CN-115768872-B - Novel Vero cell line capable of suspension culture in serum-free medium, preparation method thereof and method for preparing virus for vaccine by using novel cell line

CN115768872BCN 115768872 BCN115768872 BCN 115768872BCN-115768872-B

Abstract

The present invention relates to a sVERO C2 cell line, which is a Vero cell line derived from WHO-dispensed Vero cells (african green monkey kidney cell line) and capable of suspension culture in the absence of serum components. In addition, the present invention relates to a culture method for culturing Vero cells and a method for preparing virus for vaccine using the Vero cells.

Inventors

GUO JUNSHUO
JIN ENSHUN
JIN XUN
XU JIYUAN
LI JIANSHI
LI SHOUCHEN
HONG CHENGHUI

Assignees

SK生物科学株式会社

Dates

Publication Date: 20260508
Application Date: 20201125
Priority Date: 20191127

Claims (7)

1. A Vero cell line sVERO C2, accession number KCLRF-BP-00470.
2. The Vero cell line of claim 1, wherein the cell line is derived from WHO-dispensed Vero cells, the cell line does not require serum for cell growth, and can be cultured in suspension without an attachment carrier.
3. The Vero cell line of claim 1, wherein the cell line proliferates viruses.
4. A method of preparing a virus for a vaccine using the cell line of claim 1.
5. The method of claim 4, wherein the virus is selected from the group consisting of yellow fever virus, zika virus, rotavirus, dengue virus, influenza virus, measles virus, japanese encephalitis virus, mumps virus, rubella virus, polio virus, HSV-1, HSV-2, rabies virus, RS virus, reovirus type 3, parvovirus, coxsackievirus, adenovirus type 1 to 47, lassa virus, bullous stomatitis virus, and vaccinia virus.
6. The method of claim 4, wherein the virus is a yellow fever virus or a zika virus.
7. A method of producing a virus for a vaccine, the method comprising: (a) Inoculating the Vero cells of claim 1 into serum-free cell culture medium at a concentration of 1 x 10 5 -9×10 5 cells/mL; (b) Proliferating Vero cells in a roller bottle to a cell density of 5.0X10 5 -4.7×10 6 cells/mL, comprising the step of culturing the cells under culture conditions maintaining a stirring speed of 40-90rpm and a pH of 6.5-7.5; (c) Infecting the propagated Vero cells with yellow fever virus or zika virus; (d) Culturing the infected proliferating Vero cells, and (E) Yellow fever virus or Zika virus was isolated from the cell culture composition.

Description

Novel Vero cell line capable of suspension culture in serum-free medium, preparation method thereof and method for preparing virus for vaccine by using novel cell line Technical Field The present application claims priority based on korean patent application No. 10-2019-0154749 filed on the 10 th month 27 of 2019, and all matters disclosed in the specification and drawings of the corresponding application are incorporated herein. The present invention relates to a novel Vero-derived cell line capable of suspension culture in serum-free medium, a method for preparing the same, and a method for preparing viruses using the suspension-cultured cells. Background With commercialization of various types of vaccines, methods of manufacturing vaccines have been developed in various ways. As a conventional vaccine production method, a typical example is the production of an influenza vaccine using fertilized eggs. In the case of producing a vaccine using fertilized eggs, it is difficult to provide stable supply of fertilized eggs, and thus there is a limit in that it is necessary to regulate vaccine production. In addition, chickens suitable for production of the products must be raised in a sterile facility, thereby having a problem of increased costs, and there is also a disadvantage that egg protein allergy sufferers cannot inoculate because it is difficult to purify egg protein-derived components. As a method for overcoming the problem of producing a vaccine using fertilized eggs, there is a method for producing a vaccine by cell culture. Since the vaccine is produced by inoculating a virus after mass culture of animal cells capable of growing indefinitely, it can be supplied by mass production in a short time, and has an advantage that an egg protein allergy sufferer can be inoculated. These animal cells are typically adherent cells and are cultured using Fetal Bovine Serum (FBS), but in this case, there may be unknown animal-derived factors in the product and there may be quality differences between the products. In addition, fetal bovine serum is expensive and there is a risk of being infected with infectious proteins such as prions, viruses and mycoplasma, so that there is a disadvantage in that the manufacturing cost of the product increases and the safety cannot be ensured. Therefore, in the production of vaccines, it is preferable not to use animal-derived additives and animal-derived serum. On the other hand, the method of culturing cells in suspension has advantages such as easiness in large-scale culture, simplification of the passaging process, reduction of labor and space utilization, as another method of cell culture, compared with the adherent cell culture method. Thus, a great deal of research is being conducted on the development of serum-free suspension cells and the proliferation of viruses using them. Vero cell line is an established cell line, because it is highly sensitive to various viruses such as rotavirus, poliovirus, influenza virus, japanese encephalitis virus and dengue virus, and thus is capable of propagating various viruses. However, since Vero cell lines have very strong adhesion to surfaces, very large area culture vessels or microcarriers are required for large scale cultivation, which creates a significant cost in the vaccine manufacturing process. In addition, a step of removing the cells attached to the carrier is required. In this case, trypsin of animal origin is used, and with the use of animal-derived components, there is a possibility of cell loss and damage. Therefore, in order to produce vaccines by safe and effective animal cell culture, a Vero cell line capable of suspension culture in a serum-free medium is required, and the present inventors have previously studied and patented a Vero cell line capable of serum-free suspension culture (korean patent No. 10-1831284). However, the suspension Vero cell line Vero Sky 7458 of the previously registered patent (korean patent No. 10-1831284) shows some limited effects in terms of cell proliferation and viral infectivity. Disclosure of Invention Technical problem In the present study, in the course of developing new cells, the present inventors have tried to establish a new suspension Vero cell line having more excellent cell proliferation, cell morphology and virus proliferation than the previously registered patent cell line (Vero Sky 7458) by selecting cells having excellent functions. Accordingly, an object of the present invention is to provide a Vero cell line which can be used for virus propagation for vaccines and which can be subjected to serum-free culture and suspension culture to solve the problems of contamination or low culture efficiency caused by the use of serum and adherent culture. It is another object of the present invention to provide a more efficient virus proliferation and vaccine production method by providing a cell line having a superior effect in terms of cell proliferation or virus proliferation