A 3D cuticle-like model and a construction method and application thereof

Abstract

The application discloses a kind of 3D cutin layer model construction method and its application, the 3D cutin layer model is constituted by top surface, middle layer and bottom surface, top surface and bottom surface surface are uneven, are constituted by apoptosis cell and / or cell derivative that are closely connected with photocurable gel, middle layer is photocurable gel, wherein photocurable gel is as the skeleton for apoptosis cell and / or cell derivative provides pore structure.The construction method is to load apoptosis keratinocyte and / or cell derivative in the condition of light irradiation with photocurable gel material and form 3D cutin layer model.The 3D cutin layer model constructed by the application can be effectively applied to in vitro evaluation of the efficacy of cosmetic regulation microecology, test the efficacy of antibiotic biological preparation such as bacteriostatic, with the advantages of low cost, fast construction speed, high stability and high-throughput screening of active substances.

Description

Technical Field

[0001] This invention relates to a cuticle-like model, its construction method, and its application, and more particularly to a 3D cuticle-like model that can be colonized by various microorganisms, its construction method, and its application. Background Technology

[0002] In the development of functional skincare products that regulate the gut microbiota, it is necessary to evaluate the efficacy of active ingredients or cosmetics in regulating the gut microbiota. Previous experiments used sterile mouse models for efficacy evaluation. However, with the introduction and implementation of the 3R principle in animal testing, establishing suitable in vitro models to replace animal models for evaluating the application of cosmetic ingredients or products in regulating the gut microbiota has become essential and has significant applicability.

[0003] As the largest organ in the human body, the skin is home to a variety of microorganisms. Beneficial microorganisms act as a physical barrier against pathogens, protecting the body from external harm. Based on the different skin environments and physicochemical properties of different parts of the body, skin physiological states are divided into three categories: moist, oily, and dry. Even though the skin may lack nutrients, be acidic, and dry, it still has 10... 6 bacteria / cm 2 Most bacteria live on the surface of the stratum corneum, feeding on cross-linked proteins and lipids from keratinocytes.

[0004] When using in vitro models to study the microbiome, the impact of transitioning from the skin environment to an in vitro model on microbial growth and interactions must be considered. Currently, in vitro microbiome studies primarily utilize 3D epidermal models, constructed by differentiating human primary keratinocytes into human epidermal equivalents. In the cosmetics industry, this is mainly used for cosmetic safety evaluation. With the rise of bioprinting technology, research using 3D bioprinting to construct novel epidermal models is also increasing. Normally, bacteria remain on the skin surface, and symbiotic bacteria do not disrupt skin homeostasis. Inflammation only occurs when the skin barrier is disrupted and bacteria cross the epidermal barrier. Some researchers have used callus tissue from the soles of healthy individuals to establish stratum corneum models, demonstrating the relatively stable interaction between microbiome members and the composition and diversity of the inoculated microbiota. However, this model, made from human tissue, is difficult to widely apply and cannot maintain the stable growth of the anaerobic bacterium *Propionibacterium acnes*. Using 3D epidermal models for evaluating the efficacy of cosmetic microbiome is time-consuming and costly, and batch-to-batch variations are difficult to control, making it unsuitable for large-scale screening of active ingredients. Therefore, there is a need to develop and improve a 3D stratum corneum-like model that can simulate the physiological state of the stratum corneum for bacterial colonization. Currently, there are no patents or literature reports on in vitro methods for establishing 3D stratum corneum-like models to evaluate the microbiome-regulating efficacy of cosmetic raw materials and finished products. The 3D stratum corneum-like model established in this invention can be easily and quickly established, and can also better maintain the composition and diversity of human skin microbiota.

[0005] Currently, the main method for evaluating the regulation of the microecology in vitro is to use a 3D epidermal model inoculated with microorganisms. The 3D epidermal model used in this method is a human epidermal equivalent formed by the differentiation of human primary keratinocytes. Microorganisms are then directly inoculated onto the 3D epidermal model for the evaluation of the efficacy of regulating the microecology.

[0006] The currently used 3D epidermal models are expensive and time-consuming to create; human primary keratinocytes need to differentiate for more than ten days to form a 3D epidermal model. Therefore, this method suffers from high cost, low efficiency, and time consumption, making it difficult to achieve high-throughput screening of active ingredients. Summary of the Invention

[0007] Objectives of the Invention: The objective of this invention is to provide a rapid, stable, and high-throughput screening method for active substances, which can also be used for colonization by various microorganisms. Another objective of this invention is to provide a method for constructing a 3D stratum corneum model. A further objective of this invention is to provide the application of the aforementioned 3D stratum corneum model in in vitro efficacy evaluation of cosmetics or their raw materials, efficacy screening of active ingredients in cosmetics, or efficacy evaluation of biological agents.

[0008] Technical solution: The present invention provides a 3D stratum corneum-like model, the 3D stratum corneum-like model comprising a photocurable gel framework, the photocurable gel framework being loaded with apoptotic cells / or cell derivatives; the photocurable gel framework is composed of biomacromolecules with side chains containing double bonds that can be cross-linked by irradiation light.

[0009] Optionally, the light used for gel curing is ultraviolet, blue light, gamma rays, or near-infrared irradiation.

[0010] Preferably, the biomacromolecule includes one or more of GMHA (hyaluronic acid cross-linked methacrylated glyceryl ester), CMA (methacrylated collagen), CSMA (chondroitin sulfate methacrylate), GMA (glycidyl methacrylate), or HAMA (methacrylated hyaluronic acid).

[0011] The 3D stratum corneum model consists of a top surface, a middle layer, and a bottom surface. The top and bottom surfaces are uneven and are composed of apoptotic cells and / or cell derivatives tightly connected by a photocurable gel. The middle layer has a photocurable gel as its framework, and the photocurable gel framework forms several pores, in which apoptotic cells and / or cell derivatives are distributed.

[0012] Furthermore, the pore size of the voids formed by the photocurable gel skeleton is 1-500 μm, preferably 50-150 μm.

[0013] Furthermore, the thickness of the 3D stratum corneum model is 10μm to 1mm.

[0014] Furthermore, the cells are sebaceous gland cells and skin keratinocytes, wherein the sebaceous gland cells are human primary sebaceous gland cells or human immortalized sebaceous gland cell lines, and the keratinocytes are human primary keratinocytes or human immortalized keratinocyte cell lines; the cell derivatives are cell debris and cell metabolites.

[0015] Furthermore, skin keratinocytes account for 10% to 90% of the total number of cells in the 3D stratum corneum model.

[0016] On the other hand, the present invention provides a method for constructing the above-mentioned 3D stratum corneum model, comprising the following steps: photocurable gel is photocured with apoptotic keratinocytes and sebaceous gland cells to obtain a 3D stratum corneum model.

[0017] Furthermore, the photocuring method involves adding 0.15% to 0.3% photoinitiator to a 5-50 mg / mL photocurable gel, placing it in a well plate, adding uniformly mixed keratinocytes and sebaceous gland cells under light irradiation, and drying at a constant temperature.

[0018] Furthermore, the photocuring method involves mixing 0.15% to 0.3% of a photoinitiator with a certain number of keratinocytes and sebaceous gland cells, adding the resulting mixed solution to 5 to 50 mg / mL GMHA, placing it in a well plate, and drying it at a constant temperature under light irradiation.

[0019] Optionally, the light used for gel curing is ultraviolet, blue light, gamma rays, or near-infrared irradiation.

[0020] On the other hand, this invention provides an application of the aforementioned 3D stratum corneum model in the in vitro efficacy evaluation of cosmetics or their raw materials, the efficacy screening of active ingredients in cosmetics, or the pharmacodynamic evaluation of biological agents. The 3D stratum corneum model of this invention can be used to colonize various microorganisms and can be used to evaluate the efficacy of cosmetics and raw materials in regulating the skin microbiome in vitro or to evaluate the antibacterial efficacy of antibiotic biological agents.

[0021] Furthermore, the method of the application includes the following steps:

[0022] (1) Several types of facial bacteria were inoculated on a 3D stratum corneum model to simulate the facial microecology; facial bacteria include but are not limited to Propionibacterium acnes, Staphylococcus epidermidis, Staphylococcus aureus, Malassezia, etc.

[0023] (2) Apply the test sample to a 3D stratum corneum model that has been inoculated with facial bacteria;

[0024] (3) After a period of time, the number of bacteria or the relative abundance of each type of bacteria in the 3D stratum corneum model is detected, and the results are used to evaluate or screen test samples. The relative abundance is the proportion of each type of bacteria in the bacterial community, which represents the balance of the bacterial community.

[0025] The keratinocyte-like model of this invention is based on the photocurable hydrogel and apoptotic keratinocytes and sebaceous gland cells formed by photocuring. It has been shown to allow for the co-colonization and growth of Propionibacterium acnes, Staphylococcus epidermidis, and Staphylococcus aureus, and can be used to evaluate the efficacy of cosmetics in regulating the microecology and to test the antibacterial efficacy of antibiotics and other biological agents. The photocurable hydrogel is composed of one or more of CMA, GMHA, CSMA, GMA, or HAMA.

[0026] Beneficial Effects: Compared with existing technologies, this invention has the following significant advantages: a method for constructing a 3D stratum corneum-like model that uses methacrylamide-modified hyaluronic acid hydrogel to photopolymerize apoptotic HaCaT cells and sebaceous gland cells, forming structures resembling the stratum corneum and sebaceous glands, and providing a suitable environment for microbial colonization and growth. The established 3D stratum corneum-like model can be used to evaluate the efficacy of cosmetics in regulating the microecological environment and to test the antibacterial efficacy of antibiotics and other biological agents in vitro. It features low cost, relatively rapid processing, stability, and high-throughput screening of active substances. Attached Figure Description

[0027] Figure 1 This is a simplified diagram of the 3D stratum corneum model of the present invention;

[0028] Figure 2 It is the GMHA synthesis equation in Example 1;

[0029] Figure 3 This is a schematic diagram of the model surface under an inverted microscope at 40x magnification, based on the model made according to Scheme 1 in Example 1.

[0030] Figure 4 This is a schematic diagram of the model surface under an inverted microscope at 20x magnification, based on the model made according to Scheme 2 in Example 1.

[0031] Figure 5 This is a schematic diagram of the model surface under a scanning electron microscope at 500x magnification, showing the model made according to Scheme 1 in Example 1.

[0032] Figure 6 This is a schematic diagram of the model surface under a scanning electron microscope at 1000x magnification, showing the model made according to Scheme 1 in Example 1.

[0033] Figure 7 This is a schematic diagram of the model surface under a scanning electron microscope at 300x magnification, showing the model made according to Scheme 2 in Example 1.

[0034] Figure 8 This is a schematic diagram of the cross-sectional aperture of the model made according to Scheme 2 in Example 1, magnified 5000 times by a scanning electron microscope;

[0035] Figure 9 In Example 2, 1×10 6 Approximately CFU of Propionibacterium acnes was inoculated onto the model and cultured in a biochemical incubator at 35°C. The bacterial count was measured on days 0, 2, 3, 4, and 5 of the culture, and the bacterial count was plotted as a line graph.

[0036] Figure 10 In Example 2, the keratinoid model was observed to have a dense keratinoid layer formed by GMHA cross-linking between cells under a 400x scanning electron microscope.

[0037] Figure 11 yes Figure 10 A magnified view showing the biofilm established by Propionibacterium acnes, a common anaerobic bacterium on the face, on the surface of the stratum corneum.

[0038] Figure 12 In Example 2, 10 5 ~10 8 CFU of Propionibacterium acnes was inoculated onto the model and cultured. The total bacterial count was measured on days 1, 3, 5, 7, and 9.

[0039] Figure 13 In Example 2, 10 5 ~10 8 CFU of Staphylococcus epidermidis was inoculated onto the model and cultured. The total bacterial count was measured on days 1, 3, 5, 7, and 9.

[0040] Figure 14 In Example 2, 10 5 ~10 8 CFU of Staphylococcus aureus was inoculated onto the model and cultured. The total bacterial count was measured on days 1, 3, 5, 7, and 9.

[0041] Figure 15 In Example 2, 1×10 7 Staphylococcus aureus and Propionibacterium acnes at approximately CFU / mL were inoculated onto the model and cultured. The total bacterial count was measured on days 1, 2, 3, 4, and 6.

[0042] Figure 16 This study measured the number of three bacteria (Propionibacterium acnes, Staphylococcus aureus, and Staphylococcus epidermidis) in a facial microecology established by co-culturing these bacteria and adding different toner samples. Group 0 was a preservative-free toner; Group 1N1-0.1 was a toner containing 0.1% methylparaben; Group 1N1-0.2 was a toner containing 0.2% methylparaben; Group BEN-0.6 was a toner containing 0.6% phenoxyethanol; and Group BEN-0.8 was a toner containing 0.8% phenoxyethanol.

[0043] Figure 17 It is a mixture of CMA gel and photoinitiator, which forms a cured gel after being irradiated by a UV lamp;

[0044] Figure 18 It is a mixture of CSMA gel and photoinitiator, which forms a cured gel after being irradiated by a UV lamp;

[0045] Figure 19 (A) is a bacterial culture of Propionibacterium acnes after anaerobic culture in a 96-well plate for 48 hours, after mixing a certain concentration of CMA or CSMA with human primary keratinocytes, human primary sebaceous gland cells, human primary fibroblasts, and human immortalized fibroblasts (HSF cells); (B) is a bar graph of the absorbance of the Propionibacterium acnes suspension after culture.

[0046] Figure 20(A) is a bacterial suspension of Staphylococcus epidermidis cultured in a 96-well plate for 24 hours after mixing a certain concentration of CMA or CSMA with human primary keratinocytes, human primary sebaceous gland cells, human primary fibroblasts, and human immortalized fibroblasts (HSF cells); (B) is a bar graph of the absorbance of the Staphylococcus epidermidis suspension after culture.

[0047] Figure 21 This is a diagram showing the growth of Malassezia on the model. Detailed Implementation

[0048] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0049] In one embodiment, a 3D stratum corneum-like model is provided, such as Figure 1 As shown, the 3D keratinocyte model consists of a top surface 1, a middle layer 2, and a bottom surface 3. The surfaces of the top surface 1 and the bottom surface 3 are uneven and composed of apoptotic cells and / or cell derivatives tightly connected by a photocurable gel. The middle layer has a photocurable gel as its framework 5, which forms several pores. Cells 4, which are apoptotic cells and / or cell derivatives, are distributed within the pores. The cells 4 are tightly arranged within the framework 5 of the photocurable gel, forming a certain closed space that can support the growth of anaerobic bacteria. The pore size of the pores formed by the framework 5 of the photocurable gel is 1–500 μm, preferably 50–150 μm. The thickness of the middle layer 2 is 10 μm–1 mm.

[0050] The photocurable gel backbone is composed of biomacromolecules with double bonds in their side chains that can be cross-linked by light irradiation. Gels made from biomacromolecules with double bonds in their side chains that can be cross-linked by light irradiation can achieve the purpose of this invention. Preferably, the biomacromolecule is GMHA, CMA, CSMA, GMA, or HAMA.

[0051] Optionally, the light used for gel curing is ultraviolet, blue light, gamma rays, or near-infrared irradiation.

[0052] The preferred cells are sebaceous gland cells and HaCaT cells. HaCaT cells account for 10-90% of the total cells in the 3D keratinocyte-like model.

[0053] One embodiment provides a method for constructing a 3D stratum corneum-like model, which involves photocuring GMHA with HaCaT cells and sebaceous gland cells to obtain the 3D stratum corneum-like model.

[0054] In one embodiment, a 3D stratum corneum-like model is provided for the application of in vitro efficacy evaluation of cosmetics or their raw materials, efficacy screening of active ingredients in cosmetics, or efficacy evaluation of biological agents. The method for said application includes the following steps:

[0055] (1) Several types of facial bacteria were inoculated onto a 3D stratum corneum model to simulate the facial microecology;

[0056] (2) Apply the test sample to a 3D stratum corneum model that has been inoculated with facial bacteria;

[0057] (3) After a period of time, the number of bacteria or the relative abundance of each type of bacteria on the 3D stratum corneum model are detected, and the results are used to evaluate or screen test samples.

[0058] Facial bacteria include, but are not limited to, Propionibacterium acnes, Staphylococcus epidermidis, Staphylococcus aureus, and Malassezia.

[0059] HaCaT cells refer to human immortalized keratinocytes, which are immortalized keratinocyte lines from normal human skin without tumor origin, and have similar differentiation characteristics to normal human keratinocytes.

[0060] A fusion rate of 80% means that when cultured on a culture dish, the adherent cells form a monolayer that covers 80% of the surface area of ​​the culture dish.

[0061] PMA-qPCR quantitative detection refers to the use of propidium azide (PMA) to firmly bind to the DNA of dead cells. During the PCR reaction, the DNA bound to PMA will not amplify. Therefore, PMA-bound real-time quantitative PCR (PMA-qPCR) can eliminate interference from dead cells and quantify only the DNA of live cells.

[0062] GMHA refers to a hydrogel formed by cross-linking methacryloyl hyaluronic acid.

[0063] Example 1

[0064] This embodiment provides a method for constructing a 3D stratum corneum-like model, including the following steps:

[0065] (1) HaCaT cell and sebaceous gland cell culture steps: HaCaT cells and sebaceous gland cells were cultured in DMEM complete culture medium in a CO2 incubator at 37±0.5℃, 5% CO2 and saturated humidity. When the HaCaT cells and sebaceous gland cells reached 80%-90% confluence, the cells were collected, the original culture medium was removed, PBS buffer was added to wash twice, trypsin containing EDTA was added to digest for 6-8 min, complete culture medium was added to stop digestion, the cells were collected by centrifugation, and the cells were washed twice with PBS buffer and the culture medium was removed.

[0066] (2) Synthesis of hyaluronic acid cross-linked methylpropenyl glycerol (GMHA) hydrogel: such as Figure 2 As shown, take 1g of hyaluronic acid (molecular weight 4.1×10⁻⁶). 5 Da) was dissolved in 100 mL of PBS buffer. 7.5 mL of triethylamine, 7.5 g of tetrabutylammonium bromide, and 7.5 mL of methacrylated glycidyl ester were added sequentially to the hyaluronic acid solution. Triethylamine served as a catalyst, and tetrabutylammonium bromide served as a phase transfer catalyst. The reaction was carried out at 20 °C with constant stirring for 44 hours. After the reaction was complete, the reaction solution was transferred to a dialysis bag (with a cutoff of 3.5 × 10⁻⁶). 3 In the reaction mixture (Da), double-distilled water was used for dialysis for three days, with the water being changed every 12 hours. The dialyzed reaction solution was then freeze-dried to obtain GMHA lyophilized product.

[0067] (3) Steps for establishing a keratinocyte-like layer: Scheme 1: Take 100 μL of 5-20 mg / mL GMHA and add 0.15%-0.3% LAP, place it in a 24-well plate, irradiate under a UV lamp for 5-15 min, and add 6×10 5 1 HaCaT cells and 6×10 4 Sebaceous gland cells were dried at a constant temperature of 37°C for 24–48 hours.

[0068] The obtained 3D stratum corneum model is as follows Figure 3 , 5 As shown in Figures 7 and 8.

[0069] Option 2: Take 100 μL of 5–20 mg / mL GMHA, and add 0.15%–0.3% LAP and 6 × 10⁶ mg / mL of GMHA. 5 1 HaCaT cells and 6×10 4 Mix the sebaceous gland cells into a 24-well plate, irradiate under a UV lamp for 5–15 min, and then dry at 37°C for 24–48 h.

[0070] The obtained 3D stratum corneum model is as follows Figure 4 , 6 As shown in Figure 8.

[0071] The above methods all form a composite material containing a large number of keratinocytes. The GMHA gel tightly connects the cells and cell derivatives to form a dense keratinocyte-like layer with a thickness of 10μm to 1mm and an uneven surface. The gel has a pore size of 50 to 150μm, and the cells can be tightly arranged within the pores to form a dense, sealed space. This model can provide suitable growth environments for anaerobic and aerobic bacteria on the skin, and microorganisms can co-grow on the model.

[0072] Example 2

[0073] The method for constructing a 3D stratum corneum-like model provided in this embodiment includes the following steps:

[0074] (1) GMHA Synthesis: Dissolve 1g of hyaluronic acid in 100mL of PBS buffer. Add 7.5mL of triethylamine, 7.5g of tetrabutylammonium bromide, and 7.5mL of methacrylated glycidyl ester sequentially to the hyaluronic acid solution. Triethylamine serves as the catalyst, and tetrabutylammonium bromide serves as the phase transfer catalyst. The reaction temperature is set at 20℃, and the reaction is carried out with continuous stirring for 30–40 hours. After the reaction is complete, transfer the reaction solution to a dialysis bag (with a cutoff of 3.5 × 10⁻⁶). 3 In the reaction mixture (Da), double-distilled water was used for dialysis for three days. The dialysis solution was then freeze-dried to obtain GMHA lyophilized product.

[0075] (2) HaCaT cells and sebaceous gland cells culture and collection steps: HaCaT cells and sebaceous gland cells were cultured in DMEM complete culture medium in a CO2 incubator at 37±0.5℃, 5% CO2 and saturated humidity. The cells were collected when the HaCaT cells and sebaceous gland cells reached 80%-90% confluence. The original culture medium was removed, and the cells were rinsed twice with 2mL PBS buffer. 1.5mL of EDTA trypsin was added for digestion for 6-8min. 2mL of DMEM complete culture medium was added to stop the digestion. The cells were collected by centrifugation, rinsed twice with PBS buffer, and the supernatant was removed. The cells were then resuspended in PBS buffer and counted.

[0076] (3) Establishment of a keratinocyte-like model: Take 100 μL of 5 mg / mL GMHA and add 0.15 mg LAP. Mix well in the dark and place it into a 24-well plate containing 1 mL of 1.5% agar. Irradiate under a 405 nm UV lamp for 5–15 min. After curing, add approximately 6 × 10⁻⁶ mg LAP. 5 1 HaCaT cells and 6×10 4 Sebaceous gland cells were dried in a 37°C biochemical incubator for 12–48 hours.

[0077] The steps and results of the microbial growth detection of the stratum corneum established in step (3) are as follows:

[0078] (1) Culture steps of Propionibacterium acnes, Staphylococcus aureus, and Staphylococcus epidermidis: Propionibacterium acnes was cultured in enhanced Clostridium acnes liquid medium at 37°C for 48 h in an anaerobic gas-generating bag. After culture, the bacterial suspension was centrifuged, the supernatant was discarded, and the suspension was resuspended to prepare a bacterial suspension of a certain concentration. Staphylococcus aureus was cultured in tryptophan-soybean liquid medium at 37°C for 24 h. After culture, the bacterial suspension was centrifuged, the supernatant was discarded, and the suspension was resuspended to prepare a bacterial suspension of a certain concentration. Staphylococcus epidermidis was cultured in LB liquid medium at 37°C for 24 h. After culture, the bacterial suspension was centrifuged, the supernatant was discarded, and the suspension was resuspended to prepare a bacterial suspension of a certain concentration.

[0079] (2) Plate count method to detect whether bacteria can survive on the stratum corneum:

[0080] 1×10 6 Approximately CFU of Propionibacterium acnes was inoculated onto the model and cultured in a 35°C biochemical incubator. Bacterial counts were measured on days 0, 2, 3, 4, and 5 of culture. The specific procedure involved chopping the model into small pieces, placing them in a 15mL centrifuge tube, adding 6mL of PBS buffer, vortexing for 30 minutes to elute and isolate the bacteria. The bacterial suspension was then resuspended in PBS, diluted several times, and plated onto agar plates. After anaerobic incubation for 48 hours, the number of colonies grown on the plates was counted, and the bacterial count was calculated using the plate count method. Figure 9 It can be seen that Propionibacterium acnes can survive on the stratum corneum and maintain homeostasis within a certain range.

[0081] (3) Detection of biofilm formation by cuticle-like microbial colonization:

[0082] Using the keratinoid layer prepared in section 2.3 above, approximately 1 × 10⁻⁶ cells were inoculated. 7 Approximately CFU of *Propionibacterium acnes* was cultured at 35°C for 48 hours. After culture, the bacteria were freeze-dried, and images of the microorganisms colonizing the keratinoid layer were captured using a scanning electron microscope. The results are as follows: Figure 10 The stratum corneum model shown at 400x magnification clearly shows that the dense stratum corneum is formed by GMHA cross-linking between cells. Figure 11 for Figure 10 A magnified view shows Propionibacterium acnes establishing a biofilm on the surface of the stratum corneum and colonizing and growing there.

[0083] (4) PMA-qPCR quantitative detection of microbial growth on the cuticle:

[0084] Different concentrations of Propionibacterium acnes, Staphylococcus aureus, and Staphylococcus epidermidis were inoculated onto the model and cultured in a 35°C biochemical incubator. Bacterial counts were measured on days 0, 1, 3, 5, and 9 of culture. The model was chopped and placed in a 15mL centrifuge tube. 6mL of PBS buffer was added, and the mixture was vortexed for 30 min to elute and isolate the bacteria. The bacteria were resuspended in 490μL of PBS, and 10μL of 100μg / mL PMA solution was added. After mixing in the dark, the mixture was incubated at room temperature for 10 min, followed by irradiation under a 500W tungsten lamp for 10 min. Bacterial DNA was extracted using the Bacteria DNA Isolation Mini Kit according to the manufacturer's instructions. q-PCR detection was performed using the ChamQ SYBR qPCR Master MiX and the 16S rRNA-specific primer sequences for the bacteria listed in Table 1.

[0085] Table 1

[0086]

[0087] The cycle threshold (Ct value) calculated by real-time quantitative PCR was substituted into a pre-established standard curve relating bacterial count to Ct value to calculate the bacterial count; Figure 12 , 13 As can be seen from 14, after different concentrations of Propionibacterium acnes, Staphylococcus aureus, and Staphylococcus epidermidis were inoculated onto the stratum corneum, they were able to maintain a relatively stable state for a period of time.

[0088] Microorganisms can co-grow on the cuticle layer, and interspecies interactions between microorganisms can be detected.

[0089] 1×10 7 Staphylococcus aureus and Propionibacterium acnes at approximately CFU / mL were inoculated onto the model for growth. Separate culture groups for Staphylococcus aureus and Propionibacterium acnes, and a co-culture group for both were established. The number of viable bacteria was measured every 24 hours. Figure 15 It can be seen that when Staphylococcus aureus and Propionibacterium acnes are co-cultured, Propionibacterium acnes can promote the growth of Staphylococcus aureus. This is consistent with the literature report that Propionibacterium acnes produces a small molecule coproporphyrin III, which can promote the aggregation and biofilm formation of Staphylococcus aureus.

[0090] In summary, this invention establishes a stratum corneum-like structure that can support the growth of several common skin microorganisms, providing a new model for studying human microbiota and its preparation method. This offers new ideas and methods for further research on in vitro evaluation of skincare product efficacy, screening of active ingredient efficacy, and evaluation of antibiotic efficacy.

[0091] Example 3

[0092] The stratum corneum established in Example 2 was used to evaluate the efficacy of cosmetics containing phenoxyethanol or methylparaben.

[0093] The specific steps are as follows: The effects on several facial bacteria and the results are as follows:

[0094] (1) Facial microbial culture: Propionibacterium acnes, Staphylococcus aureus, and Staphylococcus epidermidis were selected as facial microorganisms. Propionibacterium acnes was cultured in enhanced Clostridium acnes liquid medium at 37°C for 48 h in an anaerobic gas-generating bag. After culture, the bacterial suspension was centrifuged, the supernatant was discarded, and the suspension was resuspended to prepare a bacterial suspension of a certain concentration. Staphylococcus aureus was cultured in tryptophan-soybean liquid medium at 37°C for 24 h. After culture, the bacterial suspension was centrifuged, the supernatant was discarded, and the suspension was resuspended to prepare a bacterial suspension of a certain concentration. Staphylococcus epidermidis was cultured in LB liquid medium at 37°C for 24 h. After culture, the bacterial suspension was centrifuged, the supernatant was discarded, and the suspension was resuspended to prepare a bacterial suspension of a certain concentration.

[0095] (2) Prepare the sample of the toner to be tested:

[0096] According to the formula in Table 2, weigh phases A2, A3, A4, and A5, add an appropriate amount of deionized water, and dissolve them completely in a water bath at 50–60°C. Weigh phases B2, B3, B4, and preservatives of various concentrations, dissolve them completely in a water bath at 50–60°C, then add phase B1 and mix well. Mix phases A and B thoroughly, measure the pH value, and add an appropriate amount of sodium citrate to bring the pH to 5–6. Add deionized water to bring the total volume to 100%. Add preservatives and concentrations according to Table 3.

[0097] Table 2

[0098]

[0099] Table 3

[0100]

[0101] Using the above methods, a total of five types of toner samples were prepared: a toner sample without any preservatives, a toner sample containing 0.1% methylparaben (NI-0.1), a toner sample containing 0.2% methylparaben (NI-0.2), a toner sample containing 0.6% phenoxyethanol (BEN-0.6), and a toner sample containing 0.8% phenoxyethanol (BEN-0.8).

[0102] (3) Simulate facial microecology: Take a stratum corneum model and simultaneously inoculate it with Propionibacterium acnes, Staphylococcus aureus and Staphylococcus epidermidis to simulate facial microecology.

[0103] Based on the top surface area of ​​the keratinoid model, according to the ratio of 1cm 2 Approximately 1×10 7 CFU of Propionibacterium acnes, 0.5 × 10 7 CFU of Staphylococcus epidermidis, 2.5 × 10 6The amount of CFU (Cellular Fusarium oxysporum) used to inoculate Staphylococcus aureus onto a keratinoid model was increased by simultaneously inoculating three types of bacteria. The models were then incubated at 37°C for 48 hours to obtain keratinoid models inoculated with facial bacteria. This process was repeated 15 times to obtain 15 keratinoid models inoculated with facial bacteria.

[0104] Based on the top surface area of ​​the keratinoid model, according to the ratio of 1cm 2 Approximately 50 μL of each of the five different toner samples was added to five pre-inoculated facial bacterial stratum corneum models, with three replicates. The models were incubated at 37°C for 24 hours, and the bacterial count was then determined. Figure 16 As shown, adding preservatives to toners significantly reduces the number of three types of bacteria, indicating that the addition of phenoxyethanol or methylparaben preservatives to cosmetics affects the facial flora. As the concentration of preservatives increases, the degree of inhibition of facial flora growth also increases.

[0105] In this embodiment, three common facial bacteria were inoculated onto a stratum corneum and co-cultured to evaluate the effects of toners containing phenoxyethanol or methylparaben preservatives on the facial microbiota in vitro. The experiment showed that using toners containing phenoxyethanol or methylparaben preservatives may inhibit the growth of facial microbiota and disrupt the facial microecology.

[0106] Preservatives in cosmetics can alter the balance of the skin microbiome. However, by testing different combinations of preservatives on the dynamics of the resident skin microbiome using in vitro models, we can provide a reference for the correct selection of preservatives and dosages in cosmetic formulations to maintain or restore the homeostasis of the skin microbiome.

[0107] Example 4

[0108] This embodiment provides a method for constructing a 3D stratum corneum-like model, including the following steps:

[0109] (1) Synthesis of methacrylated collagen (CMA), chondroitin sulfate methacrylate (CSMA), glycidyl methacrylate (GMA), and methacrylated hyaluronic acid (HAMA): Methacrylate groups were introduced into collagen, chondroitin sulfate (CS), gelatin, and hyaluronic acid (HA) respectively to synthesize photocurable gels CMA, CSMA, GMA, and HAMA.

[0110] (2) Culture and collection steps for human primary keratinocytes, human immortalized keratinocyte cell lines, human primary sebaceous gland cells, human immortalized sebaceous gland cell lines, human primary fibroblasts, and human immortalized fibroblast cell lines:

[0111] Cells were cultured in DMEM complete medium at 37±0.5℃ in a CO2 incubator with 5% CO2 and saturated humidity. Cells were collected when the confluence reached 80%-90%, the original culture medium was removed, and the cells were rinsed twice with PBS buffer. Then, trypsin containing EDTA was added for 6-8 min, and the digestion was stopped by adding complete culture medium. Cells were collected by centrifugation, rinsed twice with PBS buffer, and the culture medium was removed.

[0112] (3) Establishing a keratinocyte-like model: Take 100 μL of 5-50 mg / mL photocurable gels CMA, CSMA, GMA, and HAMA, add 0.15%-0.6% of photoinitiator LAP or I2959, place it in the wells of a 24-well plate containing agar gel, irradiate under UV light for 5-120 s, and after curing, add a sufficient amount of well-mixed keratinocytes, sebaceous gland cells, or fibroblasts, and dry in a 37℃ biochemical incubator for 12-48 h.

[0113] Photocurable gels CMA, GMHA, CSMA, GMA, human primary keratinocytes, human primary sebaceous gland cells, human primary fibroblasts, and human immortalized fibroblasts can also be used in 3D keratinocyte-like models, as demonstrated below:

[0114] (1) Evaluation of the gel-forming properties of photocurable gel

[0115] Take photocurable gel CMA, add a photoinitiator, and irradiate under a UV lamp for 5–120 seconds to cure and form a gel.

[0116] Take a photocurable gel CSMA, add a photoinitiator, and irradiate it under a UV lamp for 5–120 seconds to cure it into a gel.

[0117] CMA, GMHA, CSMA, and GMA are all photocurable gels that have been reported in the literature to form gels.

[0118] (2) Evaluation of the biocompatibility of gel and cells with facial bacteria

[0119] Facial microbial culture: Propionibacterium acnes, Staphylococcus aureus, and Staphylococcus epidermidis were selected as facial microorganisms. Propionibacterium acnes was cultured in enhanced Clostridium acnes liquid medium at 37°C for 48 hours in an anaerobic gas-generating bag. After culture, the bacterial suspension was centrifuged, the supernatant was discarded, and the suspension was resuspended to a specific concentration. Staphylococcus aureus was cultured in tryptophan-soybean liquid medium at 37°C for 24 hours. After culture, the bacterial suspension was centrifuged, the supernatant was discarded, and the suspension was resuspended to a specific concentration. Staphylococcus epidermidis was cultured in LB liquid medium at 37°C for 24 hours. After culture, the bacterial suspension was centrifuged, the supernatant was discarded, and the suspension was resuspended to a specific concentration.

[0120] (2.2) Mix 100 μL of a photocurable gel solution of a certain concentration with equal amounts of approximately 2 × 10⁻⁶ human primary keratinocytes, human primary sebaceous gland cells, human primary fibroblasts, and human immortalized fibroblasts (HSF cells). 4 A mixture of cells was added to a 96-well plate, with the control group containing bacterial culture medium. Several types of bacteria were resuspended in their respective culture media (OD). 600 Approximately 0.06 g of the solution was added to 100 μL of the photocurable gel solution. The mixture was co-cultured in a 96-well plate at 37°C for 24–48 h. After culturing, the plate was vortexed for 30 min, followed by centrifugation at 800 rpm for 5 min. 100 μL of the supernatant was collected and the absorbance of the bacterial culture at 600 nm was measured using a microplate reader. If the bacterial concentration in the gel solution group did not decrease significantly compared to the control group, it indicates good biocompatibility between the gel, cells, and bacteria, and it can be used to construct a 3D cuticle-like model suitable for microbial growth. Previous literature has reported that photocurable gels CMA, GMHA, CSMA, and GMA all have good cell compatibility, and cells encapsulated in these gels can grow normally.

[0121] Figure 19 This refers to the bacterial culture obtained by anaerobically culturing Propionibacterium acnes in a 96-well plate after mixing a certain concentration of CMA or CSMA with human primary keratinocytes, human primary sebaceous gland cells, human primary fibroblasts, or human immortalized fibroblasts (HSF cells) and a mixture of these with Propionibacterium acnes at a certain concentration for 48 hours.

[0122] Figure 20 This refers to the bacterial culture obtained by mixing a certain concentration of CMA or CSMA with human primary keratinocytes, human primary sebaceous gland cells, human primary fibroblasts, or human immortalized fibroblasts (HSF cells) and then culturing Staphylococcus epidermidis in a 96-well plate for 24 hours.

[0123] Example 5

[0124] The keratinoid layer established in Example 2 was used for the detection of microbial colonization and biofilm formation:

[0125] Different concentrations of Malassezia were inoculated onto the model and cultured in a 35°C biochemical incubator. Fungal counts were performed on days 0, 1, 3, 5, 7, and 9 using a plate count method. The specific procedure involved chopping the model into small pieces, placing them in a 15mL centrifuge tube, adding 6mL of PBS buffer, vortexing for 30 minutes to elute and isolate the bacteria. The bacterial suspension was then resuspended in PBS, diluted several times, and plated onto olive oil agar plates. The plates were incubated for 48 hours, and the number of colonies grown was counted using the plate count method. Figure 21 It can be seen that Malassezia can survive on the keratinoid layer and maintain homeostasis within a certain range.

Claims

1. Application of a 3D stratum corneum-like model in in vitro efficacy evaluation of cosmetics or their raw materials; The 3D stratum corneum-like model consists of a top surface, a middle layer, and a bottom surface. The top and bottom surfaces are uneven and are composed of apoptotic cells tightly connected by GMHA gel. The middle layer uses GMHA gel as a framework, and the GMHA gel framework forms several gaps in which apoptotic cells are distributed. The GMHA structure is as follows: ； The 3D stratum corneum-like model was constructed using a method that included the following steps: photopolymerizing GMHA with apoptotic HaCaT cells and sebaceous gland cells; The efficacy refers to its ability to regulate the microecological environment, and the application method includes the following steps: (1) Several types of facial bacteria are inoculated on a 3D stratum corneum model to simulate the facial microecology; the several types of facial bacteria are selected from one or more of Propionibacterium acnes, Staphylococcus epidermidis and Staphylococcus aureus. (2) Apply the test sample to a 3D stratum corneum model that has been inoculated with facial bacteria; (3) After a period of time, the number of bacteria or the relative abundance of each type of bacteria on the 3D stratum corneum model are detected, and the results are used to evaluate the test samples.

2. The application according to claim 1, characterized in that: The pore size of the voids formed by the GMHA gel skeleton is 50–150 μm.

3. The application according to claim 1, characterized in that: The thickness of the intermediate layer is 10μm to 1mm.

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