A method for constructing a human dermal microparticle skin model and its application and method in skin aging model construction and sample detection

Abstract

The present invention provides a method for constructing a human dermal microparticle skin model and its application, product and method in the construction of a skin aging model and product testing, belonging to the field of organoid culture technology. The method for constructing a human dermal microparticle skin model includes: S1. micropit array organ chip design; S2. isolation and culture of human primary fibroblasts; S3. using an external power to drive the micropit array organ chip to construct a human dermal microparticle skin model. The present invention realizes high-throughput microparticle organ model construction by using a micropit array organ chip, relying on cell autonomous assembly to form a 3D dermal organoid model close to a real skin model, and by directly constructing and testing the dermal organoid model in the chip, highly realistic detection results are achieved efficiently and accurately.

Description

Technical Field

[0001] The present invention belongs to the technical field of organoid culture, and specifically provides a method for constructing a human dermal microparticle skin model, and its application, product and method in skin aging model construction and product testing. Background Art

[0002] Skin is the largest organ in the human body. It not only protects the body from external environmental damage, but also participates in many physiological functions such as temperature regulation, sensory transmission and the synthesis of vitamin D. Skin aging is a natural biological process that involves the combined effects of multiple factors such as genetics, environment, and lifestyle. As we age, our skin gradually loses its elasticity and luster, and signs of aging such as wrinkles, spots, and sagging skin appear. These changes not only affect the appearance, but may also affect the normal function of the skin.

[0003] The biological basis of skin aging mainly includes changes at the cellular and molecular levels. At the cellular level, the proliferation ability of fibroblasts (the main cell type in the skin) and the ability to synthesize collagen decrease, resulting in reduced skin elasticity. At the molecular level, a variety of cell signaling pathways such as the transforming growth factor β (TGF-β) / Smad signaling pathway and the Wnt / β-catenin signaling pathway play an important role in skin aging. In addition, oxidative stress, inflammatory response, and cell apoptosis are also closely related to skin aging.

[0004] Microfluidic chip technology, also known as "organ chip" or "organ on a chip", is a technology that precisely controls fluids at a microscale. This technology provides a powerful platform for studying cell behavior, drug screening, and disease models by simulating the microenvironment of cells and tissues. The development of microfluidic chip technology provides new tools and methods for simulating and studying skin aging. The application of microfluidic chip technology in skin aging research mainly focuses on: simulating skin microenvironment, drug screening and toxicity testing, establishing disease models, simulating skin physiological functions, and studying anti-aging mechanisms. At present, the anti-aging research of cosmetics mainly relies on two-dimensional cell models. Although two-dimensional monolayer cell culture technology can achieve rapid and high-throughput drug screening and disease model construction, this method has some limitations, which may affect the effective detection and sensitivity of the test active substances. Two-dimensional culture cannot fully simulate the complex three-dimensional environment in which cells are located in the body, which includes factors such as cell-cell interactions, cell-extracellular matrix contact, and oxygen and nutrient gradients.

[0005] A method for constructing a skin chip and a skin model is disclosed in a Chinese patent application with publication number CN118308187A. The skin chip includes a first chip, a second chip and a porous membrane. A first flow channel and a fluid chamber are provided inside the first chip, and the fluid chamber is connected to the first flow channel. The second chip is connected to the first chip, and a second flow channel and a through hole are provided inside the second chip, and the through hole is connected to the second flow channel and corresponds to the fluid chamber. The porous membrane is provided on the first chip and / or the second chip, and is located between the through hole and the fluid chamber. The through hole is connected to the fluid chamber through the porous membrane, and the porous membrane and the through hole form a culture chamber, and the first flow channel is used for the culture medium to enter the fluid chamber, and the second flow channel is used for the culture medium to enter the culture chamber. The culture medium of the present application enters the culture chamber through the first flow channel and the second flow channel, and the contact area between the cells in the skin model and the culture medium is large. The culture medium can provide sufficient nutrients for the cells in the skin model, and the problem of insufficient nutrition of some cells can be avoided. However, the model construction method is cumbersome, the hardware structure is complex, and the practical application is limited, and there is room for further optimization. Summary of the invention

[0006] In order to solve the above problems, the present invention provides a construction and cultivation method for a full-thickness skin microparticle model based on a micropit array organ chip.

[0007] The present invention constructs a human aging dermal microparticle skin model based on a micropit array organ chip for high-throughput cosmetic or drug screening. This advanced method breaks through the limitations of traditional 2D cell culture and provides cells with a growth condition that is closer to the in vivo environment by simulating cell-cell interactions in three-dimensional space. The culture state including microfluidics enables the cell balls in the chip to achieve sufficient oxygen and nutrient exchange and timely discharge of metabolic waste. This not only allows cells to grow and differentiate in a more natural state, but also better simulates the physiological and pathological conditions inside the skin. In this three-dimensional culture environment, cells can form complex tissue structures that can more realistically reflect the microenvironment of the skin. The development and application of this technology heralds more breakthroughs and innovations in disease research and treatment in the future.

[0008] The technical solution adopted by the present invention is as follows:

[0009] In one aspect, the present invention provides a method for constructing a human dermal microparticle skin model, characterized in that it comprises the following steps:

[0010] S1. Micropit array organ chip design;

[0011] S2. Isolation and culture of primary human fibroblasts;

[0012] S3. Use external power to drive the micro-pit array organ chip to construct a human dermal microparticle skin model.

[0013] Specifically, the micropit array organ chip in step S1 includes an array of micropits, wherein the micropits are composed of a unit of cell deposition and cell spheres composed of a circular opening, a conical cavity, and a U-shaped bottom. During cell culture, cells settle and aggregate in the micropits, the top of the unit is connected to the hemispherical section, the bottom of the unit is port A for injection, and the vertex of the hemispherical surface is provided with port B for drainage.

[0014] The perfusion involved in the present invention is preferably completed by using a digital injection pump, generally injected through port A and drawn out through port B.

[0015] Specifically, in step S2:

[0016] The separation comprises: washing the human skin tissue, shearing it, digesting and dispersing it, separating the dermis from the epidermis, digesting the dermis tissue with pancreatic enzymes, filtering and collecting cells, namely, primary human fibroblasts;

[0017] The culture comprises subculturing the primary human fibroblasts to a P3 generation or above.

[0018] In some specific implementations, step S2 includes:

[0019] After HDFa cells (primary) are isolated, they are continuously passaged to P10. When the cells grow to a confluence of more than 70% (preferably 70%-80%), they are digested and the experimental operation is performed after the cells are counted.

[0020] The culture medium is preferably formulated as: DMEM / F-12 medium (Dulbecco's Modified Eagle Medium / Nutrient Mixture F-12) containing 5-15% FBS (Fetal Bovine Serum), 1-3% P / S (Penicillin-Streptomycin), and 1-3% L-Gln (L-Glutamin). More preferably, it is DMEM / F-12 medium containing 10% FBS, 1% P / S, and 1% L-Gln.

[0021] Specifically, step S3 includes:

[0022] S31. Preparation: sterilization, surface modification, and cleaning of the micropit array organ chip;

[0023] S32. Preparation of cell suspension: Digest and resuspend the cells cultured in S2 to obtain a cell density of 1×10 6 -1×10 7Cell suspension of 100 cells / mL;

[0024] S33. Cell capture: inject the cell suspension of S32 into port A of the microwell array organ chip, culture it statically, and perform subsequent operations after the cells are captured;

[0025] S34. Residual cell removal: inject the culture medium into port A of the microwell array organ chip to remove the uncaptured cells;

[0026] S35. Cell spheroid culture: The micro-pit array organ chip is cultured to obtain cell spheroids, which are human dermal microparticle skin models.

[0027] Specifically, in step S31, the sterilization conditions include: 121°C, 103.4 kPa, sterilization for 15-30 min, and oven drying; the surface modification uses 75%-100% (v / v) ethanol, and the perfusion conditions of the ethanol are 5-50 μL / min, and continuous injection for 10-30 min; the cleaning uses PBS, and the perfusion conditions of the PBS are 5-50 μL / min, and continuous injection for 10-30 min.

[0028] Preferably, in step S31, the sterilization conditions are 121°C, 103.4 kPa, sterilization for 15 to 30 minutes, and oven drying at 60°C for 12 hours;

[0029] 95% ethanol was used for surface modification and injected into port A of the microwell array organ chip through a digital syringe pump. The perfusion condition of the digital pump was 20 μl / min and the injection was continuous for 30 min. A hose was connected to port B for liquid discharge.

[0030] PBS (Phosphate Buffered Saline) was used for washing and injected into port A of the microwell array organ chip through a digital syringe pump. The perfusion condition of the digital pump was 20 μl / min and the injection was continuous for 30 min. A hose was connected to port B for liquid discharge.

[0031] Specifically, in step S32, the perfusion conditions for injecting the cell suspension are: 10-50 μL / min, continuous injection for 10-30 min.

[0032] Specifically, in step S33, the static culture time is more than 30 minutes, and the proportion of cells captured is more than 80%.

[0033] Preferably, in step S33, the cell suspension is injected into port A of the microwell array organ chip using a digital syringe pump, the perfusion condition is 20 μl / min, and the injection is continuous for 20 min. A hose is connected to port B for cell perfusion. After the perfusion is completed, the cells are incubated in an incubator for 2 h.

[0034] Specifically, in step S34, the injection condition of the culture medium is 5-50 μL / min, and the continuous injection is 10-30 min.

[0035] Preferably, in step S34, the uncaptured cells are cleared by injecting the culture medium using a digital injection pump, the perfusion condition is 10 μl / min, and the injection is continuous for 60 min, and a hose is connected to port B for liquid discharge.

[0036] In some specific implementations, the step S3 includes:

[0037] S31. sterilize the micropit array organ chip with high temperature and high pressure steam, perform surface modification, and clean it;

[0038] S32. Digest and resuspend the cultured cells to obtain a cell density of 1×10 6 -1×10 7 of cell suspension;

[0039] S33. The cell suspension is injected into port A of the microwell array organ chip through a digital syringe pump, and then transferred to an incubator and allowed to stand for a period of time, and subsequent operations are performed after 90% of the cells are captured;

[0040] S34. Inject the culture medium into port A of the microwell array organ chip through a digital syringe pump, and use a long-term low-flow perfusion method to remove uncaptured cells;

[0041] S35. Place the microwell array organ chip in an incubator for culture.

[0042] On the other hand, the present invention provides the use of the aforementioned human dermal microparticle skin model in constructing a skin aging model.

[0043] Accordingly, the present invention provides a method for constructing a skin aging model.

[0044] The method for constructing the skin aging model is based on the method for constructing the human dermal microparticle skin model, and further includes:

[0045] S4. Based on different induction methods, the model constructed in S3 is induced to undergo aging to obtain an aging model.

[0046] Preferably, the aging induction includes but is not limited to oxidative damage or photoaging.

[0047] Further preferably, the oxidative damage includes but is not limited to H2O2-induced damage, and the photoaging includes but is not limited to UVA-induced damage.

[0048] Furthermore, the conditions for H2O2-induced damage include: 200-600 μM H2O2 treatment, 1-3 h / day, continuous treatment for 2-5 days, changing the culture medium after each treatment and continuing to culture, and incubating with fluorescent probes for aging indicator detection after induction;

[0049] Furthermore, the conditions for UVA-induced damage include: UVA treatment, irradiation distance 5-15 cm, each radiation dose 1-10 J / cm 2 , irradiated continuously for 2-5 days, replaced the culture medium after each treatment and continued to culture, and after induction was completed, the fluorescent probe was continuously incubated for aging indicator detection.

[0050] In some specific embodiments, the conditions for H2O2-induced damage include: treating with 600 μM H2O2, 2 h / d, for 3 consecutive days, replacing the culture medium after each day of treatment and continuing to culture, and after three days of induction, continuously incubating with a β-gal fluorescent probe for 24 h to detect aging indicators of the organoid chip.

[0051] In some specific embodiments, the conditions for UVA-induced damage include: UVA treatment, irradiation distance 10 cm, each radiation dose 5 J / cm 2 , irradiated continuously for 3 days, replaced the culture medium after each treatment and continued to culture, and incubated with β-gal fluorescent probe for 24 hours after induction to detect aging indicators of organoid chips.

[0052] Based on the above, the present invention includes the human dermal particle skin model and skin aging model constructed by the above two construction methods, and at the same time protects the application of these two construction methods and corresponding models in functional testing or toxicity testing of skin products.

[0053] Specifically, the products include any one or more of medicines, foods, health products, and cosmetics.

[0054] Preferably, the product is a cosmetic, and the functional test of the cosmetic includes any one or more functional tests of anti-oxidation, anti-aging, firming, anti-wrinkle, and UV protection.

[0055] In yet another aspect, the present invention provides a method for functional testing or toxicity testing of a skin product.

[0056] The testing method includes using the skin aging model mentioned in claim 1 for testing.

[0057] Alternatively, the test method includes any one of the two aforementioned construction methods, followed by testing;

[0058] Alternatively, the testing method includes constructing a skin aging model using the aforementioned human dermal microparticle skin model and then conducting the test.

[0059] The aged dermal organoids constructed using HDFa in the present invention can be used as a biological model in practical applications to study skin pathophysiological processes, such as the efficacy evaluation of cosmetics on skin antioxidant, anti-aging, firming, anti-wrinkle, and ultraviolet (UVA) protection.

[0060] It is particularly important to point out that the high-throughput membrane-free micropit array organ chip used in the present invention is a 3D culture chip with one channel. It can better reconstruct the in vivo microenvironment by driving it through an external power injection system, and realize the sustainable flow of fluid in the chip by driving it through an external power injection system, so as to achieve sufficient exchange of oxygen and nutrients. The construction method of the aged dermal organoid is simple, low-cost, and easy to operate, and the formed aged dermal organoid has high stability and repeatability, and can be directly promoted and applied in related research and applications.

[0061] Beneficial effects of the present invention:

[0062] 1. High-throughput microparticle organ model construction is achieved by using micropit array organ chips.

[0063] 2. Relying on cell autonomous assembly, a 3D dermal organoid model that is close to the real skin model is formed.

[0064] 3. By directly constructing and testing the dermal organoid model within the chip, highly realistic test results can be achieved efficiently and accurately. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 This is an example of the micropit design scheme in the micropit array organ chip in Example 1, where the upper figure is a three-dimensional design diagram, the middle is a longitudinal section, and the lower figure is a top view.

[0066] Figure 2 This is the result of constructing the human dermal microparticle skin model in Example 1.

[0067] Figure 3 This is the result of constructing the human aging dermal microparticle skin model in Example 2.

[0068] Figure 4 These are the fluorescent labeling detection results of each group treated with PDRN in Example 3.

[0069] Figure 5 These are the fluorescence labeling detection results of each group treated with quercetin in Example 3. DETAILED DESCRIPTION

[0070] The present invention will be further described in detail below in conjunction with specific embodiments. The following embodiments are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following embodiments are generally conventional conditions, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified. Unless otherwise specified, the scientific and technological terms used herein are based on the understanding of general professionals in the relevant field. It should be noted that the temperatures and concentrations mentioned herein are approximate values ​​and are mainly used for explanation. Methods and materials similar to or equivalent to the methods and materials described herein can be used to implement the disclosure herein. In addition, the reaction conditions and embodiments such as the concentration and temperature of the described materials, methods, solutions are only used as examples and are not intended to limit the scope of application. In specific implementation, professional technicians can use conventional experimental means to adjust and optimize the operating parameters such as the material ratio, concentration, temperature, and order of addition involved in the method according to the content disclosed in the present invention to achieve the purpose of the present invention.

[0071] The Chinese meanings of the English abbreviations used in this article are shown in Table 1.

[0072] Table 1

[0073]

[0074] The experimental materials, biological reagents and their sources used in the following examples are shown in Table 2:

[0075] Table 2

[0076]

[0077]

[0078] The following examples use Olympus MX-35 inverted microscope and Olympus FV3000 confocal inverted microscope to detect fluorescence signals.

[0079] Example 1 A method for constructing a human dermal microparticle skin model

[0080] Step 1: Structural design and fabrication of micropit array organ chips

[0081] (1) The microwell structure is a unit formed by cell deposition and cell spheres consisting of a circular opening, a conical cavity, and a U-shaped bottom. As an example, the microwell design scheme in the microwell array organ chip is as follows: Figure 1 As shown;

[0082] (2) During cell culture, cells can be allowed to settle and aggregate in the micropits;

[0083] (3) The micro-pit chip is produced using high-precision 3D printing.

[0084] Step 2: Reagent preparation

[0085] Hank's dissection solution: Hank's buffer + 1% P / S;

[0086] DMEM / F-12 complete medium: DMEM / F-12 basal medium + 10% FBS + 1% L-Gln (L-Glutamin) + 1% P / S.

[0087] Step 3: Isolation and culture of primary human fibroblasts (HDFa)

[0088] Isolation and culture of HDFa: Place human skin tissue in a culture dish and wash it 3-5 times with 4℃ precooled Hank's dissection solution. Stop washing when there is no blood on the skin surface and in the washing solution. Carefully cut off the subcutaneous tissue with ophthalmic scissors and wash it 3-5 times with 4℃ precooled Hank's dissection solution. Use microscissors to cut the tissue into 1×2mm 2 The size of the cells was added to 2U / mL of dispase digestion solution and digested overnight (24h) at 4℃. The dermis was separated from the epidermis using toothed forceps, and the separated dermal tissue was placed in a culture dish. The cells were digested with 0.25% trypsin (containing EDTA) digestion solution at 37℃ for 30min, and an equal volume of DMEM / F-12 complete medium was added to terminate the digestion. The cells were filtered using a 200-mesh sieve, centrifuged at 1000rpm for 5min, and the supernatant was discarded to collect the precipitated cells. The cell pellet was washed and resuspended with PBS, centrifuged at 1000rpm for 5min, and the supernatant was discarded. The cell pellet was resuspended with 10mL of DMEM / F-12 complete medium, inoculated into a T75 bottle, and cultured in a 37℃, 5% CO2 incubator. The medium was changed every 24h. The cells were passaged when the plating rate was about 80%-85%, and the cells were expanded to the 7th to 10th generations for model construction.

[0089] Step 4: Construction of human dermal microparticle skin model

[0090] (1) Reagent preparation: Filter 95% ethanol, PBS, Sterilize P188 solution and DMEM / F-12 complete medium for later use.

[0091] (2) Disinfection: The chip is sterilized by high pressure, and the outer surface of the chip is sprayed with 75% alcohol. The outer packaging is torn open and the chip is taken out and placed in a clean bench under ultraviolet light for 10 min to 30 min for further disinfection.

[0092] (3) Chip modification and bubble removal: Replace the 1mL syringe with a special hose needle, extract 95% ethanol, expel the excess bubbles in the syringe, insert it into the chip A hole, and also insert a special hose needle into the B hole (drainage). Infuse ethanol into the chip until there are no bubbles in the chip.

[0093] (4) Cleaning: Replace the 1 mL syringe with a special hose needle to extract PBS, expel excess bubbles in the syringe, and insert it into hole A of the chip. Insert a special hose needle into hole B (drainage) to remove ethanol in the chip. Clean three times, taking care to avoid introducing bubbles.

[0094] (5) Cell examination: HDFa was examined under a microscope and photographed to confirm that the cell plating rate reached 80-90% and that the cells were in good growth condition and that subsequent operations could be performed.

[0095] (6) Cell preparation: discard the culture medium, add 5 mL of PBS to gently rinse the cells and discard the liquid, add 3 mL of 0.25% trypsin (containing EDTA), digest the cells at 37°C for 2 min, centrifuge at 1000 rpm for 5 min, discard the supernatant, add 3 mL of DMEM / F-12 complete medium to resuspend the cell pellet for cell counting, centrifuge at 1000 rpm for 5 min again, discard the supernatant, add an appropriate volume of DMEM / F-12 complete medium to resuspend the cells and adjust the cell density to 1×10 7 Pieces / mL.

[0096] (7) Inoculation: Replace the 1 mL syringe with a dedicated hose needle to extract the cell suspension and expel excess bubbles in the syringe. Connect the digital syringe pump and insert it into hole A of the chip. Also insert a dedicated hose needle (drainage) into hole B. The injection condition is 10 μL / min. Continue to inject until the chip is filled with cells.

[0097] (8) Cell capture and residual cell removal: The inoculated chip was placed in an incubator and incubated at 37°C, 5% CO2 for 2 h. A 1 mL syringe was replaced with a special hose needle to extract DMEM / F-12 complete medium and expel excess bubbles in the syringe. A digital injection pump was connected and inserted into hole A of the chip. A special hose needle (drainage) was also inserted into hole B. The injection condition was 20 μL / min, and the injection was continued for 20 min.

[0098] (9) Cell spheroid culture: After culturing for 12 h at 37°C and 5% CO2, cell spheroids were obtained for aging modeling.

[0099] like Figure 2 Shown are the results of constructing a human dermal microparticle skin model.

[0100] Example 2 Construction of human aging dermal microparticle skin model

[0101] The cell sphere (human dermal microparticle skin model) obtained in Example 1 was subjected to aging modeling. The modeling scheme was to subject the cell sphere to H2O2 stimulation or UVA radiation treatment, respectively. In this embodiment, three condition groups were specifically set, and the three condition groups included:

[0102] Group 1 (NC): No H2O2 treatment was performed, culture medium was injected into the chip, and fresh culture medium was replaced every day.

[0103] Group 2 (H2O2 induced group): treated with 600 μM H2O2 for 2 h for 3 consecutive days, and fresh culture medium was replaced after each treatment to continue culture.

[0104] Group 3 (UVA radiation group): irradiation distance 10cm, radiation dose 5J / cm 2 , irradiated continuously for 3 days, and replaced with fresh culture medium after each treatment and continued to culture.

[0105] The specific verification steps are as follows:

[0106] S1. Connect the digital syringe pump to a 1mL syringe and replace it with a special hose needle to extract PBS, and wash the three groups of cells at 20μL / min;

[0107] S2. Connect a 1mL syringe to a digital syringe pump and replace it with a special hose needle to extract DMEM / F-12 basal medium, and perfuse it into well A of group 1 chip at 10μL / min. Also insert a special hose needle into well B (drainage). Place the chip in a 37℃, 5% CO2 incubator for 2h / day, and replace it with DMEM / F-12 complete medium after treatment.

[0108] S3. Prepare 600 μM H2O2 using DMEM / F-12 basal medium, and use a digital syringe pump to perfuse into well A of group 2 chip at 10 μL / min, and insert a special hose needle (drainage) into well B. Place the chip in a 37°C, 5% CO2 incubator for 2 h / day, and replace with DMEM / F-12 complete medium after treatment.

[0109] S4. Connect a 1mL syringe to the digital syringe pump and replace it with a special hose needle to extract PBS, and use the digital syringe pump to perfuse it into the A hole of the group 2 chip at 10μL / min, and also insert a special hose needle into the B hole (drainage). Place the chip 10cm under the UVA lamp for irradiation treatment, and replace it with DMEM / F-12 complete culture medium after the treatment.

[0110] S5. Steps S1-S4 are performed continuously for three days.

[0111] like Figure 3 The figure shows the results of constructing a human dermal microparticle skin model. Both H2O2 treatment and UVA radiation can cause the cell spheres to show an aging state.

[0112] Example 3 Evaluation and verification of anti-aging efficacy using human aging dermal microparticle skin model

[0113] The human aging dermal microparticle skin model obtained in Example 2 was used to evaluate and verify the anti-aging efficacy.

[0114] The validation scheme is to add a certain concentration of active substances to the human aged dermal microparticle skin model in the chip and treat it for 1-2 days.

[0115] The active substances used in this example are 10% PDRN solution and 150 nM quercetin.

[0116] During the culture period, the cells were observed under an optical microscope every day. The β-gal senescence fluorescent probe (Senescence-Tracker TM Senescent cell near-infrared fluorescent probe) was incubated to mark senescent cells, and finally live cell imaging was performed. Specifically, in this embodiment, seven condition groups were set, and the seven condition groups included:

[0117] Group 1 (NC): No senescence was induced and no active substance treatment was performed. The medium was changed every 24 hours and the cells were observed and photographed.

[0118] Group 2 (oxidative aging Ctrl): H2O2 was induced, but no active substances were added. The medium was changed every 24 hours and the cells were observed and photographed.

[0119] Group 3 (oxidative aging + 10% PDRN): H2O2 induction was performed, the active substance was 10% PDRN solution, the solution was changed every 24 hours, and observation and photography were performed.

[0120] Group 4 (oxidative aging + 150 nM quercetin): H2O2 induction was performed, the active substance was 150 nM quercetin solution, the solution was changed every 24 h, and observation and photography were performed.

[0121] Group 5 (photoaging Ctrl): UVA irradiation was performed, but no active substances were added. The solution was changed every 24 hours and the samples were observed and photographed.

[0122] Group 6 (photoaging + 10% PDRN): UVA irradiation, the active substance is 10% PDRN solution, the solution is changed every 24 hours, and observation and photography are performed.

[0123] Group 7 (photoaging + 150 nM quercetin): UVA irradiation, the active substance is 150 nM quercetin solution, the solution is changed every 24 hours, and observation and photography are carried out.

[0124] The specific verification steps are as follows:

[0125] S1. Prepare a solution of the compound to be tested at a certain concentration, and use a digital syringe pump to perfuse it into the organoids on the chip. Place the chip in a 37°C, 5% CO2 incubator and incubate for 1-2 days. Replace the culture medium containing the drug to be tested once a day.

[0126] S2. 24 h before the end of drug administration, prepare the β-gal senescent cell fluorescent probe with DMEM / F-12 complete medium, inject it into the chip A hole at a rate of 10 μL / min using a digital syringe pump, and incubate it in a 37°C, 5% CO2 incubator for 24 h.

[0127] like Figure 4 As shown, under the action of PDRN, the apparent structure of the organoids in the drug-treated group was more compact than that in the control group, and the number of cells labeled with β-gal fluorescence was significantly reduced, which can be considered to have achieved a certain anti-aging effect.

[0128] like Figure 5 As shown, under the action of quercetin, the apparent structure of the organoids in the drug-treated group was denser than that in the control group, and the number of cells labeled with β-gal fluorescence was significantly reduced, which can be considered to have achieved a certain anti-aging effect.

Claims

1. A method for constructing a human dermal microparticle skin model, characterized in that: The following steps are involved: S1. Micropit array organ chip design; S2. Isolation and culture of primary human fibroblasts; S3. Use external power to drive the micro-pit array organ chip to construct a human dermal microparticle skin model.

2. The construction method according to claim 1, characterized in that: The micropit array organ chip in step S1 includes an array of micropits, wherein the micropits are units formed by cell deposition and cell balls composed of a circular opening, a conical cavity, and a U-shaped bottom; during cell culture, cells settle and aggregate in the micropits, the top of the unit is connected to the hemispherical section, the bottom of the unit is port A for injection, and the vertex of the hemispherical surface is provided with port B for drainage.

3. The construction method according to claim 2, characterized in that: In step S2: The separation comprises: washing the human skin tissue, shearing it, digesting and dispersing it, separating the dermis from the epidermis, digesting the dermis tissue with pancreatic enzymes, filtering and collecting cells, namely, primary human fibroblasts; The culture comprises subculturing the primary human fibroblasts to a P3 generation or above.

4. The construction method according to claim 3, characterized in that: Step S3 includes: S31. Preparation: sterilization, surface modification, and cleaning of the micropit array organ chip; S32. Preparation of cell suspension: Digest and resuspend the cells cultured in S2 to obtain a cell density of 1×10 6 -1×10 7 Cell suspension of 100 cells / mL; S33. Cell capture: inject the cell suspension of S32 into port A of the microwell array organ chip, culture it statically, and perform subsequent operations after the cells are captured; S34. Residual cell removal: inject the culture medium into port A of the microwell array organ chip to remove the uncaptured cells; S35. Cell spheroid culture: The micro-pit array organ chip is cultured to obtain cell spheroids, which are human dermal microparticle skin models.

5. The construction method according to claim 4, characterized in that: In step S31, the sterilization conditions include: 121°C, 103.4kPa, sterilization for 15-30min, and oven drying; the surface modification uses 75%-100% (v / v) ethanol, the perfusion conditions of the ethanol are 5-50μL / min, and continuous injection for 10-30min; the cleaning uses PBS, and the perfusion conditions of the PBS are 5-50μL / min, and continuous injection for 10-30min.

6. The construction method according to claim 4, characterized in that: In step S32, the perfusion conditions for injecting the cell suspension are: 10-50 μL / min, continuous injection for 10-30 min.

7. The construction method according to claim 4, characterized in that: In step S33, the static culture time is more than 30 minutes, and the proportion of cells captured is more than 80%.

8. The construction method according to claim 4, characterized in that: In step S34, the injection condition of the culture medium is 5-50 μL / min, and the continuous injection is 10-30 min.

9. Use of the human dermal microparticle skin model according to any one of claims 1 to 8 in constructing a skin aging model.

10. A method for constructing a skin aging model, characterized in that: Based on the construction method according to any one of claims 1 to 8, it also includes: S4. Based on different induction methods, the model constructed in S3 is induced to undergo aging to obtain an aging model.

11. The construction method according to claim 10, characterized in that: The aging induction includes oxidative damage or photoaging.

12. The construction method according to claim 11, characterized in that: The oxidative damage includes H2O2-induced damage, and the photoaging includes UVA-induced damage.

13. The construction method according to claim 12, characterized in that: The conditions for H2O2-induced damage include: 200-600 μM H2O2 treatment, 1-3 h / day, continuous treatment for 2-5 days, changing the culture medium after each treatment and continuing to culture, and incubating with fluorescent probes for aging indicator detection after induction.

14. The construction method according to claim 12, characterized in that: The conditions for UVA-induced damage include: UVA treatment, irradiation distance 5-15 cm, each radiation dose 1-10 J / cm 2 , irradiated continuously for 2-5 days, replaced the culture medium after each treatment and continued to culture, and after induction was completed, the fluorescent probe was continuously incubated for aging indicator detection.

15. A human dermal microparticle skin model constructed according to the construction method according to any one of claims 1 to 8.

16. A skin aging model constructed according to the construction method according to any one of claims 10 to 14.

17. Use of the construction method according to any one of claims 1 to 8 or the human dermal microparticle skin model according to claim 15 or the construction method according to any one of claims 10 to 14 or the skin aging model according to claim 16 in functional testing or toxicity testing of skin products.

18. The use according to claim 17, characterized in that The products include any one or more of medicines, foods, health products, and cosmetics.

19. The use according to claim 18, characterized in that The functional tests of the cosmetics include any one or more functional tests of anti-oxidation, anti-aging, firming, anti-wrinkle, and UV protection.

20. A method for functional testing or toxicity testing of a skin product, characterized in that: The method comprises using the skin aging model described in claim 16 for testing.

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