Construction of Three-Dimensional Dermo-Epidermal Skin Equivalents Using Cell Coating Technology and Their Utilization as Alternative Skin for Permeation Studies and Skin Irritation Tests
Construction of Three-Dimensional Dermo-Epidermal Skin Equivalents Using Cell Coating Technology and Their Utilization as Alternative Skin for Permeation Studies and Skin Irritation Tests
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DOI：
10.1089/ten.tea.2016.0529
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发表时间：
2017-06-01
影响因子：
4.1
通讯作者：
Akashi, Mitsuru
中科院分区：
文献类型：
--
作者：
Akagi, Takami;Nagura, Mayuka;Akashi, Mitsuru
体外生成的人类皮肤替代物作为基础研究中有前途的工具，作为动物试验的替代品，以及再生医学的临床应用，正在引起人们的兴趣。为了预测皮肤刺激和腐蚀，已经开发了由分化的人角质形成细胞（KCs）组成的三维人体皮肤等效物，并且一些模型已经被国际上接受。然而，由于缺乏含有成纤维细胞或附件的真皮，无法使用全层皮肤模型进行更精细的评估，如皮肤致敏试验。在以前的研究中，我们开发了真皮表皮人类皮肤等效物（DESE）使用细胞涂层技术，采用细胞表面涂层逐层组装的细胞外基质（ECM）薄膜。利用该技术可以很容易地制备出真皮由正常人真皮成纤维细胞（NHDFs）组成，表皮由人KC组成的DESE。在这项研究中，构建的DESE作为皮肤渗透和刺激试验的替代皮肤进行了评价。在DESE和人体皮肤数据之间观察到化学品渗透系数的良好关系。我们研究了DESE，一种新的体外皮肤模型，是否能够在20种参考化学品中识别皮肤刺激性和非刺激性物质。经证实，DESE适用于欧洲替代方法验证中心（ECVAM）性能标准（OECD试验指南439）中定义的皮肤刺激试验。我们进一步研究了使用人脐静脉内皮细胞（HUVECs）构建具有密度控制的毛细血管网络的DESE。结果表明，DESE允许纳入皮肤附件是更有前途的替代动物试验，并可应用于设计生理相关的体外皮肤模型。
In vitro generated human skin equivalents are generating interest as promising tools in basic study, as alternatives to animal testing, and for clinical applications in regenerative medicine. For prediction of skin irritation and corrosion, three-dimensional human skin equivalents consisting of differentiated human keratinocytes (KCs) have been developed and some models have been internationally accepted. However, more delicate assessments using full-thickness skin models, such as skin sensitization tests, cannot be performed due to the lack of a dermis containing fibroblasts or appendages. In a previous study, we developed dermo-epidermal human skin equivalents (DESEs) using a cell coating technique, which employs cell surface coating by layer-by-layer assembled extracellular matrix (ECM) films. The DESEs with dermis consisting of normal human dermal fibroblasts (NHDFs) and epidermis consisting of human KCs were easily fabricated by using this technology. In this study, the constructed DESEs were evaluated as an alternative skin for skin permeation and irritation tests. A good relationship of permeability coefficient of chemicals was observed between the DESEs and human skin data. We investigated whether the DESEs, a new in vitro skin model, are capable of identifying skin irritant and nonirritant substances among 20 reference chemicals. It was confirmed that the DESEs are applicable to skin irritation testing as defined in the European Centre for the Validation of Alternative Methods (ECVAM) Performance Standard (OECD Test Guideline 439). We further studied the construction of DESEs with density-controlled blood capillary networks using human umbilical vein endothelial cells (HUVECs). The results suggest that DESEs allowing incorporation of skin appendages are more promising alternatives to animal testing and can be applied to the design of physiologically relevant in vitro skin models.