72mg/L L-tryptophan, 50mg/L L-cystine, 87.6mg/L L-cysteine, 130.2mg/L L-tyrosine Sodium salt dihydrate, 324.6mg/LL-isoleucine, 376.8mg/L L-leucine, 190.6mg/L L-valine, 321.2mg/L L-arginine, 206.1mg/L L -Histidine, 425.3mg/L L-lysine, 84.7mg/L L-methionine, 196.7mg/L L-phenylalanine, 182.6mg/L L-glutamic acid, 213mg/L L-day Paragine, 13mg/L glycine, 210mg/L L-proline, 7mg/L L-alanine, 113.4mg/L L-aspartic acid, 326mg/L L-serine, 412mg/L L-threo amino acid, 584mg/L L-glutamine, 116mg/L anhydrous calcium chloride, 0.0015mg/L copper sulfate pentahydrate, 0.09mg/L ferric nitrate nonahydrate, 0.417mg/L ferrous sulfate heptahydrate, 311.8 mg/L potassium chloride, 40mg/L magnesium chloride, 62.5mg/L sodium dihydrogen phosphate monohydrate, 71mg/L anhydrous disodium hydrogen phosphate, 1mg/L zinc sulfate heptahydrate, 29.8mg/L iron citrate, 0.01 mg/L Sodium Selenite, 3000mg/L Sodium Bicarbonate, 12.5mg/L Ascorbic Acid, 2.6mg/L Pyridoxine Hydrochloride, 1.2mg/L Vitamin B ₁₂ , 4mg/L Niacinamide, 0.68mg/L Nuclear Yellow element, 3.65mg/L thiamine hydrochloride, 19.8mg/L choline chloride, 1.78mg/L folic acid, 1.6mg/L calcium pantothenate, 98mg/L sodium pyruvate, 1.25mg/L glutathione (also prototype), 12.6mg/L inositol, 0.01mg/L biotin, 4mg/L hypoxanthine, 1.8mg/L putrescine, 0.5mg/L lipoic acid, 0.08mg/L linoleic acid, 3.65mg/L Thymine, 3150mg/L glucose, 2000mg/L HEPES buffer, 35mg/L dextran sulfate and 1200mg/L Pluronic-F68.

CHO cells were inserted into the prepared medium (CHO seed cells were domesticated cells of the medium, sourced from Shanghai Fuchun Kexin Biotechnology Co., Ltd.), and the inoculation density was 5×10 ⁵ cells/ml for suspension culture. The conditions are: 37°C, 5% CO ₂ (v/v), and humidity greater than 95% for culture, and the maximum cell density reaches 7.3×10 ⁶ cells/ml.

Wherein, the CHO seed cells are domesticated and cultured according to the following method: digest the adherent CHO cells with 0.25% (w/v) trypsin, and use serum-containing medium (DMEM+10% Hyclone calf serum, purchased from (Shanghai Shiyi Biotechnology Co., Ltd.) was resuspended and then centrifuged, poured the supernatant, and resuspended the cells in the above-mentioned prepared medium of the present invention to adapt to the culture. The growth mode of CHO cells can be converted from adherent growth to suspension growth, which can CHO cells were further expanded and grown in this medium.

Example 2

On the basis of the ingredients contained in the culture medium in Example 1, add 1.5mg/L stannous chloride dihydrate, 0.08mg/L ammonium vitriol, 0.02mg/L barium acetate, 0.01mg/L silver nitrate, 0.01mg /L sodium silicate, 0.7mg/L aluminum chloride hexahydrate, 0.003mg/L cadmium sulfate octahydrate, 0.1mg/L cobalt chloride hexahydrate, 0.2mg/L manganese sulfate monohydrate, 0.033mg/L tetrahydrate Ammonium molybdate, 0.45mg/L nickel chloride hexahydrate, 0.00002mg/L potassium bromide, 0.0058mg/L sodium fluoride, 0.00023mg/L potassium iodide, 0.0035mg/L arachidonic acid, 0.52mg/L cholesterol . Among them, ferric citrate and ammonium vanitate need to be heated and dissolved in a boiling water bath before being evenly mixed with other substances; lipoic acid and stannous chloride are dissolved in absolute ethanol, then evenly mixed, and then 0.22 micron nitrocellulose membrane is used Perform filter sterilization.

The CHO cells in Example 1 were inserted into the prepared medium, and the seeding density was 5×10 ⁵ cells/ml for suspension culture. The culture conditions were: 37°C, 5% CO ₂ (v/v), humidity More than 95% of the environment is cultured, and the maximum cell density of the cells is 8.0×10 ⁶ cells/ml.

Comparative example 1

The medium used in Comparative Example 1 was GIBCO CD CHO AGT ^TM medium (Shanghai Yuanzhi Biochemical Technology Co., Ltd., this medium belongs to the medium with defined chemical composition), and CHO cells were inserted into the medium (seed cells were the culture medium for acclimation cells), the inoculation density is 5×10 ⁵ cells/ml, and the suspension culture is carried out. The culture conditions are: 37°C, 5% CO ₂ , and the humidity is greater than 95%. The maximum cell density is 4.5×10 ⁶ cells/ml.

Wherein, the CHO seed cells are domesticated and cultured according to the following method: digest the adherent CHO cells with 0.25% (w/v) trypsin, and use serum-containing medium (DMEM+10% Hyclone calf serum, purchased from (Shanghai Shiyi Biotechnology Co., Ltd.) was resuspended and centrifuged, poured the supernatant, and resuspended the cells in GIBCO CD CHO AGT ^TM medium for acclimation and adaptation culture. The growth mode of CHO cells can be converted from adherent growth to suspension growth, which can CHO cells were further expanded and grown in this medium.

Here, during the culture medium in which CHO cells were inoculated in Comparative Example 1 and Example 1, changes in cell density over time are recorded in FIG. 1 . From the comparison of Figure 1, it is found that CHO cells are cultured in the animal-free, protein-free, and chemically defined medium of Example 1. The growth of the cells is better than that of the commercially available commercial medium cultivated under the same conditions, and it can be used in Large-scale culture, and the cost is low, far lower than the price of commercial chemically determined medium, suitable for application in industrial production.

Claims (7)

1. Chinese hamster ovary cell substratum, it is characterized in that: described Chinese hamster ovary cell substratum contains following component: 9.02～141.06mg/L L-tryptophane, 31.29～148.74mg/L L-Gelucystine, 17.56～159.87mg/L L-halfcystine, 38.4～377.28mg/L L-tyrosine salt sodium monocalcium salt, 54.47～654mg/L L-Isoleucine, 59.05～874.32mg/L L-leucine, 50～420mg/L L-Xie Ansuan, 80～720mg/L L-arginine, 31～480mg/L L-Histidine, 80～852.3mg/L L-Methionin, 10～184.7mg/L L-methionine(Met), 20～438.3mg/L L-phenylalanine, 1～235.8mg/L L-L-glutamic acid, 70～540mg/L altheine, 1～40mg/L glycine, 10～620mg/L L-proline(Pro), 0.1～63mg/L L-L-Ala, 10～223.6mg/L L-aspartic acid, 20～661.2mg/L L-Serine, 40～893.4mg/L L-Threonine, 365～1168mg/L L-glutaminate, 10～160mg/L Calcium Chloride Powder Anhydrous, 0.001～0.003mg/L cupric sulfate pentahydrate, 0.05～1mg/L nine water iron nitrates, 0.1～1mg/L iron vitriol, 100～400mg/L Repone K, 20～60mg/L magnesium chloride, 5～200mg/L sodium dihydrogen phosphate-water, 50～300mg/L disodium hydrogen phosphate,anhydrous, 0.1～2mg/L Zinc Sulphate Heptahydrate, 8～50mg/L ironic citrate, 0.001～0.05mg/L Sodium Selenite, the 3000mg/L sodium bicarbonate, 2～20mg/L xitix, 0.1～4mg/L benadon, 0.2～2.72mg/L vitamins B ₁₂0.5～6.5mg/L niacinamide, 0.05～1.5mg/L riboflavin, 0.1～6.81mg/L vitamin, 2.57～26.94mg/L choline chloride 60,0.2～3mg/L folic acid, 0.1～2.5mg/L calcium pantothenate, 2～150mg/L Sodium.alpha.-ketopropionate, 0.3～2.25mg/L reduced glutathion, 5～25mg/L inositol, 0.0001～0.5mg/L vitamin H, 0.008～8mg/L xanthoglobulin, 0.001～9mg/L putrescine, 0.003～0.8mg/L Thioctic Acid, 0.003～1.2mg/L linolic acid, 0.02～6mg/L thymus pyrimidine, 2150～7000mg/L glucose, 1500～4000mg/L HEPES damping fluid, 5～70mg/L T 500 and 500～2000mg/L Pluronic-F68, all the other are water.

2. Chinese hamster ovary cell substratum as claimed in claim 1 is characterized in that: described Chinese hamster ovary cell substratum contains following component: 72mg/L L-tryptophane, 50mg/L L-Gelucystine, 87.6mg/L L-halfcystine, 130.2mg/L L-tyrosine salt sodium monocalcium salt, 324.6mg/LL-Isoleucine, 376.8mg/L L-leucine, 190.6mg/L L-Xie Ansuan, 321.2mg/L L-arginine, 206.1mg/L L-Histidine, 425.3mg/L L-Methionin, 84.7mg/L-methionine(Met), 196.7mg/L-phenylalanine, 182.6mg/L L-L-glutamic acid, the 213mg/L altheine, the 13mg/L glycine, 210mg/L L-proline(Pro), 7mg/L L-L-Ala, 113.4mg/L L-aspartic acid, 326mg/L L-Serine, 412mg/L L-Threonine, the 584mg/L L-glutaminate, the 116mg/L Calcium Chloride Powder Anhydrous, 0.0015mg/L cupric sulfate pentahydrate, 0.09mg/L nine water iron nitrates, 0.417mg/L iron vitriol, 311.8mg/L Repone K, the 40mg/L magnesium chloride, 62.5mg/L sodium dihydrogen phosphate-water, the 71mg/L disodium hydrogen phosphate,anhydrous, the 1mg/L Zinc Sulphate Heptahydrate, 29.8mg/L ironic citrate, 0.01mg/L Sodium Selenite, the 3000mg/L sodium bicarbonate, 12.5mg/L xitix, 2.6mg/L benadon, 1.2mg/L vitamins B ₁₂, 4mg/L niacinamide, 0.68mg/L riboflavin, 3.65mg/L vitamin, 19.8mg/L choline chloride 60,1.78mg/L folic acid, 1.6mg/L calcium pantothenate, 98mg/L Sodium.alpha.-ketopropionate, 1.25mg/L reduced glutathion, 12.6mg/L inositol, 0.01mg/L vitamin H, 4mg/L xanthoglobulin, 1.8mg/L putrescine, 0.5mg/L Thioctic Acid, 0.08mg/L linolic acid, 3.65mg/L thymus pyrimidine, 3150mg/L glucose, 2000mg/L HEPES damping fluid, 35mg/L T 500 and 1200mg/L Pluronic-F68, all the other are water.

3. Chinese hamster ovary cell substratum as claimed in claim 1 is characterized in that: described Chinese hamster ovary cell substratum also contains one or more in the following component: 0.5～2.5mg/L stannous chloride dihydrate, 0.02～0.18mg/L alum acid ammonium, 0.005～0.03mg/L barium acetate, 0.001～0.04mg/L Silver Nitrate, 0.005～0.03mg/L water glass, 0.1～1.0mg/L Aluminum Chloride Hexahydrate, 0.001～0.01mg/L eight water Cadmium Sulphates, 0.01～0.3mg/L CoCL2,0.07～0.5mg/L manganese sulfate monohydrate, 0.01～0.1mg/L four water ammonium molybdates, 0.1～0.9mg/L six water nickelous chlorides, 0.00001～0.0003mg/L Potassium Bromide, 0.001～0.01mg/L Sodium Fluoride, 0.00005～0.00048mg/L potassiumiodide, 0.001～0.008mg/L arachidonic acid, 0.11～0.88mg/L cholesterol, 0.035～0.28mg/L DL-alpha-tocopherol acetate, 0.003～0.05mg/L myristic acid, 0.002～0.04mg/L oleic acid, 0.005～0.04mg/L palmitinic acid alkene, 0.005～0.04mg/L palmitinic acid and 0.001～0.09mg/L stearic acid.

4. Chinese hamster ovary cell substratum as claimed in claim 3 is characterized in that: described Chinese hamster ovary cell substratum also contains one or more in the following component: the 1.5mg/L stannous chloride dihydrate, 0.08mg/L alum acid ammonium, 0.02mg/L barium acetate, 0.01mg/L Silver Nitrate, 0.01mg/L water glass, 0.7mg/L Aluminum Chloride Hexahydrate, 0.003mg/L eight water Cadmium Sulphates, 0.1mg/L CoCL2,0.2mg/L manganese sulfate monohydrate, 0.033mg/L four water ammonium molybdates, 0.45mg/L six water nickelous chlorides, 0.00002mg/L Potassium Bromide, 0.0058mg/L Sodium Fluoride, 0.00023mg/L potassiumiodide, 0.0035mg/L arachidonic acid, 0.52mg/L cholesterol, 0.11mg/L DL-alpha-tocopherol acetate, 0.018mg/L myristic acid, 0.025mg/L oleic acid, 0.019mg/L palmitinic acid alkene, 0.024mg/L palmitinic acid and 0.046mg/L stearic acid.

5. the preparation method of Chinese hamster ovary cell substratum as claimed in claim 1 or 2, it is characterized in that: it comprises the following steps: that (1) is with ironic citrate boiling water bath heating for dissolving, use dehydrated alcohol to dissolve Thioctic Acid, with all the other component water dissolution; (2) with each component uniform mixing, degerming then of step (1).

6. as the preparation method of claim 3 or 4 described Chinese hamster ovary cell substratum, it is characterized in that: it comprises the following steps: that (1) is with ironic citrate and alum acid ammonium boiling water bath heating for dissolving, use dehydrated alcohol to dissolve Thioctic Acid and tin protochloride, with all the other component water dissolution; (2) with each component uniform mixing, degerming then of step (1).

7. as the application of each described Chinese hamster ovary cell substratum of claim 1～4 in cultivating Chinese hamster ovary cell.

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