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Method for isolated culture of adipose-derived stem cells and serum-free culture medium

A serum-free medium and quality stem cell technology, applied in the field of biological cell separation, can solve the problems of low cell proliferation rate, complex composition of stem cell serum-free medium, high price, etc. Effect

Inactive Publication Date: 2018-07-20
北京汇智驰康生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The object of the present invention is to provide a method for separating and culturing adipose-derived mesenchymal stem cells and a serum-free medium to solve the technical problems of the existing stem cell serum-free medium with complex components, low cell proliferation rate and high price

Method used

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  • Method for isolated culture of adipose-derived stem cells and serum-free culture medium
  • Method for isolated culture of adipose-derived stem cells and serum-free culture medium
  • Method for isolated culture of adipose-derived stem cells and serum-free culture medium

Examples

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Effect test

Embodiment 1

[0028] Example 1 Serum-free medium of adipose-derived mesenchymal stem cells

[0029] This example is used to describe in detail the composition of the serum-free medium for culturing adipose-derived mesenchymal stem cells provided by the present invention, and the exemplary composition is shown in Table 1 below.

[0030]

Embodiment 2

[0031] Example 2 Isolation and culture method of adipose-derived mesenchymal stem cells

[0032] This example is used to describe in detail the isolation and culture method of adipose-derived mesenchymal stem cells, which includes the following steps:

[0033] ① Collect the adipose tissue from liposuction, add an equal volume of sterile PBS buffer solution to wash, put it in a centrifuge at 800rpm / min, and centrifuge for 5 minutes. After centrifugation, suck out the lower PBS buffer solution and bottom sediment with a straw and discard. Keep the floating adipose tissue on the upper layer. If there is more red in the adipose tissue, wash it again; take 10ml of the washed adipose tissue and add 2mL of 1wt.% trypsin solution (dissolve trypsin in sterile PBS buffer solution and mix well prepared), 2mL of 1wt.% type I collagenase solution (dissolved type I collagenase in sterile PBS buffer solution and mixed uniformly) and 6ml of sterile PBS, after mixing, put it on a shaker at 37°...

Embodiment 3

[0055] This example takes surgical adipose tissue as the separation object to illustrate the efficient digestion of the mixed enzyme solution in the separation and culture method of adipose-derived mesenchymal stem cells of the present invention. The method steps and experimental settings are as follows:

[0056] ①Collect 20g of surgical adipose tissue, wash it with sterile PBS buffer solution until there is no blood color, and cut it into 1mm with sterile scissors 3 The tissue blocks were divided into groups A, B and C on average, 3 groups in total. Group A was added with 1mL 1wt.% trypsin solution (prepared by dissolving trypsin in sterile PBS buffer solution and mixing uniformly), 1mL 1wt.% Type I collagenase solution (formed by dissolving type I collagenase in sterile PBS buffer solution and mixing uniformly) and 8ml sterile PBS;

[0057] Add 1mL of 1wt.% trypsin solution (prepared by dissolving trypsin in sterile PBS buffer solution and mixing uniformly) and 9ml of steril...

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Abstract

The invention relates to a method for isolated culture of adipose-derived stem cells and a serum-free culture medium, and belongs to the technical field of biological cell separation. The adopted technical scheme is a serum-free culture medium for adipose-derived stem cells, wherein the serum-free culture medium is obtained by adding ascorbic acid, basic fibroblast growth factors, platelet derivedfactors and human serum albumin into a basic culture medium, and the concentrations of the components in the serum-free stem cell culture medium are as follows: 60-80 mg / L for the ascorbic acid, 10-30 [mu]g / L for the basic fibroblast growth factors, 10-30 [mu]g / L for the platelet derived factors and 4-8 g / L for the human serum albumin. The invention has the advantages that the isolated culture method is simple, high in efficiency and fast in separation of adipose-derived stem cells, large in quantity of obtained cells, and reduced in sample usage; the serum-free culture medium provided by theinvention has scientific components and excellent proportioning, is beneficial to the growth of the adipose-derived stem cells, and greatly improves the proliferation rate of the adipose-derived stemcells.

Description

technical field [0001] The invention relates to the technical field of biological cell separation, in particular to a method for separating and culturing adipose-derived mesenchymal stem cells and a serum-free medium. Background technique [0002] Mesenchymal stem cells (MSCs) originate from the mesoderm in the early stage of development and were first discovered in the bone marrow. Later studies found that MSCs exist in various tissues, such as fat, umbilical cord, cord blood, placenta, etc. Mesenchymal stem cells positively express cell surface markers CD73, CD90, and CD105, and negatively express CD19, CD34, CD14, CD45, and HLA-DR. They are different from cells of the hematopoietic system and have multi-directional differentiation potential. Differentiate into fat, bone, cartilage, muscle, ligament, nerve, endothelial and other tissue cells. After continuous subculture and cryopreservation, it still has multi-directional differentiation potential. It can be used as ideal ...

Claims

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Application Information

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IPC IPC(8): C12N5/0775
CPCC12N5/0667C12N2500/38C12N2501/115C12N2501/135C12N2501/998C12N2509/00
Inventor 段海峰
Owner 北京汇智驰康生物科技有限公司
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