CN106635958A - CHO (Chinese Hamster Ovary) cell culture medium - Google Patents

CHO (Chinese Hamster Ovary) cell culture medium Download PDF

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CN106635958A
CN106635958A CN201611210514.5A CN201611210514A CN106635958A CN 106635958 A CN106635958 A CN 106635958A CN 201611210514 A CN201611210514 A CN 201611210514A CN 106635958 A CN106635958 A CN 106635958A
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严志海
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Abstract

The invention belongs to the field of cell culture, and relates to a CHO (Chinese Hamster Ovary) cell culture medium. The CHO (Chinese Hamster Ovary) cell culture medium provided by the invention comprises the following components: a DMEM base culture medium, a yeast extract, sodium bicarbonate, amino acids, vitamins, inorganic salts, albumins, transferrin and linoleic acid. The culture provided by the invention provides a rich nutrition environment for the growth of CHO cells; various defects with the addition of serum are avoided; various components of the culture provided by the invention act together; harmful metabolites produced by the growth and metabolism of the CHO cells are reduced; the expression of CHO cell protein can be promoted without adding an additional medium.

Description

A kind of Chinese hamster ovary cell culture medium
Technical field
The invention belongs to field of cell culture.More particularly it relates to a kind of Chinese hamster ovary cell culture medium.
Background technology
Chinese hamster ovary cell (Chinese Hamster Ovary, Chinese hamster ovary cell) is in eukaryotic The first-selected system of glycosyl protein production of recombinating at present, because compared with other expression systems, it has many advantages, such as.It is existing at present Increasing pharmaceutical protein obtains high efficient expression in Chinese hamster ovary cell, and the medicine of many of which CHO expression is Put on market, such as EPO, Enbrel etc..
International and national has many people to develop various CHO culture mediums and feed supplement formula, such as：Domestic East China University of Science The CHO culture mediums that vertical, Zhang Yuanxing et al. is announced in Chinese patent application 200410018258.0.There is commercial sale in the world Culture medium, such as JRH Biosciences companies are the public affairs for specializing in cell culture base product (cell culture media) Department, is that U.S. FDA specifies biological medicine enterprise-specific culture medium.Invitrogen companies also produce serum free medium.
But the culture base unit weight for carrying out antibody drug protein expression needs on a large scale is very big, somewhat expensive, such as JRH The price of basal medium accounts for significant proportion at per liter more than 100 yuans in cost, in China, protein drug producer Also the problem of the enormous expenditure of culture medium is faced with, and while low protein yield is also a very big problem.
Therefore, this area is in the urgent need to developing new suitable Chinese hamster ovary cell culture, high protein yield nothing Blood serum medium.
The content of the invention
The purpose of the present invention is exactly to solve the above problems, there is provided a kind of culture of new culture Chinese hamster ovary cell Base, culture medium of the present invention is to be adapted to Chinese hamster ovary cell culture, the serum free medium of high protein yield.
What culture medium of the present invention was achieved through the following technical solutions：
A kind of Chinese hamster ovary cell culture medium includes following components：DMEM basal medium 10-50g/L, yeast is taken out Extract 1-20g/L, sodium acid carbonate 0.2-3.4g/L, amino acid 5-50g/L, vitamin 1.5-10g/L, inorganic salts 2.9-15g/L, Albumin 1-20g/L, transferrins 30-50g/L and linoleic acid 1-30mg/L.
Preferably, the amino acid (g/L) includes：Arginine 0.05-1.59g/L, leucine 1.25-2.56g/L, it is different bright Propylhomoserin 0.86-3.42g/L, aspartic acid 0.12-1.27g/L, glutamic acid 0.21-4.25g/L, lysine 0.13-2.47g/L, Threonine 2.46-5.64g/L, proline 0.19-0.95g/L, glycine 0.023-0.45g/L and alanine 1.38-5.35g/ L。
Preferably, the inorganic salts (g/L) include：Ironic citrate 2.42-4.87g/L, zinc sulfate 0.13-1.24g/L, sulphur Sour copper 0.98-4.58g/L, sodium selenite 0.05-3.84g/L, magnesium sulfate 0.0042-0.05g/L and sodium metavanadate 0.00047- 0.0085g/L。
Preferably, the vitamin (g/L) includes：VitaminB10 .10-0.58g/L, vitamin B2 0.25-1.38g/L, Vitamin B5 0.02-0.65g/L, vitamin B12 0.001-0.056g/L, vitamin C 0.13-0.84g/L, vitamin E 0.23-2.41g/L and vitamin D 1.0-3.85g/L.
Preferably, the Chinese hamster ovary cell culture medium, is made up of following component and its concentration：The culture of DMEM bases Base 32g/L, yeast extract 13g/L, sodium acid carbonate 0.85g/L, albumin 16g/L, transferrins 35g/L and linoleic acid 21mg/L, arginine 1.32g/L, leucine 1.58g/L, isoleucine 2.92g/L, aspartic acid 0.73g/L, glutamic acid 2.51g/L, lysine 1.42g/L, threonine 3.65g/L, proline 0.87g/L, glycine 0.19g/L, alanine 2.13g/ L, ironic citrate 3.29g/L, zinc sulfate 1.04g/L, copper sulphate 2.97g/L, sodium selenite 1.03g/L, magnesium sulfate 0.03g/L, Sodium metavanadate 0.0022g/L, vitaminB10 .36g/L, vitamin B2 0.97g/L, vitamin B5 0.24g/L, vitamin B120.031g/L, vitamin C 0.54g/L, vitamin E 1.33g/L and vitamin D 2.67g/L.
Nutrient content of the DMEM basal mediums for needed for the growth of cell is provided, the addition of yeast extract makes culture medium Nutrition is more enriched.Amino acid as topmost nitrogen source, in being present in cell culture medium.Amino acid can synthetic proteins Matter, polypeptide and other nitrogen-containing compounds, it decomposes the energy for cell growth for producing, the amino acid added in culture medium of the present invention And its concentration, there is good facilitation to Chinese hamster ovary cell, and these amino acid therein can affect Chinese storehouse Mouse gonad cell protein expression level.Albumin is the carrier of the small-molecule substances such as lipid, inorganic salts, it is possible to resolve the indissoluble of lipid The problem of property and cytotoxicity；Also culture medium viscosity can be increased, suspension cell is protected from the damage of shearing force.
The preparation method of cell culture medium of the present invention, comprises the following steps：
S1. above-mentioned DMEM culture medium dry powders, yeast extract, amino acid, soluble vitamin, inorganic salts, white egg are weighed In vain, transferrins, injects water to the 70%-85% of cumulative volume, gentle agitation dissolving, and continuously add vitamin E, D and Linoleic acid, and 0.2% Tween 80 is added, stirring and dissolving obtains A liquid；
S2. sodium acid carbonate, gentle agitation dissolving is added to inject water to prescribed volume, obtain in step S1 gained A liquid B liquid；
S3. pH to 7.2- is adjusted with 1mol/L sodium hydroxide solutions or 1mol/L hydrochloric acid solutions in step S2 gained B liquid 7.4, obtain C liquid；
S4. will be degerming with 0.22 μm of filter membrane positive press filtration in step S3 gained C liquid, obtain final product.
The culture medium of the present invention, provides the nutrient environment for enriching, it is to avoid have blood for Chinese hamster ovary cell growth The various drawbacks of clear addition, culture medium each component collective effect of the present invention reduces Chinese hamster ovary cell growth metabolism product Raw Toxic Metabolites, under conditions of without supplemented medium, can promote Chinese hamster ovary cell functional protein Expression.The invention provides a kind of Chinese hamster ovary cell culture medium, is a kind of serum free medium, with advantages below：
1st, the serum-free basal medium of the Chinese hamster ovary cell that the present invention is provided, can promote Chinese hamster ovary Cell growth, reduces Toxic Metabolites.
2nd, culture medium of the present invention can promote the expression and accumulation of Chinese hamster ovary cell functional protein.
Specific embodiment
Below in conjunction with specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate this Bright rather than restriction the scope of the present invention.
Embodiment 1
A kind of Chinese hamster ovary cell culture medium, is made up of following component and its concentration：
DMEM basal medium 10g/L, yeast extract 1g/L, sodium acid carbonate 0.2g/L, albumin 1g/L, transferrins The linoleic acid of 30g/L and 1-30mg/L.Required addition amino acid, as shown in table 1：
The amino acid (g/L) added needed for the culture medium of table 1
Required addition inorganic salts, as shown in table 2：
The inorganic salts (g/L) added needed for the culture medium of table 2
Ironic citrate 2.42
Zinc sulfate 0.13
Copper sulphate 0.98
Sodium selenite 0.05
Magnesium sulfate 0.0042
Sodium metavanadate 0.00047
The vitamin of required addition, as shown in table 3：
The vitamin (g/L) of addition needed for the culture medium of table 3
Cobastab1 0.10
Cobastab2 0.25
Cobastab5 0.02
Cobastab12 0.001
Vitamin C 0.13
Vitamin E 0.23
Vitamin D 1.0
Culture medium preparation method：
S1. above-mentioned DMEM culture medium dry powders, yeast extract, amino acid, soluble vitamin, inorganic salts, white egg are weighed In vain, transferrins, injects water to the 70%-85% of cumulative volume, gentle agitation dissolving, and continuously add vitamin E, D and Linoleic acid, and 0.2% Tween 80 is added, stirring and dissolving obtains A liquid；
S2. sodium acid carbonate, gentle agitation dissolving is added to inject water to prescribed volume, obtain in step S1 gained A liquid B liquid；
S3. pH to 7.2- is adjusted with 1mol/L sodium hydroxide solutions or 1mol/L hydrochloric acid solutions in step S2 gained B liquid 7.4, obtain C liquid；
S4. will be degerming with 0.22 μm of filter membrane positive press filtration in step S3 gained C liquid, obtain final product.
Embodiment 2
A kind of Chinese hamster ovary cell culture medium, is made up of following component and its concentration：
DMEM basal medium 50g/L, yeast extract 20g/L, sodium acid carbonate 3.4g/L, albumin 20g/L, turn iron egg White 50g/L and linoleic acid 30mg/L.
Required addition amino acid, as shown in table 4：
The amino acid (g/L) added needed for the culture medium of table 4
Arginine 1.59
Leucine 2.56
Isoleucine 3.42
Aspartic acid 1.27
Glutamic acid 4.25
Lysine 2.47
Threonine 5.64
Proline 0.95
Glycine 0.45
Alanine 5.35
Required addition inorganic salts, as shown in table 5：
The inorganic salts (g/L) added needed for the culture medium of table 5
The vitamin of required addition, as shown in table 6：
The vitamin (g/L) of addition needed for the culture medium of table 6
Cobastab1 0.58
Cobastab2 1.38
Cobastab5 0.65
Cobastab12 0.056
Vitamin C 0.84
Vitamin E 2.41
Vitamin D 3.85
Preparation method is similar to Example 1.
Embodiment 3
A kind of Chinese hamster ovary cell culture medium, is made up of following component and its concentration：
DMEM basal medium 32g/L, yeast extract 13g/L, sodium acid carbonate 0.85g/L, albumin 16g/L, turn iron Albumen 35g/L and linoleic acid 21mg/L.
Required addition amino acid, as shown in table 7：
The amino acid (g/L) added needed for the culture medium of table 7
Arginine 1.32
Leucine 1.58
Isoleucine 2.92
Aspartic acid 0.73
Glutamic acid 2.51
Lysine 1.42
Threonine 3.65
Proline 0.87
Glycine 0.19
Alanine 2.13
Required addition inorganic salts, as shown in table 8：
The inorganic salts (g/L) added needed for the culture medium of table 8
Ironic citrate 3.29
Zinc sulfate 1.04
Copper sulphate 2.97
Sodium selenite 1.03
Magnesium sulfate 0.03
Sodium metavanadate 0.0022
The vitamin of required addition, as shown in table 9：
The vitamin (g/L) of addition needed for the culture medium of table 9
Cobastab1 0.36
Cobastab2 0.97
Cobastab5 0.24
Cobastab12 0.031
Vitamin C 0.54
Vitamin E 1.33
Vitamin D 2.67
Preparation method is similar to Example 1.
Comparative example 1
A kind of Chinese hamster ovary cell culture medium, is made up of following component and its concentration：
DMEM basal medium 32g/L, yeast extract 13g/L, disodium hydrogen phosphate 2.8g/L, albumin 16g/L, turn iron Albumen 35g/L and linoleic acid 21mg/L.
Required addition amino acid (g/L), as shown in table 10：
The amino acid added needed for the culture medium of table 10
Required addition inorganic salts, as shown in table 11：
The inorganic salts added needed for the culture medium of table 11
Ironic citrate 3.29
Zinc sulfate 1.04
Copper sulphate 2.97
Sodium selenite 1.03
Magnesium sulfate 0.03
Sodium metavanadate 0.0022
The vitamin of required addition, as shown in table 12：
The vitamin of addition needed for the culture medium of table 12
Cobastab1 0.36
Cobastab2 0.97
Cobastab5 0.24
Cobastab12 0.031
Vitamin C 0.54
Vitamin E 1.33
Vitamin D 2.67
It is to replace sodium acid carbonate with disodium hydrogen phosphate with the difference of embodiment 3.
Preparation method is similar to Example 1.
The cell culture medium of test example 1 is to Chinese hamster ovary cell growth and the research of performance
Subjects：The CHO culture mediums that embodiment 1-3 and comparative example 1 are prepared
Test method：Stream plus culture (addition supplemented medium), addition supplemented medium is JRH supplemented mediums
Test procedure：In this culture experiment, feed supplement is carried out to exponential phase mid-term in cell growth, cell exists The mid-term of plateau carries out cooling operation and starts expressing protein, and survival rate is reduced to 50% or so for culture terminal.
Culture environment：37 DEG C, 5% carbon dioxide, 95% air.
Shaking culture process：Add the culture of the embodiment 1 for having filtered into 4 125mL shaking flasks respectively in super-clean bench The culture medium of base, the culture medium of embodiment 2, the culture medium of embodiment 3 and comparative example 1, so that cell population product is after inoculation 30mL, cell density is all during inoculation：0.5x106/mL.Mended in cell growth to exponential phase mid-term (the 7th day) Material, adds corresponding μ l of supplemented medium 750 or so of concentration, cell culture fluid volume do not change much (sampling and Evaporation can lose some volumes).Cell starts cooling into after plateau mid-term, makes cell be switched to express egg by growth conditions In vain.
Experimental result：Cell growth condition and expressing protein concentration are as shown in table 13：
Upgrowth situation of the Chinese hamster ovary cell of table 13 in different culture media
Project Embodiment 1 Embodiment 2 Embodiment 3 Comparative example 1
Cell density (cell/mL) after growing seven days 2.7x106 2.3x106 3.1x106 1.8x106
Most high-density after feed supplement 12.9.x106 12.3x106 13.6x106 10.3x106
Cells survival rate during most high-density after feed supplement 97.8 97.6 98.8 92.1
Albumen titre (g/L) 1.21 1.09 1.5 0.69
Compared by experiment, the culture medium of embodiment 3 can reach higher cell compared with embodiment 1, the culture medium of embodiment 2 Density, cell density, cells survival rate and albumen titre are high than comparative example 1, and this explanation culture medium of the present invention is one stable The entirety of balance, each interaction between component collectively promotes the growth of Chinese hamster ovary cell.
Presently preferred embodiments of the present invention is the foregoing is only, the substantial technological content model of the present invention is not limited to Enclose, the substantial technological content of the present invention is broadly defined in the right of application, any technology that other people complete Entity or method, if identical with defined in the right of application, also or a kind of equivalent change, will It is considered to be covered by among the right.

Claims (6)

1. a kind of Chinese hamster ovary cell culture medium, it is characterised in that including following components and its concentration：The culture of DMEM bases Base 10-50g/L, yeast extract 1-20g/L, sodium acid carbonate 1.2-3.4g/L, amino acid 5-50g/L, vitamin 1.5-10g/ L, inorganic salts 2.9-15g/L, albumin 1-20g/L, transferrins 30-50g/L and linoleic acid 1-30mg/L.
2. Chinese hamster ovary cell culture medium according to claim 1, it is characterised in that described amino acid includes：Essence Propylhomoserin 0.05-1.59g/L, leucine 1.25-2.56g/L, isoleucine 0.86-3.42g/L, aspartic acid 0.12-1.27g/ L, glutamic acid 0.21-4.25g/L, lysine 0.13-2.47g/L, threonine 2.46-5.64g/L, proline 0.19-0.95g/ L, glycine 0.023-0.45g/L and alanine 1.38-5.35g/L.
3. Chinese hamster ovary cell culture medium according to claim 1, it is characterised in that the inorganic salts include：Lemon Sour iron 2.42-4.87g/L, zinc sulfate 0.13-1.24g/L, copper sulphate 0.98-4.58g/L, sodium selenite 0.05-3.84g/L, Magnesium sulfate 0.0042-0.05g/L and sodium metavanadate 0.00047-0.0085g/L.
4. Chinese hamster ovary cell culture medium according to claim 1, it is characterised in that described vitamin includes：Dimension Raw element B10.10-0.58g/L, Cobastab20.25-1.38g/L, Cobastab50.02-0.65g/L, Cobastab12 0.001-0.056g/L, vitamin C 0.13-0.84g/L, vitamin E 0.23-2.41g/L and vitamin D 1.0-3.85g/ L。
5. according to the arbitrary Chinese hamster ovary cell culture medium of claim 1-4, it is characterised in that by following component and its Concentration is constituted：DMEM basal medium 32g/L, yeast extract 13g/L, sodium acid carbonate 0.85g/L, albumin 16g/L, turn iron Albumen 35g/L and linoleic acid 21mg/L, arginine 1.32g/L, leucine 1.58g/L, isoleucine 2.92g/L, aspartic acid 0.73g/L, glutamic acid 2.51g/L, lysine 1.42g/L, threonine 3.65g/L, proline 0.87g/L, glycine 0.19g/ L, alanine 2.13g/L, ironic citrate 3.29g/L, zinc sulfate 1.04g/L, copper sulphate 2.97g/L, sodium selenite 1.03g/L, Magnesium sulfate 0.03g/L, sodium metavanadate 0.0022g/L, Cobastab10.36g/L, Cobastab20.97g/L, Cobastab5 0.24g/L, Cobastab120.031g/L, vitamin C 0.54g/L, vitamin E 1.33g/L and vitamin D 2.67g/L.
6. according to the arbitrary Chinese hamster ovary cell culture medium preparation method of claim 1-5, it is characterised in that include with Lower step：
S1. above-mentioned DMEM culture medium dry powders are weighed, yeast extract, amino acid, soluble vitamin, inorganic salts, albumin turns Ferritin, injects water to the 70%-85% of cumulative volume, gentle agitation dissolving, and continuously adds vitamin E, D and Asia oil Acid, and 0.2% Tween 80 is added, stirring and dissolving obtains A liquid；
S2. sodium acid carbonate, gentle agitation dissolving is added to inject water to prescribed volume, obtain B liquid in step S1 gained A liquid；
S3. pH to 7.2-7.4 is adjusted with 1mol/L sodium hydroxide solutions or 1mol/L hydrochloric acid solutions in step S2 gained B liquid, obtains C Liquid；
S4. will be degerming with 0.22 μm of filter membrane positive press filtration in step S3 gained C liquid, obtain final product.
CN201611210514.5A 2016-12-24 2016-12-24 CHO (Chinese Hamster Ovary) cell culture medium Pending CN106635958A (en)

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CN110343666A (en) * 2019-07-10 2019-10-18 通化东宝生物科技有限公司 A kind of supplemented medium and its preparation method and application of Chinese hamster ovary celI culture
CN112458049A (en) * 2020-12-17 2021-03-09 江南大学 Low-cost method for in vitro culture of pork muscle stem cells

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CN1778902A (en) * 2005-09-29 2006-05-31 华东理工大学 Non-serum culture medium for multiple animal cell large-scale culture
CN1962857A (en) * 2005-11-11 2007-05-16 上海中科伍佰豪生物工程有限公司 Serum-free medium for mammalian cell

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CN110343666A (en) * 2019-07-10 2019-10-18 通化东宝生物科技有限公司 A kind of supplemented medium and its preparation method and application of Chinese hamster ovary celI culture
CN110343666B (en) * 2019-07-10 2023-05-30 通化东宝药业股份有限公司 Feed supplement culture medium for CHO cell culture and preparation method and application thereof
CN112458049A (en) * 2020-12-17 2021-03-09 江南大学 Low-cost method for in vitro culture of pork muscle stem cells

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Application publication date: 20170510