CN117736967A - Insect culture medium with limited chemical components - Google Patents
Insect culture medium with limited chemical components Download PDFInfo
- Publication number
- CN117736967A CN117736967A CN202311605020.7A CN202311605020A CN117736967A CN 117736967 A CN117736967 A CN 117736967A CN 202311605020 A CN202311605020 A CN 202311605020A CN 117736967 A CN117736967 A CN 117736967A
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- CN
- China
- Prior art keywords
- medium
- culture medium
- insect
- chemical composition
- chemically defined
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Links
- 241000238631 Hexapoda Species 0.000 title claims abstract description 22
- 239000001963 growth medium Substances 0.000 title claims abstract description 11
- 239000000126 substance Substances 0.000 title claims abstract description 10
- 239000002609 medium Substances 0.000 claims abstract description 12
- 239000000203 mixture Substances 0.000 claims abstract description 10
- 150000001413 amino acids Chemical class 0.000 claims abstract description 9
- 229910017053 inorganic salt Inorganic materials 0.000 claims abstract description 9
- 150000001720 carbohydrates Chemical class 0.000 claims abstract description 8
- 150000002632 lipids Chemical class 0.000 claims abstract description 7
- 150000007524 organic acids Chemical class 0.000 claims abstract description 7
- 229940088594 vitamin Drugs 0.000 claims abstract description 7
- 229930003231 vitamin Natural products 0.000 claims abstract description 7
- 235000013343 vitamin Nutrition 0.000 claims abstract description 7
- 239000011782 vitamin Substances 0.000 claims abstract description 7
- 150000003722 vitamin derivatives Chemical class 0.000 claims abstract description 7
- 229930091371 Fructose Natural products 0.000 claims 2
- 239000005715 Fructose Substances 0.000 claims 2
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 claims 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims 2
- 229930006000 Sucrose Natural products 0.000 claims 2
- 239000008103 glucose Substances 0.000 claims 2
- 239000005720 sucrose Substances 0.000 claims 2
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims 1
- 239000000413 hydrolysate Substances 0.000 claims 1
- 108090000623 proteins and genes Proteins 0.000 abstract description 2
- 102000004169 proteins and genes Human genes 0.000 abstract description 2
- 238000002360 preparation method Methods 0.000 abstract 1
- 238000012258 culturing Methods 0.000 description 6
- 239000013028 medium composition Substances 0.000 description 5
- 239000012679 serum free medium Substances 0.000 description 5
- 239000012194 insect media Substances 0.000 description 4
- 229940041514 candida albicans extract Drugs 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 229960005486 vaccine Drugs 0.000 description 3
- 239000012138 yeast extract Substances 0.000 description 3
- 241000256251 Spodoptera frugiperda Species 0.000 description 2
- 239000012888 bovine serum Substances 0.000 description 2
- 239000006143 cell culture medium Substances 0.000 description 2
- 241000272470 Circus Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000202347 Porcine circovirus Species 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 238000001415 gene therapy Methods 0.000 description 1
- 239000006451 grace's insect medium Substances 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 239000012092 media component Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000004017 serum-free culture medium Substances 0.000 description 1
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to an insect chemical composition-defining medium comprising 10-20 g/L carbohydrate, 15-20.8 g/L amino acid, 6.5-10.0 g/L inorganic salt, 0.01-0.2 g/L vitamin, 0.2-2.0 g/L organic acid and 0.005-0.05g/L chemically defined lipid mixture, the medium of the invention being free of chemical composition undefined substances and therefore having a higher batch-to-batch consistency. The culture medium has the advantages of simple and definite components, easy acquisition, simple preparation process, and capability of supporting stable passage of insect cells, high-density culture and high-efficiency expression of products. Experiments show that the culture medium and the imported chemical composition limit insect culture medium can achieve similar cell density and protein expression yield.
Description
Technical Field
The present invention relates to the field of chemically defined media. In particular, the invention relates to insect cell chemistry definition media.
Background
Up to now, there are more than 800 insect cell lines established worldwide, the most widely used are spodoptera frugiperda cell line Sf-9 and spodoptera frugiperda H5 (highfive) cell lines. Insect cells are widely used in the fields of scientific research, animal vaccines, gene therapy, and the like.
The demand of the insect culture medium in China exceeds 100 ten thousand L each year, and is mainly used for producing the porcine circus vaccine. The insect medium (such as Grace insect medium) of the disclosed formulation is normally cultured with 10% bovine serum and 4g/L yeast extract. But the addition of serum increases the risk of infection and the production costs. The addition of yeast extract reduces the batch-to-batch stability of the culture medium and also increases the production cost. The addition of bovine serum and hydrolysates also has a certain impact on downstream purification.
Several companies have now developed serum-free media for culturing insect cells, such as the serum-free insect media described in the JP20060185008 patent. However, these media are relatively expensive to produce due to the recombinant proteins contained therein. Again, the insect medium described in the CN1498267A patent contains a high concentration of yeast extract, increasing costs and leading to large product lot-to-lot variations due to the presence of hydrolysates.
The need for chemically defined insect media is therefore increasing. However, the technology is mainly mastered by the Siemens and Merck company in the United states, and domestic enterprises do not have insect culture mediums with limited chemical components at present. And the price of the insect culture medium limited by the chemical components of the external enterprises is very expensive and reaches hundreds of yuan/L because the China does not have corresponding product competition, and vaccine enterprises cannot bear the cost at all.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides an insect culture medium with limited chemical components, which provides support for research and mass production of insect cell culture. This object is achieved by the solution described below.
In example 1, the present invention provides a serum-free medium for culturing insect cells comprising 16g/L carbohydrate, 15g/L amino acid, 8g/L inorganic salt, 0.13g/L vitamin, 1.16g/L organic acid, and 0.025g/L chemically defined lipid mixture.
In example 2, the present invention provides a serum-free medium for culturing insect cells comprising 10g/L carbohydrate, 20.8g/L amino acid, 8g/L inorganic salt, 0.13g/L vitamin, 1.16g/L organic acid, and 0.025g/L chemically defined lipid mixture.
In example 3, the present invention provides a serum-free medium for culturing insect cells comprising 20g/L carbohydrate, 15g/L amino acid, 10g/L inorganic salt, 0.13g/L vitamin, 1.16g/L organic acid, and 0.025g/L chemically defined lipid mixture.
In example 4, the present invention provides a serum-free medium for culturing insect cells comprising 20g/L carbohydrate, 20.8g/L amino acid, 6.5g/L inorganic salt, 0.13g/L vitamin, 1.16g/L organic acid, and 0.025g/L chemically defined lipid mixture.
In example 5, the present invention provides a serum-free medium for culturing insect cells comprising 16g/L carbohydrate, 15g/L amino acid, 8g/L inorganic salt, 0.2g/L vitamin, 2g/L organic acid, and 0.025g/L chemically defined lipid mixture.
Drawings
Fig. 1: SF9 cells in Siemens TM Growth curves in CD medium and in the medium of the invention.
Fig. 2: SF9 cells in Siemens TM CD medium and GFP protein expression patterns in the medium of the invention.
Fig. 3: h5 cells in merckCD insect cell culture medium and growth curves in the medium of the invention.
Fig. 4: h5 cells in merckCD insect cell culture medium and expression profile of porcine circovirus in the medium of the invention.
Detailed Description
For a better understanding of the example media, the following list of the example media components is detailed
TABLE 3 example 1 Medium composition Table
TABLE 4 example 2 Medium composition Table
TABLE 5 example 3 Medium composition Table
TABLE 6 example 4 Medium composition Table
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TABLE 7 example 5 Medium composition Table
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Claims (5)
1. The chemical composition-limited insect culture medium comprises 10-20 g/L of carbohydrate, 5-25 g/L of amino acid, 6-10 g/L of inorganic salt, 0.01-0.1 g/L of vitamin, 0.1-1g/L of organic acid and 0.005-0.05g/L of chemically-limited lipid mixture. Wherein the culture medium does not contain hydrolysate.
2. The chemically defined medium of claim 1, wherein: the carbohydrate of 10-20 g/L is composed of glucose, sucrose and fructose.
3. The chemically defined medium of claim 1, wherein: the glucose content is 5-10 g/L, the sucrose content is 3-8 g/L, and the fructose content is 2-5 g/L.
4. The chemically defined medium of claim 1, wherein: the amino acid content is shown in table 1.
TABLE 1 amino acid content table
5. The chemically defined medium of claim 1, wherein: the inorganic salt content is shown in table 2.
Table 2 inorganic salt content table
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311605020.7A CN117736967A (en) | 2023-11-28 | 2023-11-28 | Insect culture medium with limited chemical components |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311605020.7A CN117736967A (en) | 2023-11-28 | 2023-11-28 | Insect culture medium with limited chemical components |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117736967A true CN117736967A (en) | 2024-03-22 |
Family
ID=90258309
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311605020.7A Withdrawn CN117736967A (en) | 2023-11-28 | 2023-11-28 | Insect culture medium with limited chemical components |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117736967A (en) |
-
2023
- 2023-11-28 CN CN202311605020.7A patent/CN117736967A/en not_active Withdrawn
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| WW01 | Invention patent application withdrawn after publication | ||
| WW01 | Invention patent application withdrawn after publication |
Application publication date: 20240322 |