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Method of in-vitro amplification and purification culture of mesenchymal stem cells

A bone marrow mesenchymal and in vitro expansion technology, applied in the field of in vitro expansion, purification and culture of bone marrow mesenchymal stem cells, can solve the problems of loss of expansion ability, large amount of bone marrow, low cell viability, etc., to reduce costs, damage reduction effect

Inactive Publication Date: 2015-04-01
黄相杰
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Problems solved by technology

[0002] Bone marrow mesenchymal stem cells originate from the mesoderm in the early stage of development and are non-terminal differentiated cells. This kind of cells is easy to collect, isolate and culture, and has strong self-renewal, differentiation ability and immunosuppressive advantages in vitro, so it has an important role in the fields of tissue and organ defect diseases, tissue and organ degenerative diseases, cell therapy and tissue engineering. Application prospects. In recent years, there have been many studies at home and abroad to explore the in vitro isolation and culture methods of bone marrow mesenchymal stem cells, mainly including cell adhesion separation, density gradient centrifugation and cell sorting. The cell purity is low. Gradient centrifugation and cell sorting can obtain cells with higher purity, but the cell viability is relatively low, and the amount of bone marrow required is large, which affects subsequent research and application. At the same time, the bone marrow mesenchyme obtained by these methods Stem cells will gradually lose the ability to expand during subculture

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  • Method of in-vitro amplification and purification culture of mesenchymal stem cells
  • Method of in-vitro amplification and purification culture of mesenchymal stem cells
  • Method of in-vitro amplification and purification culture of mesenchymal stem cells

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Embodiment 1

[0029] A method for expanding, purifying and culturing bone marrow mesenchymal stem cells in vitro, comprising the following steps:

[0030] (1) Collection of bone marrow blood: 60 ml of bone marrow blood was extracted from the posterior superior iliac spine of volunteers.

[0031] (2) Separation and extraction of nucleated cells:

[0032] A. Density gradient centrifugation: inject bone marrow blood into sterile centrifuge tube a for centrifugation. After centrifugation, the bone marrow blood is divided into three layers in the centrifuge tube. The upper layer is the supernatant, and the middle layer is a thin layer of flocculent buffy coat. Nucleated cell layer, lower layer is erythrocyte layer; density gradient centrifugation is performed without adding any cell separation medium. B. Separation and extraction of the nucleated cell layer: the first separation and extraction of the nucleated cell layer: take the supernatant of the upper layer and the nucleated cell layer of t...

Embodiment 2

[0042] 1. Collection of bone marrow blood: 60ml of bone marrow blood was extracted from the volunteer's posterior superior iliac spine

[0043] 2. Separation and extraction of nucleated cells (mainly bone marrow mesenchymal stem cells): Put 60ml of bone marrow blood into the centrifuge tubes numbered ① and ② respectively, and carry out density gradient centrifugation at 200g, 6min at room temperature, After centrifugation, the bone marrow blood is divided into three layers, the upper layer is the supernatant, the middle layer is a thin white flocculent nucleated cell layer, and the lower layer is the red blood cell layer. At this time, the first separation and extraction of the nucleated cell layer is carried out: Take out the nucleated cell layer and supernatant in the centrifuge tubes numbered ① and ②. In order to obtain more nucleated cells, you can take some red blood cells and put them into the sterile centrifuge tube numbered ③. The tube was centrifuged at 1000g for 6 mi...

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Abstract

The invention relates to a method of in-vitro amplification and purification culture of mesenchymal stem cells in differentiation from mesenchymal stem cells to osteoblasts. A used cell culture medium comprises the following components: 5-20% of PL platelet lysate, 2-5U / ml of heparin, 5-10microgram / ml of doxycycline, and a basic culture medium DMEM or a basic culture medium AMEM. According to the method disclosed by the invention, the method comprising steps of separation and extraction of mesenchymal stem cells, cell inoculation, cell culture, cell culture fluid replacement and cell propagation are adopted, and the obtained mesenchymal stem cells are high in purity, high in in-vitro amplification capacity, and good in the capacity of differentiation to osteoblasts.

Description

technical field [0001] The invention relates to the technical field of collection, separation and extraction of bone marrow mesenchymal stem cells, and in vitro culture, expansion and purification of cells, in particular to a method for in vitro expansion, purification and culture of bone marrow mesenchymal stem cells. Background technique [0002] Bone marrow mesenchymal stem cells originate from the mesoderm in the early stage of development and are non-terminal differentiated cells. This kind of cells is easy to collect, isolate and culture, and has strong self-renewal, differentiation ability and immunosuppressive advantages in vitro, so it has an important role in the fields of tissue and organ defect diseases, tissue and organ degenerative diseases, cell therapy and tissue engineering. Application prospects. In recent years, there have been many studies at home and abroad to explore the in vitro isolation and culture methods of bone marrow mesenchymal stem cells, mainl...

Claims

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Application Information

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IPC IPC(8): C12N5/0775
Inventor 黄诚黄相杰姜红江王玉鹤
Owner 黄相杰
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