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Adipose-derived stem cell separation culture method

A technology for the separation and cultivation of adipose stem cells, which is applied in the field of high-purity adipose stem cells, can solve the problems of low purity of adipose stem cells, limit the comparison and repetition of research results, and make it difficult for adipose stem cells to maintain self-renewal and multi-directional differentiation potential, so as to achieve efficient acquisition , the effect of strong clone-forming ability

Active Publication Date: 2013-01-02
成都世联康健生物科技有限公司
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Problems solved by technology

So far, no specific markers of adipose-derived stem cells have been found, and no stem cell populations in adipose-derived stromal cells (ADSCs) have been found. Research at home and abroad cannot obtain high-purity adipose-derived stem cells ( High-purity adipose stem cells refer to a group of adipose tissue-derived stem cells with the same cell surface antigen markers, homogeneity, self-renewal and multidirectional differentiation capabilities)
[0005]The existing isolated adipose stem cells are not of high purity, and the in vitro culture of adipose stem cells is difficult to maintain self-renewal and multi-directional differentiation potential, which greatly limits the research results at home and abroad. contrast and repetition

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  • Adipose-derived stem cell separation culture method
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  • Adipose-derived stem cell separation culture method

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[0045] The present invention will be further described in detail below in conjunction with test examples and specific embodiments. However, it should not be understood that the scope of the above subject matter of the present invention is limited to the following embodiments, and all technologies realized based on the content of the present invention belong to the scope of the present invention.

[0046] The adipose tissue mentioned in the embodiments of the present invention is derived from mice, and the main materials, reagents and equipment used in the embodiments of the present invention are as follows:

[0047] C57 mice (provided by the West China Medical Experimental Animal Center of Sichuan University);

[0048] Green fluorescent protein transgenic C57 mice (gifted by the Stem Cell Biology Laboratory of the State Key Laboratory of Biotherapy);

[0049] Nude mice (purchased from the Institute of Experimental Animals, Chinese Academy of Medical Sciences);

[0050] Fibri...

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Abstract

The invention discloses an adipose-derived stem cell separation culture method comprising the following steps of: separating and acquiring from adipose tissues; and culturing SVF (Stromal-vascular fraction) cells. The separation culture method is characterized by also comprising the following steps of: removing Lin+cells included in the SVF cells to obtain an Lin-cell mass by using a magnetic-activated cell sorting method; gathering CD271+Sca-1+cells from the obtained Lin-cell mass to obtain the adipose-derived stem cells by using a fluorescent-activated cell sorting method; and culturing theobtained adipose-derived stem cells by using a culture medium containing LIF (Leukemia Inhibitory Factor) and FGF2 (Fibroblast Growth Factor). The invention can efficiently and fast obtain the high-purity adipose-derived stem cells by combining FACS (Fluorescent-Activated Cell Sorting) with MACS (Magnetic-Activated Cell Sorting), wherein the high-purity adipose-derived stem cells have higher clone forming ability, self-renewal ability and multi-directional differentiation potentiality; P16 generation adipose-derived stem cells obtained by the separation culture method still have higher adipose forming ability and bone forming ability; in addition, the invention provides a new method for acquiring a large quantity of seed cells with dryness in vitro to apply to tissue regeneration.

Description

technical field [0001] The invention relates to a method for separating and culturing stem cells, in particular to a method for separating and culturing high-purity fat stem cells by combining fluorescence-activated flow cytometry and magnetic-activated cell sorting. Background technique [0002] Adipose-derived Stem Cells (ASCs) are a type of adult stem cells widely used in the research fields of tissue engineering and regenerative medicine (Cowan CM,. Nat Biotechnol, 2004. 22:560-567). Adipose stem cells, like bone marrow mesenchymal stem cells, have the same multidirectional differentiation potential, and can differentiate into adipocytes, osteoblasts, chondrocytes, myoblasts, endothelial cells, and neuroblasts under specific conditions. In addition, adipose stem cells also have many advantages that other types of adult stem cells do not have, such as sufficient sources of adipose tissue, convenient material collection, slight damage during the acquisition process, and no...

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0775C12N5/077
Inventor 田卫东孙钦策肖金刚郭维华李杰黄科
Owner 成都世联康健生物科技有限公司
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