CN106520668A - Protein serum-free culture medium and preparation method thereof - Google Patents

Protein serum-free culture medium and preparation method thereof Download PDF

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Publication number
CN106520668A
CN106520668A CN201611265850.XA CN201611265850A CN106520668A CN 106520668 A CN106520668 A CN 106520668A CN 201611265850 A CN201611265850 A CN 201611265850A CN 106520668 A CN106520668 A CN 106520668A
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powder
culture medium
free medium
serum
preparation
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齐智
明恒磊
孟双有
成芳硕
孟丹丹
信健
房圆瑗
牛欣桐
朱宁
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Shandong Golden Week Biological Technology Co Ltd
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Shandong Golden Week Biological Technology Co Ltd
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Abstract

The invention discloses a protein serum-free culture medium and a preparation method thereof, and belongs to the technical field of biological chemistry. The method comprises the following steps of 1 drying, wherein raw materials are mixed and dried to obtain a mixture, and 1,500-3,000 parts by mass of sodium bicarbonate is taken and dried; 2 powdering, wherein the mixture in the step 1 is ball-milled and then screened with a 150-mesh sieve, the parts obtained after screening are taken and mixed to obtain first powder, dried sodium bicarbonate is ball-milled and then screened with a 150-mesh sieve, and the parts obtained after screening are prepared into second powder; 3 dissolving, wherein when in use, the first powder obtained in the step 2 is dissolved into 1,000 parts by mass of ultrapure water, after stirring and dissolving are conducted to be uniform, the second powder is added, stirring and dissolving are conducted to be uniform again, and the culture medium is obtained; 4 filtration sterilization, wherein the culture medium obtained in the step 3 is subjected to filtration sterilization through a 0.22-micrometer filter membrane. The protein serum-free culture medium can enable cell proliferation to be normal and enable the activity to be stable; in addition, the protein content is obviously lower than an ordinary culture medium, and the extraction technology is simplified.

Description

A kind of albumen serum-free medium and preparation method thereof
Technical field
The invention belongs to technological field of biochemistry, and in particular to a kind of albumen serum-free medium and preparation method thereof.
Background technology
In recent years, with increasingly mature, animal cell culture production vaccine, the monoclonal antibody of animal cell culture technology And the new industry of functional protein develops growth rapidly.Most of cell culture needs the serum of animal origin, serum Abundant somatomedin, hormone and other nutrient substance can be provided for the cell of isolated culture.However, due to albumen in serum Species is more, complicated component, increased the burden and cost of downstream extraction work.Meanwhile, serum is expensive, quality between batch It is unstable, be easily introduced it is zoogenous virus and mycoplasma, bring inconvenience to cell culture.
Serum-free medium is the key for solving the above problems, as serum-free medium protein content is low, steady quality, The potential danger such as virus-free is introduced, and is greatly reduced the cost that cell culture and product are extracted, is simplified production technology.Serum-free The development and application of culture medium is conducive to extensive, the industrialization culture of zooblast.
The serum-free production medium of at present external existing biology Corporation R ＆ D is successfully listed, such as Gibco, Hyclone, Lonza etc., but it is expensive；The research and development of domestic serum-free medium are still in the starting stage, rarely have ripe brand at present.
The content of the invention
The technical problem to be solved is to provide a kind of albumen serum-free medium and preparation method thereof, Neng Gouwei Cell provides good reproduction environment, and is easy to cell extraction.
To solve above-mentioned technical problem, the technical scheme is that：Invent a kind of albumen serum-free medium, its feature It is：The powder raw material of following mass parts is dissolved in the ultra-pure water of 1000 mass parts：
Preferably, the mass fraction of powder raw material is：
Preferably, the particle diameter of the powder raw material is more than 150 mesh.
Present invention also offers a kind of preparation method of above-mentioned albumen serum-free medium, it is characterised in that：Including following Step：(1) dry：
Raw material is taken by following mass fraction：
Mixing drying is carried out, compound is obtained；
Take the drying of 1500～3000 mass parts sodium bicarbonate；
(2) powder processed：150 mesh sieves will be crossed after compound ball milling in step (1), lower part of screen point blended prepared first will be taken Powder；150 mesh sieves are crossed after the sodium bicarbonate ball milling of drying, lower part of screen point is taken and the second powder is obtained；
(3) dissolve：During use, obtained first powder in step (2) is dissolved in the ultra-pure water of 1000 mass parts, is stirred The second powder is added after being uniformly dissolved, is again stirring for being uniformly dissolved, culture medium is obtained；
(4) filtration sterilization：Obtained culture medium in step (3) is crossed 0.22 μm of filter membrane carries out filtration sterilization.
Preferably, the drying temperature in step (1) is 50 DEG C, and drying time is 1～2h.
Preferably, the Ball-milling Time in step (2) is 30～60min.
Preferably, the mixing in step (2) is operated by three-dimensional stereo mixing machines, and the operating time is 30～60min.
Compared with prior art, the invention has the beneficial effects as follows：
1st, cell propagation：Albumen serum-free culture basal cell's normal proliferation of the present invention, vigor are stable, and containing 8% calf Serum finished product culture medium is compared without significant difference；
2nd, protein content is low：The albumen serum-free medium protein content of the present invention is significantly lower than conventional medium, simplifies Extraction process, reduce production cost；
3rd, steady quality：Due to known to this product composition, non-animal derived albumen, duplication of production steady quality.
4th, the present invention is applied widely, is applicable to various conventional cell lines such as Vero, BHK-21, A549, CHO, and Simple from the transition of routine serum culture medium, adaptability is fast；
5th, cell attachment speed is fast, by taking Vero cells as an example, using this product culture 6h, can adherent more than 90%；
6th, all the components of the present invention are, it is known that configuration process is simple, repeatability are preferable.
Specific embodiment
The present invention is described in further detail with reference to specific embodiment.
Embodiment one
The step of present invention prepares albumen serum-free medium is as follows：
A kind of preparation method of albumen serum-free medium, it is characterised in that：Comprise the following steps：
(1) dry：
Raw material is taken by following mass fraction：
Mixing drying is carried out, compound is obtained；
Take the drying of 2800 mass parts sodium bicarbonate；Drying temperature in this step is 50 DEG C, and drying time is 1h.
(2) powder processed：150 mesh sieves will be crossed after compound ball milling in step (1), Ball-milling Time is 45min, takes lower part of screen point And Jing three-dimensional stereo mixing machines mixing 45min is obtained the first powder；150 mesh sieves are crossed after the sodium bicarbonate ball milling of drying, is taken under sieve Part is obtained the second powder；The storage ambient of the first powder and the second powder is the place of 2 DEG C of temperature, lucifuge and drying, waits to make Used time is now with the current；
(3) dissolve：During use, obtained first powder in step (2) is dissolved in the ultra-pure water of 1000 mass parts, is stirred The second powder is added after being uniformly dissolved, is again stirring for being uniformly dissolved, culture medium is obtained；
(4) filtration sterilization：Obtained culture medium in step (3) is crossed 0.22 μm of filter membrane carries out filtration sterilization.
Embodiment two
The present embodiment with the difference of embodiment one is：Raw material of the compound in step (1) by following mass parts Mix：
Take the drying of 1500 mass parts sodium bicarbonate；Drying time in this step is 1.5h.
In step (2), Ball-milling Time is 30min, and Jing three-dimensional stereo mixing machines mixing 30min is obtained the first powder；First powder The storage ambient of end and the second powder is 5 DEG C of temperature；
Embodiment three
The present embodiment with the difference of embodiment one is：Raw material of the compound in step (1) by following mass parts Mix：
Take the drying of 3000 mass parts sodium bicarbonate；Drying time in this step is 2h.
In step (2), Ball-milling Time is 60min, and Jing three-dimensional stereo mixing machines mixing 60min is obtained the first powder；First powder The storage ambient of end and the second powder is 8 DEG C of temperature；
By taking Vero cells as an example, contrast test is carried out, test is that culture 3 days is carried out in the environment that temperature is 37 DEG C, The intake 5.0% of CO2.Wherein, in comparative example one, used medium is+8% calf serum of MEM culture medium, in comparative example two Used medium is+8% calf serum of F12 culture medium, and experimental data is as follows：
Can be seen that main performance of the serum-free medium of the present invention in terms of cell culture is complete from above-mentioned test data Entirely no less than the culture medium for having serum, or even also want more excellent, be fully able to meet the demand of cell culture.
The above, is only presently preferred embodiments of the present invention, is not the restriction for making other forms to the present invention, is appointed What those skilled in the art is combined, changes or is retrofited possibly also with the technology contents of the disclosure above and is the present invention Equivalent embodiments.But it is every without departing from technical solution of the present invention content, implement to more than according to the technical spirit of the present invention Any simple modification, equivalent variations and remodeling that example is made, still fall within the protection domain of technical solution of the present invention.

Claims (7)

1. a kind of albumen serum-free medium, it is characterised in that：The powder of following mass parts is dissolved in the ultra-pure water of 1000 mass parts Shape raw material：
2. albumen serum-free medium according to claim 1, it is characterised in that：The mass fraction of powder raw material is：
3. albumen serum-free medium according to claim 1 and 2, it is characterised in that：The particle diameter of the powder raw material is big In 150 mesh.
4. a kind of preparation method of albumen serum-free medium, it is characterised in that：Comprise the following steps：
(1) dry：
Raw material is taken by following mass fraction：
Mixing drying is carried out, compound is obtained；
Take the drying of 1500～3000 mass parts sodium bicarbonate；
(2) powder processed：150 mesh sieves will be crossed after compound ball milling in step (1), lower part of screen point blended prepared first powder will be taken End；150 mesh sieves are crossed after the sodium bicarbonate ball milling of drying, lower part of screen point is taken and the second powder is obtained；
(3) dissolve：During use, obtained first powder in step (2) is dissolved in the ultra-pure water of 1000 mass parts, stirring and dissolving The second powder is added after uniform, is again stirring for being uniformly dissolved, culture medium is obtained；
(4) filtration sterilization：Obtained culture medium in step (3) is crossed 0.22 μm of filter membrane carries out filtration sterilization.
5. the preparation method of albumen serum-free medium according to claim 4, it is characterised in that：Baking in step (1) Dry temperature is 50 DEG C, and drying time is 1～2h.
6. the preparation method of albumen serum-free medium according to claim 5, it is characterised in that：Ball in step (2) Consume time as 30～60min.
7. the preparation method of albumen serum-free medium according to claim 6, it is characterised in that：It is mixed in step (2) Close by three-dimensional stereo mixing machines to operate, the operating time is 30～60min.
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CN101988047A (en) * 2010-05-13 2011-03-23 维亚生物科技(上海)有限公司 Insect cell serum-free medium with low cost
CN102876626A (en) * 2011-07-14 2013-01-16 宁波安柯普顿生物技术有限公司 Serum-free and protein-free all-chemical-component-definition culture medium for supporting CHO high-density suspension growth
CN104293729A (en) * 2014-02-14 2015-01-21 上海美百瑞生物医药技术有限公司 Efficient serum-free culture medium
CN105441378A (en) * 2015-12-22 2016-03-30 肇庆大华农生物药品有限公司 Serum-free medium used for culturing Vero cells, and preparation method thereof
CN105985926A (en) * 2015-03-06 2016-10-05 王玉 Serum-free culture medium for CHO cell culture
CN106119186A (en) * 2016-06-24 2016-11-16 广东温氏大华农生物科技有限公司 A kind of serum-free medium for full suspension culture mdck cell and preparation method thereof

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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