Serum-free chemical component definition culture medium for EB66 cell strain suspension growth and preparation method thereof

Abstract

The invention belongs to the field of culture mediums, and particularly relates to a serum-free chemical component definition culture medium for EB66 cell strain suspension growth. The culture mediumcomprises an additive A including, according to the concentrations of the substances in the culture medium, 1-10 mg / L of insulin, 5-10 microgram / L of IGF-1, 0.5-2 mg of putrescine, 0.5-5 mg / L of glutathione, 1-5 mg / L of neovaricaine, 10-50 micromole of beta-mercaptoethanol, 1-5 mg / L of lecithin, 10-50 mg / L of vitamin C, 50-100 mg / L of taurine, 2-5 mg / L of cholesterol and 3-6 millimole of glutamine, wherein 1000-3000 mg of an amino acid mixed component is added to per liter of the culture medium. The culture medium supports rapid proliferation of EB66 cells under the serum-free and full-suspension conditions, and the doubling time is short. The cells are high in density. Besides, the invention further provides a preparation method of the culture medium.

Description

technical field [0001] The invention relates to the field of culture medium, in particular to a serum-free chemical composition-defined culture medium for suspension growth of EB66 cell lines. Background technique [0002] The EB66 cell line is a subcultured cell extracted and domesticated from duck embryonic stem cells by Valneva, a famous French biotechnology company. Headquartered in Lyon, France, Valneva is committed to vaccine technology research and development, production and commercialization innovation, and has a high international influence in the field of vaccines. Valneva selects ducks that do not detect the expression of endogenous retroviruses, collects duck embryos, extracts duck embryonic stem cells, and obtains genetically stable EB66 cell lines after domestication and passage, which are derived from embryonic stem cells and have no tumorigenicity. Any genetic modification, viral and chemical modification. [0003] The EB66 cell line is derived from duck e...

AI Technical Summary

Problems solved by technology

Restricted by these factors, the chicken embryos without specific pathogens (Spedfic Pathogen Free, SPF) have small output and high cost, which often cannot meet the needs of mass production of avian influenza vaccines

In addition, there are still many problems in the cultivation of chicken embryos. The quality of different batches of chicken embryos is often different, and it is difficult to control the quality. Chicken embryos of different quality will have different side effects on virus culture, and the source of chicken embryos is easily affected by poultry. The impact of the influenza epidemic; in addition, chicken embryos are the natural host of many bacteria, and are susceptible to bacterial infection in the environment. Some researchers have pointed out that the potential pollution brought by maternal inheritance to chicken embryos is more than artificial operation in a sterile environment in a GMP workshop. The resulting pollution rate is much higher, and this is where it is difficult to control in the chicken embryo culture process

In addition, the allantoic fluid of chicken embryos contains a large amount of animal-derived protein, which is easily brought into the virus fluid when the virus is extracted, resulting in a large amount of animal-derived protein in the finished vaccine, causing allergic reactions and other side effects

[0005] Serum can provide cells with hormones, growth factors, transfer proteins, and other nutrients needed for growth and proliferation, but it has many disadvantages: large batch-to-batch differences, unstable sources, a lot of verification work, expensive, and unclear components. It is not conducive to the separation and purification of target products such as vaccines and monoclonal antibodies, and is easy to be infected by viruses and mycoplasma, etc.

However, the above medium has not been tested to make the EB66 cell line reach 18*10 6 The concentration of cells / ml, even if as stated in the case, its amino acid concentration reaches the state of 4-10, which is used to cultivate EB66 cell lines, and the peak concentration of the cell lines is lower than 10*10 6 cells / ml

[0007] At present, there is no serum-free medium suitable for the growth of EB66 cell lines in China, and the serum-free medium for EB66 cells is still blank in China

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