WO2021036146A1

WO2021036146A1 - Phototoxicity testing method based on reconstructed skin model

Info

Publication number: WO2021036146A1
Application number: PCT/CN2019/130203
Authority: WO
Inventors: 王丹丹; 李潇; 张艳云; 胡成虎
Original assignee: 广东博溪生物科技有限公司
Priority date: 2019-08-26
Filing date: 2019-12-30
Publication date: 2021-03-04
Also published as: CN110487997A

Abstract

Disclosed is a phototoxicity testing method based on a reconstructed skin model, comprising the following steps. S1: receiving and restoring a reconstructed skin model; S2: selecting a light source; S3: determining a maximum tolerated dose of the reconstructed skin model; and S4: testing the phototoxicity of a test sample. The phototoxicity testing method based on the reconstructed skin model can be performed for water-soluble chemicals and cosmetic raw materials, as well as oily and creamy cosmetic products and formulated substances in different proportions. The present method not only simulates changes due to biological reactions of human skin in response to different types of exogenous substances, but also broadens the testing range of the substances to be tested, in that the substances to be tested are no longer limited to water-soluble chemical substances and may comprise finished cosmetics and formulations with various properties and forms.

Description

Phototoxicity detection method based on recombinant skin model

Technical field

The invention belongs to the technical field of skin phototoxicity detection and research, and specifically relates to a phototoxicity detection method based on a recombinant skin model.

Background technique

Phototoxicity refers to a skin toxic reaction caused by exposure to ultraviolet radiation after the skin is exposed to a chemical substance once, or a similar reaction that occurs when exposed to ultraviolet radiation after a systemic application of chemical substances, usually manifested as Erythema, blisters, pigmentation and thickening of the skin, etc. The current test methods used to evaluate the skin phototoxicity of cosmetics mainly include: animal test, human light patch test and in vitro test.

Animal test, the current internationally accepted animal test method is animal skin light test, that is, the back skin of the animal is dehaired, and then a certain amount of test substance is smeared on the depilated skin. After a certain time interval, UVA light is used. After irradiation, observe the skin reaction at a certain observation time after the irradiation, and judge whether the test substance has phototoxicity through the animal skin erythema and edema reaction integral. This method mostly uses guinea pigs as experimental animals, and some people use rabbits, nude mice, rats and mice. The phototoxicity test method for cosmetics specified in my country's GB 7919-1987 "Safety Evaluation Procedures and Methods for Cosmetics" and "Hygiene Standards for Cosmetics (2007 Edition)" is the test method.

Human light patch test, the results of human patch test are true, and it is an ideal test method for the safety evaluation of cosmetics phototoxicity. The human light patch test is to directly apply the test substance on the surface of the human skin, and at the same time receive a certain dose of ultraviolet radiation of the appropriate wavelength, to detect the phototoxic substances that induce phototoxicity and photoallergic dermatitis, and to test the body's resistance to certain photosensitizers. A skin test of reaction.

In vitro tests, according to different test endpoints and mechanisms of action, alternative test methods for phototoxicity in vitro can be divided into two categories: (a) Phototoxicity screening tests using cell, tissue or organ models, such as 3T3 neutral red uptake method, keratin Cell test method, hepatocyte method; (b) Tests that focus on the study of specific phototoxicity mechanisms, such as red blood cell hemolysis and histidine photooxidation.

The above three types of methods have their own advantages and disadvantages. The animal test method is a mature phototoxicity test method, and its operation is simple, but there are species differences between animals and humans. Extrapolation from animals to humans has great limitations. In addition, using a large number of animals to evaluate the safety of cosmetics is contrary to the current internationally promoted "3R" principle. The human patch test method is effective and the results are real. It is an ideal test method for the safety evaluation of the human phototoxicity of cosmetics. However, there are certain requirements for the test volunteers. The test volunteers are difficult to find. Considering humanitarian issues, this This test method is not advisable. In vitro substitution tests have become the general trend.

Patent CN107446993A discloses a skin sensitization detection method based on a three-dimensional skin model and dendritic cell co-culture mode, including the following steps: 1. Preliminary preparation of the skin model; 2. Preparation and culture of dendritic cells; 3. Skin Model and dendritic cell co-culture construction and test substance exposure; 4. Skin model activity test; 5. Skin model genome test; 6. Co-culture model lower cell secretion test; 7. Dendritic cell surface marker expression Detection; 8. Statistical methods and result prediction. Patent CN107446993A combines in vitro reconstructed 3D skin with barrier and metabolic functions and immune function dendritic cells to establish a new type of co-cultivation experimental system, which has similar functions and sensitization reactions in vivo; patent CN107446993A can Evaluate the skin sensitization effect of the test substance from both qualitative and quantitative aspects. At present, there are few reports on the evaluation of the skin phototoxicity of the test substance by the recombinant skin model.

Phototoxicity detection is not only an important part of modern chemical safety evaluation technology,

It is also the hygienic requirements of different cosmetic raw materials and finished products. Phototoxicity in vitro replacement test is the general trend. At present, the 3T3 fibroblast neutral red uptake test (3T3 NRU PT) has been approved by the European Union. It is used as an in vitro test method for skin phototoxicity test for chemical safety evaluation in EU countries. Because the test conditions are limited to water-soluble chemical substances or cosmetic raw materials, this alternative test is limited in the phototoxicity test of many cosmetic products. Therefore, the development of phototoxicity prediction methods based on in vitro recombinant human tissue engineered skin models has important practical significance.

Epikutis is a 3D recombinant epidermal model constructed by patented technology based on keratinocytes isolated from Chinese skin tissues as seed cells. The Epikutis recombinant human epidermal model has a complete epidermal structure: basal layer, spinous layer, granular layer, and stratum corneum. It is highly similar to the human epidermal structure, and it is the first industrialized 3D skin model in China.

Therefore, the development of a phototoxicity evaluation method based on the recombinant skin model Epikutis is of great significance to Chinese people.

Summary of the invention

In view of this, the purpose of the present invention is to provide a phototoxicity evaluation method based on a reorganized skin model, which can not only detect water-soluble chemicals and cosmetic raw materials, but also detect oily and creamy cosmetic products and different proportions of formula substances. .

To this end, the present invention provides the following technical solutions.

In the first aspect, the present invention provides a phototoxicity detection method based on a recombinant skin model, wherein the method includes the following steps:

S1. Receiving and resuscitating the reconstructed skin model;

S2. Select the light source;

S3. Determine the maximum tolerated dose of the recombinant skin model;

S4. Detect the phototoxicity of the test substance.

In a preferred embodiment, the receiving and resuscitating the reconstituted skin model includes:

Cultivate the reconstituted skin model in a 6-well plate, with 1 reconstituted skin model in each well, add 900μL of resuscitation fluid to each well, culture at a temperature of 36-38℃, a relative humidity of 92-95% and 4-6% CO ₂ After incubating in the box for 55-65min, replace the resuscitation solution and incubate overnight for 18-22h. During the resuscitation process, discard the damaged and reconstituted skin model with a large amount of water on the surface. After resuscitation, the appearance of the reconstituted skin model is evaluated. The surface has no bubbles, no wrinkles, and no moisture, and the follow-up operation is performed after the appearance is normal.

In a preferred embodiment, the light source is UVA with a wavelength of 320-400 nm.

In a more preferred embodiment, the dosage of the UVA is 0.5-60 J/cm ² .

In the most preferred embodiment, the highest dose irradiation time of the UVA is

Less than 120min.

In a preferred embodiment, the determination of the maximum tolerated dose of the reconstituted skin model includes: after the UVA irradiation is over, the reconstituted skin model is heated at a temperature of 36-38°C, a relative humidity of 92-95% and 4-6 Incubate in an incubator with %CO ₂ for 22 hours, use the hematoxylin-eosin staining method to detect the morphological changes of the recombinant skin model, and use the tetramethylazolium salt trace enzyme reaction colorimetric method to detect the tissue viability changes of the recombinant skin model, select There is no obvious change in morphology, and the maximum radiation dose with tissue viability above 75% is used as the maximum tolerated dose of the reconstituted skin model.

In a preferred embodiment, the detection of phototoxicity of the test substance includes:

1) Divide the reconstituted skin model with normal appearance after resuscitation in step S1 into UV+ group and UV- group;

2) Blank control group, positive control group, negative control group and test substance group are set for UV+ group and UV- group respectively;

3) According to the group, carry out the surface administration of the reorganized skin model, the volume of administration is 25-80μL, the administration time is 2h, and then use phosphate buffer to clean the remaining liquid on the surface of the reorganized skin model, and use a cotton swab to clean the reconstituted skin model Dry the surface and transfer the reconstituted skin model to a 24-well plate. The volume of the culture medium per well is 0.5-1mL;

4) Expose the UV+ group to the maximum UVA tolerable dose measured in step S3, and place the UV- group under dark conditions in the same environment. After the UV+ group has been irradiated, all reconstituted skin models in the UV+ group and UV- group Transfer to a 6-well plate containing 3.3-3.7mL culture medium and incubate for 22h;

5) Detect the tissue viability of each group and judge the potential phototoxicity of each group.

In a preferred embodiment, the test substance includes water-soluble chemicals, cosmetic raw materials, oil-like or paste-like cosmetic products, and formula substances in different proportions.

In a preferred embodiment, the apparently normal appearance is that the surface of the reconstituted skin model has no bubbles, no wrinkles, and no moisture.

In a preferred embodiment, the restructured skin model is a restructured skin model

Epikutis, a recombinant skin model Epikutis is a 3D recombinant epidermal model constructed by patented technology based on keratinocytes isolated from Chinese skin tissues as seed cells. The reorganized skin model Epikutis has a complete epidermal structure: basal layer, spinous layer, granular layer, stratum corneum, which is highly similar to the human epidermal structure.

In the present invention, the recombinant skin model adopts the recombinant skin model Epikutis produced by Guangdong Boxi Biotechnology Co., Ltd., the article number is PM1011; the culture medium adopts the culture medium produced by Guangdong Boxi Biotechnology Co., Ltd., the article number is PY3021; The resuscitation fluid is a resuscitation fluid produced by Guangdong Boxi Biological Technology Co., Ltd., and the product number is PY3031.

Compared with the prior art, the present invention has the following beneficial effects:

The phototoxicity detection method of the present invention based on the reorganized skin model can not only detect water-soluble chemicals and cosmetic raw materials, but also can detect oily and paste-like cosmetic products and different proportions of formula substances. The present invention is based on the phototoxicity of the reorganized skin model The detection method not only simulates the changes in the biological response of human skin to different types of exogenous substances, but also expands the detection range of the substance to be tested. It is no longer limited to water-soluble chemical substances, but also includes various cosmetic products and formulas. .

On the other hand, the present invention further ensures the accuracy of the phototoxicity detection method based on the recombinant skin model of the present invention by selecting UVA with a specific wavelength, selecting a specific dose of UVA, and selecting a specific UVA irradiation time. After a certain concentration of the phototoxic substance is used, the recombinant skin model Epikutis of the present invention can be used to accurately detect the phototoxicity in conjunction with the phototoxicity detection method of the present invention.

Description of the drawings

Figure 1 is a schematic diagram of the results of Epikutis, a skin model reconstituted by UVA radiation;

Figure 2 is a schematic diagram of the stratum corneum of Epikutis, a skin model reconstituted by UVA radiation;

Figure 3 is a schematic diagram of the phototoxicity evaluation results of the chemical chlorpromazine;

Figure 4 is a schematic diagram of the phototoxicity evaluation of finished cosmetics.

detailed description

Hereinafter, the present invention will be described in detail with reference to specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. Those skilled in the art should understand that these specific implementations and examples are used to illustrate the present invention, but not to limit the present invention.

Throughout the specification, unless otherwise specified, the terms used herein should be understood as the meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs. If there is a conflict, this manual takes precedence.

Hereinafter, the present invention will be explained in more detail through examples, but the present invention is not limited to these examples.

Example 1

This embodiment provides a phototoxicity evaluation method based on the recombinant skin model Epikutis, which includes the following steps:

S1. Receiving and resuscitating Epikutis, a reconstituted skin model;

S2. Select the light source;

S3. Determine the maximum tolerated dose of Epikutis in the recombinant skin model;

S4. Detect the phototoxicity of the test substance.

The step S1 includes:

Cultivate the recombinant skin model Epikutis in a 6-well plate, with 1 recombinant skin model Epikutis in each well, add 900 μL of resuscitation solution to each well _{, and incubate in an incubator at 36-38°C, 4-6% CO 2} , and 95% relative humidity for 55 -65min, replace the resuscitation fluid, incubate overnight for 18-22h. During the resuscitation process, discard the damaged and reconstituted skin model Epikutis with a lot of water on the surface. After resuscitation, evaluate the appearance of the reconstituted skin model Epikutis. After bubbles, no wrinkles, no dampness, and normal appearance, proceed to follow-up operations.

The light source selected in the step S2 is UVA with a wavelength of 320-400 nm, the dose of the UVA is 0.5-60 J/cm ² , and the maximum irradiation time of the UVA is 120 min or less.

The step S3 includes:

After UVA irradiation, the recombinant skin model Epikutis was incubated in an incubator at 36-38°C, 4-6% CO ₂ , and 95% relative humidity for 22 hours, and then hematoxylin-eosin staining was used to detect histological changes , Use the tetramethylazolyl azolyl salt micro-enzyme reaction colorimetric method to detect changes in tissue viability, and select the maximum radiation dose with no significant changes in morphology and tissue viability above 75% as the maximum tolerated dose of the recombinant skin model Epikutis.

The step S4 includes:

1) Divide the apparently normal reconstituted skin model Epikutis after step S1 resuscitation into UV+ group and UV- group;

3) According to the group, the surface of the recombinant skin model Epikutis was administered, the dosage volume was 25-80 μL, and the administration time was 2 hours. Then, the residual liquid on the surface of the recombinant skin model Epikutis was washed with phosphate buffer, and washed 15 times, and used Use a cotton swab to dry the surface of the reconstituted skin model Epikutis, and transfer the reconstituted skin model Epikutis to a 24-well plate with a volume of 0.5 mL of culture medium per well;

4) The UV+ group is exposed to the maximum UVA tolerable dose measured in step S3, and the UV- group is placed under dark conditions in the same environment; after the UV+ group is irradiated, all the reconstituted skin models of the UV+ group and the UV- group are transferred Incubate in a 6-well plate containing 3.3 mL of culture medium for 22 hours;

The said no significant change in morphology means that the skin tissue of the UV-irradiated group is compared with the tissue of the blank control group. The basal layer cells did not undergo apoptosis or vacuoles, and the stratum corneum did not thicken; the calculation of tissue viability is also Based on the blank control group.

The recombinant skin model Epikutis uses keratinocytes isolated from Chinese skin tissue as seed cells, and has a complete epidermal structure: basal layer, spinous layer, granular layer, and stratum corneum, which is highly similar to human epidermal structure.

The positive control refers to a single concentration of a chemical substance known to be phototoxic, for example, chlorpromazine (0.125-0.5mM), 8-methoxypsoralen (0.1-0.2mM), Promethazine (1.25-5mM), Typrofen (3-6mM), Isopsoralen (0.5-2mM), 6-Methylcoumarin (3-5mg/mL).

The negative control refers to a single concentration of a chemical substance known to be non-phototoxic, for example, sodium cetyl sulfonate (0.5-2mM), penicillin sodium (75-300mM), L-histidine (40-160mM).

The evaluation standard for judging the potential phototoxicity of each group is: when the chemical, cosmetic raw materials or finished products are not toxic (tissue viability>75%), the tissue viability after UVA radiation decreases more than 25%, it is judged as It has phototoxicity, that is, tissue viability (UV-)-tissue viability (UV+)>25%; if the test substance cannot be determined at a safe concentration, it is necessary to screen for a safe concentration before the phototoxicity determination.

Example 2

This embodiment provides a method for evaluating the phototoxicity of the chemical chlorpromazine based on the recombinant skin model Epikutis, which includes the following steps:

S1. Receiving and resuscitating Epikutis, a reconstituted skin model;

S2. Select the light source;

S4. Detect the phototoxicity of the test substance.

The step S1 includes:

Cultivate the recombinant skin model Epikutis in a 6-well plate, with 1 recombinant skin model Epikutis in each well, add 900 μL resuscitation solution to each well _{, and incubate in an incubator at 36-38°C, 4-6% CO 2} , and 95% relative humidity for 55 -65min, replace the resuscitation fluid, incubate overnight for 18-22h. During the resuscitation process, discard the damaged and reconstituted skin model Epikutis with a lot of water on the surface. After resuscitation, evaluate the appearance of the reconstituted skin model Epikutis. After bubbles, no wrinkles, no dampness, and normal appearance, proceed to follow-up operations.

The light source selected in the step S2 is UVA with a wavelength of 320-400 nm, the dose of the UVA is 0.5-60 J/cm2, and the maximum dose irradiation time of the UVA is less than 120 min.

The step S3 includes:

After the resuscitation is complete, transfer the reconstituted skin model Epikutis to a sterile six-well plate (add 0.9mL culture medium in advance, five models per plate), each well plate is a group, a total of 6 groups, including the control group and 5 For the irradiation groups, the irradiation doses were 5J/cm ² , 10J/cm ² , 20J/cm ² , 40J/cm ² and 60J/cm ^{2 respectively} . After the end of each group of irradiation, the irradiation instrument was turned off, and the next group of irradiation was performed 15 minutes later.

After each group of irradiation is completed, move the recombinant skin model Epikutis into another 6-well plate with fresh culture medium (3.3mL per well), and place the 6-well plate at 37℃, 5% CO ₂ , 95% relative humidity After incubating for 22 hours under the conditions of, the hematoxylin-eosin staining method was used to detect the tissue morphology changes, the tetramethylazolyzol salt trace enzyme reaction colorimetric method was used to detect the tissue viability changes, and the recombinant skin model Epikutis was selected after UVA radiation. The dose where the tissue viability is still above 75% is a safe dose, and at the same time, the maximum tolerated dose of UVA for the reconstructed skin model Epikutis is the maximum tolerated dose of the reconstituted skin model Epikutis with no significant change in the morphology of the tissue at this dose. Finally, it is determined that the maximum tolerated UVA dose of the recombinant skin model Epikutis is 40J/cm ² .

The step S4 includes:

2) Solvent control group and chlorpromazine group (3 concentrations) are set for UV+ group and UV- group respectively;

3) Reconstituted skin model Epikutis was administered to the surface according to the group. The concentration of chlorpromazine was 0.125mM, 0.25mM and 0.5mM, and the solvent was a mixed solution of propylene glycol and ethanol (propylene glycol (v): ethanol (v) ＝3:7), each concentration group has 3 parallel models, the administration volume is 25μL, the administration time is recorded, and then placed at 37℃, 5%

Incubate for 2 hours under the conditions of CO ₂ and 95% relative humidity, then wash the remaining liquid on the surface of the reconstituted skin model Epikutis with phosphate buffer for 15 times, and use a cotton swab to dry the surface of the reconstituted skin model Epikutis to dry the reconstituted skin model Epikutis Transfer to a 24-well plate, the volume of each well of the culture medium is 0.5mL;

4) The UV+ group is exposed to the maximum UVA tolerable dose measured in step S3, the irradiation dose is 40J/cm ² , the orifice cover is removed during irradiation, and the UV- group is placed in the dark condition of the same environment; waiting for the UV+ group After the irradiation, transfer all the reconstituted skin models in the UV+ group and UV- group to a 6-well plate containing 3.3 mL of culture medium and incubate for 22 hours;

5) Judge the phototoxicity of the chemical chlorpromazine

The results of phototoxicity of chlorpromazine are shown in Figure 3. It can be seen from Figure 3 that the mixed solution has almost no effect on the tissue viability of the recombinant skin model Epikutis, and the effects of 0.125mM and 0.25mM chlorpromazine on the viability of skin tissues are also Smaller (tissue vitality is above 90%), but under UVA irradiation, tissue vitality decreases significantly. When the concentration of chlorpromazine was 0.125mM, UVA caused a decrease of activity by 66.52%, and when the concentration was 0.25mM, UVA caused a decrease of activity by 80.69%. The high concentration of chlorpromazine (0.5mM) resulted in tissue activity of the recombinant skin model Epikutis It drops to 71.15%, therefore, this concentration cannot be used as a reference concentration for phototoxicity judgment. Finally, the chemical chlorpromazine has strong phototoxicity, and the lowest concentration that produces phototoxicity is 0.125mM.

Example 3

This embodiment provides a method for evaluating phototoxicity of a cosmetic product (commercially available sunscreen, abbreviated as F) based on the recombinant skin model Epikutis, which includes the following steps:

S1. Receiving and resuscitating the Epikutis skin model;

S2. Select the light source;

S4. Detect the phototoxicity of the test substance.

The step S1 includes:

Cultivate the recombinant skin model Epikutis in a 6-well plate, with 1 recombinant skin model Epikutis in each well, add 900 μL of resuscitation solution to each well _{, and incubate in an incubator at 36-38°C, 4-6% CO 2} , and 95% relative humidity for 55 After -65min, change the resuscitation solution and incubate overnight for 18-22h. During the resuscitation process, discard the damaged reconstituted skin model Epikutis with a large amount of water on the surface. After resuscitation, evaluate the appearance of the reconstituted skin model Epikutis. After bubbles, no wrinkles, no dampness, and normal appearance, proceed to follow-up operations.

The step S3 includes:

After each group of irradiation is completed, move the recombinant skin model Epikutis into another 6-well plate with fresh culture medium (3.3mL per well), and place the 6-well plate at 37℃, 5% CO ₂ , 95% relative humidity After incubating for 22 hours under the conditions of, the hematoxylin-eosin staining method was used to detect the tissue morphology changes, the tetramethylazolyzol salt trace enzyme reaction colorimetric method was used to detect the tissue viability changes, and the recombinant skin model Epikutis was selected after UVA radiation. The dose where the tissue viability is still above 75% is a safe dose, and at the same time, the maximum tolerated dose of UVA for the reconstructed skin model Epikutis is the maximum tolerated dose of the reconstituted skin model Epikutis with no significant change in the morphology of the tissue at this dose. 0-60 J/cm ² of UVA did not reduce the tissue viability of the reconstructed skin model Epikutis to less than 75%, while at 60 ^{J/cm 2} the stratum corneum of the reconstructed skin model Epikutis became thicker. The results are shown in Figure 1 and Figure 2. Finally, it is determined that the maximum tolerated UVA dose of the recombinant skin model Epikutis is 40J/cm ² . Finally, it is determined that the maximum tolerated UVA dose of the recombinant skin model Epikutis is 40J/cm ² .

The step S4 includes:

2) UV+ group and UV- group are respectively set up a control group and a cosmetic product group (3 groups in total);

3) The recombinant skin model Epikutis of the control group does not do any treatment. The finished cosmetics are composed of 3 groups, and the finished cosmetics groups with different concentrations of chlorpromazine are F group, F+CPZ (0.25mM) group and F+CPZ respectively. (0.5mM) group. There are 3 parallel models in each concentration group, the administration volume is 25μL, the administration time is recorded, and then placed at 37℃, 5% CO ₂ , 95% relative humidity, and incubated for 2 hours, and then the skin model is reconstituted with phosphate buffer Clean the finished cosmetics remaining on the surface of Epikutis, wash 15 times, and dry the surface of the reconstituted skin model Epikutis with a cotton swab, transfer the reconstituted skin model Epikutis to a 24-well plate, the volume of each well of the culture medium is 0.5mL;

4) The UV+ group was irradiated with UVA, the irradiation dose was 40J/cm ² , the orifice cover was removed during irradiation, and the UV- group was placed under dark conditions in the same environment; after the UV+ group was irradiated, the UV+ group and All the recombinant skin models Epikutis in the UV-group were transferred to a 6-well plate containing 3.3 mL of culture medium. After the well plate was incubated at 37°C, 5% CO ₂ , and 95% relative humidity for 22 hours, tetramethylazo The azole salt trace enzyme reaction colorimetric method was used to detect the tissue viability of each group of recombinant skin models Epikutis;

5) Judge the phototoxicity of finished cosmetics

The phototoxicity results of the finished cosmetics are shown in Figure 4. From Figure 4, it can be seen that the finished cosmetics are not phototoxic. However, after adding different concentrations of the strong phototoxic substance-chlorpromazine to the cosmetics, the results have certain changes. When the concentration of chlorpromazine is 0.25mM, no phototoxicity occurs. When the concentration is increased to 0.5mM, the finished cosmetics exhibit phototoxicity. UVA causes the tissue viability of the recombinant skin model Epikutis to drop by about 40%. It can be explained that if a certain concentration of phototoxic substances is added to cosmetics, it can be detected by the recombinant skin model Epikutis.

It should be understood that the disclosed invention is not limited to the specific methods, schemes and materials described, as these can be varied. It should also be understood that the terminology used herein is only for the purpose of describing specific embodiments and is not intended to limit the scope of the present invention, which is only limited by the appended claims. Those skilled in the art will also recognize, or be able to confirm, using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. These equivalents are also included in the appended claims.

Claims

A phototoxicity detection method based on a recombinant skin model, wherein the method includes the following steps:

S1. Receiving and resuscitating the reconstructed skin model;

S2. Select the light source;

S3. Determine the maximum tolerated dose of the recombinant skin model;

S4. Detect the phototoxicity of the test substance.
The method of claim 1, wherein the receiving and resuscitating the reconstituted skin model comprises:

Cultivate the reconstituted skin model in a 6-well plate, with 1 reconstituted skin model in each well, add 900μL of resuscitation fluid to each well, culture at a temperature of 36-38℃, a relative humidity of 92-95% and 4-6% CO 2 After incubating in the box for 55-65min, replace the resuscitation solution and incubate overnight for 18-22h. During the resuscitation process, discard the damaged and reconstituted skin model with a large amount of water on the surface. After resuscitation, the appearance of the reconstituted skin model is evaluated. Perform follow-up operations after the appearance is normal.
The method according to claim 1, wherein the light source is UVA with a wavelength of 320-400 nm.
The method according to claim 3, wherein the dose of the UVA is 0.5-60 J/cm 2 .
The method according to claim 4, wherein the maximum dose irradiation time of the UVA is 120 min or less.
The method according to claim 1, wherein the determining the maximum tolerated dose of the reconstituted skin model comprises: after the UVA irradiation is over, the reconstituted skin model is heated at a temperature of 36-38°C and a relative humidity of 92-95% Incubate with 4-6% CO 2 in an incubator for 22 hours, use hematoxylin-eosin staining method to detect the morphological changes of the recombinant skin model, and use the tetramethylazazole salt trace enzyme reaction colorimetric method to detect the recombinant skin model tissue For changes in vitality, the maximum radiation dose with no obvious changes in morphology and tissue vitality above 75% was selected as the maximum tolerated dose of the reconstituted skin model.
The method according to claim 1, wherein said detecting phototoxicity of the test substance comprises:

1) Divide the reconstituted skin model with normal appearance after resuscitation in step S1 into UV+ group and UV- group;

2) Blank control group, positive control group, negative control group and test substance group are set for UV+ group and UV- group respectively;

3) According to the group, carry out the surface administration of the reorganized skin model, the volume of administration is 25-80μL, the administration time is 2h, and then use phosphate buffer to clean the remaining liquid on the surface of the reorganized skin model, and use a cotton swab to clean the reconstituted skin model Dry the surface and transfer the reconstituted skin model to a 24-well plate. The volume of the culture medium per well is 0.5-1mL;

4) Expose the UV+ group to the maximum UVA tolerable dose measured in step S3, and place the UV- group under dark conditions in the same environment. After the UV+ group has been irradiated, all reconstituted skin models in the UV+ group and UV- group Transfer to a 6-well plate containing 3.3-3.7mL culture medium and incubate for 22h;

5) Detect the tissue viability of each group and judge the potential phototoxicity of each group.
The method according to claim 1, wherein the test substance includes water-soluble chemicals, cosmetic raw materials, finished cosmetic products in oil or paste form, and formula substances in different proportions.
The method according to claim 2, wherein the apparently normal appearance is that the surface of the reconstituted skin model has no bubbles, no wrinkles, and no moisture.
The use of the recombinant skin model in the preparation of a phototoxicity detection kit.