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A Technical Method for Extracting Osteoclast Precursor Cells from Mouse Skull

A technology of precursor cells and osteoclasts, applied in the biological field, can solve the problems of easily damaged osteoclast precursor cells, low bone marrow content, low efficiency, etc., achieve good application prospects, avoid grinding force, and achieve the effect of precise operation

Active Publication Date: 2022-03-04
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the characteristics of the anatomical structure and distribution of bone marrow in the mouse skull, the bone marrow content is relatively small, and the extraction of osteoclast precursor cells is mostly done by mechanical separation such as grinding, which is inefficient and easy to damage the osteoclast precursor cells.

Method used

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  • A Technical Method for Extracting Osteoclast Precursor Cells from Mouse Skull
  • A Technical Method for Extracting Osteoclast Precursor Cells from Mouse Skull
  • A Technical Method for Extracting Osteoclast Precursor Cells from Mouse Skull

Examples

Experimental program
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Effect test

Embodiment 1

[0046] Example 1 New extraction method for extracting mouse calvarial osteoclast precursor cells

[0047] 1) Dissection and clipping of the mouse skull

[0048] Six 6- to 8-week-old male mice were intraperitoneally injected with a lethal dose of pentobarbital sodium and placed in a 10 cm petri dish, and the mice were soaked in 75% ethanol for 1 minute. Then the mouse was transferred to a sterile surgical cloth, the skin of the nape of the mouse was lifted and fixed, and an incision about 12 mm in length was cut. Put the index finger against the front end of the mouse's head, roll over the skin on the surface of the skull to expose the entire skull, insert scissors into the foramen magnum and cut along the alba line between the skull and the maxilla to obtain a complete skull. Transfer to the ultra-clean bench of the laminar flow room, use micro tweezers to peel off the soft tissues such as the periosteum on the surface of the inner and outer plate of the mouse skull under a s...

Embodiment 2

[0053] Example 2 Traditional Grinding Method to Extract Osteoclast Precursor Cells from Mouse Skull

[0054] Dissect the skulls of 6 male mice aged 6-8 weeks in the same way as before, peel off the periosteum and soft tissue on the surface of the inner and outer bone plates of the skulls in an ultra-clean bench, and cut the mouse skulls into 1-2mm strip-shaped bone slices. In a sterile mortar and mortar containing 10 ml of α-MEM medium containing fetal bovine serum (10% by volume) and double antibodies (100 U / ml penicillin, 100 ug / ml streptomycin), repeatedly and gently grind the skull bone slices with a pestle , and repeatedly blow the bone slices with the culture medium. Collect the liquid in the sterile mortar, filter it with a cell filter with a pore size of 70 μm, centrifuge at 1000rmp for 5 minutes, resuspend the osteoclast precursor cells, and inoculate it in a medium containing fetal bovine serum (10% volume ratio), double antibody (100U / ml Penicillin, 100ug / ml strept...

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Abstract

The invention discloses a technical method for extracting mouse skull osteoclast precursor cells. At present, the commonly used method for extracting mouse skull osteoclast precursor cells is the grinding method. It is difficult to control the intensity of grinding during operation, it is easy to damage the cells, and often forms impurities that are not easily separated from the cells by filtration, which in turn affects the subsequent cell culture and other processes. . The present invention is based on the principle of fluid dynamics. After the mouse skull is dissected and cut, the outer plate of the mouse skull is punctured with a syringe that extracts the culture medium and is equipped with a No. 5 or No. 6 needle. After the bone marrow is flushed out from the bone marrow cavity of the mouse skull, the osteoclast precursor cells are separated and extracted. According to the distribution characteristics of the bone marrow of the mouse skull, the present invention accurately extracts the osteoclast precursor cells therein, which is not only efficient, accurate, and has less impurities, but also can ensure the activity of the osteoclast precursor cells.

Description

technical field [0001] The invention belongs to the field of biotechnology and provides a technical method for extracting mouse skull osteoclast precursor cells. Background technique [0002] Osteoclasts are large, multinucleated, TRAP-positive cells formed by the fusion of mononuclear macrophages with the ability to degrade bone and mineralize tissue. As a terminally differentiated cell with high metabolism, osteoclasts have the characteristics of being unable to be passaged and having a short survival time. The number of osteoclasts in the body is relatively small, and it is difficult to directly obtain mature osteoclasts from adult humans or animals. [0003] At present, the commonly used methods to obtain osteoclasts mainly include: newborn animal mechanical separation method, whole bone marrow induction method, peripheral blood / bone marrow mononuclear cell induction method and splenocyte induction method. Newborn animals are small, the number of cells obtained is smal...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/078
CPCC12N5/0643C12N2509/10C12N2500/84C12N2501/22C12N2501/998
Inventor 万启龙叶晓玲吴姝萱
Owner WUHAN UNIV
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