Method for suspension domestication of 293T cells

A cell, full suspension technology, applied in the field of medicine and biology, can solve the problems of contamination of exogenous microorganisms and pathogenic factors, difficulties in downstream purification processes, and biosafety risks, and achieves the goal of being conducive to separation and purification, easy to use, and simple and feasible to operate. Effect

Pending Publication Date: 2019-01-29
HRAIN BIOTECHNOLOGY CO LTD
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

293 cells belong to the adherent cell line, and can grow equally in Ca2+ or Ca2+-containing medium, and can also grow in the medium with reduced serum concentration. In the prior art, experiments have confirmed that the number of cell passages is too high for 293 cell monolayers. The growth of cells in culture has a significant effect
[0004] At present, when 293T cells are cultured in an industrialized monolayer, a certain proportion of bovine serum (including fetal bovine serum or newborn bovine serum) is added to the culture medium to allow cells to grow normally. Bovine serum is not only expensive, but also comes from natural organisms. There is a possibility of contamination of exogenous microorganisms and pathogenic factors in the process of carrying and collecting the cattle source itself, and there will be biosafety risks in the use
In addition, bovine serum is a natural mixture, and its specific components have not yet been fully analyzed. It will cause difficulties in the downstream purification process after being used in the production of biological products. If there is any residue, it will usually cause side reactions from the vaccinators and affect product quality.

Method used

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  • Method for suspension domestication of 293T cells
  • Method for suspension domestication of 293T cells

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Experimental program
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specific Embodiment

[0018] 1. Reagents used

[0019] 1) Cell line: 293T cell, purchased from ATCC cell bank;

[0020] 2) DMEM culture medium, Corning, article number: 10-013-CVR;

[0021] 3) Free style culture medium, Gibco, article number: 12338-018;

[0022] 4) Trypsin, Gibco, item number: 27250, prepared as 0.25% trypsin solution (EDTA-Na 0.3g / L);

[0023] 5) Anti clumping agent, Invitrogen, article number: 0010057AE.

[0024] 2. The equipment and utensils used

[0025] 1) Shaker, KUHNER, model: ISF1-XC;

[0026] 2) CO2 incubator, Thermo Fisher Scientific Corporation, model: 3111;

[0027] 3) Centrifuge, Eppendorf, model: 5810R;

[0028] 4) Phase contrast inverted microscope, OLYMPUS, OLYMPUS CKX41;

[0029] 5) Cell culture flasks (Corning, T25 article number: 430639, T75 article number: 430641);

[0030] 6) Conical cell flask 150ml, Haimen, Jiangsu, specification: 150ml.

Embodiment 1

[0031] Example 1. Cultivation of adherent culture type 293T cell line

[0032] Select from the company's cell bank, an adherent culture 293T cell that has been tested to be sterile, negative for mycoplasma, and foreign viruses, and cultured with 10% fetal bovine serum (the following serum concentrations are percentages by volume) DMEM, culture conditions: 37 ℃ 5% CO2 incubator culture. The viability of the cells was 98.6% after resuscitation. The cells were cultured for 48 hours to form a compact monolayer, and the cells were subcultured at a ratio of 1:4 to form a compact monolayer at 48 hours, with clear cell edges, flat morphology, and epithelial type.

Embodiment 2

[0033] Example 2. Domestication of low-serum adherent culture 293T cell line

[0034] a) The normal-growing adherent culture 293T cells described in step (1) were cultured in DMEM medium containing 10% fetal bovine serum for three generations each; the standard for each passage is to divide the bottle at a ratio of 1:4, Cultivate in 5% CO2, the cells will form a dense monolayer within 48-72h;

[0035] b) Change the culture medium to Free style medium and add 10% fetal bovine serum to continue culturing for three generations, the passage standard is the same as step a);

[0036] c) The fetal calf serum in the culture medium is reduced to 5%, and the passage ratio is reduced to 1:3 according to the method in step b, and then cultured for three generations;

[0037] d) The fetal bovine serum in the culture medium is reduced to 2%, and the volume percentage of 20% is added to the culture medium. The medium in which the cells have been cultured for 48-72 hours in the previous generation wi...

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Abstract

The invention provides a method for preparing a suspended 293T cell line cultured in low serum. The method includes the following steps: (1) resuscitating and cultivating mammalian cells to obtain theadherently cultured 293T cells, and digesting the 293T cells with trypsin; (2) culturing the adherently cultured 293T cells in a serum-free medium supplemented with a certain amount of serum, and continuously culturing the 293T cells to adapt to the serum-free medium supplemented with a certain amount of the serum; and (3) repeating the continuous cultivation procedure in the step (2) and gradually reducing serum addition amount in the serum-free medium until a serum concentration is 0 to obtain the suspension cultured 293T cell. The method of the invention has the advantages of simple operation, convenient use, low cost and good safety. An in vitro expansion culture method of 293T cell suspension culture requires no special equipment, and the operation is simple and feasible. The 293T cell suspension culture can has broad prospects in basic research and clinical applications.

Description

Technical field [0001] The invention relates to the field of medical biology, in particular to a method for culturing cells in suspension. Background technique [0002] In vitro cell culture methods include adherent culture and suspension culture, adherent culture (refers to the cultivation of cells attached to a certain solid surface; suspension culture uses shaking or rotating devices to keep the cells dispersed and suspended in the culture medium. Culture method; suspension culture can be divided into microcarrier suspension culture and full suspension culture. Compared with adherent culture, suspension culture has the following advantages: (1) Continuously expand production; (2) Conducive to cell culture The nutrient substance and gas are in full contact, and it is easy to control the culture conditions (temperature, pH, oxygen partial pressure, CO2, etc.); (3) The culture conditions are stable and tend to be uniform, which is convenient for quantitative research; (4) It is e...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/073C12N5/02
CPCC12N5/0603C12N5/0686
Inventor 黄飞金涛王海鹰何凤史子啸
Owner HRAIN BIOTECHNOLOGY CO LTD
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