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

Abstract

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. A preparation method and use of the cell are also provided. The cell can be directly used for the screening and evaluation of oil-regulating drugs or skin care products, or the cell can be further differentiated into mature sebaceous gland cells, and then the mature sebaceous gland cells are used for the screening and evaluation of oil-regulating drugs or skin care products. The advantages of the present invention are that the obtained sebaceous gland precursor cells and mature sebaceous gland cells are stable and accurate, and the expression of lipid droplet formation, lipid accumulation and androgen receptor-related genes is significantly increased, and they can be well used for the screening and evaluation of oil-regulating drugs or skin care products.

Description

Technical Field

[0001] The present invention belongs to the technical field of skin oil regulation models, and specifically relates to 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. Background Art

[0002] In recent years, the incidence of disfiguring skin diseases such as acne vulgaris, xeroderma, and seborrheic dermatitis has increased significantly, and the mechanism of their pathogenesis is closely related to the dysfunction of sebaceous glands. Sebaceous glands exist in the dermis and are one of the important accessory organs of the skin. Sebaceous gland cells exist in the sebaceous glands. Newly formed sebaceous gland cells continue to move toward the center, increase in volume, and have more and more 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 discharged at the same time as the lipid droplets, which is sebum. The secreted sebum can moisturize the skin and hair, and together with sweat, form a lipid membrane to protect the skin, playing an important 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 that affects the hair follicles and sebaceous glands. It is prone to occur on the face, chest, and back. Clinically, it mainly manifests as acne, papules, pustules, cysts, or nodules, often accompanied by enlarged pores and sebum secretion. The incidence rate among adolescents is as high as 93%, which has become the eighth largest chronic disease in the world. The incidence rate of acne in the Chinese population is 8.1%. However, studies have found that more than 95% of people will have varying degrees of acne. Without early intervention and treatment, scars are likely to remain, which seriously affects the patient's quality of life. Therefore, in order to reveal the pathophysiological mechanism of acne and develop medicines or cosmetics that inhibit abnormal sebum secretion, it is crucial to construct a cell model that can reproduce the behavior of the sebaceous glands of acne patients.

[0004] At present, as a sebaceous gland cell strain that can be used in experiments, a hamster sebaceous gland cell strain is known. This cell has the advantage of forming lipid droplets in the cell like the sebaceous gland cells of an organism, but its genomic information is not clear, so it is not suitable for research using genetic methods. Non-patent document 1 discloses that the sebaceous gland cell strain SZ95 derived from humans, which is immortalized by transfection of the giant T antigen DNA of SV40, is also commonly used in sebaceous gland-related experimental research. This cell is derived from humans, the genomic information is complete and clear, and the proliferation is high, but SZ95 lacks androgen receptors, they cannot respond to testosterone to promote fat production, and they lose the ability to form lipid droplets when cultured in vitro, and cannot fully represent sebaceous gland cells in a naturally growing state, let alone reproduce the sebaceous gland behavior of acne patients. In addition, due to the introduction of oncogenes in HPV and Epstein-Barr virus, the target cells may have a 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, which can stimulate sebum production but 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 by using microsurgical instruments for primary culture, although lipid accumulation could be seen in the cytoplasm, they could only grow for 3-6 generations, and the number of cells obtained was limited, and they could not be amplified 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 that does not use animal cells and has higher purity and yield of human sebaceous precursor 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] In order to achieve the above-mentioned object, 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 in the China Center for Type Culture Collection with a deposit number of CCTCC NO: C2023286, a classification name: human induced pluripotent stem cell-derived sebaceous gland precursor cell iPS-SBP1#, address: Wuhan University, Wuhan, China, and a deposit date of October 10, 2023.

[0009] The method for preparing the above-mentioned cell 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 and divide into 50 mL centrifuge tubes, centrifuge at 400 x g at room temperature for 10 minutes, discard the supernatant, and retain 1-2 mL of liquid;

[0013] b. Take 10 mL of the washing solution in 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 in the UrinEasy Urine Cell Isolation and Culture Kit, and inoculate it into a culture plate coated with Matrigel in advance;

[0014] c. After culturing for 24 hours, observe whether the cells are contaminated. If not, 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 4th day, observe whether there is contamination and adherent cells. If there is no contamination, discard 2 / 3 of the culture medium in the well, add 800 μL of the culture medium in the UrinEasy urine cell isolation and culture kit, and then observe whether there is contamination and adherent cells every day, and then use the culture medium to replace 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 is 80-90%, they can be passaged and expanded;

[0018] b. Discard the culture medium in the well, wash once with PBS, digest in an incubator with a mass concentration of 0.25% trypsin digestion solution at 37°C for 3 minutes, add 2 times the volume of the digestion solution to terminate the digestion, and gently blow the bottom of the culture well to make the cells fall off;

[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 using 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 in Matrigel-coated 96-well plates at cell densities of 5000 / well, 10000 / well, and 15000 / well, respectively. Three replicate wells were set for each gradient, and this was recorded as day -1.

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

[0024] c. Mix 10 μL of reprogramming additive I with the catalog number CA5002002-1 and the culture medium in 10 mL of UrinEasy urine cell expansion culture reagent to prepare "reprogramming culture medium A". Take 100 μL of reprogramming culture medium A and mix it with 10 μL of reprogramming additive II with the catalog number CA5002002-2 to prepare "reprogramming culture medium B". Discard the culture medium in the selected well and add reprogramming culture medium B. This is recorded as day 0.

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

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

[0027] f. When passaging cells, first prepare reprogramming medium C, add 10 μL of reprogramming additive III with catalog number CA5002002-3 to the medium in 10 mL of UrinEasy urine cell expansion culture kit, mix well, discard the medium in the culture wells of the cells to be digested, add PBS to wash once, add 50 μL of trypsin to the culture wells, digest at 37°C in the incubator for 3 minutes, add 100 μL of reprogramming medium C to terminate digestion, pipette the bottom of the culture wells, collect the cell suspension into a centrifuge tube containing reprogramming medium C, pipette and mix well, and evenly inoculate the cells into Matrigel-coated culture plates at a passaging ratio of 1:6-1:10;

[0028] g. On day 4, replace the culture medium in the UrinEasy urine cell expansion culture kit;

[0029] h. Observe the cell status daily. If small clusters of clones are found under the microscope, use Reproeasy Human Cell Reprogramming Basic Medium (Cat. No. CA5003050-1) to replace the medium daily;

[0030] i. If multiple clones consisting of more than 10 cells are found, replace the culture medium with PGM1 human pluripotent stem cell culture medium (catalog number CA1007500);

[0031] j. Observe the cells under a microscope, culture until the size of multiple clones can fill the 10x microscope field, cut the clones with a 1mL syringe needle, separate the clones from the bottom wall of the culture well with a 200μL sterile pipette tip, aspirate the clones and transfer them to a Matrigel-coated 24-well plate filled with the PGM1 human pluripotent stem cell culture medium, and return them to the incubator for culture at 37°C, 5% carbon dioxide by volume, and a humidity of more than 90%;

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

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

[0034] a. When the cells are in good condition under microscope and the cell confluence is about 80%, or when the clones are unevenly distributed and a single clone has a larger area, subculture can be performed;

[0035] b. Discard the culture medium in the well, wash with PBS, add DissoEasy human pluripotent stem cell digestion solution with catalog number CA1023100, digest at room temperature for 3-5 minutes, discard the digestion solution in the well, add the PGM1 human pluripotent stem cell culture medium, gently blow the bottom of the culture well in a fan-shaped manner to make the cells fall off, and collect the cell suspension into a centrifuge tube. Inoculate the cell suspension into the Matrigel-coated culture plate at a volume ratio of 1:5-1:10, and continue to culture in the incubator at 37°C, 5% carbon dioxide, and more than 90% humidity. Observe daily thereafter and change the medium with PGM1 human pluripotent stem cell culture medium every day;

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

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

[0038]

[0039] b. Replace the basic differentiation medium described in step (a) every day, and culture in an incubator at 37°C and 5% CO2 for three consecutive days;

[0040] c. After three days of cell culture, the culture medium was replaced with a sebaceous gland precursor differentiation activation medium, wherein the activation medium was the basic differentiation medium in step (a) supplemented with 20 ng / mL epidermal growth factor and 1 ng / mL bone morphogenetic protein 4;

[0041] d. Replace the sebaceous gland differentiation activation medium every other day and culture continuously for 7-8 days in an incubator at 37°C and 5% CO2;

[0042] e. Prepare sebaceous gland precursor differentiation medium, the medium consists of the following components:

[0043] EpiLife TM Culture medium: Dermacult keratinocyte expansion medium volume ratio 1:1

[0044] Epidermal growth factor 20ng / mL

[0045] Bone morphogenetic protein 4 1ng / mL

[0046] f. The cells were continuously cultured in the differentiation medium at 37°C and 5% CO2 incubator for 10-11 days, and the cells were digested with TrypLE. The digestion method was 37°C in a carbon dioxide incubator for 15 minutes, centrifuged at 200 x g for 3 minutes, and the supernatant was discarded. 10 μM dihydrochloride ATP competitive ROCK inhibitor was added to the sebaceous gland precursor differentiation medium to resuspend the cells;

[0047] g. The cells obtained in step (f) were grown in a 25 μg / mL fibronectin-coated culture plate;

[0048] 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;

[0049] i. The cells were cultured in a 37°C, 5% CO2 incubator for 7-9 days to obtain sebaceous gland precursor cells.

[0050] The present invention also provides a method for screening and evaluating oil-regulating drugs or skin care products using the precursor cells, wherein 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, and this model is used to screen and evaluate oil-regulating drugs or skin care products, or 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.

[0051] 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:

[0052] a. Prepare mature sebaceous gland basal differentiation medium, which consists of the following components:

[0053]

[0054]

[0055] b. Digest the sebaceous gland precursor cells with TrypLE: digest for 15 minutes in a 37°C, 5% CO2 incubator, centrifuge at 250 x g for 3 minutes, discard the supernatant, add 10 μM dihydrochloride ATP competitive ROCK inhibitor to the mature sebaceous gland basal differentiation medium, and then resuspend the cells at 2.5×10 4 cells / cm 2 The cells were seeded in culture plates at a density of 1.5 × 10 and cultured overnight in a 37°C, 5% CO2 incubator.

[0056] c. The medium was changed on the next day, and 10 μM TGF-β / Smad inhibitor was added to the mature sebaceous gland basal differentiation medium;

[0057] d. Change the medium every day and culture in a 37°C, 5% CO2 incubator for 3 consecutive days;

[0058] e. After 3 days, the medium was changed to mature sebaceous gland final differentiation medium and cultured in an incubator at 37°C and 5% CO2;

[0059] f. Change the medium every day and culture continuously for 3-6 days in an incubator at 37°C and 5% CO2 to obtain mature sebaceous gland cells.

[0060] Furthermore, in the step (e), the final differentiation medium of mature sebaceous glands is obtained by adding 10 μM TGF-β / Smad inhibitor to the basal differentiation medium of mature sebaceous glands; or by adding 10 μM TGF-β / Smad inhibitor and 1 μM PPAR-β / δ agonist to the basal differentiation medium of mature sebaceous glands; or by adding 10 μM TGF-β / Smad inhibitor, 1 μM GW0742 PPAR-β / δ agonist and 5 μM cyclopamine to the basal differentiation medium of mature sebaceous glands.

[0061] 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 obtained sebaceous gland precursor cells 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 androgen, 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

[0062] Figure 1 The morphology of renal epithelial cells isolated from urine samples of the present invention and cultured at different time points;

[0063] Figure 2 Morphological pictures of human induced pluripotent stem cells under an optical microscope at different time points after adding transcription factors of the present invention;

[0064] Figure 3 This is a morphological diagram of the reprogrammed human induced pluripotent stem cells by amplifying and culturing renal epithelial cells according to the present invention;

[0065] Figure 4 This is a diagram showing the alkaline phosphatase staining result of induced pluripotent stem cells by the inventor;

[0066] Figure 5 This is a diagram showing the results of immunofluorescence staining of induced pluripotent stem cells by the inventor;

[0067] Figure 6 This is a diagram showing the results of the karyotype identification of induced pluripotent stem cells by the inventor;

[0068] Figure 7 This is a staining diagram of the teratoma formation and three-germ layer differentiation of the induced pluripotent stem cells of the present invention;

[0069] Figure 8The changes in morphology during differentiation of sebaceous gland precursor cells of the present invention;

[0070] Fig. 9 This is a comparison of the expression levels of sebaceous gland precursor cell marker genes of 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;

[0071] Fig.10 This is a comparison of the expression levels of sebaceous gland precursor cell marker genes between human immortalized sebaceous gland cells (SZ95) and sebaceous gland precursor cells (SBP) of the present invention. In the figure, # P<0.05, ## P < 0.01 and ### P<0.001vs SZ95;

[0072] Fig.11 A comparison of the expression levels of sebaceous gland precursor cell marker genes of human induced pluripotent stem cells (hiPSC) and sebaceous gland precursor cells (SBP) of the present invention is shown in the figure, in the figure, & P<0.05, && P < 0.01 and &&& P<0.001vs hiPSC;

[0073] Fig.12 This is a comparison picture of lipid droplet content detected by Oil Red O staining of human keratinocytes (HaCaT), human immortalized sebaceous gland cells (SZ95), human induced pluripotent stem cells (hiPSC), and sebaceous gland precursor cells (SBP) of the present invention;

[0074] Fig.13 Comparative statistical graph of lipid droplet content detected by Oil Red O staining of human keratinocytes (HaCaT), human immortalized sebaceous gland cells (SZ95), human induced pluripotent stem cells (hiPSC), and sebaceous gland precursor cells (SBP) of the present invention;

[0075] Fig.14 This is a comparison chart of the fluorescence intensity of intracellular lipid droplets detected by Nile red fluorescence staining of human keratinocytes (HaCaT), human immortalized sebaceous gland cells (SZ95), human induced pluripotent stem cells (hiPSC), and sebaceous gland precursor cells (SBP) of the present invention;

[0076] Fig.15 This is a diagram showing the difference in gene expression between human immortalized sebaceous gland cells (SZ95) and sebaceous gland precursor cells (SBP) detected by RNA-seq of the present invention;

[0077] Fig.16Oil red O staining was used to detect the changes in lipid droplets after cells were treated with different concentrations of dihydrotestosterone (DHT);

[0078] Fig.17 Oil red O staining was used to detect the changes in lipid droplets after intervention of sebaceous gland precursor cells (SBP) with different concentrations of drugs and skin care product ingredients;

[0079] Fig.18 The morphological observation diagrams of mature sebaceous gland cells (SBM) obtained in Examples 3 to 5 of the present invention;

[0080] Fig.19 This is a diagram of lipid droplet formation and cytokeratin expression in mature sebaceous gland cells (SBM) of Example 3 of the present invention, in which arrows indicate lipid droplets and KRT7 expression;

[0081] Fig. 20 Graphs showing the responses of mature sebaceous gland cells (SBM) to dihydrotestosterone (DHT) obtained in Examples 3 to 5 of the present invention;

[0082] Fig.21 This 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 the arrow indicates lipid droplet expression;

[0083] Fig. 22 Oil Red O staining was used to detect the morphological changes of mature sebaceous gland cells (SBM) after intervention with different concentrations of drugs and skin care product ingredients. In the figure, the box indicates the morphology of mature sebaceous gland cells (SBM) that became larger in size after intervention with dihydrotestosterone (DHT). DETAILED DESCRIPTION

[0084] The specific embodiments of the present invention are described in detail below. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention. In the following experimental conditions, unless otherwise specified, room temperature conditions are used. The following reagents are 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.

[0085] Example 1: The present invention provides an immortalized human sebaceous gland precursor cell, which is deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO: C2023286. The method for preparing the cell comprises the following steps:

[0086] (1) Isolation and expansion culture of renal epithelial cells:

[0087] ① Isolation of renal epithelial cells:

[0088] a. Collect urine from acne patients and divide into 50 mL centrifuge tubes, centrifuge at 400 x g at room temperature for 10 minutes, discard the supernatant, and retain 1 mL of liquid;

[0089] b. Take 10 mL of the washing solution in 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 in the UrinEasy Urine Cell Isolation and Culture Kit, and inoculate it into a culture plate coated with Matrigel in advance;

[0090] c. After culturing for 24 hours, observe whether the cells are contaminated. If not, add 800 μL of the culture medium in the UrinEasy urine cell isolation and culture kit and repeat this step for 3 days;

[0091] d. On the 4th day, observe whether there is contamination and adherent cells. If there is no contamination, discard 2 / 3 of the culture medium in the well, add 800 μL of the culture medium in the UrinEasy urine cell isolation and culture kit, and then observe whether there is contamination and adherent cells every day, and then use the culture medium to replace half or full volume every other day;

[0092] ② Expansion and culture of renal epithelial cells:

[0093] a. After the separation in step ① above, when the confluence of renal epithelial cells reaches 80%, they can be passaged and expanded;

[0094] b. Discard the culture medium in the well, wash once with PBS, digest in an incubator with a mass concentration of 0.25% trypsin digestion solution at 37°C for 3 minutes, add 2 times the volume of the digestion solution to terminate the digestion, and gently blow the bottom of the culture well to make the cells fall off;

[0095] 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 using the UrinEasy expansion culture basal medium, gently pipette to a single cell suspension, and evenly inoculate the cells into a Matrigel-coated culture plate;

[0096] (2) Establishment and expansion of human induced pluripotent stem cell lines:

[0097] ① Establishment of human induced pluripotent stem cell lines:

[0098] a. Step (1) When passaged, urine-derived renal epithelial cells were plated in Matrigel-coated 96-well plates at cell densities of 5000 / well, 10000 / well, and 15000 / well, respectively. Three replicate wells were set for each gradient, and this was recorded as day -1.

[0099] b. The next day, observe the cell status under a microscope, select one well for each density gradient to count the cells, and select one culture well with a cell count between 10,000 and 20,000 for subsequent experiments;

[0100] c. Mix 10 μL of reprogramming additive I with the catalog number CA5002002-1 and the culture medium in 10 mL of UrinEasy urine cell expansion culture reagent to prepare "reprogramming culture medium A". Take 100 μL of reprogramming culture medium A and mix it with 10 μL of reprogramming additive II with the catalog number CA5002002-2 to prepare "reprogramming culture medium B". Discard the culture medium in the selected well and add reprogramming culture medium B. This is recorded as day 0.

[0101] d. Observe the cell morphology changes under the microscope every day and culture for 2 consecutive days;

[0102] e. On the third day, observe the cell morphology under a microscope. If the cell morphology changes significantly and the cells reach 100% confluence, they can be passaged;

[0103] f. When passaging cells, first prepare reprogramming medium C, add 10 μL of reprogramming additive III with catalog number CA5002002-3 to the medium in 10 mL of UrinEasy urine cell expansion culture kit, mix well, discard the medium in the culture wells of the cells to be digested, add PBS to wash once, add 50 μL of trypsin to the culture wells, digest at 37°C in the incubator for 3 minutes, add 100 μL of reprogramming medium C to terminate digestion, pipette the bottom of the culture wells, collect the cell suspension into a centrifuge tube containing reprogramming medium C, pipette and mix well, and evenly inoculate the cells into Matrigel-coated culture plates at a passaging ratio of 1:6;

[0104] g. On day 4, replace the culture medium in the UrinEasy urine cell expansion culture kit;

[0105] h. Observe the cell status daily. If small clusters of clones are found under the microscope, use Reproeasy Human Cell Reprogramming Basic Medium (Cat. No. CA5003050-1) to replace the medium daily;

[0106] i. If multiple clones consisting of more than 10 cells are found, replace the culture medium with PGM1 human pluripotent stem cell culture medium (catalog number CA1007500);

[0107] j. Observe the cells under a microscope, culture until the size of multiple clones can fill the 10x microscope field, cut the clones with a 1mL syringe needle, separate the clones from the bottom wall of the culture well with a 200μL sterile pipette tip, aspirate the clones and transfer them to a Matrigel-coated 24-well plate filled with the PGM1 human pluripotent stem cell culture medium, and return them to the incubator for culture at 37°C, 5% carbon dioxide by volume, and a humidity of more than 90%;

[0108] k. Observe daily and replace the medium using the PGM1 human pluripotent stem cell culture medium;

[0109] ② Expansion and culture of human induced pluripotent stem cell lines:

[0110] a. When the cells are in good condition under microscope and the cell confluence is about 80%, or when the clones are unevenly distributed and a single clone has a larger area, subculture can be performed;

[0111] b. Discard the culture medium in the well, add PBS to wash once, add DissoEasy human pluripotent stem cell digestion solution with catalog number CA1023100, digest at room temperature for 3 minutes, discard the digestion solution in the well, add the PGM1 human pluripotent stem cell culture medium, gently blow the bottom of the culture well in a fan shape to make the cells fall off, and collect the cell suspension into a centrifuge tube. Inoculate the cell suspension into the Matrigel-coated culture plate at a volume ratio of 1:5, continue to culture in the incubator, the culture conditions are 37°C, the volume concentration is 5% carbon dioxide, and the humidity is above 90%. Observe daily thereafter, and use the PGM1 human pluripotent stem cell culture medium to replace the medium daily;

[0112] (3) Differentiation of sebaceous gland precursor cells

[0113] a. The human induced pluripotent stem cells obtained in step (2) are replaced with a basic differentiation medium for differentiating human induced pluripotent stem cells into sebaceous gland precursor cells, wherein the basic differentiation medium consists of the following components:

[0114]

[0115]

[0116] b. Replace the basic differentiation medium described in step (a) every day, and culture in an incubator at 37°C and 5% CO2 for three consecutive days;

[0117] c. After three days of cell culture, the culture medium was replaced with a sebaceous gland precursor differentiation activation medium, wherein the activation medium was the basic differentiation medium in step (a) supplemented with 20 ng / mL epidermal growth factor and 1 ng / mL bone morphogenetic protein 4;

[0118] d. Replace the sebaceous gland differentiation activation medium every other day and culture continuously for 7 days in an incubator at 37°C and 5% CO2;

[0119] e. Prepare sebaceous gland precursor differentiation medium, the medium consists of the following components:

[0120] EpiLife TM Culture medium: Dermacult keratinocyte expansion medium volume ratio 1:1

[0121] Epidermal growth factor 20ng / mL

[0122] Bone morphogenetic protein 4 1ng / mL

[0123] f. The cells were continuously cultured in the differentiation medium at 37°C and 5% CO2 incubator for 10 days, and the cells were digested with TrypLE. The digestion method was 37°C in a carbon dioxide incubator for 15 minutes, centrifuged at 200 x g for 3 minutes, and the supernatant was discarded. 10 μM dihydrochloride ATP competitive ROCK inhibitor was added to the sebaceous gland precursor differentiation medium to resuspend the cells;

[0124] g. The cells obtained in step (f) were grown in a 25 μg / mL fibronectin-coated culture plate;

[0125] 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;

[0126] i. The cells were cultured in a 37°C, 5% CO2 incubator for 7 consecutive days to obtain sebaceous gland precursor cells.

[0127] The following experiments illustrate 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 well used for screening and evaluation of oil secretion regulating drugs or skin care products.

[0128] 1.1. Isolation and expansion of renal epithelial cells

[0129] 1)1) Renal epithelial cell isolation and culture process

[0130] 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).

[0131] b. Figure 1 As shown in the figure, with the passage of time and continuous fluid replacement, the suspended squamous epithelial cells and impurities gradually decreased, and multiple clusters of typical adherent cells with spindle or cobblestone morphology could be observed under the microscope, indicating that the renal epithelial cells were successfully isolated.

[0132] 2. Establishment, Culture and Identification of Human Induced Pluripotent Stem Cells

[0133] 1) Establishment of human induced pluripotent stem cells

[0134] a. Cell morphology at different time points (day 0, day 3, day 8 and day 12) after adding transcription factors was observed and photographed under an optical microscope.

[0135] b. Figure 2 As shown, compared with day 0, on day 3 after the addition of transcription factors, the morphology changed significantly, the appearance became irregular, and some dead cells were seen falling off. After continued culture until day 12, typical clonal growth was observed, and the cells within the clones were tightly connected, round, and had relatively large nuclear-cytoplasmic ratios; the edges of the clones were clear and sharp, which was consistent with the morphology of human induced pluripotent stem cells.

[0136] 2) Cultivation of human induced pluripotent stem cells

[0137] a. Use mechanical method to separate and expand the cell morphology, and observe and photograph the cell morphology under an optical microscope.

[0138] b. Figure 3 As shown, the clone morphology is typical, with sharp edges and no obvious differentiated cells; the cells in the clone are tightly connected, the cell nuclei are large, and the cells are in good condition and can be used for subsequent identification and differentiation experiments.

[0139] 3) Alkaline phosphatase staining to identify the pluripotency of human induced pluripotent stem cells

[0140] a. Evenly inoculate hiPSC cells in a 24-well plate and culture until the clone size is appropriate and there are no more clones confluent. Then the experiment can be carried out. Use the alkaline phosphatase staining kit for staining. Prepare the staining solution in the dark, mix solution A and solution B at a volume ratio of 1:1, and use within 30 minutes. Remove the cells, discard the culture medium in the wells, add PBS to wash once, and discard PBS. Add fixative at 500μL / well and fix at room temperature for 5 minutes. Add PBS to wash twice, add staining solution at 500μL / well, and incubate at room temperature in the dark for 20 minutes. After incubation, discard the staining solution in the wells, wash once with PBS, add PBS to prevent the cells from drying, and observe the staining results under a microscope.

[0141] b. Figure 4As shown, it can be observed that each clone in each field of view is stained dark blue, and the depth of color indicates the expression level, indicating that the cells in the clone highly express alkaline phosphatase.

[0142] 4) Immunofluorescence staining to identify the pluripotency of human induced pluripotent stem cells

[0143] a. Inoculate hiPSC cells evenly in a 24-well plate and culture until the clones are of appropriate size and there are no more clones confluent. Then, the experiment can be carried out. Remove the cells from the incubator, discard the culture medium in the wells, 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 500μL 0.2% Triton-100, and incubate at room temperature for 15 minutes. Discard the liquid in the wells, add 500μL 1% BSA to cover the bottom surface, and block at room temperature for 1 hour. Discard BSA, add the primary antibody solution diluted in proportion, and incubate at 4℃ overnight. The next day, remove the culture plate, discard the liquid in the wells, and wash three times with PBS, each for 5 minutes. Add the secondary antibody solution diluted 1:1000, and incubate at room temperature for 1 hour. Discard the secondary antibody, and wash three times with PBS, each for 5 minutes. Add DAPI solution and incubate at room temperature for 10 minutes. Discard DAPI and wash three times with PBS. Add PBS to cover the bottom surface to prevent the cells from drying. At this time, it can be directly observed under an inverted fluorescence microscope.

[0144] b. Figure 5 As shown, this experiment selected three protein markers, TRA-1-60, SSEA4, and Oct-4, for immunofluorescence staining detection. hiPSCs obtained by reprogramming renal epithelial cells all expressed three protein markers.

[0145] 5) Karyotype detection to identify the pluripotency of human induced pluripotent stem cells

[0146] a. When the conventional cells are cultured to a confluence of about 80%, prepare for the experiment. Add PSC culture medium containing 0.1μg / mL colchicine, continue to culture in the incubator for 1.5 hours, and then conduct subsequent experiments. Trypsin-digested cells are collected in a centrifuge tube, centrifuged at 1000rpm at room temperature for 10 minutes, and the supernatant is discarded. Add 0.075mol / L KCL solution, place in a 37℃ water bath for hypotonic treatment for 20 minutes, add 1mL pre-fixative solution (methanol: glacial acetic acid volume ratio 3:1), mix well, and centrifuge at 1000rpm at room temperature for 10 minutes. Discard the supernatant and keep the substrate, add 8mL fixative, fix for 20 minutes, and centrifuge at 1000rpm at room temperature for 10 minutes. Discard the supernatant, add fixative depending on the amount of cell precipitation, and tap the wall of the test tube with your fingers to resuspend the cells. Remove the slide from the ice water. Use a rubber-tipped dropper to draw the cell suspension and drop it about 30cm above the slide. Place the slide at 80℃ to dry for 2 hours. Dilute trypsin with PBS to a final concentration of 0.25‰. Place the slides dried at 80℃ for 2h in trypsin for 2-3 minutes. The digestion time is determined according to the degree of dryness of the slides. Rinse the slides in PBS and stain them in Giemsa stain for 5-10 minutes. Rinse the slides in tap water. Dry at room temperature and seal the slides for storage. Count and analyze the changes in chromosome structure under a microscope ( Figure 6 ).

[0147] b. Figure 6 As shown, after the cells were cultured, G-banding analysis was performed, and the number of chromosomes was 46, which was normal; the sex chromosomes were composed of XY, which was a male karyotype; and there was no abnormality in the chromosome structure.

[0148] 6) Teratoma formation assay to identify the pluripotency of human induced pluripotent stem cells

[0149] a. After culturing enough cells according to the experimental requirements, digest and centrifuge to collect the cells, and use a syringe to inject 0.3mL of hiPSCs cell suspension into the outer root of the right thigh of NOD-SCID mice. The mice were then raised normally. Tumors can be seen at the injection site in about 8-12 weeks. After taking photos, the mice were killed by cervical dislocation. Use scissors to cut the injection tumor site to expose the field of view, and use forceps and scissors to carefully peel off the tumor. Place the tumor in 10% formalin fixative, with a volume ratio of about 10:1 to the sample. After fixing for 24 hours, embed the tissue and perform HE staining. Use a slide scanner to observe the staining results and sample tissue morphology.

[0150] b. Figure 7 As shown in the figure, after 8-12 weeks of differentiation, a bulge can be observed at the injection site, and a tumor can be seen under the skin after dissection. After the tumor was peeled off and sampled, pathological HE staining was performed, and the results showed 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 the experiment have the ability to differentiate into cells and tissues of the three germ layers in vivo.

[0151] 3. Induction of differentiation of sebaceous gland precursor cells and evaluation of adipogenic ability

[0152] 1) Differentiation process of sebaceous gland precursor cells

[0153] 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).

[0154] b. The results are as follows Figure 8 As shown, compared with the morphology of human induced pluripotent stem cells, as the differentiation time goes by, the differentiating cells show a spindle-shaped morphology, similar to skin epidermal cells. In the late stage of differentiation, the cell volume can be seen to increase, and obvious vacuolar morphology can be seen.

[0155] 2) Gene expression levels of sebaceous gland precursor cell markers

[0156] a. Total RNA was extracted from cells using the Total Gold RNA Extraction Kit, and the Total Gold Reverse Transcription Kit was used to synthesize the extracted total RNA into cDNA. RT-qPCR was used to detect the gene expression levels of intracellular 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).

[0157] b. The results are as follows Figure 9-11 As shown, compared with human keratinocytes (HaCaT), human immortalized sebaceous gland cells (SZ95) and human induced pluripotent stem cells (hiPSC), the sebaceous gland precursor cells (SBP) obtained in the present invention express sebaceous gland precursor cell markers at a high level.

[0158] 3) Oil red O staining to detect lipid droplet content in sebaceous gland precursor cells

[0159] a. Inoculate cells into 24-well plates. When the cells grow to 50%, they can be stained. Remove the cells, wash twice with PBS, add 4% paraformaldehyde to fix for 20 minutes, discard the fixative, wash twice with distilled water, add 60% isopropanol to soak for 15 minutes, discard the 60% isopropanol solution, wash twice with distilled water, add the prepared Oil Red O staining solution to soak for 20 minutes, discard the staining solution, wash with water until there is no excess staining solution, counterstain the cell nucleus with hematoxylin for 2 minutes, discard the hematoxylin staining solution and wash with water until there is no excess staining solution, add distilled water to cover the cells, observe under a microscope and take pictures.

[0160] b. The results are as follows Fig.12 and 13 As shown, compared with human keratinocytes (HaCaT), human immortalized sebocytes (SZ95) and human induced pluripotent stem cells (hiPSC), 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).

[0161] 4) Detection of fluorescence intensity of lipid droplets in sebaceous gland precursor cells by Nile red fluorescence staining

[0162] a. Inoculate cells onto cell slides, and stain when cells grow to 50%. Remove cells, wash twice with PBS, add 4% paraformaldehyde to fix for 10 minutes, discard the fixative, and wash twice with PBS. Add 1 mL of prepared Nile Red and Hoechst solution to each well, incubate at room temperature and away from light for 20 minutes, observe with laser confocal microscopy to record Nile Red and Hoechst fluorescence images, and merge different fluorescence staining images. The box is the lipid droplet expression area.

[0163] b. The results are as follows Fig.14 As shown, compared with human keratinocytes (HaCaT), human immortalized sebaceous gland cells (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 were rich in lipid droplets (scale bar = 20 μm).

[0164] 5) RNA-seq detection of gene expression differences between sebaceous gland precursor cells and human immortalized sebaceous gland cells

[0165] a. Cellular RNA was extracted using the TRIzol method, and subsequent transcriptome sequencing was performed by Beijing Novogene Technology Co., Ltd.

[0166] b. The results are as follows Fig.15 As shown, the sequencing results indicate that compared with human immortalized sebaceous gland cells (SZ95), the sebaceous gland precursor cells (SBP) obtained by the present invention have significantly increased expressions of genes related to lipid droplet formation (LipidDrop), lipid accumulation (Lipid Storage) and androgen receptor (Androgen receptor).

[0167] 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.

[0168] Example 2: The present invention provides an immortalized human sebaceous gland precursor cell, which is deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO: C2023286. The method for preparing the cell comprises the following steps:

[0169] (1) Isolation and expansion culture of renal epithelial cells:

[0170] ① Isolation of renal epithelial cells:

[0171] a. Collect urine from acne patients and divide into 50 mL centrifuge tubes, centrifuge at 400 x g at room temperature for 10 minutes, discard the supernatant, and retain 2 mL of liquid;

[0172] b. Take 10 mL of the washing solution in 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 in the UrinEasy Urine Cell Isolation and Culture Kit, and inoculate it into a culture plate coated with Matrigel in advance;

[0173] c. After culturing for 24 hours, observe whether the cells are contaminated. If not, add 800 μL of the culture medium in the UrinEasy urine cell isolation and culture kit and repeat this step for 3 days;

[0174] d. On the 4th day, observe whether there is contamination and adherent cells. If there is no contamination, discard 2 / 3 of the culture medium in the well, add 800 μL of the culture medium in the UrinEasy urine cell isolation and culture kit, and then observe whether there is contamination and adherent cells every day, and then use the culture medium to replace half or full volume every other day;

[0175] ② Expansion and culture of renal epithelial cells:

[0176] d. After the separation in step ① above, when the confluence of renal epithelial cells reaches 90%, they can be passaged and expanded;

[0177] e. Discard the culture medium in the well, wash once with PBS, digest in an incubator with a mass concentration of 0.25% trypsin digestion solution at 37°C for 3 minutes, add 2 times the volume of the digestion solution to terminate the digestion, and gently blow the bottom of the culture well to make the cells fall off;

[0178] f. 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 using the UrinEasy expansion culture basal medium, gently pipet to a single cell suspension, and evenly inoculate the cells into a Matrigel-coated culture plate;

[0179] (2) Establishment and expansion of human induced pluripotent stem cell lines:

[0180] ① Establishment of human induced pluripotent stem cell lines:

[0181] a. Step (1) When passaged, urine-derived renal epithelial cells were plated in Matrigel-coated 96-well plates at cell densities of 5000 / well, 10000 / well, and 15000 / well, respectively. Three replicate wells were set for each gradient, and this was recorded as day -1.

[0182] b. The next day, observe the cell status under a microscope, select one well for each density gradient to count the cells, and select one culture well with a cell count between 10,000 and 20,000 for subsequent experiments;

[0183] c. Mix 10 μL of reprogramming additive I with the catalog number CA5002002-1 and the culture medium in 10 mL of UrinEasy urine cell expansion culture reagent to prepare "reprogramming culture medium A". Take 100 μL of reprogramming culture medium A and mix it with 10 μL of reprogramming additive II with the catalog number CA5002002-2 to prepare "reprogramming culture medium B". Discard the culture medium in the selected well and add reprogramming culture medium B. This is recorded as day 0.

[0184] d. Observe the cell morphology changes under the microscope every day and culture for 2 consecutive days;

[0185] e. On the third day, observe the cell morphology under a microscope. If the cell morphology changes significantly and the cells reach 100% confluence, they can be passaged;

[0186] f. When passaging cells, first prepare reprogramming medium C, add 10 μL of reprogramming additive III with catalog number CA5002002-3 to the medium in 10 mL of UrinEasy urine cell expansion culture kit, mix well, discard the medium in the culture wells of the cells to be digested, add PBS to wash once, add 50 μL of trypsin to the culture wells, digest at 37°C in the incubator for 3 minutes, add 100 μL of reprogramming medium C to terminate digestion, pipette the bottom of the culture wells, collect the cell suspension into a centrifuge tube containing reprogramming medium C, pipette and mix well, and evenly inoculate the cells into Matrigel-coated culture plates at a passaging ratio of 1:10;

[0187] g. On day 4, replace the culture medium in the UrinEasy urine cell expansion culture kit;

[0188] h. Observe the cell status daily. If small clusters of clones are found under the microscope, use Reproeasy Human Cell Reprogramming Basic Medium (Cat. No. CA5003050-1) to replace the medium daily;

[0189] i. If multiple clones consisting of more than 10 cells are found, replace the culture medium with PGM1 human pluripotent stem cell culture medium (catalog number CA1007500);

[0190] j. Observe the cells under a microscope, culture until the size of multiple clones can fill the 10x microscope field, cut the clones with a 1mL syringe needle, separate the clones from the bottom wall of the culture well with a 200μL sterile pipette tip, aspirate the clones and transfer them to a Matrigel-coated 24-well plate filled with the PGM1 human pluripotent stem cell culture medium, and return them to the incubator for culture at 37°C, 5% carbon dioxide by volume, and a humidity of more than 90%;

[0191] k. Observe daily and replace the medium using the PGM1 human pluripotent stem cell culture medium;

[0192] ② Expansion and culture of human induced pluripotent stem cell lines:

[0193] a. When the cells are in good condition under microscope and the cell confluence is about 80%, or when the clones are unevenly distributed and a single clone has a larger area, subculture can be performed;

[0194] b. Discard the culture medium in the well, wash with PBS, add DissoEasy human pluripotent stem cell digestion solution with catalog number CA1023100, digest at room temperature for 5 minutes, discard the digestion solution in the well, add the PGM1 human pluripotent stem cell culture medium, gently blow the bottom of the culture well in a fan shape to make the cells fall off, and collect the cell suspension into a centrifuge tube. Inoculate the cell suspension into the Matrigel-coated culture plate at a volume ratio of 1:10, and continue to culture in the incubator at 37°C, 5% carbon dioxide, and more than 90% humidity. Observe daily thereafter, and use the PGM1 human pluripotent stem cell culture medium to replace the medium daily;

[0195] (3) Differentiation of sebaceous gland precursor cells

[0196] a. The human induced pluripotent stem cells obtained in step (2) are replaced with a basic differentiation medium for differentiating human induced pluripotent stem cells into sebaceous gland precursor cells, wherein the basic differentiation medium consists of the following components:

[0197]

[0198]

[0199] b. Replace the basic differentiation medium described in step (a) every day, and culture in an incubator at 37°C and 5% CO2 for three consecutive days;

[0200] c. After three days of cell culture, the culture medium was replaced with a sebaceous gland precursor differentiation activation medium, wherein the activation medium was the basic differentiation medium in step (a) supplemented with 20 ng / mL epidermal growth factor and 1 ng / mL bone morphogenetic protein 4;

[0201] d. Replace the sebaceous gland differentiation activation medium every other day and culture continuously for 8 days in an incubator at 37°C and 5% CO2;

[0202] e. Prepare sebaceous gland precursor differentiation medium, the medium consists of the following components:

[0203] EpiLife TM Culture medium: Dermacult keratinocyte expansion medium volume ratio 1:1

[0204] Epidermal growth factor 20ng / mL

[0205] Bone morphogenetic protein 4 1ng / mL

[0206] f. The cells were continuously cultured in the differentiation medium at 37°C and 5% CO2 incubator for 10-11 days, and the cells were digested with TrypLE. The digestion method was 37°C in a carbon dioxide incubator for 15 minutes, centrifuged at 200 x g for 3 minutes, and the supernatant was discarded. 10 μM dihydrochloride ATP competitive ROCK inhibitor was added to the sebaceous gland precursor differentiation medium to resuspend the cells;

[0207] g. The cells obtained in step (f) were grown in a 25 μg / mL fibronectin-coated culture plate;

[0208] 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;

[0209] i. The cells were cultured in a 37°C, 5% CO2 incubator for 9 consecutive days to obtain sebaceous gland precursor cells.

[0210] In this example, sebaceous gland precursor cells are used to evaluate and screen oil-regulating drugs or skin care products:

[0211] 1. Changes in lipid droplets before and after intervention of different concentrations of dihydrotestosterone in sebaceous gland precursor cells (SBP) and human immortalized sebaceous gland cells (SZ95)

[0212] a. Cells were seeded in 96-well plates and treated with different concentrations (1μM, 3μM, 10μM, 30μM and 100μM) of dihydrotestosterone (DHT) for 24 hours. The culture medium was discarded, the cells were washed twice with PBS, fixed with 4% paraformaldehyde for 20 minutes, the fixative was discarded, the cells were washed twice with distilled water, immersed in 60% isopropanol for 15 minutes, the 60% isopropanol solution was discarded, the cells were washed twice with distilled water, immersed in the prepared Oil Red O staining solution for 20 minutes, the staining solution was discarded, the cells were washed with water until there was no excess staining solution, the cell nuclei were counterstained with hematoxylin for 2 minutes, the hematoxylin staining solution was discarded, and the cells were washed with tap water until there was no excess staining solution, distilled water was added to cover the cells, and the cells were observed under a microscope and photographed and recorded.

[0213] b. Fig.16 As shown, compared with human immortalized sebaceous gland cells, the sebaceous gland precursor cells obtained by this scheme responded more significantly to DHT intervention, with significantly increased lipid droplet volume and number, and can be used for subsequent drug or skin care product screening.

[0214] 2. Changes in lipid droplets after DHT stimulation of sebaceous gland precursor cells (SBP) by different drugs and skin care products

[0215] a. Cells were seeded in 96-well plates and treated with 100 μM DHT for 24 h. Various drugs of different concentrations were added and continued to be treated for 24 h. The culture medium was discarded, the cells were washed twice with PBS, fixed with 4% paraformaldehyde for 20 min, the fixative was discarded, the cells were washed twice with distilled water, immersed in 60% isopropanol for 15 min, the 60% isopropanol solution was discarded, the cells were washed twice with distilled water, immersed in the prepared Oil Red O staining solution for 20 min, the staining solution was discarded, the cells were washed with water until there was no excess staining solution, the cell nuclei were counterstained with hematoxylin for 2 min, the hematoxylin staining solution was discarded, the cells were washed with water until there was no excess staining solution, distilled water was added to cover the cells, and the cells were observed under a microscope and photographed.

[0216] b. Fig.17 As shown in the figure, the sebaceous gland precursor cells obtained by this scheme were subjected to different concentrations of various drugs intervention based on DHT stimulation, indicating that different concentrations of retinoic acid (RA), Curcuma zedoaria extract, MAT-XS TM -Clinical After intervention, the volume and number of lipid droplets of sebaceous gland precursor cells (SBP) decreased, and the effect became concentration-dependent. 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.

[0217] Example 3: The present invention provides an immortalized human sebaceous gland precursor cell, which is deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO: C2023286. The method for preparing the cell comprises the following steps:

[0218] (1) Isolation and expansion culture of renal epithelial cells:

[0219] ① Isolation of renal epithelial cells:

[0220] a. Collect urine from acne patients and divide into 50 mL centrifuge tubes, centrifuge at 400 x g at room temperature for 10 minutes, discard the supernatant, and retain 1 mL of liquid;

[0221] b. Take 10 mL of the washing solution in 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 in the UrinEasy Urine Cell Isolation and Culture Kit, and inoculate it into a culture plate coated with Matrigel in advance;

[0222] c. After culturing for 24 hours, observe whether the cells are contaminated. If not, add 800 μL of the culture medium in the UrinEasy urine cell isolation and culture kit and repeat this step for 3 days;

[0223] d. On the 4th day, observe whether there is contamination and adherent cells. If there is no contamination, discard 2 / 3 of the culture medium in the well, add 800 μL of the culture medium in the UrinEasy urine cell isolation and culture kit, and then observe whether there is contamination and adherent cells every day, and then use the culture medium to replace half or full volume every other day;

[0224] ② Expansion and culture of renal epithelial cells:

[0225] a. After the separation in step ① above, when the confluence of renal epithelial cells reaches 80%, they can be passaged and expanded;

[0226] b. Discard the culture medium in the well, wash once with PBS, digest in an incubator with a mass concentration of 0.25% trypsin digestion solution at 37°C for 3 minutes, add 2 times the volume of the digestion solution to terminate the digestion, and gently blow the bottom of the culture well to make the cells fall off;

[0227] 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 using the UrinEasy expansion culture basal medium, gently pipette to a single cell suspension, and evenly inoculate the cells into a Matrigel-coated culture plate;

[0228] (2) Establishment and expansion of human induced pluripotent stem cell lines:

[0229] ① Establishment of human induced pluripotent stem cell lines:

[0230] a. Step (1) When passaged, urine-derived renal epithelial cells were plated in Matrigel-coated 96-well plates at cell densities of 5000 / well, 10000 / well, and 15000 / well, respectively. Three replicate wells were set for each gradient, and this was recorded as day -1.

[0231] b. The next day, observe the cell status under a microscope, select one well for each density gradient to count the cells, and select one culture well with a cell count between 10,000 and 20,000 for subsequent experiments;

[0232] c. Mix 10 μL of reprogramming additive I with the product number CA5002002-1 and the culture medium in 10 mL of UrinEasy urine cell expansion culture reagent to prepare "reprogramming medium A". Take 100 μL of reprogramming medium A and mix it with 10 μL of reprogramming additive II with the product number CA5002002-2 to prepare "reprogramming medium B". Discard the culture medium in the selected well and add reprogramming medium B. This is recorded as day 0.

[0233] d. Observe the cell morphology changes under the microscope every day and culture for 2 consecutive days;

[0234] e. On the third day, observe the cell morphology under a microscope. If the cell morphology changes significantly and the cells reach 100% confluence, they can be subcultured;

[0235] f. When passaging cells, first prepare reprogramming medium C, add 10 μL of reprogramming additive III with catalog number CA5002002-3 to the medium in 10 mL of UrinEasy urine cell expansion culture kit, mix well, discard the medium in the culture wells of the cells to be digested, add PBS to wash once, add 50 μL of trypsin to the culture wells, digest at 37°C in the incubator for 3 minutes, add 100 μL of reprogramming medium C to terminate digestion, pipette the bottom of the culture wells, collect the cell suspension into a centrifuge tube containing reprogramming medium C, pipette and mix well, and evenly inoculate the cells into Matrigel-coated culture plates at a passaging ratio of 1:7;

[0236] g. On day 4, replace the culture medium in the UrinEasy urine cell expansion culture kit;

[0237] h. Observe the cell status daily. If small clusters of clones are found under the microscope, use Reproeasy Human Cell Reprogramming Basic Medium (Cat. No. CA5003050-1) to replace the medium daily;

[0238] i. If multiple clones consisting of more than 10 cells are found, replace the culture medium with PGM1 human pluripotent stem cell culture medium (catalog number CA1007500);

[0239] j. Observe the cells under a microscope, culture until the size of multiple clones can fill the 10x microscope field, cut the clones with a 1mL syringe needle, separate the clones from the bottom wall of the culture well with a 200μL sterile pipette tip, aspirate the clones and transfer them to a Matrigel-coated 24-well plate filled with the PGM1 human pluripotent stem cell culture medium, and return them to the incubator for culture at 37°C, 5% carbon dioxide by volume, and a humidity of more than 90%;

[0240] k. Observe daily and replace the medium using the PGM1 human pluripotent stem cell culture medium;

[0241] ② Expansion and culture of human induced pluripotent stem cell lines:

[0242] a. When the cells are in good condition under microscope and the cell confluence is about 80%, or when the clones are unevenly distributed and a single clone has a larger area, subculture can be performed;

[0243] b. Discard the culture medium in the well, add PBS to wash once, add DissoEasy human pluripotent stem cell digestion solution with catalog number CA1023100, digest at room temperature for 4 minutes, discard the digestion solution in the well, add the PGM1 human pluripotent stem cell culture medium, gently blow the bottom of the culture well in a fan shape to make the cells fall off, and collect the cell suspension into a centrifuge tube. Inoculate the cell suspension into the Matrigel-coated culture plate at a volume ratio of 1:6, and continue to culture in the incubator at 37°C, 5% carbon dioxide, and more than 90% humidity. Observe daily thereafter and use the PGM1 human pluripotent stem cell culture medium to replace the medium daily;

[0244] (3) Differentiation of sebaceous gland precursor cells

[0245] a. The human induced pluripotent stem cells obtained in step (2) are replaced with a basic differentiation medium for differentiating human induced pluripotent stem cells into sebaceous gland precursor cells, wherein the basic differentiation medium consists of the following components:

[0246]

[0247] b. Replace the basic differentiation medium described in step (a) every day, and culture in an incubator at 37°C and 5% CO2 for three consecutive days;

[0248] c. After three days of cell culture, the culture medium was replaced with a sebaceous gland precursor differentiation activation medium, wherein the activation medium was the basic differentiation medium in step (a) supplemented with 20 ng / mL epidermal growth factor and 1 ng / mL bone morphogenetic protein 4;

[0249] d. Replace the sebaceous gland differentiation activation medium every other day and culture continuously for 7 days in an incubator at 37°C and 5% CO2;

[0250] e. Prepare sebaceous gland precursor differentiation medium, the medium consists of the following components:

[0251] EpiLife TM Culture medium: Dermacult keratinocyte expansion medium volume ratio 1:1

[0252] Epidermal growth factor 20ng / mL

[0253] Bone morphogenetic protein 4 1ng / mL

[0254] f. The cells were continuously cultured in the differentiation medium at 37°C and 5% CO2 incubator for 10 days, and the cells were digested with TrypLE. The digestion method was 37°C in a carbon dioxide incubator for 15 minutes, centrifuged at 200 x g for 3 minutes, and the supernatant was discarded. 10 μM dihydrochloride ATP competitive ROCK inhibitor was added to the sebaceous gland precursor differentiation medium to resuspend the cells;

[0255] g. The cells obtained in step (f) were grown in a 25 μg / mL fibronectin-coated culture plate;

[0256] 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;

[0257] i. The cells were cultured in a 37°C, 5% CO2 incubator for 7 consecutive days to obtain sebaceous gland precursor cells.

[0258] 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:

[0259] a. Prepare mature sebaceous gland basal differentiation medium, which consists of the following components:

[0260]

[0261] b. Digest the sebaceous gland precursor cells with TrypLE: digest for 15 minutes in a 37°C, 5% CO2 incubator, centrifuge at 250 x g for 3 minutes, discard the supernatant, add 10 μM dihydrochloride ATP competitive ROCK inhibitor to the mature sebaceous gland basal differentiation medium, and then resuspend the cells at 2.5×10 4 cells / cm 2 The cells were seeded in culture plates at a density of 1.5 × 10 and cultured overnight in a 37°C, 5% CO2 incubator.

[0262] c. The medium was changed on the next day, and 10 μM TGF-β / Smad inhibitor was added to the mature sebaceous gland basal differentiation medium;

[0263] d. Change the medium every day and culture in a 37°C, 5% CO2 incubator for 3 consecutive days;

[0264] e. After 3 days, the medium was changed to mature sebaceous gland final differentiation medium and cultured in an incubator at 37°C and 5% CO2;

[0265] f. The medium is changed every day, and the culture is continuously cultured in an incubator at 37°C and a volume concentration of 5% CO2 for 3 days to obtain mature sebaceous gland cells, which can be used to screen and evaluate oil secretion drugs or skin care products.

[0266] 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.

[0267] Example 4: The present invention provides an immortalized human sebaceous gland precursor cell, which is deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO: C2023286. The method for preparing the cell comprises the following steps:

[0268] (1) Isolation and expansion culture of renal epithelial cells:

[0269] ① Isolation of renal epithelial cells:

[0270] a. Collect urine from acne patients and divide into 50 mL centrifuge tubes, centrifuge at 400 x g at room temperature for 10 minutes, discard the supernatant, and retain 2 mL of liquid;

[0271] b. Take 10 mL of the washing solution in 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 in the UrinEasy Urine Cell Isolation and Culture Kit, and inoculate it into a culture plate coated with Matrigel in advance;

[0272] c. After culturing for 24 hours, observe whether the cells are contaminated. If not, add 800 μL of the culture medium in the UrinEasy urine cell isolation and culture kit and repeat this step for 3 days;

[0273] d. On the 4th day, observe whether there is contamination and adherent cells. If there is no contamination, discard 2 / 3 of the culture medium in the well, add 800 μL of the culture medium in the UrinEasy urine cell isolation and culture kit, and then observe whether there is contamination and adherent cells every day, and then use the culture medium to replace half or full volume every other day;

[0274] ② Expansion and culture of renal epithelial cells:

[0275] a. After the separation in step ① above, when the confluence of renal epithelial cells reaches 90%, they can be passaged and expanded;

[0276] b. Discard the culture medium in the well, wash once with PBS, digest in an incubator with a mass concentration of 0.25% trypsin digestion solution at 37°C for 3 minutes, add 2 times the volume of the digestion solution to terminate the digestion, and gently blow the bottom of the culture well to make the cells fall off;

[0277] 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 using the UrinEasy expansion culture basal medium, gently pipette to a single cell suspension, and evenly inoculate the cells into a Matrigel-coated culture plate;

[0278] (2) Establishment and expansion of human induced pluripotent stem cell lines:

[0279] ① Establishment of human induced pluripotent stem cell lines:

[0280] a. Step (1) When passaged, urine-derived renal epithelial cells were plated in Matrigel-coated 96-well plates at cell densities of 5000 / well, 10000 / well, and 15000 / well, respectively. Three replicate wells were set for each gradient, and this was recorded as day -1.

[0281] bbThe 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 number between 10,000 and 20,000 for subsequent experiments;

[0282] c. Mix 10 μL of reprogramming additive I with the product number CA5002002-1 and the culture medium in 10 mL of UrinEasy urine cell expansion culture reagent to prepare "reprogramming medium A". Take 100 μL of reprogramming medium A and mix it with 10 μL of reprogramming additive II with the product number CA5002002-2 to prepare "reprogramming medium B". Discard the culture medium in the selected well and add reprogramming medium B. This is recorded as day 0.

[0283] d. Observe the cell morphology changes under the microscope every day and culture for 2 consecutive days;

[0284] e. On the third day, observe the cell morphology under a microscope. If the cell morphology changes significantly and the cells reach 100% confluence, they can be subcultured;

[0285] f. When passaging cells, first prepare reprogramming medium C, add 10 μL of reprogramming additive III with catalog number CA5002002-3 to the medium in 10 mL of UrinEasy urine cell expansion culture kit, mix well, discard the medium in the culture wells of the cells to be digested, add PBS to wash once, add 50 μL of trypsin to the culture wells, digest at 37°C in the incubator for 3 minutes, add 100 μL of reprogramming medium C to terminate digestion, pipette the bottom of the culture wells, collect the cell suspension into a centrifuge tube containing reprogramming medium C, pipette and mix well, and evenly inoculate the cells into Matrigel-coated culture plates at a passaging ratio of 1:8;

[0286] g. On day 4, replace the culture medium in the UrinEasy urine cell expansion culture kit;

[0287] h. Observe the cell status daily. If small clusters of clones are found under the microscope, use Reproeasy Human Cell Reprogramming Basic Medium (Cat. No. CA5003050-1) to replace the medium daily;

[0288] i. If multiple clones consisting of more than 10 cells are found, replace the culture medium with PSCeasy human pluripotent stem cell recovery medium;

[0289] j. Observe the cells under a microscope, culture until the size of multiple clones can fill the 10x microscope field, cut the clones with a 1mL syringe needle, separate the clones from the bottom wall of the culture well with a 200μL sterile pipette tip, aspirate the clones and transfer them to a Matrigel-coated 24-well plate filled with the PGM1 human pluripotent stem cell culture medium, and return them to the incubator for culture at 37°C, 5% carbon dioxide by volume, and a humidity of more than 90%;

[0290] k. Observe daily and replace the medium with PGM1 human pluripotent stem cell culture medium;

[0291] ② Expansion and culture of human induced pluripotent stem cell lines:

[0292] a. When the cells are in good condition under microscope and the cell confluence is about 80%, or when the clones are unevenly distributed and a single clone has a larger area, subculture can be performed;

[0293] b. Discard the culture medium in the well, wash with PBS, add DissoEasy human pluripotent stem cell digestion solution with catalog number CA1023100, digest at room temperature for 5 minutes, discard the digestion solution in the well, add the PGM1 human pluripotent stem cell culture medium, gently blow the bottom of the culture well in a fan-shaped manner to make the cells fall off, and collect the cell suspension into a centrifuge tube. Inoculate the cell suspension into the Matrigel-coated culture plate at a volume ratio of 1:8, and continue to culture in the incubator at 37°C, 5% carbon dioxide, and more than 90% humidity. Observe daily thereafter and use the PGM1 human pluripotent stem cell culture medium to replace the medium daily;

[0294] (3) Differentiation of sebaceous gland precursor cells

[0295] a. The human induced pluripotent stem cells obtained in step (2) are replaced with a basic differentiation medium for differentiating human induced pluripotent stem cells into sebaceous gland precursor cells, wherein the basic differentiation medium consists of the following components:

[0296]

[0297]

[0298] b. Replace the basic differentiation medium described in step (a) every day, and culture in an incubator at 37°C and 5% CO2 for three consecutive days;

[0299] c. After three days of cell culture, the culture medium was replaced with a sebaceous gland precursor differentiation activation medium, wherein the activation medium was the basic differentiation medium in step (a) supplemented with 20 ng / mL epidermal growth factor and 1 ng / mL bone morphogenetic protein 4;

[0300] d. Replace the sebaceous gland differentiation activation medium every other day and culture continuously for 8 days in an incubator at 37°C and 5% CO2;

[0301] e. Prepare sebaceous gland precursor differentiation medium, the medium consists of the following components:

[0302] EpiLife TM Culture medium: Dermacult keratinocyte expansion medium volume ratio 1:1

[0303] Epidermal growth factor 20ng / mL

[0304] Bone morphogenetic protein 4 1ng / mL

[0305] f. The cells were continuously cultured in the differentiation medium at 37°C and 5% CO2 incubator for 11 days, and the cells were digested with TrypLE. The digestion method was 37°C in a carbon dioxide incubator for 15 minutes, centrifuged at 200 x g for 3 minutes, and the supernatant was discarded. 10 μM dihydrochloride ATP competitive ROCK inhibitor was added to the sebaceous gland precursor differentiation medium to resuspend the cells;

[0306] g. The cells obtained in step (f) were grown in a 25 μg / mL fibronectin-coated culture plate;

[0307] 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;

[0308] i. The cells were cultured in a 37°C, 5% CO2 incubator for 8 consecutive days to obtain sebaceous gland precursor cells.

[0309] 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:

[0310] a. Prepare mature sebaceous gland basal differentiation medium, which consists of the following components:

[0311]

[0312] b. Digest the sebaceous gland precursor cells with TrypLE: digest for 15 minutes in a 37°C, 5% CO2 incubator, centrifuge at 250 x g for 3 minutes, discard the supernatant, add 10 μM dihydrochloride ATP competitive ROCK inhibitor to the mature sebaceous gland basal differentiation medium, and then resuspend the cells at 2.5×10 4 cells / cm 2 The cells were seeded in culture plates at a density of 1.5 × 10 and cultured overnight in a 37°C, 5% CO2 incubator.

[0313] c. The medium was changed on the next day, and 10 μM TGF-β / Smad inhibitor was added to the mature sebaceous gland basal differentiation medium;

[0314] d. Change the medium every day and culture in a 37°C, 5% CO2 incubator for 3 consecutive days;

[0315] e. After 3 days, the medium was changed to mature sebaceous gland final differentiation medium and cultured in an incubator at 37°C and 5% CO2;

[0316] f. The medium is changed every day, and the culture is continuously cultured in an incubator at 37°C and 5% CO2 for 6 days to obtain mature sebaceous gland cells, which can be used to screen and evaluate oil secretion drugs or skin care products.

[0317] 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.

[0318] Example 5: The present invention provides an immortalized human sebaceous gland precursor cell, which is deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO: C2023286. The method for preparing the cell comprises the following steps:

[0319] (1) Isolation and expansion culture of renal epithelial cells:

[0320] ① Isolation of renal epithelial cells:

[0321] a. Collect urine from acne patients and divide into 50 mL centrifuge tubes, centrifuge at 400 x g at room temperature for 10 minutes, discard the supernatant, and retain 1 mL of liquid;

[0322] b. Take 10 mL of the washing solution in 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 in the UrinEasy Urine Cell Isolation and Culture Kit, and inoculate it into a culture plate coated with Matrigel in advance;

[0323] c. After culturing for 24 hours, observe whether the cells are contaminated. If not, add 800 μL of the culture medium in the UrinEasy urine cell isolation and culture kit and repeat this step for 3 days;

[0324] d. On the 4th day, observe whether there is contamination and adherent cells. If there is no contamination, discard 2 / 3 of the culture medium in the well, add 800 μL of the culture medium in the UrinEasy urine cell isolation and culture kit, and then observe whether there is contamination and adherent cells every day, and then use the culture medium to replace half or full volume every other day;

[0325] ② Expansion and culture of renal epithelial cells:

[0326] a. After the separation in step ① above, when the confluence of renal epithelial cells reaches 80%, they can be passaged and expanded;

[0327] b. Discard the culture medium in the well, wash once with PBS, digest in an incubator with a mass concentration of 0.25% trypsin digestion solution at 37°C for 3 minutes, add 2 times the volume of the digestion solution to terminate the digestion, and gently blow the bottom of the culture well to make the cells fall off;

[0328] 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 using the UrinEasy expansion culture basal medium, gently pipette to a single cell suspension, and evenly inoculate the cells into a Matrigel-coated culture plate;

[0329] (2) Establishment and expansion of human induced pluripotent stem cell lines:

[0330] ① Establishment of human induced pluripotent stem cell lines:

[0331] a. Step (1) When passaged, urine-derived renal epithelial cells were plated in Matrigel-coated 96-well plates at cell densities of 5000 / well, 10000 / well, and 15000 / well, respectively. Three replicate wells were set for each gradient, and this was recorded as day -1.

[0332] b. The next day, observe the cell status under a microscope, select one well for each density gradient to count the cells, and select one culture well with a cell count between 10,000 and 20,000 for subsequent experiments;

[0333] c. Mix 10 μL of reprogramming additive I with the product number CA5002002-1 and the culture medium in 10 mL of UrinEasy urine cell expansion culture reagent to prepare "reprogramming medium A". Take 100 μL of reprogramming medium A and mix it with 10 μL of reprogramming additive II with the product number CA5002002-2 to prepare "reprogramming medium B". Discard the culture medium in the selected well and add reprogramming medium B. This is recorded as day 0.

[0334] d. Observe the cell morphology changes under the microscope every day and culture for 2 consecutive days;

[0335] e. On the third day, observe the cell morphology under a microscope. If the cell morphology changes significantly and the cells reach 100% confluence, they can be subcultured;

[0336] f. When passaging cells, first prepare reprogramming medium C, add 10 μL of reprogramming additive III with catalog number CA5002002-3 to the medium in 10 mL of UrinEasy urine cell expansion culture kit, mix well, discard the medium in the culture wells of the cells to be digested, add PBS to wash once, add 50 μL of trypsin to the culture wells, digest at 37°C in the incubator for 3 minutes, add 100 μL of reprogramming medium C to terminate digestion, pipette the bottom of the culture wells, collect the cell suspension into a centrifuge tube containing reprogramming medium C, pipette and mix well, and evenly inoculate the cells into Matrigel-coated culture plates at a passaging ratio of 1:9;

[0337] g. On day 4, replace the culture medium in the UrinEasy urine cell expansion culture kit;

[0338] h. Observe the cell status daily. If small clusters of clones are found under the microscope, use Reproeasy Human Cell Reprogramming Basic Medium (Cat. No. CA5003050-1) to replace the medium daily;

[0339] i. If multiple clones consisting of more than 10 cells are found, replace the culture medium with PGM1 human pluripotent stem cell culture medium (catalog number CA1007500);

[0340] j. Observe the cells under a microscope, culture until the size of multiple clones can fill the 10x microscope field, cut the clones with a 1mL syringe needle, separate the clones from the bottom wall of the culture well with a 200μL sterile pipette tip, aspirate the clones and transfer them to a Matrigel-coated 24-well plate filled with the PGM1 human pluripotent stem cell culture medium, and return them to the incubator for culture at 37°C, 5% carbon dioxide by volume, and a humidity of more than 90%;

[0341] k. Observe daily and replace the medium using the PGM1 human pluripotent stem cell culture medium;

[0342] ② Expansion and culture of human induced pluripotent stem cell lines:

[0343] a. When the cells are in good condition under microscope and the cell confluence is about 80%, or when the clones are unevenly distributed and a single clone has a larger area, subculture can be performed;

[0344] b. Discard the culture medium in the well, wash with PBS, add DissoEasy human pluripotent stem cell digestion solution with catalog number CA1023100, digest at room temperature for 3 minutes, discard the digestion solution in the well, add the PGM1 human pluripotent stem cell culture medium, gently blow the bottom of the culture well in a fan-shaped manner to make the cells fall off, and collect the cell suspension into a centrifuge tube. Inoculate the cell suspension into the Matrigel-coated culture plate at a volume ratio of 1:10, and continue to culture in the incubator at 37°C, 5% carbon dioxide, and more than 90% humidity. Observe daily thereafter, and use the PGM1 human pluripotent stem cell culture medium to replace the medium daily;

[0345] (3) Differentiation of sebaceous gland precursor cells

[0346] a. The human induced pluripotent stem cells obtained in step (2) are replaced with a basic differentiation medium for differentiating human induced pluripotent stem cells into sebaceous gland precursor cells, wherein the basic differentiation medium consists of the following components:

[0347]

[0348] b. Replace the basic differentiation medium described in step (a) every day, and culture in an incubator at 37°C and 5% CO2 for three consecutive days;

[0349] c. After three days of cell culture, the culture medium was replaced with a sebaceous gland precursor differentiation activation medium, wherein the activation medium was the basic differentiation medium in step (a) supplemented with 20 ng / mL epidermal growth factor and 1 ng / mL bone morphogenetic protein 4;

[0350] d. Replace the sebaceous gland differentiation activation medium every other day and culture continuously for 7 days in an incubator at 37°C and 5% CO2;

[0351] e. Prepare sebaceous gland precursor differentiation medium, the medium consists of the following components:

[0352] EpiLife TM Culture medium: Dermacult keratinocyte expansion medium volume ratio 1:1

[0353] Epidermal growth factor 20ng / mL

[0354] Bone morphogenetic protein 4 1ng / mL

[0355] f. The cells were continuously cultured in the differentiation medium at 37°C and 5% CO2 incubator for 10 days, and the cells were digested with TrypLE. The digestion method was 37°C in a carbon dioxide incubator for 15 minutes, centrifuged at 200 x g for 3 minutes, and the supernatant was discarded. 10 μM dihydrochloride ATP competitive ROCK inhibitor was added to the sebaceous gland precursor differentiation medium to resuspend the cells;

[0356] g. The cells obtained in step (f) were grown in a 25 μg / mL fibronectin-coated culture plate;

[0357] 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;

[0358] i. The cells were cultured in a 37°C, 5% CO2 incubator for 7 consecutive days to obtain sebaceous gland precursor cells.

[0359] 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:

[0360] a. Prepare mature sebaceous gland basal differentiation medium, which consists of the following components:

[0361]

[0362] b. Digest the sebaceous gland precursor cells with TrypLE: digest for 15 minutes in a 37°C, 5% CO2 incubator, centrifuge at 250 x g for 3 minutes, discard the supernatant, add 10 μM dihydrochloride ATP competitive ROCK inhibitor to the mature sebaceous gland basal differentiation medium, and then resuspend the cells at 2.5×10 4 cells / cm 2 The cells were seeded in culture plates at a density of 1.5 × 10 and cultured overnight in a 37°C, 5% CO2 incubator.

[0363] c. Change the medium on the next day and add 10 μM TGF-β / Smad inhibitor to the mature sebaceous gland basal differentiation medium;

[0364] d. Change the medium every day and culture in a 37°C, 5% CO2 incubator for 3 consecutive days;

[0365] e. After 3 days, the medium was changed to mature sebaceous gland final differentiation medium and cultured in an incubator at 37°C and 5% CO2;

[0366] f. The medium is changed every day, and the culture is continuously carried out in an incubator at 37°C and a volume concentration of 5% CO2 for 5 days to obtain mature sebaceous gland cells, which can be used to screen and evaluate oil secretion drugs or skin care products.

[0367] The mature sebaceous gland final differentiation medium in step (e) is obtained 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.

[0368] 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.

[0369] 1) Morphological observation of mature sebaceous gland cells in Examples 3 to 5 of the present invention

[0370] like Fig.18 As shown, 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 could be observed (scale bar = 20 μm).

[0371] 2) Fluorescence staining of BODIP and KRT7 in mature sebaceous gland cells in Example 3 of the present invention

[0372] a. Cells were seeded on an ibidi chambered eight-well slide, cells were removed, culture medium was discarded, PBS was washed twice, waste liquid was discarded, cells were fixed with 4% paraformaldehyde for 10 minutes, 4% paraformaldehyde was discarded, PBS was added for washing twice, 1% BSA solution was added, and the cells were blocked at room temperature for 1 hour. Primary antibody (KRT7-mouse; ab216016; 1:100 dilution) was added and incubated at 4°C overnight. The next day, the culture plate was removed, the liquid in the wells was discarded, PBS was washed three times, and secondary antibody (Mouse 568; 1:1000 dilution) was added and incubated at room temperature for 1 hour. The liquid in the wells was discarded, PBS was washed three times, BODIP (1:1000 dilution) & F-actin (Phantom 633, 1:1000 dilution) were added and incubated at room temperature for 30 minutes. The liquid in the wells was discarded, PBS was added for washing three times, 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.

[0373] b. Fig.19As shown, by fluorescent staining of mature sebaceous gland cells by BODIPY, it was found that the mature sebaceous gland cells obtained by this protocol have the property of forming or accumulating lipid droplets of sebaceous gland cells in the human body. In addition, because KRT7 is highly expressed in sebaceous gland cells, the expression of KRT7 protein in mature sebaceous gland cells was detected by immunofluorescence, and it was found that the mature sebaceous gland cells obtained by this protocol expressed KRT7, and the arrows in the figure indicate lipid droplets and KRT7 expression (scale bar = 20 μm).

[0374] 3) Differentiation-induced mature sebaceous gland cells under different conditions and their response to androgen (dihydrotestosterone):

[0375] like Fig. 20 As shown, the mature sebaceous gland cells of Examples 3 to 5 accelerate the maturation, differentiation and even rupture of the cells under the stimulation of dihydrotestosterone, thereby releasing more lipid droplets / sebum (scale bar = 20 μm).

[0376] 4) BODIP staining of sebaceous gland cells lipid droplets

[0377] a. Cells were seeded on an eight-well glass slide in an ibidi chamber. The experimental group was given 100 μM dihydrotestosterone (with DHT), and the control group was cultured normally (without DHT) for 24 hours. The cells were removed, the culture medium was 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 PBS was added to wash twice. BODIBY (1:1000 dilution) was added and incubated at room temperature for 30 minutes. The liquid in the wells was discarded, and PBS was added to wash three times. DAPI (1:1000 dilution) was added and incubated at room temperature for 15 minutes. The liquid in the wells was discarded, and PBS was washed three times. The images were taken using a laser confocal microscope.

[0378] b. Fig.21 As shown, 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, the mature sebaceous gland cells accelerated cell maturation and differentiation and released more lipid droplets. The arrows indicate lipid droplet expression (scale bar = 20 μm).

[0379] 5) Oil Red O staining to evaluate the screening effect of SBM drugs and skin care products

[0380] a. Inoculate mature sebaceous gland cells into a 24-well plate. When the cells grow to 50%, they can be stained. Remove the cells, wash twice with PBS, add 4% paraformaldehyde to fix for 20 minutes, discard the fixative, wash twice with distilled water, add 60% isopropanol to soak for 15 minutes, discard the 60% isopropanol solution, wash twice with distilled water, add the prepared Oil Red O staining solution to soak for 20 minutes, discard the staining solution, wash with water until there is no excess staining solution, counterstain the cell nucleus with hematoxylin for 2 minutes, discard the hematoxylin staining solution and wash with water until there is no excess staining solution, add distilled water to cover the cells, and observe and record with a microscope.

[0381] b. Fig. 22 As shown, compared with the sebaceous gland mature cells (SBM) whose volume irregularly increased 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 were reduced in volume, slowing down their maturation rate, thereby intervening in their lipid droplet release (scale bar = 20 μm), thereby being able to evaluate and screen drugs or skin care products that inhibit oil secretion.

[0382] The above results show that compared with human keratinocytes, human immortalized sebaceous gland cells 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, and have clear genetic information and clinical background, and can simulate the behavior of sebaceous glands in acne patients. Moreover, both sebaceous gland precursor cells and sebaceous gland mature cells can play an evaluation and screening role in promoting oil secretion and inhibiting oil secretion. This method can be used to provide relevant in vitro models for disease modeling and developing 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 are not 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.

[0383] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various specific technical features in any appropriate manner. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations. However, these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

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

2. A method for screening lipid regulating drugs or skin care products using the human sebaceous gland precursor cells of claim 1, characterized in that: The human sebaceous gland precursor cells are directly incubated with DHT dihydrotestosterone at room temperature for 48 hours to stimulate the human sebaceous gland precursor cells to produce more oil, which is used for screening oil-regulating drugs or skin care products.

3. An application of the human sebaceous gland precursor cells according to claim 1 for screening lipid regulating drugs or skin care products, characterized in that: The human sebaceous gland precursor cells are cultured and differentiated into mature sebaceous gland cells, and the mature sebaceous gland cells are used to screen oil regulating drugs or skin care products.

Images