Method for safe continuous enclosed cell culture, virus production/ inactivation

A cell culture, closed technology, applied in the field of bioengineering, can solve problems such as unfavorable large-scale, continuous, safe production of virus vaccines, non-closed, etc., to achieve the effect of facilitating automatic control, improving operation flexibility, and reducing pollution opportunities.

Active Publication Date: 2006-07-26
上海丽坤生物科技股份有限公司
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  • Abstract
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Problems solved by technology

[0004] However, in the above two methods, the two processes of cell culture and virus multiplication can be seen as continuous production, but the whole process is intermittent

Method used

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  • Method for safe continuous enclosed cell culture, virus production/ inactivation

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Example Embodiment

[0021] Examples:

[0022] The above description roughly explains the content of the present invention. The following examples will more directly embody the situation of the present invention. However, it should be pointed out that the examples listed here are for illustrative purposes only, rather than limiting.

Example Embodiment

[0023] Example 1 Vero cell culture production / inactivation of rabies virus

[0024] Medium: Medium 199 (Gibco), plus 5% fetal bovine serum, adjusted to pH 7.0 ~ pH 7.2 with sodium bicarbonate.

[0025] Microcarrier and its treatment: Cytodex-1 (Pharmacia) is used as the microcarrier, and the matrix is ​​cross-linked dextran. When in use, add the dry microcarrier to a silicified container with dimethyldichlorosilane, and use PBS solution at room temperature Soak thoroughly, discard the supernatant, wash twice with PBS solution, replace with new PBS, sterilize at 121°C for 30 minutes, aspirate the supernatant, wash once with Medium 199 containing 5% fetal calf serum, replace with a new culture Base, equilibrate overnight in an incubator.

[0026] 1) Primary culture of cells

[0027] Add the treated microcarriers at 3g / L into a self-made 500mL stirred bioreactor (Spinner Flasks) that has been siliconized, and connect to Vero cells. The seeding density is 1×10. 5 cells / mL, make up the...

Example Embodiment

[0036] Example 2 MDCK cell culture production / inactivation of H3N2 influenza virus

[0037] This example was carried out with reference to the steps basically the same as in Example 1, and the H3N2 influenza virus was cultured using MDCK cells.

[0038] Medium: Axcevir-MDCK serum-free medium (Excele Biotechnologies, France) adjusted to pH7.0~pH7.2 with sodium bicarbonate

[0039] Microcarrier: Cytodex-3, dosage 5g / L.

[0040] Cell culture process: seeding density 1.5×10 5 cells / mL, microcarrier Cytodex-3, dosage 5g / L, culture bioreactor is a self-made 500mL stirred bioreactor (SpinnerFlasks) siliconized, cultured at 37°C, dissolved oxygen 40%, stirring speed 50rpm, primary The rate is 600mL / day, after 2 days of inoculation, the cell density is 4.67×10 6 Cells / mL, start continuous culture, the sampling rate is 300mL / day, sampling and counting regularly within 5 days, the cell density is maintained at 4.21×10 6 About cells / mL, it was found that the continuous cell culture process wa...

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Abstract

A method of cells culture, virus production/deactivation in safe, continuum and blocked style, belonging to bioengineering technical field, compring steps of: 1) Inoculate cells and suspension culture, therebycarry out the primary culture of cells; 2)Flowing add fresh medium, simultly pump out cells suspension to maintain cell culture biological reactor stable and guarantee cell amplificatation during residence time in reactor, accordingly carry out cell's continuous cultivation; 3) Pump out cells suspension into virus-containing virus breeding reactor to make virus infect and amplificate in cells preliminary; 4)As the inflow of cells suspension and outflow of virus-containing upper clean liquid, system dynamic balancing is maintained and virus continuous cultivation is achieved; 5) Collect the upper clean liquid pumped from virus breeding reactor and deactivate the virus, accordingly achieve continuously and obturately cells cultivation and virus deactivation. This method is convenient for automatic control and standardized production, also depresses the pollution chances and heightens production assurance coefficient.

Description

technical field [0001] The invention relates to a method in the technical field of bioengineering, in particular to a method for safe continuous closed cell culture and virus production / inactivation. technical background [0002] There are two main ways to use cells to produce viruses: (1) Cells and virus seeds are cultivated together. During the process of cell proliferation, viruses are replicated to achieve the purpose of mass production of viruses. This is a way to simulate the natural living state of organisms. Method; (2) Separate the amplification process of cells and viruses, that is, a large number of cells are first amplified, and then virus seeds are added after reaching a certain level to maintain the activity of cells, and through the continuous infection and replication of progeny viruses between cells, to To achieve the purpose of mass production of viruses. With the continuous deepening of virus research, it is found that there are certain differences betwee...

Claims

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Application Information

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IPC IPC(8): C12N5/06C12N7/04A61K39/12C12N5/02C12N5/071
Inventor 罗凤山齐瀚实陈彦田孙海英耿涛
Owner 上海丽坤生物科技股份有限公司
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