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Construction of the arterial blood vessel model by layer-by-layer technique and its potential application for regenerative medicine

机译：逐层技术构建动脉血管模型及其在再生医学中的潜在应用

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Introduction: Since the establishment of induced pluripotent stem (iPS) cells, the in vitro construction of 3D tissues has been eagerly studied for potential application as regenerative medicine and drug screening assays. Especially, the arterial blood vessel is one of the most important tissues in our body as it carries the blood away from the heart to the body. Arterial diseases such as arteriosclerosis are life-ttireating issues, and thus the arterial blood vessel model has been eagerly required. In our previous study, we developed arterial blood vessel model by hierarchical cell manipulation method using Layer-by-Layer (LbL) technique . Fibronectin (FN) and gelatin (G) were alternately coated on the cells to form ECM nanofilm. Recently, we have developed cell accumulation method, which coat a single cell with ECM nanofilm and could achieve rapid construction of 3D tissues . In this study, we constructed arterial blood vessel model using these LbL and tissue construction methods and evaluated the potential application for drug screening assays. Material and Method: Arterial blood vessel model was constructed by LbL techniques using cell accumulation method . Fibroblast cells from artery were coated with FN-G nanofilm and incubated for 1 day to construct fibroblast layers. Then, smooth muscle cells (SMC) were also coated with ECM film and seeded on the fibroblast layers, and incubated for 1 day. After the top of SMC layer was coated with FN-G nanofilm, endothelial cells (EC) were seeded on it and incubated for 1 day. The structure of arterial blood vessel model was characterized by immunostaining. Result and Discussion: By employing LbL techniques, the arterial blood vessel model showed hierarchical structure made of fibroblast layers, SMC layers, and EC layer which was similar structure as the arterial blood vessels in our body. Each layer was confirmed by immunostaining of SMC and EC (Figure 1). We evaluated the formation of tight junction in EC layer, and the permeation test using small compounds was also conducted to demonstrate the barrier function of arterial model. Conclusion: Our results demonstrated that this model could be applicable for not only drug screening assays, but also tissue engineering.
机译：简介：自从建立诱导多能干（iPS）细胞以来，人们一直在热切地研究3D组织的体外构建，以作为再生医学和药物筛选测定法的潜在应用。特别是，动脉血管是我们体内最重要的组织之一，因为它可以将血液从心脏输送到身体。诸如动​​脉硬化的动脉疾病是危及生命的问题，因此迫切需要动脉血管模型。在我们先前的研究中，我们使用分层（LbL）技术通过分层细胞操作方法开发了动脉血管模型。将纤连蛋白（FN）和明胶（G）交替包被在细胞上以形成ECM纳米膜。最近，我们开发了一种细胞蓄积方法，该方法可以用ECM纳米膜覆盖单个细胞，并可以实现3D组织的快速构建。在这项研究中，我们使用这些LbL和组织构建方法构建了动脉血管模型，并评估了药物筛选测定的潜在应用。材料与方法：采用细胞蓄积法，通过LbL技术建立动脉血管模型。用FN-G纳米膜包被来自动脉的成纤维细胞，并温育1天以构建成纤维细胞层。然后，平滑肌细胞（SMC）也涂有ECM膜并接种在成纤维细胞层上，并孵育1天。在SMC层的顶部涂上FN-G纳米膜后，将内皮细胞（EC）接种在其上并孵育1天。通过免疫染色来表征动脉血管模型的结构。结果与讨论：通过采用LbL技术，动脉血管模型显示出由成纤维细胞层，SMC层和EC层组成的分层结构，其结构与人体中的血管相似。通过对SMC和EC进行免疫染色来确认每一层（图1）。我们评估了EC层中紧密连接的形成，并且还使用小分子化合物进行了渗透测试，以证明动脉模型的屏障功能。结论：我们的结果表明，该模型不仅可用于药物筛选测定，而且可用于组织工程。

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