Method for separating and extracting human subcutaneous adipose-derived mesenchymal stem cells and special culture medium for extraction

A technology of subcutaneous fat and stem cells, applied in animal cells, vertebrate cells, bone/connective tissue cells, etc., can solve the problems of low immunogenicity and low immunogenicity of stem cells, and achieve high purity, high proliferation and The effect of strong differentiation ability and vitality

Inactive Publication Date: 2013-03-13
SOUTHEAST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

(2) Low immunogenicity: Adipose-derived mesenchymal stem cells are mainly derived from adipose tissue with a high content in animals. When used for transplantation, autologous stem cells have low immunogenicity

Method used

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  • Method for separating and extracting human subcutaneous adipose-derived mesenchymal stem cells and special culture medium for extraction
  • Method for separating and extracting human subcutaneous adipose-derived mesenchymal stem cells and special culture medium for extraction
  • Method for separating and extracting human subcutaneous adipose-derived mesenchymal stem cells and special culture medium for extraction

Examples

Experimental program
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Effect test

Embodiment 1

[0033] Example 1: Extraction and culture steps of mesenchymal stem cells derived from human subcutaneous adipose tissue:

[0034] Preparation of special medium for extraction of mesenchymal stem cells derived from human subcutaneous adipose tissue: Add 50ml of fetal bovine serum, 5ml of antibiotics (penicillin and streptomycin) to 445ml of low-sugar Dulbecco's modified Eagle's medium, and reconstitute fiber Growth factor 2.5ug, epidermal growth factor 2.5ug, insulin 5mg, acetylcysteine ​​(400nM storage solution) 2.5ml, thyroxine 5ug, 0.22μm filter filter sterilization, 4℃ storage for use.

[0035] Extraction and culture steps of human mesenchymal stem cells derived from subcutaneous fat:

[0036] 1. Take the subcutaneous fat tissue taken out by human surgery, the mass is about 25g, put it into pre-cooled PBS, and use it within 24 hours. Then wash the tissue with PBS several times to wash away the residual blood, and cut it to 1-3mm 3 Small piece

[0037] 2. Transfer the chopped adipo...

Embodiment 2

[0045] Example 2: Identification of cell purity of human primary subcutaneous fat-derived mesenchymal stem cells.

[0046] 1. Digest the primary cells with 0.25% trypsin for 2 minutes, transfer them into a 2ml centrifuge tube, centrifuge at 800rpm for 5 minutes to remove the supernatant;

[0047] 2. Add 2ml PBS to each tube to resuspend, and centrifuge at 800rpm for 5 minutes to remove the supernatant;

[0048] 3. Add 1ml of PBS to resuspend the cells, count, and then add PBS to adjust the number of cells to 1×10 6 Pcs / ml;

[0049] 4. Divide the cell suspension into multiple tubes, each with 500μl, choose one tube as the flow control group;

[0050] 5. Add FITC-labeled CD29, CD44, and CD73 flow cytometry antibodies, incubate at 4°C for 30 minutes in the dark, and centrifuge at 800 rpm for 5 minutes to remove the supernatant;

[0051] 6. Add 1ml PBS to resuspend, centrifuge at 800rpm for 5 minutes to remove the supernatant;

[0052] 7. Add 500μl PBS to resuspend the cells;

[0053] 8. Detec...

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PUM

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Abstract

The invention discloses a method for separating and extracting human subcutaneous adipose-derived mesenchymal stem cells and a special culture medium for extraction. The method comprises the following steps of: cutting up the human subcutaneous adipose tissue, centrifuging at 4 DEG C to remove big adipose tissue, and adding mixed I type and II type collagenase for water-bath digestion; suspending with the special culture medium for extraction, and performing water bath at 37 DEG C; filtering the collected cells, adding the special culture medium for extraction, and culturing primary cells for 5-7 days; culturing the cells after primary culture with a common culture medium, and optionally performing continuous culture; and adding certain concentration of fetal bovine serum, recombinant fibroblast growth factor, epidermal growth factor, insulin, acetylcysteine and thyronine, wherein the special culture medium for extraction includes a low-sugar dulbecco's modified eagle's medium. Through the invention, a great quantity of adipose-derived mesenchymal stem cells can be extracted from subcutaneous adipose, and the intracellular stem cells subjected to primary culture have higher purity and stronger activity.

Description

Technical field [0001] The invention relates to a method for separating and extracting human subcutaneous fat mesenchymal stem cells and a special medium for extraction. Background technique [0002] Stem cells are divided into embryonic stem cells and adult stem cells according to the development process. Mesenchymal Stem Cells (MSCs) among adult stem cells are pluripotent stem cells derived from mesoderm and ectoderm. Mesenchymal stem cells exist in a variety of organs and tissues. At present, mesenchymal stem cells have been successfully obtained from tissues such as heart, bone marrow, fat, and muscle. In 2001, Zuk extracted a kind of mesenchymal stem cells with self-renewal and multi-differentiation ability from the adipose tissue suspension obtained from liposuction. This kind of stem cells was named adipose-derived mesenchymal stem cells ( Adipose-Derived mesenchymal StemCells, ADSCs). Adipose-derived mesenchymal stem cells have the same self-renewal ability as other st...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775
Inventor 孙博胡飞虎肖忠党
Owner SOUTHEAST UNIV
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