Miyamoto Yoshitaka, Ikeuchi Masashi, Noguchi Hirofumi, Yagi Tohru, Hayashi Shuji
Department of Advanced Medicine in Biotechnology and Robotics, Nagoya University Graduate School of Medicine, Tsurumai-cho, Showa-ku, Nagoya, Japan; †Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo, Japan.
†Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo, Japan; ‡PRESTO, Japan Science and Technology (JST), Saitama, Japan.
Cell Med. 2015 Aug 26;8(1-2):47-56. doi: 10.3727/215517915X689056. eCollection 2015 Dec 17.
In drug discovery, it is very important to evaluate liver cells within an organism. Compared to 2D culture methods, the development of 3D culture techniques for liver cells has been successful in maintaining long-term liver functionality with the formation of a hepatic-specific structure. The key to performing drug testing is the establishment of a stable in vitro evaluation system. In this article, we report a Tapered Stencil for Cluster Culture (TASCL) device developed to create liver spheroids in vitro. The TASCL device will be applied as a toxicity evaluation system for drug discovery. The TASCL device was created with an overall size of 10 mm × 10 mm, containing 400 microwells with a top aperture (500 µm × 500 µm) and a bottom aperture (300 µm diameter circular) per microwell. We evaluated the formation, recovery, and size of HepG2 spheroids in the TASCL device. The formation and recovery were both nearly 100%, and the size of the HepG2 spheroids increased with an increase in the initial cell seeding density. There were no significant differences in the sizes of the spheroids among the microwells. In addition, the HepG2 spheroids obtained using the TASCL device were alive and produced albumin. The morphology of the HepG2 spheroids was investigated using FE-SEM. The spheroids in the microwells exhibited perfectly spherical aggregation. In this report, by adjusting the size of the microwells of the TASCL device, uniform HepG2 spheroids were created, and the device facilitated more precise measurements of the liver function per HepG2 spheroid. Our TASCL device will be useful for application as a toxicity evaluation system for drug testing.
在药物研发中，评估生物体内的肝细胞非常重要。与二维培养方法相比，肝细胞三维培养技术的发展已成功通过形成肝脏特异性结构来维持长期肝脏功能。进行药物测试的关键是建立稳定的体外评估系统。在本文中，我们报告了一种用于集群培养的锥形模板（TASCL）装置，该装置用于在体外创建肝脏球体。TASCL装置将用作药物研发的毒性评估系统。TASCL装置的整体尺寸为10毫米×10毫米，每个微孔包含400个微孔，顶部孔径为（500微米×500微米），底部孔径为（直径300微米的圆形）。我们评估了TASCL装置中HepG2球体的形成、恢复和大小。形成率和恢复率均接近100%，HepG2球体的大小随着初始细胞接种密度的增加而增大。各微孔中球体的大小没有显著差异。此外，使用TASCL装置获得的HepG2球体具有活性并能产生白蛋白。使用场发射扫描电子显微镜（FE-SEM）研究了HepG2球体的形态。微孔中的球体呈现出完美的球形聚集。在本报告中，通过调整TASCL装置微孔的大小，创建了均匀的HepG2球体，并且该装置有助于更精确地测量每个HepG2球体的肝功能。我们的TASCL装置将作为药物测试的毒性评估系统很有用。
