Bionic extracellular matrix porous hydrogel for three-dimensional culture and delivery of dendritic cells and preparation method thereof

Abstract

The invention belongs to the technical field of three-dimensional culture and delivery of cells, and discloses gelatin / hyaluronic acid porous hydrogel composed of a bionic extracellular matrix and used for three-dimensional culture and delivery of dendritic cells and a preparation method of the gelatin / hyaluronic acid porous hydrogel. The method comprises the following steps: taking a semi-interpenetrating polymer network composed of methacrylic anhydride modified gelatin (GelMA) and oxidized hyaluronic acid (OHA) as a matrix, introducing temperature-responsive gelatin microspheres and dendritic cells at the same time, then raising the temperature until the gelatin microspheres are melted, and constructing a three-dimensional supramolecular hydrogel cell in-vitro culture model with decoupled aperture and rigidity. The supramolecular hydrogel has the advantages of convenience in use, simplicity in operation, biosafety and the like, and can be applied to the fields of three-dimensional culture and delivery of dendritic cells, tumor immunotherapy and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of hydrogel-based three-dimensional cell culture and delivery, and specifically relates to a bionic extracellular matrix porous hydrogel for three-dimensional culture and delivery of dendritic cells and a preparation method thereof. Background Art

[0002] Dendritic cells (DCs) are the most powerful antigen presenting cells discovered so far. The surface molecules of DCs can process and present tumor antigens, secrete a variety of cytokines in an antigen-specific form to activate T cells to recognize and kill tumor cells. At the same time, they can also stimulate immune memory protection and are the initiators of the body's anti-tumor immune response. However, DCs vaccines have problems such as low cell activity in the body, low homing efficiency, and weak immune activation ability. First, despite the high 6 DCs are adoptively infused into the tumor-bearing host, but most of the DCs die or flow to non-lesion locations after injection, and only about 1% of DCs can migrate to the draining lymph nodes. Secondly, mature DCs have a short lifespan of only a few days. If the host's own DCs cannot be recruited to continue the process of antigen uptake and presentation, the tumor-specific immune response will be further limited. It is now known that the physical properties of the tumor microenvironment, such as stiffness, dimension, and pore size, are one of the important factors affecting tumor progression and the efficacy of immunotherapy. Changes in the tumor microenvironment during tumor occurrence and development also directly affect the anti-tumor function of DCs. However, due to the lack of methods to decouple the complex interaction between stiffness and pore size, little is known about the systematic study of the behavior and function of DCs in the tumor microenvironment.

[0003] Hydrogel is a three-dimensional water-swollen polymer network constructed based on covalent bonds or physical interactions, with similar structures and properties to the extracellular matrix, such as high hydration, high permeability, adjustable biodegradability and mechanical properties. In addition, the design of hydrogel materials can also achieve minimally invasive delivery of hydrogel scaffolds, which can effectively avoid complex surgical operations and the occurrence of postoperative complications. In situ hydrogels are usually injected into the lesion site in a fluidic manner, and gels are formed in situ under the action of chemical or physical cross-linking, thereby achieving in situ fixation of cells and active molecules. Therefore, hydrogels can be used as co-delivery carriers for DCs and tumor antigens, which can effectively improve the activity of exogenous DCs and prolong their retention time at the injection site, enrich cytokines secreted by DCs, and construct an in situ inflammatory microenvironment. In addition, through the co-delivery of tumor antigens, adjuvants, chemotherapeutic drugs, etc., it is possible to further recruit and activate DCs in the host body, thereby expanding T cell immune responses and inhibiting tumor growth. However, in situ injectable hydrogels require an appropriate gelation time. If the gelation time is too fast, it is easy to cause transport blockage. If the gelation time is too slow, the precursor solution will flow to the surrounding adjacent tissues or be diluted by body fluids, thus affecting the final performance of the gel. Although the dynamic hydrogels currently constructed based on physical interactions or dynamic covalent bonds have self-healing properties and can effectively improve the performance of in situ hydrogels, the nano-sized pore structure formed is not conducive to cell-to-cell interactions and the diffusion of bioactive molecules.

[0004] Compared with in situ nanopore injectable hydrogels, the effective pore size of porous hydrogels is greater than 10 μm, and scaffold materials with different pore structures can be prepared by adjusting the preparation parameters. The porous structure can provide sufficient space for cells to migrate and proliferate inside, and is more conducive to intercellular communication, which can effectively reduce the inflammatory response in vivo. However, the existing manufacturing technologies still face many limitations in realizing pore structures. For example, bioprinting technologies (such as nozzle printing and stereolithography) can generate large-scale structures, but it is difficult to manufacture fine features smaller than nozzles or voxels; photolithography technology is limited to small-volume manufacturing due to heat accumulation and time cost. In addition, many emerging printing technologies (such as two-photon lithography) can print complex shapes, but they are difficult to apply to natural extracellular matrices, and there are problems such as material limitations, low efficiency and small construction volume. On the other hand, hydrogels can achieve the dimensions, stiffness and pore structure of biomimetic tumor extracellular matrix due to their high permeability and adjustable biophysical properties. DCs were then inoculated into biomimetic extracellular matrix hydrogels to explore the biological functions of DCs under various biophysical properties. However, as a cell culture model in vitro, it is difficult to decouple the physical properties of the extracellular matrix (stiffness, dimension, pore size) and independently explore the impact of a single physical property of the extracellular matrix on DCs. Based on this, it is urgent to develop a hydrogel that can better mimic the components of the extracellular matrix and decouple structure and stiffness. Summary of the invention

[0005] In order to solve the shortcomings and deficiencies of the prior art, the object of the present invention is to provide a biomimetic extracellular matrix porous hydrogel for three-dimensional culture and delivery of dendritic cells and a preparation method thereof. The present invention uses a semi-interpenetrating polymer network composed of methacrylic anhydride-modified gelatin (GelMA) and oxidized hyaluronic acid (OHA) as a matrix, and introduces a sacrificial gelatin microsphere template to construct an in vitro three-dimensional culture model of dendritic cells with decoupled pore size and stiffness. The hydrogel in vitro culture model has a stable double network structure, wherein the aldehyde group on OHA reacts with the amino group of GelMA to form a first Schiff base network, and the Schiff base reaction has the advantages of fast reaction speed, no need for additional cross-linking agent, and good adhesion. Subsequently, the chemical crosslinking formed by GelMA after light induction serves as the second network. The double network structure improves the mechanical strength of the hydrogel, and the network structure is similar to the three-dimensional mesh structure of the extracellular matrix. In addition, the internal pore size structure of the supramolecular porous hydrogel is conducive to the diffusion and exchange of oxygen, nutrients and metabolites, and can provide a suitable three-dimensional growth microenvironment for cells embedded therein.

[0006] In order to achieve the above object, the present invention adopts the following technical scheme:

[0007] A biomimetic extracellular matrix porous hydrogel for three-dimensional culture and delivery of dendritic cells and a preparation method thereof, comprising the following steps:

[0008] (1) Add gelatin into deionized water and stir until it is completely dissolved to obtain a gelatin solution.

[0009] The gelatin is derived from pig skin;

[0010] The stirring condition is 300-500 rpm;

[0011] The stirring temperature is 40-60°C, and the reaction time is 0.5-1h;

[0012] The mass percentage of the gelatin solution is 3% to 5%;

[0013] (2) adding anhydrous ethanol, poloxamer and gum arabic to the product, stirring overnight and then subjecting to subsequent treatment to obtain gelatin microgel.

[0014] The volume ratio of anhydrous ethanol to deionized water is 3:5 to 1:1;

[0015] The volume ratio of gum arabic to gelatin is 0.05 to 0.1 respectively;

[0016] The stirring temperature is 20°C to 25°C;

[0017] The mass ratio of poloxamer to gelatin is 0.125 to 0.25 respectively;

[0018] The subsequent treatment is to centrifuge at 300g for 5 minutes, remove the emulsion phase, add PBS buffer (pH=7.4, 0.1M) for re-suspending, centrifuge at 1000g for 5 minutes, discard the supernatant, and repeat three times.

[0019] (3) Synthesis of oxidized sodium hyaluronate polymer: Sodium hyaluronate is dissolved in deionized water, an oxidant is added, the reaction is stirred, an oxidant terminator is added, and subsequent treatment is performed to obtain an oxidized hyaluronate polymer.

[0020] The molecular weight of the hyaluronic acid is 90,000 to 1.2 million;

[0021] The oxidant is sodium periodate;

[0022] The oxidant terminator is ethylene glycol;

[0023] The mass percentage of the sodium hyaluronate is 0.1-2%;

[0024] The mass percentage of the sodium periodate is 0.1-0.5%;

[0025] The mass percentage of the ethylene glycol is 0.5-2%;

[0026] The temperature of the stirring reaction is 0°C;

[0027] The stirring reaction time is 1 to 3 hours;

[0028] The subsequent treatments include dialysis and freeze-drying.

[0029] (4) Preparation of methacrylic anhydride modified gelatin: After gelatin is added to phosphate buffer and heated to dissolve completely, methacrylic anhydride is added dropwise to react, and then treated to obtain methacrylic anhydride modified gelatin.

[0030] The reaction temperature is 50-60°C, and the reaction time is 3-5h;

[0031] The pH of the phosphate buffer is 7.4;

[0032] The mass concentration of the gelatin aqueous solution is 5-10%;

[0033] The volume percentage of the methacrylic anhydride is 1 to 20%;

[0034] The subsequent treatment is to centrifuge the reaction solution, dialyze it in deionized water, and freeze-dry it to obtain methacrylic anhydride-modified gelatin.

[0035] (5) Adding the methacrylic anhydride-modified gelatin of step (4) and the oxidized hyaluronic acid of step (3) into the basal culture medium and stirring for 3 to 5 hours to obtain a matrix gel solution.

[0036] The mass percentage of the methacrylic anhydride modified gelatin is 3% to 10%;

[0037] The mass percentage of the hyaluronic acid is 0.4% to 0.8%;

[0038] The stirring temperature is 50-60°C.

[0039] (6) adding a photoinitiator (2959) to the matrix gel solution of step (5) to obtain a hydrogel precursor solution;

[0040] The mass percentage of the photoinitiator is 0.2% to 0.5%.

[0041] (7) Add the temperature-responsive gelatin microspheres prepared in step (2) to the hydrogel precursor solution prepared in step (6) in proportion, and mix by blowing to obtain a porous hydrogel precursor solution.

[0042] The ratio of the gelatin microspheres to the matrix glue is 1:1 to 3:1.

[0043] (8) Use of gelatin / hyaluronic acid porous hydrogel for three-dimensional culture of DCs: The dendritic cells were mixed with the precursor solution in step (7), cured by ultraviolet light for 1 min, and then subjected to subsequent treatment to achieve three-dimensional uniform encapsulation of DCs.

[0044] The dendritic cells are dendritic cells induced by mouse mesenchymal stem cells via interleukin 4 (IL-4) and granulocyte macrophage colony stimulating factor (GM-CSF) for 6 days;

[0045] The cell density was 5×10 6 ~1×10 7 pcs / mL sol;

[0046] The subsequent treatment is to add complete culture medium and then culture in an incubator for 24 hours.

[0047] (9) After the culture model was placed in an incubator for 24 h in vitro, the number of DCs in the culture supernatant was measured at regular intervals every 24 h.

[0048] Compared with the prior art, the present invention has the following advantages:

[0049] The present invention introduces temperature-responsive gelatin microsphere templates of different sizes into methacrylic anhydride-modified gelatin (GelMA) and oxidized hyaluronic acid (OHA) matrices, raises the temperature after the matrix is ​​photocured, and the gelatin microspheres melt, thereby constructing a three-dimensional hydrogel in vitro culture model with the same stiffness and different pore sizes. The effective pore size of the hydrogel is precisely controlled by regulating the size of the gelatin microspheres; the stiffness of the hydrogel matrix is ​​independently controlled by adjusting the ratio of GelMA and OHA.

[0050] Compared with traditional hydrogels, the biomimetic extracellular matrix supramolecular hydrogel with decoupled pore size and rigidity is more conducive to independently exploring the influence of the biophysical properties of the extracellular matrix (pore size, rigidity) on the immunological function of DCs. In addition, the interconnected spaces inside the supramolecular porous hydrogel provide a three-dimensional microenvironment for DCs, which can effectively improve the cell activity and maturity of DCs, and the porous structure of the supramolecular hydrogel of the present invention is conducive to the sustained release of DCs wrapped therein. Based on this, the present invention can be used as a co-transport carrier of DCs and tumor antigens, effectively improving the problems of low activity of current DCs vaccine cells, low homing efficiency, and weak immune activation ability. And the supramolecular porous hydrogel has the advantages of easy use, simple operation, and biosafety, and can be used in the fields of cell culture, tissue engineering, drug delivery, and tumor immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0052] Figure 1 The morphology and particle size distribution of the temperature-responsive gelatin microgel provided by the present invention

[0053] Figure 2 and Figure 3 The internal microstructure of the supramolecular hydrogel provided by the present invention

[0054] Figure 4 Rigidity characterization of the supramolecular hydrogel provided by the present invention

[0055] Figure 5 The DCs three-dimensional culture provided by the present invention

[0056] Figure 6 The in vitro release of DCs after three-dimensional culture provided by the present invention DETAILED DESCRIPTION

[0057] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0058] The porous supramolecular hydrogel of the present invention is obtained by using methacrylic anhydride modified gelatin and oxidized hyaluronic acid as a matrix, introducing thermosensitive gelatin microspheres as a pore-forming agent, and undergoing subsequent treatment.

[0059] The specific preparation steps of methacrylic anhydride modified gelatin in the embodiment are as follows: 5 g of gelatin is weighed into a 250 mL two-necked round-bottom flask, 50 mL of phosphate buffer (pH=7.4) is taken out with a measuring cylinder and added, and after heating and stirring at 60° C. to dissolve completely, 4 mL of methacrylic anhydride is taken out with a syringe and added dropwise, and the reaction temperature is maintained at 50° C. and stirred for 3 h; the reaction solution is centrifuged at 10000 rpm for 20 h, and then placed in a dialysis bag at 8000-14000 for 5 days, and the water is changed 3 times a day; after dialysis, it is freeze-dried and stored in a -20° C. refrigerator for use.

[0060] The preparation steps of the oxidized hyaluronic acid polymer in the embodiment are as follows: take a 250mL clean round-bottom flask, weigh 1g of sodium hyaluronate, add 100mL of deionized water, and stir in an ice-water bath for 12 hours to completely dissolve the sodium hyaluronate. Slowly add 5mL of 0.5M sodium periodate dropwise, stir at 24℃ in the dark for 3 hours. Add 1mL of ethylene glycol and continue stirring for 10min to terminate the reaction. Collect the product and place it at 8000℃. The product was dialyzed in a 12000 dialysis bag for three days, with the water changed three times a day. The product was freeze-dried to obtain a white sponge-like product, which was stored in a 4°C refrigerator for later use.

[0061] Example 1

[0062] A biomimetic extracellular matrix porous hydrogel for three-dimensional culture and delivery of dendritic cells and a preparation method thereof, characterized in that it comprises the following steps:

[0063] (1) Weigh 200 mg of GelMA and 24 mg of OHA in a glass bottle, add 4 mL of basal culture medium, and stir magnetically at 300 rpm and 60°C for 4 h.

[0064] (2) Add 10 mg of photosensitizer 2959 and continue stirring for 1 h to obtain a hydrogel precursor solution;

[0065] (3) sterile filtering the hydrogel precursor solution of step (2) to obtain reaction system 1;

[0066] (4) mixing DCs with the reaction system 1 of step (3) to fully achieve three-dimensional encapsulation of DCs, thereby obtaining a complex 1;

[0067] (5) The complex 1 was placed in a cell culture device, 5 cm below a 365 nm laser, and solidified by irradiation for 1 min to obtain a dense hydrogel three-dimensional culture model.

[0068] (6) After the culture model was placed in an incubator for 24 h in vitro, the number of DCs in the culture supernatant was measured at regular intervals every 24 h.

[0069] Example 2

[0070] (1) Add 0.4 g gelatin to 10 mL deionized water, control the temperature of the thermostatic water bath to 50°C, and stir with a magnetic stirrer at 400 rpm until it is completely dissolved;

[0071] (2) adding 6 mL of anhydrous ethanol to the reaction solution of step (1), and the reaction system changes from transparent to milky white after the addition of anhydrous ethanol;

[0072] (3) Weigh 0.05 g of poloxamer and 0.02 g of gum arabic and add them to step (2). After they are completely dissolved, cool to 24° C. and continue to stir and react for 8 h.

[0073] (4) centrifuging the solution of step (3) at 300 g for 5 min to remove the emulsion and obtain gelatin microgel;

[0074] (5) resuspending the product obtained in step (4) with PBS, centrifuging at 1000 g for 5 min, repeating three times, removing the residual emulsion, resuspending the gelatin microgel precipitate with PBS and storing at 4°C for later use;

[0075] (6) 400 mg of GelMA and 32 mg of OHA were weighed into a glass bottle, 4 mL of basal culture medium was added, and 10 mg of photosensitizer 2959 was added after magnetic stirring at 300 rpm and 60°C for 4 h. Stirring was continued for 1 h to obtain reaction system 1;

[0076] (7) After sterile filtration, the reaction system 1 was mixed with the gelatin microspheres of step (5) at a ratio of 1:2 to obtain a reaction system 2;

[0077] (8) mixing the DCs with the reaction system 2 in step (7) to fully achieve three-dimensional encapsulation of the DCs, thereby obtaining a complex 1;

[0078] (9) Composite 1 was placed 5 cm directly below a 365 nm laser and cured under illumination for 1 min to obtain a hydrogel three-dimensional culture model with a pore size of 15 μm.

[0079] (10) After the culture model was placed in an incubator for 24 h in vitro, the number of DCs in the culture supernatant was measured at regular intervals every 24 h.

[0080] Example 3

[0081] (1) Add 0.4 g gelatin to 10 mL deionized water, control the temperature of the thermostatic water bath to 50°C, and stir with a magnetic stirrer at 400 rpm until it is completely dissolved;

[0082] (2) adding 10 mL of anhydrous ethanol to the reaction solution of step (1), and the reaction system changes from transparent to milky white after the addition of anhydrous ethanol;

[0083] (3) Weigh 0.1 g of poloxamer and 0.04 g of gum arabic and add them to step (2). After they are completely dissolved, cool to 24° C. and continue to stir and react for 8 h.

[0084] (4) centrifuging the solution of step (3) at 300 g for 5 min to remove the emulsion and obtain gelatin microgel;

[0085] (5) resuspending the product obtained in step (4) with PBS, centrifuging at 1000 g for 5 min, repeating three times, removing the residual emulsion, resuspending the gelatin microgel precipitate with PBS and storing at 4°C for later use;

[0086] (6) 400 mg of GelMA and 32 mg of OHA were weighed into a glass bottle, 4 mL of basal culture medium was added, and 10 mg of photosensitizer 2959 was added after magnetic stirring at 300 rpm and 60°C for 4 h. Stirring was continued for 1 h to obtain reaction system 1;

[0087] (7) After sterile filtration, the reaction system 1 was mixed with the gelatin microspheres of step (5) at a ratio of 1:2 to obtain a reaction system 2;

[0088] (8) mixing the DCs with the reaction system 2 in step (7) to fully achieve three-dimensional encapsulation of the DCs, thereby obtaining a complex 1;

[0089] (9) Composite 1 was placed 5 cm directly below a 365 nm laser and cured under illumination for 1 min to obtain a hydrogel three-dimensional culture model with a pore size of 100 μm.

[0090] (10) After the culture model was placed in an incubator for 24 h in vitro, the number of DCs in the culture supernatant was measured at regular intervals every 24 h.

[0091] The temperature-responsive gelatin microspheres prepared in this example are Figure 1 As shown in the figure, by introducing emulsion stabilizers (poloxamer and gum arabic) into the emulsification system to control the size of the emulsion droplets, two sizes of gelatin beads were prepared. Among them, the particle sizes of the two sizes of gelatin beads are mainly distributed in 15μm and 100μm, respectively. The gelatin microspheres are fully mixed with the matrix glue, the matrix glue is cross-linked by ultraviolet light curing, and then the temperature is increased to melt the gelatin microspheres to obtain a porous hydrogel. The internal structure of the supramolecular porous hydrogel is shown in Figure 2 , Figure 3 As shown in the figure, the results show that the hydrogel has a uniform and evenly distributed porous structure in both wet and freeze-dried states. The stiffness of supramolecular hydrogels with different pore sizes was characterized, such as Figure 4 As shown in the figure, the stiffness of the dense hydrogel, the hydrogel with a small pore size (15μm), and the hydrogel with a large pore size (100μm) are all 1kPa. The results show that porous hydrogels with consistent stiffness and different pore sizes have been successfully prepared. The prepared porous hydrogels were cultured in vitro for 24 hours on DCs and their morphology was characterized. Figure 5 As shown, hydrogels with pore structures are more conducive to the elongation of DCs synapses. Figure 6 After DCs were cultured in hydrogels for 24 hours, their in vitro release was measured at fixed points. The results showed that compared with dense hydrogels, hydrogels with pore structures were more conducive to the release of DCs. The effective release of DCs laid the foundation for porous hydrogels to be used as cell delivery carriers.

[0092] Conclusion: We have successfully prepared a supramolecular hydrogel with decoupled stiffness and pore size. Compared with traditional hydrogels, this hydrogel has the characteristics of controllable pore size, simulated extracellular matrix components, and double network structure. In addition, this hydrogel has the advantages of easy use, simple operation, and biosafety, and can be used in the fields of three-dimensional culture and delivery of immune cells and tumor immunotherapy.

[0093] Finally, it is noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be included therein.

Claims

1. A biomimetic extracellular matrix porous hydrogel for three-dimensional culture and delivery of dendritic cells and a preparation method thereof, characterized in that: The following steps are involved: Methacrylic anhydride-modified gelatin and sodium periodate-oxidized hyaluronic acid were used as matrix materials. Temperature-responsive gelatin microspheres of different sizes were introduced, the gelatin microspheres were fully mixed with the matrix glue, the methacrylic anhydride-modified gelatin was cross-linked by ultraviolet light curing, and then the temperature was increased to melt the gelatin microspheres to prepare supramolecular porous hydrogel.

2. A biomimetic extracellular matrix porous hydrogel for three-dimensional culture and delivery of dendritic cells and a preparation method thereof according to claim 1, characterized in that: The following steps are involved: The method for preparing the temperature-responsive gelatin microspheres comprises the following steps: (1) Add gelatin to deionized water and stir at 40-60° C. until completely dissolved to obtain solution 1; (2) adding anhydrous ethanol to the solution 1 of step (1), wherein the reaction system changes from transparent to milky white after the addition of anhydrous ethanol, to obtain a solution 2; wherein the volume ratio of anhydrous ethanol to deionized water is 3:5 to 1:1; (3) Add poloxamer and gum arabic to solution 2 of step (2) and stir until completely dissolved, then cool to 20° C. to 25° C. and stir, and obtain gelatin microgel through subsequent treatment; wherein the mass ratio of gum arabic to gelatin is 0.05 to 0.1; and the mass ratio of poloxamer to gelatin is 0.125 to 0.

25.

3. A biomimetic extracellular matrix porous hydrogel for three-dimensional culture and delivery of dendritic cells and a preparation method thereof according to claim 1, characterized in that : The method for preparing the supramolecular hydrogel with a pore structure comprises the following steps: (1) Adding methacrylic anhydride-modified gelatin and oxidized hyaluronic acid into a basal culture medium and stirring for 3 to 5 hours to obtain a matrix gel solution 1, wherein the mass percentage of methacrylic anhydride-modified gelatin is 3% to 10%; and the mass percentage of hyaluronic acid is 0.4% to 0.8%. (2) Adding a photosensitizer (2959) to the matrix glue solution 1 of step (1) to obtain a matrix glue solution 2, wherein the mass percentage of the photoinitiator is 0.2% to 0.5%. (3) Adding the temperature-responsive gelatin microspheres prepared in claim 1 to the matrix gel solution 2 of step (2) in proportion, mixing by blowing, and obtaining a porous hydrogel precursor solution; wherein the ratio of gelatin microspheres to matrix gel is 1:1 to 3:

1.

4. A biomimetic extracellular matrix porous hydrogel for three-dimensional culture and delivery of dendritic cells and a preparation method thereof according to claim 1, characterized in that : The in vitro three-dimensional culture of dendritic cells comprises the following steps: (1) At room temperature, the dendritic cells and the hydrogel precursor solution were fully mixed to achieve three-dimensional encapsulation of DCs to obtain complex 1, in which the cell density was 5×10 6 ~1×10 7 / mL sol. (2) The composite 1 was placed in a cell culture device and cured under UV light for 1 min. After adding complete culture medium, the composite was placed in a cell culture incubator for in vitro culture for 24 h.

5. A biomimetic extracellular matrix porous hydrogel for three-dimensional culture and delivery of dendritic cells and a preparation method thereof according to claim 1, characterized in that : The in vitro release of dendritic cells comprises the following steps: (1) After the DCs three-dimensional culture model was placed in an incubator and cultured for 24 hours in vitro, the DCs in the culture supernatant were collected and counted every 24 hours.

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