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Construction method of mouse bone marrow mesenchymal stem cell lines

A technology of bone marrow mesenchyme and construction method, which is applied in the field of construction of mouse bone marrow mesenchymal stem cell line, can solve the problems of poor repeatability, complicated extraction and culture methods, etc., and achieves easy operation, easy mastery and operation, and high purity. Effect

Active Publication Date: 2019-05-03
中国医科大学
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to provide a method for constructing a mouse bone marrow mesenchymal stem cell line aiming at the problems of complicated and poor reproducibility of the current methods for extracting and culturing mouse mesenchymal stem cells

Method used

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  • Construction method of mouse bone marrow mesenchymal stem cell lines
  • Construction method of mouse bone marrow mesenchymal stem cell lines
  • Construction method of mouse bone marrow mesenchymal stem cell lines

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] A method for establishing a mouse bone marrow-derived mesenchymal stem cell line, the steps are as follows:

[0027] 1) Prepare cell culture medium: take low-sugar (1500mg / L glucose) DMEM culture medium, add to the culture medium account for 20% of the total volume of cell culture medium, fetal bovine serum filtered twice, 100U / mL penicillin, 100 μg / mL streptomycin 1% glutamine and stored at 4°C for later use.

[0028] 2) Bone marrow extraction: 6-8 week-old mice were killed by cervical dislocation, the abdomen of the mice was sprayed with 75% alcohol, the bilateral leg bones were taken out, and soaked in a centrifuge tube containing 75% alcohol for 5 minutes. Wipe the leg bone with sterile gauze, carefully remove the muscle tissue adhered to the bone, and transfer it to a sterile petri dish with a diameter of 60 mm containing 5 ml DPBS. Cut off both ends of the leg bone under aseptic conditions, draw out DPBS with a 1mL syringe and repeatedly rinse the bone marrow cav...

Embodiment 2

[0032] Comparison of oxygen concentration during culture of mouse bone marrow-derived mesenchymal stem cells

[0033] 1) Oxygen concentration setting during cell culture

[0034]According to step 3) in Example 1, the extracted primary cells were cultured in a 37°C incubator with an oxygen concentration of 5%, 10% or 21%. After 4 days, the culture medium in the culture bottle was discarded, and 5 mL of step 1 was added. ) prepared cell culture medium to continue culturing. Subculture was carried out when the cells adhered to the wall and fused to the 10th day. For the specific process of subculture, refer to step 4) in Example 1.

[0035] 2) Cell growth curve drawing

[0036] In order to analyze the growth of mesenchymal stem cells cultured under different oxygen levels, the second generation cells were taken and divided according to 1×10 4 The density of cells / well was seeded in 96-well plate, and the cells were placed in 5% CO 2 , 21%O 2 (or 10%, 5% O 2 ) in an incubato...

Embodiment 3

[0038] Comparison of the time for the first medium change in the primary culture of mouse bone marrow-derived mesenchymal stem cells

[0039] 1) Time setting for the first medium change during cell culture

[0040] According to step 3) in Example 1, the extracted primary cells were placed in 5% CO 2 , 5% O 2 Culture in a 37°C incubator, discard the culture medium in the culture bottle after 1 day or 4 days, and add 5 mL of the cell culture medium prepared in step 1) to continue the culture. Subculture was carried out when the cells adhered to the wall and fused to the 10th day. For the specific process of subculture, refer to step 4) in Example 1.

[0041] 2) Cell growth curve drawing

[0042] In order to analyze the growth of mesenchymal stem cells at different times for the first medium change, the second generation cells were taken and divided according to 1×10 4 The density of cells / well was seeded in 96-well plate, and the mesenchymal stem cells with the first exchang...

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Abstract

The invention relates to mouse stem cell culture technologies, and specifically discloses a construction method of mouse bone marrow mesenchymal stem cell lines. The construction method of the mouse bone marrow mesenchymal stem cell lines comprises the following steps: extracting bone marrow; carrying out primary culture of mesenchymal stem cells; carrying out subculture; carrying out cryopreservation; carrying out resuscitation; carrying out molecular identification of cell surface; and identifying differentiation into adipocytes and osteoblasts. Cell culture fluid adopted for the construction method of the mouse bone marrow mesenchymal stem cell lines is a low-sugar-content DMEM basic cell culture medium added with fetal bovine serum. The constructed mouse bone marrow mesenchymal stem cell lines are fibroblast-like in morphology. The construction method of the mouse bone marrow mesenchymal stem cell lines disclosed by the invention is simple in operation and high in repeatability; and the constructed mouse bone marrow mesenchymal stem cell lines are feasible for continuous passage, thereby enabling supply of a large number of mesenchymal stem cells. In addition, the constructed mouse bone marrow mesenchymal stem cell lines can differentiate into adipocytes and osteoblasts, and can be directly applied in study on functional genes of mice. The construction method of the mouse bone marrow mesenchymal stem cell lines is expected to become a research platform of stem cell functions and regulation mechanisms, and is capable of promoting practical application of tissue engineering on basis of mesenchymal stem cells.

Description

technical field [0001] The invention relates to a mouse stem cell culture technology, in particular to a method for constructing a mouse bone marrow mesenchymal stem cell line. Background technique [0002] Animal models are indispensable tools in biomedical research. Research statistics show that 91% of the literature uses various strains of mice, 7.6% of the literature uses rats, and only less than 2% of the literature uses rabbits, ferrets, guinea pigs and rhesus macaques as model animals. Using the commonly used animal model - mouse, culturing and constructing cell lines is the basis for cell biology, developmental biology, toxicology, immunology and tissue engineering research. [0003] Mesenchymal stem cells (Mesenchymal stem cells, MSCs) is one of the most in-depth study of stem cells, and bone marrow (Bone marrow, BM) is an important source of MSCs. BM-MSCs are a kind of stem cells with high proliferation and differentiation potential, which are often maintained in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775
CPCY02A50/30
Inventor 胡玉鑫徐苑苑
Owner 中国医科大学
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