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A method for improving the formation rate of cell pools of stably transfected cell lines

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

Active Publication Date: 2021-03-30
WUXI BIOLOGICS CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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 cell pools of stably transfected cell lines. Under the condition of three-dimensional mixed medium, suitable cell seeding density and MSX pressurized concentration are provided, so that more positive cells can be obtained in a short time. High cell pool. The three-dimensional mixed medium is composed of three basic mediums CD Fusion, CHO Cloning Medium and GrowthA in proportion. The method provided by the invention can significantly increase the cloning rate of the cell pool, significantly reduce the screening cycle, and has good 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 Patents(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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