Osaki Tatsuya, Chow Siu Yu A, Nakanishi Yui, Hernández Joel, Kawada Jiro, Fujii Teruo, Ikeuchi Yoshiho
Institute of Industrial Science, The University of Tokyo; Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo.
Institute of Industrial Science, The University of Tokyo; Faculty of Science and Engineering, Tecnologico de Monterrey.
J Vis Exp. 2020 Sep 24(163). doi: 10.3791/61544.
A fascicle of axons is one of the major structural motifs observed in the nervous system. Disruption of axon fascicles could cause developmental and neurodegenerative diseases. Although numerous studies of axons have been conducted, our understanding of formation and dysfunction of axon fascicles is still limited due to the lack of robust three-dimensional in vitro models. Here, we describe a step-by-step protocol for the rapid generation of a motor nerve organoid (MNO) from human induced pluripotent stem (iPS) cells in a microfluidic-based tissue culture chip. First, fabrication of chips used for the method is described. From human iPS cells, a motor neuron spheroid (MNS) is formed. Next, the differentiated MNS is transferred into the chip. Thereafter, axons spontaneously grow out of the spheroid and assemble into a fascicle within a microchannel equipped in the chip, which generates an MNO tissue carrying a bundle of axons extended from the spheroid. For the downstream analysis, MNOs can be taken out of the chip to be fixed for morphological analyses or dissected for biochemical analyses, as well as calcium imaging and multi-electrode array recordings. MNOs generated with this protocol can facilitate drug testing and screening and can contribute to understanding of mechanisms underlying development and diseases of axon fascicles.
轴突束是在神经系统中观察到的主要结构模式之一。轴突束的破坏可能导致发育性和神经退行性疾病。尽管已经对轴突进行了大量研究，但由于缺乏强大的三维体外模型，我们对轴突束形成和功能障碍的理解仍然有限。在这里，我们描述了一种在基于微流控的组织培养芯片中从人诱导多能干细胞（iPS）快速生成运动神经类器官（MNO）的分步方案。首先，描述了该方法所用芯片的制作过程。从人iPS细胞中形成运动神经元球体（MNS）。接下来，将分化的MNS转移到芯片中。此后，轴突从球体中自发长出，并在芯片中配备的微通道内组装成束，从而生成携带一束从球体延伸出的轴突的MNO组织。对于下游分析，可以将MNO从芯片中取出进行固定以进行形态学分析，或进行解剖以进行生化分析，以及钙成像和多电极阵列记录。用该方案生成的MNO可以促进药物测试和筛选，并有助于理解轴突束发育和疾病的潜在机制。
