Additive for in-vitro amplification of human embryonic stem cells and amplification method

A technology for human embryonic stem cells and in vitro amplification, applied in the field of biopharmaceuticals, can solve the problems of pollution, easy to be contaminated by miscellaneous bacteria, difficult to separate, etc., and achieve the effects of improving cell expansion efficiency, facilitating mass production, and improving proliferation efficiency.

Pending Publication Date: 2021-08-31
郑州优倍得生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, in the field of human embryonic stem cell culture, there have always been problems such as high cost, unstable in vitro culture, easy to be contaminated by bacteria, etc., and the quality and quantity of embryonic stem cells cannot be guaranteed, which has caused obstacles to the clinical application of embryonic stem cells.
[0004] The traditional hESCs culture method needs to place hESCs on mitomycin-treated feeder cells (such as embryoni

Method used

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  • Additive for in-vitro amplification of human embryonic stem cells and amplification method

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Embodiment 1

[0023] An additive for in vitro expansion of human embryonic stem cells, which is used to add to the high-sugar DMEM medium for in vitro expansion of embryonic stem cells. In terms of the final concentration of the medium, the concentration of each component in the additive is: brassinolide 2.0 ng / mL, sodium mercaptosuccinate 3.5ng / mL, stigmasterol 15.8ng / mL, gentiopicroside 13.0 ng / mL, kanamycin 8ng / mL, L-glutamine 16ng / mL.

[0024] A method for expanding human embryonic stem cells in vitro, comprising the following steps:

[0025] (1) Human embryonic stem cells isolated in vitro were inoculated in high-glucose DMEM medium, and pre-cultured in a culture dish coated with fibronectin for 24 hours;

[0026] (2) Take the high-sugar DMEM medium and add the above additives to obtain the medium for expansion, inoculate the human embryonic stem cells isolated in vitro in step (1) in the medium for expansion, and the inoculation density is 7×10 4 cells / mL, at 37℃, 5%CO 2 Cultivate f...

Embodiment 2

[0028] An additive for the in vitro expansion of human embryonic stem cells, which is used to add to the high-sugar DMEM medium for the in vitro expansion of embryonic stem cells. In terms of the final concentration of the medium, the concentration of each component in the additive is: brassinolide 1.5 ng / mL, sodium mercaptosuccinate 2.8ng / mL, stigmasterol 13.3ng / mL, gentiopicroside 10.0ng / mL, kanamycin 5ng / mL, L-glutamine 15ng / mL.

[0029] A method for expanding human embryonic stem cells in vitro, comprising the following steps:

[0030] (1) Human embryonic stem cells isolated in vitro were inoculated in high-glucose DMEM medium, and pre-cultured in a culture dish coated with fibronectin for 24 hours;

[0031] (2) Take the high-sugar DMEM medium and add the above additives to obtain the medium for expansion, inoculate the human embryonic stem cells isolated in vitro in step (1) in the medium for expansion, and the inoculation density is 5×10 4 cells / mL, at 37℃, 5%CO 2 Cult...

Embodiment 3

[0033] An additive for in vitro expansion of human embryonic stem cells, which is used to add to high-sugar DMEM medium for in vitro expansion of embryonic stem cells. In terms of the final concentration of the medium, the concentration of each component in the additive is: brassinolide 4.5 ng / mL, sodium mercaptosuccinate 5.0ng / mL, stigmasterol 17.5ng / mL, gentiopicroside 14.5ng / mL, kanamycin 10ng / mL, L-glutamine 20ng / mL.

[0034] A method for expanding human embryonic stem cells in vitro, comprising the following steps:

[0035] (1) Human embryonic stem cells isolated in vitro were inoculated in high-glucose DMEM medium, and pre-cultured in a culture dish coated with fibronectin for 24 hours;

[0036] (2) Take the high-sugar DMEM medium and add the above additives to obtain the medium for expansion, inoculate the human embryonic stem cells isolated in vitro in step (1) in the medium for expansion, and the inoculation density is 8×10 4 cells / mL, at 37℃, 5%CO 2 Cultivate for 7...

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Abstract

The invention discloses an additive for in-vitro amplification of human embryonic stem cells. The additive is used for being added into a basal culture medium for in-vitro amplification of embryonic stem cells and comprises the following components: brassinolide, sodium mercaptosuccinate, stigmasterol, gentiopicroside, kanamycin and L-glutamine. The brassinolide, the sodium mercaptosuccinate, the stigmasterol and the gentiopicroside in the additive have a synergistic effect, so that the proliferation of the embryonic stem cells is remarkably promoted, the proliferation period is shortened, and the proliferation efficiency is improved. When the additive and the basic culture medium are used for performing in-vitro amplification on the embryonic stem cells, a large number of high-quality human embryonic stem cells can be obtained within a short time, animal serum is not needed, the safety is high, and scientific research and clinical requirements are fully met. The invention also provides a human embryonic stem cell in-vitro amplification method, the additive is added into the basic culture medium for cell amplification, the cell amplification efficiency is improved, the operation is simple and convenient, and the batch production of the human embryonic stem cells is facilitated.

Description

technical field [0001] The invention relates to the technical field of biopharmaceuticals, in particular to an additive for in vitro expansion of human embryonic stem cells and an expansion method. Background technique [0002] Human embryonic stem cells (hESCs) are pluripotent cells derived from the inner cell mass of blastocysts fertilized in vitro. hESCs have the ability to differentiate into ectoderm, endoderm and mesoderm, such as cardiomyocytes, neurons, glial cells, hepatocytes, islet cells, chondrocytes, skeletal muscle cells, adipocytes and endothelial cells. Therefore, embryonic stem cells can provide an ideal biological platform for scientific research and clinical applications, such as drug screening, research on the molecular regulation mechanism of cell development and differentiation, establishment of tissue engineering seed cells and gene therapy vectors, cell therapy and regeneration Medicine etc. [0003] With the deepening of research, people's understan...

Claims

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Application Information

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IPC IPC(8): C12N5/0735
CPCC12N5/0606C12N2500/32C12N2501/999C12N2533/52
Inventor 陈三飞陈友林
Owner 郑州优倍得生物科技有限公司
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