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Separating method and application of fat stem cells

An adipose stem cell and adipocyte technology, applied in animal cells, vertebrate cells, bone/connective tissue cells, etc., can solve the problems of low purity of adipose stem cells, unfavorable differentiation induction, and poor anti-aging effect.

Inactive Publication Date: 2012-06-06
CELLULAR BIOMEDICINE GRP SHANGHAI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] However, there are still many areas that need to be improved in the use of adipose stem cells for anti-aging treatment. The main reason is that the purity of the isolated adipose-derived stem cells is low, which is not conducive to differentiation induction, so the anti-aging effect is not good.

Method used

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  • Separating method and application of fat stem cells
  • Separating method and application of fat stem cells
  • Separating method and application of fat stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0110] Example 1 Separation of Adipose Stem Cells

[0111] 1. Receive adipose tissue

[0112] Wipe the outer wall of the aseptic adipose tissue collection bottle with 75% alcohol; the sterile needle draws deep adipose tissue from the human body, and moves it to the receiving container within 1 hour.

[0113] 2. Aliquot Adipose Tissue

[0114] Each 125ml storage bottle is divided into 50ml of adipose tissue, and a 10ml pipette is used to absorb the lower layer of red liquid in the fat collection bottle, discard it, and mix the remaining upper layer of fat before subpackaging.

[0115] 3. Washing Adipose Tissue

[0116] Add 50ml of sodium chloride injection into a 125ml storage bottle, tighten the cap, shake vigorously for 3 minutes to fully wash the adipose tissue and remove blood cells, then stand still for 3-5 minutes to separate the different phases, and suck off the lower aqueous phase;

[0117] Repeat the above operation three times until the lower layer is relatively c...

Embodiment 2

[0124] Embodiment 2 primary cell culture, harvest and subculture

[0125] 1. Primary Cell Culture

[0126] Place the culture bottle flat in a carbon dioxide constant temperature and humidity incubator. Culture conditions: 37±0.5°C, carbon dioxide volume fraction 5±0.2%. In the 24th hour of the primary culture, a full volume of medium was changed. After that, the medium was changed every 3 days, and the culture was carried out in a constant temperature and humidity incubator with carbon dioxide.

[0127] 2. Primary Cell Harvesting

[0128] After about 7 days, when the area percentage of the primary cultured cell clone reaches 70%-80%, digest and harvest. Add digestive enzymes (digestive enzymes are 0.125% Trypsin ~ 0.01% EDTA solution) to the culture bottle, and place it at room temperature (20-25°C) for 15-25min before use, every 75cm 2 Add 2ml of digestive enzyme solution), the digestion time is 1.5-2.5min, add 2-3ml of medium and blow the bottom of the bottle repeatedly...

Embodiment 3

[0133] Example 3 Adipogenic Induction of Adipose Stem Cells

[0134] 1. Dilute the cultured stem cells at 1.5×10 5 The density of each well was seeded in a six-well plate for adipogenic induction experiments. The specific method is: add 1 μmol / L dexamethasone, 10 μmol / L insulin, 200 μmol / L indomethacin, and 0.5 mmol / L isobutylmethacin to the basal medium (DMEM+10% fetal bovine serum) respectively. Xanthine-based xanthine was used to prepare adipogenic induction medium, and samples cultured in normal medium were used as the negative control group for adipogenic differentiation experiments. The solution was changed twice a week until the observation of adipogenic staining was carried out. All the above groups were subjected to parallel experiments (n=3).

[0135] 2. Oil Red O staining

[0136] First pour off the culture medium carefully and gently, rinse gently with D-hanks, and add 10% neutral formaldehyde to fix the cell membrane for 30 minutes. Add 0.5% Oil Red O (add 1.5...

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Abstract

The invention provides a separating method and an application of fat stem cells. The method provided by the invention comprises steps that: (1) an obtained fat cell tissue material containing fat stem cells is washed by using a washing liquid; (2) the material is digested by using collagenase, such that a digested tissue mixture is obtained; (3) the tissue mixture is filtered, such that a filtrate containing the fat stem cells is obtained; and (4) the filtrate is centrifuged, and an obtained precipitate is a fat stem cell mass. With the method provided by the invention, fat stem cells can be effectively separated; the purity of the obtained fat stem cells is high; and the proliferation speed of the fat stem cells is high. The fat stem cells separated by using the method provided by the invention can be applied in regeneration medicine, anti-aging treatments, and the like.

Description

technical field [0001] The present invention relates to the fields of biomedicine and regenerative medicine. Specifically, the present invention relates to a novel and effective method for isolating adipose stem cells and the application of the isolated adipose stem cells in anti-aging, cosmetology, regenerative medicine and the like. Background technique [0002] The entire human life process from embryonic development, life birth to maturity, aging to death always includes the interaction between human beings, the living environment and other substances, and tissue and organ damage and functional decline will inevitably occur. In the normal physiological process, a large number of cells die in the human body every day, accompanied by the regeneration of a large number of cells, in order to maintain the integrity of various tissues and organs and ensure their functions are in a normal state. If the balance of cell death and regeneration is out of balance, the increase of d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775A61L27/38A61K35/12A61P17/16A61P39/06A61K35/35
Inventor 曹卫张炳强张丽焦阳李戍
Owner CELLULAR BIOMEDICINE GRP SHANGHAI
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