Serum-free culture medium and culturing method for hair follicle stem cells

Abstract

Provided are a serum-free culture medium and a culturing method for hair follicle stem cells. The serum-free culture medium comprises an MEM non-essential amino acid solution, an MEM vitamin solution, an L-glutamine solution, L-glutathione, sodium selenite, recombinant human serum albumin, recombinant human insulin, recombinant human transferrin, recombinant human epidermal growth factor, recombinant human basic fibroblast growth factor, recombinant human platelet-derived growth factor-AB, recombinant human epiregulin, recombinant human catalase, L-ascorbic acid, (+)-α-Tocopheryl acetate, anisomycin, corticosterone, L-carnitine, D-galactose, ethanolamine, linoleic acid, linolenic acid, progesterone, putrescine, and a basal culture medium. The chemical components are clear, exogenous exosome contamination does not occur, rapid in vitro proliferation of HFSCs can be realized, and vacuolization or aging do not occur in multiple cell passagings.

Description

A serum-free culture medium and culture method for hair follicle stem cells

[0001] This application claims priority to the Chinese patent application filed with the China Patent Office on December 29, 2023, with application number 202311870364.0 and invention name “A serum-free culture medium and culture method for hair follicle stem cells”, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The invention belongs to the technical field of cell culture, and particularly relates to a serum-free culture medium and a culture method for hair follicle stem cells. Background Art

[0003] Due to the fast pace and high stress of modern life, more and more people are suffering from pathological hair loss. Studies have shown that many factors such as aging, genetics (such as androgenic alopecia), hormonal disorders (such as thyroid disease), autoimmunity (such as systemic lupus erythematosus), nutritional disorders and drugs have long-term effects on the hair growth cycle and reduce the activity of cells in the hair follicles, leading to damage or defects in the hair follicles, which are the direct causes of hair loss. Among them, androgenic alopecia accounts for the largest proportion of hair loss types, and the age of the patients is getting younger and younger, thus giving rise to a high-growth and unlimited potential hair loss prevention market.

[0004] Currently, there are limited medications available to treat male pattern baldness. Only two, finasteride and minoxidil, are approved by the US FDA. Side effects of the oral medication finasteride, such as decreased libido, sexual dysfunction, mood disorders, and possible birth defects, are a major reason for patients to exercise caution when using it. The disadvantage of the topical medication minoxidil is that it is only effective for some patients and has a long onset of action, requiring at least two months of continuous use to begin to see results. A trial period of six to 12 months is required to determine if it is suitable for you. If effective, continued use is necessary to maintain the results; if the patient stops using it, hair loss will recur.

[0005] A popular treatment for hair loss is follicular transplantation, which involves transplanting follicular units from a patient's non-hair loss area to the affected area. Although hair transplantation is relatively mature, it suffers from disadvantages such as a low survival rate. The most significant limitation is the limited donor area. This procedure is not suitable for patients with extensive hair loss, especially those with grade 6 or higher.

[0006] With the rapid development of regenerative medicine and hair tissue engineering in recent years, stem cell therapy for hair loss has become one of the most promising treatments. Currently, stem cell treatments for hair loss can be categorized into three categories: stem cell transplantation, stem cell-conditioned medium, and the use of stem cell exosomes. Existing studies have shown that transplanting MSCs from various sources (such as umbilical cord, bone marrow, and adipose tissue) in animal models can effectively prevent hair follicle degeneration, activate the vitality of various cell types in hair follicle tissue, and restore the hair follicle growth cycle. Stem cell-derived conditioned medium has also recently been widely studied and applied. Stem cell-derived conditioned medium is rich in various cell growth factors, poses no tumor risk, and is easy to harvest. Researchers have also found that it can induce hair follicle cell reactivation, development, hair cycle, and hair follicle regeneration. Exosomes are vesicles secreted by cells, approximately 30-100 nm in diameter, containing a variety of bioactive substances. They play a crucial role in intercellular signaling. The therapeutic effects of exosomes have been widely reported in various fields. In hair follicle research, studies have demonstrated that exosomes can promote the transition of mouse dorsal hair from the resting phase to the anagen phase. Stem cell-derived exosomes are small vesicles secreted by cells. They act as intercellular signal transduction factors by carrying transcription factors and cytokines. They have been shown to be important paracrine regulators. In particular, exosomes derived from hair follicle stem cells may be of great significance to hair.

[0007] The hair follicle is a complex microscopic organ. Its formation and development result from interactions between the epithelial and dermal components, and its growth and regeneration are regulated by its own stem cells. The three main stem cells residing within the hair follicle are hair follicle stem cells (HFSCs), dermal papilla cells (DPCs), and melanocyte stem cells (MSCs). DPCs and HFSCs play a crucial role in hair follicle morphogenesis and the hair cycle. HFSCs are a population of adult stem cells residing in the bulge region of the outer root sheath (ROS) of the hair follicle. They belong to the epithelial component and are characterized by slow cycling, self-renewal, undifferentiated development, and robust in vitro proliferation. HFSCs not only express surface markers of MSCs but also exhibit the ability to differentiate into adipocytes, osteoblasts, chondrocytes, hematopoietic cells, smooth muscle cells, and neurons. Furthermore, their readily accessible autologous stem cell source holds great potential for stem cell-based regenerative medicine.

[0008] Existing HFSCs in vitro culture mostly uses fetal bovine serum (FBS) or human platelet lysate (hPL). FBS has a complex composition and contains foreign proteins, making it prone to carrying viruses or being infected by mycoplasmas. hPL, as the most effective serum substitute, is nutrient-rich and contains a large number of growth factors and proteins required for multiple cell proliferation. However, the introduction of human proteins cannot be avoided during use, and the cultured cells or collected conditioned medium may cause allogeneic immune rejection when used in clinical or medical aesthetics fields. Therefore, a serum-free culture system without human or animal sources, and without the addition of serum and its derivatives, can completely avoid the shortcomings of the above-mentioned existing HFSCs culture system.

[0009] HFSCs are adult stem cells with limited in vitro expansion capacity. When HFSCs are cultured in serum-free medium, vacuolation is evident after serial passage. Cytoplasmic vacuoles increase with passage number, leading to rapid cell aging and a significant decrease in expansion capacity. As vacuoles grow larger, the cells eventually rupture and die. Therefore, inhibiting the vacuolation that occurs with serial passage of HFSCs in serum-free culture systems is key to enhancing HFSC viability, slowing aging, and improving expansion efficiency. Summary of the Invention

[0010] To address the shortcomings of existing technologies, the present invention provides a serum-free culture medium and culture method for hair follicle stem cells. The serum-free culture medium for hair follicle stem cells provided by the present invention can slow the aging of hair follicle stem cells during continuous passage and / or maintain their proliferation and passage capacity, effectively improving their in vitro expansion and / or passage capacity, and preventing vacuolation during passaged culture.

[0011] In order to achieve the above object, the present invention adopts the following technical solutions:

[0012] The first object of the present invention is to provide a serum-free culture medium for hair follicle stem cells, comprising: 0.1-2% by volume of MEM non-essential amino acid solution, 0.1-2% by volume of MEM vitamin solution, 0.1-2% by volume of L-glutamine solution, 1-8 mg / L L-glutathione, 0.1-1 μg / L sodium selenite, 0.5-5 g / L recombinant human serum albumin, 1-20 mg / L recombinant human insulin, 1-10 mg / L recombinant human transferrin, 10-50 μg / L recombinant human epidermal growth factor, 10-50 μg / L recombinant human basic fibroblast growth factor, 1-20 μg / L recombinant human platelet-derived growth factor-AB, 1-50 μg / L recombinant human epiregulin, 12.5-500 mg / L recombinant human catalase, 5-200 mg / L L-ascorbic acid, 5-200 μg / L (+)-α-tocopheryl acetate, 0.05-5 mg / L anisomycin, 0.1-10 mg / L corticosterone, 25-500 mg / L L-carnitine, 0.05-5 mg / L D-galactose, 0.1-10 mg / L ethanolamine, 25-500 μg / L linoleic acid, 25-500 μg / L linolenic acid, 0.05-5 μg / L progesterone, 50-500 mg / L putrescine, and the remainder of basal culture medium;

[0013] The final concentrations of the MEM non-essential amino acid solution, MEM vitamin solution and L-glutamine solution are all 100×.

[0014] Preferably, the hair follicle stem cell serum-free culture medium contains 1% by volume of MEM non-essential amino acid solution, 1% by volume of MEM vitamin solution, 1% by volume of L-glutamine solution, 4 mg / L L-glutathione, 0.5 μg / L sodium selenite, 2 g / L recombinant human serum albumin, 10 mg / L recombinant human insulin, 5 mg / L recombinant human transferrin, 20 μg / L recombinant human epidermal growth factor, 20 μg / L recombinant human basic fibroblast growth factor, 10 μg / L recombinant human platelet-derived growth factor-AB, 10 μg / L recombinant human epiregulin, 125 mg / L recombinant human catalase, 50 mg / L L-ascorbic acid, 50 μg / L (+)-α-tocopheryl acetate, 0.5 mg / L anisomycin, 1 mg / L corticosterone, 100 mg / L L-carnitine, 0.5 mg / L D-galactose, 2 mg / L ethanolamine, 100 ug / L linoleic acid, 100 ug / L linolenic acid, 0.5 μg / L progesterone, 200 mg / L putrescine and the remainder basal culture medium.

[0015] Preferably, the basal culture medium is a serum-free culture medium; the serum-free culture medium is IMDM or high-glucose DMEM basal culture medium.

[0016] The second object of the present invention is to provide a use of the above-mentioned serum-free culture medium for hair follicle stem cells in stem cell culture; the stem cells are hair follicle stem cells.

[0017] Preferably, the hair follicle stem cell serum-free culture medium slows down the aging of hair follicle stem cells during continuous passage and / or maintains the proliferation activity and passage ability of the hair follicle stem cells.

[0018] Preferably, the serum-free culture medium for hair follicle stem cells prevents vacuolation of the hair follicle stem cells during subculture.

[0019] Another object of the present invention is to provide a method for preparing the above-mentioned serum-free culture medium for hair follicle stem cells, comprising the steps of dissolving the components of the above-mentioned serum-free culture medium for hair follicle stem cells according to their solubility characteristics, uniformly mixing to form a 1000× solution, sterilizing by filtration with a filter membrane, and storing at a temperature below -20°C to -80°C for later use.

[0020] Another object of the present invention is to provide a method for culturing hair follicle stem cells, the method comprising the following steps:

[0021] The stem cells were inoculated into the above serum-free medium for culture;

[0022] The stem cells are hair follicle stem cells.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The present invention provides a serum-free culture medium and culture method for hair follicle stem cells (HFSCs). Using the serum-free culture medium provided by the present invention to subculture hair follicle stem cells (HFSCs) enables rapid in vitro proliferation of HFSCs, with a cell proliferation capacity greater than that achieved in serum-containing culture medium. Furthermore, the present invention achieves multiple passages of HFSCs without vacuolation or senescence, effectively inhibiting vacuolation and alleviating cell senescence, achieving comparable results to those achieved in serum-containing culture medium.

[0025] Secondly, the serum-free culture medium for culturing hair follicle stem cells (HFSCs) provided by the present invention has a clear chemical composition, is not of human or animal origin, is serum-free and its derivatives-free, and is free of exogenous exosome contamination. Compared with traditional serum-free culture systems, the present invention requires fewer types of culture medium additives when subcultured hair follicle stem cells (HFSCs), is simple and low-cost.

[0026] At the same time, the serum-free culture medium provided by the present invention is applied to the subculture of hair follicle stem cells HFSCs, which can meet the demand for the number of HFSCs in the field of hair tissue engineering research or treatment. At the same time, conditioned culture medium without exogenous exosomes can be collected or HFSCs exosomes can be extracted for the treatment of hair loss. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] FIG1 is a morphological diagram of HFSCs cultured to P3, P4, and P5 generations using the serum-free medium of Example 1;

[0028] FIG2 is a morphological diagram of HFSCs cultured to P3, P4, and P5 generations using the serum-free medium of Example 2;

[0029] FIG3 is a morphological diagram of HFSCs cultured to P3, P4 and P5 generations using the serum-free medium of Example 3;

[0030] FIG4 is a morphological diagram of HFSCs cultured to P3, P4, and P5 generations using the serum-free medium of Example 4;

[0031] FIG5 is a morphological diagram of HFSCs cultured to P3, P4, and P5 generations using the serum-free medium of Example 5;

[0032] Figure 6 shows the morphology of HFSCs cultured to P3, P4, and P5 using the complete medium of control group 1;

[0033] Figure 7 shows the morphology of HFSCs cultured to P3, P4, and P5 using the complete medium of control group 2;

[0034] Figure 8 shows the results of β-galactosidase activity staining of HFSCs senescent cells in each group (40×);

[0035] FIG9 is a graph showing the cell expansion fold of HFSCs in each group at each passage;

[0036] Figure 10 is a graph showing the results of flow cytometry detection of surface markers of HFSCs in experimental group 1;

[0037] Figure 11 is a graph showing the results of flow cytometry detection of surface markers of HFSCs in experimental group 2;

[0038] FIG12 is a graph showing the results of flow cytometry detection of surface markers of 3HFSCs in the experimental group;

[0039] FIG13 is a graph showing the results of flow cytometry detection of surface markers of 4 HFSCs in the experimental group;

[0040] FIG14 is a graph showing the flow cytometry results of surface markers of 5HFSCs in the experimental group;

[0041] Figure 15 is a graph showing the results of flow cytometry detection of surface markers of HFSCs in the control group 1;

[0042] Figure 16 is a graph showing the results of flow cytometry detection of surface markers of 2HFSCs in the control group;

[0043] Figure 17 is a diagram showing the adipogenic differentiation effects of HFSCs in experimental group 2, control group 1, and control group 2 under a 200× microscope;

[0044] FIG18 is a diagram showing the osteogenic differentiation effect of HFSCs in experimental group 2, control group 1, and control group 2 under a 40× microscope. DETAILED DESCRIPTION

[0045] The following is a further detailed description of the present invention through specific embodiments in the form of examples, but it should not be understood that the scope of the present invention is limited to the following examples.

[0046] In the present invention, all components and reagents involved are conventional commercial products. For example, recombinant human albumin (catalog number: HYC002C01) was purchased from Wuhan Heyuan Biotechnology Co., Ltd.; MEM non-essential amino acid solution and MEM vitamin solution were purchased from Thermofisher; fetal bovine serum (FBS) was purchased from Corning, catalog number 35-081-cv; IMDM complete medium was purchased from Gibco, catalog number C12440500BT; human platelet lysate (hPL) was purchased from BI, catalog number PLTGOLD0500R; L-glutamine solution, linoleic acid, linolenic acid, sodium selenite, and L-glutathione were purchased from Sigma; and various recombinant proteins were purchased from Peprotech.

[0047] The hair follicle stem cell serum-free culture medium provided by the present invention contains a variety of growth factors and nutrients, which can promote the normal growth and metabolism of hair follicle stem cells under serum-free culture conditions:

[0048] Recombinant human albumin replaces blood-derived human albumin for cell culture. It can combine with vitamins, lipids, hormones, metal ions and growth factors to stabilize and regulate the activity of the above substances in a serum-free system, while protecting cells from mechanical damage.

[0049] Recombinant human insulin can promote the synthesis of RNA, protein and fatty acids, inhibit cell apoptosis, and is an important cell survival factor.

[0050] Recombinant human transferrin is a binding protein that carries iron ions and plays an important role in cellular metabolism. Transferrin is also a crucial extracellular antioxidant. Under physiological conditions, it binds iron so tightly that there is virtually no free iron to catalyze the production of free radicals, maintaining the youthfulness of cells cultured in vitro.

[0051] Growth factors such as recombinant human epidermal growth factor, recombinant human basic fibroblast growth factor, recombinant human platelet-derived growth factor-AB and recombinant human epiregulin can bind to their corresponding receptors in cells, activate multiple signaling pathways such as MAPK-ERK and PI3K-AKT, and thus promote cell proliferation.

[0052] Recombinant human catalase, L-ascorbic acid, (+)-α-tocopheryl acetate and L-carnitine are antioxidants that can produce a synergistic effect to protect cells from damage by oxygen free radicals and slow down excessive cell aging and vacuolization.

[0053] Anisomycin is an activator of the p38 and JNK signaling pathways. Inhibition of the p38 signaling pathway can cause cytoplasmic vacuolation, rapid cell senescence, and apoptosis. Anisomycin can activate the p38 signaling pathway and prevent cell vacuolation.

[0054] The trace element selenium in sodium selenite can eliminate the damage to cells caused by peroxidase and oxygen free radicals, and is an essential trace element for cell growth.

[0055] Corticosterone and progesterone are steroid hormones that can promote cell adhesion and cell separation and promote the proliferation of hair follicle stem cells.

[0056] D-galactose is a monosaccharide composed of six carbon atoms and one aldehyde, and is one of the components required for the synthesis of cell membrane glycoproteins.

[0057] Ethanolamine is an organic compound and a precursor of phospholipid synthesis, which can provide raw materials for the synthesis of cell membrane structures.

[0058] Linoleic acid and linolenic acid are fatty acid precursors that provide cells with lipids required for membrane synthesis and growth.

[0059] Putrescine is one of the active substances required for cell metabolism. It has the function of promoting cell division and the synthesis of macromolecules such as DNA, RNA and protein. Exogenous supplementation of putrescine can also regulate the circulation of calcium and magnesium ions and maintain the balance of cell osmotic pressure.

[0060] Therefore, the chemical composition is clear, there is no exogenous exosome contamination, and it can achieve rapid proliferation of HFSCs in vitro, and the cells will not vacuolate or age after multiple passages.

[0061] Example 1 Preparation of serum-free culture medium for hair follicle stem cells of the present invention

[0062] The hair follicle stem cell serum-free culture medium of the present invention comprises: 1% by volume of MEM non-essential amino acid solution, 1% by volume of MEM vitamin solution, 1% by volume of L-glutamine solution, 1 mg / L L-glutathione, 0.1 μg / L sodium selenite, 0.5 g / L recombinant human serum albumin, 1 mg / L recombinant human insulin, 1 mg / L recombinant human transferrin, 10 μg / L recombinant human epidermal growth factor, 10 μg / L recombinant human basic fibroblast growth factor, 1 μg / L recombinant human platelet-derived growth factor-AB, 1 μg / L recombinant human epiregulin, 12.5 mg / L recombinant human catalase, 5 mg / L L-ascorbic acid, 5 μg / L (+)-α-tocopheryl acetate, 0.05 mg / L anisomycin, 0.1 mg / L corticosterone, 25 mg / L L-carnitine, 0.05 mg / L D-galactose, 0.1 mg / L ethanolamine, 25 μg / L linoleic acid, 25 μg / L linolenic acid, 0.05 μg / L progesterone, 50 mg / L putrescine, and the remainder high-glucose DMEM basal medium;

[0063] The final concentrations of the MEM non-essential amino acid solution, MEM vitamin solution and L-glutamine solution are all 100×.

[0064] The method for preparing the serum-free culture medium for hair follicle stem cells comprises the following steps: dissolving the components of the serum-free culture medium for hair follicle stem cells according to their solubility characteristics, uniformly mixing to prepare a 1000× solution, filtering through a 0.22 μm filter membrane for sterilization, and storing below -20°C for later use.

[0065] Example 2 Preparation of serum-free culture medium for hair follicle stem cells of the present invention

[0066] The hair follicle stem cell serum-free culture medium of the present invention comprises: 1% by volume of MEM non-essential amino acid solution, 1% by volume of MEM vitamin solution, 1% by volume of L-glutamine solution, 2 mg / L L-glutathione, 0.25 μg / L sodium selenite, 1 g / L recombinant human serum albumin, 5 mg / L recombinant human insulin, 2 mg / L recombinant human transferrin, 10 μg / L recombinant human epidermal growth factor, 10 μg / L recombinant human basic fibroblast growth factor, 5 μg / L recombinant human platelet-derived growth factor-AB, 5 μg / L recombinant human epiregulin, 62.5 mg / L recombinant human catalase, 10 mg / L L-ascorbic acid, 10 μg / L (+)-α-tocopheryl acetate, 0.1 mg / L anisomycin, 0.5 mg / L corticosterone, 50 mg / L L-carnitine, 0.1 mg / L D-galactose, 1 mg / L ethanolamine, 50 μg / L linoleic acid, 50 μg / L linolenic acid, 0.5 μg / L progesterone, 100 mg / L putrescine, and the remainder high-glucose DMEM basal medium;

[0067] The final concentrations of the MEM non-essential amino acid solution, MEM vitamin solution and L-glutamine solution are all 100×.

[0068] The method for preparing the serum-free culture medium for hair follicle stem cells comprises the following steps: dissolving the components of the serum-free culture medium for hair follicle stem cells according to their solubility characteristics, uniformly mixing to prepare a 1000× solution, filtering through a 0.22 μm filter membrane for sterilization, and storing below -20°C for later use.

[0069] Example 3 Preparation of serum-free culture medium for hair follicle stem cells of the present invention

[0070] The hair follicle stem cell serum-free culture medium of the present invention comprises: 1% by volume of MEM non-essential amino acid solution, 1% by volume of MEM vitamin solution, 1% by volume of L-glutamine solution, 4 mg / L L-glutathione, 0.5 μg / L sodium selenite, 2 g / L recombinant human serum albumin, 10 mg / L recombinant human insulin, 5 mg / L recombinant human transferrin, 20 μg / L recombinant human epidermal growth factor, 20 μg / L recombinant human basic fibroblast growth factor, 10 μg / L recombinant human platelet-derived growth factor-AB, 10 μg / L recombinant human epiregulin, 125 mg / L recombinant human catalase, 50 mg / L L-ascorbic acid, 50 μg / L (+)-α-tocopheryl acetate, 0.5 mg / L anisomycin, 1 mg / L corticosterone, 100 mg / L L-carnitine, 0.5 mg / L D-galactose, 2 mg / L ethanolamine, 100 μg / L linoleic acid, 100 μg / L linolenic acid, 0.5 μg / L progesterone, 200 mg / L putrescine, and the remainder IMDM basal medium;

[0071] The final concentrations of the MEM non-essential amino acid solution, MEM vitamin solution and L-glutamine solution are all 100×.

[0072] The method for preparing the serum-free culture medium for hair follicle stem cells comprises the following steps: dissolving the components of the serum-free culture medium for hair follicle stem cells according to their solubility characteristics, uniformly mixing to prepare a 1000× solution, filtering through a 0.22 μm filter membrane for sterilization, and storing below -20°C for later use.

[0073] Example 4 Preparation of serum-free culture medium for hair follicle stem cells of the present invention

[0074] The hair follicle stem cell serum-free culture medium of the present invention comprises: 1% by volume of MEM non-essential amino acid solution, 1% by volume of MEM vitamin solution, 1% by volume of L-glutamine solution, 4 mg / L L-glutathione, 0.5 μg / L sodium selenite, 2 g / L recombinant human serum albumin, 10 mg / L recombinant human insulin, 5 mg / L recombinant human transferrin, 20 μg / L recombinant human epidermal growth factor, 20 μg / L recombinant human basic fibroblast growth factor, 10 μg / L recombinant human platelet-derived growth factor-AB, 20 μg / L recombinant human epiregulin, 250 mg / L recombinant human catalase, 100 mg / L L-ascorbic acid, 100 μg / L (+)-α-tocopheryl acetate, 1 mg / L anisomycin, 2 mg / L corticosterone, 200 mg / L L-carnitine, 1 mg / L D-galactose, 4 mg / L ethanolamine, 200 μg / L linoleic acid, 200 μg / L linolenic acid, 1 μg / L progesterone, 200 mg / L putrescine, and the remainder IMDM basal medium;

[0075] The final concentrations of the MEM non-essential amino acid solution, MEM vitamin solution and L-glutamine solution are all 100×.

[0076] The method for preparing the serum-free culture medium for hair follicle stem cells comprises the following steps: dissolving the components of the serum-free culture medium for hair follicle stem cells according to their solubility characteristics, uniformly mixing to prepare a 1000× solution, filtering through a 0.22 μm filter membrane for sterilization, and storing below -20°C for later use.

[0077] Example 5 Preparation of serum-free culture medium for hair follicle stem cells of the present invention

[0078] The hair follicle stem cell serum-free culture medium of the present invention comprises: 1% by volume of MEM non-essential amino acid solution, 1% by volume of MEM vitamin solution, 1% by volume of L-glutamine solution, 8 mg / L L-glutathione, 1 μg / L sodium selenite, 5 g / L recombinant human serum albumin, 20 mg / L recombinant human insulin, 10 mg / L recombinant human transferrin, 50 μg / L recombinant human epidermal growth factor, 50 μg / L recombinant human basic fibroblast growth factor, 20 μg / L recombinant human platelet-derived growth factor-AB, 50 μg / L recombinant human epiregulin, 500 mg / L recombinant human catalase, 200 mg / L L-ascorbic acid, 200 μg / L (+)-α-tocopheryl acetate, 5 mg / L anisomycin, 10 mg / L corticosterone, 500 mg / L L-carnitine, 5 mg / L D-galactose, 10 mg / L ethanolamine, 500 μg / L linoleic acid, 500 μg / L linolenic acid, 5 μg / L progesterone, 500 mg / L putrescine, and the remainder IMDM basal medium;

[0079] The final concentrations of the MEM non-essential amino acid solution, MEM vitamin solution and L-glutamine solution are all 100×.

[0080] The method for preparing the serum-free culture medium for hair follicle stem cells comprises the following steps: dissolving the components of the serum-free culture medium for hair follicle stem cells according to their solubility characteristics, uniformly mixing to prepare a 1000× solution, filtering through a 0.22 μm filter membrane for sterilization, and storing below -20°C for later use.

[0081] Experiment 1 Effect Verification

[0082] The serum-free culture medium prepared in Examples 1-5 of the present invention was used as the experimental group, the IMDM complete culture medium containing 10% FBS was used as the control group 1, and the IMDM complete culture medium containing 5% human platelet lysate (hPL) was used as the control group 2. Hair follicle stem cells were cultured separately. P3 generation HFSCs were selected for the experiment. HFSCs were cultured at a density of 1×10 4 / cm 2 The cells were seeded in 6-well plates with 3 replicates per group.

[0083] Culture in a 5% CO2 incubator at 37°C. Subculture when cell confluence reaches 80% or higher. Repeat for at least three subcultures. Then, perform the following tests:

[0084] (1) Comparison of morphology in continuous subculture

[0085] Images were collected for HFSCs at each passage in experimental groups 1-5 and control groups 1-2. The results are shown in Figures 1-7. As shown, HFSCs in all groups grew as adherent monolayers, with the majority of cells exhibiting an irregular, elongated, spindle-shaped morphology. During serial passages, control group 1 showed no vacuolation, while control group 2 exhibited vacuolation. HFSCs in experimental groups 1 and 5 gradually developed a small number of vacuoles, but these were significantly less than those in control group 2. No vacuoles were observed in HFSCs in experimental groups 2, 3, and 4 at any passage, similar to those in the serum-treated control group 1.

[0086] (2) β-galactosidase activity staining

[0087] β-galactosidase activity staining was performed on HFSCs cultured for more than three consecutive passages in experimental groups 1-5 and control groups 1-2 to detect the senescence of HFSCs. 4 Each well was seeded with 12 cells / well in a 12-well plate and cultured in a 5% CO2 incubator at 37°C. After 48 hours, HFSCs in each group were stained using a β-galactosidase activity detection kit (purchased from Beyotime, catalog number C0602).

[0088] The experimental results are shown in Figure 8, which shows the β-galactosidase activity staining results (40×) of senescent HFSCs in each group. As shown in Control Group 2, a large number of cells were stained, indicating obvious cell senescence; a small number of cells were stained in Experimental Groups 1 and 5; and almost no cells were stained in Control Group 1, Experimental Groups 2, 3, and 4. This indicates that the serum-free culture medium for hair follicle stem cells of the present invention can effectively slow the senescence of HFSCs during serial passage, maintaining their proliferation and passage capacity.

[0089] (3) In vitro proliferation ability assay

[0090] P1 HFSCs from experimental groups 1-5 and control groups 1-2 were selected for the experiment. HFSCs were expressed as 1×10 5 pieces / cm 2 Seed the cells in T25 flasks and culture them in a 5% CO2 incubator at 37°C. Continuously subculture for at least five generations and calculate the expansion fold of each HFSC group at each generation. After 3-4 days of culture, when confluence reaches 80% or greater, harvest the cells and calculate the total harvest volume. Expansion fold = total harvest volume / seeding size.

[0091] The experimental results are shown in Table 1 and Figure 9. The results show that compared with control group 1 (serum-containing group) and control group 2 (hPL group), each experimental group can not only maintain the performance of HFSCs multi-generation expansion in vitro, but also has a higher proliferation capacity during multiple passages.

[0092] Table 1 Expansion times of HFSCs at each passage

[0093] (4) Detection of HFSCs surface markers

[0094] HFSCs that had been subcultured for more than three times in the experimental groups 1-5 and the control groups 1-2 were selected for the experiment. HFSCs were cultured at 1×10 4 / cm 2 Cells were seeded at a high density in T25 culture flasks and cultured in a 5% CO2 incubator at 37°C. After 3 days, HFSCs from each group were collected by digestion with 0.25% trypsin solution, and the expression of surface markers such as CD105, CD73, CD90, CD11b, CD45, and HLA-DR was detected by flow cytometry (Beckman, model DxFLEX B75145).

[0095] The experimental results, shown in Table 2 and Figures 10-16, demonstrate that both the experimental and control groups showed positive expression of the surface markers CD105, CD73, and CD90, with expression rates exceeding 95%, meeting the MSC reference standard (≥95.0%). On the other hand, CD11b, CD45, and HLA-DR showed negative expression, with expression rates below 2%, meeting the MSC reference standard (≤2.0%). No significant differences were observed between the groups. This indicates that the use of the serum-free culture medium for hair follicle stem cells of the present invention does not affect the expression of surface markers in HFSCs.

[0096] Table 2 Detection results of surface markers of HFSCs in each group

[0097] (5) Detection of multidirectional differentiation potential of HFSCs

[0098] HFSCs that had been subcultured for more than three times in experimental groups 1-5 and control groups 1-2 were selected for the experiment. HFSCs in experimental group 2, control group 1, and control group 2 were cultured at 1×10 5 Each well was seeded in a 6-well plate and cultured in a 5% CO2 incubator at 37°C. When the confluence of HFSCs in each group reached more than 80%, control wells and induction wells were set up to induce osteogenic and adipogenic differentiation of HFSCs.

[0099] After 7 days, cells in the adipogenic differentiation group were stained with Oil Red O, and after 21 days, cells in the osteogenic differentiation group were stained with Alizarin Red. The experimental results, shown in Figures 17-18, demonstrate that the serum-free culture medium for hair follicle stem cells provided by the present invention does not affect the adipogenic differentiation potential of HFSCs and significantly enhances their osteogenic differentiation capacity.

[0100] The serum-free culture medium for hair follicle stem cells provided by the present invention has clear chemical composition and is free of exogenous exosome contamination, and can achieve rapid proliferation of HFSCs in vitro and multiple passages of cells without vacuolation or aging.

[0101] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the present invention. Anyone skilled in the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by one of ordinary skill in the art without departing from the spirit and technical principles disclosed herein are intended to be covered by the claims of the present invention.

Claims

1. A serum-free medium for hair follicle stem cells, characterized in that, The serum-free medium for hair follicle stem cells comprises: MEM non-essential amino acid solution with a volume ratio of 0.1-2%, MEM vitamin solution with a volume ratio of 0.1-2%, L-glutamine solution with a volume ratio of 0.1-2%, 1-8 mg / L of L-glutathione, 0.1-1 μg / L of sodium selenite, 0.5-5 g / L of recombinant human serum albumin, 1-20 mg / L of recombinant human insulin, 1-10 mg / L of recombinant human transferrin, 10-50 μg / L of recombinant human epidermal growth factor, 10-50 μg / L of recombinant human basic fibroblast growth factor, 1-20 μg / L of recombinant human platelet-derived growth factor-AB, 1-50 μg / L of recombinant human epithelial regulatory protein, 12.5-500 mg / L of recombinant human catalase, 5-200 mg / L of L-ascorbic acid, 5-200 μg / L of (+)-α-tocopherol acetate, 0.05-5 mg / L of anisomycin, 0.1-10 mg / L of corticosterone, 25-500 mg / L of L-carnitine, 0.05-5 mg / L of D-galactose, 0.1-10 mg / L of ethanolamine, 25-500 μg / L of linoleic acid, 25-500 μg / L of linolenic acid, 0.05-5 μg / L of progesterone, 50-500 mg / L of putrescine, and the balance of basal medium; The final concentrations of the MEM non-essential amino acid solution, MEM vitamin solution, and L-glutamine solution are all 100×.

2. The serum-free medium for hair follicle stem cells according to claim 1, wherein The serum-free medium for hair follicle stem cells contains MEM non-essential amino acid solution with a volume ratio of 1%, MEM vitamin solution with a volume ratio of 1%, L-glutamine solution with a volume ratio of 1%, 4 mg / L of L-glutathione, 0.5 μg / L of sodium selenite, 2 g / L of recombinant human serum albumin, 10 mg / L of recombinant human insulin, 5 mg / L of recombinant human transferrin, 20 μg / L of recombinant human epidermal growth factor, 20 μg / L of recombinant human basic fibroblast growth factor, 10 μg / L of recombinant human platelet-derived growth factor-AB, 10 μg / L of recombinant human epithelial regulatory protein, 125 mg / L of recombinant human catalase, 50 mg / L of L-ascorbic acid, 50 μg / L of (+)-α-tocopherol acetate, 0.5 mg / L of anisomycin, 1 mg / L of corticosterone, 100 mg / L of L-carnitine, 0.5 mg / L of D-galactose, 2 mg / L of ethanolamine, 100 μg / L of linoleic acid, 100 μg / L of linolenic acid, 0.5 μg / L of progesterone, 200 mg / L of putrescine, and the balance of basal medium.

3. The serum-free medium for hair follicle stem cells according to claim 1 or 2, characterized in that, The basal medium is a serum-free medium; the serum-free medium is IMDM or high-glucose DMEM basal medium.

4. Use of the serum-free medium for hair follicle stem cells according to claim 1 or 2 in stem cell culture; characterized in that, The stem cells are hair follicle stem cells.

5. The application according to claim 4, characterized in that, The serum-free medium for hair follicle stem cells slows down the senescence of hair follicle stem cells during continuous passage and / or maintains the proliferation vitality and passage ability of the hair follicle stem cells.

6. The application according to claim 4, characterized in that The serum-free medium for hair follicle stem cells prevents vacuolization of the hair follicle stem cells during passage culture.

7. A method for preparing a serum-free medium for hair follicle stem cells according to claim 1 or 2, characterized in that, The preparation method comprises the following steps: dissolving according to the dissolution characteristics of each component of the serum-free medium for hair follicle stem cells described in claim 1, mixing evenly to prepare a 1000× solution, filtering and sterilizing with a filter membrane, and storing at -20 to -80 °C for later use.

8. A method for culturing hair follicle stem cells, characterized in that, The culture method comprises the following steps: Inoculating stem cells into the serum-free medium described in claim 1 or 2 for culture; The stem cells are hair follicle stem cells.

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