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Insect Cell Serum-Free Medium and Its Application

A serum-free medium and insect cell technology, applied in the field of insect cell serum-free medium, can solve the problems of unfavorable insect cell culture recombinant protein preparation, protein expression efficiency to be improved, low insect cell culture efficiency, etc. The effect of low cost, improved culture efficiency and recombinant protein expression efficiency

Active Publication Date: 2018-04-03
苏州沃美生物有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] So far, such as L.Ikonomou., etal (Appl Microbiol Biotechnol, 2003, 62:1-20), Michael S., etal (Biotechnol.Prog., 1988, 14, 573-579), R. H. Qierui Luo (CN1498267A) etc. have developed a variety of serum-free media for cultivating insect cells, but these media generally have problems such as high cost, low insect cell culture efficiency, and protein expression efficiency to be improved, which is not conducive to large-scale insects. Cell Culture and Production of Recombinant Proteins

Method used

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  • Insect Cell Serum-Free Medium and Its Application
  • Insect Cell Serum-Free Medium and Its Application
  • Insect Cell Serum-Free Medium and Its Application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 The formulation of the insect cell serum-free medium is shown in Table 2.

[0048] The formula of a kind of insect cell serum-free medium in table 2 embodiment 1

[0049]

[0050]

[0051]

[0052] And the preparation process of this culture medium of the present embodiment can comprise:

[0053] First, take or measure the raw material according to Table 3;

[0054] Second, configure the concentrate, including:

[0055] To prepare 1000× concentrate 1:

[0056] Dissolve 8 mg of ammonium molybdate tetrahydrate, 10 mg of cobalt chloride hexahydrate, 75 mg of copper chloride dihydrate, 50 mg of ferrous sulfate heptahydrate, 8 mg of manganese chloride tetrahydrate, and 10 mg of zinc chloride in 100 ml of water.

[0057] Prepare 1000× Concentrate 2:

[0058] Dissolve biotin 10mg, D-calcium pantothenate 20mg, VB12 cyanocobalamin 20mg, VB9 folic acid 20mg, i-inositol 50mg in 100ml of water.

[0059] To prepare 1000× concentrate 3:

[0060] Dissolve 5mg ...

Embodiment 2

[0073] Embodiment 2: cell culture: inoculate 1000 milliliters of cell suspensions in 3 liters of shaking flasks (select the substratum of embodiment 1 for use, wherein contain cell 0.5 * 10 6 cells / ml), placed on a shaker in a biochemical incubator at 27°C with a rotation speed of 100 rpm, when the cell density reached 5×10 6 Cells / ml, it was diluted and passaged, and the actual density of the cells was 0.5×10 6 cells / ml. Bring 3 liters to a density of 5 x 10 6 The cell suspension of cells / ml was transferred to a 42L bioreactor, and then 27 liters of fresh medium of Example 1 was added, the stirring speed was 50 rpm, the temperature was 27°C, the dissolved oxygen was controlled at 40%, and the pH value was controlled at 6.2. When the cell density reaches 5×10 6 When cells / ml, it is transferred in the bioreactor of 1000 liters, and supplements fresh embodiment 1 culture medium to 300 liters, control stirring speed 20 revs / min, temperature 27 ℃, dissolved oxygen 40%, pH valu...

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Abstract

The invention discloses an insect cell serum-free culture medium and application thereof. The culture medium comprises amino acid, inorganic salt, vitamin and carbohydrate and further comprises 0.5-15mg / L of diethylenetriamine dioleoyl phosphatidylcholine and / or 0.1-10mg / L of distearoyl phosphatidyl choline; and particularly, further comprises 0.001-0.1mg / L of barium chloride dihydrate, 0.005-0.02mg / L of lithium chloride and 0.05-7mg / L of nickel chloride. The insect cell serum-free culture medium is simple and clear in components and low in cost and is easily prepared; by the insect cell serum-free culture medium, the insect cell culture efficiency and recombinant protein expression efficiency can be significantly increased and the large-scale culture and large-scale preparation of insect cells are well achieved.

Description

technical field [0001] The invention relates to a serum-free medium, in particular to an insect cell serum-free medium suitable for large-scale cultivation of bioreactors to prepare recombinant proteins. Background technique [0002] In order to meet the increasingly wide range of application needs in basic research and applied biomedical fields such as cell biology, physiology, pharmacology and toxicology, cell culture technology has emerged as the times require. Generally speaking, cell culture technology simulates the in vivo environment of cell growth, including temperature, pH value, osmotic pressure and oxygen supply, so that cells can grow and reproduce well. One of the most critical factors is the culture medium. [0003] The traditional cell culture medium is mainly formed by adding a corresponding amount of serum or tissue extract to the basal medium. Due to the limitation of animal serum extraction, it is extremely expensive to apply to the medium, and it also st...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/07C12P21/00
Inventor 张大鹤
Owner 苏州沃美生物有限公司
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