MDCK-DERIVED CELL LINES ADAPTED TO SERUM-FREE CULTURE AND SUSPENSION CULTURE AND METHOD FOR PREPARING VACCINE VIRUS USING THE CELLS
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.
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.