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

Active Publication Date: 2022-04-26
宜明(苏州)细胞生物科技有限公司
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0011] HEK293T cells are anchorage-dependent and epithelial-like cells. The current main technology is to add serum in the cell culture process, and the cells grow on the wall according to the anchorage factors in the serum. However, it is well known that the use of serum brings various disadvantages, such as : Expensive, potential risk of pathogenic factors, difficulty in large-scale production, uncontrollable production process, etc.
[0012] However, the cells after serum-free acclimatization will change from adherent growth to suspension growth, but most of the suspension acclimated cells will still aggregate during the suspension culture process, which is not conducive to the transfection of plasmids and affects the production of viruses

Method used

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  • Rapid domestication method for HEK293T cell suspension and serum-free culture and application of rapid domestication method
  • Rapid domestication method for HEK293T cell suspension and serum-free culture and application of rapid domestication method
  • Rapid domestication method for HEK293T cell suspension and serum-free culture and application of rapid domestication method

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Experimental program
Comparison scheme
Effect test

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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Abstract

The invention provides a domestication method of HEK293T cells and application of the domestication method. The domestication method comprises a suspension and serum-free culture domestication method of the HEK293T cells, a cell line obtained by the domestication method and application of the cell line. The domestication method is few in passage times and high in domestication efficiency, and the obtained cells are high in viability, good in dispersity and stable in growth state and can meet the requirements of commercialized and large-scale production.

Description

technical field [0001] The invention relates to a HEK293T cell domestication method and application thereof, in particular to a rapid domestication method of HEK293T cell suspension and serum-free culture, a cell line obtained by the domestication method and its application, belonging to the field of biotechnology. Background technique [0002] With the development of science and technology, more and more gene therapy / cell therapy technologies are moving from the laboratory to the clinic, and from academic to commercial production. In 2017, two CAR-T drugs, Kymriah and Yescarta, were approved by the FDA for marketing. Treatment of relapsed or refractory pediatric and juvenile B-cell acute lymphoblastic leukemia (ALL), adult large B-cell lymphoma who has not responded or relapsed after at least two other lines of therapy, and certain types of non-Hodgkin Gold lymphoma patients. However, the two drugs are expensive, at US$475,000 and US$373,000, respectively. Even after bein...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12N15/867
CPCC12N5/0686C12N15/86C12N2500/90C12N2800/107C12N2740/15043C12N2740/15052Y02A50/30
Inventor 孙秀莲张玥张华
Owner 宜明(苏州)细胞生物科技有限公司
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