Hydrogel as well as preparation method and application thereof

Abstract

The invention provides hydrogel as well as a preparation method and application thereof, and the preparation method comprises the following steps: dissolving glyoxylic acid, chitosan and neutral macromolecules in water according to a mass ratio of (3-6): (6-10): (3-6), mixing to obtain a mixed solution, and carrying out heating treatment and swelling on the mixed solution to obtain the hydrogel. The hydrogel prepared by the invention can avoid damage of a cross-linking agent, an initiator or illumination and the like to cells, can ensure the survival rate and health degree of the cells, and ensures healthy proliferation and normal functions of the cells; and the hydrogel has no cytotoxicity, so that the three-dimensional cells can be cultured.

Description

Technical Field

[0001] This invention relates to the field of hydrogel technology, specifically to a hydrogel, its preparation method, and its applications. Background Technology

[0002] With the development of biomaterials and tissue engineering, three-dimensional cell culture substrates have become core tools in disease model research, drug screening, and other fields. However, existing methods for preparing three-dimensional cell culture hydrogel matrices typically require pre-mixing cells with hydrogel precursor solutions (such as collagen) and then forming a gel through chemical or photocrosslinking. The crosslinking agents or intermediates used in chemical crosslinking (such as free radicals) may cause chemical damage to cells, and the ultraviolet light used in photocrosslinking may also cause radiation damage. These factors can reduce cell viability or gene stability, affecting subsequent disease model research and drug screening. Adding additional protective agents increases the complexity of the culture system and the cost of large-scale preparation. Currently, most commercially available three-dimensional cell culture substrates are collagen-based, resulting in high costs. Summary of the Invention

[0003] The technical problem to be solved by this invention is how to prepare a hydrogel capable of culturing three-dimensional cells.

[0004] The present invention solves the above-mentioned technical problems through the following technical means:

[0005] The first aspect of the present invention provides a method for preparing a hydrogel, comprising the following steps: dissolving glyoxylic acid, chitosan and neutral polymer in water at a mass ratio of 3~6:6~10:3~6 to obtain a mixture, and then heating and swelling the mixture to obtain a hydrogel.

[0006] Beneficial effects: The hydrogel prepared by this invention can avoid damage to cells by cross-linking agents, initiators or light, and can ensure cell survival rate and health, and protect the healthy proliferation and normal function of cells; moreover, the hydrogel is non-cytotoxic, indicating that it can culture three-dimensional cells.

[0007] Preferably, the neutral polymer includes one or more of polyacrylamide, polyvinyl alcohol, and hydroxypropyl methylcellulose.

[0008] Preferably, the temperature at which it dissolves in water is 50℃~70℃.

[0009] Preferably, the mixing is ultrasonic mixing.

[0010] Preferably, the mixing temperature is 50℃~70℃.

[0011] Preferably, the heating temperature is 70℃~90℃ and the heating time is 3h~8h.

[0012] Preferably, the swelling is achieved by soaking in water for 16 to 24 hours.

[0013] A second aspect of the present invention provides a method for preparing the above-mentioned hydrogel to obtain the hydrogel.

[0014] Beneficial effects: The hydrogel of the present invention has a stable water content, which can provide nutrients to three-dimensional cells and support their healthy growth. The hydrogel has high transparency, which can enable real-time non-destructive observation of cell or cell sphere growth. The hydrogel has good mechanical properties, which is beneficial to maintaining the normal phenotype and function of cells.

[0015] The third aspect of this invention provides the application of the hydrogel prepared by the above-mentioned method in the culture of three-dimensional cells.

[0016] The fourth aspect of this invention provides the application of the hydrogel prepared by the above-mentioned method in drug screening.

[0017] Beneficial effects: After culturing tumor cells and adding anticancer drugs, the hydrogel of the present invention can clearly show cell growth inhibition and morphological changes. Therefore, the hydrogel of the present invention can be used for screening anticancer drugs or evaluating their efficacy. Attached Figure Description

[0018] Figure 1 These are water content test graphs of the hydrogels prepared in Examples 1-3 of the present invention and the chitosan gel prepared in Comparative Example 1; Figure 2 These are images of the hydrogel prepared in Example 1 of the present invention and the chitosan gel prepared in Comparative Example 1 before soaking in cell culture medium (Figure A) and after soaking for 24 h (Figure B). The arrows indicate the defects produced by the chitosan gel after soaking in deionized water. Figure 3 These are optical transparency test images of the hydrogels prepared in Examples 1-3 of this invention; Figure 4 These are mechanical property test diagrams of the hydrogels prepared in Examples 1-3 of this invention; Figure 5 These are cytotoxicity test diagrams of the hydrogels prepared in Examples 1-3 of this invention; Figure 6 This is the relative volume growth curve of cell spheres in the hydrogel cultured with three-dimensional cells prepared in Example 1 of this invention; Figure 7 These are micrographs of three-dimensional cells cultured in hydrogel prepared in Example 1 of the present invention at days 2, 6, 10, 16, and 32 (Figure A), and a DAPI-stained cell nucleus photograph after cell spheroid sections cultured to day 32 (Figure B). Figure 8 This is a drug screening test diagram of the hydrogel prepared in Example 1 of the present invention. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, 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.

[0020] Unless otherwise specified, all test materials and reagents used in the following examples are commercially available.

[0021] Unless otherwise specified in the embodiments, the techniques or conditions described in the literature in this field or in accordance with the product manual may be followed.

[0022] Example 1 This embodiment provides a method for preparing a hydrogel, the specific steps of which are as follows: Dissolve 0.24 g of glyoxylic acid in 3 mL of sterile water to obtain a glyoxylic acid solution; uniformly disperse 0.40 g of medium-viscosity chitosan (200 mPa·s~400 mPa·s) in 9 mL of sterile water and stir at 60 °C until dissolved; then add the glyoxylic acid solution and continue stirring to obtain a mixed solution; disperse 0.24 g of polyacrylamide in 6 mL of sterile water and stir at 60 °C until dissolved to obtain a polyacrylamide solution.

[0023] The mixed solution was mixed with a polyacrylamide solution and ultrasonically mixed at 60°C for 30 min to remove bubbles, and the mixture was heated at 90°C for 3 h to allow it to fully crosslink. After the reaction, the product was immersed in sterile water, with the water changed every 2 hours, and repeated 3 times. Then, it was allowed to stand in sterile water for 16 h to swell, thus obtaining the hydrogel.

[0024] Example 2 This embodiment provides a method for preparing a hydrogel, the specific steps of which are as follows: 0.15 g of glyoxylic acid and 0.30 g of high-viscosity chitosan (>400 mPa·s) were uniformly dispersed in 9 mL of sterile water and stirred at 50°C until dissolved to obtain a mixed solution. 0.30 g of polyvinyl alcohol was dispersed in 9 mL of sterile water and stirred at 60°C until dissolved to obtain a polyvinyl alcohol solution.

[0025] The mixed solution was mixed with a polyvinyl alcohol solution and ultrasonically mixed at 50°C for 30 min to remove bubbles, thus obtaining a mixed solution. The mixed solution was heated at 80°C for 8 h to allow it to fully crosslink. After the reaction, the product was immersed in sterile water, with the water changed every 2 hours, and repeated 3 times. After standing in sterile water for 16 h to swell, the hydrogel was obtained.

[0026] Example 3 This embodiment provides a method for preparing a hydrogel, the specific steps of which are as follows: 0.15 g of hydroxypropyl methylcellulose, 0.30 g of glyoxylic acid, and 0.50 g of low-viscosity chitosan (<200 mPa·s) were uniformly dispersed in 20 mL of sterile water. The mixture was stirred and sonicated at 70°C for 30 min until the solution was homogeneous and free of bubbles. The mixture was then heated at 70°C for 5 h to allow for full cross-linking. After the reaction, the product was immersed in sterile water, with the water changed every 2 hours, repeated 3 times. After standing in sterile water for 16 h to swell, the hydrogel was obtained.

[0027] Comparative Example 1 This comparative example provides a method for preparing chitosan gel, the specific steps of which are as follows: 0.12 g of glyoxylic acid was dissolved in 3 mL of deionized water at room temperature to obtain a glyoxylic acid solution. 0.20 g of high-viscosity chitosan (>400 mPa·s) was dispersed in 3 mL of deionized water. The entire glyoxylic acid solution was added while heating at 50°C with magnetic stirring. Heating and stirring were continued for 20 min to obtain a clear, slightly viscous mixed solution. The mixed solution was sonicated for 1 min to remove small air bubbles. The solution was then transferred to a 1 mL syringe, capped, and placed in a 70°C oven for 4 h. The syringe was then removed, the gel was expelled, and immersed in excess deionized water to swell, yielding chitosan gel.

[0028] Experimental Example The hydrogels prepared in Examples 1-3 and the chitosan gel prepared in Comparative Example 1 were subjected to performance characterization.

[0029] 1. Characterization of water content: The test method is as follows: The hydrogels prepared in Examples 1-3 and the chitosan gel prepared in Comparative Example 1 were placed in deionized water and allowed to swell for 24 h. After removal, one group was directly dried and the water content was calculated, while the other group was transferred to DMEM medium containing 10% FBS and 1% penicillin-streptomycin, and soaked for another 24 h. After drying, the water content in the cell culture medium was measured. The specific results are as follows: Figure 1 As shown in Table 1.

[0030] Table 1

[0031] according to Figure 1 As shown in Table 1, in deionized water, the water content of the hydrogels and chitosan gels prepared in Examples 1-3 is higher than 99%; while in cell culture medium, the water content of the hydrogels and chitosan gels prepared in Examples 1-3 decreases, but is still higher than 95%, which meets the general requirements of cell culture medium for water content.

[0032] 2. Characterization of appearance changes: The test method is as follows: The hydrogel prepared in Example 1 and the chitosan gel prepared in Comparative Example 1 were placed in deionized water and allowed to swell for 24 h. After removal, they were photographed, and then transferred to DMEM medium containing 10% FBS and 1% penicillin-streptomycin, respectively, and soaked for another 24 h before being photographed again. Figure 2 It can be seen that chitosan gel swells in deionized water and produces defects (as indicated by the arrows in Figure A), showing that chitosan gel is brittle after swelling in deionized water. This indicates that chitosan gel is prone to disintegration if used to culture three-dimensional cells. At the same time, chitosan gel shows a more significant decrease in volume and a significant reduction in gel transparency in cell culture medium, indicating that chitosan gel cannot be used for sustainable observation of three-dimensional cells. In contrast, the hydrogel prepared in Example 1 is more stable in terms of volume and transparency in cell culture medium. Therefore, the hydrogel prepared in this invention is more suitable for the culture of three-dimensional cells than chitosan gel.

[0033] 3. Characterization of optical transparency (1) The hydrogel prepared in Example 1 was placed on the printed pattern and photographed. Figure 3 As can be clearly observed in Figure A, the hydrogel itself is colorless and transparent, and the pattern beneath the hydrogel is clearly discernible without distortion or blurring. The results demonstrate the macroscopic transparency of the hydrogel.

[0034] (2) The hydrogels prepared in Examples 1-3 were scanned using a spectrophotometer in the visible light wavelength range of 400-800 nm. The results are as follows: Figure 3 As shown in B, the hydrogels maintain a high level of transmittance in the visible light range, with the hydrogel prepared in Example 1 exhibiting the highest transmittance, reaching 95% at a wavelength of 550 nm. The data confirms that the hydrogels prepared according to this invention possess high transparency.

[0035] 4. Mechanical property characterization: The hydrogels prepared in Examples 1-3 were subjected to compression tests using a texture analyzer to evaluate their mechanical properties. Hydrogels with a diameter of 1 cm and a height of 1 cm were compressed to 50% of their original height at a speed of 1 mm / s.

[0036] The results are as follows Figure 4As shown, the hydrogel exhibits typical elastomer compression behavior, with maximum compressive stress between 20 kPa and 35 kPa and elastic modulus between 10 kPa and 15 kPa. Its strength and elastic modulus match the mechanical properties of most biological soft tissues, and its elastic modulus is similar to that of solid tumor tissue. The results also indicate that the compressive yield point of the hydrogel occurs after 40% strain. Therefore, when used as a three-dimensional cell culture substrate, even if the internal cell spheres enlarge and compress the surrounding matrix, the hydrogel can maintain its structural integrity without being destroyed. Mechanically, the hydrogel is suitable as a three-dimensional cell culture substrate.

[0037] 5. Cytotoxicity characterization: The hydrogels prepared in Examples 1-3 were sterilized and then co-cultured with HeLa cells for 72 h. The cytotoxicity of the materials was detected by the MTT assay. The results are as follows: Figure 5 As shown, none of the three hydrogels exhibited significant cytotoxicity.

[0038] 6. Validation of three-dimensional cell culture performance The hydrogel prepared in Example 1 and the chitosan gel prepared in Comparative Example 1 were sterilized and then equilibrated for 24 h in DMEM medium containing 10% FBS and 1% penicillin-streptomycin. After replacing the medium with complete medium, 1×10 5 HeLa cells were injected into a hydrogel using a syringe. The cell-inoculated hydrogel was then returned to complete culture medium and placed in a cell culture incubator for further culture. During the culture process, the cell-inoculated hydrogels were removed on days 2, 6, 10, 16, and 32, and observed and photographed under bright field. The results are as follows: Figure 6 and Figure 7 As shown. The chitosan gel prepared in Comparative Example 1 completely disintegrated on day 6 and could not be used for three-dimensional cell culture; while the hydrogel prepared in Example 1 remained morphologically stable and its transparency did not change throughout the entire culture period, allowing for continuous observation. Figure 6 and Figure 7 As shown in Figure A, the formation and continuous growth of cell spheroids were clearly observed over time, demonstrating that cells can form three-dimensional cell spheroids and proliferate within the hydrogel. After culture, the cell spheroids were fixed, sectioned, and stained with DAPI to observe the distribution of cell nuclei. The staining results are shown below. Figure 7 As shown in Figure B, the cell nuclei are uniformly and densely distributed inside the cell spheres, without central necrosis or cavitation, confirming that the hydrogel can provide cells with a sufficient environment for nutrient transport and metabolic waste exchange, supporting the healthy, solid three-dimensional growth of the cell spheres.

[0039] 7. Drug Screening Validation: The hydrogel prepared in Example 1 was sterilized and equilibrated for 24 h in DMEM medium containing 10% FBS and 1% penicillin-streptomycin. After replacing the medium with complete medium, 1×10⁻⁶ g of the hydrogel was used to screen and validate the drug. 5One HeLa cell was injected into the hydrogel using a syringe. The hydrogel containing the cells was then returned to complete culture medium and placed in a cell culture incubator for further culture. On the second day, the experimental group was treated with 10... M doxorubicin was added, and the same concentration of doxorubicin was added every other day when the fluid was changed (i.e., the drug treatment group), while the control group was given phosphate buffered solution (PBS).

[0040] The results are as follows Figure 8 As shown, with increasing time, it can be clearly observed that the cell spheroids in the control group continue to grow, while the cell spheroids in the drug-treated group basically stop growing, and the edges of the cell spheroids become blurred in the later stages. The results confirm that the hydrogel of this invention can not only support three-dimensional cell culture, but its high transparency also allows for synchronous and visual observation of cell growth in drug screening.

[0041] In summary, the hydrogel of this invention achieves pre-crosslinking followed by seeding, avoiding damage to cells from chemical crosslinking agents, photoinitiators, or light exposure, thereby ensuring extremely high initial cell viability and health, and guaranteeing long-term, healthy cell proliferation and normal function. Figure 7 As shown, the cell spheres can continue to grow for 32 days without any cavitation.

[0042] The hydrogel of this invention exhibits high water content and high transparency in cell culture media: the chitosan-based amphoteric polyelectrolyte-neutral polymer semi-interpenetrating network structure solves the volume shrinkage problem of single chitosan gels in cell culture media. For example... Figure 2 As shown, the hydrogel of the present invention, after being soaked in cell culture medium, exhibits reduced volume shrinkage, no gel turbidity, and a stable water content of over 95%, while the chitosan gel undergoes drastic shrinkage and turbidity. This characteristic ensures its practicality in three-dimensional cell culture.

[0043] Hydrogels possess excellent optical transparency, enabling real-time, non-destructive observation of encapsulated cells or cell spheres: The hydrogels of this invention exhibit extremely high transmittance in the visible light range (400 nm~800 nm) (the hydrogel prepared in Example 1 achieved a transmittance of 95% at 550 nm), and its macroscopic transparency pattern is clearly discernible (e.g., Figure 3 (As shown in A). This characteristic allows for long-term, real-time, high-resolution microscopic observation of cells or cell spheres encapsulated within hydrogels, which is crucial for enabling dynamic cell monitoring and drug screening.

[0044] The mechanical properties of the hydrogel are highly compatible with those of biological soft tissues: the elastic modulus (10 kPa~15 kPa) and compressive strength (20 kPa~35 kPa) of the hydrogel of this invention are similar to the mechanical properties of solid tumors and biological soft tissues (e.g., Figure 4As shown in the figure, this helps maintain the normal phenotype and function of cells. The hydrogel compression rupture occurs after the strain reaches 40%, thus it can withstand the compression caused by the growth of internal cell spheres without rupturing, providing a stable carrier for the long-term growth and expansion of three-dimensional cell spheres.

[0045] Hydrogels are suitable for cell culture: their simple composition (chitosan, glyoxylic acid, neutral polymers) and good biocompatibility make them suitable for cytotoxicity experiments (such as...). Figure 5 (As shown) confirmed that the material had no significant cytotoxicity. Cell spheres cultured continuously for 32 days verified the solidity and healthy growth of the cell spheres at high cell density, without central necrosis (as shown). Figure 6 and Figure 7 As shown in the figure, the applicability of the hydrogel of the present invention in three-dimensional cell culture is demonstrated.

[0046] Hydrogels are suitable for drug screening: The hydrogel of this invention can be used for visualizing drug screening. For example... Figure 8 As shown, after adding anticancer drugs to hydrogels containing tumor cell spheres, the growth inhibition and morphological changes of the cell spheres can be clearly observed, providing a reliable tool for high-throughput, high-content drug screening and efficacy evaluation.

[0047] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a hydrogel, characterized in that, The process includes the following steps: dissolving glyoxylic acid, chitosan, and neutral polymer in water at a mass ratio of 3~6:6~10:3~6 to obtain a mixture, and then heating and swelling the mixture to obtain a hydrogel.

2. The method for preparing the hydrogel according to claim 1, characterized in that, Neutral polymers include one or more of polyacrylamide, polyvinyl alcohol, and hydroxypropyl methylcellulose.

3. The method for preparing the hydrogel according to claim 1, characterized in that, It dissolves in water at temperatures ranging from 50°C to 70°C.

4. The method for preparing the hydrogel according to claim 1, characterized in that, The mixture is an ultrasonic mixture.

5. The method for preparing the hydrogel according to claim 1, characterized in that, The mixing temperature is 50℃~70℃.

6. The method for preparing the hydrogel according to claim 1, characterized in that, The heating temperature is 70℃~90℃, and the heating time is 3h~8h.

7. The method for preparing the hydrogel according to claim 1, characterized in that, Swelling involves immersing the product in water for 16 to 24 hours.

8. A method for preparing a hydrogel according to any one of claims 1-7.

9. The application of a hydrogel prepared by a method according to claims 1-7 in the culture of three-dimensional cells.

10. The application of a hydrogel prepared by a method according to claims 1-7 in drug screening.

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