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293T cell culture medium for preparing retroviral vector and preparation method of 293T cell culture medium

A culture medium and lentivirus technology, applied in biochemical equipment and methods, culture process, tissue culture, etc., can solve problems such as hindering normal nutrient metabolism, affecting stable cell growth, lack of carbon source, etc., to achieve stable proliferation and purity Good, the effect of improving packaging efficiency

Inactive Publication Date: 2020-02-18
无锡生基医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The conventional medium for 293T cells is prepared with DMEM medium as the base solution and added with fetal calf serum. It lacks some key components: such as sodium pyruvate, non-essential amino acids, GlutaMax-Ⅰ, etc., which leads to the lack of nutrients in the metabolism of cells. Carbon source, which hinders normal nutrient metabolism; at the same time, the L-glutamine contained in it is easily degraded to form the accumulation of ammonia, which affects the stable growth of cells

Method used

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  • 293T cell culture medium for preparing retroviral vector and preparation method of 293T cell culture medium

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Effect test

Embodiment 1

[0030] Embodiment 1: the preparation of optimum formula 293T cell culture medium

[0031] 1) Weigh 3.50g of D-(+)-galactose, add 500mL of DMEM medium as the base solution to dissolve other components;

[0032] 2) After the D-(+)-galactose is dissolved, add 100mL fetal bovine serum, 0.90mM sodium pyruvate, 11.00mL non-essential amino acids, 2.54g Hepes and 1.50mM GlutaMax-I, mix and dissolve;

[0033] 3) Add DMEM medium to the mixed solution prepared in step 2) to make the volume to 1L, and record the pH value;

[0034] 4) Filter with a 0.22µm capsule filter (Millipore-SGEPA47HH3) to obtain sterile 293T cell culture medium.

[0035] The above-mentioned D-(+)-galactose, DMEM medium, fetal bovine serum, sodium pyruvate, GlutaMax-I and other components are examples of commercially available products, but are not limited to the above-mentioned products, and can be in accordance with the provisions of the Pharmacopoeia.

Embodiment 2

[0036] Embodiment 2: Preparation of 293T cell culture medium

[0037] 1) Weigh 3g of D-(+)-galactose, add 500mL of DMEM medium as the base solution to dissolve other components;

[0038] 2) After D-(+)-galactose is dissolved, add 80mL fetal bovine serum, 0.70mM sodium pyruvate, 7.00mL non-essential amino acids, 1.5g Hepes and 2.0mM GlutaMax-I, mix and dissolve;

[0039] 3) Add DMEM medium to the mixed solution prepared in step 2) to make the volume to 1L, and record the pH value;

[0040] 4) Filter with a 0.22µm capsule filter (Millipore-SGEPA47HH3) to obtain sterile 293T cell culture medium.

[0041] The above-mentioned D-(+)-galactose, DMEM medium, fetal bovine serum, sodium pyruvate, GlutaMax-I and other components are examples of commercially available products, but are not limited to the above-mentioned products, and can be in accordance with the provisions of the Pharmacopoeia.

Embodiment 3

[0042] Embodiment 3: Preparation of 293T cell culture medium

[0043]1) Weigh 6g of D-(+)-galactose, add 500mL of DMEM medium as the base solution to dissolve other components;

[0044] 2) After the D-(+)-galactose is dissolved, add 110mL fetal bovine serum, 1.1mM sodium pyruvate, 12.00mL non-essential amino acids, 3.0g Hepes and 3.0mM GlutaMax-Ⅰ, mix and dissolve;

[0045] 3) Add DMEM medium to the mixed solution prepared in step 2) to make the volume to 1L, and record the pH value;

[0046] 4) Filter with a 0.22µm capsule filter (Millipore-SGEPA47HH3) to obtain sterile 293T cell culture medium.

[0047] The above-mentioned D-(+)-galactose, DMEM medium, fetal bovine serum, sodium pyruvate, GlutaMax-I and other components are examples of commercially available products, but are not limited to the above-mentioned products, and can be in accordance with the provisions of the Pharmacopoeia.

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Abstract

The invention discloses a 293T cell culture medium for preparing a retroviral vector. A DMEM culture medium is used as basic liquid, fetal bovine serum, GlutaMax-I, non-essential amino acids, sodium pyruvate, D-(+)-galactose and Hepes are added to prepare the retroviral vector. The invention further discloses a preparation method of the cell culture medium. The preparation method comprises the following steps of (1) adding the D-(+)-galactose to the DMEM culture medium; (2) enabling the D-(+)-galactose to dissolve completely, sequentially adding the fetal bovine serum, the sodium pyruvate, thenon-essential amino acids, the Hepes and the GlutaMax-I, and performing mixing and dissolving; (3) adding the DMEM culture medium to the mixed liquid prepared in the step (2) until the volume is 1L;and (4) performing filtering to obtain a sterile 293T cell culture medium. Compared with a conventional culture medium, the culture medium disclosed by the invention is favorable for realization of stable proliferation of cells, the packing efficiency of viruses is improved, the virus titer is high, the purity is good, and the 293T cell culture medium is suitable for business batch production.

Description

technical field [0001] The invention relates to the field of virus biotechnology, in particular to a cell culture medium, in particular to a 293T cell culture medium for lentiviral vector preparation. In addition, the present invention also relates to a preparation method of the 293T cell culture medium for the preparation of the lentiviral vector. Background technique [0002] With the development of the immune cell therapy industry, CAR-T cell therapy has become a new international research hotspot in the field of tumor immunotherapy, favored by more and more biological companies and research institutions. 293T cells, as host cells for lentiviral packaging, are widely used in the preparation of lentiviral vectors for CAR-T technology. The cells are a human renal epithelial cell line derived from 293 cells and expressing the SV40 large T antigen. The conventional medium for 293T cells is prepared with DMEM medium as the base solution and added with fetal calf serum. It la...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
CPCC12N5/0686C12N2500/30C12N2500/32C12N2500/34C12N2500/84
Inventor 姚树元刘丹郭伟顾佩佩徐燕章贇马佳琳陈敏
Owner 无锡生基医药科技有限公司
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