Efficient separation and amplification method for human amniotic mesenchymal stem cells

A technology of amniotic mesenchymal and mesenchymal stem cells, applied in cell dissociation methods, embryonic cells, animal cells, etc., can solve the problems of incomplete separation of amniotic membrane epithelial cells, cumbersome operation, high cost, etc., and achieve convenient research and application, safety Good, simple operation effect

Inactive Publication Date: 2019-02-12
陕西九州细胞基因工程有限公司
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Problems solved by technology

The structure of amniotic membrane tissue is complex, and simple enzymatic hydrolysis cannot separate amniotic membrane mesenchymal stem cells from amniotic membrane epithelial cells, and it is difficult to obtain high-purity mesenchymal stem cell lines. The separation of mesenchymal stem cells is easy to cause pollution, and the separation of amnion epithelial cells is not complete; there is also the use of tissue separators to obtain amnion tissue homogenate, and then perform enzyme digestion, which is relatively cumbersome and expensive

Method used

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  • Efficient separation and amplification method for human amniotic mesenchymal stem cells
  • Efficient separation and amplification method for human amniotic mesenchymal stem cells

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Embodiment 1

[0050] A method for the efficient separation and expansion of human amniotic mesenchymal stem cells,

[0051] 1. Reagents

[0052] 1. Placenta preservation solution and cleaning solution

[0053] Penicillin for injection and streptomycin sulfate for injection are prepared with physiological saline for injection into a stock solution of 10,000 units per milliliter, which is 1% double antibody stock solution. 500mL of normal saline for injection is added to 5mL of double antibody storage solution, and then 2mL of gentamicin sulfate for injection is added to form a placenta preservation solution or cleaning solution;

[0054] 2. Trypsin stock solution

[0055] Weigh 0.5 g of trypsin, dissolve it in 100 mL of normal saline, and filter through a 0.22 μm filter membrane to obtain a 0.5% (m / v) trypsin stock solution;

[0056] 3. EDTA stock solution

[0057] Weigh 0.2g of disodium EDTA, dissolve it in 100mL of normal saline, and filter through a 0.22μm filter membrane to obtain a ...

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Abstract

The invention provides an efficient separation and amplification method for human amniotic mesenchymal stem cells. The efficient separation and amplification method is characterized by comprising thefollowing steps: 1, preparing a reagent; 2, separating amniotic mesenchymal stem cells through a two-step enzyme digestion method; 3, carrying out subculture on the amniotic mesenchymal stem cells. Compared with an existing amniotic mesenchymal stem cell separation method, the amniotic mesenchymal stem cells are separated by adopting the two-step enzyme digestion method; amniotic epithelial cellsare removed in the first step and two times of digestion are carried out, so that the amniotic mesenchymal stem cells are more sufficiently removed and the purity of the obtained amniotic mesenchymalstem cells is higher; the method is simple to operate and tissue and cell filtering is not needed; DNase I is used and a condition that the amniotic mesenchymal stem cell enter digestion mucus and cannot be separated is avoided; more stem cells are obtained. In a whole separation process, animal-derived components do not exist, the safety is good and subsequent research and application is convenient to realize.

Description

technical field [0001] The invention relates to a method for separating and expanding stem cells, in particular to a method for efficiently separating and expanding human amniotic mesenchymal stem cells, and belongs to the field of biotechnology. Background technique [0002] Mesenchymal stem cells have the biological characteristics of self-replication, multi-directional differentiation and immune regulation, and have become a research hotspot. Although there are many studies on bone marrow-derived mesenchymal stem cells, their sampling must be obtained through trauma, and as the individual grows, its quality and quantity will decline. At present, researchers at home and abroad have been able to obtain mesenchymal stem cells from umbilical cord, umbilical cord blood, and dental pulp, but the number of cells obtained is limited. It is an inevitable development trend to seek an efficient method for isolating mesenchyme with convenient tissue sources and no ethical constraint...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/073C12N5/0775
CPCC12N5/0605C12N5/0668C12N2509/00
Inventor 宋少锐聂苏秦朱艳丽张博
Owner 陕西九州细胞基因工程有限公司
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