Cellulose-derivative-based hydrogel, culture solution for culture and preparation method therefor and use thereof

Abstract

The present invention relates to a cellulose-derivative-based hydrogel, in which the matrix comprises a cellulose derivative, comprises a network structure formed by superfine fibers, and has a highly ordered three-dimensional micro-nano structure. The cellulose-derivative-based hydrogel is a ready-to-use hydrogel that contains no animal-derived components and has no cytotoxicity, and is suitable for 3D cell culture, tissue and organoid culture, stem cell culture, and the induced differentiation of an organoid. In the stem cell and organoid cultured by using the cellulose-derivative-based hydrogel of the present invention, a compact fused monolayer of endodermal cells is formed, which can suppress non-directed differentiation of the stem cell; and the formed organoid has a thickened and solid vesicle wall, and is a differentiated and mature organoid, which can be used in high-throughput drug screening, drug toxicity testing and regenerative medicine.

Description

Cellulose derivative-based hydrogel, culture solution for culture, and preparation method and application thereof

[0001] This application claims the priority of the following prior applications:

[0002] The priority of the following prior applications is claimed in this application: the prior application with the patent application number 202410450786.0 and the title "Cellulose derivative-based hydrogel, 3D cell culture solution, and preparation method and application thereof" filed with the China National Intellectual Property Office on April 15, 2024; the prior application with the patent application number 202410450781.8 and the title "Cellulose derivative culture solution and application thereof in culturing tissues or organoids"; and the prior application with the patent application number 202410450793.0 and the title "Cellulose derivative-based culture solution, kit, and application thereof in culturing stem cells and organoids" filed with the China National Intellectual Property Office on April 15, 2024;

[0003] The priority of the following prior application is claimed in this application: the prior application with the patent application number 202410894552.5 and the title "Cellulose derivative-based hydrogel, culture solution for culture, and preparation method and application thereof" filed with the China National Intellectual Property Office on July 4, 2024.

[0004] The above prior applications are incorporated herein by reference in their entirety. TECHNICAL FIELD

[0005] The present application belongs to the field of biotechnology and relates to a cellulose derivative-based hydrogel, a culture solution for culture, and a preparation method and application thereof, in particular to a cellulose derivative-based hydrogel, a 3D cell culture solution, a tissue and organoid culture solution, a stem cell culture solution, a culture solution for inducing differentiation of organoids, a kit, and a preparation method and application thereof. BACKGROUND

[0006] Ordinary 2D cell culture gradually loses the original properties of cells due to the proliferation of cells in an altered environment in vitro, which often does not match the in vivo situation. Animal experiments are completely performed in vivo, but the in vivo situation is complicated due to various factors and the mutual influence of the in vivo and external environments, making it difficult to study a single process and the intermediate process.

[0007] 3D cell culture technology is a technology between single-layer cell culture and animal experiments, which can maximize the simulation of in vivo environment and exhibit the advantages of direct visualization and controllability of cell culture. 3D cell culture can make up for many defects in the process of single-layer cell culture, for example:

[0008] (1) The 3D cell model can well simulate the in vivo cell microenvironment: substances such as gas, nutrients, and metabolic products exhibit gradient changes in concentration.

[0009] (2) 3D cell models can well simulate the interaction between cells: three-dimensional cell contact and direct or indirect cell-to-cell communication.

[0010] (3) 3D cell models can well simulate the biochemical and physiological responses of cells: the response of cells to internal or external stimuli is more consistent with the in vivo response.

[0011] The difficulty of 3D cell culture technology is to ensure the three-dimensional structure of cells and maintain the natural proliferation and differentiation activity. With decades of continuous development, the method of 3D cell culture is also constantly innovating, and the currently commonly used methods are mainly divided into two kinds: 3D cell culture method based on scaffold and 3D cell culture method without scaffold. The 3D cell culture method based on scaffold has a long history of development, with a large number of literature support. The materials used for cell culture scaffold include agarose, collagen, fibronectin, gelatin, laminin, etc. These composite materials simulate the natural extracellular matrix (ECM) through porosity, fiber, permeability and mechanical stability, which can well simulate the interaction between cells and the interaction between cells and extracellular matrix in the in vivo environment, while allowing cells to aggregate, proliferate and migrate on the scaffold.

[0012] Stem cells are a class of cells with proliferation and differentiation potential, which can differentiate into specific cell types of various tissues and organs. Stem cells can be divided into totipotent stem cells, pluripotent stem cells, multipotent stem cells and unipotent stem cells. Among them, pluripotent stem cells can differentiate into all cells derived from three germ layers, form all tissues and organs, and are used for various tissue and organ repair, disease treatment and drug screening, so they have become the focus of stem cell research. Culturing stem cells is also applied to the formation, maintenance and expansion of organoids. Embryonic stem cells (EC) and induced pluripotent stem cells (iPSC) are currently the most studied pluripotent stem cells. However, developing stem cells is not simple, and the main problem to be solved is how to effectively differentiate stem cells into cells with different functions. The stem cell microenvironment plays a controlling role in the differentiation of stem cells into functional cells, but the molecular mechanisms are currently poorly understood. A suitable and stable microenvironment is crucial for the culture and application of stem cells such as iPSC.

[0013] An organoid is a three-dimensional cultured cell group derived from stem cells, which has the characteristics of multi-cell assembly and self-renewal of living tissues. The tissue structure, cell type and gene characteristics of a tumor organoid are highly consistent with those of a tumor of a patient himself, and the tumor organoid can be used as a substitute for the patient to test drugs. The tumor organoid research model can maintain genomic stability and tumor heterogeneity, simulate a tumor microenvironment, and make up for the shortcomings of traditional cell and animal models.

[0014] Taking a commonly used stent material, Matrigel, as an example, the material is a basement membrane matrix extracted from an EHS mouse tumor rich in extracellular matrix proteins, and the main components include laminin, collagen IV, heparin sulfate glycoprotein, nidogen, and a variety of growth factors and matrix metalloproteinases. At room temperature, Matrigel polymerizes to form a three-dimensional matrix with biological activity, simulating the structure, composition, physical properties and functions of the basement membrane of cells in vivo, which is beneficial to the culture and differentiation of cells in vitro, and can be used for research on cell morphology, biochemical function, migration, invasion and gene expression.

[0015] The current problems of Matrigel include: 1) shortage of goods due to animal welfare issues; 2) large batch differences and difficult to control, the nutritional substances and protein components contained are greatly affected by batches; 3) animal-derived proteins and nucleic acids interfere with the drug efficacy evaluation, fluorescence detection and other links of downstream experiments; 4) the temperature-sensitive properties of Matrigel itself have strict requirements on the temperature of storage and operation. SUMMARY

[0016] To solve the above technical problems, the present application provides the following technical solutions:

[0017] A cellulose derivative-based hydrogel, the matrix of which comprises a cellulose derivative, the matrix comprising a reticular structure composed of ultrafine cellulose derivative fibers, and the reticular structure comprising ordered three-dimensional micro-nano structures.

[0018] According to some embodiments of the present application, the degree of substitution of the cellulose derivative is 0.1-3, preferably 0.5-2.5, and more preferably 1.0-2.0.

[0019] According to an embodiment of the present application, the diameter of the cellulose derivative fibers is 0.01-0.80 μm, for example, 0.01 μm, 0.02 μm, 0.03 μm, 0.04 μm, 0.05 μm, 0.06 μm, 0.07 μm, 0.08 μm, 0.09 μm or 0.10 μm.

[0020] According to embodiments of the present application, the overall particle size of the matrix in the hydrogel is 1 μm to 50 μm, for example, 1 μm, 5 μm, 8 μm, 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm, or 50 μm.

[0021] According to embodiments of the present application, the solid content of the hydrogel is 0.1 wt% to 3.0 wt%, for example, 0.1 wt%, 0.5 wt%, 1 wt%, 1.5 wt%, 2 wt%, 2.5 wt%, or 3.0 wt%.

[0022] According to embodiments of the present application, the cellulose derivative hydrogel is prepared by Scheme I or Scheme II:

[0023] Scheme I: homogeneously or heterogeneously derivatizing cellulose to obtain a cellulose derivative; preparing the cellulose derivative into a solution, pouring the solution into a coagulation bath, forming a gel, and pulverizing the gel by means of a homogenizer, colloid mill, or ball mill to obtain a cellulose derivative hydrogel;

[0024] Scheme II: preparing a cellulose solution; pouring the cellulose solution into a coagulation bath, forming a gel, and pulverizing the gel by means of a homogenizer, colloid mill, or ball mill to obtain a cellulose hydrogel, and derivatizing the cellulose hydrogel to form a cellulose derivative hydrogel.

[0025] According to embodiments of the present application, the cellulose derivative includes, but is not limited to, at least one of cellulose ester, cellulose ether, oxidized cellulose, charged cellulose, or other types of cellulose derivatives.

[0026] According to some embodiments of the present application, the cellulose ester includes, but is not limited to, cellulose monoester or cellulose mixed ester.

[0027] According to some embodiments of the present application, the cellulose monoester includes, but is not limited to, cellulose organic acid ester or cellulose inorganic acid ester.

[0028] According to some embodiments of the present application, the cellulose organic acid ester includes, but is not limited to, at least one of cellulose methyl ester, cellulose ethyl ester, cellulose propyl ester, cellulose butyl ester, saturated or unsaturated fatty acid ester such as cellulose octadecyl ester, aromatic ester such as cellulose benzoate, cellulose lactate, cellulose citrate, cellulose carbamate, cellulose cyclohexyl carboxylate, cellulose cinnamate, or cellulose 2-methylbenzoate.

[0029] According to some embodiments of the present application, the cellulose inorganic acid ester includes, but is not limited to, at least one of cellulose phosphate ester, cellulose sulfate ester, or cellulose nitrate ester.

[0030] According to some embodiments of the present application, the cellulose mixed ester includes, but is not limited to, at least one of cellulose acetate butyrate CAB, cellulose acetate propionate CAP, cellulose 2-methylbenzoate mixed ester, cellulose 2-methylbenzoate-4-trifluoromethylbenzoate mixed ester, or cellulose acetate adamantane carboxylic acid mixed ester, and the like.

[0031] According to some embodiments of the present application, the cellulose ether includes, but is not limited to, at least one of cellulose monoether or cellulose mixed ether.

[0032] According to some embodiments of the present application, the cellulose ether includes, but is not limited to, at least one of methyl cellulose MC, ethyl cellulose AC, propyl cellulose PC, or carboxymethyl cellulose CMC, and the like.

[0033] According to some embodiments of the present application, the cellulose mixed ether includes, but is not limited to, at least one of hydroxymethyl cellulose HMC, hydroxypropyl cellulose HPC, or hydroxypropyl methyl cellulose HPMC, and the like.

[0034] According to some embodiments of the present application, the oxidized cellulose includes, but is not limited to, at least one of dialdehyde cellulose, diol cellulose, or dicarboxyl cellulose, and the like.

[0035] According to some embodiments of the present application, the charged cellulose includes, but is not limited to, at least one of cationic cellulose or anionic cellulose and salts thereof, and the like.

[0036] According to some embodiments of the present application, the cationic cellulose includes, but is not limited to, at least one of quaternary ammonium group cellulose, imidazole group cellulose, or pyridine group cellulose, and the like.

[0037] According to some embodiments of the present application, the anionic cellulose includes, but is not limited to, at least one of cellulose containing carboxyl, hydroxide, sulfate, or phosphate, and the like.

[0038] According to some embodiments of the present application, the salt in the anionic cellulose salt includes at least one of sodium salt, potassium salt, calcium salt, magnesium salt, iron salt, or ammonium salt, and the like.

[0039] According to some embodiments of the present application, the other cellulose derivative includes, but is not limited to, at least one of DOPO structure containing cellulose, alkylated cellulose, aminated cellulose, chlorinated cellulose, amidated cellulose, silanized cellulose, or plasma modified cellulose, and the like.

[0040] According to some embodiments of the present application, the cellulose derivative is, for example, selected from methyl cellulose, cellulose propionate, oxidized cellulose-dialdehyde cellulose, or cellulose acrylate.

[0041] According to some embodiments of the present application, the cellulose derivative is selected from the group consisting of methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, carboxymethyl cellulose, cellulose propionate, cellulose butyrate, cellulose benzoate, cellulose cyclohexylformate, cellulose cinnamate, cellulose 2-methylbenzoate, cellulose acetate 2-methylbenzoate mixed ester, cellulose 2-methylbenzoate-4-trifluoromethylbenzoate mixed ester, cellulose adamantane carboxylic acid acetate mixed ester, oxidized cellulose-dialdehyde cellulose, or DOPO (9,10-dihydro-9-oxa-10-phosphaphenanthrene-10-oxide) cellulose derivative.

[0042] The present application also provides a method for preparing the above-mentioned cellulose derivative-based hydrogel, which is selected from the following Scheme I or Scheme II:

[0043] Scheme I: homogeneously or heterogeneously derivatizing cellulose to obtain a cellulose derivative; preparing the cellulose derivative into a solution, pouring into a coagulation bath to form a gel, and pulverizing the gel by means of a homogenizer, colloid mill or ball mill to obtain a cellulose derivative hydrogel.

[0044] Scheme II: preparing a cellulose solution; pouring the cellulose solution into a coagulation bath to form a gel, and pulverizing the gel by means of a homogenizer, colloid mill or ball mill to obtain a cellulose hydrogel; derivatizing the cellulose hydrogel to obtain a cellulose derivative hydrogel.

[0045] According to embodiments of the present application, the coagulation bath in Scheme I or Scheme II can use solvents such as water, ethanol, acetone, ethylene glycol, etc.

[0046] According to embodiments of the present application, Scheme I specifically includes:

[0047] 1) cellulose pretreatment;

[0048] 2) homogeneously or heterogeneously derivatizing the pretreated cellulose in step 1) to obtain a cellulose derivative;

[0049] 3) preparing the cellulose derivative in step 2) into a solution, pouring into a coagulation bath to form a gel, and pulverizing the gel by means of a homogenizer, colloid mill or ball mill to obtain a cellulose derivative hydrogel.

[0050] According to embodiments of the present application, in step 1), the cellulose pretreatment includes dispersing cellulose in a solvent, which can be water, DMF, ethanol or isopropanol, etc., and the cellulose can be microcrystalline cellulose, cotton pulp, wood pulp, inulin, soluble starch or dextran, etc., preferably wood pulp; or the cellulose pretreatment includes adding cellulose into a cellulose solvent to form a cellulose solution.

[0051] According to an embodiment of the present application, step 1) further comprises activating the cellulose, specifically, adding a certain amount of alkali to activate the cellulose. Exemplarily, the alkali can be sodium hydroxide, potassium hydroxide, sodium carbonate, sodium borohydride, or the like.

[0052] According to an embodiment of the present application, in step 3), the solvent used for preparing the solution can be water, ethanol, acetone, DMF, or aqueous NaOH, or the like.

[0053] According to an embodiment of the present application, the scheme two specifically comprises:

[0054] 1') preparing a cellulose solution;

[0055] 2') pouring the cellulose solution obtained in step 1') into a coagulation bath to form a gel, and crushing the gel by means of a homogenizer, a colloid mill, or a ball mill, or the like to obtain a cellulose gel;

[0056] 3') subjecting the cellulose gel of step 2') to a derivatization reaction to obtain a cellulose derivative hydrogel.

[0057] According to an embodiment of the present application, in step 1'), the cellulose is dissolved in a cellulose solvent to obtain the cellulose solution.

[0058] According to the present application, in the scheme one and the scheme two, the cellulose solvent can be selected from any excellent solvent known in the art which can dissolve (the dissolution includes complete dissolution and partial dissolution) the cellulose. Preferably, it can be selected from one or more of the following systems: a copper ammonia solution, a copper ethylenediamine solution, an organic solvent, an ionic liquid, a mixed solvent of an ionic liquid and an organic solvent, a choline type ionic liquid eutectic solvent system, an organic solvent / salt system, an amine oxide system (NMMO), a carbamate system, an alkali / water system, an alkali / urea system, an alkali / thiourea system, liquid ammonia / NH4SCN, an organic acid, a metal salt aqueous solution, an alcohol solution of a metal salt hydrate, a water-alcohol mixed solution of a metal salt hydrate, and the like.

[0059] The organic solvent can be selected from one or more of N,N-dimethyl sulfoxide (DMSO), N,N-dimethyl formamide (DMF), N,N-dimethyl acetamide (DMAc), N-methyl-2-pyrrolidone (NMP), N-methyl imidazole, imidazole, pyridine, ethylenediamine, hexafluoroacetone, hexafluoroisopropanol, glycerol, methyl isobutyl ketone, tetrahydrofuran, dioxane, and γ-valerolactone (GVL).

[0060] The organic solvent / salt system can be selected from one or more of the following: N,N-dimethylacetamide / lithium chloride (DMAc / LiCl) system, N-methyl-2-pyrrolidone / NMP, N,N-dimethyl sulfoxide / tetrabutyl ammonium fluoride system (DMSO / TBAF).

[0061] The base / water system can be selected from one or both of NaOH / H2O and KOH / H2O.

[0062] The base / urea system can be selected from NaOH / Urea.

[0063] The base / thio-urea system can be selected from NaOH / thio-urea.

[0064] The organic acid can be selected from one or more of the following: formic acid, acetic acid, propionic acid, butyric acid, succinic acid, lactic acid, glutamic acid, glycine, dichloroacetic acid, trichloroacetic acid, benzene sulfonic acid, etc.

[0065] The metal salt aqueous solution can be selected from an aqueous solution of CaCl2, ZnCl2, LiClO4, Ca(SCN)2, LiSCN, etc.

[0066] The metal salt hydrate alcohol solution can be selected from a methanol solution of CaBr2-H2O, a methanol solution of CaCl2-2H2O.

[0067] The metal salt hydrate aqueous alcohol solution can be selected from a methanol aqueous solution of CaBr2-H2O, a methanol aqueous solution of CaCl2-2H2O.

[0068] The amine oxide system can be NMMO / H2O / DMSO system, NMMO / H2O / diethyl triamine, NMMO / H2O system.

[0069] The ionic liquid can be selected from an organic molten salt formed by a cation and an anion, and having a melting point lower than 100°C, and preferably an organic molten salt capable of dissolving the natural macromolecule of the biomass.

[0070] For example, the cation of the ionic liquid can be selected from one or more of the following: substituted or unsubstituted imidazole, pyridine, pyrrole, amine, phosphine, choline, diazabicyclo, amino acid type cation; for example, the substituent can be one or more of the following: C 1-6 alkyl, C 1-6 alkenyl, phenyl or substituted phenyl; preferably one or more of the following: methyl, ethyl, butyl, allyl;

[0071] Preferably, the cation is selected from one or more of the following: 1-ethyl-3- methylimidazolium cation ([EMIM]), 3-methylimidazolium cation ([MIM]), 1- propyl-3-methylimidazolium cation ([PMIM]), 1-allyl-3-methylimidazolium cation ([AMIM]), 1-butyl-3-methylimidazolium cation ([BMIM]), 1-butyl-2,3- dimethylimidazolium cation ([BMMIM]), 1,3-dimethylimidazolium cation ([MMIM]), 1-methoxyethyl-3-methylimidazolium cation ([MeOEMIM]), 1- methoxymethyl-3-methylimidazolium cation ([MeOMMIM]), 1-hydroxy-3- methyl-imidazolium cation ([HMIM]), 1-(2-hydroxyethyl)-3-methylimidazolium cation ([HOEMIM]), 1-methyl-3-benzylimidazolium cation ([MBzIM]), 1- pentyl-3-methylimidazolium cation ([PeMIM]), 1-benzyl-3-methylimidazolium cation ([BzMIM]), 1-m-methoxybenzyl-3-methylimidazolium cation ([MeOBzMIM]), 1-m-methylbenzyl-3-methylimidazolium cation ([MeBzMIM]), N- methylimidazolium cation ([MPyr]), N-ethylpyridinium cation ([EPyr]), N- butylpyridinium cation ([BPyr]), N-n-hexylpyridinium cation ([HPyr]), 1-butyl-3- methylpyrrolidinium ion ([BMPyrr]), tris(2-hydroxyethyl)methylamine ([THEMA]), tetrabutylammonium ([TBA]), tetrabutylphosphonium ([PBu4]), glycine cation ([Gly]), choline cation ([Ch]), 1,5-diazabicyclo[4.3.0]non-5-ene ([DBNH]), and the like.

[0072] More preferably, the cation is selected from one or more of the following: 1-ethyl-3- methylimidazolium cation ([EMIM]), 1-allyl-3-methylimidazolium cation ([AMIM]), 1-butyl-3-methylimidazolium cation ([BMIM]), choline cation ([Ch]).

[0073] For example, the anion is selected from one or more of the following: halide anion, organic acid anion, organic acid ester anion, amino acid type anion, and the like.

[0074] Preferably, the anion is selected from one or more of the following anions: chloride ([CI]), bromide ([Br]), fluoride ([F]), formate ([HCOO]), acetate ([CH3COO] or [Ac]), glycolate ([HOCH2COO]), propionate ([CH3CH2COO] or [OPr]), butyrate ([CH3CH2CH2COO] or [OBu]), octoate ([Oct]), benzoate ([C6H5COO] or [PhCOO]), lactate ([CH3CH(OH)COO] or [Lac]), thioglycolate ([HSCH2COO]), hexafluorophosphate ([PF6]), triflate ([BF3]), methylphosphonate ([ (MeO)HPO2] or [MP]), dimethylphosphonate ([ (MeO)2PO2] or [DMP]), diethylphosphonate ([ (EtO)2PO2] or [DEP]), methylsulfonate ([MeOSO3]), trifluoromethylsulfonate ([CF3SO3]), glycinate ([Gly]), lysinate ([Lys]), valinate ([Val]), dicyanamide ([N(CN)2] or [DCA]), bistrifluoromethylsulfonimide ([Tf2N]), and the like.

[0075] More preferably, the anion is selected from one or more of the following anions: chloride ([CI]), formate ([HCOO]), acetate ([Ac]), methylphosphonate ([ (MeO)HPO2] or [MP]), dimethylphosphonate ([ (MeO)2PO2] or [DMP]), and dicyanamide ([N(CN)2] or [DCA]).

[0076] According to embodiments of the present application, the ionic liquid can be selected from one or more of the following ionic liquids: 1-ethyl-3-methylimidazolium chloride ionic liquid ([EMIM][CI]), 1-ethyl-3-methylimidazolium bromide ionic liquid ([EMIM][Br]), 1-ethyl-3-methylimidazolium formate ionic liquid ([EMIM][HCOO]), 1-ethyl-3-methylimidazolium acetate ionic liquid ([EMIM][Ac]), 1-ethyl-3-methylimidazolium octanoate ionic liquid ([EMIM][Oct]), 1-ethyl-3-methylimidazolium methylphosphate ionic liquid ([EMIM][MP]), 1-ethyl-3-methylimidazolium dimethylphosphate ionic liquid ([EMIM][DMP]), 1-ethyl-3-methylimidazolium diethylphosphate ionic liquid ([EMIM][DEP]), 1-ethyl-3-methylimidazolium propionate ionic liquid ([EMIM][OPr]), 1-ethyl-3-methylimidazolium terephthalate ionic liquid ([EMIM][OBu]), 1-ethyl-3-methylimidazolium glycinate ionic liquid ([EMIM][Gly]), 1-ethyl-3-methylimidazolium lysinate ionic liquid ([EMIM][Lys]), 1-allyl-3-methylimidazolium chloride ionic liquid ([AMIM][CI]), 1-allyl-3-methylimidazolium bromide ionic liquid ([AMIM][Br]), 1-allyl-3-methylimidazolium formate ionic liquid ([AMIM][HCOO]), 1-allyl-3-methylimidazolium acetate ionic liquid ([AMIM][Ac]), 1-butyl-3-methylimidazolium chloride ionic liquid ([BMIM][CI]), 1-butyl-3-methylimidazolium bromide ionic liquid ([BMIM][Br]), 1-butyl-3-methylimidazolium formate ionic liquid ([BMIM][HCOO]), 1-butyl-3-methylimidazolium acetate ionic liquid ([BMIM][Ac]), 1-butyl-3-methylimidazolium glycolate ionic liquid ([BMIM][HOCH2COO]), 1-butyl-3-methylimidazolium propionate ionic liquid ([BMIM][CH3CH2COO]), 1-butyl-3-methylimidazolium lactate ionic liquid [BMIM][Lac], 1-butyl-3-methylimidazolium butyrate ionic liquid ([BMIM][CH3CH2CH2COO]), 1-butyl-3-methylimidazolium benzoate ionic liquid ([BMIM][C6H5COO]), 1-butyl-3-methylimidazolium glycinate ionic liquid ([BMIM][H2NCH2COO]), 1-butyl-3-methylimidazolium dicyanamide ionic liquid ([BMIM][N(CN)2]), 1-butyl-3-methylimidazolium bistrifluoromethylsulfonylimide ionic liquid ([BMIM][Tf2N]),1 -butyl-3-methylimidazolium hexafluorophosphate ionic liquid ([BMIM][PF6]), 1 -butyl-3-methylimidazolium tetrafluoroborate ionic liquid ([BMIM][BF4]), 1 -butyl-3-methylimidazolium methylsulfonate ionic liquid ([BMIM][MeOSO3]), 1 -butyl-3-methylimidazolium trifluoromethylsulfonate ionic liquid ([BMIM][CF3SO3]), 1 -butyl-2,3-dimethylimidazolium tetrafluoroborate ionic liquid ([BMMIM][BF4]), 3-methylimidazolium formate ionic liquid ([MIM][HCOO]), 1,3-dimethylimidazolium chloride ionic liquid ([MMIM][CI]), 1,3-dimethylimidazolium methylphosphonate ionic liquid ([MMIM][MP]), 1,3-dimethylimidazolium dimethylphosphonate ionic liquid ([MMIM][DMP]), 1,3-dimethylimidazolium methylsulfonate ionic liquid ([MMIM][MeOSO3]), 1 -hydroxy-3-methyl-imidazolium chloride ionic liquid ([HMIM][CI]), 1 -hydroxy-3-methyl-imidazolium trifluoromethylsulfonate ionic liquid ([HMIM][CF3SO3]), 1 -(2-hydroxyethyl)-3-methylimidazolium chloride ionic liquid ([HOEMIM][CI]), 1 -methoxymethyl-3-methylimidazolium bromide ionic liquid ([MeOMMIM][Br]), 1 -methoxyethyl-3-methylimidazolium bromide ionic liquid ([MeOEMIM][Br]), N-ethylpyridinium chloride ionic liquid ([EPyr][CI]), N-ethylpyridinium bromide ionic liquid ([EPyr][Br]), N-methylpyridinium formate ionic liquid ([MPyr][HCOO]), tris(2-hydroxyethyl)methylammonium acetate ionic liquid ([THEMA][Ac]), tris(2-hydroxyethyl)methylammonium methylsulfonate ionic liquid ([THEMA][MeOSO3]), tris(2-hydroxyethyl)methylammonium trifluoromethylsulfonate ionic liquid [THEMA][CF3SO3], tetrabutylphosphonium valinate ionic liquid [PBu4][Val], tetrabutylphosphonium lysinate ionic liquid [PBu4][Lys], tetrabutylphosphonium glycinate ionic liquid [PBu4][Gly], 1 -benzyl-3-methylimidazolium chloride ionic liquid ([BzMIM][CI]), 1 -benzyl-3-methylimidazolium dicyanamide ionic liquid ([BzMIM][DCA]), 1 -m-methylbenzyl-3-methylimidazolium chloride ionic liquid ([MeBzMIM][CI]), 1 -m-methoxybenzyl-3-methylimidazolium chloride ionic liquid ([MeOBzMIM][CI]), choline chloride ionic liquid ([Ch][CI]), choline bromide ionic liquid ([Ch][Br]), choline acetate ionic liquid ([Ch][CH3COO]),ionic liquid of propionic acid choline ([Ch][CH3CH2COO]), ionic liquid of butyric acid choline ([Ch][CH3CH2CH2COO]), ionic liquid of glycine hydrochloride ([Gly][Cl]), ionic liquid of 1,5-diazabicyclo[4.3.0]non-5-ene acetate ([DBNH][Ac]), and the like.

[0077] Preferably, the ionic liquid is selected from 1-allyl-3-methylimidazolium propionate ([Amim][CH3CH2COO]), 1-ethyl-3-methylimidazolium butyrate ([Emim][CH3CH2CH2COO]), 1-butyl-3-methylimidazolium cyclohexanecarboxylate ([Bmim][ChCOO]), 1-butyl-3-methylimidazolium cinnamate ([Bmim][CCOO]), 1-butyl-3-methylimidazolium chloride (BmimCl), 1-allyl-3-methylimidazolium chloride (AmimCl), 1-allyl-3-methylimidazolium chloride (AmimCl) / N,N-dimethylacetamide (DMAc) (mass ratio of AmimCl to DMAc is 9:1), 1-ethyl-3-methylimidazolium chloride (EmimCl) / 1-methylimidazole (Mim) (mass ratio of EmimCl to Mim is 8:2).

[0078] Preferably, the choline-based deep eutectic solvent system is selected from one or more of [Ch][Cl] / urea, [Ch][Br] / urea, [Ch][Cl] / thio-urea, [Ch][Cl] / glycerol, [Ch][Cl] / lactic acid.

[0079] Preferably, the solvent system for dissolving cellulose is selected from the ionic liquid and / or NaOH / Urea system; more preferably, the ionic liquid for dissolving cellulose is selected from one or more of [AMIM][Cl], [BMIM][Cl], [EMIM][Ac], [BMIM][Ac].

[0080] The present application also provides a culture solution for 3D cell culture, which comprises the above-mentioned cellulose derivative-based hydrogel and a basic culture solution.

[0081] According to the embodiments of the present application, the basic culture solution can be selected from the basic culture solutions known in the art. Exemplarily, the basic culture solution comprises DMEM / F12 (GIBCO / 10565018), 10% FBS (GIBCO / 10091-148), and 1% P / S (GIBCO / 15140122).

[0082] The application also provides a kit comprising the above-mentioned 3D cell culture medium and instructions for use.

[0083] The application also provides a preparation method of the above-mentioned culture medium, comprising the following steps:

[0084] S1: preparing the above-mentioned cellulose derivative-based hydrogel;

[0085] S2: mixing the cellulose derivative-based hydrogel in step S1 with the base culture medium to prepare the above-mentioned 3D cell culture medium.

[0086] According to an embodiment of the application, in step S2, the volume ratio of the cellulose derivative-based hydrogel to the base culture medium is 1-10:1-10, for example 1:1, 1:5, 1:10, 5:1 or 10:1.

[0087] The application also provides the use of the above-mentioned culture medium in 3D cell culture.

[0088] The application also provides a 3D cell culture method, which is performed in the above-mentioned 3D cell culture medium.

[0089] According to an embodiment of the application, the 3D culture method comprises the following steps:

[0090] a) preparing a cell suspension: uniformly mixing the 3D cell culture medium with cells to obtain a cell suspension;

[0091] b) 3D culture: culturing the cell suspension of step a) in an incubator.

[0092] According to an embodiment of the application, in step a), the cells can also be subjected to cell resuscitation treatment and / or cell passage treatment.

[0093] Preferably, the cell resuscitation can be performed by using methods known in the art as long as the cells can be resuscitated, for example, according to the quick thawing principle for cell resuscitation. Illustratively, the cell resuscitation specifically comprises: placing the cells to be resuscitated in a 37℃ water bath, after centrifugation, resuspending the cells with the base culture medium, and then culturing in an incubator (37℃, 5% CO2), for example, for 48h.

[0094] Preferably, the cell passage can be performed by using methods known in the art as long as the desired cells can be obtained. Illustratively, the resuscitated cells are discarded, PBS is added to the culture well and gently washed twice, then a single cell suspension is prepared by using trypsin digestion, the cells are resuspended with the base culture medium and adjusted to the desired cell density (for example, 5K, 25K, 50K...).

[0095] According to an embodiment of the present application, the cells include human neuroblastoma cells, such as SH-SY5Y cells, liver cancer cells, such as HepaRG (Hepatocarcinoma-derived cell line), and the like.

[0096] According to an embodiment of the present application, in step b), the culturing can be performed by using methods known in the art, as long as the desired cells can be obtained, for example, by adding the cell suspension into a culture plate and culturing at 37°C under 5% CO2.

[0097] According to an embodiment of the present application, in step b), the culturing is performed for 1 day to 10 days, preferably for 3 days.

[0098] According to an embodiment of the present application, in step b), the culturing further includes medium replacement, for example, replacing the basal medium every 3 days.

[0099] The present application also provides a culture medium for culturing tissues and organoids, which includes the above-mentioned cellulose derivative-based hydrogel and a complex culture medium.

[0100] According to an embodiment of the present application, the complex culture medium includes a basal medium and an active component.

[0101] According to an embodiment of the present application, the mass ratio of the basal medium to the active component in 100 mL of the complex culture medium is 80-99: 1-20, for example, 97:3.

[0102] According to an embodiment of the present application, the basal medium can be selected from basal media known in the art. Illustratively, the basal medium includes DMEM / F12 (GIBCO / 10565018), 10% FBS (GIBCO / 10091-148), and 1% P / S (GIBCO / 15140122).

[0103] According to an embodiment of the present application, the complex culture medium contains Epirregulin at 100-500 ng / ml, for example, 200 ng / ml, 300 ng / ml, 400 ng / ml, or 500 ng / ml.

[0104] According to an embodiment of the present application, the active component in 100 mL of the complex culture medium at least includes:

[0105] penicillin-streptomycin (P / S) 0.1-2% (for example, 1%);

[0106] N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES) 1-20 mM (for example, 10 mM);

[0107] L-glutamine supplement (Glutamax) 0.1-2% (e.g. 1%);

[0108] Neuronal cell culture supplement (B27) 0.1-2% (e.g. 1%);

[0109] N-acetylcysteine 0.1-5 mM (e.g. 1.25 mM);

[0110] Fibroblast growth factor (FGF-7) 10-100 ng / ml (e.g. 50 ng / ml);

[0111] Noggin 50-200 ng / ml (e.g. 100 ng / ml);

[0112] R-spondin 1 100-1000 ng / ml (e.g. 500 ng / ml);

[0113] Wnt 3A 50-200 ng / ml (e.g. 100 ng / ml);

[0114] FGF 10 100-500 ng / ml (e.g. 200 ng / ml);

[0115] Gastrin 0.1-10 nM (e.g. 1 nM);

[0116] ALK inhibitor (A-83-01) 0.1-10 μΜ (e.g. 2 μΜ);

[0117] ROCK inhibitor (Y-27632) 1-20 μΜ (e.g. 10 μΜ);

[0118] p38 MAPK inhibitor (SB202190) 1-20 μΜ (e.g. 10 μΜ);

[0119] Epirregulin 100-500 ng / ml (e.g. 200 ng / ml);

[0120] Capsanthin 100-500 ng / ml (e.g. 200 ng / ml);

[0121] Nicotinamide 1-20 mM (e.g. 10 mM).

[0122] According to an exemplary aspect of the present application, the complex culture solution comprises:

[0123] P / S 1%

[0124] HEPES 10mM

[0125] Glutamax 1%

[0126] B27 1%

[0127] N-Acetylcysteine 1.25mM

[0128] FGF-7 50ng / ml

[0129] Noggin 100ng / ml

[0130] R-spondin 1 500ng / ml

[0131] Wnt 3A 100ng / ml

[0132] FGF10 200ng / ml

[0133] Gastrin 1nM

[0134] A-83-01 2μM

[0135] Y-27632 10μM

[0136] SB202190 10μM

[0137] Epirregulin 200ng / ml

[0138] Capsanthin 200ng / ml

[0139] Nicotinamide 10mM.

[0140] The present application also provides a preparation method of the culture solution for culturing the tissue and organoid, comprising the following steps:

[0141] S1': preparing the above-mentioned cellulose derivative-based hydrogel;

[0142] S2': mixing the cellulose derivative-based hydrogel in step S1' with the complex culture solution to prepare the culture solution for culturing the tissue and organoid.

[0143] According to an embodiment of the present application, in step S2', the volume ratio of the cellulose derivative-based hydrogel to the complex culture solution is 1-10: 1-10, for example, 1:1.

[0144] The present application also provides the application of the above-mentioned culture solution for culturing the tissue and organoid in culturing the tissue or organoid.

[0145] The present application also provides a culture method of a tissue or an organoid, which is carried out in the culture solution for culturing tissues and organoids.

[0146] According to an embodiment of the present application, the tissue is preferably human gastric cancer tissue / normal gastric tissue, human intestinal cancer tissue / normal intestinal tissue.

[0147] According to an embodiment of the present application, the organoid is preferably human gastric cancer organoid / normal gastric organoid, human intestinal cancer organoid / normal intestinal organoid.

[0148] According to an embodiment of the present application, the culture method comprises the following steps:

[0149] a') preparing a cell suspension of a tissue or an organoid: mixing the culture solution for culturing tissues and organoids with cells of a tissue or an organoid uniformly to obtain a cell suspension;

[0150] b') 3D culture: culturing the cell suspension of step a') in an incubator.

[0151] According to an embodiment of the present application, in step a'), the cells of a tissue or an organoid can be prepared by methods known in the art, which are not specifically limited in the present application.

[0152] According to an embodiment of the present application, in step b'), the culture can be carried out by methods known in the art, as long as the desired cells can be obtained, for example, the cell suspension is added to a culture plate and cultured at 37°C under 5% CO2.

[0153] According to an embodiment of the present application, in step b'), the culture is carried out for 1 day to 10 days, preferably for 3 days.

[0154] According to an embodiment of the present application, in step b'), the culture further comprises medium replacement, for example, the basic culture solution is replaced every 3 days.

[0155] The present application also provides a kit comprising the culture solution for culturing tissues and organoids, the complex culture solution and instructions for use.

[0156] According to an embodiment of the present application, the kit is preferably used for culturing tissues and organoids. Specifically, the tissue is preferably human gastric cancer tissue / normal gastric tissue, human intestinal cancer tissue / normal intestinal tissue. Specifically, the organoid is preferably human gastric cancer organoid / normal gastric organoid, human intestinal cancer organoid / normal intestinal organoid.

[0157] According to an embodiment of the present application, the complex culture solution contains Epirregulin 100-500 ng / ml, for example 200 ng / ml, 300 ng / ml, 400 ng / ml, 500 ng / ml.

[0158] The present application also provides a culture solution for stem cell culture, which comprises the above-mentioned cellulose derivative-based hydrogel and a basic culture solution.

[0159] According to an embodiment of the present application, the basic culture solution can be selected from the basic culture solutions known in the art. Exemplarily, the basic culture solution comprises DMEM / F12, mTeSR TM 1.

[0160] The present application also provides the use of the above-mentioned culture solution for stem cell culture in stem cell culture.

[0161] The present application also provides a method for stem cell culture, which uses the above-mentioned culture solution for stem cell culture.

[0162] According to an embodiment of the present application, the stem cell is preferably an adult stem cell or an embryonic stem cell (ESC) from mammals and primates; further preferably a pluripotent stem cell or a multipotent stem cell, such as an induced pluripotent stem cell (iPSC), a hematopoietic stem cell, a neural stem cell, a skin stem cell, a mesenchymal stem cell, an adipose stem cell, an osteogenic stem cell, a chondrogenic stem cell, a muscle stem cell, a liver stem cell, a pancreatic stem cell, an endothelial stem cell, a corneal stem cell, a hair follicle stem cell, a gastrointestinal stem cell, a mammary stem cell, a cardiac stem cell, etc.

[0163] According to an embodiment of the present application, the method for stem cell culture comprises the following steps:

[0164] a”) pretreatment before stem cell passage, dilution of cellulose derivative-based hydrogel, and standing in an incubator;

[0165] b”) digestion of extracellular matrix, digestion of extracellular matrix using Dispase;

[0166] c”) addition of mTeSR TM 1, blowing and beating the stem cells to fall off and decompose into fragments;

[0167] d”) addition of stem cell fragments to the cellulose derivative-based hydrogel culture dish for culture.

[0168] According to an embodiment of the present application, in step a”), DMEM / F12 is used to dilute the cellulose derivative-based hydrogel, which is added to the culture plate after dilution and is allowed to stand at 37°C for at least 1 hour.

[0169] According to embodiments of the present application, in step b"), the digestion using Dispase can be optionally performed using methods known in the art, for example, by covering the well plate with Dispase and placing it in a 37°C incubator until the edges of the stem cells begin to lift.

[0170] According to embodiments of the present application, in step b"), the digestion using Dispase can be optionally performed using methods known in the art, for example, by covering the well plate with Dispase and placing it in a 37°C incubator until the edges of the stem cells begin to lift.

[0171] According to embodiments of the present application, in step c"), the fragments are 1-2 mm.

[0172] According to embodiments of the present application, in step d"), the culturing can be optionally performed using methods known in the art, for example, including using mTeSR TM 1Culture medium is supplemented and incubated at 37°C, 5% CO2, and the like.

[0173] The present application also provides a culture medium for inducing differentiation of stem cells into organoids, the culture medium comprising the above-mentioned cellulose derivative-based hydrogel, a definitive endoderm differentiation culture medium, an MH differentiation culture medium, and an organoid growth culture medium.

[0174] According to embodiments of the present application, the definitive endoderm differentiation culture medium comprises the following components:

[0175] RPMI1640, containing L-glutamine, penicillin-streptomycin, and Activin A.

[0176] According to embodiments of the present application, the definitive endoderm differentiation culture medium preferably comprises the following components:

[0177] RPMI1640, containing L-glutamine 2mM,

[0178] penicillin-streptomycin 100U / ml-100g / ml, and Activin A 100ng / ml.

[0179] According to embodiments of the present application, the MH differentiation culture medium comprises the following components:

[0180] RPMI1640 containing 2% FBS, containing L-glutamine, penicillin-streptomycin, and FGF4.

[0181] According to embodiments of the present application, the MH differentiation culture medium preferably comprises the following components:

[0182] RPMI1640 containing 2% FBS, containing L-glutamine 2mM, penicillin-streptomycin 100U / ml-100g / ml, and FGF4 500ng / ml.

[0183] According to an embodiment of the application, the organoid growth medium comprises the following ingredients:

[0184] Advanced DMEM / F12 with 1 x B27, with L-glutamine, 2 mM, penicillin-streptomycin, 100 U / ml - 100 g / ml, HEPES buffer, 15 mM, and R-spondin 1, 500 ng / ml.

[0185] According to an embodiment of the application, the organoid growth medium comprises the following ingredients:

[0186] Advanced DMEM / F12 with 1 x B27, with L-glutamine, 2 mM, penicillin-streptomycin, 100 U / ml - 100 g / ml, HEPES buffer, 15 mM, and R-spondin 1, 500 ng / ml.

[0187] The application also provides the use of the above-mentioned stem cell induced differentiated organoid culture medium for stem cell induced differentiated organoids.

[0188] The application also provides a method for stem cell induced differentiated organoids, wherein the method comprises culturing a single stem cell or a population of stem cells in the stem cell induced differentiated organoid culture medium.

[0189] According to an embodiment of the application, the method for stem cell induced differentiated organoids comprises the following steps:

[0190] a'') iPSC / ES differentiation in monolayer culture, comprising definitive endoderm differentiation and mid / hindgut (MH) differentiation;

[0191] b'') organoid culture, comprising the formulation of an organoid growth medium, and incubation after mixing the cellulose derivative hydrogel with the spheroids.

[0192] According to an embodiment of the application, the definitive endoderm differentiation in step a'') comprises the formulation of a definitive endoderm differentiation medium and the definitive endoderm differentiation, wherein the definitive endoderm differentiation step comprises incubation using the definitive endoderm differentiation medium with methods known in the art, for example incubation at 37°C, 5% C02 and 95% humidity.

[0193] According to an embodiment of the application, the mid / hindgut (MH) differentiation in step a'') comprises the formulation of a MH differentiation medium and the mid / hindgut (MH) differentiation, wherein the mid / hindgut (MH) differentiation step comprises incubation using the MH differentiation medium with methods known in the art, for example incubation at 37°C, 5% C02 and 95% humidity, medium change and observation of the spheroids every 24 hours, spheroids embedded.

[0194] According to an embodiment of the present application, the cellulose derivative-based hydrogel in step b”’) is mixed with the spheres and then incubated by using methods known in the art, for example, including: incubation at 37℃ for 30 minutes, adding organoid growth medium and continuing incubation at 37℃, 5% CO2, 95% humidity, and subculturing according to the growth of the organoids.

[0195] The present application also provides a stem cell organoid obtained by the above method.

[0196] Specifically, the stem cell organoid includes an adult stem cell organoid, an induced pluripotent stem cell organoid, or an embryonic stem cell organoid.

[0197] The present application also provides an induced differentiation organoid kit, which includes the above cellulose derivative-based hydrogel.

[0198] According to an embodiment of the present application, the organoid is preferably an intestinal organoid, a gastric organoid, a liver organoid, and further preferably a small intestinal organoid.

[0199] According to an embodiment of the present application, the induced differentiation organoid kit further includes a definitive endoderm differentiation medium, an MH differentiation medium, and an organoid growth medium.

[0200] According to an embodiment of the present application, the induced differentiation organoid kit further includes components or assemblies known in the art, which are not limited in the present application.

[0201] The present application also provides an application of the induced differentiation organoid kit in inducing differentiation of an organoid.

[0202] According to an embodiment of the present application, the induced differentiation organoid is, for example, an induced differentiation intestinal organoid, a gastric organoid, a liver organoid, and preferably a small intestinal organoid.

[0203] The present application has the following advantages:

[0204] The present application uses cellulose derivatives as raw materials to prepare cellulose derivative-based hydrogels, which are ready-to-use hydrogels without animal-derived components. Specifically, the matrix of the hydrogel includes cellulose derivatives, and the matrix includes a reticular structure composed of ultrafine cellulose derivative fibers, and the reticular structure includes ordered three-dimensional micro-nano structures. Compared with animal-derived matrix glue, the cellulose derivative-based hydrogel has incomparable advantages in performance, use convenience, batch stability, process amplification stability, automation compatibility, and the like.

[0205] The cellulosic derivative-based hydrogel of the present application has better transparency, which is more conducive to observing the state of cells. It is suitable for 3D cell culture, such as SH-SY5Y and HepaRG cells, and culture of tissues and organoids, such as human gastric cancer tissue / normal gastric tissue, human intestinal cancer tissue / normal intestinal tissue, and preferably human gastric cancer organoid / normal gastric organoid, human intestinal cancer organoid / normal intestinal organoid.

[0206] The cellulosic derivative of the present application has no toxicity to cells, and the cellulosic derivative-based hydrogel has good rigidity, adhesion and porosity as a stem cell growth platform. The stem cells cultured by the cellulosic derivative-based hydrogel of the present application can form clusters. The stem cells and organoids cultured by the cellulosic derivative-based hydrogel of the present application are simple to operate, can form dense confluent endoderm cells, can inhibit the non-directional differentiation of stem cells, and the formed organoid vesicle wall is thick and presents a solid core, which is a differentiated mature organoid, and can be used for high-throughput drug screening, drug toxicity testing and regenerative medicine. BRIEF DESCRIPTION OF DRAWINGS

[0207] Figure 1a is a scanning electron microscope image of methylcellulose MC-1 hydrogel.

[0208] Figure 1a-1 is a microscopic image of gastric cancer organoid cultured for 0 days by methylcellulose hydrogel MC-1 (concentration of 0.2%).

[0209] Figure 1a-2 is a microscopic image of human pluripotent stem cell / embryonic stem cell induced differentiation intestinal organoid after 3 days in Example 7.

[0210] Figure 1b is a microscopic image of methylcellulose hydrogel MC-1 cell suspension after 1 day of culturing SH-SY5Y cells.

[0211] Figure 1b-1 is a microscopic image of gastric cancer organoid cultured for 5 days by methylcellulose hydrogel MC-1 (concentration of 0.2%).

[0212] Figure 1b-2 is a microscopic image of human pluripotent stem cell / embryonic stem cell induced differentiation intestinal organoid after 7 days in Example 7.

[0213] Figure 1c is a microscopic image of methylcellulose hydrogel MC-1 cell suspension after 3 days of culturing SH-SY5Y cells.

[0214] Figure 1c-1 is a microscopic image of gastric cancer organoid cultured for 7 days by methylcellulose hydrogel MC-1 (concentration of 0.2%).

[0215] Figure 1c-2 is a microscopic image of human pluripotent stem cell / embryonic stem cell induced differentiation intestinal organoid after 12 days in Example 7.

[0216] Figure Id Micrograph of methylcellulose hydrogel MC-1 cell suspension after 5 days of culturing SH-SY5Y cells.

[0217] Figure Id-1 Micrograph of cellulose benzoate hydrogel (0.2% concentration) culturing gastric cancer organoids for 0 days.

[0218] Figure Ie Micrograph of methylcellulose hydrogel MC-1 cell suspension after 7 days of culturing SH-SY5Y cells.

[0219] Figure Ie-1 Micrograph of cellulose benzoate hydrogel (0.2% concentration) culturing gastric cancer organoids for 5 days.

[0220] Figure If Micrograph of cellulose benzoate hydrogel (0.2% concentration) culturing gastric cancer organoids for 7 days.

[0221] Figure 2a Micrograph of methylcellulose hydrogel MC-13D culturing SH-SY5Y cells for 5 days.

[0222] Figure 2a-1 Micrograph of methylcellulose hydrogel MC-1 (0.3% concentration) culturing gastric cancer organoids for 5 days.

[0223] Figure 2a-2 Micrograph of iPSCs cultured with 0.8% cellulose derivative in Comparative Example 3.

[0224] Figure 2b Micrograph of plant cellulose matrix gel GrowDex culturing SH-SY5Y cells for 5 days.

[0225] Figure 2b-1 Micrograph of Matrigel culturing gastric cancer organoids for 5 days.

[0226] Figure 2b-2 Micrograph of Matrigel (Corning 354277) culturing iPSCs in Comparative Example 3.

[0227] Figure 2c Micrograph of plant cellulose matrix gel GrowDex culturing gastric cancer organoids for 5 days.

[0228] Figure 3a Micrograph of methylcellulose MC-1 hydrogel 3D culturing HepaRG (liver-cancer cells) for 7 days.

[0229] Figure 3a-1 Micrograph of carboxymethylcellulose hydrogel CMC-1 culturing intestinal cancer organoids for 7 days.

[0230] Figure 3a-2 Micrograph of human pluripotent stem cell / embryonic stem cell induced differentiated intestinal organoid kit containing 0.8% cellulose derivative in Comparative Example 4 culturing human pluripotent stem cell / embryonic stem cell induced differentiated intestinal organoids for 12 days.

[0231] Figure 3b Micrograph of cellulose propionate hydrogel 3D culturing HepaRG (liver-cancer cells) for 7 days.

[0232] Figure 3b-1 Micrograph of intestinal cancer organoid after 7 days of culture in adamantane acetate cellulose mixed ester hydrogel.

[0233] Figure 3b-2 Micrograph of STEMdiff TM Figure 1 Micrograph of human pluripotent stem cell / embryonic stem cell induced differentiated intestinal organoid after 12 days of culture in Intestinal Organoid Kit (stem cell, 05140).

[0234] Figure 3c Micrograph of HepaRG (liver-cancer cell) after 7 days of 3D culture in oxidized cellulose-dialdehyde cellulose hydrogel.

[0235] Figure 3c-1 Micrograph of intestinal cancer organoid after 7 days of culture in oxidized cellulose-dialdehyde cellulose hydrogel.

[0236] Figure 3d Micrograph of HepaRG (liver-cancer cell) after 7 days of 3D culture in cellulose acrylate hydrogel.

[0237] Figure 3d-1 Micrograph of intestinal cancer organoid after 7 days of culture in cellulose acrylate hydrogel.

[0238] Figure 4a Micrograph of intestinal organoid after 12 days of culture in carboxymethyl cellulose CMC-1 hydrogel.

[0239] Figure 4b Micrograph of intestinal organoid after 12 days of culture in adamantane acetate cellulose mixed ester hydrogel.

[0240] Figure 4c Micrograph of intestinal organoid after 12 days of culture in oxidized cellulose-dialdehyde cellulose hydrogel.

[0241] Figure 4d Micrograph of intestinal organoid after 12 days of culture in cellulose acrylate hydrogel. DETAILED DESCRIPTION

[0242] The technical solutions of the present application will be further described below in combination with specific examples. It should be understood that the following examples are only illustratively and explain the present application, and should not be interpreted as limiting the scope of protection of the present application. Any technology realized based on the above description of the present application is covered within the scope of protection intended by the present application.

[0243] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0244] The present application will be further described below through specific examples:

[0245] Preparation Example 1

[0246] (1) Preparation of cellulose ether hydrogel:

[0247] 1) Preparation of methylcellulose MC hydrogel

[0248] 20 g of wood pulp was dispersed in solvent, which could be water, DMF, ethanol, isopropanol. A certain amount of base was added to activate the cellulose, and the activation time was 30 min. The base could be sodium hydroxide, potassium hydroxide, sodium carbonate, sodium borohydride. Then a certain amount of dimethyl sulfate was added, and the molar ratio of dimethyl sulfate to cellulose could be 1:1, 2:1, 3:1, 4:1. The reaction was carried out at room temperature for 24 h. Acetic acid aqueous solution was added to neutralize the base in the reaction solution. The reaction solution was filtered, and the obtained filter cake was washed with solution for 6-8 times to remove residual impurities. The solution that could be used included water, ethanol, acetone, DMF, NaOH aqueous solution. The washed methylcellulose was dispersed in pure water, and the mass ratio of water to methylcellulose was 9:1. Then the gel was crushed, and was crushed to a particle size of 1-50 μm using a colloid mill, high-pressure homogenizer, etc. The solid content was 0.1-3.0 wt%. After high-pressure sterilization, hydrogel MC-1 (see FIG. 1a), MC-2, MC-3, MC-4 were obtained.

[0249] 2) Preparation of ethylcellulose EC hydrogel

[0250] 20 g of wood pulp was dispersed in solvent, which could be water, DMF, ethanol, tetrahydrofuran. A certain amount of base was added to activate the cellulose, and the activation time was 30 min. The base could be sodium hydroxide, potassium hydroxide, sodium carbonate, sodium borohydride. Then a certain amount of ethylation reagent was added, which could be bromoethane, chloroethane, chloroacetyl chloride, diethyl sulfate. The molar ratio of ethylation reagent to cellulose was set to 1:1, 2:1, 3:1, 4:1. The reaction was carried out at room temperature for 24 h. Acetic acid aqueous solution was added to neutralize the base in the reaction solution. The reaction solution was filtered, and the obtained filter cake was washed with solution for 6-8 times to remove residual impurities. The solution that could be used included water, ethanol, acetone, DMF, NaOH aqueous solution. The washed ethylcellulose was dispersed in pure water, and the mass ratio of water to ethylcellulose was 9:1. Then the gel was crushed, and was crushed to a particle size of 1-50 μm using a colloid mill, high-pressure homogenizer, etc. The solid content was 0.1-3.0 wt%. After high-pressure sterilization, hydrogel EC-1, EC-2, EC-3, EC-4 were obtained.

[0251] 3) Preparation of hydroxypropyl cellulose HPC hydrogel

[0252] The 20g wood pulp is dispersed in solvent, which can be water, DMF, ethanol, tetrahydrofuran. A certain amount of base is added to activate the cellulose, the activation time is 2h, the base can be sodium hydroxide, potassium hydroxide, sodium carbonate, sodium borohydride. Then add a certain amount of propylene oxide, set the molar ratio of propylene oxide to cellulose to 1:1, 2:1, 3:1, 4:1, react at 80°C for 8h, after the reaction is completed, cool to room temperature, then add acetic acid aqueous solution for neutralization of the base in the reaction solution. Filter the reaction solution, the obtained filter cake is washed 6-8 times using solution, the solution that can be used is water, ethanol, acetone, DMF, NaOH aqueous solution. The washed hydroxypropyl cellulose is dispersed in pure water, the mass ratio of water to hydroxypropyl cellulose is 9:1, then the gel is crushed, and then crushed to a particle size of 1-50μm using a colloid mill, high-pressure homogenizer, etc., the solid content is 0.1-3.0wt%, and the hydrogel HPC-1, HPC-2, HPC-3, HPC-4 is obtained after high-pressure sterilization.

[0253] 4) Preparation of carboxymethyl cellulose CMC hydrogel

[0254] The 20g wood pulp is dispersed in solvent, which can be water, DMF, ethanol, tetrahydrofuran. A certain amount of base is added to activate the cellulose, the activation time is 2h, the base can be sodium hydroxide, potassium hydroxide, sodium carbonate, sodium borohydride. Then add 11.6g of chloroacetic acid, set the molar ratio of base to chloroacetic acid to 0.5:1, 1:1, 1.5:1, 2:1, react at 70°C for 8h, after the reaction is completed, cool to room temperature, then add acetic acid aqueous solution for neutralization of the base in the reaction solution. Filter the reaction solution, the obtained filter cake is washed 6-8 times using solution, the solution that can be used is water, ethanol, acetone, DMF, NaOH aqueous solution. The washed carboxymethyl cellulose is dispersed in pure water, the mass ratio of water to carboxymethyl cellulose is 9:1, then the gel is crushed, and then crushed to a particle size of 1-50μm using a colloid mill, high-pressure homogenizer, etc., the solid content is 0.1-3.0wt%, and the hydrogel CMC-1, CMC-2, CMC-3, CMC-4 is obtained after high-pressure sterilization.

[0255] (2) Preparation of cellulose ester hydrogel

[0256] 1) Preparation of cellulose propionate hydrogel

[0257] Take 4.0 g of cellulose, which can be used as microcrystalline cellulose, cotton pulp, wood pulp, inulin, soluble starch, dextran, and add to 46 g of ionic liquid, which can be used as 1-allyl-3-methylimidazolium propionate ([Amim] [CH3CH2COO]), 1-ethyl-3-methylimidazolium butyrate ([Emim] [CH3CH2CH2COO]), 1-butyl-3-methylimidazolium cyclohexyl formate ([Bmim] [ChCOO]), 1-butyl-3-methylimidazolium cinnamate ([Bmim] [CCOO]), 1-butyl-3-methylimidazolium chloride (BmimCl), 80℃ stirring for 30 min to get 8wt% solution; add a certain amount of propionic anhydride to it at 80℃, the molar ratio of propionic anhydride to cellulose is 1:1, 2:1, 3:1, 4:1, stirring for 2h, pouring the reaction solution into water to precipitate, using solution to wash the product 6-8 times to remove residual impurities, the solution that can be used has water, ethanol, DMF, NaOH aqueous solution. The washed cellulose propionate gel is dispersed in pure water, the mass ratio of water to cellulose propionate gel is 9:1, then the gel is crushed, and is crushed to a particle size of 1-50μm using a colloid mill, a high-pressure homogenizer, etc., a solid content of 0.1-3.0wt%, and a high-pressure sterilization to obtain a cellulose propionate hydrogel.

[0258] 2) Preparation of cellulose butyrate hydrogel

[0259] Take 4.0 g of cellulose, which can be used as microcrystalline cellulose, cotton pulp, wood pulp, inulin, soluble starch, dextran, and add to 46 g of ionic liquid, which can be used as 1-allyl-3-methylimidazolium propionate ([Amim] [CH3CH2COO]), 1-ethyl-3-methylimidazolium butyrate ([Emim] [CH3CH2CH2COO]), 1-butyl-3-methylimidazolium cyclohexyl formate ([Bmim] [ChCOO]), 1-butyl-3-methylimidazolium cinnamate ([Bmim] [CCOO]), 1-butyl-3-methylimidazolium chloride (BmimCl), 80℃ stirring for 30 min to get 8wt% solution; add a certain amount of propionic anhydride to it at 80℃, the molar ratio of propionic anhydride to cellulose is 1:1, 2:1, 3:1, 4:1, stirring for 2h, pouring the reaction solution into water to precipitate, using solution to wash the product 6-8 times to remove residual impurities, the solution that can be used has water, ethanol, DMF, NaOH aqueous solution. The washed cellulose propionate gel is dispersed in pure water, the mass ratio of water to cellulose propionate gel is 9:1, then the gel is crushed, and is crushed to a particle size of 1-50μm using a colloid mill, a high-pressure homogenizer, etc., a solid content of 0.1-3.0wt%, and a high-pressure sterilization to obtain a cellulose propionate hydrogel.

[0260] 3) Preparation of cellulose benzoate hydrogel

[0261] Weigh 4.0 g of cellulose, which can be microcrystalline cellulose, cotton pulp, wood pulp, inulin, soluble starch, dextran, into 46 g of ionic liquid, which can be 1-allyl-3-methylimidazolium propionate ([Amim] [CH3CH2COO]), 1-ethyl-3-methylimidazolium butyrate ([Emim] [CH3CH2CH2COO]), 1-butyl-3-methylimidazolium cyclohexylcarboxylate ([Bmim] [ChCOO]), 1-butyl-3-methylimidazolium cinnamate ([Bmim] [CCOO]), 1-butyl-3-methylimidazolium chloride (BmimCl). Stir at 80°C for 30 min to obtain an 8wt% solution; add a certain amount of benzoyl chloride to it at 80°C, the molar ratio of benzoyl chloride to cellulose is 1:1, 2:1, 3:1, 4:1, stir for 2 h, pour the reaction liquid into water to precipitate, wash the product with solution for 6-8 times to remove residual impurities, which can be water, ethanol, DMF, NaOH aqueous solution. Disperse the washed cellulose benzoate gel in pure water, the ratio of water to cellulose benzoate is 9:1. Then crush the gel, and use a colloid mill, a high-pressure homogenizer, etc. to crush to a particle size of 1-50 μm, a solid content of 0.1-3.0wt%, and obtain cellulose benzoate hydrogel after high-pressure sterilization.

[0262] 4) Preparation of cellulose cyclohexylcarboxylate hydrogel

[0263] Take 4.0 g of cellulose, which can be used as microcrystalline cellulose, cotton pulp, wood pulp, inulin, soluble starch, dextran, and add it to 46 g of ionic liquid, which can be used as 1-allyl-3-methylimidazolium propionate ([Amim] [CH3CH2COO]), 1-ethyl-3-methylimidazolium butyrate ([Emim] [CH3CH2CH2COO]), 1-butyl-3-methylimidazolium cyclohexyl formate ([Bmim] [ChCOO]), 1-butyl-3-methylimidazolium cinnamate ([Bmim] [CCOO]), 1-butyl-3-methylimidazolium chloride (BmimCl), stir at 80℃ for 30 min to get an 8wt% solution; add a certain amount of cyclohexyl formyl chloride to it at 80℃, the molar ratio of cyclohexyl formyl chloride to cellulose is 1:1, 2:1, 3:1, 4:1, stir for 2h, pour the reaction solution into water to precipitate, wash the product with solution for 6-8 times to remove residual impurities, the solution that can be used has water, ethanol, DMF, NaOH aqueous solution. Disperse the washed cellulose cyclohexyl formate gel in pure water, the mass ratio of water to cellulose cyclohexyl formate gel is 9:1. Then crush the gel, and use colloid mill, high pressure homogenizer, etc. to crush to a particle size of 1-50μm, solid content of 0.1-3.0wt%, get cellulose cyclohexyl formate hydrogel after high pressure sterilization.

[0264] 5) Cellulose cinnamate hydrogel preparation

[0265] Take 4.0 g of cellulose, which can be used as microcrystalline cellulose, cotton pulp, wood pulp, inulin, soluble starch, dextran, and add it to 46 g of ionic liquid, which can be used as 1-allyl-3-methylimidazolium propionate ([Amim] [CH3CH2COO]), 1-ethyl-3-methylimidazolium butyrate ([Emim] [CH3CH2CH2COO]), 1-butyl-3-methylimidazolium cyclohexyl formate ([Bmim] [ChCOO]), 1-butyl-3-methylimidazolium cinnamate ([Bmim] [CCOO]), 1-butyl-3-methylimidazolium chloride (BmimCl), stir at 80℃ for 30 min to get an 8wt% solution; add a certain amount of cyclohexyl formyl chloride to it at 80℃, the molar ratio of cyclohexyl formyl chloride to cellulose is 1:1, 2:1, 3:1, 4:1, stir for 2h, pour the reaction solution into water to precipitate, wash the product with solution for 6-8 times to remove residual impurities, the solution that can be used has water, ethanol, DMF, NaOH aqueous solution. Disperse the washed cellulose cyclohexyl formate gel in pure water, the mass ratio of water to cellulose cyclohexyl formate gel is 9:1. Then crush the gel, and use colloid mill, high pressure homogenizer, etc. to crush to a particle size of 1-50μm, solid content of 0.1-3.0wt%, get cellulose cyclohexyl formate hydrogel after high pressure sterilization.

[0266] 6) Preparation of cellulose 2-methylbenzoate hydrogel

[0267] 8g of cellulose can be used, such as microcrystalline cellulose, wood pulp, cotton pulp, which is dissolved in 92g of ionic liquid, such as 1-allyl-3-methylimidazolium chloride ionic liquid (AmimCl), 1-butyl-3-methylimidazolium chloride ionic liquid (BmimCl), 1-allyl-3-methylimidazolium chloride ionic liquid (AmimCl) / N,N-dimethylacetamide (DMAc) (mass ratio of AmimCl to DMAc is 9:1), 1-ethyl-3-methylimidazolium chloride ionic liquid (EmimCl) / 1-methylimidazole (Mim) (mass ratio of EmimCl to Mim is 8:2), a certain amount of 2-methylbenzoyl chloride and a catalytic amount of pyridine are added, the mass ratio of cellulose to 2-methylbenzoyl chloride can be 1:5, 1:6, 1:7, and the reaction is carried out at 80°C for 2h. After the reaction is completed, pour into methanol to precipitate the precipitate, wash the product with solution for 6-8 times to remove residual impurities, and the solution that can be used includes water, ethanol, DMF, and NaOH aqueous solution. The washed cellulose 2-methylbenzoate gel is dispersed in pure water, and the mass ratio of water to cellulose 2-methylbenzoate gel is 9:1. Then the gel is crushed, and then crushed to a particle size of 1-50μm using a colloid mill, a high-pressure homogenizer, etc. After high-pressure sterilization, cellulose 2-methylbenzoate hydrogel is obtained.

[0268] 7) Preparation of cellulose acetate 2-methylbenzoate mixed ester hydrogel

[0269] 8g of cellulose can be used, such as microcrystalline cellulose, wood pulp, cotton pulp, which is dissolved in 92g of ionic liquid, such as 1-allyl-3-methylimidazolium chloride ionic liquid (AmimCl), 1-butyl-3-methylimidazolium chloride ionic liquid (BmimCl), 1-allyl-3-methylimidazolium chloride ionic liquid (AmimCl) / N,N-dimethylacetamide (DMAc) (mass ratio of AmimCl to DMAc is 9:1), 1-ethyl-3-methylimidazolium chloride ionic liquid (EmimCl) / 1-methylimidazole (Mim) (mass ratio of EmimCl to Mim is 8:2), 42.94g of 2-methylbenzoyl chloride and a catalytic amount of pyridine are added, the mass ratio of raw materials to 2-methylbenzoyl chloride can be 1:5, 1:6, 1:7, etc., and the reaction is carried out at 80°C for 2h, then 4.85g of acetyl chloride is added, and the reaction is continued at 80°C for 2h. After the reaction is completed, it is poured into methanol to precipitate and separate, the product is washed 6-8 times with a solution, such as water, ethanol, DMF, NaOH aqueous solution, etc., to remove residual impurities. The washed cellulose acetate 2-methylbenzoate mixed ester hydrogel is dispersed in pure water, and the mass ratio of water to cellulose acetate 2-methylbenzoate mixed ester hydrogel is 9:1. Then the gel is crushed, and is crushed to a particle size of 1-50μm using a colloid mill, a high-pressure homogenizer, etc. After high-pressure sterilization, cellulose acetate 2-methylbenzoate mixed ester hydrogel is obtained.

[0270] 8) Cellulose 2-methylbenzoate-4-trifluoromethylbenzoate mixed ester hydrogel preparation

[0271] 8g of cellulose is weighed and dissolved in 92g of ionic liquid. The cellulose that can be used includes microcrystalline cellulose, wood pulp, cotton pulp. The ionic liquid that can be used includes 1-allyl-3-methylimidazolium chloride ionic liquid (AmimCl), 1-butyl-3-methylimidazolium chloride ionic liquid (BmimCl), 1-allyl-3-methylimidazolium chloride ionic liquid (AmimCl) / N,N-dimethylacetamide (DMAc) (the mass ratio of AmimCl to DMAc is 9:1), 1-ethyl-3-methylimidazolium chloride ionic liquid (EmimCl) / 1-methylimidazole (Mim) (the mass ratio of EmimCl to Mim is 8:2). 42.94g of 2-methylbenzoyl chloride and a catalytic amount of pyridine are added. The mass ratio of the raw material to 2-methylbenzoyl chloride can be 1:5, 1:6, or 1:7. The reaction is carried out at 80℃ for 2h. Then 4.85g of 4-trifluoromethylbenzoyl chloride is added, and the reaction is continued at 80℃ for 2h. After the reaction is completed, the product is precipitated by pouring into methanol. The product is washed 6-8 times with a solution to remove residual impurities. The solution that can be used includes water, ethanol, DMF, and NaOH aqueous solution. The washed 2-methylbenzoic acid cellulose-4-trifluoromethylbenzoic acid mixed ester hydrogel is dispersed in pure water. The mass ratio of water to 2-methylbenzoic acid cellulose-4-trifluoromethylbenzoic acid mixed ester hydrogel is 9:1. Then the gel is crushed and pulverized to a particle size of 1-50μm using a colloid mill, a high-pressure homogenizer, etc. After high-pressure sterilization, 2-methylbenzoic acid cellulose-4-trifluoromethylbenzoic acid mixed ester hydrogel is obtained.

[0272] 9) Amantadine acetate cellulose mixed ester hydrogel

[0273] Dissolve 20 g of cellulose in 480 g of ionic liquid at 80℃ for 2 h, the cellulose can be microcrystalline cellulose, purified cotton, cotton pulp, wood pulp, and the ionic liquid can be one of allyl methyl imidazole chloride ionic liquid, butyl methyl imidazole acetate ionic liquid, ethyl methyl imidazole chloride ionic liquid. Add 34 g of co-solvent pyridine and stir for 5 min, the molar ratio of pyridine to anhydroglucose unit in cellulose is 3.5:1. Add acetic anhydride and adamantyl formyl chloride at the same time, and mechanically stir for 2 h while maintaining the temperature at 80℃. The molar ratio of adamantyl formyl chloride and acetic anhydride to anhydroglucose unit in cellulose can be 1:1, 2:1, 3:1, or 4:1. After the reaction is completed, pour the reaction solution into a coagulation bath, and the product is precipitated. Purify using DMSO, wash the gel with a solution for 6-8 times to remove residual DMSO, and the solution that can be used includes water, ethanol, acetone, and DMF. Disperse the washed cellulose mixed ester of adamantyl formate acetate gel in pure water at a mass ratio of water to cellulose mixed ester of adamantyl formate acetate gel of 9:1, then crush the gel, and use a colloid mill, high-pressure homogenizer, or the like to crush to a particle size of 1-50 μm, a solid content of 0.1-3.0 wt%, and obtain the cellulose mixed ester of adamantyl formate acetate hydrogel after high-pressure sterilization.

[0274] (3) Preparation of oxidized cellulose-dialdehyde cellulose hydrogel

[0275] Dissolve 20 g of cellulose in 480 g of ionic liquid at 80℃ for 2 h, the cellulose can be microcrystalline cellulose, purified cotton, cotton pulp, wood pulp, and the ionic liquid can be one of allyl methyl imidazole chloride ionic liquid, butyl methyl imidazole acetate ionic liquid, ethyl methyl imidazole chloride ionic liquid. Add 34 g of co-solvent pyridine and stir for 5 min, the molar ratio of pyridine to anhydroglucose unit in cellulose is 3.5:1. Add acetic anhydride and adamantyl formyl chloride at the same time, and mechanically stir for 2 h while maintaining the temperature at 80℃. The molar ratio of adamantyl formyl chloride and acetic anhydride to anhydroglucose unit in cellulose can be 1:1, 2:1, 3:1, or 4:1. After the reaction is completed, pour the reaction solution into a coagulation bath, and the product is precipitated. Purify using DMSO, wash the gel with a solution for 6-8 times to remove residual DMSO, and the solution that can be used includes water, ethanol, acetone, and DMF. Disperse the washed cellulose mixed ester of adamantyl formate acetate gel in pure water at a mass ratio of water to cellulose mixed ester of adamantyl formate acetate gel of 9:1, then crush the gel, and use a colloid mill, high-pressure homogenizer, or the like to crush to a particle size of 1-50 μm, a solid content of 0.1-3.0 wt%, and obtain the cellulose mixed ester of adamantyl formate acetate hydrogel after high-pressure sterilization.

[0276] (4) Preparation of DOPO (9,10-dihydro-9-oxa-10-phosphaphenanthrene-10-oxide) cellulose derivative hydrogel

[0277] 1) Preparation of cellulose acrylate: 10 g of cellulose was added to 190 g of ionic liquid 1-allyl-3-methylimidazolium chloride (AmimCl) and stirred mechanically for 2 h at 80 °C to dissolve the cellulose, which can be microcrystalline cellulose, purified cotton, cotton pulp, wood pulp. The cellulose solution was placed in an oil bath at 50 °C, and after half an hour of stabilization, 2.8 g of acryloyl chloride was added thereto and reacted for 2 h with mechanical stirring at 300 rpm. After the reaction was completed, the intermediate product cellulose acrylate was obtained by precipitation, washing, and drying.

[0278] 2) Preparation of DOPO type cellulose derivative matrix glue: A certain amount of DOPO was dissolved in DMSO to prepare a solution with a mass fraction of 10%, and an alkali was added as a catalyst. 10 g of cellulose acrylate in step (1) was dissolved in 190 g of DMSO, and slowly added to the DOPO solution, and reacted at 80 °C for 12 h. The molar ratio of DOPO to cellulose can be 1:1, 2:1, 3:1, 4:1, and the alkali can be sodium carbonate, triethylamine, sodium borohydride, and sodium hydride. The reaction solution was poured into methanol for precipitation, and the product was washed 6-8 times with a solution to remove residual impurities. The solution that can be used includes water, ethanol, acetone, and ethylene glycol. The washed DOPO type cellulose derivative gel was dispersed in pure water with a mass ratio of water to DOPO type cellulose derivative gel of 9:1, and then the gel was crushed and pulverized using a colloid mill, a high-pressure homogenizer, etc. to a particle size of 1-50 μm and a solid content of 0.1-3.0 wt%. After high-pressure sterilization, a DOPO type cellulose derivative hydrogel was obtained.

[0279] The first part of the cellulose derivative hydrogel was used for 3D cell culture experiments:

[0280] Example 1

[0281] The method of cell culture is as follows:

[0282] 1) Cell passage: commercially available SH-SY5Y cells were selected, resuscitated, and cultured in a 10 cm culture dish. When the confluence rate reached more than 80%, the cells were passaged. The original culture solution in the culture dish was discarded, and the culture dish was washed twice with PBS, and then trypsin was used to prepare a single cell suspension. The cells were centrifuged at 1200 rpm and room temperature for 5 min, resuspended in the base culture solution (its composition: DMEM / F12, 10% FBS, 1% P / S, and the base culture solution was used in the following examples unless otherwise specified), and adjusted to a cell density of 2 x 10^4 cells / μL to obtain SH-SY5Y cells for experiments.

[0283] 2) Preparation of methylcellulose culture solution: Take the methylcellulose hydrogel MC-1 prepared in Preparation Example 1 above, and after dilution using the base culture solution in step 1), a methylcellulose culture solution with a methylcellulose concentration of 0.2% is obtained.

[0284] 3) Cell suspension mixing: Take 500 μL of the methylcellulose culture solution in step 2), and mix with 500 μL of the base culture solution in a volume ratio of 1:1, and add 10 μL of the experimental SH-SY5Y cells in step 1), mix well to obtain a cell suspension.

[0285] 4) 3D culture: Add 1 mL of the mixed cell suspension in step 3) to a 12-well culture plate, and culture in a 37°C, 5 vol% CO2 incubator.

[0286] 5) Medium change: Change the medium every 3 days after 3 days of culture; the specific steps are as follows: centrifuge the cell-containing culture plate in step 4) at 1200 rpm at room temperature for 5 min, discard the culture solution in the upper layer of the culture plate, and add new base culture solution.

[0287] The microscopic images of the cell suspension after 1 day, 3 days, 5 days, and 7 days of culture are shown in Figures 1b, 1c, 1d, and 1e, respectively.

[0288] Comparative Example 1

[0289] The methylcellulose hydrogel MC-1 and the plant cellulose matrix gel GrowDex (UPM / 100103005) of the present application were used to culture SH-SY5Y cells in 3D for 5 days, and the results are shown in Figures 2a and 2b.

[0290] Example 2: The operation is the same as in Example 1, but the cultured cells are HepaRG (liver-cancer cells), and the hydrogels are methylcellulose MC-1 hydrogel, cellulose propionate hydrogel, oxidized cellulose-dialdehyde cellulose hydrogel, and cellulose acrylate hydrogel, respectively. The microscopic images of the above cell suspensions after 7 days of culture are shown in Figures 3a, 3b, 3c, and 3d, respectively.

[0291] Second Part: Cellulose Derivative-Based Hydrogel for Tissue and Organoid Culture Experiments

[0292] The types of raw materials involved in the following examples are as follows:

[0293] P / S Penicillin-Streptomycin, GIBCO / 15140122;

[0294] HEPES N-2-Hydroxyethylpiperazine-N-2-ethanesulfonic acid, GIBCO / 15630080;

[0295] L-glutamine additive Glutamax, GIBCO / 35050061 ;

[0296] Neuronal cell culture additive B27, GIBCO / 17504044;

[0297] N-acetylcysteine, Sigma / A9165-100G;

[0298] FGF-7, mce / HY-P7047A;

[0299] Noggin, mce / HY-P70558;

[0300] R-spondin 1, PeproTech / 120-38;

[0301] Wnt 3A, PeproTech / 315-20;

[0302] FGF10, PeproTech / 100-26;

[0303] Gastrin, mce / HY-P1097;

[0304] ALK inhibitor A-83-01, mce / HY-10432;

[0305] ROCK inhibitor Y-27632, mce / HY-10071;

[0306] p38 MAPK inhibitor SB202190, mce / HY-10295;

[0307] Epirregulin, mce / HY-P7011;

[0308] Capsanthin, mce / HY-125711;

[0309] Nicotinamide, Sigma / N0636.

[0310] Example 3

[0311] 1. The formulation of the complex culture solution is as follows: the complex culture solution is obtained by mixing the following components:

[0312] 2. Preparation of the tissue to be cultured

[0313] (1) Tissue washing

[0314] Transfer the tissue block (e.g. gastric cancer organoid, hospital surgical tissue or biopsy tissue) into a 50ml centrifuge tube, add 15ml washing solution (PBS + 2x P / S), shake and wash for no less than 5 times, 6min each time, until completely clear.

[0315] (2) Tissue dissociation

[0316] Transfer the tissue block into a 10cm culture dish, add a small amount of washing solution to ensure the surface of the tissue is wet, use a sterile scalpel and blade to mechanically dissociate the tissue until it becomes a small piece or paste about 0.5x0.5x0.5mm^3.

[0317] (3) Tissue digestion

[0318] Transfer the mechanically dissociated tissue into a 15ml sterile centrifuge tube, add 2-5ml tissue digestion solution (collagenase P (1mg / ml) + DNase (1mg / ml)) according to the amount of tissue, gently blow the tissue with a 1ml syringe to disperse it completely; place the centrifuge tube containing the digestion solution in a constant temperature air bath shaker at 37°C, 200rpm, and dissociate and digest until most of the tissue is dissociated into 3-10 cell clusters, then add 10% FBS to terminate digestion.

[0319] (4) Cell filtration

[0320] Filter the cell suspension after the above termination of digestion with a 100μm filter into a new 50ml centrifuge tube, use a 5ml syringe to grind the sample sediment until the digested tissue sample is ground to only white connective tissue, and wash the filter with DMEM / F12 for 3 times, about 5ml each time. 4°C, 300g / 1260rpm, centrifuge for 5min.

[0321] (5) Red blood cell lysis

[0322] If a large number of red blood cells are observed in the cell sediment, the following red blood cell lysis step is performed:

[0323] Remove the supernatant, add 1ml red blood cell lysis solution to resuspend the cell sediment, mix well by blowing, and place it on a 4°C shaker at 120rpm for 5min. 4°C, 300g / 1260rpm, centrifuge for 5min. Add 10ml Advanced DMEM / F12 to resuspend the cell sediment, and transfer the resuspension to a 15ml centrifuge tube. 4°C, 300g / 1260rpm, centrifuge for 5min.

[0324] (6) Cell plating

[0325] Discard the supernatant, and retain the appropriate volume (depending on the number of cells, 100,000 cells / well is recommended) of liquid, which is the tissue cell liquid to be cultured.

[0326] 3. Preparation of the cellulose derivative culture solution:

[0327] The methylcellulose hydrogel MC-1 in Preparation Example 1 was diluted with the above-mentioned composite culture solution to obtain a methylcellulose culture solution, with the concentration of methylcellulose being 0.1-0.8%.

[0328] 4. 3D culture: The tissue cell liquid to be cultured in Step 2 was gently suspended with the methylcellulose culture solution in Step 3 above, and then dropped into a preheated 24-well low-adsorption cell culture plate at 300 μl per well. The culture plate was placed in a 37°C 5% CO2 incubator for heating for 10 min, and then taken out and added with 700 μl of preheated composite culture solution per well. The culture plate was placed in a 37°C 5% CO2 incubator for culture until subculture.

[0329] Example 4

[0330] This example is basically the same as Example 3, except that the methylcellulose hydrogel MC-1 prepared in Preparation Example 1 was taken and diluted with the composite culture solution to obtain a cellulose derivative culture solution with the concentration of cellulose being 0.2%, and the gastric cancer organoids were cultured, and the culture results are shown in FIG. la-1, FIG. lb-1, FIG. lc-1, FIG. ld-1, FIG. le-1, and FIG. lf.

[0331] As can be seen from FIG. la-1, FIG. lb-1, FIG. lc-1, FIG. ld-1, FIG. le-1, and FIG. lf, after culture for different days, obvious 3D cell spheres were observed on the day of culture (0 day); the cell spheres were obviously increased in diameter and the cell walls were thickened after growth for 5 days; the maximum diameter reached 200 μm after growth for 7 days, and the 0-day gastric cancer organoids showed a typical differentiated mature morphology.

[0332] Comparative Example 2

[0333] This example is basically the same as Example 3, except that the methylcellulose hydrogel MC-1 prepared in Preparation Example 1 was taken and diluted with the composite culture solution to obtain a cellulose derivative culture solution with the concentration of cellulose being 0.2%, and the gastric cancer organoids were cultured, and the culture results are shown in FIG. la-1, FIG. lb-1, FIG. lc-1, FIG. ld-1, FIG. le-1, and FIG. lf.

[0334] After 5 days of culture, the organoid morphology and size were basically consistent, and there was no obvious difference among MC-1, Matrigel and GrowDex.

[0335] Example 5

[0336] The operation is the same as that in Example 3, except that the hydrogels and the cultured tissues are different. The hydrogels are carboxymethyl cellulose hydrogel CMC-1, adamantane formate cellulose mixed ester hydrogel, oxidized cellulose-dialdehyde cellulose hydrogel and cellulose acrylate hydrogel, respectively. The microscopic images of the intestinal cancer organoids after 7 days of culture are shown in Figures 3a-1, 3b-1, 3c-1 and 3d-1.

[0337] Part III: Cellulose derivative hydrogel for stem cell culture and induced differentiation organoid experiment

[0338] Example 6: Human induced pluripotent stem cell / embryonic stem cell culture

[0339] 1) Before the stem cell passage, dilute the methyl cellulose MC-1 hydrogel to 0.8% with DMEM / F12, add it to the 6-well plate at 1 ml / well, and place it in the 37°C incubator for at least 1 hour.

[0340] 2) Stem cell passage, about 85-90% of the cells in the culture dish are confluent and basically undifferentiated. After aspirating the stem cell culture solution mTeSR TM 1(STEMCELL, 85850), cover each well with 1 ml Dispase (1 mg / ml) and place it in the 37°C incubator until the stem cell edge begins to lift, and aspirate the Dispase.

[0341] 3) Wash 3 times with DPBS, 1 ml each time.

[0342] 4) Add 3 ml of preheated mTeSR TM 1 to the well, gently blow to make the stem cells fall off from the well plate, and repeat the blowing until the cell mass is broken into 1-2 mm fragments.

[0343] 5) Divide the stem cell fragments into the methyl cellulose hydrogel MC-1 well plate covered in step 1) at a ratio of 1:1 to 1:6, and add mTeSR TM 1 culture solution (STEMCELL, 85850) to 2 ml, gently knock the side of the 6-well plate culture dish 5 to 10 times to ensure that the stem cell colonies are evenly distributed in the air, and culture in a 37°C, 5% CO2 incubator, with daily medium change.

[0344] Example 7: Human pluripotent stem cell / embryonic stem cell induced differentiation intestinal organoid

[0345] 1. Single layer culture of iPSC / ES differentiation

[0346] The differentiation was performed in TC-treated 24-well cell culture plates. The methylcellulose hydrogel MC-1 was diluted to 0.8% with DMEM / F12 and added to the 24-well plates, 0.3 ml / well, and incubated in a 37°C incubator for at least 1 hour. Prior to the start of differentiation, the differentiation rate and starting density of the cells should be evaluated. The differentiation rate should be less than 5% and the starting density should reach 85-90% at this time.

[0347] (1) Definitive endoderm differentiation

[0348] A. Preparation of definitive endoderm differentiation medium

[0349] Day 0: Prepare the definitive endoderm differentiation medium (RPMI1640, containing L-glutamine (final concentration 2mM), penicillin-streptomycin (final concentration 100U / ml-100g / ml) and Activin A (final concentration 100ng / ml) required for day 0, 1 and 2, 0.5mL per culture well.

[0350] B. Definitive endoderm differentiation

[0351] 1) Day 0: Pre-warm the definitive endoderm differentiation medium required for day 0 (0.5mL / well) at 37°C, store the remaining medium at 2-8°C, and aspirate mTeSR from the culture well. TM 1, add 0.5mL of definitive endoderm differentiation medium dropwise along the well wall. Incubate at 37°C, 5% CO2 and 95% humidity for 24 hours.

[0352] 2) Day 1: Pre-warm the definitive endoderm differentiation medium (0.5mL / well) at 37°C, aspirate the medium from the culture well, and add 0.5mL of definitive endoderm differentiation medium dropwise along the well wall. Incubate at 37°C, 5% CO2 and 95% humidity for 24 hours.

[0353] 3) Day 2: Aspirate the medium from the culture well, and add 0.5mL of 37°C pre-warmed definitive endoderm differentiation medium dropwise along the well wall. Incubate at 37°C, 5% CO2 and 95% humidity for 24 hours.

[0354] Note: During endoderm induction, cells will undergo a large amount of death. Try to minimize the time of cells outside the 37°C incubator. After 24 hours of endoderm induction, cells are very sensitive and need to be handled carefully when changing medium. After 72 hours of incubation, a dense confluent monolayer of endoderm cells will be formed.

[0355] (2) Mid / Hindgut (MH) differentiation

[0356] A. MH differentiation medium

[0357] Day 3: Prepare MH differentiation media needed for days 3-8 (RPMI 1640 with 2% FBS, with L-glutamine (final concentration 2 mM), penicillin-streptomycin (final concentration 100 U / ml-100 g / ml), FGF4 (final concentration 500 ng / ml), 0.5 mL per culture well.

[0358] B. Mid / Posterior Intestine (MH) Differentiation

[0359] 1) Day 3: Warm sufficient amount of MH differentiation media (0.5 mL / well) to room temperature (15-25 °C), aspirate the media in the culture well, and add 0.5 mL of mid / posterior intestine (MH) differentiation media drop-wise along the well wall. Incubate at 37 °C, 5% CO2 and 95% humidity for 24 hours.

[0360] 2) Days 4-9: Change media completely and observe the spheroids every 24 hours according to the following protocol.

[0361] Note: Make sure the culture is returned to the incubator within 30 minutes of removal.

[0362] a. Observe the monolayer under the microscope. Three-dimensional structures can appear as early as day 4 of differentiation. Free-floating mid / posterior intestinal spheroids will appear

[0363] Now at day 6-9 of differentiation.

[0364] b. With a 1 mL pipette, remove 0.5 mL of media from the cells and transfer to a sterile 24-well flat bottom transparent culture plate to assess the number and concentration of mid / posterior intestinal spheroids extracted from the monolayer.

[0365] c. Add 0.5 mL of fresh MH media to the cells. Incubate at 37 °C, 5% CO2 and 95% humidity for 24 hours.

[0366] Note: Although spheroids released at day 6-9 will all generate small intestinal organoids, the length of time the cells are cultured in mid / posterior intestinal media will determine the regional identity of the small intestinal organoids that develop. For example, duodenum (shorter culture time) or ileum (longer culture time). The time at which the maximum yield of mid / posterior intestinal spheroids occurs can vary depending on the hPSC cell line. For reproducible experimental results, use mid / posterior intestinal spheroids differentiated at the same time point for consistent harvesting and initiation of human intestinal organoid culture.

[0367] 3) Spheroid Embedding: Use a 0.5% BSA rinsed pipette to suspend the spheroids in each culture well into one culture well of a 24-well plate for counting. Add the appropriate volume to a 15 mL centrifuge tube corresponding to approximately 50 or so suspended spheroids (based on previous spheroid count).

[0368] One mid / hindgut spheroid is a cell aggregate with a diameter > 75 pm, which can form a human intestinal organoid. Multiple fused spheroids should be counted as one unit, which will form a human intestinal organoid.

[0369] The remaining monolayer culture can be used to determine mid / hindgut formation or to study further differentiation of chimeric spheroids over the following days.

[0370] 2. Human intestinal organoid culture

[0371] (1) Initial culture of human intestinal organoids

[0372] A. Preparation of intestinal organoid growth medium

[0373] 1) Culture in 24-well low attachment culture plates. Prepare 4 wells (0.5 mL / well) of intestinal organoid growth medium (Advanced DMEM / F12 with 1 x B27, with L-glutamine (final concentration 2 mM), penicillin-streptomycin (final concentration 100 U / ml - 100 g / ml), HEPES buffer (final concentration 15 mM), R-spondin 1 (final concentration 500 ng / ml).

[0374] B. Mixing of methylcellulose hydrogel MC-1 with spheroids

[0375] 1) Centrifuge the spheroid suspension collected in step B of mid / hindgut (MH) differentiation at 300 x g for 5 minutes. Carefully aspirate the supernatant after centrifugation.

[0376] 2) Add 1 mL of DMEM / F-12 + 15 Mm HEPES to the spheroids. Centrifuge at room temperature (15-25 °C) at 300 x g for 5 minutes.

[0377] 3) Carefully aspirate the supernatant with 1 mL of gun tip.

[0378] 5) Add 100 pL of 0.3% methylcellulose hydrogel MC-1 in the centrifuge tube. Pipette up and down 5 times and gently dispense the spheroids into the methylcellulose hydrogel MC-1.

[0379] Note: Do not empty the gun tip completely to prevent excessive bubble formation.

[0380] 6) Using the same gun tip, gently transfer the embedded spheroids to the center of one culture well of a 24-well tissue culture dish.

[0381] 7) Incubate in a 37 °C incubator for 10-25 minutes.

[0382] 8) Warm up a sufficient volume of intestinal organoid growth medium to (15-25 °C). Store the remaining medium at 2-8 °C.

[0383] 9) Carefully add at least 0.5 mL / well of intestinal organoid growth medium along the well wall of the culture well. Incubate at 37°C, 5% CO2, 95% humidity.

[0384] 10) Replace the culture medium every 3-4 days, remove the old culture medium, and add fresh culture medium. Incubate at 37°C, 5% CO2, 95% humidity.

[0385] 11) After 10-14 days of incubation, passage according to the growth of the organoids.

[0386] The microscopic images of the human pluripotent stem cell / embryonic stem cell induced differentiation intestinal organoids after 3, 7, and 12 days of culture according to the present embodiment are shown in Figures 1a-2, 1b-2, and 1c-2.

[0387] Comparative Example 3

[0388] According to the culture method of Reference Example 6, iPSCs were cultured using 0.8% methylcellulose hydrogel MC-1 and Matrigel (Corning 354277), respectively, and the results are shown in Figures 2a-2 and 2b-2.

[0389] Both groups had clear colony edges, uniform cell size, and high nuclear-cytoplasmic ratio, representing a better stem cell state and presenting an undifferentiated cell state. This indicates that methylcellulose MC-1 hydrogel and Matrigel have comparable performance in culturing stem cells.

[0390] Comparative Example 4

[0391] According to the culture method of Reference Example 7, human pluripotent stem cells / embryonic stem cells were cultured using the induced differentiation intestinal organoid kit containing 0.3% methylcellulose hydrogel MC-1 and STEMdiff TM Intestinal Organoid Kit (Stem Cell, 05140), respectively, and the microscopic images of the human pluripotent stem cell / embryonic stem cell induced differentiation intestinal organoids after 12 days are shown in Figures 3a-2 and 3b-2.

[0392] Both groups can culture mature intestinal organoids containing multiple crypt structures, and these crypt structures are connected to each other to form a mini-intestine with an intestinal lumen. However, the kit of the present application is even better than the comparative kit, and the intestinal organoids formed have a more typical and complex crypt reentrant structure.

[0393] Example 8

[0394] The operation is the same as that in Example 7, except that the hydrogels are different, and the hydrogels are respectively: carboxymethyl cellulose hydrogel, cellulose mixed ester of adamantane carboxylic acid acetate hydrogel, oxidized cellulose-dialdehyde cellulose hydrogel and cellulose acrylate hydrogel, and Figures 4a, 4b, 4c, 4d are micrographs of the intestinal organoids after 12 days of culture in the above hydrogels, respectively.

[0395] The above describes the exemplary embodiments of the present application. However, the protection scope of the present application is not limited to the above-described embodiments. Any modification, equivalent replacement, improvement, etc. made by those skilled in the art within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A cellulose derivative-based hydrogel, characterized by, The matrix thereof comprises a cellulose derivative selected from cellulose ester, cellulose ether, oxidized cellulose, charged cellulose or other types of cellulose derivatives, preferably selected from methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, carboxymethyl cellulose, cellulose propionate, cellulose butyrate, cellulose benzoate, cellulose cyclohexylformate, cellulose cinnamate, cellulose 2-methylbenzoate, cellulose acetate 2-methylbenzoate mixed ester, cellulose 2-methylbenzoate-4-trifluoromethylbenzoate mixed ester, cellulose adamantane formic acid acetate mixed ester, oxidized cellulose-dialdehyde cellulose or DOPO (9,10-dihydro-9-oxa-10-phosphaphenanthrene-10-oxide) cellulose derivative, which comprises a network structure composed of ultrafine fibers and has a highly ordered three-dimensional micro-nano structure.

2. The cellulose derivative-based hydrogel according to claim 1, characterized by The degree of substitution of the cellulose derivative is 0.1-3, preferably 0.5-2.5, more preferably 1.0-2.0, preferably the fiber diameter of the cellulose derivative is 0.01-0.10 μm, for example 0.01 μm, 0.02 μm, 0.03 μm, 0.04 μm, 0.05 μm, 0.06 μm, 0.07 μm, 0.08 μm, 0.09 μm or 0.10 μm; the overall particle size of the matrix in the hydrogel is 1-50 μm, for example 1 μm, 5 μm, 8 μm, 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm or 50 μm; preferably the overall particle size of the matrix in the hydrogel is 1-50 μm, for example 1 μm, 5 μm, 8 μm, 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm or 50 μm, preferably the solid content of the hydrogel is 0.1-3.0 wt%, for example 0.1 wt%, 0.5 wt%, 1 wt%, 1.5 wt%, 2 wt%, 2.5 wt% or 3.0 wt%.

3. The cellulose derivative-based hydrogel according to claim 1 or 2, characterized in that, The cellulose derivative-based hydrogel is prepared by Scheme I or Scheme II: Scheme I: cellulose is subjected to homogeneous or heterogeneous derivatization to obtain a cellulose derivative; the cellulose derivative is prepared into a solution, poured into a coagulation bath to form a gel, and the gel is subjected to crushing treatment by means of a homogenizer, colloid mill or ball mill to obtain a cellulose derivative hydrogel; Scheme II: a cellulose solution is prepared; the cellulose solution is poured into a coagulation bath to form a gel, and the gel is subjected to crushing treatment by means of a homogenizer, colloid mill or ball mill to obtain a cellulose hydrogel, and the cellulose hydrogel is subjected to derivatization reaction to form a cellulose derivative hydrogel.

4. A method for preparing a cellulose derivative-based hydrogel according to any one of claims 1 to 3, characterized in that, The method is selected from the following Scheme I or Scheme II: Scheme I: cellulose is subjected to homogeneous or heterogeneous derivatization to obtain a cellulose derivative; the cellulose derivative is prepared into a solution, poured into a coagulation bath to form a gel, and the gel is subjected to crushing treatment by means of a homogenizer, colloid mill or ball mill to obtain a cellulose derivative hydrogel; Scheme two: preparing a cellulose solution; pouring the cellulose solution into a coagulation bath to form a gel, and pulverizing the gel by means of a homogenizer, colloid mill or ball mill to obtain a cellulose hydrogel; and derivatizing the cellulose hydrogel to obtain a cellulose derivative hydrogel. Preferably, the scheme one specifically comprises: 1) cellulose pretreatment; 2) derivatizing the pretreated cellulose of step 1) to obtain a cellulose derivative; 3) preparing the cellulose derivative of step 2) into a solution, pouring the solution into a coagulation bath to form a gel, and pulverizing the gel by means of a homogenizer, colloid mill or ball mill to obtain a cellulose derivative hydrogel; Preferably, in step 1), the cellulose pretreatment comprises dispersing cellulose in a solvent, which can be water, DMF, ethanol or isopropanol, and the cellulose can be microcrystalline cellulose, cotton pulp, wood pulp, inulin, soluble starch or dextran, preferably wood pulp; or the cellulose pretreatment comprises adding cellulose into a cellulose solvent to form a cellulose solution; Preferably, step 1) further comprises activating the cellulose, specifically, adding a certain amount of alkali to activate the cellulose, exemplarily, the alkali can be sodium hydroxide, potassium hydroxide, sodium carbonate or sodium borohydride; Preferably, in step 3), the solvent used for preparing the solution can be water, ethanol, acetone, DMF or NaOH aqueous solution. Preferably, the scheme two specifically comprises: 1') preparing a cellulose solution; 2') pouring the cellulose solution obtained in step 1') into a coagulation bath to form a gel, and pulverizing the gel by means of a homogenizer, colloid mill or ball mill to obtain a cellulose gel; 3') derivatizing the cellulose gel of step 2') to obtain a cellulose derivative hydrogel. Preferably, in step 1'), the cellulose is dissolved in a cellulose solvent to obtain the cellulose solution; In the scheme one or scheme two, the cellulose solvent is selected from any excellent solvent known in the art that can dissolve (the dissolution includes complete dissolution and partial dissolution) cellulose; Preferably, the solvent system for dissolving cellulose is selected from the ionic liquid and / or NaOH / Urea system; more preferably, the ionic liquid for dissolving cellulose is selected from one or more of [AMIM][Cl], [BMIM][Cl], [EMIM][Ac] and [BMIM][Ac].

5. A culture solution for 3D cell culture or for tissue and organoid culture or stem cell-induced differentiated organoid culture, characterized by, The 3D cell culture medium comprises the cellulose derivative-based hydrogel of claim 1 and a basal medium; the cellulose derivative-based medium for culturing tissues and organoids comprises the cellulose derivative-based hydrogel of claim 1 and a complex medium comprising a basal medium and active components, preferably the mass ratio of the basal medium and the active components in 100 mL of the complex medium is 80-99:1-20, for example 97:3, more preferably the complex medium contains Epirregulin 100-500 ng / ml, for example 200 ng / ml, 300 ng / ml, 400 ng / ml, 500 ng / ml; the stem cell-induced differentiation organoid culture medium comprises the cellulose derivative-based hydrogel of claim 1, a definitive endoderm differentiation medium, an MH differentiation medium, and an organoid growth medium.

6. A method for preparing the medium of claim 5, characterized in that, The method for preparing the 3D cell culture medium comprises the following steps: S1: preparing the above-mentioned cellulose derivative-based hydrogel; S2: mixing the cellulose derivative-based hydrogel in step S1 with a basal medium to prepare the 3D cell culture medium; In step S2, the volume ratio of the cellulose derivative-based hydrogel to the basal medium is 1-10:1-10, for example 1:1, 1:5, 1:10, 5:1, or 10:

1.

7. A method of culturing cells in 3D, characterized in that, The method for 3D culturing cells is carried out in the 3D cell culture medium of claim 5, and the 3D culturing method comprises the following steps: a) preparing a cell suspension: uniformly mixing the 3D cell culture medium of claim 5 with cells to obtain a cell suspension; b) 3D culturing: culturing the cell suspension of step a) in an incubator; Preferably, the cells comprise human neuroblastoma cells, for example SH-SY5Y cells and liver cancer cells, such as HepaRG cells.

8. A method of inducing differentiation of stem cells into organoids, characterized in that, The method comprises culturing a single stem cell or a group of stem cells in the stem cell-induced differentiation organoid culture medium of claim 5, and the method for inducing differentiation of stem cells into organoids comprises the following steps: a) iPSC / ES differentiation in monolayer culture, including definitive endoderm differentiation and mid / hindgut (MH) differentiation; b) organoid culture, including preparation of an organoid growth medium and incubation after mixing the cellulose derivative-based hydrogel of claim 1 with the spheroids.

9. Use of the cellulose derivative-based hydrogel according to claim 1 or the culture solution according to claim 5 in 3D cell culture, tissue and organoid culture, or stem cell culture and induced-differentiation organoid, characterized in that, Preferably, the cells comprise human neuroblastoma cells, for example SH-SY5Y cells and liver cancer cells, such as HepaRG cells, preferably the tissues are human gastric cancer tissue / normal gastric tissue, human intestinal cancer tissue / normal intestinal tissue, and the organoids are human gastric cancer organoid / normal gastric organoid, human intestinal cancer organoid / normal intestinal organoid, and the induced differentiation organoids are, for example, induced differentiation intestinal organoid, gastric organoid, liver organoid, and more preferably small intestinal organoid.

10. A kit characterized in that, The kit comprises the 3D cell culture medium or tissue and organoid culture medium or stem cell culture and induced differentiation organoid culture medium of claim 5 and instructions for use.

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