High-yield varicella virus culture method

A technique for varicella virus and a culture method, applied in the field of biomedicine, can solve problems such as low yield of varicella virus, achieve the effects of maintaining stability and oxygen supply, reducing impurities and improving safety

Inactive Publication Date: 2020-08-04
JIANGSU JINDIKE BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Aiming at the deficiencies of the prior art, the present invention provides a high-yield varicella virus culture method, which solves the problem of low varicella virus yield in the cell factory culture method

Method used

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  • High-yield varicella virus culture method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] S1. Cell resuscitation: MRC-5 cells from ATCC's working cell bank were resuscitated and passaged. The cell growth medium was M199-801 culture medium containing 10-20% newborn bovine serum, 37°C, 5% CO 2 Culture, after 3 to 4 days, the cells are covered with a single layer, ready to be passed on to the next generation;

[0027] S2. Cell subculture: Wash the cell surface twice with 0.01mol / L PBS solution, add an appropriate amount of pre-warmed EDTA-Trypsin digestion solution, and discard the digestion solution when there is a gap on the cell surface, add a small amount of cell culture solution, and pipette to make the cells Disperse evenly, passage at a ratio of 1:4 to 1:8, pass the cells to the cell factory for final passage, and add to 0.33ml / cm2 2 5-10% newborn bovine serum M199-801 culture solution, set at 37°C, 5% CO 2 nourish;

[0028] S3. Virus inoculation: when the confluence of the cell monolayer is 80-90%, take the varicella working seed batch to infect, and ...

Embodiment 2

[0032] S1. Cell resuscitation: MRC-5 cells from ATCC's working cell bank were resuscitated and passaged. The cell growth medium was M199-801 culture medium containing 10-20% newborn bovine serum, 37°C, 5% CO 2 Culture, after 3 to 4 days, the cells are covered with a single layer, ready to be passed on to the next generation;

[0033] S2. Cell subculture: Wash the cell surface twice with 0.01mol / L PBS solution, add an appropriate amount of pre-warmed EDTA-Trypsin digestion solution, and discard the digestion solution when there is a gap on the cell surface, add a small amount of cell culture solution, and pipette to make the cells Disperse evenly, passage at a ratio of 1:4 to 1:8, and finally passage the cells into the Corning HYPERStack culture chamber, add supplementation to 0.2ml / cm 25-10% newborn bovine serum M199-801 culture solution, set at 37°C, 5% CO 2 nourish;

[0034] S3. Virus inoculation: when the confluence of the cell monolayer is 80-90%, take the varicella work...

Embodiment 3

[0038] S1. Cell resuscitation: MRC-5 cells from ATCC's working cell bank were resuscitated and passaged. The cell growth medium was M199-801 culture medium containing 10-20% newborn bovine serum, 37°C, 5% CO 2 Culture, after 3 to 4 days, the cells are covered with a single layer, ready to be passed on to the next generation;

[0039] S2. Cell subculture: Wash the cell surface twice with 0.01mol / L PBS solution, add an appropriate amount of pre-warmed EDTA-Trypsin digestion solution, and discard the digestion solution when there is a gap on the cell surface, add a small amount of cell culture solution, and pipette to make the cells Disperse evenly, passage at a ratio of 1:4 to 1:8, and finally passage the cells to the Corning HYPERStack culture chamber and cell factory, and supplement the Corning HYPERStack culture chamber to 0.2ml / cm 2 5-10% newborn bovine serum M199-801 culture medium, added to the cell factory to 0.33ml / cm 2 5-10% newborn bovine serum M199-801 culture soluti...

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Abstract

The invention discloses a high-yield varicella virus culture method. The method specifically comprises the following steps of: S1, cell resuscitation: carrying out cell resuscitation passage on a working cell bank MRC-5 from ATCC, taking an M199-801 culture solution containing 10-20% of newborn calf serum as a cell growth solution, carrying out culture at 37 DEG C with 5% of CO2, and after 3-4 days, enabling cells to grow all over a single layer and preparing for passage of the next generation; S2, cell passage: washing the surfaces of the cells twice by using a 0.01 mol/L PBS (Phosphate Buffer Solution); adding a proper amount of a pre-heated EDTA-Trypsin digestive juice; observing a gap on a cell surface by naked eyes; removing digestive juice, adding a small amount of a cell culture fluid, blowing and beating to uniformly disperse cells, carrying out passage according to a ratio of 1: 4-1: 8, finally carrying out passage on the cells into a Corning HYPERStack culture room, supplementing a 5-10% newborn calf serum M199-801 culture solution with a concentration of 0.2-0.3 ml/cm<2>, and culturing at 37 DEG C with 5% CO2. The invention relates to the technical field of biological medicines. According to the high-yield varicella virus culture method, under the production scale, after the method is adopted, the production yield is increased by about 100%, and the production cost is reduced by about 40%-50%.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a high-yield varicella virus culture method. Background technique [0002] The live attenuated varicella vaccine is a freeze-dried virus product obtained by culturing and propagating varicella virus OKA strain in MRC-5 diploid cells. It is suitable for active immunization against chickenpox for healthy children over 12 months old, adolescents and adults, high-risk groups and their close contacts. The live attenuated herpes zoster vaccine is basically similar to the attenuated live varicella vaccine, and its concentration is about 10 times that of the varicella vaccine. [0003] The Food and Drug Administration strongly encourages and guides manufacturers to improve and ensure the quality of vaccines through disposable production technology. At present, the production of varicella virus vaccine generally produces virus by cultivating human diploid cells (MRC-5, 2BS cells), a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00C12R1/93
CPCC12N7/00C12N2710/16751
Inventor 杨骏宇余军李凡唐阳望朔杨文彬赵永强朱实惠
Owner JIANGSU JINDIKE BIOTECHNOLOGY CO LTD
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