Method for inducing osteoclast in vitro by mouse mononuclear macrophage leukemia cells
By passage and inducing RAW264.7 cells in DMEM high-glycemic medium of extra-grade fetal bovine serum, the addition of nuclear factor κB receptor activation factor ligand was solved, and the inefficiency of osteoclast induction and cell aging in the prior art was solved. Problem, more efficient osteoclast induction and more stable cell models are achieved.
Patent Information
- Application Number
- CN202411461119.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-18
- Publication Date
- 2025-02-14
AI Technical Summary
The existing methods of using RAW264.7 cells to induce differentiation into osteoclasts have problems such as long culture time, easy to lead to cell contamination and aging, and reduced differentiation ability due to cell stimulation.
Mouse mononuclear macrophage leukemia cells were used to pass and induce DMEM high-sugar complete medium containing extra-grade fetal bovine serum, and the induction conditions were optimized by adding nuclear factor κB receptor activation factor ligand at different concentrations and time points. Improve the induction efficiency of osteoclasts.
It improves the growth status and differentiation ability of RAW264.7 cells, shortens the cell induction time, reduces the risk of bacterial contamination, avoids cell aging, and significantly improves the induction efficiency of osteoclasts.
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Figure CN119432726A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of cell models, and in particular to a method for inducing osteoclasts from mouse mononuclear macrophage leukemia cells in vitro. Background Art
[0002] Bone is a connective tissue composed of cells, extracellular matrix and inorganic substances. Osteoblasts, osteocytes and osteoclasts are the basic functional cells that constitute bone tissue metabolism. Osteoblasts can promote bone formation, while osteoclasts are responsible for bone absorption, thereby forming bone modeling and reconstruction. The balance between osteoclast bone absorption and osteoblast bone formation is the basis for maintaining normal bone reconstruction. When the action of osteoclasts and osteoblasts is unbalanced, it will cause bone and joint lesions. In particular, the abnormal formation and activity of osteoclasts during bone absorption can lead to bone and joint lesions represented by osteoporosis, which poses a great threat to human health.
[0003] In order to prevent the occurrence of bone and joint diseases and to effectively treat bone and joint diseases, it is necessary to fully study the physiology and pathology of osteoclasts and the effects of drugs on osteoclasts. These studies need to be carried out using a stable osteoclast model. However, it is currently impossible to establish a stable osteoclast cell line, and the osteoclast model used is usually obtained by primary culture and induced differentiation. Primary culture refers to mechanical separation of the long bones of the limbs of model animals under a sterile environment, flushing the bone marrow and collecting cells, and then purifying osteoclasts. Osteoclasts cultured by induced method can be derived from a variety of cells, including primary cells such as mononuclear cells obtained from the bone marrow of the long bones of the limbs of animals, spleen cells obtained from the spleen of animals, and peripheral blood mononuclear cells, as well as mature cell lines such as RAW264.7 (mouse mononuclear macrophage leukemia cells), FDCP (mouse normal bone marrow cells), HL-60 (human promyelocytic leukemia cells), C7 (human acute T lymphocytic leukemia cells), etc. Inducers are added to the cell culture medium to induce cells to differentiate into osteoclasts. The induced osteoclast culture method is simpler to operate, has low technical difficulty, produces more osteoclasts, and has better cell uniformity, so it has been more widely used.
[0004] Existing methods for inducing differentiation of RAW264.7 cells into osteoclasts mostly use a high-glucose DMEM medium supplemented with 10% fetal bovine serum for culture, and add macrophage colony stimulating factor (M-CSF) and receptor activator for nuclear factor-κB ligand (RANKL) to the culture medium to induce RAW264.7 cells. The use of a pipetting method to collect cells not only takes a long time to culture, which easily leads to contamination of the cultured cells and aging of the osteoclasts, but also easily stimulates the cultured cells, causing the cells to grow tentacles and lose the ability to differentiate into osteoclasts, thereby affecting the efficiency of inducing osteoclasts. Summary of the invention
[0005] In order to improve the efficiency of inducing osteoclasts by RAW264.7 cells in vitro, the present invention provides a method for inducing osteoclasts by using mouse mononuclear macrophage leukemia cells in vitro.
[0006] The method for inducing osteoclasts in vitro by mouse mononuclear macrophage leukemia cells provided by the present invention comprises the following steps: S10: passaging of mouse mononuclear macrophage leukemia cells: inoculating mouse mononuclear macrophage leukemia cells into a DMEM high-glucose complete medium containing special-grade fetal bovine serum, and passaging; S20: induction of mouse mononuclear macrophage leukemia cells: collecting mouse mononuclear macrophage leukemia cells of passage 1-5, inoculating them into a DMEM high-glucose complete medium containing special-grade fetal bovine serum, and adding a nuclear factor κB receptor activating factor ligand to the medium after 20-24 hours; S30: replacing the medium after continuing to culture for 40-48 hours, and adding a nuclear factor κB receptor activating factor ligand at the same time; S40: continuing to culture for 40-48 hours to obtain mature osteoclasts.
[0007] Preferably, in S10, the mononuclear macrophage leukemia cells are RAW264.7 cells of passage 1-5.
[0008] Further preferably, in S10, the method of subculturing is: a) aspirating the culture medium on the upper part of the culture flask, leaving 1-2 mL in the flask; b) using a cell scraper to scrape the flask wall in one direction to scrape the adherent cells into the culture medium; c) using culture fluid to blow the cells remaining on the flask wall; d) inoculating the cells into a new culture medium at a subculturing ratio of 1:3-1:6.
[0009] Preferably, in S20, the mouse mononuclear macrophage leukemia cells are inoculated into the culture medium at a density of 2-3×10 4 Pieces / cm 2 .
[0010] In a preferred method, in S20, the final concentration of the NF-κB receptor activator ligand after addition is 40-60 ng / mL.
[0011] Preferably, in S30, the culture medium is replaced when the cell number reaches 50% of the culture well limit.
[0012] Further preferably, in S30, the method for replacing the culture medium is: aspirating the original culture medium from the bottom edge of the culture well, and then adding fresh DMEM high-glucose complete culture medium containing special fetal bovine serum into the culture well along the side wall of the culture well.
[0013] Preferably, in S30, the final concentration of the added receptor activator of nuclear factor κB ligand is 40-60 ng / mL.
[0014] In a preferred method, in S20, the final concentration of the NF-κB receptor activator ligand after addition is 30-50 ng / mL; in S30, the final concentration of the NF-κB receptor activator ligand after addition is 55-70 ng / mL.
[0015] Preferably, the special grade fetal bovine serum is fetal bovine serum with an endotoxin content of ≤10EU / ml.
[0016] Compared with the prior art, the use of DMEM high-glucose complete medium containing premium fetal bovine serum can improve the growth state of RAW264.7 cells, increase the proliferation rate of cells, and their ability to differentiate into osteoclasts. The culture method of using induction culture for 40-48 hours, changing the medium, and then induction culture for 40-48 hours to harvest can replenish the RANKL factor in time, so that RAW264.7 cells are continuously in a good induction environment, which is synergistic with the effect of premium fetal bovine serum on increasing the proliferation rate of cells. It can reduce the induction culture time of cells, reduce the risk of bacterial contamination caused by cell death, and avoid cell aging and apoptosis. The induction method using a lower concentration of inducer in the early stage and a higher concentration of inducer after changing the medium can better promote the conversion of RAW264.7 cells to osteoclasts and improve the induction efficiency of osteoclasts. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] Figure 1 This is a staining photograph of the second generation RAW264.7 cells cultured in Example 1 of the present invention.
[0018] Figure 2 These are photos of osteoclast staining in culture wells of different groups after 4 days of induction in Example 2 of the present invention.
[0019] Figure 3This is a comparison chart of the osteoclast counting results in each culture well of different groups after 4 days of induction in Example 2 of the present invention.
[0020] Figure 4 These are photos of osteoclast staining in culture wells of different groups after 5 days of induction in Example 2 of the present invention.
[0021] Figure 5 This is a comparison chart of the osteoclast counting results in each culture well of different groups after 5 days of induction in Example 2 of the present invention. DETAILED DESCRIPTION
[0022] The endpoints and any values of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoint values of each range, the endpoint values of each range and the individual point values, and the individual point values can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.
[0023] The present invention is described in detail below through examples. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention, and the protection scope of the present invention is not limited to the following specific embodiments.
[0024] In a specific embodiment of the present invention:
[0025] Drugs and reagents used: RAW264.7 cells were purchased from Wuhan Pronocell Life Science Co., Ltd., batch number: CL-0190; DMEM high-glucose medium was purchased from Wuhan Pronocell Life Science Co., Ltd., batch number: PM150210B; fetal bovine serum and special fetal bovine serum were both produced by CLARKBioscience (batch numbers: FB15011 and FB25015, respectively); nuclear factor κB receptor activator ligand (RANKL) was purchased from PeproTech. Other drugs and reagents are common commercial products.
[0026] The instruments and apparatus used were: cell scraper purchased from Wuxi NEST Life Science Co., Ltd. (NEST); the others were common commercially available products.
[0027] Example 1
[0028] Passaging of RAW264.7 cells:
[0029] Take two T25 cell culture flasks, add 6.3 mL of DMEM high-glucose medium to each, then add 0.7 mL of fetal bovine serum and premium fetal bovine serum to the two culture flasks respectively, mix well, and form DMEM high-glucose medium containing 10% fetal bovine serum as the control medium, and DMEM high-glucose medium containing 10% premium fetal bovine serum as the experimental medium. Take the purchased RAW264.7 cells, that is, the original RAW264.7 cells, divide them into two equal parts, inoculate them in two culture flasks respectively, place them in an incubator at a temperature of 37°C, and incubate them in CO 2 The cells were cultured at a concentration of 5%, and the medium was changed every 36 hours. The cell growth in the culture flask was observed, and when the cell density of adherent cells in the culture flask reached 60%, the RAW264.7 cells were subcultured.
[0030] When the control RAW264.7 cells were passaged, the culture medium in the culture flask was aspirated with a pipette, washed twice with phosphate buffered saline (PBS), and the same culture medium was aspirated with a curved pipette. The bottom of the flask was blown evenly and slightly vigorously to blow down the cells growing on the wall. After centrifugation, the cells were resuspended and inoculated into two identical culture flasks in equal amounts, and continued to be cultured and propagated under the same culture conditions using DMEM high-glucose culture medium containing 10% fetal bovine serum.
[0031] When the RAW264.7 cells of the experimental example are passaged, the culture medium in the culture bottle is sucked out with a pipette, washed twice with phosphate buffered saline (PBS), 1-2 mL of culture medium is added, and a cell scraper is used to scrape the culture medium in the same direction along the bottom wall of the bottle to scrape the cells that grow on the wall into the culture medium. The culture medium in the culture bottle is then sucked out with a pipette, and the remaining cells that are still attached to the wall of the culture bottle are gently blown. Since the adherent cells have been loosened after being scraped by the cell scraper, they can be easily blown into the culture medium. Take two new culture bottles, add 5-6 mL of DMEM high-glucose culture medium containing 10% special fetal bovine serum, and inoculate the scraped RAW264.7 cells into two new culture bottles in equal amounts, and continue to culture and transmit under the same culture conditions.
[0032] Compared with the blowing method for collecting RAW264.7 cells for passaging, although the cell scraper has a greater force on the cells when collecting RAW264.7 cells, usually a single action of the cell scraper on the cells can separate the adherent cells from the bottom wall of the culture flask, reducing the stimulation time of the RAW264.7 cells. The blowing method usually requires repeated blowing of the adherent cells with the culture medium, and multiple impacts are required to separate the adherent cells from the bottom wall of the culture flask, which will cause repeated stimulation of the RAW264.7 cells for a long time, which can easily cause the cells to grow tentacles and lose the ability to differentiate into osteoclasts, affecting the efficiency of inducing osteoclasts. Moreover, in the method of the present invention, after the cell scraper contacts the bottom wall of the culture flask from a position on one side edge of the bottom of the culture flask where there are fewer adherent cells, it scrapes unidirectionally toward the opposite side of the bottom of the flask, and by controlling the strength and technique of the scraper so that the scraping slides slowly and evenly along the bottom wall, the mechanical damage to the cells can be reduced to a greater extent, and the culture fluid added during harvesting allows the scraping of the cells to be carried out in the culture fluid. These measures can effectively reduce the stimulation of the cell scraper on the adherent cells, reduce the possibility of RAW264.7 cells being stimulated to grow tentacles, and ensure the ability of RAW264.7 cells to differentiate into osteoclasts.
[0033] Take a small amount of the second generation RAW264.7 cells cultured in the control example and the experimental example, respectively, stain the cells using the TRAP method, observe and compare the morphology of the RAW264.7 cells, and take pictures under a 200x microscope. Figure 1 As shown, compared with the RAW264.7 cells cultured in the control example (see Figure 1 A), RAW264.7 cells cultured in the experimental example (see Figure 1 B) has more round and quasi-round cells and fewer heteromorphic cells with pseudopodia.
[0034] Example 2
[0035] In vitro induction of osteoclasts:
[0036] Take a 96-well cell culture plate and select a certain number of culture wells as the control group, the first experimental group and the second experimental group. DMEM high-glucose medium is added to each culture well. Fetal bovine serum is added to the culture wells of the control group to form a final concentration of 10%; special grade fetal bovine serum with an endotoxin content of ≤10EU / ml is added to the culture wells of the first and second experimental groups to form a final concentration of 10%. Then 8×10 3 RAW264.7 cells cultured in the control case formed 2.5×10 4 Pieces / cm 2 Inoculation concentration; 8×10 3RAW264.7 cells were cultured in each experimental case to form 2.5×10 4 Pieces / cm 2 During inoculation, the 96-well plate was gently shaken to ensure that the RAW264.7 cells were evenly distributed in each well.
[0037] Place the 96-well plate in an incubator at 37°C in a CO 2 The cells were cultured at a concentration of 5%, and RANKL was added to each group of culture wells after 24 hours to induce the differentiation of RAW264.7 cells into osteoclasts. The amount of RANKL added to the culture wells of the control group and the first experimental group formed a final concentration of 50 ng / mL, and the amount of RANKL added to the culture wells of the second experimental group formed a final concentration of 40 ng / mL, inducing the differentiation of RAW264.7 cells into osteoclasts.
[0038] After 48 hours of continuous culture, the number of RAW264.7 cells in the culture wells of the first and second experimental groups has reached 50% of the culture well limit, and the culture medium in the culture wells of the first and second experimental groups is replaced. The specific steps are as follows:
[0039] Slowly aspirate the culture medium along the bottom edge of the culture well, avoiding contact with the adherent cells during the aspiration process. Add PBS along the side wall of the culture well to wash twice, aspirate the PBS in the culture well, and add DMEM high-glucose culture medium along the side wall of the culture well. Add special fetal bovine serum to form a final concentration of 10%. Then add RANKL to the culture wells of the first experimental group to form a final concentration of 50ng / mL, and add RANKL to the culture wells of the second experimental group to form a final concentration of 60ng / mL.
[0040] Place the 96-well plate in an incubator at 37°C and 5% CO 2 The culture was continued under the condition of concentration.
[0041] After culturing for 48 hours, the cells cultured in the culture wells of some control groups, the first experimental group and the second experimental group were stained with the TRAP method, the number of osteoclasts was counted under a microscope, and the number of osteoclasts produced in the culture wells of the first experimental group and the second experimental group was counted and compared; the morphology of the generated osteoclasts was observed and compared, and photographed under a 200x microscope.
[0042] like Figure 2 As shown, the first experimental group (see Figure 2 B) and the second experimental group (see Figure 2 C) A large number of mature osteoclasts can be seen in the cells (the cells are purple-red and the number of nuclei is ≥3). Figure 3As shown, the number of osteoclasts in the first and second experimental groups was significantly higher than that in the control group (see Figure 2 A), wherein the number of osteoclasts in the second experimental group was greater than that in the first experimental group.
[0043] The cells were cultured for 24 hours. After the total induction time reached 5 days, the cells cultured in the control group culture wells were stained with the TRAP method, the number of osteoclasts was counted under a microscope, and the number of osteoclasts produced in the control group culture wells was counted; the morphology of the generated osteoclasts was observed and compared, and photographed under a 200x microscope.
[0044] like Figure 4 and Figure 5 As shown, the first experimental group (see Figure 4 B) and the second experimental group (see Figure 4 C) The number of mature osteoblasts in the cells was also significantly higher than that in the control group (see Figure 4 A) Cells. Compared with the control group induced for 4 days (see Figure 2 A and Figure 3 ), the number of mature osteoclasts in the control group induced for 5 days increased, indicating that the 5-day induction time of the existing method to induce RAW264.7 cells to differentiate into osteoclasts is more effective. Compared with the first and second experimental groups induced for 4 days (see Figure 2 B, 2C and Figure 3 ), the number of mature osteoclasts in the first and second experimental groups induced for 5 days decreased, and a small number of cells showed apoptosis (see Figure 4 B and 4C), indicating that the optimal induction time for inducing RAW264.7 cells to differentiate into osteoclasts using the method of the present invention is 4 days. Compared with the control group induced for 5 days, the number of mature osteoblasts in the first experimental group and the second experimental group induced for 4 days was significantly greater than that in the control group, and the number of osteoclasts in the second experimental group was greater than that in the first experimental group.
[0045] It can be seen that the method of inducing osteoclasts from RAW264.7 cells in vitro of the present invention can better induce RAW264.7 cells to differentiate into mature osteoclasts, with a shorter induction time and higher osteoblast differentiation efficiency, and can establish a more stable in vitro osteoclast model, which is convenient for the study of osteoclast-related diseases. For example, it can be used to study the therapeutic effects of drugs on osteoporosis, osteoarthritis, rheumatoid arthritis, osteonecrosis of the jaw and other diseases; study the effects of drugs on osteoclast differentiation, proliferation, and apoptosis; and study the mechanism of action between cytokines in osteoclasts.
[0046] In the first and second experimental groups, the number of mature osteoclasts in the second experimental group was greater than that in the first experimental group, indicating that using a lower RANKL concentration in the early stage of induction and a higher RANKL concentration in the later stage of induction was better than using the same RANKL concentration in inducing RAW264.7 cells to differentiate into mature osteoclasts.
[0047] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.
Claims
1. A method for inducing osteoclasts from mouse mononuclear macrophage leukemia cells in vitro, characterized in that: The steps include: S10: Passaging of mouse mononuclear macrophage leukemia cells: Mouse mononuclear macrophage leukemia cells were inoculated into DMEM high-glucose complete medium containing special fetal bovine serum and passaged; S20: Induction of mouse mononuclear macrophage leukemia cells: Mouse mononuclear macrophage leukemia cells of passages 1-5 were collected and inoculated into DMEM high-glucose complete medium containing special fetal bovine serum. After 20-24 hours, nuclear factor-κB receptor activator ligand was added to the medium. S30: After continuing to culture for 40-48 hours, the medium was replaced and the ligand of receptor activator of nuclear factor-κB was added; S40: Continue culturing for 40-48 hours to obtain mature osteoclasts.
2. The method according to claim 1, characterized in that In S10, the mononuclear macrophage leukemia cells are primary RAW264.7 cells.
3. The method according to claim 2, characterized in that In S10, the method of passage is: a) Aspirate the culture medium on the top of the culture bottle, leaving 1-2 mL in the bottle; b) Use a cell scraper to scrape the flask wall in one direction to scrape the adherent cells into the culture medium; c) Use culture medium to blow the cells remaining on the flask wall; d) Inoculate the cells into new culture medium at a subculture ratio of 1:3-1:
6.
4. The method according to claim 1, characterized in that: In S20, the mouse mononuclear macrophage leukemia cells were seeded into the culture medium at a density of 2-3×10 4 Pieces / cm 2 .
5. The method according to claim 1, characterized in that In S20, the final concentration of the NF-κB receptor activator ligand after addition is 40-60 ng / mL.
6. The method according to claim 1, characterized in that In S30, the medium was changed when the cell number reached 50% of the well limit.
7. The method according to claim 6, characterized in that In S30, the method for replacing the culture medium is: sucking away the original culture medium from the bottom edge of the culture well, and then adding fresh DMEM high-glucose complete culture medium containing special fetal bovine serum into the culture well along the side wall of the culture well.
8. The method according to claim 1, characterized in that In S30, the final concentration of the NF-κB receptor activator ligand after addition is 40-60 ng / mL.
9. The method according to claim 1, characterized in that: In S20, the final concentration of the NF-κB receptor activator ligand after adding is 30-50 ng / mL; in S30, the final concentration of the NF-κB receptor activator ligand after adding is 55-70 ng / mL.
10. The method according to any one of claims 1 to 10, characterized in that The special grade fetal bovine serum is fetal bovine serum with an endotoxin content of ≤10EU / ml.