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Method for separating and culturing human adipose-derived stem cells

A technology for human adipose stem cells and adipose stem cells, applied in the field of separating and culturing human adipose stem cells, can solve the problems of adipose stem cell damage, inconsistent composition and function, virus and mycoplasma contamination, etc., and achieves maintenance of activity, good clinical application value and Potential, the effect of increasing production

Active Publication Date: 2018-01-09
SHANGHAI LIFE SCI & TECH CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] During the preparation and culture of adipose stem cells, the following problems will occur: 1. Some miscellaneous cells will be mixed in the process of isolating adipose stem cells, which will affect the attachment and growth of adipose stem cells
2. In the process of separating adipose stem cells, if the enzymatic hydrolysis time is too long, it will cause damage to adipose stem cells
3. Most scientific research or medical institutions still use animal serum to cultivate adipose stem cells, but the quality of different batches of serum varies greatly, and its components and functions cannot be kept consistent; moreover, animal serum contains low levels of substances that inhibit cell growth, and potential Virus and Mycoplasma Contamination

Method used

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  • Method for separating and culturing human adipose-derived stem cells
  • Method for separating and culturing human adipose-derived stem cells

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Experimental program
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Embodiment 1

[0062] 1. Prepare a mixed digestive enzyme solution for digesting fat:

[0063] A solution containing 0.3% (m / v) collagenase I and 0.3% (m / v) collagenase II was prepared with serum-free DMEM-F12 medium, and sterilized with a filter membrane with a pore size of 0.2 μM. Then mix the mixture of the two collagenases with ACCUTASEsolution (STEMCELL Technologies) at a volume of 1:1, and the final mixed digestive enzyme solution contains 0.15% collagenase Ⅰ, 0.15% collagenase Ⅱ and 1 / 2×ACCUTASE solution.

[0064] 2. Coat cell culture flasks with fibronectin:

[0065] When using the serum-free medium provided in this example, each culture flask should be coated with recombinant human fibronectin in advance. Taking the T75 culture flask as an example, describe the coating process: add 5 ml of recombinant human fibronectin solution diluted with normal saline to a concentration of 5.0 μg / ml in a T75 culture flask, and coat overnight at 4°C (about 12 ~16 hours). Before adding the cell...

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Abstract

The invention relates to a method for separating and culturing human adipose-derived stem cells. The method comprises the following steps: (1) digesting an adipose suction substance by adopting a mixed digestive enzyme solution which consists of collagenase I, collagenase II and ACCUTASE, neutralizing the digestive enzyme after digesting is ended, centrifuging, filtering, and then further removinghybrid cells by using a lymphocyte separating liquid; (2) culturing adipose-derived stem cells by adopting a serum-free culture medium. According to the method for separating and culturing the humanadipose-derived stem cells, on one hand, cells can be dissociated from an adipose tissue quickly and effectively, the yield of the stem cells is improved, the cell separating time is shortened, and the activity of the cells is kept to the greatest extent; on the other hand, the culture medium is clear in components, does not contain any exogenous serum component, can remarkably improve the wall adhering capacity and the multiplication capacity of the adipose-derived stem cells, is beneficial to in-vitro amplification and stemness maintenance of the adipose-derived stem cells, and has a good application prospect.

Description

technical field [0001] The invention belongs to the field of cell separation and cultivation, in particular to a method for separating and cultivating 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-Derive...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775
Inventor 李新峰
Owner SHANGHAI LIFE SCI & TECH CO LTD
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