Cutaneous sensitization detecting method based on co-culturing mode of three dimensional skin model and dendric cell

Abstract

The invention discloses a cutaneous sensitization detecting method based on a co-culturing mode of a three dimensional skin model and dendric cells. The cutaneous sensitization detecting method comprises the following steps : (1) preparing of the skin model in an early stage, (2) preparing and culturing of dendric cells, (3) constructing of co-culturing of the skin model and the dendric cells and exposing to-be-tested substances, (4) testing of activity of the skin model, (5) detecting of genome of the skin model, (6) detecting of secretion of lower layer cells of the co-culturing model, (7) detecting of expression of surface markers on the dendric cells, and (8) predicting of a statistic method and result. According to the cutaneous sensitization detecting method based on the co-culturing mode of the three dimensional skin model and the dendric cells, 3D skin which is reconstructed in vitro and has functions of barrier and metabolism and the dendric cells with the immunity function are combined together, a co-cultured novel experiment system is constructed and has similar functions and sensitization reaction with human bodies; the cutaneous sensitization of the to-be-tested substances can be evaluated from qualitative and quantitive aspects; and living animals and human bodies can be replaced through the method, and possible cutaneous sensitization caused by chemicals, cosmetics and drugs can be predicted directly.

Description

technical field

[0001] The invention belongs to the technical field of toxicity detection and research of skin sensitizers, and in particular relates to a skin sensitization detection method based on a three-dimensional skin model and a dendritic cell co-culture mode. Background technique

[0002] Skin sensitization, also known as Allergy Contact Dermatitis (ACD), is an immunogenic skin reaction of the skin to exogenous substances. Clinical manifestations of skin sensitization include itching, erythema, papules, and edema. For mass-produced industrial chemicals, regulations require testing for possible skin sensitization; similarly, for products that may come into contact with skin, including cosmetics, disinfection products, hygiene products and medical devices, etc. Skin sensitization was assessed by testing. The traditional method uses the guinea pig experiment, through the two stages of induction and excitation to smear the test substance on the skin of the animal, and...

Examples

Embodiment 1

[0134] Example 1 Chemical cinnamaldehyde skin sensitization test based on Episkin skin model and THP-1 cell co-culture

[0135] Include the following steps in turn:

[0136] 1. Skin model incubation

[0137] The commercial three-dimensional skin model Episkin was purchased, and the model of this batch was a 12-well plate. The skin model is transported to the laboratory by the courier company, and the transport temperature is 10-25°C. After the skin model arrives at the laboratory, check whether the outer packaging is intact, and check the temperature monitoring strip in the box to ensure that the transport temperature of the skin model is within the allowable fluctuation range. The outer package of the skin model was sterilized with alcohol and opened. Use tweezers to carefully move the skin model hanging basket into a 12-well culture plate to which 2 mL of phenol red-containing MEM medium had been added. Store at 37°C, 5% CO 2 Incubate for 24 hours under the culture condit...

Embodiment 2

[0169] Example 2 Skin sensitization test of plant extracts based on Altskin skin model and U937 cell co-culture

[0170] Include the following steps in turn:

[0171] 1. Skin model incubation

[0172] Use Altskin, a three-dimensional skin model made in our laboratory, which is a full-thickness skin model in a 24-well plate. The production department is responsible for transporting it to the laboratory, and after the skin model arrives at the laboratory, check whether the outer packaging is intact. Open the outer package of the skin model after disinfecting it with alcohol, use tweezers to carefully move the skin model insert dish into a 24-well culture plate to which 2 mL of phenol red-containing MEM medium has been added, and store at 37°C, 5% CO 2 Incubate for 24 hours under the culture condition of 95% humidity, and conduct the experiment after the activity of the skin model is stable.

[0173] 2. U937 cell culture:

[0174] Take out the EP tube containing U937 cells fr...

Embodiment 3

[0210] Example 3 Based on Epiderm skin model and THP-1 cell co-culture skin sensitization test of petroleum products lubricants

[0211] 1. Skin model incubation

[0212] The commercial three-dimensional skin model Epiderm was purchased, and this batch of models is a 12-well plate epidermis model. The skin model is transported to the laboratory by the courier company, and the transport temperature is 10-25°C. After the skin model arrives at the laboratory, check whether the outer packaging is intact, and check the electronic thermometer in the box to ensure that the transport temperature of the skin model is within the allowable fluctuation range. The outer package of the skin model was sterilized with alcohol and opened. Use tweezers to carefully move the skin model hanging basket into a 12-well culture plate to which 2 mL of phenol red-containing MEM medium had been added. Store at 37°C, 5% CO 2 Incubate for 24 hours under the culture condition of 95% humidity, and conduct...

technical field

[0001] The invention belongs to the technical field of toxicity detection and research of skin sensitizers, and in particular relates to a skin sensitization detection method based on a three-dimensional skin model and a dendritic cell co-culture mode. Background technique

[0002] Skin sensitization, also known as Allergy Contact Dermatitis (ACD), is an immunogenic skin reaction of the skin to exogenous substances. Clinical manifestations of skin sensitization include itching, erythema, papules, and edema. For mass-produced industrial chemicals, regulations require testing for possible skin sensitization; similarly, for products that may come into contact with skin, including cosmetics, disinfection products, hygiene products and medical devices, etc. Skin sensitization was assessed by testing. The traditional method uses the guinea pig experiment, through the two stages of induction and excitation to smear the test substance on the skin of the animal, and...

Examples

Embodiment 1

[0134] Example 1 Chemical cinnamaldehyde skin sensitization test based on Episkin skin model and THP-1 cell co-culture

[0135] Include the following steps in turn:

[0136] 1. Skin model incubation

[0137] The commercial three-dimensional skin model Episkin was purchased, and the model of this batch was a 12-well plate. The skin model is transported to the laboratory by the courier company, and the transport temperature is 10-25°C. After the skin model arrives at the laboratory, check whether the outer packaging is intact, and check the temperature monitoring strip in the box to ensure that the transport temperature of the skin model is within the allowable fluctuation range. The outer package of the skin model was sterilized with alcohol and opened. Use tweezers to carefully move the skin model hanging basket into a 12-well culture plate to which 2 mL of phenol red-containing MEM medium had been added. Store at 37°C, 5% CO 2 Incubate for 24 hours under the culture condit...

Embodiment 2

[0169] Example 2 Skin sensitization test of plant extracts based on Altskin skin model and U937 cell co-culture

[0170] Include the following steps in turn:

[0171] 1. Skin model incubation

[0172] Use Altskin, a three-dimensional skin model made in our laboratory, which is a full-thickness skin model in a 24-well plate. The production department is responsible for transporting it to the laboratory, and after the skin model arrives at the laboratory, check whether the outer packaging is intact. Open the outer package of the skin model after disinfecting it with alcohol, use tweezers to carefully move the skin model insert dish into a 24-well culture plate to which 2 mL of phenol red-containing MEM medium has been added, and store at 37°C, 5% CO 2 Incubate for 24 hours under the culture condition of 95% humidity, and conduct the experiment after the activity of the skin model is stable.

[0173] 2. U937 cell culture:

[0174] Take out the EP tube containing U937 cells fr...

Embodiment 3

[0210] Example 3 Based on Epiderm skin model and THP-1 cell co-culture skin sensitization test of petroleum products lubricants

[0211] 1. Skin model incubation

[0212] The commercial three-dimensional skin model Epiderm was purchased, and this batch of models is a 12-well plate epidermis model. The skin model is transported to the laboratory by the courier company, and the transport temperature is 10-25°C. After the skin model arrives at the laboratory, check whether the outer packaging is intact, and check the electronic thermometer in the box to ensure that the transport temperature of the skin model is within the allowable fluctuation range. The outer package of the skin model was sterilized with alcohol and opened. Use tweezers to carefully move the skin model hanging basket into a 12-well culture plate to which 2 mL of phenol red-containing MEM medium had been added. Store at 37°C, 5% CO 2 Incubate for 24 hours under the culture condition of 95% humidity, and conduct...

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