Separation culture method of human mesenchymal stem cells by density gradient centrifugation method

A technology of density gradient centrifugation and bone marrow mesenchyme, which is applied in the field of separating and culturing human bone marrow mesenchymal stem cells by density gradient centrifugation, and can solve the problem of not being able to meet the requirements of flow cytometry and BMSCs acquisition. Low rate, complex cell components, etc.

Inactive Publication Date: 2013-04-03
陆华
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Problems solved by technology

The adherent separation and screening method is based on the characteristics of BMSCs adherent growth and hematopoietic cell suspension growth, but the obtained cell components are complex, and the acquisition rate of BMSCs is low; flow cytometry separation and immunomagnetic bead separation e

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  • Separation culture method of human mesenchymal stem cells by density gradient centrifugation method

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Embodiment Construction

[0018] Specifically, such as figure 1 Shown is a flow chart of the steps of a method for separating and culturing human bone marrow mesenchymal stem cells by density gradient centrifugation according to an embodiment of the present invention. The method specifically includes the following steps:

[0019] S1. Isolation of BMSCs:

[0020] Bone marrow was collected from cancellous bone fragments during hip joint surgery or free iliac crest transplantation. All operations were carried out under sterile conditions. The collected bone marrow suspension was anticoagulated with heparin, diluted with D-Hanks solution (volume ratio 1:1), and 5ml of Ficoll separation solution (lymphocyte separation solution) was added to a 15ml centrifuge tube, and 10ml was diluted Afterwards, the bone marrow suspension was slowly added to the Ficoll separation medium, centrifuged at 1800 rpm for 15 minutes, and the interfacial layer cells rich in nucleated cells were collected, washed once with D-Hanks...

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Abstract

The invention provides a separation culture method of bone marrow mesenchymal stem cells (BMSCs) by a density gradient centrifugation method. The separation culture method comprises the steps of BMSCs separation, BMSCs primary culture, BMSCs subculture and amplification, cultured cell surface antigen detection and growing curve determination. According to the method, a density gradient centrifugation method is adopted, a lymphocyte separation medium with density of 1.077 is used for separating BMSCs, and most erythrocyte, adipocyte, soterocyte and the like are removed after gradient centrifuge to obtain BMSCs cells with relatively high purity; and the BMSCs cells are cultured for 2 to 3 days to find that the BMSCs basically grow by adhering walls according to the characteristic that the BMSCs grows by adhering to walls; therefore, BMSCs with good uniformity can be obtained in the final phase of primary culture. The method has the advantages of simpleness in operation, quickness and practicality and the like, and is an effective BMSCs separation purification method.

Description

technical field [0001] The invention relates to a method for isolating and culturing human bone marrow mesenchymal stem cells in the field of biotechnology, in particular to a method for isolating and culturing human bone marrow mesenchymal stem cells by density gradient centrifugation. Background technique [0002] Bone marrow mesenchymal stem cells (Bone Marrow Stromal Cells, BMSCs), also known as bone marrow stromal stem cells, is another type of stem cells that exist in the bone marrow. Since Friendenstein used the adhesion characteristics of BMSCs to isolate, culture and study their multi-differentiation potential in 1968, BMSCs have become a hot spot in the fields of modern biology and medicine. It is now generally believed that BMSCs are dry-stage cells of mesoderm development and have multi-directional differentiation potential. They can not only differentiate into hematopoietic parenchyma and support hematopoiesis, but also differentiate into a variety of tissue cel...

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Application Information

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IPC IPC(8): C12N5/0775
Inventor 陆华
Owner 陆华
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