Method for evaluating eye irritation in vitro and application thereof

Abstract

The invention provides a method for evaluating eye irritation in vitro and application thereof, and belongs to the technical field of non-animal test methods. According to the method provided by the invention, a recombinant human corneal epithelium model is utilized, and relative tissue activity RTV and relative cell factor expression quantity RCL are used for comprehensively evaluating the eye irritation of a to-be-detected sample; the method specifically comprises the following steps: (1) pretreating a to-be-detected sample; (2) activity determination; (3) relative tissue activity judgment; (4) measuring the expression quantity of cell factors; (5) judging the expression quantity of relative cell factors; and (6) judging an eye irritation result. According to the method, the expression quantity of cell factors is increased to serve as the evaluation standard of the eye irritation, the evaluation accuracy of the eye irritation of the sample is remarkably improved, the samples which are judged to be free of eye irritation in relative tissue activity detection can be effectively recognized, then the attention of product developers and supervisors is aroused, and the health of a user is better protected.

Description

Technical Field

[0001] The present invention relates to the technical field of non-animal testing methods, and in particular to an in vitro method for evaluating eye irritation and an application thereof. Background Art

[0002] When human eyes come into contact with agricultural products, cosmetics, medicines, medical devices and other substances, these substances may contain potentially harmful ingredients, which may cause varying degrees of damage to the eyes. The damage may range from mild eye irritation and discomfort to severe chemical corrosion, and even lead to irreversible vision loss. To ensure the health and safety of consumers, regulatory authorities conduct eye irritation tests on all products and their ingredients that may come into contact with the eyes.

[0003] Since human data related to eye hazards can usually only be obtained through accidental exposure or clinical studies, the rabbit eye irritation test first proposed by Draize in 1944 has been the main method for evaluating eye irritation. However, this test method has been widely criticized due to its poor reproducibility, strong subjectivity, and animal abuse. In addition, the differences in physiological and anatomical structures between human and rabbit eyes have questioned the reliability of the rabbit eye irritation model. Therefore, in the past 20 years, the development and implementation of methods to replace the Draize rabbit eye irritation test have been actively promoted, and many efforts have been focused on the research and development of in vitro and ex vivo test methods. At present, a variety of in vitro eye irritation assessment methods have been developed to replace the rabbit eye irritation test, such as the bovine corneal opacity and permeability test (BCOP method), the ex vivo chicken eye test (ICE method), the fluorescein leakage test (FL method), the short-term exposure test (STE method), and the recombinant human corneal epithelial model test (RhCE method).

[0004] The bovine corneal opacity and permeability test (BCOP method) uses freshly isolated bovine corneas to apply the test sample to the corneal epithelial surface. By measuring the degree to which the sample induces a decrease in corneal light transmittance (opacity) and an increase in sodium fluorescein dye permeability (permeability), the in vitro irritation score (IVIS) or LLBO irritation score (LIS) is calculated to classify the eye irritation level of the test sample.

[0005] The isolated chicken eye test (ICE method) uses short-term in vitro cultured chicken eyeballs to expose the test sample to the corneal surface. The following parameters are evaluated: corneal opacity (degree of turbidity), fluorescein retention (indicating the degree of epithelial damage), increase in corneal thickness (degree of swelling), and macroscopic morphological damage on the surface of the chicken eye. By combining these indicators, the eye irritation classification is obtained. In addition, histopathological observation of the chicken eye can be used as an additional endpoint to evaluate the depth of corneal damage and predict the reversibility of the damage, thereby improving the accuracy of the judgment results.

[0006] The Fluorescein Leakage test (FL method) is based on the ability of the test sample to cause damage to the monolayer of epithelial cells. The integrity of trans-epithelial permeability is an important function of the corneal epithelium, and the increase in corneal epithelial permeability is closely related to the development of eye irritation. This test is performed by exposing the cultured monolayer of Madin-Darby Canine Kidney (MDCK) epithelial cells to the test sample for a short time, and detecting the increase in the permeability of sodium fluorescein in MDCK epithelial cells to assess its potential eye irritation. The amount of fluorescein leakage is proportional to the degree of damage to the tight junctions, desmosome junctions and cell membranes caused by the test sample.

[0007] The short-term exposure test (STE method) is an in vitro detection method based on cytotoxicity. The cytotoxicity of the test substance to corneal epithelial cells is an important mechanism leading to eye irritation. The STE method uses a monolayer of rabbit corneal cells (Statens Seruminstitut Rabbit Cornea, SIRC) and exposes the cells to 5% and 0.05% concentrations of the test sample for 5 minutes. The relative viability of SIRC cells is then measured by the MTT method to evaluate cytotoxicity. The reduction in cell viability can be used to predict adverse effects that may cause eye damage.

[0008] The recombinant human corneal epithelium model test (RhCE method) uses primary human epidermal keratinocytes, human immortalized corneal epithelial cells or primary human corneal epithelial cells to construct a human corneal epithelial cell model (Reconstructed humanCornea-like Epithelium, RhCE). The RhCE model has a layered structure similar to that of the human cornea and highly differentiated squamous epithelium, and is highly consistent with human corneal epithelium in terms of histology, morphology, biochemistry and physiological characteristics. In the RhCE test, a certain dose of the test sample is fully contacted with the surface of the RhCE model. After exposure, the relative tissue viability of the RhCE model is measured by the MTT method, CCK-8 method or WST-1 method to predict the potential eye irritation of the test substance.

[0009] The recombinant human corneal epithelial models currently recognized by OECD include: EpiOcular TM Model, SkinEthic constructed using immortalized human corneal epithelial cells TM Models, as well as LabCyte CORNEA-MODEL24 and MCTT HCE constructed using primary human corneal epithelial cells TM Models. These models are similar to human corneal epithelium in three-dimensional structure and are constructed using human cells, thus avoiding errors introduced by species differences. This approach provides more reliable results for eye irritation assessment.

[0010] In the recombinant human corneal epithelial model test, the degree of eye irritation reaction is evaluated by directly measuring the cytotoxicity caused by the test substance. Eye irritation is mainly caused by the penetration of substances through the cornea and damage to the tissue. Tissue damage can occur through a variety of mechanisms, including cell membrane lysis, protein coagulation, lipid saponification, and alkylation of biomacromolecules. Studies have shown that regardless of the physicochemical process of tissue damage, cytotoxicity plays a major mechanistic role in determining the eye irritation reaction of substances. Generally, substances that cause eye irritation will induce cytotoxicity in the corneal epithelium, so the current RhCE model uses cytotoxicity as the detection endpoint. However, in some cases, cytotoxicity may not fully reflect eye irritation.

[0011] The recombinant human corneal epithelium model test (RhCE method) and other in vitro eye irritation detection methods can effectively supplement and replace the rabbit eye irritation test, thereby significantly reducing the use of animals. At present, in vitro eye irritation alternative tests have become an important development direction for eye irritation safety evaluation. With the continuous advancement of in vitro eye irritation models and detection methods, the application of recombinant human corneal epithelium models will continue to expand. The current single detection endpoint in the RhCE method may lead to false negative results, so other detection endpoints need to be developed to improve the predictive ability of the model. This will further improve the in vitro eye irritation alternative test, making it more scientific and effective in evaluating eye irritation. Summary of the invention

[0012] In view of this, the purpose of the present invention is to provide an in vitro method for evaluating eye irritation and its application. The method provided by the present invention incorporates the content of inflammatory cytokines secreted after RhCE model treatment into the detection endpoint of eye irritation evaluation, and uses relative tissue viability (RTV) and relative cytokine level (RCL) to comprehensively evaluate the eye irritation of the test sample, which can effectively identify those substances with no obvious cytotoxicity but still irritating, thereby significantly improving the accuracy of detection.

[0013] In order to achieve the above object, the present invention provides the following technical solutions:

[0014] An in vitro method for evaluating eye irritation, the method using a recombinant human corneal epithelial model, using relative tissue viability (RTV) and relative cytokine expression (RCL) to comprehensively evaluate the eye irritation of a sample to be tested;

[0015] The specific steps include:

[0016] (1) Pretreatment of the sample to be tested: Resuscitate the in vitro recombinant human corneal epithelial model, add the sample to be tested, CO 2 First culture in incubator, rinse, act at room temperature, CO 2 The second culture was carried out in the incubator. After the culture was completed, the supernatant was aspirated and stored for later use. The processed human corneal epithelial model was then used for later use.

[0017] (2) Activity assay: performing MTT reaction and incubation on the human corneal epithelial model treated in step (1), piercing the human corneal epithelial model to mix the solution evenly, and setting a blank control; measuring the OD value at a wavelength of 540 nm to 600 nm, and calculating the relative tissue activity; wherein the calculation formula for the relative tissue activity is:

[0018] Relative tissue viability = OD value of sample group / OD value of negative control group × 100%;

[0019] (3) Determination of relative tissue vitality:

[0020] If the relative tissue viability is greater than the set RTV threshold, the sample is judged to have no cytotoxicity and is scored as 0;

[0021] If the relative tissue viability is ≤ the set RTV threshold, the sample is judged to be cytotoxic and scored as 1;

[0022] (4) Determination of cytokine expression: Take the supernatant obtained in step (1), set up a negative control, use an ELISA detection kit to determine the cytokine content therein, and calculate the relative cytokine expression; wherein, the calculation formula of the relative cytokine expression is:

[0023] Relative cytokine expression = cytokine expression in the sample group / cytokine expression in the negative control group;

[0024] (5) Determination of relative cytokine expression:

[0025] If the relative cytokine expression level is greater than the set RCL threshold, the sample is judged to have cytokine overexpression and the score is 1;

[0026] If the relative cytokine expression is ≤ the set RCL threshold, the sample is judged to have no cytokine overexpression and the score is 0;

[0027] (6) Determination of eye irritation results: If the score of either the relative tissue viability or the relative cytokine expression level in the test sample is greater than 0, the result is determined to be irritating; if the scores of the relative tissue viability and the relative cytokine expression level in the test sample are both 0, the result is determined to be non-irritating.

[0028] In the present invention, the in vitro recombinant human corneal epithelial model is preferably a commercial in vitro recombinant human corneal epithelial model, including but not limited to Hangzhou Genentech, Guangdong Boxi Biotechnology, and Shanghai Sanofuno.

[0029] In the present invention, the cytokine is preferably IL-1β, IL-6 or IL-8, more preferably IL-6.

[0030] In the present invention, the culturing time of the first culturing in step (1) is preferably 0.5 h to 6 h, and the culturing time of the second culturing is preferably 2 h to 18 h.

[0031] In the present invention, the reaction time at room temperature in step (1) is preferably 12 min to 30 min.

[0032] In the present invention, the incubation in step (2) is preferably carried out under CO 2 Incubate in the incubator for 2h to 4h.

[0033] In the present invention, the RTV threshold in step (3) is preferably 50% or 60%. The RTV threshold needs to be specifically selected according to different in vitro recombinant human corneal epithelial models.

[0034] In the present invention, the negative control in step (4) is preferably sterile water or physiological saline.

[0035] In the present invention, the RCL threshold in step (5) is preferably >1, more preferably 1.2, 1.5 or 2.

[0036] The present invention also provides an application of the method described in the above technical solution in detecting the eye irritation of a product.

[0037] In the present invention, the products include but are not limited to agricultural products, cosmetics, medicines, and medical devices.

[0038] Beneficial technical effects: The present invention provides a method for evaluating eye irritation in vitro and its application. The method of the present invention utilizes a recombinant human corneal epithelial model, and uses relative tissue viability RTV and relative cytokine expression RCL to comprehensively evaluate the eye irritation of the sample to be tested; specifically, the following steps are included: (1) pretreatment of the sample to be tested; (2) activity determination; (3) relative tissue viability determination; (4) cytokine expression determination; (5) relative cytokine expression determination; (6) eye irritation result determination. The present invention significantly improves the accuracy of the evaluation of the eye irritation of the sample by adding cytokine expression as an eye irritation evaluation standard, and can effectively identify samples that are determined to be non-eye irritating in the relative tissue viability test, thereby attracting the attention of product developers and regulators and better protecting the health of users. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is a schematic diagram of the appearance of the in vitro recombinant human corneal epithelial model;

[0040] Figure 2 Schematic diagram of HE staining of histological sections of in vitro recombinant human corneal epithelial model;

[0041] Figure 3 The relative tissue viability and relative cytokine expression results of the eye irritation test on each sample using the model; wherein, samples 1 to 4 correspond to the test samples of Examples 1 to 4, respectively, and the control is a negative control. DETAILED DESCRIPTION

[0042] In order to better understand the present invention, the content of the present invention is further explained in conjunction with the following examples, but the content of the present invention is not limited to the following examples. The materials, reagents, etc. used in the examples and test examples of the present invention, unless otherwise specified, can be obtained from commercial channels; the methods used in the examples and test examples of the present invention, unless otherwise specified, are conventional methods.

[0043] The commercial in vitro recombinant human corneal epithelial model used in Examples 1 to 4 of the present invention is RB0404, purchased from Hangzhou Genefy, and its appearance is as follows: Figure 1 As shown, the histological sections were stained with HE. Figure 2 shown.

[0044] Example 1

[0045] Test sample: 2-hydroxyethyl acrylate, purchased from Sigma. After inquiry, this compound is found to be a serious eye irritant (https: / / pubchem.ncbi.nlm.nih.gov / compound / 13165).

[0046] (1) Model recovery: After receiving the purchased commercial in vitro recombinant human corneal epithelial model, place the in vitro recombinant human corneal epithelial model in a sterile 6-well plate with 0.9 mL of culture medium (gift from the model vendor, used together with the model, the same below) in each well, and avoid bubbles at the bottom during placement. Transfer the 6-well plate with the model to a CO 2 Incubator, 37 °C, 5% CO 2 Incubate overnight.

[0047] (2) Model preparation: Prepare a sterile 6-well plate according to the number of models required for the experiment, add 0.9 mL of maintenance culture medium to each well, and then use tweezers to move the corneal epithelial model to the 6-well plate. Make two parallel wells for each sample to be tested, methyl acetate as the positive control, and sterile water as the negative control. Both the positive and negative controls are set up with two parallel wells.

[0048] (3) Sample addition reaction: Before sample addition, 20 μL DPBS was added to the surface of the corneal epithelial model for pretreatment for 30 min, and then 40 μL of the sample to be tested, 40 μL of the positive control and the negative control were evenly added to the surface of the corneal epithelial model. The culture plate was placed at 37°C and 5% CO 2 , cultured in an incubator with a relative humidity of 95%, and the sample to be tested was exposed to it for 30 minutes.

[0049] (4) Rinse: After the reaction is completed, rinse slowly and gently with DPBS buffer to remove the sample on the surface of the corneal epithelial model, and try to avoid directly aligning it with the model surface. Each well is rinsed with 15mL DPBS, and the rinse volume is 5mL each time, and rinsed 3 times in total. After the rinse is completed, use a cotton swab to gently absorb the residual liquid on the surface of each corneal epithelial model and transfer it to a new 6-well plate, with each well containing 0.9mL of culture medium. The corneal epithelial model is exposed to room temperature for 12 minutes. After the end, the culture plate is placed at 37°C and 5% CO 2 , and incubate in an incubator at 95% relative humidity for 2 h.

[0050] (5) Storage of supernatant: After the corneal epithelial model is treated, the culture supernatant is immediately aspirated and stored at -80°C. The cytokine IL-6 content is detected using a human IL-6 ELISA kit within 1 month. Repeated freezing and thawing of the culture supernatant should be avoided.

[0051] (6) MTT reaction and incubation: Take a 24-well plate, add 300 μL of 1 mg / mL MTT working solution to each well, transfer the corneal epithelial model to the plate, and ensure that there are no bubbles between the bottom of the corneal epithelial model and the MTT solution. Place the plate at 37°C and 5% CO 2 , and cultured in an incubator at 95% relative humidity for 3 h.

[0052] (7) Color extraction of corneal epithelial model: After 3 hours of culture, the corneal epithelial model was taken out, the MTT solution on the bottom was blotted off on absorbent paper, and placed in a new 24-well plate. 2 mL of isopropanol solution was added to each well. The 24-well plate was sealed with sealing film and wrapped with tin foil to ensure light protection. The 24-well plate was placed in a 4°C refrigerator overnight.

[0053] (8) Activity assay: Puncture the corneal epithelial model to mix the solution evenly. Transfer to a 96-well plate, take two replicate wells for each tissue, add 200 μL of solution to each well, set up 4 isopropanol blank control replicate wells, and read the absorbance (OD value) at a wavelength of 570 nm.

[0054] (9) Calculation of relative tissue viability: Calculate the relative tissue viability (RTV) according to the following formula:

[0055] Relative tissue viability = OD value of sample group / OD value of negative control group × 100%

[0056] (10) Determination of relative tissue vitality

[0057] If the relative tissue viability was >60%, the sample was judged to have no cytotoxicity and scored as 0;

[0058] If the relative tissue viability is ≤60%, the sample is judged to be cytotoxic and scored as 1;

[0059] (11) Determination of cytokine IL-6 expression: According to the operating instructions of the ELISA detection kit, the cytokine IL-6 content in the culture medium supernatant stored in step (5) above was determined. The relative cytokine expression levels of the treatment group and the negative control group were calculated. The relative cytokine expression level (RCL) calculation formula is:

[0060] Relative cytokine expression = cytokine expression in sample group / cytokine expression in negative control group

[0061] (12) Determination of relative cytokine expression:

[0062] If the relative cytokine expression is greater than 1.5, the sample is judged to have cytokine overexpression and scored as 1

[0063] If the relative cytokine expression is ≤1.5, the sample is judged to have no cytokine overexpression and scored as 0

[0064] (13) Analysis of test results: The total scores obtained based on the relative tissue viability (RTV) and relative cytokine expression (RCL) were used to determine the eye irritation of the test samples. The results are shown in Table 1.

[0065] Table 1 Eye irritation results of samples in Example 1

[0066]

[0067] Example 2

[0068] Test sample: 2,2′-methylenebis[6-(2H-benzotriazole-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol]; purchased from Sigma. After inquiry, the compound is not irritating to the eyes (https: / / pubchem.ncbi.nlm.nih.gov / compound / 3571576).

[0069] (1) Model recovery: After receiving the purchased commercial in vitro recombinant human corneal epithelial model, place the in vitro recombinant human corneal epithelial model in a sterile 6-well plate with 0.9 mL of culture medium in each well. Avoid bubbles at the bottom during placement. Transfer the 6-well plate with the model to a CO 2 Incubator, 37 °C, 5% CO 2 Incubate overnight.

[0070] (2) Model preparation: Prepare a sterile 6-well plate according to the number of models required for the experiment, add 0.9 mL of maintenance culture medium to each well, and then use tweezers to move the corneal epithelial model to the 6-well plate. Make two parallel wells for each sample to be tested, methyl acetate as the positive control, and sterile water as the negative control. Both the positive and negative controls are set up with two parallel wells.

[0071] (3) Sample addition reaction: Before sample addition, 20 μL DPBS was added to the surface of the corneal epithelial model for pretreatment for 30 min, and then 40 μL of the sample to be tested, 40 μL of the positive control and the negative control were evenly added to the surface of the corneal epithelial model. The culture plate was placed at 37°C and 5% CO 2 , cultured in an incubator with a relative humidity of 95%, and the sample to be tested was exposed to it for 6 hours.

[0072] (4) Rinse: After the reaction is completed, rinse slowly and gently with DPBS buffer to remove the sample on the surface of the corneal epithelial model, and try to avoid directly aligning it with the model surface. Each well is rinsed with 15mL DPBS, and the rinse volume is 5mL each time, and rinsed 3 times in total. After the rinse is completed, use a cotton swab to gently absorb the residual liquid on the surface of each corneal epithelial model and transfer it to a new 6-well plate, with each well containing 0.9mL of culture medium. The corneal epithelial model is exposed to room temperature for 25 minutes. After the end, the culture plate is placed at 37°C and 5% CO 2 , and incubated in an incubator at 95% relative humidity for 18 h.

[0073] (5) Storage of supernatant: After the corneal epithelial model is treated, the culture supernatant is immediately aspirated and stored at -80°C. The cytokine IL-6 content is detected using a human IL-6 ELISA kit within 1 month. Repeated freezing and thawing of the culture supernatant should be avoided.

[0074] (6) MTT reaction and incubation: Take a 24-well plate, add 300 μL of 1 mg / mL MTT working solution to each well, transfer the corneal epithelial model to the plate, and ensure that there are no bubbles between the bottom of the corneal epithelial model and the MTT solution. Place the plate at 37°C and 5% CO 2 , and cultured in an incubator at 95% relative humidity for 3 h.

[0075] (7) Color extraction of corneal epithelial model: After 3 hours of culture, the corneal epithelial model was taken out, the MTT solution on the bottom was blotted off on absorbent paper, and placed in a new 24-well plate. 2 mL of isopropanol solution was added to each well. The 24-well plate was sealed with sealing film and wrapped with tin foil to ensure light protection. The 24-well plate was placed in a 4°C refrigerator overnight.

[0076] (8) Activity assay: Puncture the corneal epithelial model to mix the solution evenly. Transfer to a 96-well plate, take two replicate wells for each tissue, add 200 μL of solution to each well, set up 4 isopropanol blank control replicate wells, and read the absorbance (OD value) at a wavelength of 570 nm.

[0077] (9) Calculation of relative tissue viability: Calculate the relative tissue viability (RTV) according to the following formula:

[0078] Relative tissue viability = OD value of sample group / OD value of negative control group × 100%

[0079] (10) Determination of relative tissue vitality:

[0080] If the relative tissue viability was >60%, the sample was judged to have no cytotoxicity and scored as 0;

[0081] If the relative tissue viability is ≤60%, the sample is judged to be cytotoxic and scored as 1;

[0082] (11) Determination of cytokine IL-6 expression: According to the operating instructions of the ELISA detection kit, the cytokine IL-6 content in the culture medium supernatant stored in step (5) above was determined. The relative cytokine expression levels of the treatment group and the negative control group were calculated. The relative cytokine expression level (RCL) calculation formula is:

[0083] Relative cytokine expression = cytokine expression in sample group / cytokine expression in negative control group

[0084] (12) Determination of relative cytokine expression:

[0085] If the relative cytokine expression is >1.5, the sample is judged to have cytokine overexpression and the score is 1;

[0086] If the relative cytokine expression is ≤1.5, the sample is judged to have no cytokine overexpression and scored as 0;

[0087] (13) Analysis of test results: The total scores obtained based on the relative tissue viability (RTV) and relative cytokine expression (RCL) were used to determine the eye irritation of the test samples. The results are shown in Table 2.

[0088] Table 2 Eye irritation results of samples in Example 2

[0089]

[0090] Example 3

[0091] Test sample: Camphene, purchased from Sigma. After inquiry, this compound is found to be a severe eye irritant (https: / / pubchem.ncbi.nlm.nih.gov / compound / 6616).

[0092] (1) Model recovery: After receiving the purchased commercial in vitro recombinant human corneal epithelial model, place the in vitro recombinant human corneal epithelial model in a sterile 6-well plate with 0.9 mL of culture medium in each well. Avoid bubbles at the bottom during placement. Transfer the 6-well plate with the model to a CO 2 Incubator, 37 °C, 5% CO 2 Incubate overnight.

[0093] (2) Model preparation: Prepare a sterile 6-well plate according to the number of models required for the experiment, add 0.9 mL of maintenance culture medium to each well, and then use tweezers to move the corneal epithelial model to the 6-well plate. Make two parallel wells for each sample to be tested, methyl acetate as the positive control, and sterile water as the negative control. Both the positive and negative controls are set up with two parallel wells.

[0094] (3) Sample addition reaction: Before sample addition, 20 μL DPBS was added to the surface of the corneal epithelial model for pretreatment for 30 min, and then 40 mg of the sample to be tested, 40 μL of the positive control and the negative control were evenly added to the surface of the corneal epithelial model. The culture plate was placed at 37°C and 5% CO 2 , and cultured in an incubator at 95% relative humidity for 6 h.

[0095] (4) Rinse: After the reaction is completed, rinse slowly and gently with DPBS buffer to remove the sample on the surface of the corneal epithelial model, and try to avoid directly aligning it with the model surface. Each well is rinsed with 15mL DPBS, and the rinse volume is 5mL each time, and rinsed 3 times in total. After the rinse is completed, use a cotton swab to gently absorb the residual liquid on the surface of each corneal epithelial model and transfer it to a new 6-well plate, with each well containing 0.9mL of culture medium. The corneal epithelial model is exposed to room temperature for 25 minutes. After the end, the culture plate is placed at 37°C and 5% CO 2 , and incubated in an incubator at 95% relative humidity for 18 h.

[0096] (5) Storage of supernatant: After the corneal epithelial model is treated, the culture supernatant is immediately aspirated and stored at -80°C. The cytokine IL-6 content is detected using a human IL-6 ELISA kit within 1 month. Repeated freezing and thawing of the culture supernatant should be avoided.

[0097] (6) MTT reaction and incubation: Take a 24-well plate, add 300 μL of 1 mg / mL MTT working solution to each well, transfer the corneal epithelial model to the plate, and ensure that there are no bubbles between the bottom of the corneal epithelial model and the MTT solution. Place the plate at 37°C and 5% CO 2 , and cultured in an incubator at 95% relative humidity for 3 h.

[0098] (7) Color extraction of corneal epithelial model: After 3 hours of culture, the corneal epithelial model was taken out, the MTT solution on the bottom was blotted off on absorbent paper, and placed in a new 24-well plate. 2 mL of isopropanol solution was added to each well. The 24-well plate was sealed with sealing film and wrapped with tin foil to ensure light protection. The 24-well plate was placed in a 4°C refrigerator overnight.

[0099] (8) Activity assay: Puncture the corneal epithelial model to mix the solution evenly. Transfer to a 96-well plate, take two replicate wells for each tissue, add 200 μL of solution to each well, set up 4 isopropanol blank control replicate wells, and read the absorbance (OD value) at a wavelength of 570 nm.

[0100] (9) Calculation of relative tissue viability: Calculate the relative tissue viability (RTV) according to the following formula:

[0101] Relative tissue viability = OD value of sample group / OD value of negative control group × 100%;

[0102] (10) Determination of relative tissue vitality:

[0103] If the relative tissue viability was >60%, the sample was judged to have no cytotoxicity and scored as 0;

[0104] If the relative tissue viability is ≤60%, the sample is judged to be cytotoxic and scored as 1;

[0105] (11) Determination of cytokine IL-6 expression: According to the operating instructions of the ELISA detection kit, the cytokine IL-6 content in the culture medium supernatant stored in the above 5 was determined. The relative cytokine expression of the treatment group and the negative control group was calculated. The relative cytokine expression (RCL) calculation formula is:

[0106] Relative cytokine expression = cytokine expression in sample group / cytokine expression in negative control group

[0107] (12) Determination of relative cytokine expression:

[0108] If the relative cytokine expression is >1.5, the sample is judged to have cytokine overexpression and the score is 1;

[0109] If the relative cytokine expression is ≤1.5, the sample is judged to have no cytokine overexpression and scored as 0;

[0110] (13) Analysis of test results: The total scores obtained based on the relative tissue viability (RTV) and relative cytokine expression (RCL) were used to determine the eye irritation of the test samples. The results are shown in Table 3.

[0111] Table 3 Eye irritation results of samples in Example 3

[0112]

[0113]

[0114] Example 4

[0115] Test sample: polyoxyethylene hydrogenated castor oil, purchased from McLean. The description of the properties of this compound on the pubchem website does not contain any description of its eye irritation (https: / / pubchem.ncbi.nlm.nih.gov / substance / 363904876). However, other information indicates that it is an eye irritant (for example: https: / / redox.com / wp-content / sds / 978.pdf; https: / / www.ewg.org / cleaners / substances / 4201-PEG40HYDROGENATEDCASTOROIL / ).

[0116] (1) Model recovery: After receiving the purchased commercial in vitro recombinant human corneal epithelial model, place the in vitro recombinant human corneal epithelial model in a sterile 6-well plate with 0.9 mL of culture medium in each well. Avoid bubbles at the bottom during placement. Transfer the 6-well plate with the model to a CO 2Incubator, 37 °C, 5% CO 2 Incubate overnight.

[0117] (2) Model preparation: Prepare a sterile 6-well plate according to the number of models required for the experiment, add 0.9 mL of maintenance culture medium to each well, and then use tweezers to move the corneal epithelial model to the 6-well plate. Make two parallel wells for each sample to be tested, methyl acetate as the positive control, and sterile water as the negative control. Both the positive and negative controls are set up with two parallel wells.

[0118] (3) Sample addition reaction: Before sample addition, 20 μL DPBS was added to the surface of the corneal epithelial model for pretreatment for 30 min, and then 40 μL of the sample to be tested, 40 μL of the positive control and the negative control were evenly added to the surface of the corneal epithelial model. The culture plate was placed at 37°C and 5% CO 2 , cultured in an incubator with a relative humidity of 95%, and the sample to be tested was exposed to it for 30 minutes.

[0119] (4) Rinse: After the reaction is completed, rinse slowly and gently with DPBS buffer to remove the sample on the surface of the corneal epithelial model, and try to avoid directly aligning it with the model surface. Each well is rinsed with 15mL DPBS, and the rinse volume is 5mL each time, and rinsed 3 times in total. After the rinse is completed, use a cotton swab to gently absorb the residual liquid on the surface of each corneal epithelial model and transfer it to a new 6-well plate, with each well containing 0.9mL of culture medium. The corneal epithelial model is exposed to room temperature for 12 minutes. After the end, the culture plate is placed at 37°C and 5% CO 2 , and incubate in an incubator at 95% relative humidity for 2 h.

[0120] (5) Storage of supernatant: After the corneal epithelial model is treated, the culture supernatant is immediately aspirated and stored at -80°C. The cytokine IL-6 content is detected using a human IL-6 ELISA kit within 1 month. Repeated freezing and thawing of the culture supernatant should be avoided.

[0121] (6) MTT reaction and incubation: Take a 24-well plate, add 300 μL of 1 mg / ml MTT working solution to each well, transfer the corneal epithelial model to the plate, and ensure that there are no bubbles between the bottom of the corneal epithelial model and the MTT solution. Place the plate at 37°C and 5% CO 2 , and cultured in an incubator at 95% relative humidity for 3 h.

[0122] (7) Color extraction of corneal epithelial model: After 3 hours of culture, the corneal epithelial model was taken out, the MTT solution on the bottom was blotted off on absorbent paper, and placed in a new 24-well plate. 2 mL of isopropanol solution was added to each well. The 24-well plate was sealed with sealing film and wrapped with tin foil to ensure light protection. The 24-well plate was placed in a 4°C refrigerator overnight.

[0123] (8) Activity assay: Puncture the corneal epithelial model to mix the solution evenly. Transfer to a 96-well plate, take two replicate wells for each tissue, add 200 μL of solution to each well, set up 4 isopropanol blank control replicate wells, and read the absorbance (OD value) at a wavelength of 570 nm.

[0124] (9) Calculation of relative tissue viability: Calculate the relative tissue viability (RTV) according to the following formula:

[0125] Relative tissue viability = OD value of sample group / OD value of negative control group × 100%;

[0126] (10) Determination of relative tissue vitality:

[0127] If the relative tissue viability was >60%, the sample was judged to have no cytotoxicity and scored as 0;

[0128] If the relative tissue viability is ≤60%, the sample is judged to be cytotoxic and scored as 1;

[0129] (11) Determination of cytokine IL-6 expression: According to the operating instructions of the ELISA detection kit, the cytokine IL-6 content in the culture medium supernatant stored in step (5) above was determined. The relative cytokine expression levels of the treatment group and the negative control group were calculated. The relative cytokine expression level (RCL) calculation formula is:

[0130] Relative cytokine expression = cytokine expression in sample group / cytokine expression in negative control group

[0131] (12) Determination of relative cytokine expression:

[0132] If the relative cytokine expression is >1.5, the sample is judged to have cytokine overexpression and the score is 1;

[0133] If the relative cytokine expression is ≤1.5, the sample is judged to have no cytokine overexpression and scored as 0;

[0134] (13) Analysis of test results: The total scores obtained based on the relative tissue viability (RTV) and relative cytokine expression (RCL) were used to determine the eye irritation of the test samples. The results are shown in Table 4.

[0135] Table 4 Eye irritation results of samples in Example 4

[0136]

[0137] In previous recombinant human corneal epithelial model tests, the test criteria for evaluating the eye irritation of samples only relied on relative tissue viability based on cytotoxicity. This single evaluation criterion may lead to the omission of some samples that are not obviously cytotoxic but are actually eye irritating, thus producing false negative results. The present invention significantly improves the accuracy of the evaluation of the eye irritation of samples by introducing cytokine expression as an additional evaluation criterion. The results of relative tissue viability and relative cytokine expression of each sample in the eye irritation test using the model are shown in Figure 2. Figure 3 The results show that the eye irritation results of the samples in Examples 1 to 4 measured by the method of the present invention are consistent with the existing records.

[0138] In the process of eye irritation assessment, an important principle must be followed: analyze samples that may be irritating as strictly as possible to avoid misjudging samples with eye irritation as false negatives. Therefore, adding cytokine expression as an eye irritation evaluation criterion can effectively identify samples that are judged to be non-eye irritating in relative tissue viability testing, thereby attracting the attention of product developers and regulators and better protecting the health of users.

[0139] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for evaluating eye irritation in vitro, characterized in that: The method uses a recombinant human corneal epithelial model and uses relative tissue viability (RTV) and relative cytokine expression (RCL) to comprehensively evaluate the eye irritation of the sample to be tested; The following steps are involved: (1) Pretreatment of the sample to be tested: resuscitate the in vitro recombinant human corneal epithelial model, add the sample to be tested, culture it in a CO2 incubator for the first time, rinse it, let it stand at room temperature, and culture it in a CO2 incubator for the second time. After the culture is completed, aspirate the supernatant and save it for later use. The treated human corneal epithelial model is ready for use; (2) Activity assay: performing MTT reaction and incubation on the human corneal epithelial model treated in step (1), piercing the human corneal epithelial model to mix the solution evenly, and setting a blank control; measuring the OD value at a wavelength of 540 nm to 600 nm, and calculating the relative tissue activity; wherein the calculation formula for the relative tissue activity is: Relative tissue viability = OD value of sample group / OD value of negative control group × 100%; (3) Determination of relative tissue vitality: If the relative tissue viability is greater than the set RTV threshold, the sample is judged to have no cytotoxicity and is scored as 0; If the relative tissue viability is ≤ the set RTV threshold, the sample is judged to be cytotoxic and scored as 1; (4) Determination of cytokine expression: Take the supernatant obtained in step (1), set up a negative control, use an ELISA detection kit to determine the cytokine content therein, and calculate the relative cytokine expression; wherein, the calculation formula of the relative cytokine expression is: Relative cytokine expression = cytokine expression in the sample group / cytokine expression in the negative control group; (5) Determination of relative cytokine expression: If the relative cytokine expression level is greater than the set RCL threshold, the sample is judged to have cytokine overexpression and the score is 1; If the relative cytokine expression is ≤ the set RCL threshold, the sample is judged to have no cytokine overexpression and the score is 0; (6) Determination of eye irritation results: If the score of either the relative tissue viability or the relative cytokine expression level in the test sample is greater than 0, the result is determined to be irritating; if the scores of the relative tissue viability and the relative cytokine expression level in the test sample are both 0, the result is determined to be non-irritating.

2. The method according to claim 1, characterized in that: The cytokine is IL-1β, IL-6 or IL-8.

3. The method according to claim 1 or 2, characterized in that: The culturing time of the first culturing in step (1) is 0.5 h to 6 h, and the culturing time of the second culturing is 2 h to 18 h.

4. The method according to any one of claims 1 to 3, characterized in that: The duration of the reaction at room temperature in step (1) is 12 to 30 minutes.

5. The method according to claim 1, characterized in that The incubation described in step (2) is incubation in a CO2 incubator for 2 h to 4 h.

6. The method according to claim 1, characterized in that The RTV threshold value in step (3) is 50% or 60%.

7. The method according to claim 1, characterized in that The RCL threshold described in step (5) is >1.

8. Use of the method according to claims 1 to 7 in detecting the eye irritation of a product.

9. The use according to claim 8, characterized in that: The products include but are not limited to agricultural products, cosmetics, medicines, and medical devices.

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