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Method for in-vitro culture of HEK293 cells for transient expression of protein

A technology of transient expression and in vitro culture, applied in the field of cell culture, which can solve problems such as unfavorable enterprises, high price, and cost reduction

Inactive Publication Date: 2020-10-27
SHANGHAI OPM BIOSCI CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, although transient expression is fast, simple, and easy to operate, the protein yield is often not ideal
Currently, there is a lack of a serum-free HEK293 medium in the market for high-efficiency transient expression of proteins in vitro
In addition, the current HEK293 medium market is occupied by internationally renowned mediums such as SIGMA, Invitrogen, Hyclone, and Lonza. The price is expensive, as high as thousands of yuan per liter, which is not conducive to reducing costs for enterprises.

Method used

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  • Method for in-vitro culture of HEK293 cells for transient expression of protein
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  • Method for in-vitro culture of HEK293 cells for transient expression of protein

Examples

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Effect test

Embodiment 1

[0046] A method for culturing HEK293 in vitro of the present invention for transiently expressing protein CD20 monoclonal antibody comprises the following steps:

[0047] S1: Take the same batch of cryopreserved Expi293 cells (the original Expi293 cells were purchased from Gibco, the number of cells was 2.0×10 7 ) were revived in the original medium CD 293. Cells were placed in a 37°C incubator (120 rpm, 8% CO 2 ) to prepare for transfection and expression of target protein CD20 monoclonal antibody.

[0048] S2: When the viability of the cells is ≥95% and the growth is in the mid-log phase, the acclimatization procedure is started. In daily passage, cells are directly inoculated into HEK293 medium for culture.

[0049] S3: Subculture every 2-3 days to keep the cells in the early logarithmic growth phase. Cell seeding density is 0.3~0.6×10 6 cells / mL.

[0050] S4: When the cell density reaches 3-4×10 6 cells / mL and cell viability ≥ 95% (2-4 days), subculture the cells ag...

Embodiment 2

[0063] The difference between Example 2 and Example 1 is that the operation steps of Example 2 and Example 1 are the same, the difference is that the protein transfected and expressed in Example 2 is VEGF monoclonal antibody, and the serum-free medium formula of HEK293 cells is shown in Table 2. Show:

[0064]

[0065]

Embodiment 3

[0067] The difference between Example 3 and Example 1 is that the operation steps of Example 3 and Example 1 are the same, and the protein transfected and expressed in Example 3 is TNF-α monoclonal antibody.

[0068] The formulation of serum-free medium for HEK293 cells is shown in Table 3:

[0069]

[0070]

[0071]

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Abstract

The invention discloses a method for in-vitro culture of HEK293 for transient expression of protein. The method comprises the following steps: S1, resuscitating cryopreserved HEK293 cells in an original culture medium, and incubating the cryopreserved HEK293 cells in an incubator; S2, inoculating the cells into an HEK293 serum-free culture medium for culturing; S3, carrying out passage operation;S4, carrying out passage operation again; S5, preparing transfection plasmids; S6, acquiring data including cell density and survival rate, and diluting the cells; S7, diluting plasmid DNA; S8, diluting a transfection reagent; S9, adding the diluted transfection reagent into the diluted plasmid DNA, and incubating the mixture at room temperature; S10, slowly adding the mixture into the cells in the step S6, and performing shaking; and S11, carrying out culturing in an incubator, and harvesting the HEK293 cells and expression products. According to the invention, the expression quantity can beobviously increased, the expression efficiency is high, and more protein can be obtained in a shorter time.

Description

technical field [0001] The present invention relates to the technical field of cell culture, and more specifically relates to a method for culturing transiently expressed proteins in vitro using a serum-free HEK293 medium. Background technique [0002] HEK293 cells are an important platform for antibody and recombinant protein expression. According to the time of expression, gene product expression can be divided into transient expression and stable expression. Stable expression is the gold standard for antibody drug production systems, but the process is time-consuming and expensive, which is not conducive to high-throughput and high-efficiency screening of new antibody drugs. Transient gene expression technology means that after the exogenous gene enters the recipient cell, it exists on the free carrier and does not integrate into the chromosome. The expression product of the target gene can be obtained in a relatively short period of time. However, as the cell divides an...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12N5/073C12P21/00C07K16/00
CPCC07K16/00C12N5/0603C12N5/0686C12P21/00
Inventor 肖志华
Owner SHANGHAI OPM BIOSCI CO LTD
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