Preparation method of 3D cell culture material

Abstract

The invention relates to the technical field of 3D cell culture materials, in particular to a preparation method of a 3D cell culture material, which comprises the following steps: S1, raw material preparation: raw materials comprise sodium alginate, chitosan, polyvinyl alcohol and a phosphate buffer according to a mass ratio of 1: (0.6-0.8): (0.1-0.3): (1-3). According to the invention, the porous structure is also beneficial to enhancing the mechanical properties of the material, after the chitosan is pretreated, the molecular chain of the chitosan can be stretched, the compatibility with other components is better, the formation of a stable network structure is facilitated, and the gelatin and the genipin are subjected to a cross-linking reaction through ultraviolet irradiation to form a three-dimensional network structure; the cross-linked structure can improve the mechanical property of the material, so that the material has better stability and deformation resistance, and the use performance of the material is effectively improved.

Description

Technical Field

[0001] The present invention relates to the technical field of 3D cell culture materials, and specifically to a preparation method of a 3D cell culture material. Background Art

[0002] A 3D cell culture material is a new type of material used to simulate the in-vivo cell growth microenvironment, which can provide a three-dimensional growth space for cells, is closer to the true physiological state in vivo, helps the growth, proliferation and differentiation of cells, and has broad application prospects in the fields of cell biology research, tissue engineering, drug R & D, etc.

[0003] In the prior art, after using sodium alginate as a base material to prepare a cell culture material, its own mechanical properties are limited, and the film-forming texture is relatively soft, which will affect the overall strength of the material. Based on this, the present invention provides a preparation method of a 3D cell culture material. Summary of the Invention

[0004] The purpose of the present invention is to provide a preparation method of a 3D cell culture material. The 3D cell culture material prepared by the present invention not only has good structural stability performance, but also has excellent light transmittance performance.

[0005] To achieve the above object, the present invention provides the following technical solution: A preparation method of a 3D cell culture material, comprising the following steps: S1: Raw material preparation. The raw materials include sodium alginate, chitosan, polyvinyl alcohol, and phosphate buffer solution. The mass ratio of sodium alginate, chitosan, polyvinyl alcohol, and phosphate buffer solution is 1:(0.6 - 0.8):(0.1 - 0.3):(1 - 3); S2: Raw material pretreatment. Pretreat the sodium alginate in the raw materials to obtain a first base material, and pretreat the chitosan in the raw materials to obtain a second base material; S3: Base material preparation. Mix and prepare the first base material, the second base material, polyvinyl alcohol, and phosphate buffer solution to obtain a base material; S4: Synthetic material preparation. The synthetic materials include gelatin and genipin. The mass ratio of gelatin to genipin is 1:(0.1 - 0.3); S5: Photo-crosslinking treatment. Irradiate gelatin and genipin with an ultraviolet lamp to complete the photo-crosslinking treatment and obtain a synthetic material; S6: Main material preparation. Mix and prepare the base material and the synthetic material to obtain a main material; S7: Functionalization treatment. Chemically modify the main material and graft extracellular matrix protein on the surface of the main material to obtain a mixed material; S8: Molding preparation. Electrospinning process the mixed material, and perform molding treatment on the obtained product to obtain a 3D cell culture material.

[0006] Further, the pretreatment method of the sodium alginate is as follows: The sodium alginate is selected as a powder with a particle size of 10 - 30 μm. The sodium alginate is mixed with deionized water to prepare a mixed solution. Cation exchange resin is added to the mixed solution. The product obtained after the mixed treatment is subjected to freeze-drying treatment. The set temperature for the freeze-drying treatment is (-50) - (-60)°C, the pressure is 0.1 mbar, and the treatment time is 20 - 30 h to obtain the first base material.

[0007] Further, the mass of the deionized water is 30 - 40% of the mass of the sodium alginate, and the mass of the cation exchange resin is 2 - 4% of the mass of the sodium alginate.

[0008] Further, the pretreatment method of the chitosan is as follows: The chitosan is selected as a powder with a particle size of 10 - 30 μm. The chitosan and the acetic acid solution are added to a water bath. The water bath is set to heat water bath to 45 - 55 r / min. The water bath is connected to a magnetic stirrer. The magnetic stirrer is set to 500 - 600 r / min, and the heat preservation and stirring treatment is carried out for 10 - 20 min. After the stirring treatment is completed, the temperature is maintained at 40 - 50°C, the pH value is adjusted to 5.8 - 6, and then sodium glycerophosphate is added dropwise. The temperature is set to 50 - 60°C and the speed is 200 - 300 r / min, and the heat preservation and stirring treatment is carried out for 1 - 2 h. The obtained product is filtered and dried to obtain the second base material.

[0009] Further, the mass of the acetic acid solution is 10 - 20% of the mass of the chitosan, the mass concentration of the acetic acid solution is 1 - 3%, and the mass of the sodium glycerophosphate is 0.2 - 0.4% of the mass of the chitosan.

[0010] Further, the method of the photocrosslinking treatment is as follows: Gelatin and genipin are mixed evenly and then irradiated with an ultraviolet lamp. The ultraviolet lamp is set to 365 nm, 10 mW / cm 2 , and the treatment time is 10 - 20 min to obtain the synthetic material.

[0011] Further, the method of the functionalization treatment is as follows: The main material and the activator are added to a mixer. The mass of the activator is 4 - 6% of the mass of the main material. The mixer is set to stir at 120 - 160 r / min for 10 - 20 min. The pH value of the obtained mixture is adjusted to 7.2 - 7.4, and then it is transferred to a reaction kettle. The reaction kettle is set to a temperature of 36 - 38°C and a rotation speed of 200 - 400 r / min, and the heat preservation and stirring treatment is carried out for 4 - 6 h. The obtained product is transferred to a centrifuge tube, and the centrifuge tube is sent into a centrifuge. Under the conditions of 4 - 8°C and 3000 - 5000 r / min, the centrifugation treatment is carried out for 20 - 26 min. The supernatant of the obtained product is removed to obtain the mixed material.

[0012] Further, the activator is prepared by mixing EDC and NHS, and the mass ratio of EDC to NHS is 1:0.2.

[0013] Further, the method for forming and preparing is as follows: The mixed material is added into an electrospinning device, the set voltage is 18 - 20 kV, the feeding speed is 0.6 - 0.8 ml / h, and the receiving distance is 10 - 14 cm to obtain a fibrous material.

[0014] Further, a mold is prepared by 3D printing, the fibrous material is used to coat the mold, and after the coating is completed, the mold is dissolved to obtain a 3D cell culture material.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. In the present invention, through the freeze-drying pretreatment of sodium alginate, a porous structure can be formed, increasing the specific surface area, improving the binding force with other components, and at the same time, the porous structure is also beneficial to enhancing the mechanical properties of the material. After the pretreatment, chitosan can make its molecular chains stretch, have better compatibility with other components, and contribute to the formation of a stable network structure. Gelatin and genipin are crosslinked by ultraviolet light irradiation to form a three-dimensional network structure, and this crosslinked structure can improve the mechanical properties of the material, endowing the material with better stability and anti-deformation ability, effectively enhancing the service performance of the material.

[0016] 2. In the present invention, through the electrospinning treatment of the prepared material, the fibrous material obtained by electrospinning has a smaller diameter and a higher porosity. This microscopic structure is beneficial to the transmission of light and reduces the scattering of light, thus ensuring the light transmission performance of the material. Specific Embodiments

[0017] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0018] It should be noted that the raw materials used in the following embodiments are all commercially available raw materials.

[0019] Embodiment 1

[0020] S1: Raw material preparation. The raw materials include sodium alginate, chitosan, polyvinyl alcohol, and phosphate buffer solution. The mass ratio of sodium alginate, chitosan, polyvinyl alcohol, and phosphate buffer solution is 1:(0.6):(0.1):(1); S2: Raw material pretreatment. Pretreat the sodium alginate in the raw materials to obtain a first base material, and pretreat the chitosan in the raw materials to obtain a second base material; S3: Base material preparation. Mix and prepare the first base material, the second base material, polyvinyl alcohol, and phosphate buffer solution to obtain a base material; S4: Preparation of synthetic materials. The synthetic materials include gelatin and genipin, and the mass ratio of gelatin to genipin is 1:(0.1); S5: Photo-crosslinking treatment. Gelatin and genipin are irradiated with an ultraviolet lamp to complete the photo-crosslinking treatment, and the synthetic materials are obtained; S6: Preparation of main materials. The base materials and the synthetic materials are mixed to prepare the main materials; S7: Functionalization treatment. The main materials are chemically modified, and extracellular matrix proteins are grafted onto the surface of the main materials to obtain the mixed materials; S8: Molding preparation. The mixed materials are processed by electrospinning, and the obtained products are molded to obtain the 3D cell culture materials.

[0021] The pretreatment method of sodium alginate is as follows: The sodium alginate is selected as a powder with a particle size of 10 μm. The sodium alginate is mixed with deionized water to obtain a mixed solution. Cation exchange resin is added to the mixed solution. The obtained product after mixing treatment is freeze-dried. The set temperature of the freeze-drying treatment is (-50)°C, the pressure is 0.1 mbar, and the treatment time is 20 h to obtain the first base material.

[0022] The mass of deionized water is 30% of the mass of sodium alginate, and the mass of cation exchange resin is 2% of the mass of sodium alginate.

[0023] The pretreatment method of chitosan is as follows: The chitosan is selected as a powder with a particle size of 10 μm. The chitosan and the acetic acid solution are added to a water bath. The water bath is set to heat to 45 r / min, and the water bath is connected to a magnetic stirrer. The magnetic stirrer is set to 500 r / min, and the heat preservation and stirring treatment is carried out for 10 min. After the stirring treatment is completed, the temperature is maintained at 40°C, and the pH value is adjusted to 5.8. Then sodium glycerophosphate is added dropwise, and the set temperature is 50°C and the speed is 200 r / min, and the heat preservation and stirring treatment is carried out for 1 h. The obtained product is filtered and dried to obtain the second base material.

[0024] The mass of the acetic acid solution is 10% of the mass of chitosan, the mass concentration of the acetic acid solution is 1%, and the mass of sodium glycerophosphate is 0.2% of the mass of chitosan.

[0025] The method of photo-crosslinking treatment is as follows: After gelatin and genipin are mixed evenly, they are irradiated with an ultraviolet lamp. The ultraviolet lamp is set to 365 nm, 10 mW / cm 2 , and the treatment time is 10 min to obtain the synthetic materials.

[0026] The method of functionalization is as follows: the main material and the activator are added into a mixer. The mass of the activator is 4% of the mass of the main material. The mixer is set to stir at 120 r / min for 10 min. The pH value of the obtained mixture is adjusted to 7.2, and then it is transferred to a reaction kettle. The reaction kettle is set at a temperature of 36 °C and a rotation speed of 200 r / min, and it is kept warm and stirred for 4 h. The obtained product is transferred to a centrifuge tube, and the centrifuge tube is placed in a centrifuge and centrifuged at 4 °C and 3000 r / min for 20 min. The supernatant of the obtained product is removed to obtain a mixed material.

[0027] The activator is prepared by mixing EDC and NHS, and the mass ratio of EDC to NHS is 1:0.2.

[0028] The method of forming and preparing is as follows: the mixed material is added into an electrospinning device, the set voltage is 18 kV, the feeding speed is 0.6 ml / h, and the receiving distance is 10 cm to obtain a fibrous material.

[0029] A mold is prepared by 3D printing, the fibrous material is used to coat the mold, and after the coating is completed, the mold is dissolved to obtain a 3D cell culture material.

[0030] Example 2

[0031] S1: Raw material preparation. The raw materials include sodium alginate, chitosan, polyvinyl alcohol, and phosphate buffer solution. The mass ratio of sodium alginate, chitosan, polyvinyl alcohol, and phosphate buffer solution is 1:(0.7):(0.2):(2); S2: Raw material pretreatment. The sodium alginate in the raw materials is pretreated to obtain a first base material, and the chitosan in the raw materials is pretreated to obtain a second base material; S3: Base material preparation. The first base material, the second base material, polyvinyl alcohol, and phosphate buffer solution are mixed and prepared to obtain a base material; S4: Synthetic material preparation. The synthetic materials include gelatin and genipin, and the mass ratio of gelatin to genipin is 1:(0.2); S5: Photo-crosslinking treatment. Gelatin and genipin are irradiated with an ultraviolet lamp to complete the photo-crosslinking treatment to obtain a synthetic material; S6: Main material preparation. The base material and the synthetic material are mixed and prepared to obtain a main material; S7: Functionalization treatment. The main material is chemically modified to graft extracellular matrix protein on the surface of the main material to obtain a mixed material; S8: Forming and preparing. The mixed material is processed by electrospinning, and the obtained product is formed to obtain a 3D cell culture material.

[0032] The pretreatment method of sodium alginate is as follows: The sodium alginate used is a powder with a particle size of 20 μm. The sodium alginate is mixed with deionized water to obtain a mixed solution. Cation exchange resin is added to the mixed solution. The product obtained after the mixed treatment is subjected to freeze-drying treatment. The set temperature for the freeze-drying treatment is (-55) °C, the pressure is 0.1 mbar, and the treatment time is 25 h to obtain the first base material.

[0033] The mass of the deionized water is 35% of the mass of the sodium alginate, and the mass of the cation exchange resin is 3% of the mass of the sodium alginate.

[0034] The pretreatment method of chitosan is as follows: The chitosan used is a powder with a particle size of 20 μm. The chitosan and the acetic acid solution are added to a water bath. The water bath is set to heat water bath to 50 r / min. The water bath is connected to a magnetic stirrer, and the magnetic stirrer is set to 550 r / min. After heat preservation and stirring treatment for 15 min, after the stirring treatment is completed, the temperature is maintained at 45 °C, the pH value is adjusted to 5.9, and then sodium glycerophosphate is added dropwise. The set temperature is 55 °C and the speed is 250 r / min. After heat preservation and stirring treatment for 1.5 h, the obtained product is filtered and dried to obtain the second base material.

[0035] The mass of the acetic acid solution is 15% of the mass of the chitosan, the mass concentration of the acetic acid solution is 2%, and the mass of the sodium glycerophosphate is 0.3% of the mass of the chitosan.

[0036] The method of photo-crosslinking treatment is as follows: Gelatin and genipin are mixed evenly and then irradiated with an ultraviolet lamp. The ultraviolet lamp is set to 365 nm, 10 mW / cm 2 , and the treatment time is 15 min to obtain the synthetic material.

[0037] The method of functionalization treatment is as follows: The main material and the activator are added to a mixer. The mass of the activator is 5% of the mass of the main material. The mixer is set to stir at 140 r / min for 15 min. The pH value of the obtained mixture is adjusted to 7.3, and then it is transferred to a reaction kettle. The reaction kettle is set to a temperature of 37 °C and a rotation speed of 300 r / min. After heat preservation and stirring treatment for 5 h, the obtained product is transferred to a centrifuge tube. The centrifuge tube is sent into a centrifuge and centrifuged at 6 °C and 4000 r / min for 23 min. The supernatant of the obtained product is removed to obtain the mixed material.

[0038] The activator is prepared by mixing EDC and NHS, and the mass ratio of EDC to NHS is 1:0.2.

[0039] The method of forming and preparation is as follows: The mixed material is added to an electrospinning device. The set voltage is 19 kV, the feeding speed is 0.7 ml / h, and the receiving distance is 12 cm to obtain the fibrous material.

[0040] Prepare a mold using 3D printing, coat the mold with fiber material, dissolve the mold after coating is completed, and obtain a 3D cell culture material.

[0041] Example 3

[0042] S1: Raw material preparation. The raw materials include sodium alginate, chitosan, polyvinyl alcohol, and phosphate buffer solution. The mass ratio of sodium alginate, chitosan, polyvinyl alcohol, and phosphate buffer solution is 1:(0.8):(0.3):(3). S2: Raw material pretreatment. Pretreat the sodium alginate in the raw materials to obtain a first base material, and pretreat the chitosan in the raw materials to obtain a second base material. S3: Base material preparation. Mix and prepare the first base material, the second base material, polyvinyl alcohol, and phosphate buffer solution to obtain a base material. S4: Synthetic material preparation. The synthetic materials include gelatin and genipin. The mass ratio of gelatin to genipin is 1:(0.3). S5: Photo-crosslinking treatment. Irradiate gelatin and genipin with an ultraviolet lamp to complete the photo-crosslinking treatment and obtain a synthetic material. S6: Main material preparation. Mix and prepare the base material and the synthetic material to obtain a main material. S7: Functionalization treatment. Chemically modify the main material and graft extracellular matrix proteins on the surface of the main material to obtain a mixture. S8: Molding preparation. Electrospinning process the mixture, and perform a molding treatment on the obtained product to obtain a 3D cell culture material.

[0043] The pretreatment method of sodium alginate is as follows: Select sodium alginate powder with a particle size of 30 μm. Mix sodium alginate with deionized water to obtain a mixed solution. Add cation exchange resin to the mixed solution. After mixing treatment, the obtained product is subjected to freeze-drying treatment. The set temperature of the freeze-drying treatment is (-60)°C, the pressure is 0.1 mbar, and the treatment time is 30 h to obtain a first base material.

[0044] The mass of deionized water is 40% of the mass of sodium alginate, and the mass of cation exchange resin is 4% of the mass of sodium alginate.

[0045] The pretreatment method of chitosan is as follows: Select chitosan powder with a particle size of 30 μm. Add chitosan and acetic acid solution to a water bath. Set the water bath to heat to 55 r / min. Connect the water bath to a magnetic stirrer and set the magnetic stirrer to 600 r / min. Perform heat preservation and stirring treatment for 20 min. After the stirring treatment is completed, keep the temperature at 50°C, adjust the pH value to 6, then dropwise add sodium glycerophosphate, set at 60°C and 300 r / min, perform heat preservation and stirring treatment for 2 h, and filter and dry the obtained product to obtain a second base material.

[0046] The mass of the acetic acid solution is 20% of the mass of chitosan, the mass concentration of the acetic acid solution is 3%, and the mass of sodium glycerophosphate is 0.4% of the mass of chitosan.

[0047] The method of photocrosslinking treatment is as follows: gelatin and genipin are mixed evenly and then irradiated with an ultraviolet lamp. The ultraviolet lamp is set at 365 nm and 10 mW / cm 2 , the treatment time is 20 min, and a synthetic material is prepared.

[0048] The method of functionalization treatment is as follows: the main material and the activator are added into a mixer. The mass of the activator is 6% of the mass of the main material. The mixer is set at 160 r / min and stirred for 20 min. The pH value of the obtained mixture is adjusted to 7.4, and then it is transferred to a reaction kettle. The reaction kettle is set at a temperature of 38 °C and a rotation speed of 400 r / min, and kept warm and stirred for 6 h. The obtained product is transferred to a centrifuge tube, and the centrifuge tube is sent into a centrifuge and centrifuged at 8 °C and 5000 r / min for 26 min. The supernatant of the obtained product is removed to prepare a mixed material.

[0049] The activator is prepared by mixing EDC and NHS, and the mass ratio of EDC to NHS is 1:0.2.

[0050] The method of forming and preparing is as follows: the mixed material is added into an electrospinning device, the voltage is set at 20 kV, the feeding speed is 0.8 ml / h, and the receiving distance is 14 cm to prepare a fiber material.

[0051] A mold is prepared by 3D printing, and the fiber material is used to coat the mold. After the coating is completed, the mold is dissolved to prepare a 3D cell culture material.

[0052] Comparative Example 1. The difference between this comparative example and Example 1 is that: this comparative example does not contain a covering material.

[0053] Comparative Example 2. The difference between this comparative example and Example 1 is that: this comparative example does not perform the treatment of soaking in the film-forming solution.

[0054] Comparative Example 3. The difference between this comparative example and Example 1 is that: this comparative example does not add vanadyl sulfate when preparing the film-forming solution.

[0055] Comparative Example 4. The difference between this comparative example and Example 1 is that: this comparative example does not perform the treatment of the soaking solution.

[0056] Performance test: The 3D cell culture materials prepared in Example 1, Example 2, Example 3, Comparative Example 1, Comparative Example 2, Comparative Example 3, and Comparative Example 4 are subjected to performance tests, and the obtained test data are recorded in the following table:

[0057] By comparing and analyzing the relevant data in the table, it can be seen that the 3D cell culture material prepared by the present invention not only has good structural stability performance, but also has excellent light transmission performance. This shows that the preparation method of the 3D cell culture material provided by the present invention has a broader market prospect and is more suitable for popularization.

[0058] In the description of this specification, the description with reference to terms such as "one embodiment", "example", "specific example", etc. means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in a suitable manner in any one or more embodiments or examples.

[0059] The preferred embodiments of the present invention disclosed above are only used to help explain the present invention. The preferred embodiments do not elaborate all the details, nor do they limit the invention to the specific embodiments described. Obviously, according to the content of this specification, many modifications and changes can be made. These embodiments are selected and specifically described in this specification to better explain the principle and practical application of the present invention, so that those skilled in the art can well understand and utilize the present invention. The present invention is only limited by the claims and their full scope and equivalents.

Claims

1. A preparation method of a 3D cell culture material, characterized in that: It includes the following steps: S1: Raw material preparation. The raw materials include sodium alginate, chitosan, polyvinyl alcohol, and phosphate buffer solution. The mass ratio of sodium alginate, chitosan, polyvinyl alcohol, and phosphate buffer solution is 1:(0.6 - 0.8):(0.1 - 0.3):(1 - 3); S2: Raw material pretreatment. Pretreat the sodium alginate in the raw materials to obtain the first base material, and pretreat the chitosan in the raw materials to obtain the second base material; S3: Base material preparation. Mix and prepare the first base material, the second base material, polyvinyl alcohol, and phosphate buffer solution to obtain the base material; S4: Synthetic material preparation. The synthetic materials include gelatin and genipin. The mass ratio of gelatin to genipin is 1:(0.1 - 0.3); S5: Photo-crosslinking treatment. Irradiate gelatin and genipin with an ultraviolet lamp to complete the photo-crosslinking treatment and obtain the synthetic material; S6: Main material preparation. Mix and prepare the base material and the synthetic material to obtain the main material; S7: Functionalization treatment. Chemically modify the main material and graft extracellular matrix protein on the surface of the main material to obtain the mixed material; S8: Molding preparation. Electrospinning process the mixed material, and perform molding treatment on the obtained product to obtain the 3D cell culture material.

2. The preparation method of the 3D cell culture material according to claim 1, characterized in that, The pretreatment method of the sodium alginate is as follows: Select the sodium alginate powder with a particle size of 10 - 30μm. Mix sodium alginate with deionized water to obtain a mixed solution. Add cation exchange resin to the mixed solution. After mixing treatment, freeze-dry the obtained product. The set temperature for freeze-drying treatment is (-50) - (-60)°C, the pressure is 0.1 mbar, and the treatment time is 20 - 30 h to obtain the first base material.

3. The preparation method of the 3D cell culture material according to claim 2, wherein The mass of deionized water is 30 - 40% of the mass of sodium alginate, and the mass of cation exchange resin is 2 - 4% of the mass of sodium alginate.

4. The preparation method of the 3D cell culture material according to claim 1, characterized in that, The pretreatment method of the chitosan is as follows: Select the chitosan powder with a particle size of 10 - 30μm. Add chitosan and acetic acid solution into a water bath. Set the water bath to heat to 45 - 55 r / min. The water bath is connected to a magnetic stirrer. Set the magnetic stirrer to 500 - 600 r / min and perform heat preservation and stirring treatment for 10 - 20 min. After completing the stirring treatment, keep the temperature at 40 - 50°C, adjust the pH value to 5.8 - 6, then dropwise add sodium glycerophosphate, set at 50 - 60°C, 200 - 300 r / min, perform heat preservation and stirring treatment for 1 - 2 h. Filter and dry the obtained product to obtain the second base material.

5. The preparation method of the 3D cell culture material according to claim 4, wherein The mass of acetic acid solution is 10 - 20% of the mass of chitosan, the mass concentration of acetic acid solution is 1 - 3%, and the mass of sodium glycerophosphate is 0.2 - 0.4% of the mass of chitosan.

6. The preparation method of the 3D cell culture material according to claim 1, characterized in that, The method of the photocrosslinking treatment is as follows: gelatin and genipin are mixed evenly and then irradiated with an ultraviolet lamp. The ultraviolet lamp is set at 365 nm and 10 mW / cm 2 , and the treatment time is 10 - 20 min to obtain the synthetic material.

7. The preparation method of the 3D cell culture material according to claim 1, characterized in that, The method of the functionalization treatment is as follows: the main material and the activator are added into a mixer. The mass of the activator is 4-6% of the mass of the main material. The mixer is set to stir at 120-160 r / min for 10-20 min. The pH value of the obtained mixture is adjusted to 7.2-7.4, and then it is transferred to a reaction kettle. The reaction kettle is set at a temperature of 36-38 °C and a rotation speed of 200-400 r / min, and it is kept warm and stirred for 4-6 h. The obtained product is transferred to a centrifuge tube, and the centrifuge tube is sent into a centrifuge. It is centrifuged at 4-8 °C and 3000-5000 r / min for 20-26 min. The supernatant of the obtained product is removed to obtain a mixed material.

8. The preparation method of the 3D cell culture material according to claim 7, wherein, The activator is prepared by mixing EDC and NHS, and the mass ratio of EDC to NHS is 1:0.

2.

9. The preparation method of the 3D cell culture material according to claim 1, wherein, The method of the forming preparation is as follows: the mixed material is added into an electrospinning device. The voltage is set at 18-20 kV, the feeding speed is 0.6-0.8 ml / h, and the receiving distance is 10-14 cm to obtain a fibrous material.

10. The preparation method of the 3D cell culture material according to claim 9, characterized in that, A mold is prepared by 3D printing, and the fibrous material is used to coat the mold. After the coating is completed, the mold is dissolved to obtain a 3D cell culture material.

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