CN1696283A - Serum-free medium suitable to culturing ovary cells of Chinese hamster - Google Patents

Serum-free medium suitable to culturing ovary cells of Chinese hamster Download PDF

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CN1696283A
CN1696283A CN 200410018258 CN200410018258A CN1696283A CN 1696283 A CN1696283 A CN 1696283A CN 200410018258 CN200410018258 CN 200410018258 CN 200410018258 A CN200410018258 A CN 200410018258A CN 1696283 A CN1696283 A CN 1696283A
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substratum
acid
chinese hamster
hamster ovary
cell
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CN100537751C (en
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张立
张元兴
张芳
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East China University of Science and Technology
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East China University of Science and Technology
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Abstract

A non-serum culture medium for culturing CHO cells is prepared from DMEM/F12 as basic culture medium, transferrin, insulin, ethanolamine, etc.

Description

The serum free medium that a kind of suitable Chinese hamster ovary cell is cultivated
Technical field
The present invention relates to field of cell culture.More specifically, the present invention relates to be used for substratum and the corresponding cultural method that Chinese hamster ovary celI is cultivated.
Background technology
Because mammalian cell has the function of post transcriptional modificaiton, utilize the foreign protein of mammalian cell production unrivaled superiority to be arranged than the albumen of producing with prokaryotic organism, this technology has obtained attention more and more widely.Chinese hamster ovary cell-Chinese hamster ovary celI (Chinese hamster ovary cell) is the most widely used at present cell, be widely used in producing range gene engineered protein product, for example, human erythropoietin (rEPO), human thrombopoietin (rTPO), tissue plasminogen activator (tPA), various antibody etc.
Foreign protein can obtain expressing in Chinese hamster ovary celI usually by two kinds of different modes, i.e. DHFR defective type (Tetrahydrofolate dehydrogenase defective type) and glutamine synthetase system.By the foreign protein output height of glutamine synthetase system expression, stable, and come into one's own.
The cultivation of Chinese hamster ovary celI is normally carried out in the substratum of serum is arranged, though cell density and production concentration are higher, because albumen is too complicated with other macromolecular substance in the serum, has influenced the separation and the purifying of product, has reduced the yield and the purity of product; Simultaneously, because the interpolation of serum also makes cost improve greatly.Therefore, many researchers are devoted to develop low serum or serum free medium.
The CITTL serum free medium of exploitations such as Darfler is to be basic medium with DMEM/F12 (1: 1), add the catalase of 5mg/L, 15mg/L Regular Insulin, 1.5～3.0mg/L Transferrins,iron complexes, the 20nM Sodium Selenite, 2nM testosterone, 0.5mg/L dilinoleoylphosphatidylcholine and 1.5mg/L β-Phosphoric acid glycerol esters.This substratum can be supported the cloning growth of insect cell sf9 cell, also can be used for the cultivation of hybridoma.Yet this substratum is not too suitable to Chinese hamster ovary celI.
The serum free medium of exploitations such as Murakami also is to be basic medium with DMEM/F12, add 5mg/L Regular Insulin, 2～35mg/L Transferrins,iron complexes, 20 μ M thanomins and 1nM Sodium Selenite, the DMEM/F12-ITES prescription that Here it is now is widely used can be cultivated multiple hybridoma.
The protein-free medium of exploitations such as Cleveland (United States Patent (USP) 4767704) is mainly studied with regard to the interpolation of trace element, by interpolation, developed a kind of protein-free medium to materials such as the interpolation of elements such as copper, iron, magnesium, zinc, silicon, manganese, vanadium, nickel, tin, aluminium, calcium, bromine, chlorine, iodine, selenium, germanium and progesterone, putrescine, oleic acid, pyrimidines.This is invented topmost contribution and is to make people to recognize the importance of trace element.
Darfler equals the substratum that contains antioxidant (United States Patent (USP) 4927762) of nineteen ninety exploitation, illustrate cell and in the substratum of reductibility, grow and be more suitable for, point out that simultaneously the interpolation of materials such as N-acetylcystein, mercaptoethanol can be played oxidation resistant effect.
In sum, though the various countries scientific worker has developed many serum free mediums, but each substratum all only is applicable to a kind of or class cell growth, present most serum free medium all contains high molecular weight protein class additives such as albumin, had a strong impact on the separation and purification of product, cost is also higher simultaneously.Especially lack and be applicable to that Chinese hamster ovary celI cultivates, and culture effect and have blood serum medium quite or better substratum.
Therefore, this area presses for the new suitable Chinese hamster ovary celI of exploitation serum free medium that cultivate, low protein content.
Summary of the invention
Purpose of the present invention just provides a kind of substratum of new cultivation Chinese hamster ovary celI, this substratum can make Chinese hamster ovary celI vigorous growth, cell density and cytoactive all can compare favourably with blood serum medium is arranged, and the substratum that can substitute serum fully is used for the cultivation of Chinese hamster ovary celI.
Another object of the present invention provides the purposes of described substratum.
The inventor is through for many years extensive and deep research, found in the serum free medium of Chinese hamster ovary celI, except the general composition and Regular Insulin, Transferrins,iron complexes, thanomin and Sodium Selenite that add ordinary culture medium, add putrescine and vitamins C and can promote the growth of Chinese hamster ovary celI effectively, thereby make culture effect be equivalent to or be better than that blood serum medium is arranged.Finished the present invention on this basis.
In a first aspect of the present invention, a kind of substratum that is used for Chinese hamster ovary cell is provided, described substratum is to be made of basic medium and additive, wherein additive contains:
The 2-20mg/L Transferrins,iron complexes;
2-20mg/L Regular Insulin;
The 1-10mg/L thanomin;
0.005～0.05mg/L selenite;
The 2-10mg/L putrescine;
The 5-30mg/L vitamins C is by the cumulative volume of the substratum of Chinese hamster ovary cell.
In another preference, described basic medium is DMEM/F12, and described additive also contains the 0-2.5mg/L beta-mercaptoethanol.
In another preference, described additive also contains the trace element that is selected from down group: Cu, Fe, Zn, Mg, Se, Co, Mo, Sn, Si.
In another preference, described additive contains following trace element:
AgNO 3???????????????????????0.01～0.05mg/L
AlCl 3·6H 2O???????????????0.3～3.0mg/L
Ba(C 2H 3O 2) 2?????????????0.01～0.1mg/L
3CdSO 4·8H 2O??????????????0.001～0.010mg/L
CoCl 2·6H 2O???????????????0.03～0.30mg/L
CuSO 4·5H 2O???????????????0.001～0.005mg/L
FeSO 4·7H 2O???????????????0.2～2.0mg/L
KI??????????????????????????0.0001～0.001mg/L
KBr?????????????????????????0.0001～0.001mg/L
MnSO 4?H 2O?????????????????0.0001～0.001mg/L
NaF?????????????????????????0.001～0.01mg/L
Na 2SiO 3????????????????????0.005～0.05mg/L
NH 4VO 3?????????????????????0.01～0.1mg/L
(NH 4) 6MoO 24·4H 2O??????0.01～0.1mg/L
NiCl 2·6H 2O???????????????0.01～0.1mg/L
SnCl 2·2H 2O???????????????1.00～10.00mg/L
ZnSO 4???????????????????????0.2～2.0mg/L。
In another preference, described additive contains following amino acid:
L-L-Ala 0～30mg/L
L-aspartic acid 0～10mg/L
Altheine 20～100mg/L
L-arginine 10～50mg/L
L-L-glutamic acid 10～100mg/L
L-glutaminate 0～500mg/L
L-glycine 0～40mg/L
L-Histidine 0～50mg/L
L-Isoleucine 25～100mg/L
L-leucine 25～100mg/L
L-Methionin 10～100mg/L
L-methionine(Met) 10～50mg/L
L-phenylalanine 10～60mg/L
L-proline(Pro) 10～30mg/L
L-Serine 0～100mg/L
L-Threonine 0～100mg/L
L-tryptophane 0～50mg/L
L-Xie Ansuan 0～50mg/L
L-tyrosine 0～40mg/L.
In another preference, described additive contains following VITAMIN:
Folic acid 0～10mg/L
Vitamins B 60～0.3mg/L
Vitamins B 1200～0.88mg/L
Choline chloride 60 0～10mg/L
Scyllitol 0～30mg/L
Vitamin H 0～0.2mg/L
Niacinamide 0～5.0mg/L
Riboflavin 0～0.2mg/L
VitB1 0～5mg/L
Thioctic Acid 0～5mg/L.
In another preference, described additive contains following material:
Beta-mercaptoethanol 0～2.5mg/L
HEPES?????????????????1000～2500mg/L
Sodium.alpha.-ketopropionate 0～330mg/L
Primatone?????????????1000～5000mg/L
Lipid mixture 0.1～0.5% (v/v)
Reduced glutathion 0.3～3.0mg/L
Xanthoglobulin 0～5mg/L
VITAMIN B4 5～10mg/L
Guanine 5～10mg/L
Uridylic 5～10mg/L
Thymus pyrimidine 0.2～2mg/L
Cytosine(Cyt) 5～10mg/L
Ribose 1～10mg/L
Ribodesose 1～10mg/L
Amino sulfoxide-methionine(Met) (MSX) 9～45mg/L
In another preference, described lipid mixture contains following composition:
Arachidonic acid 2mg/L
Cholesterol 220mg/L
DL-alpha-tocopherol acetate 70mg/L
Linolic acid 10mg/L
Linolenic acid 10mg/L
Myristic acid 10mg/L
Oleic acid 10mg/L
Palm diluted acid 10mg/L
Palmitinic acid 10mg/L
Stearic acid 10mg/L
Tween 80 2200mg/L
PluronicF-68??????????100000mg/L
Cumulative volume by lipid mixture.
In a second aspect of the present invention, the purposes of substratum of the present invention is provided, it is used to cultivate Chinese hamster ovary cell.
In another preference, described Chinese hamster ovary cell is the Chinese hamster ovary cell that has the glutamine synthetase system.
In a third aspect of the present invention, a kind of method of cultivating Chinese hamster ovary celI also is provided, comprise step: at inoculation of medium Chinese hamster ovary celI of the present invention, (as 37 ± 2 ℃, under 5 ± 1% carbonic acid gas) cultivate Chinese hamster ovary celI for some time (as 5-20 days) under the condition that is fit to growth then.
Description of drawings
Fig. 1 has shown the CHO culture experiment result that blood serum medium is arranged of serum free medium of the present invention and prior art.Among the figure: ▲ adopt the cell that blood serum medium is arranged to grow; △ adopts the cell growth of serum free medium.
Embodiment
In order to develop the serum free medium of Chinese hamster ovary celI (for example passing through the Chinese hamster ovary celI of glutamine synthetase system expression foreign protein), should make protein content minimizing as far as possible in the substratum, to reduce cost, help separation and purification, be suitable for the characteristics of suitability for industrialized production.
For cell will be grown in serum free medium, essential conjugated protein, hormone, somatomedin, the lower molecular weight nutritive substance of adding is as trace element, lipid etc.In many basic mediums, nutritive substances such as many trace elements, carbohydrate have been contained.For example, DMEM/F12 is exactly a commercial basic medium commonly used in the cell cultures.This substratum is made up of multiple materials such as glucose, amino acid, VITAMIN, inorganic salt.Can buy from Sigma, GIBCO company etc. and obtain.In order to be fit to the growth of Chinese hamster ovary celI, should on the basis of DMEM/F12, add following material:
(1) Transferrins,iron complexes:
In conjunction with the glycoprotein of iron, with the single-minded receptor acting of cell surface, transmit iron ion, assist the absorption of iron, but network and harmful ion play detoxification, also have the effect of somatomedin simultaneously.
(2) hormone and somatomedin
Regular Insulin: promote glucose and amino acid by cytolemma, be beneficial to the absorption and the metabolism of cell; Promote lipid and the proteinic synthetic phosphorylation that reaches the metabolism intermediate.Regular Insulin is kept cell in the fission process neutralization and is played an important role at the physiological metabolism state aspect of health.
(3) lower molecular weight nutritive substance
Trace element: comprise Cu, Fe, Zn, Mg, Se, Co, Mo, Sn, Si or the like, mainly play a part to regulate, transmit and control.Effect in serum free medium is particularly important.
Fe: the prothetic group of enzyme and protoheme is the integral part of respiratory chain in the plastosome.
Cu: the prothetic group of superoxide-dismutase is the integral part of respiratory chain in the plastosome.
Mg: ATP enzyme, kinases etc. are played activation.
Zn: the prothetic group of enzyme.
Co:B 12Integral part.
Se: the integral part of glutathione oxidase, be present in the polypeptide chain with the form of seleno-cysteine, constitute the active centre of enzyme.Selenoperoxidase has oxidation resistant effect.
(4) VITAMIN:
In cell growth metabolism process, VITAMIN is not as the energy, and it is the cell moiety; The various Metabolic activities of cell have left VITAMIN and all can't carry out, so it are essential.Especially vitamins C, it plays the effect of effective promotion Chinese hamster ovary celI growth.
(5) other
Thanomin: be the important component part of serum free medium, synthetic relevant with phosphatide (phosphatidylethanolamine).
Putrescine: effectively promote the CHO growth.
Add above-mentioned substance on the basis of DMEM/F12, make the Chinese hamster ovary celI can vigorous growth, cell density and cytoactive be equivalent to or be better than to have the cultivation results of serum under cultivating.
In addition, in substratum of the present invention, also can add following composition:
The lipid acid cell growth all has certain promoter action.Preferred lipid acid comprises (but being not limited to): arachidonic acid, cholesterol, DL-acetic acid a-tocopherol, linolic acid, linolenic acid, myristic acid, oleic acid, physetoleic acid, palmitinic acid, stearic acid etc.Usually, the total content of lipid acid in substratum is 0.3-2mg/L.
Beta-mercaptoethanol: promote the absorption of Gelucystine, also can make gsh be in reduced state, thereby the protection cell is avoided the injury of hydrogen peroxide.
Particularly, the preferred CHO substratum of the present invention is a kind of composition, forms (addition is mg/L) by basic medium DMEM/F12 and following additive:
Transferrins,iron complexes 2-20mg/L
Regular Insulin 2-20mg/L
Thanomin 1-10mg/L
Selenite 0.005～0.05mg/L
Putrescine 2-10mg/L
Vitamins C 5-30mg/L
When Transferrins,iron complexes, Regular Insulin, thanomin, selenite, putrescine, ascorbic content are respectively in above-mentioned scope, can effectively promote the growth of Chinese hamster ovary celI synergistically.When their content during less than above-mentioned scope, its promoter action is not obvious; When greater than above-mentioned scope, though still can promote the growth of Chinese hamster ovary celI, further promoter action is not obvious, can cause wastage of material on the contrary.
More preferably, described additive comprises following composition:
First part: amino acid
The L-L-Ala ????0～30mg/L
The L-aspartic acid ????0～10mg/L
Altheine ????20～100mg/L
The L-arginine ????10～50mg/L
L-L-glutamic acid ????10～100mg/L
L-glutaminate ????0～500mg/L
The L-glycine ????0～40mg/L
The L-Histidine ????0～50mg/L
The L-Isoleucine ????25～100mg/L
The L-leucine ????25～100mg/L
L-Methionin ????10～100mg/L
The L-methionine(Met) ????10～50mg/L
The L-phenylalanine ????10～60mg/L
The L-proline(Pro) ????10～30mg/L
The L-Serine ????0～100mg/L
The L-Threonine ????0～100mg/L
The L-tryptophane ????0～50mg/L
The L-Xie Ansuan ????0～50mg/L
L-tyrosine ????0～40mg/L
The second section trace element
????AgNO 3 ?????0.01～0.05mg/L
????AlCl 3·6H 2O ?????0.3～3.0mg/L
??Ba(C 2H 3O 2) 2 ????0.01～0.1mg/L
??3CdSO 4·8H 2O ????0.001～0.010mg/L
??CoCl 2·6H 2O ????0.03～0.30mg/L
??CuSO 4·5H 2O ????0.001～0.005mg/L
??FeSO 4·7H 2O ????0.2～2.0mg/L
??KI ????0.0001～0.001mg/L
??KBr ????0.0001～0.001mg/L
??MnSO 4?H 2O ????0.0001～0.001mg/L
??Na 2SeO 3 ????0.005～0.05mg/L
??NaF ????0.001～0.01mg/L
??Na 2SiO 3 ????0.005～0.05mg/L
??NH 4VO 3 ????0.01～0.1mg/L
??(NH 4) 6MoO 24·4H 2O ????0.01～0.1mg/L
??NiCl 2·6H 2O ????0.01～0.1mg/L
??SnCl 2·2H 2O ????1.00～10.00mg/L
??ZnSO 4 ????0.2～2.0mg/L
Third part: VITAMIN
Folic acid ????0～10mg/L
Vitamins B 6 ????0～0.3mg/L
Vitamins B 12 ????0～0.88mg/L
Choline chloride 60 ????0～10mg/L
Scyllitol ????0～30mg/L
Vitamin H ????0～0.2mg/L
Niacinamide ????0～5.0mg/L
Riboflavin ????0～0.2mg/L
VitB1 ????0～5mg/L
Thioctic Acid ????0～5mg/L
The 4th part: other composition
Regular Insulin ????2～20mg/L
Transferrins,iron complexes ????2～20mg/L
Thanomin ????1～10mg/L
Beta-mercaptoethanol ????0～2.5mg/L
HEPES (N-2-hydroxyethyl piperazine-N '-2-ethanesulfonic acid) ????1000～2500mg/L
Sodium.alpha.-ketopropionate ????0～330mg/L
Primatone (Sigma company) ????1000～5000mg/L
Lipid mixture * ????0.1～0.5％(v/v)
Reduced glutathion ????0.3～3.0mg/L
Vitamins C ????5～30mg/L
Putrescine ????2～10mg/L
Xanthoglobulin ????0～5mg/L
VITAMIN B4 ????5～10mg/L
Guanine ????5～10mg/L
Uridylic ????5～10mg/L
Thymus pyrimidine ????0.2～2mg/L
Cytosine(Cyt) ????5～10mg/L
Ribose ????1～10mg/L
Ribodesose ????1～10mg/L
Amino sulfoxide-methionine(Met) (MSX) ????9～45mg/L
*: the lipid that the chemical ingredients that lipid mixture is produced for GIBCO company is determined, composition is seen embodiment 1.
The content that it is pointed out that many materials such as amino acid, inorganic salt, VITAMIN B4 all is content commonly used in the substratum field.When the content of extra matter such as amino acid, inorganic salt, VITAMIN B4 is in above-mentioned scope, can more advantageously promote the growth of cell.When the content of extra matter such as amino acid, inorganic salt, VITAMIN B4 during less than above-mentioned scope, its promoter action is not obvious; When greater than above-mentioned scope, can cause wastage of material.
Chinese hamster ovary celI substratum of the present invention and cultural method have an outstanding advantage, wherein mainly comprise following advantage:
(1) do not contain serum and protein content is low, be beneficial to the separation and the purifying of product.
(2) the Chinese hamster ovary celI culture effect is good.Chinese hamster ovary celI vigorous growth, cell density and cytoactive all can be compared favourably with blood serum medium is arranged, and the substratum that can substitute serum fully is used for the cultivation of Chinese hamster ovary celI.
Embodiment further sets forth the present invention below in conjunction with specific embodiment.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example is usually according to the normal condition or the condition of advising according to manufacturer.
Embodiment 1
Orthogonal Method is determined the optimal medium that Chinese hamster ovary celI is cultivated
On the basis of initial medium, change the concentration (seeing the following form 1) of vitamins C, putrescine, lipid, prepare different substratum.Same Chinese hamster ovary celI (2.0 * 10 with equal amts 5Cell/mL) be inoculated in the 125ml square vase is equipped with the 10ml nutrient solution in the bottle, at 37 ℃, and CO 2Cultivated about 8 days in the incubator, measure the quantity of Chinese hamster ovary celI then.
Initial medium is the mixed solution of DMEM/12 substratum (available from Sigma company) and trace element etc., and adds following composition:
Composition Content (mg/L)
Transferrins,iron complexes ????5
Regular Insulin ????5
Thanomin ????2.5
Sodium Selenite ????0.01
Table 1 orthogonal test gauge outfit
????A ????B ????C
Vitamins C (mg/L) Putrescine (mg/L) Lipid mixture *(v/v％)
????1 ????5 ????2 ????0.1
????2 ????10 ????5 ????0.2
????3 ????30 ????10 ????0.5
*Annotate: the lipid that the chemical ingredients that lipid mixture is produced for GIBCO company is determined, composition following (mg/L).
Arachidonic acid ?????2mg/L
Cholesterol ?????220mg/L
The DL-alpha-tocopherol acetate ????70mg/L
Linolic acid ????10mg/L
Linolenic acid ????10mg/L
Myristic acid ????10mg/L
Oleic acid ????10mg/L
Physetoleic acid ????10mg/L
Palmitinic acid ????10mg/L
Stearic acid ????10mg/L
Tween 80 ????2200mg/L
????Pluronic?F-68 ????100000mg/L
Orthogonal test analysis is the result show:
When vitamins C is 5-30mg/L, putrescine is 2-10mg/L, when lipid mixture is 0.1-0.5% (v/v), can promote the growth of Chinese hamster ovary celI effectively, is better than initial medium 20%-100%.
Embodiment 2
The preparation of substratum and Chinese hamster ovary celI are cultivated
(a) culture medium prescription:
DMEM/F12 substratum (available from Sigma company) also adds following additive therein simultaneously:
First part: amino acid
The L-L-Ala ????9.0mg/L
The L-aspartic acid ????13mg/L
Altheine ????85mg/L
The L-arginine ????170mg/L
L-L-glutamic acid ????75mg/L
The L-glycine ????25mg/L
The L-Histidine ????62mg/L
The L-Isoleucine ????100mg/L
The L-leucine ????150mg/L
L-Methionin ????110mg/L
The L-methionine(Met) ????50mg/L
The L-phenylalanine ????86mg/L
The L-proline(Pro) ????34mg/L
The L-Serine ????76mg/L
The L-Threonine ????43mg/L
The L-tryptophane ????47mg/L
The L-Xie Ansuan ????88mg/L
L-tyrosine ????76mg/L
The second section trace element
????AgNO 3 ????0.0194mg/L
????AlCl 3·6H 2O ????1.00mg/L
????Ba(C 2H 3O 2) 2 ????0.0186mg/L
????CaCl 2 ????77.7mg/L
????3CdSO 4·8H 2O ????0.003mg/L
????CoCl 2·6H 2O ????0.101mg/L
????CuSO 4·5H 2O ????0.0038mg/L
????Fe(NO 3) 3·9H 2O ????0.15mg/L
????FeSO 4·7H 2O ????1.25mg/L
????KCl ????311.8mg/L
????KI ????0.0002mg/L
????KBr ????0.0001mg/L
????MgCl 2 ????28.64mg/L
????MgSO 4 ????48.84mg/L
????MnSO 4?H 2O ????0.0001mg/L
????NaHCO 3 ????2438.0mg/L
????NaCl ????6400.0mg/L
????Na 2HPO 4 ????71.02mg/L
????NaH 2PO 4·2H 2O ????220mg/L
????Na 2SeO 3 ????0.01mg/L
????NaF ????0.0042mg/L
????Na 2SiO 3 ????0.01mg/L
????NH 4VO 3 ????0.092mg/L
????(NH 4) 6MoO 24·4H 2O ????0.033mg/L
????NiCl 2·6H 2O ????0.035mg/L
????SnCl 2·2H 2O ????1.5564mg/L
????ZnSO 4 ????1.295mg/L
Third part: VITAMIN
Folic acid ????2.65mg/L
Vitamins B 6 ????2.60mg/L
Vitamins B 12 ????1.12mg/L
Choline chloride 60 ????14.0mg/L
Scyllitol ????30.0mg/L
Vitamin H ????0.007mg/L
Niacinamide ????4.04mg/L
Riboflavin ????0.26mg/L
VitB1 ????4.34mg/L
Thioctic Acid ????2.50mg/L
The 4th part: other composition
Regular Insulin ????5.0mg/L
Transferrins,iron complexes ????5.0mg/L
Thanomin ????2.5mg/L
Beta-mercaptoethanol ????2.5mg/L
????HEPES ????2000mg/L
Sodium.alpha.-ketopropionate ????330mg/L
????Primatone ????1250mg/L
Lipid mixture ????0.2％(v/v)
Reduced glutathion ????0.75mg/L
Vitamins C ????6.25mg/L
Putrescine ????7.0mg/L
Xanthoglobulin ????2.0mg/L
VITAMIN B4 ????7.0mg/L
Guanine ????7.0mg/L
Uridylic ????7.0mg/L
Thymus pyrimidine ????0.6mg/L
Cytosine(Cyt) ????7.0mg/L
Ribose ????1.0mg/L
Ribodesose ????1.0mg/L
????MSX ????18.0mg/L
In 125mL cell cultures square vase, each adds the substratum that 10mL as above prepares, and then adds the Chinese hamster ovary celI of band glutamine synthase expression system, and inoculum density is 2.0 * 10 5Cell/mL was every 24 hours sampling countings.At 37 ℃, CO 2Culturing cell in the incubator.Cell cultures to the 8 days, it is the highest that viable cell density reaches, and is 1.1 * 10 6Cell/mL.
Comparative Examples 1
Adopt the cultural method identical with embodiment 2, difference only is that used substratum is the DMEM/F12 that has added 5% (wt%) foetal calf serum, and its work Chinese hamster ovary celI density is 1.0 * 10 to the maximum 6Cell/mL.
The result as shown in Figure 1, the said substratum of the present invention is compared with the substratum that serum is arranged, and does not have notable difference, the substratum that therefore can substitute serum fully is used to the cultivation with the Chinese hamster ovary celI of glutamine synthase expression system.
Embodiment 3
The wild-type Chinese hamster ovary celI is cultivated
Repeat the experiment of embodiment 2 and Comparative Examples 1, difference is to replace with the Chinese hamster ovary celI of wild-type the Chinese hamster ovary celI of band glutamine synthase expression system, and does not add amino sulfoxide-methionine(Met) in the substratum.
The highest viable cell density in serum and serum free medium are arranged is respectively 1.6 * 10 6Cells/mL and 1.8 * 10 6Cells/mL shows serum free medium of the present invention, and the substratum that therefore can substitute serum fully is used for the cultivation of Chinese hamster ovary celI.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.

Claims (10)

1. a substratum that is used for Chinese hamster ovary cell is characterized in that, described substratum is to be made of basic medium and additive, and wherein additive contains:
The 2-20mg/L Transferrins,iron complexes;
2-20mg/L Regular Insulin;
The 1-10mg/L thanomin;
0.005～0.05mg/L selenite;
The 2-10mg/L putrescine;
The 5-30mg/L vitamins C is by the cumulative volume of the substratum of Chinese hamster ovary cell.
2. substratum as claimed in claim 1 is characterized in that described basic medium is DMEM/F12, and described additive also contains the 0-2.5mg/L beta-mercaptoethanol.
3. substratum as claimed in claim 1 is characterized in that, described additive also contains the trace element that is selected from down group: Cu, Fe, Zn, Mg, Se, Co, Mo, Sn, Si.
4. substratum as claimed in claim 1 is characterized in that, described additive contains following trace element:
AgNO 3?????????????????0.01～0.05mg/L
AlCl 3·6H 2O??????????0.3～3.0mg/L
Ba(C 2H 3O 2) 2???????0.01～0.1mg/L
3CdSO 4·8H 2O?????????0.001～0.010mg/L
CoCl 2·6H 2O??????????0.03～0.30mg/L
CuSO 4·5H 2O??????????0.001～0.005mg/L
FeSO 4·7H 2O??????????0.2～2.0mg/L
KI??????????????????????0.0001～0.001mg/L
KBr?????????????????????0.0001～0.001mg/L
MnSO 4?H 2O????????????0.0001～0.001mg/L
NaF?????????????????????0.001～0.01mg/L
Na 2SiO 3??????????????0.005～0.05mg/L
NH 4VO 3???????????????0.01～0.1mg/L
(NH 4) 6MoO 24·4H 2O??0.01～0.1mg/L
NiCl 2·6H 2O???????????0.01～0.1mg/L
SnCl 2·2H 2O???????????1.00～10.00mg/L
ZnSO 4??????????????????0.2～2.0mg/L。
5. substratum as claimed in claim 1 is characterized in that, described additive contains following amino acid:
L-L-Ala 0～30mg/L
L-aspartic acid 0～10mg/L
Altheine 20～100mg/L
L-arginine 10～50mg/L
L-L-glutamic acid 10～100mg/L
L-glutaminate 0～500mg/L
L-glycine 0～40mg/L
L-Histidine 0～50mg/L
L-Isoleucine 25～100mg/L
L-leucine 25～100mg/L
L-Methionin 10～100mg/L
L-methionine(Met) 10～50mg/L
L-phenylalanine 10～60mg/L
L-proline(Pro) 10～30mg/L
L-Serine 0～100mg/L
L-Threonine 0～100mg/L
L-tryptophane 0～50mg/L
L-Xie Ansuan 0～50mg/L
L-tyrosine 0～40mg/L.
6. substratum as claimed in claim 1 is characterized in that, described additive contains following VITAMIN:
Folic acid 0～10mg/L
Vitamins B 60～0.3mg/L
Vitamins B 120～0.88mg/L
Choline chloride 60 0～10mg/L
Scyllitol 0～30mg/L
Vitamin H 0～0.2mg/L
Niacinamide 0～5.0mg/L
Riboflavin 0～0.2mg/L
VitB1 0～5mg/L
Thioctic Acid 0～5mg/L.
7. substratum as claimed in claim 1 is characterized in that, described additive contains following material:
Beta-mercaptoethanol 0～2.5mg/L
HEPES???????????????1000～2500mg/L
Sodium.alpha.-ketopropionate 0～330mg/L
Primatone???????????1000～5000mg/L
Lipid mixture 0.1～0.5% (v/v)
Reduced glutathion 0.3～3.0mg/L
Xanthoglobulin 0～5mg/L
VITAMIN B4 5～10mg/L
Guanine 5～10mg/L
Uridylic 5～10mg/L
Thymus pyrimidine 0.2～2mg/L
Cytosine(Cyt) 5～10mg/L
Ribose 1～10mg/L
Ribodesose 1～10mg/L
Amino sulfoxide-methionine(Met) 9～45mg/L.
8. substratum as claimed in claim 7 is characterized in that, described lipid mixture contains following composition:
Arachidonic acid 2mg/L
Cholesterol 220mg/L
DL-alpha-tocopherol acetate 70mg/L
Linolic acid 10mg/L
Linolenic acid 10mg/L
Myristic acid 10mg/L
Oleic acid 10mg/L
Palm diluted acid 10mg/L
Palmitinic acid 10mg/L
Stearic acid 10mg/L
Tween 80 2200mg/L
PluronicF-68??????100000mg/L
Cumulative volume by lipid mixture.
9. the purposes of the described substratum of claim 1 is characterized in that, it is used to cultivate Chinese hamster ovary cell.
10. purposes as claimed in claim 9 is characterized in that, described Chinese hamster ovary cell is the Chinese hamster ovary cell that has the glutamine synthetase system.
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