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Continuous production method of BHK21 cells by carrying out suspension culture, preservation and resuscitation in bioreactor, and application of BHK21 cells

A technology of bioreactor and suspension culture, which is applied in specific-purpose bioreactor/fermenter, biochemical equipment and method, combination of bioreactor/fermenter, etc. Generation increase and other issues, to achieve the effect of good cell state

Pending Publication Date: 2020-01-21
JINYUBAOLING BIO PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the low initial density of BHK21 cells recovered from cryopreservation in liquid nitrogen, the general initial density is only about 0.5×10 6 cell / ml, it needs to be expanded in large quantities, and it generally takes 8-15 days to meet the needs of large-scale production of seeds. During this period, the cell generation increases by 4-7 generations, so the recovery period is longer, and the recovery operation process It is cumbersome, and the operator must have strong aseptic operation ability, so the labor intensity is high; in addition, during the cell resuscitation and passage process, the shaking flask must be opened many times for operation, and the risk of contamination is high
For re-freezing, you need to put the cryopreservation tube in a refrigerator at 4°C for 30 minutes, at -20°C for 30 minutes, and at -70°C for 16-18 hours before storing it in a liquid nitrogen tank at -196°C. The preservation method is more cumbersome.
[0004] To sum up, the existing liquid nitrogen storage method for BHK21 cells has a long resuscitation production cycle, high risk of resuscitation contamination, high labor intensity, and cumbersome cryopreservation methods, which can easily lead to unstable vaccine quality

Method used

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  • Continuous production method of BHK21 cells by carrying out suspension culture, preservation and resuscitation in bioreactor, and application of BHK21 cells
  • Continuous production method of BHK21 cells by carrying out suspension culture, preservation and resuscitation in bioreactor, and application of BHK21 cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] 1) Suspension culture of BHK21 cells: Add 45L of nutrient solution to the bioreactor in advance, and complete the sterilization of the nutrient solution, and inoculate it under the conditions of rotation speed of 120rpm, temperature of 37°C, pH value of 7.2 and dissolved oxygen concentration of 40%. The cell density was 4 x 10 6 cell / ml of 5L seed solution and cultivated, after 48 hours of culture, sampled and determined the cell density was 5×10 6 cell / ml, the cell viability is 95%. Among them, the micrographs of BHK21 cells cultured for 48h are as follows: figure 1 As shown, it can be seen that the BHK21 cells obtained after 48 hours of culture have uniform size, neat edges, good refraction, and the cell state is normal;

[0042] 2) Preservation of BHK21 suspension cells at low temperature: Start the cooling mode of the bioreactor, specifically use interlayer 2-8°C frozen water to continuously cool down, and gradually reduce the stirring speed to the low temperature...

Embodiment 2

[0046] 1) Suspension culture of BHK21 cells: Add 45L of nutrient solution to the bioreactor in advance, and complete the sterilization of the nutrient solution, and inoculate it under the conditions of rotation speed of 120rpm, temperature of 37°C, pH value of 7.2 and dissolved oxygen concentration of 50%. The cell density was 4.1 x 10 6 Cell / ml 5L seed solution was cultured, and after 48 hours of culture, the density of sampling was determined to be 5.2×10 6 cell / ml. Among them, the micrographs of BHK21 cells cultured for 48h are as follows: figure 2 As shown, it can be seen that the BHK21 cells obtained after 48 hours of culture have uniform size, neat edges, good refraction, and the cell state is normal;

[0047] 2) Preservation of BHK21 suspension cells at low temperature for the first time: Start the cooling mode of the bioreactor, specifically use interlayer 2-8°C frozen water to continuously cool down, and gradually reduce the stirring speed to the low temperature st...

Embodiment 3

[0053] 1) Suspension culture of BHK21 cells: Add 45L of nutrient solution to the bioreactor in advance, and complete the sterilization of the nutrient solution, and inoculate it under the conditions of rotation speed of 120rpm, temperature of 37°C, pH value of 7.2 and dissolved oxygen concentration of 60%. The cell density was 4.8 x 10 6 cell / ml 5L seed solution and cultivated, after culturing for 48h, the first cell suspension was obtained, and the density of sampling was 5.6×10 6 cell / ml, the activity rate is 93%. Among them, the BHK21 cells cultured for 48 hours were observed under a microscope to have uniform size, neat edges, uniform dispersion, and the cell state was normal;

[0054] 2) The first cryopreservation of BHK21 suspension cells: Start the cooling mode of the bioreactor to store the first cell suspension in step 1) at a low temperature, specifically use interlayer 2-8°C frozen water to continuously cool down, and gradually reduce the stirring speed to The con...

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Abstract

The invention discloses a continuous production method of BHK21 cells by carrying out suspension culture, preservation and resuscitation in a bioreactor as well as application of the BHK21 cells, andbelongs to the field of cell preservation in biological pharmacy. According to the continuous production method of the BHK21 cells by carrying out suspension culture, preservation and resuscitation inthe bioreactor provided by the invention, BHK21 cells in the bioreactor can be preserved for up to 15 days or above by adjusting low-temperature preservation conditions in the bioreactor and / or repeating low-temperature preservation and resuscitation; and moreover, the BHK21 cells meeting cell density and cell vitality requirements for production can be continuously obtained. In addition, the obtained BHK21 cells are small in batch difference, so that product quality stability can be ensured so as to have production efficiency improved.

Description

technical field [0001] The present invention relates to a method for preserving and resuscitating suspension cells in the field of biopharmaceuticals, in particular to a method for continuously producing BHK21 cells by using a bioreactor to suspend culture, preserve and resuscitate them, and in particular to a method for using a bioreactor to preserve BHK21 at low temperature A method for suspending cells and a continuous production method for suspending culturing, preserving and resuscitating BHK21 cells using a bioreactor and applications thereof. Background technique [0002] BHK21 cells refer to BabyHamster Syrian Kidney cells, which were established in 1961. The original BHK21 cell line is a fibroblast with anchorage dependence. Its single-cell clone was obtained in 1963. After countless passages, the cells can grow in suspension and can be widely used for the propagation of various viruses and the production of veterinary vaccines. . At present, most veterinary vacci...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12M1/36C12M1/38
CPCC12N5/0686C12M41/12C12M41/26C12M41/32C12M41/00
Inventor 亢和平段剑飞赵丽霞郭建军刘潇程俊峰张耀忠王建钊王玉雯
Owner JINYUBAOLING BIO PHARMA CO LTD
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