CN112094802B - Serum-free culture medium for culturing Vero cells - Google Patents

Serum-free culture medium for culturing Vero cells Download PDF

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CN112094802B
CN112094802B CN202011046891.6A CN202011046891A CN112094802B CN 112094802 B CN112094802 B CN 112094802B CN 202011046891 A CN202011046891 A CN 202011046891A CN 112094802 B CN112094802 B CN 112094802B
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culture medium
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vero cells
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CN112094802A (en
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梁智杰
黄林
崔利凯
陈坤
谭家宇
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Boaovax Biotechnology Co ltd
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Abstract

The invention discloses a serum-free culture medium for culturing Vero cells, belonging to the technical field of biological medicines. The culture medium does not contain serum, hormone and a defoaming agent, can maintain the normal growth of cells, is suitable for large-scale culture on a reactor, and has good industrial application prospect.

Description

Serum-free culture medium for culturing Vero cells
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a Vero cell serum-free culture medium.
Background
Vero cells are African green monkey (Cercopticitech aethiops) kidney cells, cell lines were established by Yasumura and Kawakita of the university of Qianya, japan, 3 months and 27 days 1962. Vero cells are well-known excellent cell matrix which can be used for large-scale culture and preparation of virus vaccines. Vaccines produced using Vero cells include rabies vaccine, influenza vaccine, encephalitis b vaccine, hepatitis a vaccine, west nile virus vaccine, smallpox vaccine, SARS vaccine, RSV vaccine, dengue fever vaccine, chikungunya fever vaccine, and the like.
The traditional Vero cell culture adopts adherent culture, and a culture solution obtained by adding animal serum or human serum albumin into a basal culture medium is used. The animal serum and the human serum albumin have complex components, difficult sources, high price and high cost. Moreover, the added substances have the potential possibility of contamination by exogenous factors due to source and process constraints, and increase the risk of vaccine production. The importance of serum-free culture is highlighted, and the components are clear and easy to control, so that the development trend is determined.
In recent years, research on serum-free culture of Vero cells has been increasing, and for example, gibco et al have successively introduced serum-free media for serum-free culture of Vero cells. However, such a medium is expensive, and contains more or less substances such as hormones and tween 80, and the residues of these substances have an adverse effect on the human body. The invention provides a novel serum-free culture medium for Vero cell culture, which does not contain substances harmful to human bodies, such as hormone, tween 80 and the like, can support the Vero cell serum-free culture and has lower cost.
Disclosure of Invention
The invention aims to solve the problems that: provides a novel serum-free culture medium for Vero cell culture, which does not contain hormone and Tween 80, and all components are harmless to human bodies.
The technical scheme of the invention is as follows:
a serum-free medium for culturing Vero cells, the composition of the medium being as follows:
Figure BDA0002707195900000011
Figure BDA0002707195900000021
the invention also provides the application of the culture medium in culturing cells.
Further, the cell is a Vero cell.
The invention also provides a method for culturing the Vero cells, which comprises the following steps:
adding the culture medium into a cell culture container containing Vero cells, and culturing at 37 ℃.
In the method described above, the vessel is a culture flask.
The cultivation time was 4 days as described previously.
The method as described above, the vessel being a bioreactor.
The invention has the following beneficial effects:
compared with the commercial serum-free culture medium, the serum-free culture medium for culturing the Vero cells has better effect of expanding the Vero cells, and the cells have normal growth state.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Drawings
FIG. 1: pictures of cells cultured using medium No. a.
FIG. 2: pictures of cells cultured using medium No. B.
FIG. 3: pictures of cells cultured using medium No. C.
FIG. 4: pictures of cells cultured using medium No. D.
FIG. 5: pictures of cells cultured using medium No. E.
Detailed Description
EXAMPLE 1 culture Medium of the invention
The formula of the culture medium is as follows:
Figure BDA0002707195900000031
Figure BDA0002707195900000041
example 2 culture Medium of the present invention
The formula of the culture medium is as follows:
Figure BDA0002707195900000042
EXAMPLE 3 the culture Medium of the present invention
The formula of the culture medium is as follows:
Figure BDA0002707195900000043
Figure BDA0002707195900000051
the advantageous effects of the present invention are further illustrated in the form of experimental examples.
Experimental example 1 comparison of culture Effect
A cell culture comparison test was carried out on the medium (No. A, B, C) prepared in examples 1 to 3, with VP-SFM (Gibco under Thermo fisher flag, no. D) of manufacturer A and Vero SFM (Nassartight and Suzhou Biotechnology Co., ltd., no. E) of manufacturer B. The method comprises the following steps:
1. cells for assay: vero cells, purchased from ATCC.
2. Serum-free media were prepared according to examples 1 to 3, each numbered A, B, C;
3. preparing a serum-free medium according to the VP-SFM use instruction, wherein the number of the serum-free medium is D;
4. preparing a serum-free culture medium according to the Vero SFM use instruction, wherein the serial number is E;
5. taking 5T-flash 75cm 2 Respectively adding A, B, C, D, E culture mediums into a cell culture bottle; taking 5 working cell bank cells, recovering, and adding into the above 5 culture bottles respectively. The cells were cultured at 37 ℃ for 4 days, and the cell morphology and digestion count were observed.
As a result:
the cell count results are shown in the table below. It can be seen that the medium of the present invention has significantly better effect on cell expansion than the commercial medium (including the import medium VP-SFM).
Medium numbering Days of culture Cell seeding amount Cell number after cell culture
A 4 5.6×10 6 2.13×10 7
B 4 5.6×10 6 2.31×10 7
C 4 5.6×10 6 2.14×10 7
D 4 5.6×10 6 1.32×10 7
E 4 5.6×10 6 1.98×10 7
The morphology of the cells cultured in the media A to E is shown in FIGS. 1 to 5. The growth state of the cells is normal.
In conclusion, the culture medium is suitable for culturing Vero cells, has excellent propagation effect on the cells, and has normal cell growth state.

Claims (9)

1. A serum-free medium for culturing Vero cells, comprising: the culture medium does not contain hormone and Tween 80, and is used for culturing Vero cells at 37 ℃, and the components of the culture medium are as follows:
Figure FDA0003807546130000011
2. the culture medium of claim 1, wherein the culture formulation is as follows:
Figure FDA0003807546130000012
Figure FDA0003807546130000021
3. the culture medium of claim 1, wherein the culture formulation is as follows:
Figure FDA0003807546130000022
Figure FDA0003807546130000031
4. the culture medium of claim 1, wherein the culture formulation is as follows:
Figure FDA0003807546130000032
5. use of a culture medium according to any one of claims 1 to 4 for culturing cells, characterized in that: the cell is a Vero cell.
6. A method of culturing Vero cells comprising:
adding the culture medium according to any one of claims 1 to 4 into a cell culture vessel containing Vero cells, and culturing at 37 ℃.
7. The method of claim 6, wherein: the container is a culture bottle.
8. The method of claim 7, wherein: the culture time was 4 days.
9. The method of claim 6, wherein: the vessel is a bioreactor.
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Family Cites Families (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU3813597A (en) * 1996-07-26 1998-02-20 University Of Manitoba Serum-free medium for growth of anchorage-dependant mammalian cells
GB0221330D0 (en) * 2002-09-13 2002-10-23 Medi Cult As Growth factor for cell culture
CN100482783C (en) * 2007-11-27 2009-04-29 天津百若克医药生物技术有限责任公司 Culture medium used for Vero cell and cultivation method thereof
CN101864393B (en) * 2009-05-06 2012-04-04 中国人民解放军军事医学科学院生物工程研究所 Serum-free culture medium without animal origin components for culturing Vero cell micro-carrier
US8609416B2 (en) * 2009-12-18 2013-12-17 Ventria Bioscience Methods and compositions comprising heat shock proteins
CN101974481A (en) * 2010-10-29 2011-02-16 国家兽用生物制品工程技术研究中心 Serum free culture medium for growing various cells derived from kidney tissue
CN102827804B (en) * 2012-08-30 2019-02-12 苏州市沃美生物技术有限公司 Culture medium and method suitable for the amplification culture of Vero cell microcarrier suspension
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
CN110628697A (en) * 2019-09-23 2019-12-31 山东甲骨文生物科技有限公司 Serum-free culture medium for VERO serum-free cell culture and corresponding virus production

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