Mashimo Yasumasa, Yoshioka Momoko, Tokunaga Yumie, Fockenberg Christopher, Terada Shiho, Koyama Yoshie, Shibata-Seki Teiko, Yoshimoto Koki, Sakai Risako, Hakariya Hayase, Liu Li, Akaike Toshihiro, Kobatake Eiry, How Siew-Eng, Uesugi Motonari, Chen Yong, Kamei Ken-Ichiro
Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University; Department of Life Science and Technology, School of Life Science and Technology, Tokyo Institute of Technology.
Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University.
J Vis Exp. 2018 Sep 7(139):57377. doi: 10.3791/57377.
Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are well orchestrated and are crucial in regulating cell functions in a living system. Although a number of researchers have attempted to investigate the correlation between environmental factors and desired cellular functions, much remains unknown. This is largely due to the lack of a proper methodology to mimic such environmental cues in vitro, and simultaneously test different environmental cues on cells. Here, we report an integrated platform of microfluidic channels and a nanofiber array, followed by high-content single-cell analysis, to examine stem cell phenotypes altered by distinct environmental factors. To demonstrate the application of this platform, this study focuses on the phenotypes of self-renewing human pluripotent stem cells (hPSCs). Here, we present the preparation procedures for a nanofiber array and the microfluidic structure in the fabrication of a Multiplexed Artificial Cellular MicroEnvironment (MACME) array. Moreover, overall steps of the single-cell profiling, cell staining with multiple fluorescent markers, multiple fluorescence imaging, and statistical analyses, are described.
细胞微环境由多种信号组成，如生长因子、细胞外基质和细胞间相互作用。这些信号被精心编排，对调节生命系统中的细胞功能至关重要。尽管许多研究人员试图研究环境因素与所需细胞功能之间的相关性，但仍有许多未知之处。这主要是由于缺乏一种合适的方法来在体外模拟这种环境信号，同时在细胞上测试不同的环境信号。在此，我们报告了一个微流控通道和纳米纤维阵列的集成平台，随后进行高内涵单细胞分析，以研究由不同环境因素改变的干细胞表型。为了证明该平台的应用，本研究聚焦于自我更新的人类多能干细胞（hPSC）的表型。在此，我们展示了在制造多重人工细胞微环境（MACME）阵列过程中纳米纤维阵列和微流控结构的制备程序。此外，还描述了单细胞分析、用多种荧光标记物进行细胞染色、多重荧光成像和统计分析的总体步骤。
