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In-vitro skill model and keratinocyte cell long-acting culture solution

A technology of skin model and culture fluid, applied in the field of biomedicine, can solve problems such as inability to apply tests, excessive differentiation, and decreased vitality, and achieve the effect of promoting the proliferation of endothelial-like cells and good repairing effect

Inactive Publication Date: 2016-05-25
GUANGDONG BOXI BIO TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] The purpose of the present invention is to mainly aim at the problem that the vitality of the in vitro skin model will be reduced and over-differentiated during the long-term culture in vitro, which will lead to the problem that it cannot be applied to the test. The synergistic effect of culture fluid extract and glycolic acid provides a culture medium that can be used for in vitro skin models and long-term culture of keratinocytes

Method used

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  • In-vitro skill model and keratinocyte cell long-acting culture solution
  • In-vitro skill model and keratinocyte cell long-acting culture solution
  • In-vitro skill model and keratinocyte cell long-acting culture solution

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Experimental program
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Effect test

Embodiment 1

[0037] The long-acting culture of embodiment 1 epidermis skin model in vitro

[0038] Preparation of basal culture medium: select F12 and DMEM medium, prepare liquids according to the instructions, mix them according to the volume ratio of 1:3, add factors: 50μg / ml BPE, 50ng / ml EGF, 20μg / ml insulin, 0.05mmol / ml CaCl 2 ; Add embryonic stem cell culture extract 20mg / ml, glycolic acid 1×10 to the basic culture medium -7 After mol / L, it was sterilized by filtration with a 0.22 μm filter membrane and stored at 4°C for later use.

[0039] EpiKutis ? The epidermal skin model (purchased from Shaanxi Boxi Biotechnology Co., Ltd.) is used for the detection of cosmetic emulsions. The test requires samples to be loaded every day for 7 consecutive days to judge the skin irritation of cosmetic emulsions. The specific operation is as follows:

[0040] The prepared long-term culture solution (experimental group) and the construction culture solution (control group) were used to continu...

Embodiment 2

[0042] Long-acting culture of embodiment 2 keratinocytes

[0043] Basic culture solution preparation: K-SFM culture solution, add sterile factor: BPE5μg / ml to make basic culture solution; add 5ng / ml EGF, 1μg / ml insulin, 0.01mmol / ml CaCl to the basic culture solution 2 , sterile embryonic stem cell culture extract 0.01mg / ml, glycolic acid 1×10 -9 mol / L, filter and sterilize with a 0.22 μm filter membrane to make a long-term culture medium.

[0044] Human keratinocytes (purchased from the Tissue Engineering Center of Fourth Medical University) were cultured with K-SFM culture medium and long-term culture medium to P12 generation respectively, the medium was changed every day, and passaged every 48 hours, and then the β-galactosidase kit ( (purchased from Biyuntian Biotechnology Co., Ltd.) to detect cell aging.

[0045] The results showed that the keratinocytes cultured with K-SFM medium to the P12 generation had aged and died (attached Figure 7 ), the kit found that there ...

Embodiment 3

[0046] Long-acting culture of embodiment 3 in vitro full-thickness skin model

[0047] Preparation of basal culture medium: select MCDB153 medium, prepare it into liquid according to the instructions, add factors: 30μg / ml BPE, 30ng / ml EGF, 10μg / ml insulin, 0.03mmol / ml CaCl 2 ; Add embryonic stem cell culture extract 30mg / ml, glycolic acid 1×10 to the basic culture medium -5 mol / L, filter and sterilize with a 0.22 μm filter membrane.

[0048] EpiDermFT, a full-thickness skin model purchased from MatTeck TM To detect the anti-aging effect of licorice extract, the recommended test period is no more than 3 days. For this test, the full-thickness skin model that leaves the factory needs to be cultured with long-term culture medium. The experimental group is added with licorice extract (1mg / ml), and the control group is not. Added and cultured continuously for 7 days, during which the medium was changed every 24 hours. After the culture was over, the appearance of the model was ...

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Abstract

An in-vitro skill model and keratinocyte cell long-acting culture solution is composed of a basic culture solution, embryonic stem cell extracts and glycolic acid. The basic culture solution is any of K-SFM or MCDB153 or a mixture of F12 and DMEM with the volume ratio being 1:3, wherein BPE, EGF, insulin and CaCl2 are added. By means of the culture solution, the proliferation and differentiation speed of an in-vitro skill model and keratinocyte cells can be effectively balanced, the retention time of the in-vitro skill model which is successfully established can be effectively prolonged to be 7 days, and the in-vitro skill model is used for culturing the keratinocyte cells to the twelfth generation from the original generation. The time requirement of different detection conditions is greatly met, the application field of the in-vitro skill model is expanded, and meanwhile a basic condition is provided for epidermal cell aging and differentiation and other relevant mechanism research. The operating process is simple and convenient, use is convenient, and cost is low.

Description

technical field [0001] The invention belongs to the field of biomedicine and relates to an in vitro skin model and keratinocyte long-acting culture solution. Background technique [0002] The in vitro skin model is a piece of skin tissue constructed by applying tissue engineering technology to conditional culture of seed cells in vitro. This tissue has a high degree of similarity with human skin in terms of tissue structure and some biological functions, so it is widely used Applied to the efficacy and safety evaluation and testing of cosmetics, pharmaceuticals and other products and raw materials. Currently common in vitro skin models include: epidermal model, full-thickness skin model including dermis and epidermis, epidermal model containing melanocytes, etc. [0003] The commercially available in vitro skin models are directly stored in the culture medium after the construction is completed. The advertised period of use is mainly 3 days, but it is recommended to use it ...

Claims

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Application Information

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IPC IPC(8): C12N5/071
CPCC12N5/0629C12N2500/30C12N2501/11C12N2501/115C12N2501/125C12N2501/13C12N2501/15C12N2501/18C12N2501/33
Inventor 支旭勃张小波卢永波
Owner GUANGDONG BOXI BIO TECH CO LTD
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