Culture method of AD293 spherical cell cluster

A culture method and cell mass technology, which can be applied to embryonic cells, animal cells, vertebrate cells, etc., can solve the problems of human embryonic kidney cells AD293 cells, which are rarely reported, and achieve the effect of facilitating virus reproduction.

Active Publication Date: 2016-09-14
ZHAOQING DAHUANONG BIOLOGIC PHARMA +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the cells that can be cultured in suspension include BH K-21, SP20, MDCK and other cells; however, there are few reports on human embryonic kidney cell AD293 cells

Method used

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  • Culture method of AD293 spherical cell cluster
  • Culture method of AD293 spherical cell cluster
  • Culture method of AD293 spherical cell cluster

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063]This embodiment discloses a method for culturing AD293 spherical cell clusters, comprising the following steps:

[0064] 1) Prepare suspension medium: 100mL medium includes the following components:

[0065] 90mL Gibco serum-free medium,

[0066] 4mL Gibco fetal bovine serum,

[0067] 5mL Gibco Serum Replacement,

[0068] 1mL Gibco non-essential amino acid solution,

[0069] 300ng epidermal growth factor,

[0070] 200ng transforming growth factor,

[0071] 0.15 g methylcellulose;

[0072] Mix and dissolve the above substances evenly to obtain a suspension medium with a total volume of 100mL;

[0073] 2) Prepare AD293 cells: use DMEM medium containing 10% fetal bovine serum by volume to resuscitate AD293 cells in a cell flask, pass passage according to the volume ratio of 1:3, and then culture AD293 cells until the cell volume reaches 10 8 indivual;

[0074] 3) Cultivate cells: add AD293 cells treated in step 2) and suspension medium 45mL obtained in step 1) to th...

Embodiment 2

[0079] This embodiment discloses a method for culturing AD293 spherical cell clusters, comprising the following steps:

[0080] 1) Prepare suspension medium: 100mL medium includes the following components:

[0081] 90mL Gibco serum-free medium,

[0082] 4mL Gibco fetal bovine serum,

[0083] 5mL Gibco Serum Replacement,

[0084] 1mL Gibco non-essential amino acid solution,

[0085] 0.15 g methylcellulose;

[0086] Mix and dissolve the above substances evenly to obtain a suspension medium with a total volume of 100mL;

[0087] 2) Prepare AD293 cells: use DMEM medium containing 10% fetal bovine serum by volume to resuscitate AD293 cells in a cell flask, pass passage according to the volume ratio of 1:3, and then culture AD293 cells until the cell volume reaches 10 8 indivual;

[0088] 3) Cultivate cells: add AD293 cells treated in step 2) and suspension medium 45mL obtained in step 1) to the shake flask; then place it on a shaker and put it in a carbon dioxide incubator at...

Embodiment 3

[0093] 1) Prepare suspension medium: 100mL medium includes the following components:

[0094] 90mL Gibco serum-free medium,

[0095] 4mL Gibco fetal bovine serum,

[0096] 5mL Gibco Serum Replacement,

[0097] 1mL Gibco non-essential amino acid solution,

[0098] 0.15 g methylcellulose;

[0099] Mix and dissolve the above substances evenly to obtain a suspension medium with a total volume of 100mL;

[0100] 2) Prepare AD293 cells: use DMEM medium containing 10% fetal bovine serum by volume to resuscitate AD293 cells in a cell flask, pass passage according to the volume ratio of 1:3, and then culture AD293 cells until the cell volume reaches 10 8 indivual;

[0101] 3) Cultivate cells: add AD293 cells treated in step 2) and suspension medium 45mL obtained in step 1) into the shake flask; then place it on a shaker and put it in a carbon dioxide incubator at a temperature of 37°C and a rotation speed of 70rpm After culturing for 24 hours under the condition of Figure 7 As ...

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Abstract

The invention discloses a culture method of AD293 spherical cell cluster. The culture method comprises the following steps: (1) preparing a suspension culture medium: preparing the following components: a serum-free culture medium, fetal calf serum, a serum replacement, a non-essential amino acid solution, epidermal growth factor, transforming growth factor, and methyl cellulose, and evenly mixing and dissolving the components mentioned above to obtain a suspension culture medium; (2) preparing AD293 cells: using a DMEM culture medium with 10% of fetal calf serum to revive AD293 cells in a cell culture flask, and carrying out cell passage; (3) culturing the cells: adding AD293 cells obtained in the step (2) and suspension culture medium obtained in the step (1) into a shake flask, then placing the shake flask on a shaking bed, and culturing the cells for 48 to 72 hours at a temperature of 37 DEG C to obtain AD293 spherical cell clusters. Compared with the cells cultured by spherical suspension micro carriers, the cell density of obtained AD293 cell clusters is increased by 10 times or more.

Description

technical field [0001] The invention relates to the technical field of cell culture, more specifically, to a method for culturing AD293 spherical cell clusters. Background technique [0002] Cell culture is an essential process for the entire bioengineering technology, especially in the research of cell seedlings and drug screening. Traditional cell culture is to culture cells through spinner bottles or flat bottles, and then develop into bioreactors that use sheet carriers or spherical microcarriers to culture cells at high density, or culture cells through hollow fibers. There are some defects in these methods of cell culture, for example: the cell density of spinner bottle or flat bottle culture cells is low, and the average density is only 10 5 cells / ml level; and bioreactor or hollow fiber cell density can reach 10 6 per milliliter level, even higher, but with high cost and difficult expansion. At present, through methods such as cell genetic modification or domestic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/073
Inventor 孙贝贝陈瑞爱徐家华王新秋谢小雨
Owner ZHAOQING DAHUANONG BIOLOGIC PHARMA
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