[go: up one dir, main page]
More Web Proxy on the site http://driver.im/

CN100362098C - Non-serum culture medium for multiple animal cell large-scale culture - Google Patents

Non-serum culture medium for multiple animal cell large-scale culture Download PDF

Info

Publication number
CN100362098C
CN100362098C CNB2005100301989A CN200510030198A CN100362098C CN 100362098 C CN100362098 C CN 100362098C CN B2005100301989 A CNB2005100301989 A CN B2005100301989A CN 200510030198 A CN200510030198 A CN 200510030198A CN 100362098 C CN100362098 C CN 100362098C
Authority
CN
China
Prior art keywords
acid
sigma
serum
sodium
hcl
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Fee Related
Application number
CNB2005100301989A
Other languages
Chinese (zh)
Other versions
CN1778902A (en
Inventor
谭文松
朱明龙
周燕
华平
牛红星
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
East China University of Science and Technology
Original Assignee
East China University of Science and Technology
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by East China University of Science and Technology filed Critical East China University of Science and Technology
Priority to CNB2005100301989A priority Critical patent/CN100362098C/en
Publication of CN1778902A publication Critical patent/CN1778902A/en
Application granted granted Critical
Publication of CN100362098C publication Critical patent/CN100362098C/en
Expired - Fee Related legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Landscapes

  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The present invention discloses a serum-free culture medium suitable for the large-scale culture of various animal cells. The present invention is characterized in that DMEM/F12 is used as a basic culture medium, and various growth factors, hormones, essential amino acids and trace elements are added on the basis. The present invention has the characteristics that the growth of multiple cell strains or cell lines can be supported, the serum-free culture medium is close to or equivalent to culture media containing serum in the aspects of cell growth and product expression, the long term passage culture of cells is supported, the containment of low concentration protein is favorable to the separation and the purification of products and improvement in product quality, low price and suitability for large scale industrialized production are realized, etc. Above all, serum-free culture media formed by the present invention are suitable for the continuous perfusion culture of multiple cell strains or cell lines, high cell density and product concentration can be obtained, and the production process efficiency and the product output rate can be obviously increased.

Description

Be applicable to the serum free medium that multiple animal cell large-scale is cultivated
Technical field
The present invention relates to be used to produce in the animal cell large-scale high-density culture process serum free medium of biological products such as antibody, vaccine, gene recombinant protein.
Technical background
Animal cell culture has been widely used in producing all kinds of biologically active substances such as monoclonal antibody, virus vaccines, virus vector, immune-regulating factor, somatomedin, particular tumor antigens and range gene recombinant protein drug etc., usually need to add the bovine serum of a certain amount of (5%-10%) in the tradition culturing process, contain required somatomedin, hormone, carrier proteins, anchoring factor, trace element and other the nutritive substance of cell growth in the serum, can greatly promote the growth of cell and the expression of product.
Yet the application of serum also brings a lot of shortcomings:
(1) pollution of serum susceptible viral, mycoplasma or other pathogenic agent;
(2) different lot number serum differences cause the difference between product batches;
(3) existence of a large amount of serum proteins has increased the difficulty of downstream separation purifying, and its cost is raise, and the rate of recovery reduces;
(4) the part serum protein is difficult to thoroughly remove by the separation and purification means, has a strong impact on the final quality of product.
In order to overcome the various shortcomings that serum brings, the eighties plays the research and development that just has many investigators to be engaged in serum free medium, Darfler etc. have developed the CITTL serum free medium, and it is a basic medium with DMEM/F12 (1: 1), add the Regular Insulin of 1.5mg/L, 3.0mg/L Transferrins,iron complexes, the testosterone of 2nmol/L, the catalase of 5mg/L, β-Phosphoric acid glycerol esters of 1.5mg/L, 0.5mg/L two inferior oleoyl phosphatidyl cholines, be used for the cultivation of hybridoma; Murakami etc. have developed the DMEM/F12-ITES serum free medium, it is the Regular Insulin that has added 5mg/L on the basis of DMEM/F12, the Transferrins,iron complexes of 2-35mg/L, 20 μ mol/L thanomins, the 1nmol/L Sodium Selenite, this substratum is widely used in cultivating hybridoma.
In addition, United States Patent (USP) U.S.P.5,063,157 related serum free mediums are used for the mammalian cell suspension culture, having added Transferrins,iron complexes, Regular Insulin, peptone, beta-D-xylopyranose derivative, selenium and polyamines in basic medium, is a low-protein culture medium; United States Patent (USP) U.S.P.4,443,546 is the similar low-protein culture medium of another money; United States Patent (USP) U.S.P.4, some clones that 657,866 synthetic mediums that relate to a kind of whole component clear and definite (chemical defined medium) are used to cultivate the animal and human; European patent E.P.481,791 disclose a kind of serum free medium that is used for Chinese hamster ovary celI, and it contains water, osmotic pressure regulator, buffer reagent, sugar, amino acid, iron, somatomedin and other must composition.
At present, existing a large amount of commercialization serum free medium is developed and is applied to all kinds of animal cell culture, be used for CD Hybridoma substratum, Hybridoma-SFM substratum, the PFHM-IIProtein-free substratum of hybridoma as GIBCO company, be used for CD CHO substratum, the CHO-s-SFM II substratum of Chinese hamster ovary celI; Hybri-Max of SIGMA company or the like.
Above-mentioned serum free medium all has advantage separately, the growth of sustenticular cell greatly well, but also there are a lot of shortcomings in they simultaneously:
(1) because of costing an arm and a leg, use on a small scale in only suitable laboratory; And be not suitable for extensive bio-reactor;
(2) substratum is not optimized at cell, and the cell density of being supported is lower, causes the production process productive rate not high;
(3) though the sustenticular cell growth well of some substratum tends to cause the ability of cell expression product to reduce, even forfeiture;
(4) the substratum pair cell has very high specificity, and common a kind of substratum only is fit to a cell strain or clone, and is not suitable for other cell strain or clone.
Summary of the invention
The technical issues that need to address of the present invention are to disclose a kind of serum free medium that is applicable to that multiple animal cell large-scale is cultivated, to overcome the above-mentioned defective that prior art exists.
The serum free medium that is applicable to that multiple animal cell large-scale is cultivated of the present invention be with weight ratio be=1: 1 DMEM and F12 be basic medium, compositions such as interpolation Regular Insulin, Transferrins,iron complexes, thanomin, Sodium.alpha.-ketopropionate albumin, beta-mercaptoethanol, trace element, hormone, indispensable amino acid;
The component of substratum DMEM and F12 is all open on GIBCO or SIGMA company products catalogue, and the relevant personnel can consult, and the present invention repeats no more, and can adopt business-like product, as the product of GIBCO or SIGMA company;
The component and the content of the final serum free medium that forms of the present invention are as follows:
Composition The Chinese chemical name Production firm
Inorganic salt (mg/L)
CaCl 2 Calcium chloride SIGMA ?116.60
CuSO 4·5H 2O Copper sulfate SIGMA ?0.0013
?Fe(NO 3) 3·9H 2O Iron nitrate SIGMA ?0.05
?FeSO4·7H 2O Ferrous sulfate SIGMA ?0.417
?KCl Repone K SIGMA ?311.80
?MgCl 2 Magnesium chloride SIGMA ?28.64
?MgSO 4 Sal epsom SIGMA ?48.84
?NaCl Sodium-chlor SIGMA ?6995.50
?NaHCO 3 Sodium bicarbonate SIGMA ?2440
?NaH 2PO 4H 2O SODIUM PHOSPHATE, MONOBASIC SIGMA ?62.50
?Na 2HPO 4 Sodium phosphate dibasic SIGMA ?71.02
?ZnSO 4·7H 2O Zinc sulfate SIGMA ?0.432
L-amino acid (mg/L)
?Alanine L-Ala SIGMA ?4.45
?Arginine·HCl Arginine SIGMA ?147.5-210.5
?Asparagine·H 2O Asparamide SIGMA ?7.5-67.5
?Aspartic?acid Aspartic Acid SIGMA ?6.65-26.6
?Cysteine·H 2O Halfcystine SIGMA ?17.56-35.12
?Cystein·2HCl Gelucystine SIGMA ?31.29-62.58
?Glutamic?acid L-glutamic acid SIGMA ?7.35-29.4
?Glutamine Glutamine SIGMA ?584-1168
?Glycine Glycine SIGMA ?18.75-37.5
?Histidine·HCl·H 2O Histidine SIGMA ?31.48-104.93
?Isoleucine Isoleucine SIGMA ?52.4-131.0
?Leucine Leucine SIGMA ?58.95-157.2
?Lysine·HCl Methionin SIGMA ?91.25-182.5
?Methionine Methionine(Met) SIGMA ?16.39-59.6
?Phenylalanine Phenylalanine SIGMA ?33.0-82.5
?Proline Proline(Pro) SIGMA ?17.25-34.5
?Serine Serine SIGMA ?26.25-94.5
?Threonine Threonine SIGMA ?53.55-95.2
?Tryptophan Tryptophane SIGMA ?8.16-51.0
?Tyrosine·2Na·2H 2O Tyrosine SIGMA ?55.79-139.5
?Valine Xie Ansuan SIGMA ?52.65-117.0
VITAMIN/coenzyme (mg/L)
?Biotin Vitamin H SIGMA ?0.0035
?Pantothenate·Ca Calcium pantothenate SIGMA ?2.24
?Choline·Cl Choline SIGMA ?8.98
?Folic?acid Folic acid SIGMA ?2.65
?Inositol Inositol SIGMA ?12.60
?Niacinamide Niacinamide SIGMA ?2.02
?Pyridoxine·HCl Vitamin B6 SIGMA ?2.031
?Riboflavin Riboflavin SIGMA ?0.219
Thiamine·HCl Thiamines SIGMA ?2.17
Thymidine Thymidine SIGMA ?0.365
Vitamin?B 12 Vitamin B12 SIGMA ?0.68
Trace element (mg/L)
Na 2SeO 3 Sodium Selenite SIGMA ?0.0008-0.0035
MnSO 4·4H 2O Manganous sulfate SIGMA ?0.00008-0.0008
Na 2SiO 3·5H 2O Water glass SIGMA ?0.002-0.01
(NH 4) 6Mo 7O 24·4H 2O Molybdenum acid ammonia SIGMA ?0.0017-0.017
NH 4VO 3 Vanadic acid ammonia SIGMA ?0.00006-0.0006
NiCl 2·6H 2O Nickelous chloride SIGMA ?0.00002-0.00024
SnCl 2·2H 2O Tin chloride SIGMA ?0.00002-0.00023
Other composition (mg/L)
Glucose Glucose SIGMA ?1000-7200
Na?Hypoxanthine Xanthoglobulin SIGMA ?2.39
Linoleic?acid Linolic acid SIGMA ?0.042
Lipoic?acid Thioctic Acid SIGMA ?0.105
Phenol?red Phenol red SIGMA ?8.10
Sodium?Putrescine·2HCl Putrescine SIGMA ?0.081
Pyruvate·Na Sodium.alpha.-ketopropionate SIGMA ?220.00
Regular Insulin SIGMA ?5-10
Transferrins,iron complexes 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
The B-mercaptoethanol SIGMA ?0.61-1.83
Thanomin SIGMA ?1.22-12.2
The preparation method of substratum of the present invention is foolproof, can adopt conventional method that the no thermal source ultrapure water of said components dissolving can be prepared from.
Substratum of the present invention can be used for that going down to posterity of clone such as hybridoma, rCHO cell, 293 cells or cell strain cultivated and high-density continous pouring is cultivated.It has the following advantages:
1. can support the growth of a plurality of cell strains or clone;
Aspect cell growth, the product expression with contain blood serum medium near or quite;
3. the cultivation of going down to posterity for a long time of sustenticular cell;
4. the protein that contains low concentration helps the separation and purification of product, improves the quality of product;
5. cheap, be suitable for large-scale industrial production;
The most important thing is that the formed serum free medium of the present invention is applicable to the continous pouring cultivation of a plurality of cell strains or clone, can obtain higher cell density and production concentration, can significantly improve production process efficient and product yield.
Description of drawings
Fig. 1 is that the growth of HB58 hybridoma continous pouring cultured cells is expressed curve with product.
Fig. 2 is that the growth of Chinese hamster ovary celI continous pouring cultured cells is expressed curve with product.
Fig. 3 is the growth curve of 293 cells in batch cultivation.
Embodiment
Embodiment 1
Serum free medium of the present invention can be used for the high-density continous pouring of hybridoma to be cultivated, and the moiety of substratum 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) 2·9H 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
?Arginmine·HCl Arginine 210.5
?Asparagine·H 2O Asparamide 7.5
?Aspartic?acid Aspartic Acid 26.6
?Cysteine·H 2O Halfcystine 17.56
?Cystein·2HCl Gelucystine 31.29
?Glutamic?acid L-glutamic acid 14.7
?Glutamine Glutamine 1168
?Glycine Glycine 18.75
?Histidine·HCl·H 2O Histidine 104.93
?Isoleucine Isoleucine 131.0
?Leucine Leucine 157.2
?Lysine·HCl Methionin 182.5
?Methionine Methionine(Met) 16.39
?Phenylalanine Phenylalanine 82.5
?Proline Proline(Pro) 34.5
?Serine Serine 26.25
?Threonine Threonine 53.55
?Tryptophan Tryptophane 51.0
?Tyrosine·2Na·2H 2O Tyrosine 139.5
?Valine Xie Ansuan 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 0.0016
?MnSO 4·4H 2O Manganous sulfate 0.00016
?Na 2SiO 3·5H 2O Water glass 0.005
?(NH 4) 6Mo 7O 24·4H 2O Molybdenum acid ammonia 0.0034
?NH 4VO 3 Vanadic acid ammonia 0.00012
?NiCl 2·6H 2O Nickelous chloride 0.0001
?SnCl 2·2H 2O Tin chloride 0.0001
Other composition (mg/L)
?Glucose Glucose 6300
?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
Transferrins,iron complexes 10
Albumin 100
Hormone (mg/L)
Hydrocortisone 0.036
Dexamethasone 0.039
Progesterone 0.006
Estradiol 0.0027
The B-mercaptoethanol 0.61
Thanomin 12.2
Said components is mixed and the no thermal source ultrapure water of dissolving, can obtain substratum.
HB58 hybridoma (obtaining from ATCC) serum free medium of the present invention behind the adaptation of virus, is inoculated inoculum density 2.0 * 10 in 2 liters of bio-reactors of BF-2 (German B.BRAUN company) 5Cells/ml, cultivate begin after 56 hours the perfusion, irrigation rate is 0.5 (1/day), the perfusion culture base is serum free medium of the present invention, be cultured to 200 hours after cell density maintain 1.2 * 10 7About cells/ml, the monoclonal anti bulk concentration maintains 500mg/L left and right sides (see figure 1), and the result who criticizes cultivation with ordinary culture medium compares, and cell density and monoclonal antibody concentration all improve more than 8 times.Among the figure, curve 1 is a viable cell density, and curve 2 is total cell density, and curve 3 is the monoclonal antibody expression amount.
Embodiment 2
Serum free medium of the present invention can be used for the high-density continous pouring of rCHO cell to be cultivated, and the moiety of substratum 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 168.4
?Asparagine·H 2O Asparamide 67.5
?Aspartic?acid Aspartic Acid 6.65
?Cysteine·H 2O Halfcystine 17.56
?Cystein·2HCl Gelucystine 62.58
?Glutamic?acid L-glutamic acid 7.35
?Glutamine Glutamine 1168
?Glycine Glycine 37.5
?Histidine·HCl·H 2O Histidine 47.22
?Isoleucine Isoleucine 131.0
?Leucine Leucine 157.2
?Lysine·HCl Methionin 127.7
?Methionine Methionine(Met) 29.8
?Phenylalanine Phenylalanine 33.0
?Proline Proline(Pro) 34.5
?Serine Serine 94.5
?Threonine Threonine 95.2
?Tryptophan Tryptophane 20.4
?Tyrosine·2Na·2H 2O Tyrosine 105.2
?Valine Xie Ansuan 93.6
?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 0.0016
?MnSO 4·4H 2O Manganous sulfate 0.00016
?Na 2SiO 3·5H 2O Water glass 0.005
?(NH 4) 6Mo 7O 24·4H 2O Molybdenum acid ammonia 0.0034
?NH 4VO 3 Vanadic acid ammonia 0.00012
?NiCl 2·6H 2O Nickelous chloride 0.00004
?SnCl 2·2H 2O Tin chloride 0.00004
Other composition (mg/L)
?Glucose Glucose 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
?Pymvate·Na Sodium.alpha.-ketopropionate 220.00
Regular Insulin 10
Transferrins,iron complexes 10
Albumin 100
Hormone (mg/L)
Hydrocortisone 0.036
Dexamethasone 0.039
Progesterone 0.006
Estradiol 0.0027
The B-mercaptoethanol 0.61
Thanomin 12.2
(rCHO SS3 A2, expressing human anticoagulin m) behind the adaptation of virus, inoculates inoculum density 2.0 * 10 in 2 liters of bio-reactors of B.BRAUN in serum free medium of the present invention with the rCHO cell 5Cells/ml, cultivate begin after 40 hours the perfusion, irrigation rate is 0.58 (1/day), the perfusion culture base is serum free medium of the present invention, be cultured to 255 hours after cell density maintain 0.9-1.0 * 10 7About cells/ml, production concentration maintains 350-380U/L left and right sides (see figure 2), and the result who criticizes cultivation with ordinary culture medium compares, and cell density and monoclonal antibody concentration improve 6 times and 5 times respectively.Among the figure, curve 4 is a viable cell density, the total cell density of curve, and curve 6 is the product expression amount.
Embodiment 3
Serum free medium of the present invention can be used for the cultivation of 293 cells, and the moiety of substratum 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
?Arginme·HCl Arginine 168.4
?Asparagine·H 2O Asparamide 7.5
?Aspartic?acid Aspartic Acid 6.65
?Cysteine·H 2O Halfcystine 17.56
?Cystein·2HCl Gelucystine 31.29
?Glutamic?acid L-glutamic acid 7.35
?Glutamine Glutamine 584
?Glycine Glycine 18.75
?Histidine·HCl·H 2O Histidine 31.48
?Isoleucine Isoleucine 54.47
?Leucine Leucine 59.05
?Lysine·HCl Methionin 91.25
?Methionine Methionine(Met) 29.8
?Phenylalanine Phenylalanine 33.0
?Proline Proline(Pro) 34.5
?Serine Serine 26.25
?Threonine Threonine 53.45
?Tryptophan Tryptophane 20.4
?Tyrosine·2Na·2H 2O Tyrosine 55.79
?Valine Xie Ansuan 52.8
?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
?Niacimmide Niacinamide 2.02
?Pyridoxine·HCl Vitamin B6 2.031
?Riboflavin Riboflavin 0.219
?Thiarnine·HCl Thiamines 2.17
?Thymidine Thymidine 0.365
?Vitamin?B 12 Vitamin B12 0.68
Trace element (mg/L)
?Na 2SeO 3 Sodium Selenite 0.0016
?MnSO 4·4H 2O Manganous sulfate 0.00016
?Na 2SiO 3·5H 2O Water glass 0.005
?(NH 4) 6Mo 7O 24·4H 2O Molybdenum acid ammonia 0.0034
?NH 4VO 3 Vanadic acid ammonia 0.00012
?NiCl 2·6H 2O Nickelous chloride 0.00004
?SnCl 2·2H 2O Tin chloride 0.00004
Other composition (mg/L)
?Glucose Glucose 3150
?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 10
Transferrins,iron complexes 10
Albumin 100
Hormone (mg/L)
Hydrocortisone 0.036
Dexamethasone 0.039
Progesterone 0.006
Estradiol 0.0027
The B-mercaptoethanol 0.61
Thanomin 12.2
293 cells behind the adaptation of virus, are inoculated in 2 liters of bio-reactors of B.BRAUN and criticized cultivation in serum free medium of the present invention, and inoculum density is 2.45 * 10 5Cells/ml, the lag phase is almost can't see in the growth of cell as seen from Figure 3, promptly enters exponential phase of growth after the inoculation, and the average specific growth velocity is 0.46day -1, maximum viable cell density is 11.0 * 10 5Cells/ml.Among the figure, curve 7 is a viable cell density, and curve 8 is the viable cell ratio.

Claims (2)

1. a serum free medium that is applicable to that multiple animal cell large-scale is cultivated is characterized in that, final nutrient media components and the content that forms is as follows:
The final nutrient media components that forms The Chinese chemical name Content 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 168.4-210.5 Asparagine·H 2O Asparamide 7.5-67.5 Aspartic?acid Aspartic Acid 6.65-26.6 Cysteine·H 2O Halfcystine 17.56 Cystein·2HCl Gelucystine 31.29-62.58 Glutamic?acid L-glutamic acid 7.35-14.7 Glutamine Glutamine 584-1168 Glycine Glycine 18.75-37.5 Histidine·HCl·H 2O Histidine 31.48-104.93 Isoleucine Isoleucine 54.47-131.0 Leucine Leucine 59.09-157.2 Lysine·HCl Methionin 91.25-182.5 Methionine Methionine(Met) 16.39-29.8 Phenylalanine Phenylalanine 33.0-82.5 Proline Proline(Pro) 34.5 Serine Serine 26.25-94.5 Threonine Threonine 53.55-95.2 Tryptophan Tryptophane 20.4-51.0 Tyrosine·2Na·2H 2O Tyrosine 55.79-139.5
?Valine Xie Ansuan 52.8-117.0 ?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
2. the application of serum free medium according to claim 1 is characterized in that, is used for the go down to posterity cultivation and the high-density continous pouring cultivation of hybridoma, rCHO cell or 293 cells.
CNB2005100301989A 2005-09-29 2005-09-29 Non-serum culture medium for multiple animal cell large-scale culture Expired - Fee Related CN100362098C (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CNB2005100301989A CN100362098C (en) 2005-09-29 2005-09-29 Non-serum culture medium for multiple animal cell large-scale culture

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CNB2005100301989A CN100362098C (en) 2005-09-29 2005-09-29 Non-serum culture medium for multiple animal cell large-scale culture

Publications (2)

Publication Number Publication Date
CN1778902A CN1778902A (en) 2006-05-31
CN100362098C true CN100362098C (en) 2008-01-16

Family

ID=36769357

Family Applications (1)

Application Number Title Priority Date Filing Date
CNB2005100301989A Expired - Fee Related CN100362098C (en) 2005-09-29 2005-09-29 Non-serum culture medium for multiple animal cell large-scale culture

Country Status (1)

Country Link
CN (1) CN100362098C (en)

Families Citing this family (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101113432B (en) * 2007-06-25 2011-04-27 南华大学 Non-serum or low-serum-concentration cell culture method
CN101386837B (en) * 2007-09-12 2011-06-29 北京清大天一科技有限公司 Zooblast cultivation method
CN101386836B (en) * 2007-09-12 2010-11-24 北京清大天一科技有限公司 Zooblast culture medium dry powder composition, culture medium composition and preparation method thereof
CN102115728B (en) * 2009-12-31 2012-09-26 北京清大天一科技有限公司 Serum-free animal cell culture medium dry powder, liquid culture medium and preparation method thereof
EP2968544A4 (en) * 2013-03-15 2016-10-12 Hoffmann La Roche Cell culture media and methods of antibody production
CN103468638B (en) * 2013-09-23 2016-01-20 天津瑞普生物技术股份有限公司 A kind of extensive suspension culture method of 293 cells
CN104513805B (en) * 2013-10-07 2019-02-26 鲁南制药集团股份有限公司 A method of producing anti-CD 20 antibodies
CN105462916B (en) * 2015-12-22 2018-04-13 肇庆大华农生物药品有限公司 A kind of serum free medium of culture 145 cells of Marc and preparation method thereof
EP3208331A1 (en) * 2016-02-17 2017-08-23 PromoCell bioscience alive GmbH Biomedizinische Produkte Chemically defined medium for the culture of cancer stem cell (csc) containing cell populations
CN106635958A (en) * 2016-12-24 2017-05-10 严志海 CHO (Chinese Hamster Ovary) cell culture medium
CN107099508A (en) * 2017-06-23 2017-08-29 曲宝赤 A kind of Serum-free Hybridoma cell culture medium
CN107308447A (en) * 2017-07-10 2017-11-03 广州博恒生物科技有限公司 A kind of preparation method of triple inactivated vaccine
CN108384747A (en) * 2018-03-05 2018-08-10 安徽省农业科学院园艺研究所 Express the Chinese hamster ovary celI serum free suspension cultural method of Rabies virus antibody
CN109234223B (en) * 2018-11-21 2021-01-19 南京基蛋生物医药有限公司 Low-protein serum-free cell culture medium
CN110117573A (en) * 2019-04-15 2019-08-13 河北省科学院生物研究所 A kind of serum-free cell culture medium and its application
CN110894484A (en) * 2019-11-25 2020-03-20 杭州职业技术学院 Universal plant culture medium and tissue culture
CN111826341A (en) * 2020-07-23 2020-10-27 上海奥浦迈生物科技有限公司 Method for in vitro culture of HEK293 cells for transient expression of proteins
CN114736864B (en) * 2020-12-23 2024-01-26 武汉睿健医药科技有限公司 Neural stem cell induced differentiation culture medium and induced differentiation method
CN114480294B (en) * 2021-12-15 2024-01-26 上海捷门生物技术有限公司 Serum-free culture medium suitable for hybridoma cell wall-attached growth
CN114540274B (en) * 2022-02-17 2023-10-20 上海健士拜生物科技有限公司 Serum-free medium, preparation method thereof and method for culturing 2BS cells by using serum-free medium
CN116790483B (en) * 2023-08-22 2023-10-20 苏州依科赛生物科技股份有限公司 CHO cell serum-free medium and application thereof

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS61124378A (en) * 1984-11-22 1986-06-12 Agency Of Ind Science & Technol Culture medium composition for cell originated from blood stem cell
JPS62155083A (en) * 1985-12-28 1987-07-10 Hagiwara Yoshihide Novel mutant strain of human b cell lymphoblast, human/ human hybridoma thereof and antibody produced therewith
WO1996040866A1 (en) * 1995-06-07 1996-12-19 Novartis Ag Serum-free media for primitive hematopoietic cells and methods of use thereof
US6153582A (en) * 1998-11-05 2000-11-28 Bausch & Lomb Surgical, Inc. Defined serumfree medical solution for ophthalmology
CN1431299A (en) * 2003-01-17 2003-07-23 江西医学院 Serum-free medium of human epidermis cell and its cultivating method and application
CN1563365A (en) * 2004-03-12 2005-01-12 西北农林科技大学 Method for separating and purifying skin epidermal stem cells

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS61124378A (en) * 1984-11-22 1986-06-12 Agency Of Ind Science & Technol Culture medium composition for cell originated from blood stem cell
JPS62155083A (en) * 1985-12-28 1987-07-10 Hagiwara Yoshihide Novel mutant strain of human b cell lymphoblast, human/ human hybridoma thereof and antibody produced therewith
WO1996040866A1 (en) * 1995-06-07 1996-12-19 Novartis Ag Serum-free media for primitive hematopoietic cells and methods of use thereof
US6153582A (en) * 1998-11-05 2000-11-28 Bausch & Lomb Surgical, Inc. Defined serumfree medical solution for ophthalmology
CN1431299A (en) * 2003-01-17 2003-07-23 江西医学院 Serum-free medium of human epidermis cell and its cultivating method and application
CN1563365A (en) * 2004-03-12 2005-01-12 西北农林科技大学 Method for separating and purifying skin epidermal stem cells

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
一种适合骨髓瘤、杂交瘤细胞生长的无血清培养基. 邓辉南等.生物化学与生物物理进展，第20卷第1期. 1993 *
中华仓鼠卵巢（CHO）工程细胞无血清培养的研究. 李萍等.微生物学免疫学进展，第32卷第4期. 2004 *
氨基酸对中华仓鼠卵巢（CHO）细胞在无血清培养基中的作用. 胡雪梅等.华东理工大学学报，第22卷第3期. 1996 *

Also Published As

Publication number Publication date
CN1778902A (en) 2006-05-31

Similar Documents

Publication Publication Date Title
CN100362098C (en) Non-serum culture medium for multiple animal cell large-scale culture
CN102827804B (en) Culture medium and method suitable for the amplification culture of Vero cell microcarrier suspension
CN101760442A (en) Serum-free medium for MDCK cell large-scale adherent culture and single-cell suspension culture
US5122469A (en) Method for culturing Chinese hamster ovary cells to improve production of recombinant proteins
CN101418330B (en) Non protein culture medium adapted to large-scale culture of NSO cell and production of antibody
CN109337861B (en) CHO cell serum-free medium supporting high expression of product
CN102858953B (en) The cell culture medium improved
US20150329830A1 (en) Myeloma cell culture in transferrin-free low iron medium
CN112795531B (en) CHO cell serum-free and protein-free culture medium and application thereof
JPH06511389A (en) animal cell culture
JPS63267269A (en) Basal nutrition medium for cell culture
CN101603026A (en) Be suitable for the animal origin-free low-protein culture medium of zooblast products production
CN107460159A (en) Serum-free, without albumen supplemented medium and preparation method thereof and use
CN101195817A (en) Hybrid tumor cell amplification culture medium and uses thereof
CN102268403A (en) Serum-free culture medium applicable to large-scale single-cell suspension culture of baby hamster kidney cell
CN104593316A (en) Insect cell serum-free culture medium and application thereof
CN106635953A (en) Serum-free protein-free cell culture medium
CN105543163A (en) Serum-free culture medium used for full-suspension culture of MDCK (Madin Darby Canine Kidney) cells
CN106190950A (en) A kind of Chinese hamster ovary celI Serum-free and protein-free medium and preparation method thereof
CN102317440A (en) Improved culture medium additive and application method thereof
EP0659880B1 (en) Medium for culturing animal cells or antibody-producing cells
CN102021139A (en) Chinese hamster ovary culture medium as well as preparation method and application thereof
CN106399224A (en) Serum-free and protein-free cell culture medium
AU662491B2 (en) Medium for culture of mammalian cells
CN105567628A (en) Low serum medium for full-suspending culture of MDCK cells

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
C14 Grant of patent or utility model
GR01 Patent grant
C19 Lapse of patent right due to non-payment of the annual fee
CF01 Termination of patent right due to non-payment of annual fee