Anti-aging three-dimensional high-uniformity dermis evaluation model based on image analysis

Abstract

The invention discloses an anti-aging three-dimensional high-uniformity dermis evaluation model based on image analysis, and belongs to the technical field of biological medicine and cosmetic efficacy evaluation. The method comprises the following steps: mixing a prepared fibroblast suspension and a neutralized collagen solution at 0-5 DEG C, inoculating the mixture into a 24-pore plate in a 100-200L / pore manner, standing in an incubator at 37 DEG C for 20-40 minutes to form collagen gel, carrying out suspension culture to obtain a 3D dermal model, and detecting the shrinkage rate of the model and evaluating the anti-aging effect in combination with a high-content cell imaging system. The method overcomes the defect that a traditional method is large in error, an efficient and accurate evaluation system is provided for anti-aging drug screening, and the method has application value in cosmetic effect detection and skin aging mechanism research.

Description

[0001] This application is a divisional application of Chinese invention patent application filed on July 9, 2025, with application number 202510940586.8 and invention title "A method for screening anti-aging ingredients based on 3D dermis and high content imaging". Technical Field

[0002] This invention belongs to the field of biomedical and cosmetic efficacy evaluation technology, specifically involving an anti-aging three-dimensional highly uniform dermal evaluation model based on image analysis. Background Technology

[0003] Aging is a complex process involving gradual changes in the structure and function of cells, tissues, and organs. As the largest organ in the human body, the skin exhibits particularly pronounced signs of aging, specifically manifested in significant changes in viscosity and elasticity. Fibroblasts are present in various organs and tissues, including the skin, muscles, heart, lungs, and liver, and actively participate in the production and arrangement of collagen fibers. Collagen fibers significantly contribute to the tensile strength and elasticity of the skin. Aging fibroblasts show a decrease in cytoskeletal tension and a weakening of traction.

[0004] Type I collagen is the main collagen in most tissues of higher animals. It consists of two α1 chains and one α2 chain, and is uniform in diameter in natural tissues. Type I collagen is the most commonly used natural polymer component in bioengineered skin. Due to its very high water content, collagen has weak mechanical properties, and it is easily subjected to physical contraction when fibroblasts in the collagen matrix exert forces on the collagen. Therefore, in vitro models of fibroblast collagen gels can be used to simulate scar contraction and visualize the effects of different modulators on fibroblast contractile function. Compared with fibroblasts growing in a monolayer, fibroblasts dispersed in the collagen matrix behave more closely to their in vivo state and proliferate more slowly. Gels attached to the pore walls show a decrease in thickness without changing their diameter. When the collagen gel is mechanically released from the culture dish wall, the gel undergoes three-dimensional contraction, resulting in changes in thickness and diameter. These changes are caused by the random distribution of collagen fibers and the action of fibroblasts on the fibers.

[0005] The contractility of dermal fibroblasts changes during aging, with various contractility-related aspects, such as extracellular matrix remodeling, migration, and protein synthesis, experiencing a decline in age-related functions. However, NHDF from young donors exhibited significantly greater contractility in response to TGF-β1 treatment compared to NHDF from older donors, suggesting that changes in dermal fibroblast contractility can be used to assess cellular aging.

[0006] However, traditional methods for measuring collagen gel shrinkage use digital calipers to measure the gel diameter and calculate the corresponding gel area. Since the surface area of ​​a collagen gel after shrinkage is not a perfectly circular shape, calculating the surface area solely by measuring the gel diameter cannot accurately reflect the change in surface area. Furthermore, imaging collagen gels with digital cameras introduces some human error, which negatively impacts the results of collagen gel shrinkage rate measurements.

[0007] High-content cell imaging and analysis systems, while maintaining the integrity of cell structure and function, utilize automated cell imaging analysis methods to perform single-cell level analysis of cells in each well of a multi-well plate, analyzing multiple parameters and overall trends, including state and changes. The results of high-content imaging are obtained from the instrument's built-in image analysis, combining the advantages of intuitive visualization with batch statistical quantification. High-content cell imaging and analysis systems can achieve imaging and biological analysis of low-density cells and tissues in multi-well plates, including: angiogenesis, apoptosis, autophagy, cell counting, label-free cell counting, cell differentiation, endocytosis, mitotic index, lysosome detection, mitochondrial detection, and protein expression index. Using high-content cell imaging technology helps eliminate human bias. Summary of the Invention

[0008] The purpose of this invention is to provide a rapid, highly sensitive, and high-throughput method for screening anti-aging components based on 3D dermal and high-content imaging.

[0009] The technical solution adopted by the present invention to achieve the above objectives is as follows: A method for screening anti-aging ingredients includes: Fibroblast suspension was mixed with neutralized collagen solution and seeded at 100-200 µL / well in 24-well plates. The mixture was incubated at 36-38°C for 20-40 min to obtain collagen gel, which was then used to obtain a 3D dermal model through suspension culture. The collagen solution included rat tail type I collagen. The components to be screened were added to a 3D dermal model, and after cultivation, the surface area of ​​the 3D dermal model was measured and the shrinkage rate was calculated. If the average daily increase in shrinkage rate is greater than 15% after 3-4 days of cultivation, the selected components are considered to be anti-aging components.

[0010] Preferably, the shrinkage rate is calculated using the following formula: Shrinkage rate (%) = (1-S) / (2π) X / S0)×100%, where S0 is the initial surface area of ​​the 3D leather model, S X The surface area of ​​the 3D dermal model after shrinkage on day X.

[0011] Preferably, the formula for calculating the average daily shrinkage rate is: Average daily shrinkage rate (%) = Shrinkage rate / X.

[0012] Preferably, the density of fibroblasts in the fibroblast suspension is 0.9-1.1 × 10⁻⁶. 6 / mL.

[0013] Preferably, the fibroblasts in the fibroblast suspension are of generation 4-10.

[0014] Preferably, the collagen solution comprises 9-11×DMEM culture medium.

[0015] Preferably, the mass-to-volume ratio of rat tail type I collagen to 9-11×DMEM culture medium is 80-800 mg: 1-10 mL.

[0016] Preferably, the volume ratio of fibroblast suspension to collagen solution is 1-10:1-10.

[0017] Preferably, the molar volume ratio of the component to be screened to the 3D dermal model is 0.5-5µmol:50-500mL.

[0018] Preferably, the collagen solution includes functional agents and activators.

[0019] Preferably, the functional agent includes 2-hydroxy-3-isopropylsuccinic acid and 4-(3-pyrrolidinyl)butyric acid.

[0020] Preferably, the mass ratio of 2-hydroxy-3-isopropylsuccinic acid to 4-(3-pyrrolidinyl)butyric acid is 1.4-14:1.4-14.

[0021] 2-Hydroxy-3-isopropylsuccinic acid and 4-(3-pyrrolidinyl)butyric acid enhance cell-gel interactions, improve the chemical cross-linking network between collagen fibers, increase the storage modulus of collagen gel, and optimize gel mechanical properties. At the same time, the two can enhance cell compatibility by promoting fibroblast adhesion, proliferation and metabolic activities, and synergistically maintain the contractile function of fibroblasts, providing a 3D dermal model that is closer to the in vivo microenvironment for the screening of anti-aging ingredients.

[0022] Preferably, the activator comprises 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide.

[0023] Preferably, the positive medium includes TGF-β1 and DMEM complete medium.

[0024] Preferably, the mass-to-volume ratio of TGF-β1 to DMEM complete culture medium is 250-2500 ng: 50-500 mL.

[0025] More preferably, the functional agent also includes N,N-dimethyl-1,4-butanediamine, wherein the mass ratio of N,N-dimethyl-1,4-butanediamine to 4-(3-pyrrolo)butyric acid is 1.4-14:1.4-14. N,N-dimethyl-1,4-butanediamine increases the cross-linking sites between collagen fibers, strengthens the chemical cross-linking density of collagen gel under the action of activators, improves intermolecular electrostatic forces and hydrogen bond networks, and further enhances the storage modulus and mechanical rigidity of the gel; it also regulates the surface charge properties of the gel, optimizes the adhesion microenvironment of fibroblasts, promotes the proliferation and maintenance of contractile function of fibroblasts, and further optimizes the biological performance of the 3D dermal model.

[0026] This invention also provides a method for preparing a fibroblast suspension, comprising: Preparation of fibroblast suspension: Fetal bovine serum and penicillin-streptomycin were added to DMEM medium and mixed thoroughly to obtain fibroblast culture medium. Frozen fibroblasts were thawed and cultured in the medium. When the confluence of fibroblasts reached 85-90%, the medium was discarded, the cells were washed with PBS, and trypsin cell digestion solution was added. Digestion was carried out at 36.5-37.5℃ for 0.5-1.5 min. Digestion was terminated by adding culture medium, and the cells were gently pipetted to detach them. The cell suspension was transferred to centrifuge tubes and centrifuged at 900-1100 rpm for 2-4 min. The supernatant was discarded, and the cells were resuspended to a density of 0.9-1.1 × 10⁶ cells / mL. 6 / mL, to obtain a fibroblast suspension.

[0027] Preferably, the volume ratio of DMEM culture medium to fetal bovine serum is 44.5-445:5-50.

[0028] Preferably, the volume ratio of DMEM culture medium to penicillin-streptomycin is 44.5-445:0.5-5.

[0029] Preferably, the mass fraction of pancreatic enzyme in the pancreatic cell digestion solution is 0.24-0.26 wt%.

[0030] Preferably, the volume ratio of pancreatic enzyme cell digestion solution to fibroblast culture medium is 1-10:5-50.

[0031] Preferably, the volume ratio of culture medium to pancreatic enzyme cell digestion solution is 1-10:1-10.

[0032] This invention also provides a method for preparing a neutralized collagen solution, comprising: Preparation of collagen solution: At 0-4℃, type I collagen from rat tail was dissolved in 0.45-0.55 mol / L acetic acid solution to obtain collagen solution; the collagen solution was mixed with 9-11×DMEM medium, and the pH was adjusted to 7.2-7.4 with 1M NaOH to avoid the formation of bubbles. The mixture was allowed to stand for 5-15 min to obtain collagen solution.

[0033] Preferably, the mass-to-volume ratio of rat tail type I collagen to acetic acid solution is 45-450 mg: 5-50 mL.

[0034] Preferably, the volume ratio of collagen solution to 9-11×DMEM medium is 8-80:1-10.

[0035] This invention also provides a method for preparing collagen gel, comprising: Preparation of collagen gel: Mix fibroblast suspension with collagen solution and let stand at 0-4℃ for 5-10 min to obtain collagen-cell solution; add collagen-cell solution to the center of wells B1-B6 and C1-C6 in a 24-well cell culture plate, forming a uniform and regular droplet shape, let stand at room temperature for 5-10 min, and then let stand in a 36.5-37.5℃, 4.5-5.5%CO2 incubator for 20-40 min to obtain collagen gel.

[0036] Preferably, the fibroblasts in the fibroblast suspension are of 4-10 generations.

[0037] Preferably, the volume ratio of fibroblast suspension to collagen solution is 1-10:1-10.

[0038] This invention also provides a method for preparing a 3D dermal model, comprising: Preparation of 3D dermal model: DMEM complete culture medium was added to collagen gel to release the collagen gel from the bottom of the culture plate, and the gel was cultured in suspension at 36.5-37.5℃ and 4.5-5.5%CO2 for 3-5 days to obtain 3D dermal model.

[0039] Preferably, the volume of the collagen gel is measured by the volume of the collagen-cell solution therein, and the volume ratio of the collagen-cell solution to the DMEM complete culture medium is 75-750:250-2500.

[0040] This invention also provides a method for preparing a drug-treated dermal model, comprising: Preparation of drug-treated dermal models: The models were divided into blank control group, positive control group and drug-treated group according to different treatments. Different culture media were added to the 3D dermal models, mixed well, and photographed immediately using a high-content cell imaging system. After the photographs were taken, the dermal models were put back into the incubator for culture. The culture medium was changed every 1-3 days, and high-content imaging of the dermal models was performed once a day until the dermal models no longer showed significant shrinkage.

[0041] Preferably, the volume ratio of the culture medium to the 3D dermal model is 250-2500:75-750.

[0042] Preferably, the culture medium in the blank control group is DMEM complete culture medium.

[0043] Preferably, the culture medium in the positive control group is a positive culture medium, in which TGF-β1 is added to DMEM complete culture medium and diluted to obtain the positive culture medium.

[0044] Preferably, the mass-to-volume ratio of TGF-β1 to DMEM complete culture medium is 250-2500 ng: 50-500 mL.

[0045] Preferably, the culture medium in the drug treatment group is a drug culture medium, in which the drug is added to DMEM complete culture medium and diluted to obtain the drug culture medium.

[0046] Preferably, the molar volume ratio of the drug to DMEM complete culture medium is 0.5-5 µmol: 50-500 mL.

[0047] This invention also provides a method for calculating the shrinkage rate of a 3D dermal model, comprising: Calculation of shrinkage rate of 3D dermal model: The dermal model is photographed using a high-content cell imager, and the image is exported. ImageJ software is opened, the image of the dermal model is imported, the outline of the dermal model is traced, and the surface area of ​​the 3D dermal model is measured. The shrinkage rate of the model is calculated using the formula: Shrinkage rate (%) = (1-S) / (2π√ ... X / S0)×100%, where S0 is the initial surface area of ​​the 3D leather model, S X The surface area of ​​the 3D dermal model after shrinkage on day X; the shrinkage rate results of different treatment groups were analyzed for statistical significance using two-way ANOVA. Significance was expressed as *p<0.05, **p<0.01, ***p<0.001, where p≥0.05 was considered not statistically significant.

[0048] This invention also provides a method for preparing a collagen solution, comprising: Preparation of collagen solution: Dissolve the activator in deionized water and mix evenly to obtain the activator solution; dissolve rat tail type I collagen in 0.45-0.55 mol / L acetic acid solution at 2℃ to obtain the collagen solution; mix the collagen solution with 9-11×DMEM medium, add the functional agent, add the activator solution, adjust the pH to 7.2-7.4 with 1 mol / L NaOH to avoid the formation of bubbles, and let stand for 5-15 min to obtain the collagen solution.

[0049] Preferably, the activator comprises 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide.

[0050] Preferably, the mass-to-volume ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to deionized water is 1.5-15 g: 100-1000 mL.

[0051] Preferably, the mass-to-volume ratio of N-hydroxysuccinimide to deionized water is 0.3-3 g: 100-1000 mL.

[0052] Preferably, the mass-to-volume ratio of rat tail type I collagen to acetic acid solution is 45-450 mg: 5-50 mL.

[0053] Preferably, the volume ratio of collagen solution to 9-11×DMEM medium is 8-80:1-10.

[0054] Preferably, the functional agent includes 2-hydroxy-3-isopropylsuccinic acid and 4-(3-pyrrolidinyl)butyric acid.

[0055] Preferably, the mass-to-volume ratio of 2-hydroxy-3-isopropylsuccinic acid to collagen solution is 1.4-14 mg: 8-80 mL.

[0056] Preferably, the mass-to-volume ratio of 4-(3-pyrrolidinyl)butyric acid to collagen solution is 1.4-14 mg: 8-80 mL.

[0057] More preferably, the collagen solution includes N,N-dimethyl-1,4-butanediamine.

[0058] More preferably, the mass-to-volume ratio of N,N-dimethyl-1,4-butanediamine to collagen solution is 1.4-14 mg: 8-80 mL.

[0059] Preferably, the volume ratio of the activator solution to the collagen solution is 0.5-5:8-80.

[0060] This invention also provides a method for preparing collagen gel, comprising: Preparation of collagen gel: Mix fibroblast suspension with collagen solution and let stand at 0-4℃ for 5-10 min to obtain collagen-cell solution; add collagen-cell solution to the center of 12 wells B1-B6 and C1-C6 in a 24-well cell culture plate, forming a uniform and regular droplet shape, let stand at room temperature for 5-10 min, and then let stand in a 36.5-37.5℃, 4.5-5.5%CO2 incubator for 20-25 min, wash with PBS to obtain collagen gel.

[0061] Preferably, the fibroblasts in the fibroblast suspension are of 4-10 generations.

[0062] Preferably, the volume ratio of fibroblast suspension to collagen solution is 1-10:1-10.

[0063] This invention, by introducing activators and functional agents during collagen gel preparation to construct a 3D dermal model and combining it with a high-content cell imaging system to detect the model's shrinkage rate, offers the following advantages: it overcomes the large errors of traditional methods, achieves automated and precise measurement of shrinkage rate through high-content imaging, the 3D dermal model more closely resembles the in vivo microenvironment, enhances gel stability and cell activity, and improves detection efficiency and data reliability. Therefore, this invention represents a rapid, highly sensitive, and high-throughput 3D skin shrinkage combined with high-content imaging anti-aging model and its applications. Attached Figure Description

[0064] Figure 1 A schematic diagram of the process for preparing a 3D dermal model and imaging high-content cells.

[0065] Figure 2 This is a schematic diagram illustrating the different effects of the drug under test on the contraction rate of a 3D dermal model.

[0066] Figure 3 This is a schematic diagram of a 3D dermal model within a 24-well plate.

[0067] Figure 4 This is a schematic diagram of a scanning electron microscope image of collagen gel.

[0068] Figure 5 This is a schematic diagram showing the shrinkage rate of a 3D dermal model of fibroblasts of different ages.

[0069] Figure 6 This is a schematic diagram of high-content cell imaging technology used to photograph 3D dermal models after different drug treatments.

[0070] Figure 7 This is a schematic diagram showing the effects of bakuchiol and retinol on the shrinkage rate of a 3D dermal model.

[0071] Figure 8This is a schematic diagram showing the effect of three polypeptide raw materials on the shrinkage rate of a 3D dermal model. Detailed Implementation

[0072] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0073] The concepts involved in this application will first be described with reference to the accompanying drawings. It should be noted that the following descriptions of various concepts are only for the purpose of making the content of this application easier to understand and do not constitute a limitation on the scope of protection of this application; furthermore, the embodiments and features in the embodiments of this application can be combined with each other unless otherwise specified. This application will now be described in detail with reference to the accompanying drawings and embodiments.

[0074] Example 1: Preparation of fibroblast suspension: Fetal bovine serum and penicillin-streptomycin were added to DMEM medium and mixed thoroughly to obtain fibroblast culture medium. Frozen fibroblasts were thawed and cultured in the medium. When the confluence of fibroblasts reached 88%, the medium was discarded, the cells were washed with PBS, and trypsin cell digestion solution was added. Digestion was carried out at 37°C for 1 min. Digestion was terminated by adding culture medium, and the cells were detached by pipetting. The cell suspension was transferred to centrifuge tubes and centrifuged at 1000 rpm for 3 min. The supernatant was discarded, and the cells were resuspended to a density of 1 × 10⁶ cells / mL. 6 / mL, to obtain a fibroblast suspension. Fetal bovine serum was Gibco fetal bovine serum, and the volume ratio of DMEM medium to fetal bovine serum was 89:10, and the volume ratio of DMEM medium to penicillin-streptomycin was 89:1; the mass fraction of trypsin in the trypsin cell digestion solution was 0.25wt%, the volume ratio of trypsin cell digestion solution to fibroblast culture medium was 2:10, and the volume ratio of culture medium to trypsin cell digestion solution was 2:2.

[0075] Preparation of neutralized collagen solution: Type I rat tail collagen was dissolved in 0.5 mol / L acetic acid solution at 2℃ to obtain a collagen solution. The collagen solution was mixed with 10×DMEM medium, and the pH was adjusted to 7.3 with 1 mol / L NaOH, avoiding bubble formation. The mixture was allowed to stand for 10 min to obtain the final collagen solution. The mass-to-volume ratio of type I rat tail collagen to acetic acid solution was 90 mg:10 mL; the volume ratio of collagen solution to 10×DMEM medium was 16:2.

[0076] Preparation of collagen gel: Fibroblast suspension was mixed with collagen solution and incubated at 2℃ for 8 min to obtain collagen-cell solution. Collagen-cell solution was added to the center of 12 wells (B1-B6 and C1-C6) in a 24-well cell culture plate, forming a uniform, regular droplet shape. The solution was incubated at room temperature for 8 min, then incubated at 37℃ with 5% CO2 for 30 min to obtain collagen gel. The fibroblast suspension contained 4 passages of fibroblasts, and the volume ratio of fibroblast suspension to collagen solution was 2:2.

[0077] Preparation of 3D dermal models: DMEM complete medium was added to collagen gel to release it from the bottom of the culture plate. The gel was then suspended and cultured in a 37℃, 5% CO2 incubator for 4 days to obtain the 3D dermal model. The volume of the collagen gel was measured by the volume of the collagen-cell solution within it, with a volume ratio of collagen-cell solution to DMEM complete medium of 150:500.

[0078] Preparation of drug-treated dermal models: Different treatment groups were established, including a blank control group, a positive control group, and a drug-treated group. Different culture media were added to the 3D dermal models, mixed thoroughly, and immediately photographed using a high-content cell imaging system. After photography, the dermal models were returned to the incubator for further culture. The culture medium was changed every 2 days, and high-content imaging of the dermal models was performed daily until the dermal models no longer exhibited significant shrinkage. The volume ratio of culture medium to 3D dermal models was 500:150. The blank control group used DMEM complete medium; the positive control group used positive medium, with TGF-β1 added to DMEM complete medium and diluted to obtain the positive medium, with a TGF-β1 to DMEM complete medium mass-to-volume ratio of 500 ng:100 mL; the drug-treated group used drug medium, with the drug added to DMEM complete medium and diluted to obtain the drug medium, with a psoralen molar ratio of 1 µmol:100 mL.

[0079] Calculation of shrinkage rate of 3D dermal model: The dermal model is photographed using a high-content cell imager, and the image is exported. ImageJ software is opened, the image of the dermal model is imported, the outline of the dermal model is traced, and the surface area of ​​the 3D dermal model is measured. The shrinkage rate of the model is calculated using the formula: Shrinkage rate (%) = (1-S) / (2π√ ... X / S0)×100%, where S0 is the initial surface area of ​​the 3D leather model, S XThe surface area of ​​the 3D dermal model after shrinkage on day X; the shrinkage rate results of different treatment groups were analyzed for statistical significance using two-way ANOVA. Significance was expressed as *p<0.05, **p<0.01, ***p<0.001, where p≥0.05 was considered not statistically significant.

[0080] Example 2: The only difference between this example and Example 1 is that the drug used in the preparation of the drug-treated dermal model is retinol, and the molar volume ratio of retinol to DMEM complete culture medium is 1µmol:100mL.

[0081] Example 3: The only difference between this example and Example 1 is that the drug used in the preparation of the drug-treated dermal model is ZPC. ® Wrinklend013P, ZPC ® The mass-to-volume ratio of Wrinklend013P to DMEM complete culture medium was 5 mg: 100 mL, ZPC ® Wrinklend013P is from Zhejiang Paipai Biotechnology Co., Ltd.

[0082] Example 4: The only difference between this example and Example 1 is that the drug used in the preparation of the drug-treated dermal model is ZPC. ® Creasend010P, ZPC ® The mass-to-volume ratio of Creasend010P to DMEM complete culture medium was 5 mg: 100 mL, ZPC ® Creasend010P is from Zhejiang Paipai Biotechnology Co., Ltd.

[0083] Example 5: The only difference between this example and Example 1 is that the drug used in the preparation of the drug-treated dermal model is ZPC. ® Collagen005P, ZPC ® The mass-to-volume ratio of Collagen005P to DMEM complete culture medium was 5 mg: 100 mL, ZPC ® Collagen005P is from Zhejiang Paipai Biotechnology Co., Ltd.

[0084] Example 6: The only difference between this example and Example 1 is that the fibroblasts used in the preparation of the fibroblast suspension are 6-year-old fibroblasts.

[0085] Example 7: The only difference between this example and Example 1 is that the fibroblasts used in the preparation of the fibroblast suspension are 30-year-old fibroblasts.

[0086] Example 8: The only difference between this example and Example 1 is that the fibroblasts used in the preparation of the fibroblast suspension are 50-year-old fibroblasts.

[0087] Example 9: The only difference between this example and Example 1 is the preparation of the collagen solution and the preparation of the collagen gel.

[0088] Preparation of collagen solution: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were dissolved in deionized water and mixed evenly to obtain an activator solution; rat tail type I collagen was dissolved in 0.5 mol / L acetic acid solution at 2℃ to obtain a collagen solution; the collagen solution was mixed with 10×DMEM medium, 2-hydroxy-3-isopropylsuccinic acid and 4-(3-pyrrole)butyric acid were added, the activator solution was added, and the pH was adjusted to 7.3 with 1 mol / L NaOH to avoid the formation of bubbles. The solution was allowed to stand for 10 min to obtain the collagen solution. The mass-to-volume ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to deionized water was 3 g: 200 mL; the mass-to-volume ratio of N-hydroxysuccinimide to deionized water was 0.6 g: 200 mL; the mass-to-volume ratio of rat tail type I collagen to acetic acid solution was 90 mg: 10 mL; the volume ratio of collagen solution to 10×DMEM medium was 16:2; the mass-to-volume ratio of 2-hydroxy-3-isopropylsuccinic acid to collagen solution was 2.8 mg: 16 mL; the mass-to-volume ratio of 4-(3-pyrrole)butyric acid to collagen solution was 2.8 mg: 16 mL; and the volume ratio of activator solution to collagen solution was 1:16.

[0089] Preparation of collagen gel: Fibroblast suspension was mixed with collagen solution and incubated at 2℃ for 8 min to obtain collagen-cell solution. Collagen-cell solution was added to the center of 12 wells (B1-B6 and C1-C6) in a 24-well cell culture plate, forming a uniform, regular droplet shape. The solution was incubated at room temperature for 8 min, then incubated at 37℃ in a 5% CO2 incubator for 30 min. After washing with PBS, collagen gel was obtained. The fibroblast suspension contained 4 passages of fibroblasts, and the volume ratio of fibroblast suspension to collagen solution was 2:2.

[0090] Example 10: The only difference between this example and Example 9 is the preparation of the collagen solution.

[0091] Preparation of collagen solution: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were dissolved in deionized water and mixed evenly to obtain an activator solution; rat tail type I collagen was dissolved in 0.5 mol / L acetic acid solution at 2℃ to obtain a collagen solution; the collagen solution was mixed with 10×DMEM medium, 2-hydroxy-3-isopropylsuccinic acid and 4-(3-pyrrolidinyl)butyric acid were added, the activator solution was added, the pH was adjusted to 7.3 to avoid the formation of bubbles, and the mixture was allowed to stand for 10 min to obtain the collagen solution. The mass-to-volume ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to deionized water was 3 g:200 mL; the mass-to-volume ratio of N-hydroxysuccinimide to deionized water was 0.6 g:200 mL; the mass-to-volume ratio of rat tail type I collagen to acetic acid solution was 90 mg:10 mL; the volume ratio of collagen solution to 10×DMEM medium was 16:2; the mass-to-volume ratio of 2-hydroxy-3-isopropylsuccinic acid to collagen solution was 2.8 mg:16 mL; the mass-to-volume ratio of 4-(3-pyrrole)butyric acid to collagen solution was 4.2 mg:16 mL; and the volume ratio of activator solution to collagen solution was 1:16.

[0092] Example 11: The only difference between this example and Example 9 is the preparation of the collagen solution.

[0093] Preparation of collagen solution: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were dissolved in deionized water and mixed evenly to obtain an activator solution; rat tail type I collagen was dissolved in 0.5 mol / L acetic acid solution at 2℃ to obtain a collagen solution; the collagen solution was mixed with 10×DMEM medium, 2-hydroxy-3-isopropylsuccinic acid and 4-(3-pyrrole)butyric acid were added, the activator solution was added, the pH was adjusted to 7.3 to avoid the formation of bubbles, and the mixture was allowed to stand for 10 min; N,N-dimethyl-1,4-butanediamine was added, the pH was adjusted to 7.3 to avoid the formation of bubbles, and the mixture was allowed to stand for 10 min to obtain the collagen solution. The mass-to-volume ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to deionized water was 3 g:200 mL; the mass-to-volume ratio of N-hydroxysuccinimide to deionized water was 0.6 g:200 mL; the mass-to-volume ratio of rat tail type I collagen to acetic acid solution was 90 mg:10 mL; the volume ratio of collagen solution to 10×DMEM medium was 16:2; the mass-to-volume ratio of 2-hydroxy-3-isopropylsuccinic acid to collagen solution was 2.8 mg:16 mL; the mass-to-volume ratio of 4-(3-pyrrole)butyric acid to collagen solution was 2.8 mg:16 mL; the mass-to-volume ratio of N,N-dimethyl-1,4-butanediamine to collagen solution was 2.8 mg:16 mL; and the volume ratio of activator solution to collagen solution was 1:16.

[0094] Example 12: The only difference between this example and Example 9 is the preparation of the collagen solution.

[0095] Preparation of collagen solution: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were dissolved in deionized water and mixed evenly to obtain an activator solution; rat tail type I collagen was dissolved in 0.5 mol / L acetic acid solution at 2℃ to obtain a collagen solution; the collagen solution was mixed with 10×DMEM medium, 2-hydroxy-3-isopropylsuccinic acid and 4-(3-pyrrole)butyric acid were added, the activator solution was added, the pH was adjusted to 7.3 to avoid the formation of bubbles, and the mixture was allowed to stand for 10 min; N,N-dimethyl-1,4-butanediamine was added, the pH was adjusted to 7.3 to avoid the formation of bubbles, and the mixture was allowed to stand for 10 min to obtain the collagen solution. The mass-to-volume ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to deionized water was 3 g:200 mL; the mass-to-volume ratio of N-hydroxysuccinimide to deionized water was 0.6 g:200 mL; the mass-to-volume ratio of rat tail type I collagen to acetic acid solution was 90 mg:10 mL; the volume ratio of collagen solution to 10×DMEM medium was 16:2; the mass-to-volume ratio of 2-hydroxy-3-isopropylsuccinic acid to collagen solution was 2.8 mg:16 mL; the mass-to-volume ratio of 4-(3-pyrrole)butyric acid to collagen solution was 2.8 mg:16 mL; the mass-to-volume ratio of N,N-dimethyl-1,4-butanediamine to collagen solution was 4.2 mg:16 mL; and the volume ratio of activator solution to collagen solution was 1:16.

[0096] Comparative Example 1: The only difference between this comparative example and Example 9 is that 2-hydroxy-3-isopropylsuccinic acid was not used in the preparation of the collagen solution.

[0097] Comparative Example 2: This comparative example differs from Example 9 only in that 4-(3-pyrrolidinyl)butyric acid was not used in the preparation of the collagen solution.

[0098] Experimental Example 1: Microstructural characterization of collagen gel.

[0099] Test sample: Collagen gel prepared in Example 9.

[0100] Test method: After co-culturing the collagen gel for seven days, it was washed with PBS and fixed with PBS solution containing 2.5 wt% glutaraldehyde overnight at 4°C. After removing the fixative, it was washed again with PBS and dehydrated with gradient ethanol at 30%, 50%, 70%, 90% and 100% for 30 min each time. It was then air-dried for 4 h and vacuum-dried for 2 h. The surface of the gel was sputter-coated with gold and the microstructure of the cells on the collagen gel was observed using scanning electron microscopy.

[0101] Scanning electron microscope image of the collagen gel prepared by this invention is shown below. Figure 4As shown in the figure, the collagen gel was successfully obtained, in which fibroblasts adhered and proliferated on the collagen gel.

[0102] Experimental Example 2: Evaluation test of the contraction rate of dermal models of fibroblasts of three different ages.

[0103] Test sample: Shrinkage rate of the 3D dermal models obtained in Examples 6-8.

[0104] Schematic diagram of the shrinkage rate results of 3D dermal models of fibroblasts of different ages, as shown in the figure. Figure 5 As shown, there are significant differences in the contraction rate of dermal models with fibroblasts of different ages. Specifically, the contraction rate of the 6-year-old dermal model after one day of culture is significantly greater than that of the 50-year-old model. This indicates that the 6-year-old young fibroblasts can significantly promote the contraction of the dermal model compared to the 50-year-old senescent fibroblasts, meaning that the ability of senescent fibroblasts to stimulate the contraction of the dermal model is reduced. With the extension of culture time, there is no significant difference in the contraction rate among dermal models of different ages.

[0105] Experimental Example 3: Anti-aging screening test of psoralen and retinol.

[0106] Test sample: the shrinkage rate of the 3D dermal model obtained in Examples 1-2.

[0107] High-content cell imaging technology can be used to photograph 3D dermal models after different drug treatments, such as... Figure 6 As shown, the effects of bakuchiol and retinol on the shrinkage rate of the 3D dermal model are as follows: Figure 7 As shown, treatment of the dermal model with bakuchiol for 1 to 4 days significantly promoted the contraction of the dermal model compared to the blank control. The addition of retinol also promoted the contraction of the dermal model, but the stimulating effect of retinol was weaker than that of bakuchiol. The experimental results of the dermal model contraction test proved the anti-aging effects of bakuchiol and retinol.

[0108] Experimental Example 4: Anti-aging screening test of 3 different peptides.

[0109] Test sample: Shrinkage rate of the 3D dermal model obtained in Examples 3-5.

[0110] The results of the effects of three polypeptide raw materials on the shrinkage rate of the 3D dermal model are as follows: Figure 8 As shown, ZPC ® Wrinklend013P, or palmitoyl tripeptide-1, is a signal peptide that acts on the dermis, promoting the synthesis of extracellular matrix components such as collagen and glycosaminoglycans. This strengthens the dermis, making the skin thicker and firmer, reducing wrinkles, and enhancing its resistance to UV radiation, thus possessing anti-aging effects. It also utilizes peptide ZPC. ®Two and three days after treatment with Wrinklend013P, the dermal model showed significantly improved shrinkage compared to the control group, indicating that ZPC treatment significantly promoted dermal contraction. ® Wrinklend013P promotes dermal contraction by improving fibroblast senescence and enhancing their contractile ability; while the other two peptides, ZPC... ® Creasend010P and ZPC ® After three and four days of treatment with Collagen005P, the dermal model contraction was significantly inhibited compared to the blank control; the experimental results of dermal model contraction detection proved that the peptide ZPC ® The anti-aging effects of Wrinklend013P.

[0111] Experimental Example 5: Compression modulus test of collagen gel.

[0112] Test samples: Collagen gels prepared in Examples 1, 9-12, and Comparative Examples 1-2.

[0113] Test method: Collagen gel was made into cylindrical samples with a diameter of 10 mm and a thickness of 5 mm. The compressive modulus of the collagen gel was determined using a universal testing machine with a 500 N sensor and a loading speed of 1 mm / min.

[0114] The compression modulus test results of the collagen gel prepared by this invention are shown in Table 1.

[0115] Table 1. Results of compressive modulus test of collagen gel

[0116] Compared to Example 1, Examples 9-10 introduced 2-hydroxy-3-isopropylsuccinic acid and 4-(3-pyrrolidinyl)butyric acid through the action of activators, forming a chemical cross-linking network between collagen fibers, significantly improving the compressive modulus. Increasing the amount of 4-(3-pyrrolidinyl)butyric acid further improved the compressive modulus of the collagen gel, ensuring the effective expression of fibroblast contractile function and providing a more reliable 3D model basis for screening anti-aging ingredients based on contraction rate. Example 11 introduced N,N-dimethyl-1,4-butanediamine, which further improved gel rigidity by increasing cross-linking sites or improving intermolecular forces, and further increased the compressive modulus. In Example 12, the compressive modulus reached its highest value after increasing the amount of N,N-dimethyl-1,4-butanediamine. Comparative Example 1 did not use 2-hydroxy-3-isopropylsuccinic acid, and Comparative Example 2 did not use 4-(3-pyrrolidinyl)butyric acid. The cross-linking efficiency and intermolecular synergistic effect were weakened, and the compressive modulus was lower than that of Example 9, indicating that the synergistic presence of functional agents is crucial for maintaining the stability of the gel structure.

[0117] Experimental Example 6: Assay on the effect of collagen gel on promoting fibroblast proliferation.

[0118] Test samples: Collagen gels prepared in Examples 1, 9-12, and Comparative Examples 1-2.

[0119] Test method: DMEM complete medium was added to the collagen gel to release it from the bottom of the culture plate. The gel was then suspended and cultured in a 37℃, 5% CO2 incubator for 5 days. The gel was then transferred to a 24-well plate containing 50 μL of CCK-8 solution and 500 μL of DMEM complete medium and incubated for 4 h. 100 μL of supernatant was then transferred to a 96-well plate and the absorbance at 450 nm was measured using a microplate reader.

[0120] The results of the collagen gel prepared in this invention for promoting fibroblast proliferation are shown in Table 2.

[0121] Table 2 Results of the fibroblast proliferation assay using the original gel

[0122] In Example 9, 2-hydroxy-3-isopropylsuccinic acid and 4-(3-pyrrolidinyl)butyric acid enhanced cell-gel interactions, promoted fibroblast proliferation, and showed better proliferation effects than in Example 1, while also promoting fibroblast contraction. In Example 10, increasing the amount of 4-(3-pyrrolidinyl)butyric acid resulted in better cell proliferation than in Example 9. In Example 11, the introduction of N,N-dimethyl-1,4-butanediamine further improved the gel degradation rate, which was more conducive to cell metabolism, and the proliferation effect was better than in Example 9. In Example 12, increasing the amount of N,N-dimethyl-1,4-butanediamine resulted in the highest cell proliferation effect. In Comparative Examples 1-2, the absence of 2-hydroxy-3-isopropylsuccinic acid or 4-(3-pyrrolidinyl)butyric acid resulted in a weaker cell proliferation effect compared to Example 9.

[0123] The embodiments and / or implementation methods described above are merely preferred embodiments and / or implementation methods for implementing the technology of the present invention, and are not intended to limit the implementation methods of the technology of the present invention in any way. Any person skilled in the art can make some modifications or alterations to other equivalent embodiments without departing from the scope of the technical means disclosed in the content of the present invention, but they should still be regarded as the technology or embodiments that are substantially the same as the present invention.

[0124] This document uses specific examples to illustrate the principles and implementation methods of this application. The descriptions of the above embodiments are only for the purpose of helping to understand the methods and core ideas of this application. The above descriptions are only preferred embodiments of this application. It should be noted that due to the limitations of written expression, while there are objectively infinite specific structures, those skilled in the art can make several improvements, modifications, or changes without departing from the principles of this application, and can also combine the above technical features in an appropriate manner. These improvements, modifications, changes, or combinations, or the direct application of the inventive concept and technical solution to other situations without modification, should all be considered within the scope of protection of this application.

Claims

1. A three-dimensional uniform dermal assessment model, characterized by: The dermis comprises a 3D dermis model, and the evaluation model comprises image analysis, shrinkage rate calculation, and anti-aging component identification. The image analysis comprises photographing and imaging the 3D dermis model by a high-content cell imaging instrument, outlining the contour of the 3D dermis model, and measuring the surface area of the 3D dermis model. The shrinkage calculation formula is: shrinkage (%) = (1-Sx / S0) x 100%, wherein S0 is the initial surface area of the 3D dermis model, and Sx is the surface area of the 3D dermis model after shrinkage on the Xth day. X X ​​ The formula for calculating the daily average shrinkage rate is: daily average shrinkage rate (%) = shrinkage rate / X. The anti-aging component identification comprises a drug-treated dermis model, which comprises a drug-treated group, and the daily average shrinkage rate of the drug-treated group is greater than 15% after 3-4 days of culture, and the screened component is an anti-aging component.

2. A three-dimensional uniform dermal assessment model according to claim 1, characterized in that: The preparation method of the 3D dermis model comprises: mixing a fibroblast cell suspension with a neutralized collagen solution to obtain a collagen-cell solution; inoculating the collagen-cell solution into a 24-well plate at 100-200 μL / well, and standing in a 36-38 °C incubator for 20-40 minutes to obtain a collagen gel, and then culturing in suspension after adding a culture medium to obtain a 3D dermal model; the density of the fibroblasts in the fibroblast cell suspension is 0.9 x 10 6 -1.1 x 10 6 / mL, and the collagen solution comprises mouse tail type I collagen. -1.1 x 10 6 / mL, and the collagen solution comprises mouse tail type I collagen.

3. A three-dimensional uniform dermal assessment model according to claim 2, characterized in that: The passage of the fibroblasts in the fibroblast suspension is 4-10 passages, and the volume ratio of the fibroblast suspension to the collagen solution is 1-10:1-10.

4. The three-dimensional uniform dermal assessment model of claim 2, wherein: The collagen solution comprises 9-11×DMEM medium, and the mass-volume ratio of the rat tail type I collagen to the 9-11×DMEM medium is 80-800 mg:1-10 mL.

5. The three-dimensional uniform dermal assessment model of claim 2, wherein: The culture medium is DMEM complete medium, and the volume ratio of the DMEM complete medium to the collagen-cell solution is 250-2500:75-750.

6. The method for preparing a three-dimensional uniform dermal evaluation model according to claim 2, characterized in that, The suspension culture conditions of the collagen gel are as follows: culturing in a 36.5-37.5°C, 4.5-5.5% CO2 incubator for 3-5 days.

7. The three-dimensional uniform dermal assessment model of claim 1, wherein: The preparation method of the drug-treated dermis model comprises: Add the culture medium to the 3D dermis model, mix well, and immediately take a photo using a high-content cell imaging system. After the photo is taken, the dermis model is placed back in the incubator, the culture medium is replaced every 1-3 days, and high-content imaging of the dermis model is performed once a day until the dermis model no longer produces significant shrinkage. The drug-treated dermis model comprises a blank control group, a positive control group, and a drug-treated group.

8. A three-dimensional uniform dermal assessment model according to claim 7, characterized in that: The culture medium in the positive control group comprises TGF-β1 and DMEM complete medium, and the mass-volume ratio of the TGF-β1 to the DMEM complete medium is 250-2500 ng:50-500 mL.

9. A three-dimensional uniform dermal assessment model according to claim 7, wherein: The culture medium in the drug-treated group comprises a drug and DMEM complete medium, and the molar-volume ratio of the drug to the DMEM complete medium is 0.5-5 µmol:50-500 mL.

10. Use of a three-dimensional uniform dermal evaluation model according to any one of claims 1 to 9 for screening of anti-aging ingredients, characterized in that: The anti-aging component comprises a polypeptide compound or a polypeptide composition.

Images