CN105779375A - Novel serum-free culture medium - Google Patents
Novel serum-free culture medium Download PDFInfo
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- CN105779375A CN105779375A CN201410850189.3A CN201410850189A CN105779375A CN 105779375 A CN105779375 A CN 105779375A CN 201410850189 A CN201410850189 A CN 201410850189A CN 105779375 A CN105779375 A CN 105779375A
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Abstract
The invention discloses a novel serum-free culture medium. Specifically, the invention discloses a culture medium. The novel serum-free culture medium is prepared from the following components: amino acid, microelements and inorganic salt, vitamin, carbohydrates and other organic molecules including heparin. With the adoption of the novel serum-free culture medium, the difficulty that the agglomeration phenomenon is generated when CHO (Chinese Hamster Ovary) cells are subjected to high-density culture is solved; in addition, the novel serum-free culture medium does not contain serum and does not contain the proteins including animal-source growth factors, transferring and insulin, so that the pollution risk of the cells is greatly reduced, and the isolation and purification of the downstream antibody protein are benefited.
Description
Technical field
The present invention relates to biological technical field, more particularly it relates to an serum-free medium.
Background technology
Development along with biotechnology, prepare medicine with recombinant technique to be possibly realized, people can pass through clones coding antibody gene, then by this gene transformation to be expressed to suitable expression host cell, finally cultivate host cell, production and antibody purification, thus obtaining required antibody drug.By this technology, existing multiple monoclonal antibody drug gets permission listing at present, including Herceptin (Trastuzumab), Rituximab (Rituximab), adalimumab (Adalimumab), daclizumab (Daclizumab) etc., it is widely used to treatment and includes the multiple disease of tumor, autoimmune disease, graft-rejection etc., 2013, in the front ten big best-selling drugses of the whole world, antibody drug has occupied half of the country, and adalimumab sales volume is more above 10,000,000,000 dollars.
And in antibody drug preparation process, the large-scale culture of cell is a very important ring.Cell culture technology is to realize cell injuring model by analog cell tumor growth environment.Generally, the growth of zooblast all depends on the existence of serum. and in ordinary culture medium, such as not increase serum, overwhelming majority cells can not be bred.But the major drawbacks using serum is to there is potential polluter, expensive, differences between batches are big, and production and scientific research are brought inconvenience.Scientist finds to add the recruitment factor of alternative serum effect in basal medium, and such as compositions such as fibronectin, transferrins, insulin and epidermal growth factors, many cells can grow when serum-free is supplied.Serum-free medium has evaded the risk that serum brings.It is easy to product separation purification.But, general a kind of serum-free medium is only applicable to the cultivation of a class cell, and is easily subject to the impact of chemical factors.Additionally serum-free medium usually contains albumen and the lipid of alternative serum, such as transferrins, lipid additive and insulin etc., however it remains the obstacle of certain pollution risk and separation purification, cost is also higher.
CHO (ChineseHamsterOvary) cell strain built, in nineteen fifty-seven, is obtained from an Adult female Hamster Qvary separation by DrTheodoreTPuck.Hereafter, CHO engineering cell possesses the posttranscriptional modification of elaborate by it, stronger exogenous gene expands and ability to express in a large number, and be prone to the advantages such as extensive suspension culture and become the first-selected expression system that produces monoclonal antibody and recombinant protein medicine, it is currently widely used for producing various engineered protein product.Generally, the large-scale culture density of Chinese hamster ovary celI is significantly high, usually produces agglomerating phenomenon but high secret attached cell suspends in domestication or high cell densities suspension culture process, and serious cell is agglomerating up to several millimeters, thus affecting culture effect.The pockets of mechanism of current cell suspension cultures is unclear, has the DNA that scholar thinks that dead cell discharges to may result in cell at iuntercellular bridge formation agglomerating, and often changes with cellular metabolism, thus cannot continue Secondary Culture.In order to solve the agglomerating difficult problem of cell, scholars develops multiple solution route, cultivates domestication, reduction Ca as improved stirring efficiency, carrying out long-term serum free suspension2+、Mg2+Concentration, interpolation pancreatin etc..But a kind of more efficiently method that agglomerating phenomenon produces when avoiding Chinese hamster ovary celI High Density Cultivation is still required at present.
Summary of the invention
The present inventor is through substantial amounts of experimentation, the basis of DMEM/F12 culture medium (purchase of Sigma company) successfully develops a kind of serum-free medium suitable in extensive animal cell culture, this culture medium successfully solves agglomerating phenomenon during Chinese hamster ovary celI High Density Cultivation and produces a difficult problem, additionally, this culture medium is without serum, and without albumen such as the somatomedin of animal origin, transferrins and insulins, so that the contaminated risk of cell is substantially reduced, it is more beneficial for separation and the purification of the antibody protein in downstream simultaneously.Specifically, the invention discloses:
1, a kind of serum-free medium, nutrient media components includes, and aminoacid, trace element and inorganic salt, vitamin, carbohydrate and other organic molecules, including heparin.
2, according to the serum-free medium described in above-mentioned 1, it is characterised in that described heparin concentration is 1.0-50.0mg/L.
3, according to the serum-free medium described in above-mentioned 2, it is characterised in that described heparin concentration is 25.0mg/L.
4, according to the arbitrary described serum-free medium of above-mentioned 1-3, it is characterised in that described culture medium contains steroid hormone, and does not contain transferrins and insulin.
5, according to the serum-free medium described in above-mentioned 4, it is characterised in that described culture medium contains estradiol, hydrocortisone, ferric citrate, ZnSO4·7H2O, but without somatomedin, transferrins and insulin.
6, according to the Serum-free and protein-free medium described in above-mentioned 5, it is characterised in that the amino acid whose component of culture medium and consumption are as follows,
7, according to the Serum-free and protein-free medium described in above-mentioned 5, it is characterised in that component and the consumption of culture medium dry powder medium trace element and inorganic salt are as follows,
8, according to the Serum-free and protein-free medium described in above-mentioned 5, it is characterised in that in culture medium dry powder, the component of vitamin and consumption are as follows,
9, according to the Serum-free and protein-free medium described in above-mentioned 5, it is characterised in that in culture medium dry powder, carbohydrate is as follows with the component of other organic molecules and consumption,
10, according to the Serum-free and protein-free medium described in above-mentioned 8, it is characterised in that in culture medium dry powder, each component and consumption are as follows,
The component of Serum-free and protein-free medium generally comprises glucose, aminoacid, inorganic salt, vitamin, trace element, steroid hormone and associated protein substitute, wherein, aminoacid: be one of middle Major Nutrient material, synthesis for materials such as albumen, nucleic acid and lipids, TCA circulation can also be entered by several main intermediate supersession nodes, for the generation of energy, and it is associated with the metabolism of other nutrient substance；Inorganic salt: NaCl maintains osmotic balance, coordinate co-transport organic macromolecule and enter cell, K+, Ca2+, Mg2+, Zn2+ plasma is then participated in metabolism and signal transduction and promotes adherent and cell proliferation, various anion (SO42-, NO3-etc.), then it is mainly used in regulating and turns cell membrane potential energy or the precursor as sulfur-bearing or nitrogen element organic molecule；Vitamin: one of Major Nutrient material in culture medium, the amount that vitamin exists in the medium very trace, but as cell function organic catalyst in cellular metabolism, play important regulating and controlling effect；Carbohydrate: glucose is main carbohydrate, as main carbon source and energy substance；Trace element: acting primarily as adjustment, transmission and the effect controlled, the effect in serum-free medium is particularly important, as: Fe: the prothetic group of enzyme and haemachrome, is the ingredient of respiratory chain in mitochondrion；Cu: the prothetic group of superoxide dismutase, is the ingredient of respiratory chain in mitochondrion；Mg: ATP enzyme, kinases etc. are played activation；Zn: the prothetic group of enzyme；Additionally, culture medium is additionally added the phenol red indicator as medium pH, it is added with the PluronicF68 of 0.1% for the large-scale culture in applicable cell fermentation tank to eliminate the impact of shearing force.But how these compositions are carried out appropriate design, be the key of exploitation culture medium.
The present inventor by adding heparin in basal medium, thus the generation of Chinese hamster ovary celI agglomerating phenomenon when avoiding High Density Cultivation, it addition, by selecting estradiol, hydrocortisone, ferric citrate, ZnSO4·7H2The materials such as O substitute the albuminoid components such as somatomedin, transferrins and insulin, thus being beneficial to separation and the purification of downstream antibody protein.The present inventor finally determines the optimal dose of each component also by the experiment of substantial amounts of statistics.
Detailed description of the invention
The present invention is only further detailed by following example, should not be construed as limitation of the present invention.The basal medium DMEM/F12 available from Sigma of the present invention, and press related request and store.
Embodiment 1: serum-free medium is prepared
The preferred component of serum-free medium dry powder being applicable to extensive animal cell culture of the present invention and consumption are as follows:
Culture medium 1:
Culture medium 2: without heparin component, other component and consumption are identical with culture medium 1
Culture medium dry powder collocation method:
The dry powder of 1L consumption is added in 900ml ultra-pure water, temperature about 35 DEG C, after stirring half an hour, add a certain amount of sodium hydroxide hydrotropy, be subsequently adding concentrated hydrochloric acid and sodium bicarbonate, regulate PH to 6.5～7.5.Use 0.2um negative pressure filtration, be sub-packed in aseptic in the vial of 500 milliliters keeping in Dark Place.
The physicochemical property parameter of culture medium, detection method and testing result that the present invention obtains are as shown in table 1.
Table 1: culture medium testing result table
From testing result, the fluid medium result of configuration is qualified, it is possible to for the extensive suspension cell culture of CHO.
Embodiment 2: different culture media cell culture effect contrast under same culture conditions
Condition of culture is as shown in table 2 below:
Table 2: cell culture condition list
Such as above-mentioned cultural method, the above-mentioned three kinds of culture medium 300ml prepared are loaded in the shaking flask of 1000ml by the present inventor respectively, add Chinese hamster ovary celI, and inoculum density is 6.0 × 105Cell/ml, places CO2(CO in incubator2It is 5%) middle cultivation, temperature is 37 DEG C, sampled 20 μ l every 24 hours, carries out cell counting by cell counter (purchased from invitrogen), detects cell survival rate by mtt assay, and by the agglomerating situation of om observation cell.Experimental result is as shown in table 3:
Table 3: the agglomerating situation testing result table of different culture media cultured cells density, cell survival rate and cell
More than test result indicate that, the culture medium 1 of the present invention is compared commercially available DMEM/F12 culture medium and is had better cell culture effect and the culture medium 1 that with the addition of heparin of the present invention with culture medium 2, and it reaches 98.5 × 10 at cell density5During high density, have not yet to see the generation of the agglomerating phenomenon of cell, compared with the culture medium 2 not adding heparin, there is better cell culture effect.
It addition, the present inventor is also to the Chinese hamster ovary celI obtained in patent documentation disclosed in CN01132074.5, CN01132225.X, CN01132226.8, repeating above-mentioned test, final result all shows that the culture medium 1 of the present invention has cell culture effect more preferably.
Additionally, due to the culture medium of the present invention is without serum, also without albuminoids such as somatomedin, transferrins and insulins, makes the contaminated risk of cell be substantially reduced, be more beneficial for separation and the purification of the antibody protein in downstream simultaneously.
Claims (10)
1. a serum-free medium, nutrient media components includes, and aminoacid, trace element and inorganic salt, vitamin, carbohydrate and other organic molecules, including heparin.
2. serum-free medium according to claim 1, it is characterised in that described heparin concentration is 1.0-50.0mg/L.
3. serum-free medium according to claim 2, it is characterised in that described heparin concentration is 25.0mg/L.
4. according to the arbitrary described serum-free medium of claim 1-3, it is characterised in that described culture medium contains steroid hormone, and does not contain transferrins and insulin.
5. serum-free medium according to claim 4, it is characterised in that described culture medium contains estradiol, hydrocortisone, ferric citrate, ZnSO4·7H2O, but without somatomedin, transferrins and insulin.
6. Serum-free and protein-free medium according to claim 5, it is characterised in that the amino acid whose component of culture medium and consumption are as follows,
。
7. Serum-free and protein-free medium according to claim 5, it is characterised in that component and the consumption of culture medium dry powder medium trace element and inorganic salt are as follows,
。
8. Serum-free and protein-free medium according to claim 5, it is characterised in that in culture medium dry powder, the component of vitamin and consumption are as follows,
。
9. Serum-free and protein-free medium according to claim 5, it is characterised in that in culture medium dry powder, carbohydrate is as follows with the component of other organic molecules and consumption,
。
10. Serum-free and protein-free medium according to claim 8, it is characterised in that in culture medium dry powder, each component and consumption are as follows,
。
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107460159A (en) * | 2017-08-14 | 2017-12-12 | 上海多宁生物科技有限公司 | Serum-free, without albumen supplemented medium and preparation method thereof and use |
| 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 |
| CN112795531A (en) * | 2021-04-11 | 2021-05-14 | 依科赛生物科技（太仓）有限公司 | CHO cell serum-free and protein-free culture medium and application thereof |
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2014
- 2014-12-26 CN CN201410850189.3A patent/CN105779375A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107460159A (en) * | 2017-08-14 | 2017-12-12 | 上海多宁生物科技有限公司 | Serum-free, without albumen supplemented medium and preparation method thereof and use |
| CN107460159B (en) * | 2017-08-14 | 2020-12-11 | 上海多宁生物科技有限公司 | Serum-free and protein-free supplemented medium and preparation method and application thereof |
| 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 |
| CN112795531A (en) * | 2021-04-11 | 2021-05-14 | 依科赛生物科技（太仓）有限公司 | CHO cell serum-free and protein-free culture medium and application thereof |
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Application publication date: 20160720 |