CN111548991B

CN111548991B - Technical method for extracting mouse skull osteoclast precursor cells

Info

Publication number: CN111548991B
Application number: CN202010385844.8A
Authority: CN
Inventors: 万启龙; 叶晓玲; 吴姝萱
Original assignee: Wuhan University WHU
Current assignee: Wuhan University WHU
Priority date: 2020-05-09
Filing date: 2020-05-09
Publication date: 2022-03-04
Anticipated expiration: 2040-05-09
Also published as: CN111548991A

Abstract

The invention discloses a technical method for extracting mouse skull osteoclast precursor cells. The method for extracting the osteoclast precursor cells of the mouse skull is a grinding method, the grinding force is not easy to control during operation, the cells are easy to damage, impurities which are not easy to filter and separate from the cells are often formed, and then processes such as cell culture and the like which are carried out subsequently are influenced. Based on the fluid dynamics principle, after the skull of a mouse is dissected and taken out and cut, the outer plate of the skull of the mouse is punctured by an injector which extracts a culture medium and is provided with a No. 5 or No. 6 needle, the inclined plane of the needle is aligned with the marrow cavity for pressurized washing, and after the marrow is flushed out from the marrow cavity of the skull of the mouse, osteoclast precursor cells are separated and extracted. According to the distribution characteristics of the skull bone marrow of the mouse, the osteoclast precursor cells in the bone marrow are accurately extracted, so that the method is efficient and accurate, has few impurities and can ensure the activity of the osteoclast precursor cells.

Description

Technical method for extracting mouse skull osteoclast precursor cells

Technical Field

The invention belongs to the technical field of biology, and provides a technical method for extracting mouse skull osteoclast precursor cells.

Background

Osteoclasts are giant, multinucleated, TRAP-positive cells formed by fusing mononuclear macrophages with each other and have the ability to degrade bone and mineralize tissue. The osteoclast is a high-metabolism terminally differentiated cell and has the characteristics of no passage, short survival time and the like. Osteoclasts are relatively few in vivo and it is difficult to obtain mature osteoclasts directly from an adult human or animal body.

The current methods for obtaining osteoclasts are mainly as follows: mechanical separation of newborn animals, whole bone marrow induction, peripheral blood/bone marrow mononuclear cell induction and spleen cell induction. The newborn animal is small in size, the number of obtained cells is small, and osteoclasts are easily damaged in the mechanical separation process; the osteoclast obtained by the whole bone marrow induction method has lower purity; bone marrow mononuclear cells are immature monocytes, i.e., osteoclast precursor cells, and induce differentiation more efficiently than peripheral blood mononuclear cells and spleen cells. Therefore, in basic studies on osteoclasts, osteoclasts are often induced by bone marrow monocytes, i.e., osteoclast precursor cells.

More and more researches show that the osteoclast at different positions has different numbers, sizes and biological characteristics, namely the osteoclast heterogeneity. When the osteoclast heterogeneity research is involved, osteoclast precursor cells of different parts, such as skull and limb bones, need to be extracted, and the extraction method needs to be as consistent as possible. The classical method for extracting osteoclast precursor cells from mouse limb bones is to remove the metaphysis of the limb bones, extract the culture medium or PBS by syringe to wash the bone marrow from the marrow cavity of the limb bone, and isolate a large amount of osteoclast precursor cells. The skull of the mouse has relatively less bone marrow content due to the characteristics of the anatomical structure and bone marrow distribution, and the osteoclast precursor cells are extracted by mechanical separation methods such as grinding, so that the efficiency is low and the osteoclast precursor cells are easily damaged.

Therefore, establishing a simple, efficient and reliable technical method for extracting mouse skull osteoclast precursor cells, which is similar to the extraction method of the limb osteoclast precursor cells, is very important in basic research related to osteoclasts, particularly research related to osteoclast heterogeneity.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a technical method for efficiently and reliably extracting mouse skull osteoclast precursor cells, and provides a technical basis for further exploring diseases related to bone metabolism.

In order to achieve the above object, the present invention provides a technical method for extracting mouse skull osteoclast precursor cells, which is characterized in that: the method comprises the following steps:

1) dissecting and processing the skull of the mouse;

2) puncturing a needle tip of a syringe for extracting a culture medium into a marrow cavity of a skull of a mouse, and flushing out bone marrow from an inner plate and an outer plate of the skull;

3) continuously changing the flushing direction and the puncture position according to the skull bone marrow position of the mouse observed under the body type microscope, or moving the needle heads along the bone marrow distribution while flushing;

4) separating and extracting osteoclast precursor cells;

5) osteoclast precursor cells induce staining and identification of differentiation into osteoclasts.

Preferably, the skull of the mouse in the step 1) is taken from a male or female mouse with the age of 6-8 weeks.

Further, the skull dissection and treatment of the mice in the step 1) are as follows:

the skull of the mouse is dissected out in a sterile environment and then is transferred to a sterile environment; stripping the periosteum on the inner plate surface and the outer plate surface of the skull, repeatedly washing the skull of the mouse by using phosphate buffer solution containing 100U/ml penicillin and 100ug/ml streptomycin, cutting the dome-shaped skull of the mouse along a herringbone seam, and converting the three-dimensional structure into a planar structure.

Further, the step 2) is specifically: a 10ml sterile syringe for extracting the culture medium is replaced by a No. 5 or No. 6 needle head and then is punctured into the outer plate of the skull of the mouse to keep the integrity of the inner plate; the bevel of the needle is aligned with the marrow cavity to flush out the marrow.

Further, the culture medium extracted in step 2) comprises: 90% by volume of medium + 10% by volume of fetal calf serum +100U/ml penicillin +100ug/ml streptomycin.

Furthermore, the flushing direction and the puncture position are continuously changed in the step 3), and the method specifically comprises the following steps:

aligning the needle tip of the syringe needle which absorbs the culture medium to the bone marrow distribution part which can be distinguished and can not be distinguished by naked eyes in the skull of the mouse, and puncturing the bone marrow cavity of the skull of the mouse for pressurizing and washing;

the step 3) of moving the needles along the bone marrow distribution while moving and flushing specifically comprises the following steps:

and after the bone marrow area of the cut exposed part of the skull of the mouse and the needle tip of the syringe needle penetrate into the bone marrow cavity of the skull of the mouse, repeatedly washing for many times until the skull bone slices become white.

Further, the separation and extraction of the osteoclast precursor cells in the step 4) specifically comprises:

blowing and uniformly mixing the collected mouse skull bone marrow suspension; filtering with 70um cell filter, centrifuging at 1000rmp for 5 min, resuspending osteoclast precursor cells in alpha-MEM medium containing 10% by volume fetal calf serum, 100U/ml penicillin +100ug/ml streptomycin, 30ng/ml macrophage colony stimulating factor and 20ng/ml nuclear factor kappa-ligand, inoculating into 96-well plastic well plate, and culturing at 37 deg.C and 5% CO₂The incubator was left to incubate for 6 days, with the solution changed every 2 days until osteoclasts were formed.

Further, the step 5) is specifically that after the osteoclast precursor cells are cultured for 6 days in an induction manner, after the osteoclasts with obvious multinuclear fusion are observed under a microscope, the osteoclasts are stained by tartrate-resistant acid phosphatase, and the cell nuclei are counterstained by diamidino-2-phenylindole dihydrochloride, observed under a microscope, counted and photographed.

The method of the invention utilizes a No. 5 or No. 6 injection needle with thinner needle pipe diameter, firstly, the bevel of the needle tip of the injection needle is aligned to the area of exposed marrow at the periphery of the cut skull, and the bevel of the needle tip is directly aligned to the marrow for pressurized flushing by utilizing the characteristic of semi-closed marrow cavity at the periphery. When flushing, the needle tip is used to gradually enlarge the space between the inner and outer plates of the skull and move to the enlarged bone marrow distribution area. The thinner No. 5 or No. 6 injection needle can increase the pressure of the flowing out of the culture medium, is convenient for controlling the injection speed and is easy to enlarge the space between the inner plate and the outer plate of the skull; then the needle point of the syringe needle is penetrated into the closed marrow cavity of the skull, and the needle point of the syringe needle is only penetrated into the outer plate of the skull of the mouse to keep the integrity of the inner plate, so that the closed marrow cavity is opened with an opening. Aligning the needle tip bevel of the needle head to a bone marrow cavity for pressurized flushing, and flushing out bone marrow from a needle tip puncture port along with a culture medium according to the principle of fluid dynamics; finally, according to the distribution characteristics of the bone marrow in the skull of the mouse, the needle tip of the syringe is punctured into the marrow cavity for pressurized washing at different parts. The skull bone marrow of the mouse is not completely composed of red bone marrow which can be distinguished by naked eyes, and the needle tip inclined plane needs to be aligned with bone marrow distribution parts which can be distinguished by naked eyes and can not be distinguished by naked eyes for pressure washing in the washing process; after the injection of the culture medium in the injector is finished, the fresh culture medium can be not sucked, the clear part in the flushed culture medium containing bone marrow can be directly extracted, the pressurized flushing is continued, and the flushing is repeated for a plurality of times until the skull bone slice becomes white.

The invention also provides a culture and identification method for differentiating osteoclast precursor cells into mature osteoclasts under the induction of M-CSF and RANKL, which comprises the steps of identifying the osteoclasts through TRAP and DAPI staining after the osteoclast precursor cells are cultured for 6 days; compared with the osteoclast precursor cells extracted by the traditional mouse skull grinding method and the flushing method provided by the invention, the osteoclast precursor cells have the difference in inducing differentiation into mature osteoclasts.

The invention has the following advantages and beneficial effects:

the invention establishes a novel flushing method for extracting osteoclast precursor cells of mouse skull by studying anatomical structure and bone marrow distribution characteristics of mouse skull on the basis of extracting osteoclast precursor cells by using flushing method for mouse limb bones and unifying different parts osteoclast precursor cell extraction methods when considering osteoclast heterogeneity research. The method is simple and efficient to operate, can be accurately operated under direct vision, is not easy to damage cells, can obtain a large number of mouse skull osteoclast precursor cells with better activity, and has good application prospect.

In conclusion, the invention provides a novel technical method for extracting mouse skull osteoclast precursor cells, which can simply and efficiently extract the mouse skull osteoclast precursor cells on the premise of ensuring the activity of the cells. The invention provides a novel, simple, accurate, high-efficiency and practical method for osteoclast related research, and overcomes the defects that the traditional grinding method is not easy to control grinding force, is easy to damage osteoclast activity, is easy to form impurities which are not easy to separate from cells and the like. Can improve the extraction efficiency of osteoclast precursor cells in basic research related to osteoclast cells, save the experimental time and improve the experimental efficiency and success rate. In addition, the method provides a technical basis for further exploring the etiology, development and treatment of bone metabolism diseases specifically related to osteoclasts, particularly osteoclasts.

Drawings

FIG. 1 is a schematic representation of skull cutting for a mouse in accordance with the method of the present invention;

FIG. 2 is a schematic diagram showing the method of flushing the exposed bone marrow cavity in the skull margin region of the mouse after cutting and the flow direction of bone marrow in the method of the present invention;

FIG. 3 is a schematic view of the method of the present invention illustrating the pressurized irrigation of the closed bone marrow cavity with the needle tip of the syringe needle and the flow of the bone marrow;

FIG. 4 shows TRAP and DAPI staining (magnification times 200) of osteoclasts induced from osteoclasts obtained by different methods for extracting osteoclast precursor cells from mouse skull, and comparing the number of osteoclasts;

in the figure: (A) and (B) osteoclast precursor cell-induced maturation extracted from the conventional milling group and the novel extraction group, respectively Osteoclasts; (C) comparison of the number of mature osteoclasts obtained for 2 groups of different extraction methods. **: p is a radical of<0.01。

Detailed Description

The invention is further described in detail below with reference to the figures and specific examples.

Unless otherwise indicated, all reagents and methods mentioned in the examples are reagents and methods commonly used in the art.

The invention discloses a technical method for extracting mouse skull osteoclast precursor cells, which comprises the following specific steps:

1) extraction of mouse skull osteoclast precursor cells:

(1) injecting a lethal dose of sodium pentobarbital into the abdominal cavity of a male or female mouse with the age of 6-8 weeks, and soaking the mouse in 75% ethanol for 1 minute; then transferring the mouse to a sterile operation drape, lifting and fixing the skin of the back neck of the mouse, and cutting an incision with the length of about 12 mm; the forefinger supports against the foremost end of the head of the mouse, the surface skin of the skull is turned over until the whole skull is exposed, and scissors are inserted into the large hole of the occiput and cut along the white line of the boundary between the skull and the maxilla to obtain the complete skull; the whole operation must be carried out within 30 minutes after the death of the animal to ensure high cell activity;

immersing the dissected skull of a mouse in a 15ml centrifuge tube filled with an alpha-MEM culture medium (containing 200U/ml heparin), placing the centrifuge tube on ice, and transferring the centrifuge tube to a laminar flow chamber ultra-clean bench; under a body type microscope, soft tissues such as periosteum and the like on the surfaces of the skull and the outer plate of the mouse are peeled off in a sterile culture dish, and PBS containing double antibiotics (100U/ml penicillin and 100ug/ml streptomycin) is extracted by a sterile syringe to repeatedly and gently wash the skull and the outer plate for 3 times; the skull was cut along the herringbone suture and placed in a sterile petri dish containing alpha-MEM medium of heparin (200U/ml), fetal calf serum (10% by volume) and diabodies (100U/ml penicillin, 100ug/ml streptomycin);

(2)10ml sterile syringes containing fetal calf serum (10% by volume) and double antibody (100U/ml penicillin, 100ug/ml streptomycin) of α -MEM were withdrawn, the syringes fitted with 5 or 6 gauge needles; the inclined plane of the needle tip is aligned with the exposed marrow cavity at the edge of the skull shearing surface of the mouse for flushing while moving; according to the distribution characteristics of skull bone marrow, the needle tips are punctured into the outer plates of the skull of a mouse at different parts of the bone marrow distribution, the inclined surfaces of the needle heads are aligned with the marrow cavities to carry out pressurized flushing, the bone marrow is flushed out from the flat skull marrow cavities, the inner plates of the skull cannot be punctured, or the inclined surfaces are aligned to other directions; directly extracting and collecting the culture medium containing bone marrow in a culture dish for subsequent washing after the injection of the culture medium in the injector is finished until bone fragments become white;

(3) the bone marrow-containing medium collected in the sterile petri dish was transferred to a 15ml sterile centrifuge tube, mixed by pipetting, filtered through a 70 μ M pore size cell filter, centrifuged at 1000rmp for 5 minutes, osteoclast precursor cells were resuspended, seeded into 96 plastic well plates containing alpha-MEM medium containing fetal bovine serum (10% by volume), diabody (100U/ml penicillin, 100ug/ml streptomycin), M-CSF (30ng/ml) and RANKL (20ng/ml), and plated onto 37 ℃ plates with 5% CO₂Standing and culturing in an incubator for 6 days, and changing the solution every 2 days until osteoclasts are formed;

2) staining and identification of osteoclast precursor cells induced differentiation into osteoclasts

After osteoclast precursor cells were cultured for 6 days under induction, and after observing the osteoclasts with obvious multinuclear fusion under a mirror, the cells were stained with tartrate-resistant acid phosphatase (TRAP), and nuclei were counterstained with diamidino-2-phenylindole Dihydrochloride (DAPI), observed under a microscope, counted, and photographed.

EXAMPLE 1 novel extraction method for mouse skull osteoclast precursor cells

1) Dissection and cutting of mouse skull

6 male mice of 6-8 weeks old were injected intraperitoneally with a lethal dose of sodium pentobarbital and placed in a 10cm petri dish, and the mice were immersed in 75% ethanol for 1 minute. The mice were then transferred to sterile surgical drapes, the skin of the hind neck of the mice was lifted and fixed, and incisions approximately 12mm in length were cut. The forefinger supports against the foremost end of the mouse head, the surface skin of the skull is turned over until the whole skull is exposed, and scissors are inserted into the large hole of the occiput and cut along the white line of the boundary between the skull and the maxilla to obtain the complete skull. Transferring into super clean bench of laminar flow chamber, stripping soft tissues such as periosteum on the surface of the skull and outer plate of the mouse with micro forceps under a body microscope, and repeatedly washing the inside and outside of the skull with PBS containing double antibiotics (100U/ml penicillin, 100ug/ml streptomycin) for 3 times in a 6cm culture dish. The skull is cut along the herringbone suture, the three-dimensional structure is converted into a planar structure, and the cutting mode of the mouse skull is shown in figure 1.

2) Punching out the cut skull bone marrow of the mouse

The syringe was fitted with a 5-gauge needle using a 10ml sterile syringe drawn up with alpha-MEM medium containing fetal calf serum (10% by volume) and dual antibody (100U/ml penicillin, 100ug/ml streptomycin). The bevel of the needle tip is aligned with the exposed marrow cavity at the edge of the skull shearing surface of the mouse to flush while moving, and the specific flushing method and the marrow outflow path are shown in figure 2. Then according to the distribution characteristics of skull bone marrow, the needle points are pricked into the outer plate of the skull of a mouse at different parts of the bone marrow distribution, the inclined surface of the needle head is aligned with the marrow cavity to carry out pressurized flushing, the bone marrow is flushed out from the flat skull marrow cavity, the inner plate of the skull cannot be punctured, or the inclined surface is aligned to other directions, and the method for pricking the needle points of the syringe needle and the bone marrow outflow path are shown in figure 3. After the injection of the culture medium in the injector is finished, the culture medium containing bone marrow collected in a 6cm culture dish is directly extracted for subsequent washing until bone fragments become white.

3) Separation and extraction of mouse skull osteoclast precursor cell and induction culture of osteoclast precursor cell

The bone marrow-containing medium collected in 6cm dishes was transferred to a 15ml sterile centrifuge tube and mixed by pipetting, filtered through a 70 μ M pore size cell filter, centrifuged at 1000rmp for 5 minutes, osteoclast precursor cells were resuspended, seeded into 96 plastic well plates containing alpha-MEM medium containing fetal bovine serum (10% by volume), diabody (100U/ml penicillin, 100ug/ml streptomycin), M-CSF (30ng/ml) and RANKL (20ng/ml), and plated onto 37 ℃ plates with 5% CO₂And (5) standing and culturing in an incubator.

Example 2 extraction of mouse skull osteoclast precursor cells by conventional grinding method

The method comprises the steps of carrying out dissection on the skull of 6 male mice with the age of 6-8 weeks as before, stripping periosteum and soft tissues on the surfaces of internal and external bone plates of the skull in an ultra-clean bench, and shearing the skull of the mice into strip-shaped bone slices with the thickness of 1-2 mm. In a sterile mortar containing 10ml of alpha-MEM medium containing fetal calf serum (10% by volume) and diabody (100U/ml penicillin, 100ug/ml streptomycin), the skull bone fragments were gently ground repeatedly with a pestle and the bone fragments were blasted repeatedly with medium. The liquid in the sterile mortar was collected, filtered through a cell filter with a pore size of 70 μ M, centrifuged at 1000rmp for 5 minutes, the osteoclast precursor cells were resuspended, seeded in 96 plastic well plates containing alpha-MEM medium containing fetal bovine serum (10% by volume), diabody (100U/ml penicillin, 100ug/ml streptomycin), M-CSF (30ng/ml) and RANKL (20ng/ml), and cultured at 37 ℃ in 5% CO₂And (5) standing and culturing in an incubator.

Comparing the number and the activity of the mouse skull osteoclast precursor cells obtained by the two methods and the capacity of inducing mature osteoclasts:

performing cell counting and activity detection on the osteoclast precursor cells extracted from the above components by conventional grinding method and novel extraction method, and performing statistical analysis, as shown in Table 1; the osteoclast precursor cells extracted by conventional grinding method and novel extraction method are respectively inoculated into 96 plastic well plates containing alpha-MEM culture medium containing fetal calf serum (10 vol%), double antibody (100U/ml penicillin, 100ug/ml streptomycin), M-CSF (30ng/ml) and RANKL (20ng/ml), and are plated on 37 ° plate and 5% CO₂The culture was performed by standing in an incubator, and the solution was changed every 2 days. On day 6 of culture, the cells were fixed and then subjected to TRAP staining and DAPI staining, and the number of osteoclasts formed in each group was countedThe amount was counted and statistically analyzed, and the staining results and the counting results are shown in FIG. 4.

Experimental results show that the mouse skull osteoclast precursor cells extracted by the novel extraction method are large in number and good in activity, osteoclasts can be successfully induced, and the induced osteoclasts are more and larger. The technical method for extracting the mouse skull osteoclast precursor cells provides a new technical basis for the basic research related to osteoclasts.

TABLE 1 number and activity ratio of osteoclast precursor cells obtained by different methods for extracting osteoclast precursor cells from mouse skull

Claims

1. A technical method for extracting mouse skull osteoclast precursor cells is characterized in that: the method comprises the following steps:

1) dissecting and processing the skull of the mouse;

4) separating and extracting osteoclast precursor cells;

5) staining and identification of osteoclasts induced differentiation of osteoclasts by osteoclast precursor cells;

the skull of the mouse in the step 1) is taken from a male or female mouse with the age of 6-8 weeks;

the skull dissection and treatment of the mice in the step 1) are as follows:

the skull of the mouse is dissected out in a sterile environment and then is transferred to a sterile environment; stripping periosteum on the surfaces of the inner plate and the outer plate of the skull, repeatedly washing the skull of the mouse by using phosphate buffer solution containing 100U/ml penicillin and 100ug/ml streptomycin, cutting the dome-shaped skull of the mouse along a herringbone seam, and converting the three-dimensional structure into a planar structure;

the step 2) is specifically as follows: a 10ml sterile syringe for extracting the culture medium is replaced by a No. 5 or No. 6 needle head and then is punctured into the outer plate of the skull of the mouse to keep the integrity of the inner plate; aligning the bevel of the needle head to the marrow cavity, and flushing out the marrow;

the culture medium extracted in the step 2) comprises the following components: 90% by volume of culture medium + 10% by volume of fetal bovine serum +100U/ml penicillin +100ug/ml streptomycin;

the step 3) of constantly changing the flushing direction and the puncture position specifically comprises the following steps:

after a bone marrow area of a cut exposed part of the skull of the mouse and a needle point of a syringe needle are punctured into a bone marrow cavity of the skull of the mouse, repeatedly washing for many times until skull bone fragments become white;

the separation and extraction of the osteoclast precursor cells in the step 4) specifically comprises the following steps:

blowing and uniformly mixing the collected mouse skull bone marrow suspension; filtering with 70um cell filter, centrifuging at 1000rmp for 5 min, resuspending osteoclast precursor cells in alpha-MEM medium containing 10% by volume fetal calf serum, 100U/ml penicillin +100ug/ml streptomycin, 30ng/ml macrophage colony stimulating factor and 20ng/ml nuclear factor kappa-ligand, inoculating into 96-well plastic well plate, and culturing at 37 deg.C and 5% CO₂Standing and culturing for 6 days in an incubator, and changing the liquid every 2 days until osteoclasts are formed;

and the step 5) is specifically that after the osteoclast precursor cells are cultured for 6 days in an induction manner, after obvious multinuclear fused osteoclasts are observed under a microscope, tartrate-resistant acid phosphatase staining is carried out, cell nuclei are counterstained by diamidino-2-phenylindole dihydrochloride, and the cells are observed, counted and photographed under the microscope.