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A method for preparing a serum-free cultured suspension mammalian cell line, the prepared cell line and application thereof

A technology of serum-free culture and serum-free medium, which is applied in the field of cell lines, can solve the problems of increased risk of exogenous contamination, unsuitable animal vaccine production, unfavorable proliferation of influenza virus, etc., to reduce the risk of exogenous contamination, Save manpower, ensure the effect of stability and uniformity

Active Publication Date: 2018-09-28
PU LIKE BIO ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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 effects of adding serum are relatively large, and the purification process in the later stage is difficult
In addition, the addition of animal-derived serum to the culture medium greatly increases the risk of exogenous contamination in virus culture or vaccine production, and increases production costs

Method used

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  • A method for preparing a serum-free cultured suspension mammalian cell line, the prepared cell line and application thereof
  • A method for preparing a serum-free cultured suspension mammalian cell line, the prepared cell line and application thereof
  • A method for preparing a serum-free cultured suspension mammalian cell line, the prepared cell line and application thereof

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Experimental program
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Effect test

Embodiment 1

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

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

[0032] (2) After resuscitation, serially passage 3 times, use VP-SFMAGT with 5% (v / v) PAA added TM (Gibco company) serum-free medium (or MDCK cell serum-free protein-free chemical medium (Jiangyin Cambridge Biotechnology Co., Ltd.), or InVitrus TM (Cell Culture Technologies, Switzerland), or SFM4MegaVir TM (HyClone Company)) was replaced with MEM containing 5% (v / v...

Embodiment 2

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

[0039] 1. Cell culture in a bioreactor: First, directly resuscitate frozen MDCK cells adapted to serum-free suspension culture in shake flasks, change the medium for 24 hours, and culture at a cell density of more than 4 million / ml in about 72 hours. 3 times, the cell growth resumed; continue to expand the culture, inoculate Bioflo115 fermenter, the inoculation density is 500,000 / ml, and the culture conditions are 37°C, 80-110rpm, DO30%-50%, pH7.2. The medium used in this process is VP-SFMAGT TM (Gibco Company), 0.2% Pluronic F-68 and 30 μg / ml dextran sulfate were added.

[0040] 2. During the cultivation process of the bioreactor, sample and count the number of cells at 18h, 24h, 42h, 48h, 66h, 72h, 90h, 96h, and 114h after inoculation, and stop sampling when the cell viability drops below 50%. , see Table 1 for cel...

Embodiment 3

[0047] Example 3. Serum-free pure suspension culture breeding avian influenza H9 subtype

[0048] 1, bioreactor culture cell, culture method is the same as embodiment 1;

[0049] 2. When the density of the cells in the fermenter reaches the requirements for inoculating the avian influenza virus, inoculate the virus, and add the virus liquid according to the inoculation dose of MOI=0.001-0.1. In addition, because TPCK-trypsin has a great influence on the proliferation of influenza virus, Therefore, add 1.0-5.0 μg / ml after virus inoculation. The culture conditions are 33℃, 80~110rpm, DO30~50%, pH7.20;

[0050] 3. Since the suspension cells cannot observe cell lesions, samples are taken every 12 hours after inoculation, the number of cells and HA are monitored to determine the virus content, an extreme value of the virus hemagglutination value is found, and the virus liquid is harvested when the virus hemagglutination value is the highest. Cell debris was 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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Patent Type & Authority Patents(China)
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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