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A method for extracting and cultivating fat stem cells

A technology of adipose stem cells and culture methods, which is applied in the field of extraction and cultivation of adipose stem cells, which can solve the aging limitation of adipose stem cells and achieve the effects of strong multi-directional differentiation ability, fast digestion speed, and avoiding virus or mycoplasma contamination

Active Publication Date: 2017-04-12
赛尔托马斯生物科技(成都)有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Aging of adipose-derived stem cells limits further research on its application in adipose tissue engineering and other tissue engineering

Method used

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  • A method for extracting and cultivating fat stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] (1) Take 50ml of human adipose tissue, wash it repeatedly with D-Hank's balanced salt solution with a pH value of 7.2-7.4, and remove excess aqueous solution and blood by centrifugation;

[0037] (2) Mince the adipose tissue into 1-2mm 3 Small pieces were digested by adding the same volume of digestive fluid as the adipose tissue taken, and placed in a shaker at 37°C, 190rpm for 30 minutes; adding 100ml of BME medium containing 15% FBS to stop digestion; the digestive fluid was 0.1% pancreatic Enzymes - EDTA, 0.1% Collagenase Type I and 0.2% Collagenase Type IV in D-Hank's Balanced Salt Solution.

[0038] (3) Static and stratified, the bottom cells were blown repeatedly with D-Hank's balanced salt solution with a pH value of 7.2-7.4, and cleaned; the cleaned liquid was filtered through a 100-mesh sieve to remove undigested tissue, and the adipose stem cells The suspension and erythrocyte lysate were mixed at a ratio of 1:1 and incubated for 2 minutes, centrifuged at 4°...

Embodiment 2-5

[0040] Example 2-5 Effect of Digestive Liquid Formula on Adipose Stem Cells

[0041] According to the extraction and culture method in Example 1, the digestive juice was adjusted, and the condition of the finally obtained adipose stem cells was observed.

[0042] group Trypsin-EDTA(%) Type I Collagenase (%) Type IV Collagenase (%) Example 2 0.15 0.15 0.3 Example 3 - 0.7 0.3 Example 4 2.5 0.75 - Example 5 0.05 0.05 0.5

[0043] When the cells are passed to the P2 generation, the cultured cells are collected, and CD31, CD34, CD45, CD29, CD73, CD90, CD105 and CD49d are measured by flow cytometry, and the adipose stem cells are negative for CD31, CD34 and CD45, less than 1%, and CD29, CD73, CD90, CD105 and CD49d were positive, and more than 95% were regarded as qualified cells. During the passaging process, the status of miscellaneous cells was observed through a microscope, and the total amount of primary adipose stem cells extra...

Embodiment 6-10

[0045] The influence of the formula of embodiment 6-10 culture solution on adipose stem cells

[0046] According to the extraction and culture method in Example 1, the composition of the culture medium was adjusted, and the conditions of the finally obtained adipose stem cells were observed.

[0047] Example 6 Example 7 Example 8 Example 9 Example 10 L-Glutamine (mmol / L) 2 3 5 6 0.5 bFGF (ng / ml) 20 30 35 10 5 EGF (ng / ml) 10 - 20 25 15 LIF (ng / ml) 10 25 - 15 1 Glutathione (μg / ml) 10 3 5 20 35 Cordyceps Extract (μg / ml) 50 50 50 50 50

[0048] When the coverage rate of the primary cultured cells reaches 80%-90%, the cells in each culture flask are counted by using the automatic cell counter Countess of Invitrogen Company. The specific results are as follows:

[0049] Cell density (1×10 6 )

[0050] Through flow cytometric testing, the stem cells cultured in Examples 6-10 met the qua...

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Abstract

The invention belongs to the field of biotechnology and relates to a method for extracting and culturing adipose-derived stem cells. The method comprises the following steps: (1) taking human adipose tissue, and repeatedly carrying out centrifugal washing with a D-Hank's balanced salt solution with the pH value of 7.2-7.4; (2) mincing the adipose tissue into small pieces of 1-2mm<3>, and carrying out digestion by adding a digestive juice which has the same volume of the selected adipose tissue; (3) standing and layering: repeatedly blowing and beating bottom cells with a D-Hank's balanced salt solution with the pH value of 7.2-7.4 and cleaning up; filtering a cleaned liquid through a screen mesh, removing undigested tissue, centrifuging and removing a supernatant to obtain stem cells; and (4) inoculating the obtained stem cells in a culture flask according to the density of 2-3*104 / cm<2>, adding a culture solution, and culturing in an incubator. The method has advantages as follows: digestion rate is fast; insoluble tissue blocks are minimized greatly; and an activity of the stem cells is not influenced.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for extracting and cultivating fat stem cells. Background technique [0002] Adipose-derived Stem Cells (ADSCs) 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 ethical disputes. On average, about 1 ×10 6 About 2% of cells derived from adipose tissue have stem cell characteris...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0775
Inventor 张静莹
Owner 赛尔托马斯生物科技(成都)有限公司
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