CN110568173B - Method for predicting eye irritation by using combination of various corneal structural cells - Google Patents
Method for predicting eye irritation by using combination of various corneal structural cells Download PDFInfo
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Abstract
The invention discloses a method for predicting eye irritation by using a plurality of cornea structure cell combinations, which comprises the following steps: (one) prediction of corneal epithelial cell irritation; (II) predicting the stimulation of corneal stromal fibroblasts; (2.1) Neutral Red Release (NRR) test; (2.2) inflammatory factor assay; (III) prediction of corneal endothelial cell irritation; and (IV) establishing an eye irritation model based on various cornea structural cell combinations. The method can comprehensively reflect the toxic effect of a tested substance (such as an irritant or an etchant) on cells of the corneal structure, can be used for predicting the irritation degree of an eye irritation substance in a non-animal test, has rich data information, and can be widely applied.
Description
Technical Field
The invention belongs to the technical field of non-animal testing methods, and particularly relates to a method for predicting eye irritation by using various cornea structure cell combinations.
Background
In daily life, human eyes may actively contact or be accidentally exposed to exogenous substances such as medicines, daily chemical products, pesticides and the like, enter the eyes in an original or diluted form, generate local irritation and even corrosion, and possibly affect the health of people. Therefore, it is important to evaluate the possible eye irritation of foreign substances.
Eye irritation belongs to the category of health toxicology, the traditional method is a rabbit eye test proposed by Draize et al in 1944, and the simple process is to directly drip a sample to be tested into the eyes of rabbits to observe the injury reaction caused. At present, the method is still widely used in the international range. The scientificity and rationality of the Draize test has been questioned. With the continuous and deep humanity of animal welfare awareness and animal experiment 3R principles (Reduction, optimization of Refinement, Replacement of Relacement), and the development of new technologies, in-vitro Replacement methods for toxicity testing and evaluation are developed and successively verified and approved. In 2003, the european union proposed the requirement for prohibiting the animal experiments on cosmetics for the first time in the 7 th amendment (2003/15/EC) of the cosmetic directive (76/768/EEC), and from 3/11/2013, the european union performed all animal experiment ban on cosmetics and components and sale ban on cosmetics subjected to animal experiments. The official experimental REACH regulation of 6 months 2007 requires that safety assessments of all chemicals must be done with a minimum of experimental animals. The european union joint reference laboratory (EURL ECVAM) and the coordinated validation center between the us alternative methods departments (ICCVAM) proposed a series of non-animal test methods for chemicals, etc. At least 48 countries around the world have set forth regulatory requirements that prohibit the testing of cosmetic animals in general.
Corneal damage often involves three layers of structure from mechanical analysis, with the degree of damage being related to the depth of involvement. The existing in vitro experimental methods can be divided into chemical reaction, cell method, organ type method, recombinant human tissue model and the like. The chemical reaction is based on the fact that the stimulus usually has the effect of protein denaturation, a group of special proteins is designed in vitro, the test substance is reacted with the proteins, the degree of protein denaturation is quantitatively determined, and the possible eye irritation is presumed.
Organotypic methods include ex vivo corneal and chick embryo chorioallantoic membrane methods, where these ex vivo tissues can maintain normal physiological activity over a short period of time. The ex vivo cornea is obtained by exposing a chemical test substance to an eyeball or cornea of a separated cow, pig, chicken or rabbit, and observing the biological effect of the ex vivo tissue to test whether the test substance has eye irritation. Wherein, the ox horn membrane turbidity and permeability experiment (BCOP) is one of the alternative method standards of eye corrosivity and irritation approved by the verification and obtaining regulations, and the method is listed in the economic cooperation and development Organization (OECD) chemical Test Guide (TG) number 437, is suitable for identifying serious irritability/corrosivity substances and identifying nonirritant, and can test cosmetics, medicines, daily products, industrial products and the like in practice. But are less sensitive to mild to moderate eye irritation and require comprehensive evaluation in conjunction with other test methods.
Cellular methods are mainly based on assays for cytotoxicity and functionality. It has been found that certain eye-damaging subjects also elicit cytotoxic effects in various cell types, including the various epithelial, stromal, and endothelial tissues of the eyeball. Thus, ocular irritation can be detected by assays based on cytotoxicity and functionality. Such as the Neutral Red Uptake (NRU) assay, is a widely used cytotoxicity assay that is well adapted for evaluation and classification of mild ocular irritants, but does not respond to the characteristics of ocular irritation, by detecting the activity of cells in response to the ability of the test agent to inhibit the uptake of neutral Red dye by active cells. In addition, the erythrocyte hemolysis experiment is designed according to the principle that chemical substances can damage cell membranes, and can be applied to surfactant for distinguishing very mild degree from non-mild degree, but the observation index of the erythrocyte non-corneal structural cell is different from the real eye stimulation. The cell function-based test is a fluorescein leakage test (FL test) designed based on the principle that corneal epithelium has a barrier function to prevent penetration of harmful chemicals, and damage of the barrier function causes an eye irritation response to occur, and the FL test can be used to test severe irritation of surfactants but is not sensitive enough to mild irritation. The limitation of cytological methods is that the cells are not specific enough to directly measure the depth of corneal damage.
Recombinant human tissue models include EpiOcular, SkinEthic TM In the HCE model and HCE-TEP, the corneal epithelial tissue of a normal human consists of 5-6 layers of differentiated ectodermal cells, protecting the cornea and constituting his visual characteristics. The recombinant human tissue model is a model simulating corneal epithelial tissue, such as SkinEthic TM The HCE model is a human corneal epithelium model developed by a SkinEthic laboratory in France, is suitable for research experiments of cosmetics, personal care, daily chemical and chemical enterprises and the like, covers various different physicochemical properties and contains challenging raw material substances, is commercialized but cannot judge damage of a corneal endothelial layer, and has higher experimental research cost.
Eye irritation testing is one of the most active areas of research in alternative methods, most of which attempt to demonstrate their reliability and relevance by confirmatory studies, but to date there is no single in vitro test that can completely replace the in vivo Draize test, and in order to address the current situation where a single in vitro test method alone cannot cover all the lesions detected in an in vivo test, many institutions (e.g., OECD, ICCVAM, and ECVAM) recommend the use of a complete set of test procedures. Of course, there is still a need to develop new in vitro methods, which focus on not only the classification of eye irritants but also the study of functional lesion depth. In addition, the existing in vitro eye stimulation method combination evaluation has the problems of long experiment time consumption, high cost, certain operation difficulty, high laboratory variation and the like, and needs to be optimized to further improve the detection flux of the method.
Disclosure of Invention
The invention aims to provide a method for predicting the eye irritation by using a plurality of cornea structural cell combinations, which can comprehensively reflect the toxic action of a tested substance (such as an irritant or an etchant) on cornea structural cells, can predict the irritation degree of an eye irritation substance by non-animal tests, has rich data information and can be widely applied.
The above object of the present invention can be achieved by the following technical solutions: a method for predicting eye irritation by using a plurality of cornea structural cell combinations comprises the following steps:
(ii) prediction of corneal epithelial cell irritation
(1.1) determination of the highest test concentration: selecting corneal epithelial cells, and determining the concentration of a test substance with the cell activity of more than 80% by adopting an MTT (methanol to transfer) method as the highest test concentration;
(1.2) Barrier function test: performing fluorescein leakage test by adopting the highest test concentration in the step (1.1), obtaining the leakage percentage of the fluorescein sodium, and calculating the concentration FL of the test substance which causes 20 percent of the fluorescein sodium to leak out after contacting with the test substance 20 (g/100mL) and according to the concentration FL of the test substance leaked out of 20% sodium fluorescein for 4h 20 H 4 And (3) performing irritation prediction: FL 20 H 4 The stimulation is nonirritant or mild stimulation when the concentration is more than 10g/100mL, moderate stimulation is 2-10 g/100mL, and severe stimulation is less than 2g/100 mL;
(II) prediction of corneal stromal fibroblast irritation
(2.1) Neutral Red Release (NRR) test: selecting keratinocyte, and subjecting to testPreparing gradient concentration, adding neutral red dye, performing absorbance test, calculating cell activity rate, and calculating the concentration of the test substance (NRR) capable of releasing 50% of neutral red dye of angle matrix fibroblast according to dose-cell activity rate curve 50 And calculating the cell death rate at a subject concentration of 50%, i.e., the PM value, according to the dose-cell death rate curve, according to the NRR 50 And PM value to establish a model for predicting toxicity and irritation of corneal stromal fibroblasts as shown in the following Table 1:
TABLE 1 cytotoxicity determination and Ocular irritation prediction model
| NRR 50 (％) | PM | Cytotoxicity classification | Corneal stroma irritation prediction classification |
| NRR 50 >50 | PM≤20 | Has no cytotoxicity | Has no irritation |
| NRR 50 >50 | 20<PM<50 | Mild cytotoxicity | Mild irritation |
| 25≤NRR 50 ≤50 | -- | Moderate cytotoxicity | Moderate irritation |
| NRR 50 <25 | -- | Severe cytotoxicity | Severe irritation |
；
(2.2) inflammatory factor assay: when the prediction in the step (2.1) is classified as non-irritant, analyzing the expression of the inflammatory cytokines by enzyme-linked immunosorbent assay, judging as non-irritant if the expression does not cause any increase in the release of any inflammatory factor under a set concentration, judging as mild irritative if the expression causes at least one increase in the release of the inflammatory factor under a set concentration, and judging as irritative if the expression does not cause any increase in the release of the inflammatory factor under any other conditions;
(III) prediction of corneal endothelial cell irritation
The Neutral Red Release (NRR) test and the inflammatory factor test in the corneal stromal fibroblast irritation prediction in the step (II) are the same, except that the corneal endothelial cells are adopted, and the established ocular irritation prediction model and the inflammatory factor test irritation model are the same;
(IV) establishment of model for predicting eye irritation based on combination of various cornea structural cells
According to the results of the steps (one) to (three), a model for predicting the eye irritation based on various cornea structural cell combinations is established as follows:
optionally, the subject in step (1.1) comprises personal care products, household washing products, pharmaceuticals, plant extracts, medical devices and extracts thereof, biological materials, environmental pollutants, and other eye contact products.
Optionally, the personal care product comprises a shampoo, a conditioner, a hair dye, a skin care product, a sunscreen product, an eye shadow, a body wash or a body lotion, the household washing product comprises a laundry detergent, a washing powder, a soap, a detergent, a bleach, a disinfectant or a softener, and the plant extract comprises a plant crude extract, a plant fine extract and a mixture of the two; such other eye contact products include textile extract garments or undergarments.
Optionally, the percentage of sodium fluorescein leakage is calculated in step (1.2) using the following formula:
m represents the average value of the leakage amount of the fluorescein at each concentration of each test object, y represents the average value of the leakage amount of the fluorescein of a control group without the test objects, and z represents the average value of the leakage amount of the fluorescein of a control group without cell culture.
Optionally, the inflammatory factor in step (2.2) comprises one or more of TGF-beta, TGF-alpha, IL-1, IL-2, IL-8 and Platelet Derived Growth Factor (PDGF).
Optionally, in step (2.1), the test substance is prepared in a gradient concentration with gradual dilution according to the following percentages: 50%, 35%, 25%, 15%, 5%.
Optionally, the set concentration in step (2.2) is the highest assay concentration for the inflammatory factor assay selected for a concentration of the test substance having a cellular activity > 80% as determined according to the dose-cell activity rate curve of the Neutral Red Release (NRR) assay in step (2.1).
Optionally, the corneal epithelial cells of step (1.1) comprise a commercially available monolayer or multilayer culture of cell lines (rabbit corneal epithelial SIRC cells, human corneal HCE-T cells), a corneal epithelial-like cell line (e.g., canine tubular epithelial MDCK cells); primary cultures of corneal epithelial cells isolated from rabbits, cattle, or humans are also possible.
Optionally, when the highest assay concentration in step (1.1) of the present invention is determined, the method specifically includes the following steps:
preparing a group of solutions with concentration gradient for the tested objects, adding the tested objects with concentration gradient to form the tested object group when the cell density of the cells (the corneal epithelial cells cultured conventionally) is as high as 80% -90%, a positive control group (0.2g/mL zinc diethyldithiocarbamate polyurethane), a negative control group (0.2g/mL high-density polyethylene) and a blank control group (10% FBS-containing DMEM culture medium) are simultaneously arranged, MTT and DMSO solutions are respectively added into each group, the absorbance of each group is respectively measured by an enzyme labeling method, and cell death rate, which is [ 1- (test absorbance value-blank absorbance value) ÷ (negative control absorbance value-blank absorbance value) ] × 100%, was calculated according to the following formula, and 80% inhibitory concentration (IC80) of the test substance was calculated according to the cell death rate curve. The highest concentration tested was selected as the concentration of the test substance with a cell activity of > 80%.
Alternatively, MTT is known by the english name 3- (4,5) -dimethylthiohiazolo (-z-y1) -3, 5-di-phenylthiotetrazolium romide, chinese chemical name 3- (4, 5-dimethylthiazol-2) -2, 5-diphenyltetrazolium bromide, trade name: thiazole blue, a yellow dye, is a commonly used method for detecting cell survival and growth by MTT colorimetry.
Optionally, when the barrier function is tested in step (1.2) of the present invention, the method specifically includes the following steps:
fluorescein Leakage (FL) test: cells were grown in embedded dishes and cultured epithelial cells formed tight junctions and desmosomes when fused. The cell layer divides a culture medium into two parts, the cells contact a certain amount of test objects with different concentrations on the upper layer, 3 parallel holes are arranged, after 15min +/-1 min, Hank's solution is used for gently washing the surfaces of the cells to remove the test objects, the cells are placed into another culture dish containing Hank's solution, 10 mu g/mL fluorescein sodium solution is added to the cells on the upper layer, after incubation is carried out for 30min +/-5 min at 37 +/-1 ℃, a fluorescence spectrophotometer is used for detecting (excitation wavelength is 485nm, emission wavelength is 530nm) permeation into a culture plate, namely the amount of the fluorescein in the lower layer of the cells is used as an index of the damage degree of a cell barrier. Since most of the cells remained viable, the cells were washed, placed on another dish containing fresh culture medium, incubated at 37 ℃. + -. 1 ℃ for 4 hours, and then added with the sodium fluorescein solution again, and tested to see the effect of the test substance on tight cell junctions. And sequentially detecting the influence of the 24h and 72h test substances on the cells, and calculating the percentage of the leaked fluorescein sodium according to the following formula:
m represents the mean leakage of fluorescein at each concentration of each test sample, y represents the mean leakage of fluorescein of a control group without test sample (negative control), and z represents the mean leakage of fluorescein of a control group without cell culture (positive control).
Wherein the medium is DMEM containing 10% FBS. DMEM medium is commercially available, for example from GIBCO.
Embedded cell culture dishes are commercially available, such as Millcell from MILLIPORE or Transwell from corning.
Optionally, the Enzyme-linked immunosorbent assay used in step (1.3), Enzyme-linked immunosorbent assays, ELISA.
Alternatively, the ELISA kit for testing inflammatory factors in step (1.3) is commercially available, and the sample is loaded, washed, stained with reference to the ELISA kit instructions, and the OD value is measured with a microplate reader, as follows.
Optionally, the keratinocyte fibroblasts in step (2.1) include cell lines (e.g., HECE-12), transfected cells, and primary cells (rabbit, bovine, human, and equine basement-stromal fibroblast primary cultures may be isolated).
Optionally, the Neutral Red Release (NRR) test in step (2.1) may specifically include the following steps:
preparing a group of solutions with concentration gradient for a tested object, inoculating the solution to a 24-hole culture dish when the cultured cell density is 80% -90%, culturing for 18-24h conventionally, checking the cell confluence condition of each hole of the 24-hole culture dish under a microscope, adding neutral red dye into the monolayer cells which are close to or reach fusion, incubating for 3h +/-30 min at 37 +/-1 ℃, then removing the neutral red dye, and adding a culture solution to terminate for 30min +/-10 min. After the culture solution is replaced, washing the cells for 2-3 times by using about 2mL of buffer solution, adding 500 μ L of the test object with the concentration gradient to form a test object group, simultaneously setting a positive control group (0.01%, 0.05%, 0.2% Sodium Dodecyl Sulfate (SDS)), a negative control group (0.9% Sodium chloride solution) and a blank control group (DMEM culture medium containing 10% FBS), directly contacting the test object and the control group with the cells for 1-5 min, and after the exposure is finished, washing the test object (with the reference wavelength of 405nm) by using a proper solvent to measure the absorbance of the final solution.
Calculating cell activity and inhibition according to the following formula, cell activity ═ (test absorbance value-blank absorbance value) ÷ (negative control absorbance value-blank absorbance value) × 100%; cell death ═ 1- (test absorbance value-blank absorbance value) ÷ (negative control absorbance value-blank absorbance value) ] × 100%.
The concentration of the test substance that released 50% of the neutral red dye from the preloaded cells (corneal stromal fibroblasts) was calculated from the dose-cell activity rate curve, which was defined as NRR 50 。
Calculating the cell death rate, and calculating the cell death rate under the condition that the concentration of the test substance is 50% through a dose-cell death rate curve, namely the PM value (percent of cell motility observed at the 50% differentiation).
Wherein the medium was DMEM with 10% FBS, 4mmol/L L-glutamine, 100IU penicillin and 100. mu.g/mL streptomycin.
FBS (fetal bovine serum) is fetal bovine serum and is commercially available, for example, from GIBCO.
DMEM medium is a medium containing various amino acids and glucose, and is developed on the basis of MEM medium. Compared with MEM, the dosage of each component is increased, and the components are divided into a high-sugar type (lower than 4500mg/L) and a low-sugar type (lower than 1000 mg/L). The high-sugar type is favorable for the growth of cells anchored at one position, and is suitable for tumor cells which grow fast and are difficult to attach, and the like.
The buffer solution for washing the cells was DPBS (Dulbecco's phosphate buffered saline): all contain potassium chloride, potassium dihydrogen phosphate, sodium chloride and disodium hydrogen phosphate. DPBS is useful for maintaining a stable pH, is an isotonic solution of cells, and is non-cytotoxic.
Optionally, the corneal endothelial cells in step (3.1) include cell lines (e.g., HECE-12), transfected cells, and primary cells (rabbit, bovine, human, etc. endothelial cell primary cultures can be isolated).
Alternatively, in the step (iv) of establishing the model for predicting the eye irritation based on the combination of the cells in the plurality of corneal structures, in general, the weights in the toxicity of the corneal three-layer structure are sequentially from high to low: endothelial > epithelium > stroma; the injury parameter weight is, from high to low, that inflammatory factor > barrier function > cytotoxicity.
The invention has the following beneficial effects:
(1) the invention utilizes the evaluation method of predicting the eye irritation by combining various cornea structure cytotoxicity, can comprehensively embody the toxic action of a tested substance (irritant or corrosive) on corneal structure cells;
(2) the invention has wide application by carrying out mechanism research and stimulation degree prediction on the eye stimulating substances;
(3) the method has the advantages that the cytokine is qualitatively detected, the sensitivity is high, the flux is high, the detection parameters are comprehensive, the specificity is high, more detection data are provided for the prediction of eye irritation for evaluation, and the method is suitable for the accurate comparison of different tested substances;
(4) the method established by the invention can replace animals, provide quantitative and qualitative detection data, and can be directly used for predicting eye-irritating toxicity and predicting functional damage depth of products such as chemicals, medicines, cosmetics and the like.
Detailed Description
Example 1 evaluation of eye irritation of a brand of amino acid foam facial cleanser based on various methods of corneal structural cytotoxicity
1. Corneal epithelial irritation prediction
1.1 conventional culture of corneal epithelial cells: the cells used were rabbit corneal epithelial SIRC cells.
1.2 cytotoxicity assays
(1) Determination and preparation of the concentration of the test substance: 200mg of facial cleanser is weighed by an analytical balance, stock solution is prepared by taking a serum-free culture medium as a solvent, and 100mg/mL of mother solution is prepared for standby.
(2) Plate paving: when the density of corneal epithelial cells in a cell bottle reaches 80%, adding 0.25% pancreatin/0.02% EDTA to digest and separate the cells from the cell bottle, centrifuging at 1200rpm/min for 5 minutes, removing pancreatin, adding new culture medium for resuspension, diluting 1mL of cell suspension to 4mL, dropping 10 μ L of cell suspension on a cell counting plate, counting under a 40X microscope, adjusting the concentration, and counting at 1X 10 4 Cells were plated in 96-well plates and incubated in an incubator for 24 hours.
(3) Addition of test substance: the final concentration of facial cleanser was 100mg/mL, diluted to 31.6mg/mL, 10mg/mL, 3.2mg/mL, 1mg/mL, 0.32 mg/mL. Diluted facial cleanser was added to 96-well plates, with blank control, negative control, and 6 secondary wells for each concentration. A multichannel pipettor was used to reduce the inter-well variation. And placing the mixture in an incubator to incubate for 18-22 hours. After the incubation was completed, the plate was removed from the incubator, and 20. mu.l of 5mg/mL MTT solution (thiazole blue) was added to each well, protected from light. Placing in an incubator to incubate for 4 h. After incubation, DMSO (dimethyl sulfoxide) was added in an amount of 100. mu.L per well, and the mixture was placed on a shaker for 10 min.
(4) Enzyme-linked immunosorbent assay and cell activity calculation: and (4) measuring the absorbance of each well added in the 96 wells in the step (3) at 570nm by using a microplate reader to obtain the absorbance value of the surviving cells of each well. Calculating IC by formula 50 (median inhibitory concentration, i.e. the concentration of test substance at 50% cell activity):
cell activity ═ [ (test absorbance value-blank absorbance value) ÷ (negative control absorbance value-blank absorbance value) ] × 100%
(5) Cytotoxicity assay: the test results were obtained as follows: the cell survival rate at 100mg/L was 51.89%, 64.89% at 31.6mg/L, 78.91% at 10.01mg/L, 87.63% at 3.16mg/L, 88.46% at 1.00mg/L and 87.26% at 0.32 mg/L. Statistical preparation of facial cleanser, IC 80 The concentration of the test substance with a cell activity of > 80% was selected as the highest assay concentration for the fluorescein leakage assay at 14.98 mg/mL.
1.3 Fluorescein Leakage (FL) test:
(1) cells were cultured at 2X 10 5 The/well was seeded in an embedded petri dish and when the cell density reached 80% the epithelial cells formed tight junctions and desmosomes. The cell layer divides a culture medium into two parts, the concentration of a test object is set to be 14mg/mL (the concentration of the test object with the cell activity of more than 80% is selected as the highest test concentration of a fluorescein leakage test, and can be selected as long as the concentration is more than 80%), 7mg/mL and 3mg/mL are set, 3 parallel holes are arranged, 0.4mL is added on the cell surface, after 15min +/-1 min, the cell surface is washed by Hank's solution gently to remove the test object, the cell surface is placed in another culture dish containing Hank's solution, 10 mu g/mL of fluorescein sodium solution is added to cells on the upper layer, after incubation is carried out for 30min +/-5 min at 37 +/-1 ℃, and the fluorescence spectrophotometer is used for detecting (the excitation wavelength is 485nm, the emission wavelength is 530nm) to permeate into the culture plate, namely, the amount of the fluorescein in the cell lower layer is used as an index of the damage degree of the cell barrier. Since most of the cells remained viable, the cells were washed, placed on another dish containing fresh culture medium, incubated at 37 ℃. + -. 1 ℃ for 4 hours, and then added with the sodium fluorescein solution again, and tested to see the effect of the test substance on tight cell junctions. And sequentially detecting the influence of the 24h and 72h test substances on the cells, and calculating the percentage of the sodium fluorescein leaking out according to the following formula:
(2) fluorescein Leakage (FL) test results analysis:
dose-FL 20 The curve is: y is 0.0725x +0.0126, when y is 20%, x is 2.55g/100ml, namely FL 20 H 4
FL 20 H 4 The medium irritativeness is 2-10 g/100 mL.
2. Prediction of corneal stromal fibroblast irritation
2.1 corneal stromal fibroblasts are cultured conventionally, and the cells used are human primary corneal stromal fibroblasts (HCS).
2.2 sample configuration: adding 0.9% NaCl solution into facial cleanser to reach a final concentration of 50%, and diluting to 35%, 25%, 15%, 5%; the positive control is SDS, prepare 0.01%, 0.05%, 0.2%; the negative control was 0.9% NaCl solution.
2.3, paving: when the density of the corneal stroma fibroblast in the cell bottle reaches 80%, adding 0.25% pancreatin/0.02% EDTA to digest and separate the cell from the cell bottle, centrifuging at 1200rpm/min for 5 minutes, removing pancreatin, adding new culture medium for resuspension, diluting 1mL of cell suspension to 4mL, dropping 10 μ L of cell suspension onto a cell counting plate, counting under a 40X microscope, adjusting the concentration, and performing 2X 10 5 Cells were plated in 24-well plates and incubated in an incubator for 24 hours.
2.4 Neutral Red Release (NRR) test: adding 0.05mg/mL neutral red dye into a 24-well plate, incubating for 3h +/-30 min at 37 +/-1 ℃, then removing the neutral red dye, and adding culture solution to stop for 30min +/-10 min. After changing the culture solution, washing 2-3 times with about 2mL buffer solution, adding 500 μ L of the test substance with concentration gradient to form a test substance group, simultaneously setting up a positive control group, a negative control group and a blank control group, and 2 multiple wells, directly contacting the test substance and the control group with cells for 1min, after exposure, washing 2-3 times with PBS, adding 1mL of analysis solution (acetic acid: 50% ethanol is 1: 99) into each well, stirring for 15min, taking 200 μ L of each well, transferring to a 96-well plate, repeating 4 wells, and measuring the absorbance of the final solution at a reference wavelength of 405 nm.
2.5 calculating the cell activity and inhibition according to the following formula, cell activity ═ (test absorbance value-blank absorbance value) ÷ (negative control absorbance value-blank absorbance value) × 100%; cell death rate ═ 1- (test absorbance-blank absorbance) ÷ (negative control absorbance-blank absorbance)]X 100% in preloaded cells calculated by dose-cell Activity Rate CurveThe sexual red dye released 50% of the concentration of the test substance. Toxicity endpoints are commonly used with NRR 50 Expressing; the PM value was calculated as (1-cell activity) × 100% cell death rate, and the cell death rate at 50% concentration was calculated by a dose-cell death rate curve.
2.6 results:
the dose-cell activity curve is: when y is 50%, x is 63.86%, i.e., NRR is-0.7006 x +0.9472 50 。
The dose-cell death rate curve is: and y is 0.7006x +0.0528, and when x is 50%, y is 40.31%, namely PM.
Sample NRR 50 63.86% (greater than 50%), and a PM value of 40.31% (greater than 20%, less than 50%), the sample was slightly cytotoxic and slightly irritating to corneal stromal fibroblasts.
3. Prediction of corneal endothelial cell irritation
3.1 corneal endothelial cell routine culture: human primary corneal endothelial HCEC cells.
(1) Sample preparation: adding 0.9% NaCl solution into facial cleanser to reach a final concentration of 50%, and diluting to 35%, 25%, 15%, 5%; the positive control is SDS, prepare 0.01%, 0.05%, 0.2%; the negative control was 0.9% NaCl solution.
(2) Paving a plate: when the density of the corneal endothelial cells in the cell bottle reaches 80%, adding 0.25% pancreatin/0.02% EDTA to digest and separate the cells from the cell bottle, centrifuging at 1200rpm/min for 5 minutes, removing pancreatin, adding new culture medium for resuspension, diluting 1mL of cell suspension to 4mL, dropping 10 μ L of cell suspension on a cell counting plate, counting under a 40X microscope, adjusting the concentration, and counting at 2X 10 5 Wells cells were plated in 24-well plates and incubated in an incubator for 24 hours.
(3) Neutral Red Release (NRR) test: adding 0.05mg/mL neutral red dye into a 24-well plate, incubating for 3h +/-30 min at 37 +/-1 ℃, then removing the neutral red dye, and adding culture solution to stop for 30min +/-10 min. After changing the culture solution, washing 2-3 times with about 2mL buffer solution, adding 500 μ L of the test substance with concentration gradient to form a test substance group, simultaneously setting up a positive control group, a negative control group and a blank control group, and 2 multiple wells, directly contacting the test substance and the control group with cells for 1min, after exposure, washing 2-3 times with PBS, adding 1mL of analysis solution (acetic acid: 50% ethanol is 1: 99) into each well, stirring for 15min, transferring 200uL of each well to a 96-well plate, repeating 4 wells, and measuring the absorbance of the final solution at a reference wavelength of 405 nm.
(4) Calculating cell activity and inhibition according to the following formula, cell activity ═ (test absorbance value-blank absorbance value) ÷ (negative control absorbance value-blank absorbance value) × 100%; cell death rate ═ 1- (test absorbance value-blank absorbance value) ÷ (negative control absorbance value-blank absorbance value)]X 100%, the concentration of test agent that released 50% of preloaded cellular neutral red dye was calculated by the dose-cell activity curve. Toxicity endpoints are commonly used with NRR 50 Expressing; the PM value was calculated as (1-cell activity) × 100% cell death rate at 50% concentration by dose-cell death rate curve.
(5) NRR test results:
| group of | OD value | Cell viability |
| Blank control | 0.0379 | -- |
| Negative control | 0.6351 | 100.00％ |
| Positive control 0.01% | 0.6052 | 95.00％ |
| Positive control 0.05% | 0.6216 | 97.75％ |
| Positive control 0.2% | 0.2470 | 35.03％ |
| 5％dilution | 0.7458 | 103.52％ |
| 15％dilution | 0.7181 | 99.47％ |
| 25％dilution | 0.6803 | 93.95％ |
| 35％dilution | 0.6594 | 90.89％ |
| 50％dilution | 0.6245 | 85.79％ |
The dose-cell activity rate curve is: when y is 50%, x is 138.48%, i.e., NRR is-0.3976 x +1.0506 50 。
The dose-cell death rate curve is: and y is 0.7006x +0.0528, and when x is 50%, y is 14.82%, namely PM.
Sample NRR50 was 138.48% (greater than 50%) and PM 14.82% (less than 20%), which was not cytotoxic and irritating to corneal endothelial cells.
And selecting the test concentration with the cell activity of more than 80% as the highest test concentration of the cytokine test, wherein the test concentration with the cell activity of more than 80% is selected according to the NRR test dose-cell activity rate curve when the sample concentration is 63.02%.
2.3 inflammatory factor assay
(1) Sample preparation: the facial cleanser is diluted to 50% final concentration by adding 0.9% NaCl solution, and further diluted to 25%, 12.5%, blank control is 0.9% NaCl solution.
(2) Paving a plate: when the density of corneal endothelial cells in the cell bottle reaches 80%, adding 0.25% pancreatin/0.02% EDTA to digest and separate the cells from the cell bottle, centrifuging at 1200rpm/min for 5 minutes, removing pancreatin, adding new culture medium for resuspension, diluting 1mL of cell suspension to 4mL, dripping 10 μ L of cell suspension on a cell counting plate, counting under a 40 × microscope, adjusting the concentration, and counting at 2.0-3.0 × 10 5 Cells/well cells were plated in 12-well plates and placed in an incubator for 24 hours.
(3) Taking out the culture plate, discarding the original culture solution in the wells, adding 1mL of test sample containing different concentrations into each well, blank control, and culturing for 24 + -1 h.
(4) Removing the original culture solution, adding 1mL of fresh culture solution, and continuing culturing for 24 + -1 h.
(5) Supernatants were collected and assayed for the inflammatory factors TGF-. beta.as well as the growth factors PDGF, FGF-2 and EGF8 using ELISA kits.
(6) Inflammatory factor test results
The supernatants were collected and tested for the inflammatory factor EGF8, the results of which are shown in table 2 below.
TABLE 2 comparison of EGF-8 differences between different groups
*: the difference was statistically significant compared to the control group.
The release of inflammatory factors in the culture medium is not statistically significant in the sample group at the concentrations of the test substances of 50%, 25% and 12.5% compared with the blank control group, and the EGF-8 inflammatory factors are not released from the corneal endothelial cells in the sample group at the concentrations of 50%, 25% and 12.5%.
The sample was not cytotoxic, irritating to corneal endothelial cells and did not cause inflammatory factor release, with no irritation as a result.
4. Eye irritation prediction model
Example 2 evaluation of ocular irritation of a Brand of eye drops based on various corneal structural cytotoxicity methods
1. Corneal epithelial irritation prediction
1.1 corneal epithelial cells: the cells used were human corneal cell line THCE cells.
1.2 cytotoxicity assays
(1) Determination and preparation of the concentration of the test substance: 200mg of eye drop is weighed by an analytical balance, stock solution is prepared by taking a serum-free culture medium as a solvent, and mother solution of 100mg/mL is prepared for later use.
(2) Plate paving: when the density of corneal epithelial cells in a cell bottle reaches 80%, adding 0.25% pancreatin/0.02% EDTA to digest and separate the cells from the cell bottle, centrifuging at 1200rpm/min for 5 minutes, removing the pancreatin, adding new culture medium for resuspension, diluting 1mL of cell suspension to 4mL, dropping 10 μ L of the cell suspension on a cell counting plate, counting under a 40X microscope, adjusting the concentration,at 1 × 10 4 Cells were plated in 96-well plates and incubated in an incubator for 24 hours.
(3) Addition of test substance: the final concentration of the eye drop is 100mg/mL, and the eye drop is diluted to 31.6mg/mL, 10mg/mL, 3.2mg/mL, 1mg/mL and 0.32 mg/mL. Diluted facial cleanser was added to 96-well plates, with blank control, negative control, and 6 secondary wells for each concentration. A multichannel pipettor was used to reduce the inter-well variation. And placing the mixture in an incubator to incubate for 18-22 hours. After the incubation was completed, the plate was removed from the incubator, and 20. mu.l of 5mg/mL MTT solution was added to each well, protected from light. Placing in an incubator to incubate for 4 h. After incubation, DMSO was added at 100 μ L per well, and the mixture was placed on a shaker for 10 min.
(4) Enzyme-linked immunosorbent assay and cell activity calculation: and (4) measuring the absorbance of each well added in the 96 wells in the step (3) at 570nm by using a microplate reader to obtain the absorbance value of the surviving cells of each well. Calculating IC by formula 50 (median inhibitory concentration, i.e. the concentration of test substance at 50% cell activity):
cell activity ═ [ (test absorbance value-blank absorbance value) ÷ (negative control absorbance value-blank absorbance value) ] × 100%
(5) Cytotoxicity assay: the test results were obtained as follows: the cell survival rate at 100mg/L was 68.02%, 81.13% at 31.6mg/L, 85.53% at 10.01mg/L, 91.68% at 3.16mg/L, 96.22% at 1.00mg/L, and 96.38% at 0.32 mg/L. Obtaining the IC of the facial cleanser by statistics 80 The concentration of the test substance with a cellular activity of > 80% was chosen as the highest assay concentration for the fluorescein leakage assay at 49.73 mg/mL.
1.3 Fluorescein Leakage (FL) test:
(1) cells were cultured at 2X 10 5 The/well was seeded in an embedded petri dish and when the cell density reached 80% the epithelial cells formed tight junctions and desmosomes. The cell layer divides the culture medium into two parts, the concentration of the tested substance is set to be 40mg/mL (the concentration of the tested substance with the cell activity of more than 80 percent is selected as the highest test concentration of the fluorescein leakage test, and can be selected as long as the concentration is more than 80 percent), 20mg/mL and 10mg/mL, 3 parallel holes are arranged, 0.4mL is added on the cell surface, and after 15min +/-1 min, the cell surface is lightly treated by Hank's solutionThe cell surface is washed to remove the tested substance, and then put into another culture dish containing Hank's solution, 10 mu g/mL fluorescein sodium solution is added to the upper layer of cells, and after incubation for 30min +/-5 min at 37 +/-1 ℃, the amount of fluorescein permeating into the culture plate, namely the lower layer of cells, is detected by a fluorescence spectrophotometer (excitation wavelength is 485nm, emission wavelength is 530nm) and is used as an index of the damage degree of the cell barrier. Since most of the cells remained viable, the cells were washed, placed on another dish containing fresh culture medium, incubated at 37 ℃. + -. 1 ℃ for 4 hours, and then added with sodium fluorescein solution again, and examined to see the effect of the test substance on the tight junction of the cells. And sequentially detecting the influence of the 24h and 72h test substances on the cells, and calculating the percentage of the leaked fluorescein sodium according to the following formula:
(2) fluorescein Leakage (FL) test results analysis:
dose-FL 20 The curves are: when y is 0.0152x +0.0237, when y is 20%, x is 11.60g/100ml, FL is obtained 20 H 4 。
FL 20 H 4 > 10g/100mL is non/mildly irritating.
2. Corneal stromal fibroblast irritation prediction
2.1 conventional culture of corneal stromal fibroblasts: rabbit corneal anterior stromal fibroblasts RCFBF.
2.2 Neutral Red Release (NRR) test:
(1) sample preparation, adding 0.9% NaCl solution into eye drop to dilute to 50% of final concentration, and further diluting to 35%, 25%, 15%, 5%; the positive control is SDS, prepare 0.01%, 0.05%, 0.2%; the negative control was 0.9% NaCl solution.
(2) Plate paving: adding the corneal stroma fibroblast when the density of the corneal stroma fibroblast in the cell bottle reaches 80 percentDigesting and separating cells from a cell flask by 0.25% pancreatin/0.02% EDTA, centrifuging at 1200rpm/min for 5 minutes, removing pancreatin, adding new culture medium for resuspension, taking 1mL of cell suspension for dilution to 4mL, taking 10 μ L of cell suspension, dropping on a cell counting plate, counting under a 40X microscope, adjusting the concentration, and counting at 2X 10 5 Wells cells were plated in 24-well plates and incubated in an incubator for 24 hours.
(3) Adding 0.05mg/mL neutral red dye into a 24-well plate, incubating for 3h +/-30 min at 37 +/-1 ℃, then removing the neutral red dye, and adding culture solution to stop for 30min +/-10 min. After changing the culture solution, washing 2-3 times with about 2mL buffer solution, adding 500 μ L of the test substance with concentration gradient to form a test substance group, simultaneously setting up a positive control group, a negative control group and a blank control group, and 2 multiple wells, directly contacting the test substance and the control group with cells for 1min, after exposure, washing 2-3 times with PBS, adding 1mL of analysis solution (acetic acid: 50% ethanol is 1: 99) into each well, stirring for 15min, transferring 200uL of each well to a 96-well plate, repeating 4 wells, and measuring the absorbance of the final solution at a reference wavelength of 405 nm.
(4) Calculating cell activity and inhibition according to the following formula, cell activity ═ (test absorbance value-blank absorbance value) ÷ (negative control absorbance value-blank absorbance value) × 100%; cell death rate ═ 1- (test absorbance-blank absorbance) ÷ (negative control absorbance-blank absorbance)]X 100%, the concentration of test substance that released 50% NRU from preloaded cellular neutral red dye was calculated by dose-cell activity curve. Toxicity endpoints are commonly used with NRR 50 Expressing; the PM value was calculated as (1-cell activity) × 100% cell death rate at 50% concentration by dose-cell death rate curve.
(5) NRR test results:
| group of | OD value | Cellular activity |
| Blank control | 0.0363 | -- |
| Negative control | 0.8794 | 100.00％ |
| Positive control 0.01% | 0.7164 | 80.67％ |
| Positive control 0.05% | 0.3492 | 37.11％ |
| Positive control 0.2% | 0.1768 | 16.66％ |
| 5％dilution | 0.5936 | 93.06％ |
| 15％dilution | 0.5561 | 86.78％ |
| 25％dilution | 0.5908 | 92.58％ |
| 35％dilution | 0.5294 | 82.30％ |
| 50％dilution | 0.5241 | 81.42％ |
The dose-cell activity curve is: when y is 50%, x is 174.07%, i.e., NRR is-0.2514 x +0.9376 50 。
The dose-cell death rate curve is: and y is 0.2514x +0.0624, and when x is 50%, y is 18.81%, namely the PM.
Sample NRR50 was 174.07% (greater than 50%) and PM 18.81% (less than 20%), which was not cytotoxic and irritating to corneal stromal fibroblasts.
And selecting the test substance with the cell activity of more than 80% as the highest test concentration of the inflammatory factor test according to the NRR test dose-cell activity rate curve, wherein the cell activity is more than 80% when the sample concentration is 54.73%.
2.3 inflammatory factor assay
(1) The sample preparation comprises diluting eye drop with 0.9% NaCl solution to a final concentration of 50%, further diluting to 25%, 12.5%, and blank control with 0.9% NaCl solution.
(2) Plate paving: when the density of the corneal stromal fibroblasts in the cell bottle reaches 80%, adding 0.25% pancreatin/0.02% EDTA to digest and separate the cells from the cell bottle, centrifuging at 1200rpm/min for 5 minutes, removing the pancreatin, adding a new culture medium for resuspension, diluting 1mL of cell suspension to 4mL, dripping 10 μ L of the cell suspension onto a cell counting plate, counting under a 40 × microscope, adjusting the concentration to 2.0-3.0 × 10 5 Cells/well cells were plated in 12-well plates and placed in an incubator for 24 hours.
(3) Taking out the culture plate, discarding the original culture solution in the wells, adding 1mL of test sample containing different concentrations into each well, blank control, and culturing for 24 + -1 h.
(4) Removing original culture solution, adding 1mL fresh culture solution, and culturing for 24 + -1 h
(5) Supernatants were collected and assayed for the inflammatory factors TGF-. beta.as well as the growth factors PDGF, FGF-2 and EGF8 using ELISA kits.
(6) Inflammatory factor test results
Supernatants were collected and tested for inflammatory factor TGF-. beta.as shown in Table 3 below.
TABLE 3 comparison of TGF-. beta.differences between different groups
*: the difference was statistically significant compared to the control group.
The release of inflammatory factors in the culture medium was statistically significant (P <0.05) at a concentration of 50%, 25% of the test substance in the sample group compared to the blank control group, and the sample caused the release of TGF- β inflammatory factors by corneal stromal fibroblasts at a concentration of 50%, 25%.
The sample was not cytotoxic and irritating to corneal stromal fibroblasts, but caused corneal stromal fibroblasts to release an inflammatory factor, with mild irritation as a result.
3. Prediction of corneal endothelial cell irritation
3.1 conventional culture of corneal endothelial cells: rabbit corneal primary endothelial cells.
(1) Sample preparation, adding 0.9% NaCl solution into eye drop to dilute to 50% of final concentration, and further diluting to 35%, 25%, 15%, 5%; the positive control is SDS, prepare 0.01%, 0.05%, 0.2%; the negative control was 0.9% NaCl solution.
(2) Plate paving: when the density of the corneal endothelial cells in the cell bottle reaches 80%, adding 0.25% pancreatin/0.02% EDTA to digest and separate the cells from the cell bottle, centrifuging at 1200rpm/min for 5 minutes, removing pancreatin, adding new culture medium for resuspension, diluting 1mL of cell suspension to 4mL, dropping 10 μ L of cell suspension on a cell counting plate, counting under a 40X microscope, adjusting concentrationDegree, in 2X 10 5 Wells cells were plated in 24-well plates and incubated in an incubator for 24 hours.
(3) Neutral Red Release (NRR) test: adding 0.05mg/mL neutral red dye into a 24-well plate, incubating for 3h +/-30 min at 37 +/-1 ℃, then removing the neutral red dye, and adding culture solution to stop for 30min +/-10 min. After changing the culture solution, washing 2-3 times with about 2mL buffer solution, adding 500 μ L of the test substance with concentration gradient to form a test substance group, simultaneously setting up a positive control group, a negative control group and a blank control group, and 2 multiple wells, directly contacting the test substance and the control group with cells for 1min, after exposure, washing 2-3 times with PBS, adding 1mL of analysis solution (acetic acid: 50% ethanol is 1: 99) into each well, stirring for 15min, transferring 200uL of each well to a 96-well plate, repeating 4 wells, and measuring the absorbance of the final solution at a reference wavelength of 405 nm.
(4) Calculating cell activity and inhibition according to the following formula, cell activity ═ (test absorbance value-blank absorbance value) ÷ (negative control absorbance value-blank absorbance value) × 100%; cell death rate ═ 1- (test absorbance-blank absorbance) ÷ (negative control absorbance-blank absorbance)]X 100%, the concentration of test substance that released 50% of preloaded cellular neutral red dye was calculated by the dose-cell activity curve. Toxicity endpoints are commonly used with NRR 50 Expressing; the PM value was calculated as (1-cell activity) × 100% cell death rate, which was calculated by a dose-cell death rate curve at a concentration of 50%.
(5) As a result:
| group of | OD value | Cellular activity |
| Blank control | 0.0379 | -- |
| Negative control | 0.7195 | 100.00％ |
| Positive control 0.01% | 0.4555 | 61.27％ |
| Positive control 0.05% | 0.2781 | 35.24％ |
| Positive control 0.2% | 0.2168 | 26.25％ |
| 5％dilution | 0.8187 | 114.19％ |
| 15％dilution | 0.7014 | 97.03％ |
| 25％dilution | 0.6455 | 88.85％ |
| 35％dilution | 0.6276 | 86.24％ |
| 50％dilution | 0.6342 | 87.20％ |
The dose-cell activity rate curve is: y is-0.5616 x +1.093 when y is 50%, x is 105.59%, i.e., NRR 50 。
The dose-cell mortality curve was: when x is 50%, y is 18.78%, which is PM.
Sample NRR 50 105.59% (greater than 50%) and a PM value of 18.78% (less than 20%), which was not cytotoxic and irritating to corneal endothelial cells.
The test of inflammatory factors is selected, according to the NRR test dose-cell activity rate curve, when the sample concentration is 52.17%, the cell activity is more than 80%, and the highest test concentration of the test substance with the cell activity more than 80% is selected as the test concentration of the inflammatory factors.
3.2 cytokine assay
(1) The sample preparation comprises diluting eye drop with 0.9% NaCl solution to a final concentration of 50%, further diluting to 25%, 12.5%, and blank control with 0.9% NaCl solution.
(2) Plate paving: when the density of corneal endothelial cells in the cell bottle reaches 80%, adding 0.25% pancreatin/0.02% EDTA to digest and separate the cells from the cell bottle, centrifuging at 1200rpm/min for 5 minutes, removing pancreatin, adding new culture medium for resuspension, diluting 1mL of cell suspension to 4mL, dripping 10 μ L of cell suspension on a cell counting plate, counting under a 40 × microscope, adjusting the concentration, and counting at 2.0-3.0 × 10 5 Cells were plated in 12-well plates and incubated in an incubator for 24 hours.
(3) Taking out the culture plate, discarding original culture solution in the wells, adding 1mL of test sample containing different concentrations into each well, blank control, and culturing for 24 + -1 h.
(4) Removing original culture solution, adding 1mL fresh culture solution, and culturing for 24 + -1 h
(5) Collecting supernatant, detecting inflammatory factor IL-1, and determining by using an ELISA kit.
(6) Inflammatory factor test results
The supernatants were collected and tested for the inflammatory factor IL-1, the results of which are given in Table 3 below.
TABLE 3 comparison of IL-1 differences between different groups
*: the difference was statistically significant compared to the control group.
The test substance concentration of the sample group is 50%, 25% and 12.5% compared with the blank control group, the release of the inflammatory factor in the culture solution is not statistically significant, and the sample does not cause the corneal endothelial cells to release the IL-1 inflammatory factor at the concentration of 50%, 25% and 12.5%.
The sample was non-cytotoxic to corneal stromal fibroblasts, non-irritating, and did not cause, but did not cause, release of inflammatory factors by corneal endothelial cells, and was non-irritating as a result.
4. Eye irritation prediction model
Example 3 evaluation of shampoo eye irritation of certain brands based on various corneal structure cytotoxicity methods
1. Corneal epithelial irritation prediction
1.1 conventional culture of corneal epithelial cells: primary epithelial cells of human cornea.
1.2 cytotoxicity assays
(1) Determination and preparation of the concentration of a test substance: 200mg of shampoo is weighed by an analytical balance, stock solution is prepared by taking a serum-free culture medium as a solvent, and 100mg/mL of mother solution is prepared for later use.
(2) Paving a plate: when the density of corneal epithelial cells in the cell bottle reaches 80%, adding 0.25% pancreatin/0.02% EDTA to digest and separate the cells from the cell bottle, centrifuging at 1200rpm/min for 5min, removing pancreatin, adding new culture medium for resuspension, taking 1mL of fine culture medium for resuspensionDiluting the cell suspension to 4mL, dropping 10. mu.L of the cell suspension onto a cell counting plate, counting under a 40 Xmicroscope, adjusting the concentration to 1X 10 4 Cells were plated in 96-well plates and incubated in an incubator for 24 hours.
(3) Addition of test substance: the final concentration of the shampoo is 100mg/mL, and the shampoo is diluted to 31.6mg/mL, 10mg/mL, 3.2mg/mL, 1mg/mL and 0.32 mg/mL. Diluted shampoo was added to 96-well plates, with blank control, negative control, and 6 secondary wells for each concentration. A multichannel pipettor was used to reduce the inter-well variation. And placing the mixture in an incubator to incubate for 18-22 hours. After the incubation was completed, the plate was removed from the incubator, and 20. mu.l of 5mg/mL MTT solution was added to each well, protected from light. Placing in an incubator to continue hatching for 4 h. After incubation, DMSO was added at 100 μ L per well, and the mixture was placed on a shaker for 10 min.
(4) Enzyme-linked immunosorbent assay and cell activity calculation: and (4) measuring the absorbance of each well added in the 96 wells in the step (3) at 570nm by using a microplate reader to obtain the absorbance value of the surviving cells of each well. Calculating IC by formula 50 (median inhibitory concentration, i.e. the concentration of test substance at 50% cell activity):
cell activity ═ [ (test absorbance value-blank absorbance value) ÷ (negative control absorbance value-blank absorbance value) ] × 100%
(5) Cytotoxicity assay: the test results were obtained as follows: the cell survival rate at 100mg/L was 50.17%, at 31.6 mg/L65.47%, at 10.01 mg/L74.91%, at 3.16 mg/L81.41%, at 1.00 mg/L85.54%, at 0.32 mg/L87.65%. The facial cleanser IC is obtained by statistics 80 It was 7.66 mg/mL. The test substance concentration with cytotoxicity > 80% was chosen as the highest concentration tested for Fluorescein Leakage (FL) testing.
1.3 Fluorescein Leakage (FL) test:
(1) cells were cultured at 2X 10 5 The/well was seeded in an embedded petri dish and when the cell density reached 80% the epithelial cells formed tight junctions and desmosomes. The cell layer divides the culture medium into two parts, the concentration of the test substance is set to be 6mg/mL (the concentration of the test substance with the cell activity of more than 80 percent is selected as the highest test concentration of the fluorescein leakage test, and the concentration can be selected as long as the concentration is more than 80 percent), and the concentration of the test substance is set to be 3mg/mL, 2mg/mL, 3 parallel holes are arranged, 0.4mL is added on the cell surface, after 15min +/-1 min, Hank's solution is used for gently washing the cell surface to remove a tested substance, the cell surface is put into another culture dish containing Hank's solution, 10 mu g/mL fluorescein sodium solution is added to the upper layer of cells, after incubation is carried out for 30min +/-5 min at 37 +/-1 ℃, a fluorescence spectrophotometer is used for detecting (excitation wavelength is 485nm, emission wavelength is 530nm) the amount of fluorescein permeating into a culture plate, namely the lower layer of cells, and the amount of the fluorescein is used as an index of the damage degree of a cell barrier. Since most of the cells were still viable, the cells were washed, placed on another dish containing fresh medium, incubated at 37 ℃. + -. 1 ℃ for 4 hours, and added with fluorescein sodium solution again, and examined to see the effect of the test substance on tight cell junctions. And sequentially detecting the influence of the 24h and 72h test substances on the cells, and calculating the percentage of the sodium fluorescein leaking out according to the following formula:
(2) fluorescein Leakage (FL) test results analysis:
dose-FL 20 The curve is: y is 0.0771x +0.0042, when y is 20%, x is 2.54g/100ml, namely FL 20 H 4
FL 20 H 4 The medium irritation is 2-10 g/100 mL.
2. Prediction of corneal stromal fibroblast irritation
2.1 conventional culture of corneal stromal fibroblasts: rabbit corneal stroma fibroblast RCBBF.
2.2 sample preparation, namely adding the shampoo into 0.9 percent NaCl solution with the final concentration of 50 percent, and diluting the solution into 35 percent, 25 percent, 15 percent and 5 percent; the positive control is SDS, prepare 0.01%, 0.05%, 0.2%; the negative control was 0.9% NaCl solution.
2.3, paving: when the density of the corneal stromal fibroblasts in the cell bottle is up to 80 percent, addingDigesting and separating cells from a cell flask by adding 0.25% pancreatin/0.02% EDTA, centrifuging at 1200rpm/min for 5 minutes, removing pancreatin, adding new culture medium for resuspension, diluting 1mL of cell suspension to 4mL, dropping 10 μ L of cell suspension on a cell counting plate, counting under a 40X microscope, adjusting concentration, and counting at 2X 10 5 Wells cells were plated in 24-well plates and incubated in an incubator for 24 hours.
2.4 Neutral Red Release (NRR) test: adding 0.05mg/mL neutral red dye into a 24-well plate, incubating for 3h +/-30 min at 37 +/-1 ℃, then removing the neutral red dye, and adding culture solution to stop for 30min +/-10 min. After changing the culture solution, washing 2-3 times with about 2mL buffer solution, adding 500 μ L of the test object with concentration gradient to form a test object group, simultaneously setting up a positive control group, a negative control group and a blank control group, and 2 multiple wells, directly contacting the test object and the control group with cells for 1min, after the exposure is finished, washing 2-3 times with PBS, adding 1mL of analysis solution (acetic acid: 50% ethanol is 1: 99) into each well, stirring for 15min, transferring 200uL of each well to a 96-well plate, repeating 4 wells, and measuring the absorbance of the final solution at a reference wavelength of 405 nm.
2.5 calculating the cell activity and inhibition according to the following formula, cell activity ═ (test absorbance value-blank absorbance value) ÷ (negative control absorbance value-blank absorbance value) × 100%; cell death rate ═ 1- (test absorbance-blank absorbance) ÷ (negative control absorbance-blank absorbance)]X 100%, the concentration of test agent that released 50% NRU from preloaded cellular neutral red dye was calculated by dose-cell activity curve. Toxicity endpoints are commonly used with NRR 50 Expressing; the PM value was calculated as (1-cell activity) × 100% cell death rate at 50% concentration by dose-cell death rate curve.
2.6 results:
| group of | OD value | Cellular activity |
| Blank control | 0.0325 | -- |
| Negative control | 0.9962 | 100.00％ |
| Positive control 0.01% | 0.6052 | 59.43％ |
| Positive control 0.05% | 0.6216 | 61.13％ |
| Positive control 0.2% | 0.2470 | 22.26％ |
| 5％dilution | 0.9344 | 93.58％ |
| 15％dilution | 0.8231 | 82.04％ |
| 25％dilution | 0.7755 | 77.10％ |
| 35％dilution | 0.7503 | 74.48％ |
| 50％dilution | 0.7404 | 73.45％ |
The dose-cell activity curve is: y is-0.4193 x +0.9103, when y is 50%, x is 97.85%, that is NRR 50 。
The dose-cell death rate curve is: 0.4193x +0.0897, when x is 50%, 29.94%, that is PM.
Sample NRR50 was 97.85% (greater than 50%) with a PM value of 29.94% (greater than 20%, less than 50%), which was slightly cytotoxic and slightly irritating to corneal stromal fibroblasts.
3. Prediction of corneal endothelial cell irritation
3.1 conventional culture of corneal endothelial cells: human umbilical vein endothelial cell line (HUVEC)
3.2 Neutral Red Release (NRR) test:
(1) sample preparation, namely adding 0.9 percent NaCl solution into the shampoo to dilute the shampoo to a final concentration of 50 percent, and continuously diluting the shampoo to 35 percent, 25 percent, 15 percent and 5 percent; the positive control is SDS, prepare 0.01%, 0.05%, 0.2%; the negative control was 0.9% NaCl solution.
(2) Plate paving: when the density of the corneal endothelial cells in the cell bottle reaches 80%, adding 0.25% pancreatin/0.02% EDTA to digest and separate the cells from the cell bottle, centrifuging at 1200rpm/min for 5 minutes, removing pancreatin, adding new culture medium for resuspension, diluting 1mL of cell suspension to 4mL, dropping 10 μ L of cell suspension on a cell counting plate, counting under a 40X microscope, adjusting the concentration, and counting at 2X 10 5 Cells were plated in 24-well plates and incubated in an incubator for 24 hours.
(3) Adding 0.05mg/mL neutral red dye into a 24-well plate, incubating for 3h +/-30 min at 37 +/-1 ℃, then removing the neutral red dye, and adding culture solution to stop for 30min +/-10 min. After changing the culture solution, washing 2-3 times with about 2mL buffer solution, adding 500 μ L of the test substance with concentration gradient to form a test substance group, simultaneously setting up a positive control group, a negative control group and a blank control group, and 2 multiple wells, directly contacting the test substance and the control group with cells for 1min, after exposure, washing 2-3 times with PBS, adding 1mL of analysis solution (acetic acid: 50% ethanol is 1: 99) into each well, stirring for 15min, transferring 200uL of each well to a 96-well plate, repeating 4 wells, and measuring the absorbance of the final solution at a reference wavelength of 405 nm.
(4) Calculating cell activity and inhibition according to the following formula, cell activity ═ (test absorbance value-blank absorbance value) ÷ (negative control absorbance value-blank absorbance value) × 100%; cell death rate ═ 1- (test absorbance-blank absorbance) ÷ (negative control absorbance-blank absorbance)]X 100%, the concentration of test substance that released 50% NRU from preloaded cellular neutral red dye was calculated by dose-cell activity curve. Toxicity endpoints are commonly used with NRR 50 Expressing; the PM value was calculated as (1-cell activity) × 100% cell death rate, which was calculated by a dose-cell death rate curve at a concentration of 50%.
(5) NRR test results
| Group of | OD value | Cellular activity |
| Blank control | 0.0335 | -- |
| Negative control | 0.9888 | 100.00％ |
| Positive control 0.01% | 0.6052 | 59.85％ |
| Positive control 0.05% | 0.6216 | 61.57％ |
| Positive control 0.2% | 0.2470 | 22.35％ |
| 5％dilution | 0.8821 | 88.16％ |
| 15％dilution | 0.9244 | 92.55％ |
| 25％dilution | 0.8767 | 87.60％ |
| 35％dilution | 0.8116 | 80.85％ |
| 50％dilution | 0.8185 | 81.56％ |
The dose-cell activity curve is: y is-0.2229 x +0.9194, and when y is 50%, x is 94.95%, i.e., NRR 50 。
The dose-cell mortality curve was: when x is equal to 50%, y is equal to 19.21%, i.e. PM.
Sample NRR50 was 188.12% (greater than 50%) and PM 19.21% (less than 20%), which was not cytotoxic and irritating to corneal endothelial cells.
And selecting the test concentration with the cell activity of more than 80% as the highest test concentration of the test substance with the cell activity of more than 80% as the test concentration of the inflammatory factor according to the NRR test dose-cell activity rate curve when the sample concentration is 53.57%.
3.3 inflammatory factor assay
(1) The sample preparation comprises diluting eye drop with 0.9% NaCl solution to a final concentration of 50%, further diluting to 25%, 12.5%, and blank control with 0.9% NaCl solution.
(2) Plate paving: when the density of corneal endothelial cells in the cell bottle reaches 80%, adding 0.25% pancreatin/0.02% EDTA to digest and separate the cells from the cell bottle, centrifuging at 1200rpm/min for 5 minutes, removing pancreatin, adding new culture medium for resuspension, diluting 1mL of cell suspension to 4mL, dripping 10 μ L of cell suspension on a cell counting plate, counting under a 40 × microscope, adjusting the concentration, and counting at 2.0-3.0 × 10 5 Cells were plated in 12-well plates and incubated in an incubator for 24 hours.
(3) Taking out the culture plate, discarding the original culture solution in the wells, adding 1mL of test sample containing different concentrations into each well, blank control, and culturing for 24 + -1 h.
(4) Removing original culture solution, adding 1mL fresh culture solution, and culturing for 24 + -1 h
(5) The supernatant was collected and TGF- β cytokine was assayed using an ELISA kit.
(6) Inflammatory factor test results
Supernatants were collected and assayed for the inflammatory factor TGF-. beta.with the results shown in Table 4 below.
TABLE 4 comparison of TGF-. beta.Difference between different groups
*: the difference was statistically significant compared to the control group.
The release of inflammatory factors in culture medium was statistically significant (P <0.05) at a sample group subject concentration of 50%, 25% compared to the blank control group, and at a concentration of 50%, 25% the sample caused corneal endothelial cells to release TGF- β inflammatory factors.
The sample is not cytotoxic and irritating to corneal endothelial cells, but causes the corneal endothelial cells to release inflammatory factors, with a mild irritation as a final result.
4-eye irritation prediction model
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents and are included in the scope of the present invention. The present invention is exemplified by a part of cosmetic materials and products, and ophthalmic drugs. However, other non-single component systems mentioned in the present invention, such as personal care products (shampoos, conditioners, hair dyes, skin care products, sunscreens, eye shadows, body washes, body lotions, etc.), household washing products (laundry detergents, washing powders, soaps, detergents, bleaches, disinfectants, softeners, etc.), pharmaceuticals, plant extracts (crude extracts, fine extracts and mixtures thereof), medical devices and extracts thereof, biological materials, environmental pollutants, and other eye contact products (textile extracts such as clothes, underwear, etc.), etc. that may contact the eyes can also be tested for skin sensitization using the method of the present invention. This is not further illustrated.
Claims (3)
1. A method for predicting eye irritation by using a plurality of cornea structural cell combinations is characterized by comprising the following steps:
(ii) prediction of corneal epithelial cell irritation
(1.1) determination of the highest test concentration: selecting corneal epithelial cells, and determining the concentration of a test substance with the cell activity of more than 80% by adopting an MTT method as the highest test concentration;
(1.2) Barrier function test: performing fluorescein leakage test by adopting the highest test concentration in the step (1.1), obtaining the leakage percentage of the fluorescein sodium, and calculating the concentration FL of the test substance which causes 20 percent of the fluorescein sodium to leak out after contacting with the test substance 20 Concentration FL of test substance with unit g/100mL and leakage according to 4h and 20% fluorescein sodium 20 H 4 And (3) performing irritation prediction: FL 20 H 4 No irritation or slight irritation is obtained when the concentration is more than 10g/100mL, medium irritation is obtained when the concentration is 2-10 g/100mL, and heavy irritation is obtained when the concentration is less than 2g/100 mL;
(II) prediction of corneal stromal fibroblast irritation
(2.1) Neutral Red Release (NRR) test: selecting angle matrix fibroblast, preparing the test substance according to gradient concentration, adding neutral red dye, performing absorbance test, calculating cell activity rate, and calculating the concentration of the test substance which enables the neutral red dye of the angle matrix fibroblast to release 50%, namely NRR, according to a dose-cell activity rate curve 50 And calculating the cell death rate at a subject concentration of 50%, i.e., the PM value, according to the dose-cell death rate curve, according to the NRR 50 And PM value to establish a model for predicting toxicity and irritation of corneal stromal fibroblasts as shown in the following Table 1:
TABLE 1 cytotoxicity determination and Ocular irritation prediction model
；
(2.2) inflammatory factor assay: when the prediction in the step (2.1) is classified as non-irritant, analyzing the expression of the inflammatory cytokines by enzyme-linked immunosorbent assay, if the expression does not cause any increase of the release of any inflammatory factor under a set concentration, judging the expression as non-irritant, if the expression causes at least one increase of the release of the inflammatory factor under the set concentration, judging the expression as mild irritative, and under any other conditions, judging the expression as irritative regardless of the increase of the release of the inflammatory factor;
(III) prediction of corneal endothelial cell irritation
The Neutral Red Release (NRR) test and the inflammatory factor test in the corneal stromal fibroblast irritation prediction in the step (II) are the same, except that the corneal endothelial cells are adopted, and the established ocular irritation prediction model and the inflammatory factor test irritation model are the same;
(IV) establishment of model for predicting eye irritation based on combination of multiple corneal structural cells
According to the results in the steps (a) to (c), the model for predicting the eye irritation based on the combination of the plurality of corneal structural cells is established as follows:
in the step (1.2), the leakage percentage of the fluorescein sodium is calculated by adopting the following formula:
m represents the mean value of the leakage amount of the fluorescein of each concentration of each tested object, y represents the mean value of the leakage amount of the fluorescein of a control group without the tested objects, and z represents the mean value of the leakage amount of the fluorescein of a control group without cell culture;
the inflammatory factors in the step (2.2) comprise one or more of TGF-beta, TGF-alpha, IL-1, IL-2, IL-8, FGF-2, EGF-8 and platelet-derived growth factors;
the set concentration in step (2.2) is the highest test concentration for inflammatory factor testing, where the concentration of the test substance with a cellular activity > 80% is selected as determined from the dose-cellular activity rate curve of the Neutral Red Release (NRR) test in step (2.1).
2. The method of claim 1, wherein the evaluation of ocular irritancy using a plurality of corneal structural cell combinations comprises: in the step (2.1), the test substance is prepared according to the gradient concentration of gradual dilution according to the following percentage: 50%, 35%, 25%, 15%, 5%.
3. The method for evaluating the prediction of eye irritability of a patient using a combination of a plurality of corneal structural cells as set forth in claim 2, wherein: the test substances in the step (1.1) comprise personal care products, household washing products, medicines, medical instruments and extracts thereof, biological materials, environmental pollutants and other eye contact products; wherein the personal care product comprises a shampoo, a conditioner, a hair dye, a skin care product, a sunscreen product, an eye shadow or a body wash, the household washing product comprises a laundry detergent, a washing powder, a soap, a liquid detergent, a bleaching agent, a disinfectant or a softener, and the plant extract comprises a plant crude extract, a plant fine extract and a mixture of the plant crude extract and the plant fine extract; the other eye contact products include textile extract garments.
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