A method for preparing a stem cell culture preparation for repairing diabetic wounds

Abstract

This application relates to the field of cell preparation technology, and provides a method for preparing a stem cell culture preparation for repairing diabetic wounds, comprising: extracting and separating hair follicle tissue from the head to obtain a mixed tissue cell fluid of hair follicles, the mixed tissue cell fluid of hair follicles including a single-cell suspension and defatted intact hair follicle tissue; performing multiple in vitro 2D cultures of the defatted intact hair follicle tissue to obtain a supernatant to be processed, wherein the defatted intact hair follicle tissue is cultured at least 3 times; filtering and concentrating the supernatant to obtain an active supernatant, the active supernatant including VEGF, EGF, bFGF, interleukin IL10, antioxidant enzymes, and nutritional factors; adding auxiliary components to the active supernatant to obtain a stem cell culture preparation for repairing diabetic wounds. Hair follicle-derived mesenchymal stem cells exhibit strong proliferation and differentiation capabilities, and the concentrated active supernatant provides strong biological activity, improving the therapeutic effect of repairing diabetic wounds.

Description

Technical Field

[0001] The present application relates to the technical field of cell preparation preparation, and in particular to a method for preparing a stem cell culture preparation for repairing diabetic wounds. Background Art

[0002] Diabetes is a chronic metabolic disease. If the patient's body's long-term sugar and electrolyte metabolism disorders are not controlled, the condition will worsen and cause changes in nerves and blood vessels. Diabetic hand and foot disease is one of its common complications. Studies have shown that skin wounds at the extremities caused by diabetic lesions have a high infection rate and are difficult to heal. In severe cases, patients may face the risk of amputation or even death.

[0003] Stem cell technology has shown good application prospects in the treatment of diabetic foot ulcers. Mesenchymal stem cells (MSCs), as a cell with self-renewal and differentiation potential, can be isolated from various tissues such as bone marrow, fat, and umbilical cord blood, and can release a variety of cytokines, chemokines, and growth factors involved in wound healing to promote wound repair. However, stem cell technology still faces some challenges in clinical application. Studies have found that the mechanism by which stem cells regulate wound healing mainly comes from their paracrine effects rather than directly through the proliferation and differentiation of the cells themselves. Therefore, researchers continue to explore the application of stem cell-derived derivatives such as exosomes, apoptotic bodies, and stem cell culture supernatants in wound healing. Although there are some specific application methods for using cell supernatants in the treatment of diabetic wounds, these methods still face problems such as low yield and poor stability of stem cell cultures, which affect their effectiveness and promotion in preparations.

[0004] The present application provides a method for preparing a stem cell culture preparation for repairing diabetic wounds, which solves the problem that the culture method of mesenchymal stem cells is not efficient and stable enough, thus affecting the effect of the preparation in repairing diabetic wounds. Summary of the invention

[0005] In order to solve the problem that the culture method of mesenchymal stem cells is not efficient and stable enough, which affects the effect of its preparation in repairing diabetic wounds, the present application provides a method for preparing a stem cell culture preparation for repairing diabetic wounds, including: extracting and separating hair follicle tissue from the head to obtain a mixed hair follicle tissue cell fluid, wherein the mixed hair follicle tissue cell fluid includes a single cell suspension and a fat-free complete hair follicle tissue; culturing the fat-free complete hair follicle tissue in vitro for multiple times to collect a supernatant to be processed, and the number of culturing times of the fat-free complete hair follicle tissue is less than or equal to 3 times; filtering and concentrating the supernatant to be processed to obtain an active supernatant, wherein the active supernatant includes VEGF, EGF, bFGF, interleukin IL10, antioxidant enzymes and nutritional factors; adding auxiliary components to the active supernatant to obtain a stem cell culture preparation for repairing diabetic wounds, wherein the auxiliary components include chitosan, collagen, glycerol, ethylenediaminetetraacetic acid, vitamin C and vitamin E.

[0006] Hair follicle-derived mesenchymal stem cells have strong proliferation and differentiation capabilities. They can obtain a large number of cells in a short period of time, and maintain the characteristics of mesenchymal stem cells. They can differentiate into a variety of functional cells, and even form skin-like tissues during in vitro culture. At the same time, cells from this source include various types of cells, including hair papilla cells, epidermal stem cells, hair follicle mesenchymal stem cells, fibroblasts, sebaceous glands, etc. These cells are also closely related to the skin. Cells from this source are ideal seed source cells in repairing diabetic wounds. The method provided in this application accurately imitates the tissue microenvironment and cultures in 2D in vitro. It can harvest efficient, stable, and mass-producible composite stem cell derivative products for treating diabetic wounds from the same species without complicated operations, optimizes stem cell efficacy, reduces the patient's treatment pain and economic burden, and shows good clinical application prospects in promoting the healing of diabetic foot wounds. It solves the problem that mesenchymal stem cell cultures are not efficient and stable enough in culture methods, which affects the effect of their preparations in repairing diabetic wounds.

[0007] Optionally, the preparation method of the stem cell culture preparation for repairing diabetic wounds also includes: taking the hair follicle tissue from the head and washing it twice with DPBS containing 1% double antibody (penicillin / streptomycin), removing excess subcutaneous fat, retaining the sebaceous glands, and washing it again once to obtain clean hair follicle tissue. The clean hair follicle tissue is placed in a sterile test tube containing a digestion solution and digested overnight at 4°C. The next day, a stop solution is added to stop the digestion, and the cells are repeatedly blown with a pipette to disperse the cells to obtain a dispersed cell solution, wherein the digestion solution includes Dispase II enzyme, 0.05% pancreatin and DMEM culture medium, and the volume ratio of the Dispase II enzyme, 0.05% pancreatin and DMEM culture medium is 3:2:5, and the stop solution includes DPBS, FBS and EDTA. After the dispersed cell solution is filtered through a 70μm cell strainer for mixed tissue, it is centrifuged, washed, and resuspended to obtain a hair follicle mixed tissue cell solution, which includes a single cell suspension and a fat-free complete hair follicle tissue.

[0008] Optionally, the fat-free complete hair follicle tissue includes an upper tissue section, a middle tissue section and a lower tissue section, the upper tissue section includes a basal layer and sebaceous glands, the middle tissue section includes a bulge and a hair shaft, and the lower tissue section includes a hair papilla.

[0009] Optionally, the delipidated complete hair follicle tissue is subjected to multiple in vitro 2D culture to collect the supernatant to be processed, including: collecting the delipidated complete hair follicle tissue for the first time, inoculating the delipidated complete hair follicle tissue collected for the first time into a first specification culture bottle for adherent culture, inoculating the delipidated complete hair follicle tissue collected for the first time into a culture bottle of the first specification for adherent culture after subculturing and digestion, inoculating the cell fluid after the second subculturing and digestion into a culture bottle of the third specification for expanded adherent culture after subculturing and digestion, and obtaining a first supernatant, wherein the first specification culture bottle, the second specification culture bottle and the third specification culture bottle all contain culture medium, and the third specification culture bottle also contains a small molecule activator, and the small molecule activator includes a Wnt / β-catenin pathway activator and a ROCK inhibitor; collecting the delipidated complete hair follicle tissue for the second time, inoculating the delipidated complete hair follicle tissue collected for the second time into the first specification culture bottle for adherent culture, and inoculating the cell fluid after subculturing and digestion after subculturing and digestion. The hair follicle tissue is collected for the third time, and the fat-free complete hair follicle tissue collected for the third time is inoculated into the first specification culture bottle for adherence culture, subcultured and digested, and inoculated into the second specification culture bottle for adherence culture, and subcultured and digested again, and inoculated into the third specification culture bottle for expansion and adherence culture to obtain a third supernatant; the first supernatant, the second supernatant and the third supernatant are respectively subcultured, digested, centrifuged, resuspended and subcultured, and the supernatant to be processed is obtained after merging, and the supernatant to be processed includes cell substances secreted by the upper section of the tissue, the middle section of the tissue and the lower section of the tissue respectively; the specification of the first specification culture bottle is smaller than that of the second specification culture bottle, and the specification of the second specification culture bottle is smaller than that of the third specification culture bottle.

[0010] Optionally, the Wnt / β-catenin pathway activator is CHIR99021, the ROCK inhibitor is Y-27632, and the concentration of CHIR99021 in the third specification culture bottle is 3-10 µM, and the concentration of Y-27632 is 1-5 µM.

[0011] Optionally, the concentration of VEGF in the active supernatant is 204.8~212.2pg / mL, the concentration of EGF is 321.0~329.6pg / mL, the concentration of bFGF is 321.5~331.3pg / mL, and based on the total mass of the active supernatant as 100%, the mass fraction of interleukin IL10 is 5%~10%, and the mass fraction of antioxidant enzymes and nutritional factors is 2%~5%, and the antioxidant enzymes and nutritional factors include superoxide dismutase and glutathione.

[0012] Optionally, taking the mass fraction of the stem cell culture preparation for repairing diabetic wounds as 100%, the mass fraction of the chitosan is 0.5% to 2.0%, the mass fraction of the collagen is 1.0% to 3.0%, the mass fraction of the glycerol is 1.0% to 3.0%, the mass fraction of the ethylenediaminetetraacetic acid is 0.01% to 0.05%, and the mass fraction of the vitamin C and vitamin E is 0.1% to 0.5% in total.

[0013] Optionally, the repairing of diabetic wounds includes promoting angiogenesis, alleviating inflammatory response, and promoting tissue repair.

[0014] The present application provides a method for preparing a stem cell culture preparation for repairing diabetic wounds. The hair follicle-derived mesenchymal stem cells show strong proliferation and differentiation capabilities, and the concentrated active supernatant can provide strong biological activity in subsequent applications. The invention solves the problem that the mesenchymal stem cell culture is not efficient and stable enough, which affects the effect of the preparation in repairing diabetic wounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the technical solution of the present application, the drawings required for use in the embodiments are briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0016] Figure 1 This is a schematic diagram of hair follicle tissue samples from the head;

[0017] Figure 2 This is a schematic diagram of the complete hair follicle tissue structure after fat removal;

[0018] Figure 3 This is a schematic diagram of the structure of intact hair follicle tissue under a microscope;

[0019] Figure 4 Schematic diagram of obtaining skin tissue by in vitro culture and differentiation of hair follicle-derived mesenchymal stem cells;

[0020] Figure 5 Schematic diagram of RAW264.7 cells after passaging;

[0021] Figure 6 Schematic diagram of RAW264.7 cells differentiating into MI after induction treatment;

[0022] Figure 7 Schematic diagram of the inflammatory morphology of RAW264.7 cells after reversal. DETAILED DESCRIPTION

[0023] The following embodiments are described in detail, and examples thereof are shown in the accompanying drawings. When the following description refers to the drawings, unless otherwise indicated, the same numbers in different drawings represent the same or similar elements. The implementations described in the following embodiments do not represent all implementations consistent with the present application. They are only examples of systems and methods consistent with some aspects of the present application as detailed in the claims.

[0024] In the process of repairing diabetic wounds, due to the repeated infection and weak repair effect of diabetic wounds, it causes great distress and pain to patients. At the same time, the current more effective method of repairing diabetic wounds is mainly to inject cells into the wound tissue for wound repair. However, this method is very expensive, which creates great economic pressure for patients, and therefore cannot meet the actual needs of patients.

[0025] On the other hand, existing mesenchymal stem cells also have technical problems during the culture process, such as insufficient efficiency and stability, and insufficient culture volume, which further leads to high application costs.

[0026] In order to solve the above technical problems, the present application provides a method for preparing a stem cell culture preparation for repairing diabetic wounds, comprising: extracting and separating hair follicle tissue from the head to obtain a mixed hair follicle tissue cell fluid, wherein the mixed hair follicle tissue cell fluid comprises a single cell suspension and a fat-free complete hair follicle tissue; culturing the fat-free complete hair follicle tissue in vitro for multiple times in 2D, collecting a supernatant to be processed, wherein the fat-free complete hair follicle tissue is cultured for less than or equal to 3 times; filtering and concentrating the supernatant to be processed to obtain an active supernatant, wherein the active supernatant comprises VEGF, EGF, bFGF, interleukin IL10, antioxidant enzymes and nutritional factors; adding auxiliary components to the active supernatant to obtain a stem cell culture preparation for repairing diabetic wounds, wherein the auxiliary components comprise chitosan, collagen, glycerol, ethylenediaminetetraacetic acid, vitamin C and vitamin E.

[0027] In the prior art, the use of a variety of prepared stem cells, especially autologous mesenchymal stem cells, to prepare therapeutic preparations for diabetic wounds can achieve certain therapeutic effects. However, in actual applications, conventional mesenchymal stem cells, such as umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells, etc., on the one hand, their therapeutic effects cannot meet actual needs, leading to repeated attacks of diabetic wounds; on the other hand, the preparation of the above-mentioned mesenchymal stem cells is relatively inefficient and the preparation amount is small, which cannot meet actual needs. During the research and development process, the technical personnel of this application accidentally discovered that hair follicle stem cells, especially mesenchymal stem cells cultured from hair follicle tissues on the head and behind the ears, have better growth effects and inhibitory effects on M1 macrophages in diabetic wounds, thereby achieving better diabetic wound repair effects.

[0028] Hair follicles are pocket-like epithelia formed by continuous epidermal cells. They belong to the sub-organs of the skin, and their base is the dermal papilla, which is recessed in the dermis. There are many types of cells inside and around the hair follicles, which play a key role in the morphogenesis of hair follicles, the growth of hair, and the periodic renewal.

[0029] Specifically, there are mainly the following types of cells in the hair follicle: hair matrix cells (including hair papilla cells), which are responsible for the generation of hair follicles and the growth of hair. They are located at the bottom of the hair follicles and are the main driving force for the growth of hair follicles; melanocytes, which are responsible for the secretion of melanin and are responsible for the color of skin and hair. Melanocytes are distributed around the hair follicles to ensure that the hair has the appropriate color; epithelial tissue cells, which are opposite to monomeric cells, live in groups and are highly clustered. They not only constitute part of the hair follicles, but are also responsible for forming the hair shaft, that is, the main part of the hair; hair follicle stem cells, which play a leading role in the morphogenesis of hair follicles and the periodic growth of hair. They have the characteristics of slow periodicity, high cloning ability and unlimited division. Hair follicle stem cells are mainly located in the bulge of the hair follicle, which is a region that is crucial for the growth and renewal of hair follicles. In addition, the inside and outside of the hair follicle are composed of two parts: the epithelial root sheath and the surrounding hair follicle sheath. These sheath-like structures also contain a variety of cells, such as inner root sheath cells and outer root sheath cells, which together maintain the normal structure and function of the hair follicle.

[0030] Hair follicle-derived mesenchymal stem cells have strong proliferation and differentiation abilities. They can obtain a large number of cells in a short period of time and maintain the characteristics of mesenchymal stem cells. They can differentiate into a variety of functional cells and even form skin-like tissues during in vitro culture. The present application cultivates hair follicle-derived mesenchymal stem cells under in vitro 2D culture conditions, such as Figure 4 As shown, hair follicle-derived mesenchymal stem cells can differentiate and proliferate, eventually forming skin tissue. At the same time, cells from this source include various types of cells, including hair papilla cells, epidermal stem cells, hair follicle mesenchymal stem cells, fibroblasts, sebaceous glands, etc. These cells are also closely related to the skin, and cells from this source are ideal seed source cells for repairing diabetic wounds. The method provided in this application accurately imitates the tissue microenvironment and cultures in 2D in vitro. It can harvest efficient, stable, and mass-producible composite stem cell derivative products for treating diabetic wounds from the same species without complicated operations, optimizes the efficacy of stem cells, reduces the treatment pain and economic burden of patients, and shows good clinical application prospects in promoting the healing of diabetic foot wounds. It solves the problem that the culture method of mesenchymal stem cells is not efficient and stable enough, which affects the effect of its preparation in repairing diabetic wounds.

[0031] In some embodiments, the method for preparing a stem cell culture preparation for repairing diabetic wounds further includes: taking the hair follicle tissue from the head and washing it twice with DPBS containing 1% double antibody (penicillin / streptomycin), removing excess subcutaneous fat, retaining the sebaceous glands, and washing it again once to obtain clean hair follicle tissue. The clean hair follicle tissue is placed in a sterile test tube containing a digestion solution and digested overnight at 4°C. The next day, the stop solution is added to stop the digestion, and the cells are repeatedly blown with a pipette to disperse the cells to obtain a dispersed cell solution, wherein the digestion solution includes Dispase II enzyme, 0.05% pancreatin and DMEM culture medium, and the volume ratio of the Dispase II enzyme, 0.05% pancreatin and DMEM culture medium is 3:2:5, and the stop solution includes DPBS, FBS and EDTA. After the dispersed cell solution is filtered through a 70μm cell strainer for mixed tissue, at least a cell solution with complete delipidated hair follicle tissue is obtained, which is then centrifuged, washed, and resuspended to obtain a hair follicle mixed tissue cell solution.

[0032] In some embodiments, the hair follicle mixed tissue cell fluid comprises a single cell suspension and fat-free intact hair follicle tissue.

[0033] In some embodiments, the cell density of the single cell suspension is 5000 cell / cm 2 In order to better meet the tissue microenvironment requirements of the culture medium, the fat-free complete hair follicle tissue includes an upper tissue section, a middle tissue section and a lower tissue section. The upper tissue section includes a basal layer and sebaceous glands, the middle tissue section includes a bulge and a hair shaft, and the lower tissue section includes a hair papilla.

[0034] In the existing stem cell preparation methods, a single tissue is often screened and extracted to prepare specific stem cells, which is not effective when applied to diabetic wound repair. In the embodiment of the present application, the fat-free complete hair follicle tissue includes the upper tissue segment, the middle tissue segment and the lower tissue segment. In the actual application process, the technicians found that if only one or two segments of the fat-free complete hair follicle tissue are retained, the effect of the final preparation on diabetic wound repair still cannot meet the actual expectations. Therefore, the entire hair follicle tissue after removing the subcutaneous fat must be retained. In the subsequent preparation process, different segments of the hair follicle tissue will produce different cell substances. On the one hand, these different cell substances constitute a better cell growth microenvironment, greatly improve the growth efficiency of hair follicle-derived stem cells, and can ensure that a large number of hair follicle-derived stem cells are obtained in a very short time; on the other hand, these different cell substances complement each other, which can achieve better effects on diabetic wound repair, greatly improve the repair efficiency and reduce the recurrence rate.

[0035] After obtaining the complete delipidated hair follicle tissue, it is subjected to multiple in vitro 2D cultures. In traditional culture methods, the collected source tissue is often used directly for the culture of mesenchymal stem cells, and in the subsequent use process, the more active P3-P10 generations of mesenchymal stem cells are used for the preparation of biological preparations. However, in the actual application process, on the one hand, the hair follicle tissue from the head has a strong growth ability, so if it is discarded after only one use, the hair follicle tissue is not fully utilized, resulting in a waste of resources; on the other hand, due to the use of complete hair follicle tissue, and the use of a variety of cell substances harvested during its culture process to complement each other to achieve a better treatment effect of diabetic wounds, therefore, in the actual culture process, the more times the hair follicle-derived mesenchymal stem cells are passaged, the fewer the types of cells in the culture bottle, and the less cell substances are actually obtained, which reduces the treatment effect. Therefore, on the basis of obtaining a large number of hair follicle-derived mesenchymal stem cells after three passages, only collecting the first three generations of hair follicle-derived mesenchymal stem cells has the best treatment effect.

[0036] In some embodiments, the delipidated complete hair follicle tissue is subjected to multiple in vitro 2D cultures to collect the supernatant to be processed, including: collecting the delipidated complete hair follicle tissue for the first time, inoculating the delipidated complete hair follicle tissue collected for the first time into a first specification culture bottle for adherent culture, inoculating the delipidated complete hair follicle tissue collected for the first time into a culture bottle of the first specification for adherent culture after subculturing and digestion, inoculating the cell fluid after the second subculturing and digestion into a culture bottle of the second specification for expanded adherent culture after subculturing and digestion, and obtaining a first supernatant, wherein the first specification culture bottle, the second specification culture bottle and the third specification culture bottle all contain culture medium, and the third specification culture bottle also contains a small molecule activator, and the small molecule activator includes a Wnt / β-catenin pathway activator and a ROCK inhibitor; collecting the delipidated complete hair follicle tissue for the second time, inoculating the delipidated complete hair follicle tissue collected for the second time into the first specification culture bottle for adherent culture, and inoculating the second specification culture bottle after subculturing and digestion. The hair follicle tissue is collected for a third time, and the fat-free complete hair follicle tissue collected for the third time is inoculated into the first specification culture bottle for adherence culture, and is subcultured and digested, and is inoculated into the second specification culture bottle for adherence culture, and is subcultured and digested, and is inoculated into the third specification culture bottle for expansion and adherence culture to obtain a third supernatant; the first supernatant, the second supernatant and the third supernatant are respectively subcultured, digested, centrifuged, resuspended and subcultured, and the supernatant to be processed is obtained after merging, and the supernatant to be processed includes cell substances secreted by the upper section of the tissue, the middle section of the tissue and the lower section of the tissue, and the cell substances include cytokines and vesicles; the first specification culture bottle is smaller than the second specification culture bottle, and the second specification culture bottle is smaller than the third specification culture bottle.

[0037] In traditional wound treatment programs, a 3D culture program is often used to simulate the actual tissue environment of the human body and culture and differentiate mesenchymal stem cells into tissues with corresponding 3D structures, which are then applied to the lesion area to achieve the best treatment effect. However, in actual application, the above culture method is relatively complicated and has high culture costs. At the same time, the preparation efficiency of mesenchymal stem cells is also relatively low.

[0038] In the examples of the present application, technicians found that hair follicle-derived mesenchymal stem cells extracted from hair follicle tissue from the head have strong growth ability, and that a large number of hair follicle-derived mesenchymal stem cells can be quickly obtained by culturing with complete delipidated hair follicle tissue. Therefore, a large number of effective hair follicle-derived mesenchymal stem cells can be harvested by using a simpler 2D culture without using 3D culture. Therefore, in the examples of the application, the complete delipidated hair follicle tissue is subjected to multiple in vitro 2D cultures. For example, the complete delipidated hair follicle tissue is subjected to three in vitro 2D cultures in the examples of the present application.

[0039] Specifically, during the first in vitro 2D culture, the initially collected complete fat-free hair follicle tissue is first inoculated into a first specification culture bottle for adherent culture, for example, the first specification culture bottle is a T25 culture bottle, and then after subculture and digestion, it is inoculated into a second specification culture bottle for adherent culture for expansion, the second specification culture bottle is, for example, a T75 culture bottle, and after another subculture and digestion, the cell fluid after the second subculture and digestion is inoculated into a third specification culture bottle for expanded adherent culture, the third specification culture bottle is a T175 culture bottle, and the first supernatant is obtained.

[0040] It should be noted that the culture bottles of the first specification, the second specification and the third specification are all filled with culture medium in advance, for example, the culture medium is DMEM / F12 culture medium. During the first subculture and the second subculture, the conventional culture medium can be used for culture. During the third subculture, in order to obtain the most cell substances, a small molecule activator is added to the third specification culture bottle, for example, the small molecule activator includes a Wnt / β-catenin pathway activator and a ROCK inhibitor. It is not advisable to add small molecule activators during the first and second subcultures. If added, it will affect the normal growth of cells and the secretion of cell substances.

[0041] In the embodiment of the present application, the Wnt / β-catenin pathway activator is preferably CHIR99021, and the ROCK inhibitor is preferably Y-27632. In the third specification culture bottle, the concentration of CHIR99021 is 3~10 µM, and the concentration of Y-27632 is 1~5 µM. CHIR99021 (Wnt / β-catenin pathway activator), this signaling pathway plays a key role in hair follicle development and stem cell maintenance. It can not only enhance the secretion function of cells but also increase the concentration of effective factors in the supernatant. Y-27632 (ROCK inhibitor) enhances the adhesion and proliferation of cells by inhibiting Rho kinase, and has a significant effect on the survival and proliferation of hair follicle stem cells cultured in vitro. In the multiple collections of supernatant, the use of Y-27632 can improve cell tolerance, reduce apoptosis rate, and maintain long-term secretion activity.

[0042] Through the above preparation process, it can be seen that as the number of cell subcultures increases, the amount of mesenchymal stem cells continues to increase, and the cell amount is huge. After the third subculture, the cell amount can meet the actual needs, so the subculture can be stopped, while avoiding the problem of too many subcultures, the cells becoming less and less, and the resulting less and less cell material.

[0043] At this time, a second subculture is further performed. Specifically, the fat-free complete hair follicle tissue collected for the second time is cultured according to the above culture process to obtain a second supernatant. Based on the same process, a third subculture is performed to obtain a third supernatant. It should be noted that during the second subculture and the third subculture, although the same three specifications of culture bottles are used, in order to avoid cell contamination, new culture bottles are used for cell culture, which is common knowledge of those skilled in the art and will not be described in detail here.

[0044] At this time, the first supernatant, the second supernatant and the third supernatant are further subjected to subculture digestion, centrifugation, resuspending and subculture respectively, and the supernatant to be processed is obtained after fusion, and the supernatant to be processed includes cellular substances secreted by the upper section of the tissue, the middle section of the tissue and the lower section of the tissue respectively, and the cellular substances include cytokines and vesicles.

[0045] In the embodiments of the present application, the supernatant after culturing mesenchymal stem cells is used as the raw material for the preparation of biological preparations, rather than using the mesenchymal stem cells themselves as the raw material for the preparation of biological preparations, thereby greatly improving the preparation quantity and efficiency of biological preparations and reducing the preparation cost of biological preparations. In the actual implementation process, the cost of treating diabetic wounds can be reduced to one percent of the original cost, greatly reducing the treatment cost, and having great commercial promotion value and application value.

[0046] In the embodiment of the present application, by collecting and extracting the fat-free complete hair follicle tissue multiple times, it can be ensured that the same hair follicle tissue as that used in the initial culture is used in each mesenchymal stem cell culture, so that the mesenchymal stem cells obtained by subsequent subculture and the supernatant to be processed are highly uniform, ensuring the best culture effect; at the same time, by using complete hair follicle tissue for the culture of mesenchymal stem cells, the cell culture efficiency and the richness of cell substances in the supernatant are greatly promoted, which can further promote the repair effect of diabetic wounds, greatly reduce the probability of recurrence of diabetic wounds, reduce the pain of patients, and meet actual needs.

[0047] After obtaining the supernatant to be treated, the supernatant is filtered and concentrated to obtain an active supernatant, which includes VEGF, EGF, bFGF, interleukin IL10, antioxidant enzymes and nutritional factors.

[0048] In some embodiments, the active supernatant includes growth factors, cytokines, exosomes and other components.

[0049] Growth factors are a class of proteins or polypeptides that can stimulate cell proliferation and differentiation. The active supernatant of hair follicle-derived composite stem cell culture may contain a variety of growth factors, such as epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), etc. These growth factors play an important role in promoting tissue repair and regeneration.

[0050] Cytokines are a class of small molecule proteins with a wide range of biological activities that are synthesized and secreted by immune cells (such as monocytes, macrophages, T cells, B cells, NK cells, etc.) and certain non-immune cells (endothelial cells, epidermal cells, fibroblasts, etc.) upon stimulation. The active supernatant may contain a variety of cytokines, which affect cell growth and function by regulating cell-to-cell interactions and signal transduction.

[0051] Exosomes are tiny vesicles secreted by cells, with a diameter between 30 and 150 nm. Exosomes contain biologically active substances such as proteins, lipids, and nucleic acids from mother cells, which can transmit information and affect the functions of other cells. Exosomes may also be contained in the active supernatant, which may play an important role in intercellular communication and tissue repair.

[0052] In addition to the above main components, the active supernatant may also contain a variety of other biologically active substances, such as enzymes, hormones, metabolites, etc. The presence of these components makes the supernatant have complex biological functions.

[0053] In some embodiments, the concentration of VEGF is 204.8-212.2 pg / mL, the concentration of EGF is 321.0-329.6 pg / mL, the concentration of bFGF is 321.5-331.3 pg / mL, and the total mass of the active supernatant is 100%. The mass fraction of interleukin IL10 is 5%-10%, the mass fraction of antioxidant enzymes is 1.4%-4%, and the mass fraction of nutritional factors is 0.6%-1%. The antioxidant enzymes and nutritional factors include superoxide dismutase and glutathione, and the nutritional factors are, for example, trace amounts of vitamin B group, minerals, etc. It can be seen that the concentration of the above factors is much higher than the concentration of factors in the supernatant of traditional mesenchymal stem cells (such as umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells, etc.) during the culture process (generally less than 100 pg / mL).

[0054] The above-mentioned cell substances in the active supernatant can effectively inhibit and transform M1 cells in diabetic wounds, transforming them into M0 cells, that is, achieving effective treatment of diabetic wounds and preventing recurrence, greatly improving the efficacy. Those skilled in the art are aware of the meanings of M0 cells, M1 cells and M2 cells, which are common knowledge of those skilled in the art. For example, M0 cells are primitive undifferentiated macrophages, and no further elaboration is given here.

[0055] Finally, in order to further improve the use effect of the cell substances in the active supernatant, auxiliary components are added to the active supernatant to obtain a stem cell culture preparation for repairing diabetic wounds.

[0056] In some embodiments, the stem cell culture preparation is applied to diabetic wound repair, comprising: applying the stem cell culture preparation on the surface of the diabetic wound; inhibiting the M1 macrophages of the diabetic wound and converting them to M0 by the active supernatant in the stem cell culture preparation, thereby achieving diabetic wound repair.

[0057] Specifically, after the patient's autologous stem cell culture preparation is prepared, it is applied to the surface of the diabetic wound. At this time, the active supernatant components in the stem cell culture preparation inhibit the M1 macrophages in the diabetic wound and convert them to M0, so that the M1 macrophages in the diabetic wound become fewer or transform into M0 cells. At the same time, the M0 cells and other material components in the active supernatant combine with the relevant components in the auxiliary materials (such as vitamin C, etc.) to repair the diabetic wound together, so that it can achieve a rapid and stable repair effect.

[0058] In some embodiments, the auxiliary ingredients include chitosan, collagen, glycerol, ethylenediaminetetraacetic acid, vitamin C and vitamin E.

[0059] In some embodiments, taking the mass fraction of the stem cell culture preparation for repairing diabetic wounds as 100%, the mass fraction of the chitosan is 0.5% to 2.0%, the mass fraction of the collagen is 1.0% to 3.0%, the mass fraction of the glycerol is 1.0% to 3.0%, the mass fraction of the ethylenediaminetetraacetic acid is 0.01% to 0.05%, and the mass fraction of the vitamin C and vitamin E is 0.1% to 0.5% in total, wherein the mass fraction of the vitamin C is 0.06% to 0.35%, and the mass fraction of the vitamin E is 0.04% to 0.15%.

[0060] Chitosan has excellent biocompatibility and can protect the wound surface while also having antibacterial properties. Collagen is a structural support for tissue repair and can provide good extracellular matrix support. Glycerin is a moisturizer that maintains moisture on the wound surface and helps accelerate healing. EDTA (ethylenediaminetetraacetic acid) has the ability to chelate metal ions and can extend the shelf life of the product. Vitamin C and vitamin E are antioxidants that help reduce oxidative stress damage to the wound surface.

[0061] In some embodiments, the repairing of diabetic wounds includes promoting angiogenesis, reducing inflammatory response, and promoting tissue repair.

[0062] The experimental effects of the present application are described in detail below in conjunction with the embodiments.

[0063] Example 1: Collection of materials

[0064] In a professional hair transplantation institution or hospital, a doctor will take healthy hair follicle tissue from the head of a healthy cell donor, preferably, hair follicle tissue from the top of the head and / or behind the ears, such as Figure 1 As shown, the samples were washed with sterile PBS and placed directly into a sterile container containing 1% double antibody (penicillin / streptomycin) and DMEM culture medium on a sterile operating table and transported to the laboratory at 4°C.

[0065] Example 2: Extraction and separation of hair follicle tissue

[0066] (1) Wash the obtained hair follicle tissue from the head twice with DPBS containing 1% double antibody (penicillin / streptomycin), remove excess subcutaneous fat, retain the sebaceous glands, and wash again.

[0067] (2) Digestion: Place the tissue in a sterile test tube containing digestion solution (Dispase II enzyme + 0.05% trypsin + DMEM culture medium in a volume ratio of 3:2:5) at 4°C for overnight digestion. Add an equal volume of stop solution (DPBS + 5% FBS + 1mM EDTA) the next day to stop digestion; then use a pipette to repeatedly blow and beat to disperse the cells to obtain a cell dispersion.

[0068] (3) Filtration: Use a 70 μm cell strainer to filter the cell dispersion to remove impurities and large pieces of tissue and obtain a cell solution containing at least complete hair follicle tissue. At the same time, wash the culture dish with 10 ml of DPBS and further wash the cell strainer with the washing solution.

[0069] (4) Centrifugation: Centrifuge the product at 4°C and 800 rpm for 5 min to precipitate the cells and discard the supernatant.

[0070] (5) Washing: Resuspend the cells in 10 ml of DPBS, wash once, and centrifuge at 4°C, 800 rpm for 5 min.

[0071] (6) Resuspension: Since the number of cells is usually small, resuspend them in 1 ml of CNT-PR.

[0072] (7) Counting: Take 50 μl of cell suspension and put it into an EP tube. Then add trypan blue reagent in a ratio of 1:1 between cell suspension and trypan blue reagent to count the cells.

[0073] (8) Finally, the single cell suspension obtained in the previous step (cell density 5000 cell / cm 2 ) and fat-free intact hair follicle tissue were inoculated into T25 culture flasks coated with adhesive in advance, and 2 ml of stem cell complete medium (NC0103) was added to each flask and incubated at 37°C and 5% CO 2 Concentration of adherent culture.

[0074] Among them, fat-free intact hair follicle tissue Figure 2 As shown, the upper, middle and lower sections are completely preserved. The upper section of the tissue includes the basal layer and sebaceous glands, the middle section of the tissue includes the bulge and hair shaft, and the lower section of the tissue includes the hair papilla. Specifically, in the upper section of the tissue, the stem cells of the interfollicular epithelium are located here, and the division produces short-term amplified cells to form mutual communication between stem cell clusters. The adhesion between cells helps to maintain the characteristics of stem cells and prevent keratinocyte differentiation; in the middle section of the tissue, this part is multifunctional reserve stem cells, which are in a dormant period. They generally play a role in skin damage and epithelial tissue remodeling, and can reconstruct epithelial structures, hair follicles and sebaceous glands; in the lower section of the tissue, the matrix cells are short-term dividing epidermal cells, and the growth factors expressed in the dermal papilla can regulate the growth of hair follicles. As shown Figure 3 The figure shows the structure of the hair follicle tissue under a microscope, in which transparent lipid droplet-shaped sebaceous glands, inner and outer hair root sheaths, hair shafts, hair papillae, and the basal layer of hair follicles can be seen.

[0075] Example 3: Cultivation of hair follicle-derived mesenchymal stem cells

[0076] 1. Subculture of hair follicle-derived mesenchymal stem cells

[0077] (1) Observation of cell adhesion and medium replacement:

[0078] After the single cell suspension obtained in Example 2 and the fat-free complete hair follicle tissue were inoculated and cultured, the cell adhesion was observed, and half of the medium was replaced on the third day. If the cell density was less than 50% of the culture flask area, the medium was replaced twice a week; if it was higher than 50%, the medium was replaced once every 2 days. When the cell density reached 80% to 90%, the subculture operation was performed.

[0079] (2) Subculture and digestion:

[0080] Add 0.25% trypsin to the washed culture flask, about 1 mL per flask, shake gently to evenly cover the bottom of the flask with the digestion solution, and then aspirate the trypsin.

[0081] Place the culture flask in the incubator for 1 minute, then take it out and observe it under an inverted microscope. When the cells become round and most of them fall off, immediately add 2 volumes (2 mL) of complete culture medium to dilute the cell suspension and terminate digestion.

[0082] (3) Cell suspension processing and centrifugation:

[0083] Transfer the digested cell suspension to a 50 mL centrifuge tube, wash the residual cells in the culture flask with physiological saline, and transfer the washing solution to the centrifuge tube. Centrifuge at 1000 r / min for 5 minutes and discard the supernatant.

[0084] (4) Cell resuspension and counting:

[0085] Add 3-5 mL of complete medium to resuspend the cells. Take 1 mL of the cell suspension and dilute to 10 mL for cell counting and viability assessment.

[0086] (5) Subculture:

[0087] According to the actual cell number, seed the cells into a new culture flask at a density of 8000 cells / cm². Place the culture flask at 37℃ and 5% CO 2 , and in an incubator with saturated humidity. When the cell density reaches 80% to 90% again, repeat the above subculturing operation.

[0088] 2. Cell cryopreservation and recovery

[0089] Serum-free cell cryopreservation solution is used for cell cryopreservation. No programmed cooling is required. After being directly stored at -80°C for 12 hours, the cells are transferred to liquid nitrogen for storage. The cell recovery rate can reach more than 90%, and it does not contain any animal-derived components, which is suitable for subsequent cell drug development. The cryopreservation density is 1~2.7×10 7 cells / mL.

[0090] Example 4: Collection and concentration of hair follicle-derived mesenchymal stem cell supernatant

[0091] (1) Timing of collection and cell selection: The fat-free intact hair follicle tissue prepared in Example 2 was selected, and the first collection, the second collection, and the third collection were performed therefrom in chronological order.

[0092] First supernatant collection: The fat-free complete hair follicle tissue collected initially is inoculated into the first specification culture bottle (T25 culture bottle) for adherent culture. After subculture and digestion, it is inoculated into the second specification culture bottle (T75 culture bottle) for adherent culture for expansion. After subculture and digestion again, it is inoculated into the third specification culture bottle (T175 culture bottle) for expansion and adherent culture to obtain the first supernatant. Among them, the culture bottle of the first specification, the second specification culture bottle and the third specification culture bottle are all filled with culture medium in advance, and the culture medium is DMEM / F12 culture medium. When performing the first subculture and the second subculture, the conventional culture medium can be used for culture. During the third subculture, small molecule activators CHIR99021 and Y-27632 are further added to the third specification culture bottle. The concentration of CHIR99021 in the third specification culture bottle is 10µM, and the concentration of Y-27632 is 5 µM.

[0093] (2) Second supernatant collection: The fat-free intact hair follicle tissue collected for the second time is cultured according to the process of the initial collection to obtain the second supernatant.

[0094] (3) The third supernatant collection: The fat-free intact hair follicle tissue collected for the third time is cultured according to the same process as the initial collection to obtain the third supernatant.

[0095] (4) Supernatant treatment: The first supernatant, the second supernatant, and the third supernatant are respectively subjected to subculture digestion, centrifugation, resuspending, and subculture, and after merging, the supernatant to be treated is obtained. The supernatant to be treated includes cellular substances secreted by the upper tissue segment, the middle tissue segment, and the lower tissue segment, respectively.

[0096] (5) Supernatant concentration step:

[0097] Removal of cell debris: First, centrifuge at 2000 r.min for 10 minutes to remove cell debris and residual cells in the supernatant.

[0098] Filtration: The cell culture supernatant is then filtered using a 0.22 μm filter to further remove larger suspended particles and possible bacterial contamination.

[0099] Protein concentration: Ultrafiltration centrifugal concentration technology is used to concentrate growth factors and cytokines, increase the concentration of active ingredients, and obtain active supernatant. Sterile operation should be maintained during the process to avoid contamination, and low temperature centrifugation should be maintained during the centrifugation process.

[0100] Aliquoting and storage: The concentrated active supernatant is dispensed into sterile cryovials by batch and immediately stored at -80°C. Low temperature freezing can preserve bioactive molecules for a long time, but repeated freezing and thawing should be avoided to avoid reducing activity.

[0101] Example 5: Concentration of effective factors in active supernatant

[0102] The active supernatant prepared in Example 4 (i.e., P3 generation hair follicle-derived mesenchymal stem cells) was assayed for VEGF, EGF, and bFGF concentrations using enzyme-linked immunosorbent assay (ELISA). Three groups of samples were measured in parallel. The results are shown in Table 1.

[0103] Table 1 Concentrations of VEGF, EGF, and bFGF in the supernatant of P3 hair follicle-derived mesenchymal stem cells

[0104]

[0105] The optimized supernatant collection method and high-density cell culture conditions can obtain active supernatant rich in VEGF, EGF, and bFGF.

[0106] Example 6: Anti-inflammatory efficacy of hair follicle-derived mesenchymal stem cell supernatant

[0107] There are many factors that affect the healing of diabetic wounds, among which macrophage phenotype plays a key role. M1 macrophages can produce inflammatory factors to promote the development of inflammation, and M2 macrophages can produce cytokines that promote repair and thus promote wound healing. Therefore, this application selects RAW264.7 induced by lipopolysaccharide to simulate the inflammatory conditions of diabetic wounds, and co-cultures with the active supernatant obtained in Example 4 to try to evaluate the efficacy of hair follicle-derived cell supernatant in promoting diabetic wound healing by changing the macrophage phenotype or inhibiting the expression of M1.

[0108] Experimental materials and methods:

[0109] (1) Cell lines:

[0110] Cell name: RAW264.7 (mouse monocyte-macrophage cell line).

[0111] Source: National Key Laboratory of Sichuan University.

[0112] (2) Cell culture conditions:

[0113] Culture medium composition: 90% DMEM (Dulbecco's Modified Eagle Medium) + 10% FBS (Fetal Bovine Serum) + 1% PS (Penicillin-Streptomycin antibiotic mixture).

[0114] Culture environment: gas phase conditions are 95% air + 5% carbon dioxide; the temperature is maintained at a constant temperature of 37°C.

[0115] (3) Cell passaging:

[0116] Passaging ratio: Cells were passaged at a ratio of 1:2.

[0117] Passaging frequency: Cells were passaged twice a week.

[0118] (4) Experimental treatment:

[0119] Induction treatment: RAW264.7 cells were treated with 200 ng / ml lipopolysaccharide (LPS) for 24 hours to induce cell differentiation into MI (macrophage inflammatory type).

[0120] Reversal of the inflammation model: RAW264.7 cells were cultured for 48 hours using the active supernatant prepared in Example 4. Cell morphology changes were observed and recorded to evaluate whether the inflammation model was reversed.

[0121] (5) Observation of experimental results:

[0122] After the above treatment, the inflammatory morphology of RAW264.7 cells (such as antennae) gradually disappeared and began to return to the initial uninduced state. The cells showed a tendency to grow in clusters, indicating that the interaction and aggregation ability between cells were restored.

[0123] (6) Conclusion:

[0124] Figure 5 Schematic diagram of RAW264.7 cells after passage. Figure 6 Schematic diagram of RAW264.7 cells differentiated into MI after induction treatment. Figure 7 Schematic diagram of the inflammatory morphology of RAW264.7 cells after reversal. The inflammatory model was established by inducing RAW264.7 cells with LPS, and the inflammatory model was successfully reversed using the supernatant provided by the present application, indicating that the hair follicle-derived mesenchymal stem cell supernatant provided by the present application has an anti-inflammatory effect and is effective in promoting diabetic wound healing.

[0125] Example 7: Preparation of stem cell culture preparation for repairing diabetic wounds

[0126] Auxiliary components were added to the active supernatant prepared in Example 4 according to the mass fractions in Table 2 to obtain stem cell culture preparations for repairing diabetic wounds, which were the first preparation, the second preparation and the third preparation. Table 2 is based on the mass fraction of the stem cell culture preparation for repairing diabetic wounds as 100%.

[0127] Table 2 Mass fraction of auxiliary ingredients

[0128]

[0129] Example 8: Effect of hair follicle-derived mesenchymal stem cell culture preparation on the repair of diabetic wounds

[0130] 、 Experimental purpose:

[0131] The purpose of this experiment is to verify the effect of a preparation containing hair follicle-derived mesenchymal stem cell supernatant on wound repair in diabetic mice, and to evaluate the potential of this preparation in clinical application by comparing the wound healing conditions between the experimental group and the control group.

[0132] 2. Experimental Materials and Methods

[0133] (1) Experimental objects and model establishment

[0134] Experimental animals: 20 C57BL / 6 male mice aged 8-10 weeks.

[0135] Grouping and model induction: The mice were randomly divided into a control group and an experimental group, with 10 mice in each group. The diabetic mouse model was induced by injection of streptozotocin (STZ) to ensure that the blood glucose level was stable above 250 mg / dL.

[0136] Wound preparation: Under anesthesia, a full-thickness skin defect with a diameter of 6 mm was created on the back of each diabetic mouse.

[0137] (2) Treatment methods

[0138] Control group: blank matrix (basic preparation without supernatant, with normal saline as carrier) was used.

[0139] Experimental group: The first preparation prepared in Example 7 was used, with a dosage of 1.0 mL each time.

[0140] Dressing change frequency: Change the dressing once a day for 14 consecutive days.

[0141] (3) Experimental observation and indicator evaluation

[0142] Record of wound healing: record wound area, exudation, inflammatory symptoms, etc. every 2 days.

[0143] Wound area measurement: The wound area of ​​each group of mice was measured and recorded on days 0, 7, and 14, and the mean and standard deviation were calculated.

[0144] Calculation of wound healing rate:

[0145] ×100%.

[0146] Statistical analysis: The t-test was used to compare the difference in healing rate between the experimental group and the control group.

[0147] (4) Histological and molecular biological evaluation

[0148] Angiogenesis and collagen fiber reconstruction: The number of newly formed capillaries and the arrangement of collagen fibers were observed by histological staining.

[0149] 3. Experimental Results

[0150] (1) Wound healing

[0151] The wound closure rate of the experimental group was 30% to 50% higher than that of the control group. The specific data are shown in Table 3.

[0152] Table 3 Wound healing rate experimental results

[0153]

[0154] (2) Histological evaluation

[0155] The number of new capillaries in the wound of the experimental group was significantly greater than that of the control group, the collagen fibers were arranged more orderly, and the epidermal thickness increased significantly.

[0156] IV. Conclusion

[0157] The results of this experiment show that the stem cell culture preparation used to repair diabetic wounds can significantly promote the healing of wounds in diabetic mice, improve the wound closure rate, and promote angiogenesis and collagen fiber reconstruction.

[0158] This preparation has broad prospects in clinical applications and provides new ideas and methods for the treatment of diabetic wounds. At the same time, its application scope can be further expanded to the treatment of diabetic foot ulcers, chronic difficult-to-heal wounds and other complex wounds.

[0159] Although this experiment mainly focuses on diabetic wounds, the significant effect of hair follicle-derived mesenchymal stem cell supernatant preparation in promoting wound healing also suggests that it also has a positive effect on the repair of common wounds. Common wounds, such as burns and trauma, also involve complex processes such as tissue regeneration, angiogenesis, and inflammation control. Therefore, further exploration of the application of this preparation in the repair of common wounds is expected to provide a wider range of treatment options in the field of wound treatment.

[0160] Embodiment 9:

[0161] Umbilical cord stem cells were cultured according to the methods of Examples 1 to 4, and the concentrations of VEGF, EGF, and bFGF in the active supernatant were measured using the method of Example 5. Three groups of samples were measured in parallel, and the results are shown in Table 4.

[0162] Table 4 Concentrations of VEGF, EGF, and bFGF in the supernatant of umbilical cord stem cells

[0163]

[0164] Combining Table 1 and Table 4, the concentration of active ingredients in the supernatant of hair follicle-derived stem cells is higher than that in umbilical cord-derived stem cells.

[0165] Embodiment 10:

[0166] Using the method of Example 5, the concentrations of VEGF, EGF and bFGF in the active supernatant of hair follicle-derived tissue stem cells of generations P5 and P10 were measured, and the results are shown in Tables 5 and 6.

[0167] Table 5 Concentrations of VEGF, EGF, and bFGF in the supernatant of P5 hair follicle-derived mesenchymal stem cells

[0168]

[0169] Table 6 Concentrations of VEGF, EGF, and bFGF in the supernatant of P10 hair follicle-derived mesenchymal stem cells

[0170]

[0171] The active ingredients secreted by P5 hair follicle-derived mesenchymal stem cells are higher than those secreted by P10 hair follicle-derived mesenchymal stem cells.

[0172] The present application provides a method for preparing a stem cell culture preparation for repairing diabetic wounds. The hair follicle-derived mesenchymal stem cells show strong proliferation and differentiation capabilities, and the concentrated active supernatant can provide strong biological activity in subsequent applications. The problem that the mesenchymal stem cell culture is not efficient and stable enough in the culture method, which affects the effect of its preparation in repairing diabetic wounds is solved.

[0173] Similar parts between the embodiments provided in this application can be referenced to each other. The specific implementation methods provided above are only a few examples under the general concept of this application and do not constitute a limitation on the protection scope of this application. For those skilled in the art, any other implementation methods expanded based on the scheme of this application without creative work belong to the protection scope of this application.

Claims

1. A method for preparing a stem cell culture preparation for repairing diabetic wounds, characterized in that: include: Extracting and separating hair follicle tissue from the head to obtain hair follicle mixed tissue cell fluid, the hair follicle mixed tissue cell fluid includes a single cell suspension and fat-free complete hair follicle tissue, the fat-free complete hair follicle tissue includes an upper tissue section, a middle tissue section and a lower tissue section, the upper tissue section includes a basal layer and sebaceous glands, the middle tissue section includes a bulge and a hair shaft, and the lower tissue section includes a hair papilla; The fat-free complete hair follicle tissue is subjected to in vitro 2D culture for multiple times, and the supernatant to be processed is collected, wherein the number of times the fat-free complete hair follicle tissue is cultured is less than or equal to 3 times; The supernatant to be treated is filtered and concentrated to obtain an active supernatant, wherein the active supernatant includes VEGF, EGF, bFGF, interleukin IL10, antioxidant enzymes and nutritional factors, wherein the concentration of VEGF in the active supernatant is 204.8-212.2 pg / mL, the concentration of EGF is 321.0-329.6 pg / mL, the concentration of bFGF is 321.5-331.3 pg / mL, and based on the total mass of the active supernatant as 100%, the mass fraction of interleukin IL10 is 5%-10%, the mass fraction of the antioxidant enzyme is 1.4%-4%, and the mass fraction of the nutritional factors is 0.6%-1%; Auxiliary components are added to the active supernatant to obtain a stem cell culture preparation for repairing diabetic wounds. The auxiliary components include chitosan, collagen, glycerol, ethylenediaminetetraacetic acid, vitamin C and vitamin E.

2. The preparation method according to claim 1, characterized in that: Also includes: The hair follicle tissue from the head was taken and washed twice with DPBS containing 1% double antibodies, excess subcutaneous fat was removed, sebaceous glands were retained, and the hair follicle tissue was washed again to obtain clean hair follicle tissue, wherein the double antibodies were penicillin and streptomycin; The cleaned hair follicle tissue is placed in a sterile test tube containing a digestion solution at 4° C. for digestion overnight. The next day, a stop solution is added to stop the digestion. The cells are dispersed by repeatedly blowing with a pipette to obtain a dispersed cell solution. The dispersed cell fluid is filtered through a 70 μm cell filter, centrifuged, washed, and resuspended to obtain a hair follicle mixed tissue cell fluid, which includes a single cell suspension and a fat-free complete hair follicle tissue.

3. The preparation method according to claim 2, characterized in that: The digestion solution includes Dispase II enzyme, 0.05% trypsin and DMEM culture medium, the volume ratio of the Dispase II enzyme, 0.05% trypsin and DMEM culture medium is 3:2:5, and the stop solution includes DPBS, FBS and EDTA.

4. The preparation method according to claim 1, characterized in that: Taking the mass fraction of the stem cell culture preparation for repairing diabetic wounds as 100%, the mass fraction of the chitosan is 0.5% to 2.0%, the mass fraction of the collagen is 1.0% to 3.0%, the mass fraction of the glycerol is 1.0% to 3.0%, the mass fraction of the ethylenediaminetetraacetic acid is 0.01% to 0.05%, and the mass fraction of the vitamin C and vitamin E is 0.1% to 0.5% in total, wherein the mass fraction of the vitamin C is 0.06% to 0.35%, and the mass fraction of the vitamin E is 0.04% to 0.15%.

5. The preparation method according to any one of claims 1 to 4, characterized in that The repairing of diabetic wounds includes promoting angiogenesis, alleviating inflammatory response or promoting tissue repair.

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