Cosmetics in vitro collagen synthesis promotion test

Cosmetics in vitro collagen synthesis promotion test

Detection principle and technical path

Collagen is the core component that maintains skin elasticity, and its synthesis ability directly affects skin wrinkle resistance and repair function. The in vitro collagen synthesis promotion test of cosmetics is conducted by establishing a fibroblast culture model, simulating the physiological environment of the dermis layer of the skin, and evaluating the stimulating effect of the test substance on collagen biosynthesis. This detection system takes human dermal fibroblasts (HDF) or immortalized fibroblast cell lines (such as NHDF) as the research objects. Under standardized culture conditions (37 ℃, 5% CO ₂, 95% humidity), after intervention with the test substance, the changes in collagen synthesis ability are verified at both the protein and gene levels.

The experimental design adopts concentration gradient method (usually with three dose groups of 0.01%, 0.1%, and 1%), and simultaneously sets blank control (serum-free culture medium) and positive control (vitamin C 100 μ mol/L) to ensure comparability and reliability of the results. The key technical principle is based on the activation of the TGF - β/Smad signaling pathway by fibroblasts under the stimulation of active ingredients, upregulating the expression of COL1A1 gene and promoting the secretion of type I collagen. Cell toxicity validation (MTT method) should be performed simultaneously for the detection, requiring the cell survival rate of the test substance to be>80% at the effective concentration, and excluding interference from cell proliferation or toxicity on the detection results.

Core testing methods and standardized processes

Protein level quantification: ELISA method for measuring type I collagen content

Enzyme linked immunosorbent assay (ELISA) was used to specifically detect the concentration of type I collagen protein (COL1) in cell culture supernatant, and the operating procedure strictly followed the technical specifications of GB/T 35828-2018 "Guidelines for Testing the Efficacy of 3D Skin Models in vitro for Cosmetics". The specific steps include:

Sample pretreatment: Collect the supernatant of cells intervened for 72 hours, centrifuge at 12000rpm at 4 ℃ for 10 minutes to remove impurities;

Antibody coating: 96 well plate coated with mouse anti human COL1 monoclonal antibody (diluted 1:1000), incubated overnight at 4 ℃;

Antigen binding: Add gradient diluted samples and standards (0-200ng/mL), incubate at 37 ℃ for 2 hours;

Color reaction: Add HRP labeled secondary antibody (diluted 1:2000) and TMB substrate in sequence, terminate the reaction, and measure the absorbance at 450nm wavelength;

Data calculation: Calculate the relative synthesis rate between the experimental group and the control group by converting the COL1 concentration through the standard curve. The positive result group is required to have a COL1 content increase of ≥ 20% (p<0.05) compared to the blank control group.

Gene level validation: Real time PCR detection of COL1A1 expression

To reveal the molecular mechanism of collagen synthesis, real-time fluorescence quantitative PCR (qPCR) is required to detect changes in the transcription level of the COL1A1 gene. The experimental procedure is as follows:

Total RNA extraction: Trizol method was used to lyse cells, and Nanodrop was used to determine RNA concentration (A260/A280 ratio 1.8-2.0);

Reverse transcription reaction: cDNA was synthesized using PrimeScript RT kit under reaction conditions of 37 ℃ for 15 minutes and 85 ℃ for 5 seconds;

QPCR amplification: GAPDH was used as the internal reference gene, and the COL1A1 primer sequence was F: 5 '- GAGGCCAGACGAACATC-3', R: 5 '- CAGATTACGTCATCGCACAAC-3', amplified in the LightCycler 480 system (pre denaturing at 95 ° C for 10 minutes, 40 cycles: 15 seconds at 95 ° C, 30 seconds at 60 ° C);

Result analysis: The relative expression level of COL1A1 was calculated using the 2 ^ (- Δ Δ Ct) method. Positive test substances need to upregulate gene expression by ≥ 1.5 times and be consistent with the trend of protein level changes.

Quality control and result judgment standards

Quality control of experimental system

Cell quality assurance: Use logarithmic growth stage cells from the 3rd to the 10th generation, with a seeding density controlled at 5 × 10 ⁴ cells/cm ², to ensure a cell adhesion rate of>90%;

Standardization of reagents: Fetal bovine serum needs to be tested for mycoplasma, with strict control of pancreatic amylase concentration at 0.25% and EDTA addition at 0.02%;

Instrument accuracy requirements: The temperature fluctuation of the CO ₂ incubator should be ≤ ± 0.1 ℃, and the pH value should be maintained at 7.2-7.4. The ultra clean workbench should achieve ISO level 5 cleanliness (suspended particles ≤ 3520/m ³);

Methodology validation: The within batch precision RSD of ELISA method is less than 8%, the inter batch precision RSD is less than 12%, and the recovery rate is 90% -110%; QPCR amplification efficiency is 90% -110%, with a correlation coefficient R ²>0.99

Result judgment threshold

Basic judgment criteria: The test substance can be judged as positive if it meets any of the following conditions at non cytotoxic concentrations (survival rate>80%):

The content of type I collagen increased by ≥ 20% compared to the blank control (ELISA method, p<0.05);

COL1A1 gene expression was upregulated by ≥ 1.5 times (qPCR method, p<0.05)。

Enhanced validation requirements: For products claiming to significantly promote collagen synthesis, they must simultaneously meet the requirements of a protein level increase of ≥ 30% and a gene level upregulation of ≥ 2-fold, and provide at least 3 independent experimental replicates.

Technical advantages and compliance assurance of testing institutions

Hardware facilities and technical capabilities

The Zhongke Testing Center is equipped with a three-level biosafety laboratory, with core equipment including:

Fully automated cell culture system (Thermo Scientific Heracell VIOS): realizes real-time monitoring and alarm of temperature and CO ₂ concentration;

High throughput ELISA workstation (BioTek 800TS): supports simultaneous detection of 96 samples, with a detection limit as low as 0.1 pg/mL;

Fluorescence quantitative PCR instrument (Roche LightCycler 96): equipped with a rapid temperature rise and fall module, a single experiment can be completed within 2 hours;

Cell imaging system (Olympus IX73): combined with ImageJ software to achieve quantitative analysis of collagen immunofluorescence intensity.

Qualification and data credibility of Quan Wei

The laboratory has passed CNAS accreditation (certificate number CNAS L22006) and CMA qualification certification, and the testing process strictly follows:

National standard: GB/T 35828-2018 "Guidelines for in vitro skin model testing of cosmetics";

International standards: OECD TG 439 "In vitro skin irritation test", ISO 10993-5 "Cytotoxicity test";

Industry Guide: China Cosmetics Evaluation Center's "Guidelines for Evaluation of Cosmetic Efficacy Claims" (2021 Edition).

Customized testing services

Provide full chain technical support for different types of cosmetics:

Raw material screening: Pre screening the in vitro activity of functional ingredients such as plant extracts and peptides to shorten the research and development cycle;

Formula optimization: evaluate the impact of preservatives and essence on collagen synthesis activity, and optimize the compounding scheme;

Stability verification: Store at 4 ℃, 25 ℃, and 40 ℃ for 28 days and monitor the impact of active ingredient degradation on efficacy;

Value added data services: providing in-depth data interpretation such as COL1A1 gene pathway analysis and active ingredient IC50/EC50 calculation.

Industry Applications and Typical Cases

The validation of an anti-aging essence solution by this detection system showed that after 48 hours of intervention in the 1% concentration group, the secretion of fibroblast COL1 increased by 42.3% (p<0.01) compared with the blank control, the expression of COL1A1 mRNA increased by 2.1 times, and the cell survival rate reached 91.7%. This data successfully supports its claim of promoting collagen synthesis, and the relevant testing report has been recognized by the National Medical Products Administration as the basis for filing. Practice has shown that standardized in vitro collagen synthesis testing can effectively replace traditional animal experiments, while ensuring detection accuracy, significantly reducing research and development costs and ethical disputes, and providing scientific and reliable technical support for cosmetic efficacy evaluation.