Polymer gel for organoid culture and preparation method thereof

Abstract

The invention discloses a polymer gel for organoid culture and a preparation method thereof, and belongs to the technical field of biomedical materials, the gel is formed by cross-linking a phenylboronic acid grafted oxidized sodium alginate solution and a dopamine modified polyvinyl alcohol and cysteine modified gelatin mixed solution. A dynamic boric acid ester bond and enzymolysis network dual-crosslinking system is constructed, the pH-mechanical self-adaptive function is achieved, and the physical limitation of organoid growth is effectively relieved; by utilizing a phenol-sulfydryl synergistic dual anti-oxidation mechanism, active oxygen is removed, and organoid core necrosis is inhibited; a biological enzymolysis mechanism is introduced, so that the problem that gel degradation is mismatched with the cell growth rate is solved. The invention provides an excellent matrix material for standard and large-scale culture of organoids.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to a polymeric gel for organoid culture and its preparation method. Background Technology

[0002] Organoids are miniature organ structures formed by the self-assembly of stem cells in an in vitro 3D culture system, and they hold significant value in disease models, drug screening, and regenerative medicine. Currently, organoid culture primarily relies on matrix gels (such as Matrigel) extracted from mouse sarcomas. However, Matrigel suffers from serious drawbacks, including large batch-to-batch variability, the risk of carrying animal-derived pathogens, and unclear composition, which limit its clinical translation.

[0003] To replace Matrigel, researchers have developed various synthetic hydrogels (such as PEG and alginate hydrogels). However, existing synthetic hydrogel technologies suffer from the following main problems: (1) Lack of dynamic stress relaxation characteristics: Most synthetic hydrogels are static covalent cross-linked networks with constant stiffness. During the growth process, organoids expand rapidly in volume, and the static network cannot dissipate stress through rearrangement, resulting in physical constraints on organoids, growth stagnation or morphological deformities.

[0004] (2) Oxidative stress damage: Due to the high cell density, the core region of organoids is often in a state of hypoxia and high metabolism, producing a large amount of reactive oxygen species (ROS), which leads to core necrosis and limits the final size of organoids.

[0005] (3) Mismatch between degradation and growth rate: This is an objective but often overlooked problem in the existing technology. If the hydrogel degrades too quickly, the organoids will settle and fuse; if the degradation is too slow, it will hinder growth. A single degradation mechanism is difficult to adapt to the needs of different growth stages of organoids.

[0006] Therefore, developing a polymeric gel that can simultaneously address physical limitations, oxidative stress, and degradation rate matching is key to achieving standardized and large-scale organoid culture. Summary of the Invention

[0007] This invention aims to solve the aforementioned technical problems and provide a polymeric gel for organoid culture. By specifically modifying the polymeric raw materials, this invention constructs a dual cross-linking system based on a dynamic borate ester bond of "phenylboronic acid-dopamine / polyvinyl alcohol" and an enzymatic hydrolysis network of "cysteine-gelatin," achieving synergistic effects between the raw materials.

[0008] The technical solution adopted in this invention is as follows: A polymeric gel for organoid culture is formed by cross-linking a mixture of component A solution and component B solution; Component A is phenylboronic acid grafted with sodium alginate; Component B is a mixed solution of dopamine-modified polyvinyl alcohol and cysteine-modified gelatin. The polymer gel forms a dynamic borate ester bond network, a hydrogen bond network, and a disulfide bond auxiliary network.

[0009] Furthermore, in component A, the preparation method of phenylboronic acid-grafted sodium alginate is as follows: first, sodium alginate is oxidized with sodium periodate to obtain oxidized sodium alginate, and then 3-aminophenylboronic acid is grafted onto the side chain of oxidized sodium alginate through an amidation reaction.

[0010] Improved principle: By controlling the amount of raw materials used, the oxidation degree is controlled, providing sufficient grafting sites while maintaining the appropriate rigidity of the sodium alginate skeleton; at the same time, by controlling the amount of raw materials used, the grafting rate is controlled to ensure the formation of a sufficiently dense number of dynamic cross-linking points, while avoiding excessive hydrophobicity that would lead to dissolution difficulties.

[0011] Furthermore, in component B, the dopamine-modified polyvinyl alcohol is prepared by: carboxylating polyvinyl alcohol and then reacting it with dopamine via an amidation reaction.

[0012] Improvement principle: The dopamine group in dopamine-modified polyvinyl alcohol not only provides a strong binding site with phenylboronic acid (more stable than ordinary hydroxyl groups), but also provides a cell adhesion site similar to the extracellular matrix, and has a certain antioxidant capacity.

[0013] Furthermore, in component B, the method for preparing cysteine-modified gelatin is as follows: L-cysteine ​​is grafted onto the carboxyl group of the gelatin molecule using a carbodiimide chemical method.

[0014] Improvement principle: Introducing cysteine ​​endows gelatin with strong reducing properties (antioxidant), while retaining the arginine-glycine-aspartic acid (RGD) cell adhesion sequence and matrix metalloproteinase (MMP) digestion sites of gelatin.

[0015] Furthermore, the preparation method of the polymeric gel includes the following steps: Step S1: Preparation of component A solution: Dissolve phenylboronic acid-grafted sodium alginate in PBS buffer at pH 7.4 to a concentration of 15–25 mg / mL; Step S2: Preparation of component B solution: Dopamine-modified polyvinyl alcohol and cysteine-modified gelatin are dissolved in PBS buffer at pH 7.4, wherein the concentration of dopamine-modified polyvinyl alcohol is 15-25 mg / mL and the concentration of cysteine-modified gelatin is 5-15 mg / mL. Step S3: Mix component A solution and component B solution at a volume ratio of 1:1, and let stand at 37°C for 5 minutes to form a gel, i.e., form a polymer gel in situ.

[0016] The beneficial effects of this invention are: (1) A dynamic network with "pH-mechanical self-adaptation" function was constructed, which effectively relieved the physical limitations of organoid growth: The present invention constructed a main crosslinking network based on dynamic borate ester bonds by grafting sodium alginate oxidized with phenylboronic acid (component A) and dopamine modified polyvinyl alcohol (component B).

[0017] A. Verification of gelation mechanism: In Comparative Example 1, component A was replaced with ordinary sodium alginate without grafted phenylboronic acid, and the result showed "no gelation". However, in Example 2, gelation occurred within 50 seconds at the same concentration, proving that phenylboronic acid (PBA) is the key to constructing the gel skeleton.

[0018] B. Dynamic stress relaxation characteristics: Stress relaxation time (t) in Example 2 1 / 2 The time to gel formation was 95 seconds, indicating that the gel network has good dynamic rearrangement ability. In contrast, Comparative Example 2 used unmodified PVA (relying solely on PBA and hydroxyl groups), which, although able to gel, had a G′ of only 120 Pa and a time to gel formation of only 95 seconds. 1 / 2 In just 25 seconds, the structure was too loose and unstable; however, in Example 2, by introducing dopamine (DA) and utilizing the strong complexation of PBA-DA, the mechanical strength (G′ increased to 750 Pa) was significantly improved while maintaining a suitable dynamic relaxation ability.

[0019] C. pH-responsive intelligent regulation: Of particular importance, Comparative Example 5 simulated the slightly acidic environment (pH 6.6) generated by organoid metabolism. Compared with Example 2 (pH 7.4), the storage modulus G′ of the gel adaptively decreased from 750 Pa to 320 Pa, and the stress relaxation time t 1 / 2 The time was significantly reduced from 95 seconds to 32 seconds. This characteristic of "softening and relaxing faster when exposed to acid" allows it to precisely and automatically reduce physical constraints in areas of high cellular metabolism, making room for the expansion of organoid volume and solving the problem of "static network limiting growth" mentioned in the background technology.

[0020] (2) A dual antioxidant mechanism of "phenol-thiol" synergy was realized to effectively inhibit organoid core necrosis: In response to the problem of ROS accumulation caused by hypoxia in organoid core, this invention constructed a dual antioxidant system by using dopamine (containing phenolic hydroxyl groups) and cysteine ​​(containing thiol groups).

[0021] A. Dopamine’s dominant role: Example 2 showed a DPPH scavenging rate of up to 95.2%, while the scavenging rate of Comparative Example 2 (containing only cysteine) with dopamine removed dropped sharply to 42.6%, indicating that dopamine-modified polyvinyl alcohol provides basic and efficient free radical scavenging ability.

[0022] B. Synergistic effect of cysteine: Comparative Example 3 (containing only dopamine) with cysteine ​​removed had a DPPH scavenging rate of 76.5%, which was approximately 18.7% lower than that of Example 2. This indicates that the thiol groups introduced by the cysteine-modified gelatin have a synergistic effect with the phenolic hydroxyl groups of dopamine, raising the overall antioxidant capacity to a high level of over 95%. This highly efficient ROS scavenging ability can effectively protect the core cells of organoids under hypoxic conditions, prevent core necrosis, and thus support the culture of larger-sized organoids.

[0023] (3) A biological enzymatic hydrolysis mechanism was introduced to solve the problem of mismatch between gel degradation and cell growth rate: This invention introduces cysteine-modified gelatin and utilizes the sensitivity of gelatin to matrix metalloproteinases (MMPs) to endow the synthetic gel with biodegradability.

[0024] A. Verification of the necessity of enzymatic hydrolysis: Comparative Example 4 completely removed the gelatin component (containing only PVA-DA), and the degradation rate was only 5.2% in the 24-hour enzymatic degradation experiment, which is basically no degradation. This means that without gelatin, as the organoids grow, the gel will not be able to create space through enzymatic hydrolysis, eventually leading to growth stagnation.

[0025] B. Adjustable Degradation Rate: In contrast, the degradation rate increased to 45.6% after introducing gelatin in Example 2. Furthermore, comparisons with Examples 1-3 show that the degradation rate can be adjusted from 62.4% to 28.5% as the raw material concentration increases (network density increases). This indicates that the present invention can flexibly customize the degradation rate of the gel by adjusting the component concentration, precisely matching it to the expansion rate of different types of organoids, avoiding the contradictions mentioned in the background art of "degradation too fast leading to sedimentation" or "degradation too slow hindering growth."

[0026] (4) Excellent mechanical property adjustability and gelation stability: Data from Examples 1-3 show that the gelation time can be controlled between 35s and 70s by simply adjusting the raw material concentration, which is convenient for operation; the storage modulus G′ can be linearly controlled within the range of 420Pa to 1150Pa. This wide range of mechanical property adjustability allows the gel system to simulate the extracellular matrix stiffness of different tissues (such as brain, intestine, and liver), providing a universal platform for the standardized culture of various organoids. At the same time, comparing the G′ values ​​(120Pa vs 750Pa) of Comparative Example 2 (unmodified PVA) and Example 2 (dopamine-modified PVA) demonstrates that the introduction of dopamine significantly enhances the crosslinking density and structural stability of the network.

[0027] In summary, this invention, through unique raw material modification and component combination, successfully constructed a polymeric gel integrating "pH adaptive dynamic relaxation", "dual synergistic antioxidant" and "enzyme-responsive biodegradation", systematically solving the three core pain points of existing synthetic hydrogels in organoid culture: physical limitations, oxidative damage and degradation mismatch. Detailed Implementation

[0028] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Furthermore, unless otherwise specified, the raw materials, reagents, or devices used in the following embodiments can be obtained from conventional commercial channels or by existing known methods.

[0029] Preparation Example 1 Preparation of phenylboronic acid-grafted sodium alginate (OSA-PBA): Oxidation: Weigh 10g of sodium alginate (reagent grade, 90%, M / G=1:2, Maclean) and dissolve it in 200mL of distilled water. Add 6.4g of sodium periodate (reagent grade, 99.5%, Maclean) to the solution and stir in the dark for 6 hours. Then add 5mL of ethylene glycol (analytical standard, 99.9% (GC)) to terminate the reaction. Dialyze for 3 days and freeze-dry to obtain oxidized sodium alginate (OSA).

[0030] Grafting: Dissolve 2g of the above-mentioned sodium alginate (OSA) in 100mL of MES buffer (0.2M, pH 5.5, Maclean's), add 1g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, 98.0%, Sinopharm Reagent) and 0.6g of N-hydroxysuccinimide (NHS, 98.0%, Sinopharm Reagent) and activate for 30 minutes, then add 1g of 3-aminophenylboronic acid (98%, Maclean's), react at room temperature for 24 hours, dialyze (MWCO 12000Da, Sigma-Aldrich) for 3 days, and freeze-dry to obtain phenylboronic acid-grafted sodium alginate (OSA-PBA).

[0031] Preparation Example 2 Preparation of dopamine-modified polyvinyl alcohol (PVA-DA): Carboxylation: Dissolve 5g of polyvinyl alcohol (pharmaceutical grade, PVA-1788, Shanxi Jinyang Pharmaceutical Excipients) in 100mL of dimethyl sulfoxide (99.7%, Sinopharm Reagent), add 1.5g of succinic anhydride (Sinopharm Reagent) and 1mL of pyridine (AR, ≥99.5%, Sinopharm Reagent), react at 80℃ for 12 hours, and then obtain carboxylated PVA by precipitation, washing and drying.

[0032] Grafting: Carboxylated PVA was dissolved in distilled water, and 1g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, 98.0%, Sinopharm reagent) and 0.6g of N-hydroxysuccinimide (NHS, 98.0%, Sinopharm reagent) were added to activate the solution for 30 minutes. Then, 2g of dopamine hydrochloride (99%, Sinopharm reagent) was added, and the reaction was carried out under nitrogen protection for 12 hours. The solution was then dialyzed (44mm DM36, molecular weight 8000-14000, Sinopharm reagent) and lyophilized to obtain dopamine-modified polyvinyl alcohol (PVA-DA).

[0033] Preparation Example 3 Preparation of cysteine-modified gelatin (Cys-Gel): Dissolve 5g of gelatin (biotechnology grade, sourced from cowhide, Maclean's) in 200mL of warm water (50℃). Add 1.5g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, 98.0%, Sinopharm reagent) and 0.9g of N-hydroxysuccinimide (NHS, 98.0%, Sinopharm reagent) to activate the gelatin for 30 minutes. Then add 1.5g of L-cysteine ​​(Biochemical reagent BR (Shanghai Test), ≥98.5%, Sinopharm reagent), adjust the pH to 5.0, and react for 24 hours. Then, dialyze (44mm DM36, molecular weight 8000~14000, Sinopharm reagent) and freeze-dry to obtain cysteine-modified gelatin (Cys-Gel).

[0034] Example 1

[0035] A polymeric gel for organoid culture and its preparation method: Preparation of Component A solution: The phenylboronic acid-grafted sodium alginate prepared in Preparation Example 1 was dissolved in PBS buffer (China National Pharmaceutical Group Co., Ltd.) at pH 7.4 to obtain Component A solution. The concentration of phenylboronic acid-grafted sodium alginate in Component A was 15 mg / mL.

[0036] Preparation of Component B solution: The dopamine-modified polyvinyl alcohol prepared in Preparation Example 2 and the cysteine-modified gelatin prepared in Preparation Example 3 were dissolved in PBS buffer (China National Pharmaceutical Group Co., Ltd.) at pH 7.4 to obtain Component B solution. The concentration of dopamine-modified polyvinyl alcohol in Component B was 15 mg / mL and the concentration of cysteine-modified gelatin was 5 mg / mL.

[0037] Gel formation: Mix component A solution and component B solution at a volume ratio of 1:1, and let stand at 37°C for 5 minutes to form a gel, i.e., form a polymer gel in situ.

[0038] Example 2

[0039] A polymeric gel for organoid culture and its preparation method: Preparation of Component A solution: The phenylboronic acid-grafted sodium alginate prepared in Preparation Example 1 was dissolved in PBS buffer (China National Pharmaceutical Group Co., Ltd.) at pH 7.4 to obtain Component A solution. The concentration of phenylboronic acid-grafted sodium alginate in Component A was 20 mg / mL.

[0040] Preparation of Component B solution: The dopamine-modified polyvinyl alcohol prepared in Preparation Example 2 and the cysteine-modified gelatin prepared in Preparation Example 3 were dissolved in PBS buffer (China National Pharmaceutical Group Co., Ltd.) at pH 7.4 to obtain Component B solution. The concentration of dopamine-modified polyvinyl alcohol in Component B was 20 mg / mL and the concentration of cysteine-modified gelatin was 10 mg / mL.

[0041] Gel formation: Mix component A solution and component B solution at a volume ratio of 1:1, and let stand at 37°C for 5 minutes to form a gel, i.e., form a polymer gel in situ.

[0042] Example 3

[0043] A polymeric gel for organoid culture and its preparation method: Preparation of Component A solution: The phenylboronic acid-grafted sodium alginate prepared in Preparation Example 1 was dissolved in PBS buffer (China National Pharmaceutical Group Co., Ltd.) at pH 7.4 to obtain Component A solution. The concentration of phenylboronic acid-grafted sodium alginate in Component A was 25 mg / mL.

[0044] Preparation of Component B solution: The dopamine-modified polyvinyl alcohol prepared in Preparation Example 2 and the cysteine-modified gelatin prepared in Preparation Example 3 were dissolved in PBS buffer (China National Pharmaceutical Group Co., Ltd.) at pH 7.4 to obtain Component B solution. The concentration of dopamine-modified polyvinyl alcohol in Component B was 25 mg / mL and the concentration of cysteine-modified gelatin was 15 mg / mL.

[0045] Gel formation: Mix component A solution and component B solution at a volume ratio of 1:1, and let stand at 37°C for 5 minutes to form a gel, i.e., form a polymer gel in situ.

[0046] Comparative Example 1 Comparative Example 1 served as the control group for Example 2. The phenylboronic acid-grafted oxidized sodium alginate prepared in Example 1 was replaced with raw sodium alginate (reagent grade, 90%, M / G=1:2, Maclean) in component A solution, with the concentration remaining at 20 mg / mL. The rest remained unchanged, and a polymer gel was obtained.

[0047] Comparative Example 2 Comparative Example 2 served as the control group for Example 2. The dopamine-modified polyvinyl alcohol prepared in Example 2 was replaced in the component B solution with raw material polyvinyl alcohol (pharmaceutical grade, PVA-1788, Shanxi Jinyang Pharmaceutical Excipients), with the concentration remaining at 20 mg / mL and the rest unchanged, thus obtaining a polymer gel.

[0048] Comparative Example 3 Comparative Example 3 served as the control group for Example 2. The cysteine-modified gelatin prepared in Example 3 was replaced with raw material gelatin (biotechnology grade, derived from cowhide, Maclean) in the component B solution, with the concentration remaining at 10 mg / mL. All other parameters remained unchanged, thus obtaining the polymer gel.

[0049] Comparative Example 4 Comparative Example 4 served as the control group for Example 2. Solution B contained only 20 mg / mL of dopamine-modified polyvinyl alcohol prepared in Preparation Example 2, without the addition of cysteine-modified gelatin prepared in Preparation Example 3, and the rest remained unchanged, thus obtaining a polymeric gel.

[0050] Comparative Example 5 Comparative Example 5 served as the control group for Example 2. The pH of the PBS buffer (China National Pharmaceutical Group Reagent) in Component A and Component B solutions was changed from 7.4 to 6.6 (to simulate metabolic acidity), while the rest remained unchanged, thus obtaining the polymer gel.

[0051] Test Example 1 The polymer gels prepared in Examples 1-3 and Comparative Examples 1-5 were subjected to performance tests. The performance test process is as follows, and the test results are shown in Table 1: 1. Test item design principles: (1) Gel formation time: to verify the feasibility of the operation.

[0052] (2) Rheological test (storage modulus G′): to verify the mechanical strength of the gel.

[0053] (3) Stress relaxation test (key point): Verify the ability of "dynamic borate ester bond" to eliminate physical constraints.

[0054] (4) Antioxidant performance test (DPPH scavenging rate, key point): Verify the ability of "dual antioxidation" to solve core necrosis.

[0055] (5) Enzymatic hydrolysis performance test: to verify the feasibility of "biodegradation".

[0056] 2. Test Method Description: (1) Determination of gelation time: Using the test tube inversion method, component A and component B are mixed at 37°C. The test tube is inverted every 10 seconds, and the time when the flow stops is the gelation time.

[0057] (2) Rheological and mechanical properties and stress relaxation test: The test was conducted at 37°C using a rotational rheometer (e.g., Anton Paar or TA instrument).

[0058] A. Storage modulus (G′): The G′ value during the plateau period was recorded by time scanning at 1% strain and 1Hz frequency to characterize the gel stiffness.

[0059] B. Stress relaxation: Apply a constant shear strain of 10%, record the stress decay curve over time, and calculate the time (t) required for the stress to decrease to 50% of its initial value. 1 / 2 ).

[0060] This indicator directly reflects whether the gel restricts organoid growth. 1 / 2 The shorter the gel, the easier it is for dynamic rearrangement to occur, and the less physical restriction it places on cells.

[0061] (3) In vitro antioxidant performance test (DPPH free radical scavenging experiment): Prepare a 0.1 mM DPPH ethanol solution. Take a quantitative amount of gel sample and immerse it in the DPPH solution, and react in the dark for 30 minutes. Take the supernatant and measure the absorbance at 517 nm.

[0062] Calculation formula: Clearance rate (%) = [1 − (A)] sample / A control )]×100%.

[0063] This indicator corresponds to the ability to resolve "core necrosis".

[0064] (4) In vitro enzyme degradation test: Weigh the initial mass (W0) of the gel and immerse it in PBS solution containing type II collagenase (5 U / mL) (simulating the in vivo enzyme environment) and place it on a shaker at 37°C. After 24 hours, remove it, blot off the surface moisture, and weigh it (W0). t ).

[0065] Calculation formula: Degradation rate (%) = [(W0−W t ) / W0]×100%.

[0066] This indicator addresses the issue of "matching degradation and growth rates".

[0067] Table 1 Test Results

[0068] Based on the test results in Table 1, the following conclusions can be drawn: 1. Analysis of Examples 1-3: In Examples 1 to 3, the concentrations of component A (OSA-PBA) and component B (PVA-DA+Cys-Gel) increased sequentially (component A: 15→20→25 mg / mL; PVA-DA: 15→20→25 mg / mL; Cys-Gel: 5→10→15 mg / mL).

[0069] (1) Gel formation rate and mechanical strength (G′): Data trend: With increasing concentration, gel formation time shortens (70s→50s→35s), while storage modulus G′ significantly increases (420 Pa→750 Pa→1150 Pa). Analysis: Increased concentration significantly increases the density of polymer chains and crosslinking points (phenylboronic acid-dopamine / hydroxyl groups, hydrogen bonds). Higher crosslinking density leads to faster network formation and greater stiffness. This indicates that the mechanical strength of the gel can be customized by adjusting the concentration to meet the matrix stiffness requirements of different types of organoids.

[0070] (2) Dynamic stress relaxation characteristics (t) 1 / 2 Data trend: As concentration increases, stress relaxation time t 1 / 2 The duration is extended (60s→95s→135s). Analysis: Although the increased concentration improves the strength, it also makes the network denser, hindering chain segment movement and slowing down stress dissipation (t). 1 / 2 (Addition). Example 2 (95s) demonstrates a good balance between strength and dynamism, providing sufficient support while having a moderate stress relaxation capability, avoiding excessive physical constraints on the organoid.

[0071] (3) Antioxidant performance (DPPH scavenging rate): Data trend: The DPPH scavenging rate increased with increasing concentration (82.5%→95.2%→97.8%). Analysis: The total amount of antioxidant functional groups (phenolic hydroxyl groups of dopamine, thiol groups of cysteine) increased with increasing concentration, thereby enhancing the ability to scavenge free radicals. Examples 2 and 3 both maintained extremely high scavenging rates (>95%), indicating that the problem of "core necrosis" caused by oxidative stress can be effectively solved within this concentration range.

[0072] (4) Enzymatic degradation performance: Data trend: With increasing concentration, the 24-hour enzyme degradation rate decreased (62.4%→45.6%→28.5%). Analysis: The higher the concentration, the denser the gel network, and the more difficult it is for the enzyme (collagenase) to penetrate into the gel and degrade the gelatin components. This verifies that the "biodegradation" rate can be controlled by adjusting the concentration to match the expansion rate of different growth stages of organoids.

[0073] 2. Comparative analysis between the comparative example and Example 2: (1) Verification of the key role of "dynamic borate ester bonds" in construction (Comparative Example 1 vs. Example 2): Technical difference: Comparative Example 1 used ordinary sodium alginate without grafting phenylboronic acid (PBA). Result analysis: Comparative Example 1 "did not form a gel". Conclusion: This directly proves that phenylboronic acid (PBA) is the key core for forming the gel network. Without PBA, dynamic borate ester bonds cannot be formed with dopamine or hydroxyl groups in component B, and the basic three-dimensional network cannot be constructed. This verifies the necessity of component A.

[0074] (2) Verification of the dual role of dopamine in crosslinking and antioxidation (Comparative Example 2 vs. Example 2): Technical differences: Comparative Example 2 used ordinary PVA without grafted dopamine (DA). Result analysis: Gel formation and mechanics: Comparative Example 2 showed a significantly prolonged gelation time (150 s) and a drastic reduction in G′ (120 Pa vs. 750 Pa). This indicates that the borate ester bond formed between PBA and the hydroxyl groups of ordinary PVA has a weaker bonding force and a slower formation rate; while the complex formed by PBA and dopamine in Example 2 is more firmly bonded. Stress relaxation: t 1 / 2 The extremely short reaction time (25s) indicates that the network is too loose and unstable. Antioxidant performance: DPPH scavenging rate dropped sharply (42.6% vs 95.2%). This indicates that dopamine is the main contributor to antioxidant activity (a fast-responding antioxidant). Without dopamine, cysteine ​​alone (Comparative Example 2 still contains Cys-Gel) is insufficient to maintain efficient ROS scavenging capacity. Conclusion: Dopamine-modified PVA simultaneously enhances network structural stability and provides the main antioxidant capacity.

[0075] (3) Verification of the role of cysteine ​​in synergistic antioxidant and structural assistance (Comparative Example 3 vs. Example 2): Technical differences: Comparative Example 3 used ordinary gelatin without grafted cysteine ​​(Cys). Results analysis: Antioxidant: DPPH scavenging rate decreased significantly (76.5% vs. 95.2%). Although dopamine was retained, the antioxidant capacity decreased by about 19% after scavenging cysteine. This confirms the synergistic effect of the "phenol-thiol" dual antioxidant mechanism - cysteine ​​is essential as a long-acting antioxidant and buffer, and the two work together to achieve a scavenging rate of >95%. Mechanical properties: G′ decreased (580 Pa vs. 750 Pa). This indicates that the disulfide bonds or other interactions introduced by cysteine ​​have an auxiliary enhancing effect on the crosslinking density of the gel network. Conclusion: Cysteine-modified gelatin not only endows the material with synergistic antioxidant capacity (solving core necrosis) but also contributes to gel strength.

[0076] (4) Verification of the decisive role of the "gelatin component" in biodegradation (Comparative Example 4 vs. Example 2): Technical differences: Comparative Example 4 contains no cysteine-modified gelatin (Cys-Gel) at all, only PVA-DA. Results analysis: Enzymatic degradation rate: The degradation rate was extremely low (5.2% vs. 45.6%). Since PVA and sodium alginate themselves cannot be degraded by collagenase, Comparative Example 4 hardly underwent enzymatic hydrolysis. Mechanical properties: G′ decreased (480 Pa), indicating that gelatin also acts as a filler and structural support. Conclusion: This demonstrates the mechanism for "solving the degradation-growth rate mismatch"—the introduction of gelatin (and its enzymatic hydrolysis sites) is necessary to achieve the "bioenzymatic hydrolysis" mechanism in response to the secretion of MMP enzymes by cells. Without gelatin, the physical space constraints in the later stages of organoid growth cannot be resolved.

[0077] (5) Verification of the “pH-mechanical adaptation” mechanism (Comparative Example 5 vs. Example 2): Technical differences: Comparative Example 5 simulated a slightly acidic environment (pH 6.6), while Example 2 was a physiologically neutral environment (pH 7.4). Results analysis: Mechanical and dynamic properties: Under pH 6.6 conditions, G′ decreased (320 Pa), and the stress relaxation time t 1 / 2 Significantly shortened (32s vs 95s). Analysis: This validates the "pH-mechanical adaptive mechanism." In a slightly acidic environment (simulating a region of high cellular metabolism), the dynamic borate ester bond becomes more reactive (unstable), leading to gel softening (decreased G′) and accelerated stress relaxation (t). 1 / 2 (Shortening). Conclusion: This property allows the gel to automatically "soften" and "relax" in areas of rapid organoid growth and metabolic acid production, thereby freeing up growth space, while maintaining structural stability in areas far from cells. This is one of the core technologies of this invention for solving the "growth restriction" problem.

[0078] 3. Overall Summary: (1) Dual antioxidant effect: Example 2 (95.2%) was much higher than Comparative Example 2 (without dopamine, 42.6%) and Comparative Example 3 (without cysteine, 76.5%), demonstrating the synergistic antioxidant effect of PVA-DA and Cys-Gel.

[0079] (2) Dynamic adaptive growth: Physical mechanism: Through the formation of dynamic bonds between PVA-DA and OSA-PBA, the gel is endowed with suitable stress relaxation ability (Example 2 t) 1 / 2 =95s). Chemical mechanism: pH responsiveness (Comparative Example 5) demonstrates that the gel can sense a slightly acidic metabolic environment and accelerate relaxation. Biological mechanism: By introducing gelatin (Comparative Example 4), the gel is endowed with enzymatic remodeling ability (45.6% degradation rate).

[0080] (3) Structural stability: The combination of PBA and DA (Example 2) provides better gelation speed and mechanical strength than the ordinary PBA-PVA combination (Comparative Example 2).

[0081] It should be noted that, in this document, terms such as “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0082] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A polymeric gel for organoid culture, characterized in that, It is formed by cross-linking a mixture of component A solution and component B solution; Component A is phenylboronic acid grafted with sodium alginate; Component B is a mixed solution of dopamine-modified polyvinyl alcohol and cysteine-modified gelatin. The polymer gel forms a dynamic borate ester bond network, a hydrogen bond network, and a disulfide bond auxiliary network.

2. The polymeric gel for organoid culture according to claim 1, characterized in that, In component A, the preparation method of phenylboronic acid grafted oxidized sodium alginate is as follows: first, sodium alginate is oxidized with sodium periodate to obtain oxidized sodium alginate, and then 3-aminophenylboronic acid is grafted onto the side chain of oxidized sodium alginate through an amidation reaction.

3. The polymeric gel for organoid culture according to claim 1, characterized in that, The preparation method of dopamine-modified polyvinyl alcohol in component B is as follows: after carboxylating polyvinyl alcohol, it undergoes an amidation reaction with dopamine.

4. The polymeric gel for organoid culture according to claim 1, characterized in that, In component B, the preparation method of cysteine-modified gelatin is as follows: L-cysteine ​​is grafted onto the carboxyl group of gelatin molecules using a carbodiimide chemical method.

5. A method for preparing a polymeric gel for organoid culture according to any one of claims 1 to 4, characterized in that, The preparation method of the polymer gel includes the following steps: Step S1: Preparation of component A solution: Dissolve phenylboronic acid-grafted sodium alginate in PBS buffer at pH 7.4; Step S2: Preparation of component B solution: Dissolve dopamine-modified polyvinyl alcohol and cysteine-modified gelatin in PBS buffer at pH 7.4; Step S3: Mix component A solution and component B solution at a volume ratio of 1:1, and let stand at 37°C for 5 minutes to form a gel, i.e., form a polymer gel in situ.

6. The method for preparing a polymeric gel for organoid culture according to claim 5, characterized in that, The concentration of phenylboronic acid grafted onto sodium alginate in component A solution is 15–25 mg / mL.

7. The method for preparing a polymeric gel for organoid culture according to claim 5, characterized in that, The concentration of dopamine-modified polyvinyl alcohol in component B solution is 15–25 mg / mL.

8. The method for preparing a polymeric gel for organoid culture according to claim 5, characterized in that, The concentration of cysteine-modified gelatin in component B solution is 5–15 mg / mL.

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