Method for synchronous suspension culture of mammalian cells in animal cell reactor
A mammalian and animal cell technology, applied in animal cells, vertebrate cells, biochemical equipment and methods, etc., can solve the problems of toxicity, easy-to-contaminate preparation, expensive chemical reagents, etc., and achieve simple operation and large-scale production. Effect
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Embodiment 1
[0023] Example 1. Effects of 4°C Low Temperature Stress on the Survival Rate of HEK293 Cells and Marc145 Cells for Different Time
[0024] HEK293 cells were cultured by suspension culture method, and Marc145 cells were cultured by microcarrier suspension culture method.
[0025] The method is as follows:
[0026] 1. Suspension culture of HEK293 cells
[0027] Take 3×10 5 The initial density of cells / ml was inoculated in a 250ml shake flask, and the medium composition of the suspension culture was shown in the table below:
[0028]
[0029]
[0030] The volume of the medium used is 60ml, and the shake flask is placed at 37°C, 5% CO 2 On the shaker in the incubator, the cultivation speed is 60-100rpm. On the 4th day, a certain amount of HEK293 cells were inoculated into new shake flasks for passage, the passage ratio was 1 pass 3, and the culture medium was supplemented to 60ml to continue shaking suspension culture.
[0031] 2. Microcarrier suspension culture of Marc...
Embodiment 2
[0034] Example 2. Effect of 4°C low temperature stress for 1 hour on the cell cycle distribution of suspension cultured HEK293 cells and the induction of synchronized growth
[0035] HEK293 cells cultured in suspension were treated with low temperature stress at 4°C for 1 hour, and then returned to 37°C for 0h, 6h, 12h, 18h and 24h, and samples were taken regularly for cell cycle determination. The method is as follows:
[0036] 1) Centrifuge at 1000rpm for 10min to collect 2-4×10 6 Add a certain amount of phosphate buffer (PBS buffer: 8g / L NaCl, 0.2g / L KCl, 1.44g / LNa 2 HPO 4 and 0.24g / L KH 2 PO 4 , pH7.2-7.4, sterilized at 121°C for 20 minutes before use) resuspend HEK293 cells, gently blow and wash the cells, then centrifuge at 1000rpm for 10 minutes, discard the supernatant;
[0037] 2) Add 1ml of pre-cooled 70% ethanol to the cell pellet and fix overnight at 4°C. The fixed cells can be stored at 4°C for one week;
[0038] 3) Centrifuge the fixed cells obtained in step...
Embodiment 34
[0044] Example 34 ℃ low temperature stress 1 hour on the cell cycle of HEK293 cells cultured in suspension and Marc145 cells cultured in microcarrier suspension
[0045] HEK293 cells and Marc145 cells cultured in microcarrier suspension culture in 7L animal cell reactor were cultured for 24 hours, and the culture temperature was set to 4°C. At this time, the temperature control unit began to cool down, and an ice pack was used as the cooling water unit Cool down with the tank body, quickly lower the culture temperature to 4°C, and keep it at 4°C for 1 hour. Then the culture temperature was set at 37°C and quickly returned to the normal culture temperature (37°C) to continue culturing for 18h (HEK293 cells) and 16h (Marc145 cells). This can effectively control the cell cycle at the G2 / M phase arrest effect, as shown in Figure 3 (1) and Figure 3 (2), at this time it can be observed that a large number of HEK293 cells are in the G2 / M phase (Fig. Arrow in the middle), while Marc1...
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