近年來許多研究顯示細胞培養於二維與三維的環境下具有不同的形態與基因表現。將細胞培養於由胞外基質構成的三維空間擁有更貼近生物體內微環境之性質。然而如何觀察三維環境下的細胞仍然是目前研究有待克服與改進的問題。為了由光學以及電學之角度研究神經網路在三維培養環境中的動態變化，我們建立了一個結合微電極陣列與三維神經細胞培養的平台，並改良至可以。實驗細胞取自Sprague-Dawley遠交群大鼠之初生幼鼠，並與Matrigel 1:1混合構成三維細胞培養。藉由 MAP II/GFAP 免疫螢光染色與Live/Dead細胞活性測定方法可得知細胞的純度與活性。神經細胞與神經膠質細胞的比例約為1:1，細胞則在體外培養至七天後仍具有72%的存活率。三維基質直接貼附於蓋玻片並且設置在載玻片上，可以直接套用在雙光子顯微系統上。由雙光子顯微鏡產生之影像可以觀察到神經在三維環境中的生長發育變化，神經細胞在體外培養的七天中會逐漸伸展其神經突，與其周圍細胞做連結並有聚集的傾向，細胞的密度與體積亦有增加的趨勢。電特性方面則是藉由微電極陣列進行電阻抗量測，藉由阻抗分析儀量測記錄三維細胞培養於一到七天，頻率在1 kHz至1 MHz的阻抗值。結果顯示細胞於三維基質上的生長的同時可觀察到電阻抗值之增加。

There is growing number of research showed that cells culture in three-dimensions (3D) and two-dimensions (2D) have different morphologies and gene expressions. 3D cell culture has been believed to resemble closer to the in vivo microenvironment. However, the observation of 3D cell culture is challenged. To study the dynamics of neuronal network formation optically and electrically in 3D cell culture, the platform combined microelectrode array (MEA) and 3D neuronal cell culture is constructed. Cells were obtained from neonatal Sprague-Dawley (SD) rat and mixed with Matrigel 1:1 to form 3D cell culture. The purity and viability of cells is tested by MAP II/GFAP immunohistochemistry and Live/Dead cell viability kit. The ratio of neurons and glial cells is around 1:1 and Cell viability is about 72% after 7 days in vitro (DIV). Since 3D matrices were adhered on standard cover slip and setup on glass slip, it can be adapted to two-photon microscopy directly. From two-photon images of neuronal cell culture in 3D Matrigel, dynamics of neuronal cells can be observed. Our results showed that the neurons can extend their neurites, connect with other cells and aggregate in 7 DIV of 3D cell culture. The cells density and volume inside the Matrigel also have tendency to increase. Embedding the MEAs into the 3D cell culture, impedance was recorded in the frequency range between 1 kHz and 1 MHz using LCR meter during 7 DIV. Results show that impedance was increased as the cells grew in the 3D matrices.

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