A ready-to-use hydrogel, its preparation method and application

Abstract

This invention belongs to the field of hydrogel materials and relates to a method for preparing a hydrogel, comprising the following steps: dissolving bisphosphonic acid-modified pectin in water or an aqueous solution, adding a divalent metal cation solution to mix and obtain a precursor solution, adding a gelling solution to initiate cross-linking and form a hydrogel, wherein the gelling solution is a solution containing monovalent metal ions with a pH of 7-9. The hydrogel has broad application prospects in the field of ready-to-use reagents. Adding the precursor solution to a cell culture device and lyophilizing it can be used to prepare ready-to-use reagent kits. Dropping a cell suspension into the ready-to-use hydrogel reagent kit and adding cell culture medium yields a hydrogel encapsulated with cells for three-dimensional culture.

Description

Technical Field

[0001] This invention belongs to the field of hydrogel materials, specifically relating to a hydrogel for 3D cell culture and its preparation method. Background Technology

[0002] Hydrogels are three-dimensional polymer networks with high water content and excellent biocompatibility. Because hydrogels can mimic many aspects of the natural extracellular matrix, they have become a leading candidate for therapeutic drug and cell delivery carriers in biomedical engineering. Compared to covalently chemically cross-linked static hydrogels, physically cross-linked dynamic hydrogels can better mimic the dynamic structural features of the natural extracellular matrix and regulate cellular activities, including adhesion, migration, proliferation, differentiation, polarization, and apoptosis, through cellular mechanotransduction signals. Utilizing hydrogels to establish cell-cell interactions is crucial for the in vitro three-dimensional culture of stem cells and multicellular structures (such as in vitro tumors and organoids).

[0003] In tissue engineering and regenerative medicine, the culture of both stem cells and tumor cell spheres is of great significance. Stem cells, as seed cells for cell therapy and organ replacement therapy, can be used as vectors for gene therapy and for establishing drug screening platforms. Cell spheres can enhance the therapeutic efficacy of stem cells by improving cell viability, enhancing cytokine secretion, and maintaining cell pluripotency. For tumor cells, due to their morphology and spatial arrangement, cell spheres yield more accurate results in drug screening than 2D cultured cells. 3D cells can mimic several important characteristics of tumors and their microenvironment, including phenotypic heterogeneity, growth kinetics, and intercellular interactions. However, cell spheres produced using existing technologies suffer from a series of problems, such as low cell viability and inability to maintain cell stemness. These problems are often related to the materials used in the production of cell spheres. Therefore, the preparation of a hydrogel material that is simple to process, can culture stem cells, and can maintain the stemness of tumor cell spheres is an urgent problem to be solved.

[0004] Pectin is a natural polymer compound with excellent gelling and emulsifying stabilizing properties, and it has been widely used in the food, pharmaceutical, daily chemical, and textile industries. The carboxyl groups and divalent metal ions in pectin have strong ion coordination effects. Existing technologies have reported the use of free carboxyl ions on low-ester pectin to interact with free divalent calcium ions to form three-dimensional rigid hydrogels. CN114502712A discloses a pectic acid-based hydrogel suitable for cell culture, in which pectic acid or its salt is added to a crosslinking agent containing divalent ions, and a proton donor to obtain the hydrogel. The proton donor is hydrolyzed in an aqueous solution to form an acid lactone, ester, or other compound. However, existing reports primarily utilize pectin hydrogels for drug delivery; further research and development are needed to create more pectin hydrogels that are simple to prepare, easy to operate, and possess excellent mechanical properties for three-dimensional culture. Summary of the Invention

[0005] To address the needs of the prior art, the present invention aims to provide a novel method for preparing hydrogels, comprising the following steps: dissolving bisphosphonic acid-modified pectin in water or an aqueous solution, adding a divalent metal cation solution to mix and obtain a precursor solution, and adding a gelling solution to initiate cross-linking to form a hydrogel, wherein the gelling solution is a solution containing monovalent metal ions with a pH of 7-9.

[0006] The bisphosphonic acid in the bisphosphonic acid-modified pectin includes, but is not limited to, one or more of pamidrolic acid, tiludrolic acid, alendrolic acid, neriddrolic acid, opaldrolic acid, and ibandronic acid. The bisphosphonic acid-modified pectin does not undergo a gelling reaction when a divalent metal ion solution is added to form a precursor solution; however, it rapidly gels to form a hydrogel upon the addition of a solution containing monovalent metal ions.

[0007] In some embodiments, the divalent metal cation solution includes, but is not limited to, the presence of the metal cation Mg. 2+ Ca 2+ Sr 2+ Ba 2+ Mn 2+ Fe 2+ Co 2+ Ni 2+ Cu 2+ Zn 2+ One or more of the following. The solution of the monovalent metal ions includes, but is not limited to, Na+. + K + wait.

[0008] In some embodiments, the gelling solution is an aqueous solution containing monovalent metal ions, a physiological buffer solution, or a cell culture medium; the culture medium includes, but is not limited to, MEM, DMEM, αMEM, IMDM, and RPMI-1640.

[0009] In some embodiments, the grafting rate of bisphosphonic acid modified pectin is 5%-20%; in some embodiments, the mass percentage of bisphosphonic acid modified pectin in the precursor solution is 0.5-10%, preferably 1%-6%, and the concentration of divalent metal cations is not less than 50 mM; after adding the pectin-forming solution, the concentration of monovalent metal cations ranges from 100-300 mM.

[0010] In some embodiments, the method for preparing the bisphosphonic acid-modified pectin includes grafting bisphosphonic acid onto pectin molecules via an amidation reaction. Preferably, the bisphosphonic acid group is one or more of pamidronic acid, alendronic acid, neriddronic acid, or ibandronic acid. In some preferred embodiments, the method for preparing the bisphosphonic acid-modified pectin includes dissolving a bisphosphonic acid compound in a buffer solution, adding pectin and NHS, slowly adding an aqueous solution of EDC dropwise to the above pectin mixture, and continuously stirring the reaction at room temperature. After adding hydroxylamine hydrochloride to the reactants, dialyzing and then lyophilizing are performed.

[0011] Another object of the present invention is to provide a ready-to-use hydrogel kit. The kit includes a hydrogel precursor, which is a lyophilized product of a mixed solution containing bisphosphonic acid-modified pectin and divalent metal cations. Upon addition of a gelling solution to the kit, a hydrogel is formed; the gelling solution is a solution containing monovalent metal cations with a pH of 7-9.

[0012] In some embodiments, the divalent metal cations in the mixed solution include, but are not limited to, Mg. 2+ Ca 2+ Sr 2+ Ba 2+ Mn 2+ Fe 2+ Co 2+ Ni 2+ Cu 2+ Zn 2+ One or more of the following; the monovalent metal cation is Na + or K + At least one of them.

[0013] In some embodiments, the bisphosphonic acid in the bisphosphonic acid-modified pectin includes, but is not limited to, one or more of pamidronic acid, tiludronic acid, alendronic acid, neriddronic acid, opaldronic acid, and ibandronic acid.

[0014] In some embodiments, the bisphosphonic acid-modified pectin is further grafted with short peptide groups, preferably RGD groups. The hydrogel precursor is a product obtained by lyophilizing a mixed solution of bisphosphonic acid and RGD-modified pectin (PT-BP-RGD) and divalent metal cations.

[0015] In some embodiments, the pectin modified with bisphosphonic acid and RGD groups is prepared by dissolving pectin in a buffer solution and reacting it with RGD peptides in the presence of EDC and sulfo-NHS at room temperature. Bisphosphonic acid compounds are weighed and dissolved in the above reaction solution, NHS and EDC are added, and the reaction is terminated by adding hydroxylamine hydrochloride after reacting at room temperature. The resulting solution is dialyzed and lyophilized to obtain PT-BP-RGD.

[0016] Another object of this application is to provide a method for preparing a ready-to-use hydrogel kit, comprising the following steps: 1) mixing a bisphosphonic acid-modified pectin solution with a solution containing divalent metal cations to obtain a precursor solution, adding the precursor solution into a cell culture device, and lyophilizing to obtain a ready-to-use hydrogel kit.

[0017] In some embodiments, the method for preparing the bisphosphonic acid modified pectin includes grafting bisphosphonic acid onto pectin molecules via an amidation reaction, preferably the bisphosphonic acid group being pamidronic acid, alendronic acid, neriddronic acid, or ibandronic acid, etc.

[0018] Another object of this application is to provide a method for culturing cells using the ready-to-use hydrogel kit, comprising the following steps: dropping a cell suspension into the ready-to-use hydrogel kit, adding cell culture medium / culture medium, and allowing it to stand to obtain a hydrogel encapsulated with cells for three-dimensional culture.

[0019] In some implementations, the lyophilized materials in the kit are first sterilized before being used for cell culture.

[0020] In some embodiments, the method further includes the step of adding an EDTA solution to the hydrogel to degrade the hydrogel and recover the cells. For example, when an EDTA solution of not less than 10 mM is added to the hydrogel, allowing it to stand can degrade the hydrogel and recover the cells.

[0021] Beneficial technical effects

[0022] The technical solution of this application involves introducing bisphosphonic acid groups onto pectin to increase its binding capacity with divalent metal ions, thereby enhancing the coordination between pectin molecules. After the modified pectin and divalent metal ions are mixed, a gelling solution is added to initiate gelation. In some embodiments, the gelling solution contains sodium or potassium ions, and these monovalent metal ions are commonly found in buffer solutions or cell culture media. Therefore, this hydrogel can be rapidly gelled by adding conventional buffer solutions or cell culture media, showing broad application prospects in the field of ready-to-use reagents.

[0023] The hydrogel reagent kit prepared by the method described in this application has the following excellent technical effects:

[0024] 1. The hydrogel prepared by the method of this application can promote cell adhesion, proliferation and differentiation when used to culture cells, and has a better effect on promoting cell spheroidization compared with commercially available products.

[0025] 2. The preparation method of this invention is simple, the materials are widely available, and it is highly practical. It can be used as a matrix material for in vitro 3D cell culture.

[0026] 3. The ready-to-use hydrogel kit prepared by the method of this application can form a hydrogel immediately after being added to the cell culture medium, without the need for other gel-forming reagents, and is convenient to use.

[0027] 4. The content of precursors in the ready-to-use hydrogel kit of this application can be adjusted according to the needs of cell culture, adapting to the needs of multiple scenarios and different applications. Attached Figure Description

[0028] Figure 1 The 1H NMR spectra of PT-BP and PT-BP-RGD are shown.

[0029] Figure 2 a) Demonstration of PT-BP gelation initiated by DMEM, DMEM inorganic salt component solution and aqueous solution containing NaHCO3, CaCl2 and NaCl; b) Rheological test of the G′ and G″ values ​​of the hydrogel formed by the above solutions. Figure 3 This is a schematic diagram illustrating the preparation and use of the hydrogel reagent kit.

[0030] Figure 4 DMEM and Ca containing different concentrations 2+ The mechanical properties of hydrogel precursors forming hydrogels include: a) storage modulus, b) Young's modulus, and c) stress relaxation.

[0031] Figure 5 In the presence of different Ca 2+ Bright-field microscopic images of HepG2 cells cultured in a hydrogel of a certain concentration for 7 days.

[0032] Figure 6 Confocal tomographic images of HepG2 cells cultured in pectin hydrogel for 0 and 7 days.

[0033] Figure 7 Bright-field and live / dead fluorescence images of HepG2 cells cultured for 3 and 7 days in Matrigel and pectin hydrogel kits, respectively.

[0034] Figure 8 Results of cell staining experiments on HepG2 cells after 3 and 7 days of culture in a pectin hydrogel kit.

[0035] Figure 9HepG2 cells showed stemness after 3 and 7 days of culture in a pectin hydrogel kit. Figure 10 Bright-field microscopic images of human mesenchymal stem cells (hMSCs) cultured in a hydrogel kit for 3 days.

[0036] Figure 11 Images of live and dead cells after culturing various tumor cells in a hydrogel kit for 3 days. Detailed Implementation

[0037] The technical solution of the present invention is further illustrated below with specific examples, but these examples are not intended to limit the present invention. Unless otherwise stated, all reagents are commercially available products. The pectin used in the following experiments is low-ester pectin purchased from Aladdin.

[0038] The bisphosphonic acid group has a strong coordination effect with metal ions, and its structure is as follows.

[0039]

[0040] Normally, the carboxyl groups on pectin molecules bind to divalent metal ions via electrostatic forces to form a gel, while bisphosphonic acid groups can enhance this binding. The inventors of this application discovered that because the pectin is modified with bisphosphonic acid groups, it does not form a gel after mixing with divalent metal ions. However, upon further addition of a monovalent metal ion solution, hydrogel formation is initiated. Since cell culture media generally contain monovalent metal ions (such as sodium ions), the hydrogel can be initiated to form upon addition to the cell culture medium. This characteristic gives it broad application prospects in the field of ready-to-use hydrogels.

[0041] Example 1

[0042] Preparation of bisphosphonic acid modified pectin

[0043] Bisphosphonic acid-modified pectin can be prepared by introducing bisphosphonic acid groups into the side chains of pectin using conventional methods in the art. In this embodiment, bisphosphonates containing amino groups are introduced by reacting the amino group with a carboxylic acid on the pectin. This reaction is carried out in an aqueous phase under mild conditions. It should be understood that the inventive objectives of this application can also be achieved by grafting bisphosphonic acid groups using other conventional methods.

[0044] 1) Preparation of bisphosphonic acid modified pectin (PT-BP)

[0045] The method for preparing the bisphosphonic acid-modified pectin involves dissolving a bisphosphonic acid compound in MES buffer, adding pectin and NHS, and mixing. EDC is dissolved in MES buffer and slowly added dropwise to the pectin mixture, with continuous stirring at room temperature for 24 hours. Hydroxylamine hydrochloride is then added to the reactants, followed by dialyzing and lyophilization.

[0046] Specifically, 5.04 g of alendronate sodium trihydrate was added to 80 mL of MES buffer (pH = 6.5) and heated to approximately 60 °C until completely dissolved. After cooling to room temperature, 1 g of pectin and 0.89 g of NHS were added and mixed. 2.98 g of EDC was dissolved in 20 mL of MES buffer and slowly added dropwise to the pectin mixture over 30–60 minutes. The reaction was continuously stirred at room temperature for 24 hours. After adding 1.08 g of hydroxylamine hydrochloride to the reactants, dialyzing (molecular weight cutoff 3500 Da) was performed, followed by lyophilization. The degree of substitution (DS) of bisphosphonate (BP) in the resulting PT-BP was estimated to be 12.5% ​​by 1H NMR (Bruker Advance 400 MHz spectrometer).

[0047] By varying the amount of sodium alendronate added using the above method, bisphosphonic acid-modified pectin with substitution degrees of 7.5%, 10%, 12.5%, 15%, and 20% was prepared.

[0048] 2) Preparation of bisphosphonic acid and peptide-modified pectin (PT-BP-RGD)

[0049] The preparation method of the bisphosphonic acid and RPG group modified pectin is as follows: Pectin is dissolved in MES buffer, and RGD peptide and EDC are added. Sulfo-NHS is dissolved in MES buffer and added to the above solution, and the reaction is carried out at room temperature. The bisphosphonic acid compound is dissolved in the above reaction solution, and NHS is added. EDC is dissolved in MES buffer and slowly added dropwise to the previous solution, and the reaction is carried out at room temperature. Finally, hydroxylamine hydrochloride is added to terminate the reaction. The resulting solution is dialyzed and lyophilized to obtain PT-BP-RGD.

[0050] Specifically, 1 g of pectin was weighed using a balance and dissolved in 80 mL of MES buffer (pH = 6.5). After complete dissolution, 98 mg of peptide GGGYGRGDSPG and 99 mg of EDC were added. 56 mg of sulfo-NHS was weighed and dissolved in 20 mL of MES buffer and slowly added dropwise to the solution, reacting at room temperature for 24 h. 5.04 g of sodium alendronate was weighed and dissolved in the solution at 60 °C. After cooling to room temperature, 0.89 g of NHS was added. 2.98 g of EDC was dissolved in 20 mL of MES buffer and slowly added dropwise to the previous solution, reacting at room temperature for 24 h. Finally, 1.079 g of hydroxylamine hydrochloride was added to terminate the reaction. The resulting solution was dialyzed and lyophilized to obtain PT-BP-RGD. RGD is the smallest sequence recognized by cell membrane integrins in the ECM. By introducing the RGD group, the adhesion and migration of cells on the surface of hydrogel materials can be further improved. Pectin grafted with RGD can better simulate the microenvironment in tissues. This preparation method can be carried out in an aqueous phase under mild conditions and is simple to operate.

[0051] Figure 1 The 1H NMR spectra of PT-BP and PT-BP-RGD are shown respectively. PT-BP-RGD shows the characteristic peak of RGD between 7.0 and 7.5, indicating that RGD was successfully grafted onto PT-BP.

[0052] Example 2

[0053] Bisphosphonic acid modified pectin to prepare hydrogels

[0054] The bisphosphonic acid-modified pectin prepared in Example 1 was added to ultrapure water and mixed to obtain a pectin aqueous solution with a mass percentage of 6%. Simultaneously, 200 mM Ca2+ was prepared. 2+ 1 mL of aqueous solution was mixed with an equal volume of the two solutions to obtain a non-gelatinized solution, which was used as the precursor solution. Adding 50 μL of DMEM cell culture medium to the precursor solution induced rapid gelation. This example also tested other proportions of pectin aqueous solution and found that hydrogels could form in the precursor solution with a pectin concentration ranging from 1% to 10%.

[0055] Based on this, the inventors explored the mechanism by which cell culture medium induces PT-BP gelation. According to the composition of inorganic salts in the cell culture medium (as shown in Table 1), in this embodiment, 50 μl of different combinations of inorganic salt solutions were added to the precursor solution to observe the gelation process, as shown in Table 2.

[0056] Table 1. Components of inorganic salts in cell culture medium

[0057] serial number Inorganic salts Concentration (mM) A <![CDATA[CaCl2]]> 1.80 B <![CDATA[MgSO4]]> 0.81 C KCl 5.37 D <![CDATA[NaHCO3]]> 44.04 E NaCl 109.51 F <![CDATA[NaH2PO4]]> 0.91

[0058] Table 2. Gel formation of different inorganic salt solution combinations

[0059] combination Does it form a gel? combination Does it form a gel? combination Does it form a gel? A no A+C+F no A+C+D+F no A+D no A+C+D no A+C+D+E yes A+E no A+C+E no A+D+E+F yes A+F no A+E+F no B+C+E+F no A+B no A+D+E yes A+B+C+D+E yes A+C no A+D+F no A+C+D+E+F yes D+E no A+B+C+F no A+B+D+E+F yes A+B+C no A+B+D+E yes B+C+D+E+F yes A+B+D no A+B+D+F no A+B+C+D+E+F yes A+B+E no A+B+E+F no A+B+F no A+B+C+D no

[0060] By comparing the gelation effects of adding different DMEM components, the inventors discovered that the inorganic salt components in the culture medium are key to initiating PT-BP gelation. Further analysis of the various substances in the inorganic salt components revealed that the three components with the lowest gelation efficiency for PT-BP are NaHCO3, CaCl2, and NaCl. Therefore, this embodiment further compared the gelation effects of three solutions: 1. DMEM culture medium; 2. DMEM solution with inorganic salt components; 3. A solution prepared from NaHCO3, CaCl2, and NaCl. The results showed ( Figure 2 a) All three solutions can induce PT-BP to form a gel. Rheological tests of the hydrogels formed by different solutions and PT-BP showed that the hydrogels formed by DMEM culture medium and mixed solutions of NaHCO3, CaCl2, and NaCl exhibited similar properties to those formed by PT-BP (see [link to relevant documentation]). Figure 2 b). Therefore, it can be determined that due to the presence of monovalent cations in the cell culture medium, after the addition of pectin aqueous solution, these cations bind to the carboxyl groups on the pectin, reducing the electrostatic repulsion between pectins, thereby promoting calcium ion-mediated cross-linking to form a hydrogel.

[0061] This embodiment further investigated the effect of different concentrations of monovalent cations on the gelation of PT-BP, and found that when the concentration of monovalent cations was in the range of 100-300mM, gelation could be promoted.

[0062] Furthermore, this embodiment investigated the effect of different pH values ​​on gel formation. Experimental parameters and data are shown in Table 3. When the pH is in the range of 7-9, the addition of a solution containing sodium ions promotes gel formation.

[0063] Table 3 Effect of pH on gelation

[0064] ① ② ③ ④ ⑤ ⑥ ⑦ Pectin-COOH 66mM 66mM 66mM 66mM 66mM 66mM 66mM <![CDATA[Ca 2+ ]]> 1.8mM 1.8mM 1.8mM 1.8mM 1.8mM 1.8mM 1.8mM NaCl 109mM 109mM 109mM 109mM 109mM 109mM 109mM <![CDATA[NaHCO3]]> 44mM - - - - - - NaOH - 44mM - 80mM - - - <![CDATA[Na2CO3]]> - - 44mM - 33mM 22mM - <![CDATA[Na3PO4]]> - - - - - - 44mM Does it form a gel? gel gel Non-gelatinous gel gel gel Non-gelatinous pH 7-8 7-8 9-10 8-9 8-9 7-8 9-10

[0065] Example 3

[0066] Preparation of ready-to-use hydrogel kits

[0067] Based on the research in Example 2, the inventors discovered that the pectin can be used to prepare ready-to-use hydrogels; furthermore, it can also be used to prepare ready-to-use hydrogel kits, which may include, for example, one or more of the hydrogel precursors involved in this application. The kits include various forms of cavities for cell culture, or surfaces for cell culture, including various culture plates, culture dishes, tubes, or devices in various instruments for placing or culturing cells.

[0068] The components containing hydrogel precursors in the kit may be in a single container, or multiple components may be in a single container.

[0069] The procedure for preparing a ready-to-use hydrogel kit in this embodiment is as follows: Figure 3 As shown. Bisphosphonic acid-modified pectin was dissolved in water, and a solution containing Ca was added. 2+ An aqueous solution is mixed to form a precursor solution, which is then added to the culture apparatus of the kit. After lyophilization, a kit with the hydrogel precursor coated on the surface is obtained. In use, cell culture medium containing cells is directly added to the culture wells to form a hydrogel encapsulating the cells. The 96-well culture plate illustrated is only one embodiment of the culture apparatus; the precursor can be easily lyophilized and coated onto various culture surfaces.

[0070] Specifically, in one embodiment of this study, 60 mg of PT-BP-RGD is dissolved in 1 mL of ultrapure water to obtain a 6% PT-BP-RGD aqueous solution, while simultaneously preparing 200 mM Ca 2+ 1 mL of aqueous solution was mixed thoroughly to obtain a final concentration of 3% PT-BP-RGD and 100 mM Ca. 2+ 2 mL of precursor solution was prepared. To ensure the lyophilized material was fluffy and facilitated cell penetration, the precursor solution was diluted by half to obtain a final concentration of 1.5% PT-BP-RGD and 50 mM Ca. 2+ 4 mL of the precursor solution was added. The precursor solution was then aliquoted into 96-well plates at 100 μL per well and lyophilized to prepare a ready-to-use hydrogel kit. Microscopic observation revealed that the lyophilized precursor solution uniformly covered and adsorbed onto the plate surface, exhibiting a sponge-like structure.

[0071] The reagent kit obtained by the above method has a hydrogel precursor coating on the surface of the culture wells. The amount or thickness of the precursor can be adjusted by adjusting the concentration or amount of the precursor solution added to the well plate as needed.

[0072] Example 4

[0073] This embodiment examines different Ca... 2+ The effect of aqueous solution concentration on pectin formation. When Ca is mixed with a pectin aqueous solution... 2 + When the concentration of the aqueous solution is in the range of 50-200 mM, the resulting precursors can all form gels. Furthermore, Ca... 2+ The concentration of [agent / concentration] is correlated with the mechanical properties of the hydrogel. This example tested DMEM with different [agent / concentration] concentrations. 2+ Rheological and mechanical properties of PT-BP-RGD lyophilized materials at various concentrations after hydrogel formation. Figure 4 The results showed that as Ca... 2+ As the concentration of Ca increases, the energy storage capacity and Young's modulus of the hydrogel also increase, and the stress relaxation rate accelerates. Therefore, adjusting the Ca concentration in the precursor solution... 2+ The concentration can be adjusted to modify the hardness and other mechanical properties of the hydrogel according to the needs of the cells to be cultured.

[0074] Example 5

[0075] Cell culture was performed using a ready-to-use kit.

[0076] All cells used in this embodiment were purchased from ATCC (American Type Culture Collection). The cells were pre-cultured in DMEM with 10% bovine serum and 1% penicillin-dextrose antibody for 3 days. Cells were then collected using trypsin, counted, and 5 × 10⁶ cells were harvested. 5 Each cell was resuspended in 50 μL of cell culture medium.

[0077] This embodiment first examines the presence of different Ca... 2+ The kit for the concentration of precursors was used to assess the effectiveness of cell culture. 50 μL / well of a suspension containing 500,000 HepG2 cells was directly added to the 96-well hydrogel kit prepared according to Example 4. After incubation for 1 hour, 100 μL of DMEM culture medium (containing 10% fetal bovine serum and 1% penicillin antibiotics) was added, and the medium was changed every other day. After 3 to 7 days of culture, cell morphology was observed, and cell viability and immunofluorescence staining experiments were performed.

[0078] Figure 5 It shows the presence of different Ca 2+ The state of HepG2 cells after culturing in hydrogels of a specific concentration for 7 days. Electron microscopy results showed that HepG2 cells in all groups formed cell spheroids with a diameter of approximately 100 μm. In subsequent experiments, to better maintain the integrity of the hydrogel during staining, 100 mM Ca2+ was used. 2+ The cells were cultured under specific conditions for research.

[0079] Cell growth was observed using a Zeiss LSM880 confocal microscope. Figure 6 HepG2 cells were shown in a hydrogel (100 mM Ca2+). 2+ Confocal tomographic images at 0 and 7 days post-hoc. The results show that the hydrogel kit prepared by the method described in this application can effectively promote the formation of HepG2 cells into spheres.

[0080] Furthermore, this embodiment uses the commercially available Matrigel reagent kit, employing the same method for cell culture as a control, and observes bright-field and live / dead fluorescence staining during cell growth using fluorescence microscopy. The procedure for live cell staining is as follows:

[0081] 1. Prepare staining solution: Calcein AM (Beyotime) and Propidium iodide (Beyotime) are diluted 1:1000 in PBS (HyClone);

[0082] 2. The hydrogel was washed three times with PBS in a 24-well plate, 1 mL each time, and then 1 mL of staining solution was added. The plate was stained at 37°C for 30 min.

[0083] 3. Wash three times with 1 mL of PBS each time.

[0084] Figure 7 The results show the cell formation of HepG2 cells after 3 and 7 days of culture in Matrigel and hydrogel kits, respectively. The results indicate that, compared to Matrigel, the cells in the kit provided in this application exhibited more pronounced spheroidization.

[0085] This example further investigated cell proliferation in the hydrogel kit. The experimental method was fluorescent staining, and the specific procedures were as follows: The culture medium was aspirated from the hydrogel, transferred to a 24-well plate, and washed three times with 1 mL PBS for 3 min each time. 1 mL of paraformaldehyde was added, and the plate was fixed at room temperature for 2-3 h. The plate was then washed three times with 1 mL PBS for 3 min each time, infiltrated with 0.2% Triton-x-100 (Sigma) PBS solution for 30 min, and washed three times with 1 mL PBS for 3 min each time. Blocking was performed with 2.5% bovine serum albumin (BSA, Sigma) at 37°C for 3 h, followed by washing three times with 1 mL 1% BSA for 3 min each time. Primary antibodies Ki67 and OCT4 (1:500) (Proteintech) were diluted with 1% BSA and incubated overnight at 4°C. The plate was washed once with 0.1% Tween (Aladdin) PBS, and then washed three times with PBS for 3 min each time. Dilute the fluorescent secondary antibody Alexa Fluor488 (Thermo Fisher) (1:500) with 1% BSA, incubate in a dark and humid environment for 3 hours, wash once with 0.1% Tween PBS, and wash three times with PBS for 3 minutes each time. Dilute DAPI (Beyotime) (1:500) with PBS and incubate for 30 minutes, then wash three times with PBS for 3 minutes each time. Figure 8 The results show the proliferation of HepG2 cells after 3 and 7 days of culture in the hydrogel kit. The results indicate that cells in the hydrogel kit showed significant proliferation after both 3 and 7 days of culture. This demonstrates that the hydrogel reagent of this application possesses mechanical properties that promote cell proliferation and spheroidization. Figure 9 This study demonstrates the maintenance of stemness in HepG2 cells after 3 and 7 days of culture in the hydrogel kit. The results show that cells maintained stemness in the hydrogel kit after both 3 and 7 days of culture. This demonstrates that the hydrogel of this application exhibits better dynamic mechanical properties that mimic the extracellular matrix.

[0086] Example 6

[0087] This example investigated the efficacy of the hydrogel kit for culturing mesenchymal stem cells. The specific experimental method is as follows: Third-generation hMSCs (ATCC) were cultured in α-MEM with 17% bovine serum, 1% penicillin-dextrin, 1% L-glutamine, and 5 ng / mL FGF2 for 10 days. Fourth-generation hMSCs were collected via trypsin, and after cell counting, 5 × 10⁶ cells were harvested. 5 One cell was resuspended in 50 μL of cell culture medium, dropped into a hydrogel kit, incubated for 1 h, and then cultured for 3 days with 100 μL of cell culture medium. Figure 10These are bright-field microscopic images of human mesenchymal stem cells (hMSCs) cultured in a hydrogel kit for 3 days. The cell proliferation was observed, demonstrating that the stem cells spread well in the hydrogel kit.

[0088] Example 7

[0089] This embodiment examines the use of the described hydrogel kit for culturing various tumor cells (all from ATCC), using the same experimental method as the aforementioned HepG2 cells. Figure 11 The experiment showed cell viability and mortality staining of various tumor cells after 3 days of culture in the hydrogel kit. The results indicated that various tumor cells exhibited good spheroidization in the hydrogel kit, demonstrating that the hydrogel of this application possesses adaptive dynamic mechanical properties and is suitable for simulating the growth environment of various cells.

[0090] The present invention has been described above by way of example. It should be noted that any simple modifications, variations, or other implementations that can be obtained by those skilled in the art without creative effort without departing from the core of the present invention should be considered to fall within the protection scope of this application.

Claims

1. A method for preparing a hydrogel, characterized in that, Includes the following steps: Bisphosphonic acid-modified pectin is dissolved in water or an aqueous solution, and mixed with a divalent metal cation solution to obtain a precursor solution. A gelling solution is then added to initiate cross-linking and form a hydrogel. The gelling solution is a solution containing monovalent metal ions with a pH of 7-9. The three minimum components for the gelling of bisphosphonic acid-modified pectin are NaHCO3, CaCl2, and NaCl; the concentration of divalent metal cations in the precursor solution is not less than 50 mM; and the concentration of monovalent metal cations after adding the gelling solution is in the range of 100-300 mM.

2. The preparation method according to claim 1, characterized in that, The bisphosphonic acid in the bisphosphonic acid-modified pectin is selected from one or more of pamidronic acid, tiludronic acid, alendronic acid, neriddronic acid, opaldronic acid, and ibandronic acid.

3. The preparation method according to claim 1 or 2, characterized in that, The precursor solution contains 0.5-10% by mass of bisphosphonic acid-modified pectin.

4. The preparation method according to claim 1 or 2, characterized in that, The divalent metal cation solution also includes Mg. 2+ Sr 2+ Ba 2+ Mn 2+ Fe 2+ Co 2+ Ni 2+ Cu 2+ Zn 2+ One or more of the following; the monovalent metal ion solution also includes K + .

5. The application of the hydrogel prepared by the preparation method according to any one of claims 1-4 in cell culture and tissue culture.

6. A ready-to-use hydrogel kit, the kit comprising a hydrogel precursor, characterized in that, The hydrogel precursor is a product obtained by freeze-drying a mixed solution containing bisphosphonic acid-modified pectin and divalent metal cations. The mixed solution contains 0.5-10% by mass of bisphosphonic acid-modified pectin and has a concentration of not less than 50 mM for divalent metal cations.

7. The ready-to-use hydrogel kit as described in claim 6, characterized in that, At least one of the following a)-b): a) The bisphosphonic acid in the bisphosphonic acid modified pectin is selected from one or more of pamidronic acid, tiludronic acid, alendronic acid, neriddronic acid, opapadronic acid and ibandronic acid; b) The divalent metal cation is selected from Mg 2+ Ca 2+ Sr 2+ Ba 2+ Mn 2+ Fe 2+ Co 2+ Ni 2+ Cu 2+ Zn 2+ One or more of them.

8. The ready-to-use hydrogel kit as described in claim 6 or 7, characterized in that, The bisphosphonic acid-modified pectin is further grafted with short peptide groups.

9. The ready-to-use hydrogel kit as described in claim 8, characterized in that, The short peptide group is an RGD group.

10. A method for preparing a ready-to-use hydrogel kit as described in any one of claims 6-9, comprising the following steps: A bisphosphonic acid-modified pectin solution was mixed with a solution containing divalent metal cations to obtain a precursor solution. The precursor solution was then added to a cell culture device and lyophilized to obtain a ready-to-use hydrogel kit.

11. A method for culturing cells using a ready-to-use hydrogel kit as described in any one of claims 6-9, comprising the following steps: The cell suspension was dropped into the ready-to-use hydrogel kit, and cell culture medium was added to obtain a hydrogel encapsulated with cells for three-dimensional culture.

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