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Method for carrying out large-scale subculture on primary cells

A large-scale culture and primary cell technology, applied in the field of large-scale subculture of primary cells, can solve problems such as inability to obtain batches, difficulty in quality control, and restrictions on large-scale production applications, so as to improve product quality, production scale and production Efficiency, improvement of cell culture rate, and effect of quality improvement

Inactive Publication Date: 2013-03-13
李伟 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, this technology is mainly used for the cultivation of subcultured cell lines, but it is used for primary cell culture, because it is impossible to obtain high-viability cells that maintain their diploid biological properties in batches and are suitable for use in bioreactors or cell factories. , which limits its mass production application
[0004] Primary cells can be used to produce rabies vaccine, Japanese encephalitis vaccine, influenza vaccine, measles virus vaccine, mumps virus vaccine and epidemic hemorrhagic fever virus vaccine, etc. The production of these vaccines requires the use of a large number of animals, and traditional cell culture technology cannot fully Excluding the possibility of contamination by exogenous factors, the quality is difficult to control, which restricts the large-scale production of vaccine products
[0005] Based on the above reasons, it also limits the application of primary cells or their secondary cells in the production of biological products without the potential tumorigenic risk advantages of cell lines

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Example 1: Screening of Hamster Kidney Cells and Culture Using Bioreactor and Using Polyester Chips as Carriers

[0016] In an embodiment of the present invention, the cells are primary hamster kidney cells that have been subcultured for one generation through roller bottles; a bioreactor (Celligen Plus TM Bioreactor, purchased from New Brunswick Scientific Co., Inc.) and polyester chip carrier (Fibra-Cel TM Disks); the medium used was M199 medium or DMEM medium. Other reagents used include bovine serum, trypsin, and the like.

[0017] 1. Dissect the kidney cells from the hamster and digest them with trypsin to obtain primary cells; strictly control the pH value to 7.2; place the three-L spinner bottle covered with a vent stopper at 37°C and 5% CO2 for cultivation , the inner surface of the three-L spinner bottle was coated with 0.1-1 mg / ml poly-lysine, and then used Ca-free 2+ ,Mg 2+ BSS immersion balance; the time to change the medium is 4 days. When changing the ...

Embodiment 2

[0020] Example 2: Screening of chicken embryo cells and culturing by means of a cell factory

[0021] In an embodiment of the present invention, the cells are primary chick embryo cells after being subcultured in a bottle; 2 , working capacity 2000ml) to culture chicken embryo cells; the medium used is M199 medium or DMEM medium. Other reagents used include bovine serum, trypsin, and the like.

[0022] 1. Dissect the 9-day-old chicken embryos with their heads and feet removed, and digest them with trypsin to obtain primary cells; strictly control the pH value to 7.2; place the three-L spinner bottle covered with a vent stopper at 37°C for 5 Cultured in %CO2 environment, the inner surface of the three-L spinner bottle was coated with 0.1-1 mg / ml poly-lysine, and then used Ca-free 2+ ,Mg 2+ BSS immersion equilibrium; change the medium for 5 days. When changing the medium, keep a part of the old culture medium to discard 1 / 2. The volume ratio of the space above the liquid surfa...

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PUM

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Abstract

The invention provides a method for carrying out large-scale subculture on primary cells. The method comprises the following steps of: a, obtaining subculture cells which maintain biological traits and high activity of cell diploids and are suitable for a bioreactor or cell factory in batches; b, screening the cells suitable for the bioreactor or cell factory; and c, culturing the screened cells by using the bioreactor or cell factory on large scale. The method solves the problems that external pollution risk is high, the quality is difficult to control and massive production is difficultly carried out because a large quantity of animals are needed to be used when the primary cells are used in the production, and develops the advantage of no potential tumorigenicity risk of a continuous cell line of the primary cells or subculture cells during production of biological products.

Description

technical field [0001] The invention relates to a method for large-scale subculture of primary cells in the field of biological products, specifically a method for cultivating primary cells through certain technical means and screening out high-viability cells that maintain their diploid biological properties. Secondary cells (referring to the in vitro culture of no more than 5 generations of cells after isolation from tissue), and the method of using bioreactors or cell factories to achieve large-scale culture. Background technique [0002] At present, there have been many reports on the cultivation of animal cells using bioreactors, microcarriers and cell factories at home and abroad. The cell microcarrier culture method was conceived and invented by Van Wezel in 1967. After 40 years of human exploration, this technology has become increasingly mature and perfect, and is widely used in the field of bioengineering and some cell products with important value. Microcarrier c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12N5/073
Inventor 李伟苏文全
Owner 李伟