A composition containing adipose-derived stem cells, its preparation method and applications

Abstract

The present invention provides a composition containing adipose-derived stem cells, a preparation method thereof, and an application thereof, belonging to the technical field of stem cell materials. Collagen solution is prepared by enzymatic hydrolysis. Adipose-derived stem cells are added to an ionic liquid containing a surfactant and carbon dioxide, and subjected to decellularization and oscillation treatment. Then, the adipose-derived stem cells are loaded with self-assembled silk fibroin microspheres to obtain silk fibroin microsphere-modified decellularized matrix. The silk fibroin microsphere-modified decellularized matrix is mixed evenly with the collagen solution, the bubbles are removed, and the mixture is spread out and subjected to continuous dehydration lamination to form a matrix layer. Further, crosslinking is carried out under the action of NHS and EDC, followed by filtration, washing, dehydration, and sterilization to obtain a composition containing adipose-derived stem cells with a water content of 10-20%. The composition containing adipose-derived stem cells prepared by the present invention has a very soft texture, good conformability and adaptability to the tissue interface, and can maintain a tight conforming state after fitting with the tissue, thereby playing a role in isolation and preventing exudation.

Description

Technical Field

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

[0002] Compositions containing adipose stem cells have important application values in the medical field, mainly used for repairing damaged tissues, preventing tissue adhesions, promoting tissue regeneration, etc. With the development of biomaterial science and tissue engineering, various new tissue repair matrix materials have emerged continuously, providing more choices for clinical treatment.

[0003] Collagen is the most substantial tissue that constitutes connective tissue proteins, accounting for approximately one-third of the total protein content in the human body. So far, more than 20 types of collagen have been isolated. Collagen is an important material for tissue construction and tissue reconstruction, and among the biopolymer materials applied in surgery, collagen is one of the most popular materials currently under research.

[0004] Decellularized extracellular matrix (dECM) materials have shown great application potential in the fields of regenerative medicine, tissue engineering, drug delivery, etc. due to their unique biocompatibility, low immunogenicity, and biological activity. dECM materials remove immunogenic cellular components while retaining natural extracellular matrix (ECM) components and structures to have both biocompatibility and biological activity. However, traditional decellularization methods, such as using polar detergents or strong acid and strong base solutions, may lead to the loss of polysaccharides and the change of protein structures; using biological enzymes will damage bioactive substances due to too high concentration or too long action time and the cost is high. Summary of the Invention

[0005] The purpose of the present invention is to provide a composition containing adipose stem cells, a preparation method thereof, and an application thereof, which has a very soft texture, good conformability and adaptability with the tissue interface, can maintain a tightly adherent state after adhering to the tissue, so as to play a role in isolation and preventing exudation, can achieve a sealing effect without adding a bioadhesive, and has the characteristics of simple operation, no need for pressing, safer use, better biocompatibility, faster sealing speed, no gelation time, wider application range. The bioadhesive only reacts with the hydroxyl group of protein lysine, so it is not applicable to the mucosal surface and cannot be used for tissues with more fat.

[0006] The technical solution of the present invention is realized as follows:

[0007] The present invention provides a method for preparing a composition containing adipose stem cells. Collagen solution is prepared by enzymatic hydrolysis. Adipose-derived stem cells are added to an ionic liquid containing a surfactant and carbon dioxide, and subjected to decellularization by oscillation treatment. Then, they are loaded with self-assembled silk fibroin microspheres to obtain silk fibroin microsphere-modified decellularized matrix, which is mixed evenly with the collagen solution, degassed, spread out, and subjected to continuous dehydration lamination to form a matrix layer. Further cross-linking is carried out under the action of NHS and EDC, followed by filtration, washing, dehydration, and sterilization to obtain a composition containing adipose stem cells with a water content of 10-20%.

[0008] As a further improvement of the present invention, it includes the following steps:

[0009] S1. Preparation of collagen solution: Pigskin is depilated, washed, minced, added to water, added with a composite protease, subjected to enzymatic hydrolysis, filtered, inactivated by boiling, centrifuged, and the supernatant is collected to obtain collagen solution;

[0010] S2. Preparation of decellularization treatment solution: A surfactant is added to the ionic liquid, stirred and mixed evenly, and then carbon dioxide is introduced to obtain the decellularization treatment solution;

[0011] S3. Preparation of decellularized matrix: Adipose-derived stem cells are resuspended in DMEM medium containing FBS, cultured, centrifuged, the cells are collected, added with the decellularization treatment solution, subjected to oscillation treatment, centrifuged, the solid is washed, and freeze-dried to obtain the decellularized matrix;

[0012] S4. Preparation of silk fibroin microspheres: Silk fibroin peptide is added to water, added with a protease, heated and stirred for reaction, inactivated by boiling, self-assembled, and freeze-dried to obtain silk fibroin microspheres;

[0013] S5. Modification: The decellularized matrix is added to water, added with EDC and NHS, stirred for activation, added with silk fibroin microspheres, stirred for reaction, and freeze-dried to obtain silk fibroin microsphere-modified decellularized matrix;

[0014] S6. Defoaming and spreading: The collagen solution is diluted with PBS buffer, added with silk fibroin microsphere-modified decellularized matrix, stirred and mixed evenly, degassed by ultrasound, spread out in a flat mold, and subjected to continuous dehydration lamination to form a matrix layer;

[0015] S7. Cross-linking: The matrix layer is soaked in a solution containing EDC and NHS for cross-linking, filtered, washed, dehydrated, and sterilized to obtain a composition containing adipose stem cells with a water content of 10-20%.

[0016] As a further improvement of the present invention, in step S1, the mass ratio of the pigskin to the compound protease is 10 - 15:0.5 - 1. The compound protease is selected from at least two of alkaline protease, neutral protease, pepsin, trypsin, papain, ficin, and bromelain. Preferably, it is alkaline protease and trypsin, and 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 - 4 h, and the time for inactivating the enzyme with boiling water is 5 - 10 min.

[0017] As a further improvement of the present invention, in step S2, the ionic liquid is selected from at least one of 1 - hexylimidazole chloride, 1 - hexyl - 3 - methylimidazole chloride, 1 - dodecylimidazole chloride, 1 - dodecylimidazole tetrafluoroborate, 1 - dodecylimidazole hydrogen sulfate, 1 - dodecylimidazole dihydrogen phosphate, 1 - dodecylimidazole p - toluenesulfonate, 1 - dodecylimidazole trifluoromethanesulfonate, 1 - dodecylimidazole nitrate, 1 - dodecylimidazole acetate, and 1 - dodecylimidazole trifluoroacetate. The mass ratio of the ionic liquid to the surfactant is 100:2 - 3. The surfactant is Triton X - 100. The ventilation rate of carbon dioxide is 10 - 20 mL / min, and the ventilation time is 20 - 30 min.

[0018] As a further improvement of the present invention, in step S3, the content of FBS in the DMEM medium containing FBS is 8 - 12 wt%. The culture conditions are 35 - 38 °C, 4 - 7 vol% CO2, and the culture time is 2 - 4 d. The time for the oscillation treatment is 1 - 2 h.

[0019] As a further improvement of the present invention, in step S4, the mass ratio of the silk fibroin peptide to the protease 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 - 3 h, the time for inactivating the enzyme with boiling water is 5 - 10 min, and the self - assembly time is 10 - 12 h.

[0020] As a further improvement of the present invention, in step S5, the mass ratio of the acellular matrix, silk fibroin microspheres, EDC, and NHS is 12 - 15:2 - 3:1 - 2:1 - 2. The time for stirring activation is 20 - 40 min, and the time for the stirring reaction is 7 - 10 h.

[0021] As a further improvement of the present invention, in step S6, the pH value of the PBS buffer solution is 7.4 - 8, diluted to a concentration of 0.5 - 1 wt%, the mass ratio of the collagen solution to the silk fibroin microsphere-modified acellular matrix is 15 - 20:4 - 7, the power of ultrasonic bubble removal is 1500 - 2000 W, and the time is 10 - 20 min.

[0022] As a further improvement of the present invention, in step S7, the concentration of EDC in the solution containing EDC and NHS is 1 - 2 wt%, the concentration of NHS is 1 - 2 wt%, and the crosslinking time is 7 - 10 h.

[0023] The present invention further protects a composition containing adipose stem cells prepared by the above preparation method.

[0024] The present invention further protects the application of the above composition containing adipose stem cells in the preparation of tissue sealing, isolation and repair matrices.

[0025] The present invention has the following beneficial effects:

[0026] Pigs are rich in collagen. Hydrolysis can obtain small molecule collagen peptides, which have components and structures similar to human tissues. They can interact harmoniously with the cell surrounding environment, without causing obvious immune reactions or inflammatory reactions. Therefore, they have good biocompatibility in the human body and low immunogenicity at the same time. Their degradation products can serve as cell signaling molecules to regulate cell behaviors such as migration, adhesion and proliferation. Collagen peptides can interact with growth factors and cytokines to enhance their activities, 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, enabling cells to grow orderly therein, which is beneficial to tissue regeneration and repair.

[0027] The present invention prepares a decellularization treatment solution with ionic liquid as the main body. By adding the surfactant Triton X-100 and introducing carbon dioxide, Triton X-100 can reduce the stability of the cell membrane, making the carbon dioxide dissolved in the ionic liquid more easily penetrate into the cell and remove cell components. At the same time, Triton X-100 can destroy the structure of the cell membrane, dissolve the lipid layer, thereby releasing the cell organelles and cytoplasm inside the cell. By interacting with the hydrophobic regions in the lipid layer, Triton X-100 forms micelle structures, enclosing the membrane proteins therein and dissolving them in the solution. Triton X-100 can destroy the nuclear membrane structure, expose the chromatin, and dissolve the nuclear membrane proteins to achieve denucleation. In addition, Triton X-100 also reduces the surface tension of the carbon dioxide dissolved in the ionic liquid, increasing its ability to dissolve polar molecules.

[0028] Ionic liquids can penetrate and diffuse into the original tissue cells, swelling and rupturing the cells to make the cell contents flow out, leaving the ECM components and structure. They have excellent biocompatibility, low immunogenicity and biological activity. After dissolving carbon dioxide, the polarity of ionic liquids is significantly increased, enabling them to dissolve some polar substances that are usually insoluble in non-polar solvents. By using their dissolving ability, some components inside the cells are dissolved, thus achieving the removal of cell contents.

[0029] The decellularized treatment solution prepared by the method of adding surfactants and dissolving carbon dioxide in the present invention has good solubility for a variety of biomolecules, can effectively remove the cell components in tissues without damaging the structure and biological activity of the extracellular matrix (ECM), thereby retaining the integrity of the ECM. Decellularization treatment is carried out under relatively mild conditions, avoiding the damage 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 also be recycled. However, the mechanical properties of the prepared decellularized matrix are insufficient.

[0030] In the present invention, silk fibroin microspheres prepared by nano-scale self-assembly are used to modify the decellularized matrix. The nano-silk fibroin microspheres have a nanofiber microstructure, can simulate the structure and morphology of the extracellular matrix, thus promoting cell adhesion and proliferation. They have good biocompatibility, degradability and low immunogenicity, can load a large amount of cell promoting factors, and promote tissue repair. Their components and structure similar to those of the extracellular matrix can provide a good microenvironment for tissue regeneration. In addition, the silk fibroin microsphere-modified decellularized matrix also significantly enhances its mechanical properties.

[0031] In the present invention, collagen peptides and silk fibroin microsphere-modified decellularized matrix are crosslinked through protein condensation reaction, avoiding the cytotoxicity brought by using aldehyde crosslinking agents, and having high safety and being environmentally friendly.

[0032] The composition containing adipose stem cells prepared in the present invention has a very soft texture, has good conformability and adaptability with the tissue interface, can maintain a tight conforming state after fitting with the tissue, thus playing a role in isolation and preventing exudation. It can achieve a sealing effect without adding bioadhesive, and has the characteristics of simple operation, no need for pressing, safer use, better biocompatibility, faster sealing speed, no gelation time, wider application range. Bioadhesive only reacts with the hydroxyl group of protein lysine, so it is not applicable to mucosal surfaces and cannot be used for tissues with more fat. Brief Description of the Drawings

[0033] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0034] Figure 1 SEM image of the acellular matrix prepared in Example 3. Detailed implementation manners

[0035] The following will clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0036] Alkaline protease, 10,000 U / g; Trypsin, 8,000 U / g; Adipose-derived stem cells are mouse adipose mesenchymal stem cells, 5×10 5 cells / T25 cell culture flask; EDC, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide; NHS, N-hydroxysuccinimide. Example 1

[0037] This example provides a method for preparing a composition containing adipose stem cells, including the following steps:

[0038] S1. Preparation of collagen solution: Debone, wash, and crush 10 g of pigskin, add it to 100 mL of water, add 0.5 g of compound protease, at 35 °C, pH value of 8, enzymolysis for 2 h, filter, inactivate the enzyme with boiling water for 5 min, centrifuge, and collect the supernatant to obtain the collagen solution;

[0039] The compound protease is alkaline protease and trypsin, and the mass ratio is 3:2;

[0040] S2. Preparation of acellular treatment solution: Add 2 g of Triton X-100 to 100 g of 1-hexyl-3-methylimidazolium chloride, stir and mix for 15 min, introduce carbon dioxide, the ventilation rate is 10 mL / min, and the ventilation time is 20 min to obtain the acellular treatment solution;

[0041] S3. Preparation of acellular matrix: Resuspend adipose-derived stem cells in DMEM medium containing 8 wt% FBS, and the cell density is 10 4 -10 5cells / mL, at 35°C, 4 vol% CO2, cultured for 2 days, centrifuged, cells collected, 100 mL of decellularization treatment solution added, cell density 10 5 -10 6 cells / mL, subjected to shaking treatment for 1 h, centrifuged, solid washed, freeze-dried to obtain decellularized matrix;

[0042] S4. Preparation of silk fibroin microspheres: Add 15 g of silk fibroin peptide to 200 mL of water, add 1 g of alkaline protease, heat to 55°C, pH = 8, stir and react for 1 h, inactivate the enzyme in boiling water for 5 min, self-assemble for 10 h, freeze-dry to obtain silk fibroin microspheres;

[0043] S5. Modification: Add 12 g of decellularized matrix to 200 mL of water, add 1 g of EDC and 1 g of NHS, stir and activate for 20 min, add 2 g of silk fibroin microspheres, stir and react for 7 h, freeze-dry to obtain silk fibroin microsphere-modified decellularized matrix;

[0044] S6. Defoaming and spreading: Dilute 15 g of collagen solution to a concentration of 0.5 wt% with PBS buffer at pH 7.4, add 4 g of silk fibroin microsphere-modified decellularized matrix, stir and mix for 15 min, remove bubbles by ultrasonic treatment at 1500 W for 10 min, spread on a flat mold, and perform continuous dehydration lamination to form a matrix layer;

[0045] S7. Crosslinking: Immerse the matrix layer in a solution containing 1 wt% EDC and 1 wt% NHS for crosslinking for 7 h, filter, wash, dehydrate, sterilize to obtain a composition containing adipose-derived stem cells with a water content of 15%. Example 2

[0046] This example provides a method for preparing a composition containing adipose-derived stem cells, including the following steps:

[0047] S1. Preparation of collagen solution: Debark, wash, and crush 15 g of pigskin, add to 100 mL of water, add 1 g of compound protease, at 40°C, pH value 9, enzymolysis for 4 h, filter, inactivate the enzyme in boiling water for 10 min, centrifuge, collect the supernatant to obtain collagen solution;

[0048] The compound protease is alkaline protease and trypsin, with a mass ratio of 5:4;

[0049] S2. Preparation of decellularization treatment solution: Add 3 g of Triton X-100 to 100 g of 1-hexyl-3-methylimidazolium chloride, stir and mix for 15 min, introduce carbon dioxide, ventilation rate 20 mL / min, ventilation time 30 min to obtain decellularization treatment solution;

[0050] S3. Preparation of acellular matrix: Resuspend adipose-derived stem cells in DMEM medium containing 12 wt% FBS at a cell density of 10 4 -10 5 cells / mL, culture at 38 °C and 7 vol% CO2 for 4 days, centrifuge, collect the cells, add 100 mL of acellular treatment solution, and the cell density is 10 5 -10 6 cells / mL, perform oscillation treatment for 2 h, centrifuge, wash the solid, and freeze-dry to obtain the acellular matrix;

[0051] S4. Preparation of silk fibroin microspheres: Add 20 g of silk fibroin peptide to 200 mL of water, add 2 g of alkaline protease, heat to 65 °C, pH = 8.5, stir and react for 3 h, inactivate the enzyme in boiling water for 10 min, self-assemble for 12 h, and freeze-dry to obtain silk fibroin microspheres;

[0052] S5. Modification: Add 15 g of acellular matrix to 200 mL of water, add 2 g of EDC and 2 g of NHS, stir and activate for 40 min, add 3 g of silk fibroin microspheres, stir and react for 10 h, and freeze-dry to obtain silk fibroin microsphere-modified acellular matrix;

[0053] S6. Defoaming and spreading: Dilute 20 g of collagen solution with PBS buffer at pH 8 to a concentration of 1 wt%, add 7 g of silk fibroin microsphere-modified acellular matrix, stir and mix for 15 min, remove bubbles by ultrasonic treatment at 2000 W for 20 min, spread it in a flat mold, and perform continuous dehydration and lamination to form a matrix layer;

[0054] S7. Crosslinking: Immerse the matrix layer in a solution containing 2 wt% EDC and 2 wt% NHS for crosslinking for 10 h, filter, wash, dehydrate, and sterilize to obtain a composition containing adipose-derived stem cells with a water content of 15%. Example 3

[0055] This example provides a method for preparing a composition containing adipose-derived stem cells, including the following steps:

[0056] S1. Preparation of collagen solution: Debark, wash, and crush 12 g of pigskin, add it to 100 mL of water, add 0.7 g of composite protease, at 37 °C, pH value of 8.5, enzymatically hydrolyze for 3 h, filter, inactivate the enzyme in boiling water for 7 min, centrifuge, and collect the supernatant to obtain the collagen solution;

[0057] The composite protease is alkaline protease and trypsin, and the mass ratio is 4:3;

[0058] S2. Preparation of decellularized treatment solution: Add 2.5 g of TritonX-100 to 100 g of 1-hexyl-3-methylimidazolium chloride, stir and mix for 15 min, then introduce carbon dioxide with an aeration rate of 15 mL / min for 25 min to obtain the decellularized treatment solution;

[0059] S3. Preparation of decellularized matrix: Resuspend adipose-derived stem cells in DMEM medium containing 10 wt% FBS at a cell density of 10 4 -10 5 cells / mL, culture at 37 °C and 5.5 vol% CO2 for 3 d, centrifuge to collect the cells, add 100 mL of the decellularized treatment solution, and adjust the cell density to 10 5 -10 6 cells / mL, perform oscillating treatment for 1.5 h, centrifuge, wash the solid, and freeze-dry to obtain the decellularized matrix; Figure 1 SEM image of the decellularized matrix prepared in Example 3. It can be seen from the figure that the internal voids of the material are relatively obvious, the cross-linking degree of the material is good, and it further verifies that the decellularization effect is obvious and there is no cell residue.

[0060] S4. Preparation of silk fibroin microspheres: Add 17 g of silk fibroin peptide to 200 mL of water, add 1.5 g of alkaline protease, heat to 60 °C, adjust the pH to 8.2, stir and react for 2 h, inactivate the enzyme in boiling water for 7 min, perform self-assembly for 11 h, and then freeze-dry to obtain silk fibroin microspheres;

[0061] S5. Modification: Add 13 g of the decellularized matrix to 200 mL of water, add 1.5 g of EDC and 1.5 g of NHS, stir and activate for 30 min, add 2.5 g of silk fibroin microspheres, stir and react for 8 h, and then freeze-dry to obtain silk fibroin microsphere-modified decellularized matrix;

[0062] S6. Defoaming and spreading: Dilute 17 g of collagen solution with PBS buffer at pH 7.8 to a concentration of 0.7 wt%, add 5.5 g of silk fibroin microsphere-modified decellularized matrix, stir and mix for 15 min, remove bubbles by ultrasonic treatment at 1700 W for 15 min, spread it in a flat mold, and form a matrix layer after continuous dehydration and lamination;

[0063] S7. Cross-linking: Immerse the matrix layer in a solution containing 1.5 wt% EDC and 1.5 wt% NHS for cross-linking for 8 h, filter, wash, dehydrate, and sterilize to obtain a composition containing adipose-derived stem cells with a water content of 15%.

[0064] Comparative Example 1

[0065] Compared with Example 3, the difference is that carbon dioxide is not introduced in step S2.

[0066] The details are as follows:

[0067] S2. Preparation of decellularized treatment solution: Add 2.5 g of Triton X-100 to 100 g of 1-hexyl-3-methylimidazolium chloride, stir and mix for 15 min to obtain the decellularized treatment solution.

[0068] Comparative Example 2

[0069] Compared with Example 3, the difference lies in that Triton X-100 was not added in step S2.

[0070] The details are as follows:

[0071] S2. Preparation of decellularized treatment solution: Pass carbon dioxide into 100 g of 1-hexyl-3-methylimidazolium chloride at a ventilation rate of 15 mL / min for 25 min to obtain the decellularized treatment solution.

[0072] Comparative Example 3

[0073] Compared with Example 3, the difference lies in that 1-hexyl-3-methylimidazolium chloride in step S2 was replaced by water.

[0074] The details are as follows:

[0075] S2. Preparation of decellularized treatment solution: Add 2.5 g of Triton X-100 to 100 g of water, stir and mix for 15 min, then pass carbon dioxide at a ventilation rate of 15 mL / min for 25 min to obtain the decellularized treatment solution.

[0076] Comparative Example 4

[0077] Compared with Example 3, the difference lies in that steps S4 and S5 were not carried out.

[0078] The details are as follows:

[0079] S1. Preparation of collagen solution: Debone, wash, and crush 12 g of pigskin, add it to 100 mL of water, add 0.7 g of compound protease, carry out enzymatic hydrolysis at 37 °C and pH 8.5 for 3 h, filter, inactivate the enzyme with boiling water for 7 min, centrifuge, and collect the supernatant to obtain the collagen solution;

[0080] The compound protease is alkaline protease and trypsin, and the mass ratio is 4:3;

[0081] S2. Preparation of decellularized treatment solution: Add 2.5 g of Triton X-100 to 100 g of 1-hexyl-3-methylimidazolium chloride, stir and mix for 15 min, then pass carbon dioxide at a ventilation rate of 15 mL / min for 25 min to obtain the decellularized treatment solution;

[0082] S3. Preparation of decellularized matrix: The adipose-derived stem cells were resuspended in DMEM medium containing 10 wt% FBS, with a cell density of 10 4 -10 5 cells / mL. They were cultured at 37 °C with 5.5 vol% CO2 for 3 days, centrifuged, and the cells were collected. 100 mL of decellularization treatment solution was added, with a cell density of 10 5 -10 6 cells / mL. After shaking for 1.5 h, centrifugation, solid washing, and freeze-drying, the decellularized matrix was obtained;

[0083] S4. Defoaming and spreading: 17 g of collagen solution was diluted with PBS buffer at pH 7.8 to a concentration of 0.7 wt%. 5.5 g of decellularized matrix was added, and they were stirred and mixed for 15 min. Bubbles were removed by ultrasonic treatment at 1700 W for 15 min, and then spread on a flat mold. After continuous dehydration and lamination, a matrix layer was formed;

[0084] S5. Crosslinking: The matrix layer was soaked in a solution containing 1.5 wt% EDC and 1.5 wt% NHS for crosslinking for 8 h, filtered, washed, dehydrated, and sterilized to obtain a composition containing adipose-derived stem cells with a water content of 15%.

[0085] Comparative Example 5

[0086] Compared with Example 3, the difference is that silk fibroin microspheres were not added to modify the decellularized matrix in step S6.

[0087] Specifically as follows:

[0088] S6. Defoaming and spreading: 17 g of collagen solution was diluted with PBS buffer at pH 7.8 to a concentration of 0.7 wt%. Bubbles were removed by ultrasonic treatment at 1700 W for 15 min, and then spread on a flat mold. After continuous dehydration and lamination, a matrix layer was formed.

[0089] Comparative Example 6

[0090] Compared with Example 3, the difference is that EDC and NHS were replaced with glutaraldehyde of equal total mass.

[0091] Specifically as follows:

[0092] S7. Crosslinking: The matrix layer was soaked in a solution containing 3 wt% glutaraldehyde for crosslinking for 8 h, filtered, washed, dehydrated, and sterilized to obtain a composition containing adipose-derived stem cells with a water content of 15%.

[0093] Test Example 1

[0094] The compositions containing adipose stem cells prepared in Examples 1-3 and Comparative Examples 1-6 were subjected to performance tests. The sample thickness was 10 μm, and the results are shown in Table 1.

[0095] 1. Shear strength

[0096] Referring to the method in YY / T 0729.1, the sample was cut into strips with a width of 2.5 cm, and the sample was overlapped with a porcine skin graft. The overlapping area was 2.5 cm × 1.0 cm. A force of 1-2 N was applied to the overlapping area to make the sample and the porcine skin graft fit tightly, and then the shear strength was measured. The two ends of the specimen were respectively clamped on the fixtures of a tensile testing machine, with the force application direction being the long axis direction of the specimen. The specimen was loaded at a speed of 5 mm / min until the overlapping area separated, and the maximum force was recorded and the shear strength was calculated.

[0097] 2. Compressive performance

[0098] Referring to ASTM F2392-04, a fresh porcine stomach was taken, and a circular notch with a diameter of 5 mm was made on the surface. The sample was cut into a size of 2.5 cm × 2.5 cm (the distance between the sample edge and the defect area edge was at least 1 cm), and it was flatly attached to the defect in a centrally symmetric manner. One end of the sample was connected to a pressure gauge, and the other end was connected to a gas inlet to measure the maximum pressure that the defect could withstand.

[0099] 3. Degradation performance

[0100] An in vitro degradation test was carried out on the product to compare the degradation times of various samples. The sample was placed in a type I collagenase solution and put into a constant temperature incubator at 37°C. The enzyme solution was changed every 2 days, and the degradation situation of the sample was observed every 12 hours, and the time required for the sample to be completely degraded was recorded.

[0101] 4. Cytotoxicity

[0102] Referring to GB / T 16886.5-2017, the in vitro cytotoxicity of the sample was detected by the MTT method. In the cell survival rate (%), the blank control group was 100%.

[0103] Table 1

[0104]

[0105] As can be seen from the above table, the compositions containing adipose stem cells prepared in Examples 1-3 of the present invention have good mechanical properties and degradability, and low cytotoxicity. The cell viability in Comparative Examples 1-3 decreased, and their biocompatibility decreased. It can be seen that the acellular treatment solution 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 structure, and has excellent biocompatibility, low immunogenicity and biological activity. The mechanical properties of Comparative Examples 4 and 5 decreased significantly, and the mechanical properties of the acellular matrix were significantly enhanced by modifying it with silk fibroin microspheres prepared by nano-scale self-assembly. In Comparative Example 6, glutaraldehyde was used for cross-linking, and the cell viability decreased greatly, indicating that glutaraldehyde cross-linking has certain toxicity.

[0106] The above are only the preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall 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 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%.

2. The preparation method according to claim 1, 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, 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.

3. The preparation method according to claim 2, characterized in that: The composite protease is alkaline protease and trypsin, and the mass ratio is 3-5:2-4.

4. The preparation method according to claim 1, 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 1, 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 1, 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 1, 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 1, 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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