MDCK cell line, method for duplicating viruses and application of method
A cell line, virus technology, applied in the direction of viruses, animal cells, antiviral agents, etc., to achieve the effect of reducing the risk of foreign contamination
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Embodiment 1
[0037] This example provides a method for preparing MDCK cells with a deposit number of CCTCC NO: C201857, the steps are as follows:
[0038] Adherent cultured MDCK cells were introduced from ATCC by the Gansu Animal Cell Engineering Technology Research Center (GsACC) of Northwest University for Nationalities. The introduction time: February 2011, ATCC number: CCL-34, generation: P56, preservation number: 58860056 . A cell bank was established after GsACC expansion and culture, and the number of the cell bank is: GsACC2B0000090.
[0039] The MDCK cells were revived with DMEM / F12 medium containing 10% FBS, grown densely and then passaged.
[0040] Select well-grown cells and gradually domesticate and cultivate them, as follows:
[0041] Subcultured with DMEM / F12 medium containing 8% fetal bovine serum for 3 times;
[0042] Subcultured 5 times with DMEM / F12 medium containing 5% fetal bovine serum;
[0043] Subcultured 5 times with DMEM / F12 medium containing 2% fetal bovine s...
Embodiment 2
[0058] This example provides a method for culturing MDCK cells with a deposit number of CCTCC NO: C201857.
[0059] This experimental example provides the culture effect of the GJ-pyj201 medium provided by the present invention on MDCK cells with the deposit number CCTCC NO: C201857.
[0060] 1. Method
[0061] 1.1 Preparation of shake flask cells
[0062] Resuscitate the frozen suspended cells from liquid nitrogen according to the conventional method, add the cell solution into a 500ml Erlenmeyer flask with a pipette, and add filter-sterilized serum-free medium with a pH value of 7.0±0.2 to 100ml. Place the shaker flask in a shaker at 37°C, 120r / min-140r / min for cultivation and propagation. When the number of cells is sufficient, the reactor can be inoculated for culture.
[0063] 1.2 5L reactor cell culture
[0064] Take the suspended MDCK cells cultured in shake flasks, and dilute them with 1.0~2.0×10 6 The density of cells / ml is inoculated into a 5L reactor for cultur...
Embodiment 3
[0088] Cultivate the G01, G02, and G03 cells screened in Example 1 according to the culture conditions of 1.1 to 1.4 in Example 2. The medium used is 3# medium. The only difference is that in step 1.4, the dissolved oxygen is controlled at 25 -35%, thereby simulating the local anoxic environment in the bioreactor.
[0089] Samples were taken to observe the cell viability at 0h and 24h of culture.
[0090] Table 3 Cell viability of different MDCK cells in hypoxic environment
[0091]
[0092]
[0093] Among them, G01 cells obviously have stronger tolerance to hypoxic environment.
[0094] Cultivate the G01 cells screened in Example 1 according to the culture conditions of 1.1 to 1.4 in Example 2, the only difference is that in step 1.4, first use 3# medium to filter them into the corresponding reactor in a sterile manner Pre-incubation for 24 hours to make the initial cell viability in the three reactors consistent. Then during the formal cultivation, the dissolved oxy...
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