Ready-to-use sodium alginate paper-coated material for three-dimensional cell culture as well as preparation method and application of ready-to-use sodium alginate paper-coated material

Abstract

The invention discloses a ready-to-use sodium alginate paper-coated material for three-dimensional cell culture as well as a preparation method and application thereof, and belongs to the technical field of cell culture. The preparation method of the ready-to-use sodium alginate-coated paper material comprises the following steps: dropwise adding sodium alginate onto a paper material, cross-linking sodium alginate hydrogel through polyethylene glycol-diamine, 1-ethyl-(3-dimethylaminopropyl)-carbodiimide hydrochloride and N-hydroxysuccinimide, and freeze-drying to prepare the ready-to-use sodium alginate-coated paper material. Compared with the traditional hydrogel, the problems that the prepared hydrogel is difficult to dissolve, bubbles are easily generated when the hydrogel is mixed with cells, and the bubbles are difficult to eliminate are effectively avoided; besides, the ready-to-use sodium alginate and paper material has no cytotoxicity, has good stability and can be stored for 120 days at room temperature in a dark state, and compared with the currently prepared and used sodium alginate and paper material, the ready-to-use sodium alginate and paper material has no obvious difference in cell adhesion capability and cell three-dimensional culture capability.

Description

Technical Field

[0001] The present invention relates to a ready-to-use sodium alginate@paper material for three-dimensional cell culture and a preparation method and application thereof, belonging to the technical field of cell culture. Background Art

[0002] Cell culture is an indispensable fundamental reaction for studying cellular physiological and biochemical reactions. However, in most traditional cell culture experiments, cells simply adhere to the surface of the culture dish in a monolayer and grow in a two-dimensional state. Two-dimensional cell culture has the advantages of being simple and easy to operate, and easy to observe physiological processes such as cell morphology and proliferation. However, in this state, cells only interact with each other horizontally, which cannot fully simulate the actual situation in vivo and has certain limitations. Three-dimensional culture retains the three-dimensional spatial structure of cells growing in the body, making cell differentiation, proliferation and other behaviors closer to the physiological state. In recent years, three-dimensional cell culture has become increasingly popular in cell culture experiments.

[0003] Hydrogels are polymer materials that form a three-dimensional network structure using water as a dispersion medium. They are widely used in the biomedical field due to their excellent biocompatibility and biodegradability. Sodium alginate hydrogels have high gel strength, and the three-dimensional structure they form offers greater support. This makes them one of the most widely used hydrogels in cell culture.

[0004] For example, research by Liu Xincheng et al. has shown that adding sodium hyaluronate to sodium alginate can better maintain the chondrocyte phenotype and inhibit chondrocyte dedifferentiation. Xie Hang et al., based on sodium alginate, mixed sodium alginate, type I collagen, hyaluronic acid, and calcium chloride to prepare a composite hydrogel with good cell compatibility.

[0005] However, these hydrogels are usually prepared and used immediately, which may cause problems such as difficulty in dissolving and easy generation of bubbles when mixed with cells, which are difficult to eliminate. 2+ Cross-linking with sodium alginate is a reversible process. + When the hydrogel comes into contact with the solution, the cross-linking reaction will be reversed and the hydrogel will dissolve into the solution again, which is very unfavorable for the subsequent cell experiments (because PBS and other buffers are used in cell experiments, and these buffers often contain Na + ).

[0006] The crisscrossing fiber scaffold within paper provides a natural three-dimensional environment for cell growth. Within the paper, the hydrogels have interconnected pores that transport nutrients and gases necessary for cell growth. First described in 2009, the cells-in-gels-in-paper technique is a three-dimensional culture technology that uses hydrogels to encapsulate cells within paper fibers. In this technique, the paper fibers provide excellent mechanical support for the thin, soft hydrogels. Mosadegh et al. demonstrated a paper-based three-dimensional culture system that can mimic some of the interactions between cell populations during cardiac ischemia. This "cells-in-gels-in-paper" model offers a promising approach to the development of three-dimensional cell culture. However, the hydrogels must be prepared in advance and then added to the paper, which lacks portability and can easily fall off between the hydrogel and paper, hindering the widespread application of the "cells-in-gels-in-paper" model. Summary of the Invention

[0007] [Technical Issues]

[0008] In view of the problems in three-dimensional cell culture technology that sodium alginate hydrogels are difficult to dissolve when prepared and used immediately, bubbles are easily generated and difficult to eliminate when mixed with cells, and the problems in the cells-in-gels-in-paper model that sodium alginate hydrogels need to be prepared in advance and then added to the paper material, which lacks certain convenience and the hydrogels and paper materials easily fall off; the present invention aims to provide a ready-to-use sodium alginate@paper material for three-dimensional cell culture and a preparation method and application thereof, which can effectively solve the problems that sodium alginate hydrogels are difficult to dissolve when prepared and used immediately, bubbles are easily generated and difficult to eliminate when mixed with cells, and the hydrogels and paper materials easily fall off.

[0009] [Technical solution]

[0010] In order to achieve the above objectives, the technical solutions provided are as follows:

[0011] The first object of the present invention is to provide a method for preparing a ready-to-use sodium alginate@paper material for three-dimensional cell culture, the method comprising the following steps:

[0012] (1) dissolving sodium alginate in water to prepare a sodium alginate solution;

[0013] (2) adding the sodium alginate solution obtained in step (1) dropwise onto the paper material to obtain sodium alginate@paper material;

[0014] (3) A mixture of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide is first added dropwise to the sodium alginate@paper material prepared in step (2), and then a polyethylene glycol-diamine solution is added dropwise. Finally, the sodium alginate@paper material is frozen overnight and then freeze-dried to obtain a ready-to-use sodium alginate@paper material.

[0015] In one embodiment, the concentration of the sodium alginate solution in step (1) is 2-5%, g / ml.

[0016] In one embodiment, the preparation condition of step (1) is to continuously stir in a water bath at 60-80° C. to completely dissolve the mixture.

[0017] In one embodiment, the dripping method of step (2) is every 1cm 2 0.1 mL of sodium alginate solution was added dropwise onto the paper material to allow it to penetrate evenly into the paper material.

[0018] In one embodiment, the paper material is a filter paper material with a pore size of 5 to 8 μm.

[0019] In one embodiment, the mixed solution of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide in step (3) is prepared by dissolving 50 mmol / L 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 10 mmol / L N-hydroxysuccinimide in anhydrous ethanol / water mixture (4:1, v / v).

[0020] In one embodiment, the mixed solution in step (3) is added dropwise at a rate of 1 cm 2 The material was added dropwise in an amount of 0.1 mL.

[0021] In one embodiment, the mass volume concentration of the polyethylene glycol-diamine solution in step (3) is 5% to 20%, g / ml; preferably 15%, g / ml.

[0022] In one embodiment, the amount of the polyethylene glycol-diamine solution added in step (3) is equal to the amount of the mixed solution added.

[0023] In one embodiment, the overnight freezing temperature in step (3) is -60°C to -80°C.

[0024] In one embodiment, the freeze-drying conditions of step (3) are: -4 Freeze-dry at -60 to -80°C for 24 to 36 hours at kPa.

[0025] The second object of the present invention is to provide a ready-to-use sodium alginate@paper material obtained by the above-described method.

[0026] The third object of the present invention is the use of the above-mentioned ready-to-use sodium alginate@paper material in cell culture.

[0027] Beneficial effects:

[0028] The preparation of the ready-to-use sodium alginate@paper material of the present invention specifically comprises the following steps: dropping sodium alginate onto a paper material, cross-linking the sodium alginate hydrogel on the paper material using polyethylene glycol-diamine, 1-ethyl-(3-dimethylaminopropyl)-carbodiimide hydrochloride, and N-hydroxysuccinimide, and converting the sodium alginate hydrogel on the paper into an aerogel by freeze-drying to prepare the ready-to-use sodium alginate@paper material. Compared with traditional hydrogels, the ready-to-use material avoids the problems of difficulty in dissolving the prepared hydrogel and the generation of bubbles that are difficult to eliminate when mixed with cells.

[0029] In addition, this ready-to-use sodium alginate@paper material is non-cytotoxic and has good stability. It can be stored for 120 days at room temperature and away from light. There is no significant difference in cell adhesion ability and cell three-dimensional culture ability between the ready-to-use sodium alginate@paper material and the freshly prepared sodium alginate@paper material. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The swelling rate measurement data of materials prepared with different concentrations of polyethylene glycol-diamine;

[0031] Figure 2 The following are pictures of freeze-dried sodium alginate@paper materials without and with cross-linking agents added; (A) freeze-dried sodium alginate@paper materials without cross-linking agents; (B) ready-to-use sodium alginate@paper materials;

[0032] Figure 3 Scanning electron microscope images of sodium alginate@paper materials; (A) Paper; (B) Freshly prepared sodium alginate@paper materials; (C) Ready-to-use sodium alginate@paper materials;

[0033] Figure 4 Fourier transform infrared spectra of sodium alginate@paper materials; (A) Paper; (B) Freshly prepared sodium alginate@paper materials; (C) Ready-to-use sodium alginate@paper materials;

[0034] Figure 5 The results of cytotoxicity assays of two-dimensional and three-dimensional cultures of ready-to-use sodium alginate@paper materials; (A) three-dimensional cultured cells; (B) two-dimensional cultured cells;

[0035] Figure 6 Laser confocal microscopy images of cell adhesion of sodium alginate@paper materials; (A) Ready-to-use sodium alginate@paper materials; (B) Freshly prepared and ready-to-use sodium alginate@paper materials;

[0036] Figure 7Scanning electron micrographs of cells cultured on sodium alginate@paper materials; (A) Ready-to-use sodium alginate@paper materials; (B) Freshly prepared and ready-to-use sodium alginate@paper materials;

[0037] Figure 8 Data graph showing the effect of storage time of ready-to-use sodium alginate@paper materials on cytotoxicity; (A) cell culture for 24 hours; (B) cell culture for 48 hours; (C) cell culture for 72 hours;

[0038] Figure 9 Data graph showing the effect of storage time of ready-to-use sodium alginate@paper materials on cell adhesion; (A) 0 day storage; (B) 40 day storage; (C) 80 day storage; (D) 120 day storage;

[0039] Figure 10 Fourier transform infrared spectra of ready-to-use sodium alginate@paper material after 120 days of storage; (A) 120 days of storage; (B) 0 days of storage;

[0040] Figure 11 Scanning electron micrographs of ready-to-use sodium alginate@paper materials stored for 120 days; (A) Freeze-dried sodium alginate@paper materials without added cross-linking agent after 0 days of storage; (B) Freeze-dried sodium alginate@paper materials without added cross-linking agent after 120 days of storage; (C) Ready-to-use sodium alginate@paper materials after 0 days of storage; (D) Ready-to-use sodium alginate@paper materials after 120 days of storage. DETAILED DESCRIPTION

[0041] The technical solutions in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention. The following specific embodiments further describe the present invention.

[0042] The test method involved in the present invention is:

[0043] 1. Scanning electron microscopy characterization

[0044] 200 μL of 1×10 6 A logarithmically growing HEK 293 cell suspension at 10 cells / mL was dripped onto the ready-to-use sodium alginate@paper material and incubated in a cell culture incubator at 37°C and 5% CO2 for 1 hour. The ready-to-use sodium alginate@paper material was then rapidly frozen to -25°C in a dedicated temperature-controlled sample cup and photographed using a Phenom scanning electron microscope. The remaining samples were sputtered with gold at a current of 15-20 mA for 30-50 seconds and then photographed using a scanning electron microscope.

[0045] 2. Fourier transform infrared spectroscopy characterization

[0046] Place the flat surface of the sample close to the crystal and use a pressure rod (or manual pressing device) to apply uniform pressure to ensure that there is no gap between the sample and the crystal. Parameter setting: Scan range is 4000-1000cm -1 , with a resolution of 4cm -1 , the number of scans is 32.

[0047] 3. Cytotoxicity Assay

[0048] Cytotoxicity was determined using a lactate dehydrogenase release assay kit. Ready-to-use sodium alginate@paper materials were placed in a 96-well plate and sterilized overnight with UV light. HEK 293 cells in the logarithmic growth phase were then incubated at 4×10 4 Cells were seeded at a density of 10 cells / well in a 96-well plate containing ready-to-use sodium alginate@paper material. Simultaneously, the same number of cells was seeded in a 96-well plate without ready-to-use sodium alginate@paper material as a blank. Three replicate wells were set up for each group. After incubation in a cell culture incubator at 37°C and 5% CO₂ for 24, 48, and 72 hours, the 96-well plates were centrifuged at 400g for 5 minutes in a multiwell plate centrifuge. The supernatant was aspirated, and 150 μL of lactate dehydrogenase releasing reagent diluted 10-fold in PBS was added. The plate was shaken to mix thoroughly, and the cells were incubated in the cell culture incubator for another hour. The cell culture plate was then centrifuged at 400g for 5 minutes in a multiwell plate centrifuge. 120 μL of supernatant from each well was transferred to the corresponding well of a new 96-well plate and immediately assayed.

[0049] 4. Cell Adhesion Assay

[0050] The concentration was 1×10 6 cells / mL in the logarithmic growth phase HEK 293 cells were stained with SYTO TM 9 After staining with green fluorescent nucleic acid dye, 200 μL of cell suspension was added to the ready-to-use sodium alginate@paper material and cultured in a cell culture incubator at 37°C and 5% CO2 for 1 hour. The adhesion of cells on the material was then observed using a laser confocal microscope.

[0051] 5. Swelling rate test

[0052] The mass of the dry, ready-to-use sodium alginate@paper material is weighed as m1. It is then allowed to absorb water at room temperature. The water on the surface of the material is absorbed and the mass is weighed as m2. The sample is weighed three times every 20 minutes. The swelling rate is calculated using formula (1):

[0053] Swelling rate (%) = (m2-m1) / m1(1)

[0054] 6. Porosity test

[0055] The porosity of ready-to-use sodium alginate@paper material was characterized by solvent replacement method:

[0056] The mass of the dry, ready-to-use sodium alginate@paper material is weighed as m1, which is then soaked in anhydrous ethanol overnight. The anhydrous ethanol on the surface of the material is then absorbed and weighed as m2. The porosity is calculated using formula (2):

[0057] Porosity (%) = (m2 - m1) / ρv (2)

[0058] Where: ρ is the density of anhydrous ethanol, v is the volume of the material.

[0059] 7. Stability test

[0060] The ready-to-use sodium alginate@paper material was placed in a clean culture dish and stored at room temperature for 40 days, 80 days, and 120 days. The cytotoxicity, cell adhesion, porosity, infrared spectrum, and scanning electron microscopy results of the ready-to-use sodium alginate@paper material stored for 40 days, 80 days, and 120 days were compared with those of the freshly prepared ready-to-use sodium alginate@paper material (the cytotoxicity, cell adhesion, porosity, and infrared spectrum determination methods were the same as the above experimental steps) to analyze the stability of the material.

[0061] Example 1

[0062] A method for preparing a ready-to-use sodium alginate@paper material for three-dimensional cell culture comprises the following steps:

[0063] (1) Add 0.61 g of sodium alginate to 30 mL of ultrapure water and stir continuously in a 70°C water bath for about 8 h to completely dissolve it to prepare a 2% sodium alginate solution;

[0064] (2) Every 1cm 2 0.1 mL of 2% sodium alginate solution was added dropwise onto Whatman No. 2 filter paper (pore size 8 μm) to allow it to evenly penetrate the paper material to obtain ready-to-use sodium alginate@paper material;

[0065] (3) Prepare and use sodium alginate@paper material at a rate of 1 cm 2 0.1 mL of a mixture of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide was added dropwise to the material; the mixture was prepared by dissolving 50 mmol / L 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and 10 mmol / L N-hydroxysuccinimide in anhydrous ethanol / water mixture (4:1, v / v); 15% (w / v, g / ml) polyethylene glycol-diamine was then added dropwise in the same amount. Finally, the material was placed in a -80°C refrigerator and frozen overnight, and then heated at 1×10 -4kPa at -60 °C and freeze-dried for 24 h to obtain a ready-to-use sodium alginate@paper material.

[0066] Example 2

[0067] The only difference from Example 1 is that the concentrations of polyethylene glycol-diamine in step (3) are adjusted to 5%, 10% and 20% respectively. Other parameters and conditions are the same as those in Example 1.

[0068] Comparative Example 1

[0069] A method for preparing a sodium alginate@paper material for three-dimensional cell culture comprises the following steps:

[0070] (1) Add 0.61 g of sodium alginate to 30 mL of ultrapure water and stir continuously in a 70°C water bath for about 8 h to completely dissolve it to prepare a 2% sodium alginate solution;

[0071] (2) Every 1cm 2 0.1 mL of 2% sodium alginate solution was added dropwise onto the Whatman No. 2 filter paper (pore size 8 μm) to make it evenly permeate the paper material to obtain a ready-to-use sodium alginate@paper material; after freezing it in a -80°C refrigerator overnight, -4 kPa at -60 ° C and freeze-dried for 24 h to obtain freeze-dried sodium alginate@paper material.

[0072] Result Analysis

[0073] 1. The swelling rate test of ready-to-use sodium alginate@paper composites prepared with different concentrations of polyethylene glycol-diamine is as follows: Figure 1 As shown:

[0074] Depend on Figure 1 The results show that the ready-to-use sodium alginate-paper composite prepared with a 15% polyethylene glycol-diamine crosslinker concentration exhibited the lowest swelling rate and the best crosslinking effect. A high degree of crosslinking creates tighter connections between polymer chains, resulting in a more stable network structure and a correspondingly lower swelling rate.

[0075] 2. Morphological characterization of ready-to-use sodium alginate@paper materials

[0076] like Figure 2 As shown, a thin layer of white gel evenly covers the paper on the freeze-dried sodium alginate@paper material without adding a cross-linking agent ( Figure 2 A). The white gel on the ready-to-use sodium alginate@paper material becomes transparent under the action of the crosslinking agent ( Figure 2 B).

[0077] Paper is a sheet made of plant fibers. Paper materials present a three-dimensional paper fiber structure with a high specific surface area, which can provide a three-dimensional scaffold for in vitro cell culture that simulates the in vivo cell microenvironment ( Figure 3 A). Sodium alginate is added dropwise onto paper to prepare a ready-to-use sodium alginate@paper material. Sodium alginate hydrogel is retained on the paper fibers ( Figure 3 B). Sodium alginate hydrogel was also observed to be trapped on the paper fibers in the ready-to-use sodium alginate@paper material ( Figure 3 C), which has a similar morphology to the ready-made sodium alginate@paper material.

[0078] 3. Infrared spectroscopy characterization of ready-to-use sodium alginate@paper materials

[0079] The functional group composition of paper, freshly prepared sodium alginate@paper material, and ready-to-use sodium alginate@paper material was determined by Fourier transform infrared spectroscopy. Figure 4 As shown, the paper (curve A) and the prepared sodium alginate@paper material (curve B) are at 3300cm -1 , 2900cm -1 and 1600cm -1 There are absorption peaks on the left and right, which represent the stretching vibrations of -OH, -CH and -C=O respectively. Compared with paper (curve A) and ready-to-use sodium alginate@paper material (curve B), the ready-to-use sodium alginate@paper material (curve C) has a peak at 1550cm -1 An absorption peak was generated, which represented the coupling of NH bending vibration and CN stretching vibration, indicating that under the action of the cross-linker, -NH2 and -COOH were coupled to form a new amide bond, proving that sodium alginate was successfully cross-linked.

[0080] 4. Toxicity test of ready-to-use sodium alginate@paper material

[0081] Biocompatibility is an important indicator for evaluating materials for three-dimensional cell culture. Taking HEK 293 cells as an example, the cytotoxicity of cells cultured in ready-to-use sodium alginate@paper materials was measured using a lactate dehydrogenase release assay kit and compared with the cytotoxicity of cells cultured in two-dimensional culture. Figure 5 As shown in the data, the lactate dehydrogenase released by HEK293 cells cultured in the ready-to-use sodium alginate@paper material did not show an increasing trend with the extension of culture time, and the content of lactate dehydrogenase released was comparable to that of the two-dimensional cultured cells, indicating that the ready-to-use sodium alginate@paper material had no cytotoxicity.

[0082] 5. Cell Adhesion Assay of Ready-to-Use Sodium Alginate@Paper Materials

[0083] Taking HEK 293 cells as an example, SYTO TM9 fluorescent dyes were used to label cells and study the adhesion of cells on the ready-to-use sodium alginate@paper material. TM 9 Fluorescent dye-labeled cells adhered evenly to the ready-to-use sodium alginate@paper material ( Figure 6 A), and the adhesion effect is comparable to that of the ready-made sodium alginate@paper material ( Figure 6 B).

[0084] 6. Scanning electron microscopy characterization of cells cultured in ready-to-use sodium alginate@paper materials

[0085] Taking HEK 293 cells as an example, the three-dimensional culture of cells on ready-to-use sodium alginate@paper materials was studied. Cells were successfully cultured on ready-to-use sodium alginate@paper materials, maintaining the interaction between cells ( Figure 7 A), the culture effect is equivalent to that of the prepared sodium alginate@paper material ( Figure 7 B).

[0086] 7. Stability test of ready-to-use sodium alginate@paper material

[0087] Stability is another important metric for evaluating the application of ready-to-use materials. Using HEK 293 cells as an example, the cytotoxicity and cell adhesion of the ready-to-use sodium alginate@paper materials were measured after 0, 40, 80, and 120 days of storage. The porosity of the ready-to-use sodium alginate@paper materials was also measured using a solvent displacement method after 0, 40, 80, and 120 days of storage to assess their stability.

[0088] The results are as follows Figure 8 As shown in the figure, there was no significant difference in the cytotoxicity of the ready-to-use sodium alginate@paper material after storage for 40 days, 80 days, and 120 days compared with the freshly prepared ready-to-use sodium alginate@paper material.

[0089] like Figure 9 As shown, SYTO TM 9 Fluorescent dye-labeled cells evenly adhered to the ready-to-use sodium alginate@paper material after storage for 40 days, 80 days, and 120 days, and the cell adhesion ability was comparable to that of the freshly prepared ready-to-use sodium alginate@paper material.

[0090] like Figure 10 As shown in Figure 3, the chemical structure of the ready-to-use sodium alginate@paper material after storage for 120 days did not change compared with that of the freshly prepared ready-to-use sodium alginate@paper material.

[0091] Compared with freeze-dried sodium alginate@paper material without cross-linking agent stored for 0 days ( Figure 11 A) Comparison, after 120 days of storage, part of the sodium alginate hydrogel in the freeze-dried sodium alginate@paper material without adding cross-linking agent fell off from the paper fibers ( Figure 11 B), this is because during the long-term storage of 120 days, there is no cross-linking agent, and the freeze-dried sodium alginate@paper material without adding cross-linking agent is unstable. Compared with the ready-to-use sodium alginate@paper material stored for 0 days ( Figure 11 C) Comparison, the sodium alginate hydrogel in the ready-to-use sodium alginate@paper material stored for 120 days did not fall off from the paper fibers ( Figure 11 D) This is due to the cross-linking of sodium alginate hydrogel to produce amide bonds, which make the bond tighter and improve the stability of the ready-to-use sodium alginate@paper material.

[0092] The porosity test results of the ready-to-use sodium alginate@paper material are shown in Table 1. The porosity of the ready-to-use sodium alginate@paper material after storage for 40 days, 80 days, and 120 days is not significantly different from that of the freshly prepared ready-to-use sodium alginate@paper material.

[0093] Table 1 Effect of storage time of ready-to-use sodium alginate@paper materials on porosity (mean ± SD)

[0094]

[0095] The five tests above demonstrate that the properties of the ready-to-use sodium alginate@paper material remain unchanged for 120 days, demonstrating excellent stability. This is because the amide bonds formed after cross-linking possess excellent resistance to hydrolysis. Furthermore, the sodium alginate hydrogel in the paper transforms into aerogel upon freeze-drying. Compared to hydrogels, aerogels are more adaptable to environmental changes and can maintain stable properties under varying conditions of temperature, humidity, and light, further enhancing the stability of the sodium alginate hydrogel in the paper. Therefore, the ready-to-use sodium alginate@paper material exhibits excellent stability even during long-term storage.

[0096] The embodiments provided above are not intended to limit the scope of the present invention, nor are the steps described to limit their execution order. Any obvious improvements to the present invention made by those skilled in the art in combination with existing common knowledge shall fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for preparing a ready-to-use sodium alginate@paper material for three-dimensional cell culture, characterized in that: The method comprises the following steps: (1) dissolving sodium alginate in water to prepare a sodium alginate solution; (2) adding the sodium alginate solution obtained in step (1) dropwise onto the paper material to obtain sodium alginate@paper material; (3) A mixture of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide is first added dropwise to the sodium alginate@paper material prepared in step (2), and then a polyethylene glycol-diamine solution is added dropwise. Finally, the sodium alginate@paper material is frozen overnight and then freeze-dried to obtain a ready-to-use sodium alginate@paper material.

2. The method according to claim 1, characterized in that The concentration of the sodium alginate solution in step (1) is 2-5%, g / ml.

3. The method according to claim 1, characterized in that The method of adding dropwise in step (2) is to add dropwise every 1cm 2 0.1 mL of sodium alginate solution was added dropwise onto the paper material to allow it to penetrate evenly into the paper material.

4. The method according to claim 1, wherein The mixed solution of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide in step (3) is prepared by dissolving 50 mmol / L 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 10 mmol / L N-hydroxysuccinimide in an anhydrous ethanol / water mixture.

5. The method according to claim 1, wherein The mixed solution in step (3) is added dropwise at a rate of 1 cm 2 The material was added dropwise in an amount of 0.1 mL.

6. The method according to claim 1, characterized in that The mass volume concentration of the polyethylene glycol-diamine solution in step (3) is 5% to 20%, g / ml.

7. The method according to claim 1, characterized in that The amount of the polyethylene glycol-diamine solution added in step (3) is equal to the amount of the mixed solution added.

8. The method according to claim 1, characterized in that Step (3) freeze drying conditions: at 1×10 -4 Freeze-dry at -60 to -80°C for 24 to 36 hours at kPa.

9. Ready-to-use sodium alginate@paper material obtained by the method according to any one of claims 1 to 8.

10. Use of the ready-to-use sodium alginate@paper material according to claim 9 in cell culture.

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