Universal cell preparation for treating male hormone alopecia and application thereof

Abstract

The invention relates to a universal cell preparation for treating male hormone alopecia and application of the universal cell preparation, the universal cell preparation comprises hair papilla cells and normal saline, the concentration of the hair papilla cells is 105-109 cells / mL, and in flow cytometry analysis, the hair papilla cells are compared with same cells without antibody binding. And the relative expression levels of HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DP and HLA-DQ are less than 5%. The obtained human-derived hair papilla cells are identified and analyzed to be low-immunogenicity cells, and are more suitable for hair regeneration of universal cells, that is, the hair papilla cells do not need to be obtained through a body, and the application range and application scene are greatly provided. The universal cell preparation prepared from human-derived hair papilla cells and normal saline can be injected to an alopecia part and can be used for effectively treating male hormone alopecia symptoms.

Description

Technical Field

[0001] The present invention relates to the field of medical supplies, and in particular to a universal cell preparation for treating androgenic alopecia and its application in the field of hair growth. Background Art

[0002] As the pace of life accelerates, young people face increasing pressure, leading to premature hair loss. Faced with immense social and psychological pressure, hair loss has become a serious issue that must be taken seriously. Hair loss has become a global problem!

[0003] According to the "White Paper on Chinese Scalp Health," approximately 200 million people in China suffer from hair loss, with a high and increasingly severe prevalence, growing at a rapid rate of 15%-18% annually. Among those born in the 1990s and under the age of 30, the rate of hair loss has reached 36.1%, and over 50% of college students report experiencing hair loss. Scientific research indicates that there are multiple causes of hair loss, with over 90% being caused by androgenic alopecia. This is due to the sensitivity of hair follicles to dihydrotestosterone (DHT), a natural metabolite of androgens. During puberty, DHT promotes the development of male sexual characteristics (such as facial hair). However, in adulthood, excessive DHT levels can have negative effects, such as acne, blackheads, benign prostatic hyperplasia (BPH), and androgenic alopecia. Both men and women can experience hair loss due to changes in androgen metabolism, but it is more common in men. Baldness significantly impacts a person's image and significantly impacts their lives.

[0004] A survey found that 82.1% of people are very concerned about their current hair condition (hair loss or thinning), and 59.3% of these users have taken appropriate measures. Among women, 48.1% have taken appropriate measures due to hair concerns, while the proportion of men who have taken such measures is significantly higher, at 62.6%. Consequently, faced with this situation, more and more people are turning to treatments for hair loss. Among hair-saving methods, more and more people are interested in and using hair care, anti-hair loss, and hair growth products or medications. Currently, commonly used clinical medications include minoxidil and finasteride. These medications have significant side effects and can cause serious adverse effects on the body through long-term use. For example, long-term finasteride use in women can cause congenital malformations in the embryo, while in men, it can lead to serious consequences such as decreased libido and impotence. More cellular byproducts, including cytokines and exosomes, have also gradually emerged on the market, but their effectiveness has not been particularly effective. Meanwhile, some autologous stem cell products have emerged, demonstrating significant improvements in efficacy. However, due to immunogenicity issues, they cannot be used universally and can only be applied autologously, limiting their application.

[0005] However, different types of cells have different functions, and immunogenicity issues can lead to rejection reactions. Obtaining universal stem cells through appropriate means, or finding low-immunogenic cell types to make products or preparations for the purpose of treating androgenic alopecia is an urgent problem to be solved. Summary of the Invention

[0006] Technical issues

[0007] In view of this, the technical problem to be solved by the present invention is how to provide a universal cell preparation for treating androgenic alopecia that can retain hair growth ability and reduce immunogenicity, and its application in the field of hair growth.

[0008] The present invention uses hair follicle tissue as the source for primary isolation of dermal papilla cells. Through pretreatment and culture medium optimization, the purity of the cells is effectively improved, the operational difficulty of isolating pure dermal papilla cells is reduced, and the immunogenicity of the cells is also reduced, avoiding the rejection reaction caused by the cell products, which provides more feasibility for the preparation of universal cell products for hair regeneration.

[0009] Solution

[0010] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0011] In the first aspect, the present invention provides a universal cell preparation for treating androgenic alopecia, comprising hair papilla cells and physiological saline, wherein the concentration of the hair papilla cells is 10 5 ~10 9 cells / mL, among which, in flow cytometric analysis of dermal papilla cells, the expression levels of HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DP, and HLA-DQ were less than 5% when the same type of cells without antibody binding were used as controls.

[0012] Furthermore, the relative expression levels of HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DP, and HLA-DQ are <2%, optionally <1.5%.

[0013] Furthermore, the relative expression levels of HLA-A, HLA-B, HLA-C, and HLA-DQ are less than 0.3%, the relative expression level of HLA-DR is less than 1%, and the relative expression level of HLA-DP is less than 1.5%.

[0014] Furthermore, the dermal papilla cells are primary cells separated from hair follicles and subcultured for 3 to 7 generations and have the ability to germinate, and optionally are cells of 3 to 5 generations and have the ability to germinate.

[0015] Furthermore, the concentration of the hair papilla cells is 10 6 ~10 8 cells / mL.

[0016] Furthermore, the method for isolating dermal papilla cells comprises:

[0017] 1) Isolating hair follicle tissue from ex vivo human hair samples;

[0018] 2) pre-treating the hair follicle tissue to remove connective adhesion tissue and fat;

[0019] 3) The pretreated hair follicle tissue obtained in step 2) is cultured using a hair papilla cell selection medium to obtain primary hair papilla cells, which are then subcultured to obtain hair papilla cells of passage 3 to 7.

[0020] Furthermore, in step 2), the pretreatment method further comprises: removing the hair bulb tissue and part of the hair shaft tissue in the hair follicle tissue, retaining the middle section as the hair follicle middle section tissue, and optionally, the length of the hair follicle middle section tissue is 0.5-2.5 mm;

[0021] Optionally, the method of removing tissue is performed under a stereoscope.

[0022] Further: in step 1), the hair follicle tissue is a hair follicle unit tissue and / or a single hair follicle tissue;

[0023] And / or, in step 2), the hair follicle tissue is a single hair follicle tissue, optionally, the single hair follicle tissue is separated from a hair follicle unit;

[0024] Furthermore, in step 3), the hair papilla cells are isolated by culturing the pretreated hair follicle tissue in a DMEM medium containing 2.5%-12.5% ​​FBS, and the hair papilla cells are isolated after the hair papilla cells crawl out; optionally, the hair papilla cells are isolated by using a DMEM medium containing 2.5%-6.5% FBS; optionally, the hair papilla cells are isolated by using a DMEM medium containing 2.5%-5% FBS;

[0025] And / or, the subculture adopts a DMEM / F12 medium containing 7.5% to 17.5% FBS, and optionally the subculture adopts a DMEM / F12 medium containing 10% to 17.5% FBS.

[0026] In a second aspect, a method is provided for using the universal cell preparation in preparing a product with low immunogenicity for treating androgenic alopecia by transplantation and injection.

[0027] Furthermore, the product is a medicine.

[0028] Furthermore, the product is an injectable preparation for transplantation into a hair follicle area. Optionally, the hair follicle area is an area of ​​hair loss caused by androgen.

[0029] Furthermore, low immunogenicity means that: in flow cytometric analysis of dermal papilla cells, relative expression levels of HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DP, and HLA-DQ are less than 5%, optionally less than 2%, or optionally less than 1.5%, compared to the same type of cells without antibody binding as a control;

[0030] Optionally, the relative expression levels of HLA-A, HLA-B, HLA-C, and HLA-DQ are <0.3%, the relative expression level of HLA-DR is <1%, and the relative expression level of HLA-DP is <1.5%.

[0031] Beneficial effects

[0032] (1) The human dermal papilla cells obtained by the present invention have been identified as a low-immunogenic cell type, making them more suitable for hair regeneration using universal cells. This means that it is no longer necessary to obtain dermal papilla cells from the original body, greatly expanding the scope of application and application scenarios. The present invention combines human dermal papilla cells with physiological saline to produce a universal cell preparation that can be injected into the area of ​​hair loss and effectively treats the symptoms of androgenic alopecia.

[0033] (2) The present invention found that with the increase in culture passages, the purity of the isolated HDPCs gradually increases, the immunogenicity decreases, but the hair growth effectiveness also gradually decreases, which poses a great challenge to the preparation of HDPC cell preparations. However, the present invention uses a primary isolation method to obtain dermal papilla cells from hair follicle tissue. After culture for several generations, a cell type with low immunogenicity and good hair growth ability can be obtained at the P3 generation, which is more suitable for hair regeneration with universal cells. It is resuspended in physiological saline as an injectable preparation and can be directly injected into the area of ​​hair loss, showing low immunogenicity and good safety.

[0034] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and to implement it according to the contents of the specification, and to make the above and other purposes, technical features and advantages of the present invention easier to understand, one or more preferred embodiments are listed below and described in detail with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] One or more embodiments are exemplarily illustrated by the accompanying figures, and these exemplary illustrations do not limit the embodiments. The word "exemplary" is used herein to mean "serving as an example, example, or illustration." Any embodiment described herein as "exemplary" is not necessarily to be construed as superior or preferred over other embodiments.

[0036] Figure 1 These are the HE staining results of tissue sections injected with different cells in Test Example 2 of the present invention, wherein A is the HE staining result of HDPC injection (no tumor-like tissue); B is the HE staining result of HFSC injection (with tumor-like tissue).

[0037] Figure 2 Figure 3 shows the hair growth effect of AGA mice injected with different preparations according to Test Example 3 of the present invention, wherein Figure A shows the result of PBS injection, and Figure B shows the result of cell injection preparation injection according to Example 5.

[0038] Figure 3 These are the HE staining results of tissue sections injected with different preparations in Test Example 3 of the present invention, wherein Figure A shows the result of PBS injection; Figure B shows the result of cell injection preparation injection in Example 5.

[0039] Figure 4 The results of Test Example 4 of the present invention show the number of regenerated hair follicles after injection of different preparations. The statistical method is based on the comparison of the number of hair follicles at 40X magnification of the HE results, and the statistical analysis of the number of hair follicles in a skin section with a length of 15 cm is performed. Among them, Example 1 (normal saline); Comparative Example 1 preparation (DMEM-F12); Comparative Example 2 preparation (sterile water for injection); Comparative Example 3 preparation (the stem cell preparation in CN 113332313 A).

[0040] Figure 5 The following are comparison results of hair phenotype 10 days after dermal papilla cell injection. Figure A represents HDPC-P5; Figure B represents HDPC-P7; and Figure C represents HDPC-P9. DETAILED DESCRIPTION

[0041] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are 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 making creative work are within the scope of protection of the present invention. Unless otherwise expressly stated, throughout the specification and claims, the term "including" or its variations such as "comprising" or "including" will be understood to include the stated elements or components, without excluding other elements or other components.

[0042] In addition, in order to better illustrate the present invention, numerous specific details are provided in the following detailed description. It should be understood by those skilled in the art that the present invention can be practiced without certain specific details. In some embodiments, raw materials, components, methods, means, etc. that are well known to those skilled in the art are not described in detail in order to highlight the main purpose of the present invention.

[0043] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0044] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0045] In the following examples, 1% (V / V) double-antibody DDPBS / PBS solution was prepared as follows: penicillin-streptomycin (Gibico, 15140122) was added to 1×DPBS / PBS (Gibico, 8121293) at a volume ratio of penicillin-streptomycin:DPBS / PBS=1 mL:99 mL.

[0046] In the following examples, DPBS / PBS was 1×DPBS / PBS buffer (Gibico, 8121293).

[0047] The storage solution in the following examples was lactated Ringer's solution, stored at 4° C., purchased from Shandong Jieshikang, T0447.

[0048] Example 1 Preparation of universal cells:

[0049] 1) Freshly collected human hair samples were rinsed with lactated Ringer's solution and F12 culture medium (containing 1% by volume of double-streptomycin), and skin tissue samples from the back of the head were removed;

[0050] 2) The in vitro human hair sample treated in 1) was immersed in a storage solution at 4° C. and then transported in a constant temperature transport box (temperature controlled at 0-15° C.).

[0051] 3) Remove the hair follicle tissue sample from the storage solution and rinse it with 75% ethanol solution three times;

[0052] 4) Washing the isolated human hair sample treated in step 1) three times in DPBS / PBS containing 2% double antibody;

[0053] 5) Excised human hair follicle tissue was washed three times with 1% double-antibody DDPBS / PBS (Gibico, 8121293) and then placed under a stereoscope (Leica-SE9). Under a microscope (40X magnification), the hair shaft was trimmed, and the connective tissue and fat were removed. The hair follicle tissue was then washed three times in a standard DPBS / PBS solution (Gibico, 8121293) to obtain the hair follicle tissue after hair shaft removal.

[0054] 6) Finally, the hair follicle tissue after hair shaft removal was placed in DMEM (Gibico, M200PRF500) culture medium containing 5% FBS and cultured in a 37° C., 5% CO 2 incubator.

[0055] 7) Change the medium every 7 days and observe the morphology and growth of the cells under an inverted microscope until the cell confluence reaches 70-80%, which is the P0 generation of dermal papilla cells;

[0056] 8) Continue culturing P0 dermal papilla cells. When the cell confluency reaches 80-90%, perform cell subculture: discard the old culture medium from the culture flask, add 10 ml of normal saline and wash twice; discard the normal saline, add 2 ml of cell digestion solution, and place in an incubator to digest for 1 minute; add 10 ml of cell subculture medium to terminate the digestion, and collect the cells into a centrifuge tube;

[0057] 9) After centrifugation at 250 g / min for 5 min, the culture medium was discarded and 10 ml of cell culture medium was added to resuspend the cells. 4 cells / cm 2 Subculture was performed to obtain P1 dermal papilla cells; the subculture medium used for papilla cells was DMEM / F12 (Gibico, 2186796) containing 10% FBS.

[0058] 10) Continue subculturing according to the methods of 8) and 9) to culture to P3. The subculture medium used for dermal papilla cells is DMEM / F12 (Gibico, 2186796) containing 10% FBS;

[0059] 11) The collected P3 dermal papilla cells were washed three times with physiological saline, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in 1 mL of physiological saline to obtain a universal cell hair regeneration injection solution with a preparation concentration of 1×10 6 / mL.

[0060] Example 2

[0061] The difference from step 11) of Example 1 is that 2×10 P3 dermal papilla cells are taken. 6The cells were added to 1 mL of normal saline to prepare a universal cell injection preparation for the treatment of androgenic alopecia. The concentration of the preparation was 2×10 6 / mL.

[0062] Example 3

[0063] The difference from step 11) of Example 1 is that 3×10 6 The cells were added to 1 mL of normal saline to prepare a universal cell injection preparation for the treatment of androgenic alopecia. The concentration of the preparation was 3×10 6 / mL.

[0064] Example 4

[0065] The difference from step 11) of Example 1 is that 4×10 6 The cells were added to 1 mL of normal saline to prepare a universal cell injection preparation for the treatment of androgenic alopecia. The concentration of the preparation was 4×10 6 / mL.

[0066] Example 5

[0067] The difference from step 11) of Example 1 is that 5×10 P3 dermal papilla cells are taken. 6 The cells were added to 1 mL of normal saline to prepare a universal cell injection preparation for the treatment of androgenic alopecia. The concentration of the preparation was 5×10 6 / mL.

[0068] Example 6

[0069] The difference from step 11) of Example 1 is that 6×10 P3 dermal papilla cells are taken. 6 The cells were added to 1 mL of normal saline to prepare a universal cell injection preparation for the treatment of androgenic alopecia. The concentration of the preparation was 6×10 6 / mL.

[0070] Example 7

[0071] The difference from step 11) of Example 1 is that 7×10 6 The cells were added to 1 mL of normal saline to prepare a universal cell injection preparation for the treatment of androgenic alopecia. The concentration of the preparation was 7×10 6 / mL.

[0072] Example 8

[0073] The difference from step 11) of Example 1 is that 8×10 6 The cells were added to 1 mL of normal saline to prepare a universal cell injection preparation for the treatment of androgenic alopecia. The concentration of the preparation was 8×10 6 / mL.

[0074] Example 9

[0075] The difference from step 11) of Example 1 is that 9×10 6 The cells were added to 1 mL of normal saline to prepare a universal cell injection preparation for the treatment of androgenic alopecia. The concentration of the preparation was 9×10 6 / mL.

[0076] Example 10

[0077] The difference from step 11) of Example 1 is that 10×10 6 The cells were added to 1 mL of normal saline to prepare a universal cell injection preparation for the treatment of androgenic alopecia. The concentration of the preparation was 10×10 6 / mL.

[0078] Comparative Example 1

[0079] The difference from step 11) of Example 1 is that the physiological saline is replaced with an equal amount of culture medium DMEM-F12 (Gibico, 2186796).

[0080] Comparative Example 2

[0081] The difference from step 11) of Example 1 is that the normal saline is replaced with an equal amount of sterile water for injection (purchased from Sichuan Kelun Pharmaceutical, national medicine standard H20044405).

[0082] Comparative Example 3

[0083] The difference from step 11) of Example 1 is that the physiological saline is replaced with an equal amount of cell preparation (patent CN106619505A); the ratio is 100 mg of type IV collagen and 100 mg of chondroitin sulfate dissolved in 100 ml of distilled water as a cell solvent, and finally the injection preparation is prepared.

[0084] Test Example 1 Immunogenicity Analysis of Dermal Papilla Cells

[0085] In the 20th century, scientists discovered that tissue rejection can occur during tissue transplants between individuals of different species or within different lines of the same species. This rejection is essentially an immune response induced by alloantigens expressed on the cell surface. These alloantigens, representing individual specificity, are called "transplant antigens" or "histocompatibility antigens." Among these, those that can trigger strong and rapid rejection are called "major histocompatibility antigens," and in humans, they are called human leukocyte antigens (HLA).

[0086] Major histocompatibility antigens (MHCs) are a complex antigen system. The genes encoding this system are located on the same chromosomal segment, forming a tightly linked group of genes known as the major histocompatibility complex (MHC). During allogeneic tissue transplantation, if the donor and recipient have different transplant antigens, particularly if the MHC mismatch occurs, significant transplant rejection will occur in the recipient. MHC molecules are divided into class I and class II. Class I MHC molecules are distributed in all nucleated cells, while class II MHC molecules are primarily expressed in B cells, monocytes / macrophages, and dendritic cells. In humans, the major histocompatibility antigens encoded by MHC class I genes are HLA class I molecules, primarily including HLA-A, HLA-B, and HLA-C. Class II MHC genes encode HLA class II molecules, primarily including HLA-DR, HLA-DP, and HLA-DQ.

[0087] The main component of the dermal papilla cell injection preparation of the present invention was subjected to flow cytometry analysis to compare the expression levels of HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DP, and HLA-DQ to analyze the immunogenicity of the dermal papilla cells. The flow cytometry operation steps are as follows:

[0088] 1. Cell resuspension: After cell processing and counting, resuspend the cells in cell washing solution (PBS containing 2% BSA) to a cell concentration of 1×10 7 / mL;

[0089] 2. Control settings: Take 100uL of the above resuspended cells from each tube of blank control, isotype control, and test sample to make the cell count in each tube reach 1×106 / tube;

[0090] 3. Primary antibody incubation: No antibody is added to the blank control tube. 5-20uL of isotype antibody and target antibody are added to the isotype control tube and the sample tube to be tested, respectively (refer to the instructions for specific usage amounts). Mix thoroughly and incubate at 4°C for 30 minutes, or at room temperature for 30 minutes in winter. During the incubation period, shake the reaction tube every 10 minutes to allow the cells and antibodies to react fully.

[0091] 4. Cell washing: add appropriate amount of cell washing solution, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and repeat washing twice;

[0092] 5. Cell resuspension: Resuspend cells in 100 μL cell wash buffer;

[0093] 6. Secondary antibody incubation: Add appropriate amount of fluorescently labeled secondary antibody to the isotype control tube and the test tube respectively, mix thoroughly, and incubate at 4°C in the dark for 30 minutes or at room temperature in the dark for 30 minutes in winter. Shake the reaction tube every 10 minutes during the incubation period.

[0094] 7. Cell washing: add appropriate amount of cell washing solution, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and repeat washing twice;

[0095] 8. On-Cell Assay: Resuspend cells in 100-200 μL of 1× DPBS / PBS buffer (Gibico, 8121293). Measure the blank tube and isotype control tube first, followed by the tube to be assayed. Perform multiple batches of each cell generation to minimize variation. Compare expression levels of HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DP, and HLA-DQ. Non-antibody-bound isotype cells were used as controls. Results are shown in Table 1.

[0096] The results in Table 1 show that the expression levels of HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DP, and HLA-DQ in different batches of P3 generation dermal papilla cells of Example 1 are extremely low, almost absent, indicating that the P3 generation dermal papilla cells of Example 1 have low immunogenicity.

[0097] Table 1 Flow cytometry analysis results of P3 dermal papilla cells in Example 1

[0098]

[0099] Note: In Table 1, HDPC-1, HDPC-2, and HDPC-3 represent the test results of three batches of P3 HDPC cells. HFSC stands for hair follicle stem cells. The percentage represents the percentage of cells positive for the corresponding antibody (e.g., the percentage of cells positive for HLA-DR antibodies). A percentage less than 2% indicates minimal expression. A higher percentage indicates a higher probability of protein expression.

[0100] In order to further study the effect of culture passages on cell immunogenicity, dermal papilla cells of different passages were taken and the immunogenicity of dermal papilla cells of each passage from P1 to P10 was analyzed and detected. The results are shown in Table 2.

[0101] The results in Table 2 show that the purity of primary isolated HDPC cells is low, and they are mixed with many other miscells, such as hair follicle stem cells, fibroblasts and other cell types. Their immunogenicity analysis does not meet the requirements. As the culture generation increases to P10, the immunogenicity of HDPCs detected will gradually decrease, and meet the immunogenicity requirements by P3.

[0102] Table 2 Flow cytometry analysis results of dermal papilla cells from P1 to P10

[0103]

[0104] Test Example 2 Safety Analysis of Cell Injection Preparations

[0105] 1. Collect dermal papilla cells in the logarithmic growth phase, preferably with a cell density of about 80-90%. Replace the culture medium with fresh one the night before collecting the cells.

[0106] 2. After trypsin digestion, wash the cells twice with pre-cooled PBS to remove the serum from the cells.

[0107] 3. Use PBS or serum-free medium to blow the cell pellet to the appropriate concentration. Generally, the amount of cells inoculated into subcutaneous tumors is 1-5×10 6 cells / tube, the inoculation volume is 0.1 ml, so the concentration of the cell suspension is 1-5×10 6 cells / ml.

[0108] 4. After cell digestion, the cells should be inoculated subcutaneously into nude mice as soon as possible, generally within half an hour. During the process, the cell suspension should be placed on ice to reduce cell metabolism.

[0109] 5. Selected nude mice (purchased from Vital River, Nu / Nu-403) were 5 weeks old and weighed about 18-20 g;

[0110] 6. Before inoculation, use a gun to fully disperse the cell suspension to prevent cells from clumping and reducing cell survival rate.

[0111] 7. During vaccination, the needle should be inserted deeper under the skin, about 1 cm deep, to reduce the overflow of cell suspension from the needle eye after injection.

[0112] The hair papilla cell preparation (P3 generation) of Example 5 (5×10 6 The cell amount / mL (HDPC)) and hair follicle stem cells (HFSC) (Shanghai Xuanya Biotechnology, XY221109) were injected into the skin of nude mice according to the above method, and the cells were observed for 6 months, and the tissue sections were analyzed by HE.

[0113] Through observation, the dermal papilla cell preparation of Example 5 did not show tumorigenicity and the phenotype did not change; HE analysis of tissue sections showed that Figure 1 The results showed that no tumor cells appeared in the hair papilla cell preparation of Example 5, while obvious tumor-like tissue appeared in the positive control (HFSC cells).

[0114] Test Example 3 Establishment and Analysis of Pharmacological Efficacy Experimental Model

[0115] Establishment of AGA mouse model: Purchase four-week-old C57 mice (Vitamin B, C57BL-213) and adapt them to feeding for one week. Then, intraperitoneally inject 1 mg / kg / d of DHT (dissolved in corn oil). DHT injection is performed once a day. After 4 days of injection, the mice are anesthetized with 4% chloral hydrate. The back skin is selected with its long side parallel to the depilatory area of ​​the spine. The hair of the depilatory area on the back of the mouse is trimmed short with a shaver. Then, depilatory cream is evenly applied to the depilatory area according to the instructions. It is confirmed that the hair on the depilatory area on the back of the mouse is in the resting phase. As D0, DHT injection is continued for 60 days. The mice with depilated back hair have not grown hair. The effect is as follows. Figure 2 As shown in -A, when there is no hair protruding from the back, the establishment of the AGA model mouse is completed.

[0116] The AGA mice after modeling were divided into two groups, 3 mice in each group, and the following operations were performed:

[0117] Control group: any group was randomly selected and continuously injected with 1 mg / kg / d of DHT (dissolved in corn oil) intraperitoneally once a day; 50 μL of normal saline was injected intradermally;

[0118] Experimental group: any group was selected and continuously injected with 1 mg / kg / d of DHT (dissolved in corn oil) intraperitoneally, and DHT injection was once a day; 50uL of cell injection preparation was injected intradermally.

[0119] The changes in hair count of each group of AGA mice before and after using the cell preparation were observed. Figure 2 As shown: Figure 2 The results showed that 10 days after injection of the cell injection preparation of Example 5 of the present invention into AGA mice, hair regenerated in the hair loss areas, with a significant increase in hair quantity. Ten days after treatment with the cell injection preparation, new hair had grown in the hair loss areas of the AGA mice, and the texture of the newly grown hair was indistinguishable from that of healthy mice. In the control group, no significant changes were observed 10 days after injection, with no new hair growing in the hair loss areas.

[0120] The hair follicle structure in the skin tissue of the two groups of AGA mice 10 days after treatment was analyzed by pathological HE staining. Figure 3 As shown, the results showed that: it was found that the skin tissue of the untreated AGA hair loss mice in the control group had no complete hair follicles, and their hair follicle structure had completely atrophied, losing the ability to continue to provide nutrients to the hair; while the AGA hair loss mice after being treated with the cell injection preparation of Example 5 had intact hair follicle structure in their skin tissue, and had all the conditions for hair regeneration.

[0121] Therefore, after injection treatment with the dermal papilla cell preparation of the present invention, the hair follicle structure can be improved, enabling the mice to have the ability to regenerate hair, and the regeneration ability is no different from that of the hair of normal healthy mice.

[0122] In order to further study the effect of culture passages on hair growth, dermal papilla cell preparations of different passages (cell concentration of 5×10 6 / mL) were used to treat AGA mice. The results showed that the preparations made from P3 to P7 dermal papilla cells can promote hair regeneration in AGA mice. Among them, the effect was better in P3 to P6 generations, while hair regeneration decreased significantly in P7 generations and almost lost in P8 to P10 generations. Figure 5 As shown, Figure A represents HDPC-P5; Figure B represents HDPC-P7; and Figure C represents HDPC-P9.

[0123] The P3 generation dermal papilla cell preparations of Examples 1 to 10 were used to treat AGA mice according to the above method. The results showed that the P3 generation dermal papilla cell preparations of Examples 1 to 10 (concentrations of 1 to 10 × 10 6 / mL) can effectively promote hair regeneration.

[0124] Further increase the concentration of P3 dermal papilla cells to 2-10×10 7 / mL, and can also effectively promote hair regeneration.

[0125] The results show that the dermal papilla cell preparation prepared by the present invention has a good effect in different cell concentration ranges (1×10 6 ~1×10 8 / mL), the therapeutic effect on hair growth is very obvious. However, too high a cell concentration ratio will cause the cells to aggregate at the injection site, making them difficult to be absorbed and affecting the hair growth effect.

[0126] Combined with Test Example 1, although the purity of HDPC cells can be improved and the immunogenicity of HDPC cells can be reduced with the increase of culture generations, the hair growth ability will gradually be lost. Therefore, after continuous research and analysis, it is believed that primary HDPC cells separated by a specific method can obtain low-immunogenic, transplantable, and hair growth-capable dermal papilla cells at earlier culture generations. For example, the dermal papilla cells obtained at the P3 generation have low immunogenicity and good hair growth effect.

[0127] Test Example 4 Effect of Preparation Composition on Hair Growth Effect

[0128] The cells of Example 1 and Comparative Examples 1 to 3 were used to treat AGA mice according to the method of Example 3. The results are as follows: Figure 4 , the results show that:

[0129] After injection of the preparation of Comparative Example 1 (DMEM-F12 as the resuspension solvent), although AGA mice had hair growth, the phenotypic observation of hair growth was significantly reduced compared with the phenotype of Example 1 (injection of normal saline as the resuspension solvent). It is considered that some additives in the culture medium may affect the in vivo environment and no effective hair growth effect is achieved.

[0130] After injection of the formulation in Comparative Example 2 (sterile water for injection as the resuspension solvent), AGA mice showed some hair growth, but the growth phenotype was lower compared to the phenotype in Example 1 (normal saline for injection as the resuspension solvent). Sterile water for injection is water, a sterile, injectable preparation made through a special process. Normal saline specifically refers to 0.9% sodium chloride injection. Although both are commonly used liquid preparations in clinical practice, the concentration and osmotic pressure of normal saline are similar to those of plasma. Therefore, after infusion, it does not cause changes in osmotic pressure, allowing cells to maintain their original morphology, resulting in a better growth phenotype.

[0131] After injection of the preparation of Comparative Example 3, AGA mice had hair growing out, but the growth phenotype was lower than that of Example 1 (injection of physiological saline as the resuspension solvent). It is possible that certain components in the reagent affected the effect of hair follicle regeneration.

[0132] Therefore, the best effect is to use normal saline to prepare HPDC hair growth solution.

[0133] In summary, the present study found that with increasing culture passages, the purity of isolated HDPCs gradually increases, while their immunogenicity decreases, but their hair growth effectiveness also gradually decreases. This poses a significant challenge to the preparation of HDPC cell preparations. However, the present invention utilizes primary isolation to obtain dermal papilla cells from hair follicle tissue. After culture for several passages, a cell type with low immunogenicity and excellent hair growth ability can be obtained at the P3 passage, making it more suitable for universal hair regeneration. These cells can be resuspended in physiological saline as an injectable preparation, which can be injected directly into the area of ​​hair loss, demonstrating low immunogenicity and good safety.

[0134] The foregoing descriptions of specific exemplary embodiments of the present invention are for purposes of illustration and description. These descriptions are not intended to limit the invention to the precise form disclosed, and it is apparent that many changes and variations are possible in light of the foregoing teachings. The exemplary embodiments are selected and described for the purpose of explaining the specific principles of the invention and their practical application, thereby enabling those skilled in the art to realize and utilize a variety of exemplary embodiments of the invention and various options and variations. Any simple modifications, equivalent variations, and modifications made to the exemplary embodiments described above are intended to fall within the scope of protection of the present invention.

Claims

1. A universal cell preparation for treating androgenic alopecia, characterized in that: The hair papilla cells and physiological saline are included, wherein the concentration of the hair papilla cells is 10 5 ~10 9 cells / mL. In flow cytometric analysis, the relative expression levels of HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DP, and HLA-DQ in dermal papilla cells were less than 5%, using the same type of cells without antibody binding as a control.

2. The universal cell preparation according to claim 1, characterized in that The relative expression levels of HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DP, and HLA-DQ are <2%, optionally <1.5%; Optionally, the relative expression levels of HLA-A, HLA-B, HLA-C, and HLA-DQ are <0.3%, the relative expression level of HLA-DR is <1%, and the relative expression level of HLA-DP is <1.5%.

3. The universal cell preparation according to claim 1 or 2, characterized in that: The dermal papilla cells are primary cells separated from hair follicles and subcultured for 3 to 7 generations and have the ability to germinate, and optionally are cells for 3 to 5 generations and have the ability to germinate; And / or, the concentration of the hair papilla cells is 10 6 ~10 8 cells / mL.

4. The universal cell preparation according to any one of claims 1 to 3, characterized in that The method for isolating hair papilla cells comprises: 1) Isolating hair follicle tissue from ex vivo human hair samples; 2) pre-treating the hair follicle tissue to remove connective adhesion tissue and fat; 3) The pretreated hair follicle tissue obtained in step 2) is cultured using a hair papilla cell selection medium to obtain primary hair papilla cells, which are then subcultured to obtain hair papilla cells of passage 3 to 7.

5. The universal cell preparation according to claim 4, characterized in that In step 2), the pretreatment method further comprises: removing the hair bulb tissue and part of the hair shaft tissue in the hair follicle tissue, retaining the middle section as the hair follicle middle section tissue, and optionally, the length of the hair follicle middle section tissue is 0.5-2.5 mm; Optionally, the method of removing tissue is performed under a stereoscope.

6. The universal cell preparation according to claim 4 or 5, characterized in that: In step 1), the hair follicle tissue is a follicular unit tissue and / or a single hair follicle tissue; And / or, in step 2), the hair follicle tissue is a single hair follicle tissue, optionally, the single hair follicle tissue is separated from a hair follicle unit.

7. The universal cell preparation according to any one of claims 4 to 6, characterized in that: In step 3), the hair papilla cells are isolated by culturing the pretreated hair follicle tissue in a DMEM medium containing 2.5%-12.5% ​​FBS, and the hair papilla cells are isolated after the hair papilla cells crawl out; optionally, the hair papilla cells are isolated by using a DMEM medium containing 2.5%-6.5% FBS; optionally, the hair papilla cells are isolated by using a DMEM medium containing 2.5%-5% FBS; And / or, the subculture adopts a DMEM / F12 medium containing 7.5% to 17.5% FBS, and optionally the subculture adopts a DMEM / F12 medium containing 10% to 17.5% FBS.

8. Use of the universal cell preparation according to any one of claims 1 to 7 in the preparation of a product with low immunogenicity for the treatment of androgenic alopecia by transplantation and injection.

9. The use according to claim 8, characterized in that The product is a pharmaceutical; And / or, the product is an injectable preparation for transplantation into a hair follicle area, optionally, the hair follicle area is an area suffering from hair loss due to androgen.

10. The use according to claim 8 or 9, characterized in that: Low immunogenicity means that: in flow cytometric analysis of dermal papilla cells, the relative expression levels of HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DP, and HLA-DQ are less than 5%, optionally less than 2%, and optionally less than 1.5%, compared with the same type of cells without antibody binding; Optionally, the relative expression levels of HLA-A, HLA-B, HLA-C, and HLA-DQ are <0.3%, the relative expression level of HLA-DR is <1%, and the relative expression level of HLA-DP is <1.5%.

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