A method for large-scale culture of cell proliferation and its method for producing viruses
A technology for cultivating cells and cells, applied in biochemical equipment and methods, tissue culture, animal cells, etc., can solve the problems of low cell density, inability to meet large-scale production of virus vaccines, small cell lines, etc., and achieve high production efficiency , maintain the stability of the virus antigen, and improve the effect of the stability of the virus antigen
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Embodiment 1
[0056] The MDCK cells were cultured in a roller bottle, and the primary MDCK cells were obtained after the monolayer was overgrown.
[0057] Four 15,000ml primary MDCK cells covered with a single layer were digested and used as seed cells in a 5-liter cell culture reactor for suspension culture, with a stirring speed of 40 rpm, adding microcarriers, culture medium, alkali and sugar, and culturing for 40 hours , when the cells on the microcarriers are covered with a monolayer, remove the supernatant, add trypsin to digest cells 1.0 times the volume of the remaining liquid in the reactor, digest and separate the microcarriers, and then harvest the cells and concentrate the cell solution.
[0058] Amplify the cells harvested in the 5 liters of cell culture reactor into a 25 liters of cell culture reactor, the stirring speed is 40 rpm, add microcarriers, culture fluid, alkali and sugar, and cultivate for 40 hours, until the cells on the microcarriers The monolayer is covered, the ...
Embodiment 2
[0068] The MDCK cells were cultured in a roller bottle, and the primary MDCK cells were obtained after the monolayer was overgrown.
[0069] Six 15,000ml primary MDCK cells covered with a single layer were digested and used as seed cells in a 10-liter cell culture reactor for suspension culture, with a stirring speed of 60 rpm, adding microcarriers, culture medium, alkali and sugar, and culturing for 50 hours , when the cells on the microcarriers are covered with a single layer, remove the supernatant, add 2.0 times the volume of the remaining liquid in the reactor to digest cell trypsin, digest and separate the microcarriers, and then harvest the cells and concentrate the cell solution.
[0070] Amplify the cells harvested in the 10-liter cell culture reactor into a 35-liter cell culture reactor, and the stirring speed is 60 rpm, add microcarriers, culture fluid, alkali and sugar, and cultivate for 50 hours. The monolayer is covered, the supernatant is removed, and trypsin of...
Embodiment 3
[0080] The MDCK cells were cultured in a roller bottle, and the primary MDCK cells were obtained after the monolayer was overgrown.
[0081] Five 15,000ml primary MDCK cells covered with a single layer were digested and used as seed cells in a 7-liter cell culture reactor for suspension culture, with a stirring speed of 50 rpm, adding microcarriers, culture medium, alkali and sugar, and culturing for 48 hours , when the cells on the microcarriers are covered with a monolayer, remove the supernatant, add trypsin to digest cells 1.5 times the volume of the remaining liquid in the reactor, digest and separate the microcarriers, and then harvest the cells and concentrate the cell solution.
[0082] Amplify the cells harvested in the 7-liter cell culture reactor into a 30-liter cell culture reactor, with a stirring speed of 50 rpm, add microcarriers, culture fluid, alkali and sugar, and cultivate for 48 hours, until the cells on the microcarriers The monolayer is covered, the super...
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