Bone trabecula-like organ with osteoclast metabolism activity and preparation and application of bone trabecula-like organ

Abstract

The invention relates to the technical field of organoid culture, in particular to a bone trabecula organoid with osteoclast metabolism activity as well as preparation and application of the bone trabecula organoid. The method comprises the following steps: culturing bone marrow primary cells, collecting mononuclear cells and a culture medium containing M-CSF, and performing adherence and amplification to obtain osteoclast precursor cells; uniformly mixing the osteoclast precursor cells with a bone organ with autonomous mineralization ability, adding the mixture into a low-adhesion plate, and culturing the mixture in a culture medium containing M-CSF to obtain a bone trabecula organ; and finally, carrying out induction treatment on the bone trabecula-like organ by utilizing a culture medium containing RANKL and M-CSF to obtain the bone trabecula-like organ with osteoclast metabolism activity. The bone trabecula-like organ with osteoclast metabolism activity prepared by the invention can be further applied to osteoclast-osteogenesis interaction research, drug screening or target verification.

Description

Technical Field

[0001] The present invention relates to the technical field of organoid culture, and in particular to a trabecular bone organoid with osteoclast metabolic activity and the preparation and application thereof. Background Art

[0002] Bone organoids are microtissues that simulate the structure and function of real bones. Bone organoids mimic the layered structure of natural bones, including different layers such as cancellous bone, compact bone and bone marrow. This structure ensures that bone organoids are similar to real bones in terms of mechanical and biological properties. Bone organoids are mainly composed of osteoblasts, osteoclasts, osteocytes and bone marrow cells. Osteoblasts are responsible for the formation and repair of bones, while osteoclasts are involved in the absorption and remodeling of bones. Osteocytes are embedded in the bone matrix to maintain the stability and function of bones. Bone marrow cells include multiple types of stem cells, which can differentiate into various cell types and participate in bone metabolism and repair.

[0003] The local microenvironment of bone organoids includes extracellular matrix, vascular network and cytokines. The extracellular matrix provides a scaffold for cell attachment and contains a variety of bioactive molecules, such as growth factors and cytokines, which regulate cell behavior and function. The vascular network is responsible for providing nutrients and oxygen to bone organoids and removing metabolic waste.

[0004] The complexity of bone metabolism is reflected in the following aspects: bone metabolism involves the interaction of multiple cell types, including osteoblasts, osteoclasts, osteocytes and bone marrow cells, which cooperate with each other in the growth, remodeling and repair of bones; bones need to maintain dynamic balance under physiological conditions, and the activities of osteoblasts and osteoclasts must be precisely coordinated to maintain the structure and function of bones; bones not only need to withstand mechanical loads, but are also affected by multiple biological factors such as genetics, hormones, nutrition and lifestyle, which jointly affect the process of bone metabolism; bone metabolism is jointly controlled by the local microenvironment and systemic regulatory systems, such as the endocrine system and the immune system.

[0005] Currently, the research on osteogenic mineralized organoids is crucial to provide a preparation of trabecular bone organoids with osteoclastic metabolic activity. Summary of the invention

[0006] In order to solve the above problems, the purpose of the present invention is to provide a trabecular bone organoid with osteoclast metabolic activity and its preparation and use.

[0007] The purpose of the present invention can be achieved by the following technical solutions:

[0008] The first object of the present invention is to provide a method for preparing trabecular bone organoids with osteoclast metabolic activity, comprising the following steps:

[0009] (S1) After culturing primary bone marrow cells, mononuclear cells are collected and cultured in a medium containing M-CSF, and osteoclast precursor cells are obtained after adhesion;

[0010] (S2) mixing the osteoclast precursor cells prepared in step (S1) with the bone organoids having autonomous mineralization ability, adding the mixture to a low-adhesion plate, and then culturing the mixture in a culture medium containing M-CSF to obtain trabecular bone organoids;

[0011] (S3) Inducing the trabecular organoids prepared in step (S2) using a culture medium containing RANKL and M-CSF to obtain trabecular organoids with osteoclast metabolic activity.

[0012] In one embodiment of the present invention, in step (S1), the concentration of M-CSF in the culture medium containing M-CSF is 30 to 60 ng / mL.

[0013] In one embodiment of the present invention, in step (S1), the amount ratio of osteoclast precursor cells, bone organoids with autonomous mineralization ability, and culture medium containing M-CSF is 5×10 4～5 Cells: 1: 100 μL.

[0014] In one embodiment of the present invention, bone organoids with autonomous mineralization ability are prepared by the following method:

[0015] (A1) Mix Matrigel and Collagen I working solution to obtain organoid matrix working solution;

[0016] (A2) The osteoblast precursor cell line is suspended in the organoid matrix working solution prepared in step (A1) and placed in a PDMS chip. After solidification, the cell line is immersed in an α-MEM culture solution containing 10% FBS to obtain a bone organoid.

[0017] (A3) treating the bone organoids prepared in step (A2) with an osteogenic induction culture medium to obtain bone organoids with autonomous mineralization ability.

[0018] In one embodiment of the present invention, the concentration of M-CSF in the culture medium containing M-CSF is 30 to 60 ng / mL.

[0019] In one embodiment of the present invention, in step (S3), in the culture medium containing RANKL and M-CSF, the concentration of RANKL is 30 to 60 ng / mL, and the concentration of M-CSF is 30 to 60 ng / mL.

[0020] In one embodiment of the present invention, in step (S3), during the induction culture, the temperature is 37° C., 5% CO 2 , and the time is 1 to 35 days.

[0021] The second object of the present invention is to provide a trabecular bone organoid with osteoclast metabolic activity, which is prepared by the above method.

[0022] The third object of the present invention is to provide a trabecular bone organoid with osteoclast metabolic activity for use in osteoclast-osteogenesis interaction research, drug screening or target verification.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] (1) Based on mineralized bone organoids, primary bone marrow mononuclear cells are inoculated and differentiated into osteoclasts with characteristic structures, forming trabecular bone organoids with osteoclast metabolic activity.

[0025] (2) Reproduce the spatial position and structure of osteocytes, osteoblasts, and osteoclasts in vivo, and differentiate them into osteoclasts with characteristic structures, thus mimicking the physiological structure of trabecular bone in vivo.

[0026] (3) The first bone organoid that combines osteoblastic metabolism and osteoclast metabolism can be used to study osteoblast-osteoclast interactions, drug screening, target validation, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Flow chart for the preparation of trabecular bone organoids with osteoclast metabolic activity;

[0028] Figure 2 This is an immunofluorescence staining image of osteoclast Ctsk enzyme in mature trabecular bone organoids;

[0029] Figure 3 This is an immunofluorescence staining of osteoclast Trap enzyme in mature trabecular bone organoids;

[0030] Figure 4 This is a three-dimensional reconstruction of the F-actin / Ctsk / DAPI immunofluorescence staining image of the Z-stack (arrows: osteoclast pseudopods extending into the matrix; plum blossoms: F-actin rings, the characteristic structure of osteoclasts);

[0031] Figure 5 Immunohistochemical staining images of in vivo trabecular bone Trap (magnified to show local osteoclasts in vivo) and trabecular bone organoid Trap (magnified to show osteoclasts on the surface of organoids). DETAILED DESCRIPTION

[0032] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0033] The osteogenic induction solution contained the following components at the following concentrations: 10 mM β-sodium glycerophosphate, 50 μg / mL vitamin C, 0.1 μM dexamethasone in α-MEM medium containing 10% FBS;

[0034] In the following examples, unless otherwise specified, all reagents used are commercially available reagents, and all detection means and methods used are conventional detection means and methods in the art.

[0035] Example 1

[0036] This embodiment provides a method for preparing a bone organoid with autonomous mineralization ability, comprising the following steps:

[0037] (S1) Mix Matrigel and Collagen I working solution (the volume ratio of Matrigel to Collagen I working solution is 1:1) to obtain organoid matrix working solution;

[0038] (S2) Osteoblast precursor cell line (commercially available Mc3t3-e1 cell line, 5×10 6 cells) were suspended in 100 μL of the organoid matrix working solution prepared in step (S1), and mixed to obtain a mixed solution; the mixed solution was evenly added to the circular holes of the PDMS chip (wherein 10 μL of the mixed solution was added to the circular holes), and placed at 37° C. to solidify for 1 hour, and then immersed in an α-MEM culture solution containing 10% FBS to obtain bone organoids;

[0039] (S3) One bone organoid prepared in step (S2) is detached from the chip and transferred to a low-adhesion 96-well microplate, and osteogenic induction treatment is performed using 150 μL of osteogenic induction culture medium (5% CO2, temperature of 37°C, 35 days) to obtain a bone organoid with autonomous mineralization ability.

[0040] Example 2

[0041] This embodiment provides a method for preparing trabecular bone organoids with osteoclast metabolic activity (such as Figure 1 As shown), specifically comprising the following steps:

[0042] (S1) After killing mice of 4 to 8 weeks old, the mice were immersed in 75% ethanol for 5 minutes to separate the femurs; the bone marrow contents were rinsed, filtered with a cell sieve, and centrifuged at 1000 r / min for 5 minutes; incubated with 1X blood cell lysis buffer for 7 minutes, and centrifuged at 1000 r / min for 3 minutes; resuspended in α-MEM culture medium containing 10% FBS and 2% PS to obtain primary bone marrow cells, and after culturing for 24 hours, the supernatant was collected to collect the suspended cells as mononuclear cells, and the mononuclear cells were resuspended in α-MEM culture medium containing 50 ng / mL M-CSF, and cultured in α-MEM culture medium containing 50 ng / mL M-CSF, and amplified after attachment to obtain osteoclast precursor cells;

[0043] (S2) Use a scraper to hang osteoclast precursor cells, 5×10 4 The cells and mineralized bone organoids were added to a 96-well low-adhesion plate and cultured in α-MEM medium containing 50 ng / mL M-CSF, and shaken overnight in an incubator.

[0044] (S3) The medium was replaced with α-MEM containing 50 ng / mL RANKL and 50 ng / mL M-CSF. After 14 days of induction of differentiation, trabecular bone organoids with osteoclastic metabolic activity were obtained.

[0045] Trabecular bone organoids were extracted as samples, fixed with 4% paraformaldehyde for 3 h, permeabilized with 0.25% TrionX-100 PBS, blocked with 5% goat serum, and immunofluorescence staining with F-actin / DAPI / Ctsk was performed. Multinucleated giant cells were formed with characteristic F-actin rings and expressed Ctsk enzymes (such as Figure 2 ); Multinucleated giant cells can be seen to fuse, have characteristic F-actin rings and express Trap enzymes (such as Figure 3 ); laser confocal scanning was used for three-dimensional reconstruction, showing that mature osteoclasts express Ctsk enzymes and pseudopods extend into the mineralized matrix (as shown in Figure 4 shown).

[0046] Trap immunohistochemical staining was performed on the trabecular bone in mice and the trabecular bone organoids cultured in this example. It can be seen that both express Trap enzymes and osteoclasts adhere to the bone surface to perform functions (such as Figure 5 shown).

[0047] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the explanation of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A method for preparing trabecular bone organoids with osteoclast metabolic activity, characterized in that: The following steps are involved: (S1) After culturing primary bone marrow cells, mononuclear cells are collected and cultured in a medium containing M-CSF, and osteoclast precursor cells are obtained after adhesion; (S2) mixing the osteoclast precursor cells prepared in step (S1) with the bone organoids having autonomous mineralization ability, adding the mixture to a low-adhesion plate, and then culturing the mixture in a culture medium containing M-CSF to obtain trabecular bone organoids; (S3) Inducing the trabecular organoids prepared in step (S2) using a culture medium containing RANKL and M-CSF to obtain trabecular organoids with osteoclast metabolic activity.

2. The method for preparing trabecular bone organoids with osteoclast metabolic activity according to claim 1, characterized in that: In step (S1), the concentration of M-CSF in the culture medium containing M-CSF is 30 to 60 ng / mL.

3. The method for preparing trabecular bone organoids with osteoclast metabolic activity according to claim 1, characterized in that: In step (S1), the amount ratio of osteoclast precursor cells, bone organoids with autonomous mineralization ability, and culture medium containing M-CSF is 5×10 4～5 Cells: 1: 100 μL.

4. The method for preparing trabecular bone organoids with osteoclast metabolic activity according to claim 3, characterized in that: Bone organoids with autonomous mineralization ability were prepared by the following method: (A1) Mix Matrigel and Collagen I working solution to obtain organoid matrix working solution; (A2) The osteoblast precursor cell line is suspended in the organoid matrix working solution prepared in step (A1) and placed in the PDMS chip. After solidification, the cell line is immersed in α-MEM culture solution containing 10% FBS to obtain bone organoids. (A3) treating the bone organoids prepared in step (A2) with an osteogenic induction culture medium to obtain bone organoids with autonomous mineralization ability.

5. The method for preparing trabecular bone organoids with osteoclast metabolic activity according to claim 3, characterized in that: The concentration of M-CSF in the M-CSF-containing culture medium is 30 to 60 ng / mL.

6. The method for preparing trabecular bone organoids with osteoclast metabolic activity according to claim 1, characterized in that: In step (S3), in the culture medium containing RANKL and M-CSF, the concentration of RANKL is 30 to 60 ng / mL, and the concentration of M-CSF is 30 to 60 ng / mL.

7. The method for preparing trabecular bone organoids with osteoclast metabolic activity according to claim 6, characterized in that: In the medium containing RANKL and M-CSF, the concentration of RANKL was 50 ng / mL, and the concentration of M-CSF was 50 ng / mL.

8. The method for preparing trabecular bone organoids with osteoclast metabolic activity according to claim 1, characterized in that: In step (S3), during the induction culture, the temperature is 37°C, 5% CO2, and the time is 1 to 35 days.

9. A trabecular bone organoid with osteoclast metabolic activity, characterized in that: It is prepared by the method according to any one of claims 1 to 8.

10. Use of the trabecular bone organoid with osteoclast metabolic activity as claimed in claim 9 in osteoclast-osteogenesis interaction research, drug screening or target verification.

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