Composition containing adipose-derived stem cells as well as preparation method and application of composition

Abstract

The invention provides a composition containing adipose-derived stem cells as well as a preparation method and application thereof, and belongs to the technical field of stem cell materials. The preparation method comprises the following steps: preparing collagen liquid through enzymolysis, adding adipose-derived stem cells into ionic liquid containing a surfactant and carbon dioxide, carrying out decellularization oscillation treatment, then loading silk fibroin microspheres prepared through self-assembly, preparing a silk fibroin microsphere modified decellularized matrix, uniformly mixing the silk fibroin microsphere modified decellularized matrix with the collagen liquid, removing bubbles, and spreading, so as to obtain the adipose-derived stem cell decellularized matrix. Performing continuous dehydration and lamination to form a matrix layer, and further performing crosslinking, filtering, washing, dehydration and sterilization under the action of NHS and EDC to obtain the adipose-derived stem cell-containing composition with the water content of 10-20%. The composition containing the adipose-derived stem cells prepared by the invention has very soft texture, has good attachment property and adaptability to a tissue interface, and can maintain a tight attachment state after being attached to the tissue, so that the effects of isolating and preventing seepage are achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of stem cell materials, and in particular to a composition containing adipose stem cells and a preparation method and application thereof. Background Art

[0002] Compositions containing adipose stem cells have important application value in the medical field, mainly used to repair damaged tissues, prevent tissue adhesion, promote tissue regeneration, etc. With the development of biomaterials science and tissue engineering, various new tissue repair matrix materials continue to emerge, providing more options for clinical treatment.

[0003] Collagen is the largest protein in connective tissue, accounting for about one-third of the total protein in the human body. So far, more than 20 types of collagen have been isolated. Collagen is an important material for tissue construction and reconstruction. Among the biopolymer materials used in surgery, collagen is currently one of the most popular materials for research.

[0004] Decellularized extracellular matrix (dECM) materials have shown great application potential in regenerative medicine, tissue engineering, and drug delivery due to their unique biocompatibility, low immunogenicity, and bioactivity. dECM materials are biocompatible and bioactive by removing immunogenic cellular components while retaining the natural extracellular matrix (ECM) components and structures. However, traditional decellularization methods, such as the use of polar detergents or strong acid and alkali solutions, may lead to the loss of polysaccharides and changes in protein structure; the use of biological enzymes will destroy bioactive substances due to excessive concentration or prolonged action time and is costly. Summary of the invention

[0005] The purpose of the present invention is to propose a composition containing adipose stem cells and a preparation method and application thereof. The composition has a very soft texture, good adhesion and adaptability to the tissue interface, can maintain a close adhesion state after being adhered to the tissue, thereby playing a role in isolation and preventing exudation, and can achieve the same sealing effect without adding biological glue. It is easy to operate and does not require pressing. It is safer to use, has better biocompatibility, faster sealing speed, no gelation time, and a wider range of applications. The biological glue only reacts with protein lysine hydroxyl groups, so it is not suitable for mucosal surfaces and cannot be used for tissues containing more fat.

[0006] The technical solution of the present invention is achieved in this way: The invention provides a method for preparing a composition containing adipose stem cells, comprising the steps of preparing collagen liquid by enzymatic hydrolysis, adding adipose-derived stem cells into an ionic liquid containing a surfactant and carbon dioxide, carrying out a decellularization oscillation treatment, and then loading silk fibroin microspheres prepared by self-assembly to prepare a silk fibroin microsphere-modified decellularized matrix, uniformly mixing the composition with the collagen liquid, removing bubbles, paving, performing continuous dehydration and stratification to form a matrix layer, further cross-linking under the action of NHS and EDC, filtering, washing, dehydrating, and sterilizing to obtain a composition containing adipose stem cells with a water content of 10-20%.

[0007] As a further improvement of the present invention, the following steps are included: S1. Preparation of collagen solution: depilate, wash, and mince pig skin, add it into water, add compound protease, enzymolyze, filter, inactivate enzyme with boiling water, centrifuge, collect supernatant, and obtain collagen solution; S2. Preparation of a decellularization treatment solution: adding a surfactant to the ionic liquid, stirring and mixing, and then introducing carbon dioxide to obtain a decellularization treatment solution; S3. Preparation of decellularized matrix: adding adipose-derived stem cells to DMEM medium containing FBS, resuspending, culturing, centrifuging, collecting cells, adding decellularization treatment solution, shaking, centrifuging, washing solids, freeze-drying, and preparing decellularized matrix; S4. Preparation of silk fibroin microspheres: adding silk fibroin peptide to water, adding protease, heating and stirring to react, inactivating the enzyme with boiling water, self-assembling, freeze-drying, and obtaining silk fibroin microspheres; S5. modification: adding the decellularized matrix to water, adding EDC and NHS, stirring to activate, adding silk fibroin microspheres, stirring to react, and freeze-drying to obtain a silk fibroin microsphere-modified decellularized matrix; S6. Degassing and tiling: dilute the collagen solution with PBS buffer, add silk fibroin microspheres to modify the decellularized matrix, stir and mix evenly, remove bubbles with ultrasound, and tiling in a flat mold, perform continuous dehydration and stratification to form a matrix layer; S7. Cross-linking: Soak the matrix layer in a solution containing EDC and NHS for cross-linking, filter, wash, dehydrate, and sterilize to obtain a composition containing adipose stem cells with a water content of 10-20%.

[0008] As a further improvement of the present invention, the mass ratio of the pigskin and the composite protease in step S1 is 10-15:0.5-1, the composite protease is selected from at least two of alkaline protease, neutral protease, pepsin, trypsin, papain, ficin, and bromelain, preferably, alkaline protease and trypsin, the mass ratio is 3-5:2-4, the temperature of the enzymatic hydrolysis is 35-40°C, the pH value is 8-9, the time is 2-4h, and the time of the boiling water enzyme inactivation is 5-10min.

[0009] As a further improvement of the present invention, the ionic liquid in step S2 is selected from at least one of 1-hexyl imidazole chloride, 1-hexyl-3-methyl imidazole chloride, 1-dodecyl imidazole chloride, 1-dodecyl imidazole tetrafluoroborate, 1-dodecyl imidazole hydrogen sulfate, 1-dodecyl imidazole dihydrogen phosphate, 1-dodecyl imidazole p-toluenesulfonate, 1-dodecyl imidazole trifluoromethanesulfonate, 1-dodecyl imidazole nitrate, 1-dodecyl imidazole acetate, and 1-dodecyl imidazole trifluoroacetate, the mass ratio of the ionic liquid to the surfactant is 100:2-3, the surfactant is Triton X-100, the ventilation volume of the carbon dioxide is 10-20 mL / min, and the ventilation time is 20-30 min.

[0010] As a further improvement of the present invention, the content of FBS in the DMEM medium containing FBS in step S3 is 8-12wt%, and the culture conditions are 35-38°C, 4-7vol%CO 2 , culture for 2-4 days, and the shaking treatment time is 1-2 hours.

[0011] As a further improvement of the present invention, the mass ratio of the silk fibroin peptide and the protease in step S4 is 15-20:1-2, the protease is selected from at least one of alkaline protease, neutral protease, pepsin, trypsin, papain, ficin, and bromelain, the temperature of the heating and stirring reaction is 55-65°C, pH=8-8.5, the time is 1-3h, the time for the boiling water enzyme inactivation is 5-10min, and the time for the self-assembly is 10-12h.

[0012] As a further improvement of the present invention, in step S5, the mass ratio of the decellularized matrix, silk fibroin microspheres, EDC and NHS is 12-15:2-3:1-2:1-2, the stirring activation time is 20-40 min, and the stirring reaction time is 7-10 h.

[0013] As a further improvement of the present invention, the pH value of the PBS buffer in step S6 is 7.4-8, the dilution to a concentration is 0.5-1wt%, the mass ratio of the collagen solution and the silk fibroin microsphere-modified decellularized matrix is ​​15-20:4-7, the power of the ultrasonic bubble removal is 1500-2000W, and the time is 10-20min.

[0014] As a further improvement of the present invention, in the solution containing EDC and NHS in step S7, the concentration of EDC is 1-2wt%, the concentration of NHS is 1-2wt%, and the cross-linking time is 7-10h.

[0015] The present invention further protects a composition containing adipose stem cells obtained by the above preparation method.

[0016] The present invention further protects the use of the above-mentioned composition containing adipose stem cells in the preparation of tissue sealing, isolation and repair matrix.

[0017] The present invention has the following beneficial effects: Pigs are rich in collagen, and hydrolysis can obtain small molecule collagen peptides, which have similar composition and structure to human tissues, can interact harmoniously with the environment around cells, and will not cause obvious immune or inflammatory reactions. Therefore, they have good biocompatibility in the human body and low immunogenicity. Their degradation products can act as cell signaling molecules to regulate cell migration, adhesion, proliferation and other behaviors. Collagen peptides can interact with growth factors and cytokines to enhance their activity, thereby more effectively promoting tissue regeneration and repair. At the same time, they can form a three-dimensional network structure, providing a good microenvironment for cell adhesion, proliferation and differentiation, allowing cells to grow in an orderly manner, which is beneficial to tissue regeneration and repair.

[0018] The present invention prepares a decellularization treatment solution, which is based on ionic liquid, and by adding a surfactant Triton X-100 and passing carbon dioxide, Triton X-100 can reduce the stability of the cell membrane, so that the carbon dioxide dissolved in the ionic liquid can more easily penetrate into the cell and remove the cell components, and at the same time, Triton X-100 can destroy the structure of the cell membrane, dissolve the lipid layer, and thus release the organelles and cytoplasm in the cell. By interacting with the hydrophobic region in the lipid layer, Triton X-100 forms a micellar structure, wraps the membrane protein inside, and dissolves it in the solution. Triton X-100 can destroy the nuclear membrane structure, expose the chromatin, and dissolve the nuclear membrane protein to achieve denuclearization. In addition, Triton X-100 also reduces the surface tension of the carbon dioxide dissolved in the ionic liquid, and increases its ability to dissolve polar molecules.

[0019] Ionic liquids can penetrate and diffuse into the original tissue cells, swell and rupture the cells to allow the cell contents to flow out, leaving behind the ECM components and structures. They have excellent biocompatibility, low immunogenicity and biological activity. After dissolving carbon dioxide, the polarity of the ionic liquid is significantly increased, and it can dissolve some polar substances that are usually difficult to dissolve in non-polar solvents. Its dissolving ability can be used to dissolve some components in the cells, thereby removing the cell contents.

[0020] The decellularization solution prepared by adding a surfactant and dissolving carbon dioxide in the present invention has good solubility for a variety of biological molecules, can effectively remove cell components in tissues without destroying the structure and biological activity of the extracellular matrix (ECM), thereby retaining the integrity of the ECM, and performing decellularization under relatively mild conditions, avoiding the destruction of ECM components by extreme conditions such as high temperature, strong acid, and strong alkali, which helps to retain bioactive substances such as growth factors and collagen in the ECM, can better protect the three-dimensional spatial structure of the ECM, and provide an ideal microenvironment for cell adhesion, proliferation and differentiation. At the same time, it has low immunogenicity and is environmentally friendly, has good chemical stability, and can be recycled, but the mechanical properties of the prepared decellularized matrix are insufficient.

[0021] The present invention modifies the decellularized matrix with silk fibroin microspheres prepared by nanoscale self-assembly. The nano-silk fibroin microspheres have a nanofiber microstructure and can simulate the structure and morphology of the extracellular matrix, thereby promoting cell adhesion and proliferation. The microspheres have good biocompatibility, degradability and low immunogenicity, can load a large amount of cell-promoting factors, promote tissue repair, and have components and structures similar to the extracellular matrix, which can provide a good microenvironment for tissue regeneration. In addition, the decellularized matrix modified with silk fibroin microspheres also significantly enhances its mechanical properties.

[0022] The present invention cross-links the collagen peptides and the silk fibroin microsphere-modified acellular matrix through a protein condensation reaction, thereby avoiding the cytotoxicity caused by the use of aldehyde cross-linking agents, and has high safety and is environmentally friendly.

[0023] The composition containing adipose stem cells prepared by the present invention has a very soft texture, good adhesion and adaptability to the tissue interface, and can maintain a close adhesion state after being adhered to the tissue, thereby playing a role in isolation and preventing exudation. The sealing effect can also be achieved without adding biological glue, and it is easy to operate and does not require pressing. It is safer to use, has better biocompatibility, faster sealing speed, no gelation time, and a wider range of applications. The biological glue only reacts with protein lysine hydroxyl groups, so it is not suitable for mucosal surfaces and cannot be used for tissues containing more fat. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0025] Figure 1 This is the SEM image of the decellularized matrix prepared in Example 3. DETAILED DESCRIPTION

[0026] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] Alkaline protease, 10,000 U / g; trypsin, 8,000 U / g; adipose-derived stem cells are mouse adipose-derived mesenchymal stem cells, 5×10 5 cells / T25 cell culture flask; EDC, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide; NHS, N-hydroxysuccinimide. Example 1

[0028] This embodiment provides a method for preparing a composition containing adipose stem cells, comprising the following steps: S1. Preparation of collagen solution: 10 g of pig skin was dehaired, washed, minced, added to 100 mL of water, 0.5 g of compound protease was added, 35°C, pH 8, enzymatic hydrolysis for 2 h, filtered, boiled in water for 5 min, centrifuged, and the supernatant was collected to obtain collagen solution; The composite protease is alkaline protease and trypsin, with a mass ratio of 3:2; S2. Preparation of a decellularization solution: 2 g of Triton X-100 was added to 100 g of 1-hexyl-3-methylimidazolium chloride, the mixture was stirred for 15 min, and carbon dioxide was introduced at a flow rate of 10 mL / min for 20 min to obtain a decellularization solution; S3. Preparation of decellularized matrix: Adipose-derived stem cells were added to DMEM medium containing 8wt% FBS and resuspended at a cell density of 10 4 -10 5 / mL, 35℃, 4vol%CO 2 , culture for 2 days, centrifuge, collect cells, add 100 mL of decellularization solution, and the cell density is 10 5 -10 6 / mL, shaken for 1h, centrifuged, washed with solids, freeze-dried to obtain the decellularized matrix; S4. Preparation of silk fibroin microspheres: 15 g of silk fibroin peptide was added to 200 mL of water, 1 g of alkaline protease was added, the mixture was heated to 55°C, pH = 8, stirred for 1 h, the enzyme was inactivated by boiling water for 5 min, self-assembled for 10 h, and freeze-dried to obtain silk fibroin microspheres; S5. Modification: 12 g of decellularized matrix was added to 200 mL of water, 1 g of EDC and 1 g of NHS were added, and the mixture was stirred for activation for 20 min. 2 g of silk fibroin microspheres were added, and the mixture was stirred for reaction for 7 h. The mixture was freeze-dried to obtain a decellularized matrix modified with silk fibroin microspheres. S6. Degassing and tiling: 15 g of collagen solution was diluted with PBS buffer with a pH value of 7.4 to a concentration of 0.5 wt %, 4 g of silk fibroin microspheres modified with acellular matrix were added, the mixture was stirred for 15 min, bubbles were removed by ultrasound at 1500 W for 10 min, and the mixture was tiled in a flat mold, and a matrix layer was formed after continuous dehydration and stratification; S7. Cross-linking: The matrix layer was immersed in a solution containing 1wt% EDC and 1wt% NHS for cross-linking for 7 hours, filtered, washed, dehydrated, and sterilized to obtain a composition containing adipose stem cells with a water content of 15%. Example 2

[0029] This embodiment provides a method for preparing a composition containing adipose stem cells, comprising the following steps: S1. Preparation of collagen solution: 15 g of pig skin was dehaired, washed, minced, added to 100 mL of water, 1 g of compound protease was added, 40°C, pH 9, enzymatic hydrolysis for 4 h, filtered, boiled in water for 10 min, centrifuged, and the supernatant was collected to obtain collagen solution; The composite protease is alkaline protease and trypsin, with a mass ratio of 5:4; S2. Preparation of a decellularization solution: 3 g of Triton X-100 was added to 100 g of 1-hexyl-3-methylimidazolium chloride, the mixture was stirred for 15 min, and carbon dioxide was introduced at a flow rate of 20 mL / min for 30 min to obtain a decellularization solution; S3. Preparation of decellularized matrix: Adipose-derived stem cells were added to DMEM medium containing 12 wt% FBS and resuspended at a cell density of 10 4 -10 5 / mL, 38℃, 7vol%CO 2 , cultured for 4 days, centrifuged, collected the cells, added 100 mL of decellularization solution, and the cell density was 10 5 -10 6 / mL, shaken for 2h, centrifuged, washed with solids, freeze-dried to obtain the decellularized matrix; S4. Preparation of silk fibroin microspheres: 20 g of silk fibroin peptide was added to 200 mL of water, 2 g of alkaline protease was added, the mixture was heated to 65°C, pH = 8.5, stirred for 3 h, the enzyme was inactivated by boiling water for 10 min, self-assembled for 12 h, and freeze-dried to obtain silk fibroin microspheres; S5. Modification: 15 g of decellularized matrix was added to 200 mL of water, 2 g of EDC and 2 g of NHS were added, and the mixture was stirred and activated for 40 min. 3 g of silk fibroin microspheres were added, and the mixture was stirred and reacted for 10 h. The mixture was freeze-dried to obtain a decellularized matrix modified with silk fibroin microspheres. S6. Degassing and tiling: 20 g of collagen solution was diluted with PBS buffer with a pH value of 8 to a concentration of 1 wt%, 7 g of silk fibroin microspheres modified decellularized matrix was added, the mixture was stirred for 15 min, bubbles were removed by ultrasound at 2000 W for 20 min, and the matrix was tiled in a flat mold, and a matrix layer was formed after continuous dehydration and stratification; S7. Cross-linking: The matrix layer was immersed in a solution containing 2wt% EDC and 2wt% NHS for cross-linking for 10 hours, filtered, washed, dehydrated, and sterilized to obtain a composition containing adipose stem cells with a water content of 15%. Example 3

[0030] This embodiment provides a method for preparing a composition containing adipose stem cells, comprising the following steps: S1. Preparation of collagen solution: 12 g of pig skin was dehaired, washed, minced, added to 100 mL of water, 0.7 g of compound protease was added, 37°C, pH value was 8.5, enzymatic hydrolysis was performed for 3 h, filtered, enzyme was inactivated in boiling water for 7 min, centrifuged, and the supernatant was collected to obtain collagen solution; The composite protease is alkaline protease and trypsin, with a mass ratio of 4:3; S2. Preparation of decellularization solution: 2.5 g TritonX-100 was added to 100 g 1-hexyl-3-methylimidazolium chloride, stirred for 15 min, and carbon dioxide was introduced at a flow rate of 15 mL / min for 25 min to obtain a decellularization solution; S3. Preparation of decellularized matrix: Adipose-derived stem cells were added to DMEM medium containing 10wt% FBS and resuspended at a cell density of 10 4 -10 5 / mL, 37℃, 5.5vol%CO 2 , culture for 3 days, centrifuge, collect cells, add 100mL of decellularization solution, and the cell density is 10 5 -10 6 / mL, shaken for 1.5h, centrifuged, washed with solids, freeze-dried to obtain the decellularized matrix; Figure 1 This is a SEM image of the decellularized matrix prepared in Example 3. It can be seen from the image that the internal voids of the material are relatively obvious, the degree of cross-linking of the material is good, and it is further verified that the decellularization effect is obvious and no cells remain.

[0031] S4. Preparation of silk fibroin microspheres: 17 g of silk fibroin peptide was added to 200 mL of water, 1.5 g of alkaline protease was added, the mixture was heated to 60°C, pH = 8.2, stirred for 2 h, the enzyme was inactivated by boiling water for 7 min, self-assembled for 11 h, and freeze-dried to obtain silk fibroin microspheres; S5. Modification: 13 g of decellularized matrix was added to 200 mL of water, 1.5 g of EDC and 1.5 g of NHS were added, and the mixture was stirred for activation for 30 min. 2.5 g of silk fibroin microspheres were added, and the mixture was stirred for reaction for 8 h. The mixture was freeze-dried to obtain a decellularized matrix modified with silk fibroin microspheres. S6. Degassing and tiling: 17 g of collagen solution was diluted with PBS buffer with a pH value of 7.8 to a concentration of 0.7 wt %, 5.5 g of silk fibroin microspheres modified with acellular matrix were added, the mixture was stirred for 15 min, bubbles were removed by ultrasound at 1700 W for 15 min, and the mixture was tiled in a flat mold, and a matrix layer was formed after continuous dehydration and stratification; S7. Cross-linking: The matrix layer was immersed in a solution containing 1.5wt% EDC and 1.5wt% NHS for cross-linking for 8 hours, filtered, washed, dehydrated, and sterilized to obtain a composition containing adipose stem cells with a water content of 15%.

[0032] Comparative Example 1 Compared with Example 3, the difference is that no carbon dioxide is introduced in step S2.

[0033] The details are as follows: S2. Preparation of decellularization treatment solution: Add 2.5 g of Triton X-100 to 100 g of 1-hexyl-3-methylimidazolium chloride, stir and mix for 15 minutes to prepare a decellularization treatment solution.

[0034] Comparative Example 2 Compared with Example 3, the difference is that Triton X-100 is not added in step S2.

[0035] The details are as follows: S2. Preparation of decellularization treatment solution: Carbon dioxide was introduced into 100 g of 1-hexyl-3-methylimidazolium chloride at a flow rate of 15 mL / min for 25 min to obtain a decellularization treatment solution.

[0036] Comparative Example 3 Compared with Example 3, the difference is that in step S2, 1-hexyl-3-methylimidazolium chloride is replaced by water.

[0037] The details are as follows: S2. Preparation of decellularization treatment solution: Add 2.5 g of Triton X-100 to 100 g of water, stir and mix for 15 min, and introduce carbon dioxide at a ventilation volume of 15 mL / min for 25 min to obtain a decellularization treatment solution.

[0038] Comparative Example 4 Compared with Example 3, the difference is that steps S4 and S5 are not performed.

[0039] The details are as follows: S1. Preparation of collagen solution: 12 g of pig skin was dehaired, washed, minced, added to 100 mL of water, 0.7 g of compound protease was added, 37°C, pH value was 8.5, enzymatic hydrolysis was performed for 3 h, filtered, enzyme was inactivated in boiling water for 7 min, centrifuged, and the supernatant was collected to obtain collagen solution; The composite protease is alkaline protease and trypsin, with a mass ratio of 4:3; S2. Preparation of decellularization solution: 2.5 g TritonX-100 was added to 100 g 1-hexyl-3-methylimidazolium chloride, stirred for 15 min, and carbon dioxide was introduced at a flow rate of 15 mL / min for 25 min to obtain a decellularization solution; S3. Preparation of decellularized matrix: Adipose-derived stem cells were added to DMEM medium containing 10wt% FBS and resuspended at a cell density of 10 4 -10 5 / mL, 37℃, 5.5vol%CO 2 , culture for 3 days, centrifuge, collect cells, add 100mL of decellularization solution, and the cell density is 10 5 -10 6 / mL, shaken for 1.5h, centrifuged, washed with solids, freeze-dried to obtain the decellularized matrix; S4. Degassing and tiling: 17 g of collagen solution was diluted with PBS buffer with a pH value of 7.8 to a concentration of 0.7 wt %, 5.5 g of decellularized matrix was added, and the mixture was stirred for 15 min. The bubbles were removed by ultrasound at 1700 W for 15 min, and the mixture was tiled in a flat mold. After continuous dehydration and stratification, a matrix layer was formed. S5. Cross-linking: The matrix layer was immersed in a solution containing 1.5wt% EDC and 1.5wt% NHS for cross-linking for 8 hours, filtered, washed, dehydrated, and sterilized to obtain a composition containing adipose stem cells with a water content of 15%.

[0040] Comparative Example 5 Compared with Example 3, the difference is that no silk fibroin microspheres are added to modify the decellularized matrix in step S6.

[0041] The details are as follows: S6. Degassing and spreading: 17 g of collagen solution was diluted with PBS buffer with a pH value of 7.8 to a concentration of 0.7 wt %, and bubbles were removed by ultrasound at 1700 W for 15 min. The solution was spread in a flat mold and continuously dehydrated and layered to form a matrix layer.

[0042] Comparative Example 6 Compared with Example 3, the difference is that EDC and NHS are replaced by glutaraldehyde of equal total mass.

[0043] The details are as follows: S7. Cross-linking: Soak the matrix layer in a solution containing 3 wt% glutaraldehyde for cross-linking for 8 hours, filter, wash, dehydrate, and sterilize to obtain a composition containing adipose stem cells with a water content of 15%.

[0044] Test Example 1 The performance of the compositions containing adipose stem cells prepared in Examples 1-3 and Comparative Examples 1-6 was tested. The sample thickness was 10 μm. The results are shown in Table 1.

[0045] 1. Shear strength Referring to the method in YY / T 0729.1, the sample is cut into strips with a width of 2.5 cm, and the sample is overlapped with the pig skin graft, and the overlap area is 2.5 cm × 1.0 cm. A force of 1-2 N is applied to the overlap area to make the sample and the pig skin graft fit tightly, and then the shear strength is measured. The two ends of the sample are clamped on the clamp of the tensile testing machine, so that the force direction is the long axis direction of the sample, and the sample is loaded at a speed of 5 mm / min until the overlap area is separated, and the maximum force is recorded and the shear strength is calculated.

[0046] 2. Compression resistance Refer to ASTM F2392-04, take fresh pig stomach, make a 5mm diameter circular notch on the surface, cut the sample into 2.5cm×2.5cm size (the distance between the edge of the sample and the edge of the defect area is at least 1cm), and apply it flatly to the defect in a centrally symmetrical manner. Connect one end of the sample to a pressure gauge and pass gas at the other end to measure the maximum pressure that the defect can withstand.

[0047] 3. Degradation performance The product was subjected to an in vitro degradation test and the degradation time of each sample was compared. The sample was placed in a type I collagenase solution in a 37°C constant temperature incubator, the enzyme solution was replaced every 2 days, the sample degradation was observed every 12 hours, and the time required for the sample to be completely degraded was recorded.

[0048] 4. Cytotoxicity The in vitro cytotoxicity of the samples was detected using the MTT method with reference to GB / T 16886.5-2017, and the cell survival rate (%) of the blank control group was 100%.

[0049] Table 1

[0050] As can be seen from the above table, the compositions containing adipose stem cells obtained in Examples 1-3 of the present invention have good mechanical properties and degradability, and low cytotoxicity. The cell survival rate of Comparative Examples 1-3 decreased, and its biocompatibility decreased. It can be seen that the decellularization treatment fluid of the present invention can penetrate and diffuse into the original tissue cells, swell and rupture the cells to make the cell contents flow out, leaving the ECM components and structures, and has excellent biocompatibility, low immunogenicity and biological activity. The mechanical properties of Comparative Examples 4 and 5 are significantly reduced, and the decellularized matrix modified with silk protein microspheres obtained by nanoscale self-assembly significantly enhances its mechanical properties. Comparative Example 6 uses glutaraldehyde for cross-linking, and the cell survival rate is greatly reduced. It can be seen that glutaraldehyde cross-linking has certain toxicity.

[0051] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a composition containing adipose stem cells, characterized in that: The collagen solution is prepared by enzymatic hydrolysis, and the adipose-derived stem cells are added to an ionic liquid containing a surfactant and carbon dioxide, subjected to a decellularization oscillation treatment, and then loaded with silk fibroin microspheres prepared by self-assembly to obtain a silk fibroin microsphere-modified decellularized matrix, which is evenly mixed with the collagen solution, bubbles are removed, the matrix is ​​spread, and a matrix layer is formed after continuous dehydration and stratification. The matrix layer is further cross-linked under the action of NHS and EDC, filtered, washed, dehydrated, and sterilized to obtain a composition containing adipose-derived stem cells with a water content of 10-20%.

2. The preparation method according to claim 1, characterized in that: The following steps are involved: S1. Preparation of collagen solution: depilate, wash, and mince pig skin, add it into water, add compound protease, enzymolyze, filter, inactivate enzyme with boiling water, centrifuge, collect supernatant, and obtain collagen solution; S2. Preparation of a decellularization treatment solution: adding a surfactant to the ionic liquid, stirring and mixing, and then introducing carbon dioxide to obtain a decellularization treatment solution; S3. Preparation of decellularized matrix: adding adipose-derived stem cells to DMEM medium containing FBS, resuspending, culturing, centrifuging, collecting cells, adding decellularization treatment solution, shaking, centrifuging, washing solids, freeze-drying, and preparing decellularized matrix; S4. Preparation of silk fibroin microspheres: adding silk fibroin peptide to water, adding protease, heating and stirring to react, inactivating the enzyme with boiling water, self-assembling, freeze-drying, and obtaining silk fibroin microspheres; S5. modification: adding the decellularized matrix to water, adding EDC and NHS, stirring to activate, adding silk fibroin microspheres, stirring to react, and freeze-drying to obtain a silk fibroin microsphere-modified decellularized matrix; S6. Degassing and tiling: dilute the collagen solution with PBS buffer, add silk fibroin microspheres to modify the decellularized matrix, stir and mix evenly, remove bubbles with ultrasound, and tiling in a flat mold, perform continuous dehydration and stratification to form a matrix layer; S7. Cross-linking: Soak the matrix layer in a solution containing EDC and NHS for cross-linking, filter, wash, dehydrate, and sterilize to obtain a composition containing adipose stem cells with a water content of 10-20%.

3. The preparation method according to claim 2, characterized in that: The mass ratio of the pigskin and the composite protease in step S1 is 10-15:0.5-1, the composite protease is selected from at least two of alkaline protease, neutral protease, pepsin, trypsin, papain, ficin and bromelain, preferably, alkaline protease and trypsin, the mass ratio is 3-5:2-4, the enzymolysis temperature is 35-40°C, the pH value is 8-9, the time is 2-4h, and the boiling water enzyme inactivation time is 5-10min.

4. The preparation method according to claim 2, characterized in that: The ionic liquid in step S2 is selected from at least one of 1-hexyl imidazole chloride, 1-hexyl-3-methyl imidazole chloride, 1-dodecyl imidazole chloride, 1-dodecyl imidazole tetrafluoroborate, 1-dodecyl imidazole hydrogen sulfate, 1-dodecyl imidazole dihydrogen phosphate, 1-dodecyl imidazole p-toluenesulfonate, 1-dodecyl imidazole trifluoromethanesulfonate, 1-dodecyl imidazole nitrate, 1-dodecyl imidazole acetate, and 1-dodecyl imidazole trifluoroacetate, the mass ratio of the ionic liquid to the surfactant is 100:2-3, the surfactant is Triton X-100, the ventilation volume of the carbon dioxide is 10-20 mL / min, and the ventilation time is 20-30 min.

5. The preparation method according to claim 2, characterized in that: The content of FBS in the DMEM medium containing FBS in step S3 is 8-12wt%, the culture conditions are 35-38°C, 4-7vol% CO2, the culture is 2-4d, and the shaking treatment time is 1-2h.

6. The preparation method according to claim 2, characterized in that: The mass ratio of the silk fibroin peptide and the protease in step S4 is 15-20:1-2, the protease is selected from at least one of alkaline protease, neutral protease, pepsin, trypsin, papain, ficin and bromelain, the temperature of the heating and stirring reaction is 55-65°C, pH=8-8.5, the time is 1-3h, the time of the boiling water enzyme inactivation is 5-10min, and the time of the self-assembly is 10-12h.

7. The preparation method according to claim 2, characterized in that: In step S5, the mass ratio of the decellularized matrix, silk fibroin microspheres, EDC and NHS is 12-15:2-3:1-2:1-2, the stirring activation time is 20-40 minutes, and the stirring reaction time is 7-10 hours.

8. The preparation method according to claim 2, characterized in that: The pH value of the PBS buffer in step S6 is 7.4-8, the dilution concentration is 0.5-1wt%, the mass ratio of the collagen solution and the silk fibroin microsphere modified decellularized matrix is ​​15-20:4-7, the power of the ultrasonic bubble removal is 1500-2000W, and the time is 10-20min; the concentration of EDC in the solution containing EDC and NHS in step S7 is 1-2wt%, the concentration of NHS is 1-2wt%, and the cross-linking time is 7-10h.

9. A composition containing adipose stem cells obtained by the preparation method according to any one of claims 1 to 8.

10. Use of the composition containing adipose stem cells as claimed in claim 9 in preparing tissue sealing, isolation and repair matrix.

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