Bio-Acoustic MEMS in Medicine (BAMM) Laboratory, Canary Center at Stanford for Cancer Early Detection, Department of Radiology, Stanford School of Medicine, Palo Alto, CA, 94304, USA.
Bio-Acoustic MEMS in Medicine (BAMM) Laboratory, Canary Center at Stanford for Cancer Early Detection, Department of Radiology, Stanford School of Medicine, Palo Alto, CA, 94304, USA; Institute of Toxicology and Gentics, Karlsruhe Institute of Technology, Hermann-von-Helmholtz-Platz, Eggenstein-Leopoldshafen, 76344, Germany.
Biomaterials. 2019 Mar;197:171-181. doi: 10.1016/j.biomaterials.2019.01.017. Epub 2019 Jan 11.
The replication of the complex structure and three dimensional (3-D) interconnectivity of neurons in the brain is a great challenge. A few 3-D neuronal patterning approaches have been developed to mimic the cell distribution in the brain but none have demonstrated the relationship between 3-D neuron patterning and network connectivity. Here, we used photolithographic crosslinking to fabricate in vitro 3-D neuronal structures with distinct sizes, shapes or interconnectivities, i.e., milli-blocks, micro-stripes, separated micro-blocks and connected micro-blocks, which have spatial confinement from "Z" dimension to "XYZ" dimension. During a 4-week culture period, the 3-D neuronal system has shown high cell viability, axonal, dendritic, synaptic growth and neural network activity of cortical neurons. We further studied the calcium oscillation of neurons in different 3-D patterns and used signal processing both in Fast Fourier Transform (FFT) and time domain (TD) to model the fluorescent signal variation. We observed that the firing frequency decreased as the spatial confinement in 3-D system increased. Besides, the neuronal synchronization significantly decreased by irregularly connecting micro-blocks, indicating that network connectivity can be adjusted by changing the linking conditions of 3-D gels. Earlier works showed the importance of 3-D culture over 2-D in terms of cell growth. Here, we showed that not only 3-D geometry over 2-D culture matters, but also the spatial organization of cells in 3-D dictates the neuronal firing frequency and synchronicity.
大脑中神经元的复杂结构和三维（3-D）连接的复制是一个巨大的挑战。已经开发了一些 3-D 神经元图案化方法来模拟大脑中的细胞分布，但没有一种方法能够证明 3-D 神经元图案化与网络连接之间的关系。在这里，我们使用光刻交联技术制造具有不同大小、形状或连通性的体外 3-D 神经元结构，即毫块、微带、分离的微块和连接的微块，其空间限制从“Z”维度到“XYZ”维度。在 4 周的培养期间，3-D 神经元系统表现出高细胞活力、轴突、树突、突触生长和皮质神经元的神经网络活性。我们进一步研究了不同 3-D 图案中神经元的钙振荡，并使用快速傅里叶变换（FFT）和时域（TD）中的信号处理来模拟荧光信号的变化。我们观察到，随着 3-D 系统中空间限制的增加，放电频率降低。此外，不规则连接微块会导致神经元同步性显著降低，表明通过改变 3-D 凝胶的连接条件可以调整网络连接性。早期的工作表明 3-D 培养在细胞生长方面优于 2-D 培养。在这里，我们不仅表明了 3-D 几何形状优于 2-D 培养，而且 3-D 中细胞的空间组织也决定了神经元的放电频率和同步性。
