Liu Chun-Yen, Matsusaki Michiya, Akashi Mitsuru
Department of Applied Chemistry Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, 565-0871, Osaka, Japan.
Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, 565-0871, Osaka, Japan.
Chem Rec. 2016 Apr;16(2):783-96. doi: 10.1002/tcr.201500272. Epub 2016 Feb 29.
Living tissues or organ modules consist of different types of highly organized cells and extracellular matrices (ECMs) in a hierarchical manner, such as the multilayered structure of blood vessels and the radial structures of hepatic lobules. Due to animal examinations being banned in the EU since 2013 and a shortage in the demand for tissue repair or organ transplantation, the creation of artificial 3D tissues possessing specific structures and functions similar to natural tissues are key challenges in tissue engineering. To date, we have developed a simple but unique bottom-up approach, a hierarchical cell manipulation technique, with a nanometer-sized ECM matrix consisting of fibronectin (FN) and gelatin (G) on cell surfaces. About 10 nm thick FN/G ECM films on cell surfaces were coated successfully by using layer-by-layer coating methodology. Various 3D constructs with higher cell density with different types of cells were successfully constructed. In addition to the construction of tissues with higher cell densities, other tissues, such as cartilage or skin tissues, with different cell densities are also important tissue models for tissue engineering and pharmaceutical industries. Thus, we recently developed other methodologies, the collagen coating method and multiple coating method, to fabricate micrometer-sized level ECM layers on cell surfaces. Various micro- or millimeter-sized 3D constructs with lower cell densities were constructed successfully. By using these two methods, cell distances in 2D or 3D views can be controlled by different thicknesses of ECM layers on cell surfaces at the single-cell level. Both FN/G and the collagen coating method resulted in homogenous 3D tissues with a controlled layer numbers, cell type, cell location, and properties; these will be promising to achieve different goals in tissue engineering.
活组织或器官模块由不同类型的高度组织化细胞和细胞外基质（ECM）以分层方式组成，例如血管的多层结构和肝小叶的放射状结构。由于自2013年起欧盟禁止动物实验，且组织修复或器官移植的需求短缺，创建具有与天然组织相似的特定结构和功能的人工3D组织是组织工程中的关键挑战。迄今为止，我们已经开发出一种简单但独特的自下而上的方法，即一种分层细胞操作技术，在细胞表面形成由纤连蛋白（FN）和明胶（G）组成的纳米级ECM基质。通过逐层涂覆方法成功地在细胞表面涂覆了约10nm厚的FN/G ECM膜。成功构建了具有不同细胞类型且细胞密度更高的各种3D构建体。除了构建细胞密度更高的组织外，其他具有不同细胞密度的组织，如软骨或皮肤组织，也是组织工程和制药行业重要的组织模型。因此，我们最近开发了其他方法，即胶原蛋白涂覆法和多重涂覆法，以在细胞表面制备微米级水平的ECM层。成功构建了各种细胞密度较低的微米级或毫米级3D构建体。通过使用这两种方法，可以在单细胞水平上通过细胞表面ECM层的不同厚度来控制二维或三维视图中的细胞间距。FN/G法和胶原蛋白涂覆法都产生了具有可控层数、细胞类型、细胞位置和特性的均匀3D组织；这些有望在组织工程中实现不同的目标。
