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

Abstract

The invention discloses a culture solution based on a plant source as well as a preparation method, a kit and application of the culture solution. The culture solution comprises plant hydrogel and a basic culture solution, wherein the plant hydrogel is free of animal source components and comprises micro-nano biomass and water. Compared with animal-derived matrigel, the plant hydrogel disclosed by the invention has incomparable advantages in the aspects of performance, use convenience, inter-batch stability, process amplification stability, automatic compatibility and the like, and brand-new 3D culture experience is brought.

Description

[0001] This application claims the benefit of priority to prior applications filed with the State Intellectual Property Office of China on April 15, 2024, with patent application number 2024104463928, entitled “A 3D Cell Culture Medium, Preparation Method, Kit, and Application thereof,” patent application number 2024104463650, entitled “A 3D Tumor Organoid Culture Medium, Preparation Method, and Application thereof,” and patent application number 2024104463824, entitled “A Stem Cell Culture and Induced Differentiation Organoid Culture Medium, and Application thereof.” The entire contents of each of the prior applications are incorporated herein by reference. Technical Field

[0002] The present invention relates to the field of biotechnology, and in particular to a plant-based culture solution, a preparation method thereof, a kit and applications thereof. Background Art

[0003] Conventional 2D cell culture often misrepresents in vivo conditions, as cells gradually lose their original properties as they proliferate in altered in vitro environments. Animal experiments, conducted entirely in vivo, are complicated by multiple internal factors and the interplay between the internal and external environments, making it difficult to study single processes and even intermediate processes. 3D cell culture technology lies somewhere between monolayer cell culture and animal experiments, maximizing its ability to simulate the in vivo environment while also demonstrating the intuitive nature and controllable conditions of cell culture.

[0004] The emergence of 3D cell culture technology provides researchers with a cell model that is closer to the real in vivo environment. 3D cell culture can make up for many defects in the monolayer cell culture process, such as:

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

[0006] (2) 3D cell models can well simulate cell-cell interactions: three-dimensional cell-cell contacts and direct or indirect cell-cell communication.

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

[0008] Because of these advantages, 3D cell culture technology has demonstrated outstanding performance in research fields such as drug development, stem cell culture, and organ regeneration.

[0009] The difficulty of 3D cell culture technology is to ensure the three-dimensional structure of cells and maintain natural proliferation and differentiation activity. With decades of continuous development, the methods of 3D cell culture have also been constantly innovating, and the commonly used methods are mainly divided into two types: scaffold-based 3D cell culture methods and scaffold-free 3D cell culture methods. The scaffold-based 3D cell culture method has a long history of development and is supported by a large amount of literature. The materials used for cell culture scaffolds include agarose, collagen, fibronectin, gelatin, laminin, etc. These composite materials simulate the natural extracellular matrix (ECM) through porosity, fiber, permeability and mechanical stability, and can well simulate the interactions between cells and the interactions between cells and extracellular matrix in the in vivo environment, while allowing cells to aggregate, proliferate and migrate on the scaffold.

[0010] Organoids typically require matrix gel support during culture to form a 3D structure. For example, Matrigel, a commonly used scaffold material, is extracted from EHS mouse tumors, which are rich in extracellular matrix proteins. Its main components include laminin, type IV collagen, heparan sulfate glycoprotein, and entactin, as well as various growth factors and matrix metalloproteinases. At room temperature, Matrigel polymerizes to form a biologically active three-dimensional matrix that mimics the structure, composition, physical properties, and function of the in vivo cell basement membrane. This facilitates in vitro cell culture and differentiation, and can be used to study cell morphology, biochemical function, migration, invasion, and gene expression.

[0011] Stem cells are a type of cell with the potential to proliferate and differentiate, capable of developing into specific cell types in various tissues and organs. Pluripotent stem cells (pluripotent stem cells) can differentiate into all cells derived from the three germ layers, forming all tissues and organs. They are used for a variety of tissue and organ repair, disease treatment, and drug screening, making them a hot topic in stem cell research. Cultured stem cells are also used to form, maintain, and expand organoids. Embryonic stem cells (ECs) and induced pluripotent stem cells (iPSCs) are the most studied pluripotent stem cells. However, developing stem cells is not simple. A suitable and stable microenvironment is crucial for the cultivation and application of stem cells such as iPSCs. Hydrogels are porous, water-rich polymer networks with excellent biocompatibility, biochemical, and mechanical properties for 3D stem cell culture. Although some progress has been made in developing hydrogel-based stem cell cultures, it remains challenging to inhibit non-directed stem cell differentiation while mimicking the structure, composition, physical properties, and function of the in vivo basement membrane, while achieving performance comparable to Matrigel.

[0012] Take Matrigel, a commonly used scaffold material, for example. This material is extracted from EHS mouse tumors, which are rich in extracellular matrix proteins. Its main components include laminin, type IV collagen, heparan sulfate glycoprotein, and entactin, as well as various growth factors and matrix metalloproteinases. At room temperature, Matrigel polymerizes to form a biologically active three-dimensional matrix that mimics the structure, composition, physical properties, and function of the in vivo cell basement membrane. This facilitates in vitro cell culture and differentiation, and can be used to study cell morphology, biochemical function, migration, invasion, and gene expression.

[0013] The current problems with basement membrane matrix gel include: 1) supply shortages due to animal welfare issues; 2) large and difficult to control batch-to-batch differences, with the nutrients and protein content greatly affected by batches; 3) animal-derived proteins and nucleic acids interfere with downstream experiments such as efficacy evaluation and fluorescence detection; and 4) the temperature-sensitive nature of the matrix gel itself places strict temperature requirements on storage and operation. Summary of the Invention

[0014] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0015] A plant-based culture solution, comprising a plant gel and a base culture solution; wherein the plant gel is free of animal-derived components and comprises micro-nano biomass and a dispersing agent; the dispersing agent is selected from an 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 hydrogel has no animal-derived components and comprises micro-nano biomass and water.

[0017] A plant-based culture solution comprises a plant hydrogel and a basic culture solution; wherein the plant hydrogel has no animal-derived components and comprises micro-nano biomass and water.

[0018] According to the present invention, the culture solution is used for 3D cell culture, and the culture solution includes plant hydrogel and basic culture solution A; wherein the plant hydrogel does not contain animal-derived components and includes micro-nano biomass and water.

[0019] According to the present invention, the culture medium is used for 3D culture of tumor organoids, and the culture medium includes a plant hydrogel and a composite culture medium; wherein the plant hydrogel has no animal-derived components and includes micro-nano biomass and water.

[0020] According to the present invention, the culture medium is used for stem cell culture, and the culture medium includes plant hydrogel and basic culture medium C; wherein the plant hydrogel does not contain animal-derived components and includes micro-nano biomass and water.

[0021] A method for preparing the above-mentioned culture medium, wherein the method for preparing the culture medium for 3D cell culture comprises the following steps:

[0022] S1: Preparation of plant hydrogel;

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

[0024] Alternatively, the method for preparing a culture medium for 3D culture of tumor organoids comprises the following steps:

[0025] S1': Preparation of plant hydrogel;

[0026] S2': mixing the plant hydrogel prepared in step S1' with the composite culture medium to prepare a culture medium for 3D culture of tumor organoids;

[0027] Alternatively, the method for preparing a culture medium for stem cell culture comprises the following steps:

[0028] S1”: Preparation of plant hydrogel;

[0029] S2″: the plant hydrogel prepared in step S1″ is mixed with the basic culture medium C to prepare the culture medium for stem cell culture.

[0030] An application of the above-mentioned culture medium is used for the culture of cells, tissues or organoids; specifically, the application is the application of a culture medium for 3D cell culture in 3D cell culture; or, the application of a culture medium 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 a culture medium for stem cell culture in the culture of stem cells or organoids.

[0031] A method for 3D cell culture is provided, wherein the method is carried out in the culture medium, specifically in a culture medium for 3D cell culture.

[0032] A kit comprising the culture solution.

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

[0034] A use of the kit in cell culture, tissue culture, or organoid culture. Specifically, the use of the 3D cell culture kit in 3D cell culture; or the use of the tumor organoid 3D culture kit in 3D tumor organoid culture.

[0035] A method for culturing gastric cancer, lung cancer, or other tumor tissue or organoids, wherein the method is performed in the culture medium, specifically in a culture medium for 3D tumor organoid culture.

[0036] A method for culturing stem cells or organoids, wherein the method is performed in the culture medium, specifically in a culture medium for stem cell culture.

[0037] A stem cell-induced differentiation organoid culture medium, comprising a plant hydrogel, a definitive endoderm differentiation culture medium, a MH differentiation culture medium, and an organoid growth culture medium; wherein the plant hydrogel has no animal-derived components and comprises micro-nano biomass and water.

[0038] An application of the stem cell-induced differentiation organoid culture medium is used for stem cell-induced differentiation organoids.

[0039] A method for inducing stem cell differentiation into organoids, comprising culturing a single stem cell or a stem cell group in a culture medium for inducing stem cell differentiation into organoids.

[0040] A stem cell organoid prepared by the method of inducing stem cell differentiation into organoids.

[0041] A kit for inducing differentiation of organoids, comprising the stem cell-induced differentiation of organoids culture medium.

[0042] A use of the kit for inducing differentiation of organoids in inducing differentiation of organoids.

[0043] The solution of the present invention achieves the following beneficial effects:

[0044] The plant hydrogel of the present invention is free of animal-derived ingredients and mainly includes micro-nano biomass and water. Compared with animal-derived matrix glue, it has incomparable advantages in performance, ease of use, batch-to-batch stability, process amplification stability, and automation compatibility, bringing a new 3D cell culture experience. Specifically, the plant hydrogel of the present invention has micro-scale and nano-scale structures that can be fully stretched in the fluid to form a rich network lap structure. The network formed by the micro-scale structure is filled with a large number of nano-scale structures, which can form a denser lap network. This network structure is a flexible three-dimensional structure. It is easier to overlap the network support necessary for cell growth for the suspension stability function of the cell, while avoiding excessive structural rigidity. It can highly simulate the extracellular matrix in vitro, promote the formation, growth and development of tumor organoids such as SH-SY5Y 3D cells, HepaRG cells, gastric cancer, intestinal cancer, etc., and can be applied to industrial scenarios and research such as clinical, pharmaceutical, cell and stem cells. The micro-nano biomass has no toxicity to cells. The biomass-based hydrogel serves as a growth platform for stem cells and has good rigidity, adhesion and porosity. The stem cells cultured with the micro-nano biomass of the present invention can form clusters.

[0045] On the one hand, the plant hydrogel of the present invention is used to prepare the culture medium, and the suspension method can be directly used to culture SH-SY5Y cells and HepaRG cells (liver cancer cells) in 3D, which is simple to operate and has a high 3D cell sphere formation rate.

[0046] On the one hand, human gastric cancer and other tumor tissue samples are used for tissue separation, and self-developed culture medium is used to culture gastric cancer and other tumor tissues or organoids, providing an efficient, simplified operation, and low-cost standardized culture system for gastric cancer and other tumor organoids.

[0047] On the other hand, the plant hydrogel of the present invention is used to culture stem cells and organoids, which is simple to operate and forms a dense, fused monolayer of endoderm cells, which can inhibit the non-directional differentiation of stem cells. The organoid vesicles formed have thicker solid walls and are differentiated and mature organoids that can be used for high-throughput drug screening, drug toxicity testing and regenerative medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1a This is a micrograph of SH-SY5Y cells cultured in the culture medium containing 0.4% CMG for 3 days in Example 1;

[0049] Figure 1b This is a micrograph of SH-SY5Y cells cultured in the culture medium containing 0.2% CMG for 3 days in Example 1;

[0050] Figure 1c This is a micrograph of SH-SY5Y cells cultured in the culture medium containing 0.1% CMG for 3 days in Example 1;

[0051] Figure 2a This is a micrograph of SH-SY5Y cells cultured in a culture medium containing 0.2% CMG for 3 days in Example 2;

[0052] Figure 2b This is a micrograph of SH-SY5Y cells cultured for 3 days using the Matrigel 3D culture method in Example 2;

[0053] Figure 3a This is a micrograph of SH-SY5Y cells cultured in a culture medium containing 0.2% CMG for 3 days in Example 3;

[0054] Figure 3b This is a micrograph of SH-SY5Y cells cultured for 3 days using the 2D culture method in Example 3.

[0055] Figure 1a-Figure 1c In the figure, the scale bars are all 100 μm; Figure 2a and Figure 2b In the figure, the scale bars are all 200 μm; Figure 3a and Figure 3b In the figure, the scale bars are all 100 μm.

[0056] Figure 4 Scanning electron microscope photographs of the plant hydrogel of Preparation Example 1 at different magnifications, where the scales of a, b, and c are 10 μm, 5 μm, and 1 μm, respectively.

[0057] Figure 5 This is a micrograph of the culture medium in Comparative Example 1 after culturing SH-SY5Y cells for 3 days.

[0058] Figure 6 This is a micrograph of the culture medium in Comparative Example 2 after culturing SH-SY5Y cells for 3 days.

[0059] Figure 7 This is a micrograph of the comparative hydrogel in Comparative Example 3 after being used to culture SH-SY5Y cells for 3 days.

[0060] Figure 8a This is a micrograph of HepaRG (liver cancer cells) cultured in 3D culture medium containing 0.1% CMG in Example 4-1 after 7 days.

[0061] Figure 8b This is a micrograph of HepaRG (liver cancer cells) cultured in 3D culture medium containing 0.1% CMG (Preparation Example 2) in Example 4-2 after 7 days.

[0062] Figure 8cThis is a micrograph of HepaRG (liver cancer cells) cultured in 3D culture medium containing 0.1% CMG (Preparation Example 3) in Example 4-2 after 7 days.

[0063] Figure 8d This is a micrograph of HepaRG (liver cancer cells) cultured in 3D culture medium containing 0.1% CMG (Preparation Example 4) in Example 4-2 after 7 days.

[0064] Figure 8e This is a micrograph of HepaRG (liver cancer cells) cultured in 3D culture for 7 days using a culture medium containing 0.1% CMG (Preparation Example 5) in Example 4-2.

[0065] Figure 9 Microscopic image of HepaRG (liver cancer cells) cultured in 3D for 7 days in Comparative Example 4.

[0066] Figure 10a This is a micrograph of gastric cancer organoids cultured in a culture medium containing 0.8% CMG for 7 days in Example 5;

[0067] Figure 10b This is a micrograph of gastric cancer organoids cultured in a culture medium containing 0.4% CMG for 7 days in Example 5;

[0068] Figure 10c This is a micrograph of gastric cancer organoids cultured in a culture medium containing 0.2% CMG for 7 days in Example 5;

[0069] Figure 10d This is a micrograph of gastric cancer organoids cultured in a culture medium containing 0.1% CMG for 7 days in Example 5;

[0070] Figure 11a This is a micrograph of gastric cancer organoids cultured in a culture medium containing 0.2% CMG for 7 days in Example 6;

[0071] Figure 11b This is a micrograph of gastric cancer organoids cultured using the Matrigel 3D culture method in Example 6 after 7 days;

[0072] Figure 10a-Figure 10d In the figure, the scale bars are all 100 μm; Figure 11a and Figure 11b In the figure, the scale bars are all 200 μm.

[0073] Figure 12 This is a micrograph of the culture medium containing 0.2% CMG in Example 7-1 after culturing intestinal cancer organoids for 7 days.

[0074] Figure 13 This is a micrograph of the culture medium containing 0.2% CMG (Preparation Example 2) in Example 7-2 after culturing intestinal cancer organoids for 7 days.

[0075] Figure 14 This is a micrograph of the culture medium containing 0.2% CMG (Preparation Example 3) in Example 7-2 after culturing intestinal cancer organoids for 7 days.

[0076] Figure 15 This is a micrograph of the culture medium containing 0.2% CMG (Preparation Example 4) in Example 7-2 after culturing intestinal cancer organoids for 7 days.

[0077] Figure 16 This is a micrograph of intestinal cancer organoids cultured in a culture medium containing 0.2% CMG (Preparation Example 5) in Example 7-2 after 7 days of culture;

[0078] Figure 17 This is a microscopic picture of the gastric cancer tissue cultured in the cellulose-based culture medium containing different matrix gels in comparative example 5 after 7 days of culture.

[0079] Figure 18 This is a micrograph of comparative example 6 of gastric cancer tissue cultured in a cellulose-based culture medium containing different composite culture mediums for 7 days.

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

[0081] Figure 20a This is a micrograph of intestinal organoids 3 days after induction and differentiation of human pluripotent stem cells / embryonic stem cells in Example 9;

[0082] Figure 20b This is a micrograph of intestinal organoids 7 days after induction and differentiation of human pluripotent stem cells / embryonic stem cells in Example 9;

[0083] Figure 20c This is a micrograph of intestinal organoids 12 days after induction and differentiation of human pluripotent stem cells / embryonic stem cells in Example 9;

[0084] Figure 21 This is a micrograph of intestinal organoids 12 days after induction differentiation of human pluripotent stem cells / embryonic stem cells using CMG prepared in Preparation Example 2 in Example 10;

[0085] Figure 22 This is a micrograph of intestinal organoids 12 days after induction differentiation of human pluripotent stem cells / embryonic stem cells using CMG prepared in Preparation Example 3 in Example 10;

[0086] Figure 23 This is a micrograph of intestinal organoids 12 days after induction differentiation of human pluripotent stem cells / embryonic stem cells using CMG prepared in Preparation Example 4 in Example 10;

[0087] Figure 24This is a micrograph of intestinal organoids 12 days after induction differentiation of human pluripotent stem cells / embryonic stem cells using CMG prepared in Preparation Example 5 in Example 10;

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

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

[0090] Figure 26a This is a micrograph of intestinal organoids differentiated from human pluripotent stem cells / embryonic stem cells after 12 days of induction and differentiation using the intestinal organoid differentiation kit containing 0.3% plant hydrogel CMG in Comparative Example 9;

[0091] Figure 26b For STEMdiff in Comparative Example 9 TM Micrograph of intestinal organoids 12 days after induction of differentiation of human pluripotent stem cells / embryonic stem cells using the Intestinal Organoid Kit (stem cell, 05140). DETAILED DESCRIPTION

[0092] [3D cell culture medium]

[0093] As previously mentioned, the present invention provides a plant-based culture medium for 3D cell culture. Specifically, the present invention provides a 3D cell culture medium comprising a plant hydrogel and a basal culture medium A; wherein the plant hydrogel is free of animal-derived ingredients and comprises micro-nanobiomass and water.

[0094] According to an embodiment of the present invention, the basal culture fluid A can be any basal culture fluid known in the art. For example, the basal culture fluid A comprises DMEM / F12 (GIBCO / 10565018), 10% FBS (GIBCO / 10091-148) and 1% P / S (GIBCO / 15140122).

[0095] [Method for preparing culture medium for 3D cell culture]

[0096] As mentioned above, the present invention also provides a method for preparing the above-mentioned 3D cell culture medium, which comprises the following steps:

[0097] S1: Preparation of plant hydrogel;

[0098] S2: The plant hydrogel in step S1 is mixed with the basic culture medium A to prepare the culture medium for 3D cell culture.

[0099] According to an embodiment of the present invention, in step S1, the preparation method of the plant hydrogel is selected from at least one of the following methods 1 to 4:

[0100] Method 1: preparing a natural biomass-based dispersion: pouring a natural biomass solution into a coagulation bath to form a natural biomass gel; micro-nano-processing the natural biomass gel by a homogenizer, colloid mill, or ball mill to obtain the natural biomass-based dispersion, i.e., the plant hydrogel;

[0101] Method 2: Preparation of a surface-modified biomass-based dispersion: pouring a surface-modified biomass solution into a coagulation bath to form a surface-modified biomass gel; subjecting the surface-modified biomass gel to micronization by a homogenizer, colloid mill, or ball milling to obtain the surface-modified biomass-based dispersion, i.e., the plant hydrogel; alternatively, the natural biomass-based dispersion of method 1 is mixed with a modification agent or subjected to surface chemical modification to obtain the surface-modified biomass-based dispersion, i.e., the plant hydrogel;

[0102] Method 3: Preparing a substituted biomass-based dispersion: pouring a substituted biomass solution into a coagulation bath to form a substituted biomass gel; micronizing the substituted biomass gel by a homogenizer, colloid mill, or ball mill to obtain the substituted biomass-based dispersion, i.e., the plant hydrogel; alternatively, mixing the natural biomass-based dispersion in method 1 with a functionalized derivatization reagent to obtain the substituted biomass-based dispersion, i.e., the plant hydrogel;

[0103] Method 4: preparing a multi-component biomass-based dispersion: pouring a solution containing at least one of natural biomass, substituted biomass, and surface-modified biomass, and a functional component into a coagulation bath to form a gel; performing micro-nano processing on the gel by means of a homogenizer, a colloid mill, or a ball mill to obtain the multi-component biomass-based dispersion, which is the plant hydrogel; or: mixing at least one of a natural biomass-based dispersion, a substituted biomass-based dispersion, and a surface-modified biomass-based dispersion with a functional component, and performing micro-nano processing on the gel by means of a homogenizer, a colloid mill, or a ball mill to obtain the multi-component biomass-based dispersion, which is the plant hydrogel.

[0104] According to an embodiment of the present invention, in step S2, the mass volume ratio of the plant hydrogel to the basic culture solution A is 1-30:1000, for example, 5:1000, 10:1000, or 20:1000.

[0105] [Application of culture medium for 3D cell culture]

[0106] As mentioned above, the present invention also provides use of the above-mentioned 3D cell culture medium in 3D cultured cells.

[0107] As mentioned above, the present invention further provides a method for 3D cell culture, wherein the method for 3D cell culture is performed in the above-mentioned culture medium for 3D cell culture.

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

[0109] 1) Preparing a cell suspension: Evenly mix the 3D cell culture medium and cells to obtain a cell suspension;

[0110] 2) 3D culture: The cell suspension from step 1) is cultured in an incubator.

[0111] According to an embodiment of the present invention, in step 1), the cells may further undergo cell recovery treatment and / or cell passage treatment.

[0112] Preferably, the cell recovery can be performed using methods known in the art, as long as the cells can be recovered, such as recovery according to the principle of rapid cell recovery. Exemplarily, the cell recovery specifically includes: re-dissolving the cells to be recovered in a 37°C water bath, centrifuging, resuspending the cells in a basal culture medium, and then culturing in an incubator (37°C, 5% CO2) for, for example, 48 hours.

[0113] Preferably, the cell passage can be performed using methods known in the art, as long as the desired cells can be obtained. For example, the culture medium is discarded from the revived cells, PBS is added to the culture wells and gently washed twice, followed by trypsin digestion to prepare a single-cell suspension, and the cells are resuspended in basal culture medium and adjusted to the desired cell density (e.g., 5K, 25K, 50K).

[0114] According to an embodiment of the present invention, the cells are preferably human neuroblastoma cells, hepatoma cells, gastric cancer cells, such as SH-SY5Y cells and HepaRG cells.

[0115] According to an embodiment of the present invention, in step 2), the culture can be performed by methods known in the art as long as the desired cells can be obtained, for example, adding the cell suspension to a culture plate and culturing at 37° C. and 5% CO 2 .

[0116] According to an embodiment of the present invention, in step 2), the culturing time is 1 to 10 days, preferably 3 days.

[0117] According to an embodiment of the present invention, in step 2), the culturing further comprises changing the medium, for example, changing the basal culture medium every 3 days.

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

[0119] According to an embodiment of the present invention, the kit includes the 3D cell culture medium of the present invention. Specifically, the cells are, for example, at least one of SH-SY5Y cells, HepaRG cells (liver cancer cells), HCT-116 cells, HeLa cells, K562 cells, MOLM-13 cells, and the like.

[0120] According to an embodiment of the present invention, the kit further includes components or assemblies known in the art, which are not specifically limited in the present invention.

[0121] As mentioned above, the present invention also provides a 3D cell culture kit for use in 3D cell culture.

[0122] According to an embodiment of the present invention, the cells in the 3D cell culture are, for example, at least one of SH-SY5Y cells, HepaRG cells (liver cancer cells), HCT-116 cells, HeLa cells, K562 cells, MOLM-13 cells, and the like.

[0123] [Biomass-based culture medium for 3D tumor organoid culture]

[0124] As previously mentioned, the present invention provides a plant-based culture medium for 3D tumor organoid culture. Specifically, the present invention provides a biomass-based culture medium for 3D tumor organoid culture, comprising a plant hydrogel and a composite culture medium; the plant hydrogel is free of animal-derived ingredients and comprises micro-nanobiomass and water.

[0125] According to an embodiment of the present invention, the composite culture solution includes a basic culture solution B and active components.

[0126] According to an embodiment of the present invention, in every 100 mL of composite culture solution, the mass ratio of the basic culture solution B to the active component is 80-99:1-20, for example, 97:3.

[0127] According to an embodiment of the present invention, the basal culture fluid B can be any basal culture fluid known in the art. For example, the basal culture fluid B includes DMEM / F12 (GIBCO / 10565018), 10% FBS (GIBCO / 10091-148) and 1% P / S (GIBCO / 15140122).

[0128] According to an embodiment of the present invention, in every 100 mL of composite culture solution, the active components include at least:

[0129]

[0130]

[0131] According to an exemplary embodiment of the present invention, the composite culture solution comprises:

[0132]

[0133] [Method for preparing biomass-based culture medium for 3D tumor organoid culture]

[0134] As mentioned above, the present invention also provides a method for preparing the above-mentioned biomass-based culture medium for 3D culture of tumor organoids, the preparation method comprising the following steps:

[0135] S1': Preparation of plant hydrogel;

[0136] S2': mixing the plant hydrogel in step S1' with the composite culture solution to prepare the biomass-based culture solution.

[0137] According to the embodiment of the present invention, step S1' is specifically defined the same as the above step S1.

[0138] According to an embodiment of the present invention, in step S2', the volume ratio of the plant hydrogel to the composite culture solution is 1-10:1-10, for example, 1:1.

[0139] [Application of biomass-based culture medium for 3D tumor organoid culture]

[0140] As mentioned above, the present invention also provides the use of biomass-based culture medium for 3D culture of tumor organoids in the culture of tumor tissues or organoids such as gastric cancer and lung cancer.

[0141] As mentioned above, the present invention also provides a method for culturing tumor tissues or organoids such as gastric cancer and lung cancer using a biomass-based culture medium for 3D culture of tumor organoids.

[0142] According to an embodiment of the present invention, 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, or human breast cancer tissue / normal breast cancer tissue.

[0143] According to an embodiment of the present invention, the organoids are preferably human gastric cancer organoids / human normal gastric organoids, human intestinal cancer organoids / human normal intestinal organoids, human lung cancer organoids / human normal lung organoids, human pancreatic cancer organoids / human normal pancreatic cancer organoids, human breast cancer organoids / human normal breast cancer organoids.

[0144] According to an embodiment of the present invention, the culturing method comprises the following steps:

[0145] a) Preparing a cell suspension of a tissue or organoid: uniformly mixing a biomass-based culture medium for 3D tumor organoid culture with cells of the tissue or organoid to obtain a cell suspension;

[0146] b) 3D culture: The cell suspension from step a) is cultured in an incubator.

[0147] According to an embodiment of the present invention, in step a), the cells of the tissue or organoid can be prepared by methods known in the art, which are not specifically limited in the present invention.

[0148] According to an embodiment of the present invention, in step b), the culture can be performed by methods known in the art as long as the desired cells can be obtained, for example, adding the cell suspension to a culture plate and culturing at 37° C. and 5% CO 2 .

[0149] According to an embodiment of the present invention, in step b), the culturing time is 1 to 10 days, further selected from 3, 5, and 7 days.

[0150] According to an embodiment of the present invention, in step b), the culturing further comprises changing the medium, for example, changing the basal culture medium every 3 days.

[0151] As mentioned above, the present invention also provides a tumor organoid 3D culture kit.

[0152] According to an embodiment of the present invention, the kit includes the biomass-based culture medium 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.

[0153] According to an embodiment of the present invention, the kit comprises a biomass-based hydrogel and a composite culture solution.

[0154] According to an embodiment of the present invention, the kit further includes components or assemblies known in the art, which are not specifically limited in the present invention.

[0155] According to an embodiment of the present invention, the kit is preferably used for culturing gastric cancer organoids and intestinal cancer organoids.

[0156] According to an embodiment of the present invention, the kit further comprises a tissue digestion solution and / or a tissue preservation solution.

[0157] According to an embodiment of the present invention, the tissue digestion solution includes collagenase P and DNase.

[0158] According to an embodiment of the present invention, the tissue preservation solution includes Ad DMEM / F12 containing 2×P / S.

[0159] As mentioned above, the present invention also provides a tumor organoid 3D culture kit for use in tumor organoid 3D culture.

[0160] According to an embodiment of the present invention, 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, and the like.

[0161] [Biomass-based culture medium for stem cell culture]

[0162] As previously mentioned, the present invention provides a plant-based culture medium for stem cell culture, specifically for rapid stem cell culture. Specifically, the present invention provides a biomass-based culture medium for stem cell culture, comprising a plant hydrogel and a basal culture medium C; the plant hydrogel is free of animal-derived ingredients and comprises micro-nanobiomass and water.

[0163] According to an embodiment of the present invention, the basal culture medium C can be selected from basal culture medium known in the art. For example, the basal culture medium C includes DMEM / F12, mTeSR TM 1.

[0164] [Method for preparing biomass-based culture medium for stem cell culture]

[0165] As mentioned above, the present invention also provides a method for preparing the biomass-based culture medium for stem cell culture, the preparation method comprising the following steps:

[0166] S1”: Preparation of plant hydrogel;

[0167] S2″: mixing the plant hydrogel in step S1″ with the basic culture solution C to prepare the biomass-based culture solution.

[0168] According to the embodiment of the present invention, step S1″ is the same as the specific definition of step S1 above.

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

[0170] [Application of biomass-based culture medium for stem cell culture]

[0171] As mentioned above, the present invention also provides the use of the biomass-based culture medium for stem cell culture in stem cell or organoid culture.

[0172] The present invention also provides a method for using the biomass-based culture medium for stem cell culture to culture stem cells or organoids.

[0173] According to an embodiment of the present invention, the stem cells are preferably adult stem cells or embryonic stem cells (ESCs) from mammals and primates; further preferably, they are pluripotent stem cells or multipotent stem cells, such as induced pluripotent stem cells (iPSCs), hematopoietic stem cells, neural stem cells, skin stem cells, mesenchymal stem cells, adipose stem cells, osteoblastic stem cells, cartilage stem cells, muscle stem cells, liver stem cells, pancreatic stem cells, endothelial stem cells, corneal stem cells, hair follicle stem cells, gastrointestinal stem cells, mammary stem cells, cardiac stem cells, etc.

[0174] As mentioned above, the present invention also provides a stem cell culture method, which comprises the following steps:

[0175] a') Pretreatment of stem cells before passage: diluting the plant hydrogel and placing it in an incubator;

[0176] b') Digestion of extracellular matrix: Dispase was used to digest the extracellular matrix;

[0177] c') Add mTeSR TM 1. Puff the stem cells until they fall off and break into pieces;

[0178] d') Stem cell fragments were added to a culture dish containing plant hydrogel for culture.

[0179] According to an embodiment of the present invention, in step a'), the plant hydrogel is diluted with DMEM / F12, added to a culture plate after dilution, and allowed to stand at 37°C for at least 1 hour.

[0180] According to an embodiment of the present invention, in step b'), the digestion using Dispase can be performed using methods known in the art, for example, covering the well plate with Dispase and placing it in a 37°C incubator until the edges of the stem cells begin to rise.

[0181] According to an embodiment of the present invention, step b') further comprises the steps of aspirating the Dispase and washing with DPBS.

[0182] According to an embodiment of the present invention, in step c'), the fragments are 1-2 mm.

[0183] According to an embodiment of the present invention, in step d'), a method known in the art can be used for culturing, for example, using mTeSR TM 1. Replenish the culture medium, culture at 37°C, 5% CO2, and change the medium.

[0184] [Stem cell induced differentiation organoid culture medium]

[0185] As mentioned above, the present invention also provides a stem cell induced differentiation organoid culture medium, which includes plant hydrogel, definitive endoderm differentiation culture medium, MH differentiation culture medium, and organoid growth culture medium; wherein, the plant hydrogel does not contain animal-derived components and includes micro-nano biomass and water.

[0186] According to an embodiment of the present invention, the definitive endoderm differentiation culture medium comprises the following components:

[0187] RPMI1640, containing L-glutamine,

[0188] Penicillin-streptomycin,

[0189] and Activin A.

[0190] According to an embodiment of the present invention, the definitive endoderm differentiation culture medium preferably comprises the following components:

[0191]

[0192] According to an embodiment of the present invention, the MH differentiation culture medium comprises the following components:

[0193] RPMI1640 contains 2% FBS and L-glutamine.

[0194] Penicillin-streptomycin,

[0195] and FGF4.

[0196] According to an embodiment of the present invention, the MH differentiation culture medium preferably comprises the following components:

[0197] RPMI1640 containing 2% FBS, L-glutamine, 2mM,

[0198] Penicillin-streptomycin, 100U / ml-100g / ml,

[0199] and FGF4 500ng / ml.

[0200] According to an embodiment of the present invention, the organoid growth medium comprises the following components:

[0201] Advanced DMEM / F12 contains 1×B27 and L-glutamine.

[0202] Penicillin-streptomycin,

[0203] HEPES buffer,

[0204] and R-spondin 1.

[0205] According to an embodiment of the present invention, the organoid growth medium preferably comprises the following components:

[0206] Advanced DMEM / F12 contains 1×B27, L-glutamine, 2mM,

[0207] Penicillin-streptomycin, 100U / ml-100g / ml,

[0208] HEPES buffer, 15 mM,

[0209] and R-spondin 1, 500ng / ml.

[0210] [Application of stem cell-induced differentiation organoid culture medium]

[0211] As mentioned above, the present invention also provides the use of the above-mentioned stem cell-induced differentiation organoid culture medium in stem cell-induced differentiation organoids.

[0212] As mentioned above, the present invention also provides a method for inducing stem cell differentiation into organoids, wherein the method comprises culturing a single stem cell or a stem cell population in a stem cell differentiation into organoid culture medium.

[0213] According to an embodiment of the present invention, the method for inducing stem cell differentiation into organoids comprises the following steps:

[0214] a”) iPSC / ES differentiation in monolayer culture, including definitive endoderm differentiation and mid / hindgut (MH) differentiation;

[0215] b”) Organoid culture, including preparation of organoid growth medium, and mixing of the plant hydrogel and spheroids followed by incubation.

[0216] According to an embodiment of the present invention, the definitive endoderm differentiation in step a") includes preparing a definitive endoderm differentiation culture medium and performing definitive endoderm differentiation, wherein the definitive endoderm differentiation step includes incubating the culture medium using a definitive endoderm differentiation culture medium using a method known in the art, such as incubation at 37°C, 5% CO2 and 95% humidity.

[0217] According to an embodiment of the present invention, the midgut / hindgut (MH) differentiation in step a") includes the preparation of MH differentiation culture medium and midgut / hindgut (MH) differentiation, wherein the midgut / hindgut (MH) differentiation step includes incubating the spheroids using the MH differentiation culture medium using a method known in the art, such as incubating at 37°C, 5% CO2 and 95% humidity, changing the medium and observing the spheroids every 24 hours, and embedding the spheroids.

[0218] According to an embodiment of the present invention, after the plant hydrogel and spheroids are mixed in step b"), they are incubated using methods known in the art, for example, including: incubating at 37°C for less than 30 minutes, adding organoid growth medium and continuing incubation at 37°C, 5% CO2, and 95% humidity, and subculturing according to the growth of the organoids.

[0219] As mentioned above, the present invention also provides a stem cell organoid prepared by the method of inducing stem cell differentiation into organoids, such as adult stem cell organoids, induced pluripotent stem cell organoids or embryonic stem cell organoids.

[0220] As mentioned above, the present invention also provides a kit for inducing differentiation of organoids.

[0221] According to an embodiment of the present invention, the kit includes the stem cell induced differentiation organoid culture medium; specifically, the organoid is preferably at least one of intestinal organoids, gastric organoids, liver organoids, etc., and is further preferably small intestinal organoids.

[0222] According to an embodiment of the present invention, the kit further comprises a definitive endoderm differentiation culture medium, a MH differentiation culture medium, and an organoid growth culture medium.

[0223] According to an embodiment of the present invention, the kit further includes components or assemblies known in the art, which are not specifically limited in the present invention.

[0224] As mentioned above, the present invention also provides a use of a kit for inducing differentiation of organoids in inducing differentiation of organoids.

[0225] According to an embodiment of the present invention, the induced differentiated organoid is, for example, at least one of induced differentiated intestinal organoids, induced differentiated gastric organoids, induced differentiated liver organoids, etc., preferably small intestinal organoids.

[0226] [Biomass]

[0227] The biomass in the micro-nano biomass of the present invention can be at least one of natural biomass, surface-modified biomass, or substituted biomass; for example, it can be at least one of natural polymers, surface-modified natural polymers, and substituted natural polymers described in patent document CN116284844A. Specifically,

[0228] The natural polymer is, for example, selected from one or more of the following substances: cellulose, starch, lignin, chitosan, chitin, hemicellulose, glucan, low-quality cellulose or natural plant tissue containing one or more of the above components.

[0229] The cellulose is derived from cellulose extracted from plant tissue or other biomass. For example, the cellulose extracted from plant tissue can be selected from one or more of the following: microcrystalline cellulose, bacterial cellulose, cotton pulp, wood pulp, bamboo pulp, straw pulp, refined cotton, absorbent cotton, cotton linters, bagasse, wood, and straw, preferably one or more of microcrystalline cellulose, refined cotton, absorbent cotton, and wood pulp. For example, the non-extracted biomass can be a mixed biomass such as straw.

[0230] The starch is selected from at least one of amylopectin, amylose, high amylose, modified starch, and cross-linked starch; or, the starch is selected from soluble starch; or, the starch is selected from at least one of potato starch, corn starch, cassava starch, wheat starch, sweet potato starch, kudzu starch, pea starch, water chestnut starch, soybean starch, and lotus root starch.

[0231] There is no particular limitation on the selection of chitosan, chitin, lignin, hemicellulose, glucan, etc., and any of the above-mentioned ingredients can be known to those skilled in the art and can be applied to the system described in the present invention.

[0232] Preferably, the deacetylation degree of the chitosan is 50-100%; more preferably, the deacetylation degree of the chitosan is 70-95%.

[0233] Preferably, the lignin can be selected from one or more of syringyl lignin, guaiacyl lignin, p-hydroxyphenyl lignin, etc.; or, the lignin can be selected from one or more of alkaline lignin, acidic lignin, dealkalized lignin, and organic solvent-soluble lignin.

[0234] The low-quality cellulose is a plant tissue containing at least cellulose and lignin, such as herbaceous plants and / or agricultural and forestry wastes.

[0235] For example, the herbaceous plant is selected from one or more of trees, shrubs, vines, leaves, bamboo and the like.

[0236] For example, the agricultural and forestry waste is selected from one or more of bark, leaves, sawdust, crop straw, fruit shells or cores, corn cobs, sugarcane bagasse, etc.

[0237] Preferably, the crop straw can be selected from one or more of wheat straw, rice straw, corn straw, soybean straw, cotton straw, ginger stalk, and sesame straw.

[0238] The surface modified natural polymer can be a natural polymer with a surface cationization, a natural polymer with a surface anionization, or a natural polymer with a surface hydrophobic treatment. Specifically, the natural polymer in the surface modified natural polymer has the definition above. Also 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 with a modifying agent in a solvent as described below). Wherein, the modifying agent can be one or more of quaternary ammonium salts, sulfonates, phosphates, etc.; for example, sodium vinyl sulfonate or 3-chloro-2-hydroxypropyltrimethylammonium chloride.

[0239] The substituted natural polymer can be selected from one or more of the following: esterified natural polymers, acylated natural polymers, etherified natural polymers, amidated natural polymers, ammonated natural polymers, and the like. Specifically, the natural polymer in the substituted natural polymer has the definition above. 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 to 2.0, preferably 0.001 to 1.8, for example, 0.1, 0.5, 0.65, 0.86, 1.46, or 1.6. Furthermore, the substituted natural polymer can be prepared by reacting a functionalized derivatization reagent with a natural polymer. For example, the functionalized derivatization reagent can be selected from an esterification reagent, an acylation reagent, an etherification reagent, an ammoniation reagent, and / or an amidation reagent. For example, the substituted natural polymer can be selected from cellulose acetate (e.g., cellulose acetate with a degree of substitution of 0.65, 0.86, 1.46, or 1.6) or cellulose-g-PLA.

[0240] [Plant hydrogel]

[0241] As mentioned above, the culture solution of the present invention includes a plant gel, which has no animal-derived components and includes micro-nano biomass and a dispersing agent; the dispersing agent is selected from the organic solvent and / or water mentioned below, preferably water and / or alcohol.

[0242] According to an embodiment of the present invention, the culture solution of the present invention includes a plant hydrogel, which has no animal-derived components and includes micro-nano biomass and water.

[0243] According to an embodiment of the present invention, the biomass in the micro-nano biomass has the definitions listed above.

[0244] According to an embodiment of the present invention, the micro-nano biomass includes a micron-scale structure and a nano-scale structure distributed on the micron-scale structure.

[0245] According to an embodiment of the present invention, the content of the micro-nano biomass in the plant hydrogel may be 0.050-95 wt.%, illustratively 0.5-10 wt.%, and specifically 1 wt.%, 5 wt.%, 10 wt.%, 15 wt.%, 20 wt.%, 30 wt.%, 40 wt.%, 50 wt.%, 60 wt.%, 70 wt.%, 80 wt.%, 90 wt.%, or 95 wt.%. A too high concentration results in high viscosity and inconvenience in use, but facilitates transportation and saves costs. A too low concentration results in poor dispersion stability.

[0246] According to an embodiment of the present invention, the micro-nano biomass is a regenerated natural polymer having a micro-nano structure (for example, type II cellulose, or a mixture of type I cellulose and type II cellulose). The regenerated natural polymer having a micro-nano structure means that the regenerated natural polymer includes a micron-scale structure and a nanoscale structure distributed on the micron-scale structure. Specifically, the natural polymer in the regenerated natural polymer has the definition listed above.

[0247] According to an embodiment of the present invention, the size of the micron-scale structure (i.e., the volume average size) is 10 μm to 300 μm, preferably the size of the micron-scale structure is 50 μm to 250 μm, and further preferably, the size of the micron-scale structure is 100 μm to 200 μm, for example, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 105 μm, 110 μm, 115 μm, 120 μm, 125 μm, 130 μm, 135 μm, 136 μm, 137 μm, 138 μm, 139 μm, 140 μm, 141 μm, 142 μm, 143 μm, 144 μm, 145 μm, 146 μm, 147 μm, 148 μm, 149 μm, 150 μm, 151 μm, 152 μm, 153 μm, 154 μm, 156 μm, 157 μm, 158 μm, 159 μm, 160 μm, 161 μm, 162 μm, 163 μm, 164 μm, 165 μm, 166 μm, 167 μm, 168 μm, 169 μm, 170 μm, 171 μm, 172 μm, 173 μm, 174 μm, 175 μm, 5μm, 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 or any point value among the intermediate values ​​formed by any two point values.

[0248] According to an embodiment of the present invention, the length of the nanoscale structure is 500nm to 1500nm, preferably the length of the nanoscale structure is 700nm to 1200nm, and further preferably, the length of the nanoscale structure is 800nm ​​to 1000nm, for example, any value among 500nm, 600nm, 700nm, 800nm, 900nm, 1000nm, 1100nm, 1200nm, 1300nm, 1400nm, 1500nm or any point value in the intermediate value formed by any two point values.

[0249] According to an embodiment of the present invention, the diameter of the nanoscale structure is 200nm to 600nm, preferably the diameter of the nanoscale structure is 300nm to 500nm, for example, any value among 200nm, 300nm, 400nm, 500nm, 600nm or any point value in the middle value formed by any two point values.

[0250] According to an embodiment of the present invention, the aspect ratio of the nanoscale structure is 1:1 to 1:150, preferably the aspect ratio of the nanoscale structure is 1:1 to 1:100, and more preferably the aspect ratio of the nanoscale structure is 1:1 to 1:50.

[0251] According to an embodiment of the present invention, the micron-scale structure includes at least one of micron sheets, micron fibers, and micron multi-level structures.

[0252] According to an embodiment of the present invention, the micron-scale structure is in a fiber or film shape, which can also be called brooming.

[0253] According to an embodiment of the present invention, the nanoscale structures are distributed on the surface and / or edge of the microscale structure. Specifically, the nanoscale structures are densely distributed on the surface and / or edge of the microscale structure.

[0254] According to an embodiment of the present invention, the nano-scale structure is distributed on the micro-scale structure to form a flocculent shape as a whole.

[0255] According to an embodiment of the present invention, the nano-scale structure is distributed on the micro-scale structure to form an overall structure similar to a porous sponge.

[0256] According to an embodiment of the present invention, the nano-scale structure and the micro-scale structure together form a feather-like structure.

[0257] According to an embodiment of the present invention, the nanoscale structure is in the shape of hair, tentacle, whisker or other irregular structures.

[0258] According to an embodiment of the present invention, the micro-nano biomass is in the form of particles, fibers, sheets, feathers, branches, flocs or porous sponges.

[0259] According to an embodiment of the present invention, the micro-nano biomass described in the present invention is, for example, natural cellulose gum CMG.

[0260] According to an embodiment of the present invention, 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.

[0261] [Natural biomass-based dispersion and preparation method thereof]

[0262] As described above, the phytogel of the present invention can be provided by a natural biomass-based dispersion, comprising the micro-nano natural biomass and a dispersing agent, wherein the micro-nano natural biomass is distributed in the dispersing agent, and the dispersing agent is 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 dispersing agent is the continuous phase.

[0263] According to an embodiment of the present invention, the dispersing agent is selected from the group consisting of organic solvents and / or water, preferably water and / or alcohol.

[0264] The present invention also provides a method for preparing the above-mentioned natural biomass-based dispersion, comprising the following steps: pouring a natural biomass solution into a coagulation bath to form a natural biomass gel; and subjecting the natural biomass gel to micro-nano treatment by a homogenizer, colloid mill, or ball mill to obtain the biomass micro-nano dispersion, i.e., the biomass-based dispersion.

[0265] According to an embodiment of the present invention, the natural biomass has the selection as shown above.

[0266] According to an embodiment of the present invention, the coagulation bath may be the same as or different from the continuous phase, for example, the continuous phase may be the same; preferably, the continuous phase may be water and / or alcohol.

[0267] According to an embodiment of the present invention, the natural biomass-based dispersion may 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.

[0268] [Surface-modified biomass-based dispersion and preparation method thereof]

[0269] As described above, the phytogel of the present invention can be provided by a surface-modified biomass-based dispersion comprising surface-modified micro- and nano-biomass and a dispersing agent, wherein the surface-modified micro- and nano-biomass is dispersed in the dispersing agent; the dispersing agent is a solvent system capable of continuously dispersing the surface-modified micro- and nano-biomass. Specifically, in the dispersion, the surface-modified micro- and nano-biomass is the dispersed phase, and the dispersing agent is the continuous phase.

[0270] According to an embodiment of the present invention, the surface-modified micro-nano biomass is a surface-modified product of the micro-nano natural biomass; or is a product obtained by subjecting the surface-modified biomass to micro-nano treatment.

[0271] According to an embodiment of the present invention, the continuous phase of the dispersion has the meaning indicated above, preferably water and / or alcohol.

[0272] According to an embodiment of the present invention, 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.

[0273] According to an embodiment of the present invention, the natural biomass and the surface-modified biomass have the selections shown above.

[0274] According to an embodiment of the present invention, the content of the dispersed phase may be 0.050-95 wt.%, and for example, may be 0.5-10 wt.%.

[0275] According to an embodiment of the present invention, the dispersion is a micro-nano dispersion; specifically, in the dispersion, the surface-modified micro-nano biomass has a structure substantially the same as that of the micro-nano natural biomass, for example, including a micron-scale structure and a nano-scale structure distributed on the micron-scale structure.

[0276] The present invention also provides a method for preparing the 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; performing micro-nano treatment on the surface-modified biomass gel by a homogenizer, a colloid mill, or a ball mill to obtain the surface-modified biomass-based dispersion;

[0277] Alternatively, the surface-modified biomass-based dispersion is obtained by mixing the natural biomass-based dispersion with a modification agent or performing surface chemical modification.

[0278] Preferably, the surface-modified biomass and the coagulation bath have the meanings indicated above.

[0279] According to an embodiment of the present invention, the modifying agent may be one or more of quaternary ammonium salts, sulfonates, phosphates, etc.; for example, sodium vinyl sulfonate or 3-chloro-2-hydroxypropyltrimethylammonium chloride.

[0280] According to an embodiment of the present invention, the surface-modified biomass solution can be obtained by homogeneously chemically derivatizing or functionalizing natural biomass with a modification agent in a solvent as described below.

[0281] According to an embodiment of the present invention, the surface-modified biomass-based dispersion may be a homogeneous solution in which the surface-modified micro-nano biomass is completely dissolved or a heterogeneous dispersion in which the surface-modified micro-nano biomass is partially dissolved.

[0282] [Substituted biomass-based dispersion and preparation method thereof]

[0283] As described above, the phytogel of the present invention can be provided by a substituted biomass-based dispersion comprising a substituted micro-nano biomass and a dispersing agent, wherein the substituted micro-nano biomass is dispersed in the dispersing agent; the dispersing 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 dispersing agent is the continuous phase.

[0284] According to an embodiment of the present invention, the substituted micro-nano biomass is a substitute for the micro-nano natural biomass; or is a product obtained by micro-nano treatment of the substituted biomass.

[0285] According to an embodiment of the present invention, the continuous phase of the dispersion has the meaning indicated above, preferably water and / or alcohol.

[0286] According to an embodiment of the present invention, the substituted micro-nano biomass is low-substituted micro-nano biomass. For example, the substitution degree 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, or 1.6.

[0287] 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 low-substituted micro-nano natural polymer has a degree of substitution of 0.0001 to 2.0, preferably 0.001 to 1.8, for example, 0.1, 0.5, 0.65, 0.86, 1.46, or 1.6.

[0288] According to an embodiment of the present invention, the substituted micro-nano biomass can be selected from one or more of the following substances: esterified micro-nano natural polymers, acylated micro-nano natural polymers, etherified micro-nano natural polymers, amidated micro-nano natural polymers, ammonated micro-nano natural polymers, etc.

[0289] Preferably, the substituted biomass can be prepared by reacting a functionalized derivatizing agent with natural biomass. For example, the functionalized derivatizing agent can be selected from an esterification agent, an acylation agent, an etherification agent, an ammoniation agent, and / or an amidation agent.

[0290] According to an embodiment of the present invention, the substituted biomass may be selected from cellulose acetate (eg, cellulose acetate with a degree of substitution of 0.65, 0.86, 1.46, or 1.6), cellulose-g-PLA.

[0291] According to an embodiment of the present invention, the natural biomass has the selection as shown above.

[0292] According to an embodiment of the present invention, the content of the dispersed phase may be 0.050-95 wt.%, and for example, may be 0.5-10 wt.%.

[0293] According to an embodiment of the present invention, the dispersion is a micro-nano dispersion; specifically, in the dispersion, the substituted micro-nano biomass has a structure substantially the same as that of the micro-nano natural biomass, for example, including a micron-scale structure and a nano-scale structure distributed on the micron-scale structure.

[0294] The present invention also provides a method for preparing the above-mentioned substituted biomass-based dispersion, comprising the following steps: pouring a substituted biomass solution into a coagulation bath to form a substituted biomass gel; performing micro-nano treatment on the substituted biomass gel by a homogenizer, colloid mill, or ball mill to obtain the substituted biomass-based dispersion;

[0295] Alternatively, the natural biomass-based dispersion is mixed with a functionalized derivatizing agent to obtain the substituted biomass-based dispersion.

[0296] Preferably, the substituted biomass, coagulation bath and functionalized derivatization agent have the meanings indicated above.

[0297] According to an embodiment of the present invention, the substituted micro-nano biomass dispersion can be obtained by homogeneously chemically derivatizing or functionalizing micro-nano natural biomass with a functionalized derivatizing agent in the following solvent.

[0298] According to an embodiment of the present invention, the substituted micro-nano biomass dispersion may 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.

[0299] [Multi-component biomass-based dispersion and preparation method thereof]

[0300] As shown above, the plant gel of the present invention can be provided by a multi-component biomass-based dispersion, wherein the continuous phase of the multi-component biomass-based dispersion is a dispersing agent, and the dispersed phase of the dispersion contains a functional component and at least one of the following components 1) to 3):

[0301] The micro-nano natural biomass described in component 1);

[0302] Component 2) the substituted micro-nano biomass;

[0303] Component 3) The surface-modified micro-nano biomass.

[0304] According to an embodiment of the present invention, the continuous phase of the dispersion is selected as shown above, preferably water and / or alcohol. According to an embodiment of the present invention, 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 a functional component.

[0305] According to an embodiment of the present invention, 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, and gelatin.

[0306] According to an embodiment of the present invention, the functional component is selected from one or more of the following substances: nanocellulose, carbon nanotubes, graphene, fullerene, carbon black, silver nanowires, quantum dots, carbon dots, nano Fe3O4, drugs, etc.

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

[0308] The present invention also provides a method for preparing the multi-component biomass-based dispersion, which is selected from the following scheme 1 or scheme 2:

[0309] Option 1: Pour a solution containing at least one of natural biomass gel, substituted biomass gel, and surface-modified biomass gel, and a functional component into a coagulation bath to form a gel; and subject the gel to micro-nano treatment by a homogenizer, colloid mill, or ball mill to obtain the multi-component biomass-based dispersion.

[0310] Option 2: At least one of the natural biomass-based dispersion, the substituted biomass-based dispersion, and the surface-modified biomass-based dispersion is mixed with the functional component, and then micronized and nano-processed by a homogenizer, colloid mill, or ball mill to obtain the multi-component biomass-based dispersion.

[0311] 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 are all selected as shown above.

[0312] Preferably, a cross-linking agent may be added before forming the gel. Preferably, the cross-linking agent is at least one selected from boric acid, calcium chloride, acetic acid, and ethylenediaminetetraacetic acid.

[0313] According to an embodiment of the present invention, the dispersion is a homogeneous solution or a heterogeneous dispersion.

[0314] [Solvent]

[0315] In the preparation methods of the aforementioned natural biomass-based dispersions, surface-modified biomass-based dispersions, substituted biomass-based dispersions, or multi-component biomass-based dispersions, the solvent in the homogeneous solution and / or heterogeneous dispersion is not particularly limited and can be selected from any excellent solvent known in the art that can dissolve (including complete dissolution and partial dissolution) the solute (e.g., the natural polymer). Preferably, depending on 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, mixed solvent of ionic liquid and organic solvent, choline-type ionic liquid deep eutectic solvent system, organic solvent / salt system, amine oxide system (NMMO), carbamate system, alkali / water system, alkali / urea system, alkali / thiourea system, liquid ammonia / NH4SCN, organic acid, aqueous metal salt solution, alcohol solution of metal salt hydrate, water-alcohol mixed solution of metal salt hydrate, and the like.

[0316] The organic solvent may be selected from one or more of N,N-dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMAc), N-methyl-2-pyrrolidone (NMP), N-methylimidazole, imidazole, pyridine, ethylenediamine, hexafluoroacetone, hexafluoroisopropanol, glycerol, methyl isobutyl ketone, tetrahydrofuran, dioxane, and γ-valerolactone (GVL).

[0317] The organic solvent / salt system can be selected from one or more of N,N-dimethylacetamide / lithium chloride (DMAc / LiCl) system, N-methyl-2-pyrrolidone / NMP, and N,N-dimethyl sulfoxide / tetrabutylammonium fluoride system (DMSO / TBAF).

[0318] The alkali / water system can be selected from one or both of NaOH / H2O and KOH / H2O.

[0319] Wherein, the alkali / urea system can be selected from NaOH / Urea.

[0320] Wherein, the alkaline / thiourea system is selected from NaOH / thio-urea.

[0321] The organic acid can be selected from one or more of formic acid, acetic acid, propionic acid, butyric acid, succinic acid, lactic acid, glutamic acid, glycine, dichloroacetic acid, trichloroacetic acid, and benzenesulfonic acid.

[0322] Among them, the metal salt aqueous solution is preferably selected from aqueous solutions of metal salts such as CaCl2, ZnCl2, LiClO4, Ca(SCN)2, and LiSCN.

[0323] The alcohol solution of the metal salt hydrate can be selected from a methanol solution of CaBr2·H2O and a methanol solution of CaCl2·2H2O.

[0324] The water-alcohol mixed solution of the metal salt hydrate can be selected from a methanol aqueous solution of CaBr2·H2O and a methanol aqueous solution of CaCl2·2H2O.

[0325] The amine oxide system may be a NMMO / H2O / DMSO system, a NMMO / H2O / diethyltriamine system, or a NMMO / H2O system.

[0326] The ionic liquid is selected from an organic molten salt formed by cations and anions with a melting point lower than 100° C., preferably an organic molten salt that can dissolve the biomass natural polymer.

[0327] For example, the cation of the ionic liquid is selected from one or more substituted or unsubstituted imidazole, pyridine, pyrrole, amine, phosphine, choline, diazabicyclic, amino acid type cations; for example, the substituent can be C 1-6 Alkyl, C 1-6 One or more of alkenyl, phenyl or substituted phenyl; preferably one or more of methyl, ethyl, butyl and allyl;

[0328] Preferably, the cation is selected from one or more of the following cations: 1-ethyl-3-methylimidazolium cation ([EMIM]), 3-methylimidazolium cation ([MIM]), 1-propyl-3-methylimidazolium cation ([PMIM]), 1-allyl-3-methylimidazolium cation ([AMIM]), 1-butyl-3-methylimidazolium cation ([BMIM]), 1-butyl-2,3-dimethylimidazolium cation ([BMMIM]), 1,3-dimethylimidazolium cation ([MMIM]), 1-methoxyethyl-3-methylimidazolium cation ([MeOEMIM]), 1-methoxymethyl-3-methylimidazolium cation ([MeOMMIM]), 1-hydroxy-3-methylimidazolium cation ([HMIM]), 1-(2-hydroxyethyl)-3-methylimidazolium cation ([HOEMIM]), 1-methyl-3-benzylimidazolium cation ([MBzIM]), 1 cations include 1-pentyl-3-methylimidazolium cation ([PeMIM]), 1-benzyl-3-methylimidazolium cation ([BzMIM]), 1-m-methoxybenzyl-3-methylimidazolium cation ([MeOBzMIM]), 1-m-methylbenzyl-3-methylimidazolium cation ([MeBzMIM]), N-methylpyridinium cation ([MPyr]), N-ethylpyridinium cation ([EPyr]), N-butylpyridinium cation ([BPyr]), N-n-hexylpyridinium cation ([HPyr]), 1-butyl-3-methylpyrrolidinium ion ([BMPyrr]), tris(2-hydroxyethyl)methylamine ([THEMA]), tetrabutylamine ([TBA]), tetrabutylphosphine ([PBu4]), glycine cation ([Gly]), choline cation ([Ch]), and 1,5-diazabicyclo[4.3.0]one-5-ene ([DBNH]).

[0329] More preferably, the cation is selected from one or more of the following cations: 1-ethyl-3-methylimidazolium cation ([EMIM]), 1-allyl-3-methylimidazolium cation ([AMIM]), 1-butyl-3-methylimidazolium cation ([BMIM]), choline cation ([Ch]).

[0330] For example, the anion is selected from one or more of halogen anions, organic acid radical ions, organic acid ester anions, amino acid type anions, and the like.

[0331] Preferably, the anion is selected from one or more of the following anions: chloride ([Cl]), bromide ([Br]), fluoride ([F]), formate ([HCOO]), acetate ([CH3COO] or [Ac]), glycolate ([HOCH2COO]), propionate ([CH3CH2COO] or [OPr]), butyrate ([CH3CH2CH2COO] or [OBu]), octanoate ([Oct]), benzoate ([C6H5COO] or [PhCOO]), lactate ([CH3CH(OH)COO] or [Lac]), thioglycolate ([HSCH2COO]) , hexafluorophosphate ion ([PF6]), trifluoroborate ([BF3]), methyl phosphate ion ([(MeO)HPO2] or [MP]), dimethyl phosphate ion ([(MeO)2PO2] or [DMP]), diethyl phosphate ion ([(EtO)2PO2] or [DEP]), methyl sulfonate anion ([MeOSO3]), trifluoromethylsulfonate anion ([CF3SO3]), glycine anion ([Gly]), lysine anion ([Lys]), valine anion ([Val]), dicyanamide anion ([N(CN)2] or [DCA]), bistrifluoromethylsulfonimide ([Tf2N]) and the like anion or more.

[0332] More preferably, the anion is selected from one or more of the following anions: chloride ion ([Cl]), formate ion ([HCOO]), acetate ion ([Ac]), methyl phosphate ion ([(MeO)HPO2] or [MP]), dimethyl phosphate ion ([(MeO)2PO2] or [DMP]) and dicyanamide anion ([N(CN)2] or [DCA]).

[0333] According to the present invention, the ionic liquid can be selected from one or more of the following ionic liquids: 1-ethyl-3-methylimidazolium chloride ionic liquid ([EMIM][Cl]), 1-ethyl-3-methylimidazolium bromide ionic liquid ([EMIM][Br]), 1-ethyl-3-methylimidazolium formate ionic liquid ([EMIM][HCOO]), 1-ethyl-3-methylimidazolium acetate ionic liquid ([EMIM][Ac]), 1-ethyl-3-methylimidazolium octanoate ionic liquid ([EMIM][Oct]), 1-ethyl-3-methylimidazolium methyl phosphate ionic liquid ([EMIM][MP]), 1-ethyl-3-methylimidazolium dimethyl phosphate ionic liquid ([EMIM][MP]), 1-ethyl-3-methylimidazolium ... methyl phosphate ionic liquid ([EMIM][Ac]), 1-ethyl-3-methylimidazolium methyl phosphate ionic liquid ([EMIM][MP]), 1-ethyl-3-methylimidazolium methyl [EMIM][DMP]), 1-ethyl-3-methylimidazolium diethyl phosphate ionic liquid ([EMIM][DEP]), 1-ethyl-3-methylimidazolium propionate ionic liquid ([EMIM][OPr]), 1-ethyl-3-methylimidazolium top ionic liquid ([EMIM][OBu]), 1-ethyl-3-methylimidazolium glycinate ionic liquid ([EMIM][Gly]), 1-ethyl-3-methylimidazolium lysine ionic liquid ([EMIM][Lys]), 1-allyl-3-methylimidazolium chloride ionic liquid ([AMIM][Cl]), 1-allyl-3-methylimidazolium bromide ionic liquid ([AMIM][Br]), 1-ethyl-3-methylimidazolium octanoate ionic liquid ([EMIM][C1]), 1-ethyl-3-methylimidazolium lysine ionic liquid ([EMIM][C2]), 1-ethyl-3-methylimidazolium chloride ionic liquid ([AMIM][Cl]), 1-ethyl-3-methylimidazolium bromide ionic liquid ([AMIM][Br]), 1-ethyl-3-methylimidazolium octanoate ionic liquid ([EMIM][C3]), 1-ethyl-3-methylimidazolium chloride ionic liquid ([EMIM][C4]), 1-ethyl-3-methylimidazolium chloride ionic liquid ([EMIM][C5]), 1-ethyl-3-methylimidazolium chloride ionic liquid ([EMIM][C6]), 1-ethyl-3-methylimidazolium chloride ionic liquid ([EMIM][C7]), 1-ethyl-3-methylimidazolium chloride ionic liquid ([EMIM][C8]), 1-ethyl-3-methylimidazolium chloride ionic liquid ([EMIM][ Propyl-3-methylimidazolium formate ionic liquid ([AMIM][HCOO]), 1-allyl-3-methylimidazolium acetate ionic liquid ([AMIM][Ac]), 1-butyl-3-methylimidazolium chloride ionic liquid ([BMIM][Cl]), 1-butyl-3-methylimidazolium bromide ionic liquid ([BMIM][Br]), 1-butyl-3-methylimidazolium formate ionic liquid ([BMIM][HCOO]), 1-butyl-3-methylimidazolium acetate ionic liquid ([BMIM][Ac]), 1-butyl-3-methylimidazolium hydroxyacetate ionic liquid ([BMIM][HOCH2COO]), 1-butyl-3-methylimidazolium propionate ionic liquid 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 bis(trifluoromethylsulfonyl)imide 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 methanesulfonate 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][Cl]), 1,3-Dimethyl 1-(2-hydroxyethyl)-3-methylimidazolium chloride ionic liquid ([HOEMIM][Cl]), 1-methoxymethyl-3-methylimidazolium bromide ionic liquid ([MeOMMIM][Br]), 1-methoxyethyl-3-methylimidazolium chloride ionic liquid ([HMIM][Cl]), 1-hydroxy-3-methylimidazolium trifluoromethylsulfonate ionic liquid ([HMIM][CF3SO3]), 1-(2-hydroxyethyl)-3-methylimidazolium chloride ionic liquid ([HOEMIM][Cl]), 1-methoxymethyl-3-methylimidazolium bromide ionic liquid ([MeOMMIM][Br]), 1-methoxyethyl-3-methylimidazolium chloride ionic liquid ([HMIM][Cl]), 1-methoxyethyl ...methoxyethyl-3-methylimidazolium chloride 3-Methylimidazolium bromide ionic liquid ([MeOEMIM][Br]), N-ethylpyridinium chloride ionic liquid ([EPyr][Cl]), N-ethylpyridinium bromide ionic liquid ([EPyr][Br]), N-methylpicolinate ionic liquid ([MPyr][HCOO]), tris(2-hydroxyethyl)methylamine acetate ionic liquid ([THEMA][Ac]), tris(2-hydroxyethyl)methylamine methanesulfonate ionic liquid ([THEMA][MeOSO3]), tris(2-hydroxyethyl)methylamine trifluoromethanesulfonate ionic liquid [THEMA][CF3SO3], tetrabutylphosphine valine ionic liquid [PBu4][Val], tetrabutylphosphine lysate Amine salt ionic liquid [PBu4][Lys], tetrabutylphosphine glycinate ionic liquid [PBu4][Gly], 1-benzyl-3-methylimidazolium chloride ionic liquid ([BzMIM][Cl]), 1-benzyl-3-methylimidazolium dicyanamide ionic liquid ([BzMIM][DCA]), 1-m-methylbenzyl-3-methylimidazolium chloride ionic liquid ([MeBzMIM][Cl]), 1-m-methoxybenzyl-3-methylimidazolium chloride ionic liquid ([MeOBzMIM][Cl]), choline chloride ionic liquid ([Ch][Cl]), choline bromide ionic liquid (Ch][Br]), choline acetate ionic liquid ([Ch][CH3COO]),Ionic liquids include choline propionate ionic liquid ([Ch][CH3CH2COO]), choline butyrate ionic liquid ([Ch][CH3CH2CH2COO]), glycine hydrochloride ionic liquid ([Gly][Cl]), 1,5-diazabicyclo[4.3.0]keto-5-ene acetate ionic liquid ([DBNH][Ac]), etc.

[0334] Preferably, the choline-type deep eutectic solvent system is selected from one or more of [Ch][Cl] / urea, [Ch][Br] / urea, [Ch][Cl] / thio-urea, [Ch][Cl] / glycerol, and [Ch][Cl] / lactic acid.

[0335] Preferably, the solvent system for dissolving cellulose is selected from the ionic liquid and / or NaOH / Urea system; more preferably, the cellulose-dissolving ionic liquid is selected from one or more of [AMIM][Cl], [BMIM][Cl], [EMIM][Ac], and [BMIM][Ac].

[0336] Preferably, the starch-dissolving solvent is selected from one or more of the following solvent systems: aqueous solutions such as DMSO, CaCl2, ZnCl2, LiClO4, Ca(SCN)2, LiSCN, NaOH, KOH, 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 such 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], and [MMIM][MP].

[0337] 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][Cl], [BMIM][Cl], [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][Cl], [EMIM][Ac], [HMIM][Cl], [MMIM][HMIM][Ac], [MMIM][HMIM][Cl], [MMIM][HMIM ... [AMIM][Cl], [BMIM][Cl], [BMIM][Ac], [EMIM][Ac], [BMIM][CH3CH2COO], [BMIM][CH3CH2CH2COO], [BMIM][BF4], [Gly][Cl], [HMIM][Cl], [MMIM][Cl], [Ch][Cl], [Ch][CH3COO], [Ch][CH3CH2COO], [Ch][CH3CH2CH2COO], [Gly][Cl] / [BMIM][Cl] ionic liquids; more preferably, the chitosan-dissolving ionic liquid is preferably selected from: one or more of [AMIM][Cl], [BMIM][Cl], [BMIM][Ac], [EMIM][Ac], [BMIM][CH3CH2COO], [BMIM][CH3CH2CH2COO], [BMIM][BF4], [Gly][Cl], [HMIM][Cl], [MMIM][Cl], [Ch][Cl], [Ch][CH3CH2CH2COO].

[0338] Preferably, the solvent system for dissolving chitin is selected from one or more of the following solvent systems: 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 saturated methanol solution, LiCl, LiSCN, NaOH-urea aqueous solution, and [AMIM][Cl], [AMIM][Br], [AMIM][Ac], [BMIM][Ac], [BMIM][Cl], [MMIM][DMP], [ EMIM][Ac], [EMIM][DMP], [EMIM][Ac], [EMIM][OPr], [EMIM][OBu], [EMIM][Gly], [EMIM][Lys], [HOEMIM][Cl], [THEMA][Ac], [THEMA][MeOSO3], [THEMA][CF3SO3], [PBu4][Val], [PBu4][Lys], [PBu4][Gly], [Ch][Cl] / urea, [Ch][Br] / urea, [Ch][Cl] / thio-urea and other ionic liquids or mixed ionic liquid systems. More preferably, the chitin-dissolving ionic liquid is selected from one or more of: [AMIM][Ac], [BMIM][Ac], [EMIM][Ac], [BMIM][Cl], [AMIM][Cl], [AMIM][Br], [EMIM][OPr], [EMIM][OBu], [Ch][Cl] / urea, [Ch][Br] / urea, and [Ch][Cl] / thio-urea.

[0339] Preferably, the solvent for dissolving lignin is selected from DMSO / TBAF, imidazole / DMSO, and one or more ionic liquids such as [EMIM][Ac], [BMIM][Cl], [BMIM][Br], [AMIM][Cl], [HMIM][CF3SO3], [MMIM][MeOSO3], [BMIM][MeOSO3], [BMMIM][BF4], [BMIM][CF3SO3], [BzMIM][DCA], [MeOBzMIM][Cl], and [MeBzMIM][Cl]. More preferably, the ionic liquid for dissolving lignin is selected from one or more ionic liquids such as [BMIM][Cl], [EMIM][Ac], [BMIM][Br], [AMIM][Cl], [HMIM][CF3SO3], [MMIM][MeOSO3], [BMIM][MeOSO3], and [BMMIM][BF4].

[0340] According to the present invention, 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 that can completely dissolve or partially dissolve biomass polymers (such as cellulose, starch, chitosan, chitin, lignin, hemicellulose, etc.); for example, the mixed ionic liquid can be an ionic liquid that can dissolve biomass polymers (such as cellulose, starch, chitosan, chitin, lignin, hemicellulose, etc.), or it can be a mixture of ionic liquids that can dissolve biomass polymers (such as cellulose, starch, chitosan, chitin, lignin, hemicellulose, etc.) and ionic liquids that do not dissolve biomass polymers (such as cellulose, starch, chitosan, chitin, lignin, hemicellulose, etc.).

[0341] The technical solutions of the present invention will be described in further detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are encompassed within the scope of protection that the present invention is intended to protect.

[0342] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0343] The present invention will be further described below by means of specific embodiments:

[0344] The biomass-based materials described in the following preparation examples and embodiments can also be referred to as micronized cellulose, micronized chitosan, micronized starch, etc., wherein micronized cellulose, micronized chitosan, and micronized starch all include a micron-scale structural portion and a nanoscale structural portion distributed on the micron-scale structure.

[0345] Preparation Example 1

[0346] Preparation of plant hydrogel CMG:

[0347] Premix 12g of wood pulp with the ionic liquid AMIMCl. The wood pulp was oven-dried at 80°C for at least 1 hour. The mass of AMIMCl was 2388g. The premix was heated to 80°C and stirred under vacuum for 2 hours. Once completely dissolved, the solution was slowly poured into warm water at 40±5°C to form a biomass gel. The gel was washed 6-8 times with the solution to remove residual AMIMCl. The gel was then repeatedly washed with water until no precipitation formed upon addition of AgNO3 to the filtrate. The washed biomass gel was redispersed in pure water at a water:biomass gel mass ratio of 9:1. The gel was then crushed using a colloid mill or high-pressure homogenizer to a size of 5-50μm and a CMG solids content of 3.0wt%. After autoclaving, the resulting CMG emulsion, the plant hydrogel, was obtained.

[0348] Preparation Example 2

[0349] Preparation of surface-modified biomass-based dispersions as plant hydrogels (CMG):

[0350] Premix 48g of wood pulp with the ionic liquid AMIMCl. The wood pulp was oven-dried at 80°C for at least 1 hour. The mass of AMIMCl was 2352g. The premix was heated to 80°C and stirred under vacuum for 2 hours. Once completely dissolved, the solution was slowly poured into warm water at 40±5°C to form a biomass gel. The gel was washed 6-8 times with a solution of water, ethanol, DMF, or aqueous NaOH to remove residual AMIMCl. The washed biomass gel was redispersed in pure water at a water:biomass gel mass ratio of 9:1. A surface modifier (biomass gel:surface modifier molar ratio of 1:1) was added to the mixed solution. Suitable surface modifiers include xylitol, citric acid, 4-(2-aminoethyl)benzene-1,2-diol, and sorbitol. The mixture was stirred at a temperature between 0 and 50°C for 24 hours. The gel is then washed 6-8 times with a solution (e.g., water, ethanol, DMF, or aqueous NaOH) to remove any residual surface modifier. The washed, surface-modified biomass gel is dispersed in pure water at a 9:1 weight ratio. The gel is then crushed 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%. After autoclaving, the surface-modified biomass-based dispersion (CMG) is obtained.

[0351] Preparation Example 3

[0352] Preparation of substituted biomass-based dispersions as plant hydrogels (CMG):

[0353] 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.

[0354] Preparation Example 4

[0355] Preparation of multi-component biomass-based dispersion as plant hydrogel CMG:

[0356] 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.

[0357] 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.

[0358] 3. Preparation of a multi-component biomass-based dispersion: The washed biomass gel, polymer hydrogel, and dual-antibody penicillin-streptomycin are dispersed in pure water in a certain ratio (the mass ratio of biomass gel to polymer hydrogel and dual-antibody is 0.1:1:0.1). The gel is then crushed and 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 then sterilized under high pressure to obtain a multi-component biomass-based dispersion CMG.

[0359] Preparation Example 5

[0360] Preparation of multi-component biomass-based dispersion as plant hydrogel CMG:

[0361] 48g of wood pulp was mixed with a polymer and fullerene in a mass ratio of 0.1:1:0.1. This mixture was then premixed with the ionic liquid AMIMCl. The wood pulp was oven-dried at 80°C for at least 1 hour. The mass of AMIMCl was 2352g. The premixed materials were heated to 80°C and stirred under vacuum for 2 hours. The polymer was methylcellulose. After complete dissolution, the solution was slowly poured into warm water at 40±5°C to form a multi-component biomass hydrogel. The gel was washed 6-8 times with a solution (e.g., water) to remove residual AMIMCl. The washed multi-component biomass hydrogel was dispersed in pure water at a mass ratio of 9:1. The gel was then crushed and pulverized using a colloid mill or high-pressure homogenizer to a particle size of 5-50μm and a solids content of 0.1-3.0wt%. After autoclaving, the multi-component biomass-based dispersion (CMG) was obtained.

[0362] Example 1

[0363] The cell culture method is as follows:

[0364] 1) Cell Passaging: Commercially available SH-SY5Y cells were selected and revived, cultured in 10 cm culture dishes, and passaged when a confluency of 80% or greater was achieved. The original culture medium in the culture dish was discarded, and the dish was gently washed twice with PBS. A single-cell suspension was then prepared using trypsinization and centrifuged at 1200 rpm for 5 min at room temperature. The cells were resuspended in basal culture medium (composed of: DMEM / F12, 10% FBS, 1% P / S; this basal culture medium was used in all subsequent examples unless otherwise specified) and adjusted to the desired cell density of 2 × 10^4 cells / μL. This provided SH-SY5Y cells for experimental use and set aside.

[0365] 2) Preparation of plant hydrogel: The CMG emulsion prepared in Preparation Example 1 was diluted with the basal culture medium in step 1) to obtain CMG culture solutions with CMG concentrations of 0.1%, 0.2%, and 0.4%, respectively.

[0366] 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.

[0367] 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.

[0368] 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.

[0369] Microscopic images of SH-SY5Y cells cultured with plant hydrogels of different concentrations for 3 days. Figure 1a-Figure 1c As shown, Figure 1a 、 Figure 1b 、 Figure 1c The corresponding concentrations are 0.1%, 0.2% and 0.4% CMG culture medium respectively.

[0370] Example 2

[0371] 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%.

[0372] 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.

[0373] The culture results are as follows Figure 2a and Figure 2b The results of the culture method using the CMG culture medium and Matrigel 3D of this embodiment are shown in Figure 2a and Figure 2b .

[0374] Depend on Figure 2a and 2b It can be seen that the cell clusters cultured in the two groups have the same morphology and size, but the number of cell clusters in the CMG group is larger.

[0375] Example 3

[0376] 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%.

[0377] 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.

[0378] The culture results are shown in FIG3 . The results of the plant hydrogel and 2D culture methods of this embodiment are shown in FIG3 . Figure 3a and Figure 3b .

[0379] Depend on Figure 3a and 3b It can be seen that when SH-SY5Y cells are cultured in 3D using the plant hydrogel of this embodiment, the proliferation is faster when compared with ordinary 2D culture.

[0380] Comparative Example 1

[0381] 1) A plant hydrogel was prepared by referring to Preparation Example 1, except that the gel was not crushed, thereby obtaining a comparative emulsion, namely the plant hydrogel.

[0382] The control emulsion was diluted with the basal culture medium in Example 1 to obtain a control culture medium 1 having a CMG concentration of 0.2%.

[0383] 2) Referring to the cell culture method of Example 1, the difference is that: SH-SY5Y cells were cultured for 3 days using the comparative culture medium 1 of this comparative example.

[0384] The culture results are as follows Figure 5 When the comparative culture medium after cultured by the culture method of this comparative example was placed under a microscope for observation, it was difficult to observe the cell spheres in the culture medium due to its poor transparency.

[0385] Comparative Example 2

[0386] Aldehyde cellulose nanocrystals were prepared with reference to CN115232781A, and the basic culture medium in Example 1 was used for dilution to obtain a comparative culture medium 2 having an aldehyde cellulose nanocrystal concentration of 0.2%.

[0387] 2) Referring to the cell culture method of Example 1, the difference is that: SH-SY5Y cells were cultured in 3D using the comparative culture medium 2 of this comparative example for 3 days. Figure 6 shown.

[0388] The above results show that the animal-free, ready-to-use plant hydrogel prepared using the natural cellulose gum (CMG) of the present invention is made of nanofibrillar cellulose (NFC), simulates the extracellular matrix (ECM), supports cell growth and differentiation, provides solutions for automation and high-throughput detection, and can be used for 3D cell culture of cell spheroids and organoids, personalized medicine, regenerative medicine, organ-on-a-chip models, and pharmaceutical research. Moreover, compared with the formaldehyde-modified cellulose nanocrystals prepared in CN115232781A, the plant hydrogel prepared by the present invention has a greater number and size of 3D cell spheroids in 3D culture, and has better transparency when observed under a microscope.

[0389] Comparative Example 3

[0390] A CNF hydrogel was prepared with reference to the example of Chinese patent document CN103354834A as a comparative hydrogel, wherein the solid content of CNF was 0.3%. The structural scan of the fiber in the comparative hydrogel can be seen in Figure 1 of Chinese patent document CN103354834A.

[0391] The cell culture method of Example 1 was used, except that the SH-SY5Y cells were cultured in 3D using the comparative hydrogel of this comparative example for 3 days. Figure 7 shown.

[0392] Example 4-1

[0393] The cell culture method of this example is similar to that of Example 1, except that the SH-SY5Y cells in step 1) are replaced with HepaRG (liver cancer cells), and the CMG concentration in the CMG culture medium prepared in step 2) is 0.1%. The results after 7 days of culture are shown in FIG. Figure 8a shown.

[0394] Example 4-2

[0395] The cell culture method of this example is similar to that of Example 1, except that the SH-SY5Y cells in step 1) are replaced with HepaRG (liver cancer cells), and the CMG in step 2) are respectively the surface-modified biomass-based dispersion CMG with a CMG concentration of 0.1% prepared in Preparation Example 2 using xylitol as a surface modifier, the substituted biomass-based dispersion CMG with a CMG concentration of 0.1% prepared in Preparation Example 3 using pyromellitic dianhydride as a substitution reagent, the multi-component biomass-based dispersion CMG with a CMG concentration of 0.1% prepared in Preparation Example 4, and the multi-component biomass-based dispersion CMG with a CMG concentration of 0.1% prepared in Preparation Example 5. The results after 7 days of culture are as follows: Figures 8b-8e shown.

[0396] Comparative Example 4

[0397] Refer to the cell culture method of Comparative Example 3, except that the 3D cultured SH-SY5Y cells were replaced with HepaRG (liver cancer cells). The results after 7 days of culture are as follows: Figure 9 shown by Figure 9 It can be seen that the cell clusters cultured in this comparative example are somewhat scattered and have uneven boundaries.

[0398] It can be seen from the above examples that the method for culturing SH-SY5Y cells and HepaRG cells in 3D suspension culture based on natural cellulose gel (CMG) of the present invention is simple to operate and has a high 3D cell sphere formation rate.

[0399] Example 5

[0400] 1. The formula of the composite culture solution is as follows: the following components are mixed to obtain the composite culture solution:

[0401]

[0402]

[0403] 2. Prepare the tissue to be cultured

[0404] (1) Tissue cleaning

[0405] Transfer the tissue block (gastric cancer organoid, source hospital surgery / biopsy tissue) into a 50 ml centrifuge tube, add 15 ml of washing solution (PBS + 2×P / S), and shake and wash for at least 5 times, each time for 6 minutes, until completely clear.

[0406] (2) Tissue separation

[0407] Transfer the tissue block to a 10 cm culture dish, add a small amount of cleaning solution to ensure the tissue surface is moistened, and use a sterile scalpel and blade to mechanically separate the tissue until it becomes small fragments or a paste of approximately 0.5×0.5×0.5 mm^3.

[0408] (3) Tissue digestion

[0409] The mechanically separated tissue was completely transferred into a 15 ml sterile centrifuge tube. Depending on the amount of tissue, 2-5 ml of tissue digestion solution (collagenase P (1 mg / ml) + DNase (1 mg / ml)) was added. The tissue was gently pipetted with a 1 ml pipette tip to fully disperse it. The centrifuge tube containing the digestion solution was placed on a shaker in a constant temperature air bath at 37°C and 200 rpm. After most of the tissue was separated and digested into 3-10 cell clusters, 10% FBS was added to terminate the digestion.

[0410] (4) Cell filtration

[0411] Filter the cell suspension after digestion through a 100 μm filter into a new 50 ml centrifuge tube. Grind the sample pellet using a 5 ml syringe pump until only white connective tissue remains. Rinse the filter three times with DMEM / F12 (approximately 5 ml per wash). Centrifuge at 4°C, 300 g / 1260 rpm for 5 min.

[0412] (5) Lysis of red blood cells

[0413] If a large number of red blood cells are observed in the cell pellet, perform the following red blood cell lysis step:

[0414] Remove the supernatant and resuspend the cell pellet in 1 ml of red blood cell lysis buffer. Mix thoroughly by pipetting. Place the tube on a shaker at 120 rpm at 4°C for 5 minutes. Centrifuge at 300g / 1260 rpm at 4°C for 5 minutes. Resuspend the cell pellet in 10 ml of Advanced DMEM / F12 and transfer the suspension to a 15 ml centrifuge tube. Centrifuge at 300g / 1260 rpm at 4°C for 5 minutes.

[0415] (6) Cell seeding plate

[0416] Discard the supernatant and retain an appropriate volume (depending on the number of cells, 100,000 cells / well is recommended) of liquid, which is the tissue cell liquid to be cultured.

[0417] 3. Preparation of cellulose-based culture medium

[0418] The emulsion CMG prepared in Preparation Example 1 was diluted with the above composite culture solution to obtain cellulose-based culture solutions with CMG concentrations of 0.8%, 0.4%, 0.2%, and 0.1% (mass percentage concentration), respectively.

[0419] 4. 3D Culture: Gently resuspend the tissue cell suspension from step 2 with the cellulose-based culture medium from step 3 above. Add 300 μl per well of a pre-heated 24-well low-adhesion cell culture plate. Place the plate in a 37°C, 5% CO2 incubator for 10 minutes. Remove the plate and add 700 μl of pre-heated organoid culture medium per well. Incubate the plate in a 37°C, 5% CO2 incubator for 7 days.

[0420] Microscopic images of gastric cancer organoids after 7 days of culture in cellulose-based culture medium with different concentrations. Figure 10a-Figure 10d As shown, Figure 10a 、 Figure 10b 、 Figure 10c 、 Figure 10d The corresponding concentrations are 0.8%, 0.4%, 0.2% and 0.1% cellulose-based culture medium respectively.

[0421] Example 6

[0422] The composite culture solution in Example 5 was used to dilute the CMG emulsion prepared in Preparation Example 1 to obtain a cellulose-based culture solution with a CMG concentration of 0.2%.

[0423] The culture method of Example 5 was used with the exception that the cellulose-based culture medium with a CMG concentration of 0.2% and commercially available Matrigel 3D (manufacturer: Corning, model: 356231) were used to culture gastric cancer organoids for 7 days.

[0424] The culture results are as follows Figure 11a and Figure 11b The results of the culture method using the CMG culture medium and Matrigel 3D of this embodiment are shown in Figure 11a and Figure 11b .

[0425] Depend on Figure 11a and 11b It can be seen that the walls of the organoid vesicles cultured in the CMG group are thicker and solid, indicating that they are differentiated and mature organoids, while the walls of the gastric cancer organoid vesicles cultured in the Matrigel group are uneven in thickness.

[0426] Example 7-1

[0427] The culture method of this example refers to that of Example 5, except that the tissue to be cultured in step 2 is replaced with intestinal cancer organoid culture tissue, and the CMG concentration in the cellulose-based culture medium prepared in step 3 is 0.2%.

[0428] The culture results are as follows Figure 12 As shown, CMG-cultured organoids are mature intestinal organoids containing multiple typical complex crypt-shaped structures. These crypt structures are interconnected to form a mini intestine with an intestinal lumen in the middle.

[0429] Example 7-2

[0430] The operation is the same as that in 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 a surface-modified biomass-based dispersion CMG with a CMG concentration of 0.2% prepared by using the surface modifier xylitol in Preparation Example 2, a substituted biomass-based dispersion CMG with a CMG concentration of 0.2% prepared by using the substitution reagent pyromellitic dianhydride in Preparation Example 3, a multi-component biomass-based dispersion CMG with a CMG concentration of 0.2% prepared by Preparation Example 4, and a multi-component biomass-based dispersion CMG with a CMG concentration of 0.2% prepared by Preparation Example 5; the micrograph after culturing intestinal cancer organoids for 7 days is shown in FIG. Figure 13-16 shown.

[0431] Comparative Example 5

[0432] In this comparative example, a cellulose-based culture medium was prepared by referring to Example 5, except that the emulsion CMG prepared in Preparation Example 1 was replaced with plant cellulose matrix glue GrowDex (UPM / 100103005) with a mass percentage concentration of 0.2%, which was recorded as comparative culture medium 1.

[0433] Referring to step 4 of Example 5, gastric cancer tissues were cultured using the cellulose-based culture medium with a CMG concentration of 0.2% (mass percentage concentration) of Example 5 and the comparative culture medium 1 of this comparative example, respectively, and recorded as the 0.2% CMG cellulose matrix glue group and the plant cellulose matrix glue GrowDex group. The results after 7 days of culture are as follows: Figure 17 shown.

[0434] Depend on Figure 17 As shown, compared with the 0.2% CMG and GrowDex groups, gastric cancer organoids cultured in the cellulose-based medium of the present invention exhibited thicker, solid vesicle walls. In contrast, the gastric cancer organoids cultured in the medium prepared with GrowDex Matrigel (this comparative example) exhibited severe vesicle formation and a poorly differentiated state. Therefore, the cellulose-based medium of the present invention is superior for culturing gastric cancer organoids.

[0435] Comparative Example 6

[0436] In this comparative example, cellulose-based culture medium was prepared and gastric cancer tissue was cultured according to Example 5, except that the epirregulin content in the culture medium was different, including a culture medium without epirregulin, i.e., a group without epirregulin, a group with epirregulin 200 ng / ml, and a group with epirregulin 500 ng / ml. The results after 7 days of culture are as follows: Figure 18 shown.

[0437] Depend on Figure 18 The results show that compared with the group without epirregulin, the gastric cancer organoid spheres in the 200 ng / ml epirregulin group were larger and more numerous, with thicker, solid vesicle walls, indicating a significantly superior performance. Therefore, epirregulin is a key component of the culture medium. However, there was no significant difference between the 200 ng / ml epirregulin group and the 500 ng / ml epirregulin group. Therefore, the optimal epirregulin concentration is 200 ng / ml.

[0438] Comparative Example 7

[0439] This comparative example was carried out with reference to comparative example 5, except that the tissue to be cultured was replaced with intestinal cancer organoid culture tissue.

[0440] The results after 7 days of culture in plant cellulose matrix gel GrowDex group are as follows Figure 19 As shown, the results after 7 days of culture with 0.2% CMG cellulose matrix gel group (Example 7-1 Figure 12 ), the intestinal cancer organoids cultured using the cellulose-based culture medium of the present invention have advantages in size and structural complexity.

[0441] It can be seen from the above examples that the tumor organoid 3D culture kit and culture method based on natural cellulose gel (CMG) of the present invention are simple to operate and can obtain differentiated and mature organoids.

[0442] Example 8 Human induced pluripotent stem cell / embryonic stem cell culture

[0443] 1) Before passaging the stem cells, dilute the plant hydrogel CMG to 0.8% (mass percentage concentration) with DMEM / F12, add 1 ml / well to a 6-well plate, and place in a 37°C incubator for at least 1 hour.

[0444] 2) Stem cell passage: The cells in the culture dish are about 85-90% confluent and have basically not differentiated. TM After aspirating 1 (STEMCELL, 85850), each well was covered with 1 ml of Dispase (1 mg / ml) and placed in a 37°C incubator until the edge of the stem cells began to lift, and the Dispase was aspirated.

[0445] 3) Wash three times with DPBS, 1 ml each time.

[0446] 4) Add 3 ml of preheated mTeSR TM 1. Add the cells to the wells and gently pipette until the stem cells fall off the well plate. Repeat the pipetting until the cell clusters are broken down into 1-2 mm fragments.

[0447] 5) The stem cell fragments were added to the CMG plate covered with plant hydrogel in step 1) in a ratio of 1:1 to 1:6, and the mTeSR TM 1. Fill the volume to 2 ml with culture medium (STEMCELL, 85850). Gently tap the side of the 6-well culture dish 5 to 10 times to ensure that the stem cell colonies are evenly distributed in the air. Incubate the culture in a 37°C, 5% CO2 incubator, changing the medium daily.

[0448] Example 9: Using the Plant Hydrogel from Preparation Example 1 to Induce Differentiation of Human Pluripotent Stem Cells / Embryonic Stem Cells into Intestinal Organoids

[0449] 1. iPSC / ES cell differentiation in monolayer culture

[0450] To perform differentiation in a TC-treated 24-well cell culture plate, dilute the plant hydrogel CMG from Preparation Example 1 to 0.8% (mass percentage concentration) in DMEM / F12, add 0.3 ml / well to the 24-well plate, and incubate in a 37°C incubator for at least 1 hour. Before initiating differentiation, assess the cell differentiation rate and starting density. At this point, the cell differentiation rate should be less than 5% and the starting density should be 85-90%.

[0451] (1) Definitive endoderm differentiation

[0452] A. Preparation of Definitive Endoderm Differentiation Medium

[0453] Day 0: Prepare 0.5 mL of definitive endoderm differentiation medium (RPMI 1640, containing L-glutamine (final concentration 2 mM), penicillin-streptomycin (final concentration 100 U / ml-100 g / ml), and Activin A (final concentration 100 ng / ml)) required for days 0, 1, and 2 for each culture well.

[0454] B. Definitive endoderm differentiation

[0455] 1) Day 0: Preheat the definitive endoderm differentiation medium (0.5 mL / well) required for day 0 at 37°C. Store the remaining medium at 2-8°C. Aspirate the mTeSR in the culture wells. 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.

[0456] 2) Day 1: Preheat the definitive endoderm differentiation medium (0.5 mL / well) at 37°C. Aspirate the medium from the wells and add 0.5 mL of the medium dropwise along the well walls. Incubate at 37°C, 5% CO2, and 95% humidity for 24 hours.

[0457] 3) Day 2: Aspirate the culture medium from the culture wells and add 0.5 mL of 37°C preheated definitive endoderm differentiation medium dropwise along the well wall. Incubate at 37°C, 5% CO2, and 95% humidity for 24 hours.

[0458] Note: During endoderm induction, cells undergo extensive cell death. Minimize the time cells spend outside the 37°C incubator. Within 24 hours of endoderm induction, cells are very sensitive and require careful medium changes during culture. After 72 hours of incubation, a dense, confluent monolayer of endoderm cells will form.

[0459] (2) Midgut / hindgut (MH) differentiation

[0460] A.MH differentiation medium

[0461] Day 3: Prepare MH differentiation medium (RPMI1640 containing 2% FBS, L-glutamine (final concentration 2 mM), penicillin-streptomycin (final concentration 100 U / ml-100 g / ml), FGF4 (final concentration 500 ng / ml) required for days 3-8, 0.5 mL per culture well.

[0462] B. Midgut / Hindgut (MH) Differentiation

[0463] Day 3: Warm an adequate volume of MH Differentiation Medium (0.5 mL / well) to room temperature (15–25°C). Aspirate the medium from the wells and add 0.5 mL of Midgut / Hindgut Differentiation Medium dropwise along the well walls. Incubate at 37°C, 5% CO2, and 95% humidity for 24 hours.

[0464] 2) Days 4-9: Replace the medium completely and observe the spheroids every 24 hours according to the following method.

[0465] NOTE: Ensure that cultures are returned to the incubator within 30 minutes of removal.

[0466] a. Observe the monolayer under a microscope. Three-dimensional structures may appear as early as day 4 of differentiation. Free-floating mid / hindgut spheroids will appear between days 6-9 of differentiation.

[0467] b. Using a 1 mL pipette, remove 0.5 mL of culture medium from the cells and transfer to a sterile 24-well flat-bottom clear culture plate to assess the number and concentration of mid / hindgut spheroids extracted from the monolayer.

[0468] c. Add 0.5 mL of fresh MH medium to the cells and incubate at 37°C, 5% CO2, and 95% humidity for 24 hours.

[0469] Note: Although spheres released from days 6-9 can generate intestinal organoids, the length of time cells are cultured in mid / hindgut culture will determine the regional characteristics of the intestinal organoid development. For example, the duodenum (shorter culture time) or ileum (longer culture time). The time at which the highest yield of mid / hindgut spheres occurs may vary between hPSC cell lines. For reproducible experimental results, use mid / hindgut spheres differentiated at the same time point to consistently harvest and initiate human intestinal organoid culture.

[0470] 3) Spheroid Embedding: Use a pipette rinsed with 0.5% BSA to add the spheroid suspension from each well to one well of a 24-well plate for counting. Add an appropriate volume to a 15 mL centrifuge tube, corresponding to approximately 50 suspended spheres (based on the previous spheroid count).

[0471] A mid / hindgut spheroid is a cell aggregate ≥75 μm in diameter that can potentially form a human intestinal organoid. Multiple fused spheroids should be counted as one unit and will form a human intestinal organoid.

[0472] The remaining monolayer culture can be used to assay mid / hindgut formation or to study further differentiation of chimeric spheroids in subsequent days.

[0473] 2. Human intestinal organoid culture

[0474] (1) Initial culture of human intestinal organoids

[0475] A. Preparation of Intestinal Organoid Growth Medium

[0476] 1) Culture in a 24-well low-adhesion culture plate. Prepare 4 wells (0.5 mL / well) of intestinal organoid growth medium (Advanced DMEM / F12 supplemented with 1× B27, L-glutamine (final concentration 2 mM), penicillin-streptomycin (final concentration 100 U / ml-100 μg / ml), HEPES buffer (final concentration 15 mM), and R-spondin 1 (final concentration 500 ng / ml).

[0477] B. Plant hydrogel CMG mixed with spheres

[0478] 1) Centrifuge the spheroid suspension collected during midgut / hindgut (MH) differentiation described in step B at 300×g for 5 minutes. Carefully remove the supernatant after centrifugation.

[0479] 2) Add 1 mL of DMEM / F-12 + 15 mM HEPES to the spheroids. Centrifuge at 300 × g for 5 minutes at room temperature (15-25°C).

[0480] 3) Carefully remove the supernatant using a 1 mL pipette tip.

[0481] 5) Add 100 μL of 0.3% plant hydrogel CMG prepared in Preparation Example 1 to the centrifuge tube. Pipet up and down 5 times to gently distribute the spheroids into the plant hydrogel CMG.

[0482] NOTE: Do not completely empty the pipette tip to prevent excessive air bubbles.

[0483] 6) Using the same pipette tip, gently transfer the embedded spheroid to the center of a well of a 24-well tissue culture dish.

[0484] 7) Incubate in a 37°C incubator for 10-25 minutes.

[0485] 8) Warm a sufficient volume of intestinal organoid growth medium to (15-25°C). Store the remaining medium at 2-8°C.

[0486] 9) Carefully add at least 0.5 mL / well of intestinal organoid growth medium along the sides of the culture wells. Incubate at 37°C, 5% CO2, and 95% humidity.

[0487] 10) Change the culture medium every 3-4 days by aspirating the old medium and adding fresh medium. Incubate at 37°C, 5% CO2, and 95% humidity.

[0488] 11) After 10-14 days of incubation, passage the organoids depending on their growth.

[0489] The micrographs of intestinal organoids after 3, 7, and 12 days of induction differentiation of human pluripotent stem cells / embryonic stem cells in this example are as follows: Figure 20a-Figure 20c shown.

[0490] Example 10

[0491] The operation was the same as in Example 9. The xylitol surface-modified biomass-based dispersion CMG prepared in Preparation Example 2, the substituted biomass-based dispersion CMG in which the substitution reagent was pyromellitic dianhydride prepared in Preparation Example 3, the multi-component biomass-based dispersion CMG in which the polymer compound was methyl cellulose prepared in Preparation Example 4, and the multi-component biomass-based dispersion CMG in which the polymer compound was methyl cellulose prepared in Preparation Example 5 were respectively subjected to human pluripotent stem cells / embryonic stem cells for intestinal organoid differentiation. The micrograph of the intestinal organoid cultured after 12 days is shown in FIG. Figures 21-24 shown.

[0492] Comparative Example 8

[0493] Referring to the culture method of Example 8, 0.8% of the plant hydrogel CMG and Matrigel (Corning 354277) of Preparation Example 1 were used to culture iPSCs. The results are as follows: Figure 25a 、 25b shown.

[0494] Both groups showed clear colony edges, uniform cell size, and a high nuclear-cytoplasmic ratio, indicating that the stem cells were in good condition and appeared undifferentiated. This suggests that the plant hydrogel CMG and Matrigel are comparable in their performance in culturing stem cells.

[0495] Comparative Example 9

[0496] Referring to the culture method of Example 9, the intestinal organoid differentiation induction kit containing 0.3% plant hydrogel CMG of Preparation Example 1 and STEMdiff were used respectively. TM The micrograph of intestinal organoids 12 days after differentiation of human pluripotent stem cells / embryonic stem cells was shown in Figure 1. Figure 26a 、 26bshown.

[0497] Comparing the two groups, both were able to culture mature intestinal organoids containing multiple crypt structures that interconnected to form a mini-intestine with a central intestinal lumen. The kit of the present invention even outperformed the control kit, producing intestinal organoids with a more typical and complex crypt structure.

[0498] The above describes exemplary embodiments of the present invention. However, the scope of protection of this application is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc. made by those skilled in the art within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A plant-based culture solution, characterized in that The culture solution comprises a plant gel and a base culture solution; wherein the plant gel does not contain animal-derived components and comprises micro-nano biomass and a dispersing agent; the dispersing agent is selected from an organic solvent and / or water, preferably water and / or alcohol; Preferably, the culture solution comprises plant hydrogel and basic culture solution; wherein, the plant hydrogel has no 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 the culture solution includes a plant hydrogel and a basic culture solution A; wherein the plant hydrogel does not contain animal-derived components and includes micro-nano biomass and water; Alternatively, the culture medium is used for 3D culture of tumor organoids, and the culture medium comprises a plant hydrogel and a composite culture medium; wherein the plant hydrogel has no animal-derived components and comprises micro-nano biomass and water; Alternatively, the culture medium is used for stem cell culture, and the culture medium includes plant hydrogel and basic culture medium C; wherein the plant hydrogel does not contain animal-derived components and includes micro-nano biomass and water.

3. The method for preparing the culture solution according to claim 2, wherein The method for preparing a culture medium for 3D cell culture comprises the following steps: S1: Preparation of plant hydrogel; S2: mixing the plant hydrogel prepared in step S1 with basic culture medium A to prepare a culture medium for 3D cell culture; Alternatively, the method for preparing a culture medium for 3D culture of tumor organoids comprises the following steps: S1': preparing the above plant hydrogel; S2': mixing the plant hydrogel prepared in step S1' with the composite culture medium to prepare a culture medium for 3D culture of tumor organoids; Alternatively, the method for preparing a culture medium for stem cell culture comprises the following steps: S1”: Preparation of plant hydrogel; S2": The plant hydrogel prepared in step S1 is mixed with the basic culture medium C to prepare a culture medium for stem cell culture.

4. The use of the culture solution according to claim 1 or 2, characterized in that Used for the culture of cells, tissues or organoids; specifically, the application is the use of a culture medium for 3D cell culture in 3D cultured cells; or, the application of a culture medium 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 a culture medium for stem cell culture in the culture of stem cells or organoids.

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

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

7. Use of the kit according to claim 6 in cell culture, tissue culture or organoid culture; Specifically, the use of the 3D cell culture kit in 3D cell culture; or, the use of the tumor organoid 3D culture kit in 3D tumor organoid culture.

8. A method for culturing tumor tissues or organoids of gastric cancer, lung cancer, etc., characterized in that: The method is carried out in the culture medium of claim 1 or 2, specifically in a culture medium for 3D culture of tumor organoids.

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

10. A culture medium for inducing stem cell differentiation into organoids, characterized in that: The culture medium includes plant hydrogel, definitive endoderm differentiation culture medium, MH differentiation culture medium, and organoid growth culture medium; wherein the plant hydrogel does not contain animal-derived components and includes micro-nano biomass and water.

11. The use of the stem cell-induced differentiation organoid culture medium according to claim 10, characterized in that: Used to induce stem cell differentiation into organoids.

12. A method for inducing stem cell differentiation into organoids, characterized in that: The method comprises culturing a single stem cell or a stem cell group in the stem cell differentiation-inducing organoid culture medium according to claim 10.

13. A stem cell organoid prepared by the method for inducing stem cell differentiation into organoids according to claim 12.

14. A kit for inducing differentiation of organoids, characterized in that: The kit comprises the stem cell-induced differentiation organoid culture medium according to claim 10.

15. Use of the kit for inducing differentiation of organoids according to claim 14 in inducing differentiation of organoids.

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