Medium additive replacing animal serum in CHO cell culture and preparation method thereof

A medium additive and cell culture technology, applied in the field of cell engineering, can solve the problems of narrow range of applicable cell lines and high price, and achieve the effects of clear ingredients, low cost and easy preparation

Inactive Publication Date: 2014-12-17
INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the formula of the commercial serum-free medium has always been a secret core business secret. The high price and the narrow range of applicable cell lines limit its practical application to a certain extent.

Method used

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  • Medium additive replacing animal serum in CHO cell culture and preparation method thereof
  • Medium additive replacing animal serum in CHO cell culture and preparation method thereof
  • Medium additive replacing animal serum in CHO cell culture and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Example 1 Culturing K2-10-3 cells with F12 medium containing medium additives on a 24-well culture plate

[0064] K2-10-3 cells (contained from human tissue-type plasminogen) cultured in DMEM / F12 medium (Invitrogen, USA) containing 1% (v / v) newborn calf serum in T-25 culture flasks Activator target gene and expression vector of dihydrofolate reductase marker gene, transfected with dihydrofolate reductase deficient CHO cells from ATCC (American Type Culture Collection), established with stable expression of human tissue-type plasminogen activation CHO cell line) was digested with 0.25% (w / v) trypsin solution. Suspend K2-10-3 cells with F12 medium (Invitrogen, USA) and CD OPTICHO serum-free medium (Invitrogen, USA) and count the cells with Cedex AS-20 cell density and viability automatic analyzer (Innovatis, Germany) density. Press 5×10 4 Inoculate a 24-well culture plate with a cell density of cells / mL, with a culture volume of 2 mL per well, and inoculate one 24-well cul...

Embodiment 2

[0066] Example 2 Culturing K2-10-3 cells with DMEM medium containing medium additives on a 24-well culture plate

[0067] K2-10-3 cells cultured in DMEM / F12 medium containing 1% (v / v) newborn calf serum in T-25 flasks were digested with 0.25% (w / v) trypsin solution. K2-10-3 cells were suspended in DMEM medium and CD OPTICHO serum-free medium, respectively, and the cell density was counted with Cedex AS-20 cell density and viability automatic analyzer. Press 5×10 4 Inoculate a 24-well culture plate with a cell density of cells / mL, with a culture volume of 2 mL per well, and inoculate a 24-well culture plate for each medium; in each well of the inoculation with DMEM medium to suspend K2-10-3 cells, press Add 1% (v / v) of the culture volume to medium additive concentrates A, B and C. 37℃、5%CO 2 Cultivate for 2 days, use 0.25% (w / v) trypsin solution to digest 4 culture wells of K2-10-3 cells cultured in different media, and use Cedex AS-20 cell density and viability automatic analyze...

Embodiment 3

[0069] Example 3 Culturing K2-10-3 cells with DMEM / F12 medium containing medium additives on a 24-well culture plate

[0070] K2-10-3 cells cultured in DMEM / F12 medium containing 1% (v / v) newborn calf serum in T-25 flasks were digested with 0.25% (w / v) trypsin solution. K2-10-3 cells were suspended in DMEM / F12 medium and CD OPTICHO serum-free medium, respectively, and the cell density was counted with Cedex AS-20 cell density and viability automatic analyzer. Press 5×10 4 Inoculate a 24-well culture plate with a cell density of cells / mL, with a culture volume of 2 mL per well, inoculate one 24-well culture plate for each medium; suspend K2-10-3 cells in each well of the inoculation DMEM / F12 medium Add medium additive concentrates A, B, and C at 1% (v / v) of the culture volume. 37℃、5%CO 2 Cultivate for 2 days, use 0.25% (w / v) trypsin solution to digest 4 culture wells of K2-10-3 cells cultured in different media, and use Cedex AS-20 cell density and viability automatic analyzer C...

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Abstract

The invention discloses a medium additive replacing animal serum in CHO cell culture and a preparation method thereof. A formula of the medium additive comprises insulin, serotonin, putrescine, aurintricarboxylic acid, hydroxypropyl-beta-cyclodextrin, trehalose, amino acids, vitamins, microelements, and the like. According to properties of the components in the formula of the medium additive, a medium additive concentrate A, a medium additive concentrate B and a medium additive concentrate C are prepared separately, wherein each of the concentrates has a concentration 100 times of a using concentration. By adding the medium additive concentrate A, the medium additive concentrate B and the medium additive concentrate C individually according to a ratio of 1% (v / v) into a commercialized synthetic medium so that the animal serum can be replaced by the synthetic medium to support cell growth of CHO cells in static culture and suspension culture. The formula of the medium additive and the preparation method have advantages of (1) capability of supporting rapid growth of the CHO cells in static culture conditions and suspension culture conditions, (2) definite additive components and easy preparation, and (3) low cost, convenient preparation and using convenience.

Description

Technical field [0001] The invention relates to a medium additive formula for replacing animal serum in CHO cell culture and its preparation. More specifically, it is an in vitro culture of CHO cells, especially suspension culture of CHO cells, which is used to substitute animal serum for non-animal-derived medium additive formula and its preparation method, belonging to the technical field of cell engineering . Background technique [0002] Medium is the most direct and important environmental factor that affects the growth, metabolism and survival of cultured cells in vitro. According to whether animal cell culture needs to add animal serum to support the normal growth of cells in the specific implementation of animal cell culture, animal cell culture media can be divided into two categories: serum containing medium and serum free medium . Commercial synthetic media, including DMEM, F12, M199, RPMI 1640 and DMEM / F12, are basic media that consist of serum media and serum-free...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/071C12N5/02
Inventor 刘红陈昭烈叶玲玲李世崇王启伟杨振西
Owner INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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