Temperature-sensitive cell three-dimensional culture support and use method thereof

Abstract

The invention belongs to the technical field of cell culture, and particularly relates to a temperature-sensitive type three-dimensional cell culture scaffold which comprises temperature-sensitive type hydroxypropyl chitin, the molecular weight of the temperature-sensitive type hydroxypropyl chitin is 15,000-300,000 Da, and the degree of deacetylation (DD) of the temperature-sensitive type hydroxypropyl chitin is 35%-65%. The scaffold material is thermo-sensitive hydroxypropyl chitin prepared by derivatizing chitosan from a natural source through a specific process, and can be used for carrying out three-dimensional culture on cells. When the temperature-sensitive hydroxypropyl chitin is used for participating in three-dimensional culture of cells, the culture cost can be remarkably reduced, the use convenience is improved, and the activity of the cultured cells is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell culture, and particularly relates to a preparation method and a usage method of a thermosensitive aminopolysaccharide derivative applied to three-dimensional cell culture. Background Art

[0002] Cell culture is an important means for life science, medical, and pharmaceutical research. Traditional cell culture is a two-dimensional culture method marked by cell adhesion growth. However, there are still significant differences between two-dimensional cell culture and the overall growth state, structure, metabolism, and response to external stimuli of cells growing in vivo. In order to simulate the real growth environment of cells in vivo, three-dimensional cell culture has emerged.

[0003] Three-dimensional cell culture can provide three-dimensional growth conditions for cells, which is closer to the in vivo growth environment of cells. It can create a three-dimensional physiological site for cells and tissues to evenly obtain nutrients, conduct gas exchange, and discharge waste. Moreover, three-dimensional cell culture is conducive to the formation of tissue organs with reasonable morphology and physiological functions by cells and tissues. Currently, the matrix gel used in three-dimensional cell culture mainly comes from the soluble basement membrane matrix extract of Engelbreth-Holm-Swarm (EHS) mouse sarcoma, and its main components are laminin, type IV collagen, heparan sulfate proteoglycan (HSPG), nidogen, and various growth factors. The advantages of this kind of matrix gel are rich nutrition, which can provide sufficient nutrition for three-dimensional cell growth. However, the disadvantages are also obvious: the raw material source is scarce, the production and preparation cost is high, and the price is expensive; the finished product needs to be stored at low temperature throughout the process, and the transportation and storage are inconvenient; it only has a one-way thermosensitivity from liquid to solid, which affects the convenience of use. Therefore, it is urgent to develop a new three-dimensional cell culture scaffold.

[0004] Chitin and its derivatives have attracted the attention of many researchers because of their non-toxicity, biodegradability, good biocompatibility, excellent antibacterial properties, and other biological activities. They have been widely studied and prepared into new functional biomaterials and applied in the biomedical field. Hydroxypropyl chitin is a relatively common derivative among them. It is a kind of aminopolysaccharide derivative formed by introducing hydroxypropyl groups onto the hydroxyl groups of chitin. It has excellent water solubility, biodegradability, biocompatibility, and good thermo-induced gelation property, and can spontaneously form a gel at a relatively high temperature. The hydrogel formed by it consists of a three-dimensional hydrophilic network with a high water content, which is beneficial to the transportation of oxygen, nutrients, and other water-soluble metabolites. Because of its structural characteristics similar to the natural extracellular matrix and its ability to provide a suitable environment for cell growth, its application effect in three-dimensional cell culture is not very ideal at present, so it needs to be improved. Summary of the Invention

[0005] In view of the deficiencies of the prior art, one of the objectives of the present invention is to provide a temperature-sensitive three-dimensional cell culture scaffold.

[0006] The first aspect of the embodiment of the present invention provides a temperature-sensitive three-dimensional cell culture scaffold, which is characterized in that the temperature-sensitive three-dimensional cell culture scaffold includes temperature-sensitive hydroxypropyl chitosan, the molecular weight of the temperature-sensitive hydroxypropyl chitosan is 15,000 - 300,000 Da, and the degree of deacetylation (DD) is 35% - 65%. The preparation method of the hydroxypropyl chitosan includes:

[0007] S1. Using chitosan with a degree of deacetylation ≥ 85% as a raw material, reacting with propylene oxide at 0 - 5°C under alkaline conditions to obtain hydroxypropyl chitosan;

[0008] S2. Reacting the product in step S1 with acetic anhydride at low temperature under alkaline conditions to form a temperature-sensitive hydroxypropyl chitosan solution. The solution is dialyzed with triple-distilled water to remove salt particles in the solution, and then eluted with ethanol at 0 - 5°C and freeze-dried to obtain hydroxypropyl chitosan.

[0009] Further, the temperature-sensitive three-dimensional cell culture scaffold further includes a complete cell culture medium. The temperature-sensitive hydroxypropyl chitosan and the complete cell culture medium are mixed to form a composition with a hydroxypropyl chitosan concentration of 0.5% - 5%. The composition has a phase transition temperature of 10 - 15°C. When the temperature of the composition is lower than the phase transition temperature, the composition is in a liquid state; when the temperature of the composition is higher than the phase transition temperature, the composition is in a solid state.

[0010] Further, most of the free amino groups in the structure of the hydroxypropyl chitosan are blocked by acetyl groups, reducing the number of positive charges carried in the molecule, which is beneficial to the survival and amplification of cells.

[0011] Further, the alkaline condition is achieved by using an alkaline solution, and the alkaline solution includes but is not limited to a sodium hydroxide solution or a potassium hydroxide solution with a mass concentration fraction of 10% - 20%.

[0012] Further, in step S1, the mass ratio of chitosan to propylene oxide is 1:5 - 1:15, and the reaction is stirred at 0 - 25°C for 48 - 72 hours to make the degree of hydroxypropyl substitution (DS) (the number of hydroxypropyl groups on the glucosamine residue of chitosan: glucosamine residue) between 0.8 and 1.2.

[0013] Further, before adding propylene oxide in step S1, isopropanol needs to be added and stirred at 0 - 25°C for 24 - 48 h. The mass ratio of chitosan to isopropanol is 1:8 - 1:12.

[0014] Further, in step S2, the mass ratio of hydroxypropyl chitosan to acetic anhydride is between 2:1 and 2:2.

[0015] Furthermore, the three-dimensional cell culture scaffold further comprises a complete cell culture medium.

[0016] Furthermore, the composition formed by mixing the complete cell culture medium with the hydroxypropyl chitosan and having a hydroxypropyl chitosan concentration of 0.5%-5% has temperature sensitivity.

[0017] Furthermore, the three-dimensional cell culture scaffold further comprises a nutrient factor for cell culture and differentiation.

[0018] Furthermore, the cells include stem cells, differentiated cells, tumor cells, primary cells, and passage cells of all animal species.

[0019] Furthermore, the cells include, but are not limited to, mesenchymal stem cells (MSCs), induced pluripotent stem cells (iPSCs), nerve cells, cardiomyocytes, osteoblasts, chondrocytes, islet cells, and various cells and cell clusters cultured into organoids by gene editing modification.

[0020] The present invention also provides the application of the above temperature-sensitive three-dimensional cell culture scaffold in the field of three-dimensional cell culture. The present invention also provides a three-dimensional cell culture carrier, comprising the aforementioned three-dimensional cell culture scaffold.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] 1. The raw materials of the amino polysaccharide derivative provided by the present invention are rich in source, low in production and manufacturing costs, and low in price;

[0023] 2. The three-dimensional cell culture scaffold provided by the present invention can freely add and mix different growth factors according to the needs of different cell cultures; before use, it only needs to be stored at room temperature, and the transportation and storage are convenient; it has the characteristics of bidirectional temperature sensitivity in solid and liquid states, is convenient to use, and has a wider application range;

[0024] 3. The three-dimensional cell culture method provided by the present invention can promote the 3D spheroid growth of cells, enhance the cell proliferation ability, maintain the survival ability of stem cells for a long time, and enhance the cell activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is a micro-pore size structure diagram of the temperature-sensitive hydroxypropyl chitosan aqueous solution provided by the embodiment of the present invention.

[0026] Figure 2 It is the culture result of MSC by mixing different concentrations of temperature-sensitive hydroxypropyl chitosan and MSC cell complete culture medium provided by the embodiment of the present invention.

[0027] Figure 3This is a diagram showing the growth state of stem cells after mixing a temperature-sensitive hydroxypropyl chitosan solution provided by an embodiment of the present invention with an MSC cell complete medium.

[0028] Figure 4 This is a comparison diagram of the survival time of stem cells between a cell culture system supplemented with temperature-sensitive hydroxypropyl chitosan and an MSCs-3D blank culture system on the 3rd day.

[0029] Figure 5 This is the Q-PCR detection result of the expression levels of various cell secretion factor mRNAs in a cell culture system supplemented with temperature-sensitive hydroxypropyl chitosan. Detailed implementation manners

[0030] In order to make the technical problems to be solved, technical solutions and beneficial effects of the present invention clearer and more understandable, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0031] An embodiment of the present invention provides a temperature-sensitive hydroxypropyl chitosan with a molecular weight of 15,000 - 300,000 Da and a deacetylation degree of 35% - 65%. The preparation method of the hydroxypropyl chitosan includes:

[0032] S1. Using chitosan with a deacetylation degree ≥ 85% as a raw material, reacting with propylene oxide at 0 - 25°C under alkaline conditions to obtain hydroxypropyl chitosan;

[0033] S2. Reacting the product in step S1 with acetic anhydride at low temperature under alkaline conditions to generate a temperature-sensitive hydroxypropyl chitosan solution. The solution is dialyzed with triple-distilled water to remove salt particles in the solution, and then eluted with ethanol at 0 - 25°C and freeze-dried to obtain hydroxypropyl chitosan.

[0034] Chitin is a natural basic polysaccharide without biological toxicity, mainly existing in the shells of organisms such as shrimps and crabs. It has a huge reserve in nature, good biocompatibility and biodegradability, and no immunogenicity, and is an excellent biological resource. However, due to the high crystallinity of chitin itself due to hydrogen bond action, it is very difficult to dissolve in water and low-concentration acid and alkali solutions, and is not easily soluble in common organic solvents, so it cannot be well utilized. Based on this, an embodiment of the present invention designs and synthesizes a new chitin derivative. Due to the introduction of hydroxypropyl, the intramolecular hydrogen bonds and intermolecular hydrogen bonds inherent in the chitin material are damaged to a certain extent, making hydroxypropyl chitosan have certain water solubility.

[0035] Specifically, most of the free amino groups in the structure of the hydroxypropyl chitosan are blocked by acetyl groups, reducing the number of positive charges carried in the molecule, which is beneficial to the survival and amplification of cells.

[0036] Specifically, the preparation process of the hydroxypropyl chitin includes:

[0037] S1. Using chitosan as the main raw material, reacting with propylene oxide under low-temperature and alkaline conditions to obtain hydroxypropyl chitosan;

[0038] S2. Reacting the product in step S1 with acetic anhydride under low-temperature and alkaline conditions to generate a temperature-sensitive hydroxypropyl chitin solution. The solution is dialyzed with triple-distilled water to remove salt particles in the solution, and then eluted with ethanol at low temperature and freeze-dried to obtain pure solid temperature-sensitive hydroxypropyl chitin.

[0039] Specifically, the chitosan can be all commercially available chitosans with DD≥85%. The low-temperature condition refers to 0-25°C. The alkaline condition is achieved using an alkaline solution, and the alkaline solution includes but is not limited to a sodium hydroxide solution or a potassium hydroxide solution with a mass concentration fraction of 10%-20%.

[0040] Specifically, in step S1, the mass ratio of chitosan to propylene oxide is 1:5-1:15, and the stirring reaction is carried out at 0-25°C for 48-72 hours to make the degree of hydroxypropyl substitution (DS) (the number of hydroxypropyl groups on the chitosan glucosamine residue: glucosamine residue) between 1 and 1.2.

[0041] Specifically, before adding propylene oxide in step S1, isopropanol also needs to be added and stirred at room temperature for 24-48h. The mass ratio of chitosan to isopropanol is 1:8-1:12.

[0042] Specifically, in step S2, the mass ratio of hydroxypropyl chitosan to acetic anhydride is between 2:1 and 2:2.

[0043] The preparation method provided by the embodiments of the present invention can prepare hydroxypropyl chitins with different molecular weights and degrees of deacetylation by selecting chitosan substances with different molecular weights, degrees of hydroxylation, and degrees of deacetylation, and further regulate the phase transition point temperature of hydroxypropyl chitin for use in different needs.

[0044] The second aspect of the present invention provides a three-dimensional cell culture scaffold.

[0045] Three-dimensional cell culture refers to using methods such as scaffolds, gels, or suspension culture to enable cells to grow in a three-dimensional environment, thereby forming a structure similar to in vivo tissues. Three-dimensional cell culture can more realistically reproduce the interactions between cells and between cells and the extracellular matrix, more accurately simulate the actual microenvironment of cells in tissues, the cell behavior characteristics are closer to the survival state in vivo, and it is better applied to research fields such as new drug screening, tumor cell systems biology, stem cell research, functional tissue implantation, and other cell analysis.

[0046] Specifically, the three-dimensional cell culture scaffold provided by the embodiments of the present invention further includes a complete cell culture medium.

[0047] After the complete cell culture medium is mixed with the hydroxypropyl chitin, a composition with a hydroxypropyl chitin concentration of 0.5%-5% has temperature sensitivity. This composition solution has a phase transition temperature, which is 10-15°C; when the overall temperature of the solution is lower than 10-15°C, it is in a liquid state, and when the solution temperature is higher than 10-15°C, it is in a gel state.

[0048] Specifically, the three-dimensional cell culture scaffold further contains nutrient factors for cell culture and differentiation.

[0049] Specifically, the cells include stem cells, differentiated cells, tumor cells, primary cells, and passage cells of all animal species.

[0050] Specifically, the cells include, but are not limited to, mesenchymal stem cells (MSCs), induced pluripotent stem cells (iPSCs), nerve cells, cardiomyocytes, osteoblasts, chondrocytes, islet cells, and various cells and cell clusters cultured into organoids through gene editing modification, etc.

[0051] The present invention also provides a three-dimensional cell culture method using the aforementioned amino polysaccharide derivative or three-dimensional cell culture scaffold.

[0052] The following describes the present invention in detail with specific embodiments.

[0053] Example 1 Preparation of Hydroxypropyl Chitin

[0054] S1. Slowly add chitosan powder to an excessive sodium hydroxide solution with a mass-volume concentration of 10%, stir until the chitosan is fully dispersed in the aqueous solution, freeze overnight, add isopropanol (mass ratio of chitosan to isopropanol 1:12) after dissolution, stir at 10°C for 1.5 h, then slowly add propylene oxide (mass ratio of chitosan to propylene oxide 1:5), stir and react at 25°C for 48 hours, control the time and temperature to make the degree of hydroxypropyl substitution (DS) (the number of hydroxypropyl groups on the chitosan glucosamine residue: glucosamine residue) between 1 and 1.2, adjust the pH to neutral with dilute hydrochloric acid, precipitate the product with an organic solvent, filter by suction, and wash several times with absolute ethanol to obtain hydroxypropyl chitosan.

[0055] S2. Dissolve the hydroxypropyl chitosan obtained in S1 in an excessive amount of methanol aqueous solution (methanol: water = 1:1), add acetic anhydride, so that the mass ratio of hydroxypropyl chitosan to acetic anhydride is between 2:1 - 2:2. Under the condition of 15 °C, stir to completely dissolve it and let it stand overnight. After the reaction is completed, adjust the pH to neutral with dilute sodium hydroxide solution, dialyze at 4 °C with a triple-distilled water dialysis bag to remove salt ions, precipitate the dialyzed product with an organic solvent, filter by suction, wash several times with absolute ethanol, and vacuum freeze-dry to obtain hydroxypropyl chitin.

[0056] Example 2 Preparation of Hydroxypropyl Chitin

[0057] S1. Slowly add chitosan powder to an excessive amount of sodium hydroxide solution with a mass-volume concentration of 20%. After stirring until the chitosan is fully dispersed in the aqueous solution, freeze overnight. After dissolution, add isopropanol (the mass ratio of chitosan to isopropanol is 1:8), stir at 4 °C for 1 h, then slowly add propylene oxide (the mass ratio of chitosan to propylene oxide is 1:15), and stir and react at 10 °C for 72 h. Control the time and temperature to make the degree of substitution of hydroxypropyl (DS) (the number of hydroxypropyl groups on the chitosan glucosamine residue: glucosamine residue) between 1 - 1.2. Adjust the pH to neutral with dilute hydrochloric acid, precipitate the product with an organic solvent, filter by suction, wash several times with absolute ethanol to obtain hydroxypropyl chitosan.

[0058] S2. Dissolve the hydroxypropyl chitosan obtained in S1 in an excessive amount of methanol aqueous solution (methanol: water = 1:1), add acetic anhydride, so that the mass ratio of hydroxypropyl chitosan to acetic anhydride is between 2:1 - 2:2. Under the condition of 10 - 15 °C, stir to completely dissolve it and let it stand overnight. After the reaction is completed, adjust the pH to neutral with dilute sodium hydroxide solution, dialyze at 4 °C with a triple-distilled water dialysis bag to remove salt ions, precipitate the dialyzed product with an organic solvent, filter by suction, wash several times with absolute ethanol, and vacuum freeze-dry to obtain hydroxypropyl chitin.

[0059] Example 3 Preparation of Hydroxypropyl Chitin Hydrogel and Identification of Its Scaffold Structure

[0060] At 4 °C, weigh the hydroxypropyl chitin powder prepared in Example 1, dissolve it in water, and prepare hydroxypropyl chitin solutions with mass concentrations of 1%, 1.5%, 2% and 4% respectively. Heat up to 37 °C to solidify it into a gel state, and observe its structure under a scanning electron microscope. The results are as Figure 1 shown. The hydroxypropyl chitin gels of all concentrations are three-dimensional porous structures, and among them, the gel with a mass concentration of 1.5% has the most uniform pore distribution, which is more conducive to the exchange of nutrients and metabolic wastes.

[0061] Example 4 Three-dimensional Culture of Mesenchymal Stem Cells MSC

[0062] S1. Prepare the corresponding complete cell culture medium in advance according to the needs of the cultured cells. The formulation is shown in Table 1 and store it refrigerated at 4°C for later use.

[0063] Table 1 Formulation of the complete cell culture medium

[0064] Component Proportion Component Proportion DMEM or α-MEM 89％ L-Glutamine (2 mM) Trace Fetal Bovine Serum (FBS) 10％ Non-essential Amino Acids (1%) Trace 100 U / mL Penicillin or 100 μg / mL Streptomycin 1％ β-Mercaptoethanol (0.1 mM) Trace

[0065] S2. Weigh the hydroxypropyl chitosan prepared in Example 1 at room temperature and place it in the above-mentioned complete cell culture medium to make the concentrations of hydroxypropyl chitosan 1%, 1.5% and 2%, and prepare 2 portions for each concentration. Stir at 4°C for 12 - 36 hours until the hydroxypropyl chitosan is completely dissolved. Add Rock Inhibitor (purchased commercially) to one of the mixtures of hydroxypropyl chitosan and the complete cell culture medium.

[0066] S3. At 4°C, after removing the culture of mesenchymal stem cells, gently pipette and mix it with the above-mentioned complete culture medium containing hydroxypropyl chitosan to keep the MSC concentration at 10 5 -10 8 / mL. Use a pipette to transfer the liquid mixed culture containing MSC into a cell culture dish (plate).

[0067] S4. Place the cell culture dish (plate) from step S3 into a cell culture incubator at 37°C for 10 - 20 minutes. The liquid mixed culture becomes solid (gel state), and culture for 24 - 48 hours. Observe the culture status of MSC and the change in the color of the culture medium.

[0068] S5. When the MSCs start to grow in clusters (microspherical under the microscope) and the color of the culture medium turns yellow (turbid and the light transmittance becomes poor under the microscope), take out the culture dish (plate) and place it at 4°C for 10 - 20 minutes. The solid mixed culture becomes liquid, suck it out with a pipette, centrifuge, and discard the supernatant to obtain three-dimensional cultured MSCs.

[0069] The results of cell culture are as Figure 2 shown. The upper left part is static culture and the lower part is shaker culture. The results show that MSCs can grow normally under gels of the three concentrations, but grow best at a gel concentration of 1.5%. In addition, adding Rock Inhibitor can promote cell proliferation and accelerate cell spheroid growth.

[0070] Detection of cell growth status in Example 5

[0071] Perform three-dimensional cell culture according to the method described in Example 4, where the concentration of hydroxypropyl chitosan in step S2 is 1.5%, and Rock Inhibitor is not added in this example (CH group). At 1, 2, 3, and 5 days after the start of the culture, take the cell culture, centrifuge, remove the supernatant, resuspend with physiological saline, and microscopically observe the cell morphology and count. At the same time, set a control group (Ctrl, without adding hydroxypropyl chitosan), and compare the obtained results with those of the control group. As Figure 3 shown, the cells in the experimental group with added hydroxypropyl chitosan grow in spheres, with a faster growth rate, more proliferation, and longer survival.

[0072] Example 6 Cell survival time test and detection of mRNA expression of secreted factors

[0073] Set three groups: MSCs-2D (Ctrl group), MSCs-3D, and MSCs-1.5%. The MSCs-2D group is for two-dimensional cell culture, without adding hydroxypropyl chitin, and only allowing the cells to adhere and grow on the surface of the culture medium; the MSCs-3D group and the MSCs-1.5% group perform three-dimensional cell culture according to the method described in Example 3, where the MSCs-3D group adds undiluted Matrigel (100% Matrigel) at a ratio of 1:1; the MSCs-1.5% group adds 1.5% hydroxypropyl chitin. At 1, 3, 5, 7, and 9 days after the start of the culture, take the cell culture, microscopically observe the cell morphology, count, and detect the mRNA expression levels of IL-10, TGF-β1, and TSG-6 in the culture by QPCR. The results are respectively as Figure 4 、 Figure 5 shown. It can be seen that compared with the MSCs-2D group, in the two groups with added hydroxypropyl chitin (excluding the differences between liquid medium and solid culture), the cells grow in spheres, the proliferation ability is enhanced, the survival time is longer, and the mRNA expression levels of various cell secreted factors in the cells are significantly increased, and the cell activity is enhanced; compared with the MSCs-3D group, the MSCs-1.5% group has stronger cell survival time and cell viability, indicating that the three-dimensional porous structure of the cell scaffold is more conducive to the exchange of nutrients and metabolic wastes, and thus more conducive to cell survival.

[0074] In addition, the inventor also conducted similar verification on the hydroxypropyl chitin prepared in Example 2, and the results showed that it also had the same technical effects.

[0075] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A thermosensitive three-dimensional cell culture scaffold, characterized in that: The thermosensitive three-dimensional cell culture scaffold comprises thermosensitive hydroxypropyl chitosan, the molecular weight of the thermosensitive hydroxypropyl chitosan is 15,000-300,000 Da, the degree of deacetylation is 35%-65%, and the preparation method of the hydroxypropyl chitosan comprises: S1. Using chitosan with a deacetylation degree of ≥85% as a raw material, reacting with propylene oxide at 0-25°C and alkaline conditions to obtain hydroxypropyl chitosan; S2. The product in step S1 reacts with acetic anhydride under low temperature and alkaline conditions to generate a temperature-sensitive hydroxypropyl chitosan solution, the solution is dialyzed against triple distilled water to remove salt particles in the solution, and then eluted with ethanol at 0-25° C. and freeze-dried to obtain hydroxypropyl chitosan.

2. The thermosensitive three-dimensional cell culture scaffold according to claim 1, characterized in that: The temperature-sensitive three-dimensional cell culture scaffold also includes a complete cell culture medium. The temperature-sensitive hydroxypropyl chitosan and the complete cell culture medium are mixed to form a composition with a hydroxypropyl chitosan concentration of 0.5%-5%. The composition has a phase transition temperature of 10-15°C. When the temperature of the composition is lower than the phase transition temperature, the composition is liquid; when the temperature of the composition is higher than the phase transition temperature, the composition is solid.

3. The thermosensitive three-dimensional cell culture scaffold according to claim 1, characterized in that: The temperature-sensitive three-dimensional cell culture scaffold also contains nutritional factors for cell culture and differentiation.

4. The thermosensitive three-dimensional cell culture scaffold according to claim 1 or 2, characterized in that: The cells include stem cells, differentiated cells, tumor cells, primary cells and passaged cells of all animal species.

5. The thermosensitive three-dimensional cell culture scaffold according to claim 2, characterized in that: The composition is in liquid state when the temperature is above 0°C and below the phase transition temperature.

6. The thermosensitive three-dimensional cell culture scaffold according to claim 1 or 2, characterized in that: In the step S1, the chitosan and propylene oxide have a mass ratio of 1:5-1:15, and the reaction is stirred at 0-25° C. for 48-72 hours to make the degree of substitution (DS) of hydroxypropyl between 1 and 1.

2.

7. The thermosensitive three-dimensional cell culture scaffold according to claim 1 or 2, characterized in that: In the step S1, isopropanol is added before adding propylene oxide, and stirred at 0-25° C. for 24-48 hours. The mass ratio of chitosan to isopropanol is 1:8-1:

12.

8. Use of the temperature-sensitive three-dimensional cell culture scaffold according to any one of claims 1 to 7 in the field of three-dimensional cell culture.

9. A three-dimensional cell culture carrier, comprising the temperature-sensitive three-dimensional cell culture scaffold according to any one of claims 1 to 7.

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