CN106190950A - A kind of Chinese hamster ovary celI Serum-free and protein-free medium and preparation method thereof - Google Patents

A kind of Chinese hamster ovary celI Serum-free and protein-free medium and preparation method thereof Download PDF

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CN106190950A
CN106190950A CN201610503795.7A CN201610503795A CN106190950A CN 106190950 A CN106190950 A CN 106190950A CN 201610503795 A CN201610503795 A CN 201610503795A CN 106190950 A CN106190950 A CN 106190950A
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China
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free
protein
serum
hamster ovary
chinese hamster
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于红阳
吴彦卓
张苗
甘迪
甘一迪
贾东晨
徐明波
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BEIJING SHUANGLU BIOLOGICAL TECHNOLOGY Co Ltd
BEIJING SL LISHENG PHARMACEUTICAL Co Ltd
BEIJING SHUANGLU PHARMACEUTICAL Co Ltd
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BEIJING SHUANGLU BIOLOGICAL TECHNOLOGY Co Ltd
BEIJING SL LISHENG PHARMACEUTICAL Co Ltd
BEIJING SHUANGLU PHARMACEUTICAL Co Ltd
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Priority to CN201610503795.7A priority Critical patent/CN106190950A/en
Publication of CN106190950A publication Critical patent/CN106190950A/en
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Abstract

The present invention relates to the preparation of cell culture medium, specifically provide a kind of new serum-free, the preparation of cell culture medium that protein-free, chemical composition limits, this culture medium contains several amino acids, vitamin, inorganic salt, trace element, carbohydrate and other recruitment factors；Do not contain any extract or hydrolysate；Animal origin-free composition.This culture medium can support Chinese hamster ovary celI suspension culture in vitro well, meets Chinese hamster ovary celI and cultivates demand；For chemical composition defined medium, with low cost.

Description

A kind of Chinese hamster ovary celI Serum-free and protein-free medium and preparation method thereof
Technical field
The invention belongs to field of cell culture, be specifically related to a kind of serum-free being suitable to the extensive suspension culture of Chinese hamster ovary celI Protein-free medium and preparation method thereof.
Background technology
Chinese hamster ovary (chinese hamster ovary, CHO) cell is widely used in recombinant protein medicine and resists The production of body medicine, using the teaching of the invention it is possible to provide stable and the most glycosylation modified, make expression product closest to natural protein molecules, Have the advantages that safety is high.The cultivation of Chinese hamster ovary celI is interpolation 5～the tire of 10% in DMEM/F12 basal medium traditionally Ox blood serum completes, and serum, in addition to the nutritional labeling of supply cell, additionally provides cultured cell in vitro propagation necessary Somatomedin.But the use of serum exist between batch difference greatly, easily by the pollution such as mycoplasma and virus, cost is high, be unfavorable for producing Thing a lot of drawback such as isolated and purified grade, and affect the growth of cell and the quality of end product, it is unsuitable for large-scale industry metaplasia Produce.
In order to overcome the defect using serum to bring, many so-called " definition " culture medium are developed.These culture medium are past Toward being to prepare to support the cultivation of single cell type especially, do not comprise any undefined supplement and the purification of alternative serum Somatomedin, protein, lipoprotein and other materials.Owing to the component (and concentration) of this culture medium is accurately known, It is commonly called " defined medium ".Use limited culture medium to compare the culture medium containing serum or extract, be conducive to research Specific somatomedin or the effect in cytophysiology of other compositions in culture medium.Additionally, limited culture medium generally contains There is very small amount of protein, simplify product purification process, reduce purification cost.
Chinese Patent Application No. 200910241670.1 discloses a kind of serum-free cell culture medium, this culture medium be with Culture medium based on DMEM/F12 (1: 1), with the addition of the material such as insulin and transferrins, and the highest viable cell density is less than 400 Ten thousand/ml.
Chinese Patent Application No. 200710307076.9 discloses a kind of without albuminous cell culture medium, containing transferrins with Insulin substitution thing, but the highest viable cell density is less than 3,500,000/ml.
Chinese Patent Application No. 201310106385.5 discloses a kind of serum-free without albuminous cell culture medium, although do not contain There are insulin and transferrins, but containing steroid hormone.
Most of serum-free mediums of domestic-developed can support that cell grows at present, but is to cultivate mostly Adding various nutrient substance, insulin, transferrins, protolysate etc. on the basis of base such as DMEM, DMEM/F12 etc., composition is multiple Miscellaneous so that quality and the safety surface of product are tested before the examination, also bring difficulty for purification simultaneously.And the public affairs such as external Life, Thermo Although the serum-free medium of department is without protein, but it is expensive, and composition is kept secret, not only increases cell extensive The cost cultivated, reduces the space that production capacity promotes and cost reduces, and is unfavorable for carrying out culture medium according to product needed Optimize and revise, greatly reduce follow-up optimization space.The serum-free medium definite ingredients of this patent preparation, without albumen Matter, it is easy to obtain, with low cost, the limitation of commercial medium can be evaded, it is achieved the reasonable equilibrium supply of nutrient, control metabolism secondary Product accumulation, not only can support cell large-scale culture, and definite ingredients, can be optimized formula composition according to demand, Improve production capacity, save production cost.
Summary of the invention
The serum-free that it is an object of the invention to provide a kind of new support Chinese hamster ovary celI high density suspension cultivation is cultivated without albumen Base, can be suitably used for multiple different Chinese hamster ovary celI strain growth and stably passes on, specific chemical components, balanced in nutrition, with low cost, Holding Chinese hamster ovary celI preferably to grow and shorter cell doubling time, the large-scale production for biological product provides convenient.
The component of Serum-free and protein-free medium generally comprise aminoacid, vitamin, inorganic salt, trace element, glucose, These compositions are carried out rational proportion and are exploitations and optimize training by lipid, insulin and transferrins succedaneum and other materials Support the key of base.
For achieving the above object, present invention employs techniques below scheme: use zinc sulfate, ferric citrate, sodium selenite Substitute insulin and transferrins；Statistics experimental design is utilized to determine the optimal dose of each component.
Based on considerations above, the culture medium that the present invention relates to includes several amino acids, vitamin, inorganic salt, glucose, one Plant or multiple lipid, insulin substitution thing and transferrins succedaneum.
Aminoacid is the important nitrogen source of cell growth metabolism and energy substance, for materials such as albumen, nucleic acid and lipids Synthesis.Cell cultivates amino acid needed kind more up to 20 kinds, and metabolism intersects, and can pass through deaminizating, turn amino, connection Close the effects such as deaminizating to realize converting.The aminoacid ingredient of the present invention preferably includes selected from L-arginine, altheine, L-days Winter propylhomoserin, Cys, Pidolidone, L-glutaminate, glycine, L-Histidine, ILE, L-Leu, L- Lysine, METHIONINE, L-phenylalanine, L-PROLINE, Serine, L-threonine, L-Trp, TYR and L- Valine.
Vitamin participates in cell growth metabolism as the catalyst of multiple reaction；The vitamin ingredients of the present invention preferably includes Selected from biotin, choline chloride, D-VB5 calcium, folic acid, scyllitol, nicotiamide, pyridoxol, riboflavin, thiamine and vitamin B12。
Inorganic salt maintains osmotic pressure, the balance of system pH, and participates in multiple enzyme reaction as cofactor.The present invention's is inorganic Salt component preferably includes potassium chloride, multiple magnesium salt, manganese salt, sodium chloride, NaHCO3, Na2HPO4, selenium salt, vanadic salts and zinc salt.
Glucose is as the carbon source of the macromole such as the main energy sources of cellular metabolism and synthetic DNA.
The present invention uses zinc sulfate to replace insulin, and zinc sulfate can regulate the level of insulin and receptor；Use citric acid Ferrum replaces transferrins, and the TfR on cell membrane is combined, and promotes the cross-film transmission of ferrum, stablizes the work of culture medium Property；And add the clear and definite lipid material of chemical constituent, trace element, antioxidant, protect cells from hydrodynamic shear damage Material；Wherein said lipid material includes: putrescine, spermine, ethanolamine, linoleic acid, tween 80；Described trace element Including: copper sulfate, zinc sulfate, ferrous sulfate, ferric nitrate, sodium selenite, sodium silicate, inclined sodium vanadate, Nickel dichloride., stannous chloride, Ammonium molybdate, silver nitrate, manganese sulfate, cobaltous chloride, aluminum chloride, potassium bromide, potassium iodide, barium acetate, chromic sulfate, sodium fluoride, specific In concentration range, growth and protein expression to cell have facilitation；Described antioxidant includes: ascorbic acid, reduction Type glutathion；The described compound protecting cells from hydrodynamic shear damage is: Pluronic F-68；
The culture medium that the present invention relates to comprises one or more polyanions or polycationic compounds, wherein, poly-cloudy from Sub-compound is preferably many sulfonation or poly-sulfated compound, more preferably heparin, dextran sulfate, Heparan sulfate, sulphuric acid Dermatan, chondroitin sulfate, PPS, Dan Baiduotang proteoglycan PG etc., and most preferably dextran sulfate, what it preferably had divides Son amount is about 5,000 dalton.
Not same-action based on the growth of above all kinds of materials onto cells, above-mentioned substance is joined by the present invention by following concentration Ratio:
Amino acid whose component and weight portion are as follows:
Composition and the weight portion of vitamin are as follows:
Composition and the weight portion of inorganic salt are as follows:
Composition and the weight portion of trace element are as follows:
Carbohydrate is as follows with the component of other molecules and weight portion:
Preferably, above-mentioned CHO culture medium includes component and content is:
Amino acid whose composition and consumption are as follows:
The component of vitamin is as follows with consumption:
Composition and the consumption of inorganic salt are as follows:
Component and the consumption of trace element are as follows:
The composition of carbohydrate and other molecules is as follows with content:
It is further preferred that the component that includes of above-mentioned CHO culture medium and content are:
Amino acid whose composition and consumption are as follows:
The component of vitamin is as follows with consumption:
Composition and the consumption of inorganic salt are as follows:
Component and the consumption of trace element are as follows:
The composition of carbohydrate and other molecules is as follows with content:
Most preferably, above-mentioned CHO culture medium includes component and content is:
Amino acid whose composition and consumption are as follows:
The component of vitamin is as follows with consumption:
Composition and the consumption of inorganic salt are as follows:
Component and the consumption of trace element are as follows:
The composition of carbohydrate and other molecules is as follows with content:
The Serum-free and protein-free medium pH 7.0-7.4 that the present invention provides, osmotic pressure is 290-330mOsm/kg, it is possible to Support Chinese hamster ovary celI fast-growth under floating condition, without any somatomedin and protolysate, specific chemical components, With low cost, it is simple to prepare, store.
Accompanying drawing explanation
Fig. 1 is that the CHO-S cell observed under inverted microscope is outstanding in Serum-free and protein-free medium prepared by embodiment 1 The cytological map that floating life is long；
Fig. 2 is CHO-S cell viable cell density of suspension growth in Serum-free and protein-free medium prepared by embodiment 1 Change and Cell viability variation diagram；
Fig. 3 is CHO-S cell viable cell density of suspension growth in Serum-free and protein-free medium prepared by embodiment 2 Change and Cell viability variation diagram；
Fig. 4 is CHO-S cell viable cell density of suspension growth in Serum-free and protein-free medium prepared by embodiment 3 Change and Cell viability variation diagram；
Fig. 5 is that CHO-S cell suspends in embodiment 1, the Serum-free and protein-free medium of 2,3 preparations and commercially available culture medium The viable cell density variation diagram of growth；
Detailed description of the invention
The all raw material of the present invention is cell and cultivates the raw material of level, and presses related request storage.
Test method in following embodiment, if no special instructions, is conventional method.
Below in conjunction with embodiment, the present invention is described in detail.
Embodiment 1:
1) Chinese hamster ovary celI Serum-free and protein-free medium preparation:
The Chinese hamster ovary celI Serum-free and protein-free medium dry powder component of the present invention and consumption are as follows:
The dry powder of 1L consumption is added in 900ml ultra-pure water, temperature about 30 DEG C, after stirring half an hour, add a certain amount of Sodium hydroxide hydrotropy, be subsequently adding sodium bicarbonate, with salt acid for adjusting pH to 6.9～7.0.With 0.22 μm non-velum filteration, 4 DEG C Preserve.
2) cell is cultivated:
By CHO-S cell with 1 × 106The density of cell/ml is seeded in the 125ml triangle shaking flask containing 20ml culture medium, Triangle shaking flask is placed in containing 5%CO2Shaking table in 37 DEG C of cultivations, rotating speed 180rpm；When cell density about 3 × 106During cell/ml, With 1 × 106The density of cell/ml passes on, until passage more than 10 times.Examining under a microscope, cell edges is clear, shape State is full, without clustering phenomena, well-adjusted (as shown in Figure 1).
By cell good for above-mentioned domestication with 1 × 106The density of cell/ml is seeded to containing culture medium 50ml culture medium In the shaking flask of 250ml, it is placed in 37 DEG C of cultivations in 5%CO2 incubator, rotating speed 180rpm, every 24 hours sampling countings, and uses platform Expect that blue dyeing calculates Cell viability.When cell cultivates the 7th day, Cell viability more than 90%, CHO-S peak cell number is 12 × 106Cell/ml (Fig. 2)
Embodiment 2:
1) Chinese hamster ovary celI Serum-free and protein-free medium preparation
The Chinese hamster ovary celI Serum-free and protein-free medium dry powder component of the present invention and consumption are as follows:
The dry powder of 1L consumption is added in 900ml ultra-pure water, temperature about 30 DEG C, after stirring half an hour, add a certain amount of Sodium hydroxide hydrotropy, be subsequently adding sodium bicarbonate, with salt acid for adjusting pH to 6.9～7.0.With 0.22 μm non-velum filteration, 4 DEG C Preserve.
2) cell is cultivated:
By CHO-S cell with 1 × 106The density of cell/ml is seeded to the shaking flask of the 250ml containing culture medium 50ml culture medium In, it is placed in 37 DEG C of cultivations in 5%CO2 incubator, rotating speed 180rpm, every 24 hours sampling countings, and uses Trypan Blue meter Calculate Cell viability.When cell cultivates the 4th day, viable cell density reaches the highest, and CHO-S peak cell number is 5.88 × 106Carefully Born of the same parents/ml, Cell viability more than 90% (Fig. 3) after inoculating 6 days
Embodiment 3:
1) Chinese hamster ovary celI Serum-free and protein-free medium preparation
The Chinese hamster ovary celI Serum-free and protein-free medium dry powder component of the present invention and consumption are as follows:
The dry powder of 1L consumption is added in 900ml ultra-pure water, temperature about 30 DEG C, after stirring half an hour, add a certain amount of Sodium hydroxide hydrotropy, be subsequently adding sodium bicarbonate, with salt acid for adjusting pH to 6.9～7.0.With 0.22 μm non-velum filteration, 4 DEG C Preserve.
2) cell is cultivated:
By CHO-S cell with 1 × 106The density of cell/ml is seeded to the shaking flask of the 250ml containing culture medium 50ml culture medium In, it is placed in 37 DEG C of cultivations in 5%CO2 incubator, rotating speed 180rpm, every 24 hours sampling countings, and uses Trypan Blue meter Calculate Cell viability.When cell cultivates the 4th day, viable cell density reaches the highest, and CHO-S peak cell number is 6.54 × 106Carefully Born of the same parents/ml, Cell viability more than 90% (Fig. 4) after inoculating 6 days
Embodiment 4:
Embodiment 1,2, the culture medium of 3 preparations compares with the Chinese hamster ovary celI serum-free medium of commercially available producer:
Commercially available culture medium uses and embodiment 1, and CHO-S cell is cultivated by 2,3 identical cultural methods, whole cultivation Process continues 6 days, cultivates the 3rd day, CHO-S cell viable cell density in serum-free medium of the present invention and commercially available culture medium Respectively reach 10.9 × 106cells/ml、5.65×106cells/ml、6.61×106Cells/ml and 8.75 × 106cells/ Ml (Fig. 5).

Claims (5)

1. the Serum-free and protein-free medium supporting Chinese hamster ovary celI suspension culture, it is characterised in that by the thing of following weight portion Matter forms:
。
2. the Chinese hamster ovary celI Serum-free and protein-free medium described in claim 1, it is characterised in that described CHO serum-free medium In possibly together with Pluronic F-68 as anti-shearing force protective agent, the consumption of described Pluronic F-68 be: 500-2000mg/ L。
3. claim 1, the Chinese hamster ovary celI Serum-free and protein-free medium described in 2, its preferred scheme is: described culture medium Including component and content be:
。
4. claim 1, the Chinese hamster ovary celI Serum-free and protein-free medium described in 2,3, its preferred scheme is: described training Component and content that foster base includes is:
。
5. claim 1, the Chinese hamster ovary celI Serum-free and protein-free medium described in 2,3,4, its most preferred scheme is: described Component and content that culture medium includes is:
。
CN201610503795.7A 2016-07-01 2016-07-01 A kind of Chinese hamster ovary celI Serum-free and protein-free medium and preparation method thereof Pending CN106190950A (en)

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CN106399224B (en) * 2016-12-13 2021-02-19 昆明润什生物科技有限公司 Serum-free and protein-free cell culture medium
CN106635953A (en) * 2016-12-13 2017-05-10 昆明润什生物科技有限公司 Serum-free protein-free cell culture medium
CN106399224A (en) * 2016-12-13 2017-02-15 昆明润什生物科技有限公司 Serum-free and protein-free cell culture medium
CN106635953B (en) * 2016-12-13 2021-02-19 昆明润什生物科技有限公司 Serum-free and protein-free cell culture medium
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
CN110894487A (en) * 2019-12-23 2020-03-20 新乡医学院 Serum-free and protein-free CHO cell culture medium and preparation method and application thereof
CN113957042A (en) * 2020-07-20 2022-01-21 维他利肤(北京)生物科技有限公司 Preparation method of stem cell zero-protein culture medium and stem cell growth factor
CN114075540A (en) * 2020-08-21 2022-02-22 北京百普赛斯生物科技股份有限公司 Complete chemical component HEK293 cell culture medium and application thereof
CN114075540B (en) * 2020-08-21 2023-10-13 苏州新微溪生物医药有限公司 HEK293 cell culture medium with full chemical composition and application thereof
CN113846051A (en) * 2021-09-28 2021-12-28 无锡多宁生物科技有限公司 General chemical component limited CHO cell subculture medium and application thereof
CN113846051B (en) * 2021-09-28 2023-12-01 无锡多宁生物科技有限公司 Universal chemical composition limiting CHO cell subculture medium and application thereof
CN115161262A (en) * 2022-08-11 2022-10-11 无锡多宁生物科技有限公司 Novel application of dextran sulfate in basic culture medium for culturing CHO cell line cells

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Application publication date: 20161207