Method of preparing foot-and-mouth disease vaccine by using BHK-21 adherent cells
A technology for BHK-21 and foot-and-mouth disease vaccines, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problems of increasing cultivation difficulties, pollution, consumption, large culture medium, etc., to improve the quantity and quality of antigens, Simplify the process flow and the effect of simple process flow
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Embodiment 1
[0069] I. Cell Recovery and Primary Amplification
[0070] (1) Take out the BHK-21 cells frozen in liquid nitrogen, place them in a water bath at 37°C for recovery, and inoculate them into a 20ml square bottle (T-shaped bottle) containing 15% fetal bovine serum in DMEM medium, at 37 ℃, through 5% CO 2 for 2 days in a carbon dioxide incubator.
[0071] (2) The cell suspension obtained by cultivating in step (1) is digested by trypsin, and evenly distributed in 5 T-shaped bottles, and each bottle is supplemented with DMEM medium containing 10% fetal bovine serum to a total volume of 20ml , and continue to culture for 3 days by the conditions in (1).
[0072] (3) All the cells obtained after the culture in step (2) were digested with trypsin and collected.
[0073] (4) Cell count and viability were checked by trypan blue staining.
[0074] (5) The cell suspension obtained by cultivating in step (3) is digested by trypsin, and evenly distributed in 25 T-shaped bottles, and eac...
Embodiment 2
[0093] I. Cell Recovery and Primary Amplification
[0094] Same as described in Example 1.
[0095] Second, scale up the culture of cells
[0096] i. Debug and prepare the bioreactor according to the equipment instructions, perform necessary cleaning and sterilization steps, and conduct sterility tests to confirm that the bioreactor is in good production condition. Soak and sterilize the selected Cytodex1 microcarriers.
[0097] ii. DMEM medium was used as the base medium, and fetal calf serum was added to make the concentration 8%, which was used as the culture medium used in the scale-up culture. The culture fluid is injected into the bioreactor, and the microcarriers are added. Taking production in a 40L bioreactor as an example, inject 20 liters of medium, and add 750 grams of pretreated Cytodex1 microcarriers at a concentration of 25 grams per liter.
[0098] iii. Inoculate the cell suspension obtained as described in step 1 into a bioreactor, taking the production in...
Embodiment 3
[0108] I. Cell Recovery and Primary Amplification
[0109] (1) Take out the BHK-21 cells frozen in liquid nitrogen, place them in a water bath at 37°C for recovery, and inoculate them into a 20ml square bottle (T-shaped bottle) containing 15% fetal bovine serum in DMEM medium, at 37 ℃, through 5% CO 2 for 2 days in a carbon dioxide incubator.
[0110] (2) The cell suspension obtained by cultivating in step (1) is digested by trypsin, and evenly distributed in 5 T-shaped bottles, and each bottle is supplemented with DMEM medium containing 10% fetal bovine serum to a total volume of 20ml , and continue to culture for 3 days by the conditions in (1).
[0111] (3) All the cells obtained after the culture in step (2) were digested with trypsin and collected.
[0112] (4) Cell count and viability were checked by trypan blue staining.
[0113] (5) The cell suspension obtained by cultivating in step (3) is digested by trypsin, and evenly distributed in 25 T-shaped bottles, and eac...
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