CN111733126A

CN111733126A - Vero cell serum-free culture medium and application thereof

Info

Publication number: CN111733126A
Application number: CN202010720750.1A
Authority: CN
Inventors: 徐亮亮; 陈旭; 陈刚; 赵亚; 王涛; 董晓惠; 辛丽丽; 杨建国
Original assignee: Excell Biology Taicang Co ltd
Current assignee: Suzhou Ecosai Biotechnology Co.,Ltd.
Priority date: 2020-07-24
Filing date: 2020-07-24
Publication date: 2020-10-02
Anticipated expiration: 2040-07-24
Also published as: CN111733126B

Abstract

The invention relates to a Vero cell serum-free culture medium and application thereof, which comprises an amino acid component, a vitamin component, an inorganic salt component, a trace element component, a carbohydrate and other molecular compound components, wherein the trace element component comprises the following components: zinc sulfate heptahydrate, copper sulfate pentahydrate, iron nitrate nonahydrate, ferrous sulfate heptahydrate, sodium selenite, nickel chloride, stannous chloride, silver nitrate, cobalt chloride and aluminum chloride. The serum-free culture medium has clear chemical components and low cost, does not need serum, avoids the biosafety risk caused by using the serum, and provides convenience for the large-scale production of biological products, particularly vaccines. And the Vero cells grow well in the culture medium of the invention without serum, which is beneficial to improving the stability of the production process of biological products and the quality of finished products, in particular to accelerating the research and development and production of new coronavirus (SARS-CoV-2) vaccines.

Description

Vero cell serum-free culture medium and application thereof

Technical Field

The invention belongs to the technical field of cell culture, and particularly relates to a Vero cell serum-free culture medium and application thereof.

Background

Vero cells (African green kidney cells, Vero cells) are a vaccine production cell line approved by the world health organization and the national biological product code. The method is currently used for producing various vaccines such as rabies virus and influenza virus. Meanwhile, Vero cells are also extremely excellent host cells for the development approach of vaccines by obtaining inactivated strains or attenuated strains of new coronavirus.

In vaccine production, in order to realize high-density culture of cells, 5% -10% of serum is usually added into a basic culture medium, and the serum can provide various nutrients required by cell growth, such as hormones, trace elements, anchorage factors and the like. However, there are many disadvantages to the use of serum: firstly, differences exist among serum batches, and the stability and quality of the vaccine production process are influenced; secondly, the use of serum is easy to cause microbial contamination; serum components are complex, and substances for promoting cell growth and substances for inhibiting cell growth exist at the same time, so that influence is caused on cell growth and subsequent downstream purification; serum is expensive; policy and regulation and industry guiding principle are continuously strict, and the production of biological products for removing blood serum is a future trend. Therefore, the use of serum-free media has become a trend in Vero culture. Most of serum-free culture media developed at home can support cell growth at present, but various nutrients, insulin, protein hydrolysate and the like are added on the basis of the existing culture media such as DMEM, DMEM/F12 and the like, so that the components are complex, and the quality and the safety of products face the test. For example, the culture medium and the method disclosed in chinese patent CN102827804A, which is suitable for suspension amplification culture of Vero cell microcarriers, include amino acids, inorganic salts, vitamins, protein hydrolysates, lipids, buffer components and additives, however, the culture medium still needs to be added with trace serum during the Vero cell culture process, and cannot achieve true serum-free. And protein hydrolysate and the like are added into the composition of the culture medium, so that the chemical components are not clear, and the potential safety exists.

Serum-free culture media of companies such as foreign Life, Thermo, Lonza and the like are reliable and stable in quality, but are relatively expensive in price, risk exists in supply stability, and components are secret and undisplayed, so that the cost of large-scale cell culture is increased, the space for improving productivity and reducing cost is reduced, optimization and adjustment of the culture media according to product needs are not facilitated, and the subsequent optimization space is greatly reduced.

Disclosure of Invention

In order to solve the problems in the prior art, the invention aims to provide a serum-free culture medium suitable for Vero cells.

In order to achieve the purpose, the invention adopts the technical scheme that:

a Vero cell serum-free culture medium comprises amino acid component, vitamin component, inorganic salt component, microelement component, carbohydrate and other molecular compound components, wherein,

the amino acid component comprises the following components:

35-120mg/L of L-arginine;

30-95mg/L of L-aspartic acid;

30-100mg/L of L-asparagine;

15-40mg/L of L-cysteine;

20-120mg/L of L-glycine;

10-25mg/L of L-glutamic acid;

14-45mg/L of L-histidine;

25-85mg/L of L-isoleucine;

26-80mg/L of L-leucine;

30-100mg/L of L-lysine;

6-30mg/L of L-methionine;

l-phenylalanine 10-60 m/L;

15-70mg/L of L-proline;

l-serine 20-80 mg/L;

10-50mg/L of L-threonine;

10-40mg/L of L-tryptophan;

10-50mg/L of L-tyrosine;

l-valine 10-70 mg/L;

the trace element component comprises the following components:

0.5-5 mg/L of zinc sulfate heptahydrate;

0.0001-0.01 mg/L of blue vitriol;

0.01-0.45 mg/L ferric nitrate nonahydrate;

ferrous sulfate heptahydrate 0.4-15.5 mg/L;

0.01-0.8mg/L of sodium selenite;

0.00005-0.0005mg/L of nickel chloride;

stannous chloride 0.00005-0.0005 mg/L;

0.00001-0.0002mg/L silver nitrate;

cobalt chloride 0.0005-0.008 mg/L;

0.0001-0.001mg/L of aluminum chloride;

the carbohydrate and other molecular compound components include the following ingredients:

glucose 3000-9000 mg/L;

5-30mg/L of ethanolamine;

0.1-15 mg/L putrescine;

0.2-3 mg/L of lipoic acid;

linoleic acid 0.01-0.9 mg/L;

600mg/L sodium pyruvate 105-;

PVP40 100-3000mg/L；

2' -deoxyadenosine 1-30 mg/L;

1-30mg/L of 2' -deoxyguanosine;

1-30mg/L of 2' -deoxycytidine;

hypoxanthine is 1-30 mg/L;

adenosine 1-30 mg/L;

1-30mg/L uridine;

1-30mg/L of guanosine;

1-30mg/L cytidine;

5-300mg/L of insulin;

the recombinant epidermal growth factor is 0.1-100 mg/L.

According to some embodiments of the invention, the vitamin component comprises the following ingredients:

0.09-3.62 mg/L of biotin;

2-12.5 mg/L of calcium pantothenate;

folic acid 9.45-34.5 mg/L;

3.25-23mg/L of nicotinamide;

vitamin B61-10 mg/L;

thiamine hydrochloride in 3.25-13 mg/L;

120.8-15 mg/L of vitamin B;

riboflavin is 0.1-3.5 mg/L;

choline chloride 18-180 mg/L;

inositol 10-75 mg/L.

According to some embodiments of the invention, the inorganic salt component comprises the following:

10-200 mg/L of magnesium chloride;

magnesium sulfate is 30-200 mg/L;

56-232 mg/L of calcium chloride;

potassium chloride 212-936 mg/L;

sodium bicarbonate 1200 and 3600 mg/L;

800-4000 mg/L sodium chloride;

70-600 mg/L sodium phosphate.

According to some embodiment aspects of the invention, in the medium, the amino acid composition comprises:

35-120mg/L of L-arginine;

30-95mg/L of L-aspartic acid;

30-100mg/L of L-asparagine;

15-40mg/L of L-cysteine;

20-120mg/L of L-glycine;

10-25mg/L of L-glutamic acid;

14-45mg/L of L-histidine;

25-85mg/L of L-isoleucine;

26-80mg/L of L-leucine;

30-100mg/L of L-lysine;

6-30mg/L of L-methionine;

l-phenylalanine 10-60 m/L;

15-70mg/L of L-proline;

l-serine 20-80 mg/L;

10-50mg/L of L-threonine;

10-40mg/L of L-tryptophan;

10-50mg/L of L-tyrosine;

l-valine 10-70 mg/L;

the vitamin component comprises:

0.09-3.62 mg/L of biotin;

2-12.5 mg/L of calcium pantothenate;

folic acid 9.45-34.5 mg/L;

3.25-23mg/L of nicotinamide;

vitamin B61-10 mg/L;

thiamine hydrochloride in 3.25-13 mg/L;

120.8-15 mg/L of vitamin B;

riboflavin is 0.1-3.5 mg/L;

choline chloride 18-180 mg/L;

inositol 10-75 mg/L;

the inorganic salt component comprises:

10-200 mg/L of magnesium chloride;

magnesium sulfate is 30-200 mg/L;

56-232 mg/L of calcium chloride;

potassium chloride 212-936 mg/L;

sodium bicarbonate 1200 and 3600 mg/L;

800-4000 mg/L sodium chloride;

70-600 mg/L of sodium phosphate;

the trace element component comprises:

0.5-5 mg/L of zinc sulfate heptahydrate;

0.0001-0.01 mg/L of blue vitriol;

0.01-0.45 mg/L ferric nitrate nonahydrate;

ferrous sulfate heptahydrate 0.4-15.5 mg/L;

0.01-0.8mg/L of sodium selenite;

0.00005-0.0005mg/L of nickel chloride;

stannous chloride 0.00005-0.0005 mg/L;

0.00001-0.0002mg/L silver nitrate;

cobalt chloride 0.0005-0.008 mg/L;

0.0001-0.001mg/L of aluminum chloride;

the carbohydrate and other molecular compound components include:

glucose 3000-9000 mg/L;

5-30mg/L of ethanolamine;

0.1-15 mg/L putrescine;

0.2-3 mg/L of lipoic acid;

linoleic acid 0.01-0.9 mg/L;

600mg/L sodium pyruvate 105-;

PVP40 100-3000mg/L；

2' -deoxyadenosine 1-30 mg/L;

1-30mg/L of 2' -deoxyguanosine;

1-30mg/L of 2' -deoxycytidine;

hypoxanthine is 1-30 mg/L;

adenosine 1-30 mg/L;

1-30mg/L uridine;

1-30mg/L of guanosine;

1-30mg/L cytidine;

5-300mg/L of insulin;

the recombinant epidermal growth factor is 0.1-100 mg/L.

Preferably, in the culture medium,

the amino acid component comprises:

35-120mg/L of L-arginine;

30-95mg/L of L-aspartic acid;

30-100mg/L of L-asparagine;

15-30mg/L of L-cysteine;

60-100mg/L of L-glycine;

10-20mg/L of L-glutamic acid;

l-histidine 20-40 mg/L;

l-isoleucine 40-60 mg/L;

26-80mg/L of L-leucine;

30-100mg/L of L-lysine;

6-30mg/L of L-methionine;

l-phenylalanine 10-60 m/L;

15-50mg/L of L-proline;

l-serine 20-60 mg/L;

10-40mg/L of L-threonine;

10-40mg/L of L-tryptophan;

10-40mg/L of L-tyrosine;

l-valine 10-50 mg/L;

the vitamin component comprises:

0.09-3 mg/L of biotin;

2-10 mg/L of calcium pantothenate;

folic acid 9.45-34.5 mg/L;

5-20mg/L of nicotinamide;

vitamin B61-10 mg/L;

thiamine hydrochloride 5-10 mg/L;

120.8-15 mg/L of vitamin B;

0.5-2 mg/L of riboflavin;

30-150mg/L of choline chloride;

inositol 10-75 mg/L;

the inorganic salt component comprises:

10-100 mg/L of magnesium chloride;

magnesium sulfate 100-200 mg/L;

100-232 mg/L calcium chloride;

300-600mg/L potassium chloride;

sodium bicarbonate 2000-3600 mg/L;

800-2000 mg/L sodium chloride;

100mg/L of sodium phosphate;

the trace element component comprises:

0.5-3 mg/L of zinc sulfate heptahydrate;

0.0001-0.005 mg/L of copper sulfate pentahydrate;

0.01-0.2mg/L ferric nitrate nonahydrate;

ferrous sulfate heptahydrate 0.4-15.5 mg/L;

0.01-0.2mg/L of sodium selenite;

0.00005-0.0005mg/L of nickel chloride;

stannous chloride 0.00005-0.0005 mg/L;

0.00001-0.0002mg/L silver nitrate;

cobalt chloride 0.0005-0.008 mg/L;

0.0001-0.001mg/L of aluminum chloride;

the carbohydrate and other molecular compound components include:

glucose 3000-6000 mg/L;

5-30mg/L of ethanolamine;

0.1-10 mg/L putrescine;

0.2-2 mg/L of lipoic acid;

linoleic acid 0.1-0.5 mg/L;

sodium pyruvate 105-400 mg/L;

PVP40 500-1500mg/L；

2' -deoxyadenosine 1-25 mg/L;

1-25mg/L of 2' -deoxyguanosine;

1-25mg/L of 2' -deoxycytidine;

hypoxanthine is 1-25 mg/L;

adenosine 5-25 mg/L;

5-25mg/L uridine;

5-25mg/L of guanosine;

cytidine 5-25 mg/L;

50-250mg/L of insulin;

the recombinant epidermal growth factor is 0.1-100 mg/L.

More preferably, in the culture medium,

the amino acid component comprises:

45-120mg/L of L-arginine;

30-95mg/L of L-aspartic acid;

30-95mg/L of L-asparagine;

15-20mg/L of L-cysteine;

80-100mg/L of L-glycine;

10-20mg/L of L-glutamic acid;

30-40mg/L of L-histidine;

l-isoleucine 40-60 mg/L;

30-70mg/L of L-leucine;

30-100mg/L of L-lysine;

8-30mg/L of L-methionine;

l-phenylalanine 20-30 m/L;

15-35mg/L of L-proline;

15-50mg/L of L-serine;

10-30mg/L of L-threonine;

10-40mg/L of L-tryptophan;

10-40mg/L of L-tyrosine;

l-valine 10-50 mg/L;

the vitamin component comprises:

0.09-2 mg/L of biotin;

3-8 mg/L of calcium pantothenate;

10-21 mg/L folic acid;

10-20mg/L of nicotinamide;

vitamin B63-10 mg/L;

thiamine hydrochloride 5-10 mg/L;

vitamin B121-10 mg/L;

0.5-1 mg/L of riboflavin;

30-100mg/L of choline chloride;

inositol 10-50 mg/L;

the inorganic salt component comprises:

20-100 mg/L of magnesium chloride;

magnesium sulfate 100-150 mg/L;

100mg/L of calcium chloride;

300mg/L of potassium chloride;

sodium bicarbonate 2000-3000 mg/L;

sodium chloride 1500-;

sodium phosphate 200-500 mg/L;

the trace element component comprises:

0.5-2 mg/L of zinc sulfate heptahydrate;

0.0001-0.005 mg/L of copper sulfate pentahydrate;

0.01-0.1mg/L ferric nitrate nonahydrate;

5-15mg/L of ferrous sulfate heptahydrate;

0.01-0.1mg/L of sodium selenite;

0.00005-0.0005mg/L of nickel chloride;

stannous chloride 0.00005-0.0005 mg/L;

0.00001-0.0002mg/L silver nitrate;

cobalt chloride 0.0005-0.008 mg/L;

0.0001-0.001mg/L of aluminum chloride;

the carbohydrate and other molecular compound components include:

glucose 4000-5000 mg/L;

10-20mg/L of ethanolamine;

1-8 mg/L putrescine;

0.5-2 mg/L of lipoic acid;

linoleic acid 0.1-0.4 mg/L;

200-350 mg/L of sodium pyruvate;

PVP40 800-1100mg/L；

2' -deoxyadenosine 5-20 mg/L;

5-20mg/L of 2' -deoxyguanosine;

5-20mg/L of 2' -deoxycytidine;

hypoxanthine is 5-20 mg/L;

adenosine 10-20 mg/L;

10-20mg/L uridine;

guanosine is 10-20 mg/L;

10-20mg/L of cytidine;

100-250mg/L of insulin;

the recombinant epidermal growth factor is 0.1-100 mg/L.

According to an embodiment of the invention, the composition of the amino acid composition in the medium is: l-arginine 120mg/L, L-aspartic acid 95mg/L, L-asparagine 30mg/L, L-cysteine 15mg/L, L-glycine 80mg/L, L-glutamic acid 15mg/L, L-histidine 27mg/L, L-isoleucine 50mg/L, L-leucine 30mg/L, l-lysine 30mg/L, L-methionine 30mg/L, L-phenylalanine 30m/L, L-proline 15mg/L, L-serine 15mg/L, L-threonine 20mg/L, L-tryptophan 10mg/L, L-tyrosine 10mg/L and L-valine 10 mg/L;

the vitamin components comprise the following components: biotin 2.0mg/L, calcium pantothenate 5.0mg/L, folic acid 10mg/L, nicotinamide 10mg/L, vitamin B610 mg/L, thiamine hydrochloride 5mg/L, vitamin B1210 mg/L, riboflavin 1.0mg/L, choline chloride 100mg/L and inositol 50 mg/L;

the inorganic salt component comprises the following components: 100mg/L of magnesium chloride, 100mg/L of magnesium sulfate, 100mg/L of calcium chloride, 500mg/L of potassium chloride, 2400mg/L of sodium bicarbonate, 1500mg/L of sodium chloride and 500mg/L of sodium phosphate;

the trace element components comprise: 2mg/L of zinc sulfate heptahydrate, 0.0001mg/L of copper sulfate pentahydrate, 0.02mg/L of ferric nitrate nonahydrate, 10mg/L of ferrous sulfate heptahydrate, 0.1mg/L of sodium selenite, 0.00005mg/L of nickel chloride, 0.00005mg/L of stannous chloride, 0.0001mg/L of silver nitrate, 0.0005mg/L of cobalt chloride and 0.001mg/L of aluminum chloride;

the composition of the carbohydrate and other molecular compound components is: 4000mg/L glucose, 20mg/L ethanolamine, 8.0mg/L putrescine, 1.5mg/L lipoic acid, 0.2mg/L linoleic acid, 200mg/L, PVP 401000 mg/L sodium pyruvate, 20 mg/L2 ' -deoxyadenosine, 20 mg/L2 ' -deoxyguanosine, 20 mg/L2 ' -deoxycytidine, 20mg/L hypoxanthine, 20mg/L adenosine, 20mg/L uridine, 20mg/L guanosine, 20mg/L cytidine, 200mg/L insulin and 0.1mg/L recombinant epidermal growth factor.

The invention adopts another technical scheme that: the Vero cell serum-free culture medium is used for Vero cell culture.

Due to the application of the technical scheme, compared with the prior art, the invention has the following advantages:

the serum-free culture medium has clear chemical components, does not need serum, has low cost, avoids the biosafety risk caused by using the serum, and provides convenience for the large-scale production of biological products, particularly vaccines. And the Vero cells grow well in the culture medium of the invention without serum, which is beneficial to improving the stability of the production process of biological products and the quality of finished products, in particular to accelerating the research and development and production of new coronavirus (SARS-CoV-2) vaccines.

Drawings

FIG. 1 is a graph showing the results of viable cell density of Vero cells serially passaged three times using the media of examples 1-4 and control media 1-3;

FIG. 2 is a graph showing the results of the total number of viable cells after three serial passages of Vero cells using the culture media of examples 1 to 4 and control media 1 to 3;

FIG. 3 is a schematic diagram of Vero cells grown in the medium of example 1 for 72 hours.

Detailed Description

In order to clearly embody the objects, technical solutions and advantages of the present invention, the following detailed description of the embodiments of the present invention is provided with reference to the accompanying drawings. The examples are given solely for the purpose of illustration and are not intended to be limiting.

Example 1

This example provides a serum-free medium for Vero cell culture comprising an amino acid component, a vitamin component, an inorganic salt component, a trace element component, a carbohydrate, and other molecular compound components, wherein,

the amino acid component comprises the following components: l-arginine 45mg/L, L-aspartic acid 30mg/L, L-asparagine 95mg/L, L-cysteine 20mg/L, L-glycine 100mg/L, L-glutamic acid 15mg/L, L-histidine 27mg/L, L-isoleucine 50mg/L, L-leucine 66mg/L, l-lysine 100mg/L, L-methionine 8mg/L, L-phenylalanine 20m/L, L-proline 15mg/L, L-serine 50mg/L, L-threonine 20mg/L, L-tryptophan 15mg/L, L-tyrosine 35mg/L and L-valine 30 mg/L.

The vitamin component comprises the following components: biotin 0.1mg/L, calcium pantothenate 3mg/L, folic acid 10.33mg/L, nicotinamide 15mg/L, vitamin B63 mg/L, thiamine hydrochloride 5.5mg/L, vitamin B121.0 mg/L, riboflavin 0.9mg/L, choline chloride 35mg/L and inositol 10 mg/L.

The inorganic salt component comprises the following components: 20.0mg/L of magnesium chloride, 150mg/L of magnesium sulfate, 200mg/L of calcium chloride, 350mg/L of potassium chloride, 2400mg/L of sodium bicarbonate, 2000mg/L of sodium chloride and 200mg/L of sodium phosphate.

The trace element components comprise: 2.0mg/L of zinc sulfate heptahydrate, 0.005mg/L of copper sulfate pentahydrate, 0.1mg/L of ferric nitrate nonahydrate, 5.3mg/L of ferrous sulfate heptahydrate, 0.02mg/L of sodium selenite, 0.0001mg/L of nickel chloride, 0.0001mg/L of stannous chloride, 0.0001mg/L of silver nitrate, 0.003mg/L of cobalt chloride and 0.0008mg/L of aluminum chloride.

The composition of the carbohydrate and other molecular compound components is: 5000mg/L glucose, 10mg/L ethanolamine, 1.5mg/L putrescine, 0.8mg/L lipoic acid, 0.2mg/L linoleic acid, 330mg/L, PVP 401000 mg/L sodium pyruvate, 5.0 mg/L2 ' -deoxyadenosine, 5.0 mg/L2 ' -deoxyguanosine, 5.0 mg/L2 ' -deoxycytidine, 5.0mg/L hypoxanthine, 10mg/L adenosine, 10mg/L uridine, 10mg/L guanosine, 10mg/L cytidine, 100mg/L insulin and 10mg/L recombinant epidermal growth factor.

Example 2

the amino acid component comprises the following components: l-arginine 100mg/L, L-aspartic acid 30mg/L, L-asparagine 55mg/L, L-cysteine 20mg/L, L-glycine 80mg/L, L-glutamic acid 15mg/L, L-histidine 27mg/L, L-isoleucine 50mg/L, L-leucine 30mg/L, l-lysine 30mg/L, L-methionine 30mg/L, L-phenylalanine 30m/L, L-proline 35mg/L, L-serine 50mg/L, L-threonine 20mg/L, L-tryptophan 40mg/L, L-tyrosine 35mg/L and L-valine 50 mg/L.

The vitamin component comprises the following components: biotin 1.0mg/L, calcium pantothenate 8.0mg/L, folic acid 20.33mg/L, nicotinamide 10mg/L, vitamin B63 mg/L, thiamine hydrochloride 10mg/L, vitamin B1210 mg/L, riboflavin 0.9mg/L, choline chloride 35mg/L, and inositol 10 mg/L.

The inorganic salt component comprises the following components: 100mg/L of magnesium chloride, 100mg/L of magnesium sulfate, 200mg/L of calcium chloride, 350mg/L of potassium chloride, 3000mg/L of sodium bicarbonate, 1000mg/L of sodium chloride and 200mg/L of sodium phosphate.

The trace element components comprise: 0.5mg/L of zinc sulfate heptahydrate, 0.0005mg/L of copper sulfate pentahydrate, 0.01mg/L of ferric nitrate nonahydrate, 15.0mg/L of ferrous sulfate heptahydrate, 0.01mg/L of sodium selenite, 0.0001mg/L of nickel chloride, 0.0001mg/L of stannous chloride, 0.0001mg/L of silver nitrate, 0.003mg/L of cobalt chloride and 0.0001mg/L of aluminum chloride.

The composition of the carbohydrate and other molecular compound components is: 4000mg/L glucose, 20mg/L ethanolamine, 8.0mg/L putrescine, 1.5mg/L lipoic acid, 0.2mg/L linoleic acid, 200mg/L, PVP 401000 mg/L sodium pyruvate, 10 mg/L2 ' -deoxyadenosine, 10 mg/L2 ' -deoxyguanosine, 10 mg/L2 ' -deoxycytidine, 10mg/L hypoxanthine, 10mg/L adenosine, 10mg/L uridine, 10mg/L guanosine, 10mg/L cytidine, 100mg/L insulin and 100mg/L recombinant epidermal growth factor.

Example 3

the amino acid component comprises the following components: l-arginine 120mg/L, L-aspartic acid 95mg/L, L-asparagine 30mg/L, L-cysteine 15mg/L, L-glycine 80mg/L, L-glutamic acid 15mg/L, L-histidine 27mg/L, L-isoleucine 50mg/L, L-leucine 30mg/L, l-lysine 30mg/L, L-methionine 30mg/L, L-phenylalanine 30m/L, L-proline 15mg/L, L-serine 15mg/L, L-threonine 20mg/L, L-tryptophan 10mg/L, L-tyrosine 10mg/L and L-valine 10 mg/L.

The vitamin component comprises the following components: biotin 2.0mg/L, calcium pantothenate 5.0mg/L, folic acid 10mg/L, nicotinamide 10mg/L, vitamin B610 mg/L, thiamine hydrochloride 5mg/L, vitamin B1210 mg/L, riboflavin 1.0mg/L, choline chloride 100mg/L and inositol 50 mg/L.

The inorganic salt component comprises the following components: 100mg/L of magnesium chloride, 100mg/L of magnesium sulfate, 100mg/L of calcium chloride, 500mg/L of potassium chloride, 2400mg/L of sodium bicarbonate, 1500mg/L of sodium chloride and 500mg/L of sodium phosphate.

The trace element components comprise: 2mg/L of zinc sulfate heptahydrate, 0.0001mg/L of copper sulfate pentahydrate, 0.02mg/L of ferric nitrate nonahydrate, 10mg/L of ferrous sulfate heptahydrate, 0.1mg/L of sodium selenite, 0.00005mg/L of nickel chloride, 0.00005mg/L of stannous chloride, 0.0001mg/L of silver nitrate, 0.0005mg/L of cobalt chloride and 0.001mg/L of aluminum chloride.

Example 4

The trace element components comprise: 2mg/L of zinc sulfate heptahydrate, 0.0001mg/L of copper sulfate pentahydrate, 0.02mg/L of ferric nitrate nonahydrate, 10mg/L of ferrous sulfate heptahydrate, 0.1mg/L of sodium selenite, 0mg/L of nickel chloride, 0mg/L of stannous chloride, 0mg/L of silver nitrate, 0mg/L of cobalt chloride and 0mg/L of aluminum chloride.

Example 5

This example provides Vero cell culture and detection assays.

1. Preparation of culture medium for experiment

Adding 1L of culture medium into 800mL of ultrapure water, stirring at room temperature for 30min, adjusting the pH to 7.0-7.4 by using a freshly prepared 1M hydrochloric acid or sodium hydroxide solution, filtering by using a 0.22 mu M sterile membrane, and storing for later use at 4 ℃ for a long time.

The preparation of the culture medium for the experiment comprises the following steps:

1L of the culture medium of example 1 is added to 800mL of ultrapure water, stirred at room temperature for 30min, and then the pH is adjusted to 7.2 with freshly prepared 1M hydrochloric acid or sodium hydroxide solution, filtered through a 0.22 μ M sterile membrane, and stored at 4 ℃ for a long time, and the solution is marked as medium 1.

The medium of example 2 was prepared as test medium 2 in the same manner as described above.

The medium of example 3 was prepared as test medium 3 in the same manner as described above.

The medium of example 4 was prepared as test medium 4 in the same manner as described above.

Commercial medium MEM (purchased from Gibco) supplemented with 10% fetal bovine serum was used as control medium 1.

Commercial medium OptiPro SFM (purchased from Gibco) was used as control medium 2.

Commercial medium VP-SFM (purchased from Gibco) was used as control medium 3.

2. Cell culture

After 2-8 mM/L glutamine is added into the culture medium 1, the culture medium 2, the culture medium 3 and the culture medium 4 respectively, the Vero cell strain is inoculated into experimental culture media such as the culture medium 1, the culture medium 2, the culture medium 3 and the culture medium 4 respectively for culture, and the culture method and parameters are as follows: using experimental culture medium such as culture medium 1, culture medium 2, culture medium 3 or culture medium 4 in 35mm culture dish: (Purchased from Corning) at a cell seeding density of 0.5-2 × 10⁴/cm²And the culture conditions are as follows: 5-10% CO at 37 deg.C₂Culturing for 40-80 hours, digesting the cells, stopping digestion by 1-2 mL of pancreatic enzyme stop solution, collecting the cells, and then continuously subculturing twice by the same method.

The specific culture method is as follows:

1) culturing Vero cell strain in culture medium 1 in 35mm culture dish at cell inoculation density of 1 × 10⁴/cm²And the culture conditions are as follows: 37 ℃ and 5% CO₂After 72 hours of culture, the cells were digested, collected and counted, and subculture was continued twice in the same manner. In this example, 4mM/L glutamine was added.

2) Culturing the Vero cell strain in a 35mm culture dish by using a culture medium 2, wherein the inoculation density of the cells is 1 × 10⁴/cm²And the culture conditions are as follows: 37 ℃ and 5% CO₂After 72 hours of culture, the cells were digested, 1mL of trypsin stop solution was stopped from digestion and the cells were collected and counted, and subculture was continued twice in the same manner as described above. In this example, 4mM/L glutamine was added.

3) Culturing the Vero cell strain in a 35mm culture dish by using a culture medium 3, wherein the inoculation density of the cells is 1 × 10⁴/cm²And the culture conditions are as follows: 37 ℃ and 5% CO₂After 72 hours of culture, the cells were digested, 1mL of trypsin stop solution was stopped from digestion and the cells were collected and counted, and subculture was continued twice in the same manner as described above. In this example, 4mM/L glutamine was added.

4) The Vero cell line was cultured in a 35mm dish using medium 4 at a cell seeding density of 1 × 10⁴/cm²And the culture conditions are as follows: 37 ℃ and 5% CO₂After 72 hours of culture, the cells were digested, 1mL of trypsin stop solution was stopped from digestion and the cells were collected and counted, and subculture was continued twice in the same manner as described above. In this example, 4mM/L glutamine was added.

5) Vero cells were cultured in commercial medium MEM (purchased from Gibco) supplemented with 8-12% (V/V) fetal bovine serum according to the same culture method as medium 1, and passaged three times to prepare control group 1. In this example, 10% fetal bovine serum was added.

6) Vero cells were cultured in commercial medium VP-SFM (purchased from Gibco) with 2 to 8mM/L glutamine according to the same culture method as that of Medium 1, and passaged three times to prepare control group 2. In this example, 4mM/L glutamine was added.

7) Vero cells were cultured in a commercial culture medium OptiPRO (purchased from Gibco) with 2 to 8mM/L glutamine by the same culture method as that of Medium 1, and passaged three times to prepare control group 3. In this example, 4mM/L glutamine was added.

3. Detection of

After the cells are cultured in the step 2, the viable cell density of the cells subjected to the three-time serial passage by using the medium 1, the medium 2, the medium 3 and the medium 4 is compared with that of the control group, as shown in fig. 1 (in the figure, the histogram corresponding to each medium is respectively a graph of one-time passage, two-time passage and three-time passage from left to right), and it can be seen that when the Vero cells are cultured by using the medium of examples 1 to 3, the expansion capacity of the cells can reach or even be better than that of the control groups 1 to 3 (namely, the commercially available basal medium MEM, commercially available serum-free medium VP-SFM and OptiPRO SFM). A schematic diagram of cells of the Vero cells grown in the culture medium 1 for 72 hours is also given, as shown in FIG. 3, the diagram shows that the cells are in good growth state, and have a shape similar to an irregular diamond and are relatively uniform.

After the cells cultured in step 2 were cultured, the comparison results between the total number of viable cells harvested after three serial passages of the cells cultured in medium 1, medium 2, medium 3 and medium 4 and the control group are shown in fig. 2 (in the figure, the histograms corresponding to the respective media are respectively a graph of one passage, two passages and three passages from left to right), and it can be seen that when the Vero cells were cultured in the medium of examples 1 to 3, the total number of viable cells that can be harvested was about 1 to 3 times as much as the control group by three serial passages of the cells, as compared to the control groups 1 to 3 (i.e., commercially available medium MEM, commercially available basal serum-free medium VP-SFM and OptiPRO SFM).

The comparison of fig. 1 and 2 also shows that the cells cultured in medium 4 have the worst culture effect after three serial passages, indicating that the trace element components: the sodium selenite, nickel chloride, stannous chloride, silver nitrate, cobalt chloride and aluminum chloride have great influence on the cell culture effect and are indispensable in the formula.

In conclusion, the Vero cell is cultured by the culture medium provided by the invention, and the normal growth of the Vero cell can be maintained under the condition of not adding serum, the amplification efficiency of the Vero cell can be improved to a certain extent, and the research and development and production of a new coronavirus (SARS-CoV-2) vaccine can be accelerated.

The above embodiments are merely illustrative of the technical ideas and features of the present invention, and the purpose thereof is to enable those skilled in the art to understand the contents of the present invention and implement the present invention, and not to limit the protection scope of the present invention. All equivalent changes and modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.

Claims

1. A Vero cell serum-free culture medium comprises an amino acid component, a vitamin component, an inorganic salt component, a trace element component, a carbohydrate and other molecular compound components,

the amino acid component comprises the following components:

35-120mg/L of L-arginine;

30-95mg/L of L-aspartic acid;

30-100mg/L of L-asparagine;

15-40mg/L of L-cysteine;

20-120mg/L of L-glycine;

10-25mg/L of L-glutamic acid;

14-45mg/L of L-histidine;

25-85mg/L of L-isoleucine;

26-80mg/L of L-leucine;

30-100mg/L of L-lysine;

6-30mg/L of L-methionine;

l-phenylalanine 10-60 m/L;

15-70mg/L of L-proline;

l-serine 20-80 mg/L;

10-50mg/L of L-threonine;

10-40mg/L of L-tryptophan;

10-50mg/L of L-tyrosine;

l-valine 10-70 mg/L;

the trace element component comprises the following components:

0.5-5 mg/L of zinc sulfate heptahydrate;

0.0001-0.01 mg/L of blue vitriol;

0.01-0.45 mg/L ferric nitrate nonahydrate;

ferrous sulfate heptahydrate 0.4-15.5 mg/L;

0.01-0.8mg/L of sodium selenite;

0.00005-0.0005mg/L of nickel chloride;

stannous chloride 0.00005-0.0005 mg/L;

0.00001-0.0002mg/L silver nitrate;

cobalt chloride 0.0005-0.008 mg/L;

0.0001-0.001mg/L of aluminum chloride;

glucose 3000-9000 mg/L;

5-30mg/L of ethanolamine;

0.1-15 mg/L putrescine;

0.2-3 mg/L of lipoic acid;

linoleic acid 0.01-0.9 mg/L;

600mg/L sodium pyruvate 105-;

PVP40 100-3000mg/L；

2' -deoxyadenosine 1-30 mg/L;

1-30mg/L of 2' -deoxyguanosine;

1-30mg/L of 2' -deoxycytidine;

hypoxanthine is 1-30 mg/L;

adenosine 1-30 mg/L;

1-30mg/L uridine;

1-30mg/L of guanosine;

1-30mg/L cytidine;

5-300mg/L of insulin;

the recombinant epidermal growth factor is 0.1-100 mg/L.

2. The Vero cell serum-free culture medium according to claim 1, wherein the vitamin component comprises the following ingredients:

0.09-3.62 mg/L of biotin;

2-12.5 mg/L of calcium pantothenate;

folic acid 9.45-34.5 mg/L;

3.25-23mg/L of nicotinamide;

vitamin B61-10 mg/L;

thiamine hydrochloride in 3.25-13 mg/L;

120.8-15 mg/L of vitamin B;

riboflavin is 0.1-3.5 mg/L;

choline chloride 18-180 mg/L;

inositol 10-75 mg/L.

3. The Vero cell serum-free culture medium according to claim 1, wherein the inorganic salt composition comprises the following components:

10-200 mg/L of magnesium chloride;

magnesium sulfate is 30-200 mg/L;

56-232 mg/L of calcium chloride;

potassium chloride 212-936 mg/L;

sodium bicarbonate 1200 and 3600 mg/L;

800-4000 mg/L sodium chloride;

70-600 mg/L sodium phosphate.

4. The Vero cell serum-free culture medium according to claim 1, characterized in that: in the culture medium, the culture medium is a culture medium,

the amino acid component comprises:

35-120mg/L of L-arginine;

30-95mg/L of L-aspartic acid;

30-100mg/L of L-asparagine;

15-40mg/L of L-cysteine;

20-120mg/L of L-glycine;

10-25mg/L of L-glutamic acid;

14-45mg/L of L-histidine;

25-85mg/L of L-isoleucine;

26-80mg/L of L-leucine;

30-100mg/L of L-lysine;

6-30mg/L of L-methionine;

l-phenylalanine 10-60 m/L;

15-70mg/L of L-proline;

l-serine 20-80 mg/L;

10-50mg/L of L-threonine;

10-40mg/L of L-tryptophan;

10-50mg/L of L-tyrosine;

l-valine 10-70 mg/L;

the vitamin component comprises:

0.09-3.62 mg/L of biotin;

2-12.5 mg/L of calcium pantothenate;

folic acid 9.45-34.5 mg/L;

3.25-23mg/L of nicotinamide;

vitamin B61-10 mg/L;

thiamine hydrochloride in 3.25-13 mg/L;

120.8-15 mg/L of vitamin B;

riboflavin is 0.1-3.5 mg/L;

choline chloride 18-180 mg/L;

inositol 10-75 mg/L;

the inorganic salt component comprises:

10-200 mg/L of magnesium chloride;

magnesium sulfate is 30-200 mg/L;

56-232 mg/L of calcium chloride;

potassium chloride 212-936 mg/L;

sodium bicarbonate 1200 and 3600 mg/L;

800-4000 mg/L sodium chloride;

70-600 mg/L of sodium phosphate;

the trace element component comprises:

0.5-5 mg/L of zinc sulfate heptahydrate;

0.0001-0.01 mg/L of blue vitriol;

0.01-0.45 mg/L ferric nitrate nonahydrate;

ferrous sulfate heptahydrate 0.4-15.5 mg/L;

0.01-0.8mg/L of sodium selenite;

0.00005-0.0005mg/L of nickel chloride;

stannous chloride 0.00005-0.0005 mg/L;

0.00001-0.0002mg/L silver nitrate;

cobalt chloride 0.0005-0.008 mg/L;

0.0001-0.001mg/L of aluminum chloride;

the carbohydrate and other molecular compound components include:

glucose 3000-9000 mg/L;

5-30mg/L of ethanolamine;

0.1-15 mg/L putrescine;

0.2-3 mg/L of lipoic acid;

linoleic acid 0.01-0.9 mg/L;

600mg/L sodium pyruvate 105-;

PVP40 100-3000mg/L；

2' -deoxyadenosine 1-30 mg/L;

1-30mg/L of 2' -deoxyguanosine;

1-30mg/L of 2' -deoxycytidine;

hypoxanthine is 1-30 mg/L;

adenosine 1-30 mg/L;

1-30mg/L uridine;

1-30mg/L of guanosine;

1-30mg/L cytidine;

5-300mg/L of insulin;

the recombinant epidermal growth factor is 0.1-100 mg/L.

5. The Vero cell serum-free culture medium according to claim 4, characterized in that: in the culture medium, the culture medium is a culture medium,

the amino acid component comprises:

35-120mg/L of L-arginine;

30-95mg/L of L-aspartic acid;

30-100mg/L of L-asparagine;

15-30mg/L of L-cysteine;

60-100mg/L of L-glycine;

10-20mg/L of L-glutamic acid;

l-histidine 20-40 mg/L;

l-isoleucine 40-60 mg/L;

26-80mg/L of L-leucine;

30-100mg/L of L-lysine;

6-30mg/L of L-methionine;

l-phenylalanine 10-60 m/L;

15-50mg/L of L-proline;

l-serine 20-60 mg/L;

10-40mg/L of L-threonine;

10-40mg/L of L-tryptophan;

10-40mg/L of L-tyrosine;

l-valine 10-50 mg/L;

the vitamin component comprises:

0.09-3 mg/L of biotin;

2-10 mg/L of calcium pantothenate;

folic acid 9.45-34.5 mg/L;

5-20mg/L of nicotinamide;

vitamin B61-10 mg/L;

thiamine hydrochloride 5-10 mg/L;

120.8-15 mg/L of vitamin B;

0.5-2 mg/L of riboflavin;

30-150mg/L of choline chloride;

inositol 10-75 mg/L;

the inorganic salt component comprises:

10-100 mg/L of magnesium chloride;

magnesium sulfate 100-200 mg/L;

100-232 mg/L calcium chloride;

300-600mg/L potassium chloride;

sodium bicarbonate 2000-3600 mg/L;

800-2000 mg/L sodium chloride;

100mg/L of sodium phosphate;

the trace element component comprises:

0.5-3 mg/L of zinc sulfate heptahydrate;

0.0001-0.005 mg/L of copper sulfate pentahydrate;

0.01-0.2mg/L ferric nitrate nonahydrate;

ferrous sulfate heptahydrate 0.4-15.5 mg/L;

0.01-0.2mg/L of sodium selenite;

0.00005-0.0005mg/L of nickel chloride;

stannous chloride 0.00005-0.0005 mg/L;

0.00001-0.0002mg/L silver nitrate;

cobalt chloride 0.0005-0.008 mg/L;

0.0001-0.001mg/L of aluminum chloride;

the carbohydrate and other molecular compound components include:

glucose 3000-6000 mg/L;

5-30mg/L of ethanolamine;

0.1-10 mg/L putrescine;

0.2-2 mg/L of lipoic acid;

linoleic acid 0.1-0.5 mg/L;

sodium pyruvate 105-400 mg/L;

PVP40 500-1500mg/L；

2' -deoxyadenosine 1-25 mg/L;

1-25mg/L of 2' -deoxyguanosine;

1-25mg/L of 2' -deoxycytidine;

hypoxanthine is 1-25 mg/L;

adenosine 5-25 mg/L;

5-25mg/L uridine;

5-25mg/L of guanosine;

cytidine 5-25 mg/L;

50-250mg/L of insulin;

the recombinant epidermal growth factor is 0.1-100 mg/L.

6. The Vero cell serum-free culture medium according to claim 5, characterized in that: in the culture medium, the culture medium is a culture medium,

the amino acid component comprises:

45-120mg/L of L-arginine;

30-95mg/L of L-aspartic acid;

30-95mg/L of L-asparagine;

15-20mg/L of L-cysteine;

80-100mg/L of L-glycine;

10-20mg/L of L-glutamic acid;

30-40mg/L of L-histidine;

l-isoleucine 40-60 mg/L;

30-70mg/L of L-leucine;

30-100mg/L of L-lysine;

8-30mg/L of L-methionine;

l-phenylalanine 20-30 m/L;

15-35mg/L of L-proline;

15-50mg/L of L-serine;

10-30mg/L of L-threonine;

10-40mg/L of L-tryptophan;

10-40mg/L of L-tyrosine;

l-valine 10-50 mg/L;

the vitamin component comprises:

0.09-2 mg/L of biotin;

3-8 mg/L of calcium pantothenate;

10-21 mg/L folic acid;

10-20mg/L of nicotinamide;

vitamin B63-10 mg/L;

thiamine hydrochloride 5-10 mg/L;

vitamin B121-10 mg/L;

0.5-1 mg/L of riboflavin;

30-100mg/L of choline chloride;

inositol 10-50 mg/L;

the inorganic salt component comprises:

20-100 mg/L of magnesium chloride;

magnesium sulfate 100-150 mg/L;

100mg/L of calcium chloride;

300mg/L of potassium chloride;

sodium bicarbonate 2000-3000 mg/L;

sodium chloride 1500-;

sodium phosphate 200-500 mg/L;

the trace element component comprises:

0.5-2 mg/L of zinc sulfate heptahydrate;

0.0001-0.005 mg/L of copper sulfate pentahydrate;

0.01-0.1mg/L ferric nitrate nonahydrate;

5-15mg/L of ferrous sulfate heptahydrate;

0.01-0.1mg/L of sodium selenite;

0.00005-0.0005mg/L of nickel chloride;

stannous chloride 0.00005-0.0005 mg/L;

0.00001-0.0002mg/L silver nitrate;

cobalt chloride 0.0005-0.008 mg/L;

0.0001-0.001mg/L of aluminum chloride;

the carbohydrate and other molecular compound components include:

glucose 4000-5000 mg/L;

10-20mg/L of ethanolamine;

1-8 mg/L putrescine;

0.5-2 mg/L of lipoic acid;

linoleic acid 0.1-0.4 mg/L;

200-350 mg/L of sodium pyruvate;

PVP40 800-1100mg/L；

2' -deoxyadenosine 5-20 mg/L;

5-20mg/L of 2' -deoxyguanosine;

5-20mg/L of 2' -deoxycytidine;

hypoxanthine is 5-20 mg/L;

adenosine 10-20 mg/L;

10-20mg/L uridine;

guanosine is 10-20 mg/L;

10-20mg/L of cytidine;

100-250mg/L of insulin;

the recombinant epidermal growth factor is 0.1-100 mg/L.

7. The Vero cell serum-free culture medium according to claim 6, characterized in that: in the culture medium, the amino acid components comprise: l-arginine 120mg/L, L-aspartic acid 95mg/L, L-asparagine 30mg/L, L-cysteine 15mg/L, L-glycine 80mg/L, L-glutamic acid 15mg/L, L-histidine 27mg/L, L-isoleucine 50mg/L, L-leucine 30mg/L, l-lysine 30mg/L, L-methionine 30mg/L, L-phenylalanine 30m/L, L-proline 15mg/L, L-serine 15mg/L, L-threonine 20mg/L, L-tryptophan 10mg/L, L-tyrosine 10mg/L and L-valine 10 mg/L;

8. Use of a Vero cell serum-free medium according to any one of claims 1 to 7 in Vero cell culture.