Method for producing porcine circovirus type 2(PCV2) through cell pure suspension technology
A porcine circovirus and cell technology, applied in the field of cell engineering, can solve the problems of small culture system, insufficient to meet industrialization requirements, etc., and achieve the effect of reducing purification cost, saving raw material cost, and improving commercial value
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Embodiment 1
[0028] Example 1 PK15 cell suspension culture
[0029] Cells are first activated in shake flasks, and then undergo step-by-step expansion culture in bioreactors. The expansion route is as follows: figure 1 shown. Specifically, proceed in the following steps:
[0030] 1. Cell Preparation
[0031] The revived PK15 cells were placed in three 250mL shake flasks, cultured at 37°C, 5% CO 2 , 130rpm, count the cells after 3-4 days of culture, and determine the passage ratio according to the counting results. After passage, the initial cell density should be maintained at (0.8-1)×10 6 cells / mL.
[0032] 2. PK15 cells scale up in 1L bioreactor
[0033] Adjust cell density to 8 x 10 5 cells / mL, transferred to a 1L bioreactor for cultivation, the culture volume is 800mL, the culture conditions are: dissolved oxygen 40%, pH 7.2, rotation speed 130rpm, temperature 37°C, ventilation control at 0.8-1.0L / h. After 72 hours of culture, the cell density increased to 3.2×10 6 cells / mL, ce...
Embodiment 2
[0040] Example 2 Cell inoculation and subculture harvest
[0041] The PCV2 cytotoxicity was inoculated into the bioreactor according to 0.02MOI (MOI, multiplicity of infection, multiple of infection), and the PK15 cells were passaged every 72 hours after inoculation, that is, after the cell viability and density were measured by sampling, the density after dilution of the cells would be satisfied. 1×10 6 After the cell volume of cells / mL, the excess cells and medium mixture were harvested, and fresh medium was added to a cell density of 1×10 6 cells / mL, continue to culture for 72 hours, repeat the above operations until the cell viability measured by a certain sampled cell is <70%, then stop the culture, and harvest all the cells and the medium together.
[0042] In order to reflect the effect of the technical solution, an experimental example is provided below for further explanation.
experiment example 185
[0043] Determination of cell density, viability and PCV2 titer when subcultured and harvested in Experimental Example 1 85L bioreactor
[0044] 1. Method
[0045] 1.1 Cell culture
[0046] Proceed according to Example 1.
[0047] 1.2 Cell inoculation
[0048] Proceed according to Example 2.
[0049] 1.3 Sampling
[0050] After inoculation, the cultures were sampled. The sampling time points are: 48 hours (F1-48h), 72 hours (F1-72h) after inoculation, 24 hours (F2-24h), 48 hours (F2-48h), 72 hours (F2-72h) of the first passage , 24 hours after the second passage (F3-24h).
[0051] 1.4 Cell density and viability detection
[0052] The cell density and viability of the obtained samples were detected by Countstar's cell counter (model BioTech IC1000). The specific detection method is as follows:
[0053] After mixing the harvested samples with a pipette gun, take 20 μl and the same volume of 0.02% trypan blue for 1:1 mixing. After mixing, take 20 μl and evenly add it to the...
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