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Serum-free Vero cell fixed bed bioreactor high-density culture process

A technology of bioreactor and high-density culture, which is applied in the field of cell culture, can solve the problems of reducing culture shear force, achieve the effect of reducing pollution and increasing cell density

Active Publication Date: 2020-10-30
成都柏奥特克生物科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The fixed bed bioreactor can reduce the shear force of culture, and the efficiency of culturing cells is higher. Among them, the density of cultivating Vero cells can reach 1.0×10 7 cells / ml【Yang Yi, Ru Dongyu, Guo Xiuxia, Cui Guangwen, et al. Preparation of Vero cell human rabies vaccine by basket bioreactor[J]. Chinese Journal of Biological Products, May 2011, Vol. 25, No. 5], but there is still room for improvement

Method used

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  • Serum-free Vero cell fixed bed bioreactor high-density culture process
  • Serum-free Vero cell fixed bed bioreactor high-density culture process
  • Serum-free Vero cell fixed bed bioreactor high-density culture process

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Effect test

Embodiment 1

[0030] Embodiment 1 Serum-free Vero cell fixed-bed bioreactor high-density culture

[0031] 1. Production cells

[0032] The cells used for production were Vero cells Vero-SF-ACF adapted to serum-free culture, which were purchased from the American type culture collection (American Type Culture Collection), and the ATCC number was CCL-81.5.

[0033] 2. Method

[0034] 1) Fill the sheet carrier (such as figure 1 purple area).

[0035] 2) Place Serum-free Vero cells from the working cell bank in a T75 square flask and culture them with serum-free medium. When they grow into a dense monolayer, use recombinant cell digestion enzyme (instead of animal-derived trypsin) to digest the cells, and press 1: 6 Amplify and passage, digest cells with recombinant cell digestion enzyme, and then inoculate into fixed bed bioreactor, the cell density in the reactor is 6×10 5 cells / ml.

[0036] 3) After the cell suspension completely entered the reactor for 60 minutes, the cell adsorption w...

Embodiment 2

[0043] Embodiment 2 Serum-free Vero cell fixed-bed bioreactor high-density culture

[0044] 1. Production cells

[0045] The cells used for production were Vero cells Vero-SF-ACF adapted to serum-free culture, which were purchased from the American type culture collection (American Type Culture Collection), and the ATCC number was CCL-81.5.

[0046] 2. Method

[0047] 1) In the carrier filling area of ​​the fixed bed bioreactor, fill the sheet carrier (such as figure 1 purple area).

[0048] 2) Place Serum-free Vero cells from the working cell bank in a T75 square flask and culture them with serum-free medium. When they grow into a dense monolayer, use recombinant cell digestion enzyme (instead of animal-derived trypsin) to digest the cells, and press 1: 3 Amplify and passage, digest cells with recombinant cell digestion enzyme, and then inoculate into fixed bed bioreactor, the cell density in the reactor is 5×10 5 cells / ml.

[0049] 3) After the cell suspension completel...

Embodiment 3

[0057] Example 3 Serum-free Vero cells fixed bed bioreactor high density culture

[0058] 1. Production cells

[0059] The cells used for production were Vero cells Vero-SF-ACF adapted to serum-free culture, which were purchased from the American type culture collection (American Type Culture Collection), and the ATCC number was CCL-81.5.

[0060] 2. Method

[0061] 1) Fill the sheet carrier (such as figure 1 purple area).

[0062] 2) Place Serum-free Vero cells from the working cell bank in a T75 square flask and culture them with serum-free medium. When they grow into a dense monolayer, use recombinant cell digestion enzyme (instead of animal-derived trypsin) to digest the cells, and press 1: 6 Amplify and passage, digest the cells with recombinant cell digestion enzymes, and then inoculate them into a fixed bed bioreactor. The cell density in the reactor is 8×10 5 cells / ml.

[0063] 3) After the cell suspension completely enters the reactor for 60 minutes, the cell ads...

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Abstract

The invention discloses a serum-free Vero cell fixed bed bioreactor high-density culture process, and belongs to the field of cell culture. The culture process comprises the following steps of: 1) inoculating Vero cells cultured by a serum-free culture medium into a fixed bed bioreactor until a solution is clear; 2) within 12-24 hours after inoculation, setting the following parameters for cultureunder a non-perfusion condition: the temperature is 37.0 + / - 0.2 DEG C, the pH value is 7.20 + / - 0.1, the dissolved oxygen is 60 + / - 20%, and the rotating speed is 90-110 rpm; and 3) starting perfusion culture 12-24 hours after the inoculation. By adopting the method disclosed by the invention, the high-density Vero cells can be cultured, and the use of bovine serum and animal pancreatin is not involved, so that the safety of biological products obtained by the Vero cells is greatly improved.

Description

technical field [0001] The invention belongs to the field of cell culture. Background technique [0002] Cell culture is an essential prerequisite for viral vaccines and most other biological products. High-density, high-quality cells can greatly increase product yield. [0003] Vero cell is a kidney cell of African green monkey (Cercopithecus aethiops), which was established on March 27, 1962 by Yasumura and Kawakita of Chiba University (Chiba), Japan. The Vero cell line was brought to the U.S. by Dr. B. Simizu on June 15, 1964, at the laboratory of tropical virology, the National Institute of Allergy and Infectious Diseases (NIAID), and the National Institutes of Health (NIH) , the subculture level of the cells at this time is the 93rd passage. At the 113th passage level, it was submitted to the American Type Culture Collection (ATCC), where it was cultured and passaged to the 121st passage, and a bank was established. The cell bank number is ATCC CCL-81TM. [0004] Ser...

Claims

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Application Information

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IPC IPC(8): C12N5/071
CPCC12N5/0686C12N2500/30C12N2500/33C12N2500/90
Inventor 梁智杰黄林崔利凯陈坤李高军
Owner 成都柏奥特克生物科技股份有限公司
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