Method for extracting myelomonocyte and differentiating to osteoclast

A mononuclear cell and osteoclast technology, applied in the direction of bone/connective tissue cells, animal cells, vertebrate cells, etc., can solve the problems of immature osteoclast cell lines, unclear mechanism, and difficulty in obtaining

Inactive Publication Date: 2015-05-27
THE FIRST AFFILIATED HOSPITAL OF SOOCHOW UNIV
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Problems solved by technology

[0007]Technical problems to be solved: the conventional extraction and separation of bone marrow mononuclear cells, and the methods of differentiation to osteoclasts are less reported, and the only method of purification And the identification method is not clear; and there is still no mature osteoclast line, it is difficult to obtain, most of them are obtained by inducing differentiation of mouse monocyte / macrophage RAW264.7, but because RAW264.7 is a cell line Many mechanisms are not very clear for specific experimental research; therefore, the purpose of the present invention is to disclose a simple and effective method for extracting bone marrow mononuclear cells and differentiating them to osteoclasts

Method used

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  • Method for extracting myelomonocyte and differentiating to osteoclast
  • Method for extracting myelomonocyte and differentiating to osteoclast
  • Method for extracting myelomonocyte and differentiating to osteoclast

Examples

Experimental program
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Embodiment 1

[0029] Example 1 Observation of morphological characteristics of bone marrow mononuclear cells, identification of surface antigens and drawing of growth curves

[0030] Remove the bilateral femurs and tibias of C57BL / 6 mice under aseptic conditions; cut the metaphysis in an ultra-clean platform, and use a 5 mL sterile syringe to withdraw serum-free α-MEM medium to gently rinse the bone marrow cavity 4 times; 100um cell filtration After filtration, centrifuge at 1200 rpm for 5 minutes, discard the supernatant; add 10 times the cell volume (5 ml) of sterile 1X red blood cell lysate, gently pipette to mix, lyse on ice for 5 minutes, centrifuge at 1000 rpm for 5 minutes, discard the red supernatant To remove red blood cells; resuspend the pellet in serum-free α-MEM medium and wash it twice. Resuspend the bone marrow cells in 5 mL of α-MEM culture medium containing 10% fetal bovine serum and 100 U / mL antibiotics, and inoculate it on 25 cm 2 In a plastic culture flask, at 37℃, 5%...

Embodiment 2

[0041] Example 2 Identification of differentiation of bone marrow mononuclear cells into osteoclasts

[0042] The cell isolation and culture methods are the same as in Example 1. The primary bone marrow mononuclear cells in the logarithmic growth phase are scraped off with cell scraping, and the size is 1×10 4 cm 2 The density is passaged in a 12-well culture plate and placed in an incubator (37 ℃, volume fraction 5% CO 2 ) Cultivate for 4-6 days, the control group and the induction group each have 3 wells. The control group is added with 20ng / mL M-CSF, and the induction group is added with 20ng / mL M-CSF and 100ng / mL RANKL, which are changed every 2 days liquid.

[0043] 1. Osteoclasts were induced and cultured for 4-6 days. TARP staining was performed when obvious multinucleated fused osteoclasts were observed under the microscope. The cells were fixed with 4% paraformaldehyde for 20 minutes at room temperature, and then treated with naphthol AS-BI phosphate. Incubate at 37°...

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Abstract

The invention belongs to the field of cell extraction and differentiation authentication, and discloses a method for extracting myelomonocyte and differentiating to osteoclast. The method comprises the following steps: (A) performing sterile myelomonocyte separation, culturing in a complete medium of 20-100ng / mLM-CSF, replacing the complete medium every other day, and observing the morphological characteristics of monocyte; (B) when the density of monocyte is 80-90%, taking out a part of the cells for surface antigen authentication; (C) drawing a growth curve of other cells and performing differentiation induction for osteoclast on other cells. The method for extracting myelomonocyte and differentiating to osteoclast is simple, convenient and reliable, low in requested condition requirements, low in cost and short in time, the extracted monocyte is stable in property, and C57BL / 6 mice myelomonocyte which is reliable and stable in resource can be provided for medical basis, clinical research, tissue engineering study, and the like.

Description

technical field [0001] The invention belongs to the field of cell extraction and differentiation identification, and relates to a method for extracting bone marrow mononuclear cells and differentiating them into osteoclasts. Background technique [0002] In recent years, the problem of population aging has become increasingly serious, and the incidence of age-related diseases such as senile osteoporosis and osteoporotic hip fracture has increased significantly. Artificial joint replacement is an important choice for the treatment of degenerative bone and joint diseases and femoral neck fractures in the elderly. Periprosthetic osteolysis and prosthetic loosening are the main long-term complications after artificial joint replacement. How to prevent or delay its occurrence is an important topic. They are closely related to osteoclasts in terms of pathological research. Successfully obtaining osteoclasts will be the key to further study their functions. [0003] At present, os...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/077
Inventor 周龙何帆陈曦罗宗平杨惠林
Owner THE FIRST AFFILIATED HOSPITAL OF SOOCHOW UNIV
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