CN1778902A - Non-serum culture medium for multiple animal cell large-scale culture - Google Patents

Abstract

The invention opened non-serum mediums which can proper to culture many kinds of the animal cells. The character of it is to use the DMEM/F12 as the base medium, then add the growth factor, hormone, the essential amino acid and the microelement. It has the functions: (1) it can support many cells and clones; (2) it is same as the serum medium in the growth of the cell and the expression of the product; (3) it support the long heritable culture; (4) it is benefit for the isolation of the product because it contains less protein; (5) it is cheap. We can get the high density of the cells and the product concentration using the medium.

Description

Serum-free medium suitable for large-scale culture of various animal cells

technical field

The invention relates to a serum-free medium used for producing biological products such as antibodies, vaccines, and gene recombinant proteins during large-scale high-density culture of animal cells.

technical background

Animal cell culture has been widely used in the production of various biologically active substances such as monoclonal antibodies, virus vaccines, virus vectors, immune regulators, growth factors, specific tumor antigens, and various gene recombinant protein drugs. Add a certain amount (5%-10%) of bovine serum, which contains growth factors, hormones, carrier proteins, adhesion factors, trace elements and other nutrients required for cell growth, which can greatly promote cell growth and product expression.

However, the application of serum also brings many disadvantages:

(1) Serum is susceptible to contamination by viruses, mycoplasma or other pathogens;

(2) Differences between different batches of serum cause differences between product batches;

(3) The existence of a large amount of serum protein increases the difficulty of downstream separation and purification, which increases the cost and reduces the recovery rate;

(4) Some serum proteins are difficult to completely remove by means of separation and purification, which seriously affects the final quality of the product.

In order to overcome the various shortcomings brought about by serum, many researchers have been engaged in the research and development of serum-free medium since the eighties. As the basal medium, add 1.5mg/L insulin, 3.0mg/L transferrin, 2nmol/L testosterone, 5mg/L catalase, 1.5mg/L β-glycerophosphate, 0.5mg/L The dilinoleoylphosphatidylcholine of L is used for the cultivation of hybridoma cells; Murakami et al. developed DMEM/F12-ITES serum-free medium, which added 5mg/L insulin on the basis of DMEM/F12, 2 -35mg/L transferrin, 20μmol/L ethanolamine, 1nmol/L sodium selenite, this medium is widely used in culturing hybridoma cells.

In addition, the serum-free medium involved in U.S. Patent U.S.P.5,063,157 is used for suspension culture of mammalian cells, and transferrin, insulin, peptone, beta-D-xylopyranose derivative, selenium and polyamines are added to the basal medium, which is a U.S. Patent U.S.P.4,443,546 is another similar low protein medium; U.S. Patent U.S.P.4,657,866 relates to a synthetic medium with clear (chemical defined medium) for cultivating some of animals and humans Cell lines; European Patent E.P.481,791 discloses a serum-free medium for CHO cells containing water, osmotic regulators, buffers, sugars, amino acids, iron, growth factors and other essential components.

At present, a large number of commercial serum-free media have been developed and applied to various animal cell cultures, such as CD Hybridoma medium, Hybridoma-SFM medium, and PFHM-IIProtein-free medium for hybridoma cells from GIBCO. , CD CHO medium, CHO-s-SFMII medium for CHO cells; Hybri-Max from SIGMA, etc.

The above serum-free media have their own advantages, and most of them can support the growth of cells well, but they also have many disadvantages:

(1) Due to the high price, it is only suitable for small-scale use in laboratories; it is not suitable for large-scale bioreactors;

(2) The medium is not optimized for cells, and the supported cell density is low, resulting in low yield in the production process;

(3) Although some media can support cell growth well, it often leads to the reduction or even loss of the ability of cells to express products;

(4) The medium is highly specific to cells, and usually a medium is only suitable for one cell strain or cell line, but not for other cell strains or cell lines.

Contents of the invention

The technical problem to be solved in the present invention is to disclose a serum-free medium suitable for large-scale cultivation of various animal cells, so as to overcome the above-mentioned defects in the prior art.

The serum-free medium suitable for large-scale cultivation of various animal cells of the present invention is based on DMEM and F12 with a weight ratio of 1:1, and insulin, transferrin, ethanolamine, sodium pyruvate and albumin are added , β-mercaptoethanol, trace elements, hormones, essential amino acids and other ingredients;

The components of the culture medium DMEM and F12 have been disclosed in the product catalog of GIBCO or SIGMA, and relevant personnel can refer to them. The present invention will not repeat them, and commercial products can be used, such as products of GIBCO or SIGMA;

The composition and content of the serum-free medium that the present invention finally forms are as follows: Element Chinese chemical name manufacturer Inorganic salt (mg/L) _CaCl2 calcium chloride SIGMA 116.60 CuSO ₄ 5H ₂ O copper sulfate SIGMA 0.0013 Fe(NO ₃ ) ₃ 9H ₂ O Ferric nitrate SIGMA 0.05 FeSO4 7H ₂ O ferrous sulfate SIGMA 0.417 KCl potassium chloride SIGMA 311.80 MgCl ₂ magnesium chloride SIGMA 28.64 _MgSO4 magnesium sulfate SIGMA 48.84 NaCl Sodium chloride SIGMA 6995.50 NaHCO ₃ sodium bicarbonate SIGMA 2440 NaH ₂ PO ₄ H ₂ O Sodium dihydrogen phosphate SIGMA 62.50 Na ₂ HPO ₄ Disodium phosphate SIGMA 71.02 ZnSO ₄ 7H ₂ O Zinc sulfate SIGMA 0.432 L-amino acid (mg/L) Alanine Alanine SIGMA 4.45 Arginine HCl arginine SIGMA 147.5-210.5 Asparagine H ₂ O Asparagine SIGMA 7.5-67.5 Aspartic acid aspartic acid SIGMA 6.65-26.6 Cysteine H ₂ O cysteine SIGMA 17.56-35.12 Cystein·2HCl cystine SIGMA 31.29-62.58 Glutamic acid glutamic acid SIGMA 7.35-29.4 Glutamine Glutamine SIGMA 584-1168 Glycine Glycine SIGMA 18.75-37.5 Histidine·HCl·H ₂ O Histidine SIGMA 31.48-104.93 Isoleucine Isoleucine SIGMA 52.4-131.0 Leucine Leucine SIGMA 58.95-157.2 Lysine HCl Lysine SIGMA 91.25-182.5 Methionine Methionine SIGMA 16.39-59.6 Phenylalanine Phenylalanine SIGMA 33.0-82.5 Proline proline SIGMA 17.25-34.5 Serine serine SIGMA 26.25-94.5 Threonine threonine SIGMA 53.55-95.2 Tryptophan Tryptophan SIGMA 8.16-51.0 Trosine·2Na·2H ₂ O Tyrosine SIGMA 55.79-139.5 Valine Valine SIGMA 52.65-117.0 Vitamin/coenzyme (mg/L) Biotin Biotin SIGMA 0.0035 Pantothenate·Ca thbrthdrexvbdr SIGMA 2.24 Choline·Cl choline SIGMA 8.98 Folic acid folic acid SIGMA 2.65 Inositol Inositol SIGMA 12.60 Niacinamide Nicotinamide SIGMA 2.02 Pyridoxine·HCl Vitamin B6 SIGMA 2.031 Riboflavin riboflavin SIGMA 0.219 Thiamine·HCl Thiamine SIGMA 2.17 Thymidine Thymidine SIGMA 0.365 Vitamin B ₁₂ Vitamin B12 SIGMA 0.68 Trace elements (mg/L) Na ₂ SeO ₃ Sodium Selenite SIGMA 0.0008-0.0035 MnSO ₄ 4H ₂ O Manganese sulfate SIGMA 0.00008-0.0008 Na ₂ SiO ₃ 5H ₂ O Sodium silicate SIGMA 0.002-0.01 (NH ₄ ) ₆ Mo ₇ O ₂₄ 4H ₂ O Ammonium molybdate SIGMA 0.0017-0.017 NH ₄ VO ₃ Ammonium vanadate SIGMA 0.00006-0.0006 NiCl ₂ 6H ₂ O nickel chloride SIGMA 0.00002-0.00024 SnCl ₂ 2H ₂ O tin chloride SIGMA 0.00002-0.00023 Other ingredients (mg/L) Glucose glucose SIGMA 1000-7200 Na Hypoxanthine Hypoxanthine SIGMA 2.39 Linoleic acid Linoleic acid SIGMA 0.042 Lipoic acid lipoic acid SIGMA 0.105 Phenol red Phenol red SIGMA 8.10 Sodium Putrescine 2HCl Putrescine SIGMA 0.081 Pyruvate Na sodium pyruvate SIGMA 220.00 insulin SIGMA 5-10 Transferrin SIGMA 5-10 albumin SIGMA 0-100 Hormone (mg/L) Hydrocortisone SIGMA 0.036-0.362 Dexamethasone SIGMA 0.039-0.392 progesterone SIGMA 0.006-0.031 Estradiol SIGMA 0.0027-0.027 B-Mercaptoethanol SIGMA 0.61-1.83 ethanolamine SIGMA 1.22-12.2

The preparation method of the culture medium of the present invention is very simple, and can be prepared by dissolving the above-mentioned components in non-pyrogenic ultrapure water by conventional methods.

The culture medium of the invention can be used for subculture and high-density continuous perfusion culture of hybridoma cells, rCHO cells, 293 cells and other cell lines or cell strains. It has the following advantages:

1. Can support the growth of multiple cell lines or cell lines;

2. It is close to or equivalent to serum-containing medium in terms of cell growth and product expression;

3. Support the long-term subculture of cells;

4. Contains a lower concentration of protein, which is conducive to the separation and purification of the product and improves the quality of the product;

5. Low price, suitable for large-scale industrial production;

Most importantly, the serum-free medium formed by the present invention is suitable for continuous perfusion culture of multiple cell lines or cell lines, can obtain higher cell density and product concentration, and can significantly improve production process efficiency and product yield.

Description of drawings

Figure 1 is the cell growth and product expression curves of continuous perfusion culture of HB58 hybridoma cells.

Figure 2 is the cell growth and product expression curves of continuous perfusion culture of CHO cells.

Figure 3 is the growth curve of 293 cells in batch culture.

Detailed ways

Example 1

The serum-free medium of the present invention can be used for high-density continuous perfusion culture of hybridoma cells, and the composition of the medium is as follows: Element Chinese chemical name Inorganic salt (mg/L) _CaCl2 calcium chloride 116.60 CuSO ₄ 5H ₂ O copper sulfate 0.0013 Fe(NO ₃ ) ₃ 9H ₂ O Ferric nitrate 0.05 FeSO4 7H ₂ O ferrous sulfate 0.417 KCl potassium chloride 311.80 MgCl ₂ magnesium chloride 28.64 _MgSO4 magnesium sulfate 48.84 NaCl Sodium chloride 6995.50 NaHCO ³ sodium bicarbonate 2440 NaH ₂ PO ₄ H ₂ O Sodium dihydrogen phosphate 62.50 Na ₂ HPO ₄ Disodium phosphate 71.02 ZnSO ₄ 7H ₂ O Zinc sulfate 0.432 L-amino acid (mg/L) Alanine Alanine 4.45 Arginine HCl arginine 210.5 Asparagine H ₂ O Asparagine 7.5 Aspartic acid aspartic acid 26.6 Cysteine H ₂ O cysteine 17.56 Cystein·2HCl cystine 31.29 Glutamic acid glutamic acid 14.7 Glutamine Glutamine 1168 Glycine Glycine 18.75 Histidine·HCl·H ₂ O Histidine 104.93 Isoleucine Isoleucine 131.0 Leucine Leucine 157.2 Lysine HCl Lysine 182.5 Methionine Methionine 16.39 Phenylalanine Phenylalanine 82.5 Proline proline 34.5 Serine serine 26.25 Threonine threonine 53.55 Tryptophan Tryptophan 51.0 Tyrosine·2Na·2H ₂ O Tyrosine 139.5 Valine Valine 117.0 Vitamins/cofactors (mg/L) Biotin biotin 0.0035 Pantothenate·Ca thbrthdrexvbdr 2.24 Choline·Cl choline 8.98 Folic acid folic acid 2.65 Inositol Inositol 12.60 Niacinamide Nicotinamide 2.02 Pyridoxine·HCl Vitamin B6 2.03l Riboflavin riboflavin 0.219 Thiamine·HCl Thiamine 2.17 Thymidine Thymidine 0.365 Vitamin B ₁₂ Vitamin B12 0.68 Trace elements (mg/L) Na ₂ SeO ₃ Sodium Selenite 0.0016 MnSO ₄ 4H ₂ O Manganese sulfate 0.00016 Na ₂ SiO ₃ 5H ₂ O Sodium silicate 0.005 (NH ₄ ) ₆ Mo ₇ O ₂₄ 4H ₂ O Ammonium molybdate 0.0034 NH ₄ VO ₃ Ammonium vanadate 0.00012 NiCl ₂ 6H ₂ O nickel chloride 0.0001 SnCl ₂ 2H ₂ O tin chloride 0.0001 Other ingredients (mg/L) Glucose glucose 6300 Na Hypoxanthine Hypoxanthine 2.39 Linoleic acid Linoleic acid 0.042 Lipoic acid lipoic acid 0.105 Phenol red Phenol red 8.10 Sodium Putrescine 2HCl Putrescine 0.081 Pymvate Na sodium pyruvate 220.00 insulin 5 Transferrin 10 albumin 100 Hormone (mg/L) Hydrocortisone 0.036 Dexamethasone 0.039 progesterone 0.006 Estradiol 0.0027 B-Mercaptoethanol 0.61 ethanolamine 12.2

The above components are mixed and dissolved in pyrogen-free ultrapure water to obtain a culture medium.

HB58 hybridoma cells (obtained from ATCC) were subcultured and adapted in the serum-free medium according to the present invention, and then inoculated in a 2-liter bioreactor of BF-2 (Germany B.BRAUN Company) with an inoculation density of 2.0×10 ⁵ cells/ml, perfusion starts after 56 hours of culture, the perfusion rate is 0.5 (1/day), the perfusion medium is the serum-free medium of the present invention, and the cell density is maintained at 1.2×10 ⁷ cells after 200 hours of culture /ml, the monoclonal antibody concentration was maintained at about 500mg/L (see Figure 1), compared with the results of batch culture in ordinary medium, the cell density and monoclonal antibody concentration were increased by more than 8 times. In the figure, curve 1 is the living cell density, curve 2 is the total cell density, and curve 3 is the expression level of monoclonal antibody.

Example 2

The serum-free medium of the present invention can be used for high-density continuous perfusion culture of rCHO cells, and the composition of the medium is as follows: Element Chinese chemical name Inorganic salt (mg/L) _CaCl2 calcium chloride 116.60 CuSO ₄ 5H ₂ O copper sulfate 0.0013 Fe(NO ₃ ) ₃ 9H ₂ O Ferric nitrate 0.05 FeSO4 7H ₂ O ferrous sulfate 0.417 KCl potassium chloride 311.80 MgCl ₂ magnesium chloride 28.64 _MgSO4 magnesium sulfate 48.84 NaCl Sodium chloride 6995.50 NaHCO ₃ sodium bicarbonate 2440 NaH ₂ PO ₄ H ₂ O Sodium dihydrogen phosphate 62.50 Na ₂ HPO ₄ Disodium phosphate 71.02 ZnSO ₄ 7H ₂ O Zinc sulfate 0.432 L-amino acid (mg/L) Alanine Alanine 4.45 Arginine HCl arginine 168.4 Asparagine H ₂ O Asparagine 67.5 Aspartic acid aspartic acid 6.65 Cysteine H ₂ O cysteine 17.56 Cystein·2HCl cystine 62.58 Glutamic acid glutamic acid 7.35 Glutamine Glutamine 1168 Glycine Glycine 37.5 Histidine·HCl·H ₂ O Histidine 47.22 Isoleucine Isoleucine 131.0 Leucine Leucine 157.2 Lysine HCl Lysine 127.7 Methionine Methionine 29.8 Phenylalanine Phenylalanine 33.0 Proline proline 34.5 Serine serine 94.5 Threonine threonine 95.2 Tryptophan Tryptophan 20.4 Tyrosine·2Na·2H ₂ O Tyrosine 105.2 Valine Valine 93.6 Vitamins/cofactors (mg/L) Biotin Biotin 0.0035 Pantothenate·Ca thbrthdrexvbdr 2.24 Choline·Cl choline 8.98 Folic acid folic acid 2.65 Inositol Inositol 12.60 Niacinamide Nicotinamide 2.02 Pyridoxine·HCl Vitamin B6 2.031 Riboflavin riboflavin 0.219 Thiamine·HCl Thiamine 2.17 Thymidine Thymidine 0.365 Vitamin B ₁₂ Vitamin B12 0.68 Trace elements (mg/L) Na ₂ SeO ₃ Sodium Selenite 0.0016 MnSO ₄ 4H ₂ O Manganese sulfate 0.00016 Na ₂ SiO ₃ 5H ₂ O Sodium silicate 0.005 (NH ₄ ) ₆ Mo ₇ O ₂₄ 4H ₂ O Ammonium molybdate 0.0034 NH ₄ VO ₃ Ammonium vanadate 0.00012 NiCl ₂ 6H ₂ O nickel chloride 0.00004 SnCl ₂ 2H ₂ O tin chloride 0.00004 Other ingredients (mg/L) Glucose glucose 7200 Na Hypoxanthine Hypoxanthine 2.39 Linoleic acid Linoleic acid 0.042 Lipoic acid lipoic acid 0.105 Phenol red Phenol red 8.10 Sodium Putrescine 2HCl Putrescine 0.081 Pyruvate Na sodium pyruvate 220.00 insulin 10 Transferrin 10 albumin 100 Hormone (mg/L) Hydrocortisone 0.036 Dexamethasone 0.039 progesterone 0.006 Estradiol 0.0027 B-Mercaptoethanol 0.61 ethanolamine 12.2

rCHO cells (rCHO SS3 A2, expressing human anticoagulant factor III) were subcultured in the serum-free medium described in the present invention, and then seeded in a B.BRAUN 2-liter bioreactor at a seeding density of 2.0×10 ⁵ cells /ml, start perfusion after 40 hours of culture, the perfusion rate is 0.58 (1/day), the perfusion medium is the serum-free medium of the present invention, and the cell density is maintained at 0.9-1.0×10 ⁷ after 255 hours of culture The cells/ml is about 350-380U/L, and the product concentration is maintained at about 350-380U/L (see Figure 2). Compared with the results of ordinary medium batch culture, the cell density and monoclonal antibody concentration are increased by 6 times and 5 times respectively. In the figure, curve 4 is the living cell density, the curve is the total cell density, and curve 6 is the expression level of the product.

Example 3

The serum-free medium of the present invention can be used for the cultivation of 293 cells, and the composition of the medium is as follows: Element Chinese chemical name Inorganic salt (mg/L) _CaCl2 calcium chloride 116.60 CuSO ₄ 5H ₂ O copper sulfate 0.0013 Fe(NO ₃ ) ₃ 9H ₂ O Ferric nitrate 0.05 FeSO4 7H ₂ O ferrous sulfate 0.417 KCl potassium chloride 311.80 MgCl ₂ magnesium chloride 28.64 _MgSO4 magnesium sulfate 48.84 NaCl Sodium chloride 6995.50 NaHCO ₃ sodium bicarbonate 2440 NaH ₂ PO ₄ H ₂ O Sodium dihydrogen phosphate 62.50 Na ₂ HPO ₄ Disodium phosphate 71.02 ZnSO ₄ 7H ₂ O Zinc sulfate 0.432 L-amino acid (mg/L) Alanine Alanine 4.45 Arginine HCl arginine 168.4 Asparagine H ₂ O Asparagine 7.5 Aspartic acid aspartic acid 6.65 Cysteine H ₂ O cysteine 17.56 Cystein·2HCl cystine 31.29 Glutamic acid glutamic acid 7.35 Glutamine Glutamine 584 Glycine Glycine 18.75 Histidine·HCl·H ₂ O Histidine 31.48 Isoleucine Isoleucine 54.47 Leucine Leucine 59.05 Lysine HCl Lysine 91.25 Methionine Methionine 29.8 Phenylalanine Phenylalanine 33.0 Proline proline 34.5 Serine serine 26.25 Threonine threonine 53.45 Tryptophan Tryptophan 20.4 Tyrosine·2Na·2H ₂ O Tyrosine 55.79 Valine Valine 52.8 Vitamins/cofactors (mg/L) Biotin biotin 0.0035 Pantothenate·Ca thbrthdrexvbdr 2.24 Choline·Cl choline 8.98 Folic acid folic acid 2.65 Inositol Inositol 12.60 Niacinamide Nicotinamide 2.02 Pyridoxine·HCl Vitamin B6 2.031 Riboflavin riboflavin 0.219 Thiamine·HCl Thiamine 2.17 Thymidine Thymidine 0.365 Vitamin B ₁₂ Vitamin B12 0.68 Trace elements (mg/L) Na ₂ SeO ₃ Sodium Selenite 0.0016 MnSO ₄ 4H ₂ O Manganese sulfate 0.00016 Na ₂ SiO ₃ 25H ₂ O Sodium silicate 0.005 (NH ₄ ) ₆ Mo ₇ O ₂₄ 4H ₂ O Ammonium molybdate 0.0034 NH ₄ VO ₃ Ammonium vanadate 0.00012 NiCl ₂ 6H ₂ O nickel chloride 0.00004 SnCl ₂ 2H ₂ O tin chloride 0.00004 Other ingredients (mg/L) Glucose glucose 3150 Na Hypoxanthine Hypoxanthine 2.39 Linoleic acid Linoleic acid 0.042 Lipoic acid lipoic acid 0.105 Phenol red Phenol red 8.10 Sodium Putrescine 2HCl Putrescine 0.081 Pyruvate Na sodium pyruvate 220.00 insulin 10 Transferrin 10 albumin 100 Hormone (mg/L) Hydrocortisone 0.036 Dexamethasone 0.039 progesterone 0.006 Estradiol 0.0027 B-Mercaptoethanol 0.61 ethanolamine 12.2

After the 293 cells were subcultured and adapted in the serum-free medium of the present invention, they were inoculated in a B.BRAUN 2-liter bioreactor for batch culture, and the inoculation density was 2.45×10 ⁵ cells/ml, as can be seen from Figure 3 There was almost no lag phase in the growth of the cells. After inoculation, the cells entered the exponential growth phase, with an average specific growth rate of 0.46day ^-1 and a maximum viable cell density of 11.0×10 ⁵ cells/ml. In the figure, curve 7 is the living cell density, and curve 8 is the ratio of living cells.

Claims (7)

1. serum free medium that is applicable to that multiple animal cell large-scale is cultivated, it is characterized in that, be with weight ratio be 1: 1 DMEM and F12 be basic medium, add Regular Insulin, Transferrins,iron complexes, thanomin, Sodium.alpha.-ketopropionate, albumin, beta-mercaptoethanol, trace element, hormone and indispensable amino acid etc. and become to be grouped into.

2. serum free medium according to claim 1 is characterized in that, said basic medium is the commercial substratum of standard, and contained component and content are with reference to the prescription of announcing in the SIGMA company products catalogue in the substratum.

3. serum free medium according to claim 2 is characterized in that, trace element is selected from Mn and/or Mo and/or Ni and/or Se and/or Si and/or Sn and/or V.

4. according to claim 2 or 3 described serum free mediums, it is characterized in that hormone is selected from hydrocortisone, dexamethasone, estradiol or progesterone.

5. according to claim 2 or 3 described serum free mediums, it is characterized in that the indispensable amino acid that is added comprises arginine, Aspartic Acid, asparamide, halfcystine, Gelucystine, L-glutamic acid, glutamine, glycine, Histidine, leucine, Isoleucine, Methionin, methionine(Met), phenylalanine, proline(Pro), Serine, Threonine, tryptophane, tyrosine and Xie Ansuan.

6. serum free medium according to claim 5 is characterized in that, final nutrient media components and the content that forms is as follows: Composition The Chinese chemical name Inorganic salt (mg/L) CaCl ₂ Calcium chloride 116.60 CuSO ₄·5H ₂O Copper sulfate 0.0013 Fe(NO ₃) ₃·9H ₂O Iron nitrate 0.05 FeSO4·7H ₂O Ferrous sulfate 0.417 KCl Repone K 311.80 MgCl ₂ Magnesium chloride 28.64 MgSO ₄ Sal epsom 48.84 NaCl Sodium-chlor 6995.50 NaHCO ₃ Sodium bicarbonate 2440 NaH ₂PO ₄H ₂O SODIUM PHOSPHATE, MONOBASIC 62.50 Na ₂HPO ₄ Sodium phosphate dibasic 71.02 ZnSO ₄·7H ₂O Zinc sulfate 0.432 L-amino acid (mg/L) Alanine L-Ala 4.45 Arginine·HCl Arginine 147.5-210.5 Asparagine·H ₂O Asparamide 7.5-67.5 Aspartic acid Aspartic Acid 6.65-26.6 Cysteine·H ₂O Halfcystine 17.56-35.12 Cystein·2HCl Gelucystine 31.29-62.58 Glutamic acid L-glutamic acid 7.35-29.4 Glutamine Glutamine 584-1168 Glycine Glycine 18.75-37.5 Histidine·HCl·H ₂O Histidine 31.48-104.93 Isoleucine Isoleucine 52.4-131.0 Leucine Leucine 58.95-157.2 Lysine·HCl Methionin 91.25-182.5 Methionine Methionine(Met) 16.39-59.6 Phenylalanine Phenylalanine 33.0-82.5 Proline Proline(Pro) 17.25-34.5 Serine Serine 26.25-94.5 Threonine Threonine 53.55-95.2 Tryptophan Tryptophane 8.16-51.0 Tyrosine·2Na·2H ₂O Tyrosine 55.79-139.5 Valine Xie Ansuan 52.65-117.0 Vitamins/cofactors(mg/L) Biotin Vitamin H 0.0035 Pantothenate·Ca Calcium pantothenate 2.24 Choline·Cl Choline 8.98 Folic acid Folic acid 2.65 Inositol Inositol 12.60 Niacinamide Niacinamide 2.02 Pyridoxine·HCl Vitamin B6 2.031 Riboflavin Riboflavin 0.219 Thiamine·HCl Thiamines 2.17 Thymidine Thymidine 0.365 Vitamin B ₁₂ Vitamin B12 0.68 Trace element (mg/L) Na ₂SeO ₃ Sodium Selenite 0.0008-0.0035 MnSO ₄·4H ₂O Manganous sulfate 0.00008-0.0008 Na ₂SiO ₃·5H ₂O Water glass 0.002-0.01 (NH ₄) ₆Mo ₇O ₂₄·4H ₂O Molybdenum acid ammonia 0.0017-0.017 NH ₄VO ₃ Vanadic acid ammonia 0.00006-0.0006 NiCl ₂·6H ₂O Nickelous chloride 0.00002-0.00024 SnCl ₂·2H ₂O Tin chloride 0.00002-0.00023 Other composition (mg/L) Glucose Glucose 1000-7200 Na Hypoxanthine Xanthoglobulin 2.39 Linoleic acid Linolic acid 0.042 Lipoic acid Thioctic Acid 0.105 Phenol red Phenol red 8.10 Sodium Putrescine·2HCl Putrescine 0.081 Pyruvate·Na Sodium.alpha.-ketopropionate 220.00 Regular Insulin 5-10 Transferrins,iron complexes 5-10 Albumin 0-100 Hormone (mg/L) Hydrocortisone 0.036-0.362 Dexamethasone 0.039-0.392 Progesterone 0.006-0.031 Estradiol 0.0027-0.027 The B-mercaptoethanol 0.61-1.83 Thanomin 1.22-12.2

7. according to the application of each described serum free medium of claim 1～5, it is characterized in that, be used for the go down to posterity cultivation and the high-density continous pouring cultivation of hybridoma, rCHO cell or 293 cells.

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