阅读说明：本技术 一种体外细胞与3d表皮模型共培养的化妆品致敏评价方法 (Cosmetic sensitization evaluation method for in-vitro cell and 3D epidermis model co-culture ) 是由 王飞飞 高涵 王博 马骁 高绍阳 郭振宇 于 2021-07-20 设计创作，主要内容包括：本发明公开了一种体外细胞与3D表皮模型共培养的化妆品致敏评价方法,结合细胞模型和3D表皮模型检测结果,将待测物作用于所建立的致敏模型中,筛选能够抑制或促进目标分子表达的物质,从而对产品的致敏安全性进行评价,具有快速、高效的优势；为皮肤护理产品的致敏性提供快捷可靠的评价方法,为原料的复配方案提供参考依据,也为护肤品行业动物替代实验、皮肤护理产品开发及功效验证的进一步研究提供有效依据。(The invention discloses a cosmetic sensitization evaluation method by co-culturing in vitro cells and a 3D epidermal model, which combines the detection results of the cell model and the 3D epidermal model, enables an object to be tested to act on the established sensitization model, screens substances capable of inhibiting or promoting the expression of target molecules, thereby evaluating the sensitization safety of products and having the advantages of rapidness and high efficiency; the rapid and reliable evaluation method is provided for the sensitization of skin care products, a reference basis is provided for the compounding scheme of raw materials, and an effective basis is provided for further researches of animal substitution experiments, skin care product development and efficacy verification in the skin care product industry.)

1. A cosmetic sensitization evaluation method of in vitro cell and 3D epidermis model coculture is characterized by comprising the following steps:

step S1, THP-1 cell culture and 3D epidermal culture;

step S2, THP-1 cell and 3D epidermis activity detection determine the detection concentration of the experimental sample, including the following procedures:

step S21, adding an experimental sample into the 3D epidermis model with stable growth state in the step S1, wherein the experimental sample is a cosmetic stock solution to be detected, the contrast sample is a culture medium solution without any sample, and after culturing for 18-24h, detecting the tissue survival rate of the 3D epidermis model by using MTT;

step S22, screening according to the step S21 to obtain the concentration of the corresponding experimental sample as the administration concentration of the 3D epidermal model when the tissue survival rate is more than 50%;

step S3, cell plating and epidermal model combined culture step: inoculating the THP-1 cells logarithmically grown in the step S1 to a 6-hole cell culture plate, and respectively arranging a blank control group, a negative control group, a positive control group and a to-be-detected substance treatment group, wherein the blank control group is added with a complete culture medium, the negative control group is added with lactic acid, the positive control group is added with cinnamaldehyde, and the to-be-detected substance treatment group is added with a to-be-detected substance; meanwhile, the 3D epidermis model is placed above the THP-1 cells in the plate of the 6-hole cell culture plate, and air bubbles are not generated in the placing process; then the blank control, the negative control, the positive control and the substance to be detected are correspondingly coated on the 3D epidermis models of the blank control group, the negative control group, the positive control group and the substance to be detected one by one with the dosage of 100-,

step S4, subject exposure step: then transferring the 6-hole cell culture plate in the step S3 into a CO2 incubator with the temperature of 37 ℃, the CO2 percent and the relative humidity of 95 percent, and culturing for 18-24 h;

step S5, a rinsing step: taking out the 6-hole cell culture plate, and washing out the blank control, the negative control, the positive control and the substance to be detected on the surface of the 3D epidermis model by using PBS;

step S6, MTT tissue activity detection step, which comprises the following steps:

s61, the step of processing the epidermis model MTT: taking the 3D epidermis model washed in the step S5 out of the 6-hole cell culture plate, taking a 24-hole cell culture plate, putting the 24-hole cell culture plate into 200 mu L of 1mg/mL MTT working solution, transferring the 3D epidermis model onto the 24-hole cell culture plate, and putting the 3D epidermis model into a CO2 incubator with the temperature of 37 ℃, the CO2 concentration of 5% and the relative humidity of 95%, wherein the culture time is 2-3 h;

s62, detecting the activity of the epidermal model: taking a new 24-pore plate, firstly adding 1000 mu L of isopropanol into the 24-pore plate, transferring the 3D skin model treated in the step S6 into the 24-pore plate, not generating bubbles in the placing process, adding 1000 mu L of isopropanol into the 3D skin model for extraction, covering and sealing the 24-pore plate by using a sealing film, covering a cover and sealing a layer after sealing a layer, and keeping the 24-pore plate out of the sun overnight; taking out the 24-hole plate, and uniformly mixing on a shaking table for 1-2 h; mixing the solutions, detecting by enzyme labeling instrument at 570nm, wherein the blank hole of the enzyme label plate is isopropanol, and the formazan extract solution is 200 μ L/hole and 6 multiple holes; taking a 3D epidermis model with the tissue survival rate of more than 50 percent to carry out subsequent cell sensitization experimental test;

step S7, in vitro cell sensitization model establishment step, which comprises the following steps:

step S71, a step of collecting THP-1 cells, namely, after the 3D epidermal model washed in the step S5 is taken out of the 6-hole cell culture plate, respectively transferring THP-1 cells corresponding to a blank control group, a negative control group, a positive control group and a to-be-detected object treatment group into corresponding 1mL EP tubes from the 6-hole cell culture plate, centrifugally precipitating the THP-1 cells, washing the THP-1 cells once by using FACS buffer solution, and centrifugally collecting the THP-1 cells again;

step S72, FcR blocking step: preparing an FcR blocker with a proper concentration according to the number of the THP-1 cells, wherein 2.5 mu g of the FcR blocker is needed for one million cells with the volume of 50-100 mu L, and then blocking the THP-1 cells obtained by centrifugation;

step S73, antibody staining step: distributing the blocked THP-1 cells into 4 EP tubes with 1mL according to 50 mu L per tube, and adding antibodies FITC-labeled CD86 antibody, PE-labeled CD54 antibody, FITC-labeled mouse isotype control IgG1 and PE-labeled mouse isotype control IgG1 into the 4 EP tubes with 1mL in a one-to-one correspondence manner; dyeing for 30min at 4 ℃ in a dark room; washing with FACS buffer solution for 2 times, then resuspending the cells with 500. mu. LFACS buffer solution and transferring to 5mL flow tube for detection; simultaneously staining 7-AAD under each experimental condition, and determining the cell viability and corresponding antibody staining condition under the concentration;

step S74, flow cytometry: collecting and analyzing 10000 THP-1 cells, adjusting the experiment voltage of an experiment FSC vs SSC through the THP-1 cells of a blank control group without treatment so as to ensure the accuracy of the calculation result of the subsequent experiment of the THP-1 cells of the batch, obtaining the expression quantity of the target molecules on the cell surface, and establishing an in-vitro cell sensitization model taking the expression quantity of the target molecules as the reference;

and step S8, combining the in vitro cell sensitization model, evaluating the sensitization of the substance to be detected by referring to the method of step S7, calculating the change of the relative expression quantity of the target molecule caused by the substance to be detected by taking the expression quantity of the target molecule as a reference, and expressing the sensitization intensity by using the change value of the relative expression quantity of the target molecule, wherein the larger the change value of the relative expression quantity of the target molecule is, the stronger the potential sensitization of the substance to be detected is.

2. The method for evaluating the sensitization of cosmetics by CO-culturing in vitro cells and 3D epidermis model according to claim 1, wherein, in step S1, THP-1 cells are cultured in RPMI-1640+ 10% FBS complete medium, all the culture conditions are 37 ± 0.5 ℃, and the concentration of (5 ± 1)% CO2 and the saturation humidity.

3. The method for evaluating the sensitization of cosmetics by co-culturing cells in vitro and a 3D epidermal model according to claim 1, wherein the 3D epidermal model is a SkinEthic skin model in step S1.

4. The method for evaluating the sensitization of cosmetics by co-culturing in vitro cells and 3D epidermis model according to claim 1, wherein in step S3, the THP-1 cells are cultured at 10%⁶cells/mL were plated in 6 well cell culture plates at 1mL medium per well, with 3 replicate wells per well.

5. The method for evaluating the sensitization of cosmetics by co-culture of in vitro cells and a 3D epidermal model according to claim 1, wherein the lactic acid added to the negative control group is a lactic acid solution diluted to a volume concentration of 0.1% by cell culture medium in step S3; the cinnamaldehyde is added into the positive control group, and is a cinnamaldehyde solution diluted by a cell culture medium to a volume concentration of 0.5%.

6. The method for evaluating the sensitization of cosmetics by co-culture of in vitro cells and a 3D epidermis model according to claim 1, wherein in step S3, the substances to be tested are sunscreen spray, anti-aging sunscreen lotion, sunscreen gel, physical sunscreen lotion, male sunscreen lotion, whitening sunscreen spray and clear sunscreen lotion.

7. The method for evaluating the sensitization of cosmetics by co-culture of in vitro cells and 3D epidermis model according to claim 1, wherein in step S7, the target molecules are cell surface marker CD86 and cell surface marker CD 54.

Technical Field

The invention relates to a cosmetic sensitization evaluation method for in-vitro cell and 3D epidermis model co-culture, and belongs to the technical field of cosmetics.

Background

Allergy refers to the abnormal reaction of the human immune system to the exclusion of certain substances such as bacteria, viruses, pollen, dust mites, fur and drugs. After the allergic substances (namely allergens, also called allergens) enter a human body, certain tissue cells can release active substances, and the active substances can shrink smooth muscles, increase capillary permeability and increase mucous gland secretion, so that allergic people often have symptoms of red and swollen epidermis, pruritus, plaque or laryngeal, bronchial spasm, gastrointestinal spasm and the like. Allergic reactions refer to the abnormal reaction of the human body after exposure to an antigenic substance. The general manifestations of epidermal sensitization are dryness, itching, redness and swelling, pigmentation, etc. It is different from normal immune response, not only does not play a protective role, but also causes physiological dysfunction and tissue damage due to over-strong reaction.

In the induction stage of epidermal sensitization, Langerhans cells play a role in presenting and modifying antigens and initiating immune responses. After capturing antigens, Langerhans cells differentiate, mature and migrate to regional lymph nodes, delivering the antigens to T cells, and promoting T cell proliferation. During this process, langerhans cells are activated and mature, as evidenced by upregulation of expression of co-stimulatory molecules (CD86 and CD 54). The cell surface markers CD86, CD54 could be detected in induced THP-1 cells.

Traditional epidermal sensitivity prediction is mainly based on animal experiments, including guinea pig maximum value test, local closed skin test and animal local lymph node test, but on one hand, due to higher cost and low flux of animal experiments, the requirement of rapid development of the cosmetic industry is difficult to meet, and on the other hand, due to enhancement of animal welfare concept and extrapolation uncertainty of animal experiments, non-animal-based in vitro substitution methods are increasingly used for cosmetic safety evaluation.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a cosmetic sensitization evaluation method by co-culture of in vitro cells and a 3D epidermis model, which comprises the steps of establishing the in vitro epidermal cell and 3D epidermis co-culture sensitization model, acting an object to be tested on the established model, screening a substance capable of inhibiting or promoting the expression of a target molecule, and carrying out safety evaluation on the sensitization of a target product by combining the detection results of the in vitro cell model and the 3D epidermis model.

The technical scheme for realizing the purpose is as follows: a cosmetic sensitization evaluation method for in vitro cell and 3D epidermis model coculture comprises the following steps:

step S1, THP-1 cell culture and 3D epidermal culture;

step S2, THP-1 cell and 3D epidermis activity detection determine the detection concentration of the experimental sample, including the following procedures:

step S21, adding an experimental sample into the 3D epidermis model with stable growth state in the step S1, wherein the experimental sample is a cosmetic stock solution to be detected, the contrast sample is a culture medium solution without any sample, and after culturing for 18-24h, detecting the tissue survival rate of the 3D epidermis model by using MTT;

step S22, screening according to the step S21 to obtain the concentration of the corresponding experimental sample as the administration concentration of the 3D epidermal model when the tissue survival rate is more than 50%;

step S3, cell plating and epidermal model combined culture step: inoculating the THP-1 cells logarithmically grown in the step S1 to a 6-hole cell culture plate, and respectively arranging a blank control group, a negative control group, a positive control group and a to-be-detected substance treatment group, wherein the blank control group is added with a complete culture medium, the negative control group is added with lactic acid, the positive control group is added with cinnamaldehyde, and the to-be-detected substance treatment group is added with a to-be-detected substance; meanwhile, the 3D epidermis model is placed above the THP-1 cells in the plate of the 6-hole cell culture plate, and air bubbles are not generated in the placing process; then the blank control, the negative control, the positive control and the substance to be detected are correspondingly coated on the 3D epidermis models of the blank control group, the negative control group, the positive control group and the substance to be detected one by one with the dosage of 100-,

step S4, subject exposure step: the 6-well cell culture plates from step S3 were then transferred to 37 ℃ with 5% CO₂CO at a relative humidity of 95%₂Culturing for 18-24h in an incubator;

step S5, a rinsing step: taking out the 6-hole cell culture plate, and washing out blank control, negative control, positive control and substances to be detected on the surface of the 3D epidermis model by PBS (phosphate buffered saline);

step S6, MTT tissue activity detection step, which comprises the following steps:

s61, the step of processing the epidermis model MTT: taking the 3D epidermis model washed in the step S5 out of the 6-hole cell culture plate, taking a 24-hole cell culture plate, putting the 24-hole cell culture plate into 200 mu L of 1mg/mL MTT working solution, transferring the 3D epidermis model onto the 24-hole cell culture plate, and putting the 3D epidermis model into a CO2 incubator with the temperature of 37 ℃, the CO2 concentration of 5% and the relative humidity of 95%, wherein the culture time is 2-3 h;

s62, detecting the activity of the epidermal model: taking a new 24-pore plate, firstly adding 1000 mu L of isopropanol into the 24-pore plate, transferring the 3D skin model treated in the step S6 into the 24-pore plate, not generating bubbles in the placing process, adding 1000 mu L of isopropanol into the 3D skin model for extraction, covering and sealing the 24-pore plate by using a sealing film, covering a cover and sealing a layer after sealing a layer, and keeping the 24-pore plate out of the sun overnight; taking out the 24-hole plate, and uniformly mixing on a shaking table for 1-2 h; mixing the solutions, detecting by enzyme labeling instrument at 570nm, wherein the blank hole of the enzyme label plate is isopropanol, and the formazan extract solution is 200 μ L/hole and 6 multiple holes; taking a 3D epidermis model with the tissue survival rate of more than 50 percent to carry out subsequent cell sensitization experimental test;

step S7, in vitro cell sensitization model establishment step, which comprises the following steps:

step S71, a step of collecting THP-1 cells, namely, after the 3D epidermal model washed in the step S5 is taken out of the 6-hole cell culture plate, respectively transferring THP-1 cells corresponding to a blank control group, a negative control group, a positive control group and a to-be-detected object treatment group into corresponding 1mL EP tubes from the 6-hole cell culture plate, centrifugally precipitating the THP-1 cells, washing the THP-1 cells once by using FACS buffer solution, and centrifugally collecting the THP-1 cells again;

step S72, FcR blocking step: preparing an FcR blocker with a proper concentration according to the number of the THP-1 cells, wherein 2.5 mu g of the FcR blocker is needed for one million cells with the volume of 50-100 mu L, and then blocking the THP-1 cells obtained by centrifugation;

step S73, antibody staining step: distributing the blocked THP-1 cells into 4 EP tubes with 1mL according to 50 mu L per tube, and adding antibodies FITC-labeled CD86 antibody, PE-labeled CD54 antibody, FITC-labeled mouse isotype control IgG1 and PE-labeled mouse isotype control IgG1 into the 4 EP tubes with 1mL in a one-to-one correspondence manner; dyeing for 30min at 4 ℃ in a dark room; washing with FACS buffer solution for 2 times, then resuspending the cells with 500. mu. LFACS buffer solution and transferring to 5mL flow tube for detection; simultaneously staining 7-AAD under each experimental condition, and determining the cell viability and corresponding antibody staining condition under the concentration;

step S74, flow cytometry: collecting and analyzing 10000 THP-1 cells, adjusting the experiment voltage of an experiment FSC vs SSC through the THP-1 cells of a blank control group without treatment so as to ensure the accuracy of the calculation result of the subsequent experiment of the THP-1 cells of the batch, obtaining the expression quantity of the target molecules on the cell surface, and establishing an in-vitro cell sensitization model taking the expression quantity of the target molecules as the reference;

and step S8, combining the in vitro cell sensitization model, evaluating the sensitization of the substance to be detected by referring to the method of step S7, calculating the change of the relative expression quantity of the target molecule caused by the substance to be detected by taking the expression quantity of the target molecule as a reference, and expressing the sensitization intensity by using the change value of the relative expression quantity of the target molecule, wherein the larger the change value of the relative expression quantity of the target molecule is, the stronger the potential sensitization of the substance to be detected is.

In the method for evaluating cosmetic sensitization by CO-culture of in vitro cells and 3D epidermis models, in step S1, THP-1 cells are cultured by RPMI-1640+ 10% FBS complete medium, all culture conditions are 37 +/-0.5 ℃, the concentration of (5 +/-1)% CO2 and the saturation humidity.

In the method for evaluating cosmetic sensitization by co-culturing in vitro cells and 3D epidermal model, in step S1, the 3D epidermal model is a SkinEthic skin model

In the method for evaluating cosmetic sensitization by co-culturing in vitro cells and 3D epidermal model, in step S3, the THP-1 cells are counted by 10⁶The cells/mL density inoculated to 6 hole cell culture plate, each hole culture medium 1mL, each culture hole set up 3 multiple holes

In the method for evaluating cosmetic sensitization by co-culturing in vitro cells and a 3D epidermal model, in step S3, the lactic acid added to the negative control group is a lactic acid solution prepared by diluting lactic acid with a cell culture medium to a volume concentration of 0.1%; the cinnamaldehyde is added into the positive control group, and is a cinnamaldehyde solution diluted by a cell culture medium to a volume concentration of 0.5%.

In the method for evaluating cosmetic sensitization through co-culture of in vitro cells and a 3D epidermis model, in step S3, the substances to be tested are sunscreen spray, anti-aging sunscreen milk, sunscreen gel, physical sunscreen milk, male sunscreen milk, whitening sunscreen spray and clear sunscreen milk.

In the method for evaluating cosmetic sensitization by co-culturing in vitro cells and a 3D epidermal model, in step S7, the target molecules are a cell surface marker CD86 and a cell surface marker CD 54.

According to the cosmetic sensitization evaluation method by co-culturing the in-vitro cells and the 3D epidermis model, the detection results of the cell model and the 3D epidermis model are combined, the substance to be tested acts on the established sensitization model, and the active substance capable of inhibiting or promoting the expression of the target molecule is screened, so that the sensitization safety of the product is evaluated, and the method has the advantages of being rapid and efficient; the rapid and reliable evaluation method is provided for the sensitization of skin care products, a reference basis is provided for the compounding scheme of raw materials, and an effective basis is provided for further researches of animal substitution experiments, skin care product development and efficacy verification in the skin care product industry.

Drawings

FIG. 1 is a graph of the results of 3D epidermal activity under the effect of various test substances;

FIG. 2 is a graph showing the results of expression levels of cell surface markers CD86 and CD54 by different test substances.

Detailed Description

In order that those skilled in the art will better understand the technical solution of the present invention, the following detailed description is given with reference to the accompanying drawings:

the reagents and consumables referred to in the examples are shown in table 1:

TABLE 1

The test materials and numbers in the examples are shown in table 2:

numbering	Test article
		#1	Lactic acid
#2	Cinnamic aldehyde
		#3	Sunscreen spray
#4	Anti-aging sunscreen lotion
		#5	Sun-proof gel
#6	Physical sun block
		#7	Sun cream for men
#8	Whitening sunscreen spray
		#9	Clear sunscreen lotion

TABLE 2

Firstly, solution preparation:

1. preparing a FACS buffer: 100mg of BSA powder was dissolved in 100mL of 1xPBS (phosphate buffered saline), and the mixture was filtered through a 0.22 μm filter.

2. FcR blocker working solution: to 100. mu.L of FACS buffer, 0.5. mu.L of FcR blocker antibody was added.

3. 7-AAD working solution: mu.L of 7-AAD was added to 398. mu.L of FACS buffer.

II, an experimental process:

in the best embodiment of the invention, the method for evaluating the cosmetic sensitization of in-vitro cell and 3D epidermis model coculture comprises the following steps:

step S1, THP-1 cell culture and 3D epidermal culture;

(1) THP-1 cell culture:

THP-1 cells were purchased from Shanghai cell banks of Chinese academy and cultured in a 37 ℃ cell culture chamber containing 5% CO2 by adding 10% fetal bovine serum to RPMI-1640 medium according to the instructions. Passages were performed every 3-4 days, 1:3-1:4 passages. The culture is carried out by adopting RPMI-1640+ 10% FBS complete culture medium, all culture conditions are 37 +/-0.5 ℃, the concentration of (5 +/-1)% CO2 and the saturation humidity.

(2)3D epidermal culture:

the SkinEthic skin model was purchased from shanghai scanno biotechnology, ltd, and each batch contained, as indicated: skin model, maintenance medium and detection medium. After arrival of the skin model of skinnethic at the laboratory, the skin model was transferred using forceps into a 6-well plate to which 1mL of maintenance medium had been added, incubated overnight at 37 ℃, 5% CO2 and 95% humidity, and the experiment was performed after the 3D epidermal model was stabilized.

Step S2, THP-1 cell and 3D epidermis activity detection determine the detection concentration of the experimental sample, including the following procedures:

step S21, adding an experimental sample into the 3D epidermis model with stable growth state in the step S1, wherein the experimental sample is a cosmetic stock solution to be detected, the contrast sample is a culture medium solution without any sample, and after culturing for 18-24h, detecting the tissue survival rate of the 3D epidermis model by using MTT;

and step S22, screening according to the step S21 to obtain the concentration of the corresponding experimental sample as the administration concentration of the 3D epidermal model when the tissue survival rate is more than 50%.

Step S3, cell plating and epidermal model combined culture step: inoculating the logarithmic growth THP-1 cells obtained in step S1 to 6-well cell culture plate, centrifuging pre-cultured logarithmic growth THP-1 cells, removing supernatant, suspending the cells with culture medium, and making the concentration be 10⁶Pipetting 1mL of the above resuspension into 6-well cell culture plates at a concentration of 10 cells/mL, and then plating the THP-1 cells⁶Inoculating each cell/mL to a 6-well cell culture plate with 1mL of culture medium per well, and arranging 3 multiple wells per culture well; a blank control group, a negative control group, a positive control group and a substance to be detected treatment group are respectively arranged on the 6-hole cell culture plate, the blank control group is added with a complete culture medium, the negative control group is added with lactic acid, the positive control group is added with cinnamaldehyde, and the substance to be detected is added to the substance to be detected treatment group; the lactic acid added in the negative control group is a lactic acid solution with the volume concentration of 0.1 percent diluted by using a cell culture medium; adding cinnamaldehyde into the positive control group to dilute cinnamaldehyde to a cinnamaldehyde solution with a volume concentration of 0.5% by using a cell culture medium;

meanwhile, the 3D epidermis model is placed above the THP-1 cells in the plate of the 6-hole cell culture plate, and air bubbles are not generated in the placing process; then correspondingly smearing the blank control, the negative control, the positive control and the substance to be detected on the 3D epidermis models of the blank control group, the negative control group, the positive control group and the substance to be detected one by one, wherein the dosage is 150-;

the substances to be tested comprise #3 sunscreen spray, #4 anti-aging sunscreen lotion, #5 sunscreen gel, #6 physical sunscreen lotion, #7 men sunscreen lotion, #8 whitening sunscreen spray and #9 transparent sunscreen lotion.

Step S4, subject exposure step: the 6-well cell culture plates from step S3 were then transferred to 37 ℃ with 5% CO₂CO at a relative humidity of 95%₂Culturing for 20-24h in an incubator.

Step S5, a rinsing step: the 6-well cell culture plate was removed, and the blank control, the negative control, the positive control, and the test substance on the surface of the 3D epidermal model were washed away with PBS (phosphate buffered saline).

Step S6, MTT tissue activity detection step, which comprises the following steps:

s61, the step of processing the epidermis model MTT: taking the 3D epidermis model washed in the step S5 out of the 6-hole cell culture plate, taking a 24-hole cell culture plate, putting the 24-hole cell culture plate into 200 mu L of 1mg/mL MTT working solution, transferring the 3D epidermis model onto the 24-hole cell culture plate, and putting the 3D epidermis model into a CO2 incubator with the temperature of 37 ℃, the CO2 concentration of 5% and the relative humidity of 95%, wherein the culture time is 3 hours;

s62, detecting the activity of the epidermal model: taking a new 24-pore plate, firstly adding 1000 mu L of isopropanol into the 24-pore plate, transferring the 3D skin model treated in the step S6 into the 24-pore plate, not generating bubbles in the placing process, adding 1000 mu L of isopropanol into the 3D skin model for extraction, covering and sealing the 24-pore plate by using a sealing film, covering a cover and sealing a layer after sealing a layer, and keeping the 24-pore plate out of the sun overnight; taking out the 24-hole plate, and uniformly mixing on a shaking table for 1-2 h; mixing the solutions, detecting by enzyme labeling instrument at 570nm, wherein the blank hole of the enzyme label plate is isopropanol, and the formazan extract solution is 200 μ L/hole and 6 multiple holes; and (3) taking the 3D epidermal model with the tissue survival rate of more than 50% to perform subsequent cell sensitization experiment tests.

Step S7, in vitro cell sensitization model establishment step, cell surface target molecule expression quantity detection is needed, the target molecules are cell surface marker CD86 and cell surface marker CD54, and the method specifically comprises the following steps:

step S71, a THP-1 cell collection step, namely, after the 3D epidermal model washed in the step S5 is taken out of the 6-well cell culture plate, the THP-1 cells under the action of each test object are respectively transferred into corresponding 1mL EP tubes from the 6-well cell culture plate, the THP-1 cells are centrifugally precipitated, washed once by FACS buffer solution, and centrifuged again to collect the THP-1 cells;

step S72, FcR blocking step: preparing an FcR blocker with a proper concentration according to the number of the THP-1 cells, wherein 2.5 mu g of the FcR blocker is needed for one million cells with the volume of 50-100 mu L, and then blocking the THP-1 cells obtained by centrifugation;

step S73, antibody staining step: distributing the blocked THP-1 cells into 4 EP tubes with 1mL according to 50 mu L per tube, and adding antibodies FITC-labeled CD86 antibody, PE-labeled CD54 antibody, FITC-labeled mouse isotype control IgG1 and PE-labeled mouse isotype control IgG1 into the 4 EP tubes with 1mL in a one-to-one correspondence manner; dyeing for 30min at 4 ℃ in a dark room; washing with FACS buffer solution for 2 times, then resuspending the cells with 500. mu. LFACS buffer solution and transferring to 5mL flow tube for detection; simultaneously staining 7-AAD under each experimental condition, and determining the cell viability and corresponding antibody staining condition under the concentration;

step S74, flow cytometry: 10000 THP-1 cells are collected and analyzed, the THP-1 cells of a blank control group without treatment adjust the experiment voltage of the experiment FSC vs SSC to ensure the accuracy of the calculation result of the subsequent experiment of the THP-1 cells of the batch, the expression quantity of the target molecules on the cell surface is obtained, and an in-vitro cell sensitization model taking the expression quantity of the target molecules as the reference is established.

And step S8, combining the in vitro cell sensitization model, evaluating the sensitization of the substance to be detected by referring to the method of step S7, calculating the change of the relative expression quantity of the target molecule caused by the substance to be detected by taking the expression quantity of the target molecule as a reference, and expressing the sensitization intensity by using the change value of the relative expression quantity of the target molecule, wherein the larger the change value of the relative expression quantity of the target molecule is, the stronger the potential sensitization of the substance to be detected is.

Third, experimental results

1. 3D epidermal model viability test

See figure 1 and table 3 to evaluate the effect of the test subjects on the viability of the skinned model of skinning.

Numbering	3D epidermal viability (%)
		#1	110.0
#2	85.9
		#3	92.5
#4	82.1
		#5	54.5
#6	53.0
		#7	102.6
#8	54.0
		#9	51.6

TABLE 3

Referring to fig. 1, the results of the 3D epidermal viability changes caused by different subjects, and referring to table 3, the 3D epidermal viability of the subjects #5, #6, #8 and #9 is relatively low, but the 3D epidermal viability of all the subjects exceeds 50%.

THP-1 cell viability assay

The effect of different test substances on THP-1 cell survival is shown in table 4:

numbering	Cell survival rate (%)
		#1	98.2
#2	88.0
		#3	90.6
#4	93.5
		#5	94.0
#6	95.9
		#7	97.0
#8	98.7
		#9	95.4

TABLE 4

3. Detection of target molecule expression level by test substance

The expression levels of cell surface markers CD86 and CD54 by the different test agents are shown in table 5:

numbering	CD86 expression level	CD54 expression level
			#1	135	32
#2	150	219
			#3	122	82
#4	138	77
			#5	133	59
#6	187	223
			#7	118	74
#8	114	80
			#9	147	84

TABLE 5

Referring to fig. 2, the results of the variation of the expression levels of the cell surface markers CD86 and CD54 caused by different test substances, and in combination with table 4 and table 5, test substance #6 can cause the expression levels of the target molecules CD86 and CD54 to be significantly increased, and it is determined that the test substance may have potential sensitization, which requires further research. In addition, no expression level of the target molecule was increased in #3, #4, #5, #7, #8, or #9, and no sensitization was observed.

The method for screening the active substances by combining the tissue activity of the 3D epidermis and the expression of the CD86 and CD54 on the cell surface has high stability and good reproducibility, can be used for screening cosmetic products with specific purposes, and provides a new idea for subsequent research and development.

In conclusion, the cosmetic sensitization evaluation method by co-culturing the in vitro cells and the 3D epidermis model combines the detection results of the cell model and the 3D epidermis model, enables the substance to be detected to act on the established sensitization model, and screens the active substances capable of inhibiting or promoting the expression of the target molecules, so that the sensitization safety of the product is evaluated, and the method has the advantages of being rapid and efficient; the rapid and reliable evaluation method is provided for the sensitization of skin care products, a reference basis is provided for the compounding scheme of raw materials, and an effective basis is provided for further researches of animal substitution experiments, skin care product development and efficacy verification in the skin care product industry.

It should be understood by those skilled in the art that the above embodiments are only for illustrating the present invention and are not to be used as a limitation of the present invention, and that changes and modifications to the above described embodiments are within the scope of the claims of the present invention as long as they are within the spirit and scope of the present invention.