Bone organ with autonomous mineralization ability and preparation and application thereof
By suspending osteogenic precursor cells on the PDMS chip and combining with osteogenic induction culture medium to process, bone organoids with autonomous mineralization capabilities were prepared, which solved the problem that the existing technology was difficult to simulate bone complexity, and achieved the preparation of bone organoids with regular morphology and good activity, and expanded its prospects in research and application.
Patent Information
- Application Number
- CN202510035368.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-09
- Publication Date
- 2025-04-29
AI Technical Summary
The prior art is difficult to effectively simulate the complexity of bones, which limits the study of dynamic physiological development or pathological development of bones.
By suspending the osteogenic precursor cells in a mixture of Matrigel Matrigel and Collagen I, coagulation and culture using PDMS chips, and processing them in combination with osteogenic induction culture medium, bone organoids with autonomous mineralization were prepared.
It has achieved the preparation of bone organoids with regular morphology, uniform cell distribution and good activity, and has the ability to independently mineralize and deposit hydroxyapatite, which has expanded the application prospects of bone tissue regeneration engineering and preclinical drug screening.
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Figure CN119899796A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of organoid preparation, and in particular to a bone organoid with autonomous mineralization capability and the preparation and application thereof. Background Art
[0002] Bone is a rigid and dynamic tissue that performs many important functions in the body, including providing structural support, promoting mobility, regulating endocrine function, and storing minerals. Bone is continuously formed and remodeled through the highly coordinated functions of various bone-derived cells, such as osteocytes and osteoblasts. Despite the vital role of bone, research on the dynamic physiological development or pathological development of bone has been limited because traditional models cannot effectively simulate the complexity of bone.
[0003] Organoids are cells with stem cell potential that differentiate into 3D cultures with corresponding organ tissue morphology through adhesion, aggregation and self-organization. They are able to self-renew and self-organize to reproduce certain specific functions of tissues and organs in the body and form a spatial structure similar to that of organs in the body. The emergence of organoid technology complements traditional two-dimensional cell culture and animal model research strategies, promoting the development of personalized precision medicine.
[0004] Bone tissue is a hard tissue, and its organoid research is still in its infancy. The construction of its in vitro model has broad application prospects in tissue regeneration engineering, mechanism research, therapeutic target screening, and preclinical drug screening. Summary of the invention
[0005] In order to solve the above problems, the purpose of the present invention is to provide a bone organoid with autonomous mineralization ability and its preparation and application.
[0006] The purpose of the present invention can be achieved by the following technical solutions:
[0007] The first object of the present invention is to provide a method for preparing bone organoids with autonomous mineralization ability, comprising the following steps:
[0008] (S1) Mix Matrigel and Collagen I working solution to obtain organoid matrix working solution;
[0009] (S2) suspending the osteoblast precursor cell line in the organoid matrix working solution prepared in step (S1) and placing it in the PDMS chip, and after solidification, immersing and culturing it in α-MEM culture solution containing 10% FBS to obtain bone organoids;
[0010] (S3) The bone organoids prepared in step (S2) are subjected to osteogenic induction treatment using an osteogenic induction culture medium to obtain bone organoids with autonomous mineralization capability.
[0011] In one embodiment of the present invention, in step (S1), the volume ratio of the Matrigel matrix glue to the Collagen I working solution is 4:0 to 1:3;
[0012] Preferably, the volume ratio of the Matrigel matrix gel to the Collagen I working solution is 1:1.
[0013] In one embodiment of the present invention, the Collagen I working solution includes 10 μL 10×PBS, 0.2 μL 1M NaOH, and 90 μL Collagen I solution.
[0014] In one embodiment of the present invention, in step (S2), the ratio of the amount of osteoblast precursor cell line to the organoid matrix working solution is 5×10 6 Cells: 100 μL.
[0015] In one embodiment of the present invention, in step (S2), during the solidification process, the temperature is 37° C. and the time is 1 to 2 h;
[0016] In step (S2), during the immersion culture, 5% CO2, the temperature is 37°C, and the time is 1 to 2 hours;
[0017] In one embodiment of the present invention, in step (S3), the bone organoids and the osteogenic induction medium are 5×10 5 ~5×10 7 cells.
[0018] In one embodiment of the present invention, in step (S3), during the osteogenic induction treatment, 5% CO2, the temperature is 37°C, and the time is 1 to 35 days.
[0019] The second object of the present invention is to provide a bone organoid with autonomous mineralization ability, which is prepared by the above method.
[0020] The third object of the present invention is to provide a bone organoid with autonomous mineralization ability for use in the preparation of disease models, drug screening or bone tissue regeneration medicine.
[0021] Compared with the prior art, the present invention has the following beneficial effects:
[0022] (1) The present invention is based on a PDMS chip to form a bone organoid with regular morphology, uniform internal cell distribution, and good activity;
[0023] (2) The present invention screened out the matrix (Matrigel / Collagen I) ratio with the best osteogenic activity;
[0024] (3) The bone organoids prepared by the present invention have the ability to autonomously mineralize and deposit hydroxyapatite. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] Figure 1 This is a schematic diagram of the structure of the chip used in Example 1;
[0026] Figure 2 Internal F-actin / DAPI staining images of self-mineralized bone organoids prepared with different ratios (Matrigel / Collagen);
[0027] Figure 3 The protein expression of osteogenic differentiation markers (Runx2, Osx, Ocn) of self-mineralized bone organoids prepared with different ratios (Matrigel / Collagen);
[0028] Figure 4 Flow chart of the preparation of self-mineralized bone organoids based on chip templates;
[0029] Figure 5 Early cross-sectional images, internal 3D reconstructions, and surface images of self-mineralized bone organoids;
[0030] Figure 6 Micro-CT images of self-mineralized bone organoids at 7, 21, and 35 days;
[0031] Figure 7 Fourier transform infrared images of mature self-mineralized bone organoids and hydroxyapatite. DETAILED DESCRIPTION
[0032] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.
[0033] In the following examples, the PDMS chip was prepared by the following method:
[0034] The 3D chip positive film was designed using 3D CAD software Solidworks, and the negative mold was prepared using PDMS. After constant temperature curing at 70°C for 2 to 4 hours, the chip was cut out and sterilized by high pressure and air-dried to obtain a PDMS chip (such as Figure 1 shown);
[0035] in, Figure 1 The thickness of the central circular hole is 1.0 mm and the inner diameter is 2.5 mm.
[0036] Collagen I working solution includes 10 μL 10× PBS, 0.2 μL 1 M NaOH, and 90 μL Collagen I solution.
[0037] The osteoblast precursor cell line was the commercially available Mc3t3-e1 cell line;
[0038] 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;
[0039] 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.
[0040] Example 1
[0041] This embodiment provides a method for preparing a bone organoid with autonomous mineralization ability, comprising the following steps:
[0042] (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;
[0043] (S2) Osteoblast precursor 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 and cultured in an α-MEM culture solution containing 10% FBS (5% CO2, temperature of 37°C, time of 2 days) to obtain bone organoids;
[0044] Bone organoids were extracted as samples, fixed with 4% paraformaldehyde for 3 h, permeabilized with 0.25% TrionX-100 PBS, and then blocked with 5% goat serum. Finally, F-actin and DAPI staining were performed to observe the cell distribution and extension inside the bone organoids (the results are shown in Figure 2 ). Figure 2 It can be found that cell distribution and extension reach an optimal balance.
[0045] Bone organoids were extracted, lysed, quantified by BCA, and boiled. The total protein (Coomassie Brilliant Blue) was used as an internal reference, and WB was used to detect the protein expression of Runx2, Osx, and Ocn, important osteogenic differentiation markers (the results are shown in 3). Figure 3 It can be found that the expression of key osteogenic markers such as runt-related transcription factor 2 (Runx2), Osterix (Osx) and osteocalcin (Ocn) was also the highest.
[0046] (S3) The bone organoids prepared in step (S2) are removed 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, 1 to 35 days) to obtain bone organoids with autonomous mineralization ability (such as Figure 4 shown).
[0047] Early bone organoids were stained with F-actin / DAPI (same as step (S2)), and internal cross-sections and z-stack scans of the middle part were used for 3D reconstruction. The organoid surface was immunostained with Runx2 / F-actin / DAPI and laser confocal imaging was performed (such as Figure 5 shown), through Figure 5 It can be found that during the bone formation stage, osteoblasts are evenly distributed, the surface of the organoid precursors is covered with a dense layer of osteoblasts, and the osteocytes embedded in the gel are relatively sparse, evenly distributed, and well stretched. Almost all cells express the key osteogenic transcription factor Runx2.
[0048] Bone organoid samples at 7, 21, and 35 days were selected for micro-CT scanning and reconstructed using the same parameters for display and comparison (results are shown in Figure 6 ). Figure 6 It can be found that bone organoids can calcify autonomously, and the degree of calcification and density increase significantly over time.
[0049] Mature self-mineralized bone organoid samples were selected, oven-dried, and then tested by Fourier transform infrared spectroscopy. The functional groups were basically consistent with those of hydroxyapatite standards (results as shown in Figure 7 ), showing that bone organoids are able to form hydroxyapatite deposition.
[0050] Example 2
[0051] This embodiment provides a method for preparing a bone organoid with autonomous mineralization ability, comprising the following steps:
[0052] (S1) Mix Matrigel and Collagen I working solution (the volume ratio of Matrigel to Collagen I working solution is 4:0) to obtain organoid matrix working solution;
[0053] (S2) Osteoblast precursor cell line (5×10 6cells) 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;
[0054] Bone organoids were extracted as samples, fixed with 4% paraformaldehyde for 3 h, permeabilized with 0.25% TrionX-100 PBS, and then blocked with 5% goat serum. Finally, F-actin and DAPI staining were performed to observe the cell distribution and extension inside the bone organoids (the results are shown in Figure 2 ). Figure 2 It can be found that the cell distribution and extension state are worse than that of Example 1.
[0055] Bone organoids were extracted, lysed, quantified by BCA, and boiled. The total protein (Coomassie Brilliant Blue) was used as an internal reference, and WB was used to detect the protein expression of Runx2, Osx, and Ocn, important osteogenic differentiation markers (the results are shown in 3). Figure 3 It can be found that the expression of key osteogenic markers such as runt-related transcription factor 2 (Runx2), Osterix (Osx) and osteocalcin (Ocn) is worse than that in Example 1.
[0056] (S3) The bone organoids prepared in step (S2) are 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, 1 to 35 days) to obtain bone organoids with autonomous mineralization ability.
[0057] Example 3
[0058] This embodiment provides a method for preparing a bone organoid with autonomous mineralization ability, comprising the following steps:
[0059] (S1) Mix Matrigel and Collagen I working solution (the volume ratio of Matrigel to Collagen I working solution is 3:1) to obtain organoid matrix working solution;
[0060] (S2) Osteoblast precursor 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;
[0061] Bone organoids were extracted as samples, fixed with 4% paraformaldehyde for 3 h, permeabilized with 0.25% TrionX-100 PBS, and then blocked with 5% goat serum. Finally, F-actin and DAPI staining were performed to observe the cell distribution and extension inside the bone organoids (the results are shown in Figure 2 ). Figure 2 It can be found that through Figure 2 It can be found that the cell distribution and extension state are worse than that of Example 1.
[0062] Bone organoids were extracted, lysed, quantified by BCA, and boiled. The total protein (Coomassie Brilliant Blue) was used as an internal reference, and WB was used to detect the protein expression of Runx2, Osx, and Ocn, important osteogenic differentiation markers (the results are shown in 3). Figure 3 It can be found that the expression of key osteogenic markers such as runt-related transcription factor 2 (Runx2), Osterix (Osx) and osteocalcin (Ocn) is worse than that in Example 1.
[0063] (S3) The bone organoids prepared in step (S2) are 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, 1 to 35 days) to obtain bone organoids with autonomous mineralization ability.
[0064] Example 4
[0065] This embodiment provides a method for preparing a bone organoid with autonomous mineralization ability, comprising the following steps:
[0066] (S1) Mix Matrigel and Collagen I working solution (the volume ratio of Matrigel to Collagen I working solution is 1:3) to obtain organoid matrix working solution;
[0067] (S2) Osteoblast precursor 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;
[0068] Bone organoids were extracted as samples, fixed with 4% paraformaldehyde for 3 h, permeabilized with 0.25% TrionX-100 PBS, and then blocked with 5% goat serum. Finally, F-actin and DAPI staining were performed to observe the cell distribution and extension inside the bone organoids (the results are shown in Figure 2 ). Figure 2 It can be found that through Figure 2 It can be found that the cell distribution and extension state are slightly worse than that of Example 1.
[0069] Bone organoids were extracted, lysed, quantified by BCA, and boiled. The total protein (Coomassie Brilliant Blue) was used as an internal reference, and WB was used to detect the protein expression of Runx2, Osx, and Ocn, important osteogenic differentiation markers (the results are shown in 3). Figure 3 It can be found that the expression of key osteogenic markers such as runt-related transcription factor 2 (Runx2), Osterix (Osx) and osteocalcin (Ocn) is slightly worse than that in Example 1.
[0070] (S3) The bone organoids prepared in step (S2) are 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, 1 to 35 days) to obtain bone organoids with autonomous mineralization ability.
[0071] Example 5
[0072] This embodiment provides a method for preparing a bone organoid with autonomous mineralization ability, comprising the following steps:
[0073] (S1) Mix Matrigel and Collagen I working solution (the volume ratio of Matrigel to Collagen I working solution is 0:4) to obtain organoid matrix working solution;
[0074] (S2) Osteoblast precursor 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;
[0075] Bone organoids were extracted as samples, fixed with 4% paraformaldehyde for 3 h, permeabilized with 0.25% TrionX-100 PBS, and then blocked with 5% goat serum. Finally, F-actin and DAPI staining were performed to observe the cell distribution and extension inside the bone organoids (the results are shown in Figure 2 ). Figure 2 It can be found that through Figure 2 It can be found that the cell distribution and extension status are poor.
[0076] Bone organoids were extracted, lysed, quantified by BCA, and boiled. The total protein (Coomassie Brilliant Blue) was used as an internal reference, and WB was used to detect the protein expression of Runx2, Osx, and Ocn, important osteogenic differentiation markers (the results are shown in 3). Figure 3 It can be found that key osteogenic markers such as runt-related transcription factor 2 (Runx2), Osterix (Osx) and osteocalcin (Ocn) are not expressed.
[0077] (S3) The bone organoids prepared in step (S2) are 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, 1 to 35 days) to obtain bone organoids with autonomous mineralization ability.
[0078] 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 bone organoids with autonomous mineralization ability, characterized in that: The following steps are involved: (S1) Mix Matrigel and Collagen I working solution to obtain organoid matrix working solution; (S2) suspending the osteoblast precursor cell line in the organoid matrix working solution prepared in step (S1) and placing it in the PDMS chip, and after solidification, immersing and culturing it in α-MEM culture solution containing 10% FBS to obtain bone organoids; (S3) The bone organoids prepared in step (S2) are subjected to osteogenic induction treatment using an osteogenic induction culture medium to obtain bone organoids with autonomous mineralization capability.
2. The method for preparing a bone organoid with autonomous mineralization ability according to claim 1, characterized in that: In step (S1), the volume ratio of the Matrigel matrix glue to the Collagen I working solution is 4:0 to 1:
3.
3. The method for preparing a bone organoid with autonomous mineralization ability according to claim 2, characterized in that: The volume ratio of the Matrigel matrix gel to the Collagen I working solution is 1:
1.
4. The method for preparing a bone organoid with autonomous mineralization ability according to claim 2, characterized in that: The Collagen I working solution includes 10 μL 10×PBS, 0.2 μL 1M NaOH, and 90 μL Collagen I solution.
5. The method for preparing a bone organoid with autonomous mineralization ability according to claim 1, characterized in that: In step (S2), the ratio of osteoblast precursor cell line to organoid matrix working solution is 5×10 5 ~5×10 7 Cells: 100 μL.
6. The method for preparing a bone organoid with autonomous mineralization ability according to claim 1, characterized in that: In step (S2), during the solidification process, the temperature is 37° C. and the time is 1 to 2 hours.
7. The method for preparing a bone organoid with autonomous mineralization ability according to claim 1, characterized in that: In step (S3), in step (S2), during the immersion culture process, 5% CO2, the temperature is 37°C, and the time is 1.5 to 2.5 days.
8. The method for preparing a bone organoid with autonomous mineralization ability according to claim 1, characterized in that: In step (S3), during the osteogenic induction treatment, 5% CO2, the temperature is 37°C, and the time is 1 to 35 days.
9. A bone organoid with autonomous mineralization capability, characterized in that: It is prepared by the method according to any one of claims 1 to 8.
10. Use of the bone organoid with autonomous mineralization ability as claimed in claim 9 in the preparation of disease models, drug screening or bone tissue regeneration medicine.