WO2025218555A1 - Cellulose-derivative-based hydrogel, culture solution for culture and preparation method therefor and use thereof - Google Patents

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

A cellulose derivative-based hydrogel, a culture medium for culture, and a preparation method and application thereof

This application requires the applicant to:

Priority benefit of the prior application, patent application number 202410450786.0, filed with the State Intellectual Property Office of China on April 15, 2024, entitled “A cellulose derivative-based hydrogel, a culture medium for 3D cell culture, and its preparation method and application”; patent application number 202410450781.8, entitled “A cellulose derivative-based culture medium and its application in culturing tissues or organoids”; and patent application number 202410450793.0, entitled “A cellulose derivative-based culture medium, a kit, and its application in culturing stem cells and organoids”;

The priority benefit of the prior application with patent application number 202410894552.5 filed with the State Intellectual Property Office of China on July 4, 2024, and entitled “A cellulose derivative-based hydrogel, culture medium for culture, preparation method and application thereof”.

The entire contents of the above-mentioned prior applications are incorporated into the present application by reference.

Technical Field

The present invention belongs to the field of biotechnology and relates to a cellulose derivative-based hydrogel, a culture medium for culture, and a preparation method and application thereof. Specifically, it relates to a cellulose derivative-based hydrogel, a culture medium for 3D cell culture, a culture medium for tissue and organoid culture, or a culture medium for stem cell culture and its induced differentiation organoid culture, a kit, and a preparation method and application thereof.

Background Art

Conventional 2D cell culture often does not conform to the in vivo situation because cells gradually lose their original characteristics as they proliferate in a changed environment in vitro. Animal experiments are conducted entirely in vivo, but they become complicated due to the constraints of multiple factors in the body and the mutual influence between the internal and external environments. It is difficult to study a single process and it is difficult to study intermediate processes.

3D cell culture technology is a technology between monolayer cell culture and animal experiments. It can simulate the in vivo environment to the greatest extent possible while showing the advantages of intuitive cell culture and controllable conditions. 3D cell culture can make up for many defects in the monolayer cell culture process, such as:

(1) 3D cell models can well simulate the cellular microenvironment in the body: gases, nutrients, metabolites and other substances show gradient concentration changes.

(2) 3D cell models can well simulate cell-cell interactions: three-dimensional cell-cell contacts and direct or indirect cell-cell communication.

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

The difficulty of 3D cell culture technology is to ensure the three-dimensional structure of cells and maintain natural proliferation and differentiation activity. With decades of continuous development, the methods of 3D cell culture have also been constantly innovating, and the commonly used methods are mainly divided into two types: scaffold-based 3D cell culture methods and scaffold-free 3D cell culture methods. The scaffold-based 3D cell culture method has a long history of development and is supported by a large amount of literature. The materials used for cell culture scaffolds include agarose, collagen, fibronectin, gelatin, laminin, etc. These composite materials simulate the natural extracellular matrix (ECM) through porosity, fiber, permeability and mechanical stability, and can well simulate the interactions between cells and the interactions between cells and extracellular matrix in the in vivo environment, while allowing cells to aggregate, proliferate and migrate on the scaffold.

Stem cells are a type of cell with the potential to proliferate and differentiate, capable of developing into specific cell types in various tissues and organs. Stem cells can be categorized as totipotent, pluripotent, multipotent, and unipotent. Pluripotent stem cells can differentiate into all cells from the three germ layers, forming all tissues and organs. They are used for a variety of tissue and organ repair, disease treatment, and drug screening, making them a hot topic in stem cell research. Cultured stem cells are also used to form, maintain, and expand organoids. Embryonic stem cells (ECs) and induced pluripotent stem cells (iPSCs) are the most studied pluripotent stem cells. However, developing stem cells is not simple. A key challenge lies in efficiently differentiating stem cells into cells with distinct functions. The stem cell niche controls the differentiation of stem cells into functional cells, but our understanding of the molecular mechanisms is limited. A suitable and stable niche is crucial for the cultivation and application of stem cells, including iPSCs.

Organoids are three-dimensional, stem-cell-derived cell populations that possess the multicellular, self-renewing characteristics of living tissue. Tumor organoids closely resemble a patient's tumor in terms of their tissue structure, cell types, and genetic profile. They can serve as surrogate models for drug testing and are amenable to passage and expansion. They maintain genomic stability and tumor heterogeneity, mimicking the tumor microenvironment and addressing the shortcomings of traditional cell and animal models.

Take Matrigel, a commonly used scaffold material, for example. This material is extracted from EHS mouse tumors, which are rich in extracellular matrix proteins. Its main components include laminin, type IV collagen, heparan sulfate glycoprotein, and entactin, as well as various growth factors and matrix metalloproteinases. At room temperature, Matrigel polymerizes to form a biologically active three-dimensional matrix that mimics the structure, composition, physical properties, and function of the in vivo cell basement membrane. This facilitates in vitro cell culture and differentiation, and can be used to study cell morphology, biochemical function, migration, invasion, and gene expression.

The current problems with basement membrane matrix gel include: 1) supply shortages due to animal welfare issues; 2) large and difficult to control batch-to-batch differences, with the nutrients and protein content greatly affected by batches; 3) animal-derived proteins and nucleic acids interfere with downstream experiments such as efficacy evaluation and fluorescence detection; and 4) the temperature-sensitive nature of the matrix gel itself places strict temperature requirements on storage and operation.

Summary of the Invention

In order to solve the above technical problems, the present invention provides the following technical solutions:

A cellulose derivative-based hydrogel comprises a matrix comprising a cellulose derivative, a network structure consisting of ultrafine cellulose derivative fibers, and an ordered three-dimensional micro-nano structure.

According to some embodiments of the present invention, the degree of substitution of the cellulose derivative is 0.1 to 3, preferably 0.5 to 2.5, and more preferably 1.0 to 2.0.

According to an embodiment of the present invention, the diameter of the cellulose derivative fiber is 0.01 μm to 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.

According to an embodiment of the present invention, the overall particle size of the matrix in the hydrogel is 1 μm-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.

According to an embodiment of the present invention, 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%.

According to an embodiment of the present invention, the cellulose derivative hydrogel is prepared by scheme 1 or scheme 2:

Scheme 1: 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 crushed by a homogenizer, colloid mill, or ball mill to obtain a cellulose derivative hydrogel;

Option 2: Prepare a cellulose solution; pour the cellulose solution into a coagulation bath to form a gel, crush the gel by a homogenizer, colloid mill or ball mill to obtain a cellulose hydrogel, and perform a derivatization reaction on the cellulose hydrogel to form a cellulose derivative hydrogel.

According to an embodiment of the present invention, 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.

According to some embodiments of the present invention, the cellulose ester includes but is not limited to cellulose monoester or cellulose mixed ester.

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

According to some embodiments of the present invention, the cellulose organic acid ester includes but is not limited to cellulose methyl ester, cellulose ethyl ester, cellulose propyl ester, cellulose butyl ester, to cellulose octadecyl ester and other saturated and unsaturated fatty acid esters; cellulose benzoate and other aromatic esters, cellulose lactate, cellulose citrate, cellulose carbamate, cellulose cyclohexylcarboxylate, cellulose cinnamate or cellulose 2-methylbenzoate, etc.

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

According to some embodiments of the present invention, the cellulose mixed ester includes but is not limited to at least one of cellulose acetate butyrate CAB, cellulose acetate propionate CAP, cellulose acetate 2-methylbenzoate mixed ester, cellulose 2-methylbenzoate-4-trifluoromethylbenzoate mixed ester or cellulose acetate adamantane carboxylate mixed ester.

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

According to some embodiments of the present invention, 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.

According to some embodiments of the present invention, 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.

According to some embodiments of the present invention, the oxidized cellulose includes but is not limited to at least one of dialdehyde cellulose, glycol cellulose or dicarboxyl cellulose.

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

According to some embodiments of the present invention, the cationic cellulose includes but is not limited to at least one of quaternary ammonium cellulose, imidazole cellulose or pyridyl cellulose.

According to some embodiments of the present invention, the anionic cellulose includes but is not limited to cellulose containing at least one of a carboxyl group, a hydroxide group, a sulfate group or a phosphate group.

According to some embodiments of the present invention, 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.

According to some embodiments of the present invention, other cellulose derivatives include but are not limited to at least one of cellulose containing a DOPO structure, alkylated cellulose, aminated cellulose, chlorinated cellulose, amidated cellulose, silanized cellulose or plasma-modified cellulose.

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

According to some embodiments of the present invention, the cellulose derivative is selected from methylcellulose, ethylcellulose, hydroxypropyl cellulose, carboxymethyl cellulose, cellulose propionate, cellulose butyrate, cellulose benzoate, cellulose cyclohexylcarboxylate, cellulose cinnamate, cellulose 2-methylbenzoate, cellulose acetate 2-methylbenzoate mixed esters, cellulose 2-methylbenzoate-4-trifluoromethylbenzoate mixed esters, cellulose acetate adamantanecarboxylate mixed esters, oxidized cellulose-dialdehyde cellulose or DOPO (9,10-dihydro-9-oxa-10-phosphaphenanthrene-10-oxide) cellulose derivatives.

The present invention also provides a method for preparing the above-mentioned cellulose derivative-based hydrogel, which is selected from the following scheme 1 or scheme 2:

Option 1: cellulose is homogeneously or heterogeneously derivatized 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 crushed by a homogenizer, colloid mill or ball mill to obtain a cellulose derivative hydrogel.

Option 2: Prepare a cellulose solution; pour the cellulose solution into a coagulation bath to form a gel, crush the gel by a homogenizer, colloid mill or ball mill to obtain a cellulose hydrogel; and perform a derivatization reaction on the cellulose hydrogel to obtain a cellulose derivative hydrogel.

According to an embodiment of the present invention, the solvents that can be used in the coagulation bath in Scheme 1 or Scheme 2 include water, ethanol, acetone, ethylene glycol, etc.

According to an embodiment of the present invention, the first solution specifically includes:

1) Cellulose pretreatment;

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

3) The cellulose derivative prepared in step 2) is prepared into a solution, poured into a coagulation bath to form a gel, and the gel is crushed by a homogenizer, a colloid mill, or a ball mill to obtain a cellulose derivative hydrogel.

According to an embodiment of the present invention, in step 1), the cellulose pretreatment includes dispersing the 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 the cellulose to a cellulose solvent to form a cellulose solution.

According to an embodiment of the present invention, step 1) further comprises activating the cellulose, specifically by adding a certain amount of alkali to activate the cellulose. For example, the alkali may be sodium hydroxide, potassium hydroxide, sodium carbonate, or sodium borohydride.

According to an embodiment of the present invention, in step 3), the solvent used to prepare the solution can be water, ethanol, acetone, DMF or NaOH aqueous solution.

According to an embodiment of the present invention, the second solution specifically includes:

1') preparing a cellulose solution;

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

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

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

According to the present invention, in the first and second embodiments, the cellulose solvent is any excellent solvent known in the art that can dissolve cellulose (the dissolution includes complete dissolution and partial dissolution). Preferably, the solvent can be selected from one or more of the following systems: copper ammonia solution, copper ethylenediamine solution, organic solvent, ionic liquid, mixed solvent of ionic liquid and organic solvent, choline-type ionic liquid deep eutectic solvent system, organic solvent/salt system, amine oxide system (NMMO), carbamate system, alkali/water system, alkali/urea system, alkali/thiourea system, liquid ammonia/ _NH4SCN , organic acid, aqueous solution of metal salt, alcohol solution of metal salt hydrate, water-alcohol mixed solution of metal salt hydrate, and the like.

The organic solvent may be selected from one or more of N,N-dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMAc), N-methyl-2-pyrrolidone (NMP), N-methylimidazole, imidazole, pyridine, ethylenediamine, hexafluoroacetone, hexafluoroisopropanol, glycerol, methyl isobutyl ketone, tetrahydrofuran, dioxane, and γ-valerolactone (GVL).

The organic solvent/salt system can be selected from one or more of N,N-dimethylacetamide/lithium chloride (DMAc/LiCl) system, N-methyl-2-pyrrolidone/NMP, and N,N-dimethyl sulfoxide/tetrabutylammonium fluoride system (DMSO/TBAF).

The alkali/water system may be selected from one or both of NaOH/H ₂ O and KOH/H ₂ O.

Wherein, the alkali/urea system can be selected from NaOH/Urea.

Wherein, the alkaline/thiourea system is selected from NaOH/thio-urea.

The organic acid can be selected from one or more of formic acid, acetic acid, propionic acid, butyric acid, succinic acid, lactic acid, glutamic acid, glycine, dichloroacetic acid, trichloroacetic acid, and toluenesulfonic acid.

The metal salt aqueous solution is preferably selected from aqueous solutions of metal salts such as CaCl ₂ , ZnCl ₂ , LiClO ₄ , Ca(SCN) ₂ , and LiSCN.

The alcohol solution of the metal salt hydrate may be selected from a methanol solution of CaBr ₂ ·H ₂ O and a methanol solution of CaCl ₂ ·2H ₂ O.

The water-alcohol mixed solution of the metal salt hydrate may be selected from a methanol aqueous solution of CaBr ₂ ·H ₂ O and a methanol aqueous solution of CaCl ₂ ·2H ₂ O.

The amine oxide system may be a NMMO/H ₂ O/DMSO system, a NMMO/H ₂ O/diethyltriamine system, or a NMMO/H ₂ O system.

The ionic liquid is selected from an organic molten salt formed by cations and anions with a melting point lower than 100° C., preferably an organic molten salt that can dissolve the biomass natural polymer.

For example, the cation of the ionic liquid is selected from one or more of substituted or unsubstituted imidazole, pyridine, pyrrole, amine, phosphine, choline, diazabicyclic, and amino acid type cations; for example, the substituent may be one or more of C _1-6 alkyl, C _1-6 alkenyl, phenyl, or substituted phenyl; preferably one or more of methyl, ethyl, butyl, and allyl;

Preferably, the cation is selected from one or more of the following cations: 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 ([HOE MIM]), 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-methylpyridinium 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]), tetrabutylamine ([TBA]), tetrabutylphosphine ([PBu ₄ ]), glycine cation ([Gly]), choline cation ([Ch]), 1,5-diazabicyclo[4.3.0]keto-5-ene ([DBNH]) and other cations.

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

For example, the anion is selected from one or more of halogen anions, organic acid radical ions, organic acid ester anions, amino acid type anions, and the like.

Preferably, the anion is selected from one or more of the following anions: chloride ([Cl]), bromide ([Br]), fluoride ([F]), formate ([HCOO]), acetate ([CH ₃ COO] or [Ac]), glycolate ([HOCH ₂ COO]), propionate ([CH ₃ CH ₂ COO] or [OPr]), butyrate ([CH ₃ CH ₂ CH ₂ COO] or [OBu]), octanoate ([Oct]), benzoate ([C ₆ H ₅ COO] or [PhCOO]), lactate ([CH ₃ CH(OH)COO] or [Lac]), thioglycolate ([HSCH ₂ COO]), hexafluorophosphate ([PF ₆ ]), trifluoroborate ([BF ₃ ]), methylphosphate ([(MeO)HPO ₂ ] or [MP]), dimethyl phosphate ion ([(MeO) ₂ PO ₂ ] or [DMP]), diethyl phosphate ion ([(EtO) ₂ PO ₂ ] or [DEP]), methanesulfonate anion ([MeOSO ₃ ]), trifluoromethylsulfonate anion ([CF ₃ SO ₃ ]), glycine anion ([Gly]), lysine anion ([Lys]), valine anion ([Val]), dicyanamide anion ([N(CN) ₂ ] or [DCA]), bistrifluoromethylsulfonimide ([Tf ₂ N]) and the like.

More preferably, the anion is selected from one or more of the following anions: chloride ion ([Cl]), formate ion ([HCOO]), acetate ion ([Ac]), methyl phosphate ion ([(MeO)HPO ₂ ] or [MP]), dimethyl phosphate ion ([(MeO) ₂ PO ₂ ] or [DMP]) and dicyanamide anion ([N(CN) ₂ ] or [DCA]).

According to an embodiment of the present invention, the ionic liquid can be selected from one or more of the following ionic liquids: 1-ethyl-3-methylimidazolium chloride ionic liquid ([EMIM][Cl]), 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 methyl phosphate ionic liquid ([EMIM][MP]), 1-ethyl-3-methylimidazolium dimethyl phosphate ionic liquid ([EMIM][DMP]), 1-ethyl-3-methylimidazolium diethyl phosphate ionic liquid ([EMIM][DEP]), 1-ethyl-3-methylimidazolium propionate ionic liquid ([EMIM][OPr]), 1-ethyl-3-methylimidazolium octanoate ionic liquid ([EMIM][ OBu]), 1-ethyl-3-methylimidazolium glycine salt ionic liquid ([EMIM][Gly]), 1-ethyl-3-methylimidazolium lysine salt ionic liquid ([EMIM][Lys]), 1-allyl-3-methylimidazolium chloride ionic liquid ([AMIM][Cl]), 1-allyl-3-methylimidazolium bromide ionic liquid ([AMIM][Br]), 1-allyl-3-methylimidazolium formate ionic liquid ([AMIM][HCOO]), 1-allyl-3-methyl Imidazolium acetate ionic liquid ([AMIM][Ac]), 1-butyl-3-methylimidazolium chloride ionic liquid ([BMIM][Cl]), 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 hydroxyacetate ionic liquid ([BMIM][HOCH ₂ COO]), 1-butyl-3-methylimidazolium propionate ionic liquid ([BMIM][CH ₃ CH ₂ COO]), 1-butyl-3-methylimidazolium lactate ionic liquid [BMIM][Lac], 1-butyl-3-methylimidazolium butyrate ionic liquid ([BMIM][CH ₃ CH ₂ CH ₂ COO]), 1-butyl-3-methylimidazolium benzoate ionic liquid ([BMIM][C ₆ H ₅ COO]), 1-butyl-3-methylimidazolium glycinate ionic liquid ([BMIM][H ₂ NCH ₂ COO]), 1-butyl-3-methylimidazolium dicyanamide ionic liquid ([BMIM][N(CN) ₂ ]), 1-butyl-3-methylimidazolium bistrifluoromethylsulfonyl imide ionic liquid ([BMIM][Tf ₂ N]), 1-butyl-3-methylimidazolium hexafluorophosphate ionic liquid ([BMIM][PF ₆ ]), 1-butyl-3-methylimidazolium tetrafluoroborate ionic liquid ([BMIM][BF ₄ ]), 1-butyl-3-methylimidazolium methanesulfonate ionic liquid ([BMIM][MeOSO ₃ ]), 1-butyl-3-methylimidazolium trifluoromethylsulfonate ionic liquid ([BMIM][CF ₃ SO ₃ ]), 1-butyl-2,3-dimethylimidazolium tetrafluoroborate ionic liquid ([BMMIM][BF ₄ ]), 3-methylimidazolium formate ionic liquid ([MIM][HCOO]), 1,3-dimethylimidazolium chloride ionic liquid ([MMIM][Cl]), 1,3-dimethylimidazolium methyl phosphate ionic liquid ([MMIM][MP]), 1,3-dimethylimidazolium dimethyl phosphate ionic liquid ([MMIM][DMP]), 1,3-dimethylimidazolium methanesulfonate ionic liquid ([MMIM][MeOSO ₃ ]), 1-hydroxy-3-methyl-imidazolium chloride ionic liquid ([HMIM][Cl]), 1-hydroxy-3-methyl-imidazolium trifluoromethanesulfonate ionic liquid ([HMIM][CF ₃ SO ₃ ]), 1-(2-hydroxyethyl)-3-methylimidazolium chloride ionic liquid ([HOEMIM][Cl]), 1-methoxymethyl-3-methylimidazolium bromide ionic liquid ([MeOMMIM][Br]), 1-methoxyethyl-3-methylimidazolium bromide ionic liquid ([MeOEMIM][Br]), N-ethylpyridinium chloride ionic liquid ([EPyr][Cl]), N-ethylpyridinium bromide ionic liquid ([EPyr][Br]), N-methylpicolinate ionic liquid ([MPyr][HCOO]), tris(2-hydroxyethyl)methylamine acetate ionic liquid ([THEMA][Ac]), tris(2-hydroxyethyl)methylamine methanesulfonate ionic liquid ([THEMA][MeOSO ₃ ]), tris(2-hydroxyethyl)methylamine trifluoromethanesulfonate ionic liquid [THEMA][CF ₃ SO ₃ ], tetrabutylphosphine valine salt ionic liquid [PBu ₄ ][Val], tetrabutylphosphine lysine salt ionic liquid [PBu ₄ ][Lys], tetrabutylphosphine glycinate ionic liquid [PBu ₄ ][Gly], 1-benzyl-3-methylimidazolium chloride ionic liquid ([BzMIM][C1]), 1-benzyl-3-methylimidazolium dicyanamide ionic liquid ([BzMIM][DCA]), 1-m-methylbenzyl-3-methylimidazolium chloride ionic liquid ([MeBzMIM][Cl]), 1-m-methoxybenzyl-3-methylimidazolium chloride ionic liquid ([MeOBzMIM][Cl]), choline chloride ionic liquid ([Ch][Cl]), choline bromide ionic liquid (Ch][Br]), choline acetate ionic liquid ([Ch][CH ₃ COO]), choline propionate ionic liquid ([Ch][CH ₃ CH ₂ COO]), choline butyrate ionic liquid ([Ch][CH ₃ CH ₂ CH ₂ COO]), glycine hydrochloride ionic liquid ([Gly][Cl]), 1,5-diazabicyclo[4.3.0]keto-5-ene acetate ionic liquid ([DBNH][Ac]) and other ionic liquids.

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 cyclohexylcarboxylate ([Bmim][ChCOO]), 1-butyl-3-methylimidazolium cinnamate ([Bmim][CCOO]), 1-butyl-3-methylimidazolium chloride (BmimCl), 1-allyl-3-methylimidazolium chloride ionic liquid (AmimCl), 1-allyl-3-methylimidazolium chloride ionic liquid (AmimCl)/N,N-dimethylacetamide (DMAc) (the mass ratio of AmimCl to DMAc is 9:1), and 1-ethyl-3-methylimidazolium chloride ionic liquid (EmimCl)/1-methylimidazole (Mim) (the mass ratio of EmimCl to Mim is 8:2).

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

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

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

According to an embodiment of the present invention, the basal culture fluid can be a basal culture fluid known in the art. Exemplarily, the basal culture fluid includes DMEM/F12 (GIBCO/10565018), 10% FBS (GIBCO/10091-148) and 1% P/S (GIBCO/15140122).

The present invention also provides a kit, which includes the above-mentioned 3D cell culture medium and instructions for use.

The present invention also provides a method for preparing the above-mentioned culture solution, which comprises the following steps:

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

S2: The cellulose derivative-based hydrogel in step S1 is mixed with a basic culture medium to prepare the culture medium for 3D cell culture.

According to an embodiment of the present invention, in step S2, the volume ratio of the cellulose derivative-based hydrogel to the basic culture medium is 1-10:1-10, for example, 1:1, 1:5, 1:10, 5:1 or 10:1.

The present invention also provides use of the culture solution in 3D cultured cells.

The present invention also provides a method for 3D cell culture, which is carried out in the above-mentioned 3D cell culture medium.

According to an embodiment of the present invention, the 3D culture method comprises the following steps:

a) Preparing a cell suspension: uniformly mixing the 3D cell culture medium with the cells to obtain a cell suspension;

b) 3D culture: The cell suspension from step a) is cultured in an incubator.

According to an embodiment of the present invention, in step a), the cells may further be subjected to cell recovery treatment and/or cell passage treatment.

Preferably, the cell recovery can be performed by any method known in the art, as long as the cells can be recovered, for example, according to the principle of rapid cell recovery. Exemplarily, the cell recovery specifically includes: re-dissolving the cells to be recovered in a 37°C water bath, centrifuging, resuspending the cells in a basal culture medium, and then culturing in an incubator (37°C, 5% _CO2 ) for, for example, 48 hours.

Preferably, the cells can be passaged using methods known in the art, as long as the desired cells can be obtained. For example, the culture medium is discarded after the cells have recovered, and the culture wells are gently washed twice with PBS. Then, single-cell suspensions are prepared using trypsin digestion. The cells are then resuspended in basal culture medium and adjusted to the desired cell density (e.g., 5K, 25K, 50K, etc.).

According to an embodiment of the present invention, the cells include human neuroblastoma cells, such as SH-SY5Y cells, liver cancer cells, such as HepaRG (liver-cancer cell) cells, and the like.

According to an embodiment of the present invention, in step b), the culture can be performed by methods known in the art as long as the desired cells can be obtained, for example, adding the cell suspension to a culture plate and culturing at 37°C and 5% _CO2 .

According to an embodiment of the present invention, in step b), the culturing time is 1 to 10 days, preferably 3 days.

According to an embodiment of the present invention, in step b), the culturing further comprises changing the medium, for example, changing the basal culture medium every 3 days.

The present invention also provides a culture solution for culturing tissues and organoids, wherein the culture solution comprises the above-mentioned cellulose derivative-based hydrogel and a composite culture solution.

According to an embodiment of the present invention, the composite culture solution includes a basal culture solution and active components.

According to an embodiment of the present invention, in every 100 mL of composite culture solution, the mass ratio of the basal culture solution to the active component is 80-99:1-20, for example, 97:3.

According to an embodiment of the present invention, the basal culture fluid can be a basal culture fluid known in the art. Exemplarily, the basal culture fluid includes DMEM/F12 (GIBCO/10565018), 10% FBS (GIBCO/10091-148) and 1% P/S (GIBCO/15140122).

According to an embodiment of the present invention, the composite culture medium contains 100-500 ng/ml of epidermal growth factor (Epirregulin), for example, 200 ng/ml, 300 ng/ml, 400 ng/ml, 500 ng/ml.

According to an embodiment of the present invention, in every 100 mL of composite culture solution, the active components include at least:

Penicillin-streptomycin (P/S) 0.1-2% (e.g., 1%);

N-2-Hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES) 1-20 mM (e.g., 10 mM);

L-glutamine additive (Glutamax) 0.1-2% (e.g. 1%);

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

N-Acetylcysteine 0.1-5 mM (e.g., 1.25 mM);

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

Noggin 50-200 ng/ml (e.g., 100 ng/ml);

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

WNT protein (Wnt 3A) 50-200 ng/ml (e.g., 100 ng/ml);

Human fibroblast growth factor 10 (FGF10) 100-500 ng/ml (e.g., 200 ng/ml);

Gastrin 0.1-10 nM (e.g., 1 nM);

ALK inhibitor (A-83-01) 0.1-10 μM (e.g., 2 μM);

ROCK inhibitor (Y-27632) 1-20 μM (e.g., 10 μM);

p38 MAPK inhibitor (SB202190) 1-20 μM (e.g., 10 μM);

Epidermal growth factor (Epirregulin) 100-500 ng/ml (e.g., 200 ng/ml);

Carotenoids (Capsanthin) 100-500 ng/ml (e.g., 200 ng/ml);

Nicotinamide 1-20 mM (for example, 10 mM).

According to an exemplary embodiment of the present invention, the composite culture solution comprises:

P/S 1%

HEPES 10mM

Glutamax 1%

B27 1%

N-Acetylcysteine 1.25mM

FGF-7 50ng/ml

Noggin 100ng/ml

R-spondin 1 500ng/ml

Wnt 3A 100ng/ml

FGF10 200ng/ml

Gastrin 1nM

A-83-01 2μM

Y-27632 10 μM

SB202190 10μM

Epirregulin 200ng/ml

Capsanthin 200ng/ml

Nicotinamide 10mM.

The present invention also provides a method for preparing the above-mentioned culture medium for culturing tissues and organoids, the preparation method comprising the following steps:

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

S2': The cellulose derivative-based hydrogel in step S1' is mixed with the composite culture medium to prepare the culture medium for culturing tissues and organoids.

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

The present invention also provides use of the above-mentioned culture medium for culturing tissues and organoids in culturing tissues or organoids.

The present invention also provides a method for culturing tissues or organoids, wherein the culturing method is carried out in the above-mentioned culture medium for culturing tissues and organoids.

According to an embodiment of the present invention, the tissue is preferably human gastric cancer tissue/normal gastric tissue, or human intestinal cancer tissue/normal intestinal tissue.

According to an embodiment of the present invention, the organoid is preferably human gastric cancer organoid/normal gastric organoid, human intestinal cancer organoid/normal intestinal organoid.

According to an embodiment of the present invention, the culture method comprises the following steps:

a') preparing a cell suspension of tissue or organoid: uniformly mixing the culture medium for cultured tissue or organoid with cells of the tissue or organoid to obtain a cell suspension;

b’) 3D culture: Culture the cell suspension from step a’) in an incubator.

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

According to an embodiment of the present invention, 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, adding the cell suspension to a culture plate and culturing at 37°C and 5% _CO2 .

According to an embodiment of the present invention, in step b'), the culturing time is 1 to 10 days, preferably 3 days.

According to an embodiment of the present invention, in step b'), the culturing further comprises changing the medium, for example, changing the basal culture medium every 3 days.

The present invention also provides a kit, which comprises the culture medium for culturing tissues and organoids, a compound culture medium and instructions for use.

According to an embodiment of the present invention, the kit is preferably used for culturing tissues and organoids. Specifically, the tissues are preferably human gastric cancer tissue/normal gastric tissue, or human intestinal cancer tissue/normal intestinal tissue. Specifically, the organoids are preferably human gastric cancer organoids/normal gastric organoids, or human intestinal cancer organoids/normal intestinal organoids.

According to an embodiment of the present invention, the composite culture medium contains 100-500 ng/ml of epidermal growth factor (Epirregulin), for example, 200 ng/ml, 300 ng/ml, 400 ng/ml, 500 ng/ml.

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

According to an embodiment of the present invention, the basal culture fluid can be any basal culture fluid known in the art. For example, the basal culture fluid includes DMEM/F12 and mTeSR ^™ 1.

The present invention also provides use of the above-mentioned stem cell culture medium in stem cell culture.

The present invention also provides a method for culturing stem cells, wherein the method uses the above-mentioned culture medium for stem cell culture.

According to an embodiment of the present invention, the stem cells are preferably adult stem cells or embryonic stem cells (ESCs) from mammals and primates; further preferably, they are pluripotent stem cells or multipotent stem cells, such as induced pluripotent stem cells (iPSCs), hematopoietic stem cells, neural stem cells, skin stem cells, mesenchymal stem cells, adipose stem cells, osteoblastic stem cells, cartilage stem cells, muscle stem cells, liver stem cells, pancreatic stem cells, endothelial stem cells, corneal stem cells, hair follicle stem cells, gastrointestinal stem cells, mammary stem cells, cardiac stem cells, etc.

According to an embodiment of the present invention, the stem cell culture method comprises the following steps:

a”) Pretreatment of stem cells before passage: dilution of fiber derivative hydrogel and placement in an incubator;

b”) Digestion of extracellular matrix: Dispase was used to digest the extracellular matrix;

c”) Add mTeSR ^™ 1 and pipette until the stem cells are detached and broken into fragments;

d”) Stem cell fragments were added to a culture dish containing a cellulose derivative-based hydrogel for culture.

According to an embodiment of the present invention, in step a"), the cellulose derivative-based hydrogel is diluted with DMEM/F12, added to a culture plate after dilution, and allowed to stand at 37°C for at least 1 hour.

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

According to an embodiment of the present invention, step b") further comprises the steps of aspirating Dispase and washing with DPBS.

According to an embodiment of the present invention, in step c"), the fragments are 1-2 mm.

According to an embodiment of the present invention, in step d"), culture can be performed using methods known in the art, such as supplementing with mTeSR ^™ 1 culture medium, culturing at 37°C and 5% CO2, and changing the medium.

The present invention also provides a culture medium for inducing stem cell differentiation into organoids, wherein the culture medium comprises the above-mentioned cellulose derivative-based hydrogel, definitive endoderm differentiation culture medium, MH differentiation culture medium, and organoid growth culture medium.

According to an embodiment of the present invention, the definitive endoderm differentiation culture medium comprises the following components:

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

According to an embodiment of the present invention, the definitive endoderm differentiation culture medium preferably comprises the following components:

RPMI1640, containing 2 mM L-glutamine,

Penicillin-Streptomycin 100U/ml-100g/ml, and Activin A 100ng/ml.

According to an embodiment of the present invention, the MH differentiation culture medium comprises the following components:

RPMI1640 contains 2% FBS, L-glutamine, penicillin-streptomycin, and FGF4.

According to an embodiment of the present invention, the MH differentiation culture medium preferably comprises the following components:

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

According to an embodiment of the present invention, the organoid growth medium comprises the following components:

Advanced DMEM/F12 contains 1×B27, L-glutamine, penicillin-streptomycin, HEPES buffer, and R-spondin 1.

According to an embodiment of the present invention, the organoid growth culture medium preferably comprises the following components:

Advanced DMEM/F12 contains 1×B27, L-glutamine, 2mM, penicillin-streptomycin, 100U/ml-100g/ml, HEPES buffer, 15mM, and R-spondin 1, 500ng/ml.

The present invention also provides use of the above-mentioned culture medium for inducing stem cell differentiation into organoids in inducing stem cell differentiation into organoids.

The present invention also provides a method for inducing stem cell differentiation into organoids, wherein the method comprises culturing a single stem cell or a group of stem cells in a culture medium for inducing stem cell differentiation into organoids.

According to an embodiment of the present invention, the method for inducing stem cell differentiation 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 organoid growth medium, and mixing of cellulose derivative hydrogel and spheroids followed by incubation.

According to an embodiment of the present invention, the definitive endoderm differentiation in step a') includes preparing a definitive endoderm differentiation culture medium and performing definitive endoderm differentiation, wherein the definitive endoderm differentiation step includes incubating the culture medium using a definitive endoderm differentiation culture medium using a method known in the art, such as incubation at 37°C, 5% _CO2 and 95% humidity.

According to an embodiment of the present invention, the midgut/hindgut (MH) differentiation in step a') includes the preparation of MH differentiation culture medium and midgut/hindgut (MH) differentiation, wherein the midgut/hindgut (MH) differentiation step includes incubating with MH differentiation culture medium using methods known in the art, such as incubating at 37°C, 5% _CO2 and 95% humidity, changing the medium and observing the spheroids every 24 hours, and embedding the spheroids.

According to an embodiment of the present invention, the cellulose derivative-based hydrogel and the spheroids in step b″′) are mixed and incubated using methods known in the art, for example, including: incubating at 37°C for less than 30 minutes, adding organoid growth medium and continuing incubation at 37°C, 5% CO2, and 95% humidity, and subculturing according to the growth of the organoids.

The present invention also provides a stem cell organoid obtained by the above method.

Specifically, the stem cell organoids include adult stem cell organoids, induced pluripotent stem cell organoids or embryonic stem cell organoids.

The present invention also provides a kit for inducing differentiation of organoids, which comprises the above-mentioned cellulose derivative-based hydrogel.

According to an embodiment of the present invention, the organoid is preferably an intestinal organoid, a gastric organoid, a liver organoid, and more preferably a small intestinal organoid.

According to an embodiment of the present invention, the kit for inducing differentiation of organoids further comprises a definitive endoderm differentiation culture medium, a MH differentiation culture medium, and an organoid growth culture medium.

According to an embodiment of the present invention, the kit for inducing differentiation of organoids further includes components or assemblies known in the art, which are not specifically limited in the present invention.

The present invention also provides a use of the differentiation-inducing organoid kit in inducing differentiation of organoids.

According to an embodiment of the present invention, the induced differentiated organoids are, for example, induced differentiated intestinal organoids, gastric organoids, liver organoids, preferably small intestinal organoids.

Beneficial effects of the present invention:

The present invention uses cellulose derivatives as raw materials to prepare cellulose derivative-based hydrogels, which are ready-to-use hydrogels that do not contain animal-derived components. Specifically, the matrix of the hydrogel includes cellulose derivatives, and the matrix includes a network structure composed of ultrafine cellulose derivative fibers, and the network structure includes an ordered three-dimensional micro-nanostructure. Compared with animal-derived matrix glue, it has incomparable advantages in performance, ease of use, batch-to-batch stability, process amplification stability, and automation compatibility.

The cellulose derivative-based hydrogel of the present invention has superior transparency, making it easier to observe cell status. It is suitable for 3D cell culture, such as SH-SY5Y and HepaRG cells, and for the 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 organoids/normal gastric organoids, or human intestinal cancer organoids/normal intestinal organoids.

The cellulose derivatives of the present invention are non-toxic to cells. Cellulose derivative-based hydrogels, as a stem cell growth platform, exhibit excellent rigidity, adhesion, and porosity. Stem cells cultured in these hydrogels can form clusters. Using these hydrogels for stem cell and organoid cultivation is simple, forming a dense, fused monolayer of endoderm cells that inhibits non-directional stem cell differentiation. The resulting organoid vesicles have thick, solid walls, resulting in mature, differentiated organoids suitable for high-throughput drug screening, drug toxicity testing, and regenerative medicine.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1a Scanning electron microscopy image of methylcellulose MC-1 hydrogel.

Figure 1a-1 is a microscopic image of gastric cancer organoids cultured on methylcellulose hydrogel MC-1 (concentration 0.2%) on day 0.

Figure 1a-2 is a micrograph of intestinal organoids 3 days after induction of differentiation of human pluripotent stem cells/embryonic stem cells in Example 7.

Figure 1b Micrograph of MC-1 cell suspension in methylcellulose hydrogel after culturing SH-SY5Y cells for 1 day.

Figure 1b-1 is a micrograph of gastric cancer organoids cultured on methylcellulose hydrogel MC-1 (concentration 0.2%) for 5 days.

Figure 1b-2 is a micrograph of intestinal organoids 7 days after induction and differentiation of human pluripotent stem cells/embryonic stem cells in Example 7.

Figure 1c Microscopic image of MC-1 cell suspension in methylcellulose hydrogel after culturing SH-SY5Y cells for 3 days.

Figure 1c-1 is a microscopic image of gastric cancer organoids cultured on methylcellulose hydrogel MC-1 (concentration 0.2%) for 7 days.

Figure 1c-2 is a micrograph of intestinal organoids 12 days after induction of differentiation of human pluripotent stem cells/embryonic stem cells in Example 7.

Figure 1d Micrograph of MC-1 cell suspension in methylcellulose hydrogel after culturing SH-SY5Y cells for 5 days.

Figure 1d-1 Microscopic image of gastric cancer organoids cultured on cellulose benzoate hydrogel (concentration 0.2%) on day 0.

Figure 1e Microscopic image of MC-1 cell suspension in methylcellulose hydrogel after culturing SH-SY5Y cells for 7 days.

Figure 1e-1 Microscopic image of gastric cancer organoids cultured on cellulose benzoate hydrogel (concentration 0.2%) for 5 days.

Figure 1f is a micrograph of gastric cancer organoids cultured on cellulose benzoate hydrogel (concentration 0.2%) for 7 days.

Figure 2a Microscopic image of SH-SY5Y cells cultured on methylcellulose hydrogel MC-13D for 5 days.

Figure 2a-1 is a microscopic image of gastric cancer organoids cultured on methylcellulose hydrogel MC-1 (concentration 0.3%) for 5 days.

Figure 2a-2 is a micrograph of iPSCs cultured with 0.8% cellulose derivatives in Comparative Example 3.

Figure 2b is a micrograph of SH-SY5Y cells cultured on the plant cellulose matrix gel GrowDex for 5 days.

Figure 2b-1 Microscopic image of gastric cancer organoids cultured on Matrigel for 5 days.

Figure 2b-2 is a micrograph of iPSCs cultured on Matrigel (Corning 354277) in Comparative Example 3.

Figure 2c: Microscopic image of gastric cancer organoids cultured in GrowDex, a plant cellulose matrix gel, for 5 days.

FIG3 a is a micrograph of HepaRG (liver cancer cell) cells cultured in 3D methylcellulose MC-1 hydrogel for 7 days.

Figure 3a-1 Microscopic image of intestinal cancer organoids cultured on carboxymethyl cellulose hydrogel CMC-1 for 7 days.

Figure 3a-2 is a micrograph of intestinal organoids induced and differentiated from human pluripotent stem cells/embryonic stem cells after 12 days of culture using the inducing differentiation intestinal organoid kit containing 0.8% cellulose derivatives in Comparative Example 4.

Figure 3b is a micrograph of HepaRG (liver-cancer cell) cells cultured in cellulose propionate hydrogels in 3D culture for 7 days.

Figure 3b-1 Microscopic image of colorectal cancer organoids cultured on cellulose acetate adamantane carboxylate mixed ester hydrogel for 7 days.

FIG3 b-2 is a micrograph of intestinal organoids after 12 days of induction and differentiation of human pluripotent stem cells/embryonic stem cells using the STEMdiff ^™ Intestinal Organoid Kit (stem cell, 05140) in Comparative Example 4.

Figure 3c is a micrograph of HepaRG (liver cancer cell) cells cultured in 3D on oxidized cellulose-dialdehyde cellulose hydrogel for 7 days.

Figure 3c-1 Microscopic image of intestinal cancer organoids cultured on oxidized cellulose-dialdehyde cellulose hydrogel for 7 days.

Figure 3d is a micrograph of HepaRG (liver cancer cells) cultured in cellulose acrylate hydrogel 3D for 7 days.

Figure 3d-1 Microscopic image of intestinal cancer organoids cultured on cellulose acrylate hydrogel for 7 days.

Figure 4a Micrograph of intestinal organoids cultured on carboxymethyl cellulose (CMC-1) hydrogels after 12 days.

Figure 4b: Microscopic image of intestinal organoids cultured on cellulose acetate mixed ester hydrogel for 12 days.

Figure 4c Microscopic image of intestinal organoids cultured on oxidized cellulose-diacetic cellulose hydrogel for 12 days.

Figure 4d Micrograph of intestinal organoids cultured on cellulose acrylate hydrogel for 12 days.

DETAILED DESCRIPTION

The technical solutions of the present invention will be described in further detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explain the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are encompassed within the scope of protection that the present invention is intended to protect.

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

The present invention will be further described below by means of specific embodiments:

Preparation Example 1

(1) Preparation of cellulose ether hydrogel:

1) Preparation of methylcellulose MC hydrogel

20g of wood pulp was dispersed in a solvent, which could be water, DMF, ethanol, or isopropanol. A certain amount of alkali was added to activate the cellulose for 30 minutes. The alkali could be sodium hydroxide, potassium hydroxide, sodium carbonate, or sodium borohydride. Then, a certain amount of dimethyl sulfate was added. The molar ratio of dimethyl sulfate to cellulose could be 1:1, 2:1, 3:1, or 4:1. The reaction was allowed to proceed at room temperature for 24 hours. Aqueous acetic acid was then added to neutralize the alkali in the reaction mixture. The reaction mixture was filtered, and the resulting filter cake was washed 6-8 times with a solution, such as water, ethanol, acetone, DMF, or aqueous NaOH, to remove any residual impurities. The washed methylcellulose was dispersed in pure water at a water to methylcellulose mass ratio of 9:1. The gel was then crushed and pulverized using a colloid mill or high-pressure homogenizer to a particle size of 1-50μm and a solids content of 0.1-3.0wt%. After autoclaving, hydrogels MC-1 (see Figure 1a), MC-2, MC-3, and MC-4 were obtained.

2) Preparation of ethyl cellulose EC hydrogel

Disperse 20g of wood pulp in a solvent such as water, DMF, ethanol, or tetrahydrofuran. Add a suitable amount of alkali to activate the cellulose for 30 minutes. The alkali can be sodium hydroxide, potassium hydroxide, sodium carbonate, or sodium borohydride. Then, add a suitable amount of an ethylating agent such as ethyl bromide, ethyl chloride, chloroacetyl chloride, or diethyl sulfate. The molar ratio of ethylating agent to cellulose should be set at 1:1, 2:1, 3:1, or 4:1. Allow the reaction to react at room temperature for 24 hours. Aqueous acetic acid is then added to neutralize the alkali in the reaction mixture. Filter the reaction mixture, and wash the resulting filter cake 6-8 times with a solution such as water, ethanol, acetone, DMF, or aqueous NaOH to remove any remaining impurities. The washed ethyl cellulose was dispersed in pure water with a mass ratio of water to ethyl cellulose of 9:1. The gel was then crushed and pulverized using a colloid mill, a high-pressure homogenizer, or the like to a particle size of 1-50 μm and a solid content of 0.1-3.0 wt%. After high-pressure sterilization, hydrogels EC-1, EC-2, EC-3, and EC-4 were obtained.

3) Preparation of Hydroxypropyl Cellulose (HPC) Hydrogel

Disperse 20g of wood pulp in a solvent such as water, DMF, ethanol, or tetrahydrofuran. Add a suitable amount of alkali to activate the cellulose for 2 hours. The alkali can be sodium hydroxide, potassium hydroxide, sodium carbonate, or sodium borohydride. Then, add a suitable amount of propylene oxide, with a molar ratio of propylene oxide to cellulose of 1:1, 2:1, 3:1, or 4:1. The reaction is carried out at 80°C for 8 hours. After the reaction is complete, cool to room temperature and then add aqueous acetic acid to neutralize the alkali. Filter the reaction mixture and wash the resulting filter cake 6-8 times with a solution such as water, ethanol, acetone, DMF, or aqueous NaOH to remove any remaining impurities. The washed hydroxypropyl cellulose was dispersed in pure water with a mass ratio of water to hydroxypropyl cellulose of 9:1. The gel was then crushed and pulverized using a colloid mill, a high-pressure homogenizer, or the like to a particle size of 1-50 μm and a solid content of 0.1-3.0 wt%. After high-pressure sterilization, hydrogels HPC-1, HPC-2, HPC-3, and HPC-4 were obtained.

4) Preparation of carboxymethyl cellulose (CMC) hydrogel

Disperse 20g of wood pulp in a solvent such as water, isopropanol, ethanol, or tetrahydrofuran. Add a suitable amount of alkali to activate the cellulose for 2 hours. The alkali can be sodium hydroxide, potassium hydroxide, sodium carbonate, or sodium borohydride. Then, add 11.6g of chloroacetic acid, using a molar ratio of 0.5:1, 1:1, 1.5:1, or 2:1. The reaction is allowed to proceed at 70°C for 8 hours. After cooling to room temperature, aqueous acetic acid is added to neutralize the alkali. The reaction mixture is filtered, and the resulting filter cake is washed 6-8 times with a solution such as water, ethanol, acetone, DMF, or aqueous NaOH to remove any remaining impurities. The washed carboxymethyl cellulose was dispersed in pure water with a mass ratio of water to carboxymethyl cellulose of 9:1. The gel was then crushed and pulverized using a colloid mill, a high-pressure homogenizer, or the like to a particle size of 1-50 μm and a solid content of 0.1-3.0 wt%. After high-pressure sterilization, hydrogels CMC-1, CMC-2, CMC-3, and CMC-4 were obtained.

(2) Preparation of cellulose ester hydrogel

1) Preparation of cellulose propionate hydrogel

Weigh 4.0 g of cellulose, which may be microcrystalline cellulose, cotton pulp, wood pulp, inulin, soluble starch, or dextran, and add it to 46 g of ionic liquid, which may be 1-allyl-3-methylimidazole propionate ([Amim][CH3CH2COO]), 1-ethyl-3-methylimidazole butyrate ([Emim][CH3CH2CH2COO]), 1-butyl-3-methylimidazole cyclohexylcarboxylate ([Bmim][ChCOO]), 1-butyl 3-Methylimidazolium cinnamate ([Bmim][CCOO]) and 1-butyl-3-methylimidazolium chloride (BmimCl) were stirred at 80°C for 30 minutes to obtain an 8 wt% solution. A certain amount of propionic anhydride was added to the solution at a molar ratio of propionic anhydride to cellulose of 1:1, 2:1, 3:1, and 4:1 at 80°C and stirred for 2 hours. The reaction solution was poured into water for precipitation and the product was washed 6-8 times with a solution such as water, ethanol, DMF, or aqueous NaOH to remove residual impurities. The washed cellulose propionate gel was dispersed in pure water at a water to cellulose propionate gel mass ratio of 9:1. The gel was then crushed and pulverized using a colloid mill, high-pressure homogenizer, or the like to a particle size of 1-50 μm and a solid content of 0.1-3.0 wt%. The cellulose propionate hydrogel was sterilized by autoclave.

2) Preparation of cellulose butyrate hydrogel

Weigh 4.0 g of cellulose, which may be microcrystalline cellulose, cotton pulp, wood pulp, inulin, soluble starch, or dextran, and add it to 46 g of ionic liquid, which may be 1-allyl-3-methylimidazole propionate ([Amim][CH3CH2COO]), 1-ethyl-3-methylimidazole butyrate ([Emim][CH3CH2CH2COO]), 1-butyl-3-methylimidazole cyclohexylcarboxylate ([Bmim][ChCOO]), 1-butyl 3-Methylimidazolium cinnamate ([Bmim][CCOO]) and 1-butyl-3-methylimidazolium chloride (BmimCl) were stirred at 80°C for 30 minutes to obtain an 8 wt% solution. A certain amount of butyric anhydride was added to the solution at a molar ratio of butyric anhydride to cellulose of 1:1, 2:1, 3:1, and 4:1 at 80°C and stirred for 2 hours. The reaction solution was poured into water for precipitation and the product was washed 6-8 times with a solution of water, ethanol, DMF, or aqueous NaOH to remove residual impurities. The washed cellulose butyrate gel was dispersed in pure water at a water to cellulose butyrate gel mass ratio of 9:1. The gel was then crushed using a colloid mill, high-pressure homogenizer, or other equipment to a particle size of 1-50 μm and a solids content of 0.1-3.0 wt%. The cellulose butyrate hydrogel was sterilized by autoclave.

3) Preparation of cellulose benzoate hydrogel

Weigh 4.0 g of cellulose, which may be microcrystalline cellulose, cotton pulp, wood pulp, inulin, soluble starch, or dextran, and add it to 46 g of ionic liquid, which may be 1-allyl-3-methylimidazole propionate ([Amim][CH3CH2COO]), 1-ethyl-3-methylimidazole butyrate ([Emim][CH3CH2CH2COO]), 1-butyl-3-methylimidazole cyclohexylcarboxylate ([Bmim][ChCOO]), 1-butyl- 3-Methylimidazolium cinnamate ([Bmim][CCOO]) and 1-butyl-3-methylimidazolium chloride (BmimCl) were stirred at 80°C for 30 minutes to obtain an 8 wt% solution. Benzoyl chloride was added to the solution at a molar ratio of 1:1, 2:1, 3:1, or 4:1 at 80°C and stirred for 2 hours. The reaction solution was poured into water for precipitation. The product was washed 6-8 times with a solution containing water, ethanol, DMF, or aqueous NaOH to remove residual impurities. The washed cellulose benzoate gel was dispersed in pure water at a water:cellulose benzoate ratio of 9:1. The gel was then crushed using a colloid mill, high-pressure homogenizer, or other equipment to a particle size of 1-50 μm and a solids content of 0.1-3.0 wt%. The cellulose benzoate hydrogel was sterilized by autoclaving.

4) Preparation of cellulose cyclohexylcarboxylate hydrogel

Weigh 4.0 g of cellulose, which may be microcrystalline cellulose, cotton pulp, wood pulp, inulin, soluble starch, or dextran, and add it to 46 g of ionic liquid, which may be 1-allyl-3-methylimidazole propionate ([Amim][CH3CH2COO]), 1-ethyl-3-methylimidazole butyrate ([Emim][CH3CH2CH2COO]), 1-butyl-3-methylimidazole cyclohexylcarboxylate ([Bmim][Ch ... Methylimidazole cinnamate ([Bmim][CCOO]) and 1-butyl-3-methylimidazolium chloride (BmimCl) were stirred at 80°C for 30 minutes to obtain an 8 wt% solution. A certain amount of cyclohexylcarbonyl chloride was added to the solution at a molar ratio of cyclohexylcarbonyl chloride to cellulose of 1:1, 2:1, 3:1, and 4:1 at 80°C and stirred for 2 hours. The reaction solution was poured into water for precipitation. The product was washed 6-8 times with a solution of water, ethanol, DMF, or aqueous NaOH to remove residual impurities. The washed cellulose cyclohexylcarbonyl gel was dispersed in pure water at a mass ratio of 9:1. The gel was then crushed using a colloid mill or high-pressure homogenizer to a particle size of 1-50 μm and a solids content of 0.1-3.0 wt%. The solution was then sterilized by autoclave to obtain the cellulose cyclohexylcarbonyl hydrogel.

5) Preparation of cellulose cinnamate hydrogel

Weigh 4.0 g of cellulose, which may be microcrystalline cellulose, cotton pulp, wood pulp, inulin, soluble starch, or dextran, and add it to 46 g of ionic liquid, which may be 1-allyl-3-methylimidazole propionate ([Amim][CH3CH2COO]), 1-ethyl-3-methylimidazole butyrate ([Emim][CH3CH2CH2COO]), 1-butyl-3-methylimidazole cyclohexylcarboxylate ([Bmim][ChCOO]), 1-butyl- 3-Methylimidazole cinnamate ([Bmim][CCOO]) and 1-butyl-3-methylimidazole chloride (BmimCl) were stirred at 80°C for 30 minutes to obtain an 8 wt% solution. Cinnamoyl chloride was added to the solution at a molar ratio of cinnamoyl chloride to cellulose of 1:1, 2:1, 3:1, and 4:1 at 80°C and stirred for 2 hours. The reaction solution was poured into water for precipitation. The product was washed 6-8 times with a solution of water, ethanol, DMF, or aqueous NaOH to remove residual impurities. The washed cellulose cinnamoyl ester gel was dispersed in pure water at a water:cellulose cinnamoyl ester gel weight ratio of 9:1. The gel was then crushed using a colloid mill, high-pressure homogenizer, or other methods to a particle size of 1-50 μm and a solids content of 0.1-3.0 wt%. The product was then sterilized by autoclave to obtain a cellulose cinnamoyl ester hydrogel.

6) Preparation of cellulose 2-methylbenzoate hydrogel

Weigh 8g of cellulose. The cellulose that can be used includes microcrystalline cellulose, wood pulp, and cotton pulp, and dissolve it in 92g of ionic liquid. The ionic liquid can be 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- A methylimidazole chloride ionic liquid (EmimCl)/1-methylimidazole (Mim) (EmimCl:Mim mass ratio of 8:2) is added to a certain amount of 2-methylbenzoyl chloride and a catalytic amount of pyridine. The mass ratio of cellulose to 2-methylbenzoyl chloride can be 1:5, 1:6, or 1:7. The reaction is carried out at 80°C for 2 hours. After the reaction, the precipitate is poured into methanol and washed 6-8 times with a solution of water, ethanol, DMF, or aqueous NaOH to remove residual impurities. The washed cellulose 2-methylbenzoate gel is dispersed in pure water at a mass ratio of 9:1. The gel is then crushed and pulverized using a colloid mill, high-pressure homogenizer, or other equipment to a particle size of 1-50 μm. The cellulose 2-methylbenzoate hydrogel is sterilized by autoclaving.

7) Preparation of Cellulose Acetate 2-Methylbenzoate Mixed Ester Hydrogel

Weigh 8g of cellulose. The cellulose that can be used includes microcrystalline cellulose, wood pulp, and cotton pulp, and dissolve it in 92g of ionic liquid. The ionic liquid can be 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-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-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-allyl-3-methylimidazolium chloride ionic liquid (EmimCl), 1-butyl-3-methylimidazolium chloride ionic liquid (B ... To prepare a prepolymer of cellulose acetate 2-methylbenzoate (Citriloyl) and 1-methylimidazole (Mim) (the mass ratio of Citriloyl) to 1-methylimidazole (Mim) is 8:2. 42.94 g 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 continued at 80°C for 2 h. 4.85 g of acetyl chloride is then added and the reaction is continued at 80°C for 2 h. After the reaction is complete, the mixture is precipitated in methanol. The product is washed 6-8 times with a solution such as water, ethanol, DMF, or aqueous NaOH to remove residual impurities. The washed Citriloyl 2-methylbenzoate mixed ester gel is dispersed in pure water at a mass ratio of 9:1. The gel is then crushed and pulverized to a particle size of 1-50 μm using a colloid mill or high-pressure homogenizer. The cellulose acetate 2-methylbenzoate mixed ester hydrogel is sterilized by autoclaving.

8) Preparation of 2-Methylbenzoic Acid Cellulose-4-Trifluoromethylbenzoate Mixed Ester Hydrogel

Weigh 8g of cellulose and dissolve it in 92g of ionic liquid. The cellulose that can be used includes microcrystalline cellulose, wood pulp, and cotton pulp. The ionic liquid can be 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), Cl)/1-methylimidazole (Mim) (the mass ratio of EmimCl to Mim is 8:2), 42.94 g 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°C for 2 hours. 4.85 g of 4-trifluoromethylbenzoyl chloride is then added and the reaction is continued at 80°C for 2 hours. After the reaction is completed, the mixture is poured into methanol for precipitation. The product is washed 6-8 times with a solution such as water, ethanol, DMF, or aqueous NaOH to remove residual impurities. The washed cellulose 2-methylbenzoate-4-trifluoromethylbenzoate mixed ester gel is dispersed in pure water at a mass ratio of water to cellulose 2-methylbenzoate-4-trifluoromethylbenzoate mixed ester gel of 9:1. The gel is then crushed and pulverized to a particle size of 1-50 μm using a colloid mill, a high-pressure homogenizer, etc., and sterilized under high pressure to obtain a 2-methyl benzoate cellulose-4-trifluoromethyl benzoate mixed ester hydrogel.

9) Cellulose acetate and adamantane carboxylate mixed ester hydrogel

Add 20g of cellulose to 480g of an ionic liquid and mechanically stir at 80°C for 2 hours to dissolve the cellulose. The cellulose can be microcrystalline cellulose, refined cotton, cotton pulp, or wood pulp. The ionic liquid can be one of allylmethylimidazole chloride, butylmethylimidazole acetate, or ethylmethylimidazole chloride. Add 34g of pyridine as a cosolvent and stir for 5 minutes. The molar ratio of pyridine to anhydroglucose units in the cellulose is 3.5:1. Add acetic anhydride and adamantanecarbonyl chloride simultaneously and mechanically stir for 2 hours, maintaining the temperature at 80°C. The molar ratio of adamantanecarbonyl chloride to acetic anhydride to anhydroglucose units in the cellulose can be 1:1, 2:1, 3:1, or 4:1. After the reaction is complete, pour the reaction solution into a coagulation bath to precipitate the product. Purify it with DMSO. Wash the gel 6-8 times with a solution such as water, ethanol, acetone, or DMF to remove any residual DMSO. The washed cellulose acetate adamantane carboxylate mixed ester gel is dispersed in pure water with a mass ratio of water to cellulose acetate adamantane carboxylate mixed ester gel of 9:1. The gel is then crushed and then crushed using a colloid mill, a high-pressure homogenizer, or the like to a particle size of 1-50 μm and a solid content of 0.1-3.0 wt%. After high-pressure sterilization, the cellulose acetate adamantane carboxylate mixed ester hydrogel is obtained.

(3) Preparation of oxidized cellulose-dialdehyde cellulose hydrogel

Weigh 30g of cellulose and add it to 570g of an ionic liquid. Mechanically stir at 80°C for 2 hours to dissolve the cellulose. The cellulose can be microcrystalline cellulose, refined cotton, cotton pulp, or wood pulp. The ionic liquid can be one of allylmethylimidazole chloride, butylmethylimidazole acetate, and ethylmethylimidazole chloride. After tightly wrapping the reaction flask with tin foil to protect it from light, add a predetermined amount of sodium periodate and stir at 80°C for 10 hours. The molar ratio of sodium periodate to cellulose can be 0.02:1, 0.05:1, 0.1:1, or 0.2:1. After the reaction, add deionized water to precipitate and wash. Filter to obtain the crude product, which is then washed 6-8 times with a solution to remove any remaining impurities. Suitable solutions include water, ethanol, acetone, and ethylene glycol. The washed dialdehyde cellulose gel is dispersed in pure water with a mass ratio of water to dialdehyde cellulose gel of 9:1. The gel is then crushed and pulverized using a colloid mill, a high-pressure homogenizer, or the like to a particle size of 1-50 μm and a solid content of 0.1-3.0 wt%. The dialdehyde cellulose hydrogel is obtained after high-pressure sterilization.

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

1) Preparation of Cellulose Acrylate: 10 g of cellulose was added to 190 g of the ionic liquid 1-allyl-3-methylimidazolium chloride (AmimCl) and mechanically stirred at 80°C for 2 hours to dissolve the cellulose. The cellulose could be microcrystalline cellulose, refined cotton, cotton pulp, or wood pulp. The cellulose solution was placed in a 50°C oil bath and allowed to stabilize for half an hour. Then, 2.8 g of acryloyl chloride was added and mechanically stirred at 300 rpm for 2 hours. After the reaction, the intermediate product, cellulose acrylate, was obtained by precipitation, washing, and drying.

2) Preparation of DOPO-type cellulose derivative matrix gel: A certain amount of DOPO is dissolved in DMSO to prepare a solution with a mass fraction of 10%. A base is added as a catalyst. 10 g of cellulose acrylate from step (1) is dissolved in 190 g of DMSO and slowly added dropwise to the DOPO solution. The reaction is carried out at 80°C for 12 hours. The molar ratio of DOPO to cellulose can be 1:1, 2:1, 3:1, or 4:1. The base can be sodium carbonate, triethylamine, sodium borohydride, or sodium hydride. The reaction solution is poured into methanol for precipitation. The product is 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 is dispersed in pure water with a mass ratio of water to DOPO-type cellulose derivative gel of 9:1. The gel is then crushed and pulverized using a colloid mill, a high-pressure homogenizer, or the like to a particle size of 1-50 μm and a solid content of 0.1-3.0 wt%. The DOPO-type cellulose derivative hydrogel is obtained after autoclaving.

Part I: Cellulose derivative hydrogels for 3D cell culture experiments:

Example 1

The cell culture method is as follows:

1) Cell Passaging: Commercially available SH-SY5Y cells were selected and revived, cultured in 10 cm culture dishes, and passaged when a confluency of 80% or greater was achieved. The original culture medium in the culture dish was discarded, and the dish was gently washed twice with PBS. A single-cell suspension was then prepared by trypsinization and centrifugation at 1200 rpm at room temperature for 5 min. The cells were resuspended in basal culture medium (composed of: DMEM/F12, 10% FBS, 1% P/S; unless otherwise specified in the following examples, this basal culture medium was used) and adjusted to the desired cell density of 2 × 10^4 cells/μL to obtain SH-SY5Y cells for experimental use.

2) Preparation of methylcellulose culture solution: The methylcellulose hydrogel MC-1 prepared in Preparation Example 1 was diluted with the basal culture solution in step 1) to obtain a methylcellulose culture solution with a methylcellulose concentration of 0.2%.

3) Cell suspension mixing: Take 500 μL of the methylcellulose culture medium from step 2) and mix it with 500 μL of the basal culture medium at a volume ratio of 1:1. Add 10 μL of the experimental SH-SY5Y cells from step 1) and mix well to obtain a cell suspension.

4) 3D culture: 1 mL of the cell suspension mixed in step 3) was added to a 12-well culture plate and cultured in a 37°C, 5 vol% _CO2 incubator.

5) Medium exchange: The culture medium was changed after 3 days of culture, and the culture medium was changed every 3 days. Specifically, the culture plate containing cells in step 4) was centrifuged at 1200 rpm at room temperature for 5 minutes, the culture medium in the upper layer of the culture plate was discarded, and new basal culture medium was added.

Microscopic images of the cell suspension after culturing for 1 day, 3 days, 5 days, and 7 days are shown in Figure 1b, Figure 1c, Figure 1d, and Figure 1e, respectively.

Comparative Example 1

SH-SY5Y cells were 3D cultured using the methylcellulose hydrogel MC-1 of the present invention and the plant cellulose matrix gel GrowDex (UPM/100103005) for 5 days, respectively. The results are shown in Figures 2a and 2b.

Example 2: The operation was the same as that of Example 1, except that the cultured cells were HepaRG (liver cancer cells), and the hydrogels were methylcellulose MC-1 hydrogel, cellulose propionate hydrogel, oxidized cellulose-dialdehyde cellulose hydrogel, and cellulose acrylate hydrogel. Micrographs of the above cell suspensions after 7 days of culture are shown in Figures 3a, 3b, 3c, and 3d, respectively.

Part II: Cellulose derivative-based hydrogels for tissue and organoid culture experiments

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

P/S Penicillin-Streptomycin, GIBCO/15140122;

HEPESN-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid, GIBCO/15630080;

L-glutamine supplement Glutamax, GIBCO/35050061;

Neuronal Cell Culture Supplement B27, GIBCO/17504044;

N-Acetylcysteine, Sigma/A9165-100G;

Fibroblast growth factor 7FGF-7, mce/HY-P7047A;

Noggin, mce/HY-P70558;

R-spondin 1 R-spondin 1, PeproTech/120-38;

WNT protein Wnt 3A, PeproTech/315-20;

Human fibroblast growth factor 10FGF10, PeproTech/100-26;

Gastrin, mce/HY-P1097;

ALK inhibitors A-83-01, mce/HY-10432;

ROCK inhibitor Y-27632, mce/HY-10071;

p38MAPK inhibitor SB202190, mce/HY-10295;

Epirregulin, mce/HY-P7011;

Carotenoid Capsanthin, mce/HY-125711;

Nicotinamide, Sigma/N0636.

Example 3

1. The formula of the composite culture solution is as follows: the composite culture solution is obtained by mixing the following components:

2. Prepare the tissue to be cultured

(1) Tissue cleaning

Transfer the tissue block (e.g., gastric cancer organoids, hospital surgical tissue, or biopsy tissue) to a 50 ml centrifuge tube, add 15 ml of washing solution (PBS + 2×P/S), and shake and wash for at least 5 times, each time for 6 minutes, until completely clear.

(2) Tissue separation

Transfer the tissue block to a 10 cm culture dish, add a small amount of cleaning solution to ensure the tissue surface is moistened, and use a sterile scalpel and blade to mechanically separate the tissue until it becomes small fragments or a paste of approximately 0.5×0.5×0.5 mm^3.

(3) Tissue digestion

Transfer the mechanically separated tissue completely into a 15 ml sterile centrifuge tube, add 2-5 ml of tissue digestion solution (collagenase P (1 mg/ml) + DNase (1 mg/ml)) according to the amount of tissue, and gently blow the tissue with a 1 ml pipette tip to fully disperse it; place the centrifuge tube containing the digestion solution on a shaker in a constant temperature air bath at 37°C and 200 rpm. After most of the tissue is separated and digested into 3-10 cell clusters, add 10% FBS to terminate the digestion.

(4) Cell filtration

Filter the cell suspension after digestion through a 100 μm filter into a new 50 ml centrifuge tube. Grind the sample pellet using a 5 ml syringe pump until only white connective tissue remains. Rinse the filter three times with DMEM/F12 (approximately 5 ml per wash). Centrifuge at 4°C, 300 g/1260 rpm for 5 min.

(5) Lysis of red blood cells

If a large number of red blood cells are observed in the cell pellet, perform the following red blood cell lysis step:

Remove the supernatant and resuspend the cell pellet in 1 ml of red blood cell lysis buffer. Mix thoroughly by pipetting. Place the pellet on a shaker at 120 rpm at 4°C for 5 min. Centrifuge at 300g/1260 rpm at 4°C for 5 min. Resuspend the cell pellet in 10 ml of Advanced DMEM/F12 and transfer the suspension to a 15 ml centrifuge tube. Centrifuge at 300g/1260 rpm at 4°C for 5 min.

(6) Cell seeding plate

Discard the supernatant and retain an 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.

3. Preparation of cellulose derivative culture medium:

The methylcellulose hydrogel MC-1 prepared in Example 1 was diluted with the composite culture solution, and the concentration of methylcellulose was 0.1-0.8%, thereby obtaining a methylcellulose culture solution.

4. 3D Culture: Gently resuspend the tissue cell suspension from step 2 with the methylcellulose culture medium from step 3 above. Add 300 μl per well of a pre-heated 24-well low-adhesion cell culture plate. Place the plate in a 37°C, 5% _CO₂ incubator and heat for 10 minutes. Remove the plate and add 700 μl/well of the pre-heated composite culture medium. Place the plate in a 37°C, 5% _CO₂ incubator until passage.

Example 4

This example is basically the same as Example 3, except that: the methyl cellulose hydrogel MC-1 and cellulose benzoate hydrogel prepared in Preparation Example 1 were respectively diluted with a composite culture medium to obtain a cellulose derivative culture medium with a cellulose derivative concentration of 0.2%, and gastric cancer organoids were cultured. The culture results are shown in Figures 1a-1, 1b-1, 1c-1, 1d-1, 1e-1, and 1f.

As can be seen from Figures 1a-1, 1b-1, 1c-1, 1d-1, 1e-1, and 1f, after different days of culture, obvious 3D cell spheres can be observed on the day of culture (day 0); after continuing to grow to the 5th day, the diameter of these cell spheres increased significantly and the cell walls thickened; on the 7th day of growth, the maximum diameter can reach 200μm, showing the typical differentiated and mature 0-day gastric cancer organoid morphology.

Comparative Example 2

This example is basically the same as Example 3, except that the methylcellulose hydrogel MC-1 prepared in Preparation Example 1 was diluted with a composite culture medium to obtain MC-1 with a cellulose concentration of 0.3%. Matrigel (Corning/356231) and plant cellulose matrix gel GrowDex (UPM/100103005) were used in accordance with step 4 of Example 3 to culture the tissue cell fluid to be cultured, i.e., gastric cancer organoids. The culture results are shown in Figures 2a-1, 2b-1, and 2c.

After 5 days of culture, the morphology and size of organoids were basically the same among MC-1, Matrigel, and GrowDex, with no significant differences.

Example 5

The operation was the same as in Example 3, except that the hydrogels used and the cultured tissues were different. The hydrogels were: carboxymethyl cellulose hydrogel CMC-1, cellulose acetate adamantane carboxylate mixed ester hydrogel, oxidized cellulose-dialdehyde cellulose hydrogel, and cellulose acrylate hydrogel. Micrographs of intestinal cancer organoids cultured with the above hydrogels for 7 days are shown in Figures 3a-1, 3b-1, 3c-1, and 3d-1.

Part III: Cellulose derivative hydrogels for stem cell culture and organoid differentiation experiments

Example 6 Human induced pluripotent stem cell/embryonic stem cell culture

1) Before passaging stem cells, dilute methylcellulose MC-1 hydrogel to 0.8% using DMEM/F12, add 1 ml/well to a 6-well plate, and place in a 37°C incubator for at least 1 hour.

2) After passage of stem cells, the cells in the culture dish are approximately 85-90% confluent and essentially undifferentiated. After aspirating the stem cell culture medium (mTeSR ^™ 1 (STEMCELL, 85850), cover each well with 1 ml of Dispase (1 mg/ml) and place in a 37°C incubator until the edges of the stem cells begin to lift, then aspirate the Dispase.

3) Wash three times with DPBS, 1 ml each time.

4) Add 3 ml of preheated mTeSR ^™ 1 to the wells and gently pipette until the stem cells fall off the well plate. Repeat pipetting until the cell clumps are broken into 1-2 mm fragments.

5) Add the stem cell fragments in equal parts of 1:1 to 1:6 to the MC-1 well plate covered with methylcellulose hydrogel prepared in step 1). Use mTeSR ^™ 1 culture medium (STEMCELL, 85850) to make up to 2 ml. Gently tap the side of the 6-well culture dish 5 to 10 times to ensure that the stem cell colonies are evenly distributed in the air. Incubate in a 37°C, 5% CO2 incubator, changing the medium daily.

Example 7 Human pluripotent stem cells/embryonic stem cells induced differentiation of intestinal organoids

1. iPSC/ES cell differentiation in monolayer culture

To perform differentiation in TC-treated 24-well cell culture plates, dilute the methylcellulose hydrogel MC-1 to 0.8% in DMEM/F12, add 0.3 ml/well to each 24-well plate, and incubate in a 37°C incubator for at least 1 hour. Before initiating differentiation, assess the cell differentiation rate and starting density. At this point, the cell differentiation rate should be less than 5% and the starting density should be 85-90%.

(1) Definitive endoderm differentiation

A. Preparation of Definitive Endoderm Differentiation Medium

Day 0: Prepare 0.5 mL of definitive endoderm differentiation medium (RPMI 1640, containing L-glutamine (final concentration 2 mM), penicillin-streptomycin (final concentration 100 U/ml-100 g/ml), and Activin A (final concentration 100 ng/ml)) for days 0, 1, and 2 in each culture well.

B. Definitive endoderm differentiation

1) Day 0: Preheat the required Definitive Endoderm Differentiation Medium (0.5 mL/well) at 37°C. Store the remaining medium at 2-8°C. Aspirate the mTeSR ^™ 1 from the wells and add 0.5 mL of Definitive Endoderm Differentiation Medium dropwise along the well walls. Incubate at 37°C, 5% _CO₂ , and 95% humidity for 24 hours.

2) Day 1: Preheat Definitive Endoderm Differentiation Medium (0.5 mL/well) at 37°C. Aspirate the medium from the wells and add 0.5 mL of Definitive Endoderm Differentiation Medium dropwise along the well walls. Incubate at 37°C, 5% _CO₂ , and 95% humidity for 24 hours.

3) Day 2: Aspirate the culture medium from the wells and add 0.5 mL of 37°C preheated definitive endoderm differentiation medium dropwise along the well walls. Incubate at 37°C, 5% _CO₂ , and 95% humidity for 24 hours.

Note: During endoderm induction, cells undergo extensive cell death. Minimize the time cells spend outside the 37°C incubator. Within 24 hours of endoderm induction, cells are very sensitive and require careful medium changes during culture. After 72 hours of incubation, a dense, confluent monolayer of endoderm cells will form.

(2) Midgut/hindgut (MH) differentiation

A.MH differentiation medium

Day 3: Prepare MH differentiation medium (RPMI1640 containing 2% FBS, L-glutamine (final concentration 2 mM), penicillin-streptomycin (final concentration 100 U/ml-100 g/ml), FGF4 (final concentration 500 ng/ml) required for days 3-8, 0.5 mL per culture well.

B. Midgut/Hindgut (MH) Differentiation

1) Day 3: Warm an adequate amount of MH Differentiation Medium (0.5 mL/well) to room temperature (15-25°C). Aspirate the medium from the wells and add 0.5 mL of Midgut/Hindgut Differentiation Medium dropwise along the well walls. Incubate at 37°C, 5% CO2, and 95% humidity for 24 hours.

2) Days 4-9: Replace all the medium and observe the spheroids every 24 hours according to the following method.

NOTE: Ensure that cultures are returned to the incubator within 30 minutes of removal.

a. Observe the monolayer under a microscope. Three-dimensional structures may appear as early as day 4 of differentiation. Free-floating mid/hindgut spheroids will appear.

Now it is day 6-9 of differentiation.

b. Using a 1 mL pipette, remove 0.5 mL of culture medium from the cells and transfer to a sterile 24-well flat-bottom clear culture plate to assess the number and concentration of mid/hindgut spheroids extracted from the monolayer.

c. Add 0.5 mL of fresh MH medium to the cells and incubate at 37°C, 5% CO2, and 95% humidity for 24 hours.

Note: Although spheres released from days 6-9 can generate intestinal organoids, the length of time cells are cultured in mid/hindgut culture will determine the regional characteristics of the intestinal organoid development. For example, the duodenum (shorter culture time) or ileum (longer culture time). The time at which the highest yield of mid/hindgut spheres occurs may vary between hPSC cell lines. For reproducible experimental results, use mid/hindgut spheres differentiated at the same time point to consistently harvest and initiate human intestinal organoid culture.

3) Spheroid Embedding: Using a pipette rinsed with 0.5% BSA, transfer the spheroid suspension from each well to one well of a 24-well plate for counting. Add an appropriate volume to a 15 mL centrifuge tube, corresponding to approximately 50 suspended spheres (based on the previous spheroid count).

A mid/hindgut spheroid is a cell aggregate ≥75 μm in diameter that can potentially form a human intestinal organoid. Multiple fused spheroids should be counted as one unit and will form a human intestinal organoid.

The remaining monolayer culture can be used to assay mid/hindgut formation or to study further differentiation of chimeric spheroids in subsequent days.

2. Human intestinal organoid culture

(1) Initial culture of human intestinal organoids

A. Preparation of Intestinal Organoid Growth Medium

1) Culture in a 24-well low-adhesion culture plate. Prepare 4 wells (0.5 mL/well) of intestinal organoid growth medium (Advanced DMEM/F12 supplemented with 1× B27, L-glutamine (final concentration 2 mM), penicillin-streptomycin (final concentration 100 U/ml-100 μg/ml), HEPES buffer (final concentration 15 mM), and R-spondin 1 (final concentration 500 ng/ml).

B. Mixing of methylcellulose hydrogel MC-1 and spheres

1) Centrifuge the spheroid suspension collected during midgut/hindgut (MH) differentiation described in step B at 300×g for 5 minutes. Carefully remove the supernatant after centrifugation.

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

3) Carefully remove the supernatant using a 1 mL pipette tip.

5) Add 100 μL of 0.3% methylcellulose hydrogel MC-1 to the centrifuge tube. Pipet up and down five times to gently distribute the spheroids into the methylcellulose hydrogel MC-1.

NOTE: Do not completely empty the pipette tip to prevent excessive air bubbles.

6) Using the same pipette tip, gently transfer the embedded spheroid to the center of a well of a 24-well tissue culture dish.

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

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

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

10) Change the culture medium every 3-4 days by aspirating the old medium and adding fresh medium. Incubate at 37°C, 5% CO2, and 95% humidity.

11) After 10-14 days of incubation, passage the organoids depending on their growth.

Microscopic images of intestinal organoids induced to differentiate from human pluripotent stem cells/embryonic stem cells after culturing for 3, 7, and 12 days in this example are shown in Figures 1a-2, 1b-2, and 1c-2.

Comparative Example 3

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

Both groups showed clear colony margins, uniform cell size, and a high nuclear-cytoplasmic ratio, indicating optimal stem cell status and an undifferentiated cell state. This suggests that methylcellulose MC-1 hydrogel and Matrigel are comparable in stem cell culture performance.

Comparative Example 4

Referring to the culture method of Example 7, intestinal organoid differentiation kit containing 0.3% methylcellulose hydrogel MC-1 and STEMdiff ^™ Intestinal Organoid Kit (stem cell, 05140) were used for culture, respectively. Micrographs of intestinal organoids 12 days after induction and differentiation of human pluripotent stem cells/embryonic stem cells are shown in Figures 3a-2 and 3b-2.

Comparing the two groups, both were able to culture mature intestinal organoids containing multiple crypt structures that interconnected to form a mini-intestine with a central intestinal lumen. The kit of the present invention even outperformed the control kit, producing intestinal organoids with a more typical and complex crypt structure.

Example 8

The operation was the same as in Example 7, except that the hydrogels were different. The hydrogels were carboxymethyl cellulose hydrogel, cellulose acetate adamantane carboxylate mixed ester hydrogel, oxidized cellulose-dialdehyde cellulose hydrogel, and cellulose acrylate hydrogel. Figures 4a, 4b, 4c, and 4d are micrographs of intestinal organoids cultured with the above hydrogels for 12 days, respectively.

The above describes exemplary embodiments of the present invention. However, the scope of protection of this application is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc. made by those skilled in the art within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims (10)

A cellulose derivative-based hydrogel, characterized in that its matrix includes a cellulose derivative, the matrix includes a network structure composed of ultrafine fibers, and has a highly ordered three-dimensional micro-nano structure, the cellulose derivative is selected from cellulose esters, cellulose ethers, oxidized cellulose, charged cellulose or other types of cellulose derivatives, preferably, the cellulose derivative is selected from methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, carboxymethyl cellulose, cellulose propionate, cellulose butyrate, cellulose benzoate, cellulose cyclohexylcarboxylate, cellulose cinnamate, cellulose 2-methylbenzoate, cellulose acetate 2-methylbenzoate mixed ester, 2-methylbenzoate cellulose-4-trifluoromethylbenzoic acid mixed ester, cellulose acetate adamantanecarboxylate mixed ester, oxidized cellulose-dialdehyde cellulose or DOPO (9,10-dihydro-9-oxa-10-phosphaphenanthrene-10-oxide) cellulose derivatives. The cellulose derivative-based hydrogel according to claim 1, characterized in that the degree of substitution of the cellulose derivative is 0.1 to 3, preferably 0.5 to 2.5, and more preferably 1.0 to 2.0; preferably, the fiber diameter of the cellulose derivative is 0.01 μm to 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 μm-50 μm, for example, 1 μm, 5 μm, 8 μm, or 10 μm. %, 10μm, 15μm, 20μm, 25μm, 30μm, 35μm, 40μm, 45μm or 50μm; preferably, the overall particle size of the matrix in the hydrogel is 1μm-50μm, for example, 1μm, 5μm, 8μm, 10μm, 15μm, 20μm, 25μm, 30μm, 35μm, 40μm, 45μm or 50μm; preferably, the solid content of the hydrogel is 0.1-3.0wt%, for example, 0.1wt%, 0.5wt%, 1wt%, 1.5wt%, 2wt%, 2.5wt% or 3.0wt%. The cellulose derivative-based hydrogel according to claim 1 or 2, characterized in that the cellulose derivative-based hydrogel is prepared by scheme 1 or scheme 2: Scheme 1: 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 crushed by a homogenizer, colloid mill, or ball mill to obtain a cellulose derivative hydrogel; Option 2: Prepare a cellulose solution; pour the cellulose solution into a coagulation bath to form a gel, crush the gel by a homogenizer, colloid mill or ball mill to obtain a cellulose hydrogel, and perform a derivatization reaction on the cellulose hydrogel to form a cellulose derivative hydrogel. A method for preparing the cellulose derivative-based hydrogel according to any one of claims 1 to 3, characterized in that the method is selected from the following scheme 1 or scheme 2: Scheme 1: 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 crushed by a homogenizer, colloid mill, or ball mill to obtain a cellulose derivative hydrogel; Option 2: Prepare a cellulose solution; pour the cellulose solution into a coagulation bath to form a gel, and crush the gel by a homogenizer, colloid mill, or ball mill to obtain a cellulose hydrogel; and perform a derivatization reaction on the cellulose hydrogel to obtain a cellulose derivative hydrogel. Preferably, the first solution specifically includes: 1) Cellulose pretreatment; 2) homogeneously or heterogeneously derivatizing the cellulose pretreated in step 1) to obtain a cellulose derivative; 3) preparing the cellulose derivative in step 2) into a solution, pouring the solution into a coagulation bath to form a gel, and crushing the gel by a homogenizer, a colloid mill, or a ball mill to obtain a cellulose derivative hydrogel; Preferably, in step 1), the cellulose pretreatment comprises dispersing the cellulose in a solvent, which may be water, DMF, ethanol or isopropanol, and the cellulose may be microcrystalline cellulose, cotton pulp, wood pulp, inulin, soluble starch or dextran, preferably wood pulp; or the cellulose pretreatment comprises adding the cellulose to 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. For example, the alkali may be sodium hydroxide, potassium hydroxide, sodium carbonate or sodium borohydride; Preferably, in step 3), the solvent used to prepare the solution can be water, ethanol, acetone, DMF or NaOH aqueous solution. Preferably, the second solution specifically includes: 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 a homogenizer, a colloid mill, or a ball mill to obtain a cellulose gel; 3') subjecting the cellulose gel of step 2') to a derivatization reaction to obtain a cellulose derivative hydrogel. Preferably, in step 1'), cellulose is dissolved in a cellulose solvent to obtain the cellulose solution; In the first or second embodiment, the cellulose solvent is any good solvent known in the art that can dissolve (the dissolving 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 cellulose-dissolving ionic liquid is selected from one or more of [AMIM][Cl], [BMIM][Cl], [EMIM][Ac], and [BMIM][Ac]. A culture medium for 3D cell culture or a culture medium for tissue and organoids or a culture medium for induced differentiation of stem cells into organoids, characterized in that the culture medium for 3D cell culture comprises the cellulose derivative-based hydrogel and the basal culture medium according to claim 1; the cellulose derivative-based culture medium for culturing tissues and organoids comprises the cellulose derivative-based hydrogel and the composite culture medium according to claim 1, the composite culture medium comprises a basal culture medium and an active component, preferably, per 100 mL of the composite culture medium, the mass ratio of the basal culture medium to the active component is 80-99:1-20, for example, 97:3, more preferably, the composite culture medium contains 100-500 ng/ml of epidermal growth factor (Epirregulin), for example, 200 ng/ml, 300 ng/ml, 400 ng/ml, 500 ng/ml; the culture medium for induced differentiation of stem cells into organoids comprises the cellulose derivative-based hydrogel, the definitive endoderm differentiation culture medium, the MH differentiation culture medium, and the organoid growth culture medium according to claim 1. A method for preparing the culture solution according to claim 5, characterized in that: The method for preparing the culture medium for 3D cell culture 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 basic culture medium to prepare the culture medium for 3D cell culture; Wherein, in step S2, the volume ratio of the cellulose derivative-based hydrogel to the basic culture medium is 1-10:1-10, for example, 1:1, 1:5, 1:10, 5:1 or 10:1. A method for 3D cell culture, characterized in that the method is carried out in the 3D cell culture medium according to claim 5, and the 3D cell culture method comprises the following steps: a) preparing a cell suspension: uniformly mixing the 3D cell culture medium according to claim 5 with cells to obtain a cell suspension; b) 3D culture: culturing the cell suspension from step a) in an incubator; Preferably, the cells include human neuroblastoma cells, such as SH-SY5Y cells, and hepatoma cells, such as HepaRG cells. A method for inducing stem cell differentiation into organoids, characterized in that the method comprises culturing a single stem cell or a stem cell population in the stem cell differentiation organoid culture medium according to claim 5, wherein the method comprises the following steps: a) iPSC/ES differentiation in monolayer culture, including definitive endoderm differentiation and mid/hindgut (MH) differentiation; b) organoid culture, comprising preparing an organoid growth culture medium and mixing the cellulose derivative-based hydrogel according to claim 1 with the spheroids and then incubating the mixture. The use of the cellulose derivative-based hydrogel according to claim 1 or the culture medium according to claim 5 in 3D cell culture, tissue and organoid culture or stem cell culture and induced differentiation organoids, characterized in that the cells preferably include human neuroblastoma cells, such as SH-SY5Y cells and liver cancer cells, such as HepaRG cells, and the tissues are preferably human gastric cancer tissue/normal gastric tissue, human intestinal cancer tissue/normal intestinal tissue, and the organoids are preferably human gastric cancer organoids/normal gastric organs, human intestinal cancer organoids/normal intestinal organoids, and the induced differentiation organoids are preferably induced differentiation intestinal organoids, gastric organoids, liver organoids, and more preferably small intestinal organoids. A kit, characterized in that the kit comprises the culture medium for 3D cell culture or the culture medium for tissue and organoid culture or the culture medium for stem cell culture and induced differentiation of organoids according to claim 5 and instructions for use.