Large-scale suspension cultivation method of 293 cells
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A suspension culture, large-scale technology, applied in embryonic cells, tissue culture, animal cells, etc., to achieve the effect of improving utilization, easy manipulation, and tight connection
Active Publication Date: 2013-12-25
TIANJIN RINGPU BIO TECH
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[0003] In the prior art, experiments have confirmed that too many passages of cells have a significant impact on the growth of 293 cells in monolayer culture and the expression of adenovirus vectors, but for the growth of 293 cells in suspension culture and the expression of adenovirus vectors The expression level of 293 cells is indeed not affected, but there are few reports on the suspension culture process of 293 cells in the prior art
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Embodiment 1
[0013] 5L bioreactor: Produced by Biostat, low-shear paddle stirrer, spin filter (Spinfilter) and bubble-free ventilation system, and hollow fiber culture system (strengthening element); temperature, PH, stirring, dissolved oxygen PID automatic control , four-gas mixing control gas supply system, two-parameter correlation of oxygen fusion, flow rate of recovery liquid.
[0014] 250L bioreactor: Produced by Biostat, low-shear paddle stirrer, spin filter (Spinfilter) and bubble-free ventilation system, and hollow fiber culture system (strengthening element); temperature, PH, stirring, dissolved oxygen PID automatic control , four-gas mixing control gas supply system, two-parameter correlation of oxygen fusion, flow rate of recovery liquid.
[0015] Cell line: HEK-293sus cells, purchased from Shanghai Institute of Cells, Chinese Academy of Sciences; 293SFM II medium was purchased from the market.
[0016] 1.1 Acclimatization and culture of HEK-293 cells
[0017] 1.1.1 Full-susp...
Embodiment 2
[0045] 5L bioreactor: Produced by Biostat, low-shear paddle stirrer, spin filter (Spinfilter) and bubble-free ventilation system, and hollow fiber culture system (strengthening element); temperature, PH, stirring, dissolved oxygen PID automatic control , four-gas mixing control gas supply system, two-parameter correlation of oxygen fusion, flow rate of recovery liquid.
[0046] 250L bioreactor: Produced by Biostat, low-shear paddle stirrer, spin filter (Spinfilter) and bubble-free ventilation system, and hollow fiber culture system (strengthening element); temperature, PH, stirring, dissolved oxygen PID automatic control , four-gas mixing control gas supply system, two-parameter correlation of oxygen fusion, flow rate of recovery liquid.
[0047] Cell line: HEK-293sus cells, purchased from Shanghai Institute of Cells, Chinese Academy of Sciences; 293SFM II medium was purchased from the market.
[0048] 2.1 Acclimatization and culture of HEK-293 cells
[0049] 2.1.1 Full-susp...
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Abstract
The invention provides a large-scale suspension cultivation method of 293 cells. The method is characterized in that 293 cell seeds are subjected to passage amplification in a T-shaped tissue culture flask, then transferred into an agitating type cell culture flask to be subjected to further passage amplification and finally transferred into a 5L bioreactor to be cultivated; the cultivated cells are transferred and inoculated into a 250L bioreactor to be cultivated, wherein fresh culture mediums are supplemented at a fixed period during the cultivation so as to maintain a cultivation environment suitable for the growth of cells. According to the technical scheme, the large-scale suspension cultivation method of 293 cells fills a gap in 293 cell suspension cultivation technology; the selected cultivation condition is suitable for the growth of 293 cells; the cell cultivation density is maximized; the cell cultivation volume is reduced; the follow-up labor intensity is reduced; unique and stable quality is ensured; the production processes are closely joined and easy to operate and control. According to the large-scale suspension cultivation method of 293 cells, nutrition solutions are supplemented during cell cultivation and viral multiplication, so that nutrient substances are provided for cell multiplication as requirement, the utilization rate of the nutrition solutions is increased, and the cost is saved.
Description
technical field [0001] The invention relates to the technical field of cell culture, in particular to a method for large-scale suspension culture of 293 cells. Background technique [0002] 293 cells are transformed with type 5 adenovirus 75 strain, human embryonic kidney subtriploid cell line containing Ad5E1 region, and are a kind of complementary cell line with E1 region deficiency. It was constructed by F.L.Graham and J.S.Miley of McMaster University in Canada in 1976 using DNA transfection technology. 293 cells are anchorage-dependent and epithelial-like cells, exhibiting a typical phenotype of adenovirus-transformed cells, allowing Ad5 and other serotypes of adenoviruses to proliferate on them. 293 cells belong to the adherent cell line, in the absence of Ca 2+ or with Ca 2+ It can be grown in the same medium or in a medium with reduced serum concentration, [0003] In the prior art, experiments have confirmed that too many passages of cells have a significant impa...
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