CN115558126A

CN115558126A - Dynamically crosslinked hyaluronic acid hydrogels

Info

Publication number: CN115558126A
Application number: CN202111615790.0A
Authority: CN
Inventors: 边黎明; 杨伯光; 徐夏忆
Original assignee: Hainuo Biotechnology Co ltd
Current assignee: Hainuo Biotechnology Co ltd
Priority date: 2021-11-29
Filing date: 2021-12-27
Publication date: 2023-01-03

Abstract

The invention relates to a dynamic cross-linking hyaluronic acid hydrogel, which is prepared from a hydrogel prepolymer prepared by mixing hyaluronic acid modified by a hydrophobic group and cyclodextrin modified by a cross-linking group, and can be degraded through chemical or photo-cross-linking reaction, wherein the modified hyaluronic acid is formed by grafting a hydrophobic group capable of generating host-guest action with the cyclodextrin on the hyaluronic acid. The hydrophobic group is selected from butylbenzene, terpenoid or sterol molecules; the crosslinking group of the cyclodextrin is preferably a photocurable group. The invention also relates to the preparation of the dynamic cross-linked hyaluronic acid hydrogel and the application of the dynamic cross-linked hyaluronic acid hydrogel in three-dimensional cell culture and therapeutic agents. The hydrogel disclosed by the invention has good dynamic mechanical properties, so that better in-vitro regulation and control of cell behaviors are realized, and the hydrogel has positive promotion effects on the culture of embryonic stem cells, neural stem cells and immune stem cells, the in-vitro proliferation of various cancer cells and the formation of clusters with the characteristics of cancer stem cells.

Description

Dynamically crosslinked hyaluronic acid hydrogels

Technical Field

The invention relates to the technical field of tissue engineering, in particular to a degradable in-vitro 3D cell culture hydrogel material, and a preparation method and application thereof.

Background

The behavior, function and fate of cells in the human body are indistinguishable from the biochemical and biophysical signals of the microenvironment they are in. The cellular microenvironment is composed of the extracellular matrix (ECM), bioactive factors, and adjacent cells. In the in vitro 3D culture of cells, the reconstruction of the cell microenvironment and the directional regulation of the cell fate are realized, and the method has very important significance for researching determinants of the cell fate and treating diseases by using the cells. ECM has dynamic mechanical properties, and cell interaction with ECM responds to biophysical signals, followed by remodeling of the surrounding environment. Metals, ceramics, and synthetic polymers can effectively replace the mechanical functions of tissues (e.g., teeth, hips, knees, etc.), but have great limitations on the simulation of ECM dynamic mechanical properties. Hydrogel materials have dynamic mechanical properties similar to ECM, and more hydrogel materials are used in studies to induce cell behavior. The subject-guest self-assembly hydrogel takes the subject-guest self-assembly interaction as a crosslinking mode, has high dynamic mechanical properties, and is a hydrogel material which simulates the dynamic mechanical properties of ECM with great prospect.

The use of hydrogels for loading/delivering stem cells and primary cells to promote tissue regeneration is becoming a hotspot. The increase of cells is regulated and controlled by utilizing the biological physical signals of the hydrogel, such as viscoelasticity, mechanical strength, degradation performance and the likeThe reproduction, the morphology, the differentiation and the phenotypic matrix deposition have extremely important guiding effects on the 3D culture of in vitro cells and the in vivo transplantation of the cells. With natural proteins and polysaccharides (Matrigel) ^TM Alginate) is taken as a base material, and the hydrogel has good biocompatibility and viscoelasticity, and the viscoelasticity plays an important role as a dynamic mechanical property in influencing the behavior of a loaded cell. However, the uncontrollable nature of stoichiometry limits the use of natural macromolecules for cell behavior studies. The semisynthetic natural high molecular polymer is modified on the basis of a natural high molecular material, and a chemical structure with known performance is introduced into the natural high molecular material, so that the precise regulation and control of the performance of the natural high molecular material are realized, and the semisynthetic natural high molecular polymer has an important significance for deeply researching influence factors for regulating and controlling cell behaviors.

Since the cell aggregation cluster is widely applied to drug screening in vitro models, basic research of disease progression and developmental biology, clinical research fields of autologous or allogeneic organ culture and tissue regeneration and the like, the hydrogel is utilized to carry out proliferation culture, cytology research, cell release and collection on the cell cluster, and the hydrogel is used as a tissue engineering scaffold to load cells for tissue regeneration and the like, so that the hydrogel has great biomedical significance and application prospect.

In recent years, scientists have found that cancer cells with properties very similar to those of embryonic stem cells also exist in tumors, and thus are named cancer stem cells. Despite the advances in cancer therapy that have been made by scientists, the 5-year survival rate of patients with advanced cancer remains low, one reason for this is that cancer tissues in the patient's body contain cancer stem cells that are resistant to both chemotherapy and radiation therapy and that can survive as "roots" or circulate in the body, causing the cancer to recur. Cancer stem cells are a major target for the development of anti-cancer drugs, but are difficult to identify, mainly because they tend to be present in small numbers in cancer tissues. Understanding the molecular mechanisms of cancer stem cells is of great importance for the development of novel cancer therapies.

Although there have been a number of studies on the application of hydrogel systems to three-dimensional culture of cells in vitro, there are some problems to be solved by these works. Firstly, the recovery of cells from hydrogel culture systems is complicated and time-consuming. Secondly, because the dynamic performance of the existing hydrogel culture system is poor, the cells on different parts of the surface and the interior of the hydrogel are not uniformly grown, and cell clone clusters are difficult to form. Thirdly, the preparation and use costs are high, and the method is not suitable for large-scale application.

Disclosure of Invention

It is an object of the present application to provide a hydrogel with controllable dynamic properties. The raw materials of the hydrogel are hyaluronic acid grafted by hydrophobic groups and cyclodextrin modified by crosslinking groups, and the hydrogel is formed by initiating the crosslinking reaction of the crosslinking groups after the hyaluronic acid and the cyclodextrin are mixed in aqueous solution. The dynamic cross-linked hydrogel can be rapidly formed into gel under mild conditions, has good viscoelasticity, good self-healing property and shearing thinning capability, and has excellent biocompatibility.

The hydrophobic group grafted on the hyaluronic acid can be used as a guest molecule to generate host-guest action with cyclodextrin used as a host molecule.

In certain embodiments, the hydrophobic group is selected from butylbenzene, terpene, or sterol molecules.

In certain embodiments, the hydrophobic group is selected from tert-butyl benzene, ibuprofen, menthol, geraniol, cholic acid, and the like.

The crosslinking group of the modified cyclodextrin can be epoxy group, acrylate group, styrene double bond and other groups capable of generating chemical crosslinking or photocrosslinking. In one embodiment, the cyclodextrin is a photocurable group-modified cyclodextrin, preferably an acrylated cyclodextrin. In certain embodiments, the acrylated cyclodextrin has a molecular molar ratio of acrylate groups to cyclodextrin of 0.8 to 1.8. In certain embodiments, the acrylated cyclodextrin is acrylated β -cyclodextrin.

The hydrogel is formed based on the host-guest action and has dynamic adjustable performance. The subject-object recognition reaction has the characteristics of mild reaction conditions, dynamic and reversible reaction process and the like, can effectively overcome the limitation of covalent bonding, and can simulate biological functions on a molecular level. The host-guest interaction is based on non-covalent interactions, including van der waals forces, electrostatic attraction, hydrophobic interactions, hydrogen bonds and the like, and is the key to generating the host-guest recognition effect. The binding constants between different guest molecular structures and host molecules are different, and the dynamic performance of the dynamic cross-linked hydrogel can be adjusted according to the binding constants.

The hydrogel of the present invention can be obtained from hydrogels with different dynamic properties by adjusting the hyaluronic acid grafting ratio, for example, according to different cultured cell systems. The calculation method of the grafting rate can be obtained by nuclear magnetic detection of the ratio of the hyaluronic acid active sites connecting the grafting groups to the total active sites of the hyaluronic acid. The grafting ratio of the hyaluronic acid may be 5% to 80%, and in a preferred embodiment, the grafting ratio of the hyaluronic acid may be 10% to 60%, and in some embodiments, the grafting ratio of the hyaluronic acid may be 20% to 50%; in certain embodiments, the hyaluronic acid may have a grafting rate of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80%.

The hydrogel can be degraded, and can be rapidly degraded after a guest molecule with stronger binding capacity with cyclodextrin is added, and preferably amantadine hydrochloride, menthol, terpineol and structural isomers thereof, tert-butylbenzoate, triton 100X and the like can be added.

The novel hydrogel prepared by the invention has the following advantages: 1) The hydrogel scaffold raw material and the host-guest molecules are safe compounds harmless to cells. The hyaluronic acid is a medical material which is approved by FDA, and most of guest small molecules of the grafted hyaluronic acid come from natural medicines, so that the prepared hydrogel has excellent biocompatibility and biochemical activity; the introduction of partially functional guest molecules can bring more functions to the modified hydrogel, for example, ibuprofen hyaluronic acid hydrogel has anti-inflammatory effect; 2) Because of utilizing the interaction of various subjects and objects with different dynamic parameters, the novel hydrogel has good adjustable dynamic mechanical property and mechanical firmness, has good long-term stability, and is suitable for long-term in-vitro culture of various cells; 3) The reversibility of the crosslinking points endows the hydrogel network with dynamic property, so that the novel hydrogel has the capability of self-repairing and healing and can be reintegrated after mechanical rupture. The dynamic network structure of the novel hydrogel is favorable for promoting cells loaded in the hydrogel to form clusters, and can generate effective promotion effect on cell proliferation and differentiation by reediting; 4) In addition, the cyclodextrin main body structure contained in the hydrogel has a wrapping and slow-release effect on the water-insoluble drug, so that the capability of the hydrogel in regulating cell behaviors is enhanced; 5) Because the crosslinking mode of the hydrogel system belongs to weak interaction, after the guest molecules with stronger binding capacity are added, the hydrogel can be degraded and released to load cell clusters therein, so that the cell clusters are acquired and recovered in a bio-friendly manner. These properties make the novel hydrogels an ideal culture platform and can be applied in a variety of cell culture systems.

It is still another object of the present application to provide a method for preparing a dynamically crosslinked hyaluronic acid hydrogel. The method comprises the following steps:

1) Preparing acrylated beta-cyclodextrin;

2) Preparing hydrophobic group grafted hyaluronic acid;

3) Mixing the acrylated beta-cyclodextrin and the hydrophobic group grafted hyaluronic acid in an aqueous solution to obtain a hydrogel prepolymer;

4) Adding a crosslinking initiator into the hydrogel prepolymer to initiate a crosslinking reaction to obtain the degradable dynamic crosslinking hydrogel.

Preferably, in the hydrogel prepolymer, the content of the hydrophobic group grafted hyaluronic acid is 1-10% (w/v), and the content of the acrylated beta-cyclodextrin is 1-10% (w/v).

In one embodiment, the preparation method comprises the following steps:

1) The preparation method of the acrylated beta-cyclodextrin comprises the following steps:

dissolving beta-cyclodextrin in Dimethylformamide (DMF), adding triethylamine, and reducing the temperature of the system to 0 ℃; dropwise adding acryloyl chloride with the molar weight 5-10 times that of cyclodextrin into the system, and performing suction filtration to remove triethylamine hydrochloride to obtain a clear solution, namely a reaction product; concentrating the reaction product by vacuum rotary evaporation, and drying in vacuum to obtain the acrylated beta-cyclodextrin (Ac-CD);

2) Preparation of p-tert-butylbenzene grafted hyaluronic acid:

adjusting the pH value of the hyaluronic acid aqueous solution to 7.0 by using tetrabutylammonium hydroxide aqueous solution to obtain tetrabutylammonium hydroxide salt HA-TBA of the hyaluronic acid; dissolving HA-TBA in dimethyl sulfoxide, sequentially adding 2-4 times of p-tert-butyl phenylacetic acid, 0.5-1 time of 4-dimethylaminopyridine and 1.0-1.5 times of di-tert-butyl dicarbonate, and reacting at 45 ℃; freeze drying to obtain p-tert-butyl benzene modified hyaluronic acid HA-TP;

3) Mixing and dissolving p-tert-butyl benzene modified hyaluronic acid and acrylated beta-cyclodextrin in PBS buffer solution to obtain an HA-TP hydrogel prepolymer;

4) Adding a photoinitiator into the hydrogel prepolymer, and obtaining the dynamic cross-linked hyaluronic acid hydrogel under the illumination condition.

Preferably, in the hydrogel prepolymer solution, the mass percentage of the hydrophobic group modified hyaluronic acid is 1-10% (w/v), and the mass percentage of the acrylated beta-cyclodextrin is 1-10% (w/v).

Preferably, the initiators selected for use herein are: visible light initiators, such as phenyl (2,4,6-trimethylbenzoyl) lithium phosphate, phenyl (2,4,6-trimethylbenzoyl) sodium phosphate, can be used for the crosslinking reaction in the blue light range (wavelength 405 nm);

ultraviolet light initiators such as 2-hydroxy-1- [4- (2-hydroxyethoxy) phenyl ] -2-methyl-1-propanone (I2959), phenylbis (2,4,6-trimethylbenzoyl) phosphine oxide (I819), benzil dimethyl ether (I651), alpha-ketoglutaric acid, etc., and initiate crosslinking reaction (320 nm-400 nm) under illumination in the ultraviolet light range;

IR photoinitiators, such as bacteriochlorophyll (bacteriochlorophyl a), polymethines (polymethines), borate-containing dyes (dye-rate), cyanine dyes (Alkyne cyanine dyes 718), and the like, initiate crosslinking reactions in the infrared range of light.

Another object of the present application is to provide an application of culturing cells in vitro using the dynamically crosslinked hyaluronic acid hydrogel.

In one embodiment, the hydrogel is used for culturing embryonic stem cells in vitro. The dynamic cross-linked hydrogel based on the subject-object system can remarkably promote the amplification and the pluripotent maintenance of the mouse embryonic stem cell mass, and provides an excellent three-dimensional culture microenvironment for the embryonic stem cells.

In one embodiment thereof, the hydrogel is used for in vitro culture of neural stem cells.

In another embodiment, the hydrogel is used for culturing cancer cell lines in vitro, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, ewing's tumor, leiomyoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, hepatocellular carcinoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, wilm's tumor, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, angioblastoma, auditory neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma. Preferably, the cancer cell line is selected from the group consisting of a cervical cancer cell line, a human neuroblastoma cell line, and a B16 melanoma cell line.

It is still another object of the present application to provide a method for culturing cells in vitro using the dynamically crosslinked hyaluronic acid hydrogel. The method comprises the following steps:

a) Mixing hydrophobic group modified hyaluronic acid and acrylated beta-cyclodextrin, and dissolving in a PBS (phosphate buffer solution) to obtain a hydrogel prepolymer;

b) Adding a photoinitiator into the hydrogel prepolymer, mixing the photoinitiator with cells, obtaining hydrogel wrapping the cells under the condition of illumination, adding a culture solution, and standing and culturing in an incubator;

c) Adding a guest micromolecular compound with stronger binding capacity with cyclodextrin into the hydrogel wrapping the cells, degrading the hydrogel, and collecting cultured cell clusters. Preferably, amantadine hydrochloride, menthol, terpineol and its structural isomers, tert-butylbenzoate, triton 100X, and the like may be added.

The present application also provides a method of reprogramming a cancer cell in vitro to differentiate into a cancer stem cell. The method comprises the steps of loading a cancer cell line by using the dynamic cross-linked hyaluronic acid hydrogel, culturing cells, and recovering cell clusters after the hydrogel is degraded. Preferably, the dynamic cross-linked hyaluronic acid hydrogel is formed by tert-butyl benzene grafted hyaluronic acid and cyclodextrin modified by a photocuring group under the condition of a photoinitiator.

In one embodiment, by culturing cancer cells using the hydrogel of the present invention, the cancer cells loaded in the hydrogel form distinct cluster-like structures and produce the specific molecular markers SOX2, oct3/4, NANOG, etc. of cancer stem cells, indicating that these cancer cells have been reprogrammed. And (3) carrying out drug resistance detection on the released cell clusters, and finding that the clusters have obvious drug tolerance to the anticancer drug Dox, thereby further explaining that the cancer cells realize transformation to cancer stem cells.

Further provided herein are methods for rapid recovery of cells from the hydrogel three-dimensional cell culture system. The hydrogel system of the invention achieves rapid degradation by this method. The method comprises the steps of adding a guest small molecular compound with stronger binding capacity with cyclodextrin into a hyaluronic acid dynamic crosslinking hydrogel system grafted by a hydrophobic group, so that good degradation performance is realized, complete degradation of the hydrogel can be realized within 15 minutes, and the wrapped cell clusters are released, and experiments prove that the activity of the released cell clusters is good. The guest small molecule is preferably an amantadine hydrochloride aqueous solution, the concentration is 20-80mM, and the hydrogel degradation time is 5-15 minutes.

In addition, as hyaluronic acid is naturally degraded under the action of hyaluronidase in an organism, when the dynamically crosslinked hyaluronic acid hydrogel is injected or filled in the organism, loaded cells or drugs can be gradually released without adding other reagents. The dynamically crosslinked hyaluronic acid hydrogels of the present application may also be used for drug delivery into a living organism.

The hydrogel prepolymer is prepared by mixing hydrogel mixed by hyaluronic acid grafted by hydrophobic groups and cyclodextrin modified by crosslinking groups, adding gelatin to obtain hydrogel prepolymer, adding chemical crosslinking initiator or photoinitiator, and forming hydrogel under illumination. The molar ratio of the hyaluronic acid grafted by the hydrophobic groups to the gelatin in the hydrogel is 3:1-1:6, and preferably the molar ratio of the hyaluronic acid grafted by the hydrophobic groups to the gelatin in the hydrogel is 1:1-1:4.

The application further provides application of the composite dynamic cross-linked hydrogel in three-dimensional cultured cells.

It is still another object of the present application to provide use of the dynamically crosslinked hyaluronic acid hydrogel for preparing a therapeutic agent, wherein the dynamically crosslinked hyaluronic acid hydrogel is loaded with cells and/or drugs and delivered into the body, and the cells are embryonic stem cells, neural stem cells, or immune cells. Since dynamically cross-linked hyaluronic acid hydrogels are gradually degraded in vivo by hyaluronidase, the application can realize safe, efficient and long-lasting delivery of therapeutically effective cells and/or drugs into the body.

It is a further object of the present application to provide an injectable dynamically crosslinked hydrogel. The composite dynamic cross-linked hydrogel obtained by introducing the gelatin dynamic cross-linked hydrogel into the dynamic cross-linked hyaluronic acid hydrogel has injectability. Preferably, the complex dynamically crosslinked hydrogel is loaded with cells in three-dimensional culture, preferably the complex dynamically crosslinked hydrogel is loaded with cells and/or therapeutic drugs. The enzyme in vivo such as collagenase can degrade the dynamic composite crosslinking hydrogel, thereby gradually releasing the loaded cells or drugs, and realizing the effects of continuous treatment on the focus part and the like. For example, after resection of a tumor site, residual tumor cells often pose a recurrence risk. The hydrogel loaded with immune cells and/or antitumor drugs is injected into a tumor resection part, so that the hydrogel not only can play a filling role, but also can play a role in continuously inhibiting the proliferation of tumor cells and reducing relapse. Further, the present application provides use of the complex dynamically crosslinked hydrogel for the preparation of a therapeutic agent, wherein the complex dynamically crosslinked hydrogel is loaded with cells and/or drugs and delivered into the body, and the cells are embryonic stem cells, neural stem cells or immune cells.

The dynamic hydrogel developed by the invention has good dynamic performance, realizes three-dimensional culture of various cells, including but not limited to three-dimensional culture of embryonic stem cells, neural stem cells, immune cells and cancer cell lines, and the obtained cell clusters keep high activity, and can realize the recovery research of the cell clusters in a cell-friendly degradation mode. These features are of great significance for disease treatment and basic research.

The hydrogel disclosed by the invention can provide a required dynamic microenvironment for cell types growing in an agglomeration manner, such as embryonic stem cells, cancer stem cells and the like. Research results show that the self-assembled dynamic cross-linked gel can not only promote the proliferation and the dryness maintenance of embryonic stem cells in long-term three-dimensional culture, but also reprogram cancer cells of various cancer cell lines of different types to differentiate into cancer stem cells within a plurality of days. When the cancer cells of the above type are loaded in a dynamic hydrogel gel for three-dimensional culture, they will start to form cluster structures and produce specific molecular markers of cancer stem cells, such as SOX2 and Oct3/4, etc., thereby indicating that the cancer cells have been reprogrammed. The invention is a valuable culture system which can support the long-term three-dimensional culture of embryonic stem cells, and has important value on the research of the mechanism of cancer development and metastasis and the basic research of anti-cancer drugs. The invention has the beneficial effects that: compared with the traditional extracellular matrix hydrogel (such as Matrigel), the degradable dynamic hydrogel for promoting the 3D culture of the cell clusters provided by the invention has lower cost. Can be widely used for the research of various in vitro cell clusters, and is expected to promote the research of embryonic stem cells and the research and development of novel anti-cancer drugs and the process of precise treatment.

Drawings

FIG. 1 storage modulus G 'and elastic modulus G' of a dynamically crosslinked hyaluronic acid hydrogel, (A) t-butyl-phenyl hyaluronic acid; (B) ibuprofen hyaluronic acid; (C) menthol hyaluronic acid; (D) geraniol hyaluronic acid; (E) cholic acid hyaluronic acid.

FIG. 2B16 cell line schematic representation of the distribution of inner cell cluster formation in HA-TP hydrogel and Matrigel, respectively, cultures.

FIG. 3 schematic representation of the distribution of inner cell cluster formation of SH-SY5Y cell lines cultured in HA-TP hydrogel and Matrigel, respectively.

FIG. 4 is a schematic representation of the distribution of inner cell cluster formation of HeLa cell lines cultured in HA-TP hydrogel and Matrigel, respectively.

FIG. 5 immunofluorescence assay of HeLa cell clusters in HA-TP hydrogels with different contents (2%, 3% and 4%).

FIG. 6 immunofluorescence assay after incubation for 7 days with three cell lines (HeLa, B16 and SH-SY 5Y) loaded in HA-TP hydrogel.

FIG. 7 immunofluorescence assay of the surface of clusters of HeLa cells cultured in hydrogel after 36 hours of doxorubicin administration at a concentration of 0.5 nM.

FIG. 8 the appearance of cell clusters after 7 days of T cell culture in HA-TP hydrogel.

FIG. 9 expression of TNF α and IFN γ by T cell culture in HA-TP hydrogel.

FIG. 10 fluorescent staining assay results of ZAP70 and LCK in cultured T cells in HA-TP hydrogel.

FIG. 11 is an injectable performance test of the composite dynamically crosslinked hydrogel.

FIG. 12 shows the results of immunofluorescence assay and microscopic bright field observation of 3T3 cells in the composite dynamic cross-linked hydrogel, where Actin represents the fluorescent marker of Actin, and Nucleus represents the fluorescent marker of 3T3 cell Nucleus.

FIG. 13 LCK immunofluorescent staining experiments after 7 days of incubation of immune T cells in complex dynamic hydrogels, DAPI stands for nuclear fluorescent staining marker.

Fig. 14 ZAP70 immunofluorescence staining experiments after 7 days of incubation of immunot cells in complex dynamic hydrogels, DAPI stands for nuclear fluorescent staining marker.

FIG. 15 NMR spectra of hyaluronic acid grafted with different hydrophobic molecules.

Detailed Description

Apparatus and reagent

Rotational rheometer (Anton Paar MCR 01)

Laser confocal microscope (Nikon ECLIPSE TE-U)

Optical microscope (NikonDS-Fi 2)

Fluorescence microscope (LECIA THUNDER IMAGER LIVE CELL SYSTEM)

Cervical cancer cell lines (HeLa) were purchased from: american Type Culture Collection (ATCC) CCL-2; human neuroblastoma cell line (SH-SY 5Y) was purchased from: american Type Culture Collection (ATCC) CRL-2266; the B16 melanoma cell line (B16-F0) was purchased from: american Type Culture Collection (ATCC) CRL-6322. Immune CD8+ T cells, 3T3 cell source: shanghai cell bank of Chinese academy of sciences.

Other reagents were all commercially available products.

EXAMPLE 1 preparation of HA-TP hydrogel

dissolving beta-cyclodextrin in Dimethylformamide (DMF) to obtain 6% (w/v) beta-cyclodextrin/DMF solution, adding triethylamine at a ratio of 4.5% (v/v), and cooling the system to 0 deg.C; dropwise adding acryloyl chloride with the molar weight being 7 times that of cyclodextrin into the system, continuously stirring for 12 hours after the dropwise adding is finished, and performing suction filtration to remove triethylamine hydrochloride to obtain a clear solution, namely a reaction product;

concentrating the reaction product by vacuum rotary evaporation, dropwise adding the concentrated solution of the reaction product into acetone with the volume of 10 times to obtain white precipitate, washing the obtained white precipitate with acetone, and finally performing vacuum drying to obtain the acrylated beta-cyclodextrin (Ac-CD).

2) The preparation method comprises the following steps:

preparing sodium hyaluronate (molecular weight: 80,000) into 1% aqueous solution, adding 6 times of sodium hyaluronate cation exchange resin, and stirring at room temperature for 8 hr;

and (4) carrying out suction filtration to remove the resin to obtain a solution after reaction. Adjusting the pH of the obtained solution to 7.0 by using tetrabutylammonium hydroxide aqueous solution, and performing freeze drying to obtain tetrabutylammonium hydroxide salt HA-TBA of hyaluronic acid;

HA-TBA is dissolved in dimethyl sulfoxide to prepare 1% HA-TBA dimethyl sulfoxide solution. Sequentially adding 3 equivalents of p-tert-butyl phenylacetic acid, 0.75 equivalent of 4-dimethylaminopyridine and 1.2 equivalents of di-tert-butyl dicarbonate into the solution system by taking the mole number of the HA-TBA structural unit as 1 equivalent, and reacting for 24 hours at 45 ℃;

dialyzing the reaction mixture against DMSO for 3 days, dialyzing the reaction mixture against saturated saline for 1 day, dialyzing the reaction mixture against pure water for 3 days, and freeze-drying the dialysis mixture to obtain the p-tert-butyl benzene modified hyaluronic acid HA-TP.

3) The preparation method of the p-tert-butyl benzene grafted hyaluronic acid-cyclodextrin hydrogel prepolymer comprises the following steps:

mixing and dissolving p-tert-butyl benzene grafted hyaluronic acid and acrylated beta-cyclodextrin (Ac-CD) in PBS buffer solution to obtain HA-TP hydrogel prepolymer, wherein the hydrogel prepolymer solution is composed of a mixed solution of p-tert-butyl benzene grafted hyaluronic acid (HA-TP) and acrylated beta-cyclodextrin (Ac-CD), the solid content of HA-TP is 4% (w/v), and the solid content of Ac-CD is 3% (w/v).

4) Adding I2959 photoinitiator into the hydrogel prepolymer solution, mixing with cells, obtaining degradable dynamic hydrogel wrapping the cells under the condition of ultraviolet initiation, adding culture solution, and standing and culturing in an incubator. After 7 days of culture, adding amantadine hydrochloride aqueous solution into the cell-loaded hydrogel prepolymer solution, degrading the hydrogel, and collecting cultured cell clusters.

EXAMPLE 2 preparation of different kinds of dynamically crosslinked hyaluronic acid hydrogels

According to a method similar to the preparation steps of the p-tert-butyl benzene grafted hyaluronic acid, hyaluronic acid grafted by different hydrophobic molecules is respectively prepared, and HA-TBA is dissolved in dimethyl sulfoxide to prepare 1% HA-TBA dimethyl sulfoxide solution. With the mole number of HA-TBA structural units as 1 equivalent, sequentially adding 3 equivalents of corresponding hydrophobic molecules (p-ibuprofen, menthol monoester succinate, geraniol monoester succinate and cholic acid), 0.75 equivalent of 4-dimethylaminopyridine and 1.2 equivalent of di-tert-butyl dicarbonate into the solution system, and reacting at 45 ℃ for 24 hours; the reaction mixture was dialyzed against DMSO for 3 days, against saturated saline for 1 day, against pure water for 3 days, and lyophilized to obtain the objective product. The preparation methods of the hyaluronic acid grafted with different guest molecules are respectively shown in table 1.

A dynamically cross-linked hydrogel of hyaluronic acid assembled from different host and guest molecules was prepared according to the method of example 1 using hyaluronic acid grafted with different guest molecules. The nmr spectra of hyaluronic acid grafted with different hydrophobic molecules are shown in fig. 15.

Table 1 preparation of hyaluronic acid grafted with different guest molecules

Example 3 dynamic mechanical Properties of dynamically crosslinked hyaluronic acid hydrogel

The dynamic cross-linked hyaluronic acid hydrogel network has good cell adaptability and mechanical dynamics. The storage modulus and loss modulus of each hydrogel prepared in example 2 and a commercially available extracellular matrix hydrogel Matrigel hydrogel were measured using a rotational rheometer. The comparison result shows that the hydrogel prepared by the invention has better dynamic mechanical property, and the loss modulus is shown in table 2 and is larger than that of the extracellular matrix hydrogel with the same hardness.

In addition, the hydrogel of the present invention showed strong frequency dependence (see fig. 1), and as the frequency of applied shear force was gradually decreased, the storage modulus and loss modulus of the hydrogel were simultaneously decreased, indicating that the hydrogel had good dynamic properties.

The storage modulus is also called as elastic modulus, and refers to the magnitude of energy stored due to elastic (reversible) deformation when a material is deformed, and reflects the elasticity of the material; the loss modulus is also called viscous modulus, and refers to the amount of energy lost due to viscous deformation (irreversible) when a material is deformed, and reflects the amount of viscosity of the material.

TABLE 2 dynamic mechanical Properties of hydrogels

The research utilizes an object with different binding capacities with beta-cyclodextrin to modify hyaluronic acid macromolecules to obtain the macromolecular object. And the cyclodextrin modified by acrylic acid and the macromolecular object are pre-assembled, and then the dynamic cross-linked hydrogel is obtained in a photoinitiation mode. As can be seen in FIG. 1, hydrogels formed from guest macromolecules of different binding capacities have different rheological characteristics. Guest molecules with relatively strong binding capacity, such as tert-butyl benzene, cholic acid and the like, have relatively fast gelling time and relatively high storage modulus; whereas guest molecules with relatively weak binding capacity have a slower gel formation time and a relatively weak storage modulus.

Example 4HA-TP hydrogel for culturing melanoma cells of murine tumor cell line

Melanoma (B16) of murine tumor cell line at 4X 10 ⁶ And mixing the cells/mL density with the hydrogel prepolymerization solution, and initiating by ultraviolet light to obtain the cell-loaded hydrogel. To 500. Mu.l of hydrogel, a conventional cell culture medium (basimedium) was added and the mixture was subjected to static culture in an incubator.

After 7 days of culture, adding a adamantanamine hydrochloride aqueous solution into the cell-loaded hydrogel to degrade the hydrogel, and collecting cultured cell clusters. The concentration of the amantadine hydrochloride aqueous solution is 50mM, and the hydrogel degradation time is 5-15 minutes.

The murine tumor cell line melanoma (B16) was planted at the same density (4X 10) ⁶ cells/mL) was mixed with Matrigel prepolymer and formed into hydrogel as a control. The results of the clone sizes observed under the light microscope for two groups of hydrogel cultured cells are shown in FIG. 2. The results showed that B16 cell clones in the HA-TP hydrogel gradually formed from 700 μm in size with the increase of the culture time during the 7-day culture ² Gradually increased to 12500 μm ² And the clone sizes of the inner cell and the outer cell of the hydrogel are uniform. Compared with HA-TP, the Matrigel hydrogel HAs obvious clone growth of outer layer cells at the edge, the inner layer cells at the middle layer are in a spreading state, obvious cell clone cannot be formed, and the whole cell clone is unevenly distributed.

After the HA-TP hydrogel wrapping the cancer cells is cultured for 7 days, ADA (adamantanamine hydrochloride) aqueous solution is added to degrade the hydrogel to realize cell cluster recovery, and the recovered cell clusters are degraded by trypsin to obtain single cells; in contrast, after the Matrigel hydrogel encapsulating cancer cells is cultured for 7 days, the hydrogel can be degraded only by adding trypsin, while cell clusters are digested by adding trypsin, and only a single cell can be obtained because the cell clusters cannot be completely recovered. As a preferred technical scheme of the invention, the concentration of the amantadine hydrochloride aqueous solution is 50mM, and the hydrogel degradation time is 5-15 minutes.

Example 5 human neuroblastoma cell line culture

Using the experimental method of example 4, a human neuroblastoma cell line (SH-SY 5Y) was seeded in HA-TP hydrogel and Matrigel, respectively, and cultured in a conventional cell culture medium such as RPMI1640, DMEM medium, etc. The results showed that SH-SY5Y cell clones gradually formed with increasing culture time during 7 days of culture, with sizes from 2000 μm ² Gradually increased to 25000 mu m ² And the clone sizes of the inner cell and the outer cell of the hydrogel are uniform. Compared with HA-TP, the Matrigel hydrogel HAs obvious clone growth of outer layer cells at the edge, the inner layer cells at the middle layer are in a spreading state, obvious cell clone cannot be formed, and the whole cell clone is unevenly distributed. Results of the experimentAs shown in fig. 3.

Example 6HeLa cell Cluster culture

Using the experimental method of example 4, a human cervical cancer cell line (Hela) was planted in HA-TP hydrogel and Matrigel, respectively, and cultured. And cells were examined for NANOG and Oct3/4 expression by immunofluorescence staining on

days

2,4, and 7 (see fig. 4, fig. 5).

By observing the clone sizes of the cells cultured in the HA-TP hydrogel and the Matrigel of the Hela cell line, cluster structures are formed at the edge, the middle and the central parts of the HA-TP hydrogel, but the cells in the Matrigel cannot form clusters, as shown in FIG. 4; analysis of size distribution of cell clusters formed at different positions of the two hydrogels revealed that the size of the cell clusters formed from inside to outside in the HA-TP hydrogel was uniform, the size of the cell clusters formed in Matrigel was not uniform, and hydrogel cell clusters inside Matrigel were not easily formed.

Expression of homeobox proteins (NANOG) and octamer-binding transcription factor 4 (Oct 3/4) are critical for self-renewal and maintenance of sternness in stem cells. HeLa cells are loaded in HA-TP hydrogel and cultured for 7 days, and immunofluorescence staining results show that cell clones in the HA-TP hydrogel with different solid contents show a large amount of NANOG and Oct3/4 expression, so that the cell clones generated under dynamic induction of the HA-TP hydrogel have good stem cell performance and self-renewal capacity. HeLa cell clusters in HA-TP hydrogel with different contents (2%, 3% and 4%, w/v) continuously grow along with the time, which shows that the cells have good proliferation capacity in the hydrogel; the cell clusters in the HA-TP hydrogel expressed Oct3/4 and NANOG, indicating that the cancer cells in the cell clusters were reprogrammed to cancer stem cells (see figure 5).

Example 7 migration Capacity of HA-TP hydrogel-induced cell-forming clones

After three cell lines (HeLa, B16 and SH-SY 5Y) are loaded in HA-TP hydrogel and cultured for 7 days, the immunofluorescence detection result shows that HA-TP hydrogel induces the formed cell clone to express a large amount of Cdc42 (see figure 6). Cell division controlling protein 42 (Cdc 42) is a protein involved in cell cycle regulation, and plays an important role in regulating various physiological processes such as cytoskeletal changes, polarity establishment, motility, and migration. Hypoxia inducible factor 1-alpha (HIF-1 alpha) is a subunit of the heterodimeric transcription factor hypoxia inducible factor 1 encoded by the HIF1A gene and is considered as a main transcription regulator of cells and development in response to hypoxia, and three cell lines (HeLa, B16 and SH-SY 5Y) are loaded in HA-TP hydrogel and cultured for 7 days, and the result shows that the HA-TP hydrogel induces the formed cells to express a large amount of HIF-1 alpha in a clone mode (see figure 6). The above results indicate that the high expression of Cdc42 and HIF-1 alpha occurs in three cell lines (HeLa, B16 and SH-SY 5Y) loaded in HA-TP hydrogel for 7 days, indicating that the cells in the cell cluster are in a low oxygen environment and have good migration capability.

EXAMPLE 8 clonal resistance of cells

Human cervical cancer cell line (Hela) was cultured in HA-TP hydrogel and Matrigel for 7 days, respectively. The two groups of hydrogel samples were treated with 0.5nM adriamycin (Doxrubicin) for 36 hours, and after staining, the cell activity was observed by using a fluorescence microscope, and the results showed (see FIG. 7) that the HA-TP dynamicly-induced cell clones still maintained good cell activity. And Hela cells in the middle of the Matrigel cannot form cell clones, and are subjected to massive death under the action of the medicament, and compared with Hela cells at the edge of the Matrigel, hela cells can form clones, and the good cell activity can be still maintained after the medicament treatment. The experiment result shows that the Hela cell clone formed under the induction of the dynamic property of the HA-TP hydrogel HAs good resistance to anticancer drugs.

Example 9 three-dimensional culture of immune cells by HA-PT

CD8+ T cells were obtained from splenocytes from adult mouse spleen and passed through a MojoStor according to the manufacturer's instructions (biolegend) ^TM Mouse CD8+ T cell isolation kit for isolation. CD8+ T cells were collected by centrifuge and loaded into HA-TP dynamically crosslinked hydrogel according to the method of example 4.

500. Mu.l of hydrogel prepolymer mixed with CD8+ T cells (cell concentration 1-2X 10) was added to each well of a 24-well plate ⁶ /ml), UV irradiation at 365nm for 10 minutes after addition of photoinitiator, RPMI1640 medium supplemented with 5ug/ml mouseSource CD3, 5ug/mL murine CD28, L-glutamine, 1% penicillin-streptomycin, 10% FBS, and 50ng/mL IL-2.

Placing 24-well plates at 37 ℃ with 5% CO ₂ Culturing in a cell culture box. On day 3 of culture, the cell-loaded hydrogel was supplemented with half the volume of fresh medium containing 100ng/mL IL-2 to support T cell expansion.

Simultaneously, the 2D method was used to culture CD8+ T cells in vitro as a control, as follows:

1) Diluting the murine CD3 antibody to 5 mu g/ml with sterile PBS, adding the diluted antibody into a 24-well plate, wherein each well is 400 mu l, and coating the antibody at 4 ℃ overnight;

2) The medium used was composed of: RPMI1640+10% serum +5ug/ml murine CD28+ double antibody, CD8+ T cell concentration was adjusted to 1X 10 ⁶ /ml；

Taking a coating plate, discarding the coating solution, and washing for 3 times by sterile PBS; adding 500. Mu.l of cell suspension per well, standing at 37 ℃ with 5% CO ₂ Culturing in a cell culture box.

The growth morphology of the cells was observed daily using an electron microscope. Approximately 10% of the T cells appeared to cluster in the HA-TP hydrogel after 7 days of culture (see FIG. 8).

After the cells loaded in the HA-TP hydrogel were degraded by adding an amantadine hydrochloride aqueous solution, cultured T cells were recovered. Immunofluorescence staining experiments are carried out on the T cells, and the results show that the cultured T cells can be proliferated and activated in HA-PT hydrogel, and the expression of TNF alpha and IFN gamma is remarkably improved compared with that of 2D culture (figure 9). And (3) performing an immunofluorescence staining experiment on the cultured cells, and detecting the expression of activation indexes ZAP70 and LCK (sigma) in the T cells to prove that the three-dimensionally cultured immune cells have immunological activity. The indicators ZAP70 and LCK after T cell activation were both highly expressed (see figure 10).

Example 10 composite dynamically crosslinked hydrogel

The novel composite hydrogel is prepared by mixing gelatin and other cyclodextrin-based dynamic cross-linked gels with host-guest effects, and the obtained novel composite hydrogel has injectability. Ibuprofen hyaluronic acid with an anti-inflammatory effect was selected in this example. The preparation method comprises the steps of mixing ibuprofen hyaluronic acid and beta-cyclodextrin modified by a crosslinking group according to a certain proportion, adding a gelatin solution to form a prepolymer, adding a crosslinking agent initiator, and initiating crosslinking to obtain the gelatin-ibuprofen hyaluronic acid composite hydrogel. The composite dynamic cross-linked hydrogel is proved to have good injectability through experiments (see figure 11).

There are two distinct interactions within the hydrogel under physiological conditions (37 ℃): the cyclodextrin interacts with aromatic groups in the gelatin and a subject and an object of a butylbenzene structure in the ibuprofen; electrostatic interaction of amino groups and hyaluronic acid carboxyl groups in gelatin. Experiments show that the injectable gelatin-ibuprofen hyaluronic acid composite hydrogel has stable storage modulus at physiological temperature and room temperature, and the stability of the hydrogel is proved. Because the hydrogel has good adhesion promoting property and dynamic mechanical property, cells wrapped in the hydrogel can be rapidly spread.

And (3) analyzing the ratio of the gelatin/ibuprofen hyaluronic acid composite dynamic cross-linked hydrogel by using the 3T3 cells. The precursor 1 (P1) was prepared by mixing 4% ibuprofen hyaluronic acid (HA-IBF) and 3% acrylated cyclodextrin (Ac-CD), formulating 8% gelatin as precursor 2 (P2), mixing P1 and P2 according to the ratio of table 3, respectively, and adding 3T3 cells and photoinitiator, the table below lists the solids content of each component in the composite hydrogel. 3T3 cells loaded with the composite hydrogel were cultured in a culture medium, and immunofluorescence staining was performed one day later, and the results are shown in FIG. 12.

TABLE 3 compounding ratio of composite dynamically crosslinked hydrogel

According to the above 3T3 cell culture experiment, within one day, when the HA-IBF content in the hydrogel was in the range of 0.7-1.1%, the gelatin content was in the range of 5.6-6.5%, and the Ac-CD content was in the range of 0.55-0.8%, the 3T3 cells showed rapid spreading by immunofluorescent staining (see FIG. 12).

Further, the experiment shows that the hMSC is loaded by the composite dynamic cross-linked hydrogel and is cultured in three dimensions. Compounding a composite dynamic cross-linked hydrogel according to the proportion of P1 and P2=1, wherein HA-IBF is 1%, ac- β -CD is 0.75%, and gelatin is 4.8%, to obtain an injectable composite dynamic cross-linked hydrogel, the structure of which is experimentally verified to be favorable for the clonal growth and self-renewal of ESC cells.

Example 11 three-dimensional culture of immune cells with composite dynamically crosslinked hydrogel

According to the method of example 10, 4% of tert-butyl benzene grafted hyaluronic acid (HA-PT) and 3% of acrylated cyclodextrin (Ac-CD) are mixed to prepare precursor 1 (P1), 8% of gelatin is prepared as precursor 2 (P2), P1 and P2 are mixed according to the proportion of 1:3, immune cell CD8+ T cells and a photoinitiator are added, and under the action of blue light 405nm, the immune cell loaded dynamic cross-linked hydrogel is obtained. Meanwhile, tert-butyl benzene grafted hyaluronic acid-gelatin dynamic cross-linked hydrogel (PT-Gel) and ibuprofen grafted hyaluronic acid-gelatin dynamic cross-linked hydrogel (BU-Gel) are adopted to carry out conventional culture on CD8+ T cells according to the method of example 9. Cultured cells were subjected to immunofluorescence staining on day seven to detect expression of ZAP70 and LCK proteins. As shown in fig. 13 and 14, the immune T cells three-dimensionally cultured in the dynamic complex crosslinked hydrogel differentiated well on day 7, formed cell masses, and maintained immune activity. The immune cells cultured by the dynamic cross-linked hydrogel load can be directly used for in vivo injection, wherein the loaded immune cells can be gradually degraded under the action of in vivo collagenase, and the released immune cells have strong immunocompetence and can play a good therapeutic role.

Claims

1. A dynamic cross-linked hyaluronic acid hydrogel is characterized in that the hydrogel is a hydrogel prepolymer formed by mixing hydrophobic group grafted hyaluronic acid and cross-linking group modified cyclodextrin, and the hydrogel is formed by initiating a cross-linking reaction of the cross-linking group;

the hydrophobic group grafted on the hyaluronic acid can be used as a guest molecule to generate host-guest effect with cyclodextrin used as a host molecule.

2. The hydrogel of claim 1, wherein the hydrophobic groups are selected from butylbenzene, terpene or sterol groups; preferably, the hydrophobic group is selected from hydrophobic structure groups such as tert-butyl benzene, ibuprofen, menthol, geraniol and cholic acid.

3. The hydrogel according to claim 1 or 2, wherein the cyclodextrin-modifying crosslinking group is selected from an epoxy group, an acrylate-type group, or a styrene-type double bond group; preferably, the crosslinking group of the cyclodextrin is a photocurable group, preferably an acrylate group.

4. The hydrogel according to claim 1, wherein the grafting ratio of the hyaluronic acid is 5% to 80%, preferably the grafting ratio of the hyaluronic acid is 10% to 60%.

5. A method for preparing the dynamically crosslinked hyaluronic acid hydrogel of claim 1, comprising the steps of:

1) Preparing cyclodextrin modified by a crosslinking group;

2) Preparing hyaluronic acid grafted with hydrophobic groups;

3) Mixing cyclodextrin modified by a crosslinking group and hyaluronic acid grafted by a hydrophobic group in an aqueous solution to obtain a hydrogel prepolymer;

4) Adding a chemical cross-linking agent or adding a photoinitiator into the hydrogel prepolymer to initiate a cross-linking reaction under the illumination condition to obtain dynamic cross-linked hydrogel;

the hydrophobic group is selected from butylbenzene, terpenoid or sterol groups; preferably, the hydrophobic group is selected from hydrophobic structure groups such as tert-butyl benzene, ibuprofen, menthol, geraniol and cholic acid.

6. The method of claim 5, comprising the steps of:

dissolving beta-cyclodextrin in dimethylformamide, adding triethylamine, and reducing the temperature of the system to 0 ℃; dropwise adding acryloyl chloride with the molar weight 5-10 times that of cyclodextrin into the system, and performing suction filtration to remove triethylamine hydrochloride to obtain a clear solution, namely a reaction product; vacuum rotary evaporation is carried out to concentrate a reaction product, and vacuum drying is carried out to obtain the acrylated beta-cyclodextrin;

2) Preparation of p-tert-butyl benzene grafted hyaluronic acid:

3) Dissolving p-tert-butyl benzene grafted hyaluronic acid and acrylated beta-cyclodextrin in a PBS buffer solution to obtain an HA-TP hydrogel prepolymer; preferably, in the hydrogel prepolymer, the w/v content of the hyaluronic acid grafted by the hydrophobic group is 1-10%, and the w/v content of the acrylated beta-cyclodextrin is 1-10%;

4) Adding a photoinitiator into the hydrogel prepolymer solution, and obtaining the dynamically crosslinked hyaluronic acid hydrogel under the condition of illumination.

7. Use of the dynamically cross-linked hyaluronic acid hydrogel of any of claims 1-4 for culturing cells in vitro, preferably the cells are embryonic stem cells, neural stem cells, immune cells or cancer cell lines, preferably the immune cells are selected from one or more of T cells, NK cells, DC cells or CIK cells.

8. A method for culturing cells in vitro using the dynamically crosslinked hyaluronic acid hydrogel according to any of claims 1-4, comprising the steps of:

a) Mixing the hydrophobic group grafted hyaluronic acid and the acrylated cyclodextrin and dissolving in a PBS (phosphate buffer solution) to obtain a hydrogel prepolymer; preferably, in the hydrogel prepolymer solution, the w/v content of the hyaluronic acid modified by the hydrophobic group is 1-10%, and the w/v content of the acrylated beta-cyclodextrin is 1-10%;

b) Adding a photoinitiator into the hydrogel prepolymer solution, mixing the photoinitiator with cells, and obtaining cell-loaded hydrogel under the condition of illumination; adding the hydrogel loaded with cells into a culture solution, and standing and culturing in an incubator;

c) Adding small molecules which generate stronger host-guest action with hyaluronic acid grafted with the hydrophobic group to the cell-loaded hydrogel, degrading the hydrogel, and collecting cultured cell clusters, wherein the small molecules are preferably selected from amantadine hydrochloride, menthol, terpineol and structural isomers thereof, tert-butylbenzoate and Triton 100X.

9. Use of the dynamically crosslinked hyaluronic acid hydrogel of any of claims 1-4 for the preparation of a therapeutic agent, wherein the dynamically crosslinked hyaluronic acid hydrogel is loaded with cells and/or drugs and delivered into the body, the cells being embryonic stem cells, neural stem cells or immune cells.

10. A method for reprogramming cancer cells to differentiate into cancer stem cells in vitro, comprising loading cancer cell lines with the dynamically cross-linked hyaluronic acid hydrogel of any of claims 1-4, and performing cell culture in a cell culture medium.

11. A composite dynamically crosslinked hydrogel composed of the dynamically crosslinked hyaluronic acid hydrogel according to any one of claims 1 to 4 and a dynamically crosslinked gelatin hydrogel; the preparation method comprises the steps of mixing hyaluronic acid grafted by hydrophobic groups and cyclodextrin modified by crosslinking groups in aqueous solution, adding gelatin, adding a chemical crosslinking initiator or a photoinitiator, and forming hydrogel under the illumination condition.

12. The composite dynamically crosslinked hydrogel according to claim 11, wherein the molar ratio of hyaluronic acid and gelatin grafted by hydrophobic groups in the hydrogel is 3:1-1:6, preferably the molar ratio of hyaluronic acid and gelatin grafted by hydrophobic groups in the hydrogel is 1:1-1:4.

13. Use of the complex dynamically crosslinked hydrogel according to claims 11-12 for culturing cells in three dimensions.

14. Use of a composite dynamically crosslinked hydrogel according to claims 11-12 loaded with cells and/or other drugs for the preparation of an injectable hydrogel; preferably, the cell is an embryonic stem cell, a neural stem cell or an immune cell.

15. Use of the composite dynamic cross-linked hydrogel of any one of claims 11 to 12 for the preparation of a therapeutic agent, wherein the composite dynamic cross-linked hydrogel is loaded with cells and/or drugs and delivered into the body, the cells are embryonic stem cells, neural stem cells or immune cells, and preferably, the composite dynamic cross-linked hydrogel is injected or packed into the body.