Rapid domestication method for HEK293T cell suspension and serum-free culture and application of rapid domestication method
A technology of serum-free culture and serum-free medium, applied in the biological field, can solve problems such as uncontrollable production process, impact on virus yield, risk of pathogenic factors, etc., to achieve saving experimental consumables, high stability of growth state, and less passage times Effect
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Embodiment 1
[0085] Embodiment 1: HEK293TY cell line preparation method
[0086] 1. Method steps:
[0087] a. Take a 1ml cryopreserved HEK293T cell and recover it in a water bath at 37°C, add dropwise to 10ml DMEM solution containing 10% FBS;
[0088] b. Fully mix the solution in step a, centrifuge at 200rpm for 10 minutes, and discard the supernatant;
[0089] c. Resuspend and mix the precipitate obtained in step b with 10ml DMEM containing 5% FBS, and let stand for 20 minutes;
[0090] d. Transfer the cells in step c to a T25 cell culture flask for culture, and add 10 ml of DMEM basic medium with a final concentration of 0.75% FBS to each flask;
[0091] e. Put the T25 culture flask into a 37°C, 5% CO2 incubator and cultivate it for 3 days, until the suspension and adherent cells can be seen under the microscope at the same time, transfer the suspension cells in the T25 culture flask to a centrifuge tube;
[0092] f. Elute the adherent cells in the T25 culture flask with 1ml TrypLE an...
Embodiment 2
[0112] Embodiment 2: HEK293TY cell line preparation method
[0113] According to the acclimatization method of Example 1, the initial concentration and descending concentration of serum are adjusted, for example, the initial serum concentration is 12%, the second serum concentration is 7% (volume), and the third serum concentration is 3.5% (volume). For another example, the first serum concentration is 8% (volume), and the second serum concentration is 4% (volume). After multiple concentration drops, the serum concentration was greater than 1% by volume in the last processing step. In step (2), 0.5% or 0.25% low-concentration serum is used for culturing.
[0114] The performance of HEK293TY obtained by the above various serum starting and descending schemes is basically the same as that in Example 1, and it is suitable for culturing in serum-free medium, and the cells are suspended and dispersed, and cells with high viability and good dispersion are obtained.
Embodiment 3
[0115] Embodiment 3: HEK293TY is used for packaging virus
[0116] 1. Method steps:
[0117] 1. The HEK293TY cells obtained in the present invention were mixed according to 0.5~0.8×10 6 / ml inoculated cells for 2 days and prepared the cells for virus packaging
[0118] 2. Pack the GFP virus according to the PEI transfection method, collect the cell suspension 72 hours after transfection, centrifuge at 1500g for 5 minutes, collect the supernatant, and measure the virus titer.
[0119] 3. At the same time, the adherent 293T cells were used as a reference to package the GFP virus. The day before transfection, the cells were divided into 8×10 4 Cells / cm2 were inoculated. On the day of transfection, the GFP virus was also packaged according to the transfection method of PEI. After 72 hours, the cell supernatant was collected, centrifuged at 1500g for 5 minutes, and then the supernatant was collected to measure the virus titer.
[0120] 4. Use a commercial HEK293 suspension cell ...
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