A kind of autologous hair follicle stem cell culture process and its application

Abstract

The present invention relates to the technical field of autologous hair follicle stem cell culture, specifically to an autologous hair follicle stem cell culture process, comprising the following steps: S1, preliminary hair follicle analysis and healthy hair follicle marking: using a follicle scope to perform high-definition imaging of the patient's scalp, analyzing the distribution, density and health of the hair follicles, marking relatively healthy hair follicles, and after marking the healthy hair follicles, shaving the target hair follicle area to prepare for the extraction process; S2, hair follicle extraction: using a hair follicle extractor, under local anesthesia, gently extract the hair follicles from the scalp, and place the extracted hair follicle samples in a container containing a hair follicle sample preservation solution; S3, hair follicle sample processing and preservation. This process extracts stem cells from the patient's autologous hair follicles, and after culturing, amplification, identification and other steps, successfully obtains hair follicle stem cells, which have the potential to be used for the treatment of hair loss. The results of animal experiments can further verify its efficacy and provide theoretical support for clinical applications.

Description

Technical Field

[0001] The present invention relates to the technical field of autologous hair follicle stem cell culture, and in particular to an autologous hair follicle stem cell culture process and application thereof. Background Art

[0002] Hair follicles are the organs responsible for hair growth. They are distributed throughout our skin. Except for a few areas such as the palms, soles of the feet and lips, hair follicles are present in most other areas, and the head is one of the areas with the densest hair follicles.

[0003] Hair growth is cyclical, including growth phase, regression phase and resting phase. Under normal circumstances, hair follicle stem cells in the resting phase begin to differentiate into new hair follicle cells and gradually reconnect with the microcirculation system. The hair follicles regain vitality and new hair will grow, and the cycle repeats. However, due to various reasons, hair follicle stem cells cannot differentiate normally, resulting in continuous atrophy of hair follicles and various hair loss problems.

[0004] There are many reasons for hair loss, such as staying up late, excessive stress, endocrine disorders, age, genetics, diseases, etc. These external and internal factors will eventually affect the hair follicles, especially the normal function of hair follicle stem cells. Therefore, hair follicle stem cells become the key to hair growth and treatment of hair loss.

[0005] Hair follicle stem cells (HFSCs) are relatively primitive cells in hair follicles. They are mesenchymal stem cells with strong self-renewal and multidirectional differentiation abilities. They are located in the outermost layer of the bulge of the outer root sheath of the hair follicle, close to the attachment point of the arrector pili muscle. Hair follicle stem cells can not only differentiate into hair follicles and effectively treat hair loss, but also participate in the formation of epidermal tissue. At the same time, they have strong immunomodulatory effects, hematopoietic support, and promote tissue repair.

[0006] The advantages of autologous hair follicles as a source of hair follicle stem cells are: 1. Easy to obtain, a small amount of hair follicles can obtain a considerable amount of hair follicle stem cells. 2. Rich sources, the number of hair follicles in the human body is relatively abundant, even people with hair loss will have a certain amount of hair follicles left.

[0007] The traditional method of culturing hair follicle stem cells is to cut the hair follicle tissue into pieces and then culture them in cell culture medium. This method requires a large amount of hair follicle tissue, which causes great damage to the donor's tissue, and the number of primary cells obtained is small and the survival rate is low.

[0008] Therefore, an autologous hair follicle stem cell culture process and application thereof are proposed. Summary of the invention

[0009] The purpose of the present invention is to provide an autologous hair follicle stem cell culture process and its application. This process extracts stem cells from the patient's own hair follicles and successfully obtains hair follicle stem cells through the steps of culture, amplification, identification, etc., which have the potential to be used for hair loss treatment. The results of animal experiments can further verify its efficacy and provide theoretical support for clinical application to solve the problems raised in the above background technology.

[0010] To achieve the above object, the present invention provides the following technical solution: an autologous hair follicle stem cell culture process, comprising the following steps:

[0011] S1. Preliminary hair follicle analysis and healthy hair follicle labeling:

[0012] Use a follicle scope to take high-definition images of the patient's scalp, analyze the distribution, density and health of the hair follicles, mark the relatively healthy follicles, and after marking the healthy follicles, shave the target follicle area in preparation for the extraction process;

[0013] S2. Hair follicle extraction:

[0014] Using a hair follicle extractor, under local anesthesia, the hair follicles are gently extracted from the scalp, and the extracted hair follicle samples are placed in a container containing hair follicle sample preservation solution;

[0015] S3. Hair follicle sample processing and storage:

[0016] The extracted hair follicles are immediately placed in a special hair follicle preservation solution, which contains an appropriate amount of culture medium, antibiotics, and antifungal agents and is stored at 4°C;

[0017] S4. Simple flushing and preliminary treatment of hair follicles:

[0018] Under a sterile environment, the hair follicle samples were placed in physiological saline and PBS buffer for simple rinsing to remove surface blood and impurities;

[0019] S5. Hair follicle dispersion and culture:

[0020] Place the cleaned hair follicles into a 6-well plate, with one or two hair follicles in each well. Drip an appropriate amount of complete culture medium into each well. The complete culture medium should include a mixture of DMEM / F12 culture medium, 10% fetal bovine serum, and 1% penicillin-streptomycin. Place the 6-well plate in a 37°C constant temperature incubator with a CO2 concentration of 5%.

[0021] S6. Observation of hair follicle attachment and addition of culture medium:

[0022] After standing for 4 hours, take out the 6-well plate and observe whether the hair follicles have successfully attached to the wall. If the hair follicles have attached to the wall, continue to add culture medium. If the hair follicles have not attached to the wall, gently shake the plate to help the hair follicles attach;

[0023] S7, culture medium replacement:

[0024] Change the culture medium every three days to ensure sufficient nutrients and remove metabolic products. When changing the medium, gently aspirate the old culture medium to avoid damaging the hair follicles.

[0025] S8. Observation of hair follicle stem cell growth and collection of primary cells:

[0026] After 5-7 days, the growth of hair follicle stem cells is observed to ensure that the cells have sufficient proliferation, and then enzyme digestion, i.e., trypsin treatment, is performed to collect the hair follicle stem cells to obtain primary hair follicle stem cells;

[0027] S9. Cell identification and phenotype verification:

[0028] A small amount of primary hair follicle stem cells was taken for flow cytometry analysis to verify whether they expressed mesenchymal stem cell surface markers, namely CD90, CD105, and CD73. After confirming the phenotype of the cells, the cells were further used;

[0029] S10. Cell cryopreservation:

[0030] Cell cryopreservation: Store a portion of hair follicle stem cells in cryopreservation solution at -80°C or in a liquid nitrogen tank for long-term storage.

[0031] Preferably, the S10, cell cryopreservation, includes cryopreservation optimization and cell transplantation optimization.

[0032] Preferably, the cryopreservation optimization includes using a low osmotic protective agent, mainly including glycerol and dimethyl sulfoxide, with a glycerol concentration of 10% to 15% and a DMSO concentration of 5% to 10%.

[0033] Preferably, the cryopreservation optimization includes adding antioxidants glutathione and vitamin C.

[0034] Preferably, the cryopreservation optimization includes adopting a gradual cooling method and a rapid thawing method, wherein the temperature is gradually low-temperature frozen by lowering the temperature by 1°C-2°C per minute in a freezer until it reaches -80°C. When thawing, the frozen cells are quickly thawed by quickly placing them in a 37°C water bath.

[0035] Preferably, the cell transplantation is optimized by restoring stem cell activity through 48 hours of culture before stem cell transplantation, and pre-treating the stem cells, specifically using a pre-treatment medium containing collagen or fibronectin to enhance the adhesion ability of the cells, and adding hair follicle growth factors KGF and EGF before cell transplantation.

[0036] Preferably, the cell transplantation is optimized, a microneedle system is used for cell transplantation, and a three-dimensional biological scaffold, i.e., a collagen scaffold or an alginate scaffold, is used as a cell carrier to increase the stability of the cells.

[0037] Preferably, the cell transplantation is optimized by monitoring the growth and distribution of cells after transplantation through live cell imaging technology, i.e., confocal microscopy or fluorescence imaging, tracking the transplanted cells using gene labeling or fluorescence labeling technology, i.e., GFP-labeled cells, and promoting cell growth and stability by regulating the oxygen concentration, nutrient supply and temperature microenvironmental factors of the transplantation area.

[0038] Preferably, the cell transplantation is optimized and immunological monitoring is performed to evaluate whether an inflammatory response occurs in the transplanted area, and by detecting local cytokines, namely IL-6 and TNF-α levels, the use of immunosuppressants or other immunoregulatory strategies are adjusted in a timely manner.

[0039] An application of autologous hair follicle stem cell culture, mainly used for hair transplantation for hair loss on the top of the human head.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] This process extracts stem cells from the patient's own hair follicles and successfully obtains hair follicle stem cells through cultivation, amplification, and identification, which have the potential to be used in the treatment of hair loss. The results of animal experiments can further verify its efficacy and provide theoretical support for clinical application;

[0042] Improve cell survival rate and quality: By optimizing the freezing solution formula, freezing process and cell recovery method, the survival rate and activity of hair follicle stem cells can be greatly improved to ensure the optimal state of the cells.

[0043] Improve the success rate of cell transplantation: Using microneedle technology and three-dimensional scaffolds to optimize cell transplantation methods can ensure that hair follicle stem cells are accurately transplanted to the target area, thereby improving the effects of hair follicle regeneration and hair growth.

[0044] Enhance the safety and efficacy of clinical treatment: Through immune monitoring and personalized treatment strategies, ensure the safety, effectiveness and durability of cells during treatment, and reduce the occurrence of side effects and ineffective treatment.

[0045] Improve the success rate of preclinical and clinical trials: optimize the protocols for animal and human trials, accelerate the advancement of clinical trials, and ensure the success rate and effectiveness of hair follicle stem cell therapy in clinical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0047] Figure 1 The figure is a process flow chart of the autologous hair follicle stem cell culture process of the present invention. DETAILED DESCRIPTION

[0048] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0049] See also Figure 1 , the present invention provides a technical solution:

[0050] An autologous hair follicle stem cell culture process comprises the following steps:

[0051] S1. Preliminary hair follicle analysis and healthy hair follicle labeling:

[0052] Use a follicle scope to take high-definition images of the patient's scalp, analyze the distribution, density and health of the hair follicles, mark the relatively healthy follicles, and after marking the healthy follicles, shave the target follicle area in preparation for the extraction process;

[0053] S2. Hair follicle extraction:

[0054] Using a hair follicle extractor, under local anesthesia, the hair follicles are gently extracted from the scalp, and the extracted hair follicle samples are placed in a container containing hair follicle sample preservation solution;

[0055] S3. Hair follicle sample processing and storage:

[0056] The extracted hair follicles are immediately placed in a special hair follicle preservation solution, which contains an appropriate amount of culture medium, antibiotics, and antifungal agents and is stored at 4°C;

[0057] S4. Simple flushing and preliminary treatment of hair follicles:

[0058] Under a sterile environment, place the hair follicle sample in saline or PBS buffer for simple rinsing to remove surface blood and impurities;

[0059] S5. Hair follicle dispersion and culture:

[0060] Place the cleaned hair follicles into a 6-well plate, with one or two hair follicles in each well. Drip an appropriate amount of complete culture medium into each well. The complete culture medium should include a mixture of DMEM / F12 culture medium, 10% fetal bovine serum, and 1% penicillin-streptomycin. Place the 6-well plate in a 37°C constant temperature incubator with a CO2 concentration of 5%.

[0061] S6. Observation of hair follicle attachment and addition of culture medium:

[0062] After standing for 4 hours, take out the 6-well plate and observe whether the hair follicles have successfully attached to the wall. If the hair follicles have attached to the wall, continue to add culture medium. If the hair follicles have not attached to the wall, gently shake the plate to help the hair follicles attach;

[0063] S7, culture medium replacement:

[0064] Change the culture medium every three days to ensure sufficient nutrients and remove metabolic products. When changing the medium, gently aspirate the old culture medium to avoid damaging the hair follicles.

[0065] S8. Observation of hair follicle stem cell growth and collection of primary cells:

[0066] After 5-7 days, the growth of the hair follicle stem cells is observed to ensure that the cells have sufficient proliferation, and then enzyme digestion, i.e., trypsin treatment, is performed to collect the hair follicle stem cells to obtain primary hair follicle stem cells.

[0067] S9. Cell identification and phenotype verification:

[0068] A small amount of primary hair follicle stem cells was taken for flow cytometry analysis to verify whether they expressed mesenchymal stem cell surface markers, namely CD90, CD105, and CD73. After confirming the phenotype of the cells, the cells were further used.

[0069] S10. Cell cryopreservation:

[0070] Cell cryopreservation: Store a portion of hair follicle stem cells in cryopreservation solution at -80°C or in a liquid nitrogen tank for long-term storage.

[0071] Specifically, the S10, cell cryopreservation, includes cryopreservation optimization and cell transplantation optimization.

[0072] The optimization of cryopreservation and cell transplantation is a crucial link in the application of autologous hair follicle stem cells, which is directly related to cell survival rate, efficacy, and the feasibility and safety of clinical treatment. Cell damage during cryopreservation and cell adaptability after transplantation are the key issues currently faced. The following is a detailed optimization plan for cryopreservation and cell transplantation optimization.

[0073] Cell cryopreservation optimization: Cryopreservation solution formulation optimization

[0074] Current problem: When cells are frozen, they will experience a low temperature and thawing process. In particular, water crystallization during the freezing process may cause damage to the cells. The formula of the cryopreservation solution is a key factor affecting cell survival rate.

[0075] Optimization measures: Use low-penetration protective agents:

[0076] Glycerol and dimethyl sulfoxide (DMSO) are commonly used cryoprotectants, but high concentrations may be toxic to cells.

[0077] Optimized concentration: The recommended glycerol concentration is 10% to 15%, and the DMSO concentration is 5% to 10% to avoid high concentrations causing cytotoxicity. The optimal concentration can be determined through gradient experiments.

[0078] Other protective agents: such as Trehalose (a natural sugar protective agent) and Polyvinylpyrrolidone (PVP), can be used as auxiliary protective components of freezing solution to reduce the formation of ice crystals.

[0079] Added Antioxidants:

[0080] Adding antioxidants (such as glutathione, vitamin C, etc.) to the freezing solution can reduce the generation of free radicals during the freezing process and prevent cell membrane damage.

[0081] Optimizing the slow freezing process of cryopreservation solutions:

[0082] By optimizing the cell freezing program, we can ensure that the temperature of cells changes smoothly during the freezing process. Gradual cooling (such as 1℃-2℃ per minute) can reduce the formation of ice crystals and maintain the integrity of the cell structure.

[0083] Low temperature freezing can be performed by programmed freezing equipment (such as a freezer) to ensure temperature uniformity and stability.

[0084] Temperature control during freezing:

[0085] Current Problem: Temperature changes during cryopreservation directly affect cell survival rate. Rapid freezing can cause ice crystals to break, affecting the integrity of cell membranes, while slow freezing may cause cell dehydration.

[0086] Optimization measures:

[0087] Use a programmed freezing system: Use a computer-controlled programmed freezing system (such as CoolCell) to ensure that the cell cooling process is smooth and stable. For example, the temperature drops by 1°C per minute until it reaches -80°C and then is stored in liquid nitrogen.

[0088] Avoid rapid temperature fluctuations: Before transferring from -80°C to liquid nitrogen for storage, ensure that the transition temperature changes slowly to prevent cells from being damaged by rapid temperature changes.

[0089] Thawing and resuscitation optimization:

[0090] Current problem: During the thawing process of frozen cells, temperature changes that are too fast or too slow may cause cell death.

[0091] Optimization measures:

[0092] Rapid thawing method: By quickly placing the frozen cells in a 37°C water bath (about 2 to 3 minutes), they are thawed quickly to avoid prolonged exposure to lower temperatures and reduce cell damage.

[0093] Gentle recovery: After thawing, use mild culture medium to recover cells and gradually transfer cells to appropriate culture conditions to avoid rapid rewarming that may cause cell stress response.

[0094] Cell transplantation optimization

[0095] Cell processing and optimization before transplantation

[0096] Current issue: Before cell transplantation, hair follicle stem cells may be affected by environmental factors during the culture process, such as nutrient deficiency or accumulated metabolic waste, resulting in reduced cell activity.

[0097] Optimization measures:

[0098] Cell recovery: Before transplantation, cell activity can be restored by culturing for a period of time (such as 48 hours) to ensure that the cells can be transplanted in the best condition.

[0099] Cell pretreatment:

[0100] Enhance cell adhesion: Use pre-treatment culture medium containing collagen or fibronectin to enhance cell adhesion, which is crucial for the transplantation effect of hair follicle stem cells.

[0101] Growth factor addition: Before cell transplantation, add some hair follicle growth factors (such as Keratinocyte Growth Factor KGF, Epidermal Growth Factor EGF, etc.) to promote cell proliferation and directional differentiation, and improve the success rate of transplantation.

[0102] Cell transplantation methods and vector selection:

[0103] Current problem: Traditional cell transplantation mostly uses direct injection or local application, which may cause cell death during the transplantation process or make it difficult to accurately control the distribution of cells in the target area.

[0104] Optimization measures:

[0105] Microneedle technology: Using a microneedle system for cell transplantation can precisely control the injection location of cells, reduce cell loss, and promote the directional growth of cells in the target area.

[0106] Three-dimensional scaffold transplantation: Use three-dimensional biological scaffolds (such as collagen scaffolds, alginate scaffolds, etc.) as cell carriers to increase cell stability, provide more support and nutrition, and promote cell attachment and growth.

[0107] The scaffold can be customized through bioprinting technology to ensure that it matches the growth environment of hair follicle stem cells, simulating the hair follicle microenvironment and helping cells to proliferate and differentiate better in vivo.

[0108] Cell growth monitoring after cell transplantation:

[0109] Current problem: The growth status of cells after transplantation is difficult to monitor in real time, and there may be a risk of cell instability or death in the transplanted area.

[0110] Optimization measures:

[0111] Real-time imaging technology: Monitor the growth and distribution of transplanted cells through live cell imaging technology (such as confocal microscopy or fluorescence imaging) to detect problems and adjust the plan in time.

[0112] Cell tracking and labeling: Gene labeling or fluorescent labeling technology (such as GFP-labeled cells) is used to track transplanted cells, making it easier to observe the growth dynamics of cells in real time in animals.

[0113] Microenvironment regulation: Promote cell growth and stability by adjusting microenvironmental factors such as oxygen concentration, nutrient supply and temperature in the transplantation area.

[0114] Post-transplant immune response and efficacy monitoring:

[0115] Current problem: Although they are autologous cells, due to the population heterogeneity of hair follicle stem cells and changes in the microenvironment during transplantation, immune rejection and efficacy may be variable.

[0116] Optimization measures:

[0117] Immune monitoring: Immunological monitoring is performed to evaluate whether inflammatory reactions occur in the transplanted area. By detecting the levels of local cytokines (such as IL-6 and TNF-α), the use of immunosuppressants or other immunomodulatory strategies can be adjusted in a timely manner.

[0118] Biocompatibility research: Strengthen the biocompatibility research between the transplanted area and cells, especially in the observation of long-term efficacy and side effects, to ensure the stability and effectiveness of cell transplantation.

[0119] Clinical application optimization:

[0120] Preclinical trials and human trials:

[0121] Current problem: Although animal and in vitro experiments on hair follicle stem cells have achieved good results, further verification is still needed before they can be put into clinical use.

[0122] Optimization measures:

[0123] Standardization of preclinical studies: Conduct more rigorous preclinical studies, such as using multiple animal models (such as mice, rats, monkeys, etc.) to evaluate the effects of hair follicle stem cell therapy.

[0124] Optimization in the human trial stage: When conducting human trials, adopt personalized treatment strategies and adjust the number of cells, transplantation method and treatment cycle according to the specific situation of the patient to ensure efficacy and safety.

[0125] Cryopreservation and cell transplantation optimization are crucial links in the application of autologous hair follicle stem cells.

[0126] The above strategies for cryopreservation and cell transplantation optimization, after careful optimization, will bring a series of excellent results, mainly reflected in the following aspects:

[0127] Improve cell survival and quality

[0128] Enhance cryopreserved cell survival

[0129] Cryopreservation optimization: By optimizing the cryopreservation solution formula (adding low osmotic protective agents, antioxidants, etc.) and using a programmed freezing system for slow cooling, the damage to cells by ice crystals can be effectively reduced, ensuring that hair follicle stem cells maintain a high survival rate during the cryopreservation and recovery process.

[0130] Effect:

[0131] Improve cell recovery rate: The recovery rate of cells after thawing will be significantly improved to avoid excessive cell mortality, which is especially important for cells stored for a long time.

[0132] Reduce cell damage: Reduce cell membrane damage, protein and DNA damage, etc., to ensure the activity of transplanted cells in subsequent applications.

[0133] Ensure cell activity and function

[0134] Culture medium optimization: By customizing the culture medium and adding growth factors, we ensure that hair follicle stem cells can maintain optimal biological activity during the culture process and avoid cell function decline due to malnutrition or waste accumulation.

[0135] Effect:

[0136] Enhance cell proliferation and differentiation potential: The optimized culture conditions accelerate cell proliferation and better differentiate into various types of hair follicle cells, further improving the therapeutic effect of hair follicle stem cells.

[0137] Enhance immune tolerance: Optimized culture medium components help improve the immune tolerance of cells and reduce rejection reactions during transplantation.

[0138] Optimizing cell transplantation outcomes

[0139] Improve the success rate of cell transplantation

[0140] Optimization of transplantation methods: Microneedle technology and three-dimensional scaffold technology can improve the directional growth of cells, reduce cell loss during transplantation, and ensure that cells can be accurately implanted into the target area.

[0141] Effect:

[0142] Precise positioning: Microneedle technology can ensure that hair follicle stem cells are accurately injected into the hair loss area, improving the effect of hair growth.

[0143] Cell survival and functional recovery: By providing a suitable growth environment through the three-dimensional scaffold, cells can better attach, grow and differentiate, further improving the success rate after transplantation.

[0144] Promote hair follicle regeneration and hair growth

[0145] Growth factor addition: By adding hair follicle growth factors (such as EGF, KGF, etc.), the regeneration of hair follicles can be promoted, and the growth and differentiation of hair follicle stem cells can be supported by improving the local environment.

[0146] Effect:

[0147] Accelerate hair growth: Optimization of growth factors can accelerate the proliferation of hair follicle stem cells and shorten the hair regeneration cycle.

[0148] Improve hair density and quality: By precisely controlling the growth environment of cells, hair density and quality can be significantly improved, especially for patients with severe hair loss.

[0149] Improving the long-term effects of cell transplantation

[0150] Enhanced cell adaptability: By optimizing post-transplant cell growth monitoring and immune tolerance, reducing inflammatory responses and immune rejection at the transplant site, and improving long-term cell survival rate and the sustainability of hair growth.

[0151] Effect:

[0152] Lasting effect: The long-term survival of cells and the continuity of hair growth are guaranteed, avoiding the phenomenon of hair falling out soon after regeneration in traditional treatment methods.

[0153] Avoid immune rejection: Through personalized treatment and immune monitoring, immune response and rejection reaction can be reduced, ensuring the good adaptability of autologous cells.

[0154] Improved safety and efficacy in clinical applications

[0155] Improving the safety of clinical treatment

[0156] Immune response monitoring: By monitoring cellular immune rejection reactions after transplantation and combining immunomodulatory strategies, cell stability and safety during treatment can be ensured.

[0157] Effect:

[0158] Reduce side effects: By closely monitoring the immune response, possible side effects (such as local redness and swelling, allergic reactions, etc.) in clinical applications can be reduced.

[0159] Enhance cell biocompatibility: By optimizing the microenvironment during transplantation (such as nutrition, blood supply, etc.), it promotes good integration of cells and the host, and further enhances the effect of transplantation.

[0160] Improve personalization and precision of treatment

[0161] Personalized treatment strategy: According to the patient's specific type of hair loss (such as androgenic alopecia, alopecia areata, etc.), a personalized cell transplantation treatment plan is carried out.

[0162] Effect:

[0163] Precision treatment: Through personalized treatment plans, the most appropriate treatment can be provided for different types of hair loss to improve the treatment effect.

[0164] Reducing ineffective treatments: Personalized treatments can avoid blind applications, reduce ineffective treatments, and save patients time and costs.

[0165] Improved success rate of preclinical and clinical trials

[0166] Standardization of preclinical research

[0167] Optimization of animal experiments: Through verification and optimization of multiple animal models, the reliability and clinical adaptability of treatment methods are ensured, and deviations caused by differences in animal experiments are reduced.

[0168] Effect:

[0169] Reliability of experimental data: Through standardized animal experiments, more convincing efficacy data can be obtained, further improving the success rate of clinical applications.

[0170] Verify the effects on different types of hair loss: Through different animal models, the efficacy of hair follicle stem cell therapy in different types of hair loss (such as androgenic alopecia, hair loss after chemotherapy, etc.) can be verified.

[0171] Advancement of clinical trials

[0172] Optimization of human trials: When conducting human trials, ensure the safety and effectiveness of clinical treatment through personalized treatment plans and precise cell transplantation methods.

[0173] Effect:

[0174] Improve clinical efficacy: Through precise treatment and long-term monitoring, ensure the stability and durability of the treatment effect, especially for patients with hair loss symptoms can be significantly improved.

[0175] Accelerate the advancement of clinical applications: By optimizing the test plan and treatment methods, the transition from preclinical to clinical trials can be accelerated, and the promotion speed of autologous hair follicle stem cell therapy can be increased.

[0176] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A process for culturing autologous hair follicle stem cells, characterized in that: The steps include: S1. Preliminary hair follicle analysis and healthy hair follicle labeling: Use a follicle scope to take high-definition images of the patient's scalp, analyze the distribution, density and health of the hair follicles, mark the relatively healthy follicles, and after marking the healthy follicles, shave the target follicle area in preparation for the extraction process; S2. Hair follicle extraction: Using a hair follicle extractor, under local anesthesia, the hair follicles are gently extracted from the scalp, and the extracted hair follicle samples are placed in a container containing hair follicle sample preservation solution; S3. Hair follicle sample processing and storage: The extracted hair follicles are immediately placed in a special hair follicle preservation solution, which contains an appropriate amount of culture medium, antibiotics, and antifungal agents and is stored at 4°C; S4. Simple flushing and preliminary treatment of hair follicles: Under a sterile environment, the hair follicle samples were placed in physiological saline and PBS buffer for simple washing to remove surface blood and impurities; S5. Hair follicle dispersion and culture: Place the cleaned hair follicles into a 6-well plate, with one or two hair follicles in each well. Drip an appropriate amount of complete culture medium into each well. The complete culture medium should include a mixture of DMEM / F12 culture medium, 10% fetal bovine serum, and 1% penicillin-streptomycin. Place the 6-well plate in a 37°C constant temperature incubator with a CO2 concentration of 5%. S6. Observation of hair follicle adhesion and addition of culture medium: After standing for 4 hours, take out the 6-well plate and observe whether the hair follicles have successfully attached to the wall. If the hair follicles have attached to the wall, continue to add culture medium. If the hair follicles have not attached to the wall, gently shake the plate to help the hair follicles attach; S7, culture medium replacement: Change the culture medium every three days to ensure sufficient nutrients and remove metabolic products. When changing the medium, gently aspirate the old culture medium to avoid damaging the hair follicles. S8. Observation of hair follicle stem cell growth and collection of primary cells: After 5-7 days, the growth of hair follicle stem cells is observed to ensure that the cells have sufficient proliferation, and then enzyme digestion, i.e., trypsin treatment, is performed to collect the hair follicle stem cells to obtain primary hair follicle stem cells; S9. Cell identification and phenotype verification: A small amount of primary hair follicle stem cells was taken for flow cytometry analysis to verify whether they expressed mesenchymal stem cell surface markers, namely CD90, CD105, and CD73. After confirming the phenotype of the cells, the cells were further used; S10. Cell cryopreservation: Cell cryopreservation: Store a portion of hair follicle stem cells in cryopreservation solution at -80°C or in a liquid nitrogen tank for long-term storage.

2. The process for culturing autologous hair follicle stem cells according to claim 1, characterized in that: The S10, cell cryopreservation, includes cryopreservation optimization and cell transplantation optimization.

3. The process for culturing autologous hair follicle stem cells according to claim 2, characterized in that: The cryopreservation optimization includes the use of low-osmotic protective agents, mainly including glycerol and dimethyl sulfoxide, with the glycerol concentration being 10% to 15% and the DMSO concentration being 5% to 10%.

4. The process for culturing autologous hair follicle stem cells according to claim 3, characterized in that: The cryopreservation optimization includes adding antioxidants glutathione and vitamin C.

5. The autologous hair follicle stem cell culture process and application according to claim 4, characterized in that: The cryopreservation optimization includes adopting a gradual cooling method and a rapid thawing method, wherein the temperature is gradually low-temperature frozen by lowering the temperature by 1°C-2°C per minute in a freezer until it reaches -80°C. During thawing, the frozen cells are quickly thawed by quickly placing them in a 37°C water bath.

6. The process for culturing autologous hair follicle stem cells according to claim 5, characterized in that: The cell transplantation optimization is that before the stem cell transplantation, the activity of the stem cells is restored by 48 hours of culture, and the stem cells are pretreated, specifically using a pretreatment culture medium containing collagen or fibronectin to enhance the adhesion ability of the cells, and before the cell transplantation, hair follicle growth factors KGF and EGF are added.

7. The process for culturing autologous hair follicle stem cells according to claim 6, characterized in that: The cell transplantation optimization adopts a microneedle system for cell transplantation and utilizes a three-dimensional biological scaffold, namely a collagen scaffold and an alginate scaffold as a cell carrier to increase the stability of the cells.

8. The process for culturing autologous hair follicle stem cells according to claim 7, characterized in that: The cell transplantation optimization monitors the growth and distribution of cells after transplantation through live cell imaging technology, i.e., confocal microscopy or fluorescence imaging, tracks the transplanted cells using gene labeling or fluorescence labeling technology, i.e., GFP-labeled cells, and promotes cell growth and stability by regulating the oxygen concentration, nutrient supply and temperature microenvironmental factors of the transplantation area.

9. The process for culturing autologous hair follicle stem cells according to claim 8, characterized in that: The cell transplantation is optimized and immunological monitoring is performed to evaluate whether an inflammatory response occurs in the transplanted area. By detecting local cytokines, namely IL-6 and TNF-α levels, the use of immunosuppressants or other immunoregulatory strategies are adjusted in a timely manner.

10. An application of autologous hair follicle stem cell culture, characterized in that: Mainly used for hair transplantation on the top of the human head.

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