Immortalized humanized sebaceous gland precursor cell, preparation method therefor, and method for screening and evaluating grease regulation drug or skin care product by using sebaceous gland precursor cell

Abstract

An immortalized humanized sebaceous gland precursor cell, which is preserved in the China Center for Type Culture Collection and is assigned the accession number CCTCC NO: C2023286. Further provided are a preparation method for the cell and the use of same. The cell can be directly used for screening and evaluating a grease regulation drug or skin care product; and the cell can be further differentiated into a mature sebaceous gland cell, and then the mature sebaceous gland cell is used for screening and evaluating a grease regulation drug or skin care product. The sebaceous gland precursor cell and the mature sebaceous gland cell are stable and accurate, and can remarkably increase the expression of genes related to lipid droplet formation, lipid accumulation and an androgen receptor, and thus can be used for screening and evaluating grease regulation drugs or skin care products.

Description

Immortalized human sebaceous gland precursor cells, preparation method thereof, and method for screening and evaluating oil-regulating drugs or skin care products using the sebaceous gland precursor cells Technical Field

[0001] The present invention belongs to the technical field of skin oil regulation models, and specifically relates to immortalized human sebaceous gland precursor cells, a preparation method thereof, and a method for screening and evaluating oil regulating drugs or skin care products using the sebaceous gland precursor cells. Background Art

[0002] In recent years, the incidence of disfiguring skin diseases such as acne vulgaris, xeroderma, and seborrheic dermatitis has increased significantly. The pathogenesis of these diseases is closely related to abnormal function of the sebaceous glands. Sebaceous glands are located in the dermis and are one of the important accessory organs of the skin. Sebaceous cells are present in the sebaceous glands. Newly formed sebaceous cells continuously migrate toward the center, increasing in size, and increasing the number of lipid droplets in the cytoplasm. The cell nucleus shrinks, the organelles disappear, and the cytoplasm is filled with lipid droplets. Finally, the glandular cells disintegrate and are excreted along with the lipid droplets, which is sebum. The secreted sebum moisturizes the skin and hair, and together with sweat, forms a lipid membrane to protect the skin, playing a vital role in maintaining skin homeostasis. At the same time, abnormal sebaceous gland function can cause a variety of skin problems.

[0003] Acne is a chronic inflammatory skin disease affecting the hair follicles and sebaceous glands, often occurring on the face, chest, and back. Clinically, it manifests as comedones, papules, pustules, cysts, or nodules, often accompanied by enlarged pores and sebum secretion. The incidence rate among adolescents is as high as 93%, making it the eighth most common chronic disease worldwide. A cross-sectional acne incidence rate in the Chinese population is 8.1%. However, studies have found that over 95% of people will develop varying degrees of acne. Without early intervention and treatment, scarring can easily occur, seriously affecting the patient's quality of life. Therefore, in order to reveal the pathophysiological mechanisms of acne and develop pharmaceuticals or cosmetics that inhibit abnormal sebum secretion, it is crucial to construct a cell model that can reproduce the behavior of the sebaceous glands in acne patients.

[0004] Currently, hamster sebaceous gland cell lines are known as sebaceous gland cell lines that can be used in experiments. These cells have the advantage of forming lipid droplets within the cell, similar to sebaceous gland cells in living organisms, but their genomic information is unclear, making them unsuitable for research using genetic methods. Non-patent document 1 discloses that the human-derived sebaceous gland cell line SZ95, immortalized by transfection with the giant T antigen DNA of SV40, is also commonly used in sebaceous gland-related experimental research. These cells are derived from humans, have a complete and clear genomic information, and are highly proliferative. However, SZ95 lacks androgen receptors, and they cannot respond to testosterone to promote adipogenesis. When cultured in vitro, they lose the ability to form lipid droplets, and cannot fully represent sebaceous gland cells in their natural growth state, let alone replicate the sebaceous gland behavior of acne patients. In addition, due to the introduction of oncogenes from HPV and Epstein-Barr virus, target cells may have the potential risk of malignant transformation (Zouboulis CC, Seltmann H, Neitzel H, et al. Establishment and characterization of an immortalized human sebaceous gland cell line (sz95) [J]. J Invest Dermatol, 1999, 113(6): 1011-1020.). Non-patent document 2 induces SZ95 to lipidate by intervening with different plant oils. While this stimulates sebum production, it also affects cytokine secretion. Therefore, immortalized sebocyte cultures are considered an inappropriate model for key features of acne pathogenesis (Zouboulis CC, Hossini AM, Hou X, Wang C, Weylandt KH, Pietzner A. Effects of Moringa oleifera Seed Oil on Cultured Human Sebocytes In Vitro and Comparison with Other Oil Types. Int J Mol Sci. 2023 Jun 19; 24(12): 10332.).In non-patent document 3, when sebaceous gland cells were isolated from human skin using microsurgical instruments for primary culture, although lipid accumulation could be seen in the cytoplasm, they could only grow for 3-6 generations. Moreover, the number of cells obtained was limited and they could not be expanded very effectively, making it difficult to conduct continuous, large-scale and in-depth research. If the cells were obtained from multiple sources, the properties of the cells would be heterogeneous, making it difficult to avoid the resulting unstable research results, which weakened the comparability of the results between different studies (Xia LQ, Zouboulis C, Detmar M, et al. Isolation of human sebaceous glands and cultivation of sebaceous gland-derived cells as an in vitro model [J]. J Invest Dermatol, 1989, 93(3): 315-321.).

[0005] Therefore, there is an urgent need in the art for a method to generate human sebaceous progenitor cells with higher purity and yield without using animal cells, which can be applied to provide relevant in vitro models for disease modeling and the development of innovative treatments. Summary of the Invention

[0006] The purpose of the present invention is to construct humanized stable in vitro sebaceous gland cells to solve the problems in the background technology.

[0007] To achieve the above objectives, the present invention provides an immortalized human sebaceous gland precursor cell, a preparation method thereof, and a method for screening and evaluating oil-regulating drugs or skin care products using the sebaceous gland precursor cell.

[0008] The present invention provides an immortalized human sebaceous gland precursor cell, which is deposited with the China Center for Type Culture Collection with a deposit number of CCTCC NO: C2023286 and a classification name: human induced pluripotent stem cell-derived sebaceous gland precursor cell iPS-SBP1#. The address is: Wuhan University, Wuhan, China, and the deposit date is October 10, 2023.

[0009] The method for preparing the above-mentioned cells comprises the following steps:

[0010] (1) Isolation and expansion culture of renal epithelial cells:

[0011] ① Isolation of renal epithelial cells:

[0012] a. Collect urine from acne patients into 50 mL centrifuge tubes and centrifuge at 400 x g for 10 minutes at room temperature. Discard the supernatant and retain 1-2 mL of the liquid.

[0013] b. Take 10 mL of the wash buffer from the UrinEasy Urine Cell Isolation and Culture Kit, resuspend the pellet, and transfer it to another centrifuge tube. Centrifuge at 200 x g for 10 minutes at room temperature. Discard the supernatant, then resuspend the cell pellet in the medium from the UrinEasy Urine Cell Isolation and Culture Kit and inoculate it into a culture plate coated with Matrigel in advance.

[0014] c. After 24 hours of culture, observe the cells for contamination. If there is no contamination, add 800 μL of the culture medium in the UrinEasy Urine Cell Isolation and Culture Kit and repeat this step for 3 days;

[0015] d. On the fourth day, observe for contamination and adherent cells. If no contamination is found, discard 2 / 3 of the culture medium in the well and add 800 μL of the culture medium in the UrinEasy Urine Cell Isolation and Culture Kit. Observe the culture medium for contamination and adherent cells daily thereafter, and replace the medium with half or full volume every other day.

[0016] ② Expansion and culture of renal epithelial cells:

[0017] a. After the separation in step ① above, when the confluence of renal epithelial cells reaches 80-90%, they can be passaged and expanded;

[0018] b. Discard the culture medium in the wells, wash once with PBS, and digest in a 0.25% trypsin digestion solution at 37°C for 3 minutes in an incubator. Add 2 times the volume of the digestion solution to terminate the digestion with the expansion culture basal medium in the UrinEasy urine cell expansion culture kit. Gently pipette the bottom of the culture well to detach the cells.

[0019] c. Collect the cell suspension into a 15 mL centrifuge tube, centrifuge at 1200 rpm at room temperature for 3 minutes, discard the supernatant, resuspend the cell pellet in the UrinEasy expansion culture basal medium, gently pipette to a single cell suspension, and evenly inoculate the cells into a Matrigel-coated culture plate;

[0020] (2) Establishment and expansion of human induced pluripotent stem cell lines:

[0021] ① Establishment of human induced pluripotent stem cell lines:

[0022] a. Step (1) When passaged, urine-derived renal epithelial cells were plated at cell densities of 5000 / well, 10000 / well, and 15000 / well in Matrigel-coated 96-well plates. Three replicates were prepared for each gradient, and this was recorded as day -1.

[0023] b. The next day, observe the cell status under a microscope and count the cells in one well of each density gradient. Select one well with a cell count between 10,000 and 20,000 for subsequent experiments.

[0024] c. Mix 10 μL of Reprogramming Supplement I (Cat. No. CA5002002-1) with 10 mL of the culture medium in UrinEasy Urine Cell Expansion Reagent to create "Reprogramming Medium A." Then, mix 100 μL of Reprogramming Medium A with 10 μL of Reprogramming Supplement II (Cat. No. CA5002002-2) to create "Reprogramming Medium B." Discard the culture medium in the selected wells and add Reprogramming Medium B. This is recorded as Day 0.

[0025] d. Observe cell morphology changes under a microscope every day for 2 consecutive days;

[0026] e. On day 3, observe cell morphology under a microscope. If cell morphology changes significantly and the cells reach 100% confluence, they can be passaged.

[0027] f. For cell passaging, first prepare Reprogramming Medium C. Add 10 μL of Reprogramming Supplement III (Cat. No. CA5002002-3) to 10 mL of the medium in the UrinEasy Urine Cell Expansion Culture Kit and mix thoroughly. Discard the medium in the culture wells containing the cells to be digested. Wash once with PBS, then add 50 μL of trypsin to the wells. Incubate the wells at 37°C for 3 minutes. Terminate the digestion by adding 100 μL of Reprogramming Medium C. Fracture the bottom of the wells and collect the cell suspension into a centrifuge tube containing Reprogramming Medium C. Mix thoroughly by pipetting. Evenly plate the cells onto Matrigel-coated plates at a passaging ratio of 1:6-1:10.

[0028] g. On day 4, replace the culture medium with the UrinEasy Urine Cell Expansion Culture Kit;

[0029] h. Observe the cells daily. If small clones are observed under the microscope, replace the medium daily with Reproeasy Human Cell Reprogramming Basal Medium (Cat. No. CA5003050-1).

[0030] i. If multiple colonies consisting of more than 10 cells are detected, replace the culture medium with PGM1 Human Pluripotent Stem Cell Medium (Cat. No. CA1007500);

[0031] j. Observe the cells under a microscope and culture until multiple colonies fill the 10x magnification field of view. Use a 1 mL syringe needle to cut the colonies and a 200 μL sterile pipette tip to separate the colonies from the bottom wall of the culture well. Aspirate the colonies and transfer them to a Matrigel-coated 24-well plate filled with the PGM1 human pluripotent stem cell culture medium. Return the plate to the incubator and culture at 37°C, 5% carbon dioxide, and 90% or greater humidity.

[0032] k. Observe daily and replace the medium with the PGM1 human pluripotent stem cell culture medium;

[0033] ② Expansion and culture of human induced pluripotent stem cell lines:

[0034] a. Cells can be passaged when microscopic observation shows that they are in good condition and have a confluence of approximately 80%, or when there is uneven distribution of clones with a single large clone.

[0035] b. Discard the culture medium in the wells, rinse once with PBS, and add DissoEasy Human Pluripotent Stem Cell Digestion Solution (Cat. No. CA1023100). Digest at room temperature for 3-5 minutes. Discard the digestion solution in the wells and add the PGM1 Human Pluripotent Stem Cell Medium. Gently pipette the bottom of the culture wells in a fan-shaped pattern to dislodge the cells. Collect the cell suspension into a centrifuge tube. Inoculate the cell suspension onto a Matrigel-coated culture plate at a volume ratio of 1:5-1:10 and continue culturing in an incubator at 37°C, 5% carbon dioxide, and above 90% humidity. Observe daily and change the PGM1 Human Pluripotent Stem Cell Medium daily.

[0036] (3) Differentiation of sebaceous gland precursor cells

[0037] a. The human induced pluripotent stem cells obtained in step (2) were replaced with a basal differentiation medium for differentiating human induced pluripotent stem cells into sebaceous gland precursor cells, the basal differentiation medium consisting of the following components:

[0038] b. Replace the basic differentiation medium in step (a) every day and culture in an incubator at 37°C and a volume concentration of 5% CO2 for three consecutive days;

[0039] c. After three days of cell culture, the culture medium was replaced with sebaceous gland precursor differentiation activation medium, which was the basal differentiation medium in step (a) supplemented with 20 ng / mL epidermal growth factor and 1 ng / mL bone morphogenetic protein 4;

[0040] d. Replace the sebaceous gland differentiation activation medium every other day and culture continuously in a 37°C, 5% CO2 incubator for 7-8 days;

[0041] e. Prepare sebaceous gland precursor differentiation medium, which consists of the following components:

[0042] EpiLife TM Culture medium: Dermacult Keratinocyte Expansion Medium (volume ratio 1:1) Epidermal Growth Factor 20 ng / mL Bone Morphogenetic Protein 4 1 ng / mL

[0043] f. The cells were cultured in a 5% CO2 incubator at 37°C for 10-11 days using the differentiation medium. The cells were digested with TrypLE in a carbon dioxide incubator at 37°C for 15 minutes, centrifuged at 200 x g for 3 minutes, and the supernatant was discarded. The cells were resuspended in sebaceous gland precursor differentiation medium supplemented with 10 μM dihydrochloride ATP competitive ROCK inhibitor.

[0044] g. The cells obtained in step (f) were grown in 25 μg / mL fibronectin-coated culture plates;

[0045] h. On the 12th to 13th day of continuous culture in a 37°C, 5% CO2 incubator, replace the culture medium with fresh sebaceous gland precursor differentiation medium;

[0046] i. The cells were cultured daily in a 37°C, 5% CO2 incubator for 7-9 days to obtain sebaceous gland precursor cells.

[0047] The present invention also provides a method for screening and evaluating oil-regulating drugs or skin care products using the precursor cells. The sebaceous gland precursor cells are directly incubated with DHT dihydrotestosterone at room temperature for 48 hours to stimulate the sebaceous gland precursor cells to produce more oil cells. This model is used to screen and evaluate oil-regulating drugs or skin care products. Alternatively, the sebaceous gland precursor cells are cultured and differentiated into mature sebaceous gland cells, and the mature sebaceous gland cells are used to screen and evaluate drugs or skin care products.

[0048] Furthermore, sebaceous gland precursor cells are cultured and differentiated to obtain mature sebaceous gland cells, and the cells are used to screen and evaluate oil secretion drugs or skin care products. The method for culturing and differentiating sebaceous gland precursor cells to obtain mature sebaceous gland cells is:

[0049] a. Prepare mature sebaceous gland basal differentiation medium, which consists of the following components:

[0050] b. Digest the sebaceous gland precursor cells with TrypLE: Digest for 15 minutes in a 5% CO2 incubator at 37°C. Centrifuge at 250xg for 3 minutes, discard the supernatant, and add 10 μM dihydrochloride ATP competitive ROCK inhibitor to the mature sebaceous gland basal differentiation medium. Then resuspend the cells at 2.5×10 4 cells / cm 2 The cells were seeded in culture plates at a density of 100 μg / mL and cultured overnight in a 37°C incubator with a volume concentration of 5% CO2.

[0051] c. The next day, the medium was changed and 10 μM TGF-β / Smad inhibitor was added to the mature sebaceous gland basal differentiation medium;

[0052] d. Change the medium daily and incubate the cells in a 37°C, 5% CO2 incubator for 3 consecutive days.

[0053] e. After 3 days, the medium was changed to mature sebaceous gland terminal differentiation medium and cultured in a 37°C incubator with 5% CO2.

[0054] f. Change the medium daily and culture continuously at 37°C in a 5% CO2 incubator for 3-6 days to obtain mature sebaceous gland cells.

[0055] Furthermore, in the step (e), the mature sebaceous gland final differentiation medium is obtained by adding 10 μM TGF-β / Smad inhibitor to the mature sebaceous gland basal differentiation medium; or by adding 10 μM TGF-β / Smad inhibitor and 1 μM PPAR-β / δ agonist to the mature sebaceous gland basal differentiation medium; or by adding 10 μM TGF-β / Smad inhibitor, 1 μM GW0742 PPAR-β / δ agonist and 5 μM cyclopamine to the mature sebaceous gland basal differentiation medium.

[0056] In summary, the present invention has the following beneficial effects: (1) The method for preparing the immortalized human sebaceous gland precursors adopted by the present invention uses human induced pluripotent stem cells in the urine of acne patients for culture and differentiation. Acne patients themselves have strong oil secretion, which provides a basic guarantee for the subsequent acquisition of cells with good oil secretion in vitro; (2) The method for preparing the immortalized human sebaceous gland precursor cells adopted by the present invention, when using human induced pluripotent stem cells to differentiate into sebaceous gland precursor cells, the sebaceous gland precursor cells obtained have a high level of expression of sebaceous gland precursor cell markers, and the differentiation ability is stable and accurate; (3) The immortalized human sebaceous gland precursor cells obtained by the present invention have sebaceous gland precursor cell lipid droplet formation, lipid accumulation and androgen The expression of receptor-related genes is significantly increased and has strong stability, and can be well used for the screening and evaluation of oil secretion regulating drugs or skin care products; (4) When the precursor cells of the present invention are used to screen and evaluate oil secretion regulating drugs or skin care products, the precursor cells can also be further cultured and differentiated into mature sebaceous gland cells. The obtained mature sebaceous gland cells can highly express markers and have the properties of forming or accumulating lipid droplets of sebaceous gland cells in the human body. Under the stimulation of androgens, the maturation and differentiation of cells are accelerated, and more lipid droplets are released. They have clear genetic information and clinical background, are stable, and can simulate the sebaceous gland behavior of acne patients in vitro, and can be accurately and conveniently used for the screening and evaluation of oil secretion regulating drugs or skin care products. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] FIG1 shows the morphology of renal epithelial cells isolated from urine samples and cultured at different time points in the present invention;

[0058] FIG2 is a morphological picture of human induced pluripotent stem cells under an optical microscope at different time points after addition of transcription factors according to the present invention;

[0059] FIG3 is a morphological diagram of reprogrammed human induced pluripotent stem cells by expanded culture of renal epithelial cells according to the present invention;

[0060] FIG4 is a diagram showing alkaline phosphatase staining results of induced pluripotent stem cells of the present invention;

[0061] FIG5 is a diagram showing the immunofluorescence staining results of induced pluripotent stem cells of the present invention;

[0062] FIG6 is a diagram showing the results of karyotype identification of induced pluripotent stem cells of the present invention;

[0063] FIG7 is a staining diagram of teratoma formation and three-germ layer differentiation of induced pluripotent stem cells of the present invention;

[0064] FIG8 shows the morphological changes of sebaceous gland precursor cells during differentiation of the present invention;

[0065] FIG9 is a comparison of the expression levels of sebaceous gland precursor cell marker genes in human keratinocytes (HaCaT) and sebaceous gland precursor cells (SBP) of the present invention. In the figure, * P<0.05, ** P < 0.01and *** P < 0.001 vs HaCaT;

[0066] FIG10 is a graph comparing the expression levels of sebaceous gland precursor cell marker genes in human immortalized sebaceous gland cells (SZ95) and sebaceous gland precursor cells (SBP) of the present invention. # P<0.05, ## P < 0.01 and ### P < 0.001 vs SZ95;

[0067] FIG11 is a graph comparing the expression levels of sebaceous gland precursor cell marker genes in human induced pluripotent stem cells (hiPSC) and sebaceous gland precursor cells (SBP) of the present invention. & P<0.05, && P < 0.01 and &&& P < 0.001 vs hiPSC;

[0068] Figure 12 is a comparative image of lipid droplet content detected by Oil Red O staining of human keratinocytes (HaCaT), human immortalized sebocytes (SZ95), human induced pluripotent stem cells (hiPSC), and sebaceous gland precursor cells (SBP) of the present invention;

[0069] Figure 13 is a comparative statistical graph of lipid droplet content detected by Oil Red O staining of human keratinocytes (HaCaT), human immortalized sebocytes (SZ95), human induced pluripotent stem cells (hiPSC), and sebaceous gland precursor cells (SBP) of the present invention;

[0070] Figure 14 is a comparison of the fluorescence intensity of intracellular lipid droplets detected by Nile red fluorescence staining of human keratinocytes (HaCaT), human immortalized sebocytes (SZ95), human induced pluripotent stem cells (hiPSC), and sebaceous gland precursor cells (SBP) of the present invention;

[0071] FIG15 is a diagram showing the difference in gene expression between human immortalized sebaceous gland cells (SZ95) and sebaceous gland precursor cells (SBP) of the present invention detected by RNA-seq;

[0072] Figure 16 shows the changes in lipid droplets detected by Oil Red O staining after intervention of cells with different concentrations of dihydrotestosterone (DHT);

[0073] Figure 17: Oil red O staining to detect changes in lipid droplets in sebaceous gland precursor cells (SBP) after intervention with different concentrations of drugs and skin care product ingredients;

[0074] FIG18 is a morphological observation diagram of mature sebaceous gland cells (SBM) obtained in Examples 3 to 5 of the present invention;

[0075] FIG19 is a diagram showing lipid droplet formation and cytokeratin expression in mature sebaceous gland cells (SBM) according to Example 3 of the present invention. In the figure, arrows indicate lipid droplets and KRT7 expression;

[0076] FIG20 is a graph showing the responses of mature sebaceous gland cells (SBM) obtained in Examples 3 to 5 of the present invention to dihydrotestosterone (DHT);

[0077] FIG21 is a BODIP lipid droplet staining image of mature sebaceous gland cells (SBM) obtained in Examples 3 to 5 of the present invention, in which arrows indicate lipid droplet expression;

[0078] Figure 22 shows the morphological changes of mature sebaceous gland cells (SBM) detected by Oil Red O staining after intervention with different concentrations of drugs and skin care product ingredients. In the figure, the box represents the morphology of mature sebaceous gland cells (SBM) that have increased in size after intervention with dihydrotestosterone (DHT). DETAILED DESCRIPTION

[0079] The following is a detailed description of the specific embodiments of the present invention. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention and are not intended to limit the present invention. In the following experimental conditions, unless otherwise specified, all conditions were room temperature. The following reagents were purchased from manufacturers: UrinEasy urine cell isolation and culture kit, reprogramming additive I, reprogramming additive II, reprogramming additive III, Reproeasy human cell reprogramming basal medium, PGM1 human pluripotent stem cell medium, DissoEasy human pluripotent stem cell digestion solution were purchased from Beijing Saibei Biotechnology Co., Ltd., EpiLife TM The culture medium, catalog number MEPI500CA, was purchased from Thermo Fisher Scientific, and the Dermacult keratinocyte expansion medium was purchased from Jiangsu Ruijie Biotechnology Co., Ltd.

[0080] Example 1: The present invention provides an immortalized human sebaceous gland precursor cell, which is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: C2023286. The method for preparing the cell comprises the following steps:

[0081] (1) Isolation and expansion culture of renal epithelial cells:

[0082] ① Isolation of renal epithelial cells:

[0083] a. Collect urine from acne patients into 50 mL centrifuge tubes and centrifuge at 400 x g for 10 minutes at room temperature. Discard the supernatant and retain 1 mL of the liquid.

[0084] b. Take 10 mL of the wash buffer from the UrinEasy Urine Cell Isolation and Culture Kit, resuspend the pellet, and transfer it to another centrifuge tube. Centrifuge at 200 x g for 10 minutes at room temperature. Discard the supernatant, then resuspend the cell pellet in the medium from the UrinEasy Urine Cell Isolation and Culture Kit and inoculate it into a culture plate coated with Matrigel in advance.

[0085] c. After 24 hours of culture, observe the cells for contamination. If there is no contamination, add 800 μL of the culture medium in the UrinEasy Urine Cell Isolation and Culture Kit and repeat this step for 3 days;

[0086] d. On the fourth day, observe for contamination and adherent cells. If no contamination is found, discard 2 / 3 of the culture medium in the well and add 800 μL of the culture medium in the UrinEasy Urine Cell Isolation and Culture Kit. Observe the culture medium for contamination and adherent cells daily thereafter, and replace the medium with half or full volume every other day.

[0087] ② Expansion and culture of renal epithelial cells:

[0088] a. After isolation in step ① above, when the confluence of renal epithelial cells reaches 80%, they can be passaged and expanded;

[0089] b. Discard the culture medium in the wells, wash once with PBS, and digest in a 0.25% trypsin digestion solution at 37°C for 3 minutes in an incubator. Add 2 times the volume of the digestion solution to terminate the digestion with the expansion culture basal medium in the UrinEasy urine cell expansion culture kit. Gently pipette the bottom of the culture well to detach the cells.

[0090] c. Collect the cell suspension into a 15 mL centrifuge tube, centrifuge at 1200 rpm at room temperature for 3 minutes, discard the supernatant, resuspend the cell pellet in the UrinEasy expansion culture basal medium, gently pipette to a single cell suspension, and evenly inoculate the cells into a Matrigel-coated culture plate;

[0091] (2) Establishment and expansion of human induced pluripotent stem cell lines:

[0092] ① Establishment of human induced pluripotent stem cell lines:

[0093] a. Step (1) When passaged, urine-derived renal epithelial cells were plated at cell densities of 5000 / well, 10000 / well, and 15000 / well in Matrigel-coated 96-well plates. Three replicates were prepared for each gradient, and this was recorded as day -1.

[0094] b. The next day, observe the cell status under a microscope and count the cells in one well of each density gradient. Select one well with a cell count between 10,000 and 20,000 for subsequent experiments.

[0095] c. Mix 10 μL of Reprogramming Supplement I (Cat. No. CA5002002-1) with 10 mL of the culture medium in UrinEasy Urine Cell Expansion Reagent to create "Reprogramming Medium A." Then, mix 100 μL of Reprogramming Medium A with 10 μL of Reprogramming Supplement II (Cat. No. CA5002002-2) to create "Reprogramming Medium B." Discard the culture medium in the selected wells and add Reprogramming Medium B. This is recorded as Day 0.

[0096] d. Observe cell morphology changes under a microscope every day for 2 consecutive days;

[0097] e. On day 3, observe cell morphology under a microscope. If cell morphology changes significantly and the cells reach 100% confluence, they can be passaged.

[0098] f. For cell passaging, first prepare Reprogramming Medium C. Add 10 μL of Reprogramming Supplement III (Cat. No. CA5002002-3) to 10 mL of the medium in the UrinEasy Urine Cell Expansion Culture Kit and mix thoroughly. Discard the medium in the culture wells containing the cells to be digested. Wash once with PBS, then add 50 μL of trypsin to the wells. Incubate the wells at 37°C for 3 minutes. Terminate the digestion by adding 100 μL of Reprogramming Medium C. Fracture the bottom of the wells and collect the cell suspension into a centrifuge tube containing Reprogramming Medium C. Mix thoroughly by pipetting. Evenly plate the cells onto Matrigel-coated plates at a 1:6 passaging ratio.

[0099] g. On day 4, replace the culture medium with the UrinEasy Urine Cell Expansion Culture Kit;

[0100] h. Observe the cells daily. If small clones are observed under the microscope, replace the medium daily with Reproeasy Human Cell Reprogramming Basal Medium (Cat. No. CA5003050-1).

[0101] i. If multiple colonies consisting of more than 10 cells are detected, replace the culture medium with PGM1 Human Pluripotent Stem Cell Medium (Cat. No. CA1007500);

[0102] j. Observe the cells under a microscope and culture until multiple colonies fill the 10x magnification field of view. Use a 1 mL syringe needle to cut the colonies and a 200 μL sterile pipette tip to separate the colonies from the bottom wall of the culture well. Aspirate the colonies and transfer them to a Matrigel-coated 24-well plate filled with the PGM1 human pluripotent stem cell culture medium. Return the plate to the incubator and culture at 37°C, 5% carbon dioxide, and 90% or greater humidity.

[0103] k. Observe daily and replace the medium with the PGM1 human pluripotent stem cell culture medium;

[0104] ② Expansion and culture of human induced pluripotent stem cell lines:

[0105] a. Cells can be passaged when microscopic observation shows that they are in good condition and have a confluence of approximately 80%, or when there is uneven distribution of clones with a single large clone.

[0106] b. Discard the culture medium in the wells, rinse once with PBS, add DissoEasy Human Pluripotent Stem Cell Digestion Solution (Cat. No. CA1023100), digest at room temperature for 3 minutes, discard the digestion solution in the wells, add the PGM1 Human Pluripotent Stem Cell Medium, gently pipette the bottom of the culture wells in a fan-shaped pattern to detach the cells, and collect the cell suspension into a centrifuge tube. Inoculate the cell suspension onto a Matrigel-coated culture plate at a volume ratio of 1:5 and continue culturing in an incubator at 37°C, 5% carbon dioxide, and above 90% humidity. Observe daily thereafter and replace the PGM1 Human Pluripotent Stem Cell Medium daily.

[0107] (3) Differentiation of sebaceous gland precursor cells

[0108] a. The human induced pluripotent stem cells obtained in step (2) were replaced with a basal differentiation medium for differentiating human induced pluripotent stem cells into sebaceous gland precursor cells, the basal differentiation medium consisting of the following components:

[0109] b. Replace the basic differentiation medium in step (a) every day and culture in an incubator at 37°C and a volume concentration of 5% CO2 for three consecutive days;

[0110] c. After three days of cell culture, the culture medium was replaced with sebaceous gland precursor differentiation activation medium, which was the basal differentiation medium in step (a) supplemented with 20 ng / mL epidermal growth factor and 1 ng / mL bone morphogenetic protein 4;

[0111] d. Replace the sebaceous gland differentiation activation medium every other day and culture continuously in a 37°C, 5% CO2 incubator for 7 days;

[0112] e. Prepare sebaceous gland precursor differentiation medium, which consists of the following components:

[0113] EpiLife TM Culture medium: Dermacult Keratinocyte Expansion Medium (volume ratio 1:1) Epidermal Growth Factor 20 ng / mL Bone Morphogenetic Protein 4 1 ng / mL

[0114] f. The cells were cultured in the differentiation medium at 37°C and 5% CO2 in an incubator for 10 days, and the cells were digested with TrypLE. The digestion method was to digest the cells in a carbon dioxide incubator at 37°C for 15 minutes, centrifuged at 200 x g for 3 minutes, and the supernatant was discarded. The cells were resuspended in sebaceous gland precursor differentiation medium supplemented with 10 μM dihydrochloride ATP competitive ROCK inhibitor;

[0115] g. The cells obtained in step (f) were grown in 25 μg / mL fibronectin-coated culture plates;

[0116] h. On the 12th day of continuous culture in a 37°C, 5% CO2 incubator, replace the culture medium with fresh sebaceous gland precursor differentiation medium;

[0117] i. The cells were cultured daily in a 37°C, 5% CO2 incubator for 7 consecutive days to obtain sebaceous gland precursor cells.

[0118] The following experiments demonstrate that the sebaceous gland precursor cells obtained by the present invention can stably proliferate, have good lipid droplet formation and lipid accumulation capabilities, and can be used well for screening and evaluation of oil secretion regulating drugs or skin care products.

[0119] 1.1. Isolation and Expansion of Renal Epithelial Cells

[0120] 1)1) Renal epithelial cell isolation and culture process

[0121] a. Renal epithelial cell morphology was observed and photographed under an optical microscope after cells were isolated from urine samples and cultured at different time points (day 3, day 6, and day 9).

[0122] b. As shown in Figure 1, with the passage of time and continuous medium replacement, the suspended squamous epithelial cells and impurities gradually decreased, and multiple clusters of typical adherent cells with spindle or cobblestone morphology were observed under the microscope, indicating that the renal epithelial cells were successfully isolated.

[0123] 2. Establishment, Culture, and Identification of Human Induced Pluripotent Stem Cells

[0124] 1) Establishment of human induced pluripotent stem cells

[0125] a. Cell morphology at different time points (day 0, day 3, day 8, and day 12) after the addition of transcription factors was observed and photographed under an optical microscope.

[0126] b. As shown in Figure 2, compared with day 0, after adding transcription factors, the morphology of the cells changed significantly on day 3, becoming irregular in shape and with some detached dead cells. With continued culture until day 12, typical clonal growth was observed, with cells within the clones tightly connected, round in shape, and with relatively large nucleocytoplasmic ratios. The clone edges were clear and sharp, consistent with the morphology of human induced pluripotent stem cells.

[0127] 2) Culture of human induced pluripotent stem cells

[0128] a. Use mechanical separation and expansion to observe and photograph the cell morphology under an optical microscope.

[0129] b. As shown in Figure 3, the clones are typical in morphology, with sharp edges and no obvious differentiated cells. The cells within the clones are tightly connected, with large nuclei, and are in good condition, suitable for subsequent identification and differentiation experiments.

[0130] 3) Alkaline phosphatase staining to identify the pluripotency of human induced pluripotent stem cells

[0131] a. Evenly seed hiPSC cells in a 24-well plate and culture until colonies reach a suitable size and no more colonies are confluent. Experiments can be performed using an alkaline phosphatase staining kit. Prepare the staining solution in the dark by mixing Solution A and Solution B in a 1:1 volume ratio. Use within 30 minutes. Remove the cells, discard the culture medium, rinse once with PBS, and discard the PBS. Add 500 μL / well of fixative solution and fix at room temperature for 5 minutes. Wash twice with PBS, then add 500 μL / well of staining solution and incubate at room temperature for 20 minutes in the dark. After incubation, discard the staining solution, rinse once with PBS, and store in PBS to prevent the cells from drying out. Observe the staining results under a microscope.

[0132] b. As shown in Figure 4, each clone in each field of view was stained dark blue. The color depth indicated the expression level, indicating that the cells in the clone highly expressed alkaline phosphatase.

[0133] 4) Immunofluorescence staining to identify the pluripotency of human induced pluripotent stem cells

[0134] a. Plate hiPSCs evenly in a 24-well plate and culture until colonies reach a suitable size and no more colonies are confluent. Experiments can be performed. Remove cells from the incubator, discard the culture medium, and wash once with PBS. Add 4% paraformaldehyde, cover the bottom surface, and fix at room temperature for 10 minutes. Discard the fixative, wash twice with PBS, add 500 μL of 0.2% Triton-100, and incubate at room temperature for 15 minutes. Discard the liquid in the wells, cover the bottom surface with 500 μL of 1% BSA, and block at room temperature for 1 hour. Discard the BSA, add the primary antibody solution diluted proportionally, and incubate at 4°C overnight. The next day, remove the plate, discard the liquid in the wells, and wash three times with PBS for 5 minutes each. Add a secondary antibody solution diluted 1:1000 and incubate at room temperature for 1 hour. Discard the secondary antibody and wash three times with PBS for 5 minutes each. Add DAPI solution and incubate at room temperature for 10 minutes. Discard the DAPI and wash three times with PBS. Cover the bottom surface with PBS to prevent cells from drying. At this time, it can be observed directly under an inverted fluorescence microscope.

[0135] b. As shown in Figure 5, this experiment selected three protein markers: TRA-1-60, SSEA4, and Oct-4 for immunofluorescence staining. All three protein markers were expressed in hiPSCs derived from renal epithelial cell reprogramming.

[0136] 5) Karyotyping to identify the pluripotency of human induced pluripotent stem cells

[0137] a. Prepare the experiment by culturing the cells to approximately 80% confluency. Add PSC medium containing 0.1 μg / mL colchicine and continue culturing in the incubator for 1.5 hours before proceeding with subsequent experiments. Trypsinize the cells and collect them in a centrifuge tube. Centrifuge at 1000 rpm for 10 minutes at room temperature. Discard the supernatant. Add 0.075 mol / L KCl solution and place in a 37°C water bath for hypotonic treatment for 20 minutes. Add 1 mL of pre-fixative solution (methanol: glacial acetic acid, volume ratio 3:1), mix thoroughly, and centrifuge at 1000 rpm for 10 minutes at room temperature. Discard the supernatant and retain the substrate. Add 8 mL of fixative solution, fix for 20 minutes, and centrifuge at 1000 rpm for 10 minutes at room temperature. Discard the supernatant and add fixative depending on the amount of cell pellet. Resuspend the cells by tapping the tube with your finger. Remove the slide from the ice water. Use a rubber-tipped pipette to aspirate the cell suspension and place it approximately 30 cm above the slide. Dry the slide at 80°C for 2 hours. Dilute trypsin in PBS to a final concentration of 0.25‰. Dry slides at 80°C for 2 hours and place them in trypsin for 2-3 minutes, depending on the degree of dryness. Rinse the slides in PBS and stain them in Giemsa solution for 5-10 minutes. Rinse the slides in tap water. Dry at room temperature, then mount the slides for storage. Count and analyze chromosome structural changes under a microscope (Figure 6).

[0138] b. As shown in Figure 6, G-banding analysis of the cells after culture revealed a normal number of 46 chromosomes; the sex chromosomes consisted of X, Y, and had a male karyotype; no abnormalities were observed in the chromosome structure.

[0139] 6) Teratoma formation assay to identify the pluripotency of human induced pluripotent stem cells

[0140] a. After culturing a sufficient number of cells according to experimental requirements, digest and centrifuge the cells and inject 0.3 mL of the hiPSC suspension into the base of the right lateral thigh of a NOD-SCID mouse using a syringe. The mice are then housed normally. Tumors will form at the injection site around 8-12 weeks. After photographing and recording, the mice are sacrificed by cervical dislocation. Use scissors to cut the injected tumor site open to expose the field of view. Carefully excise the tumor using forceps and scissors. Fix the tumor in 10% formalin fixative at a volume ratio of approximately 10:1. After 24 hours of fixation, embed the tissue and perform hematoxylin and eosin staining. Observe the staining results and sample tissue morphology using a slide scanner.

[0141] b. As shown in Figure 7, after 8-12 weeks of differentiation, a bulge was observed at the injection site, and dissection revealed subcutaneous tumor formation. After dissection of the tumor, pathological HE staining revealed intestinal columnar epithelial tissue derived from the endoderm, cartilage-like tissue derived from the mesoderm, and rosette-like neural tissue derived from the ectoderm. The hiPSCs obtained in this experiment have the ability to differentiate into cells and tissues of all three germ layers in vivo.

[0142] 3. Induction of Differentiation of Sebaceous Gland Precursor Cells and Evaluation of Adipogenic Ability

[0143] 1) Differentiation process of sebaceous gland precursor cells

[0144] a. Cell morphology was observed and photographed under an optical microscope from day 1 to day 26 of differentiation of human induced pluripotent stem cells into sebaceous gland progenitor cells (scale bar = 20 μm).

[0145] b. The results are shown in Figure 8. Compared with the morphology of human induced pluripotent stem cells, the differentiating cells exhibited a spindle-shaped morphology over time, resembling skin epidermal cells. In the late stage of differentiation, the cells became larger and obvious vacuoles were observed.

[0146] 2) Gene expression levels of sebaceous gland precursor cell markers

[0147] a. Total RNA was extracted from cells using the Total Gold RNA Extraction Kit and synthesized into cDNA using the Total Gold Reverse Transcription Kit. RT-qPCR was used to examine the gene expression levels of sebaceous gland precursor cell markers: androgen receptor (AR), fatty acid desaturase 2 (FADS2), farnesyl diphosphate farnesyltransferase 1 (FDFT1), cytokeratin (KRT7), lipid droplet coating protein 2 (PLIN2), peroxisome proliferator-activated receptor (PPARG) type I transmembrane mucin (MUC-1), melanocortin 5 receptor (MC5R), and stearoyl-CoA desaturase (SCD).

[0148] b. The results are shown in Figures 9 to 11. Compared with human keratinocytes (HaCaT), human immortalized sebocytes (SZ95) and human induced pluripotent stem cells (hiPSC), the sebaceous gland precursor cells (SBP) obtained in the present invention expressed sebaceous gland precursor cell markers at a high level.

[0149] 3) Oil red O staining to detect lipid droplet content in sebaceous gland precursor cells

[0150] a. Seed cells into a 24-well plate. When the cells have grown to 50% of their growth rate, they can be stained. Remove the cells, wash them twice with PBS, and fix them with 4% paraformaldehyde for 20 minutes. Discard the fixative. Wash them twice with distilled water. Add 60% isopropanol and rinse for 15 minutes. Discard the 60% isopropanol solution. Wash them twice with distilled water. Add the prepared Oil Red O stain and stain for 20 minutes. Discard the stain and wash with water until there is no excess stain. Counterstain the nuclei with hematoxylin for 2 minutes. Discard the hematoxylin stain and rinse with water until there is no excess stain. Cover the cells with distilled water and observe under a microscope and take photos.

[0151] b. The results are shown in Figures 12 and 13. Compared with human keratinocytes (HaCaT), human immortalized sebocytes (SZ95) and human induced pluripotent stem cells (hiPSCs), Oil Red O staining showed that the sebaceous gland precursor cells (SBP) obtained by this protocol had a large number of red lipid droplets (scale bar = 10 μm).

[0152] 4) Detection of fluorescence intensity of lipid droplets in sebaceous gland precursor cells by Nile red fluorescence staining

[0153] a. Seed cells onto a cell slide. When cells reach 50% growth, staining is performed. Remove cells, wash twice with PBS, and fix with 4% paraformaldehyde for 10 minutes. Discard the fixative and wash twice with PBS. Add 1 mL of the prepared Nile Red and Hoechst solutions to each well and incubate at room temperature in the dark for 20 minutes. Observe with a laser confocal microscope, record Nile Red and Hoechst fluorescence images, and merge the different fluorescence staining images. The box indicates the lipid droplet expression area.

[0154] b. The results are shown in Figure 14. Compared with human keratinocytes (HaCaT), human immortalized sebocytes (SZ95) and human induced pluripotent stem cells (hiPSC), Nile red fluorescence staining showed that the sebaceous gland precursor cells (SBP) obtained in the present invention had a large amount of red fluorescence, indicating that they had rich lipid droplet content (scale bar = 20 μm).

[0155] 5) RNA-seq detection of gene expression differences between sebaceous gland precursor cells and human immortalized sebaceous gland cells

[0156] a. Cellular RNA was extracted using the TRIzol method, and subsequent transcriptome sequencing was performed by Beijing Novogene Technology Co., Ltd.

[0157] b. The results are shown in Figure 15. The sequencing results indicate that compared with human immortalized sebaceous gland cells (SZ95), the sebaceous gland precursor cells (SBP) obtained in the present invention have significantly increased expression of genes related to lipid droplet formation (Lipid Drop), lipid accumulation (Lipid Storage) and androgen receptor (Androgen receptor).

[0158] It should be noted that the above experiments are the results obtained by repeating the experiments in parallel several times. The performance of the sebaceous gland precursor cells obtained by the preparation method of the present invention is stable. The obtained sebaceous gland precursor cells have a high level of expression of sebaceous gland precursor cell markers, and the expression of sebaceous gland precursor cell lipid droplet formation, lipid accumulation and androgen receptor-related genes is significantly increased, which can be well used for the screening and evaluation of oil secretion regulating drugs or skin care products.

[0159] Example 2: The present invention provides an immortalized human sebaceous gland precursor cell, which is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: C2023286. The method for preparing the cell comprises the following steps:

[0160] (1) Isolation and expansion culture of renal epithelial cells:

[0161] ① Isolation of renal epithelial cells:

[0162] a. Collect urine from acne patients into 50 mL centrifuge tubes and centrifuge at 400 x g for 10 minutes at room temperature. Discard the supernatant and retain 2 mL of the liquid.

[0163] b. Take 10 mL of the wash buffer from the UrinEasy Urine Cell Isolation and Culture Kit, resuspend the pellet, and transfer it to another centrifuge tube. Centrifuge at 200 x g for 10 minutes at room temperature. Discard the supernatant, then resuspend the cell pellet in the medium from the UrinEasy Urine Cell Isolation and Culture Kit and inoculate it into a culture plate coated with Matrigel in advance.

[0164] c. After 24 hours of culture, observe the cells for contamination. If there is no contamination, add 800 μL of the culture medium in the UrinEasy Urine Cell Isolation and Culture Kit and repeat this step for 3 days;

[0165] d. On the fourth day, observe for contamination and adherent cells. If no contamination is found, discard 2 / 3 of the culture medium in the well and add 800 μL of the culture medium in the UrinEasy Urine Cell Isolation and Culture Kit. Observe the culture medium for contamination and adherent cells daily thereafter, and replace the medium with half or full volume every other day.

[0166] ② Expansion and culture of renal epithelial cells:

[0167] a. After isolation in step ① above, when the confluence of renal epithelial cells reaches 90%, they can be passaged and expanded;

[0168] b. Discard the culture medium in the wells, wash once with PBS, and digest in a 0.25% trypsin digestion solution at 37°C for 3 minutes in an incubator. Add 2 times the volume of the digestion solution to terminate the digestion with the expansion culture basal medium in the UrinEasy urine cell expansion culture kit. Gently pipette the bottom of the culture well to detach the cells.

[0169] c. Collect the cell suspension into a 15 mL centrifuge tube, centrifuge at 1200 rpm at room temperature for 3 minutes, discard the supernatant, resuspend the cell pellet in the UrinEasy expansion culture basal medium, gently pipette to a single cell suspension, and evenly inoculate the cells into a Matrigel-coated culture plate;

[0170] (2) Establishment and expansion of human induced pluripotent stem cell lines:

[0171] ① Establishment of human induced pluripotent stem cell lines:

[0172] a. Step (1) When passaged, urine-derived renal epithelial cells were plated at cell densities of 5000 / well, 10000 / well, and 15000 / well in Matrigel-coated 96-well plates. Three replicates were prepared for each gradient, and this was recorded as day -1.

[0173] b. The next day, observe the cell status under a microscope and count the cells in one well of each density gradient. Select one well with a cell count between 10,000 and 20,000 for subsequent experiments.

[0174] c. Mix 10 μL of Reprogramming Supplement I (Cat. No. CA5002002-1) with 10 mL of the culture medium in UrinEasy Urine Cell Expansion Reagent to create "Reprogramming Medium A." Then, mix 100 μL of Reprogramming Medium A with 10 μL of Reprogramming Supplement II (Cat. No. CA5002002-2) to create "Reprogramming Medium B." Discard the culture medium in the selected wells and add Reprogramming Medium B. This is recorded as Day 0.

[0175] d. Observe cell morphology changes under a microscope every day for 2 consecutive days;

[0176] e. On day 3, observe cell morphology under a microscope. If cell morphology changes significantly and the cells reach 100% confluence, they can be passaged.

[0177] f. For cell passaging, first prepare Reprogramming Medium C. Add 10 μL of Reprogramming Supplement III (Cat. No. CA5002002-3) to 10 mL of the medium in the UrinEasy Urine Cell Expansion Culture Kit and mix thoroughly. Discard the medium in the culture wells containing the cells to be digested. Wash once with PBS, then add 50 μL of trypsin to the wells. Incubate the wells at 37°C for 3 minutes. Terminate the digestion by adding 100 μL of Reprogramming Medium C. Fracture the bottom of the wells and collect the cell suspension into a centrifuge tube containing Reprogramming Medium C. Mix thoroughly by pipetting. Plate the cells evenly onto Matrigel-coated plates at a passage ratio of 1:10.

[0178] g. On day 4, replace the culture medium with the UrinEasy Urine Cell Expansion Culture Kit;

[0179] h. Observe the cells daily. If small clones are observed under the microscope, replace the medium daily with Reproeasy Human Cell Reprogramming Basal Medium (Cat. No. CA5003050-1).

[0180] i. If multiple colonies consisting of more than 10 cells are detected, replace the culture medium with PGM1 Human Pluripotent Stem Cell Medium (Cat. No. CA1007500);

[0181] j. Observe the cells under a microscope and culture until multiple colonies fill the 10x magnification field of view. Use a 1 mL syringe needle to cut the colonies and a 200 μL sterile pipette tip to separate the colonies from the bottom wall of the culture well. Aspirate the colonies and transfer them to a Matrigel-coated 24-well plate filled with the PGM1 human pluripotent stem cell culture medium. Return the plate to the incubator and culture at 37°C, 5% carbon dioxide, and 90% or greater humidity.

[0182] k. Observe daily and replace the medium with the PGM1 human pluripotent stem cell culture medium;

[0183] ② Expansion and culture of human induced pluripotent stem cell lines:

[0184] a. Cells can be passaged when microscopic observation shows that they are in good condition and have a confluence of approximately 80%, or when there is uneven distribution of clones with a single large clone.

[0185] b. Discard the culture medium in the wells, wash once with PBS, add DissoEasy Human Pluripotent Stem Cell Digestion Solution (Cat. No. CA1023100), digest at room temperature for 5 minutes, discard the digestion solution in the wells, add the PGM1 Human Pluripotent Stem Cell Medium, gently fan the bottom of the culture wells to detach the cells, and collect the cell suspension into a centrifuge tube. Inoculate the cell suspension onto a Matrigel-coated culture plate at a volume ratio of 1:10 and continue culturing in an incubator at 37°C, 5% carbon dioxide, and above 90% humidity. Observe daily thereafter and replace the medium with the PGM1 Human Pluripotent Stem Cell Medium daily.

[0186] (3) Differentiation of sebaceous gland precursor cells

[0187] a. The human induced pluripotent stem cells obtained in step (2) were replaced with a basal differentiation medium for differentiating human induced pluripotent stem cells into sebaceous gland precursor cells, the basal differentiation medium consisting of the following components:

[0188] b. Replace the basic differentiation medium in step (a) every day and culture in an incubator at 37°C and a volume concentration of 5% CO2 for three consecutive days;

[0189] c. After three days of cell culture, the culture medium was replaced with sebaceous gland precursor differentiation activation medium, which was the basal differentiation medium in step (a) supplemented with 20 ng / mL epidermal growth factor and 1 ng / mL bone morphogenetic protein 4;

[0190] d. Replace the sebaceous gland differentiation activation medium every other day and culture continuously at 37°C in a 5% CO2 incubator for 8 days.

[0191] e. Prepare sebaceous gland precursor differentiation medium, which consists of the following components:

[0192] EpiLife TM Culture medium: Dermacult Keratinocyte Expansion Medium (volume ratio 1:1) Epidermal Growth Factor 20 ng / mL Bone Morphogenetic Protein 4 1 ng / mL

[0193] f. The cells were cultured in a 5% CO2 incubator at 37°C for 10-11 days using the differentiation medium. The cells were digested with TrypLE in a carbon dioxide incubator at 37°C for 15 minutes, centrifuged at 200 x g for 3 minutes, and the supernatant was discarded. The cells were resuspended in sebaceous gland precursor differentiation medium supplemented with 10 μM dihydrochloride ATP competitive ROCK inhibitor.

[0194] g. The cells obtained in step (f) were grown in 25 μg / mL fibronectin-coated culture plates;

[0195] h. On the 13th day of continuous culture in a 37°C, 5% CO2 incubator, replace the culture medium with fresh sebaceous gland precursor differentiation medium;

[0196] i. The cells were cultured daily in a 37°C, 5% CO2 incubator for 9 consecutive days to obtain sebaceous gland precursor cells.

[0197] In this example, sebaceous gland precursor cells are used to evaluate and screen oil-regulating drugs or skin care products:

[0198] 1. Changes in lipid droplets before and after intervention with different concentrations of dihydrotestosterone in sebaceous gland precursor cells (SBP) and human immortalized sebaceous gland cells (SZ95)

[0199] a. Cells were seeded in 96-well plates and treated with different concentrations of dihydrotestosterone (DHT) (1 μM, 3 μM, 10 μM, 30 μM, and 100 μM) for 24 h. The culture medium was discarded, the cells were washed twice with PBS, and fixed with 4% paraformaldehyde for 20 min. The fixative was discarded, the cells were washed twice with distilled water, and then immersed in 60% isopropanol for 15 min. The 60% isopropanol solution was discarded, the cells were washed twice with distilled water, and stained with prepared Oil Red O stain for 20 min. The stain was discarded, and the cells were washed with water until no excess stain was left. The nuclei were counterstained with hematoxylin for 2 min. The hematoxylin stain was discarded, and the cells were washed with tap water until no excess stain was left. The cells were covered with distilled water and observed under a microscope, photographed, and recorded.

[0200] b. As shown in Figure 16, compared with immortalized human sebaceous gland cells, the sebaceous gland precursor cells obtained by this protocol responded more significantly to DHT intervention, with significantly larger lipid droplet volume and increased number, and can be used for subsequent drug or skin care product screening.

[0201] 2. Changes in lipid droplets after DHT stimulation of sebaceous gland precursor cells (SBP) by different drugs and skin care products

[0202] a. Cells were seeded in 96-well plates and treated with 100 μM DHT for 24 hours. Various drugs at different concentrations were then added for another 24 hours. The culture medium was discarded, cells were washed twice with PBS, fixed with 4% paraformaldehyde for 20 minutes, the fixative was discarded, cells were washed twice with distilled water, and immersed in 60% isopropanol for 15 minutes. The 60% isopropanol solution was discarded, cells were washed twice with distilled water, and stained with the prepared Oil Red O stain for 20 minutes. The stain was discarded, cells were washed with water until no excess stain was left, and nuclei were counterstained with hematoxylin for 2 minutes. The hematoxylin stain was discarded, and cells were washed with water until no excess stain was left. Cells were covered with distilled water, and observed and photographed under a microscope.

[0203] b. As shown in Figure 17, the sebaceous gland precursor cells obtained by this scheme were subjected to various drug interventions at different concentrations based on DHT stimulation, indicating that different concentrations of retinoic acid (RA), Curcuma zedoaria extract, MAT-XS TM -Clinical intervention showed that the volume and number of lipid droplets in sebaceous gland precursor cells (SBP) decreased in a concentration-dependent manner. Therefore, it can be seen that the method of the present invention can be used to screen and evaluate lipid-regulating drugs or skin care products.

[0204] Example 3: The present invention provides an immortalized human sebaceous gland precursor cell, which is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: C2023286. The method for preparing the cell comprises the following steps:

[0205] (1) Isolation and expansion culture of renal epithelial cells:

[0206] ① Isolation of renal epithelial cells:

[0207] a. Collect urine from acne patients into 50 mL centrifuge tubes and centrifuge at 400 x g for 10 minutes at room temperature. Discard the supernatant and retain 1 mL of the liquid.

[0208] b. Take 10 mL of the washing solution from the UrinEasy Urine Cell Isolation and Culture Kit, resuspend the pellet and transfer it to another centrifuge tube. Centrifuge at 200 x g at room temperature for 10 minutes, discard the supernatant, and then resuspend the cell pellet with the culture medium from the UrinEasy Urine Cell Isolation and Culture Kit and inoculate it into a culture plate coated with Matrigel in advance.

[0209] c. After 24 hours of culture, observe the cells for contamination. If there is no contamination, add 800 μL of the culture medium in the UrinEasy Urine Cell Isolation and Culture Kit and repeat this step for 3 days;

[0210] d. On the fourth day, observe for contamination and adherent cells. If no contamination is found, discard 2 / 3 of the culture medium in the well and add 800 μL of the culture medium in the UrinEasy Urine Cell Isolation and Culture Kit. Observe the culture medium for contamination and adherent cells daily thereafter, and replace the medium with half or full volume every other day.

[0211] ② Expansion and culture of renal epithelial cells:

[0212] a. After isolation in step ① above, when the confluence of renal epithelial cells reaches 80%, they can be passaged and expanded;

[0213] b. Discard the culture medium in the wells, wash once with PBS, and digest in a 0.25% trypsin digestion solution at 37°C for 3 minutes in an incubator. Add 2 times the volume of the digestion solution to terminate the digestion with the expansion culture basal medium in the UrinEasy urine cell expansion culture kit. Gently pipette the bottom of the culture well to detach the cells.

[0214] c. Collect the cell suspension into a 15 mL centrifuge tube, centrifuge at 1200 rpm at room temperature for 3 minutes, discard the supernatant, resuspend the cell pellet in the UrinEasy expansion culture basal medium, gently pipette to a single cell suspension, and evenly inoculate the cells into a Matrigel-coated culture plate;

[0215] (2) Establishment and expansion of human induced pluripotent stem cell lines:

[0216] ① Establishment of human induced pluripotent stem cell lines:

[0217] a. Step (1) When passaged, urine-derived renal epithelial cells were plated at cell densities of 5000 / well, 10000 / well, and 15000 / well in Matrigel-coated 96-well plates. Three replicates were prepared for each gradient, and this was recorded as day -1.

[0218] b. The next day, observe the cell status under a microscope and count the cells in one well of each density gradient. Select one well with a cell count between 10,000 and 20,000 for subsequent experiments.

[0219] c. Mix 10 μL of Reprogramming Supplement I (Cat. No. CA5002002-1) with 10 mL of the culture medium in UrinEasy Urine Cell Expansion Reagent to create "Reprogramming Medium A." Then, mix 100 μL of Reprogramming Medium A with 10 μL of Reprogramming Supplement II (Cat. No. CA5002002-2) to create "Reprogramming Medium B." Discard the culture medium in the selected wells and add Reprogramming Medium B. This is recorded as Day 0.

[0220] d. Observe cell morphology changes under a microscope every day for 2 consecutive days;

[0221] e. On day 3, observe cell morphology under a microscope. If cell morphology changes significantly and the cells reach 100% confluence, they can be passaged.

[0222] f. For cell passaging, first prepare Reprogramming Medium C. Add 10 μL of Reprogramming Supplement III (catalog number CA5002002-3) to 10 mL of the medium in the UrinEasy Urine Cell Expansion Culture Kit and mix thoroughly. Discard the medium in the culture wells of the cells to be digested. Wash once with PBS, then add 50 μL of trypsin to the culture wells. Incubate the cells in an incubator at 37°C for 3 minutes. Terminate the digestion by adding 100 μL of Reprogramming Medium C. Pipet the bottom of the culture wells, collect the cell suspension into a centrifuge tube containing Reprogramming Medium C, pipette and mix thoroughly. Evenly plate the cells onto Matrigel-coated culture plates at a passage ratio of 1:7.

[0223] g. On day 4, replace the culture medium with the UrinEasy Urine Cell Expansion Culture Kit;

[0224] h. Observe the cells daily. If small clones are observed under the microscope, replace the medium daily with Reproeasy Human Cell Reprogramming Basal Medium (Cat. No. CA5003050-1).

[0225] i. If multiple colonies consisting of more than 10 cells are detected, replace the culture medium with PGM1 Human Pluripotent Stem Cell Medium (Cat. No. CA1007500);

[0226] j. Observe the cells under a microscope and culture until multiple colonies fill the 10x magnification field of view. Use a 1 mL syringe needle to cut the colonies and a 200 μL sterile pipette tip to separate the colonies from the bottom wall of the culture well. Aspirate the colonies and transfer them to a Matrigel-coated 24-well plate filled with the PGM1 human pluripotent stem cell culture medium. Return the plate to the incubator and culture at 37°C, 5% carbon dioxide, and 90% or greater humidity.

[0227] k. Observe daily and replace the medium with the PGM1 human pluripotent stem cell culture medium;

[0228] ② Expansion and culture of human induced pluripotent stem cell lines:

[0229] a. Cells can be passaged when microscopic observation shows that they are in good condition and have a confluence of approximately 80%, or when there is uneven distribution of clones with a single large clone.

[0230] b. Discard the culture medium in the wells, rinse once with PBS, and add DissoEasy Human Pluripotent Stem Cell Digestion Solution (Cat. No. CA1023100). Digest at room temperature for 4 minutes. Discard the digestion solution in the wells and add the PGM1 Human Pluripotent Stem Cell Medium. Gently pipette the bottom of the wells in a fan-shaped pattern to dislodge the cells. Collect the cell suspension into a centrifuge tube. Inoculate the cell suspension onto a Matrigel-coated culture plate at a volume ratio of 1:6 and continue culturing in an incubator at 37°C, 5% carbon dioxide, and 90% humidity. Observe daily and replace the PGM1 Human Pluripotent Stem Cell Medium daily.

[0231] (3) Differentiation of sebaceous gland precursor cells

[0232] a. The human induced pluripotent stem cells obtained in step (2) were replaced with a basal differentiation medium for differentiating human induced pluripotent stem cells into sebaceous gland precursor cells, the basal differentiation medium consisting of the following components:

[0233] b. Replace the basic differentiation medium in step (a) every day and culture in an incubator at 37°C and a volume concentration of 5% CO2 for three consecutive days;

[0234] c. After three days of cell culture, the culture medium was replaced with sebaceous gland precursor differentiation activation medium, which was the basal differentiation medium in step (a) supplemented with 20 ng / mL epidermal growth factor and 1 ng / mL bone morphogenetic protein 4;

[0235] d. Replace the sebaceous gland differentiation activation medium every other day and culture continuously in a 37°C, 5% CO2 incubator for 7 days;

[0236] e. Prepare sebaceous gland precursor differentiation medium, which consists of the following components:

[0237] EpiLife TM Culture medium: Dermacult Keratinocyte Expansion Medium (volume ratio 1:1) Epidermal Growth Factor 20 ng / mL Bone Morphogenetic Protein 4 1 ng / mL

[0238] f. The cells were cultured in the differentiation medium at 37°C and 5% CO2 in an incubator for 10 days, and the cells were digested with TrypLE. The digestion method was to digest the cells in a carbon dioxide incubator at 37°C for 15 minutes, centrifuged at 200 x g for 3 minutes, and the supernatant was discarded. The cells were resuspended in sebaceous gland precursor differentiation medium supplemented with 10 μM dihydrochloride ATP competitive ROCK inhibitor;

[0239] g. The cells obtained in step (f) were grown in 25 μg / mL fibronectin-coated culture plates;

[0240] h. On the 12th day of continuous culture in a 37°C, 5% CO2 incubator, replace the culture medium with fresh sebaceous gland precursor differentiation medium;

[0241] i. The cells were cultured daily in a 37°C, 5% CO2 incubator for 7 consecutive days to obtain sebaceous gland precursor cells.

[0242] Sebaceous gland precursor cells are cultured and differentiated to obtain mature sebaceous gland cells, and the cells are used to screen and evaluate oil secretion drugs or skin care products. The method for culturing and differentiating sebaceous gland precursor cells to obtain mature sebaceous gland cells is as follows:

[0243] a. Prepare mature sebaceous gland basal differentiation medium, which consists of the following components:

[0244] b. Digest the sebaceous gland precursor cells with TrypLE: Digest for 15 minutes in a 5% CO2 incubator at 37°C. Centrifuge at 250xg for 3 minutes, discard the supernatant, and add 10 μM dihydrochloride ATP competitive ROCK inhibitor to the mature sebaceous gland basal differentiation medium. Then resuspend the cells at 2.5×10 4 cells / cm 2 The cells were seeded in culture plates at a density of 100 μg / mL and cultured overnight in a 37°C incubator with a volume concentration of 5% CO2.

[0245] c. The next day, the medium was changed and 10 μM TGF-β / Smad inhibitor was added to the mature sebaceous gland basal differentiation medium;

[0246] d. Change the medium daily and incubate the cells in a 37°C, 5% CO2 incubator for 3 consecutive days.

[0247] e. After 3 days, the medium was changed to mature sebaceous gland terminal differentiation medium and cultured in a 37°C incubator with 5% CO2.

[0248] f. The medium is changed daily and cultured in a 37°C, 5% CO2 incubator for 3 consecutive days to obtain mature sebaceous gland cells, which can be used to screen and evaluate oil-secreting drugs or skin care products.

[0249] The mature sebaceous gland final differentiation medium in step (e) is obtained by adding 10 μM TGF-β / Smad inhibitor to the mature sebaceous gland basal differentiation medium.

[0250] Example 4: The present invention provides an immortalized human sebaceous gland precursor cell, which is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: C2023286. The method for preparing the cell comprises the following steps:

[0251] (1) Isolation and expansion culture of renal epithelial cells:

[0252] ① Isolation of renal epithelial cells:

[0253] a. Collect urine from acne patients into 50 mL centrifuge tubes and centrifuge at 400 x g for 10 minutes at room temperature. Discard the supernatant and retain 2 mL of the liquid.

[0254] b. Take 10 mL of the washing solution from the UrinEasy Urine Cell Isolation and Culture Kit, resuspend the pellet and transfer it to another centrifuge tube. Centrifuge at 200 x g at room temperature for 10 minutes, discard the supernatant, and then resuspend the cell pellet with the culture medium from the UrinEasy Urine Cell Isolation and Culture Kit and inoculate it into a culture plate coated with Matrigel in advance.

[0255] c. After 24 hours of culture, observe the cells for contamination. If there is no contamination, add 800 μL of the culture medium in the UrinEasy Urine Cell Isolation and Culture Kit and repeat this step for 3 days;

[0256] d. On the fourth day, observe for contamination and adherent cells. If no contamination is found, discard 2 / 3 of the culture medium in the well and add 800 μL of the culture medium in the UrinEasy Urine Cell Isolation and Culture Kit. Observe the culture medium for contamination and adherent cells daily thereafter, and replace the medium with half or full volume every other day.

[0257] ② Expansion and culture of renal epithelial cells:

[0258] a. After isolation in step ① above, when the confluence of renal epithelial cells reaches 90%, they can be passaged and expanded;

[0259] b. Discard the culture medium in the wells, wash once with PBS, and digest in a 0.25% trypsin digestion solution at 37°C for 3 minutes in an incubator. Add 2 times the volume of the digestion solution to terminate the digestion with the expansion culture basal medium in the UrinEasy urine cell expansion culture kit. Gently pipette the bottom of the culture well to detach the cells.

[0260] c. Collect the cell suspension into a 15 mL centrifuge tube, centrifuge at 1200 rpm at room temperature for 3 minutes, discard the supernatant, resuspend the cell pellet in the UrinEasy expansion culture basal medium, gently pipette to a single cell suspension, and evenly inoculate the cells into a Matrigel-coated culture plate;

[0261] (2) Establishment and expansion of human induced pluripotent stem cell lines:

[0262] ① Establishment of human induced pluripotent stem cell lines:

[0263] a. Step (1) When passaged, urine-derived renal epithelial cells were plated at cell densities of 5000 / well, 10000 / well, and 15000 / well in Matrigel-coated 96-well plates. Three replicates were prepared for each gradient, and this was recorded as day -1.

[0264] The next day, observe the cell status under a microscope, select one well from each density gradient for cell counting, and select one culture well with a cell count between 10,000 and 20,000 for subsequent experiments;

[0265] c. Mix 10 μL of Reprogramming Supplement I (Cat. No. CA5002002-1) with 10 mL of the culture medium in UrinEasy Urine Cell Expansion Reagent to create "Reprogramming Medium A." Then, mix 100 μL of Reprogramming Medium A with 10 μL of Reprogramming Supplement II (Cat. No. CA5002002-2) to create "Reprogramming Medium B." Discard the culture medium in the selected wells and add Reprogramming Medium B. This is recorded as Day 0.

[0266] d. Observe cell morphology changes under a microscope every day for 2 consecutive days;

[0267] e. On day 3, observe cell morphology under a microscope. If cell morphology changes significantly and the cells reach 100% confluence, they can be passaged.

[0268] f. For cell passaging, first prepare Reprogramming Medium C. Add 10 μL of Reprogramming Supplement III (Cat. No. CA5002002-3) to 10 mL of the medium in the UrinEasy Urine Cell Expansion Culture Kit and mix thoroughly. Discard the medium in the culture wells containing the cells to be digested. Wash once with PBS, then add 50 μL of trypsin to the wells. Incubate the wells at 37°C for 3 minutes. Terminate the digestion by adding 100 μL of Reprogramming Medium C. Fracture the bottom of the wells and collect the cell suspension into a centrifuge tube containing Reprogramming Medium C. Mix thoroughly by pipetting. Plate the cells evenly onto Matrigel-coated plates at a passage ratio of 1:8.

[0269] g. On day 4, replace the culture medium with the UrinEasy Urine Cell Expansion Culture Kit;

[0270] h. Observe the cells daily. If small clones are observed under the microscope, replace the medium daily with Reproeasy Human Cell Reprogramming Basal Medium (Cat. No. CA5003050-1).

[0271] i. If multiple clones consisting of more than 10 cells are found, replace the culture medium with PSCeasy Human Pluripotent Stem Cell Recovery Medium;

[0272] j. Observe the cells under a microscope and culture until multiple colonies fill the 10x magnification field of view. Use a 1 mL syringe needle to cut the colonies and a 200 μL sterile pipette tip to separate the colonies from the bottom wall of the culture well. Aspirate the colonies and transfer them to a Matrigel-coated 24-well plate filled with the PGM1 human pluripotent stem cell culture medium. Return the plate to the incubator and culture at 37°C, 5% carbon dioxide, and 90% or greater humidity.

[0273] k. Observe daily and replace the medium with PGM1 human pluripotent stem cell culture medium;

[0274] ② Expansion and culture of human induced pluripotent stem cell lines:

[0275] a. Cells can be passaged when microscopic observation shows that they are in good condition and have a confluence of approximately 80%, or when there is uneven distribution of clones with a single large clone.

[0276] b. Discard the culture medium in the wells, rinse once with PBS, add DissoEasy Human Pluripotent Stem Cell Digestion Solution (Cat. No. CA1023100), digest at room temperature for 5 minutes, discard the digestion solution in the wells, add the PGM1 Human Pluripotent Stem Cell Medium, gently fan the bottom of the culture wells to detach the cells, and collect the cell suspension into a centrifuge tube. Inoculate the cell suspension onto a Matrigel-coated culture plate at a volume ratio of 1:8 and continue culturing in an incubator at 37°C, 5% carbon dioxide, and above 90% humidity. Observe daily thereafter and replace the PGM1 Human Pluripotent Stem Cell Medium daily.

[0277] (3) Differentiation of sebaceous gland precursor cells

[0278] a. The human induced pluripotent stem cells obtained in step (2) were replaced with a basal differentiation medium for differentiating human induced pluripotent stem cells into sebaceous gland precursor cells, the basal differentiation medium consisting of the following components:

[0279] b. Replace the basic differentiation medium in step (a) every day and culture in an incubator at 37°C and a volume concentration of 5% CO2 for three consecutive days;

[0280] c. After three days of cell culture, the culture medium was replaced with sebaceous gland precursor differentiation activation medium, which was the basal differentiation medium in step (a) supplemented with 20 ng / mL epidermal growth factor and 1 ng / mL bone morphogenetic protein 4;

[0281] d. Replace the sebaceous gland differentiation activation medium every other day and culture continuously at 37°C in a 5% CO2 incubator for 8 days.

[0282] e. Prepare sebaceous gland precursor differentiation medium, which consists of the following components:

[0283] EpiLife TM Culture medium: Dermacult Keratinocyte Expansion Medium (volume ratio 1:1) Epidermal Growth Factor 20 ng / mL Bone Morphogenetic Protein 4 1 ng / mL

[0284] f. The cells were cultured in the differentiation medium at 37°C and 5% CO2 in an incubator for 11 days, and the cells were digested with TrypLE. The digestion method was to digest the cells in a carbon dioxide incubator at 37°C for 15 minutes, centrifuged at 200 x g for 3 minutes, and the supernatant was discarded. The cells were resuspended in sebaceous gland precursor differentiation medium supplemented with 10 μM dihydrochloride ATP competitive ROCK inhibitor;

[0285] g. The cells obtained in step (f) were grown in 25 μg / mL fibronectin-coated culture plates;

[0286] h. On the 13th day of continuous culture in a 37°C, 5% CO2 incubator, replace the culture medium with fresh sebaceous gland precursor differentiation medium;

[0287] i. The cells were cultured daily in a 37°C, 5% CO2 incubator for 8 consecutive days to obtain sebaceous gland precursor cells.

[0288] Sebaceous gland precursor cells are cultured and differentiated to obtain mature sebaceous gland cells, and the cells are used to screen and evaluate oil secretion drugs or skin care products. The method for culturing and differentiating sebaceous gland precursor cells to obtain mature sebaceous gland cells is as follows:

[0289] a. Prepare mature sebaceous gland basal differentiation medium, which consists of the following components:

[0290] b. Digest the sebaceous gland precursor cells with TrypLE: Digest for 15 minutes in a 5% CO2 incubator at 37°C. Centrifuge at 250xg for 3 minutes, discard the supernatant, and add 10 μM dihydrochloride ATP competitive ROCK inhibitor to the mature sebaceous gland basal differentiation medium. Then resuspend the cells at 2.5×10 4 cells / cm 2 The cells were seeded in culture plates at a density of 100 μg / mL and cultured overnight in a 37°C incubator with a volume concentration of 5% CO2.

[0291] c. The next day, the medium was changed and 10 μM TGF-β / Smad inhibitor was added to the mature sebaceous gland basal differentiation medium;

[0292] d. Change the medium daily and incubate the cells in a 37°C, 5% CO2 incubator for 3 consecutive days.

[0293] e. After 3 days, the medium was changed to mature sebaceous gland terminal differentiation medium and cultured in a 37°C incubator with 5% CO2.

[0294] f. The medium is changed daily and cultured in a 37°C, 5% CO2 incubator for 6 consecutive days to obtain mature sebaceous gland cells, which can be used to screen and evaluate oil-secreting drugs or skin care products.

[0295] The mature sebaceous gland final differentiation medium in step (e) is obtained by adding 10 μM TGF-β / Smad inhibitor and 1 μM PPAR-β / δ agonist to the mature sebaceous gland basal differentiation medium.

[0296] Example 5: The present invention provides an immortalized human sebaceous gland precursor cell, which is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: C2023286. The method for preparing the cell comprises the following steps:

[0297] (1) Isolation and expansion culture of renal epithelial cells:

[0298] ① Isolation of renal epithelial cells:

[0299] a. Collect urine from acne patients into 50 mL centrifuge tubes and centrifuge at 400 x g for 10 minutes at room temperature. Discard the supernatant and retain 1 mL of the liquid.

[0300] b. Take 10 mL of the washing solution from the UrinEasy Urine Cell Isolation and Culture Kit, resuspend the pellet and transfer it to another centrifuge tube. Centrifuge at 200 x g at room temperature for 10 minutes, discard the supernatant, and then resuspend the cell pellet with the culture medium from the UrinEasy Urine Cell Isolation and Culture Kit and inoculate it into a culture plate coated with Matrigel in advance.

[0301] c. After 24 hours of culture, observe the cells for contamination. If there is no contamination, add 800 μL of the culture medium in the UrinEasy Urine Cell Isolation and Culture Kit and repeat this step for 3 days;

[0302] d. On the fourth day, observe for contamination and adherent cells. If no contamination is found, discard 2 / 3 of the culture medium in the well and add 800 μL of the culture medium in the UrinEasy Urine Cell Isolation and Culture Kit. Observe the culture medium for contamination and adherent cells daily thereafter, and replace the medium with half or full volume every other day.

[0303] ② Expansion and culture of renal epithelial cells:

[0304] a. After isolation in step ① above, when the confluence of renal epithelial cells reaches 80%, they can be passaged and expanded;

[0305] b. Discard the culture medium in the wells, wash once with PBS, and digest in a 0.25% trypsin digestion solution at 37°C for 3 minutes in an incubator. Add 2 times the volume of the digestion solution to terminate the digestion with the expansion culture basal medium in the UrinEasy urine cell expansion culture kit. Gently pipette the bottom of the culture well to detach the cells.

[0306] c. Collect the cell suspension into a 15 mL centrifuge tube, centrifuge at 1200 rpm at room temperature for 3 minutes, discard the supernatant, resuspend the cell pellet in the UrinEasy expansion culture basal medium, gently pipette to a single cell suspension, and evenly inoculate the cells into a Matrigel-coated culture plate;

[0307] (2) Establishment and expansion of human induced pluripotent stem cell lines:

[0308] ① Establishment of human induced pluripotent stem cell lines:

[0309] a. Step (1) When passaged, urine-derived renal epithelial cells were plated at cell densities of 5000 / well, 10000 / well, and 15000 / well in Matrigel-coated 96-well plates. Three replicates were prepared for each gradient, and this was recorded as day -1.

[0310] b. The next day, observe the cell status under a microscope and count the cells in one well of each density gradient. Select one well with a cell count between 10,000 and 20,000 for subsequent experiments.

[0311] c. Mix 10 μL of Reprogramming Supplement I (Cat. No. CA5002002-1) with 10 mL of the culture medium in UrinEasy Urine Cell Expansion Reagent to create "Reprogramming Medium A." Then, mix 100 μL of Reprogramming Medium A with 10 μL of Reprogramming Supplement II (Cat. No. CA5002002-2) to create "Reprogramming Medium B." Discard the culture medium in the selected wells and add Reprogramming Medium B. This is recorded as Day 0.

[0312] d. Observe cell morphology changes under a microscope every day for 2 consecutive days;

[0313] e. On day 3, observe cell morphology under a microscope. If cell morphology changes significantly and the cells reach 100% confluence, they can be passaged.

[0314] f. For cell passaging, first prepare Reprogramming Medium C. Add 10 μL of Reprogramming Supplement III (Cat. No. CA5002002-3) to 10 mL of the medium in the UrinEasy Urine Cell Expansion Culture Kit and mix thoroughly. Discard the medium in the culture wells containing the cells to be digested. Wash once with PBS, then add 50 μL of trypsin to the wells. Incubate the wells at 37°C for 3 minutes. Terminate the digestion by adding 100 μL of Reprogramming Medium C. Fracture the bottom of the wells and collect the cell suspension into a centrifuge tube containing Reprogramming Medium C. Mix thoroughly by pipetting. Plate the cells evenly onto Matrigel-coated plates at a 1:9 passaging ratio.

[0315] g. On day 4, replace the culture medium with the UrinEasy Urine Cell Expansion Culture Kit;

[0316] h. Observe the cells daily. If small clones are observed under the microscope, replace the medium daily with Reproeasy Human Cell Reprogramming Basal Medium (Cat. No. CA5003050-1).

[0317] i. If multiple colonies consisting of more than 10 cells are detected, replace the culture medium with PGM1 Human Pluripotent Stem Cell Medium (Cat. No. CA1007500);

[0318] j. Observe the cells under a microscope and culture until multiple colonies fill the 10x magnification field of view. Use a 1 mL syringe needle to cut the colonies and a 200 μL sterile pipette tip to separate the colonies from the bottom wall of the culture well. Aspirate the colonies and transfer them to a Matrigel-coated 24-well plate filled with the PGM1 human pluripotent stem cell culture medium. Return the plate to the incubator and culture at 37°C, 5% carbon dioxide, and 90% or greater humidity.

[0319] k. Observe daily and replace the medium with the PGM1 human pluripotent stem cell culture medium;

[0320] ② Expansion and culture of human induced pluripotent stem cell lines:

[0321] a. Cells can be passaged when microscopic observation shows that they are in good condition and have a confluence of approximately 80%, or when there is uneven distribution of clones with a single large clone.

[0322] b. Discard the culture medium in the wells, wash once with PBS, add DissoEasy Human Pluripotent Stem Cell Digestion Solution (Cat. No. CA1023100), digest at room temperature for 3 minutes, discard the digestion solution in the wells, add the PGM1 Human Pluripotent Stem Cell Medium, gently fan the bottom of the culture wells to detach the cells, and collect the cell suspension into a centrifuge tube. Inoculate the cell suspension into a Matrigel-coated culture plate at a volume ratio of 1:10 and continue culturing in an incubator at 37°C, 5% carbon dioxide, and above 90% humidity. Observe daily thereafter and change the medium with the PGM1 Human Pluripotent Stem Cell Medium daily.

[0323] (3) Differentiation of sebaceous gland precursor cells

[0324] a. The human induced pluripotent stem cells obtained in step (2) were replaced with a basal differentiation medium for differentiating human induced pluripotent stem cells into sebaceous gland precursor cells, the basal differentiation medium consisting of the following components:

[0325] b. Replace the basic differentiation medium in step (a) every day and culture in an incubator at 37°C and a volume concentration of 5% CO2 for three consecutive days;

[0326] c. After three days of cell culture, the culture medium was replaced with sebaceous gland precursor differentiation activation medium, which was the basal differentiation medium in step (a) supplemented with 20 ng / mL epidermal growth factor and 1 ng / mL bone morphogenetic protein 4;

[0327] d. Replace the sebaceous gland differentiation activation medium every other day and culture continuously in a 37°C, 5% CO2 incubator for 7 days;

[0328] e. Prepare sebaceous gland precursor differentiation medium, which consists of the following components:

[0329] EpiLife TM Culture medium: Dermacult Keratinocyte Expansion Medium (volume ratio 1:1)

[0330] Epidermal growth factor 20ng / mL

[0331] Bone morphogenetic protein 4 1ng / mL

[0332] f. The cells were cultured in the differentiation medium at 37°C and 5% CO2 in an incubator for 10 days, and the cells were digested with TrypLE. The digestion method was to digest the cells in a carbon dioxide incubator at 37°C for 15 minutes, centrifuged at 200 x g for 3 minutes, and the supernatant was discarded. The cells were resuspended in sebaceous gland precursor differentiation medium supplemented with 10 μM dihydrochloride ATP competitive ROCK inhibitor;

[0333] g. The cells obtained in step (f) were grown in 25 μg / mL fibronectin-coated culture plates;

[0334] h. On the 12th day of continuous culture in a 37°C, 5% CO2 incubator, replace the culture medium with fresh sebaceous gland precursor differentiation medium;

[0335] i. The cells were cultured daily in a 37°C, 5% CO2 incubator for 7 consecutive days to obtain sebaceous gland precursor cells.

[0336] Sebaceous gland precursor cells are cultured and differentiated to obtain mature sebaceous gland cells, and the cells are used to screen and evaluate oil secretion drugs or skin care products. The method for culturing and differentiating sebaceous gland precursor cells to obtain mature sebaceous gland cells is as follows:

[0337] a. Prepare mature sebaceous gland basal differentiation medium, which consists of the following components:

[0338] b. Digest the sebaceous gland precursor cells with TrypLE: Digest for 15 minutes in a 5% CO2 incubator at 37°C. Centrifuge at 250xg for 3 minutes, discard the supernatant, and add 10 μM dihydrochloride ATP competitive ROCK inhibitor to the mature sebaceous gland basal differentiation medium. Then resuspend the cells at 2.5×10 4 cells / cm 2 The cells were seeded in culture plates at a density of 100 μg / mL and cultured overnight in a 37°C incubator with a volume concentration of 5% CO2.

[0339] c. The next day, the medium was changed and 10 μM TGF-β / Smad inhibitor was added to the mature sebaceous gland basal differentiation medium;

[0340] d. Change the medium daily and incubate the cells in a 37°C, 5% CO2 incubator for 3 consecutive days.

[0341] e. After 3 days, the medium was changed to mature sebaceous gland terminal differentiation medium and cultured in a 37°C incubator with 5% CO2.

[0342] f. The medium is changed daily and cultured in a 37°C, 5% CO2 incubator for 5 consecutive days to obtain mature sebaceous gland cells, which can be used to screen and evaluate oil secretion drugs or skin care products.

[0343] The mature sebaceous gland final differentiation medium in step (e) is prepared by adding 10 μM TGF-β / Smad inhibitor, 1 μM GW0742 PPAR-β / δ agonist and 5 μM cyclopamine to the mature sebaceous gland basal differentiation medium.

[0344] The following experiments illustrate that the mature sebaceous gland cells obtained in Examples 3 to 5 of the present invention are stable and have the properties of mature sebaceous gland cells in vitro, and can be well used for screening and evaluation of oil secretion regulating drugs or skin care products.

[0345] 1) Morphological Observation of Mature Sebaceous Gland Cells in Examples 3 to 5 of the Present Invention

[0346] As shown in FIG18 , the morphology of mature sebaceous gland cells of Examples 3 to 5 was observed and photographed under an optical microscope, and an increase and enlargement of round cells were observed (scale bar = 20 μm).

[0347] 2) Fluorescent staining of BODIP and KRT7 in mature sebaceous gland cells in Example 3 of the present invention

[0348] a. Cells were seeded onto eight-well ibidi chamber slides. The cells were removed, the culture medium discarded, and the slides were washed twice with PBS. The waste solution was discarded. The cells were fixed with 4% paraformaldehyde for 10 minutes. The 4% paraformaldehyde was discarded, and the slides were washed twice with PBS. A 1% BSA solution was added and blocked at room temperature for 1 hour. A diluted primary antibody (KRT7-mouse; ab216016; 1:100 dilution) was added and incubated at 4°C overnight. The next day, the plate was removed, the wells discarded, the wells washed three times with PBS, and a diluted secondary antibody (Mouse 568; 1:1000 dilution) was added and incubated at room temperature for 1 hour. The wells were discarded, the wells washed three times with PBS, and BODIP (1:1000 dilution) and F-actin (Phanergic 633; ​​1:1000 dilution) were added and incubated at room temperature for 30 minutes. The wells were discarded, the wells washed three times with PBS, and DAPI (1:1000 dilution) was added and incubated at room temperature for 15 minutes. The liquid in the wells was discarded, and the wells were washed three times with PBS. Images were taken using a laser confocal microscope, and different fluorescent staining images were merged.

[0349] b. As shown in Figure 19 , BODIPY fluorescence staining of mature sebaceous gland cells revealed that the mature sebaceous gland cells obtained using this protocol exhibit the properties of human sebaceous gland cells that form or accumulate lipid droplets. Furthermore, because KRT7 is highly expressed in sebaceous gland cells, immunofluorescence analysis of KRT7 protein expression in mature sebaceous gland cells revealed KRT7 expression in the mature sebaceous gland cells obtained using this protocol. Arrows in the figure indicate lipid droplets and KRT7 expression (Scale bar = 20 μm).

[0350] 3) Differentiation and induction of mature sebaceous gland cells under different conditions and their response to androgen (dihydrotestosterone):

[0351] As shown in FIG20 , the mature sebaceous gland cells of Examples 3 to 5 accelerated their maturation, differentiation, and even rupture under the stimulation of dihydrotestosterone, thereby releasing more lipid droplets / sebum (scale bar=20 μm).

[0352] 4) BODIP sebaceous gland cell lipid droplet staining

[0353] a. Cells were seeded on eight-well slides in an ibidi chamber. The experimental group was treated with 100 μM dihydrotestosterone (DHT), while the control group was cultured normally (without DHT) for 24 hours. The cells were removed, the culture medium discarded, and the cells were washed twice with PBS. The waste liquid was discarded. The cells were fixed with 4% paraformaldehyde for 10 minutes. The 4% paraformaldehyde was discarded, and the cells were washed twice with PBS. BODIBY (1:1000 dilution) was added and incubated at room temperature for 30 minutes. The liquid was discarded, and the cells were washed three times with PBS. DAPI (1:1000 dilution) was added and incubated at room temperature for 15 minutes. The liquid was discarded, and the cells were washed three times with PBS. Images were taken using a laser confocal microscope.

[0354] b. As shown in Figure 21, the mature sebaceous gland cells of Examples 3 to 5 and their responses to dihydrotestosterone (DHT) were monitored by BODIP staining. Under the stimulation of dihydrotestosterone, mature sebaceous gland cells accelerated cell maturation and differentiation, releasing more lipid droplets. The arrows indicate lipid droplet expression (scale bar = 20 μm).

[0355] 5) Oil Red O staining to evaluate the screening effect of SBM drugs and skin care products

[0356] a. Mature sebaceous gland cells were seeded into 24-well plates. After the cells grew to 50% of their size, they were stained. The cells were removed, washed twice with PBS, and fixed with 4% paraformaldehyde for 20 minutes. The fixative was discarded. The cells were washed twice with distilled water. The cells were then immersed in 60% isopropanol for 15 minutes. The 60% isopropanol solution was discarded. The cells were washed twice with distilled water. The cells were immerged in the prepared Oil Red O stain for 20 minutes. The stain was discarded and the cells were washed with water until no excess stain was left. The nuclei were counterstained with hematoxylin for 2 minutes. The hematoxylin stain was discarded and the cells were washed with water until no excess stain was left. The cells were covered with distilled water and observed under a microscope and photographed.

[0357] b. As shown in Figure 22, compared with the sebaceous gland mature cells (SBM) whose volume increased irregularly after DHT intervention, after intervention with different concentrations of retinoic acid RA and Curcuma zedoaria extract, Oil Red O staining showed that the sebaceous gland mature cells (SBM) obtained by this scheme decreased in volume, slowed down their maturation rate, and thus interfered with their lipid droplet release (Scale bar = 20 μm), thereby enabling the evaluation and screening of drugs or skin care products that inhibit oil secretion.

[0358] The above results show that compared with human keratinocytes, human immortalized sebocytes and human induced pluripotent stem cells, the sebaceous gland precursor cells obtained in the present invention contain rich lipid droplets and can further differentiate into mature sebaceous gland cells. They have clear genetic information and clinical background, can simulate the behavior of sebaceous glands in acne patients, and both sebaceous gland precursor cells and sebaceous gland mature cells can play a role in evaluating and screening the promotion and inhibition of oil secretion. This method can be used to provide relevant in vitro models for disease modeling and the development of innovative treatment methods, and also provide an in vitro model for the screening of skin care products. It should be noted that in order to save space, a large number of parallel experiments have not been written in. In the actual process, the present invention has repeated several parallel experiments, and the mature sebaceous gland cells obtained are also stable and reliable. In the present invention, the mature sebaceous gland cells obtained in Examples 3 to 5 have also been verified one by one.

[0359] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, various simple variations of the technical solution of the present invention may be made, including combining the specific technical features in any suitable manner. To avoid unnecessary repetition, the present invention will not further describe various possible combinations. However, these simple variations and combinations should also be considered as disclosed in the present invention and fall within the scope of protection of the present invention.

Claims

1. An immortalized human sebaceous gland precursor cell, characterized in that: The cell line was deposited in China Center for Type Culture Collection with the deposit number CCTCC NO: C2023286.

2. A method for screening and evaluating lipid-regulating drugs or skin care products using the cell of claim 1, characterized in that: Directly incubate sebaceous gland precursor cells with DHT dihydrotestosterone at room temperature for 48 hours to stimulate the production of more oil cells. This model can be used to screen and evaluate oil-regulating drugs or skin care products. Alternatively, sebaceous gland precursor cells can be cultured and differentiated into mature sebaceous gland cells, and the mature sebaceous gland cells can be used to screen and evaluate drugs or skin care products.

3. A method according to claim 2, characterized in that: Sebaceous gland precursor cells are cultured and differentiated to obtain mature sebaceous gland cells, and the cells are used to screen and evaluate oil secretion drugs or skin care products. The method for culturing and differentiating sebaceous gland precursor cells to obtain mature sebaceous gland cells is as follows: a. Prepare mature sebaceous gland basal differentiation medium, which consists of the following components: b. Digest the sebaceous gland precursor cells with TrypLE: Digest for 15 minutes in a 5% CO2 incubator at 37°C. Centrifuge at 250xg for 3 minutes, discard the supernatant, and add 10 μM dihydrochloride ATP competitive ROCK inhibitor to the mature sebaceous gland basal differentiation medium. Then resuspend the cells at 2.5×10 4 cells / cm 2 The cells were seeded in culture plates at a density of 100 μg / mL and cultured overnight in a 37°C incubator with a volume concentration of 5% CO2. c. The next day, the medium was changed and 10 μM TGF-β / Smad inhibitor was added to the mature sebaceous gland basal differentiation medium; d. Change the medium daily and incubate the cells in a 37°C, 5% CO2 incubator for 3 consecutive days. e. After 3 days, the medium was changed to mature sebaceous gland terminal differentiation medium and cultured in a 37°C incubator with 5% CO2. f. Change the medium daily and culture continuously in a 37°C, 5% CO2 incubator for 3-6 days to obtain mature sebaceous gland cells.

4. The method according to claim 3, characterized in that The mature sebaceous gland final differentiation medium in step (e) is obtained by adding 10 μM TGF-β / Smad inhibitor to the mature sebaceous gland basal differentiation medium; or by adding 10 μM TGF-β / Smad inhibitor and 1 μM PPAR-β / δ agonist to the mature sebaceous gland basal differentiation medium; or by adding 10 μM TGF-β / Smad inhibitor, 1 μM GW0742 PPAR-β / δ agonist and 5 μM cyclopamine to the mature sebaceous gland basal differentiation medium.

Images