Hydrogel compositions for three-dimensional organoid culture and uses thereof

Abstract

The application belongs to the technical field of biological medical materials, and discloses a hydrogel composition for three-dimensional organoid culture and application thereof. The hydrogel composition comprises a hydrogel matrix and a cell adhesion peptide covalently coupled to the hydrogel matrix. The material of the hydrogel matrix is selected from one or more of carboxymethyl cellulose, hyaluronic acid, chitosan or polyethylene glycol. The concentration of the hydrogel matrix is 3wt% to 6wt%. The hydrogel composition has clear components, good biocompatibility, and the mechanical stiffness can be precisely controlled by the concentration of the hydrogel matrix material. The introduction of the cell adhesion peptide significantly improves the cell adhesion and organoid formation efficiency. The hydrogel can be widely applied to the culture of various organoids such as epidermis, tumor and nerve, and has wide application prospects in the fields of developmental biology, disease modeling, regenerative medicine and drug screening.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically, it relates to hydrogel compositions for three-dimensional organoid culture and their applications. Background Technology

[0002] The mechanical properties of the extracellular matrix (ECM) are key factors in regulating cell behavior, tissue morphogenesis, and disease progression. Organoids, as in vitro models that mimic in vivo tissues and organs, are of great value in developmental research, disease modeling, and drug screening. Currently, the mainstream scaffold material for organoid culture is matrix gel. Although matrix gel can support organoid growth, its composition is complex and unclear, and its physical and biochemical properties vary from batch to batch. The main problems are as follows: (1) The composition of matrix gel is unclear and its batch stability is poor, making it impossible to achieve standardized organoid culture, and there are safety hazards caused by animal-derived components. (2) The mechanical stiffness of existing synthetic / natural hydrogels has a narrow adjustable range, making it difficult to match the mechanical properties of ECMs of different tissues and organs, and failing to meet the diverse needs of organoid culture. (3) There is insufficient research on the synergistic regulation mechanism of mechanical force and cell adhesion signals by existing hydrogels, and it is impossible to reveal the molecular mechanism by which mechanical stiffness regulates organoid formation through cell sensing pathways. (4) There is a lack of hydrogel models that can simultaneously simulate the fibrosis of the tumor microenvironment ECM, making it difficult to accurately construct tumor organoids and conduct efficient screening of anti-tumor drugs. These issues limit the application of matrix gels in cell biology mechanism research, cell therapy, and precision drug screening.

[0003] To address these challenges, researchers have developed hydrogel scaffolds based on synthetic or natural polymers, such as alginate and hyaluronic acid hydrogels. These hydrogels can incorporate cell adhesion ligands through chemical modification; however, existing hydrogels suffer from limitations in the range of mechanical property regulation and the unclear synergistic effect between biological activity and mechanical properties. These limitations prevent existing hydrogels from accurately mimicking the dynamic mechanical microenvironment of the ECM, hindering in-depth research into the regulatory mechanisms of mechanical stiffness on cell behavior and organoid development. Summary of the Invention

[0004] The purpose of this invention is to provide hydrogel compositions for three-dimensional organoid culture and their applications.

[0005] To achieve the objectives of this invention, in a first aspect, this invention provides a hydrogel composition for three-dimensional organoid culture, comprising: a hydrogel matrix, and a cell adhesion peptide covalently coupled to said hydrogel matrix; The hydrogel matrix material can be selected from one or more of the following: natural polysaccharides such as carboxymethyl cellulose (CMC), hyaluronic acid, and chitosan, or synthetic polymers such as polyethylene glycol (PEG). The concentration of the hydrogel matrix is ​​3 wt% to 6 wt%.

[0006] The mechanical stiffness of the hydrogel composition can be adjusted by regulating the concentration of the hydrogel matrix material.

[0007] Furthermore, the hydrogel composition comprises: a carboxymethyl cellulose matrix, and a cell adhesion peptide covalently coupled to the carboxymethyl cellulose matrix.

[0008] Furthermore, the cell adhesion peptide is a peptide containing the RGD sequence (arginine-glycine-aspartic acid).

[0009] Furthermore, the hydrogel matrix is ​​made of carboxymethyl cellulose, and the optimal concentration of carboxymethyl cellulose is 4 wt%.

[0010] Furthermore, the hydrogel matrix material forms a hydrogel through calcium ion crosslinking, chemical crosslinking, or photocrosslinking.

[0011] For example, carboxymethyl cellulose matrix forms hydrogels through calcium ion crosslinking.

[0012] Furthermore, it also contains additional components such as sodium alginate and / or type I collagen to mimic the fibrotic microenvironment of the extracellular matrix. The concentration of sodium alginate is 0.6 wt% to 1.5 wt%, and the concentration of type I collagen is 2 mg / ml to 4 mg / ml.

[0013] In a preferred embodiment of the present invention, the hydrogel composition comprises RGD-modified CMC, sodium alginate, and type I collagen; Wherein, the RGD-modified CMC refers to carboxymethyl cellulose covalently coupled with RGD; The concentrations of carboxymethyl cellulose, sodium alginate, and type I collagen in the hydrogel composition are 4 wt%, 1.5 wt%, and 4 mg / ml, respectively.

[0014] In a second aspect, the present invention provides any of the following applications of the hydrogel composition: (1) Cell culture; (2) Construction of tissue-specific organoids.

[0015] Thirdly, the present invention provides a method for culturing three-dimensional organoids, comprising the following steps: 1) Prepare a precursor solution of the hydrogel composition using a culture medium; 2) Suspend the target cells in the precursor solution; 3) Add a cross-linking agent (preferably a calcium ion solution, such as a calcium chloride solution, preferably at a concentration of 0.1M) to cross-link the precursor solution to form a hydrogel encapsulating the target cells; 4) Cultivate hydrogels containing the target cells until three-dimensional organoids are formed.

[0016] Preferably, the concentration of carboxymethyl cellulose in the hydrogel composition is 3 wt% to 6 wt%; wherein, neutral stiffness (carboxymethyl cellulose concentration of 4 wt%) can promote the activation of integrin β1 (ITGB1) and phosphorylation of focal adhesion kinase (FAK), enhance the tension of the F-actin cytoskeleton, thereby inducing YAP nuclear translocation, and ultimately promoting cell proliferation and organoid expansion.

[0017] Furthermore, the target cells may be selected from epithelial cells and / or tumor cells, etc.

[0018] Preferably, the tumor cells can be selected from melanoma cells, lung cancer cells, glioblastoma cells, etc.

[0019] Fourthly, the present invention provides a method for screening candidate drugs targeting three-dimensional organoids, comprising the following steps: a) Culture three-dimensional organoids according to the above method; b) Contact the three-dimensional organoid with the candidate drug to be tested; c) Detect the biological effects of the candidate drug on the three-dimensional organoid to evaluate its efficacy.

[0020] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects: (i) The hydrogel prepared by this invention has a clear composition and good biocompatibility. Its mechanical stiffness can be precisely controlled by the concentration of the hydrogel substrate. Furthermore, the introduction of cell adhesion peptides (such as RGD) significantly improves cell adhesion and organoid formation efficiency.

[0021] (ii) This invention overcomes the problem of separation between the bioactivity and mechanical properties of existing hydrogels. Cell adhesion peptides can mediate the binding of cell integrins, and with adjustable mechanical stiffness, it can simulate the mechano-biochemical synergistic microenvironment of the ECM.

[0022] (III) This invention reveals the molecular mechanism by which mechanical stiffness regulates organoid formation and clarifies the optimal culture stiffness. Neutral stiffness hydrogels (e.g., 4 wt% carboxymethyl cellulose) can significantly promote organoid formation in various cell types, resulting in larger organoid diameters, higher expression levels of specific biomarkers, and the maintenance of organoid physiological functions, thus achieving standardized and reproducible organoid culture.

[0023] (iv) This invention clarifies the molecular mechanism by which mechanical stiffness regulates organoid formation through the ITGB1 / pFAK / F-actin / YAP pathway, providing a reliable in vitro model for elucidating the role of ECM mechanical force in tissue development and disease progression.

[0024] (v) This invention clarifies the molecular target of mechanical stiffness regulation of organoid development, and solves the problem that existing technologies cannot accurately analyze the mechanical force signal transmission mechanism.

[0025] (vi) This invention establishes a tumor organoid model with a controllable pathological microenvironment, overcoming the problem of significant differences between existing drug screening models and the in vivo tumor microenvironment. The constructed fibrotic tumor organoid model can accurately simulate the in vivo melanoma microenvironment, enabling rapid assessment of the antitumor activity of drugs during drug screening. It boasts high screening efficiency and results that more closely reflect the actual in vivo situation, providing an efficient platform for antitumor drug development.

[0026] (vii) The hydrogel of the present invention can be widely used in the culture of various organoids such as epidermis, tumors, and nerves, and has broad application prospects in the fields of developmental biology, disease modeling, regenerative medicine and drug screening. Attached Figure Description

[0027] Figure 1 To determine the concentration of CaCl2 solution by staining live and dead cells in a preferred embodiment of the present invention, scale bar: 100 μm.

[0028] Figure 2 This is an optical image of the hydrogel after gelation in a preferred embodiment of the present invention.

[0029] Figure 3 The OPA test results show that RGD has successfully connected to the CMC main chain.

[0030] Figure 4 This is a scanning electron microscope image of the hydrogel after gelation in a preferred embodiment of the present invention.

[0031] Figure 5 The storage modulus (G') and loss modulus (G'') of the hydrogel are shown in the preferred embodiment of the present invention.

[0032] Figure 6 In a preferred embodiment of the present invention, HaCat, A375, A549, and M059J cells are cultured into 3D cell organoids with tissue-like structures using hydrogels, and then subjected to light field imaging and immunofluorescence staining with specific markers.

[0033] Figure 7 Immunofluorescence staining of ITGB1, pFAK, F-actin and YAP in organoids cultured from HaCat cells in a preferred embodiment of the present invention.

[0034] Figure 8In a preferred embodiment of the present invention, sodium alginate and type I collagen are added to the hydrogel to simulate the pathogenesis of acral melanoma, regulate changes in stiffness and fibrosis, culture A375 cells into 3D cell organoids with tissue-like structures, and perform immunofluorescence staining with specific markers.

[0035] Figure 9 In a preferred embodiment of the present invention, melanoma cell organoids were treated with different concentrations of (A) methotrexate, (B) BET bromide structure inhibitor, (C) osimertinib, (D) bardocoxelone, (E) tilagagstat, (F) 5-fluoro-2'-deoxycytidine and (G) methotrexate disodium salt for 3 days, followed by staining of live and dead cells. Scale bar: 50 μm. (H) Statistical analysis of changes in cell organoid viability after treatment with different drug concentrations. Detailed Implementation

[0036] The present invention aims to provide a cell adhesion peptide (such as RGD) functionalized hydrogel with well-defined composition, adjustable mechanical stiffness, and controllable bioactivity, and to establish an organoid culture and drug screening system based on the hydrogel.

[0037] The present invention adopts the following technical solution: This invention provides a cell adhesion peptide-functionalized hydrogel that achieves synergistic regulation of mechanical stiffness and biological activity.

[0038] Specifically, cell adhesion peptides (such as RGD) are covalently coupled to a hydrogel substrate using the EDC / NHS activation method. The crosslinking density of the hydrogel is controlled by changing the concentration of the hydrogel substrate, thereby achieving precise adjustment of the storage modulus (G') and loss modulus (G'') of the hydrogel.

[0039] In one specific embodiment of the present invention, RGD cell adhesion peptides are covalently coupled to carboxymethyl cellulose (CMC) molecular chains by EDC / NHS activation method. The crosslinking density of the hydrogel is controlled by changing the concentration of CMC (3wt% - 6wt%), thereby achieving precise adjustment of the storage modulus (G') and loss modulus (G'') of the hydrogel.

[0040] Furthermore, CMC can be replaced with natural polysaccharides such as hyaluronic acid and chitosan, or synthetic polymers such as polyethylene glycol (PEG), and RGD peptides can be coupled using the same EDC / NHS activation method to prepare hydrogels with adjustable stiffness on different substrates, adapting to the organoid culture needs of different cells.

[0041] Furthermore, the hydrogel substrate is formed by calcium ion crosslinking. Physical crosslinking with calcium ions can be replaced by chemical crosslinking (such as Schiff base reaction, photocrosslinking), which further improves the mechanical stability and degradation controllability of the hydrogel, expanding its application in in vivo implantation and tissue engineering.

[0042] Furthermore, the RGD peptide can be replaced with other cell adhesion sequences (such as YIGSR, IKVAV, etc.) to target the specificity of integrin receptors for different cell types, thereby improving the adhesion of the hydrogel to target cells and the efficiency of organoid induction.

[0043] Furthermore, the mechanical stiffness of the hydrogel regulates cell behavior through the ITGB1 / pFAK / F-actin / YAP mechanotransduction pathway. Neutral stiffness (4wt% CMC concentration) can promote the activation of integrin β1 (ITGB1) and phosphorylation of focal adhesion kinase (FAK), enhance the tension of the F-actin cytoskeleton, thereby inducing YAP nuclear translocation, and ultimately promoting cell proliferation and organoid expansion.

[0044] Furthermore, different ECM components (such as fibronectin and laminin) or cytokines (such as TGF-β and VEGF) can be added to the hydrogel to construct pathological microenvironment models of different diseases (such as pulmonary fibrosis and liver cancer) and conduct targeted drug screening.

[0045] For example, sodium alginate and type I collagen were added to CMC-RGD hydrogels with optimal stiffness to simulate the ECM fibrosis and mechanical hardening characteristics of the acral melanoma microenvironment. The cellular response to the mechanical microenvironment was verified using the membrane tension probe Flipper-TR, and this model was used for in vitro screening of antitumor drugs.

[0046] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0047] The carboxymethyl cellulose (CMC) used in the following examples was purchased from Shanghai Maclean Biotechnology Co., Ltd.

[0048] RGD peptides were purchased from Shanghai Maclean Biotechnology Co., Ltd.

[0049] Unless otherwise specified, the percentage sign "%" used in this invention refers to mass percentage. However, the percentage of a solution, unless otherwise specified, refers to the number of grams of solute contained in 100 mL of solution.

[0050] Example 1: Optimization of calcium ion concentration in crosslinking agent solution To select a suitable concentration of CaCl2 solution as a cross-linking agent for hydrogel formation without causing cytotoxicity to cells, HaCat cells were treated with 0.1 M, 0.5 M, and 1 M CaCl2 solutions. First, HaCat cells were seeded into 48-well plates. When the cell density reached 80%, the culture medium was aspirated, and 100 μL of different concentrations of CaCl2 solution were added for 4 hours. The CaCl2 solution was then aspirated, and fresh culture medium was added for further culturing for 24 hours. Subsequently, live and dead cell staining was performed. 100 μL of Calcein AM / PI detection working solution (Beyotime) was added to each well, and the cells were then incubated at 37°C for 30 minutes. The results were then immediately observed under a fluorescence microscope. The results are as follows: Figure 1 As shown, 0.1 M is the optimal concentration of CaCl2 solution.

[0051] Example 2: Preparation of CMC-RGD hydrogel Synthesis of RGD-modified CMC: CMC was dissolved in deionized water at concentrations of 3 wt%, 4 wt%, 5 wt%, and 6 wt%. After mixing at room temperature, NHS and EDC were added to activate the carboxyl groups. RGD peptides were then added and reacted thoroughly, with the molar ratio of CMC to RGD being 1000:3, the molar ratio of EDC to RGD being 1:0.05, and the molar ratio of EDC to NHS being 1:0.5. The reaction solution was dialyzed through a dialysis membrane for 48 h, and then lyophilized to obtain RGD-functionalized CMC powder. Fluoroaldehyde peptide assays were performed on the functionalized CMC to confirm that RGD was successfully linked to the long chain of CMC. The results are shown below. Figure 2 As shown.

[0052] Cross-linking and molding of hydrogels: Lyophilized CMC-RGD powder and unmodified CMC powder were dissolved in culture medium (ordinary high-glucose DMEM medium without fetal bovine serum) to prepare precursor solutions of corresponding concentrations. The precursor solutions were spread on glass bottom plates, and 0.1M CaCl2 solution was added. Cross-linking was carried out at room temperature for 4 hours to obtain hydrogels of different stiffnesses, named 3CR, 4CR, 5CR, 6CR (RGD modified group) and 3C, 4C, 5C, 6C (unmodified group). The hydrogels after gelation are as follows... Figure 3 As shown. Scanning electron microscopy imaging of the hydrogel is as follows. Figure 4 As shown, the higher the concentration, the lower the porosity. The storage modulus (G') and loss modulus (G'') of the hydrogel are as follows: Figure 5 As shown, the 4CR colloidal hydrogel prepared in this invention has a storage modulus (G') of 120.87 ± 4.97 Pa at 10 rad / s and 1.512 ± 0.016 kPa at 100 rad / s, respectively, exhibiting viscoelastic response and frequency dependence.

[0053] Example 3: Organoid Culture Based on CMC-RGD Hydrogel The hydrogel prepared in Example 1 can be used to culture epithelial organoids, epithelial tumor organoids, and neural tumor organoids. Epithelial organoid culture was simulated using HaCaT (epidermal cells), epithelial tumor organoids were simulated using A375 (melanoma cells) and A549 (lung cancer cells), and neural tumor organoids were simulated using M059J (glioblastoma cells).

[0054] Cell preparation: HaCaT (epidermal cells), A375 (melanoma cells), A549 (lung cancer cells), and M059J (glioblastoma cells) were expanded and cultured according to conventional cell culture methods.

[0055] 3D organoid culture: Digested single cells were resuspended in CMC or CMC-RGD hydrogel precursor solution, and after cross-linking with 0.1M CaCl2, the culture medium was replaced with the complete culture medium for the corresponding cells. The cells were cultured at 37℃ and 5% CO2 for 7 days to form 3D organoids.

[0056] Organoid identification: Immunofluorescence staining was used to detect organoid-specific markers. Results showed that the organoids cultured in the 4CR group had the largest diameter and the highest expression levels of specific markers (such as KRT1 / 10 in HaCaT cells and HMB45 / Ki67 in A375 cells). Results are as follows: Figure 6 As shown.

[0057] Example 4: Verification of the molecular mechanism of mechanical regulation of organoid formation Immunofluorescence staining: Immunofluorescence staining of cultured HaCaT, A375, A549, and M059J organoids for ITGB1, pFAK, F-actin, and YAP was performed. Results showed that the 4CR group had the highest expression levels of ITGB1 and pFAK, and the most significant YAP nuclear translocation, demonstrating that neutral-stiff hydrogels can activate mechanotransmission pathways. Results using HaCaT cells as an example are shown below. Figure 7 As shown.

[0058] Example 5: Tumor Microenvironment Simulation and Drug Screening Construction of a fibrotic tumor organoid model: Sodium alginate and type I collagen were added to 4CR hydrogel, with concentrations of 1.5 wt% and 4 mg / ml for sodium alginate and type I collagen, respectively, to simulate fibrosis of the acral melanoma ECM. Results showed that with increasing hydrogel stiffness and collagen concentration, the volume of the A375 organoid increased, and the activation level of the ITGB1 / pFAK / F-actin / YAP pathway was enhanced. (See attached figures.) Figure 8 As shown.

[0059] Antitumor drug screening: A375 organoids were co-cultured with seven antitumor drugs, including methotrexate, a BET bromide domain inhibitor, and osimertinib. The efficacy of the drugs was evaluated by staining of live and dead cells. Results showed that 5-fluoro-2'-deoxycytidine and methotrexate disodium at a concentration of 1 mM achieved half-maximal cell killing, identifying potential drugs with high efficacy. The drug screening results are as follows: Figure 9 As shown.

[0060] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A hydrogel composition for three-dimensional organoid culture, characterized in that, It comprises: a hydrogel matrix, and cell adhesion peptides covalently coupled to the hydrogel matrix; The hydrogel matrix is ​​selected from one or more of carboxymethyl cellulose, hyaluronic acid, chitosan, or polyethylene glycol. The concentration of the hydrogel matrix is ​​3 wt% to 6 wt%.

2. The hydrogel composition according to claim 1, characterized in that, The cell adhesion peptide is a peptide containing the RGD sequence.

3. The hydrogel composition according to claim 1 or 2, characterized in that, The hydrogel matrix is ​​made of carboxymethyl cellulose, and the concentration of carboxymethyl cellulose is 4 wt%.

4. The hydrogel composition according to any one of claims 1-3, characterized in that, It also contains sodium alginate and / or type I collagen; The concentration of sodium alginate is 0.6wt% to 1.5wt%, and the concentration of type I collagen is 2mg / ml to 4mg / ml.

5. The hydrogel composition according to claim 4, characterized in that, Composed of RGD-modified CMC, sodium alginate, and type I collagen; Wherein, the RGD-modified CMC refers to carboxymethyl cellulose covalently coupled with RGD; The concentrations of carboxymethyl cellulose, sodium alginate, and type I collagen in the hydrogel composition are 4 wt%, 1.5 wt%, and 4 mg / ml, respectively.

6. Any of the following applications of the hydrogel composition according to any one of claims 1-5: (1) Cell culture; (2) Construction of tissue-specific organoids.

7. A method for culturing three-dimensional organoids, characterized in that, Includes the following steps: 1) Prepare a precursor solution from the hydrogel composition according to any one of claims 1-5 using a culture medium; 2) Suspend the target cells in the precursor solution; 3) Add a cross-linking agent to cross-link the precursor solution to form a hydrogel encapsulating the target cells; 4) Cultivate hydrogels containing the target cells until three-dimensional organoids are formed.

8. The method according to claim 7, characterized in that, The crosslinking agent is a calcium ion solution; preferably, the concentration of the calcium ion solution is 0.1 M.

9. The method according to claim 7 or 8, characterized in that, The target cells are selected from epithelial cells and / or tumor cells; Preferably, the tumor cells are selected from melanoma cells, lung cancer cells, and glioblastoma cells.

10. A method for screening candidate drugs targeting three-dimensional organoids, characterized in that, Includes the following steps: a) Culture three-dimensional organoids according to any one of claims 7-9; b) Contact the three-dimensional organoid with the candidate drug to be tested; c) Detect the biological effects of the candidate drug on the three-dimensional organoid to evaluate its efficacy.

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