Preparation method and application of collagen scaffold

Abstract

The present invention discloses a method for preparing a collagen scaffold and its application, relating to the field of biomedical materials technology. This method utilizes bovine fascia as raw material and utilizes the coordinated parameters of various steps to produce a porous, transparent collagen scaffold material suitable for tumor cell adhesion and migration. The collagen material prepared by this method significantly improves gel strength and can more effectively reveal the pathological characteristics of tumors in vivo. The method does not require the addition of traditional chemical crosslinkers, significantly reducing the risks of solvent residue, cytotoxicity, active denaturation, and calcification.

Description

Technical Field

[0001] The present 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 on tumor immune escape mechanisms has primarily been conducted using traditional in vitro two-dimensional (2D) culture systems. However, under 2D culture conditions, tumor cells typically grow in layers, restricting cell-to-cell and cell-extracellular matrix interactions to a single orientation. This leads to abnormal polarity in the distribution of cell membrane proteins, cytoskeletal organization, and intracellular signaling. Compared to in vivo cells, abnormal polarity in morphology and membrane surface molecules (such as integrins) is the most striking change in tumor cells cultured under 2D conditions. 3D culture is believed to provide cells with a 3D environment more similar to that found in vivo. Recent studies have also demonstrated that 3D-cultured tumor cells can better mimic the morphology and characteristics of in vivo tumors, thus offering greater clinical relevance for cancer research. Compared to the complex in vivo environment, 3D culture, with its simpler and more controllable conditions, meets the need for understanding in vivo cellular behavior and molecular mechanisms using a relatively simple approach. It has gradually become a third research method after 2D culture systems and in vivo experiments, serving as a bridge between in vitro experiments and the real-world in vivo environment.

[0003] Three-dimensional culture primarily involves spheroidization and scaffolding. Compared to spheroidization, scaffolding provides a certain degree of mechanical strength and spatial structure. Biomaterials also facilitate interaction between cells and the extracellular matrix, forming a specific tissue-like three-dimensional spatial structure that better simulates the in vivo environment. Therefore, it has been widely used in three-dimensional culture research. With the development of tumor engineering, a variety of new extracellular matrices have been used to prepare three-dimensional scaffolds for culturing tumor cells.

[0004] However, there are still some challenges in the clinical application of extracellular matrix to prepare three-dimensional scaffolds.

[0005] The extracellular matrix (ECM) of a tumor is derived from animal-derived gels, which contain many unidentified components, limiting its clinical application. Most existing technologies utilize decellularization techniques, removing cells from tissues through physical, chemical, or enzymatic methods, while preserving the original tissue shape and collagen composition. These decellularized ECMs are activated using specific methods and can be directly applied to tissue repair. Cells are seeded onto collagen materials, allowing for direct observation of cell proliferation, differentiation, and apoptosis. The stiffness of hydrogels derived from acellular matrix obtained using decellularization methods is far lower than that of solid ECM. Chemical crosslinking of these hydrogels with macromolecular groups in the ECM is often performed to increase the stiffness of acellular matrix scaffolds, but the use of chemical crosslinkers can cause side effects such as high cytotoxicity, active degeneration, and calcification. While the use of natural crosslinkers can reduce cytotoxicity and immunogenicity, the crosslinked scaffolds are often colored, making them difficult to observe under light microscopy, and crosslinking is time-consuming. Collagen crosslinking significantly increases mechanical strength, which is the primary reason for the increased stiffness of the tumor ECM. The hardness of ECM can regulate some property changes in the tumorigenesis process, so increasing the hardness of the collagen scaffold so that the three-dimensional culture of tumor cells on the collagen scaffold can more effectively display the pathological characteristics of tumors in the body.

[0006] In normal tissue, 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. To simulate a more realistic tumor microenvironment, it is necessary to develop new collagen fibers that can effectively enhance tumor cell survival while removing cells, lipids, and other proteins from animal gels. This is useful for exploring the key factors and molecular mechanisms that regulate tumor immune escape in a 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 combined 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 a combination of organic weak acids to treat bovine fascia to prepare collagen materials, which can significantly improve the gel strength. In addition, the method of ultra-high pressure treatment combined 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 method for producing a collagen three-dimensional scaffold material by using bovine fascia as a raw material and subjecting it to ultrahigh pressure treatment in combination with organic weak acid 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 bovine fascia after cleaning in step (1) in a combination of organic weak acids, wherein the combination of organic weak acids 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) The relaxed bovine fascia soaked in the acid treatment of step (2) is neutralized with an alkaline solution;

[0016] (4) soaking the bovine fascia after the alkali neutralization and soaking treatment in step (3) in a decellularized solution;

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

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

[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 pre-frozen and then rapidly freeze-dried. 60 Irradiation sterilization;

[0022] (10) Cut 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 material can significantly improve gel strength and regulate the changes in properties during tumor development. 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 synergistic effect of ultrahigh pressure treatment combined with organic weak acid treatment significantly reduces tumor cell apoptosis and promotes tumor cell proliferation compared to other organic weak acid treatments. This combined approach eliminates the need for traditional chemical crosslinkers, significantly reducing the risks of solvent residues, cytotoxicity, active denaturation, and calcification. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is an electron microscope image of the longitudinal section of the 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 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 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 raw material to produce collagen three-dimensional scaffold materials (under different ultrahigh pressure conditions).

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

[0034] (2) Cut the bovine fascia after cleaning 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 washing for 10 minutes;

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

[0036] (4) Soak the bovine fascia treated in step (3) in 1 L of 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;

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

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

[0039] (7) Soak the collagen material obtained in 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 with 10°C deionized water as the dialysate, and then dialyzed for 6 days until the pH value of the dialysate 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 Irradiation sterilization;

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

[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 ultrahigh pressure treatment conditions is different. The maximum tensile strength of the collagen scaffold material under the second group of ultrahigh 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 materials (different acid treatment conditions)

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

[0049] (2) Cut the bovine fascia after cleaning 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 (the solution is changed 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 washing for 10 minutes;

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

[0051] (4) Soak the bovine fascia treated in step (3) in 1 L of 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;

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

[0053] (6) The product of step (5) was subjected 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;

[0054] (7) Soak the collagen material obtained in 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 with 10°C deionized water as the dialysate, and then dialyzed for 6 days until the pH value of the dialysate 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 Irradiation sterilization;

[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 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 234g / 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 a raw material to produce a collagen three-dimensional scaffold material.

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

[0064] (2) Cut the bovine fascia after cleaning 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 (the solution was changed every 2 h); the volume ratio of acetic acid solution to citric acid solution was 1:2; then wash it with deionized water 10 times, each washing for 10 minutes;

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

[0066] (4) Soak the bovine fascia treated in step (3) in 1 L of 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) Soak the product of step (4) in a 0.15 mol / L NaCl solution and stir for 1 h;

[0068] (6) The product of step (5) was subjected 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 collagen material obtained in 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 with 10°C deionized water as the dialysate, and then dialyzed for 6 days until the pH value of the dialysate reached 7.0;

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

[0072] (10) After completing step (9), the freeze-dried collagen material was 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 micrograph 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 μm. 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 bovine fascia and then wash it three times with deionized water;

[0077] (2) Cut the bovine fascia after cleaning 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 (the solution was changed every 2 h); the volume ratio of acetic acid solution to citric acid solution was 1:2; then wash it with deionized water 10 times, each washing for 10 minutes;

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

[0079] (4) Soak the bovine fascia treated in step (3) in 1 L of 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;

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

[0081] (6) The product of step (5) was subjected 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;

[0082] (7) Soak the collagen material obtained in 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 with 10°C deionized water as the dialysate, and then dialyzed for 6 days until the pH value of the dialysate reached 7.0;

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

[0085] (10) After completing step (9), the freeze-dried collagen material was 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 bovine fascia and then wash it with deionized water three times;

[0089] (2) Cut the bovine fascia after cleaning 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 washing for 10 minutes;

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

[0091] (4) Soak the bovine fascia treated in step (3) in 1 L of 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;

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

[0093] (6) The product of step (5) was subjected 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;

[0094] (7) Soak the collagen material obtained in 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 with 10°C deionized water as the dialysate, and then dialyzed for 6 days until the pH value of the dialysate reached 7.0;

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

[0097] (10) After completing step (9), the freeze-dried collagen material was 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 instead of ultrahigh pressure treatment).

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

[0101] (2) Cut the bovine fascia after cleaning 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 washing for 10 minutes;

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

[0103] (4) Soak the bovine fascia treated in step (3) in 1 L of 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;

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

[0105] (6) Soak the collagen material obtained in 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 with 10°C deionized water as the dialysate, and then dialyzed for 6 days until the pH value of the dialysate reached 7.0;

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

[0109] (10) After completing step (9), the freeze-dried collagen material was 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] Thaw frozen MCF-7 breast cancer cells in a 36°C water bath. Transfer to a 15 mL centrifuge tube, add 10 mL of fresh cell culture medium, mix thoroughly, and centrifuge at 1200 rpm for 10 minutes. Remove the supernatant, resuspend the cells in fresh culture medium, mix again, and culture on a cell culture plate.

[0113] When breast cancer cells are 80%-90% full on the culture plate, they are washed three times with PBS solution, preheated 0.25% protease is added, digested at 37 ° C for 2 min, and then blown into a cell suspension with fresh culture medium, centrifuged at 1200 rpm for 10 min, the supernatant is discarded, and fresh culture medium is added to be blown into a single cell suspension. 8 μL of single cell suspension is added to each material, placed in a 37 ° C incubator for 1.5 hours to promote cell adhesion, and then the material is transferred to fresh culture medium for culture. After 24 hours, the culture medium is replaced, and the collagen scaffold material prepared in Example 3 of the present invention and Comparative Examples 1 to 3 is used as a three-dimensional cell material, and the culture plate is cultured in two dimensions, placed on a small shaker in the incubator for culture, and the culture medium is changed once a day.

[0114] Direct counting method was used to detect cell proliferation. The specific operation was as follows:

[0115] (1) Dissolve collagenase in 1% Hank's balanced salt solution and dilute it 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 at 37°C for 2 min, add fresh cell culture medium to terminate the enzyme action, and pipette into 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 pipette into a single-cell suspension. Count the cells directly under a microscope using a red blood cell counting plate to calculate the number of cells.

[0121] like Figure 3 As shown, the cell proliferation under the conditions of comparative examples 1 to 3 and two-dimensional culture was compared with the number of cells on the first day as the base number. Under two-dimensional culture conditions, the cells were still able to maintain proliferation on the 5th day, but continued to be cultured without passage. Due to the limited space for cell growth, the number of cells decreased significantly, and a large number of cells fell off the cell culture plate. However, the three-dimensional culture of tumor cells using the collagen scaffold materials of Example 3 and Comparative Examples 1 to 3 of the present invention showed that the number of cells continued to proliferate after the 5th day compared with the 1st day, and the proliferation rate of cells cultured on the collagen scaffold of Example 3 was significantly higher than that of the collagen scaffold of Comparative Examples 1 to 3. The results showed that the scaffold material of Example 3 was more conducive to the proliferation of tumor cells than the collagen scaffold material 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 the purpose of clearly illustrating the present invention and are not intended to limit the embodiments of the present invention. Those skilled in the art will appreciate that other variations or modifications may be made based on the above description. It is not necessary and impossible to enumerate all embodiments here. Any modifications, equivalent substitutions, and improvements derived from the present invention are intended to be within the scope of protection of the claims.

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 bovine fascia after cleaning in step (1) in a combination of organic weak acids, wherein the combination of organic weak acids 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) The relaxed bovine fascia soaked in the acid treatment of step (2) is neutralized with an alkaline solution, wherein the alkaline solution is a 0.02 mol / L Na2HPO4 solution; (4) soaking the bovine fascia after the alkali neutralization and soaking treatment in step (3) in a decellularization solution, wherein the decellularization solution comprises a mixed aqueous solution of Tween-80 and sodium secondary alkyl sulfonate; (5) washing the product of step (4) in a solution containing NaCl; (6) The product of step (5) is subjected to ultrahigh pressure treatment, wherein the ultrahigh pressure pressure is 200 MPa to 500 MPa, the ultrahigh pressure time is 5 to 15 minutes, the extraction temperature is 50°C to 60°C, and the extraction time is 4 hours; (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 pre-frozen and then rapidly freeze-dried. 60 Irradiation sterilization; (10) Cut 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; After the neutralization acid treatment in step (3), the step is washed with deionized water 10 times, each washing for 10 minutes; In step (5), the concentration of the NaCl solution 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 place 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 in 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: In the decellularization solution of step (4), 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 ultrahigh pressure treatment was performed at a pressure of 300 MPa and a time of 10 min; the extraction temperature was 55° C. and the extraction time was 4 h.

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