Thermosensitive cell three-dimensional culture scaffold and method of use thereof

Abstract

The application belongs to the technical field of cell culture, and particularly relates to a temperature-sensitive cell three-dimensional culture support, which comprises temperature-sensitive hydroxypropyl chitin, the molecular weight of the temperature-sensitive hydroxypropyl chitin is 15,000-300,000 Da, and the degree of deacetylation (DD) is 35%-65%. The support material is temperature-sensitive hydroxypropyl chitin prepared by derivatization of chitosan of a natural source through a specific process, and can be used for three-dimensional culture of cells. The temperature-sensitive hydroxypropyl chitin is used for three-dimensional culture of cells, and can significantly reduce the culture cost, improve the convenience and the viability of the cultured cells.

Description

Technical Field

[0001] This invention belongs to the field of cell culture technology, specifically relating to a method for preparing and using a thermosensitive aminopolysaccharide derivative for three-dimensional cell culture. Background Technology

[0002] Cell culture is an important tool in life science, medical, and pharmaceutical research. Traditional cell culture is a two-dimensional culture method characterized by cell adhesion and growth. However, two-dimensional cell culture differs significantly from the overall growth state, structure, metabolism, and response to external stimuli of cells in vivo. To simulate the real growth environment of cells in vivo, three-dimensional cell culture has emerged.

[0003] Three-dimensional cell culture provides a more realistic, three-dimensional environment for cell growth, closely resembling the intracellular growth environment. It creates a balanced physiological environment for cells and tissues to acquire nutrients, exchange gases, and remove waste products. Furthermore, three-dimensional cell culture facilitates the formation of organs with appropriate morphology and physiological functions. Currently, the matrix gel used in three-dimensional cell culture is primarily derived from the soluble basement membrane matrix extract of Engelbreth-Holm-Swarm (EHS) mouse sarcoma. Its main components are laminin, type IV collagen, heparan sulfate proteoglycan (HSPG), nestin, and various growth factors. While this type of matrix gel is nutrient-rich, providing ample nutrition for three-dimensional cell growth, it also has significant drawbacks: scarce raw material sources, high production costs, and high prices; the finished product requires cryogenic preservation throughout the process, making transportation and storage inconvenient; and it only exhibits unidirectional temperature sensitivity from liquid to solid state, affecting ease of use. Therefore, there is an urgent need to develop a new three-dimensional cell culture scaffold.

[0004] Chitin and its derivatives have attracted widespread attention from researchers due to their non-toxicity, biodegradability, good biocompatibility, excellent antibacterial properties, and other bioactivities. They have been extensively studied and prepared into novel functional biomaterials for application in the biomedical field. Hydroxypropyl chitin is one of the more common derivatives. It is a type of aminopolysaccharide derivative formed by introducing hydroxypropyl groups onto the hydroxyl groups of chitin. It possesses excellent water solubility, biodegradability, biocompatibility, and good thermally induced gelling properties, and can spontaneously form a gel at high temperatures. The hydrogel formed consists of a three-dimensional hydrophilic network with high water content, which is beneficial for the transport of oxygen, nutrients, and other water-soluble metabolites. Because its structural characteristics are similar to those of the natural extracellular matrix and it can provide a suitable environment for cell growth, its current application in three-dimensional cell culture is not ideal, thus requiring improvement. Summary of the Invention

[0005] To address the shortcomings of existing technologies, one of the objectives of this invention is to provide a thermosensitive three-dimensional cell culture scaffold.

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

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

[0008] S2. The product from step S1 reacts with acetic anhydride under low temperature and alkaline conditions to generate a thermosensitive hydroxypropyl chitosan solution. The solution is dialyzed with triple-distilled water to remove salt particles, and then eluted with ethanol at 0-5℃ and freeze-dried to obtain hydroxypropyl chitosan.

[0009] Furthermore, the thermosensitive three-dimensional cell culture scaffold also includes a complete cell culture medium. The thermosensitive hydroxypropyl chitin and the complete cell culture medium are mixed to form a composition with a hydroxypropyl chitin concentration of 0.5%-5%. The composition has a phase transition temperature of 10-15°C. When the temperature of the composition is below the phase transition temperature, the composition is liquid; when the temperature of the composition is above the phase transition temperature, the composition is solid.

[0010] Furthermore, most of the free amino groups in the hydroxypropyl chitin structure are blocked by acetyl groups, which reduces the number of positive charges carried in the molecule and is beneficial to cell survival and proliferation.

[0011] Furthermore, the alkaline conditions are achieved using an alkaline solution, which includes, but is not limited to, a sodium hydroxide solution or a potassium hydroxide solution with a mass concentration of 10%-20%.

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

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

[0014] Furthermore, 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 also includes complete cell culture medium.

[0016] Furthermore, the complete cell culture medium, when mixed with the hydroxypropyl chitin, forms a composition with a hydroxypropyl chitin concentration of 0.5%-5%, which is temperature sensitive.

[0017] Furthermore, the three-dimensional cell culture scaffold also contains nutrient factors for cell culture and differentiation.

[0018] Furthermore, the cells include stem cells, differentiated cells, tumor cells, primary cells, and passaged 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, pancreatic islet cells, as well as various cells and cell clusters cultured into organoids through gene editing.

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

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

[0022] 1. The aminopolysaccharide derivatives provided by this invention have abundant raw material sources, low production costs, and low prices;

[0023] 2. The three-dimensional cell culture scaffold provided by this invention allows for the addition and mixing of different growth factors according to the needs of different cell cultures; it only needs to be stored at room temperature before use, making transportation and storage convenient; it has bidirectional temperature-sensitive properties in both solid and liquid states, making it easy to use and with a wider range of applications.

[0024] 3. The three-dimensional cell culture method provided by this invention can promote the 3D spheroidization of cells, enhance cell proliferation capacity, maintain the survival capacity of stem cells for a longer period of time, and enhance cell activity. Attached Figure Description

[0025] Figure 1 This is a microstructure diagram of the temperature-sensitive hydroxypropyl chitosan aqueous solution provided in an embodiment of the present invention.

[0026] Figure 2 The results of MSC culture using different concentrations of thermosensitive hydroxypropyl chitin mixed with MSC complete culture medium provided in the embodiments of the present invention.

[0027] Figure 3The image shows the growth status of stem cells after being mixed with thermosensitive hydroxypropyl chitin solution and MSC cell complete culture medium, as provided in this embodiment of the invention.

[0028] Figure 4 The image shows a comparison of stem cell survival time on day 3 between the cell culture system with thermosensitive hydroxypropyl chitin and the MSCs-3D blank culture system.

[0029] Figure 5 The results of Q-PCR detection of the expression levels of various cell secretion factor mRNAs in a cell culture system supplemented with thermosensitive hydroxypropyl chitin. Detailed Implementation

[0030] To make the technical problem to be solved, the technical solution, and the beneficial effects of the present invention clearer, 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 merely illustrative of the present invention and are not intended to limit the present invention.

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

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

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

[0034] Chitin is a non-toxic natural alkaline polysaccharide mainly found in the shells of shrimp, crabs, and other organisms. It is abundant in nature, possesses good biocompatibility and biodegradability, and is non-immunogenic, making it an excellent biological resource. However, due to its high crystallinity caused by hydrogen bonding, chitin is difficult to dissolve in water and low-concentration acidic or alkaline solutions, and is also not readily soluble in common organic solvents, thus hindering its effective utilization. Based on this, this invention designs and synthesizes a novel chitin derivative. The introduction of hydroxypropyl groups disrupts the inherent intramolecular and intermolecular hydrogen bonds within the chitin material to a certain extent, giving hydroxypropyl chitin a certain degree of water solubility.

[0035] Specifically, most of the free amino groups in the hydroxypropyl chitin structure are blocked by acetyl groups, which reduces the number of positive charges carried in the molecule and is beneficial to cell survival and proliferation.

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

[0037] S1. Hydroxypropyl chitosan is obtained by reacting chitosan as the main raw material with propylene oxide under low temperature and alkaline conditions.

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

[0039] Specifically, the chitosan can be any commercially available chitosan with a DD ≥ 85%, the low temperature condition refers to 0-25℃, and the alkaline condition is achieved using an alkaline solution, which includes, but is not limited to, a sodium hydroxide solution or 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 mixture is stirred at 0-25°C for 48-72 hours to achieve a degree of hydroxypropyl substitution (DS) (number of hydroxypropyl residues on chitosan glucosamine residues: number of glucosamine residues) between 1 and 1.2.

[0041] Specifically, in step S1, isopropanol needs to be added before adding propylene oxide, and the mixture is stirred at room temperature for 24-48 hours. 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 in this invention can prepare hydroxypropyl chitosan with different molecular weights and degrees of deacetylation by selecting chitosan substances with different molecular weights, degrees of hydroxylation, and degrees of deacetylation. The phase transition temperature of hydroxypropyl chitosan can be further controlled to suit different needs.

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

[0045] Three-dimensional cell culture refers to the use of scaffolds, gels, or suspension culture methods to grow cells in a three-dimensional environment, thereby forming structures 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, and its cell behavior characteristics are closer to the survival state in vivo. This makes it better suited for research fields such as new drug screening, tumor cell systems biology, stem cell research, functional tissue implantation, and other cell analyses.

[0046] Specifically, the three-dimensional cell culture scaffold provided in this embodiment of the invention also includes complete cell culture medium.

[0047] The complete cell culture medium, when mixed with the hydroxypropyl chitin, forms a composition with a hydroxypropyl chitin concentration of 0.5%-5%, which is temperature sensitive. This composition solution exhibits a phase transition temperature of 10-15°C; when the overall solution temperature is below 10-15°C, it is in a liquid state; when the solution temperature is above 10-15°C, it is in a gel state.

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

[0049] Specifically, the cells include stem cells, differentiated cells, tumor cells, primary cells, and passaged 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, pancreatic islet cells, as well as various cells and cell clusters cultured into organoids through gene editing.

[0051] The present invention also provides a method for three-dimensional cell culture, utilizing the aforementioned aminopolysaccharide derivatives or three-dimensional cell culture scaffolds.

[0052] The present invention will be described in detail below with reference to specific embodiments.

[0053] Example 1: Preparation of Hydroxypropyl Chitosan

[0054] S1. Chitosan powder was slowly added to an excess of 10% sodium hydroxide solution and stirred until the chitosan was fully dispersed in the aqueous solution. The solution was then frozen overnight. After dissolution, isopropanol (chitosan to isopropanol mass ratio 1:12) was added and stirred at 10°C for 1.5 h. Then, propylene oxide (chitosan to propylene oxide mass ratio 1:5) was slowly added and stirred at 25°C for 48 h. The time and temperature were controlled to keep the degree of hydroxypropyl substitution (DS) (number of hydroxypropyl residues on chitosan glucosamine residues: glucosamine residues) between 1 and 1.2. The pH was adjusted to neutral with dilute hydrochloric acid. The product was precipitated with an organic solvent, filtered, and washed several times with anhydrous ethanol to obtain hydroxypropyl chitosan.

[0055] S2. Dissolve the hydroxypropyl chitosan obtained in S1 in an excess of methanol-water solution (methanol:water = 1:1), add acetic anhydride to make the mass ratio of hydroxypropyl chitosan to acetic anhydride between 2:1 and 2:2, stir at 15°C until completely dissolved, and let stand overnight. After the reaction is complete, adjust the pH to neutral with dilute sodium hydroxide solution, dialyze at 4°C using a triple-distilled water dialysis bag to remove salt ions, precipitate the dialysis product with an organic solvent, filter, wash several times with anhydrous ethanol, and freeze-dry under vacuum to obtain hydroxypropyl chitosan.

[0056] Example 2: Preparation of Hydroxypropyl Chitosan

[0057] S1. Chitosan powder was slowly added to an excess of 20% sodium hydroxide solution and stirred until the chitosan was fully dispersed in the aqueous solution. The solution was then frozen overnight. After dissolution, isopropanol (chitosan to isopropanol mass ratio 1:8) was added and stirred at 4°C for 1 hour. Then, propylene oxide (chitosan to propylene oxide mass ratio 1:15) was slowly added and stirred at 10°C for 72 hours. The time and temperature were controlled to keep the degree of hydroxypropyl substitution (DS) (number of hydroxypropyl residues on chitosan glucosamine residues: glucosamine residues) between 1 and 1.2. The pH was adjusted to neutral with dilute hydrochloric acid. The product was precipitated with an organic solvent, filtered, and washed several times with anhydrous ethanol to obtain hydroxypropyl chitosan.

[0058] S2. Dissolve the hydroxypropyl chitosan obtained in S1 in an excess of methanol-water solution (methanol:water = 1:1), add acetic anhydride to make the mass ratio of hydroxypropyl chitosan to acetic anhydride between 2:1 and 2:2, stir at 10-15℃ until completely dissolved, and let stand overnight. After the reaction is complete, adjust the pH to neutral with dilute sodium hydroxide solution, dialyze at 4℃ using a triple-distilled water dialysis bag to remove salt ions, precipitate the product with an organic solvent after dialysis, filter, wash several times with anhydrous ethanol, and freeze-dry under vacuum to obtain hydroxypropyl chitosan.

[0059] Example 3: Preparation of hydroxypropyl chitin hydrogel and identification of its scaffold structure

[0060] At 4°C, the hydroxypropyl chitosan powder prepared in Example 1 was weighed, dissolved in water, and prepared into hydroxypropyl chitosan solutions with mass concentrations of 1%, 1.5%, 2%, and 4%, respectively. The solutions were then heated to 37°C to solidify into a gel state, and their structure was observed under a scanning electron microscope. The results are as follows: Figure 1 As shown, all concentrations of hydroxypropyl chitin gel exhibit a three-dimensional porous structure, with the 1.5% mass concentration gel showing the most uniform pore distribution, which is more conducive to the exchange of nutrients and metabolic waste.

[0061] Example 4: Three-dimensional culture of mesenchymal stem cells (MSCs)

[0062] S1. Prepare the corresponding complete cell culture medium in advance according to the needs of the cultured cells. The preparation ratio is shown in Table 1. Store at 4℃ for later use.

[0063] Table 1. Proportions of Complete Cell Culture Medium

[0064] Components percentage Components percentage DMEM or α-MEM 89％ L-Glutamine (2mM) trace amounts Fetal bovine serum (FBS) 10％ Non-essential amino acids (1%) trace amounts 100 U / mL penicillin or 100 μg / mL streptomycin 1％ β-Mercaptoethanol (0.1mM) trace amounts

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

[0066] S3. After removing the culture medium from the mesenchymal stem cells at 4°C, gently pipette them into the complete culture medium containing hydroxypropyl chitin to maintain the MSC concentration at 10. 5 -10 8 / ml, use a pipette to transfer the liquid mixed culture containing MSCs into a cell culture dish (plate);

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

[0068] S5. When MSCs begin to grow in clusters (appearing as microspheres under a microscope) and the culture medium turns yellow (becomes turbid and has poor light transmittance under a microscope), remove the culture dish (plate) and place it at 4°C for 10-20 minutes. The solid mixed culture will become liquid. Use a pipette to remove it, centrifuge, remove the supernatant, and obtain three-dimensional cultured MSCs.

[0069] Cell culture results as follows Figure 2 As shown, the upper left half represents static culture, and the lower left half represents shaker culture. Results showed that MSCs grew normally under all three gel concentrations, but grew best at a 1.5% gel concentration. Furthermore, the addition of Rock Inhibitor promoted cell proliferation and accelerated cell spheroidization.

[0070] Example 5: Cell Growth Status Detection

[0071] Three-dimensional cell culture was performed according to the method described in Example 4, wherein step S2 used a hydroxypropyl chitosan concentration of 1.5%, and Rock Inhibitor (CH group) was not added in this example. On days 1, 2, 3, and 5 after the start of culture, cell cultures were collected, centrifuged, the supernatant was removed, and the cells were resuspended in physiological saline. Cell morphology was observed and counted under a microscope. A control group (Ctrl, without hydroxypropyl chitosan) was also set up, and the results were compared with those of the control group. Figure 3 As shown, the cells in the experimental group with added hydroxypropyl chitosan grew into spheres, with a faster growth rate, greater proliferation, and longer survival time.

[0072] Example 6: Cell viability test and detection of secretory factor mRNA expression

[0073] Three groups were set up: MSCs-2D (Ctrl group), MSCs-3D, and MSCs-1.5%. The MSCs-2D group underwent two-dimensional cell culture without the addition of hydroxypropyl chitin, allowing cells to adhere to the culture medium surface for growth. The MSCs-3D and MSCs-1.5% groups underwent three-dimensional cell culture as described in Example 3, with the MSCs-3D group receiving 1:1 undiluted Matrigel (100% Matrigel) and the MSCs-1.5% group receiving 1.5% hydroxypropyl chitin. Cell cultures were harvested on days 1, 3, 5, 7, and 9 after culture began. Cell morphology and counts were observed under a microscope, and the mRNA expression levels of IL-10, TGF-β1, and TSG-6 in the culture were detected by qPCR. The results are shown below. Figure 4 , Figure 5 As shown in the figure, compared with the MSCs-2D group, the two groups with added hydroxypropyl chitin (excluding the difference between liquid and solid culture) showed enhanced cell spheroidization, increased proliferation capacity, and longer survival time. Furthermore, the expression levels of various cell secretion factor mRNAs were significantly increased, indicating enhanced cell activity. Compared with the MSCs-3D group, the MSCs-1.5% group showed even stronger cell survival time and viability, suggesting that the three-dimensional porous structure of the cell scaffold is more conducive to the exchange of nutrients and metabolic waste, thus promoting cell survival.

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

[0075] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the principles of the present invention should 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 includes thermosensitive hydroxypropyl chitin, wherein the molecular weight of the thermosensitive hydroxypropyl chitin is 15,000-300,000 Da, and the degree of deacetylation is 35%-65%. The preparation method of the hydroxypropyl chitin includes: S1. Using chitosan with a degree of deacetylation ≥ 85% as raw material, react with propylene oxide at 0-25℃ under alkaline conditions to obtain hydroxypropyl chitosan; S2. The product from step S1 reacts with acetic anhydride at 0-15℃ under alkaline conditions to generate a thermosensitive hydroxypropyl chitosan solution. The solution is dialyzed with triple-distilled water to remove salt particles, then eluted with ethanol at 0-25℃ and freeze-dried to obtain hydroxypropyl chitosan; wherein the mass ratio of hydroxypropyl chitosan to acetic anhydride is between 2:1 and 2:

2. In step S1, the mass ratio of chitosan to propylene oxide is 1:5-1:15, and the mixture is stirred at 0-25°C for 48-72 hours to achieve a degree of hydroxypropyl substitution (DS) between 1 and 1.

2. The thermosensitive three-dimensional cell culture scaffold also includes a complete cell culture medium, wherein the thermosensitive hydroxypropyl chitin and the complete cell culture medium are mixed to form a composition with a hydroxypropyl chitin concentration of 0.5%-5%. The composition has a phase transition temperature of 10-15°C. When the temperature of the composition is below the phase transition temperature, the composition is in a liquid state; when the temperature of the composition is above the phase transition temperature, the composition is in a solid state.

2. The thermosensitive three-dimensional cell culture scaffold according to claim 1, characterized in that, The thermosensitive three-dimensional cell culture scaffold also contains nutrient factors for cell culture and differentiation.

3. 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.

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

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

12.

6. The application of the thermosensitive three-dimensional cell culture scaffold according to any one of claims 1-5 in the field of three-dimensional cell culture.

7. A three-dimensional cell culture carrier, comprising a thermosensitive three-dimensional cell culture scaffold according to any one of claims 1-5.

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