Plant source-based culture solution and preparation method therefor, kit, and use

Abstract

Disclosed are a plant source-based culture solution and a preparation method therefor, a kit, and use. The culture solution of the present invention comprises a plant hydrogel and a basic culture solution. The plant hydrogel is free of animal-derived components and comprises micro-nano biomass and water. Compared with a matrix gel of an animal source, the plant hydrogel of the present invention has the advantages in the aspects of performance, use convenience, inter-batch stability, process amplification stability, automatic compatibility, and the like.

Description

Plant source-based culture solution and preparation method, kit and application thereof

[0001] The present application claims the priority of the prior application filed on April 15, 2024 with the China National Intellectual Property Office and entitled "3D cell culture solution and preparation method, kit and application thereof", patent application number 2024104463928, patent application number 2024104463650 and entitled "Tumor organoid 3D culture solution and preparation method and application thereof", patent application number 2024104463824 and entitled "Stem cell culture and induced differentiation organoid culture solution and application thereof", and patent application number 202410894553X and entitled "Plant source-based culture solution and preparation method, kit and application thereof" filed on July 4, 2024 with the China National Intellectual Property Office. The prior application is incorporated herein by reference in its entirety. TECHNICAL FIELD

[0002] The present application relates to the field of biotechnology, in particular, to a plant source-based culture solution and preparation method, kit and application thereof. BACKGROUND

[0003] Ordinary 2D cell culture gradually loses the original characteristics due to cell proliferation in an altered environment in vitro, often not consistent with in vivo conditions. Animal experiments are completely performed in vivo, but are complicated due to various factors in vivo and the mutual influence of the in vivo and external environment, making it difficult to study single processes and intermediate processes. 3D cell culture technology is a technology between single-layer cell culture and animal experiments, which can maximize the simulation of in vivo environment and exhibit the advantages of direct visualization and controllability of cell culture.

[0004] The emergence of 3D cell culture technology provides researchers with a cell model closer to the real in vivo environment. 3D cell culture can make up for many defects in the process of single-layer cell culture, for example:

[0005] (1) 3D cell model can well simulate the in vivo cell microenvironment: gases, nutrients, metabolites and other substances show gradient changes in concentration.

[0006] (2) 3D cell model can well simulate the interaction between cells: three-dimensional cell contact and direct or indirect cell communication.

[0007] (3) 3D cell model can well simulate the biochemical and physiological reactions of cells: the response of cells to internal or external stimuli is more consistent with the real in vivo response.

[0008] Because of these advantages, 3D cell culture technology has shown excellent performance in the research fields of drug development, stem cell culture, organ regeneration, etc.

[0009] The difficulty of 3D cell culture technology is to ensure the three-dimensional structure of cells and maintain the natural proliferation and differentiation activity. With decades of continuous development, the method of 3D cell culture is also constantly innovating, and the currently commonly used methods are mainly divided into two kinds: 3D cell culture method based on scaffold and 3D cell culture method without scaffold. The 3D cell culture method based on scaffold has a long history of development and is supported by a large number 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, fibers, permeability and mechanical stability, which can well simulate the interaction between cells and the interaction between cells and extracellular matrix in the in vivo environment, while allowing cells to aggregate, proliferate and migrate on the scaffold.

[0010] Organoids usually need Matrigel support to form a 3D structure during the culture process. Taking the commonly used scaffold material Matrigel as an example, this material is a basement membrane matrix extracted from EHS mouse tumors rich in extracellular matrix proteins, and its main components include laminin, collagen type IV, heparin sulfate glycoprotein, nidogen, and also contains various growth factors and matrix metalloproteinases, etc. At room temperature, Matrigel polymerizes to form a three-dimensional matrix with biological activity, simulating the structure, composition, physical properties and functions of the basement membrane of cells in vivo, which is conducive to the culture and differentiation of cells in vitro, and can be used for research on cell morphology, biochemical function, migration, invasion and gene expression, etc.

[0011] Stem cells are a class of cells with proliferation and differentiation potential, which can differentiate into specific cell types of various tissues and organs. Among them, pluripotent stem cells can differentiate into all cells derived from three germ layers, form all tissues and organs, and are used for various tissue and organ repair, disease treatment and drug screening, so they have become the focus of stem cell research. Culturing stem cells is also applied to the formation, maintenance and expansion of organoids. Embryonic stem cells (EC) and induced pluripotent stem cells (iPSC) are currently the most studied pluripotent stem cells. However, developing stem cells is not simple, and a suitable and stable microenvironment is crucial for the culture and application of iPSC and other stem cells. Hydrogel is a porous polymer network rich in water, which has good biocompatibility, biochemical and mechanical properties for 3D stem cell culture. Although some progress has been made in the development of stem cell culture based on hydrogel, it is still challenging to inhibit the non-directional differentiation of stem cells while achieving the performance comparable to Matrigel in simulating the structure, composition, physical properties and functions of the basement membrane of cells in vivo.

[0012] Take the commonly used scaffold material basement membrane matrix (Matrigel) as an example, the material is extracted from the basement membrane matrix of EHS mouse tumor rich in extracellular matrix proteins, the main components are laminin, type IV collagen, heparan sulfate glycoprotein, nidogen, and also contain various growth factors and matrix metalloproteinases, etc. At room temperature, Matrigel polymerizes to form a three-dimensional matrix with biological activity, simulating the structure, composition, physical properties and function of the basement membrane of cells in vivo, which is beneficial to the culture and differentiation of cells in vitro, and can be used for the research on cell morphology, biochemical function, migration, invasion and gene expression, etc.

[0013] The current problems of basement membrane matrix gel are: 1) shortage of goods source caused by animal welfare problem; 2) large batch difference and difficult to control, the contained nutrients and protein components are greatly affected by batch; 3) animal source protein and nucleic acid bring interference to the drug efficacy evaluation, fluorescence detection and other links of downstream experiment; 4) the temperature sensitive characteristics of the matrix gel are strict to the temperature requirements of storage and operation links. SUMMARY

[0014] To solve the above technical problems, the technical scheme provided by the present application is as follows:

[0015] A culture solution based on plant source, the culture solution comprises plant gel and basic culture solution; wherein the plant gel is free of animal source components and comprises micro-nano biomass and dispersion reagent; the dispersion reagent is selected from organic solvent and / or water, preferably water and / or alcohol.

[0016] Preferably, the culture solution comprises plant hydrogel and basic culture solution; wherein the plant gel is free of animal source components and comprises micro-nano biomass and water.

[0017] A culture solution based on plant source, the culture solution comprises plant hydrogel and basic culture solution; wherein the plant hydrogel is free of animal source components and comprises micro-nano biomass and water.

[0018] According to the present application, the culture solution is used for 3D cell culture, and the culture solution comprises plant hydrogel and basic culture solution A; wherein the plant hydrogel is free of animal source components and comprises micro-nano biomass and water.

[0019] According to the present application, the culture solution is used for 3D culture of tumor organoids, and the culture solution comprises plant hydrogel and composite culture solution; wherein the plant hydrogel is free of animal source components and comprises micro-nano biomass and water.

[0020] According to the application, the culture solution is used for stem cell culture, and the culture solution comprises a plant hydrogel and a basic culture solution C; wherein the plant hydrogel is free of animal-derived components and comprises micro-nano biomass and water.

[0021] A preparation method of the culture solution, wherein the preparation method of the 3D cell culture solution comprises the following steps:

[0022] S1: preparing a plant hydrogel;

[0023] S2: mixing the plant hydrogel in step S1 with a basic culture solution A to prepare a 3D cell culture solution;

[0024] Alternatively, the preparation method of the tumor organoid 3D culture solution comprises the following steps:

[0025] S1': preparing a plant hydrogel;

[0026] S2': mixing the plant hydrogel in step S1' with a composite culture solution to prepare a tumor organoid 3D culture solution;

[0027] Alternatively, the preparation method of the stem cell culture solution comprises the following steps:

[0028] S1": preparing a plant hydrogel;

[0029] S2": mixing the plant hydrogel in step S1" with a basic culture solution C to prepare the stem cell culture solution.

[0030] An application of the culture solution, for culturing cells, tissues or organoids; specifically, the application is the application of the 3D cell culture solution in 3D cell culture; or the application of the tumor organoid 3D culture solution in culturing tumor tissues or organoids such as gastric cancer and lung cancer; or the application of the stem cell culture solution in culturing stem cells or organoids.

[0031] A method for 3D culturing cells, wherein the method is performed in the culture solution, specifically in the 3D cell culture solution.

[0032] A kit comprising the culture solution.

[0033] Specifically, the kit is a 3D cell culture kit comprising the 3D cell culture solution; or the kit is a tumor organoid 3D culture kit comprising the tumor organoid 3D culture solution.

[0034] The kit is applied in cell culture, tissue culture or organoid culture.

[0035] A method for culturing tumor tissues or organoids such as gastric cancer and lung cancer, which is performed in the culture solution.

[0036] A method for culturing stem cells or organoids, which is performed in the culture solution.

[0037] A stem cell induced differentiation organoid culture solution, which comprises a plant hydrogel, a definitive endoderm differentiation culture solution, an MH differentiation culture solution and an organoid growth culture solution; wherein the plant hydrogel is free of animal-derived components and comprises micro-nano biological substances and water.

[0038] An application of the stem cell induced differentiation organoid culture solution, which is used for stem cell induced differentiation organoids.

[0039] A method for culturing stem cell induced differentiation organoids, which comprises culturing single stem cells or stem cell groups in the stem cell induced differentiation organoid culture solution.

[0040] A stem cell organoid prepared by the method for culturing stem cell induced differentiation organoids.

[0041] An induced differentiation organoid kit, which comprises the stem cell induced differentiation organoid culture solution.

[0042] An application of the induced differentiation organoid kit in induced differentiation organoids.

[0043] The scheme of the present application achieves the following beneficial effects:

[0044] The plant hydrogel of the present application is free of animal-derived components and mainly comprises micro-nano biomass and water, which has incomparable advantages over animal-derived matrix glue in performance, use convenience, batch stability, process amplification stability, automation compatibility and the like, and brings a brand-new 3D cell culture experience. Specifically, the plant hydrogel of the present application has a micron-level structure and a nano-level structure which can be completely stretched in a fluid to form a rich network lapping structure, the network formed by the micron-level structure is filled with a large number of nano-level structures, and a more dense lapping network can be formed. This network structure belongs to a flexible three-dimensional structure, which is easier to lap the network support necessary for cell growth in terms of the suspension stability function of cells, and at the same time avoids excessive rigidity of the structure, can highly simulate the extracellular matrix in vitro, promotes the formation, growth and development of SH-SY5Y 3D cells, HepaRG cells, gastric cancer, intestinal cancer and other tumor organoids, and can be applied to clinical, pharmaceutical, cell and stem cell industrialization scenes and research. The micro-nano biomass has no toxicity to cells, the biomass-based hydrogel has good rigidity, adhesion and porosity as a stem cell growth platform, and the stem cells cultured by the micro-nano biomass of the present application can form clusters.

[0045] In one aspect, the plant hydrogel of the present application can be directly used to prepare a culture solution for 3D culture of SH-SY5Y cells, HepaRG cells (liver cancer cells) by a suspension method, which is simple to operate and has a high 3D cell sphere formation rate.

[0046] In one aspect, human gastric cancer and other tumor tissue samples are used for tissue separation, and a self-developed culture solution is used for culture of gastric cancer and other tumor tissues or organoids, thereby providing an efficient, simplified and low-cost gastric cancer and other tumor organoid standardized culture system.

[0047] In still another aspect, the plant hydrogel of the present application is used for culture of stem cells and organoids, which is simple to operate, forms a dense fused monolayer endoderm cell, can inhibit non-directional differentiation of stem cells, the formed organoid vesicle wall is thick and presents a solid core, is a differentiated organoid, and can be used for high-throughput drug screening, drug toxicity testing and regenerative medicine. BRIEF DESCRIPTION OF DRAWINGS

[0048] FIG. 1a is a microscopic image of the culture solution with 0.4% CMG used for culture of SH-SY5Y cells for 3 days in Example 1;

[0049] FIG. 1b is a microscopic image of the culture solution with 0.2% CMG used for culture of SH-SY5Y cells for 3 days in Example 1;

[0050] FIG. 1c is a microscopic image of the culture solution with 0.1% CMG used for culture of SH-SY5Y cells for 3 days in Example 1;

[0051] Figure 2a is a microscopic image of SH-SY5Y cells after 3 days of culture with CMG at 0.2% in Example 2;

[0052] Figure 2b is a microscopic image of SH-SY5Y cells after 3 days of culture with Matrigel 3D culture method in Example 2;

[0053] Figure 3a is a microscopic image of SH-SY5Y cells after 3 days of culture with CMG at 0.2% in Example 3;

[0054] Figure 3b is a microscopic image of SH-SY5Y cells after 3 days of culture with 2D culture method in Example 3.

[0055] In Figures 1a-1c, the scale bars are all 100 pm; in Figures 2a and 2b, the scale bars are all 200 pm; in Figures 3a and 3b, the scale bars are all 100 pm.

[0056] Figure 4 is a scanning electron microscope image of the plant hydrogel of Preparation Example 1 at different magnifications, wherein the scale bars of a, b and c are 10 pm, 5 pm and 1 pm, respectively.

[0057] Figure 5 is a microscopic image of SH-SY5Y cells after 3 days of culture with the culture solution in Comparative Example 1.

[0058] Figure 6 is a microscopic image of SH-SY5Y cells after 3 days of culture with the culture solution in Comparative Example 2.

[0059] Figure 7 is a microscopic image of SH-SY5Y cells after 3 days of culture with the comparative hydrogel in Comparative Example 3.

[0060] Figure 8a is a microscopic image of HepaRG (liver cancer cells) after 7 days of 3D culture with CMG at 0.1% in Example 4-1.

[0061] Figure 8b is a microscopic image of HepaRG (liver cancer cells) after 7 days of 3D culture with CMG (Preparation Example 2) at 0.1% in Example 4-2.

[0062] Figure 8c is a microscopic image of HepaRG (liver cancer cells) after 7 days of 3D culture with CMG (Preparation Example 3) at 0.1% in Example 4-2.

[0063] Figure 8d is a microscopic image of HepaRG (liver cancer cells) after 7 days of 3D culture with CMG (Preparation Example 4) at 0.1% in Example 4-2.

[0064] Figure 8e is a microscopic image of HepaRG (liver cancer cells) after 7 days of 3D culture with CMG (Preparation Example 5) at 0.1% in Example 4-2.

[0065] Figure 9 is a microscopic image of 3D culture of HepaRG (liver cancer cells) after 7 days in Comparative Example 4.

[0066] Figure 10a is a microscopic image of culture of gastric cancer organoids using culture medium with 0.8% CMG for 7 days in Example 5;

[0067] Figure 10b is a microscopic image of culture of gastric cancer organoids using culture medium with 0.4% CMG for 7 days in Example 5;

[0068] Figure 10c is a microscopic image of culture of gastric cancer organoids using culture medium with 0.2% CMG for 7 days in Example 5;

[0069] Figure 10d is a microscopic image of culture of gastric cancer organoids using culture medium with 0.1% CMG for 7 days in Example 5;

[0070] Figure 11a is a microscopic image of culture of gastric cancer organoids using culture medium with 0.2% CMG for 7 days in Example 6;

[0071] Figure 11b is a microscopic image of culture of gastric cancer organoids using culture method with Matrigel 3D for 7 days in Example 6;

[0072] In Figures 10a-10d, the scale bar is 100 pm; in Figures 11a and 11b, the scale bar is 200 pm.

[0073] Figure 12 is a microscopic image of culture of intestinal cancer organoids using culture medium with 0.2% CMG for 7 days in Example 7-1.

[0074] Figure 13 is a microscopic image of culture of intestinal cancer organoids using culture medium with 0.2% CMG (Preparation Example 2) for 7 days in Example 7-2.

[0075] Figure 14 is a microscopic image of culture of intestinal cancer organoids using culture medium with 0.2% CMG (Preparation Example 3) for 7 days in Example 7-2.

[0076] Figure 15 is a microscopic image of culture of intestinal cancer organoids using culture medium with 0.2% CMG (Preparation Example 4) for 7 days in Example 7-2.

[0077] Figure 16 is a microscopic image of culture of intestinal cancer organoids using culture medium with 0.2% CMG (Preparation Example 5) for 7 days in Example 7-2.

[0078] Figure 17 is a microscopic image of culture of gastric cancer tissue using cellulose-based culture medium containing different Matrigel for 7 days in Comparative Example 5.

[0079] Figure 18 is a microscopic image of culture of gastric cancer tissue using cellulose-based culture medium containing different complex culture medium for 7 days in Comparative Example 6.

[0080] Figure 19 is a micrograph of the cellulose-based medium containing GrowDex in Comparative Example 7 after 7 days of culturing intestinal cancer tissue.

[0081] Figure 20a is a micrograph of human pluripotent stem cell / embryonic stem cell induced-differentiated intestinal organoids after 3 days in Example 9;

[0082] Figure 20b is a micrograph of human pluripotent stem cell / embryonic stem cell induced-differentiated intestinal organoids after 7 days in Example 9;

[0083] Figure 20c is a micrograph of human pluripotent stem cell / embryonic stem cell induced-differentiated intestinal organoids after 12 days in Example 9;

[0084] Figure 21 is a micrograph of human pluripotent stem cell / embryonic stem cell induced-differentiated intestinal organoids after 12 days using CMG prepared in Preparation Example 2 in Example 10;

[0085] Figure 22 is a micrograph of human pluripotent stem cell / embryonic stem cell induced-differentiated intestinal organoids after 12 days using CMG prepared in Preparation Example 3 in Example 10;

[0086] Figure 23 is a micrograph of human pluripotent stem cell / embryonic stem cell induced-differentiated intestinal organoids after 12 days using CMG prepared in Preparation Example 4 in Example 10;

[0087] Figure 24 is a micrograph of human pluripotent stem cell / embryonic stem cell induced-differentiated intestinal organoids after 12 days using CMG prepared in Preparation Example 5 in Example 10;

[0088] Figure 25a is a micrograph of iPSCs cultured with 0.8% CMG in Comparative Example 8;

[0089] Figure 25b is a micrograph of iPSCs cultured with Matrigel (Corning 354277) in Comparative Example 8;

[0090] Figure 26a is a micrograph of human pluripotent stem cell / embryonic stem cell induced-differentiated intestinal organoids after 12 days of culturing with the induced-differentiated intestinal organoid kit containing 0.3% plant hydrogel CMG in Comparative Example 9;

[0091] Figure 26b is a micrograph of human pluripotent stem cell / embryonic stem cell induced-differentiated intestinal organoids after 12 days of culturing with the STEMdiff TM Intestinal Organoid Kit (stern cell, 05140) in Comparative Example 9. DETAILED DESCRIPTION

[0092] [Medium for 3D cell culture]

[0093] As described above, the present application provides a culture solution based on plant source, which is used for 3D cell culture. Specifically, the present application provides a culture solution for 3D cell culture, which comprises a plant hydrogel and a basic culture solution A; wherein the plant hydrogel is free of animal source components and comprises micro-nano biomass and water.

[0094] According to embodiments of the present application, the basic culture solution A can be selected from the basic culture solutions known in the art. Exemplarily, the basic culture solution A comprises DMEM / F12 (GIBCO / 10565018), 10% FBS (GIBCO / 10091-148) and 1% P / S (GIBCO / 15140122).

[0095] [Preparation method of the culture solution for 3D cell culture]

[0096] As described above, the present application also provides a preparation method of the culture solution for 3D cell culture as described above, which comprises the following steps:

[0097] S1: preparing a plant hydrogel;

[0098] S2: mixing the plant hydrogel in step S1 with a basic culture solution A to prepare the culture solution for 3D cell culture.

[0099] According to embodiments of the present application, in step S1, the preparation method of the plant hydrogel is selected from at least one of the following methods one to four:

[0100] Method one, preparing a natural biomass-based dispersion solution: pouring a natural biomass solution into a coagulation bath to form a natural biomass gel; and performing micro-nano processing on the natural biomass gel by means of a homogenizer, colloid mill or ball mill to obtain the natural biomass-based dispersion solution, i.e. the plant hydrogel;

[0101] Method two, preparing a surface-modified biomass-based dispersion solution: pouring a surface-modified biomass solution into a coagulation bath to form a surface-modified biomass gel; and performing micro-nano processing on the surface-modified biomass gel by means of a homogenizer, colloid mill or ball mill to obtain the surface-modified biomass-based dispersion solution, i.e. the plant hydrogel; or mixing the natural biomass-based dispersion solution in method one with a modification reagent or performing surface chemical modification to obtain the surface-modified biomass-based dispersion solution, i.e. the plant hydrogel;

[0102] Method three, preparation of a substituted biomass-based dispersion: pouring the substituted biomass solution into a coagulation bath to form a substituted biomass gel; subjecting the substituted biomass gel to micro-nano processing by means of a homogenizer, colloid mill or ball mill, etc. to obtain the substituted biomass-based dispersion, i.e. the plant hydrogel; or mixing the natural biomass-based dispersion in method one with a functional derivative reagent to obtain the substituted biomass-based dispersion, i.e. the plant hydrogel.

[0103] Method four, preparation of a multi-component biomass-based dispersion: pouring a solution containing at least one of natural biomass, substituted biomass, surface-modified biomass, and a functional component into a coagulation bath to form a gel; subjecting the gel to micro-nano processing by means of a homogenizer, colloid mill or ball mill, etc. to obtain the multi-component biomass-based dispersion, i.e. the plant hydrogel; or: mixing at least one of a natural biomass-based dispersion, a substituted biomass-based dispersion, a surface-modified biomass-based dispersion, and a functional component, and subjecting to micro-nano processing by means of a homogenizer, colloid mill or ball mill, etc. to obtain the multi-component biomass-based dispersion, i.e. the plant hydrogel.

[0104] According to an embodiment of the present application, in step S2, the mass / volume ratio of the plant hydrogel to the basal medium A is 1-30:1000, for example 5:1000, 10:1000, 20:1000.

[0105] [Application of the culture solution for 3D cell culture]

[0106] As described above, the present application also provides an application of the above-mentioned culture solution for 3D cell culture in 3D cultured cells.

[0107] As described above, the present application also provides a method for 3D cultured cells, which is carried out in the above-mentioned culture solution for 3D cell culture.

[0108] According to an embodiment of the present application, the method for 3D cultured cells comprises the following steps:

[0109] 1) Preparation of a cell suspension: uniformly mixing the culture solution for 3D cell culture with cells to obtain a cell suspension;

[0110] 2) 3D culture: culturing the cell suspension of step 1) in an incubator.

[0111] According to an embodiment of the present application, in step 1), the cells can also be subjected to cell resuscitation treatment and / or cell subculture treatment.

[0112] Preferably, the cell resuscitation can be performed by any method known in the art, as long as the cells can be resuscitated, for example, according to the quick thawing principle. Illustratively, the cell resuscitation specifically comprises: placing the cells to be resuscitated in a 37℃ water bath, after centrifugation, resuspending the cells with the basal medium, and then culturing in an incubator (37℃, 5% CO2), for example, for 48h.

[0113] Preferably, the cell passage can be performed by any method known in the art, as long as the desired cells can be obtained. Illustratively, the resuscitated cells are discarded, PBS is added to the culture well and washed gently for 2 times, then a single cell suspension is prepared by trypsin digestion, the cells are resuspended with the basal medium and adjusted to the desired cell density (for example, 5K, 25K, 50K).

[0114] According to the embodiments of the present application, the cells are preferably human neuroblastoma cells, liver cancer cells, gastric cancer cells, for example, SH-SY5Y cells, HepaRG cells.

[0115] According to the embodiments of the present application, in step 2), the culturing can be performed by any method known in the art, as long as the desired cells can be obtained, for example, the cell suspension is added to the culture plate and cultured at 37℃, 5% CO2.

[0116] According to the embodiments of the present application, in step 2), the culturing time is 1 day to 10 days, preferably 3 days.

[0117] According to the embodiments of the present application, in step 2), the culturing further comprises medium replacement, for example, the basal medium is replaced every 3 days.

[0118] As described above, the present application also provides a 3D cell culture kit.

[0119] According to the embodiments of the present application, the kit comprises the basal medium for 3D cell culture of the present application. Specifically, the cells are at least one of SH-SY5Y cells, HepaRG cells (liver cancer cells), HCT-116 cells, HeLa cells, K562 cells, MOLM-13 cells, etc.

[0120] According to the embodiments of the present application, the kit further comprises components or assemblies known in the art, which are not limited in the present application.

[0121] As described above, the present application also provides the use of a 3D cell culture kit in 3D cell culture.

[0122] According to embodiments of the present application, the cells in the 3D cell culture are at least one of SH-SY5Y cells, HepaRG cells (liver cancer cells), HCT-116 cells, HeLa cells, K562 cells, MOLM-13 cells, etc.

[0123] [biomass-based medium for 3D culture of tumor organoids]

[0124] As mentioned above, the present application provides a plant-based medium for 3D culture of tumor organoids. Specifically, the present application provides a biomass-based medium for 3D culture of tumor organoids, comprising a plant hydrogel and a complex medium; wherein the plant hydrogel is free of animal-derived components, comprising micro-nano biomass and water.

[0125] According to embodiments of the present application, the complex medium comprises a basal medium B and an active component.

[0126] According to embodiments of the present application, the mass ratio of the basal medium B and the active component in 100 mL of the complex medium is 80-99:1-20, for example, 97:3.

[0127] According to embodiments of the present application, the basal medium B can be selected from known basal media in the art. Illustratively, the basal medium B comprises DMEM / F12 (GIBCO / 10565018), 10% FBS (GIBCO / 10091-148), and 1% P / S (GIBCO / 15140122).

[0128] According to embodiments of the present application, the active component in 100 mL of the complex medium comprises at least:

[0129] penicillin-streptomycin (P / S) 0.1-2% (for example, 1%);

[0130] N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES) 1-20 mM (for example, 10 mM);

[0131] L-glutamine supplement (Glutamax) 0.1-2% (for example, 1%);

[0132] neuronal cell culture supplement (B27) 0.1-2% (for example, 1%);

[0133] N-acetylcysteine 0.1-5 mM (for example, 1.25 mM);

[0134] Fibroblast growth factor (FGF-7) 10-100 ng / ml (e.g. 50 ng / ml);

[0135] Noggin 50-200 ng / ml (e.g. 100 ng / ml);

[0136] R-spondin 1 100-1000 ng / ml (e.g. 500 ng / ml);

[0137] Wnt 3A 50-200 ng / ml (e.g. 100 ng / ml);

[0138] FGF10 100-500 ng / ml (e.g. 200 ng / ml);

[0139] Gastrin 0.1-10 nM (e.g. 1 nM);

[0140] ALK inhibitor (A-83-01) 0.1-10 μΜ (e.g. 2 μΜ);

[0141] ROCK inhibitor (Y-27632) 1-20 μΜ (e.g. 10 μΜ);

[0142] p38 MAPK inhibitor (SB202190) 1-20 μΜ (e.g. 10 μΜ);

[0143] Epirregulin 100-500 ng / ml (e.g. 200 ng / ml);

[0144] Capsanthin 100-500 ng / ml (e.g. 200 ng / ml);

[0145] Nicotinamide 1-20 mM (e.g. 10 mM).

[0146] According to an exemplary protocol of the present application, the complex culture solution comprises:

[0147] P / S 1%

[0148] HEPES 10 mM

[0149] Glutamax 1%

[0150] B27 1%

[0151] N-Acetylcysteine 1.25 mM

[0152] FGF-7 50 ng / ml

[0153] Noggin 100 ng / ml

[0154] R-spondin 1 500 ng / ml

[0155] Wnt 3A 100 ng / ml

[0156] FGF10 200 ng / ml

[0157] Gastrin 1 nM

[0158] A-83-01 2 mM

[0159] Y-27632 10 mM

[0160] SB202190 10 mM

[0161] Epirregulin 200 ng / ml

[0162] Capsanthin 200 ng / ml

[0163] Nicotinamide 10 mM.

[0164] [Preparation method of biomass-based culture solution for 3D culture of tumor organoids]

[0165] As described above, the present application also provides a preparation method of the above-mentioned biomass-based culture solution for 3D culture of tumor organoids, which comprises the following steps:

[0166] S1’: preparing a plant hydrogel;

[0167] S2’: mixing the plant hydrogel in step S1’ with a complex culture solution to prepare the biomass-based culture solution.

[0168] According to an embodiment of the present application, step S1’ is specifically defined as step S1 described above.

[0169] According to an embodiment of the present application, in step S2’, the volume ratio of the plant hydrogel to the complex culture solution is 1-10:1-10, for example, 1:1.

[0170] [Application of biomass-based culture solution for 3D culture of tumor organoids]

[0171] As described above, the present application also provides an application of the biomass-based culture solution for 3D culture of tumor organoids in the culture of tumor tissues or organoids such as gastric cancer, lung cancer, etc.

[0172] As described above, the present application also provides a method for culturing tumor tissues or organoids using the biomass-based culture solution for 3D culture of tumor organoids.

[0173] According to an embodiment of the present application, the tissue is preferably human gastric cancer tissue / normal gastric tissue, human intestinal cancer tissue / normal intestinal tissue, human lung cancer tissue / normal lung tissue, human pancreatic cancer tissue / normal pancreatic cancer tissue, human breast cancer tissue / normal breast cancer tissue.

[0174] According to an embodiment of the present application, the organoid is preferably human gastric cancer organoid / human normal gastric organoid, human intestinal cancer organoid / human normal intestinal organoid, human lung cancer organoid / human normal lung organoid, human pancreatic cancer organoid / human normal pancreatic cancer organoid, human breast cancer organoid / human normal breast cancer organoid.

[0175] According to an embodiment of the present application, the culturing method comprises the following steps:

[0176] a) preparing a cell suspension of the tissue or organoid: uniformly mixing the biomass-based culture solution for 3D culture of tumor organoids with cells of the tissue or organoid to obtain a cell suspension;

[0177] b) 3D culture: culturing the cell suspension of step a) in an incubator.

[0178] According to an embodiment of the present application, in step a), the cells of the tissue or organoid can be prepared by methods known in the art, which are not specifically limited by the present application.

[0179] According to an embodiment of the present application, in step b), the culturing can be performed by methods known in the art, as long as the desired cells can be obtained, for example, the cell suspension is added to a culture plate and cultured at 37℃ in 5% CO2.

[0180] According to an embodiment of the present application, in step b), the culturing time is 1 day to 10 days, further selected from 3, 5, 7 days.

[0181] According to an embodiment of the present application, in step b), the culturing further comprises medium replacement, for example, replacing the basic culture solution every 3 days.

[0182] As described above, the present application also provides a tumor organoid 3D culture kit.

[0183] According to an embodiment of the present application, the kit comprises the biomass-based culture solution for 3D culture of tumor organoids. Specifically, the tumor is selected from at least one of gastric cancer, lung cancer, intestinal cancer, esophageal cancer, breast cancer, etc.

[0184] According to an embodiment of the present application, the kit comprises a biomass-based hydrogel and a complex culture solution.

[0185] According to embodiments of the present application, the kit further comprises components or assemblies known in the art, without specific limitation of the present application.

[0186] According to embodiments of the present application, the kit is preferably used for culturing of gastric cancer organoids, intestinal cancer organoids.

[0187] According to embodiments of the present application, the kit further comprises a tissue digestion solution and / or a tissue preservation solution.

[0188] According to embodiments of the present application, the tissue digestion solution comprises collagenase P and DNase.

[0189] According to embodiments of the present application, the tissue preservation solution comprises Ad DMEM / F12 with 2x P / S.

[0190] As described previously, the present application also provides use of a tumor organoid 3D culture kit in tumor organoid 3D culture.

[0191] According to embodiments of the present application, the tumor organoid is selected from at least one of gastric cancer organoids, lung cancer organoids, intestinal cancer organoids, esophageal cancer organoids, breast cancer organoids, etc.

[0192] [Biomass-based culture solution for stem cell culture]

[0193] As described previously, the present application provides a plant source-based culture solution for stem cell culture, in particular for rapid stem cell culture. Specifically, the present application provides a biomass-based culture solution for stem cell culture, which comprises a plant hydrogel and a basic culture solution C; wherein the plant hydrogel is free of animal source components and comprises micro-nano biomass and water.

[0194] According to embodiments of the present application, the basic culture solution C can be selected from basic culture solutions known in the art. Exemplarily, the basic culture solution C comprises DMEM / F12, mTeSR TM 1.

[0195] [Preparation method of biomass-based culture solution for stem cell culture]

[0196] As described previously, the present application also provides a preparation method of the biomass-based culture solution for stem cell culture, which comprises the following steps:

[0197] S1”: preparing a plant hydrogel;

[0198] S2”: mixing the plant hydrogel in step S1” with a basic culture solution C to prepare the biomass-based culture solution.

[0199] According to embodiments of the present application, step S1” is the same as the specific definition of step S1 described above.

[0200] According to embodiments of the present application, in step S2”, the volume ratio of the plant hydrogel to the basal medium C is 1-10:1-10, for example, 1:1.

[0201] [Application of biomass-based medium for stem cell culture]

[0202] As described above, the present application also provides the application of the biomass-based medium for stem cell or organoid culture.

[0203] The present application also provides a method for stem cell or organoid culture using the biomass-based medium for stem cell culture.

[0204] According to embodiments of the present application, the stem cells are preferably adult stem cells or embryonic stem cells (ESCs) from mammals and primates; further preferably 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, osteogenic stem cells, chondrogenic 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 gland stem cells, cardiac stem cells, etc.

[0205] As described above, the present application also provides a stem cell culture method, which comprises the following steps:

[0206] a’) Pre-treatment before stem cell passage, dilution of plant hydrogel, and standing in an incubator;

[0207] b’) Digestion of extracellular matrix, using Dispase to digest extracellular matrix;

[0208] c’) Addition of mTeSR TM 1, blowing the stem cells to detach, disintegrate into fragments;

[0209] d’) Addition of stem cell fragments to plant hydrogel-containing culture dishes for culture.

[0210] According to embodiments of the present application, in step a’), DMEM / F12 is used to dilute the plant hydrogel, and after dilution, the diluted plant hydrogel is added to the culture plate and stands at 37°C for at least 1 hour.

[0211] According to embodiments of the present application, in step b’), the digestion using Dispase can be selected from methods known in the art, for example, covering the well plate with Dispase and placing it in a 37°C incubator until the edge of the stem cells begins to lift.

[0212] According to an embodiment of the present application, in step b'), further comprising the step of aspirating the Dispase and washing with DPBS.

[0213] According to an embodiment of the present application, in step c'), the fragments are 1-2 mm.

[0214] According to an embodiment of the present application, in step d'), the culturing can be performed using methods known in the art, for example including the use of mTeSR TM 1Culture medium is supplemented, incubated at 37°C, 5% CO2, and the like.

[0215] [Culture medium for stem cell induced differentiation organoid]

[0216] As described above, the present application also provides a culture medium for stem cell induced differentiation organoid, the culture medium comprising a plant hydrogel, a definitive endoderm differentiation medium, an MH differentiation medium, and an organoid growth medium; wherein the plant hydrogel is free of animal-derived components, and comprises micro-nano biomaterials and water.

[0217] According to an embodiment of the present application, the definitive endoderm differentiation medium comprises the following components:

[0218] RPMI1640 containing L-glutamine 2mM,

[0219] penicillin-streptomycin,

[0220] and Activin A.

[0221] According to an embodiment of the present application, preferably the definitive endoderm differentiation medium comprises the following components:

[0222] RPMI1640 containing L-glutamine 2mM,

[0223] penicillin-streptomycin 100U / ml-100g / ml,

[0224] and Activin A 100ng / ml.

[0225] According to an embodiment of the present application, the MH differentiation medium comprises the following components:

[0226] RPMI1640 containing 2% FBS, containing L-glutamine,

[0227] penicillin-streptomycin,

[0228] and FGF4.

[0229] According to an embodiment of the present application, preferably the MH differentiation medium comprises the following components:

[0230] RPMI 1640 with 2% FBS, L-glutamine, 2mM,

[0231] Penicillin-streptomycin, 100 U / ml - 100 g / ml,

[0232] and FGF4 500 ng / ml.

[0233] According to an embodiment of the application, the organoid growth medium comprises the following ingredients:

[0234] Advanced DMEM / F12 with 1 x B27, L-glutamine, 2mM,

[0235] Penicillin-streptomycin,

[0236] HEPES buffer,

[0237] and R-spondin 1.

[0238] According to an embodiment of the application, the preferred organoid growth medium comprises the following ingredients:

[0239] Advanced DMEM / F12 with 1 x B27, L-glutamine, 2mM,

[0240] Penicillin-streptomycin, 100 U / ml - 100 g / ml,

[0241] HEPES buffer, 15mM,

[0242] and R-spondin 1, 500 ng / ml.

[0243] [Use of stem cell induced differentiated organoid medium]

[0244] As mentioned previously, the application also provides the use of the above stem cell induced differentiated organoid medium in a stem cell induced differentiated organoid.

[0245] As mentioned previously, the application also provides a method for the production of a stem cell induced differentiated organoid, wherein said method comprises culturing a single stem cell or a population of stem cells in a stem cell induced differentiated organoid medium.

[0246] According to an embodiment of the application, the method for the production of a stem cell induced differentiated organoid comprises the following steps:

[0247] a") iPSC / ES differentiation in monolayer, comprising definitive endoderm differentiation and mid / hindgut (MH) differentiation;

[0248] b") organoid culture, comprising the preparation of an organoid growth medium, and the incubation of the plant hydrogel after mixing with the spheroids.

[0249] According to embodiments of the present application, the step a") said definitive endoderm differentiation comprises the preparation of a definitive endoderm differentiation medium and the definitive endoderm differentiation, wherein the step of definitive endoderm differentiation comprises incubation using the definitive endoderm differentiation medium selected from methods known in the art, for example incubation at 37°C, 5% CO2 and 95% humidity, and incubation for up to 30 minutes.

[0250] According to embodiments of the present application, the step a") said mid / hindgut (MH) differentiation comprises the preparation of a MH differentiation medium and the mid / hindgut (MH) differentiation, wherein the step of mid / hindgut (MH) differentiation comprises incubation using the MH differentiation medium selected from methods known in the art, for example incubation at 37°C, 5% CO2 and 95% humidity, medium change and observation of the spheroids once every 24 hours, spheroids embedded.

[0251] According to embodiments of the present application, the step b") said plant hydrogel is mixed with the spheroids and incubated selected from methods known in the art, for example comprising: incubation at 37°C for up to 30 minutes, addition of organoid growth medium and continued incubation at 37°C, 5% CO2, 95% humidity, and passaging according to the growth of the organoids.

[0252] As described above, the present application also provides a stem cell organoid prepared by the method of inducing differentiation of the stem cell organoid, such as an adult stem cell organoid, an induced pluripotent stem cell organoid or an embryonic stem cell organoid.

[0253] As described above, the present application also provides a kit for inducing differentiation of an organoid.

[0254] According to embodiments of the present application, the kit comprises the stem cell organoid inducing differentiation medium; in particular, the organoid is preferably at least one of an intestinal organoid, a gastric organoid, a liver organoid and the like, and further preferably a small intestinal organoid.

[0255] According to embodiments of the present application, the kit further comprises a definitive endoderm differentiation medium, a MH differentiation medium and an organoid growth medium.

[0256] According to embodiments of the present application, the kit further comprises components or assemblies known in the art, which are not specifically limited by the present application.

[0257] As described above, the present application also provides a use of the kit for inducing differentiation of an organoid.

[0258] According to embodiments of the present application, the organoid for inducing differentiation is at least one of an intestinal organoid for inducing differentiation, a gastric organoid for inducing differentiation, a liver organoid for inducing differentiation and the like, and preferably a small intestinal organoid.

[0259] [Biomass]

[0260] The biomass in the micro-nano biomass described in the present application can be at least one of natural biomass, surface-modified biomass, or substituted biomass; for example, at least one of natural polymer, surface-modified natural polymer, or substituted natural polymer in patent document CN116284844A. Specifically,

[0261] The natural polymer is, for example, selected from one or more of the following: cellulose, starch, lignin, chitosan, chitin, hemicellulose, dextran, low-quality cellulose containing one or more of the above components, or natural plant tissue.

[0262] The cellulose is derived from cellulose extracted from plant tissue or other biomass. For example, the cellulose extracted from plant tissue is selected from one or more of the following: microcrystalline cellulose, bacterial cellulose, cotton pulp, wood pulp, bamboo pulp, straw pulp, refined cotton, defatted cotton, cotton linters, and sugarcane residue, wood, and straw, etc.; preferably one or more of microcrystalline cellulose, refined cotton, defatted cotton, and wood pulp. For example, the non-extracted biomass can be mixed biomass such as straw.

[0263] The starch is selected from at least one of amylopectin, amylose, high amylose starch, modified starch, and cross-linked starch; or the starch is soluble starch; or the starch is selected from at least one of potato starch, corn starch, tapioca starch, wheat starch, sweet potato starch, kudzu starch, pea starch, water chestnut starch, soybean starch, and lotus root starch.

[0264] The selection of chitosan, chitin, lignin, hemicellulose, dextran, etc. is not particularly limited and is known to those skilled in the art and is applicable to the system described in the present application.

[0265] Preferably, the degree of deacetylation of the chitosan is 50-100%; more preferably, the degree of deacetylation of the chitosan is 70-95%.

[0266] Preferably, the lignin can be selected from one or more of syringyl lignin, guaiacyl lignin, and p-hydroxyphenyl lignin; or the lignin is selected from one or more of alkali lignin, acid lignin, dealkali lignin, and organic solvent-soluble lignin.

[0267] The low-quality cellulose is at least plant tissue containing cellulose and lignin, such as herbaceous plants and / or agricultural and forestry waste, etc.

[0268] For example, the herbaceous plants are selected from one or more of trees, shrubs, vines, leaves, and bamboo, etc.

[0269] For example, the agroforestry waste is selected from one or more of tree bark, tree leaves, sawdust, crop straw, fruit shell or pit, corn cob, sugarcane residue, and the like.

[0270] Preferably, the crop straw can be selected from one or more of wheat straw, rice straw, corn straw, soybean straw, cotton straw, ginger stem, sesame straw.

[0271] The surface-modified natural polymer can be a surface-cationized natural polymer, a surface-anionized natural polymer, or a surface-hydrophobic-treated natural polymer. Specifically, the natural polymer in the surface-modified natural polymer has the definition as above. Further specifically, the surface-modified natural polymer can be obtained by mixing the natural polymer with a modifying agent or by surface chemical modification of the natural polymer (for example, the surface-modified natural polymer can be obtained by homogeneous chemical derivatization or functionalization of the natural polymer in a solvent as below with a modifying agent). Wherein, the modifying agent can be one or more of quaternary ammonium salt, sulfonate, phosphate, and the like; for example, sodium vinyl sulfonate or 3-chloro-2-hydroxypropyl trimethyl ammonium chloride.

[0272] The substituted natural polymer can be selected from one or more of esterified natural polymer, acylated natural polymer, etherified natural polymer, amidated natural polymer, ammoniated natural polymer, and the like. Specifically, the natural polymer in the substituted natural polymer has the definition as above. Further for example, the substituted natural polymer is a low-substituted natural polymer; preferably, the degree of substitution of the low-substituted natural polymer is 0.0001-2.0, preferably 0.001-1.8, for example, 0.1, 0.5, 0.65, 0.86, 1.46, 1.6. Further specifically, the substituted natural polymer can be prepared by reaction of a functionalized derivatization agent with a natural polymer. For example, the functionalized derivatization agent can be selected from esterification agent, acylation agent, etherification agent, ammoniation agent, and / or amidation agent. Exemplarily, the substituted natural polymer can be selected from cellulose acetate (for example, cellulose acetate with a degree of substitution of 0.65, 0.86, 1.46, 1.6), cellulose-g-PLA.

[0273] [Plant hydrogel]

[0274] As mentioned above, the culture solution of the present application comprises a plant hydrogel, which is free of animal-derived ingredients, comprises micro-nano biomass and a dispersion agent; the dispersion agent is selected from an organic solvent and / or water as below, preferably water and / or alcohol.

[0275] According to an embodiment of the present application, the culture solution of the present application comprises a plant hydrogel, which is free of animal-derived ingredients, comprises micro-nano biomass and water.

[0276] According to an embodiment of the present application, the biomass in the micro-nano biomass has the definition listed above.

[0277] According to an embodiment of the present application, the micro-nano biomass comprises micro-scale structures and nano-scale structures distributed on the micro-scale structures.

[0278] According to an embodiment of the present application, the content of the micro-nano biomass in the plant hydrogel can be 0.050-95wt.%, for example, 0.5-10wt.%, specifically, 1wt.%, 5wt.%, 10wt.%, 15wt.%, 20wt.%, 30wt.%, 40wt.%, 50wt.%, 60wt.%, 70wt.%, 80wt.%, 90wt.%, or 95wt.%. Too high concentration, too viscous, inconvenient to use, but convenient to transport, save cost; too low concentration, poor dispersion stability.

[0279] According to an embodiment of the present application, the micro-nano biomass is regenerated natural polymer with micro-nano structure (for example, type II cellulose, or a mixture of type I cellulose and type II cellulose). The regenerated natural polymer with micro-nano structure means that the regenerated natural polymer comprises micro-scale structures and nano-scale structures distributed on the micro-scale structures. Specifically, the natural polymer in the regenerated natural polymer has the definition listed above.

[0280] According to an embodiment of the present application, the size (i.e. volume average size) of the micro-scale structure is 10μm-300μm, preferably the size of the micro-scale structure is 50μm-250μm, further preferably the size of the micro-scale structure is 100μm-200μm, for example, any point value in the intermediate value of any value or any two point values of 10μm, 20μm, 30μm, 40μm, 50μm, 60μm, 70μm, 80μm, 90μm, 100μm, 105μm, 110μm, 115μm, 120μm, 125μm, 130μm, 135μm, 140μm, 145μm, 150μm, 155μm, 160μm, 165μm, 170μm, 175μm, 180μm, 185μm, 190μm, 195μm, 200μm, 210μm, 220μm, 230μm, 240μm, 250μm, 260μm, 270μm, 280μm, 290μm, 300μm.

[0281] According to an embodiment of the present application, the length of the nano-scale structure is 500nm-1500nm, preferably the length of the nano-scale structure is 700nm-1200nm, more preferably the length of the nano-scale structure is 800nm-1000nm, for example any point value in the range of 500nm, 600nm, 700nm, 800nm, 900nm, 1000nm, 1100nm, 1200nm, 1300nm, 1400nm, 1500nm or any intermediate value formed by any two point values.

[0282] According to an embodiment of the present application, the diameter of the nano-scale structure is 200nm-600nm, preferably the diameter of the nano-scale structure is 300nm-500nm, for example any point value in the range of 200nm, 300nm, 400nm, 500nm, 600nm or any intermediate value formed by any two point values.

[0283] According to an embodiment of the present application, the length-diameter ratio of the nano-scale structure is 1:1-1:150, preferably the length-diameter ratio of the nano-scale structure is 1:1-1:100, more preferably the length-diameter ratio of the nano-scale structure is 1:1-1:50.

[0284] According to an embodiment of the present application, the micro-scale structure comprises at least one of micro-slices, micro-fibers and micro-multi-scale structures.

[0285] According to an embodiment of the present application, the micro-scale structure is in the form of fibers or films, which can also be referred to as whiskering.

[0286] According to an embodiment of the present application, the nano-scale structure is distributed on the surface and / or edge of the micro-scale structure. Specifically, the nano-scale structure is densely distributed on the surface and / or edge of the micro-scale structure.

[0287] According to an embodiment of the present application, the nano-scale structure is distributed on the micro-scale structure and forms a flocculent structure as a whole.

[0288] According to an embodiment of the present application, the nano-scale structure is distributed on the micro-scale structure and forms a structure similar to a porous sponge as a whole.

[0289] According to an embodiment of the present application, the nano-scale structure and the micro-scale structure together form a feather-like structure.

[0290] According to an embodiment of the present application, the nano-scale structure is in the form of villi, tentacles, whiskers or other irregular structures.

[0291] According to an embodiment of the present application, the micro-nano biomass is in the form of granular, fibrous, sheet-like, feather-like, dendritic, flocculent or porous sponge-like structures.

[0292] According to embodiments of the present application, the micro-nano biomass described in the present application is, for example, natural cellulose glue CMG.

[0293] According to embodiments of the present application, the plant hydrogel is provided by at least one of a natural biomass-based dispersion, a surface-modified biomass-based dispersion, a substituted biomass-based dispersion, and a multi-component biomass-based dispersion.

[0294] [Preparation method of natural biomass-based dispersion]

[0295] As described above, the plant gel of the present application can be provided by a natural biomass-based dispersion including the micro-nano natural biomass and a dispersion agent, the micro-nano natural biomass being distributed in the dispersion agent, and the dispersion agent being a solvent system capable of continuously dispersing the micro-nano natural biomass. Specifically, in the dispersion, the micro-nano natural biomass is the dispersed phase, and the dispersion agent is the continuous phase.

[0296] According to embodiments of the present application, the dispersion agent is selected from the organic solvents and / or water described below, and is preferably water and / or alcohol.

[0297] The present application also provides a preparation method of the natural biomass-based dispersion described above, including the following steps: pouring a natural biomass solution into a coagulation bath to form a natural biomass gel; and performing micro-nano processing on the natural biomass gel by means of a homogenizer, a colloid mill, or a ball mill to obtain the biomass micro-nano dispersion, i.e., the biomass-based dispersion.

[0298] According to embodiments of the present application, the natural biomass has the selections as shown above.

[0299] According to embodiments of the present application, the coagulation bath can be the same as or different from the continuous phase, for example, can be the same; and is preferably water and / or alcohol.

[0300] According to embodiments of the present application, the natural biomass-based dispersion can be a homogeneous solution in which the micro-nano natural biomass is completely dissolved, or a heterogeneous dispersion in which the micro-nano natural biomass is partially dissolved.

[0301] [Preparation method of surface-modified biomass-based dispersion]

[0302] As described above, the plant gel of the present application can be provided by a surface-modified biomass-based dispersion including surface-modified micro-nano biomass and a dispersion agent, the surface-modified micro-nano biomass being dispersed in the dispersion agent; and the dispersion agent being a solvent system capable of continuously dispersing the surface-modified micro-nano biomass. Specifically, in the dispersion, the surface-modified micro-nano biomass is the dispersed phase, and the dispersion agent is the continuous phase.

[0303] According to an embodiment of the present application, the surface-modified micro-nano biomass is a surface-modified product of the micro-nano natural biomass; or is a product after the surface-modified biomass is micro-nano treated.

[0304] According to an embodiment of the present application, the continuous phase of the dispersion has the meaning as indicated above, preferably water and / or alcohol.

[0305] According to an embodiment of the present application, the surface modification can be one of surface cationization, surface anionization or surface hydrophobization. For example, surface cationized micro-nano biomass, surface anionized micro-nano biomass or surface hydrophobized micro-nano biomass.

[0306] According to an embodiment of the present application, the natural biomass and the surface-modified biomass have the selection as indicated above.

[0307] According to an embodiment of the present application, the content of the dispersed phase can be 0.050-95 wt.%; for example, 0.5-10 wt.%.

[0308] According to an embodiment of the present application, the dispersion is a micro-nano dispersion; in particular, in the dispersion, the surface-modified micro-nano biomass has substantially the same structure as the micro-nano natural biomass, for example, including micro-scale structure and nano-scale structure distributed on the micro-scale structure.

[0309] The present application also provides a preparation method of the above-mentioned surface-modified biomass-based dispersion, comprising the following steps: pouring a surface-modified biomass solution into a coagulation bath to form a surface-modified biomass gel; and micro-nano treating the surface-modified biomass gel by means of a homogenizer, colloid mill or ball mill to obtain the surface-modified biomass-based dispersion.

[0310] Alternatively, the natural biomass-based dispersion is mixed with a modification reagent or is surface-chemically modified to obtain the surface-modified biomass-based dispersion.

[0311] Preferably, the surface-modified biomass and the coagulation bath have the meaning as indicated above.

[0312] According to an embodiment of the present application, the modification reagent can be one or more of quaternary ammonium salt, sulfonate, phosphate, etc.; for example, sodium vinyl sulfonate or 3-chloro-2-hydroxypropyl trimethyl ammonium chloride.

[0313] According to an embodiment of the present application, the surface-modified biomass solution can be obtained by homogenous chemical derivation or functionalization of the natural biomass in the solvent described below with the modification reagent.

[0314] According to an embodiment of the present application, the surface-modified biomass-based dispersion can be a surface-modified micro-nano biomass completely dissolved homogeneous solution or a partially dissolved heterogeneous dispersion.

[0315] [Substituted biomass-based dispersion and method for preparing the same]

[0316] As shown above, the plant gel of the present application can be provided by a substituted biomass-based dispersion, which comprises substituted micro-nano biomass and a dispersion agent, the substituted micro-nano biomass being dispersed in the dispersion agent; the dispersion agent is a solvent system capable of continuously dispersing the substituted micro-nano biomass. Specifically, in the dispersion, the substituted micro-nano biomass is the dispersed phase, and the dispersion agent is the continuous phase.

[0317] According to an embodiment of the present application, the substituted micro-nano biomass is a substitute of the micro-nano natural biomass; or is a product after the substituted biomass is micro-nano processed.

[0318] According to an embodiment of the present application, the continuous phase of the dispersion has the meaning as shown above, and is preferably water and / or alcohol.

[0319] According to an embodiment of the present application, the substituted micro-nano biomass is a low-substituted micro-nano biomass, for example, the degree of substitution of the low-substituted micro-nano biomass is 0.0001-2.0, preferably 0.001-1.8, for example, 0.1, 0.5, 0.65, 0.86, 1.46, 1.6.

[0320] Specifically, the substituted micro-nano biomass is a substituted micro-nano natural polymer; specifically, the substituted micro-nano natural polymer is a low-substituted micro-nano natural polymer. Preferably, the degree of substitution of the low-substituted micro-nano natural polymer is 0.0001-2.0, preferably 0.001-1.8, for example, 0.1, 0.5, 0.65, 0.86, 1.46, 1.6.

[0321] According to an embodiment of the present application, the substituted micro-nano biomass can be selected from one or more of the following: esterified micro-nano natural polymer, acylated micro-nano natural polymer, etherified micro-nano natural polymer, amidated micro-nano natural polymer, ammoniated micro-nano natural polymer, etc.

[0322] Preferably, the substituted biomass can be prepared by reacting a functional derivative agent with a natural biomass. For example, the functional derivative agent can be selected from an esterification agent, an acylation agent, an etherification agent, an ammoniation agent, and / or an amidation agent.

[0323] According to an embodiment of the present application, the substituted biomass can be selected from cellulose acetate (e.g. cellulose acetate with a degree of substitution of 0.65, 0.86, 1.46, 1.6), cellulose-g-PLA.

[0324] According to an embodiment of the present application, the natural biomass has a selection as shown above.

[0325] According to an embodiment of the present application, the content of the dispersed phase can be 0.050-95 wt.%; illustratively, 0.5-10 wt.%.

[0326] According to an embodiment of the present application, the dispersion is a micro-nano dispersion; specifically, in the dispersion, the substituted micro-nano biomass has substantially the same structure as the micro-nano natural biomass, e.g. including micro-scale structures and nano-scale structures distributed on the micro-scale structures.

[0327] The present application also provides a method for preparing the above-mentioned substituted biomass-based dispersion, comprising the following steps: pouring a solution of substituted biomass into a coagulation bath to form a substituted biomass gel; and subjecting the substituted biomass gel to micro-nano treatment by means of a homogenizer, colloid mill or ball mill to obtain the substituted biomass-based dispersion.

[0328] Alternatively, the natural biomass-based dispersion is mixed with a functional derivatization reagent to obtain the substituted biomass-based dispersion.

[0329] Preferably, the substituted biomass, coagulation bath and functional derivatization reagent have the meanings as shown above.

[0330] According to an embodiment of the present application, the substituted micro-nano biomass dispersion can be obtained by subjecting a micro-nano natural biomass to homogeneous chemical derivatization or functionalization in a solvent with a functional derivatization reagent.

[0331] According to an embodiment of the present application, the substituted micro-nano biomass dispersion can be a homogeneous solution in which the substituted micro-nano biomass is completely dissolved or a heterogeneous dispersion in which the substituted micro-nano biomass is partially dissolved.

[0332] [Multi-component biomass-based dispersion and method for preparing the same]

[0333] As shown above, the plant gel of the present application can be provided by a multi-component biomass-based dispersion, the continuous phase of which is a dispersion reagent, and the dispersed phase of which contains at least one of the following components 1) to 3) and a functional component:

[0334] Component 1) the micro-nano natural biomass;

[0335] Component 2) the substituted micro-nano biomass;

[0336] the surface-modified micro-nano biomass of component 3).

[0337] According to an embodiment of the present application, the continuous phase of the dispersion has the selection as shown above, preferably water and / or alcohol. According to an embodiment of the present application, the dispersed phase of the dispersion contains at least one of micro-nano natural biomass, substituted micro-nano biomass, surface-modified micro-nano biomass and functional component.

[0338] According to an embodiment of the present application, the biomass in component 1), 2) or 3) has the meaning as described above, or can be selected from at least one of methyl cellulose, ethyl cellulose, benzyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, cyanoethyl cellulose, benzyl cyanoethyl cellulose, carboxymethyl hydroxyethyl cellulose, chitosan, sodium alginate, starch, gelatin.

[0339] According to an embodiment of the present application, the functional component is selected from one or more of the following: nanocellulose, carbon nanotube, graphene, fullerene, carbon black, silver nanowire, quantum dot, carbon dot, nano Fe3O4, drug, etc.

[0340] According to an embodiment of the present application, the content of the dispersed phase can be 0.050-95 wt.%; for example, it can be 0.5-10 wt.%.

[0341] The present application also provides a preparation method of the above-mentioned multi-component biomass-based dispersion, which is selected from the following scheme one or scheme two:

[0342] Scheme one: pour a solution containing at least one of natural biomass gel, substituted biomass gel, surface-modified biomass gel and functional component into a coagulation bath to form a gel; and then micro-nanoize the gel by means of a homogenizer, colloid mill or ball mill to obtain the multi-component biomass-based dispersion;

[0343] Scheme two: mix at least one of natural biomass-based dispersion, substituted biomass-based dispersion, surface-modified biomass-based dispersion and functional component, and then micro-nanoize to obtain the multi-component biomass-based dispersion.

[0344] Preferably, the natural biomass gel, substituted biomass gel, surface-modified biomass gel, functional component, coagulation bath, natural biomass-based dispersion, substituted biomass-based dispersion and surface-modified biomass-based dispersion all have the selection as shown above.

[0345] Preferably, a crosslinking agent can also be added before forming the gel. Preferably, the crosslinking agent is selected from at least one of boric acid, calcium chloride, acetic acid, ethylenediaminetetraacetic acid.

[0346] According to an embodiment of the present application, the dispersion is a homogeneous solution or a heterogeneous dispersion.

[0347] [Solvent]

[0348] In the method for preparing the aforementioned natural biomass-based dispersion, surface-modified biomass-based dispersion, substituted biomass-based dispersion, or multi-component biomass-based dispersion, the solvent in the homogeneous solution and / or heterogeneous dispersion is not particularly limited and can be selected from any good solvent known in the art that can dissolve (the dissolution includes complete dissolution and partial dissolution) the solute (for example, the natural polymer) therein. Preferably, according to the type of natural polymer, the solvent selected can be selected from one or more of the following systems: copper ammonia solution, copper ethylenediamine solution, organic solvent, ionic liquid, ionic liquid and organic solvent mixed solvent, choline type ionic liquid eutectic solvent system, organic solvent / salt system, amine oxide system (NMMO), carbamate system, base / water system, base / urea system, base / thio-urea system, liquid ammonia / NH4SCN, organic acid, aqueous metal salt, alcohol solution of metal salt hydrate, aqueous alcohol mixed solution of metal salt hydrate, and the like.

[0349] The organic solvent can 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, gamma-valerolactone (GVL).

[0350] 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, N,N-dimethyl sulfoxide / tetrabutylammonium fluoride system (DMSO / TBAF).

[0351] The base / water system can be selected from one or both of NaOH / H2O and KOH / H2O.

[0352] The base / urea system can be selected from NaOH / Urea.

[0353] The base / thio-urea system can be selected from NaOH / thio-urea.

[0354] 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, benzenesulfonic acid, and the like.

[0355] The metal salt aqueous solution is preferably selected from the group consisting of CaCl2, ZnCl2, LiClO4, Ca(SCN)2, LiSCN and the like.

[0356] The metal salt hydrate alcohol solution is preferably selected from the group consisting of CaBr2-H2O in methanol, CaCl2-2H2O in methanol.

[0357] The metal salt hydrate alcohol solution is preferably selected from the group consisting of CaBr2-H2O in methanol, CaCl2-2H2O in methanol.

[0358] The amine oxide system is preferably selected from the group consisting of NMMO / H2O / DMSO, NMMO / H2O / diethyltriamine, NMMO / H2O.

[0359] The ionic liquid is preferably selected from the group consisting of organic molten salts with melting point lower than 100°C, and more preferably selected from the group consisting of organic molten salts which can dissolve the natural high molecular of biomass.

[0360] For example, the cation of the ionic liquid is selected from the group consisting of one or more of substituted or unsubstituted imidazole, pyridine, pyrrole, amine, phosphine, choline, diazabicyclo, amino acid type cation; for example, the substituent group can 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, allyl;

[0361] Preferably, the cation is selected from one or more of the following: 1-ethyl-3- methylimidazolium cation ([EMIM]), 3-methylimidazolium cation ([MIM]), 1- propyl-3-methylimidazolium cation ([PMIM]), 1-allyl-3-methylimidazolium cation ([AMIM]), 1-butyl-3-methylimidazolium cation ([BMIM]), 1-butyl-2,3- dimethylimidazolium cation ([BMMIM]), 1,3-dimethylimidazolium cation ([MMIM]), 1-methoxyethyl-3-methylimidazolium cation ([MeOEMIM]), 1- methoxymethyl-3-methylimidazolium cation ([MeOMMIM]), 1-hydroxy-3- methyl-imidazolium cation ([HMIM]), 1-(2-hydroxyethyl)-3-methylimidazolium cation ([HOEMIM]), 1-methyl-3-benzylimidazolium cation ([MBzIM]), 1- pentyl-3-methylimidazolium cation ([PeMIM]), 1-benzyl-3-methylimidazolium cation ([BzMIM]), 1-m-methoxybenzyl-3-methylimidazolium cation ([MeOBzMIM]), 1-m-methylbenzyl-3-methylimidazolium cation ([MeBzMIM]), N- methylimidazolium cation ([MPyr]), N-ethylpyridinium cation ([EPyr]), N- butylpyridinium cation ([BPyr]), N-n-hexylpyridinium cation ([HPyr]), 1-butyl-3- methylpyrrolidinium ion ([BMPyrr]), tris(2-hydroxyethyl)methylamine ([THEMA]), tetrabutylammonium ([TBA]), tetrabutylphosphonium ([PBu4]), glycine cation ([Gly]), choline cation ([Ch]), 1,5-diazabicyclo[4.3.0]non-5-ene ([DBNH]), and the like.

[0362] More preferably, the cation is selected from one or more of the following: 1-ethyl-3- methylimidazolium cation ([EMIM]), 1-allyl-3-methylimidazolium cation ([AMIM]), 1-butyl-3-methylimidazolium cation ([BMIM]), choline cation ([Ch]).

[0363] For example, the anion is selected from one or more of the following: halide anion, organic acid anion, organic acid ester anion, amino acid type anion, and the like.

[0364] Preferably, the anion is selected from one or more of the following anions: chloride ([CI]), bromide ([Br]), fluoride ([F]), formate ([HCOO]), acetate ([CH3COO] or [Ac]), glycolate ([HOCH2COO]), propionate ([CH3CH2COO] or [OPr]), butyrate ([CH3CH2CH2COO] or [OBu]), octoate ([Oct]), benzoate ([C6H5COO] or [PhCOO]), lactate ([CH3CH(OH)COO] or [Lac]), thioglycolate ([HSCH2COO]), hexafluorophosphate ([PF6]), triflate ([BF3]), methylphosphonate ([ (MeO)HPO2] or [MP]), dimethylphosphonate ([ (MeO)2PO2] or [DMP]), diethylphosphonate ([ (EtO)2PO2] or [DEP]), methylsulfonate ([MeOSO3]), trifluoromethylsulfonate ([CF3SO3]), glycinate ([Gly]), lysinate ([Lys]), valinate ([Val]), dicyanamide ([N(CN)2] or [DCA]), bistrifluoromethylsulfonimide ([Tf2N]), and the like.

[0365] More preferably, the anion is selected from one or more of the following anions: chloride ([CI]), formate ([HCOO]), acetate ([Ac]), methylphosphonate ([ (MeO)HPO2] or [MP]), dimethylphosphonate ([ (MeO)2PO2] or [DMP]), and dicyanamide ([N(CN)2] or [DCA]).

[0366] According to the present application, the ionic liquid can be selected from one or more of the following ionic liquids: 1-ethyl-3-methylimidazolium chloride ionic liquid ([EMIM][CI]), 1-ethyl-3-methylimidazolium bromide ionic liquid ([EMIM][Br]), 1-ethyl-3-methylimidazolium formate ionic liquid ([EMIM][HCOO]), 1-ethyl-3-methylimidazolium acetate ionic liquid ([EMIM][Ac]), 1-ethyl-3-methylimidazolium octanoate ionic liquid ([EMIM][Oct]), 1-ethyl-3-methylimidazolium methylphosphonate ionic liquid ([EMIM][MP]), 1-ethyl-3-methylimidazolium dimethylphosphonate ionic liquid ([EMIM][DMP]), 1-ethyl-3-methylimidazolium diethylphosphonate ionic liquid ([EMIM][DEP]), 1-ethyl-3-methylimidazolium propionate ionic liquid ([EMIM][OPr]), 1-ethyl-3-methylimidazolium terephthalate ionic liquid ([EMIM][OBu]), 1-ethyl-3-methylimidazolium glycinate ionic liquid ([EMIM][Gly]), 1-ethyl-3-methylimidazolium lysinate ionic liquid ([EMIM][Lys]), 1-allyl-3-methylimidazolium chloride ionic liquid ([AMIM][CI]), 1-allyl-3-methylimidazolium bromide ionic liquid ([AMIM][Br]), 1-allyl-3-methylimidazolium formate ionic liquid ([AMIM][HCOO]), 1-allyl-3-methylimidazolium acetate ionic liquid ([AMIM][Ac]), 1-butyl-3-methylimidazolium chloride ionic liquid ([BMIM][CI]), 1-butyl-3-methylimidazolium bromide ionic liquid ([BMIM][Br]), 1-butyl-3-methylimidazolium formate ionic liquid ([BMIM][HCOO]), 1-butyl-3-methylimidazolium acetate ionic liquid ([BMIM][Ac]), 1-butyl-3-methylimidazolium glycolate ionic liquid ([BMIM][HOCH2COO]), 1-butyl-3-methylimidazolium propionate ionic liquid ([BMIM][CH3CH2COO]), 1-butyl-3-methylimidazolium lactate ionic liquid [BMIM][Lac], 1-butyl-3-methylimidazolium butyrate ionic liquid ([BMIM][CH3CH2CH2COO]), 1-butyl-3-methylimidazolium benzoate ionic liquid ([BMIM][C6H5COO]), 1-butyl-3-methylimidazolium glycinate ionic liquid ([BMIM][H2NCH2COO]), 1-butyl-3-methylimidazolium dicyanamide ionic liquid ([BMIM][N(CN)2]), 1-butyl-3-methylimidazolium bistrifluoromethylsulfonylimide ionic liquid ([BMIM][Tf2N]),1 -butyl-3-methylimidazolium hexafluorophosphate ionic liquid ([BMIM][PF6]), 1 -butyl-3-methylimidazolium tetrafluoroborate ionic liquid ([BMIM][BF4]), 1 -butyl-3-methylimidazolium methylsulfonate ionic liquid ([BMIM][MeOSO3]), 1 -butyl-3-methylimidazolium trifluoromethylsulfonate ionic liquid ([BMIM][CF3SO3]), 1 -butyl-2,3-dimethylimidazolium tetrafluoroborate ionic liquid ([BMMIM][BF4]), 3-methylimidazolium formate ionic liquid ([MIM][HCOO]), 1,3-dimethylimidazolium chloride ionic liquid ([MMIM][CI]), 1,3-dimethylimidazolium methylphosphonate ionic liquid ([MMIM][MP]), 1,3-dimethylimidazolium dimethylphosphonate ionic liquid ([MMIM][DMP]), 1,3-dimethylimidazolium methylsulfonate ionic liquid ([MMIM][MeOSO3]), 1 -hydroxy-3-methyl-imidazolium chloride ionic liquid ([HMIM][CI]), 1 -hydroxy-3-methyl-imidazolium trifluoromethylsulfonate ionic liquid ([HMIM][CF3SO3]), 1 -(2-hydroxyethyl)-3-methylimidazolium chloride ionic liquid ([HOEMIM][CI]), 1 -methoxymethyl-3-methylimidazolium bromide ionic liquid ([MeOMMIM][Br]), 1 -methoxyethyl-3-methylimidazolium bromide ionic liquid ([MeOEMIM][Br]), N-ethylpyridinium chloride ionic liquid ([EPyr][CI]), N-ethylpyridinium bromide ionic liquid ([EPyr][Br]), N-methylpyridinium formate ionic liquid ([MPyr][HCOO]), tris(2-hydroxyethyl)methylammonium acetate ionic liquid ([THEMA][Ac]), tris(2-hydroxyethyl)methylammonium methylsulfonate ionic liquid ([THEMA][MeOSO3]), tris(2-hydroxyethyl)methylammonium trifluoromethylsulfonate ionic liquid [THEMA][CF3SO3], tetrabutylphosphonium valinate ionic liquid [PBu4][Val], tetrabutylphosphonium lysinate ionic liquid [PBu4][Lys], tetrabutylphosphonium glycinate ionic liquid [PBu4][Gly], 1 -benzyl-3-methylimidazolium chloride ionic liquid ([BzMIM][CI]), 1 -benzyl-3-methylimidazolium dicyanamide ionic liquid ([BzMIM][DCA]), 1 -m-methylbenzyl-3-methylimidazolium chloride ionic liquid ([MeBzMIM][CI]), 1 -m-methoxybenzyl-3-methylimidazolium chloride ionic liquid ([MeOBzMIM][CI]), choline chloride ionic liquid ([Ch][CI]), choline bromide ionic liquid ([Ch][Br]), choline acetate ionic liquid ([Ch][CH3COO]),ionic liquids such as choline propionate ionic liquid ([Ch][CH3CH2COO]), choline butyrate ionic liquid ([Ch][CH3CH2CH2COO]), glycine hydrochloride ionic liquid ([Gly][Cl]), 1,5-diazabicyclo[4.3.0]non-5-ene acetate ionic liquid ([DBNH][Ac]), and the like.

[0367] Preferably, the choline-based deep eutectic solvent system is selected from one or more of [Ch][Cl] / urea, [Ch][Br] / urea, [Ch][Cl] / thio-urea, [Ch][Cl] / glycerol, [Ch][Cl] / lactic acid.

[0368] Preferably, the cellulose-dissolving solvent system 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], [BMIM][Ac].

[0369] Preferably, the starch-dissolving solvent is selected from one or more of the following solvent systems: DMSO, CaCl2, ZnCl2, LiClO4, Ca(SCN)2, LiSCN, NaOH, KOH, aqueous solutions of NaOH / urea, ethylenediamine, pyridine, NMMO, DMAc / LiCl, and ionic liquids such as [AMIM][Cl], [EMIM][Ac], [EMIM][DEP], [BMIM][Cl], [BMIM][Ac], [BMIM][PF6], [BMIM][DCA], [MMIM][(MeO)HPO2], [AMIM][HCOO]), [MeOEMIM][Br], [MeOMMIM][Br], and aqueous solutions of the ionic liquids. More preferably, the starch-dissolving ionic liquid is selected from one or more of [AMIM][Cl], [EMIM][Ac], [BMIM][Cl], [BMIM][Ac], [BMIM][PF6], [BMIM][DCA], [EMIM][DEP], [MMIM][MP].

[0370] Preferably, the solvent for dissolving chitosan is selected from any one or more of the following solvents: formic acid, acetic acid, hexafluoroisopropanol, hexafluoroacetone, DMAc / LiCl and [AMIM][CI], [BMIM][CI], [BMIM][Ac], [BMIM][HCOO], [BMIM][CH3COO], [BMIM][CH3CH2COO], [BMIM][CH3CH2CH2COO], [BMIM][HOCH2COO], [BMIM][C6H5COO], [BMIM][CH3CH(OH)COO], [BMIM][N(CN)2], [BMIM][BF4], [EMIM][CI], [EMIM][Ac], [HMIM][CI], [MMIM][CI], [Ch][CI], [Ch][CH3COO], [Ch][CH3CH2COO], [Ch][CH3CH2CH2COO], [Gly][CI] / [BMIM][CI] ionic liquids; more preferably, the ionic liquid for dissolving chitosan is preferably selected from one or more of: [AMIM][CI], [BMIM][CI], [BMIM][Ac], [EMIM][Ac], [BMIM][CH3CH2COO], [BMIM][CH3CH2CH2COO], [BMIM][BF4], [Gly][CI], [HMIM][CI], [MMIM][CI], [Ch][CI], [Ch][CH3CH2CH2COO].

[0371] Preferably, the solvent system for dissolving chitin is selected from one or more of the following: formic acid, acetic acid, glutamic acid, lactic acid, succinic acid, dichloroacetic acid (DCA), trichloroacetic acid (TCA), N-methyl-2-pyrrolidone (NMP), hexafluoroisopropanol, hexafluoroacetone, NMP / LiCl, DMAc / LiCl, CaBr2-H2O or CaCl2-2H2O methanol saturated solution, LiCl, LiSCN, NaOH-urea aqueous solution, and [AMIM][CI], [AMIM][Br], [AMIM][Ac], [BMIM][Ac], [BMIM][CI], [MMIM][DMP], [EMIM][Ac], [EMIM][DMP], [EMIM][Ac], [EMIM][OPr], [EMIM][OBu], [EMIM][Gly], [EMIM][Lys], [HOEMIM][CI], [THEMA][Ac], [THEMA][MeOSO3], [THEMA][CF3SO3], [PBu4][Val], [PBu4][Lys], [PBu4][Gly], [Ch][CI] / urea, [Ch][Br] / urea, [Ch][CI] / thio-urea ionic liquid or mixed ionic liquid systems. More preferably, the ionic liquid for dissolving chitin is selected from one or more of the following: [AMIM][Ac], [BMIM][Ac], [EMIM][Ac], [BMIM][CI], [AMIM][CI], [AMIM][Br], [EMIM][OPr], [EMIM][OBu], [Ch][CI] / urea, [Ch][Br] / urea, [Ch][CI] / thio-urea.

[0372] Preferably, the solvent for dissolving lignin is selected from one or more of the following: DMSO / TBAF, imidazole / DMSO, and [EMIM][Ac], [BMIM][CI], [BMIM][Br], [AMIM][CI], [HMIM][CF3SO3], [MMIM][MeOSO3], [BMIM][MeOSO3], [BMMIM][BF4], [BMIM][CF3SO3], [BzMIM][DCA], [MeOBzMIM][CI], [MeBzMIM][CI] ionic liquids. More preferably, the ionic liquid for dissolving lignin is selected from one or more of the following: [BMIM][CI], [EMIM][Ac], [BMIM][Br], [AMIM][CI], [HMIM][CF3SO3], [MMIM][MeOSO3], [BMIM][MeOSO3], [BMMIM][BF4].

[0373] According to the present application, the ionic liquid can be a single ionic liquid or a mixed ionic liquid composed of multiple ionic liquids; for example, the single ionic liquid is an ionic liquid capable of completely or partially dissolving biomass polymers (such as cellulose, starch, chitosan, chitin, lignin, hemicellulose, etc.); for example, the mixed ionic liquid can be an ionic liquid capable of dissolving biomass polymers (such as cellulose, starch, chitosan, chitin, lignin, hemicellulose, etc.) or a mixture of an ionic liquid capable of dissolving biomass polymers (such as cellulose, starch, chitosan, chitin, lignin, hemicellulose, etc.) and an ionic liquid incapable of dissolving biomass polymers (such as cellulose, starch, chitosan, chitin, lignin, hemicellulose, etc.).

[0374] The technical solutions of the present application will be further described in detail below in conjunction with specific examples. It should be understood that the following examples are only illustrative and explanatory of the present application, and should not be interpreted as limiting the scope of protection of the present application. Any technology implemented based on the above description of the present application is encompassed within the scope intended to be protected by the present application.

[0375] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0376] The present application will be further described below through specific examples:

[0377] The biomass-based materials described in the following preparation examples and examples can also be referred to as micro-nano cellulose, micro-nano chitosan, micro-nano starch, etc., wherein the micro-nano cellulose, micro-nano chitosan, and micro-nano starch each include a micron-level structure part and a nano-level structure part distributed on the micron-level structure.

[0378] Preparation Example 1

[0379] Preparation of plant hydrogel CMG:

[0380] 12 g of wood pulp was pre-mixed with ionic liquid AMIMCl, the wood pulp was dried in an oven at 80°C for more than 1 h, the mass of AMIMCl was 2388 g, the pre-mixed material was heated to 80°C, and vacuum stirring was performed for 2 h. After complete dissolution, the solution was slowly poured into warm water with a temperature of 40±5°C to form a biomass gel. The gel was washed 6-8 times with the solution to remove residual AMIMCl, and repeated washing with water was performed until no precipitate was generated upon addition of AgNO3 to the filtrate. The washed biomass gel was re-dispersed in pure water with a mass ratio of water to biomass gel of 9:1, and then the gel was crushed and further crushed to a size of 5-50 μm using a colloid mill or a high-pressure homogenizer, etc. The solid content of CMG was 3.0 wt%, and after autoclaving, an emulsion CMG was obtained, which was a plant hydrogel.

[0381] Preparation Example 2

[0382] Preparation of surface-modified biomass-based dispersion, which is used as plant hydrogel CMG:

[0383] 48 g of wood pulp was pre-mixed with ionic liquid AMIMCI, the wood pulp was dried in an oven at 80°C for more than 1 h, and the mass of AMIMCI was 2352 g. The pre-mixed material was heated to 80°C, and stirred under vacuum for 2 h. After complete dissolution, the solution was slowly poured into warm water, and the water temperature was 40±5°C, to form a biomass gel. The gel was washed 6-8 times with a solution to remove residual AMIMCI, and the solution used could be water, ethanol, DMF, or NaOH aqueous solution. The washed biomass gel was re-dispersed in pure water, and the mass ratio of water to biomass gel was 9:1. A certain amount of surface modifier was added to the mixed solution (the molar ratio of biomass gel to surface modifier was 1:1), and the surface modifier used could be xylitol, citric acid, 4-(2-aminoethyl)benzene-1,2-diol, or sorbitol. The solution was stirred at a certain temperature for 24 h, and the temperature range was 0-50°C. Then the gel was washed 6-8 times with a solution to remove residual surface modifier, and the solution used could be water, ethanol, DMF, or NaOH aqueous solution. The washed surface-modified biomass gel was dispersed in pure water, and the mass ratio of water to surface-modified biomass gel was 9:1. Then the gel was crushed, and was crushed to a particle size of 1-50 μm using a colloid mill, a high-pressure homogenizer, or the like, and the solid content was 0.1-3.0 wt%. After high-pressure sterilization, a surface-modified biomass-based dispersion CMG was obtained.

[0384] Preparation Example 3

[0385] Preparation of substituted biomass-based dispersion, which is used as plant hydrogel CMG:

[0386] Premix 120g of wood pulp with the ionic liquid AMIMCl. The wood pulp was oven-dried at 80°C for at least 1 hour. The weight of AMIMCl was 2352g. The premix was heated to 80°C and stirred under vacuum for 2 hours. A certain amount of substitution reagent (10% of the wood pulp weight) was dissolved in DMF (with a DMF:AMIMCl mass ratio of 1:1). The dissolved substitution reagent (e.g., pyromellitic anhydride, 4,4'-diphthalic anhydride, 1,4,5,8-naphthalenetetracarboxylic anhydride, or 1,2,3,4-cyclopentanetetracarboxylic anhydride) was added to the dissolved wood pulp solution. A certain amount of base (e.g., sodium carbonate, triethylamine, sodium borohydride, or sodium hydride) was then added. The reaction was allowed to proceed at room temperature for 24 hours. The reaction solution was poured into warm water at 40±5°C to form a substituted biomass gel. Wash the gel 6-8 times with a solution such as water, ethanol, DMF, or aqueous NaOH to remove any remaining impurities. Disperse the washed substituted biomass gel in pure water at a 9:1 water:substituted biomass gel mass ratio. Grind the gel using a colloid mill, high-pressure homogenizer, or other similar equipment to a particle size of 1-50 μm and a solids content of 0.1-3.0 wt%. Sterilize by autoclaving to obtain the substituted biomass-based dispersion CMG.

[0387] Preparation Example 4

[0388] Preparation of multi-component biomass-based dispersion as plant hydrogel CMG:

[0389] 1. Prepare natural cellulose gel: Premix 48g of wood pulp with the ionic liquid AMIMCl. The wood pulp should have been oven-dried at 80°C for at least 1 hour. The mass of AMIMCl should be 2352g. Heat the premixed mixture to 80°C and stir under vacuum for 2 hours. Once dissolved, slowly pour the solution into warm water at 40±5°C to form a biomass gel. Wash the gel 6-8 times with a solution of water, ethanol, DMF, or aqueous NaOH to remove any residual AMIMCl.

[0390] 2. Preparation of polymer hydrogel: Dissolve the polymer compound in a solvent, such as methylcellulose, which can be water. Add an appropriate crosslinker to form a hydrogel, such as boric acid. Wash the gel 6-8 times with a solution containing water, ethanol, DMF, or aqueous NaOH to remove any residual impurities. The mass ratio of polymer to water is 0.2:1, and the mass ratio of polymer to crosslinker is 0.5:1.

[0391] 3. Preparation of multi-component biomass-based dispersion liquid: the above washed biomass gel is dispersed in pure water with a polymer hydrogel and a double-antibiotic drug penicillin-streptomycin at a certain ratio (the mass ratio of the biomass gel to the polymer hydrogel and the double-antibiotic drug is 0.1:1:0.1), the gel is then crushed, and is crushed to a particle size of 5-50 μm and a solid content of 0.1-3.0 wt% using a colloid mill, a high-pressure homogenizer, etc., and a multi-component biomass-based dispersion liquid CMG is obtained after high-pressure sterilization.

[0392] Preparation Example 5

[0393] Preparation of multi-component biomass-based dispersion liquid, which is used as a plant hydrogel CMG:

[0394] 48 g of wood pulp is mixed with a polymer compound and fullerene at a mass ratio of 0.1:1:0.1, the mixture is pre-mixed with ionic liquid AMIMCl, the wood pulp is dried in an oven at 80°C for more than 1 h, the mass of AMIMCl is 2352 g, the pre-mixed material is heated to 80°C, and is stirred under vacuum for 2 h, wherein the polymer compound is methyl cellulose. After complete dissolution, the solution is slowly poured into warm water at a temperature of 40±5°C to form a multi-component biomass hydrogel. The gel is washed 6-8 times using the solution to remove residual AMIMCl, and water can be used as the solution. The washed multi-component biomass hydrogel is dispersed in pure water at a water to multi-component biomass hydrogel mass ratio of 9:1, the gel is then crushed, and is crushed to a particle size of 5-50 μm and a solid content of 0.1-3.0 wt% using a colloid mill, a high-pressure homogenizer, etc., and a multi-component biomass-based dispersion liquid CMG is obtained after high-pressure sterilization.

[0395] Example 1

[0396] The method for culturing cells is as follows:

[0397] 1) Cell passage: commercially available SH-SY5Y cells are selected, are recovered, and are cultured in a 10 cm culture dish. When the confluence rate is more than 80%, the cells are passaged, the original culture solution in the culture dish is discarded, the culture dish is washed twice with PBS, and then is used to prepare a single cell suspension by trypsin digestion. The cells are centrifuged at 1200 rpm and room temperature for 5 min, are resuspended in a basic culture solution (its composition is: DMEM / F12, 10% FBS, 1% P / S, and the basic culture solution is used in the following examples unless otherwise specified), and are adjusted to a cell density of 2×10^4 cells / μL to obtain SH-SY5Y cells for experiments, which are ready for use.

[0398] 2) Preparation of plant hydrogel: the emulsion CMG prepared in the above Preparation Example 1 is diluted with the basic culture solution in step 1) to obtain CMG culture solutions with a CMG concentration of 0.1%, 0.2%, and 0.4%, respectively.

[0399] 3) Cell suspension mixing: Take different concentrations of CMG culture medium from step 2) and mix 500 μL of CMG culture medium with 500 μL of basal culture medium at a volume ratio of 1:1. Add 10 μL of experimental SH-SY5Y cells from step 1) and mix well to obtain a cell suspension.

[0400] 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% CO 2 incubator.

[0401] 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.

[0402] Microscopic images of SH-SY5Y cells cultured with plant hydrogels of different concentrations for 3 days are shown in Figures 1a to 1c, wherein Figures 1a, 1b, and 1c correspond to CMG culture solutions with concentrations of 0.1%, 0.2%, and 0.4%, respectively.

[0403] Example 2

[0404] The CMG emulsion prepared in Preparation Example 1 was diluted with the basic culture medium prepared in Example 1 to obtain a CMG culture medium having a CMG concentration of 0.2%.

[0405] The cell culture method of Example 1 was referred to, except that SH-SY5Y cells were cultured for 3 days using the 0.2% CMG plant hydrogel of this example and commercially available Matrigel 3D (manufacturer: Corning, model: 356231), respectively.

[0406] The culture results are shown in FIG2a and FIG2b . The results of the culture method using the CMG culture medium and Matrigel 3D of this embodiment are shown in FIG2a and FIG2b , respectively.

[0407] As shown in Figures 2a and 2b, the cell clusters cultured in the two groups were of similar morphology and size, but the number of cell clusters in the CMG group was greater.

[0408] Example 3

[0409] The emulsion CMG prepared in Preparation Example 1 was diluted with the basic culture medium in Example 1 to obtain a CMG culture medium with a CMG concentration of 0.2%.

[0410] The cell culture method of Example 1 was referred to, except that SH-SY5Y cells were cultured for 3 days using the plant hydrogel of this example and the 2D culture method, respectively.

[0411] The culture results are shown in Figure 3, and the results of the plant hydrogel and 2D culture method of the present embodiment are shown in Figures 3a and 3b, respectively.

[0412] As can be seen from Figures 3a and 3b, when the plant hydrogel of the present embodiment is used for 3D culture of SH-SY5Y cells, the cells proliferate faster than in ordinary 2D culture.

[0413] Comparative Example 1

[0414] 1) The plant hydrogel was prepared according to Preparation Example 1, except that the gel was not subjected to a crushing treatment, and a comparative emulsion was obtained, i.e., the plant hydrogel.

[0415] The comparative emulsion was diluted with the basic culture solution in Example 1 to obtain a comparative culture solution 1 with a CMG concentration of 0.2%.

[0416] 2) The cell culture method of Example 1 was used, except that the comparative culture solution 1 of the present comparative example was used to culture SH-SY5Y cells for 3 days.

[0417] The culture results are shown in Figure 5. The comparative culture solution after the culture method of the present comparative example was observed under a microscope, and it was difficult to observe the cell spheres in the culture solution due to poor transparency.

[0418] Comparative Example 2

[0419] The aldehyde-modified cellulose nanocrystals were prepared according to CN115232781A, and were diluted with the basic culture solution in Example 1 to obtain a comparative culture solution 2 with an aldehyde-modified cellulose nanocrystal concentration of 0.2%.

[0420] 2) The cell culture method of Example 1 was used, except that the comparative culture solution 2 of the present comparative example was used for 3D culture of SH-SY5Y cells, and the culture was performed for 3 days, and the results are shown in Figure 6.

[0421] As can be seen from the above results, the ready-to-use plant hydrogel prepared from the natural cellulose glue (CMG) of the present application does not contain animals, is made of nanofibrillar cellulose (NFC), simulates the extracellular matrix (ECM), supports cell growth and differentiation, provides a solution for automation and high-throughput detection, and can be used for 3D cell culture of cell spheres and organoids, personalized medicine, regenerative medicine, organ models on chips, drug research, etc. Moreover, compared with the aldehyde-modified cellulose nanocrystals prepared according to CN115232781A, the plant hydrogel prepared in the present application has more advantages in the number and size of 3D cell spheres in 3D culture, and has better transparency under a microscope.

[0422] Comparative Example 3

[0423] The CNF hydrogel was prepared according to the example of the Chinese patent document CN103354834A, as a comparative hydrogel, wherein the solid content of the CNF was 0.3%, and the structure scanning diagram of the fibers in the comparative hydrogel was shown in Figure 1 of the Chinese patent document CN103354834A.

[0424] According to the cell culture method of Example 1, except that the 3D culture of SH-SY5Y cells was carried out using the comparative hydrogel of the present comparative example, and the culture was carried out for 3 days, and the results were shown in Figure 7.

[0425] Example 4-1

[0426] The cell culture method of the present example was according to Example 1, except that the SH-SY5Y cells in step 1) were replaced by HepaRG (liver cancer cells), and the CMG concentration in the CMG culture solution prepared in step 2) was 0.1%; and the results after 7 days of culture were shown in Figure 8a.

[0427] Example 4-2

[0428] The cell culture method of the present example was according to Example 1, except that the SH-SY5Y cells in step 1) were replaced by HepaRG (liver cancer cells), and the CMG in step 2) was respectively the surface-modified biomass-based dispersion liquid CMG prepared in Preparation Example 2 using xylitol as the surface modifier, the substituted biomass-based dispersion liquid CMG prepared in Preparation Example 3 using pyromellitic dianhydride as the substitution reagent, the multi-component biomass-based dispersion liquid CMG prepared in Preparation Example 4, and the multi-component biomass-based dispersion liquid CMG prepared in Preparation Example 5, and the CMG concentration was 0.1%; and the results after 7 days of culture were shown in Figures 8b-8e, respectively.

[0429] Comparative Example 4

[0430] According to the cell culture method of Comparative Example 3, except that the 3D culture of SH-SY5Y cells was replaced by HepaRG (liver cancer cells), and the results after 7 days of culture were shown in Figure 9; and it could be seen from Figure 9 that the cell clusters cultured in the present comparative example were a little scattered, and the boundary was not smooth.

[0431] From the above examples, it could be seen that the 3D suspension culture method of SH-SY5Y cells and HepaRG cells based on the natural cellulose glue (CMG) of the present application was simple to operate, and the 3D cell sphere formation rate was high.

[0432] Example 5

[0433] 1. The formula of the composite culture solution was as follows, and the composite culture solution was obtained by mixing the following components

[0434] 2. Preparation of the tissue to be cultured

[0435] (1) Tissue washing

[0436] Transfer the tissue block (gastric cancer organoids, from hospital surgery / biopsy tissue) to a 50ml centrifuge tube, add 15ml of washing solution (PBS + 2x P / S), shake and wash for no less than 5 times, 6min each time, until completely clear.

[0437] (2) Tissue separation

[0438] Transfer the tissue block to a 10cm culture dish, add a small amount of washing solution to ensure that the tissue surface is wet, use a sterile scalpel and blade to mechanically separate the tissue until it becomes a small piece or paste about 0.5x0.5x0.5mm^3.

[0439] (3) Tissue digestion

[0440] Transfer the mechanically separated tissue into a 15ml sterile centrifuge tube, add 2-5ml of tissue digestion solution (collagenase P (1mg / ml) + DNase (1mg / ml)) according to the amount of tissue, use a 1ml syringe to gently blow the tissue to make it fully dispersed; place the centrifuge tube containing the digestion solution in a constant temperature air bath shaker at 37°C, 200rpm, and separate and digest the tissue until most of the tissue is separated and digested into 3-10 cell clusters, then add 10% FBS to terminate digestion.

[0441] (4) Cell filtration

[0442] Filter the cell suspension after the above termination of digestion with a 100μm filter into a new 50ml centrifuge tube, use a 5ml syringe to grind the sample sediment until the digested tissue sample is ground to only white connective tissue, wash the filter with DMEM / F12 for 3 times, about 5ml each time. 4°C, 300g / 1260rpm, centrifuge for 5min.

[0443] (5) Red blood cell lysis

[0444] If a large number of red blood cells are observed in the cell sediment, the following red blood cell lysis step is performed:

[0445] Discard the supernatant, add 1 ml of red blood cell lysate to resuspend the cell pellet, mix well by blowing, and place it on a 4°C shaking table at 120 rpm for 5 min. Centrifuge at 4°C, 300g / 1260 rpm for 5 min. Add 10 ml of Advanced DMEM / F12 to resuspend the cell pellet, and transfer the resuspension to a 15 ml centrifuge tube. Centrifuge at 4°C, 300g / 1260 rpm for 5 min.

[0446] (6) Cell plating

[0447] Discard the supernatant, and retain an appropriate volume of liquid (depending on the number of cells, 100,000 cells / well is recommended) as the tissue cell liquid to be cultured.

[0448] 3. Preparation of cellulose-based culture solution

[0449] The emulsion CMG prepared in Preparation Example 1 was diluted with the composite culture solution described above to obtain cellulose-based culture solutions with CMG concentrations of 0.8%, 0.4%, 0.2%, and 0.1% (mass percentage concentration), respectively.

[0450] 4. 3D culture: The tissue cell liquid to be cultured in step 2 was gently suspended with the cellulose-based culture solution in step 3 above, and then 300 μl was dropped onto a preheated 24-well low-adsorption cell culture plate. The culture plate was placed in a 37°C 5% CO2 incubator for 10 min, removed, and 700 μl of preheated organoid culture solution was added per well. The culture plate was placed in a 37°C 5% CO2 incubator for 7 days.

[0451] Micrographs of gastric cancer organoids after 7 days of culture in cellulose-based culture solutions with different concentrations are shown in FIGS. 10a-10d, where FIGS. 10a, 10b, 10c, and 10d correspond to cellulose-based culture solutions with concentrations of 0.8%, 0.4%, 0.2%, and 0.1%, respectively.

[0452] Example 6

[0453] The emulsion CMG prepared in Preparation Example 1 was diluted with the composite culture solution in Example 5 to obtain a cellulose-based culture solution with a CMG concentration of 0.2%.

[0454] The culture method of Example 5 was used as a reference, except that the cellulose-based culture solution of this example with a CMG concentration of 0.2% and commercially available Matrigel 3D (manufacturer: Corning, model: 356231) were used to culture gastric cancer organoids, and the culture time was 7 days.

[0455] The culture results are shown in FIGS. 11a and 11b, and the results of the culture methods using the CMG culture solution of this example and Matrigel 3D are shown in FIGS. 11a and 11b, respectively.

[0456] As shown in FIGS. 11a and 11b, the CMG group cultured organoid vesicle wall is thick and solid, which is a differentiated mature organoid, while the Matrigel group cultured gastric cancer organoid vesicle wall is thin and uneven.

[0457] Example 7-1

[0458] The culture method of this example is referred to Example 5, except that the tissue to be cultured in step 2 is replaced by intestinal cancer organoid culture tissue, and the concentration of CMG in the cellulose-based culture solution prepared in step 3 is 0.2%.

[0459] The culture results are shown in FIG. 12. The CMG cultured organoid is a mature intestinal organoid containing multiple typical complex crypt-shaped structures. These crypt structures are connected to each other, forming a mini-intestine with an intestinal lumen.

[0460] Example 7-2

[0461] The operation is the same as Example 5, except that: the tissue to be cultured in step 2 is replaced by intestinal cancer organoid culture tissue, and the CMG in step 3 is respectively the CMG with a concentration of 0.2% (mass percentage) prepared by using the surface modifier xylitol in Preparation Example 2, the CMG with a concentration of 0.2% (mass percentage) prepared by using the substitution reagent pyromellitic dianhydride in Preparation Example 3, the CMG with a concentration of 0.2% (mass percentage) prepared in Preparation Example 4, and the CMG with a concentration of 0.2% (mass percentage) prepared in Preparation Example 5; and the microscopic images of the intestinal cancer organoids after 7 days of culture are shown in FIGS. 13-16.

[0462] Comparative Example 5

[0463] This comparative example refers to the preparation of cellulose-based culture solution in Example 5, except that the emulsion CMG prepared in Preparation Example 1 is replaced by plant cellulose matrix gel GrowDex (UPM / 100103005) with a mass percentage concentration of 0.2%, which is recorded as Comparative Culture Solution 1.

[0464] Referring to step 4 of Example 5, gastric cancer tissue is cultured using the cellulose-based culture solution of Example 5 with a CMG concentration of 0.2% (mass percentage) and the Comparative Culture Solution 1 of this comparative example, respectively, which are recorded as 0.2% CMG cellulose matrix gel group and plant cellulose matrix gel GrowDex group. The results after 7 days of culture are shown in FIG. 17.

[0465] As shown in FIG. 17, compared with the 0.2% CMG group and the GrowDex group, the gastric cancer organoid vesicle wall cultured by the cellulose-based culture solution of the present application is thicker and presents a solid shape, while the gastric cancer organoid cultured by the culture solution prepared by the GrowDex matrix glue of the present comparative example is severely vesiculated and presents a low differentiation state. Therefore, the cellulose-based culture solution of the present application is more superior for the culture of gastric cancer organoids.

[0466] Comparative Example 6

[0467] The comparative example refers to the preparation of the cellulose-based culture solution and the culture of the gastric cancer tissue according to Example 5, except that the content of Epirregulin in the formula of the culture solution is different, including a group without Epirregulin, i.e., a group without Epirregulin, and a group of Epirregulin 200 ng / ml and a group of Epirregulin 500 ng / ml. The results after 7 days of culture are shown in FIG. 18.

[0468] As shown in FIG. 18, compared with the group without Epirregulin, the gastric cancer organoid spheres in the group of Epirregulin 200 ng / ml are larger in size, more in number, and have a thicker vesicle wall and present a solid shape, which is obviously superior, and therefore Epirregulin is a key component in the culture solution. Compared with the group of Epirregulin 200 ng / ml, the group of Epirregulin 500 ng / ml has no obvious difference, and therefore the optimal concentration of Epirregulin is 200 ng / ml.

[0469] Comparative Example 7

[0470] The comparative example refers to Comparative Example 5, except that the tissue to be cultured is replaced by intestinal cancer organoid culture tissue.

[0471] The results of the plant cellulose matrix glue GrowDex group after 7 days of culture are shown in FIG. 19. Compared with the results of the 0.2% CMG cellulose matrix glue group after 7 days of culture (FIG. 12 in Example 7-1), the intestinal cancer organoids cultured by the cellulose-based culture solution of the present application have advantages in size and structural complexity.

[0472] As shown in the above examples, the tumor organoid 3D culture kit and culture method based on the natural cellulose glue (CMG) of the present application are simple to operate and can obtain differentiated mature organoids.

[0473] Example 8 Culture of Human Induced Pluripotent Stem Cells / Embryonic Stem Cells

[0474] 1) Before passaging stem cells, dilute plant hydrogel CMG with DMEM / F12 to 0.8% (mass percent concentration) and add to 6-well plates, 1 ml / well, and place in a 37°C incubator for at least 1 hour.

[0475] 2) Passaging stem cells, cells in culture dish are about 85-90% confluent and essentially undifferentiated. After aspirating stem cell culture medium mTeSR TM 1 (STEMCELL, 85850), overlay each well with 1 ml Dispase (1 mg / ml) and place in a 37°C incubator until the stem cell edges begin to lift, aspirate the Dispase.

[0476] 3) Wash 3 times with DPBS, 1 ml each time.

[0477] 4) Add 3 ml pre-warmed mTeSR TM 1 to the well, gently pipette to dislodge the stem cells from the well plate, repeat pipetting until the cell clumps are broken up into 1-2 mm pieces.

[0478] 5) Stem cell pieces are aliquoted at a ratio of 1:1 to 1:6 into the well plates covered with plant hydrogel CMG from step 1), and supplemented with mTeSR TM 1 medium (STEMCELL, 85850) to 2 ml, gently tap the side of the 6-well plate culture dish 5 to 10 times to ensure the stem cell colonies are evenly distributed in the air, and culture in a 37°C, 5% CO2 incubator with daily medium change.

[0479] Example 9 Human pluripotent stem cell / embryonic stem cell induced differentiation of intestinal organoids using the plant hydrogel of Preparation Example 1

[0480] 1. Monolayer iPSC / ES Differentiation

[0481] Differentiate in TC-treated 24-well cell culture plates, dilute plant hydrogel CMG of Preparation Example 1 with DMEM / F12 to 0.8% (mass percent concentration) and add to 24-well plates, 0.3 ml / well, and place in a 37°C incubator for at least 1 hour. Before starting the differentiation, assess the differentiation rate and starting density of the cells. At this time, the cell differentiation rate should be less than 5% and the starting density should reach 85-90%.

[0482] (1) Definitive endoderm differentiation

[0483] A. Preparation of definitive endoderm differentiation medium

[0484] Day 0: Prepare the required definitive endoderm differentiation medium (RPMI 1640 with L-glutamine (final concentration 2 mM), penicillin-streptomycin (final concentration 100 U / ml-100 g / ml) and Activin A (final concentration 100 ng / ml), 0.5 mL per culture well) for Day 0, Day 1 and Day 2.

[0485] B. Definitive endoderm differentiation

[0486] 1) Day 0: Pre-warm the required definitive endoderm differentiation medium (0.5 mL / well) for Day 0 at 37°C, store the remaining medium at 2-8°C, aspirate the mTeSR1 in the culture well. TM 1, add 0.5 mL of definitive endoderm differentiation medium dropwise along the well wall. Incubate at 37°C, 5% CO2 and 95% humidity for 24 hours.

[0487] 2) Day 1: Pre-warm the definitive endoderm differentiation medium (0.5 mL / well) at 37°C, aspirate the medium in the culture well, add 0.5 mL of definitive endoderm differentiation medium dropwise along the well wall. Incubate at 37°C, 5% CO2 and 95% humidity for 24 hours.

[0488] 3) Day 2: Aspirate the medium in the culture well, add 0.5 mL of 37°C pre-warmed definitive endoderm differentiation medium dropwise along the well wall. Incubate at 37°C, 5% CO2 and 95% humidity for 24 hours.

[0489] Note: During the endoderm induction process, cells will undergo a large amount of death. Minimize the time the cells are outside the 37°C incubator as much as possible. After 24 hours of endoderm induction, the cells are very sensitive and require gentle medium changes when culturing. After 72 hours of incubation, a dense confluent monolayer of endoderm cells will be formed.

[0490] (2) Mid / Hindgut (MH) differentiation

[0491] A. MH differentiation medium

[0492] Day 3: Prepare the required MH differentiation medium (RPMI 1640 with 2% FBS, L-glutamine (final concentration 2 mM), penicillin-streptomycin (final concentration 100 U / ml-100 g / ml), FGF4 (final concentration 500 ng / ml), 0.5 mL per culture well) for Days 3-8.

[0493] B. Mid / Hindgut (MH) differentiation

[0494] 1) Day 3: Warm sufficient volume of MH differentiation medium (0.5 mL / well) to room temperature (15-25 °C), aspirate medium from culture wells, add 0.5 mL of mid / hindgut (MH) differentiation medium dropwise along the well walls. Incubate at 37 °C, 5% C02 and 95% humidity for 24 hours.

[0495] 2) Days 4-9: Change medium completely and observe spheroids every 24 hours according to the following protocol.

[0496] Note: Ensure that cultures are returned to the incubator within 30 minutes of removal.

[0497] a. Observe monolayer under a microscope. Three-dimensional structures can appear as early as day 4 of differentiation. Free-floating mid / hindgut spheroids will appear on days 6-9 of differentiation.

[0498] b. Using a 1 mL pipette, remove 0.5 mL of 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.

[0499] c. Add 0.5 mL of fresh MH medium to the cells. Incubate at 37 °C, 5% C02 and 95% humidity for 24 hours.

[0500] Note: Although spheroids released on days 6-9 will all generate small intestinal organoids, the length of time the cells are cultured in mid / hindgut medium will determine the regional identity of the small intestinal organoids that develop. For example, duodenum (shorter culture time) or ileum (longer culture time). The time of appearance of the highest yield of mid / hindgut spheroids can vary depending on the hPSC cell line. For reproducible experimental results, use mid / hindgut spheroids differentiated at the same time point for consistent harvesting and initiation of human intestinal organoid culture.

[0501] 3) Spheroid Embedding: Suspend spheroids in each culture well with a 0.5% BSA rinsed pipette into one culture well of a 24-well plate for counting. Add appropriate volume to a 15 mL centrifuge tube for approximately 50 suspended spheroids (based on previous spheroid count).

[0502] A mid / hindgut spheroid is a cell aggregate with a diameter > 75 pm that can form a human intestinal organoid. Multiple fused spheroids should be counted as one unit that will form a human intestinal organoid.

[0503] Remaining monolayer cultures can be used to assay for mid / hindgut formation or to study further differentiation into spheroids over the next few days.

[0504] 2. Human intestinal organoid culture

[0505] (1) Initial culture of human intestinal organoids

[0506] A. Formulation of Intestine Organoid Growth Medium

[0507] 1) Culture in 24-well low attachment culture plates. Prepare 4-well (0.5 mL / well) of Intestine Organoid Growth Medium (Advanced DMEM / F12 with 1x B27, with L-glutamine (final concentration 2 mM), penicillin-streptomycin (final concentration 100 U / ml-100 g / ml), HEPES buffer (final concentration 15 mM), R-spondin 1 (final concentration 500 ng / ml).

[0508] B. Mixing of Plant Hydrogel CMG with Spheres

[0509] 1) Centrifuge the collected sphere suspension from the mid / hindgut (MH) differentiation described in Step B at 300 x g for 5 minutes. Carefully aspirate the supernatant after centrifugation.

[0510] 2) Add 1 mL of DMEM / F-12 + 15 Mm HEPES to the spheres. Centrifuge at room temperature (15-25 °C) at 300 x g for 5 minutes.

[0511] 3) Carefully aspirate the supernatant with 1 mL of pipette tip.

[0512] 5) Add 100 μL of 0.3% Plant Hydrogel CMG of Preparation 1 to the centrifuge tube. Pipette up and down 5 times and gently dispense the spheres into the Plant Hydrogel CMG.

[0513] Note: Do not completely empty the pipette tip to prevent excessive bubble formation.

[0514] 6) Using the same pipette tip, gently transfer the embedded spheres to the center of one culture well of a 24-well tissue culture dish.

[0515] 7) Incubate in a 37 °C incubator for 10-25 minutes.

[0516] 8) Warm enough volume of Intestine Organoid Growth Medium to (15-25 °C). Store the remaining medium at 2-8 °C.

[0517] 9) Carefully add at least 0.5 mL / well of Intestine Organoid Growth Medium along the culture well well walls. Incubate at 37 °C, 5% CO2, 95% humidity.

[0518] 10) Change the medium every 3-4 days by aspirating the old medium and adding fresh medium. Incubate at 37 °C, 5% CO2, 95% humidity.

[0519] 11) After 10-14 days of incubation, passage the organoids if they have grown.

[0520] Micrographs of human pluripotent stem cell / embryonic stem cell induced differentiation intestinal organoids after 3, 7, 12 days of culture according to the present example are shown in Figures 20a-20c.

[0521] Example 10

[0522] The culture of human pluripotent stem cell / embryonic stem cell induced differentiation intestinal organoids was performed according to the culture method of Example 9, using the xylitol surface-modified biomass-based dispersion CMG prepared in Preparation Example 2, the substituted biomass-based dispersion CMG in which the substituting agent was pyromellitic dianhydride prepared in Preparation Example 3, the multi-component biomass-based dispersion CMG in which the high molecular compound was methyl cellulose prepared in Preparation Example 4, and the multi-component biomass-based dispersion CMG in which the high molecular compound was methyl cellulose prepared in Preparation Example 5, respectively. Micrographs of the intestinal organoids after 12 days of culture are shown in Figures 21-24.

[0523] Comparative Example 8

[0524] iPSCs were cultured using 0.8% of the plant hydrogel CMG prepared in Preparation Example 1 and Matrigel (Coming 354277) according to the culture method of Example 8, respectively. The results are shown in Figures 25a, 25b.

[0525] Both groups had clear colony edges, uniform cell size, and a relatively high nucleus-cytoplasm ratio, representing a better stem cell state and a state of undifferentiated cells. This indicates that the plant hydrogel CMG and Matrigel have comparable performance in culturing stem cells.

[0526] Comparative Example 9

[0527] The culture was performed according to the culture method of Example 9, using the induced differentiation intestinal organoid kit containing 0.3% of the plant hydrogel CMG prepared in Preparation Example 1 and STEMdiff TM Intestinal Organoid Kit (stern cell, 05140), respectively. Micrographs of the human pluripotent stem cell / embryonic stem cell induced differentiation intestinal organoids after 12 days are shown in Figures 26a, 26b.

[0528] Both groups could culture mature intestinal organoids containing multiple crypt structures, and these crypt structures were connected to each other to form a mini-intestine with a lumen in the middle. However, the kit of the present application is even superior to the comparative kit, and the formed intestinal organoids have a more typical and complex crypt reentrant structure.

[0529] The above describes exemplary embodiments of the present application. However, the scope of protection of the present application is not limited to the above-described embodiments. Any modifications, equivalent replacements, improvements, etc. made by those skilled in the art within the spirit and principles of the present application should be included in the scope of protection of the present application.

Claims

1. A plant source-based culture solution, characterized by, The culture solution comprises a plant gel and a basic culture solution; wherein the plant gel is free of animal-derived components and comprises micro-nano biomass and a dispersion agent; the dispersion agent is selected from organic solvents and / or water, preferably water and / or alcohol. Preferably, the culture solution comprises a plant hydrogel and a basic culture solution; wherein the plant hydrogel is free of animal-derived components and comprises micro-nano biomass and water.

2. The culture solution according to claim 1, characterized in that, The culture solution is used for 3D cell culture, and comprises a plant hydrogel and a basic culture solution A; wherein the plant hydrogel is free of animal-derived components and comprises micro-nano biomass and water. Alternatively, the culture solution is used for 3D culture of tumor organoids, and comprises a plant hydrogel and a complex culture solution; wherein the plant hydrogel is free of animal-derived components and comprises micro-nano biomass and water. Alternatively, the culture solution is used for stem cell culture, and comprises a plant hydrogel and a basic culture solution C; wherein the plant hydrogel is free of animal-derived components and comprises micro-nano biomass and water.

3. The method for preparing the culture solution according to claim 2, characterized by, The preparation method of the culture solution for 3D cell culture comprises the following steps: S1: preparing a plant hydrogel; S2: mixing the plant hydrogel in step S1 with a basic culture solution A to prepare a culture solution for 3D cell culture; Alternatively, the preparation method of the culture solution for 3D culture of tumor organoids comprises the following steps: S1': preparing the plant hydrogel; S2': mixing the plant hydrogel in step S1' with a complex culture solution to prepare a culture solution for 3D culture of tumor organoids; Alternatively, the preparation method of the culture solution for stem cell culture comprises the following steps: S1": preparing a plant hydrogel; S2": mixing the plant hydrogel in step S1" with a basic culture solution C to prepare a culture solution for stem cell culture.

4. Use of the culture solution according to claim 1 or 2, characterized in that The culture solution is used for cell, tissue or organoid culture; specifically, the application of the culture solution for 3D cell culture in 3D cell culture; or the application of the culture solution for 3D culture of tumor organoids in the culture of tumor tissues or organoids such as gastric cancer and lung cancer; or the application of the culture solution for stem cell culture in stem cell or organoid culture.

5. A method of culturing cells in 3D, characterized in that, The method of 3D cell culture is carried out in the culture solution of claim 1 or 2, specifically in the culture solution for 3D cell culture.

6. A kit characterized in that, The kit comprises the culture solution of claim 1 or 2; Specifically, the kit is a 3D cell culture kit comprising a culture solution for 3D cell culture; or the kit is a tumor organoid 3D culture kit comprising a culture solution for 3D culture of tumor organoids.

7. The kit of claim 6 is used in cell culture, tissue culture or organoid culture. Specifically, the 3D cell culture kit is used in 3D cell culture; or the tumor organoid 3D culture kit is used in 3D culture of tumor organoids.

8. A method for culturing a tumor tissue or an organoid of a gastric cancer, a lung cancer, or the like, characterized by, The method is carried out in the culture solution of claim 1 or 2, specifically in the culture solution for 3D culture of tumor organoids.

9. A method for culturing stem cells or organoids, characterized by, The method is carried out in the culture solution of claim 1 or 2, specifically in the culture solution for stem cell culture.

10. A stem cell-induced differentiated organoid culture solution, characterized by, The culture solution comprises a plant hydrogel, a definitive endoderm differentiation culture solution, an MH differentiation culture solution, and an organoid growth culture solution; wherein the plant hydrogel is free of animal-derived components and comprises micro-nano biomasses and water.

11. The use of the stem cell-induced differentiated organoid culture solution according to claim 10, characterized in that, The application discloses a stem cell-induced differentiation organoid.

12. A method of inducing differentiation of stem cells into organoids, characterized in that, The method comprises culturing a single stem cell or a stem cell group in the stem cell-induced differentiation organoid culture solution in claim 10.

13. A stem cell organoid prepared by the method for preparing a stem cell-induced differentiation organoid in claim 12.

14. A differentiation-inducing organoid kit, characterized in that, The kit comprises the stem cell-induced differentiation organoid culture solution in claim 10.

15. Application of the induction differentiation organoid kit in claim 14 to induction differentiation organoids.

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