WO2023246485A1

WO2023246485A1 - 3d stratum corneum-like model, construction method therefor and use thereof

Info

Publication number: WO2023246485A1
Application number: PCT/CN2023/098261
Authority: WO
Inventors: 吴建新; 黄庆; 王攀
Original assignee: 中国药科大学
Priority date: 2022-06-23
Filing date: 2023-06-05
Publication date: 2023-12-28
Also published as: CN115161258A; CN116286602A

Abstract

Disclosed are a construction method for a 3D stratum corneum-like model and use thereof. The 3D stratum corneum-like model is composed of a top surface, an intermediate layer, and a bottom surface. The top and bottom surfaces are uneven and are composed of apoptotic cells and/or cell derivatives closely connected to a photocurable gel. The intermediate layer is a photocurable gel, wherein the photocurable gel serves as a scaffold providing a porous structure for the apoptotic cells and/or cell derivatives. The construction method comprises loading a photocurable gel material with apoptotic keratinocytes and/or cell derivatives to form a 3D stratum corneum-like model under light irradiation conditions. The constructed 3D stratum corneum-like model can be effectively used for in vitro evaluation of the efficacy of cosmetics in regulating microecology, testing the antibacterial efficacy of biological preparations such as antibiotics, and has the advantages of low cost, fast construction speed, high stability, and being able to screen for active substances in a high-throughput manner.

Description

A 3D cuticle-like model and its construction method and application

Technical field

The present invention relates to a cuticle-like model and its construction method and application, and in particular to a 3D cuticle-like model that can be colonized by a variety of microorganisms and its construction method and application.

Background technique

In the process of developing functional skin care products that regulate microecology, it is necessary to evaluate the efficacy of active ingredients or cosmetics in regulating microecology. In the past, experiments were conducted by establishing germ-free mouse models for efficacy evaluation. However, with the proposal and implementation of the 3R principle of animal experiments, appropriate in vitro models have been established to replace animal models for evaluating the effects of cosmetic raw materials or products on regulating microecology. The application appears to be very necessary and has huge applicability.

As the largest organ of the human body, skin is home to many different types of microorganisms. Beneficial microorganisms serve as a physical barrier to prevent the invasion of pathogens and protect the body from external aggression. According to the different skin environment and physical and chemical properties of different parts of the human body, the physiological state of the skin is divided into three categories: moist, oily and dry. Although the skin lacks nutrition, is acidic, and is dry, there are still 10 ⁶ bacteria/cm ² living on it. Most of the bacteria colonize the superficial part of the stratum corneum and use the cross-linked proteins and lipids of keratinocytes as a source of nutrition.

When using in vitro models to study microbiota, the impact on microbial growth and interactions when switching from the skin environment to the in vitro model must be considered. At present, in vitro research on microecology mainly uses 3D epidermal models, which are constructed through the differentiation of human primary keratinocytes into human epidermal equivalents. In the cosmetics industry, they are mainly used for cosmetic safety evaluation; with the rise of bioprinting technology , research on using 3D bioprinting technology to construct new epidermal models is also gradually increasing. Usually bacteria stay on the surface of the skin, and symbiotic bacteria do not interfere with skin homeostasis. When the skin barrier is destroyed, bacteria penetrate the epidermal barrier and cause an inflammatory response. Some researchers have used plantar callus from healthy people to build a stratum corneum model, which can reflect the interaction between microbiome members and keep the composition and diversity of the inoculated microbiome relatively stable. However, the raw materials for making this model are derived from human tissue. It is difficult to apply widely and cannot maintain the stable growth of the anaerobic bacterium Propionibacterium acnes. If the 3D epidermal model is used to evaluate the microecological efficacy of cosmetics, it will take longer and cost more. The differences between model batches are difficult to control, and it cannot be used for large-scale screening of active substances. Therefore, it is necessary to develop and improve a model that can simulate skin. A 3D stratum corneum-like model of the stratum corneum's physiological state for bacterial colonization. Currently, there are no relevant patents or literature reports on the in vitro method of establishing a 3D stratum corneum model to evaluate the microecological regulation efficacy of cosmetic raw materials and finished products. The 3D stratum corneum model established by the present invention can be established simply and quickly, and can also better maintain the composition and diversity of human skin microorganisms.

The current method for in vitro evaluation of regulating microecology mainly uses a 3D epidermal model to inoculate microorganisms as a model. The 3D epidermal model used in this method is to use human primary keratinocytes to differentiate to form human epidermal equivalents, and the microorganisms are directly inoculated into the 3D epidermis. The model is used to evaluate the efficacy of regulating microecology.

The currently used 3D epidermal models are relatively expensive and take a long time to produce. Human primary keratinocytes need to differentiate for more than ten days to form a 3D epidermal model. Therefore, this method has the problems of high cost, low efficiency and time-consuming, and it is difficult to achieve high-throughput screening of active ingredients.

Contents of the invention

Purpose of the invention: The purpose of the present invention is to provide a 3D stratum corneum-like model that is fast, stable and capable of high-throughput screening of active substances and can be colonized by a variety of microorganisms; another purpose of the present invention is to provide a 3D stratum corneum-like model. Method for constructing a stratum corneum model; another object of the present invention is to provide the application of the above-mentioned 3D stratum corneum model in in vitro efficacy evaluation of cosmetics or their raw materials, efficacy screening of cosmetic active ingredients, or efficacy evaluation of biological preparations.

Technical solution: a 3D stratum corneum-like model of the present invention, the 3D stratum corneum-like model includes a photocurable gel skeleton, and the photocurable gel skeleton is loaded with apoptotic cells/or cell derivatives; The photocurable gel skeleton is composed of biomacromolecules with double bonds in their side chains that can be cross-linked by irradiation with light.

Optionally, the irradiation light used when the gel is cured is ultraviolet, blue light, gamma ray or near-infrared irradiation.

Preferably, the biological macromolecules include GMHA (hyaluronic acid cross-linked glyceryl methacrylate), CMA (methacrylated collagen), CSMA (chondroitin sulfate methacrylate), GMA (methacrylic acid One or more of glycidyl ester) or HAMA (hyaluronic acid methacrylate).

Wherein, the 3D stratum corneum model is composed of a top surface, a middle layer and a bottom surface. The top and bottom surfaces are uneven and composed of apoptotic cells and/or cell derivatives that are tightly connected through a photo-curable gel. The middle layer The layer uses a photocurable gel as a skeleton, and the photocurable gel skeleton constitutes several pores, and apoptotic cells and/or cell derivatives are distributed in the pores.

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

Further, the thickness of the 3D stratum corneum-like model is 10 μm to 1 mm.

Further, 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 fragments. and cellular metabolites.

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

On the other hand, the present invention provides a method for constructing the above-mentioned 3D stratum corneum-like model, which includes the following steps: light-curing a photocurable gel with apoptotic keratinocytes and sebaceous gland cells to obtain a 3D stratum corneum-like model.

Further, the photocuring method is to add 0.15% to 0.3% photoinitiator to 5-50mg/mL photocurable gel, put it into a well plate, and add evenly mixed keratinocytes under the condition of light irradiation. and sebaceous gland cells, dried at constant temperature.

Further, the photocuring method is to mix 0.15% to 0.3% photoinitiator with a certain number of keratinocytes and sebaceous gland cells, and the resulting mixed solution is added to 5 to 50 mg/mL GMHA and put into a well plate. in the light Dry at constant temperature under irradiation conditions.

On the other hand, the present invention provides an application of the above-mentioned 3D stratum corneum model in in vitro efficacy evaluation of cosmetics or their raw materials, efficacy screening of cosmetic active ingredients, or efficacy evaluation of biological preparations. The 3D stratum corneum model of the present invention can be colonized by a variety of 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 preparations.

Further, the application method includes the following steps:

(1) Inoculate several kinds of facial bacteria on the 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.;

(2) Coat the sample to be tested on the 3D stratum corneum model that has been inoculated with facial bacteria;

(3) After a period of time, detect the number of bacteria on the 3D cuticle-like model or the relative abundance of each bacteria in the bacterial community, and the results obtained are used to evaluate or screen the test samples. The relative abundance is the proportion of each type of bacteria in the flora, which represents the adjustment of the balance of the flora.

The stratum corneum model of the present invention is formed based on photocurable hydrogel and apoptotic keratinocytes and sebaceous gland cells, and has been proven to be co-colonized by Propionibacterium acnes, Staphylococcus epidermidis, and Staphylococcus aureus. Growth can be used to evaluate the efficacy of cosmetics in regulating microecology and test the antibacterial efficacy of biological agents such as antibiotics. Wherein, the component of the photocurable hydrogel is one or more of CMA, GMHA, CSMA, GMA or HAMA.

Beneficial effects: Compared with the existing technology, the present invention has the following significant advantages: it uses methacrylated hyaluronic acid hydrogel and apoptotic HaCaT cells and sebaceous gland cells to form a structure similar to the stratum corneum and sebaceous glands after light curing. and a method for constructing a 3D cuticle-like model for microbial colonization and growth. The established 3D stratum corneum-like model can be used in vitro to evaluate the efficacy of cosmetics in regulating microecology and test the antibacterial efficacy of biological agents such as antibiotics. It has the characteristics of low cost, relatively fast, stable and high-throughput screening of active substances.

Description of the drawings

Figure 1 is a simplified diagram of the 3D stratum corneum-like model of the present invention;

Figure 2 is the GMHA synthesis equation in Example 1;

Figure 3 is a schematic diagram of the surface of the model under 40 times magnification under an inverted microscope when the model was made according to Scheme 1 in Example 1;

Figure 4 is a schematic diagram of the surface of the model under 20 times magnification under an inverted microscope when the model was made according to Scheme 2 in Example 1;

Figure 5 is a schematic diagram of the model surface under a scanning electron microscope magnified 500 times in Example 1 according to Scheme 1;

Figure 6 is a schematic diagram of the model surface under a scanning electron microscope magnified 1000 times when the model was made according to Scheme 1 in Example 1;

Figure 7 is a model made according to Scheme 2 in Example 1, and the surface of the model under a scanning electron microscope is magnified 300 times. intention;

Figure 8 is a schematic diagram of the cross-sectional aperture of the model produced according to Scheme 2 in Example 1 and magnified 5000 times under a scanning electron microscope;

Figure 9 shows that in Example 2, about 1×10 ⁶ CFU of Propionibacterium acnes was inoculated into the model and cultured in a 35°C biochemical incubator. On the 0th, 2nd, 3rd, 4th and 5th days of culture respectively Line chart of the number of detected bacteria;

Figure 10 shows the stratum corneum model in Example 2. Under a scanning electron microscope at 400 times, it is seen that the cells are cross-linked by GMHA to form a dense stratum corneum;

Figure 11 is a partial enlarged view of Figure 10, showing a schematic diagram of the establishment of a biofilm on the surface of the stratum corneum by Propionibacterium acnes, a common anaerobic bacterium on the face;

Figure 12 shows that in Example 2, 10 ⁵ to 10 ⁸ CFU of Propionibacterium acnes was inoculated into the model and cultured, and the total number of bacteria was detected on days 1, 3, 5, 7, and 9;

Figure 13 shows that in Example 2, 10 ⁵ to 10 ⁸ CFU of Staphylococcus epidermidis was inoculated into the model and cultured, and the total number of bacteria was detected on days 1, 3, 5, 7, and 9;

Figure 14 shows that in Example 2, 10 ⁵ to 10 ⁸ CFU of Staphylococcus aureus was inoculated into the model and cultured, and the total number of bacteria was detected on days 1, 3, 5, 7, and 9;

Figure 15 shows that in Example 2, about 1×10 ⁷ CFU/mL of Staphylococcus aureus and Propionibacterium acnes were inoculated and cultured on the model, and the total number of bacteria was detected on days 1, 2, 3, 4, and 6;

Figure 16 shows the number of three types of bacteria after adding different lotion samples to the facial microecology established by co-culture of Propionibacterium acnes, Staphylococcus aureus, and Staphylococcus epidermidis. Group 0 is the preservative-free lotion group; NI- The 0.1 group is a lotion group containing 0.1% methylparaben; the NI-0.2 group is a lotion group containing 0.2% methylparaben; the BEN-0.6 group is a lotion group containing 0.6% phenoxyethanol; BEN- Group 0.8 is a lotion group containing 0.8% phenoxyethanol.

Figure 17 shows a mixture of CMA gel and photoinitiator, which forms a solidified gel after being irradiated with UV light;

Figure 18 shows a mixture of CSMA gel and photoinitiator, which forms a solidified gel after being irradiated with UV light;

Figure 19 shows a mixture of 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) and then mixed with Propionibacterium acnes in 96 wells. Picture of the bacteria cultured in the plate anaerobically for 48 hours; (A) is a mixture of a certain concentration of CMA or CSMA and human primary keratinocytes, human primary sebaceous gland cells, human primary fibroblasts, and human immortalized fibroblasts (HSF). Cells) were mixed with P. acnes and cultured anaerobically in a 96-well plate for 48 hours; (B) is a histogram of the absorbance of the P. acnes suspension after culture;

Figure 20 shows a mixture of 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), and then mixed with Staphylococcus epidermidis in a 96-well plate. Diagram of bacterial liquid cultured for 24 hours; (A) is a mixture of a certain concentration of CMA or CSMA and human primary keratinocytes, Human primary sebaceous gland cells, human primary fibroblasts, and human immortalized fibroblasts (HSF cells) were mixed and cultured with Staphylococcus epidermidis in a 96-well plate for 24 hours; (B) is the suspension of Staphylococcus epidermidis after culture. Liquid absorbance histogram;

Figure 21 is a diagram of the growth of Malassezia on the model.

Figure 22 is a graph of the differences between the three groups on days 0, 1, and 4 of culture. The figure shows the distribution box plot of the distance between samples in the group on day 0 and the difference between the sample on day 0 and the sample on day 1 or 4. Box plot of distances between samples on day 4. In the box plot, the meanings of each symbol are as follows: the upper and lower end lines of the box, the upper and lower interquartile range (IQR); the median line, the median; the upper and lower edges, the maximum and minimum values (1.5 times the IQR range). extreme values within); points outside the upper and lower edges represent outliers.

Figure 23 is an NMDS analysis diagram. Each point in the diagram represents a sample, and points of different shapes indicate different samples (groups). NMDS adopts hierarchical sorting, which can be approximated to mean that the closer (far) the distance between two points is, the smaller (larger) the difference in the microbial communities in the two samples is. The elliptical dotted circle is the 95% confidence ellipse (that is, 95 out of 100 samples of this sample group will fall within it).

Figure 24 is a histogram of the top twenty species compositions at the genus level relative abundance of the three groups of bacterial groups on days 0, 1 and 4 of culture. The abscissa in the figure is the name of each group, and the ordinate is the relative abundance of each taxon at the genus level.

Detailed ways

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

In one embodiment, a 3D stratum corneum-like model is provided. As shown in Figure 1, the 3D stratum corneum-like model is composed 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. It is composed of apoptotic cells and/or cell derivatives that are tightly connected through a photo-curable gel. The middle layer uses the photo-curable gel as a skeleton 5. The skeleton 5 of the photo-curable gel forms a number of gaps distributed within the gaps. Cells 4, cells 4 are apoptotic cells and/or cell derivatives; cells 4 are closely arranged within the skeleton 5 of the photocurable gel, forming a certain closed space for the growth of anaerobic bacteria; the photocurable gel The pore diameter of the voids formed by the skeleton 5 is 1 to 500 μm, preferably 50 to 150 μm; the thickness of the intermediate layer 2 is 10 μm to 1 mm.

Among them, the photocurable gel skeleton is composed of biological macromolecules whose side chains contain double bonds that can be cross-linked by irradiation of light. All can achieve the purpose of the present invention. Preferably, the biological macromolecule is GMHA, CMA, CSMA, GMA or HAMA.

The cells are preferably sebaceous gland cells and HaCaT cells. HaCaT cells account for 10-90% of the total cells in the 3D stratum corneum model.

In one embodiment, a method for constructing a 3D stratum corneum-like model is provided. GMHA, HaCaT cells and sebaceous gland cells are photocured to obtain a 3D stratum corneum-like model.

In one embodiment, the application of a 3D stratum corneum model in in vitro efficacy evaluation of cosmetics or their raw materials, efficacy screening of cosmetic active ingredients, or efficacy evaluation of biological preparations is provided. The application method described therein includes the following steps:

(1) Inoculate several kinds of facial bacteria on the 3D stratum corneum model to simulate the facial microecology;

(3) After a period of time, detect the number of bacteria on the 3D cuticle-like model or the relative abundance of each bacteria in the bacterial community, and the results obtained are used to evaluate or screen the test samples.

Facial bacteria include, but are not limited to, Propionibacterium acnes, Staphylococcus epidermidis, Staphylococcus aureus, Malassezia, etc.

HaCaT cells refer to human immortalized keratinocytes, which are immortalized keratinocytes derived from non-tumor human normal skin and have similar differentiation characteristics to normal human keratinocytes.

Reaching 80% confluence means that when cultured on a culture dish, the adherent cell monolayer covers 80% of the upper surface of the culture dish.

PMA-qPCR quantitative detection uses propidium bromide azide (PMA) to bind firmly to the DNA of dead cells. The DNA combined with PMA will not amplify during the PCR reaction. Therefore, PMA combined with real-time quantitative PCR (PMA-qPCR) can eliminate the interference of dead cells and only quantify the DNA of living cells.

GMHA is a hydrogel formed by cross-linking methacrylated hyaluronic acid.

Example 1

This embodiment provides a method for constructing a 3D stratum corneum-like model, which includes the following steps:

(1) HaCaT cells and sebaceous gland cell culture steps: HaCaT cells and sebaceous gland cells are cultured in DMEM complete culture medium in a CO ₂ incubator at 37±0.5°C, 5% CO ₂ and saturated humidity until HaCaT cells and sebaceous gland cells fuse. Collect cells when the concentration reaches 80%-90%, remove the original culture medium, add PBS buffer to rinse twice, add EDTA-containing trypsin for digestion for 6 to 8 minutes, add complete culture medium to terminate digestion, centrifuge to collect cells, and rinse with PBS buffer Rinse twice and remove culture medium.

(2) Synthesis of hyaluronic acid cross-linked glyceryl methacrylate (GMHA) hydrogel: As shown in Figure 2, take 1g of hyaluronic acid (molecular weight 4.1×10 ⁵ Da) and dissolve it in 100 mL of PBS buffer. Add 7.5 mL triethylamine, 7.5 g tetrabutylammonium bromide, and 7.5 mL glycidyl methacrylate in sequence to the acid solution. Among them, triethylamine is the catalyst and tetrabutylammonium bromide is the phase transfer catalyst. Set the reaction temperature to 20°C and react for 44 hours with constant stirring. After the reaction, the reaction solution was transferred to a dialysis bag (cutoff 3.5×10 ³ Da) and dialyzed with double-distilled water for three days, changing the double-distilled water every 12 hours. The dialyzed reaction solution is freeze-dried to obtain GMHA freeze-dried product.

(3) Steps for establishing stratum corneum: Plan 1: Take 100 μL of 5-20 mg/mL GMHA and add 0.15%-0.3% LAP, put it into a 24-well plate, irradiate it under a UV lamp for 5-15 minutes, and add evenly mixed 6 ×10 ⁵ HaCaT cells and 6 × 10 ⁴ sebaceous gland cells were dried at 37°C for 24 to 48 hours.

The obtained 3D stratum corneum-like model is shown in Figures 3, 5, and 7.

Option 2: Take 100 μL of 5-20 mg/mL GMHA, add a mixture containing 0.15%-0.3% LAP, 6×10 ⁵ HaCaT cells and 6×10 ⁴ sebaceous gland cells, and place it into a 24-well plate. Irradiate under ultraviolet lamp for 5 to 15 minutes and dry at 37°C for 24 to 48 hours.

The obtained 3D stratum corneum-like model is shown in Figures 4, 6, and 8.

The above methods will form a layer of composite material containing a large number of keratinocytes. The GMHA gel closely connects the cells and cell derivatives to form a tight keratin-like layer with a thickness of 10 μm ~ 1 mm and an uneven surface. The gel has a thickness of 50 ~ With a pore size of 150 μm, cells can be closely arranged within the pore to form a dense and certain closed space inside. This model can provide a suitable growth environment for anaerobic bacteria and aerobic bacteria on the skin, and microorganisms can grow together on the model. .

Example 2

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

(1) GMHA synthesis: Dissolve 1g hyaluronic acid in 100mL PBS buffer, add 7.5mL triethylamine, 7.5g tetrabutylammonium bromide, and 7.5mL glycidyl methacrylate to the hyaluronic acid solution in sequence . Among them, triethylamine is the catalyst and tetrabutylammonium bromide is the phase transfer catalyst. Set the reaction temperature to 20°C and react for 30 to 40 hours with constant stirring. After the reaction, the reaction solution was transferred to a dialysis bag (cutoff 3.5×10 ³ Da) and dialyzed against double-distilled water for three days. The dialyzed reaction solution is freeze-dried to obtain GMHA freeze-dried product.

(2) HaCaT cells and sebaceous gland cell culture and collection steps: HaCaT cells and sebaceous gland cells are cultured in DMEM complete culture medium in a CO ₂ incubator at 37±0.5°C, 5% CO ₂ and saturated humidity until HaCaT cells and sebaceous gland cells are Collect cells when the confluence reaches 80%-90%, remove the original culture medium, add 2 mL PBS buffer to rinse twice, add 1.5 mL EDTA-containing trypsin for digestion for 6 to 8 minutes, add 2 mL DMEM complete culture medium to terminate digestion, and centrifuge to collect The cells were washed twice with PBS buffer, and the supernatant was removed, resuspended in PBS buffer, and counted.

(3) Establish a stratum corneum model: Take 100 μL of 5 mg/mL GMHA and add 0.15 mg of LAP, mix well in the dark, then place it into a 24-well plate containing 1 mL of 1.5% agar, and irradiate it under a 405 nm UV lamp for 5 to 15 minutes. , after solidification, add approximately 6×10 ⁵ HaCaT cells and 6×10 ⁴ sebaceous gland cells that are evenly mixed, and place them in a 37°C biochemical incubator to dry for 12 to 48 hours.

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

(1) Culture steps for Propionibacterium acnes, Staphylococcus aureus, and Staphylococcus epidermidis: Propionibacterium acnes is cultured in an anaerobic gas-generating bag at 37°C for 48 hours using enhanced Propionibacterium clostridium liquid culture medium, and the bacterial liquid is taken after culture Centrifuge to remove the supernatant, and resuspend the bacterial solution to a certain concentration. Staphylococcus aureus is cultured in tryptic soybean liquid medium at 37°C for 24 hours. After culture, the bacterial liquid is centrifuged to remove the supernatant, and the bacterial liquid is resuspended to a certain concentration. Staphylococcus epidermidis The bacteria were cultured in LB liquid medium at 37°C for 24 hours. After culture, the bacterial liquid was centrifuged to remove the supernatant, and the bacterial liquid was resuspended to a certain concentration.

(2) Plate counting method to detect whether bacteria can survive on the stratum corneum:

About 1×10 ⁶ CFU of Propionibacterium acnes was inoculated into the model, cultured in a 35°C biochemical incubator, and the number of bacteria was detected on days 0, 2, 3, 4, and 5 of culture. The specific operation method is to chop the model into pieces with a knife, put it into a 15mL centrifuge tube, add 6mL PBS buffer, and vortex for 30 minutes to elute and isolate the bacteria. Suspend the bacterial liquid with PBS, dilute it several times, spread it on the culture medium plate, and incubate anaerobically for 48 hours. Count the number of bacterial colonies growing on the plate, and calculate the number of bacterial liquid according to the plate counting method. It can be seen from Figure 9 that P. acnes can survive on the stratum corneum and maintain a steady state within a certain range.

(3) Detection of colonization and formation of biofilm by cuticle-like microorganisms:

Use the stratum corneum prepared in 2.3 above and inoculate it with about 1×10 ⁷ CFU of Propionibacterium acnes and culture it at 35°C for 48 hours. After culture, they were lyophilized in a freeze dryer and images of the microorganisms colonizing the cuticle were taken using a scanning electron microscope. The results are shown in Figure 10. In the cuticle model at 400 times, it can be clearly seen that the cells are cross-linked by GMHA to form a dense cuticle. Figure 11 is a partial enlarged view of Figure 10. Propionibacterium acnes establishes a biofilm on the surface of the stratum corneum and colonizes and grows.

(4) PMA-qPCR quantitative detection of microbial growth on the stratum corneum:

Different concentrations of Propionibacterium acnes, Staphylococcus aureus, and Staphylococcus epidermidis were inoculated into the model, cultured in a 35°C biochemical incubator, and detected on days 0, 1, 3, 5, and 9 of culture respectively. Number of bacteria. Chop the model into pieces with a knife, put it into a 15mL centrifuge tube, add 6mL PBS buffer, and vortex for 30 minutes to elute and isolate the bacteria. Add 490 μL of PBS to resuspend the bacteria, add 10 μL of 100 μg/mL PMA solution, mix well in the dark, and incubate at room temperature for 10 min. Illuminate with a 500W tungsten light for 10 min. Use the Bacteria DNA isolation Mini Kit to extract bacterial DNA according to the instructions, and use ChamQ SYBR qPCR Master MiX and the bacterial 16SrRNA-specific primer sequence in Table 1 to perform q-PCR detection.

Table 1

The cycle threshold (Ct value) calculated by real-time fluorescence quantitative PCR is brought into the pre-established standard curve related to the number of bacteria and the Ct value, and the number of bacteria is calculated; as can be seen from Figures 12, 13, and 14, at different concentrations Acne C After Acidobacterium, Staphylococcus aureus, and Staphylococcus epidermidis are inoculated into the stratum corneum, they can maintain a relatively stable state for a period of time.

The stratum corneum is available for microbial co-growth and microbial interspecies interactions can be detected.

Inoculate Staphylococcus aureus and Propionibacterium acnes at about 1×10 ⁷ CFU/mL and grow them on the model. Separate culture groups for Staphylococcus aureus and Propionibacterium acnes are set up. Staphylococcus aureus and Propionibacterium acnes are cultured together. In the culture group, the number of viable bacteria was measured every 24 hours. It can be seen from Figure 15 that when Staphylococcus aureus and Propionibacterium acnes are co-cultured, Propionibacterium acnes can promote the growth of Staphylococcus aureus, which is consistent with what has been reported in the literature. Propionibacterium acnes produces a small molecule coproporphyrin III that promotes Staphylococcus aureus aggregation and biofilm formation.

In summary, the present invention establishes a stratum corneum-like layer for the growth of several common skin microorganisms, provides a new model for studying human microorganisms and its production method, and provides a useful method for in vitro evaluation of skin care product efficacy and screening of active ingredient efficacy. and provide new ideas and methods for further research on antibiotic efficacy evaluation.

Example 3

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

The specific steps are as follows: The steps and results of influencing several facial bacteria are as follows:

(1) Facial bacteria culture: Select Propionibacterium acnes, Staphylococcus aureus, and Staphylococcus epidermidis as facial bacteria; Propionibacterium acnes is cultured in an anaerobic gas-generating bag at 37°C using enhanced Clostridium propionibacterium liquid culture medium. After 48 hours of culture, take the bacterial liquid and centrifuge to remove the supernatant, then re-suspend the bacterial liquid to prepare a certain concentration. Staphylococcus aureus is cultured in tryptic soybean liquid medium at 37°C for 24 hours. After culture, the bacterial liquid is centrifuged to remove the supernatant, and the bacterial liquid is resuspended to a certain concentration. Staphylococcus epidermidis was cultured in LB liquid medium at 37°C for 24 hours. After culture, the bacterial liquid was centrifuged to remove the supernatant, and the bacterial liquid was resuspended to a certain concentration.

(2) Configure the lotion sample to be tested:

According to the formula in Table 2, weigh the _A2 , _A3 , _A4 and _A5 phases, add an appropriate amount of deionized water, and fully dissolve them in a 50-60°C water bath. Weigh B ₂ , B ₃ , B ₄ and preservatives of various concentrations, put them in a 50-60°C water bath, add phase B ₁ after they are fully dissolved, and mix well. Mix phase A and phase B, mix thoroughly, then measure the pH value, and add an appropriate amount of sodium citrate to pH 5-6. Add deionized water to 100%. Add preservatives and concentrations according to Table 3.

Table 2

table 3

Through the above method, a total of 5 kinds of lotion samples to be tested were prepared, including the lotion sample without any preservatives, the lotion sample containing methyl paraben with a concentration of 0.1% (NI-0.1), and the lotion sample with a concentration of 0.2 The lotion sample containing 0.6% methyl paraben (NI-0.2), the lotion sample containing 0.6% phenoxyethanol (BEN-0.6), and the lotion sample containing 0.8% phenoxyethanol (BEN -0.8).

(3) Simulate facial microecology: Take a cuticle-like model and inoculate it with Propionibacterium acnes, Staphylococcus aureus, and Staphylococcus epidermidis at the same time to simulate facial microecology;

Based on the top surface area of the stratum corneum model, inoculate approximately 1×10 ⁷ CFU of Propionibacterium acnes, 0.5×10 ⁷ CFU of Staphylococcus epidermidis, and 2.5×10 ⁶ CFU of Staphylococcus aureus per ¹ cm 2 amount, inoculate three kinds of bacteria on the cuticle-like model at the same time, and culture it in a 37°C incubator for 48 hours to obtain a cuticle-like model that has been inoculated with facial bacteria. Repeat 15 times to obtain 15 stratum corneum-like models inoculated with facial bacteria.

Based on the top surface area of the cuticle-like model, add the five lotion samples to be tested into five cuticle models that have been inoculated with facial bacteria at an amount of approximately 50 μL per 1 ^cm2 , and set up three replicates. Incubate at 37°C for 24 hours and detect the number of bacteria on the model. As shown in Figure 16, adding preservatives to lotion water will significantly reduce the number of three bacteria, indicating that adding phenoxyethanol or methylparaben preservatives to cosmetics will affect the facial flora. As the concentration of preservatives increases, facial bacteria The degree of growth inhibition of the group also increased.

In this example, three common facial bacteria were inoculated on the stratum corneum and co-cultured for in vitro evaluation of the impact of lotions containing phenoxyethanol or methylparaben preservatives on facial flora. The experiment showed that the use of lotions containing benzene Lotions containing oxyethanol or methylparaben preservatives may inhibit the growth of facial flora and destroy the facial microecology.

Preservatives in cosmetics can alter the balance of the skin microbiota, but the effects of different preservative combinations on the dynamics of the skin's resident flora can be tested through in vitro models, allowing for the correct selection and dosage of preservatives in cosmetic formulations to maintain or restore skin. Provide reference for the homeostasis of microorganisms.

Example 4

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

(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:

Cells were cultured in DMEM complete culture medium at 37±0.5°C, 5% _CO2 and saturated humidity in a _CO2 incubator. Culture until the cell confluence reaches 80%-90%. Collect the cells, remove the original culture medium, and add PBS. Rinse twice with buffer, add EDTA-containing trypsin for digestion for 6 to 8 minutes, add complete culture medium to terminate digestion, collect cells by centrifugation, rinse twice with PBS buffer, and remove the culture medium.

(3) Establish a stratum corneum model: Take 100 μL of 5-50 mg/mL photocurable gels CMA, CSMA, GMA, and HAMA, add 0.15% to 0.6% of the photoinitiator LAP or I2959, and put it into 24 In the hole of the well plate, irradiate it under ultraviolet light for 5 to 120 seconds. After solidification, add a sufficient amount of keratinocytes, sebaceous gland cells or fibroblasts that are evenly mixed, and place it in a 37°C biochemical incubator to dry for 12 to 48 hours.

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 stratum corneum-like models as demonstrated below:

(1) Evaluation of gel-forming properties of photocurable gel

Take the photocurable gel CMA, add a photoinitiator, and irradiate it under a UV lamp for 5 to 120 seconds to solidify to form a gel.

Take the photocurable gel CSMA, add a photoinitiator, and irradiate it under a UV lamp for 5 to 120 seconds to solidify to form a gel.

Photocurable gels CMA, GMHA, CSMA, and GMA have been reported in the literature to be able to form gels.

(2) Evaluation of biocompatibility of gel and cells with facial bacteria

Facial bacteria culture: Select Propionibacterium acnes, Staphylococcus aureus, and Staphylococcus epidermidis as facial bacteria; Propionibacterium acnes is cultured in an anaerobic gas-generating bag at 37°C for 48 hours using enhanced Clostridium propionibacterium liquid culture medium. Centrifuge the bacterial liquid to remove the supernatant, and resuspend the bacterial liquid to a certain concentration. Staphylococcus aureus is cultured in tryptic soybean liquid medium at 37°C for 24 hours. After culture, the bacterial liquid is centrifuged to remove the supernatant, and the bacterial liquid is resuspended to a certain concentration. Staphylococcus epidermidis was cultured in LB liquid medium at 37°C for 24 hours. After culture, the bacterial liquid was centrifuged to remove the supernatant, and the bacterial liquid was resuspended to a certain concentration.

(2.2) Mix 100 μL of a certain concentration of photocurable gel solution with an equal amount of human primary stratum corneum cells, human primary sebaceous gland cells, human primary fibroblasts, and human immortalized fibroblasts (HSF cells) for about 2 times each. ×10 ⁴ cells were mixed and added to a 96-well plate, and the control group was bacterial culture medium. Resuspend several bacteria in the corresponding culture medium (OD ₆₀₀ is about 0.06), add 100 μL to the photocurable gel solution, and co-culture in a 96-well plate at 37°C. 24h to 48h, vortex for 30 minutes after incubation, and then centrifuge at 800 rpm for 5 minutes. Take 100 μL of the supernatant and use a microplate reader to measure the absorbance of the bacterial solution at 600 nm. If the concentration of bacterial solution in the gel solution group is not significantly reduced compared with the control group, it means that the gel, cells and bacteria used have good biocompatibility and can be used to construct a 3D cuticle-like layer for microbial growth. Model. It has been reported in the literature that photocurable gels CMA, GMHA, CSMA, and GMA all have good cell compatibility, and cells encapsulated in the gel can grow normally.

Figure 19 shows a mixture of a certain concentration of CMA or CSMA with human primary keratinocytes, human primary sebaceous gland cells, human primary fibroblasts, human immortalized fibroblasts (HSF cells) and then mixed with Propionibacterium acnes in 96 wells. Bacterial liquid cultured anaerobically in the plate for 48 hours.

Figure 20 shows a mixture of 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) and then incubated with Staphylococcus epidermidis in a 96-well plate. Bacterial liquid cultured for 24 hours.

Example 5

The cuticle-like layer established in Example 2 was used for detection of microbial colonization and biofilm formation:

Different concentrations of Malassezia were inoculated into the model and cultured in a 35°C biochemical incubator. The number of fungi was detected by plate counting on days 0, 1, 3, 5, 7, and 9 of culture. . The specific operation method is to chop the model into pieces with a knife, put it into a 15mL centrifuge tube, add 6mL of PBS buffer, and vortex for 30 minutes to elute and isolate the bacteria. Suspend the bacterial liquid with PBS, dilute it several times, spread it on the olive oil culture medium plate, incubate it in a constant temperature incubator for 48 hours, count the number of bacterial colonies growing on the plate, and calculate the number of bacterial liquid according to the plate counting method. . It can be seen from Figure 21 that Malassezia can survive on the cuticle and maintain a steady state within a certain range.

Example 6

This embodiment provides a method for simulating the composition of microorganisms on the skin surface. This method can obtain a surface microbial composition similar to that of real skin by continuously inoculating facial flora on the stratum corneum layer multiple times, thereby simulating the composition of microorganisms on the skin surface. the goal of.

Specifically, the stratum corneum-like layer established in Example 2 was continuously inoculated with facial flora multiple times to simulate the composition of microorganisms on the skin surface:

The method of simulating the composition of microorganisms on the skin surface includes the following steps:

(1) Use cotton swabs to collect bacterial flora from the human face, inoculate the bacterial flora onto the 3D stratum corneum model multiple times, and culture for 4 consecutive days. The bacterial flora were collected from human faces and inoculated onto the model for culture.

(2) Collect bacterial colonies from the model on days 0, 1, and 4, chop the model into pieces with a knife, put it into a 15mL centrifuge tube, add 6mL PBS buffer, vortex for 30 minutes, and elute and isolate the bacteria. group. Resuspend the bacterial colony in PBS, add PMA solution to the suspension, mix well in the dark, and incubate at room temperature for 10 minutes, then illuminate with 500W tungsten light for 10 minutes.

(3) Collect the bacterial colonies by centrifugation, use the Bacteria DNA isolation Mini Kit to extract bacterial DNA according to the instructions, and use 16Sr RNA sequencing technology to detect and analyze the microbial communities (n=4).

After the bacterial flora obtained from the human face is inoculated onto the model several times in a row, it will form a group similar to the initial flora. The composition of the microbial flora changed after one day of inoculation and culture, but after four days of continuous culture, the 3D cuticle-like model formed a flora composition similar to the initial flora. As shown in Figure 22, there is a significant difference between the groups on the 0th day of culture and the first day (anosim test, P<0.05). The groups are similar on the 0th day and the fourth day of culture, and there is no significant difference (anosim test, P >0.05).

Figure 23 is the result of non-metric multidimensional scaling analysis (NMDS), showing the compositional differences of microbial communities through a two-dimensional ordination diagram. The closer (far) the distance between the two points in Figure 23 is, the smaller (larger) the difference in the microbial communities in the two samples is. It can be seen that the compositional difference in the microbial communities between day0 and day4 is small.

It can be seen from Figure 24 that compared with the bacterial flora on day 0, after one day of culture of the bacterial flora collected from the human face in the 3D cuticle-like model, the abundance of the top twenty genera with relative abundance in the bacterial flora changed significantly. Variety. When the 3D cuticle model was cultured on the fourth day, the abundance proportions of the top twenty genera with relative abundance were very similar to those on day 0.

It can be seen from Figures 22, 23 and 24 that by inoculating the facial flora multiple times on the 3D stratum corneum-like model, the skin surface flora can be reproduced on the 3D stratum corneum-like model.

Claims

A 3D stratum corneum-like model, characterized in that the 3D stratum corneum-like model includes a photocurable gel skeleton, and the photocurable gel skeleton is loaded with apoptotic cells/or cell derivatives; the photocurable gel skeleton is The solidified gel skeleton is composed of biomacromolecules with double bonds in their side chains that can be cross-linked by irradiation with light.
The 3D stratum corneum-like model according to claim 1, wherein the pores formed by the photocurable gel skeleton have a pore diameter of 1 to 500 μm.
The 3D stratum corneum-like model according to claim 1, wherein the thickness of the 3D stratum corneum-like model is 10 μm to 1 mm.
The 3D stratum corneum-like model according to claim 1, wherein the cells are sebaceous gland cells and skin keratinocytes.
The 3D stratum corneum-like model according to claim 4, wherein skin keratinocytes account for 10% to 90% of the total number of cells in the 3D stratum corneum-like model.
A method for constructing a 3D stratum corneum-like model according to any one of claims 1 to 5, characterized in that it includes the following steps: photocuring a photocurable gel with apoptotic keratinocytes and sebaceous gland cells to obtain 3D cuticle-like model.
The method for constructing a 3D stratum corneum-like model according to claim 6, characterized in that the photocuring method is to add 0.15% to 0.3% photoinitiator to 5-50 mg/mL photo-curable gel, and put In the well plate, under the condition of light irradiation, add uniformly mixed keratinocytes and sebaceous gland cells, and dry at a constant temperature.
The method for constructing a 3D stratum corneum-like model according to claim 6, characterized in that the photocuring method is to mix 0.15% to 0.3% of the photoinitiator evenly with several keratinocytes and sebaceous gland cells, and the resulting mixture The solution is added to a 5-50 mg/mL photocurable gel, placed in a well plate, and under light irradiation conditions, a sufficient amount of cells is then added and dried at a constant temperature. The initiator is any one of LAP and I2959. .
An application of the 3D stratum corneum-like model according to any one of claims 1 to 5 in in vitro evaluation of the efficacy of cosmetics or their raw materials, efficacy screening of cosmetic active ingredients, or efficacy evaluation of biological preparations.
The application according to claim 9, characterized in that the application method includes the following steps:

(1) Inoculate several kinds of facial bacteria on the 3D stratum corneum model to simulate the facial microecology;

(2) Coat the sample to be tested on the 3D stratum corneum model that has been inoculated with facial bacteria;

(3) After a period of time, detect the number of bacteria on the 3D cuticle-like model or the relative abundance of each bacteria in the bacterial community, and the results obtained are used to evaluate or screen the test samples.