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Method for preparing adipose-derived stem cells by means of extraction

A stem cell and adipose source technology, applied in the biological field, can solve the problems of slow fibroblast speed and achieve the effect of enhancing stem cell sex and enhancing metabolism

Inactive Publication Date: 2016-09-28
ANHUI HUIEN BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The microenvironment of the growth of adipose stem cells changes, and it takes a certain amount of time to adapt to the growth environment, so that the proliferation and differentiation of adipose stem cells into adipocytes and fibroblasts in the body is relatively slow

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] A method for extracting and preparing adipose-derived stem cells. First, obtain 80 g of abdominal subcutaneous normal adipose tissue, wash the adipose tissue with PBS for 5 times, cut it into pieces to a size of 2 mm, and then digest it with 0.1% type I collagenase at a mass-volume ratio at 37°C 60 minutes; after digestion, add PBS to mix and filter, then add DMEM culture solution containing human autologous serum, at 37°C, 5% volume concentration of CO 2 Incubate in the incubator, and replace the medium with fresh medium every 48 hours. At this time, the cells grow into primary cells from the tissue block;

[0020] When the primary cells reach 80% confluency, wash them twice with PBS, then add 0.25% mass concentration of trypsin and 0.04% mass concentration of EDTA digestion solution, after the cells are suspended, adjust the cell density to 3×10 5 / ml, add DMEM medium containing human autologous serum again to cultivate the second-generation cells;

[0021] When the ...

Embodiment 2

[0023] First obtain 120 g of abdominal subcutaneous normal adipose tissue, wash the adipose tissue with PBS 6 times, each time for 5 minutes, cut it into 1 mm size, and then digest it with 0.2% type I collagenase at 38°C for 50 minutes; after digestion , add 2*PBS, mix and filter, and then add DMEM culture solution containing human autologous serum, at 37°C, 5% volume concentration of CO 2 Incubate in the incubator, and replace the medium with fresh medium twice at an interval of 48 hours, at this time, the cells grow from the tissue block into primary cells;

[0024] When the primary cells reach 75% confluence, wash once more with 3*PBS, then add 0.3% mass concentration of trypsin and 0.05% mass concentration of EDTA digestion solution, after the cells are suspended, adjust the cell density to 2 ×10 5 / ml, add DMEM medium containing human autologous serum again to cultivate the second-generation cells;

[0025] When the second-generation cells reach more than 92% confluence...

Embodiment 3

[0027] A method for extracting and preparing adipose-derived stem cells. First, obtain 180 g of abdominal subcutaneous normal adipose tissue, wash the adipose tissue with 2*PBS four times, and cut it into pieces to a size of 4 mm. Digest at ℃ for 65 minutes; after digestion, add 1*PBS to mix and filter, then add DMEM culture solution containing human autologous serum, at 37℃, 5% volume concentration of CO 2 Incubate in the incubator, and replace the medium with fresh medium every 48 hours. At this time, the cells grow into primary cells from the tissue block;

[0028] When the primary cells reach 82% confluence, wash them twice with 1*PBS, then add 0.25% trypsin and 0.08% EDTA digestion solution, and adjust the cell density to 5% after the cells are suspended. ×10 5 / ml, add DMEM medium containing human autologous serum again to cultivate the second-generation cells;

[0029] When the second-generation cells reach more than 94% confluence, add normal saline, transfer to a ce...

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Abstract

The invention discloses a method for preparing adipose-derived stem cells by means of extraction. The method comprises the following steps: acquiring normal abdominal subcutaneous adipose tissue, washing the obtained adipose tissue with a phosphate buffer solution (PBS), and then shearing the adipose tissue into pieces; then, carrying out digestion on the adipose tissue for a period of time by using type I collagenase; after digestion, adding the PBS into the product, evenly mixing and filtering; adding a DMEM culture solution containing human autologous serum, putting into a culture box for culturing, and replacing the culture solution with a fresh culture solution at regular intervals, wherein the cells growing from the tissue are primary cells; when the primary cells reach a certain degree of fusion, washing the cells with the PBS again, and then adding pancreatin and ethylene diamine tetraacetic acid (EDTA) digestive juice; after the cells suspend, adjusting the density of the cells, and adding the DMEM culture solution containing the human autologous serum into the cells again to obtain second-generation cells; and when the second-generation cells reach a certain degree of fusion, adding normal saline into the cells, transferring the cells into a centrifuge tube for carrying out centrifugal filtration, taking supernatant, concentrating and extracting to obtain the adipose-derived stem cells. The adipose-derived stem cells extracted by the method contain multiple life active substances, so that the stem cell properties of the adipose-derived stem cells are further improved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for extracting and preparing adipose-derived stem cells. Background technique [0002] Adipose-derived stem cells are a type of stem cells that can self-renew and reproduce, and have the multi-lineage differentiation potential of general stem cells and the stable multi-generational proliferation ability in vitro. It can not only differentiate into adipocytes, osteoblasts, chondrocytes, muscle cells, nerve cells, etc. under the action of different inducing factors, but also secrete a certain amount of cytokines related to cell maturation and various factors that promote angiogenesis and It can act on fibroblasts through paracrine, promote the secretion of type Ⅰ, Ⅲ collagen and fibronectin, promote collagen synthesis, and play anti-inflammatory, anti-oxidation, anti-aging, damage repair and other functions. [0003] In recent years, the use of living cells for beauty has becom...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775
CPCC12N5/0667C12N2509/00
Inventor 张正亮
Owner ANHUI HUIEN BIOTECH
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