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Porcine testicular cell line st-s that can be cultured in suspension and its obtaining method and application

A ST-S, suspension culture technology, applied in the field of cell engineering, can solve the problems of limiting the application scale and production efficiency of ST cells

Active Publication Date: 2019-11-22
JINYUBAOLING BIO PHARMA CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] However, since the ST cell line was established, the ST cells used in scientific research and industrial production are all adherent cultures. So far, there have been no reports of successful suspension culture of ST cells at home and abroad, which limits the application scale of ST cells. and production efficiency

Method used

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  • Porcine testicular cell line st-s that can be cultured in suspension and its obtaining method and application
  • Porcine testicular cell line st-s that can be cultured in suspension and its obtaining method and application
  • Porcine testicular cell line st-s that can be cultured in suspension and its obtaining method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] Embodiment 1, obtaining the ST-S cell that can be cultured in suspension

[0079] In this example, by developing a personalized culture medium and optimizing the domestication approach, a pig testis cell strain that can be cultured in complete suspension under low serum conditions was finally obtained, named ST-S. Specifically, the cell strain ST-S of the present invention The method of obtaining includes the following steps:

[0080] 1. Domestication of adherent ST cells in plastic culture flasks

[0081] Usually, the subculture ratio of ST cells adherently cultured in a square culture flask is 1:3, that is, after the cells grow into a monolayer in a T75 plastic culture square flask, trypsin digestion solution (it is better to cover the cell surface, About 5 mL) of the cell suspension obtained after digestion can be evenly inoculated in three T75 plastic culture square flasks (Welter, M.W., Welter, C.J., Chambers, D.M., and Svensson, L. (1991) Adaptation and serial pa...

Embodiment 2

[0155] Embodiment 2, suspension culture ST-S cell in 5L bioreactor

[0156] The ST-S cell strain of the present invention still has the characteristics of suspension culture after cryopreservation. After the cryopreserved ST-S cell line is revived, it does not need to be acclimatized, and can be directly enlarged for suspension culture. This example provides a semi-fed-batch suspension culture method in a 5L bioreactor after recovery of the ST-S cell line.

[0157] Suspension culture of ST-S cells in a 5L reactor includes the following steps:

[0158]1) Resuscitating the frozen ST-S cell line, the method is: take out a frozen ST-S cell line (obtained in Example 1) from liquid nitrogen, thaw it quickly in a 37°C water bath, add the pre-prepared ( Put the medium in a 10mL centrifuge tube containing 5mL ST-LSM-s medium (preheated at 37°C for 10 minutes), centrifuge at 1000rpm for 5 minutes, pour off the supernatant, resuspend the cells with ST-LSM-s medium, and transfer the cel...

Embodiment 3

[0164] Embodiment 3, detect the sensitivity of ST-S cell to classical swine fever virus

[0165] This example is used to understand the susceptibility of suspension cultured ST-S cells to classical swine fever virus (CSFV), and compare it with adherent cultured ST cells.

[0166] 1. Cells infected by CSFV virus

[0167] 1. Infect adherent ST cells with CSFV virus: cryopreserved adherent ST cells were inoculated into T75 glass culture flasks after thawing and cultured in T25 glass culture flasks, at an inoculation density of 3×10 4 cells / cm 2 , the culture medium is ST-LSM-s medium containing 3% calf serum, and the culture volume is 20 mL. When the cells were cultured to a confluence of 70-80%, they were inoculated with CSFV spleen virus (purchased from Jinyu Baoling Biopharmaceutical Co., Ltd.) at 0.4% (M / V).

[0168] 2, CSFV virus infection suspension ST cells (ST-S): frozen ST-S cells (obtained by suspension culture according to the method of Example 2) were revived and i...

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Abstract

The invention discloses a swine testicular cell strain ST-S suitable for suspension culture as well as an acquisition method and an application of the swine testicular cell strain ST-S. ST cells, which are subjected to adherent culture, are domesticated into ST cells which are suitable for suspension culture and can be passaged stably, and the ST cells are named as ST-S; and the ST-S cells keep sensitivity to classical swine fever virus (CSFV) and are capable of avoiding mycoplasma contamination. The invention provides a large-scale industrial suspension culture mode on the ST-S cells, and the suspension culture mode is applicable to culture by virtue of a shake flask and a bioreactor. By virtue of the ST-S cells obtained from the suspension culture, vaccines or other bio-products can be produced, so that a virus production process is simplified, product stability is enhanced, serum dosage and production cost are reduced and production efficiency is improved.

Description

technical field [0001] The invention belongs to the technical field of cell engineering, and in particular relates to a porcine testis cell line ST-S capable of suspension culture and a method for obtaining the same, and also relates to a method for using the cell line to carry out suspension culture of ST-S cells in batches. The ST-S The cells can be used as engineering cells for industrial production of swine fever vaccine. Background technique [0002] Pig testicular cell (swine testicular cell, ST) is a fibroblast-like adherent cell that was first isolated and cultured in 1966. ST cells are sensitive to many viruses, such as classical swine fever virus, porcine parvovirus, rabies virus, pseudorabies virus, porcine transmissible gastroenteritis virus, porcine circovirus, etc. ST cells are commonly used in the isolation, cultivation and identification of the above viruses (Kemeny, L.J. (1981) Isolation of transmissible Gastroenteritis virus, pseudorabies virus, and porcin...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/071C12N1/36A61K39/187A61P31/14
CPCA61K39/12C12N1/36C12N5/0683C12N2500/84C12N2710/12034
Inventor 倪小平刘国英刘敬涛郝鹏马子敏谢雪岑李同据史林凯郝洪吉刘国瑞
Owner JINYUBAOLING BIO PHARMA CO LTD
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