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Acquisition and application of full-suspension cultured cell strain PK15

A suspension cell, PK15-U technology, applied in animal cells, vertebrate cells, urinary tract/kidney cells, etc., can solve the problems of unsuccessful domestication and other problems, and achieve good industrial application prospects, high degree of automation, and large-scale solutions. The effect of industrialization requirements

Pending Publication Date: 2019-03-01
金河佑本生物制品有限公司 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are many studies on the domestication of full suspension culture of PK-15 cell adherent culture, but there is no public report on the successful domestication of PK-15 full suspension culture cell line and its application in the production of porcine circovirus vaccine

Method used

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  • Acquisition and application of full-suspension cultured cell strain PK15
  • Acquisition and application of full-suspension cultured cell strain PK15
  • Acquisition and application of full-suspension cultured cell strain PK15

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Embodiment 1: the cultivation of the porcine kidney PK15-U suspension cell adapted to full suspension culture

[0025] ①Take well-grown porcine kidney PK15-U suspension cells, add them to shake flasks filled with nutrient solution, culture them at 36-37°C for 48-72 hours, and subculture and enlarge them according to the ratio of 1:3 to 1:5;

[0026] ② Dilute the porcine kidney PK15-U suspension cells amplified and cultured in step ① to a density of (6-10)×105 cells / ml, inoculate them into a bioreactor, culture at a temperature of 36-37°C, and a pH of 6.8-7.2. Dissolved oxygen is 40%-60%, the growth curve of porcine kidney PK15-U suspension cells is as follows figure 1 As shown, the cell morphology under the microscope in suspension culture can be seen in figure 2 ;

[0027] ③Measurement of cell density in three batches of bioreactors: three batches of porcine kidney PK15-U suspension cells were continuously cultured in bioreactors, the culture conditions were the sam...

Embodiment 2

[0030] Example 2 The method for cultivating porcine circovirus by using porcine kidney PK15-U suspension cells

[0031] Take well-grown porcine kidney PK15-U suspension cells and inoculate them into a bioreactor at a density of 6.5×105 cells / ml. The cell culture conditions are: the working volume of the reactor is 7.0 liters, the cell culture temperature is 37°C, and the pH value 7.0, dissolved oxygen was 40%, cultured for 20 hours, and when the cell density was 11×105 cells / ml, porcine circovirus was inoculated according to 8% of the volume of the cell culture medium. The virus culture conditions were: temperature 36°C, pH value 7.0, the dissolved oxygen was 40%, and the virus liquid was harvested after 144 hours of cultivation. The virus titer was judged according to the "Regulations of Veterinary Biological Products of the People's Republic of China", and the harvested virus content was 7.0 TCID50 / ml.

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Abstract

The invention provides acquisition and application of a full-suspension cultured cell strain PK15, discloses a full-suspension cultured cell strain PK15 obtained by habituated culture and an application of the full-suspension cultured cell strain PK15 to vaccine virus production and belongs to the technical field of biology. The full-suspension cultured cell strain PK15 is named pig kidney suspension cell PK15-U and preserved in China Center for Type Culture Collection on March 21, 2018 with the preservation number being CTTCC C201860. Compared with the prior art, the method for culturing vaccine viruses from the suspension cell PK15-U is high in degree of automation, does not require carrier intervention and can be subjected to suspension culture in a serum-free or low-serum medium, so that the requirement for large-scale industrialization of virus culture is met, one large-scale production method capable of meeting the technical requirement of GMP production is developed, and good industrial application prospect is achieved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a PK15 cell suitable for full suspension culture and application thereof. Background technique [0002] PK15 cells are derived from pig kidneys, which are called pig kidney epithelial cells in Chinese, and their parents are derived from adult pig kidney cells PK-2a provided by Stice in the United States in 1955. PK15 is sensitive to various viruses such as porcine circovirus (PCV), porcine parvovirus (PPV) and swine fever virus (CSFV), and has been widely used in the isolation of swine fever virus, porcine pseudorabies virus and porcine parvovirus, etc. , in vitro culture and production of related vaccines. [0003] Traditional PK15 cells grow in an anchorage-dependent manner. At present, most of the above virus cultures are cultured in spinner bottles. However, due to the large number of spinner bottles, large workshop area, manual operation, low production efficiency, ...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12N7/00C12R1/93
CPCC12N5/0686C12N7/00C12N2750/10051
Inventor 杨桂花董鹏卫广森吴孟晨赵九娥马凤龙杨严格
Owner 金河佑本生物制品有限公司
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