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Collecting method of mesenchymal stem cells

A technology of mesenchymal stem cells and stem cells, applied in the collection of mesenchymal stem cells and the collection of mesenchymal stem cells separated from microcarriers, can solve the problem of reducing the expression of the surface marker CD105 of mesenchymal stem cells and the collection rate of mesenchymal stem cells. High, low cell viability and other problems, to achieve the effect of short processing time, less damage, high cell viability

Inactive Publication Date: 2018-08-31
GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, treating mesenchymal stem cells-microcarriers with trypsin for too long, and treating cells with trypsin for a long time will reduce cell viability and cause cell damage
It has been reported in the literature that long-term exposure of cells to trypsin will reduce the expression of the surface marker CD105 of mesenchymal stem cells
At the same time, trypsin and trypsin substitutes are currently used to digest cells and process the collection of mesenchymal stem cells on microcarriers. There is also a problem that the collection rate of mesenchymal stem cells after treatment is not high.

Method used

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  • Collecting method of mesenchymal stem cells
  • Collecting method of mesenchymal stem cells
  • Collecting method of mesenchymal stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Inoculate bone marrow-derived mesenchymal stem cells P3 generation cells into 125mL microcarrier culture flasks containing 100mL medium, maintain a stirring rate of 30rpm, and store in 5% CO 2 , and cultured in a cell incubator at 37°C and 95% humidity for 5 days.

[0031] Take out the microcarrier culture bottle, let it stand for 5min, suck off the upper medium, wash it twice with 50mL PBS containing EDTA, add EDTA-trypsin twice the volume of the microcarrier, take out the stirring hammer in the microcarrier bottle, and put The microcarrier culture flask was placed on a constant temperature shaker at 37°C, shaken at 150rpm for 8min, then 200rpm for 10s, and then 50mL of medium was added to stop digestion.

[0032] Filter the cell and microcarrier mixture with a 70 μm cell mesh, collect the cell filtrate, centrifuge at 200 g for 5 min, collect the precipitate, and resuspend the cells with the medium.

[0033] After the resuspended cells were stained with trypan blue at...

Embodiment 2

[0038] The cell quantity and cell viability assay that embodiment 2 collects

[0039] The resuspended cells of Example 1 and Comparative Example 1 were stained with trypan blue at a ratio of 1:1, respectively, and placed in an automatic cell counter to count the number of collected cells and cell viability. The results are shown in figure 1 and figure 2 .

[0040] The results showed that the number and viability of the mesenchymal stem cells collected in Example 1 were higher than those in Comparative Example 1.

Embodiment 3

[0041] Example 3 Detection of surface markers of mesenchymal stem cells collected by flow cytometry

[0042] The mesenchymal stem cells collected in Example 1 and Comparative Example 1 were subjected to flow cytometric detection. Add 1×10 per tube 6For the number of cells, wash once with staining buffer, and centrifuge at 1000rpm for 5 min; discard the supernatant, blow and mix the cells with staining buffer; add 2 μL each of CD73, CD90, CD105, CD34, CD45 and HLA-DR antibodies, and set up a tube as Blank control; react in the dark for 15-20min at 4°C; wash once with staining buffer, centrifuge at 1000rpm for 5min; discard the supernatant of directly labeled cells, add 500μL of loading buffer in the dark, mix well, and filter through a 200-mesh sieve Cell samples were filtered through a mesh, and cell surface antigens were detected by flow cytometry. The test result of embodiment 1 group sees image 3 , the test results of the comparative example group 1 are shown in Figur...

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Abstract

The invention belongs to the field of stem cells, and provides a collecting method of mesenchymal stem cells separated from a micro-carrier. An upper-layer culture medium in a mesenchymal stem cell micro-carrier culture system is removed, EDTA-trypsin is added, shaking culture is carried out for 5-10 min at the temperature of 37 DEG C at the speed of 100-200 rpm, then shaking culture is carried out for 5-10 s at the speed of 200-250 rpm, cells are filtered through a screen, cell filtrate is collected, and precipitates are collected after centrifugation is completed. According to the collectingmethod, the time for the mesenchymal stem cells to be separated from the micro-carrier is shortened by shortening the treatment time of trypsin and pancreatin substitutes. The collecting method is short in treatment time, the collecting rate of the mesenchymal stem cells is high, damage of the collected mesenchymal stem cells is low, the cell activity is high, and the collecting method is widelyapplied to collecting the mesenchymal stem cells cultured in the micro-carrier.

Description

technical field [0001] The invention belongs to the field of stem cells, in particular to a method for collecting mesenchymal stem cells, in particular to a method for collecting mesenchymal stem cells separated from microcarriers. Background technique [0002] Mesenchymal stem cells (mesenchymal stem cells) are a kind of stem cells with self-renewal, strong proliferation ability and multi-directional differentiation ability existing in various tissues. Mesenchymal stem cells are adherent cells that grow adherently on the bottom of two-dimensional plates or culture flasks, and grow on the surface of the carrier or inside the carrier in three-dimensional culture. [0003] Microcarriers are microbeads with a diameter of 60-250 μm that are suitable for the growth of adherent cells. It is generally composed of various synthetic polymers such as natural dextran, collagen or polystyrene. There are more than a dozen types of microcarriers sold in the international market, includi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775
CPCC12N5/0663C12N5/0665C12N5/0667
Inventor 陈海佳葛啸虎王一飞陈婉玲马岩岩
Owner GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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