Method for separating and culturing poultry bone mesenchymal stem cells by using bone marrow tissue block

A bone marrow mesenchymal and bone marrow tissue technology, applied in the field of stem cell isolation and culture, can solve the problems of cell cloning ability, low subculture and expansion ability, poor cell differentiation potential in acquisition rate, and difficulty in BMSC separation. Good differentiation potential, reduced cell damage, and good proliferation potential

Inactive Publication Date: 2011-08-03
CHINA AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The adherence screening method generally blows the bone marrow into a single cell suspension, and adheres the whole blood cells for 4-12 hours. The BMSCs obtained by separating according to different cell adhesion characteristics are simple to operate, but the cell uniformity is poor. The essence of the cells is The mixture of various types of cells has a large difference in antigens, and the ability of cell cloning, subculture and expansion is low
The immunoseparation method can be enriched according to cell surface markers or negatively sorted according to negatively expressed antigens on the surface, which can better separate and purify BMSCs, but it has a great impact on cell activity, high cost, and requires a large sample size
[0004] At present, there are relatively few studies on poultry BMSCs. The main reason is that the isolation of poultry BMSCs is difficult. The above-mentioned existing methods are basically used, and the cost is high. At the same time, there are disadvantages such as poor BMSC acquisition rate and poor differentiation potential of the obtained cells.

Method used

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  • Method for separating and culturing poultry bone mesenchymal stem cells by using bone marrow tissue block
  • Method for separating and culturing poultry bone mesenchymal stem cells by using bone marrow tissue block
  • Method for separating and culturing poultry bone mesenchymal stem cells by using bone marrow tissue block

Examples

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Embodiment 1

[0027] Example 1 Isolation and culture of chicken bone marrow mesenchymal stem cells

[0028] (1) Isolation and primary culture of chicken bone marrow mesenchymal stem cells

[0029] Take the femur and humerus of 5-day-old white legion chicken, remove the epiphysis at both ends, cut the bone with bone scissors, take out the bone marrow and put it in a 5ml centrifuge tube, and cut it to 1-2mm with surgical scissors 3 , add 800 μl of fetal bovine serum, blow slowly with a pipette to suspend the bone marrow fragments, transfer to a 100mm petri dish, arrange with a pipette so that the bone marrow fragments are evenly distributed in the petri dish, put the petri dish into carbon dioxide culture Box, 37°C, 90% humidity, 5% CO 2 After placing it at an angle of 60 degrees for 60 minutes, take out the culture dish, suck up the serum in the culture dish, slowly add complete culture solution (low-sugar DMEM culture solution containing 10% fetal bovine serum), and cultivate for 3 days to...

Embodiment 2

[0038] Example 2 Isolation and culture of duck bone marrow mesenchymal stem cells

[0039] (1) Isolation and primary culture of duck bone marrow mesenchymal stem cells

[0040] Take the femur and humerus of a 3-day-old Peking duck, remove the epiphysis at both ends, cut the bone with bone scissors, take out the bone marrow and put it in a 5ml centrifuge tube, and cut it to 1-2mm with surgical scissors 3 , add 500 μl of fetal bovine serum, blow slowly with a pipette to suspend the bone marrow fragments, transfer to a 100mm petri dish, arrange with a pipette so that the bone marrow fragments are evenly distributed in the petri dish, put the petri dish into carbon dioxide culture Box, 37°C, 90% humidity, 5% CO 2 After placing it at an angle of 45 degrees for 40 minutes, the culture dish was taken out, the serum in the culture dish was aspirated, and complete culture solution (low-sugar DMEM culture solution, 10% fetal bovine serum) was slowly added, and cultured for 3 days to ob...

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Abstract

The invention provides a method for separating and culturing poultry bone mesenchymal stem cells by using a bone marrow tissue block, which comprises the following steps of: after obtaining primary bone mesenchymal stem cells by a bone marrow tissue block direct wall-adherence method, carrying out in-vitro subculturing, thereby obtaining the purified poultry bone mesenchymal stem cells. Compared with the traditional method, the method has the advantages of short separation time, large obtainment and higher purity of the cells, stronger in-vitro multiplication capacity of the cells and the like and can provide abundant material sources for research application and tissue engineering of the poultry bone mesenchymal stem cells.

Description

technical field [0001] The invention relates to a technique for separating and cultivating stem cells, in particular to a method for separating and cultivating poultry bone marrow mesenchymal stem cells using bone marrow tissue blocks. Background technique [0002] Bone marrow mesenchymal stem cells (BMSCs) are a group of stem cells with multi-directional differentiation potential, which can differentiate into osteoblasts, adipocytes, chondrocytes, myoblasts, nerve cells and cardiomyocytes, etc. This feature makes BMSC an ideal seed cell for tissue engineering, which is of great significance in the fields of cell engineering, gene therapy, and organ transplantation. BMSCs have a single adherent growth to form clones and become colony-forming fibroblastic units (CFU-F). CFU-F has become one of the means to identify bone marrow mesenchymal stem cells. In vitro BMSCs are spindle-shaped, express CD44, CD73, CD29, CD106, CD105, CD90, etc., and do not express CD34, CD45, and CD1...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775
Inventor 韩红兵李想连正兴
Owner CHINA AGRI UNIV
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