Method for large-scale amplification of clinical-grade hair follicle stem cells and application of clinical-grade hair follicle stem cells in preparation of products for treating alopecia

Abstract

The invention belongs to the technical field of cell biology, and particularly relates to a method for amplifying clinical-grade hair follicle stem cells on a large scale and application of the clinical-grade hair follicle stem cells in preparation of products for treating alopecia. According to the method provided by the invention, stable amplification of the hair follicle stem cells can be realized by accurately controlling passage times, and reagents used in the culture process do not contain animal sources, so that the possibility of animal source pathogen pollution does not exist. The clinical-grade hair follicle stem cells amplified in a large scale by the method provided by the invention can provide high-quality cell resources for alopecia patients, can be used for hair follicle regeneration treatment, and can be transplanted to an alopecia area to stimulate hair follicle regeneration so as to realize regeneration and growth of hair.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of cell biology, and particularly relates to a method for large-scale expansion of clinical-grade hair follicle stem cells and application thereof in preparation of a product for treating alopecia. BACKGROUND

[0002] With the improvement of living standards and the accelerated development of society, alopecia has gradually become an unignorable disease. Alopecia not only is a physiological manifestation, but also can affect the psychology of patients, who may lack confidence, feel inferior, and even be depressed, and it also has certain impact on the work and family of patients. The total number of alopecia sufferers in China is as high as 250 million, of which 130 million are males. The proportion of alopecia before the age of 30 is as high as 84%, and the problem of alopecia is gradually becoming younger. The causes of alopecia are not fully understood, and may be related to immunity, genetics, hormones, local inflammatory reaction, nerves and environmental factors, etc. Different causes lead to different types of alopecia, and there are many classification methods. Common types of alopecia include androgenetic alopecia (AGA), alopecia areata, trichotillomania, telogen effluvium, and senile alopecia, etc.

[0003] Traditional treatment of alopecia often uses hair transplantation surgery, but hair transplantation surgery is considered to be invasive, and can only redistribute hair, but cannot increase hair thickness. In addition to hair transplantation surgery, there are also treatments such as subcutaneous injection of platelet-rich plasma (PRP), use of finasteride or minoxidil tincture, traditional Chinese medicine therapy, and stem cell therapy. These treatments have achieved certain effects, but also have some negative effects. For example, finasteride, commonly used to treat seborrheic alopecia, can cause abnormal sexual function in patients; although minoxidil tincture can effectively accelerate hair regeneration, it can cause symptoms such as dermatitis and headache after stopping the drug; traditional Chinese medicine therapy is mainly used for prevention and slowing down of alopecia; stem cell therapy can significantly promote hair follicle cell proliferation, but mesenchymal stem cells derived from umbilical cords are often used, and such cells, being allogeneic stem cells, can only activate hair follicles by secreting exosomes to grow hair in the subcutaneous tissue, and cannot differentiate into cells in the hair follicle and repair the hair follicle. The treatment of alopecia areata and alopecia often has difficulties, because patients can develop resistance to the currently available treatment methods, and the use of hair follicle stem cells can be a new option for drug-resistant cases.

[0004] Adult hair follicles are divided into a constant upper part (this part generally does not undergo apoptosis and regeneration) and a lower part that is constantly remodeled during the hair cycle (this part undergoes periodic growth, generally experiencing three stages of growth, regression, and resting (such as Figure 1As shown in Figure 2), it provides the basic conditions for hair follicles to regenerate and repair themselves after being damaged (Fuchs E. Cell biology: More than skin deep[J]. J Cell Biol, 2015, 209(5): 629-631.). Studies have shown that the maintenance of the hair follicle cycle depends largely on the stem cell population in the hair follicle that can produce different epithelial components. As the precursor cells of skin-derived cells, hair follicle stem cells have the same multidirectional differentiation potential as other adult stem cells (Zhang H, Zhang S, Zhao H, et al. Ovine hair follicle stem cells derived from single vibrissae reconstitute haired skin[J]. Int J Mol Sci, 2015, 16(8): 17779-17797.). When the superficial dermis is damaged and the hair follicles are destroyed, the activated hair follicle stem cells in the bulge will proliferate using some surviving hair follicles as templates to achieve hair follicle regeneration (Su YS, Miao Y, Jiang JD, et al. A simple and rapid model for hair-follicle regeneration in the nude mouse [J]. Clin Exp Dermatol, 2015, 40(6): 653-658.). Sun Xijin et al. (Sun Xijin, Hu Zhiqi, Miao Yong. Experimental study on injection and transplantation of hair follicle cells in mice [J]. Chinese Journal of Plastic Surgery, 2012, (01): 44-49.) injected hair follicle stem cells and other types of cells into the skin of mice. New hair follicles were formed in the transplant group containing hair follicle stem cells, while no hair follicles were formed in the transplant group containing other types of cells, which proved that hair follicle stem cells have a good ability to differentiate and proliferate into hair follicles. Li et al. (Li G, Tang X, Zhang S, et al. SIRT7 activates quiescent hair follicle stem cells to ensure hair growth in mice[J]. EMBO J, 2020, 39(18): e104365.) found that upregulating SIRT7 to activate quiescent hair follicle stem cells can promote hair growth in mice.

[0005] Existing hair follicle stem cell culture technologies all have their limitations. For example, the method disclosed in patent CN112342186B requires extracting hair follicles from the donor's occipital skin and isolating the follicles within the skin. This method has the advantage of surviving intact hair follicles, but the disadvantage is that it is highly harmful to the donor. The cell culture process requires the use of fetal bovine serum (FBS) and trypsin, both of which are animal-derived. Although reagents from legitimate sources undergo rigorous inspection, they may still contain pathogens such as bovine parvovirus or Streptococcus suis type II. Similarly, the culture method described in patent CN113265374A faces similar issues. Summary of the Invention

[0006] The purpose of the present invention is to provide a method for large-scale expansion of clinical-grade hair follicle stem cells. The damage to the donor during hair follicle extraction is very minimal, the wound is almost invisible after surgery, healing is rapid, the cells proliferate rapidly, and all reagents during the culture process are free of animal origin, eliminating the possibility of contamination by animal pathogens.

[0007] The present invention provides a method for large-scale expansion of clinical-grade hair follicle stem cells, comprising the following steps: The hair follicles were extracted and washed, and then digested with a Liberase TL solution at a concentration of 150-300 μg / mL at 37°C for 1-2 hours, and then the undigested part was discarded. After centrifugation, the cells were resuspended with complete culture medium and inoculated into a T25 flask for culture, and then passaged; the passages included the first passage, the second passage, the third passage and the fourth passage; after the fourth passage, the stem cells were harvested and frozen; at the first passage, the cells were inoculated into a T175 culture flask; at the second passage, the cells were inoculated into a 5-layer cell factory; at the third passage, the cells were inoculated into a 6-layer cell factory; at the fourth passage, the cells were inoculated into a 10-layer cell factory; the complete culture medium contained 1% v / v-5% v / v human platelet lysate, 5-10 μg / mL recombinant humanized type III collagen, 10-25 μg / mL recombinant human insulin, 1-5 ng / mL recombinant human TGF-β2 in serum-free stem cell culture medium.

[0008] Preferably, the collection container used for collecting the hair follicles contains 20-30 mL of buffer solution, which is a mixture of penicillin-streptomycin-amphotericin B solution and 0.9% normal saline, and the volume ratio of the 0.9% normal saline to the penicillin-streptomycin-amphotericin B solution is 100:1-2.

[0009] Preferably, the hair follicles are stored and transported at 2°C to 8°C.

[0010] Preferably, the washing comprises the following steps: washing the hair follicle 1-2 times with a buffer solution, the buffer solution being a mixture of a penicillin streptomycin amphotericin B solution and 0.9% normal saline, the volume ratio of the 0.9% normal saline to the penicillin streptomycin amphotericin B solution being 100:1-2; and the centrifugation being performed at 300-500g for 5-10min.

[0011] Preferably, at the first passage, the inoculation density is 1×10 6 ~1.6×10 6 The cells are inoculated into T175 culture bottles, each bottle containing 20-30mL complete culture solution; at the second passage, the inoculation density is 1.8×10 7 ~3.0×10 7 The cells are inoculated into 5-layer cell factories, each bottle containing 300-450mL complete culture solution; at the third passage, the inoculation density is 2.4×10 7 ~4.5×10 7 The cells are inoculated into 6-layer cell factories, each bottle containing 400-500mL complete culture solution; at the fourth passage, the inoculation density is 5.0×10 7 ~7.0×10 7 The cells are inoculated into 10-layer cell factories, each bottle containing 700-900mL complete culture solution.

[0012] Preferably, the culture is performed at 37±1℃, 5% CO2.

[0013] Preferably, the passage comprises the following steps: adding 5-10mL TrypLE to the cells, adding an equal volume of complete culture solution to terminate the digestion after the cells become shriveled and rounded, centrifuging the cell suspension, resuspending the cells with complete culture solution, and then inoculating.

[0014] Preferably, the density of the frozen cells is 1.0×10 7 ~2.0×10 7 cells / mL.

[0015] Preferably, the freezing comprises the following steps: cooling at a rate of 1-5℃ / min for 4-16h, and then transferring the cells to a gas-phase liquid nitrogen tank for long-term storage in an environment with a temperature of-100℃-150℃.

[0016] The application also provides the use of the method described in the above technical solution in the preparation of a product for treating alopecia.

[0017] The application has the following beneficial effects: The method provided by the present invention can efficiently isolate hair follicle stem cells, reduce the interference of impurities and non-target cells, thereby improving the purity and activity of stem cells and reducing the damage to cells during the isolation process, providing a high-quality cell population for subsequent culture and expansion.

[0018] The complete culture medium provided by the present invention does not contain animal-derived products, eliminating the risk of contamination by xenoviruses, prions and unknown animal proteins; the combined use of humanized type III collagen and recombinant human insulin is beneficial to further promote cell adhesion and proliferation; recombinant human TGF-β2 can accurately inhibit the early differentiation of stem cells, so that the cells still have trilineage potential after 5 generations of culture.

[0019] By controlling the number of passages and gradually expanding the culture scale, the present invention ensures that cells are in optimal growth at each stage, avoiding overcrowding or nutrient deficiency, thereby achieving efficient expansion. Furthermore, it can reduce cell damage during operation, further improving cell survival rate and expansion efficiency.

[0020] The present invention harvests stem cells after the fourth passage, when the cells are in their optimal growth state, with high activity and differentiation potential. This ensures that the harvested stem cells are sufficient in quantity and reliable in quality, meeting the stringent quality and quantity requirements for clinical applications. The present invention optimizes the cryopreservation process, enabling long-term preservation of cell activity and function, allowing for immediate recovery for clinical treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below.

[0022] Figure 1 Schematic diagram of the hair follicle stem cell growth cycle; Figure 2 These are primary seeded hair follicle stem cells; Figure 3 Before passaging hair follicle stem cells; Figure 4 This is the result of flow cytometry detection of hair follicle stem cells; Figure 5 The results of the soft agar colony formation rate test of human hair follicle stem cells from generations P1 to P3; Figure 6 The results of the soft agar colony formation rate test of P3 human hair follicle stem cells and P4 human hair follicle stem cells after cryopreservation and resuscitation; Figure 7 These are the results of the three-lineage differentiation test of hair follicle stem cells. DETAILED DESCRIPTION

[0023] In order to further illustrate the present invention, the solutions provided by the present invention are described in detail below with reference to the accompanying drawings and embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0024] The complete culture medium used in the examples of the present invention is a serum-free stem cell culture medium containing 3% v / v human platelet lysate, 10 μg / mL recombinant humanized type III collagen, 15 μg / mL recombinant human insulin, and 5 ng / mL recombinant human TGF-β2.

[0025] The present invention has no particular limitation on the source of each reagent, and any conventional commercially available product can be used.

[0026] Example 1 Instruments and equipment: FUE hair follicle extractor, biological safety cabinet, centrifuge, constant temperature water bath, medical refrigerator, ultra-low temperature refrigerator, gas phase liquid nitrogen tank, medical transport box, liquid nitrogen transport vehicle.

[0027] Reagents: lidocaine injection, 75% alcohol, iodine tincture, 0.9% normal saline, Liberase TL, serum-free stem cell culture medium, human platelet lysate, recombinant humanized type III collagen, recombinant human TGF-β2, TrypLE, penicillin-streptomycin-amphotericin B solution.

[0028] Surgical instruments: 16cm curved scissors, 14cm forceps, disposable ophthalmic forceps.

[0029] Consumables: 15mL centrifuge tubes, 50mL centrifuge tubes, 10cm dishes, 5mL pipettes, 10mL pipettes, T25 flasks, T175 flasks, 5-10 layer cell factories.

[0030] Steps: a. Prepare a hair follicle collection container: In a biosafety cabinet, open a 50 mL centrifuge tube and add 20-30 mL of 0.9% saline solution. Add 1-2% penicillin-streptomycin-amphotericin B solution to the tube. Mix thoroughly, tighten the tube cap, seal with parafilm, apply a collection label, and store at 4°C.

[0031] b. Hair follicle collection: The hair follicle collection process is performed in the operating room. Under local anesthesia, the donor lies prone on the operating table. The hair at the back of the head is removed using 16cm curved scissors. After disinfecting the epidermis, the follicles are extracted using a Fue hair follicle extractor. An assistant places the extracted follicles into a collection container, ensuring they are immersed in the collection solution. A total of 10 to 30 follicles are collected.

[0032] c. Place the sample in a medical transport box and transport it to the laboratory as soon as possible. The sample transportation temperature should be 2~8℃.

[0033] d. Sample preparation: Wash the hair follicles 1-2 times with saline containing 1%-2% penicillin-streptomycin-amphotericin B solution. Digest the follicles with Liberase TL solution at a final concentration of 150-300µg / mL at 37°C for 1-2 hours. After digestion, discard the undigested portion and dilute the remaining cell suspension to 40-50mL with 0.9% saline. Centrifuge at 300-500g for 5-10 minutes. After centrifugation, the supernatant is sterile (soybean plate) for examination. Resuspend the cells in complete culture medium and inoculate 1-3 T25 flasks. Incubate at 37°C with 5% CO2. After 5 days of culture, the cells are as follows: Figure 2 After 11 days of culture, the first subculture can be performed. The cell status before subculture is as shown in Figure 3 shown.

[0034] e. Cell Passaging: Perform a total of four cell passages according to the procedure described in this step. The only differences between each passage are the inoculation container and inoculation density, as shown in Table 1. The quality standards for cell passage are shown in Table 2. Cells were passaged as follows: When the cell confluence reached 85%-95%, the cells were passaged: the culture medium was discarded, the bottom of the flask was washed 1-2 times with 0.9% saline, the 0.9% saline was discarded, and 5-10 mL of TrypLE was added. After the cells shrank and rounded, an equal volume of complete culture medium was added to terminate the digestion. The cell suspension was centrifuged at 300-500 g for 5-10 minutes and resuspended in 10-20 mL of complete culture medium. A sample was taken and counted, and the viability was calculated. Viable cells were seeded into the inoculation container at the density shown in Table 1, using the total culture medium volume shown in Table 1. The cells were cultured in an incubator at 37°C, 5% CO2.

[0035] Table 1 Hair follicle stem cell seeding density comparison table

[0036] Table 2 Cell passaging quality standards

[0037] f. Cell Harvest and Cryopreservation: The cryopreservation quality standards for hair follicle stem cells are shown in Table 3. Stem cells that meet these standards are cryopreserved using the following procedure: Discard the culture medium, wash once or twice with 0.9% saline, add TrypLE, digest for 1-5 minutes, add 2-3 volumes of 0.9% saline, and centrifuge at 400-600g for 5-10 minutes. Discard the supernatant, resuspend the cells in 0.9% saline, take a sample, count the cells, calculate their viability, and send them for flow cytometry. Centrifuge at 400-600g for 5-10 minutes. The supernatant after centrifugation should be tested for sterility (culture flask), mycoplasma, and endotoxin. Prepare the cryopreservation solution: Mix 5%-10% DMSO with complete culture medium and pre-chill at 2-8°C.

[0038] Resuspend the cells with the freezing solution, the density is 1.0 x 10 7 2.0 x 10 7 mL per tube. After labeling the cryopreservation tube, use the programmed cooling box and the ultra-low temperature refrigerator to cool for 4-16 hours. After cooling, use the liquid nitrogen transport vehicle to transfer the cells to the gas-phase liquid nitrogen tank for long-term storage in an environment with a temperature of -100℃ to 150℃.

[0039] Table 3: Cryopreservation standard of hair follicle stem cells

[0040] Randomly sample the P2 generation of hair follicle stem cells obtained after the first passage, and perform flow cytometry detection. The detection results are shown in Table 4 and Figure 4 Table 5.

[0041] Table 4: Flow cytometry results of P2 generation of hair follicle stem cells

[0042] Test Example 1 Human hair follicle stem cell soft agar cloning experiment Preparation of human hair follicle stem cells: Take a known concentration of stem cell suspension, add an appropriate amount of stem cell culture medium to resuspend the cells, adjust the cell concentration to 1 x 10 4 / mL, and gently blow and mix the cells.

[0043] Preparation of Hela cells: Take a known concentration of Hela cell suspension, add an appropriate amount of MEM complete medium to resuspend the cells, adjust the cell concentration to 1 x 10 4 / mL, and gently blow and mix the cells.

[0044] Preparation of MRC-5 cells: Take a known concentration of MRC-5 cell suspension, add an appropriate amount of MEM complete medium to resuspend the cells, adjust the cell concentration to 1 x 10 4 / mL, and gently blow and mix the cells. Preparation of lower gel: Mix 1.2% agarose gel (melted in an electrically heated constant temperature water tank) and soft agar cloning formation test special medium according to a mass ratio of 1:1, mix well, and then add 1.5 mL per well into a 6-well plate as the lower layer, and solidify at room temperature.

[0045] Mixing of upper gel and cells for plating: Mix 0.6% agarose gel (melted in an electrically heated constant temperature water tank) and soft agar cloning formation test special medium according to a mass ratio of 1:1, then add 1000 cells per well into the cell suspension, mix well to avoid air bubbles, and then pour the mixed solution into a 6-well plate as the upper layer at 1 mL per well, and solidify at room temperature.

[0046] Positive control group: Add 1000 Hela cells per well; Negative control group: 1000 MRC-5 cells / well were added; Experimental group: 1000 stem cells were added per well.

[0047] Label the cell information (cell batch number or name, generation number, inoculation time, etc.) on the culture container and culture it in a cell culture incubator. Add 200 μL of soft agar colony formation test culture medium every 2 to 4 days to prevent drying.

[0048] Observation results: After 2 to 3 weeks, observe clones containing more than 50 cells under a 100× microscope.

[0049] The results of soft agar colony formation rate test of human hair follicle stem cells are as follows Figure 5~Figure 6 As shown, P1 represents P1 generation human hair follicle stem cells; P2 represents P2 generation human hair follicle stem cells; P3 represents P3 generation human hair follicle stem cells; P3A represents P3 generation human hair follicle stem cells after cryopreservation and resuscitation; P4A represents P4 generation human hair follicle stem cells after cryopreservation and resuscitation.

[0050] Depend on Figure 5 and Figure 6 As can be seen, single cell outlines and translucence can be seen in MRC-5 cells cultured in soft agar. HeLa cells cultured in soft agar show clear cell division and proliferation, with round cells arranged in clusters and translucence, and clear colony formation can be seen. Single cell outlines and translucence can be seen in soft agar cultures of human hair follicle stem cells at all generations, including P1, P2, P3, P3 after cryopreservation and thawing, and P3 after cryopreservation and thawing, and P4 after cryopreservation and thawing. Preliminary results indicate that human hair follicle stem cells at all generations, before and after cryopreservation, fail to form colonies in the soft agar cloning assay, suggesting that they are not tumorigenic in vitro.

[0051] Test Example 2 Detection of residual culture medium of human hair follicle stem cells Five batches of frozen hair follicle stem cells (001, 002, 003, 004, and 005) were revived and centrifuged at 500g for 5 minutes. The supernatant was then tested for residual impurities. A serum-free complete stem cell culture medium group (complete culture medium: serum-free stem cell supplement and serum-free stem cell culture medium were prepared at a ratio of 25mL:500mL) was also established. The results are summarized as follows: (1) Basic fibroblast growth factor (bFGF) Basic fibroblast growth factor (bFGF) detection: The Human / Rat FGF basic ELISA Kit (Biyuntian / PF323) was used as described in the instructions.

[0052] The detection range of the Human / Rat FGF basic ELISA Kit is 31.25–1000 pg / mL. The relative standard deviation (RSD) of the absorbance values ​​for replicate wells at each point (excluding point 0) on the standard curve of the Human / Rat FGF basic ELISA Kit (Biyuntian / PF323) was <30%, and the correlation coefficient (R²) was 1.000, meeting the requirement of R² > 0.980. The test results are shown in Table 5.

[0053] Table 5 bFGF content determination results

[0054] The test results showed that the bFGF content in the supernatant of multiple batches of human hair follicle stem cells was below the detection limit (31.25 pg / mL). The bFGF content in serum-free complete culture medium of human hair follicle stem cells was ≥3624.39 pg / mL, and the relative standard deviation (RSD) of the absorbance values ​​of replicate wells was <30%, indicating that the human hair follicle stem cell production process effectively removes bFGF from the culture medium. The normal value of bFGF in serum is 115.98 pg / mL ± 27.02 pg / mL, and the residual bFGF level in the cell supernatant was far lower than the normal value in human serum.

[0055] (2) Transforming growth factor-β (TGF-β1) Transforming growth factor-β (TGF-β1) detection: The procedure was performed according to the instructions of the Human TGF-β1 ELISA Kit (Beyotime / PT880).

[0056] The detection range of the Human TGF-β1 ELISA Kit is 62.5–2000 pg / mL. The relative standard deviation (RSD) of the absorbance values ​​for replicate wells at each point (excluding point 0) on the standard curve of the Human TGF-β1 ELISA Kit (Beyotime / PT880) was <30%, and the correlation coefficient (R²) was 1.000, meeting the requirement of R² > 0.980. The test results are shown in Table 6.

[0057] Table 6 TGF-β1 content determination results

[0058] The test results showed that the TGF-β1 content in the supernatant of multiple batches of human hair follicle stem cells was below the detection limit (62.5 pg / mL). The TGF-β1 content in the serum-free complete culture medium of human hair follicle stem cells was higher, at 15724.4 pg / mL. The relative standard deviation (RSD) of the absorbance values ​​of replicate wells was less than 30%, indicating that the production process of human hair follicle stem cells can effectively remove TGF-β1 from the culture medium. The normal value of TGF-β1 in serum is 7.96-12.9 ng / mL, and the residual TGF-β1 level in the cell supernatant is far lower than the normal value in human serum.

[0059] (3) Insulin Insulin detection: Follow the instructions of Human Insulin ELISA Kit (Biyuntian / PI608).

[0060] The detection range of the Human Insulin ELISA Kit is 12.5–200 mIU / L. The relative standard deviation (RSD) of the absorbance values ​​of replicate wells at each point (except 0) on the standard curve of the Human Insulin ELISA Kit (Biyuntian / PI608) was <30%, and the correlation coefficient (R²) was 1.000, meeting the requirement of R²>0.980.

[0061] Table 7 Insulin content determination results

[0062] The test results show that the insulin content in the supernatant of multiple batches of human hair follicle stem cells is below the detection limit (12.5mIU / L). The insulin content in the serum-free complete culture medium of human hair follicle stem cells is ≥200mIU / L, indicating that the hair follicle stem cell production process can effectively wash out the insulin in the culture medium. The normal fasting insulin value is 5-20mIU / L.

[0063] (4) Transferrin (TF) Transferrin (TF) detection: The procedure was performed according to the instructions of the Human TF sandwich ELISA Kit (Proteintech / KE00046).

[0064] The detection range of the Human TF sandwich ELISA Kit is 1.56–100 ng / mL. The relative standard deviation (RSD) of the absorbance values ​​for replicate wells at each point (except point 0) of the standard curve for the Human TF sandwich ELISA Kit (Proteintech / KE00046) was <30%, and the correlation coefficient (R²) was 1.000, meeting the requirement of R² > 0.980. The results are shown in Table 8.

[0065] Table 8 TF content determination results

[0066] The test results showed that the TF content in the supernatant of multiple batches of human hair follicle stem cells was below the detection limit (1.56 ng / mL). The TF content in serum-free complete culture medium for human stem cells was 3.38 ng / mL, and the relative standard deviation (RSD) of the absorbance values ​​of replicate wells was <30%, indicating that the background level of TF in the complete culture medium was low and that the production process for human hair follicle stem cells effectively washed TF out of the culture medium. The normal value of TF in serum is 3.979 mg / mL ± 0.172 mg / mL, and the residual TF level in the cell supernatant was far lower than the normal value in human serum.

[0067] (5) Platelet-derived growth factor-BB (PDGF-BB) Platelet-derived growth factor-BB (PDGF-BB) detection: The procedure was performed according to the instructions of the Human / Rat PDGF-BB ELISA Kit (Biyuntian / PP775).

[0068] The detection range of the Human / Rat PDGF-BB ELISA Kit is 62.5–2000 pg / mL. The relative standard deviation (RSD) of the absorbance values ​​for replicate wells at each point (except point 0) in the standard curve of the Human / Rat PDGF-BB ELISA Kit (Biyuntian / PP775) was <30%, and the correlation coefficient (R²) was 0.997, meeting the requirement of R² > 0.980. The results are shown in Table 9.

[0069] Table 9 PDGF-BB content determination results

[0070] The test results showed that the PDGF-BB content in the supernatant, storage medium, and serum-free complete culture medium of multiple batches of human umbilical cord mesenchymal stem cell injection was below the detection limit (62.5 pg / mL), indicating that the amount of PDGF-BB added to the serum-free complete culture medium used was low and below the detection limit of the method. The washing process using human hair follicle stem cells can reduce PDGF-BB to even lower levels. The normal value of PDGF-BB in serum is 80.06-100.42 pg / mL, and the residual PDGF-BB level in the cell supernatant is lower than the normal value in human serum.

[0071] (6) Leukemia inhibitory factor (LIF) Leukemia inhibitory factor (LIF) detection: The procedure was performed according to the instructions of the Human LIF ELISA Kit (Elabscience / E-ELH0094c).

[0072] The detection range of the Human LIF ELISA Kit is 31.25–2000 pg / mL. The relative standard deviation (RSD) of the absorbance values ​​for replicate wells at each point (excluding point 0) on the standard curve of the Human LIF ELISA Kit (Elabscience / E-EL-H0094c) was <30%, and the correlation coefficient (R²) was 0.999, meeting the requirement of R² > 0.980. The test results are shown in Table 10.

[0073] Table 10 LIF content determination results

[0074] The test results show that the SCF content in multiple batches of human hair follicle stem cell supernatants and human stem cell serum-free complete culture medium was below the detection limit (31.25 pg / mL), indicating that the amount of LIF added to the human mesenchymal stem cell serum-free complete culture medium we used was low and below the detection limit of the method. The washing process for human hair follicle stem cells can reduce LIF to even lower levels. The normal value of LIF in serum is 108.47 pg / mL ± 6.66 pg / mL, and the residual LIF level in the cell supernatant is far below the normal value in human serum.

[0075] Impurities detected in the supernatants of multiple batches of human hair follicle stem cells were consistently below the limit of quantification, demonstrating that the hair follicle stem cell production process effectively removes impurities such as bFGF, TGF-β1, Insulin, PDGF-BB, and LIF from the culture medium. Detection of key components present at high concentrations in the culture medium, which may have adverse effects on subjects, further demonstrates that the human hair follicle stem cell production process effectively removes residual culture medium.

[0076] In summary, considering that the detection levels of various impurities in multiple batches of cell supernatants are lower than the quantitative limit and lower than the normal level of the human body, the risk is low. Therefore, the hair follicle stem cells produced by this process meet the clinical preparation release standards.

[0077] Test Example 3 Osteogenic differentiation assay Osteogenic differentiation was performed using the Saiye OriCell® Human Umbilical Cord Mesenchymal Stem Cell Osteogenic Differentiation Kit.

[0078] 1. Prepare differentiation medium: Remove fetal bovine serum and thaw overnight at 4°C. 30 minutes before preparation, remove the supplement and thaw in a refrigerator at 4°C. Add the supplement and serum to the basal medium, mix thoroughly, and protect from light until ready to use.

[0079] 2. Coating 6-well plate Add 1 mL of 0.1% gelatin to a 6-well plate and shake to evenly coat the bottom surface. Place the plate in a 37°C, 5% CO2 incubator for at least 30 minutes. After 30 minutes, remove the gelatin and set aside.

[0080] 3. Inoculation of cells: The experiment was divided into induced group and non-induced group. Umbilical cord mesenchymal stem cells in the logarithmic growth phase were selected and 1×10 4 Cells were seeded at a density of 100 cells / mL in pre-coated 6-well culture plates, with 2 mL of single-cell suspension added to each well. For the non-induced group, cells were seeded two days prior to staining. Osteogenic differentiation experiments were performed when cells reached 60-70% confluency.

[0081] 4. Induction differentiation experiment: Carefully discard the culture medium in the 6-well plate and add 2 mL of differentiation medium to the 6-well plate. Replace the differentiation medium every 3 days. After 2-4 weeks of induction, stain the cells with Alizarin Red based on their morphological changes and growth.

[0082] Note: To prevent osteoblasts from falling off, change the medium by half every two days after obvious calcium nodules appear during the differentiation process.

[0083] 5. Alizarin red staining: Remove the Alizarin Red solution and allow it to return to room temperature. Rinse the culture medium from the 6-well plate and gently wash twice with PBS. Add 2 mL of 4% paraformaldehyde solution to each well and fix at room temperature for 30 minutes. Aspirate the fixative and gently wash twice with PBS. Add 2 mL of Alizarin Red working solution to each well and stain at room temperature for 5-10 minutes. Aspirate the Alizarin Red solution and gently wash twice with PBS. Add 2 mL of PBS to each well and observe the 6-well plate under a microscope and take photos.

[0084] Adipogenic differentiation assay Adipogenic differentiation was performed using the Saiye OriCell® Human Umbilical Cord Mesenchymal Stem Cell Adipogenic Differentiation Kit.

[0085] 1. Prepare adipogenic differentiation medium A: Before use, thaw fetal bovine serum overnight at 4°C. At least 30 minutes before preparation, place the phagocytic differentiation induction supplement AI in a 4°C refrigerator and the supplement A-II at room temperature until completely thawed. (If particles are aspirated after thawing, briefly incubate at 37°C and pipette repeatedly until dissolved.) Place all reagents in a biosafety cabinet and add the serum, supplement AI, and supplement A-II to the basal culture medium. (To prevent precipitation of supplement A-II upon freezing, preheat the basal culture medium to 37°C.) Mix thoroughly.

[0086] 2. Prepare adipogenic differentiation medium B: Before use, thaw fetal bovine serum at 4°C overnight. Thaw Supplement B in a refrigerator at 4°C at least 30 minutes before preparation. Mix Reagent B thoroughly. Add serum and Supplement B to the basal medium and mix thoroughly.

[0087] 3. Coating six-well plate: Add 1 mL of 0.1% gelatin to a 6-well plate and shake to evenly coat the bottom surface. Place the plate in a 37°C, 5% CO2 incubator for at least 30 minutes. After 30 minutes, remove the gelatin and set aside.

[0088] 4. Cell inoculation and culture: The human umbilical cord mesenchymal stem cells to be induced were divided into 2*10 4 pieces / cm 2 Cells were seeded at a density of 100 μg / mL in a 6-well plate and 2 mL of normal complete medium was added to each well. The 6-well plate was cultured in a 37°C, 5% CO2 incubator. When the cell confluence reached 50%, the complete medium was carefully discarded and 2 mL of differentiation medium A was added to the 6-well plate. After 3 days of induction, medium A was discarded and 2 mL of differentiation medium B was added. After 1 day of induction, medium B was discarded and replaced with medium A for induction. Medium A and medium B were used alternately, and the cell status needed to be observed every day. If the cell status was not good during the induction process of medium A, medium B was replaced in time until the cell status recovered (Solution A stimulates lipid droplet formation, and solution B maintains lipid droplets and promotes lipid droplet enlargement). The induction and maintenance process was repeated until sufficient lipid droplets of appropriate size appeared.

[0089] 5. Cell identification: After adipogenic differentiation is complete, aspirate the adipogenic differentiation medium and rinse twice with PBS. Add 2 mL of 4% paraformaldehyde and fix for 30 minutes. Prepare Oil Red O working solution: Oil Red O dye solution: distilled water = 3:2, mix thoroughly, centrifuge at 250g for 4 minutes, and use the supernatant. Aspirate the paraformaldehyde and rinse twice with PBS. Add 2 mL of Oil Red O dye working solution to each well and stain for 30 minutes. Aspirate the Oil Red O dye solution and rinse twice with PBS. Take photos under a microscope. If the cells contain red droplets, they are adipocytes, demonstrating that the tested mesenchymal stem cells have the ability to be induced to differentiate into adipocytes.

[0090] Chondrogenic differentiation assay Chondrogenic differentiation was performed using the OriCell® Human Umbilical Cord Mesenchymal Stem Cell Chondrogenic Differentiation Kit.

[0091] 1. Inoculation of cells: Mesenchymal stem cells in the logarithmic growth phase were selected and routinely digested and counted, and 3×10 4 Seed cells in a six-well cell culture plate at a rate of 100 cells / well. Add 2 mL of single-cell suspension to each well, seeding a total of 6 wells. For the non-induced group, seed cells a few days before staining (approximately when cell confluency exceeds 80%). Chondrogenic differentiation experiments can be performed when cells in the induced group reach 80-90% confluency.

[0092] 2. Prepare differentiation medium: Thaw Chondrogenic Differentiation Supplement I in the kit at least 30 minutes before preparation at 4°C. Add all of Supplement I to the basal medium, mix thoroughly, and set aside. Before using the differentiation medium, add Supplement II at a ratio of 10 µL per 1 mL of differentiation medium and mix thoroughly by gently pipetting.

[0093] Note: a. Centrifuge the tube before aspirating Supplement II. b. Ensure that Supplement II is aliquoted and stored below -20°C. It is valid for 12 months. c. Prepare the differentiation medium containing Supplement II immediately before use and do not store it at 4°C for more than 12 hours.

[0094] 3. Induced differentiation: Remove the old culture medium from the six-well plate, add 2 mL of prepared chondrogenic differentiation complete medium to each well, and place in a 37°C, 5% CO 2 Continue culturing in the incubator, changing the medium every 3-4 days. Sampling cells from one well for identification using Alcian blue staining on days 7, 14, 21, and 28.

[0095] 4. Alcian blue staining: Aspirate the complete medium from the six-well plate and wash twice with PBS. Add 2 mL of 4% paraformaldehyde to each well and fix for 10 minutes. Aspirate the fixative, wash two to three times with PBS, and add 1 mL of Alcian blue stain to each well. Incubate at 37°C in the dark for 30 minutes. 4.4 Wash the cells twice with PBS to remove any residual stain. Observe under a microscope and photograph. If cells show a light blue color, they are chondrocytes, demonstrating that the tested mesenchymal stem cells have the ability to be induced to differentiate into chondrocytes.

[0096] The results of the three-lineage differentiation experiment of hair follicle stem cells are as follows Figure 7 (40x) shown by Figure 7 It can be seen that the hair follicle stem cells prepared in the present invention have good differentiation ability and can undergo osteogenic differentiation, adipogenic differentiation and chondrogenic differentiation.

[0097] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A method for large-scale expansion of clinical-grade hair follicle stem cells, characterized in that: The method comprises the following steps: The hair follicles are extracted, washed, and digested with a Liberase TL solution at a concentration of 150-300 µg / mL at 37°C for 1-2 hours. The undigested portion is then discarded, the cells are centrifuged and resuspended in complete culture medium, inoculated into T25 flasks for culture, and then passaged; the passages include the first passage, the second passage, the third passage, and the fourth passage; after the fourth passage, the stem cells are harvested and cryopreserved; At the first passage, cells were seeded into T175 culture flasks; At the second passage, cells were seeded into a 5-layer cell factory; At the third passage, cells were seeded into a 6-layer cell factory; At the fourth passage, cells were seeded into a 10-layer cell factory; The complete culture medium is a serum-free stem cell culture medium containing 1% v / v~5% v / v human platelet lysate, 5~10 μg / mL recombinant humanized type III collagen, 10~25 μg / mL recombinant human insulin, and 1~5 ng / mL recombinant human TGF-β2.

2. The method according to claim 1, characterized in that The collection container used for collecting the hair follicles contains 20-30 mL of buffer solution, which is a mixture of penicillin-streptomycin-amphotericin B solution and 0.9% normal saline, and the volume ratio of the 0.9% normal saline to the penicillin-streptomycin-amphotericin B solution is 100:1-2.

3. The method according to claim 1, characterized in that The hair follicles are stored and transported at 2°C to 8°C.

4. The method according to claim 1, wherein The cleaning comprises the following steps: washing the hair follicles 1-2 times with a buffer solution, wherein the buffer solution is a mixture of a penicillin-streptomycin-amphotericin B solution and 0.9% normal saline, and the volume ratio of the 0.9% normal saline to the penicillin-streptomycin-amphotericin B solution is 100:1-2; and the centrifugation condition is 300-500g centrifugation for 5-10 minutes.

5. The method according to claim 1, wherein At the first passage, the seeding density was 1×10 6 ~1.6×10 6 Inoculate cells into T175 culture flasks, using 20-30 mL of complete culture medium per flask; At the second passage, the seeding density was 1.8×10 7 ~3.0×10 7 Inoculate the cells into a 5-layer cell factory, using 300-450 mL of complete culture medium per bottle; At the third passage, the seeding density was 2.4×10 7 ~4.5×10 7 Inoculate the cells into a 6-layer cell factory, using 400-500 mL of complete culture medium per bottle; At the fourth passage, the seeding density was 5.0×10 7 ~7.0×10 7 Inoculate the cells into a 10-layer cell factory, using 700-900 mL of complete culture medium per flask.

6. The method according to claim 1, characterized in that The culture conditions were 37±1°C and 5% CO2.

7. The method according to claim 1, characterized in that The passaging comprises the following steps: adding 5-10 mL of TrypLE to the cells, adding an equal volume of complete culture medium to terminate digestion after the cells shrink and become round, centrifuging the cell suspension, resuspending it in complete culture medium, and then inoculating.

8. The method according to claim 1, characterized in that The frozen cell density was 1.0×10 7 ~2.0×10 7 pieces / mL.

9. The method according to claim 1, characterized in that The freezing storage comprises the following steps: cooling at a rate of 1-5°C / min for 4-16 hours, and after cooling, transferring the cells to a vapor phase liquid nitrogen tank for long-term storage at a temperature of -100°C to 150°C.

10. Use of the method according to any one of claims 1 to 9 in preparing a product for treating hair loss.

Images