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Osteoclast culturing kit and preparation method and application thereof

A technology of osteoclasts and kits, applied in the fields of medicine and biology, can solve the problems of cumbersome operation steps, time-consuming and laborious, etc., and achieve the effects of high differentiation maturity, simplified operation steps, and strong absorption capacity

Active Publication Date: 2015-02-04
HARBIN MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The purpose of the present invention is to overcome the disadvantages of cumbersome operation steps, time-consuming and labor-intensive in the prior art, and provide a simple, long-term stable, economical and practical osteoclast culture kit, preparation method and application

Method used

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  • Osteoclast culturing kit and preparation method and application thereof
  • Osteoclast culturing kit and preparation method and application thereof
  • Osteoclast culturing kit and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] The preparation of embodiment 1 osteoclast culture kit

[0033] 1. Drugs and their sources

[0034] α-MEM culture medium (α-Minimal essential medium, product number: SH30265.01B), penicillin streptomycin (product number: SV30010), fetal bovine serum (fetal bovine serum (FBS) product number: SV30087.01) were purchased from Thermo Scientific HyClone Company (USA);

[0035] M-CSF (article number: 315-02) and RANKL (article number: 315-11) were purchased from peprotech company in the United States.

[0036] 2. Method

[0037] (1) Reagent A, the preparation of frozen bone marrow mononuclear macrophages

[0038] Aseptically remove the femur and tibia from the male C57BL / 6 mouse that has been killed by neck dislocation, peel off the soft tissue above it, subtract the two ends of the bone, and insert a 5ml syringe filled with α-MEM culture solution into the bone marrow cavity from one end of the bone , wash out the red bone marrow block in the cavity; blow the bone marrow c...

Embodiment 2

[0044] Example 2 Tartrate-resistant acid phosphatase (TRAP) staining experiment of osteoclasts cultured with the kit of the present invention

[0045] When reagent A (frozen-preserved bone marrow mononuclear macrophages) was frozen at -80°C for 0 months, 3 months, 6 months, and 12 months, place the cryopreservation tubes of reagent A in a water bath at 42°C, and from time to time Shake to make it thaw and thaw quickly, then take out the cells under sterile conditions, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, add an appropriate amount of α-MEM culture medium, resuspend the cells, repeat the above steps once, to To achieve the purpose of removing the cell cryopreservation solution; the cells in the resuscitated reagent A, that is, bone marrow mononuclear macrophages, were mixed with 1×10 5 Inoculate each well in a 96-well plate, culture with reagent B for 3 days, replace the medium with reagent C, and use reagent C to change the medium every 3 days. On the...

Embodiment 3

[0049] Embodiment 3 The bone resorption experiment of the osteoclast cultured with the kit of the present invention

[0050] Bovine bone slices with a diameter of 6 mm (purchased from IDS Company, UK) were placed in the wells of a 96-well culture plate, sterilized by ultraviolet irradiation in a ultra-clean bench for 1 hour, and the culture medium was washed 3 times. Place the cryopreservation tubes of Reagent A frozen at -80°C for 0 months, March, June, and December respectively in a 42°C water bath, and shake them from time to time to make them thaw quickly, and then take them out under sterile conditions Centrifuge the cells at 1000 rpm for 5 minutes, discard the supernatant, add an appropriate amount of α-MEM culture medium, resuspend the cells, and repeat the above steps once to achieve the purpose of removing the cell freezing solution; cells, that is, bone marrow mononuclear macrophages at 1×10 5 Inoculate each well in a 96-well plate, culture with reagent B for 3 days...

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Abstract

The invention discloses osteoclast culturing kit and a manufacturing method and an application of the osteoclast culturing kit. The inventor finds out after researching that after bone marrow mononuclear macrophages are cryopreserved in -80 oC for one year, cell differentiation capability of the mononuclear macrophages does not change, namely, after obtaining a sufficient number of bone marrow mononuclear macrophages at one time, the bone marrow mononuclear macrophages can be packed separately and cryopreserved, and according to the experiment need, cells can be recovered for osteoclast culturing at any time, and the step of animal-primitive-material-taking of an existing osteoclast induction method is saved. The osteoclast culturing kit is simple and fast to operate, and a large number of osteoclast cells with bone resorption capacity can be obtained in a short time (one week). The osteoclast cells cultivated by the osteoclast culturing kit can be applied to biological and molecular biological experiments relative to the osteoclast cells, and also can be applied to research and development of screening inhibitors and promoters and other drugs of bone resorption and the study on the pathogenesis of diseases relative with the osteoclast cells.

Description

technical field [0001] The present invention relates to an osteoclast culture kit and its preparation method and application, in particular to a cell cryopreservation technique to maintain the ability of bone marrow mononuclear macrophages to differentiate into osteoclasts for a long time; on this basis, A method for inducing and culturing osteoclasts combined with cytokines. It belongs to the field of medicine and biotechnology. Background technique [0002] Currently, osteoclasts (OC) are the only cells capable of bone resorption in the body. In the process of bone metabolism, osteoclasts and osteoblasts cooperate with each other to ensure the smooth progress of bone remodeling and maintain the dynamic balance of bone metabolism. The absence or abnormal function of osteoclasts can lead to bone metabolic diseases, such as osteosclerosis and osteoporosis; in addition, osteoclasts are also involved in the occurrence and development of tumors, inflammation and autoimmune dis...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/077
Inventor 裴俊瑞孙殿军高彦辉李丙云
Owner HARBIN MEDICAL UNIVERSITY
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