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Method for separating umbilical cord MSC (mesenchymal stem cell) with high efficiency

A technology for isolating and cultivating stem cells, applied in the biological field, can solve the problems of easy aging, slow proliferation, and small number of isolated cells, and achieve the effect of improving adherent growth, small cell damage, and good differentiation ability.

Active Publication Date: 2017-07-25
GUANGDONG NO 2 PROVINCIAL PEOPLES HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to provide a method for efficiently isolating and culturing umbilical cord mesenchymal stem cells, so as to solve the technical problems of the existing umbilical cord mesenchymal stem cells, such as the small number of primary isolated cells, slow proliferation, and easy aging of expansion

Method used

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  • Method for separating umbilical cord MSC (mesenchymal stem cell) with high efficiency
  • Method for separating umbilical cord MSC (mesenchymal stem cell) with high efficiency
  • Method for separating umbilical cord MSC (mesenchymal stem cell) with high efficiency

Examples

Experimental program
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Effect test

Embodiment 1

[0030] This example specifically illustrates the method for efficiently isolating and culturing umbilical cord mesenchymal stem cells provided by the present invention. At the same time, parallel experiments are set up to compare the number of stem cells obtained with the current conventional isolation. The following methods and reagents are conventional unless otherwise specified. Methods of operation and formulation.

[0031] (1) Before the experiment, the experimental environment was aseptically treated as follows:

[0032] 1. Use 84 disinfectant to prepare water solution according to the instructions to wipe the exposed surfaces and ground of the cell room (including buffer room and storage room), and then wipe it again with 0.5% peracetic acid; soak the gauze with 75% alcohol, and wipe the operating table and incubator Clean the inner and outer walls, pipette gun, and water bath box, wipe them with 75% alcohol, and replace them with sterilized deionized water;

[0033] 2...

Embodiment 2

[0050] This example specifically illustrates the cell proliferation ability of the MSCs isolated and cultured in the present invention.

[0051] With the cells obtained in Example 1, the cell density was adjusted by means of centrifugation and resuspension steps, and planted on a 48-well cell culture plate at a density of 5000 cells / well, Groups A, B, C and D, each group was inoculated into 10 wells, of which 5 Wells were cultured in complete medium containing 10% FBS, 1% double antibody (marked as A 完全 , B 完全 、C 完全 and D 完全 ), and the other 5 wells were cultured with improved medium (marked as A 改良 , B 改良 、C 改良 and D 改良 ), the improved medium is composed of MEM basal medium supplemented with 10% FBS, 1% double antibody, 30,000 U units of gentamycin, ascorbic acid, cholesterol, and xylitol, and the contents of ascorbic acid, cholesterol, and xylitol The concentrations were 15 μg / mL, 30 μg / mL, and 70 μg / mL, respectively. Afterwards, the medium was changed every 2 days, ...

Embodiment 3

[0054] This example specifically illustrates the ability of the MSCs isolated and cultured in the present invention to differentiate into osteoblasts. The cells obtained in each group in Example 1 were cultured and passaged with ordinary complete medium (suffix mark 1) and the improved medium (suffix mark 2) in this application, respectively, and the P3 generation cells were used to investigate the development of MSCs in each group into osteoblasts. ability to differentiate.

[0055] Adjust the cell density by means of centrifugation and resuspension steps, and inoculate 10,000 cells / well in a 48-well cell culture plate (manufactured by Corning, USA). The distribution of the 48-well plate is as follows: 8 columns are A1, A2, B1, B2, C1, C2, D1, D2, 6 rows are distributed as the blank control group (500 μL of complete medium or modified medium) in the 1st and 2nd rows, and the medium with osteogenic inducer added in the 3rd to 6th rows (50 μL of osteogenic inducer plus 450 μL ...

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Abstract

The invention discloses a method for separating umbilical cord MSC (mesenchymal stem cell) with high efficiency, and belongs to the technical field of biology. The method adopts the technical scheme that when the MSC is primarily separated, a batched digestion method is combined with a tissue culture method, and the digestion time and the ratio of secondary digestion liquid are comprehensively controlled; under the interaction of digestion liquid components and cells, the separation number of the cells and the wall-attaching survival rate are greatly improved, the injury to the cells is small, and the activity and differentiation potential of the MSC are furthest protected; the components of the culture medium are further improved, the content and ratio of three added components are controlled, the synergistic promoting efficacy on wall-attaching growth, activity maintaining and proliferation ability of the umbilical cord MSC is realized, and a large amount of MSC with high survival rate, good activity and good differentiation ability are obtained at high efficiency.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for efficiently separating and culturing umbilical cord mesenchymal stem cells. Background technique [0002] Human umbilical cord mesenchymal stem cells (MSCs) are a group of adult stem cells with high self-renewal ability and differentiation potential, which can differentiate into various tissue cells such as osteoblasts, adipocytes, chondrocytes and nerve cells under appropriate induction conditions Coupled with its advantages of immune regulation, cytokine secretion, and convenient material extraction, research on the isolation, culture, differentiation, and implantation of MSCs has attracted much attention, and it has shown increasingly broad application prospects in the fields of cell therapy and tissue engineering. [0003] Since the first report of MSC application in clinical trials in 1995, currently cultured MSCs have been widely used in clinical trials, such as sp...

Claims

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Application Information

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IPC IPC(8): C12N5/0775
CPCC12N5/0665C12N2500/35C12N2500/36C12N2500/38C12N2509/00
Inventor 刘爽陈睿张青佘燕玲沈慧娟黎程
Owner GUANGDONG NO 2 PROVINCIAL PEOPLES HOSPITAL
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