Method for preparing serum-free cultured suspension mammal cell line, prepared cell line thereof and application thereof

A serum-free medium and mammalian technology, applied in animal cells, vertebrate cells, artificial cell constructs, etc., can solve the problems of increased risk of external contamination, inappropriate production of animal vaccines, unfavorable proliferation of influenza viruses, etc. , to achieve the effect of reducing the risk of external source pollution, saving manpower, ensuring stability and uniformity

Active Publication Date: 2015-06-24
PU LIKE BIO ENG
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  • Abstract
  • Description
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AI Technical Summary

Problems solved by technology

[0006] Microcarriers are expensive and expensive to produce, and are not suitable for animal vaccine production
Moreover, a certain amount of serum needs to be added in the process of culturing cells with microcarriers, which is not conducive to the proliferation of influenza virus; the side effec

Method used

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  • Method for preparing serum-free cultured suspension mammal cell line, prepared cell line thereof and application thereof
  • Method for preparing serum-free cultured suspension mammal cell line, prepared cell line thereof and application thereof
  • Method for preparing serum-free cultured suspension mammal cell line, prepared cell line thereof and application thereof

Examples

Experimental program
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Example Embodiment

[0030] Example 1. Adaptation to the domestication of single-cell serum-free pure suspension culture of MDCK cells

[0031] (1) Resuscitate one MDCK cell for adherent culture. The method of cell resuscitation is to quickly dissolve at 37°C and then inoculate it into MEM medium containing 5% PAA serum. The number of cells in each cryotube is at least 3 million. Place it in a 37°C incubator for 24 hours and replace with fresh 5% PAA's MEM medium. The cells grew into a compact monolayer at 72h, digested with 0.125% trypsin, and passaged according to the dispersion ratio of 1:3.

[0032] (2) After resuscitation, pass 3 times in succession, using VP-SFMAGT supplemented with 5% (v / v) PAA TM (Gibco) Serum-free medium (or MDCK cell serum- and protein-free chemical medium (Jiangyin Cambridge Biotechnology Co., Ltd.), or InVitrus TM (Switzerland Cell Culture Technologies), or SFM4MegaVir TM (HyClone)) Replace the MEM containing 5% (v / v) PAA, observe the cells grown into a dense monolayer for...

Example Embodiment

[0038] Example 2 The specific morphology of the MDCK cell bioreactor adapted to serum-free suspension culture under large-scale culture conditions

[0039] 1. Cell culture in bioreactor: first directly resuscitate the frozen MDCK cells adapted to serum-free suspension culture in a shake flask, change the medium for 24 hours, and then the cell density will reach more than 4 million / ml around 72 hours, subculture, continuous passage 2- After 3 times, the cell growth recovered; continue to expand the culture, inoculate the Bioflo115 fermentor, the inoculation density is 500,000 / ml, and the culture conditions are 37℃, 80~110rpm, DO 30%~50%, pH7.2. The medium used in this process is VP-SFMAGT TM (Gibco), add 0.2% Pluronic F-68 and 30μg / ml dextran sulfate.

[0040] 2. In the process of bioreactor cultivation, sample the number of cells at 18h, 24h, 42h, 48h, 66h, 72h, 90h, 96h, 114h after inoculation, and stop sampling when the cell viability drops below 50% , The cell density is shown ...

Example Embodiment

[0047] Example 3. Propagation of avian influenza H9 subtype in serum-free pure suspension culture

[0048] 1. Cells are cultured in a bioreactor, and the culture method is the same as in Example 1;

[0049] 2. When the cell density in the fermentor reaches the requirement of inoculating avian influenza virus, inoculate the virus and add the virus solution according to the inoculation dose of MOI=0.001-0.1. In addition, because TPCK-pancreatin has a great influence on the proliferation of influenza virus, So add 1.0~5.0μg / ml after virus inoculation. The culture conditions are 33℃, 80~110rpm, DO 30~50%, pH 7.20;

[0050] 3. Since the cytopathic changes of the suspended cells cannot be observed, samples are taken every 12h after inoculation to monitor the cell number and HA to determine the virus content, find an extreme value of the virus hemagglutination value, and harvest the virus liquid when the virus hemagglutination price is the highest. The cell debris is removed by filtration...

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Abstract

The invention provides a method for preparing a serum-free cultured suspension mammal cell line, which comprises the following steps: 1)recovering and culturing mammal cells to obtain adherent cultured mammal cells, digesting by pancreatin; 2)culturing the adherent cultured mammal cells by a serum-free medium added with a certain amount of serum, continuously culturing the mammal cells so as to adapt to the serum-free medium with the certain amount of serum; and 3)repeating the continuous cultivation process in the step 2), and gradually reducing the serum addition amount in the serum-free medium until the serum addition in the serum-free medium is 0 to obtain the suspension mammal cell line. According to the method, equalization nutrition of cells is obtained, vaccine stability and uniformity can be guaranteed, and the problems of side effect due to serum addition can be solved, risk due to exogenous pollution is reduced, stress reaction of animal is reduced, and subsequent processing is easy and simple.

Description

technical field [0001] The invention relates to a domestication method of a mammalian cell line adapted to single-cell pure suspension serum-free culture and a cell line prepared by the method. Background technique [0002] The culture of mammalian cells has undergone a roller bottle-microcarrier culture mode. The improvement of the culture mode not only makes it easier to achieve large-scale production, but also reduces the difficulty of the later purification process. [0003] The spinner bottle culture technology is a traditional adherent cell culture method. The cells are seeded in a rotating cylindrical spinner bottle, and the cells are attached to the glass wall. During the culture process, the spinner bottle is continuously rotated on the bearing, so that the cells can be alternately contacted and cultured. Liquid and air, to achieve the purpose of cell growth and metabolism. [0004] The roller bottle culture process cannot control and adjust the conditions of the ...

Claims

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Application Information

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IPC IPC(8): C12N5/071A61K39/145A61P31/16
Inventor 张许科孙进忠白朝勇
Owner PU LIKE BIO ENG
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