Bone organ model and construction method and application thereof

Abstract

The invention provides a bone organ model as well as a construction method and application thereof, and the method comprises the following steps: arranging a biological material with a micropore array in a cell culture plate to obtain a bone organ chip; suspending the initial stem cells in a culture medium, and promoting the cells to form a spherical structure through cell aggregation or by using a non-adhesive culture surface, so as to obtain cell spheres; and culturing the cell spheres in the bone organ chip by adopting the culture medium for constructing the bone organ model to obtain the bone organ model. The skeletal organ is successfully constructed through in-vitro self-organized 3D co-culture initial stem cells including embryonic stem cells, induced pluripotent stem cells, bone marrow mesenchymal stem cells, osteoblasts, bone cells, all levels of osteogenic precursor cells, osteogenic-like cells and MC3T3-E1 cells, and the skeletal organ can become an important tool for researching and treating bone related diseases.

Description

Technical Field

[0001] The present invention relates to the technical field of orthopedic organoids, and in particular to a bone organoid model and a construction method and application thereof. Background Art

[0002] Bone defects, especially large bone defects, are a major clinical problem. Bone defects can be caused by tumors, trauma, infection, etc., and are a common trauma in clinical practice. Existing methods for repairing large bone defects have obvious defects, and more effective methods are needed to enhance bone regeneration and accelerate healing. At the same time, severe fractures such as vertebral compression fractures also require more effective clinical solutions to relieve patients' pain and avoid the sequelae of bone cement hardening.

[0003] In recent years, adult stem cells or pluripotent stem cells have been used for three-dimensional in vitro culture to form tissue analogs with a certain spatial structure, namely organoids. Organoid technology has achieved success in the fields of intestine, brain, liver, kidney, pancreas, etc., and was named "Method of the Year" by Nature Methods magazine. Bone organoids are 3D self-organized and self-renewing micro-bone tissues (including osteoblasts, osteocytes, osteoclasts or hematopoietic cell units, etc.) with bone bionic spatial characteristics that are formed by the directed differentiation of stem cells (such as bone stem cells, embryonic stem cells, induced pluripotent stem cells, etc.) or progenitor cells. Organoids that have the basic unit structure and function of bones are also an important tool for the study of emerging bone-related diseases. A key challenge in the construction of bone organoids is to realize an interactive tissue structure system containing different cell types in a 3D tissue structure. There are many unresolved problems, such as: in vitro, the model of osteoblast-based cell line differentiation into bone cells has not yet produced a fully developed mineralized collagen matrix; the differentiation of human bone marrow-derived mesenchymal stem cells (hBMSC) into osteocytes (accounting for 90-95% of the cell part of bone tissue) has not yet been achieved in vitro, etc. These problems are still the key restrictive steps for inducing the formation of bone organoids in vitro. MC3T3-E1 cells are mouse cranial embryonic osteoblast precursor cells with good osteogenic differentiation potential and mineralization potential, and are widely used in osteogenic differentiation and mineralization deposition research. Bone organoids can be directedly differentiated from progenitor cells. They are self-organized and self-renewing tissues with 3D spatial characteristics and have basic bone-related structures and functions. Therefore, MC3T3-E1 cells can be used to construct bone organoids, and this osteoblast precursor cell has fewer differentiation steps and a clear osteogenic differentiation direction in the process of constructing osteogenic differentiation. It has significant advantages in the construction of clinical applications of bone organoids and is an ideal cell for designing bone organoids.

[0004] Therefore, it is necessary to develop a bone organoid model to address the problems in the existing orthopedic field, such as insufficient bone defect filling materials, poor effects, limited allogeneic bone resources, rejection reactions, infection problems, insufficient bone tissue organs, poor osteogenesis of inactivated bone tissue, insufficient clinical research models for bone-related diseases, and limited treatment methods for bone defects, especially large bone defects. Summary of the invention

[0005] The purpose of the present invention is to provide a bone organoid model and its construction method and application, which can realize the structurally controllable, high-throughput construction of morphologically stable bone organoids, and can observe the entire process of bone organoid differentiation and development in situ, and can be used as a research tool for bone-related diseases and the treatment of clinical bone defect-related diseases.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] In a first aspect of the present invention, a culture medium for constructing a bone organoid model is provided, wherein the culture medium for constructing a bone organoid model comprises:

[0008] Osteoblast maturation medium: Add the following final concentrations of inducing molecules to the basal medium: 40-60 μg / mL AsAP, 8-12 mM β-GP, 0.08-0.12 μM Dex.

[0009] Furthermore, the starting cells suitable for the culture medium for constructing the bone organoid model include one of the following:

[0010] Embryonic stem cells, induced pluripotent stem cells (iPSCs);

[0011] Bone marrow mesenchymal stem cells, osteoblast precursor cells at all levels, osteoblast-like cells, MC3T3-E1 cells;

[0012] osteoblast, bone cell;

[0013] The culture medium for constructing a bone organoid model using bone marrow mesenchymal stem cells, osteoblast precursor cells at various levels, osteoblast-like cells, and MC3T3-E1 cells as starting cells also includes:

[0014] Osteogenic differentiation medium: add the following induction molecules to the basal medium at the final concentration: 40-60 μg / mL AsAP, 8-12 mM β-GP, 0.08-0.12 μM Dex, 0.8-1.2 μM SAG, 0.8-1.2 μM TH;

[0015] Wherein, the culture medium for constructing the bone organoid model using embryonic stem cells and induced pluripotent stem cells (iPSCs) as starting cells also includes:

[0016] iPSCs culture medium: add the following final concentrations of induction molecules to the basal culture medium: 800-1200 units / mL LLI-leukemia inhibitory factor, 0.8-1.2 μM PD0325901, 2-4 μM CHIR99021;

[0017] Mesoderm induction medium: add the following final concentrations of induction molecules to the basal medium: 20-40 μM CHIR and 3-7 μM Cyc;

[0018] Ectodermal and endoderm induction medium: The ectoderm and endoderm induction medium is prepared by adding the following induction molecules to the basal medium at the final concentration: 3-7 μM Retinoic acid;

[0019] Osteogenic differentiation medium: Add the following final concentrations of inducing molecules to the basal medium: 40-60 μg / mL AsAP, 8-12 mM β-GP, 0.08-0.12 μM Dex, 0.8-1.2 μM SAG, and 0.8-1.2 μM TH.

[0020] Furthermore, the basal culture medium is selected from DMEM / F-12 high-glucose basal culture medium (11330-032, Gibco) and is prepared by adding neural basal culture fluid (21103-049, Gibco), N2 additive (17502-048, Gibco), B27 additive (17504044, Gibco), bovine serum albumin V (A3059, Sigma-Aldrich), and double-resistance penicillin-streptomycin solution (P4458, Sigma-Aldrich).

[0021] In a second aspect of the present invention, a set of supplementary products for constructing a bone organoid model derived from embryonic stem cells and iPSCs is provided, comprising at least one of the following:

[0022] A. Complete set of supplementary products for constructing embryonic stem cell and iPSC-derived bone organoid models:

[0023] The induction molecules used to prepare the iPSCs culture medium: 800-1200 units / mL LIF-leukemia inhibitory factor, 0.8-1.2 μM PD0325901, 2-4 μM CHIR99021;

[0024] The induction molecules used to prepare the mesoderm induction medium: 20-40 μM CHIR and 3-7 μM Cyc;

[0025] The medium for preparing ectoderm and endoderm induction: 3-7 μM Retinoic acid;

[0026] The inducing molecules used to prepare the bone differentiation medium: 40-60 μg / mL AsAP, 8-12 mM β-GP, 0.08-0.12 μM Dex;

[0027] The inducing molecules used to prepare the osteoblast maturation medium include: 40-60 μg / mL AsAP, 8-12 mM β-GP, 0.08-0.12 μM Dex;

[0028] B. A complete set of supplementary products for bone organoid models derived from bone marrow mesenchymal stem cells, osteoblast precursor cells at all levels, osteoblast-like cells, and MC3T3-E1, including:

[0029] The osteogenic differentiation medium is prepared by adding the following induction molecules at final concentrations to the basic medium: 40-60 μg / mL AsAP, 8-12 mM β-GP, 0.08-0.12 μM Dex, 0.8-1.2 μM SAG, and 0.8-1.2 μM TH;

[0030] The osteoblast maturation medium is prepared by adding the following induction molecules at final concentrations to the basal medium: 40-60 μg / mL AsAP, 8-12 mM β-GP, and 0.08-0.12 μM Dex;

[0031] C. Complete set of supplementary products for osteoblasts and osteocyte-derived bone organoids, including:

[0032] The osteoblast maturation medium is prepared by adding the following induction molecules at final concentrations to the basic medium: 40-60 μg / mL AsAP, 8-12 mM β-GP, and 0.08-0.12 μM Dex.

[0033] In a third aspect of the present invention, a method for constructing a bone organoid model is provided, the method comprising:

[0034] placing a biomaterial having a microwell array in the cell culture plate to obtain a bone organ chip;

[0035] Initial stem cells are suspended in culture medium and promoted to form spherical structures by cell aggregation or using a non-adhesive culture surface to obtain cell spheres;

[0036] The cell sphere is cultured in the bone organoid chip using the culture medium for constructing a bone organoid model to obtain a bone organoid model.

[0037] In a fourth aspect of the present invention, a bone organoid model prepared by the method is provided.

[0038] In the fifth aspect of the present invention, provided is the use of the bone organoid model in the preparation of products for treating fractures, large bone defects, rheumatoid arthritis, and ankylosing spondylitis.

[0039] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0040] 1. The present invention successfully induced the mineralization process of iPSCs and MC3T3-E1 cells in vitro: by culturing MC3T3-E1 cells in a medium containing inducers (β-GP and ascorbic acid) and a medium without inducers, it was observed that the cells in the experimental group had obvious mineralized nodules on the 21st day, while no mineralized nodules were formed in the control group. This shows that the method of the present invention can successfully induce the mineralization process of MC3T3-E1 cells. At the same time, iPSCs were cultured using a culture medium containing inducers (LIF-leukemia inhibitory factor, PD0325901, CHIR99021) and a culture medium without inducers, mesoderm induction was performed using a culture medium containing inducers (CHIR, Cyc) and a culture medium without inducers, and then osteogenic differentiation culture was performed using a culture medium containing inducers (AsAP, β-GP, Dex, SAG, TH) and a culture medium without inducers. Finally, osteoblast maturation culture was performed using a culture medium containing inducers (AsAP, β-GP, Dex) and a culture medium without inducers. It was observed that the experimental group cells began to show increased expression of mesoderm-related gene markers T and Mixl1 from the 5th day, and increased expression of osteogenic-related gene markers Runx2 and Cola1 from the 19th day, indicating that the method of the present invention can successfully induce iPSCs cells to undergo differentiation and maturation of bone organoids.

[0041] 2. Provide scientific basis for bone tissue engineering: This study simulated the internal bone environment of the human body through three-dimensional culture, explored the effects of induction factors and dynamic loads on the biological effects of iPSCs and MC3T3-E1 osteoblast-like cells, and provided new ideas and scientific basis for bone tissue engineering.

[0042] 3. In-depth understanding of the regulatory mechanism of the human internal bone environment on the biological effects of iPSCs and MC3T3-E1-like cells: The research results are expected to provide a deep understanding of the regulatory mechanism of the human internal bone environment on the biological effects of iPSCs and MC3T3-E1-like cells, which will help to develop more scientific artificial bone models and treatment plans, thereby improving their therapeutic effects and clinical application value.

[0043] 4. The effects of different induction factors, culture medium composition and mechanical strength on cell differentiation were investigated: By exploring the biological response of cells to different factor intensities under three-dimensional culture conditions, the present invention provides a theoretical basis for optimizing the culture strategy and biomaterial design of bone tissue engineering.

[0044] 5. Provide reference and reference for other osteoblast-like cells and cell culture fields: This study not only provides new ideas and scientific basis for bone tissue engineering, but also provides reference and reference for other osteoblast-like cells and cell culture fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings in the following description are some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative work.

[0046] Figure 1 Agarose microarray 3D culture induced differentiation of MC3T3-E1 cells: (A) Technical route for constructing bone organoids by 3D culture induced differentiation of MC3T3-E1 cells; (BC) Bright field microscope image of 3D culture of MC3T3-E1 cells; (DE) DAPI staining image of MC3T3-E1 cell nuclei.

[0047] Figure 2 Calcein-AM / PI staining of MC3T3-E1 cells after one week of 3D induced differentiation culture: (A, D) Calcein-AM characterizes the living cells in the micro-organoids; (B, E) PI staining characterizes the dead cells in the micro-organoids; (C, F) Merge staining.

[0048] Figure 3 Calcein-AM / PI staining of MC3T3-E1 cells after two weeks of 3D differentiation culture: (A) Calcein-AM characterizes the living cells in the microorganoids; (B) PI staining characterizes a small number of dead cells in the center of the microorganoids; (C). Merge staining.

[0049] Figure 4 Immunofluorescence staining of ALP and DMP1 in MC3T3-E1 cells after two weeks of 3D induced differentiation culture: (A) ALP immunofluorescence staining of microorganoids; (B) DAPI nucleus staining of microorganoids; (C) ALP / DAPI Merge staining; (D) DMP1 immunofluorescence staining of microorganoids; (E) DAPI nucleus staining of microorganoids; (F) DMP1 / DAPI Merge staining.

[0050] Figure 5 The morphology and mineralization characterization of MC3T3-E1 3D micro-organoids after two weeks of induced differentiation culture: (A) is the bright field 3D morphology of the micro-organoids; (B) is the Alizarin red staining experiment after two weeks of micro-organoid differentiation culture.

[0051] Figure 6 The osteogenesis-related gene Runx2 ( Figure 6 A),ALP( Figure 6 B) expression.

[0052] Figure 7 The osteogenesis-related gene Runx2 ( Figure 7 A),ALP( Figure 7 B) expression.

[0053] Figure 8 The qPCR experiment in Example 4 detected the mesoderm-related gene marker T ( Figure 8 A), Mixl1( Figure 8 B) Expression.

[0054] Fig. 9 The qPCR experiment in comparative example 2 detected the mesoderm-related gene marker T ( Fig. 9 A), Mixl1( Fig. 9 B) Expression. DETAILED DESCRIPTION

[0055] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0056] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0057] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or obtained by existing methods.

[0058] The general idea of ​​the present invention is as follows:

[0059] The present invention provides culture medium suitable for different starting cells:

[0060] (1) First culture medium

[0061] Culture media suitable for using embryonic stem cells and induced pluripotent stem cells (iPSCs) as starting cells for the construction of bone organoid models, including:

[0062] iPSCs culture medium: add the following final concentrations of induction molecules to the basal culture medium: 800-1200 units / mL LLI-leukemia inhibitory factor, 0.8-1.2 μM PD0325901, 2-4 μM CHIR99021;

[0063] Mesoderm induction medium: add the following final concentrations of induction molecules to the basal medium: 20-40 μM CHIR and 3-7 μM Cyc;

[0064] Ectodermal and endoderm induction medium: The ectoderm and endoderm induction medium is prepared by adding the following induction molecules to the basal medium at the final concentration: 3-7 μM Retinoic acid;

[0065] Osteogenic differentiation medium: add the following induction molecules to the basal medium at the final concentration: 40-60 μg / mL AsAP, 8-12 mM β-GP, 0.08-0.12 μM Dex, 0.8-1.2 μM SAG, 0.8-1.2 μM TH;

[0066] Osteoblast maturation medium: Add the following final concentrations of inducing molecules to the basal medium: 40-60 μg / mL AsAP, 8-12 mM β-GP, 0.08-0.12 μM Dex.

[0067] (2) Second culture medium

[0068] Suitable for bone marrow mesenchymal stem cells, osteoblast precursor cells at all levels, osteoblast-like cells, and MC3T3-E1 cells as starting cells for constructing bone organoid models, including:

[0069] Osteogenic differentiation medium: add the following induction molecules to the basal medium at the final concentration: 40-60 μg / mL AsAP, 8-12 mM β-GP, 0.08-0.12 μM Dex, 0.8-1.2 μM SAG, 0.8-1.2 μM TH;

[0070] Osteoblast maturation medium: Add the following final concentrations of inducing molecules to the basal medium: 40-60 μg / mL AsAP, 8-12 mM β-GP, 0.08-0.12 μM Dex.

[0071] (3) The third culture medium

[0072] Suitable for osteoblasts, osteoblasts as starting cells for the construction of bone organoid models, including:

[0073] Osteoblast maturation medium: Add the following final concentrations of inducing molecules to the basal medium: 40-60 μg / mL AsAP, 8-12 mM β-GP, 0.08-0.12 μM Dex.

[0074] The inventors of this application discovered that:

[0075] The osteogenic differentiation culture medium can successfully induce the mineralization process of MC3T3-E1 cells by containing inducers (β-GP and ascorbic acid).

[0076] iPSCs were cultured using a culture medium containing inducers (LIF-leukemia inhibitory factor, PD0325901, CHIR99021) and a culture medium without inducers, mesoderm induction was performed using a culture medium containing inducers (CHIR, Cyc) and a culture medium without inducers, and then osteogenic differentiation culture was performed using a culture medium containing inducers (AsAP, β-GP, Dex, SAG, TH) and a culture medium without inducers. Finally, osteoblast maturation culture was performed using a culture medium containing inducers (AsAP, β-GP, Dex) and a culture medium without inducers. It was observed that the experimental group cells showed increased expression of mesoderm-related gene markers T and Mixl1 from the 5th day, and increased expression of osteogenic-related gene markers Runx2 and Cola1 from the 19th day, indicating that the method of the present invention can successfully induce iPSCs cells to undergo differentiation and maturation of bone organoids.

[0077] Explanation of terms in this invention:

[0078] iPSC is the abbreviation for human induced pluripotent stem cells;

[0079] AsAP is ascorbyl phosphate, purchased from Sigma-Aldrich, catalog number A4034;

[0080] β-GP is β-glycerophosphate, purchased from Sigma-Aldrich, catalog number G9422;

[0081] Dex is dexamethasone, purchased from Wako, catalog number 41-18861;

[0082] SAG is smoothened agonist, purchased from Calbiochem, catalog number 566660;

[0083] TH is the helium oxadiazole derivative 4-(4-methoxyphenyl)pyridinyl[40,30:4,5]thieno[2,3-b]pyridine-2-carboxamide, synthesized by Takeda Chemical Co., Ltd., Japan and distributed on request;

[0084] LIF is leukemia inhibitory factor, purchased from Millipore, catalog number ESG1107;

[0085] PD0325901 is a selective, cell-permeable MEK / ERK pathway inhibitor purchased from STEMCELL Technologies with catalog numbers 72184 and 72182;

[0086] CHIR99021 is a selective GSK-3α / β inhibitor purchased from STEMCELL Technologies with catalog number 72052;

[0087] CHIR is a class of inhibitors targeting specific proteins (such as GSK-3α / β, Chk1, etc.), purchased from Wako and Axon, with catalog numbers 039-20831 and 1386;

[0088] Cyc is cyclopamine, purchased from Enzo Life Sciences, catalog number BML-GR334;

[0089] Retinoic acid is retinoic acid purchased from Yeasen with the product number 53001ES60;

[0090] The human embryonic stem cells of the present invention refer to commercially purchased human embryonic stem cells within 14 days.

[0091] The present application will be described in detail below with reference to embodiments and experimental data.

[0092] Example 1: Construction of bone organoids using MC3T3-E1 cells

[0093] (1) 3D microarray design

[0094] Use aotoCAD software to design an array with a diameter of 0.5 mm and a height of 1 mm per well on the well plate based on the size of the organoids.

[0095] (2) 3D microarray processing of polymethyl methacrylate (PMMA) plates

[0096] Use a CNC machine tool to process the PMMA plate according to the pattern designed by aotoCAD software to obtain a porous array template.

[0097] (3) Construction of polydimethylsiloxane (PDMS) 3D microarray

[0098] Use PDMS for molding, pour the degassed PDMS prepolymer onto the PMMA template, polymerize at 80°C for 60 min, remove the mold and rinse it for later use.

[0099] (4) Construction of agarose 3D microarray

[0100] Use 2wt% agarose gel for secondary molding, pour the heated and dissolved agarose solution on the PDMS array template, cool and solidify for 20 minutes, demold and use an 11mm hole puncher to punch holes, put the punched array into a 48-well plate with sterile water, sterilize under ultraviolet light for 30 minutes and set aside.

[0101] (5) Construction of MC3T3-E1 cell 3D spheroids

[0102] MC3T3-E1 cells were cultured at a cell density of 1×10 5 The cells / well were inoculated into a six-well plate, and the neural basal culture medium was added to the DMEM / F-12 high-glucose basal medium, 2% N2 additive, 2% B27 additive, 10% bovine serum albumin V + 1% double anti-penicillin-streptomycin solution (100u / mL penicillin, 100mg / mL streptomycin) medium for three days for use. The MC3T3-E1 cells were digested with trypsin and counted, and 5000 cells were inoculated into the agarose gel array in the 48-well plate in each array microwell, and the culture medium was added for two days, and the cells were centrifuged intermittently to make the cells spontaneously spherical.

[0103] (6) 3D culture directional differentiation

[0104] The target cell spheres were 3D cultured using a 3D microarray structure. Neural basal culture medium was added to DMEM / F-12 high-glucose basal culture medium, 2% N2 additive, 2% B27 additive, 10% bovine serum albumin V + 1% double-antibiotic penicillin-streptomycin solution (100u / mL penicillin, 100mg / mL streptomycin) + 50μg / mL ascorbic acid phosphate (AsAP) + 10mM β-glycerophosphate (β-GP) + 0.1μM dexamethasone (Dex) for directed induction differentiation culture, and the medium was changed every other day. After 21 days of culture, qPCR was used to detect the expression of osteogenic-related genes Runx2 and ALP in the cell spheres. Figure 6 shown.

[0105] (7) Bone organoid maturation

[0106] Supplement with sufficient nutrients and gases, place in a carbon dioxide incubator (Thermo) at 37°C, 5% CO2 and 95% humidity for differentiation culture, replace new culture medium every 3 days, continue directed induction differentiation culture for 1 month to form mature bone organoids.

[0107] (8) Calcein-AM / PI staining of bone organoids to identify organoid activity:

[0108] Prepare 1× staining buffer, add 10 μL of staining solution A and 10 μL of staining solution B to every 10 mL of 1× staining buffer and mix thoroughly to prepare the staining solution, collect 3D cultured MC3T3-E1 bone organoids, wash them three times with PBS, re-add 200 μL of staining solution to each well, incubate at 37°C in the dark for 30 min, then re-wash the micro-organoids with PBS, resuspend them with an appropriate amount of PBS, and finally use a laser confocal fluorescence microscope to detect live and dead cells at wavelengths of 490±10 nm and 545 nm, and perform Merge observation.

[0109] (9) Immunofluorescence staining of bone organoids:

[0110] MC3T3-E1 bone organoids in agarose microarray were collected and fixed with 4% paraformaldehyde for 2 hours at room temperature. The bone organoids were then washed three times with PBS, incubated with PBS containing 0.1% Triton X-100 for 60 minutes for cell permeabilization, and then washed three times with PBS. Blocking was performed with 10% goat serum and 1% BSA in PBS at room temperature for 2 hours. The primary and secondary antibodies were diluted in the blocking solution at a ratio of 1:50 and 1:200, respectively. 3D bone organoids were incubated with rabbit anti-mouse DMP1 and ALP antibodies at 4°C overnight. After washing three times with PBS, goat anti-rabbit antibodies containing Alexa Fluor 647 were added to the samples and incubated for 1 hour at room temperature. After washing three times with PBS, 100 μL of phalloidin conjugated with Alexa Fluor488 (DAPI, 10 ng / μL) was added, and fluorescence imaging was performed using a laser confocal microscope.

[0111] (10) Bone organoid morphology observation and Alizarin red assay:

[0112] The morphology and structure of bone organoids were observed using a laser confocal microscope with bright field. The samples were then fixed and stained with Alizarin Red stain (American Master Tech) for 60 min. The samples were washed with ddH2O until the washing solution was clear. The mineralized samples stained with Alizarin Red were imaged using an optical microscope (EVOS) with a multi-magnification objective.

[0113] like Figure 1As shown in A, MC3T3-E1 cells were inoculated in an agarose microarray structure, and after centrifugation, the cells were maintained and subsequently induced to differentiate in the form of 3D microspheres. As shown in Figure BE, within the first 6 hours, MC3T3-E1 cells were relatively dispersed in the agarose microarray, and after DAPI nuclear staining, it was seen that the MC3T3-E1 cells in the microarray structure were distributed in the shape of microspheres. In the subsequent differentiation culture, MC3T3-E1 cells self-assembled to form a compact spherical shape. In this project study, the cell aggregation, cohesion and differentiation of MC3T3-E1 microspheres were studied for 2 weeks.

[0114] Example 2. Construction of bone organoids by mixing MC3T3-E1 cells with human umbilical vein endothelial cells (HUVEC cells)

[0115] (1) 3D microarray design

[0116] Use aotoCAD software to design an array with a diameter of 0.5 mm and a height of 1 mm per well on the well plate based on the size of the organoids.

[0117] (2) 3D microarray processing of polymethyl methacrylate (PMMA) plate: A CNC machine tool is used to process the PMMA plate according to the pattern designed by aotoCAD software to obtain a porous array template.

[0118] (3) Construction of polydimethylsiloxane (PDMS) 3D microarrays: PDMS was used for molding. The degassed PDMS prepolymer was poured onto the PMMA template and polymerized at 80 °C for 60 min. The mold was removed and rinsed for later use.

[0119] (4) Construction of agarose 3D microarrays: 2 wt% agarose gel was used for secondary molding. The heated and dissolved agarose solution was poured onto the PDMS array template, cooled and solidified for 20 min, demolded, and punched using an 11 mm hole puncher. The punched array was placed in a 48-well plate with sterile water, sterilized under ultraviolet light for 30 min, and then set aside.

[0120] (5) Construction of MC3T3-E1 cell 3D spheroids

[0121] MC3T3-E1 cells were inoculated into six-well plates at a cell density of 1×105 cells / well, and DMEM / F-12 high-glucose basal medium was added with neural basal culture medium, 2% N2 additive, 2% B27 additive, 10% bovine serum albumin V + 1% double anti-penicillin-streptomycin solution (100u / mL penicillin, 100mg / mL streptomycin) culture medium for three days for use. Human umbilical vein endothelial cells HUVEC cells were inoculated into six-well plates at a cell density of 1×10 5The cells / well were inoculated into a six-well plate and cultured with ECM medium for two days. MC3T3-E1 cells and HUVEC cells were digested with trypsin and counted separately. The cells were inoculated into agarose gel arrays in 48-well plates at a ratio of MC3T3-E1:HUVEC=7:3, with 5000 cells per array microwell. DMEM / F-12 high-glucose basal medium:ECM=7:3 mixed medium was added and cultured for two days, followed by intermittent centrifugation to allow the cells to spontaneously spheroidize.

[0122] (6) 3D culture directional differentiation

[0123] The target cell spheres were 3D cultured using a 3D microarray structure, and neural basal culture medium was added to DMEM / F-12 high-glucose basal culture medium, 2% N2 additive, 2% B27 additive, 10% bovine serum albumin V + 1% double-antibiotic penicillin-streptomycin solution (100u / mL penicillin, 100mg / mL streptomycin) + 50μg / mL ascorbic acid phosphate (AsAP) + 10mMβ-glycerophosphate (β-GP) + 0.1μM dexamethasone (Dex) for directed induced differentiation culture, and the medium was changed every other day.

[0124] (7) Bone organoid maturation

[0125] Supplement with sufficient nutrients and gases, place in a carbon dioxide incubator (Thermo) at 37°C, 5% CO2 and 95% humidity for differentiation culture, replace new culture medium every 3-5 days, continue directed induction differentiation culture for 1 month to form mature bone organoids.

[0126] (8) Calcein-AM / PI staining of bone organoids to identify organoid activity:

[0127] Prepare 1× staining buffer, add 10μL staining solution A and 10μL staining solution B to every 10mL 1× staining buffer, mix thoroughly to prepare staining solution, collect 3D cultured MC3T3-E1 bone organoids, wash them three times with PBS, add 200μL staining solution to each well, incubate at 37℃ in the dark for 30 minutes, wash the microtissues again with PBS, resuspend them with an appropriate amount of PBS, and finally use a laser confocal fluorescence microscope to detect live and dead cells at wavelengths of 490±10nm and 545nm, and perform Merge observation.

[0128] (9) Immunofluorescence staining of bone organoids:

[0129] MC3T3-E1 bone organoids in agarose microarray were collected and fixed with 4% paraformaldehyde for 2 hours at room temperature. The bone organoids were then washed three times with PBS, incubated with PBS containing 0.1% Triton X-100 for 60 minutes for cell permeabilization, and then washed three times with PBS. Blocking was performed with 10% goat serum and 1% BSA in PBS at room temperature for 2 hours. The primary and secondary antibodies were diluted in the blocking solution at a ratio of 1:50 and 1:200, respectively. 3D bone organoids were incubated with rabbit anti-mouse DMP1 and ALP antibodies at 4°C overnight. After washing three times with PBS, goat anti-rabbit antibodies containing Alexa Fluor 647 were added to the samples and incubated for 1 hour at room temperature. After washing three times with PBS, 100 μL of phalloidin conjugated with Alexa Fluor488 (DAPI, 10 ng / μL) was added, and fluorescence imaging was performed using a laser confocal microscope.

[0130] (10) Bone organoid morphology observation and Alizarin red assay:

[0131] The morphology and structure of bone organoids were observed using a laser confocal microscope with bright field. The samples were then fixed and stained with Alizarin Red stain (American Master Tech) for 60 min. The samples were washed with ddH2O until the washing solution was clear. The mineralized samples stained with Alizarin Red were imaged using an optical microscope (EVOS) with a multi-magnification objective.

[0132] Example 3. Construction of bone organoids using BMSC cells

[0133] (1) 3D microarray design

[0134] Use aotoCAD software to design an array with a diameter of 0.5 mm and a height of 1 mm per well on the well plate based on the size of the organoids.

[0135] (2) 3D microarray processing of polymethyl methacrylate (PMMA) plates

[0136] Use a CNC machine tool to process the PMMA plate according to the pattern designed by aotoCAD software to obtain a porous array template.

[0137] (3) Construction of polydimethylsiloxane (PDMS) 3D microarray

[0138] Use PDMS for molding, pour the degassed PDMS prepolymer onto the PMMA template, polymerize at 80°C for 60 min, remove the mold and rinse it for later use.

[0139] (4) Construction of agarose 3D microarray

[0140] Use 2wt% agarose gel for secondary molding, pour the heated and dissolved agarose solution on the PDMS array template, cool and solidify for 20 minutes, demold and use an 11mm hole puncher to punch holes, put the punched array into a 48-well plate with sterile water, sterilize under ultraviolet light for 30 minutes and set aside.

[0141] (5) Construction of BMSC cell 3D spheroids

[0142] BMSCs were cultured at a cell density of 1×10 5 The cells / well were inoculated into a six-well plate, and the neural basal culture medium was added to the DMEM / F-12 high-glucose basal culture medium, 2% N2 additive, 2% B27 additive, 10% bovine serum albumin V + 1% double-antibiotic penicillin-streptomycin solution (100u / mL penicillin, 100mg / mL streptomycin) culture medium was cultured for three days for use. The BMSC cells were digested with trypsin and counted, and 5,000 cells were inoculated into the agarose gel array in the 48-well plate in each array microwell. The culture medium was added and cultured for two days, and the cells were centrifuged intermittently to allow the cells to spontaneously spheroidize.

[0143] (6) 3D culture directional differentiation

[0144] The target cell spheres were 3D cultured using a 3D microarray structure, and neural basal culture medium was added to DMEM / F-12 high-glucose basal culture medium, 2% N2 additive, 2% B27 additive, 10% bovine serum albumin V + 1% double-antibiotic penicillin-streptomycin solution (100u / mL penicillin, 100mg / mL streptomycin) + 50μg / mL ascorbic acid phosphate (AsAP) + 10mMβ-glycerophosphate (β-GP) + 0.1μM dexamethasone (Dex) for directed induced differentiation culture, and the medium was changed every other day.

[0145] (7) Bone organoid maturation

[0146] Supplement with sufficient nutrients and gases, place in a carbon dioxide incubator (Thermo) at 37°C, 5% CO2 and 95% humidity for differentiation culture, replace new culture medium every 3-5 days, continue directed induction differentiation culture for 1 month to form mature bone organoids.

[0147] (8) Calcein-AM / PI staining of bone organoids to identify organoid activity:

[0148] Prepare 1× staining buffer, add 10 μL of staining solution A and 10 μL of staining solution B to every 10 mL of 1× staining buffer and mix thoroughly to prepare the staining solution, collect 3D cultured MC3T3-E1 bone organoids, wash them three times with PBS, re-add 200 μL of staining solution to each well, incubate at 37°C in the dark for 30 min, then re-wash the micro-organoids with PBS, resuspend them with an appropriate amount of PBS, and finally use a laser confocal fluorescence microscope to detect live and dead cells at wavelengths of 490±10 nm and 545 nm, and perform Merge observation.

[0149] (9) Immunofluorescence staining of bone organoids:

[0150] BMSC bone organoids in agarose microarray were collected and fixed with 4% paraformaldehyde for 2 hours at room temperature. Bone organoids were then washed three times with PBS, incubated with PBS containing 0.1% Triton X-100 for 60 minutes for cell permeabilization, and then washed three times with PBS. Blocking was performed with 10% goat serum and 1% BSA in PBS at room temperature for 2 hours. Primary and secondary antibodies were diluted in blocking solution at a ratio of 1:50 and 1:200, respectively. 3D bone organoids were incubated with rabbit anti-mouse DMP1 and ALP antibodies at 4°C overnight. After washing three times with PBS, goat anti-rabbit antibody containing Alexa Fluor 647 was added to the samples and incubated for 1 hour at room temperature. After washing three times with PBS, 100 μL of phalloidin conjugated with Alexa Fluor 488 (DAPI, 10 ng / μL) was added, and fluorescence imaging was performed using a laser confocal microscope.

[0151] (10) Bone organoid morphology observation and Alizarin red assay:

[0152] The morphology and structure of bone organoids were observed using a laser confocal microscope with bright field. The samples were then fixed and stained with Alizarin Red stain (American Master Tech) for 60 min. The samples were washed with ddH2O until the washing solution was clear. The mineralized samples stained with Alizarin Red were imaged using an optical microscope (EVOS) with a multi-magnification objective.

[0153] Figure 2 . Calcein-AM / PI staining of MC3T3-E1 cells after 3D differentiation culture for one week: (A, D). Calcein-AM characterizes the living cells in the microorganoids; (B, E). PI staining characterizes the dead cells in the microorganoids; (C, F). Merge staining. Figure 2As shown in A and 2D, after one week of induced differentiation culture, most cells in the microtissue still have significant proliferation activity, and Calcein-AM / PI staining shows large areas of green living cells. Figure 2 As shown in C and 2F, the cells in the microtissues were still dispersed after one week, with a dispersion diameter of about 200 μm. A small amount of dead cells were also observed in the microtissues ( Figure 2 B, 2E).

[0154] Figure 3 .Calcein-AM / PI staining of MC3T3-E1 cells after two weeks of 3D induced differentiation culture: (A). Calcein-AM characterizes the living cells in the micro-organoids; (B). PI staining characterizes the small number of dead cells in the center of the micro-organoids; (C). Merge staining. After two weeks of induced differentiation culture, it can be observed that the micro-organoids are more dense, smaller in size, and become obvious 3D spherical shapes. The diameter of the micro-organoids is about 50-100μm. Calcein-AM / PI staining shows that most of the cells in the micro-organoids are viable ( Figure 3 A). At the same time, a central necrotic tissue can be observed in one of the microtissues, while the central necrosis of the other microtissue is not obvious.

[0155] Figure 4 . ALP and DMP1 immunofluorescence staining of MC3T3-E1 cells after two weeks of 3D induced differentiation culture: (A). ALP immunofluorescence staining of microorganoids; (B). DAPI nuclear staining of microorganoids; (C). ALP / DAPI Merge staining; (D). DMP1 immunofluorescence staining of microorganoids; (E). DAPI nuclei of microorganoids; (F). DMP1 / DAPI Merge staining. After two weeks of induced differentiation, osteoblasts expressing ALP and DMP1 covered most of the microorganoid surface. Microorganoid nuclear staining with DAPI showed that the microorganoids were reconstructed by a large number of cells, presenting a dense mammosphere tissue morphology, in which a large amount of ALP and DMP1 were expressed.

[0156] Figure 5 .Morphology and mineralization characterization of MC3T3-E1 3D microorganoids after two weeks of induced differentiation culture: (A). Bright field 3D morphology of microorganoids; (B) Alizarin red staining experiment after two weeks of microorganoid differentiation culture. It can be seen that the microorganoids present a clear 3D spherical structure, with dense tissue, high density in the center and slightly lower density in the periphery. The diameter of the microorganoid is about 50-100μm ( Figure 5 A). Alizarin red mineral staining experiments showed that osteoblasts almost completely covered the surface of the tissue after 2 weeks and continued to deposit mineralized substances. Dense mineralized substances were observed in the microtissues ( Figure 5 B).

[0157] Example 4 Construction of bone organoids from iPSCs through mesoderm-induced osteogenic differentiation

[0158] (1) 3D microarray design

[0159] Use aotoCAD software to design an array with a diameter of 0.5 mm and a height of 1 mm per well on the well plate based on the size of the organoids.

[0160] (2) 3D microarray processing of polymethyl methacrylate (PMMA) plates

[0161] Use a CNC machine tool to process the PMMA plate according to the pattern designed by aotoCAD software to obtain a porous array template.

[0162] (3) Construction of polydimethylsiloxane (PDMS) 3D microarray

[0163] Use PDMS for molding, pour the degassed PDMS prepolymer onto the PMMA template, polymerize at 80°C for 60 min, remove the mold and rinse it for later use.

[0164] (4) Construction of agarose 3D microarray

[0165] Use 2wt% agarose gel for secondary molding, pour the heated and dissolved agarose solution on the PDMS array template, cool and solidify for 20 minutes, demold and use an 11mm hole puncher to punch holes, put the punched array into a 48-well plate with sterile water, sterilize under ultraviolet light for 30 minutes and set aside.

[0166] (5) Construction of iPSCs 3D cell spheres

[0167] iPSCs cells were grown at a cell density of 1×10 5 The cells / well were inoculated into a six-well plate, and the neural basal culture medium was added to the DMEM / F-12 high-glucose basal medium, 2% N2 additive, 2% B27 additive, 10% bovine serum albumin V + 1% double anti-penicillin-streptomycin solution (100u / mL penicillin, 100mg / mL streptomycin) medium for three days for use. The iPSCs cells were digested with trypsin and counted, and 5,000 cells were inoculated into the agarose gel array in the 48-well plate in each array microwell, and the culture medium was added for two days, and the cells were centrifuged intermittently to make the cells spontaneously spherical.

[0168] (6) 3D culture mesoderm differentiation induction

[0169] The target cell spheres were cultured in 3D using a 3D microarray structure. DMEM / F-12 high-glucose basal medium was added with neural basal culture medium, 2% N2 additive, 2% B27 additive, 10% bovine serum albumin V + 1% double-antibiotic penicillin-streptomycin solution (100u / mL penicillin, 100mg / mL streptomycin) medium + 30μM CHIR and 5μM Cyc for mesoderm-directed differentiation. On the 5th day, qPCR experiments were performed to detect the expression of mesoderm-related gene markers T and Mixl1. Figure 8 shown.

[0170] (7) 3D culture osteogenic differentiation

[0171] Continue to use the 3D microarray structure to culture the target cell spheres in 3D, add the neural basal culture medium to the DMEM / F-12 high-glucose basal medium, add 2% N2 additives, add 2% B27 additives, 10% bovine serum albumin V + 1% double anti-penicillin-streptomycin solution (100u / mL penicillin, 100mg / mL streptomycin) medium + 50μg / mL ascorbic acid phosphate (AsAP) + 10mM β-glycerophosphate (β-GP) + 0.1μM dexamethasone (Dex) + 1μM SAG + 1μM TH for osteogenic differentiation directional induction differentiation culture, and change the medium every other day. On the 19th day, qPCR experiments were performed to detect the expression of osteogenic-related gene markers Runx2 and Cola1.

[0172] (8) 3D osteoblast maturation and differentiation culture

[0173] Continue to use the 3D microarray structure to 3D culture the target cell spheres, use DMEM / F-12 high-glucose basal culture medium to add neural basal culture medium, add 2% N2 additives, add 2% B27 additives, 10% bovine serum albumin V + 1% double-antibody penicillin-streptomycin solution (100u / mL penicillin, 100mg / mL streptomycin) culture medium + 50μg / mL AsAP + 10mMβ-GP + 0.1μM Dex for osteoblast maturation and differentiation culture, and change the medium every other day.

[0174] (9) Bone organoid maturation

[0175] Supplement with sufficient nutrients and gases, place in a carbon dioxide incubator (Thermo) at 37°C, 5% CO2 and 95% humidity for differentiation culture, replace new culture medium every 3-5 days, continue directed induction differentiation culture for 1 month to form mature bone organoids.

[0176] (10) Calcein-AM / PI staining of bone organoids to identify organoid activity:

[0177] Prepare 1× staining buffer, add 10μL staining solution A and 10μL staining solution B to every 10mL 1× staining buffer, mix thoroughly to prepare staining solution, collect 3D cultured bone organoids, wash three times with PBS, add 200μL staining solution to each well, incubate at 37℃ in the dark for 30 minutes, wash the microtissues again with PBS, resuspend with an appropriate amount of PBS, and finally use a laser confocal fluorescence microscope to detect live and dead cells at wavelengths of 490±10nm and 545nm, and perform Merge observation.

[0178] (11) Immunofluorescence staining of bone organoids:

[0179] Bone organoids in agarose microarrays were collected and fixed with 4% paraformaldehyde for 2 hours at room temperature. Bone organoids were then washed three times with PBS, incubated with PBS containing 0.1% Triton X-100 for 60 minutes for cell permeabilization, and then washed three times with PBS. Blocking was performed with 10% goat serum and 1% BSA in PBS at room temperature for 2 hours. Primary and secondary antibodies were diluted in blocking solution at a ratio of 1:50 and 1:200, respectively. 3D bone organoids were incubated with rabbit anti-mouse DMP1 and ALP antibodies at 4°C overnight. After washing three times with PBS, goat anti-rabbit antibodies containing Alexa Fluor 647 were added to the samples and incubated for 1 hour at room temperature. After washing three times with PBS, 100 μL of phalloidin conjugated with Alexa Fluor 488 (DAPI, 10 ng / μL) was added, and fluorescence imaging was performed using a laser confocal microscope.

[0180] (12) Bone organoid morphology observation and Alizarin red assay:

[0181] The morphology and structure of bone organoids were observed using a laser confocal microscope with bright field. The samples were then fixed and stained with Alizarin Red stain (American Master Tech) for 60 min. The samples were washed with ddH2O until the washing solution was clear. The mineralized samples stained with Alizarin Red were imaged using an optical microscope (EVOS) with a multi-magnification objective.

[0182] Comparative Example 1: Cultivating MC3T3-E1 cells without inducing agents to observe whether bone organoids can be constructed

[0183] (1) 3D microarray design

[0184] Use aotoCAD software to design an array with a diameter of 0.5 mm and a height of 1 mm per well on the well plate based on the size of the organoids.

[0185] (2) 3D microarray processing of polymethyl methacrylate (PMMA) plates

[0186] Use a CNC machine tool to process the PMMA plate according to the pattern designed by aotoCAD software to obtain a porous array template.

[0187] (3) Construction of polydimethylsiloxane (PDMS) 3D microarray

[0188] Use PDMS for molding, pour the degassed PDMS prepolymer onto the PMMA template, polymerize at 80°C for 60 min, remove the mold and rinse it for later use.

[0189] (4) Construction of agarose 3D microarray

[0190] Use 2wt% agarose gel for secondary molding, pour the heated and dissolved agarose solution on the PDMS array template, cool and solidify for 20 minutes, demold and use an 11mm hole puncher to punch holes, put the punched array into a 48-well plate with sterile water, sterilize under ultraviolet light for 30 minutes and set aside.

[0191] (5) Construction of MC3T3-E1 cell 3D spheroids

[0192] MC3T3-E1 cells were cultured at a cell density of 1×10 5 The cells / well were inoculated into a six-well plate, and the neural basal culture medium was added to the DMEM / F-12 high-glucose basal medium, 2% N2 additive, 2% B27 additive, 10% bovine serum albumin V + 1% double anti-penicillin-streptomycin solution (100u / mL penicillin, 100mg / mL streptomycin) medium for three days for use. The MC3T3-E1 cells were digested with trypsin and counted, and 5000 cells were inoculated into the agarose gel array in the 48-well plate in each array microwell, and the culture medium was added for two days, and the cells were centrifuged intermittently to make the cells spontaneously spherical.

[0193] (6) 3D culture directional differentiation

[0194] The target cell spheres were cultured in 3D using a 3D microarray structure. DMEM / F-12 high-glucose basal medium was added with neural basal culture medium, 2% N2 additive, 2% B27 additive, 10% bovine serum albumin V + 1% double-antibiotic penicillin-streptomycin solution (100u / mL penicillin, 100mg / mL streptomycin) for directional induction differentiation culture. The medium was changed every other day. After 21 days of culture, qPCR was used to detect the expression of osteogenic-related genes Runx2 and ALP in the cell spheres. Figure 7 As shown, no effective upregulation of Runx2 and ALP expression was observed, and there was no obvious sign of osteogenic differentiation. Subsequent experimental steps can be stopped, as effective osteogenic differentiation induction cannot be performed at this step, and effective bone organoid structures cannot be formed in subsequent steps.

[0195] Comparative Example 2: Cultivating iPSCs in basal medium without inducing agent to observe whether bone organoids can be constructed

[0196] (1) 3D microarray design

[0197] Use aotoCAD software to design an array with a diameter of 0.5 mm and a height of 1 mm per well on the well plate based on the size of the organoids.

[0198] (2) 3D microarray processing of polymethyl methacrylate (PMMA) plates

[0199] Use a CNC machine tool to process the PMMA plate according to the pattern designed by aotoCAD software to obtain a porous array template.

[0200] (3) Construction of polydimethylsiloxane (PDMS) 3D microarray

[0201] Use PDMS for molding, pour the degassed PDMS prepolymer onto the PMMA template, polymerize at 80°C for 60 min, remove the mold and rinse it for later use.

[0202] (4) Construction of agarose 3D microarray

[0203] Use 2wt% agarose gel for secondary molding, pour the heated and dissolved agarose solution on the PDMS array template, cool and solidify for 20 minutes, demold and use an 11mm hole puncher to punch holes, put the punched array into a 48-well plate with sterile water, sterilize under ultraviolet light for 30 minutes and set aside.

[0204] (5) Construction of iPSCs 3D cell spheres

[0205] iPSCs were cultured at a cell density of 1×10 5 The cells / well were inoculated into a six-well plate, and the neural basal culture medium was added to the DMEM / F-12 high-glucose basal medium, 2% N2 additive, 2% B27 additive, 10% bovine serum albumin V + 1% double anti-penicillin-streptomycin solution (100u / mL penicillin, 100mg / mL streptomycin) medium for three days for use. The iPSCs cells were digested with trypsin and counted, and 5,000 cells were inoculated into the agarose gel array in the 48-well plate in each array microwell, and the culture medium was added for two days, and the cells were centrifuged intermittently to make the cells spontaneously spherical.

[0206] (6) 3D culture mesoderm differentiation induction

[0207] The target cell spheres were cultured in 3D using a 3D microarray structure. DMEM / F-12 high-glucose basal medium was added with a neural basal culture medium, 2% N2 additive, 2% B27 additive, 10% bovine serum albumin V + 1% double-antibiotic penicillin-streptomycin solution (100u / mL penicillin, 100mg / mL streptomycin) medium for mesoderm-directed differentiation. On the 5th day, qPCR experiments were performed to detect the expression of mesoderm-related gene markers T and Mixl1. Fig. 9 As shown, no effective mesoderm-related gene expression was observed, indicating that the differentiation to the mesoderm stage was not effective. Subsequent experimental steps can be stopped, as effective bone organoids cannot be formed through the mesoderm.

[0208] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements that are inherent to such process, method, article, or apparatus.

[0209] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0210] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A culture medium for constructing a bone organoid model, characterized in that: The culture medium for constructing a bone organoid model comprises: Osteoblast maturation medium: Add the following final concentrations of inducing molecules to the basal medium: 40-60 μg / mL AsAP, 8-12 mM β-GP, 0.08-0.12 μM Dex.

2. A culture medium for constructing a bone organoid model according to claim 1, characterized in that: The starting cells suitable for the culture medium for constructing the bone organoid model include one of the following: Embryonic stem cells, induced pluripotent stem cells (iPSCs); Bone marrow mesenchymal stem cells, osteoblast precursor cells at all levels, osteoblast-like cells, MC3T3-E1 cells; osteoblast, bone cell; The culture medium for constructing a bone organoid model using bone marrow mesenchymal stem cells, osteoblast precursor cells at various levels, osteoblast-like cells, and MC3T3-E1 cells as starting cells also includes: Osteogenic differentiation medium: add the following final concentrations of induction molecules to the basal medium: 40-60 μg / mL AsAP, 8-12 mM β-GP, 0.08-0.12 μM Dex, 0.8-1.2 μM SAG, 0.8-1.2 μM TH; Wherein, the culture medium for constructing the bone organoid model using embryonic stem cells and induced pluripotent stem cells (iPSCs) as starting cells also includes: iPSCs culture medium: add the following final concentrations of induction molecules to the basal culture medium: 800-1200 units / mL LIF-leukemia inhibitory factor, 0.8-1.2 μM PD0325901, 2-4 μM CHIR99021; Mesoderm induction medium: add the following final concentrations of induction molecules to the basal medium: 20-40 μM CHIR and 3-7 μM Cyc; Ectodermal and endoderm induction medium: The ectoderm and endoderm induction medium is prepared by adding the following induction molecules to the basal medium at the final concentration: 3-7 μM Retinoic acid; Osteogenic differentiation medium: Add the following final concentrations of inducing molecules to the basal medium: 40-60 μg / mL AsAP, 8-12 mM β-GP, 0.08-0.12 μM Dex, 0.8-1.2 μM SAG, and 0.8-1.2 μM TH.

3. A complete set of supplementary products for constructing a bone organoid model derived from embryonic stem cells and iPSCs, characterized in that: Include at least one of the following: A. Complete set of supplementary products for establishing embryonic stem cell and iPSC-derived bone organoid models: The induction molecules used to prepare the iPSCs culture medium: 800-1200 units / mL LIF-leukemia inhibitory factor, 0.8-1.2 μM PD0325901, 2-4 μM CHIR99021; The induction molecules used to prepare the mesoderm induction medium: 20-40 μM CHIR and 3-7 μM Cyc; The medium for preparing ectoderm and endoderm induction: 3-7 μM Retinoic acid; The inducing molecules used to prepare the bone differentiation medium: 40-60 μg / mL AsAP, 8-12 mM β-GP, 0.08-0.12 μM Dex; The inducing molecules used to prepare the osteoblast maturation medium include: 40-60 μg / mL AsAP, 8-12 mM β-GP, 0.08-0.12 μM Dex; B. A complete set of supplementary products for bone organoid models derived from bone marrow mesenchymal stem cells, osteoblast precursor cells at all levels, osteoblast-like cells, and MC3T3-E1, including: The osteogenic differentiation medium is prepared by adding the following induction molecules at final concentrations to the basic medium: 40-60 μg / mL AsAP, 8-12 mM β-GP, 0.08-0.12 μM Dex, 0.8-1.2 μM SAG, and 0.8-1.2 μM TH; The osteoblast maturation medium is prepared by adding the following induction molecules at final concentrations to the basic medium: 40-60 μg / mL AsAP, 8-12 mM β-GP, and 0.08-0.12 μM Dex; C. Complete set of supplementary products for osteoblasts and osteocyte-derived bone organoids, including: The osteoblast maturation medium is prepared by adding the following induction molecules at final concentrations to the basic medium: 40-60 μg / mL AsAP, 8-12 mM β-GP, and 0.08-0.12 μM Dex.

4. A method for constructing a bone organoid model, characterized in that: The method comprises: Placing a biological material having a microwell array in the cell culture plate to obtain a bone organ chip; Initial stem cells are suspended in culture medium and promoted to form spherical structures by cell aggregation or using a non-adhesive culture surface to obtain cell spheres; The cell sphere is cultured in the bone organoid chip using the culture medium for constructing a bone organoid model according to any one of claims 1 to 2 to obtain a bone organoid model.

5. The method for constructing a bone organoid model according to claim 4, characterized in that: The initial stem cells are embryonic stem cells and iPSCs as starting cells, and the cell balls are cultured in the bone organ chip using the culture medium for constructing a bone organoid model, specifically comprising: On day 1, iPSCs culture medium was used for induction culture; On days 2-5, mesoderm induction medium, ectoderm induction medium, and endoderm induction medium were used for induction culture; From day 5 to day 19, osteogenic differentiation medium was used for induction culture; On days 19-26, osteoblast maturation medium was used for induction culture.

6. The method for constructing a bone organoid model according to claim 4, characterized in that: When the initial stem cells are bone marrow mesenchymal stem cells, osteoblast precursor cells at various levels, osteoblast-like cells, and MC3T3-E1, the cell spheres are cultured in the bone organ chip using the culture medium for constructing a bone organoid model, specifically comprising: The osteogenic differentiation medium is used for induction culture on days 1 to 14; and the osteoblast maturation medium is used for induction culture on days 15 to 28.

7. The method for constructing a bone organoid model according to claim 4, characterized in that: When the initial stem cells are osteoblasts or osteocytes, the cell spheres are cultured in the bone organ chip using the culture medium for constructing a bone organoid model, specifically comprising: From day 1 to day 28, the osteoblast maturation medium was used to continue the induction culture.

8. The method for constructing a bone organoid model according to claim 4, characterized in that: The biomaterial includes at least one of agarose, polyethylene glycol and sodium alginate; a plurality of micropores are provided on the biomaterial, and the plurality of micropores are evenly arranged to form the micropore array; the initial stem cells include at least one of embryonic stem cells, induced pluripotent stem cells, bone marrow mesenchymal stem cells, osteoblasts, osteocytes, osteoblast precursor cells of various levels, osteoblast-like cells, and MC3T3-E1.

9. A bone organoid model prepared by the method according to any one of claims 4 to 7.

10. Use of the bone organoid model according to claim 9 in the preparation of products for treating fractures, large bone defects, rheumatoid arthritis, and ankylosing spondylitis.

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