Use of a composite hydrogel in promoting cell spheroid culture

Abstract

The application discloses application of a composite hydrogel in promoting cell spheroid culture. The composite hydrogel is prepared from crosslinking of polyvinyl alcohol and sodium alginate. In the cell spheroid culture, a polyvinyl alcohol / sodium alginate composite hydrogel pad is arranged at the bottom of a culture solution, and cells are cultured above the polyvinyl alcohol / sodium alginate composite hydrogel. The method can make the cultured cells quickly form spheroids, and has obvious advantages compared with a currently most widely used matrix gel in the market. In addition, the technology uses polyvinyl alcohol and sodium alginate as main raw materials, both of which are mature commercial polymers, and the cost is low. Moreover, the preparation method through calcium ion crosslinking is simple and efficient.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to application of a composite hydrogel in promoting cell spheroid culture. BACKGROUND

[0002] Cancer is one of the biggest threats to public health in today's society, however, for a long time, the treatment effect of cancer in clinic is difficult to make progress. In order to solve the problem of cancer treatment, researchers culture cancer cells in vitro, establish various different in vitro cancer models in order to deepen the understanding of the mechanism of cancer occurrence and development, and also facilitate the screening of drugs for cancer treatment. In real tumor tissues, cancer cells, fibroblasts, immune cells and the like gather into dense multi-component cell balls, so that it is difficult for drugs to enter the inside of the cell ball to kill cancer cells, however, due to the traditional cell culture method is to let the cells evenly spread and adhere to the inner surface of the culture dish for growth, which is quite different from the real tumor cell growth environment, therefore, the technology of constructing multi-component cell balls in vitro is the most attractive in constructing in vitro cancer models at present, and is also the basis of constructing large tissues and complex organs. The commonly used technologies for constructing multi-component cell balls at present are as follows:

[0003] (1) Liquid coating method: the inner surface of the cell culture container is treated with a non-adhesive polymer (usually polyvinyl alcohol, methyl cellulose, etc.), which reduces the interaction between the cells and the culture container, prevents the cells from adhering to the surface, and thus promotes the formation of cell balls. This method is simple to operate, but it takes a long time and is not conducive to the formation of multi-component cell balls. In addition, the cell growth environment is still in a liquid environment, which is similar to the environment of blood circulation, but different from the microenvironment of most tumor tissues.

[0004] (2) Mechanical stirring method: by constantly stirring mechanically to prevent the cells from adhering to the surface, so as to promote the aggregation of cells to form cell balls. This method requires special equipment and needs to be connected to waste treatment equipment. Although the yield is large, the cost is high and the cycle is long.

[0005] (3) Microcarrier method: microcarriers are usually 100 to 300 nanometer microspheres prepared from different materials. According to different types of microcarriers, cells can grow on the surface of microspheres, or in the pore structure of porous microspheres. Although the cells are distributed in the form of monolayer cells in the pore structure, these cells can secrete extracellular matrix in the pore structure, thereby creating an environment similar to human internal tissues. The microcarrier method is a good solution for cells that cannot spontaneously form cell balls in a three-dimensional liquid environment, however, the main disadvantage of the method at present is that it is difficult to collect and analyze the cells after culture. In addition, the cells growing in the microporous structure are not conducive to observation under a microscope.

[0006] In summary, the new cell ball construction technology should have the advantages of simple method, high efficiency and low cost at the same time, so as to be widely applied and suitable for commercial large-scale promotion.

[0007] In the prior art, sodium alginate and polyvinyl alcohol are generally used as carriers for cell immobilization. For example, the application disclosed in CN112725327A discloses a preparation method of a cell immobilization carrier, which comprises the following steps: uniformly mixing sodium alginate solution and polyvinyl alcohol to obtain a carrier solution; wherein the mass ratio of the sodium alginate to the polyvinyl alcohol is 1:2-10; adding diatomite to the carrier solution and uniformly mixing, and then dropping the gel mixture into a crosslinking solution in a continuous stirring state for solidification to form a gel bead, and then washing the gel bead and freezing and crosslinking it at a temperature of-25 to-15 DEG C, so as to obtain the cell immobilization carrier. There is no report in the prior art that the hydrogel prepared by crosslinking sodium alginate and polyvinyl alcohol can promote cell spheroid culture. SUMMARY

[0008] In view of the above technical problems in the prior art, the application provides an application of a composite hydrogel in promoting cell spheroid culture. The application provides a technology for rapidly inducing the generation of multi-component cell balls by using a polyvinyl alcohol / sodium alginate (PVA / Alg) composite hydrogel. Different components of cells are cultured on the surface of the composite hydrogel to rapidly construct cell balls within 12 hours. The method is simple, efficient and low in cost, and greatly improves the deficiencies of the current cell ball construction technology.

[0009] The application first provides an application of a composite hydrogel in promoting cell spheroid culture, wherein the composite hydrogel is a polyvinyl alcohol / sodium alginate composite hydrogel, and when the cell spheroid culture is performed, the polyvinyl alcohol / sodium alginate composite hydrogel is placed at the bottom of a culture solution, and cells are cultured above the polyvinyl alcohol / sodium alginate composite hydrogel,

[0010] The preparation method of the polyvinyl alcohol / sodium alginate composite hydrogel comprises the following steps:

[0011] (1) polyvinyl alcohol solution, sodium alginate solution and calcium chloride solution are respectively prepared;

[0012] (2) the polyvinyl alcohol solution and the sodium alginate solution are mixed to obtain a polyvinyl alcohol / sodium alginate mixed solution;

[0013] (3) the crosslinking agent calcium chloride solution is added to the polyvinyl alcohol / sodium alginate mixed solution, and a super-large pore hydrogel composed of polyvinyl alcohol and sodium alginate is obtained by crosslinking,

[0014] The concentration of the polyvinyl alcohol solution is 10wt%, the concentration of the sodium alginate solution is 4wt%, and the polyvinyl alcohol solution and the sodium alginate solution are mixed in a volume ratio of 5:1.

[0015] Preferably, the concentration of the calcium chloride solution is 0.5M, and the volume ratio of the calcium chloride solution to the polyvinyl alcohol / sodium alginate mixed solution is 1:1.

[0016] The bubbles in the polyvinyl alcohol / sodium alginate mixed solution are removed before the calcium chloride solution is added for cross-linking.

[0017] The cross-linking process is static cross-linking for at least 12 hours.

[0018] After the cross-linking is completed, the obtained polyvinyl alcohol / sodium alginate composite hydrogel is soaked in ultrapure water to remove free calcium ions.

[0019] Preferably, the polyvinyl alcohol / sodium alginate composite hydrogel is sterilized before cell culture.

[0020] Preferably, the cells for the spheroid culture are one of the following:

[0021] (1) tumor cells;

[0022] (2) a mixture of tumor cells and tumor tissue peripheral cells;

[0023] (3) immune cells.

[0024] Further preferably, the tumor tissue peripheral cells are at least one of the following: fibroblasts, macrophages.

[0025] The application further provides a cell spheroid culture method, in which the polyvinyl alcohol / sodium alginate composite hydrogel is placed at the bottom of the culture solution, and the cells are cultured above the polyvinyl alcohol / sodium alginate composite hydrogel.

[0026] The preparation method of the polyvinyl alcohol / sodium alginate composite hydrogel comprises the following steps:

[0027] (1) preparing polyvinyl alcohol solution, sodium alginate solution and calcium chloride solution, respectively;

[0028] (2) mixing the polyvinyl alcohol solution and the sodium alginate solution to obtain a polyvinyl alcohol / sodium alginate mixed solution;

[0029] (3) adding a cross-linking agent, calcium chloride solution, to the polyvinyl alcohol / sodium alginate mixed solution to cross-link and obtain a polyvinyl alcohol / sodium alginate composite supermacroporous hydrogel,

[0030] The concentration of the polyvinyl alcohol solution is 10wt%, the concentration of the sodium alginate solution is 4wt%, and the polyvinyl alcohol solution and the sodium alginate solution are mixed in a volume ratio of 5:1.

[0031] Preferably, the concentration of the calcium chloride solution is 0.5M, and the volume ratio of the calcium chloride solution to the polyvinyl alcohol / sodium alginate mixed solution is 1:1;

[0032] The bubbles in the polyvinyl alcohol / sodium alginate mixed solution are removed before the addition of the calcium chloride solution for cross-linking;

[0033] The cross-linking process is static cross-linking for at least 12 hours;

[0034] After the completion of cross-linking, the obtained polyvinyl alcohol / sodium alginate composite hydrogel is soaked in ultrapure water to remove free calcium ions.

[0035] Preferably, the polyvinyl alcohol / sodium alginate composite hydrogel is sterilized before cell culture.

[0036] Preferably, the cells cultured in the form of spheroids are one of the following:

[0037] (1) tumor cells;

[0038] (2) a mixture of tumor cells and tumor tissue peripheral cells;

[0039] (3) immune cells.

[0040] Further preferably, the tumor tissue peripheral cells are at least one of the following: fibroblasts, macrophages.

[0041] The addition of polyvinyl alcohol in the cell culture solution reduces the adhesion between the cells and the well plate, thereby promoting the aggregation of cells into spheroids. In addition to the use of polyvinyl alcohol to reduce the adhesion between the cells and the well plate, since cells are more likely to spread on hard substrate materials and tend to aggregate on flexible substrate materials, the selection of sodium alginate and polyvinyl alcohol with weak interaction with cells to prepare flexible composite hydrogel is expected to become a new generation of multi-component spheroid construction technology. After the uniform mixing of sodium alginate and polyvinyl alcohol solution, the cross-linking of sodium alginate by calcium ions is carried out to prepare polyvinyl alcohol / sodium alginate composite hydrogel, and then the composite hydrogel is soaked in the cell culture solution, and after reaching the swelling equilibrium, the cells are added to the surface of the composite hydrogel for culture. Polyvinyl alcohol still plays a role in reducing cell adhesion, while the sodium alginate skeleton structure provides flexible support to induce rapid aggregation of cells into spheroids.

[0042] The present technology can induce KPC cells, 3T3 cells, macrophages, etc. to form spheroids rapidly within 12 hours, and can still induce the formation of cell spheroids rapidly when different cells are mixed in proportion, which has obvious advantages compared with the most widely used Matrigel in the market. In addition, the technology uses polyvinyl alcohol and sodium alginate as main raw materials, both of which are mature commercial polymers, and the cost is low. And the preparation method by calcium ion crosslinking is simple and efficient. BRIEF DESCRIPTION OF DRAWINGS

[0043] Figure 1 The preparation process of two kinds of PVA / Alg composite hydrogels (polyvinyl alcohol and sodium alginate ratio of 1:5, 1:1 and 5:1) in Example 1 and Example 2 and the schematic diagram for cell culture.

[0044] Figure 2 The surface of the composite hydrogel prepared by mixing the polyvinyl alcohol solution and the sodium alginate solution in Example 1 in a volume ratio of 1:1.

[0045] Figure 3 The state of KPC cells and 3T3 cells cultured separately and mixed in a ratio of 1:1 on 5-1 composite hydrogel for different times in Example 1. The scale is 200 microns.

[0046] Figure 4 The state of KPC cells cultured separately on 1-5 composite hydrogel for different times in Example 1. The scale is 200 microns.

[0047] Figure 5 The state of KPC cells and 3T3 cells cultured separately and mixed in a ratio of 1:1 on Matrigel for different times in Example 1. The scale is 200 microns.

[0048] Figure 6 The state of KPC cells and 3T3 cells cultured separately and mixed in a ratio of 1:1 on the well plate for different times in Example 1. The scale is 200 microns.

[0049] Figure 7 The state of macrophages cultured separately and mixed with KPC cells in a ratio of 1:1 on the composite hydrogel for different times in Example 2. The scale is 200 microns. DETAILED DESCRIPTION

[0050] As Figure 1The preparation process of PVA / Alg composite hydrogel and the schematic diagram for cell culture in the present application are shown. First, sodium alginate and polyvinyl alcohol are cross-linked to prepare PVA / Alg composite hydrogel under the action of calcium chloride, and then the PVA / Alg composite hydrogel is used for cell culture. After the PVA / Alg composite hydrogel is placed at the bottom of the culture hole of the culture device (such as culture dish, multi-well culture plate), cell culture is carried out above the PVA / Alg composite hydrogel.

[0051] Example 1

[0052] (1) Preparation of sodium alginate (Alginate) solution: 8 g of sodium alginate powder was dissolved in 192 g of ultrapure water at room temperature to obtain a 4wt% sodium alginate solution.

[0053] (2) Preparation of polyvinyl alcohol (PVA) solution: 20 g of PVA powder was dissolved in 180 g of hot water (90°C) under continuous stirring to obtain a 10wt% PVA solution.

[0054] (3) Preparation of polyvinyl alcohol / sodium alginate mixed solution: the sodium alginate solution and PVA solution in steps (1) and (2) were blended in a volume ratio of 5:1, 1:1 and 1:5, and stirred continuously at room temperature until uniform and no steam pocket.

[0055] (4) Preparation of calcium chloride solution: 11.1 g of calcium chloride particles was weighed and dissolved in 200 ml of ultrapure water to obtain a 0.5M concentration of calcium chloride solution by ultrasonic treatment.

[0056] (5) Preparation of gel mold: a 3mm thick silicone pad was prepared and cut into a frame shape (with the middle part hollowed out), and the silicone pad was clamped in the middle with two PET templates, and clamped and fixed at the bottom and edge part with a dovetail clamp to prevent leakage after adding liquid in the mold.

[0057] (6) Preparation of composite hydrogel: The mixed solutions from step (3) (polyvinyl alcohol and sodium alginate in ratios of 1:5, 1:1, and 5:1) were added to the mold from step (5). The mold was placed vertically, and the mixed solutions were added to half the mold. The mold was left at room temperature for 12 hours until the liquid level of the mixed solution was horizontal and the bubbles disappeared. Then, calcium chloride solution was added to the mold using a syringe to fill the remaining space. After the calcium chloride solution was added, the mold was placed vertically at room temperature and allowed to crosslink for 12 hours. The gel was then removed and soaked in 0.5M calcium chloride solution for 12 hours, and then soaked in ultrapure water for 24 hours to remove free calcium ions. The gel was then processed into round samples of different sizes using a punch. The sample with a solution ratio of 5:1 is abbreviated as PVA / Alg-5-1, and the sample with a ratio of 1:5 is abbreviated as PVA / Alg-1-5. The composite hydrogel prepared by mixing sodium alginate solution and PVA solution in a 1:1 volume ratio has an uneven surface and cannot be used for cell sphere construction.

[0058] (7) Gel sterilization treatment: In order to carry out cell culture, the prepared disc-shaped hydrogel needs to be sterilized by high temperature. The treatment conditions are 120℃ for 20 minutes.

[0059] (8) Treatment of the base adhesive: Take Corning base adhesive (354230) out of -20℃ and place it in a 4℃ refrigerator overnight to thaw.

[0060] (9) Prepare complete cell culture medium: Add 10% fetal bovine serum and 1% penicillin-streptomycin solution to high glucose DMEM medium.

[0061] (10) Cell culture: KPC mouse pancreatic cancer cells and 3T3 mouse fibroblasts were cultured in the complete culture medium prepared in step (9). All cells were cultured in a 37°C, 5% CO2 cell culture incubator. The culture medium was changed daily and passaged every 3 days using digestion.

[0062] (11) Preparation of cell suspension: KPC and 3T3 cells were digested with trypsin and resuspended in the corresponding fresh culture medium. After counting, the cell density was adjusted to 2 × 10⁻⁶ cells. 4 per mL.

[0063] (12) Cell culture on PVA / Alg composite hydrogel: Put three pieces of sterilized PVA / Alg-5-1 composite hydrogel in step (7) into 24-well plates for cell culture, add cell culture solution in step (9), and wait for 12 hours. Then, add the required cell types to the three pieces of hydrogel. The cells cultured are mouse pancreatic cancer cells (KPC), mouse fibroblasts (3T3), and a cell group obtained by mixing KPC and 3T3 at a ratio of 1:1.

[0064] (13) Cell culture on Matrigel: To compare the effect of the most widely used commercial Matrigel, put three pieces of Matrigel in step (8) into 24-well plates for cell culture, add cell culture solution in step (9), and wait for 12 hours. Then, add the required cell types to the three pieces of Matrigel. The cells cultured are KPC cells, 3T3 cells, and a cell group obtained by mixing KPC and 3T3 at a ratio of 1:1.

[0065] (14) Cell culture in plates: The cell growth state in plates is the blank control. Add cell culture solution in step (9) to three wells in a 24-well plate for cell culture, and then add the required cell types. The cells cultured are KPC cells, 3T3 cells, and a cell group obtained by mixing KPC and 3T3 at a ratio of 1:1.

[0066] (15) Observation of cell state: Place the cells cultured in different environments in steps (12), (13), and (14) under an optical microscope at different time points to observe the cell morphology.

[0067] Result analysis:

[0068] In Figure 2 , the composite hydrogel prepared by mixing sodium alginate solution and PVA solution at a ratio of 1:1 by volume has an uneven surface and cannot be used for cell sphere construction. In Figure 3 , KPC cells and 3T3 cells grown on PVA / Alg-5-1 composite hydrogel can quickly form cell spheres within 12 hours, and the diameter of the cell spheres can reach about 200 microns. The cell sphere structure remains stable for 24 hours. The cell group obtained by mixing the two components at a ratio of 1:1 can also achieve the same effect. In Figure 4In the study, KPC cells grown on a PVA / Alg-1-5 composite hydrogel showed only a small number of spheroids even after 24 hours, and their structure was relatively loose. This is because the increased proportion of sodium alginate caused the gel to harden, hindering cell aggregation. Furthermore, the reduced PVA content further reduced cell aggregation into spheroids. Therefore, a 5:1 ratio of polyvinyl alcohol to sodium alginate yielded the best composite hydrogel for constructing cell spheroids. Figure 5 In the study, KPC cells grown on matrix gel showed aggregated cell spheroids after 24 hours, with a significantly longer time to form spheroids compared to the composite hydrogel. Furthermore, the diameter of the KPC cell spheroids was approximately 100 micrometers, significantly smaller than those on the composite hydrogel. While 3T3 cells and KPC / 3T3 mixed cell populations grown on matrix gel showed some aggregation, they failed to form cell spheroids within 24 hours. As a blank control group, in... Figure 6 In the study, none of the cells growing within the wells showed a tendency to spheroidize or aggregate; all cells were in a dispersed, proliferating state. This data clearly demonstrates that the PVA / Alg-5-1 composite hydrogel can rapidly induce KPC and 3T3 cells to form cell spheroids, and exhibits significant advantages compared to commercially available matrix gels.

[0069] Example 2

[0070] (1) Preparation of sodium alginate solution: At room temperature, 8g of sodium alginate powder was dissolved in 192g of ultrapure water to obtain a 4wt% sodium alginate solution.

[0071] (2) Preparation of polyvinyl alcohol (PVA) solution: Dissolve 20g of PVA powder in 180g of hot water at 90℃ with continuous stirring to obtain a 10wt% PVA solution.

[0072] (3) Prepare a polyvinyl alcohol / sodium alginate mixed solution: Mix the sodium alginate solution and PVA solution from steps (1) and (2) at a volume ratio of 5:1 and stir continuously at room temperature until a uniform and air-free state is achieved.

[0073] (4) Prepare calcium chloride solution: Weigh 11.1g of calcium chloride granules, dissolve them in 200ml of ultrapure water, and obtain a 0.5M calcium chloride solution by ultrasonic treatment.

[0074] (5) Prepare the silicone mold: Prepare a 3mm thick silicone pad, cut it into the shape of a photo frame (with the middle part hollowed out), sandwich the silicone pad in the middle with two PET templates, and use dovetail clips to clamp and fix it at the bottom and edges to prevent leakage after liquid is added to the mold.

[0075] (6) Preparation of composite hydrogel: Add the mixed solution from step (3) to the mold from step (5). Place the mold vertically and fill it to half its capacity. Let it stand at room temperature for 12 hours until the liquid level of the mixed solution is horizontal and the bubbles disappear. Then, use a syringe to add calcium chloride solution to the mold to fill the remaining space. After adding the calcium chloride solution, place the mold vertically at room temperature and let it stand for crosslinking for 12 hours. Then, take out the gel and soak it in 0.5M calcium chloride solution for 12 hours, and then soak it in ultrapure water for 24 hours to remove free calcium ions. Then, use a punch to process the gel into round samples of different sizes.

[0076] (7) Gel sterilization treatment: In order to carry out cell culture, the prepared disc-shaped hydrogel needs to be sterilized by high temperature. The treatment conditions are 120℃ for 20 minutes.

[0077] (8) Prepare complete cell culture medium: Add 10% fetal bovine serum and 1% penicillin-streptomycin solution to high glucose DMEM medium.

[0078] (9) Cell culture: KPC mouse pancreatic cancer cells were cultured in the complete culture medium prepared in step (8), and RAW264.7 mouse macrophages were cultured in a special culture medium and placed in a 37°C, 5% CO2 cell culture incubator. The culture medium was changed daily and passaged every 3 days using digestion.

[0079] (10) Preparation of cell suspension: After trypsin digestion, the cells were resuspended in the appropriate fresh culture medium to obtain a cell suspension. After counting, the cell density was adjusted to 2 × 10⁶ cells / year. 4 per mL.

[0080] (11) Cell culture on PVA / Alg composite hydrogel: Place the two sterilized gels from step (7) into a 24-well plate for cell culture, add the cell culture medium from step (8), and wait for 12 hours. Then, add the desired cell types to the two gels respectively. The cultured cells are macrophages and a cell population of KPC / macrophages mixed in a 1:1 ratio.

[0081] (12) Observe cell state: Place the cells cultured in step (9) under an optical microscope at different time points to observe cell morphology.

[0082] Results analysis:

[0083] Macrophages are immune cells, and a large number of macrophages co-grow with cancer cells in tumor tissue. Traditional cell spheroidization techniques struggle to aggregate them into cell spheroids. Figure 7In this study, macrophages cultured on the PVA / Alg-5-1 composite hydrogel formed cell spheroids in 9 hours, and the largest diameter of the spheroids reached about 200 microns. When KPC cells were mixed with macrophages at a ratio of 1:1 and cultured on the composite hydrogel, a small number of cell spheroids were observed to form in 9 hours, and a large number of cell spheroids were observed to form in 24 hours. These data fully demonstrate that the PVA / Alg-5-1 composite hydrogel can rapidly construct cell spheroids from different types of cells and can be fully applied to the construction of in vitro tumor tissues or organoids.

Claims

1. The application of a composite hydrogel in promoting cell spheroidization culture, characterized in that, The composite hydrogel is a polyvinyl alcohol / sodium alginate composite hydrogel. During cell spheroidization culture, the polyvinyl alcohol / sodium alginate composite hydrogel is placed at the bottom of the culture medium, and the cells are cultured on top of the polyvinyl alcohol / sodium alginate composite hydrogel. The preparation method of the polyvinyl alcohol / sodium alginate composite hydrogel includes the following steps: (1) Prepare polyvinyl alcohol solution, sodium alginate solution and calcium chloride solution respectively; (2) Mix the polyvinyl alcohol solution and sodium alginate solution to obtain a polyvinyl alcohol / sodium alginate mixed solution; (3) Add calcium chloride solution as a crosslinking agent to the polyvinyl alcohol / sodium alginate mixed solution to obtain a hydrogel composed of polyvinyl alcohol and sodium alginate through crosslinking. The polyvinyl alcohol solution has a concentration of 10 wt%, the sodium alginate solution has a concentration of 4 wt%, and the polyvinyl alcohol solution and sodium alginate solution are mixed at a volume ratio of 5:

1. The concentration of the calcium chloride solution is 0.5M, and the volume ratio of the calcium chloride solution to the polyvinyl alcohol / sodium alginate mixed solution is 1:

1. The polyvinyl alcohol / sodium alginate composite hydrogel was sterilized before cell culture.

2. The application according to claim 1, characterized in that, Before adding calcium chloride solution for crosslinking, remove air bubbles from the polyvinyl alcohol / sodium alginate mixed solution; The crosslinking process involves static crosslinking for at least 12 hours; After cross-linking, the resulting polyvinyl alcohol / sodium alginate composite hydrogel was soaked in ultrapure water to remove free calcium ions.

3. The application according to claim 1, characterized in that, The cells cultured into spheres are of the following type: (1) Tumor cells; (2) Mixing of tumor cells with cells surrounding the tumor tissue; (3) Immune cells.

4. The application according to claim 3, characterized in that, The cells surrounding the tumor tissue are at least one of the following: fibroblasts or macrophages.

5. A method for cell spheroidization culture, characterized in that, During cell spheroidization culture, a polyvinyl alcohol / sodium alginate composite hydrogel is placed at the bottom of the culture medium, and the cells are cultured on top of the polyvinyl alcohol / sodium alginate composite hydrogel. The preparation method of the polyvinyl alcohol / sodium alginate composite hydrogel includes the following steps: (1) Prepare polyvinyl alcohol solution, sodium alginate solution and calcium chloride solution respectively; (2) Mix the polyvinyl alcohol solution and sodium alginate solution to obtain a polyvinyl alcohol / sodium alginate mixed solution; (3) Add calcium chloride solution as a crosslinking agent to the polyvinyl alcohol / sodium alginate mixed solution to obtain a macroporous hydrogel composed of polyvinyl alcohol and sodium alginate. The polyvinyl alcohol solution has a concentration of 10 wt%, the sodium alginate solution has a concentration of 4 wt%, and the polyvinyl alcohol solution and sodium alginate solution are mixed at a volume ratio of 5:

1.

6. The cell spheroidization culture method according to claim 5, characterized in that, The concentration of the calcium chloride solution is 0.5M, and the volume ratio of the calcium chloride solution to the polyvinyl alcohol / sodium alginate mixed solution is 1:

1. Before adding calcium chloride solution for crosslinking, remove air bubbles from the polyvinyl alcohol / sodium alginate mixed solution; The crosslinking process involves static crosslinking for at least 12 hours; After cross-linking, the resulting polyvinyl alcohol / sodium alginate composite hydrogel was soaked in ultrapure water to remove free calcium ions.

7. The cell spheroidization culture method according to claim 5, characterized in that, The polyvinyl alcohol / sodium alginate composite hydrogel was sterilized before cell culture.

8. The cell spheroidization culture method according to claim 5, characterized in that, The cells cultured into spheres are of the following type: (1) Tumor cells; (2) Mixing of tumor cells with cells surrounding the tumor tissue; (3) Immune cells.

9. The cell spheroidization culture method according to claim 8, characterized in that, The cells surrounding the tumor tissue are at least one of the following: fibroblasts or macrophages.

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