Autologous fat-derived mesenchymal stem cell composition, its preparation method and application

Abstract

The present invention provides a composition of mesenchymal stem cells derived from autologous fat, a preparation method thereof, and an application thereof, belonging to the technical field of medical adjuvants. Mesenchymal stem cells are isolated from autologous fat particles and cultured in a stem cell culture medium supplemented with cell stimulating particles and fetal bovine serum. Stem cells and extracellular matrix are obtained by separation. The stem cells are prepared into stem cell liposomes, and sanguinarine and curcumin are embedded in a composite shell material of chitosan and β-cyclodextrin to prepare sustained-release microspheres. The composition of mesenchymal stem cells derived from autologous fat prepared by the present invention has good air permeability, softness, certain mechanical properties and good biocompatibility, can rapidly promote wound healing, is convenient to remove, and will not cause secondary damage.

Description

Technical Field

[0001] The present invention relates to the technical field of medical dressings, and particularly relates to a composition of mesenchymal stem cells derived from autologous fat, a preparation method thereof, and an application thereof. Background Art

[0002] Wound healing refers to the process of tissue reformation under a series of regulatory effects after a body generates a wound due to various reasons, including acute wounds that can heal naturally, such as mild burn and scald wounds and superficial skin injuries, and also those that must rely on drug and surgical treatments, such as ulcerative wounds. The skin wound repair process is mainly divided into several stages: coagulation stage, inflammatory reaction stage, cell growth stage, and tissue reformation. Wound repair requires the cooperation of multiple cells. The absence or delay of any link in the whole process will cause the skin wound healing process to not proceed normally. At present, the basic treatment options for skin wounds are surgical operations and drug treatments. Surgical operations generally suture the wound surface to finally repair the skin defect, which is a commonly used method in clinical practice and has a fast effect. However, when the wound area is large and the wound is deep, performing surgical debridement is likely to aggravate the condition. For drug treatments, the general treatment effects are mostly unsatisfactory.

[0003] Human autologous adipose-derived stem cells (ADSCs) are a group of stem cells in adipose stroma. They are a type of mesenchymal stem cells (MSCs) that can self-renew and have multi-directional differentiation potential. They can differentiate into osteoblasts, chondrocytes, adipocytes, etc. in vitro and are cells with multi-potent differentiation ability. A large number of experiments have confirmed that in a certain induction environment, adipose cells can differentiate into osteocytes, adipocytes, chondrocytes, nerve cells, etc. After transplantation, they have good replacement, repair, and treatment effects, and have no tumorigenicity and do not cause immune rejection with the autologous body. Stem cells themselves have characteristics such as differentiation and proliferation. During the wound healing process, they can participate in processes related to wound healing, such as cell proliferation and differentiation, migration, and the reformation of the extracellular matrix (ECM), and play a role in accelerating wound healing in many aspects. It not only shortens the wound healing time but also solves problems such as the inability to regenerate skin appendages such as sweat glands, enabling the skin to restore its original physiological function. Adipose-derived mesenchymal stem cells are widely sourced and easy to extract. Adipose-derived mesenchymal stem cells have the ability of self-renewal and multi-directional differentiation potential. However, when using mesenchymal stem cells to treat tissue damage by transplantation, the number of cells transplanted at one time needs to reach as high as 1×10 9Only then can it effectively play a therapeutic role. To ensure the stability and comparability of experimental results, a large number of seed cells with a uniform source are required. In addition, xenotransplantation has a strong immune response. Therefore, a new method is needed to avoid the aging and even malignancy of mesenchymal stem cells caused by repeated cell passage, which is easy to obtain, convenient to store, and does not induce immune rejection after transplantation. Mesenchymal stem cells secrete a variety of bioactive factors, and it has been confirmed that the paracrine function of stem cells is the main way for them to play a role.

[0004] Traditional dressings are still widely used due to their low production cost, simple manufacturing process, and convenient transportation. With the gradual improvement of people's treatment requirements, such traditional dressings have a series of significant disadvantages. There is too much wound tissue fluid, which increases the chance of the dressing sticking to the wound, making it easy to cause secondary injury when changing the dressing; they cannot meet the adaptation to diverse wound types; they lack controllable wound hygroscopicity, resulting in the wound being too dry, and cannot promote wound healing in a timely manner. Currently, traditional dressings are only suitable for the treatment of mild extrusion wounds or as secondary dressings.

[0005] Chinese invention patent CN116687837B discloses a stem cell preparation for promoting wound healing and its preparation method. 18.75 mL of carbomer gel is mixed with 6.25 mL of concentrated supernatant of human adipose mesenchymal stem cells, and then 1562.5 μL of betaine working solution is added and shaken until evenly mixed to obtain a stem cell preparation for promoting wound healing, and the stem cell preparation is stored at 4°C. However, the stem cell preparation obtained by this patented technology has limited repair effect on wound healing, restricting its application. Summary of the Invention

[0006] The purpose of the present invention is to provide a mesenchymal stem cell composition derived from autologous fat, its preparation method and application. The preparation method is simple, has good air permeability, flexibility, certain mechanical properties and good biocompatibility, can quickly promote wound healing, is convenient to remove, has certain hemostatic and good antibacterial properties, avoids wound infection, and will not cause secondary injury.

[0007] The technical solution of the present invention is realized as follows:

[0008] The present invention provides a preparation method of a mesenchymal stem cell composition derived from autologous fat. Mesenchymal stem cells are separated from autologous fat particles and cultured in a stem cell culture medium added with cell stimulating particles and fetal bovine serum. Stem cells and extracellular matrix are separated, the stem cells are prepared into stem cell liposomes, and sanguinarine and curcumin are embedded in a composite shell material of chitosan and β-cyclodextrin to prepare a sustained-release microsphere.

[0009] As a further improvement of the present invention, it includes the following steps:

[0010] S1. Preparation of stem cell culture medium: Add cell stimulating particles and fetal bovine serum to α-MEM culture medium, and disperse evenly by ultrasonic wave to obtain stem cell culture medium;

[0011] S2. Isolation and in vitro proliferation culture of stem cells: Centrifuge the autologous fat particles extracted, collect the upper layer of adipose tissue from this centrifugation, add complex enzyme for digestion, centrifuge, collect the lower layer of cells from this centrifugation, resuspend with the stem cell culture medium in step S1, and culture under the condition of external magnetic field, then centrifuge and separate. The lower layer from this centrifugation and separation is mesenchymal stem cells derived from autologous fat, and the upper layer is extracellular matrix, which is reserved for use;

[0012] S3. Preparation of stem cell liposomes: Resuspend the mesenchymal stem cells derived from autologous fat in step S2 in PBS solution to obtain a cell suspension; Dissolve lecithin, DSPE-PEG2000, DSPE-PEG-CREKA, and cholesterol in chloroform, remove the solvent by rotary evaporation under reduced pressure to obtain liposomes, add the cell suspension and mix evenly, and repeatedly extrude with a liposome extruder using a polycarbonate membrane to obtain stem cell liposomes;

[0013] S4. Preparation of sustained-release microspheres: Dissolve chitosan in acid solution, add β-cyclodextrin, sanguinarine, and curcumin, add an octanol solution of emulsifier, stir, dropwise add an aqueous solution of sodium tripolyphosphate for crosslinking, centrifuge, wash, and freeze-dry to obtain sustained-release microspheres;

[0014] S5. Preparation of mesenchymal stem cell composition derived from autologous fat: Add N-isopropylacrylamide to the extracellular matrix in step S2, under the protection of inert gas, add initiator and 2-mercaptoethylamine, stir and react under the condition of ice-water bath, add chitosan solution, EDC, and NHS, stir and react at room temperature, then add silk fibroin peptide, EDC, and NHS, continue to stir and react, and then add stem cell liposomes and sustained-release microspheres, stir and mix evenly to obtain mesenchymal stem cell composition derived from autologous fat.

[0015] As a further improvement of the present invention, the concentration of the cell stimulating particles in step S1 is 2-4 wt%, the concentration of the fetal bovine serum is 8-12 wt%, and the preparation method of the cell stimulating particles is as follows:

[0016] T1. Preparation of UiO-66-NH2: Add zirconium chloride and 2-aminoterephthalic acid to a mixed solvent of N,N-dimethylformamide and acetic acid, carry out hydrothermal reaction, cool, centrifuge, collect the solid, wash, and dry to obtain UiO-66-NH2;

[0017] T2. Depositing magnetic iron tetroxide: Add UiO-66-NH2 into water, add ferric chloride and ferrous chloride, dropwise add ammonia water, heat and stir for reaction, centrifuge, wash, dry, and calcine to obtain Fe3O4@UiO-66-NH2;

[0018] T3. Loading TGF-β1 and TGF-β3: Add TGF-β1 and TGF-β3 into water, add NHS and EDC, stir for activation, add Fe3O4@UiO-66-NH2, stir for reaction, centrifuge, wash, dry to obtain cell-stimulating particles.

[0019] As a further improvement of the present invention, in step T1, the mass ratio of zirconium chloride to 2-aminoterephthalic acid is 13-17:11-15, the temperature of the hydrothermal reaction is 120-140 °C, and the time is 20-24 h; in step T2, the mass ratio of UiO-66-NH2, ferric chloride and ferrous chloride is 10-15:3.24:1.26, the pH value of the solution is adjusted to 10-11 by dropwise adding ammonia water, the temperature of the heating and stirring reaction is 80-90 °C, the time is 3-5 h, the calcination temperature is 550-650 °C, and the time is 1-2 h; in step T3, the mass ratio of TGF-β1, TGF-β3, NHS, EDC and Fe3O4@UiO-66-NH2 is 0.2-0.4:0.1-0.3:0.05-0.1:0.05-0.1:5-7.

[0020] As a further improvement of the present invention, in step S2, the complex enzyme is a mixture of trypsin and collagenase, the mass ratio is 2-4:1-3, the addition amount of the complex enzyme is 1-3 wt% of the mass of the upper adipose tissue, the digestion temperature is 35-38 °C, and the time is 20-40 min; the cell density after resuspension is 10 3 -10 4 cells / mL, the culture conditions are 35-38 °C, the volume concentration of carbon dioxide is 3-7%, the time is 3-5 d, and the intensity of the external magnetic field is 0.01-0.05 T.

[0021] As a further improvement of the present invention, in step S3, the cell density of the cell suspension is 10 4 -10 5 g / mL, the mass ratio of lecithin, DSPE-PEG2000, DSPE-PEG-CREKA, and cholesterol is 12-15:4-7:3-5:4-6, and the number of repeated extrusion times is 20-30 times.

[0022] As a further improvement of the present invention, in step S4, the acid solution is a 1-3 wt% acetic acid solution, and the mass ratio of chitosan, β-cyclodextrin, sanguinarine, curcumin, emulsifier and sodium tripolyphosphate is 5-7:3-5:1-2:2-3:0.5-1:0.1-0.2. The emulsifier is selected from at least one of Tween-20, Tween-40, Tween-60, Tween-80, and Tween-85.

[0023] As a further improvement of the present invention, in step S5, the initiator is selected from at least one of sodium persulfate, potassium persulfate, and ammonium persulfate. The mass ratio of N-isopropylacrylamide, initiator, 2-mercaptoethylamine, chitosan, silk fibroin peptide, stem cell liposome, and sustained-release microsphere is 18-22:3-4:10-12:10-14:7-10:8-12:5-7.

[0024] The present invention further protects an autologous fat-derived mesenchymal stem cell composition prepared by the above preparation method.

[0025] The present invention further protects the application of the above autologous fat-derived mesenchymal stem cell composition in the preparation of a drug for promoting skin healing.

[0026] The present invention has the following beneficial effects:

[0027] The present invention obtains fat-derived mesenchymal stem cells from autologous tissues, which have low immunogenicity, do not cause rejection reactions, and have a wide source of raw materials and a simple preparation method. They are added to the prepared stem cell culture medium for in vitro amplification and culture. Under the action of cell-stimulating particles in the culture medium and the action of an external magnetic field, the proliferation of stem cells is promoted. TGF-β1 and TGF-β3, TGF-β1 is a multifunctional cytokine that regulates the proliferation, differentiation, and extracellular matrix synthesis of stem cells through the Smad signaling pathway; TGF-β3 activates the Smad pathway in cells through the signal pathways mediated by integrin αvβ6 and αvβ8, promoting cell migration and tissue repair. The two work together to play an important promoting role by regulating cell proliferation, differentiation, and extracellular matrix synthesis.

[0028] The cell-stimulating particles prepared by the present invention use UiO-66-NH2 material as a carrier. -NH2 can promote the in-situ deposition of magnetic iron oxide through hydrogen bonds. Magnetic iron oxide not only has antibacterial properties and improves the resistance of stem cells, but also can promote the growth of stem cells by regulating cell proliferation and migration. UiO-66-NH2 has high porosity and good biocompatibility, can provide a stable microenvironment for stem cells, promote cell adhesion and growth, and the loaded cytokines further promote the growth and survival of stem cells.

[0029] The present invention uses the extracellular matrix after culturing stem cells in a culture medium as a solvent to prepare a hydrogel, and the cell-stimulating particles therein also have an obvious effect on promoting skin healing. Among them, magnetite can also regulate cell behavior to promote scarless healing, regulate the proliferation and migration of fibroblasts, reduce fibrosis, effectively promote wound healing, reduce inflammatory reactions, promote skin healing, and induce the regeneration of hair follicles and blood vessels. TGF-β1 can promote the transformation of fibroblasts into myofibroblasts, which promotes the wound healing process but also leads to the formation of scars. TGF-β3 can promote the synthesis of collagen and the proliferation of fibroblasts. The reduced ratio of TGF-β1 / TGF-β3 reduces the formation of scar tissue and achieves higher-quality healing.

[0030] In addition, the active components (such as extracellular vesicles and exosomes) in the extracellular matrix play a crucial role in regulating the signal transduction of cells around the wound. The hydrogel scaffold provides physical isolation for the wound surface and promotes the cell adhesion function around the wound tissue. At the same time, the addition of the hydrogel can slow down the release of the active components. The continuously released active components at the wound can better synergistically stimulate the healing of the wound and act as a "sacrificial substrate" in the wound to delay the erosion of the wound tissue by various enzymes at the wound. The combination of the two can accelerate wound healing by regulating the wound microenvironment, promoting collagen synthesis, cell migration, and angiogenesis.

[0031] The hydrogel of the present invention uses poly(N-isopropylacrylamide) (PNIPAAM) as one of the polymer materials. Since the lower critical solution temperature (about 32 °C) of its aqueous solution is close to the normal human body temperature (37 °C), when the temperature is higher than 32 °C, the hydrogen bonds in PNIPAAM itself show contraction, thereby promoting the release of the loaded drug and achieving drug controlled release. The modified scaffold structure formed by chitosan and silk fibroin peptide greatly improves the antibacterial property, low sensitization, and high biocompatibility of the hydrogel. After forming a solid hydrogel system through temperature change, the hydrogen bonds show contraction, promoting the release of stem cell liposomes and sustained-release microspheres, thereby promoting the healing of the skin.

[0032] In the present invention, stem cells are encapsulated by liposomes. On the one hand, it improves the transdermal absorption effect of stem cells, has the function of targeted release, and enables efficient delivery. On the other hand, it can protect stem cells from the influence of the external environment and extend their survival time and activity in the body. The sustained-release microspheres of the present invention encapsulate curcumin and sanguinarine, and the encapsulation layer includes cyclodextrin. The cavity of cyclodextrin can load the poorly soluble drug sanguinarine, thereby greatly increasing the loading amount. Sanguinarine has good antibacterial properties and can reduce inflammatory reactions. Curcumin has a strong antioxidant effect and can reduce the overexpression of reactive oxygen species that can cause chronic inflammation, thereby promoting the healing of skin wounds.

[0033] The present invention prepares a mesenchymal stem cell composition derived from autologous fat, which is a thermosensitive hydrogel. It presents a liquid state at room temperature. A layer of this composition is evenly dropped at the damaged part of the skin. Under the condition of body temperature, it quickly solidifies to form a hydrogel layer. As a hydrophilic gel with a three-dimensional network structure, the hydrogel has good air permeability, softness, certain mechanical properties and good biocompatibility. At the same time, the hydrogel structure can maintain a relatively moist environment at the skin, quickly promoting wound healing; the internal polymer structure has pores, enabling necessary gas exchange between the damaged tissue and the environment; at the same time, this hydrogel material can absorb cell tissue fluid, has certain hemostatic and good antibacterial properties, avoiding wound infection; it is non-toxic and simple to prepare; it has appropriate adhesiveness to the wound tissue, is convenient to remove, and will not cause secondary injury. Detailed implementation mode

[0034] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0035] NHS, N-hydroxysuccinimide; EDC, 1-ethyl-(3-dimethylaminopropyl)carbodiimide; PBS, phosphate buffer solution; TGF-β1, transforming growth factor-β1; TGF-β3, transforming growth factor-β3; DSPE-PEG2000, distearoyl phosphatidylethanolamine-polyethylene glycol 2000; DSPE-PEG-CREKA, distearoyl phosphatidylethanolamine-polyethylene glycol-tumor targeting protein. Trypsin, 2500U / mg; collagenase, 125U / mg. α-MEM culture medium, Merck Biotech.

[0036] Preparation Example 1 Preparation of cell-stimulating particles

[0037] The method is as follows:

[0038] T1. Preparation of UiO-66-NH2: 1.3 g of zirconium chloride and 1.1 g of 2-aminoterephthalic acid are added to a mixed solvent of 200 mL of N,N-dimethylformamide and acetic acid (volume ratio of 24:1), and hydrothermal reaction is carried out at 120 °C for 20 h. After cooling, centrifugation, collecting the solid, washing, and drying, UiO-66-NH2 is obtained;

[0039] T2. Deposition of magnetic iron tetroxide: Add 10 g of UiO-66-NH2 to 200 mL of water, add 3.24 g of ferric chloride and 1.26 g of ferrous chloride, dropwise add ammonia water to adjust the pH value of the solution to 10, heat to 80 °C, stir and react for 3 h, centrifuge, wash, dry, and calcine at 550 °C for 1 h to obtain Fe3O4@UiO-66-NH2;

[0040] T3. Loading of TGF-β1 and TGF-β3: Add 0.2 g of TGF-β1 and 0.1 g of TGF-β3 to 100 mL of water, add 0.05 g of NHS and 0.05 g of EDC, stir and activate for 30 min, add 5 g of Fe3O4@UiO-66-NH2, stir and react for 8 h, centrifuge, wash, dry to obtain cell-stimulating particles.

[0041] Preparation Example 2 Preparation of cell-stimulating particles

[0042] The method is as follows:

[0043] T1. Preparation of UiO-66-NH2: Add 1.7 g of zirconium chloride and 1.5 g of 2-aminoterephthalic acid to 200 mL of a mixed solvent of N,N-dimethylformamide and acetic acid (volume ratio 24:1), carry out hydrothermal reaction at 140 °C for 24 h, cool, centrifuge, collect the solid, wash, dry to obtain UiO-66-NH2;

[0044] T2. Deposition of magnetic iron tetroxide: Add 15 g of UiO-66-NH2 to 200 mL of water, add 3.24 g of ferric chloride and 1.26 g of ferrous chloride, dropwise add ammonia water to adjust the pH value of the solution to 11, heat to 90 °C, stir and react for 5 h, centrifuge, wash, dry, and calcine at 650 °C for 2 h to obtain Fe3O4@UiO-66-NH2;

[0045] T3. Loading of TGF-β1 and TGF-β3: Add 0.4 g of TGF-β1 and 0.3 g of TGF-β3 to 100 mL of water, add 0.1 g of NHS and 0.1 g of EDC, stir and activate for 30 min, add 7 g of Fe3O4@UiO-66-NH2, stir and react for 10 h, centrifuge, wash, dry to obtain cell-stimulating particles.

[0046] Preparation Example 3 Preparation of cell-stimulating particles

[0047] The method is as follows:

[0048] T1. Preparation of UiO-66-NH2: 1.5 g of zirconium chloride and 1.3 g of 2-aminoterephthalic acid were added to 200 mL of a mixed solvent of N,N-dimethylformamide and acetic acid (volume ratio 24:1), and hydrothermally reacted at 130 °C for 22 h. After cooling, centrifugation, collecting the solid, washing, and drying, UiO-66-NH2 was obtained;

[0049] T2. Deposition of magnetic iron tetroxide: 12 g of UiO-66-NH2 was added to 200 mL of water, 3.24 g of ferric chloride and 1.26 g of ferrous chloride were added, and the pH value of the solution was adjusted to 10.5 by dropping ammonia water. It was heated to 85 °C and stirred for 4 h, centrifuged, washed, dried, and calcined at 600 °C for 1.5 h to obtain Fe3O4@UiO-66-NH2;

[0050] T3. Loading of TGF-β1 and TGF-β3: 0.3 g of TGF-β1 and 0.2 g of TGF-β3 were added to 100 mL of water, 0.07 g of NHS and 0.07 g of EDC were added, and stirred for activation for 30 min. Then 6 g of Fe3O4@UiO-66-NH2 was added and stirred for reaction for 9 h, centrifuged, washed, dried, to obtain cell-stimulating particles.

[0051] Comparative Preparation Example 1

[0052] Compared with Preparation Example 3, the difference is that step T2 was not carried out.

[0053] Specifically as follows:

[0054] T1. Preparation of UiO-66-NH2: 1.5 g of zirconium chloride and 1.3 g of 2-aminoterephthalic acid were added to 200 mL of a mixed solvent of N,N-dimethylformamide and acetic acid (volume ratio 24:1), and hydrothermally reacted at 130 °C for 22 h. After cooling, centrifugation, collecting the solid, washing, and drying, UiO-66-NH2 was obtained;

[0055] T2. Loading of TGF-β1 and TGF-β3: 0.3 g of TGF-β1 and 0.2 g of TGF-β3 were added to 100 mL of water, 0.07 g of NHS and 0.07 g of EDC were added, and stirred for activation for 30 min. Then 6 g of UiO-66-NH2 was added and stirred for reaction for 9 h, centrifuged, washed, dried, to obtain cell-stimulating particles.

[0056] Comparative Preparation Example 2

[0057] Compared with Preparation Example 3, the difference is that TGF-β1 was not added in step T3.

[0058] Specifically as follows:

[0059] T3. Loading TGF-β3: Add 0.5 g of TGF-β3 into 100 mL of water, add 0.07 g of NHS and 0.07 g of EDC, stir and activate for 30 min, add 6 g of Fe3O4@UiO-66-NH2, stir and react for 9 h, centrifuge, wash, and dry to obtain cell-stimulating particles.

[0060] Comparative Preparation Example 3

[0061] Compared with Preparation Example 3, the difference lies in that TGF-β3 is not added in step T3.

[0062] Specifically as follows:

[0063] T3. Loading TGF-β1: Add 0.5 g of TGF-β1 into 100 mL of water, add 0.07 g of NHS and 0.07 g of EDC, stir and activate for 30 min, add 6 g of Fe3O4@UiO-66-NH2, stir and react for 9 h, centrifuge, wash, and dry to obtain cell-stimulating particles.

[0064] Comparative Preparation Example 4

[0065] Compared with Preparation Example 3, the difference lies in that step T3 is not carried out.

[0066] Specifically as follows:

[0067] T1. Preparation of UiO-66-NH2: Add 1.5 g of zirconium chloride and 1.3 g of 2-aminoterephthalic acid into 200 mL of a mixed solvent of N,N-dimethylformamide and acetic acid (volume ratio 24:1), carry out hydrothermal reaction at 130 °C for 22 h, cool, centrifuge, collect the solid, wash, and dry to obtain UiO-66-NH2;

[0068] T2. Deposition of magnetic iron tetroxide: Add 12 g of UiO-66-NH2 into 200 mL of water, add 3.24 g of ferric chloride and 1.26 g of ferrous chloride, dropwise add ammonia water to adjust the pH value of the solution to 10.5, heat to 85 °C, stir and react for 4 h, centrifuge, wash, dry, and calcine at 600 °C for 1.5 h to obtain Fe3O4@UiO-66-NH2, which is the cell-stimulating particle.

[0069] Example 1

[0070] This example provides a preparation method of an autologous fat-derived mesenchymal stem cell composition, including the following steps:

[0071] S1. Preparation of stem cell culture medium: Add the cell stimulation particles prepared in Preparation Example 1 and fetal bovine serum to α-MEM culture medium, and disperse them by ultrasonic wave at 1000W for 15 min to obtain the stem cell culture medium. The concentration of the cell stimulation particles is 2 wt%, and the concentration of fetal bovine serum is 8 wt%.

[0072] S2. Isolation and in vitro proliferation culture of stem cells: Take autologous fat particles, centrifuge at 1000 r / min for 5 min, collect the upper fat tissue from this centrifugation, add a composite enzyme, the addition amount of the composite enzyme is 1 wt% of the mass of the upper fat tissue, digest at 35 °C for 20 min, centrifuge at 700 g / min for 5 min, collect the lower cells from this centrifugation, resuspend them in the stem cell culture medium in step S1, and the cell density is 10 3 -10 4 cells / mL. Under the condition of an external magnetic field with an intensity of 0.01 T, at 35 °C, and a carbon dioxide volume concentration of 3%, culture for 3 d, centrifuge and separate at 700 g / min for 10 min. The lower layer after this centrifugation and separation is mesenchymal stem cells derived from autologous fat, and the upper layer is the extracellular matrix, which is reserved for use.

[0073] The composite enzyme is a mixture of trypsin and collagenase, and the mass ratio is 2:1.

[0074] S3. Preparation of stem cell liposomes: Resuspend the mesenchymal stem cells derived from autologous fat in step S2 in PBS solution to obtain a cell suspension, and the cell density is 10 4 -10 5 g / mL; Dissolve 1.2 g of lecithin, 0.4 g of DSPE-PEG2000, 0.3 g of DSPE-PEG-CREKA, and 0.4 g of cholesterol in 50 mL of chloroform, remove the solvent by rotary evaporation under reduced pressure to obtain liposomes, add 50 mL of cell suspension and mix evenly, and repeatedly extrude with a liposome extruder using a polycarbonate membrane for 20 times to obtain stem cell liposomes.

[0075] S4. Preparation of sustained-release microspheres: Dissolve 5 g of chitosan in 200 mL of 1 wt% acetic acid solution, add 3 g of β-cyclodextrin, 1 g of sanguinarine, and 2 g of curcumin, add 50 mL of octanol solution containing 0.5 g of Tween-20, stir for 30 min, dropwise add 10 mL of aqueous solution containing 0.1 g of sodium tripolyphosphate, crosslink for 1 h, centrifuge, wash, and freeze-dry to obtain sustained-release microspheres.

[0076] S5. Preparation of autologous fat-derived mesenchymal stem cell composition: Add 1.8 g of N-isopropylacrylamide to 200 mL of the extracellular matrix in step S2. Under nitrogen protection, add 0.3 g of potassium persulfate and 1 g of 2-mercaptoethylamine, and stir and react for 3 h under ice-water bath conditions. Then add 30 mL of acetic acid solution containing 1 g of chitosan (acetic acid content is 2 wt%), 0.3 g of EDC and 0.2 g of NHS, and stir and react at room temperature for 20 h. Then add 0.7 g of silk fibroin peptide, 0.3 g of EDC and 0.1 g of NHS, and continue to stir and react for 20 h. Then add 0.8 g of stem cell liposome and 0.5 g of sustained-release microspheres, and stir and mix for 30 min to obtain the autologous fat-derived mesenchymal stem cell composition.

[0077] Example 2

[0078] This example provides a method for preparing an autologous fat-derived mesenchymal stem cell composition, comprising the following steps:

[0079] S1. Preparation of stem cell culture medium: Add the cell stimulation particles and fetal bovine serum prepared in Preparation Example 2 to α-MEM culture medium, and disperse by ultrasonic wave at 1000 W for 15 min to obtain the stem cell culture medium. The concentration of the cell stimulation particles is 4 wt% and the concentration of fetal bovine serum is 12 wt%.

[0080] S2. Isolation and in vitro proliferation culture of stem cells: Take autologous fat particles, centrifuge at 1000 r / min for 5 min, collect the upper fat tissue from this centrifugation, add a composite enzyme, the addition amount of the composite enzyme is 3 wt% of the mass of the upper fat tissue, digest at 38 °C for 40 min, centrifuge at 700 g / min for 5 min, collect the lower-layer cells from this centrifugation, resuspend with the stem cell culture medium in step S1, the cell density is 10 3 -10 4 cells / mL, under the condition of an external magnetic field with an intensity of 0.05 T, at 38 °C, the carbon dioxide volume concentration is 7%, culture for 5 d, centrifuge and separate at 700 g / min for 10 min. The lower layer from this centrifugation and separation is autologous fat-derived mesenchymal stem cells, and the upper layer is extracellular matrix, which is reserved for use;

[0081] The composite enzyme is a mixture of trypsin and collagenase, and the mass ratio is 4:3;

[0082] S3. Preparation of stem cell liposome: Resuspend the autologous fat-derived mesenchymal stem cells in step S2 in PBS solution to obtain a cell suspension, and the cell density is 10 4 -10 5g / mL; Dissolve 1.5 g of lecithin, 0.7 g of DSPE-PEG2000, 0.5 g of DSPE-PEG-CREKA, and 0.6 g of cholesterol in 50 mL of chloroform. Remove the solvent by rotary evaporation under reduced pressure to obtain liposomes. Add 50 mL of cell suspension and mix evenly. Repeatedly extrude with a liposome extruder using a polycarbonate membrane 30 times to obtain stem cell liposomes;

[0083] S4. Preparation of sustained-release microspheres: Dissolve 7 g of chitosan in 200 mL of 3 wt% acetic acid solution. Add 5 g of β-cyclodextrin, 2 g of sanguinarine, and 3 g of curcumin. Add 50 mL of octanol solution containing 1 g of Tween-60 and stir for 30 min. Dropwise add 10 mL of aqueous solution containing 0.2 g of sodium tripolyphosphate, crosslink for 1 h, centrifuge, wash, and freeze-dry to obtain sustained-release microspheres;

[0084] S5. Preparation of autologous fat-derived mesenchymal stem cell composition: Add 2.2 g of N-isopropylacrylamide to 200 mL of the extracellular matrix in step S2. Under nitrogen protection, add 0.4 g of sodium persulfate and 1.2 g of 2-mercaptoethylamine, and stir and react in an ice-water bath for 5 h. Add 30 mL of acetic acid solution (acetic acid content is 2 wt%) containing 1.4 g of chitosan, 0.5 g of EDC, and 0.3 g of NHS, and stir and react at room temperature for 24 h. Then add 1 g of silk fibroin peptide, 0.4 g of EDC, and 0.2 g of NHS, and continue to stir and react for 24 h. Then add 1.2 g of stem cell liposomes and 0.7 g of sustained-release microspheres, and stir and mix for 30 min to obtain an autologous fat-derived mesenchymal stem cell composition.

[0085] Example 3

[0086] This example provides a method for preparing an autologous fat-derived mesenchymal stem cell composition, including the following steps:

[0087] S1. Preparation of stem cell culture medium: Add the cell stimulation particles prepared in Preparation Example 3 and fetal bovine serum to α-MEM culture medium, and disperse by ultrasonic wave at 1000 W for 15 min to obtain stem cell culture medium. The concentration of the cell stimulation particles is 3 wt% and the concentration of fetal bovine serum is 10 wt%;

[0088] S2. Isolation and in vitro proliferation culture of stem cells: Take autologous fat particles, centrifuge at 1000 r / min for 5 min, collect the upper fat tissue from this centrifugation, add a composite enzyme, the addition amount of the composite enzyme is 2 wt% of the mass of the upper fat tissue, digest at 37 °C for 30 min, centrifuge at 700 g / min for 5 min, collect the lower-layer cells from this centrifugation, and resuspend with the stem cell culture medium in step S1. The cell density is 10 3 -10 4at a concentration of 0 cells / mL, under the condition of an externally applied magnetic field with a strength of 0.03 T, at 37 °C, with a carbon dioxide volume concentration of 5%, cultured for 4 days, centrifuged at 700 g / min for 10 min. In this centrifugation, the lower layer is mesenchymal stem cells derived from autologous fat, and the upper layer is the extracellular matrix, which is retained;

[0089] The composite enzyme is a mixture of trypsin and collagenase, and the mass ratio is 3:2;

[0090] S3. Preparation of stem cell liposomes: Resuspend the mesenchymal stem cells derived from autologous fat in step S2 in PBS solution to obtain a cell suspension with a cell density of 10 4 -10 5 g / mL; Dissolve 1.3 g of lecithin, 0.55 g of DSPE-PEG2000, 0.4 g of DSPE-PEG-CREKA, and 0.5 g of cholesterol in 50 mL of chloroform, remove the solvent by rotary evaporation under reduced pressure to prepare liposomes, add 50 mL of the cell suspension and mix evenly, and extrude repeatedly 25 times with a liposome extruder using a polycarbonate membrane to prepare stem cell liposomes;

[0091] S4. Preparation of sustained-release microspheres: Dissolve 6 g of chitosan in 200 mL of 2 wt% acetic acid solution, add 4 g of β-cyclodextrin, 1.5 g of sanguinarine, and 2.5 g of curcumin, add 50 mL of octanol solution containing 0.7 g of Tween-85, stir for 30 min, dropwise add 10 mL of aqueous solution containing 0.15 g of sodium tripolyphosphate, crosslink for 1 h, centrifuge, wash, and freeze-dry to prepare sustained-release microspheres;

[0092] S5. Preparation of a composition of mesenchymal stem cells derived from autologous fat: Add 2 g of N-isopropylacrylamide to 200 mL of the extracellular matrix in step S2. Under nitrogen protection, add 0.35 g of ammonium persulfate and 1.1 g of 2-mercaptoethylamine, stir and react for 4 h in an ice-water bath. Add 30 mL of acetic acid solution (acetic acid content 2 wt%) containing 1.2 g of chitosan, 0.4 g of EDC, and 0.25 g of NHS, stir and react at room temperature for 22 h, then add 0.85 g of silk fibroin peptide, 0.3 g of EDC, and 0.15 g of NHS, continue to stir and react for 22 h, then add 1 g of stem cell liposomes and 0.6 g of sustained-release microspheres, stir and mix for 30 min to prepare a composition of mesenchymal stem cells derived from autologous fat.

[0093] Comparative Example 1

[0094] Compared with Example 3, the difference is that the cell-stimulating particles are prepared from Comparative Preparation Example 1.

[0095] Comparative Example 2

[0096] Compared with Example 3, the difference is that the cell stimulation particles are prepared from Comparative Preparation Example 2.

[0097] Comparative Example 3

[0098] Compared with Example 3, the difference is that the cell stimulation particles are prepared from Comparative Preparation Example 3.

[0099] Comparative Example 4

[0100] Compared with Example 3, the difference is that the cell stimulation particles are prepared from Comparative Preparation Example 4.

[0101] Comparative Example 5

[0102] Compared with Example 3, the difference is that no cell stimulation particles are added.

[0103] Specifically as follows:

[0104] S1. Preparation of stem cell medium: Fetal bovine serum was added to α-MEM culture medium and dispersed by ultrasonic wave at 1000W for 15 min to obtain stem cell medium, and the concentration of the fetal bovine serum was 10 wt%.

[0105] Comparative Example 6

[0106] Compared with Example 3, the difference is that step S3 is not carried out.

[0107] Specifically as follows:

[0108] S1. Preparation of stem cell medium: The cell stimulation particles prepared in Preparation Example 3 and fetal bovine serum were added to α-MEM culture medium and dispersed by ultrasonic wave at 1000W for 15 min to obtain stem cell medium, and the concentration of the cell stimulation particles was 3 wt% and the concentration of the fetal bovine serum was 10 wt%;

[0109] S2. Isolation and in vitro proliferation culture of stem cells: Autologous fat particles were taken, centrifuged at 1000 r / min for 5 min, the upper layer of adipose tissue from this centrifugation was collected, a composite enzyme was added, and the addition amount of the composite enzyme was 2 wt% of the mass of the upper layer of adipose tissue, digested at 37 °C for 30 min, centrifuged at 700 g / min for 5 min, the lower layer of cells from this centrifugation was collected, and resuspended with the stem cell medium in step S1, and the cell density was 10 3 -10 4 cells / mL, under the condition of an external magnetic field with an intensity of 0.03 T, at 37 °C, and a carbon dioxide volume concentration of 5%, cultured for 4 d, centrifuged and separated at 700 g / min for 10 min, the lower layer from this centrifugation and separation was mesenchymal stem cells derived from autologous fat, and the upper layer was extracellular matrix, which was reserved;

[0110] The composite enzyme is a mixture of trypsin and collagenase, and the mass ratio is 3:2;

[0111] S3. Preparation of the sustained-release microspheres: Dissolve 6 g of chitosan in 200 mL of 2 wt% acetic acid solution, add 4 g of β-cyclodextrin, 1.5 g of sanguinarine and 2.5 g of curcumin, add 50 mL of octanol solution containing 0.7 g of Tween-85, stir for 30 min, dropwise add 10 mL of aqueous solution containing 0.15 g of sodium tripolyphosphate, crosslink for 1 h, centrifuge, wash, and freeze-dry to obtain the sustained-release microspheres;

[0112] S4. Preparation of the autologous fat-derived mesenchymal stem cell composition: Add 2 g of N-isopropylacrylamide to 200 mL of the extracellular matrix in step S2. Under nitrogen protection, add 0.35 g of ammonium persulfate and 1.1 g of 2-mercaptoethylamine, stir and react for 4 h under ice-water bath conditions. Add 30 mL of acetic acid solution (acetic acid content is 2 wt%) containing 1.2 g of chitosan, 0.4 g of EDC and 0.25 g of NHS, stir and react at room temperature for 22 h, then add 0.85 g of silk fibroin peptide, 0.3 g of EDC and 0.15 g of NHS, continue to stir and react for 22 h, then add 1 g of autologous fat-derived mesenchymal stem cells in step S2 and 0.6 g of the sustained-release microspheres, stir and mix for 30 min to obtain the autologous fat-derived mesenchymal stem cell composition.

[0113] Comparative Example 7

[0114] Compared with Example 3, the difference is that sanguinarine is not added in step S4.

[0115] Specifically as follows:

[0116] S4. Preparation of the sustained-release microspheres: Dissolve 6 g of chitosan in 200 mL of 2 wt% acetic acid solution, add 4 g of β-cyclodextrin, 4 g of curcumin, add 50 mL of octanol solution containing 0.7 g of Tween-85, stir for 30 min, dropwise add 10 mL of aqueous solution containing 0.15 g of sodium tripolyphosphate, crosslink for 1 h, centrifuge, wash, and freeze-dry to obtain the sustained-release microspheres.

[0117] Comparative Example 8

[0118] Compared with Example 3, the difference is that curcumin is not added in step S4.

[0119] Specifically as follows:

[0120] S4. Preparation of sustained-release microspheres: Dissolve 6 g of chitosan in 200 mL of 2 wt% acetic acid solution, add 4 g of β-cyclodextrin and 4 g of sanguinarine, add 50 mL of octanol solution containing 0.7 g of Tween-85, stir for 30 min, dropwise add 10 mL of aqueous solution containing 0.15 g of sodium tripolyphosphate, crosslink for 1 h, centrifuge, wash, and freeze-dry to obtain the sustained-release microspheres.

[0121] Comparative Example 9

[0122] Compared with Example 3, the difference is that no sustained-release microspheres were added in step S5.

[0123] Specifically as follows:

[0124] S5. Preparation of autologous fat-derived mesenchymal stem cell composition: Add 2 g of N-isopropylacrylamide to 200 mL of the extracellular matrix in step S2. Under nitrogen protection, add 0.35 g of ammonium persulfate and 1.1 g of 2-mercaptoethylamine, stir and react for 4 h under ice-water bath conditions, add 30 mL of acetic acid solution containing 1.2 g of chitosan (acetic acid content 2 wt%), 0.4 g of EDC and 0.25 g of NHS, stir and react at room temperature for 22 h, then add 0.85 g of silk fibroin peptide, 0.3 g of EDC and 0.15 g of NHS, continue to stir and react for 22 h, and then add 1 g of stem cell liposome, stir and mix for 30 min to obtain the autologous fat-derived mesenchymal stem cell composition.

[0125] Comparative Example 10

[0126] Compared with Example 3, the difference is that no stem cell liposome was added in step S5.

[0127] Specifically as follows:

[0128] S5. Preparation of autologous fat-derived mesenchymal stem cell composition: Add 2 g of N-isopropylacrylamide to 200 mL of the extracellular matrix in step S2. Under nitrogen protection, add 0.35 g of ammonium persulfate and 1.1 g of 2-mercaptoethylamine, stir and react for 4 h under ice-water bath conditions, add 30 mL of acetic acid solution containing 1.2 g of chitosan (acetic acid content 2 wt%), 0.4 g of EDC and 0.25 g of NHS, stir and react at room temperature for 22 h, then add 0.85 g of silk fibroin peptide, 0.3 g of EDC and 0.15 g of NHS, continue to stir and react for 22 h, and then add 0.6 g of sustained-release microspheres, stir and mix for 30 min to obtain the autologous fat-derived mesenchymal stem cell composition.

[0129] Test Example 1 Determination of Gelation Temperature and Gelation Time

[0130] Take the mesenchymal stem cell composition derived from autologous fat prepared in Examples 1-3 and the commercially available poly(N-isopropylacrylamide) hydrogel in a glass test tube, place it in a constant temperature water bath, take out the test tube every 10 minutes, invert it to observe whether the hydrogel flows. If it flows, continue to heat up at a rate of 1°C. If it does not flow, define this temperature as the T of the hydrogel. gel Determine T gel After that, measure the gelation time of each group of hydrogels at 37°C. The gelation time is recorded as the time point when each group of samples does not flow. The results are shown in Table 1.

[0131] Table 1

[0132]

[0133] As can be seen from the above table, the mesenchymal stem cell composition derived from autologous fat prepared in Examples 1-3 of the present invention has a suitable gelation temperature and a short gelation time.

[0134] Test Example 2

[0135] After adaptively feeding BALB / c mice for 1 week, they were divided into a model group, Examples 1-3 groups, and Comparative Examples 1-10 groups according to the random number table method, with 6 mice in each group. A chronic skin ulcer mouse model was established in each group. The specific operation was as follows: Shave the hair on the back of the mice and depilate with 6% sodium sulfide. Anesthetize the mice by intraperitoneal injection of 0.3% pentobarbital sodium (45 mg / kg). Select a transverse incision about 5 mm long on the back, implant a metal ring at the incision, cut off the skin tissue along the inner edge of the ring to form a defective wound surface with a diameter of about 8 mm, and remove the metal ring to complete the modeling. Blank group: No treatment was given. Before each administration to other groups, the wound surface of the mice was cleaned with normal saline and furacilin solution. Model group: Only apply furacilin solution externally; In Examples 1-3 or Comparative Examples 1-10 groups, 2 mL of the corresponding drug was dropped onto the wound surface so that it could evenly cover the wound surface and form a hydrogel. Once a day for 14 consecutive days.

[0136] On the 1st and 14th days of drug administration, use Image Pro Plus 6.0 software to record the wound surface area and calculate the wound healing rate. Wound healing rate (%) = 1 - [(wound surface area on the 1st day - wound surface area on the drug administration day) / wound surface area on the 1st day] × 100%.

[0137] The results are shown in Table 2.

[0138] Table 2

[0139]

[0140] Note: Compared with the model group, P < 0.05.

[0141] As can be seen from the above table, the mesenchymal stem cell composition derived from autologous fat prepared in Examples 1-3 of the present invention has a good effect on promoting skin healing.

[0142] 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 principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for preparing a composition of autologous fat-derived mesenchymal stem cells, characterized in that, It includes the following steps: S1. Preparation of stem cell culture medium: Add cell stimulation particles and fetal bovine serum to α-MEM culture medium, and disperse evenly by ultrasonic wave to obtain stem cell culture medium; S2. Isolation and in vitro proliferation culture of stem cells: Centrifuge the autologous extracted fat particles, collect the upper layer of adipose tissue from this centrifugation, add composite enzyme for digestion, centrifuge, collect the lower layer of cells from this centrifugation, resuspend with the stem cell culture medium in step S1, and culture under an external magnetic field condition, then centrifuge and separate. The lower layer after this centrifugation and separation is mesenchymal stem cells derived from autologous fat, and the upper layer is extracellular matrix, which is reserved for use; S3. Preparation of stem cell liposomes: Resuspend the mesenchymal stem cells derived from autologous fat in step S2 in PBS solution to obtain a cell suspension; Dissolve lecithin, DSPE-PEG2000, DSPE-PEG-CREKA, and cholesterol in chloroform, remove the solvent by rotary evaporation under reduced pressure to obtain liposomes, add the cell suspension and mix evenly, and repeatedly extrude with a liposome extruder using a polycarbonate membrane to obtain stem cell liposomes; S4. Preparation of sustained-release microspheres: Dissolve chitosan in acid solution, add β-cyclodextrin, sanguinarine, and curcumin, add an emulsifier octanol solution, stir, dropwise add an aqueous sodium tripolyphosphate solution for crosslinking, centrifuge, wash, and freeze-dry to obtain sustained-release microspheres; S5. Preparation of mesenchymal stem cell composition derived from autologous fat: Add N-isopropylacrylamide to the extracellular matrix in step S2, under the protection of inert gas, add an initiator and 2-mercaptoethylamine, stir and react under an ice-water bath condition, add a chitosan solution, EDC, and NHS, stir and react at room temperature, then add silk fibroin peptide, EDC, and NHS, continue to stir and react, and then add stem cell liposomes and sustained-release microspheres, stir and mix evenly to obtain a mesenchymal stem cell composition derived from autologous fat; The preparation method of the cell stimulation particles is as follows: T1. Preparation of UiO-66-NH2: Add zirconium chloride and 2-aminoterephthalic acid to a mixed solvent of N,N-dimethylformamide and acetic acid, carry out a hydrothermal reaction, cool, centrifuge, collect the solid, wash, and dry to obtain UiO-66-NH2; T2. Deposition of magnetic iron tetroxide: Add UiO-66-NH2 to water, add ferric chloride and ferrous chloride, dropwise add ammonia water, heat and stir to react, centrifuge, wash, dry, and calcine to obtain Fe3O4@UiO-66-NH2; T3. Loading of TGF-β1 and TGF-β3: Add TGF-β1 and TGF-β3 to water, add NHS and EDC, stir and activate, add Fe3O4@UiO-66-NH2, stir and react, centrifuge, wash, and dry to obtain cell stimulation particles.

2. The preparation method according to claim 1, characterized in that, In step S1, the concentration of the cell stimulation particles is 2-4 wt%, and the concentration of the fetal bovine serum is 8-12 wt%.

3. The preparation method according to claim 1, characterized in that, In step T1, the mass ratio of zirconium chloride to 2-aminoterephthalic acid described is 13-17:11-15, the temperature of the hydrothermal reaction is 120-140 °C, and the time is 20-24 h; in step T2, the mass ratio of UiO-66-NH2, ferric chloride and ferrous chloride is 10-15: 3.24:1.26, the pH value of the solution is adjusted to 10-11 by dropping ammonia water, the temperature of the heating and stirring reaction is 80-90 °C, the time is 3-5 h, the calcination temperature is 550-650 °C, and the time is 1-2 h; in step T3, the mass ratio of TGF-β1, TGF-β3, NHS, EDC and Fe3O4@UiO-66-NH2 is 0.2-0.4:0.1-0.3:0.05-0.1:0.05-0.1:5-7.

4. The preparation method according to claim 1, characterized in that The complex enzyme described in step S2 is a mixture of trypsin and collagenase, with a mass ratio of 2 - 4:1 - 3. The addition amount of the complex enzyme is 1 - 3 wt% of the mass of the upper adipose tissue. The digestion temperature is 35 - 38 °C and the time is 20 - 40 min. The cell density after resuspension is 10 3 -10 4 cells / mL. The culture conditions are 35 - 38 °C, the volume concentration of carbon dioxide is 3 - 7%, the time is 3 - 5 d, and the intensity of the applied magnetic field is 0.01 - 0.05 T.

5. The preparation method according to claim 1, wherein, The cell density of the cell suspension described in step S3 is 10 4 -10 5 g / mL, the mass ratio of the lecithin, DSPE-PEG2000, DSPE-PEG-CREKA, and cholesterol is 12-15:4-7:3-5:4-6, and the number of times of repeated extrusion is 20-30 times.

6. The preparation method according to claim 1, wherein In step S4, the acid solution is a 1-3 wt% acetic acid solution, and the mass ratio of chitosan, β-cyclodextrin, sanguinarine, curcumin, emulsifier and sodium tripolyphosphate is 5-7:3-5:1-2:2-3:0.5-1:0.1-0.

2. The emulsifier is selected from at least one of Tween-20, Tween-40, Tween-60, Tween-80, and Tween-85.

7. The preparation method according to claim 1, characterized in that, In step S5, the initiator is selected from at least one of sodium persulfate, potassium persulfate, and ammonium persulfate. The mass ratio of N-isopropylacrylamide, initiator, 2-mercaptoethylamine, chitosan, silk fibroin peptide, stem cell liposome and sustained-release microsphere is 18-22:3-4:10-12:10-14:7-10:8-12:5-7.

8. An autologous fat-derived mesenchymal stem cell composition prepared by the preparation method according to any one of claims 1-7.

9. Use of the autologous fat-derived mesenchymal stem cell composition according to claim 8 in the preparation of a drug for promoting skin healing.

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