AU2020101687A4 - Preparation Method for Dextran-Hyaluronic Acid Hydrogel for Three-Dimensional Cell Culture and Application Thereof - Google Patents

Abstract

CN11253579A Abstract Page 1/1 The present invention relates to the field of biomedicine and relates to a preparation method for a dextran-hyaluronic acid hydrogel for three-dimensional cell culture and application thereof. The hydrogel freeze-drying scaffold synthesized by the present invention has interconnected pores and has pore size and porosity suitable for cell growth. In-vitro experiments show that cells are inoculated and cultured in the hydrogel which is non-toxic and has good cell compatibility, and the cells have a high survival rate in the hydrogel. One month after the hydrogel is injected subcutaneously into mice, the phenomena of inflammation and vascular proliferation do not occur, and the hydrogel is easily degraded without toxic or side effect. In-vivo experiments show that the hydrogel is injected subcutaneously into mice after being inoculated with cells, which can keep the cells in a favorable growth status, form tumor massive tissues and provide a necessary three-dimensional growth environment for the cells. The hydrogel can replace extracellular matrices functionally, and provide a scaffold and a three-dimensional growth environment for growth and attachment of cells. As a clinical in-vivo filling material, the hydrogel has the advantages of high bio-safety, simple and convenient operation, and low price. 1 CN111253579A Drawings of Description Page 1/7 1:44:31 PM 20.00 kV 3.0 10.0 mim 1 000 x SE Nova NanoS[ M FIG. 1 14

Description

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1:44:31 PM 20.00 kV 3.0 10.0 mim 1 000 x SE Nova NanoS[ M

FIG. 1

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Preparation Method for Dextran-Hyaluronic Acid Hydrogel for Three Dimensional Cell Culture and Application Thereof

Technical Field The present invention relates to the field of biomedicine, relates to the preparation of the biopolymer hydrogel and the application thereof in cell culture in vivo/in vitro, and particularly relates to a preparation method for a dextran-hyaluronic acid hydrogel for three-dimensional cell culture and application thereof.

Background At present, most of cell culture researches are conducted on a two-dimensional surface such as microwell plate, tissue culture flask and culture dish, and the characteristics of two-dimensional culture are simplicity, high efficiency, rapidity, and high survival rate. In a two-dimensional culture system, adherent cells grow adhering to the wall in a single layer on the surface of the solid culture plate and come into contact with the bottom surface of the culture plate with less than 50% of the surface area, while the remaining surface area comes into contact with the liquid culture medium, and only a small part is in contact with other cells or matrices; and dynamic interactions between cells and between cells and extracellular matrices in the in-vivo micro-environment are lacked, so the true growth of cells in vivo cannot be reflected accurately. In vivo, almost all the tissue cells live in extracellular matrices (ECM), and these ECMs include a plurality of complex three-dimensional fiber networks which can provide complex biological and physical signals. However, the environment provided by traditional two-dimensional cell culture is different from that for cells to grow in vivo, and it is difficult to use the environment to simulate the interactions between cells and between cells and extracellular matrices in real in-vivo physiological tissues. The three-dimensional cell culture technology makes up for these shortcomings. Almost 100% of the surface area of cells under three-dimensional culture conditions is in contact with other cells or matrices. The cultured cells have characteristic biological signal transduction, which can affect cell proliferation, adhesion, migration, gene expression and other functions. According to the latest research, under different conditions of two-dimensional culture and three-dimensional culture, many cell biological behaviors will have

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significant changes, including cell proliferation, cell apoptosis, cell differentiation, gene expression and drug metabolism. The matrices used for three-dimensional cell culture overcome the limitations of traditional two-dimensional culture, and often have porous structures which can support cells to grow, multiply and differentiate in vitro in an environment closer to the in-vivo real environment. The three-dimensional cell culture method can simulate the micro-environment for cells to grow in vivo and establish a connection between cells and between cells and extracellular matrices by using biological materials to construct scaffolds or matrices similar to in-vivo tissues, which not only provides cells with the material structure basis for matrix secretion and cell function activities required for growth micro-environment but also realizes the intuitiveness and conditional controllability of cell culture, effectively supplements the deficiencies of the two-dimensional cell culture method and facilitates molecular mechanism researches on growth, proliferation, signal transduction and the like of cells under physiological and pathological conditions. The ultimate goal of three-dimensional cell culture material design is to fully simulate the in-vivo extracellular matrix environment in an in-vitro environment. The extracellular matrix is composed of various substances produced and secreted into the tissue fluid by tissue cells, and has main components of connexin (fibronectin), fiber composition (such as elastin, collagen and gelatin) and a variety of filler molecules (such as glycosaminoglycan). These specific structures and components can provide distinctive biochemical functions such as promoting the transportation of nutrient substances, signal molecules, and wastes produced by cell metabolism, and provide mechanical performance to maintain the in-vivo structure. The action of extracellular matrices and cells is often mutual and in a dynamic environment. The extracellular matrices can promote the differentiation of cells in a specific direction, and the cells will in turn affect the environment in the extracellular matrices, and change the living environment of cells by synthesizing and degrading the main components of extracellular matrices. A three-dimensional cell culture matrix needs to have a certain scale structure, which is very important for imitating the real biological in-vivo environment, because the properties evolved in the natural environment are often in a multi-scale and multi level structure. Under normal conditions, it is stipulated that the three-dimensional cell culture matrix has three scales, including macro-scale (10-1-10-3 m), micro-scale

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(10-3-10-6 m) and nano-scale (10-610-9 in). The macro-scale structure will change the basic appearance characteristics of the matrix, including size and shape which determine the practicality and functionality for three-dimensional cell culture researches conducted in vitro. The micro-scale structure is proved to significantly change the smoothness of material and energy transportation in three-dimensional cell culture, and the controllability on this scale has an important influence on whether the micro-tissue structure in vivo can be realistically simulated, such as multicellular spatial structure widely existing in the extracellular matrix. The basic design parameters such as pore connectivity, porosity, geometric size and shape, and pore surface morphology of the micro-scale structure in the real environment are often common, which provides convenience for three-dimensional cell culture in vitro. In addition, different surface properties at micro-scale can affect the activation of specific gene expression, thereby affecting cell proliferation and differentiation. In a similar way, the nano-scale structure is also of great significance for three dimensional cell culture in vitro, because cells and extracellular matrices exchange material and energy information through nano-scale proteins so as to mediate various intracellular activities to change the extracellular matrix environment. Common macromolecules of most of extracellular matrices are nano-level. For example, the diameter of collagen fibers is usually between 50 nm and 200 nm, the diameter of fibronectin is between 60 nm and 70 nm, and the thickness is 3 nm. The characteristics of the nano-scale structure also determines the surface morphology of the cells in addition to affecting the supply of micronutrients. Because many cell signaling mechanisms determining cell surface morphology involve nano-scale molecules, a nano-scale surface structure is already proved to significantly affect cell adhesion, tissue morphology and differentiation. Biological scaffold materials include natural scaffold materials and synthetic scaffold materials. Natural scaffold materials have poor stretchability and are less stable due to existence of inherent soluble components, making it difficult to standardize the experiment. At present, many biological materials are used in the field of biomedicine, such as chitosan, sodium alginate, collagen and gelatin. The materials can be modified according to the application to meet the performance and requirements of the application. In three-dimensional adherent cell culture, various biological materials are already reported, but hydrogels that achieve good performance of blood compatibility, adjustability of scaffold gap and three

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dimensional animal culture in vivo or in vitro still need to be explored. The synthesized dextran-hyaluronic acid three-dimensional porous scaffold material has good extensibility and clear physical and mechanical properties, can be produced in a standardized manner according to the structure and shape required by the experiment, and also can control the size of cells by adjusting the size and density of the scaffold gap and achieve the requirements of three-dimensional adherent cell culture in vivo or in vitro.

Summary The purpose of the present invention is to synthesize a dextran-hyaluronic acid hydrogel which provides a scaffold and a non-toxic and easy-to-grow environment for three-dimensional cell culture in vivo and in vitro and better simulates a biological in vivo environment. The present invention adopts the following technical solution: A preparation method for a dextran-hyaluronic acid hydrogel for three dimensional cell culture comprises the specific steps as follows: (1) Preparation of dextran DEX-MA modified with methyl methacrylate Selecting dextran (DEX) with the molecular weight of 15 KD to 25 KD, dissolving with 4-dimethylaminopyridine (DMAP) in the dimethyl sulfoxide (DMSO) which is used as a solvent, controlling the mass ratio of DEX to DMAP to DMSO to (1.0-1.5):1:(15-20), adding glycidyl methacrylate (GMA) after reaction in a nitrogen environment for 15-30 min, controlling the mass ratio of DEX to GMA to 1:(1.2-1.5), stirring for reaction at 15-30°C in a nitrogen environment for 2-4 days, adjusting pH of the reaction solution to 6.5-7.2 with diluted hydrochloric acid, dialysing the mixture with a dialysis bag for 36-72 h, freezing the product in a freezing refrigerator or liquid nitrogen for 24-48 h, and then freezing and drying the product in a freeze dryer for 24-72 h to obtain DEX-MA; (2) Preparation of hyaluronic acid (HA-ADH) modified with adipic dihydrazide Respectively weighing and dissolving hyaluronic acid (HA) and 1-(3 Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) in deionized water, controlling the mass ratio of HA to EDC to deionized water to (3-4):1:(1500-2000), and stirring at room temperature for 1-2 h; then adding adipic dihydrazide (ADH) into the reaction system, controlling the mass ratio of EDC to ADH to (1.0-1.2):1, adjusting pH of the solution to 4.5-5.0 with diluted hydrochloric acid, and continuing

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stirring for 1-2 h; adding NaOH solution to adjust pH to 6.8-7.0; and dialysing the reaction product with a dialysis bag, freezing the product in the freezing refrigerator for 24-48 h, and then freezing and drying the product in the freeze dryer for 24-72 h to obtain hyaluronic acid (HA-ADH) modified with adipic dihydrazide. (3) Synthesis of dextran-hyaluronic acid hydrogel (DEX-MA-HA) Respectively dissolving DEX-MA and HA-ADH in deionized water, with the mass concentration range of 8-10% w/v; stirring and blending the two aqueous solutions, heating the two aqueous solutions according to the mass ratio of DEX-MA to HA-ADH of 8:1-6:1 in a 70-8 0 °Cwater bath for reaction for 16-24 h to obtain the dextran-hyaluronic acid hydrogel; and freezing the product in the freezing refrigerator for 24-48 h, and after freezing and drying the product in the freeze dryer for 24-72 h, and storing the product in the freezing refrigerator. A preparation method for a dextran-hyaluronic acid hydrogel for three dimensional cell culture prepared by the present invention is applied in cell culture in vivo/in vitro, and the specific application method is as follows: (1) Method for three-dimensional cell culture in vitro: first, putting DEX-HA in a test tube or culture plate and freezing in a -80°C freezing refrigerator for 1-2 h. Under aseptic conditions, adding DMEM culture solution according to the mass concentration of 30-50 mg/mL, standing at 20-40°C for about 30 min, and injecting the hydrogel into a 24-well plate or 48-well plate. Second, digesting the cultured adherent cells, inoculating the cells with the density of 2000-10000 cells/mL under aseptic conditions after centrifuging, preparing a cell hydrogel mixture, and culturing cells with 5% C02 at 37C. (2) Method for cell culture in mice: taking the cell hydrogel mixture prepared in (1), sucking out the hydrogel with a needle tube, injecting the hydrogel into the subcutaneous transplantation sites of mice, or injecting the hydrogel into the transplantation sites after dissection. The present invention has the following beneficial effects: after various indicator tests, the freeze-drying scaffold of the dextran hyaluronic acid hydrogel prepared by the present invention has better pore size and porosity, and the pores are connected to each other and are uniform in size (see Fig. 1). The hydrogel is suitable for three dimensional cell culture in vitro and has the characteristics of non-toxicity and good cell compatibility. In addition, the hydrogel is easily degraded in vivo (subcutaneously in mice) without inflammation or vascular proliferation, and can be used for slow

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release of fillers or drugs in vivo. In addition, after being mixed with the hydrogel, the cells are transplanted under the skin of mice and found to form tumor massive tissues, which can keep the cells in a favorable growth status and provide a necessary three dimensional growth environment for the cells.

Description of Drawings Fig. 1 is an SEM photograph of DEX-MA-HA prepared in embodiment 1; Fig. 2 is a micrograph of the growth status (st day) of Hela cells inoculated in the hydrogel scaffold in embodiment 1; Fig. 3 is a micrograph of the growth status ( 3 rd day) of Hela cells inoculated in the hydrogel scaffold in embodiment 1; Fig. 4 is a micrograph of the growth status ( 6 th day) of Hela cells inoculated in the hydrogel scaffold in embodiment 1; Fig. 5 is an SEM photograph of DEX-MA-HA prepared in embodiment 2; Fig. 6 shows the infrared spectroscopic analysis of DEX-MA-HA prepared in embodiment 3; Fig. 7 is a picture of Calcein staining on the 8 th day of Hela cells inoculated in the hydrogel scaffold in embodiment 4; Fig. 8 is a picture of Hoechst staining on the 8 th day of Hela cells inoculated in the hydrogel scaffold in embodiment 5.

Detailed Description The present invention is further described below in combination with the specific embodiments. It should be understood that the embodiments are only used for illustrating the present invention, not used for limiting the scope of the present invention. Methods in which specific experimental conditions are not specified in the following embodiments are carried out usually under conventional conditions or under conditions provided in the product specification. The materials and reagents used in the following embodiments are commercially available unless otherwise specified. Specific embodiments of the present invention are described below in detail in combination with the technical solution and accompanying drawings. Embodiment 1 Preparation and SEM Characterization of Dextran-Hyaluronic Acid Hydrogel and Growth Characteristics of Cells in Hydrogel

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(1) 3 g of DEX with the molecular weight of 20 KD is weighed and dissolved with 2.7 g of DMAP in 20 ml of DMSO, 4 mL of GMA is added after reaction in a nitrogen environment for 20 min, the mixture is stirred for reaction at room temperature in a nitrogen environment for 4 days, excessive DMAP in the reaction solution is neutralized with 1 mol/L hydrochloric acid until the pH of the solution is 7, the mixture is dialysed for 48 h, and after being frozen in a -80°C freezing refrigerator for 24 h, the mixture is frozen and dried for 48 h to obtain DEX-MA; (2) 800 mg of HA and 202 mg of EDC are weighed and added into a beaker containing 400 ml of deionized water, and after magnetic stirring for 1 h, 184 mg of adipic dihydrazide is added into the reaction system, PH is continuously adjusted to 4.75 with 1 mol/L hydrochloric acid, and stirring is continued for 2 h; 1 mol/L NaOH solution is added to neutralize the solution to make pH=7.0; a dialysis bag with the molecular weight cut-off of 7 KDA and 0.1 mol/L NaCl solution are used to dialyse the reaction product; and the product is frozen in the -80°C refrigerator for 24 h, and then frozen and dried in the freeze dryer for 48 h to obtainHA-ADH. (3) The dextran-hyaluronic acid hydrogel (DEX-HA) is synthesized: DEX-MA aqueous solution with the concentration of 8% and HA-ADH aqueous solution with the concentration of 8% are stirred and blended by a magnetic stirrer, wherein the mass ratio of DEX-MA to HA-ADH is 6:1; and the mixture is heated in a 70°C water bath for reaction for 20 h to obtain the dextran-hyaluronic acid hydrogel. SEM characterization of dextran-hyaluronic acid hydrogel: the surface of the swollen freeze-dried dextran-hyaluronic acid scaffold is flattened and then scanned with a scanning electron microscope, and the experimental results are observed and recorded. As shown in Fig. 1, the SEM photograph of the cross section of the hydrogel shows that the dextran-hyaluronic acid hydrogel scaffold presents a network structure, and a plurality of pores with the size of 30 pm to 80 pm exist inside the hydrogel. General mammalian cells can survive well at a pore size of 20 pm to 125 ptm. The hydrogel can achieve a favorable growth environment for cells, and cells can grow on different levels. Growth characteristics of cells in hydrogel: Hela cells are cultured by the hydrogel scaffold respectively in a 24-well plate. 30 mg of prepared hydrogel scaffold is added into each pore, 1 mL of DMEM culture solution is also added, and Hela cells are inoculated into each pore with the density of 3000/pore. The cells are cultured with 5% C02 at 37°C, and the changes of the cells are observed with a microscope;

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and as shown in Fig. 2, Fig. 3 and Fig. 4, Hela cells have good growth status and high growth rate in the hydrogel scaffold. Hela cells are uniformly distributed in the hydrogel and grow in a 3D structure in the hydrogel scaffold. After 3 days, the cells aggregate into a sphere and grow. As the number of days increases, the diameter of the sphere gradually increases, accompanied by the production of other cells with sphere 3D structures, and the contact between the cells of the sphere will be closer; and after the cells are cultured for 6 days, the hydrogel still can maintain the integrity, indicating that the new synthesized dextran-hyaluronic acid hydrogel can perform 3D cell culture well. Embodiment 2 Preparation and SEM Characterization of Dextran-Hyaluronic Acid Hydrogel In (3) of embodiment 1, the DEX-MA aqueous solution with the concentration of 8% and the HA-ADH aqueous solution with the concentration of 8% are stirred and blended by a magnetic stirrer, the mass ratio of DEX-MA to HA-ADH is adjusted to 6:1, and the mixture is heated in a 70°C water bath for reaction for 20 h to obtain the dextran-hyaluronic acid hydrogel. SEM characterization of DEX-MA-HA: as shown in Fig. 5, the morphology of the obtained dextran-hyaluronic acid hydrogel scaffold is essentially unchanged. Embodiment 3 Preparation and Infrared Spectroscopic Analysis of Dextran-Hyaluronic Acid Hydrogel In (3) of embodiment 1, the DEX-MA aqueous solution with the concentration of 8% and the HA-ADH aqueous solution with the concentration of 8% are stirred and blended by a magnetic stirrer, the mass ratio of DEX-MA to HA-ADH is adjusted to 6:1, and the mixture is heated in a 80°C water bath for reaction for 16 h to obtain the dextran-hyaluronic acid hydrogel. Infrared spectroscopic analysis of DEX-MA-ADH: the KBr pressed pellet method is used to process each intermediate product and final products, and then infrared spectroscopic analysis is performed. As shown in Fig. 6, the C=O stretching vibration absorption peak of a, P unsaturated carboxylic acid at 1707 cm-1,the C=C stretching vibration absorption peak of conjugated olefin at 1650 cm-1,and the out-of plane bending vibration absorption peak of C-H in double bond at 807 cm-1 significantly weaken, because the amino group on HA-ADH attacks the carbon carbon double bond to cause a Michael addition reaction and the double bond opens

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to form a three-dimensional cross-linked network structure, proving the occurrence of the Michael addition reaction. Embodiment 4 Preparation and Cell Compatibility Analysis of Dextran-Hyaluronic Acid Hydrogel The products obtained in (1) and (2) of embodiment 1 are prepared into DEX MA aqueous solution with the concentration of 10% and HA-ADH aqueous solution with the concentration of 10% which are stirred and blended by a magnetic stirrer, the mass ratio of DEX-MA to HA-ADH is adjusted to 8:1, and the mixture is heated in a °C water bath for reaction for 24 h to obtain the dextran-hyaluronic acid hydrogel. Cell compatibility analysis of hydrogel: a 24-well cell culture plate is taken, 30 mg of prepared hydrogel scaffold is added into each pore, 1 mL of DMEM culture solution is also added, and Hela cells are inoculated into each pore with the density of 3000/pore. The cells are cultured with 5% C02 at 37°C, and the changes of the cells are observed with a microscope; According to the steps on the live/dead cell staining kit, the cells are stained with esterified calcein (Calcein-Am, CA) and Hoechst, incubated at 4°C under dark conditions for about 20 min, rinsed twice with the PBS buffer solution, and observed under a fluorescence microscope. As shown in Fig. 7, live Hela cells are stained with calcein after being cultured for 8 days, which can show intracellular lipase activity. More than 90% of Hela cells in the dextran hyaluronic acid hydrogel are labeled with green fluorescence, indicating that the Hela cells have a high survival rate in the hydrogel. Fig. 6 shows Hoechst cell nucleus staining after the Hela cells are cultured for 8 days. Hoechst is a live cell dye. In the picture, more than 90% of Hela cells are labeled with blue fluorescence, indicating that the Hela cells have a higher survival rate in the hydrogel. Embodiment 5 Preparation and Degradability and Histocompatibility Detection in Vivo of Dextran-Hyaluronic Acid Hydrogel In (1) and (2) of embodiment 1, the DEX-MA aqueous solution with the concentration of 10% and the HA-ADH aqueous solution with the concentration of 10% are stirred and blended by a magnetic stirrer, the mass ratio of DEX-MA to HA-ADH is adjusted to 8:1, and the mixture is heated in a 70°C water bath for reaction for 24 h to obtain the dextran-hyaluronic acid hydrogel. A 24-well cell culture plate is taken, 30 mg of prepared hydrogel scaffold is

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added into each pore, 1 mL of DMEM culture solution is also added, and Hela cells are inoculated into each pore with the density of 3000/pore. The cells are cultured with 5% C02 at 37C, and the changes of the cells are observed with a microscope; According to the steps on the live/dead cell staining kit, the cells are stained with Hoechst, incubated at 4°C under dark conditions for 10 min, rinsed three times with the PBS buffer solution, and observed under a fluorescence microscope. As shown in Fig. 8, more than 90% of Hela cells are labeled with blue fluorescence, indicating that the Hela cells have a higher survival rate in the hydrogel. Degradability and histocompatibility detection in vivo of hydrogel: 20 mg of hydrogel is weighed and divided into two parts injected into a 24-well plate, 10 mg for each pore. 0.5 mL of DMEM solution is added into each pore and is standing for about 30 min. 6 SPF-level mice are taken, the hydrogel is sucked out with a needle tube, and 0.5 ml of hydrogel is injected into the subcutaneous site of the right abdomen of each mouse. The mice are cultured normally for 30 days. One month after subcutaneous injection of the dextran-hyaluronic acid hydrogel, the mice do not show any abnormality, and it is found that the mice have no inflammation or vascular proliferation and the hydrogel block already disappears, indicating that the synthesized dextran-hyaluronic acid hydrogel is non-toxic and has good biodegradability. Hydrogel cell culture in mice: 20 mg of hydrogel is weighed and divided into two parts injected into a 24-well plate, 10 mg for each pore. 0.5 mL of DMEM solution containing 5x105 Hela cells is added into each pore and is standing for about min. 6 SPF-level mice are taken, and the hydrogel cell compound is sucked out with a needle tube and injected into the subcutaneous site of the right abdomen of each mouse. The mice are observed for morphological change and put back into the cage after 60 min. For the next three days, the wound is cleaned with alcohol for disinfection. The mice are normally cultured for 30 days. One month after subcutaneous injection of the dextran-hyaluronic acid hydrogel and 5x105 Hela cells into the mice, the status of cell tumor formation under the skin is observed, and it is found that the cells grow well in the hydrogel and can grow and form tumor massive tissues under the skin of the mice and provide a necessary three-dimensional growth environment for cells in mice.

Claims (3)

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1. A preparation method for a dextran-hyaluronic acid hydrogel for three dimensional cell culture, characterized in that the specific steps are as follows: (1) preparation of dextran DEX-MA modified with methyl methacrylate selecting dextran DEX with the molecular weight of 15 KD to 25 KD, dissolving with 4-dimethylaminopyridine DMAP in the dimethyl sulfoxide DMSO which is used as a solvent, controlling the mass ratio of DEX to DMAP to DMSO to (1.0-1.5):1:(15-20), adding glycidyl methacrylate GMA after reaction in a nitrogen environment for 15-30 min, controlling the mass ratio of DEX to GMA to 1:(1.2-1.5), stirring for reaction at 15-30°C in a nitrogen environment for 2-4 days, adjusting pH of the reaction solution to 6.5-7.2 with diluted hydrochloric acid, dialysing the mixture with a dialysis bag for 36-72 h, freezing the product in a freezing refrigerator or liquid nitrogen for 24-48 h, and then freezing and drying the product in a freeze dryer for 24-72 h to obtain DEX-MA; (2) preparation of hyaluronic acid HA-ADH modified with adipic dihydrazide respectively weighing and dissolving hyaluronic acid HA and 1-(3 Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride EDC in deionized water, controlling the mass ratio of HA to EDC to deionized water to (3-4):1:(1500-2000), and stirring at room temperature for 1-2 h; then adding adipic dihydrazide ADH into the reaction system, controlling the mass ratio of EDC to ADH to (1.0-1.2):1, adjusting pH of the solution to 4.5-5.0 with diluted hydrochloric acid, and continuing stirring for 1-2 h; adding NaOH solution to adjust pH to 6.8-7.0; dialysing the reaction product with a dialysis bag; and freezing the product in the freezing refrigerator for 24-48 h, and then freezing and drying the product in the freeze dryer for 24-72 h to obtain the hyaluronic acid HA-ADH modified with adipic dihydrazide; (3) synthesis of dextran-hyaluronic acid hydrogel DEX-MA-HA respectively dissolving DEX-MA and HA-ADH in deionized water, with the mass concentration range of 8-10% w/v; stirring and blending the two aqueous solutions, heating the two aqueous solutions according to the mass ratio of DEX-MA to HA-ADH of 8:1-6:1 in a 70-80°Cwater bath for reaction for 16-24 h to obtain the dextran-hyaluronic acid hydrogel; and freezing the product in the freezing refrigerator for 24-48 h, and after freezing and drying the product in the freeze dryer for 24-72 h, and storing the product in the freezing refrigerator. 2. The dextran-hyaluronic acid hydrogel prepared according to claim 1 is applied in cell culture in vivo/in vitro.

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3. The application of the dextran-hyaluronic acid hydrogel for three-dimensional cell culture according to claim 2, characterized in that the specific method is as follows: (1) method for three-dimensional cell culture in vitro: first, putting DEX-HA in a test tube or culture plate and freezing in a -80°C freezing refrigerator for 1-2 h; under aseptic conditions, adding DMEM culture solution according to the mass concentration of 30-50 mg/mL, standing at 20-40C for 30 min, and injecting the hydrogel into a 24-well plate or 48-well plate; second, digesting the cultured adherent cells, inoculating the cells with the density of 2000-10000 cells/mL under aseptic conditions after centrifuging, preparing a cell hydrogel mixture, and culturing with 5% C02 cells at 37°C; (2) method for cell culture in mice: taking the cell hydrogel mixture prepared in (1), sucking out the hydrogel with a needle tube, injecting the hydrogel into the subcutaneous transplantation sites of mice, or injecting the hydrogel into the transplantation sites after dissection.

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FIG. 5 Transmittance (%)

Wave Number (cm-1) FIG. 6

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