Method for extracting stem cells from skin tissue

Abstract

The invention relates to the field of biological medicine, and discloses a method for extracting stem cells in skin tissue, which comprises the following steps: treating the skin tissue by using a dissociation mixed solution of recombinant trypsin-normal saline-antibiotic, and separating an epidermal layer from other layers; the method comprises the following steps: respectively digesting an epidermal layer and other layers in different enzyme solutions to respectively obtain an epidermal stem cell suspension and an adipose-derived stem cell suspension, modifying the epidermal stem cell suspension, filling the adipose-derived stem cell suspension and the modified epidermal stem cell suspension into electrophoresis equipment, separating, respectively collecting, centrifuging, and culturing to obtain the adipose-derived stem cell. And respectively obtaining epidermal stem cells and adipose tissue-derived stem cells. According to the method, double-layer enzymolysis and electrophoretic separation are combined, so that the accuracy and cell activity of extracting epidermal stem cells and adipose-derived mesenchymal stem cells are ensured, the cells are not damaged, the separation efficiency is high, the purity is high, the cell phenotypic stability is ensured, the differentiation tendency in cell culture is reduced, and the method is used for subsequent clinical or experimental research.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a method for extracting stem cells from skin tissue, and more specifically to extracting epidermal stem cells and adipose mesenchymal stem cells from skin tissue. Background Art

[0002] Epidermal stem cells (ESCs) can differentiate into keratinocytes and participate in skin repair and cosmetic regeneration; adipose-derived mesenchymal stem cells (ADSCs) have multi-directional differentiation potential and can be used for the repair of bone, cartilage and adipose tissue. Discarded skin tissue usually contains the epidermis, dermis and subcutaneous adipose tissue. Skin tissue contains both epidermal stem cells and adipose mesenchymal stem cells. Therefore, it would be of great value if epidermal stem cells and adipose mesenchymal stem cells could be extracted and separated from skin tissue at the same time.

[0003] Conventional stem cell extraction methods are mostly for single-source stem cells. It is rare to extract and separate epidermal stem cells and adipose-derived mesenchymal stem cells from skin tissue at the same time. Generally, mechanical cutting and enzymatic hydrolysis are used. Mechanical cutting is to wash the discarded skin tissue with physiological saline or sterile PBS to remove blood and impurities, and cut the skin tissue into small pieces, usually controlled at 2-3mm. 3 The size of the cells is then separated by different enzymes for enzymatic hydrolysis of epidermal stem cells and adipose mesenchymal stem cells to obtain stem cells. However, this method can easily damage the stem cells, resulting in low separation efficiency and low separation purity.

[0004] Patent document CN 108103008 A discloses a method for preparing stem cells from human amniotic membrane, wherein human amniotic epithelial cells are separated after collecting the placenta, human amniotic membrane is cut, applied, sprayed and infiltrated, and a mixed solution of type II collagenase and DNA enzyme is used for pre-digestion, and then trypsin is added for digestion, and then the digestion is terminated and washed to collect the lower layer of human amniotic epithelial cells; human amniotic mesenchymal stem cells are separated by adding trypsin to the human amniotic membrane after separating human amniotic epithelial cells, and then washed after digestion, and then type IV collagenase is added to digest until the amniotic tissue melts, centrifuged, and the lower layer of human amniotic mesenchymal stem cells are collected, and then cell culture and cryopreservation are performed. The human amniotic epithelial cells and human amniotic mesenchymal stem cells prepared by the above method have high purity and proliferate rapidly after culture, but are not suitable for extracting and separating epidermal stem cells and adipose mesenchymal stem cells from skin tissue, because the purity of epidermal stem cells and adipose mesenchymal stem cells in skin tissue is lower than that of human amniotic membrane, and the above method cannot effectively guarantee the activity of stem cells, and the separation purity is low.

[0005] Therefore, it is necessary to develop a method for simultaneously extracting epidermal stem cells and adipose-derived mesenchymal stem cells. Summary of the invention

[0006] The purpose of the present invention is to provide a method for extracting stem cells from skin tissue, in order to address the problems that existing methods for extracting epidermal stem cells and adipose mesenchymal stem cells are prone to cell damage, have low separation efficiency, and have low separation purity. The method utilizes skin tissue to efficiently separate epidermal stem cells and adipose mesenchymal stem cells while retaining their biological activity.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: A method for extracting stem cells from skin tissue comprises the following steps: S1. Treating the skin tissue with a dissociation mixture of recombinant trypsin, physiological saline, and antibiotics to dissociate the epidermis from the skin tissue and separate the epidermis into the epidermis and other layers; the mass concentration of the recombinant trypsin in the dissociation mixture is 0.8-1.5%; S2, cutting the epidermal layer obtained in step S1 into pieces and placing them in a trypsin-EDTA solution, incubating for 30 to 45 minutes, stirring, filtering and centrifuging after terminating the digestion to obtain epidermal stem cell-rich material, and resuspending to obtain an epidermal stem cell material suspension; Cut the other layers obtained in step S1 into pieces and digest them with a solution containing type I collagenase and / or type II collagenase, filter and centrifuge to obtain adipose-derived mesenchymal stem cell-rich material, and resuspend them to obtain adipose-derived mesenchymal stem cell suspension; S3, adding a positive charge modifier to the epidermal stem cell suspension for treatment; placing the modified epidermal stem cell suspension and the adipose mesenchymal stem cell suspension into different chambers of an electrophoresis device for separation, wherein the temperature of the electrophoresis buffer is controlled between 4°C and 10°C, the voltage is set to 50-100V, the duration is 20-40min, the epidermal stem cell solution is collected at the cathode, and the adipose mesenchymal stem cell solution is collected at the anode; S4. Centrifuge the collected epidermal stem cell solution and adipose mesenchymal stem cell solution respectively, and culture the cells obtained after centrifugation respectively to obtain epidermal stem cells and adipose mesenchymal stem cells respectively.

[0008] In the above technical scheme, the epidermis and other layers are first separated by a dissociation mixture prepared by recombinant trypsin, and the epidermis is easily peeled off by using a dissociation mixture containing recombinant trypsin. The other layers include dermal tissue and adipose tissue, so that epidermal stem cells and adipose mesenchymal stem cells can be extracted separately later. Then, the epidermis is cut into pieces and digested with a trypsin-EDTA solution to obtain a substance rich in epidermal stem cells, and the substance is resuspended to obtain adipose mesenchymal stem cell suspension. The other layers are cut into pieces and digested with a solution containing type I collagenase and / or type II collagenase to obtain a substance rich in adipose mesenchymal stem cells, and the substance is resuspended to obtain adipose mesenchymal stem cell suspension. The above double-layer enzymatic hydrolysis method is adopted, the first layer of enzymatic hydrolysis is to separate the epidermis and other layers, and the second layer of enzymatic hydrolysis is to digest the epidermis and other layers respectively with different enzymatic hydrolysis solutions to obtain suspensions rich in epidermal stem cells and adipose mesenchymal stem cells, respectively. Different types of enzymes are used for gradual dissociation, and the concentration and time in the double-layer enzymatic hydrolysis process are controlled to ensure the activity of epidermal stem cells and adipose mesenchymal stem cells. The double-layer enzymatic hydrolysis method avoids the mixing of different cell populations, separates the epidermis and dermis by physical and enzymatic digestion, and maximizes the retention of the activity of the two stem cells. In addition, by designing the separation step in the double-layer enzymatic hydrolysis, cross contamination is reduced, the purity of the cell population is ensured, and the independent release and enrichment of epidermal stem cells and adipose mesenchymal stem cells are ensured. At the same time, considering the purity of the stem cells after the double-layer enzymatic hydrolysis, electrophoretic cell separation is adopted, and charge labeling and electrophoretic parameter control are used to effectively distinguish epidermal stem cells and adipose mesenchymal stem cells, and achieve efficient separation. The present invention combines double-layer enzymatic hydrolysis with electrophoretic separation, ensures the accuracy and cell activity of epidermal stem cells and adipose mesenchymal stem cells in the extraction process, ensures the accuracy and cell activity of separation and purification, and provides a basis for further cell application research.

[0009] As a preferred embodiment of the present invention, the skin tissue is eyelid tissue discarded after double eyelid surgery. In double eyelid surgery, discarded eyelid tissue is not only medical waste, but also contains huge potential for regenerative medicine. These eyelid tissues contain abundant epidermal stem cells and adipose mesenchymal stem cells, which have wide application prospects in tissue repair and regenerative medicine. As a non-traditional source of adipose and epidermal stem cells, eyelid tissue has a special physiological structure and cell environment, providing a new option for local tissue repair.

[0010] As a preferred embodiment of the present invention, the mass concentration of recombinant trypsin in the dissociation mixture is 0.9-1.2%, preferably, the mass concentration of recombinant trypsin is 1%. The mass concentration of recombinant trypsin in the dissociation mixture is 0.8-1.5%, and the dissociation mixture containing recombinant trypsin makes the epidermis easier to peel off. The selection of a suitable recombinant trypsin concentration can ensure the activity of stem cells in the skin tissue. Therefore, the mass concentration of recombinant trypsin is preferably 0.9-1.2%, more preferably 1%, and a lower concentration of recombinant trypsin is used to treat the skin tissue.

[0011] As a preferred embodiment of the present invention, in step S2, the mass concentration of trypsin in the trypsin-EDTA solution is 0.08-0.25%; more preferably, the mass concentration of trypsin is 0.15%. An appropriate trypsin concentration can gently and effectively separate cells, which is crucial for maintaining cell activity and function. In order to ensure the efficiency of cell separation and cell activity, the mass concentration of trypsin in the trypsin-EDTA solution is selected to be 0.08-0.25%; more preferably 1%, which has good cell separation efficiency and maintains the activity of epidermal stem cells.

[0012] As a preferred embodiment of the present invention, in step S2, the collagenase mass concentration in the solution containing type I collagenase and / or type II collagenase is 0.3~0.7%, and more preferably, the collagenase mass concentration is 0.5%. The damage to adipose mesenchymal stem cells containing type I collagenase and / or type II collagenase is small, and the collagen fibers in the intercellular matrix can be gently digested to release cells, and tissues can be efficiently digested, reducing the impact on cell differentiation and improving cell survival rate. Therefore, the collagenase mass concentration in the solution containing type I collagenase and / or type II collagenase is selected to be 0.3~0.7%.

[0013] As a preferred embodiment of the present invention, the positive charge modifier is at least one of a polylysine modifier and a polypeptide, and the polypeptide is Arg-Gly-Asp, which is a sequence composed of three amino acids: arginine (Arg), glycine (Gly) and aspartic acid (Asp). In order to increase the charge difference, biocompatible charge-modified molecules are used to make epidermal stem cells and adipose mesenchymal stem cells carry different charges. Epidermal stem cells carry positive charges and migrate to the cathode. By detecting the migration of cells in the electric field, cell populations with different charges are separated, and adipose mesenchymal stem cells carry negative charges and migrate to the anode, thereby achieving the purpose of simultaneous separation and purification.

[0014] As a preferred embodiment of the present invention, in step S3, the adipose-derived mesenchymal stem cell suspension is treated with a positive charge modifier to modify a small amount of epidermal stem cells in the adipose-derived mesenchymal stem cell suspension. The adipose-derived mesenchymal stem cell suspension is obtained by cutting up other layers and digesting them, and is rich in adipose-derived mesenchymal stem cells and also contains a small amount of epidermal stem cells. In order to separate the epidermal stem cells, a positive charge modifier is used to modify them to be positively charged, but the amount of the positive charge modifier added should not be too much to prevent the influence on the adipose-derived mesenchymal stem cells.

[0015] As a preferred embodiment of the present invention, the method of using a positive charge modifier is as follows: the positive charge modifier is added to a cell suspension so that the concentration of the positive charge modifier in the cell suspension is 1-5 μg / mL to treat epidermal stem cells, incubate for 15 minutes, centrifuge for 5 minutes after incubation, and wash twice with an electrophoresis buffer to remove unbound positive charge modifiers.

[0016] As a preferred embodiment of the present invention, before electrophoresis, electrophoresis buffer is added to the modified epidermal stem cell suspension and adipose mesenchymal stem cell suspension to adjust the cell concentration to 0.5-2×10 5 cells / mL.

[0017] As a preferred embodiment of the present invention, the electrophoresis buffer is a Tris-HCl buffer with a pH of 7.0 to 7.8. During the electrophoresis process, the pH and liquid temperature of the electrophoresis buffer are controlled between 4°C and 10°C to avoid high temperature affecting cell activity.

[0018] As a preferred embodiment of the present invention, the detailed steps of the extraction method include: In step S1, the skin tissue is eyelid tissue discarded after double eyelid surgery; the treatment is overnight treatment at 4°C; In step S2, the epidermis layer is incubated at 36-38°C for 10-15 minutes, and the digestion is terminated with a DEME solution containing serum; the digestion of the other layers is performed at 4°C overnight.

[0019] As a preferred embodiment of the present invention, in step S4, adipose-derived mesenchymal stem cells are cultured in a first culture medium, wherein the first culture medium is based on DMEM / F12 and further comprises 50-200 μg / mL of Ligustrum lucidum extract and a first additional component, wherein the first additional component is as follows: 8~15% KOSR (Knockout Serum Replacement); 5~20μg / mL recombinant human insulin; 20~100 ng / mL EGF (recombinant human epidermal growth factor); 10~40 ng / mL FGF-2 (recombinant fibroblast growth factor); 20~100 ng / mL SDF-1 (stromal cell-derived factor-1); 2~10 ng / mL TGF-β (recombinant human transforming growth factor β); 0.5-1 g / L of an amino acid complex, wherein the amino acid complex comprises arginine, glycine, alanine and glutamic acid; 1–3.5 mM, L-glutamine.

[0020] In the above technical scheme, the first culture medium uses DMEM / F12 (Dulbecco's Modified Eagle Medium / Nutrient Mixture F-12) as the basal medium, and other ingredients are added to provide richer nutrients and promote cell growth and proliferation to ensure the success rate of stem cell culture. Among them, the extract of the leaves of the ginger contains rich antioxidant active ingredients, such as quercetin, kaempferol, proanthocyanidins, etc., which help protect cells from oxidative damage and delay the cell aging process. It is suitable for the growth of adipose mesenchymal stem cells. Experimental data show that when the extract of the leaves of the ginger is in the range of 50~200μg / mL, the first culture medium of the present invention is used to culture adipose mesenchymal stem cells. The proportion of live cells reaches 90%, and the cell proliferation rate reaches more than 115%, which significantly improves the proportion of live cells, cell proliferation rate, and ROS level; KOSR is an animal-free cell culture supplement, and recombinant human insulin has anti-apoptosis and mitotic effects. Splitting effect; EGF can promote the proliferation and migration of epidermal cells. FGF-2 plays a key role in the proliferation and differentiation of adipose-derived mesenchymal stem cells, and can promote cell proliferation and enhance the differentiation potential of adipose-derived mesenchymal stem cells; SDF-1 promotes cell migration and proliferation and supports tissue regeneration; TGF-β can inhibit the proliferation of immunocompetent cells and regulate cell phenotype; the use of specific amino acid complexes in the culture medium can increase the metabolic activity and proliferation capacity of cells. These amino acids play a key role in the growth and differentiation of adipose-derived mesenchymal stem cells and can promote the overall health and function of cells. L-glutamine serves as an energy source for cells.

[0021] As a preferred embodiment of the present invention, the preparation method of the extract of Zingiber officinale is as follows: Step 1: collecting mature leaves of the Ligusticum chuanxiong plant, washing them, and drying them in a dryer at a temperature not exceeding 40° C. until the moisture content is less than 10%; and crushing the dried leaves into a particle size of 0.2 to 5 mm to obtain Ligusticum chuanxiong powder; Step 2: Mix the powder of the Ligustrum lucidum leaf with 60-75v% ethanol solution in a weight ratio of 1:10-25, soak and extract in a water bath at 15-30°C for 10-40min, and filter after ultrasonication; wash the filtrate with n-hexane for 2-5 times, and extract with ethyl acetate for 2-5 times after washing; concentrate the extract by evaporation, filter through a membrane, and then freeze-dry to obtain a Ligustrum lucidum leaf concentrate; Step 3: Pass the Zingiber officinale concentrated solution through a chromatography column to concentrate and obtain the Zingiber officinale extract.

[0022] As a preferred embodiment of the present invention, in step S4, epidermal stem cells are cultured using a second culture medium, wherein the second culture medium is based on RPMI-1640 and further includes 50-150 μg / mL of Ligustrum lucidum extract and a second added component, wherein the second added component is as follows: 1-3.5 mM L-glutamine; 5-20% B-27 supplement; 5~30μg / mL recombinant human insulin; 20~100 ng / mL EGF (recombinant human epidermal growth factor); 5~20 ng / mL KGF (recombinant human keratinocyte growth factor); 2~10 ng / mL TGF-β (recombinant human transforming growth factor β); 0.5-1g / L collagen; 0.5~2% rBSA (recombinant bovine serum albumin); 50~200μg / mL recombinant human ascorbic acid; 0.5~5 mg / L vitamin mixture.

[0023] In the above technical scheme, the second culture medium is suitable for the growth of epidermal stem cells, and is based on RPMI (Roswell Park Memorial Institute)-1640. The extract of the leaves of the ginger contains rich antioxidant active ingredients, such as quercetin, kaempferol, proanthocyanidins, etc., which help protect cells from oxidative damage and delay the cell aging process. It is suitable for the growth of epidermal stem cells. Experimental data show that when the extract of the leaves of the ginger is in the range of 50-150 μg / mL, epidermal stem cells are cultured with the culture medium of the present invention. The ROS level is between 75% and 45%, which greatly reduces the ROS level. The cell proliferation rate is better than that of the blank group at 24 hours, 48 ​​hours, and 72 hours. The proliferation rate is more than 2 times within 3 days, which can significantly improve the cell proliferation rate and ROS level; L-glutamine is used as an energy source for cells; B-27 supplements provide certain nutrients required by cells. B-27 supplements contain a variety of growth factors and antioxidants. The agent can provide comprehensive nutritional support for epidermal stem cells; recombinant human insulin has anti-apoptotic and mitotic effects; EGF can promote the proliferation and migration of epidermal cells, and KGF specifically promotes the proliferation and differentiation of keratinocytes, which is essential for maintaining the characteristics of epidermal stem cells; adding matrix components such as collagen to the culture medium can simulate the environment of the extracellular matrix, provide a more suitable growth environment for epidermal stem cells, and help them attach, proliferate and differentiate; TGF-β can inhibit the proliferation of immunocompetent cells and regulate cell phenotypes; collagen can provide a matrix for cell attachment and proliferation, and promote cell growth and differentiation; recombinant human ascorbic acid acts as an antioxidant in cell culture, helps maintain the redox balance of cells, and promotes cell growth and differentiation; the vitamin mixture provides a variety of vitamins required by cells, and plays a role in cell metabolism, growth and differentiation. The use of the above-mentioned formula culture medium can significantly improve the survival rate and growth efficiency of epidermal stem cells, especially in a serum-free environment.

[0024] As a preferred embodiment of the present invention, the preparation method of the extract of Zingiber officinale is as follows: Step 1: collecting mature leaves of the Ligusticum chuanxiong plant, washing them, and drying them in a dryer at a temperature not exceeding 40° C. until the moisture content is less than 10%; and crushing the dried leaves into a particle size of 0.2 to 5 mm to obtain Ligusticum chuanxiong powder; Step 2: Mix the powder of the Ligustrum lucidum leaf with 60-75v% ethanol solution in a weight ratio of 1:10-25, soak and extract in a water bath at 15-30°C for 10-40min, and filter after ultrasonication; wash the filtrate with n-hexane for 2-5 times, and extract with ethyl acetate for 2-5 times after washing; concentrate the extract by evaporation, filter through a membrane, and then freeze-dry to obtain a Ligustrum lucidum leaf concentrate; Step 3: Pass the Zingiber officinale concentrated solution through a chromatography column to concentrate and obtain the Zingiber officinale extract.

[0025] The present invention also provides a method for culturing stem cells in skin tissue, comprising the following steps: Adopting the above-mentioned method for extracting stem cells from skin tissue to obtain adipose mesenchymal stem cells and epidermal stem cells respectively; The culture medium of the adipose-derived mesenchymal stem cells is configured as a first culture medium; the culture medium of the epidermal stem cells is configured as a second culture medium; Adipose-derived mesenchymal stem cells are placed in the first culture medium, and epidermal stem cells are placed in the second culture medium, using microcarrier 3D FloTrix ® Adipose-derived mesenchymal stem cells and epidermal stem cells were expanded and cultured separately.

[0026] In the above technical solution, the above culture method is used to effectively improve the culture efficiency of adipose mesenchymal stem cells and epidermal stem cells, providing a more reliable cell source for their application in the fields of regenerative medicine, ophthalmology and skin repair.

[0027] Compared with the prior art, the present invention has the following beneficial effects: 1. The extraction method of the present invention adopts double-layer enzymolysis, the first layer of enzymolysis is to separate the epidermis and other layers, and the second layer of enzymolysis is to digest the epidermis and other layers respectively using different enzymolysis solutions, so as to obtain suspensions rich in epidermal stem cells and adipose mesenchymal stem cells respectively, and gradually dissociate by using different types of enzymes, control the concentration and time during the double-layer enzymolysis process, and ensure the activity of epidermal stem cells and adipose mesenchymal stem cells. The double-layer enzymolysis method avoids the mixing of different cell populations, separates the epidermis and dermis by physical and enzymatic digestion, and maximizes the retention of the activity of the two stem cells. In addition, by designing the separation step in the double-layer enzymolysis, cross contamination is reduced, the purity of the cell population is ensured, and the independent release and enrichment of epidermal stem cells and adipose mesenchymal stem cells are ensured. At the same time, considering the purity of the stem cells after the double-layer enzymolysis, electrophoretic cell separation is adopted, and charge labeling and electrophoresis parameter control are used to effectively distinguish epidermal stem cells and adipose mesenchymal stem cells, and achieve efficient separation. The present invention combines double-layer enzymatic hydrolysis with electrophoretic separation, thereby ensuring the accuracy and cell activity of epidermal stem cells and adipose mesenchymal stem cells during the extraction process.

[0028] 2. During the amplification process of the adipose mesenchymal stem cells extracted by the method of the present invention, the expression rates of CD73, CD29, and CD90 of the adipose mesenchymal stem cells are maintained at more than 90%, with high expression; during the amplification process of the epidermal stem cells, the expression rates of CD73, CD29, and CD90 are maintained at more than 90%, with high expression, and the CK15 and p63 markers of the epidermal stem cells are expressed. The method of the present invention ensures the stability of the cell phenotype, reduces the differentiation tendency in cell culture, and ensures its application in subsequent clinical or experimental research. It can be seen that the method of the present invention can simultaneously extract epidermal stem cells and adipose mesenchymal stem cells without damaging the cells, and has high separation efficiency and high purity.

[0029] 3. The present invention provides a first culture medium suitable for culturing adipose-derived mesenchymal stem cells, which uses DMEM / F12 as a basic culture medium and is supplemented with different components, wherein the addition amount of the extract of Alpinia zeylanicum is 50-200 μg / mL. After being used for culturing adipose-derived mesenchymal stem cells, the proportion of living cells reaches more than 90%, and the proliferation rate reaches more than 2 times within 3 days, significantly improving the proportion of living cells, cell proliferation rate, and ROS level.

[0030] 4. The present invention provides a second culture medium suitable for culturing epidermal stem cells, which uses RPMI-1640 as a basic culture medium and adds different ingredients, wherein the addition amount of the extract of the leaves of Alpinia officinalis is 50-150 μg / mL, which greatly reduces the ROS level and increases the proliferation rate. The proliferation rate reaches more than 2 times within 3 days, significantly improving the cell proliferation rate and ROS level.

[0031] 5. The present invention provides a method for simultaneously extracting adipose-derived mesenchymal stem cells and epidermal stem cells from eyelid tissue. Since the stem cells derived from eyelid tissue have good biocompatibility with periocular tissue, the cell line faces a lower immune rejection reaction when treating eye diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Flow cytometry of ADSCs-specific surface antigens: (a) control group; (b) expression of CD31, CD29, and CD73; (c) expression of CD34, CD90, and CD45; Figure 2 Flow cytometry of specific surface antigens of ESCs: (a) control group; (b) expression of CD31, CD29, and CD73; (c) expression of CD34, CD90, and CD45; Figure 3 The results of cell proliferation rate under different concentrations of Zingiber officinale extract in Test Example 2; Figure 4 Microscopic images of epidermal stem cells added with 100 μg / mL of Alpinia zeylanicum extract in Test Example 2: (a) P0; (b) P1; Figure 5 Microscope images of adipose-derived mesenchymal stem cells isolated with 1% recombinant trypsin in Test Example 3: (a) P0; (b) P1; Figure 6 Microscope images of adipose-derived mesenchymal stem cells isolated with 2% recombinant trypsin in Test Example 3: (a) P0; (b) P1. DETAILED DESCRIPTION

[0033] In order to more clearly describe the invention purpose, technical scheme and technical effect advantages in the specific implementation case of the present invention, the scheme in the specific embodiment will be described in detail in combination with the drawings of the specification of the present invention. The specific technical scheme involved in the following specific embodiments is only for a clear and complete description of the innovative technical scheme of the present invention. It is only a part of the specific implementation scheme that can be adopted by the present invention, not all embodiments, and should not be understood as a limitation on the innovative scheme of the present invention. Any scheme adopting the same inventive concept of the present invention should be included in the protection scope of the present invention.

[0034] Secondly, the description of the drawings in the specific embodiments of the present invention is only for the convenience of technical personnel to understand the solution of the present invention. The partial details in the drawings are for the convenience of clearly presenting the technical solution. It should not be considered that all technical features in the drawings must be included in the specific implementation cases, and the detailed features in the drawings should not be identified as additional limitations on the innovative technical solution of the present invention. The components in the various embodiments described and shown in the drawings can be combined and arranged according to different configurations, and these changes in combination and arrangement should be identified as part of the entire embodiment of the innovative solution of the present invention and included in the scope of protection of the present invention.

[0035] In summary, the schemes or descriptions presented in the specific embodiments and drawings of the present invention are not intended to limit the scope of protection claimed, but are merely selected embodiments / cases to help technicians understand the relevant innovative solutions. Based on these embodiments, all other equivalent or parallel embodiments obtained by those skilled in the art without creative work are within the scope of protection claimed by the present invention.

[0036] Example 1 This embodiment provides a method for extracting stem cells from skin tissue. The skin tissue is waste eyelid tissue after double eyelid surgery. The extraction method includes the following steps: Separation of the epidermis and other layers: Use a low concentration of recombinant trypsin (1%)-normal saline-antibiotic (2%) dissociation mixture to pretreat the epidermis in the double eyelid discarded tissue at 4°C overnight, gently dissociate the epidermis, release it from the skin tissue, and use tweezers to peel off the epidermis from the other layers of the skin tissue, including the dermis and adipose tissue.

[0037] The obtained epidermal layer was placed in a DEME (dulbecco's modified eagle medium) solution, which contains various amino acids and glucose. The epidermal layer was cut into minced meat while maintaining cell activity. The obtained suspension was added to a 0.25% trypsin-EDTA solution, where EDTA is ethylenediaminetetraacetic acid, and incubated at 37°C for 30-45 minutes. The solution was gently stirred and digested with a serum-containing DEME solution. The solution was filtered through a 100 μm filter to collect the epidermal stem cell suspension. The epidermal stem cell suspension was centrifuged at 1500 rpm for 5 minutes, and the supernatant was removed to obtain a precipitate rich in epidermal stem cell material. The epidermal stem cell material was resuspended in a PBS solution to obtain an epidermal stem cell suspension.

[0038] The remaining layers were placed in DEME solution and chopped into minced meat while maintaining cell activity. The resulting suspension was further digested overnight at 4°C with a solution containing type I collagenase or type II collagenase (concentration 0.5%) to release ADSCs in the adipose matrix. The digested tissue was filtered through a 70μm filter to remove incompletely digested tissue blocks and retain the adipose mesenchymal stem cell suspension containing ADSCs. The adipose mesenchymal stem cell suspension was centrifuged at 1000 rpm for 10 minutes, the supernatant was removed, and the precipitate was the matrix vascular part rich in adipose mesenchymal stem cells. The enzymatic hydrolysis at this stage was time-optimized to ensure that the adipose tissue was effectively dissociated without destroying the active stem cells in the extracellular matrix. The adipose mesenchymal stem cells were resuspended in a PBS solution to obtain an adipose mesenchymal stem cell suspension.

[0039] Cell charge labeling: Epidermal stem cells and adipose-derived mesenchymal stem cells are purified and separated simultaneously based on their charge differences. To increase the charge difference, epidermal stem cells are modified with positively charged peptides to make their charge significantly positive, such as poly-lysine modification, Poly-L-lysine, Sigma #P4832. Adipose-derived mesenchymal stem cells are naturally rich in negatively charged lipids, so no charge amplification treatment is required. Add the positive charge modifier to the cell suspension to make the concentration of the positive charge modifier in the cell suspension 1~5 μg / mL to treat the epidermal stem cell suspension or the adipose-derived mesenchymal stem cell suspension, incubate for 15 minutes, centrifuge for 5 minutes after incubation, and wash twice with electrophoresis buffer to remove unbound positive charge modifiers.

[0040] Prepare electrophoresis buffer, such as Tris-HCl buffer, pH 7.4, Gibco, electrophoresis buffer: 0.01 M phosphate buffer (PBS), 5 mM sodium chloride (NaCl), 1 mM potassium chloride (KCl); adjust pH to 7.4, filter with a 0.22 μm filter to remove particulate impurities, and precool the buffer to 4 °C and ensure constant temperature before use.

[0041] In this embodiment, the electrophoresis device adopts a microfluidic electrophoresis system, including an electric field generation, cell separation and collection module; specifically, a Bio-Rad Model 3000 Power Supply (Bio-Rad #1645050); the electrophoresis chamber is used for cell migration and separation in the electric field; specifically, a Bio-Rad Criterion Cell Electrophoresis Chamber (Bio-Rad #1656001), the electrophoresis system is connected to a cooling circulator, the liquid temperature in the electrophoresis chamber is set to 4°C, the cell suspension is respectively loaded into different chambers of the electrophoresis equipment, and during the electrophoresis process, the liquid temperature is ensured to be controlled between 4°C and 10°C to avoid high temperature affecting cell activity; the electrophoresis is performed in a Bio-Rad Criterion (Bio-Rad #1656001) in an electrophoresis pool, with a voltage of 50V and a duration of 20-40min. Different voltages and separation times were adjusted in a timely manner according to the separation effect of the experimental samples. Cell populations with different charges were separated by detecting the migration of cells in the electric field: epidermal stem cells (positively charged) migrated to the cathode, and adipose mesenchymal stem cells (negatively charged) migrated to the anode. After electrophoresis, the electrophoresis power supply was turned off, and the cell suspension was collected from the cathode and anode chambers respectively with a pipette; the cells were washed twice with pre-cooled PBS solution to remove the peptide and buffer residues, and epidermal stem cell solution and adipose mesenchymal stem cell solution were obtained respectively.

[0042] The collected epidermal stem cell solution and adipose mesenchymal stem cell solution were centrifuged again at 1500 rpm for 5 min, and the electrophoresis buffer was removed to obtain epidermal stem cells and adipose mesenchymal stem cells, which were resuspended in culture medium for culture.

[0043] The epidermal stem cells and adipose-derived mesenchymal stem cells were cultured separately, and the cells were identified by flow cytometry after the first passage. Cell surface markers were detected by flow cytometry, and adipose-derived mesenchymal stem cells such as Figure 1As shown, CD73-PE: 93.64%, CD29-APC: 98.61%, CD90-FITC: 96.06%, CD73, CD31-FITC: 0.00%, CD34-PE: 0.00%, CD45-Per: 0.02%; it was observed that the expression rates of CD73, CD29, and CD90 of adipose mesenchymal stem cells were maintained above 90% during the expansion process, with high expression; among them, epidermal stem cells such as Figure 2 As shown, CD73-PE: 98.38%, CD29-APC: 98.95%, CD90-FITC: 91.06%, CD31-FITC: 0.06%, CD34-PE: 0.02%, CD45-Per: 0.02%, and the expression rates of CD73, CD29, and CD90 of epidermal stem cells are maintained at more than 90% during the amplification process, with high expression, and CK15 and p63 markers of epidermal stem cells are expressed. The method of the present invention ensures the stability of cell phenotype, reduces the differentiation tendency in cell culture, and ensures its application in subsequent clinical or experimental research. The above method not only provides a new source of stem cell extraction, but also the obtained epidermal stem cells and adipose mesenchymal stem cells have high cell purity and biological activity, which can play an important role in the fields of ophthalmology and regenerative medicine.

[0044] The adipose-derived mesenchymal stem cells cultured by the above method have shown the ability to promote corneal epithelial cell proliferation and accelerate corneal wound healing in in vitro experiments, and are particularly suitable for the treatment of corneal damage, dry eye and other ocular surface diseases. The cultured epidermal stem cells can be used for skin repair and anti-aging treatment, and are particularly suitable for the treatment of sensitive areas such as the face and periocular area, with significant aesthetic and regenerative potential.

[0045] Example 2 The present embodiment provides a first culture medium for adipose-derived mesenchymal stem cells, wherein the first culture medium is based on DMEM / F12 and further includes the following components: 5% KOSR; 10 μg / mL recombinant human insulin; 50 ng / mL EGF; 20 ng / mL FGF-2; 50 ng / mL SDF-1; 5 ng / mL TGF-β; 0.5-1 g / L amino acid complex, wherein the amino acid complex includes arginine, glycine, alanine and glutamic acid; 2 mM, L-glutamine; and 100 μg / mL extract of Ligusticum chuanxiong.

[0046] The DMEM / F12 basal medium is a 1:1 mixture of DMEM and Hams F-12, 500 mL each, and also includes the following components: 100 mL KOSR; 10 mg recombinant human insulin; 50 μg EGF; 20 μg FGF-2; 50 μg SDF-1; 5 μg TGF-β; 0.5 g amino acid complex; 292 mg L-glutamine; 100 mg Ligustrum lucidum extract. The other components added above are purchased externally, and the Ligustrum lucidum extract is prepared using Example 4. In the preparation of the first culture medium, the basal culture medium is prepared first, and the added components are added one by one according to the above dosage. 2~4 g / L sodium bicarbonate (NaHCO3) is used to adjust the pH to 7.2~7.4, and the pH value of the culture medium is checked with a pH meter; sterilized by filtration through a 0.22 μm filter membrane to ensure sterility. The prepared culture medium is stored at 4°C and is recommended to be used within two weeks.

[0047] In the above culture medium, the extract of Alpinia zeylanicum is used as an antioxidant component, which is suitable for the culture of adipose-derived mesenchymal stem cells. The microcarrier 3D FloTrix produced by Huakan Biotechnology is used in the culture process. ® To expand the culture of adipose-derived mesenchymal stem cells, the microcarriers were disinfected according to the instructions provided by Huakan Biotechnology to ensure a sterile environment. The use of microcarriers significantly increased the adhesion rate of ADSCs and epidermal stem cells on 3D microcarriers, making the adhesion process more efficient.

[0048] Test Example 1 In order to test the antioxidant and proliferation-promoting effects of different extracts of Ligusticum chuanxiong on adipose-derived mesenchymal stem cells culture, the culture medium of Example 2 was used, except that different concentrations of Ligusticum chuanxiong extract (0 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL) were added to the culture medium, and the other components were the same as those in Example 2. The adipose-derived mesenchymal stem cells extracted in Example 1 were cultured to ensure that the cells were in good condition. The cells were cultured in an incubator at 37°C and 5% CO2, and the culture time was set to 3 days (proliferation experiment) and 7 days (antioxidant experiment). When sampling, the cells and culture supernatant were collected for detection respectively. At least 3 biological replicates were set for each condition to ensure the reliability of the experimental data. Then the test was performed. The test methods were as follows: the OD value was determined by CCK-8 method, the proliferation rate was calculated by counting the number of cells daily to evaluate the effect of microcarrier surface modification and periodic speed change; the reactive oxygen species (ROS) level was detected by using DCFH-DA fluorescent probe to determine the intracellular ROS content after 3 days of culture; the antioxidant factor SOD and the secretion-promoting factors VEGF and TGF-β in the culture medium were detected by ELISA. The test results are shown in Table 1. The increase rate of proliferation rate and the increase rate of living cell ratio were obtained by comparing with the blank group.

[0049] Table 1 Culture test results of adipose-derived mesenchymal stem cell culture medium Note: “ / ” indicates that there is no experimental data under this condition.

[0050] The live cell ratio is required to be ≥90% in the cell culture process. From the data in the table above, we can see that the blank group has no addition of the extract of Alpinia zingiberis, and the live cell ratio is only 83%, which is far below 90% and cannot meet the requirement. With the increase of the concentration of the extract of Alpinia zingiberis, when the concentration of the extract of Alpinia zingiberis is 50 μg / mL, the proportion of live cells reached 90%, the proliferation rate reached more than 2 times within 3 days, the ROS level decreased, VEGF increased, and TGF-β increased. Due to the blank group and green tea extract, when the extract of Alpinia zingiberensis was in the range of 50-200μg / mL, the cell proliferation rate reached more than 115%, compared with the blank group, the proliferation rate increased by more than 17%, and the proportion of live cells increased by more than 8%; when the concentration of Alpinia zingiberensis extract was 100μg / mL, the performance was best, and the effect of 200μg / mL was slightly improved, but the cost may increase or produce an inhibitory effect of excessive concentration; from the above results, it can be seen that the extract of Alpinia zingiberensis is in the range of 50-200μg / mL, which can be used for the culture of adipose mesenchymal stem cells to significantly increase the proportion of live cells, cell proliferation rate, and ROS level.

[0051] Example 3 The present embodiment provides a second culture medium for epidermal stem cells, which uses RPMI-1640 as a basic culture medium and also includes the following components: 2 mM L-glutamine; 10% B-27 supplement; 10 μg / mL recombinant human insulin; 50 ng / mL EGF; 10 ng / mL KGF; 5 ng / mL TGF-β; 0.5~1g / L collagen; 1% rBSA; 100 μg / mL recombinant human ascorbic acid; 2 mg / L vitamin mixture; 50~150 μg / mL Ligustrum lucidum extract.

[0052] RPMI-1640 solution is 1L, 292 mg L-glutamine; 100 mL B-27 supplement; recombinant human insulin; 50μg EGF; 10μg KGF; 5μg TGF-β; 0.5 g collagen; 10g rBSA (recombinant human bovine serum albumin); 100μg / mL, recombinant human ascorbic acid; 2 mg / L, vitamin mixture (vitamin A, vitamin E, vitamin B group); 100μg / mL Ligustrum lucidum extract. The other ingredients added above are purchased externally, and the Ligustrum lucidum extract is prepared by Example 4. In the preparation of the culture medium, the basic culture medium is first prepared, and the added ingredients are added one by one according to the above dosage. 2~4g / L sodium bicarbonate (NaHCO3) is used to adjust the pH to 7.2~7.4, and the pH value of the culture medium is checked with a pH meter; sterilized by filtration through a 0.22μm filter membrane to ensure sterility. The prepared culture medium is stored at 4°C and is recommended to be used within two weeks.

[0053] In the above culture medium, the extract of Alpinia zeylanicum leaves is used as an antioxidant component, which is suitable for the culture of epidermal stem cells. The microcarrier 3D FloTrix of Huakan Biotechnology is used in the culture process. ® Carry out the expanded culture of epidermal stem cells and disinfect the microcarriers according to the instructions provided by Huakan Biotechnology to ensure a sterile environment.

[0054] Test Example 2 In order to test the antioxidant and proliferation-promoting effects of different extracts of Zingiber officinale on epidermal stem cell culture, the culture medium of Example 2 was used, except that different concentrations of Zingiber officinale extract (0 μg / mL, 50 μg / mL, 100 μg / mL, 150 μg / mL) were added to the culture medium, and the other components were the same as those in Example 3. The epidermal stem cells extracted in Example 1 were cultured to ensure that the cells were in good condition. The cells were cultured in an incubator at 37°C and 5% CO2. The CCK-8 method was used to detect the cell proliferation rate at different time points (24 hours, 48 ​​hours, and 72 hours); and the intracellular reactive oxygen level was measured after 24 hours of culture. The results of ROS level detection are shown in Table 2, and the proliferation rate is shown in Table 2. Figure 3 shown.

[0055] Table 2 RPMI-1640 culture results From the above data, we can see that as the addition concentration of the extract of the leaves of the ginger increased from 50 μg / mL to 150 μg / mL, the ROS level was between 75% and 45%, indicating that the addition of the extract of the leaves of the ginger greatly reduced the ROS level; as the addition concentration increased from 50 μg / mL to 150 μg / mL, the proliferation rate was improved, and the increase rate of the proliferation rate was relatively stable from 100μg / mL to 150 μg / mL. When the extract of the leaves of the ginger was in the range of 50-150μg / mL, the proliferation rate reached more than 2 times within 3 days, and the cell proliferation rate was better than that of the blank group at 24 hours, 48 ​​hours, and 72 hours. When the concentration of the extract of the leaves of the ginger was 100μg / mL, the performance was the best. The epidermal stem cell microscope showed Figure 4 Therefore, the extract of Alpinia officinalis leaf in the range of 50~150μg / mL can be used for the culture of epidermal stem cells to significantly increase the cell proliferation rate and ROS level.

[0056] Test Example 3 This example is to test the effect of different concentrations of recombinant trypsin dissociation mixture on the extraction of stem cells. This test example adopts the extraction method of Example 1, wherein the recombinant trypsin in the dissociation mixture is respectively 1% and 2%, and then the culture medium of Example 2 is used to culture adipose mesenchymal stem cells to obtain the original generation (P0) and the cells after the first passage (P1). The microscope images are as follows: Figure 5 , Figure 6 .

[0057] From the figure, we can see that when the recombinant trypsin in the dissociation mixture used is 1%, the cell consistency of P0 adipose-derived mesenchymal stem cells is good, the bottom of the culture dish is clean after culture of P1 adipose-derived mesenchymal stem cells, the cells have good light transmittance, proliferate quickly, and the cell morphology and consistency under the microscope are good; when the concentration of recombinant trypsin is increased to 2%, the bottom of the culture dish is dirty after culture of P0 adipose-derived mesenchymal stem cells, the cell state is poor, and the cell proliferation of P1 adipose-derived mesenchymal stem cells is poor. Therefore, choosing 1% concentration of recombinant trypsin is more conducive to maintaining cell activity than 2% concentration, because the lower concentration of enzyme action can reduce the damage to the cell membrane and cell structure, thereby improving the survival rate of cells after the digestion process. However, due to the high enzyme activity, the 2% concentration of recombinant trypsin may cause over-digestion, resulting in reduced cell activity.

[0058] Test Example 4 This example is to test the effect of different voltages on the cell purification effect in step S3 of Example 1. The direction of Example 1 is adopted, except that different voltages of 50V, 75V, and 100V are used to test the cell survival rate and cell migration rate of epidermal stem cells and adipose mesenchymal stem cells, respectively. Trypan Blue staining is used to detect the cell survival rate, and the target is ≥ 90% survival. The test results are shown in Table 3 below.

[0059] Table 3 Test results at different voltages As can be seen from the table above, when the voltage is 50V in electrophoresis separation, the cell survival rate and cell migration rate of epidermal stem cells and adipose-derived mesenchymal stem cells are both above 90%. As the voltage increases from 50V to 120V, the cell survival rate of epidermal stem cells and adipose-derived mesenchymal stem cells decreases, while the cell migration rate can reach above 90%. At a voltage of 100V, the cell survival rate of epidermal stem cells and adipose-derived mesenchymal stem cells can reach above 70%, meeting the separation requirements, while at a voltage of 120V, the cell survival rate of adipose-derived mesenchymal stem cells is less than 70%. Therefore, the voltage is set to 50~100V during electrophoresis separation.

[0060] Example 4 This embodiment provides a method for preparing an extract of Zingiber officinale, comprising the following steps: Step 1. Select well-growing Ligustrum lucidum plants and give priority to collecting mature leaves. Immediately put them in an ice bag after collection to reduce the degradation of active ingredients; use distilled water to thoroughly wash the leaves to remove surface dirt and pesticide residues. After washing, pat dry gently and absorb excess water with sterile paper towels. Dry the above-mentioned Ligustrum lucidum leaves in a dryer, set the temperature not to exceed 40°C, and dry until the moisture content is less than 10%; use a stainless steel grinder to crush the dried leaves to a particle size of about 1~2mm to increase the surface area to improve the extraction efficiency; after crushing, sieve the sample to remove larger particles to ensure uniformity and obtain Ligustrum lucidum powder.

[0061] Step 2: 69v% ethanol can effectively dissolve a variety of polar and non-polar components. Mix the powder of Ligustrum lucidum and 69% ethanol in a weight ratio of 1:18, soak and extract in a 25°C water bath for 18.4 minutes, the extraction temperature is 25°C, and the ultrasonic power is 450W; filter; wash the filtrate with n-hexane for 3 times to remove fat; after washing, extract with 250ml ethyl acetate for 3 times; concentrate by rotary evaporation at 45°C to obtain a concentrated solution, filter through a 0.22μm filter membrane, and then freeze-dry to obtain a sterilized Ligustrum lucidum concentrated solution.

[0062] Step 3: Pass the concentrated solution of Zingiber officinale leaves through a chromatography column to concentrate and obtain the Zingiber officinale leaves extract.

[0063] The treated AB-8 macroporous adsorption resin was loaded into a common chromatography column (16mm×500mm) with a filling height of 40cm and a column bed height of 35cm. Then, 2 to 3 times the column bed volume of H2O was used for elution to balance the column. The concentrated solution of Ligusticum chuanxiong was pumped into the resin with an adsorption flow rate of 0.5 mL / min. After the adsorption was completed, it was first washed with H2O until the effluent was colorless, and the flushing flow rate was 2.0 mL / min. Then, 50% ethanol was used for desorption until the effluent was colorless, and the desorption flow rate was 1.0 mL / min. The collected desorbed solution was vacuum condensed and frozen at -80°C, and then freeze-dried to obtain the Ligusticum chuanxiong extract.

[0064] For those skilled in the art, when understanding the solutions described in the specific embodiments of the present invention, they can refer to the conventional technical manuals in the field. At the same time, for the places where the above-mentioned terms appear, they can make appropriate understandings or adjustments for reference, and deduce the implementation of the same or similar technical solutions without paying any creative work.

[0065] The above embodiments describe only the basic principles, main features and / or advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and the invention content of the specification only describe the principles or specific cases of the present invention. Without departing from the essence of the innovative idea of ​​the present invention, the innovative scheme of the present invention may be subject to various changes and improvements, and these changes and improvements all fall within the scope of protection required by the present invention.

Claims

1. A method for extracting stem cells from skin tissue, characterized in that: The following steps are involved: S1. Using a dissociation mixture of recombinant trypsin, saline and antibiotics to treat the skin tissue and separate it into the epidermis and other layers; the mass concentration of the recombinant trypsin in the dissociation mixture is 0.8-1.5%; S2, cutting the epidermal layer obtained in step S1 into pieces and placing them in a trypsin-EDTA solution, incubating for 30 to 45 minutes, stirring, filtering and centrifuging after terminating the digestion to obtain epidermal stem cell-rich material, and resuspending to obtain an epidermal stem cell material suspension; Cut the other layers obtained in step S1 into pieces and digest them with a solution containing type I collagenase and / or type II collagenase, filter and centrifuge to obtain adipose-derived mesenchymal stem cell-rich material, and resuspend them to obtain adipose-derived mesenchymal stem cell suspension; S3, adding a positive charge modifier to the epidermal stem cell suspension for treatment; placing the adipose-dermal mesenchymal stem cell suspension and the modified epidermal stem cell suspension into different chambers of an electrophoresis device for separation, wherein the temperature of the electrophoresis buffer is controlled at 4-10°C, the voltage is set at 50-100V, the duration is 20-40min, the epidermal stem cell solution is collected at the cathode, and the adipose-dermal mesenchymal stem cell solution is collected at the anode; S4. Centrifuge the collected epidermal stem cell solution and adipose mesenchymal stem cell solution respectively, and culture the cells obtained after centrifugation respectively to obtain epidermal stem cells and adipose mesenchymal stem cells respectively.

2. The method for extracting stem cells from skin tissue according to claim 1, characterized in that: The skin tissue is the eyelid tissue discarded after double eyelid surgery.

3. The method for extracting stem cells from skin tissue according to claim 1, characterized in that: The mass concentration of recombinant trypsin in the dissociation mixture is 0.9-1.2%.

4. The method for extracting stem cells from skin tissue according to claim 1, characterized in that: The mass concentration of trypsin in the trypsin-EDTA solution is 0.08-0.25%.

5. The method for extracting stem cells from skin tissue according to claim 1, characterized in that: The mass concentration of collagenase in the solution containing type I collagenase and / or type II collagenase is 0.3-0.7%.

6. The method for extracting stem cells from skin tissue according to any one of claims 1 to 5, characterized in that: The detailed steps of the extraction method include: In step S1, the skin tissue is eyelid tissue discarded after double eyelid surgery; the treatment is overnight treatment at 4°C; In step S2, the epidermis layer is incubated at 36-38°C for 30-45 min, and the digestion is terminated with a DEME solution containing serum; the digestion of the other layers is performed at 4°C overnight.

7. The method for extracting stem cells from skin tissue according to any one of claims 1 to 5, characterized in that: In step S4, the adipose-derived mesenchymal stem cells are cultured in a first culture medium, wherein the first culture medium is based on DMEM / F12 and is characterized in that it further comprises 50-200 μg / mL of Ligustrum lucidum extract and a first additive, wherein the first additive is as follows: 8~15% KOSR; 5~20μg / mL recombinant human insulin; 20-100 ng / mL EGF; 10-40 ng / mL FGF-2; 20-100 ng / mL SDF-1; 2-10 ng / mL TGF-β; 0.5-1 g / L of an amino acid complex, wherein the amino acid complex comprises arginine, glycine, alanine and glutamic acid; 1–3.5 mM, L-glutamine.

8. The method for extracting stem cells from skin tissue according to claim 7, characterized in that: The preparation method of the extract of Zingiber officinale is as follows: Step 1: collecting mature leaves of the Ligusticum chuanxiong plant, washing them, and drying them in a dryer at a temperature not exceeding 40° C. until the moisture content is less than 10%; and crushing the dried leaves into a particle size of 0.2 to 5 mm to obtain Ligusticum chuanxiong powder; Step 2: Mix the powder of Ligustrum lucidum with 60-75v% ethanol solution in a weight ratio of 1:10-25, soak and extract in a water bath at 15-30°C for 10-40min, and filter after ultrasonication; wash the filtrate with n-hexane for 2-5 times, and extract with ethyl acetate for 2-5 times after washing; evaporate and concentrate the extract, filter through a membrane, and then freeze-dry to obtain Ligustrum lucidum concentrate; Step 3: Pass the Zingiber officinale concentrated solution through a chromatography column to concentrate and obtain the Zingiber officinale extract.

9. The method for extracting stem cells from skin tissue according to any one of claims 1 to 5, characterized in that: In step S4, epidermal stem cells are cultured in a second culture medium, wherein the second culture medium is based on RPMI-1640, characterized in that it further comprises 50-150 μg / mL of Alpinia zeylanicum extract and a second added component, wherein the second added component is as follows: 1-3.5 mM L-glutamine; 5-20% B-27 supplement; 5~30μg / mL recombinant human insulin; 20-100 ng / mL EGF; 5-20 ng / mL KGF; 2-10 ng / mL TGF-β; 0.5~1g / L collagen; 0.5~2% rBSA; 50~200μg / mL recombinant human ascorbic acid; 0.5~5 mg / L vitamin mixture.

10. The method for extracting stem cells from skin tissue according to claim 9, characterized in that: The preparation method of the extract of Zingiber officinale is as follows: Step 1: collecting mature leaves of the Ligusticum chuanxiong plant, washing them, and drying them in a dryer at a temperature not exceeding 40° C. until the moisture content is less than 10%; and crushing the dried leaves into a particle size of 0.2 to 5 mm to obtain Ligusticum chuanxiong powder; Step 2: Mix the powder of Ligustrum lucidum with 60-75v% ethanol solution in a weight ratio of 1:10-25, soak and extract in a water bath at 15-30°C for 10-40min, and filter after ultrasonication; wash the filtrate with n-hexane for 2-5 times, and extract with ethyl acetate for 2-5 times after washing; evaporate and concentrate the extract, filter through a membrane, and then freeze-dry to obtain Ligustrum lucidum concentrate; Step 3: Pass the Zingiber officinale concentrated solution through a chromatography column to concentrate and obtain the Zingiber officinale extract.

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