Preparation method and application of collagen scaffold

Abstract

The invention discloses a preparation method and application of a collagen scaffold, and relates to the technical field of biomedical materials. According to the preparation method, the porous collagen scaffold material with a transparent structure is prepared by taking the bovine fascia as a raw material and utilizing synergistic cooperation of parameters in each step, and is suitable for adhesion and migration of tumor cells; the collagen material prepared by the invention can obviously improve the gel strength, and can more effectively show the pathological characteristics of tumors in vivo; according to the method, a traditional chemical cross-linking agent does not need to be added, and the risks of solvent residues, cytotoxicity, activity denaturation, calcification and the like are greatly reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical materials, and in particular relates to a preparation method of a collagen scaffold and application thereof. Background Art

[0002] Previous research results on the mechanism of tumor immune escape were mainly obtained under the traditional in vitro two-dimensional culture system. However, under two-dimensional culture conditions, tumor cells mostly grow in layers, and the interactions between cells and cells and the extracellular matrix are limited to a certain direction of the cells, resulting in abnormal polar distribution of cell membrane proteins, cytoskeleton construction, and intracellular signal transduction. Compared with cells in vivo, the abnormal polar distribution of morphology and membrane surface molecules (such as integrins) is the most significant change in tumor cells under two-dimensional culture conditions. Three-dimensional culture is believed to provide cells with a three-dimensional environment more similar to that in vivo. Recent studies have also shown that tumor cells cultured in three dimensions can better simulate the morphology and characteristics of tumors in vivo, so they have better clinical relevance for tumor research. Compared with the complex environment in vivo, its conditions are single and controllable. Three-dimensional culture meets people's needs to understand the in vivo cell behavior and molecular mechanism research with a relatively simple method. It has gradually become the third research method after the two-dimensional culture system and in vivo experiments, and also a bridge connecting in vitro experiments and the real state in vivo.

[0003] There are two main methods of three-dimensional culture: spheroid culture and biological scaffold material culture. Compared with spheroid culture, scaffold culture can provide certain mechanical strength and spatial structure. With the help of biomaterials, it can also provide interaction between cells and extracellular matrix to form a specific tissue-like three-dimensional spatial structure, which can better simulate the real environment in the body. Therefore, it has a wide range of applications in three-dimensional culture research. With the development of tumor engineering, a variety of new extracellular matrices have been used in the preparation of three-dimensional scaffolds for culturing tumor cells.

[0004] However, there are still some challenges in the clinical application of extracellular matrix-based 3D scaffolds.

[0005] The extracellular matrix of the tumor is derived from animal gel, which contains many uncertain components, resulting in its clinical application being limited. Most of the existing technologies use decellularization technology to process. By physical, chemical or enzymatic methods, the cells in the tissue are removed, and the original tissue shape and collagen components can be completely maintained. These decellularized extracellular matrices are activated by certain methods and can be directly applied to tissue damage repair. Cells are inoculated on collagen materials, and cell proliferation, differentiation and apoptosis can be directly observed. The rigidity of the decellularized matrix-derived hydrogel obtained by the decellularization method is much lower than that of the solid extracellular matrix. The hydrogel is usually chemically cross-linked with the macromolecular groups in the extracellular matrix to increase the rigidity of the decellularized matrix scaffold, but the use of chemical cross-linking agents can cause side effects such as high cytotoxicity, active degeneration and calcification. Although the use of natural cross-linking agents can reduce cytotoxicity and immunogenicity, the cross-linked scaffold is usually colored, which is not conducive to light microscopy observation and the cross-linking takes a long time. The mechanical strength of collagen is significantly increased after cross-linking, which is the main reason for the increase in the hardness of the tumor extracellular matrix. The hardness of ECM can regulate some property changes in the tumor occurrence process. Therefore, increasing the hardness of the collagen scaffold can more effectively display the pathological characteristics of tumors in vivo by culturing tumor cells in three dimensions on the collagen scaffold.

[0006] In normal tissues, collagen is curved and uneven. However, during tumorigenesis, many collagens aggregate into linear structures and become denser. Changes in collagen structure can affect the diffusion of drugs into tumor cells. In order to simulate a more realistic tumor microenvironment, it is necessary to develop a new collagen fiber that can effectively improve tumor cell survival and remove cells, lipids and miscellaneous proteins in animal gels, so as to explore the main factors and molecular mechanisms of tumor immune escape regulated by three-dimensional environment. Summary of the invention

[0007] In view of the current state of the art, the technical problem to be solved by the present invention is to overcome the problem that the existing collagen scaffold uses chemical cross-linking agents to improve the mechanical strength, which leads to side effects such as cytotoxicity, active degeneration and calcification. By using the method of ultra-high pressure coordinated with organic weak acid combination, not only the mechanical strength of the collagen scaffold is effectively increased, but also the purity of the collagen is improved, and the application is reduced. The present invention uses organic weak acid combination to treat bovine fascia to prepare collagen material, which can significantly improve the gel strength. In addition, the method of ultra-high pressure treatment coordinated with organic weak acid combination treatment synergistically enhances the effect, which is more conducive to the proliferation of tumor cells.

[0008] The purpose of the present invention is to provide a collagen three-dimensional scaffold material which is prepared by using bovine fascia as raw material and undergoing ultrahigh pressure treatment in conjunction with organic weak acid combined treatment.

[0009] Another object of the present invention is to provide the use of the collagen scaffold material in three-dimensional culture of tumor cells.

[0010] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0011] A method for preparing a collagen scaffold material comprises the following steps:

[0012] (1) Remove and clean the beef fascia;

[0013] (2) soaking the cleaned beef fascia in a weak organic acid combination, wherein the weak organic acid combination is acetic acid and citric acid;

[0014] The concentration of the acetic acid is 0-0.03 mol / L, and the concentration of the citric acid is 0-0.03 mol / L; the volume ratio of the organic weak acid combination acetic acid solution to the citric acid solution is 1:(1-3);

[0015] (3) treating the relaxed beef fascia soaked in the acid treatment in step (2) with an alkaline solution to neutralize the acid treatment;

[0016] (4) soaking the bovine fascia treated with alkali neutralization and soaking in step (3) in a decellularizing solution;

[0017] (5) washing the product of step (4) in a solution containing NaCl;

[0018] (6) subjecting the product of step (5) to ultrahigh pressure treatment, wherein the ultrahigh pressure pressure is 200 MPa to 500 MPa, the ultrahigh pressure time is 5 to 15 min, the extraction temperature is 50°C to 60°C, and the extraction time is 4 h;

[0019] (7) Soaking the product of step (6) in MES buffer;

[0020] (8) dialyzing the product of step (7);

[0021] (9) The dialyzed product of step (8) is first pre-frozen and then rapidly freeze-dried. 60 Sterilization by irradiation;

[0022] (10) Cutting the freeze-dried collagen material.

[0023] The above technical solution provided by the present invention has at least the following beneficial effects compared with the prior art:

[0024] 1. The present invention selectively utilizes different condition parameters in each step to prepare a porous, structurally transparent collagen scaffold material suitable for tumor cell adhesion and migration.

[0025] 2. Using a combination of organic weak acids to treat bovine fascia to prepare collagen materials can significantly improve gel strength and regulate the changes in the properties of the tumor process. The three-dimensional culture of tumor cells on this collagen scaffold can more effectively display the pathological characteristics of tumors in vivo.

[0026] 3. The method of ultra-high pressure treatment and organic weak acid combined treatment has synergistic effects. Compared with other combinations of organic weak acid treatment, it can significantly reduce tumor cell apoptosis and is more conducive to tumor cell proliferation. This combined method does not require the addition of traditional chemical cross-linking agents, greatly reducing the risks of solvent residues, cytotoxicity, active denaturation and calcification. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is an electron micrograph of a longitudinal section of collagen material, showing that the pores are transparent in the longitudinal direction, which is conducive to guiding cells to achieve directional growth.

[0028] Figure 2 This is an electron microscope image of a cross-section of the collagen material, showing that the material is a porous scaffold structure with irregular rectangular or polygonal pores and a large number of filaments inside, providing sufficient three-dimensional space for cell adhesion and proliferation.

[0029] Figure 3 It is the proliferation of tumor cells cultured under different conditions. DETAILED DESCRIPTION

[0030] The embodiments of the present invention are described in detail below. The embodiments provided by the present invention are exemplary and are intended to be used to explain the present invention, but should not be understood as limiting the present invention.

[0031] Example 1

[0032] In this embodiment, bovine fascia is used as a raw material to produce a collagen three-dimensional scaffold material (under different ultrahigh pressure conditions).

[0033] (1) First, remove other tissues on the beef fascia and then wash it with deionized water three times;

[0034] (2) Cut the cleaned bovine fascia in step (1) into pieces, soak it in 0.01 mol / L acetic acid solution, and store it in a refrigerator at 4-8°C for 2 hours; then soak it in 0.01 mol / L citric acid solution and store it in a refrigerator at 4-8°C for 4 hours (the solution is changed every 2 hours); the volume ratio of acetic acid solution to citric acid solution is 1:2; then wash it with deionized water 10 times, each time for 10 minutes;

[0035] (3) soaking the relaxed bovine fascia obtained in step (2) in a 0.02 mol / L Na2HPO4 solution for 3 h, and then washing it with deionized water for 15 times, each time for 10 min;

[0036] (4) Soaking the bovine fascia treated in step (3) in 1 L of a decellularization solution for 10 h, wherein the decellularization solution is a mixed aqueous solution of 0.05 mol / L Tween-80 and 0.03 mol / L sodium secondary alkyl sulfonate in a volume ratio of 2:3;

[0037] (5) Soaking the product of step (4) in a 0.15 mol / L NaCl solution and stirring for 1 h;

[0038] (6) subjecting the product of step (5) to ultrahigh pressure treatment, wherein the ultrahigh pressure pressure is 200 MPa to 500 MPa, the ultrahigh pressure time is 5 to 15 min, the extraction temperature is 50°C to 60°C, and the extraction time is 4 h;

[0039] (7) Soak the product of step (6) in 0.05 mol / L MES buffer for 3 h.

[0040] (8) The product of step (7) was placed in a dialysis bag, the dialysis fluid was 10°C deionized water, and then dialyzed for 6 days until the pH value of the dialysis fluid reached 7.0;

[0041] (9) The dialyzed product of step (8) was pre-frozen at -50°C and then rapidly freeze-dried for 24 hours. 60 Sterilization by irradiation;

[0042] (10) After completing step (9), the tensile strength of the freeze-dried collagen material is tested.

[0043] Table 1 Tensile strength of collagen materials prepared under different ultrahigh pressure conditions

[0044]

[0045] Figure 1 The tensile strength of collagen scaffolds prepared under different ultrahigh pressure conditions is shown. Figure 1 It can be seen that with the increase of tensile strain of the scaffold material, the maximum tensile strength of the collagen scaffold material prepared under different ultra-high pressure treatment conditions is different. The maximum tensile strength of the collagen scaffold material under the second group of ultra-high pressure conditions can reach 25 Mpa, and the tensile effect is the best.

[0046] Example 2

[0047] In this example, bovine fascia was used as raw material to prepare collagen three-dimensional scaffold material (different acid treatment conditions)

[0048] (1) First, remove other tissues on the beef fascia and then wash it with deionized water three times;

[0049] (2) Cut the cleaned bovine fascia in step (1) into pieces, soak it in 0-0.03 mol / L acetic acid solution, and store it in a refrigerator at 4-8°C for 2 hours; then soak it in 0-0.03 mol / L citric acid solution, and store it in a refrigerator at 4-8°C for 4 hours (change the solution every 2 hours); the volume ratio of acetic acid solution to citric acid solution is 1:(1-3); then wash it with deionized water 10 times, each time for 10 minutes;

[0050] (3) soaking the relaxed bovine fascia obtained in step (2) in a 0.02 mol / L Na2HPO4 solution for 3 h, and then washing it with deionized water for 15 times, each time for 10 min;

[0051] (4) Soaking the bovine fascia treated in step (3) in 1 L of a decellularization solution for 10 h, wherein the decellularization solution is a mixed aqueous solution of 0.05 mol / L Tween-80 and 0.03 mol / L sodium secondary alkyl sulfonate in a volume ratio of 2:3;

[0052] (5) Soaking the product of step (4) in a 0.15 mol / L NaCl solution and stirring for 1 h;

[0053] (6) subjecting the product of step (5) to ultrahigh pressure treatment, wherein the ultrahigh pressure treatment pressure is 300 MPa, the ultrahigh pressure treatment time is 10 min, the extraction temperature is 55° C., and the extraction time is 4 h;

[0054] (7) Soak the product of step (6) in 0.05 mol / L MES buffer for 3 h.

[0055] (8) The product of step (7) was placed in a dialysis bag, the dialysis fluid was 10°C deionized water, and then dialyzed for 6 days until the pH value of the dialysis fluid reached 7.0;

[0056] (9) The dialyzed product of step (8) was pre-frozen at -50°C and then rapidly freeze-dried for 24 hours. 60 Sterilization by irradiation;

[0057] (10) After completing step (9), the freeze-dried collagen material is tested for gel strength.

[0058] Table 2 Gel strength of collagen materials prepared under different acid treatment conditions

[0059]

[0060] Table 2 shows the gel strength of collagen materials prepared under different acid treatment conditions. It can be seen from the table that the gel strength of the collagen materials prepared under the second group of acid treatment conditions (0.01 mol / L acetic acid solution; 0.01 mol / L citric acid solution, volume ratio of 1:2) can reach 390.1 g / cm 2 The gel strength of collagen materials prepared by conventional methods is generally 234 g / cm 2 It can be seen that the preparation of collagen material by treating bovine fascia with the organic weak acid combination of the present invention can significantly improve the gel strength.

[0061] Example 3

[0062] In this embodiment, bovine fascia is used as raw material to produce a collagen three-dimensional scaffold material.

[0063] (1) First, remove other tissues on the beef fascia and then wash it with deionized water three times;

[0064] (2) Cut the cleaned bovine fascia in step (1) into pieces, soak it in 0.01 mol / L acetic acid solution, and store it in a refrigerator at 4-8°C for 2 h; then soak it in 0.01 mol / L citric acid solution and store it in a refrigerator at 4-8°C for 4 h (change the solution every 2 h); the volume ratio of acetic acid solution to citric acid solution is 1:2; then wash it with deionized water 10 times, each time for 10 min;

[0065] (3) soaking the relaxed bovine fascia obtained in step (2) in a 0.02 mol / L Na2HPO4 solution for 3 h, and then washing it with deionized water for 15 times, each time for 10 min;

[0066] (4) Soak the bovine fascia treated in step (3) in 1 L of a decellularization solution for 10 h. The decellularization solution is a mixed aqueous solution of 0.05 mol / L Tween-80 and 0.03 mol / L sodium secondary alkyl sulfonate in a volume ratio of 2:3;

[0067] (5) Soaking the product of step (4) in a 0.15 mol / L NaCl solution and stirring for 1 h;

[0068] (6) subjecting the product of step (5) to ultrahigh pressure treatment at an ultrahigh pressure treatment pressure of 300 MPa, an ultrahigh pressure treatment time of 10 min, an extraction temperature of 55°C, and an extraction time of 4 h;

[0069] (7) Soak the product of step (6) in 0.05 mol / L MES buffer for 3 h.

[0070] (8) The product of step (7) was placed in a dialysis bag, the dialysis fluid was 10°C deionized water, and then dialyzed for 6 days until the pH value of the dialysis fluid reached 7.0;

[0071] (9) The dialyzed product of step (8) was pre-frozen at -50°C and then rapidly freeze-dried for 24 h. 60 Sterilization by irradiation;

[0072] (10) After completing step (9), the freeze-dried collagen material is cut into pieces of 1 cm × 1 cm × 1 mm in size for use in three-dimensional culture of tumor cells.

[0073] Figure 2 The cross-section scanning electron microscope image of the collagen scaffold material prepared in this embodiment is shown: Figure 2 It can be seen that the scaffold material of this embodiment is a porous structure with a pore size of 50-80 um. The porous structure is permeable and suitable for cell adhesion and migration.

[0074] Comparative Example 1

[0075] In this comparative example, bovine fascia was used as a raw material to prepare a collagen three-dimensional scaffold material (acetic acid was replaced by a carbonic acid solution).

[0076] First, remove other tissues on the beef fascia and then wash it three times with deionized water;

[0077] (2) Cut the cleaned bovine fascia in step (1) into pieces, soak it in 0.01 mol / L carbonic acid solution, and store it in a refrigerator at 4-8°C for 2 h; then soak it in 0.01 mol / L citric acid solution and store it in a refrigerator at 4-8°C for 4 h (change the solution every 2 h); the volume ratio of acetic acid solution to citric acid solution is 1:2; then wash it with deionized water 10 times, each time for 10 min;

[0078] (3) soaking the relaxed bovine fascia obtained in step (2) in a 0.02 mol / L Na2HPO4 solution for 3 h, and then washing it with deionized water for 15 times, each time for 10 min;

[0079] (4) Soaking the bovine fascia treated in step (3) in 1 L of a decellularization solution for 10 h, wherein the decellularization solution is a mixed aqueous solution of 0.05 mol / L Tween-80 and 0.03 mol / L sodium secondary alkyl sulfonate in a volume ratio of 2:3;

[0080] (5) Soaking the product of step (4) in a 0.15 mol / L NaCl solution and stirring for 1 h;

[0081] (6) subjecting the product of step (5) to ultrahigh pressure treatment, wherein the ultrahigh pressure treatment pressure is 300 MPa, the ultrahigh pressure treatment time is 10 min, the extraction temperature is 55° C., and the extraction time is 4 h;

[0082] (7) Soak the product of step (6) in 0.05 mol / L MES buffer for 3 h.

[0083] (8) The product of step (7) was placed in a dialysis bag, the dialysis fluid was 10°C deionized water, and then dialyzed for 6 days until the pH value of the dialysis fluid reached 7.0;

[0084] (9) The dialyzed product of step (8) was pre-frozen at -50°C and then rapidly freeze-dried for 24 h. 60 Sterilization by irradiation;

[0085] (10) After completing step (9), the freeze-dried collagen material is cut into pieces of 1 cm × 1 cm × 1 mm in size for use in three-dimensional culture of tumor cells.

[0086] Comparative Example 2

[0087] In this comparative example, bovine fascia was used as a raw material to prepare a collagen three-dimensional scaffold material (citric acid was replaced by oxalic acid solution).

[0088] (1) First, remove other tissues on the beef fascia and then wash it with deionized water three times;

[0089] (2) Cut the cleaned bovine fascia in step (1) into pieces, soak it in 0.01 mol / L acetic acid solution, and store it in a refrigerator at 4-8°C for 2 h; then soak it in 0.01 mol / L oxalic acid solution and store it in a refrigerator at 4-8°C for 4 h (the solution was changed every 2 h); the volume ratio of acetic acid solution to oxalic acid solution was 1:2; then wash it with deionized water 10 times, each time for 10 min;

[0090] (3) soaking the relaxed bovine fascia obtained in step (2) in a 0.02 mol / L Na2HPO4 solution for 3 h, and then washing it with deionized water for 15 times, each time for 10 min;

[0091] (4) Soaking the bovine fascia treated in step (3) in 1 L of a decellularization solution for 10 h, wherein the decellularization solution is a mixed aqueous solution of 0.05 mol / L Tween-80 and 0.03 mol / L sodium secondary alkyl sulfonate in a volume ratio of 2:3;

[0092] (5) Soaking the product of step (4) in a 0.15 mol / L NaCl solution and stirring for 1 h;

[0093] (6) subjecting the product of step (5) to ultrahigh pressure treatment, wherein the ultrahigh pressure treatment pressure is 300 MPa, the ultrahigh pressure treatment time is 10 min, the extraction temperature is 55° C., and the extraction time is 4 h;

[0094] (7) Soak the product of step (6) in 0.05 mol / L MES buffer for 3 h.

[0095] (8) The product of step (7) was placed in a dialysis bag, the dialysis fluid was 10°C deionized water, and then dialyzed for 6 days until the pH value of the dialysis fluid reached 7.0;

[0096] (9) The dialyzed product of step (8) was pre-frozen at -50°C and then rapidly freeze-dried for 24 h. 60 Sterilization by irradiation;

[0097] (10) After completing step (9), the freeze-dried collagen material is cut into pieces of 1 cm × 1 cm × 1 mm in size for use in three-dimensional culture of tumor cells.

[0098] Comparative Example 3

[0099] In this comparative example, bovine fascia was used as a raw material to produce a collagen three-dimensional scaffold material (cross-linking agent was used to replace ultrahigh pressure treatment).

[0100] (1) First, remove other tissues on the beef fascia and then wash it with deionized water three times;

[0101] (2) Cut the cleaned bovine fascia in step (1) into pieces, soak it in 0.01 mol / L acetic acid solution, and store it in a refrigerator at 4-8°C for 2 hours; then soak it in 0.01 mol / L citric acid solution and store it in a refrigerator at 4-8°C for 4 hours (the solution is changed every 2 hours); the volume ratio of acetic acid solution to citric acid solution is 1:2; then wash it with deionized water 10 times, each time for 10 minutes;

[0102] (3) soaking the relaxed bovine fascia obtained in step (2) in a 0.02 mol / L Na2HPO4 solution for 3 h, and then washing it with deionized water for 15 times, each time for 10 min;

[0103] (4) Soaking the bovine fascia treated in step (3) in 1 L of a decellularization solution for 10 h, wherein the decellularization solution is a mixed aqueous solution of 0.05 mol / L Tween-80 and 0.03 mol / L sodium secondary alkyl sulfonate in a volume ratio of 2:3;

[0104] (5) Soaking the product of step (4) in a 0.15 mol / L NaCl solution and stirring for 1 h;

[0105] (6) Soak the product of step (5) in 0.05 mol / L MES buffer for 3 h.

[0106] (7) Add 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide (EDC) at a concentration of 0.6 mg / mL and N-hydroxysuccinimide (NHS) at a concentration of 0.3 mg / mL to the product of step (6) and cross-link the collagen material for 3 h;

[0107] (8) The product of step (7) was placed in a dialysis bag, the dialysis fluid was 10°C deionized water, and then dialyzed for 6 days until the pH value of the dialysis fluid reached 7.0;

[0108] (9) The dialyzed product of step (8) was pre-frozen at -50°C and then rapidly freeze-dried for 24 h. 60 Sterilization by irradiation;

[0109] (10) After completing step (9), the freeze-dried collagen material is cut into pieces of 1 cm × 1 cm × 1 mm in size for use in three-dimensional culture of tumor cells.

[0110] Experimental Example 1

[0111] The effects of three-dimensional cell culture scaffolds prepared under different conditions on tumor cell proliferation were detected.

[0112] The frozen breast cancer cell line MCF-7 was quickly thawed in a 36°C water bath. Then it was transferred to a 15 mL centrifuge tube, 10 mL of fresh cell culture medium was added, mixed thoroughly, centrifuged at 1200 rpm for 10 min, the supernatant was removed, the cells were resuspended in fresh culture medium, mixed again, and cultured on a cell culture plate.

[0113] When breast cancer cells grow 80%-90% on the culture plate, wash with PBS solution 3 times, add preheated 0.25% protease, digest at 37 ° C for 2 min, then use fresh culture medium to blow into cell suspension, centrifuge at 1200rpm for 10min, discard the supernatant, add fresh culture medium to blow into single cell suspension. Add 8 μL of single cell suspension to each material, place it in a 37 ° C incubator for 1.5h to promote cell adhesion, and then transfer the material to fresh culture medium for culture. After 24 h, replace the culture medium, and use the collagen scaffold material prepared in Example 3 of the present invention and Comparative Examples 1 to 3 as a three-dimensional cell material, as well as the culture plate two-dimensional culture, and place it on a small shaker in the incubator for culture, and change the culture medium once a day.

[0114] The cell proliferation was detected by direct counting method. The specific operation is as follows:

[0115] (1) Dissolve collagenase in 1% Hank's balanced salt solution and dilute to 0.1% cell digestion solution with cell culture medium for later use.

[0116] (2) Take 5 mL of collagenase digestion solution and add it to a 15 mL centrifuge tube. Add the three-dimensional cultured cells to the centrifuge tube and digest on a shaker at 37°C for 0.5-1.5 h, blowing once every 15 minutes until the material is completely degraded.

[0117] (3) Centrifuge at 900 rpm for 5 min; discard the supernatant, resuspend the cells in PBS, and centrifuge again at 900 rpm for 10 min. Repeat 4 times.

[0118] (4) Add 1 mL of 0.25% trypsin for digestion and digest at 37°C for 2 min. Add fresh cell culture medium to terminate the enzyme action and blow to form a single-cell suspension.

[0119] (5) Centrifuge at 1000 rpm for 5 min and discard the supernatant.

[0120] (6) Add fresh cell culture medium and blow to form a single-cell suspension. Calculate the number of cells directly under a microscope using a red blood cell counting plate.

[0121] like Figure 3 As shown, the number of cells on the first day is used as the base number, and the cell proliferation under the conditions of comparative examples 1 to 3 and two-dimensional culture is compared. Under two-dimensional culture conditions, the cells can still maintain proliferation on the 5th day, but if they continue to be cultured without passage, the cell number will drop significantly due to the limited space for cell growth, and a large number of cells will fall off the cell culture plate. However, the three-dimensional culture of tumor cells in the collagen scaffold materials of Example 3 and Comparative Examples 1 to 3 of the present invention, compared with the first day, the number of cells continues to proliferate after the 5th day, and the proliferation rate of cells cultured in the collagen scaffold of Example 3 is significantly higher than that of the collagen scaffold of Comparative Examples 1 to 3. The results show that the scaffold materials of Example 3 are more conducive to the proliferation of tumor cells than the collagen scaffold materials prepared under comparative examples 1 to 3 and two-dimensional culture conditions. In summary, the ultra-high pressure synergistic organic weak acid combination of the present invention has a synergistic effect.

[0122] The above embodiments are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the embodiments here. Any modifications, equivalent substitutions and improvements derived from the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A method for preparing a collagen scaffold material, characterized in that: The following steps are involved: (1) Remove and clean the beef fascia; (2) soaking the cleaned beef fascia in a weak organic acid combination, wherein the weak organic acid combination is acetic acid and citric acid; The concentration of the acetic acid is 0-0.03 mol / L, and the concentration of the citric acid is 0-0.03 mol / L; the volume ratio of the organic weak acid combination acetic acid solution to the citric acid solution is 1:(1-3); (3) treating the relaxed beef fascia soaked in the acid treatment in step (2) with an alkaline solution to neutralize the acid treatment; (4) soaking the bovine fascia treated with alkali neutralization and soaking in step (3) in a decellularizing solution; (5) washing the product of step (4) in a solution containing NaCl; (6) subjecting the product of step (5) to ultrahigh pressure treatment, wherein the ultrahigh pressure pressure is 200 MPa to 500 MPa, the ultrahigh pressure time is 5 to 15 min, the extraction temperature is 50°C to 60°C, and the extraction time is 4 h; (7) Soaking the product of step (6) in MES buffer; (8) dialyzing the product of step (7); (9) The dialyzed product of step (8) is first pre-frozen and then rapidly freeze-dried. 60 Sterilization by irradiation; (10) Cutting the freeze-dried collagen material.

2. The method for preparing the collagen scaffold material according to claim 1, characterized in that: The step (1) is washed with deionized water three times; The alkaline solution in step (3) is 0.02 mol / L Na2HPO4 solution, and then washed with deionized water 10 times, each washing for 10 minutes; The concentration of the NaCl solution in step (5) is 0.15 mol / L, and the soaking and stirring treatment time is 1 h; In step (7), the concentration of MES buffer is 0.05 mol / L and the treatment is performed for 3 h; The dialysis treatment in step (8) is to put the product of step (7) into a dialysis bag, the dialysis fluid is 10° C. deionized water, and then dialyze for 6 days until the pH value of the dialysis fluid reaches 7.0; The pre-freezing temperature in step (9) is -50°C, followed by rapid freeze-drying for 24 hours.

3. The method for preparing the collagen scaffold material according to claim 2, characterized in that: The organic weak acid combination of step (2) is acetic acid and citric acid, the concentration of the acetic acid is 0.01 mol / L, and the concentration of the citric acid is 0.01 mol / L; the volume ratio of the organic weak acid combination acetic acid solution to the citric acid solution is 1:

2.

4. The method for preparing the collagen scaffold material according to claim 3, characterized in that: The decellularization solution of step (4) includes a mixed aqueous solution of Tween-80 and sodium secondary alkyl sulfonate, wherein the concentration of Tween-80 is 0.05 mol / L, the concentration of sodium secondary alkyl sulfonate is 0.03 mol / L, and the volume ratio is 2:

3.

5. The method for preparing the collagen scaffold material according to claim 4, characterized in that: The ultra-high pressure treatment was carried out under a pressure of 300 MPa and for a time of 10 min; the extraction temperature was 55° C. and the extraction time was 4 h.

6. The collagen scaffold material obtained by the preparation method according to any one of claims 1-5.

7. Use of the collagen scaffold material according to claim 6 in three-dimensional culture of tumor cells.

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