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Method for separating and culturing animal bone marrow mesenchymal stem cell

A technology for the isolation and cultivation of stem cells, applied in animal cells, vertebrate cells, bone/connective tissue cells, etc., can solve the problems of high experimental conditions, limited wide application, and large amount of bone marrow

Active Publication Date: 2015-06-17
INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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  • Application Information

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Problems solved by technology

Although these two methods can obtain cells with higher purity, the separation process has a greater impact on the activity of the cells, and even causes the cells to lose their activity completely.
Moreover, the experimental conditions are demanding, a large amount of bone marrow is required, and the cost and technical difficulty limit the wide application of these two methods to some extent.

Method used

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  • Method for separating and culturing animal bone marrow mesenchymal stem cell
  • Method for separating and culturing animal bone marrow mesenchymal stem cell
  • Method for separating and culturing animal bone marrow mesenchymal stem cell

Examples

Experimental program
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Effect test

Embodiment 1

[0040] Embodiment 1, the method for isolating and culturing animal bone marrow mesenchymal stem cells

[0041] Three methods of isolating and culturing animal bone marrow mesenchymal stem cells were used in this implementation, namely whole bone marrow adherent method (abbreviated as BM), bone fragment digestion method (abbreviated as Bone) and bone marrow plus bone fragment method (abbreviated as BM+Bone). The inoculum for all three methods was derived from the femur of the same C57BL / 6 mouse. The inoculum of the whole bone marrow adherence method is the bone marrow of the isolated mouse femur, and the inoculum of the bone chip digestion method is the bone chip of the mouse femur. The bone chip is prepared as follows: remove the bone marrow in the whole bone marrow The remaining femur (the remaining part after removing the bone marrow of the complete femur) was cut into 1 cubic millimeter particles in 0.01M, pH7.2-7.4 PBS, added 0.1% type II collagenase, and placed at 37°C, ...

Embodiment 2

[0067] Embodiment 2, the method for isolating and culturing animal bone marrow mesenchymal stem cells

[0068] Three methods for isolating and culturing animal bone marrow mesenchymal stem cells were used in this implementation, namely the whole bone marrow adherent method, the bone slice digestion method and the bone marrow plus bone slice method. The inoculum for all three methods was derived from the tibia of the same C57BL / 6 mouse. The experimental methods of these three methods are the same as in Example 1 except that the femur is replaced by the tibia. The experimental results of these three methods are not significantly different from those in Example 1.

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Abstract

The invention discloses a method for separating and culturing an animal bone marrow mesenchymal stem cell (MSC). The method comprises the following steps: inoculating an inoculum into an animal cell culturing medium for primary culture, and subcultring cells obtained after the primary culture to obtain the animal MSC, wherein the inoculum is a precipitate obtained after centrifuging a long bone breaking liquid, and the long bone breaking liquid is a mixture obtained by breaking in-vitro animal long bones in a buffer solution to 0.5-2mm<3> particles. The method does not comprise a collagenase digestion step or an animal long bone marrow removal step. The method has the advantages of full utilization of the marrow and sclerite, simple processing, and obtaining of enough-quantity, good-growth-state and strong-proliferation-capability MSCs within a short time, and is a convenient, fast and efficient MSC separating and culturing method.

Description

technical field [0001] The invention relates to a method for isolating and culturing animal bone marrow mesenchymal stem cells. Background technique [0002] Mesenchymal stem cell (MSC) is another important stem cell besides hematopoietic stem cell in bone marrow. MSC has the ability of self-renewal, multi-directional differentiation, immune regulation and hematopoietic support, and it has a wide range of sources, and is easy to culture and expand in vitro. It has been used clinically and has shown good therapeutic effect. Although MSCs are widely used clinically, many key issues such as the specific mechanism of immune regulation have not been resolved. Mice are one of the important experimental animals and are widely used in basic experimental research. The successful isolation and culture of MSCs in vitro and the obtaining of a large number of purified MSCs are the prerequisites for the study of their differentiation and functional regulation mechanisms. MSC is mainly ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0775
Inventor 江小霞毛宁杨燕美李红党瑞杰张雷李萍刘元林
Owner INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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