Amplification culture medium and culture method for human adult epidermal stem cells

Abstract

The invention discloses an amplification culture medium and a culture method of human adult epidermal stem cells, and relates to the technical field of stem cell in-vitro culture. Comprising the following components: a fibroblast growth factor, an interleukin-6, an insulin-like growth factor-1, an epidermal growth factor, insulin, a growth hormone, hydrocortisone, Y-27632, A83-01, Forsklin, PY60, CHIR99021, linoleic acid, linolenic acid, alanine, asparagine, glutamic acid, glycine, proline, serine, arginine, N-acetyl-L-cysteine, beta-mercaptoethanol and CaCl2. The culture medium can be used for continuously and stably culturing human adult epidermal stem cells for more than 20 generations, and a more stable epidermal stem cell long-term in-vitro amplification system is established; the epidermal stem cells can maintain high proliferation rate and multiple differentiation potential after continuous passage, and have the ability to construct multilayer skin.

Description

Technical Field

[0001] This invention relates to the field of in vitro stem cell culture technology, and more specifically to an expansion culture medium and culture method for human adult epidermal stem cells. Background Technology

[0002] Human epidermal stem cells (hEpiSCs) are a key cell population for maintaining skin homeostasis, repairing trauma, and regulating the hair cycle. Traditionally, these cells can be obtained through biopsies of the hair follicle protuberance or basal layer, and possess the potential for long-term self-renewal and directed differentiation into structures such as the epidermis, hair follicles, and sebaceous glands. Therefore, hEpiSCs are not only the core seed cells for regenerative medicine of large-area skin defects such as burns and chronic ulcers, but are also considered an important tool for gene therapy, cosmeceutical screening, and the construction of in vitro skin models.

[0003] However, hEpiSCs are highly susceptible to "stem cell depletion" during in vitro expansion—that is, they rapidly enter terminal differentiation or senescence, leading to a decrease in clonogenic rate and loss of pluripotent markers (such as p63, ITGA6, and KRT15), ultimately resulting in an inability to obtain a sufficient quantity of functionally stable cell products. This bottleneck directly limits their clinical translation and industrial application.

[0004] Existing technologies mainly use commercially available serum-free epidermal keratinocyte culture medium (D-KSFM) and a culture medium based on DMEM / F12 with added B27 and 5% serum (referred to as "DMEM / F12+"). The following common defects exist in this type of culture medium: (1) It depends on animal-derived components, such as bisphenol analog BPE, fetal bovine serum FBS, and cholesterol-rich lipid mix, which brings batch differences, exogenous contamination and ethical regulatory risks; (2) The growth factor concentration window is narrow, which easily leads to "over-differentiation" or "senescence arrest". After 3-4 generations of expansion, the purity of stem cells drops to below 30%; (3) It requires feeder cells, such as mouse embryonic fibroblasts 3T3-J2, dermal fibroblasts HDF or a high-oxygen environment (40% O2) to maintain clonality. The operation process is complicated, costly and difficult to achieve GMP-level scale-up culture; (4) It lacks precise regulation of ROS stress, DNA damage repair and epigenetic homeostasis, resulting in a survival rate of <60% after cryopreservation and thawing and a significant decrease in in vivo regeneration capacity.

[0005] In summary, current technologies have not yet solved the challenge of large-scale expansion of adult human epidermal stem cells (hEpiSCs) that are "adult-derived, animal-free, feeder-free, low-cost, and high-fidelity." Therefore, there is an urgent need for a novel culture medium formulation that can maintain the long-term self-renewal and pluripotency of hEpiSCs under GMP-grade conditions with fully defined chemical compositions, while also ensuring cryopreservation stability and in vivo regenerative function, to meet the pressing needs of clinical-grade skin regeneration, gene therapy, and the construction of in vitro skin models. Summary of the Invention

[0006] In view of this, the present invention provides an expansion culture medium and a culture method for human adult epidermal stem cells.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: An expansion culture medium for human adult epidermal stem cells, characterized by comprising the following components: Fibroblast growth factor 5-20 ng / mL, interleukin-6 5-20 ng / mL, insulin-like growth factor-1 5-20 ng / mL, epidermal growth factor 5-40 ng / mL, insulin 5-20 μg / mL, growth hormone 5-200 ng / mL, hydrocortisone 0.5-2 μg / mL, Y-27632 1-10 μM, A83-01 5-20 μM, Forsklin 5-100 ng / mL, PY60 1-10 μM, CHIR99021 1-10 μM, linoleic acid 0.5-1 mg / mL, linolenic acid 0.5-1 mg / mL mg / mL, alanine 5-20 μg / mL, asparagine 10-25 μg / mL, glutamic acid 10-25 μg / mL, glycine 7.5-30 μg / mL, proline 10-30 μg / mL, serine 10-30 μg / mL, arginine 10-40 μg / mL, N-acetyl-L-cysteine ​​0.5-2.0 mM, β-mercaptoethanol 1-100 μM, CaCl2 0.1-1.0 mM.

[0008] Preferably, it includes the following ingredients: Fibroblast growth factor 10 ng / mL, interleukin-6 10 ng / mL, insulin-like growth factor-1 10 ng / mL, epidermal growth factor 20 ng / mL, insulin 10 μg / mL, growth hormone 100 ng / mL, hydrocortisone 1 μg / mL, Y-27632 5 μM, A83-01 10 μM, Forsklin 50 ng / mL, PY60 5 μM, CHIR99021 1 μM, linoleic acid 0.5 mg / mL, linolenic acid 0.5 mg / mL, alanine 8.9 μg / mL, asparagine 13.3 μg / mL, glutamic acid 14.7 μg / mL, glycine 7.5 μg / mL, proline 11.5 μg / mL, serine 10.5 μg / mL, arginine 21 μg / mL, N-acetyl-L-cysteine ​​1 mM, β-mercaptoethanol 50 μM, CaCl2 0.2 mM.

[0009] Furthermore, the amplification medium uses calcium-free DMEM / F12 as a solvent.

[0010] A method for preparing an expansion culture medium for human adult epidermal stem cells includes the following steps: (1) Fibroblast growth factor, interleukin-6, insulin-like growth factor-1, epidermal growth factor, alanine, asparagine, glutamic acid, glycine, proline, serine, arginine, N-acetyl-L-cysteine, and calcium chloride were dissolved in calcium-free DMEM / F12 to prepare a stock solution. Human insulin was dissolved in 0.01M HCl to prepare a stock solution; Hydrocortisone, Y-27632, A-83-01, Forsklin, linoleic acid, and linolenic acid were dissolved in DMSO to prepare a mother liquor; (2) Add each mother liquor from step (1) to calcium-free DMEM / F12 according to the final concentration of the culture medium.

[0011] Furthermore, the method for preparing the mother liquor in step (1) is as follows: Fibroblast growth factor was dissolved in calcium-free DMEM / F12 to prepare a stock solution of 50ug / mL; Interleukin-6 was dissolved in calcium-free DMEM / F12 to prepare a stock solution of 10 μg / mL; Insulin-like growth factor-1 was dissolved in calcium-free DMEM / F12 to prepare a stock solution of 100 ug / mL; Growth hormone was prepared into a stock solution of 100 ug / mL in DMEM / F12 without calcium ions; Epidermal growth factor was dissolved in calcium-free DMEM / F12 to prepare a stock solution of 50ug / mL; Alanine was dissolved in calcium-free DMEM / F12 to prepare a stock solution of 8.9 mg / mL; Asparagine was dissolved in calcium-free DMEM / F12 to prepare a stock solution of 13.3 mg / mL; Glutamic acid was dissolved in calcium-free DMEM / F12 to prepare a stock solution of 14.7 mg / mL; Glycine was dissolved in calcium-free DMEM / F12 to prepare a stock solution of 7.5 mg / mL; Proline was dissolved in calcium-free DMEM / F12 to prepare a stock solution of 11.5 mg / mL; Serine was dissolved in calcium-free DMEM / F12 to prepare a stock solution of 10.5 mg / mL; Arginine was dissolved in calcium-free DMEM / F12 to prepare a stock solution of 21 mg / mL; β-mercaptoethanol was dissolved in calcium-free DMEM / F12 to prepare a 50 mM stock solution; N-acetyl-L-cysteine ​​was dissolved in calcium-free DMEM / F12 to prepare a 100mM stock solution; Calcium chloride was dissolved in DMEM / F12, a solution free of calcium ions, to prepare a 100 mM stock solution; Human insulin was dissolved in 0.01M HCl overnight to prepare a stock solution of 10 mg / ml; Hydrocortisone was dissolved in DMSO to prepare a stock solution of 1 mg / mL; Y-27632 was dissolved in DMSO to prepare a 100mM stock solution; A-83-01 was dissolved in DMSO to prepare a 10mM stock solution; Forsklin was dissolved in DMSO to prepare a stock solution of 1 mg / mL; PY60 was dissolved in DMSO to prepare a 100 mM stock solution; CHIR99021 was dissolved in DMSO to prepare a 10 mM stock solution; Linoleic acid was dissolved in DMSO to prepare a stock solution of 5 mg / mL; Linolenic acid was dissolved in DMSO to prepare a stock solution of 5 mg / mL.

[0012] A method for culturing human adult epidermal stem cells, comprising the following steps: 1) Human primary epidermal stem cells were cultured in a culture dish coated with rat tail collagen to allow human adult epidermal stem cells to adhere to the dish. 2) Collect adherent human adult epidermal stem cells and culture them in the aforementioned expansion medium to obtain human adult epidermal stem cells.

[0013] Furthermore, in step 2), half of the medium is replaced every other day during the culture process.

[0014] A type of human adult epidermal stem cell, cells obtained by culturing using the aforementioned expansion culture medium or the aforementioned culture method.

[0015] Application of a human adult epidermal stem cell expansion culture medium in the preparation of expanded epidermal stem cell products.

[0016] Applications of human adult epidermal stem cells for non-diagnostic and therapeutic purposes, wherein the application is any one of the following: A: In vitro study of the regulatory mechanisms of epidermal stem cell proliferation, differentiation, and death; B: Evaluation of the efficacy of skin cell therapy; C: Construction of functional skin organoids; D: Drug development, screening, and pharmacological efficacy evaluation for skin diseases; E: Research on skin aging mechanisms and evaluation of cosmetic efficacy.

[0017] As can be seen from the above technical solution, compared with the prior art, the beneficial effects of the present invention are as follows: The culture medium of this invention can continuously and stably culture human adult epidermal stem cells for more than 20 generations. Compared with the existing culture systems in the world, it establishes a more stable long-term in vitro expansion system for epidermal stem cells. After continuous passage, epidermal stem cells can maintain a high proliferation rate and multiple differentiation potential, and have the ability to construct multilayer skin. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0019] Figure 1 This is a morphological diagram of the P1 generation human adult epidermal stem cells cultured in Example 1 of the present invention. Figure 2 Morphological images of human adult epidermal stem cells P1-P3 generation cultured in commercial culture medium according to Example 1 of the present invention; Figure 3 This is a graph showing the in vitro expansion rate of human adult epidermal stem cells cultured in Example 2 of the present invention; Figure 4 This is a cell morphology diagram of human adult epidermal stem cells cultured in Example 2 of the present invention expanded to generation P20. Figure 5Identification of marker genes in P1 and P20 generation human adult epidermal stem cells cultured in Example 3 of this invention; Figure 6 The images show the cell morphology of human adult epidermal stem cells cultured at different passages in Example 4 of this invention. Figure 7 Identification of stem cell markers for P1 and P10 generation human adult epidermal stem cells cultured in Example 5 of this invention. Figure 8 The differentiation capacity of P20 generation human adult epidermal stem cells cultured in Example 6 of this invention. Detailed Implementation

[0020] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] Source of experimental materials: Fibroblast growth factor: MCE; FGF basic / bFGF protein; Human (154a.a), HY-P7331; Interleukin-6: MCE; IL-6 protein, Human; HY-P7044; Insulin-like growth factor-1: MCE; IGF-I / IGF-1 protein, Human (70a.a); HY-P7018; Epidermal growth factor: MCE; EGF protein, Human; HY-P7109; Insulin: Yisheng; Insulin human recombinant; 40112ES25; Growth hormone: MCE; GH / Somatotropin protein, Human (CHO); HY-P7360; Hydrocortisone: MCE; Hydrocortisone; HY-N0583; Y-27632:MCE;Y-27632;HY-10071; A83-01:MCE;A 83-01;HY-10432; Forsklin:MCE;Forskolin;HY-15371; PY60:MCE;PY-60;HY-141644; CHIR99021: MCE; laduviglusib; HY-10182; Linoleic acid: MERK; α-Linoleic acid; L1376; Linolelaidic acid: Yisheng; 718568ES50; β-Mercaptoethanol: MCE; 2-Mercaptoethanol; HY-Y0326; Calcium-free DMEM / F12: Shanghai Yuanpei Biotechnology; Calcium-free DMEM / F12; K421316 D-KSFM: ThermoFisher; Keratinocyte SFM (1X); 17005042; DMEM / F12: ThermoFisher; DMEM / F-12; 11320033; B27: ThermoFisher; B-27™ Additive (50X); 17504044; Serum: ThermoFisher; Fetal bovine serum, premium grade, New Zealand; 10091148.

[0022] Unless otherwise specified, all reagents involved in the examples can be purchased through commercial channels. Methods not mentioned are conventional experimental methods and will not be described in detail here.

[0023] Example 1 A method for preparing an expansion culture medium for human adult epidermal stem cells Step (1) Preparation of the stock solutions of each component in the culture medium Fibroblast growth factor was dissolved in calcium-free DMEM / F12 to prepare a stock solution of 50ug / mL; Interleukin-6 was dissolved in calcium-free DMEM / F12 to prepare a stock solution of 10 μg / mL; Insulin-like growth factor-1 was dissolved in calcium-free DMEM / F12 to prepare a stock solution of 100 ug / mL; Growth hormone was prepared into a stock solution of 100 ug / mL in DMEM / F12 without calcium ions; Epidermal growth factor was dissolved in calcium-free DMEM / F12 to prepare a stock solution of 50ug / mL; Alanine was dissolved in calcium-free DMEM / F12 to prepare a stock solution of 8.9 mg / mL; Asparagine was dissolved in calcium-free DMEM / F12 to prepare a stock solution of 13.3 mg / mL; Glutamic acid was dissolved in calcium-free DMEM / F12 to prepare a stock solution of 14.7 mg / mL; Glycine was dissolved in calcium-free DMEM / F12 to prepare a stock solution of 7.5 mg / mL; Proline was dissolved in calcium-free DMEM / F12 to prepare a stock solution of 11.5 mg / mL; Serine was dissolved in calcium-free DMEM / F12 to prepare a stock solution of 10.5 mg / mL; Arginine was dissolved in calcium-free DMEM / F12 to prepare a stock solution of 21 mg / mL; β-mercaptoethanol was dissolved in calcium-free DMEM / F12 to prepare a 50 mM stock solution; N-acetyl-L-cysteine ​​was dissolved in calcium-free DMEM / F12 to prepare a 100mM stock solution; Calcium chloride was dissolved in DMEM / F12, a solution free of calcium ions, to prepare a 100 mM stock solution; Human insulin was dissolved in 0.01M HCl overnight to prepare a stock solution of 10 mg / ml; Hydrocortisone was dissolved in DMSO to prepare a stock solution of 1 mg / mL; Y-27632 was dissolved in DMSO to prepare a 100mM stock solution; A-83-01 was dissolved in DMSO to prepare a 10mM stock solution; Forsklin was dissolved in DMSO to prepare a stock solution of 1 mg / mL; PY60 was dissolved in DMSO to prepare a 100mM stock solution; CHIR99021 was dissolved in DMSO to prepare a 10mM stock solution; Linoleic acid was dissolved in DMSO to prepare a stock solution of 5 mg / mL; Linolenic acid was dissolved in DMSO to prepare a stock solution of 5 mg / mL.

[0024] Step (2) Preparation of culture medium The stock solutions from step (1) were sequentially added to calcium-free DMEM / F12 to prepare the final culture medium. The final culture medium consisted of: fibroblast growth factor (10 ng / mL), interleukin-6 (10 ng / mL), insulin-like growth factor-1 (10 ng / mL), epidermal growth factor (20 ng / mL), insulin (10 μg / mL), growth hormone (100 ng / mL), hydrocortisone (1 μg / mL), Y-27632 (5 μM), A83-01 (10 μM), Forsklin (50 ng / mL), and PY60. (5 μM), CHIR99021 (1 μM), linoleic acid (0.5 mg / mL), linolenic acid (0.5 mg / mL), alanine (8.9 μg / mL), asparagine (13.3 μg / mL), glutamic acid (14.7 μg / mL), glycine (7.5 μg / mL), proline (11.5 μg / mL), serine (10.5 μg / mL), arginine (21 μg / mL), N-acetyl-L-cysteine ​​(1 mM), β-mercaptoethanol (75 μM), CaCl2 (0.2 mM).

[0025] Step (3) Expansion and culture of human adult epidermal stem cells Adult foreskin tissue was trimmed into small pieces of approximately 1 mm × 1 mm using sterile scissors. Subsequently, the dermis and epidermis were separated by digestion with 2 mg / mL Dispase II enzyme at 37°C for 2 hours. The separated epidermal tissue was collected and further digested with 0.25% trypsin at 37°C for 15 minutes. After digestion, DMEM / F12 containing 20% ​​fetal bovine serum (FBS) was added to terminate the reaction. The cell suspension was then filtered and centrifuged to obtain a single-cell suspension of epidermal cells.

[0026] The resulting cells were seeded into culture dishes pretreated with rat tail collagen and cultured using the medium from step (2). Initial culture for 48 hours was allowed to promote epidermal stem cell adhesion, after which the medium was changed every two days. The first passage was performed on day 14. In subsequent cultures, when cells covered 80% of the bottom area of ​​the culture dish, passages were performed at a ratio of 1:5.

[0027] Figure 1 The cell morphology observed at different time points when P1 generation human adult epidermal stem cells were cultured using the culture medium provided in Example 1 of the present invention is shown.

[0028] like Figure 1As shown, throughout the culture process, the cells consistently exhibited typical small spindle-shaped and round morphologies and maintained good cell refractivity, which is a direct morphological indicator that these stem cells have not differentiated and are in a healthy state. Most importantly, no morphological changes indicating cell senescence, such as cell enlargement, spreading, or vacuolization, were observed during the culture period.

[0029] To evaluate the performance of the culture medium provided in Example 1 of this invention, two control culture media were set up for parallel comparison: commercial epidermal keratinocyte serum-free culture medium (D-KSFM) and a culture medium based on DMEM / F12 with added B27 and 5% serum (DMEM / F12+).

[0030] like Figure 2 As shown, compared with the culture medium of the present invention, the two control culture media are significantly insufficient in maintaining the stemness and proliferation capacity of epidermal stem cells. Specifically, cells cultured using D-KSFM and DMEM / F12+ control culture media showed obvious signs of cell senescence or differentiation by the second generation, including changes in cell morphology, the appearance of tentacled-like protrusions at the edges, significant spreading and thinning of the cell body, and an increase in specific surface area.

[0031] Furthermore, when the culture entered the third generation, the cells in both control culture media lost their ability to adhere to the culture medium, resulting in a large number of cells floating and making it virtually impossible to form a stable adherent cell layer, thus making effective passage culture difficult.

[0032] The results of this control experiment clearly demonstrate that existing commercial culture media and conventional formulations containing serum supplementation cannot support long-term in vitro expansion of epidermal stem cells, thus proving the significant advantages and inventiveness of the culture medium of this invention in maintaining cell stemness, promoting stable proliferation, and extending the culture period.

[0033] Example 2 A method for preparing an expansion culture medium for human adult epidermal stem cells: Step (1) Preparation of the stock solutions of each component in the culture medium Fibroblast growth factor was dissolved in calcium-free DMEM / F12 to prepare a stock solution of 75ug / mL; Interleukin-6 was dissolved in calcium-free DMEM / F12 to prepare a stock solution of 15ug / mL; Insulin-like growth factor-1 was dissolved in calcium-free DMEM / F12 to prepare a stock solution of 150 ug / mL; Growth hormone was prepared into a stock solution of 150 ug / mL in DMEM / F12 without calcium ions; Epidermal growth factor was dissolved in calcium-free DMEM / F12 to prepare a stock solution of 75ug / mL; Alanine was dissolved in calcium-free DMEM / F12 to prepare a stock solution of 15 mg / mL; Asparagine was dissolved in calcium-free DMEM / F12 to prepare a stock solution of 18 mg / mL; Glutamic acid was dissolved in calcium-free DMEM / F12 to prepare a stock solution of 20 mg / mL; Glycine was dissolved in calcium-free DMEM / F12 to prepare a stock solution of 15 mg / mL; Proline was dissolved in calcium-free DMEM / F12 to prepare a stock solution of 20 mg / mL; Serine was dissolved in calcium-free DMEM / F12 to prepare a stock solution of 20 mg / mL; Arginine was dissolved in calcium-free DMEM / F12 to prepare a stock solution of 30 mg / mL; β-mercaptoethanol was dissolved in calcium-free DMEM / F12 to prepare a 75 mM stock solution; N-acetyl-L-cysteine ​​was dissolved in calcium-free DMEM / F12 to prepare a 150mM stock solution; Calcium chloride was dissolved in DMEM / F12, a solution free of calcium ions, to prepare a 150 mM stock solution. Human insulin was dissolved in 0.01M HCl overnight to prepare a stock solution of 15 mg / ml; Hydrocortisone was dissolved in DMSO to prepare a stock solution of 2 mg / mL; Y-27632 was dissolved in DMSO to prepare a 150mM stock solution; A-83-01 was dissolved in DMSO to prepare a 15mM stock solution; Forsklin was dissolved in DMSO to prepare a stock solution of 2 mg / mL; PY60 was dissolved in DMSO to prepare a 150mM stock solution; CHIR99021 was dissolved in DMSO to prepare a 15mM stock solution; Linoleic acid was dissolved in DMSO to prepare a stock solution with a concentration of 10 mg / mL; Linolenic acid was dissolved in DMSO to prepare a stock solution of 10 mg / mL.

[0034] Step (2) Preparation of culture medium The stock solutions from step (1) were sequentially added to calcium-free DMEM / F12 to prepare the final culture medium. The final culture medium consisted of: fibroblast growth factor (15 ng / mL), interleukin-6 (15 ng / mL), insulin-like growth factor-1 (15 ng / mL), epidermal growth factor (30 ng / mL), insulin (15 μg / mL), growth hormone (150 ng / mL), hydrocortisone (1.5 μg / mL), Y-27632 (7.5 μM), A83-01 (15 μM), Forsklin (75 ng / mL), and PY60. (7.5 μM), CHIR99021 (1.5 μM), linoleic acid (0.75 mg / mL), linolenic acid (0.75 mg / mL), alanine (12.5 μg / mL), asparagine (17.5 μg / mL), glutamic acid (20 μg / mL), glycine (10 μg / mL), proline (15 μg / mL), serine (15 μg / mL), arginine (30 μg / mL), N-acetyl-L-cysteine ​​(1.5 mM), β-mercaptoethanol (75 μM), CaCl2 (0.5 mM).

[0035] Step (3) Expansion and culture of human adult epidermal stem cells Adult foreskin tissue was trimmed into small pieces of approximately 1 mm × 1 mm using sterile scissors. Subsequently, the dermis and epidermis were separated by digestion with 1 mg / mL Dispase II enzyme at 37°C for 4 hours. The separated epidermal tissue was collected and further digested with 0.15% trypsin at 37°C for 30 minutes. After digestion, DMEM / F12 containing 20% ​​fetal bovine serum (FBS) was added to terminate the reaction. The cell suspension was then filtered and centrifuged to obtain a single-cell suspension of epidermal cells.

[0036] The resulting cells were seeded into culture dishes pretreated with rat tail collagen and cultured using the medium from step (2). Initial culture was performed for 48 hours to promote epidermal stem cell adhesion, followed by changing the medium every two days. The first passage was performed on day 14. In subsequent cultures, when cells covered 80% of the bottom area of ​​the culture dish, passages were performed at a ratio of 1:3.

[0037] Figure 3 This is a graph showing the in vitro expansion rate of human adult epidermal stem cells cultured in Example 2 of the present invention. As can be seen from the graph, the culture medium of the present invention can maintain stable in vitro expansion of human adult epidermal stem cells for at least 20 generations.

[0038] Figure 4This is a cell morphology diagram of human adult epidermal stem cells cultured in Example 2 of the present invention, expanded to passage P20. As can be seen from the figure, after 20 passages of culture, the cells still maintain the typical small spindle-shaped or round stem cell morphology and exhibit excellent refractive properties, with no obvious signs of cell senescence observed.

[0039] The above results demonstrate that the culture medium of the present invention can effectively support the long-term in vitro expansion of human adult epidermal stem cells.

[0040] Example 3 A method for preparing an expansion culture medium for human adult epidermal stem cells: Step (1) Preparation of the stock solutions of each component in the culture medium Fibroblast growth factor was dissolved in calcium-free DMEM / F12 to prepare a stock solution of 10ug / mL; Interleukin-6 was dissolved in calcium-free DMEM / F12 to prepare a stock solution of 5 μg / mL; Insulin-like growth factor-1 was dissolved in calcium-free DMEM / F12 to prepare a stock solution of 10 μg / mL; Growth hormone was prepared as a stock solution of 10ug / mL in DMEM / F12 without calcium ions; Epidermal growth factor was dissolved in calcium-free DMEM / F12 to prepare a stock solution of 10ug / mL; Alanine was dissolved in calcium-free DMEM / F12 to prepare a stock solution of 5 mg / mL; Asparagine was dissolved in calcium-free DMEM / F12 to prepare a stock solution of 10 mg / mL; Glutamic acid was dissolved in calcium-free DMEM / F12 to prepare a stock solution of 10 mg / mL; Glycine was dissolved in calcium-free DMEM / F12 to prepare a stock solution of 10 mg / mL; Proline was dissolved in calcium-free DMEM / F12 to prepare a stock solution of 10 mg / mL; Serine was dissolved in calcium-free DMEM / F12 to prepare a stock solution of 10 mg / mL; Arginine was dissolved in calcium-free DMEM / F12 to prepare a stock solution of 10 mg / mL; β-mercaptoethanol was dissolved in calcium-free DMEM / F12 to prepare a 20 mM stock solution; N-acetyl-L-cysteine ​​was dissolved in calcium-free DMEM / F12 to prepare a 20mM stock solution; Calcium chloride was dissolved in DMEM / F12, a solution free of calcium ions, to prepare a 10 mM stock solution; Human insulin was dissolved in 0.01M HCl overnight to prepare a stock solution of 5 mg / ml; Hydrocortisone was dissolved in DMSO to prepare a stock solution of 0.5 mg / mL; Y-27632 was dissolved in DMSO to prepare a 10mM stock solution; A-83-01 was dissolved in DMSO to prepare a 10mM stock solution; Forsklin was dissolved in DMSO to prepare a stock solution of 1 mg / mL; PY60 was dissolved in DMSO to prepare a 50mM stock solution; CHIR99021 was dissolved in DMSO to prepare a 5mM stock solution; Linoleic acid was dissolved in DMSO to prepare a stock solution with a concentration of 1 mg / mL; Linolenic acid was dissolved in DMSO to prepare a stock solution of 1 mg / mL.

[0041] Step (2) Preparation of culture medium The mother solutions from step (1) were sequentially added to calcium-free DMEM / F12 to prepare the final culture medium. The final culture medium consisted of: fibroblast growth factor (5 ng / mL), interleukin-6 (5 ng / mL), insulin-like growth factor-1 (5 ng / mL), epidermal growth factor (10 ng / mL), insulin (5 μg / mL), growth hormone (10 ng / mL), hydrocortisone (1 μg / mL), Y-27632 (5 μM), A83-01 (5 μM), Forsklin (5 ng / mL), and PY60. (2 μM), CHIR99021 (2 μM), linoleic acid (0.5 mg / mL), linolenic acid (0.5 mg / mL), alanine (10 μg / mL), asparagine (10 μg / mL), glutamic acid (10 μg / mL), glycine (10 μg / mL), proline (10 μg / mL), serine (10 μg / mL), arginine (10 μg / mL), N-acetyl-L-cysteine ​​(1 mM), β-mercaptoethanol (10 μM), CaCl2 (0.2 mM).

[0042] Step (3) Expansion and culture of human adult epidermal stem cells Adult foreskin tissue was trimmed into small pieces of approximately 1 mm × 1 mm using sterile scissors. Subsequently, the dermis and epidermis were separated by digestion with 2 mg / mL Dispase II enzyme at 37°C for 2 hours. The separated epidermal tissue was collected and further digested with 0.25% trypsin at 37°C for 15 minutes. After digestion, DMEM / F12 containing 20% ​​fetal bovine serum (FBS) was added to terminate the reaction. The cell suspension was then filtered and centrifuged to obtain a single-cell suspension of epidermal cells.

[0043] The resulting cells were seeded into culture dishes pretreated with rat tail collagen and cultured using the medium from step (2). Initial culture for 48 hours was allowed to promote epidermal stem cell adhesion, after which the medium was changed every two days. The first passage was performed on day 14. In subsequent cultures, when cells covered 80% of the bottom area of ​​the culture dish, passages were performed at a ratio of 1:4.

[0044] Figure 5 This invention relates to the identification of marker genes in P1 and P20 generation human adult epidermal stem cells cultured in Example 3 of this invention. As shown in the figure, the human adult epidermal stem cells cultured in vitro for the 20th generation still expressed multiple epidermal stem cell genes, indicating that the epidermal stem cells cultured in this medium have high differentiation potential.

[0045] The specific method for identifying the expression of epidermal stem cell genes in in vitro amplified human adult epidermal stem cells using real-time quantitative PCR was as follows: Cells cultured for passages 1 and 20, and human skin tissue were thoroughly lysed with Trizol. Half a volume of chloroform was added, and the mixture was centrifuged at 12,000 rpm for 15 minutes at 4°C. The supernatant was collected, and an equal volume of isopropanol was added and thoroughly mixed. The mixture was allowed to stand at room temperature for 10 minutes, then centrifuged at 12,000 rpm for 15 minutes at 4°C. The precipitate was then thoroughly washed with 75% ethanol and air-dried at room temperature until translucent. RNA was dissolved in water. Reverse transcription of RNA was performed using a reverse transcription kit (Vazyme, #R323) according to the manufacturer's instructions. The expression levels of the corresponding genes were detected by real-time quantitative PCR. The reaction system for real-time quantitative PCR is shown in Table 1, the reaction conditions in Table 2, and the primer sequences in Table 3.

[0046] Table 1. Reaction system for real-time quantitative PCR

[0047] Table 2. Reaction conditions for real-time quantitative PCR

[0048] Table 3. Primer sequences for real-time quantitative PCR

[0049] Example 4 A method for preparing an expansion culture medium for human adult epidermal stem cells: Step (1) Preparation of the stock solutions of each component in the culture medium Fibroblast growth factor was dissolved in calcium-free DMEM / F12 to prepare a stock solution of 100 ug / mL; Interleukin-6 was dissolved in calcium-free DMEM / F12 to prepare a stock solution of 100 μg / mL; Insulin-like growth factor-1 was dissolved in calcium-free DMEM / F12 to prepare a stock solution of 100 ug / mL; Growth hormone was dissolved in calcium-free DMEM / F12 to prepare a stock solution of 100 ug / mL; Epidermal growth factor was dissolved in calcium-free DMEM / F12 to prepare a stock solution of 100ug / mL; Alanine was dissolved in calcium-free DMEM / F12 to prepare a stock solution of 20 mg / mL; Asparagine was dissolved in calcium-free DMEM / F12 to prepare a stock solution of 20 mg / mL; Glutamic acid was dissolved in calcium-free DMEM / F12 to prepare a stock solution of 20 mg / mL; Glycine was dissolved in calcium-free DMEM / F12 to prepare a stock solution of 20 mg / mL; Proline was dissolved in calcium-free DMEM / F12 to prepare a stock solution of 20 mg / mL; Serine was dissolved in calcium-free DMEM / F12 to prepare a stock solution of 20 mg / mL; Arginine was dissolved in calcium-free DMEM / F12 to prepare a stock solution of 20 mg / mL; β-mercaptoethanol was dissolved in calcium-free DMEM / F12 to prepare a 50 mM stock solution; N-acetyl-L-cysteine ​​was dissolved in calcium-free DMEM / F12 to prepare a 200mM stock solution; Calcium chloride was dissolved in DMEM / F12, a solution free of calcium ions, to prepare a 200 mM stock solution; Human insulin was dissolved in 0.01M HCl overnight to prepare a stock solution of 20 mg / ml; Hydrocortisone was dissolved in DMSO to prepare a stock solution of 5 mg / mL; Y-27632 was dissolved in DMSO to prepare a 200mM stock solution; A-83-01 was dissolved in DMSO to prepare a 20mM stock solution; Forsklin was dissolved in DMSO to prepare a stock solution of 5 mg / mL; PY60 was dissolved in DMSO to prepare a 100mM stock solution; CHIR99021 was dissolved in DMSO to prepare a 10mM stock solution; Linoleic acid was dissolved in DMSO to prepare a stock solution of 5 mg / mL; Linolenic acid was dissolved in DMSO to prepare a stock solution of 5 mg / mL.

[0050] Step (2) Preparation of culture medium The stock solutions from step (1) were sequentially added to calcium-free DMEM / F12 to prepare the final culture medium. The final culture medium consisted of: fibroblast growth factor (20 ng / mL), interleukin-6 (20 ng / mL), insulin-like growth factor-1 (20 ng / mL), epidermal growth factor (40 ng / mL), insulin (20 μg / mL), growth hormone (200 ng / mL), hydrocortisone (2 μg / mL), Y-27632 (10 μM), A83-01 (20 μM), Forsklin (100 ng / mL), and PY60. (5 μM), CHIR99021 (5 μM), linoleic acid (1 mg / mL), linolenic acid (1 mg / mL), alanine (20 μg / mL), asparagine (20 μg / mL), glutamic acid (20 μg / mL), glycine (20 μg / mL), proline (20 μg / mL), serine (20 μg / mL), arginine (20 μg / mL), N-acetyl-L-cysteine ​​(2 mM), β-mercaptoethanol (100 μM), CaCl2 (1 mM).

[0051] Step (3) Expansion and culture of human adult epidermal stem cells Adult foreskin tissue was trimmed into small pieces of approximately 1 mm × 1 mm using sterile scissors. Subsequently, the dermis and epidermis were separated by digestion with 2 mg / mL Dispase II enzyme at 37°C for 2 hours. The separated epidermal tissue was collected and further digested with 0.25% trypsin at 37°C for 15 minutes. After digestion, DMEM / F12 containing 20% ​​fetal bovine serum (FBS) was added to terminate the reaction. The cell suspension was then filtered and centrifuged to obtain a single-cell suspension of epidermal cells.

[0052] The resulting cells were seeded into culture dishes pretreated with rat tail collagen and cultured using the medium from step (2). Initial culture for 48 hours was allowed to promote epidermal stem cell adhesion, after which the medium was changed every two days. The first passage was performed on day 14. In subsequent cultures, when cells covered 80% of the bottom area of ​​the culture dish, passages were performed at a ratio of 1:5.

[0053] Figure 6 The figures show the cell morphology of human adult epidermal stem cells cultured at different passages in Example 4 of this invention. As can be seen from the figures, the culture medium and method support the stable expansion and culture of human adult epidermal stem cells in vitro to more than 20 passages. The cells maintain the typical small spindle-shaped or round stem cell morphology and exhibit excellent refractive properties; no obvious signs of cell senescence were observed.

[0054] Example 5 A method for preparing an expansion culture medium for human adult epidermal stem cells: Step (1) Preparation of the stock solutions of each component in the culture medium Consistent with that in Example 2.

[0055] Step (2) Preparation of culture medium The stock solutions from step (1) were sequentially added to calcium-free DMEM / F12 to prepare the final culture medium. The final culture medium consisted of: fibroblast growth factor (10 ng / mL), interleukin-6 (10 ng / mL), insulin-like growth factor-1 (10 ng / mL), epidermal growth factor (10 ng / mL), insulin (10 μg / mL), growth hormone (50 ng / mL), hydrocortisone (1 μg / mL), Y-27632 (5 μM), A83-01 (10 μM), Forsklin (50 ng / mL), and PY60. (5 μM), CHIR99021 (1 μM), linoleic acid (1 mg / mL), linolenic acid (1 mg / mL), alanine (15 μg / mL), asparagine (25 μg / mL), glutamic acid (25 μg / mL), glycine (30 μg / mL), proline (30 μg / mL), serine (30 μg / mL), arginine (40 μg / mL), N-acetyl-L-cysteine ​​(2 mM), β-mercaptoethanol (50 μM), CaCl2 (0.5 mM).

[0056] Step (3) Expansion and culture of human adult epidermal stem cells Consistent with that in Example 1.

[0057] Figure 7 This invention provides an identification of stem cell markers for P1 and P10 generation human adult epidermal stem cells cultured in Example 5 of this invention. As shown in the figure, human adult epidermal stem cells amplified to generation 10 using the culture medium of this invention still stably express key stem cell markers, including transcription factor P63 (a marker of proliferative potential cells in the basal layer of the epidermis), KRT14 (a marker of basal stem cells), ITGA6 (CD49f), and ITGB1 (CD29). ITGA6 and ITGB1 are integrin molecules, enriched on the stem cell membrane surface, demonstrating that this culture medium system can effectively support the maintenance of undifferentiated state and multi-lineage differentiation potential of human adult epidermal stem cells during long-term in vitro culture.

[0058] The specific steps for biomarker identification are as follows: Cells passaged 10 times consecutively were selected. When the cells reached approximately 80% confluence, the culture medium was discarded, and the cells were gently washed with pre-cooled PBS. Then, 4% paraformaldehyde solution was added, and the cells were fixed at room temperature for 30 minutes. After fixation, the cells were washed again with PBS. Next, specific primary antibodies targeting key biomarkers of epidermal stem cells, including transcription factors P63, KRT14, ITGA6, and ITGB1, were added. The cells and primary antibodies were incubated overnight at 4°C. The next day, the cells were washed three times thoroughly with PBS to remove unbound primary antibodies. Then, a fluorescently labeled secondary antibody matching the species of the primary antibody was added, and the cells were incubated at room temperature in the dark for 1 hour. After incubation, the cells were washed three times again with PBS. Finally, the cell nuclei were counterstained with DAPI, incubated at room temperature for 5 minutes, washed with PBS, and observed and images were acquired under a fluorescence microscope.

[0059] Example 6 A method for preparing an expansion culture medium for human adult epidermal stem cells: Step (1) Preparation of the stock solutions of each component in the culture medium Consistent with that in Example 1.

[0060] Step (2) Preparation of culture medium The stock solutions from step (1) were sequentially added to calcium-free DMEM / F12 to prepare the final culture medium. The final culture medium consisted of: fibroblast growth factor (10 ng / mL), interleukin-6 (10 ng / mL), insulin-like growth factor-1 (5 ng / mL), epidermal growth factor (5 ng / mL), insulin (5 μg / mL), growth hormone (5 ng / mL), hydrocortisone (0.5 μg / mL), Y-27632 (1 μM), A83-01 (5 μM), Forsklin (5 ng / mL), and PY60. (10 μM), CHIR99021 (3 μM) Linoleic acid (1 mg / mL), Linolenic acid (1 mg / mL), Alanine (5 μg / mL), Asparagine (20 μg / mL), Glutamic acid (20 μg / mL), Glycine (7.5 μg / mL), Proline (20 μg / mL), Serine (20 μg / mL), Arginine (20 μg / mL), N-acetyl-L-cysteine ​​(0.5 mM), β-mercaptoethanol (20 μM), CaCl2 (0.1 mM).

[0061] Step (3) Expansion and culture of human adult epidermal stem cells Consistent with that in Example 1.

[0062] Figure 8The differentiation capacity of P20 generation human adult epidermal stem cells cultured in Example 6 of this invention. As shown in the figure, after 20 generations of long-term in vitro expansion, the human adult epidermal stem cells cultured in this medium still retain the ability to differentiate into functional epidermal cells, including terminally differentiated epidermal keratinocytes (KRT10) and the outermost keratinocytes (Loricrin and Filaggrin-labeled keratin capsule), confirming that their excellent multi-lineage differentiation potential can be maintained for a long time.

[0063] Identification of the differentiation potential of epidermal stem cells: Cells passaged 20 times were selected for differentiation induction experiments. On day 3 of cell passage, differentiation was induced using the gas-liquid interface contact method, and the cells were cultured for another 14 days in differentiation induction medium to stimulate their potential to differentiate into different epidermal lineages. After induction, the cells were fixed with 4% paraformaldehyde solution at room temperature for 30 minutes. After fixation, the cells were thoroughly washed with PBS. Subsequently, specific antibodies KRT10, Loricrin, and Filagrin, which are labeled for different terminally differentiated epidermal cells, were added. After incubating the antibodies and cells overnight at 4°C, the cells were washed three times with PBS; then, the corresponding fluorescently labeled secondary antibodies were added, and the cells were incubated at room temperature in the dark for 1 hour; after washing three more times with PBS, the cell nuclei were counterstained with DAPI for 5 minutes; after a final three washes with PBS, the cells were observed and images were acquired under a fluorescence microscope.

[0064] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. An expansion medium for human adult epidermal stem cells, characterized in that, fibroblast growth factor 5-20 ng / mL, interleukin-6 5-20 ng / mL, insulin-like growth factor-1 5-20 ng / mL, epidermal growth factor 5-40 ng / mL, insulin 5-20 μg / mL, growth hormone 5-200 ng / mL, hydrocortisone 0.5-2 μg / mL, Y-27632 1-10 μM, A83-01 5-20 μM, Forsklin 5-100 ng / mL, PY60 1-10 μM, CHIR99021 1-10 μM, linoleic acid 0.5-1 mg / mL, linolenic acid 0.5-1 mg / mL, alanine 5-20 μg / mL, asparagine 10-25 μg / mL, glutamic acid 10-25 μg / mL, glycine 7.5-30 μg / mL, proline 10-30 μg / mL, serine 10-30 μg / mL, arginine 10-40 μg / mL, N-acetyl-L-cysteine 0.5-2.0 mM, β-mercaptoethanol 1-100 μM, CaCl2 0.1-1.0 mM. fibroblast growth factor 5-20 ng / mL, interleukin-6 5-20 ng / mL, insulin-like growth factor-1 5-20 ng / mL, epidermal growth factor 5-40 ng / mL, insulin 5-20 μg / mL, growth hormone 5-200 ng / mL, hydrocortisone 0.5-2 μg / mL, Y-27632 1-10 μM, A83-01 5-20 μM, Forsklin 5-100 ng / mL, PY60 1-10 μM, CHIR99021 1-10 μM, linoleic acid 0.5-1 mg / mL, linolenic acid 0.5-1 mg / mL, alanine 5-20 μg / mL, asparagine 10-25 μg / mL, glutamic acid 10-25 μg / mL, glycine 7.5-30 μg / mL, proline 10-30 μg / mL, serine 10-30 μg / mL, arginine 10-40 μg / mL, N-acetyl-L-cysteine 0.5-2.0 mM, β-mercaptoethanol 1-100 μM, CaCl2 0.1-1.0 mM.

2. The expansion medium of claim 1, wherein, The expansion medium uses calcium ion-free DMEM / F12 as a solvent. The method comprises the following steps:

3. The expansion medium according to any one of claims 1 or 2, characterized in that, (1) respectively dissolve fibroblast growth factor, interleukin-6, insulin-like growth factor-1, epidermal growth factor, alanine, asparagine, glutamic acid, glycine, proline, serine, arginine, N-acetyl-L-cysteine and calcium chloride in calcium ion-free DMEM / F12 to prepare a mother liquor; 4. The method of claim 1, wherein the preparation of the expansion medium for human somatic epidermal stem cells is characterized by, dissolve human insulin in 0.01M HCl to prepare a mother liquor; dissolve hydrocortisone, Y-27632, A-83-01, Forsklin, linoleic acid and linolenic acid in DMSO to prepare a mother liquor; ​ ​ (2) Add each stock solution in step (1) to the calcium ion-free DMEM / F12 according to the final concentration of the medium.

5. The preparation method according to claim 4, characterized in that, The preparation method of the stock solution in step (1) is as follows: Fibroblast growth factor is dissolved in calcium ion-free DMEM / F12 to prepare a stock solution of 50 ug / mL; Interleukin-6 is dissolved in calcium ion-free DMEM / F12 to prepare a stock solution of 10 ug / mL; Insulin-like growth factor-1 is dissolved in calcium ion-free DMEM / F12 to prepare a stock solution of 100 ug / mL; Growth hormone is dissolved in calcium ion-free DMEM / F12 to prepare a stock solution of 100 ug / mL; Epidermal growth factor is dissolved in calcium ion-free DMEM / F12 to prepare a stock solution of 50 ug / mL; Alanine is dissolved in calcium ion-free DMEM / F12 to prepare a stock solution of 8.9 mg / mL; Asparagine is dissolved in calcium ion-free DMEM / F12 to prepare a stock solution of 13.3 mg / mL; Glutamic acid is dissolved in calcium ion-free DMEM / F12 to prepare a stock solution of 14.7 mg / mL; Glycine is dissolved in calcium ion-free DMEM / F12 to prepare a stock solution of 7.5 mg / mL; Proline is dissolved in calcium ion-free DMEM / F12 to prepare a stock solution of 11.5 mg / mL; Serine is dissolved in calcium ion-free DMEM / F12 to prepare a stock solution of 10.5 mg / mL; Arginine is dissolved in calcium ion-free DMEM / F12 to prepare a stock solution of 21 mg / mL; β-Mercaptoethanol is dissolved in calcium ion-free DMEM / F12 to prepare a stock solution of 50 mM; N-Acetyl-L-cysteine is dissolved in calcium ion-free DMEM / F12 to prepare a stock solution of 100 mM; Calcium chloride is dissolved in calcium ion-free DMEM / F12 to prepare a stock solution of 100 mM; Human insulin is dissolved in 0.01 M HCl overnight to prepare a stock solution of 10 mg / mL; Hydrocortisone is dissolved in DMSO to prepare a stock solution of 1 mg / mL; Y-27632 is dissolved in DMSO to prepare a stock solution of 100 mM; A-83-01 is dissolved in DMSO to prepare a stock solution of 10 mM; Forsklin is dissolved in DMSO to prepare a stock solution of 1 mg / mL; PY60 is dissolved in DMSO to prepare a stock solution of 100 mM; CHIR99021 is dissolved in DMSO to prepare a stock solution of 10 mM; Linoleic acid is dissolved in DMSO to prepare a stock solution of 5 mg / mL; Linolenic acid is dissolved in DMSO to prepare a stock solution of 5 mg / mL.

6. A method for culturing human adult epidermal stem cells, characterized by, The method comprises the following steps: 1) Culturing human epidermal stem cells in a mouse tail collagen-coated culture dish to make the human epidermal stem cells adhere to the wall; 2) Collecting the adherent human epidermal stem cells and culturing them with the expansion medium according to any one of claims 1 or 2 to obtain human epidermal stem cells.

7. The culture method according to claim 6, characterized by, The step 2) culture process is carried out every other day with half-volume medium replacement.

8. A human adult epidermal stem cell, characterized in that, The obtained cells are cultured using the expansion medium according to any one of claims 1 or 2 or the culture method according to any one of claims 6 or 7.

9. Use of the expansion medium according to any one of claims 1 or 2 in the preparation of an epidermal stem cell preparation.

10. The use of human somatic epidermal stem cells according to claim 8 for non-diagnostic therapeutic purposes, characterized in that, The use is any of the following: A: In vitro study of epidermal stem cell proliferation, differentiation, and death regulation mechanism; B: Evaluation of skin cell treatment efficacy; C: Construction of functional skin organoids; D: Development, screening, and pharmacological potency evaluation of skin disease drugs; E: Study of skin aging mechanisms and evaluation of cosmetic effects.

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