Mdck-derived cell lines adapted to serum-free culture and suspension culture and method for preparing vaccine virus using the cells

Abstract

Disclosed is a Madin-Darby canine kidney (MDCK)-derived cell line. The MDCK-derived cell line is derived from MDCK cells deposited under accession number ATCC CCL-34. The MDCK-derived cell line can be prepared by serum-free culture and suspension culture. Preferably, the MDCK-derived cell line has low or no tumorigenicity. The MDCK-derived cell line is preferably selected from MDCK Sky1023, MDCK Sky10234 and MDCK Sky3851. Further disclosed are a culture method for growing the MDCK-derived cells and a method for producing a vaccine virus using the MDCK-derived cells.

What is claimed is: 
     
         1 . A Madin-Darby canine kidney (MDCK)-derived cell line that is derived from MDCK cells deposited under accession number ATCC CCL-34, does not require serum for cell growth and is prepared by suspension culture without the need to be attached to carriers. 
     
     
         2 . A method for producing a vaccine virus using the MDCK-derived cell line according to  claim 1 . 
     
     
         3 . The method according to  claim 2 , wherein the virus is selected from the group consisting of influenza viruses, measles viruses, Japanese encephalitis viruses, mumps viruses, rubella viruses, polio viruses, HSV-1, HSV-2, rabies viruses, RS viruses, reovirus type 3, yellow fever virus, parvoviruses, coxsackie viruses, adenovirus types 1 to 47, Lassa viruses and vacciniaviruses. 
     
     
         4 . The method according to  claim 3 , wherein the virus is an influenza virus. 
     
     
         5 . The MDCK-derived cell line according to  claim 1 , wherein the MDCK-derived cell line has low or no tumorigenicity as compared to the original MDCK cell line. 
     
     
         6 . A method for producing a vaccine virus using the MDCK-derived cell line according to  claim 5 . 
     
     
         7 . The method according to  claim 6 , wherein the virus is selected from the group consisting of influenza viruses, measles viruses, Japanese encephalitis viruses, mumps viruses, rubella viruses, polio viruses, HSV-1, HSV-2, rabies viruses, RS viruses, reovirus type 3, yellow fever virus, parvoviruses, coxsackie viruses, adenovirus types 1 to 47, Lassa viruses and vacciniaviruses. 
     
     
         8 . The method according to  claim 7 , wherein the virus is an influenza virus. 
     
     
         9 . The MDCK-derived cell line according to  claim 1 , wherein the MDCK-derived cell line is MDCK Sky1023 (DSM ACC3112), MDCK Sky10234 (DSM ACC3114) or MDCK Sky3851 (DSM ACC3113). 
     
     
         10 . A method for producing a vaccine virus using the MDCK-derived cell line according to  claim 9 . 
     
     
         11 . The method according to  claim 10 , wherein the virus is selected from the group consisting of influenza viruses, measles viruses, Japanese encephalitis viruses, mumps viruses, rubella viruses, polio viruses, HSV-1, HSV-2, rabies viruses, RS viruses, reovirus type 3, yellow fever virus, parvoviruses, coxsackie viruses, adenovirus types 1 to 47, Lassa viruses and vacciniaviruses. 
     
     
         12 . The method according to  claim 11 , wherein the virus is an influenza virus. 
     
     
         13 . A method for producing an influenza virus from a cell culture, the method comprising:
 (a) inoculating a serum-free culture medium with the MDCK-derived cell according to  claim 1  at a concentration of 1×10 4  to 1×10 6  cells/ml;   (b) allowing the MDCK-derived cells to grow in a disposable bioreactor system until the cell density reaches at least 5×10 6  cells/ml, comprising culturing the MDCK-derived cells while maintaining one or more culture conditions selected from the group consisting of a stirring rate of 40 to 100 rpm, a pH of 6.5 to 7.5 and a dissolved oxygen (DO) concentration of 35 to 100%;   (c) infecting the grown MDCK-derived cells with an influenza virus;   (d) culturing the infected grown MDCK-derived cells under conditions allowing cloning of the influenza virus; and   (e) isolating the influenza virus from the cell culture composition.   
     
     
         14 . The method according to  claim 13 , further comprising adding a fresh medium to the cell culture or replacing a portion of the medium with a fresh medium in step (b). 
     
     
         15 . A virus or a virus antigen produced by the method according to  claim 13 . 
     
     
         16 . A virus or a virus antigen produced by the method according to  claim 14 . 
     
     
         17 . A method for producing an influenza virus from a cell culture, the method comprising:
 (a) inoculating a serum-free culture medium with the MDCK-derived cell according to  claim 5  at a concentration of 1×10 4  to 1×10 6  cells/ml;   (b) allowing the MDCK-derived cells to grow in a disposable bioreactor system until the cell density reaches at least 5×10 6  cells/ml, comprising culturing the MDCK-derived cells while maintaining one or more culture conditions selected from the group consisting of a stirring rate of 40 to 100 rpm, a pH of 6.5 to 7.5 and a dissolved oxygen (DO) concentration of 35 to 100%;   (c) infecting the grown MDCK-derived cells with an influenza virus;   (d) culturing the infected grown MDCK-derived cells under conditions allowing cloning of the influenza virus; and   (e) isolating the influenza virus from the cell culture composition.   
     
     
         18 . The method according to  claim 17 , further comprising adding a fresh medium to the cell culture or replacing a portion of the medium with a fresh medium in step (b). 
     
     
         19 . A virus or a virus antigen produced by the method according to  claim 17 . 
     
     
         20 . A virus or a virus antigen produced by the method according to  claim 18 . 
     
     
         21 . A method for producing an influenza virus from a cell culture, the method comprising:
 (a) inoculating a serum-free culture medium with the MDCK-derived cell according to  claim 9  at a concentration of 1×10 4  to 1×10 6  cells/ml;   (b) allowing the MDCK-derived cells to grow in a disposable bioreactor system until the cell density reaches at least 5×10 6  cells/ml, comprising culturing the MDCK-derived cells while maintaining one or more culture conditions selected from the group consisting of a stirring rate of 40 to 100 rpm, a pH of 6.5 to 7.5 and a dissolved oxygen (DO) concentration of 35 to 100%;   (c) infecting the grown MDCK-derived cells with an influenza virus;   (d) culturing the infected grown MDCK-derived cells under conditions allowing cloning of the influenza virus; and   (e) isolating the influenza virus from the cell culture composition.   
     
     
         22 . The method according to  claim 21 , further comprising adding a fresh medium to the cell culture or replacing a portion of the medium with a fresh medium in step (b). 
     
     
         23 . A virus or a virus antigen produced by the method according to  claim 21 . 
     
     
         24 . A virus or a virus antigen produced by the method according to  claim 22 . 
     
     
         25 . A method for preparing an MDCK-derived cell line that does not require serum for cell growth and is prepared by suspension culture without the need to be attached to carriers, the method comprising:
 (a) preparing original MDCK cells deposited under accession number ATCC CCL-34;   (b) adapting the original MDCK cells to a serum-free medium to allow the original MDCK cells to grow in the serum-free medium; and   (c) adapting the adherent MDCK cells adapted in step (b) to a serum-free medium to allow the MDCK cells to grow in a suspension state without the need for carriers.