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Bone-marrow-derived osteoclast culturing method

A technology derived from osteoclasts and bone marrow, applied in the direction of bone/connective tissue cells, animal cells, vertebrate cells, etc., can solve the problems of low purity of mononuclear cells of osteoclasts, many induction factors, and cumbersome steps. Achieve the effect of saving energy, simplifying the experimental process, and streamlining the experimental steps

Inactive Publication Date: 2019-05-28
NANJING UNIVERSITY OF TRADITIONAL CHINESE MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The above-mentioned technical solution has obvious insurmountable shortcomings: long time-consuming: it takes eleven days to achieve the effect; low efficiency: the current steps are cumbersome and energy-consuming; many inducing factors are used, which consumes funds; the mononuclear cells of osteoclasts obtained The cells (osteoclast precursor cells) are not pure

Method used

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  • Bone-marrow-derived osteoclast culturing method
  • Bone-marrow-derived osteoclast culturing method
  • Bone-marrow-derived osteoclast culturing method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] 1. Configuration of complete medium

[0054] It is made by adding 15% fetal bovine serum FBS and 1% separating liquid double antibody solution to MEM Alpha basic medium.

[0055] 2. Isolation and culture of mouse bone marrow mononuclear cells

[0056] The 5-week-old ICR mice were killed by dislocation, soaked in 75% alcohol for 5 minutes, and the femur and tibia of the mouse were taken under aseptic conditions, and the bone marrow in the inner cavity was washed out by drawing 6ml of homogenate flushing solution with a 1mL syringe. Until the bone marrow cavity turns white. Filter the resulting cell suspension through a 100 μm cell mesh into a 10 mL centrifuge tube. Centrifuge at 450g for 10min. Discard the supernatant and add 0.6ml sample diluent to resuspend the cells. Take a 5mL glass blood collection tube, carefully add 4mL of separation solution 1, 1.33mL of separation solution 2 and cell suspension in sequence to form a gradient interface, and the liquid layer m...

Embodiment 2

[0060] 1. Configuration of complete medium

[0061] It is made by adding 10% fetal bovine serum FBS and 1% separating liquid double antibody solution to MEM Alpha basic medium.

[0062] 2. Isolation and culture of mouse bone marrow mononuclear cells

[0063] Take the femur and tibia of two 5-week-old ICR mice, and use a 1 mL sterile syringe to draw an appropriate amount of serum-free α-MEM culture solution to wash out the bone marrow in the inner cavity until the bone marrow cavity turns white. Pipette repeatedly to form a cell suspension, filter through a 100μm cell mesh into a 10mL centrifuge tube. Centrifuge at 1200r / min for 5min. Discard the supernatant, add 5mL red blood cell lysate, mix gently by pipetting, lyse on ice for 5min, and centrifuge at 1000r / min for 5min. Discard the supernatant, add 5mL 10% FBS, mix the cells, and centrifuge at 1000r / min for 5min. After discarding the supernatant, add an appropriate amount of 10% FBS to resuspend the cells, pipette evenly...

Embodiment 3

[0068] Use the microscope AXIO vert A1 type inverted microscope (ZEISS) to photograph (50 times) the cells in each well of the 96-well plate of embodiment 1 and embodiment 2, and take about 20 pictures in each well, and put the pictures together to form The overall picture of the whole hole, such as figure 1 shown. (Group A is the culture method of Example 2, and Group B is the culture method of Example 1) Image J software calculates the ratio of the osteoclast growth area to the total hole area, and the osteoclast growth area of ​​group A accounts for 64.56% of the orifice plate area, Group B accounted for 88.48%, and there was a significant difference between Group B and Group A (P figure 2 shown. The osteoclasts cultured by the optimized culture method have a wider growth area on the well plate, as shown in the figure below, in the osteoclasts cultured at the same seed plate density, the number of osteoclasts is more and the growth is denser, showing Mosaic.

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Abstract

The invention discloses a bone-marrow-derived osteoclast culturing method, belonging to the technical field of cell culturing, comprising the steps of: subjecting an extracted bone marrow to treatmentby a separation solution to obtain monocytes, washing, adding a complete culture medium and macrophage colony stimulating factor (M-CSF) to the obtained monocytes, inoculating into a 96-pore plate, and adding a culture medium containing RANKL and M-CSF for culturing on a second day, wherein the complete culture medium is prepared by adding fetal bovine serum and double antibodies into an MEM Alpha basic medium. The bone-marrow-derived osteoclast culturing method has the characteristics of shortened culturing time and reduced culturing cost, so that the monocytes (osteoclast precursor cells) which can be differentiated into osteoclasts and obtained by bone marrow separation have higher purity, the cultured osteoclasts have comparative or even better effect, and the osteoclasts have a widerrange at the respect of enzyme activity and quantity, for providing a finer, more rapid and convenient culture method for screening drugs.

Description

technical field [0001] The invention relates to a bone marrow-derived osteoclast culture method, which belongs to the technical field of cell culture. Background technique [0002] Osteoclast (OC) is the main functional cell of bone resorption and plays an important role in bone development, growth, repair and reconstruction. Osteoclasts originate from the mononuclear-macrophage system of the blood lineage and are a special terminally differentiated cell, which can form huge multinucleated cells from their mononuclear precursor cells through fusion in various ways. Osteoclasts are a major object of study in the field of bone diseases. The extraction and culture of osteoclasts is one of the most commonly used experimental techniques in the field of bone disease research. How to obtain osteoclasts efficiently and quickly is the primary task of research. [0003] The traditional osteoclast culture method is to take two 5-week-old ICR mouse femurs and tibias on the first day, ...

Claims

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Application Information

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IPC IPC(8): C12N5/077A61K35/32A61P19/10
Inventor 李伟东陆金兰谢辉吴丽夏晨洁周雅倩
Owner NANJING UNIVERSITY OF TRADITIONAL CHINESE MEDICINE
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