Separation and cultivation method of human adipose stem cells

A technology of human adipose stem cells and a culture method, which is applied in the field of separation and culture of human adipose stem cells to achieve high purity, good therapeutic effect, and time-saving effects

Active Publication Date: 2018-06-29
SHANGHAI LIFE SCI & TECH CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to provide a method for separating and culturing human adipose stem cells, to solve the problems in the above-mentioned existing adipose stem cell separation methods

Method used

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  • Separation and cultivation method of human adipose stem cells
  • Separation and cultivation method of human adipose stem cells
  • Separation and cultivation method of human adipose stem cells

Examples

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Embodiment 1

[0054] 1. Preparation of digestive enzyme solution:

[0055] Use serum-free medium (DMEM-F12) to prepare a solution containing 0.01-0.4WU / mL Liberase digestive enzyme, and sterilize it with a filter membrane with a pore size of 0.2 μM.

[0056] 2. Coated cell culture flasks:

[0057] When using the serum-free medium provided by the invention, each culture bottle should be coated in advance. The digested human adipose stem cells were cultured with 1-10 μg / mL recombinant human fibronectin and 1-10 μg / mL hyaluronic acid-coated culture flasks for the first time, and only 1-10 μg / mL recombinant human fibronectin was used later. Culture flasks coated with human fibronectin. Add 5 ml of coating solution diluted with normal saline to a T75 culture flask and aspirate the coating solution before adding the cells.

[0058] 3. Isolation and culture of human adipose stem cells:

[0059] Add the lipoaspirate to a 250ml centrifuge tube, then add an equal volume of normal saline; shake th...

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Abstract

The invention discloses a separation and cultivation method of human adipose stem cells. The method comprises the following steps: first, digesting a fat suction matter by using a Liberase digestive enzyme solution, neutralizing digestive enzyme after digestion is ended, centrifuging, filtering, and further removing parenchyma cells by using a lymphocyte separation solution so as to obtain the human adipose stem cells; second, cultivating the human adipose stem cells by using a serum-free medium, wherein following components are added in the serum-free medium: a recombinant human epidermal growth factor, a recombinant human basic fibroblast growth factor, a recombinant human transforming growth factor-beta, a recombinant human platelet-derived growth factor-BB, a recombinant human stem cell factor, reduced glutathione, coenzyme A, biotin, MEM vitamin solution, MEM amino acid solution, MEM non-essential amino acid solution, a GlutaMAX additive, insulin-transferrin-selenium solution andgentamicin. The method can remarkably increase the yield of adipose stem cells and improve the activity of the adipose stem cells, so as to obtain more adipose stem cells with proliferation ability.

Description

technical field [0001] The invention relates to the separation and cultivation of stem cells, in particular to a method for the separation and cultivation of human adipose stem cells. Background technique [0002] Mesenchymal stem cells (Mesenchymal Stem Cells, MSCs) is a branch of stem cells, a type of cells with self-replication and multi-directional differentiation capabilities, widely present in a variety of tissues, such as bone marrow, umbilical cord blood and umbilical cord tissue, placental tissue and adipose tissue, etc. Mesenchymal stem cells have three notable characteristics: 1. In vitro cultured mesenchymal stem cells are adherent growth; 2. Mesenchymal stem cells highly express CD73, CD90 and CD105, but do not express CD31, CD34, CD45, HLA- Markers such as DR, CD14, CD19, and CD11b; 3. Under appropriate stimulating factors, mesenchymal stem cells can differentiate into cells of various tissues such as osteoblasts, adipocytes, and nerve cells. [0003] Adipose...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775
CPCC12N5/0667C12N2500/32C12N2500/38C12N2500/25C12N2501/11C12N2501/115C12N2501/125C12N2501/135C12N2500/90C12N2509/00C12N2533/80C12N2533/52C12N2501/999C12N2501/15
Inventor 李文荣李新峰周雁冰
Owner SHANGHAI LIFE SCI & TECH CO LTD
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