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Method for improving formation rate of cell pool of stably transfected cell strain

A cell line and cell technology, applied in the field of cell culture, can solve problems such as inconvenient operation

Active Publication Date: 2021-01-15
WUXI BIOLOGICS CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

However, semi-solid medium is used in this screening step, which is inconvenient to operate when mixed with cell suspension, and needs to be further improved.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Embodiment 1 A kind of cultivation method of stably transfected cell line

[0046] In this example:

[0047] EX-CELL® CD CHO Fusion was purchased from Sigma, the article number is 24365C;

[0048] ExcellCHO Cloning Medium was purchased from Sigma, the article number is C6366;

[0049] BalanCD CHO Growth A was purchased from Irvine, Cat. No. 91128.

[0050] The empty vector of the plasmid to be transfected is pcGS3 from Merck; the plasmid to be transfected expresses the Fc fusion protein.

[0051] (1) Cell preparation

[0052] The day before electroporation, the host cell CHOZN® CHO K1 cells (purchased from SAFC) were sampled and counted, and 0.8×10 6 The cells / mL cell density was inoculated in shake flasks.

[0053] (2) Electrostaining

[0054]I. After counting the host cells in the logarithmic growth phase in step (1), count them with 1×10 7 cells / transfection calculates the cell volume required for each electroporation reaction. Centrifuge the cell suspension ...

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PUM

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Abstract

The invention provides a method for improving the formation rate of a cell pool of a stably transfected cell strain. The method is used for providing proper cell inoculation density and MSX pressurized concentration under the condition of a three-dimensional mixed culture medium, so that relatively more cell pools with high positive rate can be obtained in a short time. The three-dimensional mixedculture medium is prepared from three basic culture media, namely CD Fusion, CHO Cloning Medium and Growth A in proportion. The method provided by the invention can significantly improve the clone rate of the cell pool and significantly reduce the screening period, and has relatively high practical application significance.

Description

technical field [0001] The invention belongs to the technical field of cell culture, and in particular relates to a method for increasing the formation rate of cell pools of stable cell lines. Background technique [0002] With the development of biotechnology, the expression and production of antibodies and vaccines occupy an important position in biopharmaceuticals. Cell line screening can obtain stable and high-yielding cell lines. The construction of stable transfected cell lines is one of the important means of expressing antibody vaccines. The current cell line screening process mainly includes: construction of expression plasmids, host cell transfection, cell pool pressurization screening, monoclonal screening, and obtaining high-expression cell lines. [0003] Merck's CHO-K1 expression system adds glutamate synthetase (glutaminesythetase, GS) inhibitor aminosulfomethionine (methionine sulfoximine, MSX) during cell selection, so that the GS gene and the target gene co...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/85
CPCC12N5/0682C12N15/85C12N2510/02C12N2513/00
Inventor 刘晓旺陶佳林董艳秋周义千王永忠
Owner WUXI BIOLOGICS CO LTD
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