Construction method of sebaceous gland model
Sebaceous gland microspheres with hollow structure are constructed through 3D printing or cell self-assembly technology, and induce them to mature into sebaceous gland models with high expression of oil secretion function, inflammatory factor secretion function and lipid synthesis-related genes in a specific culture medium, solving the problem that it is difficult to fully reflect the oil control effect of cosmetics on sebaceous gland function in the prior art, and achieving effective simulation and evaluation under conditions closer to in vivo.
Patent Information
- Application Number
- CN202510139351.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-08
- Publication Date
- 2025-05-06
AI Technical Summary
The prior art is difficult to fully reflect the oil control effect of cosmetics on sebaceous gland function under two-dimensional in vitro culture conditions, and it is unable to effectively simulate the complex sebaceous gland system in the human body.
Sebaceous gland microspheres with hollow structures are constructed through 3D printing or cell self-assembly technology, and induced to mature into sebaceous gland models with high expression of oil secretion, inflammatory factor secretion, and lipid synthesis-related genes in a specific culture medium.
Simulating physiological skin lipid metabolism in conditions closer to in vivo can more effectively screen and evaluate the efficacy of modulating active ingredients to balance sebum excess or lack of sebum, and obtain more predictive and representative therapeutic results.
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Figure CN119931922A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of medical model manufacturing and application, and in particular to a method for constructing a sebaceous gland model. Background Art
[0002] Sebaceous glands are holocrine skin appendages that can secrete a variety of lipids. They are mainly composed of alveoli and ducts, and the alveoli are filled with lipid droplets. A large amount of evidence shows that sebaceous gland dysfunction and disordered sebum composition can lead to certain skin diseases, including skin aging, acne vulgaris, rosacea, androgenic alopecia and scarring alopecia, as well as sebaceous gland tumors. Among them, excessive sebum secretion can cause problems such as oily face and clogged pores, affecting appearance and skin health. Therefore, controlling sebum secretion has become an important research direction in the field of cosmetics and medical cosmetology.
[0003] In the cosmetics field, oil control products have always been an important market segment. Traditional oil control products mainly achieve oil control effects by adsorbing or absorbing excess sebum, but this method can only achieve short-term effects.
[0004] With the advancement of science and technology, functional oil-control products that use active ingredients to regulate sebaceous gland activity and inhibit sebum secretion are gaining more and more attention. Existing research on oil-control efficacy is mainly based on two-dimensional cultured sebaceous gland cells in vitro, and the oil-control effect of cosmetics is evaluated by observing the secretion of cell lipid droplets. However, the sebaceous glands in the human body are a complex system, and the oil-control effect of cosmetics cannot be fully reflected by only two-dimensional cultured sebaceous gland cells in vitro. Summary of the invention
[0005] The purpose of the present invention is to provide a method for constructing a sebaceous gland model with oil secretion function, inflammatory factor secretion function and high expression of lipid synthesis related genes.
[0006] In order to achieve the above object, the present invention is implemented by the following technical solutions:
[0007] A method for constructing a sebaceous gland model comprises constructing obtained sebaceous gland cells into sebaceous gland microspheres with a hollow structure by means of a 3D printer or cell self-assembly, and then placing the sebaceous gland microspheres in a complete culture medium, culturing them in a 37°C, 5% CO2 incubator for 1-3 days, and then replacing them with a sebaceous gland induction culture medium to induce the sebaceous gland microspheres into a sebaceous gland model with oil secretion.
[0008] The sebaceous gland cells include but are not limited to primary sebaceous gland cells and sebaceous gland cell lines such as SZ95.
[0009] Furthermore, the method for obtaining sebaceous gland cells is as follows: collecting a full-thickness human skin specimen, cutting it into pieces, and 2+ and Mg2+ The skin was washed in phosphate buffered saline and placed in 4°C, 2.4 U / ml separation enzyme. After 20 hours, the epidermis and dermis were separated to ensure that the hair follicles on the epidermis were intact and connected to the sebaceous glands.
[0010] Then, the epidermis was placed in DNA enzyme at 37°C for 15 min, and then the intact sebaceous glands were separated from the epidermis, the sebaceous gland ducts were removed, and the sebaceous glands were inoculated into a culture dish coated with matrix gel;
[0011] Complete culture medium was added and cultured in an incubator at 37°C containing 5% CO2. The medium was changed three times a week. After 5-8 days of inoculation, cells growing outwards were seen around the glands. After three weeks, cell clusters with a diameter of 1-2 cm were formed. Cells were incubated in 0.25% trypsin and 0.02% EDTA at 37°C for 3-15 minutes to separate the cells. Complete culture medium was added again to inactivate trypsin, and single cell suspension was obtained by mechanical disaggregation for 1:3 subculture amplification.
[0012] Furthermore, the culture dish for inoculating sebaceous glands requires 2 The number of sebaceous glands is 2-3, and the complete culture medium is added to cover the surface of the sebaceous glands.
[0013] Furthermore, the components of the complete culture medium include, but are not limited to, 0.1-1 ng / ml human recombinant epidermal growth factor, 1-10 ng / ml hepatocyte growth factor, 1-10 ng / ml keratinocyte growth factor, 1-10 μg / ml insulin, 1-10 μg / ml hydrocortisone, 10-100 μg / ml gentamicin, 1-10 mM HEPES, 1-10 mM glutamine, 0.01-0.1 μg / ml atrimycin B and 0.1-1% bovine pituitary extract.
[0014] Furthermore, the method for constructing sebaceous gland microspheres with a hollow structure based on 3D printing technology is: after mixing the obtained sebaceous gland cells with a biological material with a certain viscosity, the hollow microsphere model is introduced based on a biological 3D printer or a spotting device to construct sebaceous gland microspheres with a hollow structure.
[0015] Furthermore, the method for constructing sebaceous gland microspheres with a hollow structure based on 3D printing technology is as follows: a biological material with a certain viscosity is used to construct material microspheres through a biological 3D printer or a spotting device, and then the obtained sebaceous gland cells are printed into the material microspheres through a biological 3D printer or a spotting device to construct sebaceous gland microspheres with a hollow structure.
[0016] The biomaterials include, but are not limited to, collagen, agar, GelMA, matrigel, F127, gelatin, alginate, silk fibroin and mixtures thereof in various concentrations.
[0017] Furthermore, the method for constructing hollow structured sebaceous gland microspheres based on cell self-assembly is as follows: in a well plate, 0.1-1 μL of biomaterial droplets are added, and then 3,000-50,000 obtained sebaceous gland cells are added, complete culture medium is added, and cultured for 24 hours. The sebaceous gland cells grow along the biomaterial droplets to form cell spheres.
[0018] The orifice plates include but are not limited to U-shaped low-adsorption orifice plates, V-shaped low-adsorption orifice plates and various orifice plates that can promote cell aggregation.
[0019] The construction methods of the biomaterial droplets include but are not limited to 3D printing, pipette, pipette, spotter, and droplet injection.
[0020] Furthermore, a certain concentration of microorganisms can be added to the biomaterial or biomaterial droplets to construct a co-culture model of sebaceous gland cells and microorganisms to simulate the formation of skin diseases.
[0021] Furthermore, microorganisms that can be added to the biomaterial or biomaterial droplets include, but are not limited to, Propionibacterium acnes, Malassezia, Staphylococcus albus, Pityrosporum ovale, and the like.
[0022] Furthermore, the components of the sebaceous gland induction medium include but are not limited to: 0.1-1ng / ml human recombinant epidermal growth factor, 1-10ng / ml hepatocyte growth factor, 1-10ng / ml keratinocyte growth factor, 1-10μg / ml insulin, 1-10μg / ml hydrocortisone, 10-100μg / ml gentamicin, 1-10mM HEPES, 1-10mM glutamine, 0.01-0.1μg / ml atrimycin B, 0.1-1% bovine pituitary extract and 1-50μg testosterone, 10-500μM linoleic acid, 10-500μM palmitic acid.
[0023] Compared with the prior art, the present invention has the following advantages:
[0024] The present invention provides a method for constructing a sebaceous gland model, which can construct a sebaceous gland model with oil secretion function, inflammatory factor secretion function and high expression of lipid synthesis related genes. Specifically, physiological skin lipid metabolism is simulated, and the efficacy of regulating active ingredients to balance excess or deficiency of sebum is screened and evaluated under conditions closer to the body, so as to obtain more predictive and representative efficacy results; the sebaceous gland model with Propionibacterium acnes simulates acne-prone skin, can detect lipid overproduction and metabolic activity, and can be used to study active substances and skin microbiota under the condition of skin dysbiosis; the sebaceous gland model with Malassezia can simulate the formation of seborrheic dermatitis. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] Figure 1 It is the sebaceous gland microsphere with a hollow structure constructed in Example 1 of the present invention.
[0026] Figure 2 It is a schematic diagram comparing the secretion results of the inflammatory factors IL6 and IL8 of the sebaceous gland model constructed in Example 2 of the present invention.
[0027] Figure 3 It is a schematic diagram comparing the expression results of lipid-related synthetic genes DGA2, FASN, SCD1, and PPARγ in the sebaceous gland model constructed in Example 2 of the present invention.
[0028] Figure 4 It is a schematic diagram comparing the oil secretion results of the sebaceous gland model constructed in Example 2 of the present invention. DETAILED DESCRIPTION
[0029] The embodiments of the present invention are described in further detail below in conjunction with the accompanying drawings.
[0030] Example 1 Construction of hollow sebaceous gland microspheres based on 3D printing technology
[0031] S1) Cut the human back skin into 3×5 mm pieces and place them in a Ca-free 2+ and Mg 2+ The cells were washed three times in phosphate buffered saline, and 5 mL of dissociation enzyme (2.4 U / ml) was added at 4°C. After 20 hours, the epidermis and dermis were separated with fine forceps to ensure that the epidermis had intact hair follicles connected to sebaceous glands. The complete sebaceous glands were separated from the epidermis under a dissecting microscope using microsurgical instruments, and the sebaceous gland ducts were removed. The sebaceous glands were inoculated into a culture dish coated with matrix gel. Six days after inoculation, cells growing outwards were seen around the glands. After two weeks of continuous culture, the cells were separated by incubation in 0.25% trypsin and 0.02% EDTA for 5 minutes, and 10 5 cell / mL suspension for later use.
[0032] S2) Assemble 5 mg / mL collagen solution onto the 3D printer, and set the printer nozzle to 4°C; place a 96-well plate on the printer platform, and set the temperature to 37°C; set the printing parameters, select a 210 μm needle to extrude 0.5 μL of collagen droplets per well; then add 100 μL of the cell suspension in step S1) into the well plate per well;
[0033] After two days of culture in a 37°C, 5% CO2 incubator, the sebaceous gland microspheres with hollow structures were formed. Figure 1 shown.
[0034] Example 2 Construction of sebaceous gland model
[0035] S1) culturing primary sebaceous gland cells using a complete medium, wherein the complete medium comprises: 0.1-1 ng / ml human recombinant epidermal growth factor, 1-10 ng / ml hepatocyte growth factor, 1-10 ng / ml keratinocyte growth factor, 1-10 μg / ml insulin, 1-10 μg / ml hydrocortisone, 10-100 μg / ml gentamicin, 1-10 mM HEPES, 1-10 mM glutamine, 0.01-0.1 μg / ml atrimycin B and 0.1-1% bovine pituitary extract;
[0036] When the primary sebaceous gland cells grow to 90% confluence, they are digested by incubation with 0.25% trypsin and 0.02% EDTA for 5 min. 10 5 cell / mL cell suspension.
[0037] S2) adding 100 μL of the cell suspension in step S1) into each well of a 96-well plate containing collagen microspheres and culturing for one day.
[0038] S3) replacing the complete culture medium of the sebaceous gland microspheres in step S2) with sebaceous gland induction medium, and continuing culturing for two days, wherein the induction medium comprises: 0.1-1 ng / ml human recombinant epidermal growth factor, 1-10 ng / ml hepatocyte growth factor, 1-10 ng / ml keratinocyte growth factor, 1-10 μg / ml insulin, 1-10 μg / ml hydrocortisone, 10-100 μg / ml gentamicin, 1-10 mM HEPES, 1-10 mM glutamine, 0.01-0.1 μg / ml atrimycin B and 0.1-1% bovine pituitary extract, as well as 1-50 μg testosterone, 10-500 μM linoleic acid, and 10-500 μM palmitic acid.
[0039] The constructed sebaceous gland model was used to detect oil secretion, inflammatory factors IL6, IL8, and lipid-related synthesis genes DGA2, FASN, SCD1, and PPARγ.
[0040] Specifically, in order to verify the advantages of the sebaceous gland model constructed by the present invention, four groups of comparative experiments were designed as follows. (1) Blank group: complete culture medium was used throughout the whole process; (2) Modeling group: after one day of complete culture medium culture, it was replaced with induction culture medium cultured for two days, and then replaced with complete culture medium cultured for one day; (3) Isotretinoin group: after one day of complete culture medium cultured, it was replaced with induction culture medium cultured for two days, and then replaced with complete culture medium containing 0.01mM isotretinoin cultured for one day; (4) Finasteride group: after one day of complete culture medium cultured, it was replaced with induction culture medium cultured for two days, and then replaced with complete culture medium containing 0.1mg / mL finasteride cultured for one day. Among them, isotretinoin and finasteride, as positive controls, can inhibit the oil secretion function of sebaceous glands. The culture medium of the four comparative experiments was collected at the end of time to detect the inflammatory factors IL6 and IL8. Figure 2 As shown, cell RNA was collected to detect lipid synthesis-related genes DGA2, FASN, SCD1, and PPARγ, as Figure 3 As shown, the results of detecting oil secretion using a fixed sebaceous gland model are shown. Figure 4 It is shown that the method of constructing the sebaceous gland model of the present invention is effective and applicable.
[0041] Example 3 Construction of co-culture model of Propionibacterium acnes and sebaceous gland cells
[0042] S1) culturing primary sebaceous gland cells using a complete medium, wherein the complete medium comprises: 0.1-1 ng / ml human recombinant epidermal growth factor, 1-10 ng / ml hepatocyte growth factor, 1-10 ng / ml keratinocyte growth factor, 1-10 μg / ml insulin, 1-10 μg / ml hydrocortisone, 10-100 μg / ml gentamicin, 1-10 mM HEPES, 1-10 mM glutamine, 0.01-0.1 μg / ml atrimycin B and 0.1-1% bovine pituitary extract;
[0043] When the primary sebaceous gland cells grow to 90% confluence, incubate with 0.25% trypsin and 0.02% EDTA for 5 min to digest the cells, and prepare 10 5 cell / mL cell suspension.
[0044] S2) Mix 0.5 mg / mL collagen with Propionibacterium acnes to prepare a 10 6 CFU / ml suspension was assembled on the 3D printer, and the printer nozzle was set to 4°C for temperature control; a 96-well plate was placed on the printer platform and the temperature was set to 37°C; printing parameters were set, and a 210μm needle was selected to extrude 0.5μL of a mixed droplet of collagen and Propionibacterium acnes per well;
[0045] The cell suspension in step S1) was added to a 96-well plate containing collagen microspheres at 100 μL per well and cultured for one day.
[0046] S3) replacing the complete culture medium of the sebaceous gland microspheres in step S2) with a sebaceous gland induction medium, and continuing culturing for two days, wherein the induction medium comprises: 0.1-1 ng / ml human recombinant epidermal growth factor, 1-10 ng / ml hepatocyte growth factor, 1-10 ng / ml keratinocyte growth factor, 1-10 μg / ml insulin, 1-10 μg / ml hydrocortisone, 10-100 μg / ml gentamicin, 1-10 mM HEPES, 1-10 mM glutamine, 0.01-0.1 μg / ml atrimycin B and 0.1-1% bovine pituitary extract, as well as 1-50 μg testosterone, 10-500 μM linoleic acid, and 10-500 μM palmitic acid, thereby constructing a co-culture model of Propionibacterium acnes and sebaceous gland cells.
[0047] Example 4 Construction of Malassezia and sebaceous gland cell co-culture model
[0048] S1) culturing primary sebaceous gland cells using a complete medium, wherein the complete medium comprises: 0.1-1 ng / ml human recombinant epidermal growth factor, 1-10 ng / ml hepatocyte growth factor, 1-10 ng / ml keratinocyte growth factor, 1-10 μg / ml insulin, 1-10 μg / ml hydrocortisone, 10-100 μg / ml gentamicin, 1-10 mM HEPES, 1-10 mM glutamine, 0.01-0.1 μg / ml atrimycin B and 0.1-1% bovine pituitary extract;
[0049] When the primary sebaceous gland cells grow to 90% confluence, they are digested by incubation with 0.25% trypsin and 0.02% EDTA for 5 min. 10 5 cell / mL cell suspension.
[0050] S2) Mix 0.5 mg / mL collagen with Malassezia to prepare a mixture containing 5*10 5 CFU / ml suspension was assembled on the 3D printer, and the printer nozzle was set to 4°C for temperature control; a 96-well plate was placed on the printer platform and the temperature was set to 37°C; the printing parameters were set, and a 210μm needle was selected to extrude 0.5μL of the mixed droplet of collagen and Malassezia per well;
[0051] The cell suspension in step S1) was added to a 96-well plate containing collagen microspheres at 100 μL per well and cultured for one day.
[0052] S3) replacing the complete culture medium of the sebaceous gland microspheres in step S2) with sebaceous gland induction medium, and continuing culturing for two days, wherein the induction medium is: 0.1-1 ng / ml human recombinant epidermal growth factor, 1-10 ng / ml hepatocyte growth factor, 1-10 ng / ml keratinocyte growth factor, 1-10 μg / ml insulin, 1-10 μg / ml hydrocortisone, 10-100 μg / ml gentamicin, 1-10 mM HEPES, 1-10 mM glutamine, 0.01-0.1 μg / ml atrimycin B and 0.1-1% bovine pituitary extract, as well as 1-50 μg testosterone, 10-500 μM linoleic acid, and 10-500 μM palmitic acid, thereby constructing a Malassezia and sebaceous gland cell co-culture model.
[0053] The above description is only a preferred embodiment of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and modifications without departing from the concept of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.
Claims
1. A method for constructing a sebaceous gland model, characterized in that: The obtained sebaceous gland cells are constructed into sebaceous gland microspheres with a hollow structure through 3D printing or cell self-assembly. The sebaceous gland microspheres are then placed in complete culture medium and cultured in a 37°C, 5% CO2 incubator for 1-3 days. The culture medium is then replaced with sebaceous gland induction culture medium to induce the sebaceous gland microspheres into a sebaceous gland model with oil secretion.
2. The method for constructing a sebaceous gland model according to claim 1, characterized in that: The method for obtaining sebaceous gland cells is as follows: a full-thickness human skin specimen is collected, cut into pieces, and then separated into two pieces in a Ca-free 2+ and Mg 2+ The skin was washed in phosphate buffered saline and placed in 4°C, 2.4 U / ml separation enzyme. After 20 hours, the epidermis and dermis were separated to ensure that the hair follicles on the epidermis were intact and connected to the sebaceous glands. Then, the epidermis was placed in DNA enzyme at 37°C for 15 min, and then the intact sebaceous glands were separated from the epidermis, the sebaceous gland ducts were removed, and the sebaceous glands were inoculated into a culture dish coated with matrix gel; Complete culture medium was added and cultured in an incubator at 37°C containing 5% CO2. The medium was changed three times a week until cell clusters with a diameter of 1-2 cm were formed. Cells were incubated in 0.25% trypsin and 0.02% EDTA at 37°C for 3-15 min to separate the cells. Complete culture medium was added again to inactivate trypsin, and single cell suspension was obtained by mechanical disaggregation for 1:3 subculture amplification.
3. The method for constructing a sebaceous gland model according to claim 2, characterized in that: The culture dish for inoculating sebaceous glands requires 1000mm 2 The number of sebaceous glands is 2-3, and the complete culture medium is added to cover the surface of the sebaceous glands.
4. A method for constructing a sebaceous gland model according to claim 1, 2 or 3, characterized in that: The components of the complete culture medium include, but are not limited to, 0.1-1 ng / ml human recombinant epidermal growth factor, 1-10 ng / ml hepatocyte growth factor, 1-10 ng / ml keratinocyte growth factor, 1-10 μg / ml insulin, 1-10 μg / ml hydrocortisone, 10-100 μg / ml gentamicin, 1-10 mM HEPES, 1-10 mM glutamine, 0.01-0.1 μg / ml atrimycin B and 0.1-1% bovine pituitary extract.
5. The method for constructing a sebaceous gland model according to claim 4, characterized in that: The method for constructing sebaceous gland microspheres with a hollow structure based on 3D printing technology is as follows: after mixing the obtained sebaceous gland cells with a biological material with a certain viscosity, the hollow microsphere model is introduced based on a biological 3D printer or a spotting device to construct sebaceous gland microspheres with a hollow structure.
6. The method for constructing a sebaceous gland model according to claim 4, characterized in that: The method for constructing sebaceous gland microspheres with a hollow structure based on 3D printing technology is as follows: biological materials with a certain viscosity are used to construct material microspheres through a biological 3D printer or a spotting device, and then the obtained sebaceous gland cells are printed into the material microspheres through a biological 3D printer or a spotting device to construct sebaceous gland microspheres with a hollow structure.
7. The method for constructing a sebaceous gland model according to claim 4, characterized in that: The method for constructing hollow structured sebaceous gland microspheres based on cell self-assembly is as follows: 0.1-1 μL of biomaterial droplets are added to the well plate, followed by 3,000-50,000 obtained sebaceous gland cells, complete culture medium, and culture for 24 hours. The sebaceous gland cells grow along the biomaterial droplets to form cell spheres.
8. A method for constructing a sebaceous gland model according to claim 5, 6 or 7, characterized in that: A certain concentration of microorganisms can be added to the biomaterial or biomaterial microdroplets to construct a co-culture model of sebaceous gland cells and microorganisms to simulate the formation of skin diseases.
9. A method for constructing a sebaceous gland model according to claim 5, 6 or 7, characterized in that: Microorganisms that can be added to the biomaterial or biomaterial droplets include, but are not limited to, Propionibacterium acnes, Malassezia, Staphylococcus albus, Pityrosporum ovale, and the like.
10. The method for constructing a sebaceous gland model according to claim 1, characterized in that: The components of the sebaceous gland induction medium include, but are not limited to, 0.1-1 ng / ml human recombinant epidermal growth factor, 1-10 ng / ml hepatocyte growth factor, 1-10 ng / ml keratinocyte growth factor, 1-10 μg / ml insulin, 1-10 μg / ml hydrocortisone, 10-100 μg / ml gentamicin, 1-10 mM HEPES, 1-10 mM glutamine, 0.01-0.1 μg / ml atrimycin B, 0.1-1% bovine pituitary extract, 1-50 μg testosterone, 10-500 μM linoleic acid, and 10-500 μM palmitic acid.