Method for culturing hair epithelial stem cells
Through specific hair epithelial stem cell culture methods, including cleaning, digestive enzyme treatment and epithelial cell stacking culture methods, problems such as inefficiency of existing methods and carcinogenicity of materials are solved, and efficient amplification of hair epithelial stem cells and maintenance of dry properties are achieved.
Patent Information
- Application Number
- CN202510233132.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-28
- Publication Date
- 2025-06-03
AI Technical Summary
The existing hair epithelial stem cell proliferation methods are complex in operation and inefficient, which are difficult to meet the needs of clinical application. Commonly used materials have a risk of carcinogenicity, and the growth factor is expensive and is not suitable for large-scale production.
Specific culture methods are adopted, including cleaning and isolation of occipital hair follicles, preparing hair follicle fragments with digestive enzyme treatment, cell culture in epithelial cell culture medium, and passage amplification by epithelial cell stacking culture method to maintain the stem properties of the cells.
It realizes efficient expansion of hair epithelial stem cells and maintains stem cell characteristics, reduces operational complexity and cost, is suitable for large-scale production, and the stemness characteristics of cells are maintained.
Smart Images
Figure CN120082505A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biotechnology, and particularly to a method for culturing hair follicle epithelial stem cells. Background Art
[0002] With the influence of social pressure and environmental factors, the problem of hair loss has become increasingly serious. Although drug treatments (such as minoxidil and finasteride) and hair transplantation surgeries provide certain relief solutions for patients, these methods cannot fundamentally solve the problem of hair loss, with limited efficacy and easy recurrence after drug withdrawal. Therefore, the development of more effective and long-term stable hair regeneration medical technologies is regarded as the fundamental way to solve the problem of hair loss.
[0003] In the process of hair regeneration, the activation, proliferation, and functional maintenance of hair follicle stem cells are crucial. However, existing methods for proliferating hair follicle epithelial stem cells face many challenges, including complex operations and low efficiency, making it difficult to meet the requirements of clinical applications. When using the hydrogel encapsulation technique to culture hair follicle epithelial stem cells, although it mimics the in vivo microenvironment to a certain extent, experiments have found that the cells hardly proliferate at all. In addition, Chacón Martínez et al. pointed out in the article "Hair follicle stem cell cultures reveal self-organizing plasticity of stem cells and their progeny" in 2017 that materials used in the process of culturing hair follicle stem cells, such as Matrigel, are derived from animal tumor tissues and have potential carcinogenicity, and the cells cultured are difficult to be applied to human experiments. And when using the method of adding growth factors, for example, using the Rho-associated protein kinase inhibitor (ROCK inhibitor) Y-27632 can promote cell proliferation to a certain extent, but it cannot completely restore the stemness of the cells. In addition, the high cost of growth factors makes it inapplicable to large-scale production. Summary of the Invention
[0004] The purpose of the present invention is to provide a method for culturing hair follicle epithelial stem cells, including:
[0005] (1) Cleaning and separating the occipital hair follicles;
[0006] (2) Cutting the hair follicles and spreading the upper part of the hair follicles in dispase solution for incubation in a culture dish;
[0007] (3) Treating the incubated hair follicles with digestive enzymes to obtain hair follicle fragments, and adding epithelial cell medium to make a hair follicle epithelial stem cell suspension for cell culture;
[0008] (4)When the hair follicle epithelial stem cells proliferate to 80% of the culture dish area, single hair follicle epithelial stem cells are obtained by digestion with digestive enzymes and transferred to a new culture dish at a ratio of 1:3 for subculture and continued amplification;
[0009] (5)The hair follicle epithelial stem cells in the new culture dish are cultured by the epithelial cell stacking culture method.
[0010] Further, the specific process of the epithelial cell stacking culture method is as follows:
[0011] The hair follicle epithelial stem cells are inoculated on the bottom surface of the culture dish in a monolayer adherent form;
[0012] The hair follicle epithelial stem cells are cultured in an environment of 37°C and 5% CO 2 The epithelial cell culture medium is changed daily for 20 - 50 days.
[0013] Further, the occipital hair follicles are washed with PBS solution containing 1% penicillin.
[0014] Further, the hair follicles are cut open 2 mm from the bottom, and the upper part is transferred to Dispase II enzyme solution at 2 U / mL and incubated at 37°C and 5% CO 2 for 5 hours.
[0015] Further, the epithelial cell culture medium is prepared from 0.004 mL / mL of BPE, 0.125 ng / mL of rhEGF, 5 μg / mL of rh - Insulin, 0.33 μg / mL of Hydrocortisone, 0.39 μg / mL of Epinephrine, 10 μg / mL of Transferrin, and 0.06 mM of CaCl 2 and formulated.
[0016] Further, the cell culture is carried out in a culture dish without a feeder layer and cultured in an environment of 37°C and 5% CO 2 The epithelial cell culture medium is changed daily for 20 - 50 days.
[0017] Through long - term culture and specific culture conditions, the present invention can efficiently amplify hair follicle epithelial stem cells while maintaining their stem cell characteristics.
[0018] The present invention will be further described below with reference to the accompanying drawings of the specification. Description of the Drawings
[0019] Figure 1 is a flow chart of the culture method of the present invention.
[0020] Figure 2 is a schematic diagram of cell counting at different days.
[0021] Figure 3 It is a schematic diagram of the cell number proliferation rate.
[0022] Figure 4 It is a schematic diagram of the relative expression level increase of the cell marker CD200.
[0023] Figure 5 It is a schematic diagram of the relative expression level increase of the cell marker KRT15.
[0024] Figure 6 It is a schematic diagram of the count of the number of new hairs on different days.
[0025] Figure 7 It is a schematic diagram of the comparison of hair growth quantity. Detailed implementation manners
[0026] A method for culturing hair epithelial stem cells, comprising the following steps:
[0027] Step S100, cleaning the occipital follicles;
[0028] Step S200, trimming the follicles, and spreading the upper part of the follicles in Dispase II enzyme solution for incubation;
[0029] Step S300, treating the incubated follicles with digestive enzymes to prepare follicle fragments, adding epithelial cell medium and pipetting to make a hair epithelial stem cell suspension and culturing it;
[0030] Step S400, when the hair epithelial stem cells proliferate to 80% of the culture dish area, obtaining single hair epithelial stem cells by digestion with digestive enzymes and transferring them to a new culture dish at a ratio of 1:3 for subculture and continuous amplification;
[0031] Step S500, culturing the hair epithelial stem cells in the new culture dish by the epithelial cell stacking culture method.
[0032] In step S200, the purpose of trimming the follicles is to distinguish hair epithelial stem cells. The follicles contain two types of cells, namely the upper hair epithelial stem cells and the bottom dermal papilla cells. Hair epithelial stem cells are usually located in the bulge area of the follicle and are responsible for the hair growth cycle, such as being activated during the growth phase to promote hair regeneration. This part is clearly stem cells and participates in the regeneration and repair of the follicle. Dermal papilla cells, located at the bottom of the follicle, are responsible for transmitting signals and regulating the growth and differentiation of the follicle. The purpose of this embodiment is to culture hair epithelial stem cells. Therefore, the follicles are cut open, and the upper part of the hair epithelial stem cells is transferred to 2 U / mL Dispase II enzyme solution and incubated in an environment of 37°C and 5% CO 2 for 5 hours. After observation, if the hair epithelial stem cells have been dispersed, the incubation is ended in advance.
[0033] The epithelial cell medium in step S300 consists of 0.004 mL / mL BPE, 0.125 ng / mL rhEGF, 5 μg / mL rh-Insulin, 0.33 μg / mL Hydrocortisone, 0.39 μg / mL Epinephrine, 10 μg / mL Transferrin, 0.06 mM CaCl 2 and 100 μg Normocin. The epithelial cell medium can also be the Cnt-PR medium from CELLnTEC Company. The cell culture in step S300 is carried out in a feeder-free culture dish, and 0.5×10 6 -5×10 6 cells are seeded, and cultured under the conditions of 37°C and 5% CO 2 environment, and the epithelial cell medium is changed daily.
[0034] The digestive enzyme in step S400 is trypsin-EDTA (0.25%). The ratio of 1:3 means that after the cells grow and divide to 80% of the culture dish area, they are digested and recovered with the digestive enzyme to obtain a certain number of cells. After dividing the cells into three parts, they are respectively seeded into 3 culture dishes to achieve the purpose of expanded culture.
[0035] The epithelial cell stacking culture method in step S500 is an in vitro culture strategy used to promote the proliferation, stratification and maintenance of stemness of epithelial cells (such as hair follicle epithelial stem cells, keratinocytes). This method regulates cell density, culture time and culture conditions to enable the cells to gradually form a multi-layer stacking structure, simulating the layered characteristics of in vivo epithelial tissues. The process of the epithelial cell stacking culture method is as follows:
[0036] Step S501, inoculate hair follicle epithelial stem cells on the bottom surface of the culture dish in a monolayer adherent form;
[0037] Step S502, culture the hair follicle epithelial stem cells under the conditions of 37°C and 5% CO 2 environment, and the epithelial cell medium is changed daily for 20 - 50 days.
[0038] In step S502, the culture of hair follicle epithelial stem cells includes the following three stages:
[0039] (1) Cell adhesion and proliferation stage
[0040] Around the 1st - 4th day of culture, under appropriate culture conditions, hair follicle epithelial stem cells rapidly proliferate and spread on the surface of the culture dish, forming a monolayer cell layer. During the cell adhesion and proliferation stage, cells interact with each other through tight junctions and adherens junctions; around the 5th - 15th day of culture, cells proliferate massively. Due to the high proliferative activity state of the cells, their hair induction ability rapidly declines, which is related to the impairment of stem cell characteristics, changes in gene expression patterns, and reduced interaction with the cell microenvironment at high division rates.
[0041] (2) Stacking stage
[0042] Around the 16th - 20th day of culture, cells continue to proliferate, leading to an increase in local cell density and then a vertical growth trend. Due to space limitations, some cells detach from the bottom surface of the culture dish and start to arrange in layers, mimicking the growth pattern of epithelial cells in vivo. The cell morphology gradually transitions from adherent growth to multiple layers (2 - 3 layers).
[0043] (3) Stratification and differentiation stage
[0044] Through continuous culture, the hair - forming ability lost by the cells during the proliferation stage is restored. As the culture time extends to 20 - 30 days, the cells gradually enter the stacking culture stage, forming an epithelial - like layer structure and restoring their stem cell characteristics. The expression levels of stem cell markers such as CD200 and KRT15 are significantly up - regulated, indicating that the self - renewal ability of the cells is maintained.
[0045] Example 1
[0046] Take the occipital follicles of the patient and wash them with PBS solution containing 1% penicillin for 5 minutes.
[0047] Cut the follicles at 2 mm from the bottom and transfer the upper part to Dispase II enzyme solution at 2 U / mL, and incubate at 37°C and 5% CO 2 environment for 5 hours.
[0048] After incubation, treat the follicle fragments with digestive enzymes, add epithelial cell medium, pipette and suspend them, and make a hair follicle epithelial cell suspension.
[0049] Use epithelial cell medium Keratinocyte Growth Medium 2 (PromoCell) + 100 µg Normocin for culture, and the specific components are as follows:
[0050] - Basic components
[0051] - Bovine Pituitary Extract: 0.004 mL / mL
[0052] -Epidermal Growth Factor (recombinant human): 0.125 ng / mL
[0053] -Insulin (recombinant human): 5μg / mL
[0054] -Hydrocortisone: 0.33g / mL
[0055] -Epinephrine: 0.39μg / mL
[0056] -Transferrin (recombinant human): 10μg / mL
[0057] -CaCl 2 : 0.06 mM。
[0058] The basal components used the high-glucose DMEM medium.
[0059] In a 10-cm culture dish (Corning) without a feeder layer, 1×10 6 cells were seeded and cultured in an environment of 37°C and 5% CO 2 The culture medium was changed daily for 30 days.
[0060] On the 1st, 3rd, 5th, 10th, 15th, 20th, 25th, and 30th days, 0.25% trypsin solution was used for cell recovery for cell counting. As Figure 2 、 Figure 3 shown. On the 10th day of culture, the cells covered the culture dish. After the 15th day, they began to stack. After the 20th day, obvious stratification formed. The cell number increased by about 12 times within 30 days.
[0061] During the culture by the stacking method, the expression levels of the hair follicle epithelial stem cell markers CD200 and KRT15 also increased sharply. As Figure 4 、 Figure 5 shown, by measuring the expression levels of the hair follicle epithelial stem cell markers CD200 and KRT15, the expression levels of CD200 and KRT15 increased significantly by 12 times and 22 times respectively with the culture time, indicating that this method can maintain the stemness of cells.
[0062] Example 2
[0063] This example detected the hair growth ability of long-term cultured cells.
[0064] The hair follicle epithelial stem cells cultured on the 5th day and the 30th day were respectively mixed with 1×10 5 hair follicle epithelial stem cells and 1×105 After mixing neonatal mouse dermal cells (mesenchymal cells), they were implanted subcutaneously into the back of immunodeficient mice. After 3 weeks of breeding, the skin tissues were recovered and the number of newly generated hairs was counted. The experimental results are as Figure 6 , Figure 7 shown. The hair growth ability of hair follicle epithelial stem cells cultured for 30 days was more than 36 times higher than that of the cells on the 5th day.
Claims
1. A method for culturing hair epithelial stem cells, characterized in that: include: (1) Clean the hair follicles in the occipital area; (2) Cutting the hair follicles and placing the upper part of the hair follicles in a dispase solution for incubation in a culture dish; (3) treating the incubated hair follicles with digestive enzymes to obtain hair follicle fragments, adding epithelial cell culture medium to prepare a hair epithelial stem cell suspension for cell culture; (4) When the hair epithelial stem cells proliferate to 80% of the culture dish area, single hair epithelial stem cells are obtained by digestion with digestive enzymes and transferred to a new culture dish at a ratio of 1:3 for continued proliferation; (5) Cultivate hair epithelial stem cells in a new culture dish using the epithelial cell stack culture method.
2. The method according to claim 1, characterized in that The specific process of epithelial cell stack culture method is as follows: The hair epithelial stem cells are seeded on the bottom of the culture dish in the form of a monolayer adherent; Hair epithelial stem cells were cultured at 37°C, 5% CO2, with epithelial cell culture medium replaced daily for 20-50 days.
3. The method according to claim 1 or 2, characterized in that: The occipital hair follicles were washed with 1% penicillin in PBS.
4. The method according to claim 1 or 2, wherein the hair follicle is cut open 2 mm from the bottom, the upper part is transferred to 2 U / mL Dispase II enzyme solution, and incubated at 37°C and 5% CO2 for 5 hours.
5. The method according to claim 1 or 2, characterized in that: The epithelial cell culture medium components include 0.004 mL / mL BPE, 0.125 ng / mL rhEGF, 5 μg / mL rh-Insulin, 0.33 μg / mL Hydrocortisone, 0.39 μg / mL Epinephrine, 10 μg / mL Transferrin and 0.06 mm CaCl2.
6. The method according to claim 5, characterized in that Cells were cultured in culture dishes without a feeder layer at 37°C, 5% CO2, and the epithelial cell culture medium was changed daily for 20-50 days.