Method for preparing oncolytic virus by using 293 cells

A technology of oncolytic virus and oncolytic rhabdovirus, which is applied in the field of biomedicine, can solve the problems of enhancing virus cytolytic activity, etc., and achieve the effects of reducing costs, reducing the possibility of product contamination, and saving labor costs

Inactive Publication Date: 2019-10-08
FANTASIA BIOPHARMA ZHEJIANG CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At the same time, the rupture of tumor cells leads to the release of tumor antigens from tumor cells, which

Method used

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  • Method for preparing oncolytic virus by using 293 cells
  • Method for preparing oncolytic virus by using 293 cells
  • Method for preparing oncolytic virus by using 293 cells

Examples

Experimental program
Comparison scheme
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Embodiment 1

[0072] Example 1 Vero, 293 adherent cells, and 293sus suspension production cells were infected with inoculated doses of different MOI values, and the changes in the total amount of virus titers obtained from production amplification were compared.

[0073] In Vero and 293 adherent cells, replace the original complete medium with opti-MEM, then infect Vero / 293 adherent cells with VSV virus at different MOI, and replace with complete medium for 48 hours after 2h-3h; 293sus Suspended cells were centrifuged to replace the medium, counted and spread into 6-well plates according to E5 / well, and infected for 24 hours according to different MOI; the supernatant was collected to detect the prepared virus titer (TCID50 ) changes, the overall experimental process refers to figure 1 The specific implementation described in .

[0074] The specific steps of the above experimental process are as follows:

[0075] 1. Vero-E6: Add 2 mL of Vero-E6 cell suspension to each well of a 6-well cul...

Embodiment 2

[0086] Example 2 Further, in order to enhance the infectivity of the virus to cells, PBS, DMEM-0, and opti-MEM were selected as solvents for cell infection as the dilution of U400 virus infection (Vero selects MOI=5, and 293 adherent cells select MOI =0.1)

[0087] Such as Figure 3A , after the cells were congested, replace the original culture medium with PBS, DMEM-0 and opti-MEM in Vero and 293 adherent cells respectively, and then add VSV virus infection at MOI=5 and 0.1 respectively for 2 hours and replace with complete medium culture medium, the supernatant of Vero cells was collected after 48 hours, and the supernatant of 293 adherent cells was collected after 24 hours to detect the titer (TCID50) of the virus produced by the virus strain.

[0088] The specific operation steps of detecting the titer of above-mentioned virus are as follows:

[0089] 1. Vero-E6: Add 2 mL of Vero-E6 cell suspension to each well of a 6-well culture plate to make the cell volume reach 4×10...

Embodiment 3

[0098] Example 3 Effect of Serum Concentration on Virus Production in Culture Medium

[0099] In the process of traditional virus preparation, it will be found that during the amplification process of some viruses, the serum concentration in the medium will seriously affect the increase of virus titer. In order to solve this problem and further increase the virus titer, optimize the virus amplification process, the Vero ( Figure 4A ) and 293 adherent cells ( Figure 4B ), replace the original medium with DMEM-0, then infect VSV virus according to MOI=5 and 0.1 respectively, and after 2h-3h, use volume percentages of 0%, 1.5%, 3%, 6%, 9% The DMEM medium prepared with different concentrations of FBS was used for virus amplification. After the cells were completely lysed, the supernatant was collected to detect the virus titer (TCID50) under different serum concentrations.

[0100] The concrete steps of detecting the titer of above-mentioned virus are as follows:

[0101] 1. ...

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Abstract

The invention discloses a method for preparing an oncolytic virus by using 293 adherent cells and 293sus suspension cells. The method utilizes adherent or suspension 293 cells to prepare oncolytic virus, and utilizes 293 adherent cells to optimize the optimal conditions for virus amplification and production, wherein, the optimal conditions comprise multiplicity of infection (MOI), a virus weak solution, a serum concentration during virus amplification, a virus harvest time, and a virus amplification temperature; further, a method for preparing the oncolytic virus by optimizing 293sus suspension cells includes optimizing the multiplicity of infection (MOI), the virus harvest time, and the like, and the single cell production amount reaches E4 power. Further, when the high cell density expansion is carried out, the virus yield can reach 4E10 pfu/mL. Both 293 cells can produce the high-titer virus efficiently and stably, which provides technical support for the large-scale production ofsubsequent viruses.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a method for producing and preparing oncolytic VSV virus by using 293 adherent cells and 293sus suspension cells. Background technique [0002] At present, the cells approved to be used as vaccine preparation and virus production substrates mainly include Vero, 293, BHK, CHO, etc.; there are three main methods of large-scale culture of mammalian cells: adherent culture, microcarrier culture, and serum-free suspension culture. All three methods can be used for large-scale culture of 293 cells. In the previous experiment, we used Vero cells for the production and preparation of VSV virus, and the virus titer was E9 pfu / mL. In order to obtain higher virus production and reduce production costs, the process is more economical and more competitive in the industry In order to carry out the force and subsequent purification process, here we try to use adherent cultured 293 cells and suspensi...

Claims

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Application Information

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IPC IPC(8): C12N7/00A61P35/00C12R1/93
CPCA61P35/00C12N7/00C12N2760/20221C12N2760/20251
Inventor 吴飞韦治明秦晓峰
Owner FANTASIA BIOPHARMA ZHEJIANG CO LTD
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