A kind of construction method of 3D epidermis model

Abstract

The invention belongs to the technical field of tissue engineering and particularly relates to a construction method of a 3D epidermis model. According to the method, a fibroblast treated by using mitomycin C (Mitomycin C, MC) is adopted as a trophoblast and inoculated with a keratinocyte, various factors and proteins secreted by the fibroblast are supplied to the keratinocyte for growth requirements, and meanwhile, low-concentration serum, EGF, insulin and CaCl2 are matched to construct the 3D epidermis model through two phases of submerged culture and gas-liquid surface culture. The constructed 3D epidermis model is highly consistent with a human epidermis structure, the production cost is reduced, and the economical, effective and efficient requirements required by large-scale production of the 3D epidermis model are met.

Description

technical field

[0001] The invention belongs to the technical field of tissue engineering, and in particular relates to a method for constructing a 3D epidermis model. Background technique

[0002] Traditional cosmetic raw material screening and product development verification are mainly done through animal experiments. Due to species differences and individual differences, the results of animal experiments cannot accurately evaluate their safety and efficacy, and the reproducibility of the results is poor. Animal welfare organizations are also increasingly vocal about boycotting animal experiments. In view of the above reasons, the European Union, the United States and other countries have successively legislated to prohibit the sale of cosmetics or raw materials tested on animals in their markets. In order to meet the needs of cosmetics development, effectively evaluate their safety and efficacy, and promote the upgrading of the cosmetics industry, animal substitution tes...

Examples

Embodiment 1

[0033] The construction method of the 3D epidermis model of the present embodiment comprises the following steps:

[0034] Step 1. Resuscitate P2 generation human fibroblasts, perform routine culture on the revived fibroblasts, digest and passage after the cell plating rate reaches about 85%, and inoculate the culture medium containing serum at a density of 6×104 / cm2 after counting the cells After cultivating in a 37°C, 5% CO2 incubator for 24 hours, the culture solution was discarded, and then the culture solution containing mitomycin C was added, treated in a carbon dioxide incubator for 2 hours, and the culture solution was discarded. The hot PBS buffer solution gently washes the cells 3 times to obtain a fibroblast trophoblast; the serum-containing culture medium is based on a low-sugar DMEM culture medium, and fetal bovine serum is added, and the fetal bovine serum in the serum-containing culture medium is The volume percentage of serum is 10%; the culture fluid containin...

Embodiment 2

[0038] The construction method of the 3D epidermis model of the present embodiment comprises the following steps:

[0039] Step 1. Resuscitate the P2 generation human fibroblasts, perform routine culture on the revived fibroblasts, digest and passage after the cell plating rate reaches about 85%, and inoculate the cultured cells containing serum at a density of 3×104 / cm2 after counting the cells After cultivating in a 37°C, 5% CO2 incubator for 18 hours, the culture solution was discarded, and then the culture solution containing mitomycin C was added, treated in a carbon dioxide incubator for 3 hours, and the culture solution was discarded. Gently wash the cells with hot PBS buffer solution 5 times to obtain a fibroblast trophoblast; the serum-containing culture medium is based on α-MEM culture medium, and fetal bovine serum is added, and the fetal bovine serum in the serum-containing culture medium is The volume percentage of serum is 5%; the culture solution containing mito...

Embodiment 3

[0043] The construction method of the 3D epidermis model of the present embodiment comprises the following steps:

[0044] Step 1. Resuscitate P2 generation human fibroblasts, perform routine culture on the revived fibroblasts, digest and passage after the cell plating rate reaches about 85%, and inoculate the cultured cells containing serum at a density of 5×104 / cm2 after counting the cells After cultivating in a 37°C, 5% CO2 incubator for 20 hours, the culture solution was discarded, and then the culture solution containing mitomycin C was added, treated in a carbon dioxide incubator for 6 hours, and the culture solution was discarded. The hot PBS buffer solution gently washed the cells 4 times to obtain a fibroblast trophoblast; the serum-containing culture medium was based on DF12 culture medium, and fetal bovine serum was added, and the content of fetal bovine serum in the serum-containing culture medium was The volume percent content is 15%; the culture solution containi...

technical field

[0001] The invention belongs to the technical field of tissue engineering, and in particular relates to a method for constructing a 3D epidermis model. Background technique

[0002] Traditional cosmetic raw material screening and product development verification are mainly done through animal experiments. Due to species differences and individual differences, the results of animal experiments cannot accurately evaluate their safety and efficacy, and the reproducibility of the results is poor. Animal welfare organizations are also increasingly vocal about boycotting animal experiments. In view of the above reasons, the European Union, the United States and other countries have successively legislated to prohibit the sale of cosmetics or raw materials tested on animals in their markets. In order to meet the needs of cosmetics development, effectively evaluate their safety and efficacy, and promote the upgrading of the cosmetics industry, animal substitution tes...

Examples

Embodiment 1

[0033] The construction method of the 3D epidermis model of the present embodiment comprises the following steps:

[0034] Step 1. Resuscitate P2 generation human fibroblasts, perform routine culture on the revived fibroblasts, digest and passage after the cell plating rate reaches about 85%, and inoculate the culture medium containing serum at a density of 6×104 / cm2 after counting the cells After cultivating in a 37°C, 5% CO2 incubator for 24 hours, the culture solution was discarded, and then the culture solution containing mitomycin C was added, treated in a carbon dioxide incubator for 2 hours, and the culture solution was discarded. The hot PBS buffer solution gently washes the cells 3 times to obtain a fibroblast trophoblast; the serum-containing culture medium is based on a low-sugar DMEM culture medium, and fetal bovine serum is added, and the fetal bovine serum in the serum-containing culture medium is The volume percentage of serum is 10%; the culture fluid containin...

Embodiment 2

[0038] The construction method of the 3D epidermis model of the present embodiment comprises the following steps:

[0039] Step 1. Resuscitate the P2 generation human fibroblasts, perform routine culture on the revived fibroblasts, digest and passage after the cell plating rate reaches about 85%, and inoculate the cultured cells containing serum at a density of 3×104 / cm2 after counting the cells After cultivating in a 37°C, 5% CO2 incubator for 18 hours, the culture solution was discarded, and then the culture solution containing mitomycin C was added, treated in a carbon dioxide incubator for 3 hours, and the culture solution was discarded. Gently wash the cells with hot PBS buffer solution 5 times to obtain a fibroblast trophoblast; the serum-containing culture medium is based on α-MEM culture medium, and fetal bovine serum is added, and the fetal bovine serum in the serum-containing culture medium is The volume percentage of serum is 5%; the culture solution containing mito...

Embodiment 3

[0043] The construction method of the 3D epidermis model of the present embodiment comprises the following steps:

[0044] Step 1. Resuscitate P2 generation human fibroblasts, perform routine culture on the revived fibroblasts, digest and passage after the cell plating rate reaches about 85%, and inoculate the cultured cells containing serum at a density of 5×104 / cm2 after counting the cells After cultivating in a 37°C, 5% CO2 incubator for 20 hours, the culture solution was discarded, and then the culture solution containing mitomycin C was added, treated in a carbon dioxide incubator for 6 hours, and the culture solution was discarded. The hot PBS buffer solution gently washed the cells 4 times to obtain a fibroblast trophoblast; the serum-containing culture medium was based on DF12 culture medium, and fetal bovine serum was added, and the content of fetal bovine serum in the serum-containing culture medium was The volume percent content is 15%; the culture solution containi...

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