A method for screening anti-aging ingredients based on 3D dermal and high-content imaging

Abstract

The present invention discloses a method for screening anti-aging ingredients based on 3D dermis and high-content imaging, which belongs to the technical field of biomedicine and cosmetic efficacy evaluation. After preparing a fibroblast suspension and mixing it with a neutralized collagen solution at 0-5°C, the suspension is inoculated in a 24-well plate at 100-200µL / well, and the collagen gel is formed by standing in a 37°C incubator for 20-40min. A 3D dermis model is obtained by suspension culture, and the shrinkage rate of the model is detected by a high-content cell imaging system to evaluate the anti-aging efficacy. At the same time, an activator and a functional agent are introduced into the preparation of the collagen gel, and the functional agent includes 2-hydroxy-3-isopropylsuccinic acid and 4-(3-pyrrolyl)butyric acid to optimize the mechanical properties of the collagen gel and cell compatibility. This method overcomes the defect of large errors in traditional methods, provides an efficient and accurate evaluation system for anti-aging drug screening, and has application value in cosmetic efficacy testing and skin aging mechanism research.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine and cosmetic efficacy evaluation, and specifically relates to a method for screening anti-aging ingredients based on 3D dermis and high-content imaging. Background Art

[0002] Aging is a complex process involving gradual changes in the structure and function of cells, tissues, and organs. Skin, the largest organ in the human body, is particularly susceptible to aging, manifested by significant changes in viscosity and elasticity. Fibroblasts, present in various organs and tissues, including skin, muscle, heart, lungs, and liver, actively participate in the production and arrangement of collagen fibers, which contribute significantly to the tensile strength and elasticity of the skin. Aging fibroblasts exhibit decreased cytoskeletal tension and weakened traction.

[0003] Type I collagen is the predominant collagen in most tissues of higher animals. It consists of two α1 chains and one α2 chain and has a uniform diameter in native tissue. Type I collagen is the most commonly used natural polymer component in bioengineered skin. However, due to its very high water content, collagen has weak mechanical properties and is susceptible to physical contraction when fibroblasts within the collagen matrix exert forces on the collagen. Therefore, the fibroblast-collagen gel in vitro model can be used to simulate scar contraction and visualize the effects of different modulators on fibroblast contractile function. Compared to fibroblasts grown in a monolayer, fibroblasts dispersed within the collagen matrix behave more like in vivo and proliferate more slowly. Gels attached to the pore wall exhibit a decrease in thickness without changing in diameter. When the collagen gel is mechanically released from the culture dish wall, it undergoes three-dimensional contraction, resulting in changes in thickness and diameter. This change is attributed to the random distribution of collagen fibers and the action of fibroblasts on the fibers.

[0004] The contractility of dermal fibroblasts changes during aging, and various aspects of their contractility, such as extracellular matrix remodeling, migration, and protein synthesis, show a decline in aging-related functions. NHDFs from young donors exhibit significantly greater contractility in response to TGF-β1 treatment compared to NHDFs from older donors, suggesting that changes in dermal fibroblast contractility can be used to assess cell aging.

[0005] However, traditional methods for measuring collagen gel shrinkage use digital calipers to measure the gel diameter and calculate the corresponding gel area. However, the surface area of ​​a shrinking collagen gel is not a regular circle, so calculating the surface area by measuring the gel diameter alone fails to accurately reflect the change in surface area. Furthermore, imaging collagen gels with digital cameras is subject to certain human errors, which can affect the results of collagen gel shrinkage.

[0006] A high-content cell imaging and analysis system uses automated cell imaging and analysis methods to analyze the state, changes, and overall trends of cells in each well of a multi-well plate at the single-cell level, using automated cell imaging and analysis methods while maintaining the integrity of cell structure and function. High-content imaging results are derived from the instrument's built-in image analysis, combining the advantages of intuitive visualization with batch statistical quantification. The high-content cell imaging and analysis system enables imaging and biological analysis of low-density cells and tissues in 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

[0007] The purpose of the present invention is to provide a rapid, highly sensitive and high-throughput method for screening anti-aging ingredients based on 3D dermal and high-content imaging.

[0008] The technical solutions adopted by the present invention to achieve the above-mentioned purpose are:

[0009] A method for screening anti-aging ingredients, comprising:

[0010] The fibroblast suspension is mixed with a neutralized collagen solution, seeded into a 24-well plate at 100-200 µL / well, and allowed to stand at 36-38°C for 20-40 minutes to form a collagen gel, which is then cultured in suspension to form a 3D dermal model. The collagen solution includes rat tail type I collagen;

[0011] Add the ingredients to be screened into the 3D dermal model, measure the surface area of ​​the 3D dermal model after incubation, and calculate the shrinkage rate;

[0012] The average increase in the average daily shrinkage rate on day 3-4 of culture is greater than 15%, and the screened ingredient is an anti-aging ingredient.

[0013] Preferably, the shrinkage rate calculation formula is: Shrinkage rate (%) = (1-S X / S0)×100%, where S0 is the initial surface area of ​​the 3D dermal model, S X is the surface area of ​​the 3D dermal model after contraction on day X.

[0014] Preferably, the calculation formula for the average daily shrinkage rate is: average daily shrinkage rate (%) = shrinkage rate / X.

[0015] Preferably, the density of fibroblasts in the fibroblast suspension is 0.9-1.1×10 6 / mL.

[0016] Preferably, the passage number of the fibroblasts in the fibroblast suspension is 4-10.

[0017] Preferably, the collagen solution comprises 9-11×DMEM medium.

[0018] Preferably, the mass volume ratio of rat tail type I collagen to 9-11×DMEM culture medium is 80-800 mg:1-10 mL.

[0019] Preferably, the volume ratio of the fibroblast suspension to the collagen solution is 1-10:1-10.

[0020] Preferably, the molar volume ratio of the component to be screened to the 3D dermal model is 0.5-5 µmol: 50-500 mL.

[0021] Preferably, the collagen solution includes a functional agent and an activating agent.

[0022] Preferably, the functional agent includes 2-hydroxy-3-isopropylsuccinic acid and 4-(3-pyrrolyl)butanoic acid.

[0023] Preferably, the mass ratio of 2-hydroxy-3-isopropylsuccinic acid to 4-(3-pyrrolyl)butyric acid is 1.4-14:1.4-14.

[0024] 2-Hydroxy-3-isopropylsuccinic acid and 4-(3-pyrrolyl)butyric acid enhance cell-gel interactions, improve the chemical cross-linking network between collagen fibers, increase the storage modulus of collagen gel, and optimize the mechanical properties of the gel; at the same time, the two can promote fibroblast adhesion, proliferation and metabolic activity, enhance cell compatibility, 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.

[0025] Preferably, the activating agent comprises 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide.

[0026] Preferably, the positive culture medium comprises TGF-β1 and DMEM complete medium.

[0027] Preferably, the mass volume ratio of TGF-β1 to DMEM complete culture medium is 250-2500 ng:50-500 mL.

[0028] More preferably, the functional agent also includes N,N-dimethyl-1,4-butanediamine, with the mass ratio of N,N-dimethyl-1,4-butanediamine to 4-(3-pyrrolyl)butyric acid being 1.4-14:1.4-14. N,N-dimethyl-1,4-butanediamine increases the crosslinking sites between collagen fibers, strengthening the chemical crosslinking density of the collagen gel under the action of the activator, improving the intermolecular electrostatic forces and hydrogen bond network, and further enhancing the gel's storage modulus and mechanical rigidity. It also regulates the gel's surface charge properties, optimizes the fibroblast adhesion microenvironment, promotes fibroblast proliferation, and maintains contractile function, further optimizing the biological properties of the 3D dermal model.

[0029] The present invention also provides a method for preparing a fibroblast suspension, comprising:

[0030] Preparation of fibroblast suspension: Add fetal bovine serum and penicillin-streptomycin to DMEM medium and mix well to obtain fibroblast medium. Resuscitate frozen fibroblasts and culture in the medium. When the fibroblast confluence reaches 85-90%, discard the medium, wash with PBS, add trypsin cell digestion solution, and digest at 36.5-37.5°C for 0.5-1.5 minutes. Add culture medium to terminate digestion, gently blow the cells to remove them, transfer the cell suspension to a centrifuge tube, centrifuge at 900-1100 rpm for 2-4 minutes, discard the supernatant, and resuspend the cells to a density of 0.9-1.1×10 6 / mL to obtain a fibroblast suspension.

[0031] Preferably, the volume ratio of DMEM culture medium to fetal bovine serum is 44.5-445:5-50.

[0032] Preferably, the volume ratio of DMEM culture medium to penicillin-streptomycin is 44.5-445:0.5-5.

[0033] Preferably, the mass fraction of pancreatic enzyme in the pancreatic cell digestion solution is 0.24-0.26 wt %.

[0034] Preferably, the volume ratio of trypsin cell digestion solution to fibroblast culture medium is 1-10:5-50.

[0035] Preferably, the volume ratio of the culture medium to the trypsin cell digestion solution is 1-10:1-10.

[0036] The present invention also provides a method for preparing a neutralized collagen solution, comprising:

[0037] Preparation of collagen solution: Dissolve rat tail type I collagen in 0.45-0.55 mol / L acetic acid solution at 0-4°C to obtain a collagen solution; mix the collagen solution with 9-11× DMEM medium, adjust the pH to 7.2-7.4 with 1 M NaOH to avoid the formation of bubbles, and let it stand for 5-15 minutes to obtain the collagen solution.

[0038] Preferably, the mass volume ratio of rat tail type I collagen to acetic acid solution is 45-450 mg:5-50 mL.

[0039] Preferably, the volume ratio of the collagen solution to the 9-11×DMEM medium is 8-80:1-10.

[0040] The present invention also provides a method for preparing collagen gel, comprising:

[0041] Preparation of collagen gel: Mix the fibroblast suspension with the collagen solution and let it stand at 0-4°C for 5-10 minutes to obtain a collagen-cell solution; add the collagen-cell solution to the center of wells B1-B6 and C1-C6 in a 24-well cell culture plate to form a uniform, regular droplet, let it stand at room temperature for 5-10 minutes, and let it stand in a 36.5-37.5°C, 4.5-5.5% CO2 incubator for 20-40 minutes to obtain a collagen gel.

[0042] Preferably, the fibroblasts in the fibroblast suspension are of passage 4-10.

[0043] Preferably, the volume ratio of the fibroblast suspension to the collagen solution is 1-10:1-10.

[0044] The present invention also provides a method for preparing a 3D dermal model, comprising:

[0045] Preparation of 3D dermal model: DMEM complete medium was added to the collagen gel to release the collagen gel from the bottom of the culture plate. The collagen gel was suspended and cultured in a 36.5-37.5°C, 4.5-5.5% CO2 incubator for 3-5 days to obtain a 3D dermal model.

[0046] 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 medium is 75-750:250-2500.

[0047] The present invention also provides a method for preparing a drug-treated dermal model, comprising:

[0048] 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, and immediately photographed using a high-content cell imaging system. After photography, the dermal models were returned to the incubator for culture. The culture media was replaced every 1-3 days, and high-content imaging of the dermal models was performed daily until the dermal models no longer showed significant contraction.

[0049] Preferably, the volume ratio of the culture medium to the 3D dermal model is 250-2500:75-750.

[0050] Preferably, the culture medium in the blank control group is DMEM complete medium.

[0051] Preferably, the culture medium in the positive control group is a positive culture medium, and TGF-β1 is added to a DMEM complete culture medium and diluted to obtain a positive culture medium.

[0052] Preferably, the mass volume ratio of TGF-β1 to DMEM complete culture medium is 250-2500 ng:50-500 mL.

[0053] Preferably, the culture medium in the drug treatment group is a drug culture medium, and the drug is added to a DMEM complete culture medium and diluted to obtain the drug culture medium.

[0054] Preferably, the molar volume ratio of the drug to the DMEM complete medium is 0.5-5 µmol:50-500 mL.

[0055] The present invention also provides a method for calculating the shrinkage rate of a 3D dermal model, comprising:

[0056] Calculation of shrinkage rate of 3D dermal model: Take a picture of the dermal model using a high-content cell imager and export the image of the dermal model; open Image J software, import the image of the dermal model, trace the outline of the dermal model, and measure the surface area of ​​the 3D dermal model; calculate the shrinkage rate of the model according to the formula: Shrinkage rate (%) = (1-S X / S0)×100%, where S0 is the initial surface area of ​​the 3D dermal model, S X The shrinkage rate of the 3D dermal model on day X was calculated. The shrinkage rate of the different treatment groups was analyzed for statistical significance using two-way ANOVA. The significance was indicated by *p<0.05, **p<0.01, and ***p<0.001, with p≥0.05 being considered as not statistically significant.

[0057] The present invention also provides a method for preparing a collagen solution, comprising:

[0058] Preparation of collagen solution: Dissolve the activator in deionized water and mix evenly to obtain an activator solution; dissolve rat tail type I collagen in 0.45-0.55 mol / L acetic acid solution at 2°C to obtain a collagen solution; mix the collagen solution with 9-11× DMEM culture 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 it stand for 5-15 minutes to obtain a collagen solution.

[0059] Preferably, the activating agent comprises 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide.

[0060] Preferably, the mass volume ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to deionized water is 1.5-15 g:100-1000 mL.

[0061] Preferably, the mass volume ratio of N-hydroxysuccinimide to deionized water is 0.3-3 g:100-1000 mL.

[0062] Preferably, the mass volume ratio of rat tail type I collagen to acetic acid solution is 45-450 mg:5-50 mL.

[0063] Preferably, the volume ratio of the collagen solution to the 9-11×DMEM medium is 8-80:1-10.

[0064] Preferably, the functional agent includes 2-hydroxy-3-isopropylsuccinic acid and 4-(3-pyrrolyl)butanoic acid.

[0065] Preferably, the mass volume ratio of 2-hydroxy-3-isopropylsuccinic acid to collagen solution is 1.4-14 mg:8-80 mL.

[0066] Preferably, the mass volume ratio of 4-(3-pyrrolyl)butyric acid to collagen solution is 1.4-14 mg:8-80 mL.

[0067] More preferably, the collagen solution includes N,N-dimethyl-1,4-butanediamine.

[0068] More preferably, the mass volume ratio of N,N-dimethyl-1,4-butanediamine to collagen solution is 1.4-14 mg:8-80 mL.

[0069] Preferably, the volume ratio of the activator solution to the collagen solution is 0.5-5:8-80.

[0070] The present invention also provides a method for preparing collagen gel, comprising:

[0071] Preparation of collagen gel: Mix the fibroblast suspension with the collagen solution and let it stand at 0-4°C for 5-10 minutes to obtain a collagen-cell solution; add the collagen-cell solution to the center of 12 wells B1-B6 and C1-C6 in a 24-well cell culture plate to form a uniform, regular droplet, let it stand at room temperature for 5-10 minutes, let it stand in a 36.5-37.5°C, 4.5-5.5% CO2 incubator for 20-25 minutes, add PBS for washing, and obtain the collagen gel.

[0072] Preferably, the fibroblasts in the fibroblast suspension are of passage 4-10.

[0073] Preferably, the volume ratio of the fibroblast suspension to the collagen solution is 1-10:1-10.

[0074] This invention utilizes activators and functional agents during collagen gel preparation to construct a 3D dermal model, and combines this with a high-content cell imaging system to detect shrinkage. This method overcomes the significant errors associated with traditional methods, enabling automated and precise shrinkage measurement through high-content imaging. The 3D dermal model more closely resembles the in vivo microenvironment, enhancing gel stability and cell activity, and improving detection efficiency and data reliability. Therefore, this invention provides a rapid, highly sensitive, and high-throughput 3D skin contraction combined with high-content imaging anti-aging model and its applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] Figure 1 Schematic diagram of the process for 3D dermal model preparation and high-content cell imaging.

[0076] Figure 2 Schematic diagram showing the different effects of the tested drugs on the shrinkage rate of the 3D dermal model.

[0077] Figure 3 Schematic diagram of the 3D dermal model in a 24-well plate.

[0078] Figure 4 Schematic diagram of the scanning electron microscopy image of collagen gel.

[0079] Figure 5 Schematic diagram of the contraction rate results of the 3D dermal model of fibroblasts of different ages.

[0080] Figure 6 Schematic diagram of high-content cell imaging technology used to photograph 3D dermal models after treatment with different drugs.

[0081] Figure 7 Schematic diagram of the effects of bakuchiol and retinol on the shrinkage rate of the 3D dermal model.

[0082] Figure 8Schematic diagram of the effects of three polypeptide raw materials on the shrinkage rate of the 3D dermal model. DETAILED DESCRIPTION

[0083] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0084] The following first describes the concepts involved in this application with reference to the accompanying drawings. It should be noted that the following description of each concept is intended only to make the content of this application easier to understand and does not limit the scope of protection of this application. At the same time, the embodiments and features in the embodiments of this application can be combined with each other unless there is a conflict. The following detailed description of this application will be made with reference to the accompanying drawings and in conjunction with the embodiments.

[0085] Example 1:

[0086] Preparation of fibroblast suspension: Fetal bovine serum and penicillin-streptomycin were added to DMEM medium and mixed well to obtain fibroblast culture medium. Frozen fibroblasts were revived and cultured in the culture medium. When the confluence of fibroblasts reached 88%, the culture medium was discarded, the cells were washed with PBS, and trypsin cell digestion solution was added. The cells were digested at 37°C for 1 min. The culture medium was added to terminate the digestion, and the cells were blown to detach. The cell suspension was transferred to a centrifuge tube and centrifuged at 1000 rpm for 3 min. The supernatant was discarded and the cells were resuspended to a density of 1×10 6 / mL to obtain a fibroblast suspension. The fetal bovine serum was Gibco fetal bovine serum, 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.

[0087] Preparation of neutralized collagen solution: Dissolve rat tail type I collagen in 0.5 mol / L acetic acid at 2°C to obtain a collagen solution. Mix the collagen solution with 10× DMEM medium, adjust the pH to 7.3 with 1 mol / L NaOH to avoid air bubbles, and let it stand for 10 minutes to obtain the collagen solution. The mass-to-volume ratio of rat tail type I collagen to acetic acid solution is 90 mg:10 mL; the volume ratio of collagen solution to 10× DMEM medium is 16:2.

[0088] Preparation of collagen gel: Fibroblast suspension and collagen solution were mixed and allowed to stand at 2°C for 8 minutes to obtain a collagen-cell solution. The collagen-cell solution was then added to the center of 12 wells (B1-B6 and C1-C6) of a 24-well cell culture plate, forming a uniform, regular droplet. The solution was allowed to stand at room temperature for 8 minutes and then incubated in a 37°C, 5% CO2 incubator for 30 minutes to obtain a collagen gel. The fibroblasts in the fibroblast suspension were of passage 4, and the volume ratio of fibroblast suspension to collagen solution was 2:2.

[0089] Preparation of the 3D dermal model: DMEM complete medium was added to the collagen gel to release the collagen gel from the bottom of the culture plate. The gel was suspended and cultured in a 37°C, 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. The volume ratio of the collagen-cell solution to DMEM complete medium was 150:500.

[0090] Preparation of drug-treated dermal models: Different culture media were added to the 3D dermal models, mixed thoroughly, and immediately imaged using a high-content cell imaging system. After imaging, the dermal models were returned to the incubator for culture. The culture media were changed every two days, and high-content imaging of the dermal models was performed daily until the dermal models no longer exhibited significant contraction. The volume ratio of culture media to 3D dermal models was 500:150. The culture medium in the blank control group consisted of complete DMEM medium. The culture medium in the positive control group consisted of positive culture medium diluted with TGF-β1 to complete DMEM medium at a mass volume ratio of 500 ng:100 mL. The culture medium in the drug-treated groups consisted of drug medium diluted with bakuchiol to complete DMEM medium at a molar volume ratio of 1 µmol:100 mL.

[0091] Calculation of shrinkage rate of 3D dermal model: Take a picture of the dermal model using a high-content cell imager and export the image of the dermal model; open Image J software, import the image of the dermal model, trace the outline of the dermal model, and measure the surface area of ​​the 3D dermal model; calculate the shrinkage rate of the model according to the formula: Shrinkage rate (%) = (1-S X / S0)×100%, where S0 is the initial surface area of ​​the 3D dermal model, S XThe shrinkage rate of the 3D dermal model on day X was calculated. The shrinkage rate of the different treatment groups was analyzed for statistical significance using two-way ANOVA. The significance was indicated by *p<0.05, **p<0.01, and ***p<0.001, with p≥0.05 being considered as not statistically significant.

[0092] Example 2: This example differs from Example 1 only in 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 medium is 1 µmol:100 mL.

[0093] Example 3: This example is different from Example 1 only in that the drug used in the preparation of the drug-treated dermal model is ZPC ® Wrinklend013P, ZPC ® The mass volume ratio of Wrinklend013P to DMEM complete medium is 5 mg:100 mL, ZPC ® Wrinklend013P was obtained from Zhejiang Paipeptide Biotechnology Co., Ltd.

[0094] Example 4: This example is different from Example 1 in that the drug used in the preparation of the drug-treated dermal model is ZPC ® Creasend010P, ZPC ® The mass volume ratio of Creasend010P to DMEM complete medium is 5 mg:100 mL, ZPC ® Creasend010P was obtained from Zhejiang Paipeptide Biotechnology Co., Ltd.

[0095] Example 5: This example is different from Example 1 in that the drug used in the preparation of the drug-treated dermal model is ZPC ® Collagen005P, ZPC ® The mass volume ratio of Collagen005P to DMEM complete medium is 5 mg:100 mL, ZPC ® Collagen005P was obtained from Zhejiang Paipeptide Biotechnology Co., Ltd.

[0096] Example 6: This example is different from Example 1 only in that the fibroblast suspension is prepared in which the fibroblasts are 6-year-old fibroblasts.

[0097] Example 7: Compared with Example 1, the only difference between this example and Example 1 is that in the preparation of the fibroblast suspension, the fibroblasts are 30-year-old fibroblasts.

[0098] Example 8: This example is different from Example 1 only in that the fibroblast suspension is prepared in which the fibroblasts are 50-year-old fibroblasts.

[0099] Example 9: Compared with Example 1, the only difference between this example is the preparation of the collagen solution and the preparation of the collagen gel.

[0100] 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°C to obtain a collagen solution; the collagen solution was mixed with 10×DMEM medium, 2-hydroxy-3-isopropylsuccinic acid and 4-(3-pyrrolyl)butyric acid were added, and the activator solution was added. The pH was adjusted to 7.3 with 1 mol / L NaOH to avoid the formation of bubbles, and the mixture was allowed to stand for 10 minutes to obtain a collagen solution. The mass volume ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to deionized water is 3 g:200 mL, and the mass volume ratio of N-hydroxysuccinimide to deionized water is 0.6 g:200 mL; the mass volume ratio of rat tail type I collagen to acetic acid solution is 90 mg:10 mL; the volume ratio of collagen solution to 10×DMEM culture medium is 16:2, the mass volume ratio of 2-hydroxy-3-isopropylsuccinic acid to collagen solution is 2.8 mg:16 mL, the mass volume ratio of 4-(3-pyrrolyl)butyric acid to collagen solution is 2.8 mg:16 mL, and the volume ratio of activator solution to collagen solution is 1:16.

[0101] Preparation of collagen gel: Fibroblast suspension and collagen solution were mixed and allowed to stand at 2°C for 8 minutes to obtain a collagen-cell solution. The collagen-cell solution was then added to the center of 12 wells (B1-B6 and C1-C6) of a 24-well cell culture plate, forming a uniform, regular droplet. The solution was allowed to stand at room temperature for 8 minutes, then incubated in a 37°C, 5% CO2 incubator for 30 minutes. The solution was then washed with PBS to obtain a collagen gel. The fibroblasts in the fibroblast suspension were of passage 4, and the volume ratio of fibroblast suspension to collagen solution was 2:2.

[0102] Example 10: Compared with Example 9, this example is different only in the preparation of the collagen solution.

[0103] 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°C to obtain a collagen solution; the collagen solution was mixed with 10×DMEM medium, 2-hydroxy-3-isopropylsuccinic acid and 4-(3-pyrrolyl)butyric acid were added, and 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 minutes to obtain a collagen solution. The mass volume ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to deionized water is 3 g:200 mL, and the mass volume ratio of N-hydroxysuccinimide to deionized water is 0.6 g:200 mL; the mass volume ratio of rat tail type I collagen to acetic acid solution is 90 mg:10 mL; the volume ratio of collagen solution to 10×DMEM culture medium is 16:2, the mass volume ratio of 2-hydroxy-3-isopropylsuccinic acid to collagen solution is 2.8 mg:16 mL, the mass volume ratio of 4-(3-pyrrolyl)butyric acid to collagen solution is 4.2 mg:16 mL, and the volume ratio of activator solution to collagen solution is 1:16.

[0104] Example 11: Compared with Example 9, the only difference between this example is the preparation of the collagen solution.

[0105] 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°C to obtain a collagen solution; the collagen solution was mixed with 10×DMEM culture medium, 2-hydroxy-3-isopropylsuccinic acid and 4-(3-pyrrolyl)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 minutes; 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 minutes to obtain a collagen solution. The mass volume ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to deionized water is 3 g:200 mL, the mass volume ratio of N-hydroxysuccinimide to deionized water is 0.6 g:200 mL; the mass volume ratio of rat tail type I collagen to acetic acid solution is 90 mg:10 mL; the volume ratio of collagen solution to 10×DMEM medium is 16:2, the mass volume ratio of 2-hydroxy-3-isopropylsuccinic acid to collagen solution is 2.8 mg:16 mL, the mass volume ratio of 4-(3-pyrrolyl)butyric acid to collagen solution is 2.8 mg:16 mL, the mass volume ratio of N,N-dimethyl 1,4-butanediamine to collagen solution is 2.8 mg:16 mL, and the volume ratio of activator solution to collagen solution is 1:16.

[0106] Example 12: Compared with Example 9, the only difference between this example is the preparation of the collagen solution.

[0107] 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°C to obtain a collagen solution; the collagen solution was mixed with 10×DMEM culture medium, 2-hydroxy-3-isopropylsuccinic acid and 4-(3-pyrrolyl)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 minutes; 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 minutes to obtain a collagen solution. The mass volume ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to deionized water is 3 g:200 mL, the mass volume ratio of N-hydroxysuccinimide to deionized water is 0.6 g:200 mL; the mass volume ratio of rat tail type I collagen to acetic acid solution is 90 mg:10 mL; the volume ratio of collagen solution to 10×DMEM medium is 16:2, the mass volume ratio of 2-hydroxy-3-isopropylsuccinic acid to collagen solution is 2.8 mg:16 mL, the mass volume ratio of 4-(3-pyrrolyl)butyric acid to collagen solution is 2.8 mg:16 mL, the mass volume ratio of N,N-dimethyl 1,4-butanediamine to collagen solution is 4.2 mg:16 mL, and the volume ratio of activator solution to collagen solution is 1:16.

[0108] Comparative Example 1: This comparative example is different from Example 9 only in that 2-hydroxy-3-isopropylsuccinic acid is not used in the preparation of the collagen solution.

[0109] Comparative Example 2: This comparative example is different from Example 9 only in that 4-(3-pyrrolyl)butyric acid is not used in the preparation of the collagen solution.

[0110] Experimental Example 1: Microstructural characterization of collagen gel.

[0111] Test sample: collagen gel prepared in Example 9.

[0112] Test method: After the collagen gel was co-cultured for seven days, it was washed with PBS and fixed with a PBS solution containing 2.5wt% glutaraldehyde at 4°C overnight. After the fixative was aspirated, it was washed again with PBS and dehydrated with gradient ethanol at a gradient of 30%, 50%, 70%, 90% and 100%, each for 30 minutes. It was then naturally dried for 4 hours and vacuum dried for 2 hours. The surface of the gel was gold-sprayed and the microscopic appearance of the cells on the collagen gel was observed using a scanning electron microscope.

[0113] The scanning electron microscope image of the collagen gel prepared by the present invention is as follows: Figure 4As shown, it was shown that the collagen gel as shown in the figure was successfully obtained, wherein fibroblasts adhered and proliferated on the collagen gel.

[0114] Experimental Example 2: Evaluation test of the shrinkage rate of a dermal model using fibroblasts of three different ages.

[0115] Test sample: Shrinkage rate of the 3D leather model obtained in Examples 6-8.

[0116] Schematic diagram of the contraction rate of the 3D dermal model of fibroblasts of different ages Figure 5 As shown, there are significant differences in the contraction rates of dermal models with fibroblasts of different ages. That is, 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 fibroblasts, indicating that the 6-year-old fibroblasts can significantly promote the contraction of the dermal model compared with the 50-year-old fibroblasts, that is, the ability of the aged fibroblasts to stimulate the contraction of the dermal model decreases; with the extension of the culture time, there is no significant difference in the contraction rate between the dermal models of different ages.

[0117] Test Example 3: Anti-aging screening test of bakuchiol and retinol.

[0118] Test sample: Shrinkage rate of the 3D leather model obtained in Example 1-2.

[0119] High-content cell imaging technology was used to take pictures of 3D dermal models after treatment with different drugs. Figure 6 The results of the effects of bakuchiol and retinol on the shrinkage rate of the 3D dermal model are shown in Figure 7 As shown, adding bakuchiol to treat the dermis model for 1 to 4 days can significantly promote the contraction of the dermis model compared with the blank control; adding retinol can also promote the contraction of the dermis model, but the stimulating effect of retinol is weaker than that of bakuchiol; the experimental results of dermis model contraction detection prove the anti-aging effect of bakuchiol and retinol.

[0120] Experimental Example 4: Anti-aging screening test of 3 different peptides.

[0121] Test sample: Shrinkage rate of the 3D leather model obtained in Examples 3-5.

[0122] The results of the effects of three kinds of peptide raw materials on the shrinkage rate of 3D dermal models are as follows Figure 8 As shown, ZPC ® Wrinklend013P, or palmitoyl tripeptide-1, is a signal peptide that acts on the dermis and promotes the synthesis of extracellular matrix such as collagen and glycosaminoglycans, strengthening the dermis, making the skin thicker and firmer, soothing wrinkles, and enhancing its ability to resist ultraviolet radiation, thus having anti-aging effects. ®After two and three days of treatment, Wrinklend013P significantly promoted the contraction of the dermis model compared with the blank control, indicating that ZPC ® Wrinklend013P improves the aging of fibroblasts and enhances their contraction ability, thereby promoting the contraction of the dermal model; while the other two peptides ZPC ® Creasend010P and ZPC ® After three and four days of treatment, Collagen005P significantly inhibited the contraction of the dermis model compared with the blank control; the experimental results of the dermis model contraction test proved that the peptide ZPC ® The anti-aging effects of Wrinklend013P.

[0123] Test Example 5: Compression modulus test of collagen gel.

[0124] Test samples: collagen gels prepared in Example 1, Examples 9-12, and Comparative Examples 1-2.

[0125] Test method: Collagen gel was made into a cylindrical sample with a diameter of 10 mm and a thickness of 5 mm. The compression modulus of the collagen gel was measured using a universal material testing machine with a 500N sensor and a loading speed of 1 mm / min.

[0126] The compression modulus test results of the collagen gel prepared in the present invention are shown in Table 1.

[0127] Table 1 Compression modulus test results of collagen gel

[0128]

[0129] Compared with Example 1, in Examples 9-10, 2-hydroxy-3-isopropylsuccinic acid and 4-(3-pyrrolyl)butyric acid were introduced through the action of an activator, and a chemical cross-linked network was formed between the collagen fibers, and the compression modulus was significantly improved. Increasing the amount of 4-(3-pyrrolyl)butyric acid was beneficial to increasing the compression modulus of the collagen gel, ensuring the effective expression of the contractile function of fibroblasts, and providing a more reliable 3D model basis for the screening of anti-aging ingredients based on shrinkage rate; Example 11 introduced N,N-dimethyl-1,4-butanediamine, and further improved the gel rigidity by increasing the cross-linking sites or improving the intermolecular force, and the compression modulus was further improved; After increasing the amount of N,N-dimethyl-1,4-butanediamine in Example 12, the compression modulus reached the highest; Comparative Example 1 did not use 2-hydroxy-3-isopropylsuccinic acid, and Comparative Example 2 did not use 4-(3-pyrrolyl)butyric acid. The cross-linking efficiency and intermolecular synergy were weakened, and the compression modulus was lower than that of Example 9, indicating that the synergistic presence of functional agents is crucial to maintaining the stability of the gel structure.

[0130] Test Example 6: Fibroblast proliferation test using collagen gel.

[0131] Test samples: collagen gels prepared in Example 1, Examples 9-12, and Comparative Examples 1-2.

[0132] Test method: Add DMEM complete medium to the collagen gel to release the collagen gel from the bottom of the culture plate, culture it in suspension, and culture it in a 37°C, 5% CO2 incubator for 5 days. Move it to a 24-well plate containing 50 μL of CCK-8 solution and 500 μL of DMEM complete medium and incubate it for 4 hours. Pipette 100 μL of supernatant into a 96-well plate and measure the absorbance at 450 nm using a microplate reader.

[0133] The test results of the collagen gel prepared by the present invention on promoting fibroblast proliferation are shown in Table 2.

[0134] Table 2 Results of the fibroblast proliferation test on the original gel

[0135]

[0136] In Example 9, 2-hydroxy-3-isopropylsuccinic acid and 4-(3-pyrrolyl)butyric acid can enhance cell-gel interaction and promote fibroblast proliferation. The proliferation effect is better than that of Example 1, and the contraction of fibroblasts is promoted. In Example 10, after increasing the dosage of 4-(3-pyrrolyl)butyric acid, the cell proliferation effect is better than that of Example 9. In Example 11, N,N-dimethyl-1,4-butanediamine is introduced to further improve the gel degradation rate, which is more conducive to cell metabolism, and the proliferation effect is better than that of Example 9. In Example 12, after increasing the dosage of N,N-dimethyl-1,4-butanediamine, the cell proliferation effect reaches the highest. Comparative Example 1-2 lacks 2-hydroxy-3-isopropylsuccinic acid or 4-(3-pyrrolyl)butyric acid, and the cell proliferation effect is weakened compared with Example 9.

[0137] The embodiments and / or implementation methods described above are only used to illustrate the preferred embodiments and / or implementation methods for realizing the technology of the present invention, and do not impose any form of limitation on the implementation methods of the technology of the present invention. Any person skilled in the art may make slight changes or modifications 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 technologies or embodiments that are essentially the same as the present invention.

[0138] This article uses specific examples to illustrate the principles and implementation methods of this application. The description of the above embodiments is only used to help understand the method and core ideas of this application. The above is only the preferred implementation method of this application. It should be pointed out that due to the limitations of textual expression, there are objectively infinite specific structures. For ordinary technicians in this technical field, without departing from the principles of this application, they can also make several improvements, modifications or changes, 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 occasions without improvement, should be regarded as the scope of protection of this application.

Claims

1. A method for screening anti-aging ingredients, comprising: The fibroblast suspension was mixed with the neutralized collagen solution, inoculated into a 24-well plate at 100-200 μL / well, and placed in a 36-38°C incubator for 20-40 minutes to obtain a collagen gel, which was then suspended and cultured to obtain a 3D dermal model. The density of the fibroblasts in the fibroblast suspension was 0.9-1.1×10 6 / mL, the collagen solution includes rat tail type I collagen; Add the ingredients to be screened into the 3D dermal model, measure the surface area of ​​the 3D dermal model after incubation, and calculate the shrinkage rate; If the average daily shrinkage rate on day 3-4 of culture is greater than 15%, the ingredient to be screened is an effective anti-aging ingredient; The shrinkage calculation formula is: Shrinkage (%) = (1-S X / S0)×100%, where S0 is the initial surface area of ​​the 3D dermal model, S X is the surface area of ​​the 3D dermal model after contraction on day X; The collagen solution comprises a functional agent and an activator, wherein the functional agent comprises 2-hydroxy-3-isopropylsuccinic acid and 4-(3-pyrrolyl)butyric acid, and the activator comprises 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide.

2. The method for screening anti-aging ingredients according to claim 1, characterized in that: The passage number of the fibroblasts in the fibroblast suspension is 4-10.

3. The method for screening anti-aging ingredients according to claim 1, characterized in that: The collagen solution includes 9-11×DMEM culture medium, and the mass volume ratio of the rat tail type I collagen to the 9-11×DMEM culture medium is 80-800 mg:1-10 mL.

4. The method for screening anti-aging ingredients according to claim 1, characterized in that: The volume ratio of the fibroblast suspension to the collagen solution is 1-10:1-10.

5. The method for screening anti-aging ingredients according to claim 1, characterized in that: The molar volume ratio of the component to be screened to the 3D dermal model is 0.5-5µmol:50-500mL.

6. The method for screening anti-aging ingredients according to claim 1, characterized in that: The mass ratio of the 2-hydroxy-3-isopropylsuccinic acid to the 4-(3-pyrrolyl)butyric acid is 1.4-14:1.4-14.

7. The method for screening anti-aging ingredients according to claim 1, characterized in that: The mass ratio of the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide is 1.5-15:0.3-3.

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