Mdck-derived Cell Lines Adapted To Serum-free Culture And Suspension Culture And Method For Preparing Vaccine Virus Using The Cells
一种无血清培养基、悬浮培养的技术，应用在制备疫苗病毒领域，能够解决致瘤性危险、培养过程复杂等问题，达到极低致瘤性的效果
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Embodiment 1
[0034] Prepare the cell line derived from MDCK by serum-free culture and suspension culture
[0035] CCL-34 in MDCK cell line provided by ATCC. In T-25 flasks, in EMEM medium supplemented with 10% serum at 37 °C and 5% CO 2 CCL-34 was cultured under. After cell expansion, the cells were cultured in a medium consisting of EMEM medium and serum-free medium (50%). During the culture process, confirm whether the growth of the cells is normal. When normal cell growth was confirmed, the grown cells were cultured in a medium containing serum-free medium (75%). This process was repeated to obtain cell lines adapted to serum-free medium (100%). EX-CELL MDCK (Sigma), UltraMDCK (Lonza) and VP-SFM (Invitrogen) can be used as medium for serum-free culture.
[0036] Cell lines adapted to serum-free media were extensively expanded in T-flasks. Then, in a spinner flask (Corning) at 37 °C and 5% CO 2 The expanded cell line was adapted to suspension culture with agitation at a rate of 4...
Embodiment 2
[0037] Evaluation of Cell Line Proliferation Potential
[0038] The prepared MDCK-derived cell lines cultured in a serum-free medium were cultured under the conditions shown in Table 1. The proliferative potential of MDCK-derived cell lines was assessed. MDCK cell line (ATCC CCL-34) was cultured in medium supplemented with 10% serum as a negative control.
[0039] Table 1
[0040] [Table 1]
[0041] [surface]
[0042]
[0043] At the beginning of the culture, the cell culture concentration was about 1.0×10 5 cells / ml. When the cell concentration reaches about 1×10 6 cells / ml or after 3-4 days of culture, the cells were passaged. Adjust the cell concentration at the initial stage of subculture to 1×10 5 cells / ml.
[0044] Each of the MDCK-derived cell lines grown in serum-free media exhibited comparable cell growth rates to MDCK cells grown in serum media.
Embodiment 3
[0045] Evaluation of proliferation curve and passage stability of cell lines
[0046] After adaptation to serum-free culture and suspension culture, the three cell lines were further cultured in spinner flasks, and their proliferation curves and passage stability were evaluated. Adjust the concentration of cells at the initial stage of culture to approximately 4×10 5 cells / ml. After about 3-4 days of culture, the cell concentration reached about 2×10 6 cells / ml or more. Culture was performed under the following conditions. The result is as figure 1 shown.
[0047] Starting cell concentration: 4 x 10 5 cells / ml
[0048] Culture scale: 50ml spinner bottle
[0049] Spinner rotation rate: 60rpm
[0050] Culture conditions: 37°C, 5% CO 2 , wet
[0051] Subculture conditions: 3-4 days after culture
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