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Artificial three-dimensional epidermis model, and construction method and construction medium thereof

A culture medium and three-dimensional technology, applied in artificial cell constructs, tissue culture, cell culture active agents, etc., can solve the problems of skin irritation test verification, insufficient attention and lag in research on alternative methods, and achieve obvious effects of stratum corneum differentiation

Pending Publication Date: 2018-05-11
SHANGHAI JAHWA UNITED
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] The 3R principle (Reduce, Replace, Refine) has promoted global research on in vitro skin models, but domestic animal experiments are cheap and widely available, resulting in insufficient research on alternative methods to replace animal experiments in my country Pay attention to, relatively lag behind
However, the skin irritation tests of the chemicals in the above studies have not been verified according to the technical guidelines issued by ECVAM (European Center for Alternative Methods)

Method used

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  • Artificial three-dimensional epidermis model, and construction method and construction medium thereof
  • Artificial three-dimensional epidermis model, and construction method and construction medium thereof
  • Artificial three-dimensional epidermis model, and construction method and construction medium thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0103]

[0104] Human epidermal keratinocytes prepared as above were used as seed cells, digested and separated as above, and then proliferated and differentiated by the above-mentioned gas-liquid phase culture.

[0105] Wherein, the differentiation medium used is containing 10% Fetal Clone II serum, 10ng / ml EGF1, 10 -10 M cholera toxin, 1 μg / ml hydrocortisone, 5 μg / ml bovine insulin, 10 -10 M triiodothyronine, and 5 μg / ml gentamicin in DMEM / F12 medium.

[0106] After being replaced with differentiation medium, the culture was continued for 30 days, and then taken out for paraffin section staining. The slice dyeing figure of the artificial three-dimensional epidermis model of embodiment 1 is as follows figure 1 shown.

[0107] figure 1 The stratum corneum constructed by normal human primary keratinocytes is very obvious and multi-layer differentiated, and the thickness of the stratum corneum is obviously enhanced after 30 days of air-liquid phase culture. Many experimen...

Embodiment 2

[0108]

[0109] Human epidermal keratinocytes prepared as above were used as seed cells, digested and separated as above, and then proliferated and differentiated by the above-mentioned gas-liquid phase culture.

[0110] Wherein, the differentiation medium used is containing 12% Fetal Clone II serum, 12ng / ml EGF1, 10 -11 M cholera toxin, 1.2 μg / ml hydrocortisone, 4 μg / ml bovine insulin, 10 -10 M triiodothyronine, and 5 μg / ml gentamicin in DMEM / F12 medium.

[0111] After being replaced with differentiation medium, the culture was continued for 30 days, and then taken out for paraffin section staining. figure 2 The stratum corneum constructed by normal human primary keratinocytes is relatively obvious, and the thickness of the stratum corneum is obviously enhanced after 30 days of gas-liquid phase culture. figure 2 The black arrows in indicate the thickness of the constructed epidermis.

Embodiment 3

[0112]

[0113] Human epidermal keratinocytes prepared as above were used as seed cells, digested and separated as above, and then proliferated and differentiated by the above-mentioned gas-liquid phase culture.

[0114] Wherein, the differentiation medium used is containing 8% Fetal Clone II serum, 8ng / ml EGF1, 10 -9 M cholera toxin, 0.8 μg / ml hydrocortisone, 6 μg / ml bovine insulin, 10 -10 M triiodothyronine, and DMEM / F12 medium of 5 μg / ml gentamicin.

[0115] After being replaced with differentiation medium, the culture was continued for 30 days, and then taken out for paraffin section staining. The section dyeing figure and the artificial three-dimensional epidermis model of embodiment 1 figure 1 similar.

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Abstract

The invention relates to an artificial three-dimensional epidermis model, and a construction method and a construction medium thereof, and concretely relates to a method for constructing an artificialthree-dimensional epidermis model by separated normal human epidermis cutin cells and an artificial scaffold, and the artificial three-dimensional epidermis model constructed by the method. The method for constructing the artificial three-dimensional epidermis model adopts the medium containing cholera toxins and Fetal Clone II serum.

Description

technical field [0001] The invention relates to a method for constructing an artificial three-dimensional epidermal model and the artificial three-dimensional epidermal model constructed by the method. Specifically, it relates to a method for constructing an artificial three-dimensional epidermal model from isolated normal human epidermal keratinocytes and an artificial scaffold, and the artificial three-dimensional epidermal model constructed by the method. The stratum corneum of the artificial three-dimensional epidermis model constructed by the method of the invention is obviously differentiated, and there are multiple layers of stratum corneum differentiated. Background technique [0002] Skin medicines, daily chemicals (detergents, cleaners), and cosmetics are closely related to life, and their quality and safety should be guaranteed. For these products, skin irritation is one of the most common adverse reactions in daily use, so the skin irritation test is the main it...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
CPCC12N5/0629C12N2501/01C12N2533/30
Inventor 冯冰魏少敏马来记
Owner SHANGHAI JAHWA UNITED
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