Organ-like construction method and application thereof

Abstract

The invention relates to the technical field of biology and new medicine, in particular to an organoid construction method and application thereof. The construction method of the organ is based on double factors of mechanics and biochemistry, spiral polyisocyanide serves as an extracellular matrix, and effective regulation and control of differentiation paths of cartilage organs and bone organs are achieved by adjusting the chain length and modifying RGD and cooperating with a cartilage generating cell population or an osteoblast population for differentiation.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and new medicine, specifically to a method for constructing organoids and its applications. Background Technology

[0002] Organoids are functional cell-tissue complexes constructed using in vitro three-dimensional culture technology, designed to mimic the structure and function of natural organs. Compared to traditional two-dimensional cell culture, they provide physiological responses and microenvironments that more closely resemble those in vivo; compared to animal models, they better simulate and reflect human-specific physiological functions. Therefore, organoids are now widely used in regenerative medicine, disease model construction, and drug screening.

[0003] The preparation of organoids primarily relies on the differentiation and tissue formation of stem cells in a three-dimensional environment. Specifically, the preparation process typically begins with induced blastocysts or pluripotent stem cells, promoting their differentiation into specific cell types by adding specific biochemical signals and growth factors. The extracellular matrix (ECM) provides a three-dimensional environment for cell adhesion, growth, and differentiation. Under suitable conditions, its mechanical and biochemical properties influence cell growth and differentiation behavior, supporting stem cell differentiation into specific cell types, ultimately forming organoids.

[0004] In the process of organoid preparation, researchers can further subdivide cartilage organoids and bone organoids by adjusting the cell differentiation pathways and culture conditions for different tissue types. Cartilage organoids are formed by differentiating stem cells into chondrocytes and further organizing them in a three-dimensional environment; they are commonly used in cartilage injury repair and joint disease model research. Bone organoids, on the other hand, are created by inducing stem cells to differentiate into osteoblasts, mimicking the bone tissue generation and mineralization process, and are used for research related to bone regeneration and osteoporosis.

[0005] Precise regulation of cell differentiation is crucial in the generation of these two organoids, such as controlling whether cells differentiate into chondrocytes or osteoblasts. However, this process is highly complex, typically influenced by multiple factors including biochemical signals, cell-cell interactions, and microenvironmental factors. Although the addition of growth factors or regulation of signaling pathways can partially guide differentiation, these mechanisms are highly intertwined, and precise spatial and temporal control of signal transduction is difficult to achieve in a three-dimensional culture system. Therefore, current technologies cannot effectively regulate the differentiation pathways of cartilage and bone organoids, resulting in limitations in their functionality and tissue maturity, which restricts their ability to simulate real tissue functions and achieve regenerative capacity. Summary of the Invention

[0006] This invention uses spiral polyisocyanate as an extracellular matrix, and combines it with chondrogenic cell populations or osteoblast populations for differentiation, to provide a method for constructing organoids and its applications.

[0007] Specifically, the present invention provides the following technical solutions:

[0008] In a first aspect, the present invention provides a method for constructing organoids, comprising: S1, dissolving helical polyisocyanates with adjustable chain length in an induction culture medium to obtain a helical polyisocyanate hydrogel solution; and S2, embedding chondrogenic cell populations or osteoblast populations in the helical polyisocyanate hydrogel solution, and obtaining the organoids by gelation.

[0009] In some embodiments, the spiral polyisocyanate in step S1 is an RGD-functionalized spiral polyisocyanate.

[0010] In some embodiments, in step S2, the chondrogenic cell population or the osteoblast population includes human bone marrow mesenchymal stem cells.

[0011] In some embodiments, the induction medium used in step S1 is a chondrogenic differentiation induction medium; in step S2, the chondrogenic cell population further includes one or more of chondrogenic progenitor cells and chondrocytes, and the obtained organoid is a cartilage organoid.

[0012] In some embodiments, the induction medium used in step S1 is an osteogenic induction medium; in step S2, the osteoblast population further includes one or more of bone progenitor cells and osteocytes, and the obtained organoid is a bone organoid.

[0013] In some embodiments, in step S2, the cell embedding density of the chondrogenic cell population or the osteoblast population is 10-1. 5 Up to 10 7 The concentration of the spiral polyisocyanate hydrogel solution is 1 to 3 mg / mL.

[0014] A second aspect of the present invention provides an organoid obtained by the above-described organoid construction method.

[0015] A third aspect of the present invention provides the application of the above-described organoid construction method in disease modeling, drug screening, and / or preparation of tissue repair drugs.

[0016] The beneficial effects of this invention are that, based on both mechanical and biochemical factors, this invention uses helical polyisocyanide (PIC) as an extracellular matrix, and through adjusting the chain length and RGD modification, and in conjunction with chondrogenic cell populations or osteoblast populations to differentiate, it achieves effective regulation of the differentiation pathways of cartilage organoids and bone organoids.

[0017] First, by adjusting the chain length of helical polyisocyanate molecules, the stress stiffening properties of polyisocyanate hydrogels can be effectively controlled, thereby regulating the growth and differentiation pathways of organoids in three-dimensional matrices. Second, azidated polyisocyanates increase the efficiency and selectivity of chemical reactions, especially in click chemistry, where azido groups can rapidly bind to specific molecules via the SPAAC reaction (copper-free azido-alkyne cycloaddition reaction), ensuring high efficiency of the functionalization process and material stability. This functionalization reaction not only improves the cell compatibility of the material but also optimizes the cell differentiation microenvironment through precise regulation. Third, by using click chemistry to modify the side chains of polyisocyanates with RGD peptides, abundant cell adhesion sites are provided, thereby enhancing the interaction between cells and the matrix, optimizing cell attachment and diffusion on the matrix, and thus achieving biochemical regulation of organoid construction. Fourth, using human bone marrow mesenchymal stem cells as seed cells, the differentiation rate and maturity of organoids can be further controlled by adjusting differentiation induction conditions to meet the needs of different research and clinical applications.

[0018] Furthermore, the polyisocyanate hydrogel used in this invention not only possesses many advantages of artificially synthesized biomaterials, such as small batch-to-batch variation, high reproducibility, and strong controllability, but also has biomimetic micron-sized pores and collagen-like stress stiffening phenomenon. This provides a more ideal biomimetic matrix for the construction of cartilage organoids, while also making the stress stiffening behavior more controllable.

[0019] Meanwhile, the construction method of this invention is not only simple and rapid, but also highly adjustable, making it suitable for a wide range of tissue engineering applications. Specifically, the cell embedding density and cell passage number can be adjusted according to experimental needs, and the culture period of organoids can also be flexibly set to optimize experimental conditions for different research directions. Furthermore, the concentration of the polyisocyanate hydrogel can be precisely controlled to adapt to the growth requirements and differentiation pathways of different cell types. This flexibility enables this invention to be adapted to various tissue engineering applications, including the repair and regeneration of cartilage and bone tissue, as well as the construction and research of organoids for other complex organs. Attached Figure Description

[0020] Figure 1 This is a flowchart of an organoid construction method in one embodiment;

[0021] Figures 2A to 2F This is a graph showing the qPCR experimental results in one embodiment, including two sets of results based on PIC-N3 with chain lengths of 1k and 5k, wherein... Figure 2A , 2B 2C, 2D, 2E, and 2F used COLI, COLII, COLII / COLI, Aggrecan, SOX9, and RUNX-2 as target genes, respectively.

[0022] Figures 3A to 3F This is a graph showing the qPCR experimental results in one embodiment, including two sets of results based on PIC-N3 and PIC-RGD with a chain length of 1k, wherein... Figure 3A , 3B 3C, 3D, 3E, and 3F were targeted by COLI, COLII, COLII / COLI, Aggrecan, SOX9, and RUNX-2, respectively.

[0023] Figures 4A to 4F This is a graph of qPCR experimental results from one embodiment, including two sets of results based on PIC-N3 and PIC-RGD with a chain length of 5k, wherein... Figure 4A , 4B 4C, 4D, 4E, and 4F used COLI, COLII, COLII / COLI, Aggrecan, SOX9, and RUNX-2 as target genes, respectively. Detailed Implementation

[0024] The technical solution of this patent will be further described in detail below with reference to specific embodiments. It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0025] like Figure 1 As shown, this invention provides a method for constructing organoids, comprising: S1, dissolving helical polyisocyanates with adjustable chain length in an induction culture medium to obtain a helical polyisocyanate hydrogel solution; and S2, embedding chondrogenic cell populations or osteoblast populations in the helical polyisocyanate hydrogel solution, heating to form a gel, and obtaining organoids through induction culture for a certain period of time. Specifically, as follows:

[0026] Example 1: Construction of cartilage organoids based on PIC-N3 and hBMSC

[0027] (1) Spiral polyisocyanate (PIC-N3) was dissolved in the culture medium to obtain a PIC-N3 hydrogel solution. 3.3% of PIC-N3 modified with azide groups (N3) was dissolved at a concentration of 2 mg / mL in chondrogenic differentiation induction medium. After dissolving at 4°C for 24 h, the solution was vigorously shaken and allowed to stand at 4°C to defoam the next day, thus obtaining the PIC-N3 hydrogel solution. The chondrogenic differentiation induction medium was prepared by adding 1% P / S, 2% FBS, 0.1 μmol / L dexamethasone, 50 μg / mL L-ascorbic acid, and 1% P / S to high-glucose DMEM medium.

[0028] ITS, 40 μg / mL proline and 10 ng / mL LTGF-β1 were obtained.

[0029] (2) hBMSCs were embedded in PIC-N3 hydrogel solution and gelled to obtain cartilage organoids. hBMSCs with cell passage number P7 were then encapsulated in PIC-N3 hydrogel solution at 10... 6 The cell embedding density was determined by mixing the PIC-N3 hydrogel solution obtained in step (2) at a volume ratio of 1:1 on ice. The mixture was then added dropwise to 200 μL of each well in a 48-well plate for low-adhesion cell culture. After gelation at 37°C for half an hour, 200 μL of chondrogenic differentiation induction medium was added to construct chondrogenic organoids. The differentiation induction culture period was 21 days, with medium changes performed every three days (removing the medium and adding fresh chondrogenic differentiation induction medium). Samples were harvested on day 21 of differentiation induction, the medium was discarded, and the cells were washed twice with cold PBS. The cells were incubated on ice for at least 30 minutes, and the solution was collected in centrifuge tubes. The centrifuge tubes were then washed twice with cold PBS to obtain the chondrogenic organoids. As a control, samples were harvested on days 0, 3, 7, and 14 of differentiation induction, with day 0 referring to the harvesting of cells before embedding.

[0030] (3) qPCR experiments were performed on cartilage organoids. Total RNA was extracted from the cells of the cartilage organoids obtained in step (3) using a total RNA extraction kit. After the concentration was found to be qualified, it was reverse transcribed into cDNA using a reverse transcription kit. qPCR technology was used with GAPDH as an internal control to obtain the expression of cartilage-related genes (COLII, Aggrecan, SOX9) and osteogenic-related genes (COLI, RUNX-2). The primers used were designed based on the relevant gene sequences published by NCBI, as shown in Table 1.

[0031] Table 1. PCR Primer Sequence List

[0032]

[0033] result: Figures 2A to 2F The results of qPCR experiments based on PIC-N3 with chain lengths of 1k and 5k are presented, where the horizontal axis represents time and the vertical axis represents the mRNA expression of the target gene. Figures 2A to 3F The target genes are COLI, COLII, COLII / COLI, Aggrecan, SOX9, and RUNX-2.

[0034] It can be seen that within 0 to 21 days, the fold changes of COLI, COLII, Aggrecan, and SOX9 in the groups with chain lengths of 1k and 5k all exceeded 10-fold. This indicates that chondrogenic differentiation induction by embedding hBMSCs in PIC-N3 hydrogels resulted in chondrogenic differentiation at certain chain lengths and differentiation times.

[0035] Furthermore, comparing the results of the 1k and 5k chain length groups, the expression of COLII / COLI, AGGRECAN, and SOX9 in the 1k group was higher than that in the 5k group, while the expression of RUNX-2 was lower in the 1k group. Firstly, COLII is a major component of the cartilage matrix, while COLI is mainly found in osteoblasts or fibroblasts. The higher COLII / COLI expression ratio in the 1k group compared to the 5k group suggests that cells in the 1k group are more likely to differentiate into chondrocytes, while the 5k group may exhibit more fibroblast or osteoblast characteristics. Secondly, AGGRECAN is one of the important proteoglycans in chondrocyte formation and a core component of the cartilage matrix, typically highly expressed during cartilage differentiation. Therefore, the higher AGGRECAN expression in the 1k group indicates more mature chondrocyte formation and more active cartilage matrix production. Third, SOX9 is a key transcription factor for chondrocyte differentiation. SOX9 expression in the 1k group was higher than in the 5k group, indicating that the 1k group played a stronger regulatory role in chondrocyte differentiation, further supporting the view that the 1k group was more inclined towards chondrocyte differentiation. Fourth, RUNX-2 is a key transcription factor for osteogenic differentiation. RUNX-2 expression in the 1k group was lower than in the 5k group, indicating that the 1k group differentiated less into osteoblasts, while the 5k group may have a higher tendency towards osteogenic differentiation. In summary, the 1k group showed a stronger tendency towards chondrocyte differentiation, while the 5k group showed a stronger tendency towards osteogenic differentiation.

[0036] In summary, the results of this embodiment indicate that a lower stress stiffening threshold in a matrix without RGD leads to better chondrogenic differentiation and is more suitable for the construction of cartilage organoids.

[0037] Example 2: Construction of cartilage organoids based on PIC-RGD and hBMSC

[0038] In Example 1, cartilage organoids were prepared based on PIC-N3 hydrogel and hBMSC. In this example, the following steps were added between step (1) and step (2): GRGDS peptide was modified into the side chain of helical polyisocyanate PIC-N3 by click chemistry at a modification ratio of 1% to obtain RGD-functionalized polyisocyanate PIC-RGD; and PIC-RGD was used for embedding and testing in subsequent steps.

[0039] result: Figures 3A to 3F The results of qPCR experiments based on PIC-N3 and PIC-RGD with a chain length of 1k are presented.Figures 4A to 4F The results of qPCR experiments based on PIC-N3 and PIC-RGD with a chain length of 5k are presented. It can be seen that within 0-21 days, the gene expression levels of COLI, COLII, Aggrecan, and SOX9 were increased more than 10-fold in the PIC-N3 and PIC-RGD groups with different chain lengths. This indicates that chondrogenic differentiation induction induced by PIC-N3 hydrogel embedding of hBMSCs resulted in chondrogenic differentiation at certain chain lengths and differentiation times.

[0040] Furthermore, regarding COLII / COLI expression, from day 0 to 21, the expression of COLII / COLI was significantly higher in the group using PIC-N3 as the extracellular matrix than in the group using PIC-RGD. This indicates that modifying the cell adhesion peptide RGD on the side chain of PIC hydrogel can provide adhesion sites for cells, thereby affecting the construction of different organoids, such as having a negative impact on the construction of cartilage organoids and a positive impact on the construction of bone organoids.

[0041] Meanwhile, regarding RUNX-2 expression, in the PIC-N3 group with a chain length of 1k, the expression level was lower than that in the PIC-RGD group. However, in the PIC-N3 group with a chain length of 5k, the expression level was mostly higher than that in the PIC-RGD group. This indicates that biochemical factors dominated by RGD side chain modification and mechanical factors dominated by polymer chain length jointly influence organoid formation.

[0042] What needs to be understood is:

[0043] Firstly, the above embodiments use chondrogenic differentiation induction medium to prepare hydrogel solutions and perform embedding operations, while simultaneously using human bone marrow mesenchymal stem cells to construct chondrogenic organoids. In practical applications, osteogenic induction medium can also be used to construct bone organoids or to explore the effects of mechanical and biochemical factors on bone organoid construction. Accordingly, other chondrogenic cell populations or osteoblast populations, such as chondrocyte progenitors and chondrocytes, or osteoblast progenitors and osteoblasts, can be adaptively used to construct chondrogenic or osteoblastic organoids. That is, the above embodiments use chondrogenic differentiation induction medium combined with human bone marrow mesenchymal stem cells as an example to illustrate the specific implementation of this application. In practical applications, other induction mediums and cells can be selected based on the purpose of the steps, which should not be construed as a limitation of this application.

[0044] Secondly, in the above embodiments, a cell embedding density of 102 is used. 6 A cell suspension composed of human bone marrow mesenchymal stem cells (cells / mL) was mixed with a 2 mg / mL helical polyisocyanate hydrogel solution. In practical applications, the cell embedding density can also be adjusted to 102. 5-10 7 The concentration of polyisocyanate in the system can be changed to adapt to the growth requirements and differentiation pathways of different cell types, with the concentration of the hydrogel solution being 1-3 mg / mL. However, this should not be considered a limitation of this application.

[0045] Third, the above embodiments utilize the DBCO group modified at the end of the GRGDS peptide and the azide group of PIC-N3 to undergo a SPAAC reaction to functionalize the GRGDS peptide onto the polyisocyanate side chain, thereby obtaining PIC-RGD. In practical applications, other methods such as modifying double bonds on the helical polyisocyanate can also be used to achieve RGD functionalization, which should not be construed as a limitation of this application.

[0046] Fourth, in the above embodiments, the group with a chain length of 1k refers to the group that uses a catalyst:monomer ratio of 1:1000 to obtain a lower chain length spiral polyisocyanate for the experiment; the group with a chain length of 5k refers to the group that uses a catalyst:monomer ratio of 1:5000 to obtain a lower chain length spiral polyisocyanate for the experiment.

[0047] Example 3: Application of organoid construction methods

[0048] This embodiment provides an application of the above-mentioned organoid construction method in disease modeling, drug screening, and preparation of tissue repair drugs.

[0049] Based on the research objectives, specific genetic mutations or exogenous stimuli were introduced into the organoids prepared above to simulate the pathological processes of various diseases. For example, adding pro-inflammatory factors such as IL-1β and TNF-α to cartilage organoids can effectively simulate cartilage degeneration and inflammatory responses, thereby allowing observation of cartilage matrix degradation and the gradual decline in chondrocyte function. Similarly, by using cells carrying specific gene mutations or regulating the mineralization process of the organoids prepared above, typical pathological features of osteoporosis can be simulated, including decreased bone density and increased bone fragility. Furthermore, the introduction of RG D groups during the construction of these organoids may exacerbate cartilage inflammation in disease models, thus providing a reliable organoid model for further research on the mechanisms of cartilage inflammation.

[0050] Furthermore, based on the constructed organoids as in vitro disease models, candidate drugs were introduced to evaluate their effects on cell behavior, matrix formation, and pathological changes within the organoids. Specifically, qPCR was used to detect changes in gene expression related to cartilage or bone formation to assess the drug's regulatory effect on organoid cell differentiation; cell proliferation, migration, and survival rates were measured to assess the drug's impact on cell health in the organoids. In addition, based on the efficacy evaluation results of the drugs in the organoid models, the drug formulation and dosage were further optimized to ensure effective promotion of cartilage or bone tissue repair and regeneration. The optimal treatment regimen was determined by adjusting the drug's release rate, concentration, and duration of action.

[0051] It can be seen that, based on the above organoid construction methods, not only can the pathological processes of cartilage and bone-related diseases be accurately simulated, but also a reliable experimental platform can be provided for new drug development, efficacy evaluation and personalized treatment. This greatly promotes the research and application of tissue engineering and regenerative medicine, helps to accelerate the development of tissue repair drugs, and brings new solutions to the field of personalized medicine.

[0052] In summary, this application starts from the stress stiffening properties of the extracellular matrix, focuses on controlling the critical stress of stress stiffening by adjusting the chain length of polyisocyanate, and cooperates with the differentiation of chondrogenic cell groups or osteoblast groups to achieve effective regulation of the differentiation pathways of cartilage organoids and bone organoids.

[0053] Specifically, collagen is a crucial component of the extracellular matrix, serving as the core of the fibrin network and playing a vital role in the extracellular matrix of both cartilage and bone cells. The main components of the in vivo cartilage matrix include collagen, proteoglycans, and hyaluronic acid, while the bone cell matrix also contains collagen. Mechanical analysis shows that collagen and fibrin hydrogels typically exhibit constant stiffness or storage modulus under minute deformations, and this stiffness can be adjusted by modifying polymer concentration or preparation conditions. However, when stress or strain exceeds a certain critical value, these biogels exhibit a significant nonlinear mechanical enhancement, known as strain stiffening or stress stiffening, with stiffness reaching hundreds of times the original low stress modulus. Although collagen or fibrin hydrogels are frequently used as matrices for in vitro cell culture, their main drawback—changes in matrix stiffness—are usually accompanied by alterations in other physicochemical properties, such as protein concentration, pore size, and ligand density, which also affects their practical application.

[0054] Furthermore, the widely used extracellular matrix Matrigel is a mouse-derived extract containing a variety of bioactive components. This complex and variable composition makes Matrigel unsuitable for precise studies of cell-matrix interactions. Additionally, the high content of glycoproteins and viscoproteins in Matrigel alters the structure of collagen fibers, inhibiting the typical nonlinear stress stiffening response associated with fiber structure.

[0055] This application, starting from the stress-rigidification properties of the extracellular matrix, utilizes polyisocyanates to prepare organoids. Compared to traditional collagen hydrogels, its mechanical properties can be optimized by adjusting the critical stress for stress rigidification, without affecting other properties such as the gel's pore size or polyisocyanate concentration. Furthermore, polyisocyanates have a relatively large micrometer scale, and the porous structure of their fibrous network facilitates the efficient transport of metabolic molecules between cells.

[0056] Furthermore, polyisocyanate gels exhibit a distinct stress stiffening mechanism, achieving enhanced "static" stiffness even at relatively low stress or strain, and their critical stress can be adjusted by polymer concentration and polymer chain length. This application focuses on controlling the critical stress for stress stiffening by adjusting the polymer chain length without altering the gel's network structure, thereby optimizing its mechanical properties and biological applicability.

[0057] The relevant scientific terms used in this application are explained below:

[0058] Culture medium related. High-glucose DMEM (Dulbecco's Modified Eagle Medium): A high-nutrient medium that provides ample carbon source for cells, promoting cell proliferation and differentiation; P / S (Penicillin / Streptomycin): A mixed antibiotic solution of penicillin and streptomycin to prevent bacterial contamination; FBS (Fetal Bovine Serum): Fetal bovine serum used to support cell growth, proliferation, and maintain cell function; Dexamethasone: Dexamethasone affects cell proliferation and differentiation by regulating gene expression in the cell nucleus, especially stimulating osteoblast formation; L-Ascorbic Acid: L-Ascorbic acid promotes collagen synthesis in the extracellular matrix, helping cells generate and maintain the structural integrity of the extracellular matrix; ITS (Insulin-Transferrin-Selenium): A combination of insulin, transferrin, and zinc used to support cell growth; Proline: Proline promotes the growth and differentiation of chondrocytes and osteoblasts; TGF-β1 (Transforming Growth Factor Beta-β1)... 1) Transforming growth factor-β1 promotes the differentiation of fibroblasts, chondrocytes and osteoblasts, and stimulates the synthesis of extracellular matrix.

[0059] Embedding-related. hBMSC (human Bone Marrow-derived Mesenchymal Stem Cells): Human bone marrow mesenchymal stem cells, a type of pluripotent stem cell derived from human bone marrow, possessing the ability to self-renew and differentiate into various cell types, and widely used in tissue engineering and regenerative medicine; PBS (Phosphate-Buffered Saline): Phosphate-buffered saline, used to maintain pH and osmotic pressure balance in biological samples and cell experiments.

[0060] Test-related. RNA (Ribonucleic Acid): Ribonucleic acid, a carrier of genetic information found in biological cells and some viruses and viroids, mainly used to guide protein synthesis; cDNA (Complete DNA): Complementary DNA, specifically referring to a DNA strand that is complementary to RNA after reverse transcription in vitro. Unlike genomic DNA, its sequence has only exons and no introns; qPCR (Quantitative Real-time PCR): Real-time quantitative PCR, a method that uses fluorescent chemicals to measure the total amount of product after each polymerase chain reaction cycle in a DNA amplification reaction, i.e., internal or external control method to quantitatively analyze specific DNA sequences in the test sample.

[0061] PCR primer related. GAPDH (glyceraldehyde-3-phosphate dehydrogenase): Glyceraldehyde-3-phosphate dehydrogenase, an enzyme in glycolysis, widely distributed in cells of various tissues; COLI (Collagentype I): Type I collagen, often forming relatively thick fiber bundles, widely distributed, mainly found in skin, tendons, ligaments and bones, with strong tensile strength, accounting for about 90% of the collagen content in the human body; COLII (Collagen type... II): Type II collagen, a high-molecular-weight protein. Filamentous collagen fibers intertwine with elastin and polysaccharide proteins to form a network structure, generating a certain mechanical strength. It is mainly found in cartilage. Aggrecan: Aggregated proteoglycan, a proteoglycan initially isolated from cartilage tissue. Its core protein is composed of multiple structural domains and is attached with approximately 100 chondroitin sulfate chains. SOX9: SRY-box transcription factor 9, a homologous gene of the SRY gene (male sex-determining gene) located on the Y chromosome. Its encoded product is a transcription factor related to cartilage formation, sex differentiation, nervous system, and heart development. It plays a key role in chondrocyte differentiation and bone development. RUNX-2: Runt-related transcription factor 2, an important transcription factor regulating the initiation of mesenchymal stem cell differentiation into osteoblasts.

[0062] Side chain modification is relevant. SPAAC (Strain-Promoted Alkyne-Azide Cycloaddition): Copper-free ring-strain-promoted cycloaddition of azide-alkyne, utilizing the stress between the cycloalkyne and the azide group to drive the cycloaddition reaction; DBCO (Dibenzylcyclooctyne): Dibenzylcyclooctyne, a molecule with a cycloalkyne structure, often used in bioorthogonal chemical reactions; GRGDS peptide: A short peptide sequence composed of five amino acids, specifically glycine-arginine-glycine-aspartic acid-serine; RGD group: A short peptide sequence composed of arginine, glycine, and aspartic acid, also a common cell adhesion recognition sequence.

[0063] The above descriptions are merely some embodiments of the present invention. Those skilled in the art can make various modifications and improvements without departing from the inventive concept of the present invention, and these all fall within the scope of protection of the present invention.

Claims

1. A method for constructing organoids, characterized in that, include: S1. Dissolve the helical polyisocyanate with adjustable chain length in the induction medium to obtain a helical polyisocyanate hydrogel solution. and S2. The chondrogenic cell population or osteoblast population is embedded in the spiral polyisocyanate hydrogel solution and gelled to obtain the organoid.

2. The organoid construction method according to claim 1, characterized in that, The spiral polyisocyanate in step S1 is an RGD-functionalized spiral polyisocyanate.

3. The organoid construction method according to claim 1, characterized in that, In step S2, the chondrogenic cell population or the osteoblast population includes human bone marrow mesenchymal stem cells.

4. The organoid construction method according to claim 3, characterized in that, The induction medium used in step S1 is a chondrogenic differentiation induction medium; In step S2, the chondrogenic cell population further includes one or more of chondrogenic progenitor cells and chondrocytes, and the obtained organoid is a chondrogenic organoid.

5. The organoid construction method according to claim 3, characterized in that, The induction medium used in step S1 is an osteogenic induction medium; In step S2, the osteoblast population also includes one or more of bone progenitor cells and osteocytes, and the obtained organoid is a bone organoid.

6. The method for constructing organoids according to any one of claims 1-5, characterized in that, In step S2, the cell embedding density of the chondrogenic cell population or the osteoblast population is 10. 5 Up to 10 7 The concentration of the spiral polyisocyanate hydrogel solution is 1 to 3 mg / mL.

7. An organoid obtained by the organoid construction method according to any one of claims 1-6.

8. The use of the organoid construction method according to any one of claims 1-6 in disease modeling, drug screening and / or preparation of tissue repair drugs.

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